EMPTY POROUS PARTICLES FOR USE IN TREATMENT, PREVENTION AND/OR POSTPONEMENT OF DEGENERATION OF NEURODEGENERATIVE DISEASES, NEURONS AND GLIA

Abstract

The present invention relates to empty porous particles having a diameter between 0.1 and 1000 μm, as measured by e.g. SEM, for use in diagnosis, prevention and/or postponement of neurodegenerative diseases, or for prevention and/or postponement of degeneration of neurons and glia. The invention also relates to a method of identifying biomarkers for use in diagnosis.

Claims

1. Empty mesoporous silica particles having a diameter between 0.1 and 1000 μm, for use in prevention and/or postponement of degeneration of neurodegenerative diseases (NDDs) in a mammal, wherein co-transplantation of stem cells is disclaimed, and the empty particles have no surface bound molecules or agents, and wherein the neurodegenerative diseases is selected from the group consisting of Alzheimer's disease (AD), Parkinson's disease (PD), amyotrophic lateral sclerosis (ALS), Huntington's disease (HD), dementia with Lewy bodies (DLB), progressive supranuclear palsy (PSP), multiple system atrophy (MSA), corticobasal degeneration (CBD), frontotemporal dementia (FTD), spinocerebellar ataxia (SCA) disorders and spinal muscular atrophy.

2. Empty porous particles for use according to claim 1, wherein the neurodegenerative disease is amyotrophic lateral sclerosis (ALS).

3. Empty porous particles for use according to claim 1, for use in prevention and/or postponement of degeneration of neurons and glia in a mammal.

4. Empty porous particles for use according to claim 1, for use in prevention and/or postponement of NDDs in a mammal, whereby the empty particles are administered before, during or after treatment of the mammal with, all or at least a portion of the particles loaded with trophic factors selected from peptide mimetics of Glial cell-derived neurotrophic factor (GDNF) and/or Ciliary neurotrophic factor (CNTF) and/or Stem Cells.

5. Empty porous particles for use according to claim 1, wherein the empty particles are mesoporous silica particles having a pore size between 1 and 100 nm, a pore volume between 0.1 and 3 cm.sup.3/g and a surface area between 40 and 1500 m.sup.2/g.

6. Empty porous particles for use according to claim 1, wherein the empty particles have a pore size between 0.3 and 20 nm, a pore volume between 0.5 and 1.5 cm.sup.3/g and a surface area between 50 and 800 m.sup.2/g.

7. Empty porous particles for use according to claim 1, wherein the empty particles have a diameter between 0.1 and 500 μm, or between 0.1 and 250 μm, or between 0.1 and 100 μm, or between 0.2 and 50 μm, or between 0.3 and 25 μm, or between 0.3 and 20 μm, or between 0.3 and 12 μm, or between 0.3 and 6 μm.

8. Empty porous particles for use according to claim 1, wherein the empty particles are administered by injection in the cerebrospinal fluid (CSF), brain or spinal cord parenchyma.

9. Empty porous particles for use according to claim 1, wherein an incubator liquid is loaded in or on all or at least a portion of the empty particles, and the incubator liquid is a buffered solution having a physiological pH suitable for administration by injection.

10. A method for diagnosis of a neurodegenerative diseases (NDDs) using empty porous particles having a diameter between 0.1 and 1000 μm.

11. A method of identifying biomarkers for diagnosis and/or treatment of NDDs using empty porous particles having a diameter between 0.1 and 1000 μm, comprising the steps of: a) administering the empty porous particles into the cerebrospinal fluid (CSF); b) retrieving a portion of said porous particles after a period of time from the CSF; c) determining biomolecules that have been loaded into the porous particles during their presence in the CSF; and d) establishing if the compounds can be used as biomarkers.

12. A method of identifying biomarkers for diagnosis and/or treatment of NDDs using empty porous particles having a diameter between 0.1 and 1000 μm, comprising the steps of: a) providing cerebrospinal fluid (CSF) from a mammal; b) adding the empty porous particles to the fluid; c) retrieving a portion of said porous particles after a period of time, from the CSF; d) determining biomolecules that have been loaded into the porous particles during their presence in the CSF; and e) establishing if the compounds can be used as biomarkers.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0070] The invention will now be explained more closely by the description of different embodiments of the invention and with reference to the appended figures.

[0071] FIG. 1 shows Scanning Electron Microscopy (SEM) of empty mesoporous silica particles used for the sequestering of biomolecules and loading mimetics. The particles consist of aggregates in the size range of 1-10 microns made up from several primary particles of the size of 500 nm

[0072] FIG. 2 shows pore size distribution of empty mesoporous silica particles obtained by Nitrogen (N.sub.2) Adsorption-Desorption technique using Micromeritics's TriStar II 3020. The pore size was determined to be 23 nm using a Density Functional Theory (DFT) model. The Brunauer-Emmett-Teller (BET) surface area of nanoporous silica was determined to be 722 m.sup.2/g.

[0073] FIG. 3 shows that treatment with empty mesoporous silica particles or mimetic loaded mesoporous particles extends the survival of mutant SOD1G93A mouse model of ALS. Ages of disease end stage (determined as the time when the animal could not right itself within 20 seconds when placed on its side) of SOD1G93A and SOD1G93A littermates injected with mesoporous particles loaded with mimetics (MesoMIM) (0.1 μg/ul particles), or with empty particles (Meso). (0.1 μg/ul particles) Mean ages±S.D. is provided.

[0074] FIG. 4 shows that treatment with empty mesoporous silica particles or mimetic loaded mesoporous particles or bNCSCs (Stem Cells) extends weight progression of mutant SOD1G93A mouse model of ALS. SOD1G93A littermates injected with mesoporous particles loaded with mimetics (MesoMIM) (0.1 ug/μl particles), or with empty particles (Meso) (0.1 ug/μl particles) or with bNSCs (.sup.˜13,000 cells/μl). Mean ages±S.D is provided.

[0075] FIG. 5 shows a graph of averaged hindlimb grip strength for SOD1G93A injected with empty mesoporous particles or injected with mesoporous particles loaded with mimetics, or with bNCSCs cells at indicated age. The results are the means±S.D.

[0076] FIG. 6 shows a graph of averaged forelimb grip strength for SOD1G93A injected with empty mesoporous particles or injected with mesoporous particles loaded with mimetics, or with bNCSCs cells at indicated age. The results are the means±S.D.

[0077] FIG. 7 shows immunoblot for mSOD-1 and TDP-43 of particles unbound and bound fractions obtained following incubation of the porous silica particles with spinal cord extracts obtained from Tg(SOD1*G93A) 1Gur/J mice (4 mice) and WT blac6 mice (4 mice), followed by separation of particles bound and unbound fractions.

DETAILED DESCRIPTION OF VARIOUS EMBODIMENTS OF THE INVENTION

Definitions and Measurement Methods

[0078] As used herein, the term “disease” is intended to include disorder, condition or any equivalent thereof.

[0079] The term “loading” means that the bioactive compound may be loaded into and/or onto the particle, without being fixedly bound to the particle.

[0080] As used herein, the term “substantially” or “about” refers to a deviation of a value around the number mentioned, e.g. substantially or about 5 means that the value may be between 4 and 6.

[0081] As used herein, the term “coating” refers to coverage of a surface, which may include blockage of the pores or not.

[0082] The term “extended release” means any release of a bioactive compound that is not immediate, i.e. not a release of at least 80% of the compound within 30 minutes.

[0083] As used herein, the term “patient” refers to a mammal, for example, a human.

[0084] As used herein, the term “at least a portion” means that at least 50 w/w %, or at least 75 w/w %, or at least 90 w/w % of all empty particles.

[0085] In the context of the present specification, the term “therapy” also includes “prophylaxis” unless there are specific indications to the contrary. The term “therapeutic” and “therapeutically” should be construed accordingly. The term “therapy” within the context of the present invention further encompasses to administer an effective amount of a compound of the present invention, to mitigate either a pre-existing disease state, acute or chronic, or a recurring condition. This definition also encompasses prophylactic therapies for prevention of recurring conditions and continued therapy for chronic diseases.

[0086] As used herein, the term “optional” or “optionally” means that the subsequently described event or circumstance may but need not occur, and that the description includes instances where the event or circumstance occurs and instances where it does not.

[0087] As used herein, the term “mimetics” refers to a small protein-like chain designed to mimetic a peptide. They typically arise either from modification of an existing peptide, or by designing similar systems that mimetic peptides, such as peptoids and (3-peptides. Irrespective of the approach, the altered chemical structure is designed to advantageously adjust the molecular properties such as, stability or biological activity. This can have a role in the development of drug-like compounds from existing peptides.

[0088] As used herein, the term “postponement” refers to a delay of onset of a disease.

[0089] As used herein, the term “cervical and/or spinal fluid” refers to cerebrospinal fluid, brain or spinal cord parenchyma.

[0090] As used herein, the term “neural cells” or “neuronal cells” means all cells in the central nervous system (CNS), such as neurons and glia, such as astrocytes, oligodendrocytes and microglia.

[0091] Mercury (Hg) intrusion is a standard method for measuring porosity. In this method mercury is pushed into pores under applied pressure. However, there is a lower pore size limit of around 3.2 nm, below which, the mercury cannot penetrate. For porous materials with pore sizes in the mesoscale range of 1 to 50 nm, nitrogen (N.sub.2) sorption is commonly used to estimate pore size and pore volumes. The nitrogen sorption technique measures the available surface area of the porous materials. An empirical model is used to calculate the pore volume and pore size. The BET (Brunauer-Emmett-Teller) and BJH (Barrett, Joyner and Halenda) models are used to calculate porosity for the pores of silica particles.

[0092] Scanning electron microscope (SEM) can be used to provide images of the porous particles. The diameter of the particles can be determined using SEM.

[0093] The empty porous particles may be manufactured by cooperative self-assembly of silica species and organic templates, such as cationic surfactants, such as alkyltrimethylammonium templates with varying carbon chain lengths, and counterions, such as cetyltrimethylammonium chloride (CTA+Cl− or CTAC) or cetyltrimethylammonium bromide (CTA+Br− or CTAB) or non-ionic species, such as diblock and triblock polymer species such as copolymers of Polyethylene Oxide and Polypropylene Oxide for example Pluronic 123 surfactant. The formation of mesoporous particles occurs following the hydrolysis and condensation of silica precursor, which can include alkylsilicates, such as tetraethylorthosilicate TEOS or teramethylorthosilicate TMOS in solution or sodium silicate solution. The mesoporous silica particle size can be controlled by adding suitable additive agents e.g. inorganic bases, alcohols including methanol, ethanol, propanol and organic solvents, such as acetone, which affect the hydrolysis and condensation of silica species. The pore size can be influenced by hydrothermal treatment of the reaction mixture, such as heating up to 100° C. or even above, and also with the additional of swelling agents in the form of organic oils and liquids that expand the surfactant micelle template. After condensation of the silica matrix, the templating surfactant can be removed by calcination typically at temperatures from 500° C. up to 650° C. for several hours, which burns away the organic template resulting in a porous matrix of silica. The template may alternatively be removed by extraction and washing with suitable solvents, such as organic solvents or acidic of basic solutions.

[0094] The empty porous particles may be manufactured by a sol-gel method comprising of a condensation reaction of a silica precursor solution, such as sodium silicate or an aqueous suspension of silica nanoparticles as an emulsion, with a non-miscible organic solution, oil, or liquid polymer in which droplets are formed by for example stirring or spraying the solution followed by gelation of the silica by means of change in pH and or evaporation of the aqueous phase. The porosity of the empty particles here are formed either by exclusion due to the presence of the non-miscible secondary phase or by the jamming of the silica nanoparticles during evaporation. The empty particles may further be treated by heating to induce condensation of the silica matrix and washing to remove the non-miscible secondary phase. Furthermore, the empty particles may be treated by calcination to strengthen the silica matrix.

[0095] The empty porous particles may be manufactured as porous glass through a process of phase separation in borosilicate glasses (such as SiO.sub.2—B.sub.2O.sub.3—Na.sub.2O), followed by liquid extraction of one of the formed phases through the sol-gel process; or simply by sintering glass powder. During a thermal treatment, typically between 500° C. and 760° C. an interpenetration structure is generated, which results from a spinodal decomposition of the sodium-rich borate phase and the silica phase.

[0096] The porous empty particles may be manufactures using fumed process. In this method, fumed silica was produced by burning silicon tetrachloride in an oxygen-hydrogen flame producing microscopic droplets of molten silica, which fuse into amorphous silica particles in three-dimensional secondary particles which then agglomerate into tertiary particles. The resulting powder has an extremely low bulk density and high surface area.

[0097] The empty porous particles may have a diameter between 0.1 and 1000 μm, as measured by SEM, or between 0.1 and 500 μm, or between 0.1 and 250 μm, or between 0.1 and 100 μm, or between 0.2 and 50 μm, or between 0.3 and 25 μm, or between 0.3 and 20 μm, or between 0.3 and 12 μm, or between 0.3 and 6 μm. The empty particles may have nano- and/or mesopores.

[0098] The empty particles may have a pore size between 1 and 100 nm, or between 1 and 80 nm, or between 2 and 50 nm, or between 2 and 25 nm, or between 5 and 15 nm, or substantially 12 nm.

[0099] The empty particles may have a pore volume between 0.1 and 3 cm.sup.3/g, or between 0.2 and 2 cm.sup.3/g, or between 0.5 and 1.5 cm.sup.3/g, or between 0.7 and 1.2 cm.sup.3/g, or between 0.5 and 1.0 cm.sup.3/g, or substantially 8.5 cm.sup.3/g.

[0100] The empty particles may have a diameter between 0.1 and 1000 μm, a pore size between 1 and 100 nm, a pore volume between 0.1 and 3 cm.sup.3/g and a surface area between 40 and 1500 m.sup.2/g. The empty particles may have a pore size between 0.3 and 20 nm, a pore volume between 0.5 and 1.5 cm.sup.3/g and a surface area between 50 and 800 m.sup.2/g. The empty particles as used in the description may have any combination of the intervals mentioned above.

[0101] The empty particles may have different shapes. The shapes of the empty particles can be controlled by the process and may be spheres, pseudo-spheres, cylinders, gyroids, rods, fibres, core-shell shape of empty particles, or mixtures thereof. The empty particles may be substantially spherical or pseudo-spheres.

[0102] The empty porous particles may be porous silica particles. The silica may be any silica. The silica may be biodegradable and/or dissolvable. Examples of silica is amorphous silica or synthetic amorphous silica.

[0103] The empty porous particles may be manufactured by a process comprising or consisting of the steps of providing the silica particles, preferably by manufacturing by a sol-gel method or by a spray drying method, [0104] separating the empty particles. Separation may be done by air classifier, or cyclonic separation, or elutriation, or sedimentation and/or sieving using one or more sieves.

[0105] An incubator liquid may be loaded in or on all or at least a portion of the empty particles. Examples of such liquids may be any buffered liquid having a physiological pH of about 6.8, adapted for administration to a mammal, such as a human. Administration may be by injection.

[0106] Additional compounds, such as one or more mimetics or additional agents, or any combination thereof, may be loaded in or on all or at least a portion of the empty particles without being fixedly bound to the surface of the particles. Fixedly bound means that the agent or mimetic is permanently attached to the surface of the particles.

[0107] Examples of other mimetics may be, Gliafin and/or Cintrofin.

[0108] Examples of one or more additional agents may be growth factors, pH regulators, stabilizers, antibiotics, anti-inflammatory drugs and/or immune-suppressors.

[0109] The empty particles may be loaded using different techniques, such as solvent evaporation, impregnation, spray-drying, melting, supercritical CO.sub.2 loading or freeze-drying, and the like. Solvent evaporation involves combining a concentrated solution of the bioactive compound with the silica particles, then removing the solvent and/or drying the sample prior to further processing.

[0110] The loading of the porous silica particles may be about 5 w/w % or more, or about 15, 20, 25%, 30, 40, 50, 75, 80, 85, 90 w/w %.

[0111] The empty porous particles may be biotinylated. The empty porous particles may be chemically modified by having a surface chemistry of terminal hydroxyl groups —OH, or having a hydrophobic surface chemistry, or having chemical functionalities including —COOH, —NH.sub.3, OCH.sub.3, —OCH.sub.2CH.sub.3, or mixtures thereof. A mixture of particles having different chemical modification may also be used.

[0112] Surface modification may be done prior to loading. Surface modification may be a chemical modification, such as etching. Etching may be done by boiling the silica particles in a base, and then in an acid to form —S—OH bonds on the outer surface of the particle. Examples of suitable bases may be a solution of KOH, a solution of NaOH or ammonia solution. Examples of suitable acids may be HNO.sub.3, HCl and H.sub.2SO.sub.4.

[0113] The porous particles may be formulated into a pharmaceutical composition adapted for administration of the particles to a mammal, such as a human. The composition may comprise or consist of a plurality of porous particles together with a solvent, such as water or a biologically acceptable liquid adapted for administration to a mammal. The physiological pH is about 6.8 (physiological pH). The composition may further comprise or consist of pharmaceutical acceptable additives. Conventional procedures for the selection and preparation of suitable pharmaceutical compositions are described in, for example, “Pharmaceuticals—The Science of Dosage Form Designs”, M. E. Aulton, Churchill Livingstone, 1988.

[0114] The pharmaceutical composition can be used for controlled or extended release of one or more mimetics or additional agents, or any combination thereof, loaded in or onto the porous particles. An extended release means a release of the loaded ingredient from the particles by less than 50% within 30 minutes from the start of a standard dissolution test, such as FDA Paddle Method (USP apparatus 2).

[0115] Medical Use.

[0116] As shown in the results, 10-week-old mice on B6/SJC background (B6SJL-TgN-SOD1-G93A-1Gur) were subjected to double or triple intracervical laminectomy surgery injection (2 μl) on the left side of the cervical spinal cord (C3-C4) after. Injection of empty nanoparticles or nanoparticles containing mimetics significantly delayed the disease course of SOD1 mutant mice.

[0117] The empty porous particles having a diameter between 0.1 and 1000 μm, as measured by SEM, or the pharmaceutical composition as defined above, may be used in treatment, prevention and/or postponement of degeneration of neurons and glia.

[0118] The empty porous particles or the pharmaceutical composition may be administered by injection in the spinal and/or cervical fluid. The composition can be injected anywhere in the spinal fluid or cervical fluid. The empty particles may be administered by injection in the cerebrospinal fluid (CSF), or in the brain or spinal cord parenchyma. The composition may be administered by injection in the spinal fluid between the third and fourth vertebrae.

[0119] The empty porous particles or the pharmaceutical composition as defined above, may be used in prevention and/or postponement of neurodegenerative diseases (NDDs), selected from the group comprising or consisting of Alzheimer's disease (AD), Parkinson's disease (PD), amyotrophic lateral sclerosis (ALS), Huntington's disease (HD), dementia with Lewy bodies (DLB), progressive supranuclear palsy (PSP), multiple system atrophy (MSA), corticobasal degeneration (CBD), frontotemporal dementia (FTD), spinocerebellar ataxia (SCA) disorders, and spinal muscular atrophy, or other NDDs.

[0120] The empty porous particles or the pharmaceutical composition as defined above, may be used in sequestering SOD1, TDP-43, amyloid-beta, alpha-synuclein, tau, ELAVL4, FUS and other biomolecules that affect degeneration of neurons and glia.

[0121] The empty porous particles or the pharmaceutical composition as defined above, may be used in sequestering SOD1, TDP-43, amyloid-beta, alpha-synuclein, tau, ELAVL4, FUS and other biomolecules that contribute to progression of neurodegenerative diseases (NDDs), selected from the group comprising or consisting of Alzheimer's disease (AD), Parkinson's disease (PD), amyotrophic lateral sclerosis (ALS), Huntington's disease (HD), dementia with Lewy bodies (DLB), progressive supranuclear palsy (PSP), multiple system atrophy (MSA), corticobasal degeneration (CBD), frontotemporal dementia (FTD), spinocerebellar ataxia (SCA) disorders, and spinal muscular atrophy and other NDDs

[0122] The empty porous particles or the pharmaceutical composition as defined above, may be used in sequestering SOD1, TDP-43, amyloid-beta, alpha-synuclein, tau, ELAVL4, FUS and other biomolecules that contribute to progression of neurodegenerative diseases (NDDs) before, during or after treatment of a mammal with porous particles at least partially loaded with trophic factors, such as peptide mimetics of Glial cell-derived neurotrophic factor (GDNF) and/or Ciliary neurotrophic factor (CNTF) and/or Stem Cells.

[0123] The empty porous particles or the pharmaceutical composition as defined above, may be used sequestering SOD1, TDP-43, amyloid-beta, alpha-synuclein, tau, ELAVL4, FUS and other biomolecules that contribute to progression of neurodegenerative diseases (NDDs) before, during or after treatment of a mammal with Stem Cells.

[0124] The empty porous particles may be used in prevention and/or postponement of NDDs in a mammal, whereby the empty particles are injected before, during or after treatment of the mammal with the particles loaded with trophic factors selected from peptide mimetics of Glial cell-derived neurotrophic factor (GDNF) and/or Ciliary neurotrophic factor (CNTF) and/or Stem Cells. The empty particles may be injected before said treatment. A mammal may be first treated by injection of empty particles and subsequently (within 1 to 24 hours) be treated by injection of particles that are at least partially loaded with trophic factors selected from peptide mimetics of Glial cell-derived neurotrophic factor (GDNF) and/or Ciliary neurotrophic factor (CNTF) and/or Stem Cells. Alternatively, a mammal may be simultaneously treated by injection of empty particles and by injection of particles that are at least partially loaded with trophic factors selected from peptide mimetics of Glial cell-derived neurotrophic factor (GDNF) and/or Ciliary neurotrophic factor (CNTF) and/or Stem Cells. Or, a mammal may be first treated by injection of particles that are at least partially loaded with trophic factors selected from peptide mimetics of Glial cell-derived neurotrophic factor (GDNF) and/or Ciliary neurotrophic factor (CNTF) and/or Stem Cells and subsequently (within 1 to 24 hours) be treated by injection of empty particles. The particles may be administered by injection in the cerebrospinal fluid (CSF), or in the brain or spinal cord parenchyma.

[0125] A dosage regime for administration of the pharmaceutical composition may be administration by injection in the spinal fluid at least once every month, or at least once every two months, or at least once every three weeks, or once every two weeks, or once every week.

[0126] Diagnosis of a Neurodegenerative Diseases

[0127] The empty porous particles may be used in diagnosis of a neurodegenerative diseases (NDDs), selected from the group comprising or consisting of Alzheimer's disease (AD), Parkinson's disease (PD), amyotrophic lateral sclerosis (ALS), Huntington's disease (HD), dementia with Lewy bodies (DLB), progressive supranuclear palsy (PSP), multiple system atrophy (MSA), corticobasal degeneration (CBD), frontotemporal dementia (FTD), spinocerebellar ataxia (SCA) disorders and spinal muscular atrophy, or other NDDs.

[0128] The empty porous particles may be used in a method of

[0129] identifying biomarkers for diagnosis and/or treatment of NDDs, such as ALS comprising the steps of:

[0130] a) administering the empty porous particles having a diameter between 0.1 and 1000 μm, as measured by SEM, as defined above into the cerebrospinal fluid (CSF);

[0131] b) retrieving a portion of said porous particles after a period of time, such as 1 to 36 hours, from the CSF;

[0132] c) determining biomolecules that have been loaded into the porous particles during their presence in the CSF;

[0133] d) establishing if the compounds can be used as biomarkers.

[0134] The period of time in step b) may be 1 to 24 hours or 1 to 72 hours.

[0135] All or at least a portion of the empty particles may be loaded with an incubation liquid as mentioned above.

[0136] Administration means in vivo administration as well as in vitro addition of empty particles to CSF retrieved from a mammal.

[0137] Thus, step a) may be adding the empty porous particles as defined herein, to the cerebrospinal fluid (CSF) retrieved from a mammal in vitro.

[0138] The identified biomarkers may be used in for diagnosis, prevention, treatment and/or postponement of NDDs, such as ALS; or NDDs selected from the group comprising or consisting of Alzheimer's disease (AD), Parkinson's disease (PD), Huntington's disease (HD), dementia with Lewy bodies (DLB), progressive supranuclear palsy (PSP), multiple system atrophy (MSA), corticobasal degeneration (CBD), frontotemporal dementia (FTD), spinocerebellar ataxia (SCA) disorders and spinal muscular atrophy.

Experimental

[0139] Synthesis of Mesoporous Silica Particles.

[0140] Pluronic 123 (triblock co-polymer, EO.sub.20PO.sub.70EO.sub.20, Sigma-Aldrich) (4 g) as a templating agent and, 1,3,5-trimethylbenzene (TMB) (Mesitylene, Sigma-Aldrich) (3.3 g) as swelling agent were dissolved in 127 ml distilled H.sub.2O and 20 ml hydrochloric acid (HCl, 37%, Sigma-Aldrich) while stirring at room temperature (RT) for 3 days. The solution was preheated to 40° C. before adding 9.14 ml TEOS (Tetraethyl orthosilicate, Sigma-Aldrich). The mixture was stirred for another 10 mins at the speed of 500 rpm and then kept at 40° C. for 24 hours, then hydrothermally treated in the oven at 100° C. for another 24 hours. Finally, the mixture was filtered, washed and dried at room temperature. The product was calcined to remove the surfactant template and swelling agent. The calcination was conducted by heating to 600° C. with a heating rate of 1.5° C./min and kept at 600° C. for 6 hours followed by cooling to ambient conditions. The resulting product is a white powder comprising of nanoporous silica particles.

[0141] Synthesis of Peptide Mimetics

[0142] The peptides Cintrofin and Gliafin, derived from the ciliary neurotrophic factor (148-DGGLFEKKLWGLKV-161; UniProtKB entry no. P26441) and glial cell line-derived neurotrophic factor (153-ETMYDKILKNLSRSR-167; UniProtKB entry no. Q07731), respectively, were synthesized using the solid-phase Fmoc protection strategy, and purity was estimated to be at least 80% by high performance liquid chromatography. All of the peptides were synthesized by Schafer-N AS (Copenhagen, Denmark, http://www.schafer-n.com) as dendrimers composed of four monomers coupled to a lysine backbone. During the synthesis of biotinylated peptides, only N-terminal amino acids were biotin labeled amino acids. Thus, each tetrameric dendrimer contained four biotin residues.

[0143] Loading Peptide Mimetics Gliafin and Cintrofin.

[0144] The peptide mimetics were loaded via impregnation in water. Mesoporous silica particles (50 mg) were added to 0.5 ml of Gliafin in a water solution at a concentration of 8.87 mg/ml and stirred at 4° C. for 16 hours. Mesoporous silica particles (25 mg) were added to a 0.4-ml Cintrofin water solution at a concentration of 4.4 mg/ml and stirred at 4° C. for 16 hours. The water in both solutions was evaporated under atmospheric conditions. The loading amount of peptide was determined by thermogravimetric analysis (PerkinElmer). Scanning was performed from 20° C. to 900° C. at a heating rate of 20° C./minute. The plug-in gas atmosphere was dry air (flow rate, 20 ml/minute). The sample weights varied from 5 to 10 mg. Loading efficiencies of mesopores were 8.3 and 11.8 wt % for Cintrofin and Gliafin, respectively.

[0145] Preparation of Particle Solutions

[0146] The solutions were prepared on the day of the start of the experiments.

[0147] Dispersion of Mesoporous Particle Solution

[0148] A 1 μg/μl concentration was made by mixing an equal volume of solvent or injection solution to dry powder of mesoporous silica particles. The mixture was mixed thoroughly. 100 μl of the final solution was put in a sterile tube for injection.

[0149] Mimetic Solution

[0150] 1 μg/μl of each concentrated Gliafin, Cintrofin loaded mesoporous silica particle solution was prepared as above. 100 μl from each tube was added to a sterile tube and 900 μl of solvent was added. The mixture was mixed thoroughly. 100 μl of the final solution was put in a sterile tube for injection. Final concentration of each mimetic was 0.1 μg/μl.

[0151] Mice Injections and Survival

[0152] At 79 days (about 10 week) B6/SJL background (B6SJL-TgN-SOD1-G93A-1Gur) mice were anesthetized with 3% isoflurane at the beginning of the procedure and decreasing over the time till reaching 0.8% at a flow rate of 500-480 ml/min. A partial laminectomy was made over the left cervical spinal cord, and double or triple intracervical injection was made into the ventral horn on the left side of spinal cord segments C3-C4. A 2 μl intracervical injection was performed in the three groups; empty porous silica particles (0.1 μg/μl), mimetic group (Gliafin, Cintrofin loaded mesoporous silica particles, 0.1 μg/μl), bNCSCs (boundary cap neural crest stem cells, about 13,000 cells/up. A 10 μl Hamilton syringe with a point style AS needle was attached to a micro syringe pump controller (Micro 4, WPI), set at an infusion pace of 4 μl/min.

[0153] For survival experiments, SOD1G93A mice injected with mimetics or boundary cap neural crest stem cells (bNCSCs) were always compared with their littermates injected with empty particles. Time of disease onset was retrospectively determined as the time when mice reached peak body weight. Early disease was defined at the time when denervation-induced muscle atrophy had produced a 10% loss of maximal weight. End-stage was determined by paralysis so severe that the animal could not right itself within 20 seconds when placed on its side, an endpoint frequently used for SOD1 mutant mice and one that was consistent with the requirements of the Animal Care and Use Committee of Ben-Gurion University of the Negev.

[0154] Grip strength test was performed with Chatillon force measurement device, Ametek, measurement of each member of the groups included two measurements (forelimb pulling and total limb pulling). Measurements were performed twice a week beginning two weeks before the surgery.

[0155] Cell Death Analysis

[0156] Cells are incubated with the indicated mimetics in the appropriate serum-free growth medium at 37° C., harvested, washed twice with PBS and cell death is analyzed by propidium iodide (PI) staining and flow cytometry.

[0157] Determination of Neurite Outgrowth Intersections Between Neurites and Survival

[0158] Stereological estimation of neurite length to evaluate neurite outgrowth in cells in culture was carried out as described previously (Florin L C, Ralets I, Hartz B P, Bech M, Berezin A, Berezin V, Møller A, Bock E. Asimple procedure for quantification of neurite outgrowth based on stereological principles. J Neurosci Methods. 2000 Jul. 31; 100(1-2):25-32). The total neurite length per cell was estimated by counting the number of intersections between neurites and test lines of an unbiased counting frame superimposed on images of cell cultures obtained by conventional computer-assisted microscopy. The absolute length (L) of neurites per cell was subsequently estimated from the number of neurite intersections (I) per cell by means of the equation L=(πd/2)I describing the relationship between the number of neurite intersections and the vertical distance d between the test lines used.

[0159] In vivo experiment is done using a mouse model for ALS (mouse heterozygous for the mutant SOD1 transgene). In addition, the effect of the selected mimetics on neurite outgrowth, MN density and survival is tested.

[0160] Treatment with Empty Nanoparticles, Mimetics or bNCSCs Extends the Survival of Mutant SOD1G93A Mice

[0161] It was examined how injection of mesoporous silica particles containing mimetics or direct injection of bNCSCs will affect disease course in SOD1 mutant mice. To do this, the B6SJL-TgN-SOD1-.sup.G93A-1Gur (SOD1G93A) mice, heterozygous for the mutant SOD1 transgene, was used. These mice develop progressive motor neuron disease and have a median survival of 128.9±9.1 days (Gurney et al., 1994). 79 days old mice were injected with nanoparticles containing mimetics (n=10) or with empty nanoparticles (n=10) (FIG. 1). A simple and objective measure of disease onset and early disease progression was applied by determining the initiation of weight loss, which reflects denervation-induced muscle atrophy.

[0162] Surprisingly, while timing of disease end stage was extended by injection of mesoporous silica particles loaded with mimetics (140±5 days), the injection of empty nanoparticles had the strongest survival effect (158±4 days) (FIG. 1), demonstrating that these porous particles improved the disease course.

[0163] In addition, the ability of boundary cap neural crest stem cells (bNCSCs) to affect disease course was tested. Injection of bNCSCs cells to the SOD1G93A mice extended survival (142±3.5 days) (FIG. 2), at a similar extent as the nanoparticles loaded with mimetics.

[0164] This strong effect of the empty porous particles can be clearly observed by the results from measurements of grip strength. The empty porous particles group showed a significant (p=≤0.01, multi t-test) enhancement in the pulling strength from hindlimbs (28.8 g) (FIG. 3) and forelimbs (49.61 g) (FIG. 4) at 137 days compared to the group of mimetics or the bNCSC group. Overall, the empty porous particles group showed a significant improvement (p=≤0.01, multi t-test) in the forelimbs throughout the development of the disease at 133 days (29 g), 137 days (28.8 g) and 151 days (19.16) compared to the other groups (mimetics or bNCSCs) (FIG. 4). These results suggest that mice injected with empty particles retain their muscular strength mainly in the forelimbs. This can be explained by the proximity of the forelimb innervating motor neurons to the injection site (C3-C4).

[0165] Mesoporous Silica Particles Sequester Proteins/Peptides Including Proteins Considered as Hallmarks for NDDs.

[0166] Spinal cord extracts were obtained from four 12 weeks old mice, mutant SOD1-.sup.G93A-B6SJL-Tg(SOD1*G93A) 1Gur/J mice (4 mice) and WT blac6 mice (4 mice) by homogenization in a buffer (210 mM mannitol, 70 mM sucrose, 10 mM Tris, pH 7.4 and a protease inhibitors), centrifuged (12,000 g, 10 min). The mitochondria-free supernatants (50 μg protein) from SOD1*G93A and wt mice were incubated for 5 h with 10 μg MSPs particles (37° C. 300 RPM), followed by centrifugation (12,000 g, 10 min). The obtained supernatants (unbound fraction) were subjected to immunoblotting. The obtained mesoporous silica particles pellets were washed with 10 mM Tris, 50 mM NaCl, pH 7.4 and the resulting pellet was resuspended in a 50 μl of lysis buffer (100 mM Tris-HCl, pH 8.0, 5 mM DTT 4% SDS and a protease inhibitor cocktail), incubated for 15 min at 50° C., 300 RPM and centrifuged (20 min, 12,000 g) to obtain the bound fraction. Unbound and bound fractions were subjected to SDS-PAGE, and immunoblotting using the indicated antibodies. (FIG. 5).

[0167] The results demonstrate the porous particles high capacity to absorb proteins as mutant SOD1, and TDP-43. We expect the particles absorbed other proteins, although their entity has not been identified yet. Using proteomics and bioinformatics analysis, we expect to identify the key proteins that are absorbed by MSPs, thereby, eliminating their toxic effects associated with the pathology conditions of ALS and other NDDs. These proteins/factors can be used as biomarker for early detection and/or as new drug targets.

[0168] Development and remodeling of neuronal extensions (neurite outgrowth) in the presence of empty particles (Ivert et al, Ivert P, Otterbeck A, Panchenko M, Hoeber J, Vasylovska S, Zhou C, Garcia Bennett A, Kozlova E N. The Effect of Mesoporous Silica Particles on Stem Cell Differentiation. J Stem Cell Res Ther. 2017; 2(3):73-78. DOI: 10.15406/jsrt.2017.02.00063) in cell culture was determined by tracing neurites and their branches using the stereological procedure as previously described (Ronn et al., A simple procedure for quantification of neurite outgrowth based on stereological principles. J Neurosci Methods. 2000 Jul. 31; 100(1-425-32.).

[0169] The present invention is not limited to the embodiments disclosed but may be varied and modified within the scope of the following claims.