Treatment of mitochondrial-related diseases and disorders, including symptoms thereof, with pridopidine

文档序号：197088 发布日期：2021-11-02 浏览：32次中文

阅读说明：本技术 使用普利多匹定治疗线粒体相关疾病和病症，包含其症状 (Treatment of mitochondrial-related diseases and disorders, including symptoms thereof, with pridopidine ) 是由迈克尔·海登米甲·杰瓦安娜·克里斯蒂娜·卡瓦略雷戈于 2020-03-15 设计创作，主要内容包括：本发明提供了一种用于治疗患有与线粒体功能障碍相关的疾病或病症的受试者的方法,所述方法包括向所述受试者施用包括普利多匹定或其药学上可接受的盐的组合物。(The present invention provides a method for treating a subject having a disease or disorder associated with mitochondrial dysfunction, the method comprising administering to the subject a composition comprising pridopidine or a pharmaceutically acceptable salt thereof.)

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 (pridopidine) or a pharmaceutically acceptable salt thereof, thereby treating the subject.

2. The method of claim 1, wherein the disease, disorder, or any symptom thereof associated with mitochondrial dysfunction is a disease, disorder, or any symptom associated with mitochondrial myopathy.

3. The method of claim 2, wherein the Mitochondrial myopathy is selected from the group consisting of MELAS Syndrome, MERRF Syndrome, reishi Disease (Leigh Disease), Chronic Progressive extraocular paralysis (Chronic Progressive External opthalmoplegia, C/PEO), diabetes-associated deafness (mid or DAD), cahns-seoul Syndrome (keys-Sayre Syndrome, KSS), Alpers Syndrome (Alpers Syndrome), Mitochondrial DNA depletion Syndrome (mitocholord DNA depletion Syndrome, MDS), Mitochondrial neurogastrointestinal encephalomyopathy (mitochordronous neuropathogenic intrinsic pathophysiological rp), MNGIE, Neuropathy, ataxia and retinitis pigmentosa (neuropathic, retinal Atrophy), retinal Dominant Atrophy (retinopathy, Optic Atrophy), retinal Atrophy (Optic Atrophy ), retinal Atrophy (Optic Atrophy), retinal Atrophy, Optic nerve Atrophy, Optic nerve degeneration, nerve degeneration, nerve degeneration, nerve, Wolfrom Syndrome (Wolfram Syndrome), Friedrich's Ataxia (FRDA), mitochondrial neurogastrointestinal encephalomyopathy (MNGIE), and any combination thereof.

4. The method of claim 1, wherein the disease, disorder, or any symptom thereof associated with mitochondrial dysfunction is a disease, disorder, or any symptom associated with a lysosomal storage disease.

5. The method of claim 4, wherein the lysosomal storage Disease is selected from the group consisting of Glycogenosis Type II (Pompe Disease), Multiple sulfatase Deficiency (Multiple sulfophase Deficiency, MSD), Mucopolysaccharidosis (MPS), Mucolipidosis (ML) from Type I to Type III, G (M1) -Gangliosidosis (G (M1) -gangliosis), Fabry Disease (Fabry Disease), Faber Disease (Faber Disease), Gaucher Disease (Gaucher Disease), Niemann-Pick Disease (Niemann-Pick Disease), Mucolipidosis (ML) Type IV, Cystinosis (Cystinosis), Neuronal lipofuscosis (Ceroid), and any combination thereof.

6. The method of claim 1, wherein the disease, disorder, or any symptom thereof associated with mitochondrial dysfunction is bipolar disorder (bipolar disorder).

7. The method of any one of claims 1-6, wherein the pridopidine is in its neutral/basic form.

8. The method of any one of claims 1-6, wherein the pridopidine is in the form of a pharmaceutically acceptable salt.

9. The method of any one of claims 1-6, wherein the pridopidine is pridopidine hydrochloride.

10. The method or composition of any one of claims 1-9, wherein the composition is administered by systemic administration.

11. The method of claim 10, wherein the composition is administered by oral administration.

12. The method of any one of the preceding claims, wherein the composition is administered in the form of an inhalable powder, injection, liquid, gel, solid, capsule, eye drop, or tablet.

13. The method of any one of the preceding claims, wherein the composition is administered periodically.

14. The method of claim 11, wherein the composition is administered once daily, twice daily, three times daily, or less than once daily.

15. The method of claim 11, wherein the composition is administered in one dose per day, two doses per day, or three doses per day.

16. The method of any one of the preceding claims, wherein pridopidine is administered at a daily dose of between 1 mg/day and 400 mg/day.

17. The method of claim 16, wherein pridopidine is administered at a daily dose of between 1 mg/day and 300 mg/day.

18. The method of claim 16, wherein pridopidine is administered at a daily dose of between 1 mg/day and 90 mg/day.

19. The method of claim 16, wherein pridopidine is administered at a daily dose of between 20 mg/day and 90 mg/day.

20. The method of claim 16, wherein pridopidine is administered at a daily dose of between 45 mg/day and 90 mg/day.

21. The method of claim 16, wherein pridopidine is administered at a daily dose of between 20 mg/day and 50 mg/day.

Technical Field

The present invention provides a method for treating a subject having a disease or disorder associated with mitochondrial dysfunction, comprising administering to the subject a composition comprising pridopidine (pridopidine) or a pharmaceutically acceptable salt thereof.

Background

Mitochondria are double-membrane organelles found in most eukaryotic cells and perform many metabolic functions, including ATP synthesis through oxidative phosphorylation (OXPHOS). Mitochondria are also involved in the synthesis of biomolecules, maintenance of calcium homeostasis, production of Reactive Oxygen Species (ROS), and activation of apoptosis. Mitochondria are structurally complex and highly dynamic, mobile organelles. Mitochondria undergo constant morphological changes through a continuous cyclic process of fusion and fission, which determines their morphology and most mitochondrial function.

Given its central role in cellular homeostasis, mitochondrial dysfunction is associated with a number of age-related disorders, including mitochondrial diseases, cancer, metabolic diseases and diabetes, inflammatory conditions, neurodegenerative disorders, neuropathy, and neurodegenerative diseases, such as alzheimer's disease, parkinson's disease, and huntington's disease. Pridopidine (4- [3- (methylsulfonyl) phenyl)]-1-propyl-piperidine) (formerly known as ACR16,TV-7820) is under clinical development for the treatment of HD and ALS. Polypridine is shown to play a neuroprotective role in animal and cellular models of neurodegenerative diseases, including models of HD, PD, ALS and AD (Francardo, Veronica, Michal Geva, France score, Quentin Denis, Lilach Steiner, Michael R.Hayden and M.Angela Cenci.2019. "Primolidine Induces Functional neurological recovery in a Mouse Model of Parkinson's Disease Via the Sigma-1 Receptor (Primolidine industries Via the Sigma-1 Receptor in the Mouse Model of Parkinson's Disease" (Neurothelials) 16(2): 465-79; Ryskamp, Daniel Wyal, Jiun Wyal, Kingan Wvabin, Geuch, Miuchai, and HamiltoniStabilization of mushrooms in a Mouse model of Alzheimer's Disease by Acting on Sigma-1 receptors (Primary stabitizes Mushroom spores in Mouse Models of Alzheimer's Disease) Neurobiology of the Disease (Neurobiology of Disease), 124, 489-504; ryskamp, Daniel, Jun Wu, Michal Geva, Rebecca Kusko, Iris Grossman, Michael Hayden, and Ilya Bezprozvanny.2017. "Sigma-1 Receptor Mediates The Beneficial Effects of Pridopidine in Huntington's Disease Mouse models (The Sigma-1 receptors Mediates The Benefit Effects of Pridopidine in a Mouse Model of Mouse of Huntington's Disease neurobiology 97 (part A): 46-59; ionescu, Ariel, Tal Gradus, Topaz Altman, Roy Maimon, Noi Saraf Avraham, Michal Geva, Michael Hayden, and Eran Perlson.2019. "improving the core characteristics of ALS Pathology in the SOD1G93A Model by Targeting Pridopridine to the Sigma-1 Receptor (Targeting the Sigma-1 Receptor is primer amino acids complexes catalysts of ALS Pathology in a SOD1G93A Model.)" Cell Death and disease (Cell Death)&Disease) 10, (3) 210; garcia-mirales, Marta, Michal Geva, lacing Yin Tan, Nur Amirah Bite Mohammad Yuso, Yoonjeong Cha, Rebecca Kusko, Liang Juin Tan et al 2017, "Early Pruli Do-P.treatment Improves the behavior and Transcriptional Deficits in YAC128 Huntington Disease Mice (Early Pridopidine Treatment Impropens beer and Transcriptional Deficits in YAC128 Huntington Disease Mice.)" journal of clinical investigation (JCI Insight.) "). Mitochondrial dysfunction has been demonstrated in the pathology of each of these neurodegenerative diseases.

Disclosure of Invention

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.

In a further aspect, the 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.

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.

In other embodiments, the Mitochondrial myopathy is selected from MELAS Syndrome, MERRF Syndrome, leiomycosis (Leigh Disease), Chronic Progressive extraocular paralysis (Chronic Progressive External opthalmoplegia, C/PEO), diabetes-associated deafness (mid or DAD), cahn-seoul Syndrome (Kearns-Sayre Syndrome, KSS), Alpers Syndrome (Alpers Syndrome), Mitochondrial DNA depletion Syndrome (mitochondroidal DNA depletion Syndrome, MDS), Mitochondrial neurogastrointestinal encephalomyopathy (mitochondrogenic neuropathogenic neuropathophathy, MNGIE), Neuropathy, ataxia and Pigmentary retinitis pigmentosa (neuropathic and Pigmentary, corticospinal syndromous), dermatosis (neurovegetative paranoid), neurodystrophy (neurovegetative Atrophy), neuroretinal Atrophy (neurovegetative Atrophy), neurovegetative Atrophy (neurovegetative Atrophy), neurovegetative Atrophy, Optic Atrophy Syndrome, neuronopathy (neuronopathy), neuroretinopathy (neuroretinopathy, Optic Atrophy), neuroretinopathy (neuronopathy, Optic Atrophy Syndrome), neuroretinopathy, neuroleptic Atrophy (neuroretinopathy, neurolysis, neuroretinopathy, neurolysis, neuroretinopathy, neuro, Friedrich's Ataxia (FRDA), mitochondrial neurogastrointestinal encephalomyopathy (MNGIE), and any combination thereof.

In further embodiments, the disease, disorder, or any symptom thereof associated with mitochondrial dysfunction is a disease, disorder, or any symptom associated with a lysosomal storage disease.

In other embodiments, the lysosomal storage Disease is selected from the group consisting of Glycogenosis Type II (Pompe Disease), Multiple Sulfatase Deficiency (MSD), Mucopolysaccharidosis (MPS), Mucolipidosis (ML) types I to III, G (M1) -Gangliosidosis (G (M1) -Gangliosidosis), Fabry Disease (Fabry Disease), Fabry Disease (Farber Disease), Gaucher Disease, Niemann-Pick Disease (niema-Pick Disease), Mucolipidosis (ML) Type IV, Cystinosis (Cystinosis), Neuronal Ceroid lipofuscinosis (cerulosis), and any combination thereof.

In some embodiments, the disease, disorder, or any symptom thereof associated with mitochondrial dysfunction is a disease, disorder, or any symptom associated with a neurodegenerative disease.

In some embodiments, the neurodegenerative disease is selected from parkinson's disease, huntington's disease, amyotrophic lateral sclerosis, alzheimer's disease, frontotemporal dementia (FTD), peroneal muscular atrophy (CMT), and any combination thereof.

In some embodiments, the disease, disorder, or any symptom thereof associated with mitochondrial dysfunction is bipolar disorder.

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

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

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

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

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

Drawings

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

figures 1A-1G show that pridopidine rescues abnormal mitochondrial morphology and restores mitochondrial-ER contact in Y128(YAC128, HD) neurons. FIG. 1A: visual representation of mitochondrial networks in WT and Y128(HD) cortical/striatal neurons stained with Mitotracker dye. FIG. 1B: the number of mitochondria between quantified-WT from 1A and Y128(HD) cortical/striatal neurons was similar before and after pridopidine treatment (n-4). Fig. 1C and 1D: y128 HD mitochondria showed morphologic impairment with an increased percentage of round mitochondria (1C) and a decreased percentage of elongated mitochondria (1D). Pridopidine treatment (1 μ M) corrected the Y128 morphology to normal levels. (n-4). FIG. 1E: WT and Y128 neurons were stained with mitochondrial markers (column 1E) and ER markers (column 1E right) and co-localization of ER and mitochondria was analyzed (column 1E left) (n ═ 15 projections/pathology). FIG. 1F: quantification of 1E showed reduced Mito-ER contact in Y128 neurons compared to WT. Pridopidine (1 μ M) significantly increased the Mito-ER contact. FIG. 1G: aspect ratio, the ratio between the mitochondrial axes indicates mitochondrial health, (see methods, paragraph 86 and results, paragraph 103) is reduced in HD neurons. Pridopidine treatment rescued the aspect ratio of the Y128 neuron (1G). P <0.05 and p <0.001 by kruskal-vories test and dunn multiple comparison test.

Figures 2A-2B show that pridopidine improves impaired mitochondrial dynamics in Y128 HD neurons. FIG. 2A: MitoDSRed transfected mitochondria were tracked in rotating disc confocal for 12 minutes and velocity and directional transport were quantified using a waverecorder (n ═ 7-9 projections/pathology). WT-upper plot shows mitochondrial motion in both retrograde and antegrade directions. Y128 — intermediate image shows no mitochondrial motion in either direction in HD compared to WT. Y128 treated with 1 μ M pridopidine-the lower panel shows that pridopidine treatment restored mitochondrial movement in both retrograde and antegrade, similar to WT. FIG. 2B: the quantitative, transport-oriented analysis of figure 2A shows that the percentage of quiescent mitochondria in Y128 neurons is increased, while both retrograde and anterograde transport are decreased. Pridopidine treatment reduced the percentage of quiescent mitochondria to WT levels and increased the percentage of both retrograde and antegrade transport to WT levels. FIG. 2C: the overall velocity of the Y128 neuron decreases. Pridopidine treatment increases bus mitochondrial speed. By 2-way ANOVA, then by Tukey's multiple comparison test, # p <0.05, $ p <0.05 (for antegrade Y128 basis).

Figures 3A-3H show that pridopidine rescues impaired mitochondrial respiration in the HD cell model. FIGS. 3A-3D: oxygen consumption and ATP production were assessed in WT and Y128 cortical/striatal neurons treated with 1 and 5 μ M pridopidine for 24 hours, with both doses showing rescue of basal and maximal respiration, and increased ATP production (n-3). FIGS. 3E-3H: oxygen consumption and ATP production of HD Neural Stem Cells (NSCs) are reduced. Pridopidine treatment at 1 μ M for 24 hours saved basal and maximal breathing and ATP production. P <0.05 and p <0.01 by kruskal-vories test and dunn multiple comparison test.

Figures 4A-4C show that pridopidine treatment protected Y128 neurons and HD lymphoblasts from induced mitochondrial dysfunction. Cortex (FIG. 4A-left and FIG. 4B) and striatum (FIG. 4A-right and FIG. 4C) WT and Y128 neurons treated with 0.1 or 1 μ M pridopidine were evaluated for mitochondrial membrane potential (MMP,. DELTA.Ψ) following depolarization with oligomycin plus FCCP (carbonyl cyanide-4- (trifluoromethoxy) phenylhydrazone, an oxidative phosphorylation uncoupler)_m) (n-7-10). Pridopidine (0.1 and 1 μ M) increased MMP, which was decreased in Y128 neurons, and returned to levels comparable to WT in both cortical and striatal neurons. FIGS. 4D-4E: using H₂O₂Induction of oxidative stress in Y128 cortical/striatal co-cultures, resulting in significant reduction of MMP (FIG. 4E) and reduced cell viability(FIG. 4F). Pridopidine treatment (5 μ M) rescued MMP (fig. 4D) and increased cell survival (fig. 4E), fig. 4F: human lymphoblasts from HD patients or healthy controls were treated with 1, 5 or 10 μ M pridopidine and/or H₂O₂And (4) preprocessing. All doses of pridopidine increased Δ Ψ_mThe most significant effect was achieved with 5 μ M (n ═ 4). P by Kruskal-Wals test and Dunn multiple comparison test<0.05，**p<0.01，***p<0.001，****p<0.0001. "Pri" refers to pridopidine.

Figures 5A-5E show that pridopidine restores oxidative challenge-induced Reactive Oxygen Species (ROS) production in Y128 neurons and HD NSCs and lymphoblasts. FIGS. 5A-5C: FIG. 5A is a schematic view for recording H₂O₂Representative images of cortical/striatal neuron cultures treated with MitoPY fluorescent probe at level. As indicated, mitochondrial complex III inhibitor antimycin a (AntA, 2 μ M) was added to induce mitochondrial dysfunction (left panel-before AntA treatment, middle panel-untreated neurons after Ant a treatment, right panel-pridopidine treated neurons after AntA treatment). FIG. 5B: fig. 5A quantification in cortical neurons. FIG. 5C: fig. 5A quantification in striatal neurons. Cortical and striatal neurons treated with 1 μ M pridopidine showed mitochondrial H recorded by MitoPY fluorescent probes after AntA₂O₂The levels decreased, as indicated (n-4, considering about 20 cells/pathology for striatal neurons and about 10 cells/pathology for cortical neurons). Scale bar 30 μ M. FIG. 5D: treatment of human NSC with mitochondrial Complex II inhibitor Thiazolidine (Myxo, 3 μ M), which also induces mitochondrial dysfunction and increases mitochondrial H₂O₂And (4) horizontal. Treatment with pridopidine (1. mu.M) for 24 hours reduced mitochondrial H₂O₂Horizontal (n-4). FIG. 5E: h for control and HD lymphoblastoid cells₂O₂Elicitation to induce mitochondrial dysfunction, resulting in increased ROS levels (using CellRox staining to determine ROS levels). Pretreatment with 5 μ M pridopidine for 24 hours significantly reduced ROS levels. (n-4). Multiple comparison tests by Tukey, by 2-way ANOVA,^*p<0.05，^**p<0.01，^*＊＊*p<0.0001. in fig. 5E, by the kruskal-wallis test and dunne multiple comparison test,^*＊*p<0.001，^*＊＊*p<0.0001。

FIGS. 6A-6M show that pridopidine treatment delayed the onset of Y128 mouse motor deficits, normalized mitochondrial Complex Activity and reduced H in isolated Y128 striatal mitochondria₂O₂And (4) generating. FIG. 6A: schematic representation of in vivo experimental design: mice were treated with vehicle or pridopidine for 45 days. Rotarod behavioral testing (RR) was measured before treatment on day 0 (1.5 months of age) and on day 44 (3 months of age); FIG. 6B: at 1.5 months of age, Y128 mice had no impairment in locomotor ability compared to WT prior to treatment. FIG. 6C: at 3 months of age, significant lesions were observed in vehicle-treated Y128 mice. In the rotarod exercise test, pridopidine treatment significantly increased the Y128 mice drop delay time compared to vehicle treated controls. FIGS. 6D-6H: measurement of oxygen consumption rate using hippocampal XF (OCR) the electron flow in striatal mitochondria from wild type and Y128 mice treated with vehicle or treated with pridopidine was evaluated. Mitochondrial complex inhibitors and substrates, 2 μ M rotenone, 10mM succinate, 4 μ M antimycin A, and 1mM ascorbate/100 mM TMPD were injected sequentially to calculate the activity of mitochondrial complex I, complex II, complex III, and complex IV, respectively. Complexes II, III and IV showed increased OCR in Y128 mice, indicating that an early compensatory mechanism exists and pridopidine rescued this effect. FIGS. 6I-6K: antimycin A (2. mu.M) was used to inhibit mitochondrial complex III. Mitochondrial H in mitochondria isolated from Y128 mice compared to WT mice₂O₂The level increased. Pridopidine reduced mitochondrial H in mice₂O₂And (4) horizontal. XY lines show the time-dependent change in fluorescence after addition of antimycin a. FIGS. 6L-6M: calcium uptake in mitochondria from pridopidine-treated Y128 mice was also improved. Extracellular calcium levels decreased in response to pridopidine treatment (fig. 6L), which is evidence of increased mitochondrial calcium regulation (fig. 6M), by non-parametric kruskal-walis assay,^*p<0.05，^**p<0.01。

FIGS. 7A-7B show that pridopidine reduces mHtt-induced ER stress. H2a-GFP was transiently co-expressed with the mutations Htt96Q-mCherry (HD exon 1) (FIG. 7A) or Htt20Q-mCherry (WT) (FIG. 7B) in STHdhQ7/7 cells. H2a-GFP aggregation is a marker of ER stress. Aggregation of mCherry and GFP was evaluated and images were acquired using confocal microscopy. Htt96Q enhanced ER stress compared to untreated cells, and treatment with pridopidine at 0.03 μ M, 0.3 μ M, and 3 μ M reduced ER stress in these cells (fig. 7A). Htt20Q (wt) did not induce ER stress (fig. 7B). For comparison purposes, 100% represents the relative intensity of H2a-GFP in untreated cells exhibiting Htt96Q-mCherry aggregates, and 0% is the relative intensity of H2a-GFP in untreated cells without Htt96Q-cherry aggregates. The graph is the average of 3 experiments + -SE. P <0.05 and p <0.01 compared to untreated cells.

Figure 8 shows that pridopidine reduces elevated levels of phosphorylated eIF 2a (eIF2 a-P) in the HD model (a measure of ER stress). eIF2 α -P levels were determined in HEK293 cells transfected with either mutant Htt (Htt96Q, solid line) or WT Htt (Htt20Q, dashed line) and treated with 0.3 and 3 μ M pridopidine for 24 hours. The ratio of eIF2 α -P to total eIF2 α was quantified by immunoblotting. Mutant Htt increased phosphorylated eIF2 α levels compared to WT, and both pridopidine concentrations decreased eIF2 α -P levels. (. p <0.05)

Detailed Description

In the following detailed description, numerous specific details are set forth in order to provide a thorough understanding of the present invention. However, it will be understood by those skilled in the art 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.

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

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.

In other embodiments, the mitochondrial myopathy is selected from MELAS syndrome, MERRF syndrome, reith's disease, alper's syndrome, chronic progressive extraocular paralysis (C/PEO), diabetes with deafness (mid or DAD), cahns-seoul 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, waldens's syndrome, friedreich's ataxia (FRDA), mitochondrial neurogastrointestinal encephalomyopathy (MNGIE), and any combination thereof.

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 genetic mutations in nuclear DNA, the gene product of which is imported into mitochondria (mitochondrial proteins) and acquired mitochondrial pathology. Mitochondrial diseases have unique characteristics, both because of the genetic pattern of the disease and because mitochondria are critical to cellular function. A subclass of these diseases with symptoms of neuromuscular diseases is commonly referred to as mitochondrial myopathy.

Mitochondrial myopathy diseases and disorders include, but are not limited to:

MELAS syndrome (mitochondrial myopathy, encephalopathy, lactic acidosis and stroke). Progressive neurodegenerative disorders caused by mutations in mitochondrial DNA. As the disease is poorly understood and difficult to diagnose, it is currently unknown how many people worldwide have MELAS. The syndrome affects all ethnic groups as well as both men and women. Affected individuals typically begin to exhibit symptoms between the ages of 4 and 40. Poor prognosis; the disease is often fatal. MELAS syndrome is not curable; healthcare is primarily supportive. Symptoms are: due to the presence of defective mitochondria in all cells of patients with MELAS syndrome, a variety of symptoms can occur, often debilitating. Stroke can cause brain damage, resulting in seizures, numbness, or partial paralysis. Encephalopathy (brain disease) can result in tremors, muscle spasms, blindness, deafness, and may lead to dementia. Myopathy (muscle disease) causes difficulty in walking, exercising, eating, and speaking.

MERRF syndrome (or myoclonic epilepsy with broken red fibers): extremely rare conditions that begin in childhood and affect the nervous system and skeletal muscles, as well as other body systems. MERRF is characterized by myoclonus, consisting of sudden, transient, and tic 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 slow deterioration of mental function (dementia). Short stature, optic nerve degeneration (optic atrophy), hearing loss, cardiomyopathy, and paresthesia caused by nerve damage (peripheral neuropathy) are also common symptoms. When trichrome stained with modified Gomori and viewed under a microscope, abnormal muscle cells were present and appeared as broken red fibers (RRF). MERRF is caused by mitochondrial dna (mtdna) mutations.

The prevalence of Raynaud's disease at birth is estimated to be approximately one 36000. The typical onset of symptoms occurs before 12 months of age, but in rare cases the disease may manifest during puberty or even early adulthood. Loss of motor milestones, hypotonia with poor head control, repeated vomiting, and dyskinesia are common initial symptoms. Pyramidal tracts and extrapyramidal signs, nystagmus, respiratory disorders, ophthalmoplegia and peripheral neuropathy are often noted later. Epilepsy is relatively rare. There is no specific treatment for reishi disease.

Chronic progressive external ophthalmoplegia (C/PEO) is characterized by slow progressive paralysis of the external ocularis. Patients often experience bilateral symmetrical progressive ptosis, followed by ophthalmoplegia after months to years. 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 are often associated with skeletal muscle weakness. However, individuals with similar clinical presentations may have various mitochondrial defects.

Diabetes with deafness (MIDD or DAD): MIDD accounts for 1% of people with diabetes. More than 85% of people carrying mitochondrial DNA mutations at position 3243 present symptoms of diabetes. Persons diagnosed with mid are typically diagnosed with an average age of 37 years, but are also considered to range in age between 11 and 68 years. In 75% of these diabetic patients carrying mitochondrial DNA mutations at position 3243 experienced sensorineural hearing loss. In these cases, hearing loss usually occurs before the onset of diabetes and is marked by a decline in perception of high tonal frequencies. The hearing loss associated with diabetes is generally more common in men than women and declines more rapidly.

Kanssier syndrome (KSS) onset: before the age of 20 years. The prevalence of cahns-seoul syndrome is approximately 1 to 3 per 100,000 individuals. Rare neuromuscular disorders. An important clinical symptom is the presence of unilateral or bilateral ptosis (partial closure of the eyelids). The disease is primarily characterized by three main findings: progressive paralysis of certain ocular muscles (chronic progressive external ophthalmoplegia [ CPEO ]); abnormal accumulation of pigmented material on the nerve-rich membranes lining the eye (atypical retinitis pigmentosa), leading to chronic inflammation, progressive degeneration and abrasion of certain eye structures (retinitis pigmentosa); and heart diseases (cardiomyopathy), such as heart block. Other findings may include muscle weakness, short stature, hearing loss, and/or loss of ability to coordinate voluntary movement (ataxia) due to problems affecting a part of the brain (cerebellum). In some cases, KSS may be associated with other disorders and/or conditions.

Alper's Syndrome (Alpers-Huttenlocher Syndrome)): attack: several weeks to years after birth. Symptoms are: psychomotor degeneration (dementia), epilepsy, and liver disease. Severe and persistent seizures can lead to death within the first decade of life.

Onset of mitochondrial DNA depletion syndrome (MDS): symptoms in infancy: such conditions often lead to muscle weakness and/or liver failure, and more rarely to brain abnormalities. "flaccidity", feeding difficulties and developmental delay are common symptoms; PEO and seizures are less common.

Onset of mitochondrial neurogastrointestinal encephalomyopathy (MNGIE): usually 20 years ago. Symptoms are: this condition can lead to PEO, ptosis (drooping eyelids), weakness in limbs, and gastrointestinal (digestive) problems, including chronic diarrhea and abdominal pain. Another common symptom is peripheral neuropathy (neurological dysfunction that can lead to sensory damage and muscle weakness).

Neuropathy, ataxia, and retinitis pigmentosa (NARP) attacks: from infancy to adulthood. Symptoms are: NARP can lead to neuropathy (neurological dysfunction that can lead to sensory damage and muscle weakness), ataxia, and retinitis pigmentosa (degeneration of the retina of the eye, which leads to loss of vision). NARP may also lead to developmental delay, epilepsy and dementia.

Pearson syndrome onset: in infancy. Symptoms are: this syndrome can lead to severe anemia and pancreatic dysfunction. Children who survive the disease often develop the cahns-seoul syndrome.

Leber's Hereditary Optic Neuropathy (LHON), the first maternally inherited ophthalmic disorder associated with mitochondrial DNA point mutations, is characterized by the appearance of acute and painless central vision loss in both eyes in a continuous fashion over a period of days with months. The generally accepted prevalence of LHON in the uk and other parts of europe is estimated to be one in 25,000. Three mtDNA point mutations in the mitochondrial respiratory chain complex I subunit gene (G11778A in ND4, G3460A in ND1, and T14484C in ND 6) collectively led to 95% of LHON cases. Other pathogenic mtDNA mutations continue to be identified, particularly in the non-caucasian population, as the recently identified mtDNA T12338C mutation in ND5 appears to be common in the han nationality.

Dominant Optic Atrophy (DOA): DOA is an inherited disease that primarily 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 drops to an average of 20/80 to 20/120 over the first two decades of life. Neural retinal limbic thinning appears to be a common finding of DOA, with occasional findings involving disc "dishing", cup-to-disc ratios in excess of 0.5, and peri-optic atrophy. Early optic nerve appearance is often characterized by pale sector of the optic nerve.

Pigmentary retinopathy and other ophthalmic problems: pigmentary retinopathy is a non-specific finding that may be found in several mitochondrial diseases. The most detailed primary mtDNA diseases seen in pigmented retinopathy are neurogenic weakness, ataxia and retinitis pigmentosa (NARP), which are caused by a T8993C mtDNA mutation in the mitochondrial complex V subunit gene ATPase 6.

Walflemm's syndrome: a genetic condition commonly associated with childhood onset insulin-dependent diabetes mellitus and progressive optic nerve atrophy. In addition, many people with walflem's syndrome also develop diabetes insipidus and sensorineural hearing loss. The old name of the syndrome is didoad, which refers to diabetes insipidus, diabetes, optic nerve atrophy and deafness. Some people have mutations in the same gene that causes walflemm's syndrome, but they do not acquire all the features of the syndrome, and are therefore said to have WFS 1-related diseases. The main symptoms of walflerm syndrome (diabetes, optic atrophy, diabetes insipidus and hearing loss) can occur at different ages and change at different rates.

Friedreich ataxia (FRDA): hereditary progressive neurodegenerative movement disorders, with a typical age of onset between 10 and 15 years. Initial symptoms may include postural instability, frequent falls, and progressive difficulty walking due to impaired ability to coordinate voluntary movements (ataxia). Affected individuals often 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 (arrhythmias). About one third of people with FRDA develop diabetes. The symptoms and clinical findings associated with FRDA are primarily due to degenerative changes in sensory nerve fibers at the point where they enter the spinal cord in a structure known as the dorsal root ganglion. This can lead to secondary degeneration of nerve fibers in the spinal cord, resulting in a lack of sensory signals in the cerebellum, the part of the brain that helps coordinate voluntary movements.

Mitochondrial neurogastrointestinal encephalomyopathy (MNGIE): progressive metabolic disorders caused by Thymidine Phosphorylase (TP) enzyme deficiency. The lack of TP results in systemic accumulation of the deoxyribonucleosides thymidine (dThd) and deoxyuridine (dUrd). In these patients, clinical features include mental deterioration, ophthalmoplegia, and fatal gastrointestinal complications. The accumulation of nucleosides also leads to 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 is still unknown.

In further embodiments, the disease, disorder, or any symptom thereof associated with mitochondrial dysfunction is a disease, disorder, or any symptom associated with a lysosomal storage disease.

In other embodiments, the lysosomal storage disease is selected from the group consisting of glycogen storage disease type II (pompe disease), Multiple Sulfatase Deficiency (MSD), Mucopolysaccharidosis (MPS), Mucolipidosis (ML) types I to III, G (M1) -gangliosidosis, fabry disease, gaucher disease, niemann-pick disease, Mucolipidosis (ML) type IV, cystinosis, neuronal ceroid lipofuscinosis, and any combination thereof.

When referring to "Lysosomal Storage Disease (LSD)," it should be understood to encompass any disease, disorder or condition characterized by the gradual accumulation of undigested macromolecules in lysosomes. The massive accumulation of material can affect lysosomal function and impair autophagy flux, which can affect cellular mass control of organelles such as mitochondria. LSDs exhibit signs of mitochondrial dysfunction including changes in mitochondrial morphology, a decrease in mitochondrial membrane potential (Δ Ψ m), a decrease in ATP production, and an increase in Reactive Oxygen Species (ROS) production. Furthermore, a decrease in autophagy 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), mucopolysaccharide storage disease (MPS), Mucolipidosis (ML) type I-III, G (M1) -gangliosidosis, Fabry's disease, gaucher's disease, Niemann-pick's disease, Mucolipidosis (ML) type IV, cystinosis, neuronal ceroid lipofuscinosis.

In some embodiments, the disease, disorder, or any symptom thereof associated with mitochondrial dysfunction is a disease, disorder, or any symptom associated with a neurodegenerative disease.

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

In neurodegenerative diseases such as parkinson's disease, huntington's disease, amyotrophic lateral sclerosis, frontotemporal dementia (FTD), peroneal amyotrophic lateral sclerosis (CMT), and alzheimer's disease, mitochondrial function is impaired.

In some embodiments, the neurodegenerative disease is selected from parkinson's disease, huntington's disease, amyotrophic lateral sclerosis, frontotemporal dementia (FTD), peroneal muscular atrophy (CMT), alzheimer's disease, and any combination thereof.

In some embodiments, the disease, disorder, or any symptom thereof associated with mitochondrial dysfunction is white matter disappearance (VWM) disease. Leukopenia (VWM) is one of more than 50 conditions collectively known as leukodystrophy, which affects the white matter or myelin of the brain. VWM, also known as childhood ataxia with central nervous system hypomyelination (CACH), is a very rare neurological condition that is the destruction of myelin, white matter of the brain, or myelin sheath. In doing so, the 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: childhood ataxia with diffuse CNS hypomyelination (CACH), leukoleukopenia (VWM), critikoencephalopathy, leukodystrophy with ovarian failure, and any combination thereof.

In a further embodiment, 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.

Bipolar disorder: one of the major mental disorders, presents with manic and depressive episodes, often accompanied by psychotic symptoms. Mitochondrial DNA mutations and mitochondrial dysfunction account for a subset of patients with the disorder.

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 symptom of the disease or disorder associated with mitochondrial dysfunction comprises any one or more of: dysplasia, loss of muscle coordination, muscle weakness, neurological deficit, epilepsy, autism spectrum, autism-like features, learning disorders, heart diseases, liver diseases, kidney diseases, gastrointestinal diseases, severe constipation, diabetes, increased risk of infection, thyroid dysfunction, adrenal dysfunction, autonomic dysfunction, confusion, disorientation, memory loss, growth disorders, developmental retardation, coordination disorders, sensory (vision, hearing) problems, mental decline, organ diseases, dementia, respiratory problems, hypoglycemia, apnea, lactic acidosis, seizures, dysphagia, developmental delays, movement disorders (dystonia, muscle spasm, tremor, chorea), stroke, and brain atrophy.

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.

For the methods and uses disclosed herein, the route of administration can be, for example, oral. Routes of administration can also be classified by whether the effect is local (e.g., topical administration) or systemic (e.g., enteral or parenteral administration). As used herein, "topical administration" shall mean the administration of a compound or composition directly to the site in need of its action, and specifically excludes systemic administration. As used herein, "topical administration" of a compound or composition shall mean application of the compound or composition to a bodily surface, such as skin or a mucosal membrane, such as the eye. As used herein, "ocular administration" shall mean applying a compound or composition to the eye of a subject or to the skin surrounding the eye (periocular skin) or to the mucosa surrounding the eye, particularly the conjunctiva of a subject, i.e., topical administration. The amounts of pridopidine and pharmaceutical compositions of the present invention may be administered by oral administration, topical administration, systemic administration, topical administration or ocular administration.

In some embodiments, the pridopidine is administered orally.

In further embodiments, the pridopidine is administered in the form of an inhalable powder, an injectable formulation, a liquid, a gel, a solid, eye drops, an eye ointment, a capsule, or a tablet.

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

Examples of pridopidine derivatives are pridopidine and salts enriched with deuterium. Examples of deuterium enriched pridopidine and salts and methods of making the same can be found in U.S. application publications nos. 2013-0197031, 2016-0166559, and 2016-0095847, the disclosures of each of which are incorporated herein by reference in their entirety.

By "deuterium enriched" is meant that the abundance of deuterium at any relevant site of a compound is greater than the abundance of deuterium in the amount of the compound naturally occurring at that site. The naturally occurring distribution of deuterium is about 0.0156%. Thus, in a "deuterium-enriched" compound, the abundance of deuterium at any of the deuterium-associated sites exceeds 0.0156%, and may range from more than 0.0156% to 100%. Deuterium enriched compounds may be obtained by exchanging hydrogen for deuterium or by synthesizing said compounds from deuterium enriched starting materials.

The invention also encompasses any salt of pridopidine, including any pharmaceutically acceptable salt, wherein pridopidine has a net charge (positive or negative) 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 salts" means those salts of the compounds of the present invention that are safe and effective for pharmaceutical use in mammals and that have the desired biological activity. Pharmaceutically acceptable salts comprise salts of acidic or basic groups present in the compounds of the invention. Pharmaceutically acceptable acid addition salts include, but are not limited to, the hydrochloride, hydrobromide, hydroiodide, nitrate, sulfate, bisulfate, phosphate, acid phosphate, isonicotinate, acetate, lactate, salicylate, citrate, tartrate, pantothenate, bitartrate, ascorbate, succinate, maleate, gentisate, fumarate, gluconate, glucuronate, gluconate, formate, benzoate, glutamate, methanesulfonate, ethanesulfonate, benzenesulfonate, p-toluenesulfonate, and pamoate salts (i.e., 1,1' -methylene-bis- (2-hydroxy-3-naphthoate)). Certain compounds of the invention may form pharmaceutically acceptable salts with various amino acids. Suitable base salts include, but are not limited to, aluminum, calcium, lithium, magnesium, potassium, sodium, zinc, and diethanolamine salts. For a review of pharmaceutically acceptable salts see BERGE et al, 66 journal of pharmaceutical science (j.p. arm.sci.) -1-19 (1977), which is incorporated herein by reference. In another embodiment, the pridopidine salt of the invention is the hydrochloride salt.

In some embodiments, the methods of the invention utilize a combination of pridopidine, or a pharmaceutically acceptable salt thereof, and one or more analogs, or pharmaceutically acceptable salts, thereof, or a pharmaceutically acceptable salt thereof.

In one embodiment, the analogue of pridopidine is represented by the following structure:

in other embodiments, the methods of the invention utilize a combination of pridopidine, or a pharmaceutically acceptable salt thereof, and an analog of compound (1), or a pharmaceutically acceptable salt thereof.

In other embodiments, the methods of the invention utilize 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.

Thus, the present invention also relates to pharmaceutical compositions comprising the agents of the present invention in admixture with pharmaceutically acceptable auxiliary agents and optionally other therapeutic agents. An adjuvant must be "acceptable" in the sense of being compatible with the other ingredients of the composition and not deleterious to the recipient thereof.

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 may be prepared by any method known in the art of pharmacy.

Such methods comprise the step of introducing the associative compound or combination thereof used in the present invention with any adjuvant. Adjuvants, also known as co-ingredients, include those conventional in the art, such as carriers, fillers, binders, diluents, disintegrating agents, lubricants, coloring agents, flavoring agents, antioxidants, and wetting agents.

Pharmaceutical compositions suitable for oral administration may 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 may also be presented in the form of a bolus or paste. The composition may be further processed into suppositories or enemas for rectal administration.

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

For parenteral administration, suitable compositions include aqueous and non-aqueous sterile injections. The compositions may be presented in unit-dose or multi-dose containers, for example sealed vials and ampoules, and may be stored in a freeze-dried (lyophilized) condition requiring only the addition of the sterile liquid carrier, for example water, prior to use. For transdermal administration, for example, gels, patches or sprays can be considered. Compositions or formulations suitable for pulmonary administration comprise a fine dust or mist, for example by nasal inhalation, which may be produced by a metered dose pressurised aerosol, nebuliser or insufflator.

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.

As used herein, the term "treatment" refers to the administration of a therapeutic amount of a composition of the present invention effective to ameliorate an undesirable disease, condition, including symptoms associated with a disease or condition, to prevent the manifestation of such disease, condition, including symptoms associated with a disease or condition prior to its occurrence, to slow the progression of a disease, slow the worsening of symptoms, promote the onset of remission, slow the irreversible damage inflicted during the progressive chronic phase of a disease, delay the onset of the progressive phase, lessen the severity or cure of a disease, improve survival or faster recovery, or prevent the occurrence of a form of a disease or a combination of two or more of the foregoing. An "effective amount" for the purposes disclosed herein is determined by such considerations as are known in the art. The amount must be effective to achieve the desired therapeutic effect as described above, depending on, inter alia, 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 400mg, daily, twice daily, three times daily, or less than once daily. It is generally well known that an effective amount depends on a variety of factors, including the affinity of the ligand for the receptor, its profile in vivo, a variety of pharmacological parameters such as half-life in vivo, undesirable side effects, if any, with respect to age and sex.

Experimental part

Materials and methods

The hemizygous YAC128(Y128) [ HD 53; mHTT high expressor ] and WT mice with FVB/N background were housed under controlled temperature (22-23 ℃) conditions and a 12 hour light/12 hour dark cycle. Food and water were available ad libitum. All mouse experiments were performed according to institutional animal care and use committee guidelines and european community directive (2010/63/EU) and protocols approved by the local animal care committee. All efforts were made to minimize animal suffering and reduce the number of animals used.

Primary neuronal cultures: primary cortical, striatal and cortical-striatal co-cultures were generated from cross-offspring between Wild Type (WT) mice (used as controls) or hemizygous Y128 mouse [ line HD53] males and WT females from the same genetic background (FVB/N). Embryos from the chronologically pregnant female were collected on days E15.5-16.5 of gestation. For cortical-striatal co-cultures, the cortex and striatum were microdissected, then sectioned and each genotype pooled. The tissue is then dissociated and ground. Cells were seeded on poly-D-lysine coated plates in enriched neurobasal medium. Cells were fed every five days with 1/3 fresh medium.

To obtain pure cortical and striatal neurons, the tissues were microdissected and mechanically digested. Neurons were cultured in enriched Neurobasal medium and at 130 × 10³Individual cell/cm²(high Density) or 85X 10³(Low Density) Density was plated on poly-D-lysine (0.1mg/ml) coated plates. Cultures were maintained at 95/5% air/CO₂The incubator was maintained at 37 ℃. On day 3 in vitro (DIV3), 5. mu.M 5-fluoro-2' -deoxyuridine (5-FdU) was added to reduce dividing non-neuronal cells. Fresh media was added at DIV7 and cells were used at DIV 12.

Neuron transfection: striatal neurons were transfected with pDsRed2-Mito vector at 8DIV using the calcium phosphate precipitation method. The transfected neurons were then washed with neurobasal medium and transferred back to their original dishes containing conditioned medium until DIV 12.

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

Human neural stem cell culture: neural Stem Cells (NSCs) were derived from heterozygous human Induced Pluripotent Stem Cells (iPSCs) HD 4-iPSCs (with normal (19 CAG repeats) and amplified alleles (7)2 CAG repeats)) and control AMS 4-iPSC. IPSC is maintained at(Sermer Feishell science, Inc. (Thermo Fisher Sci.), Cat. No.: A1413202) until it reached 90% confluence, at which time the neural induction protocol was applied. Neural differentiation is based on dual SMAD inhibition of SB431542 (left-handed/activin/transforming growth factor beta-TGF β inhibitor), doxorphin (bone morphogenetic protein-BMP inhibitor) and XAV-939 (beta-catenin transcription inhibitor and axin stabilizer). Neural induction occurs between day 0 and day 12 to day 15. From day 0 to day 5, cells were maintained in the absence of FGF₂And incubated with 5 μ M doxofine and 10 μ M SB 431542. 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 day 12 and day 15, the region filled with rosettes became morphologically clearly visible. For differentiation, cells were reseededCoated 12-well plates. Expression of the neural lineage markers nestin and SOX2 was confirmed by immunocytochemistry at each differentiation process.

Pridopis for temperature raising: unless otherwise indicated, pridopidine incubations were performed for 24 hours in all cell models used. The final concentrations are depicted in the figures and legend.

Co-localization of mitochondrial network and ER: cortical-striatal cocultures were labeled with Mitotracker dark Red fm (Mitotracker Deep Red fm) dye for 30 minutes. Stained cells were washed at room temperature before fixation with ice-cold methanol for 15 minutes.

MitoDsRed transfected striatal neurons were fixed with 4% paraformaldehyde, permeabilized and blocked, then reacted with IP₃The R3 antibody (1:1000, Merck Millipore (EMD Millipore), Cat. No. AB9076) was incubated together. For nuclear detection, neurons were incubated with Hoechst 33342And then installed.

Confocal images were obtained in the form of stacks separated by 0.46 μm along the z-axis using a 63-fold lens of a zeiss confocal microscope with LSM 710 software. FIJI software was used for image analysis. Z-stacked images were normalized to background, the peak intensity regions representing mitochondrial specific fluorescence were identified using Find Foci ("FindFoci: a Focus Detection Algorithm with Automated Parameter Training That Closely Matches manual Assignments, Reduces manual Inconsistencies, and Increases Analysis Speed (FindFoci: A Focus Detection with Automated Parameter Training That Human interference Analysis, reduce Human Inconsite and incorporated specifications of Analysis"), and were best resolved by Michael Lichten, American public library of sciences Integrated (PLoS ONE) 9(12), and by filter application. The mitochondrial contour was followed using analytical particles. The aspect ratio (the ratio between the major axis and the minor axis of mitochondria) is used as an indicator of the length of mitochondria. For IP₃R3 fluorescence, setting of threshold values similar to those described above, and calculating integrated density within the mitochondrial region of interest (ROI) to obtain co-localization with ER of mitochondria.

Mitochondrial motility analysis: MitoDsRed transfected striatal neurons were washed and plated in Na⁺The medium was incubated and mitochondrial motility studies were performed at 37 ℃. Neuronal projections were imaged every 5 seconds using a rotating disk zeiss inverted confocal 63-fold objective for a total of 145 frames. Mitochondrial motility analysis was performed using Kymograph Macro from feij. The ROI follows the mitochondrial trajectory across the projections using segmentation line designation. A waveform recorder generated in the x-y dimension (distance versus time) is used to obtain the slope to calculate mitochondrial velocity.

Hippocampal oxygen respirometry: oxygen Consumption Rates (OCR) in WT and hemizygous Y128 cortical/striatal cocultures and NSCs were measured using a hippocampal XFe-24/96 flux analyzer (hippocampal Bioscience). Cortical-striatal primary neurons were cultured at a density of 20,000 cells/well in hippocampal XF 96V 3 cell culture microplates. NSCs were seeded at 30,000 cells/wellTo be coated withXF24 cell culture microplates and allowed to adhere at 37 ℃ for 24 hours. Pridopidine (0.1, 1 and/or 5 μ M) was added 24 hours prior to the experiment, as indicated in the graph. non-CO at 37 ℃ with sensor cartridge plate and immersion sensor₂Incubate in the incubator for about 16 hours. Three baseline measurements for OCR were sampled before the sequential injections of mitochondrial complex V inhibitor oligomycin (1mM), proton carrier FCCP (carbacyn-4- (trifluoromethoxy) phenylhydrazone, oxidative phosphorylation uncoupler) (neuron 0.5mM, NSC 0.3mM) and antimycin a (neuron 0.5mM, NSC 1 μ M) plus rotenone (neuron 0.5 μ M and NSC 1 μ M) to completely inhibit mitochondrial respiration. Therefore, mitochondrial basal respiration, maximal respiration and ATP production were automatically calculated and recorded by hippocampal software. Data were normalized to protein levels.

Mitochondrial membrane potential: positively charged fluorescent probe tetramethyl rhodamine methyl ester (TMRM) is used under quenching condition⁺) Assessment of mitochondrial membrane potential (MMP,. DELTA.. psi.) in cortical and striatal neurons_m) And its accumulation in mitochondria was assessed following mitochondrial depolarization with oligomycin plus mitochondrial respiratory uncoupler FCCP. In cortical/striatal co-cultures and lymphoblasts, MMPs use an equivalent probe (tetramethylrhodamine ethyl ester (TMRE)⁺) Fluorescent probes) whose accumulation in mitochondria was assessed directly by flow cytometry. When indicated, cortical and striatal neurons previously treated with pridopidine (0.1 and 1. mu.M; 24 hours) were incubated with 150nM TMRM (quenching conditions) in Na⁺The medium was incubated at 37 ℃ for 30 minutes. Under these conditions, mitochondrial retention of TMRM was investigated to estimate the change in MMP/Δ ψ. Basal fluorescence (503nm excitation and 525nm emission) was recorded for 4 minutes, then 2.5 μ M FCCP and 2.5 μ g/mL oligomycin were added to achieve maximum mitochondrial depolarization and mitochondrial probe release. TMRM release was calculated based on the difference in fluorescence before and after addition of oligomycin/FCCP.

Primary neuron or gonorrheaThe blast cells were cultured in 6-well plates. Pridopidine and hydrogen peroxide (H) with/without experimental conditions₂O₂) The cells were pretreated and then incubated with 25nM TMRE methyl ester at 37 ℃ for 15 minutes. After TMRE incubation, cells were collected for FACS analysis.

Mitochondria H₂O₂Measurement of level: cortical and striatal neurons were pretreated with pridopidine (0.1 and 1 μ M) for 24 hours and probed with mitochondrial peroxyyellow 1(MitoPY1) probe (8 μ M) in Na⁺The medium was incubated at 37 ℃ for 30 minutes. MitoPY1 was washed away and neurons in the same experimental medium were imaged every 1 minute for 30 minutes using a 63-fold lens of a zeiss inverted confocal spinning disk microscope with Zen Black 2012 software. Fluorescence was recorded by 503nm excitation and 528nm enhanced emission (Dickinson, Bryan C, Vivian S Lin and Christopher J Chang.2013. "Preparation of MitoPY1 and Use for Imaging of Hydrogen Peroxide in living cell Mitochondria (Preparation and Use of MitoPY1 for Imaging Hydrogen Peroxide in Mitochlor of Live Cells.)" Nature Protocols (Nature Protocols) 8 (6)). 10 minutes after basal reading, neurons were stimulated with antimycin A (2. mu.M). Specific MitoPY1 fluorescence in mitochondria was confirmed by co-incubation of cells with MitoTracker dark red (300 nM). The fluorescence intensity at each time point was analyzed using a time series analyzer plug-in (v 3.0) at fijic.

NSC are plated at 30,000/wellIn a 96-well assay plate at 37 ℃ for 24 hours. Thereafter, NSCs were incubated with 1. mu.M pridopidine for an additional 24 hours. Prior to harvest, cells were washed and incubated at 37 ℃ and 5% CO₂Next, the mixture was incubated with 10. mu.M MitoPY1 for 20 minutes. The basal level of MitoPY1 fluorescence was measured for 10-15 minutes, then exposed to myxothiazole (3 μ M mitochondrial complex III inhibitor) and measured for another 30 minutes. The results were calculated as Relative Fluorescence Units (RFU) per 30,000 cells. Among the isolated mitochondria, by resuspending 5. mu.g of the isolated mitochondria in Amplex Red reagent containing horseradish peroxidase (0.5 units/mL),and measuring the fluorescence at 570nm excitation and 585nm emission to measure H₂O₂And (4) horizontal. After a basal reading of 10 minutes, mitochondria were challenged with antimycin a (2 μ M) and measured for an additional 10 minutes. The results were analyzed as time-dependent changes in fluorescence.

Reactive Oxygen Species (ROS) assay: primary neurons and lymphoblasts attached to PDL-coated plates with H₂O₂(0-1mM) for up to 6 hours. Cells were treated with 5 μ M CellRox red reagent in complete medium and then incubated for 30 minutes. After washing, oxidative stress was measured by imaging all samples using the same exposure setting on a zeiss inverted microscope using a 40-fold objective. Eight random fields were sampled and fluorescence intensity was measured using ImageJ software.

In vivo study design: 1.5 month old WT and hemizygous Y128 mice (equal proportion of males and females) were divided into four groups. Mice received pridopidine (30mg/kg, 100 μ L/25g) or an equal volume of sterile water by oral gavage over 45 consecutive days until 4 months of age. Mice were housed in cages rich in maize hull nesting material and paper rolls, 4 animals per cage, each cage representing a separate experiment, for a total of 9 animals per group. Animals were weighed weekly and treatment volumes adjusted accordingly. Mice were behaviorally tested in rotarod immediately prior to treatment and the day before treatment was complete. The test was performed blindly at a fixed time of day. 24 hours after the last gavage, mice were sacrificed and mitochondria were dissected from the striatum.

Rotating rod analysis: exercise learning and coordination, and evaluation on a rotating device. In this test, the mouse must learn to run while being placed on a constant rotating wand to prevent it from falling. Once the task is learned, the acceleration rotor can be used to assess the motor coordination and balance. Mice were allowed to acclimate to the behavior chamber for 2 hours. The procedure was consistent for all subjects and the tests were performed at the lowest noise level. Training consisted of four traces per day (120 seconds per trace) with 1 hour intervals and a fixed speed of 14 rpm. The test phase was carried out on the following day in accelerated speed bars at 4 to 40rpm in 5 minutes, consisting of 3 trials, spaced 2 hours apart. Rotarod scores are the average of 3 trials. Blind experiments were performed for genotype and treatment. Exercise coordination scores were measured after training and fall delay times were quantified over 5 minutes with an accelerated rotarod at 5 to 40 rpm.

Separation of functional mitochondria: striatum was dissected from mouse brains washed in mitochondrial isolation buffer. Striatal mitochondria were isolated after homogenization using a discontinuous percoll density gradient centrifugation. The protein content of the isolated mitochondria was quantified by the Bio-Rad assay.

Mitochondrial complex activity: complex activity was assessed by measuring Oxygen Consumption Rate (OCR) using the hippocampal XF method (Agilent). Mu.g of isolated mitochondria diluted in mitochondrial assay solution were seeded in 450. mu.L of poly (ethylenimine) -coated XF24 hippocampal plates and the plates were allowed to stand at 37 ℃ in the absence of CO₂The humidified incubator was equilibrated for 10-12 minutes. Sequential electron flow through the electron transport chains was assessed by OCR measurements after sequential injection of rotenone (2. mu.M; complex I inhibitor), succinate (10 mM; complex II substrate), antimycin A (4. mu.M; complex III inhibitor) and ascorbate/TMPD (10 mM/100. mu.M; cytochrome C/electron donor of complex IV).

Mitochondrial calcium regulation: using calcium (Ca)²⁺) Sensitive probe Calcium Green-5N for measuring and separating mitochondria pair Ca²⁺The intake of (1). Briefly, 5 μ g of mitochondria were incubated with 1 μ M oligomycin and 150nM Calcium Green-5N and fluorescence measured in a fluorescence spectrophotometer microplate reader (excitation 506nM, emission 523 nM). After a baseline of 2 minutes, pulses of 10 μ M CaCl2 were added to the mitochondria at 4 minute intervals. Mitochondrial Ca²⁺Regulation was calculated by the area under the curve after CaCl2 pulse, indicating mitochondrial uptake of extra-mitochondrial Ca²⁺The amount of (c).

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

Cell lysis and immunoblotting: cells were lysed and phosphatase inhibitor cocktail 2 and 3 and 10mM β -glycerophosphate were added to the lysis buffer to inhibit phosphatase to detect phosphorylated proteins. After SDS-PAGE and transfer to nitrocellulose membranes, the membranes were blocked and immunoblotted with primary antibody overnight at 4 ℃ followed by washing with secondary antibody and blotting. After washing, enhanced chemiluminescence assays were performed, and the membranes were exposed and quantified.

Statistical analysis: results are expressed as mean ± SEM (standard error of the mean) of the number of independent experiments or animals indicated in the legend. Comparisons between groups were made by non-parametric one-way analysis of variance (ANOVA) using the kruskal-vories test. Correction for multiple comparisons was done by two-way ANOVA and Tukey post hoc testing. The comparison between the two groups was performed by either the nonparametric Mann-Whitney test or the parametric student's t-test. An F-test is performed to analyze the interactive item. Significance was accepted at p < 0.05. All analyses were performed using Prism software (graphpad version 8.0). Mitochondrial parameters were assessed 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 ex vivo models.

The results are

Insight into mitochondrial function can be gained by studying morphology and transport. Since fission and fusion events are required for mitochondrial quality control, both mitochondrial aspect ratio (which is equivalent to the ratio between the major and minor axes of the ellipse of the mitochondria) and mitochondrial transport, which are necessary to nourish synaptic terminals for high energy demand, are measures of mitochondrial function. Cortical striatum primary neurons collected from HD model YAC128(Y128) and Wild Type (WT) mice were assessed for staining with MitoTracker (fig. 1A) and mitochondrial number (fig. 1B) and morphology (fig. 1C and 1D). HD neurons show impaired mitochondrial morphology: a significant increase in the number of round mitochondria (fig. 1C) and a decrease in the elongated mitochondria (p <0.05) was observed compared to age-matched wild-type neurons (fig. 1D). Under these conditions, although there was no change in mitochondrial mass (fig. 1B), mitochondrial fragmentation (fission) was favored, indicating a decrease in mitochondrial function. Pridopidine (1 μ M) treatment rescued the number of both round and elongated mitochondria (p < 0.05).

In transfection with mitoDsRed (mitochondrial side) and anti-IP₃Poor co-localization of mitochondria and ER in Y128 HD neurons for visualization of both cell populations compared to WT was observed in R (ER side) antibody stained striatal neurons (fig. 1E). Pridopidine (1. mu.M) highly increased mitochondrion-ER co-localization in Y128 striatal neurons (FIGS. 1E and 1F, p)<0.001). This result may explain ATP production and increased mitochondrial transport and velocity (observed in fig. 2A-2B). MitoDsRed-labeled mitochondria from Y128 striatal neurons also showed reduced aspect ratios (p)<0.05) (fig. 1G), thereby confirming the previous results (fig. 1A-D). Treatment with pridopidine (1. mu.M) reduced the number of fragmented mitochondria (p)<0.05) (FIGS. 1E, 1G).

Mitochondrial antegrade transport in HD neurons is also greatly reduced, with approximately 90% of mitochondria in Y128 striatal neurons appearing quiescent (p < 0.05). Pridopidine reduces the percentage of quiescent mitochondria, increasing both antegrade and retrograde transport (p < 0.05). (FIGS. 2A and 2B-quantitative analysis). Mitochondrial trafficking velocity of Y128 neurons was also reduced compared to wild-type neurons, moving at half velocity (p < 0.05). This reduction was improved (p <0.05) after pridopidine treatment (1 μ M) (fig. 2A, 2C).

Y128 neurons showed reduced basal and maximal respiration and impaired ATP production (fig. 3A-3H). The reduction in respiration may be the result of the impairment of mitochondrial dynamics and morphology demonstrated in HD neurons (fig. 1A-1G and 2A-2B). Treatment with pridopidine at 1 μ M and 5 μ M doses rescued basal and maximal respiration (p <0.01), and ATP production (p <0.05) in HD Y128 cortical striatal neurons (fig. 3B (basal), 3C (maximal) 3D (ATP production)). Pridopidine 1 μ M also increased basal (p <0.001) and maximal (p <0.05) mitochondrial respiration, as well as ATP production from human iPSC-derived Neural Stem Cells (NSCs) from heterozygous HD patients (HD-ipscs) (fig. 3E, 3F (basal), 3G (maximal), 3H (ATP production)).

Mitochondrial membrane potential (MMP,. DELTA.. psi.)_m) Directly affects ATP production and is affected by mitochondrial Ca²⁺Signal transduction effects. Y128 cortical and striatal neurons exhibit lower Δ Ψ than wild-type cortical neurons_mThis is due to oligomycin and FCCP induced mitochondrial dysfunction (p)<0.05), indicating a lower mitochondrial retention of TMRM (fig. 4A, 4B). Pridopidine (0.1 and 1 μ M) increased Δ Ψ in Y128 cortical and striatal neurons_m(cortical neuron p)<0.05; 0.1 μ M pridopidine p in striatal neurons<0.05, 1. mu.M Pridopidine p<0.01) (fig. 4A-4C).

Hydrogen peroxide (H)₂O₂) Is used as a more effective oxidative stimulus to assess Δ Ψ_m. In response to 0.1mM H₂O₂Treatment, Δ Ψ observed in Y128 neurons_mIs obviously reduced (p)<0.0001). Treated with pridopidine (5. mu.M) and then exposed to 0.1mM H₂O₂Y128 cortical/striatal neurons lasting 6 hours were shown to be composed of H₂O₂Induced Δ Ψ_mComplete recovery of losses (p)<0.001) (FIG. 4D), and restored cell viability (p)<0.01) (fig. 4E). In lymphoblasts from HD patients, 0.1mM H₂O₂Treatment results in Δ Ψ_mThe reduction is 50%. At 0.1mM H₂O₂After treatment, pridopidine doses of 1. mu.M pridopidine, 5. mu.M pridopidine and 10. mu.M pridopidine all increased Δ Ψ_mWherein the 5 μ M dose shows the greatest protective effect (p)<0.01) (fig. 4F).

Mitochondrial pair H from Y128 neurons and HD lymphoblasts₂O₂Higher sensitivity indicates that these cells exhibit increased oxidative stress. To test this, local H was measured with the fluorescent probe MitoPY1₂O₂Flux. Complex inducing production of Reactive Oxygen Species (ROS)Compound III inhibitor antimycin A (AntA) stimulates cortex (p)<0.01) and striatal (p ═ 0.0001) Y128 neurons to show mitochondrial driven H compared to WT neurons₂O₂The level was significantly increased by 2-fold. In both cortical and striatal neurons, 1 μ M pridopidine reversed AntA-induced mito-H₂O₂Increased levels (in cortical neurons, p)<0.0001, in striatal neurons, for 0.1. mu.M, p<0.01, for 1. mu.M, p<0.0001) (fig. 5A-5C).

In HD-NSC treated with another mitochondrial Complex III inhibitor, mucothiazole (Myxo, 3. mu.M), cells showed mito-H₂O₂The level is greatly increased (p)<0.0001). Pridopidine (1. mu.M) rescues H caused by Complex III inhibition₂O₂Abnormal increase in level (p)<0.01) (fig. 5D). Under basal conditions, HD lymphoblasts show increased ROS production when compared to control lymphoblasts; when using H₂O₂Upon stimulation, pridopidine treatment reduced the basal condition and H₂O₂ROS levels (p) under both challenge conditions<0.001) (fig. 5E). Thus, pridopidine reduced ROS levels in three HD cell models.

The neuroprotective effects of pridopidine reappear in vivo. 1.5 months old WT and Y128 mice (with equal proportion of male and female prior to symptoms) were treated with pridopidine 30mg/kg or water for 45 consecutive days. Y128 mice exhibited motor deficits at 3 months of age in the rotarod performance test, so this test was applied before and after treatment to test the efficacy of pridopidine at the motor level. At 1.5 months (pretreatment), Y128 mice exhibited the same motor coordination as wild-type mice (fig. 6A, 6B). At 3 months of age, vehicle-treated Y128 mice exhibited motor deficits as observed by reduced drop delay time during accelerated rotarod testing compared to vehicle-treated wild-type mice (p)<0.05) (fig. 6C). In contrast, HD mice treated with pridopidine showed significant motor performance improvement in accelerated rolling bars (p) compared to vehicle-treated HD mice<0.05) (fig. 6C). After behavioral analysis, from all smallsFunctional mitochondria were isolated from the striatum of the murine group and their activity was assessed by assessing the sequential electron flow through the electron transport chains by measuring OCR after sequential injection of rotenone, succinate, antimycin a and ascorbate/TMPD (N, N' -tetramethylp-phenylenediamine) which induces individual stimulation or inhibition of mitochondrial complexes I, II, III and IV, respectively (fig. 6D). Striatal mitochondria from vehicle-treated Y128 mice showed higher activity of complexes I, II, III and IV (p) compared to vehicle-treated wild-type mice<0.01), indicating the presence of an early compensation mechanism (fig. 6D, 6F). Interestingly, the increase in complex activity in the striatum of Y128 HD mice was accompanied by mitochondrial H before and after repression of complex III with AntA₂O₂Increase in production (p)<0.05) (fig. 6D, 6G). These results suggest that abnormal complex activity may underlie mitochondrial ROS production by increasing electron leakage, leading to impaired ATP production. Pridopidine treatment in Y128 mice normalized mitochondrial Complex Activity, H₂O₂Levels are normalized to the level of WT vehicle-treated mice (p)<0.05) (FIGS. 6I-6K) and increased mitochondrial Ca²⁺Buffering capacity (fig. 6L, 6M).

Mitochondria and the Endoplasmic Reticulum (ER) are both functionally and physically associated. Physical contact between mitochondria and the ER occurs at the site of the mitochondria-associated membrane (MAM), a highly specialized structure rich in S1R. MAM acts as a protein, lipid, signaling molecule and, importantly, Ca²⁺A pipeline of exchanges. Thus, ER Stress is closely related to mitochondrial dysfunction (Morris, Gerwyn, basal K.puri, Ken Walder, Michael Berk, Bredon Stubbs, Michael Maes and andre F.Carvalho. "Endoplasmic Reticulum Stress Response in neurodegenerative Diseases: Emerging Pathophysiological effects and transforming Implications (The Endoplasmic Reticulum Stress Response in neurological Diseases: emergent neurological disease and transforming Implications)", "Molecular Neurobiology (Molecular Neurobiology) (2018)55: 8765-8787).

Pridopidine reduces mHtt-induced ER stress: visible aggregates of Htt96Q-mCherry (usually one large aggregate per cell) along with high levels of accumulated H2a-GFP indicative of ER stress were observed in STHdhQ7/7 cells transfected with the mutant Htt96Q-mCherry (expanded) construct. STHdhQ7/7 cells expressing Htt20Q-mCherry (normal) or Htt96Q-mCherry without visible aggregates showed low levels of H2a-GFP (no ER stress). Pridopidine significantly reduced H2a-GFP accumulation in mHtt aggregate-positive cells in a dose-dependent manner (fig. 7A) and did not alter H2a-GFP levels in cells without aggregates or cells expressing Htt20Q-mCherry (fig. 7B). Thus, pridopidine reduces Htt-induced ER stress in a dose-dependent manner.

Phosphorylation of eIF2 α is a hallmark of ER stress. The level of eIF2 α -phosphorylation (eIF2 α -p) in STHdhQ7/7 cells expressing Htt96Q-mCherry was 3.5 times the level of eIF2 α -p in cells expressing Htt 20Q-mCherry. Pridopidine treatment resulted in a significant reduction in eIF2 α -P (measured by the ratio of eIF2 α -P to total eIF2 α), indicating a reduction in cellular ER stress (figure 8).

In summary, preclinical results indicate that mitochondrial dysfunction is a hallmark of HD in both in vitro and in vivo/ex vivo HD models, excluding effective antioxidant responses to oxidative stimuli. This dysfunction may affect synaptic integrity and cell survival. Pridopidine demonstrates rescue of various aspects of mitochondrial function in the HD model, including decreased ROS levels and increased mitochondrial velocity and percentage of elongated mitochondria in both the HD human and mouse models, all of which indicate mitochondrial dysfunction. Thus, pridopidine is effective in repairing mitochondrial dysfunction. Administration of pridopidine also delayed the appearance of first motor symptoms in Y128 mice, increased cell viability and rescued impaired oxidative phosphorylation. Additionally, in the in vitro HD model system, pridopidine relieved ER stress closely associated with mitochondrial dysfunction because S1R localized to MAM (mitochondrial associated membrane) sites of the ER membrane. These highly specialized sites have a key role in mitochondrial function, including mitochondrial fission, Ca²⁺Shuttle and oxidative stress.

47页详细技术资料下载

上一篇：一种医用注射器针头装配设备

下一篇：稳定的含水血液滤过置换基础液、透析溶液和相应的套件

Treatment of mitochondrial-related diseases and disorders, including symptoms thereof, with pridopidine

相关技术

网友询问留言