WO2025008625A1

WO2025008625A1 - Danegaptide for use in treating synucleinopathies

Info

Publication number: WO2025008625A1
Application number: PCT/GB2024/051728
Authority: WO
Inventors: Saifur Rahman; Nataly HASTINGS
Original assignee: Cambridge Enterprise Ltd
Current assignee: Cambridge Enterprise Ltd
Priority date: 2023-07-03
Filing date: 2024-07-03
Publication date: 2025-01-09
Anticipated expiration: 2026-01-03
Also published as: GB202310195D0

Abstract

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

Description

THERAPIES

TECHNICAL FIELD

The present disclosure relates to therapies for treating a synucleinopathy disease.

BACKGROUND

Parkinson's Disease (PD), is an alpha-synucleinopathy and is an incurable neurodegenerative disorder with increasing prevalence. Reportedly, there are up to 10 million people suffering from PD worldwide, including arxound 150,000 people in the UK. Moreover, PD is likely to become more prevalent in ageing populations; PD is more frequently encountered by people over 65 years of age. Contemporarily, approved pharmacological treatments for PD are used to correct for dopamine deficiency in brains of PD sufferers, wherein dopamine is decreased following cell damage and subsequent death of brain cells, in particular dopaminergic neurones in a midbrain region of the human brain. However, these pharmacological treatments are not disease-modifying and often cause serious side effects including involuntary movements and hallucinations. Moreover, the underlying cause behind dopaminergic neurone death is not well-understood, wherein abnormal concentrations of calcium, alpha-synuclein (a-syn) aggregation, and an inflammatory environment are prominent examples of key proposed factors in PD pathogenicity.

Therefore, in light of the foregoing discussion, there exists a need to provide improved methods and pharmaceutical compounds for treating synucleinopathy diseases, particularly PD, and further mitigating their symptoms.

SUMMARY

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.

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

In a second aspect, the present disclosure provides a kit for treating or preventing a synucleinopathy disease, according to claim 12.

In a third aspect, the present disclosure provides a method of treating or preventing a synucleinopathy disease, according to claim 15. In a fourth aspect, the present disclosure provides a use of a compound for treating or preventing a synucleinopathy disease, according to claim 16.

Throughout the description and claims of this specification, the words "comprise" , "include", "have", and "contain" and variations of these words, for example "comprising" and "comprises" , mean "including but not limited to", and do not exclude other components, items, integers or steps not explicitly disclosed also to be present. Moreover, the singular encompasses the plural unless the context otherwise requires. In particular, where the indefinite article is used, the specification is to be understood as contemplating plurality as well as singularity, unless the context requires otherwise.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGs. 1A-1C illustrate the HPLC and electrochemical detection analysis of striatal monoamines (dopamine metabolites: 3,4-Dihydroxyphenylacetic acid, DOPAC; and homovanillic acid, HVA); and tyrosine hydroxylase (TH, marker of dopaminergic neurones) immunostaining in the striatum from the in vivo study conducted in Example 1;

FIGs. 2A-2B illustrate the Ibal immunostaining from the in vivo study conducted in Example 1;

FIGs. 3A-3B illustrate the cerebrospinal fluid (CSF) cytokine I chemokine profile analysis from the in vivo study conducted in Example 1;

FIGs. 4A-4C illustrate a reduction in a-syn aggregation and phosphorylation of a serine amino acid residue located at position 129 of the a-syn protein sequence, which is associated with pathological a-syn in human PD, in rat and human astrocytes grown in the presence of other cell types such as neurons due to treatment with 30nM DG;

FIGs. 5A-D show 3 aspects of therapeutic action of 30nM DG in cell models of synucleinopathy;

FIGs. 6A-6B illustrate a pharmacodynamic effect of DG in the rat brain, namely a preservation of astrocytic coupling; and a change, namely a decrease, in inflammatory cytokine and chemokine release in the rat CSF due to treatment with lOmg/kg DG under inflammatory challenge induced by LPS injection intraperitoneally;

FIGs. 7A-D and 8A-B illustrate changes, namely the downregulation, in Cx43 puncta or Cx43 fluorescence intensity per astrocyte, or per cell, in cell culture models upon various challenges relevant to the pathogenesis of PD over a period ranging from two days to 2.5 weeks, in rat and human cells; FIGs. 9A-9E and 10A-10C illustrate changes, namely the downregulation, in 0x43 puncta or 0x43 fluorescence intensity per cell, in two rat models of a-syn aggregation relevant to human PD pathogenesis, in several brain regions relevant to the motor symptom development in PD, over a period of 12-18 months and 9 months, respectively;

FIGs. 11A-11C illustrate functional changes in cortical rat astrocytes in cell culture showing decreased gap junctional (GJ) coupling in astrocytic networks upon inflammatory and a-syn challenges relevant to human PD pathogenesis; and

FIGs. 12A-12D illustrate changes, namely the downregulation, in 0x43 puncta per cell or 0x43 protein levels, in human PD from several brain regions relevant to motor and nonmotor symptom development.

DETAILED DESCRIPTION OF EMBODIMENTS

The following detailed description illustrates embodiments of the present disclosure and ways in which they may be implemented. Although some modes of carrying out the present disclosure have been disclosed, those skilled in the art would recognize that other embodiments for carrying out or practicing the present disclosure are also possible.

In a first aspect, the present disclosure provides a compound of formula I for treating or preventing a synucleinopathy disease, wherein the compound of formula I is:

(I)

or a pharmaceutically acceptable salt or hydrate thereof.

By the term "treatment" or "treating" as used herein, we refer to therapeutic (curative) treatment, which includes stopping or slowing the disease from progressing. By the term "prevention" or "preventing" as used herein, we refer to "prophylactic" treatment, which includes administering the compound of the invention to a patient in a prodromal or early disease phase of Parkinson's Disease.

"Patient" and "subject" are used interchangeably and refer to the subject that is to be administered the compound of the invention. Preferably the subject is a human. Suitably the compound of formula I is for administration via a non-invasive route. Suitably the non-invasive route is oral, intranasal, sublingual, inhalation, rectal or transdermal.

Suitably the synucleinopathy disease is selected from Parkinson's Disease, dementia with Lewy bodies (DLB), and multiple system atrophy (MSA). Preferably the disease is Parkinson's Disease.

Suitably the compound of formula I is for use in mitigating and/or reducing the progression of one or more symptoms associated with a synucleinopathy disease, particularly associated with Parkinson's Disease. Suitably the one or more symptoms are selected from motor symptoms (such as tremors, stiffness and slowness of movement), memory disorders, depression, psychotic symptoms (such as hallucinations and delusions), sleep disturbance, and dementia.

Suitably the patient is exhibiting one or more symptoms selected from motor symptoms (such as tremors, stiffness and slowness of movement), memory disorders, depression, psychotic symptoms (such as hallucinations and delusions), sleep disturbance, dementia.

In a second aspect, there is provided a kit for treating or preventing a synucleinopathy disease, the kit comprising: a compound of formula I:

or a pharmaceutically acceptable salt or hydrate thereof.

Beneficially, the disclosed kit is configured to provide effective delivery of the therapeutically effective amount ofthe drug, for example danegaptide (DG), to the subject. Suitably delivery is via a non-invasive route, such as by using a delivery system whereby DG is encapsulated within a phospholipid-based delivery vehicle, namely liposomes. Such a delivery method is expected to enhance drug delivery across the blood-brain barrier of the subject.

Optionally, the system is configured to provide mitigation when the synucleinopathy disease is characterised by an increase in at least one of: inflammatory cytokines/chemokines, accumulation of misfolded a -syn. It will be appreciated that inflammatory cytokines and chemokines are small signalling proteins that play a crucial role in immune response and regulation of inflammation. Optionally, the inflammatory cytokines include, but are not limited to, tumour necrosis factor-alpha (TNF-a), interleukin-1 beta (IL-ip), interleukin-6 (IL-6), tumour necrosis factor receptor superfamily member 21 / death receptor 6 (TNR21 1 TNFRSF21 1 DR6), and interferon gamma induced protein 10 (IP-10 I CXCL10). An increase in inflammatory cytokines/chemokines suggests a state of chronic inflammation within the central nervous systems (CNS), namely, neuroinflammation, that may contribute to symptoms such as neuronal damage, inflammation-mediated cell death, disruption of normal brain function, memory disorders, depression, psychotic symptoms, hallucinations, sleep disturbance, dementia, etc. Typically, neuroinflammation is a complex process involving activation of immune cells, including microglia (the resident immune cells of the CNS), and reactive or atrophic changes in astrocytes, that are involved in the pathogenesis and progression of various neurological disorders. Similarly, the accumulation of the misfolded a-syn protein, which is abundant in the brain, particularly in presynaptic terminals (where it can be involved in the regulation of synaptic function), leads to the formation of protein deposits called Lewy bodies (LB), resulting in LB disease. The inflammatory environment can contribute to a-syn misfolding and aggregation. The misfolded a-syn may disrupt cellular processes, impair neuronal function, increase intracellular calcium, and contribute to neurodegeneration. Moreover, the misfolded a-syn aggregates may further promote an inflammatory response, triggering the release of pro-inflammatory cytokines and activating immune cells in the CNS.

Without wishing to be bound by theory, the interaction between misfolded a-syn and inflammation is complex and may create a self-perpetuating cycle of symptoms that need to be mitigated. Inflammatory processes may promote an accumulation and increased distribution of misfolded a-syn, and, in turn, misfolded a-syn may induce an inflammatory response. Such an interplay between inflammation and a-syn pathology is thought to contribute to a progressive neurodegeneration of dopaminergic neurons and associated motor and non-motor symptoms of synucleinopathies, for example as observed in PD. In another aspect, there is provided a method of treating or preventing a synucleinopathy disease, the method comprises administering, to a subject, a therapeutically effective amount of a compound of formula I:

or a pharmaceutically acceptable salt or hydrate thereof.

Herein, the term "compound" as used throughout the present disclosure, refers to a therapeutically active ingredient or an active pharmaceutical ingredient (API) or pharmaceutically acceptable salts or hydrates (or esters and prodrugs) thereof, that may be administered in the form of suitable pharmaceutical compositions, alone or in combination with any other ingredients (such as with one or more pharmaceutically acceptable carriers, diluents, buffer, preservative, stabiliser or excipients) known in the art, to the subject for their effective and sustained release at the target site. Herein, the term "effective and sustained release" refers to a mechanism of delivery of the compound to the target area, such as the brain parenchyma, over many hours or days after administration of the compound via a suitable route of administration thereof. It will be appreciated that the effective and sustained release at the target site may be followed by subsequent administration (after the first administration) of the aforementioned compound.

In an alternative embodiment there is also disclosed the AAP10 peptide, which consists of the sequence, Ac-DTyr-DPro-DHyp-Gly-DAIa-Gly-NH2, and/or the drug called Rotigaptide, for use in treating or preventing a synucleinopathy disease as described herein; or a pharmaceutically acceptable salt or hydrate thereof.

Optionally, the pharmaceutically acceptable salts of danegaptide (DG) include danegaptide (DG) hydrochloride.

In an alternative embodiment, the present invention is directed to a composition comprising the compound of formula I, or a pharmaceutically acceptable salt or hydrate thereof, for use in the treatment or prevention of a synucleinopathy disease, wherein the compound of formula I is the only active agent in the composition. By only active agent it is meant that the composition does not contain other components which may be used in the treatment or prevention of a synucleinopathy disease, preferably Parkinson's Disease.

The compositions of the invention comprising the compound of formula I may further contain a pharmaceutically acceptable carrier forming a pharmaceutical composition. By "pharmaceutically acceptable carrier" is meant any diluent or excipient, such as fillers or binders, that is compatible with the other ingredients of the composition, and which is not deleterious to the recipient. The pharmaceutically acceptable carrier can be selected on the basis of the desired route of administration, in accordance with standard pharmaceutical practices.

Without wishing to be bound by theory, it is believed that a technical effect of using danegaptide as a gap junctional intercellular communication (GJIC) modulator is that these compounds may provide better coupling between cells, enable cells to share available energy (in the form of ATP), and enable intercellular signaling, including calcium signaling. Additionally, DG would act to change the 0x43 conformation in such a way as to prevent hemichannel (HC) opening as well as enhance gap junction (GJ) opening, thus restricting inflammasome activation and inflammatory mediator release from the cells such as astrocytes.

Optionally, the compound is encapsulated in the liposome. Liposomes are vesicles composed of lipid bilayers, which are able to encapsulate the compound within their aqueous core or lipid membrane. When used as a sustained release formulation, liposomes allow the compound to cross the blood brain barrier and engage its target that is located within the brain parenchyma.

A technical benefit of liposomes is that liposomal encapsulation of the compound (namely danegaptide, preferably danegaptide hydrochloride) results in controlled release of the compound namely, danegaptide, preferably danegaptide hydrochloride, over an extended period of time, providing sustained and controlled drug delivery to maintain a consistent therapeutic concentration of the compound at the target site that is located within the brain parenchyma. Moreover, liposomal membranes act as a barrier, shielding the compound from external factors and enzymatic degradation or inactivation, improving its stability, prolonging its shelf life and effectiveness. Furthermore, liposomes may be designed to exhibit specific targeting properties. For example, functionalization of the liposomal surface with ligands or antibodies, may result in targeted delivery of the encapsulated compound to specific cells or tissues, enhancing its therapeutic efficacy and reducing potential side effects on non-targeted cells. Additionally, liposomes may help mitigate the toxicity of the compound by reducing its exposure to non-targeted tissues or organs and improve the therapeutic index of the compound.

It will be appreciated that, in addition to liposomes, other sustained release formulations mentioned, such as niosomes, microspheres, emulsions, or micelles, and liquid stabilisers, may potentially offer similar advantages of controlled release of the compound and improved stability thereof, depending on the specific properties and requirements of the compound.

Herein, the term "pharmaceutically acceptable salts" refers to compounds having an acidic moiety formed using organic or inorganic bases or a basic moiety formed using organic or inorganic acids. Optionally, the pharmaceutically acceptable salts having an acidic moiety include: metal salts, for example, alkali metal or alkaline earth metal salts, for example, sodium, potassium or magnesium salts; ammonia salts and organic amine salts formed with morpholine, thiomorpholine, piperidine, pyrrolidine, mono-, di-or tri-basic lower alkylamines such as ethyl tert-butylamine, diethylamine, diisopropylamine, triethylamine, tributylamine or dimethylpropylamine, or mono-, di-or tri-basic hydroxy lower alkylamines such as mono-, di- or triethanolamine; internal salts. Optionally, the pharmaceutically acceptable salts having a basic moiety include: 1- (2-aminoacetyl) -4- benzoylamino- pyrrolidine-2-carboxylic acid and its enumerated diastereomers. Such pharmaceutically acceptable salts having a basic moiety may be formed from the following acids: acetic, propionic, lactic, citric, tartaric, succinic, fumaric, maleic, malonic, mandelic, malic, phthalic, hydrochloric, hydrobromic, phosphoric, nitric, sulfuric, methanesulfonic, naphthalenesulfonic, benzenesulfonic, toluenesulfonic, camphorsulfonic acid, etc.

The term "therapeutically effective amount" or "dosage" as used herein refers to an amount of a free unbound compound that is capable of alleviating a symptom of a given synucleinopathy disease (or pathology) and, preferably, is capable of partially or completely normalising the physiological response, such as astrocytic network coupling, of a subject suffering from the given synucleinopathy disease (or pathology). Optionally, the therapeutically effective amount is determined based on various factors including, but not limited to, the potency of the compound, the age and constitution of the subject, the body weight of the subject, pharmacokinetic characteristics of the compound, and the route of administration, by a person skilled in the art. Suitably the dosage of the compound administered to the patient is (e.g., such as the pharmaceutical composition comprises) of from 10 to 350 mg, preferably of from 20 to 300 mg, more preferably of from 30 to 250 mg, even more preferably of from 40 to 225 mg, yet more preferably of from 50 to 200 mg, yet more preferably of from 60 to 175 mg, such as of from 75 to 150 mg, for example of from 100 to 130 mg of the compound of formula (I).

Suitably the dosage of the compound administered to the patient is of from 0.05 to 5 mg/kg, preferably of from 0.1 to 4.5 mg/kg, more preferably of from 0.3 to 4 mg/kg, even more preferably of from 0.5 to 3.5 mg/kg, yet even more preferably of from 0.7 to 3.5 mg/kg, still more preferably of from 0.9 to 3 mg/kg, such as of from 1 to 2.5 mg/kg, for example of from 1.1 to 2 mg/kg of the compound of formula (I).

Optionally, the dosage of the free compound in the brain or cerebrospinal fluid (CSF) that is predicted to be "therapeutically effective" is in a range of 10 to 100 nM, optionally substantially 30 nM. Optionally, the dosage of the compound is 10, 20, 30, 40, 50, 60, 70,

80 or 90 nM up to 20, 30, 40, 50, 60, 70, 80, 90 or 100 nM, preferably 30 nM. Generally, the compounds are administered in the range of about 10 to 100 nM.

A technical effect of the aforementioned dosage of the compound into the brain in the range of 10 to 100 nM, optionally substantially 30 nM, is that, at this specified dosage range, the compound may effectively engage with its target molecule or receptor, and exhibit the desired therapeutic outcome. The dose will be adjusted according to the body weight of the patient.

Optionally, the compound is formulated in a form of liquid phospholipid formulations, by processes that may be known to a person skilled in the art.

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

The intranasal route of administration typically involves delivering the compound (namely, drug or medication) through the nasal cavity into the olfactory epithelium region. In this regard, the compound is usually formulated as a spray, solution, or powder, which is administered using a suitable device and implemented as a nasal spray device. In the intranasal route of administration, the compound (or drug or medication) may penetrate into the brain parenchyma through the olfactory epithelium and/or may be absorbed through the nasal mucosa and enter the systemic circulation, from where the compound can cross the blood brain barrier directly.

A technical effect of the intranasal route includes efficient absorption of the compound into the brain and rapid onset of action. Moreover, via the intranasal route of administration, the compound bypasses the gastrointestinal (GI) tract and liver, to avoid the first-pass metabolism that occurs with oral administration. Additionally, the intranasal administration is relatively simple and non-invasive, making it more convenient for patients.

Optionally, administration of the compounds or pharmaceutically acceptable salts or hydrates thereof may be carried out as a continuous therapy with multiple doses over time, as determined by a healthcare professional associated with the subject. Alternatively, a continuous dosage system or a slow-release (sustained release) depot may be employed.

A technical benefit of the non-invasive route implemented as the intranasal administration of the compound is that it is patient-friendly, it does not require assistance from a trained professional, it provides better quality of life to the patient, and it is well-suited for chronic dosing. Additionally, the aforementioned route of administration ensures the effective therapeutic amount of the compound to be delivered at the target site, namely, the brain parenchyma and across the blood-brain barrier, to result in a desired therapeutic effect, in addition to the potency of the compound.

Moreover, notably, release of cytokines, and chemokines to the extracellular environment and cerebrospinal fluid excessively may contribute to neuroinflammation and neuronal dysfunction. The compound is configured to decrease the release of such signalling molecules from astrocytes into the extracellular environment, to help mitigate neuroinflammation and maintain a more balanced signalling environment.

Moreover, inflammatory activation of astrocytes for which excessive nuclear phosphorylated STAT3 at amino acid tyrosine at position 705 serves as a marker, can provoke the release of pro-inflammatory cytokines and chemokines, in response to certain stimuli, which contribute to neuroinflammation. The compound is designed to attenuate the inflammatory activation of astrocytes to help modulate the immune response in the central nervous system and promote a more balanced and regulated homeostatic state.

The present disclosure also relates to the system described above. Various embodiments and variants disclosed above, with respect to the aforementioned method, apply mutatis mutandis to the system. EXAMPLES AND DETAILED DESCRIPTION OF THE DRAWINGS

EXAMPLE 1 - in vivo proof of concept (PoC) using Danegaptide (DG) in an inflammatory rat model of Parkinson's disease (PD)

Model: "inflammatory Parkinsonism" (as described in Beier EE, et al., Neurobiol Dis. 2017 Dec;108: 115-127. PMID: 28823928).

Sprague-Dawley rats, male, 8 weeks at delivery, weight around 180-200g on delivery, approx. 250g at the start of the experiment (Janvier's Lab), 12h light/dark cycle, free access to food I water.

Lesion: lipopolysaccharide I endotoxin (LPS) from Salmonella abortus equi or saline (control) injected intraperitoneally (IP) at a dose of 1 mg/kg/day in a total volume of lOOpI at the same time of day on 4 subsequent days.

Treatment: Danegaptide (DG) encapsulated liposomally, administered intranasally (IN) in awake rats twice daily to approx. lOmg/kg in lOOpI total volume, 50pl per nostril per administration. Treatment commenced 1 day after the lesion protocol finished and lasted for 14 days. Control - sterile PBS.

N = 12-15 per group.

Objective: determine efficacy related to translationally-valid PD biomarkers.

Endpoints and outcomes:

1. High-performance liquid chromatography (HPLC) analysis and electrochemical detection of striatal monoamines (dopamine metabolites) - Fig. l

Reduction in dopamine metabolites has been noted in human PD and various models in correlation with motor symptom development. Therefore, this outcome represents evidence of inflammation-induced PD-relevant 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. Outliers (as detected by the Tukey's fences method) are included. *p<0.05, error bars: SEM.

Significant outcomes:

• DOPAC (3,4-Dihydroxyphenylacetic acid) content - significant decrease in the "PD" group compared to the control group, significantly rescued by the DG treatment (Fig.lA). • HVA (homovanillic acid) content - significant decrease in the "PD" group compared to the control group, partially rescued by the DG treatment (Fig. IB).

2. Striatal tyrosine hydroxylase (TH) immunostaining - Fig.l

TH is an enzyme involved in dopamine production; its downregulation has been associated with Parkinsonian motor symptoms. This corroborates the inflammation-induced PD- relevant nigrostriatal pathway damage in the model. TH staining was performed using the DAB agent and analysed with light microscopy.

Statistics: one-way ANOVA with Tukey-Kramer post-hoc test, Shapiro-Wilk test to test the normality of the distribution. Outliers (as detected by the Tukey's fences method) are included. *p<0.05, error bars: SEM.

Significant outcome:

• Moderate (p=0.0848) reduction in the TH optical density in the "PD" group compared to the control group; statistically significant preservation of TH in the DG treated group compared to the "PD" group (Fig.lC).

3. Ibal immunostaining - Fig.2

Ibal is a marker protein of a cell type called microglia, which are associated with an inflammatory response and immunoregulation in the brain. Microglial "reactivity" (i.e. a pro-inflammatory phenotype) can be assessed via its morphological features, two of which were measured in this study:

• Form Factor - also called "circularity", ranges from 0 to 1, relates area to perimeter with the formula 4n x area I perimeter² with the value of 1 representing more circular (presumed to be more reactive) cells. Higher values = more pro-inflammatory phenotype.

• Solidity - also called "density", ranges from 0 to 1, relates the area of the cell with its convex hull (the "territory" covered). When higher Solidity correlates with a higher Form Factor, this indicates hypertrophic cells associated with tissue damage. However if not correlated with a higher Form Factor, it could also indicate a quiescent highly-ramified cell.

Statistics: one-way ANOVA with Tukey-Kramer post-hoc test, Shapiro-Wilk test to test the normality of the distribution. Outliers (as detected by the Tukey's fences method) are included. *p<0.05, **p<0.005, ****p<0.0001, error bars: SEM. Significant outcomes:

• Striatal Ibal Form Factor (per rat) - significant increase in the "PD" group compared to the control group, significantly rescued by the DG treatment (Fig.2A).

• Striatal Ibal Solidity (per rat) - significant increase in the "PD" group compared to the control group, significantly rescued by the DG treatment (Fig.2B). Trends between the Form Factor and Solidity correlate, suggesting reactive changes in microglia in the "PD" group that are reversed or prevented by the DG treatment.

4. CSF cytokine I chemokine profile analysis - Fig.3

CSF cytokines / chemokines of inflammation are translationally-valuable biomarkers of inflammatory changes in the CNS, which have been described in PD and other neurodegenerative and psychiatric conditions. CSF screening is available in human trials as a surrogate marker of mechanism engagement of / pharmacodynamic response to a drug.

Proteomics screening presented here was performed using microarrays functionalised with antibodies.

Statistics: a multi-factorial linear model was fitted via least squares regression with LIMMA, resulting in a two-sided t-test or F-test based on moderated statistics. Next to the sample group, a subgroup factor, defined from the distribution of samples in the principal component analysis, was used as an additional factor in the linear model. All presented p values were adjusted for multiple testing by controlling the false discovery rate according to Benjamini and Hochberg. Differences in protein abundance between different samples or sample groups are presented as log-fold changes (logFC) calculated for the basis 2. Proteins with a |logFC| > 0.5 and an adjusted p value < 0.05 were defined as differential and displayed in black in the following volcano plots. Proteins reaching reduced thresholds as defined individually for each comparison, are defined as noteworthy and displayed in grey.

Significant outcomes:

• LIF, lactoferrin, IL13, and IL34 were found to be elevated in association with control and treated groups (Fig.3A). This is consistent with a more antiinflammatory profile of the cytokines in these groups.

• TNR.21 and prion protein were upregulated in the "PD" group compared to the control and treated groups (Fig.3B), this indicated a pro-inflammatory cytokine environment in the CSF relevant to the human Parkinson's Disease.

13

RECTIFIED SHEET (RULE 91) ISA/EP Conclusions

Example 1 shows that DG reversed phenotypes associated with Parkinson's Disease in an in vivo model. As such, it has been demonstrated that DG may be effective for treating and preventing Parkinson's Disease.

EXAMPLE 2 - in vitro models associated with Parkinson's Disease (PD)

The following experiments have been performed using rat and human cells. The results between these rat and human cell cultures were consistent demonstrating translational conservation of the underlying mechanism identified from rat studies to human studies. A summary of the findings of the following experiments is that o-syn aggregation and increase in inflammatory cytokine and chemokine release has been shown to be relevant to human PD pathogenesis. The experiments also show that treatment with Danegaptide prevents the o-syn aggregation and increase in inflammatory cytokine and chemokine release.

Referring to FIGs. 4A-4C, due to treatment with 30nM DG, there is shown a reduction in a- syn aggregation and of phosphorylated serine residues located at position 129 of the a-syn protein, which is associated with pathological a-syn in human PD, in rat and human astrocytes grown in the presence of other cell types such as neurons. Statistics: t- test or ANOVA with Tukey's multiple comparisons test; error bars: SEM.

Referring to FIGS 5A-D, there are shown 3 aspects of therapeutic action of 30nM DG in cell models of synucleinopathy. A: IP-10 cytokine released in the medium by rat cortical astrocyte- iNeurone co-cultures lesioned with a-syn PFF over 3 weeks; B-C: calcium oscillations in rat cortical astrocytes lesioned with a-syn PFF for 3 days; baseline calcium levels in human cortical astrocytes lesioned with a-syn PFF for 3 days; D: nuclear STAT3 in rat cortical astrocyte cultures lesioned with a-syn PFF for 3 days. Statistics: ANOVA with Tukey's multiple comparisons test; error bars: SEM. These demonstrate an anti-inflammatory action of DG (reduced nuclear STAT3 as a marker of reactive astrocytes; reduced cytokine release in the medium) and normalisation of functional cell signalling, which correlates with the reduction in a-syn aggregation. The inflammation markers also provide continuity with the in vivo biomarkers in the CSF and the rationale for using those.

Referring to FIG. 6A-6B, there is shown an increase in GJ communication measured by the Lucifer Yellow (LY) dye spread in the brain after a stereotactic injection into the striatum, and a decrease in inflammatory cytokine and chemokine release in the rat CSF under inflammatory challenge (relevant to the pathogenicity of PD) induced by LPS injection intraperitoneally at Img/kg, 1.5 days total lesion duration, upon treatment with DG at 10 mg/kg DG delivered intranasally twice a day when encapsulated in liposomes. Statistics: ANOVA with Tukey's multiple comparisons test or t- tests with FDR control; error bars: SEM.

Now referring to FIGs. 7, and 8, there is shown downregulation, in 0x43 puncta or 0x43 fluorescence intensity per astrocyte, or per cell, in cell culture models upon various challenges relevant to the pathogenesis of PD over a period ranging from two days to 2.5 weeks, in rat and human cells thus demonstrating translational conservation of the mechanism. Statistics: t-test or ANOVA with Tukey's multiple comparisons test; error bars: SEM.

Referring to FIGs. 7A-7D, there are shown changes in 0x43 puncta per astrocyte upon various challenges over two days up to two weeks. 0x43 protein levels using 0x43 fluorescence intensity and the number of 0x43 puncta per cell (analysed via fluorescent microscopy) are studied. Herein, the various challenges are LPS 100 ng/ml and mechanical injury for 48 hours (7A, Left Panel), and a-syn pre-formed fibrils (PFFs) and amyloid-p (Am- b) PFFs at O.lng per 10,000 cells in 72 hours (namely, over 3 days), i.e., misfolded protein challenges (7B, Right Panel). Referring to FIGs. 8A-8B, there are shown changes in 0x43 puncta per astrocyte upon a-syn challenge in rat cortical astrocyte-iNeurone co-cultures over 2.5 weeks.

Now referring to FIGs. 9A-9E and 10A-10C, there are shown changes, namely the downregulation, in 0x43 puncta or 0x43 fluorescence intensity per cell, in two rat models of a-syn aggregation relevant to human PD pathogenesis, in several brain regions relevant to the motor symptom development in PD, over a period of 12-18 months and 9 months, respectively. Statistics: t-test or ANOVA with Tukey's multiple comparisons test; error bars: SEM.

The first model is generated via peripheral injection of a-syn PFF alongside a carrier protein RVG9R into the tail vein; the rats subsequently develop progressive dopaminergic and cholinergic cell loss, several NMS such as olfactory loss and digestive slow-down, as well as mild motor symptoms, over 6-18 months post-injection. 0x43 puncta and fluorescence intensity per cell is measured in two brain regions relevant to PD pathology: midbrain substantia nigra (SN, A9 group of dopaminergic neurones identified with tyrosine hydroxylase staining) as shown FIG. 9A-9C, and striatum as shown in FIG. 9D-9E. Significant downregulation of 0x43 puncta and fluorescence intensity is observed in a-syn- lesioned animals at 12-18 months post-lesion compared to non-lesioned controls of the same age in the midbrain SN, while only 0x43 fluorescence intensity per cell is significantly reduced in the striatum as shown in FIG. 9D and 9E. A trend towards downregulation of 0x43 puncta per cell is seen in the striata as shown in FIG. 9D and 9E.

Referring to FIGs. 10A-10C, there are shown changes in 0x43 protein expression in a rat model of PD generated via a-syn PFF injection into the Nucleus Basalis of Meynert. Brain samples are analysed 9 months post-injection, by which point these animals start developing motor dysfunction and dopaminergic loss. It is observed that there is a significant reduction in 0x43 fluorescence intensity per cell, and a trend towards the downregulation of 0x43 puncta per cell, in the striata of the PFF-lesioned animals compared to the sham-lesioned controls (as shown in FIG. 10B and IOC).

Referring to FIG. 11A-11C, there are shown functional changes in cortical rat astrocytes in cell culture showing decreased GJ coupling in astrocytic networks upon inflammatory and a-syn challenges relevant to human PD pathogenesis. Statistics: ANOVA with Tukey's multiple comparisons test; error bars: SEM.

To assess the functional consequences of changes in Cx43 protein levels and punctate distribution on astrocyte network coupling, dye microinjection assays using Lucifer Yellow (LY) - a GJ-permeant dye that can be transferred between astrocytes through 0x43, are conducted. HCs composed of 0x43 protein are blocked during the injection procedure using a HC blocker GAP19. As shown, a significant downregulation in coupling of cortical astrocytes with both inflammatory and a-syn PFF challenges over 48h is visible, and similar results are obtained using midbrain and striatal astrocytes. Only fibrilized a-syn (both sonicated, representing "oligomers", and unsonicated, representing "fibrils"), but not monomeric a-syn, is able to induce GJ block (FIG. 11C). Sonicated a-syn PFFs are used where "a-syn PFF" is mentioned in other studies.

Referring to FIG. 12A-12D, there are shown changes, namely downregulation, in 0x43 puncta per cell or 0x43 protein levels, in human PD from several brain regions relevant to motor and non-motor symptom development. Statistics: ANOVA with FDR correction; error bars: SEM.

FIGs. 12B (Top panel) shows representative changes in TritonX-soluble 0x43 protein levels in a human post-mortem brain in PD as compared to controls. FIG. 12B (Bottom panel) shows the total protein stain of the same membrane as the blot. FIG. 12D is the quantification of the 0x43 protein level analysis. Western blot analysis of the TritonX- soluble fraction is used to complement the microscopic study (FIGs. 12A, C); grey matter of the parietal cortex, striatum, and midbrain SN are included for analysis. The TritonX- soluble fraction of 0x43 is proposed to represent 0x43 en route to the membrane in the Golgi and the endoplasmic reticulum, thus serving as a marker of the cell's ability to produce Cx43 protein, although Cx43 in HC may also be captured. Due to the high number of samples, western blot gels are split into four batches with a parietal cortex sample serving as a loading control among all batches for semi-quantitative analysis of combined protein levels. This protein analysis revealed a similar pattern of significant Cx43 reduction in the parietal cortex, while striatum and midbrain samples showed a non-significant trend towards Cx43 downregulation in PD. Referring to FIGs. 12C, there are shown regional 0x43 protein expression in a human postmortem brain in control and PD, respectively. Midbrain samples present with 0x43 protein distribution that differed in appearance to the punctate staining indicative of GJ plaques.

Quantitative microscopic examination in photon-counting mode reveals a significant reduction in 0x43 puncta per cell (i.e., per nucleus) in PD compared to controls in the frontal, parietal, and insular cortical grey matter samples as well as in the globus pallidus. Significant regional differences were also detected with cortical regions exhibiting markedly higher 0x43 puncta levels; this pattern was altered in PD as significant differences between cortex and midbrain disappeared.

Claims

CLAIMS:

1. A compound of formula I for use in the treatment or prevention of a synucleinopathy disease, wherein the compound of formula I is:

or a pharmaceutically acceptable salt or hydrate thereof.

2. A compound for use according to claim 1, wherein the use is treatment.

3. A compound for use according to claim 1 or 2, wherein the synucleinopathy disease is selected from Parkinson's Disease, dementia with Lewy bodies (DLB), and multiple system atrophy (MSA).

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

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

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

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

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

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

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

11. A compound for use according to any of the preceding claims, wherein the patient is exhibiting one or more symptoms selected from motor symptoms (such as tremors, stiffness and slowness of movement), memory disorders, depression, psychotic symptoms (such as hallucinations and delusions), sleep disturbance, and dementia.

12. A kit for treating or preventing a synucleinopathy disease, the kit comprising: a compound of formula I:

(I); or a pharmaceutically acceptable salt or hydrate thereof.

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

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

15. A method of treating or preventing a synucleinopathy disease comprising administering a patient a therapeutically effective amount of a compound of formula I:

or a pharmaceutically acceptable salt or hydrate thereof.

16. Use of a compound of formula I for the manufacture of a medicament for treating or preventing a synucleinopathy disease, wherein the compound of formula I is:

or a pharmaceutically acceptable salt or hydrate thereof.

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