Ionic liquid-based formulations for the prevention or treatment of neurological disorders

Abstract

The present disclosure relates to ionic liquid (IL)-based formulations comprising inhibitors of NADPH oxidase enzyme (Nox), preferably isoforms 1 and 4, in particular the specific inhibitor 3-cyclohexyl-5-(2,4-dihydroxybenzylidene)-1-methyl-2-thiohydantoin, for the treatment, management, or prevention of neurological disorders, in particular Parkinson's disease.

Description

[Technical field]

[0001] The present disclosure relates to ionic liquid (IL) formulations (ionic liquid formulations) comprising inhibitors of the NADPH oxidase enzyme (Nox), preferably isoforms 1 and 4, in particular the specific inhibitor 3-cyclohexyl-5-(2,4-dihydroxybenzylidene)-1-methyl-2-thiohydantoin, for the treatment, management, or prevention of neurological disorders, in particular Parkinson's disease (PD). [Background technology]

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

[0003] According to a study by Dorsey et al. entitled "The Emerging Evidence of the Parkinson Pandemic" (Journal of Parkinson's Disease 2018; 8: S3-S8), among neurological disorders, Parkinson's disease (PD) is the fastest growing disease, with the number of patients doubling between 1990 and 2015 to more than 6 million. The study also reveals that due to the aging of the population, the number of patients will reach more than 12 million by 2040. Moreover, as the same authors emphasize, taking into account other factors (e.g., increased life expectancy, decreased smoking rates, and increasing industrialization), the burden associated with PD could exceed 17 million by 2040. The costs associated with PD are therefore substantial.

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

[0005] Currently, there is no cure for PD, and the available treatments are symptomatic, allowing only to manage symptoms. The current scenario of PD therapy is mainly based on the prescription of drugs that act as dopamine precursors, dopamine agonists or drugs with the aim of inhibiting key enzymes in the catabolic pathway of dopamine. However, the effectiveness of such treatments decreases over time as the disease progresses to more debilitating stages, making limited therapeutic options available to patients. Thus, prevention of disease progression is increasingly considered to be a promising solution for PD patients. However, the lack of preventive treatments to stop the progression of PD is one of the most important unmet needs in this area, as highlighted by a market study conducted by Research and Markets ("The Parkinson's Disease Market: Pipeline Review, Developer Landscape and Competitive Insights", pp. Report ID: 4586296). Therefore, the development of preventive treatments against PD progression is essential.

[0006] In the central nervous system (CNS), oxidative stress is associated with several diseases and aging, is a strong contributor to Parkinson's disease, and may therefore represent a suitable target to halt the progression of neurological diseases.

[0007] Until recently, mitochondria were thought to be the main source of ROS in the CNS, but recent studies have revealed that homologs of NADPH oxidase (Nox) enzymes are also located in the CNS and that they 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 are responsible for leading to cell death associated with the pathological processes of several neurological diseases, such as Parkinson's disease and amyotrophic lateral sclerosis (ALS). In this field, previous studies have shown that Nox1-mediated oxidative stress plays a crucial role in the degeneration of dopaminergic neurons in PD and have validated the role of Nox (nitric oxide) 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 studies further demonstrated that it was possible to reduce the negative effects of oxidative stress produced by some isoforms of Nox in neurons by using apocynin (Apo), a nonspecific inhibitor of Nox.However, Apo is only soluble in dimethyl sulfoxide (DMSO), and as a result, it can induce direct or indirect neurotoxicity, as previously shown 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). Some antioxidant molecules and other compounds with the ability to inhibit Nox share a common problem of insolubility, which leads to poor bioavailability and thus affects their therapeutic efficacy and application. This problem is also common to other active pharmaceutical ingredients (APIs) that are generally used in solid state. Solid-state APIs suffer from recurrent problems of polymorphism and poor aqueous solubility, which severely impair their bioavailability and therapeutic efficiency, and are the main reason for the failure of drug candidate molecules in phase 2 of clinical trials 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). Furthermore, their poor performance in crossing the blood-brain barrier (BBB) ​​is also a major issue, especially in the context of CNS-directed drugs. For this reason, the intranasal delivery route is becoming a route of intense interest for the delivery of pharmaceuticals to the brain. However, this cannot be achieved with traditional solid formulations of the majority of drugs. Therefore, to avoid concerns regarding BBB penetration that are particularly relevant for neurodegenerative diseases like PD, new formulations need to be developed that facilitate intranasal delivery while preventing issues related to the polymorphism and poor solubility of these drugs.

[0008] ILs are salts consisting of organic cations and organic or inorganic anions. Due to the large size of the ions, these salts do not have a regular crystalline structure and are liquid at lower temperatures than normal salts. The majority of known ILs are liquid at room and body temperatures, which contributes to overcoming the polymorphic scenarios of most drugs and improving the solubility of APIs, since there is no need to overcome the energy associated with the enthalpy of fusion. In addition, since they are salts consisting of ions, strong interactions with water are established, leading to a significant increase in their solubility. The development of ILs containing APIs for pharmacological applications has already been suggested and is emerging as a new era for 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 studies have successfully reported the conversion of various types of APIs, including antibiotics, analgesics, and nonsteroidal anti-inflammatory drugs (NSAIDS), into ILs (Pedro et al. Ionic Liquids in Drug Delivery. Encyclopedia 2021; 1: 324-339).

[0010] Conversion of an analgesic (lidocaine) and anti-inflammatory drug (NSAID) into a dual-functional IL allowed for an increase in water solubility up to 470-fold compared to the analgesic and anti-inflammatory precursors without affecting the 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). Similarly, the conversion of different NSAIDs into ILs, namely ibuprofen, ketoprofen, and (S)-naproxen, allowed increasing their bioavailability by improving their aqueous solubility by 100-fold (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] Besides these APIs, phenolic antioxidants have already been successfully converted into ILs. In the study "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, namely gallate, caffelate, vanillate, syringate, and ellagate, were combined with the cholinium cation. The resulting salts were significantly more water-soluble (about three orders of magnitude higher) than the corresponding phenolic acids. Moreover, they generally showed higher antioxidant and anti-inflammatory activity than their precursors, as well as comparable cytotoxicity and lower ecotoxicity profiles.

[0012] Based on these promising results, as a proof of concept of the present invention, a non-specific inhibitor of Nox's, apocynin (Apo), was reformulated into cholinium apocynate ([Chol][Apo]), as disclosed in the study 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 showed a 50-fold increase in aqueous solubility, overcoming the poor solubility of Apo. The neuroprotective potential of the new formulation in the context of PD was further confirmed by a significant reduction in 6OHDA-induced toxicity in dopaminergic neurons in a PD model. These promising results highlight the potential use of new IL-based pharmaceutical formulations in neurodegenerative diseases and PD. However, Apo is a broad-spectrum inhibitor of NADPH oxidase enzymes that can inhibit 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). It is mostly used as an NADPH oxidase inhibitor for research purposes because it does not specifically inhibit the 1 isoform (Bedard & Krause. The NOX family of ROS-generating NADPH oxidases: physiology and pathophysiology. Physiological Reviews 2007; 87: 245-313). This lack of target specificity is a strong limitation for its use as a therapeutic approach.Because inhibition of multiple Nox isoforms in different cell types may result in undesired and uncontrollable biological effects, highlighting the need to develop specific Nox inhibitors that exhibit high bioavailability and efficacy. Considering the potential therapeutic role of targeting Nox for several pathologies, the synthesis of specific inhibitors against these enzymes has attracted great attention. A previous study (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 the formulation of several 3-substituted 5-benzylidene-1-methyl-2-thiohydantoin-based compounds to inhibit Nox1(4). 3-Cyclohexyl-5-(2,4-dihydroxybenzylidene)-1-methyl-2-thiohydantoin (N1(4)inh) was shown to be a potent inhibitor of Nox1 and a partial inhibitor of Nox4 through lucigenin-based chemiluminescence assays in Drosophila.

[0013] The findings previously generated with [Chol][Apo] helped to build an undeniable proof of concept for the application of ILs in the context of PD and made it possible to extrapolate the approach to specific inhibitors of Nox.

[0014] The Nox1(4) specific inhibitor 3-cyclohexyl-5-(2,4-dihydroxybenzylidene)-1-methyl-2-thiohydantoin (N1(4)inh) has been 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) and showed high neuroprotective effects in experimental models of PD (Cristovao et al. 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 However, N1(4)inh is only soluble in solvents consisting of buffer, ethanol and Triton-X and has cytotoxic effects on dopaminergic neurons by itself, precluding the use of this highly promising inhibitor as a future therapeutic strategy in the context of neurodegenerative diseases.

[0015] The identification of the problems described in the state of the art, as well as the approaches taken to address them, well describe the problem that the present invention seeks to solve.

[0016] Document KR20080051246(A) "Pharmaceutical composition for prevention of treatment of neurodegenerative disease and inhibition of NADPH OXIDASE" refers to NADPH oxidase inhibitors, including fluoxetine or norfluoxetine, which are useful for the prevention and treatment of neurodegenerative diseases and can prevent the death of dopaminergic neurons through microglial reduction toxicity. Although the present invention is about the same target enzyme as the present invention, the inhibitor of the present invention shows a different formulation, since it is an ionic liquid based on 3-substituted 5-benzylidene-1-methyl-2-thiohydantoin.

[0017] The following series of documents aim to prevent or treat neurological diseases through modulation / inhibition of various pathological cellular mechanisms, including prevention of oxidative stress, by using chemical molecules or genes, but neither the pharmaceutical preparations nor the molecular targets are the same as those of the present invention. For example, US2020289671(A1)-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;WO2018129421(A1)‐A Promising Drug Candidate For Parkinson's Disease;US2013109714(A1)‐Neurodegenerative Disease Therapeutic Agent.

[0018] The document CN111138376(A)-3,5-Disubstituted Phenyl-1,2,4-Oxadiazole Derivative, And Preparation Method And Application Thereof discloses 3,5-disubstituted phenyl-1,2,4-oxadiazole derivatives, as well as their preparation methods and applications.

[0019] The documents: WO2020205937(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 comprehending NADPH oxidases inhibitors contain different formulations and applications. In these cases, the above documents aim to inhibit NADPH oxidases with different APIs such as hyaluronic acid or iodonium analogs used in cancer treatment. The documents do not mention the use of our molecules, even in neurodegenerative diseases.

[0020] The following documents describe various synthesis methods of various NADPH oxidase inhibitors, using either chemical or non-chemical methods, aiming to target a wide range of pathologies such as metabolic and neurological disorders. Nevertheless, these inhibitors differ from our [Chol][N1(4)inh] in both chemistry and formulation methods. Despite targeting the NADPH oxidase enzyme, they diverge in terms of inhibitors, use different Nox, and have diverse applications and formulations when compared to the present disclosure: 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] Reference SG10201808940W(A) - NOX Inhibitor And NFКB 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. For this purpose, a specific methoxyflavone is used.

[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 when compared to our disclosure and is not targeted to NOX's; and WO2010078258(A1) - Compounds Comprising Two Or More Biologically Functional Ions And Method Of Treating Parkinson's Disease, which comprises an active compound totally different and with completely different therapeutic target from the one used in our disclosure. The APIs used in the ILs of document WO2010078258(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 (oxolinate), and (2S,5R,6R)-6-((R)-2-amino-2-phenylacetamido)-3,3-dimethyl-7-oxo-4-thia-l-azabicyclo[3.2.0]heptane-2-carboxylic acid (ampicillinate); whereas ours is a 3-substituted 5-benzylidene-1-methyl-2-thiohydantoin. Moreover, our ILs and formulations specifically target Nox1 and Nox4 isoforms.

[0023] Known solutions in the state of the art present a technical problem to be solved by the present disclosure.

[0024] These facts are disclosed to explain the technical problem addressed by the present disclosure. Summary of the Invention

[0025] The present disclosure relates to the development of new IL-based formulations of certain NOX inhibitors. Their negligible solubility in aqueous solvents is a problem shared by several compounds that can inhibit Nox, and this low solubility negatively impacts their bioavailability, reducing their efficacy and therapeutic potential. The present disclosure is intended to develop non-toxic alternative formulations that allow improved solubilization of Nox inhibitors in aqueous solutions, thus improving their bioavailability, efficacy and therapeutic potential. One of the objectives of the present disclosure is to increase the solubility and consequent bioavailability and efficacy of certain Nox inhibitors for application in neurological diseases.

[0026] Considering the shortcomings of the prior art, the technical problem underlying the present invention was to develop an IL-based formulation of N1(4)inh for use in the prevention, treatment to slow disease progression or therapy of neurological diseases, in particular Parkinson's disease (PD).

[0027] Because Nox is an ideal target for specific antioxidant therapeutic strategies, the present disclosure is useful for the development of new therapeutic approaches aimed at reducing / halting the progression of neurodegenerative diseases over time, including PD.

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

[0029] [ka]

[0030] In one embodiment, IL formulations of the inhibitors showed increased solubility, neurotoxicity, and increased neuroprotective potential in the context of PD compared to the non-reformulated inhibitors (precursors). Thus, the disclosed compounds and formulations overcome the aqueous solubility issues of Nox inhibitors, thereby increasing their potential as neuroprotective therapies to slow PD progression and representing a related advance in the field of specific antioxidant therapy.

[0031] One embodiment of the present disclosure relates to specific inhibitors of Nox in ILs and formulations and their therapeutic application in neurological diseases, namely PD.

[0032] In one embodiment, the inhibitors disclosed herein are specific for NADPH oxidase enzyme isoforms 1 and 4. Thus, the present disclosure relates to new pharmaceutical preparations that specifically target NADPH oxidases 1 and 4 by inhibition, which are used to address neurological disorders, i.e., to reduce / halt PD progression after it has been diagnosed.

[0033] One aspect of the present disclosure is a compound of the formula: [ka] where R is an alkyl or cycloalkyl group; a cation selected from the list consisting of the cholinium, tetraalkylammonium, tetraalkylphosphonium, or 1-alkyl-3-methylimidazolium cation families; The present invention relates to an ionic liquid comprising:

[0034] In one embodiment, the molar ratio of anion to cation ranges from 1:2 to 2:1 (mol:mol), preferably from 1:1.5 to 1.5:1 (mol:mol).

[0035] In one embodiment, the molar ratio of anion to cation is 1:1 (mol:mol).

[0036] In one embodiment, R is a C3-C7 cycloalkyl group.

[0037] In one embodiment, R is an unsubstituted cycloalkyl group from C3 to C7.

[0038] In one embodiment, R is a cyclohexyl group.

[0039] In one embodiment, the cation is cholinium.

[0040] 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.

[0041] Another aspect of the present disclosure relates to the use of the ionic salts of the present disclosure, preferably the use of ionic liquids and formulations in pharmaceuticals or as medicaments.

[0042] In one embodiment, the ionic liquids, preferably the ionic liquids and formulations (formulations) of the present disclosure can be used in the prevention or treatment of a disease, disorder or condition of the central nervous system.

[0043] In one embodiment, the ionic liquids and formulations of the present disclosure can be used to prevent or treat neurodegeneration, cognitive impairment, dementia, or multiple system atrophy.

[0044] In one embodiment, the ionic liquids and formulations, preferably the ionic liquids and combinations, of the present disclosure can be used in the prevention or treatment of Parkinson's disease.

[0045] In one embodiment, the ionic liquids and formulations of the present disclosure can be used to slow down or delay the progression of Parkinson's disease.

[0046] 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 pharma- ceutically acceptable carrier.

[0047] In one embodiment, the pharma- ceutically acceptable carrier is saline buffer, PBS, water or a mixture thereof.

[0048] In one embodiment, the amount of ionic liquid ranges from 0.005 mM to 10 mM, preferably from 0.1 to 5 mM, and more preferably from 1 to 2 mM.

[0049] In one embodiment, the composition is in an injectable, intranasal, intrathecal or intracerebroventricular form, preferably the composition is in an intranasal, intrathecal or intracerebroventricular form.

[0050] In one embodiment, the composition is administered daily, preferably for 30 days or more, to a person with a neurodegenerative disease or disorder of the central nervous system. In one embodiment, the dosage is up to 1000 mg / day, preferably in the range of 0.05-1000 mg / day, more preferably in the range of 0.05-5 mg / day, especially in intranasal or intrathecal (intracerebroventricular) form.

[0051] In one embodiment, the daily dosage form consists of a tablet, suppository, ampoule or intranasal or intrathecal form containing a pharma- ceutical effective amount of the composition of the present disclosure, the entirety of which is intended to be administered as a single dose in a daily regimen.

[0052] The present disclosure also relates to the development of novel formulations of specific inhibitors of Nox1 and Nox4 (N1(4)inh), comprising the steps of: 1) Conversion of the specific inhibitor [N1(4)inh] into an IL-based formulation, i.e., a mixture (1:1) of cholinium 3-cyclohexyl-5-(2,4-dihydroxybenzylidene)-1-methyl-2-thiohydantonate and 3-cyclohexyl-5-(2,4-dihydroxybenzylidene)-1-methyl-2-thiohydantonate ([Chol]2[N1(4)inh]+N1(4)inh, 1:1), comprising the following steps: 1a) Selection of IL anion and cation. 1b) Synthesis of [Chol]2[N1(4)inh] by metathesis reaction using cholinium salt as the cation source and 3-cyclohexyl-5-(2,4-dihydroxybenzylidene)-1-methyl-2-thiohydantoin (N1(4)inh) as the anion source. 1c) Nuclear magnetic resonance ( 1 H NMR and 13 Characterization of the purity of [Chol]2[N1(4)inh]+N1(4)inh and N1(4)inh by C NMR, their solubility in water and phosphate buffered saline (PBS) solutions, and their thermal stability by thermogravimetric analysis (TGA). 2) In vitro and in vivo evaluation of the biological effects of [Chol]2[N1(4)inh]+N1(4)inh on dopaminergic neurons by: 2a) Evaluation of the cytotoxicity of [Chol]2[N1(4)inh]+N1(4)inh in an immortalized rat dopaminergic neuronal cell line (N27). 2b) Evaluating the biological effects of remodeling in the context of the treatment of PD by assessing its dopaminergic neuroprotective potential in in vitro models of PD 2c) Assessment of [Chol]2[N1(4)inh]+N1(4)inh neurodopaminergic toxicity in the substantia nigra of mice. 2d) Evaluation of the toxicity of [Chol]2[N1(4)inh]+N1(4)inh in mice and rats. 2e) Evaluation of the biological effects of remodeling in the context of the treatment of PD by evaluating its dopaminergic neuroprotective effects and its ability to prevent the progression of motor dysfunction in in vivo models of PD.

[0053] In one embodiment, considering the pathological role of oxidative stress originating from Nox enzymes in PD and other neurological diseases, the use of inhibitors of the activity of these enzymes plays a major role in the development of future therapies aimed at reducing the rate of progression and disability of this disease over time. However, most, if not all, of these inhibitors have negligible solubility in aqueous solutions, which reduces their efficacy due to low bioavailability, and also requires the use of organic solvents as solvents, which results in high cytotoxicity. Furthermore, reformulation of these inhibitors into liquid compounds is important for intranasal administration, circumventing the high selectivity of the blood-brain barrier (BBB) ​​and allowing their concentration in the brain (target organ).

[0054] The present disclosure also relates to the development of novel IL-based formulations of inhibitors of NADPH oxidase enzymes (Nox), in particular inhibitors specific for Nox1 and Nox4 (Nox1(4)), for use as therapeutic agents in neurological disorders, particularly Parkinson's disease.

[0055] The present disclosure also relates to a new IL-based formulation of a specific inhibitor of Nox1(4), comprising the steps of: 1) Conversion of N1(4)inh to IL-based preparations, i.e., cholinic acid 3-cyclohexyl-5-(2,4-dihydroxybenzylidene)-1-methyl-2-thiohydantonate and 3-cyclohexyl-5-(2,4-dihydroxybenzylidene)-1-methyl-2-thiohydantonate [Chol]2[N1(4)inh]+N1(4)inh, comprising the steps of: 1a) Selection of IL anion and cation. 1b) Synthesis of [Chol]2[N1(4)inh]. 1c) Chemical characterization of [Chol]2[N1(4)inh]+N1(4)inh and N1(4)inh, i.e. purity, solubility and thermal stability. 2) Evaluation of the biological effects of [Chol]2[N1(4)inh]+N1(4)inh in dopaminergic neurons in vitro and in vivo, including: 2a) Evaluation of the cytotoxicity of [Chol]2[N1(4)inh]+N1(4)inh in an immortalized rat dopaminergic neuronal cell line (N27). 2b) Validating the biological effects of remodeling in relation to the treatment of PD and evaluating its dopaminergic neuroprotective capacity in in vitro models of PD. 2c) Evaluation of [Chol]2[N1(4)inh]+N1(4)inh neurodopaminergic toxicity in the black subcutaneous tissue (SN) of mice. 2d) Evaluation of the toxicity of [Chol]2[N1(4)inh]+N1(4)inh in mice and rats. 2e) Evaluation of the biological effects of remodeling in the context of the treatment of PD by assessing its dopaminergic neuroprotective effects and its ability to prevent the progression of motor dysfunction in in vivo models of PD.

[0056] In one embodiment, for the first part of the method, a cholinium salt was used as the cation source and 3-cyclohexyl-5-(2,4-dihydroxybenzylidene)-1-methyl-2-thiohydantoin (N1(4)inh) was used as the anion source. [Chol]2[N1(4)inh]+N1(4)inh was synthesized by metathesis reaction and characterized in terms of purity, thermal stability and solubility. The purity of [Chol]2[N1(4)inh]+N1(4)inh and N1(4)inh was 1 H and 13 This was confirmed by C NMR (Figures 1-2). The solubility results reveal that [Chol]2[N1(4)inh]+N1(4)inh is more soluble in water and PBS than its precursor alone -N1(4)inh (Figures 3-4). The thermal stability results show that under the defined conditions, the decomposition temperature of the formulation is 200 °C (Figure 5). At room temperature (approximately 23 °C), the IL-based formulation is a highly viscous liquid, meaning that its melting temperature is below room temperature. Thus, the IL-based formulation does not exhibit a well-ordered crystalline structure, making it possible to overcome the challenges associated with polymorphism of solid-state formulations.

[0057] In one embodiment, with regard to the evaluation of the biological effects of [Chol]2[N1(4)inh]+N1(4)inh, the obtained results disclose that reformulation of a specific Nox1(4) inhibitor into [Chol]2[N1(4)inh]+N1(4)inh does not cause changes in the viability of the dopaminergic neuronal cell line N27 cells, indicating no potential cytotoxicity for these cells (Figure 6). With regard to its dopaminergic neuroprotective potential, pretreatment of N27 cells with [Chol]2[N1(4)inh]+N1(4)inh significantly inhibited the neurotoxic effects of 6OHDA, and this neuroprotective effect was not enhanced by pretreatment with cholinium, the cation used in the synthesis of [Chol]2[N1(4)inh]+N1(4)inh (Figure 7). Furthermore, pretreatment of N27 cells with [Chol]2[N1(4)inh]+N1(4)inh could also significantly prevent the neurotoxic effects of MPP+ toxins, and this neuroprotective effect was not enhanced by pretreatment with cholinium chloride (Figure 8). Since 6OHDA and MPP+ induce dopaminergic neurotoxicity by inducing different intracellular pathological mechanisms, these results are an indication that [Chol]2[N1(4)inh]+N1(4)inh can prevent neuronal cell death induced by two different toxins and thus modulate two different pathological mechanisms. These data are important because the goal is to use [Chol]2[N1(4)inh]+N1(4)inh as a treatment to stop the progression of multifactorial diseases in terms of pathological mechanisms.

[0058] In one embodiment, the dopaminergic neuroprotective ability of [Chol]2[N1(4)inh]+N1(4)inh was evaluated in an animal model of PD induced by intrastriatal injection of 10 μg of 6OHDA. As shown in Figure 9, right ventricular infusion of [Chol]2[N1(4)inh]+N1(4)inh at 0.2 mg / kg / day for 7 days did not induce dopamine neurotoxicity in the SN of mice. Moreover, this dose of [Chol]2[N1(4)inh]+N1(4)inh could prevent 6OHDA-induced degeneration of dopaminergic neurons in the SN (Figure 10).

[0059] In one embodiment, the toxicity of [Chol]2[N1(4)inh]+N1(4)inh was evaluated when administered to mice via the intranasal route. These administrations did not induce toxic effects in mice. As shown in Figures 11 and 12, daily intranasal administration of different doses of IL-based formulations for 14 days did not induce changes in the body weight (Figure 11) or motor performance (Figure 12) of the animals. Moreover, as shown in Figure 13, daily administration of the high dose tested (0.16 mg / kg / day) did not induce olfactory dysfunction.

[0060] In one embodiment, the toxicity of [Chol]2[N1(4)inh]+N1(4)inh was evaluated when administered to rats via the intracerebroventricular route. No toxic effects were observed in rats exposed to 0.007 mg / kg / day. No changes in the body weight and motor performance of the animals were observed, as shown in Figures 14 and 15, respectively.

[0061] In one embodiment, [Chol]2[N1(4)inh]+N1(4)inh was evaluated for toxicity when administered to rats via intranasal route. These administrations did not induce toxic effects in rats. As shown in Figures 16 and 17, when the IL-based formulation was administered intranasally at 0.062 mg / kg / day for 30 days, no changes in the animals' weight (Figure 16) or motor performance (Figure 17) were observed. Furthermore, daily administration of [Chol]2[N1(4)inh]+N1(4)inh did not induce olfactory impairment, as shown in Figure 18.

[0062] In one embodiment, the neuroprotective effect of [Chol]2[N1(4)inh]+N1(4)inh, which can prevent motor dysfunction in the PD setting when administered via the intracerebroventricular route, was evaluated in an animal model of PD induced by chronic exposure of rats to low doses (2.5 mg / kg / day) of paraquat (PQ). Infusion of 0.007 mg / kg / day of [Chol]2[N1(4)inh]+N1(4)inh into the right ventricle for 30 days was able to prevent the progression of motor dysfunction induced by PQ (Figures 19, 20 and 21).

[0063] In one embodiment, the neuroprotective effect of [Chol]2[N1(4)inh]+N1(4)inh, which can prevent motor dysfunction in the PD setting when administered via intranasal route, was evaluated in an animal model of PD induced by chronic exposure to low doses (2.5 mg / kg / day) of paraquat (PQ) in rats. 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).

[0064] These results are good indications that novel formulations of Nox inhibitors into IL-based formulations may be used as novel therapeutic approaches for PD or other neuropathologies, and that [Chol]2[N1(4)inh]+N1(4)inh may be used as novel therapeutic approaches for PD or other neuropathologies, and that these enzymes play important pathological roles.

[0065] The following figures provide preferred embodiments to illustrate the disclosure and should not be considered as limiting the scope of the invention. [Brief description of the drawings]

[0066] [Figure 1]FIG. 1 shows the 1H NMR spectra of a) a specific Nox1(4) inhibitor (N1(4)inh) and b) an IL formulation of a specific Nox1(4) inhibitor ([Chol]2[N1(4)inh]+N1(4)inh). [Diagram 2] Figure 2 shows the 13C NMR spectra of a) a specific Nox1(4) inhibitor (N1(4)inh) and b) an IL formulation of a specific Nox1(4) inhibitor ([Chol]2[N1(4)inh]+N1(4)inh). [Diagram 3] FIG. 3 shows the solubility of the Nox1(4) inhibitor N1(4)inh in water and PBS relative to its IL-based formulation ([Chol]2[N1(4)inh]+N1(4)inh) in mg / mL. [Figure 4] Figure 4 shows the solubility of the Nox1(4) inhibitor N1(4)inh in water and PBS relative to its ionic liquid-based formulation ([Chol]2[N1(4)inh]+N1(4)inh) in mol / L. [Diagram 5] Figure 5 shows the decomposition temperatures of [Chol]2[N1(4)inh]+N1(4)inh evaluated by thermogravimetric analysis (TGA). [Figure 6] Figure 6 shows the effect of two concentrations of [Chol]2[N1(4)inh]+N1(4)inh on the viability of N27 dopaminergic neuronal cells: (1) untreated cells; (2) cells expeosed with 20 μM [Chol]2[N1(4)inh]+N1(4)inh; (3) cells expeosed with 30 μM [Chol]2[N1(4)inh]+N1(4)inh. [Figure 7]Figure 7 shows that pretreatment of dopaminergic neurons (N27) with [Chol]2[N1(4)inh]+N1(4)inh significantly inhibited the neurotoxic effects of 6OHDA: (1) untreated cells; (2) cells exposed to 20 mM [Chol]2[N1(4)inh]+N1(4)inh; (3) cells exposed to 50 mM 6OHDA; (4) cells exposed to 50 mM 6OHDA and 20 mM [Chol]2[N1(4)inh]+N1(4)inh; (5) cells exposed to 50 mM 6OHDA and 20 mM Choline-chloride. [Figure 8] Figure 8 shows that pretreatment of dopaminergic neurons (N27) with [Chol]2[N1(4)inh]+N1(4)inh significantly inhibited the neurotoxic effects of the neurotoxin MPP+: (1) untreated cells, (2) cells exposed to 20 mM [Chol]2[N1(4)inh]+N1(4)inh, (3) cells exposed to 10 mM MPP+, (4) cells exposed to 10 mM MPP+ and 20 mM [Chol]2[N1(4)inh]+N1(4)inh, and (5) cells exposed to 10 mM MPP+ and 20 mM choline chloride. [Figure 9] Figure 9 shows the effect of intracerebroventricular injection of [Chol]2[N1(4)inh]+N1(4)inh on dopaminergic neuronal viability in the substantia nigra (SN) of mice: (1) untreated mice, (2) mice exposed to 0.2 mg / kg / day of [Chol]2[N1(4)inh]+N1(4)inh (1:1) for 7 days. [Figure 10] Figure 10 shows that intracerebroventricular infusion of [Chol]2[N1(4)inh]+N1(4)inh prevented dopaminergic neuronal death in the substantia nigra (SN) induced by intravenous injection of 6OHDA, an animal model of Parkinson's disease: (1) untreated mice, (2) mice exposed to 10 mg 6OHDA, and (3) mice exposed to 10 mg 6OHDA and 0.2 mg / kg / day [Chol]2[N1(4)inh]+N1(4)inh (1:1). [Figure 11]FIG. 11 shows the effect of twice-daily intranasal administration of 0.02, 0.04, 0.08, or 0.16 mg / kg / day of [Chol]2[N1(4)inh]+N1(4)inh for 14 days on mouse body weight. [Figure 12] FIG. 12 shows the effect of intranasal administration of [Chol]2[N1(4)inh]+N1(4)inh (1) vehicle, (2) 0.02, (3) 0.04, (4) 0.08, or (5) 0.16 mg / kg / day twice daily for 14 days on the motor performance of mice. [Figure 13] Figure 13 shows the effect of twice-daily intranasal administration of 0.16 mg / kg / day [Chol]2[N1(4)inh]+N1(4)inh for 14 days on olfactory function in mice: (1) vehicle (untreated group), (2) 0.16 mg / kg / day [Chol]2[N1(4)inh]+N1(4)inh-treated group. [Figure 14] FIG. 14 shows the effect of intracerebroventricular administration of 0.007 mg / kg / day of [Chol]2[N1(4)inh]+N1(4)inh once daily for 30 days on rat body weight. [Figure 15] FIG. 15 shows the effect of intracerebroventricular administration of 0.007 mg / kg / day of [Chol]2[N1(4)inh]+N1(4)inh once daily for 30 days on the motor ability of rats. [Figure 16] FIG. 16 shows the effect of intranasal administration of 0.062 mg / kg / day of [Chol]2[N1(4)inh]+N1(4)inh once daily for 30 days on rat weight. [Figure 17] Figure 17 shows the effect of intranasal administration of 0.062 mg / kg / day of [Chol]2[N1(4)inh]+N1(4)inh once daily for 30 days on the motor performance of rats: (1) vehicle (untreated group), (2) 0.062 mg / kg / day of [Chol]2[N1(4)inh]+N1(4)inh treated group. [Figure 18]Figure 18 shows the effect of intranasal administration of 0.062 mg / kg / day of [Chol]2[N1(4)inh]+N1(4)inh once daily for 30 days on olfactory function in rats: (1) vehicle (untreated group), (2) 0.062 mg / kg / day of [Chol]2[N1(4)inh]+N1(4)inh-treated group. [Figure 19] Figure 19 shows that intracerebroventricular administration of 0.007 mg / kg / day of [Chol]2[N1(4)inh]+N1(4)inh once daily for 30 days prevents the progression of PQ-induced motor dysfunction in rats, an animal model of PD: (1) control group (saline only), (2) PQ-treated group, and (3) PQ and [Chol]2[N1(4)inh]+N1(4)inh co-treated group. [Figure 20] Figure 20 shows that intracerebroventricular administration of 0.007 mg / kg / day of [Chol]2[N1(4)inh]+N1(4)inh once daily for 30 days prevents the decrease in distance traveled induced by PQ in a rat animal model of PD: (1) control group (saline only), (2) PQ-treated group, and (3) PQ and [Chol]2[N1(4)inh]+N1(4)inh co-treated group. [Figure 21] Figure 21 shows that intracerebroventricular administration of 0.007 mg / kg / day of [Chol]2[N1(4)inh]+N1(4)inh once daily for 30 days prevents PQ-induced slowing of animal speed in a rat animal model of PD: (1) control group (saline only), (2) PQ-treated group, (3) PQ and [Chol]2[N1(4)inh]+N1(4)inh co-treated group. [Figure 22] Figure 22 shows that intranasal administration of 0.062 mg / kg / day of [Chol]2[N1(4)inh]+N1(4)inh once daily for 30 days prevents the progression of PQ-induced motor dysfunction in rats, an animal model of PD: (1) control group (saline only), (2) PQ-treated group, and (3) PQ and [Chol]2[N1(4)inh]+N1(4)inh co-treated group. [Diagram 23] FIG. 23 is a schematic diagram of the present invention. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0067] The present disclosure relates to the development of IL-based formulations of specific inhibitors of NADPH oxidase enzymes (Nox), in particular Nox1 and Nox4, for therapeutic applications in Parkinson's disease (PD) or other neurological disorders. The strategy used consisted of converting the Nox1(4) specific inhibitor 3-cyclohexyl-5-(2,4-dihydroxybenzylidene)-1-methyl-2-thiohydantoin (N1(4)inh) into a formulation containing cholinium 3-cyclohexyl-5-(2,4-dihydroxybenzylidene)-1-methyl-2-thiohydantonate and 3-cyclohexyl-5-(2,4-dihydroxybenzylidene)-1-methyl-2-thiohydantonate ([Chol]2[N1(4)inh]+N1(4)inh, 1:1).

[0068] The present specification refers to the conversion of Nox1(4) specific inhibitors to IL-based formulations, [Chol]2[N1(4)inh]+N1(4)inh, for use in the context of PD and other neurological disorders, comprising the steps of: (1) preparing an IL-based formulation of the specific inhibitor N1(4)inh, i.e., cholinium 3-cyclohexyl-5-(2,4-dihydroxybenzylidene)-1-methyl-2-thiohydantonate and 3-cyclohexyl-5-(2,4-dihydroxybenzylidene)-1-methyl-2-thiohydantonate; to 2-thiohydantonate ([Chol]2[N1(4)inh]+N1(4)inh, 1:1, mol:mol), comprising the steps of: a) selection of IL cation source and selection of anion source; b) synthesis of [Chol]2[N1(4)inh] by metathesis reaction using cholinium salt as cation source and 3-cyclohexyl-5-(2,4-dihydroxybenzylidene)-1-methyl-2-thiohydantoin (N1(4)inh) as anion source; c) nuclear magnetic resonance (NMR) analysis. 1 H NMR and 13characterization of [Chol]2[N1(4)inh]+N1(4)inh purity by 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 thermogravimetric analysis (TGA). (2) Evaluation of the biological effect 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 an immortalized rat dopaminergic neuronal cell line (N27); b) evaluation of the biological effect of the reformulation in the treatment of PD by evaluation of its dopaminergic neuroprotective potential in an in vitro model of PD.

[0069] Unless otherwise stated, the [Chol]2[N1(4)inh] + N1(4)inh mixtures used in the examples herein were in a ratio of 1:1 (mol:mol) [Chol]2[N1(4)inh] + N1(4)inh.

[0070] In this disclosure, [Chol]2[N1(4)inh]+N1(4)inh may also be referred to as [Chol]2[N1(4)inh]+Nox1(4)inh or [Chol]2[N1(4)inh]+Nox1(4).

[0071] Conversion of the specific inhibitor N1(4)inh into an IL-based formulation: In one embodiment, the synthesis of an IL-based formulation begins with the selection of the components, namely the cation source and the anion source.

[0072] In one embodiment, the cation source should belong to the class of cholinium salts, preferably cholinium bicarbonate.

[0073] In one embodiment, the anion source should belong to the group of 3-substituted 5-benzylidene-1-methyl-2-thiohydantoin based compounds, preferably 3-cyclohexyl-5-(2,4-dihydroxybenzylidene)-1-methyl-2-thiohydantoin (N1(4)inh).

[0074] In one embodiment, the IL-based formulation ([Chol]2[N1(4)inh]+N1(4)inh) was synthesized by metathesis reaction of a 1:1 molar ratio solution of cholinium bicarbonate (80% (m / v) in water) and N1(4)inh prepared in a minimum amount of absolute ethanol.

[0075] In one embodiment, N1(4)inh was mixed by dropwise addition to the cholinium bicarbonate solution under dark and chilled conditions (with an ice bath at ∼5°C) with continuous stirring.

[0076] In one embodiment, the reaction mixture was further kept at ∼5°C under continuous stirring for 2 hours.

[0077] In one embodiment, excess solvent and water were removed under a continuous stream of nitrogen gas until complete dryness (nitrogen gas flow for approximately 2-3 hours while the IL-containing vial was kept in an ice-cold water bath (~10 °C)).

[0078] In one embodiment, the mixture containing the synthesized [Chol]2[N1(4)inh] and N1(4)inh was finally collected from the ice-cold water bath and stored under dry, cooled and dark conditions.

[0079] In one embodiment, the purity of [Chol]2[N1(4)inh] and N1(4)inh is as disclosed in Figures 1 and 2, respectively: 1 H NMR and 13 C NMR was used to characterize the structure.

[0080] In one embodiment, the NMR spectrum obtained made it possible to confirm that [Chol]2[N1(4)inh]+N1(4)inh was successfully synthesized and that it was pure.

[0081] In one embodiment, the solubility of [Chol]2[N1(4)inh]+N1(4)inh in water and phosphate buffered saline (PBS) solutions was measured.

[0082] In one embodiment, mixtures containing solutes N1(4)inh and [Chol]2[N1(4)inh]+N1(4)inh were added to a volume (500 μL) or more of water and PBS solution. These mixtures were incubated at 37°C under constant agitation at 1150 rpm and for a minimum of 72 hours using an Eppendorf Thermomixer Comfort instrument. Throughout this process, solutes were added to the mixture as needed, i.e., until solution saturation was reached. All samples were filtered using a syringe filter (0.45 mm) to remove possible suspended solid particles.

[0083] In one embodiment, quantification of N1(4)inh and [Chol]2[N1(4)inh] was performed by UV spectroscopy using UV spectrophotometry (SYNERGY|HT microplate reader, BioTek) at wavelengths of 416 nm and 480 nm, respectively. Interference of PBS with the quantification method was also checked, and blank control samples were always used.

[0084] In one embodiment, the mixture [Chol]2[N1(4)inh]+N1(4)inh has a solubility in water of 0.405±0.053 mg / mL, which is 36 times higher than N1(4)inh alone (0.011±0.002 mg / mL). The increase in solubility of the IL-based formulation was also notable in PBS, with a 21-fold increase (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).

[0085] These results clearly demonstrated that the reformulation of N1(4)inh to [Chol]2[N1(4)inh]+N1(4)inh overcomes one of the most important problems associated with specific inhibitors of Nox, namely low solubility, and strengthens the potential of this new reformulation to be used in the context of PD and other neurological disorders.

[0086] In one embodiment, the decomposition temperature of [Chol]2[N1(4)inh]+N1(4)inh was evaluated by thermogravimetric analysis (TGA), and the results, as disclosed in FIG. 5, disclose that the mixture is thermally stable up to 200° C.

[0087] (2) Evaluation of the biological effects of [Chol]2[N1(4)inh]+N1(4)inh in dopaminergic neurons in vitro In one embodiment, the cytotoxicity of [Chol]2[N1(4)inh]+N1(4)inh was evaluated in an immortalized rat dopaminergic neuronal cell line (N27).

[0088] Cells were exposed to 20 and 30 μM [Chol]2[N1(4)inh]+N1(4)inh or 20 μM cholinium chloride. Cholinium chloride was dissolved in saline, and [Chol]2[N1(4)inh]+N1(4)inh was dissolved in PBS 1x (phosphate-saline buffer).

[0089] The cells were kept under stimulation for 24 hours, and cell viability was assessed using a Cell Counting Kit-CCK-8 (Dojindo Molecular Technologies).

[0090] [Chol]2[N1(4)inh]+N1(4)inh did not show any toxic effects on N27 dopaminergic cells when exposed to 20 or 30 μM of the mixture, as no statistically significant differences were observed between cell viability in cultures exposed to [Chol]2[N1(4)inh]+N1(4)inh (20 μM FIG. 6(2) or 30 μM FIG. 6(3)) compared to untreated cells (CTR FIG. 6(1)).

[0091] In one embodiment, the dopaminergic neuroprotective potential of [Chol]2[N1(4)inh]+N1(4)inh was evaluated in an in vitro model of PD.

[0092] To exclude the possibility that neuroprotection was due to the cholinium salt rather than the IL-based formulation, neuroprotection was assessed using 20 μM [Chol]2[N1(4)inh]+N1(4)inh or 20 μM cholinium chloride.

[0093] The neurotoxins 6-hydroxydopamine (6OHDA) and 1-methyl-4-phenylpyridinium (MPP+) were added 2 h 30 min after [Chol]2[N1(4)inh]+N1(4)inh or cholinium chloride, respectively, and stimulation was maintained for 24 h before cell viability was assessed using a CCK-8 kit.

[0094] The working concentration of [Chol]2[N1(4)inh]+N1(4)inh was 20 μM, based on results previously obtained with the non-reformulated inhibitor (N1(4)inh).

[0095] In one embodiment, when comparing cell conditions treated with 6OHDA, a 45% decrease in dopaminergic neuronal viability was observed, which is statistically different from the control condition (CTR(1)) (Figure 7). Pretreatment with [Chol]2[N1(4)inh]+N1(4)inh for 2.5 hours prior to exposure to 50 μM 6OHDA completely prevented toxin-induced neurotoxicity, with no statistically significant difference when comparing cell viability values ​​between CTR vs. 6OHDA +[Chol]2[N1(4)inh]+N1(4)inh conditions, but rather between the latter and the 6OHDA condition alone (Figure 7).

[0096] In one embodiment, the results shown in Figure 7 also show that the 6OHDA-induced decrease in N27 viability was not prevented by pretreatment with 20 μM cholinium chloride, indicating that the protective effect of [Chol]2[N1(4)inh]+N1(4)inh described above is due to the specific efficacy of the Nox1 inhibitor anion and not due to the presence of cholinium in the formulation.

[0097] In one embodiment, [Chol]2[N1(4)inh]+N1(4)inh also plays a neuroprotective role in preventing MPP+ dopaminergic neurotoxicity, since it significantly prevented a 45% loss of N27 cell viability upon exposure to this neurotoxin (Figure 8). As confirmed by 6OHDA, pretreatment with cholinium chloride did not provide any protection against MPP+, emphasizing that the protection observed in the presence of [Chol]2[N1(4)inh]+N1(4)inh results from the action of the inhibitor and not from that of cholinium (Figure 8).

[0098] The biological effects of [Chol]2[N1(4)inh]+N1(4)inh were then evaluated in vivo. All animal experiments were performed in our in-house animal house and in accordance with national and European Community regulations (86 / 609 / ECC; 2010 / 63 / EU).

[0099] First, the toxicity of [Chol]2[N1(4)inh]+N1(4)inh (1:1) was evaluated in healthy mice using two different administration routes: 1) 0.2 mg / kg / day [Chol]2[N1(4)inh]+N1(4)inh by intracerebroventricular infusion for 7 days; 2) 0.16, 0.08, 0.04, or 0.02 mg / kg / day [Chol]2[N1(4)inh]+N1(4)inh by intranasal administration twice daily for 14 days.

[0100] In one embodiment, 8-12 week old male C57BL / 6 mice were housed in a temperature / humidity controlled environment under a 12 hour light / dark cycle with free access to water and food. 1) Mice were anesthetized with an intraperitoneal (ip) injection of saline containing ketamine (0.67 mL per kg of mouse weight) and xylazine (0.33 mL per kg of mouse weight) and then placed in a digital stereotaxic frame (51900 Stoelting). Once the mouse skull was exposed, the injection site was determined using a digital coordinate system with 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 was performed by direct intraventricular infusion for 7 days using an Alzet osmotic pump (ref. 1007D) connected to an Alzet catheter (ref. Brain infusion Kit 3) at the following coordinates: medial-lateral (ML): -1.1 mm; anterior-posterior (AP): 0.5 mm, and dorsoventral (DV): -2.5. On day 7, animals were anesthetized by intraperitoneal (ip) injection with saline containing ketamine (0.67 mL per kg of mouse weight) and xylazine (0.33 mL per kg of mouse weight) and after transcardial perfusion first with saline and then with buffered formalin (experimental end point). Brains were frozen in liquid nitrogen and kept at -80 °C. Brains were then embedded in ideal cutting temperature (OCT) gel and cut on a cryostat (Leica CM 3050S, Leica Microsystems). Coronal sections 30 μm thick from the front pole to the midbrain terminal were taken at −20°C. Sections corresponding to the ST and SN of each animal were taken and stored sequentially 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).Appropriately identified plates were kept at -20°C 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 cell marker tyrosine hydroxylase (TH). Results shown in Figure 9 indicated that administration of [Chol]2[N1(4)inh]+N1(4)inh did not significantly reduce the number of dopaminergic (TH+) neurons in the substantia nigra (SN) 7 days after intracerebroventricular infusion of 0.2 mg / kg / day of [Chol]2[N1(4)inh]+N1(4)inh, indicating the non-toxic profile of the IL-based formulation. 2) To evaluate the general putative toxicity of [Chol]2[N1(4)inh]+N1(4)inh when administered intranasally to mice, general anesthesia was induced by inhaling 3–4% (v / v) isoflurane for 4 min, allowing mice to receive four different doses (0.16, 0.08, 0.04 or 0.02 mg / kg / day) of [Chol]2[N1(4)inh]+N1(4)inh intranasally twice daily for 14 days. In-line, mice lying supine with their nose elevated were infused with 6 μL of [Chol]2[N1(4)inh]+N1(4)inh per 30 g of mouse body weight using a polyurethane catheter (Introcan Safety®, 24G, 0.7 × 19 mm) attached to a 50 μL microliter syringe (Hamilton®, USA). The catheter was inserted approximately 0.3 mm deep into one of the nostrils to allow delivery of the formulation towards the roof of the nasal cavity. The animals were kept in the above position until they sat up to prevent the IL-based formulation from entering the airways and to remain longer in the nasal cavity. The day of first administration was designated as 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 reach a consistent performance. Training was performed on two consecutive days before the first administration, with four test trials each day assessed, each trial lasting 5 min. Mice were trained on day 1 at 12 RPM (fixed mode), followed by two test trials at 24 RPM (fixed mode) and 4–40 RPM (accelerating mode). The animals were allowed to rest for approximately 30 min between each test.The rotarod test was performed for 5 min under an accelerating protocol (4–40 RPM), and the latency to fall (latency) was recorded using specific software. The trial was stopped when the mouse fell (activating a switch that automatically stopped the timer) or when 5 min had elapsed. Four independent trials with an intertrial period of approximately 30 min were performed for each animal to reduce stress and fatigue. To examine olfactory function, a food discovery test (FFT) olfactory paradigm was performed. Mice were food restricted for 16 h prior to testing. On the test day, animals were placed in clean cages with only a filter top lid, with approximately 4 cm of clean bedding evenly distributed throughout the cage, for habituation for 30–40 min (no water, no food). After habituation, the animal was returned to its home cage. Then, one food pellet was buried on one side of the cage and covered with bedding. To start the test, the animal was placed on the opposite side of the buried pellet, the timer was started, and the top lid was placed. The timer was stopped when the animal removed the pellet cover and started to eat it. If the animal failed to find the pellet within 5 min, the test was terminated with a score of 5 min. After the test was completed, the animal was returned to its home cage. In summary, the toxicity of [Chol]2[N1(4)inh]+N1(4)inh administered to mice via the intranasal route was evaluated by analyzing the animals' body weight, motor ability, and ability to find the food pellet. Inline, mice were weighed daily for 14 days, subjected to a rotarod motor behavior assay on day 14, and an olfactory test on day 14. As shown in Figures 11, 12, and 13, no changes in body weight, motor, and olfactory ability were observed in mice administered [Chol]2[N1(4)inh]+N1(4)inh via the intranasal route, reinforcing the non-toxic profile of the IL-based formulation.

[0101] Second, the toxicity of [Chol]2[N1(4)inh]+N1(4)inh was evaluated in healthy rats using two different routes of administration: 1) intracerebroventricular infusion of [Chol]2[N1(4)inh]+N1(4)inh at 0.007 mg / kg / day for 30 days, and 2) intranasal administration of [Chol]2[N1(4)inh]+N1(4)inh at 0.062 mg / kg / day for 30 days.

[0102] In one embodiment, 8-12 week 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) Rats were anesthetized with an intraperitoneal (ip) injection of ketamine (90 mg / kg) and xylazine (10 mg / kg) in saline and then placed in a digital stereotaxic frame (51900 Stoelting). Once exposed, the rat skull was located using a digital coordinate system with the injection site set to zero at the bregma point. Delivery of [Chol]2[N1(4)inh]+N1(4)inh (1:1) was performed at a dose of 0.007 mg / kg / day by direct intracerebroventricular infusion for 30 days using an Alzet osmotic pump (ref. 2004) connected to an Alzet catheter (Ref. Brain infusion Kit 2) at coordinates medial-lateral (ML): 1.5 mm; anterior-posterior (AP): -1.0 mm and dorsoventral (DV): -4.0. Toxicity was assessed by analyzing the weight of the animals on days 0 and 30 and their behavioral motor performance on 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 reach a consistent performance. Training was performed on 2 consecutive days and a test trial was assessed on each 4th day, with each trial lasting 5 min. Rats were first trained at 12 RPM (fixed mode), followed by two test trials at 24 RPM (fixed mode) and then another two trials at 4–40 RPM (accelerated mode). The animals were allowed to rest for approximately 30 min between each trial. The rotarod test was performed for 5 min under an accelerating protocol (4–40 RPM) and the latency to fall was recorded using specific software. The trial was stopped when the mouse fell (activating the switch that automatically stops the timer) or when 5 min had elapsed. To reduce stress and fatigue, four independent trials with an intertrial period of approximately 30 min were performed on each animal. The results shown in Figures 14 and 15 showed that administration of [Chol]2[N1(4)inh]+N1(4)inh did not induce significant changes in body weight and motor performance, indicating the non-toxic profile of the IL-based formulation.2) To evaluate the general putative toxicity of [Chol]2[N1(4)inh]+N1(4)inh administered intranasally to rats, general anesthesia was induced by 4-min inhalation of 3-4% (v / v) isoflurane, allowing the rats to receive 0.062 mg / kg / day of [Chol]2[N1(4)inh]+N1(4)inh intranasally once a day for 14 days. In-line, rats lying supine with their nose elevated were infused with 50 μL of [Chol]2[N1(4)inh]+N1(4)inh in a microliter syringe (Hamilton®, USA) at 40 μL per 260 g of rat body weight using a polyurethane catheter (Introcan Safety®, 24 G, 0.7 × 19 mm). Catheters were inserted into the left and right nostrils at a depth of approximately 1.7 mm, allowing delivery of the formulation towards the roof of the nasal cavity. The animals were kept in the above position until they sat up, to prevent the IL-based formulation from entering the airways and to remain longer in the nasal cavity. 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, learned and reached a consistent performance. Training was performed on two consecutive days, with a test trial assessed on each fourth day, each trial lasting 5 min. Rats were first trained at 12 RPM (fixed mode), followed by two test trials at 24 RPM (fixed mode) and then another two trials at 4–40 RPM (accelerating mode). The animals were allowed to rest for approximately 30 min between each trial. The rotarod test was performed for 5 min under an accelerating protocol (4–40 RPM) and the duration to fall was recorded using specific software. A trial was stopped when the mouse fell (activating a switch that automatically stopped the timer) or when 5 min had elapsed. Four independent trials with an inter-trial period of approximately 30 min were performed on each animal to reduce stress and fatigue. To examine olfactory function, the food discovery test (FFT) olfactory paradigm was performed. Rats were food restricted for 16 h prior to testing.On the test day, animals were placed in clean cages with only a filter top cover, with approximately 4 cm of clean bedding evenly distributed throughout the cage, for habituation (familiarization) for 30-40 min (no water, no food). After habituation, animals were returned to their home cage. One food pellet was then buried on one side of the cage and covered with bedding. To start the test, the animal was placed on the other side of the buried pellet, a timer was started, and the top cover was placed. The timer was stopped when the animal uncovered the pellet and began to eat it. If the animal did not find the pellet within 5 min, the test was terminated with a score of 5 min. After the test, the animal was returned to its home cage. Overall, toxicity was assessed by analyzing the rats' body weight, behavioral motor performance, and ability to find food pellets hidden in the cage. Inline, rats were weighed daily for 30 days, and a rotarod motor behavior assay was performed on the 30th day, followed by an olfactory test on the 30th day. As shown in Figure 16, Figure 17 and Figure 18, rats administered [Chol]2[N1(4)inh]+N1(4)inh intranasally showed no changes in body weight, motor ability and olfactory ability, respectively, enhancing the non-toxicity profile of the IL-based formulation.

[0103] In one embodiment, the overall results obtained in relation to the in vivo toxicity of [Chol]2[N1(4)inh]+N1(4)inh demonstrate that the IL-based formulation is not toxic whether administered via the intracerebroventricular or intranasal route.

[0104] The neuroprotective potential of [Chol]2[N1(4)inh]+N1(4)inh was then evaluated in two in vivo models of PD induced by intracerebral injection of 6OHDA and by exposure to chronic low-dose paraquat (PQ).

[0105] In one embodiment, the in vivo neuroprotective effect of [Chol]2[N1(4)inh]+N1(4)inh when administered via intracerebroventricular infusion route was evaluated in a mouse model for PD induced by intracerebral infusion of 6OHDA, and the number of dopaminergic neurons in the substantia nigra was counted.

[0106] In one embodiment, male C57BL / 6 mice were anesthetized and placed in a digital stereotaxic frame. Once the mouse skull was exposed, the injection and infusion sites were zeroed at the bregma point using a digital coordinate system. In this regard, injections of 6-OHDA (10 μg / 2 μL of ascorbic acid 0.1% v / v) were administered to the right striatum (ST) of each animal using the following coordinates: medial-lateral (ML): -2 mm; anterior-posterior (AP): 0.6 mm; dorsoventral (DV): -3.0 mm, using a Hamilton syringe at a rate of 0.2 μL / min. Delivery of 0.2 mg / kg / day of [Chol]2[N1(4)inh]+N1(4)inh was performed as described above. On day 7, animals were anesthetized by intraperitoneal (i.p.) injection of saline containing ketamine (0.67 mL / kg mouse body weight) and xylazine (0.33 mL / kg mouse body weight) and perfused transcardially, first with saline and then with buffered formalin (experimental end point). Brains were frozen in liquid nitrogen and kept at −80°C. They were then embedded in ideal cutting temperature (OCT) gel and sectioned on a cryostat (Leica CM 3050S, Leica Microsystems). Coronal sections 30 μm thick from the front pole to the midbrain terminal were taken at −20°C. Sections corresponding to the ST and SN of each animal were taken and stored sequentially in free-floating 24-well plate compartments (Orange Scientific) containing a cryopreservation medium of 30% (v / v) glycerol and 30% (v / v) ethylene glycol in phosphate buffer (PB). Plates were kept at −20 °C where appropriate identification was made for later use in immunohistochemistry to assess the number of dopaminergic neurons in the SN by stereological counting of TH+ neurons. Two experimental groups were 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 lateral ventricle, and [Chol]2[N1(4)inh]+N1(4)inh in the lateral ventricle. The saline-injected hemisphere served as a control for 6OHDA in group 1 and [Chol]2[N1(4)inh]+N1(4)inh in group 2. In group 2, intraventricular and stereotaxic surgeries were performed on the same day.As shown in Figure 10, exposure to 6-OHDA induced a statistically significant decrease of 45% in the number of SN dopaminergic neurons when compared to the control group (Figure 10, (1) vs. (2)). Regarding the dopaminergic neuroprotective effect of [Chol]2[N1(4)inh]+N1(4)inh, the results showed that the presence of the IL-based formulation had neuroprotective properties by preventing 37% of SN dopaminergic neuron degeneration induced by 6-OHDA in the mouse SN (Figure 10, (3) vs. (2)).

[0107] In one embodiment, the bioneuroprotective effect of [Chol]2[N1(4)inh]+N1(4)inh when administered via intracerebroventricular infusion route was evaluated in a rat model of PD induced by exposure to low doses of PQ by analysis of the progression of behavioral dysfunction. In this model, animals were exposed to the toxin for 30 days (4 weeks) and then allowed to survive for an additional 30 days to allow the disease to progress (total of 8 weeks). Administration of [Chol]2[N1(4)inh]+N1(4)inh began after the first 30 exposure to PQ via intracerebroventricular or intranasal route and continued for a second 30 days. At week 8, animals were euthanized.

[0108] In one embodiment, male Wistar rats, 8-12 weeks of age, were housed in a temperature / humidity controlled environment under a 12-h light / dark cycle with free access to water and food, and were administered PQ subcutaneously and chronically for 4 weeks at a dose of 2.5 mg / kg / day at a fluid delivery rate of 0.25 μL / hr using osmotic minipumps (Alzet Direct, Cupertino, CA) (Alzet model 2004, large pumps). A control group was implanted with minipumps filled with sterile saline, the vehicle used to dissolve PQ. Rats were anesthetized with intraperitoneal (ip) injection of ketamine (90 mg / kg) and xylazine (10 mg / kg), and pumps were implanted subcutaneously on the back, slightly posterior to the scapula (scapularis dorsalis). After 4 weeks of exposure to PQ, intraventricular infusion of [Chol]2[N1(4)inh]+N1(4)inh was initiated and continued for an additional 4 weeks. The experimental paradigm consisted of a total of 8 weeks, the first 4 weeks of PQ exposure and the last 4 weeks of IL-based formulation administration. The rats were anesthetized and placed in a digital stereotaxic frame (51900 Stoelting). Once the rat skull was exposed, the injection site was determined using a digital coordinate system with zero set at the bregma point. [Chol]2[N1(4)inh]+N1(4)inh (1:1) was delivered at a dose of 0.007 mg / kg / day by direct intracerebroventricular infusion for a further 4 weeks (30 days) using an Alzet osmotic pump (ref. 2004) connected to an Alzet catheter (Ref. Brain infusion Kit 2) at coordinates of medial-lateral (ML): 1.5 mm; anterior-posterior (AP): -1.0 mm and dorsoventral (DV): -4.0. To assess the neuroprotective function of the IL-based formulation when injected into the ventricles, 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, learned and reached a consistent performance. Training was performed on two consecutive days, with a test trial assessed on each fourth day, and each trial lasted 5 min.Rats were first trained at 12 RPM (fixed mode), followed by two test trials at 24 RPM (fixed mode) and then another two trials at 4–40 RPM (accelerated mode). The animals were allowed to rest for approximately 30 min between each trial. The rotarod test was performed for 5 min under an accelerating protocol (4–40 RPM) and the latency to fall was recorded using specific software. The trial was stopped when the mouse fell (activating a switch that automatically stops the timer) or when 5 min had elapsed. Four independent trials with an intertrial period of approximately 30 min were performed for each animal to reduce stress and fatigue. Exploratory behavior and general activity were measured using the open field test. The animals were transported to the testing room and left undisturbed for 30 min–1 h before testing. There is a beam-breaking system around the arena, which is interrupted by the movement of the animal in the arena. This information is processed with specific software to analyze several parameters, such as distance, speed, and rear, which can be entered at the center. Each animal was placed in the center of the rectangular arena and allowed to explore freely for 10 min. The timer was started exactly at the same time that the animal was placed in the arena. The operator 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 results presented showed that intracerebroventricular [Chol]2[N1(4)inh]+N1(4)inh administration significantly prevented motor dysfunction (Figure 19), as well as the reduction in distance (Figure 20) and speed (Figure 21) induced by PQ. These highlight that IL-based formulations are capable of reducing the progression of motor dysfunction associated with PD.

[0109] As shown in Figure 19, exposure to PQ statistically significantly reduced the fall latency of animals exposed to PQ by 43% compared to the control group (Figure 19, (2) vs. (1)). Regarding the neuroprotective effect of [Chol]2[N1(4)inh]+N1(4)inh, the results indicate its ability to prevent the development of motor dysfunction, as the fall latency of animals exposed to PQ increased by 37% compared to animals exposed to PQ alone (Figure 19, (3) vs. (2)).

[0110] Regarding the results of distance traveled and speed in rats shown in Figures 20 and 21, PQ induced a 55% decrease in distance traveled (Figure 20, (2) vs. (1)) and a 50% decrease in speed (Figure 21, (2) vs. (1)). Regarding the neuroprotective effect of [Chol]2[N1(4)inh]+N1(4)inh, the results showed that it has the ability to prevent the development of motor dysfunction, as the distance traveled increased by 50% (Figure 20, (3) vs. (2)) and the speed increased by 37% (Figure 21, (3) vs. (2)) in animals exposed to PQ compared to animals exposed to PQ only.

[0111] In one embodiment, the bioneuroprotective effect of [Chol]2[N1(4)inh]+N1(4)inh, when administered via the intranasal route, was evaluated in a rat model of PD induced by chronic exposure to low doses of PQ, and the progression of behavioral dysfunction was analyzed. In this model, animals were exposed to the toxin for 30 days (4 weeks) and then allowed to survive for an additional 30 days to allow disease progression (8 weeks in total). Administration of [Chol]2[N1(4)inh]+N1(4)inh was initiated after the first 30 exposures to PQ, via either the intracerebroventricular or intranasal route, and continued for the second 30 days. At week 8, animals were euthanized.

[0112] In one embodiment, 8-12 week old male Wistar rats were subjected to chronic subcutaneous administration of PQ for 4 weeks (Alzet model 2004, large pumps) at a dose of 2.5 mg / kg / day using osmotic minipumps (Alzet Direct, Cupertino, CA) at a fluid delivery rate of 0.25 μL / hr. A control group was implanted with minipumps filled with sterile saline, the vehicle used to dissolve PQ. Pumps were implanted subcutaneously on the back, slightly posterior to the scapula (dorsal scapular region) under anesthesia induced with ketamine (90 mg / kg) and xylazine (10 mg / kg). Intranasal administration of [Chol]2[N1(4)inh]+N1(4)inh was initiated 4 weeks after exposure to PQ and continued daily for an additional 4 weeks. The experimental paradigm lasted a total of 8 weeks, with the first 4 weeks of PQ exposure and the last 4 weeks of IL-based formulation administration. General anesthesia was induced by inhalation of 3–4% (v / v) isoflurane for 4 min, and rats were administered 0.062 mg / kg / day of [Chol]2[N1(4)inh]+N1(4)inh intranasally once daily for an additional 4 weeks (30 days). Inline, rats were positioned lying supine with their nose elevated and infused with 40 μL of [Chol]2[N1(4)inh]+N1(4)inh per 260 g of rat body weight using a polyurethane catheter (Introcan Safety®, 24 G, 0.7 × 19 mm) attached to a 50 μL microliter syringe (Hamilton®, USA). The catheters were inserted into the left and right nostrils at a depth of approximately 1.7 mm, allowing delivery of the formulation towards the roof of the nasal cavity. The animals maintained the above position until they sat up, preventing the IL-based formulation from entering the airways and allowing it to remain longer in the nasal cavity. 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[N1(4)inh]+N1(4)inh. To evaluate the neuroprotective function of the IL-based formulations when administered via the intranasal route, behavioral functions were 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, learned, and reached a consistent performance. Training was performed on two consecutive days, and test trials were assessed on each fourth day, with each trial lasting 5 min. Rats were first trained at 12 RPM (fixed mode), followed by two test trials at 24 RPM (fixed mode), and then another two trials at 4–40 RPM (accelerating mode). The animals were allowed to rest for approximately 30 min between each trial. The rotarod test was performed for 5 min under an accelerating protocol (4–40 RPM), and the latency to fall was recorded using specific software. The trial was stopped when the mouse fell (when the switch that automatically stopped the timer was activated) or when 5 min had elapsed. To reduce stress and fatigue, each animal was subjected to four independent trials with an intertrial period of approximately 30 min. Behavioral testing was performed at week 8. The results show that intranasal administration of [Chol]2[N1(4)inh]+N1(4)inh significantly suppressed motor dysfunction (Figure 22). These highlight that IL-based formulations are capable of reducing the progression of motor dysfunction in PD conditions.

[0113] As shown in Figure 22, exposure to PQ statistically significantly reduced the fall latency of animals exposed to PQ by 47% compared to the control group (Figure 22, (2) vs. (1)). Regarding the neuroprotective effect of [Chol]2[N1(4)inh]+N1(4)inh, the results indicate its ability to prevent the development of motor dysfunction, as the fall latency of animals exposed to PQ increased by 40% compared to animals exposed to PQ alone (Figure 22, (3) vs. (2)).

[0114] In one embodiment, these results demonstrate that [Chol]2[N1(4)inh]+N1(4)inh, unlike its insoluble precursor, is not toxic but exerts the same neuroprotective effects in the context of PD as exerted by N1(4)inh alone.

[0115] In one embodiment, these results demonstrate that [Chol]2[N1(4)inh]+N1(4)inh reduces the progression of motor dysfunction associated with PD.

[0116] These results clearly show that the present disclosure allows the conversion of toxic molecules into non-toxic mixtures. This reformulation does not impede the efficacy of the precursors, fundamentally transforming their potential use as a differentiated therapeutic approach to reduce / halt the progression of pathological conditions associated with PD and reduce the rate of increase in the degree of disability over time.

[0117] The present disclosure enables currently available treatments to modulate symptoms more consistently over a longer period of time, significantly increasing the quality of life for patients and their families while reducing the negative socio-economic impact of the disease.

[0118] Example: In the central nervous system (CNS), oxidative stress is one of the main contributors to the development of disease and aging. Recent studies have revealed that several isoforms of NADPH oxidase (Nox) enzymes, whose sole function is to produce reactive oxygen species (ROS), are also present in the CNS. There, they play a key role in regulating ROS-dependent cellular mechanisms, but when at high levels, they lead to cellular dysfunction and cell death, accelerating the aging process characteristic of the pathological processes of neurodegenerative diseases.

[0119] Previous studies have demonstrated the importance of Nox-mediated oxidative stress in inducing neuronal loss in various neurological diseases, as in PD, amyotrophic lateral sclerosis (ALS) and stroke. These studies have highlighted the importance of developing specific inhibitors for these enzymes, reinforcing the goals of several laboratories, as well as the goals of pharmaceutical companies such as the Swiss company GenKyoTex, which is dedicated to developing Nox inhibitors for future therapeutic applications. For example, the synthesis of an inhibitor specifically inhibiting Nox isoforms 1 and 4 has been recently reported (Bae et al., Synthesis and biological evaluation of 3-substituted 5-benzylidene-1-methyl-2-thiohydantoins as potent NADPH oxidase (NOX) inhibitors. Bioorg Med Chem 2016; 24: 4144-4151), which was found to be soluble only in solvents containing Triton-X / ethanol / PBS and was a vehicle for inducing toxicity in dopaminergic neurons. This disclosure integrates ionic liquid technology and Nox inhibition to develop a new therapeutic strategy that targets specific harmful ROS production involved in the pathological process of neurodegeneration. Below are three examples of applications of the present invention:

[0120] Example 1: Parkinson's Disease (PD) In one embodiment, PD is a chronic neurodegenerative disease that affects over 6.1 million people and tends to become less effective as the disease progresses to more debilitating stages, resulting in fewer treatment options. According to the 2018 and 2020 reports on PD (The Parkinson's Disease Market: Pipeline 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 absence of preventive therapies to stop PD progression is one of the major unmet needs in the field. In view of this, a paradigm shift is taking place towards the exploration / development of treatments to prevent progression of the disease. In fact, 53% of drugs under development by pharmaceutical companies are disease-modifying drugs aimed at preventing PD progression, and only 32% are symptomatic. The pathogenesis of PD is strongly influenced by oxidative stress. Nox1‐ROS generation plays a key role in the dopaminergic cell death and alpha‐synucleinopathy processes that occur in the disease, and as described in the literature, its inhibition can prevent disease progression in animal models.(Cristovao et al., The role of NADPH oxidase 1-derived reactive oxygen species in paraquat-mediated dopaminergic cell death. Antioxidants & redox signaling 2009; 11:9: 2105-18. Choi et al., NADPH Oxidase 1-Mediated Oxidative Stress Leads to Dopamine Neuron Death in Parkinson's Disease. Antioxidants & redox signaling 2012; 16(10):1033-45. Cristovao et al., NADPH oxidase 1 mediates α-synucleinopathy in Parkinson's disease. Journal of Neurosciences 2012; 32(42):14465-77). Furthermore, Nox4 has a crucial involvement in PD dementia, as shown in the paper "The Role of NOX4 in Parkinson's Disease with Dementia" (Choi et al. International Journal of Molecular Sciences 2019; 20(3):696), which shows that increased expression of Nox4 in the hippocampal dentate gyrus associated with PD induces Aβ expression and oligomeric A11 production, thereby impairing cognitive function. These documents validate Nox1 and Nox4 as important targets for the development of new therapeutic approaches against this disease. In this context, the present disclosure is of benefit for its possible application in the reformulation of Nox1-Nox4 specific inhibitors, which, although currently available, have low solubility and therefore low pharmacological availability. Increasing their solubility would make them more bioavailable and therefore lead to a higher therapeutic effect against PD.

[0121] Example 2: Stroke In one embodiment, neurovascular diseases are the leading cause of death worldwide. In terms of costs, stroke alone costs 64.1 billion euros (Europe) and 44.2 billion euros (USA) per year. Strokes occur due to the restriction of blood flow to the brain, and are caused by ischemia or hemorrhage. While about 20% of patients die within one month after stroke, survivors often develop severe neurological dysfunction and chronic disability, which imposes a significant socio-economic burden. Current treatments only affect a minority of patients and can cause significant side effects.

[0122] Administration of anticoagulants such as aspirin has limited preventive power, while thrombolytic therapy such as recombinant tissue plasminogen activator (rtPA) has a narrow therapeutic window and may 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 effects of surgical procedures such as angioplasty are still unclear, highlighting the importance and necessity of developing more efficient and safe therapies to treat these patients.

[0123] The paper "Oxidative stress and pathophysiology of ischemic stroke: novel therapeutic opportunities" (CNS & neurological disorders drug targets 2013, 12, 698-714) demonstrates that elevated oxidative stress levels are associated with brain damage following ischemic stroke. Furthermore, the paper "Biochemistry, physiology, and pathophysiology of NADPH oxidases in the cardiovascular system" (Circulation research 2012, 110(10), 1364-1390) reveals the involvement of different isoforms of Nox, namely isoforms 1, 2, 4, and 5. In stroke, ROS derived from Nox's may have protective or detrimental functions depending on the isoforms involved as proposed (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). Considering this and specifically inhibiting the isoforms involved in the pathological processes associated with stroke, the development of an antioxidant therapy for this disease by inhibition of Nox is presented as having great therapeutic potential to address this pathology. Thus, there is another potential application of the present disclosure.

[0124] Example 3: Amyotrophic lateral sclerosis (ALS) Amyotrophic lateral sclerosis (ALS) is characterized by progressive degeneration of motor neurons and subsequent activation of glial cells, which leads to muscle weakness and disability and ultimately to the development of fatal respiratory and cardiac disorders within 3 to 5 years after diagnosis. Despite intensive research to date, only one drug has been approved for the treatment of ALS (riluzole), with only a modest effect on survival (de Jongh ADet 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 the manifestation of this condition remain largely unknown. However, the contribution of oxidative stress is again a driving factor in the development of the disease and the involvement of Nox in this process has also been demonstrated (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).

[0125] Previous studies investigating the role of nitric oxide in ALS patients showed that patients with low isoform 2 activity in peripheral blood showed a significant increase in survival (Marrali et al. NADPH oxidase (NOX2) activity is a modifier of survival in ALS. Journal of neurology 2014, 261(11), 2178-2183). The same was observed in ALS mice by Marden et al. (Redox modifier genes in ALS in mice. The Journal of clinical investigation 2007, 117(10), 2913-2919). However, knockout of Nox isoforms 1 and 2 showed increased survival and delayed disease onset. On the other hand, treatment with the broad-spectrum inhibitor apocynin (Apo) in an in vitro model of ALS improved motor neuron survival 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), further highlighting that Nox inhibition may play a neuroprotective role in ALS. Pharmacological inhibition of Nox also showed benefit in an animal model of ALS, extending survival by almost 50% and increasing the number of motor neurons in the spinal cord (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). In this case, reformulation of Nox inhibitors could lead to increased efficacy and extend the neuroprotective effects already described in Apo, further strengthening the potential applications of the present disclosure.

[0126] Table 1 shows the samples used in the assays of the present disclosure.

[0127] [Table 1]

[0128] In one embodiment, cholinium salts were used as cation sources and 3-cyclohexyl-5-(2,4-dihydroxybenzylidene)-1-methyl-2-thiohydantoin (N1(4)inh) as anion sources for the synthesis of [Chol]2[N1(4)inh]. [Chol]2[N1(4)inh] was synthesized by metathesis reaction of cholinium bicarbonate (80% in water, m / v) with 1:1.05 molar ratio of N1(4)inh solution prepared in minimum amount of absolute ethanol. N1(4)inh was added dropwise and mixed into the cholinium bicarbonate solution under dark cooling condition (using ice bath at ∼5 °C) with continuous stirring. The reaction mixture was further kept under continuous stirring at ∼5 °C for 2 h. Excess solvent and water were removed under a continuous flow of nitrogen gas until complete drying (~2-3 h nitrogen gas flow with the IL-containing bottle kept in an ice-cold water bath (~10 °C)). The synthesized [Chol]2[N1(4)inh] and N1(4)inh mixture (1:1, mol:mol) were finally recovered from the ice-cold water bath and stored under dry, cooled and dark conditions.

[0129] In one embodiment, FIG. 1 H NMR spectra are shown for: 2a) a specific Nox1(4) inhibitor (N1(4)inh) and 2b) an IL-based formulation of a specific Nox1(4) inhibitor ([Chol]2[N1(4)inh]+N1(4)inh).

[0130] [Chol]2[N1(4)inh]+N1(4)inh purity 1 The compound was evaluated by H NMR. 1 H NMR spectrum and respective peak identification of specific Nox1(4) inhibitor (N1(4)inh), FIG. 1b shows 1We reveal the H NMR spectra and the respective peak identifications of an IL-based formulation of a specific Nox1(4) inhibitor ([Chol]2[N1(4)inh]+N1(4)inh) and disclose that [Chol]2[N1(4)inh] has been successfully synthesized.

[0131] In one embodiment, FIG. 13 C NMR spectra are shown for: 3a) a specific Nox1(4) inhibitor (+N1(4)inh) and 3b) an IL-based formulation of a specific Nox1(4) inhibitor ([Chol]2[N1(4)inh]+N1(4)inh).

[0132] [Chol]2[N1(4)inh]+N1(4)inh and N1(4)inh purity 13 The results were evaluated by C NMR. 13 C NMR spectrum and the respective peak identification of a specific Nox1(4) inhibitor (N1(4)inh) are disclosed, FIG. 2b shows 13 We reveal the C NMR spectrum and the respective peak identifications of an IL-based formulation of a specific Nox1(4) inhibitor ([Chol]2[N1(4)inh]+N1(4)inh) and disclose that [Chol]2[N1(4)inh]+N1(4)inh has been successfully synthesized.

[0133] In one embodiment, FIG. 3 shows the solubility of a Nox1(4) inhibitor (N1(4)inh) and an IL-based formulation ([Chol]2[N1(4)inh]+N1(4)inh) in water and PBS in mg / mL.

[0134] In one embodiment, solutes (N1(4)inh and [Chol]2[N1(4)inh]+N1(4)inh) were added to a fixed volume (500 μL) of water and PBS solutions. These mixtures were incubated at 37 °C under constant agitation at 1150 rpm and for a minimum of 72 hours using an Eppendorf Thermomixer Comfort instrument. Throughout the process, solutes were added to the mixtures as needed, i.e., until solution saturation was reached. All samples were filtered using a syringe filter (0.45 μm) to remove possible suspended solid particles. Quantification of N1(4)inh and [Chol]2[N1(4)inh]+N1(4)inh was performed by ultraviolet spectroscopy using UV spectrophotometry (SYNERGY|HT microplate reader, BioTek) at wavelengths of 416 nm and 480 nm, respectively. Interference of the quantification method with PBS was also checked and blank control samples were always used. The results obtained revealed a 36-fold increase in the solubility of the IL-based formulation in water and a 21-fold increase in solubility in PBS.

[0135] In one embodiment, FIG. 4 shows the solubility in water and PBS in mol / L of a Nox1(4) inhibitor (N1(4)inh) and an IL-based formulation ([Chol]2[N1(4)inh]+N1(4)inh).

[0136] In one embodiment, solutes (N1(4)inh and [Chol]2[N1(4)inh]+N1(4)inh) were added to a fixed volume (500 μL) of water and PBS solutions. These mixtures were incubated at 37 °C under constant agitation at 1150 rpm and for a minimum of 72 h using an Eppendorf Thermomixer Comfort instrument. Throughout this process, solutes were added to the mixtures as needed, i.e., until solution saturation was reached. All samples were filtered using a syringe filter (0.45 μm) to remove possible suspended solid particles. The results were analyzed by UV spectrophotometry (SYNERGY|HT microplate reader, 0.4 μm) at wavelengths of 416 nm and 480 nm, respectively. Quantification of N1(4)inh and [Chol]2[N1(4)inh]+N1(4)inh was performed by UV spectroscopy using a ELISA kit (Dilution Kit, BioTek). Interference of the quantification method with PBS was also checked and a blank control sample was always used. The results obtained reveal that the solubility of the IL-based formulation in water increased 28-fold, whereas the solubility in PBS increased 16-fold.

[0137] In one embodiment, FIG. 5 shows the decomposition temperature of [Chol]2[N1(4)inh]+N1(4)inh as assessed by thermogravimetric analysis (TGA).

[0138] In one embodiment, the decomposition temperature was determined by thermogravimetric analysis (TGA). The TGA curves show a sharp decrease in the decomposition temperature around 200 °C, indicating that the decomposition of [Chol]2[N1(4)inh]+N1(4)inh starts at 200 °C, as also shown by the percursor.

[0139] In one embodiment, FIG. 6 shows the effect of [Chol]2[N1(4)inh]+N1(4)inh on the viability of N27 dopaminergic neuronal cells.

[0140] In one embodiment, the potential toxicity of [Chol]2[N1(4)inh]+N1(4)inh on N27 dopaminergic neurons was assessed 24 hours after exposure to 20 μM or 30 μM [Chol]2[N1(4)inh]+N1(4)inh. No statistically significant differences were observed between treated and control cells (CTR / untreated cells), demonstrating that the IL-based formulation, [Chol]2[N1(4)inh]+N1(4)inh, is not toxic to dopaminergic neurons. Data are expressed as percentage of CTR and shown as mean ± SEM of at least five replicates from 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. (1) Control cells; (2) Cells exposed to 20 μM [Chol]2[N1(4)inh] + N1(4)inh, (3) cells exposed to 30 μM [Chol]2[N1(4)inh] + N1(4)inh.

[0141] In one embodiment, FIG. 7 shows that pretreatment of dopaminergic neuronal cells (N27) with [Chol]2[N1(4)inh]+N1(4)inh significantly inhibited the neurotoxic effects of 6OHDA. (1) Control untreated cells. (2) Cholinium control. (3) 6OHDA significantly reduced the viability of dopaminergic neurons compared to control cells. (4) Pretreatment with [Chol]2[N1(4)inh]+N1(4)inh significantly reduced dopaminergic neurotoxicity induced by 6OHDA compared to 6OHDA-treated cells in (3). (5) Cholinium chloride did not prevent 6OHDA-induced neurotoxicity compared to 6OHDA-treated cells in (3). Cell viability was measured using the WST-8 assay in N27 cells pretreated with 20 μM [Chol]2[N1(4)inh]+N1(4)inh or cholinium chloride for 2.5 h and then exposed to 50 μM 6OHDA for 24 h. Data are presented as percentage of control and shown as mean ± SEM of at least five replicates from three independent experiments (n = 3). Statistical analysis was performed using one-way ANOVA (nonparametric analysis) followed by Kruskal-Wallis test and Dunn's multiple comparison test. **p<0.01 compared to cells exposed to CTR or cholinium chloride only, +++p<0.001 compared to 6OHDA. (B). (1) control cells, (2) cells exposed to cholinium chloride (20 μM), (3) cells exposed to 6OHDA (50 μM), (4) cells exposed to 6OHDA and [Chol]2[N1(4)inh]+N1(4)inh (20 μM), (5) cells exposed to 6OHDA (50 μM) and cholinium chloride (20 μM).

[0142] In one embodiment, FIG. 8 shows that pretreatment of dopaminergic neuronal cells (N27) with [Chol]2[N1(4)inh]+N1(4)inh significantly inhibited the neurotoxic effects of the neurotoxin MPP. (1) Control untreated cells. (2) Cholinium chloride control. (3) MPP+ significantly reduced the viability of dopaminergic neurons. (4) Pretreatment with [Chol]2[N1(4)inh]+N1(4)inh significantly reduced MPP+-induced dopamine neurotoxicity. (5) Cholinium chloride did not prevent MPP+-induced neurotoxicity. Cell viability was measured using the WST-8 assay in N27 cells pretreated with 20 μM [Chol]2[N1(4)inh]+N1(4)inh or cholinium chloride for 2.5 hours, followed by exposure to 10 μM MPP+ for 24 hours. Data are presented as the mean ± SEM of at least five replicates from three independent experiments (n = 3) and as percentage of control. Statistical analysis was performed using one-way ANOVA (non-parametric analysis), Kruskal-Wallis test followed by 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 cells exposed to CTR or cholinium chloride only. (1) Control cells, (2) cells exposed to cholinium chloride (20 μM), (3) cells exposed to MPP+ (10 μM), (4) cells exposed to MPP+ (10 μM) and [Chol]2[N1(4)inh]+N1(4)inh (20 μM), (5) cells exposed to MPP+ (10 μM) and cholinium chloride (20 μM).

[0143] In one embodiment, FIG. 9 shows the effect of intracerebroventricular injection of [Chol]2[N1(4)inh]+N1(4)inh on dopaminergic neuronal survival in mouse substantia nigra (SN).

[0144] In one embodiment, the change in the number of tyrosine hydroxylase (TH) immunoreactive neurons in the SN of mice 7 days after intracerebroventricular injection of 0.2 mg / kg / day [Chol]2[N1(4)inh]+N1(4)inh: (1) control group (saline only); (2) [Chol]2[N1(4)inh]+N1(4)inh treatment group. The number of TH positive neurons in the SN of mice treated with [Chol]2[N1(4)inh]+N1(4)inh was not statistically different from that quantified in the SN of the control group ((2) compared to (1)), indicating that an IL-based formulation of [Chol]2[N1(4)inh]+N1(4)inh is not toxic to dopaminergic neurons in vivo. Results are expressed as a percentage of saline (group (1) being animals exposed to saline only) and are presented as the mean ± SEM of at least three independent experiments (n = 3–4). Statistical analysis was performed using one-way ANOVA followed by multiple comparison analysis using the Bonferroni test. No statistical differences were observed between both groups.

[0145] In one embodiment, FIG. 10 shows that intracerebroventricular infusion of [Chol]2[N1(4)inh]+N1(4)inh prevents the death of dopaminergic neurons in the substantia nigra (SN) induced by intravenous injection of 6OHDA, an animal model of Parkinson's disease. Changes in the number of tyrosine hydroxylase (TH) immunoreactive neurons in the SN of mice 7 days after 6OHDA treatment with or without [Chol]2[N1(4)inh]+N1(4)inh. (1) Control group (saline only), (2) 6-OHDA treatment group, (3) 6OHDA and [Chol]2[N1(4)inh]+N1(4)inh co-treatment group. The number of TH-positive neurons was significantly reduced in animals treated with 6OHDA ((2) compared to (1)). This decrease was significantly suppressed in mice co-treated with 6OHDA and [Chol]2[N1(4)inh]+N1(4)inh ((3) compared with (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 that observed in the control group ((1) compared with (3)). Results are expressed as percentage of saline (group (1) being animals exposed to saline only) and as mean ± SEM of at least three independent experiments (n=3–4). Statistical analysis was performed using one-way ANOVA followed by multiple comparison analysis using the Bonferroni test. **p<0.01 when compared with the control group of animals ((2) compared with (1)) and ##p<0.01 when compared with the group of animals exposed to 6OHDA only ((3) compared with (2)).

[0146] In the embodiment, FIG. 11 shows the effect of intranasal administration of [Chol]2[N1(4)inh]+N1(4)inh for 14 days on mouse body weight. Time course of body weight change induced by intranasal administration of four different doses of [Chol]2[N1(4)inh]+N1(4)inh, i.e., 0.02, 0.04, 0.08, and 0.16 mg / kg / day twice daily for 14 days. Results are expressed as the mean of at least five independent experiments (n=5-6). Statistical analysis was performed using one-way ANOVA followed by multiple comparison analysis using Tukey's test. No statistical differences were observed between groups.

[0147] Figure 12 shows the effect of intranasal administration of [Chol]2[N1(4)inh]+N1(4)inh for 14 days on the motor performance of mice. Motor performance changes induced by intranasal administration of four different doses of [Chol]2[N1(4)inh]+N1(4)inh, namely (1) vehicle, (2) 0.02, (3) 0.04, (4) 0.08, and (5) 0.16 mg / kg / day twice daily for 14 days. Results are expressed as mean ± SEM of at least five independent experiments (n=5–6). Statistical analysis was performed using one-way ANOVA, followed by multiple comparison analysis using the Bonferroni test. No statistical differences were observed between groups.

[0148] Figure 13 shows the effect of intranasal administration of [Chol]2[N1(4)inh]+N1(4)inh for 14 days on mouse olfactory function. Olfactory function changes induced by intranasal administration of 0.16 mg / kg / day of [Chol]2[N1(4)inh]+N1(4)inh twice daily for 14 days. (1) Vehicle (untreated group), (2) 0.016 mg / kg / day [Chol]2[N1(4)inh]+N1(4)inh treatment group. Results are expressed as mean ± SEM of at least five independent experiments (n=5-6). Statistical analysis was performed using Unpaired Student T-test. No statistical differences were observed between groups.

[0149] Figure 14 shows the effect of intracerebroventricular administration of [Chol]2[N1(4)inh]+N1(4)inh for 30 days on rat body weight. Time course of body weight change induced by intracerebroventricular injection of 0.007 mg / kg / day of [Chol]2[N1(4)inh]+N1(4)inh once a day for 30 days. Results are expressed as the mean of at least four independent experiments (n=4-5). Statistical analysis was performed using one-way ANOVA, followed by multiple comparison analysis using the Bonferroni test. No statistical differences were observed between groups. Figure 15 shows the effect of intracerebroventricular administration of [Chol]2[N1(4)inh]+N1(4)inh for 30 days on motor performance in rats. Motor performance changes induced by intracerebroventricular injection of 0.007 mg / kg / day [Chol]2[N1(4)inh]+N1(4)inh once daily for 30 days. (1) Vehicle (untreated group), (2) 0.007 mg / kg / day [Chol]2[N1(4)inh]+N1(4)inh-treated group. Results are expressed as the mean of at least four independent experiments (n=4-5). Statistical analysis was performed using the Unpaired Student T-test. No statistical differences were observed between groups.

[0150] Figure 16 shows the effect of intranasal administration of [Chol]2[N1(4)inh]+N1(4)inh for 30 days on rat body weight. Body weight change induced by intranasal administration of 0.062 mg / kg / day [Chol]2[N1(4)inh]+N1(4)inh once a day for 30 days. Results are expressed as the mean of at least four independent experiments (n=4-5). Statistical analysis was performed using one-way ANOVA, followed by multiple comparison analysis using Tukey's test. No statistical differences were observed between groups.

[0151] Figure 17 shows the effect of intranasal administration of [Chol]2[N1(4)inh]+N1(4)inh for 30 days on the motor performance of rats. Motor performance changes induced by intranasal administration of 0.062 mg / kg / day [Chol]2[N1(4)inh]+N1(4)inh once a day for 30 days. (1) Vehicle (untreated group), (2) 0.062 mg / kg / day [Chol]2[N1(4)inh]+N1(4)inh-treated group. Results are expressed as the mean of at least four independent experiments (n=4-5). Statistical analysis was performed using the Unpaired Student T-test. No statistical differences were observed between groups.

[0152] Figure 18 shows the effect of intranasal administration of [Chol]2[N1(4)inh]+N1(4)inh for 30 days on olfactory function in rats. Olfactory function changes induced by intranasal administration of 0.062 mg / kg / day of [Chol]2[N1(4)inh]+N1(4)inh once a day for 30 days. (1) Vehicle (untreated group), (2) 0.062 mg / kg / day [Chol]2[N1(4)inh]+N1(4)inh treatment group. Results are expressed as mean ± SEM of at least four independent experiments (n=5-6). Statistical analysis was performed using Unpaired Student T-test. No statistical differences were observed between groups.

[0153] Figure 19 shows that intracerebroventricular injection of [Chol]2[N1(4)inh]+N1(4)inh inhibited the progression of PQ-induced motor dysfunction in rats, an animal model of PD. Changes in fall latency (latency to fall) in animals exposed to PQ for 30 days in the presence or absence of 0.007 mg / kg / day [Chol]2[N1(4)inh]+N1(4)inh were recorded for 30 days using the rotarod test. (1) Control group (saline only), (2) PQ-treated group, (3) PQ and [Chol]2[N1(4)inh]+N1(4)inh co-treated group. Animals treated with PQ showed a significant decrease in fall latency (latency to fall) (compared to (1) in (2)). This decrease was significantly prevented in rats co-treated with PQ and [Chol]2[N1(4)inh]+N1(4)inh ((2) compared with (3)). Falling latencies in rats co-treated with PQ and [Chol]2[N1(4)inh]+N1(4)inh were not statistically different from those observed in the control group ((1) compared with (3)). Results are expressed as mean ± SEM of at least four independent experiments (n = 4–6). Statistical analysis was performed using one-way ANOVA, followed by multiple comparison analysis using the Bonferroni test. ***p<0.001 when compared with the control animal group ((2) vs. (1)), ###p<0.001 when compared with the animal group exposed to PQ only ((3) vs. (2)).

[0154] Figure 20 shows that intracerebroventricular injection of [Chol]2[N1(4)inh]+N1(4)inh inhibited the progression of PQ-induced motor dysfunction in rats, an animal model of PD. Changes in the distance traveled in animals exposed to PQ for 30 days in the presence or absence of 0.007 mg / kg / day [Chol]2[N1(4)inh]+N1(4)inh were recorded for an additional 30 days using the open field test. (1) Control group (saline only), (2) PQ-treated group, (3) PQ and [Chol]2[N1(4)inh]+N1(4)inh co-treated group. Distance traveled was significantly decreased in animals treated with PQ (compared to (1) in (2)). This decrease was significantly prevented in rats co-treated with PQ and [Chol]2[N1(4)inh]+N1(4)inh ((2) compared with (3)). The distance traveled in rats co-treated with PQ and [Chol]2[N1(4)inh]+N1(4)inh was not statistically different from that observed in the control group ((1) compared with (3)). Results are expressed as the mean ± SEM of at least four independent experiments (n = 4–6). Statistical analysis was performed using one-way ANOVA, followed by multiple comparison analysis using the Bonferroni test. **p<0.01 when compared with the control animal group ((2) vs. (1)), #p<0.05 when compared with the animal group exposed to PQ only ((3) vs. (2)).

[0155] Figure 21 shows that intracerebroventricular infusion of [Chol]2[N1(4)inh]+N1(4)inh inhibited the progression of PQ-induced motor dysfunction in rats, an animal model of PD. Changes in speed in animals exposed to PQ for 30 days in the presence or absence of 0.007 mg / kg / day [Chol]2[N1(4)inh]+N1(4)inh were recorded for an additional 30 days using the open field test: (1) control group (saline only), (2) PQ-treated group, (3) PQ and [Chol]2[N1(4)inh]+N1(4)inh co-treated group. Speed ​​was significantly decreased in animals treated with PQ ((1) compared to (2)). This decrease was significantly prevented in rats co-treated with PQ and [Chol]2[N1(4)inh]+N1(4)inh ((2) compared to (3)). The speed of rats co-treated with PQ and [Chol]2[N1(4)inh]+N1(4)inh was not statistically different from that observed in the control group ((1) compared with (3)). Results are expressed as the mean ± SEM of at least four independent experiments (n = 4–6). Statistical analysis was performed using one-way ANOVA, followed by multiple comparison analysis using the Bonferroni test. **p<0.01 when compared with the control animal group ((2) vs. (1)), #p<0.05 when compared with the animal group exposed to PQ only ((3) vs. (2)).

[0156] Figure 22 shows that intranasal administration of [Chol]2[N1(4)inh]+N1(4)inh inhibited the progression of PQ-induced motor dysfunction in rats, an animal model of PD. Changes in fall latency (time to fall) in animals exposed to PQ for 30 days in the presence or absence of 0.062 mg / kg / day [Chol]2[N1(4)inh]+N1(4)inh were recorded for an additional 30 days using the Rotarod test. (1) Control group (saline only), (2) PQ-treated group, (3) PQ and [Chol]2[N1(4)inh]+N1(4)inh co-treated group. Animals treated with PQ showed a significant decrease in the time to fall (time to fall) ((2) compared to (1)). This decrease was significantly prevented in rats co-treated with PQ and [Chol]2[N1(4)inh]+N1(4)inh ((2) compared with (3)). Falling latencies in rats co-treated with PQ and [Chol]2[N1(4)inh]+N1(4)inh were not statistically different from those observed in the control group ((1) compared with (3)). Results are expressed as the mean ± SEM of at least four independent experiments (n = 4–6). Statistical analysis was performed using one-way ANOVA, followed by multiple comparison analysis using the Bonferroni test. **p<0.01 when compared with the control animal group ((2) vs. (1)); #p<0.05 when compared with the animal group exposed to PQ only ((3) vs. (2)).

[0157] Whenever used in this document, the term "comprising" is intended to indicate the presence of stated features, integers, steps, components, but does not exclude the presence or addition of one or more other features, integers, steps, components, or groups thereof.

[0158] When the singular form of an element or feature is used in the description of the claims, the plural form is also included, and vice versa, unless specifically excluded. For example, the term "an ionic liquid" or "the ionic liquid" includes the plural forms of "ionic liquid" or "the ionic liquid", and vice versa. In the claims, articles such as "a", "an" and "the" may mean one or more than one, unless stated otherwise or otherwise clear from the context. A claim or specification containing "or" between one or more members of a group is considered to be satisfied if one, more than one, or all of the group members are present in, used in, or otherwise related to a given product or process, unless stated otherwise or otherwise clear from the context. The invention includes embodiments in which exactly one member of the group is present in, used in, or otherwise related to a given product or process. The invention also includes embodiments in which two or more or all of the group members are present in, used in, or otherwise related to a given product or process.

[0159] Furthermore, when a claim recites a composition, it is understood that it includes methods of using that composition for any of the purposes disclosed herein, and methods of making that composition according to any of the methods of making disclosed herein or other methods known in the art, unless otherwise stated or where a contradiction or inconsistency would arise is apparent to one of ordinary skill in the art.

[0160] The present disclosure is not limited to the described embodiments, and those skilled in the art foresee many possibilities for modifying it.

[0161] The above-described embodiments can be combined.

[0162] The following claims further describe certain embodiments of the present disclosure.

Claims

1. formula: 【Chemistry 1】 an ionic liquid comprising an anion having the formula: R is an alkyl or cycloalkyl group, and An ionic liquid wherein the cation is selected from the list consisting of cholinium, tetraalkylammonium, tetraalkylphosphonium, or 1-alkyl-3-methylimidazolium cation families.

2. 2. The ionic liquid of claim 1, wherein the molar ratio of the anion to the cation is in the range of 1:2 to 2:1 (mol:mol), preferably 1:1.5 to 1.5:1 (mol:mol).

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

4. 2. The ionic liquid according to claim 1, wherein R is a C3 to C7 cycloalkyl group.

5. 2. The ionic liquid according to claim 1, wherein R is an unsubstituted cycloalkyl group from C3 to C7.

6. 2. The ionic liquid of claim 1, wherein R is a cyclohexyl group.

7. 2. The ionic liquid of claim 1, wherein the cation is cholinium.

8. 2. The ionic liquid of claim 1, comprising 3-substituted 5-benzylidene-1-methyl-2-thiohydantoin or 3-cyclohexyl-5-(2,4-dihydroxybenzylidene)-1-methyl-2-thiohydantoin.

9. 10. The ionic liquid of claim 1 for use as a pharmaceutical or drug.

10. 10. The ionic liquid of claim 1 for use in preventing, slowing the progression of, or treating a disease or disorder of the central nervous system.

11. 10. The ionic liquid of claim 1 for use in the prevention or treatment of neurodegenerative diseases, cognitive impairment, dementia, or multiple system atrophy.

12. 12. The ionic liquid for use according to claim 11, wherein the neurodegenerative disease is Parkinson's disease.

13. 10. The ionic liquid of claim 1 for use in slowing the progression of Parkinson's disease.

14. 10. The ionic liquid of claim 1 for use in preventing motor dysfunction in Parkinson's disease.

15. 10. The ionic liquid according to claim 1 for use in the prevention or treatment of amyotrophic lateral sclerosis.

16. 10. The ionic liquid of claim 1 for use in the prevention or treatment of neurovascular diseases, preferably stroke.

17. A pharmaceutical composition comprising a therapeutically effective amount of the ionic liquid according to any one of claims 1 to 16 and a pharmaceutically acceptable carrier.

18. 18. The pharmaceutical composition of claim 17, wherein the pharmaceutically acceptable carrier is saline buffer, PBS, or water, or a mixture thereof.

19. 18. The pharmaceutical composition according to claim 17, comprising the ionic liquid in an amount ranging from 0.005 mM to 10 mM, preferably from 0.1 to 5 mM.

20. 18. The pharmaceutical composition according to claim 17, in an injectable, intranasal, intrathecal or intracerebroventricular form, preferably in intranasal, intracerebroventricular or intrathecal form.

21. 18. A pharmaceutical composition according to claim 17, for administration in a daily dose to a person with a disease or disorder of the central nervous system, preferably for a period of 30 days or more.

22. 22. The pharmaceutical composition of claim 21, wherein the amount administered is 1000 mg / day or less.

23. 23. The pharmaceutical composition of claim 22, wherein the amount administered ranges from 0.05 to 1000 mg / day.

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