WO2021052962A1 - Traitement de lésion de la moelle épinière - Google Patents

Traitement de lésion de la moelle épinière Download PDF

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WO2021052962A1
WO2021052962A1 PCT/EP2020/075769 EP2020075769W WO2021052962A1 WO 2021052962 A1 WO2021052962 A1 WO 2021052962A1 EP 2020075769 W EP2020075769 W EP 2020075769W WO 2021052962 A1 WO2021052962 A1 WO 2021052962A1
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spinal cord
senescent cells
injury
senolytic agent
individual
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PCT/EP2020/075769
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English (en)
Inventor
Maria Leonor Tavares SAÚDE
Diogo de Abreu Paramos-de- CARVALHO
António Alfredo Coelho JACINTO
Isaura Vanessa Antunes MARTINS
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Instituto de Medicina Molecular João Lobo Antunes
Universidade Nova De Lisboa
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Publication of WO2021052962A1 publication Critical patent/WO2021052962A1/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/63—Compounds containing para-N-benzenesulfonyl-N-groups, e.g. sulfanilamide, p-nitrobenzenesulfonyl hydrazide
    • A61K31/635—Compounds containing para-N-benzenesulfonyl-N-groups, e.g. sulfanilamide, p-nitrobenzenesulfonyl hydrazide having a heterocyclic ring, e.g. sulfadiazine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
    • A61K31/35—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom
    • A61K31/352—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom condensed with carbocyclic rings, e.g. methantheline 
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/41—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with two or more ring hetero atoms, at least one of which being nitrogen, e.g. tetrazole
    • A61K31/425—Thiazoles
    • A61K31/428—Thiazoles condensed with carbocyclic rings
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
    • A61K31/506—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim not condensed and containing further heterocyclic rings
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/65—Tetracyclines
    • 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
    • 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
    • A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia

Definitions

  • the present invention relates to methods of treating spinal cord injury and compounds for use in such methods.
  • SC injury interrupts the autonomic nervous system and induces dysfunction or failure in multiple organs because of the critical role in coordinating body functions.
  • Complications following SC injury can occur in the nervous system (neurogenic pain and depression), lungs (pulmonary edema and respiratory failure), cardiovascular system (rthostatic hypotension and autonomic dysreflexia), spleen (splenic atrophy and leukopenia), urinary system (neurogenic bladder, kidney damage, and urinary tract infection), skeletal muscle (muscle spasticity and atrophy), bone and soft tissue (osteoporosis and heterotopic ossification), and skin (pressure sores) and include sexual dysfunction, hepatic pathology, neurogenic bowel dysfunction, syringomyelia, and increased susceptibility to infection (Sun et al., 2016). Therefore, therapeutic interventions that ameliorate post SC injury complications may be as important for prolonging life expectancy and improving life quality as those interventions that promote neuro-regeneration and motor
  • the limited regeneration of the mammalian SC stems from several obstructing factors that have been studied in mammalian models.
  • the ependymal region of an adult SC contains neural stem cells (NSCs) that in vivo form mostly astrocytes that form a glial scar (Meletis et al, 2008; Barnabe-Heider et al, 2010). This scar acts as a physical barrier for axonal crossing and contains molecules that inhibit sensory and motor function restoration.
  • NSCs neural stem cells
  • senescent cells through their senescence-associated secretory phenotype (SASP), were shown to induce cellular plasticity and reprogramming in neighbouring cells (Mosteiro et al, 2016; Ritschka et al, 2017).
  • salamander limbs known for their ability to regenerate, show a burst of senescent cells upon amputation that is efficiently cleared from the regenerating tissues by macrophages possibly recruited by the SASP itself (Yun et al, 2015).
  • the zebrafish and the neonatal mouse hearts are also known to regenerate, and again transient senescence states were recently reported in these organs, further reinforcing potential beneficial effects (Sarig et al., 2019).
  • the present inventors have discovered that the elimination of senescent cells improves recovery and healing after spinal cord (SC) injury in mammals.
  • the elimination of senescent cells for example using a senolytic agent, may therefore be useful in the treatment of patients with spinal cord injury.
  • a first aspect of the invention provides a method of treating a spinal cord injury in an individual comprising depleting or reducing the number of senescent cells in the individual.
  • a second aspect of the invention provides a method of improving locomotor function or reducing autonomic dysfunction in an individual following spinal cord injury comprising depleting or reducing the number of senescent cells in the individual.
  • a method of the first or second aspect may comprise depleting or reducing the number of senescent cells at the site of the SC injury and/or at peripheral sites in the individual.
  • a method of the first or second aspect may comprise administering a therapeutically effective amount of a senolytic agent to the individual.
  • a third aspect of the invention provides a senolytic agent for use in a method of treating a spinal cord injury in an individual, for example a method of the first aspect, or improving locomotor function or reducing autonomic dysfunction in an individual following spinal cord injury, for example a method of the second aspect.
  • a fourth aspect of the invention provides the use of a senolytic agent in the manufacture of a medicament for use in a method of treating a spinal cord injury in an individual, for example a method of the first aspect, or improving locomotor function or reducing autonomic dysfunction in an individual following spinal cord injury, for example a method of the second aspect.
  • Preferred senolytic agents include Navitoclax (ABT-263).
  • a fifth aspect of the invention provides a method of screening for a candidate compound for use in the treatment of spinal cord injury comprising; contacting a population of senescent cells and a control population of non-senescent cells with a test compound and; determining the apoptosis of the senescent cells relative to the non-senescent cells, wherein increased apoptosis of the senescent cells relative to non-senescent cells is indicative that the test compound is a candidate compound for the treatment of spinal cord injury.
  • the population and control population of cells may be spinal cord cells for example spinal cord neurons. Other aspects and embodiments of the invention are described in more detail below.
  • Figure 1 shows the number of SAp-gal+ cells overtime post-injury in zebrafish (1A) and mouse (1B).
  • Figure 2 shows the identity of senescent cells in injured spinal cords in zebrafish and in mice.
  • Figure 2A shows the proportion of senescent (SAp-gal+) cells in SC injured zebrafish and mouse that are p-gal+ HuC/D+ neurons (Zebrafish) or p-gal+ NeuN+ neurons (mouse) overtime after injury.
  • Figure 2B shows the number of senescent p-gal+ HuC/D+ neurons (Zebrafish) or p-gal+ NeuN+ neurons (mouse) overtime after injury.
  • Figure 3 shows the impact of eliminating senescent cells on motor and sensory recovery following a spinal cord injury in mice.
  • Figures 3A and 3B show the BMS scores and BMS subscores at several time-points for a period of 60 days in the open field test.
  • Figures 3E and 3F show the results of the horizontal ladder test at several time-points for a period of 60 days.
  • Figures 3G and 3H show the results of thermal allodynia tests to evaluate the sensory performance of SC-injured mice at 30 and 60 dp.
  • Figure 4 shows the impact of eliminating senescent cells on autonomic recovery following a spinal cord injury in mice.
  • Figure 4A shows the effect on bladder function overtime and
  • Figure 4B shows the effect on liver weight overtime.
  • Figure 5 shows the impact of eliminating senescent cells on myelin preservation following a spinal cord injury in mice at 15 dpi (5A), 30 dpi (5B) and 60 dpi (5C).
  • FIG. 6 shows that ABT-263 administration increases the number of GAP43+ fibers.
  • Axon number was calculated at 30 days post-injury (dpi) as a ratio of the total GAP43+ fibers at 4 mm rostral (-4) from the lesion epicentre (0).
  • n 3.
  • Data are presented as mean ⁇ SEM. *p ⁇ 0.05, **p ⁇ 0.01 , ***p ⁇ 0.001 , ABT-263 versus Vehicle.
  • Figure 7 shows the impact of eliminating senescent cells on fibrotic scar area/extension following a spinal cord injury in mice at 15 dpi (7A & 7B), 30 dpi (7A & 7C) and 60 dpi (7A &7D).
  • FIG 8 shows the injury force and displacement applied by the IH Impactor in different experimental groups
  • Figure 9 shows the number of senescent cells in the SC at 15 dpi in ABT-263 and vehicle treated mice in the rostal (9A), caudal (9B) and lumbar-sacral (9C) regions.
  • Figure 10 shows the Impact of eliminating senescent cells on motor recovery following spinal cord injury in p16-3MR transgenic mice.
  • Figure 10A shows the BMS score overtime in ABT-263 and vehicle treated mice.
  • Figure 10B shows the variation in impact force and displacement between experimental groups.
  • FIG 11 shows that ABT-263 treatment decreases the number of macrophages at the injury site.
  • the area of F4/80+ tissue was measured at lesion epicenter and 600 pm rostral and caudal from the epicenter. Measurements are expressed as a percentage of the total cross-sectional area.
  • Data are presented as mean ⁇ SEM. *p ⁇ 0.05, **p ⁇ 0.01 , ***p ⁇ 0.001 , ABT-263 versus Vehicle.
  • Figure 12 shows the elimination of senescent cells with dasatinib (D) and quercetin (Q) promotes motor and sensory recovery following a spinal cord injury in mice.
  • D dasatinib
  • Q quercetin
  • This invention relates to the treatment of spinal cord (SC) injury by eliminating, depleting or reducing the number of senescent cells at the injury site, for example by administration of a senolytic agent. This is shown herein to improve recovery from the injury.
  • SC spinal cord
  • a spinal cord injury in an individual may be treated as described herein by eliminating, depleting or reducing the number of senescent cells at the injury site. For example, 10% or more, 20% or more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more or 90% or more of the senescent cells at the injury site may be eliminated.
  • senescent cells may reduce or inhibit the formation of a fibrotic scar; promote the survival or recovery of white matter; increase myelin preservation and/or reduce pericyte recruitment at the site of spinal cord injury.
  • a senescent cell is a post-replicative cell that is no longer capable of mitosis or division.
  • Senescent cells at a site of spinal cord injury may comprise or consist of senescent neurons.
  • Senescent cells may display a senescence associated secretory phenotype (SASP).
  • SASP senescence associated secretory phenotype
  • a SASP may comprise the secretion from the cell of a combination of factors including cytokines, such as I L- 1 a , 11-1 b, IL-6, IL-7, IL-13 IL-8, growth factors, such as bFGF, GM-CSF and VEGF, and proteases, such as MMP-1 and Cathepsin B (Coppe et al Ann. Rev Pathol. 2010; 5: 99-118).
  • Senescent cells may be characterised by the expression of senescence associated b- galactosidase (SA-p-gal), p21 and/or p16 (p16INK4a, cyclin-dependent kinase inhibitor 2A).
  • Senescent cells may be eliminated, depleted or reduced in number at the site of the spinal cord injury through the induction of apoptosis.
  • senescent cells may be eliminated, depleted or reduced in number by administering a therapeutically effective amount of a senolytic agent to the individual.
  • a senolytic agent is a compound or treatment that selectively induces the apoptosis of senescent cells relative to non- senescent cells i.e. it induces apoptosis in senescent cells but not non-senescent (i.e. replicative) cells or induces apoptosis to a greater extent in senescent cells compared to non-senescent cells.
  • Suitable senolytic agents may inhibit anti-apoptotic factors, such as BCL2 (Gene ID: 596) and/or BCL-XL (also called BCL2L1 ; Gene ID: 598).
  • Suitable senolytic agents may include Navitoclax (ABT-263; 4-[4-[[2-(4- chlorophenyl)-5,5-dimethylcyclohexen-1-yl]methyl]piperazin-1-yl]-N-[4-[[(2R)-4-morpholin-4-yl-1- phenylsulfanylbutan-2-yl]amino]-3-(trifluoromethylsulfonyl)phenyl]sulfonylbenzamide), A1331852 (3-(1- (((3r,5r,7r)-adamantan-1-yl)methyl)-5-methyl-1 H-pyrazol-4-yl)-6-(8-(benzo[d]thiazol-2-y
  • Suitable senolytic agents may inhibit Src kinases, such as BCR-ABL (also called ABL1 ; Gene ID: 25).
  • Suitable senolytic agents may include dasatinib (N-(2-chloro-6-methylphenyl)-2-(6-(4-(2-hydroxyethyl) piperazin-1-yl)-2-methylpyrimidin-4-ylamino) thiazole-5-carboxamide).
  • Suitable senolytic agents may include anti-oxidants, for example plant polyphenol anti-oxidants, such as quercetin (3, 3', 4',5,7-Pentahydroxyflavone).
  • anti-oxidants for example plant polyphenol anti-oxidants, such as quercetin (3, 3', 4',5,7-Pentahydroxyflavone).
  • Suitable senolytic agents may include PI3K/AKT/mTOR pathway inhibitors, for example sirtuin activators, such as fisetin (7, 3’,4’-flavon-3-ol).
  • a combination of senolytic agents such as a combination of dasatinib, fisetin and quercetin (DFQ) or dasatinib and quercetin (DQ) may be employed.
  • DFQ dasatinib, fisetin and quercetin
  • DQ dasatinib and quercetin
  • the senolytic agent may be an agent other than quercetin.
  • a method may comprise determining or monitoring the number of senescent cells at the injury site following administration of the senolytic agent.
  • a senolytic agent as described above may be administered alone or may be formulated into a pharmaceutical composition.
  • a pharmaceutical composition is a formulation comprising one or more active agents and one or more pharmaceutically acceptable excipients. The pharmaceutical composition may be capable of eliciting a therapeutic effect.
  • a suitable pharmaceutical composition for use as described herein may comprise a senolytic agent and a pharmaceutically acceptable excipient.
  • pharmaceutically acceptable relates to compounds, materials, compositions, and/or dosage forms which are, within the scope of sound veterinary or medical judgement, suitable for use in contact with the tissues of a subject (e.g. human or other mammal) without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio.
  • a subject e.g. human or other mammal
  • Each carrier, excipient, etc. must also be “acceptable” in the sense of being compatible with the other ingredients of the formulation.
  • Suitable excipients and carriers include, without limitation, water, saline, buffered saline, phosphate buffer, alcoholic/aqueous solutions, emulsions or suspensions. Other conventionally employed diluents, adjuvants, and excipients may be added in accordance with conventional techniques.
  • Such carriers can include ethanol, polyols, and suitable mixtures thereof, vegetable oils, and injectable organic esters. Buffers and pH- adjusting agents may also be employed, and include, without limitation, salts prepared from an organic acid or base.
  • Representative buffers include, without limitation, organic acid salts, such as salts of citric acid (e.g., citrates), ascorbic acid, gluconic acid, carbonic acid, tartaric acid, succinic acid, acetic acid, phthalic acid, Tris, trimethylamine hydrochloride, or phosphate buffers.
  • Parenteral carriers can include sodium chloride solution, Ringer's dextrose, dextrose, trehalose, sucrose, lactated Ringer's, or fixed oils.
  • Intravenous carriers can include fluid and nutrient replenishers, electrolyte replenishers, such as those based on Ringer's dextrose, and the like.
  • Preservatives and other additives such as, for example, antimicrobials, antioxidants, chelating agents (e.g., EGTA; EDTA), inert gases, and the like may also be provided in the pharmaceutical carriers.
  • chelating agents e.g., EGTA; EDTA
  • inert gases e.g., inert gases, and the like
  • the pharmaceutical compositions described herein are not limited by the selection of the carrier.
  • compositions from the above-described components, having appropriate pH, isotonicity, stability and other conventional characteristics, is within the skill of the art.
  • Suitable carriers, excipients, etc. may be found in standard pharmaceutical texts, for example, Remington’s Pharmaceutical Sciences and The Handbook of Pharmaceutical Excipients, 4th edit., eds. R. C. Rowe et al, APhA Publications, 2003.
  • a pharmaceutical composition may conveniently be presented in unit dosage form and may be prepared by any methods well-known in the art of pharmacy. Such methods include the step of bringing the one or more isolated immunogenic polypeptides into association with a carrier or excipient as described above which may constitute one or more accessory ingredients. In general, the formulations are prepared by uniformly and intimately bringing into association the active compound with liquid carriers or finely divided solid carriers or both.
  • compositions described herein may be produced in various forms, depending upon the route of administration.
  • the pharmaceutical compositions may be prepared for administration to subjects in the form of, for example, liquids, powders, aerosols, tablets, capsules, enteric-coated tablets or capsules, or suppositories.
  • Pharmaceutical compositions may also be in the form of suspensions, solutions, emulsions in oily or aqueous vehicles, pastes, and implantable sustained-release or biodegradable formulations.
  • Compositions for sustained release or implantation may comprise pharmaceutically acceptable polymeric or hydrophobic materials, such as an emulsion, an ion exchange resin, a sparingly soluble polymer, or a sparingly soluble salt.
  • compositions may be made in the form of sterile aqueous solutions or dispersions, suitable for injectable use, or made in lyophilized forms using freeze-drying techniques. Lyophilized pharmaceutical compositions are typically maintained at about 4°C, and can be reconstituted in a stabilizing solution, e.g., saline or HEPES, with or without adjuvant. Pharmaceutical compositions can also be made in the form of suspensions or emulsions.
  • compositions may be presented in unit-dose or multi-dose sealed containers, for example, ampoules and vials, and may be stored in a freeze-dried (lyophilised) condition requiring only the addition of the sterile liquid carrier, for example water for injections immediately prior to use.
  • sterile liquid carrier for example water for injections immediately prior to use.
  • the pharmaceutical composition may be administered to a subject by any convenient route of administration.
  • administration is by systemic routes, including oral, or more preferably parenteral routes.
  • the pharmaceutical composition may be administered by intravenous, intraperitoneal or subcutaneous injection.
  • a senolytic agent or pharmaceutical composition comprising a senolytic agent may be useful in treating a spinal cord injury (SCI) as described herein.
  • SCI spinal cord injury
  • a spinal cord injury may be a contusion or other injury that damages the spinal cord and, in particular the nerve fibres therein, and temporarily or permanently alters its function.
  • the SCI may be in the cervical (C1 to C8), thoracic (T1 to T12), lumbar (L1 to L5) or sacral (S1 to S5) spine.
  • An SCI may be traumatic or non-traumatic.
  • An SCI may be a complete injury in which all functions mediated by nerves below the site of injury are lost, or an incomplete injury, in which some function, such as sensory or motor function, mediated by nerves below the site of injury is preserved.
  • Treatment may be any treatment and therapy, whether of a human or an animal (e.g. in veterinary applications), in which some desired therapeutic effect is achieved, for example, improving or ameliorating one or more symptoms of spinal cord injury or post spinal cord injury complications.
  • some desired therapeutic effect is achieved, for example, improving or ameliorating one or more symptoms of spinal cord injury or post spinal cord injury complications.
  • locomotor function, sensory function; and autonomic function may be improved in the individual following spinal cord injury by depletion of senolytic cells as described herein.
  • Post spinal cord injury complications may include neural complications, such as neurogenic pain and depression, pulmonary complications, such as pulmonary edema and respiratory failure, cardiovascular complications, such as rthostatic hypotension and autonomic dysreflexia, splenic complications, such as splenic atrophy and leukopenia, urinary complications, such as neurogenic bladder, kidney damage, and urinary tract infection, skeletal muscle complications, such as muscle spasticity and atrophy, bone and soft tissue complications, such as osteoporosis and heterotopic ossification, and dermal complications, such as pressure sores.
  • Other post SC injury complications may include sexual dysfunction, hepatic pathology, neurogenic bowel dysfunction, syringomyelia, and increased susceptibility to infection.
  • An individual suitable for treatment as described above may be a mammal, such as a rodent (e.g. a guinea pig, a hamster, a rat, a mouse), murine (e.g. a mouse), canine (e.g. a dog), feline (e.g. a cat), equine (e.g. a horse), a primate, simian (e.g. a monkey or ape), a monkey (e.g. marmoset, baboon), an ape (e.g. gorilla, chimpanzee, orang-utan, gibbon), or a human.
  • a rodent e.g. a guinea pig, a hamster, a rat, a mouse
  • murine e.g. a mouse
  • canine e.g. a dog
  • feline e.g. a cat
  • equine e.g. a horse
  • the individual is a human.
  • non-human mammals especially mammals that are conventionally used as models for demonstrating therapeutic efficacy in humans (e.g. murine, primate, porcine, canine, or rabbit animals) may be employed.
  • a SCI may be treated as described herein within 6 hours, within 12 hours, within 24 hours, within 48 hours, or within 96 hours of the occurrence of the SCI.
  • the senolytic agent may be administered by any appropriate route of administration.
  • the senolytic agent may be administered directly to the site of injury or systemically, e.g., by oral or parenteral routes.
  • Parenteral routes include, for example, intravenous, intrarte rial, intracranial, intraorbital, opthalmalic, intraventricular, intraspinal (e.g., into the cerebrospinal fluid), intracisternal, intramuscular, intradermal, subcutaneous, intranasal and intraperitoneal routes. It is contemplated that local modes of administration may reduce or eliminate the incidence of potential side effects (e.g., systemic toxicity) that may occur during systemic administration.
  • side effects e.g., systemic toxicity
  • the senolytic agent may be blood-spinal cord barrier (BSCB) permeant (i.e. it may cross the BSCB).
  • BSCB permeant senolytic agent may be administered, for example, by oral or intraperitoneal routes.
  • the senolytic agent may be BSCB impermeant (i.e. it may not cross the spinal-cord barrier).
  • a BSCB impermeant senolytic agent may be administered, for example, by intrathecal routes.
  • the senolytic agent may comprise an agent or moiety for enhancing the permeability of the BSCB or for permitting the delivery of the senolytic agent through the BSCB.
  • Suitable agents are well known in the art.
  • appropriate dosages of the senolytic agent can vary from patient to patient. Determining the optimal dosage will generally involve the balancing of the level of therapeutic benefit against any risk or deleterious side effects of the treatments of the present invention.
  • the selected dosage level will depend on a variety of factors including, but not limited to, the activity of the particular senolytic agent, the route of administration, the time of administration, the rate of loss or inactivation of the senolytic agent, the duration of the treatment, other drugs, compounds, and/or materials used in combination, and the age, sex, weight, condition, general health, and prior medical history of the patient.
  • the dosage of senolytic agent and the route of administration will ultimately be at the discretion of the physician, although generally the dosage will be to achieve local concentrations at the site of action which achieve the desired effect without causing substantial harmful or deleterious side-effects.
  • the senolytic agent may be administered at a dosage that is effective in reducing the number of senescent cells at the injury site.
  • Prescription of treatment is within the responsibility of general practitioners and other medical doctors and may depend on the severity of the symptoms and/or progression of a disease being treated.
  • Appropriate doses of therapeutic polypeptides are well known in the art (Ledermann J.A. et al. (1991) Int. J. Cancer 47: 659-664; Bagshawe K.D. et al. (1991) Antibody, Immunoconjugates and Radiopharmaceuticals 4: 915-922).
  • Specific dosages may be indicated herein or in the Physician's Desk Reference (2003) as appropriate for the type of medicament being administered may be used.
  • a therapeutically effective amount or suitable dose of a senolytic agent described herein may be determined by comparing its in vitro activity and in vivo activity in an animal model. Methods for extrapolation of effective dosages in mice and other test animals to humans are known.
  • Treatment may comprise the administration of a therapeutically effective amount of a senolytic agent or pharmaceutical composition to the individual.
  • “Therapeutically effective amount” relates to the amount of a senolytic agent or pharmaceutical composition that is effective for producing some desired therapeutic effect, commensurate with a reasonable benefit/risk ratio.
  • a suitable amount of a senolytic agent or pharmaceutical composition for administration to an individual may be an amount that generates a therapeutic effect in the individual.
  • a therapeutic effect may be at least amelioration of at least one symptom.
  • a treatment as described herein may have a duration of up to 3 weeks, up to 6 weeks, up to 3 months, up to 6 months or up to 12 months.
  • the treatment schedule for an individual may be dependent on the pharmocokinetic and pharmacodynamic properties of the senolytic agent, the route of administration and the nature of the condition being treated.
  • Treatment may be in one dose, continuously or intermittently (e.g., in divided doses at appropriate intervals). Treatment may be periodic, and the period between administrations may be about 12 hours or more, 24 hours or more, 36 hours or more, 48 hours or more, 96 hours or more, or one week or more. Suitable formulations and routes of administration are described above and may be readily determined by a physician for any individual patient.
  • a senolytic agent as described herein may be administered in combination with one or more other therapies, either simultaneously or sequentially dependent upon the circumstances of the individual to be treated.
  • Other therapies may include treatment with therapeutic agents that diminish neurological tissue destruction and ameliorate functional recovery, such as riluzole and minocycline.
  • the compounds When the therapeutic agents are used in combination with additional therapeutic agents, the compounds may be administered either sequentially or simultaneously by any convenient route.
  • a therapeutic agent When a therapeutic agent is used in combination with an additional therapeutic agent active against the same disease, the dose of each agent in the combination may differ from that when the therapeutic agents are used alone. Appropriate doses will be readily appreciated by those skilled in the art.
  • Other aspects of the invention relate to methods of screening to identify compounds as candidate compounds for use in the treatment of spinal cord injury.
  • a method of screening for a candidate compound for use in the treatment of spinal cord injury may comprise; contacting a population of senescent cells and a control population of non-senescent cells with a test compound and; determining the apoptosis of the senescent cells relative to the non-senescent cells.
  • Increased apoptosis of the senescent cells relative to non-senescent cells may be indicative that the test compound is a candidate compound for the treatment of spinal cord injury.
  • the senescent and non-senescent cells are spinal cord cells, such as neurons, astrocytes, microglia, oligodendrocytes, endothelial cells, pericytes, fibroblasts or Schwann cells.
  • the senescent and non-senescent cells are spinal cord neurons.
  • test compound may be an isolated molecule or may be comprised in a sample, mixture or extract, for example, a biological sample.
  • Compounds which may be screened using the methods described herein may be natural or synthetic chemical compounds used in drug screening programmes. Extracts of plants, microbes or other organisms, which contain several characterised or uncharacterised components may also be used.
  • Suitable test compounds include analogues, derivatives, variants and mimetics of known senolytic agents, such as Navitoclax, for example compounds produced using rational drug design to provide test candidate compounds with particular molecular shape, size and charge characteristics suitable for the selective apoptosis of senescent cells.
  • known senolytic agents such as Navitoclax
  • Combinatorial library technology provides an efficient way of testing a potentially vast number of different compounds for ability to selectively apoptose senescent cells.
  • Such libraries and their use are known in the art, for all manner of natural products, small molecules and peptides, among others. The use of peptide libraries may be preferred in certain circumstances.
  • test compound which may be added to an assay of the invention will normally be determined by trial and error depending upon the type of compound used. Typically, from about 0.001 nM to 1 mM or more concentrations of putative inhibitor compound may be used, for example from 0.01 nM to 100pM, e.g. 0.1 to 50 mM, such as about 10 pM. Even a compound which has a weak effect may be a useful lead compound for further investigation and development.
  • a test compound identified as selectively apoptosing senescent cells may be investigated further.
  • the selectivity of a compound for senescent cells, such as senescent neurons, at a site of SC injury may be determined in animal models. Suitable methods for determining the effect of a compound on senescent cells are well known in the art.
  • test compound identified as a senolytic agent may be isolated and/or purified or alternatively, it may be synthesised using conventional techniques of recombinant expression or chemical synthesis. Furthermore, it may be manufactured and/or used in preparation, i.e. manufacture or formulation, of a composition such as a medicament, pharmaceutical composition or drug. Methods described herein may thus comprise formulating the test compound in a pharmaceutical composition with a pharmaceutically acceptable excipient, vehicle or carrier for therapeutic application.
  • a method may further comprise modifying the compound to optimise its pharmaceutical properties. Suitable methods of optimisation, for example by structural modelling, are well known in the art. Further optimisation or modification can then be carried out to arrive at one or more final compounds for in vivo or clinical testing.
  • Zebrafish (Danio rerio) were bred, grown and maintained at 28°C following the standard guidelines for fish care and maintenance protocols. Male and female fish were used in the experiments.
  • mice Male C57BL/6J mice (Mus musculus) were purchased from Charles River Laboratory.
  • the p16-3MR transgenic line (Demaria et al., 2014) was kindly provided by Marco Demaria (European Research Institute for the Biology of Aging, Groningen, Netherlands). Animals were housed in the iMM animal facility under conventional conditions on a 12 hour light-dark cycle with ad libitum access to food and water.
  • ABT263 (Selleckchem, S1001 , 50 mg/kg/day) was administered by oral gavage, as described previously (Chang et al., 2015), for 10 consecutive days starting at 5 days post-injury (dpi) until 15 dpi.
  • mice After two weeks-period of handling and acclimatization, body weight was assessed to ensure ideal weight (18-20 g) and animals were assigned to spinal cord injury. Mice (9-11 week old) were anesthetized using a cocktail of ketamine (120 mg/kg) and xylazine (16 mg/kg) administered by ip injection.
  • ketamine 120 mg/kg
  • xylazine (16 mg/kg) administered by ip injection.
  • spinal contusion injuries a laminectomy of the ninth thoracic vertebra (T9), identified based on anatomical landmarks, was first performed (Harrison et al., 2013) followed by a moderate to severe (75 kdyne) contusion using the Infinite Horizons Impactor (Precision Systems and Instrumentation, LLC.) (Scheff et al., 2003). The mean applied force and tissue displacement for each experimental group are shown in Figure 8. There were no differences in injury parameters between experimental groups.
  • mice After SCI, the muscle and skin was closed with 4.0 polyglycolic absorbable sutures (Safil, G1048213). In control uninjured mice (sham), the wound was closed and sutured after the T9 laminectomy and the spinal cord was not touched. Animals were injected with saline (0.5 ml) subcutaneously (sq) then placed into warmed cages until they recovered from anaesthesia and for the following recovery period (3 days). To prevent dehydration mice were supplemented with daily saline (0.5 ml, sq) for the first 5 dpi. Bladders were manually voided twice daily for the duration of experiments. Body weight was monitored weekly.
  • saline 0.5 ml
  • sq subcutaneously
  • mice and zebrafish were sacrificed and spinal cords were isolated at similar experimental time-points (3, 7, 15, 30 and 60 dpi).
  • BMS Basso Mouse Scale
  • the total number of mistakes was averaged across the three trials per mouse and quantified as mistakes per centimetre.
  • the total number of positive and negative events for each rung in each attempt were also quantified and are divided as singular positive events (plantar step, toe step and skip) or singular negative events (slip, miss and drag). Mice were tested on the HL at 15, 30 and 60 dpi.
  • Hot/Cold Incremental Plate To determine the effect of ABT263 noxious heat/cold threshold temperature (thermal allodynia) in mice, the IITC’s Incremental Hot Cold Plate (IITC Inc. Life Science) was used. In this test, each mouse was placed into the observation chamber on the plate that had a starting temperature of 37°C. For hot reaction, the plate was heated up to 49°C at a rate of 6°C per minute until the animal showed nocifensive behaviour involving either hindpaw. For cold stimulation, the the plate was cooled until 0°C at the same rate. The typical response was hindpaw licking, shaking and lifting of the paw, jumping and extensor spasm.
  • the plate temperature evoking any of these nocifensive reactions confined to any paw was regarded as the noxious heat/cold threshold of the animal. Following recording of the threshold temperature, the animal was immediately removed from the plate. The threshold measurement was repeated after 30 minutes and the mean of the two thresholds was considered as the control noxious heat/cold threshold of the animal. For this specific test, animals were only assessed at the experimental endpoint of 30 or 60 dpi.
  • the vertebral column of adult zebrafish was dissected and fixed in 4% paraformaldehyde (PFA) at 4°C overnight (ON). After fixation the spinal cord was isolated from the vertebral column. The samples were washed 3 times in phosphate-buffered saline (PBS) during the day and incubated O/N with senescence- associated b-galactosidase (SA-p-gal) staining solution (see details below).
  • PFA paraformaldehyde
  • samples were cryoprotected in 30% sucrose/0.12 M phosphate buffer (PB) for a minimum of 72 hours at 4°C or until the tissue sinks to the bottom of the vial, followed by another embedding in 7.5% gelatin (Sigma, G6144)/15% sucrose/0.12 M PB and subsequently frozen.
  • PB phosphate buffer
  • the samples were cryosectioned in 12 pm-thick longitudinal slices using a Cryostat LEICA CM 3050S and either processed for immunohistochemistry or counterstained with eosin for SA-p-gal quantifications.
  • mice were anesthetized with ketamine/xylazine mix (120 mg/kg + 16 mg/kg, ip) and then transcardially perfused with 0.9% sodium chloride followed by 4% paraformaldehyde. Post-mortem anatomical assessment of the T9 was confirmed to ensure correct thoracic contusion. Spinal cords were removed, post-fixed in 4% PFA for 2 hours and then incubated ON with SA-p-gal staining solution (see details below). Samples were then submitted to the same cryoprotection/embedding procedure as for zebrafish spinal cords.
  • mice spinal cords were divided in two segments: a thoracic segment centred on the impact site and a lumbar-sacral segment immediately following the thirteenth thoracic vertebra (T13). Both segments measured 1 cm in length. Tissue sections were cut in series either transversally (10 pm thick, 10 slides per series) or longitudinally (10 pm thick, 6 slides per series). For each time-point samples were distributed as equally as possible in cuts along the coronal (rostral-caudal) axis and horizontal (dorsal-ventral) axis. Slides were stored at -20°C until needed. Livers were dissected, weighted and post-fixed in formaldehyde until processed for histology.
  • SA-p-gal activity was determined in isolated spinal cords using the SA-p-gal kit (Cell Signalling, #9860) according to manufacturer’s instructions, with minor adaptations.
  • Spinal cords were fixed ON in 4% PFA, washed three times in PBS and stained ON at 37°C using the SA-p-gal staining solution (pH 5.9-6.1 , prepared according to kit’s instructions). The samples were then washed in PBS, fixed in 4% PFA for 4 hours, washed 3 x 5 minutes in PBS and embedded in sucrose as described above.
  • SA-p-gal+ cells were manually quantified (using a Cell Counter plugin in Fiji) and averaged across 4 (zebrafish) or 8 (mouse) longitudinal sections imaged at the lesion periphery (from 0.5 to 2.5 mm laterally to the lesion) or laterally to the injury segment (for Sham- injured animals) at 3, 7, 15, 30 and 60 dpi.
  • SA-p-gal+ cells were quantified in the gray matter but not in the white matter and normalized to the total area covered (cells/mm 2 ).
  • SA-p-gal+ cells were quantified in the gray matter but not in the white matter and normalized to the total area covered (cells/mm2). Two distinct quantifications were performed: one in the total sectional grey matter and other only in the ventral horn.
  • the gelatin was removed from the cryosections using PBS heated to 37°C (4 x 5 minutes washes). After incubation with blocking solution for 2 hours at room temperature (RT), the sections were incubated ON with primary antibody solution at 4°C. Sections were then washed in PBS/0.1 % Triton X-100 (PBSTx) and incubated with the secondary antibody (1 :500) and 1 mg ml-1 DAPI (Sigma, D9564) for 2 hours at RT. Details on the blocking solutions, primary and secondary antibodies used are described in the supplementary tables T1 and T2. After incubation with the secondary antibodies, the sections were washed in PBS and mounted in Mowiol mounting medium.
  • GAP43+ axons were counted based on previously described methods (Hata et al., 2006; Almutiri et al., 2018). Quantifications of GAP43+ fibers were performed only in the white matter in 3 longitudinal spinal sections (per biological sample) of the dorsal horn region using a custommade macro in Fiji that, after manually establishing a threshold value and defining the lesion epicenter, determined the number of positive fibers every 1 mm from 4 mm rostral to 4 mm caudal to the lesion site and normalized it to the tissue length covered in each measurement. Axon number was calculated as a percentage towards the ratio (fibers/mm) obtained 4 mm above (rostral) the lesion, where the spinal tracts were intact.
  • a distinct set of sections was stained with anti-PDGFRp and anti-GFAP in order to identify the fibrotic scar area and border.
  • the percentage of fibrotic scar area at lesion epicenter was calculated by manually outlining the PDGFRp+ area and normalizing it to the total crosssectional area. Measurements were performed using Fiji tools.
  • the rostral and caudal extents of PDGFRp+ fibrosis were determined for each lesion, and total lesion length was calculated by multiplying the number of sections containing fibrotic tissue by the distance between each section (0.1 mm).
  • GraphPad Prism 7 was used for data visualization and SigmaPlot 14 for statistical analysis.
  • the senescence profile after SCI was analyzed using a one-way ANOVA followed by a Bonferroni’s post hoc test (zebrafish) or a non-parametric Kruskal-Wallis one-way ANOVA test (mouse).
  • BMS and Bladder Score data were analyzed using a two-way repeated-measures ANOVA, followed by a Bonferroni’s post hoc test.
  • HL, ITP, white matter sparing, axonal preservation, fibrotic area and inflammation data were analyzed using a normal two-way ANOVA, followed by a Bonferroni’s post hoc test. All data were expressed as mean ⁇ SEM, with statistical significance determined at p-values ⁇ 0.05.
  • Senescent cells are induced in the spinal cord in response to an injury
  • SApgal senescence-associated b-galactosidase
  • mice are unable to efficiently eliminate senescent cells in the spinal cord following an injury.
  • Senescent cells present in the spinal cord after injury are mainly neurons
  • senescent cells are induced in the SC in response to an injury in both zebrafish and mice.
  • In injured SCs we detected a co-localization between SAp-gal+ cells and the pan-neuronal markers HuC/D in zebrafish and NeuN in mouse.
  • In sham controls and injured animals at all time-points studied the majority of senescent cells are neurons in zebrafish and mice (Fig. 2A).
  • mice In zebrafish, the percentage of total neurons that are SA-p-gal+ reaches a peak of 8.9% at 15 dpi and then returns to basal levels (2.3%) at 60 dpi. On the other hand, mice display 25.3% of senescent neurons at 15 dpi ( Figure 2B) and strikingly, this number keeps increasing until 60 dpi, reaching 35.3% of total neurons.
  • mice were habituated to the behavioural apparatus and were injured with a moderate-severe contusion injury (75 kdynes). Mice were randomly distributed to each experimental group (SCI + vehicle and SCI + ABT-263). Both vehicle and ABT-263 were administered via oral gavage, beginning at 5 dpi, and then daily until 15 dpi (Fig. 3A, B). There were no differences in injury force or displacement applied by the IH Impactor between experimental groups (Fig. 8).
  • ABT-263 significantly improved locomotion, as observed in the total BMS score (Fig. 3A). This effect is extended up to 30 dpi, even after the administration protocol has ceased. Although no differences were seen between vehicle and ABT-263-treated mice in the BMS score from 45 dpi onwards, a statistically significant improvement of the BMS subscore was still achieved until 60 dpi (Fig. 3B). By 30 dpi, all ABT-263-treated mice achieved frequent plantar stepping with 93% (14 out of 15) of mice displaying parallel placement both hindpaws at initial contact and 40% (6 out of 15) also at lift off. 33% (5 out of 15) of mice exhibited consistent plantar stepping and mild trunk stability.
  • mice were analysed using the horizontal ladder test. Although performance on the ladder at 15 dpi was not different between vehicle- and ABT-263-treated mice, animals treated with the senolytic made significantly less stepping mistakes (Fig. 3C, 3E) and displayed significantly more positive stepping events (Fig. 3D, 3F) at 30 and 60 dpi.
  • SC injuries are characterized by allodynia i.e., hypersensitivity to normally innocuous stimuli.
  • allodynia i.e., hypersensitivity to normally innocuous stimuli.
  • thermal allodynia tests to evaluate the sensory performance of SC-injured mice at 30 and 60 dpi (Fig. 3G,
  • the senolytic ABT-263 does not cross the blood-SC barrier. For this reason, we were only able to administer this senolytic within the first 15 days, a period during which the blood-SC barrier is kept open following a SC injury.
  • a fusion protein with functional domains of luciferase (LUC), a red fluorescent protein (mRFP) and a truncated herpes simplex virus 1 thymidine kinase (HSV-TK) are expressed under the control of p16 promoter.
  • LUC allows luminescence detection of senescent cells
  • mRFP permits their immunohistochemistry detection and sorting
  • HSV-TK allows their selective killing by Ganciclovir (GCV).
  • GCV Ganciclovir
  • TK converts GCV into a toxic DNA chain terminator causing death by apoptosis in pieexpressing cells (i.e in senescent cells). In contrast to ABT-263, GCV is known to cross blood-SC barrier.
  • Chronic liver pathology is also associated with SC injury and is known to contribute to systemic inflammation, cardiovascular disease, and metabolic syndrome (Sauerbeck et al. 2015).
  • SC-injured animals treated with ABT-263 we saw a significant reduction of the liver weight at 15 dpi, when compared with injured animals treated with vehicle (Fig. 4B).
  • Fig. 4B We used a simple histological analysis to quantify lipid accumulation in hepatocytes in the mouse thoracic contusion mouse model.
  • the white matter spared was larger over 0.5 mm caudally to the lesion site in ABT-263-treated mice.
  • 60 dpi 0.3 mm rostrally and 0.5 mm caudally at 60 dpi from the epicentre of the lesion (Fig. 5A-C).
  • GAP43 neuronal growth-associated protein 43
  • Macrophage numbers at the injury site are reduced following ABT-263 treatment
  • a spinal cord lesion in mice elicits a strong and long-lasting inflammatory response that potentiates secondary injury (Blight, 1985; Popovich, Wei and Stokes, 1997).
  • Macrophages are the most abundant inflammatory cells in a spinal lesion, infiltrating the injury core and releasing several molecules, namely nitrogen/oxygen metabolites, cytokines, proteases and chondroitin sulphate proteoglycans that can cause cellular damage and inhibit axonal growth (Fitch and Silver, 1997).
  • depletion of macrophages was demonstrated to promote repair and partial motor recovery after spinal cord injury in rats (Popovich et al., 1999).
  • SCs through their SASP, can secrete a plethora of immune modulators and proinflammatory cytokines like TNF-a and CCL2 (two potent macrophage recruiters), interleukin-6 (IL-6), IL-8 and IL-1 a (Coppe et al., 2010). Therefore, the accumulation/persistence of SCs in tissues is usually associated with chronic inflammation.
  • IL-6 interleukin-6
  • IL-8 interleukin-1 a
  • F4/80 pan-macrophage marker
  • SCs we describe the induction of SCs as a new cellular response triggered by an injury in the spinal cord.
  • Our data shows that the majority of senescent cells, quantified in the grey matter located at the lesion periphery of injured spinal cords, are neurons.
  • SCs start to accumulate essentially at the lesion periphery but are eventually cleared and returned to basal levels.
  • Senolytic drugs selectively eliminate SCs by transiently disabling the pro-survival networks.
  • One of such drugs is ABT-263, a specific inhibitor of anti-apoptotic proteins BCL-2 and BCL-xL, already shown to selectively and efficiently kill senescent cells in vivo in mice (Demaria et al., 2014; Chang et al., 2016).
  • ABT-263 a specific inhibitor of anti-apoptotic proteins BCL-2 and BCL-xL
  • Persistent senescent fibroblasts and myogenic cells through their SASP were shown to promote a pro- fibrotic response and to limit tissue repair in fibrotic lung disease (Schafer et al., 2017) and injured muscles (Le Roux et al., 2015), respectively. Accordingly, we showed that the effect of ABT-263 on SCs depletion was translated into a consistently reduced fibrotic scar area and length. In addition, SCs depletion with ABT- 263 results in a higher myelin preservation overtime.

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Abstract

La présente invention concerne des méthodes de traitement d'une lésion de la moelle épinière chez un individu qui consistent à dépléter ou à réduire le nombre de cellules sénescentes chez l'individu, par exemple par administration d'une quantité thérapeutiquement efficace d'un agent sénolytique, tel que le Navitoclax (ABT-263) à l'individu. L'invention concerne également des procédés de traitement, des utilisations et des composés destinés à être utilisés dans des méthodes de traitement.
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