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WO2017036852A1 - Modulateurs du récepteur erbb 4 pour une utilisation dans le traitement de maladies associées à la dystrophine - Google Patents

Modulateurs du récepteur erbb 4 pour une utilisation dans le traitement de maladies associées à la dystrophine Download PDF

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WO2017036852A1
WO2017036852A1 PCT/EP2016/069877 EP2016069877W WO2017036852A1 WO 2017036852 A1 WO2017036852 A1 WO 2017036852A1 EP 2016069877 W EP2016069877 W EP 2016069877W WO 2017036852 A1 WO2017036852 A1 WO 2017036852A1
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erbb4
dystrophin
intracellular domain
free
variant
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Nadesan Gajendran
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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
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/18—Growth factors; Growth regulators
    • A61K38/1883—Neuregulins, e.g.. p185erbB2 ligands, glial growth factor, heregulin, ARIA, neu differentiation factor
    • 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

  • the present invention relates to novel means for regulating dystrophin expression for treating diseases in a subject and more particularly to novel means for regulating dystrophin expression for treating dystrophin associated diseases via modulators of ErbB4 receptor and/or free ErbB4 intracellular domain.
  • Duchenne and Becker's muscular dystrophy is caused by the absence or truncation respectively of dystrophin, a 427 kDa protein that forms a vital part of the dystrophin-associated protein complex (DAPC) (Matsumura, K., et al., Association of dystrophin-related protein with dystrophin associated proteins in mdx mouse muscle. Nature, 1992 360(6404): p. 588-591).
  • DAPC links the intracellular cytoskeleton to the extracellular basal lamina in skeletal and cardiac muscle fibers and absence of one of the components forming DAPC in most cases results in a dystrophic phenotype characterized by a progressive degeneration of skeletal and cardiac muscle.
  • the DAPC is also formed with the 427 kDa utrophin instead of dystrophin.
  • DAPC has been gaining increasing interest for its role in signaling functions and providing a scaffold for signaling molecules to interact.
  • the DAPC provides structural support by linking F-actin via dystrophin or utrophin in the subsarcolemrnal cytoskeleton to ⁇ - dystroglycan, sarcoglycans and sarcospan in the sarcolemma which in turn associate with a-dystroglycan and laminin in the extracellular matrix.
  • D/BMD patients also exhibit cardiomyopathy (Cox, G.F., Kunkel, L.M., Dystrophies and heart disease . Curr. Opin. Cardiol, 1997. 12(3): p. 329-343).
  • DOT1L Disruptor of Telomeric Silencing
  • DCM dilated cardiomyopathy
  • NRG1 signaling through ErbB2 phosphorylates a-DBl and revealed the highly dynamic tyrosine phosphorylation and dephosphorylation of a- DB1 (Schmidt, N., et al., Neuregulin/ErbB regulate neuromuscular junction development by phosphorylation of a-dystrobrevin. The Journal of Cell Biology, 2011. 195(7): p. 1171-1184).
  • Signaling from NRG1 , through ErbB receptors has major functions in several organs such as heart, breast, and nervous system including central and peripheral synapses.
  • NRG signaling for normal cardiac development in mice was firmly established by the fact that ablation of NRG, ErbB4, or ErbB2 resulted in premature death during midgestation (Meyer, D. and C. Birchmeier, Multiple essential functions of neuregulin in development. Nature, 1995. 378(6555): p. 386-390; Gassmann, M., et al., Aberrant neural and cardiac development in mice lacking the ErbB4 neuregulin receptor. Nature, 1995. 378(6555): p. 390-394).
  • NRG/ErbB4 signaling was shown to be sufficient for cardiomyocyte proliferation and repair of heart injury (Bersell, K., et al., Neuregulinl/ErbB4 Signaling Induces Cardiomyocyte Proliferation and Repair of Heart Injury. Cell, 2009. 138(2): p. 257-270) although the exact mechanisms through which this is achieved remains to be elucidated.
  • dystrophin expression In spite of intense investigations, very little is known regarding the regulation of dystrophin expression, especially regarding regulating dystrophin expression by signaling from ligands acting via surface receptors and/or by Protein Kinase C activators.
  • a ligand for a surface receptor a Protein Kinase C activator and/or other means for regulating dystrophin expression for use in the treatment of dystrophin associated diseases in a subject.
  • the present invention provides such means, ligands, protein kinase C activators, receptors, receptor domains, and signaling pathways to regulate dystrophin expression, in particular, to upregulate expression of dystrophin, for use in the treatment of dystrophin associated diseases in a subject.
  • the present invention relates to an ErbB4 receptor modulator for use in the treatment of dystrophin associated diseases in a subject, wherein the modulator upregulates dystrophin expression.
  • the present invention relates to an ErbB4 receptor modulator according the preceding embodiment, wherein the receptor is (a) ErbB4, ErbB4 homodimers, or ErbB4 heterodimers or (b) an isoform, a variant, a fragment, a homologue, or a derivative of ErbB4, ErbB4 homodimers, or ErbB4 heterodimers or (c) any combinations of (a) and/or (b).
  • the receptor is (a) ErbB4, ErbB4 homodimers, or ErbB4 heterodimers or (b) an isoform, a variant, a fragment, a homologue, or a derivative of ErbB4, ErbB4 homodimers, or ErbB4 heterodimers or (c) any combinations of (a) and/or (b).
  • the present invention relates to an ErbB4 receptor modulator for use in the treatment of dystrophin associated diseases in a subject, wherein the modulator upregulates dystrophin expression and the isoform of ErbB4 receptor is JM-a CYT-1 , JM-a CYT-2, JM-d, or combinations thereof.
  • the present invention provides an ErbB4 receptor modulator according to any of the preceding embodiments, wherein said ErbB4 heterodimers receptor is ErbB4/l, ErbB4/2, or ErbB4/3, or combinations thereof.
  • the present invention provides an ErbB4 receptor modulator according to any of the preceding embodiments, wherein the modulator is Neuregulin, NRG1 , NRG2, NRG3, NRG4, NRG-like polypeptide, Neuregulin mutant, HB-EGF, Epiregulin, ⁇ -cellulin, TPA, vitamin D3, TACE, Presenilin-dependent gamma- secretase, platelet-derived growth factor, ionomycin, diacylglycerol, antibodies to ErbB4 receptor, antibody against ErbB4 receptor fused to TACE, chimeric heteromultimer adhesin, aptamers to ErbB4 receptor, or an isoform, a variant, a fragment, a homologue, or
  • the present invention provides an ErbB4 receptor modulator according to any of the preceding embodiments, wherein the isoform of NRG1 is NRG-1- , a variant thereof, a fragment thereof, a homologue thereof, or a derivative thereof or NRG-1- ⁇ , a variant thereof, a fragment thereof, a homologue thereof, or a derivative thereof.
  • the present invention provides an ErbB4 receptor modulator according to any of the preceding embodiments, wherein the isoform of NRG2 is NRG-2- ⁇ , a variant thereof, a fragment thereof, a homologue thereof, or a derivative thereof.
  • the present invention provides an ErbB4 receptor modulator according to any of the preceding embodiments, wherein the modulator induces or promotes activity of the ErbB4 receptor according to any of claims 1-4 and thereby upregulates dystrophin expression.
  • the present invention provides an ErbB4 receptor modulator according to any of the preceding embodiments, wherein the modulator further induces or promotes release of ErbB4 intracellular domain, an isoform thereof, or a variant thereof, a fragment thereof, a homologue thereof, or a derivative thereof resulting in the generation of free ErbB4 intracellular domain, an isoform thereof, a variant thereof, a fragment thereof, a homologue thereof, or a derivative thereof.
  • the ErbB4 intracellular domain isoform is 4ICD1 , a variant thereof, a fragment thereof, a homologue thereof, or a derivative thereof or 4ICD2, a variant thereof, a fragment thereof, a homologue thereof, or a derivative thereof.
  • the ErbB4 receptor modulator according to any of the preceding claims, wherein the modulator upregulates dystrophin expression in skeletal, cardiac, smooth muscle tissue, and/or in central nervous system tissue of the subject.
  • the present invention relates to a pharmaceutical composition for use in the treatment of dystrophin associated diseases in a subject, comprising an effective amount of the modulator of any of the preceding embodiments, in admixture with a pharmaceutically acceptable carrier.
  • the pharmaceutical composition of the present invention comprises an effective amount of the modulator of any of the preceding embodiments, in admixture with a pharmaceutically acceptable carrier, wherein the modulator upregulates dystrophin expression.
  • the present invention relates to a method of treatment of dystrophin associated diseases in a subject, comprising administering the modulator of any of the preceding embodiments to a subject.
  • the method of the present invention comprises administering the modulator of any of the preceding embodiments to a subject, wherein the modulator upregulates dystrophin expression.
  • the present invention relates to free ErbB4 intracellular domain, an isoform thereof, a variant thereof, a fragment thereof, a homologue thereof, or a derivative thereof for use in the treatment of dystrophin associated diseases in a subject, wherein the free ErbB4 intracellular domain upregulates dystrophin expression.
  • the free ErbB4 intracellular domain upregulates dystrophin expression in skeletal, cardiac, smooth muscle tissue, and/or in central nervous system tissue of the subject.
  • the present invention relates to a pharmaceutical composition for treatment of dystrophin associated diseases in a subject, comprising an effective amount of free ErbB4 intracellular domain, a variant thereof, a fragment thereof, a homologue thereof, or a derivative thereof or an effective amount of a nucleic acid encoding free ErbB4 intracellular domain, a variant thereof, a fragment thereof, a homologue thereof, or a derivative thereof, in admixture with a pharmaceutically acceptable carrier.
  • the present invention relates to a method of treatment of dystrophin associated diseases in a subject comprising administering free ErbB4 intracellular domain, an isoform thereof, a variant thereof, a fragment thereof, a homologue thereof, or a derivative thereof to a subject.
  • the present invention relates to a method of treatment of dystrophin associated diseases in a subject comprising administering a nucleic acid encoding free ErbB4 intracellular domain, an isoform thereof, a variant thereof, a fragment thereof, a homologue thereof, or a derivative thereof to a subject, whereby the nucleic acid is expressed in the subject and free ErbB4 intracellular domain, an isoform thereof, a variant thereof, a fragment thereof, a homologue thereof, or a derivative thereof is released.
  • the free ErbB4 intracellular domain, an isoform thereof, a variant thereof, a fragment thereof, a homologue thereof, or a derivative thereof according to the pharmaceutical composition and methods of present invention upregulates dystrophin expression.
  • the present invention relates to a ErbB4 receptor modulator or free ErbB4 intracellular domain according to any of the preceding embodiments, wherein the disease is selected from a group consisting of muscular dystrophy, Duchenne Muscular Dystrophy, Becker Muscular Dystrophy, myopathy due to a lack of or truncation of or insufficient dysferlin including miyoshi myopathy, limb girdle muscular dystrophy, and distal myopathy of the anterior tibialis, autosomal recessive muscular dystrophy, progressive muscular dystrophy due to lack of a - sarcoglycan, severe muscular dystrophy and cardiomyopathy including cardiomyopathy due to lack of or insufficient dystrophin, dysferlin, or delta- sarcoglycan, schizophrenia, sarcopenia, cachexia including cancer cachexia, or aberrant vasoregulation, or cancer, or myasthenia gravis or combinations thereof.
  • the disease is selected from a group consisting of muscular dyst
  • the present invention relates to the ErbB4 receptor modulator or the free ErbB4 intracellular domain according to any of the preceding claims, wherein the disease is characterized by reduction or loss of dystrophin.
  • Figure 1 Illustrates dystrophin and utrophin levels in db-/- relative to wt C2C12 myotubes.
  • Figures la and lb show qPCR data of dystrophin and utrophin expression levels respectively relative to wt C2C12.
  • Figures lc and Id show ECL developed Western blots of immunoprecipitated (IP) proteins and precipitated proteins from supernatant (SN) detected with anti-dystrobrevin (c) and anti-utrophin (d) antibodies.
  • Control refers to non-transfected myotubes.
  • Figures lg and lh show Western blots of IP proteins detected with dystrophin (g) and utrophin (h) antibodies.
  • Figure li shows a similar Western blot as in Figure lg and Figure lh detected with an anti-a-DBl antibody showing expression of GFP- -DBland GFP-a- DB1-P3 as well as the endogenous 75 KDa a-DB l band in wt C2C12 myotubes.
  • Lower panels incubated with anti-a-syntrophin antibody to show similar loading levels.
  • Figure 2 Illustrates ErbB4 and ErbB2 expression levels in db 7" and wt C2C12 myotubes expressing GFP-a-DBl and GFP-a-DB l-P3 relative to wt C2C12 myotubes. Control refers to non-transfected myotubes.
  • Figures 2a and 2b show qPCR data.
  • Figures 2c and 2d show ECL developed Western blots of myotube lysates incubated with anti-ErbB4 (c) and anti-ErbB2 (d) antibodies.
  • Figure 3 Illustrates dystrophin and utrophin expression levels in wt C2C12 relative to erbb2/4 " ' ⁇ myotubes.
  • Figures 3a and 3b show qPCR data of dystrophin and utrophin expression levels respectively relative to erbb2/4 " ' " .
  • Figures 3c and 3d show Western blots of IP proteins from myotubes with loxP flanked exons of Erbb2 and Erbb4 genes (lanes 1 to 3) and cre-mediated knock-out of Erbb2 and Erbb4 genes (lanes 4 to 6) detected with dystrophin (c) and utrophin (d). Lanes 1 to 3 and 4 to 6 each represent the same experiment performed in triplicate.
  • FIG. 3e and 3f show qPCR data of ErbB4 and ErbB2 expression levels in db ' ' and C2C12 myoblasts expressing GFP-a- DBl and GFP-a-DBl -P3 relative to wt C2C12 myoblasts.
  • Figures 3g and 3h show qPCR data of dystrophin and utrophin expression levels in db " ' " and C2C12 myoblasts expressing GFP-a-DBl and GFP-a-DBl-P3 relative to wt C2C12 myoblasts.
  • Figure 3i shows qPCR data of ErbB2 expression levels relative to ErbB4 expression levels in db- /- myotubes.
  • Figure 3j shows, qPCR data of dystrophin expression levels in db-/- myotubes relative to db-/- myoblasts. Control refers to non-transfected myotubes.
  • an ErbB4 receptor modulator for use in the treatment of dystrophin associated diseases in a subject, wherein the modulator upregulates dystrophin expression is provided.
  • ErbB4 receptor or "ErbB4 receptors” shall include but not limited to ErbB4, isoforms of ErbB4, variants of ErbB4, fragments of ErbB4, homologues of ErbB4, or derivatives of ErbB4, ErbB4 homodimers, ErbB4 isoforms homodimers, ErbB4 variants of homodimers, fragments of ErbB4 homodimers, homologues of ErbB4 homodimers, or derivatives of ErbB4 homodimers, ErbB4 heterodimers, ErbB4 isoforms heterodimers, ErbB4 variants of heterodimers, fragments of ErbB4 heterodimers, homologues of ErbB4 heterodimers, or derivatives of ErbB4 heterodimers, or their combinations thereof.
  • the isoforms of ErbB4 receptor according to the present invention are, preferably, JM- a CYT-1 , JM-a CYT-2, or JM-d.
  • JM-d can be JM-d CYT-1 and/or JM-d CYT-2.
  • An ErbB4 receptor can heterodimerize with an ErbBl receptor (referred herein as "ErbB4/l” or "ErbBl/4"), with an ErbB2 receptor (referred herein as "ErbB4/2" or "ErbB2/4"), and/or with an ErbB3 receptor (referred herein as "ErbB4/3" or "ErbB3/4").
  • the modulator of the present invention binds to and/or activates ErbB4/l , ErbB4/2, or ErbB4/3 heterodimers, or any combination of ErbB4, ErbB4 homodimers, and ErbB4 heterodimers and their isoforms, variants, fragments, homologues, or derivatives. More preferably, the modulator of the present invention can bind to and/or activate ErbB4/2 receptor.
  • the ErbB4, ErbB4 homodimers, ErbB4 heterodimers and their isoforms, variants, fragments, homologues, or derivatives according to the present invention can be screened and obtained by any route known in art.
  • modulator or “modulators” according to the present invention is any naturally occurring or non-naturally occurring ligand or any naturally occurring or non- naturally occurring protein kinase C activator that induces or promotes activity of any of the ErbB4 receptors according to the present invention thereby upregulates dystrophin expression in a subject.
  • the modulator according to the present invention is any naturally occurring or non-naturally occurring ligand or any naturally occurring or non-naturally occurring protein kinase C activator, which induces or promotes release of ErbB4 intracellular domain thereby generating free ErbB4 intracellular domain which thereby induces or promotes upregulation of dystrophin expression in a subject.
  • module shall include any of its isoforms, variants, fragments, homologues, or derivatives.
  • the term “induce” or “promote” shall be understood to refer to a modulator which directly or indirectly increases or accelerates activity of the ErbB4 receptors.
  • the term induce or promote shall be understood to refer to a modulator which directly or indirectly increases or accelerates release of ErbB4 intracellular domain thereby generating free ErbB4 intracellular domain which subsequently increases or accelerates upregulation of dystrophin expression.
  • the term “induce” or “promote” may also be referred to as “stimulate”, “cause”, “activate” and the like.
  • the ErbB4 receptor modulator according to the present invention includes but is not limited to NRG1 (Neuregulin 1), isoforms of NRG1 , variants of NRG1 , fragments of NRG1 , homologues of NRG1 , or derivatives of NRG1; NRGl-a isoform, variants of NRGl-oc, fragments of NRGl-a, homologues of NRGl-a, or derivatives of NRGl-a; NRGl- ⁇ isoform, variants of NRGl- ⁇ isoform, fragments of NRGl- ⁇ isoform, homologue of NRGl- ⁇ isoform, or derivatives of NRGl- ⁇ isoform; NRG2 (Neuregulin 2), isoforms of NRG2, variants of NRG2, fragments of NRG2, homologues of NRG2, or derivatives of NRG2; ⁇ 1 ⁇ 2- ⁇ isoform, variants of NRG2- ⁇ isoform, fragments of NRG2 ⁇ isoform
  • the ErbB4 receptor modulator according to the present invention includes but not limited to:
  • modulator or series of modulators as defined and disclosed above can be used in the treatment of dystrophin associated diseases in a subject, wherein the modulator or series of modulators upregulates dystrophin expression.
  • NRGl Neuregulin 1
  • NRGl- ⁇ and NRGl- as endogenous ligand of ErbB4 are described, for example, in Plowman, G.D., et al., Heregulin induces tyrosine phosphorylation of HER4/pl80erbB4. Nature, 1993. 366(6454): p. 473-475; Hobbs, S.S., et al., Neuregulin isoforms exhibit distinct patterns of ErbB family receptor activation. Oncogene, 2002. 21(55): p. 8442-52..
  • Neuregulin 2 (NRG2) and its isoforms especially Neuregulin2 ⁇ as endogenous ligand of ErbB4 is described, for example, in Carraway Iii, .L., et al., Neuregulin-2, a new ligand of ErbB3/ErbB4-receptor tyrosine kinases. Nature, 1997. 387(6632): p. 512- 516.
  • Neuregulin 3 as endogenous ligand of ErbB4 is described, for example, in Zhang, D., et al., Neuregulin-3 (NRG3): A novel neural tissue-enriched protein that binds and activates ErbB4. Proceedings of the National Academy of Sciences, 1997. 94(18): p. 9562-9567.
  • Neuregulin 4 as endogenous ligand of ErbB4 is disclosed, for example, in Harari, D., et al., Neuregulin-4: a novel growth factor that acts through the ErbB-4 receptor tyrosine kinase. Oncogene, 1999. 18(17): p. 2681-9.
  • Heparin-binding EGF-like growth factor as endogenous ligand of ErbB4 is described, for example, in Elenius, K., et al., Activation of HER4 by heparin-binding EGF-like growth factor stimulates chemotaxis but not proliferation. Embo J, 1997. 16(6): p. 1268-78.
  • Epiregulin as endogenous ligand of ErbB4 is described, for example, in Komurasaki, T., et al., Epiregulin binds to epidermal growth factor receptor and ErbB-4 and induces tyrosine phosphorylation of epidermal growth factor receptor, ErbB -2, ErbB-3 and ErbB-4. Oncogene, 1997. 15(23): p. 2841-8.
  • ⁇ -cellulin as endogenous ligand of ErbB4 is described, for example, in Riese, D.J., 2nd, et al., ⁇ -cellulin activates the epidermal growth factor receptor and erbB-4, and induces cellular response patterns distinct from those stimulated by epidermal growth factor or neuregulin- ⁇ . Oncogene, 1996. 12(2): p. 345-53.
  • TPA (12-0-tetradecanoylphorbol-13-acetate) as Protein kinase C activator is disclosed in, for example, Vecchi, M., et al., Selective Cleavage of the Heregulin Receptor ErbB- 4 by Protein Kinase C Activation. Journal of Biological Chemistry, 1996. 271(31): p. 18989-18995.
  • Vitamin D3 natural product that increases ErbB4 expression levels (protein and mRNA) is disclosed, for example, in Ojfterdinger, M., et al., Expression of c-erbB- 41HER4 Is Regulated in T47D Breast Carcinoma Cells by Retinoids and Vitamin D3. Biochemical and Biophysical Research Communications, 1999. 258(3): p. 559-564.
  • TACE Tumor necrosis factor a converting enzyme
  • ECD extracellular domain
  • Presenilin-dependent gamma-secretase cleaves and releases free ErbB4 ICD (intracellular domain) after ECD release as described, for example, in Ni, C.-Y., et al., ⁇ -Secretase Cleavage and Nuclear Localization of ErbB-4 Receptor Tyrosine Kinase. Science, 2001. 294(5549): p. 2179-2181.
  • the modulators of ErbB4 receptor are also disclosed in patent publications US2010135957 (Chimeric Heteromultimer Adhesins- soluble chimeric heteromultimers comprising the extracellular domains of a heteromultimeric receptor monomers especially ErbB4-IgG; CN101397337 (Neuroregulation protein mutant and uses thereof -Neuregulin mutant with higher affinity for ErbB4); and US2009203595 Neuregulin variants and methods of screening and using thereof -polypeptide variants of neuregulin- 1 ⁇ (NRG- ⁇ ) that have enhanced or decreased binding affinity to ErbB3 and/or ErbB4.
  • NSG- ⁇ neuregulin- 1 ⁇
  • the ErbB4 modulator is a small molecule, a nucleic acid, a compound, a synthetic compound, or a drug having the same activity or substantially the same activity as the above-defined ErbB4 receptor modulator.
  • a small molecule can be a synthetic peptide that binds to and activates the ErbB4 receptor.
  • Such small peptides can be identified using Biacore as described, for example, in Leonard, P., et al., Measuring protein-protein interactions using Biacore. Methods Mol Biol. 2011, 681: p403-18.
  • a synthetic compound can be identified using a high throughput screening strategy as described in Cabrera, P.V., et al., High Throughput Screening for Compounds That Alter Muscle Cell Glycosylation Identifies New Role for N-Glycans in Regulating Sarcolemmal Protein Abundance and Laminin Binding. Journal of Biological Chemistry, 2012. 287(27): p. 22759-22770; or by using radioligand binding assay using a method reviewed in Sweetnam, P.M., et al., The Role of Receptor Binding in Drug Discovery. Journal of Natural Products, 1993. 56(4): p. 441-455.
  • the modulator of the present invention upregulates dystrophin expression in a skeletal, cardiac, smooth muscle, and/or central nervous system tissue, in particular, brain or neurons of the subject.
  • the present invention relates to a pharmaceutical composition for use in the treatment of dystrophin associated diseases in a subject, comprising an effective amount of the modulator of any of the preceding embodiments, in admixture with a pharmaceutically acceptable carrier.
  • the modulator of said pharmaceutical composition upregulates dystrophin expression in a subject.
  • the present invention provides a method of treatment of dystrophin associated diseases in a subject, comprising administering the modulator of any of the preceding embodiments to a subject.
  • the modulator of said method upregulates dystrophin expression in a subject.
  • the administration route of the modulator may include but not limited to administration by oral, intramuscular, intravenous (IV), intraperitoneal, transmucosal, or transdermal.
  • IV intravenous
  • transmucosal transdermal.
  • the method according to the invention can be performed in vivo and in vitro.
  • ErbB4 intracellular domain shall mean the intracellular domain of intact ErbB4 receptor or the intracellular domain of membrane anchored ErbB4 receptor after the extracellular domain has been cleaved off.
  • the term "ErbB4 intracellular domain” according to the present invention shall also include any of the ErbB4 intracellular domain isoforms, variants, fragments, homologues, or derivatives.
  • the ErbB4 intracellular domain isoforms are 4ICD1 including variants of 4ICD1 , fragments of 4ICD1, homologues of 4ICDl ,or derivatives of 4ICDland 4ICD2 including variants of 4ICD2, fragments of 4ICD2, homologues of 4ICD2,or derivatives of 4ICD2.
  • free ErbB4 intracellular domain shall refer to the ErbB4 intracellular domain which is not attached to the membrane and/or to the membrane spanning domain of the ErbB4 receptor. Free ErbB4 intracellular domain may be generated by cleavage of the ErbB4 receptors according to the present invention or by synthesis, purification or by expression.
  • free ErbB4 intracellular domain shall refer to naturally occurring or non-naturally occurring forms.
  • free ErbB4 intracellular domain shall include any of the free ErbB4 intracellular domain isoforms, variants, fragments, homologues, or derivatives.
  • the free ErbB4 intracellular domain isoforms are free 4ICD1 including variants of free 4ICD1 , fragments of free 4ICD1 , homologues of free 4ICDl,or derivatives of free 4ICD1 and free 4ICD2 including variants of free 4ICD2, fragments of free 4ICD2, homologues of free 4ICD2,or derivatives of free 4ICD2.
  • the free 4ICD1 comprises an amino acid sequence defined by SEQ ID NO. 1 , 2, or 3 and the free 4ICD2, preferably, comprises an amino acid sequence defined by SEQ ID NO. 4, 5, or 6.
  • the free 4ICD1 preferably, comprises an amino acid sequence defined by SEQ ID NO. 7, 8, or 9 and the free 4ICD2, preferably, comprises an amino acid sequence defined by SEQ ID NO. 10, 11 , 12.
  • Neuregulin or Heregulin dependent cleavage is described, for example, in Zhou, W., et al., Heregulin-dependent Trafficking and Cleavage of ErbB-4. Journal of Biological Chemistry, 2000. 275(44): p. 34737-34743.
  • the ErbB4 ectodomain is cleaved by a metalloprotease (Rio, C, et al., Tumor necrosis factor-alpha-converting enzyme is required for cleavage of erbB4IHER4. J Biol Chem, 2000. 275(14): p.
  • the Free ErbB4 intracellular domain can also translocate to the mitochondria and endoplasmic reticulum following apoptotic stimulus in breast cancer cells Naresh, A., et al.
  • the ERBB4 Intracellular Domain is a BH3-Only Protein Promoting Apoptosis of Breast Cancer Cells. Cancer Research, 2006. 66(12): p. 6412-6420) and it is a constitutively active tyrosine kinase that can phosphorylate substrates in the cytoplasm or nucleus (Linggi, B., et al., The ErbB-4 s80 intracellular domain is a constitutively active tyrosine kinase. Oncogene, 2005. 25(1): p. 160-163).
  • ErbB4 JM-d can be cleaved sequentially to release 4ICD1 and 4ICD2 (Gilbertson, R., et al, Novel ERBB4 juxtamembrane splice variants are frequently expressed in childhood medulloblastoma. Genes Chromosomes Cancer, 2001. 31(3): p. 288-94). ErbB4 isoforms JM-a and JM-d undergo cleavage of their extracellular domain first and then undergo cleavage of their membrane bound intracellular domain.
  • TACE Tumor necrosis factor a converting enzyme
  • ECD extracellular domain
  • the second cleavage step is made by a presenilin-dependent gamma-secretase which releases free ErbB4 intracellular domain after ECD release (Ni, C.-Y., et ah, ⁇ -Secretase Cleavage and Nuclear Localization of ErbB-4 Receptor Tyrosine Kinase, Science, 2001. 294(5549): p. 2179-2181).
  • TACE and presenilin-dependent gamma-secretase enzymes promote release of free ErbB4 intracellular domain together.
  • TACE may act first before presenilin-dependent gamma-secretase can cleave and release free ErbB4 intracellular domain.
  • the present invention relates to free ErbB4 intracellular domain for use in the treatment of dystrophin associated diseases in a subject, wherein free ErbB4 intracellular domain upregulates dystrophin expression.
  • the free ErbB4 intracellular domain of the present invention can be synthesized in presence or absence of a modulator of the present invention.
  • the Free ErbB4 intracellular domain upregulates dystrophin expression in skeletal, cardiac, smooth muscle, and/or in central nervous system tissue, in particular brain or neurons of a subject.
  • the present invention relates to a pharmaceutical composition for treatment of dystrophin associated diseases in a subject, comprising an effective amount of free ErbB4 intracellular domain, an isoform thereof, a variant thereof, a fragment thereof, a homologue thereof, or a derivative thereof or an effective amount of a nucleic acid encoding free ErbB4 intracellular domain, an isoform thereof, a variant thereof, a fragment thereof, a homologue thereof, or a derivative thereof, in admixture with a pharmaceutically acceptable carrier.
  • the present invention relates to a method of treatment of dystrophin associated diseases in a subject comprising administering free ErbB4 intracellular domain, an isoform thereof, a variant thereof, a fragment thereof, a homologue thereof, or a derivative thereof to a subject.
  • the present invention relates to a method of treatment of dystrophin associated diseases in a subject comprising administering a nucleic acid encoding free ErbB4 intracellular domain, an isoform thereof, a variant thereof, a fragment thereof, a homologue thereof, or a derivative thereof to a subject, whereby the nucleic acid is expressed in the subject and free ErbB4 intracellular domain, an isoform thereof, a variant thereof, a fragment thereof, a homologue thereof, or a derivative thereof is released.
  • the free ErbB4 intracellular domain of said pharmaceutical composition and said methods upregulates dystrophin expression in a subject.
  • disease or “diseases” according to the present invention shall cover medical conditions, disorders, illnesses, syndromes, and the like.
  • dystrophin associated diseases also covers dystrophin associated medical conditions.
  • the dystrophin associate diseases shall include any disease that can be treated by upregulating expression of dystrophin.
  • the disease is selected from a group consisting of muscular dystrophy, Duchenne Muscular Dystrophy, Becker Muscular Dystrophy, myopathy due to a lack of or truncation of or insufficient dysferlin including miyoshi myopathy, limb girdle muscular dystrophy, and distal myopathy of the anterior tibialis, autosomal recessive muscular dystrophy, progressive muscular dystrophy due to lack of a -sarcoglycan, severe muscular dystrophy and cardiomyopathy including cardiomyopathy due to lack of or insufficient dystrophin, dysferlin, or delta-sarcoglycan, schizophrenia, sarcopenia, cachexia including cancer cachexia, or aberrant vasoregulation, or cancer, or myasthenia gravis or combinations thereof.
  • the present invention provides an ErbB4 receptor modulator for use in the treatment of a subject, wherein the treatment causes the muscle of the subject to be healthier by upregulating dystrophin expression.
  • dystrophin expression or "upregulation of dystrophin” and the like according to the present invention shall mean increase in expression of the dystrophin gene, isoforms of dystrophin gene, variants of dystrophin gene, fragments of dystrophin gene, homologues of dystrophin gene, or derivatives of dystrophin gene.
  • the term “upregulates dystrophin expression” or “upregulation of dystrophin” and the like according to the present invention shall mean upregulation of the expression of a nucleic acid encoding the dystrophin gene, isoforms of dystrophin gene, variants of dystrophin gene, fragments of dystrophin gene, homologues of dystrophin gene, or derivatives of dystrophin gene resulting in an increase in the level of the 427 Kilo Dalton dystrophin protein, a variant of the dystrophin protein, or one of the isoforms of the dystrophin or a truncated dystrophin protein or one of the derivatives and homologues of dystrophin or a truncated dystrophin protein.
  • Upregulation of dystrophin also refers to an increase in expression of the dystrophin gene resulting in an increased level of dystrophin mRNA or increase in level of dystrophin protein or a variant thereof, an isoform thereof, a fragment thereof, a derivative thereof, or a homologue thereof due to the modulator of the present invention.
  • the dystrophin isoforms according to the present invention preferably are Dp427, Dp260, Dpl40, Dpi 16, Dp71 (numbers represent mass in kDa).
  • treatment refers to obtaining a desired pharmacologic and/or physiologic effect.
  • the effect may be prophylactic in terms of completely or partially preventing a disease thereof and/or may be therapeutic in terms of a partial or complete cure for a disease and/or adverse effect attributable to the disease.
  • Treatment also covers any treatment of a disease in a subject, and includes: (a) preventing the disease or the disorder from occurring in a subject which may be predisposed to the disease or disorder but has not yet been diagnosed as having it; (b) inhibiting the disease, i.e., preventing its development; (c) relieving the disease, i.e., causing regression of the disease; and (d) ameliorating or alleviating one or more symptom(s) of the disease.
  • the "treatment” also encompasses any pharmaceutical use of the compositions herein.
  • ⁇ ел ⁇ ество As used herein, “effective amount” refers to that amount that is sufficient for the treatment of dystrophin associated diseases in a subject.
  • administering refers to any suitable method of providing a modulator or free ErbB4 intracellular domain to a subject. Repeated administration may be required to achieve the desired treatment.
  • Amelioration or alleviating of a symptom of a disease refers to any lessening of symptoms, whether permanent or temporary, that can be attributed to or associated with administration of the modulator, free ErbB4 intracellular domain and their compositions according to the present invention.
  • the subject according to the present invention is a mammal.
  • the subject is a human.
  • the subject can be a mammalian muscle, mammalian muscle fiber, mammalian myoblasts, mammalian satellite cells or myosatellite cells (muscle stem cells), mammalian induced pluripotent stem cells, or mammalian embryonic stem cells.
  • compositions and the methods of the present invention for use in the treatment of dystrophin associated diseases in a subject, the skilled person in the art will ascertain suitable screening and obtaining routes, proper effective amount of the modulator, acceptable pharmaceutical carrier, and the administration routes considering, for example, the desired effect, duration of the treatment, and therapeutic context, age, and general health of the subject.
  • Dystrobrevin suppresses dystrophin but not utrophin expression
  • dystrobrevin knock-out (db " ' " ) myotubes were analyzed by qPCR and immunoprecipitation of DAPC followed by Western blot analysis. Analysis by qPCR clearly showed a six fold increase in dystrophin expression in db " ' " myotubes compared to wild type (wt) C2C12 myotubes demonstrating that dystrobrevin or signaling from it, under normal conditions downregulated dystrophin expression to physiological levels (Fig. la). Utrophin expression on the other hand was not changed in db " ' " myotubes compared to wt C2C12 myotubes (Fig. lb) clearly demonstrating specificity in the regulatory effect of dystrobrevin.
  • Skeletal muscle contains three isoforms of a-dystrobrevin, a-DBl , a-DB2 and a-DB3 (Enigk, R.E. and MM. Maimone, Differential expression and developmental regulation of a novel a-dystrobrevin isoform in muscle. Gene, 1999. 238(2): p. 479-488) which have distinct carboxy termini but otherwise identical.
  • Dystrobrevin (not the a-DBl isoform) downregulated ErbB4 expression andErbB4 in db '1' myotubes is processed to generate ErbB4 intracellular domain. Since ErbB2 phosphorylates a-DBl and ErbB4 can heterodimerize with ErbB2 upon ligand binding, expression levels of ErbB4/2 were examined to find out if there was a feedback on expression of these two receptors due to signaling from a-DBl . Essentially the same experiments as above were performed for qPCR but for the Western blot analysis, instead of performing an Immunoprecipitation, myotubes lysates were loaded on the gel.
  • dystrophin levels are not high, this essentially meant that dystrophin expression is downregulated to almost detection limits as confirmed by the Western blot analysis of Immunoprecipitated samples performed in triplicate (Fig. 3c). Re-incubating the Western blot, after stripping the bound antibodies, with an anti-Utrophin antibody confirmed the PCR data for Utrophin levels (Fig. 3d). Detection of a -syntrophin confirmed similar amounts of sample were loaded in each lane (Fig. 3d).
  • GFP-a-DBl-P3 downregulated ErbB4 expression to a lesser extent compared to GFP- a-DBl (Fig. 3e) thus phosphorylation of aDBl enhanced downregulation of ErbB4 expression in myoblasts but not in myotubes (see Fig. 2a).
  • Downregulation of dystrophin by GFP-a-DBl and GFP-a-DB l-P3 in myoblasts (Fig. 3e) was consistent with that observed in myotubes (Fig. le) except that expression of dystrophin was much higher in myoblasts in the absence of dystrobrevin.
  • dystrophin levels in db-/- myoblasts were about 350 fold compared to wt C2C12 myotubes, this apparent increased expression is due to less dystrophin being expressed in myoblasts compared to myotubes in wt C2C12. This was obvious when dystrophin levels in myoblasts were compared to myotubes in db " ' " which showed similar amounts in both (Fig. 3j). Results:
  • the association between the modulators of the present invention, preferably, NRGl signaling through ErbB4 receptors of the present invention, especially via free ErbB4 intracellular domain to increase dystrophin expression is disclosed.
  • the advantage of increasing dystrophin expression is that it is naturally present throughout the sarcolemma (cytoplasm) and, apart from muscle structural support, provides a signaling scaffold so that other signaling events can take place.
  • Increasing utrophin on the other hand may not serve the same function as it is usually found at the postsynaptic site of the neuromuscular synapse.
  • Utrophin can rescue dystrophic muscle to some extent since utrophin, although it shares sequence homology with dystrophin, differs in sequence and therefore in binding sites for proteins.
  • Dystrophin is the largest gene in humans and is estimated to take about 16hrs to transcribe. Hence, the six fold increase in dystrophin expression as illustrated by qPCR in db " ' ⁇ myotubes according to the present invention is a great achievement. In DMD the muscle upregulates utrophin by 40% (a 2 fold increase by qPCR is 100%) but this is not sufficient to prevent dystrophy and subsequent death by the age of 25-30.
  • an increased ErbB4 expression in myoblasts and myotubes formed from these myoblasts results in an increased amount of free ErbB4 intracellular domain and at the same time an increased amount of dystrophin expression.
  • ErbB2 levels were not increased to any significant amounts in myotubes and free ErbB2 Intracellular domain was not detected as expected since it is not known to be cleaved.
  • dystrophin expression levels are reduced to very low levels and barely detected on a Western blot confirming a direct relation between free ErbB4 intracellular domain and dystrophin expression since similar changes in ErbB2 levels was not observed and free ErbB2 intracellular domain was not detected on Westerns.
  • the data disclosed here demonstrates that increased expression of the epidermal growth factor NRGl receptor, ErbB4, leads to higher free ErbB4 intracellular domain levels driving increased dystrophin expression. Elimination of all NRGl signaling through ablation of ErbB4 and ErbB2 receptors, results in an absence of free ErbB4 intracellular domain and consequently a specific reduction in dystrophin expression.
  • a-dystrobrevin (a-DBl), a component of DAPC, downregulates dystrophin expression but not utrophin expression and a-DBl positively regulates ErbB4 but not ErbB2 expression.
  • a-DBl a -dystrobrevin
  • the data here shows for the first time that NRGl regulates dystrophin expression with the implication that regulation of dystrophin expression underlies the cause of several disease states or beneficial effects assigned to NRGl signaling.
  • the data demonstrates that the expressed ErbB4 is cleaved to generate the free ErbB4 intracellular domain.
  • ablation of ErbB2/4 reduces dystrophin expression confirming the above results that NRG signaling through ErbB2/4, through free ErbB4 intracellular domain, stimulated dystrophin expression.
  • dystrophin causes myopathy, including cardiomyopathy
  • results shown here demonstrate a mechanism through which NRG/ErbB4 signaling repairs heart injury and ameliorate symptoms in muscular dystrophy as well as other disease states associated with NRG signaling or dystrophin, as well as other diseases, for example, diseases due to lack of dsytroferlin (dysferlinopathies), through regulating dystrophin expression.
  • the data disclosed here demonstrate a mechanism through which dystrophin expression is regulated by NRG signaling through ErbB2/4 and by a-DBl .
  • This is supported by (i) ablation of dystrobrevin removes the downregulating effect on dystrophin expression in db " ' " myotubes; (ii) expression of a-DBl in db-/- myotubes restores dystrophin expression levels to wt C2C12 levels; (iii) higher expression of dystrophin in db " ' " myotubes are accompanied by higher expression of ErbB4; and (iv) ablation of ErbB2/4 downregulates dystrophin levels to detection limits.
  • free ErbB4 intracellular domain stimulated dystrophin expression either upon translocating to the nucleus or by phosphorylating a substrate in the cytoplasm that translocates to the nucleus activating dystrophin expression as free ErbB4 intracellular domain is a constitutively active tyrosine kinase (Linggi, B ., et al., The ErbB-4 s80 intracellular domain is a constitutively active tyrosine kinase. Oncogene, 2005. 25(1): p. 160-163).
  • Neuregulinl/ErbB4 signaling was shown to induce cardiomyocyte proliferation and repair heart injury (Bersell, K., et al., NeuregulinllErbB4 Signaling Induces Cardiomyocyte Proliferation and Repair of Heart Injury. Cell, 2009. 138(2): p. 257- 270).
  • dystrophin deficiency results in skeletal and cardiomyopathy.
  • Restoration of dystrophin in cardiomyopathy rescues the phenotype (Nguyen, A.T., et al., DOT1L regulates dystrophin expression and is critical for cardiac function. Genes & Development, 2011. 25(3): p. 263-274).
  • the evidence presented according to the present invention provides means for the reported beneficial effects of ErbB4 in cardiomyopathy where NRG signaling through ErbB2/4 increases dystrophin expression, mediated through free ErbB4 intracellular domain. Furthermore the results demonstrate how dystrophin expression is regulated by oc-DB l and NRG signaling to maintain physiological levels, a mechanism that can be utilized to ameliorate symptoms in muscular dystrophy.
  • dystrophin compensates to some extent loss of dystrophin, even though under normal conditions utrophin is localized at the postsynaptic sites of the neuromuscular synapse, thus increased dystrophin expression will rescue dystrophic phenotypes caused by a lack of or due to mutations in proteins, for example, dysferlin resulting in dysferlinopathies, by compensation. In dysferlinopathies, even increasing levels of a truncated dysferlin rescues the phenotype to some extent, similar to increasing utrophin or truncated dystrophin in muscular dystrophy.
  • NRG signaling through ErbB2/4 has a very broad scope as a therapeutic in treating myopathy, for example.
  • C2C12 and a-db-/- myoblasts (kind gift from B. Pawlikowski and M. Maimone (Upstate Medical University, State University of New York, Syracuse, NY) were cultured on laminin (Roche) coated dishes and upon reaching 70-80% confluency, were allowed to form myotubes by changing to differentiation media (2% horse serum, 1% penicillin/streptomycin [sigma], DMEM [sigma]).
  • Myotubes from 10 cm culture dishes were harvested in 600 ul of lysis buffer and protein complexes were purified essentially as described previously (Kramarcy, NR., et al., Association ofutrophin and multiple dystrophin short forms with the mammalian M(r) 58,000 dystrophin- associated protein (syntrophin). Journal of Biological Chemistry, 1994. 269(4): p. 2870-2876) with modifications.
  • myotubes harvested in icecold lysis buffer (10 mM Na3P04, pH 7.8, 150 mM NaCl, 5 mM EDTA, 1 mM EGTA, 1% Triton X-100, protease inhibitor mixture [Roche] , and phosphatase inhibitors Picl and Pic2 [Sigma- Aldrich]) were homogenized in a Dounce homogenizer and incubated for 3 h at 4°C with protein G-coupled mouse monoclonal syntrophin antibody 1351 (abeam).
  • 3 x SDS gel loading buffer 150 mM Tris-HCl [pH 6.8], 300 mM dithiothreitol [added just before use], 6% SDS, 0.3% bromophenol blue, and 30% glycerol.
  • 3 x SDS gel loading buffer 150 mM Tris-HCl [pH 6.8], 300 mM dithiothreitol [added just before use], 6% SDS, 0.3% bromophenol blue, and 30% glycerol.
  • 600 ul of 3 x SDS gel loading buffer was added to 10 cm culture dishes, myoblasts/myotubes were scraped together, put in a 2 ml eppendorf tube and pipetted repeatedly to reduce viscosity. Samples were then treated as above and loaded on SDS PAGE gels. Gels were transferred onto PVDF membranes (Millipore) and subject to ECL (Thermo Fisher Scientific) development after incubation with primary and secondary antibodies.
  • BSA (%) was used as a blocking reagent.
  • the following primary antibodies were used: mouse monoclonal Neu/ErbB2 (3B5; sc33684, 1 :300), rabbit polyclonal ErbB4 (s.c-283, 1:300), rabbit anti-dystrophin polyclonal (1: 400) and mouse anti-utrophin monoclonal (1:400) were all from Santa Cruz Biotechnology, Inc., mouse monoclonal anti-syntrophin 1351 (abeam, 4 ⁇ 1/10 cm dish of myotubes), rabbit anti-a-syntrophin 259 (5 ⁇ g/ml for Westerns; a kind gift from Stanly C.
  • Transfected myoblasts were then sorted for EGFP+ cells using the influx cell sorter (Becton Dickinson). To obtain more than 90% positive EGFP+ population, transfected myoblasts were sorted at least twice with a cell culture phase (3-4 passages) between each sort.
  • RNA isolation and qPCR were performed as previously described (Gajendran, N., et al., Neuregulin Signaling Is Dispensable for NMD A- and GABAA-Receptor Expression in the Cerebellum In Vivo. The Journal of Neuroscience, 2009. 29(8): p. 2404-2413) with modifications.
  • RNA from myoblast or myotube cultures was isolated with TRJzol (Invitrogen) according to their protocol.
  • DNase I (Promega) treatment and reverse transcription was performed on 1-5 mg total RNA with random primers and superscript reverse transcriptase from Invitrogen according to their protocol. For a given set of experiments, the same amount of total RNA from each sample was used for cDNA synthesis.
  • cDNA was diluted 1 :5 before use in qPCR.
  • Quantitative PCR was performed with SyBR Green mix (Applied Biosystems) using the applied biosystems StepOne machine with a two-step PCR (60°C, 1 min and 95°C 15 s) for 40 cycles using the standard program.
  • the quantitative PCR mix was prepared as follows: 12.5 ul SyBR Green mix, 2.5 ul of a 3 uM solution each of forward and reverse primer, 1 ul of diluted cDNA synthesized according to the reverse transcription kit protocol from Invitrogen and made up to 25 ⁇ total volume with sterile water. Each sample for real time PCR was done in triplicate and the mean of the resulting three values were taken.
  • ErbB2 forward 5 '- ATGTGTGGACCTGGACG AAC-3 ' and reverse 5'- CAGCCTACGCATGGTATACTTC-3';

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Abstract

La présente invention concerne de nouveaux moyens de régulation de l'expression de la dystrophine pour le traitement de maladies chez un sujet, et plus particulièrement de nouveaux moyens de régulation de l'expression de la dystrophine pour le traitement de maladies associées à la dystrophine, par l'intermédiaire de modulateurs du récepteur ErbB 4 et/ou par l'intermédiaire du domaine intracellulaire libre d'ErbB4.
PCT/EP2016/069877 2015-09-03 2016-08-23 Modulateurs du récepteur erbb 4 pour une utilisation dans le traitement de maladies associées à la dystrophine Ceased WO2017036852A1 (fr)

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WO2019115812A1 (fr) * 2017-12-14 2019-06-20 Universite De Strasbourg Peptides pour le traitement et la prévention d'une stéatose hépatique non alcoolique et d'une fibrose
JP2021506764A (ja) * 2017-12-14 2021-02-22 ユニヴェルシテ・ドゥ・ストラスブール 非アルコール性脂肪肝疾患及び線維症の治療及び予防のためのペプチド
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JP7362611B2 (ja) 2017-12-14 2023-10-17 ユニヴェルシテ・ドゥ・ストラスブール 非アルコール性脂肪肝疾患及び線維症の治療及び予防のためのペプチド
IL275188B1 (en) * 2017-12-14 2023-11-01 Univ Strasbourg Use of cyclic tripeptide to improve cellular energy metabolism
AU2018383035B2 (en) * 2017-12-14 2023-11-09 INSERM (Institut National de la Santé et de la Recherche Médicale) Peptides for treatment and prevention of nonalcoholic fatty liver disease and fibrosis
IL275188B2 (en) * 2017-12-14 2024-03-01 Univ Strasbourg Peptides for treatment and prevention of nonalcoholic fatty liver disease and fibrosis

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