GB2563899A - Inorganic polyphosphate formulations for use in the treatment of Alzheimer disease - Google Patents

Abstract

A pharmaceutical composition comprising inorganic polyphosphate for use in the prevention or treatment of Alzheimer disease or other neurodegenerative disorders in a mammal or for the improvement of memory function of the ageing brain. The polyphosphate may be in the form of nanoparticles or microparticles consisting of salts of said polyphosphate with divalent cations, wherein they are produced by: dissolving sodium polyphosphate in an aqueous medium and adjusting the pH to about 10.0 with sodium hydroxide solution to form a polyphosphate solution; dissolving a salt of a suitable divalent cation in an aqueous medium or an aqueous ethanol solution to form a divalent cation solution; slowly adding said polyphosphate solution to said divalent cation salt solution while keeping the pH between about 8.2 to 10.5; forming nanoparticles or microparticles, preferably by stirring of the suspension overnight at room temperature; and collecting said nanoparticles or microparticles, preferably by filtration and washing and drying said particles. The divalent cation may be selected from calcium, magnesium and strontium.

Description

INORGANIC POLYPHOSPHATE FORMULATIONS FOR USE IN THE TREATMENT OF ALZHEIMER DISEASE

This invention concerns a novel method towards therapy of Alzheimer disease (AD) a degenerative disorder characterized by an impaired energy homeostasis of brain tissue. The inventive method is based on the use of inorganic polyphosphate (polyP) which physiologically exists both extracellularly and intracellularly, and measures to enhance the level of this high-energy polymer in brain tissue. The inventor demonstrates that treatment of neuronal cells with polyP protects these cells against the neurotoxic effect of the Alzheimer peptide Αβ25/35. The strongest effect is observed with amorphous microparticles prepared from the calcium salt of polyP (Ca-polyP-MP). The soluble sodium salt, Na-polyP, in the presence of calcium ions is also, but less, active. Ca-polyP microparticles and Na-polyP (plus calcium ions) markedly increase the cellular ATP level. The neuroprotective effect of polyP and Ca-polyP-MP according to this invention is based on the recovery of the compromised energy state in neuronal cells of AD patients caused by the β-amyloid-induced decrease of ATP level but not on the interaction of polyP with the Αβ25/35 filament formation as shown in pre-incubation experiments. PolyP and its nontoxic and biodegradable Ca-salts, as well as drugs increasing the polyP level of the aging brain, provide a promising strategy for treatment of AD.

Background of the Invention

There is a dramatic increase in age-dependent neurodegenerative disorders, in particular Alzheimer’s disease (AD). The number of AD patients has been estimated to be around 25 million worldwide and it is predicted to reach around 115 million in 2050. Current medications of AD relying on drugs targeting the cholinergic system turned out to have only a limited efficacy. Moreover, 98% of drugs which entered in clinical trials for AD have failed in the last 20 years. These numbers demonstrate that there is an urgent need for new effective therapies and drug candidates. AD is characterized by deposition of amyloid-β (Αβ) peptides in the brain and leads to progressive memory loss and dementia. The two proteins Αβ and Tau are characteristic signatures of AD (reviewed in: A, Bauer B, Hartz AM (2012) ABC transporters and the Alzheimer's disease enigma. Front Psychiatry 3:54). Αβ is a small peptide fragment generated through cleavage from the amyloid precursor protein (APP). The most common Αβ isoforms in AD are the neurotoxic peptides Αβ40 and Αβ42. Tau proteins are microtubule-associated proteins that interact with tubulin by promoting the insertion into microtubules resulting in a modulation of the stability as well as the flexibility of axonal microtubules. In AD the hyperphosphorylation of Tau causes a collapse of the microtubular system and formation of neurofibrillary tangles.

Due to the fact that the etiologic agent(s) and the metabolic targets for AD have not been identified unequivocally no causative cure can be offered. In turn, only agents have been developed that temporarily ameliorate the disease especially with respect to memory and thinking problems, while their clinical effect remains modest (Cummings J, Aisen PS, DuBois B, Frolich L, Jack CR Jr, Jones RW, Morris JC, Raskin J, Dowsett SA, Scheltens P (2016) Drug development in Alzheimer's disease: the path to 2025. Alzheimers Res Ther 8:39). Five agents have been accepted with sufficient safety and efficacy to allow marketing approval: four cholinesterase inhibitors (Chase TN (2015) High-dose cholinesterase inhibitor treatment of Alzheimer's disease. Alzheimers Dement 1 l(Supplement):466-467) and memantine (Informed Health Online (2016) Alzheimer's disease: Does memantine help? Internet https://www.ncbi.nlm.nih.gov/pubmedhealth/PMH0072540/). With respect to the action of the latter drug the inventor could demonstrate that memantine prevents neuronal apoptosis due to modulation of the NMDA [A-methyl-D-aspartate] receptor (Muller WEG, Schroder HC, Ushijima H, Dapper J, Bormann J (1992) Gpl20 of HIV-1 induces apoptosis in rat cortical cell cultures: Prevention by memantine. Europ J Pharmacol 226: 209-214).

In general neurons and glia cells consume high levels of ATP in the brain. Since no energy storage system, like fat or glucose, is available in the central nervous system, there is a high demand for a continuous ATP supply that must be guaranteed to maintain energy homeostasis. A decrease and impairment of energy supply and a disruption in bioenergy homeostasis have been implicated in various neuropathological conditions, like ischemia, stroke, and AD. It has been firmly described that oxidative stress in brain is associated with aging in general and AD pathogenesis in particular; it plays a crucial role in the progression of the disease (Ansari MA, Scheff SW (2010) Oxidative stress in the progression of Alzheimer disease in the frontal cortex. J Neuropathol Exp Neurol 69:155-167).

The inventor previously reported that the level of inorganic polyphosphate [polyP] in the brain of rats decrease with age (Lorenz B, Mtinkner J, Oliveira MP, Kuusksalu A, Leitao JM, Muller WEG, Schroder HC (1997) Changes in metabolism of inorganic polyphosphate in rat tissues and human cells during development and apoptosis. Biochim Biophys Acta 1335: 51-60). Now, very recently this concept has been extended by the hypothesis that the decline of polyP might contribute to the known pre-disposition of the elderly to neurodegenerative amyloid diseases (Cremers CM, Knoefler D, Gates S, Martin N, Dahl JU, Lempart J, Xie L, Chapman MR, Galvan V, Southworth DR, Jakob U (2016) Polyphosphate: A conserved modifier of amyloidogenic processes. Mol Cell 63:768-780). This polymer, polyP, consisting of up to 1,000 phosphoanhydride bond-linked phosphate monomers, has been detected in all prokaryotic and eukaryotic organisms; the concentration in mammalian tissues is approximately 50 μΜ (in terms of Pi residues), equivalent to 5 mg/g of tissue. Especially high is the concentration of polyP in blood platelets, where polyP is encapsulated into dense granules at approximately 130 mM. Intracellularly polyP is present in the cytosol and in 100-200 nm sized acidocalcisomes and is secreted into the extracellular space by platelets, astrocytes, and bacteria; there a steady-state concentration of 1 to 3 μΜ polyP can be calculated (Morrissey JH, Choi SH, Smith SA (2012) Polyphosphate: An ancient molecule that links platelets, coagulation, and inflammation. Blood 119:5972-5979).

Recently, it has been reported that polyP accelerates the fibril formation of both bacterial and human proteins, e.g. α-synuclein, Αβ1-40/42, and Tau, and contributes to the fibril stability without affecting the fibril yield (Cremers CM, Knoefler D, Gates S, Martin N, Dahl JU, Lempart J, Xie L, Chapman MR, Galvan V, Southworth DR, Jakob U (2016) Polyphosphate: A Conserved modifier of amyloidogenic processes. Mol Cell 63:768-780).

The inventive method concerns the neuroprotective effect of two polymer preparations on neuronal cells: (i) amorphous microparticles fabricated from the Ca2+ salt of polyP (Ca-polyP-MP) and (ii) the soluble Na+ salt of polyP (Na-polyP); both preparations contain polyP with an average chain length of 40 Pi. Crystalline Ca-phosphate nanoparticles (Ca-phosphate-NP), prepared from sodium orthophosphate and tested for comparison, were ineffective.

Both rat pheochromocytoma cells [PC 12 cells] as well as primary cortical neurons have been used. As an inducer of Αβ-related cell toxicity an Αβ fragment, displaying amyloidogenic ability, Αβ25/35, was used. This peptide retains the full neurotoxicity of the full-length Αβ peptide (Sato K, Wakamiya A, Maeda T, Noguchi K, Takashima A, Imahori K (1995) Correlation among secondary structure, amyloid precursor protein accumulation, and neurotoxicity of amyloid beta(25-35) peptide as analyzed by single alanine substitution. J Biochem 118:1108-1111) and has the same amphiphilic nature and ability to form β-sheet-rich fibrils. In a previous study we reported that the Αβ25/35 sample needs to stay in distilled water in a stock solution (900 μΜ) for 5 d to develop full toxicity (Muller WEG, Romero FJ, Perovic S, Pergande G, Pialoglou P (1997) Protection of flupirtine on B-amyloid-induced apoptosis in neuronal cells in vitro. Prevention of amyloid-induced glutathione depletion. J Neurochem 68: 2371-2377; Muller WEG, Pialoglou P, Romero FJ, Perovic S, Pergande G (1996) Protective effect of the drug flupirtine on β-amyloid-induced apoptosis in primary neuronal cells in vitro. J Brain Res 37: 575-577).

The technology for the fabrication of a morphogenetically active amorphous calcium-polyP microparticles (Ca-polyP MP) that unexpectedly turned out to suppress the neurotoxic effect of the Alzheimer peptide in neuronal cell has been described by the inventors. This material is obtained by co-precipitating of Na-polyP with CaCk; under those conditions amorphous calcium polyP microparticles (Ca-polyP MP) with a size range of 150 to 250 nm are formed (Muller WEG, Tolba E, Schroder HC, Wang S, GlaBer G, Munoz-Espi R, Link T, Wang XH (2015) A new polyphosphate calcium material with morphogenetic activity. Mater Lett 148:163-166). These microparticles turned out to be biologically active (Wang XH, Schroder HC, Muller WEG (2016) Polyphosphate as a metabolic fuel in Metazoa: A foundational breakthrough invention for biomedical applications. Biotechnol. J 11:11-30).

The preparation of Na-polyP complexed in a stoichiometric ratio to Ca (molar ratio of 2 [with respect to the phosphate monomer]; abbreviated as Na-polyP[Ca ]) has been described in (Muller WEG, Wang XH, Diehl-Seifert B, Kropf K, SchloBmacher U, Lieberwirth I, Glasser G, Wiens M, Schroder HC (2011) Inorganic polymeric phosphate/polyphosphate as an inducer of alkaline phosphatase and a modulator of intracellular Ca level in osteoblasts (SaOS-2 cells) in vitro. Acta Biomater 7:2661-2671).

The state-of-the-art of polyP has been described, for example, in: Muller WEG, Tolba E, Schroder HC, Wang XH (2015) Polyphosphate: a morphogenetically active implant material serving as metabolic fuel for bone regeneration. Macromolec Biosci 15:1182-1197.

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Here, the inventor applied three phosphate formulations, Na-polyP, complexed to Ca (Na-polyP[Ca ]), orthophosphate nanoparticles (Ca-phosphate-NP), and finally Ca-polyP microparticles (Ca-polyP-MP). While Na-polyP[Ca ] is highly soluble, the two particle preparations, Ca-phosphate-NP and Ca-polyP-MP, are less soluble. The orthophosphate particles are crystalline, while the polyP particles are amorphous.

The inventor demonstrated that the three phosphate formulations did not cause any significant adverse effect on viability of PC12 cells. Further, the inventor showed that exposure of the cells to the neurotoxic Alzheimer Αβ25/35 peptide fragment (after activation of this fragment by aggregate formation during a several-hours pre-incubation period), resulted in a strong reduction of cell survival; after 6 h incubation, an about 50% reduction was measured. In addition, the inventor showed that addition of the polyP preparations to the Αβ25/35 peptide during the pre-incubation period (aggregate/fibril formation), before the exposure of the cells, has, if at all, only a small effect on reduction of the cell viability by this fragment.

Surprising and basically unexpected is the finding of the inventor that the Aβ25/35-mediated cell toxicity is significantly reduced by polyP if the polymer is added to the cells 24 h prior to the addition of the Αβ fragment. The reduction by Na-polyP[Ca2+] amounted to 20% (PC12 cells). Even no toxic effect of Αβ25/35 is measured if the cells were pre-incubated with Ca-polyP-MP for 24 h before exposed to the toxic fragment.

The cytoprotective effect of Ca-polyP-MP against Αβ25/35 exposure was confirmed with primary embryonic rat cortex neurons.

In subsequent experiments, the inventor found that the Ca-polyP preparations induces ATP synthesis in brain-like cells (PC 12 cells) as well as brain cells (primary cortex neurons). Based on these finding the inventor concluded that the Ca-polyP preparations protect brain cells against the toxic effect elicited by Αβ25/35 through upregulation of the ATP pool.

By contrast, all formulations containing monomeric phosphate (orthophosphate) instead of polyP did not cause any significant alteration.

Most likely, in the extracellular space the upregulation of the intracellular as well as the extracellular ATP is caused by degradation of polyP by the alkaline phosphatase (ALP; Lorenz B, Schroder HC (2001) Mammalian intestinal alkaline phosphatase acts as highly active exopolyphosphatase. Biochim Biophys Acta 1547:254-261). Intracellularly polyP transfers the metabolic energy stored in its energy-rich phosphoanhydride bonds to AMP or ADP; the additional enzyme required for this process, the adenylate kinase, exists in both in the intracellular and extracellular compartments.

The data underlying this invention qualify the polyP and its various formulations for application in therapy in Alzheimer disease or related disorders of the aging brain, which are characterized by an energy deficiency.

The following patent applications on polyP are deemed relevant: GB1420363.2. Morphogenetically active calcium polyphosphate nanoparticles. Inventor: Muller WEG; GB1502116.5. Synergistically acting amorphous calcium-polyphosphate nanospheres containing encapsulated retinol for therapeutic applications. Inventor: Muller WEG; and GB1403899.6. Synergistic composition comprising quercetin and polyphosphate for treatment of bone disorders. Inventors: Muller WEG, Schroder HC, Wang XH.

This invention concerns a method for the protection of neuronal cells against the neurotoxic effect of Alzheimer peptides based on the use of inorganic polyP in various formulations.

Thus, the object is solved by providing a pharmaceutical composition comprising inorganic polyphosphate for use in the prevention or treatment of Alzheimer disease or other neurodegenerative disorders in a mammal, or for the improvement of memory function of the ageing brain.

Unexpectedly, the inventor found that polyP nanoparticles or microparticles, prepared from Ca-polyP, effectively block the toxic effect elicited by the toxic Alzheimer peptide fragment Αβ25/35. This inhibition is attributed to an elevation of intracellular ATP. A decreased ATP level has been described to be one factor for neuronal cell death in AD.

The increase the ATP level by the polyP nanoparticles or microparticles is relevant both intracellularly and extracellularly. In the extracellular space, ATP, or equivalent metabolic energy, is required for the heat shock protein-mediated folding of the AD amyloid precursor protein.

The method for the preparation of the amorphous calcium-polyP nanoparticles or microparticles (Ca-polyP-MP) and the properties of these particles have been disclosed in a previous patent application GB1420363.2. Only the use as a bone regeneration material has been described (e.g., GB 1406840.7. Morphogenetically active hydrogel for bioprinting of bioartificial tissue [Inventors: Muller WEG, Schroder HC, Wang XH]; and GB 1403899.6. Synergistic composition comprising quercetin and polyphosphate for treatment of bone disorders [Inventors: Muller WEG, Schroder HC, Wang XH]).

Based on the known effects of polyP on bone formation, an application of polyP for therapy of AD was unthinkable. It could not be anticipated that Ca-polyP-MP, as well as, at a lower extent, the soluble sodium salt Na-polyP (in the presence of calcium ions) strongly reduce the toxic effect of Αβ25/35 on neuronal cells. This effect was only found after pre-incubation of the cells with the polyP preparation before exposure to Αβ25/35 but not after pre-incubation of Αβ25/35 with polyP before cell exposure. In addition, only the amorphous polyP microparticles but not crystalline Ca-phosphate nanoparticles were effective. A further aspect of this invention is that, besides Ca-polyP-MP, polyP nanoparticles or microparticles consisting of salts of polyP with other divalent cations can be used, which are prepared according to the following method: a) Dissolution of sodium polyphosphate in distilled water and adjustment to pH 10.0 with sodium hydroxide solution. b) Dissolution of a salt of a divalent cation in a suitable aqueous medium or aqueous ethanol. c) Dropwise addition of the polyphosphate solution to the divalent cation salt solution and keeping the pH at around pH 8.2 to 10.5 with sodium hydroxide solution. d) Formation of the particles preferably by stirring of the suspension overnight at room temperature e) Collection of the particles preferably by filtration, washing with aqueous ethanol, and drying. A further aspect of this invention relates to the pharmaceutical composition for use according to the present invention, wherein said polyphosphate is used in the form of nanoparticles or microparticles consisting of salts said polyphosphate with divalent cations, wherein said nanoparticles or microparticles are produced according to a method comprising the steps of a) Dissolving of sodium polyphosphate in an aqueous medium, e.g. in distilled water, and adjusting to pH of about 10.0 with a suitable buffer, e.g. a sodium hydroxide solution, to form a polyphosphate solution; b) Dissolving of a salt of a suitable divalent cation in a suitable aqueous medium or an aqueous ethanol solution, to form a divalent cation salt solution; c) Slowly (e.g. dropwise) adding said polyphosphate solution to said divalent cation salt solution while keeping the pH at between about 8.2 to 10.5; d) Forming of nanoparticles or microparticles, preferably by stirring of the suspension overnight at room temperature; and e) Collecting said nanoparticles or microparticles, preferably by filtration, and washing and drying said particles.

Preferred is the pharmaceutical composition for use according to the present invention, wherein said divalent cation is selected from calcium, magnesium and strontium.

The chain length of the polyP can be in the range of about 3 to about 1000 phosphate units, preferably in the range of about 10 to about 100 phosphate units, and most preferred about 40 phosphate units.

The preferred composition of the polyP nanoparticles or microparticles with calcium or another divalent cation (magnesium or strontium) used in the inventive method is a stoichiometric ratio between about 4 to about 0.8 (calcium to phosphate, or divalent cation to phosphate), preferably between about 3 and about 1.5, and most preferred by a stoichiometric ratio of about 2. Preferably, said polyphosphate nanoparticles or microparticles are characterized by a stoichiometric ratio of between about 4 to about 0.8 (cation to phosphate), preferably of between about 3 and about 1.5, and most preferred of about 2.

The preferred average size of the calcium polyphosphate nanoparticles or microparticles is in the range of about 100 to about 1000 nm, and most preferred about 150 to about 250 nm.

In addition to the polyP nanoparticles of microparticles consisting of a divalent cation (preferably calcium) salt of polyP, the polymer can also be used in the form of the soluble sodium salt in the presence of calcium ions.

In the context of the present invention, the term “about” shall mean +/- 10 % of a given value, unless indicated otherwise. A further aspect of the inventive method concerns the application of the polyP formulations for the delivery of polyP to brain via the blood-brain barrier, preferably as Ca-polyP nanoparticles or microparticles. A further aspect of the inventive method concerns the pharmaceutical composition for use according to the invention, which is suitable for the delivery of said polyphosphate via the blood-brain barrier, such as, for example, selected from liposomes, micelles, dendrimers, comprising β-cyclodextrin carriers, carbon nanotubes, and chitosan-based nanomers. That is, in addition, a state-of-the-art drug delivery system can be used for the delivery of polyP to brain via the blood-brain barrier. This delivery system can consist, for example, of liposomes, micelles, dendrimers, β-cyclodextrin carriers, carbon nanotubes, or chitosan based nanomers.

The delivery of polyP nanoparticles/microparticles across the blood-brain barrier appears to be facilitated, since this structural barrier undergoes dysfunction during the AD progression (Zenaro E, Piacentino G, Constantin G (2016) The blood-brain barrier in Alzheimer's disease. Neurobiol Dis. 2016 Jul 15). A further aspect of the inventive method is the application of the polyP formulations for the delivery of polyP to brain via the blood-brain barrier together with drugs that modulate (activate) the brain ALP activity, which is involved in ADP or ATP formation from polyP, either together or by administration of the drugs via a separate state-of-the-art drug delivery system. A further aspect of the inventive method concerns the pharmaceutical composition for use according to the invention, wherein said pharmaceutical composition further comprises a drug that modulates the activity of the brain alkaline phosphatase involved in ADP or ATP formation from polyphosphate. A further aspect of the inventive method concerns the pharmaceutical composition for use according to the invention, wherein said pharmaceutical composition is administered simultaneously or sequentially with a pharmaceutical composition comprising a drug that modulates the activity of the brain alkaline phosphatase involved in ADP or ATP formation from polyphosphate. A further aspect of the inventive method concerns the pharmaceutical composition for use according to the invention, wherein said mammal is a human patient.

The method according to this invention can be applied for the therapy of Alzheimer disease or other neurodegenerative disorders. A further aspect of this invention is that the method according to this invention can also be applied for the improvement of memory function of the ageing brain. A further aspect of the inventive method thus concerns a method for treating or preventing Alzheimer disease or other neurodegenerative disorders in a mammal, or for improving the memory function of the ageing brain, comprising administering to said mammal an effective amount of the pharmaceutical composition according to the invention. Preferably, said mammal is a human patient. Further preferred is a method according to the invention, wherein said pharmaceutical composition is administered simultaneously or sequentially with a pharmaceutical composition comprising a drug that modulates the activity of the brain alkaline phosphatase involved in ADP or ATP formation from polyphosphate.

The energy delivered by the high energy phosphodiester bonds in polyP or the ADP/ATP generated during ALP-mediated metabolization of this inorganic polymer in the extracellular space can be available for folding of misfolded proteins such as misfolded Αβ which has been implicated in the initiation of apoptotic neuronal cell death (Muller WEG, Romero FJ, Perovic S, Pergande G, Pialoglou P (1997) Protection of flupirtine on β-amyloid-induced apoptosis in neuronal cells in vitro. J Neurochem 68: 2371-2377). It has been demonstrated that heat shock protein(s) which require energy in form of ATP can reduce the neurotoxic activity of Tau and Αβ (Wu Y, Cao Z, Klein WL, Luo Y (2010) Heat shock treatment reduces beta amyloid toxicity in vivo by diminishing oligomers. Neurobiol Aging 1055-1058).

The invention will now be described further in the following preferred examples, nevertheless, without being limited thereto. For the purposes of the present invention, all references as cited herein are incorporated by reference in their entireties.

Figure 1 shows the micrographs of Ca-phosphate-NP and of Ca-polyP-MP; A and B: optical microscopy; C to F: SEM. (A, C, E) The Ca-phosphate-NP appear as homogeneous material and as spherical particles of a size around 35 nm at high magnification. (B, D and F) The Ca-polyP-MP particles are a likewise homogenous powder at lower magnification and spherical particles at high power SEM.

Figure 2 shows the characterization of the (A and B) Ca-phosphate-NP and (C and D) Ca-polyP-MP particles. The analyses were performed by (A and C) FTIR and (B and D) XRD.

Figure 3 shows the viability of PC12 cells incubated in 48-well plates for a period of 72 h. Cell viability was determined using the MTT assay. The concentrations of Na-polyP[Ca ], Ca-phosphate-NP and Ca-polyP-MP in the assays were identical (30 pg/mL); the controls did not contain additional phosphate or polyP. Data represent mean ± SD of ten independent experiments; no significant differences are calculated (P > 0.05).

Figure 4 shows the viability of PC12 cells incubated with the Αβ fragment Αβ25/35. The peptide had been pre-incubated in distilled water for 6 to 24 hrs, or was immediately used after solubilization. During the pre-incubation period Αβ25/35 remained untreated or was treated with 30 pg/mL of Na-polyP[Ca ] (minus/plus Na-polyP[Ca ]), which was prepared as described (Muller WEG, Wang XH, Diehl-Seifert B, Kropf K, SchloBmacher U, Lieberwirth I, Glasser G, Wiens M, Schroder HC (2011) Inorganic polymeric phosphate/polyphosphate as an inducer of alkaline phosphatase and a modulator of intracellular Ca level in osteoblasts (SaOS-2 cells) in vitro. Acta Biomater 7:2661-2671). After that, the respective peptide sample was added to the cell culture (8 x 104 PC12 cells/mL) at a concentration of 5 pM and incubated for 12 h. Then the viability of the cells was determined by the MTT assay; the cell survival rate was calculated and is given as % to the corresponding control culture. Data (± SD) have been based on ten independent experiments; (*) the significance has been calculated (P < 0.05).

Figure 5 shows the protective effect of polyP against the toxic effect displayed by the Αβ fragment; the experiments were performed with (A) PC 12 cells or with (B) primary rat cortex neurons. The cells remained either untreated, or were pre-incubated with 30 pg/mL of the phosphate/polyP samples (either Na-polyP[Ca ], Ca-phosphate-NP, or Ca-polyP-MP) for 24 h. Then the cells, at a concentration of 8 x 104 cells/mL, were incubated with the 6 h preincubated Αβ25/35 fragment. After a following period of 12 h the viability of the cells in the respective assays was determined with the MTT assay. From those values the cell survival rate was calculated and the values are given in % of the respective controls (without phosphate/polyP and Αβ). Data ± SD (ten independent experiments) are give; the significance (*) is calculated P < 0.05.

Figure 6 shows the frequency of cells in a microscopic view-frame pre-treated with phosphate/polyP (24 h) and treated with Αβ25/35 (6 h). The PC12 cell cultures remained untreated (A) or were pre-treated with 30 pg/mL of Na-polyP[Ca ] (B), Ca-phosphate-NP (C), or Ca-polyP-MP (D). After staining with Calcein AM the cells were inspected by fluorescence microscopy.

Figure 7 shows the change of the ATP pool in PC 12 cells in dependence on the exposure to Αβ25/35 and phosphate/polyP. In the first series the cells were not exposed to phosphate/polyP and not to Αβ25/35 (Minus Αβ25/35); open bar. In the Αβ25/35 test series (closed bars) the cells were treated with 5 μΜ of the peptide and remained either untreated (control) or were exposed to 30 pg/mL of Na-polyP[Ca ], Ca-phosphate-NP, or Ca-polyP -MP, as indicated. The values came from 5 parallel assays; the means as well as the standard deviations are given. The significant differences between the values (control [minus phosphate/polyP] but plus Αβ25/35) and the test samples (Na-polyP[Ca ] and Ca-polyP-MP; both together with Αβ25/35) are indicated (*) P < 0.01.

Examples

In the following examples, the inventive method described only for polyP molecules with a chain length of 40 phosphate units. Similar results can be obtained by using polyP molecules with lower and higher chain lengths, such as between about 10 to about 100 units.

Fabrication and morphology of the particles

The particles were produced using a previously developed precipitation method from CaCk and an aqueous (poly)phosphate solution at an approximate weight ratio of 2:1 (Muller WEG, Tolba E, Schroder HC, Wang S, GlaBer G, Munoz-Espi R, Link T, Wang XH (2015) A new polyphosphate calcium material with morphogenetic activity. Materials Letters 148:163-166). Under these weight conditions polyP was found to form microspheres. This procedure was applied both for Na-polyP, with a chain length of ~40 Pi units, and tri-sodium (ortho)-phosphate. The CaCk solution was added dropwise to the respective phosphate solution.

The produced particles, both Ca-phosphate-NP and Ca-polyP-MP, had a powder like consistency (Fig. 1A and B). At a higher magnification they appear as homogeneous grains (Fig. 1C and D). At the nanoscale the Ca-phosphate-NP show a largely homogeneous morphology with a diameter of the particles of 35±8 nm [n=20] (Fig. IE). In contrast, the spherical Ca-polyP-MP showed an average size of 170±87 nm (Fig. IF).

Characterization by FTIR and XRD

The FTIR spectra of the Ca-phosphate-NP and the Ca-polyP-MP show characteristic differences (Fig. 2A). The Ca-phosphate-NP exhibit a spectrum indicative for carbonated apatite. The spectrum comprises the typical V4 bending vibrations of PO4 " at 557 cm' and 600 cm' , the Vi symmetric PO4 ' stretching at 960 cm' (not shown) as well as the V3 asymmetric stretching at 1018 cm'1. The occurrence of the latter band is also proven to be a marker for ortho-phosphate. Additionally, bands originating from carbonate are visible at 877 cm'1 (V2 bending vibration) and 1415 cm'1 as well as 1455 cm'1 (V3 asymmetric stretching vibration; double band). The occurrence of these CO3 ' bands is characteristic for type B apatite. In contrast, the spectrum of the Ca-polyP-MP (Fig. 2C) shows only the characteristic signals for polyP, as described previously (Muller WEG, Tolba E, Schroder HC, Wang S, GlaBer G, Munoz-Espi R, Link T, Wang XH (2015) A new polyphosphate calcium material with morphogenetic activity. Materials Letters 148:163-166). These can be ascribed with 1245 cm'1 for vas (PO2)', 1104 cm'1 for vas (PO3)2', 997 cm'1 for vsym (PO3)2', 905 cm'1 for vas (P-O-P) and 735 cm'1 for Vsym (P-O-P). Vibrations indicative for carbonate are not present.

The XRD pattern for Ca-phosphate-NP shows that the mineral is crystalline (Fig. 2B). This must be deduced from the recorded pattern between 20° and 57°; there, sharp reflections are seen with the maximum peak at 26.4°. In contrast, the XRD pattern for Ca-polyP-MP indicates that this material is amorphous (Fig. 2D). The signals have been recorded between 0 and 57°.

Cell viability after exposure to phosphate or polyP preparations PC12 cells were exposed to three different phosphate preparations (concentration 30 pg/mL), first against Na-polyP[Ca ], then against Ca-phosphate-NP and finally against Ca-polyP-MP (Fig. 3). In the controls no phosphate sample was added. The incubation in the 48-well plates was for 72 h; the seeding concentration was 2 χ 104 cells/mL. At the end of the incubation period the cells were harvested and subjected to the MTT assay; the amount of formazan crystals was quantitated as described under “Methods”. As seen the viability, measured on the basis of the enzymatic reaction, was found to be for all three preparations not significantly different if compared to the control (without phosphate or polyP); Fig. 3. In more detailed viability tests the different phosphate and polyP samples were tested in the concentration range of 3 pg/mL to 100 pg/mL. In none of the assays the viability in the controls were significantly different than those in the test series (data not shown).

Induced toxicity by Αβ peptide

The Αβ25/35 peptide was dissolved in distilled water (900 pM). Then the peptide was added either immediately to the cells or was pre-incubated for 6 to 24 h in water and then added to the PC12 cells; the final concentration of the Αβ25/35 peptide was 5 pM. The concentration of cells was adjusted to 8 χ 104 cells/mL. Subsequently, the cells were incubated for 12 h followed by the determination of the viability, using the MTT assay system. The results (Fig. 4) show that the Αβ25/35 peptide, pre-incubated for 6 h or longer caused a significant reduction of the cell number [measured on the basis of the viability of the cells in the assay].

In a parallel series of experiments the PC 12 cells were exposed to Αβ25/35 peptide, pre-incubated for 0 to 24 h in the presence of 30 pg/mL of Na-polyP[Ca ] in the 900 pM stock solution. Then the samples were diluted down to 5 pM and the assays were continued to be incubated for 12 h, followed by the MTT measurements. In this series only the values for the 6 h pre-incubation period showed a significant difference between the polyP-untreated and polyP-pretreated fragment. The results show that the Αβ25/35 fragment pre-incubated with polyP is about 15% more toxic (Fig. 4).

The calculated cell survival rates, calculated in correlation to the growth rate in the respective controls, not containing Αβ or polyP (added as Na-polyP[Ca ]) were almost identical irrespectively of the duration of the pre-incubation period for Αβ.

Protection against Αβ-caused toxicity by polyP

Based on the preceding results we determined the (potential) cytoprotective effect of polyP, by using an Αβ25/35 sample that had been pre-incubated for 6 h in aqueous solution. In parallel the PC12 cells were pre-incubated with 30 pg/mL of the phosphate/polyP samples (either Na-polyP[Ca2+], Ca-phosphate-NP, or Ca-polyP-MP) for 24 h. Then, the phosphate-pre-incubated cells assays were mixed with Αβ (5 μΜ Αβ25/35 [final concentration]). In the controls to these experiments, the cells were only exposed to the toxic peptide.

The results with PC12 cells (8 x 104 cells/mL) show that in the assays with 5 μΜ Αβ25/35, the phosphate (Ca-phosphate-NP) pre-treated cells did not show any significant increase in cell survival compared to the control. However, the cells that had been pre-incubated with Na-polyP[Ca ] or Ca-polyP-MP show a significant resistance against the toxic effect of Αβ25/35 (Fig. 5A). Striking is the effect of Ca-polyP-MP on cells during the pre-incubation period. Those cells reached the same survival rate, compared to cells that are not incubated with the Αβ fragment.

In order to support and express the cytoprotective effect of the Ca-polyP-MP towards the peptide the PC12 cells remained either untreated or were pre-treated with 30 pg/mL of Na-polyP[Ca2+], Ca-phosphate-NP or Ca-polyP-MP (24 h) Then the cells were treated with 5 μΜ Αβ25/35 for 6 h followed by staining with Calcein AM. The fluorescence images from the untreated (Fig. 6A) and Ca-phosphate-NP-treated cells (Fig. 6C) exhibit only relatively few cells, while the cultures treated with Na-polyP[Ca ] (Fig. 6B) and especially those incubated with Ca-polyP-MP (Fig. 6D) display markedly more cells in the microscopic view-frame.

The results of these experiments were corroborated with primary rat cortex neurons (Fig. 5B). Also those neurons (8 x 104 cells/mL), pre-incubated with 30 pg/mL of Na-polyP[Ca2-] or Ca-polyP-MP for 24 h, showed a significant higher survival rate after exposure to Αβ25/35, compared to the non-treated controls (not pre-treated). While cells, which remained untreated or had been pre-treated with 30 pg/mL of Ca-phosphate-NP showed a survival rate towards

'Λ I the toxic effect of Αβ of only -50%, this value increased to 69% (for Na-polyP[Ca ]) or even 92% (Ca-polyP-MP).

Modulation of the intracellular ATP pool in cells by Αβ and phosphate/polyP

The PC12 cells (8 χ 104 cells/mL) incubated in the absence of Αβ25/35 and phosphate/polyP for 24 h in the standard assay system contained 2.27±0.28 pmol of ATP (103 cells); Fig. 7. If the cells were incubated for 24 h in the presence of 5 μΜ Αβ25/35 the ATP pool dropped to 0.87±0.09 pmol (10 cells). If the cultures were treated together with the peptide and 30 pg/mL of Ca-phosphate-NP no significant change was measured. However, if they were treated with polyP and together with the peptide a significant increase was seen; with Na-polyP[Ca2+] the level increases to 1.26±0.02 pmol (103 cells), and with Ca-polyP-MP to 1.94±0.21 pmol (103 cells).

Methods

Materials

Sodium polyphosphate (Na-polyP of an average chain of 40 phosphate units) was obtained from Chemische Fabrik Budenheim (Budenheim; Germany).

Phosphate/polyphosphate sample fabrication

The polyP particles are prepared as described (Muller WEG, Tolba E, Schroder HC, Wang S, GlaBer G, Munoz-Espi R, Link T, Wang XH (2015) A new polyphosphate calcium material with morphogenetic activity. Materials Letters 148:163-166). In brief, Na-polyP (2 g) is dissolved in 100 mL of distilled water; the resulting pH value is increased from pH 3.4 to pH 10 with 2 N NaOH. Then 60 mL of a CaCk solution (5.6 g CaCk x 2H2O; #C3306 Sigma, Taufkirchen; Germany) are added dropwise to the polyP solution. After stirring for 12 h the particles formed are collected by filtration through Nalgene Filter Units (pore size 0.45 pm; Cole-Parmer, Kehl/Rhein; Germany). Then the particles are washed twice with ethanol to remove the unbound Ca2+. Finally the amorphous microparticles, “Ca-polyP-MP”, were dried at 60° C overnight.

The particles formed from sodium orthophosphate (tri-sodium phosphate; Sigma #342483) are prepared in the same way. 2 g of tri-sodium phosphate are dissolved in 100 mL of water; the pH value was adjusted to pH 10; then 60 mL of the CaCk solution was added. The resulting crystalline particles, Ca-phosphate-NP, are processed as described above. 2+

Na-polyP complexed in a stoichiometric ratio to Ca (molar ratio of 2 [with respect to the phosphate monomer]; abbreviated as Na-polyP[Ca ]) is prepared as described in (Muller WEG, Wang XH, Diehl-Seifert B, Kropf K, SchloBmacher U, Lieberwirth I, Glasser G, Wiens M, Schroder HC (2011) Inorganic polymeric phosphate/polyphosphate as an inducer of alkaline phosphatase and a modulator of intracellular Ca level in osteoblasts (SaOS-2 cells) in vitro. Acta Biomater 7:2661-2671).

Fourier transformed infrared spectroscopy and X-ray diffraction

Fourier transformed infrared (FTIR) spectroscopy can be performed, for example, with a Varian 660-IR spectrometer with Golden Gate ATR auxiliary (Agilent). X-ray diffraction (XRD) of the dried powder samples can be conducted, for example, in a Philips PW1820 diffractometer with monochromatic Cu-Κα radiation (λ= 1.5418 A,40 kV, 30 mA, 5s, Δ0=Ο.Ο2).

Microscopy

Scanning electron microscopy (SEM) can be conducted, for example, with a HITACHI SU8000 electron microscope (Hitachi High-Technologies Europe). Light microscopy can be performed, for example, with a VHX-600 Digital Microscope from KEYENCE. PC12 cells

Rat pheochromocytoma cells (PC12 cells) used in the Examples are obtained from Sigma (#88022401) and cultivated in RPMI 1640 medium (Sigma) and heat-inactivated horse serum (10%)/heat-inactivated fetal bovine serum (5%) together with gentamicin; incubation is performed in a humidified atmosphere of air (95%) and CO2 (5%). In the Examples, the cells are seeded at a density 1.5 x 104 cells per well of a 96-well/or 6-well plate for the indicated periods of time.

Three different phosphate preparations are added to the culture: (z) Na-polyP complexed in a stoichiometric ratio to Ca2+ (molar ratio of 2 [with respect to the phosphate monomer]; abbreviated as Na-polyP[Ca2+]) as described (Muller WEG, Wang XH, Diehl-Seifert B, Kropf K, SchloBmacher U, Lieberwirth I, Glasser G, Wiens M, Schroder HC (2011) Inorganic polymeric phosphate/polyphosphate as an inducer of alkaline phosphatase and a modulator of intracellular Ca level in osteoblasts (SaOS-2 cells) in vitro. Acta Biomater 7:2661-2671); (if) Ca-phosphate-NP; and (z'z'z) Ca-polyP-MP (40 phosphoanhydride bond-linked phosphate units). The concentrations are given with the respective experiments. In the Examples, they are usually 30 pg/mL.

For microscopic visualization the cells can be stained, for Example, with 5 pM Calcein AM (Sigma). Then they are inspected by fluorescence microcopy at an excitation wavelength of 494 nm and an emission of 540 nm.

Cell viability assays

In the Examples, the toxicity of the phosphate preparations is determined after an incubation period of the PC 12 cells in medium/serum for 72 h. The cells are incubated in 48-well plates at an initial density 2 x 104 cells/mL. Then the viability of the cells is determined with the 3-[4,5-dimethyl thiazole-2-yl]-2,5-diphenyl tetrazolium (MTT; #M2128, Sigma) assay. The cells are detached and a 2 mL cell suspension is aspirated and subsequently incubated with fresh medium containing 200 pL of MTT for 4 h in the dark. Subsequently the remaining MTT dye is removed and 200 pL of DMSO is added to solubilize the formazan crystals. Finally, the optical densities (OD) at 570 nm are measured using an ELISA reader/spectrophotometer. Cell viability is expressed as a percentage of the untreated control (without phosphate/polyP). In the Examples, 10 parallel experiments are performed. Αβ-induced cell toxicity

Previously, the inventor found that the Αβ25/35 sample needs to stay in distilled water in a stock solution (900 μΜ) for 5 d to develop the full toxicity (Muller WEG, Romero FJ, Perovic S, Pergande G, Pialoglou P (1997) Protection of flupirtine on B-amyloid-induced apoptosis in neuronal cells in vitro. Prevention of amyloid-induced glutathione depletion. JNeurochem 68: 2371-2377; Muller WEG, Pialoglou P, Romero FJ, Perovic S, Pergande G (1996) Protective effect of the drug flupirtine on B-amyloid-induced apoptosis in primary neuronal cells in vitro. J Brain Res 37: 575-577). In the experiments underlying the present invention, time kinetics for the pre-incubation of the Αβ25/35 in distilled water for a period between 6 and 24 h is performed. The Αβ25/35 sample is diluted down to a concentration of 5 μΜ in the cell assays. Then the cells are incubated for 12 h (seeding concentration of 8 χ 104 cells/mL). After termination, the viability of the cells in the culture is determined by the MTT assay (see above). From those results the cell survival rate, in % of the controls (without Αβ), can be calculated.

For testing the toxicity of the Αβ25/35 peptide the stock solution is diluted to a final concentration in the toxicity assays of 5 μΜ (Muller WEG, Pialoglou P, Romero FJ, Perovic S, Pergande G (1996) Protective effect of the drug flupirtine on β-amyloid-induced apoptosis in primary neuronal cells in vitro. J Brain Res 37: 575-577). Prior to the addition of Αβ the cells are pre-incubated for 24 h in medium/serum at a final concentrations of 30 pg/mL with Na-polyP[Ca ], Ca-phosphate-NP or Ca-polyP-MP; the controls are incubated in parallel and did not contain any phosphate sample. Then the medium is removed and the Αβ25/35 sample is added. Incubation is performed for 12 h; then the viability of the cells is determined with MTT. Cell viability is given in percent of the untreated control (without Αβ25/35 and without phosphate/polyP).

Primary culture of cortical neurons

Primary rat cortex neurons are isolated from 18-day old rat embryos; in the examples, they have been obtained from GIBCO/Thermo Fisher Scientific. They are cultivated in Neurobasal Medium (from GIBCO/Thermo Fisher Scientific) as described (Perovic S, Schleger C, Pergande G, Iskric S, Ushijima H, Rytik P, Muller WEG (1994) The triaminopyridine Flupirtine prevents cell death in rat cortical cells induced by N-methyl-D-aspartate and gpl20 of HIV-1. Europ J Pharmacol 288:27-33). Five days old cultures are used for the studies. The experiments are performed in 96-well/or 6-well plates.

The assay regimen with Αβ25/35 as well as the pre-incubation protocol of the cells with the phosphate samples is the same as for PC 12 cells.

Cell toxicity is determined after 12 h with the MTT assay following the procedure described in (Wang X, Mori T, Sumii T, Lo EH (2002) Hemoglobin-induced cytotoxicity in rat cerebral cortical neurons: caspase activation and oxidative stress. Stroke 33:1882-1888).

Determination of the ATP level in PC12 cells PC12 cells are cultivated in 12-well plates until they reached about 75% of confluency. Then ATP is extracted using published procedures (Stanley PE (1986) Extraction of adenosine triphosphate from microbial and somatic cells. Methods Enzymol 133:14-22; Marcaida G, Minana MD, Grisolia S, Felipo V (1997) Determination of intracellular ATP in primary cultures of neurons. Brain Res Brain Res Protoc 1:75-78), and its concentration is determined by using an ATP luminescence kit (for example, no. LL-100-1, Kinshiro, Toyo Ink; Japan) as described (Muller WEG, Tolba E, Feng Q, Schroder HC, Markl JS, Kokkinopoulou M and Wang XH (2015) Amorphous Ca polyphosphate nanoparticles regulate the ATP level in bone-like SaOS-2 cells. J Cell Sci 128:2202-2207). After establishing a standard curve for given ATP concentrations, the absolute amount of ATP is extrapolated and is given as pmol/10 cells.

Prior to the ATP determination the cells are pre-incubated for 24 h with 30 pg/mL of Na-polyP[Ca ], Ca-phosphate-NP or Ca-polyP-MP; again, the controls are incubated in parallel and they do not contain any phosphate sample.

Statistical analyses

After verification that the respective values follow a standard normal Gaussian distribution and that the variances of the respective groups are equal, the results are statistically evaluated using the independent two-sample Student’s t-test.

Claims (13)

1. A pharmaceutical composition comprising inorganic polyphosphate for use in the prevention or treatment of Alzheimer disease or other neurodegenerative disorders in a mammal, or for the improvement of memory function of the ageing brain.

2. The pharmaceutical composition for use according to claim 1, wherein said polyphosphate is used in the form of nanoparticles or microparticles consisting of salts said polyphosphate with divalent cations, wherein said nanoparticles or microparticles are produced according to a method comprising the steps of a) Dissolving of sodium polyphosphate in an aqueous medium, and adjusting to pH of about 10.0 with sodium hydroxide solution, to form a polyphosphate solution; b) Dissolving of a salt of a suitable divalent cation in an aqueous medium or an aqueous ethanol solution, to form a divalent cation salt solution; c) Slowly adding said polyphosphate solution to said divalent cation salt solution while keeping the pH at between about 8.2 to 10.5; d) Forming of nanoparticles or microparticles, preferably by stirring of the suspension overnight at room temperature; and e) Collecting said nanoparticles or microparticles, preferably by fdtration, and washing and drying said particles.

3. The pharmaceutical composition for use according to claim 2, wherein said divalent cation is selected from calcium, magnesium and strontium.

4. The pharmaceutical composition for use according to any one of claims 1 to 3, wherein the chain length of said polyphosphate is in the range of about 3 to about 1000 phosphate units, preferably in the range of about 10 to about 100 phosphate units, and most preferred about 40 phosphate units.

5. The pharmaceutical composition for use according to any one of claims 2 to 4, wherein said polyphosphate nanoparticles or microparticles are characterized by a stoichiometric ratio of between about 4 to about 0.8 (cation to phosphate), preferably of between about 3 and about 1.5, and most preferred of about 2.

6. The pharmaceutical composition for use according to any one of claims 2 to 5, wherein the average size of said calcium polyphosphate nanoparticles or microparticles is in the range of about 100 to about 1000 nm, and most preferred about 150 to about 250 nm.

7. The pharmaceutical composition for use according to any one of claims 1 to 6, which is suitable for the delivery of said polyphosphate via the blood-brain barrier, such as, for example, selected from liposomes, micelles, dendrimers, comprising β-cyclodextrin carriers, carbon nanotubes, and chitosan-based nanomers.

8. The pharmaceutical composition for use according to any one of claims 1 to 7, wherein said pharmaceutical composition further comprises a drug that modulates the activity of the brain alkaline phosphatase involved in ADP or ATP formation from polyphosphate.

9. The pharmaceutical composition for use according to any one of claims 1 to 7, wherein said pharmaceutical composition is administered simultaneously or sequentially with a pharmaceutical composition comprising a drug that modulates the activity of the brain alkaline phosphatase involved in ADP or ATP formation from polyphosphate.

10. The pharmaceutical composition for use according to any one of claims 1 to 9, wherein said mammal is a human patient.

11. A method for treating or preventing Alzheimer disease or other neurodegenerative disorders in a mammal, or for improving the memory function of the ageing brain, comprising administering to said mammal an effective amount of the pharmaceutical composition according to any one of claims 1 to 8.

12. The method according to claim 11, wherein said mammal is a human patient.

13. The method according to claim 11 or 12, wherein said pharmaceutical composition is administered simultaneously or sequentially with a pharmaceutical composition comprising a drug that modulates the activity of the brain alkaline phosphatase involved in ADP or ATP formation from polyphosphate.