WO2024028324A1 - Molecules for the prevention and treatment of neuromuscular disorders - Google Patents

Molecules for the prevention and treatment of neuromuscular disorders Download PDF

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Publication number
WO2024028324A1
WO2024028324A1 PCT/EP2023/071291 EP2023071291W WO2024028324A1 WO 2024028324 A1 WO2024028324 A1 WO 2024028324A1 EP 2023071291 W EP2023071291 W EP 2023071291W WO 2024028324 A1 WO2024028324 A1 WO 2024028324A1
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Prior art keywords
hydrochloride
whole organism
neuromuscular
acetylcholine receptor
compound
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PCT/EP2023/071291
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French (fr)
Inventor
Benoît BORDIGNON
Léa LESCOUZÈRES
Pascale BOMONT
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Institut National De La Sante Et De La Recherche Medicale
Centre National De La Recherche Scientifique
Université Claude Bernard Lyon 1
Université De Montpellier
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Publication of WO2024028324A1 publication Critical patent/WO2024028324A1/en

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    • A61K31/7042—Compounds having saccharide radicals and heterocyclic rings
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    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
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    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
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    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/94—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving narcotics or drugs or pharmaceuticals, neurotransmitters or associated receptors
    • G01N33/9406—Neurotransmitters
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    • G01N2800/2878—Muscular dystrophy

Definitions

  • the present invention relates to the field of neuromuscular disorders and corresponding quantification methods. More particularly, it provides new compounds suitable for treating and/or preventing a neuromuscular disorder in an individual in need thereof, in particular Charcot-Marie-Tooth disease (CMT) and/or Giant Axonal Neuropathy (GAN), as well as a method to identify such compounds.
  • CMT Charcot-Marie-Tooth disease
  • GAN Giant Axonal Neuropathy
  • NMDs Neuromuscular disorders encompass a large spectrum of diseases with more than 150 different types described.
  • the most common feature of NMDs is muscle weakness due to an impaired neurotransmission in the peripheral nervous system (PNS) or damages muscles.
  • NMDs can be classified based on the primarily affected neuromuscular unit, as motor neuron, skeletal muscle, neuromuscular junction or peripheral nerve diseases.
  • CMTs Charcot-Marie-Tooth Disease hereditary neuropathies are the most prevalent genetic conditions in the pediatric patient population (Jani-Acsadi, A. et al. Pediatric Clinics of North America 62, 767-786 (2015)).
  • CMTs are characterized by defects in the myelinating Schwann cell (CMT1 type), nerve axon degeneration (CMT2 type) or dominant intermediate form (DI-CMT).
  • CMT diseases are characterized by distal limb weakness and atrophy, sensory loss, and decreased or even absent tendon reflexes. Even though the underlying symptoms of CMTs refers to the PNS, central nervous system (CNS) involvement has been reported in some cases. Interestingly, the rare neurodegenerative disease Giant Axonal Neuropathy (GAN) shares at early-stage overlapping clinical and histopathological features with several forms of CMT diseases. Thus, it is likely that the incidence of the disease is largely underestimated, because of the complexity of the differential diagnosis between the two diseases (Boizot, A. et al., acta neuropathol commun 2, 47 (2014)).
  • GAN GAN
  • GAN GAN
  • GAN GAN
  • GAN GAN
  • 75 disease-causing mutations have been described in the GAN gene (Lescouzeres and Bomont 2020).
  • the inventors created and described the gan zebrafish model through repression of the zebrafish gigaxonin expression.
  • This first gan zebrafish model has been a unique opportunity to decipher physiopathological mechanism causing locomotion defects. For the time, it closely mimics the severe motor deficits seen in patients, with a penetrant loss of locomotion.
  • the inventors showed that gigaxonin is essential for specifying neuronal fate and axonal outgrowth, and to sustain locomotor activity in vivo.
  • the gan zebrafish model has helped demonstrate that Gigaxonin- E3 ligase loss-of-function inhibits the Sonic Hedgehog (Shh) pathway.
  • the Shh path is essential for neuron and muscle fates in vertebrates.
  • the impaired Shh pathway is coupled with impaired motor neuron stability and somitogenesis and suppressed neuromuscular junction (NMJ) formation with a typical denervation profile.
  • NMJ neuromuscular junction
  • a first object of the present invention relates to a compound for use in the prevention and/or treatment of a neuromuscular disorder in an individual in need thereof, the said compound being selected from the group consisting of:
  • the inventors indeed managed to identify several compounds useful in the prevention and/or in the treatment of a neuromuscular disease.
  • the inventors moreover developed a novel method allowing for the automated identification of compounds of interest according to the present invention.
  • the present invention also relates to a compound for use in the partial or complete restoration of the motility of an individual affected by a neuromuscular disorder, the said compound being selected from the group consisting of:
  • the compound for use according to the invention may more particularly be selected from the group consisting of: Phentolamine hydrochloride, Bicalutamide, Valdecoxib, Theobromine, lopamidol, Dipyrone, Benzoxiquine, lopromide, Clomiphene citrate (Z,E), Chlorphensin carbamate, Xylometazoline hydrochloride, Iproniazide phosphate, Tetrahydrozoline hydrochloride, Dydrogesterone, Dexfenfluramine hydrochloride, Cefotetan, Clidinium bromide, Tropicamide, Alverine citrate salt, Aceclofenac, Azacytidine- 5, Ketanserin tartrate hydrate, Oxantel pamoate, Methylhydantoin-5-(L), Novobiocin sodium salt, Ifenprodil tartrate, Methylhydantoin- 5- (D), Hyoscyamine (L), Lomeflox
  • the compound for use according to the invention may in particular be selected from the group consisting of:
  • the compound for use according to the invention may in particular be selected from the group consisting of:
  • the compound for use according to the invention may more particularly be selected from the group consisting of:
  • Valdecoxib Dichlorphenamide, Xylometazoline hydrochloride, Tetrahydrozoline hydrochloride, Benzthiazide, Clidinium bromide, Prochlorperazine dimaleate, Tropicamide, Alverine citrate salt, Phentolamine hydrochloride, Haloperidol, Aceclidine Hydrochloride, Hyoscyamine (L), Scopolamin-N-oxide hydrobromide, Digitoxigenin, Silodosin, Trichlormethiazide and Oxymetazoline hydrochloride.
  • the compound for use according to the invention may more particularly be selected from the group consisting of:
  • Valdecoxib Xylometazoline hydrochloride, Tetrahydrozoline hydrochloride, Clidinium bromide, Tropicamide, Alverine citrate salt, Phentolamine hydrochloride, Hyoscyamine (L), Scopolamin-N-oxide hydrobromide, Silodosin and Oxymetazoline hydrochloride.
  • the compound for use according to the invention may even more particularly be selected from the group consisting of:
  • Phentolamine hydrochloride Trichlormethiazide, Aceclidine Hydrochloride, Oxymetazoline hydrochloride and Digitoxigenin.
  • the compound for use according to the invention may even more particularly be selected from the group consisting of:
  • Phentolamine hydrochloride and Oxymetazoline hydrochloride are Phentolamine hydrochloride and Oxymetazoline hydrochloride.
  • the neuromuscular disorder according to the invention may in particular be selected from the group consisting of:
  • a muscular disorder in particular a muscular dystrophy or a myopathy.
  • the neuromuscular disorder according to the invention may more particularly be selected from the group consisting of:
  • CMS Congenital myasthenic syndromes
  • LEMS Lambert-Eaton myasthenic syndrome
  • MG Charcot-Marie-Tooth disease
  • CMT Charcot-Marie-Tooth disease
  • GAN Giant Axonal Neuropathy
  • ALS Amyotrophic Lateral Sclerosis
  • SBMA Spinal-bulbar muscular atrophy
  • SMA Spinal muscular atrophy
  • BMD Becker muscular dystrophy
  • CMD Congenital muscular dystrophies
  • DMD Duchenne muscular dystrophy
  • EDMD Emery- Dreifuss muscular dystrophy
  • FSHD Facioscapulohumeral muscular dystrophy
  • EGMD Eimb-girdle muscular dystrophies
  • DM Myotonic dystrophy
  • OPMD Oculopharyngeal muscular dystrophy
  • the neuromuscular disorder according to the invention may even more particularly be selected from the group consisting of:
  • CMS Congenital myasthenic syndromes
  • LEMS Eambert-Eaton myasthenic syndrome
  • MG Myasthenia gravis
  • MG Charcot-Marie-Tooth disease
  • GAN Giant Axonal Neuropathy
  • ALS Amyotrophic Lateral Sclerosis
  • SBMA Spinal-bulbar muscular atrophy
  • SMA Spinal muscular atrophy
  • the compound for use according to the invention may in particular be comprised in a composition, the said composition further comprising a physiologically acceptable medium.
  • the said composition may in particular comprise one or at least two different compounds from any one of the lists of compounds of the present invention.
  • the individual mentioned above is preferably a mammal and is more particularly be a human being.
  • the present invention further relates to a method for automating the quantification of axons and/or neuromuscular junctions in a whole organism or in a biological sample, the method comprising the steps of:
  • step (c) screening the stained whole organism or biological sample of step (b) using a high content screening system, by:
  • (6) quantify axons and/or neuromuscular junctions by automatically calculating a coefficient of co-localization of the axonal region automatically detected in point (4) and the acetylcholine receptor clusters number, area and intensity automatically detected in (5).
  • the whole organism of the method may in particular be selected from the group consisting of a zebrafish, a Caenorhabditis elegans and a Diptera of the genus Drosophila, in particular Drosophila melanogaster, and may preferably be a zebrafish Danio rerio, said organism being optionally affected by a neuromuscular disorder, more particularly selected from the group consisting of:
  • a muscular disorder in particular a muscular dystrophy or a myopathy.
  • the axons and/or neuromuscular junctions quantification of the method may in particular relate to a whole organism with the method comprising the steps of:
  • step (b) placing the stained whole organism in side position in a well plate reader; (c) screening the stained whole organism of step (b) using a high content screening system comprising a high-content analysis software, by:
  • (9) quantify axons and/or neuromuscular junctions by obtaining the coefficient of co-localization of the axonal region detected in point (6) and the acetylcholine receptor clusters number, area and intensity automatically detected in point (8), using “Calculate Position Properties - Cross population” module of the high- content analysis software in a fraction of the whole organism myotome; then optionally,
  • the present invention further relates to a method for the identification of a compound able to prevent and/or treat a neuromuscular disorder in an individual in need thereof comprising:
  • a neuromuscular disorder in particular a neuromuscular disorder selected from the group consisting of a neuromuscular junction disorder; a peripheral nerve disease; a motor neuron disease and a muscular disorder, in particular a muscular dystrophy or a myopathy.
  • Figure 1 represents pictures of video tracking of total distance travelled by quadruplicate fish with the establishment of z-score scale ranging from 0 (WT Control, left square) to negative values ⁇ -2 (MO Control, right square).
  • a nonparametric Mann Whitney U test was applied; medians with range, minimum, and maximum values are represented.
  • Figure 3 represents the automated High-Throughput imaging- screen pipeline identifying novel regulators of NMJ development in gan MO zebrafish
  • Figure 3b represents images of detection filters segmenting AchR clusters from a-bungarotoxin staining, pMNs (primary motor neurons) axons area from znpl straining, and NMJ overlapping compounds within the spinal cord of control larvae.
  • Figure 3c represents images of smoothing filter permitting to extract the dorsal spinal cord staining as a specific region of interest to individualize axons.
  • Figure 3d is a summary of the 3 parameters (1), (2) and (3) allowing effective quantification of NMJ indicated in the examples.
  • the present invention relates to the identification of compounds suitable for treating and/or preventing a neuromuscular disorder in an individual in need thereof.
  • the compounds described in the present invention are able to restore locomotion in the closest and most relevant animal model of neuromuscular disorder, and in particular of Giant Axonal Neuropathy (GAN) animal model. More particularly, in these models, these compounds are also able to restore the number of clusters of Acetylcholine receptors and/or the number of neuromuscular junctions and/or the length of axons to levels seen in corresponding WT animals.
  • GAN Giant Axonal Neuropathy
  • the inventors moreover developed an automated high-throughput imagingscreening method, according to the invention, allowing the fast, automatic and reliable visualization and quantification of these parameters and the identification of compounds able to prevent and/or treat a neuromuscular disorder.
  • the present invention relates to a compound for use in the prevention and/or treatment of a neuromuscular disorder in an individual in need thereof, the said compound being selected from the group consisting of:
  • a neuromuscular disorder (NMD) relates to diseases affecting the peripheral nervous system, which consists of all the motor and sensory nerves that connect the brain and spinal cord to the rest of the body, and the muscle cells that it innervates.
  • a neuromuscular disorder according to the invention may in particular be selected from the group consisting of:
  • the neuromuscular disorder according to the invention may more particularly be selected from the group consisting of Congenital myasthenic syndromes (CMS), Lambert-Eaton myasthenic syndrome (LEMS), Myasthenia gravis (MG), Charcot-Marie- Tooth disease (CMT), Giant Axonal Neuropathy (GAN), Amyotrophic Lateral Sclerosis (ALS), Spinal-bulbar muscular atrophy (SBMA), Spinal muscular atrophy (SMA), Becker muscular dystrophy (BMD), a Congenital muscular dystrophies (CMD), Duchenne muscular dystrophy (DMD), Emery-Dreifuss muscular dystrophy (EDMD), Facioscapulohumeral muscular dystrophy (FSHD), Limb-girdle muscular dystrophies (LGMD), Myotonic dystrophy (DM), Oculopharyngeal muscular dystrophy (OPMD), Fredreich’s Ataxia, Mitochondrial myasthenic syndromes (CMS), Lambert-Eaton myasthenic
  • the neuromuscular disorder according to the invention may even more particularly be selected from the group consisting of:
  • CMS Congenital myasthenic syndromes
  • LEMS Lambert-Eaton myasthenic syndrome
  • MG Myasthenia gravis
  • CMT Charcot-Marie-Tooth disease
  • GAN Giant Axonal Neuropathy
  • ALS Amyotrophic Lateral Sclerosis
  • SBMA Spinal-bulbar muscular atrophy
  • SMA Spinal muscular atrophy
  • the neuromuscular disorder according to the invention may more particularly be Giant Axonal Neuropathy (GAN).
  • GAN Giant Axonal Neuropathy
  • a compound for use according to the invention may be comprised in a composition further comprising a physiologically acceptable medium.
  • composition may be prepared by conventional techniques, e.g. as described in Remington: The Science and Practice of Pharmacy 2005, Lippincott, Williams & Wilkins.
  • physiologically acceptable medium is intended to denote a medium which is compatible with the body of the individual to whom said composition must be administered. It is, for example, a non-toxic solvent such as water. In particular, said medium is compatible with oral, sublingual, subcutaneous, intramuscular, intravenous, topical, local, intratracheal, intranasal or rectal administration, and more particularly with oral, subcutaneous, intravenous, topical or local administration.
  • the physiologically acceptable medium can be either solid or liquid. Solid form preparations include powders, tablets, pills, capsules, cachets, suppositories, and dispersible granules.
  • a solid medium can be one or more excipients which may also act as diluents, flavoring agents, solubilizers, lubricants, suspending agents, binders, preservatives, wetting agents, tablet disintegrating agents, or an encapsulating material. Also included are solid form preparations which are intended to be converted, shortly before use, to liquid form preparations for oral administration. Such liquid forms include solutions, suspensions, and emulsions. These preparations may contain, in addition to the active component, colorants, flavors, stabilizers, buffers, artificial and natural sweeteners, dispersants, thickeners, solubilizing agents, and the like.
  • compositions for use of the present invention may in particular be formulated for parenteral administration and may be presented in unit dose form in ampoules, pre-filled syringes, small volume infusion or in multi-dose containers, optionally with an added preservative.
  • the compositions may take such forms as suspensions, solutions, or emulsions in oily or aqueous vehicles, for example solutions in aqueous polyethylene glycol.
  • oily or non-aqueous mediums, diluents, solvents or vehicles examples include propylene glycol, polyethylene glycol, vegetable oils (e.g., olive oil), and injectable organic esters (e.g., ethyl oleate), and may contain agents such as preserving, wetting, emulsifying or suspending, stabilizing and/or dispersing agents.
  • the active ingredient may be in powder form, obtained by aseptic isolation of sterile solid or by lyophilisation from solution for constitution before use with a suitable vehicle, e.g., sterile, pyrogen-free water.
  • composition for use of the present invention may be formulated for oral administration.
  • Oral administration forms include solid form preparations including powders, tablets, drops, capsules, cachets, lozenges, and dispersible granules.
  • Other forms suitable for oral administration may include liquid form preparations including emulsions, syrups, elixirs, aqueous solutions, aqueous suspensions, toothpaste, gel dentrifrice, chewing gum, or solid form preparations which are intended to be converted shortly before use to liquid form preparations, such as solutions, suspensions, and emulsions.
  • the medium is a finely divided solid which is a mixture with the finely divided active component.
  • composition for use as described herein may be formulated in a tablet or capsule.
  • the active component is mixed with the medium having the necessary binding capacity in suitable proportions and compacted in the shape and size desired.
  • Suitable mediums may be magnesium carbonate, magnesium stearate, talc, sugar, lactose, pectin, dextrin, starch, gelatin, tragacanth, methylcellulose, sodium carboxymethylcellulose, a low melting wax, cocoa butter and the like.
  • Drops may comprise sterile or non-sterile aqueous or oil solutions or suspensions, and may be prepared by dissolving the active ingredient in a suitable aqueous solution, optionally including a bactericidal and/or fungicidal agent and/or any other suitable preservative, and optionally including a surface active agent.
  • suitable solvents for the preparation of an oily solution include glycerol, diluted alcohol and propylene glycol.
  • Emulsions may be prepared in solutions in aqueous propylene glycol solutions or may contain emulsifying agents such as lecithin, sorbitan monooleate, or acacia.
  • Aqueous solutions can be prepared by dissolving the active component in water and adding suitable colorants, flavors, stabilizing and thickening agents.
  • Aqueous suspensions can be prepared by dispersing the finely divided active component in water with viscous material, such as natural or synthetic gums, resins, methylcellulose, sodium carboxymethylcellulose, and other well-known suspending agents.
  • compositions for use of the present invention may also be formulated in a wide variety of formulations for parenteral administration.
  • the formulations may take such forms as suspensions, solutions, or emulsions in oily or aqueous vehicles, for example solutions in aqueous polyethylene glycol.
  • the composition may be in powder form, obtained by aseptic isolation of sterile solid or by lyophilisation from solution for constitution before use with a suitable vehicle, e.g., sterile, pyrogen-free water.
  • the formulations can be presented in unit-dose or multi-dose sealed containers, such as ampoules, vials, pre- filled syringes, infusion bags, or can be stored in a freeze-dried (lyophilized) condition requiring only the addition of the sterile liquid excipient, for example, water, for injections, immediately prior to use.
  • Extemporaneous injection solutions and suspensions can be prepared from sterile powders, granules, and tablets.
  • oily or non-aqueous mediums, diluents, solvents or vehicles examples include propylene glycol, polyethylene glycol, vegetable oils, and injectable organic esters, and may contain formulatory agents such as preserving, wetting, emulsifying or suspending, stabilizing and/or dispersing agents.
  • the formulations for injection will typically contain from about 0.5 to about 25% by weight of the active ingredient in solution.
  • Regions for topical administration include the skin surface and also mucous membrane tissues of the vagina, rectum, nose, mouth, and throat.
  • the topical composition will typically include a pharmaceutically acceptable carrier adapted for topical administration.
  • the composition may take the form of a suspension, solution, ointment, lotion, sexual lubricant, cream, foam, aerosol, spray, suppository, implant, inhalant, tablet, capsule, dry powder, syrup, balm or lozenge, for example. Methods for preparing such compositions are well known in the pharmaceutical industry.
  • Formulations for use in nasal, pulmonary and/or bronchial administration are normally administered as aerosols in order to ensure that the aerosolized dose actually reaches the mucous membranes of the nasal passages, bronchial tract or the lung.
  • aerosol particle is used herein to describe the liquid or solid particle suitable for nasal, bronchial or pulmonary administration, i.e., that will reach the mucous membranes.
  • Transdermal administration typically involves the delivery of a pharmaceutical agent for percutaneous passage of the drug into the systemic circulation of the patient.
  • the skin sites include anatomic regions for transdermally administering the drug and include the forearm, abdomen, chest, back, buttock, mastoidal area, and the like.
  • Transdermal delivery is accomplished by exposing a source of the complex to a patient's skin for an extended period of time.
  • Transdermal patches have the added advantage of providing controlled delivery of a pharmaceutical agent-chemical modifier complex to the body.
  • dosage forms can be made by dissolving, dispersing, or otherwise incorporating the pharmaceutical agent-chemical modifier complex in a proper medium, such as an elastomeric matrix material.
  • Absorption enhancers can also be used to increase the flux of the compound across the skin. The rate of such flux can be controlled by either providing a rate-controlling membrane or dispersing the compound in a polymer matrix or gel.
  • a simple adhesive patch can be prepared from a backing material and an acrylate adhesive.
  • a compound for use according to the invention is administered in a therapeutically effective amount to an individual in need thereof.
  • a therapeutically effective amount is an amount that produces a therapeutic response or desired effect in the person taking it.
  • a compound for use according to the invention may for example be administered in a dosage of from 1 pg/kg - 30,000 pg/kg body weight, such as 1 pg/kg - 7,500 pg/kg, such as 1 pg/kg - 5,000 pg/kg, such as 1 pg/kg - 2,000 pg/kg, such as 1 pg/kg - 1 ,000 pg/kg, such as 1 pg/kg - 700 pg/kg, such as 5 pg/kg - 500 pg/kg, such as 10 pg/kg to 100 pg/kg body weight.
  • the compound as described herein is to be administered in a dosage of from 1 pg/kg -1 ,000 pg/kg body weight, such as 1 pg/kg - 500 pg/kg, such as 1 pg/kg - 250 pg/kg, such as 1 pg/kg - 100 pg/kg, such as 1 pg/kg - 50 pg/kg, such as 1 pg/kg to 10 pg/kg body weight.
  • the compound as described herein is to be administered in a dosage of from 10 pg/kg - 30,000 pg/kg body weight, such as 10 pg/kg - 7,500 pg/kg, such as 10 pg/kg - 5,000 pg/kg, such as 10 pg/kg - 2,000 pg/kg, such as 10 pg/kg - 1 ,000 pg/kg, such as 10 pg/kg - 700 pg/kg, such as 10 pg/kg - 500 pg/kg, such as 10 pg/kg to 100 pg/kg body weight.
  • a compound for use according to the invention may comprise at least two, or at least three, or at least four or at least five different compounds according to the invention as listed above.
  • An individual in need thereof to which a compound or a composition of the invention is administered may in particular be a mammal, and more particularly a human being.
  • the present invention further relates to a method for automating the quantification of axons and/or neuromuscular junctions in a whole organism or in a biological sample, the method comprising the steps of:
  • step (b) placing the stained whole organism or biological sample in a predefined position in a container, in particular in a lateral position; (c) screening the stained whole organism or biological sample of step (b) using a high content screening system, by:
  • (6) quantify axons and/or neuromuscular junctions by automatically calculating a coefficient of co-localization of the axonal region automatically detected in point (4) and the acetylcholine receptor clusters number, area and intensity automatically detected in (5).
  • a biological sample is from an organism and may in particular be a tissue sample, and more particularly a muscle biopsy, from an organism having a nervous system, such as for example a mammal, and in particular a human being; a zebrafish Danio rerio; a Caenorhabditis elegans or a Diptera of the genus Drosophila.
  • the whole organism may be selected from the group consisting of a zebrafish Danio rerio, a Caenorhabditis elegans and a Diptera of the genus Drosophila, in particular Drosophila melanogaster, and is preferably a zebrafish.
  • Immunostaining has its usual meaning, i.e. that an antibody conjugated with a dye, in particular a fluorescent dye, is caused to bind to an antigen of interest or that a first antibody is caused to bind to an antigen of interest and that a second antibody with a dye, in particular a fluorescent dye, is caused to bind to the first antibody.
  • a fluorescent dye is excited by excitation light and emits a fluorescence. The fluorescence emitted due to the excitation light has a wavelength range on the longer wavelength side compared to the wavelength range of the excitation light.
  • the antigen of interest in the present invention are antigens present on axons of the motor neurons or on acetylcholine receptor clusters of the whole organism or biological sample, and may in particular respectively be znpl/SV2 and alpha-bungarotoxin.
  • the staining, and in particular immuno staining, of the motor neurons and acetylcholine receptor clusters must be different from one another in a way allowing to discriminate one from the other.
  • step (a) of the method accordingly comprises immunostaining axons of the motor neurons comprising the implementation of anti- znpl/SV2 antibodies and immuno staining acetylcholine receptor clusters comprising the implementation of anti-alpha-bungarotoxin antibodies, of a whole organism, in particular selected from the group consisting of a zebrafish Danio rerio, a Caenorhabditis elegans and a Diptera of the genus Drosophila, in particular Drosophila melanogaster, and preferably a zebrafish.
  • a container according to step (b) can be any container appropriate for the present method, i.e. any container appropriate for performing a screening using a high content screening system.
  • Such container can for example be a well plate reader.
  • a high content screening system may be selected from known high-content screening systems, such as for example Opera PhenixTM, Opera LXTM or IN Cell 6000TM high content screening imagers, and is in particular Opera PhenixTM high content screening imager. It allows the screening and analyzing of the stained whole organism or biological sample using appropriate high content imaging and analysis software. Such software may for example be v4.9, PerkinElmer®.
  • step (c) of a method according to the invention comprises:
  • a pre-scan is a scan of the entirety of the stained whole organism or of the entirety of the biological sample intended to ensure that the image of the whole organism or of the biological sample used in the following steps of step (c) is appropriate and can be analyzed in order to obtain the needed information.
  • this pre-scan may comprise pre-scanning the whole organism or biological sample at 5X or 10X magnification to cover the entire well surface in order to automatically find the whole organism or biological sample position in the well using the corresponding tool available in the implemented high content imaging and analysis software.
  • This step may in particular be performed only on a channel appropriate to the immunostaining used for the immunostaining of acetylcholine receptor clusters, with a signal distributed throughout the whole organism or biological sample.
  • step (c) (1) may comprise (1) performing a pre-scan, in particular at 5X magnification to cover the entire well surface in order to automatically find the whole organism position in the well, only on a channel appropriate to the immuno staining used for the immunostaining of acetylcholine receptor clusters, with a signal distributed throughout the whole organism.
  • Said appropriate channel may for example be a red channel (561nm).
  • step (c)(2) may comprise obtaining a global image of the whole organism or biological sample, in particular whole organism, by using an on-the-fly image analysis set, in particular by using the corresponding tool available in the implemented high content imaging and analysis software, and setting the appropriate intensity threshold to detect the strained whole organism or biological sample using a “Find Image Region” module of the high-content analysis software of the high content screening system.
  • step (c)(3) may comprise:
  • This step may in particular comprise applying “Find Image Region” module of the high-content analysis software on the axons of the previously obtained motor neurons channel global image.
  • This step may in particular comprise:
  • This step may in particular comprise quantifying axons and/or neuromuscular junctions by obtaining the coefficient of co-localization of the axonal region detected in point (4) and the acetylcholine receptor clusters number, area and intensity automatically detected in point (5), using “Calculate Position Properties - Cross population” module of the high- content analysis software in a fraction of the whole organism or biological sample, and in particular in a fraction of the whole organism, more particularly in a fraction of the whole organism myotome.
  • This step (6) may optionally comprise the following additional steps performed afterwards:
  • the method according to the invention may in particular be such that:
  • step (c) screening the strained whole organism of step (b) using a high content screening system comprising a high-content analysis software, by:
  • (9) quantify axons and/or neuromuscular junctions by obtaining the coefficient of co-localization of the axonal region detected in point (6) and the acetylcholine receptor clusters number, area and intensity automatically detected in point (8), using “Calculate Position Properties - Cross population” module of the high-content analysis software in a fraction of the whole organism myotome; then optionally, (10) conducting confocal re-scan at 20X magnification on the images obtained in points (2) and (3) and obtain a global image with Maximum Intensity Projections (MIP) as indicated in point (4);
  • MIP Maximum Intensity Projections
  • Such method may be particularly useful for the identification of a compound able to prevent and/or treat a neuromuscular disorder in an individual in need thereof.
  • the present invention also relates to a method for the identification of a compound able to prevent and/or treat a neuromuscular disorder in an individual in need thereof, comprising:
  • step (c) comparing measurements and quantifications performed in steps (a) and (b) in order to determine if the candidate compound is able to prevent and/or treat a neuromuscular disorder in an individual in need thereof, and in particular is able:
  • a neuromuscular disorder in particular a neuromuscular disorder selected from the group consisting of a neuromuscular junction disorder; a peripheral nerve disease; a motor neuron disease and a muscular disorder, in particular a muscular dystrophy or a myopathy.
  • the neuromuscular disorder may more particularly be selected from the group consisting of Congenital myasthenic syndromes (CMS), Lambert-Eaton myasthenic syndrome (LEMS), Myasthenia gravis (MG), Charcot-Marie-Tooth disease (CMT), Giant Axonal Neuropathy (GAN), Amyotrophic Lateral Sclerosis (ALS), Spinal-bulbar muscular atrophy (SBMA), Spinal muscular atrophy (SMA), Becker muscular dystrophy (BMD), a Congenital muscular dystrophies (CMD), Duchenne muscular dystrophy (DMD), Emery- Dreifuss muscular dystrophy (EDMD), Facioscapulohumeral muscular dystrophy (FSHD), Limb-girdle muscular dystrophies (LGMD), Myotonic dystrophy (DM), Oculopharyngeal muscular dystrophy (OPMD), Fredreich’s Ataxia, Mitochondrial myopathies, Congenital myopathies, Distal myopathies, Dystrophies, Multiple sclerosis
  • the neuromuscular disorder may even more particularly be selected from the group consisting of:
  • CMS Congenital myasthenic syndromes
  • LEMS Lambert-Eaton myasthenic syndrome
  • MG Myasthenia gravis
  • CMT Charcot-Marie-Tooth disease
  • GAN Giant Axonal Neuropathy
  • ALS Amyotrophic Lateral Sclerosis
  • SBMA Spinal-bulbar muscular atrophy
  • SMA Spinal muscular atrophy
  • the neuromuscular disorder may more particularly be Giant Axonal Neuropathy (GAN).
  • GAN Giant Axonal Neuropathy
  • step (b) when a first whole organism is used in step (a) and has been previously exposed to a candidate compound, then a second whole organism not previously exposed to a candidate compound is used in step (b).
  • step (a) when a first biological sample is used in step (a), the first biological sample originating from an organism which has not previously been exposed to a candidate compound, then a second biological sample originating from an organism which has not previously been exposed to a candidate compound is used in step (b).
  • the first and second biological samples can originate from the same organism, the second biological sample being collected before the organism is exposed to a candidate compound, the first sample being collected after the organism is exposed to a candidate compound.
  • Steps (a) and (b) of this method according to the invention can be performed in any order, i.e. step (a) can be performed before step (b), simultaneously to step (b) or after step (b).
  • the method further comprises comparing measurements and quantifications performed in steps (a) and (b) of the method in order to determine if the candidate compound is able to prevent and/or treat a neuromuscular disorder in an individual in need thereof.
  • a compound tested in a method according to the invention will be considered as being able to prevent and/or treat a neuromuscular disorder in an individual in need thereof if the comparison step of the method indicates that said compound:
  • - increases the length of the axons. and accordingly, is able: - to restore neuromuscular junctions structure by improving acetylcholine receptor clustering; and/or
  • Zebrafish models indeed provide a strong therapeutic potential for neurologic diseases, and offers the best alternative to mammalian screening for phenotype-based in vivo drug discovery.
  • This species has many common advantages. Can for instance be mentioned the high conservation of genes and similarity to human, the external fertilization and rapid development through well-defined stages, facilitating the observation and experimental manipulation of embryos. Moreover, the statistical power of the screens is ensured by the high fecundity and small size of embryos. This feature also allows to place them in 96 wellplates and easily treat them by balneation. Finally, the transparency of the embryos facilitates the investigation at the physiological level within tissues, especially of neuronal and neuromuscular integrity in the case of NMDs. Indeed, zebrafish neuromuscular system has been well characterized (Beattie, C. E. Brain Research Bulletin 53, 489-500 (2000)).
  • Zebrafish embryos were treated with pronase (Img/ml, Roche; Ref : 10165921001) at 6 hpf (hours post fertilization) for 7 minutes under agitation and washed three times in E3 medium.
  • pronase Img/ml, Roche; Ref : 10165921001
  • a first gan zebrafish model is characterized by the depletion of gigaxonin using a transient approach (by injecting morpholino oligonucleotides antisense) which recapitulates the severe locomotor symptoms seen in patients.
  • the repression of z-gigaxonin induces both the shortening of axons and decreased production of motor neurons and impedes the formation of stable neuromuscular junction (NMJ).
  • Embryos were then distributed manually in 200pL of E3 medium, 4 per well in a 96-well plate (Nunclon, NuncTM, ThermoFisher). Plates of drugs stock solutions (ImM) were thawed and diluted in H20 to a 2X concentrated solution of 20pM 2% DMSO using a liquid handling robot (FreedomEV0200, Tecan). At 8 hpf, lOOpL was removed from each well and lOOpl of 2X drug solutions were mechanically loaded (FreedomEV0200, Tecan). No mix at this step was done to avoid aspirating embryos and treatment was done at 3mm from the well bottom to avoid any contact with embryos.
  • the final concentration was lOpM with 1% DMSO in a total volume of 200pl per well.
  • Drugs were automatically washed 5 times with E3 medium (FreedomEV0200, Tecan) at 48 hpf. These washes were done by a suction of 150pL and redistribution of 150pL medium with 2 slow mixes (lOpL/sec) and a soaking for 5 min without agitation.
  • the aim was to identify small molecules able to rescue loss-of-locomotion by monitoring the spontaneous locomotion of 5 dpf (days post fertilization) treated gan zebrafish, compared to WT larvae.
  • a behavioral scoring relying on a z-score mean value of 4 biological replicates that measure the total distance travelled over an hour was generated
  • the locomotion score scale ranges from null (WT animals) to negative values, indicative of a lack of locomotion, characteristic of untreated gan MO.
  • the Search tool was used for Multiple Protein and all the identified targets were indicated in the List of Name, the chosen organism being Homo Sapiens.
  • the Network can be refined in the Settings by changing the basic Settings in order to activate all the Active interaction sources, while also selecting a high confidence as minimum required interaction score.
  • Tetrahydrozoline hydrochloride Tetrahydrozoline hydrochloride, Benzthiazide, Clidinium bromide, Prochlorperazine dimaleate, Tropicamide, Alverine citrate salt, Phentolamine hydrochloride, Haloperidol, Hyoscyamine (L), Scopolamin-N-oxide hydrobromide, Silodosin, Trichlormethiazide and Oxymetazoline hydrochloride.
  • Cell imaging analysis was then performed following the development, by the inventors, of a high-throughput tailored imaging method in zebrafish to understand and treat neuromuscular diseases of a method comprising automating the quantification of axons and neuromuscular junctions in said animal.
  • Zebrafish were treated with 75pM l-Phenyl-2-thiourea (PTU, Sigma) from 24 hpf to prevent pigmentation. After drug-wash, they were anesthetized at appropriate developmental stages with 0,0168% tricaine (MS-222, Sigma -E10521-50G), fixed in 4% PFA for 4h at RT, and permeabilized in IX PBS-l%TritonX-100 for 2h on an orbital shaker. Subsequently, embryos were incubated in blocking buffer (1% DMSO, 1% normal donkey serum, 1% BSA, 0.7% TritonX-100, PBS) for Ih at RT and incubated in primary antibodies overnight at 4°C.
  • blocking buffer 1% DMSO, 1% normal donkey serum, 1% BSA, 0.7% TritonX-100, PBS
  • mice IgG2a anti- synaptotagmin (1:100, Znp-1, DSHB), anti-a-bungarotoxin (1:50, B35451, Invitrogen).
  • mice IgG2a anti- synaptotagmin (1:100, Znp-1, DSHB)
  • anti-a-bungarotoxin (1:50, B35451, Invitrogen).
  • TritonX-100 PBS washes, embryos were incubated in secondary antibodies (Alexa 488, 1:500, Jackson Labs 200-542-211) overnight at 4°C and subsequently washed in PBS prior to imaging.
  • each fluorescent zebrafish is manually cut in the anterior part of the vitellus and placed in side position in individual wells of 96 well black bottom plates (pClear, Greiner Bio -one).
  • a pre-scan at 5X magnification was used to cover the entire well surface (9 fields per well), only on the red channel (561nm), with a signal distributed throughout the embryo.
  • the “Find Image Region” module of the Harmony® High-content analysis software (v4.9, PerkinElmer) was used to set the appropriate intensity threshold to detect the fish.
  • the “Determine Well Layout” module was then used to define a re-scan magnification, of 20X with an overlap of 6% between fields, covering the entire object.
  • PreciScan re-scan automated imaging, and analysis: neuromuscular junctions Well areas containing whole zebrafish larvae were automatically imaged in confocal mode at 20X magnification on green (znp-1) and red (a-bungaro toxin) channels. A z-stack of 90pm (5pm interval) was applied, creating an on-the-fly image analysis to obtain a global image with Maximum Intensity Projections (MIP). From AChR-channel global image, the “Find Image Region and Select Region” modules allowed to detect fish body and subtract 7 pixels around to restrict the analysis to the region of interest.
  • MIP Maximum Intensity Projections
  • “Find Image Region” module was then applied on the green channel global image to detect and measure size and area of the Axonal region, i.e., spine and axons (Fig. 6.b).
  • the “Find spots” module (method D) was applied with a specific intensity threshold to locate AChR clusters on the red channel global image.
  • AChR quantification AChR number, area and intensity were measured using the “Calculate Position Properties - Cross population” module. The coefficient of colocalization was obtained using the same module in a fraction of the myotome to quantify NMJ.
  • Second High-content analysis axons length
  • confocal re-scan at 20X magnification another automated analysis was conducted. After creating a global image with Maximum Intensity Projections for the two channels, a smoothing with a median filter of 20px was applied on the green channel global image to bring out the densest region of the global image.
  • the spinal cord area was subtracted from the fish body area.
  • a new region of interest was then created using the “Modify Population” module, corresponding to axonal region without the spinal cord. This specific region was used to measure axonal length, width, area, and the ratio of body length to mean axonal length.
  • All the 59 compounds of interest identified above allowed for the restoration of at least one of these parameters, i.e. the said parameter was restored to a level similar to the one observed in the WT zebrafish.

Abstract

The present invention relates to the field of neuromuscular disorders (NMDs) and corresponding quantification methods. There indeed remains a strong need for novel drugs for the treatment and prevention of neuromuscular disorders. The inventors showed that the compounds according to the invention allow restoring locomotion and cellular related parameters: neuromuscular junctions structure, in particular by improving AChR clustering, and/or by increasing the co-localization of presynaptic nerve terminals and AChR microclusters, and/or by rescuing axonal outgrowth, in an individual with a neuromuscular disorder. In particular, the present invention relates to new compounds suitable for treating and/or preventing a neuromuscular disorder in an individual in need thereof. The present invention also relates to a method for automating the quantification of axons and/or neuromuscular junctions in a whole organism or in a biological sample and to a method for the identification of a compound able to prevent and/or treat a neuromuscular disorder in an individual in need thereof.

Description

MOLECULES FOR THE PREVENTION AND TREATMENT OF NEUROMUSCULAR DISORDERS
FIELD OF THE INVENTION
The present invention relates to the field of neuromuscular disorders and corresponding quantification methods. More particularly, it provides new compounds suitable for treating and/or preventing a neuromuscular disorder in an individual in need thereof, in particular Charcot-Marie-Tooth disease (CMT) and/or Giant Axonal Neuropathy (GAN), as well as a method to identify such compounds.
BACKGROUND OF THE INVENTION
Neuromuscular disorders (NMDs) encompass a large spectrum of diseases with more than 150 different types described. The most common feature of NMDs is muscle weakness due to an impaired neurotransmission in the peripheral nervous system (PNS) or damages muscles. NMDs can be classified based on the primarily affected neuromuscular unit, as motor neuron, skeletal muscle, neuromuscular junction or peripheral nerve diseases. Among peripheral forms, Charcot-Marie-Tooth Disease (CMTs) hereditary neuropathies are the most prevalent genetic conditions in the pediatric patient population (Jani-Acsadi, A. et al. Pediatric Clinics of North America 62, 767-786 (2015)). CMTs are characterized by defects in the myelinating Schwann cell (CMT1 type), nerve axon degeneration (CMT2 type) or dominant intermediate form (DI-CMT).
CMT diseases are characterized by distal limb weakness and atrophy, sensory loss, and decreased or even absent tendon reflexes. Even though the underlying symptoms of CMTs refers to the PNS, central nervous system (CNS) involvement has been reported in some cases. Interestingly, the rare neurodegenerative disease Giant Axonal Neuropathy (GAN) shares at early-stage overlapping clinical and histopathological features with several forms of CMT diseases. Thus, it is likely that the incidence of the disease is largely underestimated, because of the complexity of the differential diagnosis between the two diseases (Boizot, A. et al., acta neuropathol commun 2, 47 (2014)). GAN, and more widely some forms of CMTs, are characterized by the spreading of symptoms across neuronal tissues in both peripheral and extensively in central nervous system. GAN (MIM#256850) is an autosomal recessive and rare neurodegenerative disease, caused by mutations in the GAN gene (Bomont, P. et al., Nat Genet 26, 370-374 (2000)), encoding for Gigaxonin-E3-ligase. GAN . To date, 75 disease-causing mutations have been described in the GAN gene (Lescouzeres and Bomont 2020). Different mutation types (a majority of missense, truncations, nonsense mutations and deletions/insertions) lead to a generalized instability of the mutated-gigaxonin (Boizot, A. et al., acta neuropathol commun 2, 47 (2014)).
In the most severe form of GAN, patients are diagnosed during early infancy while they develop PNS -associated symptoms (Johnson-Kerner, B. L. et al. Muscle Nerve 50, 467-476 (2014)). Starting with decrease of deep tendon reflexes, areflexia, and amyotrophy, the disease evolves rapidly towards sensory and motor damages in teens, resulting in a loss of deep and superficial sensitivity and loss of ambulation. In young adults, the initial sensory-motor damages subsequently spread to the CNS, causing severe symptoms including intellectual disability, ataxia, nystagmus, dysarthria and epileptic seizures (Kuhlenbaumer, G. et al., GeneReviews® (eds. Adam, M. P. et al.) (University of Washington, Seattle, 1993)). Overall, amongst NMDs, the most frequent forms of GAN have extremely broad and severe set of phenotypes, and there is no cure for this fatal disease in young adult patients.
In this context, it is essential to develop effective therapies. Indeed, despite the great expansion of gene therapies (Ravi, B. et al., Human Molecular Genetics 28, R55-R64 (2019)), pharmacological access to treat NMDs are still limited.
Several cellular and mouse models have been generated for GAN (Lescouzeres and Bomont, 2020). They provided crucial insights into Gigaxonin functions in controlling the turn-over of the cytoskeletal IF family (Mahammad, S. et al., J. Clin. Invest. 123, 1964- 1975 (2013)) and regulating autophagosome production in neurons (Scrivo, A. et al., Nat Commun 10, 780 (2019)). Unfortunately, GAN mice failed to reproduce the severity of symptoms, possibly because of genic compensation due to the activation of the nonsense- induced transcriptional compensation (NITC) pathway.
Using both transient and stable knockout approaches, the inventors created and described the gan zebrafish model through repression of the zebrafish gigaxonin expression. This first gan zebrafish model has been a unique opportunity to decipher physiopathological mechanism causing locomotion defects. For the time, it closely mimics the severe motor deficits seen in patients, with a penetrant loss of locomotion. The inventors showed that gigaxonin is essential for specifying neuronal fate and axonal outgrowth, and to sustain locomotor activity in vivo. The gan zebrafish model has helped demonstrate that Gigaxonin- E3 ligase loss-of-function inhibits the Sonic Hedgehog (Shh) pathway. Interestingly, the Shh path is essential for neuron and muscle fates in vertebrates. In gan zebrafish model, the impaired Shh pathway is coupled with impaired motor neuron stability and somitogenesis and suppressed neuromuscular junction (NMJ) formation with a typical denervation profile. This model reflects for the first time the loss of ambulation and the severity of symptoms seen in GAN patients. This robust and disease-relevant biological system provides the first hope to develop therapeutic strategies for the fatal GAN disease.
However, there is currently no method allowing a reproductible, fast, automatic and reliable visualization and quantification of neuro-muscular phenotypes in a GAN model, in particular after administration of a tested compound to the GAN model, and in particular allowing:
(i) the automatic detection of single AChRs clusters, axonal region, and the percentage of co-localization area between pre- and post-synaptic NMJ components in such model;
(ii) the automatic demarcation of the spinal cord as a specific region of interest allowing axonal length measurement by in particular individualizing each axon; and/or
(iii) quantification of (a) AChRs clusters, (b) percentage of co-localization area between pre- and post-synaptic NMJ components and (c) axonal outgrowth.
Accordingly, there remains a need in the art for the identification of molecules able to restore locomotion and associated cellular deficits in a patient with a neuromuscular disorder.
There also remains a need for the identification of molecules able to restore motor function in a patient with a neuromuscular disorder.
There moreover also remains a need for the identification of molecules able to partially or completely restore the motility of an individual affected by a neuromuscular disorder. There further remains a need for the identification of molecules having a protective effect on the NMJ, in particular in an individual with, or at risk of developing, a neuromuscular disorder.
There further remains a need for the identification of molecules able to stabilize and/or restore neuromuscular junctions, in particular in an individual with, or at risk of developing, a neuromuscular disorder.
There is moreover a need for an imaging-based screening method allowing to automatically identify compounds rescuing NMJs.
More particularly, there remains a need for a method for the identification of molecules able to restore NMJ structure, in particular by improving AChR clustering, and/or by increasing the co-localization of presynaptic nerve terminals and AchR microclusters, and/or by rescuing axonal outgrowth, in an individual with a neuromuscular disorder.
There accordingly remains a need in the art for a method allowing a reproductible, fast and reliable, and more particularly automatic, visualization and/or quantification of neuro-muscular phenotypes in a neuromuscular disorder model, allowing the identification of compounds of interest according to the invention.
There also remains a need in the art for a method allowing automatically:
(i) detecting single AChRs clusters and/or axonal region and/or the percentage of co-localization area between pre- and post-synaptic NMJ components; and/or
(ii) measuring axonal length; and/or
(iii) quantifying (a) AChRs clusters and/or (b) the percentage of co-localization area between pre- and post-synaptic NMJ components and (c) axonal outgrowth.
SUMMARY OF THE INVENTION
A first object of the present invention relates to a compound for use in the prevention and/or treatment of a neuromuscular disorder in an individual in need thereof, the said compound being selected from the group consisting of:
Phentolamine hydrochloride, Trichlormethiazide, Aceclidine Hydrochloride, Oxymetazoline hydrochloride, Digitoxigenin, Bicalutamide, Valdecoxib, Theophylline monohydrate, Indapamide, Theobromine, Roxithromycin, lopamidol, Dipyrone, Benzoxiquine, lopromide, Clomiphene citrate (Z,E), Chlorphensin carbamate, Dichlorphenamide, Xylometazoline hydrochloride, Iproniazide phosphate, Tetrahydrozoline hydrochloride, Dydrogesterone, Dexfenfluramine hydrochloride, Tranexamic acid, Benzthiazide, Cefotetan, Amikacin hydrate, Clidinium bromide, Prochlorperazine dimaleate, Nimesulide, Tropicamide, Pargyline hydrochloride, Alverine citrate salt, Aceclofenac, Mephenesin, Azacytidine- 5, Ketanserin tartrate hydrate, Oxantel pamoate, Methylhydantoin-5-(L), Novobiocin sodium salt, Haloperidol, Ifenprodil tartrate, Etoposide, Methylhydantoin-5-(D), Hyoscyamine (L), Lomefloxacin hydrochloride, Clemizole hydrochloride, Spectinomycin dihydrochloride, Diperodon hydrochloride, loversol, Formoterol fumarate, Scopolamin-N-oxide hydrobromide, Kanamycin A sulfate, Pentobarbital, Silodosin, Carbinoxamine maleate salt, Clopidogrel, Methimazole and Fenbufen.
The inventors indeed managed to identify several compounds useful in the prevention and/or in the treatment of a neuromuscular disease. The inventors moreover developed a novel method allowing for the automated identification of compounds of interest according to the present invention.
The present invention also relates to a compound for use in the partial or complete restoration of the motility of an individual affected by a neuromuscular disorder, the said compound being selected from the group consisting of:
Phentolamine hydrochloride, Trichlormethiazide, Aceclidine Hydrochloride, Oxymetazoline hydrochloride, Digitoxigenin, Bicalutamide, Valdecoxib, Theophylline monohydrate, Indapamide, Theobromine, Roxithromycin, lopamidol, Dipyrone, Benzoxiquine, lopromide, Clomiphene citrate (Z,E), Chlorphensin carbamate, Dichlorphenamide, Xylometazoline hydrochloride, Iproniazide phosphate, Tetrahydrozoline hydrochloride, Dydrogesterone, Dexfenfluramine hydrochloride, Tranexamic acid, Benzthiazide, Cefotetan, Amikacin hydrate, Clidinium bromide, Prochlorperazine dimaleate, Nimesulide, Tropicamide, Pargyline hydrochloride, Alverine citrate salt, Aceclofenac, Mephenesin, Azacytidine- 5, Ketanserin tartrate hydrate, Oxantel pamoate, Methylhydantoin-5-(E), Novobiocin sodium salt, Haloperidol, Ifenprodil tartrate, Etoposide, Methylhydantoin-5-(D), Hyoscyamine (L), Lomefloxacin hydrochloride, Clemizole hydrochloride, Spectinomycin dihydrochloride, Diperodon hydrochloride, loversol, Formoterol fumarate, Scopolamin-N-oxide hydrobromide, Kanamycin A sulfate, Pentobarbital, Silodosin, Carbinoxamine maleate salt, Clopidogrel, Methimazole and Fenbufen. The compound for use according to the invention may more particularly be selected from the group consisting of: Phentolamine hydrochloride, Bicalutamide, Valdecoxib, Theobromine, lopamidol, Dipyrone, Benzoxiquine, lopromide, Clomiphene citrate (Z,E), Chlorphensin carbamate, Xylometazoline hydrochloride, Iproniazide phosphate, Tetrahydrozoline hydrochloride, Dydrogesterone, Dexfenfluramine hydrochloride, Cefotetan, Clidinium bromide, Tropicamide, Alverine citrate salt, Aceclofenac, Azacytidine- 5, Ketanserin tartrate hydrate, Oxantel pamoate, Methylhydantoin-5-(L), Novobiocin sodium salt, Ifenprodil tartrate, Methylhydantoin- 5- (D), Hyoscyamine (L), Lomefloxacin hydrochloride, Clemizole hydrochloride, Spectinomycin dihydrochloride, Diperodon hydrochloride, loversol, Scopolamin-N-oxide hydrobromide, Kanamycin A sulfate, Pentobarbital, Silodosin, Carbinoxamine maleate salt, Clopidogrel and Fenbufen
The compound for use according to the invention may in particular be selected from the group consisting of:
Bicalutamide, Valdecoxib, Theophylline monohydrate, Indapamide, Theobromine, Roxithromycin, lopamidol, Dipyrone, Benzoxiquine, lopromide, Clomiphene citrate (Z,E), Chlorphensin carbamate, Dichlorphenamide, Xylometazoline hydrochloride, Iproniazide phosphate, Tetrahydrozoline hydrochloride, Dydrogesterone, Dexfenfluramine hydrochloride, Tranexamic acid, Benzthiazide, Cefotetan, Amikacin hydrate, Clidinium bromide, Prochlorperazine dimaleate, Nimesulide, Tropicamide, Pargyline hydrochloride, Alverine citrate salt, Aceclofenac, Mephenesin, Azacytidine- 5, Ketanserin tartrate hydrate, Oxantel pamoate, Methylhydantoin-5-(L), Phentolamine hydrochloride, Novobiocin sodium salt, Haloperidol, Ifenprodil tartrate and Etoposide.
The compound for use according to the invention may in particular be selected from the group consisting of:
Bicalutamide, Valdecoxib, Theobromine, lopamidol, Dipyrone, Benzoxiquine, lopromide, Clomiphene citrate (Z,E), Chlorphensin carbamate, Xylometazoline, Iproniazide phosphate, Tetrahydrozoline hydrochloride, Dydrogesterone, Dexfenfluramine hydrochloride, Cefotetan, Clidinium bromide, Tropicamide, Alverine citrate salt, Aceclofenac, Azacytidine- 5, Ketanserin tartrate hydrate, Oxantel pamoate, Methylhydantoin-5-(L), Novobiocin sodium salt and Ifenprodil tartrate. The compound for use according to the invention may more particularly be selected from the group consisting of:
Valdecoxib, Dichlorphenamide, Xylometazoline hydrochloride, Tetrahydrozoline hydrochloride, Benzthiazide, Clidinium bromide, Prochlorperazine dimaleate, Tropicamide, Alverine citrate salt, Phentolamine hydrochloride, Haloperidol, Aceclidine Hydrochloride, Hyoscyamine (L), Scopolamin-N-oxide hydrobromide, Digitoxigenin, Silodosin, Trichlormethiazide and Oxymetazoline hydrochloride.
The compound for use according to the invention may more particularly be selected from the group consisting of:
Valdecoxib, Xylometazoline hydrochloride, Tetrahydrozoline hydrochloride, Clidinium bromide, Tropicamide, Alverine citrate salt, Phentolamine hydrochloride, Hyoscyamine (L), Scopolamin-N-oxide hydrobromide, Silodosin and Oxymetazoline hydrochloride.
The compound for use according to the invention may even more particularly be selected from the group consisting of:
Phentolamine hydrochloride, Trichlormethiazide, Aceclidine Hydrochloride, Oxymetazoline hydrochloride and Digitoxigenin.
The compound for use according to the invention may even more particularly be selected from the group consisting of:
Phentolamine hydrochloride and Oxymetazoline hydrochloride.
The neuromuscular disorder according to the invention may in particular be selected from the group consisting of:
(a) a neuromuscular junction disorder; and/or
(b) a peripheral nerve disease; and/or
(c) a motor neuron disease; and/or
(d) a muscular disorder, in particular a muscular dystrophy or a myopathy.
The neuromuscular disorder according to the invention may more particularly be selected from the group consisting of:
Congenital myasthenic syndromes (CMS), Lambert-Eaton myasthenic syndrome (LEMS), Myasthenia gravis (MG), Charcot-Marie-Tooth disease (CMT), Giant Axonal Neuropathy (GAN), Amyotrophic Lateral Sclerosis (ALS), Spinal-bulbar muscular atrophy (SBMA), Spinal muscular atrophy (SMA), Becker muscular dystrophy (BMD), a Congenital muscular dystrophies (CMD), Duchenne muscular dystrophy (DMD), Emery- Dreifuss muscular dystrophy (EDMD), Facioscapulohumeral muscular dystrophy (FSHD), Eimb-girdle muscular dystrophies (EGMD), Myotonic dystrophy (DM), Oculopharyngeal muscular dystrophy (OPMD), Fredreich’s Ataxia, Mitochondrial myopathies, Congenital myopathies, Distal myopathies, Dystrophies, Multiple sclerosis, Myasthenic syndrome and Centronuclear myopathy 1.
The neuromuscular disorder according to the invention may even more particularly be selected from the group consisting of:
Congenital myasthenic syndromes (CMS), Eambert-Eaton myasthenic syndrome (LEMS), Myasthenia gravis (MG), Charcot-Marie-Tooth disease (CMT), Giant Axonal Neuropathy (GAN), Amyotrophic Lateral Sclerosis (ALS), Spinal-bulbar muscular atrophy (SBMA) and Spinal muscular atrophy (SMA), and is in particular selected from the group consisting of Charcot-Marie-Tooth disease (CMT) and Giant Axonal Neuropathy (GAN), more particularly Giant Axonal Neuropathy (GAN).
The compound for use according to the invention may in particular be comprised in a composition, the said composition further comprising a physiologically acceptable medium. The said composition may in particular comprise one or at least two different compounds from any one of the lists of compounds of the present invention.
The individual mentioned above is preferably a mammal and is more particularly be a human being.
The present invention further relates to a method for automating the quantification of axons and/or neuromuscular junctions in a whole organism or in a biological sample, the method comprising the steps of:
(a) staining, in particular immuno staining, (i) axons of the motor neurons and (ii) acetylcholine receptor clusters of said whole organism or biological sample;
(b) placing the stained whole organism or biological sample in a predefined position in a container, in particular in side position;
(c) screening the stained whole organism or biological sample of step (b) using a high content screening system, by:
(1) performing a pre-scan covering the entirety of the stained whole organism or biological sample with a signal distributed throughout the stained whole organism or biological sample and obtaining a global image of the entirety of the stained whole organism or biological sample;
(2) set an appropriate intensity threshold to detect said stained whole organism or biological sample;
(3) automatically obtain an image of the stained whole organism or biological sample on channels allowing the detection of (i) the staining of the axons of the motor neurons and (ii) the staining of the acetylcholine receptor clusters;
(4) automatically locate then measure the size and area of the axonal region using the staining of the axons of the motor neurons;
(5) automatically locate, then quantify the stained acetylcholine receptor clusters by automatically measuring the acetylcholine receptor clusters number, area and intensity;
(6) quantify axons and/or neuromuscular junctions by automatically calculating a coefficient of co-localization of the axonal region automatically detected in point (4) and the acetylcholine receptor clusters number, area and intensity automatically detected in (5).
The whole organism of the method may in particular be selected from the group consisting of a zebrafish, a Caenorhabditis elegans and a Diptera of the genus Drosophila, in particular Drosophila melanogaster, and may preferably be a zebrafish Danio rerio, said organism being optionally affected by a neuromuscular disorder, more particularly selected from the group consisting of:
(a) a neuromuscular junction disorder; and/or
(b) a peripheral nerve disease; and/or
(c) a motor neuron disease; and/or
(d) a muscular disorder, in particular a muscular dystrophy or a myopathy.
The axons and/or neuromuscular junctions quantification of the method may in particular relate to a whole organism with the method comprising the steps of:
(a) immunostaining axons of the motor neurons comprising the implementation of anti-znpl, SV2 antibodies and immuno staining acetylcholine receptor clusters comprising the implementation of anti-alpha-bungarotoxin antibodies, of the whole organism;
(b) placing the stained whole organism in side position in a well plate reader; (c) screening the stained whole organism of step (b) using a high content screening system comprising a high-content analysis software, by:
(1) performing a pre-scan, in particular at 5X magnification to cover the entire well surface in order to automatically find the whole organism position in the well, only on a channel appropriate to the immuno staining used for the immunostaining of acetylcholine receptor clusters, with a signal distributed throughout the whole organism;
(2) obtaining a global image of the whole organism by using an on-the-fly image analysis set and setting the appropriate intensity threshold to detect the stained whole organism using a “Find Image Region” module of the high-content analysis software of the high content screening system;
(3) automatically imaging the stained whole organism by implementing channels appropriate to the immuno staining used for the immuno staining of the axons of the motor neurons and of the acetylcholine receptor clusters;
(4) applying z-stack of 90pm (5pm interval) in order to perform an on-the- fly image analysis to obtain a global image with Maximum Intensity Projections (MIP);
(5) applying the “Find Image Region and Select Region” modules of the high-content analysis software from the acetylcholine receptor clusters global image to detect the stained whole organism and subtract 7 pixels around to restrict the analysis to the region of interest;
(6) then applying “Find Image Region” module of the high-content analysis software on the axons of the motor neurons channel global image to detect and measure size and area of the axonal region;
(7) applying “Find spots” module of the high-content analysis software with a specific intensity threshold to locate stained acetylcholine receptor clusters on the channel of the acetylcholine receptor clusters global image;
(8) then measuring acetylcholine receptor number, area and intensity to quantify stained acetylcholine receptors using “Calculate Position Properties - Cross population” module of the high-content analysis software;
(9) quantify axons and/or neuromuscular junctions by obtaining the coefficient of co-localization of the axonal region detected in point (6) and the acetylcholine receptor clusters number, area and intensity automatically detected in point (8), using “Calculate Position Properties - Cross population” module of the high- content analysis software in a fraction of the whole organism myotome; then optionally,
(10) conducting confocal re-scan at 20X magnification on the images obtained in points (2) and (3) and obtain a global image with Maximum Intensity Projections (MIP) as indicated in point (4);
(11) then applying a smoothing of the motor neurons channel global image with a median filter of 20px to bring out the densest region of the global image;
(12) then using “Find Surrounding Region” module of the high-content analysis software in order to subtract the spinal cord area of the whole organism;
(13) then creating a new region of interest using “Modify Population” module of the high-content analysis software, the new region corresponding to axonal region without the spinal cord;
(14) using the new region created in point (13) to measure axonal length and/or width and/or area and/or the ratio of body length to mean axonal length.
The present invention further relates to a method for the identification of a compound able to prevent and/or treat a neuromuscular disorder in an individual in need thereof comprising:
(a) implementing the method according to any one of claims 12 to 14 with a first whole organism or biological sample wherein the whole organism, or the organism from which the biological sample has been taken, has previously been exposed to a candidate compound, in order to (i) measure the size and area of the axonal region, (ii) measure the acetylcholine receptor clusters number, area and intensity and (iii) quantify axons and/or neuromuscular junctions
(b) implementing the method according to any one of claims 12 to 14 with a second whole organism or biological sample, identical to the said first whole organism or biological sample to the exception that the organism, or the organism from which the biological has been taken, has not previously been exposed to a candidate compound, in order to (i) measure the size and area of the axonal region, (ii) measure the acetylcholine receptor clusters number, area and intensity and (iii) quantify axons and/or neuromuscular junctions; (c) comparing measurements and quantifications performed in steps (a) and (b) in order to determine if the candidate compound is able to prevent and/or treat a neuromuscular disorder in an individual in need thereof, and in particular is able:
- to restore neuromuscular junction structure by improving acetylcholine receptor clustering; and/or
- to increase the co-localization of presynaptic nerve terminals and acetylcholine receptor clusters; and/or
- to rescue axonal outgrowth; and/or
- improve post-synaptic acetylcholine receptor clustering number and percentage of co-localization area between pre- and post-synaptic neuromuscular junction components; the whole organisms, or the organism(s) from which the biological samples have been taken from, of steps (a) and (b) being affected by a neuromuscular disorder, in particular a neuromuscular disorder selected from the group consisting of a neuromuscular junction disorder; a peripheral nerve disease; a motor neuron disease and a muscular disorder, in particular a muscular dystrophy or a myopathy.
BRIEF DESCRIPTION OF THE FIGURES
Figure 1 represents pictures of video tracking of total distance travelled by quadruplicate fish with the establishment of z-score scale ranging from 0 (WT Control, left square) to negative values <-2 (MO Control, right square).
Figure 2 represents the distance travelled by gan-MO 5 dpf zebrafish larvae during the spontaneous locomotion test over an hour compared to noninjected WT controls. Each dot represents an individual larva; n = 48 (WT), n = 48 (MO); ****P < 0.0001. A nonparametric Mann Whitney U test was applied; medians with range, minimum, and maximum values are represented.
Ordinate: total distance (m)
Abscissa: left WT (n=48); right MO zebrafish (MO; n=48).
Figure 3 represents the automated High-Throughput imaging- screen pipeline identifying novel regulators of NMJ development in gan MO zebrafish
Figure 3a represents a schematic overview of 5 dpf larvae array in 96 well-plates for fully automated detection, ROI (region of interest) segmentation and Rescan imaging. Representative image of NMJ staining (znpl in green, a-bungarotoxin in red) within the spinal cord of control larvae. Scale bar= 1mm. Figure 3b represents images of detection filters segmenting AchR clusters from a-bungarotoxin staining, pMNs (primary motor neurons) axons area from znpl straining, and NMJ overlapping compounds within the spinal cord of control larvae. Figure 3c represents images of smoothing filter permitting to extract the dorsal spinal cord staining as a specific region of interest to individualize axons. Figure 3d is a summary of the 3 parameters (1), (2) and (3) allowing effective quantification of NMJ indicated in the examples.
DETAILED DESCRIPTION OF THE INVENTION
The present invention relates to the identification of compounds suitable for treating and/or preventing a neuromuscular disorder in an individual in need thereof.
As previously mentioned, there is a strong need for molecules able to in particular restore locomotion and/or motor function and/or the motility in a patient with a neuromuscular disorder.
The inventors discovered that the compounds described in the present invention are able to restore locomotion in the closest and most relevant animal model of neuromuscular disorder, and in particular of Giant Axonal Neuropathy (GAN) animal model. More particularly, in these models, these compounds are also able to restore the number of clusters of Acetylcholine receptors and/or the number of neuromuscular junctions and/or the length of axons to levels seen in corresponding WT animals.
The inventors moreover developed an automated high-throughput imagingscreening method, according to the invention, allowing the fast, automatic and reliable visualization and quantification of these parameters and the identification of compounds able to prevent and/or treat a neuromuscular disorder.
To the inventor’s knowledge, the advantageous properties of the compounds of the invention in the treatment and/or prevention of a neuromuscular disorder in an individual in need thereof and an automated imaging screening method allowing the fast, automatic and reliable visualization and quantification of the above-mentioned parameters, and accordingly the identification of compounds relevant in the treatment and/or prevention of said disorder, has never been described.
Figure imgf000015_0001
to the invention
As previously mentioned, the present invention relates to a compound for use in the prevention and/or treatment of a neuromuscular disorder in an individual in need thereof, the said compound being selected from the group consisting of:
Phentolamine hydrochloride, Trichlormethiazide, Aceclidine Hydrochloride, Oxymetazoline hydrochloride, Digitoxigenin, Bicalutamide, Valdecoxib, Theophylline monohydrate, Indapamide, Theobromine, Roxithromycin, lopamidol, Dipyrone, Benzoxiquine, lopromide, Clomiphene citrate (Z,E), Chlorphensin carbamate, Dichlorphenamide, Xylometazoline hydrochloride, Iproniazide phosphate, Tetrahydrozoline hydrochloride, Dydrogesterone, Dexfenfluramine hydrochloride, Tranexamic acid, Benzthiazide, Cefotetan, Amikacin hydrate, Clidinium bromide, Prochlorperazine dimaleate, Nimesulide, Tropicamide, Pargyline hydrochloride, Alverine citrate salt, Aceclofenac, Mephenesin, Azacytidine- 5, Ketanserin tartrate hydrate, Oxantel pamoate, Methylhydantoin-5-(L), Novobiocin sodium salt, Haloperidol, Ifenprodil tartrate, Etoposide, Methylhydantoin-5-(D), Hyoscyamine (L), Lomefloxacin hydrochloride, Clemizole hydrochloride, Spectinomycin dihydrochloride, Diperodon hydrochloride, loversol, Formoterol fumarate, Scopolamin-N-oxide hydrobromide, Kanamycin A sulfate, Pentobarbital, Silodosin, Carbinoxamine maleate salt, Clopidogrel, Methimazole and Fenbufen.
A neuromuscular disorder (NMD) according to the invention relates to diseases affecting the peripheral nervous system, which consists of all the motor and sensory nerves that connect the brain and spinal cord to the rest of the body, and the muscle cells that it innervates.
A neuromuscular disorder according to the invention may in particular be selected from the group consisting of:
(a) a neuromuscular junction disorder; and/or
(b) a peripheral nerve disease; and/or
(c) a motor neuron disease; and/or
(d) a muscular disorder, in particular a muscular dystrophy or a myopathy. The neuromuscular disorder according to the invention may more particularly be selected from the group consisting of Congenital myasthenic syndromes (CMS), Lambert-Eaton myasthenic syndrome (LEMS), Myasthenia gravis (MG), Charcot-Marie- Tooth disease (CMT), Giant Axonal Neuropathy (GAN), Amyotrophic Lateral Sclerosis (ALS), Spinal-bulbar muscular atrophy (SBMA), Spinal muscular atrophy (SMA), Becker muscular dystrophy (BMD), a Congenital muscular dystrophies (CMD), Duchenne muscular dystrophy (DMD), Emery-Dreifuss muscular dystrophy (EDMD), Facioscapulohumeral muscular dystrophy (FSHD), Limb-girdle muscular dystrophies (LGMD), Myotonic dystrophy (DM), Oculopharyngeal muscular dystrophy (OPMD), Fredreich’s Ataxia, Mitochondrial myopathies, Congenital myopathies, Distal myopathies, Dystrophies, Multiple sclerosis, Myasthenic syndrome and Centronuclear myopathy 1.
The neuromuscular disorder according to the invention may even more particularly be selected from the group consisting of:
Congenital myasthenic syndromes (CMS), Lambert-Eaton myasthenic syndrome (LEMS), Myasthenia gravis (MG), Charcot-Marie-Tooth disease (CMT), Giant Axonal Neuropathy (GAN), Amyotrophic Lateral Sclerosis (ALS), Spinal-bulbar muscular atrophy (SBMA) and Spinal muscular atrophy (SMA), and may in particular selected from the group consisting of Charcot-Marie- Tooth disease (CMT) and Giant Axonal Neuropathy (GAN).
The neuromuscular disorder according to the invention may more particularly be Giant Axonal Neuropathy (GAN).
A compound for use according to the invention may be comprised in a composition further comprising a physiologically acceptable medium.
The composition may be prepared by conventional techniques, e.g. as described in Remington: The Science and Practice of Pharmacy 2005, Lippincott, Williams & Wilkins.
The term “ physiologically acceptable medium” is intended to denote a medium which is compatible with the body of the individual to whom said composition must be administered. It is, for example, a non-toxic solvent such as water. In particular, said medium is compatible with oral, sublingual, subcutaneous, intramuscular, intravenous, topical, local, intratracheal, intranasal or rectal administration, and more particularly with oral, subcutaneous, intravenous, topical or local administration. The physiologically acceptable medium can be either solid or liquid. Solid form preparations include powders, tablets, pills, capsules, cachets, suppositories, and dispersible granules. A solid medium can be one or more excipients which may also act as diluents, flavoring agents, solubilizers, lubricants, suspending agents, binders, preservatives, wetting agents, tablet disintegrating agents, or an encapsulating material. Also included are solid form preparations which are intended to be converted, shortly before use, to liquid form preparations for oral administration. Such liquid forms include solutions, suspensions, and emulsions. These preparations may contain, in addition to the active component, colorants, flavors, stabilizers, buffers, artificial and natural sweeteners, dispersants, thickeners, solubilizing agents, and the like.
The compositions for use of the present invention may in particular be formulated for parenteral administration and may be presented in unit dose form in ampoules, pre-filled syringes, small volume infusion or in multi-dose containers, optionally with an added preservative. The compositions may take such forms as suspensions, solutions, or emulsions in oily or aqueous vehicles, for example solutions in aqueous polyethylene glycol. Examples of oily or non-aqueous mediums, diluents, solvents or vehicles include propylene glycol, polyethylene glycol, vegetable oils (e.g., olive oil), and injectable organic esters (e.g., ethyl oleate), and may contain agents such as preserving, wetting, emulsifying or suspending, stabilizing and/or dispersing agents. Alternatively, the active ingredient may be in powder form, obtained by aseptic isolation of sterile solid or by lyophilisation from solution for constitution before use with a suitable vehicle, e.g., sterile, pyrogen-free water.
The composition for use of the present invention may be formulated for oral administration. Oral administration forms include solid form preparations including powders, tablets, drops, capsules, cachets, lozenges, and dispersible granules. Other forms suitable for oral administration may include liquid form preparations including emulsions, syrups, elixirs, aqueous solutions, aqueous suspensions, toothpaste, gel dentrifrice, chewing gum, or solid form preparations which are intended to be converted shortly before use to liquid form preparations, such as solutions, suspensions, and emulsions. In powders, the medium is a finely divided solid which is a mixture with the finely divided active component.
The composition for use as described herein may be formulated in a tablet or capsule. In tablets, the active component is mixed with the medium having the necessary binding capacity in suitable proportions and compacted in the shape and size desired. Suitable mediums may be magnesium carbonate, magnesium stearate, talc, sugar, lactose, pectin, dextrin, starch, gelatin, tragacanth, methylcellulose, sodium carboxymethylcellulose, a low melting wax, cocoa butter and the like.
Drops may comprise sterile or non-sterile aqueous or oil solutions or suspensions, and may be prepared by dissolving the active ingredient in a suitable aqueous solution, optionally including a bactericidal and/or fungicidal agent and/or any other suitable preservative, and optionally including a surface active agent. Suitable solvents for the preparation of an oily solution include glycerol, diluted alcohol and propylene glycol.
Emulsions may be prepared in solutions in aqueous propylene glycol solutions or may contain emulsifying agents such as lecithin, sorbitan monooleate, or acacia. Aqueous solutions can be prepared by dissolving the active component in water and adding suitable colorants, flavors, stabilizing and thickening agents. Aqueous suspensions can be prepared by dispersing the finely divided active component in water with viscous material, such as natural or synthetic gums, resins, methylcellulose, sodium carboxymethylcellulose, and other well-known suspending agents.
The compositions for use of the present invention may also be formulated in a wide variety of formulations for parenteral administration.
For injections and infusions the formulations may take such forms as suspensions, solutions, or emulsions in oily or aqueous vehicles, for example solutions in aqueous polyethylene glycol. Alternatively, the composition may be in powder form, obtained by aseptic isolation of sterile solid or by lyophilisation from solution for constitution before use with a suitable vehicle, e.g., sterile, pyrogen-free water. The formulations can be presented in unit-dose or multi-dose sealed containers, such as ampoules, vials, pre- filled syringes, infusion bags, or can be stored in a freeze-dried (lyophilized) condition requiring only the addition of the sterile liquid excipient, for example, water, for injections, immediately prior to use. Extemporaneous injection solutions and suspensions can be prepared from sterile powders, granules, and tablets.
Examples of oily or non-aqueous mediums, diluents, solvents or vehicles include propylene glycol, polyethylene glycol, vegetable oils, and injectable organic esters, and may contain formulatory agents such as preserving, wetting, emulsifying or suspending, stabilizing and/or dispersing agents. The formulations for injection will typically contain from about 0.5 to about 25% by weight of the active ingredient in solution.
The compounds may also be administered topically. Regions for topical administration include the skin surface and also mucous membrane tissues of the vagina, rectum, nose, mouth, and throat.
The topical composition will typically include a pharmaceutically acceptable carrier adapted for topical administration. Thus, the composition may take the form of a suspension, solution, ointment, lotion, sexual lubricant, cream, foam, aerosol, spray, suppository, implant, inhalant, tablet, capsule, dry powder, syrup, balm or lozenge, for example. Methods for preparing such compositions are well known in the pharmaceutical industry.
Formulations for use in nasal, pulmonary and/or bronchial administration are normally administered as aerosols in order to ensure that the aerosolized dose actually reaches the mucous membranes of the nasal passages, bronchial tract or the lung. The term "aerosol particle" is used herein to describe the liquid or solid particle suitable for nasal, bronchial or pulmonary administration, i.e., that will reach the mucous membranes.
The compounds for use according to the invention, as well as can be administered transdermally. Transdermal administration typically involves the delivery of a pharmaceutical agent for percutaneous passage of the drug into the systemic circulation of the patient. The skin sites include anatomic regions for transdermally administering the drug and include the forearm, abdomen, chest, back, buttock, mastoidal area, and the like.
Transdermal delivery is accomplished by exposing a source of the complex to a patient's skin for an extended period of time. Transdermal patches have the added advantage of providing controlled delivery of a pharmaceutical agent-chemical modifier complex to the body. Such dosage forms can be made by dissolving, dispersing, or otherwise incorporating the pharmaceutical agent-chemical modifier complex in a proper medium, such as an elastomeric matrix material. Absorption enhancers can also be used to increase the flux of the compound across the skin. The rate of such flux can be controlled by either providing a rate-controlling membrane or dispersing the compound in a polymer matrix or gel. For example, a simple adhesive patch can be prepared from a backing material and an acrylate adhesive. A compound for use according to the invention is administered in a therapeutically effective amount to an individual in need thereof. A therapeutically effective amount is an amount that produces a therapeutic response or desired effect in the person taking it.
A compound for use according to the invention may for example be administered in a dosage of from 1 pg/kg - 30,000 pg/kg body weight, such as 1 pg/kg - 7,500 pg/kg, such as 1 pg/kg - 5,000 pg/kg, such as 1 pg/kg - 2,000 pg/kg, such as 1 pg/kg - 1 ,000 pg/kg, such as 1 pg/kg - 700 pg/kg, such as 5 pg/kg - 500 pg/kg, such as 10 pg/kg to 100 pg/kg body weight. In another embodiment the compound as described herein is to be administered in a dosage of from 1 pg/kg -1 ,000 pg/kg body weight, such as 1 pg/kg - 500 pg/kg, such as 1 pg/kg - 250 pg/kg, such as 1 pg/kg - 100 pg/kg, such as 1 pg/kg - 50 pg/kg, such as 1 pg/kg to 10 pg/kg body weight. In yet another embodiment the compound as described herein is to be administered in a dosage of from 10 pg/kg - 30,000 pg/kg body weight, such as 10 pg/kg - 7,500 pg/kg, such as 10 pg/kg - 5,000 pg/kg, such as 10 pg/kg - 2,000 pg/kg, such as 10 pg/kg - 1 ,000 pg/kg, such as 10 pg/kg - 700 pg/kg, such as 10 pg/kg - 500 pg/kg, such as 10 pg/kg to 100 pg/kg body weight.
A compound for use according to the invention may comprise at least two, or at least three, or at least four or at least five different compounds according to the invention as listed above.
An individual in need thereof to which a compound or a composition of the invention is administered may in particular be a mammal, and more particularly a human being.
Methods according to the invention
As previously mentioned, the present invention further relates to a method for automating the quantification of axons and/or neuromuscular junctions in a whole organism or in a biological sample, the method comprising the steps of:
(a) staining, in particular immuno staining, (i) axons of the motor neurons and (ii) acetylcholine receptor clusters of said whole organism or biological sample;
(b) placing the stained whole organism or biological sample in a predefined position in a container, in particular in a lateral position; (c) screening the stained whole organism or biological sample of step (b) using a high content screening system, by:
(1) performing a pre-scan covering the entirety of the stained whole organism or biological sample with a signal distributed throughout the stained whole organism or biological sample and obtaining a global image of the entirety of the stained whole organism or biological sample;
(2) set an appropriate intensity threshold to detect said stained whole organism or biological sample;
(3) automatically obtain an image of the stained whole organism or biological sample on channels allowing the detection of (i) the staining of the axons of the motor neurons and (ii) the staining of the acetylcholine receptor clusters;
(4) automatically locate then measure the size and area of the axonal region using the staining of the axons of the motor neurons;
(5) automatically locate, then quantify the stained acetylcholine receptor clusters by automatically measuring the acetylcholine receptor clusters number, area and intensity;
(6) quantify axons and/or neuromuscular junctions by automatically calculating a coefficient of co-localization of the axonal region automatically detected in point (4) and the acetylcholine receptor clusters number, area and intensity automatically detected in (5).
A biological sample is from an organism and may in particular be a tissue sample, and more particularly a muscle biopsy, from an organism having a nervous system, such as for example a mammal, and in particular a human being; a zebrafish Danio rerio; a Caenorhabditis elegans or a Diptera of the genus Drosophila.
The whole organism may be selected from the group consisting of a zebrafish Danio rerio, a Caenorhabditis elegans and a Diptera of the genus Drosophila, in particular Drosophila melanogaster, and is preferably a zebrafish.
Staining and immuno staining methods are well known by the man skilled in the art. Immunostaining has its usual meaning, i.e. that an antibody conjugated with a dye, in particular a fluorescent dye, is caused to bind to an antigen of interest or that a first antibody is caused to bind to an antigen of interest and that a second antibody with a dye, in particular a fluorescent dye, is caused to bind to the first antibody. A fluorescent dye is excited by excitation light and emits a fluorescence. The fluorescence emitted due to the excitation light has a wavelength range on the longer wavelength side compared to the wavelength range of the excitation light.
The antigen of interest in the present invention are antigens present on axons of the motor neurons or on acetylcholine receptor clusters of the whole organism or biological sample, and may in particular respectively be znpl/SV2 and alpha-bungarotoxin. The staining, and in particular immuno staining, of the motor neurons and acetylcholine receptor clusters must be different from one another in a way allowing to discriminate one from the other.
In a particular embodiment, step (a) of the method accordingly comprises immunostaining axons of the motor neurons comprising the implementation of anti- znpl/SV2 antibodies and immuno staining acetylcholine receptor clusters comprising the implementation of anti-alpha-bungarotoxin antibodies, of a whole organism, in particular selected from the group consisting of a zebrafish Danio rerio, a Caenorhabditis elegans and a Diptera of the genus Drosophila, in particular Drosophila melanogaster, and preferably a zebrafish.
A container according to step (b) can be any container appropriate for the present method, i.e. any container appropriate for performing a screening using a high content screening system. Such container can for example be a well plate reader.
A high content screening system according to step (c) of the method of the invention may be selected from known high-content screening systems, such as for example Opera Phenix™, Opera LX™ or IN Cell 6000™ high content screening imagers, and is in particular Opera Phenix™ high content screening imager. It allows the screening and analyzing of the stained whole organism or biological sample using appropriate high content imaging and analysis software. Such software may for example be v4.9, PerkinElmer®.
As mentioned above, step (c) of a method according to the invention comprises:
(1) performing a pre-scan covering the entirety of the stained whole organism or entirety of the biological sample with a signal distributed throughout the stained whole organism or biological sample and obtaining a global image of the entirety of the stained whole organism or biological sample. A pre-scan is a scan of the entirety of the stained whole organism or of the entirety of the biological sample intended to ensure that the image of the whole organism or of the biological sample used in the following steps of step (c) is appropriate and can be analyzed in order to obtain the needed information.
For example, this pre-scan may comprise pre-scanning the whole organism or biological sample at 5X or 10X magnification to cover the entire well surface in order to automatically find the whole organism or biological sample position in the well using the corresponding tool available in the implemented high content imaging and analysis software. This step may in particular be performed only on a channel appropriate to the immunostaining used for the immunostaining of acetylcholine receptor clusters, with a signal distributed throughout the whole organism or biological sample.
More particularly, step (c) (1) may comprise (1) performing a pre-scan, in particular at 5X magnification to cover the entire well surface in order to automatically find the whole organism position in the well, only on a channel appropriate to the immuno staining used for the immunostaining of acetylcholine receptor clusters, with a signal distributed throughout the whole organism. Said appropriate channel may for example be a red channel (561nm).
(2) setting an appropriate intensity threshold to detect said stained whole organism or biological sample.
In particular, step (c)(2) may comprise obtaining a global image of the whole organism or biological sample, in particular whole organism, by using an on-the-fly image analysis set, in particular by using the corresponding tool available in the implemented high content imaging and analysis software, and setting the appropriate intensity threshold to detect the strained whole organism or biological sample using a “Find Image Region” module of the high-content analysis software of the high content screening system.
(3) automatically obtaining an image of the stained whole organism or biological sample on channels allowing the detection of (i) the staining of the axons of the motor neurons and (ii) the staining of the acetylcholine receptor clusters.
In particular, step (c)(3) may comprise:
- automatically imaging the strained whole organism or biological sample, in particular whole organism, by implementing channels (for example red and green channels) appropriate to the immuno staining used for the immuno staining of the axons of the motor neurons and of the acetylcholine receptor clusters, in particular using the corresponding tool available in the implemented high content imaging and analysis software;
- then applying z-stack of about 90pm (5pm interval) in order to perform an on- the-fly image analysis to obtain a global image with Maximum Intensity Projections (MIP);
- then applying the “Find Image Region and Select Region” modules of the high- content analysis software from the acetylcholine receptor clusters global image to detect the stained whole organism and subtract pixels, for example 7 pixels, around to restrict the analysis to the region of interest;
(4) automatically locate then measure the size and area of the axonal region using the staining of the axons of the motor neurons.
This step may in particular comprise applying “Find Image Region” module of the high-content analysis software on the axons of the previously obtained motor neurons channel global image.
(5) automatically locating, then quantifying the stained acetylcholine receptor clusters by automatically measuring the acetylcholine receptor clusters number, area and intensity.
This step may in particular comprise:
- applying “Find spots” module of the high-content analysis software with a specific appropriate intensity threshold, in particular as mentioned above, to locate stained acetylcholine receptor clusters on the channel of the previously obtained acetylcholine receptor clusters global image;
- then measuring acetylcholine receptor number, area and intensity to quantify stained acetylcholine receptors using “Calculate Position Properties - Cross population” module of the high-content analysis software.
(6) quantifying axons and/or neuromuscular junctions by automatically calculating a coefficient of co-localization of the axonal region automatically detected in point (4) and the acetylcholine receptor clusters number, area and intensity automatically detected in (5).
This step may in particular comprise quantifying axons and/or neuromuscular junctions by obtaining the coefficient of co-localization of the axonal region detected in point (4) and the acetylcholine receptor clusters number, area and intensity automatically detected in point (5), using “Calculate Position Properties - Cross population” module of the high- content analysis software in a fraction of the whole organism or biological sample, and in particular in a fraction of the whole organism, more particularly in a fraction of the whole organism myotome.
This step (6) may optionally comprise the following additional steps performed afterwards:
- conducting confocal re-scan at 20X magnification on the images obtained in points (2) and (3) and obtain a global image with Maximum Intensity Projections (MIP) as indicated in point (3);
- then applying a smoothing of the motor neurons channel global image with a median filter of 20px to bring out the densest region of the global image;
- then using “Find Surrounding Region” module of the high-content analysis software in order to subtract the spinal cord area of the whole organism or biological sample, in particular of the whole organism;
- then creating a new region of interest using “Modify Population” module of the high-content analysis software, the new region corresponding to axonal region without the spinal cord; and
- using the new region created to measure axonal length and/or width and/or area and/or the ratio of body length to mean axonal length.
The method according to the invention may in particular be such that:
- the axons and/or neuromuscular junctions quantification relates to a whole organism; and
- it comprises the steps of:
(a) immunostaining axons of the motor neurons comprising the implementation of anti-znpl antibodies and immuno staining acetylcholine receptor clusters comprising the implementation of anti-alpha-bungarotoxin antibodies, of the whole organism;
(b) placing the stained whole organism in a lateral position in a well plate reader;
(c) screening the strained whole organism of step (b) using a high content screening system comprising a high-content analysis software, by:
(1) performing a pre-scan, in particular at 5X magnification to cover the entire well surface in order to automatically find the whole organism position in the well, only on a channel appropriate to the immunostaining used for the immuno staining of acetylcholine receptor clusters, with a signal distributed throughout the whole organism;
(2) obtaining a global image of the whole organism by using an on-the-fly image analysis set and setting the appropriate intensity threshold to detect the strained whole organism using a “Find Image Region” module of the high-content analysis software of the high content screening system;
(3) automatically imaging the strained whole organism by implementing channels appropriate to the immunostaining used for the immuno staining of the axons of the motor neurons and of the acetylcholine receptor clusters;
(4) applying z-stack of 90pm (5pm interval) in order to perform an on-the-fly image analysis to obtain a global image with Maximum Intensity Projections (MIP);
(5) applying the “Find Image Region and Select Region” modules of the high- content analysis software from the acetylcholine receptor clusters global image to detect the stained whole organism and subtract 7 pixels around to restrict the analysis to the region of interest;
(6) then applying “Find Image Region” module of the high-content analysis software on the axons of the motor neurons channel global image to detect and measure size and area of the axonal region;
(7) applying “Find spots” module of the high-content analysis software with a specific intensity threshold to locate stained acetylcholine receptor clusters on the channel of the acetylcholine receptor clusters global image;
(8) then measuring acetylcholine receptor number, area and intensity to quantify stained acetylcholine receptors using “Calculate Position Properties - Cross population” module of the high-content analysis software;
(9) quantify axons and/or neuromuscular junctions by obtaining the coefficient of co-localization of the axonal region detected in point (6) and the acetylcholine receptor clusters number, area and intensity automatically detected in point (8), using “Calculate Position Properties - Cross population” module of the high-content analysis software in a fraction of the whole organism myotome; then optionally, (10) conducting confocal re-scan at 20X magnification on the images obtained in points (2) and (3) and obtain a global image with Maximum Intensity Projections (MIP) as indicated in point (4);
(11) then applying a smoothing of the motor neurons channel global image with a median filter of 20px to bring out the densest region of the global image;
(12) then using “Find Surrounding Region” module of the high-content analysis software in order to subtract the spinal cord area of the whole organism;
(13) then creating a new region of interest using “Modify Population” module of the high-content analysis software, the new region corresponding to axonal region without the spinal cord;
(14) using the new region created in point (13) to measure axonal length and/or width and/or area and/or the ratio of body length to mean axonal length.
Such method may be particularly useful for the identification of a compound able to prevent and/or treat a neuromuscular disorder in an individual in need thereof.
Accordingly, the present invention also relates to a method for the identification of a compound able to prevent and/or treat a neuromuscular disorder in an individual in need thereof, comprising:
(a) implementing the method according to any one of claims 12 to 14 with a first whole organism or biological sample wherein the whole organism, or the organism from which the biological sample has been taken, has previously been exposed to a candidate compound, in order to (i) measure the size and area of the axonal region, (ii) measure the acetylcholine receptor clusters number, area and intensity and (iii) quantify axons and/or neuromuscular junctions
(b) implementing the method according to any one of claims 12 to 14 with a second whole organism or biological sample, identical to the said first whole organism or biological sample to the exception that the organism, or the organism from which the biological has been taken, has not previously been exposed to a candidate compound, in order to (i) measure the size and area of the axonal region, (ii) measure the acetylcholine receptor clusters number, area and intensity and (iii) quantify axons and/or neuromuscular junctions; T1
(c) comparing measurements and quantifications performed in steps (a) and (b) in order to determine if the candidate compound is able to prevent and/or treat a neuromuscular disorder in an individual in need thereof, and in particular is able:
- to restore neuromuscular junction structure by improving acetylcholine receptor clustering; and/or
- to increase the co-localization of presynaptic nerve terminals and acetylcholine receptor clusters; and/or
- to rescue axonal outgrowth; and/or
- improve post-synaptic acetylcholine receptor clustering number and percentage of co-localization area between pre- and post-synaptic neuromuscular junctions components.
The whole organisms, or the organism(s) from which the biological samples have been taken from, of steps (a) and (b) of the method of identification of the invention must be affected by a neuromuscular disorder, in particular a neuromuscular disorder selected from the group consisting of a neuromuscular junction disorder; a peripheral nerve disease; a motor neuron disease and a muscular disorder, in particular a muscular dystrophy or a myopathy.
The neuromuscular disorder may more particularly be selected from the group consisting of Congenital myasthenic syndromes (CMS), Lambert-Eaton myasthenic syndrome (LEMS), Myasthenia gravis (MG), Charcot-Marie-Tooth disease (CMT), Giant Axonal Neuropathy (GAN), Amyotrophic Lateral Sclerosis (ALS), Spinal-bulbar muscular atrophy (SBMA), Spinal muscular atrophy (SMA), Becker muscular dystrophy (BMD), a Congenital muscular dystrophies (CMD), Duchenne muscular dystrophy (DMD), Emery- Dreifuss muscular dystrophy (EDMD), Facioscapulohumeral muscular dystrophy (FSHD), Limb-girdle muscular dystrophies (LGMD), Myotonic dystrophy (DM), Oculopharyngeal muscular dystrophy (OPMD), Fredreich’s Ataxia, Mitochondrial myopathies, Congenital myopathies, Distal myopathies, Dystrophies, Multiple sclerosis, Myasthenic syndrome and Centronuclear myopathy 1.
The neuromuscular disorder may even more particularly be selected from the group consisting of:
Congenital myasthenic syndromes (CMS), Lambert-Eaton myasthenic syndrome (LEMS), Myasthenia gravis (MG), Charcot-Marie-Tooth disease (CMT), Giant Axonal Neuropathy (GAN), Amyotrophic Lateral Sclerosis (ALS), Spinal-bulbar muscular atrophy (SBMA) and Spinal muscular atrophy (SMA), and may in particular be selected from the group consisting of Charcot-Marie- Tooth disease (CMT) and Giant Axonal Neuropathy (GAN).
The neuromuscular disorder may more particularly be Giant Axonal Neuropathy (GAN).
In a method of the invention, when a first whole organism is used in step (a) and has been previously exposed to a candidate compound, then a second whole organism not previously exposed to a candidate compound is used in step (b).
On the contrary, when a first biological sample is used in step (a), the first biological sample originating from an organism which has not previously been exposed to a candidate compound, then a second biological sample originating from an organism which has not previously been exposed to a candidate compound is used in step (b). In particular, the first and second biological samples can originate from the same organism, the second biological sample being collected before the organism is exposed to a candidate compound, the first sample being collected after the organism is exposed to a candidate compound.
Steps (a) and (b) of this method according to the invention can be performed in any order, i.e. step (a) can be performed before step (b), simultaneously to step (b) or after step (b).
The method further comprises comparing measurements and quantifications performed in steps (a) and (b) of the method in order to determine if the candidate compound is able to prevent and/or treat a neuromuscular disorder in an individual in need thereof.
A compound tested in a method according to the invention will be considered as being able to prevent and/or treat a neuromuscular disorder in an individual in need thereof if the comparison step of the method indicates that said compound:
- increases the number of clusters of Acetylcholine receptors; and/or
- increases the number of neuromuscular junctions; and/or
- increases the length of the axons. and accordingly, is able: - to restore neuromuscular junctions structure by improving acetylcholine receptor clustering; and/or
- to increase the co-localization of presynaptic nerve terminals and acetylcholine receptor clusters; and/or - to rescue axonal outgrowth; and/or
- to improve post-synaptic acetylcholine receptor clustering number and percentage of co-localization area between pre- and post-synaptic neuromuscular junction components.
The present invention is further illustrated by, without in any way being limited to, the examples herein.
Identification of small molecules
Figure imgf000031_0001
locomotion in
Figure imgf000031_0002
zebrafish model a. To identify small molecules restoring locomotion in a first gan zebrafish model, FDA-approved drugs were screened and tested at a single concentration of lOpM. This concentration provides a good balance between biological effect and toxicity, as described in the literature (Rennekamp, A. J. & Peterson, R. T. Current Opinion in Chemical Biology 24, 58-70 (2015)).
Zebrafish models indeed provide a strong therapeutic potential for neurologic diseases, and offers the best alternative to mammalian screening for phenotype-based in vivo drug discovery. This species has many common advantages. Can for instance be mentioned the high conservation of genes and similarity to human, the external fertilization and rapid development through well-defined stages, facilitating the observation and experimental manipulation of embryos. Moreover, the statistical power of the screens is ensured by the high fecundity and small size of embryos. This feature also allows to place them in 96 wellplates and easily treat them by balneation. Finally, the transparency of the embryos facilitates the investigation at the physiological level within tissues, especially of neuronal and neuromuscular integrity in the case of NMDs. Indeed, zebrafish neuromuscular system has been well characterized (Beattie, C. E. Brain Research Bulletin 53, 489-500 (2000)).
Zebrafish embryos were treated with pronase (Img/ml, Roche; Ref : 10165921001) at 6 hpf (hours post fertilization) for 7 minutes under agitation and washed three times in E3 medium.
As described in Arribat, Y. et al. (Journal of Clinical Investigation 129, 5312— 5326 (2019)) and known to the man skilled in the art, a first gan zebrafish model is characterized by the depletion of gigaxonin using a transient approach (by injecting morpholino oligonucleotides antisense) which recapitulates the severe locomotor symptoms seen in patients. At the cellular level, the repression of z-gigaxonin induces both the shortening of axons and decreased production of motor neurons and impedes the formation of stable neuromuscular junction (NMJ). Altogether, these alterations in motor neurons and NMJ development induce a severe and penetrant locomotor deficit, characterized by a decrease of 85% of the mean of total distance travelled within an hour, and a complete loss of locomotion in 79,2 % of gan morpholino-injected (MO) (see Figures 1 and 2).
Embryos were then distributed manually in 200pL of E3 medium, 4 per well in a 96-well plate (Nunclon, Nunc™, ThermoFisher). Plates of drugs stock solutions (ImM) were thawed and diluted in H20 to a 2X concentrated solution of 20pM 2% DMSO using a liquid handling robot (FreedomEV0200, Tecan). At 8 hpf, lOOpL was removed from each well and lOOpl of 2X drug solutions were mechanically loaded (FreedomEV0200, Tecan). No mix at this step was done to avoid aspirating embryos and treatment was done at 3mm from the well bottom to avoid any contact with embryos. Therefore, the final concentration was lOpM with 1% DMSO in a total volume of 200pl per well. Drugs were automatically washed 5 times with E3 medium (FreedomEV0200, Tecan) at 48 hpf. These washes were done by a suction of 150pL and redistribution of 150pL medium with 2 slow mixes (lOpL/sec) and a soaking for 5 min without agitation.
The aim was to identify small molecules able to rescue loss-of-locomotion by monitoring the spontaneous locomotion of 5 dpf (days post fertilization) treated gan zebrafish, compared to WT larvae. A behavioral scoring relying on a z-score mean value of 4 biological replicates that measure the total distance travelled over an hour was generated The locomotion score scale ranges from null (WT animals) to negative values, indicative of a lack of locomotion, characteristic of untreated gan MO.
The systematic locomotion scoring of the tested drugs allowed to identify compounds efficient in gan MO, i.e. able to restore motor function in at least half of the treated gan MO replicates, offering at least 50% efficacy in this specific model. b. To ensure reproducibility of results across different z-gigaxonin depleted models, the same strategy was adopted to rescreen the compounds identified as being efficient in the gan knockout zebrafish line (gandel/del zebrafish).
As previously described in Arribat, Y. et al. (Journal of Clinical Investigation 129, 5312-5326 (2019)), the gandel/del zebrafish presents severe defects in spontaneous locomotion assay, with a strong decrease on travelled distance.
The total distances traveled by treated gan KO versus WT were normalized and a scale from 0-20 % (gan KO) to 100% (WT) Normal Percentage Activation was established, to provide the most appropriate scoring method in identifying sub-population of compounds among efficient hits set.
With this methodology, 59 hit compounds were identified as being able to rescue at least 50% of the locomotion in the treated gan KO zebrafish model, as illustrated in Table 1.
Figure imgf000033_0001
Figure imgf000034_0001
Table 1 c. A systems biology bioinformatic analysis was then performed on the basis of these 59 compounds using the targets known for each of these compounds in order to create a corresponding Drug-target list of 93 different targets.
On this basis, a Functional Enrichment Analysis of the Protein-Protein Interaction Networks was performed using the bioinformatic tools available to the man skilled in the art, such as for example the online tool STRING® V.11 (https://string-db.org/), and following the recommendations of the program.
In particular, the Search tool was used for Multiple Protein and all the identified targets were indicated in the List of Name, the chosen organism being Homo Sapiens. This directly provides a network. The Network can be refined in the Settings by changing the basic Settings in order to activate all the Active interaction sources, while also selecting a high confidence as minimum required interaction score.
This allows the identification of the most recurring targets (i.e. the targets targeted by at least 3 different compounds).
Identifying the compounds who target these recurring targets led to a list of “Hit Favorites”: Valdecoxib, Dichlorphenamide, Xylometazoline hydrochloride,
Tetrahydrozoline hydrochloride, Benzthiazide, Clidinium bromide, Prochlorperazine dimaleate, Tropicamide, Alverine citrate salt, Phentolamine hydrochloride, Haloperidol, Hyoscyamine (L), Scopolamin-N-oxide hydrobromide, Silodosin, Trichlormethiazide and Oxymetazoline hydrochloride. d. Cell imaging analysis was then performed following the development, by the inventors, of a high-throughput tailored imaging method in zebrafish to understand and treat neuromuscular diseases of a method comprising automating the quantification of axons and neuromuscular junctions in said animal.
Zebrafish were treated with 75pM l-Phenyl-2-thiourea (PTU, Sigma) from 24 hpf to prevent pigmentation. After drug-wash, they were anesthetized at appropriate developmental stages with 0,0168% tricaine (MS-222, Sigma -E10521-50G), fixed in 4% PFA for 4h at RT, and permeabilized in IX PBS-l%TritonX-100 for 2h on an orbital shaker. Subsequently, embryos were incubated in blocking buffer (1% DMSO, 1% normal donkey serum, 1% BSA, 0.7% TritonX-100, PBS) for Ih at RT and incubated in primary antibodies overnight at 4°C.
Primary antibodies were from the following sources: mouse IgG2a anti- synaptotagmin (1:100, Znp-1, DSHB), anti-a-bungarotoxin (1:50, B35451, Invitrogen). Following 0.1% TritonX-100: PBS washes, embryos were incubated in secondary antibodies (Alexa 488, 1:500, Jackson Labs 200-542-211) overnight at 4°C and subsequently washed in PBS prior to imaging.
Then, each fluorescent zebrafish is manually cut in the anterior part of the vitellus and placed in side position in individual wells of 96 well black bottom plates (pClear, Greiner Bio -one).
High content screening was then performed using the Opera Phenix™ high content screening system "High Content Screening System confocal" (PerkinElmer).
Then the following steps were performed.
1. PreciScan pre-scan, to identify zebrafish position in 96 well plate
To automatically find zebrafish larvae positions in wells, a pre-scan at 5X magnification was used to cover the entire well surface (9 fields per well), only on the red channel (561nm), with a signal distributed throughout the embryo. Using the on-the-fly image analysis, a global image of the whole well was created, and the “Find Image Region” module of the Harmony® High-content analysis software (v4.9, PerkinElmer) was used to set the appropriate intensity threshold to detect the fish. The “Determine Well Layout” module was then used to define a re-scan magnification, of 20X with an overlap of 6% between fields, covering the entire object.
2. PreciScan re-scan, automated imaging, and analysis: neuromuscular junctions Well areas containing whole zebrafish larvae were automatically imaged in confocal mode at 20X magnification on green (znp-1) and red (a-bungaro toxin) channels. A z-stack of 90pm (5pm interval) was applied, creating an on-the-fly image analysis to obtain a global image with Maximum Intensity Projections (MIP). From AChR-channel global image, the “Find Image Region and Select Region” modules allowed to detect fish body and subtract 7 pixels around to restrict the analysis to the region of interest. “Find Image Region” module was then applied on the green channel global image to detect and measure size and area of the Axonal region, i.e., spine and axons (Fig. 6.b). The “Find spots” module (method D) was applied with a specific intensity threshold to locate AChR clusters on the red channel global image. For AChR quantification, AChR number, area and intensity were measured using the “Calculate Position Properties - Cross population” module. The coefficient of colocalization was obtained using the same module in a fraction of the myotome to quantify NMJ.
3. Second High-content analysis: axons length On the same images batch, confocal re-scan at 20X magnification, another automated analysis was conducted. After creating a global image with Maximum Intensity Projections for the two channels, a smoothing with a median filter of 20px was applied on the green channel global image to bring out the densest region of the global image. Using the “Find Surrounding Region” module, the spinal cord area was subtracted from the fish body area. A new region of interest was then created using the “Modify Population” module, corresponding to axonal region without the spinal cord. This specific region was used to measure axonal length, width, area, and the ratio of body length to mean axonal length.
See Figure 3.
The establishment of this method interestingly allowed the observation and study of three parameters:
(1) the number of clusters of Acetylcholine receptors;
(2) the overlap between the two markings, allowing the quantification of neuromuscular junctions; and
(3) the axonal length.
The following results were obtained when this method was applied to WT nontreated zebrafish (control), non-treated gan MO zebrafish (control) and to gan MO zebrafish treated with the different identified compounds of interest mentioned above.
These three parameters are reduced in non-treated gan MO zebrafish compared to the WT zebrafish.
All the 59 compounds of interest identified above allowed for the restoration of at least one of these parameters, i.e. the said parameter was restored to a level similar to the one observed in the WT zebrafish.
5 compounds were more particularly interesting as they allowed for the restoration of at least above parameters (1) and (2): Phentolamine hydrochloride, Trichlormethiazide, Aceclidine Hydrochloride, Oxymetazoline hydrochloride and Digitoxigenin.

Claims

1. A compound for use in the prevention and/or treatment of a neuromuscular disorder in an individual in need thereof, the said compound being selected from the group consisting of:
Phentolamine hydrochloride, Trichlormethiazide, Aceclidine Hydrochloride, Oxymetazoline hydrochloride, Digitoxigenin, Bicalutamide, Valdecoxib, Theophylline monohydrate, Indapamide, Theobromine, Roxithromycin, lopamidol, Dipyrone, Benzoxiquine, lopromide, Clomiphene citrate (Z,E), Chlorphensin carbamate, Dichlorphenamide, Xylometazoline hydrochloride, Iproniazide phosphate, Tetrahydrozoline hydrochloride, Dydrogesterone, Dexfenfluramine hydrochloride, Tranexamic acid, Benzthiazide, Cefotetan, Amikacin hydrate, Clidinium bromide, Prochlorperazine dimaleate, Nimesulide, Tropicamide, Pargyline hydrochloride, Alverine citrate salt, Aceclofenac, Mephenesin, Azacytidine- 5, Ketanserin tartrate hydrate, Oxantel pamoate, Methylhydantoin-5-(L), Novobiocin sodium salt, Haloperidol, Ifenprodil tartrate, Etoposide, Methylhydantoin-5-(D), Hyoscyamine (L), Lomefloxacin hydrochloride, Clemizole hydrochloride, Spectinomycin dihydrochloride, Diperodon hydrochloride, loversol, Formoterol fumarate, Scopolamin-N-oxide hydrobromide, Kanamycin A sulfate, Pentobarbital, Silodosin, Carbinoxamine maleate salt, Clopidogrel, Methimazole and Fenbufen.
2. A compound for use in the partial or complete restoration of the motility of an individual affected by a neuromuscular disorder, the said compound being selected from the group consisting of:
Phentolamine hydrochloride, Trichlormethiazide, Aceclidine Hydrochloride, Oxymetazoline hydrochloride, Digitoxigenin, Bicalutamide, Valdecoxib, Theophylline monohydrate, Indapamide, Theobromine, Roxithromycin, lopamidol, Dipyrone, Benzoxiquine, lopromide, Clomiphene citrate (Z,E), Chlorphensin carbamate, Dichlorphenamide, Xylometazoline hydrochloride, Iproniazide phosphate, Tetrahydrozoline hydrochloride, Dydrogesterone, Dexfenfluramine hydrochloride, Tranexamic acid, Benzthiazide, Cefotetan, Amikacin hydrate, Clidinium bromide, Prochlorperazine dimaleate, Nimesulide, Tropicamide, Pargyline hydrochloride, Alverine citrate salt, Aceclofenac, Mephenesin, Azacytidine- 5, Ketanserin tartrate hydrate, Oxantel pamoate, Methylhydantoin-5-(L), Novobiocin sodium salt, Haloperidol, Ifenprodil tartrate, Etoposide, Methylhydantoin-5-(D), Hyoscyamine (L), Lomefloxacin hydrochloride, Clemizole hydrochloride, Spectinomycin dihydrochloride, Diperodon hydrochloride, loversol, Formoterol fumarate, Scopolamin-N-oxide hydrobromide, Kanamycin A sulfate, Pentobarbital, Silodosin, Carbinoxamine maleate salt, Clopidogrel, Methimazole and Fenbufen.
3. The compound for use according to claim 1 or 2, wherein the said compound is selected from the group consisting of:
Valdecoxib, Dichlorphenamide, Xylometazoline hydrochloride, Tetrahydrozoline hydrochloride, Benzthiazide, Clidinium bromide, Prochlorperazine dimaleate, Tropicamide, Alverine citrate salt, Phentolamine hydrochloride, Haloperidol, Aceclidine Hydrochloride, Hyoscyamine (E), Scopolamin-N-oxide hydrobromide, Digitoxigenin, Silodosin, Trichlormethiazide and Oxymetazoline hydrochloride, and in particular from the group consisting of Valdecoxib, Xylometazoline hydrochloride, Tetrahydrozoline hydrochloride, Clidinium bromide, Tropicamide, Alverine citrate salt, Phentolamine hydrochloride, Hyoscyamine (L), Scopolamin-N-oxide hydrobromide, Silodosin and Oxymetazoline hydrochloride.
4. The compound for use according to any one of claims 1, 2 or 3, wherein the said compound is selected from the group consisting of:
Phentolamine hydrochloride, Trichlormethiazide, Aceclidine Hydrochloride, Oxymetazoline hydrochloride and Digitoxigenin, and in particular from the group consisting of Phentolamine hydrochloride and Oxymetazoline hydrochloride.
5. The compound for use according to any one of claims 1 to 4, wherein the said neuromuscular disorder is selected from the group consisting of:
(a) a neuromuscular junction disorder; and/or
(b) a peripheral nerve disease; and/or
(c) a motor neuron disease; and/or
(d) a muscular disorder, in particular a muscular dystrophy or a myopathy. 6. The compound for use according to any one of claims 1 to 5, wherein the said neuromuscular disorder is selected from the group consisting of:
Congenital myasthenic syndromes (CMS), Lambert-Eaton myasthenic syndrome (LEMS), Myasthenia gravis (MG), Charcot-Marie-Tooth disease (CMT), Giant Axonal Neuropathy (GAN), Amyotrophic Lateral Sclerosis (ALS), Spinal-bulbar muscular atrophy (SBMA), Spinal muscular atrophy (SMA), Becker muscular dystrophy (BMD), a Congenital muscular dystrophies (CMD), Duchenne muscular dystrophy (DMD), Emery- Dreifuss muscular dystrophy (EDMD), Facioscapulohumeral muscular dystrophy (FSHD), Limb-girdle muscular dystrophies (LGMD), Myotonic dystrophy (DM), Oculopharyngeal muscular dystrophy (OPMD), Fredreich’s Ataxia, Mitochondrial myopathies, Congenital myopathies, Distal myopathies, Dystrophies, Multiple sclerosis, Myasthenic syndrome and Centronuclear myopathy 1.
7. The compound for use according to any one of claims 1 to 7, wherein the said neuromuscular disorder is selected from the group consisting of:
Congenital myasthenic syndromes (CMS), Lambert-Eaton myasthenic syndrome (LEMS), Myasthenia gravis (MG), Charcot-Marie-Tooth disease (CMT), Giant Axonal Neuropathy (GAN), Amyotrophic Lateral Sclerosis (ALS), Spinal-bulbar muscular atrophy (SBMA) and Spinal muscular atrophy (SMA), and is in particular selected from the group consisting of Charcot-Marie-Tooth disease (CMT) and Giant Axonal Neuropathy (GAN), more particularly Giant Axonal Neuropathy (GAN).
8. The compound for use according to any one of claims 1 to 7, wherein the said compound is comprised in a composition, the said composition further comprising a physiologically acceptable medium.
9. The compound for use according to claim 9, wherein the said composition comprises at least two different compounds from the list according to any one of claims 1 to 4.
10. The compound for use according to any one of claims 1 to 9, wherein the individual is a mammal, and is in particular a human being. 11. A method for automating the quantification of axons and/or neuromuscular junctions in a whole organism or in a biological sample, the method comprising the steps of:
(a) staining, in particular immunostaining, (i) axons of the motor neurons and (ii) acetylcholine receptor clusters of said whole organism or biological sample;
(b) placing the stained whole organism or biological sample in a predefined position in a container, in particular in a side position;
(c) screening the stained whole organism or biological sample of step (b) using a high content screening system, by:
(1) performing a pre-scan covering the entirety of the stained whole organism or biological sample with a signal distributed throughout the stained whole organism or biological sample and obtaining a global image of the entirety of the stained whole organism or biological sample;
(2) set an appropriate intensity threshold to detect said stained whole organism or biological sample;
(3) automatically obtain an image of the stained whole organism or biological sample on channels allowing the detection of (i) the staining of the axons of the motor neurons and (ii) the staining of the acetylcholine receptor clusters;
(4) automatically locate then measure the size and area of the axonal region using the staining of the axons of the motor neurons;
(5) automatically locate, then quantify the stained acetylcholine receptor clusters by automatically measuring the acetylcholine receptor clusters number, area and intensity;
(6) quantify axons and/or neuromuscular junctions by automatically calculating a coefficient of co-localization of the axonal region automatically detected in point (4) and the acetylcholine receptor clusters number, area and intensity automatically detected in (5).
12. The method according to claim 11, wherein the whole organism is selected from the group consisting of a zebrafish Danio rerio, a Caenorhabditis elegans and a Diptera of the genus Drosophila, in particular Drosophila melanogaster, and is preferably a zebrafish, said organism being optionally affected by a neuromuscular disorder, more particularly selected from the group consisting of:
(a) a neuromuscular junction disorder; and/or
(b) a peripheral nerve disease; and/or
(c) a motor neuron disease; and/or
(d) a muscular disorder, in particular a muscular dystrophy or a myopathy.
13. The method according to claim 11 or 12, wherein the axons and/or neuromuscular junctions quantification relates to a whole organism and wherein the method comprises the steps of:
(a) immuno staining axons of the motor neurons comprising the implementation of anti-znpl and SV2 antibodies and immuno staining acetylcholine receptor clusters comprising the implementation of anti-alpha-bungarotoxin antibodies, of the whole organism;
(b) placing the stained whole organism in side position in a well plate reader;
(c) screening the stained whole organism of step (b) using a high content screening system comprising a high-content analysis software, by:
(1) performing a pre-scan, in particular at 5X magnification to cover the entire well surface in order to automatically find the whole organism position in the well, only on a channel appropriate to the immunostaining used for the immuno staining of acetylcholine receptor clusters, with a signal distributed throughout the whole organism;
(2) obtaining a global image of the whole organism by using an on-the-fly image analysis set and setting the appropriate intensity threshold to detect the stained whole organism using a “Find Image Region” module of the high-content analysis software of the high content screening system;
(3) automatically imaging the stained whole organism by implementing channels appropriate to the immuno staining used for the immuno staining of the axons of the motor neurons and of the acetylcholine receptor clusters;
(4) applying z-stack of 90pm (5pm interval) in order to perform an on-the-fly image analysis to obtain a global image with Maximum Intensity Projections (MIP); (5) applying the “Find Image Region and Select Region” modules of the high- content analysis software from the acetylcholine receptor clusters global image to detect the stained whole organism and subtract 7 pixels around to restrict the analysis to the region of interest;
(6) then applying “Find Image Region” module of the high-content analysis software on the axons of the motor neurons channel global image to detect and measure size and area of the axonal region;
(7) applying “Find spots” module of the high-content analysis software with a specific intensity threshold to locate stained acetylcholine receptor clusters on the channel of the acetylcholine receptor clusters global image;
(8) then measuring acetylcholine receptor number, area and intensity to quantify stained acetylcholine receptors using “Calculate Position Properties - Cross population” module of the high-content analysis software;
(9) quantify axons and/or neuromuscular junctions by obtaining the coefficient of co-localization of the axonal region detected in point (6) and the acetylcholine receptor clusters number, area and intensity automatically detected in point (8), using “Calculate Position Properties - Cross population” module of the high-content analysis software in a fraction of the whole organism myotome; then optionally,
(10) conducting confocal re- scan at 20X magnification on the images obtained in points (2) and (3) and obtain a global image with Maximum Intensity Projections (MIP) as indicated in point (4);
(11) then applying a smoothing of the motor neurons channel global image with a median filter of 20px to bring out the densest region of the global image;
(12) then using “Find Surrounding Region” module of the high-content analysis software in order to subtract the spinal cord area of the whole organism;
(13) then creating a new region of interest using “Modify Population” module of the high-content analysis software, the new region corresponding to axonal region without the spinal cord;
(14) using the new region created in point (13) to measure axonal length and/or width and/or area and/or the ratio of body length to mean axonal length. 14. A method for the identification of a compound able to prevent and/or treat a neuromuscular disorder in an individual in need thereof comprising:
(a) implementing the method according to any one of claims 12 to 14 with a first whole organism or biological sample wherein the whole organism, or the organism from which the biological sample has been taken, has previously been exposed to a candidate compound, in order to (i) measure the size and area of the axonal region, (ii) measure the acetylcholine receptor clusters number, area and intensity and (iii) quantify axons and/or neuromuscular junctions
(b) implementing the method according to any one of claims 12 to 14 with a second whole organism or biological sample, identical to the said first whole organism or biological sample to the exception that the organism, or the organism from which the biological has been taken, has not previously been exposed to a candidate compound, in order to (i) measure the size and area of the axonal region, (ii) measure the acetylcholine receptor clusters number, area and intensity and (iii) quantify axons and/or neuromuscular junctions;
(c) comparing measurements and quantifications performed in steps (a) and (b) in order to determine if the candidate compound is able to prevent and/or treat a neuromuscular disorder in an individual in need thereof, and in particular is able:
- to restore neuromuscular junctions structure by improving acetylcholine receptor clustering; and/or
- to increase the co-localization of presynaptic nerve terminals and acetylcholine receptor clusters; and/or
- to rescue axonal outgrowth; and/or
- improve post-synaptic acetylcholine receptor clustering number and percentage of co-localization area between pre- and post-synaptic neuromuscular junctions components; the whole organisms, or the organism(s) from which the biological samples have been taken from, of steps (a) and (b) being affected by a neuromuscular disorder, in particular a neuromuscular disorder selected from the group consisting of a neuromuscular junction disorder; a peripheral nerve disease; a motor neuron disease and a muscular disorder, in particular a muscular dystrophy or a myopathy.
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