WO2014066964A1 - Compositions pharmaceutiques contenant un agoniste du récepteur mas pour le traitement de maladies dégénératives musculaires - Google Patents

Compositions pharmaceutiques contenant un agoniste du récepteur mas pour le traitement de maladies dégénératives musculaires Download PDF

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Publication number
WO2014066964A1
WO2014066964A1 PCT/BR2013/000444 BR2013000444W WO2014066964A1 WO 2014066964 A1 WO2014066964 A1 WO 2014066964A1 BR 2013000444 W BR2013000444 W BR 2013000444W WO 2014066964 A1 WO2014066964 A1 WO 2014066964A1
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WIPO (PCT)
Prior art keywords
mas
muscle
ang
receptor agonist
mice
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PCT/BR2013/000444
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English (en)
Portuguese (pt)
Inventor
Robson Augusto Souza Dos Santos
Enrique Brandan
Maria José ACUNA
Claudio CABELLO-VERRUGIO
Anderson José FERREIRA
Walkyria Neyde De Oliveira Sampaio
Ivana SILVA LULU
Maria José CAMPAGNOLE-SANTOS
Adelina Martha Dos Reis
Marcelo De Castro Leal
Fréderic Jean Georges FRÉZARD
Rubén Dario Sinisterra Millan
Sérgio VELOSO BRANT PINHEIRO
Luiz Renato FRANÇA
Frederico Barros De Sousa
Lenice KAPPES BECKER OLIVEIRA
Raphael De Faria E Silva
Original Assignee
Universidade Federal De Minas Gerais - Ufmg
Pontificia Universidad Cátolica De Chile
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Application filed by Universidade Federal De Minas Gerais - Ufmg, Pontificia Universidad Cátolica De Chile filed Critical Universidade Federal De Minas Gerais - Ufmg
Publication of WO2014066964A1 publication Critical patent/WO2014066964A1/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
    • A61K38/08—Peptides having 5 to 11 amino acids
    • A61K38/085—Angiotensins
    • 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

  • DMD Duchene Muscular Dystrophy
  • TGF transforming growth factor
  • Dystrophin, the dystrophin-associated glycoprotein complex, and alpha-2 laminin connect the extracellular matrix to the intracellular cytoskeleton and are therefore crucial for maintaining the structural integrity of muscle fibers (Blake, DJ, Weir, A., Newey, SE and Davies, KE Physiological Reviews 82, 291-329, 2002).
  • the main consequences of the lack of dystrophin in the skeletal muscle are sarcolemma instability and increased fiber vulnerability to mechanical stress, which results in fiber degeneration, followed by partial regeneration. Complete regeneration, however, is precluded by fibrosis (connective tissue proliferation), which progressively replaces muscle tissue (Mann, CJ, et al.
  • Fibrosis is a complex process whose mechanisms have not yet been fully described. It is characterized by excessive accumulation of collagen and other extracellular matrix components and occurs under chronic disease conditions that can affect various tissues and organs (Wynn, TA J Pathol 214, 199-210, 2008; Wynn, TA J Clin / wesf 17, 524-529, 2007).
  • angiotensin-1-7 [Ang- (1-7)] in the treatment of this type of muscular dystrophy.
  • Angiotensin-1-7 [Ang- (1-7)] is an alternative renin-angiotensin system (RAS) metabolite, with opposite effects to angiotensin II (Ang II).
  • RAS renin-angiotensin system
  • Ang- (1-7) is involved in several important processes such as vasodilation, inhibition of cell proliferation, antihypertensive and antiarrhythmic effect (Marangoni, RA, et al.
  • Ang- (1 - 7) is involved in protecting tissues from chronic damage through direct effects on tissue fibrogenesis (Iwata, M., et al., American Journal Of Physiology. Heart And Circulatory Physiology 289, H2356 -2363, 2005; Grobe, JL, Mecca, AP, Mao, H. and Katovich, MJ, American Journal of Physiology, Heart And Circulatory Physiology 290, H2417-2423, 2006).
  • Ang- (1-7) are mediated by the Mas receptor.
  • the receptor Ang- (1 - 7) 1 axis But it appears to represent a counter-regulatory mechanism of RAS capable of protecting chronically damaged tissues from the harmful effects of locally produced Ang II (Santos, RA, et al.) Proceedings of the National Academy of Sciences of the United States of America 100, 8258-8263, 2003; Warner, FJ, Lubel, JS, McCaughan, GW and Angus, PW Clin Sci (Lond) 11, 109-118, 2007).
  • TGF- ⁇ is a central mediator of fibrogenesis, regulated and stimulated in fibrotic diseases, acting directly or through other growth factors (Wynn, TA J Pathol 214, 199-210, 2008; Prud'homme, GJ Laboratory Investigation; The Journal Of Technical Methods And Pathology 87, 1077-1091, 2007; Vial, C, et al. J Cell Physiol 215, 410-421, 2008).
  • Losartan an Ang II type 1 receptor blocker
  • inhibits increased TGF- ⁇ signaling activity which transduces its signal through the Smad intracellular signaling cascade and promotes muscle remodeling in a murine model of the murine model.
  • Marfan DMD21 syndrome providing protection against muscle disuse or atrophy in humans with sarcopenia (Massague, J. Nat. Rev. Mol Cell Biol 1, 169-178, 2000; Burks, TN, et al. Science Translational Medicine 3, 82ra37, 201 1 ).
  • Treatment with Losartan after muscle damage induction also improved skeletal muscle regeneration in a normal adult murine model by reducing fibrotic tissue (Bedair, HS, Karthikeyan, T., Quintero, A., Li, Y. and Huard, J. The American Journal of Sports Medicine 36, 1548-1554, 2008).
  • WO2009054725 describes a means and method for alleviating one or more symptoms of Duchenne muscular dystrophy and or Becker muscular dystrophy, which is based on administering to the patient a pharmaceutical composition comprising, within other compounds, Losartan, which acts by inhibiting angiotensin II type I receptor.
  • Losartan which acts by inhibiting angiotensin II type I receptor.
  • WO2005040791 protects the use of a pharmaceutical composition for the treatment of muscle diseases, among others, consisting of a therapeutic agent that binds to the angiotensin and somatostin receptor polypeptide.
  • a pharmaceutical composition for the treatment of muscle diseases consisting of a therapeutic agent that binds to the angiotensin and somatostin receptor polypeptide.
  • this document does not specify the type of muscle disease and the therapeutic agent. Therefore, there are no prior art studies demonstrating the use of MAS receptor agonists, such as Ang- (1-7), in the treatment of muscular dystrophy.
  • Figure 2 shows Ang- (1-7) infusion under TGF- ⁇ on muscle architecture and fibrosis in GM muscle of aged mdx mice, (a) comparison of deteriorated histology of untreated mdx mice with mdx mice eight-month-old infants who received Ang- (1-7) by infusion, (b) total collagen after infusion of elderly mdx mice with Ang- (1-7) evidenced by Sirius red staining (darker portions) compared to untreated mdx mice. (c) fibronectin in the muscle of Ang- (1-7) -treated animals compared to untreated. (d) quantification of type III collagen levels in Ang- (1-7) and untreated mdx mice after PR Western blot analysis.
  • Figure 3 shows the effect of A-779, a potent Ang- (1-7) antagonist, on dystrophic muscle histology, fibrosis, TGF- ⁇ signal and skeletal muscle strength of mdx mice.
  • A-779 a potent Ang- (1-7) antagonist
  • Fibrotic proteins (evidenced by collagen type I, type III and fibronectin) in antagonist-infused mdx mice compared to muscles of untreated mdx mice.
  • the present invention consists in the use of a pharmaceutical composition containing a Mas receptor agonist such as Angiotensin- (1-7) for the treatment of degenerative muscular diseases such as Duchene Muscular Dystrophy.
  • a pharmaceutical composition containing a Mas receptor agonist such as Angiotensin- (1-7) for the treatment of degenerative muscular diseases such as Duchene Muscular Dystrophy.
  • Such a composition comprises at least one pharmaceutically acceptable excipient such as water, saline, phosphate buffered solutions, Ringer's solution, dextrose solution, Hank's solution, biocompatible saline solutions containing or not polyethylene glycol combined with the receptor agonist. But free or coupled to controlled release systems.
  • Controlled release systems may comprise liposomes, cyclodextrins, biodegradable polymers, capsules, micro- and nano-capsules, micro- and nano-particles, bolus preparations, osmotic pumps, diffusion devices, lipospheres and / or transdermal delivery systems.
  • Other controlled release compositions of the present invention include liquids which, when subjected to temperature changes, form a solid or gel in situ.
  • Non-aqueous vehicles such as fixed oils, sesame oil, ethyl oleate, or triglyceride may also be used as excipients.
  • Other useful formulations include agents capable of increasing viscosity, such as sodium carboxymethylcellulose, sorbitol, or dextran.
  • Excipients may also contain minor amounts of additives, such as substances that increase isotonicity and chemical stability of the substance or buffers.
  • buffers include phosphate buffer, bicarbonate buffer and Tris buffer
  • condoms include thimerosal, m- or o-cresol, formalin and benzyl alcohol.
  • compositions may be in liquid or solid state, in preparations for oral, topical, intramuscular, intravenous, subcutaneous, inhalation or implantable device administration.
  • GM gastrocnemius
  • TA anterior tibial skeletal muscle
  • DPG diaphragm skeletal muscle
  • Treatment with Angiotensin- (1-7) was performed with wild mice, Mas-KO, mdx and mdx-Mas-KO, and treatment was started at 12 weeks of age using osmotic minipumps (Alzet, Durect co, model 1004) sc with release of Ang- (1-7) (100 ng / kg * min) for 28-56 days.
  • fibrosis levels and muscle strength were measured.
  • Mas antagonism by treatment with A-779 (D Ala-Angiotensin- (1-7), Phoenix Pharmaceuticals, USA), a potent Mas receptor antagonist, was accomplished by administration via osmotic minipumps (100 ng / kg *). min) for 2 months.
  • Taqman quantitative real-time PCR was performed in duplicate on the Stratagene MX 3005 thermal cycler (Agilent Technology) using pre-designed Mas murine primers and GAPDH housekeeping gene (Taqman Assays-on-Demand, Applied Biosystems, USA) .
  • MRNA expression was quantified by the dCt comparative method (2-AACT) using GAPDH as the reference gene. MRNA levels were expressed relative to the wild mouse.
  • Immunoblot analysis was performed with DPG or GM muscles, which were homogenized in 10 volumes of Tris-EDTA pH 7.4 with 1 mM PMSF, plus a buffer volume containing 2% glycerol, 4% SDS, Tris 0.125M pH 6.8. Aliquots were subjected to 10% polyacrylamide gel electrophoresis, electrophoretically transferred to PVDF membranes (Millipore, USA) and labeled with specific antibodies to fibronectin, tubulin (Sigma-Aldrich, USA), collagen III (Rockland, USA), and GAPDH (Chemicon, USA).
  • muscle cryopathies (7 ⁇ ) were fixed in 4% paraformaldehyde, blocked for 1 h in 10% goat serum in PBS, and incubated for 1 h at room temperature with anti-fibronectin, anti-collagen antibodies.
  • III anti-collagen I and anti-phosphoSmad3 (invitrogen, USA) (Cabello-Verrugio, C., Morales, G., Cabrera, D., Vio, CP. And Brandan, E. Journal of Cellular and Molecular Medicine, 20 1 Morales, G., Cabello-Verrugio, C., Cabrera, D., Goldschmeding, R. and Brandan, EJ Pathol., 225 490-501, 2011).
  • Alexa Fluor 568 conjugated anti IgGs were used.
  • slices were incubated with 1 ⁇ g / ml Hoechst 33258 in PBS for 10 min.
  • pSmad3 positive nuclei were counted using the ImageJ v1, 43u plug-in cell counter in 10 different windows for each muscle analyzed, with 40x enlarged photographs. The data obtained were corrected for the total area.
  • mice were sacrificed and the DPG was quickly removed and dipped in oxygenated Krebs-Ringer solution. Muscle strength was determined as previously described (Cabello-Verrugio, C., Morales, G., Cabrera, D., Vio, CP. And Brandan, E. Journal of Cellular and Molecular Medicine, 2011; Morales, G., Cabello-Verrugio, C., Cabrera, D., Goldschmeding, R. and Brandan, E. J. Pathol., 225 490-501, 2011).
  • Optimal muscle length (Lo) and stimulus voltage were determined through manipulations of the length of the muscle to produce the maximum contraction isometric force.
  • the maximum specific tetanic isometric force was determined from the curve plateau that relates the specific isometric force (mN / mm2) to the stimulus frequency (Hz) from 1 to 200 Hz for 450 ms, with 2 min interval between stimuli. . Muscle mass and Lo were used to calculate the specific resultant force (normalized force per total muscle fiber cross-sectional area (CSA), mN / mm2).
  • CSA normalized force per total muscle fiber cross-sectional area
  • mice For the running test, wild Mas-KO, mdx and mdx-Mas-KO mice were subjected to the treadmill running test for 5 min at 15 m / min. The number of times mice were detained on the first third of the moving platform was counted.
  • Ang- (1-7) was infused by osmotic pumps from 12 weeks of age. After four weeks of infusion, histological analysis showed an improvement in skeletal muscle morphology, indicating a marked decrease in the amount of inflammatory tissue and fibrosis around the individual fibers in the gastrocnemius (GM) muscle (Fig. 1a).
  • GM gastrocnemius
  • EXAMPLE 4 EFFECTS OF ANGIOTENSIN- (1-7) ON ELDERLY ANIMALS Fibrosis and muscle damage increase with age, as previously described. For this reason, the effect of Ang- (1-7) in elderly animals was evaluated. Eight-month-old mdx mice received Ang- (1-7) for two months. Histological analysis of GM and TA revealed an important structural improvement compared to untreated mdx mice (Fig. 2a). Total collagen, type III collagen and fibronectin levels decreased significantly (Fig. 2b, c, d). In elderly animals, Ang- (1-7) also decreased TGF-beta signaling, determined by nuclear translocation of pSmad3 (Fig 2e).
  • Ang- (1-7) performs its activities primarily through the Mas receiver.
  • mdx mice were infused with A-779, a potent Mas antagonist receptor agonist (Santos, RA, et al. Proceedings of the National Academy of Sciences of the United States of America 100, 8258-8263, 2003).
  • A-779 a potent Mas antagonist receptor agonist
  • Treatment of mdx mice with A-779 significantly deteriorated GM and TA, as demonstrated by histological analysis (Fig. 3a).
  • An increase in total collagen type I, III collagen and fibronectin was observed in GM from A-779 treated mdx mice compared to untreated mice (Fig. 3 b, c, d).
  • EXAMPLE 6 EFFECTS OF ANGIOTENSIN- (1-7) ON KNOCK OUT ANIMALS FOR THE MAS RECEIVER
  • mdx-Mas-KO GM and TA showed significant increases in deteriorated architecture, inflammation and damaged areas compared to mdx mouse muscles (Fig. 4b).
  • Non-expressing Mas and GM dystrophic muscles showed an increase in total collagen and collagen type I, III and fibronectin (Fig. 4 c, d, e).
  • TGF-beta signaling was also increased in mdx-Mas-KO as evidenced by the increase of muscle-translocated pSmad3 in GM (Fig. 4f) and TA (data not shown).
  • Mdx-Mas-KO performance in the running test decreased by Comparison with mdx mice. No differences were observed between wild mouse and Mas-KO (Fig. 4g).
  • any compound acting as this receptor agonist can have beneficial effects for the treatment of degenerative muscle diseases.

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Abstract

Le présent certificat d'addition concerne l'utilisation de compositions pharmaceutiques contenant un agoniste du récepteur Mas, tel que l'angiotensine-(1-7), dans le traitement de maladies dégénératives musculaires.
PCT/BR2013/000444 2012-10-31 2013-08-25 Compositions pharmaceutiques contenant un agoniste du récepteur mas pour le traitement de maladies dégénératives musculaires WO2014066964A1 (fr)

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BRBR1320120280050 2012-10-31
BR132012028005-0A BR132012028005E2 (pt) 2012-10-31 2012-10-31 Composições farmacêuticas contendo um agonista do receptor mas para o tratamento de doenças degenerativas musculares.

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2016011420A1 (fr) * 2014-07-17 2016-01-21 University Of Southern California Méthodes, composés et compositions pour le traitement de maladies musculo-squelettiques
EP2986341A4 (fr) * 2013-04-19 2016-11-30 Univ Iowa Res Found Angiotensines dans la dystrophie musculaire

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2003039434A2 (fr) * 2001-11-05 2003-05-15 Universidade Federal De Minas Gerais - Ufmg Procede de preparation de compositions du peptide angiotensine (1-7) et de ses analogues, agonistiques et antagonistes au moyen de cyclodextrines, de liposomes et de polymeres biodegradables et/ou de melanges et de produits de ces derniers
WO2007000036A2 (fr) * 2005-06-28 2007-01-04 Universidade Federal De Minas Gerais Utilisation d'antagonistes et d'agonistes de recepteurs couples a la proteine g mas utilises en tant que modulateurs de l'activite apoptotique dans l'etude, la prevention et le traitement de maladies

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2003039434A2 (fr) * 2001-11-05 2003-05-15 Universidade Federal De Minas Gerais - Ufmg Procede de preparation de compositions du peptide angiotensine (1-7) et de ses analogues, agonistiques et antagonistes au moyen de cyclodextrines, de liposomes et de polymeres biodegradables et/ou de melanges et de produits de ces derniers
WO2007000036A2 (fr) * 2005-06-28 2007-01-04 Universidade Federal De Minas Gerais Utilisation d'antagonistes et d'agonistes de recepteurs couples a la proteine g mas utilises en tant que modulateurs de l'activite apoptotique dans l'etude, la prevention et le traitement de maladies

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
SANTOS RAS ET AL.: "Characterization of a new selective antagonist for angiotensin-(1-7), d-Pro7-angiotensin-(1-7).", HYPERTENSION, vol. 41, 2003, pages 737 - 743 *
SILVA MB ET AL.: "Influencia do bloqueador de receptor de angiotensina (Losartana potassica) na -funçao renal e pressao arterial em caes GRMD.", PESQ. VE. BRAS., vol. 29, no. 4, 2009, pages 322 - 326 *

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US9943509B2 (en) 2013-03-15 2018-04-17 University Of Southern California Methods, compounds, and compositions for the treatment of musculoskeletal diseases
EP2986341A4 (fr) * 2013-04-19 2016-11-30 Univ Iowa Res Found Angiotensines dans la dystrophie musculaire
WO2016011420A1 (fr) * 2014-07-17 2016-01-21 University Of Southern California Méthodes, composés et compositions pour le traitement de maladies musculo-squelettiques

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