Use of N-(3-(4-(3-(diisobutylamino)propyl)piperazin-1-yl)propyl)-1H-benzo[d]imidazol-2-amine sulfate and its solvates for treating motor neuron diseases and neuromuscular junction disorders

Abstract

The present invention relates to a sulfate salt of N-(3-(4-(3-(diisobutylamino)propyl)piperazine-1-yl)propyl)-1H-benzo[d]imidazole-2-amine and a pharmaceutically acceptable solvate thereof for use in the treatment and / or prevention of motor neuron diseases and neuromuscular junction disorders.

Description

Technical Field

[0001] The present invention relates to a novel use of N-(3-(4-(3-(diisobutylamino)propyl)piperazin-1-yl)propyl)-1H-benzo[d]imidazol-2-amine sulfate and a pharmaceutically acceptable solvate thereof in the treatment and / or prevention of motor neuron diseases and neuromuscular junction disorders. Background Art

[0002] Motor neurone disease (MND) and neuromuscular junction disorder (NJD) are both considered neuromuscular disorders that result in muscle weakness and fatigue that become more pronounced over time.

[0003] Motor neurons are neuronal cells found in the central nervous system (motor cortex, brainstem, spinal cord…) and the peripheral nervous system that are responsible for the control of organs such as muscles and glands. Motor neurons are classified as either upper motor neurons or lower motor neurons. Upper motor neurons (UMN) are located in the cerebral cortex and brainstem, and they transmit signals to interneurons and lower motor neurons (LMN) via glutamatergic neurotransmission. LMNs are located in the spinal cord and innervate skeletal muscle fibers (neuromuscular junctions), where acetylcholine is released to carry signals across the muscle cell membrane, signaling the muscle to contract or relax. Both UMNs and LMNs are necessary for voluntary movements, balance, body posture regulation, and muscle control in general.

[0004] Progressive motor neuron degeneration is the hallmark of motor neuron disease, which ultimately leads to motor neuron apoptosis and fasciculation of motor units (units that define motor neurons - their axons - neuromuscular junctions - the individual muscle fibers they innervate together), preventing the transmission of neural influx that controls muscle. Death of motor neuron cell bodies is the primary process in MND, and it occurs along with deposition of aggregated proteins in motor neurons and oligodendrocytes, and neuroinflammation. These diseases, which can affect either the UMN or the LMN or even both, cause weakness, hypotonia, hypokinesia, impaired reflexes, and muscle atrophy.

[0005] There are many motor neuron diseases, and amyotrophic lateral sclerosis (ALS) is the most common acquired motor neuron disease that can affect the UMN and LMN. Sporadic ALS is the most common form of ALS diagnosed in patients without family members known to be affected by the disease (≥90% of cases). Familial ALS is of genetic origin and exists in families, and several gene mutations have been identified and associated with it. The most common is chromosome 9 open reading frame 72 (C9ORF72), and can also cause patients to suffer from cognitive deficits: ALS-FTD is a specific form of ALS in which patients also suffer from frontotemporal dementia (FTD). Amyotrophic lateral sclerosis with Parkinson's-dementia complex 1 (ALS-PDC) or Lytico-bodig disease is a form of ALS in which patients experience symptoms of ALS, dementia, and Parkinson's disease (PD). Other known genes associated with familial and sporadic ALS include those encoding superoxide dismutase 1 (SOD1), TAR DNA binding protein of 43-kDa (TDP-43), RNA binding protein (FUS / TLS: fusion in sarcoma / translocation in sarcoma), and ubiquitin 2 (UBQLN2). These mutations can cause toxic accumulation of these proteins in motor neurons and astrocytes, which subsequently leads to neuronal death. TAR DNA binding protein of 43-kDa (TDP-43) was shown to accumulate in the cytoplasm of motor neurons in most ALS cases. TDP-43 is a nuclear RNA binding protein involved in several aspects of RNA processing that actively shuttles between the nucleus and cytoplasm. In ALS, TDP-43 is excluded from the nucleus, but this cytoplasmic mislocalization is common in neuronal injury or stress, and TDP-43-positive inclusions may represent secondary pathology in motor neuron disease.

[0006] Other motor neuron diseases may affect only UMNs (such as primary lateral sclerosis (PLS)) or only LMNs (such as progressive muscular atrophy (PMA)). In addition, LMN degeneration and ALS can affect neuromuscular junctions (Dupuis L et al Curr Drug Targets 2010; 11 (10): 1250-1261-Gromova A et al, Trends Neurosci. 2020 Sep; 43 (9): 709-724-Hashizume A et al, J Neurol Neurosurg Psychiatry 2020 Oct; 91 (10): 1085-1091). NJD targets this key area and blocks nerve impulses that are normally transmitted to muscles to promote movement or their contraction. Myasthenic syndromes (myasthenia gravis and Eaton-Lambert syndrome) affect the efficacy of synaptic transmission through immune or genetic processes. In these pathologies, the absolute number of NMJs remains roughly the same, but their efficacy in firing muscle action potentials in response to motor neuron stimulation decreases, leading to fatigable muscle weakness (Gilhus NE et al Curr Opin Neurol. 2012 Oct; 25(5): 523-9). Furthermore, it is recognized that designing treatments to strengthen and stabilize the remaining NMJs (a common final pathway in diseases with LMN degeneration) will be equally beneficial to patients with ALS, spinal muscular atrophy (SMA), and spinal bulbar muscular atrophy (SBMA).

[0007] Currently, the U.S. Food and Drug Administration (FDA) and the European Medicines Agency (EMA) have only approved one treatment for ALS patients. Riluzole is an oral drug that blocks glutamatergic neurotransmission in the CNS. It is believed that these effects may be partly due to the inactivation of voltage-dependent sodium channels on glutamatergic nerve endings. Riluzole also blocks some postsynaptic effects of glutamate by non-competitively blocking N-methyl-D-aspartate (NMDA) receptors. However, riluzole has no effect on motor and respiratory function and is not suitable for advanced forms of ALS or other motor neuron diseases.

[0008] In a phase 3 clinical trial, the intravenously injected free radical scavenger Edavarone showed efficacy in a small subgroup of people with ALS. The study showed that the decline in the revised score of the ALS Functional Rating Scale was significantly smaller than that of placebo. So far, there is no indication that Edavarone may be effective in a wider group of ALS patients who do not meet the criteria (Abe et al., Lancet Neurol. 2017, 16 (7), 505-512), and the EMA withdrew its marketing authorization application for the treatment of ALS.

[0009] There are no specific treatments available for neuromuscular junction disorders other than corticosteroids or immunosuppressants, which can have serious side effects.

[0010] N-(3-(4-(3-(diisobutylamino)propyl)piperazin-1-yl)propyl)-1H-benzo[d]imidazol-2-amine sulfate and solvates thereof previously disclosed in WO2014 / 102339 can be used to treat and / or prevent neurodegenerative diseases, amyloid diseases, tauopathies and developmental diseases. These compounds are of particular interest for Alzheimer's disease, Parkinson's disease and tauopathies: a Phase 2A clinical trial of N-(3-(4-(3-(diisobutylamino)propyl)piperazin-1-yl)propyl)-1H-benzo[d]imidazol-2-amine disulfate is ongoing for patients with progressive supranuclear palsy (PSP). These salts are able to modulate Tau phosphorylation and increase progranulin (PGRN) neurotrophic factor levels, a defect known to accelerate Tau deposition and phosphorylation, as demonstrated in mice expressing human tau (J. Neuropathol. Exp. Neurol. 74, 158–165 (2015)). Therefore, these compounds are particularly useful for treating tauopathies, such as frontotemporal dementia, and are therefore proposed to be used specifically for the treatment of ALS-FTD.

[0011] Patients suffering from motor neuron diseases or neuromuscular junction disorders have limited treatments available and there remains a need in the art for new chemical entities that can be used to treat or prevent these diseases, so the applicants have investigated the potential of using N-(3-(4-(3-(diisobutylamino)propyl)piperazin-1-yl)propyl)-1H-benzo[d]imidazol-2-amine sulfate and its solvates for this purpose. SUMMARY OF THE INVENTION

[0013] The present invention is based on the unexpected discovery that the sulfate salt of N-(3-(4-(3-(diisobutylamino)propyl)piperazin-1-yl)propyl)-1H-benzo[d]imidazol-2-amine has a neuroprotective effect on motor neurons and neuromuscular junctions.

[0014] The present invention therefore relates to N-(3-(4-(3-(diisobutylamino)propyl)piperazin-1-yl)propyl)-1H-benzo[d]imidazol-2-amine sulfate and solvates thereof for use in the treatment and / or prevention of motor neuron diseases and neuromuscular junction disorders.

[0015] Furthermore, the applicants have shown that N-(3-(4-(3-(diisobutylamino)propyl)piperazin-1-yl)propyl)-1H-benzo[d]imidazol-2-amine sulfate and its solvates are able to reduce the abnormal translocation of TDP43 from the nucleus to the cytoplasm in motor neurons, a pathological feature observed in most ALS cases. DETAILED DESCRIPTION OF THE INVENTION

[0017] Applicants have shown that N-(3-(4-(3-(diisobutylamino)propyl)piperazin-1-yl)propyl)-1H-benzo[d]imidazol-2-amine sulfate and pharmaceutically acceptable solvates thereof have an inhibitory effect on the survival of wild-type rats and SOD1 G93A The beneficial effects of sulfate on primary cultures of tg rat spinal motor neurons. In addition, the abnormal translocation of TDP-43 from the nucleus to the cytoplasm of motor neurons is a pathological feature observed in most ALS cases. The results presented in the Examples section show that the sulfate can also reverse the abnormal translocation of TDP-43 from the nucleus to the cytoplasm under glutamatergic stress.

[0018] Furthermore, N-(3-(4-(3-(diisobutylamino)propyl)piperazin-1-yl)propyl)-1H-benzo[d]imidazol-2-amine sulfate and its pharmaceutically acceptable solvates successfully protected neuromuscular junctions and neurite networks against glutamatergic stress in co-culture of myoblasts and spinal cord explants.

[0019] All these findings support that N-(3-(4-(3-(diisobutylamino)propyl)piperazin-1-yl)propyl)-1H-benzo[d]imidazol-2-amine sulfate and pharmaceutically acceptable solvates thereof may be effective therapies for improving motor neuron diseases such as ALS, SMA and SBMA, as well as neuromuscular junction disorders such as myasthenia gravis and Eaton-Lambert syndrome.

[0020] Motor neuron diseases and neuromuscular junction disorders include, but are not limited to, non-FTD amyotrophic lateral sclerosis, primary lateral sclerosis (PLS), hereditary spastic paraplegia (HSP), neurological coma, Konzo, Tay-Sachs disease, Sandhoff disease, progressive muscular atrophy (PMA), monosomic muscular atrophy, spinal muscular atrophy (SMA), spinal-bulbar muscular atrophy (SBMA), progressive bulbar palsy (PBP), post-polio syndrome, post-irradiation syndrome, stiff-person syndrome, motor unit disorders caused by accidents, myasthenia gravis, and Eaton-Lambert syndrome.

[0021] Non-FTD ALS refers to forms of sporadic or familial ALS that are not associated with frontotemporal dementia and includes ALS with Parkinson-dementia complex 1 (ALS-PDC), familial ALS caused by mutations in one of the genes encoding superoxide dismutase 1 (SOD1), TAR DNA-binding protein of 43 kDa (TDP-43), RNA-binding protein (FUS / TLS: fusion in sarcoma / translocation in sarcoma), and ubiquitin 2 (UBQLN2). In ALS, patients with bulbar onset progress more rapidly than those with limb-onset or LMN manifestations. Recent descriptions of regional variants suggest that some patients have ALS isolated to a single spinal cord region, including brachial amyotrophic bilateral paresis, leg amyotrophic bilateral paresis, and isolated bulbar palsy, all of which are considered variants of non-FTD ALS in this context. Non-FTD ALS also includes breathing-onset ALS, a rare variant that accounts for about 3% of all ALS cases, in which the initial symptom is difficulty breathing on effort at rest.

[0022] Primary lateral sclerosis (PLS) is a motor neuron disease that causes the slow breakdown of nerves in the brain. This makes the nerves unable to activate the motor neurons in the spinal cord that control the muscles.

[0023] Hereditary spastic paraplegia (HSP), also known as familial spastic paraplegia (FSP), is caused by slowly degenerating upper motor neurons, resulting in progressive spasticity and leg weakness. This leads to difficulty walking. As degeneration continues, symptoms worsen and can include impaired vision, ataxia, seizures, cognitive impairment, peripheral neuropathy, and / or deafness.

[0024] Neuromyxotoxicity is caused by consumption of large amounts of toxins from certain legumes of the genus Lathyrus containing high concentrations of the glutamate analog neurotoxin β-oxalyl-L-α,β-diaminopropionic acid (ODAP). ODAP is a mitochondrial poison that causes excessive cell death, especially in motor neurons; the toxin causes paralysis characterized by lack of strength or inability to move the lower limbs and may involve the pyramidal tract, producing signs of upper motor neuron damage.

[0025] Progressive muscular atrophy (PMA), also known as Duchenne muscular atrophy, is clinically characterized by signs of lower motor neuron dysfunction and can progress to ALS. Symptoms of PMA include atrophy, muscle weakness, lack of reflexes, and lack of spasticity, which can be limited to the arms, legs, or both.

[0026] Monosomic muscular dystrophy, also known as benign focal muscular atrophy, juvenile segmental atrophy, and Hirayama disease, is a rare, benign lower motor neuron disease characterized by weakness and wasting of the distal upper limbs during adolescence, followed by spontaneous cessation of progression and stabilization of symptoms.

[0027] Spinal muscular atrophy (SMA) is a disease that disables people by affecting the motor nerve cells in the spinal cord, taking away the ability to walk, eat or breathe. SMA is caused by mutations in the survival motor neuron 1 (SMN1) gene, which is critical for nerve function that controls muscles. Without it, nerve cells cannot function properly and eventually die, leading to debilitating and sometimes fatal muscle weakness.

[0028] Spinal-bulbar muscular atrophy (SBMA), also known as bulbospinal muscular atrophy and Kennedy disease, is an inherited condition in which the loss of motor neurons affects voluntary muscle movements, particularly the facial and swallowing muscles, as well as the arm and leg muscles, especially those closest to the center of the body.

[0029] Progressive bulbar palsy (PBP) involves both upper and lower movements. This form of MND often causes speech or swallowing difficulties. If the lower motor neurons are affected, the tongue tends to atrophy, with visible fasciculations and reduced activity. This results in a rather nasal type of speech. If the upper motor neurons are affected, the tongue is spastic and tends to cause dysarthria, a difficulty with the mechanism of speech.

[0030] Catalepsy syndrome (also called catalepsy spectrum disorder) is a neurological disorder affecting the brain and spinal cord that causes fluctuating rigidity of the trunk and limbs, painful muscle spasms, task-specific phobias, exaggerated startle responses, and spastic deformities such as fixed lumbar kyphosis.

[0031] As used herein, "accident-induced motor unit disorder" refers to a disorder in which the motor unit is damaged due to neuropathy following an accident, such as a domestic or traffic accident.

[0032] Myasthenia gravis is an autoimmune disease that impairs acetylcholine transmission at the neuromuscular junction, i.e. antibodies attack acetylcholine receptors causing muscle weakness. The most common symptoms of myasthenia gravis are weakness, drooping eyelids, weak eye muscles that cause double vision, and excessive weakness of the affected muscles after use.

[0033] Eaton-Lambert syndrome is an autoimmune disease in which antibodies interfere with the release of the neurotransmitter acetylcholine receptors at the neuromuscular junction. It results in muscle weakness that tends to start in the hip and thigh muscles and then typically spreads to the shoulder muscles and then down the arms and legs to the hands and feet. The nerves that connect the head, face, eyes, nose, muscles, and ears to the brain (cranial nerves) are the last to be affected.

[0034] In one embodiment, the sulfate salts of N-(3-(4-(3-(diisobutylamino)propyl)piperazin-1-yl)propyl)-1H-benzo[d]imidazol-2-amine are those of Formula I.

[0035]

[0036] wherein x is 0.5-4, preferably x is 0.5-3.5, and more preferably x is 0.9-3.

[0037] In other words, for one molecule of N-(3-(4-(3-(diisobutylamino)propyl)piperazin-1-yl)propyl)-1H-benzo[d]imidazol-2-amine, the sulfate salt of N-(3-(4-(3-(diisobutylamino)propyl)piperazin-1-yl)propyl)-1H-benzo[d]imidazol-2-amine contains 0.5-4 equivalents, preferably 0.5-3.5 equivalents, and more preferably 0.9-3 equivalents of sulfuric acid.

[0038] In a preferred embodiment, x is 1.7-2.3, preferably x is 1.9-2.1, more preferably x is about 2 or x is 2.

[0039] In a particularly preferred embodiment, the sulfate salt is N-(3-(4-(3-(diisobutylamino)propyl)piperazin-1-yl)propyl)-1H-benzo[d]imidazol-2-amine disulfate.

[0040] In one embodiment, the sulfate of formula I is in the form of a pharmaceutically acceptable solvate, preferably a hydrate. For 1 molecule of the sulfate of formula I, the solvate stoichiometry is 0.5-5, preferably 1-4, more preferably 1.5-2.5, still more preferably 1.8-2.2, even more preferably 2 or about 2 molecules of solvate.

[0041] Therefore, N-(3-(4-(3-(diisobutylamino)propyl)piperazin-1-yl)propyl)-1H-benzo[d]imidazol-2-amine sulfate and pharmaceutically acceptable solvates can be used as medicaments, in particular for the treatment or prevention of motor neuron diseases, neuromuscular junction disorders and all diseases in which abnormal translocation of TDP43 from the nucleus to the cytoplasm in motor neurons is observed.

[0042] Therefore, the present invention also relates to a sulfate salt of N-(3-(4-(3-(diisobutylamino)propyl)piperazin-1-yl)propyl)-1H-benzo[d]imidazol-2-amine as defined herein, or a pharmaceutically acceptable solvate thereof, for use in the treatment and / or prevention of motor neuron diseases and neuromuscular junction disorders, in particular selected from non-FTD amyotrophic lateral sclerosis, primary lateral sclerosis (PLS), hereditary spastic paraplegia (HSP), neuromuscular poisoning, Konzo, Tay-Sachs disease, Sandhoff disease, progressive muscular atrophy (PMA), monosomic muscular atrophy, spinal muscular atrophy (SMA), spinal-bulbar muscular atrophy (SBMA), progressive bulbar palsy (PBP), post-polio syndrome, post-irradiation syndrome, stiff-person syndrome, motor unit disorders caused by accidents, myasthenia gravis, Eaton-Lambert syndrome. Preferably, the disease is selected from non-FTD amyotrophic lateral sclerosis, primary lateral sclerosis (PLS), hereditary spastic paraplegia (HSP), progressive muscular atrophy (PMA), monosomic muscular atrophy, spinal muscular atrophy (SMA), spinal-bulbar muscular atrophy (SBMA), progressive bulbar palsy (PBP), myasthenia gravis, Eaton-Lambert syndrome, motor unit disorders caused by accidents. More preferably, the disease is selected from non-FTD amyotrophic lateral sclerosis, primary lateral sclerosis (PLS), hereditary spastic paraplegia (HSP), spinal muscular atrophy (SMA), spinal-bulbar muscular atrophy (SBMA), progressive bulbar palsy (PBP). Even more preferably, the disease is non-FTD amyotrophic lateral sclerosis.

[0043] In other aspects, the present invention also provides methods and embodiments thereof for treating and / or preventing motor neuron diseases or neuromuscular junction disorders, in particular those cited above, comprising administering to a patient in need thereof a pharmaceutically effective amount of N-(3-(4-(3-(diisobutylamino)propyl)piperazin-1-yl)propyl)-1H-benzo[d]imidazol-2-amine sulfate or a pharmaceutically acceptable solvate thereof as described herein. In specific embodiments, the disease is selected from non-FTD amyotrophic lateral sclerosis, primary lateral sclerosis (PLS), hereditary spastic paraplegia (HSP), spinal muscular atrophy (SMA), spinal-bulbar muscular atrophy (SBMA), progressive bulbar palsy (PBP).

[0044] In other aspects, the present invention also provides the use of N-(3-(4-(3-(diisobutylamino)propyl)piperazin-1-yl)propyl)-1H-benzo[d]imidazol-2-amine sulfate or a pharmaceutically acceptable solvate thereof as described herein in the preparation of a medicament for the treatment and / or prevention of motor neuron diseases or neuromuscular junction disorders, in particular those cited above, and embodiments thereof. In a specific embodiment, the disease is selected from non-FTD amyotrophic lateral sclerosis, primary lateral sclerosis (PLS), hereditary spastic paraplegia (HSP), spinal muscular atrophy (SMA), spinal-bulbar muscular atrophy (SBMA), progressive bulbar palsy (PBP).

[0045] In a particular embodiment, the present invention also relates to N-(3-(4-(3-(diisobutylamino)propyl)piperazin-1-yl)propyl)-1H-benzo[d]imidazol-2-amine sulfate as defined herein or a pharmaceutically acceptable solvate thereof for use in delaying the onset of motor neuron diseases and neuromuscular junction disorders in a patient, wherein the motor neuron diseases and neuromuscular junction disorders are particularly selected from non-FTD amyotrophic lateral sclerosis, primary lateral sclerosis (PLS), hereditary spastic paraplegia (HSP), neuromuscular poisoning, Konzo, Tay-Sachs disease, Sandhoff disease, progressive muscular atrophy (PMA), monosomic muscular atrophy, spinal muscular atrophy (SMA), spinal-bulbar muscular atrophy (SBMA), progressive bulbar palsy (PBP), post-polio syndrome, post-irradiation syndrome, stiff-person syndrome, motor unit disorders caused by accidents, myasthenia gravis, Eaton-Lambert syndrome. Preferably, the disease is selected from non-FTD amyotrophic lateral sclerosis, primary lateral sclerosis (PLS), hereditary spastic paraplegia (HSP), progressive muscular atrophy (PMA), monosomic muscular atrophy, spinal muscular atrophy (SMA), spinal-bulbar muscular atrophy (SBMA), progressive bulbar palsy (PBP), myasthenia gravis, Eaton-Lambert syndrome, motor unit disorders caused by accidents. More preferably, the disease is selected from non-FTD amyotrophic lateral sclerosis, primary lateral sclerosis (PLS), hereditary spastic paraplegia (HSP), spinal muscular atrophy (SMA), spinal-bulbar muscular atrophy (SBMA), progressive bulbar palsy (PBP). Even more preferably, the disease is non-FTD amyotrophic lateral sclerosis.

[0046] In other aspects, the present invention provides methods and embodiments thereof for delaying the onset of a motor neuron disease or a neuromuscular junction disorder, particularly those cited above, in a patient, the method comprising administering to a patient in need thereof a pharmaceutically effective amount of N-(3-(4-(3-(diisobutylamino)propyl)piperazin-1-yl)propyl)-1H-benzo[d]imidazol-2-amine sulfate or a pharmaceutically acceptable solvate thereof. In specific embodiments, the disease is selected from non-FTD amyotrophic lateral sclerosis, primary lateral sclerosis (PLS), hereditary spastic paraplegia (HSP), spinal muscular atrophy (SMA), spinal-bulbar muscular atrophy (SBMA), progressive bulbar palsy (PBP).

[0047] In other aspects, the present invention also provides the use of N-(3-(4-(3-(diisobutylamino)propyl)piperazin-1-yl)propyl)-1H-benzo[d]imidazol-2-amine sulfate or a pharmaceutically acceptable solvate thereof as described herein for the preparation of a medicament for delaying the onset of motor neuron disease or neuromuscular junction disorders, particularly those cited above, in a patient and embodiments thereof. In specific embodiments, the disease is selected from non-FTD amyotrophic lateral sclerosis, primary lateral sclerosis (PLS), hereditary spastic paraplegia (HSP), spinal muscular atrophy (SMA), spinal-bulbar muscular atrophy (SBMA), progressive bulbar palsy (PBP).

[0048] According to another feature of the present invention, there is provided a method for reducing the abnormal translocation of TDP43 from the nucleus to the cytoplasm in motor neurons in a patient, preferably a warm-blooded animal, and even more preferably a human, in need of such treatment, the method comprising administering to the patient an effective amount of N-(3-(4-(3-(diisobutylamino)propyl)piperazin-1-yl)propyl)-1H-benzo[d]imidazol-2-amine sulfate or a pharmaceutically acceptable solvate thereof.

[0049] According to one embodiment, the sulfate salt of N-(3-(4-(3-(diisobutylamino)propyl)piperazin-1-yl)propyl)-1H-benzo[d]imidazol-2-amine and its pharmaceutically acceptable solvates may be administered as part of a combination therapy. Thus, embodiments comprising co-administration of compositions and medicaments are included within the scope of the present invention, wherein the compositions and medicaments contain, in addition to the sulfate salt of N-(3-(4-(3-(diisobutylamino)propyl)piperazin-1-yl)propyl)-1H-benzo[d]imidazol-2-amine or its pharmaceutically acceptable solvates as active ingredients, additional therapeutic agents and / or active ingredients. Such multi-drug regimens, often referred to as "combination therapy", may be used to treat and / or prevent any motor neuron disease or neuromuscular junction disorder. The use of such a combination of therapeutic agents is particularly relevant to the treatment of the above-mentioned motor neuron diseases in patients in need of treatment or in patients at risk of becoming such patients.

[0050] In addition to the requirements of therapeutic efficacy that may require the use of active agents other than the sulfate salt of N-(3-(4-(3-(diisobutylamino)propyl)piperazin-1-yl)propyl)-1H-benzo[d]imidazol-2-amine or a pharmaceutically acceptable solvate thereof, there are additional reasons that compel or highly recommend the combined use of drugs involving active ingredients that represent adjunctive therapy, i.e., they supplement and augment the functions performed by the sulfate salt of N-(3-(4-(3-(diisobutylamino)propyl)piperazin-1-yl)propyl)-1H-benzo[d]imidazol-2-amine or a pharmaceutically acceptable solvate thereof. Suitable complementary therapeutic agents for adjunctive therapy purposes include drugs that do not directly treat and / or prevent diseases or conditions mediated by or associated with motor neuron degeneration, neuromuscular junction degeneration and / or pathological accumulation of TDP-43 in the motor neuron cytoplasm, but rather treat diseases or conditions that are directly produced by or indirectly associated with said degeneration.

[0051] According to another feature of the present invention, the succinate of N-(3-(4-(3-(diisobutylamino)propyl)piperazine-1-yl)propyl)-1H-benzo[d]imidazole-2-amine, its pharmaceutically acceptable solvate can be used in combination with other drugs for the treatment of motor neuron diseases (such as ALS, SMA, SBMA) and neuromuscular junction disorders (such as myasthenia gravis) and Eaton-Lambert syndrome. More particularly, the compound of formula I and its pharmaceutically acceptable solvate can be used as an adjuvant therapy with riluzole, edaravone, pyridostigmine, glucosylceramide degradation inhibitors (such as ambroxol and cycloheptanol B epoxide), acetylcholine release inducers (such as guanidine), corticosteroids (such as prednisolone), antiepileptic drugs (such as carbamazepine and phenytoin), or as disclosed in Van Eijk et al., Current opinion in neurology 2020, 33(5), 655, which are currently being used in clinical trials for the treatment of ALS.

[0052] Thus, the therapeutic methods and pharmaceutical compositions of the present invention can use N-(3-(4-(3-(diisobutylamino)propyl)piperazin-1-yl)propyl)-1H-benzo[d]imidazol-2-amine or a pharmaceutically acceptable solvate thereof in monotherapy for motor neuron disease and neuromuscular junction disorders. However, the methods and compositions can also be used in multitherapy, wherein one or more N-(3-(4-(3-(diisobutylamino)propyl)piperazin-1-yl)propyl)-1H-benzo[d]imidazol-2-amine or a pharmaceutically acceptable salt or solvate thereof is co-administered in combination with one or more other therapeutic agents.

[0053] In the above embodiments, the combination of N-(3-(4-(3-(diisobutylamino)propyl)piperazin-1-yl)propyl)-1H-benzo[d]imidazol-2-amine or a pharmaceutically acceptable salt or solvate thereof and other therapeutically active agents can be administered separately or in combination with each other in terms of dosage form and in terms of their administration time, sequentially or simultaneously. Thus, administration of one component agent can be before, at the same time, or after administration of the other component agents.

[0054] Typically, for pharmaceutical use, a sulfate salt of N-(3-(4-(3-(diisobutylamino)propyl)piperazin-1-yl)propyl)-1H-benzo[d]imidazol-2-amine or a pharmaceutically acceptable solvate thereof can be formulated into a pharmaceutical composition comprising at least one sulfate salt of N-(3-(4-(3-(diisobutylamino)propyl)piperazin-1-yl)propyl)-1H-benzo[d]imidazol-2-amine or a pharmaceutically acceptable solvate thereof and at least one pharmaceutically acceptable carrier, diluent, excipient and / or adjuvant and optionally one or more other therapeutic agents and / or active ingredients.

[0055] As non-limiting examples, the pharmaceutical composition may be in a dosage form suitable for oral administration, parenteral administration (e.g., by intravenous, intramuscular or subcutaneous injection or intravenous infusion), topical administration, by inhalation, by skin patch, by implant, by suppository, etc. Such suitable administration forms (which may be solid, semi-solid or liquid, depending on the mode of administration) and methods and carriers, diluents and excipients for their preparation will be clear to the skilled person; reference is made to the latest edition of Remington's Pharmaceutical Sciences. The pharmaceutical composition may be formulated in solid form and redispersed or suspended prior to use.

[0056] Some preferred but non-limiting examples of dosage forms include tablets, pills, powders, lozenges, sachets, cachets, elixirs, suspensions, emulsions, solutions, syrups, aerosols, ointments, creams, lotions, soft and hard capsules, suppositories, drops, sterile injectable solutions for bolus administration and / or continuous administration, and sterile packaged powders (which are normally reconstituted prior to use), which can be formulated with carriers, excipients and diluents which are themselves suitable for such formulations, such as lactose, glucose, sucrose, sorbitol, mannitol, starch, agar, gum arabic, calcium phosphate, alginates, tragacanth, gelatin, calcium silicate, microcrystalline cellulose, polyvinyl pyrrolidone, polyethylene glycol, cellulose, (sterile) water, methylcellulose, methyl- and propylhydroxybenzoates, talc, magnesium stearate, edible oils, vegetable oils and mineral oils, or suitable mixtures thereof. The pharmaceutical composition may optionally contain other substances commonly used in pharmaceutical preparations, such as lubricants, wetting agents, emulsifiers and suspending agents, dispersants, disintegrants, stabilizers, isotonic agents, expanders, fillers, preservatives, sweeteners, flavoring agents, fragrances, colorants, antibacterial and / or antifungal agents such as parabens, chlorobutanol, phenol, sorbic acid, partitioning agents, flow regulators, release agents, etc. The composition may also be formulated so as to provide quick release, sustained release or delayed release of the active substance contained therein.

[0057] The pharmaceutical composition of the present invention is preferably in unit dosage form and can be suitably packaged in, for example, boxes, blisters, vials, bottles, sachets, ampoules or any other suitable single-dose or multi-dose reservoirs or containers (which can be appropriately labeled); optionally with one or more leaflets containing product information and / or instructions for use. Typically, such a unit dose will contain 0.05-1000 mg, typically 1-500 mg of at least one compound of the present invention, for example about 10, 25, 50, 100, 200, 300 or 400 mg / unit dose.

[0058] Usually, depending on the condition to be prevented or treated and the route of administration, the active compound of the invention is usually administered at 0.01-100 mg / kg, more usually 0.1-50 mg, for example 1-25 mg, such as about 0.5, 1, 2, 5, 10, 15, 20 or 25 mg / kg patient body weight / day, which can be administered as a single daily dose, divided into one or more daily doses, or essentially continuously, for example by drip infusion.

[0059] All references to compounds of formula I include reference to solvates, in particular hydrates, multicomponent complexes and liquid crystals thereof.

[0060] The compounds disclosed throughout this application employ Ultra version 11.0 (CambridgeSoft, Cambridge, MA, USA).

[0061] The free base of N-(3-(4-(3-(diisobutylamino)propyl)piperazin-1-yl)propyl)-1H-benzo[d]imidazol-2-amine can be obtained as disclosed in WO2006 / 051489, and its sulfate salt and solvates thereof are prepared according to the procedure reported in WO2014 / 102339.

[0062] definition

[0063] The following definitions and explanations are for terms used throughout this application, including the specification, drawings, and claims.

[0064] The term "administration" or variations thereof (e.g., "administering") refers to providing an active agent or ingredient (e.g., N-(3-(4-(3-(diisobutylamino)propyl)piperazin-1-yl)propyl)-1H-benzo[d]imidazol-2-amine), alone or as part of a pharmaceutically acceptable composition, to a patient to be treated or prevented a condition, symptom, or disease.

[0065] The term "human" refers to subjects of both sexes and at any stage of development (ie, neonates, infants, young children, adolescents, adults).

[0066] The term "patient" refers to a warm-blooded animal, more preferably a human, who is awaiting or receiving medical care or is / will be the subject of a medical procedure.

[0067] "Pharmaceutically acceptable" means that the ingredients of a pharmaceutical composition are compatible with each other and not deleterious to the patient thereof.

[0068] As used herein, the terms "prevent," "preventing," and "prevention" refer to a method of delaying or arresting the onset of a disorder or disease and / or its attendant symptoms, preventing a patient from acquiring a disorder or disease, or reducing a patient's risk of acquiring a disorder or disease.

[0069] The expression "reduced" as used herein means partially reduced or completely reduced.

[0070] The term "solvate" is used herein to describe compounds of the invention that contain stoichiometric or substoichiometric amounts of one or more pharmaceutically acceptable solvent molecules (e.g., ethanol). When the solvent is water, the term "hydrate" is used. Pharmaceutically acceptable solvent molecules may co-crystallize with the compounds of the invention, and / or be present in the crystalline and / or amorphous phase of the solid, and / or adsorbed thereon.

[0071] The term "therapeutically effective amount" (or more simply "effective amount") as used herein refers to the amount of an active agent or active ingredient (e.g., N-(3-(4-(3-(diisobutylamino)propyl)piperazin-1-yl)propyl)-1H-benzo[d]imidazol-2-amine) sufficient to achieve the desired therapeutic or prophylactic effect in the patient to which it / he is administered.

[0072] As used herein, the terms "treat," "treating," and "treatment" are meant to include alleviating, lessening or eliminating a disorder or disease and / or its attendant symptoms.

[0073] The present invention will be better understood with reference to the following examples. These examples are intended to represent specific embodiments of the present invention and are not intended to limit the scope of the invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0074] Figure 1 Shown is the effect of incubation of N-(3-(4-(3-(diisobutylamino)propyl)piperazin-1-yl)propyl)-1H-benzo[d]imidazol-2-amine) disulfate for 48 hours after glutamate insult for 20 minutes on the survival (A), neurite network (B) and extranuclear TDP-43 (eTDP43) (C) of MAP-2 positive MNs in primary cultures of rat spinal motor neurons. Results are expressed as mean ± SEM (n = 5-6 / group) as a percentage of control. One-way ANOVA analysis followed by PLSD Fisher's test.

[0075] Figure 2Shown are the effects of pre-incubation with N-(3-(4-(3-(diisobutylamino)propyl)piperazin-1-yl)propyl)-1H-benzo[d]imidazol-2-amine) disulfate for 24 hours before glutamate injury plus incubation for 24 hours after injury on the survival (A), neurite network (B) and extranuclear TDP-43 (eTDP43) (C) of MAP-2 positive MNs. Results are expressed as mean ± SEM (n = 5-6 / group) as a percentage of control. One-way ANOVA analysis followed by PLSD Fisher's test.

[0076] Figure 3 Compared with riluzole (Ril) and edavarone (Eda), N-(3-(4-(3-(diisobutylamino)propyl)piperazin-1-yl)propyl)-1H-benzo[d]imidazol-2-amine) disulfate has an inhibitory effect on SOD1 lesions damaged by glutamate. G93A Effects of Tg rat spinal motor neurons in primary culture. (A) Number of neurons (A), integrity of neurite networks (B) and translocation of TDP43 (C). Results are expressed as mean + / - SEM (n = 4-6) as a percentage of control. One-way ANOVA analysis followed by PLSD Fisher test.

[0077] Figure 4 Shown are the effects of N-(3-(4-(3-(diisobutylamino)propyl)piperazin-1-yl)propyl)-1H-benzo[d]imidazol-2-amine) disulfate in co-cultures of spinal cord explants and myoblasts injured with glutamate: (A) number of NMJs, (B) size of NMJs, and (C) neurite networks of NFH(+) MNs compared to riluzole (Ril). One-way ANOVA analysis followed by PLSD Fisher's test. DETAILED DESCRIPTION

[0078] Example 1 :Effects of N-(3-(4-(3-(diisobutylamino)propyl)piperazin-1-yl)propyl)-1H-benzo[d]imidazol-2-amine) disulfate on primary motor neurons of rats injured with glutamate

[0079] Culture of primary motor neurons

[0080] Rat motor neurons (MN) were cultured as described previously by Martinou et al., Neuron. 1992, Apr; 8(4), 737-44 and Wang et al. Hum. Mol. Genet., 2013 Dec 1; 22(23), 4706-19. Pregnant female rats (Rats Wistar; Janvier Labs France) at 14 days of gestation were deeply anesthetized with CO2 and then killed by cervical dislocation. Then, the fetuses were removed from the uterus and immediately placed in ice-cold L15 Leibovitz medium containing 2% penicillin (10,000 U / mL) and streptomycin (10 mg / mL) solution (PS) and 1% bovine serum albumin (BSA). The spinal cord was treated with trypsin-ethylenediaminetetraacetic acid (EDTA) at a final concentration of 0.05% trypsin and 0.02% EDTA for 20 minutes at 37°C. Dissociation was stopped by adding Dulbecco's modified Eagle's medium (DMEM) containing 4.5 g / liter glucose, containing grade II DNase I (final concentration 0.5 mg / mL) and 10% fetal calf serum (FCS). Cells were mechanically dissociated by three forced passes through a 10-mM pipette tip. The cells were then centrifuged at 180 x g for 10 min at +4°C on a BSA (3.5%) layer in L15 medium. The supernatant was discarded and the pellet was resuspended in a defined medium consisting of Neurobasal medium containing 2% B27 supplement solution, 2 mmol / liter L-glutamine, 2% PS solution and 10 ng / mL brain-derived neurotrophic factor (BDNF). Viable cells were counted in a Neubauer cell counter using a trypan blue exclusion test. The cells were seeded at a density of 20,000 / well in a 96-well plate pre-coated with poly-L-lysine (immunostaining) and cultured at 37°C in an air (95%)-CO2 (5%) incubator. The culture medium was changed every 2 days. After 13 days of culture, the motor neurons were injured by glutamate.

[0081] Compound treatment

[0082] On day 13 of culture, glutamate was added to a final concentration of 5 μM, diluted in control medium in the presence of sulfate for 20 min. After 20 min, glutamate was washed out and fresh medium containing sulfate was added for 48 h.

[0083] Endpoint evaluation - 48-hour co-culture

[0084] After 14 or 15 days in culture (48 hours after glutamate injury), cell culture supernatants were collected and spinal cord MNs were fixed with a cold solution of ethanol (95%) and acetic acid (5%) for 5 minutes at -20°C. After permeabilization with 0.1% saponin, cells were blocked with PBS containing 1% fetal bovine serum for 2 hours. Then, cells were incubated with:

[0085] a) Mouse monoclonal antibody anti-microtubule-associated protein 2 (MAP-2) diluted 1 / 400 in PBS containing 1% fetal bovine serum and 0.1% saponin. This antibody specifically binds to MAP-2 present in the cell bodies and neurites of all MNs. The antibody was visualized with Alexa Fluor 488 goat anti-mouse IgG. The nuclei of neurons were labeled by fluorescent labeling (Hoechst solution).

[0086] b) Rabbit polyclonal antibody anti-TDP43 diluted 1 / 100 in PBS containing 1% fetal calf serum and 0.1% saponin. The cytoplasmic localization of TDP43 was investigated. The antibody was visualized with Alexa Fluor 568 goat anti-rabbit IgG.

[0087] For each condition, 30 pictures per well (representing 90% of the well area) were automatically taken at 20x magnification using ImageXpress (Molecular Devices). All images were taken under the same conditions (exposure time, gain, and laser intensity). Analysis of different endpoints was automated using a custom module editor (Molecular Devices). The following morphological criteria were used to distinguish motor neurons (MAP-2) from interneurons (stained for MAP-2): cell body diameter >15 mm and the presence of a minimum of three neuritic processes (Ferraiuolo et al. Brain 2011: 134; 2627–2641).

[0088] The end point is:

[0089] -MN survival (number of MNs)

[0090] -Total neurite outgrowth of MN (expressed in μm)

[0091] -TDP43 in MNs (extranuclear, eTDP43) (expressed as area eTDP43-μm 2 / Number of MNs)

[0092] Statistical analysis

[0093] All values ​​are expressed as mean + / - SEM. Statistical analysis was performed by one-way ANOVA followed by PLS D Fisher test. Neuro-Sys graphics and statistical analysis of different conditions were performed using GraphPad Prism software version 7.04. *p < 0.05 was considered significant.

[0094] result

[0095] Motor neuron survival: As expected, glutamate intoxication significantly reduced cell survival compared to controls (mean survival: 55%; Figure 1 A). Low doses of N-(3-(4-(3-(diisobutylamino)propyl)piperazin-1-yl)propyl)-1H-benzo[d]imidazol-2-amine)disulfate (10 nM to 30 nM) showed a positive and significant effect on survival compared to glutamate conditions. The maximal effect was obtained with a dose of 30 nM (mean survival rate: 82%).

[0096] Neurite network integrity: Glutamate significantly reduced neurite network integrity ( Figure 1 B). N-(3-(4-(3-(diisobutylamino)propyl)piperazin-1-yl)propyl)-1H-benzo[d]imidazol-2-amine) disulfate administration was able to improve neurite networks after glutamate injury at all doses studied, with the maximal effect at 30 nM (mean length: 80%).

[0097] Extranuclear TDP-43: Glutamate administration significantly increased abnormal cytoplasmic TDP-43 signaling ( Figure 1 C) Disulfate at concentrations of 10 and 30 nM prevents TDP-43 accumulation in the cytoplasm.

[0098] Example 2 Effects of N-(3-(4-(3-(diisobutylamino)propyl)piperazin-1-yl)propyl)-1H-benzo[d]imidazol-2-amine) disulfate pretreatment on rat primary motor neurons injured with glutamate: 24h pretreatment-injury-24h treatment

[0099] Culture of primary motor neurons

[0100] Cultivation was performed as described in Example 1.

[0101] Compound treatment - 24h pre-incubation + 24h co-incubation

[0102] On day 12 of culture, primary motor neurons were treated with sulfate for 24 hours. On day 13 of culture, glutamate was added to a final concentration of 5 μM, diluted in control medium in the presence of sulfate for 20 minutes. After 20 minutes, glutamate was washed out, and fresh medium containing sulfate was added for 24 hours.

[0103] End point evaluation

[0104] End point evaluations were performed as described in Example 1.

[0105] Statistical analysis

[0106] Statistical analysis was performed as described in Example 1.

[0107] result

[0108] Motor neuron survival: As expected, glutamate significantly reduced cell survival compared to controls (mean survival: 65%; Figure 2 A). Low doses of sulfate (3 nM to 30 nM) showed a positive and significant effect on survival compared to glutamate conditions. The maximal effect was obtained with a sulfate dose of 30 nM (mean survival: 87%).

[0109] Neurite network integrity: Glutamate strongly impairs the neurite network of spinal motor neurons ( Figure 1 B) Sulfate protected neurite networks from glutamate damage at all doses studied, with a maximal effect at 30 nM (mean length: 91%).

[0110] Extranuclear TDP-43: Glutamate application significantly increased cytoplasmic TDP43 signal (mean eTDP-43 signal: 128% compared to control, Figure 1 C). Sulfate (at doses of 10 nM to 30 nM) was also able to completely prevent the abnormal distribution of TDP-43.

[0111] Example 3 :Effects of N-(3-(4-(3-(diisobutylamino)propyl)piperazin-1-yl)propyl)-1H-benzo[d]imidazol-2-amine) disulfate on the maturation of primary SOD1 motor neurons in rats after glutamate injury

[0112] Genetic rodent models have been used to study the pathogenesis of ALS, including rats (e.g., SOD1G93A rats) that overexpress human superoxide dismutase 1 (SOD1) with mutations known to cause human familial ALS. ALS rat models expressing mutant forms of hSOD-1G93A show features that closely recapitulate the clinical and histopathological features of human disease (Nagai et al., The Journal of Neuroscience, December 1, 2001, 21(23): 9246–9254). In human or rodent studies (SOD1 models), MN loss is preceded by increased excitability. While increased neuronal excitability is associated with structural changes in dendritic structure and spines, dendritic atrophy and spine loss are observed in striatal medium spiny neurons (MSNs) and submedullary MNs of the lumbar spinal cord (Ferrucci et al., Neurobiology of Disease 37 (2010) 370-383; Avossa et al., Neuroscience 138 (2006) 1179-1194).

[0113] Genotyping of SOD1 motor neuron embryos

[0114] On the day of dissection (from pregnant females at gestation day 14), each piece of embryonic head (~3 mm 3 ) was placed in a 2 mL tube free of DNA enzyme. DNA was extracted using the SYBR Green Extract-N-Amp Tissue PCR Kit (Sigma Aldrich). Briefly, 120 μL of extraction solution was placed on each embryonic head. They were then incubated at room temperature for 10 minutes. At the end of this incubation period, the embryonic heads were incubated at 95°C for 5 minutes. 100 μL of neutralization solution was added immediately after the last incubation; each DNA extract was diluted 1 / 40 and stored at +4°C until use. SOD1 was determined using a genomic fragment with human SOD1 primers (5'-CATCAGCCCTAATCCATCTGA-3'; 5'-CGCGACTAACAATCAAAGTGA-3') G93A Gene. Dilute SOD1 primers at 3 μM in sterile ultrapure water. Briefly, prepare a mixture for PCR with ultrapure water (4 μL / sample), 3 μM primers (2 μL / sample) and master mix (10 μL / sample). In a PCR 96-well plate, add 16 μL of PCR mix to each well. Add 4 μL of each diluted DNA according to the planned reserve.

[0115] RT-PCR was performed using a CFX96 Biorad RT-PCR system using the following program:

[0116] - Start: 95℃ - 20 seconds

[0117] -45 cycles: 95℃-10 sec, 65℃-10 sec, 72℃-30 sec (data acquisition)

[0118] - Melting curve: 95℃-15 seconds, 64℃-1 minute, 90℃-30 seconds (continuous data collection), 60℃-15 seconds

[0119] Amplification and melting curves were analyzed using Biorad software.

[0120] The results of each sample were compared with the negative control (ultrapure water) and the positive control to summarize the genotype (WT or Tg) of each embryo.

[0121] Culture of spinal cord SOD1 motor neurons

[0122] Rat spinal cord (SC) motor neurons were cultured as described by Martinou et al., Neuron. 1992, Apr; 8(4), 737-44 and Wang et al. Hum. Mol. Genet., 2013 Dec 1; 22(23), 4706-19. Pregnant female rats at 14 days of gestation were killed by cervical dislocation. Fetuses were collected and immediately placed in ice-cold L15 Leibovitz medium containing 2% penicillin (10,000 U / mL) and streptomycin (10 mg / mL) solution (PS) and 1% bovine serum albumin (BSA). Each fetus was assigned to a numbered petri dish (35 mm diameter). The fetal tail was cut and placed on a 1.5 ml tube free of DNA enzyme; DNA was extracted using the Extract-N-Amp tissue kit.

[0123] Genotyping of SOD tg fetuses was performed using the kit Fast SYBR Green Master Mix. This genotyping was performed during spinal cord dissection, thus at the end of the dissected culture of SOD Tg and WT spinal cords. The spinal cords were removed and placed in ice-cold Leibovitz medium (L15).

[0124] SCs were treated with trypsin-EDTA solution at 37°C for 20 minutes at a final concentration of 0.05% trypsin and 0.02% EDTA. Dissociation was stopped by adding Dulbecco's modified Eagle's medium (DMEM) containing 4.5 g / liter glucose, which contained grade II DNase I (final concentration 0.5 mg / mL) and 10% fetal calf serum (FCS). Cells were mechanically dissociated by three forced passes through a 10-mM pipette tip. Then, cells were centrifuged at 180 x g for 10 min on a BSA (3.5%) layer in L15 culture medium at +4°C. The supernatant was discarded and the pellet was resuspended in a defined medium consisting of Neurobasal culture medium containing 2% B27 supplement solution, 2 mmol / liter L-glutamine, 2% PS solution and 10 ng / mL brain-derived neurotrophic factor (BDNF). Live cells were counted in a Neubauer cell counter using a trypan blue exclusion test. The cells were seeded at a density of 20,000 / well in a 96-well plate pre-coated with poly-L-lysine (immunostaining) and cultured in an air (95%)-CO2 (5%) incubator at 37° C. The culture medium was changed every 2 days.

[0125] Compound treatment - 48h co-incubation

[0126] On day 13, the medium was removed and the cultures were exposed to the test compounds: N-(3-(4-(3-(diisobutylamino)propyl)piperazin-1-yl)propyl)-1H-benzo[d]imidazol-2-amine) disulfate, riluzole and edavarone, and glutamate (5 μM) in defined medium for 20 minutes at 37° C. After glutamate exposure, the cultures were washed with defined medium and then placed in fresh medium containing the test compounds for another 48 hours at 37° C.

[0127] End point evaluation

[0128] After 15 days of culture (48 hours after glutamate injury), the cell culture supernatant was removed and the spinal motor neurons were fixed by a cold solution of ethanol (95%) and acetic acid (5%) at -20°C for 5 minutes.

[0129] After permeabilization with 0.1% saponin, cells were incubated for 2 h with:

[0130] - Mouse monoclonal antibody anti-microtubule-associated protein 2 (MAP-2) (this antibody stains all motor neurons, mature and early differentiated) at 1 / 400 dilution in PBS containing 1% fetal calf serum and 0.1% saponin, revealed with Alexa Fluor 488 goat anti-mouse IgG.

[0131] - Rabbit polyclonal antibody anti-TDP43 (this antibody binds to TDP43, a protein that is abnormally translocated in ALS) diluted 1 / 100 in PBS containing 1% fetal bovine serum and 0.1% saponin. Consider TDP-43 (nuclear and cytoplasmic). The antibody was visualized with Alexa Fluor 568 goat anti-rabbit IgG. The nuclei of neurons were labeled with a fluorescent marker (Hoechst solution).

[0132] The end point is:

[0133] - Overall survival of MNs (number of MAP-2 positive MNs),

[0134] - total neurite network of MAP-2 positive neurites (length in μm),

[0135] - Cytoplasmic TDP43 (extranuclear, eTDP-43) in MNs is expressed as area (μm 2 ) / the number of MN.

[0136] Statistical analysis

[0137] Statistical analysis was performed as described in Example 1.

[0138] result

[0139] Application of glutamate significantly reduced the number of MNs, decreased the length of their neurite networks and triggered an abnormal distribution of TDP-43 to the cytoplasm (see Figure 3 ).

[0140] N-(3-(4-(3-(diisobutylamino)propyl)piperazin-1-yl)propyl)-1H-benzo[d]imidazol-2-amine) disulfate was neuroprotective at all doses studied. The protective effects on neuronal survival and TDP-43 distribution were dose-dependent. At a dose of 300 nM, the protective effect of N-(3-(4-(3-(diisobutylamino)propyl)piperazin-1-yl)propyl)-1H-benzo[d]imidazol-2-amine) disulfate was greater than that of riluzole or edaravone, even at higher concentrations of 1 μM and 5 μM, respectively.

[0141] Embodiment 4: Effects of N-(3-(4-(3-(diisobutylamino)propyl)piperazin-1-yl)propyl)-1H-benzo[d]imidazol-2-amine) disulfate on glutamate-damaged spinal cord / muscle neuromuscular junctions.

[0142] Culture of human myoblasts and rat spinal cord explants

[0143] All experiments were performed according to national institutional health guidelines for the care and use of laboratory animals and in compliance with current European Union regulations (Directive 2010 / 63 / EU).

[0144] Human muscle cell lines were established from dissociated cells (22,000 cells per well). They were seeded on 48-well plates in a 0.1% aqueous solution of gelatin and grown in a proliferation medium consisting of a mixture of 62% MEM medium and 25% M199 medium supplemented with glutamine 2 Mm, human insulin 10 μg / mL, human recombinant epidermal growth factor 10 ng / mL (EGF), human recombinant fibroblast growth factor basic 2 ng / mL (bFGF), fetal calf serum 10% (FCS) and 2% penicillin 10.000 U / mL and streptomycin 10.000 μg / mL (PS). The culture medium was replaced every 2 days.

[0145] 5 days after the start of culture, the entire transverse section of the spinal cord attached with 4 dorsal root ganglia (DRG) taken from 13-day-old rat Wistar embryos (JanvierLabs, France) was placed on a muscle monolayer (one explant per hole in the central region) immediately after satellite cell fusion. The presence of DRG is necessary for achieving a good nerve distribution ratio. The innervated culture was maintained in a mixed (67% / 25%) culture medium consisting of MEM and culture medium 199 supplemented with 5% FCS, insulin 5 μg / ml, glutamine 2mM and 2% PS. After 24 hours of co-cultivation, it is usually observed that the neurites from the spinal cord explants extend. These neurites contact with myotubes and induce the formation of neuromuscular junctions, and the first contraction is observed after about 8 days of co-cultivation. Soon thereafter, the innervated muscle fibers located near the spinal cord explants actually contract continuously. The innervated fibers are different from the non-innervated fibers in morphology and space, and can be easily distinguished from them. The plates were maintained at 37° C. in a humidified incubator in an atmosphere of air (95%)-CO 2 (5%).

[0146] Compound treatment - 48h co-incubation

[0147] On day 27 (co-culture), co-cultures were incubated with N-(3-(4-(3-(diisobutylamino)propyl)piperazin-1-yl)propyl)-1H-benzo[d]imidazol-2-amine) disulfate and glutamate application. Glutamate was added to a final concentration of 60 μM and diluted in control medium for 20 minutes in the presence of disulfate.

[0148] After 20 min of intoxication, the supernatant was removed and fresh medium containing N-(3-(4-(3-(diisobutylamino)propyl)piperazin-1-yl)propyl)-1H-benzo[d]imidazol-2-amine) disulfate was added for 48 h.

[0149] End point evaluation

[0150] Immunostaining

[0151] 48 hours after intoxication, cells were incubated with 500 nM α-bungarotoxin conjugated to Alexa 488 in culture medium for 15 min at 37°C to detect motor endplates. After washing twice in PBS, cells were fixed with 4% paraformaldehyde in PBS (pH=7.3) for 20 min at room temperature.

[0152] The cells were washed twice in PBS. A PBS solution containing 0.1% saponin and 1% FCS was placed at room temperature for 15 minutes to permeabilize the cells and block nonspecific sites.

[0153] The co-cultures were then incubated for 2 hours at room temperature with a mouse monoclonal anti-neurofilament 200KD antibody (NFH) diluted 1 / 400 in PBS containing 1% FCS, 0.1% saponin. The anti-NFH antibody stains the neurites and axons of motor neurons. The antibody was revealed with Alexa Fluor 568 goat anti-mouse IgG diluted 1 / 400 in PBS containing 1% FCS, 0.1% saponin for 1 hour at room temperature. The nuclei of the neurons were labeled with Hoechst solution, which is a nuclear fluorescent marker at 1 μm / ml in the same solution.

[0154] analyze

[0155] Co-culture was performed once (6 wells per condition). For each condition, 20 pictures per well were automatically taken at 10x magnification using ImageXpress (Molecular devices). All images were taken under the same conditions.

[0156] Automatically measures the following endpoints:

[0157] (1) The number of NMJs,

[0158] (2) Average size of NMJ (NMJ area in μm) 2 express),

[0159] (3) Total neurite length (μm) (=nerve distribution network).

[0160] Statistical analysis

[0161] Statistical analysis was performed as described in Example 1.

[0162] result

[0163] Administration of glutamate (60 μM, 20 minutes) resulted in a significant reduction in the number (A) and total area (B) of NMJs in the cultures. In addition, the injury resulted in a significant loss of neurites (C). Disulfate (300 nM) showed neuroprotection in a dose-dependent manner, as it increased the number, total area and total neurite network of NMJs, which successfully protected the neuromuscular junction and neurite network against glutamatergic stress.

Claims

1. Use of the sulfate salt of N-(3-(4-(3-(diisobutylamino)propyl)piperazin-1-yl)propyl)-1H-benzo[d]imidazol-2-amine in the preparation of a medicament for treating and / or preventing motor neuron diseases and neuromuscular junction disorders selected from the following: non-FTD amyotrophic lateral sclerosis, primary lateral sclerosis (PLS), hereditary spastic paraplegia (HSP), neuromyositis, Tay-Sachs disease, Sandhoff disease, progressive muscular atrophy (PMA), monosomic muscular atrophy, spinal-bulbar muscular atrophy (SBMA), progressive bulbar palsy (PBP), post-irradiation syndrome, myasthenia gravis, Eaton-Lambert syndrome.

2. Use of N-(3-(4-(3-(diisobutylamino)propyl)piperazin-1-yl)propyl)-1H-benzo[d]imidazol-2-amine sulfate in the preparation of a medicament for delaying the onset of motor neuron diseases and neuromuscular junction disorders in patients selected from the following: non-FTD amyotrophic lateral sclerosis, primary lateral sclerosis (PLS), hereditary spastic paraplegia (HSP), neuromyositis, Tay-Sachs disease, Sandhoff disease, progressive muscular atrophy (PMA), monosomic muscular atrophy, spinal-bulbar muscular atrophy (SBMA), progressive bulbar palsy (PBP), post-irradiation syndrome, myasthenia gravis, Eaton-Lambert syndrome.

3. The use according to claim 1 or 2, characterized in that The sulfate has the formula I where x is 0.5-4.

4. The use according to claim 3, characterized in that The sulfate salt is N-(3-(4-(3-(diisobutylamino)propyl)piperazin-1-yl)propyl)-1H-benzo[d]imidazol-2-amine disulfate.

5. The use according to claim 1 or 2, characterized in that The motor neuron disease and neuromuscular junction disorder is selected from non-FTD amyotrophic lateral sclerosis, primary lateral sclerosis (PLS), hereditary spastic paraplegia (HSP), progressive muscular atrophy (PMA), monosomic muscular atrophy, spinal-bulbar muscular atrophy (SBMA), progressive bulbar palsy (PBP), myasthenia gravis, and Eaton-Lambert syndrome.

6. The use according to claim 5, characterized in that The motor neuron disease is selected from non-FTD amyotrophic lateral sclerosis, primary lateral sclerosis (PLS), hereditary spastic paraplegia (HSP), spinal-bulbar muscular atrophy (SBMA), progressive bulbar palsy (PBP).

7. The use according to claim 6, characterized in that The motor neurone disease is non-FTD amyotrophic lateral sclerosis.

Images