Phosphatidylinositol 3,5-bisphosphates for use in the treatment of neurodegenerative diseases

Abstract

The present invention relates to Phosphatidylinositol 3,5-bisphosphate (PI(3,5)P2) of formula (I), preferably formulated in LNP, preferably SANP, and optionally together with a miRNA or antagomir, for use in the treatment of neurodegenerative disorders, like AD, PD and ALS, characterized by impaired / blocked autophagy. It also relates to a LNP, preferably SANP, containing PI(3,5)P2 and optionally a miRNA or antagomir.

Description

[0001] INDUSTRIAL INVENTION PATENT APPLICATION TITLED:

[0002] PHOSPHATIDYLINOSITOL 3,5-BISPHOSPHATES FOR USE IN THE TREATMENT OF NEURODEGENERATIVE DISEASES

[0003] FIELD OF THE INVENTION

[0004] The present invention relates to pharmacological therapies for the treatment of neurodegenerative diseases.

[0005] STATE OF THE ART

[0006] Neurodegenerative diseases refer to the diseases targeting the nervous system (brain, spine or nerves) that often impair memory, cognition and movement. They are the second leading cause of death, and the first cause of severe long-term disability in the world. The prevalence of most neurological diseases increases sharply with age, and age also modulates the impact of risk factors, clinical presentation and the natural course of these diseases. However, some other neurodegenerative diseases occur during the life before aging and can be linked to genetic mutations, including juvenile encephalopathies and some forms of Alzheimer’s disease (AD), Parkinson’s disease (PD), Amyotrophic Lateral Sclerosis (ALS) Huntington’s disease and Multiple Sclerosis.

[0007] Unfortunately, most of these diseases (e.g. ALS and Huntington’s disease) are considered as an unmet medical need since no pharmacological therapies are currently available.

[0008] Neurodegenerative diseases are characterized by several dysfunctional pathways and mechanisms -most of which in common- including dysregulation in synaptic function, neurotransmission, free radical production, lipid metabolism, ionic homeostasis, mRNA and protein metabolism, etc.

[0009] Several mechanisms contributing to the previously indicated dysfunctional pathways have been identified as putative druggable targets. For instance, dysfunction in dopamine metabolism and acetylcholine neurotransmission, glutamate receptor dysregulation, reactive oxygen species (ROS) overproduction, organellar calcium dyshomeostasis have been targeted by the following drugs: Amantadine (SYMMETREL), Apomorphine (APOKYN), Baclofen (LIORESAL), Carbidopa (LODOSYN), Carbidopa / levodopa (SINEMET, ATAMET, others; orally disintegrating tablet, PARCOPA), Dantrolene (DANTRIUM), Donepezil (ARICEPT), Entacapone (COMTAN; fixed combination with carbidopa / levodopa, STAVELO), Galantamine (NIVALIN, others), Levodopa (L-DOPA, LARODOPA), Memantine (NAMENDA), Pramipexole (MIRAPEX), Rasagiline (AZILECT), Riluzole (RILUTEK), Rivastigmine (EXELON), Ropinirole (REQUIP), Selegiline (ELDEPRYL; oral disintegrating tablet, EMSAM; transdermal patch, ZELAPAR), Tacrine (COGNEX) — discontinued in the United States, Tetrabenazine

[0010] (XENAZINENITOMAN), Tizanidine (ZANAFLEX), Tolcapone (TASMAR).

[0011] Among the mechanisms involved in the etiology of neurodegenerative diseases, calcium (Ca2+) dyshomeostasis represents the most common causative pathway. Moreover, the concerted work of several mechanisms contributes to the regulation of intracellular calcium homeostasis. For this reason, the identification of the causative mechanism contributing to the pathogenesis of each neurological disease may be hard to reach. For instance, an impairment of plasma membrane voltagedependent channels, pumps, transporters, ionic receptors and Gq-coupled receptors can be found in encephalopathies, stroke, PD, AD, ALS. Moreover, intracellular receptors, pumps and channels can be also considered important dysfunctional targets mainly involved in the etiopathogenesis of the same diseases. The dysfunctional activity of these ionic players triggering calcium dyshomeostasis is mainly due to their gain- or loss-of-function mutations or to the aberrant levels of their endogenous modulators. For instance, mutations in FIG4 gene, a component of the PI(3,5)P2 regulatory complex, is now considered a possible causative mechanism in familial ALS as well as in Charcot-Marie-Tooth disease (Ferguson et al., Autophagy, 2010, 6(1 ), 170-171 ). Under physiological conditions, PI(3,5)P2 is able to modulate the activity of one of the most important lysosomal channel moving calcium named TRPML1.

[0012] The levels of PI(3,5)P2 are exceedingly low to act and to be measured. In fact, PI(3,5)P2 is approximately 0.05 % ~ 0.1 % of total phosphatidylinositol lipids. Recent observations that mutations in Fig4 cause defects with striking similarities to lysosomal storage disorders may also provide insight into the links between these lipids and some neurodegenerative disease. The severity of these pathologies under-scores the importance of uncovering the molecular mechanisms that regulate the Vac14 / PIKfyve / Fig4 complex, as well as the discovery of new molecules that may enhance or mimic PI(3,5)P2 action. PI(3,5)P2 is now considered the endogenous activator of a particular lysosomal channel that represents the first member of the mammalian mucolipin transient receptor potential (TRP) subfamily named TRPML1 or mucolipin-1. TRPML1 is a cation-permeable channel predominantly localized on the membrane of lysosome. It is a non-selective cation channel mainly involved in Ca2+signalling during lysosomal fusion with other membranes. Interestingly, a deficiency of TRPML1 / mucolipin-1 causes mucolipidosis IV, a lysosomal storage disease characterized by neurodegeneration. Importantly, TRPML1 activation leads to lysosomal Ca2+release and calcineurin- dependent TFEB nuclear translocation. In addition, it has been recently demonstrated that TRPML1 is also involved in lysosomal biogenesis.

[0013] Inositol phospholipids are a minor class of phospholipids that play pivotal roles in the control of Ca2+-dependent signalling pathways. In particular, within the cells, they control calcium homeostasis modulating specific ionic proteins, including PI4P for the activity of plasmalemmal 0RAI1 , PI(4,5)P2 for sustained Kir2.1 activity, and such regulation of Kir2.1 channels mediates electrical signalling during neurovascular coupling (Harraz et al., 2018a, 7:e38689).

[0014] The term ‘autophagy' refers to relevant cellular processes involved in the clearance of dysfunctional proteins and organelles via the lysosomal system. In addition, autophagy recycles their components for other biosynthetic pathways.

[0015] Based on several researchers, autophagy activation may represent a valuable therapeutic approach for several neurodegenerative diseases including Azheimer disease, PD and ALS. For example, in case of insufficient autophagy (e.g. lack of crucial factors linked to haploinsufficiency associated with loss-of-function mutations; etc.), it might be useful to stimulate the remaining functional wild type allele, or 'redundant' factors to restore autophagy (Chua, J.P., et al. Autophagy 2022, 18, 254-282).

[0016] In general, there are only few drugs stimulating autophagy that are relatively poorly specific. Among these drugs there are lithium, ML-SA1 and trehalose (Damri, 0., et al. Int. J. Mol. Sci. 2020, 22, 189-206; Costa, A. J., et al. Pharmaceutics 2021 , 13, 135-148; Tedeschi V, et al. Sci Rep. 2019 Jul 24;9(1 ): 10743; Pasquali, L., et al. Muscle Nerve 2009, 40, 173-194; Motoi, Y., et al. ACS Chem. Neurosci. 2014, 5, 434-442).

[0017] Therefore, the identification of a proper target to be tackled by drugs capable of modulating the autophagic pathways is still challenging.

[0018] It is emerging that lysosomal calcium channels could help in triggering autophagic flux. However, a fine modulation of these channels is auspicial. microRNAs (miRNAs) are short non-coding RNAs that contribute to multiple aspects of brain development and function playing key regulatory roles in the development, differentiation, survival, and gene expression of neuronal cells. Furthermore, they play an important role in many human diseases including cancer and neurodegenerative disorders such as Alzheimer's disease, amyotrophic lateral sclerosis, and Parkinson's disease (Khezri MR et al. Mol Neurobiol. 2022, 59(3), 1589-1603.; Li B, et al. FASEB J. 2021 , 35(10), e21916). Of note, some circulating miRNAs can be used as biomarkers of prognosis for many neurological disorders and may help new drug development. Of interest, miR-132-5p, miR-138-5p, miR- 129-5p are upregulated in AD brain (Dobricic V. et al. Translational Psychiatry 2022, 12, Article number: 352); miR-124-3p increases in the symptomatic phase of ALS, while miR-155 upregulation has been observed in the early stages of the disease (Cunha C., et al. Mol. Neurobiol. 2018, 55, 4207-4224); miR-181 a and miR-181 c are involved in the pathogenesis of acute ischemic stroke promoting both inflammatory responses and neuronal apoptosis (Ma, Q., et al. Aging Dis. 2016, 7, 705-714). Under physiological conditions, some of the above-mentioned miRNAs are involved in the regulation of neuronal homeostasis and functions; however, some others such as miR-181 a and miR-181 c control glial functions. In this respect, as an example, miR-124-3p is involved in maintaining neuronal cell identity and synaptic plasticity. Therefore, miRNAs modifications may impact on neuronal and glial homeostasis disturbing differentiation and proliferation, and increasing inflammatory mediators release, etc. In this consideration, the proposal of antagomiRs as new drugs able to interfere with relevant pathophysiological process in cancer could help to develop new treatments also for neurodegenerative diseases. For instance, miR-124-3p results to be highly upregulated in the cerebrospinal fluid (CSF)in ALS patients(Waller R, et al.. Front. Neurosci. 2018, 11 , 731 ).

[0019] The use of nucleic acids, e.g. miRNA and antagomir, in therapy has been largely explored. Actually despite the promising therapeutic potential of nucleic acids, their administration requires strategies to overcome the poor biopharmaceutical profile, mainly due to the rapid degradation in biological fluids and poor crossing of cell membranes. Moreover, in the case of diseases associate to the central nervous system, the blood brain barrier represents a further biological barrier to overcome. Currently, the majority of the pharmaceutical products based on nucleic acids (especially plasmid DNA) involves the use of viral vectors. However, the approval of ONPATTRO in 2018, followed by the approval of two mRNA-based vaccines Comirnaty and SpikeVax, opens a new era for the delivery of nucleic acids, in particular RNA, by using non-viral vectors and in particular, lipid nanoparticles (LNP) (Jeong Met al. Adv Drug Deliv Rev. 2023 Sep;200:114990). The use of non-viral nanovectors, especially based on lipids, has been largely investigated for the delivery of RNA, with different product on the market for in vitro transfection purposes. However, only an evolution of the lipids used for RNA delivery (S.C. Semple, et al. Nat Biotechnol. 28 (2010) 172-176; C. Hald Albertsen, et al. Adv Drug Deliv Rev. 188 (2022) 114416) as well as the development of more reproducible and scalable manufacturing methods (M. Maeki, e al. Journal of Controlled Release. 344 (2022) 80-96), allowed to achieve formulations with high in vivo RNA delivery; this speeded up the clinical development of new medicinal products based on RNA drugs and lipid nanovectors, with the above mentioned three approved products and many other in advanced stage of clinical development. In these context, further advancements have been proposed to overcome issues such as the poor stability of the LNP-based products that require very low temperatures (as in the case of the RNA-based vaccines). Campani V, et al. (Int J Pharm. 2020 Oct 15;588:119693.) recently reported that self-assembling nanoparticles (SANP) allowed the delivery of RNA to the central nervous system, said SANP consisting of a lipid shell surrounding a core of colloidal calcium phosphate and miR603.

[0020] WO20231 47351 describes a LNP comprising one or more phosphoinositides e.g. PI(3,5)P2. Aim of the present invention is to provide a new pharmacological treatment of neurodegenerative diseases, in particular for the treatment of AD, PD and ALS, by fine and specific upregulation of autophagy.

[0021] DEFINITION AND ABBREVIATIONS

[0022] AD: Alzheimer’s Disease

[0023] ALS: Amyotrophic Lateral Sclerosis

[0024] HD: Huntington’s Disease

[0025] MS: Multiple Sclerosis

[0026] PIP or PI(3,5)P2: Phosphatidylinositol 3,5-bisphosphate.

[0027] SANP: Self-Assembling Nanoparticle

[0028] SUMMARY OF THE INVENTION

[0029] Subject matter of the present invention is a phosphatidylinositol 3,5-bisphosphate (PI(3,5)P2) of formula (I) for use in the treatment of neurodegenerative diseases, like AD, PD, HD, MS and ALS, characterized by impaired or blocked autophagy; wherein formula (I) is wherein

[0030] R is a C6-20 fatty acid; and

[0031] R’ is H or a C6-20 fatty acid equal or different from R.

[0032] Surprisingly it has been found that a PI(3,5)P2 of formula (I), in particular PI(3,5)P2 dioctanoyl, is a neuroprotective agent, by way of upregulation of autophagy, in the neurodegeneration occurring in several neurodegenerative diseases (including ALS).

[0033] Surprisingly, PI(3,5)P2 of formula (I) was advantageously used as lipid ingredient to prepare lipid nanoparticles (LNPs), which are self-assembling lipid nanoparticles (SANPs) composed by cationic or ionizable lipids and neutral lipids, inorganic salts and a PI(3,5)P2 of formula (I) which acts both as active pharmaceutical ingredient and as lipid excipient.. Even more surprisingly it has been found that a PI(3,5)P2 of formula (I) within a lipid nanoparticle (LNP), preferably a SANP, is a neuroprotective agent more potent than PI(3,5)P2 of formula (I) alone as such.

[0034] Surprisingly it has also been found that LNPs comprising a PI(3,5)P2 of formula (I) and anti-miR124, encapsulated within the LNP, displayed a synergistic effect in an in vitro model of ALS.

[0035] Further subject matter of the present invention is a LNP comprising a PI(3,5)P2 of formula (I) acting both as pharmaceutical active ingredient and as lipid ingredient of the LNP, and optionally a miRNA or antagomiR.

[0036] Further subject matter of the present invention is a LNP as above described for use as a medicament, in particular for use in the treatment of neurodegenerative diseases characterized by impaired or blocked autophagy.

[0037] DETAILED DESCRIPTION OF THE INVENTION

[0038] The neurodegenerative diseases according to the invention are those characterized by impaired or blocked autophagy and include and preferably are AD, PD, ALS, Charcot-Marie-Tooth disease, HD, lysosomal storage disorders, encephalopathies, MS.

[0039] According to a preferred embodiment of the present invention the PI(3,5)P2 of formula (I) has R=R’.

[0040] The LNPs according to the invention are preferably self-assembling lipid nanoparticles (SANPs) composed by cationic or ionizable lipids and neutral lipids, inorganic salts and a PI(3,5)P2 of formula (I) which acts both as active pharmaceutical ingredient and as lipid excipient.

[0041] Preferably the lipids for a LNPs according to the invention are selected in the group consisting of 1 ,2-dioleoyl-3-trimethylammonium-propane chloride (DOTAP chloride or DOTAP), N-[1 -(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA), 3s-[N-(N',N'-dimethylaminoethane)-carbamoyl]cholesterol hydrochloride (DC-chol), 1 ,2-dioleoyl-3-dimethylammonium-propane (DODAP), O-(Z,Z,Z,Z- heptatriaconta-6,9,26,29-tetraen-19-yl)-4-(N,N-dimethylamino) (DLinMC3), 2,2- dilinoleyl-4-dimethylam inoethyl-[1 ,3]-dioxolane (DLin-KC2-DMA), [(4-

[0042] Hydroxybutyl)azanediyl]di(hexane-6, 1 -diyl) bis(2-hexyldecanoate) (ALC-0315), (heptadecane-9-yl 8-[2-hydroxyethyl-(6-oxo-6-undecoxyhexyl)amino]octanoate (SM-102), cholesterol (CHOL), dioleoylphosphatidylethanolamine (DOPE), N- palmitoylsphingosine-1 {succinyl[methoxy(polyethylene glycol)2000]} (cene- PEG2000) (cer-PEG) and 1 ,2-distearoyl-sn-glycero-3-phosphoethanolamine-N- [amino(polyethylene glycol)-2000] (DSPE-PEG2000).

[0043] More preferably the lipids are added in form of PEGylated liposomes consisting of DOTAP, DOTMA or Dc-chol and DSPE-PEG or Ceri6-PEG2ooo and optionally CHOL or DOPE; more preferably DOTAP and Cene-PEG2ooo. even more preferably DOTAP and Cene-PEG2ooo in mM ratio 1 :0.125.

[0044] The SANP according to the invention are PEGylated liposomes encapsulating inorganic salts and a PI(3,5)P2 of formula (I) and optionally a miRNA or an antagomir; wherein the miRNA is one of those downregulated in patients affected by one of the above mentioned neurodegenerative diseases, and wherein the antagomir is against is one of those RNA upregulated in patients affected by one of the above mentioned neurodegenerative diseases.

[0045] The inorganic salts are selected in the group consisting of Na2HPO4 or sodium citrate tribasic, CaCL, Cerium(lll) nitrate . Preferably Na2HPO4 together with CaCL to form Ca3(PO4)2, more preferably together also with sodium citrate which stabilizes the phosphate. Ce(NO3)3can be used in alternative to CaCL.

[0046] The miRNA or antagomir encapsulated in the SANPs is preferably selected in the group consisting of miR-124-3p antagomir (anti-miR124).

[0047] The present invention will be better understood in light of the following experimental section.

[0048] BRIEF DESCRIPTION OF THE FIGURES

[0049] Figure 1. Z-average diameter and PDI (a) and zeta potential (b) of control SANP formulations (SANP) and of SANP formulations encapsulating PIP (SANP PIP). For each formulation the z-average diameter, PDI, and zeta potential were calculated as mean ± standard deviation of measurements from two independent batches. Figure 2. Z-average diameter and PDI (a) and zeta potential (b) of SANP formulations encapsulating PIP (SANP PIP), anti-miR124 (SANP miR) or both (SANP PIP-miR). (c) Encapsulation efficiency of anti-miR124 measured via the Quant-iT RiboGreen RNA Assay for SANP formulations encapsulating PIP (SANP PIP), anti-miR124 (SANP miR) or both (SANP PIP-miR). The total concentration of anti-miR124 was 98 pg / mL (= 16 pM). For each formulation the z-average diameter, PDI, zeta potential, and anti-miR124 encapsulation efficiency were calculated as mean ± standard deviation of measurements from two independent batches.

[0050] Figure 3 - shows MTT assay to measure cell viability of motor neurons exposed to L-BMAA (ALS) in absence or presence of PIP administrated together of before L- BMAA. *p<0.05 vs control; **p<0.05 vs L-BMAA alone

[0051] Figure 4 - shows the quantification of Western blotting analysis of LC3II protein expression in motor neurons under control conditions or in the presence of PI(3,5)P2 alone (incubated for 1 min). LC3 II has been normalized for tubulin expression or its precursor LC3 I. *p<0.05 vs control

[0052] Figure 5 - shows the detrimental effect of the activation of PI(3,5)P2 receptor, TRPML1 , during ischemic conditions in neurons exposed to hypoxic conditions. In A western blotting and quantification of LC3 II expression under control condition, during ischemic conditions and in the presence of TRPML1 activation (Ac TRPML1 ) under ischemic conditions. *p<0.05 vs control (normoxic conditions); **p<0.05 vs ischemic conditions (CH / Rx; chemical hypoxia plus reoxygenation). In B increased expression of the ER stress marker CHOP under ischemic conditions (CH / Rx). *p<0.05 vs control (normoxic conditions); **p<0.05 vs ischemic conditions (CH / Rx; chemical hypoxia plus reoxygenation).

[0053] Figure 6 - shows patch-clamp experiments on TRPML1 channel modulated by PI(3,5)P2 (DC8-PIP) (A,B,C) and SANP-PIP (D) in motor neurons. *p<0.05 vs control; **p<0.05 vs all. The effect of the synthetic agonist of TRPML1 , MLSA1 , has been reported in E as well as the AUC of the elicited current by each of these molecules has been reported in F. *p<0.05 vs PI(3,5)P2 alone (DC8-PIP).

[0054] Figure 7: shows Fura-2 experiments on TRPML1 activity measure as [Ca2+]i elicited by SANP-PIP (F1 ) and SANP-free (F9) in motor neurons. *p<0.05 vs all; **p<0.05 vs all

[0055] Figure 8 - shows the quantification of Western blotting analysis of LC3II protein expression in motor neurons under control conditions (CTL), in the presence of PI(3,5)P2 alone (incubated for 1 min) or PI(3,5)P2 + a synthetic activator TRPML1 and, finally, after incubation of SANP-PIP (incubated for 1 min). LC3 II has been normalized for tubulin expression or its precursor LC3 I. *p<0.05 vs control (CTL); **p<0.05 vs PIP

[0056] Figure 9 shows the survival rate in ALS mice treated SANP-PIP for 6 weeks at two different dosages (1 OX and 100X). *p<0.05 ALS mice (TG) treated with physiological solution.

[0057] Figure 10 shows the grip performances of ALS mice treated with physiological solution, SANP-PIP at 10X or SANP-PIP at 100X. Muscle strength progressively decreased in ALS mice treated with physiological solution while this process resulted reduced in ALS mice treated with the highest dosage of SANP-PIP.

[0058] Figure 11 - shows the Western blotting p-GSK3b expression.Ap<0.05 vs control; *p<0.05 vs L-BMAA; **p<0.05 vs SANP-PIP anti-miR124

[0059] Figure 12 - shows the quantification of p-GSK3b expression. *p<0.05 vs anti- miR124+ L-BMAA; *p<0.05 vs L-BMAA; **p<0.05 vs SANP anti-miR124+L-BMAA Figure 13 - shows Western blot and quantification of phospho- GSK3beta in motor neurons under control conditions, after L-BMAA treatment (24 h), after L-BMAA + PI(3,5)P2 and L-BMAA+ PI(3,5)P2+antimiR.

[0060] *p<0.05 vs control

[0061] Figure 14 - shows the effect of PI(3,5)P2 (alone in free form, in SANP and in SANP together with anti-miRNA) at different time-point of incubation on p-GSK3b expression. All treatments have been preincubated 30’ before L-BMAA. *p<0.05 vs control and L-BMAA + PI(3,5)P2; **p<0.05 vs control, L-BMAA, L-BMAA + SANP PIP and L-BMAA + PI(3,5)P2 (all).

[0062] EXPERIMENTAL SECTION

[0063] SANPs PREPARATION

[0064] Materials and methods

[0065] Materials

[0066] N-palmitoyl-sphingosine-1 -{succinyl[methoxy(polyethylene glycol)2000]} (Cene- PEG2000) and 1 ,2-dioleoyl-3-trimethylammonium-propane (chloride salt) (DOTAP) were purchased from Avanti Polar Lipids (Alabaster, USA). Calcium chloride (CaCL), sodium phosphate dibasic (Na2HPO4), sodium hydroxide (NaOH), and RNAse-free water were purchased from Sigma-Aldrich (St. Louis, MO, USA). Phosphatidylinositol 3,5-bisphosphate dioctanoyl (PIPdiC8) was purchased from Tebu-Bio (Magenta (Ml), Italy). The miR-124 -3p antagomir (anti-miR124) was purchased from Tema Ricerca S.r.l. (Castenaso, Italy) and has the following sequence: 5’-GGCAUUCACCGCGUGCCUU-3’. The Quant-iT RiboGreen RNA Assay was purchased from ThermoFisher Scientific (Milan, Italy).

[0067] Methods

[0068] CaP NPs synthesis

[0069] CaP NPs were prepared by adding an aqueous solution of 10.8 mM Na2HPO4 (pH 9.5) to an aqueous solution of 18 mM CaCL (pH 9.5) in a 1 : 1 v / v ratio dropwise while stirring. Both solutions were prepared by using RNAse-free water. The CaP NPs suspension was left to stir for 10 min and filtered through 0.22 pm pore-sized polycarbonate filters (MF-Millipore, MicroglassHeim, Italy). The CaP NPs suspension was then stored at 4 °C.

[0070] Liposome preparation

[0071] PEGylated liposomes were prepared by the thin film hydration method followed by extrusion. Briefly, the lipids in the appropriate ratios were dissolved in a chlorofomrmethanol mixture (2:1 v / v) and placed in a 50 mL round bottom flask. The organic solvent mixture was removed by rotary evaporation (Laborata 4010 digital, Heidolph, Schwabach, Germany) and the obtained lipid film was hydrated with RNAse-free water for 2 hours at 65 °C. The vesicle suspension was extruded through pore-sized polycarbonate membranes (Nucleopore Track-Etched 25 mm membrane, Whatman, Brentford, UK) by using a thermobarrel extruder (Lipex Extruder, Evonik, Essen, Germany) at 65 °C. More specifically, the vesicle suspension was forced to pass sequentially through 400 nm membranes (3 passages), 200 nm membranes (3 passages), and 100 nm membranes (5 passages). The lipid composition of the PEGylated liposomes was DOTAP:Cer16- PEG2000 94:6 mol%.

[0072] Preparation of PI P-loaded SANP formulations

[0073] In order to form CaP-PIP complexes, 100 pL of an aqueous solution containing PIPdiC8 at 1 mg / mL was added to 100 pL of CaP NPs; the suspension was vortexed and allowed to react at room temperature for 10 minutes. The CaP-PIP suspension was then added to 100 pL of PEGylated liposomes; the formulation was vortexed and allowed to react for 25 minutes obtaining PIP-loaded SANP formulations at a final PIP concentration of 390 pM. Control formulations were obtained by replacing the PIP solution with water.

[0074] Physico-chemical characterization of SANP formulations

[0075] Control SANP formulations and PIP-loaded SANP formulations were characterized in terms of colloidal dimensions, polydispersity index (PDI), and surface charge by using a Zetasizer Ultra (Malvern, UK). Prior to the measurements, samples were diluted to 1 % in filtered deionized water.

[0076] Results

[0077] The physico-chemical characterization of control SANP formulations and PIP- loaded SANP formulations is shown in Figure 1.

[0078] Preparation of SANP formulations co-encapsulating PIP and anti -mi R 124

[0079] In order to form CaP-PIP complexes, 60 pL of an aqueous solution containing PIPdiC8 at 1 mg / mL was added to 60 pL of CaP NPs; the suspension was vortexed and allowed to react at room temperature for 10 minutes. The CaP-PIP suspension was then added to 16 pL of an aqueous solution of anti-miR124 200 pM, vortexed and allow to react at room temperature for 10 minutes. The 136 pL of CaP-PIP-anti- miR124 complex were added to 60 pL of PEGylated liposomes; the formulation was vortexed and allowed to react for 25 minutes obtaining PIP-anti-miR124 SANP. The final concentrations of anti-miR124 and PIP were 16 pM and 358 pM, respectively. Control formulations were obtained by replacing the anti-miR124 or the PIP solution with RNAse-free water.

[0080] Physico-chemical characterization of liposomes and SANP

[0081] SANP formulations encapsulating either PIP, anti-miR124 or a mixture of PIP and anti-miR124 were characterized in terms of colloidal dimensions, polydispersity index (PDI), and surface charge by using a Zetasizer Ultra (Malvern, UK). Prior to the measurements, samples were diluted to 1 % in filtered deionized water. The concentration of encapsulated anti-miR124 was determined by measuring the amount of unencapsulated anti-miR124 by the Quant-iT RiboGreen RNA Assay. The encapsulation efficiency was calculated as:

[0082] Encapsulation efficiency (%) = where [anti - miR124]totalis the total anti-miR124 concentration in the formulation and [anti - miR124]bufferis the concentration of unencapsulated miRNA.

[0083] Results

[0084] The physico-chemical characterization of liposomes is reported in table 1:

[0085] Table 1. Physico-chemical characterization of the liposomes used for the preparation of SANP formulations.

[0086] Z-average Diameter (nm) PDI Zeta Potential (mV)

[0087] 123.3 ± 2.01 nm 0.046 ± 0.035 49.22 ± 1.46 mV

[0088] The physico-chemical characterization of SANP formulations encapsulating PIP, anti-miR124 or both is shown in Figure 2.

[0089] Biological Tests

[0090] Cell cultures and treatments

[0091] NSC-34 motor neurons, displaying a multipolar neuron-like phenotype and expressing either biochemical or functional markers of primary motor neurons (Petrozziello et al., Cell Death Differ, 2017), were grown in Dulbecco's Modified Eagles Medium with 4.5 g / l glucose, 10% fetal bovine serum (FBS), 2 mM L- glutamine, 100 lll / ml penicillin and 100 pg / ml streptomycin. NSC-34 motor neurons were plated in Petri dishes and kept in a 5% CO2 and 95% air atmosphere at 37 °C as previously described (Petrozziello et al., Cell Death Differ, 2017; Tedeschi et al., Sci Rep. 2019). Cells were differentiated with 10 pM retinoic acid for 48 h; then culture medium was replaced with fresh medium containing the neurotoxin L-BMAA (300 pM / 24 h) (Tedeschi et al., Sci Rep. 2019).

[0092] ALS / PDC cellular model

[0093] The cyanobacterial [3-N-methylamino-L-alanine (L-BMAA) is described as a low- potency excitotoxin increasing the incidence of amyotrophic lateral sclerosis (ALS) and Parkinsonism-dementia complex (PDC) in Guam. This neurotoxin could be produced by a variety of genera cyanobacteria and it is widely present in water sources contaminated by cyanobacteria. Therefore, L-BMAA may threaten human health through drinking water since it is able to cross BBB. Furthermore, aerosolization may lead to human BMAA exposure through the olfactory bulb (Pierozan P, Piras E, Brittebo E, Karlsson 0. Arch Toxicol. 2020 Aug;94(8):2799- 2808). To mimic ALS / PDC, NSC-34 motor neurons were plated in multiple well cluster plates and differentiated in 10 pM retinoic acid for 48 h. After differentiation, the culture medium was replaced with fresh medium containing L-BMAA (300 pM / 48 h). Under these conditions, L-BMAA promotes oxidative stress and ER stress (Petrozziello T, Secondo A, Tedeschi V, Esposito A, Sisalli M, Scorziello A, Di Renzo G, Annunziato L.Cell Death Differ. 2017 Mar;24(3):511 -522), and impairs autophagic flux in motor neuronal cells (Tedeschi V, Petrozziello T, Sisalli MJ, Boscia F, Canzoniero LMT, Secondo A. Sci Rep. 2019 Jul 24;9(1 ): 10743).

[0094] Ischemic conditions and Chemical hypoxia

[0095] Chemical hypoxia was reproduced by adding 5 pg / ml oligomycin plus 2 mM 2-DG in glucose-free medium to the cells. This combination produces a fall in cell ATP content (Schmid-Antomarchi et al., 1987; De Weille et al., 1989; Amoroso et al., 1990) because oligomycin inhibits oxidative phosphorylation (Lehninger, 1975; Dubinski and Rothman, 1991 ), whereas 2-DG causes inhibition at the first step of the glycolytic pathway, that is, at the reaction catalyzed by hexokinase (Devlin, 1982). The composition of the medium was 145 mM NaCI, 5 mM KCI, 1.2 mM MgSO4, 10 mM HEPES, 1.5 mM CaCI2, and 0.2% bovine serum albumin, pH adjusted to 7.4 with 1 M Tris (standard buffer).

[0096] Western Blotting

[0097] Following the indicated treatments, NSC-34 cells under control conditions, treated with PIPdiC8 alone, with the formulation of the invention (e.g. SANP encapsulating both PIPdiC8 and antagomir-124), with the nanoparticles encapsulating only antagomiR-124 or with antagomiR alone were lysed using the buffer containing 100 mM NaCI, 50 mM Tris-HCI (pH 7.4), 0.2% SDS, Nonidet P- 40 0.5%, EGTA 1 mM, PMSF 1 mM, NasVO4 1 mM, NaF 1 mM, and a mixture of protease inhibitors (30 minutes, 4°C). Samples were then centrifuged at 13,000 rpm for 20 minutes; protein concentration in the supernatants was determined according to the Bradford method (Bradford, Anal Biochem, 1976). For each sample, 10-20 pg of total proteins was separated by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS- PAGE) and subsequently electrotransferred onto a 0.2 pm pore nitrocellulose membrane. After 2 hours blocking at room temperature with a solution containing 3% BSA, the membranes were incubated with a solution (overnight at 4°C) containing one of the following rabbit polyclonal primary antibodies: anti-p-GSK3b antibody (Cell Signaling #5558) and anti-GSK3b (Cell Signalling #12456) antibodies (1 :1000 dilution for both the antibodies). Subsequently, the membranes were reblotted with mouse monoclonal primary antibody directed against 0-actin (dilution 1 :10000; 1 h at room temperature).

[0098] SOD1(G93A) ALS mice and treatments

[0099] All procedures were performed in accordance with European Guidelines for the use of animals in research (2010 / 63 / EU) and the requirements of Italian laws (D.L. 26 / 2014) and were approved by the Animal Care Committee of “Federico II” University of Naples and by the Animal welfare office, Department of Public Health and Veterinary, Nutrition and Food Safety, General Management of Animal Care and Veterinary Drugs of the Italian Ministry of Health (protocol n° 589 / 2020-PR). All efforts were made to minimize animal suffering and to reduce the number of animals used.

[0100] B6 / SJL-Tg(SOD1 *G93A)1 Gur / J mice were purchased from Jackson Laboratories (Bar Harbor, USA); mice were bred and housed in a virus / antigen-free facility at constant temperature and humidity (between 40 and 70%). Then, B6 / SJL males harbouring the SOD1 G93A transgene were crossed with wild-type B6 / SJL females. Transgenic hemizygous SOD1 G93A males were crossbred with C57BL / 6 (RRID:MGI:5657312) females and transgenic progeny were genotyped by PCR as previously described (Stifanese R, et al. Adaptive modifications in the calpain / calpastatin system in brain cells after persistent alteration in Ca2+ homeostasis. J. Biol. Chem. 2010;285:631-643). Disease onset was defined at 70 days of age by evaluation of grip strength, which provides an index of neuromuscular function (Ge, X., et al. Pathobiology of Aging & age-Related Diseases, 2016, 6(1 ), 32981 ). To monitor disease progression, behavioural scores and body weight were recorded from 49 days of age (Apolloni et al., 2019; Migliarini et al., 2021 ). Disease end stage was defined by full paralysis of hind limbs and loss of the ability to turn within 30 seconds of being positioned on their back. At this stage mice were sacrified. We assigned presymptomatic and late symptomatic terms at 1 ,5-months and 4,5 months, respectively.

[0101] Electrophysiology of TRPML1 -mediated currents

[0102] The whole-endolysosome configuration was achieved by a modified patch-clamp method (Wang et al. 2012; Dong et al. 2010; Zhong and Dong 2008; Zang et al. 2019). Whole-endolysosome recordings were performed on enlarged vacuoles, obtained treating NSC-34 cells with 1 mM vacuolin-1 overnight, using the pipette (luminal) solution contained (in mM): 145 NaCI, 5 KCI, 2 CaCI2, 1 MgCI2, 10 HEPES, 10 MES and 10 glucose (pH 4.6 with NaOH) and bath (cytoplasmic) solution contained (in mM): 140 K-Gluconate, 4 NaCI, 1 EGTA, 2 Na2-ATP, 2 MgCI2, 0.39 CaCI2, 0.1 GTP and 10 HEPES (pH 7.2 with KOH and [Ca2+]i of 100 nM). Cells were held at 0 mV and the currents were elicited by repeated voltage ramps (from -140 to +140 mV; 400 ms) with a 4-s interval between ramps. Bath application of 10 pM MLSA1 induced the activation of TRPML1 -mediated currents (ITRPMLI ). ITRPMLI were recorded using the commercially available amplifier Axopatch 200 B and Digidata 1322 A interface (Molecular Devices). Data were acquired and analyzed using the pCIamp software (version 9.0, Molecular Devices). Whole- endolysosome currents were digitized at 10 kHz and filtered at 2 kHz. All experiments were conducted at room temperature (21 -23°C) and all recordings were analysed in pCLAMP (Molecular Devices).

[0103] [Ca2+]i measurement

[0104] [Ca2+]i was detected by single-cell Fura-2 / AM computer assisted video-imaging (Tedeschi et al. Sci Rep 2019). Results are presented as cytosolic Ca2+concentration calculated by the equation of Grynkiewicz (Grynkiewicz et al. J. Biol. Chem. 260, 3440-3450).

[0105] Statistical analysis

[0106] Data are expressed as mean ± S.E.M. Statistical analysis was performed with either unpaired non-parametric or paired non-parametric t-tests and ANOVA. Unpaired t- test or one-way analysis of variance were followed by Newman-Keuls test. Statistical significance was accepted at the 95% confidence level (p < 0.05).

[0107] Effect of PI(3,5)P2 in an in vitro ALS / PDC cellular model Beta-Methylamino-L-alanine (L-BMAA)-treated motor neurons has been used to reproduce in vitro a Guamanian form of ALS called ALS / PDC sharing several features with Parkinson disease. Of note in both these pathologies as well as in the rest of neurodegenerative diseases with relatively slow rate of progression, autophagic flux is blocked determining the aggregation of dysfunctional proteins and organelles. In this ALS / PDC model PI(3,5)P2 prevented cell death induced by L- BMAA neurotoxin in different experimental paradigms (Figure 3). This significant neuroprotection has been correlated with the ability of PI(3,5)P2 to induce autophagy as demonstrated by the increased protein expression of the specific marker LC3II (Figure 4). This marker increases in the early phase of autophagy indicating the increase in the fusion of lysosome with autophagosome.

[0108] The induction of the autophagic flux in neurons is due to the ability of PI(3,5)P2 to open TRPML1 at lysosomal level thus inducing a release of Ca2+and the specific activation of the transcription factor TFEB, the master regulator of autophagy.

[0109] Therefore the activation of TRPML1 by PI(3,5)P2 could be the principal mechanism able to produce neuroprotection during the neurodegenerative process.

[0110] Interestingly, this mechanism is protective only in the neurodegenerative process associated to the classical neurodegenerative diseases in which autophagy is blocked but not in the acute neurological diseases such as stroke, in which autophagy is already hyperfunctional (Tedeschi et al. Neurobiol of Disease 2023). Accordingly, the activation of TRPML1 channel in ischemia-like conditions characterized by hypoxia produced significant detrimental effect in neurons (Fig. 5). In fact, the activation of TRPML1 worsened neuronal death during hypoxic conditions since it further enhanced autophagy - as measured by the increased expression of the autophagic marker LC3 II (Figure 5A)- as well as ER stress -as measured by the increased level of ER stress marker CHOP (Figure 5B). ER stress is a detrimental mechanism already described as one of the main causes of stroke.

[0111] Effect of SANP containing PI(3,5)P2 in electrophysiology of TRPML1 -mediated currents

[0112] The efficacy of SANP containing PI(3,5)P2 (SANP-PIP named F1 in the figures) has been measured as electrophysiological activity on TRPML1 channel in motor neurons. Patch-clamp experiments on enlarged lysosomes -produced in NSC-34 motor neuronal cells by vacuolin- showed that SANP-PIP was more effective than PI(3,5)P2 alone in eliciting a larger inwardly rectifying currents encoded by TRPML1 (Figure 6). In particular, SANP-PIP opened TRPML1 channel for more time compared with PI(3,5)P2 alone (see t reported for each agonist) and it was as effective as the synthetic agonist ML-SA1 (Figure 3). Moreover, SANP free -without PI(3,5)P2- failed to modulate the inwardly rectifying current. Being TRPML1 considered a cation channel located only at lysosomal level, lysosomal Ca2+release was elicited by SANP-PIP in the presence or absence of the channel and measured either as A% of basal [Ca2+]i increase or as AUC of the Ca2+responses (Fig. 7). SANP-PIP promoted lysosomal Ca2+release only in NSC-34 cells expressing TRPML1 while it failed to produce the same effect in motor neurons treated with siRNA against this lysosomal channel (Fig. 7). SANP lacking PI(3,5)P2 failed to induce [Ca2+]i (Fig. 4). These experiments showed the specificity of the preparation of the invention SANP-PIP for TRPML1 channel as well as its ameliorative biophysical properties compared with the progenitor molecule PI(3,5)P2. The better pharmacodynamic features of SANP-PIP enhanced the ability of PI(3,5)P2 to induce autophagy. In fact SANP-PIP increased LC3 II overexpression better then PI(3,5)P2 alone or PI(3,5)P2+ a synthetic activator of TRPML1 (Figure 8), thus suggesting the highest propensity of the preparation of the invention to induce a powerful autophagy in neurodegeneration.

[0113] Effect of SANP containing PI(3,5)P2 in in vivo experiments

[0114] Having SANP-PIP a better pharmacological profile than that of the progenitor molecule (PI(3,5)P2), the preclinical tests in vivo have been performed only with the formulation SANP-PIP at two different dosages (10 times or 100 times higher than the ECso of PI(3,5)P2 used in vitro).

[0115] In vivo experiments confirmed that SANP-PIP administrated to asymptomatic SOD- G93A mice for 6 weeks increased lifespan of these ALS animals compared with SOD-G93A mice treated only with physiological solution (Figure 9). In fact, ALS mice treated only with physiological solution died at 127 days while animals exposed to the preparation oof the invention died after 140 days, a significant increase of their lifespan. Furthermore, SANP-PIP 100X increased their muscle strength measured by Grip test (Figure 10). Effect of SANP containing PI(3,5)P2 and anti-miR124 in an in vitro ALS cellular model

[0116] Many studies indicate that the inactivation of the serine / threonine-protein kinase GSK3[3 plays a neuroprotective role in ALS pathogenesis. Indeed, GSK3[3 activity is increased in different preclinical models of neurodegenerative diseases and in ALS patients (Recent Advances on the Role of GSK3[3 in the Pathogenesis of Amyotrophic Lateral Sclerosis Hyun-Jun Choi, Sun Joo Cha, Jang-Won Lee, Hyung- Jun Kim, and Kiyoung Kim. Brain Sci. 2020 10(10): 675). Recent studies have also suggested a possible role of GSK3[3 in other neurodegenerative diseases, such as Parkinson's disease (PD) and Alzheimer's disease (AD) [Kockeritz L., Doble B., Patel S., Woodgett J.R. Glycogen synthase kinase-3-an overview of an overachieving protein kinase. Curr. Drug Targets. 2006;7:1377-1388; Frame S., Cohen P. GSK3 takes center stage more than 20 years after its discovery. Biochem. J. 2001 ;359:1-16], As it inhibits several pathogenic pathways common in neurodegenerative diseases, GSK3[3 could be considered as a potential target for the development of novel therapies for neurodegenerative diseases.

[0117] On the light of this evidence, were performed in vitro experiments on a cellular model of Guamanian ALS. Once differentiated, NSC-34 cells were exposed to the neurotoxin beta-Methylamino-L-alanine (L-BMAA; 300 pM for 24 h). Under these conditions, motor neuron cells expressed high levels of the phosphorylated form (i.e. the active form) of the GSK3[3 protein so that it could be considered a neuronal marker of ALS.

[0118] The results obtained from the protein preparations of motor neuron cells pretreated with antagomiR-124 in free form, with nanoparticles encapsulating only antagomiR- 124 or with nanoparticles containing both antagomiR-124 and PI(3,5)P2diC8 showed different results on the expression of GSK3[3 marker (Figure 11 ). Surprisingly, it was observed a synergic reduction of GSK3[3 active form only in cells treated with the formulation co-encapsulating PI(3,5)P2diC8 and antagomiR-124, (Figure 12). However, nanoparticles containing PI(3,5)P2diC8 were able to produce a reduction of GSK3[3 active form to a lesser extent than the formulation coencapsulating both PI(3,5)P2diC8 and antagomiR-124 thus confirming a synergic effect of the nanoparticle preparation encapsulating both antagomiR-124 and PI(3,5)P2diC8 (Fig. 13)..

[0119] On the other hand, when administered together with L-BMAA, PI(3,5)P2diC8 alone or in combination with antagomiR-124, both in free form, failed to modulate GSK3[3 activation induced by L-BMAA. However, when preincubated for 30 minutes, PI(3,5)P2diC8 alone in free form or in SANP or in combination with antagomiR-124 in SANP reduced GSK3[3 expression induced by L-BMAA. Interestingly, the nanoparticle preparation encapsulating both antagomiR-124 and PI(3,5)P2diC8 exerted a synergic effect compared with the other treatments (Fig. 14).

Claims

CLAIMS1 . A phosphatidylinositol 3,5-bisphosphate (PI(3,5)P2) of formula (l)for use in the treatment of a neurodegenerative disease characterized by impaired or blocked autophagy, wherein formula (I) iswhereinR is a C6-20 fatty acid; andR’ is H or a C6-20 fatty acid equal or different from R.

2. The PI(3,5)P2 for use according to claim 1 wherein R=R’=octanoyl.

3. The PI(3,5)P2 for use according to claim 1 or 2 wherein the neurodegenerative disease is selected in the group consisting of AD, PD, ALS, Charcot-Marie-Tooth disease, HD, lysosomal storage disorders, encephalopathies, MS.

4. A lipid nanoparticle (LNP) encapsulating a PI(3,5)P2 of formula (I) as defined in any one of claims 1-2, said LNP is a self-assembly nanoparticle (SANP) composed by cationic or ionizable lipids and neutral lipids, inorganic salts and a PI(3,5)P2 of formula (I) which acts both as active pharmaceutical ingredient and as lipid excipient.

5. A LNP encapsulating a PI(3,5)P2 of formula (I) as defined in any one of claims 1-2 and further encapsulating a miRNA or an antagomir; wherein the miRNA is one of those downregulated in patients affected by a neurodegenerative disease characterized by impaired or blocked autophagy, and wherein the antagomir is against is one of those RNA upregulated in patients affected by said neurodegenerative disease.

6. The LNP according to any one of claims 4-5 which is a Self-Assembling Nanoparticle (SANP) composed by cationic or ionizable and neutral lipids selected in the group consisting of 1 ,2-dioleoyl-3-trimethylammonium-propane chloride (LIPOID DOTAP-CL or DOTAP), N-[1 -(2,3-dioleyloxy)propyl]-N,N,N- trimethylammonium chloride (DOTMA) or 3s-[N-(N',N'-dimethylaminoethane)- carbamoyl]cholesterol hydrochloride (DC-chol), 1 ,2-dioleoyl-3-dimethylammonium- propane (DODAP), O-(Z,Z,Z,Z-heptatriaconta-6,9,26,29-tetraen-19-yl)-4-(N,N- dimethylamino) (DLinMC3), 2,2-dilinoleyl-4-dimethylaminoethyl-[1 ,3]-dioxolane (DLin-KC2-DMA), [(4-Hydroxybutyl)azanediyl]di(hexane-6, 1 -diyl) bis(2- hexyldecanoate) (ALC-0315), (heptadecane-9-yl 8-[2-hydroxyethyl-(6-oxo-6- undecoxyhexyl)amino]octanoate (SM-102), cholesterol (CHOL), dioleoylphosphatidylethanolamine (DOPE), N-palmitoylsphingosine-1 {succinyl[methoxy(polyethylene glycol)2000]} (cene-PEG2ooo) (cer-PEG) and 1 ,2- distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethylene glycol)-2000] (DSPE-PEG2000); and inorganic salts selected in the group consisting of Na2HPO4, sodium citrate tribasic, CaCL and Cerium(lll) nitrate.

7. The LNP according to any one of claims 4-6 is a SANP composed by PEGylated liposomes, consisting of DOTAP and Cen6-PEG2ooo, encapsulating colloidal calcium phosphate and PI(3,5)P2 di octanoyl.

8. The LNP according to claim 7 further encapsulating anti-miR124.

9. A LNP according to any one of claims 4-8 for use as neuroprotective agent in the treatment of a neurodegenerative disease characterized by impaired or blocked autophagy.

10. The LNP for use according to claim 9 wherein the neurodegenerative disease is selected in the group consisting of AD, PD, ALS, Charcot-Marie-Tooth disease, HD, lysosomal storage disorders, encephalopathies, MS.11 . The LNP according to any one of claim 7-8 for use in the treatment of ALS.

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