WO2022157381A1

WO2022157381A1 - Phloretin for use in the treatment of neurodegenerative and demyelinating diseases

Info

Publication number: WO2022157381A1
Application number: PCT/EP2022/051607
Authority: WO
Inventors: Jerome HENDRIKS; Tess DIERCKX; Dany Bylemans; Marijke JOZEFCZAK
Original assignee: Universiteit Hasselt
Priority date: 2021-01-25
Filing date: 2022-01-25
Publication date: 2022-07-28

Abstract

The present application is related to compositions comprising phloretin and/or a derivative thereof for use in the treatment and/or prevention of a neurodegenerative disease, more specifically a neurodegenerative and demyelinating disease. Further, the use of phloretin and/or a derivative thereof in in vitro, ex vivo and in vivo assays is disclosed.

Description

PHLORETIN FOR USE IN THE TREATMENT OF NEURODEGENERATIVE AND DEMYELINATING

DISEASES

FIELD OF THE INVENTION

The present application is related to compositions comprising phloretin and/or a derivative

5 thereof for use in the treatment and/or prevention of a neurodegenerative disease, more specifically a neurodegenerative and demyelinating disease. Further, the use of phloretin and/or a derivative thereof in in vitro, ex vivo and in vivo assays is disclosed.

BACKGROUND TO THE INVENTION

Neurodegenerative diseases and in particular multiple sclerosis (MS) are often characterized by chronic inflammation, demyelination and neurodegeneration. Current therapies for MS are able to reduce the inflammatory burden but are unable to stop neurological decline as the disease advances. In this progressive phase, a failure of endogenous remyelination occurs in part due5 to the inability of oligodendrocyte precursor cells (OPCs) to differentiate into mature myelinating oligodendrocytes (OLS). This inability to restore myelin after injury renders axons susceptible to degeneration by the lack of trophic support and enhanced vulnerability to toxic mediators.

Remyelination is a process that is influenced by several aspects such as the level of inhibitory, inflammatory and trophic mediators in the microenvironment of the lesion, as well as the age of0 the patient. Current strategies to enhance remyelination involve approaches such as stimulating resident CNS microglia and peripherally-derived infiltrated macrophages towards a phenotype with wound healing and reparative features. This phenotype is able to accelerate remyelination by clearing inhibitory factors such as myelin debris and obtaining a disease-resolving characteristic associated with trophic factors and reduced inflammatory mediator production. 5 Another strategy to enhance repair is by directly manipulating signaling pathways that regulate OPC differentiation such as PPARgamma, LINGO-1 , Notch, RXR, and Wnt. Overall, therapeutics that influence both immune and repair processes for the treatment of neurodegenerative and remyelinating disorders such as MS are currently still lacking.

Dietary components, such as flavonoids, have been shown to be important stimuli in driving macrophage function and neuro-inflammation. In particular, the flavonoid family is increasingly being acknowledged to contain promising compounds that influence pathogenic pathways and drive the phenotype of immune cells such as macrophages (Thorburn, A.N., L. Macia, and C.R. Mackay, Diet, metabolites, and "western-lifestyle" inflammatory diseases. Immunity, 2014. 5 40(6): p. 833-42; Odegaard, A.O., et al., Western-style fast food intake and cardiometabolic risk in an Eastern country. Circulation, 2012. 126(2): p. 182-8). Flavonoids form one of the largest phytonutrient families that contains over 80000 phenolic compounds with diverse bioactivity. Several members of the flavonoid family display anti-inflammatory and anti-oxidative effects on macrophages. The flavonoid member phloretin is a member of the dihydrochalcones and is present in commonly consumed fruits such as apples and strawberries. Phloretin is known to exert immunomodulatory features and is widely used as a skinceutical due to its anti-oxidative characteristic (Sheldon R. Pinnell, J.Z., Isabelle Hansenne Anti-aging composition containing phloretin. 2006, LOreal SA: US; Kum, H., et al., Evaluation of anti-acne properties of phloretin in vitro and in vivo. Int J Cosmet Sci, 2016. 38(1 ): p. 85-92; Oresajo, C., et al., Protective effects of a topical antioxidant mixture containing vitamin C, ferulic acid, and phloretin against ultraviolet-induced photodamage in human skin. J Cosmet Dermatol, 2008. 7(4): p. 290-7.).

In the present application, the inventors show that phloretin also plays a crucial role in CNS repair, in particular by promoting and accelerating remyelination. The enhanced repair was addressed to the ability of phloretin to stimulate OPC maturation by using in vitro OPC cultures and microglia-depleted ex vivo brain slices.

SUMMARY OF THE INVENTION

The present application discloses a composition comprising phloretin and/or a derivative thereof for use in the treatment and/or prevention of a neurodegenerative disease in a subject.

In a particular aspect, the neurodegenerative disease is selected from Alzheimer’s disease, dementia, multiple sclerosis, Parkinson disease, ALS, Charcot-Marie-Tooth disease, Huntington disease, multiple system atrophy, traumatic nerve injury, diabetic neuropathy, chronic inflammatory polyradiculoneuropathy, Guillain Barre syndrome.

In another aspect, the present invention discloses a composition comprising phloretin and/or a derivative thereof for use in the treatment and/or prevention of a neurodegenerative and demyelinating diseases in a subject.

In a further embodiment, the neurodegenerative and demyelinating disease is a neurodegenerative and demyelinating disease of the central nervous system. Even more preferred, the neurodegenerative and demyelinating disease of the central nervous system is multiple sclerosis; in particular progressive multiple sclerosis.

In yet another embodiment, the present invention provides a composition comprising phloretin and/or a derivative thereof for use in the treatment and/or prevention of a neurodegenerative and demyelinating disease of the central nervous system in a subject. Even more preferred, the neurodegenerative and demyelinating disease of the peripheral nervous system is selected from diabetic neuropathy, Marie-Charcot tooth disease or traumatic nerve injury, chronic inflammatory polyradiculoneuropathy, Guillain Barre syndrome. In a further embodiment, the subject in the present invention is a non-human animal or a human. In a further preferred embodiment, the subject is a human subject.

In another aspect of the present invention, the composition for use according to any of the embodiments, is administered to the subject by oral, intravenous, intraperitoneal or subcutaneous administration. In a preferred embodiment, the composition is administered by oral administration.

In a further aspect, the use of phloretin and/or derivatives thereof is provided in in vitro, ex vivo and in vivo assays selected from remyelination assays, oligodendrocyte progenitor cell (OPC) differentiation assays, OPC myelination assays, OPC migration assays, OPC proliferation assays. In a preferred embodiment, the use of phloretin and/or derivatives thereof is provided in in vitro, ex vivo and in vivo remyelination assays.

In a further aspect, the present invention provides the non-therapeutic use of phloretin and/or derivative thereof in improving physical and/or cognitive ability, fatigue and/or in improving neurological functioning.

In a further aspect, the present application provides a method for preventing and/or treating a neurodegenerative disease in a subject; preferably in a human subject. Said method comprises administering a composition comprising phloretin and/or a derivative thereof to the subject. In a further embodiment, the neurodegenerative disease is selected from Alzheimer’s disease, dementia, multiple sclerosis, Parkinson disease, ALS, Charcot-Marie-Tooth disease, Huntington disease, multiple system atrophy, traumatic nerve injury, diabetic neuropathy, chronic inflammatory polyradiculoneuropathy, Guillain Barre syndrome.

In another embodiment, the neurodegenerative disease is a neurodegenerative and demyelinating disease; in particular a neurodegenerative and demyelinating disease of the central nervous system or a neurodegenerative disease of the peripheral nervous system.

In a further embodiment, a method is provided for preventing and/or treating multiple sclerosis; in particular progressive multiple sclerosis, wherein in said method a composition comprising phloretin and/or a derivative thereof is administered to the subject.

In another embodiment, a method is provided for preventing and/or treating a neurodegenerative and demyelinating disease of the central nervous system and selected from diabetic neuropathy, Marie-Charcot tooth disease or traumatic nerve injury, chronic inflammatory polyradiculoneuropathy, Guillain Barre syndrome, wherein a composition comprising phloretin and/or a derivative thereof is administered to the subject. BRIEF DESCRIPTION OF THE DRAWINGS

Fig 1 : Phloretin induces repair after cuprizone-induced demyelination. A. Representative images of immunofluorescence MBP staining and transmission electron microscopy (TEM) analysis of corpus callosum (CC) from vehicle- or phloretin-treated mice after cuprizone-induced demyelination (6w) and subsequent remyelination (6+1 w). The outer border of the CC is defined by a dotted line. B. Remyelination efficacy (ratio of the amount of myelination at 6+1 w over the amount myelination at 6w using the MBP staining) in CC from vehicle- or phloretin-treated mice. C-D. Analysis of the G-ratio (ratio of the inner axonal diameter to the total outer diameter) and G-ratio in function of the axon diameter in CC from vehicle- or phloretin-treated mice. E-F. mRNA expression of myelin proteins MBP and PLP in the CC of vehicle- or phloretin-treated mice at 6w and 6+1 w. Dotted line represents animals of the same age without cuprizone. G-H. mRNA expression of inflammatory mediators in the CC of vehicle- or phloretin-treated mice at 6w and 6+1 w.

Fig 2: Phloretin stimulates OPC maturation in vitro. A. Representative images of MBP/O4 staining on OPCs treated with phloretin or vehicle. Area positive for MBP and 04 is represented as overlay in white. B-C. Sholl-analysis (related to process complexity and branching of OPCs) treated with phloretin or vehicle as a read-out parameter of OPC differentiation. D. Ratio of the mature oligodendrocyte (OLN) MBP marker over the pre-mature OLN marker 04 as a read-out parameter of OPC differentiation. E. mRNA levels of myelin proteins MBP and PLP in vehicle- or phloretin-treated OPCs.

Fig 3: Phloretin stimulates OPC-mediated repair after LPC-induced demyelination in ex vivo brain slices. A. Representative images of vehicle- or phloretin-treated cerebellar brain slice cultures (BSC) stimulated with or without clodronate liposomes. B. Remyelination index of vehicle- or phloretin-stimulated BSC. C. Remyelination index of vehicle- or phloretin-treated BSC stimulated with clodronate liposomes.

Fig 4: Phloretin stimulates OPC maturation in PPARy-dependent manner. A. Luciferase assay illustrating a dose-response activation of PPARy in cos7 cells treated with different phloretin concentrations or the PPARy-agonist rosiglitazone. B. mRNA expression of PPARy response-genes in OPCs treated with vehicle or phloretin. C. mRNA expression of mature oligodendrocyte (OLN) markers MPB/PLP in OPCs treated with phloretin or phloretin and the PPARy-antagonist GW9662 together. Dotted line represents OPCs treated with vehicle. D. Representative images of a MBP/O4 staining on OPCs treated with phloretin or phloretin and the PPARy-antagonist GW9662 together. Area positive for MBP and 04 is represented as overlay in white. E-F. Sholl-analysis related to process complexity and branching of OPCs treated with phloretin or phloretin and PPARy-antagonist GW9662 as a read-out parameter of OPC differentiation. G. Ratio of the mature OLN MBP marker over the pre-mature OLN marker 04 of OPCs treated with phloretin or phloretin and PPARy-antagonist GW9662 as a read-out parameter of OPC differentiation. H. mRNA levels of PPARy-response genes in the CC of vehicle-or phloretin-treated mice at timepoint 6w in the cuprizone experiment. I. mRNA levels of PPARy-response genes in the CC of vehicle-or phloretin-treated mice at timepoint 6+1 w in the cuprizone experiment. J. mRNA levels of PPARy-response genes in vehicle- or phloretin-treated brain slices.

Fig. 5: Transcriptional changes associated with phloretin treatment of macrophages. RNA sequencing was performed to establish the anti-inflammatory effects of phloretin and identify the underlying mechanisms. Differentially expressed genes were used as input for the core analysis in Ingenuity Pathway Analysis (IPA) (n=5, cut-off criteria p<0.05, see supplementary Fig. 1 ). A. Pathway analysis of activated macrophages stimulated with phloretin demonstrates a downregulation in the expression of genes associated with pro-inflammatory canonical pathways. In addition, pathway analysis of phloretin-treated macrophages illustrates that the Nrf2 pathway, among other pathways, is activated. -Log (P-value of overlap) and down- or upregulated canonical pathways with corresponding z-score are indicated at x- and y-axis, respectively. B-C. Heat map representing the normalized counts of differentially expressed genes associated to the pro-inflammatory canonical pathways (INOS-, toll-like receptor-, acute phase response- and interferon-signaling) and the Nrf2 pathway. A color gradient was used to indicate the normalized counts and corresponding fold change (Fc) differences per sample and gene, respectively. Negative fold changes are represented by in their box. D. Upstream analysis of the RNA-seq data predicts that phloretin lowered the activation of pro-inflammatory transcription regulators (IRF1 , IRF7 and STAT1 , and stimulated the activation Nrf2 (NFE2L2) transcription factor. E. Downstream analysis of the RNA-seq samples in IPA illustrated that phloretin upregulated the expression of a set of genes involved in the regulation of ROS levels as one of the main downstream functional effects (z-score: 2.008). Ctrl, control; phi, phloretin.

Fig. 6: The Nrf2 pathway controls the phenotype of phloretin-treated macrophages A. ROS production in vehicle- or phloretin-treated bone marrow-derived macrophages (BMDMs) stimulated with PMA (n=9). B. NO production in vehicle- or phloretin-treated BMDMs stimulated with LPS (n=9-11 ) C. mRNA levels of the pro-inflammatory genes IL-6, NOS2, C0X2 and IL-12 in vehicle- or phloretin-treated BMDMs stimulated with LPS (n=13-16). D. mRNA levels of Nrf2- response genes HO1 and NQ01 in LPS-stimulated WT and Nrf2 KO BMDMs (n=9-10). Dotted line represents corresponding control cells stimulated with LPS. E. ROS production (n=5-9) in vehicle- or phloretin-treated WT and Nrf2 KO BMDMs after PMA stimulation. F. Pro- inflammatory gene expression of NOS2, IL-6, C0X2 and IL-12 (n=9-10) in phloretin-treated WT and LW2 KO BMDMs after LPS stimulation. Dotted line represents the corresponding control cells stimulated with LPS. Ctrl, control; phi, phloretin. Data are represented as mean ± s.e.m. *p < 0.05, “p < 0.01 , *“p < 0.001 and **“p < 0.0001 . Fig. 7: Phloretin promotes AMPK activation. A-B. Western blot quantification and representative bands of pAMPK and AMPK in LPS-activated BMDMs stimulated with phoretin or phloretin and the AMPK-inhibitor BML-275 together (n=3). Dotted line represents control cells stimulated with LPS. C. ROS production in phloretin-treated or phloretin and AMPK-inhibitor- treated BMDMs (n=10). Dotted line represents control cells stimulated with PMA. Ctrl, control; phi, phloretin. Data are represented as mean ± s.e.m. *p < 0.05, “p < 0.01 and ***p < 0.001 .

Fig. 8: Phloretin stimulates autophagy in an AMPK-dependent manner. A. Downstream analysis of the RNA-seq data obtained from phloretin treated BMDMs stimulated with LPS illustrated autophagy as one of the downstream biological processes that is activated by phloretin (z-score: 0.779). Data are represented by a heat map containing the normalized counts of genes associated with autophagy. A color gradient was used to indicate the normalized counts and corresponding Fold change (Fc) differences, per sample and gene respectively. Negative fold changes are represented by in their box . B-D Quantification and representative images of LC3 and p62 staining in phloretin-treated BMDMs stimulated with bafilomycin A1 (n=5). Dotted line represents cells without bafilomycin A1 treatment. E-F. Western blot quantification and representative bands of the autophagy markers LC3II and p62 in phloretin-treated BMDMs stimulated with bafilomycin A1 alone or bafilomycin and the AMPK-inhibitor together (n=2). Dotted line represents cells treated with bafilomycin A1. Ctrl, control; phi, phloretin; baf, bafilomycin A1 . Data are represented as mean ± s.e.m. *p < 0.05 and **p < 0.01 .

Fig. 9: Phloretin activates the Nrf2 pathway through autophagy-mediated Keapl degradation. A-B. Representative images of Keapl and p62 staining and quantification of their colocalisation (Pearson coefficient) on control or phloretin-treated BMDMs (90+ cells per well, 3 wells). Area positive for Keapl and p62 is represented as overlay in white. C. Quantification of Keapl positive counts in phloretin-treated BMDMs treated with or without bafilomycin A1 (90+ cells per well, 3 wells). Dotted line represents the corresponding control cells (stimulated with or without bafilomycin A1 ). D-E. Western blot quantification and representative bands of phloretin- treated BMDMs treated with or without bafilomycin A1 (n=4). Dotted line represents the corresponding control cells (stimulated with or without bafilomycin A1 ). Ctrl, control; phi, phloretin; baf, bafilomycin A1 . Data are represented as mean ± s.e.m. *p < 0.05 and “p < 0.01 .

Fig. 10: Phloretin reduces neuroinflammation in the EAE model. A. Disease scores of EAE mice treated 6 days post immunization with vehicle or phloretin on a daily basis (prophylactic setting, 50 mg/kg ip, n=5) B. Disease scores of mice in which EAE was induced. Treatment of vehicle or phloretin on a daily basis started after disease onset (disease score>1 ) (therapeutic setup, 50 mg/kg, n=5). C-D. Quantification and representative images of F4/80 staining of spinal cord tissue obtained from EAE animals treated with vehicle or phloretin in the prophylactic setting. E-G. Quantitative PCR was used to determine the mRNA levels of the pro-inflammatory genes TNFa, Nos2, IL-6, Ccl4, Ccl5 and CXCL2, the anti-inflammatory and neurotrophic genes IL-4, CNTF and IGF-1 and genes related to the Nrf2 pathway (Nrf2, NQO1, GPX1) in the spinal cord of phloretin-treated EAE animals (prophylactic setting). Gene expression was corrected for the number of F4/80+ cells. Ctrl, control; phi, phloretin. Data are represented as mean ± s.e.m. *p < 0.05 and **p < 0.01 .

DETAILED DESCRIPTION OF THE INVENTION

The present invention is typically characterized in that it provides compositions comprising phloretin and/or a derivative thereof for use in the prevention and/or treatment of a neurodegenerative disease. In a preferred embodiment, said compositions are provided for use in the treatment and/or prevention of a neurodegenerative and demyelinating disease.

The inventors of the present application surprisingly found that the dihydrochalcone phloretin is able to accelerate remyelination. In particular, the inventors found that phloretin stimulated OPC maturation in in vitro OPCs cultures and improved (re)myelination in microglia-depleted ex vivo demyelinated brain slices. Furthermore, it is shown here that phloretin is able to drive OPC differentiation via PPARy activation.

Because of these effects on remyelination and oligodendrocyte differentiation and maturation, the present application provides in a first aspect a composition comprising phloretin and/or a derivative thereof for use in the treatment and/or prevention of neurodegenerative diseases. Those neurodegenerative diseases are typically characterized by reduced or even loss of function of nerve cells in the brain or peripheral nervous system. Examples of such neurodegenerative diseases are Alzheimer’s disease, dementia, multiple sclerosis, Parkinson disease, ALS, Charcot-Marie-Tooth disease, Huntington disease, multiple system atrophy, traumatic nerve injury, diabetic neuropathy, chronic inflammatory polyradiculoneuropathy, Guillain Barre syndrome.

In a further embodiment, the present invention provides a composition comprising phloretin and/or a derivative thereof for use in the prevention and/or treatment of a neurodegenerative and demyelinating disease. It is accordingly an objective of the present invention to provide a composition comprising phloretin and/or a derivative thereof for use in the prevention and/or treatment of a neurodegenerative and demyelinating disease of the central or peripheral nervous system. In one embodiment, the neurodegenerative and demyelinating disease is a neurodegenerative and demyelinating disease of the central nervous system; preferably said disease is multiple sclerosis; even more preferably said disease is progressive multiple sclerosis. In another embodiment, the neurodegenerative and demyelinating disease is a neurodegenerative and demyelinating disease of the peripheral nervous system; in particular a neurodegenerative and demyelinating disease selected from diabetic neuropathy, Marie- Charcot tooth disease or traumatic nerve injury, chronic inflammatory polyradiculoneuropathy, Guillain Barre syndrome.

As used herein, the term “demyelinating disease”, is a disease condition in which the myelin sheath which surrounds neurons in nervous tissue is lost or damaged, leading to axonal degeneration and impaired signal transduction in the affected nerves. A demyelinating disease of the central nervous system is a disease in which the myelin sheaths of neurons in the central nervous system are lost or damaged. Examples of demyelinating diseases of the central nervous systems are multiple sclerosis, neuromyelitis optic (Devic’s disease), inflammatory demyelinating diseases, central nervous system neuropathy, central pontine myelinolysis, myelopathy, leukoencephalopathy, or leukodystrophy.

A demyelinating disease of the peripheral nervous system is a disease condition in which the myelin sheaths of neurons in the peripheral nervous system are lost or damaged. Examples of demyelinating diseases of the peripheral nervous system are Guillain-Barre syndrome, chronic inflammatory demyelinating polyneuropathy, anti-MAG peripheral neuropathy, Charcot-Marie tooth disease, hereditary neuropathy with liability to pressure palsy; copper deficiency- associated conditions such as peripheral neuropathy, myelopathy, optic neuropathy; progressive inflammatory neuropathy, diabetic neuropathy or traumatic nerve injury.

As used herein, the term “multiple sclerosis” or “MS” entails an autoimmune-mediated process in which an abnormal response of the body’s immune system is directed against the central nervous system (CNS), which is made up of the brain, spinal cord and optic nerves. The immune reaction results in death of oligodendrocytes, demyelination, and eventually loss of axons, featured by a physical and cognitive disability.

As used herein, the term “progressive multiple sclerosis” or “pMS” is featured by an accumulation of chronic demyelinated lesions and is subdivided in Primary progressive MS (PPMS), Secondary progressive MS (SPMS) and relapse remitting MS (RRMS).

Primary progressive MS (PPMS) is characterized by worsening neurologic function (accumulation of disability) from the onset of symptoms, without early relapses or remissions. PPMS can be further characterized at different points in time as either active (with an occasional relapse and/or evidence of new MRI activity) or not active, as well as with progression (evidence of disease worsening on an objective measure of change over time, with or without relapse or new MRI activity) or without progression. Secondary progressive MS (SPMS) follows an initial relapsing-remitting course. Most people who are diagnosed with a relapse remitting MS (RRMS) will eventually transition to a secondary progressive course in which there is a progressive worsening of neurologic function (accumulation of disability) over time. SPMS can be further characterized at different points in time as either active (with relapses and/or evidence of new MRI activity) or not active, as well as with progression (evidence of disease worsening on an objective measure of change over time, with or without relapses) or without progression.

The subject may be a non-human animal or a human. Preferably, the subject is a human.

Furthermore, the invention provides the use of phloretin and/or derivatives thereof in in vitro, ex vivo and in vivo assays, selected from remyelination assays, oligodendrocyte progenitor cell (OPC) differentiation assays, OPC myelination assays, OPC migration assays or OPC proliferation assays. In a preferred embodiment, the use of phloretin and/or derivatives thereof in in vitro, ex vivo and in vivo remyelination assays is provided.

Said in vitro, ex vivo and in vivo remyelination assays may for example be characterized by OPC differentiation assays (in vitro), brain slices (ex vivo) and cuprizone modelling with a molecular and functional readout (in vivo).

In a further aspect, the composition for use according to the different embodiments is administered to the subject by oral, intravenous, intraperitoneal or subcutaneous administration; preferably by oral administration.

Generally, for pharmaceutical use, the compounds of the inventions may be formulated as a pharmaceutical preparation or pharmaceutical composition comprising at least one compound of the invention and at least one pharmaceutically acceptable carrier, diluent or excipient and/or adjuvant, and optionally one or more further pharmaceutically active compounds.

By means of non-limiting examples, such a formulation may be in a form suitable for oral administration, for parenteral administration (such as by intravenous, intramuscular or subcutaneous injection or intravenous infusion), etc.. Such suitable administration forms - which may be solid, semi-solid or liquid, depending on the manner of administration - as well as methods and carriers, diluents and excipients for use in the preparation thereof, will be clear to the skilled person; reference is again made to for instance US-A-6,372,778, US-A-6,369,086, US-A-6,369,087 and US-A-6,372,733, as well as to the standard handbooks, such as the latest edition of Remington’s Pharmaceutical Sciences. Some preferred, but non-limiting examples of such preparations include tablets, pills, powders, lozenges, sachets, cachets, elixirs, suspensions, emulsions, solutions, syrups, aerosols, ointments, creams, lotions, soft and hard gelatin capsules, suppositories, eye drops, sterile injectable solutions and sterile packaged powders (which are usually reconstituted prior to use) for administration as a bolus and/or for continuous administration, which may be formulated with carriers, excipients, and diluents that are suitable per se for such formulations, such as lactose, dextrose, sucrose, sorbitol, mannitol, starches, gum acacia, calcium phosphate, alginates, tragacanth, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, polyethylene glycol, cellulose, (sterile) water, methylcellulose, methyl- and propylhydroxybenzoates, talc, magnesium stearate, edible oils, vegetable oils and mineral oils or suitable mixtures thereof. The formulations can optionally contain other pharmaceutically active substances (which may or may not lead to a synergistic effect with the compounds of the invention) and other substances that are commonly used in pharmaceutical formulations, such as lubricating agents, wetting agents, emulsifying and suspending agents, dispersing agents, disintegrants, bulking agents, fillers, preserving agents, sweetening agents, flavoring agents, flow regulators, release agents, etc.. The compositions may also be formulated so as to provide rapid, sustained or delayed release of the active compound(s) contained therein, for example using liposomes or hydrophilic polymeric matrices based on natural gels or synthetic polymers. In order to enhance the solubility and/or the stability of the compounds of a pharmaceutical composition according to the invention, it can be advantageous to employ a-, - or y- cyclodextrins or their derivatives. An interesting way of formulating the compounds in combination with a cyclodextrin or a derivative thereof has been described in EP-A-721 ,331 . In particular, the present invention encompasses a pharmaceutical composition comprising an effective amount of a compound according to the invention with a pharmaceutically acceptable cyclodextrin.

In addition, co-solvents such as alcohols may improve the solubility and/or the stability of the compounds. In the preparation of aqueous compositions, addition of salts of the compounds of the invention can be more suitable due to their increased water solubility.

Particular reference is made to the compositions, formulations (and carriers, excipients, diluents, etc. for use therein), routes of administration etc., such as those described in WO2015121212. More in particular, the compositions may be formulated in a pharmaceutical formulation comprising a therapeutically effective amount of particles consisting of a solid dispersion of the compounds of the invention and one or more pharmaceutically acceptable water-soluble polymers. The term "a solid dispersion" defines a system in a solid state (as opposed to a liquid or gaseous state) comprising at least two components, wherein one component is dispersed more or less evenly throughout the other component or components. When said dispersion of the components is such that the system is chemically and physically uniform or homogenous throughout or consists of one phase as defined in thermodynamics, such a solid dispersion is referred to as "a solid solution". Solid solutions are preferred physical systems because the components therein are usually readily bioavailable to the organisms to which they are administered.

It may further be convenient to formulate the compounds in the form of nanoparticles which have a surface modifier adsorbed on the surface thereof in an amount sufficient to maintain an effective average particle size of less than 1000 nm. Suitable surface modifiers can preferably be selected from known organic and inorganic pharmaceutical excipients. Such excipients include various polymers, low molecular weight oligomers, natural products and surfactants. Preferred surface modifiers include nonionic and anionic surfactants.

Yet another interesting way of formulating the compounds according to the invention involves a pharmaceutical composition whereby the compounds are incorporated in hydrophilic polymers and applying this mixture as a coat film over many small beads, thus yielding a composition with good bio-availability which can conveniently be manufactured and which is suitable for preparing pharmaceutical dosage forms for oral administration. Materials suitable for use as cores in the beads are manifold, provided that said materials are pharmaceutically acceptable and have appropriate dimensions and firmness. Examples of such materials are polymers, inorganic substances, organic substances, and saccharides and derivatives thereof.

The preparations may be prepared in a manner known per se, which usually involves mixing at least one compound according to the invention with the one or more pharmaceutically acceptable carriers, and, if desired, in combination with other pharmaceutical active compounds, when necessary under aseptic conditions. Reference is again made to US-A-6,372,778, US-A- 6,369,086, US-A-6,369,087 and US-A-6,372,733 and the further prior art mentioned above, as well as to the standard handbooks, such as the latest edition of Remington’s Pharmaceutical Sciences.

The pharmaceutical preparations of the invention are preferably in a unit dosage form, and may be suitably packaged, for example in a box, blister, vial, bottle, sachet, ampoule or in any other suitable single-dose or multi-dose holder or container (which may be properly labeled); optionally with one or more leaflets containing product information and/or instructions for use. Generally, such unit dosages will contain between 0.01 and 1000 mg, usually between 0.05 and 500 mg, of at least one compound of the invention, e.g. about 0.05, 1 , 2.5, 5, 10, 20, 50, 100, 150, 200, 250 or 500 mg per unit dosage.

The compounds can be administered by a variety of routes including the oral, rectal, ocular, transdermal, subcutaneous, intravenous, intramuscular or intranasal routes, depending mainly on the specific preparation used and the condition to be treated or prevented, and with oral and intravenous administration usually being preferred. The at least one compound of the invention will generally be administered in an “effective amount”, upon suitable administration, that is sufficient to achieve the desired therapeutic or prophylactic effect in the individual to which it is administered.

Usually, depending on the condition to be prevented or treated and the route of administration, such an effective amount will usually be between 0.01 to 1000 mg per day, more often between 0.05 and 500 mg, such as for example about 0.05, 1 , 2.5, 5, 10, 20, 50, 100, 150, 200, 250 mg or 500mg, which may be administered as a single daily dose, divided over one or more daily doses, or essentially continuously, e.g. using a drip infusion. The amount(s) to be administered, the route of administration and the further treatment regimen may be determined by the treating clinician, depending on factors such as the age, gender and general condition of the patient and the nature and severity of the disease/symptoms to be treated. Reference is again made to USA-6, 372, 778, US-A-6, 369, 086, US-A-6,369,087 and US-A-6,372,733 and the further prior art mentioned above, as well as to the standard handbooks, such as the latest edition of Remington’s Pharmaceutical Sciences.

In accordance with the method of the present invention, said pharmaceutical composition can be administered separately at different times during the course of therapy or concurrently in divided or single combination forms. The present invention is therefore to be understood as embracing all such regimes of simultaneous or alternating treatment and the term "administering" is to be interpreted accordingly.

For an oral administration form, the compositions of the present invention can be mixed with suitable additives, such as excipients, stabilizers, or inert diluents, and brought by means of the customary methods into the suitable administration forms, such as tablets, coated tablets, hard capsules, aqueous, alcoholic, or oily solutions. Examples of suitable inert carriers are gum arabic, magnesia, magnesium carbonate, potassium phosphate, lactose, glucose, or starch, in particular, corn starch. In this case, the preparation can be carried out both as dry and as moist granules. Suitable oily excipients or solvents are vegetable or animal oils, such as sunflower oil or cod liver oil. Suitable solvents for aqueous or alcoholic solutions are water, ethanol, sugar solutions, or mixtures thereof. Polyethylene glycols and polypropylene glycols are also useful as further auxiliaries for other administration forms. As immediate release tablets, these compositions may contain microcrystalline cellulose, dicalcium phosphate, starch, magnesium stearate and lactose and/or other excipients, binders, extenders, disintegrants, diluents and lubricants known in the art.

For subcutaneous administration, the compound according to the invention, if desired with the substances customary therefore such as solubilizers, emulsifiers or further auxiliaries are brought into solution, suspension, or emulsion. The compounds of the invention can also be lyophilized and the lyophilizates obtained used, for example, for the production of injection or infusion preparations. Suitable solvents are, for example, water, physiological saline solution or alcohols, e.g. ethanol, propanol, glycerol, in addition also sugar solutions such as glucose or mannitol solutions, or alternatively mixtures of the various solvents mentioned. The injectable solutions or suspensions may be formulated according to known art, using suitable non-toxic, parenterally-acceptable diluents or solvents, such as mannitol, 1 ,3-butanediol, water, Ringer's solution or isotonic sodium chloride solution, or suitable dispersing or wetting and suspending agents, such as sterile, bland, fixed oils, including synthetic mono- or diglycerides, and fatty acids, including oleic acid.

The compositions are of value in the veterinary field, which for the purposes herein not only includes the prevention and/or treatment of diseases in animals, but also - for economically important animals such as cattle, pigs, sheep, chicken, fish, etc. - enhancing the growth and/or weight of the animal and/or the amount and/or the quality of the meat or other products obtained from the animal. Thus, in a further aspect, the invention relates to a composition for veterinary use that contains at least one compound of the invention and at least one suitable carrier (i.e. a carrier suitable for veterinary use). The invention also relates to the use of a compound of the invention in the preparation of such a composition.

As detailed herein above, demyelinating diseases are particularly characterized by impaired physical ability, fatigue, cognitive ability, in particular due to impaired neurological functioning. Accordingly, the present invention provides the non-therapeutic use of phloretin and/or derivatives thereof in improving physical and/or cognitive ability and/or in improving neurological functioning.

The compounds of the present invention (i.e. phloretin and derivatives thereof) may thus be used as nutritional supplements (e.g. as nutraceutical), particularly for people who want to promote their cognitive function and / or psychosocial support. A non-exhaustive list of people who would benefit from enhanced cognitive function would include: elderly people, students or persons who are preparing for exams, children who are engaged in a great deal of learning, i.e. infants, toddlers, pre-school children and school children, construction workers, or those operating potentially dangerous machinery, truck drivers, pilots, train drivers, or other transportation professionals, air traffic controllers, salespeople, executives, and other "high performance professionals" police officers and military personnel, housewives, or for anyone exposed to high amounts of stress in their daily work or who needs especially high attention/concentration/high mental and psychological performance in their daily work, such as those participating in sports, chess players, golfers, professional performers (e.g. actors, musicians).

The term "nutraceutical" as used herein denotes usefulness in both nutritional and pharmaceutical fields of application. Thus, novel nutraceutical compositions can be used as supplements to food and beverages and as pharmaceutical formulations for enteral or parenteral application which may be solid formulations, such as capsules or tablets, or liquid formulations, such as solutions or suspensions.

Examples of food are dairy products including, for example, margarines, spreads, butter, cheese, yoghurts or milk-drinks.

Examples of fortified food are sweet corn, bread, cereal bars, bakery items, such as cakes and cookies, and potato chips or crisps.

Beverages encompass non-alcoholic and alcoholic drinks as well as liquid preparations to be added to drinking water and liquid food. Non-alcoholic drinks are e.g. soft drinks, sports drinks, fruit juices, lemonades, teas and milk-based drinks. Liquid foods are e.g. soups and dairy products. The nutraceutical composition containing phloretin or its derivates may be added to a soft drink, an energy bar, or a candy.

In another aspect, the application provides the use of a composition comprising phloretin and/or a derivative thereof as food supplement or in the food industry. More specifically, the composition comprising phloretin and/or a derivative thereof can be use in dietary supplements, dietary food additives and/or nutraceuticals.

In one aspect of the present application, the composition comprises phloretin and/or a derivative thereof. In a specific aspect, the composition of the present invention comprises phloretin. In another aspect, the composition comprises one or more derivatives of phloretin. In another aspect the composition comprises phloretin in combination with one or more derivatives thereof.

Phloretin is the aglucone portion of phlorizin and is a polyphenolic compound, also known as 3- (4-Hydroxyphenyl)-1 -(2,4,6-trihydroxyphenyl)propan-1 -one. It is split from phlorizin by acid hydrolysis. Alternatively, phloretin can be completely synthesized directly by known processes. Suitable phloretin derivatives include, but are not limited to, dihydrochalcone derivatives, such as those disclosed in U.S. Pat. No. 6,448,232, the entire contents of which is hereby incorporated by reference. Also glycosylated derivatives, such as phlorizin, or phosphorylated derivatives, such as 2-phosphophloretin, may suitably be used within the context of the invention.

In the context of the present invention, the term “derivative” is meant to be an analog of phloretin, which has substantially similar characteristics/functions as phloretin itself, i.e. it may be a functional analogue of phloretin.

The invention will now be illustrated by means of the following synthetic and biological examples, which do not limit the scope of the invention in any way.

EXAMPLES

EXAMPLE 1 : Effects of phloretin on remyelination

Materials and methods

Antibodies and chemical reagents

Phloretin (Sigma Aldrich) was dissolved in 50 mM KOH to a 15mM stock solution and stored at -20°C. For in vivo treatment, phloretin was dissolved in 1 N NaOH (pH was readjusted to 7.2 with 1 N HCL) and further diluted in physiological water to obtain a concentration of 50 mg/kg. The following antibodies were used for immunofluorescence: rat anti-MBP (1/500, MAB386, Millipore, brain cryosections and in vitro OPC cultures), rat anti-MBP (1/250, MCA409S, Millipore, cerebellar brain slices), mouse anti-04 (1/1000, MAB1326, R&Dsystems), Mouse anti- CC1 (1/50, ab16794, Abeam), Goat anti-olig2 (1/50, AF2418, R&Dsystems). Appropriate secondary antibodies were purchased from Invitrogen.

Mice

Wild type (WT) C57BL/6JOIaHsd mice (purchased from Envigo) were fed a regular diet and housed in the animal facility of the Biomedical Research Institute of Hassell University. All experiments were performed according to institutional guidelines and were approved by the ethical committee for animal experiments of Hassell University.

Cell lines

Coss/Oli-neu cells were cultured in RPM1640 (Gibco) supplemented with 1% penicillin/streptomycin (P/S, Invitrogen), 10% fetal calf serum (FCS, Invitrogen), and 1 % L- glutamine (Sigma-Aldrich).

Bone marrow derived macrophages

Bone marrow-derived macrophages (BMDMs) were isolated from WT purchased from Envigo. BMDMs were obtained as described previously (Bogie, J.F., et al., Scavenger receptor collectin placenta 1 is a novel receptor involved in the uptake of myelin by phagocytes. Sci Rep, 2017. 7: p. 44794). In short, tibial and femoral bone marrow cells from 12-week-old WT mice were cultured in 10 cm petri plates at a concentration of 10 x 106 cells/plate, in RPMI-1640 medium supplemented with 10% FCS, 1% P/S, and 15% L929-conditioned medium (LCM). After differentiation, BMDMs were detached at 37°C with 10 mM EDTA in PBS (Gibco) and cultured (0.5 x 10⁶ cells/ml) in RPMI 1640 supplemented with 10% FCS, 1 %P/S, and 5% LCM at 37°C and 5% CO2.

OPC isolation and culturing

OPCs were isolated from pooled P0-P2 C57BU6JOIaHsd neonatal mice cerebral cortices. Cortices were isolated, meninges removed, minced and dissociated for 20 min at 37°C with papain and DNase I (both 20 pg/mL, Sigma-Aldrich). The resulting mixed glial cultures were seeded in poly-L-lysine (PLL, 50 ug/ml, Sigma-Aldrich)-coated T75 flasks and cultured (37°C, 8.5% CO2) in DMEM (Invitrogen) supplemented with 10% FCS and 1% P/S. After day 7, the medium was supplemented with insulin (5 pg/ml , Sigma-Aldrich). Medium changes were performed on day 4, 7, 1 1 and 14. Mixed glial cultures were separated after 14 days by mechanical shaking at 75 rpm for 45min (to remove microglia) followed by additional 18h shaking at 250 rpm at 37°C. Medium containing the detached cells was then transferred to petri dishes to further remove microglia and astrocytes based on differential adhesion characteristics (20 min, 37°C, 8.5% CO2). Afterwards, the enriched OPCs were collected and plated in PLL-coated wells. OPCs were cultured in sato medium (DMEM, 100 pg/ml apo-transferrin, 16 pg/ml Putrescine, 5 pg/ml insulin, 60 ng/ml progesterone, 40 ng/ml sodium selenite, 30 ng/ml triiodothyronine, 40 ng/ml L-Thyroxine, 1% P/S, 2% horse serum, 2% B-27; all from Sigma- Aldrich) supplemented with PDGF and bFGF (both 10 ug/ml, Peprotech) for the first two days to reset their cell cycle. Afterwards, OPCs were cultured in normal sato medium and medium was changed every two days.

Luciferase-based nuclear receptor reporter assay.

To determine the activation of PPARa, PPARp/6, and PPARy, luciferase-based reporter assays were performed using the ONE-GloTM Luciferase Assay System kit (Promega). Coss cells were transfected with bacterial plasmid constructs expressing luciferase under the control of the ligand-binding domain for PPARa, PPARp/6, or PPARy, which were kindly provided by prof. dr. Bart Staels (Univ. Lille, Inserm, France). Cells were grown to 60% confluency in 60 mm plates, transfected with 1.8 pg of plasmid DNA including 0.2 pg pGAL4hPPARa, pGAL4hPPARp/6 or pGAL4hPPARy, 1 pg pG5-TK-GL3, and 0.6 pg of pCMV-p-galactosidase. JetPEI (Polyplustransfection SA, France) was used as transfection reagent. Transfected cells were treated with vehicle or phloretin for 24 h. Following treatment, cells were lysed in lysis buffer (25 mM Glycyl- Glycine, 15 mM MgSO4, 4 mM EGTA, and 1x Triton; all from Sigma-Aldrich). To correct for transfection efficacy, p-galactosidase activity was measured using cell lysate (10%) in B-gal buffer, consisting of 20% 2-Nitrophenyl p-D-galactopyranoside (ONGP; Sigma-Aldrich) and 80% Buffer-Z (0.1 M Na2HPO4, 10 mM KCI, 1 mM MgSO4, and 3.4 pl/ml 2-mercaptoethanol; all from Sigma-Aldrich). Luminescence and absorbance (410 nm) were measured using the FLUOstar Optima (BMG Labtech).

Quantitative PCR (qPCR)

Cell lysis was performed by using Qiazol Lysis reagent (Qiagen). RNA was extracted using the RNeasy mini kit (Qiagen). RNA concentration and quality were determined with a Nanodrop spectrophotometer (Isogen Life Science). cDNA synthesis was performed by using the Quanta qScript cDNA SuperMix (Quanta Biosciences) per manufacturer’s instructions. qPCR was conducted on a StepOnePlus™ Real-Time PCR system (Applied biosystems) using a SYBR green mix containing 1 x SYBR green (Applied Biosystems), 0.3 pM primers (Integrated DNA Technologies), 12.5 ng cDNA and nuclease free water. The comparative Ct method was used to quantify gene expression. Data were normalized to the most stable reference genes cyclin A (cyca) and hypoxanthine phosphoribosyltransferase 1 (hprf). Primer sequences are available on request.

Immunofluorescence and analysis

Frozen brain cryosections were air-dried and fixed in ice cold acetone for 10 min at -20°C. Cerebellar brain slices were fixed in 4% paraformaldehyde (PFA) for 15 min. Mouse OPCs were cultured on PLL-coated glass cover slides and fixed in 4% PFA for 30 min. Brain sections, cerebellar slices and OPCs were blocked by using either Dako protein block (Agilent, 30 min), blocking buffer containing 1 % BSA and 0.1% Triton X-100 in PBS (1 hour) or blocking buffer containing 1% BSA in 0.1 % PBS-Tween (30 min), respectively. Afterwards, they were incubated overnight at 4°C with primary antibodies, washed, and incubated with the appropriate secondary antibodies for one hour at RT. Images of brain cryosections and OPCs were taking using a Nikon eclipse 80i microscope (1 Ox objective) and NIS Elements BR 3.10 software (Nikon). To quantify the level of OPC maturation in vitro, MBP/NF ratios per cell and Sholl analysis parameters related to process complexity and branching (sum intersections and average intersections/sholl ring) were used as readouts. Sholl analysis was performed as described previously (Murtie, J.C., W.B. Macklin, and G. Corfas, Morphometric analysis of oligodendrocytes in the adult mouse frontal cortex. J Neurosci Res, 2007. 85(10): p. 2080-6.; Rajasekharan, S., et al., Netrin 1 and Dec regulate oligodendrocyte process branching and membrane extension via Fyn and RhoA. Development, 2009. 136(3): p. 415-26.). Images of cerebellar brain slices were made on the LSM880 confocal microscope (Zeiss). The level of remyelination in brain slice were represented by the myelinating index, which was calculated by dividing the colocalized area of MBP and NF by the total NF area. Colocalization was calculated using the ‘colocalize threshold’ plugin in Imaged. Three-dimensional rendering of cerebellar brain slices was done in the vaa3d software (Peng, H., et al., Extensible visualization and analysis for multidimensional images using Vaa3D. Nat Protoc, 2014. 9(1 ): p. 193-208.). The images shown in the figures are digitally enhanced.

Transmission Electron Microscope (TEM)

Brain samples were isolated and fixed with 2% glutaraldehyde. Afterwards, the samples were post-fixed in 2% osmiumtetroxide in 0.05 M sodium cacodylate buffer for 1 h at 4°C. Samples were dehydrated by ascending concentrations of acetone and impregnated overnight in a 1 :1 mixture of acetone and araldite epoxy resin. Thereafter, the samples were embedded in araldite epoxy resin at 60°C and cut in slices of 70 nm (perpendicular to the CC, Leica EM UC6 microtome) and transferred to 0.7% formvar-coated copper grids (Aurion). The samples were contrasted with 0.5% uranyl acetate and lead citrate using a Leica EM AC20. Analysis was performed by using the Philips EM208 S electron microscope (Philips) equipped with a Morada Soft Imaging System camera with iTEM-FEI software (Olympus SIS). Imaged was used to calculate the g-ratio (the ratio of the inner axonal diameter to the total outer diameter), using between four-eight images/animal. Cerebellar slice cultures

Cerebellar slices were obtained from C57BL/6JOIaHsd mouse pups at the age of P9 or P10, as described previously (Hussain et al., 201 1 ; Meffre et al., 2015). To induce demyelination, slices were treated with lysolecithin (LPC, 0.5 mg/ml, Sigma-Aldrich) three days after isolation for 16 hours. After demyelination, slices were treated daily with phloretin (50 pM) or vehicle for 6 days. For microglia depletion, slices were treated with clodronate or empty liposomes (0.5 mg/ml, LIPOSOMA) immediately after isolation for 24 hours. After three days, slices were treated with LPC for 16 hours followed by daily treatment of phloretin or vehicle.

In vivo cuprizone-induced demyelination

To induce demyelination, 10-week-old male mice (n=10) were fed ad libitum a diet of 0.3% cuprizone (bis[cyclohexanone]oxaldihydrazone, Sigma-Aldrich) mixed in powdered standard chow for 6 weeks (6w). Mice were daily intraperitoneal injected with vehicle or phloretin (50 mg/kg) starting from the first day of cuprizone diet. Upon withdrawal of the cuprizone diet (6w), spontaneous remyelination occured. Tissue was collected at 6w and after 1 week of recovery (6+1 w).

Statistical analysis

GraphPad Prism was used to statistically analyse the data, which are represented as mean ± standard error of mean (SEM). D’Agostino and Pearson omnibus normality test was used to test for normal distribution. Two-tailed unpaired student T-test (with Welch’s correction if necessary) was used for normally distributed data. The Mann-Whitney analysis was used for data that did not pass the normality test. P-values <0.05 were considered to demonstrate significant differences (*p < 0.05, “p < 0.01 , *“p < 0.001 and ““p<0.0001 ).

Results

Phloretin induces repair after cuprizone-induced demyelination.

We previously found that phloretin alters the inflammatory phenotype of macrophages and alleviates neuroinflammation. As macrophages also play a crucial role in repair processes, we investigated whether phloretin is also able to enhance remyelination. Remyelination was studied in vivo by using the cuprizone-induced de- and remyelination model. Cuprizone feeding results in toxic demyelination of diverse brain regions such as the corpus callosum (CC), and switching to normal chow leads to spontaneous remyelination. After 6 weeks of cuprizone (6w), demyelination was observed in both vehicle- and phloretin-treated mice, as determined by MBP staining and TEM analysis (G-ratio= inner axonal diameter over total outer diameter) of the CC (Fig. 1 A-C). One week after cuprizone termination (6+1 w), phloretin-treated mice obtained increased MBP protein levels and lower G-ratios in the CC compared to vehicle-treated mice, illustrating that phloretin enhanced remyelination (Fig. 1 A-C). In addition, the small diameter axons of the phloretin-treated mice showed thicker myelin sheaths compared with control mice (Fig. 1 D). In line with these findings, increased gene expression levels of the myelin proteins MBP and PLP were observed in phloretin-treated mice at 6w and 6+1 w (Fig. 1 E-F). Interestingly, no significant differences on gene expression levels of CCL5, CCL4, IL6, IL1 b and INOS were observed on 6w and 6+1 w cuprizone animals. As phagocytes are the main producers of these inflammatory mediators, this suggests that the observed effect on remyelination by phloretin may not be mediated by the induction of disease-resolving phagocytes. Altogether, phloretin enhances remyelination after cuprizone-induced demyelination without significantly affecting the local inflammatory response.

Phloretin stimulates OPC maturation in vitro.

As phloretin stimulates remyelination without affecting the gene expression of inflammatory mediators, we investigated whether phloretin influences OPC differentiation directly. Phloretin- treated OPCs show enhanced OPC differentiation compared to vehicle-treated OPCs, as determined by the read-out parameters of the sholl-analysis and the ratio of the mature-OLs marker MBP over the pre-OLs marker 04 per cell (Fig. 2A-D). In line with these findings, elevated mRNA levels of the mature OLs markers MBP and PLP were observed in phloretin- treated OPC compared to vehicle treated-OPCs (Fig. 2E). Overall, results demonstrate that phloretin enhances OPC differentiation in vitro.

Phloretin stimulates OPC-mediated repair after LPC-induced demyelination in ex vivo brain slices.

To determine whether phloretin promotes remyelination independently of its immunomodulatory effects, we studied the role of both OPC and microglia in phloretin-mediated remyelination by using ex vivo cerebellar brain slices (BSC) demyelinated with lysolecithin. In agreement with our in vivo findings, phloretin-treated BSC showed more remyelination compared to vehicle-treated BSC, as demonstrated by a higher myelination index (Fig 3A-B). To elucidate the role of microglia (the resident immune cells of the CNS) in these effects, we used brain slices that were microglia depleted by clodronate liposomes. Administration of clodronate liposomes show increased mortality in bone marrow derived macrophages (BMDMs) in vitro and reduced F4/80+ microglia in BSC compared to both empty liposomes and negative control (data not shown). Less remyelination was observed in microglia-depleted BSC compared to normal BSC (data not shown), confirming the importance of microglia in remyelination. Importantly, phloretin promoted remyelination in microglia-depleted BSC. These findings demonstrate that phloretin promotes remyelination independently of microglia. All together, our findings indicate that phloretin promotes remyelination by inducing OPC differentiation.

Phloretin stimulates OPC maturation in PPARy-dependent manner.

Here, we show that phloretin enhances OPC differentiation in a PPARy-dependent manner. The luciferase assay was used to assess the level of PPARy activation upon phloretin treatment. Findings demonstrate a dose-response effect of phloretin on PPARy activation in cos cells (Fig 4A). Remarkably, the level of PPARy activation upon phloretin treatment with 50 uM was as high as treatment with the PPARy agonist rosiglitazone, emphasizing the potential of phloretin to activate the PPARy pathway. Activation of the PPARy isotype was considered specific, as no significant activation of PPARa or PPARb/d was observed (suppl Fig). Moreover, increased gene expression of the PPARy response genes CD36, APOE, ABCA1 and CPTA1 in OPC treated with phloretin was observed, illustrating phloretin-induced PPARy activation in OPCs (Fig 4B). To establish whether phloretin stimulates OPC differentiation via PPARy, OPCs were treated with the antagonist GW9662. Elevated MBP and PLP mRNA levels observed upon phloretin treatment were counteracted when OPCs were additionally treated with the PPARy antagonist GW9662 (Fig 4C). In line with these findings, higher MBP/O4 ratios and sholl analysis parameters found upon phloretin treatment were diminished upon additional treatment with GW9662, demonstrating that PPARy drives phloretin-mediated OPC differentiation (Fig 4D-G). In agreement with these in vitro findings, elevated mRNA levels of PPARy response genes CD36, APOE, ABCA1 and CPTA1 were observed in brain tissue derived from phloretin-treated mice of the cuprizone experiment at 6w (Fig 4H) and 6+1w (Fig. 4I) and in phloretin-treated ex vivo brain slices (Fig 4J). In conclusion, PPARy activation underlies phloretin-induced OPC maturation.

EXAMPLE 2: Phloretin suppresses neuroinflammation by autophagy-mediated Nrf2 activation in macrophages

Materials and Methods

Antibodies and chemical reagents

Phloretin (Sigma Aldrich) was dissolved in 50 mM KOH to a 15mM stock solution and stored at -20°C. Further dilutions were made in RPMI1640 (Gibco) medium. For in vivo treatment, phloretin was dissolved in 1 N NaOH, whereafter the pH was readjusted to 7.2 with 1 N HCL, and the solution further diluted in physiological water to obtain a concentration of 50 mg/kg. BML-275 (1 pM, Santa Cruz Biotechnology) was used to inhibit AMPK. Bafilomycin A1 (baf, 0.1 pM, InvivoGen) used to block the fusion of autophagosomes and lysosomes. Lipopolysaccharide (LPS, 100 ng/ml, Sigma-Aldrich) was used to stimulate cells for inflammatory phenotyping. Phorbol 12-myristate 13-acetate (PMA, 100 ng/ml, Sigma-Aldrich) was used to induce ROS production. The following antibodies were used for western blot: mouse anti-p-actin (1 :10 000; sc-47778, Santa Cruz Biotechnology), mouse anti-GAPDH (1 :10 000; AB_2537659, Invitrogen), rabbit anti-AMPK (1 :1000; 5831 S, Cell Signaling Technology), rabbit anti-phosphorylated AMPK (1 :1000; 2535S, Cell Signaling Technology), rabbit anti-LC3 (1 :1000; L7543, Sigma-Aldrich), rabbit anti-p62 (1 :1000; 23214, Cell Signaling Technology). The following antibodies were used for immunofluorescence: rat anti-CD3 (1 :150; MCA500G, BioRad), rat anti-F4/80 (1 :100; MCA497G, Bio-Rad), rabbit anti-LC3 (1 :1000; L7543, Sigma- Aldrich), rabbit anti-p62 (1 :500; 23214, Cell Signaling Technology), rabbit anti-Keap1 (1 :500; 60027-1 -Ig, Proteintech Europa). Appropriate secondary antibodies were purchased from Invitrogen.

Mice

Wild type (WT) C57BL/6JOIaHsd mice were purchased from Envigo. Animals were fed a regular diet and housed in the animal facility of the Biomedical Research Institute of Hassell University. All experiments were performed according to institutional guidelines and were approved by the ethical committee for animal experiments of Hassell University.

Cell Culture

Bone marrow-derived macrophages (BMDMs) were isolated from WT and Nrf2 KO C57BL/6JOIaHsd mice, purchased from Envigo and provided by the RIKEN BRC according to an MTA to Prof S. Kerdine-Rbmer respectively (Helou, D.G., et al., Cutting Edge: Nrf2 Regulates Neutrophil tact Hypersensitivity. J Immunol, 2019. 202 Mat heterodimer mediates the induction

antioxidant response elements. Biochem Biophys Res Commun, 1997. 236(2): p. 313-22.). BMDMs were obtained as described previously (Bogie, J.F., et al., Scavenger receptor collectin placenta 1 is a novel receptor involved in the uptake of myelin by phagocytes. Sci Rep, 2017. 7: p. 44794.). In short, tibial and femoral bone marrow cells from 12-week-old WT and Nrf2 KO C57BU6JOIaHsd mice were cultured in 10 cm petri plates at a concentration of 10 x 10⁶ cells/plate, in RPMI-1640 medium (Gibco) supplemented with 10% fetal calf serum (FCS, Gibco), 50 U/ml penicillin (Invitrogen), 50 U/ml streptomycin (Invitrogen), and 15% L929-conditioned medium (LCM). After differentiation, BMDMs were detached at 37°C with 10 mM EDTA in PBS (Gibco) and cultured (0.5 x 10⁶ cells/ml) in RPMI 1640 supplemented with 10% FCS, 50 U/ml penicillin, 50 U/ml streptomycin, and 5% LCM (37°C, 5% CO2).

RNA sequencing (RNA-seq)

Cells were pretreated with phloretin (50 pM) for 20 hours and LPS-stimulated (100 ng/ml) for 6 hours. Cell lysis was performed using Qiazol Lysis reagent (Qiagen). RNA was extracted from cells using the RNeasy mini kit (Qiagen). Samples were then processed by the Genomics Core Leuven (Belgium). Library preparation was performed with the Lexogen’s QuantSeq kit to generate Illumina compatible libraries. Libraries were sequenced on the Illumina HiSeq4000 sequencing system. Splice-aware alignment was performed with STAR v2.6.1 b (Dobin, A., et al., STAR: ultrafast universal RNA-seq aligner. Bioinformatics, 2013. 29(1 ): p. 15-21.). Quantification of reads per gene was performed with HT-seq Count v2.7.14. Count-based differential expression analysis was done with R-based (The R Foundation for Statistical Computing, Vienna, Austria) Bioconductor package DESeq2. List of differentially expressed genes were selected at a p<0.05 and used as an input for the core analysis by QIAGEN’S Ingenuity Pathway Analysis (IPA).

Quantitative reverse transcription PCR (RT-qPCR)

Cells were pretreated with phloretin (50 pM) for 20 hours and LPS-stimulated (100 ng/ml) for 6 hours. Lysis was performed by using Qiazol Lysis reagent (Qiagen). RNA was extracted using the RNeasy mini kit (Qiagen). RNA concentration and quality were determined with a Nanodrop spectrophotometer (Isogen Life Science). cDNA synthesis was conducted using the Quanta qScript cDNA SuperMix (Quanta Biosciences) per manufacturer’s instructions. qPCR was performed on a StepOnePlus™ Real-Time PCR system (Applied biosystems) using a SYBR green mix containing 1 x SYBR green (Applied Biosystems), 0.3 pM primers (Integrated DNA Technologies), 12.5 ng cDNA, and nuclease free water. The comparative Ct method was used to quantify gene expression. Data were normalized to the most stable reference genes cyclin A (cyca) and hypoxanthine phosphoribosyltransferase 1 (hprf). Primer sequences are available in the supplementary Tablet .

Determination of reactive oxygen species (ROS)

Cells were pretreated with phloretin (50 pM) for 2 hours. Afterwards, cells were stimulated with PMA (15min, 100 ng/ml) and ROS production was measured using the fluorescent probe 2', 7'- Dichlorodihydrofluorescein diacetate (DCDHF-DA) at 10 pM in PBS for 30 min. Fluorescence was measured using the fluorescence FLUOstar optima microplate reader (BMG Labtech, Ortenberg, Germany) (excitation: 495nm, emission: 529nm).

Measurements of NO

Cells were pretreated with phloretin (50 pM) for 2 hours. Afterwards, cells were stimulated with LPS (24h, 100 ng/ml). NO was indirectly monitored using the Griess reagent nitrite measurement kit (Abeam). Briefly, nitrite reacts with sulfanilamide and N-(1 -naphthyl)ethylenediamine dihydrochloride (NED) to produce a pink azo dye. Absorbance of this azo derivative was then measured at 540 nM using microplate reader (IMark, Bio-Rad). Western Blot

Cells were lysed using RIPA-buffer (150 mM NaCI, 1 % Triton X-100, 0.5% sodium deoxycholate, 1% SDS, 50 mM Tris), and separated via sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Gels were transferred to a PVDF-membrane (VWR) and blots were blocked for one hour in 5% bovine serum albumin (BSA) in Tris-buffered saline containing 0.1% Tween-20 (TBS-T). Membranes were probed with primary antibodies overnight at 4°C, washed with TBS-Tween and incubated with the corresponding secondary horseradish peroxidase-labeled antibody for one hour at room temperature (RT). Immunoreactive signals were detected with enhanced chemiluminescence (ECL Plus, Thermo Fisher)using the Amersham Imager 680 (GE Healthcare Life Sciences, Diegem, Belgium. The densities of the bands were determined using Imaged.

Immunofluorescence

Spinal cord cryosections were air-dried and fixed in ice cold acetone for 10 min at -20°C. Mouse BMDMs were cultured on glass cover slides and fixed in ice cold methanol for 10 min at -20°C. Sections and BMDMs were blocked for 30 min using Dako protein block (Agilent). Afterwards, they were incubated overnight at 4°C with primary antibodies, washed, and incubated with the appropriate secondary antibodies for one hour at RT. Images of the spinal cord tissue were taking using a Nikon eclipse 80i microscope (10x objective) and NIS Elements BR 3.10 software (Nikon). Images of BMDMs stained for p62, LC3 and Keapl were taken using the Zeiss LSM 880 confocal microscope and were airyscan corrected (63x objective). P62 and LC3 positive puncta were determined by semi-automated puncta analysis imaged. In short, after the image was made binary and cells were selected by hand, puncta were analysed per cell. P62 and Keapl colocalisation was performed using a colocalisation pipeline in the CellProfiler software (McQuin, C., et al., CellProfiler 3.0: Next-generation image processing for biology. PLoS Biol, 2018. 16(7): p. e2005970.). The images shown in the figures are digitally enhanced.

Experimental autoimmune encephalomyelitis (EAE) model

Eleven-week-old C57BL/6JOIaHsd mice were immunized subcutaneously with 200 ng of myelin oligodendrocyte glycoprotein peptide (MOG35-55) emulsified in 100 pl complete Freund's supplemented with 4 mg/ml of Mycobacterium tuberculosis (EK21 10, Hooke Laboratories). Immediately after MOG immunization and after 24 hours, mice were intraperitoneally injected with 50 ng pertussis toxin (EK21 10 kit, Hooke Laboratories) to induce EAE. Mice were weighed and scored daily for neurological signs of the disease according to manufacturer's mouse EAE scoring guide: 0: no clinical symptoms, 0.5: tip of tail is limp, 1 : limp tail, 1 .5: limp tail and hind leg inhibition 2: limp tail and weakness of hind legs, 2.5: limp tail and dragging of hind legs, 3: limp tail and complete paralysis of the hind legs, 3.5: limp tail and complete paralysis of hind legs and mouse is unable to right itself when placed on its side, 4: paralysis to the diaphragm, 5: death by EAE.

Statistical analysis

GraphPad Prism was used to statistically analyse the data, which are represented as mean ± s.e.m. D’Agostino and Pearson omnibus normality test was used to test for normal distribution. Two-tailed unpaired student T-test (with Welch’s correction if necessary) was used for normally distributed data. The Mann-Whitney analysis was used for data that did not pass the normality test. P-values <0.05 were considered to demonstrate significant differences (*p < 0.05, “p < 0.01 , *“p < 0.001 and and ““p<0.0001 ).

RESULTS

Transcriptional changes associated with phloretin treatment of macrophages.

To establish potential anti-inflammatory effects and identify underlying mechanisms of phloretin treatment on macrophages, we performed bulk RNA sequencing. Pathway analysis of activated macrophages treated with phloretin showed that differentially expressed genes were overrepresented in canonical pathways related to inflammation, such as iNOS (z-score: -2.449), toll-like receptor (z-score: -2.236), interferon signaling (z-score: -2) and acute phase response pathway (z-score: -1.633) (Fig. 5A,B). Similar to canonical pathway analysis, upstream analysis of the RNA-seq data predicted that phloretin reduced the activation of key pro-inflammatory transcription regulators such as IRF7 (z-score: -2.229), IRF1 (z-score: -2.025), and STAT1 (z- score: -2.022) (Fig. 5D). Next to downregulating macrophage pro-inflammatory pathways, RNA- seq analysis of phloretin-treated macrophages showed that, among other pathways, phloretin potently activated the Nrf2 pathway (z-score: 1.897), evidenced by an upregulation of Nrf2- associated genes such as mafG and prdxl (Fig. 5A,C). Even more, Nrf2 (NFE2L2/was identified as the most activated upstream transcriptional regulator (z-score: 2.801 ) and regulation of ROS levels was identified as one of the most upregulated biological functions in phloretin-treated BMDMs (z-score: 2.008) (Fig. 5D,E). Collectively, findings show that phloretin activates Nrf2 and suppresses the inflammatory phenotype of macrophages.

The Nrf2 pathway controls the phenotype of phloretin-treated macrophages.

Next, we validated the anti-inflammatory effect of phloretin and confirmed the role of Nrf2 in driving the phenotype of macrophages exposed to phloretin. Reduced ROS levels were observed in phloretin-treated WT BMDMs stimulated with PMA. Moreover, reduced NO- production and mRNA levels of pro-inflammatory genes NOS2, IL-6, COX2 and IL-12 were observed in phloretin-treated WT BMDMs stimulated with LPS (Fig. 6B,C). In relation to the crucial role of Nrf2 in inducing a less-inflammatory phenotype, our data show that phloretin activates Nrf2-response genes HO1 and NQO1 in WT but not Nrf2 KO BMDMs stimulated with LPS (Fig. 6D). Furthermore, phloretin treatment reduced ROS production in WT but not in Nrf2 KO BMDMs. Aside from controlling anti-oxidative responses, Nrf2 is reported to suppress the inflammatory phenotype of macrophages (Kobayashi, E.H., et al., Nrf2 suppresses macrophage inflammatory response by blocking proinflammatory cytokine transcription. Nat Commun, 2016. 7: p. 11624.). In support of the latter, phloretin was unable to reduce the expression of pro- inflammatory genes NOS2, IL-6, COX2 and iL-12 'm activated Nrf2 deficient BMDMs (Fig. 6F). Altogether, these results indicate that Nrf2 drives the phloretin-mediated inflammatory phenotype shift of macrophages.

Phloretin promotes AMPK activation.

Phloretin is a well-defined glucose transporter (GLUT) inhibitor and recent studies highlight the importance of glucose uptake by GLUTs as a way to modulate macrophage activation (Freemerman, A.J., et al., Metabolic reprogramming of macrophages: glucose transporter 1 (GLUT1 )-mediated glucose metabolism drives a proinflammatory phenotype. J Biol Chem, 2014. 289(11 ): p. 7884-96.; Freemerman, A.J., et al., Myeloid Slc2a1 -Deficient Murine Model Revealed Macrophage Activation and Metabolic Phenotype Are Fueled by GLUT 1. J Immunol, 2019. 202(4): p. 1265-1286.). Hence, we determined whether phloretin activates the energy sensor AMPK, which is activated upon low energy/glucose levels. Our results show that phloretin treatment leads to AMPK phosphorylation and activation, and that addition of the AMPK-inhibitor BML-275 largely prevents AMPK-activation in phloretin-treated BMDMs (Fig. 7A,B). Even more, our findings demonstrate that AMPK activation is essential for phloretin to suppress ROS production, as shown by higher ROS levels in BMDMs treated with both phloretin and AMPK- inhibitor compared to BMDMs treated only with phloretin (Fig. 70). In conclusion, our data suggest that phloretin-induced AMPK activation is crucial for driving macrophages towards a less-inflammatory phenotype.

Phloretin stimulates autophagy in an AMPK-dependent manner.

Since AMPK activation is related to the activation of catabolic processes in response to nutrient deprivation (Hardie, D.G., F.A. Ross, and S.A. Hawley, AMPK: a nutrient and energy sensor that maintains energy homeostasis. Nat Rev Mol Cell Biol, 2012. 13(4): p. 251 -62.), we investigated whether phloretin can activate autophagy. Autophagy is a conserved catabolic process that is induced upon starvation and other stress responses, which promotes the lysosomal degradation of intracellular cargo sequestered in vesicles termed autophagosomes. RNA-seq analysis predicted autophagy as one of the downstream biological processes showing an increased activity in phloretin-treated BMDMs (z-score: 0.779) (Fig. 8A). Upon induction of autophagy, MAP1 LC3/LC3 (microtubule associated protein 1 light chain 3) is converted from the LC3I form to the lipidated LC3II form, which correlates with the number of autophagosomes. Immunocytochemical analysis of BMDMs treated with phloretin and the autophagy inhibitor bafilomycin A1 displayed increased accumulation of LC3 and the autophagy marker p62 positive puncta compared to bafilomycin A1 -treated BMDMs (Fig. 8B-D), indicating increased autophagic flux upon phloretin treatment (Klionsky, D.J., et al., Guidelines for the use and interpretation of assays for monitoring autophagy (3rd edition). Autophagy, 2016. 12(1): p. 1 -222.). These results were further confirmed by western blot (Fig. 8E-F). Interestingly, this increase in p62 and LC3II by phloretin was prevented by treating BMDMs with the AMPK-inhibitor BML-275 (Fig. 8B-F). Collectively, our data suggest that phloretin stimulates autophagy in an AMPK-dependent manner.

Phloretin activates the Nrf2 pathway through autophagy-mediated Keapl degradation.

Our data show that phloretin activates Nrf2 and stimulates autophagy in macrophages. Interestingly, the autophagy receptor p62 can compete with Nrf2 for binding to Keapl , the adaptor that facilitates Nrf2 ubiquitination and degradation under normal condition. This p62- mediated dissociation of Keapl /Nrf2 will therefore prevent Nrf2 degradation and eventually lead to Nrf2 activation (Kansanen, E., et al., The Keapl -Nrf2 pathway: Mechanisms of activation and dysregulation in cancer. Redox Biol, 2013. 1 : p. 45-9.). For this reason, we determined whether phloretin promotes p62/Keap1 interaction, thereby activating the Nrf2 pathway. Here, we show that phloretin activates the Nrf2 pathway through p62-mediated Keapl degradation in macrophages. By using high-resolution Airyscan confocal microscopy combined with colocalisation analysis, we show that phloretin stimulates the interaction of p62 and Keapl , as demonstrated by an increased value of the colocalisation parameter (Pearson coefficient) in phloretin-treated BMDMs (Fig. 9A,B). Moreover, our findings show that lower protein levels of Keapl are present in phloretin-treated BMDMs, confirming degradation of Keapl (Fig. 9C). To confirm that the degradation is autophagy-dependent, we added the autophagy inhibitor bafilomycin A1 to phloretin-treated BMDMs. Interestingly, this reduction in Keapl was reversed by bafilomycin A1 treatment (Fig. 9C). These results were confirmed by western blot, showing a reduction in Keapl protein levels in phloretin-treated BMDMs which was prevented by bafilomycin A1 (Fig. 9D,E).

Phloretin reduces neuroinflammation in the EAE model.

To validate our findings in vivo, we investigated the impact of phloretin on the EAE model that is characterized by a pronounced macrophage-mediated inflammatory response in the CNS (Bogie, J.F., P. Stinissen, and J. J. Hendriks, Macrophage subsets and microglia in multiple sclerosis. Acta Neuropathol, 2014. 128(2): p. 191 -213.). EAE animals treated with phloretin before disease onset showed reduced clinical scores compared to vehicle-treated animals (Fig. 10A). Importantly, even in a therapeutic setup, in which phloretin treatment was started after disease onset (clinical score >1 ), phloretin reduced disease severity (Fig. 10B). Reduced disease severity in phloretin-treated animals was paralleled by a decreased expression of inflammatory genes Nos2, TNFa, IL-6, CCL4, CCL5 and CXCL2 in the spinal cord (Fig. 10E). Moreover, a reduced amount of F4/80 positive cells was found in the spinal cord of phloretin- treated animals (Fig. 10C,D). In addition to reducing the expression of inflammatory genes, phloretin increased the expression of anti-inflammatory and neurotrophic factors, i.e. IL-4, CNTF and IGF-1 in the spinal cord of EAE animals (Fig. 10F). These findings strongly suggest that phloretin reduces EAE disease severity by driving macrophages towards a disease-resolving phenotype. In line with our in vitro findings we detected elevated Nrf2 signalling in the CNS of phloretin-treated EAE animals as indicated by increased mRNA expression of Nrf2 and NQO1, together with an increased trend of the Nrf2-response gene GPX1 (Fig. 10G). Altogether, these data indicate that phloretin suppresses neuroinflammation in both a prophylactic and therapeutic setting, and suggest that phloretin is able to reduce neuroinflammation by suppressing the inflammatory features of macrophages through Nrf2 activation.

Claims

-28-CLAIMS

1 . A composition comprising phloretin and/or a derivative thereof for use in the treatment and/or prevention of a demyelinating disease.

2. The composition for use according to claim 1 , wherein the demyelinating disease is a neurodegenerative and demyelinating disease of the central nervous system.

3. The composition for use according to claim 2, wherein the neurodegenerative and demyelinating disease of the central nervous system is multiple sclerosis; in particular progressive multiple sclerosis (pMS), more in particular Primary progressive MS (PPMS), Secondary progressive MS (SPMS) or relapse remitting MS (RRMS).

4. The composition for use according to claim 1 wherein the demyelinating disease is a neurodegenerative and demyelinating disease of the peripheral nervous system.

5. The composition for use according to claim 4 wherein the neurodegenerative and demyelinating disease of the peripheral nervous system is selected from diabetic neuropathy, Marie-Charcot tooth disease or traumatic nerve injury, chronic inflammatory polyradiculoneuropathy, Guillain Barre syndrome.

6. The composition for use according to any of the preceding claims, wherein the subject is a non-human animal or a human.

7. The composition for use according to any of the preceding claims wherein the composition is administered to the subject by oral, intravenous, intraperitoneal or subcutaneous administration; preferably by oral administration.

8. Use of phloretin and/or derivatives thereof in in vitro, ex vivo and in vivo assays selected from remyelination assays, oligodendrocyte progenitor cell (OPC) differentiation assays, OPC. OPC myelination assays, OPC migration assays, OPC proliferation assays.

9. Use of phloretin and/or derivatives thereof in in vitro, ex vivo and in vivo remyelination assays.

10. The non-therapeutic use of phloretin and/or derivative thereof in improving physical and/or cognitive ability, fatigue and/or in improving neurological functioning.

11 . A method for preventing and/or treating a demyelinating disease in a subject in need thereof; said method comprises administering a composition comprising phloretin and/or a derivative thereof to the subject.

12. The method according to claim 11 , wherein the demyelinating disease is a neurodegenerative and demyelinating disease of the central nervous system.

13. The method according to claim 12, wherein the neurodegenerative and demyelinating disease of the central nervous system is multiple sclerosis; in particular progressive multiple sclerosis (pMS), more in particular Primary progressive MS (PPMS), Secondary progressive MS (SPMS) or relapse remitting MS (RRMS).

14. The method according to claim 11 wherein the demyelinating disease is a neurodegenerative and demyelinating disease of the peripheral nervous system.

15. The method according to claim 14 wherein the neurodegenerative and demyelinating disease of the peripheral nervous system is selected from diabetic neuropathy, Marie-Charcot tooth disease or traumatic nerve injury, chronic inflammatory polyradiculoneuropathy, Guillain Barre syndrome.