Use of Pridopidine or Analogues Thereof for Treating Rett Syndrome

Abstract

The present invention provides a method for treating a subject suffering from Rett syndrome (RTT), comprising the step of administering to the subject a composition comprising pridopidine or a pharmaceutically acceptable salt thereof and at least one of compounds 1 to 8 or a pharmaceutically acceptable salt thereof, thereby treating the subject.

Description

[Technical Field]

[0001] (Sequence Listing Statement) A 2,997 byte ASCII file entitled "P-609324-PC-SQL-15SEP17.txt" created on September 15, 2017, filed concurrently with this application, is incorporated herein by reference. [Background technology]

[0002] Rett syndrome

[0003] Rett syndrome (RTT) is a neurodevelopmental disorder estimated to occur in 1 in 10,000–15,000 live births among girls of all races and ethnicities (Amaral 2007).

[0004] In 95–97% of cases, RTT is caused by mutations in the methyl-CpG-binding protein 2 (MeCP2) gene, located on the X chromosome (Isaias 2014). Mutations are usually random and spontaneous. In less than 1% of documented cases, the mutations are inherited and passed on to subsequent generations. The MeCP2 gene is responsible for producing the methylcystine-binding protein 2 (MeCP2) protein. The MeCP2 protein binds to methylcytosine and 5-hydroxymethylcytosine at CpG sites within the promoter regions of target genes and regulates their transcription by recruiting corepressors and coactivators (Pozzo-Miller 2015).

[0005] Rarely, RTT is also caused by partial gene deletions or mutations in other genes, such as cyclin-dependent kinase-like 5 (CDKL5), forkhead box protein G1 (FOXG1), and as yet unidentified genes.

[0006] RTT is an early-onset neurodevelopmental autism spectrum disorder that begins in infancy and is divided into four stages. In the first stage, which occurs between 6 and 18 months of age, developmental maturation stagnates after seemingly normal development. In the second stage, between 1 and 4 years of age, a regression phase begins, during which acquired skills are lost. During this stage, purposeful hand movements are replaced by stereotyped behaviors, such as hand twisting and hand clapping. Social withdrawal also begins, leading to a diagnosis of autism. During this stage, gait disorders, including ataxia and apraxia, become evident. Other symptoms include respiratory arrhythmias, sleep disorders, teeth grinding, and inappropriate bouts of laughing or crying.

[0007] In the third stage, between the ages of two and ten, deterioration plateaus. Motor function stabilizes and social interaction may improve, although seizures may occur. In the fourth stage, from age ten through adulthood, motor function declines further. Patients often develop parkinsonism (rigidity, bradykinesia, and tremor), muscle weakness, and osteoporosis (Sandweiss 2020).

[0008] Patients with RTT exhibit neuromorphological abnormalities and reduced brain size, and develop impaired coordination, intellectual decline, gait abnormalities, and seizures (Weng 2011).

[0009] Currently, there is no cure for RTT, and only supportive care is available.

[0010] Pridopidine

[0011] Pridopidine (4-[3-(methylsulfonyl)phenyl]-1-propyl-piperidine) (formerly ACR16) is a drug under development for the treatment of Huntington's disease. Pridopidine's chemical name is 4-(3-(methylsulfonyl)phenyl)-1-propyl-piperidine, and its chemical registration number is CAS 346688-38-8 (CSID: 7971505, 2016). Pridopidine hydrochloride's chemical registration number is 882737-42-0 (CSID: 25948790, 2016).

[0012] Pridopidine selectively binds to the sigma 1 receptor (S1R) with high affinity (Ki = 0.057 μM), and binds with low affinity to other receptors in the central nervous system, including dopamine D2 / D3 receptors, adrenergic α2C receptors, and serotonin 5-HT1A receptors. Pridopidine is approximately 28-fold more selective for sigma 1 receptors (S1R) than for α2C and dopamine D3 receptors (Ki = 1.28 and Ki = 1.63 μM, respectively), 64-fold more selective for 5-HT1A receptors (Ki = 3.63 μM), 100-fold more selective for sigma 2 receptors (S2R) (Ki = 5.45 μM), and 500-fold more selective for dopamine D2 receptors (Ki = 29.5 μM) (Table 1) (Johnston et al., 2019).

[0013] [Table 1]

[0014] S1R is an endoplasmic reticulum (ER) protein involved in cell differentiation, neuroplasticity, neuroprotection, and cognitive function in the brain. Activation of S1R by pridopidine leads to the upregulation of pathways known to promote neuronal plasticity and survival.

[0015] Pridopidine upregulates the secretion and downstream signaling of brain-derived neurotrophic factor (BDNF), which has neuroprotective effects ( Geva et al., 2016 ).

[0016] Decreased BDNF is associated with the pathogenesis of Rett syndrome. Homeostatic synaptic plasticity (HSP), a process that maintains the stability of neuronal networks and underlies learning and cognitive abilities, is regulated by BDNF (Smith-Dijak et al., 2019). HSP is also impaired in Rett syndrome. Mecp2-deficient neurons exhibit impaired homeostatic synaptic plasticity (Xin xu and Pozzo-Miller, J Physiolo 2017). Pridopidine restores impaired HSP in cultured cortical neurons of the HDYAC128 mouse model (Smith-Dijak et al., 2019).

[0017] Regulation of the BDNF pathway is a major component of the S1R-mediated neuroprotective effects of pridopidine. Summary of the Invention [Means for solving the problem]

[0018] The present invention provides a method for treating a subject suffering from Rett syndrome (RTT), comprising the step of administering to the subject a composition comprising pridopidine or a pharmaceutically acceptable salt thereof and at least one of the following compounds 1 to 8 or a pharmaceutically acceptable salt thereof, thereby treating the subject.The present invention provides a method for increasing the concentration of brain-derived neurotrophic factor (BDNF) in a subject suffering from Rett syndrome (RTT), comprising the step of administering to the subject a composition comprising pridopidine or a pharmaceutically acceptable salt thereof and at least one of the following compounds 1 to 8 or a pharmaceutically acceptable salt thereof, thereby increasing the concentration of serum BDNF.

[0019] [ka] [Brief explanation of the drawings]

[0020] [Figure 1]Figure 1: Functional recovery rate as a measure of therapeutic drug efficacy in Mecp2-KO Rett syndrome model mice. This figure explains the analysis method for the data in Figures 4, 5, and 12. Left: "Cloud graph": Visualization of the relationship between the control group, disease group, and (disease + treatment) group in the optimal discriminant feature space. The cloud is plotted in two-dimensional space, and the two coordinates are the highest-ranked uncorrelated features analyzed by the automated NeuroCube system (details of NeuroCube are described in Examples 1 and 3). The drug administration effect can be expressed as a combination of two components: a component along the direction of the "recovery line" (the line connecting the center of the control (WT) and disease (MECP2-KO) clouds, indicated by a thick left-pointing arrow with a diagonal line drawn above it) and a component perpendicular to that direction (extending upward from the origin of the recovery line) (indicated by an upward-pointing arrow with a parallel line drawn within it). The relative length of the "recovery" arrow relative to the control-disease distance can be interpreted as "drug-induced recovery," while the relative length of the "other effects" arrow (upward-pointing arrow with a parallel line through it) represents the characteristic changes that separate the diseased + treated group from the control group. A summary of this analysis can be effectively represented as a bar graph (right panel of Figure 1), commonly referred to as the recovery signature. Right: "Recovery Signature" graph: This bar graph represents a summary of the recovery analysis. The sum of overlap and discrimination probability equals 100%. Recovery rates range from 0 to the discrimination probability value. The smaller the overlap, the better the quality of the disease model and the greater the discrimination power between WT and Rett mice. "Other behavioral effects" have the same relative units (relative to the length of the distance between WT and Rett mice). [Figure 2]Figure 2: Pridopidine rescues the hindlimb clasping phenotype in 8-week-old Rett model mice. Muscle strength in the limbs was assessed using clasping. Hindlimb clasping was measured by grasping the mouse's tail and gently lifting it until its front legs were just off the counter surface. Experimenters then observed the mice's legs for clasping or splaying. Wild-type (WT, n = 24) mice did not exhibit clasping (0%) at 8 weeks of age. Rett mice (female MECP2 heterozygotes, "RETT" mice, n = 20) exhibited significant clasping (approximately 17%) at 8 weeks of age (p < 0.05 vs. placebo). Rett mice ("RETT") treated with pridopidine (30 mg / kg, bid, n=20) were rescued at week 8 (i.e., showed no clasping (0%)), p<0.06 compared to the RETT-vehicle group. Data are mean ± SEM, N-1 two-group comparison test. Source: DPR-2016-061. [Figure 3] Figure 3: Pridopidine improves the mean startle response to acoustic stimuli in 8- and 12-week-old RETT mice. The acoustic startle assay was used to measure unconditioned reflex responses to external auditory stimuli. Prepulse inhibition (PPI) consists of the suppression of the startle response to an auditory stimulus after the presentation of a weak auditory stimulus, or prepulse. Acoustic startle is measured by placing a mouse in a sound-attenuating startle chamber and measuring the strength of the mouse's movements. The amount of suppression after the acoustic prepulse is expressed as a percentage of the baseline startle response (relative to the startle-alone test), excluding the startle response from the first habituation block. Vehicle-treated RETT mice (n = 20) showed significant suppression of the startle response by approximately 65% ​​and 75% at 8 and 12 weeks, respectively, compared to WT mice (n = 24) (p < 0.05). Pridopidine showed significant rescue effects of approximately 40% and 50% at weeks 8 and 12, respectively (n=24, p<0.05). Data are expressed as mean ± SEM. Compared with the WT-vehicle group, #p<0.05. Compared with the Rett-vehicle group, *p<0.05. [Figure 4]Figure 4: Pridopidine restores gait function in 8-week-old female Rett model mice. Summary of analysis of the recovery of gait function in 8-week-old female Rett model mice by pridopidine (30 mg / kg, administered twice daily). Untreated Rett mice (bottom right cloud) and WT mice (upper cloud) can be distinguished from each other by their gait function, as their clouds are completely separated. The bar graph shows that administration of 30 mg / kg pridopidine twice daily significantly improves gait function in 8-week-old Rett mice by 45% (p=0.0181) (darkest color). The cloud graph is used to visualize the relationship between WT (upper cloud), Rett female mice (lower right cloud), and Rett female mice + pridopidine (left cloud) in the optimal discriminant feature space (see legend for Figure 1). [Figure 5] Figure 5: Pridopidine restores locomotor function in 12-week-old female Rett model mice. Summary of analysis of locomotor function recovery in 12-week-old female Rett mice treated with pridopidine (30 mg / kg, administered twice daily). Untreated Rett mice (lower left cloud) and WT mice (upper cloud) can be distinguished from each other by their locomotor function, as their clouds are completely separated. Pridopidine (30 mg / kg, administered twice daily) significantly restored locomotor function in 12-week-old Rett mice by 55% (p = 0.0022) (darkest color). Cloud graphs are used to visualize the relationship between WT (upper cloud), Rett mice (lower left cloud), and Rett + pridopidine (right cloud) in the optimal discriminant feature space (see legend for Figure 1). [Figure 6A]Figures 6A-6C, 7, 8, 9, and 10 show the mRNA levels of BDNF transcripts measured in the brains of heterozygous MeCP2(Rett) female mice. Column A represents vehicle-treated WT mice; column B represents vehicle-treated Rett female mice; column C represents pridopidine-treated Rett female mice (3 mg / kg, twice daily); and column D represents pridopidine-treated Rett female mice (30 mg / kg, twice daily). The relative abundance of the target genes was normalized to the geometric mean of the relative abundance of the housekeeping genes APT5B, GAPDH, and RPK12A. The relative abundance of the target genes was then normalized to the WT vehicle group. All data are presented as mean ± SEM. ANOVA followed by Tukey's multiple comparison test was performed. Figure 6A: Pridopidine does not affect the levels of the housekeeping genes ATP5B, GAPDH, and RPK13A. Relative mRNA expression levels of whole-brain control housekeeping genes: ATP5B (Figure 6A), GAPDH (Figure 6B), and RPL13A (Figure 6C); each normalized to the geometric mean of the other two genes. Pridopidine does not affect the expression of control housekeeping genes. [Figure 6B] Figure 6B: Pridopidine does not affect the levels of the housekeeping genes ATP5B, GAPDH, and RPL13A. Relative mRNA expression levels of whole-brain control housekeeping genes: ATP5B (Figure 6A), GAPDH (Figure 6B), and RPL13A (Figure 6C); each normalized to the geometric mean of the other two genes. Pridopidine does not affect the expression of the control housekeeping genes. [Figure 6C] Figure 6C: Pridopidine does not affect the levels of the housekeeping genes ATP5B, GAPDH, and RPL13A. Relative mRNA expression levels of whole-brain control housekeeping genes: ATP5B (Figure 6A), GAPDH (Figure 6B), and RPL13A (Figure 6C); each normalized to the geometric mean of the other two genes. Pridopidine does not affect the expression of the control housekeeping genes. [Figure 7]Figure 7: Pridopidine does not affect BDNF-I mRNA levels. Relative BDNF-I mRNA expression in the whole brain. In Rett mice, BDNF-I levels are reduced by approximately 20% (p<0.001). Pridopidine does not affect BDNF-I levels in Rett mice. [Figure 8] Figure 8: Pridopidine rescues BDNF-IV mRNA levels. Relative BDNF-IV mRNA expression in the whole brain. In Rett mice, BDNF-IV levels are reduced by approximately 15% (p<0.001). Pridopidine significantly increases BDNF-IV mRNA levels by approximately 30% at doses of 3 mg / kg bid and 30 mg / kg bid (p<0.001). [Figure 9] Figure 9: Pridopidine does not affect BDNF-VI mRNA levels. Relative BDNF-VI mRNA expression in the whole brain. In Rett mice, BDNF-VI levels are reduced by approximately 25% (p<0.05). Pridopidine does not affect BDNF-VI levels in Rett mice. [Figure 10] Figure 10: Pridopidine rescues BDNF-IV mRNA levels. Relative BDNF-IV mRNA expression in the whole brain. In Rett mice, BDNF-IV levels are reduced by approximately 15% (p<0.0001). Pridopidine significantly increases BDNF-IV mRNA levels to WT levels at doses of 3 mg / kg bid and 30 mg / kg bid (p<0.001). [Figure 11A]Figures 11A and 11B: Feature discrimination in gait analysis using NeuroCube. Male B6.129P2-Mecp2tm2Bird / J (Rett-KO) mice were used in this study. This figure shows a discrimination plot, an output from gait analysis using NeuroCube, an automated tool for evaluating the effects of pridopidine. The columns represent the various behavioral features and their values ​​evaluated by NeuroCube. The curves with rectangular outlines indicate the ranking of each behavioral feature. The rankings relate to the ability of a particular feature to discriminate between the control and disease groups. The relative difference (%) between feature values ​​in the two different sets was calculated and plotted in an order corresponding to the feature rank, ranging from 0 to 100%. Feature names are a combination of the parameter name and the paw name. STRL: stride length, STPL: step length, BSWD: base width, STRD: stride time, STND: stand time, SWGD: swing time, Avg_speed: average running speed, FR: forelimb (right), FL: forelimb (left), HR: hindlimb (right), HL: hindlimb (left). Features are arranged from top to bottom in the figure based on their rank. Details of the NeuroCube are described in Examples 1 and 3. Figure 11A shows the discrimination plot of Rett-KO male mice compared to WT mice at 6 weeks of age. [Figure 11B] FIG. 11B is a discrimination plot of Rett-KO male mice compared to WT mice at 7 weeks of age. [Figure 12]Figure 12: Pridopidine restores gait function in 6- and 7-week-old male Rett mice. Summary of the analysis of recovery from the effects of Rett syndrome in B6.129P2-Mecp2tm2Bird / J (Rett-KO) male mice (see legend to Figure 1). Top: Bar graph showing the recovery effect of pridopidine (30 mg / kg, administered twice daily) in Rett-KO model mice. Bottom: Cloud graph visualizing the relationship between WT, Rett, and Rett + pridopidine in the optimal discriminant feature space. Pridopidine restores 44% (left, p<0.05) and 100% (right, p<0.05) of gait impairment in 6- and 7-week-old Rett model mice, respectively. One-way ANOVA. [Figure 13A] Figures 13A-B: Pridopidine 45 mg twice daily improved gait and balance in early HD (TFC 7-13) patients at weeks 26 and 52. Figure 13A shows the change from baseline in UHDRS TMS gait and balance at week 26 in early HD patients (TFC 7-13 at baseline) from the PRIDE-HD study. [Figure 13B]Figure 13B shows the change from baseline in UHDRS-TMS gait and balance at week 52 in early HD patients (TFC 7-13 at baseline) in the PRIDE-HD study. Placebo-treated patients experienced a worsening of gait and balance (Δ from baseline = 0.14, positive values ​​indicate worsening). The efficacy of pridopidine was assessed using a mixed-model repeated measures (MMRM) analysis of the change from baseline in Unified Huntington's Disease Rating Scale Total Motor Score gait and balance (UHDRS-TMS) over the 52-week period. Pridopidine 45 mg twice daily improved gait function compared with placebo at week 26 (Δ from placebo = -0.48, negative values ​​indicate improvement; p = 0.0563). Table 2, below, accompanies this figure. Pridopidine 45 mg twice daily showed a trend toward improvement compared with placebo at week 52 (delta from placebo was -0.41, negative values ​​indicate improvement). [Table 2] [Figure 14A] Figures 14A and 14B: Pridopidine 45 mg twice daily improved gait and balance in HD1 (TFC11-13) patients at weeks 26 and 52. Figure 14A shows the change from baseline in UHDRS-TMS gait and balance at week 26 in HD1 patients (baseline TFC11-13) from the PRIDE-HD study. [Figure 14B]Figure 14B shows the change from baseline in UHDRS-TMS gait and balance at week 52 in HD1 patients (baseline TFC 11-13) from the PRIDE-HD trial. The efficacy of pridopidine was assessed using a Mixed Models Repeated Measures (MMRM) analysis of the change from baseline in Unified Huntington's Disease Rating Scale Total Motor Score gait and balance (UHDRS-TMS; gait and balance) over the 52-week period. Patients receiving placebo experienced a worsening of gait and balance at week 26. Pridopidine treatment demonstrated a trend toward improvement (Δ from placebo: -0.31). Table 3 accompanies this figure. Patients in the placebo group experienced a worsening of gait and balance at week 52. Pridopidine 45 mg twice daily showed significant improvement compared with placebo (delta from placebo was -0.94, p=0.0445, negative values ​​indicate improvement). [Table 3] [Figure 15A] Figures 15A and 15B: Pridopidine 45 mg twice daily improved gait and balance in HD2 patients (baseline TFC 7-10) from the PRIDE-HD study at weeks 26 and 52. Figure 15A shows the change from baseline in UHDRS-TMS gait and balance at week 26 in HD2 patients (baseline TFC 7-10) from the PRIDE-HD study. [Figure 15B]Figure 15B shows the change from baseline in UHDRS-TMS gait and balance at week 52 in HD2 patients (baseline TFC 7-10) from the PRIDE-HD study. The efficacy of pridopidine was assessed using a Mixed Models Repeated Measures (MMRM) analysis of the change from baseline in Unified Huntington's Disease Rating Scale Total Motor Score gait and balance (UHDRS-TMS; gait and balance) over the 52-week period. At both weeks 26 and 52, pridopidine 45 mg twice daily demonstrated a trend toward improvement compared with placebo (Δ from placebo was −0.53 and −0.18 at weeks 26 and 52, respectively; negative values ​​indicate improvement). This figure is accompanied by Table 4 below. [Table 4] [Figure 16] Figure 16: Synergistic effect of pridopidine and Compound 4 on BDNF release in B104 rat neuroblastoma cells. B104 neuroblastoma cells were incubated with the test compounds for 5 days, and BDNF levels were assessed using an in-situ ELISA. Figure 16(A): Synergistic effect of pridopidine at a concentration of 0.001 μM combined with Compound 4 at a concentration of 0.001 μM. Pridopidine alone increased BDNF secretion by 13.5%. Compound 4 alone had no effect on BDNF secretion (-1.5%). Pridopidine combined with Compound 4 increased BDNF secretion by 59.1%, an effect greater than the sum of the effects of both compounds administered alone. Figure 16(B): Synergistic effect of pridopidine at a concentration of 0.005 μM combined with Compound 4 at a concentration of 0.001 μM. Pridopidine alone increased BDNF secretion by 26.0%. Compound 4 alone had no effect on BDNF secretion (-1.5%). The combination of pridopidine and compound 4 increased BDNF secretion by 80.7%, an effect greater than the sum of the effects of either compound administered alone. [Figure 17] Figure 17: Synergistic effect of pridopidine and Compound 1 on BDNF release in B104 cells. B104 neuroblastoma cells were cultured with test compounds for 5 days, and BDNF levels were assessed using an in-situ ELISA. 0.01 μM pridopidine alone increased BDNF secretion by 3.4%. 1 μM Compound 1 alone increased BDNF secretion by 12.5%. The combination of pridopidine and Compound 1 increased BDNF secretion by 53.1%, an effect greater than the sum of the effects of either compound administered alone. DETAILED DESCRIPTION OF THE INVENTION

[0021] The present invention provides a method for treating a subject suffering from Rett syndrome (RTT), comprising administering to the subject a pharmaceutical composition comprising pridopidine or a pharmaceutically acceptable salt thereof, thereby treating the subject.

[0022] The present invention provides a method for treating a subject suffering from Rett syndrome (RTT), the method comprising the step of administering to the subject a composition comprising pridopidine or a pharmaceutically acceptable salt thereof and at least one of compounds 1 to 8 below or a pharmaceutically acceptable salt thereof, thereby treating the subject.

[0023] [ka]

[0024] In one embodiment, the present invention provides a method for delaying the onset of, preventing the worsening of, delaying the worsening of, or ameliorating at least one symptom associated with Rett syndrome (RTT) in a subject suffering from Rett syndrome, the method comprising administering to the subject a composition comprising pridopidine, or a pharmaceutically acceptable salt thereof, and at least one of compounds 1-8, or a pharmaceutically acceptable salt thereof, as described above.

[0025] In one embodiment, the subject is a human patient. In one embodiment, the human patient is female. In another embodiment, the human patient is male.

[0026] In one embodiment, the subject has a mutation in the methyl-CpG binding protein 2 (MeCP2) gene. In one embodiment, the subject has a mutation in the cyclin-dependent kinase-like 5 (CDKL5) gene. In one embodiment, the subject has a mutation in the forkhead box protein G1 (FOXG1) gene.

[0027] In some embodiments, the method of the present invention comprises administering a pharmaceutical composition comprising pridopidine, or a pharmaceutically acceptable salt thereof, and at least one of Compounds 1-8, or a pharmaceutically acceptable salt thereof. In other embodiments, the pharmaceutically acceptable salt of pridopidine is hydrochloride. In another embodiment, the pharmaceutically acceptable salt of pridopidine is hydrochloride, hydrobromide, nitrate, perchlorate, phosphate, sulfate, formate, acetate, aconate, ascorbate, benzenesulfonate, benzoate, cinnamate, citrate, embonate, enantate, fumarate, glutamate, glycolate, lactate, maleate, malonate, mandelate, methanesulfonate, naphthalene-2-sulfonate, phthalate, salicylate, sorbate, stearate, succinate, tartrate, or toluene-p-sulfonate.

[0028] In some embodiments, the method of the present invention comprises administering a pharmaceutical composition comprising pridopidine, or a pharmaceutically acceptable salt thereof, and at least one of the above compounds 1-8, or a pharmaceutically acceptable salt thereof. In other embodiments, the pharmaceutically acceptable salt of compounds 1-8 is a hydrochloride salt. In another embodiment, the pharmaceutically acceptable salt of compounds 1-8 is a hydrochloride, hydrobromide, nitrate, perchlorate, phosphate, sulfate, formate, acetate, aconate, ascorbate, benzenesulfonate, benzoate, cinnamate, citrate, embonate, enanthate, fumarate, glutamate, glycolate, lactate, maleate, malonate, mandelate, methanesulfonate, naphthalene-2-sulfonate, phthalate, salicylate, sorbate, stearate, succinate, tartrate, or toluene-p-sulfonate.

[0029] In one embodiment, the pharmaceutical composition of the present disclosure is administered orally, nasally, by inhalation, subcutaneous injection, intravenously, intraperitoneally, intramuscularly, intranasally, bucally, intravaginally, rectally, intraocularly, intrathecally, topically, or intradermally. In one embodiment, the pharmaceutical composition of the present disclosure is administered orally.

[0030] In one embodiment, the pharmaceutical composition of the present disclosure is administered in the form of an aerosol, inhalable powder, injectable, liquid, gel, solid, capsule, or tablet.

[0031] In one embodiment, the pharmaceutical composition of the present disclosure is administered orally, and is formulated as a tablet, capsule, pill, powder, multiparticulates in a capsule or sachet, a liquid solution, or a liquid suspension.

[0032] In one embodiment, the pharmaceutical compositions of the present disclosure are administered periodically.

[0033] In one embodiment, the pharmaceutical composition of the present disclosure is administered less than once a day. In one embodiment, the pharmaceutical composition of the present disclosure is administered every day. In one embodiment, the pharmaceutical composition of the present disclosure is administered once a day. In another embodiment, the pharmaceutical composition of the present disclosure is administered more than once a day. In one embodiment, the pharmaceutical composition of the present disclosure is administered twice a day.

[0034] In some embodiments, the pharmaceutical compositions of the present disclosure, comprising pridopidine or a pharmaceutically acceptable salt thereof and at least one of compounds 1-8 or a pharmaceutically acceptable salt thereof, used in the methods of the present disclosure, are administered in a daily dose of 0.5-315 mg of pridopidine or a pharmaceutically acceptable salt thereof. In another embodiment, the pharmaceutical compositions of the present disclosure are administered in a daily dose of 0.5-10 mg of pridopidine or a pharmaceutically acceptable salt thereof. In another embodiment, the pharmaceutical compositions of the present disclosure are administered in a daily dose of 10-22.5 mg of pridopidine or a pharmaceutically acceptable salt thereof. In another embodiment, the pharmaceutical compositions of the present disclosure are administered in a daily dose of 22.5-315 mg of pridopidine or a pharmaceutically acceptable salt thereof. In another embodiment, the pharmaceutical compositions of the present disclosure are administered in a daily dose of 10-315 mg of pridopidine or a pharmaceutically acceptable salt thereof. In another embodiment, the pharmaceutical composition of the present disclosure is administered in a daily dose comprising 0.5 to 50 mg of pridopidine or a pharmaceutically acceptable salt thereof. In another embodiment, the pharmaceutical composition of the present disclosure is administered in a daily dose comprising 22.5 to 315 mg of pridopidine or a pharmaceutically acceptable salt thereof. In another embodiment, the pharmaceutical composition of the present disclosure is administered in a daily dose comprising 45 to 250 mg of pridopidine or a pharmaceutically acceptable salt thereof. In another embodiment, the pharmaceutical composition of the present disclosure is administered in a daily dose comprising 45 to 135 mg of pridopidine or a pharmaceutically acceptable salt thereof. In another embodiment, the pharmaceutical composition of the present disclosure is administered in a daily dose comprising 90 to 315 mg of pridopidine or a pharmaceutically acceptable salt thereof.

[0035] In another embodiment, the amount of pridopidine administered is about 1 mg / day, about 5 mg / day, about 10 mg / day, about 20 mg / day, about 22.5 mg / day, about 45 mg / day, about 67.5 mg / day, about 90 mg / day, about 100 mg / day, about 112.5 mg / day, about 125 mg / day, about 135 mg / day, about 150 mg / day, about 180 mg / day, about 200 mg / day, about 225 mg / day, about 250 mg / day, or about 315 mg / day. In one embodiment, the amount of pridopidine administered is 45 mg / day. In one embodiment, the amount of pridopidine administered is 90 mg / day. In one embodiment, the amount of pridopidine administered is 180 mg / day. In one embodiment, the amount of pridopidine administered is 225 mg / day.

[0036] In one embodiment, the pharmaceutical composition of the present disclosure is administered once daily. In one embodiment, the pharmaceutical composition of the present disclosure is administered twice daily.

[0037] In one embodiment, the pharmaceutical composition of the present disclosure is administered in a dose comprising pridopidine in an amount of about 1 mg, about 5 mg, about 10 mg, about 22.5 mg, about 45 mg, about 67.5 mg, about 90 mg, about 100 mg, about 112.5 mg, about 125 mg, about 135 mg, about 150 mg, about 180 mg, about 200 mg, about 250 mg, or about 315 mg. In one embodiment, the pharmaceutical composition of the present disclosure comprises pridopidine in an amount of 45 mg. In one embodiment, the pharmaceutical composition of the present disclosure comprises pridopidine in an amount of 10-45 mg.

[0038] In one embodiment, the pharmaceutical composition of the present disclosure is administered twice daily, and the pharmaceutical composition of the present disclosure comprises 45 mg of pridopidine per dose. In one embodiment, the pharmaceutical composition of the present disclosure is first administered as early as one day after the subject's birth. In one embodiment, the pharmaceutical composition of the present disclosure is first administered to the subject within one day of birth. In one embodiment, the pharmaceutical composition of the present disclosure is first administered to the subject within one week of birth. In one embodiment, the pharmaceutical composition of the present disclosure is first administered to the subject within one month of birth. In one embodiment, the pharmaceutical composition of the present disclosure is first administered to the subject within three months of birth. In one embodiment, the pharmaceutical composition of the present disclosure is first administered to the subject within six months of birth. In one embodiment, the pharmaceutical composition of the present disclosure is first administered to the subject within nine months of birth. In one embodiment, the pharmaceutical composition of the present disclosure is first administered to the subject within twelve months of birth. In one embodiment, the pharmaceutical composition of the present disclosure is first administered to the subject within eighteen months of birth. In one embodiment, the pharmaceutical composition of the present disclosure is first administered to the subject within three years of birth. In one embodiment, the pharmaceutical composition of the present disclosure is first administered to the subject within the first five years of life. In one embodiment, the pharmaceutical composition of the present disclosure is first administered to the subject within the first ten years of life. In one embodiment, the pharmaceutical composition of the present disclosure is first administered to the subject within the first fifteen years of life. In one embodiment, the pharmaceutical composition of the present disclosure is first administered to the subject within the first twenty years of life. In one embodiment, the pharmaceutical composition of the present disclosure is first administered to the subject within the first twenty-five years of life. In one embodiment, the pharmaceutical composition of the present disclosure is first administered to the subject within the first thirty years of life. In one embodiment, the pharmaceutical composition of the present disclosure is first administered to the subject more than thirty years of age.

[0039] In one embodiment, the regular administration of the pharmaceutical composition of the present disclosure continues 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 more.

[0040] In one embodiment, the pharmaceutical compositions of the present disclosure treat a subject by delaying the onset of symptoms in the subject.

[0041] In one embodiment, the pharmaceutical composition of the present disclosure treats a subject by delaying the onset, preventing the worsening, delaying the worsening, or ameliorating at least one symptom in a subject suffering from Rett syndrome (RTT). In one embodiment, the pharmaceutical composition of the present disclosure delays or ameliorate the worsening of at least one symptom in a subject suffering from RTT. In one embodiment, the pharmaceutical composition of the present disclosure treats a subject by ameliorating at least one symptom in a subject suffering from RTT.

[0042] In some embodiments, the present invention relates to a method for delaying the onset, preventing the worsening, delaying the worsening, or ameliorating at least one symptom of Rett syndrome (RTT) by administering a pharmaceutical composition comprising pridopidine, or a pharmaceutically acceptable salt thereof, and at least one of compounds 1-8, or a pharmaceutically acceptable salt thereof, including abnormal gait, ataxia, impaired gait initiation, delayed acquisition of purposeful manual motor function, partial or complete loss of acquired purposeful manual motor function, abnormal hand movements, startle response, delayed crawling and / or walking, impaired ability to crawl and / or walk, or eye movement abnormalities.

[0043] In one embodiment, the symptom is delayed acquisition of motor function. In one embodiment, the symptom is delayed sitting, crawling, and / or walking. In one embodiment, the symptom is partial or complete loss of acquired motor function. In one embodiment, the symptom is decreased ability to sit, crawl, and / or walk. In one embodiment, the motor function is coordination.

[0044] In one embodiment, the symptom is abnormal gait. In one embodiment, the symptom is ataxia. In one embodiment, the symptom is apraxia. In one embodiment, the symptom is muscle weakness. In one embodiment, the symptom is spasticity. In one embodiment, the symptom is rigidity. In one embodiment, the symptom is impaired gait initiation.

[0045] In one embodiment, the symptom is dystonosis. In one embodiment, the symptom is hypotonia. In one embodiment, the symptom is peripheral vasomotor disorder. In one embodiment, the symptom is scoliosis. In one embodiment, the symptom is impaired gait initiation.

[0046] In one embodiment, the symptom is delayed acquisition of purposeful hand motor function. In one embodiment, the symptom is partial or complete loss of acquired purposeful hand motor function. In one embodiment, the symptom is abnormal hand movements. In one embodiment, the abnormal hand movements are hand twisting, hand wringing, clapping, hand washing, hand smacking, hand rubbing, and / or repetitive hand-to-mouth movements.

[0047] In one embodiment, the symptom is delayed acquisition of communication skills. In one embodiment, the symptom is partial or complete loss of acquired communication skills. In one embodiment, the communication skills are language skills. In one embodiment, the language skills are spoken language skills. In one embodiment, the communication skill is eye contact.

[0048] In one embodiment, the symptom is eye movement abnormalities, hi one embodiment, the eye movement abnormalities are prolonged staring, excessive blinking, crossed eyes, and / or closing one eye at a time.

[0049] In one embodiment, the symptom is disordered breathing. In one embodiment, the disordered breathing occurs when the subject is awake. In one embodiment, the disordered breathing is apnea. In one embodiment, the disordered breathing is hyperventilation.

[0050] In one embodiment, the symptom is bruxism when the subject is awake.

[0051] In one embodiment, the symptom is increased irritability, decreased attention span, and / or decreased concentration span. In one embodiment, the symptom is inappropriate laughter and / or crying.

[0052] In one embodiment, the symptom is a seizure.

[0053] In one embodiment, the symptom is a cardiac abnormality. In one embodiment, the cardiac abnormality is bradycardia. In one embodiment, the cardiac abnormality is tachycardia.

[0054] In one embodiment, the symptom is decreased response to pain. In one embodiment, the symptom is delayed development. In one embodiment, the symptom is microcephaly. In one embodiment, the symptom is disturbed sleep patterns. In one embodiment, the symptom is enlarged, cold, pale feet.

[0055] In one embodiment, the pharmaceutical composition of the present disclosure improves symptoms by at least 5%. In one embodiment, the pharmaceutical composition of the present disclosure improves symptoms by at least 10%. In one embodiment, the pharmaceutical composition of the present disclosure improves symptoms by at least 20%. In one embodiment, the pharmaceutical composition of the present disclosure improves symptoms by at least 30%. In one embodiment, the pharmaceutical composition of the present disclosure improves symptoms by at least 50%. In one embodiment, the pharmaceutical composition of the present disclosure improves symptoms by at least 80%. In one embodiment, the pharmaceutical composition of the present disclosure improves symptoms by 100%.

[0056] In one embodiment, the pharmaceutical compositions of the present disclosure are used to treat a subject by improving the subject's ability to perform daily activities, household chores, manage finances, and / or perform a job. In one embodiment, the pharmaceutical compositions of the present disclosure are used to treat a subject by reducing the level of nursing care required for the subject.

[0057] In one embodiment, the pharmaceutical compositions of the present disclosure are used to treat a subject by maintaining the subject's ability to perform daily activities, household chores, manage finances, and / or perform a career.

[0058] In one embodiment, the pharmaceutical composition of the present disclosure is effective for increasing the serum BDNF concentration of a subject. In one embodiment, the pharmaceutical composition of the present disclosure is effective for increasing the brain BDNF concentration of a subject. In one embodiment, the pharmaceutical composition of the present disclosure is effective for maintaining the serum BDNF concentration of a subject.

[0059] The present invention also provides a pharmaceutical composition comprising an amount of pridopidine for use in treating a subject suffering from Rett Syndrome (RTT).

[0060] The present invention also provides pharmaceutical compositions in unit dosage form useful for treating a subject suffering from Rett Syndrome (RTT).

[0061] In one embodiment, the amount of pridopidine administered is 0.5-315 mg / day. In one embodiment, the amount of pridopidine is 10-315 mg. In one embodiment, the amount of pridopidine is 90-315 mg. In one embodiment, the amount of pridopidine is 90-225 mg. In another embodiment, the amount of pridopidine is about 22.5 mg, about 45 mg, about 67.5 mg, about 90 mg, about 100 mg, about 112.5 mg, about 125 mg, about 135 mg, about 150 mg, about 180 mg, about 200 mg, about 225 mg, about 250 mg, or about 315 mg. In one embodiment, the amount of pridopidine is 45 mg. In one embodiment, the amount of pridopidine is 90 mg. In one embodiment, the amount of pridopidine is 180 mg. In one embodiment, the amount of pridopidine is 225 mg.

[0062] The present invention also provides the use of the pharmaceutical composition in the manufacture of a medicament for treating a subject suffering from Rett Syndrome (RTT).

[0063] The present invention also provides the use of an amount of pridopidine for treating a subject suffering from Rett Syndrome (RTT).

[0064] The present invention also provides a method for increasing serum BDNF levels in a subject suffering from Rett syndrome (RTT), comprising administering to the subject a pharmaceutical composition of the present disclosure, thereby increasing the subject's serum BDNF levels.The present invention also provides a method for increasing brain BDNF levels in a subject suffering from RTT, comprising administering to the subject a pharmaceutical composition of the present disclosure, thereby increasing the subject's brain BDNF levels.

[0065] With respect to the above embodiments, each embodiment disclosed herein is considered applicable to each other embodiment disclosed herein. In addition, elements recited in embodiments of the method of the present invention can be used in embodiments of the pharmaceutical composition, package, and use described herein, and vice versa.

[0066] Pharmaceutical compositions for use in the methods of the present invention:

[0067] In some embodiments, the methods of the present invention use a pharmaceutical composition comprising pridopidine, or a pharmaceutically acceptable salt thereof, and at least one of compounds 1-8 below, or a pharmaceutically acceptable salt thereof:

[0068] [ka]

[0069] In another embodiment, the methods of the present invention employ a pharmaceutical composition comprising pridopidine, or a pharmaceutically acceptable salt thereof, and Compound 1, as described above, or a pharmaceutically acceptable salt thereof.

[0070] In another embodiment, the methods of the present invention employ a pharmaceutical composition comprising pridopidine, or a pharmaceutically acceptable salt thereof, and compound 2, as described above, or a pharmaceutically acceptable salt thereof.

[0071] In another embodiment, the methods of the present invention employ a pharmaceutical composition comprising pridopidine, or a pharmaceutically acceptable salt thereof, and compound 3, as described above, or a pharmaceutically acceptable salt thereof.

[0072] In another embodiment, the methods of the present invention employ a pharmaceutical composition comprising pridopidine, or a pharmaceutically acceptable salt thereof, and compound 4, above, or a pharmaceutically acceptable salt thereof.

[0073] In another embodiment, the methods of the present invention employ a pharmaceutical composition comprising pridopidine, or a pharmaceutically acceptable salt thereof, and compound 5, above, or a pharmaceutically acceptable salt thereof.

[0074] In another embodiment, the methods of the present invention employ a pharmaceutical composition comprising pridopidine, or a pharmaceutically acceptable salt thereof, and compound 6 above, or a pharmaceutically acceptable salt thereof.

[0075] In another embodiment, the methods of the present invention employ a pharmaceutical composition comprising pridopidine, or a pharmaceutically acceptable salt thereof, and compound 7, above, or a pharmaceutically acceptable salt thereof.

[0076] In another embodiment, the methods of the present invention employ a pharmaceutical composition comprising pridopidine, or a pharmaceutically acceptable salt thereof, and compound 8 above, or a pharmaceutically acceptable salt thereof.

[0077] In other embodiments, the methods of the present invention use a pharmaceutical composition comprising pridopidine or a pharmaceutically acceptable salt thereof and at least one of compound 1 above or a pharmaceutically acceptable salt thereof, compound 4 above or a pharmaceutically acceptable salt thereof, or any combination thereof.

[0078] In another embodiment, the methods of the present invention employ a pharmaceutical composition comprising pridopidine, or a pharmaceutically acceptable salt thereof, and Compound 1, as described above, or a pharmaceutically acceptable salt thereof.

[0079] In another embodiment, the methods of the present invention employ a pharmaceutical composition comprising pridopidine, or a pharmaceutically acceptable salt thereof, and compound 4, above, or a pharmaceutically acceptable salt thereof.

[0080] In another embodiment, the methods of the present invention use a pharmaceutical composition comprising pridopidine or a pharmaceutically acceptable salt thereof, compound 1 described above or a pharmaceutically acceptable salt thereof, and compound 4 described above or a pharmaceutically acceptable salt thereof.

[0081] In another embodiment, the pharmaceutical composition used in the methods of the invention comprises a salt of pridopidine, including the hydrochloride, hydrobromide, nitrate, perchlorate, phosphate, sulfate, formate, acetate, aconate, ascorbate, benzenesulfonate, benzoate, cinnamate, citrate, embonate, enanthate, fumarate, glutamate, glycolate, lactate, maleate, malonate, mandelate, methanesulfonate, naphthalene-2-sulfonate, phthalate, salicylate, sorbate, stearate, succinate, tartrate, or toluene-p-sulfonate salt.

[0082] In another embodiment, the methods of the present invention use pharmaceutical compositions comprising at least one of the salts of compounds 1-8 above, including the hydrochloride, hydrobromide, nitrate, perchlorate, phosphate, sulfate, formate, acetate, aconate, ascorbate, benzenesulfonate, benzoate, cinnamate, citrate, embonate, enanthate, fumarate, glutamate, glycolate, lactate, maleate, malonate, mandelate, methanesulfonate, naphthalene-2-sulfonate, phthalate, salicylate, sorbate, stearate, succinate, tartrate, or toluene-p-sulfonate salts.

[0083] In another embodiment, the pharmaceutical composition used in the methods of the present invention is administered in an oral dosage form comprising 0.5 to 315 mg of pridopidine or a pharmaceutically acceptable salt thereof. In another embodiment, the oral dosage form comprises 0.5 to 10 mg of pridopidine. In another embodiment, the oral dosage form comprises 10 to 22.5 mg of pridopidine. In another embodiment, the oral dosage form comprises 22.5 to 45 mg of pridopidine. In another embodiment, the oral dosage form comprises 45 to 250 mg of pridopidine. In another embodiment, the oral dosage form comprises 45 to 135 mg of pridopidine. In another embodiment, the oral dosage form comprises 90 to 315 mg of pridopidine.

[0084] In another embodiment, the method of the present invention uses a pharmaceutical composition comprising pridopidine, or a pharmaceutically acceptable salt thereof, and at least one of compounds 1-8, or a pharmaceutically acceptable salt thereof, wherein the weight ratio of pridopidine to at least one of compounds 1-8 is in the range of 1:0.0001 to 1:0.1. In another embodiment, the weight ratio of pridopidine to at least one of compounds 1-8 is in the range of 1:0.005 to 1:0.1. In another embodiment, the weight ratio of pridopidine to at least one of compounds 1-8 is in the range of 1:0.001 to 1:0.005.

[0085] In another embodiment, the concentration of Compound 1, 2, 3, 4, 5, 6, 7, or 8 or a pharmaceutically acceptable salt thereof in the pharmaceutical composition is 0.01 to 10% by weight. In another embodiment, the concentration of Compound 1, 2, 3, 4, 5, 6, 7, or 8 or a pharmaceutically acceptable salt thereof in the pharmaceutical composition is 0.01 to 0.05% by weight. In another embodiment, the concentration of Compound 1, 2, 3, 4, 5, 6, 7, or 8 or a pharmaceutically acceptable salt thereof in the pharmaceutical composition is 0.01 to 0.5% by weight. In another embodiment, the concentration of Compound 1, 2, 3, 4, 5, 6, 7, or 8 or a pharmaceutically acceptable salt thereof in the pharmaceutical composition is 0.01 to 0.15% by weight. In another embodiment, the concentration of Compound 1, 2, 3, 4, 5, 6, 7, or 8 or a pharmaceutically acceptable salt thereof in the pharmaceutical composition is 0.01 to 0.15% by weight. In another embodiment, the concentration of Compound 1, 2, 3, 4, 5, 6, 7, or 8 or a pharmaceutically acceptable salt thereof in the pharmaceutical composition is 0.01 to 0.5% by weight. In another embodiment, the concentration of Compound 1, 2, 3, 4, 5, 6, 7, or 8 or a pharmaceutically acceptable salt thereof in the pharmaceutical composition is 0.01 to 1% by weight. In another embodiment, the concentration of Compound 1, 2, 3, 4, 5, 6, 7, or 8 or a pharmaceutically acceptable salt thereof in the pharmaceutical composition is 0.05 to 0.2% by weight. In another embodiment, the concentration of Compound 1, 2, 3, 4, 5, 6, 7, or 8 or a pharmaceutically acceptable salt thereof in the pharmaceutical composition is 0.05 to 0.3% by weight. In other embodiments, the concentration of the above compound 1, 2, 3, 4, 5, 6, 7, or 8 or a pharmaceutically acceptable salt thereof in the pharmaceutical composition ranges from 0.05 to 0.4% by weight.

[0086] While the active compounds for use in accordance with the present invention may be administered in the form of raw compounds, it is preferred that the active compound or its pharmaceutically acceptable salts be included in a pharmaceutical composition together with one or more adjuvants, excipients, carriers, buffers, diluents, and / or other common pharmaceutical auxiliary agents. In one embodiment, the present invention provides a pharmaceutical composition comprising the active compound or a pharmaceutically acceptable salt or derivative thereof, one or more pharmaceutically acceptable carriers, and, optionally, other therapeutic and / or prophylactic ingredients known and used in the art. A carrier must be "acceptable" in the sense of being compatible with the other ingredients of the formulation and not harmful to the subject to which the pharmaceutical composition of the present disclosure is administered.

[0087] The pharmaceutical compositions of the present disclosure can be administered by any convenient route suitable for the desired treatment. Preferred routes of administration include oral administration, particularly in tablet, capsule, multiparticulate, powder, or liquid form, and parenteral administration, particularly by cutaneous, subcutaneous, intramuscular, or intravenous injection. The pharmaceutical composition used in the methods of the present invention can be an oral dosage unit formulated as a tablet, capsule, pill, powder, multiparticulate in a capsule or sachet, liquid solution, or liquid suspension.

[0088] term

[0089] As used herein, unless otherwise stated, each of the following terms shall have the definition set forth below.

[0090] As used herein, "pridopidine" refers to pridopidine base or a pharmaceutically acceptable salt or derivative thereof, e.g., deuterium-enriched versions of pridopidine and salts. Examples of deuterium-enriched versions of pridopidine and salts, and methods for their preparation, are described in U.S. Patent Application Publication Nos. 2013-0197031, 2016-0166559, and 2016-0095847, the entire disclosures of which are incorporated herein by reference.

[0091] "Deuterium-enriched" means that, for a given amount of compound, the abundance of deuterium at any relevant site of the compound is greater than the abundance of deuterium naturally occurring at that site. The natural abundance of deuterium is approximately 0.0156%. Thus, in a "deuterium-enriched" compound, the abundance of deuterium at any relevant site is greater than 0.0156% and can range from 0.0156 to 100%. Deuterium-enriched compounds can be obtained by exchanging hydrogen for deuterium or by synthesizing the compound using deuterium-rich starting materials.

[0092] The active compounds for use in accordance with the present invention may be provided in any form suitable for the intended administration. Suitable forms include pharmaceutically acceptable salts of the compounds of the invention, and pre-drug or pro-drug forms of the compounds of the invention.

[0093] "Salt of" refers to a salt of the present compound modified by making an acid salt or base salt of the compound. In this regard, the term "pharmaceutically acceptable salt" refers to a relatively non-toxic, inorganic or organic acid addition salt or base addition salt of the compound of the present invention that is suitable for pharmaceutical use. Pharmaceutically acceptable salts can be prepared by methods well known and described in the art. One method for preparing such salts is to treat the compound of the present invention with an inorganic base.

[0094] Examples of acid addition salts of the compounds of the present invention include, but are not limited to, hydrochloride, hydrobromide, nitrate, perchlorate, phosphate, sulfate, formate, acetate, aconate, ascorbate, benzenesulfonate, benzoate, cinnamate, citrate, embonate, enanthate, fumarate, glutamate, glycolate, lactate, maleate, malonate, mandelate, methanesulfonate, naphthalene-2-sulfonate, phthalate, salicylate, sorbate, stearate, succinate, tartrate, and toluene-p-sulfonate. In certain embodiments, pridopidine is a pharmaceutically acceptable salt, such as an HCl salt or a tartrate salt. Preferably, in any of the embodiments of the present invention described herein, pridopidine is in the form of its hydrochloride salt.

[0095] As used herein, an "amount" or "dose" of pridopidine measured in milligrams refers to the milligrams (mg) of pridopidine (4-[3-(methylsulfonyl)phenyl]-1-propyl-piperidine) contained in the formulation, regardless of the formulation. For example, a unit dose containing "90 mg pridopidine" means that the amount of pridopidine contained in the formulation is 90 mg, regardless of the formulation. Thus, in the case of a salt form such as pridopidine hydrochloride, the weight of the salt form required to provide a 90 mg dose of pridopidine will be greater than 90 mg due to the presence of the salt.

[0096] As used herein, "unit dose" and "unit dosage form" refer to a single drug administration entity. "Unit dose" and "unit dosage form" can be prepared for oral dosage forms such as tablets, capsules, pills, powders, and granules.

[0097] As used herein, "about" in the context of a numerical value or numerical range means 90-110% of the stated or claimed numerical value or range.

[0098] As used herein, "administration to a subject" or "administration to a (human) patient" means the administration, dosing, or application of a pharmaceutical, drug, or therapeutic agent to a subject / patient to delay, alleviate, cure, or relieve symptoms associated with a condition, such as a pathological condition. Oral administration is one method of administering the compounds of the present invention to a subject.

[0099] The compounds according to the invention can be administered in base form or in the form of a pharmaceutically acceptable salt, preferably in a pharmaceutical composition together with one or more adjuvants, excipients, carriers, buffers, diluents, and / or other conventional pharmaceutical auxiliary agents.

[0100] A "pharmaceutically acceptable carrier" refers to a carrier or excipient suitable for human and / or animal use that does not cause excessive side effects (such as toxicity, irritation, or allergic response) commensurate with a reasonable benefit / risk ratio. A pharmaceutically acceptable carrier can be a pharmaceutically acceptable solvent, suspending agent, or vehicle for delivering a compound of the invention to a subject.

[0101] Administration may be regular administration. As used herein, "regular administration" refers to repeated / repeated administration separated by a predetermined period. The period between administrations is preferably constant from time to time. Regular administration may include, for example, administration once a day, twice a day, three times a day, four times a day, once a week, twice a week, three times a week, four times a week, etc.

[0102] As used herein, "treatment" includes alleviating, alleviating, lessening the severity of, eliminating, substantially eliminating, or ameliorating the physical, mental, or emotional limitations in a subject afflicted with Rett Syndrome (RTT). Treatment also refers to delaying or preventing symptoms or reducing disability associated with the disease.

[0103] As used herein, "effective," such as an amount effective for a particular purpose, refers to the amount of an ingredient that, when used in the disclosed methods, produces an indicated therapeutic response without causing excessive side effects (such as toxicity, irritation, or allergic reaction) commensurate with a reasonable benefit / risk ratio. For example, an amount effective to treat the symptoms of Rett Syndrome (RTT). The specific effective amount will vary depending on factors such as the particular condition being treated, the patient's physical condition, the species of mammal being treated, the duration of treatment, the nature of any concomitant therapy, the particular formulation used, and the structure of the compound or its derivatives.

[0104] Where a parameter range is provided, it is understood that all integers within that range, and tenths of those integers, are also provided by the present invention. For example, "22 to 300.0 mg" includes 22.0 mg, 22.1 mg, 22.2 mg, 22.3 mg, 22.4 mg, ... up to 300.0 mg.

[0105] The present invention may be better understood by reference to the "Experimental Details" section below. However, those skilled in the art will readily appreciate that the specific experiments described in detail are merely illustrative of the invention as more fully described in the claims that follow.

[0106] Exam details

[0107] Example 1: Evaluation of the efficacy of pridopidine in a heterozygous MeCP2 female mouse model of Rett syndrome

[0108] The aim of this study was to evaluate the effects of pridopidine in the female MeCP2(BIRD) mouse model of Rett syndrome (Guy 2001).

[0109] material:

[0110] Pridopidine (30 mg / kg or 30 mg / kg) was orally administered at a volume of 10 ml / kg twice daily (6 hours between doses). On the test day, pridopidine was administered 30 minutes before the test.

[0111] Pridopidine treatment began when the mice were approximately 5.5 weeks of age and continued until the end of behavioral testing, which was performed at 8 and 12 weeks of age.

[0112] Female MeCP2 (MeCP2_HET, Rett) mice and wild-type (MeCP2_WT, WT) littermates were housed under a 12 / 12 light / dark cycle at 50% relative humidity. Food and water were available ad libitum. All tests were performed during the light phase. To ensure adequate health and fitness and minimize nonspecific stress associated with the tests, all animals were examined and weighed before and during the test period. A 12 / 12 light / dark cycle was maintained throughout the test period. Room temperature was maintained between 20 and 23°C, with relative humidity maintained at approximately 50%. Food and water were available ad libitum throughout the study period. Tests were performed during the animals' light phase.

[0113] method:

[0114] Treatment group: WT mice: vehicle (saline), n=24 Rett HET MeCP2 mice: vehicle (saline), n=24 Rett HET MeCP2 mice: pridopidine (3 mg / kg; orally twice daily), n=20 Rett HET MeCP2 mice: pridopidine (30 mg / kg; orally twice daily), n=20

[0115] Behavioral testing:

[0116] (1) Gait analysis using the NeuroCube (registered trademark) system

[0117] The NeuroCube® system is a platform that uses computer vision to detect changes in gait morphology and dynamics in rodent models of neurological disease, pain, and neurological disorders. This platform is unique to gait testing for the following reasons: · The system is fully automated, eliminating bias and subjectivity. The system captures both gait morphology and dynamics (stance, swing, propulsion, etc.).

[0118] Mice were placed in the NeuroCube and tested for 5 minutes. The most dominant collected features defining the disease phenotype (symptom descriptors) were identified and ranked. Complex bioinformatic algorithms were used to calculate the probability of discrimination between WT and Rett HET MeCP2 mice and to detect the ability of test compounds to reverse the disease phenotype. The discrimination between mutant and wild-type mice and the recovery of disease features in Rett HET MeCP2 mice treated with the test compounds were calculated.

[0119] (2) Clasping

[0120] Clasping was used to assess limb muscle strength. The mouse was grasped by the tail and gently lifted until its front paws were just off the counter surface. The experimenter observed the mouse's paws for clasping or splaying of the limbs. After testing, the animal was returned to its test or home cage. The percentage of hind limb clasping was measured and reported.

[0121] (3) Startle response / prepulse inhibition (PPI)

[0122] The acoustic startle technique was used to measure unconditioned reflex responses to external auditory stimuli. Prepulse inhibition (PPI), a reduction in the amplitude of the startle response to an auditory stimulus after the presentation of a weak auditory stimulus or prepulse, has been used as a tool to assess sensorimotor gating deficits, such as those seen in schizophrenia.

[0123] Mice were placed in a PPI chamber (Med Associates) and allowed to acclimate to white noise (70 dB) for 5 min. After the acclimation period, the test session began automatically. The test session began with a habituation block of six startle-only presentations, followed by a 10-trial PPI block of six trials.

[0124] Trial types included null (no stimulus), startle (120 dB), startle + prepulse (4 dB, 8 dB, or 12 dB above background noise, i.e., 74 dB, 78 dB, or 82 dB), and prepulse alone (82 dB). Trial types were presented randomly within each block. Each trial began with a 50-ms null period during which baseline movement was recorded. A prepulse stimulus was then presented, and responses to the prepulse were recorded for 20 ms. After an additional 100 ms, a startle stimulus was presented for 40 ms, and responses to the startle onset were recorded for 100 ms. Responses were sampled every 1 ms. The intertrial interval averaged 15 s (range, 10–20 s).

[0125] In the startle-alone test, the baseline acoustic startle was measured, and in the prepulse + startle test, the amount of inhibition of normal startle was measured and expressed as a percentage of the baseline startle response (relative to the startle-alone test), excluding the startle response of the first habituation block.

[0126] Brain collection:

[0127] After completing all behavioral tests, brain samples were collected 60 minutes after pridopidine administration. Mice were euthanized by cervical dislocation and decapitation. Whole brains were collected from 10 mice per treatment group, weighed, and frozen on dry ice. Brain samples were stored at -80°C until analysis of brain-derived neurotrophic factor (BDNF).

[0128] BDNF analysis:

[0129] Total RNA extraction:

[0130] Tissues (whole brain) were homogenized, RNA extracted and quantified, and an aliquot was reverse transcribed into cDNA.

[0131] Up to three independent RT reactions were performed for each RNA sample. qPCR was performed using primers detailed in Table 5 below.

[0132] [Table 5]

[0133] qPCR data analysis:

[0134] Whole brain cDNA obtained from pooled samples of WT vehicle-treated animals was used as a calibrator (calibrator diluted identically to sample cDNA) to normalize interplate variation.

[0135] Each cDNA sample (diluted 1:10) was analyzed in triplicate and Ct values ​​were averaged. Values ​​greater than 0.5 standard deviations from the mean were discarded.

[0136] The relative amount of PCR product (relative to the calibrator) was calculated as follows:

[0137]

number

[0138] The geometric mean of the housekeeping genes was calculated as follows:

[0139]

number

[0140] The relative levels of the target genes were calculated as follows:

[0141]

number

[0142] The relative levels of the target genes were then normalized to the WT vehicle group.

[0143] Statistical analysis:

[0144] Data from standard tests were analyzed by genotype (t-test) and treatment (ANOVA), followed by post-hoc comparisons where appropriate. For some indices, repeated measures ANOVA was performed. For clasping data, an N-1 2-proportional test was performed. Effects were considered significant if p<0.05. All data were expressed as mean and standard error of the mean (sem). Values ​​±2 standard deviations from the mean were considered outliers.

[0145] Data analysis from NeuroCube:

[0146] The output of NeuroCube is a set of dozens of behavioral features that are submitted for analysis by machine learning techniques used in bioinformatics. Many of these features are correlated (e.g., number of rearings and number of supported rearings). Therefore, PGI forms statistically independent combinations of the original features that more effectively discriminate between the two groups (also called uncorrelated features).

[0147] Each uncorrelated feature extracts information from the entire cluster of original features, so the new feature space has lower dimensionality. PGI then applies a proprietary feature ranking algorithm to score each feature's discriminatory power (its ability to separate two groups, e.g., control and disease).

[0148] Ranking is an important part of the analysis because it weights each feature change by its relevance. If there is a large change in an irrelevant feature measured for a particular phenotype, a lower-ranked feature automatically reduces the impact of such change in the analysis, eliminating the need to rely on traditional "feature selection" approaches to discard information embedded in less informative features. Ranking algorithms can be applied to either the original features or the new features to gain insight into key control-disease differences.

[0149] Signature analysis: quantitative assessment of disease phenotypes

[0150] In the new feature space, the overlap between "clouds" (Gaussian distributions approximating mouse groups in the ranked, uncorrelated feature space) serves as a quantitative measure of the separability ("distinguishability") between the two groups. For visualization purposes, each cloud was plotted with a semi-axis equal to one standard deviation along the corresponding dimension.

[0151] result:

[0152] Behavioral testing:

[0153] (1) Clasping

[0154] Patients with Rett syndrome lose or are unable to acquire purposeful hand motor skills, which are replaced by stereotyped movements such as twisting the hand. In the Rett mouse model, this symptom correlates with a hindlimb clasping phenotype. Rett model mice exhibit significantly more clasping than WT mice (Figure 2). As shown in Figure 2, pridopidine improves clasping at 8 weeks. Vehicle-treated Rett mice exhibit significantly more clasping than WT mice. Administration of pridopidine (30 mg / kg, twice daily) normalized this behavior at 8 weeks (p<0.06). At 8 weeks, clasping was 0% in both vehicle-treated WT mice and pridopidine-treated animals (30 mg / kg, twice daily). This suggests that administration of pridopidine (30 mg / kg, twice daily) may be effective in treating this symptom and delay its onset.

[0155] (2) Startle response / PPI

[0156] The symptoms of Rett syndrome, including impaired attention and concentration span, were reproduced in a mouse model and assessed using acoustic startle response. Vehicle-treated Rett mice showed a significant suppression of startle response compared to WT mice, approximately 65% ​​at week 8 and approximately 75% at week 12 (p<0.05). Pridopidine (3 mg / kg, bid) had a significant beneficial effect on startle response, approximately 40% at week 8 and approximately 50% at week 12 (p<0.05), as shown in Figure 3.

[0157] (3) NeuroCube (registered trademark)

[0158] The probability of discrimination between WT and Rett mice was 90% at 8 weeks of age and 94% at 12 weeks of age. Some of the top gait characteristics that distinguished WT and Rett mice were a larger stride length, a narrower base width, and lower paw strength in WT mice compared with Rett mice.

[0159] The effect of pridopidine on walking ability at week 8 is shown in Figure 4. The effect of pridopidine on walking ability at week 12 is shown in Figure 5. Administration of pridopidine (30 mg / kg, twice daily) showed significant recovery of overall walking function at both week 8 and week 12 (45% at week 8 and 55% at week 12).

[0160] Further analysis revealed significant differences in several gait domains, as shown in Table 6 below. Rett mice were significantly different from WT control mice in all gait functions. Data at 8 weeks show that pridopidine (3 mg / kg or 30 mg / kg, bid) improved physical activity or gait in Rett mice. Administration of pridopidine (3 mg / kg, bid) significantly improved gait and physical activity at 12 weeks. Administration of pridopidine (30 mg / kg, bid) had significant effects on gait, physical activity, and paw positioning.

[0161] [Table 6]

[0162] BDNF analysis

[0163] The effect of pridopidine on relative BDNF expression in brain samples from WT and Rett mice is shown in Figures 6-10.

[0164] Whole brain control housekeeping gene mRNA expression levels did not change among the animal treatment groups examined (see Figures 6(A)-(C)).

[0165] Compared with WT (vehicle), BDNF-I mRNA expression was significantly reduced in Rett mice (vehicle) by approximately 20% (p<0.001). Pridopidine administration (3 mg / kg or 30 mg / kg, administered twice daily) did not affect BDNF-I mRNA levels in Rett mice (see Figure 7).

[0166] Compared to WT (vehicle), BDNF-IV mRNA expression was significantly reduced in Rett mice (vehicle) by approximately 15% (p<0.001). Pridopidine administration (3 mg / kg or 30 mg / kg, administered twice daily) rescued the downregulated BDNF-IV mRNA in Rett mice to levels close to WT levels, approximately 30% (p<0.001) (Figure 8).

[0167] Compared with WT (vehicle), BDNF-VI mRNA expression was significantly reduced in Rett mice (vehicle) by approximately 25% (p<0.001). Pridopidine administration (3 mg / kg or 30 mg / kg, administered twice daily) did not affect BDNF-VI mRNA levels in Rett mice (see Figure 9).

[0168] Compared with WT (vehicle), BDNF-IX mRNA expression was significantly reduced in Rett mice (vehicle) by approximately 20% (p<0.001). Pridopidine administration (3 mg / kg or 30 mg / kg, administered twice daily) rescued the downregulated BDNF-IX mRNA in Rett mice to levels close to those of WT (p<0.001) (see Figure 10).

[0169] conclusion

[0170] In this study, we evaluated the effects of chronic administration of pridopidine on locomotion, hindlimb clasping, and startle / PPI in Rett mice.

[0171] Rett mice exhibit distinct alterations in gait measurements, which distinguish them from WT mice. Rett mice also exhibit a stereotyped hindlimb immobilization phenotype. Furthermore, Rett mice exhibit a suppressed acoustic startle response compared to WT mice. Pridopidine treatment significantly rescued hindlimb clasping at 8 weeks of age (30 mg / kg, bid) and significantly improved startle response at 8 and 12 weeks compared to vehicle-treated Rett mice (3 mg / kg, bid). Rett mice treated with pridopidine (30 mg / kg, bid) showed significant recovery of locomotor function at 8 and 12 weeks.

[0172] Administration of both doses of pridopidine (3 mg / kg or 30 mg / kg twice daily) completely rescued the downregulated mRNA levels of BDNF-IV and BDNF-IX. The positive effect of pridopidine on BDNF mRNA expression was consistent with the improvements observed in behavioral paradigms.

[0173] Example 2: RNA analysis of MeCP2 mice treated with pridopidine

[0174] method:

[0175] Approximately 5.5-week-old female Rett model mice (MeCP2 heterozygous) and wild-type (WT) littermates were orally administered either pridopidine or vehicle. Pridopidine (3 mg / kg or 30 mg / kg) was orally administered at a dose of 10 ml / kg twice daily with a 6-hour interval between doses. Four treatment groups were established: 1. WT mice: vehicle; 2. Rett mice: vehicle; 3. Rett mice: pridopidine (3 mg / kg; orally administered twice daily); and 4. Rett mice: pridopidine (30 mg / kg; orally administered twice daily).

[0176] In this study, we evaluated whether pridopidine reversed the abnormal transcription observed in Rett mice by examining whether pridopidine restored the expression of genes disrupted in the disease state to WT levels. Additionally, we evaluated the effects of pridopidine on gene expression in a mouse model of Rett syndrome.

[0177] result:

[0178] Pridopidine reversed the Rett syndrome disease gene expression signature in the striatum and cortex as analyzed by gene set enrichment analysis (GSEA).

[0179] [Table 7]

[0180] [Table 8]

[0181] A comparison of global gene expression patterns revealed that pridopidine potently reversed gene expression patterns in both the striatum and cortex of Rett mice.

[0182] [Table 9]

[0183] Table 9 shows that pridopidine 3 mg / kg administered twice daily reversed the Rett gene expression pattern in the striatum in both directions (up-regulating genes that were down in Rett mice vs. WT and down-regulating genes that were up in Rett mice vs. WT). Pridopidine 30 mg / kg administered twice daily significantly up-regulated genes that were down in Rett mice compared to WT mice.

[0184] [Table 10-1]

[0185] [Table 10-2]

[0186] Table 10 shows that when pridopidine was administered at 3 mg / kg or 30 mg / kg twice daily, the cortical Rett gene expression pattern was reversed in both directions (up-regulating genes that were down in Rett mice vs. WT, and up-regulating genes that were down in Rett mice vs. WT).

[0187] The effect of pridopidine on gene expression downstream of the BDNF-TrkB pathway was evaluated. Administration of pridopidine at 30 mg / kg twice daily significantly increased gene expression downstream of BDNF (Table 11).

[0188] [Table 11]

[0189] Example 3: Pridopidine improves locomotor function in male MeCP2 knockout (KO) Rett syndrome mouse model (Rett-KO)

[0190] method:

[0191] A colony of Rett model mice (Jackson Laboratories, Bar Harbor, ME; B6.129P2-Mecp2tm2Bird / J | Stock Number: 003890) was established by mating heterozygous (HET) females with wild-type (WT) males (C57Bl / 6J). Heterozygous MeCP2 Rett model mice (Rett-KO) and their wild-type (WT) littermates were housed in a temperature-controlled room at 20–23°C with 50% humidity and a 12 / 12 light / dark cycle. The room temperature was maintained at 20–23°C, and the relative humidity was kept around 50%. Food and water were available ad libitum throughout the study period. Additionally, mice were provided with hydrogel daily upon first observation of hindlimb splay and / or signs of motor difficulty.

[0192] After weaning, mice were housed singly in OPTI mouse cages. All animals were housed singly for the remainder of the study. Baseline weight and grip strength measurements taken before the start of the study were used to balance mice and assign them to treatment groups. All studies were performed during the light phase.

[0193] Pridopidine was evaluated at 30 mg / kg twice daily. Pridopidine was dissolved in sterile DDW and administered orally twice daily (bid) at a volume of 10 mL / kg.

[0194] NeuroCube® - Gait Analysis

[0195] The NeuroCube® (NRC) system is a proprietary technology from PsychoGenics. This system is a platform that uses computer vision to detect changes in gait morphology and dynamics in rodents. This platform is unique for gait testing because: · The system is fully automated, eliminating bias and subjectivity. The system captures both gait morphology and dynamics (stance, swing, propulsion, etc.). The system uses the sensitivity of computer vision and bioinformatics to capture disease model symptoms faster and more accurately.

[0196] Gait analysis was performed on mice at 5, 6, and 7 weeks of age. The mice were placed in the NeuroCube® and subjected to a 5-minute test. The most prominent features (symptom descriptors) defining the disease phenotype were identified and ranked. Complex bioinformatic algorithms were used to calculate the probability of discrimination between WT and Rett mice and to detect the ability of test compounds to reverse the disease phenotype.

[0197] Functional analysis

[0198] Ranking is an important part of the analysis because it weights each functional change by its relevance: if there is a large change in an unrelated function measured for a particular phenotype, a lower ranking of this function automatically reduces the influence of such a change in the analysis.

[0199] The relative difference (%) between the feature values ​​of the two different sets was calculated and plotted in an order corresponding to the feature rank, with the rank ranging from 0 to 100%.

[0200] Feature analysis - a list of the features analyzed

[0201] (1) Average velocity: A measure of the average speed traveling the length of the NRC. (2) Body posture: Paw image parameters were used to measure the XY coordinates of the body, the XY coordinates of the paw, and the direction vector of the paw, which are related to the subject's body movements. (3) Gait: Measurement of gait morphology (e.g., stride width, step width, base width) and gait kinetics (e.g., stride duration, step duration, swing duration). (4) Image processing: Measurement of paw contact area, perimeter of contact area, and paw diameter (horizontal / vertical). (5) Paw position: The position of the contact area of ​​each paw relative to the center of the body was recorded. By superimposing all recorded relative positions of the four paws, four clusters of points were created (one for each paw). For each paw, the coordinates of the cluster center, the cluster size, the number of paw prints, and the relative geometry of the cluster position were measured. (6) Rhythm: correlation coefficients between the walking signal of each leg and the walking signals of all other legs: RF-LF, RF-LH, RF-RH, LH-RH, LH-RF, LF-RH, LH-RH (F-forelimb; H-hindlimb; R-right; L-left).

[0202] Signature analysis: quantitative assessment of disease phenotypes

[0203] In feature space, the overlap between "clouds" (Gaussian distributions approximating mouse groups in the ranked, uncorrelated feature space) serves as a quantitative measure of the separability ("distinguishability") of WT and Rett-KO mice (see Figure 1). For visualization purposes, each cloud was plotted with a semi-axis equal to one standard deviation along the corresponding dimension.

[0204] Feature Analysis: Drug-Induced Relapse

[0205] In studies of "drug-induced reinstatement," data are typically presented in three classes: WT, Rett-KO, and Rett-KO + pridopidine ("pridopidine-treated group"). It is therefore instructive to consider (and plot) a third group, the pridopidine-treated group, in the same coordinate system that best distinguishes the other groups (WT and Rett), as shown in Figure 1A.

[0206] Treatment group

[0207] The following treatment groups were used in this study: 1. WT mice - vehicle 2. Rett-KO (B6.129P2-Mecp2tm2Bird / J) mice - vehicle 3. Rett-KO (B6.129P2-Mecp2tm2Bird / J) mice - pridopidine (30 mg / kg, twice daily)

[0208] statistical analysis

[0209] Data were analyzed by repeated measures analysis of variance (ANOVA), followed by post-hoc comparisons where appropriate. Effects were considered significant when p<0.05. Data are expressed as means and standard errors of the means (sem).

[0210] result:

[0211] NeuroCube®

[0212] 1. Walking function

[0213] Discrimination plots for 6- and 7-week-old Rett-KO versus WT mice are shown in Figures 11A and 11B. Feature names are a combination of the parameter name and the paw name: FR - forelimb (right); FL - forelimb (left); HR - hindlimb (right); HL - hindlimb (left).

[0214] Analysis of gait function revealed that at both 6 and 7 weeks of age, Rett-KO mice exhibited gait impairments compared with WT mice, with discrimination probabilities of 100% and 99%, respectively. At both 6 and 7 weeks of age, Rett-KO mice exhibited impairments in measures of gait function compared with WT mice, primarily in stride length, base width, and step width.

[0215] The effect of pridopidine (30 mg / kg, administered twice daily) on gait impairment in Rett-KO mice was evaluated in 6- and 7-week-old mice. A summary of recovery is shown in Figure 12. Pridopidine reversed gait impairment by 44% and 100% in 6- and 7-week-old Rett model mice (both p<0.05, ANOVA).

[0216] conclusion

[0217] Genotype comparison revealed that Rett-KO mice exhibited significant impairments in gait measurements compared with WT mice. Chronic administration of pridopidine (30 mg / kg, twice daily) to Rett-KO mice resulted in significant recovery of gait at 6 and 7 weeks.

[0218] Example 4: Pridopidine 45 mg twice daily improves gait and balance in early HD patients at 52 weeks

[0219] Change from baseline in UHDRS TMS gait and balance scales at weeks 26 and 52 of early HD (baseline TFC 7-13). Table 2 (see Brief Description of Figures) and Figures 13A and 13B show the trend for improvement in UHDRS TMS gait and balance at weeks 26 and 52 in early HD patients receiving pridopidine 45 mg twice daily. Early HD included HD1 (TFC 11-13) and HD2 (TFC 7-10). Figure 14B and Table 3 (see Brief Description of Figures) show a significant effect on change from baseline in gait and balance at week 52 in patients receiving pridopidine 45 mg twice daily (p=0.0445). Figure 14A shows the trend for improvement at week 26 in HD1 patients receiving pridopidine. Figures 15A, 15B and Table 4 (see Brief Description of Figures section) show the trend for improvement in change from baseline in gait and balance at both Week 52 and Week 26 in HD2 patients receiving pridopidine 45 mg twice daily.

[0220] Example 5: Synergistic effect of pridopidine and compound 1 or pridopidine and compound 4 on BDNF secretion

[0221] Both Compound 1 and Compound 4 exhibit synergistic effects with pridopidine on BDNF secretion from B104 neuroblastoma cells.

[0222] As shown in Table 12, Compound 1 and Compound 4 selectively bind to sigma-1 receptors (S1R) (Ki = 0.37 μM for Compound 1, Ki = 2.9 μM for Compound 4) but do not bind to sigma-2 receptors (S2R) (Ki > 100 μM for both Compound 1 and Compound 4).

[0223] [Table 12]

[0224] Thus, both Compound 1 and Compound 4 exhibit high affinity for the sigma-1 receptor (S1R), but no affinity for the sigma-2 receptor (S2R) (Ki>100).

[0225] Decreased brain-derived neurotrophic factor (BDNF) levels play an important role in the pathogenesis of neurodegenerative and neurodevelopmental disorders, such as Huntington's disease (HD), Parkinson's disease, Alzheimer's disease (Zuccato and Cattaneo 2009), and Rett syndrome (Katz 2014).

[0226] Pridopidine dose-dependently increased BDNF secretion in rat neuroblastoma cells as measured by in situ ELISA, suggesting that its effect is mediated by S1R activation, as pharmacological inhibition of S1R abolished the effect (Geva, Birnberg, et al. 2016).

[0227] When we evaluated the synergistic effects of Compound 1 or Compound 4 with pridopidine, we found an unexpected synergistic effect, which was observed by in-situ ELISA of BDNF (Geva, Kusko, et al. 2016).

[0228] Therefore, the synergistic effect on BDNF release shown below is directly related to the therapeutic efficacy of pridopidine with Compound 1 or Compound 4.

[0229] The data below show that surprisingly and unexpectedly, the combination of pridopidine with Compound 4 or Compound 1 exerts a synergistic effect on BDNF release.

[0230] Synergistic effect of compound 4 and pridopidine on BDNF release

[0231] Pridopidine alone increased BDNF release by +13.5% at 0.001 μM and +26% at 0.005 μM compared to untreated control cells. Compound 4 alone at 0.001 μM did not affect BDNF release (-1.5%) compared to untreated control cells. However, the combination of pridopidine and compound 4 produced an unexpected synergistic effect on BDNF release. · The combination of 0.001 μM pridopidine with 0.001 μM compound 4 increased BDNF release by 59.1% compared to control untreated cells (Figure 16(A)). · 0.001 μM pridopidine in combination with 0.005 μM compound 4 increased BDNF release by 80.7% compared to control untreated cells (Figure 16(B)).

[0232] The effect of the combination of pridopidine and Compound 4 was greater than the sum of the individual effects of each compound, indicating that the combination of pridopidine and Compound 4 has a surprising synergistic effect on BDNF secretion. The results are shown as a percentage change compared to untreated control cells.

[0233] Synergistic effect of compound 1 and pridopidine on BDNF release

[0234] Pridopidine alone at a concentration of 0.01 μM increased BDNF release by +3.4% compared to control untreated cells. Compound 1 alone at a concentration of 1 μM increased BDNF release by +12.5% ​​compared to control untreated cells. However, the combination of pridopidine and compound 1 had a synergistic effect on BDNF release (+53.1%). Pridopidine (0.01 μM) in combination with compound 1 (1 μM) increased BDNF release by 53.1% compared to control untreated cells (FIG. 17).

[0235] As with the combination of pridopidine and Compound 4, the effect of the combination of pridopidine and Compound 1 was greater than the sum of the effects of each compound individually, indicating that the combination of pridopidine and Compound 1 has a surprising and unexpected synergistic effect on BDNF secretion.

[0236] Thus, the present inventors have demonstrated that Compounds 1 and 4 have selective binding affinity for the sigma-1 receptor (S1R) and exhibit surprising and unexpected synergistic effects with pridopidine on BDNF release.

[0237] Example 6: Evaluation of the efficacy of pridopidine in treating patients with Rett syndrome (RTT)

[0238] Regular (e.g., daily or twice daily) intravenous or oral administration of pridopidine is effective in treating patients with Rett syndrome (RTT).

[0239] Administration of pridopidine effectively delays the onset of symptoms in patients with RTT.

[0240] Administration of pridopidine effectively prevents, delays, or ameliorates at least one symptom in RTT patients.

[0241] Administration of pridopidine effectively prevents, slows, or improves motor function in RTT patients. Administration of pridopidine effectively prevents partial or complete loss of acquired motor function in RTT patients.

[0242] Administration of pridopidine effectively prevents, slows, or improves ambulation in RTT patients.

[0243] Administration of pridopidine effectively prevents, delays, or ameliorates ataxia, apraxia, muscle weakness, spasticity, and / or rigidity in RTT patients. Administration of pridopidine effectively prevents, delays, or ameliorates gait initiation disorders in RTT patients.

[0244] Administration of pridopidine effectively prevents, delays, or improves dystonia, peripheral vasomotor disorders, and / or scoliosis in RTT patients.

[0245] Administration of pridopidine effectively prevents, delays, or improves the deterioration of purposeful hand motor function in RTT patients. Administration of pridopidine effectively prevents, delays, or improves abnormal hand movements, including, but not limited to, hand twisting, hand wringing, clapping, hand washing, hand smacking, hand rubbing, and / or repetitive hand-to-mouth movements. Administration of pridopidine effectively prevents partial or complete loss of acquired purposeful hand motor function in RTT patients.

[0246] Administration of pridopidine effectively prevents, slows, or improves communication skills in RTT patients, including but not limited to the ability to speak and maintain normal eye contact. Administration of pridopidine effectively prevents partial or complete loss of acquired communication skills in RTT patients.

[0247] Administration of pridopidine effectively prevents, delays, or ameliorates developmental delays, seizures, cardiac abnormalities, irregular breathing, disturbed sleep patterns, teeth grinding while awake, decreased response to pain, enlarged, cold, pale feet, increased excitability, decreased attention span, inappropriate laughter, and / or inappropriate crying.

[0248] (Reference) “Amaral, M.D., et al. (2007) "TRPC channels as novel effectors of BDNF signaling: Potential implications for Rett syndrome". Pharmacol Ther, 113(2):394-409.” “Cheng, Yung-Chi, and William H. Prusoff. 1973. "Relationship between the Inhibition Constant (KI) and the Concentration of Inhibitor Which Causes 50 per Cent Inhibition (I50) of an Enzymatic Reaction." Biochemical Pharmacology. https: / / doi.org / 10.1016 / 0006-2952(73)90196-2.” “CSID:25948790, www.chemspider.com / Chemical-Structure.25948790.html (accessed 23:27, Jul 15, 2016).” “CSID:7971505, www.chemspider.com / Chemical-Structure.7971505.html (accessed 23:33, Jul 15, 2016).” “Geva, Michal, et al. "Pridopidine activates neuroprotective pathways impaired in Huntington Disease." Human molecular genetics 25.18 (2016): 3975-3987.? ” 「 Guy J, Hendrich B, Holmes M, Martin JE, Bird A. (2001) A mouse MeCP2-null mutation causes neurological symptoms that mimic Rett syndrome. Nat Genet. 27(3):322-326.」 「Isaias, I.U., et al. (2014). "Gait Initiation in Children with Rett Syndrome." PLoS One, 9(4): e92736.」 「 Johnston, Tom H., Michal Geva, Lilach Steiner, Aric Orbach, Spyros Papapetropoulos, Juha-Matti Savola, Ian J. Reynolds, et al. 2019. "Pridopidine, a Clinic-Ready Compound, Reduces 3,4-Dihydroxyphenylalanine-Induced Dyskinesia in Parkinsonian Macaques." Movement Disorders, December. https: / / doi.org / 10.1002 / mds.27565.」 「 Katz, DM. 2014. "Brain-Derived Neurotrophic Factor and Rett Syndrome." Handbook of Experimental Pharmacology 220: 481-95. https: / / doi.org / 10.1007 / 978-3-642-45106-5_18.」 「 Pozzo-Miller, L., Pati S., & Percy, A.K. (2015). "Rett Syndrome: Reaching for Clinical Trials." Neurotherapeutics, 12(3):631-40.」 「 Smith-Dijak, A. I., Nassrallah, W. B., Zhang, L. Y., Geva, M., Hayden, M. R., & Raymond, L. A. (2019). Impairment and restoration of homeostatic plasticity in cultured cortical neurons from a mouse model of huntington disease. Frontiers in cellular neuroscience, 13, 209.」 「 Sandweiss AJ, Brandt VL, Zoghbi HY. (2020) "Advances in understanding of Rett syndrome and MECP2 duplication syndrome: prospects for future therapies". Lancet Neurol. Aug;19(8):689-698.」 「 Weng, S.M. et al. (2011). "Rett Syndrome: From Bed to Bench." Pediatrics and Neonatology, 52:309-316.」 「 Xu X, Pozzo-Miller L. EEA1 restores homeostatic synaptic plasticity in hippocampal neurons from Rett syndrome mice. J Physiol. 2017 Aug 15;595(16):5699-5712.」 「 Zuccato, Chiara, and Elena Cattaneo. 2009. "Brain-Derived Neurotrophic Factor in Neurodegenerative Diseases." Nature Reviews Neurology 5 (6): 311-22. https: / / doi.org / 10.1038 / nrneurol.2009.54.」

Claims

1. 1. A pharmaceutical composition for treating Rett Syndrome in a subject in need thereof, comprising: A pharmaceutical composition comprising pridopidine or a pharmaceutically acceptable salt thereof and at least one of the following compounds 1 or 4 or a pharmaceutically acceptable salt thereof:

2. 2. The pharmaceutical composition of claim 1, Symptoms of Rett Syndrome include abnormal gait, ataxia, impaired gait initiation, delayed acquisition of purposeful manual motor skills, partial or complete loss of acquired purposeful manual motor skills, abnormal hand movements, startle response, delayed crawling and / or walking, impaired ability to crawl and / or walk, or eye movement abnormalities; The pharmaceutical composition delays the onset, prevents the worsening, delays the worsening, or ameliorates at least one of the symptoms.

3. 3. The pharmaceutical composition according to claim 1 or 2, The pharmaceutically acceptable salt of pridopidine comprises hydrochloride, hydrobromide, nitrate, perchlorate, phosphate, sulfate, formate, acetate, aconate, ascorbate, benzenesulfonate, benzoate, cinnamate, citrate, embonate, enanthate, fumarate, glutamate, glycolate, lactate, maleate, malonate, mandelate, methanesulfonate, naphthalene-2-sulfonate, phthalate, salicylate, sorbate, stearate, succinate, tartrate, or toluene-p-sulfonate.

4. 3. The pharmaceutical composition according to claim 1 or 2, The pharmaceutical composition includes a pharmaceutically acceptable salt of compound 1 or 4, such as hydrochloride, hydrobromide, nitrate, perchlorate, phosphate, sulfate, formate, acetate, aconate, ascorbate, benzenesulfonate, benzoate, cinnamate, citrate, embonate, enanthate, fumarate, glutamate, glycolate, lactate, maleate, malonate, mandelate, methanesulfonate, naphthalene-2-sulfonate, phthalate, salicylate, sorbate, stearate, succinate, tartrate, or toluene-p-sulfonate.

5. 2. The pharmaceutical composition of claim 1, The composition is administered orally, nasally, by inhalation, subcutaneous injection, intravenously, intraperitoneally, intramuscularly, intranasally, bucally, intravaginally, intrarectally, intraocularly, intrathecally, topically, or intradermally.

6. 6. The pharmaceutical composition of claim 5, The composition is a pharmaceutical composition for oral administration.

7. 2. The pharmaceutical composition of claim 1, The composition is administered in the form of an aerosol, inhalable powder, injectable, liquid, gel, solid, capsule, or tablet.

8. 7. The pharmaceutical composition of claim 6, The composition is administered orally; The composition is formulated as a tablet, capsule, pill, powder, multiparticulates in a capsule or sachet, a liquid solution, or a liquid suspension.

9. 2. The pharmaceutical composition of claim 1, The pharmaceutical composition is administered less frequently than once a day.

10. 2. The pharmaceutical composition of claim 1, The pharmaceutical composition is administered once daily or twice daily.

11. 2. The pharmaceutical composition of claim 1, A pharmaceutical composition wherein the daily dose of the pharmaceutical composition comprises said pridopidine in an amount of 0.5 to 315 mg.

12. 2. The pharmaceutical composition of claim 1, A pharmaceutical composition wherein the daily dose of the pharmaceutical composition comprises said pridopidine in an amount of 0.5 to 45 mg.

13. 2. The pharmaceutical composition of claim 1, A pharmaceutical composition wherein the daily dose of the pharmaceutical composition comprises said pridopidine in an amount of 10 to 100 mg.

14. 2. The pharmaceutical composition of claim 1, A pharmaceutical composition wherein the daily dose of the pharmaceutical composition comprises said pridopidine in an amount of 45 to 90 mg.

15. 2. The pharmaceutical composition of claim 1, A pharmaceutical composition wherein the daily dose of the pharmaceutical composition comprises said pridopidine in an amount of 45 to 180 mg.

16. 2. The pharmaceutical composition of claim 1, The pharmaceutical composition wherein the composition is administered in one or two doses per day.

17. The pharmaceutical composition according to any one of claims 1 to 16, The composition comprises: the pridopidine or a pharmaceutically acceptable salt thereof; A pharmaceutical composition comprising at least one of Compound 1 or a pharmaceutically acceptable salt thereof, Compound 4 or a pharmaceutically acceptable salt thereof, and a combination thereof.

18. The pharmaceutical composition according to any one of claims 1 to 16, The composition comprises: the pridopidine or a pharmaceutically acceptable salt thereof; and a pharmaceutical composition comprising Compound 1 or a pharmaceutically acceptable salt thereof.

19. The pharmaceutical composition according to any one of claims 1 to 16, The composition comprises: the pridopidine or a pharmaceutically acceptable salt thereof; Compound 1 or a pharmaceutically acceptable salt thereof; and a pharmaceutical composition comprising the compound 4 or a pharmaceutically acceptable salt thereof.

20. 2. The pharmaceutical composition of claim 1, A pharmaceutical composition, wherein the weight ratio of said pridopidine to said compound 1 and / or 4 is in the range of 1:0.0001 to 1:0.

1.

21. 21. The pharmaceutical composition of claim 20, A pharmaceutical composition, wherein the weight ratio of said pridopidine to said compound 1 and / or 4 is in the range of 1:0.005 to 1:0.

1.

22. 21. The pharmaceutical composition of claim 20, A pharmaceutical composition, wherein the weight ratio of said pridopidine to said compound 1 and / or 4 is in the range of 1:0.001 to 1:0.

005.

23. 3. The pharmaceutical composition of claim 2, The abnormal hand movements are hand twisting movements, hand wringing movements, clapping movements, hand washing movements, hand smacking movements, hand rubbing movements, and / or repetitive hand-to-mouth movements.

24. 3. The pharmaceutical composition of claim 2, The eye movement abnormality is prolonged staring, excessive blinking, crossed eyes, and / or closing one eye at a time.

25. 3. The pharmaceutical composition of claim 2, The composition improves the symptoms by at least 20%, at least 30%, at least 50%, at least 80%, or 100%.

26. 2. The pharmaceutical composition of claim 1, The composition is effective for increasing or maintaining serum BDNF levels in the subject, or for increasing brain BDNF levels in the subject.

27. 2. The pharmaceutical composition of claim 1, The subject has a mutation in at least one of the methyl-CpG binding protein 2 (MeCP2) gene, the cyclin-dependent kinase-like 5 (CDKL5) gene, or the forkhead box protein G1 (FOXG1) gene.

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