Low dose pridopidine for Parkinson's disease and other diseases associated with Parkinson's disease

Abstract

The present invention relates to low doses of pridopidine for use in Parkinson's disease and other diseases associated with Parkinson's disease. The present invention provides a method for treating Parkinson's disease or a symptom thereof with a low dose of pridopidine.

Description

[0001] This application is a divisional application of the application with the application number 202080012531.6 and the title "Low dose pridopidine for Parkinson's disease and other diseases related to Parkinson's disease" filed on 04 / 02 / 2020. TECHNICAL FIELD

[0002] The present invention provides a method for treating a subject suffering from Parkinson's Disease or Parkinsonism related to other diseases, the method comprising administering to the subject a low dose of pridopidine or a pharmaceutically acceptable salt thereof. BACKGROUND

[0003] Pridopidine (4-[3-(methylsulfonyl)phenyl]-1-propyl-piperidine) (formerly known as ACR16) has been found to improve motor function in Huntington's Disease (HD) patients and animal models of HD. Although its motor effects were initially attributed to its low affinity antagonism of dopamine receptors, in vivo PET studies in rats showed that behaviorally relevant doses of pridopidine occupy sigma-1 receptors (S1R) rather than dopamine D2 / D3R. This is consistent with in vitro binding affinity assays showing much higher binding affinity at S1R (Ki of about 100 nM) compared to D2R (Ki of 10-30 μΜ) and D3R (Ki of about 1.6 uM). S1R is a ligand-operated chaperone protein that is enriched at the mitochondria-associated endoplasmic reticulum membrane (MAM), supporting several cellular defense and survival pathways. S1R modulates a variety of cellular mechanisms, including upregulation of anti-apoptotic genes, reduction of microglial activation, reduction of reactive oxygen species (ROS) and nitric oxide (NO) production, and increase of trophic factor secretion.

[0004] Parkinson's Disease (PD) is an age-related neurodegenerative disease in which bidirectional interactions between alpha-synuclein aggregation, oxidative stress, mitochondrial dysfunction, and neuroinflammation play central pathogenic roles. SUMMARY

[0005] In one aspect, the present application provides a method for preventing, treating, or slowing the progression of Parkinson's disease or another disease associated with Parkinson's disease or another disease associated with Parkinson's disease or its symptoms in a subject in need thereof, the method comprising administering to the subject at least one pharmaceutical composition per day, wherein the pharmaceutical composition comprises pridopidine or a pharmaceutically acceptable salt thereof, and wherein the total administered dose of the pridopidine or the pharmaceutically acceptable salt thereof is from 1 mg to 100 mg per day.

[0006] In certain embodiments, the method comprises preventing, treating, slowing, or reversing functional decline in the subject.

[0007] In certain embodiments, the functional decline in the subject is manifested by a symptom selected from the group consisting of tremor, bradykinesia, rigidity, postural instability, decline according to the Unified Parkinson's Disease Rating Scale part II (UPDRS part II) including activities of daily living, and decline according to the Modified Hoehn and Yahr Staging of PD.

[0008] In certain embodiments, the method comprises preventing, treating, slowing, or reversing cognitive decline in the subject.

[0009] In certain embodiments, the cognitive decline in the subject is manifested by a symptom selected from the group consisting of impaired intelligence, thought disorder, depression, reduced motivation, decreased initiative, speech disorder, increased rescue, swallowing disorder, writing disorder, and increased pain sensation.

[0010] In certain embodiments, the method comprises preventing, treating, slowing, or reversing neurodegeneration in the subject.

[0011] In certain embodiments, the neurodegeneration in the subject is manifested by reduced fibroblast GCase activity, increased ER stress, neuronal mitochondrial dysfunction, increased neuronal mitochondrial ROS production, reduced autophagic flux, reduced neuronal mitochondrial velocity, reduced mitochondrial autophagy, reduced plasma BDNF levels, reduced cerebrospinal fluid (CSF) BDNF levels, reduced neuronal and BDNF axonal transport, increased protein aggregates in the brain or cerebrospinal fluid (CSF), increased brain inflammation, increased microglial activation, increased astrocyte activation, reduced brain volume, increased plasma levels of neurofilament light chain (NFL), or increased cerebrospinal fluid (CSF) levels of neurofilament light chain (NFL).

[0012] In some embodiments, the method includes administering a pharmaceutical composition comprising pridopidine or a pharmaceutically acceptable salt thereof to the subject at least twice daily.

[0013] In some embodiments, the method includes administering a pharmaceutical composition comprising pridopidine or a pharmaceutically acceptable salt thereof to the subject twice daily.

[0014] In some embodiments, the method includes administering a pharmaceutical composition comprising pridopidine or a pharmaceutically acceptable salt thereof to the subject three times daily.

[0015] In some embodiments, the method includes administering a pharmaceutical composition comprising pridopidine or a pharmaceutically acceptable salt thereof to the subject four times daily.

[0016] In some embodiments, the total dose of pridopidine or a pharmaceutically acceptable salt thereof is 10 mg to 100 mg per day.

[0017] In some embodiments, the total dose of the pridopidine or a pharmaceutically acceptable salt thereof is 50 mg to 100 mg per day.

[0018] In some embodiments, the total dose of the pridopidine or a pharmaceutically acceptable salt thereof is 80 mg to 100 mg per day.

[0019] In some embodiments, the total dose of pridopidine or a pharmaceutically acceptable salt thereof is 80 mg per day.

[0020] In some embodiments, the total dose of pridopidine or a pharmaceutically acceptable salt thereof is 90 mg per day.

[0021] In some embodiments, the total dose of pridopidine or a pharmaceutically acceptable salt thereof is 100 mg per day.

[0022] In some embodiments, the method includes administering, twice daily, a pharmaceutical composition comprising pridopidine or a pharmaceutically acceptable salt thereof to the subject, wherein the total dose of pridopidine or a pharmaceutically acceptable salt thereof is 80 mg to 100 mg per day.

[0023] In some embodiments, the method includes administering, twice daily, a pharmaceutical composition comprising pridopidine or a pharmaceutically acceptable salt thereof to the subject, wherein the total dose of pridopidine or a pharmaceutically acceptable salt thereof is 90 mg per day.

[0024] In some embodiments, the method includes administering, twice daily, to the subject a pharmaceutical composition comprising pridopidine or a pharmaceutically acceptable salt thereof, wherein each of the two pharmaceutical compositions comprises a different dose of pridopidine or a pharmaceutically acceptable salt thereof, wherein the total dose of pridopidine or a pharmaceutically acceptable salt thereof administered is 90 mg per day.

[0025] In some embodiments, the method includes administering to the subject twice daily a pharmaceutical composition comprising 45 mg of pridopidine or a pharmaceutically acceptable salt thereof, wherein the total dose of pridopidine or a pharmaceutically acceptable salt thereof is 90 mg per day.

[0026] In some embodiments, the method includes systemic administration of the pharmaceutical composition comprising pridopidine or a pharmaceutically acceptable salt thereof to the subject.

[0027] In some embodiments, the method includes orally administering the pharmaceutical composition comprising pridopidine or a pharmaceutically acceptable salt thereof to the subject.

[0028] In some embodiments, the pharmaceutical composition comprising pridopidine or a pharmaceutically acceptable salt thereof is administered in a form selected from the group consisting of: inhalable powder, injectable, liquid, gel, solid, capsule, or tablet.

[0029] In some embodiments, the pharmaceutically acceptable pridopidine salt is pridopidine hydrochloride.

[0030] In some embodiments, the Parkinson's disease or its symptoms are part of or associated with a neurodegenerative condition selected from the group consisting of: Parkinson's disease (PD), multiple system atrophy (MSA), Lewy body dementia (DLB), amyotrophic lateral sclerosis (ALS), Parkinson's dementia (PDD), Huntington's disease (HD), Alzheimer's disease, progressive supranuclear palsy (PSP), corticobasal degeneration, frontotemporal dementia, Gerstmann–Sträussler–Scheinker syndrome, Lytico-bodig disease (Guam-type ALS complex), neuroacanthosis, neuronal ceroid lipofuscin deposition, olivopontocerebellar atrophy, pantothenic acid kinase-associated neurodegeneration, and Wilson's disease. Corticobasal degeneration (CBD) and Pick's disease.

[0031] In some embodiments, the Parkinson's disease or its symptoms are part of or related to Parkinson's disease (PD).

[0032] In some embodiments, the PD is associated with glucocerebrosidase mutation (PD-GBA).

[0033] In some embodiments, the Parkinson's disease or its symptoms are part of or related to multiple system atrophy (MSA).

[0034] In some embodiments, the Parkinson's disease or its symptoms are part of or related to Lewy body dementia (DLB).

[0035] In some embodiments, the Parkinson's disease or its symptoms are part of or related to amyotrophic lateral sclerosis (ALS).

[0036] In one embodiment, the present invention relates to a method for treating Parkinson's disease (PD) in a subject in need, the method comprising administering a low dose of pridopidine or a pharmaceutically acceptable salt thereof to the subject, thereby treating the subject.

[0037] In one embodiment, the present invention relates to a method for slowing functional decline in a subject with PD, the method comprising administering to the subject a low dose of pridopidine or a pharmaceutically acceptable salt thereof, thereby slowing functional decline in the subject with PD.

[0038] In one embodiment, the present invention relates to a method for inducing / promoting functional neurorepair in a subject with PD, the method comprising administering a low dose of pridopidine or a pharmaceutically acceptable salt thereof to the subject, thereby inducing / promoting functional neurorepair in the subject with PD.

[0039] In another embodiment, the Parkinson's disease is associated with Parkinson's disease associated with glucocerebrosidase (GBA) deficiency (PD-GBA), multiple system atrophy (MSA), or Lewy body dementia (LBD).

[0040] In another embodiment, the pridopidine is pridopidine hydrochloride.

[0041] In another embodiment, the pridopidine is administered orally.

[0042] In another embodiment, the pridopidine is administered in the form of an inhalable powder, injectable, liquid, gel, solid, capsule, or tablet.

[0043] In another embodiment, the pridopidine is administered periodically.

[0044] In another embodiment, the pridopidine is administered less than once a day.

[0045] In another embodiment, the pridopidine is administered once, twice, or three times daily.

[0046] In another embodiment, pridopidine is administered at a daily dose between 10 mg / day and 100 mg / day.

[0047] In another embodiment, pridopidine is administered at a daily dose between 20 mg / day and 90 mg / day.

[0048] In another embodiment, pridopidine is administered at a daily dose between 45 mg / day and 90 mg / day.

[0049] In another embodiment, pridopidine is administered at a daily dose between 20 mg / day and 50 mg / day. BRIEF DESCRIPTION OF DRAWINGS

[0050] The concluding section of the specification specifically points out and explicitly claims protection for the subject matter considered to be the present invention. However, when read in conjunction with the accompanying drawings, both the organization and operation of the invention, together with its objects, features, and advantages, can be well understood by referring to the following detailed description, in which: FIG. 1A-1C Behavioral improvements were demonstrated following treatment with low doses of pridopidine. Spontaneous spinning was assessed weekly in mice treated with 0.3 mg / kg or 1 mg / kg pridopidine (Pridop 0.3 mg / kg or Pridop 1 mg / kg, respectively) or saline solution (n = 8–12 per group). FIG. 1A ) and asymmetry in forelimb use (cylinder test, B and stepping test, C). Results are expressed as the number of spontaneous ipsilateral rotations during the 10-minute test phase ( FIG. 1A ), or can be represented as support wall contact performed using the claw (left claw) located on the opposite side of the lesion ( FIG. 1B ) and adjusting steps ( FIG. 1C The percentage of [unspecified percentage]. Lower doses of pridopidine (0.3 mg / kg) improved animal performance in all tests. Improvement in forelimb use ( FIG. 1B and FIG. 1C This gradually occurred and became apparent after 5 weeks of treatment. In all these tests using repeated measures ANOVA and the post-hoc Bonferroni test, significant overall differences were found in mice treated with a low dose of 0.3 mg / kg pridopidine, but no significant overall difference was found in mice treated with a higher dose of 1 mg / kg pridopidine: ( FIG. 1ASpontaneous rotation: ANOVAp < 0.0001. Time, treatment, interaction; time F(4,160) = 50.46, treatment F(3,40) = 16.97, interaction F(12,160) = 5.43; FIG. 1B Cylindrical test: ANOVA p > 0.05 for interaction; < 0.0001 for time and treatment; time F(4,160) = 8.74, treatment F(3,40) = 20.96, interaction F(12,160) = 1.52; FIG. 1C Step-by-step test: ANOVA p = 0.006 interaction; p = 0.0004 time, p < 0.0001 treatment, time F(4,160) = 5.46, treatment F(340) = 75.44, interaction F(12,160) = 2.44; p < 0.05, asterisk for "saline"; hash for "sham surgery"; and sum for "Pridop 1".

[0051] FIG. 2A-2K Long-term treatment with a low dose of pridopidine (0.3 mg / kg) (instead of a high dose of pridopidine 1 mg / kg) induced neural tissue repair. FIG. 2A Stereoscopic counting of TH-positive cells was performed in the substantia nigra pars compacta (SNc) (dashed lines delineate the areas included in the analysis). Data show the total number of cells from the SNc on the same side as the lesion (mean ± SEM of 7 slices per animal throughout the SNc). One-way ANOVA p < 0.0001; F(3,39) = 44.29; post-hoc Tukey test, p < 0.05, asterisks relative to "saline"; hash marks relative to sham surgery. FIG. 2B-2E Low-magnification photomicrographs of immunostained sections of the substantia nigra of TH, where the low-magnification photomicrographs represent different groups. Scale bar: 100 μm. FIG. 2F-2K Low-magnification micrograph of a striatal section; dashed lines indicate areas included in the TH fiber density analysis. FIG. 2F ′-2H′) Magnified illustrations from the same slice show the differences in fiber density and morphology between different groups ( FIG. 2F ′, cases treated with saline; FIG. 2G ′: Mice treated with 0.3 mg / kg pridopidine, FIG. 2H ′: Mice treated with 1 mg / kg pridopidine. Scale bar: 200 μm. FIG. 2IImage segmentation analysis of TH-positive fibers performed on random sample regions at high magnification. Values ​​are given as fiber pixels / sample region, with whiskers covering the entire range of values ​​in each group (median values ​​are shown as horizontal lines). Kruskal–Wallis and post-hoc Dunn's test, p = 0.0091; asterisk, p < 0.05 relative to saline. FIG. 2J-2K ) to the ventrolateral striatum ( FIG. 2J ) or dorsolateral striatum ( FIG. 2K Density analysis of TH immunostaining across the entire cross-sectional area; density values ​​from the ipsilateral side of the lesion are expressed as a percentage of values ​​from the contralateral intact side in each animal (mean of 4 sections per animal across the entire striatum ± SEM). FIG. 2J (K) VL striatum, ANOVA p = 0.0002, F(2,33) = 11.35; (K) DL striatum, ANOVA p = 0.039, F(2,33) = 3.56. Post-hoc Tucker test, p < 0.05, asterisk relative to "saline"; sum relative to Pridop 1.

[0052] Figure 3: Low-dose (0.3 mg / kg) rather than high-dose (1 mg / kg) treatment with pridopidine reduced microglial activation in the nigrostriatal pathway. SNc ( FIG. 3A ) and ventrolateral striatum ( FIG. 3F The counts of CD68-positive microglia and corresponding photomicrographs (3B-J′) in mice were observed. Compared with the saline-treated control group, mice receiving 0.3 mg / kg pridopidine showed significantly lower numbers of CD68-positive cells at both the substantia nigra and striatum levels. Low-magnification photomicrographs were taken under a ×4 objective lens. FIG. 3B-3J The illustrations (B′-J′) were taken with a ×20 objective lens. The scale bar is 100 μm. FIG. 3A One-way ANOVA: p = 0.0002, F(3,28) = 9.005; FIG. 3F One-way ANOVA yielded p = 0.0005 and F(3,28) = 8.049. Post-hoc Tucker tests showed p < 0.05, with asterisks representing "saline" and hash marks representing sham surgeries.

[0053] Figure 4: Low-dose (0.3 mg / kg) pridocipidine did not improve motor deficits in S1R-deficient 6-OHDA lesion mice. Spontaneous rotation was assessed weekly in S1R KO mice with 6-OHDA lesions or sham-operated lesions. FIG. 4A ) and forelimbs use asymmetry (cylinder test, FIG. 4Band step test, FIG. 4C ), followed by treatment with 0.3 mg / kg pridopidine (Pridop 0.3) or saline solution (n = 8-10 per group). Results are expressed as the number of spontaneous ipsilateral rotations (A) during the 10-minute test phase or as the number of support wall contacts performed with the claw located on the contralateral side of the lesion (left claw). FIG. 4B ) and adjusting steps ( FIG. 4C The percentage of ). Repeated measures ANOVA and post-hoc Bonferroni test: ( FIG. 4A Spontaneous rotation: ANOVA p < 0.0001. Time, treatment, and interaction: Time F(4,104) = 45.36, Treatment F(2,26) = 15.54, Interaction F(8,104) = 11.15; FIG. 4B Cylindrical test: ANOVA p > 0.05 for time and interaction, p < 0.0001 for treatment, time F(4,104) = 1.23, treatment F(2,26) = 1.76, interaction F(8,104) = 0.32; FIG. 4C Step-by-step test: ANOVA p > 0.05 for time and interaction, p < 0.0001 for treatment, time F(4,104) = 0.46, treatment F(2,26) = 346.4, interaction F(8,104) = 1.35; p < 0.05, hash sign relative to "sham surgery".

[0054] Figure 5: In 6-OHDA lesion mice lacking S1R, low-dose pridopidine (0.3 mg / kg) had no neuroreparative effect. FIG. 5A The stereotactic count of TH-positive cells in the SNc of S1R KO mice and ( FIG. 5B-5D Low-magnification micrographs of immunostained sections of TH substantia nigra (dashed lines delineate the areas included in the analysis). Scale bar is 100 μm. Data in A show the total number of cells from the SNc on the same side as the lesion (mean ± SEM of 7 sections per animal throughout the SNc). One-way ANOVA p < 0.0001, F(2,26) = 41.73; post-hoc Bonferroni test; p < 0.05, hash relative to sham-operated group. (Fig. 5E) Image segmentation analysis of TH-positive fibers performed on random sample regions via the lateral striatum at high magnification. Values ​​are represented as fiber pixels / sample region, whiskers cover the entire range of values ​​in each group, and the median is shown as a bar. Mann–Whitney test, p = 0.084. (Fig. 5F- FIG. 5G ) ventrolateral striatum (Fig. 5F) or dorsolateral striatum ( FIG. 5GThe optical density (OD) of TH immunostaining across the entire cross-sectional area of ​​the striatum. The optical density values ​​from the ipsilateral side of the lesion are expressed as a percentage of the values ​​from the intact contralateral side in each animal (mean of 4 sections per animal across the entire striatum ± SEM). (Fig. 5F) Ventral lateral (VL) striatum, unpaired t-test, p = 0.516; FIG. 5G The dorsolateral (DL) striatum, p = 0.219. FIG. 5H-5I ′) from brine treatment ( FIG. 5H S1R KO mice treated with 5H′ and pridopidine-0.3 mg / kg ( FIG. 5I Representative micrographs of TH immunostained striatal sections (5I′). Scale bar: 200 μm.

[0055] Figure 6: Treatment with 0.3 mg / kg pridopidine upregulates nutritional factors and phosphorylated ERK1 / 2. Striatal samples were used from wild-type 6-OHDA-affected mice treated for 5 weeks with either 0.3 mg / kg pridopidine (n = 9) or saline solution (n = 11). FIG. 6A-6C ) and melanin samples ( FIG. 6D-6E Western blot analysis was performed on GDNF, BDNF, and phosphorylated ERK1 / 2 (pERK). Results were normalized for β-actin levels, and data from animals treated with pridopidine are shown as a percentage of values ​​from saline-treated controls. Unpaired t-test, striatum: ( FIG. 6A GDNF p = 0.003; FIG. 6B ) BDNF p = 0.013, ( FIG. 6C pERK1 / 2 p = 0.049; Substance: ( FIG. 6D GDNF p = 0.72; FIG. 6E ) BDNF p = 0.0008; FIG. 6F pERK1 / 2 p = 0.106.

[0056] FIG. 7Pridolpicin reduced the dose-dependent early mHtt-induced ER stress. H2a-GFP was transiently co-expressed with Htt20Q-mCherry or Htt96Q-mCherry (exon 1) in STHdhQ7 / 7 cells. H2a-GFP aggregation is a marker of cellular stress. Cells were treated with primidopyridine at constant or increased concentrations (from 0.03 μM to 3 μM, equivalent to from 0.01 μg / ml to 1 μg / ml) starting 4 hours post-transfection, and imaged under a confocal microscope 24 hours post-transfection. Images of single cells (approximately 150 cells per experiment) with Htt96Q-mCherry aggregates or Htt20Q-mCherry aggregates were quantified compared to untreated cells with or without aggregates. For comparative purposes, 100% represents the relative intensity of H2a-GFP in untreated cells showing Htt96Q-mCherry aggregates, and 0% represents the relative intensity of H2a-GFP in untreated cells without Htt96Q-cherry aggregates. The graph is the average of the three experiments + and -SE. An asterisk indicates a p-value compared to untreated cells, < 0.05 (*) and < 0.01 (**).

[0057] FIG. 8 Pridopidine reduced eIF2α-phosphorylation, as measured by ER stress using a bell-shaped dose-response curve. HEK293 cells were transfected with myc-Htt96Q (diamond) or myc-Htt20Q (square) and then treated with pridopidine at progressively increasing concentrations (from 0.03 μM to 150 μM, equivalent to increasing from 0.01 µg / ml to 50 µg / ml) for 24 hours. The ratio of eIF2α-P to total eIF2α was quantified by Western blotting.

[0058] FIG. 9 Prilidopidine increased mitochondrial membrane potential (ΔΨm) in cortical HD neurons after oxidative stress. Striatal WT and YAC128 neurons expressing mutant huntingtin were treated with prilidopidine for 24 hours, and changes in ΔΨm after depolarization with oligomycin plus FCCP were assessed using TMRM (n = 7–10). A two-way ANOVA analysis revealed the effect of prilidopidine treatment in striatal neurons [F(2,107) = 3.257; p = 0.0423].

[0059] FIG. 10Pridolpicine reduced reactive oxygen species (ROS) in YAC128 HD striatal neurons. Striatal neurons treated with or without 1 μM pridolpicine were incubated with the MitoPY1 fluorescent probe. Mitochondrial H2O2 was recorded in a confocal disc before and after antimycin A (Ant A, 2 μM), as indicated (n = 4, considering approximately 20 cells / condition). Scale bar = 30 μM. Two-dimensional ANOVA revealed the rescue effect of pridolpicine treatment on mitochondrial ROS production [F(1,389 = 15.24; p < 0.0001).]

[0060] FIG. 11 Pridolpicine increased mitochondrial velocity. Quantitative analysis of mitochondrial velocity in axons revealed accelerated axonal transport of mitochondria in WT neurons after application of 0.1 μM primidopidine (n = particle step number) (modified from Ionescu et al., 2019, Fig. 4F).

[0061] FIG. 12 Pridopidine enhanced BDNF secretion in the B104 neuroblastoma cell line. Data are expressed in pg / ml (mean 6 SEM; *P < 0.05; **P < 0.01; ***P < 0.001; one-way ANOVA followed by Dunnett's test). NE100, a σ-1 receptor antagonist (1 µM). Source: Gomes et al., 2016.

[0062] FIG. 13Pridopidine enhances BDNF axonal transport in ALS motor neurons through an S1R-mediated mechanism. To visualize and analyze axonal transport, spinal cord extracts were plated in the proximal compartment of a microfluidic chamber, allowing axons to grow through grooves into the distal compartment. Qdot-BDNF particles were introduced only into the distal compartment, and the particles were visualized via retrograde axonal transport using a confocal rotary microscope. Bar graphs of the instantaneous velocity values ​​of Qdot-BDNF particles in WT (column 1 from left) or SOD1G93A (columns 2-4 from left) MN show the slower velocities in SOD1G93A MN. Application of pridopidine accelerated the instantaneous velocities in both SOD1G93A MNs (0.1 μM and 1 μM). S1R− / − MN (columns 5-7 from left) reveal defects in BDNF axonal transport. Prilidopildidine at 0.1 μM or 1 μM failed to recover these defects (n = qDot - number of steps in BDNF). Data are presented as mean ± SEM. *p < 0.05; **p < 0.01; ***p < 0.001 (n = 6 independent experiments; sample size for each experiment is represented on the bar chart; Student t-test) (modified from Ionescu et al., 2019). FIG. 3C ). DETAILED DESCRIPTION

[0063] In one aspect, the present invention provides a method for preventing, treating, or slowing the progression of Parkinson's disease or another disease associated with Parkinson's disease or its symptoms in a subject in need, the method comprising administering to the subject daily at least one pharmaceutical composition, wherein the pharmaceutical composition comprises pridopidine or a pharmaceutically acceptable salt thereof, and wherein the total dose of said pridopidine or a pharmaceutically acceptable salt thereof is from 1 mg to 100 mg per day.

[0064] In some embodiments, the method is used to prevent or treat Parkinson's disease or slow its progression. In some embodiments, the method is used to prevent or treat diseases associated with Parkinson's disease or slow its progression. In some embodiments, the method is used to prevent or treat symptoms of Parkinson's disease or slow its progression. In some embodiments, the method is used to prevent or treat symptoms of diseases associated with Parkinson's disease or slow its progression. In some embodiments, the method is used to prevent or treat symptoms associated with Parkinson's disease or slow its progression.

[0065] In some embodiments, the method includes preventing, treating, slowing, or reversing the functional decline of the subject. In some embodiments, the method includes preventing the functional decline of the subject. In some embodiments, the method includes treating the functional decline of the subject. In some embodiments, the method includes slowing the functional decline of the subject. In some embodiments, the method includes reversing the functional decline of the subject.

[0066] In some embodiments, the functional decline of the subject is manifested by symptoms selected from the group consisting of: tremor, bradykinesia, rigidity, postural instability, decline according to the Unified Parkinson's Disease Rating Scale Part II (UPDRS Part II), which includes activities of daily living, and decline according to the modified PD-Hoya staging. In some embodiments, the functional decline of the subject is manifested as tremor. In some embodiments, the functional decline of the subject is manifested as bradykinesia. In some embodiments, the functional decline of the subject is manifested as rigidity. In some embodiments, the functional decline of the subject is manifested as postural instability. In some embodiments, the functional decline of the subject is manifested as decline according to the Unified Parkinson's Disease Rating Scale Part II (UPDRS Part II), which includes activities of daily living. In some embodiments, the functional decline of the subject is manifested as decline according to the modified PD-Hoya staging.

[0067] In some embodiments, the method includes preventing, treating, slowing down, or reversing the cognitive decline of the subject. In some embodiments, the method includes preventing the cognitive decline of the subject. In some embodiments, the method includes treating the cognitive decline of the subject. In some embodiments, the method includes slowing down the cognitive decline of the subject. In some embodiments, the method includes reversing the cognitive decline of the subject.

[0068] In some embodiments, the cognitive decline in the subject manifests as symptoms selected from the group consisting of: intellectual impairment, thinking disorder, depression, decreased motivation, decreased initiative, speech impairment, increased need for help, swallowing disorder, writing disorder, and increased pain sensation. In some embodiments, the cognitive decline in the subject manifests as intellectual impairment. In some embodiments, the cognitive decline in the subject manifests as thinking disorder. In some embodiments, the cognitive decline in the subject manifests as depression. In some embodiments, the cognitive decline in the subject manifests as decreased motivation. In some embodiments, the cognitive decline in the subject manifests as decreased initiative. In some embodiments, the cognitive decline in the subject manifests as speech impairment. In some embodiments, the cognitive decline in the subject manifests as increased need for help. In some embodiments, the cognitive decline in the subject manifests as swallowing disorder. In some embodiments, the cognitive decline in the subject manifests as writing disorder. In some embodiments, the cognitive decline in the subject manifests as increased pain sensation.

[0069] In some embodiments, the method includes preventing, treating, slowing, or reversing neurodegenerative changes in the subject. In some embodiments, the method includes preventing neurodegenerative changes in the subject. In some embodiments, the method includes treating neurodegenerative changes in the subject. In some embodiments, the method includes slowing neurodegenerative changes in the subject. In some embodiments, the method includes reversing neurodegenerative changes in the subject.

[0070] In some embodiments, the neurodegenerative changes in the subject are manifested as decreased fibroblast GCase activity, increased ER stress, neuronal mitochondrial dysfunction, increased neuronal mitochondrial ROS production, decreased autophagy flux, decreased neuronal mitochondrial velocity, decreased mitophagy, decreased plasma BDNF levels, decreased cerebrospinal fluid (CSF) BDNF levels, decreased neuronal and BDNF axonal transport, increased protein aggregates in the brain or CSF, brain inflammation, increased microglia activation, increased astrocyte activation, decreased brain volume, and increased plasma or CSF levels of neurofilament light chains (NFL).

[0071] In some embodiments, the neurodegeneration in the subject is manifested as decreased fibroblast GCase activity. In some embodiments, the neurodegeneration in the subject is manifested as increased ER stress. In some embodiments, the neurodegeneration in the subject is manifested as neuronal mitochondrial dysfunction. In some embodiments, the neurodegeneration in the subject is manifested as increased neuronal mitochondrial ROS production. In some embodiments, the neurodegeneration in the subject is manifested as decreased autophagy flux. In some embodiments, the neurodegeneration in the subject is manifested as decreased neuronal mitochondrial velocity. In some embodiments, the neurodegeneration in the subject is manifested as decreased mitophagy. In some embodiments, the neurodegeneration in the subject is manifested as decreased plasma BDNF levels. In some embodiments, the neurodegeneration in the subject is manifested as decreased cerebrospinal fluid (CSF) BDNF levels. In some embodiments, the neurodegeneration in the subject is manifested as decreased neuronal and BDNF axonal transport. In some embodiments, the neurodegeneration in the subject is manifested as an increase in protein aggregates in the brain or cerebrospinal fluid (CSF). In some embodiments, the neurodegeneration in the subject is manifested as brain inflammation. In some embodiments, the neurodegeneration in the subject is manifested as increased microglial activation. In some embodiments, the neurodegeneration in the subject is manifested as increased astrocyte activation. In some embodiments, the neurodegeneration in the subject is manifested as decreased brain volume. In some embodiments, the neurodegeneration in the subject is manifested as increased plasma levels of neurofilament light chains (NFL). In some embodiments, the neurodegeneration in the subject is manifested as increased levels of neurofilament light chains (NFL) in cerebrospinal fluid (CSF).

[0072] In some embodiments, the method includes administering a pharmaceutical composition comprising pridopidine or a pharmaceutically acceptable salt thereof to the subject at least twice daily. In some embodiments, the method includes administering a pharmaceutical composition comprising pridopidine or a pharmaceutically acceptable salt thereof to the subject at least three times daily. In some embodiments, the method includes administering a pharmaceutical composition comprising pridopidine or a pharmaceutically acceptable salt thereof to the subject at least four times daily.

[0073] In some embodiments, the method includes administering a pharmaceutical composition comprising pridopidine or a pharmaceutically acceptable salt thereof to the subject twice daily.

[0074] In some embodiments, the method includes administering a pharmaceutical composition comprising pridopidine or a pharmaceutically acceptable salt thereof to the subject three times daily.

[0075] In some embodiments, the method includes administering a pharmaceutical composition comprising pridopidine or a pharmaceutically acceptable salt thereof to the subject four times daily.

[0076] In some embodiments, the total dose of pridopidine or a pharmaceutically acceptable salt thereof is 10 mg to 100 mg daily. In some embodiments, the total dose of pridopidine or a pharmaceutically acceptable salt thereof is 20 mg to 100 mg daily. In some embodiments, the total dose of pridopidine or a pharmaceutically acceptable salt thereof is 30 mg to 100 mg daily. In some embodiments, the total dose of pridopidine or a pharmaceutically acceptable salt thereof is 40 mg to 100 mg daily. In some embodiments, the total dose of pridopidine or a pharmaceutically acceptable salt thereof is 50 mg to 100 mg daily. In some embodiments, the total dose of pridopidine or a pharmaceutically acceptable salt thereof is 60 mg to 100 mg daily. In some embodiments, the total dose of pridopidine or a pharmaceutically acceptable salt thereof is 70 mg to 100 mg daily. In some embodiments, the total dose of pridopidine or a pharmaceutically acceptable salt thereof is 80 mg to 100 mg daily. In some embodiments, the total dose of pridopidine or a pharmaceutically acceptable salt thereof is 90 mg to 100 mg per day.

[0077] In some embodiments, the total dose of pridopidine or a pharmaceutically acceptable salt thereof is 80 mg daily. In some embodiments, the total dose of pridopidine or a pharmaceutically acceptable salt thereof is 85 mg daily. In some embodiments, the total dose of pridopidine or a pharmaceutically acceptable salt thereof is 90 mg daily. In some embodiments, the total dose of pridopidine or a pharmaceutically acceptable salt thereof is 95 mg daily. In some embodiments, the total dose of pridopidine or a pharmaceutically acceptable salt thereof is 100 mg daily.

[0078] In some embodiments, the method includes administering, twice daily, a pharmaceutical composition comprising pridopidine or a pharmaceutically acceptable salt thereof to the subject, wherein the total dose of pridopidine or a pharmaceutically acceptable salt thereof is 80 mg to 100 mg daily. In some embodiments, the method includes administering, twice daily, a pharmaceutical composition comprising pridopidine or a pharmaceutically acceptable salt thereof to the subject, wherein the total dose of pridopidine or a pharmaceutically acceptable salt thereof is 85 mg to 95 mg daily.

[0079] In some embodiments, the method includes administering, twice daily, a pharmaceutical composition comprising pridopidine or a pharmaceutically acceptable salt thereof to the subject, wherein the total dose of pridopidine or a pharmaceutically acceptable salt thereof is 90 mg per day.

[0080] In some embodiments, the method includes administering, twice daily, to the subject a pharmaceutical composition comprising pridopidine or a pharmaceutically acceptable salt thereof, wherein each of the two pharmaceutical compositions comprises a different dose of pridopidine or a pharmaceutically acceptable salt thereof, wherein the total dose of pridopidine or a pharmaceutically acceptable salt thereof administered is 90 mg per day.

[0081] In some embodiments, the method includes administering to the subject twice daily a pharmaceutical composition comprising 45 mg of pridopidine or a pharmaceutically acceptable salt thereof, wherein the total dose of pridopidine or a pharmaceutically acceptable salt thereof is 90 mg per day.

[0082] In some embodiments, the method includes systemic administration of the pharmaceutical composition comprising pridopidine or a pharmaceutically acceptable salt thereof to the subject.

[0083] In some embodiments, the method includes orally administering the pharmaceutical composition comprising pridopidine or a pharmaceutically acceptable salt thereof to the subject.

[0084] In some embodiments, the pharmaceutical composition comprising pridopidine or a pharmaceutically acceptable salt thereof is administered in a form selected from the group consisting of: inhalable powder, injectable formulation, liquid, gel, solid, capsule, or tablet. In some embodiments, the pharmaceutical composition comprising pridopidine or a pharmaceutically acceptable salt thereof is administered in powder form. In some embodiments, the pharmaceutical composition comprising pridopidine or a pharmaceutically acceptable salt thereof is administered in injectable material form. In some embodiments, the pharmaceutical composition comprising pridopidine or a pharmaceutically acceptable salt thereof is administered in liquid form. In some embodiments, the pharmaceutical composition comprising pridopidine or a pharmaceutically acceptable salt thereof is administered in gel form. In some embodiments, the pharmaceutical composition comprising pridopidine or a pharmaceutically acceptable salt thereof is administered in solid form. In some embodiments, the pharmaceutical composition comprising pridopidine or a pharmaceutically acceptable salt thereof is administered in capsule form. In some embodiments, the pharmaceutical composition comprising pridopidine or a pharmaceutically acceptable salt thereof is administered in tablet form.

[0085] In some embodiments, the pharmaceutically acceptable pridopidine salt is pridopidine hydrochloride.

[0086] In some embodiments, the Parkinson's disease or its symptoms are part of or associated with a neurodegenerative condition selected from the group consisting of: Parkinson's disease (PD), multiple system atrophy (MSA), Lewy body dementia (DLB), amyotrophic lateral sclerosis (ALS), Parkinson's disease dementia (PDD), Huntington's disease (HD), Alzheimer's disease (AD), progressive supranuclear palsy (PSP), corticobasal degeneration, frontotemporal dementia, Gerstmann-Straussler-Schönck syndrome, Parkinson's disease-dementia-amyotrophic lateral sclerosis complex (Guam-type ALS complex), neuroacanthosis, neuronal ceroid lipofuscin deposition, olivopontocerebellar atrophy, pantothenic acid kinase-associated neurodegeneration, Wilson's disease, corticobasal degeneration (CBD), and Pick's disease.

[0087] In some embodiments, the Parkinson's disease or its symptoms are part of or related to PD. In some embodiments, the Parkinson's disease or its symptoms are part of or related to MSA. In some embodiments, the Parkinson's disease or its symptoms are part of or related to DLB. In some embodiments, the Parkinson's disease or its symptoms are part of or related to ALS. In some embodiments, the Parkinson's disease or its symptoms are part of or related to PDD. In some embodiments, the Parkinson's disease or its symptoms are part of or related to HD. In some embodiments, the Parkinson's disease or its symptoms are part of or related to AD. In some embodiments, the Parkinson's disease or its symptoms are part of or related to PSP. In some embodiments, the Parkinson's disease or its symptoms are part of or related to corticobasal degeneration. In some embodiments, the Parkinson's disease or its symptoms are part of or related to frontotemporal dementia. In some embodiments, the Parkinson's disease or its symptoms are part of or related to Gerstmann-Straussler-Schönke syndrome. In some embodiments, the Parkinson's disease or its symptoms are part of or related to the Parkinson's disease-dementia-amyotrophic lateral sclerosis complex. In some embodiments, the Parkinson's disease or its symptoms are part of or related to neuroacanthosis. In some embodiments, the Parkinson's disease or its symptoms are part of or related to neuronal ceroid lipofuscin deposition. In some embodiments, the Parkinson's disease or its symptoms are part of or related to olivopontocerebellar atrophy. In some embodiments, the Parkinson's disease or its symptoms are part of or related to pantothenic acid kinase-associated neurodegeneration. In some embodiments, the Parkinson's disease or its symptoms are part of or related to Wilson's disease. In some embodiments, the Parkinson's disease or its symptoms are part of or related to CBD. In some embodiments, the Parkinson's disease or its symptoms are part of or related to Picker's disease.

[0088] In some embodiments, the Parkinson's disease or its symptoms are part of or related to Parkinson's disease (PD). In some embodiments, the PD is PD-GBA.

[0089] In some embodiments, the Parkinson's disease or its symptoms are part of or related to multiple system atrophy (MSA).

[0090] In some embodiments, the Parkinson's disease or its symptoms are part of or related to Lewy body dementia (DLB).

[0091] In some embodiments, the Parkinson's disease or its symptoms are part of or related to amyotrophic lateral sclerosis (ALS).

[0092] The present invention further provides a method for treating, inhibiting or reducing PD decline in a subject in need, the method comprising administering a low dose of pridopidine or a pharmaceutically acceptable salt thereof to the subject, thereby treating the subject.

[0093] The present invention further provides a method for slowing functional decline in a subject with PD, the method comprising administering a low dose of pridopidine or a pharmaceutically acceptable salt thereof to the subject, thereby slowing functional decline in the subject with PD.

[0094] The present invention further provides a method for inducing / promoting functional neurological repair in a subject with PD, the method comprising administering a low dose of pridopidine or a pharmaceutically acceptable salt thereof to the subject, thereby inducing / promoting functional neurological repair in the subject with PD.

[0095] In another embodiment, the present invention further provides a method for treating Parkinson's disease (PD) associated with glucocerebrosidase mutation (GBA) mutation, GBA deficiency, multiple system atrophy (MSA), or Lewy body dementia (LBD). In another embodiment, the present invention further provides a method for treating PD associated with GBA mutation. In another embodiment, the present invention further provides a method for treating PD associated with GBA deficiency. In another embodiment, the present invention further provides a method for treating Parkinson's disease associated with multiple system atrophy (MSA). In another embodiment, the present invention further provides a method for treating Parkinson's disease associated with Lewy body dementia (LBD).

[0096] Mutations in the GBA gene, which encodes the lysosomal hydrolase glucocerebrosidase (GCase), are the most common risk factor for developing Parkinson's disease (PD), and between 5% and 20% of PD patients are carriers. Compared to the general PD patient population, PD-GBA patients have a younger age of onset and more associated cognitive changes.

[0097] There is a clinical link between PD and Gaucher disease (GD), a lysosomal storage disease caused by recessive mutations in the GBA gene. Patients with GD and gene vectors have an increased risk of developing PD. Several cellular mechanisms contributing to GBA-related neurodegeneration have been proposed, including (1) ER stress, (2) mitochondrial dysfunction, (3) autophagy defects, and (4) defects in BDNF levels and axonal transport.

[0098] Parkinson's disease is an early feature of both MSA and LBD. MSA symptoms include motor disorders (loss or limitation of muscle control or movement or restricted activity), which may include tremors, rigidity and / or loss of muscle coordination similar to PD, as well as difficulties with speech and gait (the way a person walks).

[0099] MSA is a rare disease affecting both men and women, and all ethnic groups. Symptoms often appear in people in their 50s and progress rapidly over 5 to 10 years, accompanied by gradual loss of motor function and eventually bedriddenness. Currently, there are no drugs that can slow the progression of the disease, nor is there a cure. MSA includes conditions historically known as Shy-Drager syndrome, olivopontocerebellar atrophy, and striatal substantia nigra degeneration.

[0100] Lewy body dementia (LBD) is a progressive brain disorder in which Lewy bodies (abnormal deposits of proteins called alpha-synuclein) accumulate in areas of the brain that regulate behavior, cognition, and movement, similar to PD.

[0101] In one embodiment, the subject is a human patient.

[0102] In one embodiment, the pridopidine is administered orally.

[0103] In one embodiment, the pridopidine is administered in the form of an inhalable powder, injectable, liquid, gel, solid, capsule, or tablet.

[0104] In one embodiment, the pridopidine is administered periodically.

[0105] In one embodiment, primidopidine is administered less than once a day. In one embodiment, primidopidine is administered daily. In one embodiment, primidopidine is administered once a day. In another embodiment, primidopidine is administered more than once a day. In one embodiment, primidopidine is administered two or three times a day.

[0106] In one embodiment, pridopidine is administered at a low dose between 10 mg / day and 100 mg / day. In one embodiment, pridopidine is administered at a low dose between 20 mg / day and 90 mg / day. In one embodiment, the dosage of pridopidine is 45 mg / day to 90 mg / day. In one embodiment, the dosage of pridopidine is 20 mg / day to 50 mg / day. In another embodiment, the dosage of pridopidine is about 20 mg / day, 22.5 mg / day, about 45 mg / day, about 67.5 mg / day, about 90 mg / day, or about 100 mg / day. In one embodiment, the dosage of pridopidine is 45 mg / day. In one embodiment, the dosage of pridopidine is 90 mg / day.

[0107] In one embodiment, pridopidine is administered once daily. In another embodiment, pridopidine is administered twice daily.

[0108] In one embodiment, the dosage of a single dose of pridopidine is about 10 mg, about 22.5 mg, about 45 mg, about 67.5 mg, or about 90 mg.

[0109] In one embodiment, pridopidine is administered twice daily, with each dose being 45 mg.

[0110] In one embodiment, the periodic administration of pridopidine lasts for at least 3 days, at least 30 days, at least 42 days, at least 8 weeks, at least 12 weeks, at least 24 weeks, at least 6 months, at least 1 year, at least 2 years, at least 5 years, at least 10 years, at least 15 years, at least 20 years, at least 25 years, or 30 years or longer.

[0111] In one embodiment, pridopidine treats the subject by delaying the onset of symptoms.

[0112] With respect to the foregoing embodiments, each embodiment disclosed herein is to be considered applicable to each embodiment of the other disclosed embodiments. Furthermore, elements described in the method embodiments may be used in the pharmaceutical compositions, uses, and packaging embodiments described herein, and vice versa.

[0113] the term As used herein and unless otherwise stated, each of the following terms shall have the definition set forth below.

[0114] As used in this article, "pridopidine" refers to pridopidine base or its pharmaceutically acceptable salt.

[0115] The active compounds used according to the invention can be provided in any form suitable for intended administration. Suitable forms include pharmaceutically acceptable salts and prodrug (predrug or prodrug) forms of the compounds of the invention.

[0116] "Salts" refer to salts of the compounds of the present invention that are modified by preparing acid salts or base salts of the compounds. In this regard, the term "pharmaceutically acceptable salt" refers to a relatively non-toxic inorganic or organic acid or base addition salt of the compounds of the present invention suitable for pharmaceutical use. Pharmaceutically acceptable salts can be formed by procedures well known and described in the art. One way to prepare such salts is by treating the compounds of the present invention with an inorganic base.

[0117] Examples of acid addition salts of the compounds of the present invention include, but are not limited to, hydrochlorides, hydrobroms, nitrates, perchlorates, phosphates, sulfates, formates, acetates, aconates, ascorbic acid salts, benzenesulfonates, benzoates, cinnamates, citrates, bis(hydroxynaphthyl) salts, heptanoates, fumarates, glutamates, glycolates, lactates, maleates, malonates, mandelates, methanesulfonates, naphthalene-2-sulfonates, phthalates, salicylates, sorbates, stearates, succinates, tartrates, p-toluenesulfonates, etc. In some embodiments, pridopidine is a pharmaceutically acceptable salt, such as an HCl salt or a tartrate salt. Preferably, in any embodiment of the invention as described herein, pridopidine is in its hydrochloride form.

[0118] As used herein, the “amount” or “dosage” of pridopidine measured in milligrams refers to the number of milligrams of pridopidine (4-[3-(methanesulfonyl)phenyl]-1-propyl-piperidine) present in the formulation, regardless of the form of the formulation. For example, a unit dose containing “90 mg pridopidine” means that the amount of pridopidine in the formulation is 90 mg, regardless of the form of the formulation. Therefore, when in the form of a salt such as pridopidine hydrochloride, the weight of the salt form required to provide a dose of 90 mg pridopidine will be greater than 90 mg due to the presence of said salt.

[0119] As those skilled in the art will understand, when referring to any type of symptoms in a patient, the terms “increase” or “decrease” are used in comparison to the presence and / or severity of the symptoms in a corresponding healthy subject, the corresponding group of healthy subjects, or the patient’s own medical history.

[0120] In some embodiments, the term "functional decline" refers to one or more of a subject's ability to maintain a profession, manage finances, or perform household chores, where such functional decline requires a low level of care and activities of daily living (ADL). In some embodiments, the method of the present invention provides a way to prevent, treat, slow down, or reverse a subject's ability to maintain one or more of a profession, manage finances, or perform household chores, where such decline requires a low level of care and activities of daily living (ADL).

[0121] Additional assessments of functional impairment / disability are incorporated into the UDysRS scale for patients with movement disorders. This scale provides a direct observation of the patient's ability to perform ADL (Activities of Daily Living). Parts 3 and 4 of the UDysRS describe how the patient's ADL function is affected.

[0122] The term “functional decline” includes, for example, (in the case of PD patients) a decline according to Part II of the Unified Parkinson’s Disease Rating Scale (UPDRS Part II), which includes activities of daily living. For example, the modified Hoya staging of PD includes: Grade 0 (no signs of disease), Grade 1 (unilateral disease), Grade 1.5 (unilateral plus axial involvement), Grade 2 (bilateral disease, no balance disorder), Grade 2.5 (mild bilateral disease, recoverable by tread test), Grade 3 (mild to moderate bilateral disease; some postural instability; independent), Grade 4 (severe disability; still able to walk or stand independently), and Grade 5 (must be wheelchair-bound or bedridden unless assisted).

[0123] The term "Activities of Daily Living" further includes a decline according to the Schwab and England Activities of Daily Living Scale, which includes: 100% (Completely independent. Able to do all household chores without slowness, difficulty, or obstruction. Generally normal. No awareness of any difficulty.), 90% (Completely independent. Able to do all household chores with some degree of slowness, difficulty, or obstruction. May take twice as long. Beginning to be aware of difficulties.), 80% (Completely independent in most household chores. Takes twice as long. Aware of difficulties and slowness.), 70% (Not completely independent. More difficult when doing some household chores. Takes three to four times as long in some household chores. Must spend most of the day doing...). Regarding housework: 60% (some degree of dependence. Can do most housework, but extremely slowly and with more effort. Errors; some impossible to complete.), 50% (more dependent. Helps with half, even slower, etc. Difficulty doing everything.), 40% (very dependent. Can assist with all housework, but rarely does housework alone.), 30% (with effort, occasionally does a small amount of housework alone or begins to do a small amount of housework alone. Needs more help.), 20% (Cannot do anything alone. Can help a little with some housework. Severely ineffective.), 10% (completely dependent, cannot help with housework. Completely ineffective.), and 0% (e.g., vegetative-like functions such as swallowing, bladder and bowel function are abnormal. Bedridden.).

[0124] As used herein, “unit dose,” “unit doses,” and “unit dosage form” refer to a single entity / entity of drug administration. “Unit dose,” “unit dosage,” and “unit dosage form” can refer to oral dosage forms such as tablets, capsules, pills, powders, and granules.

[0125] As used herein, “about” in the context of numerical values ​​or ranges means 90-110% of the numerical values ​​or ranges listed or claimed.

[0126] "Administered to a subject" or "administered to a (human) patient" means to give, dispense, or apply a medicine, drug, or remedy to a subject / patient to delay, alleviate, cure, or reduce symptoms associated with, for example, a symptom (e.g., a case symptom). Oral administration is one method of administering the compounds of the present invention to a subject.

[0127] The compounds according to the invention can be administered in the form of a base or a pharmaceutically acceptable salt, preferably in the form of a pharmaceutical composition used together with one or more adjuvants, excipients, carriers, buffers, diluents and / or other commonly used pharmaceutical adjuvants.

[0128] "Pharmaceutically acceptable carrier" refers to a carrier or excipient suitable for use with humans and / or animals without excessive adverse side effects (such as toxicity, irritation, and allergic reactions) commensurate with a reasonable benefit / risk ratio. The pharmaceutically acceptable carrier may be a pharmaceutically acceptable solvent, suspension, or mediator for delivering the compounds of the present invention to a subject.

[0129] Application can be periodic. As used herein, "periodic application" means repeated / repeated application at intervals. The time intervals between applications are preferably always consistent. Periodic application can include, for example, once a day, twice a day, three times a day, four times a day, weekly, twice a week, three times a week, four times a week, etc.

[0130] As used herein, “treat” or “treating” encompasses alleviating, reducing, or decreasing the severity of physical, mental, or emotional limitations in a subject with PD, eliminating or substantially eliminating or improving said physical, mental, or emotional limitations. Treatment also refers to delaying or preventing disease-related symptoms or reducing disease-related deficits.

[0131] As used herein, “effective” as a quantity effective for a given purpose means an amount of component that, when used in the manner disclosed herein, is sufficient to produce the indicated therapeutic response without excessive adverse side effects (such as toxicity, irritation, or allergic reactions) commensurate with a reasonable benefit / risk ratio. For example, an effective amount for treating symptoms of PD. The specific effective amount varies with factors such as the particular condition being treated, the patient’s physical condition, the type of mammal being treated, the duration of treatment, the nature of any concurrent therapies, and the structure of the specific formulation and compound or its derivatives used.

[0132] It should be understood that, while providing a parameter range, the present invention also provides all integers within the range and one-tenth of them. For example, "22 mg-300.0 mg" includes 22.0 mg, 22.1 mg, 22.2 mg, 22.3 mg, 22.4 mg, etc., and at most 300.0 mg.

[0133] The invention will be better understood by referring to the following experimental details, but those skilled in the art will readily understand that the specific examples, as more fully described in the following claims, are merely illustrative of the invention.

[0134] Example The effects of pridopidine as a potential S1R agonist in rodents with hemilateral Parkinson's disease were investigated. Mice with striatal 6-hydroxydopamine (6-OHDA) lesions were treated with pridopidine for 5 weeks. The results surprisingly showed that at low doses (e.g., 0.3 mg / kg), pridopidine produced behavioral and neurohistochemical repair, accompanied by upregulation of neuroplastic pathways in the striatum. Pridopidine treatment in mice with S1R-deficient 6-OHDA lesions did not show any of these beneficial effects. Pharmacokinetic data indicated that at the effective dose (0.3 mg / kg), the brain maximum concentration (Cmax) of pridopidine was 110 ng / ml, a concentration sufficient to selectively bind S1R but not to selectively bind dopamine receptors. These results demonstrate for the first time that, acting as an S1R agonist, pridopidine can prevent dopaminergic neuronal degeneracy and restore significant levels of function in dopaminergic fibers in the motor striatum.

[0135] method: Animals The study was conducted in male C57BL6J mice (approximately 25 g in weight and 8 to 9 weeks old at the start of the experiment). A total of 65 wild-type mice and 52 S1R knockout (KO) mice were used. S1R knockout mice and wild-type littermates (bisexual) were bred in a C57BL6J background. The S1R KO line was derived from well-characterized Sigmar1 mice distributed by the MutantMouse Resource Regional Centre (MMRRC). Gt(OST422756)Lex Mouse strain. Mice were housed in captivity under a 12-hour light / dark cycle with free access to food and water. Household conditions and experimental treatments were approved by the Malmo-Lund Ethical Committee on Animal Research.

[0136] 6-OHDA lesion The procedure was performed according to a previously described protocol [Francardo V, Bez F, Wieloch T, Nissbrandt H, Ruscher K, Cenci 33. MA. Pharmacological stimulation of sigma-1 receptors has neurorestorative effects in experimental Parkinson's disease and parkinsonism. Brain: A Journal of Neurology. 2014; 137(Pt 7):1998-2014]. In short, mice were anesthetized with isoflurane (Isoba®vet, Apoteksbolaget, Solna, Stockholm, Sweden) and placed in a stereotactic frame in the flat-skull position. 6-OHDA-HCl (Sigma-Aldrich AB, Stockholm, Sweden) was freshly dissolved in 0.02% ascorbate-saline at a concentration of 3.2 mg free base per mL. One μL of the toxin solution was injected at each site into the right striatum at the following coordinates (given in millimeters relative to the anterior fontanelle, sagittal suture, and dura mater surface; see Paxinos and Franklin, 2001): AP +1.0, ML -2.1, and DV -2.9, site 1; and AP +0.3, ML -2.3, and DV -2.9, site 2. The solution was injected at a rate of 0.5 L / min through a glass capillary (tip diameter approximately 50 μm), and the capillary was left in place for 2 min after each injection.

[0137] Treatment Prior to administration, pridopidine was dissolved in physiological saline and administered as a single subcutaneous (sc) injection daily at a dose of 0.1 mL / 10 g body weight. The first injection was given immediately after lesion surgery. In the first experiment, pridopidine was administered at either 0.3 mg / kg or 1.0 mg / kg. This dose was selected for the remainder of the study after it was determined that only 0.3 mg / kg pridopidine produced function-related neuronal repair. Pridopidine was always administered at approximately 2 p.m., while behavioral tests were always performed between 9:00 and 10:30 a.m. (this time lag was allowed to exclude confounding factors due to the potential motor effects of pridopidine).

[0138] Behavioral tests All mice participating in this study underwent three behavioral tests once a week.

[0139] i） Spontaneous rotation activity The spontaneous rotation test performed in an open field was used to assess postural and motor asymmetry resulting from unilateral nigrostriatal denervation. In this test, in response to the novelty of the test environment, animals temporarily turned to the side ipsilateral to the 6-OHDA lesion, and the turning response gradually decreased in all animals upon test repetition [Francardo V, Bez F, Wieloch T, Nissbrandt H, Ruscher K, Cenci 33. MA. Impact of the lesion procedure on the profiles of motor impairment and molecular responsiveness to L-DOPA in the 6-hydroxydopamine mouse model of Parkinson's disease. Neurobiology of Disease. 2011; 42(3):327-340.]

[0140] In short, each mouse was placed individually in the center of an open field (50 x 50 cm) and immediately video-recorded for 10 minutes, the time corresponding to the period of maximum activity [Francardo V, Recchia A, Popovic N, Andersson D, Nissbrandt H, Cenci MA. Effects of lesion program on motor dysfunction and L-DOPA molecular response profile in a 6-hydroxydopamine mouse model of Parkinson's disease. Neurobiology of Disease. 2011; 42(3):327-340]. Complete rotations (360 rotations in succession) were counted offline by experimentally blinded researchers. Data are expressed as the net total number of complete rotations on the ipsilateral side of the lesion at each test phase.

[0141] ii) Cylinder test Asymmetric forelimb use during vertical exploration provides an effective measure of forelimb akinesia in rodents with unilateral Parkinson's disease [Lundblad M, Andersson M, Winkler C, Kirik D, Wierup N, Cenci MA. Pharmacological validation of behavioral measures of akinesia and dyskinesia in a rat model of Parkinson's disease. The European Journal of Neuroscience. 2002; 15(1):120-32]. The test was performed as follows: [Francardo V, Bez F, Wieloch T, Nissbrandt H, Ruscher K, Cenci MA. Briefly, mice were placed alone in a glass cylinder 161 (10 cm in diameter and 14 cm in height) and video recorded for 3 to 5 minutes.] The number of support wall contacts performed independently by the claw located on the opposite side of the lesion is expressed as the percentage of all support wall contacts in each phase.

[0142] iii) Step test Impaired ability to perform regulated gait during human-induced movement (stepping test) reflects both dyskinesia and postural deficits associated with PD [Blume SR, Cass DK, Tseng KY. Stepping test in mice: a reliable approach in determining forelimb akinesia in MPTP-induced Parkinson's Disease. Experimental Neurology. 2009; 219(1):208-11.] Winkler C, Bentlage C, Nikkhah G, Samii M, Bjorklund A. Intranigral transplants of GABA-rich striatal tissue induce behavioral recovery in the rat Parkinson's Disease model and promote the effects obtained by intrastriatal dopaminergic transplants. *Experimental Neurology*. 1999;155(2):165-86. Mice were placed at the entrance of a custom-made plastic corridor (7 cm wide, 1 m long, with walls 10 cm high on both sides), with their tails gently raised and pulled back at a fixed speed (1 m / 4 sec). Each experiment was video-recorded, and the number of modulated steps performed by each forelimb was counted using a camera. Experiments in which mice turned their bodies 90° to the walls of the corridor (attempting to escape or explore) were abandoned. Mice were tested in each phase until three valid trials were obtained per animal. Results are expressed as the percentage of total steps taken by the forelimb (left side) located on the side opposite the lesion (average of three trials per phase).

[0143] Brain preparation Mice assigned to immunohistochemistry were perfused and fixed the day after the last small carpet / saline administration (approximately 20 hours after the last injection). Animals were deeply anesthetized with sodium pentobarbital (240 mg / kg, intraperitoneally) and their hearts were perfused with 0.9% saline solution, followed by perfusion with ice-cold buffered 4% paraformaldehyde (pH 7.4). After rapid extraction, the brain was immersed in the same fixative for 2 hours and then cryoprotected overnight in ice-cold phosphate-buffered 25% sucrose solution. Coronal sections of 30 μm thickness were cut on a sliding microtome and stored at -20°C in a cryoprotectant (30% ethylene glycol and 30% glycerol in 0.1 M phosphate buffer).

[0144] Animals prepared for Western blot analysis were euthanized by cervical dislocation; the brains of these animals were rapidly extracted and frozen on crushed dry ice. Tissue samples were dissected in a cryostat chamber (-14°C). Coronal brain sections were extracted using a mouse brain-shaped container, spanning the cephalothorax (+1.18 mm to -0.34 mm) relative to the anterior fontanelle. The striatum was dissected using a scalpel. Tissue samples were collected from the substantia nigra using a 2 mm diameter tissue perforator, spanning the cephalothorax (-2.70 mm to -3.80 mm) relative to the anterior fontanelle. The samples were cryopreserved until further analysis.

[0145] Quantitative immunohistochemistry According to the established protocol [Francardo V, Bez F, Wieloch T, Nissbrandt H, Ruscher K, Cenci 33. MA. Disease Neurobiology. 2011; 42(3):327-340], a primary antibody against tyrosine hydroxylase [Mysona BA, Zhao J, Smith S, Bollinger KE. Relationship between sigma-1 receptor and BDNF in the visual system. Experimental Eye Research. 2018; 167:25-30] (rabbit anti-TH antiserum from Pel-Freez, Rogers, AR, 1:1000) and differentiation cluster 68 (CD68) (rat anti-CD68 antiserum from AbD Serotec, Kidlington, Oxfordshire, UK) was used. Immunohistochemical and quantitative analyses were performed using a 1:1000 ratio (Oxfordshire, UK). Quantitative analyses were performed by blinded researchers using the following methods.

[0146] The number of TH-positive cell bodies in the substantia nigra pars compacta (SNc) was determined using unbiased stereology based on the optical fractionator method described below [West MJ. Stereological methods for estimating the total number of neurons and synapses: issues of precision and bias. Trends in Neurosciences. 1999; 22(2):51-61].

[0147] Using software controlled by NewCAST (Visiopharm). x-yAnalysis was performed using a Nikon 80i microscope with an electric stage. Following the established protocol [Francardo V, Recchia A, Popovic N, Andersson D, Nissbrandt H, Cenci MA. Effects of pathological procedures on motor impairment and L-DOPA molecular response profiles in a 6-hydroxydopamine mouse model of Parkinson's disease. Neurobiology of Disease. 2011; 42(3):327-340], sampling fractions were selected to count at least 100 neurons per side per animal, and the total number of TH-positive neurons in the SNc was then estimated using the optical fractionator formula, i.e., number of neurons = 1 / ssf (slice sampling fraction) x 1 / asf (area sampling fraction) x 1 / tsf (thickness sampling fraction) x E (number of counted objects) [West MJ. Stereoscopic methods for estimating total number of neurons and synapses: problems of precision and bias. Trends in Neuroscience. 1999; 22(2):51-61].

[0148] The density of TH immunoreactive fibers in the highly denervated lateral striatum was measured using image segmentation software (VIS, Visiopharm IntegratorSystem; Visiopharm, H0rsholm, Denmark). For this purpose, the lateral striatum was delineated at four head-tail levels per animal using well-defined anatomical landmarks at low magnification (x4 objectives) in a Nikon Eclipse 80i microscope (e.g., in [Francardo V, Recchia A, Popovic N, Andersson D, Nissbrandt H, Cenci MA. Effects of lesioning procedures on motor dysfunction and L-DOPA molecular response profiles in a 6-hydroxydopamine mouse model of Parkinson's disease. Neurobiology of Disease. 2011; 42(3):327-340]. Automated random sampling was then applied under x100 objectives to cover a fixed percentage (10% of the lateral striatum cross-sectional area) of the structures of interest in all sections. Images were captured using a digital camera (Olympus DP72) to obtain 25 to 30 sample regions per mouse (region size 5742 pixels). 2On each image, a pixel classifier based on a Bayesian algorithm was used to separate different TH-immunopositive fibers from background objects [Westin JE, Lindgren HS, Gardi J, Nyengaard JR, Brundin P, Mohapel P et al. Endothelial proliferation and increased blood-brain barrier permeability in the basal ganglia in a rat model of 3,4-dihydroxyphenyl-L-alanine-induced dyskinesia. The Journal of Neuroscience: The Official Journal of the Society for Neuroscience. 2006;26(37):9448-61.]

[0149] The results are expressed as the total number of TH immunopositive pixels per sample region (average across regions). This analysis was not performed in animals with sham-operated lesions due to the presence of a fine network of dopaminergic fibers in this group (Type I fibers, [Song DD, Haber SN. Striatal responses to partial dopaminergic lesion: evidence for compensatory sprouting. Journal of Neuroscience: Official Journal of the Society for Neuroscience. 2000; 20(13):5102-14]), thus excluding the resolution of different axonal structures. Magnification analysis of TH fibers in random sample regions was supplemented by optical density (OD) measurements of TH immunostaining across the entire dorsolateral and ventrolateral striatum. Although this method is far less sensitive than actual fiber analysis [Francardo V, Bez F, Wieloch T, Nissbrandt H, Ruscher K, Cenci 33. MA. Pharmacological stimulation of sigma-1 receptors has neurorestorative effects in experimental parkinsonism. Brain: Journal of Neurology. 2014; 137(Pt 7):1998-2014], the method provides an indication of the overall neural distribution density of the entire region of interest.

[0150] use NIH Image J softwareOD analysis was performed at the same head-to-tail level as described in the following: / [Francardo V, Recchia A, Popovic N, Andersson D, Nissbrandt H, Cenci MA. Effects of pathological procedures on motor dysfunction and L-DOPA molecular response profiles in a 6-hydroxydopamine mouse model of Parkinson's disease. Disease Neurobiology. 2011; 42(3):327-340]. After subtracting background values ​​(measured in the corpus callosum of each animal), the OD on the ipsilateral side of the lesion is expressed as a percentage of the OD on the intact side of the same animal (values ​​from the intact side represent normal innervation density) [Francardo V, Recchia A, Popovic N, Andersson D, Nissbrandt H, Cenci MA. Effects of lesion program on motor dysfunction and L-DOPA molecular response profiles in a 6-hydroxydopamine mouse model of Parkinson's disease. Neurobiology of Disease. 2011; 42(3):327-340].

[0151] CD68 immunopositive cells exhibiting the morphology of active microglia [Cicchetti F, Brownell AL, Williams K, Chen YI, Livni E, Isacson O. Neuroinflammation of thenigrostriatal pathway during progressive 6-OHDA dopamine degeneration in rats monitored by immunohistochemistry and PET imaging. European Journal of Neuroscience. 2002;15(6):991-8; Perry VH, Teeling J. Microglia and macrophages of the central nervous system: the contribution of microglia priming and systemic inflammation to chronic neurodegeneration. Seminars [in Immunopathology]. 2013; 35(5):601-12] Manual counting was performed in the body of the SNC (corresponding to the cephalothorax level of 3.40 to 3.64 posterior to the anterior fontanelle) and the ventrolateral striatum (0.26–0.02 anterior to the anterior fontanelle). For this purpose, a mask depicting the region of interest was defined at x4 magnification and the region was controlled using NewCAST software (Visiopharm). x-y The electric stage randomly selects a sample area (36,921 m) within this mask under an x40 objective lens. 2 Size). The number of CD68 immunopositive cells on the same side as the lesion is expressed as a percentage of the number of CD68 immunopositive cells on the contralateral side.

[0152] Western blot analysis Tissue homogenates were prepared in cell lysis buffer (20 mmol / L Tris (pH 7.5), 150 mmol / L NaCl, 1 mmol / L EDTA, 1 mmol / L EGTA, 1% Triton X-100, 2.5 mmol / L sodium pyrophosphate, 1 mmol / L 3-glycerophosphate, 1 mmol / L Na3VO4, 11 g / mL leucine inhibitor, and 1 mmol / L benzyl sulfonyl fluoride). Twenty micrograms of protein were isolated on a 10% SDS-PAGE polyacrylamide gel. The protein was transferred to a polyvinyl fluoride membrane, which was incubated in blocking buffer (20 mM Tris, 136 mM NaCl, pH 7.6, 0.1% Tween 20, 5% skim milk powder). Subsequently, the membrane was incubated overnight at 4°C using one of the following primary antibodies: rabbit polyclonal anti-GDNF (Santa Cruz Biotechnology, Inc., Santa Cruz, CA; 1:1000) and monoclonal anti-BDNF (Santa Cruz Biotechnology, Inc., 1:1000); or rabbit polyclonal antibody against Thr202 / Tyr204-phosphorylated ERK1 / 2 (p44 / 42-MAPK; CellSignaling Technology Inc., Danvers, MA, 1:2000). After appropriate washing steps, the membrane was incubated together with an HRP-linked secondary antibody (Sigma-Aldrich, Deisenhofen, Germany, 1:15000). The signal was visualized using a chemiluminescence reagent kit (Merck Millipore, Watford, Hertfordshire, UK) and images were acquired using a CCD camera (LAS1000 system, Fuji Films, Tokyo, Japan). NIH Image J The optical density on the specific immune response band was measured using software. The membrane was then peeled off and re-probened with a |-actin antibody (Sigma-Aldrich, 1:50,000). The optical density of the specific band was then normalized to the |-actin band in the same lane.

[0153] Pridopidine brain tissue binding According to the manufacturer's protocol, rapid equilibrium dialysis using a rapid equilibrium dialysis (RED) device insert from Thermo Scientific utilizes fresh mouse brain homogenate in...In vitro To assess the brain binding properties of pridopidine.

[0154] Statistical analysis Using repeated measures analysis of variance (ANOVA) and Post hoc The Bonferroni test compared behavioral data recorded during long-term treatment. All remaining analyses used unpaired data. t Test or 1-factor ANOVA and Post hoc The Tucker test was performed. Because the data are not normally distributed, nonparametric statistics (Kruskal-Wallis test and...) were used. Post hoc Dunn test) analyzes striatal fiber density. Unless otherwise stated, data are presented as group mean ± standard mean error (SEM). The exact ANOVA value is reported in the legend. p Value and F The value was determined by subsequent paired comparisons, which were reported as significant or insignificant. In all comparisons, the statistical significance level was set to [value missing]. p < 0.05.

[0155] ER stress measurement H2a-GFP can be used as a protein marker in the early stages of ER stress to measure ER stress levels. H2a-GFP is a misfolded secreted protein that accumulates in response to ER stress. STHdhQ7 / 7 is a striatum-derived cell line from wild-type knock-in transgenic mice containing a homozygous humanized huntingtin gene (HTT) with seven polyglutamine repeats. STHdhQ7 / 7 cells were transfected with either the Htt96Q-mCherry (mutant mimicking typical pathogenic expression of Htt in HD patients) construct or the Htt20Q-mCherry (normal) construct. When exon 1 of the polyQ-extended Htt protein (96Q) fused with the fluorescent mCherry protein is expressed, the levels and aggregation of the protein in individual cells can be monitored using fluorescence microscopy.

[0156] Mitochondrial dysfunction induction and measurement Cortical and striatal neurons from YAC128 HD mice were treated with 1 μM pridopidine and then stimulated with antimycin A (Ant A, a fish killer that inhibits cellular respiration, particularly oxidative phosphorylation and ATP production) to induce mitochondrial reactive oxygen species (ROS) production. Mitochondrial function was assessed by measuring the level of the fluorescent probe MitoPY1, which detects H2O2 levels in mitochondria. Mitochondrial membrane potential (MMP) was assessed in cortical and striatal neurons using the positively varying fluorescent probe TMRM (tetramethylrhodamine methyl ester). Neurons were subjected to 150 nM TMRM (quenching condition) at 37°C in Na+. + Incubate in culture medium for 30 minutes. Under these conditions, mitochondrial retention of TMRM was investigated to estimate Dy m Changes were observed. Bottom fluorescence (503 nm excitation and 525 nm emission) was recorded for 4 minutes using a Gemini EM spectrophotometer (Molecular Devices, USA). Then, 2.5 μM FCCP and 2.5 μg / mL oligomycin were added to induce maximum mitochondrial depolarization and mitochondrial probe release. TMRM release was calculated based on the difference in fluorescence before and after the addition of oligomycin / FCCP.

[0157] Visualization and analysis of axonal transport To visualize and analyze axonal transport (AT), spinal cord extracts from WT or SOD1G93A embryos were plated in the proximal compartment of a microfluidic chamber. Retrograde axonal transport of Qdot-BDNF particles, specifically introduced into the distal compartment of the axon, was tracked using real-time imaging.

[0158] Example 1: Behavioral recovery induced by pridopidine in mice with 6-OHDA lesions Wild-type mice with striatal 6-OHDA lesions or sham-operated lesions were treated daily for 35 days with pridopidine (0.3 mg / kg or 1.0 mg / kg) or a mediator solution (“saline”). In the final statistical analysis, sham-operated lesion animals were pooled into one group (“sham-operated”) after determining that the sham-operated lesion animals treated with pridopidine or saline produced very similar results. Comparisons of spontaneous turning behavior between groups and test phases revealed significant overall differences between treatment modalities and time points. FIG. 1A). All groups injected with 6-OHDA showed significant ipsilateral rotational bias, which was most pronounced in the first week and then gradually decreased in all groups, as is usually the case in this test [Francardo V, Bez F, Wieloch T, Nissbrandt H, Ruscher K, Cenci 33. MA. Pharmacological stimulation of σ-1 receptors has neurodegenerative effects in experimental Parkinson's disease. Brain: Journal of Neurology. 2014; 137(Pt 7):1998-2014; Francardo V, Recchia A, Popovic N, Andersson D, Nissbrandt H, Cenci MA. Effects of pathological procedures on motor dysfunction and L-DOPA molecular response profiles in a 6-hydroxydopamine mouse model of Parkinson's disease. Disease Neurobiology. 2011; 42(3):327-340. However, there were highly significant differences between the groups in the degree of this reduction (regarding the interaction between treatment and time). p < 0.0001, see FIG. 1A In fact, from week one to week five and the last week, mice with 6-OHDA lesions treated with saline remained significantly different from those with sham-operated lesions, while by the end of the treatment period, mice treated with pridopidine were no longer different from the sham-operated lesion control group. FIG. 1A Regarding the dosage of the two drugs in week 5 relative to the sham surgery, p > 0.05).

[0159] Furthermore, a difference was noted between the two doses of pridopidine, as mice receiving the 0.3 mg / kg dose differed significantly from saline-treated animals during most testing phases (for weeks 1–3, 0.3 mg / kg pridopidine versus saline). *p < 0.05), while mice treated with 1.0 mg / kg pridopidine differed from the saline group only during the first test phase (see FIG. 1A In the first week of treatment, for 1 mg / kg pridopidine relative to saline, p < 0.05). In the cylinder test, 6-OHDA lesions produced similar defects in contralateral forelimb use in all treatment groups (see...). FIG. 1B Data from week 1 showed approximately 40% difference between mice treated with saline and those treated with pridopidine compared to sham surgery. p < 0.05). This significant difference from the sham-operated lesion control group was maintained in all groups during week three. FIG. 1BSubsequently, mice treated with 0.3 mg / kg pridopidine showed visible improvement, reaching performance levels comparable to the sham-operated control group at weeks 4 and 5. FIG. 1B Week 4, relative to saline solution, p < 0.05 and relative to sham surgery, p > 0.05; Week 5, relative to both saline and 1.0 mg / kg doses, p < 0.05, p relative to sham > 0.05). In contrast, treatment with 1.0 mg / kg pridopidine did not restore use of the contralateral forelimb in the cylinder test (see [link]). FIG. 1B At week 5, the difference between the pridopidine-1 group and the saline group relative to the sham surgery was approximately 33%. p <0.05. Significant overall differences were also found between the groups and the test phases in the step test (…). FIG. 1C In all groups, the lesion-induced walking defect was initially severe (see...). FIG. 1C Data from week 1 showed approximately 60% difference compared to sham surgery in mice treated with saline and pridopidine. p < 0.05). Regardless of treatment allocation, all mice with 6-OHDA lesions continued to exhibit significant defects during the first 2 weeks. Subsequently, animals treated with 0.3 mg / kg pridopidine showed gradual improvement, significantly different from the saline-treated group at weeks 4 and 5. Although not reaching the values ​​of the sham-operated control group, by the end of the treatment period, mice treated with 0.3 mg / kg pridopidine showed approximately 94% better performance than the median (saline) (see [link]). FIG. 1C In week 5, pridopidine-0.3 was administered relative to both saline and pridopidine-1. p < 0.05, compared to sham surgery, p > 0.05). Animals receiving 1.0 mg / kg pridopidine showed a trend toward improved walking performance over the past 2 weeks, but no significant difference was observed compared to mice treated with saline. FIG. 1C See week 5 for the pridopidine-1 group and saline group, which showed differences of approximately 49% and 63% respectively compared to the sham surgery. For both comparisons, p <0.05).

[0160] Example 2: Pridolpiride has neuroprotective and neuroreparative effects on dopamine neurons in the substantia nigra striatum. Dopaminergic cell bodies in the substantia nigra (SNc) were counted using unbiased stereometry. On the ipsilateral side of the lesion, mice treated with saline showed a reduction of approximately 65% ​​in TH-positive neurons in the substantia nigra (i.e., approximately 1960 cells in the saline-treated mice compared to approximately 5076 cells in the sham-operated lesion control group). p < 0.05, FIG. 2A Mice treated with 0.3 mg / kg pridopidine showed a significantly greater number of TH-positive neurons (approximately 3034 cells), an increase of approximately 55% compared to the saline-treated mice. FIG. 2A , *p < 0.05). However, higher doses of pridopidine (1.0 mg / kg) did not significantly protect dopaminergic cell bodies in SNc (approximately 1901 cells counted; relative to saline, p > 0.05), but it appears to generate a greater density of TH immunopositive neurites in the SN reticular formation (see FIG. 2B and FIG. 2D ).

[0161] To examine the effect of pridopidine treatment on striatal DA nerve innervation density, an estimated 10% cross-sectional area (by...) within the lateral caudate putamen was analyzed. FIG. 2F Random sampling was performed to measure TH-immunopositive axonal fibers at high magnification (surrounded by shaded lines in the image). Due to the inability to resolve different fibers in the intact striatum, this analysis could not be applied to sham-operated lesion mice [Francardo V, Bez F, Wieloch T, Nissbrandt H, Ruscher K, Cenci 33.MA., SongDD, Haber SN. Striatal response to partial dopaminergic lesions: evidence of compensatory budding. Journal of Neuroscience: Official Journal of the Society for Neuroscience. 2000; 20(13):5102-14; Granado N, Ares-Santos S, Tizabi Y, Moratalla R. Striatal Reinnervation Process after Acute Methamphetamine-Induced Dopaminergic Degeneration in Mice. Neurotoxicity Research. 2018.].

[0162] Long-term treatment with 0.3 mg / kg pridopidine significantly increased the number of TH fiber spectra (see [link]). FIG. 2F(and Figure 4G), thus increasing the median value by approximately 15 times relative to the median value of animals injected with saline (and Figure 4G). FIG. 2I , p < 0.05. All animals treated with 0.3 mg / kg pridopidine showed an abundance of dense and highly differentiated axons with enlarged varicose veins, indicating a budding response. FIG. 2G [Song DD, Haber SN. Striatal response to partial dopaminergic lesions: evidence of compensatory budding. Journal of Neuroscience: Official Journal of the Society for Neuroscience. 2000; 20(13):5102-14; Granado N, Ares-Santos S, Tizabi Y, Moratalla R. Striatal reinnervation following acute methamphetamine-induced dopaminergic degeneration in mice. Neurotoxicity Research. 2018]. Although significantly less abundant, similar morphological features were also encountered in mice treated with 1.0 mg / kg pridopidine. FIG. 2H However, the effect of this larger dose did not reach statistical significance. FIG. 2I Regarding pridopidine-1 relative to saline, p > 0.05).

[0163] A rough estimate of the level of DA innervation was obtained by measuring the OD of TH immunostaining in the ventrolateral striatum and dorsolateral striatum throughout these regions. FIG. 2J , FIG. 2K In the ventrolateral striatum, treatment with 0.3 mg / kg pridopidine resulted in a more than twofold increase in THOD compared to the saline control. （p < 0.05) FIG. 2J Mice treated with 1 mg / kg pridopidine did not show a statistically significant trend of increase. FIG. 2J Animals treated with 0.3 mg / kg pridopidine also showed a significant but minor increase in TH OD in the dorsolateral striatum, while animals treated with 1 mg / kg pridopidine again showed only a trend ( FIG. 2KIn summary, these results indicate that treatment with 0.3 mg / kg pridopidine provides partial protection for the substantia nigra dopaminergic cell bodies, accompanied by significant dopaminergic re-innervation of the highly depleted lateral striatum. The latter effect is particularly prominent in the ventrolateral quadrant, which is the region that controls the use of the forelimbs in rodents [Chang JW, Wachtel SR, Young D, Kang UJ. Biochemical and anatomical characterization of forepaw adjusting steps in rat models of Parkinson's disease: studies on medial forebrain bundle and striatal lesions. Neuroscience. 1999; 88(2):617-28; Cousins ​​MS, Salamone JD. Skilled motor deficits in rats induced by ventrolateral striatal dopamine depletion: behavioral and pharmacological characterization. Brain Research. 1996; [732(1-2):186-94]. Higher doses of pridopidine (1 mg / kg) did not produce significant effects on any parameters.

[0164] The study investigated whether the neuroreparative effect of pridopidine treatment was consistent with a reduction in microglial activation in the affected areas. To this end, the number of CD68-immunopositive microglia in the SNc and ventrolateral striatum was counted. In mice treated with 0.3 mg / kg pridopidine, this number was significantly reduced in both regions ( FIG. 3A , FIG. 3F Regarding pridopidine-0.3 relative to saline, p < 0.05, compared to sham surgery ，p > 0.05 ）。 Mice treated with higher doses of pridopidine showed a tendency to produce effects in SNc mice. FIG. 3A Compared to sham surgery, p> 0.05), but no trend toward producing an effect was shown in the ventrolateral striatum ( FIG. 3F Compared to sham surgery, p < 0.05, relative to saline, p > 0.05).

[0165] Example 3: Treatment with pridopidine was ineffective in σ-1KO mice. The effective dose of pridopidine (0.3 mg / kg) in S1R KO mice with 6-OHDA lesions or sham-operated lesions was investigated. Throughout the 5-week treatment period, S1R KO lesion mice treated with 0.3 mg / kg pridopidine or saline showed efficacy in a cylinder test (…). FIG. 4B ) and step test ( FIG. 4C The spontaneous rotation exhibits a similar level in ) FIG. 4A ) and forelimb asymmetry (in all tests and phases, for pridopidine relative to saline, p > 0.05). Furthermore, treatment with pridopidine reduced the density of substantia nigra dopamine neurons (5A) or striatal TH fibers (Figure 5E-). FIG. 5G There was no effect (on all parameters, for pridopidine relative to saline), p > 0.05). These data clearly demonstrate that, in the absence of S1R, pridopidine cannot produce any repair effect on the damaged substantia nigra striatum DA pathway.

[0166] An interesting side finding of this experiment is that S1R KO mice respond to 6-OHDA lesions with a unique substantia nigra-striatal damage pattern. Indeed, compared to wild-type animals, S1R KO mice with 6-OHDA lesions are characterized by less severe DA cell body loss in the SNc (approximately 22% instead of approximately 61% loss, see [reference needed]). FIG. 2A Compared to FIG. 5A (in the saline group), but with lower TH fiber density in the lateral striatum (899 vs. 5842 fiber pixels / sample area median in KO mice relative to wild-type mice, see [reference]). FIG. 2I (Compared to the saline group in Figure 5E).

[0167] The more severe degeneration of striatal dopaminergic fibers compared to the substantia nigra cell bodies in S1R KO mice may explain the significant difference in lesion-induced motor dysfunction relative to the wild-type genotype, particularly the less severe rotational bias occurring concurrently with more severe forelimb walking defects (see Figures 1 and 4).

[0168] Example 4: Pridolpiride upregulates neurotrophic factors and activates ERK1 / 2 in the striatum. The gradual improvement in forelimb use induced by 0.3 mg / kg pridopidine and the accompanying increase in striatal DA fiber density indicate neurorepair mediated by trophic factors, and brain levels can be increased under pharmacological stimulation of S1R [Penas C, Pascual-Font A, Mancuso R, Fores J, Casas C, Navarro X. Sigma receptor agonist 2-(4-morpholinethyl)1-phenylcyclohexanecarboxylate (Pre084) increases GDNF and BiP expression and promotes neuroprotection after root avulsion injury. Journal of Neurotrauma. 2011; 28(5):831-40; Francardo V, Bez F, Wieloch T, Nissbrandt H, Ruscher K, Cenci33, MA, Fujimoto M, Hayashi T, Urfer R, Mita S, Su TP. Sigma-1 receptor chaperones regulate the secretion of brain-derived neurotrophic factor. Synapse. 2012; 66(7):630-9.

[0169] Western blot analysis of GDNF, BDNF, and phosphorylated ERK1 / 2 (major intracellular effectors of neurotrophic protein signaling [Garcia-Martinez JM, Perez-Navarro E, Gavalda N, Alberch J. Glial cell line-derived neurotrophic factor promotes the arborization of cultured striatal neurons through the p42 / p44 mitogen-activated protein kinase pathway. Journal of Neuroscience Research 2006; 83(1):68-79]) was therefore performed in a separate group of wild-type mice with striatal 6-OHDA lesions, followed by 35 days of treatment with 0.3 mg / kg pridopidine or saline. Pridopidine significantly upregulated GDNF and BDNF levels in the striatum (+37.6% and +80.8% relative to saline, respectively, for both markers). p <0.05, FIG. 6A , FIG. 6C In SN, only BDNF levels showed a significant increase (+88.5% relative to saline). FIG. 6D , p <0.05. Treatment with 0.3 mg / kg pridopidine also increased the levels of phosphorylated ERK1 / 2 in the striatum and substantia nigra. FIG. 6E , FIG. 6F The treatment was significant only in the striatum (+71% relative to saline). p < 0.05, FIG. 6E ).

[0170] The efficacy spectrum of a lower drug dose (0.3 mg / kg) includes both protection of DA cell bodies in the substantia nigra and restoration of dopaminergic fiber density in the lateral striatum. In this region, the TH-positive fiber network exhibits the typical morphological features of regenerating axons. With these morphological observations, the gradual recovery of forelimb use (which only became significant after 4 weeks of pridopidine treatment) suggests that stimulation of DA axonal budding is a key mechanism for functional recovery. This interpretation is further supported by the significant upregulation of GDNF, BDNF, and phosphorylated ERK1 / 2 in the striatum by 0.3 mg / kg pridopidine. These factors strongly stimulate neurogenesis from DA neurons in the substantia nigra striatum both in vitro and in vivo.

[0171] Higher doses of pridopidine (1 mg / kg) were generally ineffective for most behavioral and neurohistochemical endpoints. The neuroreparative effect of 0.3 mg / kg pridopidine, assessed in the S1R, was determined to be contingent on definitive evidence in the S1R.

[0172] Example 5: Pharmacokinetics of Pridolpicin in Mice Pridopidine pharmacokinetics in mice In the first pharmacokinetic (PK) study, pridopidine was administered orally (po, the route of administration for human use). In this study, male C57Bl6 mice were administered four doses of pridopidine (0.3 mg / kg, 3 mg / kg, 10 mg / kg, and 30 mg / kg per dose) via oral force-feed. N = 24), lasting for 7 days. Blood and brain samples were collected from 3 different mice at each time point in each dose group at the following time points: before administration (t = -10 min) and 15 min, 30 min, 1 hour, 2 hours, 4 hours, 6 hours, and 8 hours after administration. A second study was conducted to compare the drug pharmacokinetic profile between oral (po) and subcutaneous (sc) administration. Here, pharmacokinetics were examined after 7 daily oral or subcutaneous administrations (N = 24 mice per route of administration) of 30 mg / kg pridocipidine (HCl salt equivalent in water). Blood and brain samples were collected from 3 animals at each time point per route of administration at the following time points: before administration, 15 min, 30 min, 1 hour, 2 hours, 4 hours, 6 hours, and 8 hours after the last administration.

[0173] Plasma samples were placed in K3-EDTA tubes (Becton-Dickinson, Mississauga, ON, Canada) and centrifuged at 1500 g for 5 minutes. Brain tissue was collected and frozen without further processing. Prior to analysis, pre-weighed brain samples were diluted and homogenized with 0.01 M phosphate buffer (pH 7.4) containing 0.32 M sucrose at a ratio of 1 g of 330 brain samples to 4 mL of homogenate solution.

[0174] Quantification of pridopidine plasma and brain concentrations and pharmacokinetic analysis Pridopidine and its internal standard ACR354 (4-(3-methanesulfonyl)phenyl)-1-(propyl7)-piperidinium chloride) were extracted from EDTA plasma and mouse brain homogenate (1:3 in phosphate-buffered saline, pH 7.4) into acetonitrile using liquid-liquid extraction. Briefly, LC-MS / MS analysis was performed using a Shimadzu LC-20AD pump equipped with a Shimadzu SIL-20AC autosampler and a Shimadzu CTO-20AC column oven. The MS / MS system was an MDS Sciex API-5000 mass spectrometer with an electrospray ionization probe (Toronto, Canada).

[0175] Chromatographic separation of the analyte was achieved on a Phenomenex Synergi 2.5 μm Hydro-RP column. Linearity in plasma ranged from 2 ng / mL to 3000 ng / mL, with a lower limit of quantitation (LLOQ) of 2 ng / mL. Linearity in brain homogenate ranged from 2 ng / mL to 3000 ng / mL at the 0.3-mg / kg and 3-mg / kg dose groups, with an LLOQ of 2 ng / mL. For higher doses, linearity in plasma ranged from 25 ng / mL to 20,000 ng / mL, and linearity in brain homogenate ranged from 75 ng / mL to 60,000 ng / mL, with LLOQs of 2 ng / mL and 75 ng / mL, respectively. For all calibration curves, the deviation of pridocipidine concentration from the nominal concentration (accuracy value) of the quality control (QC) sample was less than 15%. PK parameters were calculated using group-mean concentration-time data based on nominal time (i.e., within ±5% of the predetermined time point) and non-compartmental modeling of extravascular administration using WinNonlin 6.3. Pridopidine exposure after a 1 mg / kg dose was estimated using dose-normalized exposure (see Table 1).

[0176] Table 1: Plasma and brain exposures following administration of pridopidine to C57Bl6 mice. The concentrations of pridopidine in plasma and brain are shown after administration of escalating doses (0.3 mg / kg, 1 mg / kg, 3 mg / kg, 10 mg / kg, and 30 mg / kg). Maximum Q3 concentration (Cmax) is expressed in nanograms per milliliter and micromoles. AUC (area under the curve, corresponding to the amount of pridopidine reaching systemic circulation after a specific dose) is expressed in nanograms per hour per milliliter and picometers per hour. Each data point represents the mean obtained from 3 mice. Parameters for 1-mg / kg doses are inferred based on the mean Cmax / dose and AUC0-last / dose value in the 0.3 mg / kg, 3 mg / kg, 10 mg / kg, and 30 mg / kg groups.

[0177]

[0178] 1) Estimated value Following single oral and subcutaneous administration, pridopidine exhibited similar plasma and cerebral PK, with a relative oral / subcutaneous exposure ratio of 117% for Cmax in plasma and an AUC of [missing value]. 0-t The Cmax in brain tissue was 90%, while the AUC was 88%. 0-t The figure was 96%. Therefore, the pridopidine exposure data following oral administration fairly represent the exposure achieved via subcutaneous administration. Dose-response PK studies following oral administration showed that plasma and brain exposure to the drug were dose-proportional (Table 1). Measurements of the brain-to-plasma ratio indicated that, for a dose of 0.3 mg / kg, the concentration of pridopidine in the brain was four to five times higher than that in plasma, and for a dose range of 1 mg / kg to 30 mg / kg, it was two to three times higher (see Cmax and AUC values ​​for brain relative to plasma, Table 1).

[0179] Administration of 0.3 mg / kg and 1 mg / kg pridopidine produced brain Cmax values ​​of 110 ng / mL and 331 ng / mL (corresponding to 390 nM and 1200 nM), respectively, which occurred at the median Tmax at 0.25 hours and the corresponding AUC values ​​were... 0-last The values ​​were 120 ng / h / mL and 392 ng / h / mL (corresponding to 0.43 and 1.4 h / µm, respectively). Pridopidine was rapidly cleared after 3 hours, with total brain levels approaching 3 ng / mL (corresponding to 11 nM, of which only about 2 nM was free). The free fraction of pridopidine was obtained using commercially available fractions. In vitroThe protein binding assay was used to determine the concentration. At doses of 0.3 and 1 mg / kg, the estimated unbound (free) fraction of pridopidine in brain tissue was approximately 50%. Therefore, for doses of 0.3 mg / kg and 1 mg / kg, the estimated concentrations of free pridopidine in the brain at Cmax correspond to 55 ng / mL and 165.5 ng / mL (or 195 nM and 600 nM), respectively. Based on the reported binding affinity of pridopidine to rat S1R (Ki 69.7 nM, [Sahlholm K, Armem P, Fuxe K, Marcelino D. The dopamine stabilizers ACR16 and (-)-OSU6162 display nanomolar affinities at 42. the sigma-1 receptor. Molecular Psychiatry. 2013; 18(1):12-4.]), it seems reasonable that both doses of pridopidine, 0.3 mg / kg and 1 mg / kg, would produce 100% S1R occupancy at Cmax. This concentration suggests that at this dose, selective binding to the sigma-1 receptor is present only.

[0180] Example 6: Decreased GCase activity in GBA-related PDs GBA encodes the lysosomal hydrolase glucocerebrosidase (GCase). GCase activity was measured in human control fibroblasts, fibroblasts from PD patients with heterozygous GBA mutations (N370S / wt and L444P / wt), and fibroblasts from idiopathic (sporadic) PD patients (iPD). GCase activity was significantly reduced by 32% and 35% in N370S / wt and L444P / wt fibroblasts, respectively. No loss of GCase activity was observed in iPD fibroblasts, suggesting that PD associated with GBA deficiency involves a different mechanism of action (Sanchez-Martinez et al., 2016).

[0181] Example 7: ER Stress Accumulation of misfolded proteins leads to ER stress in mutant GBA dopaminergic neurons The accumulation of misfolded proteins in the ER can overload the cellular capacity for protein refolding and induce ER stress. In Fernandes et al., dopaminergic neurons were differentiated from human induced pluripotent stem cells (iPSCs) from healthy controls and PD patients with the GBA-N370S mutation. Compared with control-derived dopaminergic cultures, the levels of two ER resident chaperone proteins (Bip / GRP78 and calreticulin) were significantly upregulated in GBA-N370S dopaminergic neuron cultures, indicating increased ER stress (Fernandes et al. 2016).

[0182] Pridopidine reduces mHtt-induced ER stress STHdhQ7 / 7 cells transfected with the mutant Htt96Q-mCherry (extended) construct exhibited visible Htt96Q-mCherry aggregates (typically one large aggregate per cell) along with high levels of accumulated H2a-GFP indicating ER stress. STHdhQ7 / 7 cells expressing Htt20Q-mCherry (normal) or Htt96Q-mCherry without visible aggregates showed low levels of H2a-GFP (no ER stress). Pridolpicin significantly reduced H2a-GFP accumulation in mHtt aggregate-positive cells in a dose-dependent manner and did not alter H2a-GFP levels in cells without aggregates or in cells expressing Htt20Q-mCherry. FIG. 7 Therefore, pridopidine reduces Htt-induced ER stress in a dose-dependent manner.

[0183] The effect of pridopidine on ER stress was further investigated by assessing the phosphorylation level of the translation initiation factor eIF2α. eIF2α phosphorylation is a marker of ER stress response. STHdhQ7 / 7 cells expressing Htt96Q-mCherry showed a 3.5-fold increase in eIF2α-phosphorylation (eIF2α-p). Pridopidine eIF2α-p (the eIF2α-p / eIF2α ratio) was significantly decreased, indicating reduced cellular ER stress, but higher concentrations did not reduce it. FIG. 8 Effective concentrations of 0.03–3 μM are associated with human doses of 45 mg BID and below (Cmax is approximately 2 μM).

[0184] Example 8: Mitochondrial dysfunction Mitochondrial function is impaired in mutant GBA neurons Mitochondrial function was assessed in mutant GBA and WT neurons by measuring mitochondrial membrane potential (MMP, ΔΨm) using MitoTracker Red (MitoRed). Total mitochondrial content was determined using MitoTracker Green (MitoGreen). Compared with WT controls, mutant GBA neurons exhibited lower baseline MMP but increased total mitochondrial content. The ratio of MitoRed to MitoGreen was significantly lower in mutant GBA neurons compared with controls, supporting impaired mitochondrial function (Li et al., 2019).

[0185] MitoSOX red (a mitochondrial indicator of ROS) was also used to assess mitochondrial ROS levels in live mutant GBA neurons. Compared with controls, mutant GBA neurons showed increased MitoSOX red fluorescence, indicating the production of higher levels of superoxide (Li et al., 2019).

[0186] Furthermore, mitochondrial transport is impaired due to GBA deficiency, which may result from increased levels of α-synuclein and its aggregates. Mitochondrial movement was analyzed in hippocampal neurons genetically introduced with exogenous wild-type (WT) or mutant (A35T) α-synuclein. Overexpression of WT or A53T α-synuclein significantly reduced the mean velocity of mitochondria along neuronal neurites (Xie and Chung, 2012).

[0187] Due to GBA defects, the clearance of abnormal or nonfunctional mitochondria (also known as mitophagy) is disrupted. Mitophagy was induced in mutant GBAs using CCCP (carbonyl cyanide-m-chlorophenylhydrazine, an uncoupling agent for mitochondrial oxidative phosphorylation), and assessed by measuring the colocalization of MitoGreen-labeled mitochondria with LysoTracker Red-labeled lysosomes. Compared to WT, mutant GBA neurons exhibited less colocalization of mitochondria and lysosomes, indicating impaired mitophagy (Li et al., 2019).

[0188] Pridopidine reduces mitochondrial ROS production HD mouse neurons exhibited increased sensitivity to oxidative challenges, leading to increased ROS levels and inadequate antioxidant response. Prior to ROS generation induction, striatal neurons from YAC128 HD mice were treated with 1 μM pridopidine (associated with human exposure at <= 45 mg BID). Pridopidine showed a robust and significant reduction in mitochondrial-generated ROS. FIG. 10 ).

[0189] Pridolpicine increases mitochondrial velocities. Assessment of mitochondrial transport in axons of wild-type motor neurons with and without topical pridolpicine treatment revealed that pridolpicine accelerates mitochondrial transport in axons. An effective concentration of 0.1 μM was associated with exposure at human doses < 45 mg bid (Ionescu et al., 2019). FIG. 11 ).

[0190] Deletion of the S1R gene leads to impaired mitophagy. In wild-type (WT) retinal explants, both mitochondrial proteins VDAC1 and TIMM23 are significantly reduced in similar proportions during mitophagy. However, in S1R- / - retinal explants, these two markers are not reduced but rather increase 2-3 times after induction of mitophagy, indicating impaired mitochondrial clearance in S1R- / - cells compared to WT (Yang et al., 2019). Therefore, S1R activity positively regulated by pridopidine is essential for mitophagy.

[0191] Example 9: Autophagy Defect In SH-SY5Y cells with GBA deleted from the siRNA gene, autophagy flux was reduced. Autophagy flux was measured by LC3B-II levels. High levels compared to controls indicated impaired autophagosome clearance in GBA-deficient cells (Magalhaes et al., 2016).

[0192] Autophagy reduction leads to alpha-synuclein aggregation in primary rat cortical neurons The accumulation and aggregation of α-synuclein plays a crucial role in the pathogenesis of Parkinson's disease (PD). A significant increase in α-synuclein levels was observed in insoluble fractions in rat cortical cells, indicating its accumulation in aggregates, where autophagy is reduced by the application of phospholipase D inhibitors. α-synuclein aggregation is a hallmark of PD and PD-GBA and is directly associated with neuronal cell death (Bae et al., 2014).

[0193] Activation of S1 R enhances autophagy activity Assess autophagy flux in HeLa cells treated with S1R agonists. S1R activation significantly induced autophagy flux compared to control conditions (Christ et al., 2019).

[0194] Example 10: BDNF Decreased plasma BDNF levels in Gaucher disease (GD, GBA mutations) Brain-derived neurotrophic factor (BDNF) is important for the development and survival of the nervous system and is also key to synaptic plasticity. Decreased BDNF levels are associated with neurodegenerative diseases such as Parkinson's disease (PD) and Alzheimer's disease (AD). Plasma BDNF levels are also significantly reduced in patients with Graves' disease (GD) (Vairo et al., 2015).

[0195] Pridopidine enhances BDNF secretion and axonal transport in S1 R-mediated mechanisms Compared to untreated control cells, 100 nM and 1 μM of pridopidine enhanced BDNF secretion in the B104 neuroblastoma cell line. BDNF secretion was inhibited when these cells were co-incubated with the known S1R antagonist NE100 (Geva et al., 2016). FIG. 12 The effective concentration of 1 μM is associated with a human dose of ≤ 45 mg bid.

[0196] Pridopidine rescues BDNF axonal transport in ALS neurons in S1 R-mediated mechanisms BDNF axonal transport velocity was significantly reduced in ALS SOD1G93A motor neurons. Treatment with cultures containing 0.1 μM and 1 μM pridopidine significantly accelerated the velocity in SOD1G93A motor neurons in a dose-dependent manner. 1 μM pridopidine was associated with human doses <= 45 mg bid. This effect was eliminated in motor neurons with S1R gene deletion, suggesting an S1R-dependent mechanism (Ionescu et al., 2019). FIG. 13 ).

Claims

1. Use of the pharmaceutical composition in the preparation of a medicament for the prevention, treatment or slowing of the progression of Parkinson's disease, wherein the Parkinson's disease is Parkinson's disease associated with glucocerebrosidase mutation (PD-GBA); wherein the pharmaceutical composition comprises pridopidine or a pharmaceutically acceptable salt thereof, wherein the dose of pridopidine or a pharmaceutically acceptable salt thereof is between 1 mg and 100 mg per day.

2. The use according to claim 1, wherein the drug is used to prevent, treat, slow down or reverse neurodegenerative changes in a subject.

3. The use according to claim 2, wherein the neurodegenerative changes in the subject are manifested as increased ER stress, neuronal mitochondrial dysfunction, decreased autophagy flux, decreased plasma BDNF levels, decreased cerebrospinal fluid (CSF) BDNF levels, and reduced neuronal and BDNF axonal transport.

4. The use according to any one of claims 1 to 3, wherein the drug is applied at least twice daily.

5. The use according to claim 4, wherein the drug is administered twice daily.

6. The use according to claim 4, wherein the drug is administered three times daily.

7. The use according to claim 4, wherein the drug is applied four times daily.

8. The use according to any one of claims 1 to 7, wherein the total dose of said pridopidine or a pharmaceutically acceptable salt thereof is 10 mg to 100 mg per day.

9. The use according to claim 8, wherein the total dose of said pridopidine or a pharmaceutically acceptable salt thereof is 50 mg to 100 mg per day.

10. The use according to claim 9, wherein the total dose of said pridopidine or a pharmaceutically acceptable salt thereof is 80 mg to 100 mg per day.

Images