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WO2006053923A1 - Utilisation de la serine-protease connue sous le nom d'ancrod dans le traitement de la dystrophie musculaire de duchenne - Google Patents

Utilisation de la serine-protease connue sous le nom d'ancrod dans le traitement de la dystrophie musculaire de duchenne Download PDF

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Publication number
WO2006053923A1
WO2006053923A1 PCT/ES2005/000619 ES2005000619W WO2006053923A1 WO 2006053923 A1 WO2006053923 A1 WO 2006053923A1 ES 2005000619 W ES2005000619 W ES 2005000619W WO 2006053923 A1 WO2006053923 A1 WO 2006053923A1
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WO
WIPO (PCT)
Prior art keywords
ancrod
treatment
muscle
muscular dystrophy
duchenne muscular
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PCT/ES2005/000619
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English (en)
Spanish (es)
Inventor
Purificación MUÑOZ CÀNOVES
Mónica SUELVES ESTEBAN
Mercè JARDÍ RIPOLL
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Centre De Regulació Genòmica
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Application filed by Centre De Regulació Genòmica filed Critical Centre De Regulació Genòmica
Publication of WO2006053923A1 publication Critical patent/WO2006053923A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y304/00—Hydrolases acting on peptide bonds, i.e. peptidases (3.4)
    • C12Y304/21—Serine endopeptidases (3.4.21)
    • C12Y304/21074—Venombin A (3.4.21.74)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/43—Enzymes; Proenzymes; Derivatives thereof
    • A61K38/46—Hydrolases (3)
    • A61K38/48—Hydrolases (3) acting on peptide bonds (3.4)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P21/00—Drugs for disorders of the muscular or neuromuscular system

Definitions

  • This invention is related to the field of human medicine, and specifically with a new use of a compound for the treatment of a specific human disease.
  • Duchenne muscular dystrophy is one of the most frequent and lethal X-linked diseases, and affects 1 in 3,500 boys. Because it is recessive and linked to the X chromosome, the DMD mainly affects men who inherit the disease through their mothers. Women may be carriers of DMD but do not have symptoms. The disease is usually diagnosed by muscle biopsy. It is characterized by a generalized weakness and a muscle loss that first affects the muscles of the hip, pelvic area, thighs and shoulders. As the disease progresses, the DMD affects the heart and muscles of the breath over time. The onset of the disease occurs in early childhood, between 2 and 6 years, and survival is uncommon beyond 30 years.
  • the DMD is caused by mutations in the gene that codes for the dystrophin protein, which is located in the internal part of the muscular sarcolenma.
  • Dystrophin is associated with a large set of membrane proteins, known as the dystrophin glycoprotein complex (DGC), which is important for the integrity of the cell membrane (cf. EP. Hoffman, et al. , "Dystrophin: The protein product of the duchenne muscular dystrophy locus", CeI1 1987, vol. 51, pp. 919-28; JM Ervasti, et al., "Membrane organization of the dystrophin-glycoprotein complex", CeI1 1991, vol 66, pp. 1121-31).
  • DGC dystrophin glycoprotein complex
  • the mdx mouse strain is the most widely used animal model of DMD, harboring a nonsense mutation in exon 23 that eliminates dystrophin expression (cf. P. Sicinski, et al., "The molecular basis of muscular dystrophy in the mdx mouse: A point mutation ", Science 1989, vol. 244, pp. 1578-80).
  • the inventors have demonstrated a significant accumulation of extravascular fibrin in regenerating muscle of mice deficient in uPA (uPA ';' ) and in mice deficient in plasminogen (PIg ' ' ' ) after experimentally induced muscle degeneration (damage by glycerol injection) .
  • the administration of ancrod reduces plasma fibrinogen levels in uPA-deficient mice and plasminogen-deficient mice, resulting in a substantial restoration of the normal muscle regeneration process (cf. F. Llu ⁇ s et al., "Urokinase-dependent plasminogen activation is required for efficient skeletal muscle regeneration in vivo ", Blood 2001, vol. 97, pp. 1703-11; M.
  • fibrin deposits clearly detect fibrin deposits and that deposition of fibrin specifically correlates with mdx dystrophinopathy.
  • the inventors have also found that the administration of ancrod leads to a decrease in muscle degeneration and a functional improvement of the mdx mouse.
  • the invention relates to the use of the ancrod for the preparation of a medicament for the treatment of Duchenne muscular dystrophy in a mammal.
  • the ancrod is a serine proteinase from Agkistrodon (registration number CAS 9046-56-4), a defibringing enzyme isolated from the poison of the Malaysian pit viper Agkistrodon rhodostoma Boie (Calloselasma rhodostoma Boie). It is a glycosylated serine protease of 234 amino acid residues. It cuts the fibrinogen to form soluble, non-polymerized fibrin and increases the local release of the tissue activator of the plasminogen.
  • the ancrod is being developed as a neuroprotective agent by Neurobiological Technologies to minimize the extent of cerebral infarction. It was previously released as Arvin by Knoll (now Abbott) as an anticoagulant. It has been approved in Canada for use as an antithrombotic in patients with heparin-induced thrombocytopenia.
  • Another aspect of the invention relates to a method of treating a mammal suffering from Duchenne muscular dystrophy, which comprises the administration of a therapeutically effective amount of ancrod, together with appropriate amounts of pharmaceutically acceptable diluents or carriers.
  • the invention provides a pharmaceutical composition for the treatment of Duchenne muscular dystrophy in a mammal, which comprises a therapeutically effective amount of ancrod, together with appropriate amounts of pharmaceutically diluents or carriers. acceptable.
  • the mammal is a human.
  • the ancrod treatment results in a significant reduction of the accumulation of fibrin in the muscles.
  • the muscle fibers appear much healthier and the muscle degeneration is significantly attenuated because there are fewer groups of degenerating myofibers and fewer cycles of degeneration and regeneration.
  • these result in a functional improvement of the state of the pathological state of the whole skeletal musculature.
  • an expert in the field will select an appropriate route of administration of the ancrod, such as subcutaneous or intravenous, among others.
  • an appropriate route of administration of the ancrod such as subcutaneous or intravenous, among others.
  • the person skilled in the art will also choose a suitable release system for the selected route of administration.
  • the figures show how the systemic depletion of the fibrin improves the muscular dystrophy of the rndx mouse.
  • the 12-day-old mdx mice were injected subcutaneously with ancrod (1 U / d for 18 days) or with a saline solution until 30 days of age.
  • FIG. 1A shows an increase in fibrin deposition in muscle of mdx mice, by western blot analysis of muscle extracts from normal (WT) and mdx mice, 14 and 30 days old, with an anti-fibrin antibody. ⁇ -Tubulin is a control.
  • FIG. 1B shows as the Ancrod treatment reduces fibrin levels in the mdx muscle by western blotting with an anti-fibrin antibody.
  • FIG. 1C represents a hematoxylin / eosin (HE) staining of muscle sections of treated (ancrod) and untreated (saline) mice. 50 ⁇ m bar
  • FIG. 2A-B shows how treatment with ancrod reduces the percentage of total area of muscle degeneration (% DA in FIG. 2A) and the average number of degenerative groups (DG in FIG. 2B) that contain more than 10 fibers per muscle section
  • FIG. 2C shows the effect of ancrod treatment, reducing the percentage of fibers with central nuclei (CNFs) of the muscles.
  • FIG. 3A represents the increase in muscular endurance time on a Rota rod apparatus of mice treated with ancrod.
  • RRT is the time in the Rota rod and is expressed in seconds (s).
  • FIG. 3B shows the reduction of serum creatine kinase concentration (CK, in U / 1) as a result of ancrod treatment.
  • Mdx mice were purchased from Jackson Laboratories (USA). The 12-day mdx mice were injected subcutaneously with ancrod (Sigma Chemical Co .; 1 U / d for 18 days) or with a saline solution until 30 days of life. After cervical dislocation, the muscles were isolated, frozen and stored at -80 ° C until analysis. Preparation of muscle extracts
  • Muscle extracts were prepared from gastrocnemius muscles in a 100 mM Tris-HCI buffer, pH 7.6, with 200 mM NaCI, 100 mM CaCI 2 and 0.4% Triton X-100.
  • the 10 ⁇ m sections were obtained from the central part of the muscles and stained with hematoxylin / eosin (HE). All analyzes and photographs were performed with a Leica DC 500 microscope equipped with a video camera.
  • HE hematoxylin / eosin
  • Muscle degeneration (%) was determined microscopically and expressed as a percentage of total muscle area. The number of DGs (degenerating groups) containing more than 10 degenerating fibers in muscle cross sections of mdx mice (saline or ancrod) was counted.
  • the regeneration of muscle fiber was determined microscopically and expressed as the percentage of total muscle fibers containing central nuclei (CNF) in an entire cross section of the muscle.
  • Assay of serum creatine kinase activity was determined microscopically and expressed as the percentage of total muscle fibers containing central nuclei (CNF) in an entire cross section of the muscle.
  • CK creatine kinase
  • blood samples were obtained from mdx mice treated with ancrod and saline (6 mice). Blood was allowed to clot for 1 hour Ia at 37 0 C before centrifuging at 150Ox g for 3 minutes. The serum was collected and the analyzes of the creatine kinase activity were done using the ThermoTrace enzyme assay following the manufacturer's instructions. The absorbance was measured at 340 nm every 20 seconds for 3 minutes at 37 0 C to calculate the enzymatic activity. The amount of serum CK is presented in units (U) of enzyme activity per liter (I) of serum.
  • the Rota rod test is used to assess the motor capacity of mice (maintenance of the ability to grip and suspend against gravity).
  • the Rota rod test was applied to mdx mice treated with ancrod and saline, using a Rota rod device (Rota rod LE8500, Panlab SA.)
  • fibrin accumulated in the muscle of mdx mice Before the onset of the disease (14 days of age), the fibrin deposits were detectable in the muscles of mdx mice. However, at the first peak of the disease (30 days of age), fibrin deposits were clearly noticeable in the muscles of mdx mice (cf. FIG. 1A). Since fibrin was not detected in muscles of wild and healthy mice of the same age, fibrin deposition correlates specifically with mdx dystrophinopathy. Ancrod was administered to mdx mice. producing systemic fibrinogen consumption and dramatically reducing plasma fibrinogen levels (cf. FIG. 1B) (cf. W.
  • the Rota rod test was performed to assess the muscular strength of the entire body.
  • the mice treated with ancrod Ia performed better than the controls (cf. FlG. 3A), which is consistent with the observed reduction in muscle degeneration.
  • serum creatine kinase (CK) was analyzed. High concentrations of CK were consistently detected in mdx mice because of sarcolenma damage.
  • Ancrod treatment resulted in a marked decrease in serum CK concentration compared to that in saline treated mice (cf. FIG. 3B).
  • the decrease in muscle degeneration and serum CK levels offers histological and biochemical evidence of a functional improvement of the muscle of mdx mice produced by the reduction of fibrin levels in vivo.

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  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • General Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Organic Chemistry (AREA)
  • Public Health (AREA)
  • Medicinal Chemistry (AREA)
  • Veterinary Medicine (AREA)
  • Animal Behavior & Ethology (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Zoology (AREA)
  • Physical Education & Sports Medicine (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Neurology (AREA)
  • Genetics & Genomics (AREA)
  • Orthopedic Medicine & Surgery (AREA)
  • Wood Science & Technology (AREA)
  • Biochemistry (AREA)
  • General Engineering & Computer Science (AREA)
  • Gastroenterology & Hepatology (AREA)
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  • Proteomics, Peptides & Aminoacids (AREA)
  • Epidemiology (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)

Abstract

La dystrophie musculaire de Duchenne (DMD) est une des maladies liées au chromosome X les plus fréquentes et mortelles. Cette maladie touche 1 enfant de sexe masculin sur 3500. Jusqu'à présent, il n'existe aucun traitement qui prolonge sensiblement la durée de vie ou qui guérisse la maladie. L'invention concerne l'utilisation de la sérine-protéase de Agkistrodon (ancrod) pour la préparation d'un médicament destiné au traitement de la dystrophie musculaire de Duchenne chez un mammifère, notamment un humain. L'administration d'ancrod entraîne une diminution de la dégénérescence du muscle et une amélioration fonctionnelle du modèle de souris de DMD mdx.
PCT/ES2005/000619 2004-11-16 2005-11-15 Utilisation de la serine-protease connue sous le nom d'ancrod dans le traitement de la dystrophie musculaire de duchenne WO2006053923A1 (fr)

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ES200402804 2004-11-16
ESP200402804 2004-11-16

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WO2006053923A1 true WO2006053923A1 (fr) 2006-05-26

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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1998015292A1 (fr) * 1996-10-08 1998-04-16 Lefebvre Jean Marie Composition visqueuse hemostatique, notamment a l'etat de gel
US20030219431A1 (en) * 2002-05-24 2003-11-27 Empire Pharmaceuticals, Inc. Treatment of neuronal and neurological damage associated with spinal cord injury

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1998015292A1 (fr) * 1996-10-08 1998-04-16 Lefebvre Jean Marie Composition visqueuse hemostatique, notamment a l'etat de gel
US20030219431A1 (en) * 2002-05-24 2003-11-27 Empire Pharmaceuticals, Inc. Treatment of neuronal and neurological damage associated with spinal cord injury

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
ROMA CASTANE JOSEP.: "Identificado i caracteritzacio de factor implicats en la proteccio i en la regenaracio del muscul esqueletic de ratoli sotmes a necrosi-regeneracio cronica.", TESIS DOCTORAL., 20 June 2003 (2003-06-20), Retrieved from the Internet <URL:http://www.tdx.cesca.es/TDX-0123104-172103> *

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