Treatment of mitochondrial related diseases and conditions, including symptoms thereof, using pridopidine

Abstract

The present application relates to the treatment of mitochondrial related diseases and disorders, including symptoms thereof, using pridopidine. The present invention relates to the use of a composition comprising pridopidine or a pharmaceutically acceptable salt thereof and at least one pridopidine analogue represented by the structure of Compound 1 and / or Compound 4 or a pharmaceutically acceptable salt thereof in the manufacture of a composition for treating a disease associated with mitochondrial dysfunction in a subject in need thereof, the invention also relates to a use in medicines for treating diseases or any symptoms thereof.

Description

[0001] This application is a divisional application of the application with the filing date of March 15, 2020, application number 202080021165.0, and invention title "Treatment of mitochondrial-related diseases and conditions, including their symptoms, using pridopidine". Technical Field

[0002] The present invention provides a method for treating a subject suffering from a disease or condition associated with mitochondrial dysfunction, the method comprising administering to the subject a composition comprising pridopidine or a pharmaceutically acceptable salt thereof. Background Art

[0003] Mitochondria are double-membrane organelles found in most eukaryotic cells and performing many metabolic functions, including ATP synthesis by oxidative phosphorylation (OXPHOS). Mitochondria are also involved in the synthesis of biomolecules, the maintenance of calcium homeostasis, the production of reactive oxygen species (ROS), and the activation of apoptosis. Mitochondria are structurally complex and highly dynamic motile organelles. Mitochondria undergo continuous morphological changes through a continuous cycle of fusion and fission processes, which determine their morphology and most mitochondrial functions.

[0004] Given their central role in cellular homeostasis, mitochondrial dysfunction is associated with many age-related conditions, including mitochondrial diseases, cancer, metabolic diseases and diabetes, inflammatory conditions, neurodegenerative conditions, neuropathies, and neurodegenerative diseases such as Alzheimer's disease, Parkinson's disease, and Huntington's disease. Pridopidine (4-[3-(methylsulfonyl)phenyl]-1-propyl-piperidine) (previously known as ACR16, TV-7820) is in clinical development for the treatment of HD and ALS. Pridopidine has been shown to exert neuroprotective effects in animal and cell models of neurodegenerative diseases (including models of HD, PD, ALS, and AD) (Francardo, Veronica, Michal Geva, Francesco Bez, Quentin Denis, Lilach Steiner, Michael R. Hayden, and M. Angela Cenci. 2019. “Pridopidine Induces Functional Neurorestoration Via the Sigma-1 Receptor in a Mouse Model of Parkinson's Disease.” Neurotherapeutics 16(2):465-79; Ryskamp, Daniel A., Lili Wu, Jun Wu, Dabin Kim, Gerhard Rammes, Michal Geva, Michael Hayden, and Ilya Bezprozvanny. 2019 “Pridopidine Stabilizes Mushroom Spines in Mouse Models of Alzheimer's Disease by Acting on the Sigma-1 Receptor.” Neurobiology of Disease, 124, 489-504; Ryskamp, Daniel, Jun Wu, Michal Geva, Rebecca Kusko, Iris Grossman, Michael Hayden, and Ilya Bezprozvanny. 2017. “The Sigma-1 Receptor Mediates the Beneficial Effects of Pridopidine in a Mouse Model of Huntington Disease.)”Neurobiology of Disease 97(Part A):46 - 59; Ionescu, Ariel, Tal Gradus, Topaz Altman, Roy Maimon, Noi Saraf Avraham, Michal Geva, Michael Hayden, and Eran Perlson. 2019. “Targeting the Sigma - 1 Receptor via Pridopidine Ameliorates Central Features of ALS Pathology in a SOD1G93A Model.” Cell Death and Disease 10(3):210; Garcia - Miralles, Marta, Michal Geva, Jing Ying Tan, Nur Amirah Binte Mohammad Yusof, Yoonjeong Cha, Rebecca Kusko, Liang Juin Tan, et al. 2017. “Early Pridopidine Treatment Improves Behavioral and Transcriptional Deficits in YAC128 Huntington Disease Mice.” JCI Insight. Mitochondrial dysfunction has been demonstrated in the pathology of each of these neurodegenerative diseases. SUMMARY OF THE INVENTION

[0005] In a first aspect, the present invention provides a method for treating a disease, disorder, or any symptom thereof associated with mitochondrial dysfunction in a subject in need thereof, the method comprising administering to the subject an effective dose of a composition comprising pridopidine or a pharmaceutically acceptable salt thereof, thereby treating the subject.

[0006] In a further aspect, the present invention provides a composition comprising pridopidine or a pharmaceutically acceptable salt thereof for use in a method for treating a disease, disorder, or any symptom thereof associated with mitochondrial dysfunction.

[0007] In some embodiments, the disease, disorder, or any symptom thereof associated with mitochondrial dysfunction is a disease, disorder, or any symptom associated with mitochondrial myopathy.

[0008] In other embodiments, the mitochondrial myopathy is selected from MELAS syndrome, MERRF syndrome, Leigh Disease, Chronic Progressive External Ophthalmoplegia (C / PEO), MIDD or DAD (Diabetes with Deafness), Kearns-Sayre syndrome (KSS), Alpers Syndrome, Mitochondrial DNA depletion syndrome (MDS), Mitochondrial neurogastrointestinal encephalomyopathy (MNGIE), Neuropathy, ataxia and retinitis pigmentosa (NARP), Pearson syndrome, Leber's Hereditary Optic Neuropathy (LHON), Dominant Optic Atrophy (DOA), Pigmentary retinopathy, Wolfram Syndrome, Friedrich's Ataxia (FRDA), Mitochondrial neurogastrointestinal encephalomyopathy (MNGIE), and any combination thereof.

[0009] In additional embodiments, the disease, disorder or any symptom thereof related to mitochondrial dysfunction is a disease, disorder or any symptom related to lysosomal storage disease.

[0010] In other embodiments, the lysosomal storage disease is selected from glycogenosis type II (Pompe disease), multiple sulphatase deficiency (MSD), mucopolysaccharidoses (MPS), mucolipidoses (ML) types I to III, G(M1)-gangliosidosis, Fabry disease, Farber disease, Gaucher disease, Niemann-Pick disease, mucolipidosis (ML) type IV, cystinosis, neuronal ceroid-lipofuscinoses, and any combination thereof.

[0011] In some embodiments, the disease, disorder or any symptom thereof related to mitochondrial dysfunction is a disease, disorder or any symptom related to neurodegenerative diseases.

[0012] In some embodiments, the neurodegenerative disease is selected from Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis, Alzheimer's disease, frontotemporal dementia (FTD), Charcot-Marie-Tooth disease (CMT), and any combination thereof.

[0013] In some embodiments, the disease, disorder or any symptom thereof related to mitochondrial dysfunction is bipolar disorder.

[0014] In some embodiments, pridopidine is in its neutral / basic form. In some embodiments, pridopidine is in the form of a pharmaceutically acceptable salt. In some additional embodiments, pridopidine is pridopidine hydrochloride.

[0015] In some embodiments, the composition comprising pridopidine is administered orally.

[0016] In other embodiments, the composition comprising pridopidine is administered in the form of an inhalable powder, injection, liquid, gel, solid, capsule, eye drops or tablet.

[0017] In some embodiments, the composition comprising pridopidine is administered periodically (i.e., the pridopidine is administered at regular predetermined time intervals such as daily, hourly, weekly, monthly, and each cycle also optionally defines the dose to be administered and the number of administrations per time period). In additional embodiments, the composition comprising pridopidine is administered once a day, twice a day, or three times a day. In additional embodiments, the composition comprising pridopidine is administered less than once a day. In some embodiments, the composition comprising pridopidine is administered in one dose per day, two doses per day, or three doses per day.

[0018] In some embodiments, pridopidine is administered at a daily dose between 1 mg / day and 400 mg / day. In some embodiments, pridopidine is administered at a daily dose between 1 mg / day and 300 mg / day. In other embodiments, pridopidine is administered at a daily dose between 1 mg / day and 90 mg / day. In other embodiments, pridopidine is administered at a daily dose between 20 mg / day and 90 mg / day. In additional embodiments, pridopidine is administered at a daily dose between 45 mg / day and 90 mg / day. In other embodiments, pridopidine is administered at a daily dose between 20 mg / day and 50 mg / day. In additional embodiments, pridopidine is administered at a daily dose between 1 mg / day and 10 mg / day. In additional embodiments, pridopidine is administered at a daily dose between 10 mg / day and 20 mg / day. In additional embodiments, pridopidine is administered at a daily dose between 20 mg / day and 30 mg / day. In additional embodiments, pridopidine is administered at a daily dose between 30 mg / day and 40 mg / day. In additional embodiments, pridopidine is administered at a daily dose between 40 mg / day and 50 mg / day. In additional embodiments, pridopidine is administered at a daily dose between 50 mg / day and 60 mg / day. In additional embodiments, pridopidine is administered at a daily dose between 60 mg / day and 70 mg / day. In additional embodiments, pridopidine is administered at a daily dose between 70 mg / day and 80 mg / day. In additional embodiments, pridopidine is administered at a daily dose between 80 mg / day and 90 mg / day. In additional embodiments, pridopidine is administered at a daily dose between 90 mg / day and 100 mg / day. In additional embodiments, pridopidine is administered at a daily dose between 100 mg / day and 150 mg / day. In additional embodiments, pridopidine is administered at a daily dose between 150 mg / day and 200 mg / day. In additional embodiments, pridopidine is administered at a daily dose between 200 mg / day and 250 mg / day. In additional embodiments, pridopidine is administered at a daily dose between 250 mg / day and 300 mg / day. In additional embodiments, pridopidine is administered at a daily dose between 300 mg / day and 350 mg / day. In additional embodiments, pridopidine is administered at a daily dose between 350 mg / day and 400 mg / day. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] The subject matter regarded as the invention is particularly pointed out and distinctly claimed in the concluding portion of this specification. However, both as to the organization and method of operation, together with objects, features, and advantages thereof, the invention, when read in conjunction with the accompanying drawings, may be better understood by reference to the following detailed description, in which:

[0020] Figures 1A - 1G Shown is that pridopidine rescues abnormal mitochondrial morphology and restores mitochondrial-ER contacts in Y128 (YAC128, HD) neurons. Figure 1A : Visual representation of mitochondrial network in WT and Y128 (HD) cortical / striatal neurons stained with Mitotracker dye. Figure 1B : Quantification from 1A - the number of mitochondria was similar between WT and Y128 (HD) cortical / striatal neurons before and after pridopidine treatment (n = 4). Figure 1C and Figure 1D : Y128 HD mitochondria showed impaired morphology, with an increased percentage of round mitochondria ( Figure 1C ) and a decreased percentage of elongated mitochondria ( Figure 1D ). Pridopidine treatment (1 μM) corrected Y128 morphology to normal levels. (n = 4). Figure 1E : WT and Y128 neurons were stained with a mitochondrial marker ( Figure 1E middle column) and an ER marker ( Figure 1E right column), and co - localization of ER and mitochondria was analyzed ( Figure 1E left column) (n = ~15 projections / lesion). Figure 1F : Figure 1E Quantification showed that Mito - ER contacts were reduced in Y128 neurons compared to WT. Pridopidine (1 μM) significantly increased Mito - ER contacts. Figure 1G : The aspect ratio, the ratio between the mitochondrial axes indicating mitochondrial health, (see Methods, paragraph 86 and Results, paragraph 103) was decreased in HD neurons. Pridopidine treatment rescued the aspect ratio in Y128 neurons ( Figure 1G ). By Kruskal - Wallis test and Dunn's multiple comparison test, *p < 0.05, ***p < 0.001.

[0021] Figures 2A - 2B Shown is that pridopidine improves impaired mitochondrial dynamics in Y128 HD neurons. Figure 2A : MitoDSRed - transfected mitochondria were tracked in spinning disk confocal for 12 minutes, and velocity and directed transport were quantified using a kymograph (n = 7 - 9 projections / lesion). WT - the upper panel shows mitochondrial movement in both retrograde and anterograde directions. Y128 - the middle image shows no mitochondrial movement in either direction in HD compared to WT. Y128 treated with 1 μM pridopidine - the lower panel shows that pridopidine treatment restored mitochondrial movement in both retrograde and anterograde, similar to WT. Figure 2B : Figure 2AQuantification and directional analysis of transport showed an increase in the percentage of stationary mitochondria in Y128 neurons, with a decrease in both retrograde and anterograde transport. Pledidopidine treatment reduced the percentage of stationary mitochondria to WT levels and increased the percentage of both retrograde and anterograde transport to WT levels. Figure 2C : The total velocity of Y128 neurons was reduced. Pledidopidine treatment increased the total mitochondrial velocity. By two-way ANOVA, then by Tukey's multiple comparison test, *p < 0.05, $p < 0.05 (versus anterograde Y128 basal).

[0022] Figures 3A - 3H showed that pridopidine rescued impaired mitochondrial respiration in the HD cell model. Figures 3A - 3D : Oxygen consumption and ATP production were evaluated in WT and Y128 cortical / striatal neurons treated with 1 and 5 μM pridopidine for 24 h, and both doses showed rescue of basal and maximal respiration, as well as increased ATP production (n = 3). Figures 3E - 3H : Oxygen consumption and ATP production were reduced in HD neural stem cells (NSCs). Pridopidine treatment at 1 μM for 24 h rescued basal and maximal respiration as well as ATP production. By the Kruskal-Wallis test and Dunn's multiple comparison test, *p < 0.05, **p < 0.01.

[0023] Figures 4A - 4C showed that pridopidine treatment protected Y128 neurons and HD lymphoblasts from induced mitochondrial dysfunction. Cortical ( Figure 4A -left and Figure 4B ) and striatal ( Figure 4A -right and Figure 4C ) WT and Y128 neurons were evaluated for changes in their mitochondrial membrane potential (MMP, ΔΨ m ) after depolarization with oligomycin plus FCCP (carbonyl cyanide-4-(trifluoromethoxy)phenylhydrazone, an oxidative phosphorylation uncoupler) (n = 7 - 10). Pridopidine (0.1 and 1 μM) increased MMP, which was reduced in Y128 neurons and restored to WT-equivalent levels in both cortical and striatal neurons. Figures 4D - 4E : Oxidative stress was induced in Y128 cortical / striatal co-cultures using H 2 O 2 , resulting in a significant decrease in MMP ( Figure 4E ) and cell viability ( Figure 4F ). Pridopidine treatment (5 μM) rescued MMP ( Figure 4D ) and increased cell survival ( Figure 4E ), Figure 4F: Human lymphoblasts from HD patients or healthy controls were pretreated with 1, 5 or 10 μM pridopidine and / or H 2 O 2 All doses of pridopidine increased ΔΨ m , with the most significant effect achieved with 5 μM (n = 4). By Kruskal-Wallis test and Dunn's multiple comparison test, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. "Pri" refers to pridopidine.

[0024] Figures 5A - 5E Show that pridopidine restores oxidative stress-induced reactive oxygen species (ROS) production in Y128 neurons and HD NSCs and lymphoblasts. Figures 5A - 5C : Figure 5A Is a representative image of cortical / striatal neuron cultures treated with the MitoPY fluorescent probe for recording H 2 O 2 levels. As indicated, the mitochondrial complex III inhibitor antimycin A (AntA, 2 μM) was added to induce mitochondrial dysfunction (left panel - before AntA treatment, middle panel - untreated neurons after AntA treatment, right panel - pridopidine-treated neurons after AntA treatment). Figure 5B : Figure 5A Quantification in cortical neurons. Figure 5C : Figure 5A Quantification in striatal neurons. Cortical and striatal neurons treated with 1 μM pridopidine showed a decrease in mitochondrial H 2 O 2 levels recorded by the MitoPY fluorescent probe after AntA, as indicated (n = 4, considering approximately 20 cells / condition for striatal neurons and approximately 10 cells / condition for cortical neurons). Scale bar = 30 μM. Figure 5D : Human NSCs were treated with the mitochondrial complex II inhibitor myxothiazol (Myxo, 3 μM), which also induces mitochondrial dysfunction and increases mitochondrial H 2 O 2 levels. Treatment with pridopidine (1 μM) for 24 hours decreased mitochondrial H 2 O 2 levels (n = 4). Figure 5E : Control and HD lymphoblasts were treated with H 2 O 2Stimulation was used to induce mitochondrial dysfunction, leading to an increase in ROS levels (determined using CellRox staining). Pretreatment with 5 μM pridopidine for 24 hours significantly reduced ROS levels. (n = 4). By two-way ANOVA, followed by Tukey's multiple comparison test, * p < 0.05, ** p < 0.01, **** p < 0.0001. In Figure 5E , by the Kruskal-Wallis test and Dunn's multiple comparison test, *** p < 0.001, **** p < 0.0001.

[0025] Figures 6A - 6M It was shown that pridopidine treatment delayed the onset of motor deficits in Y128 mice, normalized mitochondrial complex activity, and reduced H 2 O 2 production in isolated Y128 striatal mitochondria. Figure 6A : Schematic illustration of the in vivo experimental design: Mice were treated with vehicle or pridopidine for 45 days. Rotarod behavior test (RR) was measured before treatment on day 0 (1.5 months old) and on day 44 (3 months old); Figure 6B : At 1.5 months old, the motor ability of pre-treatment Y128 mice was not impaired compared to WT. Figure 6C : At 3 months old, significant impairment was observed in vehicle-treated Y128 mice. In the rotarod motor test, pridopidine treatment significantly increased the fall latency time of Y128 mice compared to vehicle-treated controls. Figures 6D - 6H : Oxygen consumption rate (OCR) was measured using Seahorse XF to evaluate electron flow in striatal mitochondria from vehicle-treated or pridopidine-treated wild-type and Y128 mice. Mitochondrial complex inhibitors and substrates were sequentially injected, 2 μM rotenone, 10 mM succinate, 4 μM antimycin A, and 1 mM ascorbate / 100 mM TMPD to calculate the activities of mitochondrial complex I, complex II, complex III, and complex IV, respectively. Complexes II, III, and IV showed increased OCR in Y128 mice, indicating the presence of an early compensatory mechanism, and pridopidine rescued this effect. Figures 6I - 6K : Antimycin A (2 μM) was used to inhibit mitochondrial complex III. Mitochondrial H 2 O 2 levels were increased in mitochondria isolated from Y128 mice compared to WT mice. Pridopidine reduced mitochondrial H 2 O 2 levels in mice. The XY line shows the time-dependent change in fluorescence after addition of antimycin A. Figures 6L - 6M: Calcium uptake in mitochondria from pridopidine-treated Y128 mice was also improved. Extracellular calcium levels decreased in response to pridopidine treatment ( Figure 6L ), which is evidence of increased mitochondrial calcium regulation ( Figure 6M ), by non-parametric Kruskal-Wallis test, * p < 0.05, **p < 0.01.

[0026] Figures 7A - 7B Show that pridopidine reduces mHtt-induced ER stress. H2a-GFP was transiently co-expressed with mutant Htt96Q-mCherry (HD exon 1) ( Figure 7A ) or Htt20Q-mCherry (WT) ( Figure 7B ) in STHdhQ7 / 7 cells. Aggregation of H2a-GFP is a marker of ER stress. Aggregation of mCherry and GFP was evaluated and images were acquired using confocal microscopy. Compared with untreated cells, Htt96Q enhanced ER stress, and pridopidine treatment at 0.03 μM, 0.3 μM, and 3 μM reduced ER stress in these cells ( Figure 7A ). Htt20Q (wt) did not induce ER stress ( Figure 7B ). For comparison purposes, 100% represents the relative intensity of H2a-GFP in untreated cells showing Htt96Q-mCherry aggregates, and 0% is the relative intensity of H2a-GFP in untreated cells without Htt96Q-cherry aggregates. The graphs are the mean of 3 experiments +- SE. *p < 0.05 and **p < 0.01 compared with untreated cells.

[0027] Figure 8 Show that pridopidine reduces the elevated phosphorylated eIF2α (eIF2α-P) levels (a measure of ER stress) in the HD model. eIF2α-P levels were measured in HEK293 cells transfected with mutant Htt (Htt96Q, solid line) or WT Htt (Htt20Q, dashed line) and treated with 0.3 and 3 μM pridopidine for 24 hours. The ratio of eIF2α-P to total eIF2α was quantified by immunoblotting. Compared with WT, mutant Htt increased the phosphorylated eIF2α level, and both pridopidine concentrations reduced the eIF2α-P level. (*p < 0.05) Detailed Description

[0028] In the following specific embodiments, numerous specific details are set forth in order to provide a thorough understanding of the present invention. However, those skilled in the art will understand that the present invention may be practiced without these specific details. In other instances, well-known methods, procedures, and components have not been described in detail so as not to obscure the present invention.

[0029] In a first aspect, the present invention provides a method for treating a disease, disorder, or any symptom thereof associated with mitochondrial dysfunction in a subject in need thereof, the method comprising administering to the subject an effective dose of a composition comprising pridopidine or a pharmaceutically acceptable salt thereof, thereby treating the subject.

[0030] In a further aspect, the present invention provides a composition comprising pridopidine or a pharmaceutically acceptable salt thereof, the composition for use in a method for treating a disease, disorder, or any symptom thereof associated with mitochondrial dysfunction.

[0031] When referring to "a disease, disorder, or any symptom thereof associated with mitochondrial dysfunction", it should be understood to encompass any type of medical condition that endangers the health of the subject, in which impairment of the function of the mitochondria or any part thereof plays a direct or indirect role.

[0032] In some embodiments, the disease, disorder, or any symptom thereof associated with mitochondrial dysfunction is a disease, disorder, or any symptom associated with mitochondrial myopathy.

[0033] In other embodiments, the mitochondrial myopathy is selected from the group consisting of MELAS syndrome, MERRF syndrome, Leigh's disease, Alpers' syndrome, chronic progressive external ophthalmoplegia (C / PEO), diabetes with deafness (MIDD or DAD), Kearns-Sayre syndrome (KSS), mitochondrial DNA depletion syndrome (MDS), mitochondrial neurogastrointestinal encephalomyopathy (MNGIE), neuropathy, ataxia, and retinitis pigmentosa (NARP), Pearson syndrome, Leber's hereditary optic neuropathy (LHON), dominant optic atrophy (DOA), retinitis pigmentosa, Wolfram syndrome, Friedreich's ataxia (FRDA), mitochondrial neurogastrointestinal encephalomyopathy (MNGIE), and any combination thereof.

[0034] When referring to "mitochondrial myopathy", it should be understood to encompass any disease or disorder or any symptom caused by dysfunctional mitochondria. Mitochondrial diseases are sometimes (about 15% of the time) caused by mutations in mitochondrial DNA that affect mitochondrial function. Other mitochondrial diseases are caused by gene mutations in nuclear DNA, the gene products of which are imported into the mitochondria (mitochondrial proteins), as well as acquired mitochondrial pathologies. Mitochondrial diseases have unique characteristics, both because of the mode of inheritance of the diseases and because mitochondria are essential for cell function. Subclasses of these diseases with neuromuscular disease symptoms are commonly referred to as mitochondrial myopathies.

[0035] Mitochondrial myopathy diseases and disorders include but are not limited to:

[0036] MELAS syndrome (mitochondrial myopathy, encephalopathy, lactic acidosis, and stroke). A progressive neurodegenerative disorder caused by a mutation in mitochondrial DNA. Since little is known about the disease and it is difficult to diagnose, it is not currently known how many people worldwide have MELAS. The syndrome affects all racial groups and both males and females. Affected individuals typically begin to exhibit symptoms between the ages of 4 and 40. The prognosis is poor; the disease is often fatal. MELAS syndrome is incurable; healthcare is mainly supportive. Symptoms: Since there are defective mitochondria in all cells of patients with MELAS syndrome, a variety of symptoms may occur, often debilitating. Strokes can cause brain damage, leading to seizures, numbness, or partial paralysis. Encephalopathy (brain disease) can cause tremors, muscle spasms, blindness, deafness, and may lead to dementia. Myopathy (muscle disease) causes difficulty walking, moving, eating, and speaking.

[0037] MERRF syndrome (or myoclonic epilepsy with ragged red fibers): A very rare disorder that begins in childhood and affects the nervous system, skeletal muscle, and other body systems. The hallmark of MERRF is myoclonus, which consists of sudden, brief, jerking spasms that can affect the arms and legs or the entire body. In addition, individuals with MERRF syndrome may have muscle weakness (myopathy), impaired ability to coordinate movement (ataxia), seizures, and a slow deterioration of intellectual function (dementia). Short stature, degeneration of the optic nerve (optic atrophy), hearing loss, cardiomyopathy, and abnormal sensations (peripheral neuropathy) caused by nerve damage are also common symptoms. When stained with modified Gomori trichrome and viewed under a microscope, abnormal muscle cells are present and appear as ragged red fibers (RRF). MERRF is caused by a mutation in mitochondrial DNA (mtDNA).

[0038] The prevalence of Leigh's disease at birth is estimated to be approximately 1 in 36,000. The typical onset of symptoms occurs before 12 months of age, but in rare cases, the disease may manifest during adolescence or even early adulthood. Loss of motor milestones, hypotonia with poor head control, repeated vomiting, and movement disorders are common initial symptoms. Pyramidal and extrapyramidal signs, nystagmus, respiratory disturbances, ophthalmoplegia, and peripheral neuropathy are often noted later. Epilepsy is relatively rare. There is no specific treatment for Leigh's disease.

[0039] Chronic progressive external ophthalmoplegia (CPEO) is characterized by slowly progressive paralysis of the extraocular muscles. Patients typically experience bilateral symmetric progressive ptosis, followed by ophthalmoplegia months to years later. The ciliary and iris muscles are not involved. CPEO is the most common manifestation of mitochondrial myopathy. CPEO associated with mitochondrial DNA (mtDNA) mutations may occur in the absence of any other clinical symptoms, but is usually associated with skeletal muscle weakness. However, individuals with a similar clinical presentation may have various mitochondrial defects.

[0040] Diabetes with deafness (MIDD or DAD): MIDD accounts for 1% of people with diabetes. More than 85% of people carrying a mutation in mitochondrial DNA at position 3243 present with diabetes symptoms. The average age of people typically diagnosed with MIDD is 37 years, but it is also considered to range from 11 to 68 years of age. Among these diabetic patients carrying the mitochondrial DNA mutation at position 3243, 75% experience sensorineural hearing loss. In these cases, the hearing loss usually occurs before the onset of diabetes and is marked by a decrease in the perception of high-pitched frequencies. Hearing loss associated with diabetes is generally more common in men than in women and progresses more rapidly.

[0041] Onset of Kearns-Sayre syndrome (KSS): Before 20 years of age. The prevalence of Kearns-Sayre syndrome is approximately 1 to 3 in every 100,000 individuals. A rare neuromuscular disorder. The important clinical symptom characteristic is the presence of unilateral or bilateral ptosis (partial closure of the eyelids). This disease is mainly characterized by three main findings: progressive paralysis of certain eye muscles (chronic progressive external ophthalmoplegia [CPEO]); abnormal accumulation of a colored (pigmented) substance on the nerve-rich membrane lining the eye, leading to chronic inflammation, progressive degeneration, and wear of certain eye structures (retinal pigmentation); and heart disease (cardiomyopathy), such as heart block. Other findings may include muscle weakness, short stature, hearing loss, and / or loss of the ability to coordinate voluntary movements (ataxia) due to problems affecting a part of the brain (cerebellum). In some cases, KSS may be associated with other disorders and / or conditions.

[0042] Alpers syndrome (Alpers-Huttenlocher Syndrome): Onset: A few weeks to several years after birth. Symptoms: Psychomotor regression (dementia), epilepsy, and liver disease. Severe and persistent seizures can lead to death within the first decade of life.

[0043] Mitochondrial DNA depletion syndrome (MDS) Onset: Infancy Symptoms: This disorder usually causes muscle weakness and / or liver failure, and more rarely, brain abnormalities. "Flaccidity", feeding difficulties, and developmental delay are common symptoms; PEO and seizures are less common.

[0044] Mitochondrial neurogastrointestinal encephalomyopathy (MNGIE) Onset: Usually before the age of 20. Symptoms: This disorder causes PEO, ptosis (drooping of the eyelids), limb weakness, and gastrointestinal (digestive) problems, including chronic diarrhea and abdominal pain. Another common symptom is peripheral neuropathy (nerve dysfunction that can cause sensory impairment and muscle weakness).

[0045] Neuropathy, ataxia, and retinitis pigmentosa (NARP) Onset: From infancy to adulthood. Symptoms: NARP causes neuropathy (nerve dysfunction that can cause sensory impairment and muscle weakness), ataxia, and retinitis pigmentosa (degeneration of the retina of the eye, which leads to loss of vision). NARP can also cause developmental delay, epilepsy, and dementia.

[0046] Pearson syndrome Onset: Infancy. Symptoms: This syndrome causes severe anemia and pancreatic dysfunction. Children who survive the disease usually develop into Kearns-Sayre syndrome.

[0047] Leber's hereditary optic neuropathy (LHON) is characterized by acute and painless central vision loss in both eyes in a sequential manner over a period of days to months. LHON is the first maternally inherited ophthalmic disorder associated with a mitochondrial DNA point mutation. The estimated prevalence of the recognized disease of LHON in the UK and other parts of Europe is 1 in 25,000. Three mtDNA point mutations in the genes of the subunits of mitochondrial respiratory chain complex I (G11778A in ND4, G3460A in ND1, and T14484C in ND6) together cause 95% of LHON cases. Other pathogenic mtDNA mutations continue to be identified, especially in non-Caucasian ethnic groups. For example, the recently identified mtDNA T12338C mutation in ND5 seems to be common in the Han Chinese.

[0048] Dominant optic atrophy (DOA): DOA is a hereditary disease that mainly affects retinal ganglion cells (RGCs) and the retinal nerve fiber layer. In Northern Europe, the prevalence of DOA is estimated to be 1 in every 35,000 individuals. Visual acuity typically declines to an average of 20 / 80 to 20 / 120 within the first two decades of life. Thinning of the neuroretinal rim appears to be a common finding in DOA, where occasional findings include disc "cupping", a cup-to-disc ratio exceeding 0.5, and peripapillary atrophy. The early appearance of the optic nerve is typically characterized by sectoral pallor of the optic nerve.

[0049] Retinitis pigmentosa and other ophthalmic problems: Retinitis pigmentosa is a non-specific finding that may be found in several mitochondrial diseases. The most well-described primary mtDNA disease in which retinitis pigmentosa can be seen is neurogenic weakness, ataxia, and retinitis pigmentosa (NARP), which is caused by a T8993C mtDNA mutation in the gene for subunit of mitochondrial complex V, ATPase 6.

[0050] Wolfram syndrome: A genetic condition that is typically associated with insulin-dependent diabetes mellitus with onset in childhood and progressive optic atrophy. Additionally, many individuals with Wolfram syndrome also develop diabetes insipidus and sensorineural hearing loss. The old name for the syndrome was DIDMOAD, referring to diabetes insipidus, diabetes mellitus, optic atrophy, and deafness. Some individuals have mutations in the same gene that causes Wolfram syndrome, but they do not acquire all the features of the syndrome and are thus said to have a WFS1-related disorder. The major symptoms of Wolfram syndrome (diabetes, optic atrophy, diabetes insipidus, and hearing loss) can appear at different ages and change at different rates.

[0051] Friedreich ataxia (FRDA): A hereditary progressive neurodegenerative movement disorder in which the typical age of onset is between 10 and 15 years. Initial symptoms can include postural instability, frequent falls, and a progressive difficulty in walking due to impaired ability to coordinate voluntary movements (ataxia). Affected individuals typically develop slurred speech (dysarthria), characteristic foot deformities, and irregular curvature of the spine (scoliosis). FRDA is often associated with cardiomyopathy, a disease of the heart muscle that can lead to heart failure or irregular heart rhythm (arrhythmia). Approximately one-third of individuals with FRDA develop diabetes. The symptoms and clinical findings associated with FRDA are mainly due to degenerative changes in sensory nerve fibers at the point where they enter the spinal cord in a structure called the dorsal root ganglion. This leads to secondary degeneration of nerve fibers in the spinal cord, resulting in a lack of sensory signals in the cerebellum, a part of the brain that helps coordinate voluntary movements.

[0052] Mitochondrial neurogastrointestinal encephalomyopathy (MNGIE): A progressive metabolic disorder caused by the deficiency of thymidine phosphorylase (TP) enzyme. The deficiency of TP leads to the systemic accumulation of deoxyribonucleoside thymidine (dThd) and deoxyuridine (dUrd). In these patients, the clinical features include mental degeneration, ophthalmoplegia, and fatal gastrointestinal complications. The accumulation of nucleosides also causes an imbalance of mitochondrial DNA (mtDNA) deoxyribonucleoside triphosphates (dNTPs), which may play a direct or indirect role in mtDNA depletion / deletion abnormalities, although the exact underlying mechanism remains unknown.

[0053] In additional embodiments, the disease, disorder, or any symptom thereof related to mitochondrial dysfunction is a disease, disorder, or any symptom related to lysosomal storage disease.

[0054] In other embodiments, the lysosomal storage disease is selected from glycogen storage disease type II (Pompe disease), multiple sulfatase deficiency (MSD), mucopolysaccharidosis (MPS), mucolipidosis (ML) types I to III, G(M1)-gangliosidosis, Fabry disease, Farber disease, Gaucher disease, Niemann-Pick disease, mucolipidosis (ML) type IV, cystinosis, neuronal ceroid lipofuscinosis, and any combination thereof.

[0055] When referring to "lysosomal storage disease (LSD)", it should be understood to encompass any disease, disorder, or symptom characterized by the progressive accumulation of undigested macromolecules in lysosomes. The massive accumulation of substances affects the function of lysosomes and impairs autophagic flux, which may affect the cellular quality control of organelles such as mitochondria. LSD exhibits signs of mitochondrial dysfunction, including mitochondrial morphological changes, decreased mitochondrial membrane potential (ΔΨm), reduced ATP production, and increased production of reactive oxygen species (ROS). In addition, the reduction of autophagic flux may lead to the persistence of mitochondrial dysfunction. Examples of lysosomal storage diseases include, but are not limited to: glycogen storage disease type II (Pompe disease), multiple sulfatase deficiency (MSD), mucopolysaccharidosis (MPS), mucolipidosis (ML) types I-III, G(M1)-gangliosidosis, Fabry disease, Farber disease, Gaucher disease, Niemann-Pick disease, mucolipidosis (ML) type IV, cystinosis, neuronal ceroid lipofuscinosis.

[0056] In some embodiments, the disease, disorder, or any symptom thereof related to mitochondrial dysfunction is a disease, disorder, or any symptom related to neurodegenerative disease.

[0057] Neurodegenerative diseases are associated with any type of disabling disease, disorder, or symptom of the nervous system and are characterized by the relatively selective death of neuronal subtypes. Impaired mitochondrial function is key to the development of these diseases, such as impaired mitochondrial dynamics (shape, size, fission fusion, distribution, movement, etc.), abnormal mitochondrial membrane potential, oxygen consumption rate, ROS levels.

[0058] In neurodegenerative diseases such as Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis, frontotemporal dementia (FTD), Charcot-Marie-Tooth disease (CMT), and Alzheimer's disease, mitochondrial function is impaired.

[0059] In some embodiments, the neurodegenerative disease is selected from Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis, frontotemporal dementia (FTD), Charcot-Marie-Tooth disease (CMT), Alzheimer's disease, and any combination thereof.

[0060] In some embodiments, the disease, disorder, or any symptom thereof associated with mitochondrial dysfunction is vanishing white matter (VWM) disease. Vanishing white matter disease (VWM) is one of more than 50 conditions collectively known as leukodystrophies that affect the white matter or myelin of the brain. VWM, also known as ataxia with cerebral hypomyelination (CACH) in children, is an extremely rare neurological condition that disrupts the myelin sheath, the white matter of the brain, or both. In doing so, VWM permanently affects the transmission of brain signals to the rest of the body. Clinical conditions identified under VWM disease include, but are not limited to: ataxia with diffuse CNS hypomyelination (CACH) in children, vanishing white matter leukodystrophy (VWM), Krabbe leukodystrophy, vanishing white matter leukodystrophy with ovarian failure, and any combination thereof.

[0061] In further embodiments, the present invention provides a method for treating a disease, disorder, or any symptom thereof associated with mitochondrial dysfunction in a subject in need thereof, the method comprising administering to the subject a composition comprising pridopidine or a pharmaceutically acceptable salt thereof, wherein the mitochondrial dysfunction is bipolar disorder.

[0062] Bipolar disorder: A major mental disorder characterized by manic and depressive episodes, often accompanied by psychotic symptoms. Mitochondrial DNA mutations and mitochondrial dysfunction account for a subset of patients with the disorder.

[0063] In additional embodiments, the present invention provides a method for treating a disease, disorder, or any symptom thereof associated with mitochondrial dysfunction in a subject in need thereof, the method comprising administering to the subject a composition comprising pridopidine or a pharmaceutically acceptable salt thereof, wherein the symptoms of the disease or disorder associated with mitochondrial dysfunction comprise any one or more of the following: dysplasia, loss of muscle coordination, muscle weakness, neurological deficits, epilepsy, autism, autism spectrum, autistic-like features, learning disabilities, heart disease, liver disease, kidney disease, gastrointestinal disease, severe constipation, diabetes, increased risk of infection, thyroid dysfunction, adrenal dysfunction, autonomic dysfunction, confusion, disorientation, memory loss, poor growth, developmental arrest, poor coordination, sensory (vision, hearing) problems, decline in mental function, organ disease, dementia, respiratory problems, hypoglycemia, apnea, lactic acidosis, seizures, dysphagia, developmental delay, movement disorders (dystonia, muscle spasm, tremor, chorea), stroke, and brain atrophy.

[0064] In some embodiments, the pridopidine is in its neutral / basic form. In other embodiments, the pridopidine is in the form of a pharmaceutically acceptable salt. In some embodiments, the pridopidine is pridopidine hydrochloride.

[0065] For the methods and uses disclosed herein, the route of administration can be, for example, oral. The route of administration can also be classified as being local (e.g., topical administration) or systemic (e.g., enteral or parenteral administration). As used herein, "topical administration" shall mean the direct administration of a compound or composition to the site where its action is desired and specifically excludes systemic administration. As used herein, "topical administration" of a compound or composition shall mean the administration of the compound or composition to a body surface, such as the skin or mucosa, such as the eye. As used herein, "ocular administration" shall mean the administration of a compound or composition to the eye or the skin around the eye (periorbital skin) or the mucosa around the eye, particularly the conjunctiva of the subject, i.e., topical administration. A quantity of pridopidine and pharmaceutical composition of the present invention can be administered by oral administration, topical administration, systemic administration, local administration, or ocular administration.

[0066] In some embodiments, the pridopidine is administered orally.

[0067] In additional embodiments, the pridopidine is administered in the form of an inhalable powder, an injectable, a liquid, a gel, a solid, an eye drop, an eye ointment, a capsule, or a tablet.

[0068] As used herein, "pridopidine" means pridopidine base, a pharmaceutically acceptable salt thereof, a derivative thereof, an analogue thereof, or a combination of pridopidine and an analogue thereof.

[0069] Examples of pridopidine derivatives are deuterium-rich pridopidine and salts. Examples of deuterium-rich pridopidine and salts and their preparation methods can be found in U.S. Application Publications Nos. 2013-0197031, 2016-0166559, and 2016-0095847, the entire contents of each of which are incorporated herein by reference.

[0070] "Deuterium-rich" means that the abundance of deuterium at any relevant site of the compound is greater than the abundance of deuterium in a given amount of the compound that occurs naturally at that site. The natural abundance distribution of deuterium is about 0.0156%. Thus, in a "deuterium-rich" compound, the abundance of deuterium at any relevant site among the deuterium-related sites exceeds 0.0156% and can range from more than 0.0156% to 100%. Deuterium-rich compounds can be obtained by exchanging hydrogen with deuterium or by synthesizing the compound from deuterium-rich starting materials.

[0071] The present invention also encompasses any salts of pridopidine, including any pharmaceutically acceptable salts, where pridopidine has a net charge (positive or negative charge) and at least one counterion (having a counter negative or positive charge) is added thereto to form the salt. As used herein, the phrase "pharmaceutically acceptable salt" means those salts of the compounds of the present invention that are safe and effective for pharmaceutical use in mammals and have the desired biological activity. Pharmaceutically acceptable salts include salts of acidic or basic groups present in the compounds of the present invention. Pharmaceutically acceptable acid addition salts include, but are not limited to, hydrochloride, hydrobromide, hydroiodide, nitrate, sulfate, bisulfate, phosphate, acid phosphate, isonicotinate, acetate, lactate, salicylate, citrate, tartrate, pantothenate, bitartrate, ascorbate, succinate, maleate, gentisate, fumarate, gluconate, glucuronate, galacturonate, formate, benzoate, glutamate, mesylate, esylate, benzenesulfonate, p-toluenesulfonate, and pamoate (i.e., 1,1'-methylene-bis-(2-hydroxy-3-naphthoate)). Certain compounds of the present invention can form pharmaceutically acceptable salts with various amino acids. Suitable base salts include, but are not limited to, aluminum salts, calcium salts, lithium salts, magnesium salts, potassium salts, sodium salts, zinc salts, and diethanolamine salts. For a review of pharmaceutically acceptable salts, see BERGE et al., 66 JOURNAL OF PHARMACEUTICAL SCIENCES 1-19 (1977), which is incorporated herein by reference. In another embodiment, the pridopidine salt of the present invention is hydrochloride.

[0072] In some embodiments, the method of the present invention utilizes pridopidine or a pharmaceutically acceptable salt thereof in combination with one or more analogs or pharmaceutically acceptable salts of an analog thereof.

[0073] In one embodiment, an analog of pridopidine is represented by the following structure:

[0074]

[0075]

[0076] In other embodiments, the method of the present invention utilizes a combination of pridopidine or a pharmaceutically acceptable salt thereof and an analog of compound (1) or a pharmaceutically acceptable salt thereof.

[0077] In other embodiments, the method of the present invention utilizes a combination of pridopidine or a pharmaceutically acceptable salt thereof, an analog of compound (1), and an analog of compound (4) or a pharmaceutically acceptable salt thereof.

[0078] Accordingly, the present invention also relates to a pharmaceutical composition comprising an agent of the present invention admixed with a pharmaceutically acceptable adjuvant and optionally other therapeutic agents. The adjuvant must be "acceptable" in the sense of being compatible with the other ingredients of the composition and not injurious to its recipient.

[0079] The pharmaceutical compositions include those suitable for oral, rectal, nasal, topical (including transdermal, buccal, and sublingual), vaginal, or parenteral (including subcutaneous, intramuscular, intravenous, and intradermal) administration or administration by implant. The compositions can be prepared by any method well known in the art of pharmacy.

[0080] The pharmaceutical compositions include those suitable for oral, rectal, nasal, topical (including transdermal, buccal, and sublingual), vaginal, or parenteral (including subcutaneous, intramuscular, intravenous, and intradermal) administration or administration by implant. The compositions can be prepared by any method well known in the art of pharmacy.

[0081] Such methods include the step of incorporating the associating compounds or combinations thereof for use in the present invention with any adjuvant. Adjuvants, also known as auxiliary ingredients, include those conventional in the art, such as carriers, fillers, binders, diluents, disintegrants, lubricants, colorants, flavorants, antioxidants, and wetting agents.

[0082] Pharmaceutical compositions suitable for oral administration can be presented as discrete dosage units, such as pills, tablets, dragees, or capsules, or as powders or granules, or as solutions or suspensions. The active ingredient can also be presented in the form of a bolus or paste. The compositions can be further processed into suppositories or enemas for rectal administration.

[0083] The present invention further comprises a pharmaceutical composition as described above in combination with a packaging material, comprising instructions for use of the composition for the uses described above.

[0084] For parenteral administration, suitable compositions comprise aqueous and non-aqueous sterile injectables. The compositions may be presented in unit-dose or multi-dose containers, such as sealed vials and ampoules, and may be stored under lyophilized (freeze-dried) conditions that require only the addition of a sterile liquid carrier, such as water, immediately prior to use. For transdermal administration, for example, gels, patches or sprays may be considered. Compositions or formulations suitable for pulmonary administration, for example, comprise fine dusts or mists for nasal inhalation, which may be generated by metered-dose pressurized aerosols, nebulizers or insufflators.

[0085] The exact dosage and regimen of administration of the composition will necessarily depend on the therapeutic or nutritional effect to be achieved and may vary with the formulation, the route of administration, and the age and condition of the individual subject to which the composition is to be administered.

[0086] As used herein, the term "treatment" refers to the administration of a therapeutically effective amount of a composition of the present invention, which is effective to ameliorate an undesirable disease, disorder, including symptoms associated with the disease or disorder, to prevent the manifestation of such disease, disorder, including symptoms associated with the pre-occurrence of the disease or disorder, to slow the progression of the disease, to slow the worsening of symptoms, to promote the onset of a remission period, to slow the irreversible damage caused during the progressive chronic phase of the disease, to delay the onset of the progressive phase, to reduce the severity or to cure the disease, to increase survival rate or to recover more rapidly, or to prevent the occurrence of a disease form or a combination of two or more of the above. The "effective amount" for the purposes disclosed herein is determined by such considerations known in the art. The amount must be effective to achieve the desired therapeutic effect as described above, particularly depending on the type and severity of the disease to be treated and the treatment regimen. In some embodiments, the composition comprising pridopidine or a pharmaceutically acceptable salt thereof is between 1 and 400 mg, daily, twice daily, three times daily or less than once a day. It is generally well known that the effective amount depends on a variety of factors, including the ligand's affinity for the receptor, its distribution profile in the body, various pharmacological parameters such as in vivo half-life, undesirable side effects, if any, and factors such as age and gender.

[0087] In some embodiments, pridopidine is administered at a daily dose between 1 mg / day and 400 mg / day. In some embodiments, pridopidine is administered at a daily dose between 1 mg / day and 300 mg / day. In other embodiments, pridopidine is administered at a daily dose between 1 mg / day and 90 mg / day. In other embodiments, pridopidine is administered at a daily dose between 20 mg / day and 90 mg / day. In additional embodiments, pridopidine is administered at a daily dose between 45 mg / day and 90 mg / day. In other embodiments, pridopidine is administered at a daily dose between 20 mg / day and 50 mg / day. In additional embodiments, pridopidine is administered at a daily dose between 1 mg / day and 10 mg / day. In additional embodiments, pridopidine is administered at a daily dose between 10 mg / day and 20 mg / day. In additional embodiments, pridopidine is administered at a daily dose between 20 mg / day and 30 mg / day. In additional embodiments, pridopidine is administered at a daily dose between 30 mg / day and 40 mg / day. In additional embodiments, pridopidine is administered at a daily dose between 40 mg / day and 50 mg / day. In additional embodiments, pridopidine is administered at a daily dose between 50 mg / day and 60 mg / day. In additional embodiments, pridopidine is administered at a daily dose between 60 mg / day and 70 mg / day. In additional embodiments, pridopidine is administered at a daily dose between 70 mg / day and 80 mg / day. In additional embodiments, pridopidine is administered at a daily dose between 80 mg / day and 90 mg / day. In additional embodiments, pridopidine is administered at a daily dose between 90 mg / day and 100 mg / day. In additional embodiments, pridopidine is administered at a daily dose between 100 mg / day and 150 mg / day. In additional embodiments, pridopidine is administered at a daily dose between 150 mg / day and 200 mg / day. In additional embodiments, pridopidine is administered at a daily dose between 200 mg / day and 250 mg / day. In additional embodiments, pridopidine is administered at a daily dose between 250 mg / day and 300 mg / day. In additional embodiments, pridopidine is administered at a daily dose between 300 mg / day and 350 mg / day. In additional embodiments, pridopidine is administered at a daily dose between 350 mg / day and 400 mg / day.

[0088] Experimental Section

[0089] Materials and Methods

[0090] Colonies of hemizygous YAC128 (Y128) [strain HD53; mHTT overexpressor] and WT mice with an FVB / N background were housed under controlled temperature (22 - 23 °C) conditions and a 12-hour light / 12-hour dark cycle. Food and water were available ad libitum. All mouse experiments were conducted in accordance with the guidelines of the Institutional Animal Care and Use Committee and the European Community Directive (2010 / 63 / EU) and an approved protocol by the local animal care committee. All efforts were made to minimize animal suffering and reduce the number of animals used.

[0091] Primary neuron cultures: Primary cortical, striatal, and corticostriatal co-cultures were generated from the offspring of a cross between wild-type (WT) mice (used as controls) or hemizygous Y128 mice [strain HD53] males and WT females (FVB / N) from the same genetic background. Timed pregnant females were sacrificed on embryonic day E15.5 - E16.5 of gestation. For corticostriatal co-cultures, the cortex and striatum were microdissected, then chopped and pooled for each genotype. The tissue was then dissociated and homogenized. Cells were seeded onto poly-D-lysine-coated plates in enriched neurobasal medium. Cells were fed with 1 / 3 fresh medium every five days.

[0092] To obtain pure cortical and striatal neurons, the tissue was microdissected and mechanically digested. Neurons were cultured in enriched Neurobasal medium and plated at a density of 130×10 3 cells / cm 2 (high density) or 85×10 3 (low density) on poly-D-lysine (0.1 mg / ml)-coated plates. Cultures were maintained at 37 °C in a 95 / 5% air / CO 2 incubator. On day in vitro 3 (DIV3), 5 μM 5-fluoro-2'-deoxyuridine (5-FdU) was added to reduce dividing non-neuronal cells. Fresh medium was added at DIV7, and cells were used at DIV12.

[0093] Neuron transfection: Striatal neurons were transfected with the pDsRed2-Mito vector using the calcium phosphate precipitation method at 8 DIV. The transfected neurons were then washed with neurobasal medium and transferred back to their original culture dishes containing conditioned medium until DIV12.

[0094] Lymphoblast culture and transfection: Lymphoblasts from healthy controls (GM02174) and HD patients (NA04724) obtained from the Coriell Institute were grown in supplemented RPMI medium. Lymphoblasts were passaged at a 1:3 ratio every 5 - 6 days.

[0095] Human neural stem cell culture: Neural stem cells (NSCs) were differentiated from heterozygous human induced pluripotent stem cells (iPSCs) HD4 - iPSCs (with normal (19 CAG repeats) and expanded alleles (72 CAG repeats)) and control AMS4 - iPSCs. The iPSCs were maintained in (Thermo Fisher Sci., catalog number: A1413202) - coated 6 - well plates until they reached 90% confluence, at which point the neural induction protocol was applied. Neural differentiation was based on dual SMAD inhibition of SB431542 (lefty / activin / transforming growth factor β - TGFβ inhibitor), dorsomorphin (bone morphogenetic protein - BMP inhibitor), and XAV - 939 (β - catenin transcriptional inhibitor and axin stabilizer). Neural induction occurred between day 0 and days 12 - 15. From day 0 to day 5, the cells were maintained in iPSC medium without FGF 2 and incubated with 5 μM dorsomorphin and 10 μM SB431542. The medium was changed every other day. From day 5 to day 12, the medium was gradually replaced with medium with an increasing percentage of N2 medium. Between days 12 and 15, the regions filled with rosettes became morphologically clearly visible. For differentiation, the cells were re - plated in coated 12 - well plates. The expression of the neural lineage marker proteins nestin and SOX2 was confirmed by immunocytochemistry at each stage of differentiation.

[0096] Pridopidine incubation: Pridopidine incubation was performed for 24 hours in all cell models used unless otherwise stated. The final concentrations are described in the figures and legends.

[0097] Mitochondrial network and ER co - localization: Cortico - striatal co - cultures were labeled with Mitotracker Deep Red FM dye for 30 minutes. The stained cells were washed before fixation with ice - cold methanol for 15 minutes at room temperature.

[0098] MitoDsRed - transfected striatal neurons were fixed, permeabilized, and blocked with 4% paraformaldehyde, and then incubated with IP 3Incubated with R3 antibody (1:1000, EMD Millipore, catalog number AB9076). For nuclear detection, neurons were incubated with Hoechst 33342 and then mounted.

[0099] Confocal images in the form of stacks separated by 0.46 μm along the z-axis were acquired using a 63x objective of a Zeiss confocal microscope with LSM 710 software. FIJI software was used for image analysis. The z-stack images were normalized for background, and regions of peak intensity representing mitochondrial-specific fluorescence were identified using Find Foci (Herbert, Alex D., Antony M. Carr, and Eva Hoffmann. 2014. “FindFoci: A Focus Detection Algorithm with Automated Parameter Training That Closely Matches Human Assignments, Reduces Human Inconsistencies and Increases Speed of Analysis.” Edited by Michael Lichten. PLoS ONE 9(12)), and optimally resolved by filter application. Mitochondrial profiles were traced using Analyze Particles. The aspect ratio (the ratio between the long and short axes of the mitochondria) was used as an indicator of mitochondrial length. For IP 3 For R3 fluorescence, the threshold was set similarly to that described above, and the integrated density was calculated within the region of interest (ROI) of the mitochondria of interest to obtain co-localization with the ER of the mitochondria.

[0100] Mitochondrial motility analysis: Striatal neurons transfected with MitoDsRed were washed and incubated in Na + medium and mitochondrial motility studies were performed at 37 °C. Neuronal projections were imaged every 5 seconds for a total of 145 frames using a 63x objective of a spinning disk Zeiss inverted confocal. Mitochondrial motility analysis was performed using the Kymograph Macro in Fiji. The ROI was specified using a dividing line, following the mitochondrial tracks across the projection. The kymograph generated in the x-y dimension (distance vs. time) was used to obtain the slope to calculate mitochondrial velocity.

[0101] Seahorse oxygen respiration measurement: The oxygen consumption rate (OCR) in WT and hemizygous Y128 cortical / striatal co-cultures and NSCs was measured using a Seahorse XFe-24 / 96 Flux Analyzer (Seahorse Bioscience). Cortical-striatal primary neurons were cultured at a density of 20,000 cells / well in a Seahorse XF96 V3 cell culture microplate. NSCs were seeded at 30,000 cells / well onto an XF24 cell culture microplate coated with and allowed to adhere at 37 °C for 24 h. When indicated in the graph, pridopidine (0.1, 1, and / or 5 μM) was added 24 h before the experiment. The sensor cartridge plate was incubated with the immersion sensor in a non-CO 2 incubator at 37 °C for approximately 16 h. Three baseline measurements of OCR were sampled before sequential injection of the mitochondrial complex V inhibitor oligomycin (1 mM), the protonophore FCCP (carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone, an oxidative phosphorylation uncoupler) (0.5 mM for neurons, 0.3 mM for NSCs), and antimycin A (0.5 mM for neurons, 1 μM for NSCs) plus rotenone (0.5 μM for neurons and 1 μM for NSCs) to completely inhibit mitochondrial respiration. Thus, mitochondrial basal respiration, maximal respiration, and ATP production were automatically calculated and recorded by Seahorse software. Data were normalized to the protein level.

[0102] Mitochondrial membrane potential: The mitochondrial membrane potential (MMP, Δψ + ) was evaluated in cortical and striatal neurons using the positively charged fluorescent probe tetramethylrhodamine methyl ester (TMRM m ) under quenching conditions, and its accumulation in mitochondria was evaluated after mitochondrial depolarization with oligomycin plus the mitochondrial uncoupler FCCP. In cortical / striatal co-cultures and lymphoblasts, MMP was evaluated using an equivalent probe (tetramethylrhodamine ethyl ester (TMRE + ) fluorescent probe), and its accumulation in mitochondria was directly evaluated by flow cytometry. When indicated, cortical and striatal neurons previously treated with pridopidine (0.1 and 1 μM; 24 h) were incubated with 150 nM TMRM (quenching conditions) in Na + medium at 37 °C for 30 min. Under these conditions, mitochondrial retention of TMRM was studied to estimate changes in MMP / Δψ. Basal fluorescence (excitation at 503 nm and emission at 525 nm) was recorded for 4 min, then 2.5 μM FCCP and 2.5 μg / mL oligomycin were added to achieve maximal mitochondrial depolarization and mitochondrial probe release. TMRM release was calculated based on the fluorescence difference before and after addition of oligomycin / FCCP.

[0103] Primary neurons or lymphoblasts were cultured on 6-well plates. Cells were pretreated with / without pridopidine and hydrogen peroxide (H 2 O 2 ) according to the experimental conditions, and then incubated with 25 nM TMRE methyl ester at 37 °C for 15 minutes. After TMRE incubation, cells were collected for FACS analysis.

[0104] Measurement of mitochondrial H 2 O 2 levels: Cortical and striatal neurons were pretreated with pridopidine (0.1 and 1 μM) for 24 hours and incubated with the mitochondrial peroxiredoxin 1 (MitoPY1) probe (8 μM) in Na + medium at 37 °C for 30 minutes. MitoPY1 was washed off, and neurons in the same experimental medium were imaged every 1 minute for 30 minutes using a 63× lens of a Zeiss inverted confocal spinning disk microscope with Zen Black 2012 software. Fluorescence was recorded by 503 nm excitation and 528 nm emission enhancement (Dickinson, Bryan C., Vivian S. Lin, and Christopher J. Chang. 2013. “Preparation and Use of MitoPY1 for Imaging Hydrogen Peroxide in Mitochondria of Live Cells.” *Nature Protocols* 8(6)). Ten minutes after the baseline reading, neurons were stimulated with antimycin A (2 μM). Specific MitoPY1 fluorescence in mitochondria was confirmed by co-incubating cells with MitoTracker Deep Red (300 nM). Fluorescence intensity at each time point was analyzed using the Time Series Analyzer plugin (v 3.0) in Fiji.

[0105] NSCs were plated at 30,000 cells / well in 96-well assay plates coated with and incubated at 37 °C for 24 hours. Thereafter, NSCs were incubated with 1 μM pridopidine for another 24 hours. Before harvesting, cells were washed and incubated at 37 °C and 5% CO 2Incubate with 10 μM MitoPY1 for 20 minutes below. Measure the basal level of MitoPY1 fluorescence for 10 - 15 minutes, then expose to myxothiazol (3 μM mitochondrial complex III inhibitor) and measure for an additional 30 minutes. Results are calculated as relative fluorescence units (RFU) per 30,000 cells. In isolated mitochondria, measure H 2 O 2 levels by resuspending 5 μg of isolated mitochondria in Amplex Red reagent containing horseradish peroxidase (0.5 units / mL) and measuring fluorescence at 570 nm excitation and 585 nm emission. After a 10-minute basal reading, stimulate the mitochondria with antimycin A (2 μM) and measure for an additional 10 minutes. Results are analyzed as the time-dependent change in fluorescence.

[0106] Reactive oxygen species (ROS) assay: Primary neurons and lymphoblasts attached to PDL-coated plates are treated with H 2 O 2 (0 - 1 mM) for up to 6 hours. Treat the cells with 5 μM CellRox Red reagent in complete medium, then incubate for 30 minutes. After washing, oxidative stress is measured by imaging all samples using the same exposure settings with a 40x objective on a Zeiss inverted microscope. Sample eight random fields and measure the fluorescence intensity using ImageJ software.

[0107] In vivo study design: 1.5-month-old WT and hemizygous Y128 mice (equal proportions of males and females) are divided into four groups. Mice receive pridopidine (30 mg / kg, 100 μL / 25 g) or an equal volume of sterile water by oral gavage for 45 consecutive days until 4 months old. House the mice in cages enriched with corncob nesting material and paper rolls, with 4 animals per cage, and each cage represents a separate experiment, with a total of 9 animals per group. Weigh the animals weekly and adjust the treatment volume accordingly. Conduct behavioral tests on the mice in a rotarod immediately before treatment and one day before the end of treatment. The tests are performed blindly at a fixed time during the day. Twenty-four hours after the last gavage, sacrifice the mice and isolate mitochondria from the striatum.

[0108] Rotarod analysis: Motor learning and coordination are evaluated on a rotating device. In this test, mice must learn to run while being placed on a constantly rotating rod to prevent them from falling. Once the task is learned, an accelerating rotarod can be used to evaluate motor coordination and balance. Mice are allowed to acclimate to the behavioral chamber for 2 hours. The procedure is consistent for all subjects and the tests are conducted at the lowest noise level. Training consists of four trials per day (each trial for 120 seconds), spaced 1 hour apart, at a fixed speed of 14 rpm. The testing phase is conducted on the next day on an accelerating rotarod from 4 to 40 rpm within 5 minutes and consists of 3 trials, spaced 2 hours apart. The rotarod score is the average of the 3 trials. Experiments are blinded to genotype and treatment. Motor coordination scores are measured after training, and the fall latency time is quantified on an accelerating rotarod from 5 to 40 rpm within 5 minutes.

[0109] Isolation of functional mitochondria: The striatum is dissected from mouse brains washed in mitochondrial isolation buffer. Striatal mitochondria are isolated using discontinuous Percoll density gradient centrifugation after homogenization. The protein content of the isolated mitochondria is quantified by Bio-Rad assay.

[0110] Mitochondrial complex activity: Complex activity is evaluated by measuring the oxygen consumption rate (OCR) using the Seahorse XF method (Agilent). 5 μg of isolated mitochondria diluted in mitochondrial assay solution are seeded in a 450 μL poly(ethyleneimine)-coated Seahorse XF24 plate, and the plate is allowed to equilibrate in a humidified incubator at 37 °C for 10 - 12 minutes. After sequential injection of rotenone (2 μM; complex I inhibitor), succinate (10 mM; complex II substrate), antimycin A (4 μM; complex III inhibitor), and ascorbate / TMPD (10 mM / 100 μM; electron donors for cytochrome C / complex IV), the sequential electron flow through the electron transport chain is evaluated by OCR measurement. 2 After sequential injection of rotenone (2 μM; complex I inhibitor), succinate (10 mM; complex II substrate), antimycin A (4 μM; complex III inhibitor), and ascorbate / TMPD (10 mM / 100 μM; electron donors for cytochrome C / complex IV), the sequential electron flow through the electron transport chain is evaluated by OCR measurement.

[0111] Mitochondrial calcium regulation: Calcium (Ca 2+ ) uptake by isolated mitochondria is measured using the calcium (Ca 2+ )-sensitive probe Calcium Green-5N. Briefly, 5 μg of mitochondria are incubated with 1 μM oligomycin and 150 nM Calcium Green-5N, and fluorescence (excitation 506 nm, emission 523 nm) is measured in a fluorescence spectrophotometer microplate reader. After a 2-minute baseline, pulses of 10 μM CaCl2 are added to the mitochondria at 4-minute intervals. Mitochondrial Ca 2+ regulation is calculated by the area under the curve after the CaCl2 pulse, which indicates the amount of extracellular Ca 2+ absorbed by the mitochondria.

[0112] ER stress measurement: The level of ER stress can be measured using H2a-GFP as a protein indicator of the early stage of ER stress. H2a-GFP is a misfolded secretory protein that accumulates in response to ER stress. STHdhQ7 / 7 is a striatum-derived cell line from a knock-in transgenic mouse (wild type) containing a homozygous humanized Huntington gene (HTT) with 7 polyglutamine repeats. STHdhQ7 / 7 cells were transfected with an Htt96Q-mCherry (mutant mimicking the typical pathogenic expression of Htt in HD patients) construct or an Htt20Q-mCherry (WT) construct. When the exon 1 of the polyQ-expanded Htt protein (96Q) fused with the fluorescent mCherry protein is expressed, the level and aggregation of the protein in individual cells can be monitored using a fluorescence microscope.

[0113] Cell lysis and immunoblotting: Cells were lysed and phosphatase inhibitor mixtures 2 and 3 and 10 mM β-glycerophosphate were added to the lysis buffer to inhibit phosphatases for the detection of phosphorylated proteins. After SDS-PAGE and transfer to a nitrocellulose membrane, the membrane was blocked and immunoblotted with the primary antibody overnight at 4 °C, then washed with the secondary antibody and blotted. After washing, an enhanced chemiluminescence assay was performed, and the membrane was exposed and quantified.

[0114] Statistical analysis: Results are expressed as the mean ± SEM (standard error of the mean) of the number of independent experiments or animals indicated in the legend. Comparisons between multiple groups were performed by nonparametric one-way analysis of variance (ANOVA) using the Kruskal-Wallis test. Correction for multiple comparisons was done by two-way ANOVA and Tukey post hoc test. Comparisons between two groups were performed by nonparametric Mann-Whitney test or parametric Student's t-test. An F-test was performed to analyze the interaction term. Significance was accepted at p < 0.05. All analyses were performed using Prism software (version 8.0, GraphPad). Mitochondrial parameters were evaluated in vitro using primary neurons isolated from Y128 HD mouse embryos, human HD lymphoblasts, and neural stem cells (NSCs). Striatal mitochondria isolated from Y128 mice treated with pridopidine or vehicle were used as an ex vivo model.

[0115] Results—

[0116] Insights into mitochondrial function can be obtained by studying morphology and transport. Since mitochondrial quality control requires fission and fusion events, both the mitochondrial aspect ratio (equivalent to the ratio between the major and minor axes of the mitochondrial ellipse), which is necessary for the high energy demands of the trophic synaptic terminal, and mitochondrial transport are metrics of mitochondrial function. Primary cortical striatal neurons collected from the HD model YAC128 (Y128) and wild-type (WT) mice were stained with MitoTracker( Figure 1A ) and evaluated for mitochondrial number( Figure 1B ) and morphology( Figure 1C and Figure 1D ). HD neurons showed impaired mitochondrial morphology: a significant increase in the number of round mitochondria( Figure 1C ) and a decrease in elongated mitochondria (p < 0.05) were observed compared to age-matched wild-type neurons( Figure 1D ). Under these conditions, although mitochondrial mass did not change( Figure 1B ), there was a bias towards mitochondrial fragmentation (fission), indicating a decline in mitochondrial function. Treatment with pridopidine (1 μM) rescued the number of both round and elongated mitochondria (p < 0.05).

[0117] In striatal neurons transfected with mitoDsRed (mitochondrial side) and stained with anti-IP 3 R (ER side) antibody, poor co-localization of mitochondria with the ER for visualizing the two cell populations was observed in Y128 HD neurons compared to WT( Figure 1E ). Pridopidine (1 μM) highly increased mitochondrial-ER co-localization in Y128 striatal neurons( Figure 1E and Figure 1F , p < 0.001). This result could explain the increase in ATP production as well as mitochondrial transport and velocity (observed in Figures 2A - 2B ). MitoDsRed-labeled mitochondria from Y128 striatal neurons also showed a decreased aspect ratio (p < 0.05)( Figure 1G ), thus confirming the previous results( Figures 1A - 1D ). Treatment with pridopidine (1 μM) reduced the number of fragmented mitochondria (p < 0.05)( Figure 1E 、 Figure 1G ).

[0118] Mitochondrial anterograde transport was also greatly reduced in HD neurons, with approximately 90% of the mitochondria in Y128 striatal neurons being stationary (p < 0.05). Pridopidine reduced the percentage of stationary mitochondria and increased both anterograde and retrograde transport (p < 0.05).( Figure 2A 、 Figure 2B—Quantitative). Compared to wild-type neurons, the mitochondrial transport rate of Y128 neurons was also reduced, moving at half speed (p<0.05). After pridopidine treatment (1 μM), this reduction was improved (p<0.05)( Figure 2A 、 Figure 2C ).

[0119] Y128 neurons showed reduced basal and maximal respiration and impaired ATP production( Figures 3A - 3H ). The reduced respiration may be a result of impaired mitochondrial dynamics and morphology demonstrated in HD neurons( Figures 1A - 1G and Figures 2A - 2B ). Treatment with pridopidine at 1 μM and 5 μM doses rescued basal and maximal respiration (p<0.01), and ATP production (p<0.05) in HD Y128 corticostriatal neurons( Figure 3B (basal), Figure 3C (maximal) Figure 3D (ATP production)). Pridopidine 1 μM also increased basal (p<0.001) and maximal (p<0.05) mitochondrial respiration, and ATP production from human iPSC-derived neural stem cells (NSCs) from heterozygous HD patients (HD-iPSCs)( Figure 3E 、 Figure 3F (basal), Figure 3G (maximal), Figure 3H (ATP production)).

[0120] Mitochondrial membrane potential (MMP, ΔΨ m ) directly affects ATP production and is affected by mitochondrial Ca 2+ signaling. Compared to wild-type cortical neurons, Y128 cortical and striatal neurons showed lower ΔΨ m , which was due to oligomycin- and FCCP-induced mitochondrial dysfunction (p<0.05), indicating lower mitochondrial retention of TMRM( Figure 4A 、 Figure 4B ). Pridopidine (0.1 and 1 μM) increased ΔΨ m in Y128 cortical and striatal neurons (cortical neurons p<0.05; 0.1 μM pridopidine in striatal neurons p<0.05, 1 μM pridopidine p<0.01)( Figures 4A - 4C ).

[0121] Hydrogen peroxide (H 2 O 2 ) was used as a more potent oxidative stimulus to evaluate ΔΨ m . In response to 0.1 mM H 2 O 2 treatment, a decrease in ΔΨ was observed in Y128 neuronsm Significantly reduced (p < 0.0001). Treatment with pridopidine (5 μM) and then exposure to 0.1 mM H 2 O 2 -induced ΔΨ loss in Y128 cortical / striatal neurons exposed for 6 h was completely restored (p < 0.001)( 2 O 2 ), as well as restored cell viability (p < 0.01)( m ). In lymphoblasts from HD patients, 0.1 mM H Figure 4D -treatment resulted in a 50% decrease in ΔΨ. After 0.1 mM H Figure 4E -treatment, pridopidine doses of 1 μM pridopidine, 5 μM pridopidine, and 10 μM pridopidine all increased ΔΨ 2 O 2 , with the 5 μM dose showing the greatest protective effect (p < 0.01)( m ). 2 O 2 m 2 2 Figure 4F )

[0122] The higher sensitivity of mitochondria from Y128 neurons and HD lymphoblasts to H 2 O 2 suggests that these cells exhibit increased oxidative stress. To test this, local H 2 O 2 flux was measured using the fluorescent probe MitoPY1. The complex III inhibitor antimycin A (AntA), which induces the production of reactive oxygen species (ROS), stimulated mitochondrial dysfunction in cortical (p < 0.01) and striatal (p = 0.0001) Y128 neurons, showing a significant 2-fold increase in mitochondrial-driven H 2 O 2 levels compared to WT neurons. In both cortical and striatal neurons, 1 μM pridopidine reversed the AntA-induced increase in mito-H 2 O 2 levels (in cortical neurons, p < 0.0001, in striatal neurons, for 0.1 μM, p < 0.01, for 1 μM, p < 0.0001)( Figures 5A - 5C ).

[0123] In HD-NSCs treated with another mitochondrial complex III inhibitor, myxothiazol (Myxo, 3 μM), cells showed a substantial increase in mito-H 2 O 2 levels (p < 0.0001). Pridopidine (1 μM) rescued the H2 O 2 Horizontal abnormal increase (p < 0.01) ( Figure 5D ). Under basal conditions, HD lymphoblasts showed increased ROS production when compared to control lymphoblasts; when stimulated with H 2 O 2 , pridopidine treatment reduced ROS levels under both basal conditions and H 2 O 2 -stimulated conditions (p < 0.001) ( Figure 5E ). Thus, pridopidine reduced ROS levels in three HD cell models.

[0124] The neuroprotective effect of pridopidine was reproduced in vivo. 1.5-month-old WT and Y128 mice (pre-symptomatic, equal male and female ratio) were treated with pridopidine 30 mg / kg or water for 45 consecutive days. Y128 mice showed motor deficits in the rotarod performance test at 3 months of age, so this test was applied before and after treatment to test the efficacy of pridopidine at the motor level. At 1.5 months (pre-treatment), Y128 mice showed the same motor coordination as wild-type mice ( Figure 6A , Figure 6B ). At 3 months of age, vehicle-treated Y128 mice showed motor deficits compared to vehicle-treated wild-type mice, as observed by a reduced latency to fall during the accelerating rotarod test (p < 0.05) ( Figure 6C ). In contrast, HD mice treated with pridopidine showed significant improvement in motor performance in the accelerating rotarod compared to vehicle-treated HD mice (p < 0.05) ( Figure 6C ). After behavioral analysis, functional mitochondria were isolated from the striatum of all mouse groups, and the activity of mitochondrial complexes was evaluated by measuring OCR after sequential injection of rotenone, succinate, antimycin A, and ascorbate / TMPD (N,N,N′,N′-tetramethyl-p-phenylenediamine) that individually stimulated or inhibited mitochondrial complexes I, II, III, and IV, respectively, thereby allowing calculation of their activity ( Figure 6D ). Compared to vehicle-treated wild-type mice, striatal mitochondria from vehicle-treated Y128 mice showed higher activity of complexes I, II, III, and IV (p < 0.01), indicating the presence of an early compensatory mechanism ( Figure 6D , Figure 6F ). Interestingly, the increase in complex activity in the striatum of Y128 HD mice was accompanied by an increase in mitochondrial H 2 O 2 production (p < 0.05) before and after inhibition of complex III with AntA (Figure 6D , Figure 6G ). These results suggest that abnormal complex activity may underlie mitochondrial ROS production by increasing electron leakage, leading to impaired ATP production. Treatment with pridopidine in Y128 mice normalizes mitochondrial complex activity and normalizes the H 2 O 2 level to that of WT vehicle-treated mice (p < 0.05)( Figures 6I - 6K ) and increases mitochondrial Ca 2+ buffering capacity( Figure 6L , Figure 6M ).

[0125] Mitochondria and the endoplasmic reticulum (ER) are functionally and physically connected. Physical contact between mitochondria and the ER occurs at sites of mitochondria-associated membranes (MAM), which are highly specialized structures enriched in S1R. MAM serves as a conduit for the exchange of proteins, lipids, signaling molecules, and importantly, Ca 2+ . Thus, ER stress and mitochondrial dysfunction are closely related (Morris, Gerwyn, Basant K. Puri, Ken Walder, Michael Berk, Brendon Stubbs, Michael Maes, and André F. Carvalho. "The Endoplasmic Reticulum Stress Response in Neuroprogressive Diseases: Emerging Pathophysiological Role and Translational Implications." Molecular Neurobiology, (2018) 55:8765 - 8787).

[0126] Pridopidine reduces mHtt-induced ER stress: In STHdhQ7 / 7 cells transfected with the mutant Htt96Q-mCherry (extended) construct, visible Htt96Q-mCherry aggregates (usually one large aggregate per cell) can be observed along with high levels of accumulated H2a-GFP indicating ER stress. STHdhQ7 / 7 cells expressing Htt20Q-mCherry (normal) or Htt96Q-mCherry without visible aggregates show low levels of H2a-GFP (no ER stress). Pridopidine significantly reduces the accumulation of H2a-GFP in mHtt aggregate-positive cells in a dose-dependent manner( Figure 7A), and does not change the H2a-GFP levels in cells without aggregates or cells expressing Htt20Q-mCherry Figure 7B ). Thus, pridopidine reduces Htt-induced ER stress in a dose-dependent manner.

[0127] Phosphorylation of eIF2α is a marker of ER stress. The level of eIF2α-phosphorylation (eIF2α-p) in STHdhQ7 / 7 cells expressing Htt96Q-mCherry is 3.5-fold that in cells expressing Htt20Q-mCherry. Treatment with pridopidine led to a significant decrease in eIF2α-P (measured by the ratio of eIF2α-P to total eIF2α), indicating reduced cellular ER stress Figure 8 ).

[0128] In summary, preclinical results indicate that in in vitro and in vivo / ex vivo HD models, mitochondrial dysfunction is a hallmark of HD, excluding an effective antioxidant response to oxidative stimuli. This dysfunction may affect synaptic integrity and cell viability. Pridopidine demonstrated rescue of different aspects of mitochondrial function in HD models, including reduced ROS levels, increased mitochondrial velocity, and percentage of elongated mitochondria in HD human and mouse models, all of which indicate mitochondrial dysfunction. Thus, pridopidine can effectively repair mitochondrial dysfunction. Administration of pridopidine also delayed the onset of the first motor symptoms in Y128 mice, increased cell viability, and rescued impaired oxidative phosphorylation. Additionally, in an in vitro HD model system, pridopidine alleviated ER stress closely related to mitochondrial dysfunction because S1R is localized at the MAM (mitochondria-associated membrane) sites of the ER membrane. These highly specialized sites play a key role in mitochondrial function, including mitochondrial fission, Ca 2+ shuttling, and oxidative stress.

[0129] This application also relates to the following items:

[0130] 1. A method for treating a disease, disorder, or any symptom thereof associated with mitochondrial dysfunction in a subject in need thereof, the method comprising administering to the subject a composition comprising pridopidine or a pharmaceutically acceptable salt thereof, thereby treating the subject.

[0131] 2. The method according to item 1, wherein the disease, disorder, or any symptom thereof associated with mitochondrial dysfunction is a disease, disorder, or any symptom associated with mitochondrial myopathy.

[0132] 3. The method according to item 2, wherein the mitochondrial myopathy is selected from MELAS syndrome, MERRF syndrome, Leigh Disease, Chronic Progressive External Ophthalmoplegia (C / PEO), diabetes with deafness (MIDD or DAD), Kearns-Sayre syndrome (KSS), Alpers Syndrome, Mitochondrial DNA depletion syndrome (MDS), Mitochondrial neurogastrointestinal encephalomyopathy (MNGIE), Neuropathy, ataxia and retinitis pigmentosa (NARP), Pearson syndrome, Leber's Hereditary Optic Neuropathy (LHON), Dominant Optic Atrophy (DOA), Pigmentary retinopathy, Wolfram Syndrome, Friedrich's Ataxia (FRDA), Mitochondrial neurogastrointestinal encephalomyopathy (MNGIE), and any combination thereof.

[0133] 4. The method according to item 1, wherein the disease, disorder, or any symptom thereof related to mitochondrial dysfunction is a disease, disorder, or any symptom related to lysosomal storage disease.

[0134] 5. The method according to item 4, wherein the lysosomal storage disease is selected from glycogenosis type II (Pompe disease), multiple sulphatase deficiency (MSD), mucopolysaccharidoses (MPS), mucolipidoses (ML) types I to III, G(M1)-gangliosidosis, Fabry disease, Farber disease, Gaucher disease, Niemann-Pick disease, mucolipidosis (ML) type IV, cystinosis, neuronal ceroid-lipofuscinoses, and any combination thereof.

[0135] 6. The method according to item 1, wherein the disease, disorder or any symptom thereof associated with mitochondrial dysfunction is bipolar disorder.

[0136] 7. The method according to any one of items 1 to 6, wherein pridopidine is in its neutral / basic form.

[0137] 8. The method according to any one of items 1 to 6, wherein pridopidine is in the form of a pharmaceutically acceptable salt.

[0138] 9. The method according to any one of items 1 to 6, wherein pridopidine is pridopidine hydrochloride.

[0139] 10. The method or composition according to any one of items 1 to 9, wherein the composition is administered by systemic administration.

[0140] 11. The method according to item 10, wherein the composition is administered orally.

[0141] 12. The method according to any one of the foregoing items, wherein the composition is administered in the form of an inhalable powder, injection, liquid, gel, solid, capsule, eye drop or tablet.

[0142] 13. The method according to any one of the foregoing items, wherein the composition is administered periodically.

[0143] 14. The method according to item 11, wherein the composition is administered once a day, twice a day, three times a day or less than once a day.

[0144] 15. The method according to item 11, wherein the composition is administered in one dose per day, two doses per day or three doses per day.

[0145] 16. The method according to any one of the preceding items, wherein pridopidine is administered at a daily dose between 1 mg / day and 400 mg / day.

[0146] 17. The method according to item 16, wherein pridopidine is administered at a daily dose between 1 mg / day and 300 mg / day.

[0147] 18. The method according to item 16, wherein pridopidine is administered at a daily dose between 1 mg / day and 90 mg / day.

[0148] 19. The method according to item 16, wherein pridopidine is administered at a daily dose between 20 mg / day and 90 mg / day.

[0149] 20. The method according to item 16, wherein pridopidine is administered at a daily dose between 45 mg / day and 90 mg / day.

[0150] 21. The method according to item 16, wherein pridopidine is administered at a daily dose between 20 mg / day and 50 mg / day.

Claims

1. Use of a composition comprising pridopidine or a pharmaceutically acceptable salt thereof and at least one pridopidine analogue represented by the structure of Compound 1 and / or Compound 4 or a pharmaceutically acceptable salt thereof in the manufacture of a medicament for treating a disease, disorder or any symptom thereof associated with mitochondrial dysfunction in a subject in need thereof:

2. The use according to claim 1, wherein the composition comprises pridopidine or a pharmaceutically acceptable salt thereof and Compound 1 or a pharmaceutically acceptable salt thereof.

3. The use according to claim 1, wherein the composition comprises pridopidine or a pharmaceutically acceptable salt thereof and Compound 4 or a pharmaceutically acceptable salt thereof.

4. The use according to claim 1, wherein the composition comprises pridopidine or a pharmaceutically acceptable salt thereof and Compound 1 and Compound 4 or a pharmaceutically acceptable salt thereof.

5. The use according to any one of claims 1-4, wherein the disease, disorder or any symptom thereof associated with mitochondrial dysfunction is a disease, disorder or any symptom associated with mitochondrial myopathy, lysosomal storage disease, bipolar disorder, Charcot-Marie-Tooth disease (CMT), vanishing white matter (VWM) disease or any combination thereof.

6. The use according to any one of claims 1-5, wherein the disease, disorder or any symptom thereof associated with mitochondrial dysfunction is vanishing white matter (VWM) disease.

7. The use according to any one of claims 1-5, wherein the disease, disorder or any symptom thereof associated with mitochondrial dysfunction is a disease, disorder or any symptom associated with mitochondrial myopathy.

8. The use according to claim 7, wherein the mitochondrial myopathy is selected from MELAS syndrome, MERRF syndrome, Leigh's disease, chronic progressive external ophthalmoplegia (CPEO), diabetes with deafness (MIDD or DAD), Kearns-Sayre syndrome (KSS), Alpers' syndrome, mitochondrial DNA depletion syndrome (MDS), mitochondrial neurogastrointestinal encephalomyopathy (MNGIE), neuropathy, ataxia and retinitis pigmentosa (NARP), Pearson syndrome, Leber's hereditary optic neuropathy (LHON), dominant optic atrophy (DOA), retinitis pigmentosa, Wolfram syndrome, Friedreich's ataxia (FRDA), mitochondrial neurogastrointestinal encephalomyopathy (MNGIE) and any combination thereof.

9. The use according to any one of claims 1-5, wherein the disease, disorder or any symptom thereof associated with mitochondrial dysfunction is a disease, disorder or any symptom associated with lysosomal storage disease.

10. Use according to claim 9, wherein the lysosomal storage disease is selected from glycogen storage disease type II (Pompe disease), multiple sulfatase deficiency (MSD), mucopolysaccharidosis (MPS), mucolipidosis (ML) types I to III, G(M1)-gangliosidosis, Fabry disease, Farber disease, Gaucher disease, Niemann-Pick disease, mucolipidosis (ML) type IV, cystinosis, neuronal ceroid lipofuscinosis, and any combination thereof.

11. Use according to any one of claims 1-5, wherein the disease, disorder or any symptom related to mitochondrial dysfunction is a disease, disorder or any symptom related to Charcot-Marie-Tooth disease (CMT).

12. Use according to any one of claims 1-11, wherein pridopidine is in its neutral / basic form.

13. Use according to any one of claims 1-11, wherein pridopidine is in the form of a pharmaceutically acceptable salt.

14. Use according to any one of claims 1-11 and 13, wherein pridopidine is pridopidine hydrochloride.

15. Use according to any one of claims 1-14, wherein the composition is administered by systemic administration.

16. Use according to any one of claims 1-15, wherein the composition is administered orally.

17. Use according to any one of claims 1-16, wherein the composition is administered in the form of an inhalable powder, injection, liquid, gel, solid, capsule, eye drop or tablet.

18. Use according to any one of claims 1-17, wherein the composition is administered periodically.

19. Use according to any one of claims 1-18, wherein the composition is administered once a day, twice a day, three times a day or less than once a day.

20. Use according to any one of claims 1-19, wherein the composition is administered one dose a day, two doses a day or three doses a day.

21. Use according to any one of claims 1-20, wherein pridopidine is administered at a daily dose between 1 mg / day and 400 mg / day.

22. Use according to claim 21, wherein pridopidine is administered at a daily dose between 1 mg / day and 90 mg / day.

23. Use according to claim 21, wherein pridopidine is administered at a daily dose between 45 mg / day and 90 mg / day.

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