Ionic liquid-based formulations for the prevention or treatment of nervous system diseases

Abstract

The present disclosure relates to an ionic liquid (IL)-based formulation for the treatment, therapy or prevention of a nervous system disease, in particular Parkinson's disease, said formulation comprising an inhibitor, in particular a specific inhibitor, of NADPH oxidase (Nox), preferably isoforms 1 and 4, 3-cyclohexyl-5-(2,4-dihydroxybenzylidene)-1-methyl-2-thiohydantoin.

Description

TECHNICAL FIELD

[0001] The present disclosure relates to an ionic liquid (IL) formulation for the treatment, therapy or prevention of a nervous system disease, in particular Parkinson's Disease (PD), said formulation comprising an inhibitor, in particular a specific inhibitor, of NADPH oxidase (Nox), preferably isoforms 1 and 4, 3-cyclohexyl-5-(2,4-dihydroxybenzylidene)-1-methyl-2-thiohydantoin. BACKGROUND

[0002] Nervous system disorders are a leading cause of disability and the second largest cause of death worldwide. According to the Global Burden of Diseases, Injuries, and Risk Factors Study (GBD) data published in the Journal Lancet Neurology (2019; 18(5):459-80) in 2016, nervous system disorders are the leading cause of disability-adjusted life years - DALYs (276 million) and the second largest cause of death (9 million).

[0003] According to the study by Dorsey et al. (Journal of Parkinson's Disease 2018; 8:S3-S8) entitled "The Emerging Evidence of the Parkinson Pandemic", Parkinson's Disease (PD) is the fastest growing disease among nervous system disorders, more than doubling in the number of people affected from 1990 to 2015, exceeding 60 million. The same study discloses that, due to population aging, this number will further increase to more than 120 million by 2040. Moreover, as highlighted by the same authors, if other factors are taken into account (e.g. increased longevity, decreased smoking rates and increased industrialization), the PD-related burden could increase to more than 170 million by 2040. Therefore, the costs associated with PD are enormous.

[0004] According to the study by Yang et al. (Current and projected future economic burden of Parkinson's disease in the U.S. npj Parkinson's Disease 2020; 6(1): 15), in 2017, the total economic burden of PD in the U.S. alone was $51.9 billion, including $25.4 billion in direct medical costs and $26.5 billion in indirect and non-medical costs.

[0005] Currently, there is no cure for PD and the available therapies are symptomatic and can only control symptoms. The current regimen of PD therapies is mainly based on the prescription of drugs as dopamine precursors, dopamine agonists or agents that aim to inhibit key enzymes in the catabolic pathway of dopamine. However, as the disease progresses to more debilitating stages, the effectiveness of this therapy decreases over time, leaving patients with limited treatment options. Therefore, preventing disease progression is increasingly being considered as a promising solution for PD patients. However, as highlighted by a market research conducted by Research and Markets ("The Parkinson's Disease Market: Pipeline Review, Developer Landscape and Competitive Insights", pp. Report ID: 4586296), the lack of preventive therapies that block PD progression remains one of the main unmet needs in this field. Therefore, the development of preventive therapies for PD progression is of paramount importance.

[0006] In the central nervous system (CNS), oxidative stress is associated with multiple diseases and aging, is a major predisposing factor for Parkinson's disease, and therefore it can be a suitable target to block the progression of neurological diseases.

[0007] Until recently, mitochondria were also considered the main source of ROS in the CNS, but recent studies have revealed that homologues of NADPH oxidases (Nox) are also located in the CNS, which play a crucial role in the production of ROS, which are necessary for processes such as development, memory, neuronal signaling and vascular hemostasis. However, the ROS produced by these enzymes cause cell death associated with the pathological processes of several neurological disorders, such as Parkinson's disease and amyotrophic lateral sclerosis (ALS). In this field, previous studies have shown that Noxl-mediated oxidative stress plays a crucial role in the degeneration of dopaminergic neurons in PD and the role of Nox has been validated in experimental models of PD (Cristovao et al. The role of NADPH oxidase 1 -derived reactive oxygen species in paraquat-mediated dopaminergic cell death. Antioxidants & Redox Signaling 2009; 11 : 2105-2118; Choi et al. NADPH Oxidase 1 -Mediated Oxidative Stress Leads to Dopamine Neuron Death in Parkinson's Disease. Antioxidants & Redox Signaling 2012; 16(10): 1033-1045; Cristovao et al. NADPH oxidase 1 mediates alpha-synucleinopathy in Parkinson's disease. Journal of Neuroscience 2012; 32: 14465-14477). These works further demonstrate that the negative effects of oxidative stress produced by several Nox isoforms in neurons can be reduced by using aporaphin (Apo), a non-specific inhibitor of Nox.However, Apo is only soluble in dimethyl sulfoxide (DMSO), which in turn induces direct or indirect neurotoxicity, as previously demonstrated by Hanslick et al. (Dimethyl sulfoxide (DMSO) produces widespread apoptosis in the developing central nervous system; Neurobiology of Disease 2009; 34: 1-10) and Yuan et al. (Dimethyl sulfoxide damages mitochondrial integrity and membrane potential in cultured astrocytes; PLoS One 2014; 9: e107447). This solubility problem exists for several antioxidant molecules or other compounds that are capable of inhibiting Nox, affecting their therapeutic efficacy / application due to low bioavailability. This problem is also common to other active pharmaceutical ingredients (APIs) that are usually used in solid state. The recurring problem of polymorphism and low aqueous solubility in solid state of APIs severely impairs their bioavailability and therapeutic efficiency, which is the main reason for the failure of drug-candidates in phase II of clinical studies, according to the Tufts Center for the Study of Drug Development (Trial watch: Phase II failures: 2008-2010. Nature Reviews Drug Discovery 2011; 10: 328-329). Moreover, in the particular case of CNS-directed drugs, their low ability to cross the blood brain barrier (BBB) is also a major problem. For this reason, the intranasal route of delivery is becoming an interesting route for the delivery of drugs into the brain. However, this is not achievable with the traditional solid-state formulations of most drugs. Therefore, there is a need to develop new formulations that prevent the problems associated with polymorphism and low solubility of these drugs, while promoting intranasal delivery to avoid problems regarding BBB penetration, which is particularly relevant in the context of neurodegenerative diseases such as PD.

[0008] ILs are salts composed of organic cations and organic or inorganic anions. Due to the large size of the ions, these salts do not have an ordered crystalline structure and are therefore liquid at temperatures lower than those of conventional salts. Most of the known ILs are liquid at room and body temperature, which helps to overcome the polymorphic situation of most drugs and increases the solubility of the API, since the energy associated with the fusion enthalpy does not need to be overcome. In addition, since they are salts composed of ions, they form strong interactions with water, making their solubility significantly increase. The development of ILs including APIs for pharmacological applications has been proposed and is becoming a new era of therapeutic strategies (Shamshina et al. Chemistry: Develop ionic liquid drugs. Nature 2015; 528: 188-189; Egorova et al. Biological Activity of Ionic Liquids and Their Application in Pharmaceutics and Medicine. Chemical Reviews 2017; 117(10): 7132-7189; Pedro et al. Ionic Liquids in Drug Delivery. Encyclopedia 2021; 1: 324-339).

[0009] Previous works have successfully reported the transformation of different types of APIs into ILs, including antibiotics, analgesics and non-steroidal anti-inflammatory drugs (NSAIDS) (Pedro et al. Ionic Liquids in Drug Delivery. Encyclopedia 2021; 1: 324-339).

[0010] Compared to analgesics and anti-inflammatory precursors, the conversion of analgesics (lidocaine) and anti-inflammatory drugs (NSAIDs) into bifunctional ILs can increase their water solubility by 470 times without affecting their cytotoxicity profile (Abednejad et al. Polyvinylidene fluoride-hyaluronic acid wound dressing comprised of ionic liquids for controlled drug delivery and dual therapeutic behavior. Acta Biomaterialia 2019; 100: 142-157). Likewise, the conversion of different NSAIDs into ILs, namely ibuprofen, ketoprofen, and (S)-naproxen, can improve their bioavailability by increasing their water solubility by 100 times (Chantereau et al. Design of Nonsteroidal Anti-Inflammatory Drug-Based Ionic Liquids with Improved Water Solubility and Drug Delivery. ACS Sustainable Chemistry & Engineering 2019; 7: 14126-14134).

[0011] In addition to these APIs, phenolic antioxidants have been successfully converted into ILs. In the work “Enhancing the antioxidant characteristics of phenolic acids by their conversion into cholinium salts” (ACS Sustainable Chemistry & Engineering 2015; 3: 2558-2565), five anions with antioxidant and anti-inflammatory properties (i.e., gallic, caffeic, vanillic, syringic, and ellagic acids) were conjugated with the choline cation. The obtained salts were significantly more soluble in water (about 3 orders of magnitude higher) than the corresponding phenolic acids. Moreover, they generally presented higher antioxidant and anti-inflammatory activities, comparable cytotoxicity, and lower ecotoxicity profiles than their precursors.

[0012] Based on these promising results, as a proof of concept behind the present invention, Apo, a non-specific inhibitor of Nox, was reformulated into [Chol][Apo], as disclosed in the work entitled “Testing the application of new antioxidant chemical formulations to prevent neuronal degeneration” (Afonso, 2017, MSc thesis, University of Beira Interior). The reformulated ionic liquid overcame the low solubility of Apo, with a 50-fold increase in water solubility. In a PD model, 6OHDA-induced dopaminergic neuronal toxicity was significantly reduced, further confirming the neuroprotective potential of the new formulation in the context of PD. These encouraging results highlight the potential use of new IL-based drug formulations in neurodegenerative diseases and PD. However, Apo is a broad inhibitor of NADPH oxidases, capable of inhibiting several Nox isoforms by blocking the association of p47phox and p67phox with gp91phox (Stolk et al. Characteristics of the inhibition of NADPH oxidase activation in neutrophils by apocynin, a methoxy-substituted catechol. American Journal of Respiratory Cell and Molecular Biology 1994; 11 : 95-102). Since it does not specifically inhibit one isoform, it is mainly used as a NADPH oxidase inhibitor for research (Bedard and Krause. The NOX family of ROS-generating NADPH oxidases: physiology and pathophysiology. Physiological Reviews 2007; 87: 245-313).This lack of target specificity severely limits its use as a therapeutic approach, since in different cell types, due to the inhibition of more than one Nox isoform, it can cause unwanted and uncontrolled biological effects, highlighting the necessity of developing specific Nox inhibitors that exhibit high bioavailability and efficacy. Given the potential therapeutic effect of targeting Nox for several pathologies, the synthesis of specific inhibitors of these enzymes has attracted wide attention. Previous work (Bae et al. Synthesis and biological evaluation of 3-substituted 5-benzylidene-1-methyl-2-thiohydantoins as potent NADPH oxidase (NOX) inhibitors. Bioorganic & Medicinal Chemistry 2016; 24: 4144-4151) reported several formulations of 3-substituted 5-benzylidene-1-methyl-2-thiohydantoins-based compounds for the inhibition of Noxl (4). 3-cyclohexyl-5-(2,4-dihydroxybenzylidene)-1-methyl-2-thiohydantoin (N1(4)inh) was demonstrated to be an effective inhibitor of Noxl and a partial inhibitor of Nox4 by a lucigenin-based chemiluminescence assay in Drosophila.

[0013] The knowledge previously acquired using [Chol][Apo] helped to establish an irrefutable proof of concept about the application of IL in the context of PD, from which it can be extrapolated a method for Nox-specific inhibitors.

[0014] The Noxl (4)-specific inhibitor 3-cyclohexyl-5-(2,4-dihydroxybenzylidene)-1-methyl-2- thiohydantoin (N1(4)inh) described in the publication “Synthesis and biological evaluation of 3-substituted 5-benzylidene-1-methyl-2-thiohydantoins as potent NADPH oxidase (NOX) inhibitors” (Bioorganic & Medicinal Chemistry 2016; 24: 4144-4151) showed high neuroprotective potential in experimental models of PD (Correia-Branco V et al. Neuroprotective effects of 3-cyclohexyl-5-(2,4-dihydroxybenzylidene)-1-methyl-2-thiohydantoin in a mouse model of Parkinson’s disease. Neuropharmacology 2017; 122: 1-11). Nox1 / Nox4 inhibitor protects dopaminergic neurons from degeneration: new candidate for Parkinson disease therapeutic? 14th International Conference on Alzheimer's and Parkinson's Diseases and related neurological disorders AD / PD TM 2019 (14th International Conference on Alzheimer's and Parkinson's Diseases and related neurological disorders AD / PD TM 2019)). However, N1(4)inh is only soluble in a solvent consisting of a buffer, ethanol and triton-X, which is itself cytotoxic to dopaminergic neurons, preventing the potential use of this promising inhibitor as a future therapeutic strategy in the context of neurodegenerative diseases.

[0015] The identification of the problem described in the state of the art and the method to solve it are a good illustration of the problem our invention wants to solve.

[0016] Document KR20080051246(A) "Pharmaceutical composition for prevention or treatment of neurodegenerative disease and inhibition of NADPH OXIDASE" refers to an NADPH oxidase inhibitor that can prevent the death of dopaminergic neurons by reducing the toxicity of microglial cells, which can be used to prevent and treat neurodegenerative diseases, including fluoxetine or norfluoxetine. This invention, although it targets the same target enzyme as our invention, shows a different formulation, since our inhibitor is based on an ionic liquid of 3-substituted 5-benzylidene-1-methyl-2-thiohydantoin.

[0017] By using chemical molecules or genes, the following set of documents aims to prevent or treat neurological disorders by modulating / inhibiting different pathological cellular mechanisms, including oxidative stress prevention, such as our invention, however the drug formulations and molecular targets are not the same. For example: US2020289671(A1) - Pharmaceutical composition comprising Aimp2-Dx2 for preventing or treating neuronal diseases and use thereof (Pharmaceutical Composition Comprising Aimp2-Dx2 For Preventing Or Treating Neuronal Diseases And Use Thereof); US2020385342(A1) - Methods of making deuterium-enriched N-acetylcysteine amide (D-NACA) and (2R,2R')-3,3'-disulfanediyl BIS(2-acetamidopropanamide) (DINACA) and using D-NACA and DINACA to treat diseases involving oxidative stress (Methods of Making Deuterium-Enriched N-acetylcysteine Amide (D-NACA) and (2R,2R')-3,3'-Disulfanediyl BIS(2-Acetamidopropanamide) (DINACA) and Using D-NACA and DINACA to Treat Diseases Involving Oxidative Stress); WO 2018129421(A1) - A promising drug candidate for Parkinson's disease (A Promising Drug Candidate For Parkinson's Disease); US2013109714(A1) - Neurodegenerative disease therapeutic agent (Neurodegenerative Disease Therapeutic Agent).

[0018] Document CN111138376(A) - 3,5-disubstituted phenyl-1,2,4-oxadiazole derivative, and preparation method and application thereof (3,5-Disubstituted Phenyl-1,2,4-Oxadiazole Derivative, And Preparation Method And Application Thereof) discloses 3,5-disubstituted phenyl-1,2,4-oxadiazole derivative, and preparation method and application thereof.

[0019] Documents: WO 2020205937 (A1) - Hyaluronic Acid Nanoparticles Comprising NADPH Oxidases Inhibitors And Uses In Treating Cancer and US2019048001 (A1) - Iodonium Analogs As Inhibitors Of NADPH Oxidases And Other Flavin Dehydrogenases; Formulations Thereof; And Uses Thereof. Despite comprising NADPH oxidase inhibitors, they refer to different formulations and applications. In these cases, the above documents aim to use different APIs for cancer treatment (such as hyaluronic acid or iodonium analogs) to inhibit NADPH oxidase. The documents do not mention the use of our molecules, not even in neurodegenerative diseases.

[0020] The following documents specified below describe different synthesis methods of different NADPH oxidase inhibitors, using chemical or non-chemical methods, aiming to target a wide range of pathologies, such as metabolic and neurological disorders. However, these inhibitors are different from our [Chol][N1(4)inh] in terms of chemical and formulation methods. Despite targeting NADPH oxidase, they differ in terms of inhibitors compared to the present disclosure, utilizing different Nox, with different applications and formulations: for example, US2020270214 (A1) - NADPH Oxidase Inhibitors and Uses Thereof; JP2020063283 (A) - Nox Inhibitor And Nfκb Inhibitor Including Methoxy Flavone; KR20200022193 (A) - Pharmaceutical composition for preventing or treating tuberculous pleural fibrosis, comprehending a different NOX.

[0021] Document SG10201808940W (A) - NOX Inhibitor And NFKB Inhibitor Containing Methoxyflavone aims at providing NOX inhibitors and NFKB inhibitors having superior actions, as well as agents for preventing or treating Nox- or NFicB-associated diseases that utilize such inhibitors. To this end, a specified methoxyflavone is employed.

[0022] The following documents disclose IL-based formulations targeting PD: MX2016011152(A)- Pramipexole-Containing Transdermal Patch For Treatment Of Neurodegenerative Disease, which discloses a different compound and does not target NOX, compared to the present disclosure; and WO 2010078258(A1)- Compounds Comprising Two Or More Biologically Functional Ions And Method Of Treating Parkinson's Disease, which includes active compounds that are completely different from the active compounds used in the present disclosure and have completely different therapeutic targets. The APIs used in the IL of document WO 2010078258(A1) are 2-(diethylamino)-N-(2,6-dimethylphenyl)acetamide (lidocaine), (2S,3S)-5-[2-(dimethylamino)ethyl]-2-(4-methoxyphenyl)-oxo-2,3,4,5-tetrahydrobenzo[b][l,4]thiazepin-3-yl acetate, 2-(2,6-dichloro-3-methylphenylamino)benzoic acid (meclofenamate), 5-ethyl-8-oxo-5,8-dihydro-[l,3]dioxolo[4,5-g]quinoline-7-carboxylic acid (oxolinic acid), and (2S,5R,6R)-6-((R)-2-amino-2-phenylacetylamino)-3,3-dimethyl-7-oxo-4-thia-l- azabicyclo[3.2.0]heptane-2-carboxylic acid (ampicillanic acid); while our API is 3- substituted 5-benzylidene-l-methyl-2-thiohydantoin. Moreover, our IL and formulation specifically target Noxl and Nox4 isoforms.

[0023] The technical problem addressed by the present disclosure is solved by the known solutions in the prior art.

[0024] The disclosure of these facts is intended to illustrate the technical problem addressed by the present disclosure. SUMMARY

[0025] The present disclosure relates to the development of novel IL-based formulations of specific Nox inhibitors. Their negligible solubility in aqueous solvents is a common problem for several compounds capable of inhibiting Nox, and this low solubility negatively affects their bioavailability, reducing their efficacy and therapeutic potential. The present disclosure contemplates the development of a non-toxic alternative formulation that allows to improve the solubility of Nox inhibitors in aqueous solutions, and thus to increase bioavailability, efficacy and therapeutic potential. One of the objectives of the present disclosure is to increase the solubility of specific Nox inhibitors applied to neurological diseases and thereby to increase bioavailability and efficacy.

[0026] In view of the drawbacks of the prior art, the technical problem of the present invention is the development of an IL-based formulation of N1(4)inh to be applied to neurological diseases, particularly for the prevention, slowing of disease progression therapy or treatment of Parkinson's disease (PD).

[0027] Since Nox is an ideal target for specific antioxidant therapeutic strategies, the present disclosure can be used to develop a new therapeutic method aimed at reducing / stopping the progression of neurodegenerative diseases, including PD, over time.

[0028] In an embodiment, the novel IL formulation of N1(4)inh was developed using the following methods: (1) synthesis of the specific inhibitor 3-cyclohexyl-5-(2,4-dihydroxybenzylidene)-1- methyl-2-thioxohydantoin (N1(4)inh) into an IL-based formulation (i.e., 3-cyclohexyl-5-(2,4- dihydroxybenzylidene)-1-methyl-2-thioxohydantoin choline ([Chol]2[N1(4)inh] + N1(4)inh)) and its chemical characterization, and (2) in vitro evaluation of its biological effects on dopaminergic neurons.

[0029]

[0030] In an embodiment, the IL formulation of the inhibitor shows higher solubility, no neuronal toxicity, and high neuroprotective potential in the context of PD compared to the non- reformulated inhibitor (precursor). Thus, the compounds and formulations of the present disclosure overcome the solubility problem of Nox inhibitors in aqueous solvents, increasing their potential as neuroprotective therapies to slow down the progression of PD, representing a relevant advance in the field of specific antioxidant therapies.

[0031] One aspect of the present disclosure relates to the Nox-specific inhibitor in IL and formulation, and its therapeutic application to neurological diseases, i.e., PD.

[0032] In one embodiment, the inhibitors disclosed in the present disclosure are specific for isoforms 1 and 4 of NADPH oxidase. Thus, the present disclosure relates to a novel pharmaceutical formulation that specifically targets NADPH oxidase 1 and 4 by inhibition for the treatment of a nervous system disorder, i.e. to reduce / stop the progression of PD after diagnosis.

[0033] One aspect of the present disclosure relates to an ionic liquid comprising: an anion of the following formula:

[0034]

[0035] wherein R is an alkyl or a cycloalkyl group;

[0036] and a cation selected from the list consisting of: choline, tetraalkylammonium, tetraalkylphosphonium or the 1-alkyl-3-methylimidazolium cation family.

[0037] In one embodiment, the molar ratio of the anion and the cation ranges from 1 :2 to 2:1 (mol:mol), preferably from 1 :1.5 to 1.5:1 (mol:mol).

[0038] In one embodiment, the molar ratio of the anion and the cation is 1 :1 (mol:mol).

[0039] In one embodiment, R is a cycloalkyl group from C3-C7.

[0040] In one embodiment, R is an unsubstituted cycloalkyl group from C3-C7.

[0041] In one embodiment, R is a cyclohexyl group.

[0042] In one embodiment, the cation is choline.

[0043] In one embodiment, the compound is 3-substituted 5-benzylidene-1-methyl-2- thiohydantoin or 3-cyclohexyl-5-(2,4-dihydroxybenzylidene)-1-methyl-2- thiohydantoin.

[0044] Another aspect of the present disclosure relates to the use of the ionic salt, preferably ionic liquid, and formulation of the present disclosure in medicine or as a pharmaceutical.

[0045] In one embodiment, the ionic liquid, preferably ionic liquid, and formulation of the present disclosure can be used for the prevention or treatment of a disease, disorder or condition of the central nervous system.

[0046] In one embodiment, the ionic liquid and formulation of the present disclosure can be used for the prevention or treatment of neurodegeneration, cognitive dysfunction, dementia or multiple system atrophy.

[0047] In an embodiment, the ionic liquid and formulation of the present disclosure, preferably the ionic liquid and formulation, can be used for preventing or treating Parkinson's disease.

[0048] In an embodiment, the ionic liquid and formulation of the present disclosure can be used for slowing down or delaying the progression of Parkinson's disease.

[0049] Another aspect of the present disclosure relates to a pharmaceutical composition comprising a therapeutically effective amount of the ionic liquid of the present disclosure and a pharmaceutically acceptable carrier.

[0050] In an embodiment, the pharmaceutically acceptable carrier is a saline buffer, PBS, water or a mixture thereof.

[0051] In an embodiment, the amount of the ionic liquid is in the range of 0.005 mM - 10 mM, preferably 0.1 - 5 mM, more preferably 1 - 2 mM.

[0052] In an embodiment, the composition is in injectable form, intranasal form, intrathecal form or intracerebroventricular form; preferably, the composition is in intranasal form, intracerebroventricular form or intrathecal form of administration.

[0053] In an embodiment, the composition comprises administration of a daily dose to a person suffering from a neurodegenerative disease or a central nervous system disorder, preferably over a period of not less than 30 days. In an embodiment, the dose is less than 1000 mg / day, preferably in the range of 0.05 - 1000 mg / day, more preferably in the range of 0.05 - 5 mg / day, in particular by intranasal form or intrathecal (intracerebroventricular).

[0054] In an embodiment, the daily form consists of tablets, suppositories, ampoules or intranasal form or intrathecal form comprising a pharmaceutically effective amount of the composition of the present disclosure, all intended to be administered in a single dose in a daily regimen.

[0055] The present disclosure also relates to the development of novel formulations of specific inhibitors of Noxl and Nox4 (N1(4)inh) comprising the following steps:

[0056] 1) Conversion of the specific inhibitor [N1(4)inh] into an IL-based formulation, i.e. a mixture of 3-cyclohexyl-5-(2,4-dihydroxybenzylidene)-l-methyl-2-thiohydantoin choline and 3-cyclohexyl-5-(2,4-dihydroxybenzylidene)-l-methyl-2-thiohydantoin salt (1:1) ([Chol]2[N1(4)inh] + N1(4)inh, 1:1), comprising the following procedure:

[0057] 1a) Selection of IL anions and cations.

[0058] 1 b) Synthesis of [Chol]2[N1(4)inh] by metathesis reaction using choline salt as source of cations and 3-cyclohexyl-5-(2,4-dihydroxybenzylidene)-1 -methyl-2- thiohydantoin (N1(4)inh) as source of anions.

[0059] 1 c) Characterization of [Chol]2[N1(4)inh] + N1(4)inh and N1(4)inh by nuclear magnetic resonance (NMR) and carbon nuclear magnetic resonance (C NMR) for purity; solubility in water and in phosphate buffered saline (PBS) solution; and thermal stability by thermogravimetric analysis (TGA). 1 H NMR and 13 C NMR) for purity; solubility in water and in phosphate buffered saline (PBS) solution; and thermal stability by thermogravimetric analysis (TGA).

[0060] 2) Evaluation of the biological effects of [Chol]2[N1(4)inh] + N1(4)inh in dopaminergic neurons in vitro and in vivo by:

[0061] 2a) Evaluation of the cytotoxicity of [Chol]2[N1(4)inh] + N1(4)inh in immortalized rat dopaminergic neuronal cell line (N27).

[0062] 2b) Evaluation of the biological effects of the reformulation in the context of therapy for PD by assessing its dopaminergic neuroprotective capacity in an in vitro model of PD.

[0063] 2c) Evaluation of the neuronal dopaminergic toxicity of [Chol]2[N1(4)inh] + N1(4)inh in the substantia nigra of mice.

[0064] 2d) Evaluation of the toxicity of [Chol]2[N1(4)inh] + N1(4)inh in mice and rats.

[0065] 2e) Evaluation of the biological effects of the reformulation in the context of therapy for PD by assessing its dopaminergic neuroprotective effect and its ability to prevent the progression of motor dysfunction in an in vivo model of PD.

[0066] In an embodiment, considering the pathological role of oxidative stress originating from Nox enzymes in PD and other neurological diseases, the use of inhibitors of these enzymes activities plays an important role in the development of future therapies aiming at reducing the rate of progression and disability of such diseases over time. However, most if not all of these inhibitors have negligible solubility in aqueous solutions and reduce efficacy due to low bioavailability and also require the use of organic solvents as vehicles which in turn have high cytotoxic effects. In addition, the re-formulation of these inhibitors into liquid compounds is essential for intranasal administration and bypasses the high selectivity of the blood brain barrier (BBB) allowing their concentration in the brain (target organ).

[0067] The present disclosure also relates to the development of new IL-based formulations of inhibitors of NADPH oxidases (Nox), in particular specific for Noxl and Nox4 (Noxl(4)), to be used as therapeutic agents for neurological diseases, in particular Parkinson's disease.

[0068] The present disclosure also relates to the development of new IL-based formulations of specific inhibitors of Noxl(4), comprising the following steps:

[0069] 1) Conversion of Nl(4)inh into IL-based formulations, i.e. 3-cyclohexyl-5-(2,4- dihydroxybenzylidene)-l-methyl-2-thiohydantoin choline and 3-cyclohexyl-5-(2,4- dihydroxybenzylidene)-l-methyl-2-thiohydantoin salt [Chol]2[Nl(4)inh] + Nl(4)inh), which comprises the following procedure:

[0070] 1a) Selection of IL anions and cations.

[0071] 1b) Synthesis of [Chol]2[Nl(4)inh].

[0072] 1c) Chemical characterization of [Chol]2[Nl(4)inh] + Nl(4)inh and Nl(4)inh, i.e. purity, solubility and thermal stability.

[0073] 2) Evaluation of the biological effects of [Chol]2[Nl(4)inh] + Nl(4)inh in dopaminergic neurons in vitro and in vivo, which comprises:

[0074] 2a) Evaluation of the cytotoxicity of [Chol]2[Nl(4)inh] + Nl(4)inh in immortalized rat dopaminergic neuronal cell line (N27).

[0075] 2b) Validate the biological effects of the reformulated compound in the context of therapy for PD, and evaluate its dopaminergic neuroprotective capacity in an in vitro model of PD.

[0076] Evaluation of neuronal dopaminergic toxicity of 2c)[Chol]2[N1(4)inh]+N1(4)inh in the substantia nigra (SN) of mice.

[0077] Evaluation of the toxicity of 2d)[Chol]2[N1(4)inh]+N1(4)inh in mice and rats.

[0078] 2e) The bioefficacy of the reformulated compound in the context of therapy for PD was evaluated by assessing its dopaminergic neuroprotective effects and its ability to prevent the progression of motor dysfunction in in vivo models of PD.

[0079] In one embodiment, regarding the first part of the method, a choline salt is used as the cation source, and 3-cyclohexyl-5-(2,4-dihydroxybenzylmethyl)-1-methyl-2-thiohydantoin (N1(4)inh) is used as the anion source. [Chol]2[N1(4)inh]+N1(4)inh is synthesized via a metathesis reaction, and its purity, thermal stability, and solubility are characterized. 1 H and 13 C10 NMR confirmed the purity of [Chol]2[N1(4)inh]+N1(4)inh and N1(4)inh (Figures 1-2). Solubility results revealed that [Chol]2[N1(4)inh]+N1(4)inh has higher solubility in water and PBS than its precursor -N1(4)inh alone. Figures 3-4 Thermal stability results indicate that, under specified conditions, the decomposition temperature of the formulation is 200℃. Figure 5 At room temperature (approximately 23°C), IL-based formulations are highly viscous liquids, meaning their melting temperature is below room temperature. Therefore, IL-based formulations do not exhibit a well-ordered crystal structure, thus overcoming the problems associated with polymorphism in solid-state formulations.

[0080] In one embodiment, regarding the assessment of the biological effects of [Chol]2[N1(4)inh]+N1(4)inh, the results obtained revealed that reformulating the specific Nox1(4) inhibitor to [Chol]2[N1(4)inh]+N1(4)inh did not induce changes in the viability of the dopaminergic neuronal line N27 cells, indicating that there was no cytotoxicity to these cells. Figure 6). In terms of its dopaminergic neuroprotective capacity, pre-treatment of N27 cells with [Chol]2[Ni(4)inh] + Ni(4)inh significantly prevented the neurotoxic effects of 6OHDA, while pre-treatment with the cationic choline used to synthesize [Chol]2[Ni(4)inh] + Ni(4)inh did not promote this neuroprotection Figure 7 ). In addition, pre-treatment of N27 cells with [Chol]2[Ni(4)inh] + Ni(4)inh also significantly prevented the neurotoxic effects of MPP+toxin, while pre-treatment with choline chloride did not promote this neuroprotection Figure 8 As 6OHDA and MPP+induce their dopaminergic neuronal toxicity by inducing different intracellular pathomechanisms, these later results indicate that [Chol]2[Ni(4)inh] + Ni(4)inh is able to modulate two different pathomechanisms, as it can prevent neuronal death induced by two different toxins. These data are important as the goal is to use [Chol]2[Ni(4)inh] + Ni(4)inh as a therapy to stop the progression of the multifactorial disease from a pathomechanistic point of view.

[0081] In an embodiment, the dopaminergic neuroprotective capacity of [Chol]2[Ni(4)inh] + Ni(4)inh was assessed in a PD animal model induced by intrastriatal injection of 10 pg of 6OHDA. As shown in Figure 9 infusion of 0.2 mg / kg / day of [Chol]2[Ni(4)inh] + Ni(4)inh into the right ventricle for 7 days did not induce dopaminergic neuronal toxicity in the SN of mice. In addition, this dose of [Chol]2[Ni(4)inh] + Ni(4)inh was able to prevent 6OHDA-induced degeneration of dopaminergic neurons in the SN Figure 10

[0082] In an embodiment, the toxicity of [Chol]2[Ni(4)inh] + Ni(4)inh was assessed when administered via the intranasal route in mice. These administrations did not induce toxic effects in mice. As shown in Figure 11 and 12 Intranasal administration of different doses of the IL-based formulation every day during 14 days did not induce changes in the body weight of the animals Figure 11 nor in the motor performance Figure 12 In addition, as shown in Figure 13 the administration of a higher tested dose (0.16 mg / kg / day) every day did not induce olfactory dysfunction.

[0083] ​In an embodiment, the toxicity of [Chol]2[N1(4)inh] + N1(4)inh was evaluated in rats when administered via the intracerebroventricular route. No toxic effects were observed in rats exposed to 0.007 mg / kg / day. As shown in Figure 14 and 15 , no changes in animal body weight and motor performance, respectively, were observed.

[0084] In an embodiment, the toxicity of [Chol]2[N1(4)inh] + N1(4)inh was evaluated in rats when administered via the intranasal route. These administrations did not induce toxic effects in rats. As shown in Figure 16 and 17 , the daily intranasal administration of 0.062 mg / kg / day of the IL-based formulation did not induce changes in animal body weight Figure 16 , nor in motor performance Figure 17 , during the 30-day period. Moreover, as shown in Figure 18 , the daily administration of [Chol]2[N1(4)inh] + N1(4)inh did not induce olfactory dysfunction.

[0085] In an embodiment, the neuroprotective effect of [Chol]2[N1(4)inh] + N1(4)inh to prevent motor dysfunction in the context of PD was evaluated when administered via the intracerebroventricular route in a PD animal model induced by long-term exposure of rats to low doses (2.5 mg / kg / day) of paraquat (PQ). The infusion of 0.007 mg / kg / day of [Chol]2[N1(4)inh] + N1(4)inh into the right cerebral ventricle for 30 days was able to prevent the progression of motor dysfunction induced by PQ Figure 19 , 20 and 21).

[0086] In an embodiment, the neuroprotective effect of [Chol]2[N1(4)inh] + N1(4)inh to prevent motor dysfunction in the context of PD was evaluated when administered via the intranasal route in a PD animal model induced by long-term exposure of rats to low doses (2.5 mg / kg / day) of paraquat (PQ). The administration of 0.062 mg / kg / day of [Chol]2[N1(4)inh] + N1(4)inh for 30 days was able to prevent the progression of motor dysfunction induced by PQ Figure 22 ).

[0087] These results demonstrate well that Nox inhibitors formulated as novel IL-based formulations can be used as novel therapeutic approaches for PD or other neurological pathologies, and [Chol]2[N1(4)inh] + N1(4)inh can be used as novel therapeutic approaches for PD or other neurological pathologies in which these enzymes play an important pathological role. BRIEF DESCRIPTION OF DRAWINGS

[0088] The following drawings provide a further understanding of the preferred embodiments of the present disclosure and are not meant to limit the scope of the application.

[0089] Figure 1 shows the following 1 H NMR spectra: a) specific Noxl(4) inhibitor (N1(4)inh) and b) IL formulation of specific Noxl(4) inhibitor ([Chol]2[N1(4)inh] + N1(4)inh).

[0090] Figure 2 shows the following 13 C NMR spectra: a) specific Noxl(4) inhibitor (N1(4)inh) and b) IL formulation of specific Noxl(4) inhibitor ([Chol]2[N1(4)inh] + N1(4)inh).

[0091] Figure 3 Solubility of Noxl(4) inhibitor N1(4)inh in water and PBS vs. solubility of its IL-based formulation ([Chol]2[N1(4)inh] + N1(4)inh) in water and PBS (in mg / mL).

[0092] Figure 4 Solubility of Noxl(4) inhibitor N1(4)inh in water and PBS vs. solubility of its IL-based formulation ([Chol]2[N1(4)inh] + N1(4)inh) in water and PBS (in mol / L).

[0093] Figure 5 Decomposition temperature of [Chol]2[N1(4)inh] + N1(4)inh evaluated by thermogravimetric analysis (TGA).

[0094] Figure 6 Effect of [Chol]2[N1(4)inh] + N1(4)inh at two concentrations on N27 dopaminergic neuronal cell viability. (1) Untreated cells; (2) cells exposed to 20 mM [Chol]2[N1(4)inh] + N1(4)inh; (3) cells exposed to 30 mM [Chol]2[N1(4)inh] + N1(4)inh.

[0095] Figure 7 It is shown that pre-treatment of dopaminergic neuronal cells (N27) with [Chol]2[Ni(4)inh]+Ni(4)inh significantly prevents the neurotoxic effects of 6OHDA. (1) Untreated cells; (2) cells exposed to 20 mM of [Chol]2[Ni(4)inh]+Ni(4)inh; (3) cells exposed to 50 mM of 6OHDA; (4) cells exposed to 50 mM of 6OHDA and 20 mM of [Chol]2[Ni(4)inh]+Ni(4)inh; (5) cells exposed to 50 mM of 6OHDA and 20 mM of choline chloride.

[0096] Figure 8 It is shown that pre-treatment of dopaminergic neuronal cells (N27) with [Chol]2[Ni(4)inh]+Ni(4)inh significantly prevents the neurotoxic effects of the neurotoxin MPP+. (1) Untreated cells; (2) cells exposed to 20 mM of [Chol]2[Ni(4)inh]+Ni(4)inh; (3) cells exposed to 10 mM of MPP + +; (4) cells exposed to 10 mM of MPP + + and 20 mM of [Chol]2[Ni(4)inh]+Ni(4)inh; (5) cells exposed to 10 mM of MPP + + and 20 mM of choline chloride.

[0097] Figure 9 It is shown that intracerebroventricular infusion of [Chol]2[Ni(4)inh]+Ni(4)inh has an effect on the viability of dopaminergic neurons in the substantia nigra (SN) of mice. (1) Untreated mice; (2) mice exposed to 7 to 0.2 mg / kg / day of [Chol]2[Ni(4)inh]+Ni(4)inh (1 : 1).

[0098] Figure 10 It is shown that intracerebroventricular infusion of [Chol]2[Ni(4)inh]+Ni(4)inh prevents the death of dopaminergic neurons in the substantia nigra (SN) (animal model of Parkinson's disease) induced by intrastriatal injection of 6OHDA. (1) Untreated mice; (2) mice exposed to 10 mg of 6OHDA; (3) mice exposed to 10 mg of 6OHDA and 0.2 mg / kg / day of [Chol]2[Ni(4)inh]+Ni(4)inh (1 : 1).

[0099] Figure 11The effect of twice daily intranasal administration of 0.02, 0.04, 0.08, 0.16 mg / kg / day of [Chol]2[Ni(4)inh] + Ni(4)inh for 14 days on mouse body weight is shown.

[0100] Figure 12 The effect of twice daily intranasal administration of (1) vehicle, (2) 0.02, (3) 0.04, (4) 0.08, (5) 0.16 mg / kg / day of [Chol]2[Ni(4)inh] + Ni(4)inh for 14 days on mouse motor performance is shown.

[0101] Figure 13 The effect of twice daily intranasal administration of 0.16 mg / kg / day of [Chol]2[Ni(4)inh] + Ni(4)inh for 14 days on mouse olfactory function is shown. (1) vehicle (untreated group); (2) 0.16 mg / kg / day of [Chol]2[Ni(4)inh] + Ni(4)inh treated group.

[0102] Figure 14 The effect of once daily intracerebroventricular administration of 0.007 mg / kg / day of [Chol]2[Ni(4)inh] + Ni(4)inh for 30 days on rat body weight is shown.

[0103] Figure 15 The effect of once daily intracerebroventricular administration of 0.007 mg / kg / day of [Chol]2[Ni(4)inh] + Ni(4)inh for 30 days on rat motor performance is shown.

[0104] Figure 16 The effect of once daily intranasal administration of 0.062 mg / kg / day of [Chol]2[Ni(4)inh] + Ni(4)inh for 30 days on rat body weight is shown.

[0105] Figure 17 The effect of once daily intranasal administration of 0.062 mg / kg / day of [Chol]2[Ni(4)inh] + Ni(4)inh for 30 days on rat motor performance is shown. (1) vehicle (untreated group); (2) 0.062 mg / kg / day of [Chol]2[Ni(4)inh] + Ni(4)inh treated group.

[0106] Figure 18The effect of intranasal administration of 0.062 mg / kg / day of [Chol]2[Ni(4)inh] + Ni(4)inh once a day for 30 days on olfactory function in rats is shown. (1) Vehicle (non-treated group); (2) group treated with [Chol]2[Ni(4)inh] + Ni(4)inh at 0.062 mg / kg / day.

[0107] Figure 19 The prevention of the progression of motor dysfunction induced by PQ in rats (PD animal model) by intracerebroventricular administration of 0.007 mg / kg / day of [Chol]2[Ni(4)inh] + Ni(4)inh once a day for 30 days is shown. (1) Control group (saline only); (2) group treated with PQ; (3) group co-treated with PQ and [Chol]2[Ni(4)inh] + Ni(4)inh.

[0108] Figure 20 The prevention of the reduction of distance traveled induced by PQ in rats (PD animal model) by intracerebroventricular administration of 0.007 mg / kg / day of [Chol]2[Ni(4)inh] + Ni(4)inh once a day for 30 days is shown. (1) Control group (saline only); (2) group treated with PQ; (3) group co-treated with PQ and [Chol]2[Ni(4)inh] + Ni(4)inh.

[0109] Figure 21 The prevention of the reduction of animal speed induced by PQ in rats (PD animal model) by intracerebroventricular administration of 0.007 mg / kg / day of [Chol]2[Ni(4)inh] + Ni(4)inh once a day for 30 days is shown. (1) Control group (saline only); (2) group treated with PQ; (3) group co-treated with PQ and [Chol]2[Ni(4)inh] + Ni(4)inh.

[0110] Figure 22 The prevention of the progression of motor dysfunction induced by PQ in rats (PD animal model) by intranasal administration of 0.062 mg / kg / day of [Chol]2[Ni(4)inh] + Ni(4)inh once a day for 30 days is shown. (1) Control group (saline only); (2) group treated with PQ; (3) group co-treated with PQ and [Chol]2[Ni(4)inh] + Ni(4)inh.

[0111] Figure 23 The figure showing an overview of the present invention. DETAILED DESCRIPTION

[0112] The present disclosure relates to the development of IL-based formulations of specific inhibitors of NADPH oxidases (Nox), in particular Noxl and Nox4, for therapeutic applications in Parkinson's Disease (PD) or other neurological disorders. The strategy used consists of the conversion of the Noxl(4) specific inhibitor 3-cyclohexyl-5-(2,4-dihydroxybenzylidene)-l-methyl-2-thiohydantoin (N1(4)inh) into a formulation comprising 3-cyclohexyl-5-(2,4-dihydroxybenzylidene)-l-methyl-2-thiohydantoin choline and 3-cyclohexyl-5-(2,4-dihydroxybenzylidene)-l-methyl-2-thiohydantoin salt ([Chol]2[N1(4)inh] + N1(4)inh, 1 : 1).

[0113] The present disclosure relates to the conversion of Noxl(4) specific inhibitors into IL-based formulations [Chol]2[N1(4)inh] + N1(4)inh for use in the context of PD and other neurological disorders and comprises the following steps: (1) Conversion of the specific inhibitor N1(4)inh into an IL-based formulation, i.e. 3-cyclohexyl-5-(2,4-dihydroxybenzylidene)-l-methyl-2-thiohydantoin choline and 3-cyclohexyl-5-(2,4-dihydroxybenzylidene)-l-methyl-2-thiohydantoin salt ([Chol]2[N1(4)inh] + N1(4)inh, 1 : 1, mol:mol) which comprises the following steps: a) selection of the source of IL cations and selection of the source of anions; b) synthesis of [Chol]2[N1(4)inh] by metathesis reaction using choline salt as source of cations and 3-cyclohexyl-5-(2,4-dihydroxybenzylidene)-l-methyl-2-thiohydantoin (N1(4)inh) as source of anions; c) characterization of the purity of [Chol]2[N1(4)inh] + N1(4)inh and N1(4)inh by H NMR and C NMR; d) determination of the solubility of [Chol]2[N1(4)inh] + N1(4)inh in water and PBS; e) characterization of the thermal stability of [Chol]2[N1(4)inh] + N1(4)inh by thermal gravimetric analysis (TGA). (2) Evaluation of the biological effects of [Chol]2[N1(4)inh] + N1(4)inh in dopaminergic neurons in vitro by: a) evaluation of the cytotoxicity of [Chol]2[N1(4)inh] + N1(4)inh in immortalized rat dopaminergic neuronal cell line (N27); b) evaluation of the biological effects of the re-formulation in the context of therapy for PD by evaluating its dopaminergic neuroprotective capacity in a PD in vitro model. 1 H NMR and 13 C NMR) characterization of the purity of [Chol]2[N1(4)inh] + N1(4)inh and N1(4)inh; d) determination of the solubility of [Chol]2[N1(4)inh] + N1(4)inh in water and PBS; e) characterization of the thermal stability of [Chol]2[N1(4)inh] + N1(4)inh by thermal gravimetric analysis (TGA). (2) Evaluation of the biological effects of [Chol]2[N1(4)inh] + N1(4)inh in dopaminergic neurons in vitro by: a) evaluation of the cytotoxicity of [Chol]2[N1(4)inh] + N1(4)inh in immortalized rat dopaminergic neuronal cell line (N27); b) evaluation of the biological effects of the re-formulation in the context of therapy for PD by evaluating its dopaminergic neuroprotective capacity in a PD in vitro model.

[0114] The ratio of [Chol]2[N1(4)inh] + N1(4)inh mixture used in the examples of the present disclosure is 1 : 1 (mol:mol) ([Chol]2[N1(4)inh]:N1(4)inh), unless otherwise stated.

[0115] In the present disclosure, [Chol]2[N1(4)inh] + N1(4)inh can also be referred to as [Chol]2[N1(4)inh] + Nox1(4)inh or [Chol]2[N1(4)inh] + Nox1(4).

[0116] Converting the specific inhibitor N1(4)inh into an IL-based formulation:

[0117] In an embodiment, the synthesis of the IL-based formulation starts with the selection of the components (i.e., the cation source and the anion source).

[0118] In an embodiment, the cation source should belong to the class of choline salts, preferably choline bicarbonate.

[0119] In an embodiment, the anion source should belong to the group of 3-substituted 5- phenylmethylene-1-methyl-2-thiohydantoin-based compounds, preferably it should be 3- cyclohexyl-5-(2,4-dihydroxyphenylmethylene)-1-methyl-2-thiohydantoin (N1(4)inh).

[0120] In an embodiment, the IL-based formulation ([Chol]2[N1(4)inh] + N1(4)inh) is synthesized by a double decomposition reaction with a 1 : 1 molar ratio of choline bicarbonate (80% (m / v) in water) and N1(4)inh solution prepared in a minimum amount of absolute ethanol.

[0121] In an embodiment, N1(4)inh is mixed by dropwise addition to the choline bicarbonate solution under dark and cold conditions (using an ice bath of ~5°C) and under continuous stirring.

[0122] In an embodiment, the reaction mixture is further kept at ~5°C for 2 hours under continuous stirring.

[0123] In an embodiment, the excess solvent and water are removed under a continuous flow of nitrogen until complete drying (~2-3 hours of nitrogen flow, the vial containing the IL is kept in an ice-cold water bath (~10°C)).

[0124] In an embodiment, the mixture comprising the synthesized [Chol]2[N1(4)inh] and N1(4)inh is finally collected from the ice-cold water bath and stored under dry, cool, and dark conditions.

[0125] In an embodiment, the purity of [Chol]2[Ni(4)inh] and Ni(4)inh was evaluated by H NMR and C NMR as disclosed in Figures 1 and 2, respectively. 1 H NMR and 13 C NMR were performed.

[0126] In an embodiment, the obtained NMR spectra allowed confirming that [Chol]2[Ni(4)inh] + Ni(4)inh were successfully synthesized and pure.

[0127] In an embodiment, the solubility of [Chol]2[Ni(4)inh] + Ni(4)inh in water and phosphate buffered saline (PBS) solutions was determined.

[0128] In an embodiment, the solutes Ni(4)inh and the mixtures including [Chol]2[Ni(4)inh] + Ni(4)inh were added in excess to a fixed volume (500 pL) of water and PBS solutions. Using an Eppendorf Thermomixer Comfort device, these mixtures were incubated at 37°C under constant stirring at 1150 rpm for at least 72 hours. Throughout this process, solutes were added to the mixtures whenever necessary (i.e. before saturation of the solution). All samples were filtered using a syringe filter (0.45 mm) to remove possible suspended solid particles.

[0129] In an embodiment, the quantification of Ni(4)inh and [Chol]2[Ni(4)inh] was performed by UV spectroscopy (using UV spectrophotometry (SYNERGY | HT microplate reader, BioTek) at wavelengths of 416 nm and 480 nm, respectively). The interference of PBS on the quantification method was also determined and blank control samples were always used.

[0130] In an embodiment, the solubility of the mixture [Chol]2[Ni(4)inh] + Ni(4)inh in water was 0.405 ± 0.053 mg / mL, which is 36 higher than that of Ni(4)inh alone (0.011 ± 0.002 mg / mL). In PBS, the solubility of the IL-based formulation also increased significantly, by 21-fold (from 0.008 ± 0.001 mg / mL to 0.168 ± 0.002 mg / mL) Figure 3 ). Similar results were obtained for the increase in solubility provided in mol / L Figure 4 ).

[0131] These results clearly demonstrate that reformulating N1(4)inh as [Chol]2[N1(4)inh] + N1(4)inh allows to overcome one of the most important problems associated with Nox-specific inhibitors (i.e. low solubility), thus enhancing the potential of this new reformulation for use in the context of PD and other neurological disorders.

[0132] In an embodiment, the decomposition temperature of [Chol]2[N1(4)inh] + N1(4)inh was evaluated by thermal gravimetric analysis (TGA) and the results revealed that the mixture is thermally stable up to 200 °C, as Figure 5 revealed.

[0133] (2) Evaluation of the biological effects of [Chol]2[N1(4)inh] + N1(4)inh in dopaminergic neurons in vitro.

[0134] In an embodiment, the cytotoxicity of [Chol]2[N1(4)inh] + N1(4)inh was evaluated in an immortalized rat dopaminergic neuronal cell line (N27).

[0135] Cells were exposed to 20 and 30 mM of [Chol]2[N1(4)inh] + N1(4)inh, or to 20 mM of choline chloride. Choline chloride was dissolved in saline and [Chol]2[N1(4)inh] + N1(4)inh was dissolved in PBS 1x (Phosphate-Saline Buffer).

[0136] Cells were kept under stimulation for 24 hours and cell viability was evaluated using a CCK-8 kit (Cell Counting Kit-CCK-8; Dojindo Molecular Technologies).

[0137] [Chol]2[N1(4)inh] + N1(4)inh did not produce toxic effects on N27 dopaminergic cells when exposed to 20 or 30 mM of the mixture, as no statistically significant differences between cell viability values were observed in cultures exposed to [Chol]2[N1(4)inh] + N1(4)inh (20 mM ② or 30 mM ③, Figure 6 ), compared to untreated cells (CTR ①, Figure 6 ).

[0138] In an embodiment, the dopaminergic neuroprotective capacity of [Chol]2[N1(4)inh] + N1(4)inh was evaluated in a PD in vitro model.

[0139] The neuroprotection evaluation was performed using 20 μΜ [Chol]2[N1(4)inh] + N1(4)inh or 20 μΜ choline chloride to rule out the possibility that the neuroprotection effect was caused by the choline salt and not by the IL-based formulation.

[0140] The neurotoxins 6-hydroxydopamine (6OHDA) and 1 -methyl-4-phenylpyridinium (MPP+) were added alone at 2 hours and 30 minutes after [Chol]2[N1(4)inh] + N1(4)inh or choline chloride and the stimulation was maintained for 24 hours, after which the cell viability was evaluated using the CCK-8 kit.

[0141] Based on the results obtained previously using the non-reformulated inhibitor (N1(4)inh), the working concentration of [Chol]2[N1(4)inh] + N1(4)inh selected was 20 μΜ.

[0142] In an embodiment, when comparing the conditions of cells treated with 6OHDA, a 45% decrease in the viability of dopaminergic neurons can be observed, which is statistically different from the control condition (CTR1) Figure 7 ). The pre-treatment with [Chol]2[N1(4)inh] + N1(4)inh 2.5 hours before the exposure to 50 μΜ of 6OHDA prevented the toxin-induced neurotoxicity and when comparing the cell viability values between the conditions CTR and 6OHDA + [Chol]2[N1(4)inh] + N1(4)inh, there was no statistically significant difference, whereas there was a statistically significant difference between the latter and the 6OHDA alone condition Figure 7

[0143] In an embodiment, Figure 7 The results presented in Table 2 also show that pre-treatment with 20 μΜ choline chloride does not prevent the decrease in N27 viability induced by 6OHDA, which indicates that the protective effect of [Chol]2[N1(4)inh] + N1(4)inh described above is due to the specific efficacy of the Noxl inhibitor anion and not to the presence of choline in the formulation.

[0144] In an embodiment, [Chol]2[N1(4)inh] + N1(4)inh also exerts a neuroprotective effect in the prevention of MPP+ dopaminergic neurotoxicity, since it significantly prevents a 45% loss of N27 cell viability exposed to this neurotoxin Figure 8 ). As verified for 6OHDA, pre-treatment with choline chloride does not confer any protective effect against MPP+, which reinforces the fact that the protection observed in the presence of [Chol]2[N1(4)inh] + N1(4)inh comes from the action of the inhibitor and not of choline.​Figure 8 ).

[0145] The biological effects of [Chol]2[Ni(4)inh] + Ni(4)inh were then evaluated in vivo. All animal experiments were performed in accordance with the institutional animal house, national and European Community regulations (86 / 609 / ECC; 2010 / 63 / EU).

[0146] First, the toxicity of [Chol]2[Ni(4)inh] + Ni(4)inh (1 : 1) was evaluated in healthy mice using two different routes of administration: 1) 7-day intracerebroventricular infusion of 0.2 mg / kg / day of [Chol]2[Ni(4)inh] + Ni(4)inh; 2) twice daily intranasal administration of 0.16, 0.08, 0.04 or 0.02 mg / kg / day of [Chol]2[Ni(4)inh] + Ni(4)inh during a 14-day period.

[0147] In an embodiment, 8-12 weeks old male C57BL / 6 mice are housed in a temperature / humidity controlled environment under a 12 hours light / dark cycle with free access to water and food. 1) Mice are anesthetized by intraperitoneal (i.p.) injection of a saline containing ketamine (0.67 mL / kg of mouse weight) and xylazine (0.33 mL / kg of mouse weight) before placing them in a digital stereotaxic frame (51900, Stoelting). Once the skull of the mouse is exposed, a digital coordinate system is used to determine the infusion site with the zero set at the bregma point. Delivery of [Chol]2[N1(4)inh] + N1(4)inh (1 : 1) at a dose of 0.2 mg / kg / day is performed by its direct intracerebroventricular infusion over a 7-day period using an Alzet osmotic pump (reference number 1007D) connected to an Alzet catheter (reference brain infusion kit 3) with coordinates: mediolateral (ML): -1.1 mm; anteroposterior (AP): 0.5 mm and dorsoventral (DV): -2.5. On day 7, animals are anesthetized by intraperitoneal (i.p.) injection of a saline containing ketamine (0.67 mL / Kg of mouse weight) and xylazine (0.33 mL / Kg of mouse weight) and first heart-perfused with saline and then with buffered formaline (end of experiment). The brain is frozen in liquid nitrogen and kept at -80°C. Subsequently, the brain is incorporated in a gel of ideal cutting temperature (OCT) to be sectioned in a cryostat (Leica CM 3050S, Leica Microsystems). Coronal sections of 30 pm thickness from the anterior pole to the end of the midbrain are collected at -20°C. Sections corresponding to the ST and SN of each animal are collected and stored in succession in free-floating 24-well plate compartments (Orange Scientific) containing a cryopreservation solution of 30% glycerol (v / v) and 30% (v / v) ethylene glycol in phosphate buffer (PB) at -20°C. Plates are kept at -20°C, properly identified for later use in immunohistochemistry to assess the number of dopaminergic neurons in the substantia nigra (SN) by stereological counting of neurons immunopositive for the specific cellular marker tyrosine hydroxylase (TH). Figure 9The results presented show that the administration of [Chol]2[Ni(4)inh] + Ni(4)inh did not significantly reduce the number of dopaminergic (TH+) neurons in the substantia nigra (SN) after 7 days of intracerebroventricular infusion of 0.2 mg / kg / day of [Chol]2[Ni(4)inh] + Ni(4)inh. Non-toxic profile of the IL-based formulation was shown. 2) To assess the general putative toxicity of [Chol]2[Ni(4)inh] + Ni(4)inh when administered via the intranasal route in mice, general anesthesia was induced by inhalation of 3-4% (v / v) isoflurane for 4 min to enable twice daily intranasal administration of 4 different doses (0.16, 0.08, 0.04 or 0.02 mg / kg / day) of [Chol]2[Ni(4)inh] + Ni(4)inh to mice over a 14-day period. Consistently, polyurethane catheters (Introcan® USA) attached to 50 μL microsyringes (Hamilton® 24G; 0.7 x 19 mm) instilled with 6 pL of [Chol]2[N1(4)inh] + N1(4)inh per 30 g of mouse body weight, nose up. The catheter was inserted about 0.3 mm deep into one nostril, enabling delivery to the top of the nasal cavity. The animals were kept in the above-mentioned position until they woke up to prevent the IL-based formulation from entering the respiratory tract and staying longer in the nasal cavity. The day of the first administration was considered day 1. Motor coordination, balance, and grip strength were analyzed using a mouse rotarod apparatus, model 47600 (Ugo Basile, Comerio, Italy). All mice were pre-trained on the rotarod to learn and achieve consistent performance. Training was performed for 2 consecutive days before the first administration, with 4 test trials assessed each day for 5 minutes. Mice were trained at 12 RPM (fixed mode), 24 RPM (fixed mode) for 2 test trials, and 4-40 RPM (accelerating mode) on day 1. The animals were allowed to rest for approximately 30 minutes between each trial. Rotarod testing was performed for 5 minutes under the accelerating protocol (4-40 RPM) and drop latency was recorded using specific software. The trial stopped when the mouse fell (activating a switch that automatically stopped the timer) or after 5 minutes. Each animal performed four individual trials with an inter-trial period of approximately 30 minutes to reduce stress and fatigue. To investigate olfactory function, a food finding test (FFT) olfactory paradigm was performed. Before the test, mice were food-restricted for a period of 16 hours. On the test day, the animals were placed in a clean cage with only a filter top cap, with clean bedding evenly distributed throughout the cage (~4 cm), for habituation for 30-40 minutes (no water, no food). After habituation, the animals were returned to their home cage. Then, a food pellet was buried on one side of the cage, covered by the bedding. To start the test, the animals were placed on the opposite side of the buried pellet, the timer was started, and the top cap was placed. When the animal uncovered the pellet and started eating it, the timer stopped. In case the animal did not find the pellet within 5 minutes, the trial ended with a score of 5 minutes. After the trial, the animals were returned to their home cage. In summary, the toxicity of [Chol]2[N1(4)inh] + N1(4)inh administered via the intranasal route in mice was evaluated by analyzing animal body weight, behavioral motor performance, and the ability to find a food pellet. Consistently, mice were weighed every day for 14 days, performed the rotarod motor behavior assay on day 14, and the olfactory test on day 14. As shown in Figures 1-3, no changes in body weight, motor, and olfactory performance were observed in mice administered [Chol]2[N1(4)inh] + N1(4)inh via the intranasal route, which reinforces the non-toxic profile of its IL-based formulation. Figure 11 , 12 and 13, no changes in body weight, motor, and olfactory performance were observed in mice administered [Chol]2[N1(4)inh] + N1(4)inh via the intranasal route, which reinforces the non-toxic profile of its IL-based formulation.

[0148] Second, the toxicity of [Chol]2[N1(4)inh]+N1(4)inh was evaluated in healthy rats using two different routes of administration: 1) 30-day intracerebroventricular infusion of 0.007 mg / kg / day of [Chol]2[N1(4)inh]+N1(4)inh; and 2) daily intranasal administration of 0.062 mg / kg / day of [Chol]2[N1(4)inh]+N1(4)inh during 30 days.

[0149] In an embodiment, 8-12 weeks old male Wistar rats were housed in a temperature / humidity controlled environment under a 12-hour light / dark cycle with free access to water and food. 1) Before placing the rats in a digital stereotaxic frame (Stoelting, ref. 51900), rats were anesthetized by intraperitoneal (i.p.) injection of ketamine (90 mg / kg) and xylazine (10 mg / kg) in saline. Once the rat skull was exposed, the infusion site was determined using a digital coordinate system setting the zero point at the bregma. The delivery of [Chol]2[N1(4)inh]+N1(4)inh (1 : 1) at a dose of 0.007 mg / kg / day was performed by its direct intracerebroventricular infusion during 30 days using an Alzet osmotic pump (ref. 2004) connected to an Alzet catheter (reference brain infusion kit 2) with coordinates: mediolateral (ML): 1.5 mm; anteroposterior (AP): -1.0 mm and dorsoventral (DV): -4.0. Toxicity was assessed by analyzing the animal body weight at day 0 and day 30 and the behavioral motor performance at day 21. Motor coordination, balance and grip strength were analyzed using a rat rotarod apparatus model 47700 (Ugo Basile, Comerio, Italy). All rats were pre-trained on the rotarod to learn and achieve consistent performance. Training was performed for 2 consecutive days, with 4 test trials assessed each day, each lasting 5 minutes. Rats were first trained at 12 RPM (fixed mode), 24 RPM (fixed mode) for 2 test trials and 4-40 RPM (accelerated mode) for the other 2 test trials. Animals were allowed to rest approximately 30 minutes between each trial. Rotarod test was performed for 5 minutes under the accelerated protocol (4-40 RPM) and the fall latency was recorded using specific software. The trial stopped when the mouse fell (activating a switch that automatically stopped the timer) or after 5 minutes. Four individual trials were performed for each animal with approximately 30 minutes inter-trial interval to reduce stress and fatigue. Figure 14 and 15The results presented show that administration of [Chol]2[Ni(4)inh] + Ni(4)inh did not significantly cause changes in body weight and motor performance, which shows the non-toxic profile of the IL-based formulation. 2) To assess the general putative toxicity of [Chol]2[Ni(4)inh] + Ni(4)inh when administered via the intranasal route in rats, general anesthesia was induced by inhalation of 3-4% (v / v) isoflurane for 4 minutes to enable the intranasal administration of 0.062 mg / kg / day of [Chol]2[Ni(4)inh] + Ni(4)inh to rats once a day during a 14-day period. Consistently, rats were laid face-up with the nose elevated and a polyurethane catheter (Introcan® USA) attached to a 50 μL microsyringe (Terumo®, HT-125-50G) was used to administer the drug. The catheter was inserted into the nostril of the rat and the drug was administered by pushing the plunger of the microsyringe. The drug was administered in a volume of 50 μL of saline. The rats were observed for 5 minutes after the administration of the drug to ensure that they were not in distress. The rats were then returned to their home cage. 24G; 0.7 x 19 mm) instillation, where 40 pL of [Chol]2[N1(4)inh] + N1(4)inh per 260 g of rat body weight. The catheter was inserted about 1.7 mm deep into the left and right nostrils, enabling the delivery of the formulation to the roof of the nasal cavity. The animals were kept in the above-mentioned position until they woke up to prevent the IL-based formulation from entering the respiratory tract and staying in the nasal cavity for a longer period of time. Motor coordination, balance, and grip strength were analyzed using a rat rotarod apparatus, model 47700 (Ugo Basile, Comerio, Italy). All rats were pre-trained on the rotarod to learn and achieve consistent performance. Training was performed for 2 consecutive days, with 4 test trials assessed each day, each lasting 5 minutes. The rats were first trained at 12 RPM (fixed mode), 24 RPM (fixed mode) for 2 test trials, and 4-40 RPM (accelerating mode) for another 2 test trials. The animals were allowed to rest for approximately 30 minutes between each trial. The rotarod test was performed for 5 minutes under the accelerating protocol (4-40 RPM) and the fall latency was recorded using specific software. The trial stopped when the mouse fell (activating a switch that automatically stopped the timer) or after 5 minutes. Each animal performed four individual trials with an inter-trial period of approximately 30 minutes to reduce stress and fatigue. To investigate olfactory function, a food finding test (FFT) olfactory paradigm was performed. Prior to the test, the rats were restricted from food for a period of 16 hours. On the test day, the animals were placed in a clean cage with only a filter top cap, with clean bedding evenly distributed throughout the cage at ~4 cm, for habituation for 30-40 minutes (no water, no food). After habituation, the animals were returned to their home cage. Then, one food pellet was buried on one side of the cage, covered by the bedding. To start the test, the animals were placed on the opposite side of the buried pellet, the timer was started, and the top cap was placed. When the animal uncovered the pellet and started to eat it, the timer stopped. In the case where the animal did not find the pellet within 5 minutes, the trial ended with a score of 5 minutes. After the trial, the animals were returned to their home cage. Overall, toxicity was assessed by analyzing the body weight of the rats, their behavioral motor performance, and their ability to find a food pellet hidden in the cage. Consistently, the rats were weighed every day for 30 days, the rotarod motor behavior assay was performed on day 30, and the olfactory test was performed on day 30. As shown in FIGS. 1-3, no changes in body weight, motor, and olfactory performance were observed in rats administered [Chol]2[N1(4)inh] + N1(4)inh via the intranasal route, which enhanced the non-toxic profile of their IL-based formulation. Figure 16 , 17 and 18, no changes in body weight, motor, and olfactory performance were observed in rats administered [Chol]2[N1(4)inh] + N1(4)inh via the intranasal route, which enhanced the non-toxic profile of their IL-based formulation.

[0150] In an embodiment, the overall results obtained in relation to the in vivo toxicity of [Chol]2[Ni(4)inh]+Ni(4)inh demonstrate that the IL-based formulations are non-toxic when administered via intracerebroventricular or intranasal routes.

[0151] Subsequently, the neuroprotective ability of [Chol]2[Ni(4)inh]+Ni(4)inh was evaluated in two PD in vivo models, one induced by intracerebral injection of 6OHDA and the other induced by exposure to chronic low doses of paraquat (PQ).

[0152] In an embodiment, the biological neuroprotective effect of [Chol]2[Ni(4)inh]+Ni(4)inh+Ni(4)inh when administered via intracerebroventricular infusion route was evaluated in a PD mouse model induced by intracerebral injection of 6OHDA and the number of dopaminergic neurons in the substantia nigra was counted.

[0153] In an embodiment, male C57BL / 6 mice are anesthetized and placed in a digital stereotaxic frame. Once the mouse skull is exposed, the injection and infusion sites are determined using a digital coordinate system, setting the zero point at the bregma point. Immediately after, using the coordinates: Medial-Lateral (ML): -2 mm; Anterior-Posterior (AP): 0.6 mm; Dorsal-Ventral (DV): -3.0 mm, 6-OHDA (10 pg / 2 pL ascorbic acid 0.1% v / v) is injected in the right striatum (ST) of each animal at a rate of 0.2 microliter / minute using a Hamilton syringe. Delivery of [Chol]2[N1(4)inh] + N1(4)inh at 0.2 mg / kg / day is performed as described above. On day 7, animals are anesthetized by intraperitoneal (i.p.) injection of a saline solution containing ketamine (0.67 mL / kg mouse weight) and xylazine (0.33 mL / kg mouse weight) and first perfused with saline through the heart and then with buffered formaline through the heart (end of experiment). The brain is frozen with liquid nitrogen and kept at -80 °C. Subsequently, the brain is incorporated in a gel of optimal cutting temperature (OCT) for sectioning in a cryostat (Leica CM 3050S, Leica Microsystems). Coronal sections of 30 pm thickness from the anterior pole to the end of the midbrain are collected at -20 °C. Sections corresponding to the ST and SN of each animal are collected and sequentially stored in free-floating 24-well plate compartments (Orange Scientific) containing a cryopreservation solution of 30% (v / v) glycerol and 30% (v / v) ethylene glycol in phosphate buffer (PB) at -20 °C. Plates are kept at -20 °C, properly identified for later use in immunohistochemistry to assess the number of dopaminergic neurons in the SN by stereological counting of TH+ neurons. Two experimental groups are used: 1) mice injected with 6-OHDA in the right striatum and saline (0.9% m / v NaCl) in the left striatum, and 2) mice injected with 6-OHDA in the right striatum, saline in the left striatum and [Chol]2[N1(4)inh] + N1(4)inh in the lateral ventricle. The saline-injected hemisphere is considered a control for 6OHDA in group 1 and for [Chol]2[N1(4)inh] + N1(4)inh in group 2. In group 2, the intracerebroventricular and stereotaxic procedures are performed on the same day. As Figure 10 As depicted, exposure to 6-OHDA induced a statistically significant decrease of 45% in the number of SN dopaminergic neurons compared to the control group Figure 10; 2) [Chol]2[N1(4)inh] + N1(4)inh. As for the dopaminergic neuroprotective effect of [Chol]2[N1(4)inh] + N1(4)inh, the results prove that it has neuroprotective capacity, since the presence of the IL-based formulation prevented 37% of the SN dopaminergic neurodegeneration induced by 6-OHDA in the mouse SN Figure 10 ; 3) [Chol]2[N1(4)inh] + N1(4)inh.

[0154] In one embodiment, the biological neuroprotective effect of [Chol]2[N1(4)inh] + N1(4)inh when administered via the intracerebroventricular route was evaluated in a rat model of PD induced by exposure to low chronic doses of PQ by analyzing the progression of behavioral dysfunction. In this model, the animals are exposed to the toxin during 30 days (4 weeks) and then kept alive during 30 more days to allow the progression of the condition (8 weeks in total). The administration of [Chol]2[N1(4)inh] + N1(4)inh via the intracerebroventricular or intranasal route started 30 days after the first exposure to PQ and lasted for a second 30 days. The animals were euthanized at week 8.

[0155] In an embodiment, 8-12 week old male Wistar rats are housed in a temperature / humidity controlled environment under a 12 hour light / dark cycle with free access to water and food and PQ is chronically administered subcutaneously at a dose of 2.5 mg / kg / day and a fluid delivery rate of 0.25 microliter / hour using osmotic minipumps (Alzet Durect, Cupertino, CA) for a period of four weeks (Alzet model 2004, large pump). Control groups are implanted with minipumps filled with sterile saline (the vehicle used to dissolve PQ). Pumps are implanted subcutaneously in the dorsal side of the scapula (shoulder blade) posteriorly, for which the rats are anesthetized by intraperitoneal (i.p.) injection of ketamine (90 mg / kg) and xylazine (10 mg / kg). Four weeks after exposure to PQ, intracerebroventricular infusion of [Chol]2[N1(4)inh] + N1(4)inh is initiated and continued for more than 4 weeks. The experimental paradigm involves 8 weeks in total, with the first 4 weeks for PQ exposure and the last 4 weeks for IL-based formulation administration. For this, rats are anesthetized and placed in a digital stereotaxic frame (51900, Stoelting). Once the rat skull is exposed, the infusion site is determined using a digital coordinate system with the zero point set at the bregma. Delivery of [Chol]2[N1(4)inh] + N1(4)inh (1 : 1) at a dose of 0.007 mg / kg / day is performed by its direct intracerebroventricular infusion during more than four weeks (30 days) using an Alzet osmotic pump (reference number 2004) connected to an Alzet catheter (reference brain infusion kit 2) with coordinates: mediolateral (ML): 1.5 mm; anteroposterior (AP): -1.0 mm and dorsoventral (DV): -4.0. To assess the neuroprotective function of IL-based formulations when infused in the ventricles, behavioral functions are assessed. Motor coordination, balance and grip strength are analyzed using a rat rotarod apparatus (Ugo Basile, Como, Italy) model number 47700. All rats are pre-trained on the rotarod to learn and achieve consistent performance. Training is performed for 2 consecutive days with 4 test trials per day, each lasting 5 minutes. Rats are first trained at 12 RPM (fixed pattern), 24 RPM (fixed pattern) for 2 test trials and 4-40 RPM (accelerating pattern) for another 2 test trials. Animals are allowed to rest for approximately 30 minutes between each trial. Rotarod testing is performed for 5 minutes under an accelerating protocol (4-40 RPM) and fall latency is recorded using specific software. The trial stops when the mouse falls (activating a switch that automatically stops the timer) or after 5 minutes. Each animal performs four separate trials with an inter-trial period of approximately 30 minutes to reduce stress and fatigue. An open field test is used to measure exploratory behavior and general activity.Animals were transported to the test room and left undisturbed for 30 min to 1 h before testing. The arena was surrounded by a break beam system that was broken by the animal movements inside the arena. This information was processed in a specific software allowing the analysis of several parameters such as distance, speed, rear and entrance to the center. Each animal was placed in the center of the rectangular arena and allowed to freely explore for 10 min. A timer was started at the exact same time as the animal was placed in the arena. The operator had left the room. Between each animal, the arena was wiped with 70% (v / v) ethanol and the next animal was placed. All behavioral tests were performed at week 8. The presented results show that intracerebroventricular administration of [Chol]2[N1(4)inh] + N1(4)inh significantly prevented the motor dysfunction induced by PQ ( Figure 19 ) as well as the reduction of the distance traveled ( Figure 20 ) and the speed ( Figure 21 ). These effects highlight the ability of IL-based formulations to reduce the progression of motor dysfunction in the context of PD.

[0156] As depicted in Figure 19 , exposure to PQ induced a statistically significant reduction of 43% of the fall latency of the animals exposed to PQ compared to the control group ( Figure 19 ; 2 vs. 1). In terms of neuroprotective effect of [Chol]2[N1(4)inh] + N1(4)inh, the results demonstrate its ability to prevent the progression of motor dysfunction as the fall latency of the animals exposed to PQ increased by 37% compared to the animals exposed to PQ only ( Figure 19 ; 3 vs. 2).

[0157] In terms of the results of the distance traveled and the speed of the rats respectively depicted in Figure 20 and 21 , PQ induced a 55% reduction of the distance traveled ( Figure 20 ; 2 vs. 1) and a 50% reduction of the speed ( Figure 21 : 2 vs. 1). In terms of neuroprotective effect of [Chol]2[N1(4)inh] + N1(4)inh, the results demonstrate its ability to prevent the progression of motor dysfunction as the distance traveled of the animals exposed to PQ increased by 50% compared to the animals exposed to PQ only ( Figure 20 ; 3 vs. 2) and the speed increased by 37% ( Figure 21 ; 3 vs. 2).

[0158] In an embodiment, the bio-neuroprotective effects of [Chol]2[N1(4)inh] + N1(4)inh when administered via the intranasal route are evaluated in a rat model of PD induced by exposure to low chronic doses of PQ by analyzing the progression of behavioral dysfunction. In this model, animals are exposed to the toxin during 30 days (4 weeks) and then kept alive during 30 more days to allow the progression of the condition (8 weeks in total). The administration of [Chol]2[N1(4)inh] + N1(4)inh via the intracerebroventricular or intranasal route starts after the first 30 days of exposure to PQ and continues for the second 30 days. Animals are euthanized at week 8.

[0159] In an embodiment, 8-12 week old male Wistar rats are used for PQ chronic subcutaneous administration using osmotic minipumps (Alzet Durect, Cupertino, CA) at a dose of 2.5 mg / kg / day and a fluid delivery rate of 0.25 microliters / hour for a period of four weeks (Alzet model 2004, large pump). Control groups are implanted with minipumps filled with sterile saline (the vehicle used to dissolve PQ). Pumps are implanted subcutaneously in the dorsal side of the scapular (shoulder blade) hump under anesthesia induced by ketamine (90 mg / kg) and xylazine (10 mg / kg). Four weeks after exposure to PQ, intranasal delivery of [Chol]2[N1(4)inh] + N1(4)inh is initiated and administration is performed daily for more than 4 weeks. The experimental paradigm involves 8 weeks in total, the first 4 weeks being used for PQ exposure and the last 4 weeks for IL-based formulation administration. General anesthesia is induced by inhalation of 3-4% (v / v) isoflurane for 4 minutes to enable the intranasal administration of 0.062 mg / kg / day of [Chol]2[N1(4)inh] + N1(4)inh to rats once daily during the additional 4 weeks (30 days). Consistently, rats are laid face up and the nose is elevated and a polyurethane catheter (Introcan USA) is used to deliver the drug. The catheter is attached to a 50 μL microliter syringe (Becton Dickinson, Franklin Lakes, NJ) and the drug is delivered by gently pressing the syringe plunger. The syringe is then disconnected from the catheter and the catheter is removed from the nose. The rats are then placed in a cage with a heating lamp to recover from anesthesia. 24G; 0.7 x 19 mm) instillation, where 40 pL of [Chol]2[Ni(4)inh] + Ni(4)inh per 260 g of rat body weight. The catheter was inserted about 1.7 mm deep into the left and right nostrils, enabling the delivery of the formulation to the roof of the nasal cavity. The animals were kept in the above-mentioned position until they woke up, to prevent the IL-based formulation from entering the respiratory tract and staying in the nasal cavity for a longer period of time. The following experimental groups were used: 1) rats exposed to saline (control group); 2) rats exposed to PQ; and 3) rats exposed to PQ and [Chol]2[Ni(4)inh] + Ni(4)inh. To assess the neuroprotective function of the IL-based formulation when administered via the intranasal route, the behavioral function was evaluated. Motor coordination, balance, and grip strength were analyzed using a rat rotarod apparatus, model 47700 (Ugo Basile, Comerio, Italy). All rats were pre-trained on the rotarod to learn and achieve consistent performance. Training was performed for 2 consecutive days, with 4 test trials assessed each day, each lasting 5 minutes. The rats were first trained at 12 RPM (fixed mode), 24 RPM (fixed mode) for 2 test trials, and 4-40 RPM (accelerated mode) for another 2 test trials. The animals were allowed to rest for approximately 30 minutes between each trial. The rotarod test was performed for 5 minutes under the accelerated protocol (4-40 RPM) and the fall latency was recorded using specific software. The trial stopped when the mouse fell (activating a switch that automatically stopped the timer) or after 5 minutes. Each animal performed four individual trials with an inter-trial period of approximately 30 minutes to reduce stress and fatigue. The behavioral test was performed at week 8. The results showed that intranasal administration of [Chol]2[Ni(4)inh] + Ni(4)inh significantly prevented motor dysfunction Figure 22 ) in the context of PD.

[0160] As Figure 22 depicted, exposure to PQ induced a statistically significant reduction of 47% in the fall latency of the animals exposed to PQ compared to the control group Figure 22 ; 2) compared to 1). As for the neuroprotective effect of [Chol]2[Ni(4)inh] + Ni(4)inh, the results demonstrate its ability to prevent the progression of motor dysfunction, as the fall latency of the animals exposed to PQ increased by 40% compared to the animals exposed to PQ only Figure 22 ; 3) compared to 2).

[0161] In an embodiment, these results demonstrate that [Chol]2[Ni(4)inh] + Ni(4)inh, unlike its insoluble precursor, is non-toxic, however, exerts the same neuroprotective efficacy as Ni(4)inh alone in the context of PD.

[0162] In one embodiment, these results demonstrate that [Chol]2[N1(4)inh]+N1(4)inh reduces the progression of motor dysfunction in the context of PD.

[0163] These results clearly demonstrate that the present disclosure allows the conversion of toxic molecules into non-toxic mixtures. The repackaging does not interfere with the effectiveness of the precursors, thus radically changing their potential applicability as a differentiated therapeutic approach, which allows to reduce / stop the progression of the pathologies associated with PD and thus reduce the rate of increase in disability over time.

[0164] The present disclosure allows the currently available symptomatic therapies to act more consistently over longer periods of time, greatly improving the quality of life of patients and their families and reducing the negative socio-economic impact of the disease.

[0165] Example: In the central nervous system (CNS), oxidative stress is one of the main factors that contribute to the development of diseases and aging. Recent studies have revealed that several isoforms of NADPH oxidases (Nox), whose only function is to produce reactive oxygen species (ROS), are also present in the CNS. There, they play a key role in modulating ROS-dependent cellular mechanisms, but also, at high levels, cause cell dysfunction and death, thus accelerating the aging process characteristic of the pathological processes of neurodegenerative diseases.

[0166] Previous studies have demonstrated the importance of Nox-mediated oxidative stress in inducing neuronal loss in various neurological conditions, such as PD, amyotrophic lateral sclerosis (ALS) and stroke. These studies have reinforced the importance of developing specific inhibitors of these enzymes, as well as the objective of several research laboratories and pharmaceutical companies, such as GenKyoTex, a Swiss company dedicated to the development of Nox inhibitors for future therapeutic applications. For example, the synthesis of an inhibitor that specifically inhibits Nox isoforms 1 and 4 has recently been reported (Ba et al. Synthesis and biological evaluation of 3-substituted 5-phenylmethylene-1-methyl-2-thiohydantoins as potent NADPH oxidase (NOX) inhibitors. Bioorganic & Medicinal Chemistry 2016; 24: 4144-4151), but it is only soluble in solvents containing Triton-X / ethanol / PBS, which was found to be the vehicle that induces toxicity in dopaminergic neurons. The present disclosure combines ionic liquid technology and inhibition of Nox to develop a new therapeutic strategy that targets specific harmful ROS production involved in the neurodegenerative pathological process. Below are three examples of the applicability of the present invention:

[0167] Example 1 : Parkinson's disease (PD)

[0168] In an embodiment, PD is a chronic neurodegenerative disorder that affects 6.1 million people and whose efficacy tends to decrease as the disease progresses to more debilitating stages, reducing the treatment options. According to the reports on PD in 2018 (Parkinson's Disease Market: Channel Review, Developer Landscape, and Competitive Insights, 2018, pp. Report ID: 4586296. Global Parkinson's Disease Market and Competitive Landscape, 2020, pp. Report ID: 5023386), the lack of preventive therapies that stop the progression of PD remains one of the main unmet needs in the field. In view of this, the paradigm is shifting towards the research / development of progression-preventing therapies for said disease. In fact, of the drugs being developed by pharmaceutical companies, 53% are disease-modifying agents aimed at preventing the progression of PD and only 32% are symptomatic. Oxidative stress has a great influence on the pathogenesis of PD. The production of Noxl-ROS plays a crucial role in the process of dopaminergic cell death and in the a-synucleinopathy that occurs in this disease and its inhibition in animal models prevents the progression of the disease, as described in the literature (Christov et al., Role of NADPH Oxidase 1-Derived Reactive Oxygen Species in Paraquat-Mediated Dopaminergic Cell Death. Antioxidants & Redox Signaling 2009; 11 : 9: 2105-18; Cui et al., NADPH Oxidase 1-Mediated Oxidative Stress Causes Dopaminergic Neuron Death in Parkinson's Disease. Antioxidants & Redox Signaling 2012; 16(10): 1033-45; Christov et al., NADPH Oxidase 1 Mediates a-Synucleinopathy in Parkinson's Disease. Journal of Neuroscience 2012; 32(42): 14465-77). Furthermore, as demonstrated in the literature "The Role of NOX4 in Parkinson's Disease with Dementia" (Cui et al., International Journal of Molecular Sciences 2019; 20(3): 696), which shows that the increased expression of Nox4 in the hippocampal dentate gyrus in the context of PD induces the expression of Ab and the production of oligomers A11 and, as a result, reduces cognitive function, Nox4 plays a crucial role in PD dementia. These documents confirm that Noxl and Nox4 are important targets for the development of new disease treatment methods. In this context, the present disclosure is beneficial for its possible application to the repurposing of Noxl-Nox4 specific inhibitors, which are currently available, but have poor solubility and low pharmacological suitability.Increasing their solubility, making them more bioavailable, and thus having a higher therapeutic efficacy on PD.

[0169] Example 2: Stroke

[0170] In one embodiment, neurovascular diseases are a major cause of death worldwide. In terms of costs, only stroke amounts to a total of 64.1 billion euros (Europe) and 44.2 billion euros (United States of America) per year. The occurrence of stroke is due to an inhibition of cerebral blood flow caused by ischemia or hemorrhage. While approximately 20% of patients die within the first month after stroke, survivors often present severe neurological dysfunction and chronic disability, which represents a huge socio-economic burden. Current therapies only affect a small proportion of patients and can lead to serious side effects.

[0171] The preventive effect of the administration of anticoagulant drugs such as aspirin is limited, while the therapeutic window of thrombolytic therapy such as recombinant tissue plasminogen activator (rtPA) is narrow and can induce cerebral hemorrhage, edema and ischemic cell death, as described by Suzuki et al. (Novel situations of endothelial injury in stroke--mechanisms of stroke and strategy of drug development: intracranial bleeding associated with the treatment of ischemic stroke: thrombolytic treatment of ischemia-affected endothelial cells with tissue-type plasminogen activator. Journal of pharmacological sciences 2011, 116, 25-29). The beneficial effect of surgical procedures such as angioplasty is not well established, which underlines the importance and the necessity to develop more efficient and safe therapies to treat these patients.

[0172] The document "Oxidative stress and pathophysiology of ischemic stroke: novel therapeutic opportunities" (CNS & neurological disorders drug targets 2013, 12, 698-714) indicates that an increase in the levels of oxidative stress is associated with the brain damage that occurs after ischemic stroke. In addition, the document "Biochemistry, physiology, and pathophysiology of NADPH oxidases in the cardiovascular system" (Circulation research 2012, 110(10), 1364-1390) reveals the different isoforms of Nox, i.e. involving isoforms 1, 2, 4 and 5. In stroke, Nox-derived ROS can have a protective or deleterious function, depending on the isoform proposed to be involved (Kleikers et al. NADPH oxidases as a source of oxidative stress and molecular target in ischemia / reperfusion injury. Journal of molecular medicine 2012, 90(12), 1391-1406; Gray et al. Reactive Oxygen Species Can Provide Atheroprotection via NOX4-Dependent Inhibition of Inflammation and Vascular Remodeling. Arteriosclerosis, thrombosis, and vascular biology 2016 36(2), 295-307). Taking this into account, and given the specificity of the isoform involved in the pathologies associated with stroke, the development of antioxidant therapies for this disease by inhibiting Nox presents a great therapeutic potential for the management of this pathology.Thus, the present disclosure has another potential application.

[0173] Example 3: Amyotrophic Lateral Sclerosis (ALS)

[0174] Amyotrophic Lateral Sclerosis (ALS) is characterized by progressive degeneration of motor neurons and subsequent activation of glial cells, leading to the development of muscle weakness and disability, and ultimately to fatal respiratory and cardiac defects 3 to 5 years after diagnosis. Despite intensive research so far, only one drug has been approved for the treatment of ALS (riluzole) and its impact on survival is modest (de Jongh AD et al. Evidence for a multimodal effect of riluzole in patients with ALS? Journal of Neurology, Neurosurgery & Psychiatry 2019; 90: 1183-1184). The pathological mechanisms underlying this pathology are largely unknown; however, the role of oxidative stress again comes into play as a factor promoting disease progression and Nox involvement in this process is also documented (Harraz et al. SOD1 mutations disrupt redox-sensitive Rac regulation of NADPH oxidase in a familial ALS model. The Journal of clinical investigation 2008, 118(2), 659-670).

[0175] Previous studies investigating the role of Nox in ALS patients found that patients with lower activity of isoform 2 in peripheral blood had a significantly increased survival time (Marrali et al. NADPH oxidase (NOX2) activity is a modifier of survival in ALS. Journal of neurology 2014, 261(11), 2178-2183). Marden et al. (Redox modifier genes in ALS in mice. Journal of clinical investigation 2007, 117(10), 2913-2919) also observed the same in mice with ALS. However, the knock-out of Nox isoforms 1 and 2 showed an increased survival rate and delayed the onset of disease progression. On the other hand, the treatment with the broad inhibitor Apo in an in vitro model of ALS improved the survival of motor neurons when co-cultured with astrocytes carrying mutations associated with disease development (Marrali et al. NADPH oxidase (NOX2) activity is a modifier of survival in ALS. Journal of neurology 2014, 261(11), 2178-2183), thus further reinforcing the idea that the inhibition of Nox can have a neuroprotective effect in ALS. The pharmacological inhibition of Nox also showed benefits in animal models of ALS. It increased survival time by almost 50% and increased the number of motor neurons in the spinal cord (Hara et al. SOD1 mutation disrupts redox-sensitive Rac regulation of NADPH oxidase in a familial ALS model. Journal of clinical investigation 2008, 118(2), 659-670). In this case, the potential application of the present disclosure becomes even stronger, since the repurposing of Nox inhibitors can increase their effectiveness and expand the neuroprotective effect of Apo that has been described so far.

[0176] Table 1 depicts the samples used in the assays of the present disclosure.

[0177] Table 1 - Sample identification

[0178]

[0179] In an embodiment, for the synthesis of [Chol]2[Ni(4)inh], choline salt was used as the cation source and 3-cyclohexyl-5-(2,4-dihydroxybenzylidene)-l-methyl-2- thiohydantoin (Ni(4)inh) was used as the anion source. [Chol]2[Ni(4)inh] was synthesized by a metathesis reaction with a 1 : 1.05 molar ratio of choline bicarbonate (80% in water, m / v) and a solution of Ni(4)inh prepared in a minimum amount of absolute ethanol. Ni(4)inh was mixed by dropwise addition to the choline bicarbonate solution under dark and cold conditions (using an ice bath of ~5°C) and under continuous stirring. The reaction mixture was further kept at ~5°C for 2 hours under continuous stirring. The excess solvent and water were removed under a continuous flow of nitrogen until complete drying (~2-3 hours of nitrogen flow, vial containing IL kept in an ice-cold water bath (~10°C)). The synthesized [Chol]2[Ni(4)inh] and Ni(4)inh mixture (1 : 1, mol:mol) was finally collected from the ice-cold water bath and stored under dry, cool, and dark conditions.

[0180] In an embodiment, Figure 2 shows the following 1 H NMR spectra: 2a) specific Noxl(4) inhibitor (Ni(4)inh) and 2b) IL-based formulation of specific Noxl(4) inhibitor ([Chol]2[Ni(4)inh] + Ni(4)inh).

[0181] The purity of [Chol]2[Ni(4)inh] + Ni(4)inh was evaluated by 1 H NMR. Figure 1a reveals the H NMR spectrum of specific Noxl(4) inhibitor (Ni(4)inh) and the identification of the corresponding peaks, while Figure 1b reveals the H NMR spectrum of IL-based formulation of specific Noxl(4) inhibitor ([Chol]2[Ni(4)inh] + Ni(4)inh) and the identification of the corresponding peaks, which reveal that [Chol]2[Ni(4)inh] was successfully synthesized. 1 H NMR spectrum and the identification of the corresponding peaks, while Figure 1b reveals the H NMR spectrum of IL-based formulation of specific Noxl(4) inhibitor ([Chol]2[Ni(4)inh] + Ni(4)inh) and the identification of the corresponding peaks, which reveal that [Chol]2[Ni(4)inh] was successfully synthesized. 1 H NMR spectrum and the identification of the corresponding peaks, which reveal that [Chol]2[Ni(4)inh] was successfully synthesized.

[0182] In an embodiment, Figure 2 shows the following 13 C NMR spectra: 3a) specific Noxl(4) inhibitor (Ni(4)inh) and 3b) IL-based formulation of specific Noxl(4) inhibitor ([Chol]2[Ni(4)inh] + Ni(4)inh).

[0183] The purity of [Chol]2[Ni(4)inh] + Ni(4)inh and Ni(4)inh was evaluated by 13C NMR was performed to evaluate. Figure 2a reveals the C NMR spectrum and the identification of the corresponding peaks of the specific Noxl(4) inhibitor (N1(4)inh) 13 C NMR spectrum and the identification of the corresponding peaks, while Figure 2b reveals the C NMR spectrum and the identification of the corresponding peaks of the IL-based formulation of the specific Noxl(4) inhibitor ([Chol]2[N1(4)inh] + N1(4)inh) 13 C NMR spectrum and the identification of the corresponding peaks, which reveals that [Chol]2[N1(4)inh] + N1(4)inh was successfully synthesized.

[0184] In an embodiment, Figure 3 The solubility of the Noxl(4) inhibitor (N1(4)inh) and the IL-based formulation ([Chol]2[N1(4)inh] + N1(4)inh) in water and PBS is shown in mg / mL.

[0185] In an embodiment, the solute (N1(4)inh and [Chol]2[N1(4)inh] + N1(4)inh) was added in excess to a fixed volume (500 μί) of water and PBS solution. These mixtures were incubated at 37 °C under constant stirring at 1150 rpm using an Eppendorf comfort thermomixer device for at least 72 h. Throughout this process, the solute was added to the mixture whenever necessary (i.e. before reaching the saturation of the solution). All samples were filtered using a syringe filter (0.45 μιη) to remove possible suspended solid particles. The quantification of N1(4)inh and [Chol]2[N1(4)inh] + N1(4)inh was performed by UV spectroscopy (UV spectrophotometry at 416 nm and 480 nm, respectively, using a SYNERGY | HT microplate reader, Biotek). The interference of PBS on the quantification method was also determined and a blank control sample was always used. The results obtained revealed that the IL-based formulation increased the solubility in water by 36-fold and in PBS by 21-fold.

[0186] In an embodiment, Figure 4 The solubility of the Noxl(4) inhibitor (N1(4)inh) and the IL-based formulation ([Chol]2[N1(4)inh] + N1(4)inh) in water and PBS is shown in mg / mL.

[0187] In an embodiment, solutes (N1(4)inh and [Chol]2[N1(4)inh] + N1(4)inh) were added in excess to a fixed volume (500 pL) of water and PBS solutions. These mixtures were incubated at 37 °C under constant stirring at 1150 rpm for at least 72 h using an Eppendorf comfort temperature mixer device. Throughout this process, solutes were added to the mixtures whenever necessary (i.e. before reaching solution saturation). All samples were filtered using a syringe filter (0.45 pm) to remove possible suspended solid particles. Quantification of N1(4)inh and [Chol]2[N1(4)inh] + N1(4)inh was performed by UV spectroscopy (UV spectrophotometry at 416 nm and 480 nm wavelengths, respectively, using a SYNERGY | HT microplate reader, Biotek). Interference of PBS to the quantification method was also determined and blank control samples were always used. The results obtained revealed that the solubility of IL-based formulations in water increased 28-fold, while in PBS it increased 16-fold.

[0188] In an embodiment, Figure 5 The decomposition temperature of [Chol]2[N1(4)inh] + N1(4)inh is shown to be evaluated by thermal gravimetric analysis (TGA).

[0189] In an embodiment, the decomposition temperature was determined by thermal gravimetric analysis (TGA). The TGA curve reveals a sharp decrease in the degradation temperature at about 200 °C, indicating that [Chol]2[N1(4)inh] + N1(4)inh starts to decompose at 200 °C and also as shown for the precursor.

[0190] In an embodiment, Figure 6 The effect of [Chol]2[N1(4)inh] + N1(4)inh on N27 dopaminergic neuronal cell viability is shown.

[0191] In an embodiment, the potential toxicity of [Chol]2[Ni(4)inh] + Ni(4)inh on N27 dopaminergic neurons was assessed after 24 hours of exposure to 20 or 30 mM of [Chol]2[Ni(4)inh] + Ni(4)inh. No statistically significant differences were observed between treated and control cells (CTR / untreated cells), indicating that the IL-based formulation of [Chol]2[Ni(4)inh] + Ni(4)inh was not toxic to dopaminergic neurons. Data are expressed in percentage relative to CTR and shown as the mean ± SEM of at least five replicates of three independent experiments (n=3). Statistical analysis was performed using one-way ANOVA (non-parametric analysis) followed by the Kruskal-Wallis test followed by Dunn's multiple comparison test. 1 Control cells; 2 cells exposed to 20 mM of [Chol]2[Ni(4)inh] + Ni(4)inh; 3 cells exposed to 30 mM of [Chol]2[Ni(4)inh] + Ni(4)inh.

[0192] In an embodiment, Figure 7It is shown that pre-treatment of dopaminergic neuronal cells (N27) with [Chol]2[N1(4)inh] + N1(4)inh significantly prevented the neurotoxic effects of 6OHDA. 1 Control - untreated cells. 2 Choline control. 3 6OHDA significantly reduced the viability of dopaminergic neurons compared to control cells 1. 4 Pre-treatment with [Chol]2[N1(4)inh] + N1(4)inh significantly reduced 6OHDA-induced dopaminergic neurotoxicity compared to 3 cells treated with 6OHDA. 5 Choline chloride did not prevent 6OHDA-induced neurotoxicity compared to 3 cells treated with 6OHDA. Cell viability was measured using WST-8 assay in N27 cells pre-treated with 20 mM of [Chol]2[N1(4)inh] + N1(4)inh or choline chloride for 2.5 hours and then exposed to 50 mM of 6OHDA for 24 hours. Data are expressed as percentage relative to control and are presented as mean ± SEM of at least five replicates of three independent experiments (n = 3). Statistical analysis was performed using one-way ANOVA (non-parametric analysis) followed by Kruskal-Wallis test followed by Dunn's multiple comparison test. ** p < 0.01 compared to CTR or cells exposed to choline chloride only, +++ p < 0.001 compared to 6OHDA. (B). 1 Control cells; 2 cells exposed to choline chloride (20 mM); 3 cells exposed to 6OHDA (50 mM); 4 cells exposed to 6OHDA and [Chol]2[N1(4)inh] + N1(4)inh (20 mM); 5 cells exposed to 6OHDA (50 mM) and choline chloride (20 mM).

[0193] In an embodiment, Figure 8It is shown that pre-treatment of dopaminergic neuronal cells (N27) with [Chol]2[N1(4)inh] + N1(4)inh significantly prevented the neurotoxic effects of the neurotoxin MPP. 1 Control - untreated cells. 2 Choline chloride control. 3 MPP+ significantly reduced the viability of dopaminergic neurons. 4 Pre-treatment with [Chol]2[N1(4)inh] + N1(4)inh significantly reduced MPP+-induced dopaminergic neurotoxicity. 5 Choline chloride did not prevent MPP+-induced neurotoxicity. Cell viability was measured using the WST-8 assay in N27 cells pre-treated with 20 mM of [Chol]2[N1(4)inh] + N1(4)inh or choline chloride for 2.5 hours and then exposed to 10 mM MPP+ for 24 hours. Data are expressed as percentage relative to control as the mean of at least five replicates of three independent experiments (n=3) ± SEM. Statistical analysis was performed using one-way ANOVA (non-parametric analysis) followed by the Kruskal-Wallis test and then the Dunn's multiple comparison test. ** p<0.01 compared to CTR (untreated cells); +++ p<0.001 compared to cells treated with MPP+; *** p<0.001 compared to CTR or cells exposed only to choline chloride. 1 Control cells; 2 Cells exposed to choline chloride (20 mM); 3 Cells exposed to MPP+ (10 mM); 4 Cells exposed to MPP+ (10 mM) and [Chol]2[N1(4)inh] + N1(4)inh (20 mM); 5 Cells exposed to MPP+ (10 mM) and choline chloride (20 mM).

[0194] In an embodiment, Figure 9 It is shown that [Chol]2[N1(4)inh] + N1(4)inh intracerebroventricular infusion had an effect on the viability of dopaminergic neurons in the substantia nigra (SN) of mice.

[0195] In an embodiment, the number of tyrosine hydroxylase (TH)-immunoreactive neurons in the mouse SN was altered 7 days after intracerebroventricular infusion of 0.2 mg / kg / day of [Chol]2[N1(4)inh] + N1(4)inh. 1 Control group (saline only); 2 Group treated with [Chol]2[N1(4)inh] + N1(4)inh. There was no statistical difference in the number of TH-positive neurons in the SN of mice treated with [Chol]2[N1(4)inh] + N1(4)inh compared to the number of TH-positive neurons quantified in the SN of the control group (2 vs. 1), indicating that the IL-based formulation of [Chol]2[N1(4)inh] + N1(4)inh was not toxic to dopaminergic neurons in vivo. Results are expressed as a percentage relative to saline (group 1, animals exposed to saline only) and are represented as the mean ± SEM of at least 3 independent experiments (n = 3-4). Statistical analysis was performed using one-way ANOVA followed by Bonferroni test for multiple comparison analysis. No statistical difference was found between the two groups.

[0196] In an embodiment, Figure 10 It is shown that intracerebroventricular infusion of [Chol]2[N1(4)inh] + N1(4)inh prevents the death of dopaminergic neurons in the substantia nigra (SN) (animal model of Parkinson’s disease) induced by intrastriatal injection of 6OHDA. The number of tyrosine hydroxylase (TH)-immunoreactive neurons in the mouse SN was altered 7 days after treatment with 6OHDA in the presence or absence of [Chol]2[N1(4)inh] + N1(4)inh. 1 Control group (saline only); 2 Group treated with 6-OHDA; 3 Group co-treated with 6OHDA and [Chol]2[N1(4)inh] + N1(4)inh. The number of TH-positive neurons was significantly reduced in animals treated with 6OHDA (2 vs. 1). This reduction was significantly prevented in mice co-treated with 6OHDA and [Chol]2[N1(4)inh] + N1(4)inh (3 vs. 2). The number of TH-positive neurons in the SN of mice co-treated with 6OHDA and [Chol]2[N1(4)inh] + N1(4)inh was not statistically different from the number observed in the control group (3 vs. 1). Results are expressed as a percentage relative to saline (group 1, animals exposed to saline only) and are represented as the mean ± SEM of at least 3 independent experiments (n = 3-4). Statistical analysis was performed using one-way ANOVA followed by Bonferroni test for multiple comparison analysis. ** p < 0.01 compared to the control animals group (2 vs. 1); ## p < 0.01 compared to the animals group exposed to 6OHDA only (3 vs. 2).

[0197] In one embodiment, Figure 11 The effect of 14 days of [Chol]2[N1(4)inh] + N1(4)inh intranasal administration on mouse body weight is shown. Body weight change over time was induced by twice daily intranasal administration of four different doses of [Chol]2[N1(4)inh] + N1(4)inh (i.e. 0.02, 0.04, 0.08, 0.16 mg / kg / day) for 14 days. Results are expressed as the mean of at least 5 independent experiments (n=5-6). Statistical analysis was performed using two-way ANOVA followed by Tukey test for multiple comparison analysis. No statistical differences were found between groups.

[0198] Figure 12 The effect of 14 days of [Chol]2[N1(4)inh] + N1(4)inh intranasal administration on mouse motor performance is shown. Motor performance change was induced by twice daily intranasal administration of four different doses of [Chol]2[N1(4)inh] + N1(4)inh (i.e. ① vehicle, ② 0.02, ③ 0.04, ④ 0.08, ⑤ 0.16 mg / kg / day) for 14 days. Results are expressed as the mean ± SEM of at least 5 independent experiments (n=5-6). Statistical analysis was performed using one-way ANOVA followed by Bonferroni test for multiple comparison analysis. No statistical differences were found between groups.

[0199] Figure 13 The effect of 14 days of [Chol]2[N1(4)inh] + N1(4)inh intranasal administration on mouse olfactory function is shown. Olfactory function change was induced by twice daily intranasal administration of 0.16 mg / kg / day of [Chol]2[N1(4)inh] + N1(4)inh for 14 days. ① vehicle (non-treated group); ② 0.016 mg / kg / day of [Chol]2[N1(4)inh] + N1(4)inh treated group. Results are expressed as the mean ± SEM of at least 5 independent experiments (n=5-6). Statistical analysis was performed using unpaired Student's T test. No statistical differences were found between groups.

[0200] Figure 14 The effect of 30 days of [Chol]2[N1(4)inh] + N1(4)inh intracerebroventricular administration on rat body weight is shown. Body weight change over time was induced by once daily intracerebroventricular infusion of 0.007 mg / kg / day of [Chol]2[N1(4)inh] + N1(4)inh for 30 days. Results are expressed as the mean of at least 4 independent experiments (n=4-5). Statistical analysis was performed using one-way ANOVA followed by Bonferroni test for multiple comparison analysis. No statistical differences were found between groups.

[0201] Figure 15 The effect of 30 days of [Chol]2[N1(4)inh] + N1(4)inh intranasal administration on rat motor performance is shown. Changes in motor performance were induced by 0.062 mg / kg / day of [Chol]2[N1(4)inh] + N1(4)inh intranasally administered once daily for 30 days. 1 Vehicle (non-treated group); 2 0.062 mg / kg / day of [Chol]2[N1(4)inh] + N1(4)inh treated group. Results are expressed as the mean of at least 4 independent experiments (n=4-5). Statistical analysis was performed using a two-way ANOVA followed by a Tukey test for multiple comparison analysis. No statistical differences were found between groups.

[0202] Figure 16 The effect of 30 days of [Chol]2[N1(4)inh] + N1(4)inh intranasal administration on rat body weight is shown. Changes in body weight were induced by 0.062 mg / kg / day of [Chol]2[N1(4)inh] + N1(4)inh intranasally administered once daily for 30 days. Results are expressed as the mean of at least 4 independent experiments (n=4-5). Statistical analysis was performed using a two-way ANOVA followed by a Tukey test for multiple comparison analysis. No statistical differences were found between groups.

[0203] Figure 17 The effect of 30 days of [Chol]2[N1(4)inh] + N1(4)inh intranasal administration on rat motor performance is shown. Changes in motor performance were induced by 0.062 mg / kg / day of [Chol]2[N1(4)inh] + N1(4)inh intranasally administered once daily for 30 days. 1 Vehicle (non-treated group); 2 0.062 mg / kg / day of [Chol]2[N1(4)inh] + N1(4)inh treated group. Results are expressed as the mean of at least 4 independent experiments (n=4-5). Statistical analysis was performed using a two-way ANOVA followed by a Tukey test for multiple comparison analysis. No statistical differences were found between groups.

[0204] Figure 18 The effect of 30 days of [Chol]2[N1(4)inh] + N1(4)inh intranasal administration on rat olfactory function is shown. Changes in olfactory function were induced by 0.062 mg / kg / day of [Chol]2[N1(4)inh] + N1(4)inh intranasally administered once daily for 30 days. 1 Vehicle (non-treated group); 2 0.062 mg / kg / day of [Chol]2[N1(4)inh] + N1(4)inh treated group. Results are expressed as the mean ± SEM of at least 4 independent experiments (n=5-6). Statistical analysis was performed using a two-way ANOVA followed by a Tukey test for multiple comparison analysis. No statistical differences were found between groups.

[0205] Figure 19 Intracerebroventricular infusion of [Chol]2[N1(4)inh] + N1(4)inh is shown to prevent the progression of motor dysfunction induced by PQ in rats (model of PD). Changes in the fall latency of animals exposed to PQ for 30 days were recorded using the rotarod test for an additional 30 days in the presence or absence of 0.007 mg / kg / day [Chol]2[N1(4)inh] + N1(4)inh. 1 control group (saline only); 2 group treated with PQ; 3 group co-treated with PQ and [Chol]2[N1(4)inh] + N1(4)inh. In animals treated with PQ, the fall latency was significantly reduced (2 vs. 1). This reduction was significantly prevented in rats co-treated with PQ and [Chol]2[N1(4)inh] + N1(4)inh (3 vs. 2). The fall latency of rats co-treated with PQ and [Chol]2[N1(4)inh] + N1(4)inh was not statistically different from the fall latency observed in the control group (3 vs. 1). Results are expressed as the mean ± SEM of at least 4 independent experiments (n = 4-6). Statistical analysis was performed using one-way ANOVA followed by Bonferroni's test for multiple comparison analysis. *** p < 0.001 compared to the control group of animals (2 vs. 1); ### p < 0.001 compared to the group of animals exposed to PQ only (3 vs. 2).

[0206] Figure 20Intracerebroventricular infusion of [Chol]2[N1(4)inh] + N1(4)inh is shown to prevent the progression of motor dysfunction induced by PQ in rats (PD animal model). Changes in velocity were recorded in animals exposed to PQ for 30 days using the open field test in the presence or absence of 0.007 mg / kg / day [Chol]2[N1(4)inh] + N1(4)inh for an additional 30 days. 1 Control group (saline only); 2 PQ-treated group; 3 co-treated with PQ and [Chol]2[N1(4)inh] + N1(4)inh. Velocity was significantly reduced in animals treated with PQ (2 vs. 1). This reduction was significantly prevented in rats co-treated with PQ and [Chol]2[N1(4)inh] + N1(4)inh (3 vs. 2). Velocity in rats co-treated with PQ and [Chol]2[N1(4)inh] + N1(4)inh was not statistically different from the velocity observed in the control group (3 vs. 1). Results are expressed as mean ± SEM of at least 4 independent experiments (n = 4-6). Statistical analysis was performed using one-way ANOVA followed by Bonferroni's test for multiple comparison analysis. ** p < 0.01 compared to control animals group (2 vs. 1); # p < 0.05 compared to animals exposed to PQ only (3 vs. 2).

[0207] Figure 21 Intracerebroventricular infusion of [Chol]2[N1(4)inh] + N1(4)inh is shown to prevent the progression of motor dysfunction induced by PQ in rats (PD animal model). Changes in velocity were recorded in animals exposed to PQ for 30 days using the open field test in the presence or absence of 0.007 mg / kg / day [Chol]2[N1(4)inh] + N1(4)inh for an additional 30 days. 1 Control group (saline only); 2 PQ-treated group; 3 co-treated with PQ and [Chol]2[N1(4)inh] + N1(4)inh. Velocity was significantly reduced in animals treated with PQ (2 vs. 1). This reduction was significantly prevented in rats co-treated with PQ and [Chol]2[N1(4)inh] + N1(4)inh (3 vs. 2). Velocity in rats co-treated with PQ and [Chol]2[N1(4)inh] + N1(4)inh was not statistically different from the velocity observed in the control group (3 vs. 1). Results are expressed as mean ± SEM of at least 4 independent experiments (n = 4-6). Statistical analysis was performed using one-way ANOVA followed by Bonferroni's test for multiple comparison analysis. ** p < 0.01 compared to control animals group (2 vs. 1); # p < 0.05 compared to animals exposed to PQ only (3 vs. 2).

[0208] Figure 22 Intranasal administration of [Chol]2[Ni(4)inh] + Ni(4)inh was shown to prevent the progression of motor dysfunction induced by PQ in rats (PD animal model). Changes in the fall latency of animals exposed to PQ for 30 days were recorded using the rotarod test for an additional 30 days in the presence or absence of 0.062 mg / kg / day [Chol]2[Ni(4)inh] + Ni(4)inh. 1 control group (saline only); 2 PQ-treated group; 3 co-treated with PQ and [Chol]2[Ni(4)inh] + Ni(4)inh. In animals treated with PQ, the fall latency was significantly reduced (2 vs. 1). This reduction was significantly prevented in rats co-treated with PQ and [Chol]2[Ni(4)inh] + Ni(4)inh (3 vs. 2). The fall latency of rats co-treated with PQ and [Chol]2[Ni(4)inh] + Ni(4)inh was not statistically different from the fall latency observed in the control group (3 vs. 1). Results are expressed as the mean ± SEM of at least 4 independent experiments (n = 4-6). Statistical analysis was performed using one-way ANOVA followed by Bonferroni's test for multiple comparison analysis. ** p < 0.01 compared to the control animals group (2 vs. 1); # p < 0.05 compared to the group of animals exposed to PQ only (3 vs. 2).

[0209] The term "comprising", used in this document whenever it is used, is intended to mean that the features, integers, steps, components that follow the term are present, but not to the exclusion of one or more other features, integers, steps, components or groups thereof that are also described herein.

[0210] If a singular form of an element or feature is used in the description of claims, plural forms are also contemplated unless otherwise indicated. For example, the terms "an ion liquid" or "the ion liquid" also encompass the plural form "ion liquids" or "the ion liquids," and vice versa. In claims, the articles "a," "an" and "the" can mean one or more, unless indicated otherwise or otherwise apparent from the context. Thus, for example, reference to "a member of a group" or "the member of a group" can mean one, more than one, or all of the members of the group unless indicated otherwise or otherwise apparent from the context. The disclosure includes embodiments in which exactly one member of the group is present in, used in, or otherwise associated with a given product or process. The disclosure also includes embodiments in which more than one, or all, of the members of the group are present in, used in, or otherwise associated with a given product or process.

[0211] Furthermore, where a composition is recited in a claim, it is understood that, unless indicated to the contrary in the specification or unless otherwise apparent from context, methods involving use of the composition for any of the purposes disclosed herein are included, and methods of making the composition according to any of the preparation methods disclosed herein or other methods known in the art are included.

[0212] The present disclosure should not be considered limited to the described embodiments and the person of ordinary skill in the art will foresee many possibilities of modification thereof.

[0213] The above embodiments are combinable.

[0214] The following claims further particularize the present disclosure.

Claims

1. An ionic liquid comprising anion having the following formula: Where R is cyclohexyl; And cations, wherein the cation is choline.

2. The ionic liquid according to claim 1, wherein the molar ratio of the anion to the cation is in the range of 1:2 to 2:1 (mol:mol).

3. The ionic liquid according to claim 1, wherein the molar ratio of the anion to the cation is 1:1 (mol:mol).

4. The ionic liquid according to claim 1, comprising 3-cyclohexyl-5-(2,4-dihydroxybenzylmethyl)-1-methyl-2-thiohydantoin.

5. Use of the ionic liquid according to claim 1 in the preparation of a medicament for the prevention or treatment of Parkinson's disease.

6. Use of the ionic liquid according to claim 1 in the preparation of a medicament for slowing the progression of Parkinson's disease.

7. Use of the ionic liquid according to claim 1 in the preparation of a medicament for the prevention of motor dysfunction in Parkinson's disease.

8. Use of the ionic liquid according to claim 1 in the preparation of a medicament for the prevention or treatment of amyotrophic lateral sclerosis (ALS).

9. Use of the ionic liquid according to claim 1 in the preparation of a medicament for the prevention or treatment of stroke.

10. A pharmaceutical composition comprising a therapeutically effective amount of the ionic liquid according to claim 1 and a pharmaceutically acceptable carrier.

11. The pharmaceutical composition of claim 10, wherein the pharmaceutically acceptable carrier is phosphate-buffered saline (PBS) or water or a mixture thereof.

12. The pharmaceutical composition according to claim 10, wherein the amount of the ionic liquid included therein is 0.005 mM to 10 mM.

13. The pharmaceutical composition of claim 10, wherein the composition is in an injectable, intranasal, intrathecal, or intraventricular form.

14. Use of the pharmaceutical composition according to claim 10 in the preparation of a medicament for administration of a daily dose to a person suffering from Parkinson's disease, amyotrophic lateral sclerosis, or stroke.

15. The use according to claim 14, wherein the dose is less than 1000 mg / day.

16. The use according to claim 15, wherein the dosage ranges from 0.05 to 1000 mg / day.

Images