EA043430B1 - METHOD FOR TREATING AMYOTROPHIC LATERAL SCLEROSIS WITH PRIDOPIDINE - Google Patents

Description

Cross reference to related applications

This invention claims the benefit of U.S. Provisional Application No. 62/445,315, filed August 14, 2017, the contents of which are incorporated herein by reference in their entirety.

Prior Art

Amyotrophic lateral sclerosis.

Amyotrophic lateral sclerosis (ALS) is a devastating degenerative disease characterized by the progressive loss of motor neurons in the motor cortex, brainstem, and spinal cord (Peters 2015). This rapidly fatal disease begins locally and spreads, leading to weakness of the limb muscles, respiratory muscles and eyeball muscles. Immediately before death, there is almost complete loss of limb and respiratory function, as well as loss of the ability to chew, swallow, and speak.

ALS is a rare disease with an average incidence rate of 2.8 per 100,000 in Europe and 1.8 per 100,000 in North America, and an average prevalence rate of 5.40 per 100,000 in Europe and 3.40 per 100,000 in North America (Bozzoni, 2016).

Approximately 10% of ALS cases are classified as familial ALS (FALS), while in the remaining 90% the disease is sporadic (SALS) and occurs randomly in the community (Riva, 2016). More than 60% of patients die within 3 years of disease onset, usually from respiratory failure, and about 10% survive for more than 10 years (Zou, 2016). There is currently no disease-modifying therapy for ALS known in the art, although riluzole slows the rate of disease progression and prolongs survival by 2 or 3 months.

Clinical manifestations of ALS include muscle weakness and wasting, fasciculations and spasms, spastic hypertonicity and hyperreflexia, which are the main clinical manifestations. Some patients also experience dysarthria, dysphagia, and respiratory weakness. Non-motor symptoms include behavioral disturbances, executive dysfunction, and frontotemporal dementia.

Neuropathological features of ALS include muscle atrophy, loss of anterior horn cells, and sclerosis of the lateral columns of the spinal cord gray matter (Martel, 2016). Gliosis, defined as activation of astrocytes and microglia, is also a hallmark of ALS.

Pridopidine.

Pridopidine (formerly ACR16, Huntexil®) is a unique compound developed for the treatment of patients with Huntington's disease. The chemical name of pridopidine is 4-(3-(methylsulfonyl)phenyl)1-propylpiperidine and its registration number is CAS 346688-38-8 (CSID: 7971505, 2016). The registration number of pridopidine hydrochloride is CAS 882737-42-0 (CSID: 25948790 2016). Methods for the synthesis of pridopidine and its pharmaceutically acceptable salt are disclosed in US Patent No. 7923459 and PCT Application Publication No. WO 2017/015609. US Patent No. RE46117 discloses pridopidine for the treatment of various diseases and disorders.

Pridopidine has been shown to modulate locomotor activity by inhibiting hyperactivity or enhancing hypoactivity. Pridopidine also exhibits neuroprotective properties, which are believed to be explained by its high affinity for the sigma-1 receptor (S1R, binding IC ₅₀ ~70-80 nM) (Sahlholm, 2013), while the motor activity of pridopidine is thought to be mediated primarily turn its low affinity, antagonistic activity towards the dopamine D2 receptor (D2R) ( _IC50 binding ~10 µM) (Ponten, 2010, Sahlholm, 2015). Pridopidine exhibits low to moderate binding affinity to additional receptors in the micromolar range.

S1R is an endoplasmic reticulum (ER) chaperone protein involved in cellular differentiation, neuroplasticity, neuroprotection, and cognitive function in the brain. Recently, transcriptomic analysis of the rat striatum showed that pridopidine treatment activates the expression of BDNF, dopamine 1 receptor (D1R), glucocorticoid receptor (GR), and serine-threonine-specific protein kinase B (Akt)/phosphoinositide 3-kinase (PI3K) pathways. Pridopidine has been shown to enhance the secretion of the neuroprotective brain-derived neurotrophic factor (BDNF) in a neuroblastoma cell line in an S1R-dependent manner (Geva, 2016) and restore spinal cord abnormalities and aberrant calcium signaling by activating S1R (Ryskamp, 2017).

PCT International Patent Application Publication No. WO 2016/138135 discloses the use of S1R agonists for the treatment of, inter alia, familial adult amyotrophic lateral sclerosis (ALS) and juvenile amyotrophic lateral sclerosis (ALS).

There remains an unmet need to provide effective treatment for ALS, particularly sporadic ALS.

Summary of the invention

The present invention is based, at least in part, on the unexpected experimental discovery that treatment with pridopidine improves axonal transport deficits, enhances ERK (extracellular regulated kinase) activation, and restores neuromuscular activity.

- 1 043430 unity (NMJ) in co-cultures of SOD1-deficient muscle cells reduces mutant SOD1 aggregates in the spinal cord and attenuates NMJ disruption and subsequent muscle wasting in SOD1-deficient mice.

The invention provides a method of treating a subject suffering from amyotrophic lateral sclerosis (ALS), comprising periodically administering to the subject an amount of pridopidine effective to treat the human subject.

The invention also provides pridopidine for use in the treatment of a human suffering from ALS.

The invention also provides a method of treating ALS, comprising periodically administering to a subject in need thereof an amount of pridopidine effective to treat ALS.

In some embodiments, the subject suffers from sporadic ALS.

The invention also provides a pharmaceutical composition for treating a human suffering from ALS, containing an effective amount of pridopidine.

The invention also provides the use of pridopidine in the production of a medicinal product for the treatment of ALS.

In addition, a package is provided containing pridopidine and, if necessary, instructions for use of pridopidine for the treatment of ALS.

In one embodiment, pridopidine is used as monotherapy in a subject suffering from ALS. In other embodiments, a subject suffering from ALS is treated with pridopidine and one or more additional agents.

Further provided is a method of treating a subject suffering from ALS, comprising periodically administering to the subject an amount of riluzole and an amount of pridopidine.

The following is a method of treating a subject suffering from ALS, comprising periodically administering to the subject an amount of edaravone and an amount of pridopidine.

The following is a method of treating a subject suffering from ALS, comprising periodically administering to the subject an amount of dextromethorphan/quinidine and an amount of pridopidine.

Further packaging is provided, including:

(a) a first pharmaceutical composition containing an amount of an agent that is riluzole, edaravone or dextromethorphan/quinidine; and (b) a second pharmaceutical composition containing an amount of pridopidine and a pharmaceutically acceptable carrier.

In a further embodiment, the package also contains:

(c) instructions for use for the first and second pharmaceutical compositions together for the treatment of a subject suffering from ALS.

In some embodiments, the second pharmaceutical composition is riluzole, edaravone, a dextromethorphan/quinidine combination, or laquinimod.

In addition, pridopidine is provided for use as adjunctive therapy or in combination with riluzole in the treatment of a subject suffering from ALS. In addition, pridopidine is provided for use as adjunctive therapy or in combination with edaravone in the treatment of a subject suffering from ALS. Also provided is pridopidine for use as adjunctive therapy or in combination with dextromethorphan/quinidine in the treatment of a subject suffering from ALS.

The present invention also relates to a pharmaceutical composition containing an amount of riluzole, edaravone or dextromethorphan/quinidine and an amount of pridopidine and at least one pharmaceutically acceptable carrier.

This invention also provides the use of:

(a) an amount of riluzole, edaravone or dextromethorphan/quinidine; and (b) an amount of pridopidine in the preparation of a combination for treating a subject suffering from ALS, wherein the amount of riluzole, edaravone or dextromethorphan/quinidine and the amount of pridopidine are administered simultaneously or sequentially.

The present invention also provides a pharmaceutical composition comprising an amount of riluzole, edaravone or dextromethorphan/quinidine, for use in treating a subject suffering from a movement disorder, in combination with an amount of pridopidine, by periodically administering the pharmaceutical composition and amount of pridopidine to the subject.

The present invention also provides a pharmaceutical composition containing an amount of pridopidine for use in treating a subject suffering from a movement disorder, in combination with an amount of riluzole, edaravone or dextromethorphan/quinidine, by periodically administering to the subject the pharmaceutical composition and the amount of riluzole, edaravone or dextromethorphan/quinidine, respectively.

The present invention also provides riluzole and pridopidine for treating a subject suffering from a movement disorder, wherein riluzole and pridopidine are administered simultaneously or sequentially.

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The present invention also provides a product containing an amount of riluzole and an amount of pridopidine for simultaneous or sequential use in the treatment of a subject suffering from ALS.

In another aspect, the invention provides a combination of pridopidine and riluzole, edaravone or dextromethorphan/quinidine for use as a medicament for the treatment, prevention or relief of ALS.

In another aspect, the invention provides a combination of pridopidine and riluzole, edaravone, laquinimod or dextromethorphan/quinidine for use as a drug.

In another aspect, the invention relates to a pharmaceutical composition comprising a therapeutically effective amount of pridopidine and a therapeutically effective amount of riluzole, edaravone, laquinimod or dextromethorphan/quinidine, together with one or more adjuvants, excipients, carriers and/or diluents.

In another aspect, the invention relates to a method of treating ALS in an animal, including a human, which includes the step of administering to such animal in need thereof a therapeutically effective amount of pridopidine; in combination therapy with riluzole, edaravone, laquinimod or dextromethorphan/quinidine.

In another aspect, the invention provides a kit of parts comprising at least two separate unit dosage forms (A) and (B), wherein (A) contains pridopidine; a (B) contains riluzole, edaravone, or dextromethorphan/quinidine; and, as appropriate, (C) instructions for simultaneous or sequential administration of pridopidine (A) and riluzole, edaravone, or dextromethorphan/quinidine (B) to a patient in need thereof.

In another aspect, the invention provides a method of treating a subject suffering from ALS, comprising administering to the subject a combination of a therapeutically effective amount of pridopidine and a therapeutically effective amount of riluzole, edaravone, laquinimod, or dextromethorphan/quinidine, wherein these amounts, taken together, are effective to treat the human patient.

Brief description of drawings

Fig. 1a-1c. Analysis of axonal transport.

Fig. 1a. Experimental design for the analysis of axonal transport.

Fig. 1b. Schematic illustration of the experimental system for tracking axonal transport in motor neurons (MNs).

Fig. 1s. Time-lapse images of Qdot-BDNF axonal transport (bright orange).

Fig. 2a, 2b. Graphs showing the effect of pridopidine on the instantaneous velocity and number of stops of Qdot-BDNF particles along the axon (LM (littermates) = WT (wild type)).

Fig. 2a. Effect of pridopidine on instantaneous velocity values (μm/s) for Qdot-BDNF particles in WT (blue), SOD1G93A (red), or Sigma-1 receptor knockout (S1R-/-) motor neurons (gray).

Fig. 2b. Effect of pridopidine on the number of particle stops (number of counted Qdot-BDNF stops per second).

Fig. 3. Schematic illustration of the experimental procedure for neuromuscular cell co-culture techniques with measurements of muscle innervation and neuromuscular junction (HMC) function. The spinal explant is cultured in the proximal compartment, and primary myocytes are cultured in the distal compartment.

Fig. 4. Graph showing the effect of pridopidine on axonal growth for WT and SOD1G93A, which measures the number of cavities with axons crossing the muscle compartment.

Fig. 5a, 5b. Results of co-culture assays in a microfluidic compartment (MFC).

Fig. 5a. Top panel: phase image of a myocyte in a distal compartment connected by axons (green arrows). Bottom panel: High magnification images of myocytes: MN contact points.

Fig. 5b. Muscle contraction traces taken from measurements of muscle contraction intensity over time.

Fig. 6. Graph showing the effect of pridopidine on innervation frequency in WT and SOD1G93A myocytes.

Fig. 7a, 7b. Graphs showing the level of myocyte contraction in a co-culture of MN-myocytes.

Fig. 7a. Percentage of contracting myoblasts in co-cultures of ventral spinal cord slices (WT or SOD1G93A) and primary myocytes (SOD1G93A or LM controls).

Fig. 7b. Effects of pridopidine on the neuromuscular junction (NMJ) in muscle tissue from WT or SOD1G93A and motor neurons from WT or S1R-/- mice.

Fig. 8a, 8b. Effect of pridopidine on ERK levels.

Fig. 8a. Effect of pridopidine on total ERK (tERK) and phosphorylated ERK (pERK) levels shown by Western blotting of WT, SOD1G93A and S1R-/- motor neuron culture extracts.

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Fig. 8b. Quantification of the ERK activation effect of pridopidine as measured by tERK/pERK.

Fig. 9a-9c. Effect of pridopidine on SOD1 aggregates.

Fig. 9a. Imaging and quantification of fluorescently labeled spinal cord cells using NC500 to label mutant SOD1 aggregates in WT and SOD1G93A spinal cords, with or without pridopidine treatment.

Fig. 9b. Quantitative analysis of the number of aggregates per area identified in gray matter. Fig. 9s. Quantitative analysis of the number of aggregates per area identified in white matter.

Fig. 10a, 10b. Effect of pridopidine on muscle tissue.

Fig. 10a. Exemplary images of hematoxylin-eosin-stained cross-sections of the gastrocnemius muscle of WT or SOD1G93A mice treated or not treated with pridopidine.

Fig. 10b. Evaluation of the effect of pridopidine on muscle fiber wasting: quantitative analysis of the effect of pridopidine on muscle fiber diameter.

Fig. 11a, 11b. Effect of pridopidine on NMS.

Fig. 11a. The effect of pridopidine on the preservation of NMJ in vivo.

Fig. 11b. Quantitative analysis of the effect of pridopidine on the percentage of innervated NMJs.

Detailed Description of the Invention

The invention provides a method of treating a subject suffering from amyotrophic lateral sclerosis (ALS), comprising periodically administering to the subject an amount of pridopidine effective to treat the human subject.

In one embodiment of the invention, the ALS is sporadic ALS.

In one embodiment of the invention, the ALS is familial ALS (FALS). In some embodiments, the ALS is juvenile ALS (JALS).

In some embodiments, the BAS is not FALS. In some embodiments, the ALS is non-juvenile ALS (JALS).

In one embodiment, the type of ALS is classic, bulbar, arm laxity, leg laxity, pyramidal and respiratory ALS, progressive muscular atrophy, primary lateral sclerosis, or progressive bulbar palsy.

In one embodiment of the invention, the subject carries a mutant version of a gene that causes or contributes to the pathogenesis of ALS. In some embodiments, the mutant version of the gene is selected from the group of genes consisting of the genes superoxide dismutase 1 (SOD1), TAR DNA binding protein (TARDBP), encoding TDP-43 fused to sarcoma (FUS), p62 (SQSTM1), ubiquin- 2 (UBQLN2), TANK-binding kinase 1 (TBK1), profilin 1 (PFN1), VCP or p97 (VCP), angiogenin (ANG), optineurin (OPTN), C9orf72, sigma-1 receptor (S1R), tubulin alpha- 4A (TUBA4A), dynactin (DCTN1), hnRNPA1 (HNRNPA1), matrine 3 (MATR3), coiled-coil-supercoil-helix domain-containing protein 10 (CHCHD10), and any combination thereof.

In one embodiment of the invention, the amount of pridopidine is effective to inhibit or reduce the progression of an ALS symptom in a subject.

In an embodiment of the invention, the ALS symptom is a clinical symptom of ALS.

In one embodiment of the invention, the symptoms of ALS include muscle weakness and wasting, fasciculations and cramps, spastic hypertonicity, hyperreflexia, dysarthria, dysphagia and respiratory weakness, behavioral disorders, impairment of executive functions, or frontotemporal dementia.

In one embodiment of the invention, the ALS symptom is a neuropathological symptom.

In some embodiments, the symptom is bulbar palsy or pseudobulbar affect (PBA).

In one embodiment, the symptom of ALS is muscle atrophy, motor neuron loss, anterior horn cell loss, lateral column sclerosis of the spinal cord gray matter, or gliosis.

In one embodiment, the symptom of ALS is the rate of decline in (a) pulmonary function, (b) functional disability, or (c) measures of lower extremity ability. In one embodiment of the invention, the amount of pridopidine is effective to provide survival of the subject or neuroprotection in the subject.

In some embodiments, treatment of a subject with pridopidine results in amelioration of impairment or improvement in the subject's condition in one or more of the following areas: (1) speech, (2) salivation, (3) swallowing, (4) handwriting, (5) food cutting and handling utensils (with or without a gastrostomy tube), (6) dressing and hygiene, (7) turning over in bed and handling bedding, (8) walking, (9) climbing stairs, (10) breathing, (11) dyspnea, (12) dyspnea when lying down, and (13) failure.

In some embodiments, patients are monitored for changes in the above

- 4 043430 areas using a rating scale, such as the Amyotrophic Lateral Sclerosis Functional Rating Scale (ALSFRS) or the ALSFRS-Revised (ALSFRS-R), and monitors the patient's functional changes over time.

In some embodiments, patients are monitored for pseudobulbar affect (PBA). In some embodiments, the severity and/or frequency of emotional outbursts in subjects experiencing PBA are reduced by treatment with pridopidine.

In one embodiment of the invention, pridopidine is administered daily.

In one embodiment of the invention, pridopidine is administered more frequently than once daily.

In one embodiment of the invention, pridopidine is administered twice daily.

In one embodiment of the invention, pridopidine is administered three times daily.

In one embodiment of the invention, pridopidine is administered less than once per day, for example on alternating days, three times a week, twice a week, or once a week.

In one embodiment of the invention, pridopidine is administered orally.

In some embodiments, a single dose of the pharmaceutical composition contains 10-250 mg of pridopidine. In some embodiments, the composition contains 45, 67.5, 90, or 112.5 mg of pridopidine.

In one embodiment, the patient is administered 10 to 225 mg of pridopidine per day. In another embodiment, the patient is administered 45 to 180 mg of pridopidine per day. In another embodiment, the patient is administered 10, 22.5, 45, 67.5, 90, 100, 112.5, 125, 135, 150, or 180 mg of pridopidine per day.

In one embodiment, the pharmaceutical composition is administered twice daily. In another embodiment, an equal amount of the pharmaceutical composition is used with each administration. In one embodiment, two doses are administered at least 6 hours, at least 7 hours, at least 8 hours, at least 9 hours, at least 10 hours, at least 11 hours apart. In some embodiments, pharmaceutical the composition is administered for at least 12 weeks, at least 20 weeks, at least 24 weeks, at least 26 weeks, at least 52 weeks, or at least 78 weeks.

In one embodiment of the invention, pridopidine is pridopidine hydrochloride.

In one embodiment of the invention, the subject is a human.

The invention also provides pridopidine for use in the treatment of a human suffering from ALS.

The invention also provides a pharmaceutical composition for use in the treatment of a person suffering from ALS, containing an effective amount of pridopidine.

The invention further provides a method of treating ALS, comprising periodically administering to a subject in need thereof an amount of pridopidine effective to treat ALS.

In one embodiment, the pharmaceutical composition contains an amount of pridopidine and an amount of a second compound, for example a compound useful in treating patients with ALS.

In some embodiments, the second compound is riluzole, edaravone, a combination of dextromethorphan and quinidine, or laquinimod.

In one embodiment, a pharmaceutical composition for use in treating a subject suffering from ALS comprises pridopidine and a second compound, which are formulated for simultaneous or sequential administration.

In one embodiment, the pharmaceutical composition is in a unit dosage form useful for treating a subject suffering from ALS, which includes:

(a) amount of pridopidine;

(b) an amount of a second compound, wherein corresponding amounts of said second compound and said pridopidine in said composition are effective when simultaneously administered to said subject one or more of said unit dosage forms of said composition to treat the subject.

In some embodiments, the second compound is riluzole, edaravone, or dextromethorphan/quinidine.

In one embodiment, the pharmaceutical composition contains an amount of pridopidine for use in treating a subject suffering from ALS as adjunctive therapy to a second compound, which is riluzole. In another embodiment, the pharmaceutical composition contains an amount of pridopidine for use in treating a subject suffering from ALS as adjunctive therapy to a second compound, which is edaravone. In another embodiment, the pharmaceutical composition contains an amount of pridopidine for use in treating a subject suffering from ALS as adjunctive therapy to a second compound which is dextromethorphan/quinidine.

In one embodiment, the pharmaceutical composition contains an amount of pridopidine for use in treating a subject suffering from ALS, simultaneously or sequentially with second

- 5 043430 eye compound, which is riluzole. In another embodiment, the pharmaceutical composition contains an amount of pridopidine for use in treating a subject suffering from ALS, simultaneously or sequentially with a second compound, which is edaravone. In another embodiment, the pharmaceutical composition contains an amount of pridopidine for use in treating a subject suffering from ALS, simultaneously or sequentially with a second compound, which is dextromethorphan/quinidine. In another embodiment, the pharmaceutical composition contains an amount of pridopidine for use in treating a subject suffering from ALS, simultaneously or sequentially with a second compound, which is laquinimod. In one embodiment, the pharmaceutical composition contains an amount of a compound that is riluzole for use in treating a subject suffering from ALS as an adjunctive therapy to pridopidine. In another embodiment, the pharmaceutical composition contains an amount of a compound that is edaravone, for use in the treatment of a subject suffering from ALS, as an adjunctive therapy to pridopidine. In another embodiment, the pharmaceutical composition contains an amount of a compound that is dextromethorphan/quinidine, for use in treating a subject suffering from ALS, as an adjunctive therapy to pridopidine. In another embodiment, the pharmaceutical composition contains an amount of a compound that is laquinimod, for use in the treatment of a subject suffering from ALS, as an adjunctive therapy to pridopidine.

In one embodiment, the pharmaceutical composition contains an amount of a compound that is riluzole for use in treating a subject suffering from ALS, simultaneously or sequentially with pridopidine. In another embodiment, the pharmaceutical composition contains an amount of a compound that is edaravone, for use in treating a subject suffering from ALS, simultaneously or sequentially with pridopidine. In another embodiment, the pharmaceutical composition contains an amount of a compound that is dextromethorphan/quinidine, for use in treating a subject suffering from ALS, simultaneously or sequentially with pridopidine.

The invention also provides a compound that is riluzole for use as an adjunctive therapy to pridopidine in the treatment of a subject suffering from ALS.

The invention also provides a compound that is edaravone for use as an adjunctive therapy to pridopidine in the treatment of a subject suffering from ALS.

The invention also provides a dextromethorphan/quinidine compound for use as an adjunctive therapy to pridopidine in the treatment of a subject suffering from ALS.

The invention also provides pridopidine for use as an adjunctive therapy to the compound riluzole in the treatment of a subject suffering from ALS.

The invention also provides pridopidine for use as an adjunctive therapy to a compound that is edaravone in the treatment of a subject suffering from ALS.

The invention also provides pridopidine for use as an adjunctive therapy to a dextromethorphan/quinidine compound in the treatment of a subject suffering from ALS.

The invention also provides pridopidine for use as an adjunctive therapy to the compound that is laquinimod for the treatment of a subject suffering from ALS.

In one embodiment, the adjunctive therapy is for treating, preventing, or alleviating a symptom of ALS.

The invention also provides a combination of a compound that is riluzole and pridopidine for use in the treatment, prevention or alleviation of ALS symptoms.

The invention also provides a combination of a compound that is edaravone and pridopidine for use in the treatment, prevention, or symptomatic relief of ALS.

The invention also provides a combination of a compound that is dextromethorphan/quinidine and pridopidine for use in the treatment, prevention or alleviation of ALS symptoms.

The invention also provides the use of pridopidine in the production of a medicinal product for the treatment of ALS.

The method, use and composition further include reducing the rate of neurological deterioration in the subject.

In one embodiment, the methods of the present invention further comprise administering to the subject a therapeutically effective amount of a second compound, which is riluzole or edaravone. In one embodiment, the methods of the present invention further comprise administering to the subject a therapeutically effective amount of a second compound, which is dextromethorphan/quinidine. In one embodiment, the second compound is riluzole. In another embodiment, the second connection is

- 6 043430 is edaravone. In another embodiment, the second compound is dextromethorphan/quinidine. In another embodiment, the second compound is laquinimod.

In an embodiment of the invention, pridopidine and the second compound are administered in one unit. In another embodiment, pridopidine and the second compound are administered in more than one unit.

In one embodiment, the amount of pridopidine and the amount of the second compound are administered simultaneously. In one embodiment, the amount of pridopidine and the amount of the second compound are administered sequentially.

In another embodiment, administration of the second compound precedes administration of pridopidine. In another embodiment, administration of pridopidine precedes administration of the second compound.

In one embodiment, the subject receives edaravone therapy prior to initiation of pridopidine therapy. In another embodiment, the subject receives riluzole prior to initiation of pridopidine therapy. In another embodiment, the subject receives laquinimod prior to initiation of pridopidine therapy. In another embodiment, the subject receives dextromethorphan/quinidine to initiate treatment with pridopidine.

In another embodiment, the subject has been receiving edaravone therapy for at least 24, 28, 48, or 52 weeks before starting pridopidine therapy. In another embodiment, the subject has been receiving riluzole therapy for at least 24, 28, 48, or 52 weeks before starting treatment with pridopidine. In another embodiment, the subject receives dextromethorphan/quinidine therapy for at least 1, 2, 4, or 6 weeks before starting pridopidine therapy.

In one embodiment, the subject receives pridopidine therapy prior to initiation of edaravone therapy. In another embodiment, the subject has been receiving pridopidine therapy for at least 24, 28, 48, or 52 weeks before starting edaravone therapy.

In one embodiment, the subject receives pridopidine therapy prior to initiation of riluzole therapy. In another embodiment, the subject has received pridopidine therapy for at least 24, 28, 48, or 52 weeks prior to initiation of riluzole therapy.

In one embodiment, the subject receives pridopidine therapy prior to initiation of laquinimod therapy. In another embodiment, the subject has received pridopidine therapy for at least 24, 28, 48, or 52 weeks prior to initiation of laquinimod therapy.

In one embodiment, the subject receives pridopidine therapy prior to initiation of dextromethorphan/quinidine therapy. In another embodiment, the subject has been receiving pridopidine therapy for at least 24, 28, 48, or 52 weeks prior to initiating dextromethorphan/quinidine therapy.

In one embodiment, the patient is administered 0.5 to 1.5 mg of laquinimod per day. In another embodiment, the patient is administered 0.5 or 1.0 mg of laquinimod per day. In one embodiment, laquinimod is administered orally.

In one embodiment, the patient is administered 10 to 200 mg of riluzole per day. In another embodiment, the patient is administered 50, 100, or 200 mg of riluzole per day.

In one embodiment, riluzole is administered orally. In one embodiment, dextromethorphan/quinidine is administered orally.

In one embodiment, the patient is administered 5 to 60 mg of edaravone per day. In another embodiment, the patient is administered 30 or 60 mg of edaravone per day.

In one embodiment, edaravone is administered by intravenous infusion. In another embodiment, edaravone is administered once daily for 10 days, followed by a 14-day drug-free period. In another embodiment, edaravone is administered once daily for 14 days, followed by a 14-day drug-free period.

In one embodiment, each of the amount of the second compound when taken alone and the amount of pridopidine when taken alone is effective for treating a subject. In another embodiment, either the amount of the second compound when taken alone or the amount of pridopidine when taken alone is less effective for treating the subject. In another embodiment, either the amount of the second compound when taken alone or the amount of pridopidine when taken alone is ineffective for treating the subject.

In one embodiment, pridopidine is co-administered with a second compound. In another embodiment, the second compound is administered in addition to pridopidine.

In one embodiment, a saturating dose in an amount different from the intended dose is administered over a period of time at the beginning of intermittent use.

In one embodiment, a method of enhancing axonal transport of BDNF in motor neurons in a subject suffering from ALS is provided, comprising administering to the subject an amount of pridopidine effective to enhance axonal transport of BDNF in motor neurons of the subject.

In one embodiment, a method of enhancing ERK activation in motor neurons is provided.

- 7 043430 to a subject suffering from ALS, comprising administering to the subject an amount of pridopidine effective to enhance ERK activation in motor neurons of the subject.

In one embodiment, a method of preserving neuromuscular junction (NMJ) structure in muscle cells of a subject suffering from ALS is provided, comprising administering to the subject an amount of pridopidine effective to preserve neuromuscular junction structure in the subject's muscles.

In addition, a method of improving muscle contraction in a subject suffering from ALS is provided, comprising administering to the subject an amount of pridopidine effective to improve muscle contraction function in the subject.

In addition, a method is provided for improving the degree of innervation of muscle tissue in a subject suffering from ALS, comprising administering to the subject an amount of pridopidine effective to improve the degree of innervation in the subject.

In one embodiment, a method is provided for enhancing axonal growth of motor neurons in a subject suffering from ALS, comprising administering to the subject an amount of pridopidine effective to enhance axonal growth of motor neurons in the subject.

In one embodiment, a method of increasing muscle cell survival in a subject suffering from ALS is provided, comprising administering to the subject an amount of pridopidine effective to increase muscle cell survival in the subject.

In one embodiment, a method of reducing the progression of muscle fiber wasting in a subject suffering from ALS is provided, comprising administering to the subject an amount of pridopidine effective to reduce the progression of muscle fiber wasting in the subject.

In one embodiment, a method of reducing axonal degeneration in a subject suffering from ALS is provided, comprising administering to the subject an amount of pridopidine effective to reduce axonal degeneration in the subject.

In one embodiment, a method of preserving NMJ formation in a subject suffering from ALS is provided, comprising administering to the subject an amount of pridopidine effective to preserve NMJ formation in the subject.

In one embodiment, a method of preserving the structure and function of the NMJ in a subject suffering from ALS is provided, comprising administering to the subject an amount of pridopidine effective to preserve the structure and function of the NMJ in the subject.

In one embodiment, a method of reducing protein aggregation in a subject suffering from ALS is provided, comprising administering to the subject an amount of pridopidine effective to reduce protein aggregation in the subject.

In one embodiment, a method of attenuating the progression of pseudobulbar syndrome in a subject suffering from ALS is provided, comprising administering to the subject an amount of pridopidine effective to attenuate the progression of pseudobulbar syndrome in the subject.

The invention also provides a package containing:

a pharmaceutical composition comprising an amount of pridopidine; and, if necessary, instructions for use of the pharmaceutical composition for treating a subject suffering from ALS.

The invention also provides a therapeutic package for dispensing to a subject or for use in dispensing to a subject, which includes:

one or more unit doses, each such unit dose containing an amount of said pridopidine, wherein the amount of said pridopidine in said unit dose is effective when administered to said subject for treating ALS in the subject; and a ready-made pharmaceutical container therefor containing said dosage unit or dosages, wherein said container optionally further contains or includes instructions indicating the use of said package in treating a subject suffering from ALS.

Such a unit dose contains 10-250 mg pridopidine or, for example, 10, 22.5, 45, 67.5, 90 or 112.5 mg pridopidine.

The invention also provides a package containing:

a first pharmaceutical composition comprising an amount of pridopidine and a pharmaceutically acceptable carrier;

a second pharmaceutical composition comprising an amount of a second compound that is riluzole, edaravone or dextromethorphan/quinidine and a pharmaceutically acceptable carrier; and, if necessary, instructions for using the first and second pharmaceutical compositions together to treat a subject suffering from ALS.

In one embodiment, the amount of the second compound and the amount of pridopidine are prepared for simultaneous or sequential administration.

The invention also provides a therapeutic package for dosing to a subject suffering from

- 8 043430 general BAS, or for use in dosing, which includes:

one or more unit doses, wherein each such unit dose comprises: an amount of pridopidine and an amount of a second compound that is riluzole or edaravone or dextromethorphan/quinidine, wherein respective amounts of said pridopidine and the second compound at said unit dose are effective when administered concomitantly to a specified subject for treating the subject; and a ready-made pharmaceutical container therefor containing said unit dose or units, optionally further containing or including instructions indicating the use of said package in the treatment of said subject.

The invention also provides a method of combination therapy for the treatment of ALS, comprising administering to a subject in need thereof a therapeutically effective amount of pridopidine and a therapeutically effective amount of riluzole, edaravone or dextromethorphan/quinidine.

For the above embodiments, each embodiment disclosed herein is considered to apply to each of the other disclosed embodiments. For example, the elements listed in the method embodiments may be used in the pharmaceutical composition, packaging and use embodiments described herein, and vice versa.

All combinations, subcombinations and permutations of various elements of the methods and applications described herein are intended and are within the scope of the invention.

Pharmaceutically acceptable salts.

As used herein, the term pridopidine means pridopidine base or a pharmaceutically acceptable salt thereof, as well as derivatives, for example, a deuterium-enriched version of pridopidine and its salts. Examples of deuterium-enriched pridopidine and its salts and methods for their preparation can be found in US Application Publications No. 2013-0197031, 2016-0166559 and 2016-0095847, each of which is incorporated herein by reference in its entirety. In some embodiments, pridopidine is a pharmaceutically acceptable salt, such as an HCl salt or a tartrate salt. Preferably, in any embodiments of the invention as described herein, pridopidine is in the form of its hydrochloride salt.

Examples of pharmaceutically acceptable addition salts include non-toxic addition salts of inorganic and organic acids such as hydrochloride, hydrobromide, nitrate, perchlorate, phosphate, sulfate, formate, acetate, aconitate, ascorbate, benzenesulfonate, benzoate, cinnamate, citrate, embonate, enanthate, fumarate, glutamate, glycolate, lactate, maleate, malonate, almond, methanesulfonate, naphthalene-2-sulfonate, phthalate, salicylate, sorbate, stearate, succinate, tartrate, toluene-p-sulfonate and the like, and the like. Such salts can be prepared using procedures well known and described in the art.

Deuterium-enriched means that the deuterium content at any relevant site of the compound exceeds the amount of deuterium naturally occurring in that region in the amount of the compound. The distribution of deuterium in nature is about 0.0156%. Thus, in a deuterium-enriched compound, the deuterium content in any of its respective regions is greater than 0.0156% and can vary from greater than 0.0156 to 100%. Deuterium-enriched compounds can be prepared by exchanging hydrogen with deuterium or by synthesizing the compound with deuterium-enriched starting materials.

Pharmaceutical compositions.

Although pridopidine for use according to the invention can be administered in the form of the parent compound, the preferred administration of pridopidine, if necessary in the form of a physiologically acceptable salt, is carried out in a pharmaceutical composition together with one or more adjuvants, excipients, carriers, buffering agents, diluents and/or other conventional pharmaceutical agents. excipients.

In one embodiment, the invention relates to pharmaceutical compositions containing pridopidine or pharmaceutically acceptable salts or derivatives thereof together with one or more pharmaceutically acceptable carriers and, if necessary, other therapeutic and/or prophylactic ingredients known and used in the art, including riluzole , edaravone or Nuedexta® (dextromethorphan/quinidine), but not limited to.

The carrier(s) must be acceptable in terms of compatibility with the other ingredients of the formulation and suitable for administration to humans.

Combination therapy.

When the invention involves a combination of an active compound and an additional one or more therapeutic and/or prophylactic ingredients, the combination of the invention may be formulated for simultaneous or sequential administration with at least a pharmaceutically acceptable carrier, additive, adjuvant or vehicle. This means that the combination

- 9 043430 tion of two active compounds can be introduced:

as a combination, which is part of the same dosage form, where two active compounds are administered simultaneously, or as a combination of two units, each with one of the active substances, allowing simultaneous or sequential administration.

The effects of any individual drug are related to its absorption, distribution, and elimination. When two drugs are administered to the body, each drug can affect the absorption, distribution, and elimination of the other, and therefore alter the effect of the other. For example, one drug may inhibit, activate, or induce the production of enzymes involved in the metabolic pathway for eliminating another drug (Guidance for Industry, 1999).

Not only can the interaction between two drugs affect the intended therapeutic activity of each drug, but the interaction can also increase levels of toxic metabolites (Guidance for Industry, 1999). Interactions may also increase or decrease the side effects of each drug. Therefore, after administering two drugs to treat a disease, it is impossible to predict what change will occur in the negative side profile of each drug.

In addition, it is difficult to accurately predict when the effects of interactions between two drugs will occur. For example, metabolic interactions between drugs may become apparent upon initial administration of a second drug, after the two drugs reach a constant concentration, or after discontinuation of one drug (Guidance for Industry, 1999).

One example is the combined administration of GA and interferon (IFN), which abrogates the clinical effectiveness of either therapy (Brod, 2000). Another experiment reported that the addition of prednisone in combination therapy with IFN-β counteracted its up-regulation effect. Thus, when two drugs are administered to treat the same condition, it cannot be predicted whether each will complement, be unaffected by, or interfere with the therapeutic activity of the other drug in a person.

Application.

The pharmaceutical composition of the invention can be administered by any convenient route that is suitable for the desired therapy. Preferred routes of administration include oral administration, in particular tablets, capsules, dragees, powder or liquid form, intranasal administration, intradermal administration and parenteral administration, in particular dermal, subcutaneous, intramuscular or intravenous injection. The pharmaceutical composition of the invention can be prepared by a skilled artisan using standard methods and conventional techniques suitable for the desired composition. If necessary, compositions adapted for sustained release of the active ingredient may be used.

Tablets may contain suitable binders, lubricants, disintegrants, coloring agents, flavoring agents, flow improvers and freezing point depressants. For example, for oral administration in unit dose tablet or capsule form, the active drug component can be combined with an oral, non-toxic, pharmaceutically acceptable inert carrier such as lactose, gelatin, agar, starch, sucrose, glucose, methylcellulose, dicalcium phosphate, calcium sulfate, mannitol , sorbitol, microcrystalline cellulose, etc. Suitable binders include starch, gelatin, natural sugars such as glucose or beta-lactose, corn starch, natural and synthetic gums such as acacia gum, gum tragacanth or sodium alginate, povidone, carboxymethylcellulose, polyethylene glycol, waxes and the like. Lubricants used in these dosage forms include sodium oleate, sodium stearate, sodium benzoate, sodium acetate, sodium chloride, stearic acid, sodium stearyl fumarate, talc and the like. Disintegrants include, but are not limited to, starch, methylcellulose, agar, bentonite, xanthan gum, croscarmellose sodium, sodium starch glycolate, and the like.

General procedures and compositions for the preparation of dosage forms useful in the present invention are described in the following references: Modern Pharmaceutics, Chapters 9 and 10 (Banker & Rhodes, Editors, 1979); Pharmaceutical Dosage Forms: Tablets (Lieberman 1981); Ansel, Introduction to Pharmaceutical Dosage Forms ^2nd Edition (1976); Remington's Pharmaceutical Sciences, ^17th ed. (Mack Publishing Company, Easton, Pa., 1985); Advances in Pharmaceutical Sciences (David Ganderton, Trevor Jones, Eds., 1992); Advances in Pharmaceutical Sciences Vol 7. (David Ganderton, Trevor Jones, James McGinity, Eds., 1995); Aqueous Polymeric Coatings for Pharmaceutical Dosage Forms (Drugs and the Pharmaceutical Sciences, Series 36 (James McGinity, Ed., 1989); Pharmaceutical Particulate Carriers: Therapeutic Applications: Drugs and the Pharmaceutical Sciences, Vol 61 (Alain Rolland, Ed., 1993) ; Drug Delivery to the Gastrointestinal Tract (Ellis Horwood Books in the Biological Sciences. Series in Pharmaceutical Technology; JG Hardy, SS Davis, Clive G. Wilson, Eds); Modern Pharmaceutics Drugs and the Pharmaceutical Sciences, Vol. 40 (Gilbert S. Banker, Christopher T. Rhodes, Eds. These sources are incorporated herein in their entirety.

- 10 043430 by means of reference.

Terms.

As used herein, and unless otherwise specified, each of the following terms shall have the definition set forth below.

As used herein, the term riluzole means riluzole or a pharmaceutically acceptable salt thereof, as well as derivatives, such as a deuterium-enriched version of riluzole and its salts. Riluzole is described in Preservers' Digital Reference, which is incorporated herein by reference (Riluzole PDR 2017).

As used herein, the term edaravone means edaravone or a pharmaceutically acceptable salt thereof, as well as derivatives, such as the deuterium-enriched version of edaravone and its salts. Edaravone is described in the Prescribers' Digital Reference, which is incorporated herein by reference (Edaravone PDR 2017).

Dextromethorphan and quinidine combination or dextromethorphan/quinidine or dextromethorphan hydrobromide/quinidine sulfate refers to a combination of dextromethorphan hydrobromide (20 mg) and quinidine sulfate (10 mg), such as Nuedexta®. Nuedexta® is a drug that is currently marketed for the treatment of pseudobulbar affect (PBA), among others, in patients with ALS. Nuedexta® has been shown to act on sigma-1 and NMDA receptors in the brain. Recent evidence suggests that the combination affects bulbar function in ALS but does not affect other aspects of motor function (Smith, 2017). Dextromethorphan hydrobromide/quinidine sulfate is described in the Prescribers' Digital Reference, which is incorporated herein by reference (Dextromethorphan hydrobromide/quinidine sulfate PDR 2017).

As used herein, the term amount or dose of pridopidine, measured in milligrams, refers to the milligrams of non-derivatized pridopidine base present in the formulation, regardless of the formulation form. A 45 mg dose of pridopidine means that the amount of pridopidine in the preparation is sufficient to provide 45 mg of underrivatized pridopidine base having a natural isotope distribution, regardless of the form of the preparation. Thus, in a salt form, such as pridopidine hydrochloride, the mass of salt in the form required to provide a 45 mg dose of underrivatized pridopidine base will be greater than 45 mg due to the presence of an additional salt ion. Likewise, when it is in the deuterium-enriched derivative form, the mass of the derivatized form required to provide a 45 mg dose of underrivatized pridopidine base having a natural isotope distribution will be greater than 45 mg due to the presence of additional deuterium.

By any range disclosed herein, it is meant that all hundredths, tenths, and whole number units within the range are specifically disclosed as part of the invention. So, for example, from 0.01 to 50 mg means that 0.02; 0.03, ..., 0.09; 0.1; 0.2, ..., 0.9 and 1; 2, ..., 49 mg unit amounts are included as embodiments of the present invention. By any time range disclosed herein (ie, weeks, months or years), it is meant that all time periods of days and/or weeks within that range are specifically disclosed as part of the invention. Thus, for example, 3-6 months means that 3 months and 1 day, 3 months and 1 week, and 4 months are included as embodiments of the invention.

As used herein, the term approximately in the context of a numerical value or range means ±10% of the numerical value or range specified or claimed.

As used herein, the term monotherapy means treatment with a single active agent, for example, treatment with pridopidine alone.

As used herein, the term adjuvant means the treatment or use of an additional compound with the primary compound, for example, to enhance the effectiveness or safety of the primary compound or to facilitate its activity.

As used herein, the term intermittent administration means repeated/multiple administrations separated by a period of time. The time period between administrations is preferably constant in time. Periodic administration may include administration, for example, once a day, twice a day, three times a day, four times a day, weekly, twice a week, three times a week, four times a week, etc.

As used herein, the term combination means a combination of reagents for use in therapy by simultaneous or sequential administration. Co-administration refers to the administration of a mixture (whether a true mixture, suspension, emulsion or other physical combination) of pridopidine and a second compound (eg, riluzole). In this case, the combination may be a mixture or separate containers of pridopidine and the second compound, which are combined immediately before administration. Sequential administration or concomitant administration refers to the separate administration of pridopidine and a second compound (eg, riluzole) simultaneously or sometimes close enough to each other such that additive or preferably synergistic activity is observed relative to the activity of either pridopidine or the second compound alone, or to administration at a time close enough to allow the individual therapeutic effects of each agent to overlap.

As used herein, the term adjunct or adjunctive therapy means a set of reagents for use in therapy, wherein the subject receiving the therapy begins a first treatment regimen with one or more reagents before starting a second treatment regimen with one or more different reagents in addition to the first treatment regimen, such that not all reagents used in therapy are started at the same time. For example, adding pridopidine therapy to a patient already receiving riluzole therapy.

As used herein, the term effective in relation to an amount of pridopidine refers to an amount of pridopidine that is sufficient to produce the desired therapeutic response. In a preferred embodiment, the amount of pridopidine administered does not result in unwanted side effects (such as toxicity, irritation or allergic reaction).

Administration to a subject or administration to a patient (human) means the provision, dispensing or use of drugs, drugs or medical devices to a subject/patient to alleviate, cure or reduce symptoms associated with a disease, disorder or condition, such as a pathological condition.

As used herein, treatment includes inducing inhibition, regression, or arrest of a disease or disorder, or reduction, suppression, inhibition, amelioration, elimination, or substantial elimination or amelioration of a symptom of a disease or disorder.

Inhibiting the progression of a disease or complication of a disease in a subject means preventing or reducing the progression of a disease and/or complication of a disease in a subject.

A symptom associated with a disease or disorder includes any clinical or laboratory manifestation associated with a disease or disorder and is not limited to what the subject may feel or observe.

As used herein, the term subject suffering from a disease, disorder or condition means a subject who has been clinically diagnosed with a disease, disorder or condition.

Glial cell-derived neurotrophic factor (GDNF) is a protein encoded by the GDNF gene and is thought to promote the survival of many types of neurons.

Brain-derived neurotrophic factor (BDNF) is a protein produced by neurons that serves to maintain function and promote neuronal growth and neurogenesis.

The following numbered paragraphs define various aspects and features of the present invention:

1. A method of treating a subject suffering from amyotrophic lateral sclerosis (ALS), comprising periodically administering to the subject an amount of pridopidine effective to treat the subject.

2. The method of claim 1, wherein the amount of pridopidine is effective to inhibit or reduce the progression of an ALS symptom in the subject.

3. The method according to claim 2, wherein the symptom is muscle stiffness, muscle weakness, muscle wasting, muscle spasms, difficulty speaking, difficulty swallowing, difficulty breathing, difficulty chewing, difficulty walking, fasciculations and/or poor posture.

4. The method according to any one of claims 1 to 3, wherein the ALS is sporadic ALS.

5. The method according to any one of claims 1 to 4, wherein the amount of pridopidine is administered daily or wherein the amount of pridopidine is administered more than once a day.

6. The method according to any one of claims 1 to 4, in which the amount of pridopidine is administered twice a day.

7. The method according to any one of claims 1-4, in which the amount of pridopidine is administered less frequently than once a day.

8. Method according to any one of claims 1 to 7, wherein the amount of pridopidine is administered orally.

9. The method according to any one of claims 1 to 8, in which the amount of pridopidine administered is from 22.5 to 225 mg per day.

10. The method according to claim 9, in which the amount of pridopidine administered is from 45 to 180 mg per day.

11. The method according to claim 9, in which the amount of pridopidine administered is 5, 10, 22.5, 45, 67.5, 90, 100, 112.5, 125, 135, 150 or 18o mg per day.

12. Method according to any one of claims 1 to 11, wherein intermittent administration is continued for at least 24 weeks.

13. The method according to any one of claims 1 to 12, in which pridopidine is pridopidine hydrochloride.

14. The method according to any one of claims 1 to 13, wherein the subject is a human.

15. The method according to any one of claims 1 to 14, further comprising administering to a subject therapeutically

- 12 043430 effective amount of the second compound.

16. The method of claim 15, wherein the second compound is riluzole, edaravone or dextromethorphan/quinidine.

17. The method according to claims 15, 16, in which pridopidine and the second compound are administered in one unit.

18. The method according to claims 15, 16, wherein pridopidine and the second compound are administered in more than one unit.

19. The method according to any one of claims 15 to 18, wherein the second compound is riluzole.

20. The method according to claim 19, characterized in that the subject is administered 10-200 or 50, 100 or 200 mg of riluzole per day.

21. The method according to any one of claims 15 to 20, wherein riluzole is administered orally.

22. The method according to any one of claims 15 to 18, wherein the second compound is edaravone.

23. The method of claim 22, wherein 5-60, or 30, or 60 mg of edaravone is administered to the subject per day.

24. The method according to any one of claims 15-18 and 22, 23, in which edaravone is administered by intravenous infusion.

25. The method according to any one of claims 15-18 and 22-24, wherein edaravone is administered once daily for 14 or 10 days, followed by a 14-day drug-free period.

26. The method according to any one of claims 15 to 18, wherein the second compound is dextromethorphan/quinidine.

27. The method of claim 26, wherein 10, 20 or 40 mg of dextromethorphan is administered to the subject per day and 5, 10 or 20 mg of quinidine is administered to the subject per day.

28. The method according to any one of claims 26, 27, in which dextromethorphan/quinidine is administered orally.

29. The method according to any one of claims 15 to 28, wherein the amount of pridopidine and the amount of the second compound are administered simultaneously.

30. The method according to any one of claims 15 to 28, wherein the administration of the second compound substantially precedes the administration of pridopidine.

31. The method according to any one of claims 15 to 28, wherein the administration of pridopidine substantially precedes the administration of the second compound.

32. The method of any one of claims 15 to 28, wherein the subject receives edaravone therapy, dextromethorphan/quinidine therapy, or riluzole therapy prior to initiation of pridopidine therapy.

33. The method of claim 32, wherein the subject has received edaravone therapy, dextromethorphan/quinidine therapy, or riluzole therapy for at least 24, 28, 48, or 52 weeks before starting pridopidine therapy.

34. The method of any one of claims 15 to 28, wherein the subject receives pridopidine therapy prior to initiation of edaravone therapy, dextromethorphan/quinidine therapy, or riluzole therapy.

35. The method of claim 34, wherein the subject has received pridopidine therapy for at least 24, 28, 48, or 52 weeks prior to initiating edaravone therapy, dextromethorphan/quinidine therapy, or riluzole therapy.

36. The method according to any one of claims 15 to 35, wherein each of the amount of the second compound taken separately and the amount of pridopidine taken separately is effective for treating the subject.

37. The method of any one of claims 15 to 35, wherein either the amount of the second compound when taken alone, the amount of pridopidine when taken alone, or each such amount when taken alone is ineffective for treating the subject.

38. The method of any one of claims 15 to 35, wherein either the amount of the second compound when taken alone, the amount of pridopidine when taken alone, or each such amount when taken alone is less effective in treating the subject .

39. Method according to any one of claims 15 to 38, wherein pridopidine is administered in addition to the second compound.

40. The method according to any one of claims 15 to 38, wherein the second compound is administered in addition to pridopidine.

41. The method according to any one of claims 1 to 40, wherein the saturating dose in an amount different from the intended dose is administered over a period of time at the beginning of the periodic administration.

42. A method of enhancing axonal transport of BDNF in motor neurons in a subject suffering from ALS, comprising administering to the subject an amount of pridopidine effective to enhance axonal transport of BDNF in motor neurons of the subject.

43. A method of improving NMJ formation and function in a subject suffering from ALS, comprising administering to the subject an amount of pridopidine effective to improve NMJ formation and muscle contractile function in the subject.

44. A method of improving the degree of innervation of muscle tissue in a subject suffering from ALS, comprising administering to the subject an amount of pridopidine effective to improve the degree of innervation in the subject.

45. A method of enhancing axonal growth of motor neurons in a subject suffering from ALS, comprising administering to the subject an amount of pridopidine effective to enhance axonal growth of motor neurons in the subject.

46. A method of enhancing muscle contraction in a subject suffering from ALS, comprising administering

- 13 043430 to the subject an amount of pridopidine effective to enhance muscle contraction in the subject.

47. A method of restoring muscle contraction in a subject suffering from ALS, comprising administering to the subject an amount of pridopidine effective to improve muscle contraction in the subject.

48. A pharmaceutical composition containing an effective amount of pridopidine for use in treating a subject suffering from ALS.

49. Use of an amount of pridopidine to produce a medicament for treating a subject suffering from ALS.

50. Packaging containing:

a) a pharmaceutical composition comprising an amount of pridopidine; and if necessary

b) instructions for use of the pharmaceutical composition for the treatment of a subject suffering from ALS.

51. A therapeutic package for dosing to a subject or for use in dosing to a subject, which includes:

a) one or more unit doses, each such unit dose containing an amount of pridopidine, wherein the amount of said pridopidine in said unit dose is effective when administered to said subject for treating ALS in the subject; And

b) a ready-made pharmaceutical container therefor containing said unit dose or units, wherein said container further includes or contains instructions indicating the use of said package in the treatment of a subject suffering from ALS.

52. Packaging containing:

a) a first pharmaceutical composition comprising an amount of pridopidine and a pharmaceutically acceptable carrier;

b) a second pharmaceutical composition comprising an amount of a second compound that is riluzole, edaravone or dextromethorphan/quinidine, and a pharmaceutically acceptable carrier; and if necessary

c) instructions for using the first and second pharmaceutical compositions together to treat a subject suffering from ALS.

53. The package according to claim 52, wherein the amount of the second compound and the amount of pridopidine are prepared for administration simultaneously or sequentially.

54. A therapeutic package for dosing to a subject suffering from ALS or for use in dosing to a patient, which includes:

a) one or more unit doses, including:

i) an amount of pridopidine; and ii) an amount of a second compound which is riluzole, edaravone or dextromethorphan/quinidine;

wherein appropriate amounts of said pridopidine and a second compound in said unit dose are effective when administered concomitantly to said subject for treating the subject, and

b) a ready-made pharmaceutical container therefor containing said unit dose or units, wherein said container further contains or includes instructions indicating the use of said package in the treatment of said subject.

55. A pharmaceutical composition containing an amount of pridopidine and an amount of a second compound which is riluzole, edaravone or dextromethorphan/quinidine.

56. The pharmaceutical composition according to claim 55 for use in treating a subject suffering from ALS, wherein pridopidine and the second compound are prepared for simultaneous, sequential or concomitant administration.

57. A pharmaceutical composition in unit dosage form suitable for treating a subject suffering from ALS, which includes:

a) amount of pridopidine;

b) an amount of a second compound that is riluzole, edaravone, or dextromethorphan/quinidine, wherein respective amounts of said second compound and said pridopidine in said composition are effective when simultaneously administered to said subject by one or more of said unit dosage forms of said composition for treating the subject.

58. A pharmaceutical composition comprising an amount of pridopidine for use in the treatment of a subject suffering from ALS as adjunctive therapy to a second compound which is riluzole, edaravone or dextromethorphan/quinidine.

59. A pharmaceutical composition containing an amount of pridopidine for use in treating a subject suffering from ALS, simultaneously, sequentially or in conjunction with a second compound that is riluzole, edaravone or dextromethorphan/quinidine.

60. A pharmaceutical composition containing an amount of a compound that is riluzole, edaravone or dextromethorphan/quinidine, for use in the treatment of a subject suffering from

- 14 043430 general ALS, as an additional therapy to pridopidine.

61. A pharmaceutical composition containing an amount of a compound that is riluzole, edaravone or dextromethorphan/quinidine, for use in the treatment of a subject suffering from ALS, simultaneously, sequentially or in parallel with pridopidine.

62. A compound that is riluzole, edaravone or dextromethorphan/quinidine for use as an adjunctive therapy to pridopidine in the treatment of a subject suffering from ALS.

63. Pridopidine for use as an adjunctive therapy to a compound that is riluzole, edaravone or dextromethorphan/quinidine for the treatment of a subject suffering from ALS.

64. An adjunctive therapy as defined in claim 63 for the treatment, prevention, or symptomatic relief of ALS.

65. A combination of pridopidine with a compound that is riluzole, edaravone, or dextromethorphan/quinidine, for use in the treatment, prevention, or symptomatic relief of ALS.

Certain publications and patent application publications are referenced in this document. Full references to publications can be found immediately before the claims. The disclosures of these publications and patent application publications are incorporated herein by reference in their entirety to more fully describe the prior art to which this invention relates.

This invention will be better understood by reference to the experimental details that follow, but those skilled in the art will readily understand that the detailed specific experiments are only illustrative of the invention, as more fully described in the claims that follow.

Experiment details

Experiment 1. Analysis of axonal transport.

Healthy motor neurons (MNs) extend axons over long distances and through various extracellular microenvironments to form synapses with muscles. The ability of a neuron to maintain this specialized morphology depends on cytoskeletal elements and the continuous transport of proteins and organelles into and out of the cell body. Cytoskeletal changes are a major pathway involved in the pathogenesis of ALS, affecting axonal transport, growth and function of the neuromuscular junction (NMJ) (Eykens and Robberecht, 2015). Changes in axonal transport are among the first cellular processes to occur in neurodegenerative diseases, including ALS. Axonal transport was assessed using an in vitro compartmental microfluidic chamber system that separates neuronal cell bodies from their axons and synapses, allowing the study of retrograde/anterograde transport through specific monitoring and manipulation of cellular microenvironments (Figure 1b; Zahavi, 2015; Ionescu, 2016) .

Quantum dot-labeled BDNF (Qdot-BDNF) is retrogradely transported in the axons of motor neuron explants grown in a microfluidic chamber (MCC). MPA was used to analyze the axonal transport of Qdot-BDNF. Axonal transport of BDNF has been studied in the SOD1 (SOD1G93A) model for ALS (Bilsland, 2010; Perlson, 2009; De Vos, 2007). The effect of pridopidine on Qdot-BDNF transport along motor neuron axons was assessed in spinal cord explants of SOD1 and WT (LM) mice. Experimental progress for axonal transport assay (from left to right, Fig. 1a): SFC1G93A or wild-type (LM) spinal cord explants were placed in the MFC. Approximately 5 days after insertion into the chamber, axons began to move into the distal compartment. On the 6th day after placement in the chamber, an amount of pridopidine was added to both compartments. On day 7, Qdot-BDNF was added to the distal compartment and axonal transport images were obtained. Schematic representation of the microfluidic chamber system (Figure 1b): Spinal cord (SC) explants from WT or SOD1G93A E12.5 mouse embryos were placed in the proximal compartment of the microfluidic chamber. MHs from the spinal cord send their axons to the distal region. At this point, Qdot-BDNF was used exclusively for this compartment. Axonal transport of Qdot-BDNF was then visualized and recorded using a high-resolution spinning-disk confocal imaging system.

Spinning-disk confocal microscopy was used to track Qdot-BDNF along the axons of motor neuron explant cultures. Time-lapse images of Qdot-BDNF axonal transport were acquired at 60× magnification (Fig. 1c). Yellow arrows indicate a single Qdot-BDNF particle that is retrogradely transported (left) to the cell body. Scale bar: 10 µm. The bottom panel shows the complete Qdot-BDNF time-lapse recording. Scale bars: horizontal 10 µm; vertically for 100 s (Fig. 1c).

Control and pridopidine were added to both sides of the chamber at two concentrations (0.1 and 1 μM) on experimental day 6, and Qdot-BDNF was added to the distal compartment after incubation with pridopidine overnight (Fig. 1a and 1b). Six independent biological replicates from six different cultures were tested such that from each culture and neuronal/glial explant, ∼250 BDNF particles were traced along the axons in the cavities. Speed n refers

- 15 043430 to the movement of one BDNF particle. The experiment was repeated with MH from sigma-1 receptor knockout (S1R KO or S1R-/-) mice (Langa, 2003). Ventral spinal cord sections from S1R mouse embryos

KO were cultured and placed in MFC as described above, and axonal transport was analyzed

Qdot-BDNF.

SOD1G93 and S1R KO explants with or without pridopidine were compared with littermate controls (LM).

Qdot-BDNF particle tracking was performed on Bitplane Imaris using the semi-automated point tracking function. Inclusion criteria for particle analysis: track duration >10 frames; average speed >0.2 µm/s; stop duration: speed <0.1 µm/s for 3 frames. The data were then exported to MATLAB for further analysis of particle transport, including instantaneous velocities (Fig. 2a) from 6 independent cultures; and counting stops (Fig. 2b).

Results.

In fig. Figure 2a shows that pridopidine enhances the instantaneous rate of axonal transport of BDNF in SOD1G93A motor neurons. The instantaneous rate of retrograde transport of BDNF is generally reduced in SOD1G93A motor neurons. SOD1G93A MHs show slower rates compared to wild-type MHs. Pridopidine treatment accelerated instantaneous velocities in both wild-type (0.1 μM) and SOD1G93A motor neurons (0.1 and 1 μM). Application of 25 or 100 μM riluzole to MH SOD1G93A had no effect on instantaneous rates. MH S1R−/− showed defects in axonal transport of BDNF. Pridopidine at 0.1 or 1 μM failed to rescue these defects in MH S1R KO (Fig. 2a).

The number of particle stalls (number of Qdot-BDNF stalls counted per second) shows that pridopidine reduced the number of pauses during axonal transport in WT MH (blue) (0.1 μM only) and SOD1G93A MH (red) (both 0.1 and and 1 µM). Axonal transport parameters of Qdot-BDNF in S1R−/− MHs show that they do not respond to pridopidine at any of the concentrations tested (Fig. 2b).

These results indicate that pridopidine enhances axonal transport of BDNF in SOD1G93A motor neurons and has the ability to correct ALS-associated deficits.

Experiment 2: Axon-muscle growth/degeneration assays.

An early event in the pathogenesis of ALS is axonal degeneration. A compartmental microfluidic co-culture chamber system was used to determine whether pridopidine reversed axonal degeneration (Figure 3). Primary muscle cells from asymptomatic (P60) WT or SOD1G93A mice were cultured. On the sixth day, primary skeletal myoblasts were cultured in the distal IFC. Approximately six days later (day 12), ventral spinal cord explants from WT or SOD1G93A E12.5 mouse embryos that express HB9-GFP (a specific marker of motor neurons) were placed in the proximal compartment, followed by injection of pridopidine or control solution into both compartments. Pridopidine was renewed every other day. Two days after explant placement in the chamber (day 14), motor axon growth and degeneration were assessed using real-time imaging on a spinning-disc confocal system. Axonal growth was monitored by imaging every 10 min for 8 h. Experiments were repeated three times.

Results.

The data show that pridopidine enhanced neurite outgrowth (Fig. 4). Myocytes carrying the SOD1G93A mutation have a reduced number of healthy axons that are able to penetrate the distal microfluidic compartment compared to WT myocytes (LM). Treatment with 1 μM pridopidine (far right column) showed an increase in the number of axons extending into the distal compartment (compartment with muscle cells). (The Y axis is the average number of cavities with axons extending into the muscle compartment.)

These results indicate that pridopidine enhances axonal growth and has the ability to correct ALS-related deficits.

Experiment 3: Measurement of neuromuscular junction (NMJ) formation and function.

Synaptic destruction is the earliest disruption of the cellular compartment in ALS. To test the ability of pridopidine to influence synaptic function in the ALS model, cultures from experiment 2 were grown for approximately four additional days (day 18), when axons extend into the distal compartment and generate NMJ connections. In this co-culture, primary myocytes were fully differentiated and formed connections with MN axons, and these connections were postsynaptic markers (AChR (acetylcholine R)) colocalizing with HB9:GFP neurons. Fig. 5a: Top panel: phase image of a myocyte in the distal compartment connected by axons (green arrows). Scale bar: 20 µm. Bottom panel: High power images of myocytes: MH contact points show NMJ formation as seen by co-localization of postsynaptic AChR with HB9::GFP axons and three-dimensional co-localization of pre- and postsynaptic markers (coloc). To assess NMJ function, muscle contraction recordings were acquired at 30 frames/s for 1000 frames.

- 16 043430 (Fig. 5b). Muscle contraction traces extracted from time intensity measurements of muscle contraction show a flat trace of a non-contracting, stationary myocyte (top) and a contracting myocyte trace showing multiple bursts of activity (bottom).

To study the effect of pridopidine on MH and NMJ formation and function, either 0.1 or 1 μM pridopidine or control solution was added. Measurements of % innervation and innervation-induced contraction in myotubes were assessed using live cell imaging as previously reported (Ionescu, 2015; Zahavi, 2015). Briefly, contractile activity of muscles in the distal portion of the IFC that were exposed to at least one axon was examined. Muscles were divided into two groups: contracting or non-contracting, depending on their motor activity during recording. Muscle mobility was confirmed by plotting intensity plots for each muscle (Fig. 5b). The number of contracting muscle fibers per chamber was divided by the total number of muscle fibers analyzed in the same chamber to obtain the percentage of contracting myotubes as the output for NMJ activity.

Results.

Treatment of muscle cells with pridopidine was able to induce innervation, resulting in a higher likelihood of a saltatory muscle contraction profile. The level of innervation of muscles carrying the SOD1 mutation is lower compared to WT (wild type) muscles (20% innervation compared to ~40% in WT). Pridopidine at a concentration of 1 μM restores the level of innervation of muscles carrying the SOD1 mutation to a level close to WT (Fig. 6).

The percentage of muscle fibers contracting in co-cultures shows that only ~10% of muscles contracted in the absence of MH versus 74% in cultures including MH. Co-culture combinations that included at least one of the cell types expressing SOD1G93A showed a significantly lower percentage of contracting myocytes (Fig. 7a). Treatment of myocytes co-cultured with SOD1G93A with pridopidine (0.1 and 1 μM) increases the percentage of myocyte contraction and restores neuromuscular activity to WT levels. The combination of MH S1R−/− with WT myocytes results in a low number of contracting myotubes compared to MH WT. Application of 0.1 μM pridopidine to S1R −/− co-cultures did not restore neuromuscular activity as seen for the same concentration of pridopidine in co-cultures with WT neurons. The increase in the percentage of contracting myocytes observed after treatment with 1 μM pridopidine is significantly lower than the percentage of contracting myocytes in untreated and treated WT cultures. Data are shown as mean ± SEM (standard error of the mean). * p-value <0.05; ** p-value <0.01, *** p-value <0.001, **** p-value <0.0001. (Student's T-test).

Experiment 4: ERK activation in MH WT and SOD1G93A.

The ERK pathway contributes to numerous cellular functions, including proliferation and differentiation. Phosphorylation (activation) of ERK in neurons is associated with neurotrophic signaling, such as the BDNF stimulus, which in turn promotes neuroprotection and neuronal survival (Bonni 1999). Previously, pridopidine was found to enhance BDNF signaling in the rat striatum through S1R, which in turn enhances ERK activation (Geva, 2016). Primary MH cultures at 2DIV were maintained overnight in neurotrophin- and serum-free (PNB) medium. The next day, cultures were treated for 30 min with pridopidine or BDNF as a positive control.

Results.

Western blot testing for phosphorylated ERK (pERK) shows significant activation of ERK by pridopidine (0.1 and 1 μM) as early as 30 min in cultures in MH WT (left panel) and SOD1G93A (middle panel), but not in MH S1R-/- (right panel) (Fig. 8a). Quantification of pERK shows ∼3.5- and ∼4-fold increases in WT MH abundance after treatment with 0.1 and 1 μM pridopidine, respectively. SOD1G93A shows ∼2.9- and ∼8.5-fold increases in pERK after treatment with 0.1 and 1 μM pridopidine, respectively. Pridopidine did not induce ERK phosphorylation in MH S1R−/−. Data are presented as mean pERK/ERK ratio ± SEM. *p-value <0.05, ~p-value <0.1 (Student's t test) (Fig. 8b).

Experiment 5. Effect of pridopidine on mutant SOD1 aggregates in the spinal cord of SOD1G93A mice.

Pridopidine induced neuroprotective properties through activation of S1R, as demonstrated by its effects on axonal transport, axonal degeneration, NMJ function, and ERK activation. S1R is located on the ER membrane in close proximity to the outer mitochondrial membrane, where mutant SOD1 protein tends to aggregate in the spinal cord of SOD1G93A mice (Millecamps and Julien, 2013). Asymptomatic SOD1G93A mice (5 weeks old) and WT control mice were treated with saline, 3 or 30 mg/kg pridopidine, daily by subcutaneous injection for 11 weeks (until 16 weeks of age). At the end of the experiment, the lumbar spinal cord (L1-L6) was removed, fixed, and embedded in paraffin for cryosection. Then

- 17043430 µm sections were prepared and stained with NSC500 to visualize SOD1 aggregates (Hammarstrom, 2010). The effect of pridopidine treatment in vivo on the number of mutant SOD1 aggregates in the gray and white matter of the spinal cord was assessed.

Results.

Fig. 9a - left panel: representative images of the spinal cord with a fluorescent label at low magnification for 4 groups of mice. Right panel: High magnification images for the areas marked with a square in the left panel. Scale scale: left panel: 500 µm; right panel 50 µm. From top to bottom: WT with control solution, SOD1G93A with control solution, SOD1G93A with 0.3 mg/kg pridopidine, SOD1G93A with 30 mg/kg pridopidine, all stained with NSC500 dye to label SOD1 mutant protein aggregates. A significant increase in the number of mSOD1 aggregates was observed in both gray and white matter of the spinal cord of SOD1G93A mice compared to WT mice. Pridopidine at a dose of 30 mg/kg significantly reduced the number of aggregates in both the gray and white matter of the SOD1G93A spinal cord by ~50% (Fig. 9a-9c). Data are presented as mean ± SEM. * p-value <0.05; **p-value <0.01 (one-way ANOVA followed by Fisher's least significant difference post hoc tests), (Fig. 9b, 9c. Y-axis - number of NSC500-positive SOD1 aggregates per mm ² ).

Experiment 6: In vivo assessment of muscle fiber atrophy and NMJ preservation.

Destruction of the NMJ and subsequent wasting of skeletal muscle are the two main pathologies of ALS. The effect of pridopidine on muscle fiber atrophy and NMJ preservation was assessed in vivo. Asymptomatic SOD1G93A mice and WT control mice (5 weeks) were treated with either saline or pridopidine 3 or 30 mg/kg by daily subcutaneous administration for 11 weeks. Gastrocnemius muscles from mice treated with control solution or pridopidine (30 mg/kg sc) were isolated from SOD1G93A and WT mice at 16 weeks of age. Muscle cross sections were stained with hematoxylin and eosin (H&E), and the average muscle fiber diameter was quantified for each group (Fig. 10a). NMJ retention was assessed by confocal imaging of the co-localization of presynaptic (NFH + synapsin-I; green) and postsynaptic (AChR (BTX; red) markers and counting the number of fully innervated NMJs in gastrocnemius muscles (Fig. 11a).

Results.

Fig. 10a: Representative images of HE-stained transverse sections of gastrocnemius muscles from mice from 4 groups: WT control, 30 mg/kg pridopidine WT, control SOD1G93A, and 30 mg/kg pridopidine SOD1G93A mice. The muscle histology of control solution-treated SOD1G93A mice was poor and showed smaller muscle fiber diameter compared to control solution-treated WT mice and 30 mg/kg pridopidine-treated WT mice (Figures 10a, 10b). Pridopidine (30 mg/kg, daily subcutaneous administration) resulted in a significant increase in muscle fiber diameter by ∼4 μm in SOD1G93A mice and by ∼5 μm in muscle from WT mice.

The preservation of NMJ in muscle of SOD1G93A mice treated with control solution indicates the expected mass loss of NMJ of ~60% and morphological changes in the postsynaptic apparatus in muscle of SOD1G93A mice compared to WT mice (Fig. 11a, 11b). Strikingly, pridopidine treatment limited NMJ loss in SOD1G93A mice to ∼20%. Data are shown as mean ± SEM. * p-value <0.05; **p-value <0.01; ***p-value <0.001 (double-blind Student's t test).

Overall, these results demonstrate that pridopidine has neuroprotective effects in cellular and animal models of ALS. In vitro, in MH SOD1G93A, pridopidine enhances axonal BDNF transport, up-regulates ERK activation, accelerates axonal growth, restores muscle innervation, and improves NMJ formation and function. These neuroprotective effects were mediated by S1R. In vivo treatment of ALS SOD1G93 A mice with pridopidine reduced the aggregation of mutant SOD1 in the spinal cord (one of the characteristic phenotypic manifestations of the disease), increased the diameter of muscle fibers reduced in ALS, and preserved the degenerated NMJs observed in the affected tissue. These data support the use of pridopidine as a neuroprotective agent and S1R as a therapeutic target for the treatment of patients with ALS.

Additional animal models of ALS useful for testing pridopidine are described, for example, in McGoldrick (2013).

The following abbreviations are used in the figures: Geno = genotype (ie wild type (LM), SOD1 mutant), Prido = pridopidine, mpk = mg/kg.

Experiment 7. Treatment of ALS in humans.

Intermittent oral administration of pridopidine provides clinically significant benefit in reducing ALS symptoms in people with ALS. Pridopidine therapy provides effective treatment for the patient without unwanted side effects and is at least effective

- 18 043430 in one of the following embodiments:

1) therapy is effective in improving ALS symptoms;

2) therapy is effective in enhancing axonal transport of BDNF in motor neurons and/or enhancing ERK activation;

3) therapy is effective for improving the formation and preservation of the NMJ, preserving the structure of the NMJ, preserving the function of the NMJ and/or improving the degree of innervation of muscle tissue;

4) the therapy is effective in enhancing motor neuron axonal growth and/or reducing axonal degeneration, including motor neuron axonal degeneration;

5) therapy is effective in increasing muscle cell survival, increasing muscle fiber diameter and function, reducing the progression of muscle fiber wasting, and/or improving muscle contraction and/or

6) therapy is effective in reducing SOD1 aggregation and/or reducing the progression of pseudobulbar syndrome.

In some patients, the physician prescribes pridopidine and a second compound, where the second compound is riluzole, edaravone, dextromethorphan/quinidine. In some embodiments, the second compound is laquinimod.

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   ФОРМУЛА ИЗОБРЕТЕНИЯ

   1. Применение придопидина для лечения субъекта, страдающего боковым амиотрофическим склерозом (БАС), причем придопидин применяется в терапевтически эффективном количестве.
2. 2. Применение по п.1, где БАС является спорадическим БАС.
3. 3. Применение по п. 1 или 2, где количество придопидина является эффективным для ингибирования или уменьшения прогрессирования симптома БАС у субъекта.
4. 4. Применение по п.3, где симптомом является ригидность мышц, мышечная слабость, истощение мышц, мышечные спазмы, затруднение речи, затруднение глотания, затруднение дыхания, затруднение жевания, затруднение ходьбы, фасцикуляции и/или ухудшение осанки.
5. 5. Применение по любому из пп.1-4, где количество придопидина является эффективным для уси

   -