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    • 专利摘要:
    The invention relates to 20-hydroxyecdysone and its derivatives, for use in the treatment of myopathy, more particularly in the treatment of conditions resulting from an impairment of muscle function caused either by genetic myopathy or by acquired myopathy
    公开号:FR3065642A1
    申请号:FR1758071
    申请日:2017-08-31
    公开日:2018-11-02
    发明作者:Pierre Dilda；Rene Lafont；Mathilde Latil；Maria Serova；Onnik Agbulut；Stanislas Veillet
    申请人:Universite Pierre et Marie Curie Paris 6；Biophytis SA；
    IPC主号:
    专利说明:

    Holder (s): BIOPHYTIS Public limited company, UNIVERSITE PIERRE ET MARIE CURIE (PARIS 6) Public establishment.
    Extension request (s)
    Agent (s): IPSIDE.
    L4 / USE OF 20-HYDROXYECDYSONE AND ITS DERIVATIVES IN THE TREATMENT OF MYOPATHIES.
    (üç) The invention relates to 20-hydroxyecdysone and its derivatives, intended for use in the treatment of myopathy, more particularly in the treatment of conditions resulting from an impairment of muscle function caused either by genetic myopathy either by an acquired myopathy
    FR 3 065 642 - A1
    TECHNICAL FIELD OF THE INVENTION
    The present invention relates to the use of purified natural 20-hydroxyecdysone (20E) or synthetic derivatives for the treatment of myopathies and in particular muscular dystrophies of genetic origin.
    STATE OF THE ART
    Myopathies are diseases that directly affect the muscle. There are different forms depending on the manifestations and mechanisms of muscle damage. Two categories exist: genetic myopathies and acquired myopathies.
    Genetic myopathies are divided into:
    - progressive muscular dystrophies (Duchenne and Becker muscular dystrophies, muscular dystrophies of the belts and facioscapulo humeral dystrophy), or congenital dystrophies,
    - metabolic myopathies such as glycogenoses and lipidoses as well as mitochondrial myopathies,
    - myotonic dystrophies such as Steinert myopathy (DM1),
    - congenital central core, rod, multi mini core or centronuclear and myotubular myopathy,
    Acquired myopathies include:
    - toxic myopathies,
    - inflammatory myopathies,
    - endocrine myopathies.
    Myopathies are manifested by progressive or stable muscle weakness. They are generally characterized by a loss of muscle mass (atrophy). During the course of the disease, muscle tissue is gradually replaced by fibrous tissue (fibrosis).
    There are more than thirty forms of muscular dystrophies which differ in particular in the nature of the muscles affected. They appear more or less early and affect the skeletal muscles of different parts of the body. In some cases progressive damage to the respiratory and cardiac muscles reduces the life expectancy of patients.
    Several dozen different genes are involved in muscular dystrophies. Most often, these are genes responsible for the synthesis of proteins located in the membrane of muscle cells or linked to it and essential for the maintenance of architecture and muscle function. For exemple :
    - dystrophin is involved in Duchenne muscular dystrophy (DMD) and Becker muscular dystrophy (BMD),
    - calpain, dysferline, sarcoglycans are involved in muscular dystrophies of the belts,
    - merosine, alpha-dystroglycan or selenoprotein N are involved in the case of congenital muscular dystrophies (DMC).
    DMD is the most common muscular dystrophy. It affects 1 in 3,500 boys and results from mutations affecting the dystrophin gene, located on the X chromosome. A less severe form, BMD, also involves the dystrophin gene and affects 1 in 18,000 boys. DMD is a condition serious genetics that affects the entire musculature. In this disease, the fragility of muscle fibers leads to their destruction resulting in necrosis of muscle tissue. When regeneration mechanisms are exceeded, degeneration prevails, leading to a loss of muscle strength and intolerance to exercise (Barnabei et al. 2011). The muscle fibers are then replaced by connective tissue (fibrosis). Muscle weakness gradually affects the lower limbs of children over the age of 3. The disease subsequently develops in the back muscles, the upper limbs and finally the respiratory muscles.
    There is currently no curative treatment for DMD and BMD, but palliative, orthopedic and respiratory treatments improve quality of life and prognosis. The care of patients is currently based on optimizing their muscular capacities as well as on the prevention and treatment of cardiac and respiratory complications. The use of corticosteroids prolongs the walking period by two years on average. However, some children do not respond to this treatment, which is also responsible for side effects, including significant bone weakening. Partial cardiac protection is achieved through a combination of ACE inhibitors and beta blockers.
    New treatments are in the clinical development stage. The exon jump involves forcing the cell to produce a shorter version of the dystrophin than the normal but still functional protein. Another similar approach is to bypass a mutation that prematurely interrupts the synthesis of dystrophin. However, this type of therapy is only intended for a small number of patients, depending on the exact nature of the mutations that cause their disease. Finally, gene therapy, which offers the possibility of synthesizing short versions of dystrophin (mini-dystrophins or micro-dystrophins) in patients, is faced with a major problem of immune response against these proteins considered foreign by the body.
    In addition to an irreversible decrease in appendicular muscle strength and the appearance of exercise intolerance, one of the major complications of the disease is the installation of fibrosis which affects the heart in particular, leading to heart failure (accompanied by '' enlarged hypertrophy). These attacks are life-threatening for patients. The installation of fibrosis is therefore an irreversible development that must be counteracted in order to maintain muscle function. In this sense, therapeutic approaches must focus their efforts on:
    - maintenance of exercise tolerance
    - maintenance of muscle strength
    - and on the prevention of the onset of fibrosis.
    Phytoecdysones represent an important family of polyhydroxylated sterols. These molecules are produced by various species of plants (ferns, gymnosperms, angiosperms) and participate in the defense of these plants against pests.
    Patent FR 3 021 318 discloses that phytoecdysones, and more particularly 20-hydroxyecdysone (20E), have been the subject of numerous pharmacological studies. These studies have highlighted the anti-diabetic and anabolic properties of this molecule. Its stimulating effects on protein synthesis in muscles are observed in rats in vivo (Syrov et al., 2000; Tôth et al., 2008; Lawrence et al., 2012) and on murine C2C12 myotubes in vitro (GorelickFeldman and al., 2008). This is an effect at the level of translation, which involves the phosphorylation of the ribosomal protein p70S6K, at the end of a cascade in which the protein kinase Akt / PkB intervenes, a route also used by IGF- 1 to stimulate proteosynthesis.
    Some of the effects described above in animal models have been found in clinical studies, still few in number. Thus, 20hydroxyecdysone increases muscle mass in young athletes (Simakin et al., 1988).
    Finally, French patent FR3021318 describes the use of 20-hydroxyecdysone and its derivatives, for the treatment and prevention of sarcopenia and sarcopenic obesity (Lafont et al. 2017).
    OBJECT OF THE INVENTION
    The inventors have discovered that 20-hydroxyecdysone and its derivatives significantly improve physical performance in toto as well as muscle strength in situ of mammals affected by myopathy. In toto physical performance and in situ muscle strength are determined by the maximum distance traveled and the absolute isometric maximum strength of the anterior tibial muscle, respectively. These effects improve mobility in mammals suffering from myopathy and in particular muscular dystrophies.
    The present invention relates to 20-hydroxyecdysone and its derivatives intended for use in the treatment of myopathy.
    In the following description, the term 20-hydroxyecdysone and its derivatives means 20-hydroxyecdysone, its derivatives, plant extracts rich in 20hydroxyecdysone and its derivatives, and compositions comprising as active agent 20-hydroxyecdysone , its derivatives and / or plant extracts rich in 20 hydroxyxydysone and its derivatives.
    The 20-hydroxyecdysone derivatives are obtained by hemisynthesis.
    The 20-hydroxyecdysone and its derivatives are advantageously purified at the pharmaceutical grade.
    More particularly, the invention relates to 20-hydroxyecdysone and its derivatives, intended for use in the treatment of conditions resulting from an impairment of muscle function caused either by a genetic myopathy or by an acquired myopathy.
    Muscle function is that of the skeletal striated muscle or the myocardium.
    More specifically, impaired muscle function is an enlarged myocardium.
    Even more particularly the invention relates to 20-hydroxyecdysone and its derivatives, intended to be used in the treatment of any myopathy in which the muscular function is at least partly altered by the progressive installation of a fibrosis.
    In particular, the invention relates to 20-hydroxyecdysone and its derivatives, intended for use in the treatment of myopathy resulting from genetic damage.
    By genetic alteration is meant a mutation, an insertion of nucleotide (s) or a deletion of nucleotide (s).
    The invention relates to 20-hydroxyecdysone and its derivatives, intended for use in the treatment for example of Duchenne muscular dystrophy (DMD) and / or Becker muscular dystrophy (BMD).
    According to the invention, 20-hydroxyecdysone and its derivatives are intended for use in the treatment of any myopathy resulting from a mutation in the dystrophin gene.
    According to one characteristic, 20-hydroxyecdysone is a compound of formula (I):
    OH
    Advantageously, the compound of formula (I) is purified to the pharmaceutical grade.
    In particular, 20-hydroxyecdysone is an extract from a plant or part of a plant, said plant being chosen from plants containing at least 0.5% of 20-hydroxyecdysone of formula (I) by dry weight of said plant, said extract comprising at least 95%, and preferably at least 97%, of 20-hydroxyecdysone of formula (I).
    We are talking about purification at the pharmaceutical grade.
    The extract is subsequently called BIO101. It remarkably comprises between 0 and 0.05%, by dry weight of the extract, of impurities, such as minor compounds, capable of affecting the safety, availability or effectiveness of a pharmaceutical application of said extract.
    According to a characteristic of the invention, the impurities are compounds with 19 or 21 carbon atoms, such as Rubrosterone, Dihydrorubrosterone or Poststerone.
    The plant from which BIO101 is produced is advantageously chosen from Stemmacantha carthamoides (also called Leuzea carthamoides), Cyanotis arachnoidea and Cyanotis vaga.
    The invention is particularly interested in the use of an extract of roots of Stemmacantha carthamoides comprising at least 95%, and preferably at least 97%, of 20-hydroxyecdysone of formula (I).
    In this extract, 20-hydroxyecdysone is purified to pharmaceutical grade.
    More particularly, the extract is administered at a rate of 3 to 15 mg / kg * day.
    In particular, the extract is administered at a rate of 200 to 1000 mg / day, in one or more doses, in an adult patient, and a dose of 70 to 350 mg / day, in one or more doses, in children .
    In addition, the invention relates to a composition comprising, as active agent BIO101.
    The composition preferably contains between 200 and 1000 mg of active agent (BIO101).
    According to another characteristic, a derivative of 20-hydroxyecdysone is a compound of general formula (II):
    in which :
    V-U is a carbon-to-carbon single bond and Y is a hydroxyl group or a hydrogen, or V-U is an ethylene bond C = C;
    X is oxygen,
    Q is a carbonyl group;
    R 1 is chosen from: a group (CrC6) W (CrC6); a group (Cr C6) W (Ci-C6) W (Ci-C 6 ); a group (Ci-C6) W (Ci-C6) CO2 (Ci-C 6 ); a group (CrC 6 ) A, A representing a heterocycle optionally substituted by a group of type OH, OMe, (Ο-ι-Οθ), Ν (Οι-Οθ), 002 (0ι-0β); a CH 2 Br group;
    W being a heteroatom chosen from N, O and S, preferably O and even more preferably S.
    More particularly, in formula (II):
    Y is a hydroxyl group;
    R 1 is chosen from: a group (Ci-C6) W (Ci-C 6 ); a group (C r C6) W (Ci-C6) W (Ci-C 6 ); a group (Ci-C6) W (Ci-C6) CO2 (Ci-C 6 ); a group (Οι-Οθ) Α, A representing a heterocycle optionally substituted by a group of type OH, OMe, (Ο-ι-Οθ), Ν (Οι-Οθ), 002 (0ι-0β);
    W being a heteroatom chosen from N, O and S, preferably O and more preferably S.
    In particular, the compounds of formula (II) are chosen from:
    - No. 1: (2S, 3R, 5R, 10R, 13R, 14S, 17S) -2,3,14-trihydroxy-10,13-dimethyl-17 (2-morpholinoacetyl) -2,3,4,5, 9,11,12,15,16,17-decahydro-1 Hcyclopenta [a] phenanthren-6-one,
    - n ° 2: (2S, 3R, 5R, 10R, 13R, 14S, 17S) -2,3,14-trihydroxy-17- [2- (3hydroxypyrrolidin-1 -yl) acetyl] -10,13-dimethyl- 2,3,4,5,9,11,12,15,16,17decahydro-1 H-cyclopenta [a] phenanthren-6-one;
    - n ° 3: (2S, 3R, 5R, 10R, 13R, 14S, 17S) -2,3,14-trihydroxy-17- [2- (4-hydroxy-1 piperidyl) acetyl] -10,13-dimethyl -2,3,4,5,9,11,12,15,16,17-decahydro-1 Hcyclopenta [a] phenanthren-6-one;
    - n ° 4: (2S, 3R, 5R, 10R, 13R, 14S, 17S) -2,3,14-trihydroxy-17- [2- [4- (2hydroxyethyl) -1 -piperidyl] acetyl] -10, 13-dimethyl-2,3,4,5,9,11,12,15,16,17decahydro-1 H-cyclopenta [a] phenanthren-6-one;
    - No. 5: (2S, 3R, 5R, 10R, 13R, 14S, 17S) -17- [2- (3dimethylaminopropyl (methyl) amino) acetyl] -2,3,14-trihydroxy-10,13dimethyl-2, 3,4,5,9,11,12,15,16,17-decahydro-1 Hcyclopenta [a] phenanthren-6-one;
    - n ° 6: 2- [2-oxo-2 - [(2S, 3R, 5R, 10R, 13R, 14S, 17S) -2,3,14-trihydroxy-10,13dimethyl-6-oxo-2,3 , 4,5,9,11,12,15,16,17-decahydro-1 Hcyclopenta [a] phenanthren-17-yl] ethyl] ethyl sulfanylacetate;
    - n ° 7: (2S, 3R, 5R, 10R, 13R, 14S, 17S) -17- (2-ethylsulfanylacetyl) -2,3,14 trihydroxy-10,13-dimethyl-2,3,4,5,9 , 11,12,15,16,17-decahydro-1 Hcyclopenta [a] phenanthren-6-one;
    - No. 8: (2S, 3R, 5R, 10R, 13R, 14S, 17S) -2,3,14-trihydroxy-17- [2- (2hydroxyethylsulfanyl) acetyl] -10,13-dimethyl-2,3, 4,5,9,11,12,15,16,17 decahydro-1 H-cyclopenta [a] phenanthren-6-one.
    In particular, phytoecdysone is a compound of formula (III):
    (III)
    This compound is subsequently called BIO103.
    BRIEF DESCRIPTION OF THE FIGURES
    Other advantages, aims and particular characteristics of the present invention will emerge from the following non-limiting description of at least one particular embodiment of the devices which are the subject of the present invention, with reference to the appended drawings, in which:
    FIG. 1A is a representative diagram of the exercise tolerance of groups of mice of genetic background C57BL10: healthy (C57) and mdx (mutated on the dystrophin gene) untreated,
    FIG. 1B is a representative diagram of the exercise tolerance of groups of mice: mdx not treated, mdx treated with BIO101 and mdx treated with BIO103 after two months of treatment,
    FIG. 2A is a diagram representing the absolute maximum isometric force of the anterior tibial muscle of groups of mice of genetic background C57BL10: healthy (C57) and untreated mdx,
    FIG. 2B is a diagram representing the absolute maximum isometric force of the anterior tibial muscle of the groups of mice: mdx not treated, mdx treated with BIO101 and mdx treated with BIO103 after two months of treatment,
    - Figures 3A and 3B are diagrams representative of the gene expression (mRNA) of the fibrosis markers CTGF (connective tissue growth factor) and Collai (collagen 1) of the heart of different groups of mice with genetic background C57BL10: healthy (C57), mdx not treated, mdx treated with BIO101 and mdx treated with BIO103, after two months of treatment,
    FIGS. 3C and 3D are diagrams representative of the gene expression (mRNA) of the hypertrophy markers myh7 (beta heavy chain of myosin) and BMP4 (bone morphogenetic protein 4) of the heart of different groups of mice of genetic background C57BL10: healthy (C57), mdx not treated, mdx treated with BIO101 and mdx treated with BIO103, after two months of treatment,
    FIG. 4A is a representative image of a histological section of the anterior tibial muscle of mice of healthy C57BL10 genetic background (C57), stained with hematoxylin eosin (HE),
    FIG. 4B is a representative image of a histological section of the anterior tibial muscle of mice of C57BL10 mdx untreated genetic background (mdx), stained with hematoxylin eosin (HE),
    FIG. 4C is a representative image of a histological section of the anterior tibial muscle of mice of genetic background C57BL10 mdx mdx treated with BIO101 (mdx + BIO101), stained with hematoxylin eosin (HE)
    FIG. 4D is a representative image of a histological section of the anterior tibial muscle of mice of genetic background C57BL10 mdx treated with BIO103 (mdx + BIO103), stained with hematoxylin eosin (HE),
    FIGS. 5A and 5B are histological sections representative of anterior tibial muscle stained with sirius red (RS), respectively, groups of mice of healthy background C57BL10 (C57) and untreated mdx (mdx),
    FIG. 5C is a representative diagram of the quantification of the fibrosis zones of groups of healthy C57BL10 genetic background mice (C57) and untreated mdx (mdx),
    FIGS. 5D and 5E are histological sections representative of anterior tibial muscle stained with sirius red (RS) of the groups of mdx mice treated with BIO101 (mdx BIO101) and mdx treated with BIO103 (mdx BIO103),
    FIG. 5F is a representative diagram of the quantification of the zones of fibrosis of the groups of mdx mice treated with BIO101 (mdx BIO101) and mdx treated with BIO103 (mdx BIO103),
    FIG. 6A shows the protein expression, detected by western blot, of the marker for collagen fibrosis 1 (Collai) of the gastrocnemius muscle of the groups of mouse of genetic background C57BL10: healthy (C57), untreated mdx (mdx),
    FIG. 6B presents the protein expression, detected by western blot, of the same marker of fibrosis of the gastrocnemius muscle of the mice groups of genetic background C57BL10: untreated mdx (mdx), mdx treated with BIO101 and mdx treated with BIO103, after two months of treatment.
    DETAILED DESCRIPTION OF EMBODIMENTS OF
    THE INVENTION
    This description is given without limitation.
    1. Purification process of BIO1Q1
    BIO101 is prepared from a preparation of pure 20-hydroxyecdysone in the order of 90%, according to the following steps:
    a) Hot dissolution of pure 20-hydroxyecdysone in the order of 90% in methanol, filtration and partial concentration,
    b) Addition of 3 volumes of acetone,
    c) Cooling to a temperature between 0 and 5 ° C, with stirring,
    d) Filtration of the precipitate obtained,
    e) successive rinses with acetone and water, and
    f) Drying.
    This purification involves a recrystallization process suitable for this molecule and capable of being carried out on an industrial scale.
    The filtration in step a) is carried out using a 0.2 μm particle filter.
    The partial concentration of step a) is advantageously carried out by vacuum distillation, at a temperature of the order of 50 ° C., in the presence of MeOH. The drying step f) is carried out under vacuum at a temperature of the order of
    50 ° C.
    2. BIQ103 synthesis process
    BIO103 is obtained by hemi-synthesis from 20hydroxyecdysone then purification at the pharmaceutical grade according to the following preparation process:
    BIO103
    BIO103 synthesis scheme in 3 steps:
    1) oxidative cleavage of the side chain of 20-hydroxyecdysone between carbons C20 and C22 to obtain the poststerone (protocol known to those skilled in the art),
    2) introduction of a bromine atom in position C21,
    3) reaction of the brominated derivative with ethane-thiol.
    3. Biological activity of BIO1Q1 and BIQ103
    If the anabolic effects of 20E present in commercial preparations have already been demonstrated in young animals, we do not know the effect of 20E in a context of mammals suffering from myopathy in which the progressive installation of a fibrosis contributes impaired muscle function. The animal model of Duchenne muscular dystrophy using mdx mice, of genetic background C57BL10, which have a mutation on the dystrophin gene, has been implemented (Bulfield et al. 1984; Sicinski et al. 1989).
    Twelve-week-old male mice C57BL10 (wild mice named "C57" in the figures) and C57BL10 mdx (murine model of Duchenne muscular dystrophy, called "mdx" in the figures), produced at Charles Rivers, were used. Two groups of mdx mice were chronically exposed orally to either BIO101 (number of mice n = 9) or to BIO103 (number of mice n = 9) at a dose of 50 mg / kg * day. A group of C57 mice (number of mice n = 5) and a group of mdx mice (number of mice n = 15) received the vehicle, ie no treatment. The animals of all groups were tested for their functional capacity (exercise tolerance test) after two months of treatment. The effort tolerance test consists of measuring the maximum distance traveled (activity in toto; Figures 1A and 1B). In addition, measurements of the absolute maximum isometric strength of the anterior tibial muscle (in situ activity; Figures 2A and 2B) were made after two months of treatment.
    The two-month oral treatment consists of force-feeding for five days a week and in drinking water two days a week.
    Exercise tolerance test (functional study in toto: Figures 1A and 1B)
    The exercise tolerance test is a forced exercise on a motorized treadmill. It is a non-invasive method to assess the function of skeletal muscle in toto. It is the benchmark method in the field (Ferry et al. 1992, 1993; HadjSaid et al. 2012).
    The acclimatization period for animals is at least 48 hours before the session during which the maximum running distance is measured. During the test, the mouse is placed in a running lane on a motorized treadmill which allows you to do an exercise controlled by the experimenter in its intensity and duration. The treadmill ends with a horizontal grid which administers an electric shock (0.4 mA) if the animal is in contact for more than a second with the grid.
    The running session begins with a 2 minute warm-up period during which the speed is brought from 0 to 20 cm / s. Thereafter, the running speed is increased by 5 cm / s every 10 minutes until the limit of the maximum capacities of the mouse. When it undergoes 5 shocks in less than 10 seconds, the test stops. The distance traveled is noted.
    As expected, it is found that animals which have a mutation on the dystrophin gene (mdx) run significantly less (75.4%, p <0.001, Mann Whitney test) than healthy animals (C57) (Figure 1A ). After two months of daily exposure (Figure 1B), mdx animals who received BIO101 or BIO103 run significantly more than mdx animals who received the vehicle. BIO101 and BIO103 significantly improve (respectively p <0.001 and p <0.05, unpaired t-test) the distance covered by + 136% and + 67% respectively compared to that of untreated mdx animals (vehicle). Importantly, we demonstrate here that the treatment of mdx animals with BIO101 or BIO103 partially compensates for the significant functional loss observed in animals mutated on the dystrophin gene (mdx). The overall physical performance (total activity) of animals suffering from myopathy is very markedly improved by BIO101 (2.4 times) and BIO103 (1.7 times).
    This study demonstrates that treatment with BIO101 or BIO103 results in a functional improvement characterized by a significant improvement in exercise tolerance in an animal model of myopathy (Figures 1A and 1B).
    Absolute maximum isometric force of the anterior tibial muscle (in situ functional study: Figure 2)
    An evaluation of the contractility in situ of the anterior tibial muscle is carried out at the end of the protocol, ie after two months of treatment.
    On the day of the sacrifice, the mouse is anesthetized with an intraperitoneal injection of pentobarbital (55 mg / kg, 0.1 ml / 10 g of body weight) before the in situ strength measurement of the anterior tibial muscle (AT). The skin on the top of the leg is incised, revealing the tendon which is cut at its distal end. The distal tendon of the TA is attached to the actuator lever (305B DualMode Lever, Aurora Scientific). The skin on the lateral side of the thigh is incised, which reveals the sciatic nerve, between 2 muscle groups. The sciatic nerve is stimulated with a bipolar electrode (supra-maximal square wave of 10V, 0.1 ms). The force is measured during contractions in response to electrical stimulation (frequency of 75-150 Hz, duration of 500 ms). The mouse temperature is maintained at 37 ° C using a radiant lamp. The absolute isometric maximum tetanus force is measured.
    As expected, it is found that the mdx animals have a significantly lower absolute isometric maximum contraction force (p <0.05, unpaired t-test) than that of healthy animals C57 (Figure 2A). After two months of daily treatment, a significant increase in the maximum isometric absolute strength of the anterior tibial muscle is observed in mdx animals treated with BIO101 (+ 15.3%, p <0.05, unpaired t-test) and with BIO103 (+ 22.5%, p <0.001, unpaired t-test) at a dose of 50 mg / kg * day compared to the mdx animals that received the vehicle (Figure 2B).
    This study demonstrates that treatment with BIO101 or BIO103 results in a functional improvement characterized by a significant increase in the absolute maximum isometric strength of the anterior tibial muscle in an animal model of myopathy (Figures 2A and 2B).
    Myocardial fibrosis and cardiac hypertrophy (molecular study: figures
    3A, 3B, 3C, 3D)
    An assessment of markers of fibrosis and cardiac hypertyrophy is carried out at the end of the protocol, ie after two months of treatment. On the day of sacrifice, the mice are euthanized by decapitation and the heart is removed and immediately frozen in liquid nitrogen. Total RNA is extracted with TRIzol® lysis
    Reagent (Life technologies) and a tissue homogenizer (Bio-Gen PRO200). The extracted RNAs are quantified by spectrophotometry then their quality is verified and validated by the Experion RNA StdSens Analysis Kit (Bio-Rad). The cDNA is then synthesized with the RevertAid First Strand cDNA Synthesis Kit (Thermo Fisher Scientific). Finally, the semi-quantitative PCR analysis was performed using SYBR® Green (Roche), a DNA interlayer and a LightCycler® 480 Real Time PCR device (Roche) on 384-well plates. The molecular markers measured are as follows:
    - CTGF and collagen I: the connective tissue growth factor (CTGF) is associated with scarring and many pathological fibrotic processes including DMD (Brigstock 2010; Song Y. 2017). An increase in CTGF gene expression is associated with cardiac fibrosis in mdx mice (Au 2011). CTGF stimulates the synthesis of collagen (including collagen I) in cardiac fibroblasts and contributes to myocardial fibrosis (Wang 2010),
    - myh7: an increase in the expression of mRNA of the beta myosin heavy chain (myh7) is linked to an enlarged heart. This embryonic isoform is expressed in the myocardium of adults in response to the establishment of heart failure (Yin et al. 2014). On the other hand, an increase in the expression of myh7 is notably demonstrated in DMD (Murphy et al. 2016) and in arrhythmogenic cardiomyopathy which are both characterized by a loss of cardiomyocyte and the installation of myocardial fibrosis. (Gerçek 2017).
    - BMP4: the bone morphogenetic protein 4 (BMP4) is an important repressor of myogenic differentiation. It interferes with the muscle regeneration process in DMD (Shi 2011).
    Analysis of molecular markers linked to the installation of cardiac fibrosis in DMD confirms a significant increase in the gene expression of CTGF and collagen I (Collai) in mdx animals compared to healthy animals (respectively p <0.001 and p <0.05, unpaired t-test, Figure 3A). The two-month treatment with BIO101 at a dose of 50 mg / kg * day prevents (p = 0.0506, unpaired t-test) gene expression of CTGF in mdx animals treated with BIO101 compared to mdx control animals , having received the vehicle (Figure 3B).
    Treatment of mdx animals with BIO103 at the same dose tends to prevent Collai gene expression compared to mdx control animals (Figure 3B).
    The myh7 gene expression is significantly increased (p <0.05, unpaired t-test) in mdx animals compared to wild animals C57 (Figure 3C). Treatment of mdx animals with BIO101 and BIO103 at a dose of 50 mg / kg * day significantly reduced the expression of myh7 (p <0.01, unpaired t-test) compared to mdx animals that received the vehicle (Figure 3D). Furthermore, BIO101 reduced the gene expression of BMP4 (p = 0.0529, unpaired t-test) compared to the mdx control animals, which received the vehicle (Figure 3D).
    Skeletal muscle fibrosis (histological study: Figures 4 and 5)
    An evaluation of muscular fibrosis by histological study is carried out on the anterior tibial muscle (AT). Histological sections (7pm) are made and stained either with hematoxylin eosin (HE) or with sirius red (RS).
    An anatomopathological study of the sections of anterior tibial muscle stained by HE makes it possible to assess the level of affection of the muscle fibers. Representative images of HE stained anterior tibial muscle sections from animals C57 (Figure 4A), mdx (Figure 4B), mdx treated with BIO101 (Figure 4C) and mdx treated with BIO103 (Figure 4D) are presented. The scale bars correspond to 200pm. The TA muscles of C57 mice show no muscle damage (Figure 4A). The LDs of untreated mdx mice show a wide variety of lesional profiles: moderate anisocytosis with numerous necrotic fibers. Certain muscles, for their part, have a marked to severe, multifocal anisocytosis, with atrophy of a large part of the myocytes, as well as large chronic inflammatory foci associating fibrosis (FIG. 4B) and infiltrates of mononuclear cells (mainly macrophages ). The sign Δ indicates the inflammatory foci with which fibrosis is associated. Finally, some muscles have large areas of acute necrosis. The muscles of the mice treated with BIO101 exclusively exhibit two types of lesion profile: a slight lesion profile with little anisocytosis, necrosis or inflammation (37.5%, 3 of 8 of the BP) and a marked lesion profile with a anisocytosis, dispersed necrotic fibers, and variable inflammation (62.5%, 5 of 8 BP) (Figure 4C). Interestingly, the muscles of mice treated with BIO103 show fewer muscle lesions with a slight lesion profile.
    In fact, 67% (6 out of 9 LDs) of the muscles treated with BIO103 can be classified in this category. From a histological point of view, the vast majority of myocytes have central nuclei (witnesses of necrosis-regeneration phenomena), a slight to slight variation in size (anisocytosis), rare necrotic and hyper-contracted myocytes (initial phase of necrosis; black arrows, Figure 4D), and rare inflammatory cells in the endomysium. The other muscles treated with BIO103 have either a marked lesion profile with a rather necrotic tendency (11% of the TA, 1 in 9 of the TA) with anisocytosis, dispersed necrotic fibers, and acute necrosis or a marked lesion profile tending towards atrophy with strong anisocytosis, without necrotic fiber (11%, 1 in 9 of LD). Finally, one of the TA presents a severe lesion profile with large areas of acute necrosis without regeneration (11%, 1 in 9 of the TA).
    An anatomopathological analysis of the sections of anterior tibial muscle stained by RS makes it possible to quantify the area of fibrosis. The RS staining makes it possible to visualize the zones of fibrosis on the histological sections (Rittié 2017). Representative images of the anterior tibial muscle sections stained with RS from animals C57 (Figure 5A), mdx (Figure 5B), mdx treated with BIO101 (Figure 5D) and C57 mdx treated with BIO103 (Figure 5E) are presented. The scale bar represents 200pm. The percentage of surface area corresponding to fibrosis is significantly increased (p <0.001, Mann Withney test) in the mdx group compared to the C57 group (Figure 5C). The black arrows indicate the areas with strong muscular fibrosis. Treatment of mdx animals with BIO103 clearly tends to prevent (p = 0.0709, unpaired t-test) the appearance of fibrosis compared to the mdx control group, which received the vehicle (Figure 5F).
    Skeletal muscle fibrosis (biochemical study: Figure 6)
    In addition to the observations made on the anterior tibial muscle, an evaluation of a muscle fibrosis marker is performed on the gastrocnemius muscle. The muscle is dissociated with moderate agitation for 16 h at 4 ° C. in RIPA lysis buffer. The proteins are assayed, resolved into SDS PAGE gel before being transferred to a PVDF membrane. Collagen I is detected by western blot, revealed by chemiluminescence and then quantified by densitometry after normalization with respect to the detection of GAPDH. As expected, the protein expression of collagen I is significantly increased (p <0.01; unpaired t-test) in the gastrocnemius muscles of mdx mice compared to the muscles of healthy mice (C57). The protein expression of collagen I is increased by 27 times (Figure 6A). Treatment with BIO103 of mdx mice makes it possible to observe a significant reduction (p <0.01; unpaired t-test) in the protein expression of collagen I compared to untreated mdx mice (vehicle) (FIG. 6B).
    Conclusion
    Given the properties of BIO101 and BIO103 on muscle function and on the establishment of myocardial and skeletal muscle fibrosis in mammals affected by myopathy, the use of BIO101 and BIO103 can therefore be proposed, alone or in combination. complement of a treatment aiming at correcting the effects of a gene alteration, to preserve the muscular function, in particular the muscular force and the tolerance to the effort and thus to slow down the evolution of myopathies which result in the deterioration of said function muscle, and more particularly the installation of fibrosis. These myopathies include, acquired and genetic myopathies, including Duchenne muscular dystrophy.
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    权利要求:
    Claims (17)
    [1" id="c-fr-0001]
    1. 20-hydroxyecdysone and its derivatives, intended for use in the treatment of myopathy.
    [2" id="c-fr-0002]
    2. 20-hydroxyecdysone and its derivatives, intended for use in the treatment of conditions resulting from an impairment of muscle function caused either by genetic myopathy or by acquired myopathy.
    [3" id="c-fr-0003]
    3. 20-Hydroxyecdysone and its derivatives intended to be used according to the preceding claim in which the muscular function is that of the skeletal striated muscle or of the myocardium.
    [4" id="c-fr-0004]
    4. 20-Hydroxyecdysone and its derivatives intended to be used according to the preceding claim in which the alteration of the muscular function is an enlarged myocardium.
    [5" id="c-fr-0005]
    5. 20-hydroxyecdysone and its derivatives intended to be used according to any one of claims 2 to 4, characterized in that the muscular function is at least partly altered by the progressive installation of a fibrosis.
    [6" id="c-fr-0006]
    6. 20-hydroxyecdysone and its derivatives, intended for use in the treatment of myopathy resulting from genetic damage.
    [7" id="c-fr-0007]
    7. 20-hydroxyecdysone and its derivatives, for use in the treatment of myopathy resulting from a mutation in the dystrophin gene.
    [8" id="c-fr-0008]
    8. 20-hydroxyecdysone and its derivatives, intended for use in the treatment of Duchenne muscular dystrophy (DMD) and / or Becker muscular dystrophy (BMD).
    [9" id="c-fr-0009]
    9. 20-hydroxyecdysone and its derivatives for use according to any one of the preceding claims, characterized in that 20hydroxyecdysone is a compound of formula (I):
    OH
    [10" id="c-fr-0010]
    10. 20-hydroxyecdysone and its derivatives for use according to the preceding claim, characterized in that the 20-hydroxyecdysone is an extract of a plant or part of a plant, said plant being chosen from plants containing at least 0.5 % of 20-hydroxyecdysone of formula (I), by dry weight of said plant, said extract comprising at least 95%, and preferably at least 97%, of 20-hydroxyecdysone of formula (I).
    [11" id="c-fr-0011]
    11.20- hydroxyecdysone and its derivatives for use according to the preceding claim, characterized in that the extract comprises between 0 and 0.05% by dry weight of the extract of impurities liable to affect the safety, availability or the effectiveness of a pharmaceutical application of said extract.
    [12" id="c-fr-0012]
    12.20- hydroxyecdysone and its derivatives for use according to any one of claims 5 to 6, characterized in that the plant is chosen from Stemmacantha carthamoides, Cyanotis arachnoidea and Cyanotis vaga.
    [13" id="c-fr-0013]
    13.20- hydroxyecdysone and its derivatives for use according to any one of claims 5 to 7, characterized in that the 20-hydroxyecdysone is an extract from the roots of Stemmacantha carthamoides comprising at least 95%, and preferably at least 97%, of 20-hydroxyecdysone of formula (I).
    [14" id="c-fr-0014]
    14.20-hydroxyecdysone and its derivatives for use according to any one of claims 1 to 3, characterized in that a derivative is a compound of general formula (II):
    in which :
    V-U is a carbon-to-carbon single bond and Y is a hydroxyl group or a hydrogen, or V-U is an ethylene bond C = C;
    X is oxygen,
    Q is a carbonyl group;
    R 1 is chosen from: a group (CrC6) W (CrC6); a group (Cr C6) W (Ci-C6) W (Ci-C 6 ); a group (Ci-C6) W (Ci-C6) CO2 (Ci-C 6 ); a group (CrC 6 ) A, A representing a heterocycle optionally substituted by a group of OH, OMe, (Ο-ι-Οθ), Ν (Οι-Οθ), CO2 (CiC 6 ) type; a CH 2 Br group;
    W being a heteroatom chosen from N, O and S.
    [15" id="c-fr-0015]
    15.20-hydroxyecdysone and its derivatives for use according to the preceding claim, characterized in that in formula (II):
    Y is a hydroxyl group;
    R 1 is chosen from: a group (Ci-C6) W (Ci-C 6 ); a group (C r C 6 ) W (Ci-C 6 ) W (Ci-C 6 ); a group (CrC6) W (Ci-C6) CO2 (Ci-C6); a group (CrCejA, A representing a heterocycle optionally substituted by a group of OH, OMe, (Ci-C 6 ), N (CrC 6 ), CO 2 (CiC 6 ) type.
    W being a heteroatom chosen from N, O and S.
    [16" id="c-fr-0016]
    16.20- hydroxyecdysone and its derivatives for use according to any one of claims 10 to 11, characterized in that they are chosen from:
    - n ° 1: (2S, 3R, 5R, 10R, 13R, 14S, 17S) -2,3,14-trihydroxy-10,13-dimethyl-17- (2morpholinoacétyl) -2,3,4,5,9 , 11,12,15,16,17-decahydro-1 Hcyclopenta [a] phenanthren-6-one;
    - n ° 2: (2S, 3R, 5R, 10R, 13R, 14S, 17S) -2,3,14-trihydroxy-17- [2- (3hydroxypyrrolidin-1 -yl) acetyl] -10,13-dimethyl- 2,3,4,5,9,11,12,15,16,17decahydro-1 H-cyclopenta [a] phenanthren-6-one;
    - n ° 3: (2S, 3R, 5R, 10R, 13R, 14S, 17S) -2,3,14-trihydroxy-17- [2- (4-hydroxy-1 piperidyl) acetyl] -10,13-dimethyl -2,3,4,5,9,11,12,15,16,17-decahydro-1 Hcyclopenta [a] phenanthren-6-one;
    - n ° 4: (2S, 3R, 5R, 10R, 13R, 14S, 17S) -2,3,14-trihydroxy-17- [2- [4- (2hydroxyethyl) -1 -piperidyl] acetyl] -10, 13-dimethyl-2,3,4,5,9,11,12,15,16,17decahydro-1 H-cyclopenta [a] phenanthren-6-one;
    - No. 5: (2S, 3R, 5R, 10R, 13R, 14S, 17S) -17- [2- (3dimethylaminopropyl (methyl) amino) acetyl] -2,3,14-trihydroxy-10,13-dimethyl2, 3,4,5,9,11,12,15,16,17-decahydro-1 H-cyclopenta [a] phenanthren-6-one;
    - n ° 6: 2- [2-oxo-2 - [(2S, 3R, 5R, 10R, 13R, 14S, 17S) -2,3,14-trihydroxy-10,13dimethyl-6-oxo-2,3 , 4,5,9,11,12,15,16,17-decahydro-1 Hcyclopenta [a] phenanthren-17-yl] ethyl] ethyl sulfanylacetate;
    - n ° 7: (2S, 3R, 5R, 10R, 13R, 14S, 17S) -17- (2-ethylsulfanylacetyl) -2,3,14 trihydroxy-10,13-dimethyl-2,3,4,5,9 , 11,12,15,16,17-decahydro-1 Hcyclopenta [a] phenanthren-6-one;
    - No. 8: (2S, 3R, 5R, 10R, 13R, 14S, 17S) -2,3,14-trihydroxy-17- [2- (2hydroxyethylsulfanyl) acetyl] -10,13-dimethyl-2,3, 4,5,9,11,12,15,16,17 decahydro-1 H-cyclopenta [a] phenanthren-6-one.
    [17" id="c-fr-0017]
    17.20- hydroxyecdysone and its derivatives for use according to any one of claims 10 to 12, characterized in that the derivative is of formula (III):
    (III)
    1/5
    Maximum force Distance traveled (m) absolute isometric (g)
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    同族专利:
    公开号 | 公开日
    CN110753547A|2020-02-04|
    WO2018197708A1|2018-11-01|
    US20200148718A1|2020-05-14|
    BR112019022392A2|2020-05-19|
    JP2020517697A|2020-06-18|
    EP3615035A1|2020-03-04|
    FR3065644B1|2020-02-21|
    PT3615035T|2021-08-20|
    FR3065644A1|2018-11-02|
    CN110769832A|2020-02-07|
    EP3615036A1|2020-03-04|
    KR20200005574A|2020-01-15|
    BR112019022379A2|2020-05-19|
    JP2020517712A|2020-06-18|
    PL3615035T3|2021-12-20|
    EP3615035B1|2021-06-09|
    FR3065642B1|2020-02-28|
    ES2882571T3|2021-12-02|
    US20200179407A1|2020-06-11|
    KR20200012871A|2020-02-05|
    WO2018197731A1|2018-11-01|
    引用文献:
    公开号 | 申请日 | 公开日 | 申请人 | 专利标题
    FR3021318A1|2014-05-20|2015-11-27|Inst Biophytis|20-HYDROXYECDYSONE-DERIVED PRODUCTS AND THEIR USE IN THE PREPARATION OF MEDICAMENTS|
    CZ309080B6|2018-12-19|2022-01-19|Karel SLÁMA|Method of determining the risk of malignant tumours|
    FR3093640B1|2019-03-15|2021-10-01|Biophytis|Phytoecdysones and their derivatives for their use in the treatment of neuromuscular diseases|
    FR3093641A1|2019-03-15|2020-09-18|Biophytis|Phytoecdysones and their derivatives for use in the treatment of impaired respiratory function|
    FR3108504A1|2020-03-30|2021-10-01|Biophytis|Phytoecdysones and their derivatives for use in the treatment of impaired respiratory function during viral infection|
    法律状态:
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    2020-03-13| TQ| Partial transmission of property|Owner name: BIOPHYTIS, FR Effective date: 20200131 Owner name: SORBONNE UNIVERSITE, FR Effective date: 20200131 |
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    FR1753775A|FR3065644B1|2017-04-28|2017-04-28|PHARMACEUTICAL QUALITY 20-HYDROXYECDYSONE EXTRACT, ITS USE AND ITS PREPARATION|
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    BR112019022379A| BR112019022379A2|2017-04-28|2018-04-27|20-hydroxyecdysone and its derivatives|
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    PCT/EP2018/060975| WO2018197708A1|2017-04-28|2018-04-27|Use of 20-hydroxyecdysone and the derivatives thereof in the treatment of myopathies|
    EP18719583.9A| EP3615035B1|2017-04-28|2018-04-27|Use of 20-hydroxyecdysone and the derivatives thereof in the treatment of myopathies|
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    JP2019558383A| JP2020517697A|2017-04-28|2018-04-30|Pharmaceutical grade 20-hydroxyecdysone extract, its use and its preparation|
    BR112019022392A| BR112019022392A2|2017-04-28|2018-04-30|method for preparing an extract, extract of a plant and composition|
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