WO2006053923A1

WO2006053923A1 - Use of serine-proteinase known as ancrod in the treatment of duchenne muscular dystrophy

Info

Publication number: WO2006053923A1
Application number: PCT/ES2005/000619
Authority: WO
Inventors: Purificación MUÑOZ CÀNOVES; Mónica SUELVES ESTEBAN; Mercè JARDÍ RIPOLL
Original assignee: Centre De Regulació Genòmica
Priority date: 2004-11-16
Filing date: 2005-11-15
Publication date: 2006-05-26

Abstract

The invention relates to the use of serine-proteinase known as ancrod in the treatment of Duchenne muscular dystrophy. Duchenne muscular dystrophy (DMD) is one of the most frequent fatal chromosome X-linked diseases and affects 1 in 3500 boys. Until now there has been no treatment which significantly extends life or cures the disease. The invention relates to the use of serine-proteinase of Agkistrodon (ancrod) for the preparation of a medicament for the treatment of Duchenne muscular dystrophy in mammals, particularly humans. The administration of ancrod gives rise to a decrease in muscle degeneration and a functional improvement to the DMD mdx mouse model.

Description

serine proteinase known as ANCROD in the treatment of Duchenne muscular dystrophy

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.

STATE OF THE PREVIOUS TECHNIQUE

Duchenne muscular dystrophy (DMD) 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). If the dystrophin complex cannot bind the actin cytoskeleton inside the muscle cell to the extracellular matrix, the forces generated by the muscle fiber result in ruptures of the sarcolenma that lead to muscle damage (cf. KP Campbell, "Three muscle dystrophies: loss of cytoskeleton-extracellular matrix linkage ", CeH 1995, vol. 80, pp. 675-9). 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). Humans with DMD and mdx mice suffer progressive muscle degeneration. The loss of myofibers is initially compensated by proliferation and fusion of satellite cells with pre-existing myofibers, giving an increase in muscle size. After repetitive cycles of degeneration and regeneration, dystrophic muscle damage can no longer be repaired and dystrophic myofibers are gradually replaced, initially by fibrotic infiltrates and subsequently by adipose tissue (cf. HH Stedman, et al., "The mdx mouse diaphragm reproduces the degenerative changes of Duchenne muscular dystrophy ", Nature 1991, vol. 352, pp. 536-9; JF Watchko, et al.," Functional characteristics of dystrophic skeletal muscle: insights from animal models ", J Appl Phvsiol 2002, vol. 93, pp. 407-17). Although mdx mice and patients with DMD share many genetic, biochemical and histological similarities, clinical manifestations are less severe in mdx mice than in humans. For example, DMD muscles have high fibrosis and patients suffer premature death, while mdx muscles have lower fibrosis and mice have a normal lifespan.

Currently, the only relatively effective treatment for DMD is the management of each case and the symptoms. Although there is no treatment so far that significantly prolongs life or cures the disease, there are numerous clinical trials underway. Among the compounds that are being tested or have been tested, albuterol, a beta adrenergic receptor agonist has resulted in an increase in strength and muscle mass. Research is also underway with utrophin that is similar to structurally and functionally dystrophin. Corticosteroids, such as prednisone and deflazacort, appear in numerous studies in these last 30 years to prolong the ability to walk and delay the course of the disease. However, they have numerous side effects, so they should be closely monitored and, if possible, avoided.

In addition, there are research and clinical trials that study the efficacy of Embryonic human stem cell transplants. It is expected that injecting non-specialized and healthy stem cells in patients with DMD will lead to the specialization of the stem cells to produce a correct dystrophin structurally and functionally. If dystrophin could occur, it could delay the progression of the disease and even cure it.

Based on this strategy, the research also focuses on the gene transfer for the treatment of DMD. So far, replacement therapies for dystrophin have not been successful. One of the main drawbacks is the particular size of the gene and the complexity of the protein it produces. It is also necessary to produce vehicles for gene therapy that meet the requirements regulate us for human use.

Thus, it is very desirable to provide new therapeutic agents for the treatment of Duchenne muscular dystrophy.

EXPLANATION OF THE INVENTION

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. Lluís et al., "Urokinase-dependent plasminogen activation is required for efficient skeletal muscle regeneration in vivo ", Blood 2001, vol. 97, pp. 1703-11; M. Suelves et al.," Plasmin activity is required for myogenesis in vitro and skeletal muscle regeneration ", Blood 2002 , vol. 99, pp. 2835-44). Mice deficient in PIg or uPA show defects in regeneration after experimentally induced muscle degeneration, but these models are far from the complex scenario present in a patient suffering from DMD or in an earlier step, in the mdx mouse model of DMD .

Now, the inventors have surprisingly found that 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.

Thus, 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.

Phase III clinical trials in an ischemic condition are under way (Press relée, Neurobiological Technologies, August 19, 2004). The ancrod promises to be an alternative to tPA (tissue plasminogen activator) which is the only FDA approved treatment for acute ischemic conditions.

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.

In addition, 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. In particular embodiments of the invention, the mammal is a human.

The ancrod treatment results in a significant reduction of the accumulation of fibrin in the muscles. After the treatment with ancrod, 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. In addition to the morphological and biochemical improvements, these result in a functional improvement of the state of the pathological state of the whole skeletal musculature.

Depending on the circumstances, an expert in the field will select 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.

Throughout the description and the claims, the word "comprises" and its variants are not intended to exclude other technical characteristics, additives, components or steps. The summary of this application is incorporated here by reference. For those skilled in the art, other objects, advantages and characteristics of the invention will emerge partly from the description and partly from the practice of the invention. The following embodiments and drawings are provided by way of illustration, and are not intended to be limiting of the present invention.

DESCRIPTION OF THE DRAWINGS

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. Α-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 μm 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. The results represent the mean ± the standard error; n = 6 animals per group; 10 sections per muscle; an asterisk means statistically significant from the values of saline treatment (P <0.05).

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. The results represent the mean ± the standard error; n = 6 animals per group; an asterisk means statistically significant from the values of saline treatment (P <0.05).

DETAILED EXHIBITION OF REALIZATION MODES

Ancrod invention

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.

Western blot analysis

50 μg of total protein (from muscle extracts) was analyzed by SDS-PAGE and transferred to PVDF membranes. Antibody dilutions: anti-fibrin (provided by Dr. K. Damage, Finsenlab, Denmark; 1: 1000) and α-tubulin (DM1 A, Sigma; 1/4000). After incubations with the appropriate secondary antibodies, the signals were detected using amplified chemiluminescence (Amersham Pharmacia Biotech).

Morphometric analysis

The 10 μm 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.

Analysis of the degeneration of muscle fibers

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.

Assessment of the regeneration of muscle fiber

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

Muscle damage such as that occurs in DMD (or rndx) results in high levels of creatine kinase (CK) in the blood. To measure the CK activity, 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.

Rota rod test

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.)

Results

First, it was evaluated if 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. Bell, et al., "The effects of ancrod, the coagulating enzyme from the venom of Malayan pit viper ( A. Rhodostoma) or prothrombin and fibrinogen metabolism and fibrinopeptide A relée ¡n man ", Journal of Laboratorv and Clinical Medicine 1978, vol. 9, pp. 592-604; F. Lluis, et al., "Urokinase-dependent plasminogen activation is required for efficient skeletal muscle regeneration in vivo", Blood 2001, vol. 97, pp. 1703-11).

Ancrod (1 U / day) or saline was administered daily to mdx mice, starting at 12 days after birth and continuing for the next 18 days. As shown in FIG. 1B, the treatment with ancrod resulted in a significant reduction in the accumulation of fibrin in the muscles (compare the rails ancrod versus saline). Staining with hematoxylin / eosin revealed that after administration of ancrod to mdx mice, muscle fibers appeared much healthier (cf. FIG. 1C) and that muscle degeneration was significantly attenuated. When the degenerated muscle area was quantified, as a percentage of the total muscle area, muscle degeneration was reduced by 40% in mdx mice treated with ancrod (cf. FIG. 2A). In addition, there were fewer groups of degenerating myofibers, which contained more than 10 degenerating fibers, after treatment with ancrod (cf. FIG. 2B). The percentage of myofibres with central nuclei (CNF), indicative of previous cycles of degeneration and regeneration, was also reduced by 25% in mdx mice treated with ancrod versus those treated with saline (cf. FIG. 2C).

To determine if the morphological improvement was functional, 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. To detect an improvement in the pathological state of skeletal musculature, 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.

Claims

1. Use of the ancrod for the preparation of a medicament for the treatment of Duchenne muscular dystrophy in a mammal.

2. Use according to claim 1, wherein the mammal is a human.

3. Method of treatment of a mammal suffering from Duchenne muscular dystrophy, which comprises the administration of a therapeutically effective amount of the ancrod together with appropriate amounts of pharmaceutically acceptable diluents or carriers.

4. Method according to claim 3, wherein the mammal is a human.

5. Pharmaceutical composition for the treatment of Duchenne muscular dystrophy in a mammal, which comprises a therapeutically effective amount of the ancrod together with appropriate amounts of pharmaceutically acceptable diluents or carriers.

6. Composition according to claim 5, wherein the mammal is a human.