EP3442553A1 - Utilisation d'un extrait de withania pour le traitement de maladies neuromusculaires - Google Patents

Utilisation d'un extrait de withania pour le traitement de maladies neuromusculaires

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Publication number
EP3442553A1
EP3442553A1 EP16720888.3A EP16720888A EP3442553A1 EP 3442553 A1 EP3442553 A1 EP 3442553A1 EP 16720888 A EP16720888 A EP 16720888A EP 3442553 A1 EP3442553 A1 EP 3442553A1
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EP
European Patent Office
Prior art keywords
als
disease
composition
web
extract
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Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
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Application number
EP16720888.3A
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German (de)
English (en)
Inventor
Chérif RABHI
Jamal Ouazzani
Guillaume ARCILE
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Ethnodyne
Centre National de la Recherche Scientifique CNRS
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Ethnodyne
Centre National de la Recherche Scientifique CNRS
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Publication of EP3442553A1 publication Critical patent/EP3442553A1/fr
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/18—Magnoliophyta (angiosperms)
    • A61K36/185—Magnoliopsida (dicotyledons)
    • A61K36/81—Solanaceae (Potato family), e.g. tobacco, nightshade, tomato, belladonna, capsicum or jimsonweed
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/18—Magnoliophyta (angiosperms)
    • A61K36/185—Magnoliopsida (dicotyledons)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/18—Magnoliophyta (angiosperms)
    • A61K36/185—Magnoliopsida (dicotyledons)
    • A61K36/28—Asteraceae or Compositae (Aster or Sunflower family), e.g. chamomile, feverfew, yarrow or echinacea
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/18—Magnoliophyta (angiosperms)
    • A61K36/185—Magnoliopsida (dicotyledons)
    • A61K36/47—Euphorbiaceae (Spurge family), e.g. Ricinus (castorbean)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/18—Magnoliophyta (angiosperms)
    • A61K36/185—Magnoliopsida (dicotyledons)
    • A61K36/67—Piperaceae (Pepper family), e.g. Jamaican pepper or kava
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/18—Magnoliophyta (angiosperms)
    • A61K36/185—Magnoliopsida (dicotyledons)
    • A61K36/80—Scrophulariaceae (Figwort family)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/18—Magnoliophyta (angiosperms)
    • A61K36/88—Liliopsida (monocotyledons)
    • A61K36/906—Zingiberaceae (Ginger family)
    • A61K36/9066—Curcuma, e.g. common turmeric, East Indian arrowroot or mango ginger
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P21/00—Drugs for disorders of the muscular or neuromuscular system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the invention relates to the use of a composition from a plant extract of Withania somnifera, to treat or limit development o f neuromuscular diseases, including motor neuron diseases like amyotrophic lateral sclerosis.
  • Neuromuscular diseases are diseases that affect the muscles and/or their direct nervous system control. A large proportion o f these neurological disorders leads to problems with movement. Neuropathies invo lve dysfunction of the peripheral nerves, called motor neurons, which carry the electrical signals directly from the spinal cord and brain stem to activate muscle movement; the sensory neurons which convey sensory information such as pain, temperature, light touch, vibration and position to the brain; and the autonomic neurons which go to the internal organs and control blood vessel reflexes .
  • motor neurons which carry the electrical signals directly from the spinal cord and brain stem to activate muscle movement
  • the sensory neurons which convey sensory information such as pain, temperature, light touch, vibration and position to the brain
  • autonomic neurons which go to the internal organs and control blood vessel reflexes .
  • Motor neuron diseases or disorders are characterized by progressive loss o f motor neurons o f the spinal cord ( ' lower motor neurons ' (MN)) or motor neurons o f the brain ( 'upper motor neurons ' ), or both, leading to atrophy and/or spasticity o f the associated musculature.
  • SMA spinal muscular atrophy
  • ALS amyotrophic lateral sclerosis
  • HSP hereditary spastic paraplegia
  • MNDs occur in adults and children. In children, particularly in inherited or familial forms o f the disease, symptoms can be present at birth or appear before the child learns to walk. In adults, MNDs occur more commonly in men than in women, with symptoms appearing after age 40. The causes o f mo st MNDs are not known. In sporadic or non- inherited MNDs, environmental, toxic, viral, or genetic factors may be implicated.
  • MNDs are classified according to whether they are inherited or sporadic, and to whether degeneration affects upper motor neurons, lower motor neurons, or both.
  • ALS affects both upper and lower motor neurons . It has inherited and sporadic forms and can affect the arms, legs, or facial muscles. Although the majority o f ALS cases are sporadic, up to 10% are inherited (Robberecht & Philips, 20 13) and the mo st common familial forms o f ALS in adults are caused by mutations of the superoxide dismutase gene, or SOD 1 , located on chromosome 21 . There are also rare juvenile-onset forms of familial ALS .
  • Primary lateral sclerosis is a disease of the upper motor neurons, while progressive muscular atrophy affects only lower motor neurons in the spinal cord.
  • SMA Spinal muscular atrophy
  • Hereditary spastic paraplegia is the co llective term for a group of clinically and genetically heterogeneous neurodegenerative disorders characterized by progressive spasticity and weakness in the lower limbs due to loss o f upper motor neurons (Harding, 1983 ).
  • the clinical heterogeneity o f HSP is related to a notable genetic heterogeneity.
  • MNDs There is no cure or standard treatment for the MNDs .
  • Symptomatic and supportive treatment can help people be more comfortable while maintaining their quality o f life .
  • Research has provided evidence about the role of excitotoxicity in the pathophysio lo gy o f sporadic amyotrophic lateral sclerosis and suggests that glutamate receptors activation contributes greatly in mediating injury to motor neurons .
  • the drug riluzole (Rilutek®), the only prescribed drug approved by the U. S . Food and Drug Administration to treat ALS , prolongs life by 2-3 months but does not relieve symptoms.
  • the drug reduces the body's natural production o f the neurotransmitter glutamate, which carries signals to the motor neurons.
  • Muscle relaxants and the benzodiazepines may reduce spasticity.
  • Anticonvulsants and nonsteroidal anti-inflammatory drugs may help relieve pain, and antidepressants may be helpful in treating depression.
  • Some individuals may eventually require stronger medicines such as morphine to cope with musculo skeletal abnormalities or pain, and opiates are used to provide comfort care in terminal stages of the disease.
  • the applicant has found that, by combining an extraction step and a fermentation step using filamentous fungi on the extracts of the plant Withania somnifera, it is possible to use the detoxified extract to treat MNDs and other patho logies o f progressive neurological disorders.
  • the purpose o f the invention is therefore to use a non-toxic composition based on extracts o f Withania somnifera, having a protective effect against motor neuron diseases to treat or limit development of ALS and related neuron disorders.
  • the invention is directed to a composition containing a Withania somnifera extract for its use to treat or limit development of motor neuron diseases in a mammal.
  • the mammal is a human.
  • the Withania somnifera extract has been fermented by its incubation with a filamentous fungus in a suitable environment.
  • the Withania somnifera plant is obtained from India.
  • the root of this plant is marketed by Alp Erbo (Marseille) .
  • the process o f production o f extracts according to the invention can be found in WO 2014/202469. Briefly, the plants are fermented in presence o f a filamentous fungus o f the family Cordycipitaceae, preferably the genus Beauveria. More preferably, the filamentous fungus is derived from the strain Beauveria bassiana, more particularly the strain having reference ATCC 7159. Preferably, the roots of the plant are used.
  • the controlled fermentation detoxifies the Withania Somnifera extract by a series of biocatalysis of various mo lecules contained in this extract and, more particularly, the chemical family o f withano lide aglycones, the substances mainly responsible for the toxicity o f the extract.
  • detoxification is used to mean elimination by the microorganism of potentially toxic mo lecules in the medium.
  • the medium is then subj ected to sterilization steps, preferably by ultrafiltration, in order to obtain the solution which constitutes the plant extract.
  • the plant extract of the invention contains Withania somnifera but may also contain at least one o f the fo llowing extracts : Emblica officinalis, originating from India and marketed by Infrag, Bengalore), Bacopa monnieri (India) marketed by Alp Erbo (Marseille), Punica granatum (China) (Shanghai Brightol International Co, Ltd (Shanghai), Curcuma longa (India) (Omnipharm, Chambery), Piper longum (Thailand) (Omnipharm, Chambery), or Calendula officinalis (China) (Shanghai Brightol International Co , Ltd (Shanghai), using the same procedure), by independent extraction steps for each plant extract used in the realization of the said preparation.
  • the composition used in this invention includes between 5 and 100 g/L o f Withania somnifera, preferably 20 g/L .
  • this composition also includes one o f the fo llowing extracts :
  • Punica granatum preferably 10 g/L
  • the composition used in this invention comprises an extract of the plants Withania somnifera, Emblica officinalis and Bacopa monnieri. More preferably, the composition according to the invention comprises Withania somnifera at a concentration of 20 g/L, of Emblica officinalis at a concentration o f 15 g/L and o f Bacopa monnieri at a concentration o f 15 g/L .
  • compositions according to the invention are used to treat or limit development o f MN diseases like ALS (amyotrophic lateral sclerosis), PBP (progressive bulbar palsy) , PMA (progressive muscular atrophy), PLS(primary lateral sclerosis), SMA (spinal muscular atrophy), Kennedy's disease, PPS (Post-polio syndrome), PPMA (Post- Polio Muscular Atrophy), MMN (Multifo cal motor neuropathy), MMA (Monomelic amyotrophy), paraneoplastic motor neuron disease, LEMS (Lambert-Eaton Myasthenic Syndrome), MG (Myasthenia gravis) and botulism, in particular by limiting degeneration of motor neurons.
  • ALS amotrophic lateral sclerosis
  • PBP progressive bulbar palsy
  • PMA progressive muscular atrophy
  • PLS primary lateral sclerosis
  • SMA spinal muscular atrophy
  • Kennedy's disease PPS (Post-polio syndrome),
  • Amyotrophic lateral sclerosis also called Lou Gehrig ' s disease or classical motor neuron disease
  • ALS Lou Gehrig ' s disease or classical motor neuron disease
  • This form of the disease is characterized by weakness and wasting in the limbs. Muscle weakness and atrophy occur on both sides of the body. Affected individuals lose strength and the ability to move their arms and legs, and to ho ld the body upright. Other symptoms include spasticity, spasms, muscle cramps, and fasciculations. Speech can become slurred or nasal. When muscles of the diaphragm and chest wall fail to function properly, individuals lose the ability to breathe without mechanical support.
  • PBP Progressive bulbar palsy
  • Symptoms include pharyngeal muscle weakness (invo lved with swallowing), weak j aw and facial muscles, progressive lo ss o f speech, and tongue muscle atrophy.
  • Limb weakness with both lower and upper motor neuron signs is almo st always evident but less prominent.
  • Affected persons have outbursts o f laughing or crying (called emotional lability) .
  • early symptoms begin with bulbar invo lvement.
  • Some 75 % o f individuals with classic ALS eventually show some bulbar involvement. Life expectancy is between six months and three years from onset of symptoms .
  • PMA Progressive muscular atrophy
  • PLS Primary lateral sclerosis
  • SMA Spinal muscular atrophy
  • Kennedy' s Disease also known as progressive spinobulbar muscular atrophy, is an X-linked recessive progressive disorder o f the motor neurons caused by mutations in the gene for the androgen receptor. Symptoms include weakness and atrophy o f the facial, j aw, and tongue muscles, leading to problems with chewing, swallowing, and changes in speech. Early symptoms may include muscle pain and fatigue. Individuals with Kennedy' s disease also develop sensory loss in the feet and hands. It only affects men, but women may carry the mutation. The course of the disorder is generally slowly progressive. Individuals tend to remain ambulatory until late in the disease. The life expectancy for individuals with Kennedy disease is usually normal.
  • Post-polio syndrome is a condition that can strike polio survivors decades after their recovery from poliomyelitis. Polio is an acute viral disease that destroys motor neurons. PPS and Post-Polio Muscular Atrophy (PPMA) are thought to occur when the surviving motor neurons are lost in the aging process or through injury or illness. Symptoms include fatigue, slowly progressive muscle weakness, muscle atrophy, fasciculations, co ld into lerance, and muscle and joint pain. These symptoms appear most often among muscle groups affected by the initial disease, and may consist o f difficulty breathing, swallowing, or sleeping. PPS is not usually life threatening. Doctors estimate that 25 to 50 percent of survivors o f paralytic poliomyelitis usually develop PPS .
  • Multifocal motor neuropathy is a progressively worsening condition where muscles in the extremities gradually weaken. MMN is thought to be autoimmune and invo lves only lower motor nerves . MMN usually invo lves very little pain however muscle cramps, spasms and twitches can cause pain for some sufferers. MMN is not fatal, and does not diminish life expectation.
  • MMA Monomelic amyotrophy
  • Paraneoplastic motor neuron disease is a disease affecting the motor neurons .
  • Lambert-Eaton Myasthenic Syndrome is a rare autoimmune disorder characterized by muscle weakness o f the limbs. Around 60% of those with LEMS have an underlying malignancy, mo st commonly small cell lung cancer; it is therefore regarded as a paraneoplastic syndrome.
  • Myasthenia gravis leads to fluctuating muscle weakness and fatigue.
  • muscle weakness is caused by circulating antibodies that block acetylcho line receptors at the postsynaptic neuromuscular junction, inhibiting the excitatory effects of the neurotransmitter acetylcho line on nicotinic receptors at neuromuscular junctions.
  • muscle weakness is caused by a genetic inherited defect in some portion o f the neuromuscular junction.
  • Botulism a rare and potentially fatal illness caused by a toxin produced by the bacterium Clostridium botulinum, prevents muscle contraction by blo cking the release o f acetyl cho line, thereby halting postsynaptic activity of the neuromuscular junction.
  • the methods and compositions treat, limit development or reduce the progression o f ALS in particular.
  • treating ALS means providing any clinical benefit to a subject with ALS .
  • the clinical benefit may be temporary or long- lasting.
  • the treatm ent results in one or more clinical outcome selected from the group consisting o f:
  • reducing the progression" or “limiting development” of ALS means providing a limitation in development o f symptoms or disease in a subj ect that is at risk of developing ALS .
  • there is a method of treating or limiting development o f a neuromuscular disease in a subj ect comprising the step of administering to the subj ect a therapeutic amount of a plant extract composition, su ch that said neuromu scu lar disease in a subj ect is treated or its developm ent limited, wherein said composition contains a non toxic plant extract of Withania somnifera .
  • the neuromuscular diseases comprise MN diseases, ALS, PBP, PMA, PLS, SMA, Kennedy's disease, PPS , PPMA, MMN, MMA, paraneoplastic motor neuron disease, LEMS , MG and botulism.
  • the subj ect is a human.
  • composition according to the invention is formulated for oral or parenteral administration.
  • compositions may be in liquid, gel, emulsion, solid or inj ectable form.
  • composition used may additionally include suspensions, emulsions, syrups containing conventionally used inert diluents, and possibly other substances such as wetting agents, sweeteners, preservatives, thickeners, co lourings or any other substance known to a person skilled in the art suitable for oral administration, in particular ((sodium sorbate (E201 ) (Sigma-Aldrich), anthocyanin (E 163) (FBC Industries, USA), sodium metabisulphite (E223) (Sigma-Aldrich), alpha- tocopherol (E307) (FBC Industries, USA) .
  • E201 sodium sorbate
  • anthocyanin E 163
  • E223 sodium metabisulphite
  • E307 alpha- tocopherol
  • composition used may also comprise so lvents or other excipients such as water, propylene glyco l, vegetable oils or other suitable organic solvents .
  • excipient is used to mean any compound which does not interfere with the effectiveness o f the bio logical activity o f the composition according to the invention, and which is not toxic to the host to which it is administered.
  • composition used may also contain adjuvants, such as wetting agents, isotoning agents, emulsifiers, salts or any other substances known to a person skilled in the art that can be used as adjuvants (Polydimethylsiloxane, polyvinyl alcoho l (PVA), hydrogels (Carbopol), polyvinylpyrrolidone, hydroxypropyl cellulose (HPC), poloxamer 1 88 , EDTA, chlorobutanol) (Lubrizol, France, Dow Corning, USA) .
  • adjuvants such as wetting agents, isotoning agents, emulsifiers, salts or any other substances known to a person skilled in the art that can be used as adjuvants (Polydimethylsiloxane, polyvinyl alcoho l (PVA), hydrogels (Carbopol), polyvinylpyrrolidone, hydroxypropyl cellulose (HPC), polox
  • composition may comprise other substances such as vitamins, mineral salts, pharmaceutically acceptable vectors, stabilizers, antioxidants, or any other substance known to a person skilled in the art and intended to be integrated into a drug.
  • the composition is liquid, orally administrable and contains at least a non-toxic extract of Withania somnifera, some preservatives, vitamins, water and salt.
  • the preservatives are potassium sorbate or benzoate.
  • the vitamin is ribo flavin (vitamin B2) .
  • the therapeutic composition used in the method of the invention is administered in a pharmaceutically acceptable vehicle.
  • compositions according to the invention are used to mean any vehicle which does not interfere with the effectiveness o f the bio logical activity o f the composition according to the invention and which is not toxic to the host to which it is administered.
  • the composition obtained is usable as a medicinal product for a mammal, and more particularly for humans, to assist in the treatment or limitation o f development of MNDs and in particular ALS .
  • the term "medicinal product” is used to mean a product containing an accurate dose of said preparation according to European directive 65/65/EC, namely any substance or composition described as possessing curative or preventive properties with respect of human or animal disease.
  • the medicinal product containing said preparation at therapeutic doses can be administered orally as a capsule or a tablet, or inj ected via any other route to confer the beneficial effects .
  • An appropriate dosage o f the therapeutic composition can be determined by one o f skill in the art, taking into consideration the findings described herein together with typical factors such as the body mass of the patient, the physical condition o f the patient, and so on.
  • the dosage should contain the therapeutic composition in an amount that is effective for treating or limiting development of MNDs and in particular ALS .
  • the drug can be administered daily, weekly, or on an intermittent basis .
  • the drug can be administered for three weeks on, fo llowed by one week o ff, or for two weeks on, fo llowed by one week off, or under other dosing schedules as can be determined by one skilled in the field.
  • each unit dose may be larger than when daily dosages are provided.
  • the daily dose o f the compositions used may vary according to the needs and severity o f symptoms o f the patient and according to the route .
  • the daily dose is between 10 mg/mL and 300 mg/mL o f the composition after fermentation.
  • the daily dose for an adult human is between 30 and 100 mg/mL of the composition after fermentation.
  • Example 1 Composition WEB- 1 before fermentation
  • the composition WEB- 1 contains a commercial extract o f Withania Somnifera at a concentration o f 20 g/L, o f Emblica officinalis at a concentration o f 15 g/L, o f Bacopa monnieri at a concentration o f 15 g/L .
  • Example 2 Strain of filamentous fungus Beauveria bassiana
  • the strain Beauvaria Bassiana (reference ATCC 71 59) has been cultivated in a medium containing 0,5 g/L KH 2 P0 4 ; 1 g/L KH 2 P0 4 ; 1 g/L MgS0 4 ; 2 g/L NaN0 3 ; 0,5 g/L KC1 ; 0,02 g/L FeS0 4 ; 30 g/L glucose (all reagents from Sigma-Aldrich, France) and 10 g/L o f corn steep liquor (Roquette, France) .
  • the culture was then agitated at 200 rotations per minute, for 72 hours at 27 °C . It was then filtered by non-sterile methods on a filter paper to separate the fungal biomass from the culture medium. The fungal biomass was then washed thoroughly with water.
  • Example 3 Composition WEB-2 used in the invention
  • composition WEB- 1 as in example 1 is added to the fresh fungal biomass of example 2 using 60 g o f biomass per liter o f composition WEB- 1 containing 50g of glucose.
  • this seeded composition was agitated at 200 rpm for 5 days at a temperature of 27 °C . After 5 days, the incubation medium was filtered on a filter paper, the samples for HPLC assay were also filtered using a 0.45 micron filter (Ait-France, ref: SFNY 013045N) .
  • Example 4 Composition WE-2 used in the invention
  • composition WE- 1 contains commercial extracts of Withania Somnifera at a concentration o f 20 g/L, and o f Emblica officinalis at a concentration o f 15 g/L .
  • the markers identified in the composition WE-2 were:
  • Example 5 Composition WB-2 used in the invention
  • the composition WB- 1 contains an extract of Withania Somnifera at a concentration o f 20 g/L, and of Bacopa Monnieri at a concentration o f 15 g/L .
  • composition WB-2 used in the invention
  • the composition BE-2 contains an extract of Bacopa Monnieri at a concentration o f 15 g/L, and o f Emblica officinalis at a concentration of 15 g/L . To 100 mL of such a so lution, are added 5 g of glucose and 6 g of biomass of example 2.
  • the markers identified in the composition BE-2 were Bacopaside
  • Example 7 Composition WEB-4 according to the invention
  • the composition WBE-4 contains an extract of Withania Somnifera at a concentration of 40 g/L, of Bacopa Monnieri at a concentration of 1 5 g/L, and of Emblica officinalis at a concentration o f 15 g/L .
  • Example 8 Composition WEB-6 used in the invention
  • composition WEB-6 contains an extract of Withania Somnifera at a concentration of 20 g/L, of Bacopa Monnieri at a concentration of 1 5 g/L, and of Emblica officinalis at a concentration o f
  • Example 9 Composition WEB-8 used in the invention
  • the composition WEB-8 contains an extract of Withania Somnifera at a concentration of 20 g/L, of Bacopa Monnieri at a concentration of 30 g/L, and of Emblica officinalis at a concentration o f 15 g/L .
  • Example 10 primary motor neuron culture survival
  • the aim of this study was to study the effect of WEB-2 on primary motor neuron culture from rat spinal cord (SC) injured by glutamate exposure (a well validated in vitro ALS model and model of motor neuron diseases).
  • SC Spinal cord
  • Rat SC motor neurons were cultured as described by Martinou et al., Neuron. 1992 Apr;8(4):737-44 and Wang et al., Hum Mol Genet. 2013 Dec 1;22(23):4706-19. Briefly, pregnant female rats (Wistar, Janvier labs) of 14 days gestation were killed by cervical dislocation. Foetuses were collected and immediately placed in ice- cold L15 Leibovitz medium (Batch: 4001014, Pan Biotech) with a 2% penicillin (10,000 U/mL) and streptomycin (10 mg/mL) solution (PS; Pan Biotech, batch: 3090914) and 1% bovine serum albumin (BSA; Pan Biotech, batch: hl40603).
  • PS penicillin
  • streptomycin 10 mg/mL
  • BSA bovine serum albumin
  • SC were treated for 20 min at 37°C with a trypsin- EDTA (Pan Biotech, batch: 5890314) solution at a final concentration of 0.05% trypsin and 0.02% EDTA.
  • the dissociation was stopped by addition of Dulbecco's modified Eagle's medium (DMEM) with 4.5 g/liter of glucose (Pan Biotech, batch: 6031214), containing DNAse I grade II (final concentration 0.5 mg/mL; Pan Biotech, batch: H140508) and 10% fetal calf serum (FCS; Invitrogen, batch: 41Q7218K).
  • DMEM Dulbecco's modified Eagle's medium
  • FCS 10% fetal calf serum
  • the cells were seeded at a density of 20,000 per well in 96-well plates precoated with poly-L-lysine (Biocoat, batch: 21614030) and were cultured at 37°C in an air (95%)-C02 (5%) incubator. The medium was changed every day. b) WEB-2 and glutamate exposure
  • WEB-2 was pre-incubated with cells for 1 hour before glutamate exposure. Then, glutamate (Sigma, Batch: SLBL7326V) was added into cell culture to a final concentration of 10 ⁇ diluted in control medium in presence of WEB-2 for 20 min. After 20 min, glutamate was washed and fresh culture medium with WEB-2 was added for additional 48 hours.
  • glutamate Sigma, Batch: SLBL7326V
  • the aim o f this study was to test the effect o f WEB-2 on nerve /muscle co-culture injured by glutamate exposure.
  • Riluzo le (5 ⁇ ) was used as reference compound.
  • Evaluation o f neuromuscular junction (NMJ) integrity (number and mean size) and neurite network innervating the muscular cells were assessed in presence of the treatment.
  • NMJ neuromuscular junction
  • a. Primary cultures of nerve muscle co-culture Human muscle (Promocell, Batch: 3061107) was prepared according to a previously described method from portions of a biopsy from a healthy subject (Braun et al., 1996, J Neurol Sci. 136: 17-23, Combes et al, 2015, J Neurosci Res. 93(4):633-43).
  • the human muscle cell line was established from dissociated cells (21 000 cells per wells), plated in gelatin-coated 0.1% (Sigma, Batch: slbkl410v) in water on 48 wells plate (Greiner, Batch: E13111ME) and grown in a proliferation medium consisting of mix of 62 % of MEM medium (PAN, Batch: 7410215) and 25 % of M199 medium (PAN, Batch: 4581114) supplemented with glutamine 2mM (PAN, Batch: 6620314), human insulin l( ⁇ g/mL (PAN, Batch: 4480413), Human recombinant Epidermal growth factor lOng/mL (EGF, GIBCO, Batch: 1291552A), human recombinant Fibroblast growth factor basic 2ng/mL (bFGF, PAN, Batch: H080113), foetal calf serum 10% (FCS, GIBCO, Batch: 41Q7218K), 2% of Penicillin 1
  • the medium was changed every 2 days. Five days after the start of culture, immediately after satellite cell fusion, whole transverse slices of 13-day-old rat Wistar embryos (Janvier, France) spinal cords with 4 dorsal root ganglia (DRG) attached are placed on the muscle monolayer (one explant per well in the central area). DRG are necessary to achieve a good ratio of innervation. Innervated cultures were maintained in a mixed (67%/25%) medium composed of MEM and medium 199, supplemented with 5% FCS, insulin 5 ⁇ g/mL, glutamine 2mM and 2% PS. After 24h of co-culture, neurites were observed growing out of the spinal cord explants.
  • a mixed (67%/25%) medium composed of MEM and medium 199, supplemented with 5% FCS, insulin 5 ⁇ g/mL, glutamine 2mM and 2% PS.
  • glutamate Sigma, Batch: SLBL7326V
  • glutamate Sigma, Batch: SLBL7326V
  • glutamate was added into co-culture to a final concentration of 60 ⁇ diluted in control medium in presence of WEB-2 or riluzole for 20 min. After 20 min, glutamate was washed and fresh culture medium with WEB-2 or riluzole was added for additional 48 hours.
  • the cells were washed 2 times in PBS and then permeabilized and non-specific sites were blocked with a solution of PBS containing 0.1% of saponin (Sigma-Aldrich, Batch: BCBJ8417V) and 1% FBS (Gibco, Batch: 41Q1613K) for 15 min at room temperature, co-cultures were incubated with a mouse monoclonal anti-neurofilament 200 KD antibody (NF, Sigma Aldrich, Batch: 053M4756) at the dilution of 1/400 in PBS containing 1% FCS, 0.1 % saponin, for 2 h at room temperature. Antibody against NF stained the axon of motor neuron.
  • saponin Sigma-Aldrich, Batch: BCBJ8417V
  • FBS Gabco, Batch: 41Q1613K
  • co-cultures were incubated with a mouse monoclonal anti-neurofilament 200 KD antibody (NF, Sigma Aldrich, Batch:
  • WEB-2 (all concentrations tested except the lowest dose : 1 00 ng/mL) showed a protective effect. A similar effect was observed with riluzo le (5 ⁇ ) used as reference compound.
  • WEB-2 (from 500 ng/mL to 5 ⁇ g/mL) showed a significant protective effect of the neuritic network.
  • the aim o f this study was to study the effect of WEB-2 on primary motor neuron culture survival and neurite outgrowth (as a model of neurodegeneration of spinal cord MN) .
  • SC Spinal cord
  • Rat SC motor neurons were cultured as previously described in example 1 0. Viable cells were counted in a Neubauer cytometer, using the trypan blue exclusion test. The cells were seeded at a density o f 20,000 per well in 96-well plates precoated with po ly-L-lysine and were cultured at 37°C in an air (95 %)-C02 (5 %) incubator. b. Immunostaining of Motor neurons
  • This antibody was revealed with Alexa Fluor 488 goat anti- mouse IgG (Molecular probe, batch: 1613346) at the dilution of 1/400 in PBS containing 1% foetal calf serum and 0.1% of saponin for 1 hour at room temperature.
  • the survival was increase by 30 % .
  • the WEB-2 neuroprotective effect was increased compared to 3 days treatment.
  • the neurite network was increased by -30 % in presence of WEB-
  • WEB-2 induced a large increase of neurite network (WEB-2 promotes the neurite outgrowth) of spinal cord motor neurons .
  • Example 13 primary motor neuron culture survival
  • the aim o f this study was to study the effect of WEB-2 on primary motor neuron culture from rat spinal cord (SC) injured by glutamate exposure.
  • the culture o f Spinal cord (SC) motor neurons has been performed in the same conditions as in example 10. a) WEB-2 and glutamate exposure
  • glutamate Sigma, Batch: SLBL7326V
  • WEB-2 was added into cell culture or glutamate and riluzole were added into cell culture. After 20 min, glutamate was washed and fresh culture medium with either WEB-2 of riluzole was added for additional 48 hours .
  • WEB-2 is even better than riluzole to protect the motor neurons from glutamate-induced cell death.
  • the aim of this study was to study the effect of WEB-2 or riluzole on primary motor neuron culture survival.
  • the protocol is the same as in example 11. Briefly, after culture of Spinal cord (SC) motor neurons and immunostaining of Motor neurons, WEB-2 was added on the culture, immediately after seeding
  • This antibody was revealed with Alexa Fluor 488 goat anti- mouse IgG (Molecular probe, batch: 1613346) at the dilution of 1/400 in PBS containing 1% foetal calf serum and 0.1% of saponin for 1 hour at room temperature.
  • MAP-2 motor neuron survival

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Abstract

L'invention concerne l'utilisation d'un extrait de withania pour le traitement de maladies neuromusculaires. Elle concerne l'utilisation d'une composition à partir d'un extrait végétal de Withania somnifera, pour traiter ou limiter le développement de maladies neuromusculaires, y compris des maladies des neurones moteurs comme la sclérose latérale amyotrophique.
EP16720888.3A 2016-04-13 2016-04-13 Utilisation d'un extrait de withania pour le traitement de maladies neuromusculaires Withdrawn EP3442553A1 (fr)

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EP2266586A1 (fr) * 2004-03-23 2010-12-29 Lifeline Nutraceuticals Corporation Composition et méthode pour alléger l'inflammation et stress oxydatif dans les Mammifèrs.
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