Armoclol for the treatment of glucocarbonidase-related diseases

Abstract

The present invention relates to the use of an active pharmaceutical ingredient N-[2-hydroxy-3-(1-piperidinyl)-propoxy]-pyridine-1-oxide-3-methylenimine acyl chloride and an acid addition salt thereof in the preparation of an oral medicament for the treatment of glucocerebrosidase (GBA)-related Parkinson's disease or GBA-related Parkinson's syndrome. The invention also relates to the use of the N-[2-hydroxy-3-(1-piperidinyl)-propoxy]-pyridine-1-oxide-3-methylenimine acyl chloride and the acid addition salt thereof.

Description

[0001] This application is a divisional application of the application entitled "arimoclomol for treating glucocerebrosidase-related diseases", application number 201780026445.9, filed on April 28, 2017. Technical Field

[0002] This invention relates to an active pharmaceutical ingredient selected from N-[2-hydroxy-3-(1-piperidinyl)-propoxy]-pyridine-1-oxide-3-carboximidoyl chloride, its stereoisomers and their acid addition salts (arimoclomol), for the treatment of glucocerebrosidase (GBA)-related diseases other than Gaucher's disease (GD), including GBA-related α-synucleinopathies such as GBA-related Parkinson's disease (PD), GBA-related Lewy body dementia (DLB) and GBA-related multiple system atrophy (MSA). Background of the Invention

[0003] Gaucher disease (GD) is the most common lysosomal storage disease characterized by the accumulation of glucocerebrosides. It is a form of sphingolipid disease because it involves dysfunction of sphingolipids. To date, up to 300 mutations in the GBA gene have been identified and are associated with Gaucher disease. GBA mutations can be classified as mild (causing type I GD, non-neurotic) or severe (causing type II and III GD). Homozygous GBA mutations and complex heterozygous mutations lead to GD. Some common mutations are predominant; the most common in type I GD is a missense mutation that substitutes asparagine for serine at amino acid residue 370 (N370S), while the most common in type II and III is L444P (codon numbering starts from the first codon of the mature protein, i.e., without a signal peptide).

[0004] Many of these mutations have also been found in patients with Parkinson's disease (PD). Heterozygous mutations found in GBA mutation carriers (those with a mutated GBA gene) have been found to predispose to the development of Parkinson's disease (Gan-Or et al., Neurology, 2015). Mutations in the GBA are now considered one of the major genetic risk factors for Parkinson's disease. It is estimated that at least 8% of Parkinson's disease patients have GBA gene mutations (mild and severe GBA mutations), including L444P heterozygotes. Secondary defects in GBA activity may also be associated with Parkinson's disease.

[0005] The main pathology of Parkinson's disease caused by GBA deficiency has not yet been elucidated, but preclinical experiments have shown an inverse relationship with α-synuclein.

[0006] Carriers of GBA gene mutations also appear to have an increased risk of developing Lewy body dementia (DLB) and possible multiple system atrophy (MSA), which links GBA deficiency to at least some α-synucleinopathies.

[0007] WO 2014 / 071282 discloses a recombinant self-complementary adeno-associated virus vector encoding human glucocerebrosidase (AAV-GBA1) in a model to support glucocerebrosidase-enhanced therapy for PD and associated synucleinopathies and tau proteinopathies.

[0008] WO 2013 / 148333 discloses salicylic acid derivatives as glucocerebrosidase activators for the treatment of Gaucher disease and for the suppression of Gaucher disease symptoms in patients with GBA gene mutations and for the treatment of Parkinson's disease.

[0009] WO 2009 / 155936 discloses heat shock protein 70 and its inducers for the treatment of lysosomal storage diseases, including Gaucher disease.

[0010] WO 2005 / 041965 discloses the use of the heat shock inducer arimoclomol for the protection of neurons in neurodegenerative diseases, including Parkinson's disease.

[0011] Overview

[0012] Arimoclomol is a heat shock protein amplifier that is currently being evaluated for the treatment of pediatric lysosomal storage diseases and amyotrophic lateral sclerosis (ALS).

[0013] The inventors have now discovered that arimoclomol increases GBA levels and activity, not only in GBA homozygotes (exhibiting Gaucher disease and significantly reduced GBA activity) but also in mutant GBA heterozygotes (carriers). Specifically, arimoclomol increases GBA activity in GBA homozygotes (Gaucher disease patients) to clinically unaffected levels. Furthermore, arimoclomol increases GBA activity in GBA heterozygotes (clinically unaffected) and increases GBA enzyme levels (total levels and mature / ER-bound GBA).

[0014] The inventors also demonstrate herein that arimoclomol increases GBA activity in Parkinson's disease patients with mutated GBA alleles (heterozygous or homozygous, clinically unaffected by Gaucher disease).

[0015] One aspect provides an active pharmaceutical ingredient selected from N-[2-hydroxy-3-(1-piperidinyl)-propoxy]-pyridine-1-oxide-3-methyliminoyl chloride (arimoclomol), its stereoisomers and its acid addition salts, for the treatment of glucocerebrosidase (GBA)-related disorders.

[0016] In one embodiment, the GBA-related condition is associated with reduced GBA enzyme levels and / or reduced GBA enzyme activity. In another embodiment, the GBA-related condition is associated with one or more GBA gene mutations, including heterozygous and homozygous GBA gene mutations.

[0017] In one embodiment, the GBA-related condition is a GBA-related alpha-synucleinopathic disease, such as GBA-related Parkinson's disease (PD), GBA-related Lewy body dementia (DLB), and GBA-related multiple system atrophy (MSA).

[0018] In one implementation, the GBA-associated Parkinson's disease is associated with a genetically high-risk GBA phenotype of Parkinson's disease.

[0019] It also provides an active pharmaceutical ingredient selected from N-[2-hydroxy-3-(1-piperidinyl)-propoxy]-pyridine-1-oxide-3-methyliminoyl chloride, its stereoisomers and its acid addition salts, for increasing GBA levels and / or GBA activity. Attached Figure Description

[0020] Figure 1 Arimoclomol-induced dose-dependent increase in ER Hsp70 (BiP) in primary cells (human fibroblasts) from individuals (carriers, clinically unaffected by Gaucher disease) possessing heterozygous GBA alleles containing cis-L444P, A456P, and V460V mutations. See Example 1.

[0021] Figure 2 Arimoclomol induced a dose-dependent increase in GBA enzyme levels in primary cells (human fibroblasts) from individuals (carriers, unaffected by Gaucher disease) possessing heterozygous GBA alleles containing cis-L444P, A456P, and V460V mutations. See Example 1.

[0022] Figure 3 Arimoclomol-induced dose-dependent increase in GBA activity in L444P / L444P, A456P, and V460V Gaucher disease TII patients. The activity level increased to a clinically unaffected level (dashed line). See Example 2.

[0023] Figure 4Arimoclomol-induced dose-dependent increase in GBA activity was observed in L444P, A456P, and V460V heterozygotes (carriers; clinically unaffected parents of Gaucher disease patients; high-risk Parkinson's disease genotypes). Levels increased more than two-fold. See Example 2.

[0024] Figure 5 Arimoclomol-induced dose-dependent increase in GBA activity in primary cells of patients with type I (N370S / V394L and N370S / 1-BP ins 84G), type II (E326K, L444P / E326K, L444P and G325R / C342G and P415R / L444P), or type III (L444P / L444P) Gaucher disease. See Example 3.

[0025] Figure 6 Arimoclomol-induced dose-dependent increase in GBA activity in primary cells of Parkinson's disease patients with heterozygous GBA alleles containing the N370S mutation (N370S / +). See Example 4.

[0026] Figure 7 Arimoclomol-induced dose-dependent increase in GBA activity in human fibroblasts from asymptomatic healthy individuals without GBA mutations (+ / +). See Example 5.

[0027] Figure 8 Increased ME569 response to active GBA-labeled Arimoclomol induced by primary cells from patients with type I (N370S / V394L), type II (G325R / C342G), and type III (L444P / L444P) Gaucher disease. See Example 6.

[0028] Figure 9 Arimoclomol-induced dose-dependent increase in ER Hsp70 (BiP) from primary cells of patients with type I Gaucher disease (N370S / V394L). Vinculin was used as a loading control. See Example 7.

[0029] Figure 10 Arimoclomol-induced dose-dependent increase in ER Hsp70 (BiP) from primary cells of patients with type I Gaucher disease (N370S / 1-BP ins 84G). RPA was used as a loading control. See Example 7.

[0030] Figure 11Arimoclomol-induced dose-dependent increase in GBA protein levels was observed in primary cells from patients with type I Gaucher disease (N370S / 1-BP ins 84G). RPA was used as a loading control. See Example 7.

[0031] Figure 12 Arimoclomol-induced dose-dependent increase in ER Hsp70 (BiP) from primary cells of patients with type II Gaucher disease (L444P / P415R). RPA was used as a loading control. See Example 8.

[0032] Figure 13 Arimoclomol-induced increase in ER Hsp70 (BiP) in primary cells from patients with type II Gaucher disease (G325R / C342G). Follicle adhesion protein was used as a loading control. See Example 8.

[0033] Figure 14 Arimoclomol-induced dose-dependent increase in GBA protein levels in primary cells from patients with type II Gaucher disease (L444P / P415R). Focal adhesion protein was used as a loading control. See Example 8.

[0034] Figure 15 Arimoclomol-induced dose-dependent increase in GBA protein levels in primary cells from patients with type II Gaucher disease (G325R / C342G). RPA was used as a loading control. See Example 8.

[0035] Figure 16 Arimoclomol-induced dose-dependent increase in ER Hsp70 (BiP) from primary cells of patients with type III Gaucher disease (L444P / L444P). Follicle adhesion protein was used as a loading control. See Example 9.

[0036] Figure 17 Arimoclomol-induced dose-dependent increase in GBA protein levels in primary cells from patients with type III Gaucher disease (L444P / L444P). RPA was used as a loading control. See Example 9.

[0037] Figure 18 Arimoclomol-induced dose-dependent increase in ER Hsp70 (BiP) from primary cells of PD-GBA (N370S / N370S) individuals. Focal adhesion protein was used as a loading control. See Example 10.

[0038] Figure 19: Arimoclomol does not affect neuronal differentiation in MASCs from GD individuals with the shown GBA mutation. Cells were treated with either a mimicry (PBS) or 400 μM arimoclomol (Ari) for 9 days. Neuronal markers such as tubulin β3 were assessed by immunostaining. Figure 19A ) and NeuN( Figure 19B The expression is shown in Example 11.

[0039] Figure 20 Increased GDA activity induced by Arimoclomol in primary neuron-like cells of GD individuals with the indicated GDA mutation. Skin-derived fibroblasts from individuals with GDTIII (L444P / L444P) were included as controls. Cells were treated with either a mimic (PBS) or 400 μM arimoclomol (Ari). See Example 11.

[0040] Detailed Explanation

[0041] β-glucocerebrosidase or glucocerebrosidase (UniProt entry P04062, GLCM_HUMAN, also known as glucocerebrosidase, acidic β-glucosidase, D-glucosyl-N-acylsphingosine glucosylhydrolase, GCase or GBA) is an enzyme with glucocerebrosidase activity that cleaves the β-glucosidic bond of glucocerebroside, an intermediate in glycolipid metabolism, through hydrolysis.

[0042] D-glucosyl-N-acylsphingosine + H2O = D-glucose + N-acylsphingosine.

[0043] GBA activity requires sphingolipid-activating protein C and anionic phospholipids. It is located in lysosomes. It is encoded by the GBA gene (official name: acid glucosidase β; gene / locus MIM number 606463; EC 3.2.1.45). Alternative splicing results in a variety of transcript variants.

[0044] Mutations in the GBA gene, which encodes a defective lysosomal enzyme in Gaucher disease, are an important and common risk factor for Parkinson's disease and related conditions. This association was first recognized clinically, where Parkinson's syndrome is rare in patients with Gaucher disease but more common in relatives of confirmed carriers (individuals who are clinically unaffected but whose gene mutations must be present based on family history).

[0045] GBA gene mutations are continuously updated in the LOVD CCHMC Molecular Genetics Laboratory Mutation Database, Gaucher Disease; Acid Glucosidase β (GBA), https: / / research.cchmc.org / LOVD2 / home.php?select_db=GBA.

[0046] Subsequently, large-scale studies showed that patients with Parkinson's disease and related Lewy body symptoms had an increased frequency of GBA mutations compared to controls. Patients with GBA-associated Parkinson's syndrome exhibited different Parkinsonian phenotypes, but tended to have earlier age of onset and more associated cognitive alterations compared to Parkinson's syndrome patients without GBA mutations. Hypotheses proposed to explain this association include gain of function due to glucocerebrosidase mutations promoting α-synuclein aggregation; substrate accumulation due to loss of enzyme function affecting α-synuclein processing and clearance; and a bidirectional feedback loop.

[0047] Alpha-synuclein is a synuclein protein with an unknown function, primarily found in neural tissue. It can aggregate to form insoluble fibrils in pathological conditions characterized by Lewy bodies, such as Parkinson's disease, Lewy body dementia, and multiple system atrophy. Alpha-synuclein is a major structural component of Lewy body fibrils.

[0048] Arimoclomol is a small molecule inducer of heat shock proteins, including Hsp70. It is currently being investigated for the treatment of amyotrophic lateral sclerosis (ALS) and lysosomal storage disease, Niemann-Pick type C. Induction of heat shock proteins, including Hsp70, protects the lysosomal membrane and increases the activity of lysosomal enzymes responsible for the degradation of lysosomal substrates.

[0049] The inventors here demonstrate that arimoclomol increases GBA activity in cells from patients with type III Gaucher disease (e.g., L444P / L444P) to clinically unaffected levels (in some cases, the same levels as GBA mutation carriers). This paper also shows that arimoclomol surprisingly increases GBA activity in cells from GBA mutation carriers (e.g., L444P heterozygotes) by more than two times to clinically unaffected levels. Furthermore, arimoclomol increases N370S GBA activity in cells from PD patients. Therefore, GBA activity (and levels) can also be increased in cells from heterozygous mutant GBA (carriers) and from cells from clinically unaffected homozygous mutant GBA.

[0050] Therefore, the increase in GBA levels and / or activity induced by Arimoclomol can be used to treat a range of protein disorders in which GBA levels and / or activity are impaired.

[0051] Arimoclomol is defined in this paper as an active pharmaceutical ingredient selected from N-[2-hydroxy-3-(1-piperidinyl)-propoxy]-pyridine-1-oxide-3-methyliminoyl chloride, its stereoisomers and its acid addition salts.

[0052] This document describes the use of arimoclomol for the treatment of GBA deficiency. In one embodiment, the GBA deficiency does not include Gaucher disease (GD) itself / aspects.

[0053] This invention provides arimoclomol for the treatment of glucocerebrosidase (GBA)-related diseases other than Gaucher disease (GD).

[0054] In one embodiment, the treatment is preventative, curative, or alleviating. In one specific embodiment, the treatment is preventative. In another embodiment, the treatment is curative. In yet another embodiment, the treatment is alleviating.

[0055] The present invention also provides the use of arimoclomol in the preparation of medicaments for treating glucocerebrosidase (GBA)-related diseases other than Gaucher disease (GD).

[0056] This article also provides methods for treating glucocerebrosidase (GBA)-related conditions other than Gaucher disease (GD), methods comprising administering an effective amount of arimoclomol to individuals in need.

[0057] The term "individual" or "subject" refers to a member of a vertebrate, particularly a mammalian species, preferably including primates. In a preferred embodiment, the individual used herein is a human of any age, male or female.

[0058] "Individuals in need" refers to individuals who can benefit from the present invention. In one embodiment, the individual in need is a diseased individual, wherein the disease is a GBA-related condition.

[0059] GBA-related symptoms

[0060] In one embodiment, a compound selected from (+)-RN-[2-hydroxy-3-(1-piperidinyl)-propoxy]-pyridine-1-oxide-3-methyliminoyl chloride citrate; (-)-SN-[2-hydroxy-3-(1-piperidinyl)-propoxy]-pyridine-1-oxide-3-methyliminoyl chloride citrate; (+)-RN-[2-hydroxy-3-(1-piperidinyl)-propoxy]-pyridine-1-oxide-3-methyliminoyl chloride maleate; and (-)-SN-[2-hydroxy-3-(1-piperidinyl)-propoxy]-pyridine-1-oxide-3-methyliminoyl chloride maleate is provided for the treatment of GBA-related conditions.

[0061] As defined in this article, mentioning the use of arimoclomol for the treatment of GBA-related conditions includes any of the following conditions.

[0062] As defined herein, a GBA-related condition can refer to any condition associated with GBA levels and / or GBA activity. Therefore, a decrease in GBA levels and / or activity is associated with a GBA-related condition as defined herein. In one implementation, association implies susceptibility (or increased risk of development; or risk of performance).

[0063] In one implementation, the GBA-related symptoms are not Gaucher disease. In one implementation, the GBA-related symptoms are not type I Gaucher disease. In one implementation, the GBA-related symptoms are not type II Gaucher disease. In one implementation, the GBA-related symptoms are not type III Gaucher disease. In one implementation, the GBA-related symptoms are neither type II nor type III Gaucher disease.

[0064] In one implementation, GBA-related symptoms are associated with reduced GBA enzyme levels.

[0065] In one implementation, GBA-related symptoms are associated with reduced GBA enzyme activity.

[0066] Reduced GBA enzyme levels and / or GBA activity can also be defined as impaired GBA enzyme levels and / or GBA activity; insufficient GBA enzyme levels and / or GBA activity; or defective GBA enzyme levels and / or GBA activity.

[0067] In one implementation, GBA-related conditions are referred to as GBA-deficiency.

[0068] In one embodiment, the GBA-associated disease has GBA activity and / or enzyme levels that are reduced but sufficient to remain clinically unaffected by Gaucher disease (i.e., without Gaucher disease and not diagnosed with Gaucher disease). In another embodiment, the GBA-associated disease has reduced GBA activity and / or enzyme levels compared to wild-type activity levels.

[0069] In one implementation, GBA-related disease is associated with one or more individual GBA gene mutations. In another implementation, GBA-related disease is defined as an individual with one or more GBA gene mutations who is clinically unaffected by Gaucher disease.

[0070] In one implementation, GBA-related conditions are associated with one or more mild GBA gene mutations (associated with type I GD (TI)).

[0071] In another implementation, GBA-related conditions are associated with one or more severe GBA gene mutations (associated with type II GD (TII) and type III GD (TIII)).

[0072] In one implementation, GBA-related symptoms are associated with one or more heterozygous GBA gene mutations, wherein the heterozygous GBA gene mutations do not cause or contribute to the development of Gaucher disease.

[0073] In one implementation, GBA-related disease refers to an individual with one or more heterozygous GBA gene mutations who is clinically unaffected by Gaucher disease.

[0074] In one implementation, GBA-related conditions are associated with one or more homozygous GBA gene mutations and / or compound heterozygous GBA gene mutations, wherein the GBA gene mutations do not cause or contribute to the development of Gaucher disease.

[0075] In one implementation, GBA-related disease refers to an individual who has one or more homozygous and / or compound heterozygous GBA gene mutations but is clinically unaffected by Gaucher disease.

[0076] GBA gene-specific mutations that may affect GBA protein activity include L444P, D409H, D409V, E235A, E340A, E326K, N370S, N370S / 1-BP ins 84G, V394L, A456P, V460V, C342G, G325R, P415R, Y133*, F213I, N188S, and IVS2+1G>A / N188S.

[0077] In one implementation, GBA-related conditions are associated with (or contain, express said mutations) one or more mutations in the GBA gene, said mutations being selected from the group consisting of: L444P, D409H, D409V, E235A, E340A, E326K, N370S, N370S / 1-BP ins 84G, V394L, A456P, V460V, C342G, G325R, P415R, Y133*, F213I, N188S, and IVS2+1G>A / N188S. The one or more mutations in the GBA gene can be heterozygous, compound heterozygous, or homozygous mutations.

[0078] In one implementation, GBA-related disease is defined as an individual who remains clinically unaffected by Gaucher disease and has one or more mutations selected from the following groups: L444P, D409H, D409V, E235A, E340A, E326K, N370S, N370S / 1-BP ins 84G, V394L, A456P, V460V, C342G, G325R, P415R, Y133*, F213I, N188S, and IVS2+1G>A / N188S. The aforementioned mutations in the GBA gene can be heterozygous, compound heterozygous, or homozygous.

[0079] In one implementation, GBA-related conditions are associated with the L444P GBA gene mutation (L444P / , L444P / +, or L444P / L444P). A heterozygous GBA allele containing the L444P mutation can be designated L444P / +.

[0080] In one implementation scheme, GBA-related conditions are associated with mutations in the D409H GBA gene.

[0081] In one implementation scheme, GBA-related conditions are associated with mutations in the D409V GBA gene.

[0082] In one implementation, GBA-related conditions are associated with mutations in the E235A GBA gene.

[0083] In one implementation, GBA-related conditions are associated with mutations in the E340A GBA gene.

[0084] In one implementation, GBA-related conditions are associated with mutations in the E326K GBA gene.

[0085] In one implementation, GBA-related conditions are associated with the N370S GBA gene mutation. A homozygous GBA allele containing the N370S mutation is designated N370S / N370S. A heterozygous GBA allele containing the N370S mutation is designated N370S / +.

[0086] In one implementation, GBA-related disease is associated with mutations in the N370S / 1-BP ins 84G GBA gene.

[0087] In one implementation, GBA-related conditions are associated with mutations in the V394L GBA gene.

[0088] In one implementation, GBA-related conditions are associated with mutations in the A456P GBA gene.

[0089] In one implementation scheme, GBA-related conditions are associated with mutations in the V460V GBA gene.

[0090] In one implementation, GBA-related conditions are associated with mutations in the C342G GBA gene.

[0091] In one implementation, GBA-related conditions are associated with mutations in the G325R GBA gene.

[0092] In one implementation, GBA-related conditions are associated with mutations in the P415R GBA gene.

[0093] In one implementation, GBA-related conditions are associated with mutations in the Y133*GBA gene.

[0094] In one implementation, GBA-related conditions are associated with mutations in the F213I GBA gene.

[0095] In one implementation, GBA-related conditions are associated with mutations in the N188S and / or IVS2+1G>A / N188S GBA genes.

[0096] In one implementation, GBA-related conditions are associated with mutations in one or more GBA genes without a decrease in GBA enzyme activity.

[0097] In one embodiment, GBA-related symptoms are associated with reduced GBA enzyme activity and the GBA gene is wild-type. In another embodiment, GBA-related symptoms are associated with idiopathic reduced GBA enzyme activity. Wild-type GBA alleles may be designated (+ / +) (without GBA mutations).

[0098] In one implementation, GBA-related symptoms are associated with reduced activity of the protein due to inhibition of GBA protein activity.

[0099] In one implementation, GBA-related disorders are associated with reduced GBA activity due to repression of gene / protein transcription or translation.

[0100] In one implementation, the GBA-related condition is associated with reduced GBA activity, and the GBA gene is wild-type, and the reduced GBA activity is due to inhibition of the protein's activity or repression of the gene / protein's transcription or translation.

[0101] In one implementation, GBA-related disease is an individual with a heterozygous GBA allele containing one or more mutations selected from the group consisting of: L444P, D409H, D409V, E235A, E340A, E326K, N370S, N370S / 1-BP ins 84G, V394L, A456P, V460V, C342G, G325R, P415R, Y133*, F213I, N188S, and IVS2+1G>A / N188S.

[0102] In one implementation, GBA-related disease refers to an individual with heterozygous GBA alleles containing cis-L444P, A456P, and V460V mutations.

[0103] In one implementation, the GBA-related disease is a heterozygote of L444P, A456P, and V460V.

[0104] In one implementation, the GBA-related disease is heterozygous for the composite GBA alleles L444P, A456P, V460V.

[0105] In one implementation, GBA-related disease is defined as being a GBA mutation carrier. In another implementation, GBA-related disease is defined as being an obligate carrier. In yet another implementation, GBA mutation carriers are clinically unaffected by Gaucher disease.

[0106] In one implementation plan, GBA-related conditions are the clinically unaffected grandparents, parents, siblings, or children of a patient with Gaucher disease.

[0107] In one implementation, GBA-related conditions are clinically unaffected parents or siblings of patients with Gaucher disease.

[0108] In one implementation, GBA-related disease is an individual having a homozygous or compound heterozygous GBA allele containing one or more mutations selected from the group consisting of: L444P, D409H, D409V, E235A, E340A, E326K, N370S, N370S / 1-BP ins 84G, V394L, A456P, V460V, C342G, G325R, P415R, Y133*, F213I, N188S, and IVS2+1G>A / N188S, wherein the individual remains clinically unaffected by Gaucher disease.

[0109] In one implementation, GBA-related disease refers to an individual with a homozygous GBA allele containing the N370S / N370S mutation.

[0110] In one embodiment, an active pharmaceutical ingredient selected from N-[2-hydroxy-3-(1-piperidinyl)-propoxy]-pyridine-1-oxide-3-methyliminoyl chloride, its stereoisomers and its acid addition salts are provided for a method of treating glucocerebrosidase (GBA)-related conditions such as glucocerebrosidase (GBA)-related conditions other than Gaucher disease (GD).

[0111] In one implementation, glucocerebrosidase (GBA)-related disease is GBA-associated Parkinson's syndrome.

[0112] In one implementation, the GBA-related condition is a GBA-related Lewy body condition, such as GBA-related Parkinson's disease (PD), GBA-related Lewy body dementia, and GBA-related multiple system atrophy.

[0113] In one embodiment, an active pharmaceutical ingredient selected from N-[2-hydroxy-3-(1-piperidinyl)-propoxy]-pyridine-1-oxide-3-methyliminoyl chloride, its stereoisomers and its acid addition salts are provided for a method of treating GBA-associated α-synucleinopathy.

[0114] GBA-associated alpha-synucleinopathy is defined herein as alpha-synucleinopathy associated with the level and / or activity of the GBA enzyme. In one embodiment, alpha-synucleinopathy is characterized by decreased GBA levels and / or activity, which is associated with an increase in alpha-synuclein. In one embodiment, treatment with arimoclomol reduces alpha-synuclein aggregation. In one embodiment, treatment with arimoclomol increases GBA activity and / or levels.

[0115] In one embodiment, an active pharmaceutical ingredient selected from N-[2-hydroxy-3-(1-piperidinyl)-propoxy]-pyridine-1-oxide-3-methyliminoyl chloride, its stereoisomers, and its acid addition salts are provided for a method of treating GBA-associated α-synucleinosis, wherein the GBA-associated α-synucleinosis is selected from GBA-associated Parkinson's disease (PD), GBA-associated Lewy body dementia (DLB), and GBA-associated multiple system atrophy (MSA).

[0116] In one embodiment, an active pharmaceutical ingredient selected from N-[2-hydroxy-3-(1-piperidinyl)-propoxy]-pyridine-1-oxide-3-methylimine acyl chloride, its stereoisomers and its acid addition salts are provided for the treatment of Parkinson's disease, particularly GBA-associated Parkinson's disease.

[0117] In one implementation, GBA-associated Parkinson's disease is Parkinson's disease associated with reduced GBA enzyme levels and / or activity.

[0118] In one implementation, GBA-associated Parkinson's disease is Parkinson's disease associated with one or more GBA gene mutations. In one implementation, an individual with GBA-associated Parkinson's disease remains clinically unaffected by Gaucher disease.

[0119] In one implementation, GBA-associated Parkinson's disease is Parkinson's disease associated with a heterozygous GBA gene mutation. In another implementation, GBA-associated disease is an individual with one or more heterozygous GBA gene mutations who is clinically unaffected by Gaucher disease.

[0120] In one implementation, GBA-associated Parkinson's disease is Parkinson's disease associated with a homozygous GBA gene mutation. In another implementation, GBA-associated disease is an individual with one or more homozygous and / or compound heterozygous GBA gene mutations who is clinically unaffected by Gaucher disease.

[0121] In one implementation, GBA-associated disease is a genetically high-risk Parkinson's disease GBA phenotype. In one implementation, GBA-associated disease is GBA-deficient Parkinson's disease (PD-GBA). In one implementation, GBA-associated disease is a Parkinson's disease patient with a heterozygous GBA allele. In one implementation, GBA-associated disease is a Parkinson's disease patient clinically unaffected by Gaucher disease with a homozygous GBA allele.

[0122] In one implementation, GBA-associated Parkinson's disease is Parkinson's disease associated with mutations in the GBA gene selected from the following groups: L444P, D409H, D409V, E235A, E340A, E326K, N370S, V394L, A456P, V460V, C342G, G325R, P415R, Y133*, F213I, N188S, and IVS2+1G>A / N188S. The aforementioned one or more mutations in the GBA gene can be heterozygous, compound heterozygous, or homozygous. In one implementation, individuals exhibiting GBA gene mutations are clinically unaffected by Gaucher disease.

[0123] In one implementation, an individual with GBA-associated Parkinson's disease has a GBA gene mutation selected from the following groups: L444P, D409H, D409V, E235A, E340A, E326K, N370S, V394L, A456P, V460V, C342G, G325R, P415R, Y133*, F213I, N188S, and IVS2+1G>A / N188S.

[0124] In one implementation, GBA-associated Parkinson's disease is Parkinson's disease associated with mutations in the N370S GBA gene.

[0125] In one implementation, GBA-associated Parkinson's disease is Parkinson's disease associated with heterozygous N370S GBA gene mutations (N370S / +).

[0126] In one implementation, GBA-associated Parkinson's disease is Parkinson's disease associated with homozygous N370S GBA gene mutations (N370S / N370S).

[0127] In one implementation, GBA-associated Parkinson's disease is Parkinson's disease associated with a heterozygous L444P GBA gene mutation.

[0128] In one implementation, GBA-associated Parkinson's disease is Parkinson's disease associated with a heterozygous A456P GBA gene mutation.

[0129] In one implementation, GBA-associated Parkinson's disease is Parkinson's disease associated with a heterozygous V460V GBA gene mutation.

[0130] In one implementation, GBA-associated Parkinson's disease is Parkinson's disease associated with a heterozygous E326K GBA gene mutation.

[0131] In one embodiment, GBA-related symptoms are Parkinson's disease associated with idiopathic reduced GBA enzyme activity and / or levels. In another embodiment, GBA-related symptoms are Parkinson's disease associated with idiopathic reduced GBA enzyme activity and / or levels, wherein no GBA gene mutation has been identified.

[0132] This article also provides active pharmaceutical ingredients selected from N-[2-hydroxy-3-(1-piperidinyl)-propoxy]-pyridine-1-oxide-3-methyliminoyl chloride, its stereoisomers, and its acid addition salts, for use in one or more of the following methods:

[0133] -Increases GBA activity

[0134] -Increase the level (or amount) of GBA.

[0135] -Increase the amount of active mutant GBA,

[0136] -Increase the amount of active wild-type GBA,

[0137] -Enhancing the folding of ER-preserved mutant GBAs.

[0138] -Increase the amount of processed / mature GBAs.

[0139] - Increase the amount of mature (post-ER) GBA, and / or

[0140] - Increase the amount of mature GBA reaching the lysosomes.

[0141] In one implementation, arimoclomol is used to increase GBA levels and / or activity in individuals with GBA-related conditions, such as GBA-associated alpha-synucleinopathies, such as GBA-associated Parkinson's disease.

[0142] In one embodiment, the GBA activity is increased to 50% or more of the assumed wild-type activity level, for example, 50-60%, 60-70%, 70-80%, 80-90%, 90-100%, 100-110%, 110-120%, 120-130%, 130-140%, or 140-150% of the assumed wild-type activity level.

[0143] In one implementation, the GBA activity is increased to the assumed wild-type activity level or more.

[0144] In one embodiment, the GBA activity is increased by at least 10%, for example, at least 20%, for example, at least 30%, for example, at least 40%, for example, at least 50%, for example, at least 60%, for example, at least 70%, for example, at least 80%, for example, at least 90%, for example, at least 100%, for example, at least 110%, for example, at least 120%, for example, at least 130%, for example, at least 140%, for example, at least 150%, for example, at least 160%, for example, at least 170%, for example, at least 180%, for example, at least 190%, for example, at least 200%, for example, at least 200%, for example, at least 210%, for example, at least 220%, for example, at least 230%, for example, at least 240%, for example, at least 250%, for example, at least 260%, for example, at least 200%, for example, at least 270%, for example, at least 280%, for example, at least 290%, for example, at least 300%.

[0145] In one embodiment, the GBA level (or amount) is increased to 50% or more of the assumed wild-type level, for example, 50-60%, 60-70%, 70-80%, 80-90%, 90-100%, 100-110%, 110-120%, 120-130%, 130-140%, or 140-150% of the assumed wild-type level.

[0146] In one implementation, the GBA level is increased to the presumed wild-type level or more.

[0147] In one embodiment, the GBA level and / or activity is increased by at least 1.5 times, for example at least 2 times, for example at least 2.5 times, for example at least 3 times.

[0148] In one embodiment, the GBA level (or amount) is increased by at least 10%, for example, at least 20%, for example, at least 30%, for example, at least 40%, for example, at least 50%, for example, at least 60%, for example, at least 70%, for example, at least 80%, for example, at least 90%, for example, at least 100%, for example, at least 110%, for example, at least 120%, for example, at least 130%, for example, at least 140%, for example, at least 150%, for example, at least 160%, for example, at least 170%, for example, at least 180%, for example, at least 190%, for example, at least 200%, for example, at least 210%, for example, at least 220%, for example, at least 230%, for example, at least 240%, for example, at least 250%, for example, at least 260%, for example, at least 200%, for example, at least 270%, for example, at least 280%, for example, at least 290%, for example, at least 300%.

[0149] This article also provides a method for reducing α-synuclein aggregation using active pharmaceutical ingredients selected from N-[2-hydroxy-3-(1-piperidinyl)-propoxy]-pyridine-1-oxide-3-methyliminoyl chloride, its stereoisomers and its acid addition salts.

[0150] Preventive use

[0151] On the other hand, an active pharmaceutical ingredient selected from N-[2-hydroxy-3-(1-piperidinyl)-propoxy]-pyridine-1-oxide-3-methyliminoyl chloride, its stereoisomers and their acid addition salts are provided for reducing the risk of an individual developing glucocerebrosidase (GBA)-related conditions other than Gaucher disease, wherein the individual has reduced GBA enzyme levels and / or activity.

[0152] In one implementation, the individual has lower GBA levels and / or activity than assumed wild-type levels.

[0153] In one implementation, the individual has a lower level (or amount) of GBA than the assumed wild-type level.

[0154] In one implementation, the individual has lower GBA activity than the assumed wild-type activity level.

[0155] In one implementation, the individual has higher levels and / or activity of GBA than patients with clinically affected levels and / or activity of Gaucher disease.

[0156] In one implementation, the individual has GBA levels and / or activity that are lower than those of the assumed wild type but higher than those of clinically affected patients with Gaucher disease.

[0157] In one embodiment, the individual has the same degree of reduced GBA activity as a GBA gene mutation carrier (heterozygous GBA mutation), such as a clinically unaffected carrier, such as a confirmed carrier.

[0158] In one implementation, the individual has the same reduced GBA levels as a GBA gene mutation carrier (heterozygous GBA mutation), such as a clinically unaffected carrier, such as a confirmed carrier.

[0159] In one implementation, the individual having reduced GBA levels and / or activity has one or more heterozygous GBA gene mutations.

[0160] In one implementation, the individual having reduced GBA levels and / or activity has one or more homozygous or compound heterozygous GBA gene mutations.

[0161] In one implementation, the individual has reduced GBA activity and / or levels to a degree lower than that of the assumed wild-type.

[0162] In one embodiment, the individual has GBA activity and / or level that is approximately 5 to 95% or 10 to 90% of the assumed wild-type level, for example, 5 to 10% of the assumed wild-type activity and / or level, for example, 10 to 20%, for example, 20 to 30%, for example, 30 to 40%, for example, 40 to 50%, for example, 50 to 60%, for example, 60 to 70%, for example, 70 to 80%, for example, 80 to 90%, for example, 90 to 95%.

[0163] In one embodiment, the individual has approximately 25% to 75% of the presumed wild-type level of GBA activity and / or level. In another embodiment, the individual has approximately 50% of the presumed wild-type level of GBA activity and / or level.

[0164] In one embodiment, the individual has approximately 10% of the presumed wild-type level of GBA activity and / or level, for example, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the presumed wild-type activity and / or level.

[0165] In one embodiment, the GBA-related disease is a GBA-related alpha-synucleinosis, such as a GBA-related alpha-synucleinosis selected from the group consisting of GBA-related Parkinson's disease (PD), GBA-related Lewy body dementia (DLB), and GBA-related multiple system atrophy (MSA).

[0166] In one embodiment, an active pharmaceutical ingredient selected from N-[2-hydroxy-3-(1-piperidinyl)-propoxy]-pyridine-1-oxide-3-methyliminoyl chloride, its stereoisomers, and its acid addition salts are provided for reducing the risk of an individual developing Parkinson's disease, particularly GBA-associated Parkinson's disease, wherein the individual has reduced GBA enzyme levels and / or activity.

[0167] In one embodiment, the individual has one or more heterozygous GBA gene mutations. In one embodiment, the individual has a heterozygous GBA gene mutation selected from the group consisting of: L444P, D409H, D409V, E235A, E340A, E326K, N370S, N370S / 1-BP ins 84G, V394L, A456P, V460V, C342G, G325R, P415R, Y133*, F213I, N188S, and IVS2+1G>A / N188S. In one embodiment, the individual has a heterozygous L444P GBA gene mutation. In one embodiment, the individual has a heterozygous E326K GBA gene mutation. In one embodiment, the individual has a heterozygous N370S GBA gene mutation.

[0168] In one embodiment, the individual has one or more homozygous GBA gene mutations. In one embodiment, the individual has a homozygous GBA gene mutation selected from the following: L444P, D409H, D409V, E235A, E340A, E326K, N370S, N370S / 1-BP ins 84G, V394L, A456P, V460V, C342G, G325R, P415R, Y133*, F213I, N188S, and IVS2+1G>A / N188S. In one embodiment, the individual has a homozygous N370S GBA gene mutation.

[0169] In one embodiment, an active pharmaceutical ingredient selected from N-[2-hydroxy-3-(1-piperidinyl)-propoxy]-pyridine-1-oxide-3-methyliminoyl chloride, its stereoisomers, and its acid addition salts are provided for reducing the risk of an individual developing GBA-related Parkinson's disease, wherein the individual is a patient with Gaucher disease, such as type I, II, or III Gaucher disease.

[0170] GBA activity

[0171] Glucocerebroside lipase activity can be assessed using methods known in the art. For example, glucocerebroside lipase activity can be measured from the cerebrospinal fluid of mammals. In some embodiments, the mammal is wild-type for the GBA gene. The term "wild-type" refers to a gene or protein without any known detectable mutations that could affect protein levels and / or enzyme activity.

[0172] When a gene is found to be wild-type, but decreased glucocerebrosidase activity is observed, the decrease in activity may be due to inhibition of protein activity or repression of gene / protein transcription or translation. These mechanisms are well known in the art. For example, protein production may be repressed by anomalous cellular mechanisms. Alternatively, proteins may be modified in the cell, leading to decreased or lost enzyme activity.

[0173] Arimoclomol

[0174] Arimoclomol, as mentioned herein, includes active pharmaceutical ingredients (APIs) selected from N-[2-hydroxy-3-(1-piperidinyl)-propoxy]-pyridine-1-oxide-3-methyliminoyl chloride (arimoclomol), its stereoisomers, and its acid addition salts. Arimoclomol is further described, for example, in WO 00 / 50403.

[0175] Arimoclomol refers to the basic compound N-[2-hydroxy-3-(1-piperidinyl)-propoxy]-pyridine-1-oxide-3-methyliminoyl chloride, its optically active (+) or (-) enantiomers, mixtures of enantiomers in any proportion, and racemic compounds. Furthermore, acid addition salts formed from any of the above compounds with inorganic or organic acids are also objects of this invention. All possible geometric isomers of N-[2-hydroxy-3-(1-piperidinyl)-propoxy]-pyridine-1-oxide-3-methyliminoyl chloride are within the scope of this invention. The term "stereoisomers of N-[2-hydroxy-3-(1-piperidinyl)-propoxy]-pyridine-1-oxide-3-methyliminoyl chloride" refers to all possible optical and geometric isomers of this compound.

[0176] If necessary, N-[2-hydroxy-3-(1-piperidinyl)-propoxy]-pyridine-1-oxide-3-methylimine acyl chloride or one of its optically active enantiomers can be converted by known methods into an acid addition salt that forms with an inorganic or organic acid.

[0177] In one embodiment, the active pharmaceutical ingredient is a racemic mixture of N-[2-hydroxy-3-(1-piperidinyl)-propoxy]-pyridine-1-oxide-3-methyliminoyl chloride.

[0178] In one embodiment, the active pharmaceutical ingredient is an optically active stereoisomer of N-[2-hydroxy-3-(1-piperidinyl)-propoxy]-pyridine-1-oxide-3-methyliminoyl chloride.

[0179] In one embodiment, the active pharmaceutical ingredient is an enantiomer of N-[2-hydroxy-3-(1-piperidinyl)-propoxy]-pyridine-1-oxide-3-methyliminoyl chloride.

[0180] In one embodiment, the active pharmaceutical ingredient is selected from the group consisting of (+)-RN-[2-hydroxy-3-(1-piperidinyl)-propoxy]-pyridine-1-oxide-3-methyliminoyl chloride and (-)-(S)-N-[2-hydroxy-3-(1-piperidinyl)-propoxy]-pyridine-1-oxide-3-methyliminoyl chloride.

[0181] In one embodiment, the active pharmaceutical ingredient is an acid addition salt of N-[2-hydroxy-3-(1-piperidinyl)-propoxy]-pyridine-1-oxide-3-methyliminoyl chloride.

[0182] In one embodiment, the active pharmaceutical ingredient is selected from the group consisting of: N-[2-hydroxy-3-(1-piperidinyl)-propoxy]-pyridine-1-oxide-3-methyliminoyl chloride citrate (also known as BRX-345) and N-[2-hydroxy-3-(1-piperidinyl)-propoxy]-pyridine-1-oxide-3-methyliminoyl chloride maleate (also known as BRX-220).

[0183] In one embodiment, the active pharmaceutical ingredient is selected from the group consisting of: (+)-RN-[2-hydroxy-3-(1-piperidinyl)-propoxy]-pyridine-1-oxide-3-methyliminoyl chloride citrate; (-)-SN-[2-hydroxy-3-(1-piperidinyl)-propoxy]-pyridine-1-oxide-3-methyliminoyl chloride citrate; (+)-RN-[2-hydroxy-3-(1-piperidinyl)-propoxy]-pyridine-1-oxide-3-methyliminoyl chloride maleate; and (-)-SN-[2-hydroxy-3-(1-piperidinyl)-propoxy]-pyridine-1-oxide-3-methyliminoyl chloride maleate.

[0184] Composition

[0185] Although active pharmaceutical ingredients can be administered as feedstock chemicals, they are preferably provided in the form of pharmaceutical formulations in some embodiments. Therefore, compositions, such as pharmaceutical compositions, i.e., pharmaceutically safe compositions, comprising an active pharmaceutical ingredient as defined herein. In one embodiment, the composition comprises a pharmaceutically and / or physiologically acceptable carrier or excipient.

[0186] Pharmaceutical compositions containing the bioactive agents of this invention can be prepared using conventional techniques, for example, as described in Remington: The Science and Practice of Pharmacy, 20 th As described in Edition, Gennaro, Ed., Mack Publishing Co., Easton, PA, 2000.

[0187] Therefore, one aspect provides a composition, such as a pharmaceutical composition, comprising an active pharmaceutical ingredient selected from N-[2-hydroxy-3-(1-piperidinyl)-propoxy]-pyridine-1-oxide-3-methylimine acyl chloride (arimoclomol), its stereoisomer, and its acid addition salt, for the treatment of glucocerebrosidase (GBA)-related conditions other than Gaucher disease (GD) as defined herein.

[0188] Application and dosage

[0189] In one implementation, an active pharmaceutical ingredient or a composition comprising it, as defined herein, is administered to an individual in need at a pharmaceutically effective dose or therapeutically effective amount.

[0190] In one implementation, for a given disease or condition and its complications, a therapeutically effective amount of the active pharmaceutical ingredient is an amount sufficient to cure, prevent, reduce risk, or alleviate or partially prevent clinical manifestations. The amount effective for a specific therapeutic purpose will depend on the severity and type of the condition, as well as the subject's weight and general condition. An amount sufficient to achieve this purpose is defined as a "therapeutically effective amount."

[0191] In one embodiment, the dose is 1 μg / day to 100 mg / day; for example, 1 μg / day to 10 μg / day, for example, 10 μg / day to 100 μg / day, for example, 100 μg / day to 250 μg / day, for example, 250 μg / day to 500 μg / day, for example, 500 μg / day to 750 μg / day, for example, 750 μg / day to 1 mg / day, for example, 1 mg / day to 2 mg / day, for example, 2 mg / day to 5 mg / day, or for example, 5 mg / day to 10 mg / day, for example, 10 mg / day to 20 mg / day, for example, 20 mg / day to 30 mg / day, for example, 30 mg / day to 40 mg / day, for example, 40 mg / day to 50 mg / day, for example, 5 ... Dosage composition of 0 mg / day to 75 mg / day, for example 75 mg / day to 100 mg / day, for example 100 mg / day to 150 mg / day, for example 150 mg / day to 200 mg / day, or for example 200 mg / day to 250 mg / day, for example 250 mg / day to 300 mg / day, for example 300 mg / day to 400 mg / day, for example 400 mg / day to 500 mg / day, for example 500 mg / day to 600 mg / day, for example 600 mg / day to 700 mg / day, for example 700 mg / day to 800 mg / day, for example 800 mg / day to 900 mg / day, for example 900 mg / day to 1000 mg / day.

[0192] In one embodiment, the active pharmaceutical ingredient or composition is administered at a dose of 1 μg / kg body weight to 100 mg / kg body weight, for example, 1 to 10 μg / kg body weight, for example, 10 to 100 μg / day, for example, 100 to 250 μg / kg body weight, for example, 250 to 500 μg / kg body weight, for example, 500 to 750 μg / kg body weight, for example, 750 μg / kg body weight to 1 mg / kg body weight, for example, 1 mg / kg body weight to 2 mg / kg body weight, for example, 2 to 5 mg / kg body weight, for example, 5 to 10 mg / kg body weight, for example, 10 to 20 mg / kg body weight, for example, 20 to 30 mg / kg body weight, for example, 30 to 40 mg / kg body weight, for example, 40 to 50 mg / kg body weight, for example, 50 to 75 mg / kg body weight, or for example, 75 to 100 mg / kg body weight.

[0193] In one implementation, the dose is administered once or several times a day, for example, 1 to 6 times a day, for example, 1 to 5 times a day, for example, 1 to 4 times a day, for example, 1 to 3 times a day, for example, 1 to 2 times a day, for example, 2 to 4 times a day, for example, 2 to 3 times a day. In another implementation, the dose is administered less than once a day, for example, once every other day or once a week.

[0194] Application route

[0195] It should be understood that the preferred route of administration depends on the general condition and age of the subject to be treated, the nature of the disease to be treated, the location of the tissue to be treated in the body, and the active ingredient selected.

[0196] Systemic treatment

[0197] In one implementation, the route of administration allows the bioactive agent to be introduced into the bloodstream to ultimately target the site of desired action.

[0198] In one implementation, the route of administration is any suitable route, such as enteral routes (including oral, rectal, nasal, pulmonary, oral, sublingual, transdermal, intracisional, and intraperitoneal administration) and / or parenteral routes (including subcutaneous, intramuscular, intrathecal, intravenous, and intradermal administration).

[0199] Suitable dosage forms for this application can be prepared using conventional techniques.

[0200] External application

[0201] Parenteral administration is any route of administration other than oral / enteral, in which the bioactive agent avoids first-pass degradation in the liver. Therefore, parenteral administration includes any injection and infusion, such as bolus injection or continuous infusion, such as intravenous, intramuscular, or subcutaneous administration. Furthermore, parenteral administration includes inhalation and topical administration.

[0202] Therefore, in one embodiment, the active pharmaceutical ingredient or composition is applied externally to penetrate any mucous membrane of the animal, such as mucous membranes in the nose, vagina, eyes, mouth, reproductive tract, lungs, gastrointestinal tract, or rectum, such as the nasal or oral mucosa. Thus, parenteral administration may also include oral, sublingual, nasal, rectal, vaginal, and intraperitoneal administration, as well as administration via inhalation or via an implanted pulmonary or bronchial tube. In some embodiments, the bioactive agent is applied externally to penetrate the skin.

[0203] In one implementation, parenteral administration is performed in intravenous, subcutaneous, and intramuscular forms.

[0204] Local treatment

[0205] In one embodiment, the active pharmaceutical ingredient or composition is used as a local treatment, i.e., directly introduced to the site of action. Therefore, the active pharmaceutical ingredient can be applied directly to the skin or mucous membranes, or injected into the site of action, such as into diseased tissue or directly into the terminal artery of the diseased tissue.

[0206] Combination therapy

[0207] One aspect also provides an active pharmaceutical ingredient selected from N-[2-hydroxy-3-(1-piperidinyl)-propoxy]-pyridine-1-oxide-3-methyliminoyl chloride, its stereoisomers and its acid addition salt (arimoclomol), in combination with other therapeutic modalities, for the treatment of glucocerebrosidase (GBA)-related conditions other than Gaucher disease (GD).

[0208] Therefore, in one embodiment, the active pharmaceutical ingredient is combined with at least one other treatment modality and administered to an individual in need, said at least one other treatment modality being a conventional or known treatment for (GBA)-related conditions, including GBA-related alpha-synucleinopathies such as GBA-related Parkinson's disease (PD), GBA-related Lewy body dementia (DLB), and GBA-related multiple system atrophy (MSA).

[0209] Combining multiple treatment modalities can occur simultaneously or sequentially. Simultaneous application can be two compounds contained in the same composition or in separate compositions, or it can be one composition and another treatment modality applied substantially simultaneously. Sequential application means applying more than one treatment modality at different time points, for example, applying one treatment modality first, followed by a second treatment modality. The timeframe for sequentially applying more than one treatment modality to achieve optimal effects can be determined by those skilled in the art, and in one embodiment, the timeframe can be from 30 minutes to 72 hours.

[0210] Treatments in the form of chemical compounds can be administered together or separately at their most effective doses. Administering more than one compound can have a synergistic effect, thus effectively reducing the required dose of each drug.

[0211] Another aspect also provides a composition comprising, independently or together, i) an active pharmaceutical ingredient selected from N-[2-hydroxy-3-(1-piperidinyl)-propoxy]-pyridine-1-oxide-3-methyliminoyl chloride, its stereoisomers and their acid addition salts (arimoclomol); and ii) other therapeutic agents for treating GBA-related conditions, including GBA-related α-synucleinopathies such as GBA-related Parkinson's disease (PD), GBA-related Lewy body dementia (DLB), and GBA-related multiple system atrophy (MSA).

[0212] In one implementation, other treatment methods, or conventional or known treatment methods, are referred to as other active ingredients.

[0213] In one embodiment, an active pharmaceutical ingredient selected from N-[2-hydroxy-3-(1-piperidinyl)-propoxy]-pyridine-1-oxide-3-methyliminoyl chloride, its stereoisomers and their acid addition salts (arimoclomol), is combined with one or more other active ingredients and / or formulated as a combination product for application.

[0214] In one embodiment, the other active ingredients are selected from one or more known and / or used to treat (GBA)-related conditions, including GBA-related alpha-synucleinopathies such as GBA-related Parkinson's disease (PD), GBA-related Lewy body dementia (DLB), and GBA-related multiple system atrophy (MSA).

[0215] In one embodiment, the other active ingredient is a compound for treating Parkinson's disease. In one embodiment, the compound for treating Parkinson's disease is selected from dopamine, L-DOPA, levodopa, dopamine receptor agonists, carboxylase inhibitors such as carbidopa or benserazide, NMDA antagonists such as amatoline (Symmetrel), catechol-O-methyltransferase (COMT) inhibitors such as tolcapone and entacapone, MAO-B inhibitors such as selegiline and rasagiline, carbidopa-levodopa, anticholinergic drugs, and amantadine.

[0216] In one embodiment, the other active ingredient is a compound used to treat Gaucher disease. In one embodiment, the other active ingredient is selected from enzyme replacement therapy, allosteric chaperones, pharmacological chaperones, and substrate reduction therapy. In one embodiment, the other active ingredient is selected from the group consisting of: zebufo (Zavesca), imiglucerase (Cerezyme), eliglustat (Cerdelga), VPRIV, taliglucerase alfa (Elelyso), and velaglurase α. Example

[0217] Example 1: Dose-dependent response in Gaucher disease type II heterozygotes (Parkinson's disease genotype) - BiP and GBA induction

[0218] Materials and Methods

[0219] Cell culture

[0220] Primary human fibroblast lines were cultured in DMEM supplemented with non-essential amino acids (NEAA), 1% Pen-Strep, and 12% FCS under standard cell culture conditions (37°C and 5% CO2). They were passaged 1-2 times per week at a ratio of 1:2 or 1:3. Cells from approximately passages 16-26 without observed signs of replication senescence (visual inspection) were used for experiments.

[0221] cell lines GBA mutation Age at the time of sampling GD type GM00877 L444P / L444P, A456P, V460V 1Y II GM00878 L444P, A456P, V460V - carrier

[0222] Protein blot

[0223] Cells were collected in PBS and centrifuged at 3500 rpm for 5 min at 4 °C. The cell pellet was lysed in 1X extraction buffer (Enzo Life Science) containing protease inhibitors, sonicated, and clarified by centrifugation at 13000 rpm for 10 min at 4 °C. Protein concentration was measured by BCA assay. Samples containing approximately 10–20 μg of protein were diluted in glycoprotein denaturation buffer (New England Biolabs) and denatured by incubation at 100 °C for 10 min. Samples were incubated at 37 °C for 1 h with or without EndoH (New England Biolabs), and Laemmli sample buffer was added. SDS-PAGE was performed using the TGX gel system (Bio-Rad). After transfer to nitrocellulose membranes (Trans-Blot Turbo, Bio-Rad), the membranes were briefly stained with Ponceau S and then blocked in PBS containing 5% skim milk + 0.1% Tween (PBS-T). Incubate the membrane overnight at 4°C with primary antibody (1:500 to 1:2000 dilution) on a glass plate covered with plastic wrap. After washing in PBS-T, incubate the membrane with secondary antibody diluted 1:10,000 in PBS-T containing 5% skim milk for 1 hour. Use SuperSignal. TM West DuraExtended Duration Substrate (Life technologies) makes the imprint colored and visualizes it using the G-box system (Syngene).

[0224] result

[0225] Arimoclomol induces a dose-dependent increase in ER Hsp70 (BiP) in primary cells.

[0226] Arimoclomol has been reported to increase the expression levels of heat shock proteins such as heat shock protein 70 (HSP70) (Kieran et al., Nature Medicine, 2004).

[0227] To assess the effect of arimoclomol on ER Hsp70 (BiP) expression levels in primary cells, human fibroblasts from individuals (carriers, clinically unaffected by Gaucher disease) possessing heterozygous GBA alleles containing cis-L444P, A456P, or V460V mutations were treated for 14 days with 0, 10, 50, or 200 μM arimoclomol. Cells were then harvested for Western blot analysis. Lysates from untreated normal human fibroblasts served as controls.

[0228] Our results indicate that arimoclomol dose-dependently increased BiP expression levels in human fibroblast cell lines that are heterozygous for the composite GBA alleles L444P, A456P, and V460V. This suggests that arimoclomol can lead to a fold increase in ER-retaining mutant GBA through BiP upregulation.

[0229] Arimoclomol induces a dose-dependent increase in GBA enzyme levels in primary cells.

[0230] The effect of arimoclomol on GBA protein levels was also evaluated in human fibroblast cell lines that are heterozygous for the composite GBA alleles L444P, A456P, and V460V. Consistent with the upregulation of the ER chaperone protein BiP, a dose-dependent increase in total GBA levels was observed in arimoclomol-treated cells.

[0231] Example 2: Dose-dependent response to GBA activity in homozygous and heterozygous individuals with type II Gaucher disease (GTII and high-risk Parkinson's disease genotypes).

[0232] Materials and Methods

[0233] Cell culture

[0234] Primary human fibroblast lines were cultured in DMEM supplemented with non-essential amino acids (NEAA), 1% Pen-Strep, and 12% FCS under standard cell culture conditions (37°C and 5% CO2). They were passaged 1-2 times per week at a ratio of 1:2 or 1:3. Cells at approximately passages 16-26 without observed signs of replicative senescence (visual inspection) were used for experiments.

[0235] GBA activity assay

[0236] GBA activity was measured using the "intact cell" GBA assay with 4-methylumbelliferyl ketone β-D-glucopyranoside (4-MUG) as a substrate (Mu et al., Cell, 2008). Briefly, fibroblasts were seeded in 12-well plates and treated with arimoclomol biologically at indicated concentrations in triplicate for 4 weeks. The medium was replenished with fresh compound every 2–3 days, and cells were aliquoted twice during the experiment. After 4 weeks of treatment, cells were transferred to 96-well plates, and GBA activity was measured at pH 4.0 using 4-MUG as a substrate. The released 4-MU fluorophores were quantified as fluorescent units (FLUs), and cell density was normalized using crystal violet staining of parallel plates. Normalized data are reported in arbitrary units (mean ± SD).

[0237] result

[0238] The effect of arimoclomol on GBA activity in primary cells with GBA mutations was evaluated in fibroblasts from patients with type II Gaucher disease possessing genotypes L444P / L444P, A456P, and V460V. We observed that arimoclomol treatment increased GBA activity in a dose-dependent manner. Notably, the increase in GBA activity induced by 50 μM arimoclomol corresponded to the activity level in cells from asymptomatic individuals that were heterozygous for the L444P, A456P, and V460V alleles (as determined by...). Figure 3 (The gray line markings in the text).

[0239] Importantly, arimoclomol also increased GBA activity in primary fibroblasts that were heterozygous for the L444P, A456P, and V460V alleles. This result indicates that GBA activity can be increased even in cells derived from heterozygous mutant GBA (carriers).

[0240] Example 3: Dose-dependent response to GBA activity in homozygous individuals with Gaucher disease type I, II, and III.

[0241] Materials and Methods

[0242] Cell culture

[0243] Primary human fibroblast lines were cultured in DMEM supplemented with non-essential amino acids (NEAA), 1% Pen-Strep, and 12% FCS under standard cell culture conditions (37°C and 5% CO2). They were passaged 1-2 times per week at a ratio of 1:2 or 1:3. Cells at approximately passages 16-26 without observed signs of replication senescence (visual inspection) were used for experiments.

[0244] cell lines GBA mutation Age at the time of sampling disease GM08760 L444P,E326K / L444P,E326K 1Y TII GD GM10915 L444P / L444P 7Y TIII GD GM01607 N370S / V394L 30Y TI GD GM00372 N370S / 1-BP ins 84G 29Y TI GD GM02627 G325R / C342G 3Y TII GD GM01260 L444P / P415R 11M TII GD

[0245] GBA activity assay

[0246] GBA activity was measured using the "intact cell" GBA assay with 4-methylumbelliferyl ketone β-D-glucopyranoside (4-MUG) as a substrate (Mu et al., Cell, 2008). Briefly, fibroblasts were seeded in 96-well plates and treated with arimoclomol biologically at indicated concentrations in triplicate for 5 days. The medium was replenished with fresh compound every 2–3 days. GBA activity was measured at pH 4.0 using 4-MUG as a substrate. The released 4-MU fluorophores were quantified as fluorescent units (FLUs), and cell concentrations were normalized using crystal violet staining of parallel plates. Normalized data are reported as fold change (mean ± SD) relative to control cells treated with the simulant.

[0247] result

[0248] The effect of arimoclomol on GBA activity in primary cells with additional GBA mutations was assessed by treating cells of the indicated genotypes with arimoclomol. Our data showed that arimoclomol dose-dependently increased GBA activity in two type I GD cell lines: N370S / V394L and N370S / 1-BP ins 84G. Importantly, since 1-BP ins84G is considered a null allele, these results indicate that arimoclomol increased the activity of the N370S mutation.

[0249] A dose-dependent effect of arimoclomol was also observed on GBA activity in primary cells from patients who were homozygous for L444P (L444P / L444P) or complex heterozygous for GBA mutations G325R / C342G or P415R / L444P. A less pronounced increase in GBA activity was observed in arimoclomol-treated type II GD cells that were homozygous for the E326K and L444P alleles.

[0250] Example 4: Dose-dependent response of GBA activity in primary cells of Parkinson's disease patients with heterozygous GBA alleles containing the N370S mutation.

[0251] Materials and Methods

[0252] Cell culture

[0253] Primary human fibroblast cell lines were cultured in DMEM supplemented with non-essential amino acids (NEAA), 1% Pen-Strep, and 12% FCS under standard cell culture conditions (37°C and 5% CO2). Cells were passaged weekly at a ratio of 1:2. Cells at approximately passages 16-26 without observed signs of replication senescence (visual inspection) were used for experiments.

[0254] cell lines GBA mutation Age at the time of sampling disease ND34263 N370S 65Y PD

[0255] GBA activity assay

[0256] GBA activity was measured using the "intact cell" GBA assay with 4-methylumbelliferyl ketone β-D-glucopyranoside (4-MUG) as a substrate (Mu et al., Cell, 2008). Briefly, fibroblasts were seeded in 96-well plates and treated with arimoclomol biologically at indicated concentrations in triplicate for 5 days. The medium was replenished with fresh compound every 2–3 days. GBA activity was measured at pH 4.0 using 4-MUG as a substrate. The released 4-MU fluorophores were quantified as fluorescent units (FLUs), and cell concentrations were normalized using crystal violet staining of parallel plates. Normalized data are reported as fold change (mean ± SD) relative to control cells treated with the simulant.

[0257] result

[0258] To investigate the effect of arimoclomol on the N370S mutation in Parkinson's disease (PD), primary cells from PD patients with the N370S mutation were treated with arimoclomol. Our data showed that arimoclomol dose-dependently increased N370S GBA activity in PD patient cells.

[0259] Example 5: Dose-dependent response of GBA activity in primary (+ / +) cells of healthy individuals without GBA mutations

[0260] Materials and Methods

[0261] Cell culture

[0262] Primary human fibroblast lines were cultured in DMEM supplemented with non-essential amino acids (NEAA), 1% Pen-Strep, and 12% FCS under standard cell culture conditions (37°C and 5% CO2). They were passaged 1-2 times per week at a ratio of 1:2 or 1:3. Cells at approximately passages 16-26 without observed signs of replication senescence (visual inspection) were used for experiments.

[0263] cell lines GBA mutation Age at the time of sampling disease GM00498 - 3Y - GM05659 - 1Y - GM08401 - 75Y -

[0264] GBA activity assay

[0265] GBA activity was measured using the "intact cell" GBA assay with 4-methylumbelliferyl ketone β-D-glucopyranoside (4-MUG) as a substrate (Mu et al., Cell, 2008). Briefly, fibroblasts were seeded in 96-well plates and treated with arimoclomol biologically at indicated concentrations in triplicate for 5 days. The medium was replenished with fresh compound every 2–3 days. GBA activity was measured at pH 4.0 using 4-MUG as a substrate. The released 4-MU fluorophores were quantified as fluorescent units (FLUs), and cell concentrations were normalized using crystal violet staining of parallel plates. Normalized data are reported as fold change (mean ± SD) relative to control cells treated with the simulant.

[0266] result

[0267] To investigate the effect of arimoclomol on WT GBA protein, primary cells (+ / +) from healthy individuals without the GBA mutation were treated with arimoclomol. We observed that arimoclomol treatment increased WT GBA activity to varying degrees in a dose-dependent manner in all three cell lines.

[0268] Example 6: Increase in active GBA-based probe labeling in Arimoclomol-induced Gaucher disease TI / TII / TIII

[0269] Materials and Methods

[0270] Cell culture

[0271] Primary human fibroblast lines were cultured in DMEM supplemented with non-essential amino acids (NEAA), 1% Pen-Strep, and 12% FCS under standard cell culture conditions (37°C and 5% CO2). They were passaged 1-2 times per week at a ratio of 1:2 or 1:3. Cells at approximately passages 16-26 that showed no signs of replication senescence (visual inspection) were used for experiments.

[0272] cell lines GBA mutation Age at the time of sampling disease GM01607 N370S / V394L 30Y TI GD GM02627 G325R / C342G 3Y TII GD GM10915 L444P / L444P 7Y TIII GD

[0273] GBA is labeled ME569

[0274] Active GBA can be selectively labeled using the fluorescence-based bioprobe (ABP) ME569 (Witte et al, 2010). Briefly, fibroblasts were seeded in dishes and treated with arimoclomol biologically at the indicated concentration in duplicate for 5 days. The culture medium was replenished with fresh compound every 2–3 days. Cells were collected in PBS, and protein was extracted and its concentration determined using the BCA assay. Equal volumes of total protein were incubated with ME569 at 37°C for 30 min. Loading buffer was added, and the sample was incubated at 98°C for 5 min, followed by SDS-PAGE using a TGX gel system (Bio-Rad). After gel electrophoresis, fluorescence was detected using a red LED / 705M filter (G-box, Syngene). The amount of labeled GBA was quantified using GeneTools v.4.03.01.0 software from Syngene. Normalized data are reported as fold change relative to control cells treated with the simulant (mean ± SEM, n = 3–4).

[0275] result

[0276] The effect of arimoclomol on the amount of GBA labeled with fluorescent ABP was evaluated in primary cells from GD patients with the indicated genotypes. Our data showed that arimoclomol dose-dependently increased GBA labeling in the TI GD cell line (N370S / V394L) and the TII GD cell line (G325R / C342G). Only high doses of arimoclomol were evaluated in the TII GD cell line (homozygous for L444P), and in this cell line, arimoclomol increased the amount of GBA that could be labeled with fluorescent ABP.

[0277] In summary, these data show that arimoclomol increases the amount of active mutant GBA in primary cells from all three types of Gaucher disease (TI1, TIIII, and TIII III).

[0278] Example 7: Dose-dependent response in type I Gaucher disease - BiP and GBA induction

[0279] Materials and Methods

[0280] Cell culture

[0281] Primary human fibroblast lines were cultured in DMEM supplemented with non-essential amino acids (NEAA), 1% Pen-Strep, and 12% FCS under standard cell culture conditions (37°C and 5% CO2). They were passaged 1-2 times per week at a ratio of 1:2 or 1:3. Cells at approximately passages 16-26 without observed signs of replication senescence (visual inspection) were used for experiments.

[0282] cell lines GBA mutation Age at the time of sampling GD type GM00372 N370S / 1-BP ins 84G 29Y I GM01607 N370S / V394L 30Y I

[0283] Protein blot

[0284] Cells were collected in PBS and centrifuged at 3500 rpm for 5 min at 4 °C. The cell pellet was lysed in lysis buffer (Enzo Life Science) containing protease inhibitors, sonicated, and clarified by centrifugation at 13000 rpm for 10 min at 4 °C. Protein concentration was measured by BCA assay. Samples containing approximately 10–20 μg of protein were diluted in glycoprotein denaturation buffer (New England Biolabs) and denatured by incubation at 100 °C for 10 min. Samples were incubated at 37 °C for 1 h with or without EndoH (New England Biolabs), and Laemmli sample buffer was added. SDS-PAGE was performed using the TGX gel system (Bio-Rad). After transfer to a nitrocellulose membrane (Trans-Blot Turbo, Bio-Rad), the membrane was briefly stained with Ponceau S and then blocked in PBS containing 5% skim milk + 0.1% Tween (PBS-T). Incubate the membrane overnight at 4°C with primary antibody (1:500 to 1:2000 dilution) on a glass plate covered with plastic wrap. After washing in PBS-T, incubate the membrane with secondary antibody diluted 1:10,000 in PBS-T containing 5% skim milk for 1 hour. Use SuperSignal. TM West DuraExtended Duration Substrate (Life technologies) makes the imprint colored and visualizes it using the G-box system (Syngene).

[0285] result

[0286] Arimoclomol induces a dose-dependent increase in ER Hsp70 (BiP) in primary cells of type I GD.

[0287] Arimoclomol has been reported to increase the expression levels of heat shock proteins such as heat shock protein 70 (HSP70) (Kieran et al., Nature Medicine, 2004). To assess the effect of arimoclomol on the expression levels of ER Hsp70 (BiP) in primary cells, human fibroblasts from patients with TI GD were treated for 5 days with 0, 25, 100, 200, or 400 μM arimoclomol (N370S / V394L) or 0, 100, 200, or 400 μM arimoclomol (N370S / 1-BP ins 84G). Cells were then harvested for Western blot analysis.

[0288] Our results indicate that arimoclomol dose-dependently increased BiP expression levels in human fibroblast cell lines derived from individuals with type I Gaucher disease. This suggests that arimoclomol, through BiP upregulation, can lead to enhanced folding of the ER-retaining mutant GBA.

[0289] Arimoclomol induces a dose-dependent increase in GBA enzyme levels in primary type I GD cells.

[0290] The effects of arimoclomol on GBA protein levels were also evaluated in human fibroblast cell lines from patients with type I Gaucher disease possessing the N370S / 1-BP ins 84G genotype. Consistent with the upregulation of the ER chaperone protein BiP, a dose-dependent increase in total GBA levels was observed in cells treated with arimoclomol from this individual. Furthermore, the increased EndoH resistance fraction indicated that arimoclomol increased the amount of processed / mature GBA in the cells.

[0291] Example 8: Dose-dependent response in type II Gaucher disease - BiP and GBA induction

[0292] Materials and Methods

[0293] Cell culture

[0294] Primary human fibroblast lines were cultured in DMEM supplemented with non-essential amino acids (NEAA), 1% Pen-Strep, and 12% FCS under standard cell culture conditions (37°C and 5% CO2). They were passaged 1-2 times per week at a ratio of 1:2 or 1:3. Cells at approximately passages 16-26 without observed signs of replication senescence (visual inspection) were used for experiments.

[0295] cell lines GBA mutation Age at the time of sampling GD type GM01260 L444P / P415R 11M II GM02627 G325R / C342G 3Y II

[0296] Protein blot

[0297] Cells were collected in PBS and centrifuged at 3500 rpm for 5 min at 4 °C. The cell pellet was lysed in lysis buffer (Enzo Life Science) containing protease inhibitors, sonicated, and clarified by centrifugation at 13000 rpm for 10 min at 4 °C. Protein concentration was measured by BCA assay. Samples containing approximately 10–20 μg of protein were diluted in glycoprotein denaturation buffer (New England Biolabs) and denatured by incubation at 100 °C for 10 min. Samples were incubated at 37 °C for 1 h with or without EndoH (New England Biolabs), and Laemmli sample buffer was added. SDS-PAGE was performed using the TGX gel system (Bio-Rad). After transfer to a nitrocellulose membrane (Trans-Blot Turbo, Bio-Rad), the membrane was briefly stained with Ponceau S and then blocked in PBS containing 5% skim milk + 0.1% Tween (PBS-T). Incubate the membrane overnight at 4°C with primary antibody (1:500 to 1:2000 dilution) on a glass plate covered with plastic wrap. After washing in PBS-T, incubate the membrane with secondary antibody diluted 1:10,000 in PBS-T containing 5% skim milk for 1 hour. Use SuperSignal. TM West DuraExtended Duration Substrate (Life technologies) makes the imprint colored and visualizes it using the G-box system (Syngene).

[0298] result

[0299] Arimoclomol induces a dose-dependent increase in ER Hsp70 (BiP) in primary type II GD cells.

[0300] To assess the effect of arimoclomol on ER Hsp70 (BiP) expression levels in primary cells, human fibroblasts from patients with type II Gaucher disease were treated with the indicated concentration of arimoclomol for 5 days. Cells were then harvested for Western blot analysis.

[0301] Our results indicate that arimoclomol dose-dependently increased BiP expression levels in human fibroblast cell lines of TII GD individuals with the L444P / P415R or G325R / C342G genotypes. This suggests that arimoclomol can lead to enhanced folding of the ER-retaining mutant GBA in TII GD cells through BiP upregulation.

[0302] Arimoclomol induces a dose-dependent increase in GBA enzyme levels in TII GD primary cells.

[0303] The effects of arimoclomol on GBA protein levels and maturation were also evaluated in primary TII GD cell lines. Consistent with the upregulation of the ER chaperone protein BiP, a dose-dependent increase in total GBA levels was observed in cells from these individuals treated with arimoclomol. Furthermore, the increase in the EndoH resistance fraction indicated that arimoclomol increased the amount of mature (post-ER) GBA in type II GD cells.

[0304] Example 9: Dose-dependent response in type III Gaucher disease - BiP and GBA induction

[0305] Materials and Methods

[0306] Cell culture

[0307] Primary human fibroblasts were cultured in DMEM supplemented with non-essential amino acids (NEAA), 1% Pen-Strep, and 12% FCS under standard cell culture conditions (37°C and 5% CO2). They were passaged 1-2 times per week at a ratio of 1:2 or 1:3. Cells at approximately passages 16-26 without observed signs of replication senescence (visual inspection) were used for experiments.

[0308] cell lines GBA mutation Age at the time of sampling GD type GM10915 L444P / L444P 7Y III

[0309] Protein blot

[0310] Cells were collected in PBS and centrifuged at 3500 rpm for 5 min at 4 °C. The cell pellet was lysed in lysis buffer (Enzo Life Science) containing protease inhibitors, sonicated, and clarified by centrifugation at 13000 rpm for 10 min at 4 °C. Protein concentration was measured by BCA assay. Samples containing approximately 10–20 μg of protein were diluted in glycoprotein denaturation buffer (New England Biolabs) and denatured by incubation at 100 °C for 10 min. Samples were incubated at 37 °C for 1 h with or without EndoH (New England Biolabs), and Laemmli sample buffer was added. SDS-PAGE was performed using the TGX gel system (Bio-Rad). After transfer to a nitrocellulose membrane (Trans-Blot Turbo, Bio-Rad), the membrane was briefly stained with Ponceau S and then blocked in PBS containing 5% skim milk + 0.1% Tween (PBS-T). Incubate the membrane overnight at 4°C with primary antibody (1:500 to 1:2000 dilution) on a glass plate covered with plastic wrap. After washing in PBS-T, incubate the membrane with secondary antibody diluted 1:10,000 in PBS-T containing 5% skim milk for 1 hour. Use SuperSignal. TM West DuraExtended Duration Substrate (Life technologies) makes the imprint colored and visualizes it using the G-box system (Syngene).

[0311] result

[0312] Arimoclomol induces a dose-dependent increase in ER Hsp70 (BiP) in TIII GD primary cells.

[0313] To assess the effect of arimoclomol on ER Hsp70 (BiP) expression levels in primary cells, human fibroblasts from patients with type III Gaucher disease (L444P / L444P) were treated with the indicated concentration of arimoclomol for 5 days. Cells were then harvested for Western blot analysis.

[0314] Our results indicate that arimoclomol dose-dependently increased BiP expression levels in this cell line, which is homozygous for L444P. This suggests that arimoclomol can lead to enhanced folding of the ER-retaining mutant GBA through BiP upregulation.

[0315] Arimoclomol induces a dose-dependent increase in GBA enzyme levels in TIII GD primary cells.

[0316] The effects of arimoclomol on GBA protein levels and maturation were also evaluated in the L444P / L444P TIII GD primary cell line. Consistent with the upregulation of the ER chaperone protein BiP, a dose-dependent increase in total GBA levels was observed in arimoclomol-treated cells. Furthermore, the increased EndoH resistance fraction of GBA indicated that arimoclomol increased the amount of mature GBA in TIII GD cells.

[0317] In summary, these results suggest that arimoclomol may increase GBA activity due to the arrival of more mature GBA in the lysosomes.

[0318] Example 10: Dose-dependent response of BiP expression in GBA-deficient Parkinson's disease

[0319] Materials and Methods

[0320] Cell culture

[0321] Primary human fibroblasts were cultured in DMEM supplemented with non-essential amino acids (NEAA), 1% Pen-Strep, and 12% FCS under standard cell culture conditions (37°C and 5% CO2). They were passaged 1-2 times per week at a ratio of 1:2 or 1:3. Cells at approximately passages 16-26 without observed signs of replication senescence (visual inspection) were used for experiments.

[0322] cell lines GBA mutation Age at the time of sampling ND34263 N370S / N370S 65Y PD-GBA

[0323] Protein blot

[0324] Cells were collected in PBS and centrifuged at 3500 rpm for 5 min at 4 °C. The cell pellet was lysed in lysis buffer (Enzo Life Science) containing protease inhibitors, sonicated, and clarified by centrifugation at 13000 rpm for 10 min at 4 °C. Protein concentration was measured by BCA assay, and SDS-PAGE was performed on the samples using the TGX gel system (Bio-Rad). After transfer to nitrocellulose membranes (Trans-Blot Turbo, Bio-Rad), the membranes were briefly stained with Ponceau S and then blocked in PBS containing 5% skim milk + 0.1% Tween (PBS-T). The membranes were incubated overnight at 4 °C with primary antibody (1:500 to 1:2000 dilution) on glass plates covered with plastic wrap. After washing in PBS-T, the membranes were incubated for 1 h with secondary antibody diluted 1:10,000 in PBS-T containing 5% skim milk. SuperSignal was used. TM West Dura Extended Duration Substrate (Life technologies) makes the imprint colored and visualizes it using the G-box system (Syngene).

[0325] result

[0326] Arimoclomol induces a dose-dependent increase in ER Hsp70 (BiP) in primary PD-GBA cells.

[0327] To assess the effect of arimoclomol on ER Hsp70 (BiP) expression levels in primary cells from individuals with GBA-deficient Parkinson's disease (PD-GBA), human fibroblasts from individuals with PD-GBA (N370S / N370S) were treated with the indicated concentration of arimoclomol for 5 days. Cells were then harvested for Western blot analysis.

[0328] Our results indicate that arimoclomol dose-dependently increases BiP expression levels in primary cells from individuals with PD-GBA (N370S / N370S). This suggests that arimoclomol induces enhanced folding of ER-retaining mutant GBA through BiP upregulation.

[0329] Example 11: Effects of arimoclomol on GBA activity in primary neuron-like cells of individuals with TI GD and TIII GD

[0330] Materials and Methods

[0331] Cell culture

[0332] Human pluripotent adult stem cells (MASCs) were isolated from GD individuals obtained from skin biopsies. The genotypes of the MASCs are shown below. As described in Bergamin et al., *Orphanet Journal of Rare Diseases*, 2013, these cells were induced to differentiate along neuronal fates. The surface immunophenotype of the stem cells was analyzed by FACS. The expression of stem cell and neuronal markers was assessed by immunofluorescence.

[0333]

[0334] An asterisk indicates a frame shift and the corresponding new stop codon.

[0335] MASCs were induced to differentiate along the neuronal lineage on day 0. On day 1, cells were treated with either a neuron mimic (PBS) or 400 μM Marimoclomol. Treatment continued for a total of 9 days throughout the differentiation process. On day 9, differentiation was assessed by immunofluorescence of neuronal markers. GBA activity was measured using the fluorescent substrate 4-MUG.

[0336] result

[0337] Arimoclomol does not affect neuronal differentiation of skin-derived human pluripotent adult stem cells from individuals with TI GD and TIII GD.

[0338] To assess the effects of arimoclomol on neuronal differentiation, MASCs from GD individuals were induced to differentiate upon treatment with either arimoclomol or arimoclomol. Our results indicate that the expression of neuronal markers tubulin β3 and NeuN was unaffected by arimoclomol.

[0339] Arimoclomol induced an increase in GBA activity in neurons of individuals with T1 GD and TIII GD.

[0340] We found that arimoclomol increased mutant GBA activity in neurons from individuals with type I GD (N370S / Y133*) and three individuals with type III GD (F213I / L444P, L444P / L444P, or IVS2+1G>A / N188S).

[0341] In summary, our results indicate that arimoclomol increases mutant GBA activity in neurons from individuals with TI GD and TIII GD without affecting neuronal differentiation.

[0342] Various technical solutions

[0343] 1. An active pharmaceutical ingredient selected from N-[2-hydroxy-3-(1-piperidinyl)-propoxy]-pyridine-1-oxide-3-carboximide chloride, its stereoisomers and its acid addition salts, for the treatment of glucocerebrosidase (GBA)-related diseases.

[0344] 2. The active pharmaceutical ingredient according to item 1, wherein the GBA-related condition is not Gaucher disease (GD).

[0345] 3. The active pharmaceutical ingredient according to item 1, wherein the GBA-related disease is GBA-associated α-synucleinosis.

[0346] 4. The active pharmaceutical ingredient according to any one of the preceding claims, wherein the GBA-associated α-synucleinopathy is selected from GBA-associated Parkinson's disease (PD), GBA-associated Lewy body dementia (DLB), and GBA-associated multiple system atrophy (MSA).

[0347] 5. The active pharmaceutical ingredient according to any one of the preceding items, wherein the GBA-related condition is GBA-related Parkinson's syndrome.

[0348] 6. The active pharmaceutical ingredient according to any one of the preceding items, wherein the GBA-related condition is GBA-related Parkinson's disease.

[0349] 7. The active pharmaceutical ingredient according to any one of the preceding items, wherein the GBA-related symptoms are associated with decreased GBA enzyme levels.

[0350] 8. The active pharmaceutical ingredient according to any one of the preceding items, wherein the GBA-related condition is associated with decreased GBA enzyme activity.

[0351] 9. The active pharmaceutical ingredient according to any one of the preceding claims, wherein the GBA-related condition is associated with reduced GBA enzyme activity and / or levels, and the GBA gene is wild-type, and the reduction in GBA activity is due to inhibition of the protein activity or repression of the transcription or translation of the gene / protein, or is idiopathic.

[0352] 10. The active pharmaceutical ingredient according to any one of the preceding items, wherein the GBA-related condition is associated with one or more individual GBA gene mutations.

[0353] 11. The active pharmaceutical ingredient according to any one of the preceding items, wherein an individual suffering from GBA-related conditions remains clinically unaffected by Gaucher disease.

[0354] 12. The active pharmaceutical ingredient according to any one of the preceding items, wherein the GBA-related disease is associated with one or more heterozygous GBA gene mutations.

[0355] 13. The active pharmaceutical ingredient according to any one of the preceding claims, wherein the GBA-related disease is associated with a homozygous GBA gene mutation, and wherein the GBA-related disease is not Gaucher disease.

[0356] 14. The active pharmaceutical ingredient according to any one of the preceding items, wherein the one or more individual GBA gene mutations are mild (associated with type I GD).

[0357] 15. The active pharmaceutical ingredient according to any one of the preceding items, wherein the one or more individual GBA gene mutations are severe (associated with type II and type III GD).

[0358] 16. The active pharmaceutical ingredient according to any one of the preceding claims, wherein the GBA gene mutation is selected from the group consisting of: L444P, D409H, D409V, E235A, E340A, E326K, N370S, N370S / 1-BP ins 84G, V394L, A456P, V460V, C342G, G325R, P415R, Y133*, F213I, N188S and IVS2+1G>A / N188S.

[0359] 17. The active pharmaceutical ingredient according to any one of the preceding items, wherein the GBA gene mutation is L444P.

[0360] 18. The active pharmaceutical ingredient according to any one of the preceding items, wherein the GBA gene mutation is E326K.

[0361] 19. The active pharmaceutical ingredient according to any one of the preceding items, wherein the GBA gene mutation is N370S.

[0362] 20. The active pharmaceutical ingredient according to any one of the preceding claims, wherein the GBA-related disease is a heterozygote of L444P, A456P, and V460V.

[0363] 21. The active pharmaceutical ingredient according to any one of the preceding claims, wherein the GBA-related condition is a GBA mutation carrier, such as a confirmed carrier, such as a carrier clinically unaffected by Gaucher disease.

[0364] 22. The active pharmaceutical ingredient according to any one of the preceding claims, wherein the GBA-related condition is a clinically unaffected parent or sibling of a patient with Gaucher disease.

[0365] 23. The active pharmaceutical ingredient according to any one of the preceding claims, wherein the GBA-related Parkinson's disease is associated with reduced GBA enzyme levels and / or reduced GBA enzyme activity.

[0366] 24. The active pharmaceutical ingredient according to any one of the preceding claims, wherein the GBA-related Parkinson's disease is associated with one or more GBA gene mutations.

[0367] 25. The active pharmaceutical ingredient according to any one of the preceding claims, wherein the GBA-associated Parkinson's disease is associated with one or more heterozygous GBA gene mutations.

[0368] 26. The active pharmaceutical ingredient according to any one of the preceding claims, wherein the GBA-associated Parkinson's disease is associated with one or more homozygous or complex heterozygous GBA gene mutations.

[0369] 27. The active pharmaceutical ingredient according to any one of the preceding claims, wherein the GBA-related Parkinson's disease is associated with one or more GBA gene mutations selected from the group consisting of: L444P, D409H, D409V, E235A, E340A, E326K, N370S, N370S / 1-BP ins 84G, V394L, A456P, V460V, C342G, G325R, and P415R.

[0370] 28. The active pharmaceutical ingredient according to any one of the preceding claims, wherein the GBA-related Parkinson's disease is associated with heterozygous or homozygous N370S / N370S GBA gene mutations.

[0371] 29. The active pharmaceutical ingredient according to any one of the preceding claims, wherein the GBA-associated Parkinson's disease is associated with the genetically high-risk GBA genotype of Parkinson's disease.

[0372] 30. The active pharmaceutical ingredient according to any one of the preceding claims, wherein the GBA-associated Parkinson's disease is associated with idiopathic reduced GBA enzyme activity and / or levels, and no accompanying GBA gene mutations have been found.

[0373] 31. The active pharmaceutical ingredient according to any one of the preceding claims, wherein the active pharmaceutical ingredient is a racemic derivative of N-[2-hydroxy-3-(1-piperidinyl)-propoxy]-pyridine-1-oxide-3-carboximide chloride.

[0374] 32. The active pharmaceutical ingredient according to any one of the preceding claims, wherein the active pharmaceutical ingredient is an optically active stereoisomer of N-[2-hydroxy-3-(1-piperidinyl)-propoxy]-pyridine-1-oxide-3-carboximide chloride.

[0375] 33. The active pharmaceutical ingredient according to any one of the preceding claims, wherein the active pharmaceutical ingredient is an enantiomer of N-[2-hydroxy-3-(1-piperidinyl)-propoxy]-pyridine-1-oxide-3-carboximide chloride.

[0376] 34. The active pharmaceutical ingredient according to any one of the preceding claims, wherein the active pharmaceutical ingredient is selected from the group consisting of:

[0377] (+)-RN-[2-hydroxy-3-(1-piperidinyl)-propoxy]-pyridine-1-oxide-3-carboximide chloride, and

[0378] (-)-(S)-N-[2-hydroxy-3-(1-piperidinyl)-propoxy]-pyridine-1-oxide-3-formimide chloride.

[0379] 35. The active pharmaceutical ingredient according to any one of the preceding claims, wherein the active pharmaceutical ingredient is an acid addition salt of N-[2-hydroxy-3-(1-piperidinyl)-propoxy]-pyridine-1-oxide-3-carboximide chloride.

[0380] 36. The active pharmaceutical ingredient according to any one of the preceding claims, wherein the active pharmaceutical ingredient is selected from the group consisting of:

[0381] N-[2-hydroxy-3-(1-piperidinyl)-propoxy]-pyridine-1-oxide-3-carboximide citrate, and

[0382] N-[2-hydroxy-3-(1-piperidinyl)propoxy]-pyridine-1-oxide-3-formimide chloride maleate.

[0383] 37. The active pharmaceutical ingredient according to any one of the preceding claims, wherein the active pharmaceutical ingredient is selected from the group consisting of:

[0384] (+)-RN-[2-hydroxy-3-(1-piperidinyl)propoxy]-pyridine-1-oxide-3-carboximide chloride citrate;

[0385] (-)-SN-[2-hydroxy-3-(1-piperidinyl)-propoxy]-pyridine-1-oxide-3-formimide chloride citrate;

[0386] (+)-RN-[2-hydroxy-3-(1-piperidinyl)-propoxy]-pyridine-1-oxide-3-carboximide maleate chloride, and

[0387] (-)-SN-[2-hydroxy-3-(1-piperidinyl)-propoxy]-pyridine-1-oxide-3-formimide chloride maleate.

[0388] 38. The active pharmaceutical ingredient according to any one of the preceding items, wherein the treatment is preventive, curative or alleviating.

[0389] 39. An active pharmaceutical ingredient selected from N-[2-hydroxy-3-(1-piperidinyl)-propoxy]-pyridine-1-oxide-3-carboximide chloride, its stereoisomers and their acid addition salts, for reducing the risk of an individual developing glucocerebrosidase (GBA)-related diseases other than Gaucher disease, wherein the individual has reduced GBA levels and / or reduced GBA activity.

[0390] 40. The active pharmaceutical ingredient according to claim 39, wherein the individual having reduced GBA levels and / or activity has one or more GBA gene mutations, such as heterozygous GBA gene mutations or homozygous GBA gene mutations.

[0391] 41. The active pharmaceutical ingredient according to any one of items 39-40, wherein the GBA-related condition is a GBA-related α-synucleinosis, such as a GBA-related α-synucleinosis selected from GBA-related Parkinson's syndrome, GBA-related Parkinson's disease (PD), GBA-related Lewy body dementia (DLB), and GBA-related multiple system atrophy (MSA).

[0392] 42. The active pharmaceutical ingredient according to item 40, wherein the GBA-related condition is GBA-related Parkinson's disease.

[0393] 43. The active pharmaceutical ingredient according to any one of claims 39-42, wherein said individual has about 5 to 95% of the presumed wild-type activity and / or level of GBA activity and / or level, for example 5 to 10%, 10 to 20%, 20 to 30%, 30 to 40%, 40 to 50%, 50 to 60%, 60 to 70%, 70 to 80%, 80 to 90%, or 90 to 95% of the presumed wild-type activity and / or level.

[0394] 44. An active pharmaceutical ingredient selected from N-[2-hydroxy-3-(1-piperidinyl)-propoxy]-pyridine-1-oxide-3-carboximide chloride, its stereoisomers and its acid addition salts, used in one or more of the following methods:

[0395] a. Increase GBA activity,

[0396] b. Increase the level (or amount) of GBA.

[0397] c. Increase the amount of active mutant GBA.

[0398] d. Increase the amount of active wild-type GBA.

[0399] e. Enhance the doubling of ER-preserving mutant GBA,

[0400] f. Increase the amount of processed / mature GBA.

[0401] g. Increase the amount of mature (post-ER) GBA.

[0402] h. Increase the amount of mature GBA reaching the lysosomes, and / or

[0403] i. Reduce α-synuclein aggregation.

[0404] 45. A composition, such as a pharmaceutical composition, comprising, independently or together with, an active pharmaceutical ingredient selected from N-[2-hydroxy-3-(1-piperidinyl)-propoxy]-pyridine-1-oxide-3-carboximide chloride, its stereoisomers and its acid addition salt (arimoclomol); and one or more other active ingredients; for the treatment of glucocerebrosidase (GBA)-related conditions other than Gaucher disease (GD), including GBA-related α-synucleinopathies such as GBA-related Parkinson's disease (PD), GBA-related Lewy body dementia (DLB) and GBA-related multiple system atrophy (MSA).

[0405] References

[0406] Kieran,D.,Kalmar,B.,Dick,JRT,Riddoch-Contreras,J.,Burnstock,G.,&Greensmith,L.(2004).Treatment with arimoclomol,a coinducer of heat shockproteins,delays disease progression in ALS mice.Nature Medicine,10(4),402–405

[0407] Mu, T., Ong, DST, Wang, Y., Balch, WE, Yates, JR, Segatori, L., & Kelly, JW (2008). Chemical and biological approaches synergize to ameliorate protein-folding diseases. Cell, 134(5),769–81

[0408] Bergamin,N.,Dardis,A.,Beltrami,A.,Cesselli,D.,Rigo,S.,Zampieri,S.,...Beltrami,CA(2013).Orphanet Journal ofRare Diseases,8(1),34.

[0409] Witte,MD,Kallemeijn,WW,Aten,J.,Li,K.-Y.,Strijland,A.,Donker-Koopman,WE,Aerts,JMFG(2010).Ultrasensitive in situ visualization ofactive glucocerebrosidase molecules.Nature Chemical Biology,6(12),907–13.

Claims

1. Use of an active pharmaceutical ingredient selected from N-[2-hydroxy-3-(1- piperidinyl)-propoxy]-pyridine-1 -oxide-3-carboximidoyl chloride and acid addition salts thereof, for the manufacture of an oral medicament for the treatment of a glucocerebrosidase (GBA)-related Parkinson's disease (PD) or a GBA-related Parkinsonism in an individual.

2. Use according to claim 1 for the manufacture of a medicament for the treatment of a GBA-related Parkinsonism.

3. Use according to claim 1 for the manufacture of a medicament for the treatment of a GBA-related Parkinson's disease.

4. Use according to claim 1, wherein the GBA-related Parkinsonism or GBA-related Parkinson's disease is associated with a decrease in GBA enzyme level and / or GBA enzyme activity.

5. Use according to claim 1, wherein the GBA-related Parkinsonism or GBA-related Parkinson's disease is associated with a decrease in GBA enzyme activity and / or level and the GBA gene is wild type and the decrease in GBA activity is due to inhibition of the protein activity or repression of the gene / protein transcription or translation or is idiopathic.

6. Use according to claim 1, wherein the GBA-related Parkinsonism or GBA-related Parkinson's disease is associated with one or more individual GBA gene mutations.

7. Use according to claim 1, wherein the individual having the GBA-related Parkinsonism or GBA-related Parkinson's disease is clinically unaffected by Gaucher disease.

8. Use according to claim 1 or 6, wherein the GBA-related Parkinsonism or GBA- related Parkinson's disease is associated with one or more heterozygous GBA gene mutations.

9. Use according to claim 1, wherein the GBA-related Parkinsonism or GBA- related Parkinson's disease is associated with homozygous GBA gene mutations, wherein the GBA-related disorder is not Gaucher disease.

10. Use according to claim 6, wherein the one or more individual GBA gene mutations are mild associated with Type I GD or severe associated with Type II and III GD.

11. Use according to claim 6, wherein the one or more individual GBA gene mutations are selected from the group consisting of L444P, D409H, D409V, E235A, E340A, E326K, N370S, N370S / 1-BP ins 84G, V394L, A456P, V460V, C342G, G325R, P415R, Y133*, F213I, N188S and IVS2+1 G>A / N188S.

12. Use according to claim 11, wherein the one or more individual GBA gene mutations are L444P, E326K and / or N370S.

13. The use of claim 1, wherein the individual with GBA-associated parkinsonism or GBA- associated Parkinson’s disease is L444P, A456P, V460V heterozygote.

14. The use of claim 1, wherein the individual with GBA-associated parkinsonism or GBA- associated Parkinson’s disease is a GBA mutation carrier.

15. The use of claim 1, wherein the individual with GBA-associated parkinsonism or GBA- associated Parkinson’s disease is a definite carrier.

16. The use of claim 1, wherein the individual with GBA-associated parkinsonism or GBA- associated Parkinson’s disease is a clinically unaffected carrier of GBA.

17. The use of claim 1, wherein the individual with GBA-associated parkinsonism or GBA- associated Parkinson’s disease is a clinically unaffected parent or sibling of a GBA patient.

18. The use of claim 3, wherein the GBA-associated Parkinson’s disease is associated with: reduced GBA enzyme levels and / or reduced GBA enzyme activity; one or more GBA gene mutations; one or more heterozygous GBA gene mutations; one or more homozygous or compound heterozygous GBA gene mutations; and / or genetic high-risk Parkinson’s disease GBA genotype.

19. The use of claim 3, wherein the GBA-associated Parkinson’s disease is associated with one or more GBA gene mutations selected from the group consisting of L444P, D409H, D409V, E235A, E340A, E326K, N370S, N370S / 1-BP ins 84G, V394L, A456P, V460V, C342G, G325R, and P415R.

20. The use of claim 3, wherein the GBA-associated Parkinson’s disease is associated with a heterozygous or homozygous N370S / N370S GBA gene mutation.

21. The use of claim 3, wherein the GBA-associated Parkinson’s disease is associated with idiopathic reduced GBA enzyme activity and / or levels without a concomitant GBA gene mutation found.

22. The use of claim 1, wherein the treatment is prophylactic, curative, or palliative.

23. The use of claim 22, wherein the prophylactic treatment reduces the risk of developing a glucocerebrosidase (GBA)-associated disorder other than Gaucher disease, wherein the individual has reduced GBA levels and / or reduced GBA activity.

24. The use of claim 23, wherein the individual has 5 to 95% of GBA activity and / or levels of putative wild-type activity and / or levels.

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