WO2020234496A1 - Moxifloxacine destinée à être utilisée dans le traitement de l'amyotrophie spinale - Google Patents

Moxifloxacine destinée à être utilisée dans le traitement de l'amyotrophie spinale Download PDF

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WO2020234496A1
WO2020234496A1 PCT/ES2020/070311 ES2020070311W WO2020234496A1 WO 2020234496 A1 WO2020234496 A1 WO 2020234496A1 ES 2020070311 W ES2020070311 W ES 2020070311W WO 2020234496 A1 WO2020234496 A1 WO 2020234496A1
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moxifloxacin
muscular atrophy
spinal muscular
treatment
pharmaceutically acceptable
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PCT/ES2020/070311
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English (en)
Spanish (es)
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Rubén Artero Allepuz
Piotr Tadeusz KONIECZNY
Arturo LÓPEZ CASTEL
Manuel PÉREZ ALONSO
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Universitat De València
Fundación Incliva
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/16—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing nitrogen, e.g. nitro-, nitroso-, azo-compounds, nitriles, cyanates
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system

Definitions

  • the present invention relates to moxifloxacin, or a pharmaceutically acceptable salt or solvate thereof, as well as pharmaceutical compositions comprising it, for use in the prevention and / or treatment of spinal muscular atrophy.
  • SMA Spinal muscular atrophy
  • OMIM # 253300 is a rare genetic neuromuscular disease (with an occurrence of 1/6000 births) mainly caused by the loss of ⁇ -motor neurons from the spinal cord and brainstem nucleus.
  • Five clinical types of spinal muscular atrophy (0, 1, 2, 3, and 4) have been described, ranging from a complete absence of motor function and infant mortality, to minor defects that do not significantly reduce life expectancy.
  • Spinal muscular atrophy is caused by mutations in the SMN1 gene that encodes a 38 kDa protein that is highly expressed in the cytoplasm and in the nucleus.
  • the SMN locus contains an additional, human-specific copy called SMN2. More than 98% of spinal muscular atrophy patients have a homozygous deletion, mutation, or other type of change in the SMN1 gene, and all spinal muscular atrophy patients nevertheless carry one or more copies of the functional SMN2 gene.
  • the SMN1 and SMN2 genes are almost identical, with a difference in the coding region of only one nucleotide: a change from C- (cytidine) to T- (thymidine) at position +6 of exon 7 (Ex7) causing exclusion of exon 7 in most of the SMN2 transcripts (Al SMN2), giving rise to a truncated SMN in the C-terminal part and the generation of protein with less stability and activity.
  • a small fraction of the SMN2 mRNA includes exon 7 (FL-SMN2) which generates a fully functional protein.
  • the nucleotide change C to U is found in an exonic splicing enhancer region (Exonic Splicing Enhancer region, for its acronym in English ESE), which in SMN1 transcripts favors normal splicing of Ex7.
  • ESE Exonic Splicing Enhancer region
  • the splicing factor rich in serine and arginine SRSF1 also known as SF2 / ASF
  • CAGACAA is transforms into an exonic splicing silencer (ESS) (UAGACAA) and becomes a heterogeneous ribonucleoprotein A1 / A2 (hnRNP A1 / A2) recognition site.
  • SMN proteins are known to inhibit splicing, since they operate on ESSs and bind to UAG sites. The reduction of splicing inhibiting factors restores the splicing pattern of SMN1, suggesting that there is a competition between enhancers and repressors of splicing at the regulatory sites.
  • SMN proteins are dispersed in the cytoplasm, but in the nucleus they aggregate in punctiform structures close to Cajal bodies, and are called gems, which are aggregates of the SMN protein.
  • gems which are aggregates of the SMN protein.
  • These nuclear SMN aggregates are believed to play a direct role in small ribonucleoprotein (snRNP) maturation and pre-mRNA splicing. SMN has been suggested to play a role in the formation of snRNPs in various cell types. Therefore, the appearance of said SMN protein aggregates is directly related to the existence of adequate levels of said protein.
  • snRNP small ribonucle
  • SMA Spinal muscular atrophy
  • SMN2 transcription and / or seeking to enhance the inclusion of exon 7 in the mature transcripts resulting in both cases in an increase in the amount of SMN protein.
  • Particularly relevant is the recent commercialization of the drug Spinraza® (Nusinersen) by the company Biogen, a drug that consists of a chemically modified antisense oligonucleotide that favors the inclusion of exon 7 of SMN2.
  • Spinraza is administered by lumbar puncture, which is invasive and not without risk, and the costs of the drug range from 400,000-600,000 euros per patient per year.
  • the degree of activity of Nusinersen outside the central nervous system and its consequent side effects is also under debate.
  • the present invention relates to moxifloxacin, or a pharmaceutically acceptable salt or solvate thereof, as well as to a pharmaceutical composition comprising it, for use in the treatment of spinal muscular atrophy.
  • FIG. 1 Toxicity test. Fibroblasts of the GM03813 line (Corriel Cell Repository) were seeded in 96-well plates with 1.0 x 10 4 cells per well and incubated 24 hours. Moxifloxacin was added at various concentrations (1000 mM, 100 pM, 10 pM and 1 pM) in complete Dulbecco's Modified Eagle Medium (DMEM) and the cells were incubated for 48 h.
  • DMEM Dulbecco's Modified Eagle Medium
  • FIG. 1 Quantification of SMN protein levels in fibroblasts derived from patients with spinal muscular atrophy, (2A) treated with moxifloxacin at the maximum non-toxic concentration: 500 pM, for 72 h. SMN levels were measured by western blot and the results obtained were compared to those of a sample treated with solvent dimethylsulfoxide (DMSO 0.5%, dotted line). (2B) Representative images processed with ImageJ of the results obtained by western blot. Statistical values were calculated using Student's t test (* p ⁇ 0.05, ** p ⁇ 0.01, **** p ⁇ 0.001).
  • FIG. 3 Levels of SMN2 FL and D7 isoforms in fibroblasts from patients with spinal muscular atrophy treated with moxifloxacin. The fibroblasts were treated with four different concentrations for 48 h.
  • FIG. 4 Confocal microscopy image of aggregates of SMN and Cajal's bodies. HeLa cells were treated with 500 pM moxifloxacin for 72 h and cells were double stained for SMN and Coilin. The nuclei were stained with Hoechst 33258 diluted in mounting medium (1: 10,000). Images were taken with a LSM 800 confocal microscope (Zeiss).
  • FIG. 6 Fusion index of myoblasts derived from patients with SMA type 2 (SMA; ref. KM1150SMAII7PV) and healthy individuals as controls (CON; ref. AB1190). Both lines have been obtained from the Institut de Myologie, Paris. (6A) Cells initially differentiated for 3 days and were subsequently treated with moxifloxacin, for the next 72 h. Solvent treated SMA and healthy myoblasts (0.5% DMSO) were used to establish a difference in the differentiation process. Risdiplam at 1 mM was used as a positive control.
  • Nuclei were labeled with DAPI (4 ', 6-diamino-2-phenylindole) and myoblasts and myotubes were detected using an anti-Desmin antibody. Multinucleated myotubes are marked with white arrows.
  • the fusion index (defined as the percentage of nuclei in myotubes with at least two nuclei, with respect to the total nuclei in the preparation) of the differentiated healthy myoblasts and of AME treated with DMSO and the indicated compounds was calculated. Data were collected after analysis of at least 100 nuclei and statistical comparisons were made using Student's t-test (* p ⁇ 0.05, ** p ⁇ 0.01, *** p ⁇ 0.001). (6B) Representative images of the experiment taken on a confocal microscope. DESCRIPTION OF THE INVENTION
  • the present invention relates to moxifloxacin (1-cyclopropyl-6-fluoro-7 - ((4aS, 7aS) -hexahydro-1 H-pyrrolo [3,4-b] pyridin-6 (2H) -yl acid) -8 -methoxy-4-oxo-1,4-dihydroquinoline-3-carboxylic) for use in the prevention and / or treatment of spinal muscular atrophy.
  • Moxifloxacin is an antibiotic with a quinolone structural core that exhibits antibacterial activity.
  • Moxifloxacin has been found to significantly increase SMN protein levels, promoting the inclusion of exon 7 of SMN2 in a concentration-dependent manner, an activity that, as described above, is a treatment and cure route for atrophy spinal muscle (SMA).
  • Moxifloxacin is part of the family of fluoroquinolones (Fábrega et al., Microb Biotechnol. 2009 Jan; 2 (1): 40-61), compounds with antibiotic activity that comprise a quinolone bicyclic nucleus that comprises, among other substituents, a fluorine and a carboxylate group.
  • the present invention refers to the use of moxifloxacin in the prevention and / or treatment of spinal muscular atrophy, but also refers to the use of structural variants of said compound belonging to the family of fluoroquinolones, that is, to use of compounds with said bicyclo quinolone comprising, among other substituents, a fluorine and a carboxylate group.
  • Moxifloxacin was evaluated in fibroblasts from patients with spinal muscular atrophy, as described in Example 1, by measuring SMN transcript levels in fibroblasts treated with the candidate compound versus SMN transcript levels from vehicle-treated control fibroblasts. .
  • moxifloxacin was also evaluated in HeLa cells to determine the existence of SMN aggregates together or co-localized with Cajal's bodies, data indicative of the presence of functional SMN in the organism. For this, immunofluorescence was used, determining the overlap of the signal corresponding to SMN aggregates and of the signal corresponding to Cajal Bodies, in HeLa cells treated with moxifloxacin and in cells treated with vehicle.
  • Example 1 shows how moxifloxacin increased the levels of the SMN protein in fibroblasts derived from patients with spinal muscular atrophy, causing the inclusion of exon 7 SMN2 in a concentration-dependent manner, and showed the ability to modulate splicing of SMN2 favoring the inclusion of Ex7.
  • Example 2 shows that administration of moxifloxacin to HeLa cells resulted in an almost double increase compared to the DMSO control ( Figure 5). These results demonstrate that the increase in SMN levels after treatment with moxifloxacin is beneficial for cells that do not have the function of the SMN1 gene.
  • moxifloxacin promotes the inclusion of exon 7 of SMN2 in fibroblasts from patients with type II spinal muscular atrophy. Furthermore, the activity of moxifloxacin results in an increase in the expression of the SMN protein and in a greater expression of SMN2 in the form of subcellular structures, or SMN aggregates, a characteristic way in which said protein accumulates under normal conditions. The recovery of normal protein levels could be confirmed by the localization of nuclear SMN aggregates (or gems) next to Cajal Bodies ( Figure 4).
  • moxifloxacin was also evaluated in myoblasts from patients with spinal muscular atrophy type II (SMA type 2), as described in example 3.
  • SMA patients have myoblasts deficient in SMN, and have fusion capacities (forming myotubes with more than two cores) deteriorated.
  • treatment with moxifloxacin allows recovery of fusion ability in myoblasts to levels similar to that of healthy myoblasts.
  • One embodiment of the invention thus relates to moxifloxacin, or a pharmaceutically acceptable salt or solvate thereof, for use in the prevention and / or treatment of spinal muscular atrophy.
  • a type 0 spinal muscular atrophy is defined as that which affects fetuses. It is the most severe form of the disease and is characterized by movement poor fetus, joint abnormalities, difficulty swallowing, and respiratory failure. It can be fatal before birth and before the first year of life.
  • a type 1 spinal muscular atrophy is defined as infantile spinal muscular atrophy or Werdnig-Hoffmann disease. It is the most common type of SMA and occurs during birth or in the first few months of life, with developmental delays and problems raising the head, turning, breathing, and swallowing.
  • a type 2 spinal muscular atrophy, or intermediate SMA is defined, that which appears between 7 and 18 months of age, with an inability to walk in children who suffer from it.
  • a type 3 spinal muscular atrophy, or juvenile SMA is defined as that which shows obvious symptoms before the age of three or even during puberty, and which causes problems in standing and walking, worsening said symptoms with age.
  • a type 4 spinal muscular atrophy appears in adults, after the age of 30, and carries only mild or moderate symptoms.
  • One embodiment of the invention relates to moxifloxacin, or a pharmaceutically acceptable salt or solvate thereof, for use in the prevention and / or treatment of spinal muscular atrophy, wherein said spinal muscular atrophy is of type 0, 1, 2, 3 , 4.
  • the spinal muscular atrophy is type 0. In another embodiment of the invention, the spinal muscular atrophy is type 1. In another embodiment of the invention, the spinal muscular atrophy is type 2. In another In one embodiment of the invention, the spinal muscular atrophy is type 3. In another embodiment of the invention, the spinal muscular atrophy is type 4.
  • the term “comprises” indicates that it includes a group of certain characteristics (for example, a group of characteristics A, B and C) is interpreted to mean that it includes those characteristics (A, B and C), but that it does not exclude the presence of other characteristics (for example, characteristics D or E), provided that they do not make the claim impracticable. Additionally, the terms “contains”, “includes”, “has” or “encompasses”, and the plural forms thereof, should be taken synonymously with the term “comprises” for the purposes of the present invention. On the other hand, if the expression "consists (s) of” is used, then no additional features are present in the apparatus / method / product, apart from those that follow said expression.
  • the term “comprises” may be replaced by any of the terms “consist of”, or “consists essentially of”. Accordingly, “comprises” can refer to a group of characteristics A, B and C, which may additionally include other characteristics, such as E and D, provided that said characteristics do not make the claim impracticable, but said term “comprises ”Also includes the situation where the feature group“ consists of ”or“ consists essentially ”of A, B, and C.
  • pharmaceutically acceptable salt refers to any salt that, when administered to the patient, is capable of directly or indirectly providing the described compound.
  • solvate refers to any form of the active compound, moxifloxacin, that is non-covalently bound to another molecule (usually a polar solvent), and especially refers to hydrates and alcoholates.
  • the salt is a metal salt.
  • a salt of an alkali metal or of an alkaline earth metal more preferably Na + , K + and Ca 2+ .
  • the pharmaceutically acceptable salt is an ammonium salt.
  • said ammonium salt comprises an ammonium cation substituted with one or more groups independently selected from H, alkyl, alkenyl, alkynyl, hydroxyalkyl, poly (hydroxy) alkyl, cycloalkyl, alkylated, aminoaryl, aminoalkyl, aminoalkenyl, aminoalkynyl, arylalkylaminoalkyl and alkylaminoaryl; or is a cyclic ammonium cation.
  • the salt is a guanidine salt.
  • the pharmaceutically salt is a salt of an amino acid. Said amino acid is preferably L-lysine or L-arginine.
  • the pharmaceutically acceptable salt is a salt of an inorganic acid, preferably a hydrochloride, hydrobromide, sulfate, or phosphate.
  • the pharmaceutically acceptable salt of moxifloxacin for use according to the present invention is a salt of an organic acid, preferably a citrate, oxalate, salicylate, benzoate, acetate, fumarate or maleate.
  • the pharmaceutically acceptable salt of moxifloxacin is a salt formed with the carboxylic acid group or with a basic amine of moxifloxacin, or with both groups.
  • the moxifloxacin for use in accordance with the present invention is a basic addition salt. Said salt can also be hydrated.
  • a preferred embodiment of the present invention relates to a moxifloxacin hydrochloride salt for use in the prevention and / or treatment of spinal muscular atrophy.
  • Another preferred embodiment of the present invention relates to a moxifloxacin hydrochloride monohydrate salt, for use in the prevention and / or treatment of spinal muscular atrophy.
  • Another embodiment of the invention relates to the use of moxifloxacin, or a pharmaceutically acceptable salt or solvate thereof, for the manufacture of a medicament for use in the treatment of spinal muscular atrophy.
  • the salt is a hydrochloride.
  • An embodiment of the present invention also relates to a pharmaceutical composition
  • a pharmaceutical composition comprising an effective amount of moxifloxacin, or a pharmaceutically acceptable salt or solvate thereof, and at least one excipient or vehicle, for use in the prevention and / or treatment of spinal muscular atrophy.
  • an effective amount for the purposes of the present invention, is understood as that which provides a therapeutic effect without providing unacceptable toxic effects in the patient.
  • the effective amount or dose of the drug depends on the compound and the condition or disease treated and on, for example, the age, weight and clinical condition of the patient treated, the method of administration, the clinical history of the patient, the severity of the disease. and the potency of the administered compound.
  • a preferred embodiment of the present invention relates to a pharmaceutical composition comprising an effective amount of moxifloxacin hydrochloride, for use in the prevention and / or treatment of spinal muscular atrophy.
  • Another preferred embodiment of the present invention relates to a pharmaceutical composition
  • a pharmaceutical composition comprising an effective amount of moxifloxacin hydrochloride monohydrate, for use in the prevention and / or treatment of spinal muscular atrophy.
  • moxifloxacin can be used alone or in conjunction with another active compound.
  • an active compound is understood to be a chemical entity or an active principle that exerts therapeutic effects when administered to a human or an animal.
  • a preferred embodiment of the invention relates to a pharmaceutical composition comprising an effective amount of moxifloxacin, or a pharmaceutically acceptable salt or solvate thereof, at least one excipient or vehicle, and at least one other active compound, for use in the prevention and / or treatment of spinal muscular atrophy.
  • Another preferred embodiment of the invention refers to moxifloxacin, or to a pharmaceutical composition comprising it, for use in the prevention and / or treatment of spinal muscular atrophy, wherein said use is characterized in that it comprises the administration of a second additional active compound together with, before, or after administration of moxifloxacin.
  • said additional active compound is a compound for use in the prevention and / or treatment of spinal muscular atrophy.
  • said additional active compound for use in the prevention and / or treatment of spinal muscular atrophy is nusinersen (Spinraza®) or risdiplam. Even more preferably said additional active compound for use in the prevention and / or treatment of spinal muscular atrophy is nusinersen.
  • the present invention describes a method of prevention and / or treatment of spinal muscular atrophy that comprises the administration of an effective amount of moxifloxacin, or a pharmaceutically acceptable salt or solvate thereof, or of a pharmaceutical composition comprising it, to a patient in need.
  • the pharmaceutically acceptable salt is a hydrochloride.
  • said spinal muscular atrophy is of type 0, 1, 2, 3, 4.
  • the spinal muscular atrophy is type 0. In another embodiment of the invention, the spinal muscular atrophy is type 1. In another embodiment of the invention, the spinal muscular atrophy is type 2. In another In one embodiment of the invention, the spinal muscular atrophy is type 3. In another embodiment of the invention, the spinal muscular atrophy is type 4.
  • compositions of the invention include, together with the compounds described in the present invention at least one pharmaceutically acceptable excipient, which can be a carrier or diluent.
  • a diluent is such that it does not modify the biological action of the compound of interest.
  • compositions can be included in capsules, tablets, sachets or sachets or any other type of presentation.
  • the compound of interest may be mixed with a vehicle or diluted in a vehicle or contained in a vehicle in the form of an ampoule, capsule, tablet, sachet, sachet or other container.
  • the carrier serves as a solvent, it can be solid, semi-solid or liquid and act as an excipient or medium for said active compound.
  • the compound of interest can be adsorbed onto a solid granular medium.
  • Suitable vehicles are water, saline solutions, alcohols, polyethylene glycols, polyhydroxyethoxylated castor oil, peanut oil, olive oil, lactose, terra alba, sucrose, cyclodextrins, amylose, magnesium stearate, talc, gelatin, agar, pectin , acacia, stearic acid, cellulose alkyl ethers, silicon acid, fatty acids, fatty acid amines, fatty acid monoglycerides and diglycerides, pentaerythrole fatty esters, polyethylene, hydroxymethylcellulose and polyvinylpyrrolidone.
  • the vehicle or support can include sustained release materials known in the state of the art, such as glyceryl monostearate or diesterate alone or mixed with a wax.
  • the formulations may also include wetting, emulsifying, suspending, preserving, sweetening or flavoring agents.
  • the compositions can be formulated to provide rapid, sustained or delayed release of the active agent after it is administered to the patient using methods known in the art.
  • compositions can be sterilized and mixed, if desired, with additional agents, emulsifiers, salt to influence osmotic pressure, buffers and / or coloring substances that do not react adversely with the active compounds.
  • An embodiment of the invention relates to the mode of administration, which can be any mode that effectively transports the compound of interest to the desired site of action, such as oral, rectal, or parenteral, eg, subcutaneous, intravenous, intraurethral, intramuscular. , intranasal or as an ophthalmic solution.
  • one embodiment of the invention relates to a composition of moxifloxacin, or a pharmaceutically acceptable salt or solvate thereof, for use in accordance with the present invention, wherein said composition is administered intrathecally, orally, rectally, ophthalmic. or parenteral.
  • the pharmaceutical composition for use according to the present invention is a tablet, an injectable solution, or an ophthalmic solution.
  • both solid and liquid dosage forms can be prepared.
  • the compound of interest it is mixed in a formulation with other conventional ingredients such as talc, magnesium stearate, dicalcium phosphate, magnesium aluminum silicate, starch, lactose, acacia, methyl cellulose and functionally similar materials such as pharmaceutical carriers and diluents.
  • Capsules can be prepared by mixing the compound of interest with a pharmaceutically inert solvent and filling the mixture into an appropriately sized hard gelatin.
  • Soft capsules are prepared with machines for encapsulating suspensions of the compound of interest with an acceptable vegetable oil, a light paraffin or an inert oil.
  • Liquid dosage forms such as syrups, elixirs and suspensions can also be prepared.
  • the water-soluble forms can be dissolved in an aqueous vehicle along with sugar, flavorings, and preservatives to form a syrup.
  • An elixir is prepared using a hydroalcoholic vehicle (eg ethanol) with suitable sweeteners such as sugar or saccharin, along with flavoring aromatic agents.
  • Suspensions can be prepared with an aqueous vehicle and the aid of a suspending agent such as acacia, tragacanth, methylcellulose, and the like.
  • the preparation may contain the compound of interest dissolved or suspended in a liquid vehicle, in particular an aqueous vehicle, for application as an aerosol.
  • the carrier may contain additives such as solubilizing agents, for example propylene glycol, surfactants, absorption enhancers such as lecithin (phosphatidylcholine) or cyclodextrin, or preservatives such as parabens.
  • the compound of interest is formulated in solutions, suspensions and ointments suitable for use in the eye.
  • the concentrations are usually the same as in preparations for local use.
  • injectable solutions or suspensions for intradermal, intramuscular, intravascular, intrathecal and subcutaneous use, are obvious to those skilled in the art.
  • compositions may include other pharmaceutically acceptable non-toxic diluents and excipients, including vehicles commonly used in pharmaceutical compositions commonly used in humans or animals.
  • the diluent is selected so that it does not affect the biological activity of the composition.
  • compositions can include additives such as other excipients, adjuvants, non-therapeutic and non-immunogenic stabilizers, and the like.
  • excipients examples include, but are not limited to cosolvents, surfactants, oils, humectants, emollients, preservatives, stabilizers, and antioxidants. Any physiologically acceptable buffer can be used, such as Tris or phosphate buffers. Effective amounts of diluents or additives or excipients are those that are effective to obtain a pharmaceutically acceptable formulation in terms of solubility and biological activity.
  • unit dose refers to physically discrete units suitable as unit doses for an individual, eg, mammal, human, dog, cat, rodent, etc. wherein each unit contains a predetermined amount of active material calculated to produce the appropriate therapeutic effect in association with the appropriate diluent, carrier or vehicle.
  • RNA extraction from cells was carried out with the ReliaPrep TM RNA Cell Miniprep System (Promega), following the manufacturer's instructions. This extraction system includes DNase I treatment and therefore RNA reverse transcription from cells does not include this step.
  • RNA concentration and salt contamination was measured with the NanoDrop spectrophotometer (ThermoFisher). The samples were stored at -80 ° C. 1 pg of total RNA was reverse transcribed using SuperScript III reverse transcriptase (Invitrogen) following the manufacturer's instructions. Genomic DNA contamination was monitored in the RNA samples without the addition of reverse transcriptase.
  • PCR Reactions Polymerase Chain Reaction
  • Probe qPCR Mix (SolisBiodyne) was used for quantification of SMN2 FL and D7 transcripts. The reaction was carried out in a multiplex fashion, in which the signal from the SMN2 probe and GAPDH were read in a single reaction. 10 ng of cDNA was used as a standard and the concentration of both probes was 250 nM.
  • the primer sets for the detection of the SMN2 FL or D7 isoforms (SEQ ID NO. 1 to 4) were used at a final concentration of 500 nM while the concentration of the primer set used for GAPDH was 150 nM. All reactions were carried out using a Step-One Real-Time PCR thermal cycler (Applied Biosystems). In all cases the experiment included three biological samples and three replicates for each sample.
  • PSI spliced-in percentages
  • Fibroblasts derived from SMA patients were cultured at 37 ° C in cell culture bottles with Dulbecco's Modified Eagle Medium (DMEM; Gibco) standard medium and 4.5 g / ml glucose. The medium was supplemented with penicillin (100 U / ml) and streptomycin (100 mg / ml) [1% W / S; Gibco] and 10% inactivated fetal bovine serum (FBS; Gibco).
  • DMEM Dulbecco's Modified Eagle Medium
  • FBS inactivated fetal bovine serum
  • HeLa cells were cultured under the same conditions except DMEM medium with 1 g / ml of glucose was used.
  • RNA extraction 400,000 GM03813 fibroblasts were seeded in 60 mm culture plates in 4 ml of supplemented DMEM medium. The following day the medium was changed and the candidate compounds were added at their maximum non-toxic concentration. The cells were incubated for 48 hours and then washed with 1x PBS and collected in sterile 1.5 ml tubes. The same was done for the evaluation of the effect of the compounds on the SMN protein level with the difference that 1,000,000 fibroblasts were used in a 100 mm cell culture plate and that the cells were harvested after 2 days with a RIPA protein extraction buffer supplemented with a protease inhibitor cocktail (cOmplete; Roche).
  • a protease inhibitor cocktail cOmplete
  • HeLa cells were seeded in a 400 ⁇ l 24-well plate with supplemented DMEM medium followed by medium exchange with treatment of the corresponding candidate compound. The cells were incubated for 72 h with the candidate compounds and fixed in 4% paraformaldehyde for 10 minutes. All cell cultures were incubated at 37 ° C with 5% CO2.
  • Myoblasts derived from SMA type II patients (KM1150SMAII7PV; Institut de Myologie, Paris) and control myoblasts (AB1 190; Institut de Myologie, Paris) were cultured at 37 ° C with 5% CO2 in cell culture bottles with medium.
  • KMEM composed of 4 vol of DMEM Glutamax 4.5 g (Gibco) + 1 vol of 199 medium (Gibco) supplemented with 20% inactivated fetal bovine serum (FBS; Gibco), fetuin (25 pg / mL; Life Technologies), hEGF (5 ng / mL; Life Technologies), bFGF (0.5 ng / mL; Life Technologies), insulin (5 g / mL; Sigma), dexamethasone (0.2 pg / mL; Sigma), penicillin (100 U / ml) and streptomycin (100 mg / ml) [1% W / S; Gibco]
  • 40,000 cells were seeded in a 24-well plate (previously covered with collagen) of 400 ⁇ l with KMEM medium and were incubated overnight. The following day the medium was exchanged for DMEM Glutamax 4.5 g differentiation medium (Gibco) supplemented with 2% horse serum (Sigma); penicillin (100 U / ml) and streptomycin (100 mg / ml) [1% P / S; Gibco], gentamicin (50 pg / mL; Sigma) and insulin (10 pg / mL; Sigma).
  • the medium was changed and the corresponding compound treatment was administered (50 pM moxifloxacin; 1 pM risdiplam).
  • the cells were incubated for 72 h with the compounds and fixed in 4% paraformaldehyde for 10 minutes. All cell cultures were incubated at 37 ° C with 5% CO2.
  • Example 1 Evaluation of moxifloxacin in SMA fibroblasts.
  • Moxifloxacin was evaluated in fibroblasts derived from patients with type II spinal muscular atrophy. The dose used in the study was determined experimentally in normal fibroblasts.
  • the toxicity test was carried out using CelITiter 96® Aqueous Non-Radioactive Cell Proliferation Assay (Promega). Fibroblasts of the GM03814 line were seeded in 96-well plates with 1.0 x 10 4 cells per well and incubated for 24 hours. Moxifloxacin was added at various concentrations in complete DMEM medium and the cells were incubated for 48 h. Cell viability was measured with the MTS tetrazolium salt that was added to each well and incubated for 4 h at 37 ° C in a chamber moistened with 5% CO2. The conversion of MTS in soluble form, by metabolically active cells, was measured by absorbance at 490 nm.
  • Fibroblasts derived from SMA type II patients were incubated with moxifloxacin at the maximum non-toxic concentration previously established for 72 h.
  • Chemiluminescence was detected using SuperSignal West Femto Maximum Sensitivity Substrate (Pierce) and recorded with ImageQuant LAS 4000 (GE Healthcare).
  • the selected SMA fibroblast line has 3 copies of the SMN2 gene and a homozygous deletion of Ex7-Ex8 in SMN1, and thus only transcripts of the SMN2 gene were measured.
  • Moxifloxacin increases the levels of SMN protein and changes the ratio of the SMN2FL and D7 (Delta7) isoforms, promoting the inclusion of exon 7 in the final transcript, as can be seen in Figure 3B.
  • the FL isoform which includes exon 7
  • the D7 isoform an isoform that excludes said exon, decreases.
  • Moxifloxacin showed the ability to modulate the result of SMN2 splicing, favoring the inclusion of Ex7. It was then checked whether the increase in the FL transcript of SMN2 was translated into stable and functional SMN protein. For this, HeLa cells were treated with 500 mM moxifloxacin for 72 h and the cells were doubly stained for SMN and Coilin. Coilin is a molecular component of the Cajal Bodies (CB) and is responsible for the formation of a bridge between SMN and CB that is only formed with complete and functional SMN protein.
  • CB Cajal Bodies
  • HeLa cells were seeded in 24-well plates in the presence of 400 pL of complete DMEM, with 40,000 cells per well, and incubated overnight at 37 ° C. The following day the cells were treated with DMSO (0.5%) and moxifloxacin at four different concentrations (500, 375, 250 and 50 mM; diluted with 0.5% DMSO). Cells were incubated for 72 h and fixed with 4% paraformaldehyde for 10 minutes. The membrane was permeabilized with 1x PBST (1x PBS, 0.3% Triton X-100) for 15 min with subsequent incubation in blocking buffer (1x PBST, 4% normal goat serum) for 1 hr at room temperature.
  • 1x PBST (1x PBS, 0.3% Triton X-100
  • SMN-deficient myoblasts have been reported to have impaired fusion abilities.
  • moxifloxacin for its ability to restore the ability of myoblasts to form multinucleated myotubes upon fusing.
  • SMA SMA type 2 patient-derived line
  • CON healthy control line
  • myoblasts were treated with 50 mM moxifloxacin for 72 h and the cells were stained for Desmin, a muscle cell specific protein. Nuclei were stained with DAPI.
  • the myoblasts were seeded in 24-well plates (previously treated with collagen) in the presence of 400 pL of KMEM, with 40,000 cells per well, and incubated overnight at 37 ° C. The following day the cells were administered differentiation medium, in which they were incubated for 3 days. The following day the cells were changed to medium, and they were treated with DMSO (0.5%), moxifloxacin at 50 mM or risdiplam at 1 mM (positive control) diluted with DMSO at 0.5%. Cells were incubated for 72 h and fixed with 4% paraformaldehyde for 10 minutes.
  • the membrane was permeabilized with 1x PBST (1x PBS, 0.3% Triton X-100) for 15 min with subsequent incubation in blocking buffer (1x PBST, 1% normal goat serum) for 1 hr at room temperature.
  • Anti-Desmin primary monoclonal antibody (mouse, Millipore) was diluted with blocking buffer (1:50) and incubated overnight at 4 ° C.
  • the cells were washed three times with PBS-T (5 min) and incubated for 1 hr at room temperature with biotin-conjugated anti-mouse secondary antibody diluted in blocking buffer (1: 200).
  • the fusion index was calculated for healthy cells treated with DMSO 0, 5% (CON 0.5%), cells from patients treated with 0.5% DMSO, cells from patients treated with risdiplam positive control (1 mM), and cells from patients treated with moxifloxacin (50 mM).

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Abstract

L'invention concerne de la moxifloxacine, ou un sel ou solvate pharmaceutiquement acceptable de celle-ci, ainsi qu'une composition pharmaceutique la comprenant, destinée à être utilisée dans la prévention et/ou le traitement de l'amyotrophie spinale (SMA).
PCT/ES2020/070311 2019-05-17 2020-05-14 Moxifloxacine destinée à être utilisée dans le traitement de l'amyotrophie spinale WO2020234496A1 (fr)

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Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
CHOI SUNGWOON ET AL.: "Optimization of a series of heterocycles as survival motor neuron gene transcription enhancers", BIOORGANIC & MEDICINAL CHEMISTRY LETTERS PERGAMON, vol. 27, no. 23, December 2017 (2017-12-01), Amsterdam, Nl, pages 5144 - 5148, XP085261314, ISSN: 0960-894X, DOI: 10.1016/j.bmcl. 2017.10.06 6 *
HAMMOND C J ET AL., ALZHEIMER'S & DEMENTIA: THE JOURNAL OF THE ALZHEIMER'SASSOCIATION, vol. 2, no. 3, 1 July 2006 (2006-07-01), New York, Ny, Us, pages S232 - S233, XP005548665, ISSN: 1552-5260, DOI: 10.1016/j.jalz. 2006.05.8 40 *
PATON D M: "Nusinersen: antisense oligonucleotide to increase SMN protein production in spinal muscular atrophy", DRUGS OF TODAY, vol. 53, no. 6, June 2017 (2017-06-01), pages 327 - 337, XP055761866, ISSN: 1699-3993, DOI: 10.1358/dot.2017.53.6.2652413 *
RIETZ ANNE ET AL.: "Discovery of a Small Molecule Probe that Post-Translationally Stabilizes the Survival Motor Neuron Protein for the Treatment of Spinal Muscular Atrophy", JOURNAL OF MEDICINAL CHEMISTRY, vol. 60, no. 11, 8 June 2017 (2017-06-08), pages 4594 - 4610, XP055761863, ISSN: 0022-2623, DOI: 10.1021/acs.jmedchem.6b01885 *

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