Treatment

Abstract

A compound of formula I for use in a method for treating or preventing synucleinopathy is provided, comprising administering a therapeutically effective amount of said compound (I): or a pharmaceutically acceptable salt or hydrate thereof, to a subject.

Description

Technical Field

[0001] The present disclosure relates to a treatment for treating synucleinopathy diseases.

Background Art

[0002] Parkinson's disease (PD) is an alpha-synucleinopathy disease and an incurable neurodegenerative disorder with an increasing prevalence. According to reports, up to 10 million people worldwide, including approximately 150,000 in the UK, are affected by PD. Furthermore, PD is more likely to be seen in the elderly population and is more frequently encountered by people over 65 years old. At the same time, the approved pharmacological treatments for PD are used to correct dopamine deficiency in the brains of PD patients, and dopamine decreases after cell damage and subsequent death of dopaminergic neurons in the midbrain region of the human brain. However, these pharmacological treatments are not disease-modifying and often cause severe side effects including involuntary movements and hallucinations. Furthermore, the underlying causes behind dopaminergic neuron death are not well understood, and abnormal calcium concentrations, alpha-synuclein (α-syn) aggregation, and an inflammatory environment are prominent examples of important proposed factors in PD pathogenesis.

[0003] Therefore, in light of the foregoing considerations, there is a need to provide improved methods and pharmaceutical compounds for treating synucleinopathy diseases, particularly PD, and further reducing its symptoms.

Summary of the Invention

[0004] As demonstrated by the in vitro and in vivo data presented herein, the inventors have found that the compounds described herein have utility for treating and preventing synucleinopathy diseases such as PD.

[0005] In a first aspect, the present disclosure provides a compound for treating or preventing a synucleinopathy disease according to claim 1.

[0006] In a second aspect, the present disclosure provides a kit for treating or preventing a synucleinopathy disease as described in claim 12.

[0007] In a third aspect, the present disclosure provides a method for treating or preventing a synucleinopathy disease as described in claim 15.

[0008] In a fourth aspect, the disclosure provides the use of a compound for treating or preventing the synucleinopathy disease described in claim 16.

[0009] Throughout this description and the claims, the terms “comprise,” “include,” “have,” and “contain,” as well as variations thereof, such as “comprising” and “comprises,” mean “including but not limited to,” and do not exclude the existence of other components, items, integers, or steps not expressly disclosed. Furthermore, the singular form includes the plural form unless the context requires otherwise. In particular, where the indefinite article is used, this specification is understood to intend both the plural and the singular unless the context requires otherwise. [Brief explanation of the drawing]

[0010] [Figure 1]Figure 1A illustrates HPLC and electrochemical detection analysis of striatal monoamines (dopamine metabolites: 3,4-dihydroxyphenylacetic acid, DOPAC, and homovanylic acid, HVA) in the striatum from an in vivo study performed in Example 1, as well as tyrosine hydroxylase (TH, a marker of dopaminergic neurons) immunostaining. Figure 1B illustrates HPLC and electrochemical detection analysis of striatal monoamines (dopamine metabolites: 3,4-dihydroxyphenylacetic acid, DOPAC, and homovanylic acid, HVA) in the striatum from an in vivo study performed in Example 1, as well as tyrosine hydroxylase (TH, a marker of dopaminergic neurons) immunostaining. Figure 1C illustrates HPLC and electrochemical detection analysis of striatal monoamines (dopamine metabolites: 3,4-dihydroxyphenylacetic acid, DOPAC, and homovanylic acid, HVA) in the striatum from an in vivo study performed in Example 1, as well as tyrosine hydroxylase (TH, a marker of dopaminergic neurons) immunostaining. [Figure 2] Figure 2A: Illustration of Iba1 immunostaining from an in vivo study performed in Example 1. Figure 2B: Illustration of Iba1 immunostaining from an in vivo study performed in Example 1. [Figure 3] Figure 3A: Illustrates the cerebrospinal fluid (CSF) cytokine / chemokine profile analysis from the in vivo study performed in Example 1. Figure 3B: Illustrates the cerebrospinal fluid (CSF) cytokine / chemokine profile analysis from the in vivo study performed in Example 1. [Figure 4-1] Figure 4A: Exemplifies the reduction in α-syn aggregation and phosphorylation of the serine amino acid residue located at position 129 of the α-syn protein sequence, associated with pathological α-syn in human PD, in rat and human astrocytes grown in the presence of other cell types such as neurons, resulting from treatment with 30 nM DG. Figure 4B: Exemplifies the reduction in α-syn aggregation and phosphorylation of the serine amino acid residue located at position 129 of the α-syn protein sequence, associated with pathological α-syn in human PD, in rat and human astrocytes grown in the presence of other cell types such as neurons, resulting from treatment with 30 nM DG. [Figure 4-2] Figure 4C illustrates the reduction in α-syn aggregation and phosphorylation of the serine amino acid residue located at position 129 of the α-syn protein sequence, associated with pathological α-syn in human PD, in rat and human astrocytes proliferated in the presence of other cell types such as neurons, resulting from treatment with 30 nM DG. [Figure 5] Figure 5A: Illustrates three aspects of the therapeutic effect of 30 nM DG in a cell model of synucleinopathy. Figure 5B: Illustrates three aspects of the therapeutic effect of 30 nM DG in a cell model of synucleinopathy. Figure 5C: Illustrates three aspects of the therapeutic effect of 30 nM DG in a cell model of synucleinopathy. Figure 5D: Illustrates three aspects of the therapeutic effect of 30 nM DG in a cell model of synucleinopathy. [Figure 6] Figure 6A: Illustrates the pharmacodynamic effects of DG in rat brain resulting from treatment with 10 mg / kg DG under intraperitoneal inflammatory load induced by LPS injection, namely preservation of astrocyte junctions and changes in the release of inflammatory cytokines and chemokines in rat CSF, namely a decrease. Figure 6B: Illustrates the pharmacodynamic effects of DG in rat brain resulting from treatment with 10 mg / kg DG under intraperitoneal inflammatory load induced by LPS injection, namely preservation of astrocyte junctions and changes in the release of inflammatory cytokines and chemokines in rat CSF, namely a decrease. [Figure 7-1] Figure 7A: Changes in Cx43 point or Cx43 fluorescence intensity per astrocyte or per cell in cell culture models subjected to various stresses related to the pathogenesis of PD over a period ranging from 2 days to 2.5 weeks in rat and human cells, illustrating downregulation. [Figure 7-2]Figure 7B: Exemplifies the change in Cx43 point or Cx43 fluorescence intensity per astrocyte or per cell, i.e., downregulation, in cell culture models of rat and human cells subjected to various stresses related to the pathogenesis of PD over a period ranging from 2 days to 2.5 weeks. Figure 7C: Exemplifies the change in Cx43 point or Cx43 fluorescence intensity per astrocyte or per cell, i.e., downregulation, in cell culture models of rat and human cells subjected to various stresses related to the pathogenesis of PD over a period ranging from 2 days to 2.5 weeks. Figure 7D: Exemplifies the change in Cx43 point or Cx43 fluorescence intensity per astrocyte or per cell, i.e., downregulation, in cell culture models of rat and human cells subjected to various stresses related to the pathogenesis of PD over a period ranging from 2 days to 2.5 weeks. [Figure 8] Figure 8A: Illustrates changes in Cx43 point or Cx43 fluorescence intensity per astrocyte or per cell, i.e., downregulation, in cell culture models of rat and human cells subjected to various stresses related to the pathogenesis of PD over a period ranging from 2 days to 2.5 weeks. Figure 8B: Illustrates changes in Cx43 point or Cx43 fluorescence intensity per astrocyte or per cell, i.e., downregulation, in cell culture models of rat and human cells subjected to various stresses related to the pathogenesis of PD over a period ranging from 2 days to 2.5 weeks. [Figure 9-1] Figure 9A: Illustrates changes in Cx43 points or Cx43 fluorescence intensity per cell, i.e., downregulation, in two rat models of α-syn aggregation related to the pathogenesis of human PD in several brain regions associated with the onset of motor symptoms in PD, over periods of 12–18 months and 9 months, respectively. [Figure 9-2]Figure 9B: Illustrates changes in Cx43 points or Cx43 fluorescence intensity per cell, i.e., downregulation, in two rat models of α-syn aggregation related to the pathogenesis of human PD in several brain regions associated with the onset of motor symptoms in PD, over periods of 12–18 months and 9 months, respectively. Figure 9C: Illustrates changes in Cx43 points or Cx43 fluorescence intensity per cell, i.e., downregulation, in two rat models of α-syn aggregation related to the pathogenesis of human PD in several brain regions associated with the onset of motor symptoms in PD, over periods of 12–18 months and 9 months, respectively. [Figure 9-3] Figure 9D: Illustrates changes in Cx43 points or Cx43 fluorescence intensity per cell, i.e., downregulation, in two rat models of α-syn aggregation related to the pathogenesis of human PD in several brain regions associated with the onset of motor symptoms in PD, over periods of 12–18 months and 9 months, respectively. Figure 9E: Illustrates changes in Cx43 points or Cx43 fluorescence intensity per cell, i.e., downregulation, in two rat models of α-syn aggregation related to the pathogenesis of human PD in several brain regions associated with the onset of motor symptoms in PD, over periods of 12–18 months and 9 months, respectively. [Figure 10]Figure 10A: Exemplifies changes in Cx43 points or Cx43 fluorescence intensity per cell, i.e., downregulation, in two rat models of α-syn aggregation related to the human PD pathogenesis in several brain regions associated with the onset of motor symptoms in PD, over periods of 12–18 months and 9 months, respectively. Figure 10B: Exemplifies changes in Cx43 points or Cx43 fluorescence intensity per cell, i.e., downregulation, in two rat models of α-syn aggregation related to the human PD pathogenesis in several brain regions associated with the onset of motor symptoms in PD, over periods of 12–18 months and 9 months, respectively. Figure 10C: Exemplifies changes in Cx43 points or Cx43 fluorescence intensity per cell, i.e., downregulation, in two rat models of α-syn aggregation related to the human PD pathogenesis in several brain regions associated with the onset of motor symptoms in PD, over periods of 12–18 months and 9 months, respectively. [Figure 11-1] Figure 11A: Illustrates functional changes in cortical rat astrocytes in cell culture, showing a decrease in gap junctions (GJs) within the astrocyte retinoplasm in response to inflammation and α-syn loading, which are associated with the pathogenesis of human Parkinson's disease. [Figure 11-2] Figure 11B: Illustrates functional changes in cortical rat astrocytes in cell culture, showing a decrease in gap junctions (GJs) within the astrocyte reticular network in response to inflammation and α-syn loading, which are associated with the pathogenesis of human Parkinson's disease. Figure 11C: Illustrates functional changes in cortical rat astrocytes in cell culture, showing a decrease in gap junctions (GJs) within the astrocyte reticular network in response to inflammation and α-syn loading, which are associated with the pathogenesis of human Parkinson's disease. [Figure 12-1] Figure 12A: Illustrations of changes in per-cell Cx43 points or Cx43 protein levels, i.e., downregulation, in human PD from several brain regions associated with the development of motor and non-motor symptoms. Figure 12B: Illustrations of changes in per-cell Cx43 points or Cx43 protein levels, i.e., downregulation, in human PD from several brain regions associated with the development of motor and non-motor symptoms. [Figure 12-2]Figure 12C: Illustrates the change in Cx43 puncta or Cx43 protein level per cell in human PD from several brain regions associated with the onset of motor and non-motor symptoms, i.e., downregulation. Figure 12D: Illustrates the change in Cx43 puncta or Cx43 protein level per cell in human PD from several brain regions associated with the onset of motor and non-motor symptoms, i.e., downregulation.

Mode for Carrying Out the Invention

[0011] The following detailed description illustrates embodiments of the present disclosure and the ways in which they may be implemented. Although several modes of executing the present disclosure are disclosed, those skilled in the art will recognize that other embodiments for executing or practicing the present disclosure are also possible.

[0012] In a first aspect, the present disclosure provides a compound of formula I for treating or preventing synucleinopathy diseases, wherein the compound of formula I is

Chemical Formula

[0013] As used herein, the term "treatment" or "treating" refers to a therapeutic (curative) treatment, including halting or slowing the progression of a disease. As used herein, the term "prevention" or "preventing" refers to a "preventive" treatment, including administering a compound of the present invention to a patient in the prodromal or early disease stage of Parkinson's disease.

[0014] "Patient" and "subject" are used synonymously and refer to a subject to whom a compound of the present invention is administered. Preferably, the subject is human.

[0015] Preferably, the compound of formula I is for administration via a non-invasive route. Preferably, the non-invasive route is oral, intranasal, sublingual, inhalation, rectal, or transdermal.

[0016] Preferably, the synucleinopathic disease is selected from Parkinson's disease, Lewy body dementia (DLB), and multiple system atrophy (MSA). Preferably, the disease is Parkinson's disease.

[0017] Preferably, the compounds of formula I are intended for use in reducing and / or mitigating the progression of one or more symptoms associated with synucleinopathy, particularly Parkinson's disease. Preferably, the one or more symptoms are selected from motor symptoms (such as tremors, rigidity, and bradykinesia), memory impairment, depression, psychotic symptoms (such as hallucinations and delusions), sleep disorders, and dementia.

[0018] Preferably, the patient presents with one or more symptoms selected from motor symptoms (such as tremors, rigidity, and bradykinesia), memory impairment, depression, psychotic symptoms (such as hallucinations and delusions), sleep disturbances, and dementia.

[0019] In a second embodiment, a kit for treating or preventing synucleinopathy, comprising a compound of formula I: [ka] A kit is provided containing either a pharmaceutically acceptable salt or hydrate thereof.

[0020] Beneficially, the disclosed kit is configured to provide effective delivery of a therapeutically effective dose of a drug, such as danegaptide (DG), to a target. Preferably, delivery is via a non-invasive route, such as by using a delivery system, in which case DG is encapsulated within a phospholipid delivery vehicle, i.e., a liposome. Such a delivery method is expected to enhance drug delivery across the blood-brain barrier of the target.

[0021] Optionally, this system is configured to provide relief when synucleinopathy is characterized by an increase in at least one of the following: inflammatory cytokines / chemokines, or misfolded α-syn accumulation.

[0022] Inflammatory cytokines and chemokines are understood to be small signaling proteins that play a crucial role in regulating immune responses and inflammation. Selectively, inflammatory cytokines include, but are not limited to, tumor necrosis factor α (TNF-α), interleukin-1 beta (IL-1β), interleukin-6 (IL-6), tumor necrosis factor receptor superfamily member 21 / death receptor 6 (TNR21 / TNFRSF21 / DR6), and interferon-gamma-inducible protein 10 (IP-10 / CXCL10). Increased levels of inflammatory cytokines / chemokines suggest a state of chronic inflammation in the central nervous system (CNS), i.e., neuroinflammatory conditions, which can contribute to symptoms such as nerve damage, inflammation-mediated cell death, impaired normal brain function, memory impairment, depression, psychotic symptoms, hallucinations, sleep disturbances, and dementia. Typically, neuroinflammation is a complex process involving the activation of immune cells, including microglia (resident immune cells of the CNS), as well as astrocyte responses or atrophic changes, which are involved in the pathogenesis and progression of various neurological disorders. Similarly, the accumulation of misfolded α-syn proteins, which are abundant in the brain, particularly in presynaptic terminals (which can be involved in regulating synaptic function), leads to the formation of protein deposits called Lewy bodies (LBs), resulting in LB diseases. An inflammatory environment may contribute to the misfolding and aggregation of α-syn. Misfolded α-syn can disrupt cellular processes, impair neuronal function, increase intracellular calcium, and contribute to neurodegeneration. Furthermore, misfolded α-syn aggregates may further promote the inflammatory response, induce the release of pro-inflammatory cytokines, and activate immune cells in the CNS.

[0023] While we do not wish to be constrained by theory, the interaction between misfolded α-syns and inflammation is complex and can generate a self-persistent cycle of symptoms that need to be alleviated. Inflammatory processes can promote the accumulation and increased distribution of misfolded α-syns, and consequently, misfolded α-syns can induce an inflammatory response. Such interactions between inflammation and α-syn pathology are thought to contribute, for example, to the progressive neurodegeneration of dopaminergic neurons observed in Parkinson's disease (PD), as well as to the associated motor and non-motor symptoms of synucleinopathies.

[0024] In another embodiment, a method for treating or preventing synucleinopathy, comprising a therapeutically effective amount of a compound of formula I: [ka] A method is provided which includes administering to a patient a pharmaceutically acceptable salt or hydrate thereof.

[0025] In this specification, the term “compound” as used throughout this disclosure means a therapeutic active ingredient or pharmaceutically active ingredient (API) or a pharmaceutically acceptable salt or hydrate (or ester and prodrug) thereof that can be administered to a target for effective and sustained release at a target site, either alone or in combination with any other component known in the art (such as one or more pharmaceutically acceptable carriers, diluents, buffers, preservatives, stabilizers, or excipients) in the form of a preferred pharmaceutical composition. In this specification, the term “effective and sustained release” means the mechanism of delivery of the compound to the target area over several hours or days after administration of the compound via its preferred route of administration. It will be understood that subsequent administrations of the compound (after the initial administration) may occur following the effective and sustained release at the target site.

[0026] In alternative embodiments, the AAP10 peptide, consisting of the sequence Ac-DTyr-DPro-DHyp-Gly-DAla-Gly-NH2, and / or a drug called rotigaptide, or a pharmaceutically acceptable salt or hydrate thereof, are also disclosed for use in the treatment or prevention of the synucleinopathy diseases described herein.

[0027] Optionally, pharmaceutically acceptable salts of danegaptide (DG) include danegaptide (DG) hydrochloride.

[0028] In alternative embodiments, the present invention relates to compositions comprising a compound of formula I, or a pharmaceutically acceptable salt or hydrate thereof, for use in the treatment or prevention of synucleinopathy, wherein the compound of formula I is the sole activator in the composition. Being the sole activator means that the composition does not contain any other components that may be used in the treatment or prevention of synucleinopathy, preferably Parkinson's disease.

[0029] The compositions of the present invention comprising the compound of formula I may further contain a pharmaceutically acceptable carrier that forms a pharmaceutical composition. “pharmaceutically acceptable carrier” means any diluent or excipient, such as a filler or binder, that is compatible with the other components of the composition and is not harmful to the recipient. The pharmaceutically acceptable carrier can be selected based on the desired route of administration, in accordance with standard pharmaceutical practice.

[0030] While we do not wish to be constrained by theory, the technical effect of using danegaptides as gap junction intercellular signaling (GJIC) regulators is thought to be that these compounds may provide better intercellular bonding, allow cells to share available energy (in the form of ATP), and enable intercellular signaling, including calcium signaling. Additionally, DG acts to alter the Cx43 conformation in a way that prevents hemichannel (HC) opening and enhances gap junction (GJ) opening, thus limiting inflammasome activation and the release of inflammatory mediators from cells such as astrocytes.

[0031] Optionally, the compound is encapsulated in a liposome. A liposome is a vesicle composed of a lipid bilayer that can encapsulate a compound within its aqueous core or lipid membrane. When used as a sustained-release formulation, liposomes allow the compound to cross the blood-brain barrier and engage with its target located within the brain parenchyma.

[0032] The technological benefit of liposomes is that liposomal encapsulation of a compound (i.e., danegaptide, preferably danegaptide hydrochloride) results in controlled release of the compound, i.e., danegaptide, preferably danegaptide hydrochloride, over extended periods, providing sustained and controlled drug delivery to maintain consistent therapeutic concentrations of the compound at target sites located within the brain parenchyma. Furthermore, the liposome membrane acts as a barrier, protecting the compound from external factors and enzymatic degradation or inactivation, improving its stability and extending its shelf life and efficacy. Moreover, liposomes can be designed to exhibit specific targeting properties. For example, functionalization of the liposome surface with ligands or antibodies can result in targeted delivery of the encapsulated compound to specific cells or tissues, enhancing its therapeutic efficacy and reducing potential side effects on non-target cells. Additionally, liposomes may help reduce the toxicity of the compound by reducing its exposure to non-target tissues or organs, thereby improving the therapeutic index of the compound.

[0033] In addition to liposomes, other sustained-release formulations mentioned, such as niosomes, microspheres, emulsions, or micelles, and liquid stabilizers, may potentially offer similar advantages, such as controlled release of compounds and improved stability, depending on the specific properties and requirements of the compounds.

[0034] In this specification, the term “pharmaceutically acceptable salt” refers to a compound having an acidic moiety formed with an organic or inorganic base, or a basic moiety formed with an organic or inorganic acid. Optionally, pharmaceutically acceptable salts having an acidic moiety include metal salts, e.g., alkali metal or alkaline earth metal salts, e.g., sodium, potassium, or magnesium salts; ammonia salts and organic amine salts formed with mono, di, or tribasic lower alkylamines such as morpholine, thiomorpholine, piperidine, pyrrolidine, ethyl tert-butylamine, diethylamine, diisopropylamine, triethylamine, tributylamine, or dimethylpropylamine, or mono, di, or tribasic hydroxy lower alkylamines such as mono, di, or triethanolamine; and internal salts. Optionally, pharmaceutically acceptable salts having a basic moiety include 1-(2-aminoacetyl)-4-benzoylaminopyrrolidine-2-carboxylic acid and its listed diastereomers. Such pharmaceutically acceptable salts having a basic moiety can be formed from the following acids: acetic acid, propionic acid, lactic acid, citric acid, tartaric acid, succinic acid, fumaric acid, maleic acid, malonic acid, mandelic acid, malic acid, phthalic acid, hydrochloric acid, hydrobromic acid, phosphoric acid, nitric acid, sulfuric acid, methanesulfonic acid, naphthalenesulfonic acid, benzenesulfonic acid, toluenesulfonic acid, camphorsulfonic acid, and the like.

[0035] As used herein, the terms “therapeutic dose” or “dosage” refer to the amount of a free, unbound compound that is capable of alleviating the symptoms of a given synucleinopathy (or pathology), and preferably, partially or completely normalizing physiological responses, such as astrocyte reticular junctions, in a subject suffering from the given synucleinopathy (or pathology). Optionally, the therapeutic dose is determined by those skilled in the art based on a variety of factors, including but not limited to the potency of the compound, the age and size of the subject, the body weight of the subject, the pharmacokinetic properties of the compound, and the route of administration.

[0036] Preferably, the dosage of the compound administered to the patient is 60 to 175 mg, such as 100 to 130 mg of the compound of formula (I), including 10 to 350 mg, preferably 20 to 300 mg, more preferably 30 to 250 mg, even more preferably 40 to 225 mg, even more preferably 50 to 200 mg, and even more preferably 75 to 150 mg (for example, as contained in a pharmaceutical composition).

[0037] Preferably, the dosage of the compound administered to the patient is 0.05 to 5 mg / kg, preferably 0.1 to 4.5 mg / kg, more preferably 0.3 to 4 mg / kg, even more preferably 0.5 to 3.5 mg / kg, even more preferably 0.7 to 3.5 mg / kg, and still more preferably 1 to 2.5 mg / kg, such as a compound of formula (I) at a dose of 0.9 to 3 mg / kg, for example, 1.1 to 2 mg / kg.

[0038] Optionally, the dose of the free compound in the brain or cerebrospinal fluid (CSF) that is expected to be "therapeutably effective" is in the range of 10 to 100 nM, and optionally substantially 30 nM. Optionally, the dose of the compound is 10, 20, 30, 40, 50, 60, 70, 80, or 90 nM to a maximum of 20, 30, 40, 50, 60, 70, 80, 90, or 100 nM, preferably 30 nM. Generally, the compound is administered in the range of about 10 to 100 nM.

[0039] The technical effect of the aforementioned doses of the compound into the brain, ranging from 10 to 100 nM, and optionally substantially 30 nM, is that within this specified dose range, the compound can effectively engage with its target molecule or receptor and produce the desired therapeutic outcome. The dose is adjusted according to the patient's body weight.

[0040] Optionally, the compound may be formulated in the form of a liquid phospholipid preparation by a process that may be known to those skilled in the art.

[0041] Optionally, a non-invasive route is intranasal. Healthcare professionals can consider these factors to determine the most appropriate and effective route of administration for a particular drug and patient.

[0042] Intranasal administration routes typically involve delivering compounds (i.e., drugs or medications) into the olfactory epithelial region through the nasal cavity. In this regard, compounds are usually administered using a suitable device and are formulated as sprays, solutions, or powders, often implemented as nasal spray devices. Through intranasal administration routes, compounds (or drugs or medications) can penetrate the brain parenchyma through the olfactory epithelium and / or be absorbed through the nasal mucosa, entering the systemic circulation, from which the compound can directly cross the blood-brain barrier.

[0043] The technical advantages of the intranasal route include efficient absorption of the compound into the brain and rapid onset of action. Furthermore, via the intranasal route, the compound bypasses the gastrointestinal (GI) tract and liver, avoiding the first-pass metabolism that occurs with oral administration. In addition, intranasal administration is relatively simple and non-invasive, making administration more convenient for the patient.

[0044] Optionally, administration of the compound or a pharmaceutically acceptable salt or hydrate may be carried out as a series of doses over time, as determined by the healthcare professional concerned with the patient. Alternatively, a series of dosing systems or sustained-release depots may be employed.

[0045] The technical advantages of the non-invasive route implemented as intranasal administration of the compound are that it is patient-friendly, does not require the assistance of a trained specialist, provides a better quality of life for the patient, and is suitable for chronic medication. In addition, the aforementioned route of administration ensures that an effective therapeutic amount of the compound is delivered to the target site, i.e., the brain parenchyma and across the blood-brain barrier, in order to produce the desired therapeutic effect, in addition to the potency of the compound.

[0046] Furthermore, in particular, excessive release of cytokines and chemokines into the extracellular environment and cerebrospinal fluid can contribute to neuroinflammation and neurological dysfunction. The compound is formulated to reduce the release of such signaling molecules from astrocytes into the extracellular environment, thereby mitigating neuroinflammation and helping to maintain a more balanced signaling environment.

[0047] Furthermore, inflammatory activation of astrocytes, markered by excessive nuclear phosphorylation of STAT3 at the amino acid tyrosine at position 705, can induce the release of pro-inflammatory cytokines and chemokines in response to certain stimuli, contributing to neuroinflammation. The compound is designed to attenuate inflammatory activation of astrocytes, thereby regulating the immune response in the central nervous system and promoting a more balanced and regulated state of homeostasis.

[0048] This disclosure also relates to the system described above. With respect to the aforementioned methods, the various embodiments and modifications disclosed above are applicable to this system with necessary modifications.

[0049] Detailed description of the examples and drawings Example 1 - In vivo proof of concept (PoC) using danegaptide (DG) in an inflammatory rat model of Parkinson's disease (PD). Model: "Inflammatory Parkinson's disease" (as described in Beier EE, et al., Neurobiol Dis. 2017 Dec;108:115-127. PMID:28823928).

[0050] Sprague-Dawley rat, male, 8 weeks old upon delivery, weighing approximately 180-200g upon delivery, approximately 250g at the start of the experiment (Janvier's Lab), 12-hour light / dark cycle, free access to food / water.

[0051] Lesions: Intraperitoneal (IP) injection of either lipopolysaccharide / endotoxin (LPS) derived from Salmonella abortus equi or saline (control) at a dose of 1 mg / kg / day in a total volume of 100 μl at the same time over the following four days.

[0052] Treatment: Liposome-encapsulated danegaptide (DG) was administered intranasally (IN) twice daily to awake rats up to approximately 10 mg / kg in a total volume of 100 μl, with 50 μl per nostril per dose. Treatment was initiated one day after completion of the lesion protocol and continued for 14 days. Control - Sterile PBS. N=12-15 per group

[0053] Objective: To determine the efficacy of translationally valid PD biomarkers.

[0054] Endpoints and results: 1. High-performance liquid chromatography (HPLC) analysis and electrochemical detection of striatal monoamines (dopamine metabolites) - Figure 1 A reduction in dopamine metabolites has been noted in human Parkinson's disease (PD) and various models in correlation with the onset of motor symptoms. Therefore, these results represent evidence of pro-inflammatory PD-related nigrostriatal pathway damage in the model. Statistics: One-way ANOVA with Tukey-Kramer post-hoc test, Shapiro-Wilk test to test the normality of the distribution. Includes outliers (if detected by Tukey's fence method). *p<0.05, error bars: SEM.

[0055] Important results: ●DOPAC (3,4-dihydroxyphenylacetic acid) content - Significant reduction in the "PD" group compared to the control group, significantly reduced by DG treatment (Figure 1A) ●HVA (homovanylic acid) content - Significant reduction in the "PD" group compared to the control group, partially rescued by DG treatment (Figure 1B).

[0056] 2. Striatal tyrosine hydroxylase (TH) immunohistochemistry - Figure 1 Thyroid hormone (TH) is an enzyme involved in dopamine production, and its downregulation is associated with motor symptoms in Parkinson's disease. This supports pro-inflammatory PD-related nigrostriatal pathway damage in the model. TH staining was performed using a DAB agent and analyzed by light microscopy. Statistics: One-way ANOVA with Tukey-Kramer post-hoc test, Shapiro-Wilk test to test the normality of the distribution. Includes outliers (if detected by Tukey's fence method). *p<0.05, error bars: SEM.

[0057] Important results: ● Moderate reduction (p=0.0848) in TH optical density in the "PD" group compared to the control group, and statistically significant preservation of TH in the DG treatment group compared to the "PD" group (Figure 1C).

[0058] 3. Iba1 immunohistochemical staining - Figure 2 Iba1 is a marker protein for a cell type called microglia, which is associated with inflammatory responses and immunomodulation in the brain. The "reactivity" of microglia (i.e., pro-inflammatory phenotype) can be assessed through their morphological characteristics, two of which were measured in this study. ●Shape Factor - Also called "Roundness," it ranges from 0 to 1 and is expressed by the formula 4π × Area / Perimeter. 2 The area is correlated to the surrounding area, with a value of 1 representing a more circular (presumably more reactive) cell. Higher values ​​= more pro-inflammatory phenotype. ● Solidity – also called "density," ranges from 0 to 1 and relates the cell's area to its convex hull (covered "territory"). When higher solidity correlates with a higher shape factor, this indicates hypertrophic cells associated with tissue damage. However, if it does not correlate with a higher shape factor, it may also indicate quiescent, highly branched cells. Statistics: One-way ANOVA with Tukey-Kramer post-hoc test, Shapiro-Wilk test to test the normality of the distribution. Includes outliers (if detected by Tukey's fence method). *p<0.05, **p<0.005, ****p<0.0001, error bars: SEM.

[0059] Important results: ● Striatal Iba1 shape factor (per rat) - Significantly increased in the "PD" group compared to the control group, significantly rescued by DG treatment (Figure 2A). ● Striatal Iba1 solidity (per rat) - Significantly increased in the "PD" group compared to the control group, significantly rescued by DG treatment (Figure 2B). The trend between shape factor and solidity correlates, suggesting changes in microglial responsiveness in the "PD" group that are reversed or prevented by DG treatment.

[0060] 4. Analysis of CSF cytokine / chemokine profiles - Figure 3 Inflammatory CSF cytokines / chemokines are translationally useful biomarkers of inflammatory changes in the CNS, which are described in PD and other neurodegenerative and psychiatric disorders. CSF screening is available in human trials as a surrogate marker of drug mechanism involvement / pharmacodynamic response to drugs. We performed the proteomics screening presented here using antibody-functionalized microarrays.

[0061] Statistics: A multifactor linear model was fitted via least-squares regression using LIMMA, yielding a two-sided t-test or F-test based on adjusted statistics. Subgroup factors, defined from the distribution of samples in principal component analysis, were used as additional factors in the linear model, following the sample groups. All presented p-values ​​were adjusted for multiple tests by controlling for false detection rates according to Benjamini and Hochberg. Differences in protein abundance between different samples or sample groups are presented as log-folded changes (logFC) calculated for base 2. Proteins with |logFC| > 0.5 and adjusted p-value < 0.05 are defined as derivatives and are shown in black in the volcano plot below. Proteins reaching the reduced threshold, when defined individually for each comparison, are defined as noteworthy and are shown in gray.

[0062] Important results: ●LIF, lactoferrin, IL13, and IL34 were found to be elevated in both the control and treatment groups (Figure 3A). This is consistent with the more anti-inflammatory cytokine profiles in these groups. ●TNR21 and prion proteins were upregulated in the "PD" group compared to the control and treatment groups (Figure 3B), indicating a pro-inflammatory cytokine environment in CSF associated with human Parkinson's disease.

[0063] conclusion Example 1 demonstrates that DG reversed Parkinson's disease-related phenotypes in an in vivo model. Therefore, it is demonstrated that DG may be effective in treating and preventing Parkinson's disease.

[0064] Example 2 - In vitro model related to Parkinson's disease (PD) The following experiments were conducted using rat and human cells. The results between these rat and human cell cultures were consistent, demonstrating translational conservation of the underlying mechanisms identified from rat studies to human studies. In summary, the findings of the following experiments indicate that α-syn aggregation and increased release of inflammatory cytokines and chemokines are associated with the pathogenesis of human Parkinson's disease (PD). The experiments also show that treatment with danegaptide prevents α-syn aggregation and increased release of inflammatory cytokines and chemokines.

[0065] As shown in Figures 4A-4C, treatment with 30 nM DG resulted in a reduction in α-syn aggregation and a reduction in the phosphorylated serine residue at position 129 of the α-syn protein sequence in rat and human astrocytes grown in the presence of other cell types, such as neurons, which are associated with pathological α-syn in human PD. Statistics: ANOVA with t-test or Tukey's multiple comparison test; Error bars: SEM.

[0066] Referring to Figures 5A-D, three aspects of the therapeutic effect of 30 nM DG in a cell model of synucleinopathy are shown. A: IP-10 cytokine released into the culture medium by lesioning rat cortical astrocytes-iNeurone co-culture with a-syn PFF for 3 weeks; B-C: calcium oscillations in lesioning rat cortical astrocytes with a-syn PFF for 3 days; baseline calcium levels in lesioning human cortical astrocytes with a-syn PFF for 3 days; D: nuclear STAT3 in lesioning rat cortical astrocytes with a-syn PFF for 3 days. Statistics: ANOVA with Tukey's multiple comparison test; error bars: SEM. These demonstrate the anti-inflammatory effect of DG (reduced nuclear STAT3 as a marker of reactive astrocytes, reduced cytokine release into the culture medium) and normalization of functional cell signaling correlated with reduced a-syn aggregation. Inflammatory markers also provide continuity with in vivo biomarkers in CSF and a rationale for their use.

[0067] Referring to Figures 6A-6B, we see increased GJ signaling as measured by Lucifer Yellow (LY) dye spread throughout the brain after stereotactic injection into the striatum, and a decrease in inflammatory cytokine and chemokine release in rat CSF subjected to inflammatory load (related to PD pathogenicity) induced intraperitoneally by 1 mg / kg LPS injection during treatment with 10 mg / kg DG delivered intranasally twice daily when encapsulated in liposomes, over a total lesion duration of 1.5 days. Statistics: ANOVA with Tukey's multiple comparison test or t-test with FDR control; Error bars: SEM.

[0068] Referring here to Figures 7 and 8, we see the downregulation of Cx43 points or Cx43 fluorescence intensity per astrocyte or per cell in rat and human cells subjected to various stresses related to the pathogenesis of PD over a period ranging from 2 days to 2.5 weeks, thus demonstrating translational conservation of the mechanism. Statistics: ANOVA with t-test and Tukey's multiple comparison test; Error bars: SEM.

[0069] Referring to Figures 7A–7D, changes in Cx43 points per astrocyte under various loads over 2 days to up to 2 weeks are shown. Cx43 protein levels and the number of Cx43 points per cell (analyzed via fluorescence microscopy) are studied using Cx43 fluorescence intensity. Hereinafter, the various loads are 100 ng / ml LPS and mechanical damage over 48 hours (7A, left panel), as well as α-syn preformative fibril (PFF) and amyloid-β (Am-β)PFF, i.e., misfold protein loads, at 0.1 ng per 10,000 cells over 72 hours (i.e., over 3 days) (7B, right panel). Referring to Figures 8A–8B, changes in Cx43 points per astrocyte under α-syn loads in rat cortical astrocyte-iNeurone coculture over 2.5 weeks are shown.

[0070] Referring here to Figures 9A–9E and 10A–10C, changes in Cx43 points or Cx43 fluorescence intensity per cell, i.e., downregulation, are shown in two rat models of α-syn aggregation related to the pathogenesis of human PD in several brain regions associated with the onset of motor symptoms in PD, over periods of 12–18 months and 9 months, respectively. Statistics: ANOVA with t-tests and Tukey's multiple comparison test; Error bars: SEM.

[0071] The first model was induced via peripheral injection of α-syn PFF along with the carrier protein RVG9R into the tail vein, after which rats developed several NMS symptoms, including progressive dopaminergic and cholinergic cell loss, loss of smell, and slowed digestion, as well as mild motor symptoms, over a period of 6–18 months post-injection. Cx43 points and fluorescence intensity per cell were measured in two brain regions associated with PD pathology: the substantia nigra (SN, group A9 of dopaminergic neurons identified by tyrosine hydroxylase staining) as shown in Figures 9A–9C, and the striatum as shown in Figures 9D–9E. A significant downward regulation of Cx43 points and fluorescence intensity was observed in the midbrain SN in α-syn-pathogenic animals at 12–18 months post-infection compared to non-pathogenic controls of the same age, while only Cx43 fluorescence intensity per cell was significantly reduced in the striatum, as shown in Figures 9D and 9E. A downward regulation of Cx43 points per cell is observed in the striatum, as shown in Figures 9D and 9E.

[0072] Referring to Figures 10A–10C, changes in Cx43 protein expression in a rat model of PD induced by α-syn PFF injection into the nucleus basalis of Meynert are shown. Brain samples were analyzed 9 months after injection, by which time these animals had begun to develop motor dysfunction and dopaminergic loss. Compared to sham lesion controls (as shown in Figures 10B and 10C), a significant reduction in Cx43 fluorescence intensity per cell and a tendency toward downregulation of Cx43 points per cell were observed in the striatum of PFF lesion animals.

[0073] Referring to Figures 11A–11C, functional changes in cortical rat astrocytes in cell culture are shown, indicating a decrease in GJ junctions within the astrocyte retinoplasm in response to inflammation and α-syn loading, which are associated with the pathogenesis of human Parkinson's disease. Statistics: ANOVA with Tukey's multiple comparison test; Error bars: SEM.

[0074] To evaluate the functional effects of changes in Cx43 protein levels and punctate distribution on astrocyte reticular junctions, a dye microinjection assay is performed using Lucifer Yellow (LY), a GJ-permeable dye that can be transported between astrocytes via Cx43. HC, composed of Cx43 protein, is blocked during the injection procedure using the HC blocker GAP19. As shown, a significant downregulation of cortical astrocyte junctions with both inflammation and α-syn PFF loading over 48 hours is visible, and similar results are obtained using midbrain and striatal astrocytes. Only fibrillated α-syn (both sonicated representing "oligomers" and unsonicated representing "fibrillaries"), and not monomeric α-syn, can induce GJ block (Figure 11C). Sonicated α-syn PFFs are used when "α-syn PFFs" are referred to in other studies.

[0075] Referring to Figures 12A–12D, changes in per-cell Cx43 points or Cx43 protein levels, i.e., downregulation, are shown in human PD from several brain regions associated with the development of motor and non-motor symptoms. Statistics: ANOVA with FDR correction, Error bars: SEM.

[0076] Figure 12B (upper panel) shows representative changes in TritonX-soluble Cx43 protein levels in postmortem human brain in PD compared to a control. Figure 12B (lower panel) shows total protein staining of the same membrane as the blot. Figure 12D is the quantification of Cx43 protein level analysis. Western blot analysis of the TritonX-soluble fraction is used to complement the microscopic study (Figures 12A, C). Gray matter from the parietal cortex, striatum, and midbrain SN is included for analysis. The TritonX-soluble fraction of Cx43 has been proposed to represent Cx43 on its way to the membrane in the Golgi apparatus and endoplasmic reticulum, and therefore serves as a marker of the ability of cells to produce Cx43 protein, although Cx43 in HC may also be captured. Due to the large number of samples, the Western blot gel is divided into four batches, with the parietal cortex sample serving as a loading control between all batches for semi-quantitative analysis of complex protein levels. This protein analysis revealed a similar pattern of significant Cx43 reduction in the parietal cortex, while striatal and midbrain samples showed less significant trends toward Cx43 downregulation in PD.

[0077] Referring to Figure 12C, local Cx43 protein expression in postmortem human brains is shown for control and PD, respectively. Midbrain samples present a Cx43 protein distribution that differs in appearance from punctate staining showing GJ plaques.

[0078] Quantitative microscopy in photon counting mode reveals a significant reduction in Cx43 points per cell (i.e., per nucleus) in PD compared to controls in frontal, parietal, and insular cortical gray matter samples, as well as in the globus pallidus. Significant local differences were also detected in cortical regions showing markedly high Cx43 point levels. This pattern altered in PD as the significant difference between the cortex and midbrain disappeared.

Claims

1. A compound of formula I for use in the treatment or prevention of synucleinopathy, wherein the compound of formula I is 【Chemistry 1】 A compound that is either a pharmaceutically acceptable salt or hydrate thereof.

2. The compound for use according to claim 1, wherein the use is therapeutic.

3. The compound for use according to claim 1 or 2, wherein the synucleinopathic disease is selected from Parkinson's disease, Lewy body dementia (DLB), and multiple system atrophy (MSA).

4. The compound for use according to claim 3, wherein the synucleinopathy is Parkinson's disease.

5. The compound for use according to any one of the prior claims, wherein the compound is danegaptide (DG) hydrochloride.

6. The compound for use according to any one of the prior claims, wherein the compound is for administration via an invasive route, preferably by injection.

7. The compound for use according to claim 6, wherein the injection is administered intraperitoneally, subcutaneously, intravenously, or intramuscularly.

8. The compound for use according to any one of claims 1 to 5, wherein the compound is for administration via a non-invasive route.

9. The compound for use according to claim 8, wherein the non-invasive route is oral, intranasal, sublingual, inhalation, rectal, or transdermal.

10. The compound for use according to any one of the prior claims, wherein the dosage of the compound of formula (I) is (a) 50 to 200 mg, such as 10 to 350 mg, more preferably 75 to 150 mg, and / or (b) 0.05 to 5 mg / kg, more preferably 0.7 to 3.5 mg / kg, for example 1.1 to 2 mg / kg.

11. The compound for use according to any one of the prior claims, wherein the patient exhibits one or more symptoms selected from motor symptoms (such as tremors, rigidity, and bradykinesia), memory impairment, depression, psychotic symptoms (such as hallucinations and delusions), sleep disorders, and dementia.

12. A kit for treating or preventing synucleinopathy, Compound of formula I: 【Chemistry 2】 A kit containing either a pharmaceutically acceptable salt or hydrate thereof.

13. The kit according to claim 12, wherein the kit further comprises a delivery system.

14. The kit according to claim 13, wherein the delivery system is configured to administer the drug non-invasively or invasively.

15. A method for treating or preventing synucleinopathy, comprising a therapeutically effective amount of a compound of formula I: 【Transformation 3】 A method comprising administering to a patient a pharmaceutically acceptable salt or hydrate thereof.

16. The use of a compound of formula I for the manufacture of a drug for treating or preventing synucleinopathy, wherein the compound of formula I is 【Chemistry 4】 or a pharmaceutically acceptable salt or hydrate thereof, for use.

17. A kit, method, or use according to any one of claims 12 to 16, having any one of the features of claims 1 to 11.

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