WO2016119856A1 - Molécules induisant une autophagie permettant d'augmenter la libération d'insuline - Google Patents

Molécules induisant une autophagie permettant d'augmenter la libération d'insuline Download PDF

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WO2016119856A1
WO2016119856A1 PCT/EP2015/051818 EP2015051818W WO2016119856A1 WO 2016119856 A1 WO2016119856 A1 WO 2016119856A1 EP 2015051818 W EP2015051818 W EP 2015051818W WO 2016119856 A1 WO2016119856 A1 WO 2016119856A1
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molecule
cells
autophagy
peptide
insulin
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Romeo Ricci
Alexander GOGINASHVILI
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Centre National de la Recherche Scientifique CNRS
Institut National de la Sante et de la Recherche Medicale INSERM
Universite de Strasbourg
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Centre National de la Recherche Scientifique CNRS
Institut National de la Sante et de la Recherche Medicale INSERM
Universite de Strasbourg
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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
    • A61K38/1703—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • 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
    • A61K38/1703—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • A61K38/1709—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • A61K38/1761—Apoptosis related proteins, e.g. Apoptotic protease-activating factor-1 (APAF-1), Bax, Bax-inhibitory protein(s)(BI; bax-I), Myeloid cell leukemia associated protein (MCL-1), Inhibitor of apoptosis [IAP] or Bcl-2
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P5/00—Drugs for disorders of the endocrine system
    • A61P5/48—Drugs for disorders of the endocrine system of the pancreatic hormones

Definitions

  • the present invention relates to the field of medicine, in particular to the treatment of hyperglycemia and diabetes or any conditions necessitating an increase in insulin secretion.
  • Hyperglycemia is another metabolic disorder that poses a challenge to the health care system.
  • the liver is the primary organ for glucose and lipid metabolism.
  • blood glucose is maintained by the breakdown of hepatic glycogen followed by gluconeogenesis.
  • the insulin action in the liver turns off both glycogenolysis and gluconeogenesis and activates glycogenesis and lipogenesis to store the excess glucose.
  • Tight insulin-mediated regulation of hepatic glucose production is essential to maintain normoglycemia, and dysregulation caused by insulin resistance is the most prevalent metabolic abnormality in the developed world.
  • hyperglycemia is associated with increased mortality.
  • wound infections and other morbidities are increased among patients having diabetes or uncontrolled hyperglycemia.
  • Strict glycemic control during the perioperative time period can thus reduce morbidities and mortality.
  • Insulin is a hormone involved in glucose homeostasis. Insulin is produced by pancreatic beta-cells ( ⁇ -cells) located in the islets of Langerhans, and they secrete it in response to rising levels of blood glucose. Glucose is taken up from the blood by hepatocytes, muscle cells, and adipocytes used either as energy source or for storage as glycogen and triglycerides. Insulin also inhibits lipolysis, preventing fatty acid release from the fat tissue. On the contrary, low blood glucose levels result both in a reduced production and release of insulin. Together with glucagon action, it results in glucose release into blood stream.
  • ⁇ -cells pancreatic beta-cells
  • pancreatic ⁇ -cells can be increased by using autophagy- inducing molecules on pancreatic ⁇ -cells. Indeed, they showed that an autophagy-inducing molecule is able to dramatically increase the insulin release independently of glucose concentrations.
  • the present invention relates to an autophagy-inducing molecule for use to increase insulin release or secretion. It also relates to the use of an autophagy-inducing molecule for the manufacture of a medicament for increasing insulin release or secretion. It further relates to a method for increasing insulin release or secretion in a subject in need thereof, comprising administering a therapeutically efficient amount of an autophagy-inducing molecule, thereby increasing insulin release or secretion.
  • the increase of insulin release or secretion is aiming to treat hyperglycemia.
  • hyperglycemia is caused by or associated to a pre-diabetic condition, diabetes, preferably type 2 diabetes, hyperglycemia associated to medications, to acute stress such as stroke or myocardial infarction, to dysfunction of the thyroid, adrenal and pituitary glands, to pancreas disease such as pancreatitis and pancreas cancer, or to intracranial diseases.
  • the molecule inducing autophagy is a peptide derived from Beclin- 1 protein.
  • the peptide comprises Beclin 1 residues 269-283 (VFNATFHr HSGQF, SEQ ID No 1), more preferably comprises the sequence TNVFNATFH VHSGQFGT, SEQ ID No 2), wherein up to six of said residues may be substituted.
  • the peptide comprises or consists on a peptide comprising the amino acid sequence V-F-N-A-T-F-E/H-I- W-H-D/S (SEQ ID No 3), T-N-V-F-N-A-T-F-E/H-I-W-H-D/S-G-Q/E-F-G-T (SEQ ID No 4) or the D-retro-inverso sequence thereof, wherein up to 6 of said residues may be substituted.
  • the peptide is selected in the group consisting of YGRKKRRQRRRGGTNVFNATFHIWHSGQFGT (SEQ ID No 12); YGRKKRRQRRRGGTNVFNATFEIWHDGEFGT (SEQ ID No 13); and the D-retro-inverso amino acid sequence RRRQRRKKRGYGGTGFEGDHWIEFTANFVNT (SEQ ID No 14).
  • the molecule inducing autophagy is an antibody or a fragment or derivative thereof, an aptamer, a Spiegelmer or a chemical compound.
  • the molecule may comprise a cell penetrating moiety or a moiety targeting the pancreatic ⁇ cell.
  • the moiety targeting the pancreatic ⁇ cell is selected from the group consisting of glucagon- like peptide- 1 (GLP-1), glucagon- like peptide-2 (GLP-2), peptide YY (PYY), neuropeptide Y (NPY), pancreatic peptide (PPY), exendin-4, naphthylalanine, and naphthylalanine derivatives, , or is a moiety that that selectively binds a protein selected from the group consisting of DiGeorge syndrome critical region gene 2 (DGCR2), golgi brefeldin A resistant guanine nucleotide exchange factor 1 (GBFl), orphan G protein-coupled receptor GPR44 (GPR44), SerpinBlO (PI- 10), FXYD domain containing ion transport regulator 2 (FXYD2), Tetraspanin-7
  • the moiety targeting the pancreatic ⁇ cell is an antibody or a fragment thereof, an aptamer, or a ligand.
  • the moiety targeting the pancreatic ⁇ cell is a combination of several moieties targeting the pancreatic ⁇ cell which have different binding partners.
  • the molecule inducing autophagy is used in combination with an additional active drug, preferably an anti-diabetic drug, a hypo-glycemic drug or an anti-hyperglycemic drug.
  • an additional active drug preferably an anti-diabetic drug, a hypo-glycemic drug or an anti-hyperglycemic drug.
  • this product is for use in the treatment of a pre-diabetic condition, diabetes, preferably type 2 diabetes, hyperglycemia associated to medications, to acute stress such as stroke or myocardial infarction, to dysfunction of the thyroid, adrenal and pituitary glands, to pancreas disease such as pancreatitis and pancreas cancer, or to intracranial diseases.
  • diabetes preferably type 2 diabetes
  • hyperglycemia associated to medications to acute stress such as stroke or myocardial infarction
  • pancreas disease such as pancreatitis and pancreas cancer
  • intracranial diseases e.glycemia associated to medications
  • FIG. 1A Immunofluorescence (IF) of LC3B-GFP puncta (white arrows) in INSl cells under growing conditions (GC), without amino acids and fetal calf serum (no AA/FCS) or glucose (no Glc/FCS) for 2 and 6 hours. Quantification of LC3B-GFP puncta per cell (mean + s. e. m.). **P ⁇ 0.01.
  • FIG IB LC3B- GFP puncta in iNSl LC3B GFPendo cells under GC and no AA/FCS for 1 hour in absence and presence of Bafilomycin Al (BafAl), 10 nM.
  • FIG 1C CLEM of ptfLC3 expressing INSl cells under GC and no AA/FCS for 2 hours. EM, corresponding fluorescent and merged pictures are shown. Regions of interest (ROI) are indicated with labelled dashed squares. Yellow and black arrows indicate autophagosomes and autolysosomes, respectively.
  • FIG ID Western blot of LC3B and p62 using soluble and insoluble fractions of lysates of INSl cells under GC and no AA/FCS for 1.5 hours, non-treated or treated with BafAl, 1 nM for last 1 hour of incubation. GAPDH served as a loading control.
  • FIG IE IF of LC3B-GFP puncta (white arrows) and insulin (red) in ⁇ cells in islets of fed and fasted LC3B-GFP expressing mice. The nucleus was stained with DAPI. Quantification of LC3B-GFP puncta per 1600 ⁇ m 2 (mean + s. e. m.). **P ⁇ 0.01.
  • FIG. 2A Left: Immunofluorescence (IF) of Phogrin and Lampl in INSl cells under growing conditions (GC) or without amino acids and fetal calf serum (no AA/FCS) for 2 hours. Regions of interest (ROI) are indicated with dashed squares. White arrows indicate co-localization of Phogrin with Lampl. A Golgi marker (pGolgi-CFP) was used. Quantification of co-localization of Phogrin and Lampl per cell (mean + s. e. m.). **P ⁇ 0.01.
  • FIG 2C Western blot of Lamp2, Phogrin and LC3B using lysates of indicated fractions from INSl cells under GC and no AA/FCS for 30 minutes. Right: EM of GCLs in shifted fractions (red dashed boxes).
  • FIG 2D Western blot of proinsulin using lysates of INSl cells treated or not with lysosomal inhibitors (LI) under GC and no AA/FCS for 6 hours. GAPDH was used as a loading control.
  • FIG 2E Left: EM of Golgi areas in primary murine islets under GC and no AA/FCS for 2 hours. Yellow arrows indicate granule-containing lysosomes (GCLs). Right: Quantification of GCLs (N per cell view) in Golgi areas of ⁇ cells in primary murine islets under GC and no AA/FCS for 2 hours (mean + s.e.m.). **P ⁇ 0.01.
  • FIG 2F Left: IF of proinsulin/insulin ((Pro)insulin) and Lamp2 (left panel) and (Pro)insulin and LC3B-GFP in ⁇ cells in islets of fed and fasted LC3B-GFP expressing mice.
  • the nuclei were stained with DAPL; Right: Enhanced GCL formation in ⁇ cells in fasted as compared to fed mice.
  • FIG. 3A Granule containing lysosomes (GCLs) increase in INSl cells upon starvation. Electron microscopy of Golgi areas in INSl cells under growing culture (GC) conditions and without AA and fetal calf serum (no AA/FCS) for 2 hours. Regions of interest (ROI) highlighted with white dashed squares. Yellow asterisks indicate GCLs. Quantification of GCLs (mean + s.e.m.). **P ⁇ 0.01.
  • FIG. 3B Identification of granule containing lysosomes (GCLs) in starved LNSl cells by immunogold-labeling.
  • Fig. 4 Co-localization of LC3B-GFP and Phogrin did not increase in INSl cells upon starvation.
  • DAPI was used to stain nuclei. Merged, LC3B-GFP and Phogrin signals are shown separately. Quantification of Phogrin/LC3B-GFP puncta per cell view (mean + s.e.m.). ns: not significant.
  • FIG. 5A Western blotting of Beclinl or ATG5 using lysates of INSl cells transfected with non-silencing (NS) siRNA, siRNA against Beclinl (siBeclinl) or siRNA against ATG5 (siATG5). GAPDH was used as a loading control.
  • FIG 5B Immunofluorescence of Lampl and Phogrin in INSl cells in media without amino acids and fetal calf serum for 2 hours. Cells have been either transfected with NS siRNA, siATG5 or siBeclinl (lower panel) or treated or not with the autophagy inhibitor 3-methyladenine (3-MA).
  • DAPI was used to stain nuclei.
  • White arrows point at yellow puncta indicating co-localization of Phogrin with Lampl.
  • Quantifications of Phogrin/Lampl puncta per cell view (mean + s.e.m.). ns: not significant.
  • FIG. 6A IF of LC3B- GFP puncta (white arrows) in INSl cells under growing conditions (GC) or without amino acids and fetal calf serum (no AA/FCS) for 2 hours treated or not with rapamycin, 100 nM or torin-1, 250 nM as indicated. Quantification of LC3B-GFP puncta per cell (mean + s. e. m.). **P ⁇ 0.01.
  • FIG 6B Immunofluorescence (IF) of mTOR in INSl cells co-expressing Phogrin- GFP and Lampl-RFP under GC and no AA/FCS for 2 hours.
  • FIG 6D Upper panel: IF of phosphorylated ULK1 (S757-ULK1) in INSl cells co-expressing Phogrin-GFP and Lampl-RFP under GC and no AA/FCS for 2 hours. ROI are indicated with dashed squares. White arrows in ROI indicate co-localization of Phogrin- GFP with Lampl-RFP and S757-ULK1. A Golgi marker (Giantin) was used. Lower panel: Starvation led to formation of large bright S757-ULK1 puncta in INSl cells.
  • FIG. 6E Insulin in supernatants of INSl cells treated with non-stimulatory 2.8 mM Glc in presence of DMSO or rapamycin. Insulin concentrations are expressed as a percentage of DMSO control (mean + s. e. m.).
  • FIG. 6F Insulin in supernatants of human islets treated as indicated. Insulin concentrations are expressed as a percentage of insulin upon 16.7 mM stimulatory Glc (mean + s. e. m.). ***P ⁇ 0.05.
  • FIG. 7A Electron microscopy of ⁇ cells in isolated murine islets treated either with scrambled (scr.) tat-beclinl or tat-beclinl. The yellow asterisk indicates an autophagosome. Quantification of autophagic compartments (AC) per 100 ⁇ 2 cytoplasm area (mean + s.e.m.). ***P ⁇ 0.001.
  • FIG. 7B Viability of islets treated as indicated expressed as a percentage of viability of islets in 2.8 mM glucose (Glc) (mean + s.e.m.). ns: not significant.
  • FIG 7C Insulin of supernatants of islets treated as indicated. Insulin concentrations are expressed as a percentage of insulin upon 16.7 mM stimulatory Glc.
  • FIG 7D Insulin in supernatants of islets treated as indicated normalized to insulin concentrations in supernatants of islets treated with DMSO (mean + s.e.m.). ***P ⁇ 0.001.
  • FIG. 8A Electron microscopy of ⁇ cells in isolated murine islets treated either with scrambled (scr.) tat-beclinl or tat-beclinl. The yellow asterisk indicates an autophagosome. Quantification of autophagic compartments (AC) per 100 ⁇ 2 cytoplasm area (mean + s.e.m.). *P ⁇ 0.05.
  • FIG 8B Viability of islets treated as indicated expressed as a percentage of viability of islets in 2.8 mM glucose (Glc) (mean + s.e.m.). ns: not significant.
  • PKD 1 controls SINGD.
  • FIG 9A Left: Western blot of proinsulin using lysates of INS 1 cells treated with CID755673 for indicated times. GAPDH was used as a loading control.
  • FIG 9B Left: EM of Golgi areas in non-silenced and PKDl-depleted (shPKDl) INSl cells. Yellow arrows indicate secretory granules-containing lysosomes (GCLs). Right: Increased granule containing lysosomes (GCLs) in INSl cells depleted of PKDl. Quantification of GCLs in INSl cells transfected with short hairpin RNA against PKDl (shPKDl) compared to cells transfected with non- silencing shRNA (NS) (mean + s.e.m.). ***P ⁇ 0.001.
  • FIG 9C Immunofluorescence (IF) of mTOR and Lamp2 in non-silenced and PKDl-depleted (shPKDl) INSl cells. White arrows indicate co- localization of mTOR with Lamp2. The nucleus was stained with DAPI.
  • FIG 9D Western blot of indicated proteins using lysates of non-silenced and PKDl-depleted (shPKDl) INS l cells. GAPDH was used as a loading control.
  • FIG 9E LC3B-GFP puncta in iNSl LC3B GFPendo cells treated with CID755673 or DMSO for 4 hours in presence and absence of BafAl, 10 nM.
  • FIG 9F Live-cell FRET assay in INSl cells expressing G-PKDrep-live under growing culture (GC) conditions, without amino acids and fetal calf serum (no AA/FCS) or without glucose/FCS (no Glc/FCS). FRET was measured and expressed as normalized YFP-C/CFP ratios during a time course as indicated.
  • FIG 9G Left: Upper panel : EM of Golgi areas of fasted ⁇ cells in primary islets of ⁇ 38 ⁇ +,+ and ⁇ 38 ⁇ ⁇ ' ⁇ mice. Yellow arrows indicate secretory GCLs. Lower panel : EM of cytoplasm of fasted ⁇ cells in primary islets of ⁇ 38 ⁇ +,+ and ⁇ 38 ⁇ ⁇ ' ⁇ mice. The yellow arrow indicates an autophagosome.
  • GCLs Granule containing lysosomes
  • AC autophagic compartments
  • PKD1 knockdown in INSl cells decreases accumulation of newly formed insulin.
  • Autoradiography of total input was used as a loading control.
  • Fig. 11 Identification of granule containing lysosomes (GCLs) in PKDl-depleted INSl cells by immunogold-labeling.
  • FIG. 12 Identification of GCLs in lysosomal fractions. Western blotting of Lamp2, Phogrin and LC3B using lysates of indicated density gradient fractions from top to bottom from INS 1 cells expressing non-silencing (NS) short hairpin RNA (shRNA) or PKD1 -silencing shRNA (shPKDl) as indicated. Electron microscopy of GCLs in shifted fractions 7 to 9 (red dashed boxes) (right panel).
  • NS non-silencing
  • shPKDl PKD1 -silencing shRNA
  • Fig. 13 Inhibition of PKD markedly increased Phogrin and Lampl co-localization in INSl cells. From left to right: Immunofluorescence of LAMP1 and Phogrin of INS 1 cells treated with DMSO or the PKD inhibitor CID755673 for 4 hours in presence and absence of lysosomal inhibitors (LI). DAPI was used to stain nuclei. Quantification of co-localized Lampl- and Phogrin-positive puncta in INSl cells treated with DMSO or CID755673 in presence and absence of LI (mean + s.e.m.). **P ⁇ 0.01.
  • Fig. 14 Co-localization of Phogrin with LC3B-GFP remained unchanged in PKD-inhibited INSl cells.
  • DAPI was used to stain nuclei. Merged, LC3B-GFP and Phogrin signals are shown separately.
  • Fig. 15. Reduced autophagic flux in INSl cells depleted for PKD1.
  • the present invention relates to an autophagy-inducing molecule for increasing insulin release or secretion, in particular by pancreatic ⁇ cells. Indeed, the inventors surprisingly observed that a molecule inducing pancreatic ⁇ cell autophagy directly increases the insulin release/secretion, even at high blood glucose concentration.
  • the present invention relates to an autophagy-inducing molecule for increasing insulin release or secretion, in particular by pancreatic ⁇ cells.
  • Hyperglycemia is a condition in which an excessive amount of glucose circulates in the blood plasma. Generally, it is a glucose level higher than 11.1 mmol/1. A subject with a consistent range between 5.6 a,d 7 mmol/1 is considered as hyperglycemic, even if symptoms may not start to become noticeable until 15-20 mmol/1. Chronic levels exceeding 7 mmol/1 can produce organ damage such as kidney damage, neurological damage, cardiovascular damage, retinal damage and feet and leg damage. Acute hyperglycemia involving glucose levels that are extremely high is a medical emergency. Ketoacidosis, a life-threatening condition, may occur with untreated hyperglycemia.
  • the molecule inducing autophagy is useful for controlling blood glucose levels in hyperglycemic conditions.
  • Hyperglycemia can be due to different conditions. It can be caused by a pre-diabetic condition or diabetes. Indeed, hyperglycemia is caused by low insulin levels in the context of Type I diabetes or by resistance at the cellular level for type II diabetes.
  • any stage of Type II diabetes can be of interest, i.e. stage 1, stage 2, stage 3, stage 4 and stage 5.
  • treatment of prediabetic and late diabetic stage is contemplated.
  • the molecule is suitable for treating or delaying the progression or onset of diabetes mellitus, insulin resistance, diabetic retinopathy, diabetic neuropathy, diabetic nephropathy, insulin resistance, and obesity. Indeed, decreasing hyperglycemia by increasing insulin release or secretion could treat or delay the progression or onset of these diseases.
  • Hyperglycemia can be due to certain medications including corticosteroids, octreotide, beta blockers, epinephrine, thiazide diuretics, niacin, pentamidine, protease inhibitors, L- asparaginase and some antipsychotic agents.
  • corticosteroids including corticosteroids, octreotide, beta blockers, epinephrine, thiazide diuretics, niacin, pentamidine, protease inhibitors, L- asparaginase and some antipsychotic agents.
  • An acute stress such as stroke, or myocardial infarction may lead to hyperglycemia, even in the absence of diabetes.
  • Other conditions can cause hyperglycemia, in particular in the absence of diabetes. They include dysfunction of the thyroid, adrenal and pituitary glands, especially hyperthyroidism and Cushing's syndrome; numerous diseases of the pancreas such as pancreatitis and a pancreatic cancer; sepsis and certain infections; intracranial diseases such as encephalitis, brain tumors (especially those located near the pituitary gland), brain bleeds and meningitis; convulsions and terminal stages of many diseases.
  • an initial oral drug monotherapy with insulin-sensitizing metformin is used to treat hyperglycemia in type 2 diabetes.
  • this monotherapy is not sufficient in long- term. Therefore, this treatment is followed by two- or three-drug combinations.
  • metformin is either combined with sulfonylureas or meglitinides targeting ATP- dependent potassium channels of the ⁇ cell.
  • the latter drugs can provoke hypoglycemia and cause a weight gain. In long term, they can lead to ⁇ cell failure.
  • insulin- sensitizing thiazolidinediones TDZs
  • TDZs For pioglitazone, bladder cancer has been reported. Therefore, in many countries, TDZs have been withdrawn from the market. More recently, very promising drugs targeting the incretin axis, such as glucagon-like peptide 1 receptor agonists (GLP-1RA) and dipeptidyl peptidase-4 (DPP-IV) inhibitors are in clinical use. These drugs seem to have good effects in controlling glycemia, lead to body weight reduction and do not trigger hypoglycemia. Moreover, they evoke favorable effects on cardiovascular risk factors and biomarkers.
  • GLP-1RA glucagon-like peptide 1 receptor agonists
  • DPP-IV dipeptidyl peptidase-4
  • GLP-1 -based therapeutics will cause inflammation of the pancreas (pancreatitis) or even pancreatic cancer.
  • postmortem histology revealed that the use of GLP-1RA or DPP-IV inhibitors was associated with pancreatic exocrine cell dysplasia accompanied by hyperplasia of glucagon-secreting a-cells.
  • treat or “treatment” is intended that the disease or condition is cured, alleviated or delayed. It includes the preventive or curative treatment.
  • treatment designates in particular the correction, retardation, or reduction of an impaired glucose homeostasis.
  • treatment also designates an improvement in the insulin secretion or release.
  • controlling the blood glucose level or “the control of blood glucose level” refer to the normalization or the regulation of the blood or plasma glucose level in a mammalian subject having abnormal levels (i.e., levels that are below or above a known reference, median, or average value for a corresponding mammalian subject with a normal glucose homeostasis).
  • the present invention relates to the pharmaceutical or veterinary use of the molecule.
  • the subject may be any mammal, preferably a human subject, such as an adult or a child.
  • the subject may be a subject suffering of obesity.
  • the subject has no detectable anti-islet antibodies, and ultrasonography revealed no pancreatic abnormalities.
  • the subject can be an animal, preferably a mammal, in particular a pet animal such as a dog, a cat or a horse.
  • induce or “promote” autophagy is intended to refer to the occurrence of autophagy in a cell population increased by at least 10, 20, 30, 40, 50, 60, 70, 80 or 90 % when compared to the autophagy measured in absence of the molecule in the same conditions.
  • the method suitable for measuring autophagy is detailed below and in the example section.
  • the capacity to induce or promote autophagy is determined by the method disclosed in Orvedahl et al (2011, Nature, 480, 113-117, the disclosure of which being incorporated herein by reference).
  • the autophagy-inducing molecule is a peptide derived from Beclin- 1 protein.
  • the peptide derived from Beclin- 1 protein is a variant of human Beclin- 1 in which the threonine at position 119 is substituted with a phospho-mimicking residue, preferably a glutamic acid or an aspartic acid as disclosed in WO 10/064250 (the disclosure of which being incorporated herein by reference).
  • the peptide derived from Beclin- 1 protein is a variant of human Beclin- 1 in which Ser234, Ser295, Tyr229, Tyr233 or Try352 is substituted with a phospho-silencing residue, preferably an alanine as disclosed in WO14/046966 (the disclosure of which being incorporated herein by reference).
  • the molecule is a polynucleotide encoding such a variant of Beclin- 1.
  • the autophagy-inducing molecule is a peptide comprising Beclin 1 fragment as disclosed in WO2013/119377 and US8, 802,633 (the disclosure of which being incorporated herein by reference).
  • the autophagy-inducing peptide comprises Beclin 1 residues 269-283 (VFNATFHIWHSGQF, SEQ ID No 1), more preferably comprises the sequence TNVFNATFHIWHSGQFGT, SEQ ID No 2), wherein up to six of said residues may be substituted.
  • the peptide comprises at least one of F270, F274 and W277.
  • the peptide comprises at least one, two or three of substitutions: H275E, S279D and Q281E.
  • the autophagy-inducing peptide comprises or consists on a peptide comprising the amino acid sequence V-F-N-A-T-F-E/H-I-W-H-D/S (SEQ ID No 3), T-N-V-F- N-A-T-F-E/H-I-W-H-D/S-G-Q/E-F-G-T (SEQ ID No 4) or the D-retro-inverso sequence thereof, wherein up to 6 of said residues may be substituted.
  • the F residues are not substituted.
  • the peptide comprises the amino acid sequence VFNATFEr HD (SEQ ID No 5) or TNVFNATFEIWHDGEFGT (SEQ ID No 6) or the D-retro-inverso sequence thereof.
  • the peptide comprises the D-retro-inverso amino acid sequence TGFEGDHWIEFTANFVNT (SEQ ID No 7).
  • the peptide can be linked to additional moiety, optionally through a linker or spacer (e.g., diglycine).
  • the additional moiety can be a moiety facilitating its cellular uptake or entry, in particular a PTD (protein transduction domain); a homing peptide; a stabilizing agent such as PEG (polyethyleneglycol), oligo-N-methoxy-ethylglycine (NMEG), albumin, an albumin- binding protein or an immunoglobulin Fc domain; an affinity tag such as an immune-tag, biotin, lectin, or chelator; a detectable label such as an optical tag, a chelated lanthamide, a fluorescent dye, or a FRET acceptor/donor; a targeting moiety or a combination thereof.
  • PTD protein transduction domain
  • NMEG oligo-N-methoxy-ethylglycine
  • albumin an albumin- binding protein or an immunoglobulin Fc domain
  • the peptide can be part of a protein fusion.
  • PTD generally comprises a certain amino acid sequence of 10 to 20 amino acids (Matsushita and Matsui, (2005), J Mol Med 83, 324-328; Vives et al, Biochimic et Biophysica Acta, 2008, 1786, 126-138).
  • PTD is mainly composed of basic amino acids such as arginine or lysine
  • representative examples of the PTD include arginine rich peptides such as poly R 8 (RRRRRRRR, SEQ ID No 8) or (RRPRRPRRPRRPRRP, SEQ ID No 9), antennapedia or penetratin peptide such as (RQIKIWFQNRRMKWKK, SEQ ID No 10) or HIV-Tat (YGRKKRRQRRR, SEQ ID No 11).
  • Tat-Beclin 1 peptides have been disclosed as powerful inducer of autophagy (Shoji-Kawata et al, 2013, Nature, 494, 201-206; US 8,802,633).
  • Tat-Beclin 1 peptides are the followings: the wild-type Tat- Beclin 1 peptide of sequence YGRKKRRQRRRGGTNVFNATFITiWHSGQFGT (SEQ ID No 12); the mutated Tat-Beclin 1 peptide of sequence
  • YGRKKRRQRRRGGTNVFNATFEr HDGEFGT (SEQ ID No 13); and the D-retro-inverso amino acid sequence RRRQRRKKRGYGGTGFEGDHWIEFTANFVNT (SEQ ID No 14).
  • the diglycine linker in these peptides could be replaced by another amino acid linkers.
  • up to 6, 5, 4, 3, 2 or 1 residue substitutions can be introduced.
  • one or both F residues can be substituted or linked by other moieties as disclosed in US 8,802,633 (the disclosure of which being incorporated herein by reference), for instance crosslinkable moieties, by homocysteines for preparing cyclic peptides.
  • the peptide can comprise one or more D-amino acids, L-P-homo amino acids, D-P-homo amino acids, or N-methylated amino acids.
  • the peptide can be cyclized. It can be acetylated, acylated, formylated, amidated, phosphorylated, sulfated or glycosylated. In particular, it can comprise an N-terminal acetyl, formyl, myristoyl, palmitoyl, carboxyl or 2-furosyl group, and/or a C- terminal hydroxyl, amide, ester or thioester group.
  • the peptide can comprise the D-retro-inverso amino acid sequence of one peptide described above.
  • the peptide may have between 5 and 50 amino acids. More preferably, it has between 5 and 20 amino acids.
  • Telmisartan (2-(4- ⁇ [4-methyl-6-(l-methyl-lH-l,3-benzodiazol-2-yl)-2-propyl-lH- 1,3-benzodiazol-l-yl] methyl ⁇ phenyl) benzoic acid;
  • R being selected from the group consisting of dihydroxyacetone, glucose, galactose, glyceraldehyde, threose, xylose, mannose, ribose, ribulose, tagatose, psicose, fructose, sorbose, rhamnose, erythrose, erthrulose, arabinose, lyxose, allose, altrose, gulose, idose, talose, sucrose, lactose, maltose, lactulose, trehalose, cellobose, isomaltotriose, nigerotriose, maltotriose, melezitose, maltotriulose, raffinose, kestose and a combination thereof (US 2014296175);
  • Urolithin preferably selected from the group consisting of urolithin A, urolithin B, urolithin C, urolithin D, and any combination thereof as disclosed in WO14/004902 (the disclosure of which being incorporated herein by reference);
  • carbomezepine carbomezepine, tamoxifen, minoxidil, erapumil, or clonidine ;
  • Ri R 2 , R 3 , R4, R5, R6, Rn, Ri 2 and Ri 3 are each independently selected from hydrogen, hydroxyl, halogen, Cl-6 alkyl and Cl-6 haloalkyl;
  • R 7 and Rs are each independently selected from methoxyl and hydroxyl;
  • R9 and Rio are each independently selected from hydrogen, hydroxyl, halogen, Cl-6 alkyl as disclosed in WO12126390 (the disclosure of which being incorporated herein by reference);
  • a ceramide such as N-acylsphingosine or as disclosed in WO09076598 (the disclosure of which being incorporated herein by reference); an mTOR inhibitor such as rapamycin (sirolimus), rapamycin derivatives, CI-779, everolimus (CerticanTM), ABT-578, tacrolimus (FK 506), ABT- 578, AP-23675, BEZ-235, OSI-027, QLT-0447, ABI-009, BC-210, salirasib, TAFA-93, deforolimus (AP-23573), AP-23841, and temsirolimus (ToriselTM); temozolomide; arsenic trioxide; an Akt inhibitor such as perifosine or an Akt inhibitor disclosed in WO12/087336; a lithium salt such as lithium chloride, lithium carbonate, lithium citrate, lithium sulfate, lithium aspartate, lithium orotate; a BH3 mi
  • a calpain inhibitor such as calpastatin, ALLM, calpeptin, leupeptin, a-dicarbonyls, quinolinecarboxamides, sulfonium methyl ketones, diazomethyl ketones, Leu-Abu- CONHEt (AK275), 27-mer calpastatin peptide, Cbz-Val-Phe-H (MDL28170), calpeptin (Z-Leu-Nle-H), a- mercaptoacrylic acids, phosphorus derivatives, epoxysuccinates, acyloxymethyl ketones, halomethylketones and E64 as disclosed in WO07/003941 (the disclosure of which being incorporated herein by reference);
  • X H, CN, CI. OMe. or NMe 2 e, or NMe 2
  • These molecules might be selected from the group consisting of peptides, polypeptides, peptide mimetics, antibodies, fragments or derivatives thereof, aptamers, Spiegelmers, and chemical compounds.
  • the molecule is an antibody, a fragment thereof or a derivative thereof.
  • antibody and “immunoglobulin” have the same meaning and are used indifferently in the present invention.
  • the term “antibody” refers to immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, i.e., molecules that contain an antigen-binding site that immune-specifically binds an antigen.
  • Antibodies include any kind of antibodies, preferably monoclonal. They can be for instance IgG (immunoglobulin G) or VHH (heavy chain variable domain antibody from camelids).
  • Antibodies fragments or derivatives thereof include Fab, Fab', F(ab')2, scFv, (scFv)2, dAb, complementarity determining region (CDR) fragments, linear antibodies, single- chain antibody molecules, minibodies, nanobodies, diabodies, and multispecific antibodies formed from antibody fragments.
  • aptamer means a molecule of nucleic acid or a peptide. It refers to a class of molecule that represents an alternative to antibodies in term of molecular recognition. Aptamers are oligonucleotide or oligopeptide sequences with the capacity to recognize virtually any class of target molecules with high affinity and specificity. Such ligands may be isolated through Systematic Evolution of Ligands by Exponential enrichment (SELEX) of a random sequence library, as described in Tuerk C.
  • SELEX Systematic Evolution of Ligands by Exponential enrichment
  • the random sequence library is obtainable by combinatorial chemical synthesis of DNA.
  • each member is a linear oligomer, eventually chemically modified, of a unique sequence. Possible modifications, uses and advantages of this class of molecules have been reviewed in Jayasena S.D., Clin. Chem., 1999, 45(9): 1628-50.
  • Peptide aptamers consist of a conformationally constrained antibody variable region displayed by a platform protein, such as E. coli Thioredoxin A that are selected from combinatorial libraries by two hybrid methods (Colas et al., Nature, 1996,380, 548-50).
  • Spiegelmers have been disclosed for instance in WO 98/08856. They are molecules similar to aptamers. However, aptmers consist either completely or mostly of L-nucleotides rather than D-nucleotides in contrast to aptamers. Otherwise, particularly with regard to possible lengths of aptamers, the same applies to aptmers as outlined in connection with aptamers.
  • Chemical compounds refers to a molecule of less than about 1500 Daltons, 1000 Daltons, 800 Daltons, or even less than about 500 Daltons, in particular organic or inorganic compounds. Structural design in chemistry should help to find such a molecule. The molecule may have been identified by a screening method disclosed in the present invention.
  • Synthetic compound libraries are commercially available from a number of companies including Maybridge Chemical Co. (Trevillet, Cornwall, UK), Comgenex (Princeton, N. J.), Brandon Associates (Merrimack, N.H.), and Microsource (New Milford, Conn.). Combinatorial libraries are available or can be prepared according to known synthetic techniques. Alternatively, libraries of natural compounds in the form of bacterial, fungal, plant and animal extracts are available from e.g., Pan Laboratories (Bothell, Wash.) and MycoSearch (NC), or are readily producible by methods well known in the art. Additionally, natural and synthetically produced libraries and compounds can be further modified through conventional chemical and biochemical techniques.
  • a screening method refers to an in vitro and in vivo method allowing to measure or assess the effect of a candidate molecule on autophagy, and especially autophagy on ⁇ cells.
  • the in vitro method can comprise the use of insulin secreting cell lines, comprising HIT, RIN, HIT, beta TC, MIN6, INS-1 and INS-2 cells lines.
  • the in vivo method can comprise the use of animal model as Obese mouse, Diabetic mouse, Sand mouse [Psammomys obesus], Spiny mouse [Acomys cahirinus], BB rats, KK mouse, Yellow mouse, Yellow KK mouse, New Zealand obese mouse, Tuco-tuco [clenomys talarum], Chinese hamster [Cricetulus griseus], NOD mouse, Japanese wistar rat [Goto rat] and C57BL/6 mice.
  • animal model as Obese mouse, Diabetic mouse, Sand mouse [Psammomys obesus], Spiny mouse [Acomys cahirinus], BB rats, KK mouse, Yellow mouse, Yellow KK mouse, New Zealand obese mouse, Tuco-tuco [clenomys talarum], Chinese hamster [Cricetulus griseus], NOD mouse, Japanese wistar rat [Goto rat] and C57BL/6 mice.
  • the in vivo method may comprise the use of chemical agents capable of inducing diabetes, as Alloxan, Steptozocin, Diphenyl thiocarbazine, Oxine-9- hydroxyquinolone, Vacor, 6- aminonicotinamide, 1-asparginase, Azide, Cyanide, Cyproheptadine, Phenyloin, Thiazides, Malonates, Anti insulin antibodies Somatostatins, Catecholamines, Glucocorticoids or Glucagon.
  • chemical agents capable of inducing diabetes as Alloxan, Steptozocin, Diphenyl thiocarbazine, Oxine-9- hydroxyquinolone, Vacor, 6- aminonicotinamide, 1-asparginase, Azide, Cyanide, Cyproheptadine, Phenyloin, Thiazides, Malonates, Anti insulin antibodies Somatostatins, Catecholamines, Glucocorticoids or Glucagon.
  • the screening method can comprise the measure of RNA expression level or protein level expression and/or localization of beclin-1, ATG1 (also known as ULK1), lysosome-associated membrane proteins 2 (LAMP2) and cathepsin B, D, F, H, Atg8, Atg7 and micro tubule-associated proteinl light chain 3 (LC3).
  • Methods like quantitative RT-PCR or by Western blotting or immunocytochemistry can be used to determine the accumulation of autophagic vacuoles and autophagosomes in cells.
  • the screening method comprise the next steps (1) treating insulin secreting cell lines with a test substance; (2) measuring the amount of the accumulation of autophagic vacuoles and autophagosomes ; (3) optionally, measuring the amount of the insulin release or secretion ; (4) selecting the test substance if it increases the amount of the accumulation of autophagic vacuoles and autophagosomes and/or, optionally if it increases the insulin release.
  • the capacity to induce or promote autophagy is determined by the method disclosed in Orvedahl et al (2011, Nature, 480, 113-117, the disclosure of which being incorporated herein by reference). ⁇ cells targeting.
  • Autophagy-inducing molecules can be linked, covalently or not, to a moiety targeting the pancreatic tissue and more specifically ⁇ cells.
  • Several targeting molecules have already been disclosed in the art.
  • the pancreatic ⁇ cells targeting moiety can comprise an antibody, receptor, receptor ligand and the like, which preferentially and/or specifically bind to pancreatic ⁇ cells.
  • One or more targeting moieties can be attached to the autophagy inducing molecule to provide a composition that is capable targeting the composition to pancreatic ⁇ cells.
  • targeting moieties include, but are not limited to peptides that preferentially bind pancreatic ⁇ cells, antibodies that bind pancreatic ⁇ cells, and receptor ligands that bind pancreatic ⁇ cells.
  • aptamers are good affinity tools for cell surface proteins.
  • Alternatives that can be coupled to autophagy-inducing moieties include antibody fragments (scFvs) or Nanobodies (VHHs).
  • the pancreatic ⁇ cells specific targeting moiety comprises one or more peptides or other moieties that preferentially bind pancreatic ⁇ cells, selected from the group consisting of glucagon- like peptide- 1 (GLP-1), glucagon- like peptide-2 (GLP-2), peptide YY (PYY), neuropeptide Y (NPY), pancreatic peptide (PPY), exendin-4, naphthylalanine, naphthylalanine derivatives, and a combination thereof.
  • GLP-1 glucagon- like peptide- 1
  • GLP-2 glucagon- like peptide-2
  • PYY peptide YY
  • NPY neuropeptide Y
  • PPY pancreatic peptide
  • exendin-4 naphthylalanine, naphthylalanine derivatives, and a combination thereof.
  • the pancreatic ⁇ cells specific targeting moiety can be a moiety that selectively binds a protein selected from the group consisting of DiGeorge syndrome critical region gene 2 (DGCR2), golgi brefeldin A resistant guanine nucleotide exchange factor 1 (GBF1), orphan G protein-coupled receptor GPR44 (GPR44), SerpinBlO (PI- 10), FXYD domain containing ion transport regulator 2 (FXYD2), Tetraspanin-7 (TSPAN7), gap junction protein, delta 2, 36kDa (GJD2), solute carrier family 18 (vesicular monoamine), member 2 (SLC18A2), prokineticin receptor 1 (PROKR1), glutamate receptor, metabotropic 5 (GRM5), neuropeptide Y receptor Y2 (NPY2R), glucagon- like peptide 1 receptor (GLP1R), transmembrane protein 27 (TMEM27) and a combination thereof.
  • DGCR2 DiGeorge syndrome critical region
  • the moiety that selectively binds a protein as listed can be a ligand of the protein, an antibody specific for the protein or an aptamer specific for the protein. Details of such targeting strategies are disclosed in WO14140113, the disclosure of which being incorporated herein by reference.
  • a molecule which binds the transmembrane protein 27 (TMEM27) which is a surface N-glycoprotein that is highly expressed on ⁇ cells could be used, e.g. an antibody or fragment thereof or an aptamer.
  • TMEM27 transmembrane protein 27
  • an antibody 8/9-mAb is disclosed in Vats et al (2012, Diabetologia, 55, 2407-2416, the disclosure of which being incorporated herein by reference).
  • Hart et al (2010, FASEB J., 24, 816.9; 2014, Chembiochem. 15 ⁇ 135- 145) discloses a ligand composed of Glucagon-like Peptide 1 and Glibenclamide, this ligand displaying a high specificity for ⁇ cells.
  • the molecules according to the invention can be used in combination with one or more additional active drugs, preferably anti-diabetic or hypo-glycemic/anti-hyperglycemic drugs, in particular for treating, delaying or preventing hyperglycemia and associated disorders and diseases.
  • additional active drugs preferably anti-diabetic or hypo-glycemic/anti-hyperglycemic drugs, in particular for treating, delaying or preventing hyperglycemia and associated disorders and diseases.
  • the present invention also relates to a pharmaceutical composition
  • a pharmaceutical composition comprising a molecule according to the present invention and one or more additional active drugs, preferably an anti-diabetic drug, a hypo-glycemic drug or an anti-hyperglycemic drug.
  • a product or kit containing a molecule according to the invention and one or more additional active drugs, preferably an anti-diabetic drug, a hypo-glycemic drug or an anti- hyperglycemic drug, as a combined preparation for simultaneous, separate or sequential use, or a combined preparation which comprises a molecule according to the invention and one or more additional active drugs, preferably anti-diabetic drug, an hypo-glycemic drug or an anti- hyperglycemic drug, for simultaneous, separate or sequential use, in particular for treating, delaying or preventing hyperglycemia and associated disorders and diseases.
  • additional active drugs preferably an anti-diabetic drug, a hypo-glycemic drug or an anti- hyperglycemic drug, as a combined preparation for simultaneous, separate or sequential use, or a combined preparation which comprises a molecule according to the invention and one or more additional active drugs, preferably anti-diabetic drug, an hypo-glycemic drug or an anti- hyperglycemic drug, for
  • It relates to a molecule according to the invention for use for treating, delaying or preventing hyperglycemia and associated disorders and diseases in combination with one or more additional active drugs, preferably an anti-diabetic drug, a hypo-glycemic drug or an anti- hyperglycemic drug.
  • additional active drugs preferably an anti-diabetic drug, a hypo-glycemic drug or an anti- hyperglycemic drug.
  • a molecule according to the invention preferably an anti-diabetic drug, a hypo-glycemic drug or an anti-hyperglycemic drug, for the manufacture of a medicament, in particular for treating, delaying or preventing hyperglycemia and associated disorders and diseases.
  • additional active drugs preferably an anti-diabetic drug, a hypo-glycemic drug or an anti-hyperglycemic drug
  • a therapeutic effective amount of a molecule according to the invention is administered in combination with a therapeutic or sub -therapeutic effective amount of one or more additional active drugs, preferably an anti-diabetic drug, a hypo-glycemic drug or an anti-hyperglycemic drug.
  • additional active drugs preferably an anti-diabetic drug, a hypo-glycemic drug or an anti-hyperglycemic drug.
  • sub-therapeutic is intended to refer to an amount can be for instance 90, 80, 70, 60, 50, 40, 30, 20 or 10 % of the conventional therapeutic dosage (in particular for the same indication and the same administration route).
  • the additional active drug is a drug used for treating or delaying the progression or onset of diabetes mellitus, diabetic retinopathy, diabetic neuropathy, diabetic nephropathy, and hyperglycemia.
  • the additional drug can be an anti-diabetic drug such as a hypoglycemic agent or an antihyperglycemic agent.
  • insulin may be selected in the non-exhaustive list comprising insulin, metformin, sulfonylureas such as tolbutamide, acetohexamide, tolazamide, chlorpropamide, glyburide (also called glibenclamide), glimepiride, glipizide, glicazide, glycopyramide and gliquidone, alpha-glucosidase inhibitors such as acarbose, miglitol and voglibose, thiazolidinediones such as pioglitazone and rosiglitazone, a meglitinide such as repaglinide and nateglinide, incretin mimetics, glucagon-like peptide analogs and agonists such as exenotide, taspoglutide and liraglutide, dipeptidyl peptidase-4 inhibitors such as vildagliptin, sitagliptin, sax
  • compositions of the invention can be formulated for a topical, oral, parenteral, intranasal, intravenous, intramuscular, subcutaneous or intraocular administration and the like.
  • the molecule used in the pharmaceutical composition of the invention is present in a therapeutically effective amount.
  • therapeutically effective amount as used in the present application is intended to an amount of therapeutic agent, administered to a patient that is sufficient to constitute a treatment of diabetes mellitus, diabetic retinopathy, diabetic neuropathy, diabetic nephropathy, hyperglycemia, as defined above.
  • composition comprising the molecule is formulated in accordance with standard pharmaceutical practice (Lippincott Williams & Wilkins, 2000 and Encyclopedia of Pharmaceutical Technology, eds. J. Swarbrick and J. C. Boylan, 1988-1999, Marcel Dekker, New York) known by a person skilled in the art.
  • the composition can be formulated into conventional oral dosage forms such as tablets, capsules, powders, granules and liquid preparations such as syrups, elixirs, and concentrated drops.
  • Non-toxic solid carriers or diluents may be used which include, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, talcum, cellulose, glucose, sucrose, magnesium, carbonate, and the like.
  • binders which are agents which impart cohesive qualities to powdered materials, are also necessary.
  • starch, gelatine, sugars such as lactose or dextrose, and natural or synthetic gums can be used as binders.
  • Disintegrants are also necessary in the tablets to facilitate break-up of the tablet.
  • Disintegrants include starches, clays, celluloses, algins, gums and crosslinked polymers.
  • lubricants and glidants are also included in the tablets to prevent adhesion to the tablet material to surfaces in the manufacturing process and to improve the flow characteristics of the powder material during manufacture.
  • Colloidal silicon dioxide is most commonly used as a glidant and compounds such as talc or stearic acids are most commonly used as lubricants.
  • composition can be formulated into ointment, cream or gel form and appropriate penetrants or detergents could be used to facilitate permeation, such as dimethyl sulfoxide, dimethyl acetamide and dimethylformamide.
  • nasal sprays for transmucosal administration, nasal sprays, rectal or vaginal suppositories can be used.
  • the active compound can be incorporated into any of the known suppository bases by methods known in the art. Examples of such bases include cocoa butter, polyethylene glycols (carbowaxes), polyethylene sorbitan monostearate, and mixtures of these with other compatible materials to modify the melting point or dissolution rate.
  • compositions according to the invention may be formulated to release the active drug substantially immediately upon administration or at any predetermined time or time period after administration.
  • compositions according to the invention can comprise one or more molecule of the present invention associated with pharmaceutically acceptable excipients and/or carriers. These excipients and/or carriers are chosen according to the form of administration as described above.
  • the pharmaceutical composition according to the invention comprises 0.001 mg to 10 g of the molecule of the invention.
  • pharmaceutical composition according to the invention comprises 0.01 mg to 1 g of the molecule of the invention.
  • SINGD Starvation-Induced Nascent Granule Degradation
  • PKI Protein Kinase 1
  • SG Secretory Granule
  • mTOR mechanistic Target Of Rapamycin Switching from autophagy to SINGD is important to keep insulin secretion low under nutrient-poor conditions.
  • ⁇ cells employ a PKDl -dependent mechanism to adapt to nutrient availability coupling autophagy flux to secretory function.
  • pancreatic ⁇ cell catabolizes nutrients to secrete insulin that acts to induce anabolic pathways. Inversely, upon fasting insulin secretion has to be maintained at a low level. Shortage of nutrients is also expected to induce autophagy. During autophagy, cellular components are sequestered into double-membrane autophagosomes, which fuse with lysosomes (autolysosomes), where degradation occurs. Resulting catabolites maintain cells metabolically active ensuring cell survival. In fact, catabolism of nutrients also triggers insulin release, which should be avoided during fasting.
  • microtubule- associated protein 1 light chain 3 B incorporates into membranes of autophagosomes appearing as punctate structures.
  • INS1 cells a rat insulinoma- derived ⁇ cell line
  • LC3B- GFP GFP
  • INS1 cells deprived of serum and amino acids (AA) or glucose (Glc) dramatically decreased LC3B- GFP puncta (Fig. 1A).
  • Deprivation from serum alone did not change the amount of LC3B-GFP puncta.
  • AA amino acids
  • Glc glucose
  • INS 1 cells endogenously expressing LC3B-GFP INS 1 LC3B - GFPendo cells.
  • INS 1 LC3B - GFPendo cells starved human embryonic kidney 293
  • Time-lapse fluorescent microscopy of INS1 cells expressing ptf-LC3 revealed multiple RFP- GFP puncta that were converted into RFP only puncta upon onset of AA/serum or Glc/serum deprivation indicating the delivery of RFP-GFP-positive autophagosomes to autolysosomes. Subsequently, no reappearance of GFP-RFP puncta was observed and the number of RFP puncta was reduced.
  • Correlative Light and Electron Microscopy confirmed the autophagic origin of RFP-GFP and RFP-only puncta (Fig. 1C).
  • the inventors next assessed endogenous autophagy flux by Western Blotting (WB). They observed decreased accumulation of lipidated autophagosomal LC3B (LC3B-II) in AA/serum- starved INS 1 cells non-treated or treated with Baf A 1 as compared to non-starved cells (Fig ID). In contrast, starvation markedly increased LC3B-II in HEK293 cells. p62 binds polyubiquitinylated substrates for subsequent targeting to autophagosomes and was therefore used as another autophagy marker. Total p62 levels were moderately increased during starvation independent of BafAl (Fig. ID).
  • IF Immunofluorescence
  • ATG16L1- and ATG16Ll/LC3B-GFP-positive puncta moderately decreased upon AA/serum deprivation of iNSi LC3B - GFPendo cells.
  • QEM Quantitative EM
  • the inventors next used Quantitative EM (QEM) in ⁇ cells of non-starved and AA/serum-deprived murine primary islets.
  • QEM confirmed reduced autophagic compartments in starved as compared to non- starved primary ⁇ cells.
  • the inventors next used LC3B-GFP expressing mice et al, I Mol Biol Cell. 15, 1101-11 (2004)) to address autophagy levels in ⁇ cells in vivo. Strikingly, they found reduced LC3B-GFP puncta in ⁇ cells of fasted as compared to fed mice (Fig. IE). Overall, these data strongly suggest that autophagy is suppressed in fasted ⁇ cells.
  • SGs are generated at the trans-Golgi network (TGN).
  • TGN trans-Golgi network
  • the inventors thus investigated whether nascent granules are specifically targeted to lysosomes.
  • Readily formed SGs contain proinsulin, which is subsequently converted into insulin.
  • a 6-hour depletion from AA/serum led to an almost complete loss of proinsulin (Fig. 2D).
  • the decrease in proinsulin was partially restored by lysosomal protease inhibitors suggesting that nutrient deprivation leads to lysosomal degradation of de novo synthesized SGs in ⁇ cells.
  • Abundant GCLs upon starvation were confirmed ex vivo by QEM of primary murine islets (Fig. 2E).
  • Lysosomal AAs derived from degraded proteins induce translocation of mechanistic Target Of Rapamycin Complex 1 (mTORCl) to lysosomal membranes, mTORCl activation and subsequent suppression of autophagy.
  • mTORCl mechanistic Target Of Rapamycin Complex 1
  • rapamycin and torin 1 increased the number of LC3B-GFP puncta in AA/serum-depleted INSl cells as compared to control treated cells (Fig. 6A). This indicates that mTORCl activity is required for suppression of autophagy upon starvation.
  • PKD1 inactivation could have an effect on the turnover of de novo formed SGs.
  • treatment of INS1 cells with the PKD inhibitor CID755673 markedly decreased the amount of proinsulin under nutrient-rich conditions (Fig. 9A).
  • the rate of proinsulin biosynthesis was unchanged in INS1 cells stably expressing shRNA against PKD1 as compared to non-silenced cells (Fig. 9A).
  • PKDl largely co- localized with lysosomal Lampl in cells depleted of PKDl (Fig. 9C).
  • mTOR- mediated ULK1-S757 phosphorylation was markedly increased (Fig. 9D).
  • PKDl knockdown led to decreased accumulation of LC3-II in presence of BafAl (Fig. 15).
  • PKD inhibition considerably decreased LC3B-GFP puncta in presence and absence of BafAl (Fig. 9E).
  • inactivation of PKD is sufficient to trigger SINGD and to attenuate autophagy even under nutrient-rich conditions.
  • PKD TGN-localized Fluorescence Resonance Energy Transfer
  • G-PKDrep-live TGN-localized Fluorescence Resonance Energy Transfer reporter of PKDl activity
  • Fig. 9F time-lapse Fluorescence Resonance Energy Transfer
  • PKDl rapid inactivation of PKDl evokes SINGD, localized activation of mTOR and suppression of autophagy, which is critical to prevent insulin release during fasting.
  • PKD 1 activity is required for proper SG biogenesis and subsequent insulin secretion (Fig. 9H).
  • SINGD-mediated suppression of autophagy is an optimal strategy to counteract insulin secretion, at the same time providing sufficient nutrients. Since SINGD-mediated suppression of autophagy depends on an abundant nascent SG pool, its depletion will likely derepress autophagy without increasing insulin release.
  • Timing when depletion occurs in the course of starvation may vary substantially depending on the models and protocols used and may thus explain previously described inconsistencies (C. Ebato et ah, supra). Accordingly, prolonged fasting of mice was reported to induce autophagy in ⁇ cells (K. Fujimoto et ah, J. Biol. Chem. 284, 27664-73 (2009)). The inventors' observations point at the fact that triggering autophagy results in increased secretion of insulin. While this should be avoided during fasting, it may entail beneficial effects in conditions, where insulin demands are high, for example after a meal or in a diabetic state.

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Abstract

La présente invention concerne l'utilisation de molécules induisant une autophagie afin d'augmenter la libération ou la sécrétion d'insuline par les cellules bêta du pancréas. Cette invention est utile pour traiter l'hyperglycémie, associée ou non au diabète.
PCT/EP2015/051818 2015-01-29 2015-01-29 Molécules induisant une autophagie permettant d'augmenter la libération d'insuline Ceased WO2016119856A1 (fr)

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WO2017093330A1 (fr) * 2015-12-03 2017-06-08 Genethon Compositions et procédés permettant d'améliorer l'efficacité de vecteurs viraux
CN107674902A (zh) * 2017-11-02 2018-02-09 中国科学院兰州化学物理研究所 一种具有降血糖功能的驼血多肽及其制备方法
WO2018143403A1 (fr) * 2017-02-03 2018-08-09 国立大学法人東北大学 Composé hétérocyclique
US20210363205A1 (en) * 2020-04-30 2021-11-25 Larimar Therapeutics, Inc. Methods for treating myelin associated diseases and mitochondria associated diseases
WO2022038638A1 (fr) * 2020-08-19 2022-02-24 Translational Health Science And Technology Institute Peptides induisant l'autophagie
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US12411145B2 (en) 2019-11-25 2025-09-09 Larimar Therapeutics, Inc. Methods for quantifying frataxin activity
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US20240343766A1 (en) * 2020-04-30 2024-10-17 Larimar Therapeutics, Inc. Methods for treating myelin associated diseases and mitochondria associated diseases
US20210363205A1 (en) * 2020-04-30 2021-11-25 Larimar Therapeutics, Inc. Methods for treating myelin associated diseases and mitochondria associated diseases
WO2022038638A1 (fr) * 2020-08-19 2022-02-24 Translational Health Science And Technology Institute Peptides induisant l'autophagie
CN114316065B (zh) * 2021-10-29 2023-09-22 新乡医学院 小分子肽及其应用、细胞培养基、核酸分子
CN114316065A (zh) * 2021-10-29 2022-04-12 新乡医学院 小分子肽及其应用、细胞培养基、核酸分子

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