Use of pridopidine and its analogues in the treatment of RETT syndrome

Abstract

This invention provides a method for treating a subject suffering from Rett syndrome, comprising administering a pharmaceutical composition to the subject comprising pridopidine or a pharmaceutically acceptable salt thereof and at least one of compounds 1-8 disclosed herein or a pharmaceutically acceptable salt thereof, in order to treat the subject.

Description

[0001] Sequence list declaration

[0002] An ASCII file entitled P-609324-PC-SQL-15SEP17.txt, consisting of 2,997 bytes, which was created on September 15, 2017 and filed concurrently with this application, is incorporated herein by reference.

[0003] background

[0004] Rett syndrome

[0005] Rett syndrome (RTT) is a neurodevelopmental disorder that is estimated to affect 1 in 10,000 to 15,000 live births across all racial and ethnic groups (Amaral 2007).

[0006] In 95%–97% of cases, RTT is caused by a mutation 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 recorded cases, the mutation is hereditary or passed from one generation to the next. The MeCP2 gene is involved in the production of the methyl-cysteine-binding protein 2 (MeCP2). The MeCP2 protein binds methylcytosine and 5-hydroxymethylcytosine at CpG sites in the promoter regions of target genes, controlling their transcription by recruiting co-repressors and co-activators (Pozzo-Miller 2015).

[0007] In rare cases, RTT may also be caused by partial gene deletions or mutations in other genes (such as cyclin-dependent kinase-like 5 (CDKL5), forkhead box protein G1 (FOXG1)) and other possible genes that have not yet been identified.

[0008] RTT is 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 stalls after a seemingly normal developmental period. In the second stage, between 1 and 4 years of age, a period of regression begins, during which the child begins to lose acquired skills. In this stage, purposeful hand movements are replaced by stereotyped movements, such as hand-twisting or clapping, and social withdrawal begins, leading to a diagnosis of autism. Gait disturbances become apparent in this stage, such as ataxia and apraxia. Other symptoms include respiratory rhythm disturbances, sleep disturbances, bruxism, and inappropriate laughing or crying for a period of time.

[0009] In the third stage, between the ages of 2 and 10, the deterioration plateaus. Motor function may stabilize, and social interactions may improve, but these may be accompanied by seizures. In the fourth stage, from age 10 to adulthood, further motor deterioration occurs. Patients typically develop Parkinson's syndrome (rigidity, bradykinesia, and tremor), as well as myasthenia gravis and osteoporosis (Sandweiss 2020).

[0010] RTT patients exhibit abnormal neuronal morphology and reduced brain size, which manifests as incoordination, intellectual decline, gait abnormalities, and seizures (Weng 2011).

[0011] Currently, there is no treatment for RTT, only supportive care.

[0012] Pridopidin

[0013] Pridopidine (4-[3-(methanesulfonyl)phenyl]-1-propylpiperidine) (formerly known as ACR16) is a drug under development for the treatment of Huntington's disease. The chemical name of pridopidine is 4-(3-(methanesulfonyl)phenyl)-1-propylpiperidine, and its chemical registry number is CAS 346688-38-8 (CSID:7971505 2016). The chemical registry number for pridopidine hydrochloride is 882737-42-0 (CSID:25948790 2016).

[0014] Pridolpicine binds selectively to the sigma-1 receptor (S1R, Ki = 0.057 μM) with high affinity. It binds with low affinity to other receptors in the central nervous system, including dopamine D2 / D3 receptors, adrenergic α2C receptors, and serotonin 5-HT1A. Compared to adrenergic α2C receptors and dopamine D3 receptors (Ki = 1.28 μM and 1.63 μM, respectively), pridolpicine exhibits ~28×-fold higher selectivity for S1R, ~64-fold higher selectivity relative to the 5-HT1A receptor (Ki = 3.63 μM), ~100-fold higher selectivity relative to the sigma-2 receptor (S2R, Ki = 5.45 μM), and ~500-fold higher selectivity relative to the dopamine D2 receptor (Ki = 29.5 μM) (Table 1) (Johnston et al., 2019).

[0015] Table 1: Binding affinity and selectivity of pridopidine to CNS receptors

[0016]

[0017]

[0018] Source: Johnston et al., 2019.

[0019] 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 upregulation of pathways known to promote neuronal plasticity and survival.

[0020] Pridolpicine upregulates the secretion of neuroprotective brain-derived neurotrophic factor (BDNF) and downstream signal transduction (Geva et al., 2016).

[0021] Decreased BDNF is associated with the pathogenesis of Rett syndrome. Homeostatic synaptic plasticity (HSP), a process that maintains the stability of neural networks and forms the basis of learning and cognitive abilities, is regulated by BDNF (Smith-Dijak et al., 2019). HSP is also disrupted 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 from the HD YAC128 mouse model (Smith-Dijak et al., 2019).

[0022] Regulation of the BDNF pathway is a major component of the S1R-mediated neuroprotective effect of pridopidine. Invention Overview

[0024] This invention provides a method for treating a subject suffering from Rett syndrome (RTT), comprising administering a pharmaceutical composition to the subject, said pharmaceutical composition comprising pridopidine or a pharmaceutically acceptable salt thereof and at least one of compounds 1-8:

[0025]

[0026]

[0027] Or its pharmaceutically acceptable salt, in order to treat the subject.

[0028] The present invention also provides a method for increasing brain-derived neurotrophic factor (BDNF) levels in subjects suffering from RTT, comprising administering a pharmaceutical composition to the subject, said pharmaceutical composition comprising pridopidine or a pharmaceutically acceptable salt thereof and at least one of compounds 1-8 or a pharmaceutically acceptable salt thereof, so as to thereby increase the subject's serum BDNF levels.

[0029] Attached Figure Description

[0030] Figure 1Feature recovery as a measure of the therapeutic effect of drugs in a Mecp2-KO Rett syndrome model mouse. This figure is intended to provide information about the data in... Figure 4 , Figure 5 and Figure 12 Explanation of how it is analyzed.

[0031] Left side: “Cloud Map”: Visualization of the control-disease-(disease + treatment) group relationship in the best discriminative feature space. The cloud is plotted in two-dimensional space, with the two coordinates being the highest-rated decorrelational features analyzed by the automated NeuroCube system (more information about NeuroCube can be found in Examples 1 and 3).

[0032] The therapeutic effect of the drug can be represented as a combination of two components: one along the “recovery line” (the line connecting the centers of the control (WT) and disease (MECP2-KO) clouds, shown as a thick left-pointing arrow with a diagonal line at the top), and the component orthogonal to this direction (“pointing away” from it), shown as an upward arrow with a parallel line inside. The relative length of the “recovery” arrow with respect to the control-disease distance can then be interpreted as “recovery due to the drug,” while the relative length of the “other effects” arrow (orthogonal to the inner parallel line) represents the characteristic changes in moving the diseased mice + treatment group away from the control group. This summary of the analysis can be effectively represented as a bar graph (right), which is commonly referred to as… Restore Mark .

[0033] Right side: "Recovery Marker" plot: The bar chart represents a summary of the recovery analysis. Overlap probabilities and distinguishing probabilities are summed up to 100%. Recovery ranges from 0 to the distinguishing probability value. Lower overlap indicates better quality of the disease model and higher distinguishability between the WT group and the Rett mouse group. "Other behavioral effects" are in the same relative units (the length of the distance between the WT group and the Rett mouse group).

[0034] Figure 2: Prilidopidine rescues hindlimb clasping phenotype in 8-week-old Rett model mice. Muscle strength of limb muscles was assessed using clasping. Hindlimb clasping was measured in mice by grasping the tail and gently lifting until the forepaws were just lifted off a table surface. The experimenter observed the leg and determined whether the limb was clasped or open. Wild-type (WT, n=24) mice showed no clasping at 8 weeks of age (0%). Rett mice (female MECP2 heterozygous, “RETT” mice, n=20) showed significant clasping at 8 weeks of age (~17%) (#p<0.05 compared to placebo). Rett mice (“RETT”) treated with prilidopidine (30 mg / kg bid, n=20) showed rescue at 8 weeks (i.e., no clasping, 0%) compared to the RETT mediator group (^p<0.06). Data are mean ± SEM, N-1 two-proportion test. Source: DPR-2016-061.

[0035] Figure 3 Prilidopidine improved the mean startle response to acoustic stimuli in RETT mice at 8 and 12 weeks of age. Acoustic startle measures the unconditioned reflex response to external auditory stimuli. Prepulse inhibition (PPI) consists of the inhibited startle response to an auditory stimulus following the presentation of a weak auditory stimulus or a prepulse. Acoustic startle was measured by placing mice in a startle chamber with attenuated sound, where the intensity of movement performed by the mice was measured. The amount of inhibition after the acoustic prepulse is expressed as a percentage of the baseline startle response (from a single startle test), excluding the startle response of the initial habituation module. Compared with WT mice (n=24), mediator-treated RETT mice (n=20) showed significant inhibition of the startle response at 8 and 12 weeks, respectively (p<0.05). Prilidopidine had significant rescue effects of ~40% and ~50% at 8 and 12 weeks, respectively (n=24, p<0.05). Data are presented as mean ± SEM. #p < 0.05 compared to the WT media group. *p < 0.05 compared to the Rett media group.

[0036] Figure 4Prilidopidine restores gait characteristics in 8-week-old female Rett model mice. Summary of the analysis of gait characteristic restoration of prilidopidine (30 mg / kg, twice daily (bid)) in 8-week-old female Rett model mice. Untreated Rett mice (bottom right - largest cloud) can be distinguished from WT mice (top cloud) by gait characteristics, as the two clouds are completely separated. The bar graph shows that prilidopidine 30 mg / kg bid significantly improved the gait of Rett mice at 8 weeks by 45% (p = 0.0181) (darkest color). The relationship between WT Rett female mice (bottom right - largest cloud) and Rett female mice + prilidopidine (bottom left - largest cloud) in the best distinguishing feature space is visualized using cloud plots (see [link]). Figure 1 (Explanation in the text).

[0037] Figure 5 Prilidopidine restored gait characteristics in 12-week-old female Rett model mice. Summary of the analysis of gait characteristic recovery in 12-week-old female Rett mice with prilidopidine (30 mg / kg bid). Untreated Rett mice (bottom left - largest cloud) can be distinguished from WT mice (top cloud) by gait characteristics, as the two clouds are completely separated. Prilidopidine (30 mg / kg bid) showed a significant 55% recovery of gait characteristics in Rett mice at 12 weeks (p = 0.0022) (darkest color). The relationship between WT (top cloud), Rett mice (bottom left - largest cloud), and Rett + prilidopidine (bottom right - largest cloud) in the best distinguishing feature space is visualized using cloud plots (see [link]). Figure 1 (Explanation in the text).

[0038] Figures 6A-6C , Figure 7 , Figure 8 , Figure 9 , Figure 10 These figures show the mRNA levels of BDNF transcripts measured in the brains of female heterozygous MeCP2 (Rett) mice. Column A represents vector-treated WT mice, column B represents vector-treated female Rett mice, column C represents pridopidine-treated female Rett mice (3 mg / kg bid), and column D represents pridopidine-treated female Rett mice (30 mg / kg bid). The relative amounts of gene targets were normalized to the geometric mean against the relative amounts of housekeeping genes ATP5B, GAPDH, and RPL13A. The relative levels of target genes were then normalized against the WT vector group. All data are presented as mean ± SEM. ANOVA was followed by Tukey's multiple comparison test.

[0039] Figures 6A-6CPridopidine does not affect the levels of housekeeping genes ATP5B, GAPDH, and RPL13A. Relative mRNA expression of whole-brain control housekeeping genes: ATP5B (6A), GAPDH (6B), and RPL13A (6C); each normalized to the geometric mean against the other two genes. Pridopidine does not affect the expression of control housekeeping genes.

[0040] Figure 7 Prilidopidine had no effect on BDNF I mRNA levels. Relative mRNA expression of BDNF I in the whole brain. In Rett mice, BDNF I levels were reduced by ~20% (p<0.001). Prilidopidine did not affect BDNF I levels in Rett mice.

[0041] Figure 8 Prilidopidine rescues BDNF IV mRNA levels. Relative mRNA expression of BDNF IV in the whole brain. In Rett mice, BDNF IV levels were reduced by ~15% (p<0.001). Prilidopidine at doses of 3 mg / kg bid and 30 mg / kg bid significantly increased BDNF IV mRNA levels by ~30% (p<0.001).

[0042] Figure 9 Prilidopidine had no effect on BDNF VI mRNA levels. Relative mRNA expression of BDNF VI in the whole brain. In Rett mice, BDNF VI levels were reduced by ~25% (p<0.05). Prilidopidine did not affect BDNF VI levels in Rett mice.

[0043] Figure 10 Prilidopidine rescues BDNF IV mRNA levels. Relative mRNA expression of BDNF IV in the whole brain. In Rett mice, BDNF IV levels were reduced by ~15% (p<0.0001). Prilidopidine at doses of 3 mg / kg bid and 30 mg / kg bid significantly increased BDNF IV mRNA levels back to WT levels (p<0.001).

[0044] Figures 11A-11B Distinguishing features through Neurocube gait analysis

[0045] In these experiments, male B6.129P2-Mecp2tm2Bird / J (Rett-KO) mice were used. The figure shows discrimination plots output from gait analysis using Neurocube, an automated tool for evaluating the effects of pridopidine. Bars represent different behavioral traits assessed by Neurocube along with their values. Curves with square outlines show the rating for each behavioral trait. The rating is correlated with the ability of a particular trait to distinguish between control and disease groups. The relative difference (%) between trait values ​​in two different sets was calculated and plotted in order corresponding to the trait rating along with its variation from 0% to 100%. The feature name is a combination of parameter name and claw name: STRL: stride length; STPL: step length; BSWD: base width; STRD: stride duration; STND: standing duration; SWGD: swing duration; Avg_speed: average running speed; FR - right forelimb; FL - left forelimb; HR - right hindlimb; HL - left hindlimb.

[0046] Features are arranged from top to bottom in the figure based on their ratings. More information about Neurocube can be found in Examples 1 and 3. Distinguishing features of Rett-KO male mice compared to WT mice at 6 weeks of age (A) and 7 weeks of age (B).

[0047] Figure 12 Pridolpiride restored gait characteristics in male Rett mice at 6 and 7 weeks of age. A summary of the recovery analysis of Rett syndrome effects in male B6.129P2-Mecp2tm2Bird / J (Rett-KO) mice (see [link to relevant documentation]). Figure 1 (Explanation in the text). Top: Bar graph showing the recovery effect of pridopidine (30 mg / kg bid) in Rett-KO model mice. Bottom: Contour plot visualizing the relationship between WT, Rett, and Rett+pridopidine in the optimal distinguishing feature space (WT, Rett, Rett+pridopidine). Pridopidine showed 44% (left, p<0.05) and 100% (right, p<0.05) gait defect recovery in 6-week-old and 7-week-old Rett model mice, respectively. One-way ANOVA.

[0048] Figures 13A-13BPrilidopidine 45 mg bid improved gait and balance in patients with early-stage HD (TFC 7-13) at weeks 26 and 52. Changes in gait and balance relative to baseline were measured at weeks 26 (A) and 52 (B) in patients with early-stage HD (baseline TFC 7-13) using the UHDRSTMS method in the PRIDE-HD study. Patients receiving placebo showed worsening of gait and balance (Δ0.14 relative to baseline, a positive value indicating worsening). The potency of prilidopidine was assessed by analyzing changes in gait and balance relative to baseline (UHDRS TMS; gait and balance) using the Mixed Model Repeated Measures (MMRM) method with the Unified Huntington's Disease Rating Scale total motor score throughout the 52-week period.

[0049] At week 26, pridopidine 45 mg bid improved gait function compared to placebo (Δ -0.48, a negative value indicating improvement, p = 0.0563). See Table 2 below with accompanying figures. At week 52, pridopidine 45 mg bid treatment showed a trend toward improvement compared to placebo (Δ -0.41, a negative value indicating improvement).

[0050] Table 2: Improvement of gait and balance in early-stage HD patients (HD1+HD2, TFC 7-13) of PRIDE-HD at weeks 26 and 52.

[0051]

[0052] Figures 14A-14B Prilidopidine 45 mg bid improved gait and balance in HD1 (TFC 11-13) patients at weeks 26 and 52. The PRIDE-HD study showed changes in UHDRS TMS gait and balance relative to baseline in HD1 (baseline TFC 11-13) patients at weeks 26 (A) and 52 (B). The efficacy of prilidopidine was assessed by analyzing changes in gait and balance relative to baseline (UHDRS TMS; gait and balance) using a mixed-model repeated measures (MMRM) approach with the Unified Huntington's Disease Rating Scale total motor score throughout the 52-week period.

[0053] At week 26, patients receiving placebo showed a worsening of gait and balance. Prilidopidine showed a trend toward improvement (Δ-0.31 relative to placebo). See Table 3 below with accompanying figures. At week 52, patients receiving placebo showed a worsening relative to baseline. Prilidopidine 45 mg bid treatment showed a significant improvement compared to placebo (Δ-0.94 relative to placebo, p = 0.0445, negative values ​​indicate improvement).

[0054] Table 3: Improvement of gait and balance in HD1 (TFC 11-13) patients in PRIDE-HD at weeks 26 and 52 by pridopidine 45 mg bid.

[0055]

[0056] Figures 15A-15B Prilidopidine 45 mg bid improved gait and balance in HD2 (TFC 7-10) patients at weeks 26 and 52. Changes in UHDRSTMS gait and balance relative to baseline in HD2 (baseline TFC 7-10) patients at weeks 26 (A) and 52 (B) in the PRIDE-HD study. The efficacy of prilidopidine was assessed by analyzing changes in gait and balance relative to baseline (UHDRS TMS; gait and balance) using mixed-model repeated measures (MMRM) with the Unified Huntington's Disease Rating Scale total motor score throughout the 52-week period. At both weeks 26 and 52, prilidopidine 45 mg bid treatment showed a trend toward improvement compared to placebo (Δ vs. placebo at weeks 26 and 52 were -0.53 and -0.18, respectively; negative values ​​indicate improvement). See Table 4 below with accompanying figures.

[0057] Table 4: Improvement of gait and balance in HD2 patients (TFC 7-10) in PRIDE-HD at weeks 26 and 52 by pridopidine 45 mg bid.

[0058]

[0059] Figures 16A-16B Pridolpicine and compound 4 had a synergistic effect 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 in situ ELISA. Figure 16A The study compared 0.001 μM of pridopidine and 0.001 μM of compound 4. Pridopidine alone increased BDNF secretion by 13.5%. Compound 4 alone had no effect on BDNF secretion (-1.5%). Together, pridopidine and compound 4 increased BDNF secretion by 59.1%, an effect greater than the additive effect of the two compounds administered individually. Figure 16B The study compared 0.005 μM of pridopidine and 0.001 μM of compound 4. Pridopidine alone increased BDNF secretion by 26.0%. Compound 4 alone had no effect on BDNF secretion (-1.5%). Together, pridopidine and compound 4 increased BDNF secretion by 80.7%, an effect greater than the additive effect of the two compounds administered individually.

[0060] Figure 17 Pridopidine and Compound 1 have a synergistic effect on BDNF release from B104 cells. The synergistic effect of pridopidine and Compound 1 on BDNF release from B104 cells was investigated. B104 neuroblastoma cells were incubated with the test compounds for 5 days, and BDNF levels were assessed using in situ ELISA. Pridopidine alone at a concentration of 0.01 μM increased BDNF secretion by 3.4%. Compound 1 alone at a concentration of 1 μM increased BDNF secretion by 12.5%. The combined effect of pridopidine and Compound 1 increased BDNF secretion by 53.1%, which is greater than the additive effect of either compound alone. Invention Details

[0062] The present invention provides a method for treating a subject suffering from Rett syndrome (RTT), comprising administering a pharmaceutical composition to the subject, said pharmaceutical composition comprising pridopidine or a pharmaceutically acceptable salt thereof, so as to treat the subject.

[0063] This invention provides a method for treating a subject suffering from Rett syndrome (RTT), comprising administering a pharmaceutical composition to the subject, said pharmaceutical composition comprising pridopidine or a pharmaceutically acceptable salt thereof and at least one of compounds 1-8:

[0064]

[0065] In order to treat the subject.

[0066] In one embodiment, the present invention provides a method for delaying the onset of at least one symptom associated with Rett syndrome in a subject suffering from Rett syndrome, preventing the exacerbation of at least one symptom associated with Rett syndrome in a subject suffering from Rett syndrome, delaying the exacerbation of at least one symptom associated with Rett syndrome in a subject suffering from Rett syndrome, or improving at least one symptom associated with Rett syndrome in a subject suffering from Rett syndrome, wherein the method 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.

[0067] In one implementation, the subject is a human patient. In one implementation, the human patient is female. In another implementation, the human patient is male.

[0068] 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.

[0069] In some embodiments, the method of the present invention includes 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 pridopidine salt is pridopidine hydrochloride. In yet another embodiment, pridopidine is a hydrochloride, hydrobromide, nitrate, perchlorate, phosphate, sulfate, formate, acetate, aconate, ascorbate, benzenesulfonate, benzoate, cinnamate, citrate, embonate, heptanoate, fumarate, glutamate, glycolate, lactate, maleate, malonate, mandelate, methanesulfonate, naphthalene-2-sulfonate, phthalate, salicylate, sorbate, stearate, succinate, tartrate, or p-toluenesulfonate.

[0070] In some embodiments, the method of the present invention includes 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 salt of at least one of compounds 1-8 is a hydrochloride. In yet another embodiment, the salt of at least one of compounds 1-8 is a hydrochloride, hydrobromide, nitrate, perchlorate, phosphate, sulfate, formate, acetate, aconate, ascorbate, benzenesulfonate, benzoate, cinnamate, citrate, embosate, heptanoate, fumarate, glutamate, glycolate, lactate, maleate, malonate, mandelate, methanesulfonate, naphthalene-2-sulfonate, phthalate, salicylate, sorbate, stearate, succinate, tartrate, or p-toluenesulfonate.

[0071] In one embodiment, the pharmaceutical composition disclosed herein is administered orally, nasally, by inhalation, by subcutaneous injection, or via intravenous, intraperitoneal, intramuscular, intranasal, sublingual, vaginal, rectal, intraocular, intrathecal, local, or intradermal routes. In one embodiment, pridopidine is administered orally.

[0072] In one embodiment, the pharmaceutical composition disclosed herein is administered in the form of an aerosol, inhalable powder, injectable, liquid, gel, solid, capsule, tablet, or multigranule.

[0073] In one embodiment, the pharmaceutical composition disclosed herein is administered orally and formulated as a multiplying, liquid solution, or liquid suspension in tablets, capsules, pills, powders, capsules, or sachets.

[0074] In one embodiment, the pharmaceutical composition disclosed herein is applied periodically.

[0075] In one embodiment, the pharmaceutical composition disclosed herein is administered less frequently than once daily. In one embodiment, pridopidine is administered daily. In one embodiment, pridopidine is administered once daily. In another embodiment, pridopidine is administered more frequently than once daily. In one embodiment, pridopidine is administered twice daily.

[0076] In some embodiments, the pharmaceutical compositions disclosed herein comprise pridopidine or a pharmaceutically acceptable salt thereof, and at least one of compounds 1-8 or a pharmaceutically acceptable salt thereof, for use in the methods of the invention, administered at a daily dose of pridopidine or a pharmaceutically acceptable salt thereof between 0.5 mg and 315 mg. In another embodiment, the composition is administered at a daily dose of 0.5 mg to 10 mg pridopidine or a pharmaceutically acceptable salt thereof. In another embodiment, the composition is administered at a daily dose of 10 mg to 22.5 mg pridopidine or a pharmaceutically acceptable salt thereof. In another embodiment, the composition is administered at a daily dose of 22.5 mg to 315 mg pridopidine or a pharmaceutically acceptable salt thereof. In another embodiment, the composition is administered at a daily dose of 10 mg to 315 mg pridopidine or a pharmaceutically acceptable salt thereof. In another embodiment, the composition is administered at a daily dose of 0.5 mg to 50 mg pridopidine or a pharmaceutically acceptable salt thereof. In another embodiment, the composition is administered at a daily dose of 22.5 mg to 315 mg pridopidine or a pharmaceutically acceptable salt thereof. In another embodiment, the composition is administered at a daily dose of 45 mg to 250 mg pridopidine or a pharmaceutically acceptable salt thereof. In another embodiment, the composition is administered at a daily dose of 45 mg to 135 mg pridopidine or a pharmaceutically acceptable salt thereof. In another embodiment, the composition is administered at a daily dose of 90 mg to 315 mg pridopidine or a pharmaceutically acceptable salt thereof.

[0077] In another embodiment, the dosage of pridopidine administered is about 1 mg / day, about 5 mg / day, about 10 mg / day, 20 mg / day, 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 another embodiment, the dosage of pridopidine administered is 45 mg / day. In another embodiment, the dosage of pridopidine administered is 90 mg / day. In another embodiment, the dosage of pridopidine administered is 180 mg / day. In another embodiment, the dosage of pridopidine administered is 225 mg / day.

[0078] In one embodiment, the pharmaceutical composition disclosed herein is administered once daily. In one embodiment, the pharmaceutical composition disclosed herein is administered twice daily.

[0079] In one embodiment, the pharmaceutical composition disclosed herein is administered in doses 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 of pridopidine. In another embodiment, the amount of pridopidine administered in the composition is 45 mg. In yet another embodiment, the amount of pridopidine administered in the composition disclosed herein is between 10 mg and 45 mg.

[0080] In one embodiment, the compositions disclosed herein are administered twice daily, wherein each composition comprises an amount of 45 mg pridopidine per dose.

[0081] In one embodiment, the pharmaceutical composition disclosed herein is first administered as early as 1 day or more after birth. In one embodiment, the pharmaceutical composition disclosed herein is first administered within 1 day after the subject's birth. In one embodiment, the pharmaceutical composition disclosed herein is first administered within 1 week after the subject's birth. In one embodiment, the pharmaceutical composition disclosed herein is first administered within 1 month after the subject's birth. In one embodiment, the pharmaceutical composition disclosed herein is first administered within 3 months after the subject's birth. In one embodiment, the pharmaceutical composition disclosed herein is first administered within 6 months after the subject's birth. In one embodiment, the pharmaceutical composition disclosed herein is first administered within 9 months after the subject's birth. In one embodiment, the pharmaceutical composition disclosed herein is first administered within 12 months after the subject's birth. In one embodiment, the pharmaceutical composition disclosed herein is first administered within 18 months after the subject's birth. In one embodiment, the pharmaceutical composition disclosed herein is first administered within 3 years after the subject's birth. In one embodiment, the pharmaceutical composition disclosed herein is first administered within 5 years after the subject's birth. In one embodiment, the pharmaceutical composition disclosed herein is first administered within 10 years after the subject's birth. In one embodiment, the pharmaceutical composition disclosed herein is first administered within 15 years of the subject's birth. In one embodiment, the pharmaceutical composition disclosed herein is first administered within 20 years of the subject's birth. In one embodiment, the pharmaceutical composition disclosed herein is first administered within 25 years of the subject's birth. In one embodiment, the pharmaceutical composition disclosed herein is first administered within 30 years of the subject's birth. In one embodiment, the pharmaceutical composition disclosed herein is first administered 30 years or more after the subject's birth.

[0082] In one embodiment, the periodic application of the pharmaceutical composition disclosed herein lasts for at least 3 days, at least 30 days, at least 42 days, at least 8 weeks, at least 12 weeks, at least 24 weeks, at least 6 months, at least 1 year, at least 2 years, at least 5 years, at least 10 years, at least 15 years, at least 20 years, at least 25 years, or 30 years or longer.

[0083] In one embodiment, the pharmaceutical composition disclosed herein treats the subject by delaying the onset of symptoms.

[0084] In one embodiment, the pharmaceutical composition disclosed herein is used to treat a subject suffering from Rett syndrome by: delaying the onset of at least one symptom in the subject, preventing the worsening of at least one symptom in the subject, delaying the worsening of at least one symptom in the subject, or improving at least one symptom in the subject. In one embodiment, the pharmaceutical composition disclosed herein improves at least one symptom in a subject suffering from Rett syndrome or delays the worsening of at least one symptom in a subject suffering from Rett syndrome. In one embodiment, the pridopidine pharmaceutical composition disclosed herein treats a subject by improving at least one symptom in the subject.

[0085] In some embodiments, the present invention relates to a method for delaying the onset of at least one RTT symptom in a subject, preventing the worsening of at least one RTT symptom in a subject, delaying the worsening of at least one RTT symptom in a subject, or improving at least one RTT symptom in a subject, 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, wherein the RTT symptom is abnormal gait, ataxia, impaired gait initiation, delay in acquiring purposeful hand skills or partial or complete loss of acquired purposeful hand skills, or the symptom is abnormal hand movements, startle response or delayed crawling and / or walking; reduced crawling and / or walking ability; or abnormal eye movements.

[0086] In one embodiment, the symptom is a delay in acquiring motor skills. In one embodiment, the symptom is a delayed sitting, crawling, and / or walking. In one embodiment, the symptom is partial or complete loss of acquired motor skills. In one embodiment, the symptom is a reduced ability to sit, crawl, and / or walk. In one embodiment, the motor skill is a motor coordination skill.

[0087] In one implementation, the symptom is abnormal gait. In one implementation, the symptom is ataxia. In one implementation, the symptom is apraxia. In one implementation, the symptom is muscle weakness. In one implementation, the symptom is spasticity. In one implementation, the symptom is rigidity. In one implementation, the symptom is impaired gait initiation.

[0088] In one embodiment, the symptom is abnormal muscle tone. In one embodiment, the symptom is hypotonia. In one embodiment, the symptom is peripheral vasomotor dysfunction. In one embodiment, the symptom is scoliosis. In one embodiment, the symptom is impaired gait initiation.

[0089] In one embodiment, the symptom is a delay in acquiring purposeful hand skills. In one embodiment, the symptom is partial or complete loss of acquired purposeful hand skills. In one embodiment, the symptom is abnormal hand movements. In one embodiment, abnormal hand movements are twisting, clenching, clapping, washing hands, tapping hands, rubbing hands, and / or repeatedly placing hands near the mouth.

[0090] In one embodiment, the symptom is a delay in acquiring communication skills. In one embodiment, the symptom is partial or complete loss of acquired communication skills. In one embodiment, the communication skill is a language skill. In one embodiment, the language skill is a verbal skill. In one embodiment, the communication skill is eye contact.

[0091] In one implementation, the symptom is abnormal eye movement. In one implementation, abnormal eye movement is prolonged staring, excessive blinking, strabismus, and / or closing one eye at a time.

[0092] In one implementation, the symptom is irregular breathing. In one implementation, the irregular breathing occurs while the subject is awake. In one implementation, the irregular breathing is apnea.

[0093] In one implementation, irregular breathing is hyperventilation.

[0094] In one implementation, the symptom is bruxism (teeth grinding) when the subject is awake.

[0095] In one implementation, symptoms are increased irritability, decreased alertness, and / or reduced attention span. In another implementation, symptoms are inappropriate laughing and / or screaming.

[0096] In one implementation, the symptom is a seizure.

[0097] In one implementation, the symptom is a cardiac abnormality. In one implementation, the cardiac abnormality is bradycardia. In one implementation, the cardiac abnormality is tachycardia.

[0098] In one implementation, the symptom is a reduced response to pain. In one implementation, the symptom is growth retardation. In one implementation, the symptom is microcephaly. In one implementation, the symptom is impaired sleep patterns. In one implementation, the symptom is malnutrition-related cold blue feet.

[0099] In one embodiment, the pharmaceutical composition disclosed herein improves symptoms by at least 5%. The composition improves symptoms by at least 10%. In one embodiment, the composition improves symptoms by at least 20%. In one embodiment, the composition improves symptoms by at least 30%. In one embodiment, the composition improves symptoms by at least 50%. In one embodiment, the composition improves symptoms by at least 80%. In one embodiment, the composition improves symptoms by 100%.

[0100] In one embodiment, the pharmaceutical composition disclosed herein is used to treat a subject by improving the subject's ability to perform daily living activities, perform domestic chores, manage finances, and / or perform anoccupation. In one embodiment, the pharmaceutical composition disclosed herein is used to treat a subject by reducing the level of care required by the subject.

[0101] In one embodiment, the pharmaceutical composition disclosed herein is used to treat a subject by maintaining the subject's ability to perform daily living activities, do housework, manage finances, and / or engage in occupation.

[0102] In one embodiment, the pharmaceutical composition disclosed herein is effective in increasing serum BDNF levels in a subject. In one embodiment, the composition is effective in increasing BDNF levels in the brain of a subject. In one embodiment, the composition is effective in maintaining serum BDNF levels in a subject.

[0103] The present invention also provides a pharmaceutical composition comprising a certain amount of pridolpicin for use in treating subjects with RTT.

[0104] The present invention also provides a pharmaceutical composition in unit dosage form for use in treating subjects with RTT.

[0105] In one embodiment, the dosage of pridopidine administered is 0.5 mg / day to 315 mg / day. In one embodiment, the dosage of pridopidine is 10 mg to 315 mg. In one embodiment, the dosage of pridopidine is 90 mg to 315 mg. In one embodiment, the dosage of pridopidine is 90 mg to 225 mg. In another embodiment, the dosage 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 dosage of pridopidine is 45 mg. In one embodiment, the dosage of pridopidine is 90 mg. In one embodiment, the dosage of pridopidine is 180 mg. In the implementation plan, the dosage of pridopidine is 225 mg.

[0106] The present invention also provides the use of the pharmaceutical compositions disclosed herein in the manufacture of a medicament for treating a subject suffering from RTT.

[0107] The present invention also provides the use of a certain amount of pridopidine for the treatment of subjects with RTT.

[0108] The present invention also provides a method for increasing serum BDNF levels in subjects with RTT, comprising administering the pharmaceutical composition disclosed herein to the subject to thereby increase the subject's serum BDNF levels. The present invention also provides a method for increasing brain BDNF levels in subjects with RTT, comprising administering the pharmaceutical composition disclosed herein to the subject to thereby increase the subject's brain BDNF levels.

[0109] For the foregoing embodiments, each embodiment disclosed herein is considered applicable to each of the other disclosed embodiments. Furthermore, elements described in the method embodiments can be used in the pharmaceutical composition, use, and packaging embodiments described herein, and vice versa.

[0110] Pharmaceutical compositions for use in the methods of the present invention:

[0111] In some embodiments, the method of the present invention utilizes a pharmaceutical composition comprising pridopidine or a pharmaceutically acceptable salt thereof and at least one of compounds 1-8.

[0112]

[0113] Or its pharmaceutically acceptable salt.

[0114] In other embodiments, the method of the present invention utilizes a pharmaceutical composition comprising pridopidine or a pharmaceutically acceptable salt thereof and compound 1 or a pharmaceutically acceptable salt thereof.

[0115] In other embodiments, the method of the present invention utilizes a pharmaceutical composition comprising pridopidine or a pharmaceutically acceptable salt thereof and compound 2 or a pharmaceutically acceptable salt thereof.

[0116] In other embodiments, the method of the present invention utilizes a pharmaceutical composition comprising pridopidine or a pharmaceutically acceptable salt thereof and compound 3 or a pharmaceutically acceptable salt thereof.

[0117] In other embodiments, the method of the present invention utilizes a pharmaceutical composition comprising pridopidine or a pharmaceutically acceptable salt thereof and compound 4 or a pharmaceutically acceptable salt thereof.

[0118] In other embodiments, the method of the present invention utilizes a pharmaceutical composition comprising pridopidine or a pharmaceutically acceptable salt thereof and compound 5 or a pharmaceutically acceptable salt thereof.

[0119] In other embodiments, the method of the present invention utilizes a pharmaceutical composition comprising pridopidine or a pharmaceutically acceptable salt thereof and compound 6 or a pharmaceutically acceptable salt thereof.

[0120] In other embodiments, the method of the present invention utilizes a pharmaceutical composition comprising pridopidine or a pharmaceutically acceptable salt thereof and compound 7 or a pharmaceutically acceptable salt thereof.

[0121] In other embodiments, the method of the present invention utilizes a pharmaceutical composition comprising pridopidine or a pharmaceutically acceptable salt thereof and compound 8 or a pharmaceutically acceptable salt thereof.

[0122] In other embodiments, the method of the present invention utilizes a pharmaceutical composition comprising pridopidine or a pharmaceutically acceptable salt thereof, and at least one of compound 1, compound 4, a pharmaceutically acceptable salt thereof, or a combination thereof.

[0123] In other embodiments, the method of the present invention utilizes a pharmaceutical composition comprising pridopidine or a pharmaceutically acceptable salt thereof and compound 1 or a pharmaceutically acceptable salt thereof.

[0124] In other embodiments, the method of the present invention utilizes a pharmaceutical composition comprising pridopidine or a pharmaceutically acceptable salt thereof and compound 4 or a pharmaceutically acceptable salt thereof.

[0125] In other embodiments, the method of the present invention utilizes a pharmaceutical composition comprising pridopidine or a pharmaceutically acceptable salt thereof, compound 1 and compound 4 or a pharmaceutically acceptable salt thereof.

[0126] In other embodiments, the method of the present invention utilizes a pharmaceutical composition comprising pridopidine salt, wherein the salt is a hydrochloride, hydrobromide, nitrate, perchlorate, phosphate, sulfate, formate, acetate, aconate, ascorbate, benzenesulfonate, benzoate, cinnamate, citrate, embosate, heptanoate, fumarate, glutamate, glycolate, lactate, maleate, malonate, mandelate, methanesulfonate, naphthalene-2-sulfonate, phthalate, salicylate, sorbate, stearate, succinate, tartrate, or p-toluenesulfonate.

[0127] In other embodiments, the method of the present invention utilizes a pharmaceutical composition comprising at least one of the salts of compounds 1-8, wherein the salt is a hydrochloride, hydrobromide, nitrate, perchlorate, phosphate, sulfate, formate, acetate, aconate, ascorbate, benzenesulfonate, benzoate, cinnamate, citrate, embosate, heptanoate, fumarate, glutamate, glycolate, lactate, maleate, malonate, mandelate, methanesulfonate, naphthalene-2-sulfonate, phthalate, salicylate, sorbate, stearate, succinate, tartrate, or p-toluenesulfonate.

[0128] In other embodiments, the method of the present invention utilizes a pharmaceutical composition, wherein the composition is an oral dose unit comprising 0.5 mg to 315 mg of pridopidine or a pharmaceutically acceptable salt thereof. In other embodiments, the oral dose unit comprises 0.5 mg to 10 mg of pridopidine. In other embodiments, the oral dose unit comprises 10 mg to 22.5 mg of pridopidine. In other embodiments, the oral dose unit comprises 22.5 mg to 45 mg of pridopidine. In other embodiments, the oral dose unit comprises 45 mg to 250 mg of pridopidine. In other embodiments, the oral dose unit comprises 45 mg to 135 mg of pridopidine. In other embodiments, the oral dose unit comprises 90 mg to 315 mg of pridopidine.

[0129] In other embodiments, the method of the present invention utilizes 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 other embodiments, 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 other embodiments, 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.

[0130] In other embodiments, the concentration of compound 1, compound 2, compound 3, compound 4, compound 5, compound 6, compound 7, or compound 8, or a pharmaceutically acceptable salt thereof, in the composition is between 0.01% w / w and 10% w / w. In other embodiments, the concentration of compound 1, compound 2, compound 3, compound 4, compound 5, compound 6, compound 7, or compound 8, or a pharmaceutically acceptable salt thereof, in the composition is between 0.01% w / w and 0.05% w / w. In other embodiments, the concentration of compound 1, compound 2, compound 3, compound 4, compound 5, compound 6, compound 7, or compound 8, or a pharmaceutically acceptable salt thereof, in the composition is between 0.01% w / w and 0.5% w / w. In other embodiments, the concentration of compound 1, compound 2, compound 3, compound 4, compound 5, compound 6, compound 7, or compound 8, or a pharmaceutically acceptable salt thereof, in the composition is between 0.01% w / w and 0.15% w / w. In other embodiments, the concentration of compound 1, compound 2, compound 3, compound 4, compound 5, compound 6, compound 7, or compound 8, or a pharmaceutically acceptable salt thereof, in the composition is between 0.01% w / w and 0.15% w / w. In other embodiments, the concentration of compound 1, compound 2, compound 3, compound 4, compound 5, compound 6, compound 7, or compound 8, or a pharmaceutically acceptable salt thereof, in the composition is between 0.01% w / w and 0.5% w / w. In other embodiments, the concentration of compound 1, compound 2, compound 3, compound 4, compound 5, compound 6, compound 7, or compound 8, or a pharmaceutically acceptable salt thereof, in the composition is between 0.01% w / w and 1% w / w. In other embodiments, the concentration of compound 1, compound 2, compound 3, compound 4, compound 5, compound 6, compound 7, or compound 8, or a pharmaceutically acceptable salt thereof, in the composition is between 0.05% w / w and 0.2% w / w. In other embodiments, the concentration of compound 1, compound 2, compound 3, compound 4, compound 5, compound 6, compound 7, or compound 8, or a pharmaceutically acceptable salt thereof, in the composition is between 0.05% w / w and 0.3% w / w. In other embodiments, the concentration of compound 1, compound 2, compound 3, compound 4, compound 5, compound 6, compound 7, or compound 8, or a pharmaceutically acceptable salt thereof, in the composition is between 0.05% w / w and 0.4% w / w.

[0131] While the compounds according to the invention can be administered in their original form, it is preferred that the active ingredient (optionally in the form of a physiologically acceptable salt) be introduced into the pharmaceutical composition together with one or more excipients, carriers, buffers, diluents, and / or other commonly used pharmaceutical adjuvants. In embodiments, the invention provides pharmaceutical compositions comprising an active compound or a pharmaceutically acceptable salt or derivative thereof, together with one or more pharmaceutically acceptable carriers, and optionally other therapeutic and / or prophylactic ingredients known and used in the art. One or more carriers must be "acceptable" in the sense of compatibility with other components of the formulation and harmless to the recipient.

[0132] The pharmaceutical compositions of the present invention can be administered via any convenient route suitable for the desired therapy. Preferred routes of administration include oral administration, particularly in tablet, capsule, multigranule, powder, or liquid form, and parenteral administration, particularly by skin, subcutaneous, intramuscular, or intravenous injection. The pharmaceutical compositions used in the methods of the present invention are oral dosage units, formulated as multigranules, liquid solutions, or liquid suspensions in tablets, capsules, pills, powders, capsules, or sachets.

[0133] the term

[0134] As used herein, and unless otherwise stated, each of the following terms should have the definition set forth below.

[0135] As used herein, “pridopidine” means pridopidine base or a pharmaceutically acceptable salt thereof, and its derivatives or analogues, such as deuterium-rich pridopidine and its salts. Examples of deuterium-rich pridopidine and its salts and methods of their preparation can be found in U.S. Patent Application Publications Nos. 2013-0197031, 2016-0166559, and 2016-0095847, the entire contents of each of which are incorporated herein by reference.

[0136] "Deuterium-rich" means that the abundance of deuterium at any relevant site in a compound is greater than the abundance of naturally occurring deuterium at that site in a given amount of the compound. The naturally occurring distribution of deuterium is approximately 0.0156%. Therefore, in "deuterium-rich" compounds, the abundance of deuterium at any relevant site is greater than 0.0156%, and can range from greater than 0.0156% to 100%. Deuterium-rich compounds can be obtained by exchanging hydrogen with deuterium or by synthesizing compounds from deuterium-rich starting materials.

[0137] The active compounds according to the invention can be provided in any form suitable for the intended administration. Suitable forms include pharmaceutically acceptable salts and prodrug or prodrug forms of the compounds of the invention.

[0138] "Its salt" refers to the salt of the compound of the present invention, which has been modified by preparing 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 or base addition salt of the compound of the present invention suitable for pharmaceutical use. Pharmaceutically acceptable salts can be formed by procedures well known and described in the art. One means of preparing such a salt is by treating the compound of the present invention with an inorganic base.

[0139] Examples of acid addition salts of the compounds of the present invention include, but are not limited to, hydrochlorides, hydrobroms, nitrates, perchlorates, phosphates, sulfates, formates, acetates, aconates, ascorbic acid salts, benzenesulfonates, benzoates, cinnamates, citrates, embolates, heptanoates, fumarates, glutamates, glycolates, lactates, maleates, malonates, mandelates, methanesulfonates, naphthalene-2-sulfonates, phthalates, salicylates, sorbates, stearates, succinates, tartrates, p-toluenesulfonates, etc. In some embodiments, pridopidine is a pharmaceutically acceptable salt, such as an HCl salt or a tartrate salt. Preferably, in any embodiment of the invention as described herein, pridopidine is in the form of its hydrochloride salt.

[0140] As used herein, the “amount” or “dosage” of pridopidine, measured in milligrams, refers to the number of milligrams of pridopidine (4-[3-(methylsulfonyl)phenyl]-1-propyl-piperidine) present in the product, regardless of the form of the product. For example, a unit dose containing “90 mg pridopidine” means that the amount of pridopidine in the product is 90 mg, regardless of the form of the product. Therefore, when in a salt form, such as pridopidine hydrochloride, the weight of the salt form necessary to provide a dose of 90 mg pridopidine will be greater than 90 mg due to the presence of the salt.

[0141] As used herein, “unit dose,” “unit doses,” and “unit dosage form(s)” refer to a single entity for drug administration. “Unit dose,” “unit doses,” and “unit dosage form(s)” can be prepared for oral dosage forms such as tablets, capsules, pills, powders, and granules.

[0142] As used herein, in the context of numerical values ​​or ranges, “about” means 90%–110% of the numerical value or range described or claimed.

[0143] "Administered to a subject" or "administered to a (human) patient" means to give, dispense, or apply medicines, drugs, or remedies to a subject / patient to delay, alleviate, cure, or reduce symptoms associated with a condition (e.g., a pathological condition). Oral administration is one method of administering the compounds of the present invention to a subject.

[0144] The compounds invented according to this subject matter can be administered in the form of an alkaline base or a pharmaceutically acceptable salt, preferably as a pharmaceutical composition together with one or more excipients, excipients, carriers, buffers, diluents and / or other commonly used pharmaceutical adjuvants.

[0145] "Pharmaceutically acceptable carrier" means a carrier or excipient suitable for use in humans and / or animals without excessive adverse side effects (such as toxicity, irritation, and allergic reactions) and commensurate with a reasonable benefit / risk ratio. It can be a pharmaceutically acceptable solvent, suspending agent, or medium for delivering the compounds of the present invention to a subject.

[0146] Application can be periodic. As used herein, "periodic application" means repeated / repeated application separated by certain time intervals. The time intervals between applications are preferably consistent from time to time. Periodic application can include, for example, 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.

[0147] As used herein, “treat” or “treating” encompasses the reduction, lessening, decrease in severity, elimination, or substantial elimination or improvement of physical, mental, or emotional limitations in a subject suffering from RTT. Treatment also refers to delaying or preventing the reduction of disease-related symptoms or deficits.

[0148] As used herein, “effective” as a quantity that effectively achieves the endpoint means, when used in the manner disclosed herein, an amount of a component sufficient to produce the indicated therapeutic response without excessive adverse side effects (such as toxicity, irritation, or allergic reactions) in proportion to a reasonable benefit / risk ratio. For example, an effective quantity for treating symptoms of Rett syndrome. The specific effective quantity varies depending on factors such as the particular condition being treated, the patient's physical condition, the type of mammal being treated, the duration of treatment, the nature of concurrent therapies (if any), and the specific formulation and structure of the compound or its derivatives used.

[0149] It should be understood that, while providing a parameter range, the present invention also provides all integers within that range and numbers up to one-tenth of that range. For example, "22mg-300.0mg" includes 22.0mg, 22.1mg, 22.2mg, 22.3mg, 22.4mg, etc., up to 300.0mg (including the endpoints).

[0150] The invention will be better understood by referring to the experimental details below, but those skilled in the art will readily understand that the detailed experiments are merely illustrative of the invention, which is more fully described in the claims below.

[0151] Experimental details

[0152] Example 1: Evaluation of the efficacy of pridopidine in a heterozygous female MeCP2 mouse model of Rett syndrome

[0153] The aim of this study was to evaluate the role of pridopidine in a female Rett syndrome MeCP2-Het (BIRD) mouse model (Guy 2001).

[0154] Material:

[0155] Pridopidine (3 mg / kg and 30 mg / kg) is administered orally twice daily at a dose volume of 10 ml / kg (6 hours apart). On the day of the test, pridopidine is administered 30 minutes before the test.

[0156] Drug administration was initiated when mice were approximately 5.5 weeks old and continued until the end of behavioral testing. Behavioral testing was conducted at 8 and 12 weeks of age.

[0157] Female MeCP2 (MeCP2_HET, Rett) mice and wild-type (MeCP2_WT, WT) littermates were housed at 20°C–23°C with 50% relative humidity and a 12 / 12 light / dark cycle. Food and water were provided at all times. All tests were conducted during the light phase. Animals were examined and weighed throughout the study to ensure adequate health and fitness and to minimize operation-related nonspecific stress. All animals were examined and weighed before the start of the study and throughout the study to ensure adequate health and fitness and to minimize operation-related nonspecific stress. A 12 / 12 light / dark cycle was maintained during the study. Room temperature was maintained between 20°C and 23°C, and relative humidity was maintained at approximately 50%. Food and water were provided at all times during the study. Tests were conducted during the animals' light cycle phases.

[0158] method:

[0159] Treatment group:

[0160] • WT mice - vector (saline), n = 24

[0161] • Rett HET MeCP2 mice - mediator (saline), n=24

[0162] • Rett HET MeCP2 mice - Pridopidine (3 mg / kg; orally twice daily, bid), n = 20

[0163] • Rett HET MeCP2 mice - Pridopidine (30 mg / kg; orally twice daily, bid), n = 20

[0164] Behavioral testing:

[0165] (1) Use The system performs gait analysis.

[0166] The system is a platform that uses computer vision to detect changes in gait geometry and gait dynamics in rodent models of neurological disorders, pain, and neuropathy. This platform is unique for gait testing for the following reasons:

[0167] The platform is fully automated, and therefore eliminates any bias or subjectivity.

[0168] The system captures both gait geometry and gait dynamics (standing posture, swaying, propulsion, etc.).

[0169] Mice were placed in a NeuroCube for 5 minutes for testing. The most important features of the collected disease phenotypes (symptom descriptors) were identified and rated. Complex bioinformatics algorithms were used to calculate the distinguishing probability between WT and Rett HET MeCP2 mice and to test the ability of the test compound to reverse the disease phenotype. The distinguishing factor between mutants and wild-type mice and the recovery of disease features in Rett HET MeCP2 mice treated with the test compound were calculated.

[0170] (2) Holding hands

[0171] Use the grasping technique to assess the muscle strength of a limb. Hold the mouse by its tail and gently lift it until the front paws are just off the table surface. The experimenter observes the legs and determines whether the limb is grasping or open. After the test, return the animal to its test or living cage. Determine and report the percentage of grasping in the hind limbs.

[0172] (3) Startle response / pre-pulse inhibition (PPI)

[0173] Acoustic startle measurement measures the unconditioned reflex response to external auditory stimuli. Prepulse inhibition (PPI), consisting of a suppressed startle response (a reduction in amplitude) to an auditory stimulus following the presentation of a weak auditory stimulus or a prepulse, has been used as a tool to assess sensorimotor gating deficits, such as those observed in schizophrenia.

[0174] Mice were placed in a PPI chamber (Med Associates) for a 5-minute period of white noise (70 dB) acclimatization. After the acclimatization period, the testing phase began automatically. This phase started with a habituation module of six individually presented startle stimuli, followed by ten PPI modules of six different types of tests.

[0175] The trial types were: zero (no stimulus), startle (120 dB), startle plus pre-pulse (4 dB, 8 dB, and 12 dB above background noise, i.e., 74 dB, 78 dB, or 82 dB), and pre-pulse alone (82 dB). Trial types were presented randomly within each module. Each trial began with a 50 ms zero period during which baseline shift was recorded. This was followed by a 20 ms period during which the pre-pulse stimulus was presented and the response to the pre-pulse was measured. After another 100 ms, the startle stimulus was presented for 40 ms, and the response was recorded from the start of the startle for a 100 ms duration. Responses were sampled every millisecond. The intervals between trials were variable, averaging 15 s (range 10 s to 20 s).

[0176] In the individual startle test, basic auditory startle was measured, and in the pre-pulse plus startle test, the amount of inhibition of normal startle was determined and expressed as a percentage of the basic startle response (from the individual startle test), excluding the startle response of the first habituation module.

[0177] Brain collection:

[0178] Brain samples were collected 60 minutes after administration of pridopidine following completion of all behavioral tests. Mice were euthanized and decapitated via cervical dislocation. Whole brains were collected from 10 mice / treatment group, weighed, and then frozen on dry ice. Samples were stored at -80°C until analysis of brain-derived neurotrophic factor (BDNF).

[0179] BDNF Analysis:

[0180] Total RNA extraction:

[0181] The tissue (whole brain) was homogenized, and RNA was extracted and quantified. Aliquots were then reverse transcribed into cDNA.

[0182] Perform up to three independent RT reactions for each RNA sample. Use the primers detailed in Table 5 below for qPCR.

[0183] Table 5: qPCR and primer / probe information

[0184]

[0185]

[0186] qPCR data analysis:

[0187] Whole-brain cDNA prepared from collected WT-treated animal samples was used as a calibrator (the calibrator was diluted identically to the sample cDNA) to normalize the differences between plates.

[0188] Each cDNA sample (1:10 dilution) was tested in triplicate, and the Ct values ​​were averaged. Values ​​greater than 0.5 standard deviations from the average were discarded.

[0189] The relative amounts of PCR products (relative to calibrators) are calculated as follows:

[0190] relative amount of target genes

[0191] = (PCR efficiency target) (Ct校准品-Ct样品)

[0192] The relative amount of the household gene 1

[0193] =(PCR efficiency maintenance 1) (Ct校准品-Ct样品)

[0194] The relative amount of the household gene 2

[0195] =(PCR efficiency maintenance 2) (Ct校准品-Ct样品)

[0196] The relative amount of the home-keeping gene 3

[0197] =(PCR efficiency maintenance 3) (Ct校准品-Ct样品)

[0198] The geometric mean of the three household-management genes is calculated as follows:

[0199] Geometric Mean

[0200] = (Relative amount of household management gene 1 * Relative amount of household management gene 2 * Relative amount of household management gene 3) (1 / 3)

[0201] The relative levels of target genes are calculated as follows:

[0202] Relative amount of target genes ÷ Geometric mean of housekeeping genes

[0203] The relative levels of target genes were then normalized for the WT mediator group.

[0204] Statistical analysis:

[0205] Data from standard tests were analyzed by genotype (t-test) and by treatment (ANOVA), followed by post-hoc comparisons where appropriate. Repeated measures ANOVA was performed for some measures. For hold data, a two-proportion N-1 test was performed. An effect was considered significant if p < 0.05. All data are expressed as mean and standard error of the mean (sem). Values ​​within ± 2 standard deviations from the mean were considered outliers.

[0206] Data analysis from NeuroCube:

[0207] NeuroCube's output is a collection of behavioral features submitted for analysis using machine learning techniques employed in bioinformatics. Many of these features are correlated (e.g., reclining count and support count). Therefore, the PGI forms statistically independent combinations of the original features (also known as decorrelated features), thus more effectively distinguishing the two groups.

[0208] Each decorrelation feature extracts information from the entire cluster of the original features, resulting in a new feature space with lower dimensionality. Next, PGI applies a proprietary feature rating algorithm to score the discriminative power of each feature (the ability to separate two groups, such as a control group and a disease group).

[0209] Rating is a crucial part of the analysis because it measures the relevance of each feature change: if there are significant changes in some irrelevant features that measure a particular phenotype, a low rating for that feature will automatically reduce the role of such changes in the analysis, thus eliminating the need to resort to conventional "feature selection" methods and discard information hidden in less informative features. Rating algorithms can be applied to original or new features to gain insights into key control-disease differences.

[0210] Feature analysis: Quantitative assessment of disease phenotypes

[0211] In the new feature space, the overlap between “clouds” (approximately the Gaussian distribution of the mouse groups in the rated decorrelation feature space) is used as a quantitative measure of separability (“distinguishability”) between the two groups. For visualization purposes, for each cloud plot, its semi-axis is equal to one standard deviation along the corresponding dimension.

[0212] result:

[0213] Behavioral testing:

[0214] (1) Hug

[0215] Rett syndrome patients lose or fail to acquire purposeful hand movements, and these movements are replaced by stereotyped movements such as hand wringing. In the Rett mouse model, this symptom is associated with a hindlimb grasping phenotype. Compared to WT mice, Rett model mice show significantly more grasping ( Figure 2 ).like Figure 2 As shown, pridopidine improved holding at 8 weeks. RETT mice treated with the vector showed significantly more holding compared to WT mice. At 8 weeks, pridopidine (30 mg / kg bid) normalized this behavior (p<0.06). At 8 weeks, the column representing WT mice treated with the vector and those treated with pridopidine (30 mg / kg bid) was zero. This suggests that pridopidine 30 mg / kg bid is effective in treating this symptom and may delay its onset.

[0216] (2) Fright response / PPI

[0217] The reduced alertness and attention duration associated with Rett syndrome symptoms were reproduced in a mouse model and assessed using acoustic startle response. Compared to WT mice, mediator-treated Rett mice showed a significant inhibition of startle response at 8 and 12 weeks, respectively (p<0.05). At 8 and 12 weeks, pridopidine (3 mg / kg bid) had a significant beneficial effect on startle response of ~40% and ~50%, respectively (p<0.05). Figure 3 As shown in the image.

[0218] (3)

[0219] The distinguishing probabilities between WT and Rett mice were 90% and 94% at 8 and 12 weeks of age, respectively. Some key gait features that differentiate WT from Rett mice include a longer stride and stride length, a narrower base width, and less claw strength compared to Rett mice.

[0220] At 8 weeks, the effect of primidopidin on gait performance was shown to be... Figure 4 In the study, at 12 weeks, the effect of primidopinol on gait performance was shown to be... Figure 5 In the mean, pridopidine (30 mg / kg bid) showed significant recovery of overall gait characteristics at weeks 8 and 12 (45% and 55%, respectively).

[0221] Further analysis revealed significant differences in specific gait domains, as shown in Table 6 below. Rett mice were significantly different from WT control mice overall in all gait characteristics. Week 8 data showed that pridopidine (3 mg / kg and 30 mg / kg bid) improved body movement and individual gait in Rett mice. At week 12, pridopidine treatment (3 mg / kg bid) significantly improved...

[0222] Gait and body movement alone. At 12 weeks, pridopidine (30 mg / kg bid) was also observed to have a significant effect on gait, body movement and paw position alone.

[0223] Table 6: Effects of pridopidine on gait at 8 and 12 weeks.

[0224]

[0225] BDNF Analysis

[0226] The effect of pridopidine on relative BDNF expression in WT and Rett mouse brain samples is shown in Figure 6- Figure 10 middle.

[0227] The expression level of the housekeeping gene mRNA in the whole brain control group did not change between the different animal groups examined for treatment (see [link]). Figures 6A-6C ).

[0228] Compared with WT (carrier), BDNF I mRNA expression was significantly reduced by ~20% in the Rett (carrier) treatment group (p<0.001). Pridolpicine treatment (3 mg / kg or 30 mg / kg bid) did not affect BDNF I mRNA levels in Rett mice (see [link to original text]). Figure 7 ).

[0229] Compared with the WT (carrier) group, BDNF IV mRNA expression was significantly reduced by ~15% in the Rett (carrier) treatment group (p<0.01). Pridolpicine treatment (3 mg / kg or 30 mg / kg bid) rescued ~30% of the downregulated BDNF IV mRNA in Rett mice, approaching WT levels (p<0.001) (see [link to original text]). Figure 8 ).

[0230] Compared with WT (carrier), BDNF VI mRNA expression was significantly reduced by ~25% in the Rett (carrier) treatment group (p<0.05). Pridolpicine treatment (3 mg / kg or 30 mg / kg bid) did not affect BDNF VI mRNA levels in Rett mice (see [link to original text]). Figure 9 ).

[0231] Compared with the WT (carrier) group, BDNF IX mRNA expression was significantly reduced by ~20% in the Rett (carrier) treatment group (p<0.0001). Pridolpicine treatment (3 mg / kg or 30 mg / kg bid) rescued downregulated BDNF IX mRNA in Rett mice to near WT levels (p<0.001) (see [link to original text]). Figure 10 ).

[0232] in conclusion

[0233] This study evaluated the effects of long-term administration of pridopidine on gait, hindlimb gripping, and startle / PPI in Rett model mice.

[0234] Rett mice exhibited significant changes in gait measurements, distinguishing them from WT mice. They also displayed a stereotyped hindlimb holding phenotype. Additionally, Rett mice showed suppressed acoustic startle responses compared to WT mice. Prilidopidine treatment significantly salvaged hindlimb holding at 8 weeks of age (30 mg / kg bid) and significantly improved startle responses at 8 and 12 weeks (3 mg / kg bid) compared to mediator-treated Rett mice. Rett mice treated with prilidopidine (30 mg / kg bid) showed significant recovery of gait characteristics at 8 and 12 weeks.

[0235] Treatment with two doses of pridopidine (3 mg / kg and 30 mg / kg bid) completely salvaged the downregulated mRNA levels of BDNF IV and BDNF IX. The positive effect of pridopidine on BDNF mRNA expression is consistent with the improvements observed in the behavioral paradigm.

[0236] Example 2: RNA analysis of MeCP2 mice treated with pridopidine

[0237] method:

[0238] Female Rett model mice (MeCP2 heterozygotes) and wild-type (WT) littermates aged approximately 5.5 weeks were treated orally (per os, PO) with pridopidine or a carrier. Pridopidine (3 mg / kg and 30 mg / kg bid) was administered orally twice daily at a dose volume of 10 ml / kg (bid, 6 hours apart). Four treatment groups were established: 1. WT mice - carrier, 2. Rett mice - carrier, 3. Rett mice - pridopidine (3 mg / kg; bid), 4. Rett mice - pridopidine (30 mg / kg; bid).

[0239] This study assessed whether pridopidine reversed the aberrant transcription observed in Rett mice. This was accomplished by testing whether pridopidine restored the expression of disrupted genes in the disease context back to WT levels. Additionally, the effects of pridopidine on gene expression in a Rett syndrome mouse model were evaluated.

[0240] result:

[0241] Gene set enrichment analysis (GSEA) showed that pridopidine reversed the expression characteristics of Rett syndrome genes in the striatum and cortex.

[0242] Table 7: Pridopidin reversed gene expression signals in the striatum of Rett model mice.

[0243]

[0244] Table 8: Priligopidin reversed gene expression signals in the cortex of Rett model mice.

[0245]

[0246] A comparison of broad gene expression patterns revealed that pridopidine strongly reversed gene expression patterns in both the striatum and cortex of Rett mice.

[0247] Table 9: Pridopidin reversed the Rett disease gene in the striatum.

[0248]

[0249] NES-Normalized Enrichment Score

[0250] Table 9 shows that pridopidine 3 mg / kg bid reversed the striatal effect in both directions.

[0251] Rett gene expression pattern (upregulation of genes downregulated relative to WT in Rett mice, and downregulation of genes upregulated relative to WT in Rett mice). Pridolpicin 30 mg / kg bid significantly upregulated genes downregulated relative to WT in Rett mice.

[0252] Table 10: Priligopidin reversed the Rett disease gene in the cortex.

[0253]

[0254]

[0255] NES-Normalized Enrichment Score

[0256] Table 10 shows that pridopidine at 3 mg / kg and 30 mg / kg bid reversed the Rett gene expression pattern in the cortex in two directions (upregulating genes downregulated relative to WT in Rett mice and downregulating genes upregulated relative to WT in Rett mice).

[0257] The effects of pridopidine on the expression of downstream genes in the BDNF-TrkB pathway were evaluated. Pridopidine 30 mg / kg bid significantly increased the expression of downstream genes in the BDNF pathway (Table 11).

[0258] Table 11: Pridolpiride increased the expression of downstream genes of BDNF.

[0259]

[0260] NES-Normalized Enrichment Score

[0261] Example 3: Pridolpiride improved gait function in a male Rett syndrome MeCP2 knockout (KO) mouse model (Rett-KO).

[0262] method:

[0263] A population of Rett model mice (Jackson Laboratories, Bar Harbor, ME; B6.129P2-Mecp2tm2Bird / J | Accession No.: 003890) was established by crossing 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 room with a temperature controlled between 20°C and 23°C, 50% humidity, and a 12 / 12 light / dark cycle. Room temperature was maintained between 20°C and 23°C, and relative humidity was maintained at approximately 50%. Food and water were provided continuously throughout the study. Additionally, the mice were given hydrogel daily upon initial observation of signs of hind limb extension and / or dyskinesia. After weaning, the mice were housed individually in OPTIMice cages. All animals remained individually housed for the remainder of the study. Mice were balanced and assigned to the treatment group using baseline weight and grip strength measurements taken before the start of the study. All tests were performed during the animals' photoperiod.

[0264] Pridopidine was evaluated at 30 mg / kg bid. The compound was dissolved in sterile DDW and administered orally twice daily (BID) at a dose volume of 10 mL / kg.

[0265] Gait analysis

[0266] The (NRC) system is one of PsychoGenics' proprietary technologies. It is a platform that uses computer vision to detect changes in rodent gait geometry and gait dynamics. This platform is unique for gait testing for the following reasons:

[0267] The platform is fully automated, and therefore eliminates any bias or subjectivity.

[0268] This system captures both gait geometry and gait dynamics (stance, sway, propulsion, etc.).

[0269] The sensitivity of computer vision and bioinformatics allows PsychoGenics to capture symptoms of disease models earlier and more accurately.

[0270] Gait analysis was performed on mice at 5, 6, and 7 weeks of age. Mice were placed... In the test, a 5-minute test was conducted. The most important features defining the disease phenotype (symptom descriptive symbols) were identified and rated. Complex bioinformatics algorithms were used to calculate the distinguishing probability between WT and Rett mice, and the ability of the test compound to reverse the disease phenotype was also tested.

[0271] Feature Analysis

[0272] Rating is an important part of the analysis because it measures the relevance of each feature change: if there are significant changes in some unrelated features for a particular phenotype, a low rating for that feature will automatically reduce the role of such changes in the analysis.

[0273] Calculate the relative difference (%) between eigenvalues ​​in two different sets and plot them together with their ratings as they vary from 0% to 100%, in order corresponding to the eigenvalue ratings.

[0274] Feature analysis - a list of features being analyzed

[0275] 1) Average speed: Measurement of the average speed over the traveled NRC length.

[0276] 2) Body position: The X and Y body coordinates, X and Y claw coordinates, and claw direction vector are measured using claw imaging parameters, as they are related to the subject's body movement.

[0277] 3) Gait: Measurement of the geometry (e.g., stride length, step length, base width) and dynamics (e.g., stride duration, step duration, swing duration) of gait.

[0278] 4) Imaging: Measurement of claw contact area, contact area perimeter and claw diameter (horizontal / vertical).

[0279] 5) Claw Positions: Record the position of each claw print relative to the body center. The superposition of all recorded relative positions for the four claws creates four point clusters (one for each claw). For each claw, measure the coordinates of the cluster center, its size, the number of claw prints, and the relative geometry of the cluster positioning.

[0280] 6) Rhythmality: The correlation coefficient between the gait signals of each claw and all other claws:

[0281] RF-LF, RF-LH, RF-RH, LH-RH, LH-RF, LF-RH, LH-RH; (F – forelimb; H – hindlimb; R – right; L – left).

[0282] Feature analysis: Quantitative assessment of disease phenotypes

[0283] In the feature space, the overlap between “clouds” (approximately the Gaussian distribution of the mouse groups in the rated decorrelational feature space) is used as a quantitative measure of the separability (“distinguishability”) between WT and Rett-KO mice (see [link to feature space]). Figure 1 For visualization purposes, we plot each cloud with its semi-axis equal to one standard deviation along the corresponding dimension.

[0284] Feature analysis: Drug-induced recovery

[0285] In the “Recovery Due to Drugs” trial, the data are typically presented in three categories: WT, Rett-KO, and Rett-KO plus pridopidine treatment (“treated”).

[0286] Therefore, it is beneficial to consider (and plot) the third group (treated) in the same coordinate system that best distinguishes it from the other groups (WT and Rett), such as Figure 1 As shown in the image.

[0287] Treatment group

[0288] The following treatment groups were used in this study.

[0289] 1. WT mice - vector

[0290] 2. Rett-KO (B6.129P2-Mecp2tm2Bird / J) mice - vector

[0291] 3. Rett-KO (B6.129P2-Mecp2tm2Bird / J) mice - Pridopidine (30 mg / kg bid)

[0292] Statistical analysis

[0293] Data were analyzed using repeated measures ANOVA, followed by post-hoc comparisons where appropriate. An effect was considered significant if p < 0.05. Data are expressed as mean and standard error of the mean (sem).

[0294] result:

[0295]

[0296] 1. Gait characteristics

[0297] The illustrations showing the differentiation between Rett-KO mice and WT mice at 6 and 7 weeks of age are shown in the figure. Figures 11A-11B In Chinese, the characteristic name is a combination of the parameter name and the claw name. FR – right forelimb; FL – left forelimb; HR – right hindlimb; HL – left hindlimb.

[0298] Gait feature analysis indicated that Rett-KO mice showed gait defects at both 6 and 7 weeks of age compared to WT mice, with distinguishing probabilities of 100% and 99%, respectively. At both 6 and 7 weeks of age, Rett-KO mice showed defects in gait geometry measurements (primarily stride length, base width, and stride length) compared to WT mice.

[0299] The effect of pridopidine (30 mg / kg bid) on gait defects in Rett-KO mice was evaluated at 6 and 7 weeks of age. A summary of the recovery is shown in... Figure 12 In Rett model mice at 6 and 7 weeks of age, pridopidine showed 44% and 100% recovery of gait deficits, respectively (both p < 0.05, ANOVA).

[0300] in conclusion

[0301] Genotypic comparisons revealed that Rett-KO mice exhibited significant deficiencies in gait measurements compared to WT mice. Long-term administration of pridopidine (30 mg / kg / bid) to Rett-KO mice showed significant gait recovery at weeks 6 and 7.

[0302] Example 4: At 52 weeks, pridopidine 45 mg bid improved gait and balance in patients with early-stage HD.

[0303] Changes in UHDRS TMS gait and mean metric scale relative to baseline (baseline TFC 7–13) at weeks 26 and 52 in early HD. Table 2 (in the attached figure description) and Figures 13A-13B The study showed a trend toward improved UHDRS TMS gait and balance in patients with early-stage HD treated with pridolpidine 45 mg bid compared to placebo at 26 and 52 weeks.

[0304] Early HDs included HD1 (TFC 11-13) and HD2 (TFC 7-10). Figure 14B Table 3 (in the figure description) shows the significant effect of pridopidine 45 mg bid on changes in gait and balance relative to baseline at 52 weeks compared with placebo (p = 0.0445). Figure 14A This study demonstrates a trend toward improvement in HD1 patients treated with pridopidine at 26 weeks. Figures 15A-15B Table 4 (in the figure description) shows the trend toward improvement in gait and balance relative to baseline in HD2 patients at 52 and 26 weeks compared with placebo.

[0305] Example 5 - Synergistic effect of pridopidine and compound 1, or pridopidine and compound 4 on BDNF secretion

[0306] Compounds 1 and 4, together with pridopidine, showed a synergistic effect on BDNF secretion from B104 neuroblastoma cells.

[0307] Compounds 1 and 4 showed selective binding to the Sigma-1 receptor (S1R) (Ki = 0.37 μM for compound 1 and Ki = 2.9 μM for compound 4) but not to the Sigma-2 receptor (S2R) (Ki > 100 μM for both compounds 1 and 4), as shown in Table 12.

[0308] Table 12: Binding affinity of pridopidine, compound 1 and compound 4 to Sigma-1 and Sigma-2 receptors

[0309] compound S1R Ki(μM) S2R Ki(μM) S1R selectivity ratio (S2R / S1R) Pridopidin 0.057 5.45 96 Compound 1 0.37 >100 >270 Compound 4 2.9 >100 >35

[0310] In vitro binding assays were performed at Eurofins Panlabs Taiwan, Ltd. Specific ligand binding was determined in the presence of excess unlabeled ligands. The inhibition constant (Ki) was calculated from the in vitro binding assays using the Cheng Prusoff equation (Cheng and Prusoff 1973). Source: Johnston et al., 2019 and NC20-PHARM-2.

[0311] Therefore, both compounds 1 and 4 have a high affinity for S1R, but no affinity for S2R (Ki>100).

[0312] Reduced levels of brain-derived neurotrophic factor (BDNF) play a key role in the pathogenesis of neurodegenerative and neurodevelopmental disorders, and its levels are reduced in 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).

[0313] Using in situ ELISA, pridopidine showed a dose-dependent increase in BDNF secretion in rat neuroblastoma cells. This effect was mediated by S1R activation, as pharmacological inhibition of S1R eliminated the effect of pridopidine (Geva, Birnberg, et al. 2016).

[0314] In evaluating the effects of compound 1 or compound 4 with pridopidine, the applicant identified an unexpected synergistic effect. This effect was observed in a BDNF in situ ELISA assay (Geva, Kusko, et al. 2016).

[0315] Therefore, the synergistic effect on BDNF release shown below is directly related to the therapeutic effects of pridopidine and compounds 1 and 4.

[0316] The following data surprisingly and unexpectedly show that pridopidine, together with compound 4 or compound 1, exhibits a synergistic effect on BDNF release.

[0317] Synergistic effect of compound 4 and pridopidine on BDNF release

[0318] Compared to untreated control cells, pridopidine alone induced a +13.6% increase in BDNF release at a concentration of 0.001 μM and a +26% increase at a concentration of 0.005 μM. Compound 4 alone had no effect on BDNF release (-1.5%) compared to untreated control cells at a concentration of 0.001 μM. However, pridopidine and compound 4 together exhibited a surprising synergistic effect on BDNF release.

[0319] Compared with untreated control cells, 0.001 μM pridopidine + 0.001 μM compound 4 induced a 59.1% increase in BDNF release. Figure 16A ).

[0320] Compared with untreated control cells, 0.005 μM pridopidine + 0.001 μM compound 4 induced an 80.7% increase in BDNF release. Figure 16B ).

[0321] The combined effect of pridopidine and compound 4 was greater than the sum of the effects of each compound alone, indicating a surprising synergistic effect on BDNF secretion. Results are shown as percentage changes (%) compared to the untreated control.

[0322] Synergistic effect of compound 1 and pridopidine on BDNF release

[0323] Compared with untreated control cells, pridopidine alone at a concentration of 0.01 μM induced a +3.4% increase in BDNF release. Compared with control, compound 1 alone at a concentration of 1 μM induced a +12.5% ​​increase in BDNF release. However, pridopidine and compound 1 together had a synergistic effect on BDNF release (+53.1%).

[0324] Compared with untreated control cells, pridopidine (0.01 μM) + compound 1 (1 μM) induced a 53.1% increase in BDNF release. Figure 17 ).

[0325] Similarly, these results indicate that pridopidine and compound 1 have a surprising and unexpected synergistic effect on BDNF secretion, as their combined effect (+53.1%) is greater than the sum of the effects of each compound alone.

[0326] Therefore, the applicant demonstrates that compounds 1 and 4 have selective binding affinity for S1R, and a surprising and unexpected synergistic effect with pridopidine on BDNF release.

[0327] Example 6: Evaluation of the efficacy of pridopidine in treating patients with RTT

[0328] Periodic intravenous or oral administration of pridopidine and at least one of compounds 1-8 (e.g., once or twice daily) to patients with Rett syndrome is effective in treating patients.

[0329] Administration of pridopidine effectively delayed the onset of symptoms in Rett patients.

[0330] Administration of pridopidine effectively prevented or delayed the exacerbation of at least one symptom in Rett patients, or improved at least one symptom in Rett patients.

[0331] Administration of pridopidine effectively prevented or delayed the deterioration of motor skills in Rett patients, or improved their motor skills. Administration of pridopidine effectively prevented the partial or complete loss of acquired motor skills in Rett patients.

[0332] Administration of pridopidine effectively prevented or delayed the deterioration of gait in Rett patients, or improved their gait.

[0333] Administration of pridopidine effectively prevented, delayed, or improved ataxia, apraxia, muscle weakness, spasticity, and / or rigidity in Rett patients. Administration of pridopidine effectively prevented, delayed, or improved impaired gait initiation in Rett patients.

[0334] Administration of pridopidine effectively prevents, delays or improves abnormal muscle tone, peripheral vasomotor dysfunction and / or scoliosis in Rett patients.

[0335] Administration of pridopidine effectively prevented or delayed the deterioration of purposeful hand skills in Rett patients, or improved their purposeful hand skills. Pridopidine effectively prevented, delayed, or improved abnormal hand movements, including but not limited to twisting, clenching, clapping, washing hands, tapping hands, rubbing hands, and repeatedly placing hands to the mouth. Pridopidine effectively prevented the partial or complete loss of acquired purposeful hand skills in Rett patients.

[0336] Administration of pridopidine effectively prevented or delayed the deterioration of communication skills in Rett patients, or improved their communication skills, including but not limited to verbal and normal eye contact. Administration of pridopidine effectively prevented the partial or complete loss of acquired communication skills in Rett patients.

[0337] Prilitopidine has been shown to effectively prevent, delay, or improve growth retardation, seizures, cardiac abnormalities, irregular breathing, impaired sleep patterns, bruxism during wakefulness, reduced response to pain, malnutrition-related cold feet, increased irritability, decreased alertness, reduced attention span, inappropriate laughter, and / or inappropriate screaming.

[0338] References

[0339] -Amaral, MD, et al. (2007) "TRPC channels as novel effectors of BDNFsignaling: Potential implications for Rett syndrome". Pharmacol Ther, 113(2):394-409.

[0340] -Cheng,Yung-Chi,and William H.Prusoff.1973.“Relationship between theInhibition Constant(KI)and the Concentration of Inhibitor Which Causes 50perCent Inhibition(I50)of an Enzymatic Reaction.”BiochemicalPharmacology.https: / / doi.org / 10.1016 / 0006-2952(73)90196-2.

[0341] -CSID:25948790,www.chemspider.com / Chemical-Structure.25948790.html(accessed 23:27,Jul 15,2016).

[0342] -CSID:7971505,www.chemspider.com / Chemical-Structure.7971505.html(accessed 23:33,Jul15,2016).

[0343] -Geva,Michal,et al."Pridopidine activates neuroprotective pathwaysimpaired in Huntington Disease."Human molecular genetics 25.18(2016):3975-3987.

[0344] -Guy J,Hendrich B,Holmes M,Martin JE,Bird A.(2001)A mouse MeCP2-nullmutation causes neurological symptoms that mimic Rett syndrome.Nat Genet.27(3):322–326.

[0345] -Isaias,I.U.,et al.(2014).“Gait Initiation in Children with RettSyndrome.”PLoS One,9(4):e92736.

[0346] -Johnston,Tom H.,Michal Geva,Lilach Steiner,Aric Orbach,SpyrosPapapetropoulos,Juha-Matti Savola,Ian J.Reynolds,et al.2019.“Pridopidine,aClinic-Ready Compound,Reduces3,4-Dihydroxyphenylalanine-Induced Dyskinesia inParkinsonian Macaques.”Movement Disorders,December.https: / / doi.org / 10.1002 / mds.27565.

[0347] -Katz,DM.2014.“Brain-Derived Neurotrophic Factor and Rett Syndrome.”Handbook ofExperimental Pharmacology 220:481–95.https: / / doi.org / 10.1007 / 978-3-642-45106-5_18.

[0348] -Pozzo-Miller,L.,Pati S.,&Percy,A.K.(2015).“Rett Syndrome:Reachingfor Clinical Trials.”Neurotherapeutics,12(3):631-40.

[0349] -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 incultured cortical neurons from amouse model of huntington disease.Frontiersin cellular neuroscience,13,209.

[0350] -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.

[0351] -Weng, SMet al. (2011). "Rett Syndrome: From Bed to Bench." Pediatrics and Neonatology, 52: 309-316.

[0352] -Xu

[0353] -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. sequence list <110> Prinia Neurotherapy Co., Ltd. Mija Jewa Michael Hayden <120> Use of pridopidine and its analogues in the treatment of RETT syndrome <130> P-609324-PC <150> 17 / 498,075 <151> 2021-10-11 <150> 16 / 952,123 <151> 2020-11-19 <160> 14 <170> PatentIn version 3.5 <210> 1 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> 5' primer sequence <400> 1 ggcacaatgc aggaaagg 18 <210> 2 <211> twenty one <212> DNA <213> Artificial Sequence <220> <223> 3' primer sequence <400> 2 tcagcaggca catagatagc c 21 <210> 3 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> 5' primer sequence <400> 3 ttgtggccaa gcaggtact 19 <210> 4 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> 3' primer sequence <400> 4 gttgatgcct tcacagcgta 20 <210> 5 <211> twenty one <212> DNA <213> Artificial Sequence <220> <223> 5' primer sequence <400> 5 caatgtgtcc gtcgtggatc t 21 <210> 6 <211> twenty two <212> DNA <213> Artificial Sequence <220> <223> 3' primer sequence <400> 6 gtcctcagtg tagcccaaga tg 22 <210> 7 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> 5' primer sequence <400> 7 agtctccagg acagcaaagc 20 <210> 8 <211> twenty three <212> DNA <213> Artificial Sequence <220> <223> 3' primer sequence <400> 8 tgcaaccgaa gtatgaaata acc 23 <210> 9 <211> twenty two <212> DNA <213> Artificial Sequence <220> <223> 5' primer sequence <400> 9 gctgccttga tgtttacttt ga 22 <210> 10 <211> twenty three <212> DNA <213> Artificial Sequence <220> <223> 3' primer sequence <400> 10 aaggatggtc atcactcttc tca 23 <210> 11 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> 5' primer sequence <400> 11 ccgagagctt tgtgtggac 19 <210> 12 <211> twenty one <212> DNA <213> Artificial Sequence <220> <223> 3' primer sequence <400> 12 tcatgcaacc gaagtatgaa a 21 <210> 13 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> 5' primer sequence <400> 13 gcctttggag cctcctctac 20 <210> 14 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> 3' primer sequence <400> 14 gcggcatcca ggtaatttt 19

Claims

1. Use of the composition in the preparation of a medicament for treating Rett syndrome in a subject with a corresponding need, wherein the composition comprises pridopidine or a pharmaceutically acceptable salt thereof and at least one of compound 1 or 4: (Compound 1) (Compound 4); Or a pharmaceutically acceptable salt thereof, wherein the composition is administered when the drug is used in order to treat the subject.

2. The use according to claim 1, wherein the drug further delays the onset of at least one symptom of Rett syndrome in the subject, prevents the worsening of at least one symptom of Rett syndrome in the subject, delays the worsening of at least one symptom of Rett syndrome in the subject, or improves at least one symptom of Rett syndrome in the subject, wherein the symptom is abnormal gait, ataxia, impaired gait initiation, delay in acquiring purposeful hand skills or partial or complete loss of acquired purposeful hand skills, or the symptom is abnormal hand movements, startle response or delayed crawling and / or walking; reduced crawling and / or walking ability; or abnormal eye movements.

3. The use according to claim 1 or claim 2, wherein the pharmaceutically acceptable salt of said pridopidine is hydrochloride, hydrobromide, nitrate, perchlorate, phosphate, sulfate, formate, acetate, aconate, ascorbate, benzenesulfonate, benzoate, cinnamate, citrate, embosate, heptanoate, fumarate, glutamate, glycolate, lactate, maleate, malonate, mandelate, methanesulfonate, naphthalene-2-sulfonate, phthalate, salicylate, sorbate, stearate, succinate, tartrate, or p-toluenesulfonate.

4. The use according to claim 1 or claim 2, wherein the pharmaceutically acceptable salt of said compound 1 or 4 is a hydrochloride, hydrobromide, nitrate, perchlorate, phosphate, sulfate, formate, acetate, aconate, ascorbate, benzenesulfonate, benzoate, cinnamate, citrate, embosate, heptanoate, fumarate, glutamate, glycolate, lactate, maleate, malonate, mandelate, methanesulfonate, naphthalene-2-sulfonate, phthalate, salicylate, sorbate, stearate, succinate, tartrate, or p-toluenesulfonate.

5. The use according to claim 1, wherein the composition is administered orally, nasally, by inhalation, by subcutaneous injection, or by intravenous, intraperitoneal, intramuscular, intranasal, sublingual, vaginal, rectal, intraocular, intrathecal, local, or intradermal routes.

6. The use according to claim 5, wherein the composition is administered orally.

7. The use according to claim 1, wherein the composition is administered in the form of an aerosol, an inhalable powder, an injectable, a liquid, a gel, a solid, a capsule, or a tablet.

8. The use according to claim 6, wherein the composition is formulated as a multiplying substance, liquid solution, or liquid suspension in a tablet, capsule, pill, powder, capsule or sachet.

9. The use according to claim 1, wherein when the drug is used, the pridopidine is administered less than once daily.

10. The use according to claim 1, wherein when the drug is used, the pridopidine is administered once or twice daily.

11. The use according to claim 1, wherein when the drug is used, the pridopidine is administered at a daily dose between 0.5 mg / day and 315 mg / day.

12. The use according to claim 1, wherein when the drug is used, the pridopidine is administered at a daily dose between 0.5 mg / day and 45 mg / day.

13. The use according to claim 1, wherein when the drug is used, the pridopidine is administered at a daily dose between 10 mg / day and 100 mg / day.

14. The use according to claim 1, wherein when the drug is used, the pridopidine is administered at a daily dose of 45 mg / day to 90 mg / day.

15. The use according to claim 1, wherein when the drug is used, the pridopidine is administered at a daily dose of 45 mg / day to 180 mg / day.

16. The use according to claim 1, wherein when the drug is used, the composition is administered at one or two doses per day.

17. The use according to any one of claims 5-16, wherein the composition comprises pridopidine or a pharmaceutically acceptable salt thereof and compound 1 or a pharmaceutically acceptable salt thereof.

18. The use according to any one of claims 5-16, wherein the composition comprises pridopidine or a pharmaceutically acceptable salt thereof, compound 1 and compound 4 or a pharmaceutically acceptable salt thereof.

19. The use according to claim 1, wherein the weight ratio of pridopidine to at least one of compounds 1 and 4 is in the range of 1:0.0001 to 1:0.

1.

20. The use according to claim 1, wherein the weight ratio of pridopidine to at least one of compounds 1 and 4 is in the range of 1:0.0005 to 1:0.

1.

21. The use according to claim 1, wherein the weight ratio of pridopidine to at least one of compounds 1 and 4 is in the range of 1:0.0005 to 1:0.

005.

22. The use according to claim 2, wherein the abnormal hand movements are twisting, clenching, clapping, washing hands, tapping hands, rubbing hands, and / or repeatedly placing hands near the mouth.

23. The use according to claim 2, wherein the abnormal eye movement is prolonged staring, excessive blinking, strabismus, and / or closing one eye at a time.

24. The use according to claim 2, wherein when the drug is used, the drug improves the symptoms by at least 20%.

25. The use according to claim 2, wherein when the drug is used, the drug improves the symptoms by at least 30%.

26. The use according to claim 2, wherein when the drug is used, the drug improves the symptoms by at least 50%.

27. The use according to claim 2, wherein when the drug is used, the drug improves the symptoms by at least 80%.

28. The use according to claim 2, wherein when the drug is used, the drug improves the symptoms by 100%.

29. The use according to claim 1, wherein when the drug is used, the drug is effective in increasing or maintaining the serum BDNF level of the subject and / or increasing the brain BDNF level of the subject suffering from Rett syndrome.

30. The use according to claim 1, wherein 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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