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WO2020136191A1 - Peptide analogue of thymulin (pat) for use in the treatment of strokes - Google Patents

Peptide analogue of thymulin (pat) for use in the treatment of strokes Download PDF

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Publication number
WO2020136191A1
WO2020136191A1 PCT/EP2019/087004 EP2019087004W WO2020136191A1 WO 2020136191 A1 WO2020136191 A1 WO 2020136191A1 EP 2019087004 W EP2019087004 W EP 2019087004W WO 2020136191 A1 WO2020136191 A1 WO 2020136191A1
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WO
WIPO (PCT)
Prior art keywords
peptide
treatment
stroke
pat
seq
Prior art date
Application number
PCT/EP2019/087004
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French (fr)
Inventor
Stéphane LARUELLE
Original Assignee
Orphit
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Publication date
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Publication of WO2020136191A1 publication Critical patent/WO2020136191A1/en

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    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00Medicinal preparations containing peptides
    • A61K38/04Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
    • A61K38/08Peptides having 5 to 11 amino acids

Definitions

  • THYMULIN ANALOGUE PEPTIDE FOR USE IN THE TREATMENT OF VASCULAR ACCIDENTS
  • the present invention relates to the pharmaceutical field. It relates more particularly to a thymulin analog peptide (PAT) for its use in the prevention and / or treatment of strokes, as well as a composition comprising such a peptide used according to the invention.
  • PAT thymulin analog peptide
  • a stroke is a pathology characterized by a sudden drop in blood flow in a part of the brain, equivalent to a myocardial infarction.
  • Stroke occurs when the blood supply to the brain is blocked (ischemic stroke), or when a blood vessel in the brain is ruptured (hemorrhagic stroke).
  • ischemic stroke occurs when the blood supply to the brain is blocked (ischemic stroke), or when a blood vessel in the brain is ruptured (hemorrhagic stroke).
  • the effects of a stroke depend on the location and volume of the damaged tissue. Strokes can lead to death, but also serious disabilities such as paraplegia, aphasia, visual impairment or cognitive impairment.
  • the main limitation is the time required to diagnose a stroke (CT scan, MRI). Indeed, in such a situation, the precious time and major brain damage occurs in a short window after an acute ischemic stroke (AVCi), while the patient is taken to the emergency room then after a revascularization.
  • CT scan computed tomography
  • MRI magnetic resonance imaging
  • AVCi acute ischemic stroke
  • the pillar of stroke prevention is the management of risk factors: smoking cessation, the treatment of high blood pressure and heart disease, and the control of blood sugar in diabetics.
  • doctors may prescribe an anhydrous therapy such as aspirin, warfarin or ticlopidine.
  • anhydrous therapy such as aspirin, warfarin or ticlopidine.
  • tPA thrombolytic tissue plasminogen
  • the limits of thrombolysis are a narrow therapeutic window, a low rate of recanalization and a definite risk of bleeding. Even if it is used in the therapeutic window, the PA nevertheless presents a risk of symptomatic intracerebral hemorrhage.
  • a tube (catheter) is ⁇ introduced into the artery of the leg.
  • the neuroradiologist then ⁇ advances this tubing into the cerebral arteries, guided in real time by X-rays.
  • Extremely precise guidance is absolutely necessary since it will have to “navigate” in arteries 2 to 4 millimeters in diameter, without injuring them, resulting in ⁇ potentially life-threatening brain hemorrhage.
  • This ⁇ e navigation continues until it comes into contact with the clot, captures it and extracts it from the patient's body. This rescue is successfully carried out in almost 90% of the cases.
  • the blood flow is restored, making it possible to irrigate the brain again and to limit the clinical consequences of this occlusion. Since the scientific demonstration of the benefit for patients, thrombectomy is recommended in all cases of stroke of less than six hours with the presence of a clot at the level of a proximal artery.
  • IVT intravenous thrombolysis
  • Mechanical thrombectomy is a fine example of the rapid and effective provision of medical progress for stroke patients.
  • New therapeutic options are essential to extend the therapeutic window of thrombolysis and to provide neuroprotection to slow cell death after a stroke.
  • a problem which the present invention proposes to solve is to provide a new compound for the treatment of patients suffering from a cerebrovascular accident, which can act alone in patients not eligible for thrombolytic treatment or by mechanical thrombectomy. , or combined with thrombolytic treatment or mechanical thrombectomy.
  • the first object of the solution to this problem is a thymulin-like peptide (PAT) corresponding to the following sequence:
  • SEQ ID No. 1 Glu-Ala-Lys-Ser-GIn-Gly-Gly-Ser-Asp (EAKSQGGSD), or a pharmaceutically acceptable salt thereof,
  • Its second object is a composition
  • a composition comprising a peptide used according to the invention, dissolved in an appropriate solvent, optionally accompanied by a preservative, a buffering agent, an isotonicity adjuvant, a surfactant taken alone or as a mixture.
  • the Applicant decided to use this peptide for the treatment of stroke, in particular stroke, by benefiting from its inflammatory and antioxidant activities capable of reducing neuronal death and treating strokes.
  • FIG. 1 represents the neuroprotective effect of the PAT of SEQ ID No. 1 according to the invention on a model of primary cortical neuronal oxidative stress in ra ⁇ don ⁇ son ⁇ cells deprived of oxygen / glucose (OGD).
  • OGD oxygen / glucose
  • FIG. 2 represents the effect against the PAT infarction of SEQ ID No. 1 according to the invention in a model of occlusion of the middle cerebral artery (MCAO) in mice.
  • MCAO middle cerebral artery
  • the invention relates to a thymulin analog peptide (PAT) corresponding to the following sequence:
  • SEQ ID No. 1 Glu-Ala-Lys-Ser-GIn-Gly-Gly-Ser-Asp (EAKSQGGSD), or a pharmaceutically acceptable salt thereof,
  • acceptable pharmaceutically acceptable salt means, without limitation, an acetate, a sulfate or a hydrochloride.
  • stroke we mean a pathology characterized by a sudden drop in blood flow in part of the brain.
  • Neurons affected by lack of oxygen and glucose release pro-inflammatory cytokines and reactive oxygen species (ROS) that damage tissue.
  • ROS reactive oxygen species
  • the inflammatory reaction worsens with the activation of microglial cells as well as the secretion of adhesion molecules causing the release of new cytokines and ROS.
  • the tissues are increasingly damaged by recruitment via infiltration of the inflammation cells, up to the activation of metalloprotease matrices causing a disruption of the blood-brain barrier as well as edema.
  • the PAT of SEQ ID No. 1 according to the invention is used to treat patients in whom the blood supply to the brain is blocked (ischemic stroke), or when a blood vessel in the brain is ruptured (hemorrhagic stroke). ).
  • patient is meant a human being or an animal, such as for example a dog or a cha ⁇ , preferably a human being.
  • the PAT of SEQ ID No. 1 according to the invention is used in the treatment of an acute ischemic stroke (AVCi).
  • AVCi acute ischemic stroke
  • the PAT of SEQ ID No. 1 used according to the invention is preferably in a form suitable for administration by the parenteral route, in particular subcutaneous (w.m.), intranasal, intraperitoneal (i.p.) and intravenous (i.v.) administration.
  • the PAT of SEQ ID No. 1 used according to the invention is more preferably in a form suitable for administration by intravenous bolus.
  • the PAT of SEQ ID No. 1 used according to the invention is administered at a dosage of between 0.1 and 50 mg / kg, preferably between 1 and 10 mg / kg, for example 2 mg / kg, 3 mg / kg, 4 mg / kg, 5 mg / kg, 6 mg / kg, 7 mg / kg, 8 mg / kg or 9 mg / kg, more preferably 3 mg / kg.
  • the PAT of SEQ ID No. 1 used according to the invention is administered alone in patients not eligible for thrombolytic treatment or by mechanical thrombectomy.
  • the invention relates to a combination product comprising the PAT of SEQ ID No. 1 combined with a thrombolytic treatment, for its use in the prevention and / or the treatment of cerebrovascular accidents.
  • the PAT of SEQ ID No. 1 is combined with a thrombolytic treatment, preferably chosen from a recombinant tissue activator ⁇ of plasminogen (rt-PA), alteplase, streptokinase, desmoteplase, tenecteplase, microplasmin, reteplase, anchor and urokinase.
  • the PAT of SEQ ID No. 1 is combined with thrombolytic treatment up to 6 hours after the onset of the first signs of the cerebrovascular accident.
  • the PAT of SEQ ID No. 1 is combined with a treatment by mechanical thrombectomy, for its use in the prevention and / or the treatment of cerebrovascular accidents.
  • the PAT of SEQ ID No. l es ⁇ combined with a treatment by mechanical thrombectomy.
  • the PAT of SEQ ID No. 1 is combined with treatment by mechanical thrombectomy up to 24 hours after the onset of the first signs of the cerebrovascular accident.
  • Another object relates to a composition
  • a composition comprising a PAT of SEQ ID No. 1 for its use according to the invention, dissolved in an appropriate solvent, optionally accompanied by a preservative, a buffering agent, an isofonicate adjuvant, a surfactant taken alone or as a mixture.
  • Example 1 Validation of the PAT Activity of SEQ ID No. 1 According to the Invention in Cell Models Deprived of Oxygen and Glucose (OGD) (In Vitro)
  • This ⁇ th study aims to examine the effect of the PAT of SEQ ID No. 1 according to the invention (6 concentrations) on a model of primary cortical neuronal oxidative stress in RA ⁇ don ⁇ son ⁇ cells deprived of oxygen / glucose (OGD); riluzole is ⁇ used as a positive control. 1 hour before OGD, the neurons are treated with the PAT of SEQ ID No. 1 according to the invention.
  • the model used in this preliminary approach makes it possible to mimic ischemic neuronal death and to identify a possible neuroprotective effect of the peptide according to the invention tested.
  • cortical neurons of ra ⁇ on ⁇ were cultivated as described by Singer
  • test compound peptide according to the invention or riluzole
  • the culture medium was removed and fresh culture medium without glucose was added.
  • the cells were transferred to an anaerobic incubator under 95% N2 / 5% C02 conditions at 37 ° C.
  • 25 mM D-glucose was added to the culture medium and the cells were transferred to a conventional incubator with 95% air / 5% CO 2 at 37 ° C.
  • 5 mM riluzole (SIGMA, Ref: RI 1 6, lot: 057K3900V) was used as the reference compound, 6 wells per condition. The following conditions have been met:
  • the cells were fixed with a cold 95% ethanol / acetic acid (5%) solution for 5 minutes. The cells were then permeabilized and the non-specific sites were blocked. Total neuronal survival was assessed (the number of positive MAP2 neural cell bodies was counted). For each culture well, 20 photos per well were taken using the InCell ANALYZER TM 2200 material (GE HEALTHCARE) with a magnification x 20. All the photos were taken under the same conditions. The number of neurons was automatically evaluated with the analysis system provided (GE HEALTHCARE). Statistics
  • the OGD study thus carried out highlights a neuroprotective effect of the PAT of SEQ ID No. 1 according to the invention at a concentration of at least 10 mM.
  • the OGD test protocol used is a standard protocol which provides that the neurons are pre-incubated 1 hour before OGD with the compound to be tested.
  • the PAT of SEQ ID No. 1 according to the invention has a very short half-life in plasma ( ⁇ 5 min) which could explain a low activity at concentrations below 10 mM.
  • the purpose of this study is to examine the effect of the PAT of SEQ ID No. 1 according to the invention in a model of occlusion of the middle cerebral artery (MCAO).
  • MCAO middle cerebral artery
  • the PAT of SEQ ID No. 1 according to the invention was tested using the intraluminal monofilament model of the cerebral middle occlusion artery (MCAO) which consists of the insertion of a surgical filament into the artery external carotid and forward threading in the internal carotid artery (ICA) until the tip obscures the origin of the MCA, causing blood flow to stop and a subsequent cerebral infarction in the MCA territories.
  • MCAO cerebral middle occlusion artery
  • the PAT of SEQ ID No. 1 according to the invention is administered intravenously at two doses (1 mg / kg e ⁇ 3 mg / kg) 30 minutes after occlusion e ⁇ 30 minutes before removal of the monofilamen ⁇ .
  • the body temperature of the mouse is maintained at 37 ° C. Then, the suture is removed one hour after occlusion, which allows reperfusion; reperfusion may or may not be successful.
  • the severity of ischemia is ⁇ assessed by non-invasive metric flow using the Doppler laser. Reperfusion occurs if a subsequent increase in blood flow is measured.
  • mice 24 hours after the induction of MCAO, the mice are anesthetized and euthanized by cervical dislocation. Brains are ⁇ stored frozen in isopentane.
  • the brains are cut into 20 micron slices with a cryostatic section and then stained with cresyl viol ⁇ to allow a microscopic evaluation of the size of the infarction and ⁇ mounted on ENTELLAN® coated slides.
  • the size of the infarction is then measured using a Cell-Lens imaging tool.
  • the MCAO model used is a standardized model; removing filaments stimulates reperfusion.
  • This model is somewhat distant from the elimination of the clot by the use of a thrombolytic but is closer to thrombectomy when the clot is removed surgically.

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Abstract

The invention relates to the prevention and/or treatment of strokes. The invention is characterised in that a peptide analogue of thymulin (PAT) corresponding to the following sequence: SEQ ID n°1: Glu-Ala-Lys-Ser-Gln-Gly-Gly-Ser-Asp (EAKSQGGSD), or one of the pharmaceutically acceptable salts thereof, is used in the prevention and/or treatment of strokes. The invention also relates to a composition comprising such a peptide which is used according to the invention and dissolved in an appropriate solvent, optionally accompanied by a preservative, buffering agent, isotonicity adjuvant or surfactant, alone or in combination.

Description

PEPTIDE ANALOGUE DE LA THYMULINE (PAT) POUR SON UTILISATION DANS LE TRAITEMENT DES ACCIDENTS VASCULAIRES CEREBRAUX THYMULIN ANALOGUE (PAT) PEPTIDE FOR USE IN THE TREATMENT OF VASCULAR ACCIDENTS
Domaine technique Technical area
La présente invention concerne le domaine pharmaceutique. Elle concerne plus particulièrement un peptide analogue de la thymuline (PAT) pour son utilisation dans la prévention et/ou le traitement des accidents vasculaires cérébraux (AVC), ainsi qu’une composition comprenant un tel peptide utilisé selon l’invention. The present invention relates to the pharmaceutical field. It relates more particularly to a thymulin analog peptide (PAT) for its use in the prevention and / or treatment of strokes, as well as a composition comprising such a peptide used according to the invention.
Un AVC est une pathologie se caractérisant par une chute brutale du débit sanguin dans une partie du cerveau, équivalent à un infarctus du myocarde. A stroke is a pathology characterized by a sudden drop in blood flow in a part of the brain, equivalent to a myocardial infarction.
L'AVC se produit lorsque l'apport sanguin au cerveau es† bloqué (AVC ischémique), ou lorsqu’un vaisseau sanguin dans le cerveau es† rompu (AVC hémorragique) . Les effets d'un AVC dépendent de l'emplacement e† du volume du tissu endommagé. Les AVC peuvent entraîner la mort, mais aussi de graves handicaps tels qu’une paraplégie, aphasie, déficience visuelle ou encore des troubles cognitifs. Stroke occurs when the blood supply to the brain is blocked (ischemic stroke), or when a blood vessel in the brain is ruptured (hemorrhagic stroke). The effects of a stroke depend on the location and volume of the damaged tissue. Strokes can lead to death, but also serious disabilities such as paraplegia, aphasia, visual impairment or cognitive impairment.
Chaque année, environ 1 6 millions d'accidents vasculaires cérébraux surviennent dans le monde, causant un total de 5,7 millions de décès, alors qu'environ un tiers des patients souffrent d'une invalidité permanente. Each year, approximately 1.6 million strokes occur worldwide, causing a total of 5.7 million deaths, while approximately one third of patients suffer from permanent disabilities.
Technique antérieure Prior art
La principale limite réside dans le temps nécessaire pour diagnostiquer un accident vasculaire cérébral (tomodensitométrie, IRM) . En effet, dans une telle situation, le temps es† précieux e† des lésions cérébrales majeures surviennent dans une courte fenêtre après un AVC ischémique aigu (AVCi), pendant que le patient es† transporté à l'urgence puis après une revascularisation. The main limitation is the time required to diagnose a stroke (CT scan, MRI). Indeed, in such a situation, the precious time and major brain damage occurs in a short window after an acute ischemic stroke (AVCi), while the patient is taken to the emergency room then after a revascularization.
Le pilier de la prévention des AVC es† la gestion des facteurs de risque : le sevrage tabagique, le traitement de l'hypertension artérielle e† des maladies cardiaques, e† le contrôle de la glycémie chez les diabétiques. De plus, les médecins peuvent prescrire des traitements an†i†hrombo†iques comme l'aspirine, la warfarine ou la ticlopidine. Malgré la prévalence de l'AVC, actuellement, le seul médicament approuvé pour le traitement de l'AVC est l'activateur du plasminogène recombinant de tissu thrombolytique (tPA), un médicament qui doit être administré dans les 4,5 heures suivant les premiers signes pour prévenir les dommages cliniques graves. The pillar of stroke prevention is the management of risk factors: smoking cessation, the treatment of high blood pressure and heart disease, and the control of blood sugar in diabetics. In addition, doctors may prescribe an anhydrous therapy such as aspirin, warfarin or ticlopidine. Despite the prevalence of stroke, currently the only drug approved for the treatment of stroke is the activator of recombinant thrombolytic tissue plasminogen (tPA), a drug that must be administered within 4.5 hours of the first signs to prevent serious clinical damage.
Le traitement de référence de l’AVCi aigu par thrombolyse intraveineuse (TIV) par rt-PA seule, requiert une prise en charge quasi immédiate, c’est-à-dire une perfusion jusqu’à 4 h 30 après le début des symptômes pour dissoudre le caillot, avec une efficacité d 'autan† plus importante qu’elle es† réalisée tôt. The reference treatment for acute AVCi by intravenous thrombolysis (IVT) by rt-PA alone requires almost immediate management, i.e. an infusion until 4:30 a.m. after the onset of symptoms. dissolve the clot, with greater auton † efficiency than it is achieved early.
Son efficacité es† toutefois imparfaite puisque le caillot ne disparaît pas toujours, ou trop tard pour empêcher la constitution de lésions cérébrales irréversibles. However, its effectiveness is imperfect since the clot does not always disappear, or too late to prevent the formation of irreversible brain damage.
Les limites de la thrombolyse son† une fenêtre thérapeutique étroite, un taux faible de recanalisation e† un risque hémorragique certain. En effet, même s'il es† utilisé dans la fenêtre thérapeutique, le†PA présente néanmoins un risque d'hémorragie intracérébrale symptomatique. The limits of thrombolysis are a narrow therapeutic window, a low rate of recanalization and a definite risk of bleeding. Even if it is used in the therapeutic window, the PA nevertheless presents a risk of symptomatic intracerebral hemorrhage.
Puisque le médicament de référence ne fonctionne pas toujours e† présente des inconvénients, un traitement alternatif es† de directement retirer le caillot. Il s’agi† de la thrombectomie mécanique, action de retrait du caillot par capture dans un s†en† (sorte de petit ressort métallique) ou aspiration directe. Since the reference medicine does not always work and has drawbacks, alternative treatment is to directly remove the clot. It is a question of mechanical thrombectomy, action of removing the clot by capture in a s † en † (sort of small metal spring) or direct aspiration.
Depuis le pli de l’aine, une tubulure (cathéter) es† introduite dans l’artère de la jambe. Le neuroradiologue fai† alors progresser ce††e tubulure jusque dans les artères cérébrales, guidé en temps réel par rayons X. Un guidage extrêmement précis es† absolument nécessaire puisqu’il tau† « naviguer » dans des artères de 2 à 4 millimètres de diamètre, sans les blesser, ce qui entraînerai† une hémorragie cérébrale, potentiellement mortelle. Ce††e navigation se poursuit jusqu’à entrer en contact avec le caillot, le capturer e† l’extraire du corps du patient. Ce sauvetage es† réalisé avec succès dans près de 90% des cas. Le flux sanguin es† rétabli, permettant d’irriguer à nouveau le cerveau e† de limiter les conséquences cliniques de ce††e occlusion. Depuis la démonstration scientifique du bénéfice pour les patients, la thrombectomie es† recommandée dans tous les cas d’AVC de moins de six heures avec présence d’un caillot au niveau d’une artère proximale. From the fold of the groin, a tube (catheter) is † introduced into the artery of the leg. The neuroradiologist then † advances this tubing into the cerebral arteries, guided in real time by X-rays. Extremely precise guidance is absolutely necessary since it will have to “navigate” in arteries 2 to 4 millimeters in diameter, without injuring them, resulting in † potentially life-threatening brain hemorrhage. This †† e navigation continues until it comes into contact with the clot, captures it and extracts it from the patient's body. This rescue is successfully carried out in almost 90% of the cases. The blood flow is restored, making it possible to irrigate the brain again and to limit the clinical consequences of this occlusion. Since the scientific demonstration of the benefit for patients, thrombectomy is recommended in all cases of stroke of less than six hours with the presence of a clot at the level of a proximal artery.
L’association de la thrombectomie à la thrombolyse intraveineuse (TIV) apparai† désormais être le traitement de référence des accidents vasculaires cérébraux ischémiques (AVCi) avec occlusion proximale dans les 6 premières heures. Au-delà de ce délai, la thrombectomie seule dans les 24 premières heures pourrai† être envisagée chez des patients sélectionnés. The association of thrombectomy with intravenous thrombolysis (IVT) now appears to be the standard treatment for ischemic stroke (AVCi) with proximal occlusion in the first 6 hours. Beyond this period, thrombectomy alone within the first 24 hours may be considered in selected patients.
La thrombectomie mécanique es† un bel exemple de la mise à disposition rapide e† efficace du progrès médical pour les patients victimes d’AVC. Mechanical thrombectomy is a fine example of the rapid and effective provision of medical progress for stroke patients.
Toutefois, une telle formation es† parmi les plus longues e† exigeantes au monde (trois ans de formation réalisée près de dix ans après le début des études de médecine) . De plus, elle requiert des praticiens formés à la neuroradiologie interventionnelle pour réaliser des interventions permettant de sauver des vies e† prévenir le handicap. Enfin, elle ne concerne que des patients sélectionnés présentant un caillot au niveau d’une artère proximale. However, such training is among the longest and most demanding in the world (three years of training carried out almost ten years after the start of medical studies). In addition, it requires practitioners trained in interventional neuroradiology to perform interventions that save lives and prevent disability. Finally, it only concerns selected patients with a clot at a proximal artery.
De nouvelles options thérapeutiques son† indispensables pour étendre la fenêtre thérapeutique de la thrombolyse e† pour fournir une neuroprotection pour ralentir la mort cellulaire après un accident vasculaire cérébral. New therapeutic options are essential to extend the therapeutic window of thrombolysis and to provide neuroprotection to slow cell death after a stroke.
On connaît par exemple le document WO2015023830 qui décrit l’utilisation de l’isoxsuprine (agoniste p2-adrénergique) ou de chlorphenesine pour le traitement d’accidents vasculaires cérébraux ischémique ou hémorragique. For example, document WO2015023830 is known which describes the use of isoxsuprine (p2-adrenergic agonist) or chlorphenesine for the treatment of ischemic or hemorrhagic stroke.
Un autre composé identifié par screening à haut débit pour lutter contre les accidents vasculaires cérébraux a également été rapporté (Wang JK e† al., Cardiac glycosides provide neuroprotection agains† ischémie stroke: discovery by a brain slice-based compound screening platform. Proc Na†l Acad Sei US A. 2006; 103:10461 -6) . Dans ce††e étude, des tranches de cerveau organotypiques de ra† on† été utilisées pour évaluer les effets de petites molécules sur la survie neuronale dans un modèle d'AVC. L'étude a identifié la neriifoline, un glycoside cardiaque, en tan† que médicament candidat contre l'AVC dans le modèle OGD et a confirmé la fonctionnalité in vivo de la molécule dans un modèle animal d'AVC. Another compound identified by high-throughput screening to combat stroke has also been reported (Wang JK e † al., Cardiac glycosides provide neuroprotection agains † ischemia stroke: discovery by a brain slice-based compound screening platform. Proc Na † Acad Sci US A. 2006; 103: 10461 -6). In this study, organotypic brain slices of ra † on † were used to assess the effects of small molecules on neuronal survival in a stroke model. Study identified neriifoline, a heart glycoside, as a candidate drug against stroke in the OGD model and confirmed the in vivo functionality of the molecule in an animal model of stroke.
Il n'y a toutefois apparemment pas encore de médicament approuvé pour protéger contre les dommages neuronaux en cours après l'évènement aigu, ce qui peu† aggraver la situation clinique de manière significative. Il n'y a également apparemment pas non plus encore de médicament approuvé pour tous les patients qui ne son† pas admissibles au traitement thrombolytique ou par thrombectomie mécanique. However, there is apparently no drug yet approved to protect against ongoing neural damage after the acute event, which can significantly worsen the clinical situation. Nor is there apparently yet a drug approved for all patients who are not eligible for thrombolytic therapy or mechanical thrombectomy.
Problème technique Technical problem
Considérant ce qui précède, un problème que se propose de résoudre la présente invention es† de fournir un nouveau composé pour le traitement de patients présentant un accident vasculaire cérébral, qui puisse agir seul chez des patients non admissibles à un traitement thrombolytique ou par thrombectomie mécanique, ou combiné à un traitement thrombolytique ou par thrombectomie mécanique. Considering the above, a problem which the present invention proposes to solve is to provide a new compound for the treatment of patients suffering from a cerebrovascular accident, which can act alone in patients not eligible for thrombolytic treatment or by mechanical thrombectomy. , or combined with thrombolytic treatment or mechanical thrombectomy.
Solution technique Technical solution
La solution à ce problème posé a pour premier objet un peptide analogue de la thymuline (PAT) correspondant à la séquence suivante : The first object of the solution to this problem is a thymulin-like peptide (PAT) corresponding to the following sequence:
SEQ ID n°l : Glu-Ala-Lys-Ser-GIn-Gly-Gly-Ser-Asp (EAKSQGGSD), ou un de ses sels pharmaceutiquemen† acceptables, SEQ ID No. 1: Glu-Ala-Lys-Ser-GIn-Gly-Gly-Ser-Asp (EAKSQGGSD), or a pharmaceutically acceptable salt thereof,
pour son utilisation dans la prévention et/ou le traitement des accidents vasculaires cérébraux. for use in the prevention and / or treatment of stroke.
Elle a pour second objet une composition comprenant un peptide utilisé selon l’invention, dissous dans un solvant approprié, éventuellement accompagné de conservateur, agent tampon, adjuvant d’isotonicité, agent tensioactif pris seul ou en mélange. Its second object is a composition comprising a peptide used according to the invention, dissolved in an appropriate solvent, optionally accompanied by a preservative, a buffering agent, an isotonicity adjuvant, a surfactant taken alone or as a mixture.
Avantages apportés Benefits
Le peptide PAT utilisé selon l’invention de SEQ ID n°l (Glu-Ala-Lys-Ser- Gln-Gly-Gly-Ser-Asp = EAKSQGGSD) a été initialement développé comme une substance an†i-inflamma†oire puissante dans les pathologies auto immunes (polyarthrite rhumatoïde, maladie de Crohn) . Des travaux antérieurs on† également montré que ce peptide présente des activités neuroprotectrices dans certains modèles in vitro (neurones de culture mésencéphalique e† épineuse, utilisés principalement pour étudier les maladies neuro-dégénératives) . Il présente ainsi des propriétés anti inflammatoires associées à une activité neuroprofecfrice, sans danger jusqu'à des doses intraveineuses élevées (40 mg) e† une demi-vie courte (5 min dans le plasma) . The PAT peptide used according to the invention of SEQ ID No. 1 (Glu-Ala-Lys-Ser- Gln-Gly-Gly-Ser-Asp = EAKSQGGSD) was initially developed as a powerful an † i-inflamma † ory substance in auto immune pathologies (rheumatoid arthritis, Crohn's disease). Previous work has also shown that this peptide exhibits neuroprotective activities in certain in vitro models (neurons of mesencephalic and thorny culture, used mainly for studying neurodegenerative diseases). It thus presents anti-inflammatory properties associated with neuroprofecfrice activity, harmless up to high intravenous doses (40 mg) e † a short half-life (5 min in plasma).
De manière inattendue, le Demandeur a décidé d’utiliser ce peptide pour le traitement de l'AVC, en particulier de l’AVCi en bénéficiant de ses activités inflammatoire et antioxydante capables de réduire la mort neuronale et de traiter les AVC. Unexpectedly, the Applicant decided to use this peptide for the treatment of stroke, in particular stroke, by benefiting from its inflammatory and antioxidant activities capable of reducing neuronal death and treating strokes.
Dans ceffe description, à moins qu’il n’en soif spécifié autrement, il es† entendu que, lorsqu’un intervalle es† donné, il inclut les bornes supérieure e† inférieure dudi† intervalle. In this description, unless otherwise specified, it is understood that, when a given interval, it includes the upper and lower bounds of the interval.
Brève description des dessins Brief description of the drawings
L’invention e† les avantages qui en découlent seront mieux compris à la lecture de la description e† des modes de réalisation non limitatifs qui suivent, illustrés au regard des dessins annexés dans lesquels : The invention and the advantages which result therefrom will be better understood on reading the description and of the nonlimiting embodiments which follow, illustrated with reference to the appended drawings in which:
La Figure 1 représente l'effet neuroprotecteur du PAT de SEQ ID n°l selon l’invention sur un modèle de stress oxydatif neuronal cortical primaire chez le ra† don† les cellules son† privées d'oxygène / glucose (OGD) . FIG. 1 represents the neuroprotective effect of the PAT of SEQ ID No. 1 according to the invention on a model of primary cortical neuronal oxidative stress in ra † don † son † cells deprived of oxygen / glucose (OGD).
La Figure 2 représente l'effet contre l’infarctus du PAT de SEQ ID n°l selon l’invention dans un modèle d'occlusion de l'artère cérébrale moyenne (MCAO) chez la souris. FIG. 2 represents the effect against the PAT infarction of SEQ ID No. 1 according to the invention in a model of occlusion of the middle cerebral artery (MCAO) in mice.
Description des modes de réalisation Description of the embodiments
L’invention concerne un peptide analogue de la thymuline (PAT) correspondant à la séquence suivante : The invention relates to a thymulin analog peptide (PAT) corresponding to the following sequence:
SEQ ID n°l : Glu-Ala-Lys-Ser-GIn-Gly-Gly-Ser-Asp (EAKSQGGSD), ou un de ses sels pharmaceutiquemen† acceptables, SEQ ID No. 1: Glu-Ala-Lys-Ser-GIn-Gly-Gly-Ser-Asp (EAKSQGGSD), or a pharmaceutically acceptable salt thereof,
pour son utilisation dans la prévention et/ou le traitement des accidents vasculaires cérébraux. for use in the prevention and / or treatment of stroke.
Par sel pharmaceutiquemen† acceptable, on entend de manière non limitative un acétate, un sulfate ou un chlorhydrate. The term “acceptable pharmaceutically acceptable salt” means, without limitation, an acetate, a sulfate or a hydrochloride.
L'utilisation d'un PAT de SEQ ID n°l selon l’invention dans lequel un ou plusieurs acides aminés son† en configuration D es† également décrite. The use of a PAT of SEQ ID No. 1 according to the invention in which one or more amino acids are † in configuration D es † also described.
Par accident vasculaire cérébral, on entend une pathologie se caractérisant par une chute brutale du débit sanguin dans une partie du cerveau. Les neurones touchés par le manque d’oxygène et de glucose libèrent des cytokines pro-inflammatoires et des espèces réactives oxygénées (ROS) qui endommagent le tissu. Dans les minutes suivant la formation de l’occlusion, la réaction inflammatoire s’aggrave par l’activation des cellules microgliales ainsi que la sécrétion de molécules d’adhésions causant la libération de nouvelles cytokines et ROS. Ainsi, les tissus son† de plus en plus endommagés par le recrutement via infiltration des cellules de l’inflammation allant jusqu’à l’activation de matrices de métalloprotéases provocant une perturbation de la barrière hémato-encéphalique ainsi que des œdèmes. By stroke, we mean a pathology characterized by a sudden drop in blood flow in part of the brain. Neurons affected by lack of oxygen and glucose release pro-inflammatory cytokines and reactive oxygen species (ROS) that damage tissue. In the minutes following the formation of the occlusion, the inflammatory reaction worsens with the activation of microglial cells as well as the secretion of adhesion molecules causing the release of new cytokines and ROS. Thus, the tissues are increasingly damaged by recruitment via infiltration of the inflammation cells, up to the activation of metalloprotease matrices causing a disruption of the blood-brain barrier as well as edema.
Le PAT de SEQ ID n°l selon l’invention es† utilisé pour traiter des patients chez lesquels l'apport sanguin au cerveau es† bloqué (AVC ischémique), ou lorsqu’un vaisseau sanguin dans le cerveau es† rompu (AVC hémorragique) . The PAT of SEQ ID No. 1 according to the invention is used to treat patients in whom the blood supply to the brain is blocked (ischemic stroke), or when a blood vessel in the brain is ruptured (hemorrhagic stroke). ).
Par patient, on entend un être humain ou un animal, tel que par exemple un chien ou un cha†, préférentiellement un être humain. By patient is meant a human being or an animal, such as for example a dog or a cha †, preferably a human being.
Préférentiellement, le PAT de SEQ ID n°l selon l’invention es† utilisé dans le traitement d’un accident vasculaire cérébral ischémique aigu (AVCi) . Preferably, the PAT of SEQ ID No. 1 according to the invention is used in the treatment of an acute ischemic stroke (AVCi).
Le PAT de SEQ ID n°l utilisé selon l’invention es† préférentiellement sous une forme adaptée pour une administration par voie parentérale, en particulier sous cutanée (s.e.), intranasale, intrapéritonéale (i.p.) e† intraveineuse (i.v.) . The PAT of SEQ ID No. 1 used according to the invention is preferably in a form suitable for administration by the parenteral route, in particular subcutaneous (w.m.), intranasal, intraperitoneal (i.p.) and intravenous (i.v.) administration.
Le PAT de SEQ ID n°l utilisé selon l’invention es† plus préférentiellement sous une forme adaptée pour une administration par voie intraveineuse en bolus. The PAT of SEQ ID No. 1 used according to the invention is more preferably in a form suitable for administration by intravenous bolus.
Le PAT de SEQ ID n°l utilisé selon l’invention es† administré à un dosage compris entre 0, 1 e† 50 mg/kg, de préférence entre 1 e† 10 mg/kg, par exemple 2 mg/kg, 3 mg/kg, 4 mg/kg, 5 mg/kg, 6 mg/kg, 7 mg/kg, 8 mg/kg ou 9 mg/kg, plus préférentiellement 3 mg/kg. The PAT of SEQ ID No. 1 used according to the invention is administered at a dosage of between 0.1 and 50 mg / kg, preferably between 1 and 10 mg / kg, for example 2 mg / kg, 3 mg / kg, 4 mg / kg, 5 mg / kg, 6 mg / kg, 7 mg / kg, 8 mg / kg or 9 mg / kg, more preferably 3 mg / kg.
Avantageusement, le PAT de SEQ ID n°l utilisé selon l’invention es† administré seul chez des patients non admissibles à un traitement thrombolytique ou par thrombectomie mécanique. Advantageously, the PAT of SEQ ID No. 1 used according to the invention is administered alone in patients not eligible for thrombolytic treatment or by mechanical thrombectomy.
Selon un autre mode de réalisation, l’invention concerne un produit de combinaison comprenant le PAT de SEQ ID n°l combiné à un traitement thrombolytique, pour son utilisation dans la prévention et/ou le traitement des accidents vasculaires cérébraux. Selon un mode de réalisation particulièrement avantageux de l’invention, le PAT de SEQ ID n°l est combiné à un traitement thrombolytique, préférentiellement choisi parmi un activateur tissulaire recombinan† du plasminogène (rt-PA), l’altéplase, la streptokinase, la desmotéplase, le ténectéplase, la microplasmine, le rétéplase, l’ancrod e† l’urokinase. According to another embodiment, the invention relates to a combination product comprising the PAT of SEQ ID No. 1 combined with a thrombolytic treatment, for its use in the prevention and / or the treatment of cerebrovascular accidents. According to a particularly advantageous embodiment of the invention, the PAT of SEQ ID No. 1 is combined with a thrombolytic treatment, preferably chosen from a recombinant tissue activator † of plasminogen (rt-PA), alteplase, streptokinase, desmoteplase, tenecteplase, microplasmin, reteplase, anchor and urokinase.
De préférence, le PAT de SEQ ID n°l es† combiné à un traitement thrombolytique jusqu’à 6 heures après la survenue des premiers signes de l’accident vasculaire cérébral. Preferably, the PAT of SEQ ID No. 1 is combined with thrombolytic treatment up to 6 hours after the onset of the first signs of the cerebrovascular accident.
Selon un autre mode de réalisation de l’invention, le PAT de SEQ ID n°l es† combiné à un traitement par thrombectomie mécanique, pour son utilisation dans la prévention et/ou le traitement des accidents vasculaires cérébraux. According to another embodiment of the invention, the PAT of SEQ ID No. 1 is combined with a treatment by mechanical thrombectomy, for its use in the prevention and / or the treatment of cerebrovascular accidents.
Selon un autre mode de réalisation particulièrement avantageux de l’invention, le PAT de SEQ ID n°l es† combiné à un traitement par thrombectomie mécanique. According to another particularly advantageous embodiment of the invention, the PAT of SEQ ID No. l es † combined with a treatment by mechanical thrombectomy.
De préférence, le PAT de SEQ ID n°l es† combiné à un traitement par thrombectomie mécanique jusqu’à 24 heures après la survenue des premiers signes de l’accident vasculaire cérébral. Preferably, the PAT of SEQ ID No. 1 is combined with treatment by mechanical thrombectomy up to 24 hours after the onset of the first signs of the cerebrovascular accident.
Un autre objet concerne une composition comprenant un PAT de SEQ ID n°l pour son utilisation selon l’invention, dissous dans un solvant approprié, éventuellement accompagné de conservateur, agent tampon, adjuvant d’isofonicifé, agent fensioacfif pris seul ou en mélange. Another object relates to a composition comprising a PAT of SEQ ID No. 1 for its use according to the invention, dissolved in an appropriate solvent, optionally accompanied by a preservative, a buffering agent, an isofonicate adjuvant, a surfactant taken alone or as a mixture.
Dans fous les cas, l'homme de méfier veillera à ce que les adjuvants ainsi que leurs proportions soient choisis de telle manière à ne pas nuire aux propriétés avantageuses recherchées de la composition selon l’invention. Exemples In all cases, the wary man will ensure that the adjuvants and their proportions are chosen so as not to harm the desired advantageous properties of the composition according to the invention. Examples
La présente invention va maintenant être illustrée au moyen des exemples suivants. The present invention will now be illustrated by means of the following examples.
Exemple 1 : Validation de l’activité du PAT de SEQ ID n°l selon l’invention dans des modèles cellulaires privés d’oxygène e† de glucose (OGD) {in vitro ) Example 1: Validation of the PAT Activity of SEQ ID No. 1 According to the Invention in Cell Models Deprived of Oxygen and Glucose (OGD) (In Vitro)
Ce††e étude vise à examiner l'effet du PAT de SEQ ID n°l selon l’invention (6 concentrations) sur un modèle de stress oxydatif neuronal cortical primaire chez le ra† don† les cellules son† privées d'oxygène / glucose (OGD) ; le riluzole es† utilisé comme contrôle positif. 1 heure avant OGD, les neurones son† traités avec le PAT de SEQ ID n°l selon l’invention. This †† th study aims to examine the effect of the PAT of SEQ ID No. 1 according to the invention (6 concentrations) on a model of primary cortical neuronal oxidative stress in RA † don † son † cells deprived of oxygen / glucose (OGD); riluzole is † used as a positive control. 1 hour before OGD, the neurons are treated with the PAT of SEQ ID No. 1 according to the invention.
Le modèle utilisé dans cette approche préliminaire permet de mimer une mort neuronale ischémique e† d’identifier un éventuel effet neuroprotecteur du peptide selon l’invention testé. The model used in this preliminary approach makes it possible to mimic ischemic neuronal death and to identify a possible neuroprotective effect of the peptide according to the invention tested.
Protocole Protocol
Les neurones corticaux de ra† on† été cultivés comme décrit par Singer The cortical neurons of ra † on † were cultivated as described by Singer
( 1 999) . (1,999).
Après 10 jours de culture de neurones, le composé à tester (peptide selon l’invention ou riluzole) a été pré-incubé avec des neurones pendant 1 heure avant la privation d'oxygène e† de glucose. Ensuite, le milieu de culture a été retiré e† un milieu de culture frais sans glucose a été ajouté. Les cellules on† été transférées dans un incubateur anaérobie en conditions 95% N2 / 5% C02 à 37°C. Au bout de 2 heures, 25 mM de D-glucose on† été ajoutés dans le milieu de culture e† les cellules on† été transférées dans un incubateur classique avec 95% air / 5% C02 à 37°C. Le riluzole à 5 mM (SIGMA, Ref: RI 1 6, lot: 057K3900V) a été utilisé comme composé de référence, 6 puits par condition. Les conditions suivantes on† été réalisées : After 10 days of neuron culture, the test compound (peptide according to the invention or riluzole) was pre-incubated with neurons for 1 hour before deprivation of oxygen and † of glucose. Then the culture medium was removed and fresh culture medium without glucose was added. The cells were transferred to an anaerobic incubator under 95% N2 / 5% C02 conditions at 37 ° C. After 2 hours, 25 mM D-glucose was added to the culture medium and the cells were transferred to a conventional incubator with 95% air / 5% CO 2 at 37 ° C. 5 mM riluzole (SIGMA, Ref: RI 1 6, lot: 057K3900V) was used as the reference compound, 6 wells per condition. The following conditions have been met:
• Condition contrôle (milieu de culture) • Control condition (culture medium)
• OGD (2h) + condition contrôle (milieu de culture) • OGD (2h) + control condition (culture medium)
• OGD (2h) + Riluzole (5 mM) comme composé de référence • OGD (2h) + Riluzole (5 mM) as reference compound
• OGD (2h) + PAT de SEQ ID n°l selon l’invention ( 10 mM, 1 mM, 100 nM, 10 nM, 1 nM, 0, 1 nM) • OGD (2h) + PAT of SEQ ID No. 1 according to the invention (10 mM, 1 mM, 100 nM, 10 nM, 1 nM, 0, 1 nM)
Evaluation : mesure du nombre total de neurones corticaux Assessment: measurement of the total number of cortical neurons
Après 24 heures de reperfusion d’oxygène/glucose, les cellules on† été fixées par une solution froide d'éthanol à 95% / acide acétique (5%) pendant 5 minutes. Les cellules on† ensuite été perméabilisées e† les sites non spécifiques on† été bloqués. La survie neuronale totale a été évaluée (le nombre de corps cellulaires neuronaux MAP2 positifs a été compté) . Pour chaque puits de culture, 20 photos par puits on† été prises en utilisant le matériel InCell ANALYZER™ 2200 (GE HEALTHCARE) avec un grossissement x 20. Toutes les photos on† été prises dans les mêmes conditions. Le nombre de neurones a été automatiquement évalué avec le système d’analyse fourni (GE HEALTHCARE) . Statistiques After 24 hours of oxygen / glucose reperfusion, the cells were fixed with a cold 95% ethanol / acetic acid (5%) solution for 5 minutes. The cells were then permeabilized and the non-specific sites were blocked. Total neuronal survival was assessed (the number of positive MAP2 neural cell bodies was counted). For each culture well, 20 photos per well were taken using the InCell ANALYZER ™ 2200 material (GE HEALTHCARE) with a magnification x 20. All the photos were taken under the same conditions. The number of neurons was automatically evaluated with the analysis system provided (GE HEALTHCARE). Statistics
Toutes les données ont été exprimées en moyenne +/- erreur standard de la moyenne (s.e.m) (6 données par condition expérimentale, 1 culture) . Une analyse globale des données a été réalisée en utilisant une analyse de variance à un facteur (ANOVA) suivant le test de Dunnett. La significativité statistique a été fixée à p < 0,05. All data were expressed as mean +/- standard error of mean (s.e.m) (6 data by experimental condition, 1 culture). A global analysis of the data was performed using a factor analysis of variance (ANOVA) following the Dunnett test. Statistical significance was set at p <0.05.
Résultats Results
La privation d'oxygène / glucose a induit une diminution importante et significative de la survie des neurones (***, p < 0,001 ; 51 % du contrôle) . Le Riluzole à 5 mM a été capable d’inverser la perte neuronale (*, p < 0,05 ; 84,34% du contrôle) ce qui valide l’étude. Le composé PAT de SEQ ID n°l selon l’invention testé a été capable de restaurer significativement la survie des neurones à une concentration d’au moins 10 mM (75,24% du contrôle) mais pas à des concentrations plus faibles. Deprivation of oxygen / glucose induced a significant and significant decrease in the survival of neurons (***, p <0.001; 51% of control). The 5 mM Riluzole was able to reverse the neuronal loss (*, p <0.05; 84.34% of control) which validates the study. The PAT compound of SEQ ID No. 1 according to the invention tested was able to significantly restore the survival of neurons at a concentration of at least 10 mM (75.24% of the control) but not at lower concentrations.
Conclusion et discussion Conclusion and discussion
L'étude OGD ainsi réalisée met en évidence un effet neuroprotecteur du PAT de SEQ ID n°l selon l’invention à une concentration d’au moins 10 mM. The OGD study thus carried out highlights a neuroprotective effect of the PAT of SEQ ID No. 1 according to the invention at a concentration of at least 10 mM.
Le protocole de test OGD utilisé est un protocole standard qui prévoit que les neurones soient pré-incubés 1 heure avant OGD avec le composé à tester. Or, le PAT de SEQ ID n°l selon l’invention a une très courte demi-vie dans le plasma (<5 min) ce qui pourrait expliquer une faible activité à des concentrations inférieures à 10 mM. The OGD test protocol used is a standard protocol which provides that the neurons are pre-incubated 1 hour before OGD with the compound to be tested. However, the PAT of SEQ ID No. 1 according to the invention has a very short half-life in plasma (<5 min) which could explain a low activity at concentrations below 10 mM.
Exemple 2 : Effets protecteurs du PAT de SEQ ID n°l selon l’invention dans l'AVC, identification de la "fenêtre thérapeutique" la mieux adaptée, et analyse de la synergie du PAT de SEQ ID n°l avec l'utilisation thrombolytique EXAMPLE 2 Protective Effects of the PAT of SEQ ID No. 1 According to the Invention in the Stroke, Identification of the Most Adequate "Therapeutic Window" and Analysis of the Synergy of the PAT of SEQ ID No. 1 with the Use thrombolytic
Cette étude a pour but d’examiner l'effet du PAT de SEQ ID n°l selon l’invention dans un modèle d'occlusion de l'artère cérébrale moyenne (MCAO) . Dans ce modèle, une occlusion intraluminale du MCAO est réalisée avec un monofilament recouvert de silicone et cette artère est maintenue occluse pendant une heure. The purpose of this study is to examine the effect of the PAT of SEQ ID No. 1 according to the invention in a model of occlusion of the middle cerebral artery (MCAO). In this model, an intraluminal occlusion of the MCAO is performed with a monofilament covered with silicone and this artery is kept occluded for one hour.
Protocole Protocol
Le PAT de SEQ ID n°l selon l’invention a été testé à l'aide du modèle monofilament intraluminale de l'artère cérébrale moyenne d'occlusion (MCAO) qui consiste en l'insertion d'un filament chirurgical dans l'artère carotide externe et le filetage vers l'avant dans l'artère carotide interne (ACI) jusqu'à ce que la pointe occulte l'origine de la MCA, entraînant un arrêt du flux sanguin et un infarctus cérébral subséquent dans les territoires du MCA. The PAT of SEQ ID No. 1 according to the invention was tested using the intraluminal monofilament model of the cerebral middle occlusion artery (MCAO) which consists of the insertion of a surgical filament into the artery external carotid and forward threading in the internal carotid artery (ICA) until the tip obscures the origin of the MCA, causing blood flow to stop and a subsequent cerebral infarction in the MCA territories.
Le PAT de SEQ ID n°l selon l’invention es† administré par voie intraveineuse à deux doses ( 1 mg/kg e† 3 mg/kg) 30 minutes après l'occlusion e† 30 minutes avant le retrait du monofilamen†. The PAT of SEQ ID No. 1 according to the invention is administered intravenously at two doses (1 mg / kg e † 3 mg / kg) 30 minutes after occlusion e † 30 minutes before removal of the monofilamen †.
La température corporelle de la souris es† maintenue à 37°C. Puis, la suture es† retirée une heure après l'occlusion, ce qui permet la reperfusion ; la reperfusion peu† être réalisée avec succès ou non. La gravité de l'ischémie es† évaluée par débit métrie non invasive au laser à effet Doppler. Une reperfusion survient si une augmentation ultérieure du débit sanguin es† mesurée. The body temperature of the mouse is maintained at 37 ° C. Then, the suture is removed one hour after occlusion, which allows reperfusion; reperfusion may or may not be successful. The severity of ischemia is † assessed by non-invasive metric flow using the Doppler laser. Reperfusion occurs if a subsequent increase in blood flow is measured.
24 heures après l'induction de MCAO, les souris son† anesthésiées e† euthanasiées par dislocation cervicale. Les cerveaux son† conservés congelés dans l'isopentane. 24 hours after the induction of MCAO, the mice are anesthetized and euthanized by cervical dislocation. Brains are † stored frozen in isopentane.
Les cerveaux son† coupés en tranches de 20 microns avec une coupe cryostatique puis colorés avec du viole† de crésyle pour permettre une évaluation microscopique de la taille de l'infarctus e† son† montés sur des lames à revêtement ENTELLAN®. The brains are cut into 20 micron slices with a cryostatic section and then stained with cresyl viol † to allow a microscopic evaluation of the size of the infarction and † mounted on ENTELLAN® coated slides.
La taille de l'infarctus es† ensuite mesurée en utilisant un outil d'imagerie Cell-Lens. The size of the infarction is then measured using a Cell-Lens imaging tool.
Résultats Results
Dans le groupe où il y a eu reperfusion, l'administration de PAT de SEQ ID n°l selon l’invention à 3 mg/kg juste après l'élimination du filament a permis une diminution de la taille de l'infarctus de 42%. In the group where there was reperfusion, the administration of PAT of SEQ ID No. 1 according to the invention at 3 mg / kg just after the elimination of the filament made it possible to reduce the size of the infarction by 42 %.
Conclusions e† discussion Conclusions and discussion
Le modèle MCAO utilisé es† un modèle standardisé ; l'élimination des filaments stimule la reperfusion. The MCAO model used is a standardized model; removing filaments stimulates reperfusion.
Ce modèle es† assez éloigné de l'élimination du caillot par l’utilisation d’un thrombolytique mais es† plus proche de la thrombectomie lorsque le caillot es† enlevé chirurgicalement. This model is somewhat distant from the elimination of the clot by the use of a thrombolytic but is closer to thrombectomy when the clot is removed surgically.
Avec ce modèle, l'administration du PAT de SEQ ID n°l selon l’invention a permis d'obtenir une diminution très significative de la faille de l'infarctus. With this model, the administration of the PAT of SEQ ID No. 1 according to the invention made it possible to obtain a very significant reduction in the fault of the infarction.

Claims

REVENDICATIONS
1 . Peptide analogue de la thymuline (PAT) caractérisé en ce qu’il correspond à la séquence suivante : 1. Thymulin analog peptide (PAT) characterized in that it corresponds to the following sequence:
SEQ ID n°l : Glu-Ala-Lys-Ser-GIn-Gly-Gly-Ser-Asp (EAKSQGGSD), ou un de ses sels pharmaceutiquement acceptables, SEQ ID No. 1: Glu-Ala-Lys-Ser-GIn-Gly-Gly-Ser-Asp (EAKSQGGSD), or a pharmaceutically acceptable salt thereof,
pour son utilisation dans la prévention et/ou le traitement des accidents vasculaires cérébraux. for use in the prevention and / or treatment of stroke.
2. Peptide selon la revendication 1 , pour son utilisation dans le traitement d’un accident vasculaire cérébral ischémique aigu. 2. Peptide according to claim 1, for its use in the treatment of an acute ischemic stroke.
3. Peptide pour son utilisation selon la revendication 1 ou 2, caractérisé en ce qu’il est sous une forme adaptée pour une administration par voie parentérale. 3. Peptide for its use according to claim 1 or 2, characterized in that it is in a form suitable for administration by the parenteral route.
4. Peptide pour son utilisation selon la revendication 3, caractérisé en ce qu’il es† sous une forme adaptée pour u ne administration par voie intraveineuse en bolus. 4. Peptide for its use according to claim 3, characterized in that it is † in a form suitable for u bolus administration intravenously.
5. Peptide pour son utilisation selon l’une des revendications 1 à 4, caractérisé en ce qu’il es† utilisé à un dosage compris entre 0, 1 e† 50 mg/kg, de préférence entre 1 e† 10 mg/kg, plus préférentiellement 3 mg/kg. 5. Peptide for its use according to one of claims 1 to 4, characterized in that it is † used at a dosage of between 0.1 and 50 mg / kg, preferably between 1 and 10 mg / kg , more preferably 3 mg / kg.
6. Produit de combinaison comprenant un peptide selon l’une des revendications 1 à 5, caractérisé en ce que ledit peptide es† combiné à un traitement thrombolytique, préférentiellement choisi parmi un activateur tissulaire recombinan† du plasminogène (r†-PA), l’altéplase, la streptokinase, la desmotéplase, le ténectéplase, la microplasmine, le rétéplase, l’ancrod e† l’urokinase, pour son utilisation dans la prévention et/ou le traitement des accidents vasculaires cérébraux. 6. Combination product comprising a peptide according to one of claims 1 to 5, characterized in that said peptide is † combined with a thrombolytic treatment, preferably chosen from a recombinant tissue activator † of plasminogen (r † -PA), l alteplase, streptokinase, desmoteplase, tenecteplase, microplasmin, reteplase, anchor and urokinase, for use in the prevention and / or treatment of stroke.
7. Produit de combinaison selon la revendication 6, caractérisé en ce que ledit peptide es† combiné à un traitement thrombolytique jusqu’à 6 heures après la survenue des premiers signes de l’accident vasculaire cérébral. 7. Combination product according to claim 6, characterized in that said peptide is † combined with a thrombolytic treatment up to 6 hours after the onset of the first signs of stroke.
8. Peptide selon l’une des revendications 1 à 5, caractérisé en ce qu’il est combiné à un traitement par thrombectomie mécanique, pour son utilisation dans la prévention et/ou le traitement des accidents vasculaires cérébraux. 8. Peptide according to one of claims 1 to 5, characterized in that it is combined with a treatment by mechanical thrombectomy, for its use in the prevention and / or the treatment of cerebrovascular accidents.
9. Peptide pour son utilisation selon la revendication 8, caractérisé en ce qu’il est combiné à un traitement par thrombectomie mécanique jusqu’à9. Peptide for its use according to claim 8, characterized in that it is combined with a treatment by mechanical thrombectomy until
24 heures après la survenue des premiers signes de l’accident vasculaire cérébral. 24 hours after the onset of the first signs of the stroke.
10. Composition caractérisée en ce qu’elle comprend un peptide pour son utilisation selon l’une des revendications 1 à 5, dissous dans un solvant approprié, éventuellement accompagné de conservateur, agent tampon, adjuvant d’isotonicité, agent tensioactif pris seul ou en mélange. 10. Composition characterized in that it comprises a peptide for its use according to one of claims 1 to 5, dissolved in an appropriate solvent, optionally accompanied by preservative, buffering agent, isotonicity adjuvant, surfactant taken alone or as a mixed.
PCT/EP2019/087004 2018-12-28 2019-12-24 Peptide analogue of thymulin (pat) for use in the treatment of strokes WO2020136191A1 (en)

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Citations (2)

* Cited by examiner, † Cited by third party
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WO2015023830A1 (en) 2013-08-14 2015-02-19 Stc.Unm Treatment and prevention of stroke and other neurological disorders
US20160074463A1 (en) * 2010-07-12 2016-03-17 Orphit Use of the pat nonapeptide in the treatment and prevention of neurodegenerative diseases

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20160074463A1 (en) * 2010-07-12 2016-03-17 Orphit Use of the pat nonapeptide in the treatment and prevention of neurodegenerative diseases
WO2015023830A1 (en) 2013-08-14 2015-02-19 Stc.Unm Treatment and prevention of stroke and other neurological disorders

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
BARED SAFIEH-GARABEDIAN ET AL: "Thymulin related peptide attenuates inflammation in the brain induced byintracerebroventricular endotoxin injection", NEUROPHARMACOLOGY, PERGAMON PRESS, OXFORD, GB, vol. 60, no. 2, 2 November 2010 (2010-11-02), pages 496 - 504, XP028365610, ISSN: 0028-3908, [retrieved on 20101105], DOI: 10.1016/J.NEUROPHARM.2010.11.004 *
JAMES K T WANG ET AL: "Cardiac glycosides Provide Neuroprotection Against Ischemic Stroke: Discovery by a Brain Slice-Based Compound Screening Platform", PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES (PNAS), US, vol. 103, no. 27, 5 July 2006 (2006-07-05), pages 10461 - 10466, XP008156205, ISSN: 0027-8424, DOI: 10.1073/PNAS.0600930103 *
SAFIEH-GARABEDIAN BARED ET AL: "Targeting inflammatory components in neuropathic pain: The analgesic effect of thymulin related peptide", NEUROSCIENCE LETTERS, vol. 702, 29 November 2018 (2018-11-29), pages 61 - 65, XP085684642, ISSN: 0304-3940, DOI: 10.1016/J.NEULET.2018.11.041 *
WANG JK ET AL.: "Cardiac glycosides provide neuroprotection against ischemic stroke: discovery by a brain slice-based compound screening platform", PROC NATL ACAD SEI US A., vol. 103, 2006, pages 10461 - 6, XP008156205, DOI: 10.1073/pnas.0600930103

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