Peptides targeting GIP and GLP-2 receptors for the treatment of bone diseases

Abstract

The present invention relates to an isolated peptide and the use of the isolated peptide in treating bone diseases, wherein the peptide comprises a consensus amino acid sequence SEQ ID NO: 1: HGEGSFX 7 SDX 10 SX 12 X 13 LDKLAARDFVNWLLQTK (SEQ ID NO: 1) wherein X 7 is any amino acid residue, X 10 is any amino acid residue, X 12 is any amino acid residue, and X 13 is any amino acid residue.

Description

Technical Field

[0001] The present invention relates to the treatment of bone disorders. Background Art

[0002] Bone fragility is currently an important public health problem. In fact, with the aging of the population, the number of fractures continues to increase (377,000 fractures occurred in France in 2013), with serious socioeconomic consequences.

[0003] Bone tissue is a tissue that continuously remodels to adapt to mechanical stress (gravity, movement, etc.) and metabolic stress (release or storage of calcium, phosphate, protein, etc.), representing a system of balance between bone formation and bone resorption. Bone remodeling depends on several factors, such as calcium phosphate metabolic hormones, mechanical loads or local factors.

[0004] Bone fragility occurs when there is an imbalance between bone formation and bone resorption during bone remodeling, or when osteocyte activity is disturbed. This results in a decrease in the amount of bone tissue or changes in the quality of the bone matrix, or both, leading to increased bone fragility and a higher risk of fracture.

[0005] Several therapeutic options for the treatment of bone fragility are already available (vitamin D, bisphosphonates, anti-RANKL, recombinant calcitonin, intermittent injections of recombinant parathyroid hormone, modulators of the estrogen receptor) or will soon be available (anti-cathepsin K, anti-sclerostin, PTHrP analogs). However, these molecules show limitations of application and cannot be applied to all patients suffering from bone fragility. In addition, some of these molecules have side effects (osteonecrosis of the jaw, atypical fractures of the femur, risk of cancer) that require strict monitoring and regular follow-up of the patients. In addition, the efficiency of these molecules is still moderate and, despite their application, it is estimated that only 50% of bone fragility can be prevented with continuous treatment, justifying the need to find new therapeutic pathways. Summary of the invention

[0006] The present invention satisfies this need.

[0007] Gut hormones released after food enters the intestine have recently been shown to act on bone remodeling.

[0008] Among the numerous bioactive peptides secreted by the gastrointestinal tract, a class of peptides called incretins has emerged as an important regulator of energy metabolism. Incretins are hormones that are secreted from the intestine in response to glucose and stimulate insulin release in a glucose-dependent manner. Although several hormones with insulinotropic effects are secreted by the intestine, only two physiological incretins have been identified so far: glucose-dependent insulinotropic polypeptide (GIP) and glucagon-like peptide-1 (GLP-1). Recently, based on knockout preclinical animal models, the potential beneficial effects of both GIP and GLP-1 have been prominently demonstrated in diabetes-induced bone fragility.

[0009] The present invention arises from the unexpected discovery of the inventors that a peptide carrying a specifically designed consensus sequence is able to bind to and activate both the glucose-dependent insulinotropic polypeptide (GIP) receptor and the glucagon-like peptide 2 (GLP-2) receptor, similar to the respective natural peptides of GIP and GLP-2, thereby controlling bone remodeling. In particular, the inventors have shown that this dual-target peptide increases the enzymatic cross-linking of the collagen matrix produced by osteoblasts at a higher level than the individual natural peptides, and reduces the number of osteoclasts produced in a more significant manner than the individual natural peptides.

[0010] Therefore, the present invention relates to an isolated peptide comprising the consensus amino acid sequence SEQ ID NO: 1: HGEGSFX7SDX 10 SX 12 X 13 LDKLAARDFVNWLLQTK (SEQ ID NO: 1) wherein X7 is any amino acid residue, X 10 is any amino acid residue, X 12 is any amino acid residue, and X 13 is any amino acid residue.

[0011] The present invention also relates to a pharmaceutical composition comprising the peptide of the present invention.

[0012] The present invention further relates to an implantable medical device comprising a peptide according to the present invention, in particular the implantable medical device is coated with a peptide according to the present invention.

[0013] Another object of the invention is the in vitro use of the peptides according to the invention for coating implantable medical devices.

[0014] A further object of the present invention relates to the use of a peptide according to the present invention or a pharmaceutical composition according to the present invention for use in a method for the treatment and / or prevention of a bone disease in a subject.

[0015] In specific embodiments, the bone disorder is a manifestation of a metabolic disorder or a hormonal disorder in the bone.

[0016] The present invention also relates to a bone filling biomaterial comprising the peptide of the present invention.

[0017] The present invention further relates to the use of a bone filling biomaterial comprising the peptide of the present invention for bone regeneration. DETAILED DESCRIPTION

[0018] definition

[0019] In the context of the present invention, the term "peptide" refers to natural peptides (protein hydrolysates or synthetically synthesized peptides), and further refers to mimetic peptides, such as peptidomimetics and hemi-peptidomimetics that are peptide analogs, which may have modifications that, for example, make the peptide more stable or more immunogenic in vivo. Such modifications include, but are not limited to, cyclization, N-terminal modification, C-terminal modification, acylation, PEGylation, peptide bond modification, including, but not limited to, CH2-NH, CH2-S, CH2-S=O, O=C-NH, CH2-O, CH2-CH2, S=C-NH, CH=CH or CF=CH, backbone modification and residue modification. Methods for preparing mimetic peptide compounds are well known in the art and are specifically described in Quantitative Drug Design, CA. Ramsden Gd., Chapter 17.2, F. Choplin Pergamon Press (1992).

[0020] The peptides of the present invention are preferably composed of at least 30 amino acids, at least 31 amino acids, at least 32 amino acids or at least 33 amino acids, and preferably less than 50 amino acids. In a specific embodiment, the peptides of the present invention, in addition to the at least 30, 31, 32 or 33 amino acids, further comprise at least a peptide tag as defined below.

[0021] As used herein, the term "amino acid" is understood to include: the 20 natural amino acids, i.e., alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine and valine; amino acids carrying post-translational modifications that can be found in vivo, such as hydroxyproline, phosphoserine and phosphothreonine; and other unnatural amino acids, including, but not limited to, 2-aminoadipic acid, hydroxylysine, isodesmosine, norvaline, norleucine and ornithine. In addition, the term "amino acid" includes both D- and L-amino acids.

[0022] By "non-polar amino acids" is meant herein a class of amino acids in which the variable R groups are mostly composed of hydrocarbons. Non-polar amino acids include glycine, alanine, proline, valine, leucine, isoleucine, methionine, tryptophan, and phenylalanine.

[0023] As used herein, the term "isolated peptide" refers to any peptide, regardless of its method of synthesis, which differs locally from the native protein sequence of which the peptide may form a part in nature.

[0024] Peptides

[0025] As indicated above and shown in the examples below, the inventors designed a specific consensus amino acid sequence that enables peptides carrying this sequence to bind to and activate both the glucose-dependent insulinotropic polypeptide (GIP) receptor and the glucagon-like peptide 2 (GLP-2) receptor, similar to the respective native peptides of GIP and GLP-2.

[0026] Therefore, the present invention relates to an isolated peptide comprising or consisting of the consensus amino acid sequence SEQ ID NO: 1:

[0027] HGEGSFX7SDX 10 SX 12 X 13 LDKLAARDFVNWLLQTK(SEQ ID NO:1)

[0028] Where X7 is any amino acid residue, X 10 is any amino acid residue, X 12 is any amino acid residue, and X 13 is any amino acid residue.

[0029] In a specific embodiment, X7 is an amino acid selected from the group consisting of glycine, valine, threonine and serine, preferably an amino acid selected from the group consisting of glycine, valine and threonine.

[0030] In another specific embodiment, X 10 It is an amino acid selected from the group consisting of methionine, leucine and phenylalanine.

[0031] In another specific embodiment, X 12 It is an amino acid selected from the group consisting of isoleucine, valine and threonine, preferably an amino acid selected from the group consisting of isoleucine and valine.

[0032] In another specific embodiment, X 13 It is a non-polar amino acid, preferably an amino acid selected from the group consisting of alanine, valine and isoleucine, and particularly an amino acid selected from the group consisting of alanine and valine.

[0033] In a specific embodiment, X7 is an amino acid selected from the group consisting of glycine, valine, threonine and serine, X 10is an amino acid selected from the group consisting of methionine, leucine and phenylalanine, X 12 is an amino acid selected from the group consisting of isoleucine, valine and threonine, X 13 It is a non-polar amino acid.

[0034] In another specific embodiment, X7 is an amino acid selected from the group consisting of glycine, valine, threonine and serine, X 10 is an amino acid selected from the group consisting of methionine, leucine and phenylalanine, X 12 is an amino acid selected from the group consisting of isoleucine, valine and threonine, X 13 It is an amino acid selected from the group consisting of alanine, valine and isoleucine.

[0035] In another specific embodiment, X7 is an amino acid selected from the group consisting of glycine, valine and threonine, X 10 is an amino acid selected from the group consisting of methionine, leucine and phenylalanine, X 12 is an amino acid selected from the group consisting of isoleucine and valine, X 13 It is an amino acid selected from the group consisting of alanine and valine.

[0036] In a preferred embodiment, the C-terminal lysine of the peptides of the invention is amidated.

[0037] In a specific embodiment, the peptide of the present invention comprises or consists of the consensus amino acid sequence SEQ ID NO: 2: HGEGSFX7SDX 10 SX 12 X 13 LDKLAARDFVNWLLQTKITD(SEQ ID NO:2)

[0038] Among them, X7, X 10 , X 12 and X 13 As defined above.

[0039] In a specific embodiment, in the peptide of the present invention, K 16 and / or K 30 Further with modification, typically post-translational modification. In a more specific embodiment, in the peptide of the present invention, K 16 and / or K 30 Acylated or PEGylated.

[0040] The peptide according to the present invention may further comprise a peptide tag at its N-terminus or C-terminus, in particular at its C-terminus.

[0041] In the present invention, any of a variety of peptide tags recognized in the prior art can be used. For example, suitable peptide tags include: FLAG peptide, short FLAG peptide, His-6 peptide, glutathione-S-transferase (GST), staphylococcal protein A, streptococcal protein G, calmodulin, calmodulin binding peptide, thioredoxin, β-galactosidase, ubiquitin, chloramphenicol acetyltransferase S-peptide (Chloramphenicol cetyltransferasel S-peptide, ribonuclease A, residues 1-20), myosin heavy chain, DsbA, biotin subunit, avidin, streptavidin, Sfrp-tag, c-Myc, dihydrofolate reductase, CKS, polyarginine, polycysteine, polyphenylalanine, lac repressor, N-terminus of growth hormone, maltose binding protein, galactose binding protein, cyclomaltodextrin glucanotransferase, cellulose binding domain, hemolysin A, or Protein kinase site, BAI epitope, Btag, VP7 region of bluetongue virus, green fluorescent protein or any fluorescent dye.

[0042] The peptide tag may include one or more specific protease cleavage sites.

[0043] In a preferred embodiment, the peptide tag is a peptide consisting of the sequence GAADDDDDD (SEQ ID NO: 13).

[0044] In a specific embodiment, the peptide of the present invention is selected from the group consisting of:

[0045] - GL-0001 of sequence HGEGSFGSDMSIALDKLAARDFVNWLLQTKITD (SEQ ID NO: 3)

[0046] - GL-0007 of sequence HGEGSFGSDFSIALDKLAARDFVNWLLQTK-NH2 (SEQ ID NO: 4)

[0047] - GL-0001-tag of the sequence HGEGSFGSDMSIALDKLAARDFVNWLLQTKITDGAADDDDDD (SEQ ID NO: 5)

[0048] - GL-0002 of sequence HGEGSFVSDMSIVLDKLAARDFVNWLLQTK-NH2 (SEQ ID NO: 6)

[0049] - GL-0003 of sequence HGEGSFVSEMSIVLDKLAARDFVNWLLQTK-NH2 (SEQ ID NO: 7)

[0050] - GL-0004 of sequence HGEGSFVSDMSVVLDKLAARDFVNWLLQTK-NH2 (SEQ ID NO: 8)

[0051] - GL-0005 of sequence HGEGSFVSDLSVVLDKLAARDFVNWLLQTK-NH2 (SEQ ID NO: 9)

[0052] - GL-0006 of sequence HGEGSFVSDFSVVLDKLAARDFVNWLLQTK-NH2 (SEQ ID NO: 10)

[0053] - GL-0008 of sequence HGEGSFTSDFSIALDKLAARDFVNWLLQTK-NH2 (SEQ ID NO: 11), and

[0054] - GL-0009 of sequence HGEGSFVSDFSIALDKLAARDFVNWLLQTK-NH2 (SEQ ID NO: 12).

[0055] In a preferred embodiment, the peptide of the present invention is selected from the group consisting of:

[0056] - GL-0001 of sequence HGEGSFGSDMSIALDKLAARDFVNWLLQTKITD (SEQ ID NO: 3) and

[0057] GL-0007 of sequence HGEGSFGSDFSIALDKLAARDFVNWLLQTK-NH2 (SEQ ID NO: 4).

[0058] In a specific embodiment, the peptide of the invention is peptide GL-0001 of the sequence HGEGSFGSDMSIALDKLAARDFVNWLLQTKITD (SEQ ID NO: 3).

[0059] In a specific embodiment, the peptide of the present invention is modified so that its stability (especially in vivo stability) and / or its circulation time are increased compared to the unmodified peptide. Potential modifications that can be performed include those defined in the "peptide" chapters above, such as PEGylation, acylation, biotinylation, acetylation, formylation, ubiquitination, amidation, enzyme labeling or radiolabeling. For example, varying degrees of PEGylation can be used to change the half-life of the peptide, and increased PEGylation corresponds to an increase in half-life. Modifications can occur at any position on the peptide, including the peptide backbone, amino acid side chains, and amino or carboxyl termini.

[0060] In embodiments, the peptides of the present invention may be modified by the addition of TAT (transactivator of transcription) peptide, a well-known peptide commonly used for delivering peptides and known to facilitate in vivo administration of short peptides.

[0061] In another embodiment, the peptides of the present invention can be modified by adding the Fc domain of an antibody. The Fc domain of an antibody is a relatively constant region that is responsible for biological activity rather than antigen binding. Various therapeutic polypeptides have been established using the Fc domain to increase the half-life of a polypeptide or to add certain biological functions such as the ability to bind to specific receptors. The connection of the Fc domain to the peptides of the present invention is likely to increase the half-life of the peptide. The Fc domain may comprise a portion of a digested natural antibody, or it may be derived from a recombinant or humanized antibody.

[0062] The peptides of the present invention are capable of activating both GIP and GLP-2 receptors.

[0063] "GIP" or "glucose-dependent insulinotropic polypeptide" means herein an inhibitory hormone of the secretin family of hormones, which belongs to the incretin type and stimulates insulin secretion. In humans, it is derived from a 153 amino acid proprotein encoded by the GIP gene and circulates as a biologically active 42 amino acid peptide. It is synthesized by K cells, which are found in the mucosa of the duodenum and jejunum of the gastrointestinal tract.

[0064] "GIP receptor" or "GIP-R" herein refers to the protein encoded by the GIPR gene in humans, which is a member of the seven-transmembrane protein family and is a type of G protein-coupled receptor found on pancreatic beta cells.

[0065] "Activation of the GIP receptor" means herein that binding of the peptide to the GIP-R results in activation of intracellular signaling pathways, such as, but not limited to, an increase in cyclic adenosine monophosphate (cAMP).

[0066] Activation of the GIP receptor can be detected by any technique known to the skilled artisan. In particular, activation of the GIP receptor can be detected by evaluating cAMP by biochemical and / or imaging assays, such as, but not limited to, enzyme-linked immunosorbent assay or Fractional resonance energy transfer (FRET).

[0067] "GLP-2" or "glucagon-like peptide 2" means herein a 33 amino acid peptide formed by specific post-translational proteolytic cleavage of proglucagon in a process that also releases the related glucagon-like peptide- 1. GLP-2 is produced by enteroendocrine L cells and a variety of neurons in the central nervous system.

[0068] "GLP-2 receptor" or "GLP-2R" means the protein encoded by the GLP2R gene in humans, which is a member of the G protein-coupled receptor family that is closely related to the GLP1 receptor.

[0069] "Activation of the GLP-2 receptor" means herein that binding of the peptide to the GLP2-R results in activation of intracellular signaling pathways, such as, but not limited to, an increase in cAMP.

[0070] Activation of the GLP-2 receptor can be detected by any technique known to the skilled person. In particular, activation of the GLP-2 receptor can be detected by evaluating cAMP by biochemical and / or imaging assays, such as, but not limited to, enzyme-linked immunosorbent assay or Fractional resonance energy transfer (FRET).

[0071] Pharmaceutical compositions and medical devices

[0072] The present invention also relates to a pharmaceutical composition comprising a peptide as defined above in the section "Peptides" and optionally a pharmaceutically acceptable excipient.

[0073] The term "pharmaceutically acceptable" refers to properties and / or substances that are acceptable for administration to a subject from a pharmacological or toxicological point of view. In addition, "pharmaceutically acceptable" refers to factors known to pharmaceutical chemists such as formulation, stability, patient acceptance and bioavailability from a physical / chemical point of view.

[0074] As used herein, "pharmaceutically acceptable excipient" refers to any substance in a pharmaceutical composition that is different from the active ingredient. The excipient can be a sterile liquid, such as, for example, water and oils, including those of gasoline, animal, plant or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil, and the like, disintegrants, wetting agents, solubilizers, antioxidants, antimicrobial agents, isotonic agents, stabilizers or diluents. Suitable excipients and / or pharmaceutical carriers are described in "Remington's Pharmaceutical Sciences" by EW Martin.

[0075] The pharmaceutical compositions of the invention can be formulated for parenteral, e.g., intravenous, intradermal, intracerebroventricular, subcutaneous, intramuscular, intraperitoneal, oral (e.g., buccal, inhalation, nasal and pulmonary spray), intradermal, transdermal (topical), transmucosal, or intraocular administration.

[0076] The peptide of the present invention is particularly useful when used with implantable medical devices because it is able to, among other things, increase and improve integration into bone.

[0077] The present invention therefore also relates to an implantable medical device comprising a peptide according to the invention, in particular an implantable medical device coated with a peptide according to the invention.

[0078] As used herein, "implantable medical device" refers to any type of appliance that is introduced in whole or in part into a subject's body surgically or medically, or into a natural cavity through medical intervention, and is intended to remain there after the procedure. The duration of implantation may be substantially permanent, i.e., intended to remain in place for the remainder of the subject's life; until the device biodegrades; or until it is physically removed. Examples of implantable medical devices include orthopedic prostheses and dental implants.

[0079] The invention further relates to the in vitro use of the peptides of the invention for coating an implantable medical device, as defined above.

[0080] Medical indications

[0081] The present inventors have demonstrated that the peptides of the present invention increase the enzymatic cross-linking of the collagen matrix produced by osteoblasts at a higher level than GIP and GLP-2, respectively, and reduce the number of osteoclasts produced in a more important manner than GIP and GLP-2. Therefore, the peptides of the present invention are particularly promising for the treatment of bone diseases.

[0082] Therefore, the present invention also relates to the use of a peptide as defined in the above section "Peptides" or a pharmaceutical composition as defined in the above section "Pharmaceutical Compositions" in a method for treating and / or preventing a bone disease in a subject.

[0083] The present invention also relates to the use of a peptide as defined in the above section "Peptides" or a pharmaceutical composition as defined in the above section "Pharmaceutical Compositions" for the preparation of a medicament intended for the treatment and / or prevention of bone diseases.

[0084] The present invention also relates to a method for treating and / or preventing a bone disease in a subject, comprising administering to a subject in need thereof a therapeutically effective amount of a peptide as defined in the above section "Peptides" or a pharmaceutical composition as defined in the above section "Pharmaceutical Compositions".

[0085] "Bone disease" means herein a condition in which bone formation, deposition or resorption is abnormal. Bone diseases include, but are not limited to, any type of primary or secondary osteoporosis (postmenopausal osteoporosis, glucocorticoid-induced osteoporosis, disuse osteoporosis and senile osteoporosis), osteopenia, diabetic bone disease, osteomalacia and rickets, osteodystrophy such as Paget's disease of bone, hypercalcemia of malignancy, osteopenia due to bone metastasis, osteosarcoma, Ewing's osteoma, osteochondroma, bone abnormalities caused by cancer treatment, osteogenesis imperfecta, osteomyelitis, achondroplasia, avascular necrosis, osteosclerosis, myositis ossificans, periodontal disease, hyperparathyroidism, periarticular erosions in rheumatoid arthritis, bone erosions in ankylosing spondylitis and bone loss in anorexia nervosa.

[0086] Thus, in specific embodiments, the bone disease is selected from primary or secondary osteoporosis (including postmenopausal osteoporosis, glucocorticoid-induced osteoporosis, disuse osteoporosis and senile osteoporosis), osteopenia, diabetic bone disease, osteomalacia and rickets, bone dystrophies such as Paget's disease of bone, hypercalcemia of malignancy, osteopenia due to bone metastasis, osteosarcoma, Ewing's osteoma, osteochondroma, bone abnormalities caused by cancer treatment, osteogenesis imperfecta, osteomyelitis, achondroplasia, avascular necrosis, osteopetrosis, myositis ossificans, periodontal disease, hyperparathyroidism, periarticular erosions in rheumatoid arthritis, bone erosions in ankylosing spondylitis and bone loss in anorexia nervosa.

[0087] In a more specific embodiment, said bone disease is osteogenesis imperfecta.

[0088] In specific embodiments, the bone disorder is a manifestation of a metabolic disorder or a hormonal disorder in the bone.

[0089] "Metabolic disorder" or "metabolic disease" herein means any condition or disease that changes the normal metabolic process induced by abnormal chemical reactions in the body. Metabolic disorders include acid-base imbalance, metabolic encephalopathy, calcium metabolism disorders, DNA repair defect disorders, glucose metabolism disorders, hyperlactatemia, iron metabolism disorders, lipid metabolism disorders, malabsorption syndrome, metabolic syndrome X, inborn errors of metabolism, mitochondrial diseases, phosphorus metabolism disorders, porphyria, protein homeostasis defects, metabolic skin diseases, wasting syndromes and water-electrolyte disorders.

[0090] Preferably, the metabolic disorder is selected from the group consisting of a calcium metabolism disorder, a glucose metabolism disorder and a phosphorus metabolism disorder.

[0091] "Hormonal disorders" or "hormonal diseases" herein means a condition, disorder or disease that occurs in mammals due to overproduction and / or underproduction of any soluble molecule that acts at a distance from its site of production by circulating in the bloodstream and is associated or correlated with serum concentrations of insulin, glucagon, estrogen, testosterone and / or sex hormone binding globulin, thyroid hormone, parathyroid hormone, calcitonin, fibroblast growth factor 23, calcitriol that are below or above those of young healthy disease-free mammals. Hormonal disorders include disorders of glucose homeostasis, such as diabetes, hypoglycemia and glucagonoma; thyroid diseases, such as goiter, hyperthyroidism, hypothyroidism, thyroiditis and thyroid hormone resistance; calcium homeostasis disorders and metabolic bone diseases, such as parathyroid disease, osteoporosis, osteitis deformans, rickets and osteomalacia; pituitary gland diseases, such as diabetes insipidus and hypopituitarism.

[0092] Preferably, the hormonal disorder is selected from the group consisting of diabetes, hyperthyroidism, hypothyroidism, thyroiditis, thyroid hormone resistance, calcium homeostasis disorders, parathyroid disease, osteoporosis, osteitis deformans, rickets and osteomalacia.

[0093] The peptide of the present invention is particularly useful when used with bone filling biomaterials, as it is particularly capable of increasing and improving their integration into the bone.

[0094] The present invention therefore also relates to bone filling biomaterials comprising the peptides of the present invention.

[0095] "Bone filling biomaterial" means in this article a biomaterial that can fill bone defects (e.g., liquid or paste) in the early stage and solidify (e.g., solidify or semi-solidify) over time. Then, "bone filling material" is a material that can fill bone defects on bones (place the material at the bone defect by fusion, absorption, replacement, organization, etc.) (also including bone improvement such as promoting and increasing bone union in addition to bone union).

[0096] The present invention further relates to a bone filling biomaterial comprising the peptide of the present invention, said bone filling biomaterial being used for bone regeneration.

[0097] In specific embodiments, the bone filling biomaterial is used in, but not limited to, neurosurgery, orthopedics, or orbital / bone defect filling.

[0098] In a specific embodiment, the peptides of the present invention improve the bone integration of the bone filling material when used for bone regeneration.

[0099] "Subject" herein means a mammal, such as a rodent, a feline, a canine or a primate. Preferably, the subject according to the present invention is a human.

[0100] In specific embodiments, the subject has an implantable medical device, such as a prosthesis.

[0101] In the context of the present invention, the terms "treat" or "treatment" means reversing, alleviating, inhibiting the progress of the disorder or condition to which such terms apply or one or more symptoms of such disorder or condition.

[0102] In the context of the present invention, the term "prevention" or "prevention" refers to prophylactic treatment of a subject at risk of developing a condition, which results in a reduced likelihood that the subject will develop the condition or a delay in the onset of the condition.

[0103] A "therapeutically effective amount" of a peptide or pharmaceutical composition of the invention is meant to be an amount of the peptide or composition sufficient to treat or prevent a particular disease at a reasonable benefit / risk ratio applicable to any medical treatment. However, it is to be understood that the total daily dosage of the peptide or composition of the invention will be determined by the attending physician within the scope of reasonable medical judgment. The specific therapeutically effective dosage level for any particular subject will depend on a variety of factors, including the condition being treated and the severity of the condition, the activity of the specific peptide or composition employed, the specific combination employed, the age, weight, general health, sex and diet of the subject, the time of administration, the route of administration and the excretion rate of the specific peptide employed, the duration of treatment, the drugs used in combination or concurrently with the specific peptide employed, and similar factors well known in the medical field. For example, it is within the ability of the art to start the peptide at a level lower than the dose required to achieve the desired therapeutic effect, and gradually increase the dose until the desired effect is achieved.

[0104] The form of the pharmaceutical composition, route of administration, dosage and dosing regimen generally depend on the condition to be treated or prevented, the severity of the disease, the age, weight and sex of the patient, and the like.

[0105] The peptides and pharmaceutical compositions of the invention can be administered by any suitable route, in particular by parenteral, e.g., intravenous, intradermal, intracerebroventricular, subcutaneous, intramuscular, intraperitoneal, oral (e.g., buccal, inhalation, nasal and pulmonary spray), intradermal, transdermal (topical), transmucosal or intraocular administration.

[0106] Throughout this application, the term "comprising" is to be interpreted as covering all specifically mentioned features as well as optional, additional, unspecified features. As used herein, the use of the term "comprising" also discloses embodiments in which features other than those specifically mentioned are present (i.e., "consisting of ...").

[0107] The present invention will be further illustrated by means of the following drawings and examples.

[0108] A brief description of the sequence

[0109]

[0110] Sequence Listing <110> University of Angers Centre Hospitalier Universitaire de Angers (CHU d'Angers) <120> Peptides targeting GIP and GLP-2 receptors for the treatment of bone diseases <130> BET18P4591 <150> EP19305210.7 <151> 2019-02-21 <160> 16 <170> PatentIn version 3.5 <210> 1 <211> 30 <212> PRT <213> Artificial sequence <220> <223> consensus sequence <220> <221> MISC_FEATURE <222> (7) <223> Xaa = any amino acid residue <220> <221> MISC_FEATURE <222> (10)..(10) <223> Xaa = any amino acid residue <220> <221> MISC_FEATURE <222> (12)..(12) <223> Xaa = any amino acid residue <220> <221> MISC_FEATURE <222> (13) <223> Xaa = any amino acid residue <400> 1 His Gly Glu Gly Ser Phe Xaa Ser Asp Xaa Ser Xaa Xaa Leu Asp Lys 1 5 10 15 Leu Ala Ala Arg Asp Phe Val Asn Trp Leu Leu Gln Thr Lys 20 25 30 <210> 2 <211> 33 <212> PRT <213> Artificial sequence <220> <223> Longer consensus sequence <220> <221> MISC_FEATURE <222> (7) <223> Xaa = any amino acid <220> <221> MISC_FEATURE <222> (10)..(10) <223> Xaa = any amino acid <220> <221> MISC_FEATURE <222> (12)..(12) <223> Xaa = any amino acid <220> <221> MISC_FEATURE <222> (13) <223> Xaa = any amino acid <400> 2 His Gly Glu Gly Ser Phe Xaa Ser Asp Xaa Ser Xaa Xaa Leu Asp Lys 1 5 10 15 Leu Ala Ala Arg Asp Phe Val Asn Trp Leu Leu Gln Thr Lys Ile Thr 20 25 30 Asp <210> 3 <211> 33 <212> PRT <213> Artificial sequence <220> <223> Peptide GL-0001 <400> 3 His Gly Glu Gly Ser Phe Gly Ser Asp Met Ser Ile Ala Leu Asp Lys 1 5 10 15 Leu Ala Ala Arg Asp Phe Val Asn Trp Leu Leu Gln Thr Lys Ile Thr 20 25 30 Asp <210> 4 <211> 30 <212> PRT <213> Artificial sequence <220> <223> Peptide GL-0007 <220> <221> MOD_RES <222> (30)..(30) <223> Amidation <400> 4 His Gly Glu Gly Ser Phe Gly Ser Asp Phe Ser Ile Ala Leu Asp Lys 1 5 10 15 Leu Ala Ala Arg Asp Phe Val Asn Trp Leu Leu Gln Thr Lys 20 25 30 <210> 5 <211> 42 <212> PRT <213> Artificial sequence <220> <223> Peptide GL-0001-Tag <400> 5 His Gly Glu Gly Ser Phe Gly Ser Asp Met Ser Ile Ala Leu Asp Lys 1 5 10 15 Leu Ala Ala Arg Asp Phe Val Asn Trp Leu Leu Gln Thr Lys Ile Thr 20 25 30 Asp Gly Ala Ala Asp Asp Asp Asp Asp Asp 35 40 <210> 6 <211> 30 <212> PRT <213> Artificial sequence <220> <223> Peptide GL-0002 <220> <221> MOD_RES <222> (30)..(30) <223> Amidation <400> 6 His Gly Glu Gly Ser Phe Val Ser Asp Met Ser Ile Val Leu Asp Lys 1 5 10 15 Leu Ala Ala Arg Asp Phe Val Asn Trp Leu Leu Gln Thr Lys 20 25 30 <210> 7 <211> 30 <212> PRT <213> Artificial sequence <220> <223> Peptide GL-0003 <220> <221> MOD_RES <222> (30)..(30) <223> Amidation <400> 7 His Gly Glu Gly Ser Phe Val Ser Glu Met Ser Ile Val Leu Asp Lys 1 5 10 15 Leu Ala Ala Arg Asp Phe Val Asn Trp Leu Leu Gln Thr Lys 20 25 30 <210> 8 <211> 30 <212> PRT <213> Artificial sequence <220> <223> Peptide GL-0004 <220> <221> MOD_RES <222> (30)..(30) <223> Amidation <400> 8 His Gly Glu Gly Ser Phe Val Ser Asp Met Ser Val Val Leu Asp Lys 1 5 10 15 Leu Ala Ala Arg Asp Phe Val Asn Trp Leu Leu Gln Thr Lys 20 25 30 <210> 9 <211> 30 <212> PRT <213> Artificial sequence <220> <223> Peptide GL-0005 <220> <221> MOD_RES <222> (30)..(30) <223> Amidation <400> 9 His Gly Glu Gly Ser Phe Val Ser Asp Leu Ser Val Val Leu Asp Lys 1 5 10 15 Leu Ala Ala Arg Asp Phe Val Asn Trp Leu Leu Gln Thr Lys 20 25 30 <210> 10 <211> 30 <212> PRT <213> Artificial sequence <220> <223> Peptide GL-0006 <220> <221> MOD_RES <222> (30)..(30) <223> Amidation <400> 10 His Gly Glu Gly Ser Phe Val Ser Asp Phe Ser Val Val Leu Asp Lys 1 5 10 15 Leu Ala Ala Arg Asp Phe Val Asn Trp Leu Leu Gln Thr Lys 20 25 30 <210> 11 <211> 30 <212> PRT <213> Artificial sequence <220> <223> Peptide GL-0008 <220> <221> MOD_RES <222> (30)..(30) <223> Amidation <400> 11 His Gly Glu Gly Ser Phe Thr Ser Asp Phe Ser Ile Ala Leu Asp Lys 1 5 10 15 Leu Ala Ala Arg Asp Phe Val Asn Trp Leu Leu Gln Thr Lys 20 25 30 <210> 12 <211> 30 <212> PRT <213> Artificial sequence <220> <223> Peptide GL-0009 <220> <221> MOD_RES <222> (30)..(30) <223> Amidation <400> 12 His Gly Glu Gly Ser Phe Val Ser Asp Phe Ser Ile Ala Leu Asp Lys 1 5 10 15 Leu Ala Ala Arg Asp Phe Val Asn Trp Leu Leu Gln Thr Lys 20 25 30 <210> 13 <211> 9 <212> PRT <213> Artificial sequence <220> <223> Peptide tag <400> 13 Gly Ala Ala Asp Asp Asp Asp Asp Asp 1 5 <210> 14 <211> 33 <212> PRT <213> Artificial sequence <220> <223> Peptide Co-3 of WO2018 / 069442 <400> 14 His Ala Asp Gly Thr Phe Ile Ser Asp Tyr Ser Thr Ile Leu Asp Asn 1 5 10 15 Leu Ala Ala Arg Asp Phe Ile Asn Trp Leu Ile Gln Thr Lys Ile Thr 20 25 30 Asp <210> 15 <211> 33 <212> PRT <213> Artificial sequence <220> <223> Peptide Co-7 of WO2018 / 069442 <400> 15 His Ala Glu Gly Thr Phe Ile Ser Asp Tyr Ser Ile Ala Met Asp Lys 1 5 10 15 Leu Ala Ala Arg Asp Phe Ile Asn Trp Leu Ile Gln Thr Lys Ile Thr 20 25 30 Asp <210> 16 <211> 33 <212> PRT <213> Artificial sequence <220> <223> Peptide Co-19 of WO2018 / 069442 <400> 16 His Ala Asp Gly Thr Phe Ile Ser Asp Tyr Ser Thr Ile Leu Asp Asn 1 5 10 15 Leu Ala Ala Arg Asp Phe Ile Asn Trp Leu Ile Gln Thr Lys Gly Lys 20 25 30 Lys BRIEF DESCRIPTION OF THE DRAWINGS

[0111] Figure 1 : Bioinformatics (in silico) strategy for designing dual GIP / GLP-2 analogs. The ultimate goal is to obtain 1-30 or a consensus sequence with at least 50% homology between human GLP-2. The lowercase letters in step 3 represent: n: negatively charged amino acid, p: polar amino acid, a: aliphatic amino acid.

[0112] Figure 2-3 :Effects of combined administration of GIP and GLP-2 on collagen maturation and the number of osteoclasts.

[0113] Figure 2 : In MC3T3-E1 cultures, the cells were cultured in the presence of vehicle (CTRL) or 200 nM [D-Ala 2 ]-GIP 1-30NH2 (GIP), [Gly 2 Collagen maturation was assessed in the presence of [D-Ala]-GLP-2 (GLP-2) or both molecules (GIP+GLP-2). *: p<0.05 and ***: p<0.001 relative to vehicle-treated cultures; $$: p<0.01 relative to [D-Ala]-GLP-2 (GLP-2) or both molecules (GIP+GLP-2). 2 ]GIP 1-30NH2 -treated cultures p<0.01, #: relative to [Gly 2 ]-GLP-2-treated cultures p<0.05.

[0114] Figure 3 : The number of newly generated osteoclasts per well (N.Oc / well) was evaluated in human peripheral blood mononuclear cells isolated from healthy individuals and cultured in the presence of 25 ng / ml human M-CSF and 50 ng / ml human soluble RANKL (MR). This parameter was also evaluated in the presence of 1 nM [D-Ala 2 ]-GIP 1-30NH2 (GIP), [Gly 2 ]-GLP-2 (GLP-2) or both molecules (GIP + GLP-2) were evaluated in MR-treated cultures. ***: p<0.001 vs. MR; $: p<0.001 vs. [D-Ala 2 ]GIP 1-30NH2 -treated cultures p<0.05 and $$$: relative to [D-Ala 2 ]GIP 1-30NH2 -treated cultures p<0.001, and ###: relative to [Gly 2 ]-GLP-2-treated cultures p<0.001.

[0115] Figure 4: Effect of GL-0001 on osteoblast cell death. -6 Murine MC3T3-E1 cells were cultured for 24 hours in the presence of M rosiglitazone. *: p<0.05 relative to saline-treated cultures.

[0116] Figure 5-7 : Effect of the dual analogs on collagen maturation in vitro. The dashed line corresponds to the baseline level of collagen maturation.

[0117] Figure 8 : Effect of GL-0001 or GL-0007 on body weight. Body weight of ovariectomized animals treated with vehicle (OVX+Veh), 25 nmol / kg / day GL-0001 (OVX+GL-0001), 25 nmol / kg / day GL-0007 (OVX+GL-0007_25), 100 nmol / kg / day GL-0007 (OVX+GL-0007_100), or once with 100 μg / kg zoledronic acid (OVX+Zol) is presented at the end of 8 weeks of treatment. *: p<0.05 vs. OVX+Veh and **: p<0.01 vs. OVX+Veh.

[0118] Fig. 9 : Effect of GL-0001 or GL-0007 on cortical bone strength. Cortical bone strength was evaluated at the femoral shaft in ovariectomized animals treated with vehicle (OVX+Veh), 25 nmole / kg / day GL-0001 (OVX+GL-0001), 25 nmole / kg / day GL-0007 (OVX+GL-0007_25), 100 nmole / kg / day GL-0007 (OVX+GL-0007_100), or once with 100 μg / kg zoledronic acid (OVX+Zol) by 3-point bending assay. *: p<0.05 relative to OVX+Veh and **: p<0.01 relative to OVX+Veh.

[0119] Figure 10-11Effect of GL-0001 or GL-0007 on cortical bone microarchitecture. Cortical bone microarchitecture was evaluated by micro-computed tomography 4 mm above the distal femoral growth plate (white rectangle on 3D model). Animals were treated with vehicle (OVX+Veh), 25 nmol / kg / day GL-0001 (OVX+GL-0001), 25 nmol / kg / day GL-0007 (OVX+GL-0007_25), 100 nmol / kg / day GL-0007 (OVX+GL-0007_100), or once with 100 μg / kg zoledronic acid (OVX+Zol). The characteristics studied are as follows: Tt.Ar: total cross-sectional area, Ct.Th: cortical thickness, Ma.Ar: bone marrow area, J: polar moment of inertia, Ct.Ar: cortical bone area, Ct.Ar / Tt.At: fraction of cortical area, Iap: moment of inertia of the section about the anterior-posterior axis, Iml: moment of inertia of the interface about the mediolateral axis. *: p < 0.05 vs. OVX+Veh and **: p < 0.01 vs. OVX+Veh.

[0120] Figure 12-13 : Effects of GL-0001 or GL-0007 on trabecular bone strength and microarchitecture.

[0121] Fig.12 :Evaluation of trabecular bone strength by compression testing of the second lumbar vertebral body.

[0122] Fig.13 : Trabecular microarchitecture was evaluated in the vertebral body of the fifth lumbar vertebra. Animals were treated with vehicle (OVX+Veh), 25 nmol / kg / day GL-0001 (OVX+GL-0001), 25 nmol / kg / day GL-0007 (OVX+GL-0007_25), 100 nmol / kg / day GL-0007 (OVX+GL-0007_100), or once with 100 μg / kg zoledronic acid (OVX+Zol). The characteristics studied were as follows: BV / TV: bone volume fraction, Tb.N: trabecular number, Tb.Th: trabecular thickness, Tb.Sp: trabecular separation. *: p<0.05 vs. OVX+Veh and **: p<0.01 vs. OVX+Veh.

[0123] Figure 14-15: Effect of GL-0001 or GL-0007 on bone matrix composition. Tissue material properties were evaluated by Fourier transform infrared imaging (FTIRI) in the posterior quarter of the femoral shaft. Animals were treated with vehicle (OVX+Veh), 25 nmol / kg / day GL-0001 (OVX+GL-0001), 25 nmol / kg / day GL-0007 (OVX+GL-0007_25), 100 nmol / kg / day GL-0007 (OVX+GL-0007_100) or once with 100 μg / kg zoledronic acid (OVX+Zol). CCL: collagen maturity, XST: mineral crystallinity, Crystal_size: crystal size index, P / A: phosphate / amide ratio, C / P: carbonate / phosphate ratio, AcP: acid phosphate content. The mean and heterogeneity (width) of each parameter are given. *: p<0.05 vs. OVX+Veh and **: p<0.01 vs. OVX+Veh.

[0124] Figure 16-18 :Activation of GIPr and GLP-2r.

[0125] Fig.16 HEK-293 cells were transfected with a plasmid encoding human GIPr. Cyclic AMP production was recorded by FRET using the H74 probe. An increase in the FRET 470 / 530nm ratio indicates higher cAMP. 2 ]GIP 1-30NH2 , [Gly 2 ]GLP-2, GL-0001 or vehicle was added to the culture, and cAMP levels were recorded 30 minutes later. ***: p<0.001 relative to vehicle; ###: p<0.001 relative to [Gly 2 ]GLP-2, p<0.001.

[0126] Fig.17 HEK-293 cells were transfected with a plasmid encoding human GLP-2r. Cyclic AMP production was recorded by FRET using the H74 probe. An increase in the FRET 470 / 530nm ratio indicates higher cAMP. 2 ]GIP 1-30NH2 , [Gly 2 ]GLP-2, GL-0001 or vehicle was added to the culture, and cAMP levels were recorded 30 minutes later. **: p<0.01 relative to vehicle, ***: p<0.001 relative to vehicle; $: p<0.01 relative to [D-Ala 2 ]GIP 1-30NH2 , p<0.05, $$: relative to [D-Ala 2 ]GIP1-30NH2 , p<0.01.

[0127] Fig.18 HEK-293 cells were transfected with plasmids encoding human GIPr and human GLP-2r. Cyclic AMP production was recorded by FRET using the H74 probe. An increase in the FRET 470 / 530nm ratio indicates higher cAMP. 2 ]GIP 1-30NH2 , [Gly 2 ]GLP-2, GL-0001 or vehicle was added to the culture, and cAMP levels were recorded 30 minutes later. ***: p<0.001 relative to vehicle; ###: p<0.001 relative to [Gly 2 ]GLP-2, p<0.001; $$$: relative to [D-Ala 2 ]GIP 1-30NH2 , p<0.001.

[0128] Fig.19 : Sequence alignment of the peptides of the present invention and the peptides disclosed in WO2018 / 069442.

[0129] Fig. 20 : Collagen maturation index as a function of peptide concentration. The dashed line represents the collagen maturation index observed in cultures without any peptide.

[0130] Fig.21 :Evolution of the combination index (CI) within the dose range of collagen maturation.

[0131] Fig. 22 : Osteoclast responses of Raw 264.7 cells exposed to several concentrations of the dual GIP / GLP-2 analog. The dashed line represents the half-maximal effect used to compete for the IC50.

[0132] Fig.23 : Dose response of the inhibitory effect of the dual GIP / GLP-2 analogs. The dashed line represents the half-maximal effect (EC50).

[0133] Fig.24 : For osteoclastogenesis, the combined action index (CI) at the EC50 of GIP and GLP-2. The gray area between the dashes represents the CI value indicating additivity. The values ​​above the gray area indicate antagonism, while the values ​​below the gray area indicate synergy.

[0134] Example

[0135] Example 1 : Design of the peptide of the present invention

[0136] Materials and methods

[0137] Full details of all steps are in Figure 1 The amino acid sequences of human GIP (accession number #: P09681) and human GLP-2 (accession number #: P01275.3) were collected from the NCBI website (www.ncbi.nlm.nih.gov / protein). The amino acid sequences of known GLP-2 analogs, namely Teduglutide (PubChem CID: 16139605), Elsiglutide, ZP1848 and FE 203799, were also collected (Wisniewski et al., (2016) J. Med. Chem. 59: 3129-3139). The protein sequences were manually aligned using Word 2013 software. The analysis of the homology percentage was performed in Matlab R2016b using the Needleman-Wunsch algorithm. The sequence homology threshold for verification was set to GIP 1-30 or GLP-2 with >50% homology.

[0138] result

[0139] Figure 1 The bioinformatics strategy used to design dual GIP / GLP-2 analogs is presented.

[0140] In step 1, manually perform GIP in Word 2013 1-30 Sequence alignment between α-GLP-2 and human GLP-2 generated a consensus sequence 1 (CS1) corresponding to the same amino acid at the same sequence position. 2 , Gly 4 ,Phe 6 , Asp 15 , Asp 21 ,Phe 22 , Asn 24 , Trp 25 ,Leu 26 and Lys 30 composition.

[0141] In step 2, four known GLP-2 analogs, namely teduglutide, elsiglutide, ZP1848 and FE203799, were also manually aligned to establish consensus sequence 2 (CS2). CS2 consists of His 1 , Gly 2 , Gly 4 ,Phe 6 , Ser 7Glu 9 , Thr 12 ,Ile 13 ,Leu 14 , Asp 15 ,Leu 17 , Ala 18 , Ala 19 , Arg 20 , Asp 21 ,Phe 22 ,Ile 23 , Trp 25 ,Leu 26 ,Ile 27 , Thr 29 , Lys 30 ,Ile 31 , Thr 32 and Asp 33 composition.

[0142] In step 3, human GIP1-30 and human GLP-2 were manually aligned to determine the amino acid type in the missing position of CS1. When an amino acid from the same type was encountered in both sequences at the same position, its type was indicated in the consensus sequence 3 (CS3). It appears that negatively charged amino acids, polar amino acids, negatively charged amino acids, polar amino acids, aliphatic amino acids, aliphatic amino acids, aliphatic amino acids, aliphatic amino acids, aliphatic amino acids, and polar amino acids were found in both peptide sequences at positions 3, 5, 9, 11, 13, 14, 17, 23, 27, and 29, respectively.

[0143] In step 4, the inventors compared CS1 and CS2 to establish a sequence that could form a GLP-2 analog. From this comparison, consensus sequence 4 (CS4) was established, which contained His 1 , Gly 4 ,Phe 6 , Ser 7 Glu 9 , Thr 12 ,Ile 13 ,Leu 14 , Asp 15 ,Leu 17 , Ala 18 , Ala 19 , Arg 20 , Asp 21 ,Phe 22 ,Ile 23 , Asn 24 , Trp 25 ,Leu 26 ,Ile27 , Thr 29 , Lys 30 ,Ile 31 , Thr 32 , Asp 33 .

[0144] In step 5, the inventors aligned CS4 and CS3 to verify whether the amino acids at positions 3, 5, 9, 11, 13, 14, 17, 23, 27 and 29 were of the correct type. CS4 was then verified.

[0145] Next, the inventors carried out human GIP 1-30 The present inventors have demonstrated that CS4 has a high sequence homology (76%) with human GLP-2 and a low sequence homology (76%) with human GIP-2. 1-30 Has low homology (27%).

[0146] Next they adjusted CS4 at positions 2, 3, 5, 8, 10, 11, 16, and 28, resulting in consensus sequence 7 (CS7) as follows: His 1 , Gly 2 Glu 3 , Gly 4 , Thr 5 ,Phe 6 , Ser 7 , Ser 8 Glu 9 , Ser 11 , Thr 12 ,Ile 13 ,Leu 14 , Asp 15 , Lys 16 ,Leu 17 , Ala 18 , Ala 19 , Arg 20 , Asp 21 ,Phe 22 ,Ile 23 , Asn 24 , Trp 25 ,Leu 26 ,Ile 27 , Ala 28 , Thr 29 , Lys 30 ,Ile 31 , Thr 32 and Asp 33 It is worth noting that the choice of Gly2 To confer resistance to dipeptidyl-peptidase-4.

[0147] The present inventors have re-examined the 1-30 The sequence homology with human GLP-2 was found to be 45% and 76%, respectively. However, because their goal was to obtain a molecule with at least 50% homology to both humans, they re-modified CS7 to include the GIP 1-30 More homology. Modifications were made at positions 5, 7, 9, 12, 13, 23, 27, 28, and 31-33. This resulted in consensus sequence 9 (CS9), which is His 1 , Gly 2 Glu 3 , Gly 4 , Ser 5 ,Phe 6 , Ser 8 , Asp 9 , Ser 11 ,Leu 14 , Asp 15 , Lys 16 ,Leu 17 , Ala 18 , Ala 19 , Arg 20 , Asp 21 ,Phe 22 , Val 23 , Asn 24 , Trp 25 ,Leu 26 ,Leu 27 , Asn 28 , Thr 29 and Lys 30 , and has terminal amidation. From the literature, it seems that if Lys 30 is amidated, then Ile 31 、Thr 32 and Asp 33 It is not necessary (Wisniewski et al. (2016) J. Med. Chem. 59: 3129-3139). They also judged that any amino acid can be present at positions 7, 10, 12, and 13 to contribute to receptor binding ability and biological activity.

[0148] They next compared CS9 with human GIP 1-30 and human GLP-2 and were found to have more than 50% homology with both molecules, which validated CS9.

[0149] Embodiment 2:Effects of the peptides of the present invention on bone remodeling

[0150] Materials and methods

[0151] 1. Reagents

[0152] All analogs were purchased from GeneCust Europe with a purity of >95% (Dudelange, Luxembourg). Purity was verified by HPLC and peptide composition by mass spectrometry. Murine MC3T3-E1 subclone 4, murine Raw 264.7, and human HEK-293 cells were purchased from the American Type Culture Collection (ATCC, Teddington, UK). Buffy coats from healthy individuals were obtained from Etablissement du sang (Angers, France). Human and mouse receptor activators of nuclear factor kB ligand (RANKL) and human macrophage colony stimulating factor (M-CSF) were purchased from R&D Systems Europe (Abingdon, UK). Human GIP receptor and human GLP-2 receptor cDNA were purchased from Addgene (plasmid 14942, a gift from B. Thorens) and PlasmID Repository (plasmid HsCD00346244), respectively. All other chemicals were purchased from Sigma-Aldrich (Lyon, France) unless otherwise specified.

[0153] 2. Cell Culture

[0154] Murine MC3T3-E1 subclone 4 cells were grown and expanded in propagation medium containing alpha minimal essential medium (αMEM) supplemented with 5% fetal bovine serum (FBS), 5% calf serum, 100 U / mL penicillin and 100 μg / mL streptomycin at 37°C in a humidified atmosphere enriched with 5% CO2.

[0155] Murine Raw 264.7 and human HEK-293 cells were grown and expanded in propagation medium containing Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 100 U / mL penicillin and 100 μg / mL streptomycin in a humidified atmosphere enriched with 5% CO2 at 37°C.

[0156] 3. Cell Death Assay

[0157] The increase in plasma membrane permeability that occurs during cell death can be visualized using trypan blue, a dye that is excreted from living cells and incorporated into cells as they undergo death (Bellido and Plotkin (2008) Methods Mol. Biol. 455:51-75).

[0158] Briefly, MC3T3-E1 cells were seeded in 24-well plates and incubated in the presence of saline, various concentrations of GL-0001, or 10 -6 M rosiglitazone, a drug known to increase osteoblast cell death (Mieczkowska et al. (2012) J. Biol. Chem 287: 23517-23526). After 24 hours, the cell culture supernatant containing the floating cells was collected and placed in a pre-labeled eppendorf tube. Each well was washed in PBS before adding trypsin to dissociate the adherent cells. The mixture containing the dissociated adherent cells was collected and concentrated in the eppendorf containing the cell culture supernatant. The cells were centrifuged at 1,500 revolutions per minute (rpm) for 10 minutes, the supernatant was carefully removed, and the cells were incubated with 0.04% trypan blue and transferred to a hemacytometer. The live cells (clear) and dead cells (blue) were counted under light microscopy, and the percentage of dead cells was determined for each condition as follows:

[0159] % dead cells = 100 x (number of dead cells) / (number of dead cells + number of live cells)

[0160] 4. Osteoclast Assay

[0161] Human peripheral blood mononuclear cells were isolated from 3 buffy coats obtained at the Etablissement Francais du Sang (Angers, France) as described in Mabilleau and Sabokbar (2009) PLoS One 4:e4173.

[0162] Blood was diluted 1:1 in α-Minimum Essential Medium (MEM) (Invitrogen, Paisley, UK), layered on neutrophil separation medium (Histopaque), and centrifuged (700× g) for 20 minutes. The interface layer was resuspended in MEM and then centrifuged (600× g) for another 10 minutes, after which the resulting cells were resuspended in medium supplemented with 10% heat-inactivated fetal calf serum (FCS, Invitrogen, Paisley, UK) and counted in a hemocytometer after lysing red blood cells using a 5% (v / v) acetic acid solution.

[0163] To assess the extent of osteoclast formation, isolated human PBMCs were cultured in MEM (osteoclast culture medium) containing 100 UI / ml penicillin, 100 μg / ml streptomycin and 10% FCS in 24-well plates (Mabilleau et al. (2011) J Biol Chem 286: 3242-3249). After 2 hours of incubation, the cultures were vigorously rinsed in the culture medium to remove non-adherent cells and then maintained in 1 ml MEM / FCS containing 25 ng / ml recombinant human M-CSF, 50 ng / ml recombinant human sRANKL (added on day 7) and various concentrations of gastrointestinal hormone analogs (added on day 7). Culture was terminated after 14 days to assess the extent of osteoclast formation (TRAcP staining was performed as described below). All factors were updated every 2-3 days.

[0164] Rat Raw 264.7 cells were scraped from plastic dishes and cultured at 1.25 × 10 4 cells / cm 2 Cells were plated at a density of 100 μg / ml and grown in propagation medium enriched with 10 ng / ml soluble murine RANKL. After 110 h, cells were fixed with formalin (10% in PBS buffer) for 10 min and rinsed in distilled water before TRAcP staining.

[0165] 5.TRAcP Staining

[0166] Using naphthol AS-BI-phosphate as substrate and Fast violet B as diazonium salt, tartrate-resistant acid phosphatase (TRAcP) was histochemically developed by synchronous coupled reaction at 37°C in the dark for 90 minutes. The culture was rinsed three times in distilled water and the residual activity was inhibited by 4% NaF for 30 minutes. The cells were then rinsed in distilled water, counterstained with DAPI for 20 minutes, and dried before being sealed with an aqueous medium. TRAcP-positive cells with more than three nuclei were identified as osteoclasts. The number of newly generated cells was evaluated using optical microscopy.

[0167] 6. Collagen Maturity Determination

[0168] For the collagen maturation assay, cells were dissociated with trypsin-EDTA and cultured at 1.5 × 10 4 cells / cm 2 The cells were plated at a density of 100 μg / mL and grown to confluence in propagation medium. At confluence, the propagation medium was replaced with differentiation medium containing αMEM supplemented with 5% FBS, 5% calf serum, 100 U / mL penicillin, 100 μg / mL streptomycin, 50 μg / ml ascorbic acid, and various concentrations of analogs. This day was considered day 1. The differentiation medium was changed every two days.

[0169] On day 14, osteoblast cultures were decellularized by incubation for 4 hours on an orbital shaker in 0.2 M sodium cacodylate buffer (pH 7.4) containing 0.1% triton X100. Cultures were rinsed at least 6 times with milliQ water, fixed in absolute ethanol, scraped from the culture dishes, and transferred to BaF2 windows, where they were air-dried.

[0170] The integrity of the collagen extracellular matrix was verified by comparing the acquired FTIR spectra with those of commercially available collagen.

[0171] Spectroscopic analysis was performed using a Bruker Vertex 70 spectrometer (Bruker optics, Ettlingen, Germany) interfaced to a Bruker Hyperion 3000 infrared microscope equipped with a standard single-element mercury cadmium telluride (MCT) detector. -1 The infrared spectra were recorded in transmission mode with an average of 32 scans at a resolution of 100 nm. Background spectral images were collected from the same BaF2 window at the beginning and end of each experiment under the same conditions to ensure instrument stability. At least 20 spectra were acquired for each condition and analyzed in Matlab R2016b (The Mathworks, Natick, MA) using a laboratory-made routine script. The collagen maturity index was determined as the relative ratio of mature pyridinium to immature dehydrodihydroxylysine norleucine collagen cross-links using their respective 1660 cm-1 peaks at amide I. -1 and 1690cm -1 The sub-band at is determined.

[0172] 7. Binding Assay

[0173] Human HEK-293 cells were cultured at 2 x 10 5 cells / cm 2 The cells were plated at a density of 100 μg in a 10 cm Petri dish. After 24 hours, the cells were transfected with 15 μg of plasmid DNA encoding human GIP receptor or human GLP-2 receptor using the optimized calcium phosphate method. 24 hours after transfection, the transfected cells were dissociated and plated at 6 x 10 4 cells / cm 2 The cells were plated at a density of 10 μg / mL in black 96-well plates with a clear bottom (Ibidi GmbH, Martinsried, Germany). After 24 h, 10 μg / mL of αMEM supplemented with 0.1% bovine serum albumin (BSA) were added to each well. -7 M Fam-[D-Ala2 ]-GIP 1-30 or 10 -6 M Fam-[Gly 2 Various concentrations of analogs were added in the presence of ]-GLP-2. Equilibration binding was allowed to complete overnight at 37°C. Cells were then washed twice with assay buffer and lysed in 0.1 M NaOH.

[0174] Fluorescence was read using an M2 microplate reader (Molecular devices, Wokingham, UK) with the excitation wavelength set at 490 nm and the emission wavelength set at 525 nm.

[0175] Binding at the human GIP receptor or human GLP-2 receptor was performed by nonlinear regression analysis in GraphPad Prism 6.0.

[0176] 8. Cyclic AMP Assay

[0177] Human HEK-293 cells were cultured at 2 x 10 5 cells / cm 2 The cells were plated at a density of 10 μg on 4-well micro slides (ibidi GmbH, Martinsried, Germany). After 24 h, 10 μg of plasmid DNA encoding human GIP receptor or human GLP-2 receptor and 5 μg of plasmid DNA encoding mTurquoise-EPAC- cp173 Plasmid DNA of the Venus-Venus H74 probe (gift from Prof. K. Jalink (Netherland Cancer Institute, Amsterdam, Netherland)) was used to transfect cells using the optimized calcium phosphate method. In co-transfection experiments, cells were transfected with 5 μg of plasmid DNA encoding the human GIP receptor, 5 μg of plasmid DNA encoding the human GLP-2 receptor, and 5 μg of plasmid DNA encoding the H74 probe. 24 hours after transfection, the transfected cells were rinsed in phosphate-buffered saline and incubated in HEPES-buffered saline (containing 140 mM NaCl, 5 mM KCl, 1 mM MgCl2, 1 mM CaCl2, 10 mM glucose, 10 mM HEPES) in a chamber containing a Leica imaging system (DMI6000 inverted microscope equipped with an SP8 confocal head) and a controlled atmosphere (37°C, 5% CO2). Images were acquired using a 63X, 1.4NA oil immersion objective and The images were collected after 30 minutes using a detector (Leica). The donor was excited using a 458 nm Ar laser and the donor emission was collected between 460-505 nm and the acceptor emission between 520-600 nm by setting the SP8 spectrometer accordingly. FRET was expressed as the ratio between the donor and acceptor signals. The FRET value was set to 1 at the beginning of the experiment. The solvent, 10 -9 M[D-Ala 2 ]-GIP 1-30 , 10 -9 M[Gly 2 ]-GLP-2 or 10-9M GL-0001 was added to the assay medium and the FRET signal was measured as above.

[0178] 9. Animals

[0179] BALB / c (BALB / cJRj) mice were obtained from Janvier Labs (Saint-Berthevin, France). All animal experiments were approved by the French ministry of higher education, research and innovation, license number 6154-201607211130415v1.

[0180] Mice were housed in an institutional animal facility (protocol number E49007002) at 24°C + / - 2°C with a 12-hour day / night cycle, with tap water and normal diet (Diet A04, Safe, Augy, France) provided ad libitum until sacrificed by cervical dislocation. All procedures were performed in accordance with the French Animal Scientific Procedures Act 2013-118.

[0181] Bilateral ovariectomy (OVX) was performed in 40 BALB / c mice at 12 weeks of age under general anesthesia supplemented with a β2 adrenergic receptor agonist. At 16 weeks of age, ALZET osmotic pumps (model 2006, Durect Corp., Cupertino, CA) filled with saline or peptides as described below were implanted subcutaneously between the two shoulder blades in 32 mice under general anesthesia. Successful filling of the pumps was verified by weighing the pumps before and after filling. At 20 weeks of age, the ALZET osmotic pumps were replaced with similar pumps to ensure that delivery continued for another 4 weeks. Mice were randomly assigned to 4 groups:

[0182] (i) once daily vehicle (OVX+Veh, n=8),

[0183] (ii) 25 nmol / kg / day GL-0001 (OVX+GL-0001, n=8),

[0184] (iii) 25 nmol / kg / day GL-0007 (OVX+GL-0007_25, n=8),

[0185] (iv) 100 nmol / kg / day GL-0007 (OVX+GL-0007_100, n=8).

[0186] The doses of these analogs were based on the inventors' combined knowledge of gut hormone analogs (Mabilleau et al. (2014) Bone 63:61-68; Mansur et al. (2015) J Cell Physiol 230:3009-3018; Mieczkowska et al. (2015) Bone 74:29-36; Mabilleau et al. (2016) Bone 91:102-112; Mansur et al. (2016) Bone 87:102-113; Pereira et al. (2017) Front Endocrinol (Lausanne) 8:327; Mabilleau et al. (2018) J Endocrinol.). Unfortunately, one mouse on 25 nmol / kg / day died during the osmotic pump change.

[0187] Eight additional OVX mice were used as positive controls (OVX+Zol) and administered a single 100 μg / kg zoledronic acid (reference number 6111, batch number 1A / 203523, Tocris Bioscience, Bristol, UK) by intravenous injection in the tail vein. This dose and dosing regimen of zoledronic acid is equivalent to the 5 mg infusion approved in humans for the treatment of postmenopausal osteoporosis.

[0188] Using a precision linear ruler (Adventurer TM Body weight was monitored once a week by ELISA (Pro, Ohaus, Nanikon, Switzerland). All mice were also administered calcein (10 mg / kg; ip) 12 days and 2 days before being sacrificed at 24 weeks of age. After necropsy, femurs, second and fifth lumbar vertebrae, livers and pancreases were collected and processed as detailed below.

[0189] 10. Biomechanical testing

[0190] After necropsy, the soft tissues of the femur and the second lumbar vertebra (L2) were cleaned and wrapped in saline-soaked gauze and immediately frozen at -20° C. After thawing the bones overnight at 4° C., a three-point bending test was performed on the femur.

[0191] Femoral length was measured using a digital caliper (Mitutoyo, Roissy en France, France). No significant differences in femoral length were observed between the groups.

[0192] The femur was loaded to failure in three-point bending at 2 mm / min using a servohydraulic materials testing system (Instron 5942, Instron, Elancourt, France). The lower span length was 10 mm. The femur was positioned so that the front quarter was downward and subjected to a tensile load. The load and displacement of the upper crosshead were digitally recorded at a sampling rate of 100 Hz and measured using a 500 N load cell (Instron). Stiffness, ultimate load, yield load, post-yield displacement (PYD), and fracture work were calculated from the load-displacement curve. PYD is defined as the displacement at failure minus the displacement at yield. Yield is defined as the point at which the regression line representing a 0.2% reduction in stiffness intersects the load-displacement curve.

[0193] Compression experiments were performed on L2. Briefly, the vertebral body was carefully excised and glued to a Plexiglas plate with cyanoacrylate, then incubated in saline at 4°C until use the next day. The L2 vertebral body was compressed to failure at a displacement rate of 1 mm / min using an Instron 5942 device. The load and displacement of the upper platform were recorded at a sampling rate of 100 Hz. The maximum load and stiffness were calculated by computer from the load-displacement curve.

[0194] 11. Micro / Nano-Computed Tomography (MicroCT)

[0195] MicroCT analysis of the femoral shaft was performed using a Skyscan 1076 microcomputed tomography scanner (Bruker MicroCT, Kontich, Belgium) operated at 50 kV, 200 μA, and 2000 ms integration time. The isotropic pixel size was fixed at 9 μm, the rotation step value was 0.5°, and the exposure was completed using a 0.5-mm aluminum filter. Each 3D reconstructed image dataset was binarized using a global threshold. The target cortical volume (VOI) was located 4 mm above the distal growth plate and extended 1 mm further superiorly.

[0196] The vertebral body of the fifth lumbar vertebra (L5) was analyzed using a Nanotom nano-computed tomography scanner (Phoenix, GE, USA) at 85 kV, 220 μA, and 1000 ms integration time. The isotropic pixel size was fixed at 4 μm, the rotation step value was 0.25 °, and the exposure was completed using a 0.1 mm copper filter. Each 3D reconstructed image data set was binarized using a global threshold. The target trabecular volume was calculated by computer on the coronal slices of the L5 vertebral body. The trabecular VOI was distributed throughout the vertebral body, except for the first and last 80 μm from the anterior and posterior cortical walls. All micro-CT / nano-CT parameters were determined according to the guidelines and nomenclature proposed by the American Society for Bone and Mineral Research (Bouxsein et al., 2010).

[0197] 12. Bone composition assessment

[0198] After the three-point bending test, the femur was embedded in pMMA at 4°C without decalcification. One micron cross-sectional slices of the femoral shaft were sandwiched between BaF2 optical windows and Fourier transform infrared imaging (FTIRI) was evaluated in the posterior quarter. FTIRI was performed using a vertex 70 spectrometer (Bruker, Ettlingen, Germany) interfaced with a Hyperion 3000 microscope and a focal plane array detector (64 x 64 pixels) covering a field of view of 180 x 180 μm. Nine fields of view were stitched together to allow enough bone to be analyzed. 8 cm -1 Spectral resolution scanning slice (spectral range 900-2000cm -1 Each spectrum was corrected for Mie scattering using the RMieS-EMSC_v5 algorithm (gift from Prof. Peter Gardner, University of Manchester, UK) before pMMA subtraction.

[0199] Evaluation of spectral images was performed in Matlab R2016b (The Mathworks, Natick, MA) using a laboratory-made routine script as described in Aguado et al. (2017) Calcif Tissue Int 100:332-340.

[0200] The calculated FTIR bone parameters (Paschalis (2012) Methods Mol Biol 816:517-525) are: (1) phosphate / amide ratio (the area of ​​phosphates in v1, v3 / the area of ​​amide 1); (2) acidic phosphate content (1127 cm -1 / 1096cm -1intensity ratio) (Spevak et al. (2013) Calcif Tissue Int 92:418-428); (3) mineral crystallinity (1030 cm -1 / 1020cm -1 intensity ratio), which reflects the crystal size and integrity; (4) crystal size index (1075cm -1 / 1055cm -1 intensity ratio), which reflects the crystal size in the 002, 211, 200, and 202 directions (Gadaleta et al. (1996) Calcif Tissue Int 58:9-16); and (5) collagen maturity (1660 cm -1 / 1690cm -1 strength ratio).

[0201] The carbonate / phosphate ratio (at ~1415 cm -1 / 1030cm -1 v3 carbonate intensity at (Ou-Yang et al. (2001) J Bone Miner Res 16:893-900). For each component parameter, the mean and half-maximum width of the pixel distribution (except for the zero background value) were calculated and expressed as mean and heterogeneity.

[0202] 13. Histology

[0203] The liver and pancreas were collected at autopsy and immediately fixed in formalin. After paraffin embedding, 4-μm thick sections were cut and stained with hematoxylin / phloxin staining. Histological observations were performed by experienced histologists to evaluate the presence of tissue abnormalities.

[0204] 14. Statistical Analysis

[0205] One-way ANOVA with a single pooled variance using the Holm-Sidak multiple comparison test was used to compare Figure 2-3 Collagen maturity and osteoclast number in Figure 16-18 Differences in cAMP levels in the

[0206] One-way ANOVA with Dunnett's multiple comparison test was used to test the significance between OVX+Veh, OVX+GL-0001, OVX+GL-0007_25 and OVX+GL-0007_100 groups.

[0207] Differences with p equal to or less than 0.05 were considered significant.

[0208] result

[0209] Based on the consensus sequence obtained as disclosed in Example 1, the present inventors tested the following peptides.

[0210] Table 1: Bis-analog peptides

[0211]

[0212] 1. Effects of combined administration of GIP and GLP-2 on collagen maturation and osteoclast number

[0213] Figure 2 The solvent (CTRL), [D-Ala 2 ]-GIP 1-30NH2 , [Gly 2 ]-GLP-2 or co-administered with [D-Ala 2 ]-GIP 1-30NH2 and [Gly 2 ]-Effect of GLP-2 on collagen maturation.

[0214] [D-Ala 2 ]-GIP 1-30NH2 or [Gly 2 ]-GLP-2 was able to increase collagen maturation by approximately 82% (p=0.074) or 111% (p=0.0246), respectively. [D-Ala 2 ]-GIP 1-30NH2 and [Gly 2 The combined administration of [D-Ala]-GLP-2 resulted in a significant increase in this parameter (255%, p<0.001). This increase was also significantly greater than that of [D-Ala]-GLP-2 alone. 2 ]GIP 1-30NH2 or [Gly 2 ]-GLP-2.

[0215] Figure 3 The solvent (CTRL), [D-Ala 2 ]-GIP 1-30NH2 , [Gly 2 ]-GLP-2 or co-administered with [D-Ala 2 ]-GIP 1-30NH2 and [Gly 2 ]-GLP-2 on the formation of human osteoclasts in vitro. Here again, [D-Ala 2 ]-GIP 1-30NH2 and [Gly 2]-GLP-2 was effective in significantly reducing osteoclast formation by 16% (p<0.001) and 34% (p<0.001), respectively. Combined administration of these molecules resulted in a significant reduction in osteoclast formation of 66% (p<0.001).

[0216] 2. Effect of GL-0001 on osteoblast cell death.

[0217] Figure 4 Results are presented for MC3T3-E1 cell death in the presence of vehicle, GL-0001 or rosiglitazone.The inventors have previously found that administration of rosiglitazone results in increased cell death and as such was used as a positive inducer of cell death in this assay.

[0218] This figure shows that GL-0001 has a -6 M concentration did not induce cell death, in contrast to rosiglitazone.

[0219] 3. Binding affinities of the bis-analogs at the human GIP receptor and their respective IC50s.

[0220] The present inventors determined [D-Ala 2 ]GIP 1-30NH2 Or the binding affinity of the bi-analog peptides disclosed in Table 1 above at the human GIP receptor. The corresponding IC50 determined for each compound is as follows:

[0221] Table 2: IC50 for GIP receptor

[0222] IC50(nM) <![CDATA[[D-Ala 2 ]GIP 1-30NH2 ]]> 0.97±0.29 GL-0001 1.19±0.53 GL-0002 2.78±0.82 GL-0003 0.72±0.15 GL-0004 37.85±14.8 GL-0005 0.14±0.04 GL-0006 4.60±1.25 GL-0007 1.05±0.41 GL-0008 0.91±0.25 GL-0009 13.05±3.14

[0223] From the IC50 values, the potency of the different analogs tested appears to be GL-0005>GL-0003>GL-0008>[D-Ala 2 ]GIP 1-30NH2 >GL-0007>GL-0001>GL-0002>GL-0006>GL-0009>GL-0004.

[0224] 4. Binding affinities of the bis-analogs at the human GLP-2 receptor and their respective IC50s.

[0225] The present inventors determined [Gly 2 ] The binding affinity of GLP-2 or the bi-analog peptides disclosed in Table 1 above at the human GLP-2 receptor. The corresponding IC50 determined for each compound is as follows:

[0226] Table 3: IC50 for GLP-2 receptor

[0227] IC50(nM) <![CDATA[[Gly 2 ]GLP-2]]> 0.19±0.04 GL-0001 0.44±0.12 GL-0002 15.16±3.00 GL-0003 22.86±3.67 GL-0004 0.07±0.02 GL-0005 15.05±4.93 GL-0006 1.04±0.20 GL-0007 0.57±0.17 GL-0008 14.74±5.69 GL-0009 53.01±11.9

[0228] From the IC50 values, the potency of the different analogs tested appears to be GL-0004>[Gly 2 ]GLP-2>GL-0001>GL-0007>GL-0006>GL-0008>GL-0005>GL-0002>GL-0003>GL-0009.

[0229] 5. Effects of the dual analogs on osteoclast formation in vitro and their respective IC50s.

[0230] The present inventors determined [D-Ala 2 ]-GIP 1-30NH2 , [Gly 2 Effects of ]-GLP-2 or the bi-analog peptides disclosed in Table 1 on Raw264.7 cell-mediated in vitro osteoclastogenesis.

[0231] The corresponding IC50 determined for each compound is as follows:

[0232] Table 4: IC50 for osteoclast formation

[0233]

[0234]

[0235] From the IC50 values, the potency of the different analogs tested appears to be GL-0006>GL-0005>GL-0004>GL-0008>GL-0001>[Gly 2 ]GLP-2>GL-0007>[D-Ala 2 ]GIP 1-30NH2 >GL-0009>GL-0002>GL-0001-Tag>GL-0003.

[0236] 6. Effects of dual analogs on collagen maturation in vitro.

[0237] Figure 5-7 Given [D-Ala 2 ]-GIP 1-30NH2 , [Gly 2 Effect of -GLP-2 or the bi-analog peptides disclosed in Table 1 on collagen maturation in in vitro MC3T3-E1 cultures. The dashed line corresponds to basal collagen maturation.

[0238] Some analogs are given as biphasic spectra, ie GL-0001-label, GL-0002, GL-0004.

[0239] The inventors previously found that at 100-200 pM [D-Ala2 ]GIP1-42 concentration to obtain the maximum collagen maturation (Mieczkowska et al. (2015) Bone 74:29-36). In this way, the average collagen maturation between 100-200pM concentrations was determined for each analog, and the potency of all these molecules was GL-0007>GL-0001>[Gly 2 ]GLP-2>GL-0008>GL-0006 and GL-0009>[D-Ala 2 ]GIP 1-30NH2 >GL-0001-Tag>GL-0002>GL-0004 and GL-0005>GL-0003.

[0240] Although positive effects were observed with each of the peptides tested, the inventors identified peptides GL-0001 and GL-0007 as peptides of particular interest. Based on in vitro osteoclast formation and in vitro collagen maturation, GL-0001 and GL-0007 were the only two peptides that were more potent than each of the native peptides and were therefore selected for the following experiments.

[0241] 7. Effects of GL-0001 or GL-0007 on body weight

[0242] Figure 8 The body weights of the animals at the end of the 8-week treatment are given. It is noteworthy that the zoledronic acid injected animals, which did not receive any ALZET pump implants, weighed approximately 1.3-1.5 g. None of the treatments caused a significant change in body weight compared to vehicle.

[0243] 8. Effect of GL-0001 or GL-0007 on cortical bone strength

[0244] Fig. 9 Data are presented for cortical bone strength in the appendicular skeleton during ovariectomy-induced bone loss in mice as a model of postmenopausal osteoporosis. Ovariectomized animals were treated with vehicle, 25 nmol / kg / day GL-0001, 25 nmol / kg / day GL-0007, or 100 nmol / kg / day GL-0007. Zoledronic acid, which represents one of the most commonly used molecules for the treatment of postmenopausal osteoporosis, was used as a positive comparator after a single intravenous administration (100 μg / kg).

[0245] Although none of the bis-analogs or zoledronic acid effectively altered ultimate load, yield load, or stiffness, GL-0001 was able to significantly increase ultimate displacement (34%, p=0.047), displacement after yield (70%, p=0.038), and work of fracture (37%, p=0.022), the latter representing the energy required to break the bone. Higher work of fracture means a more resistant bone. GL-0007 at 100 nmol / kg / day increased ultimate displacement (33%, p=0.054) and displacement after yield (62%, p=0.074), ultimately leading to a significant increase in work of fracture (33%, p=0.049).

[0246] 9. Effects of GL-0001 or GL-0007 on cortical bone microstructure.

[0247] Figure 10-11 Data are shown on the cortical bone microarchitecture of the appendicular skeleton in ovariectomized mice. Animals were treated with vehicle, 25 nmol / kg / day GL-0001, 25 nmol / kg / day GL-0007, 100 nmol / kg / day GL-0007, or zoledronic acid (100 μg / kg iv once). Cortical bone microarchitecture was studied in the femur 4 mm above the distal growth plate. None of the vehicle, bis-analog, or zoledronic acid was able to affect cortical bone microarchitecture.

[0248] 10. Effects of GL-0001 or GL-0007 on trabecular bone strength and microstructure

[0249] Figure 12-13 Data on trabecular bone characteristics in the axial skeleton and more precisely in the lumbar spine are reported. Trabecular bone strength was assessed in the second lumbar vertebra by compression testing ( Fig.12 ). Both concentrations of GL-0001, GL-0007, and zoledronic acid significantly reduced bone stiffness compared to vehicle-treated animals. Because compression testing is destructive, trabecular microarchitecture was studied in the fifth lumbar vertebra ( Fig.13 As expected, zoledronic acid increased trabecular mass (BV / TV) by 15% (p=0.013) and trabecular number by 12% (p=0.036). GL-0007 at a concentration of 25 nmol / kg / day caused a moderate but significant (p=0.009) decrease in trabecular spacing by 17%.

[0250] 11. Effects of GL-0001 or GL-0007 on bone matrix composition

[0251] Figure 14-15The effects of dual analog administration on the tissue material properties of cortical bone of the appendicular skeleton were studied. Compared with vehicle, GL-0001 significantly increased mean collagen maturity (10%, p = 0.017), collagen maturity heterogeneity (28%, p = 0.008), acid phosphate content heterogeneity (21%, p = 0.016) and reduced mean phosphate / amide ratio (6%, p = 0.016). GL-0007 at 100 nmole / kg / day significantly reduced mean phosphate / amide ratio (5%, p = 0.031) and increased acid phosphate content heterogeneity (18%, p = 0.044). Zoledronic acid had a modest effect, as only mineral crystallinity heterogeneity increased significantly (21%, p = 0.002).

[0252] 12. Histological study of liver and pancreas structure

[0253] The inventors performed histological analysis on the liver and pancreas. The liver and pancreas were processed, embedded, sliced, stained and examined for any signs of edema, inflammation or necrosis in the presence of vehicle, 25 nanomoles / kg / day of GL-0001 or 25 nanomoles / kg / day of GL-0007. No morphological changes or signs of necrosis were observed between the three groups.

[0254] 13. Activation of GIPr and GLP-2r

[0255] The present inventors studied the response of solvent, [D-Ala 2 ]GIP 1-30NH2 , [Gly 2 ]GLP-2 or GL-0001 increased cAMP levels. 2 ]GIP 1-30NH2 In HEK 293 cells transfected with GL-0001, the intracellular level of cAMP increased, while that of vehicle or [Gly 2 ] GLP-2 transfected cells did not increase ( Fig.16 ). In the case of using only human GLP-2r, [Gly 2 In HEK 293 cells transfected with GLP-2 and GL-0001, the intracellular level of cAMP increased, while that of vehicle or [D-Ala 2 ]GIP 1-30NH2 Transfected cells did not increase ( Fig.17 ). In the use of human GIPr and human GLP-2r, [D-Ala 2 ]GIP 1-30NH2 , [Gly 2 In HEK 293 cells co-transfected with GLP-2 and GL-0001, the intracellular level of cAMP increased, whereas the vehicle did not ( Fig.18 ). It is noteworthy that the magnitude of the increase in cAMP in cells co-transfected with GL-0001 was greater than that of each native peptide.

[0256] in conclusion

[0257] All these data demonstrate that the peptides of the present invention increase the enzymatic cross-linking of the collagen matrix produced by osteoblasts at a higher level than the respective natural peptides in vitro and in vivo, reduce the number of osteoclasts produced in a more important manner than the respective natural peptides, increase the resistance of bones to fracture better than the gold standard zoledronic acid, and are therefore effective agents for the treatment of bone diseases.

[0258] Example 3 : Comparison of the peptides of the present invention with the peptides of the prior art

[0259] The aim of this study was to compare the potential of the dual GIP / GLP-2 analogs of the present invention, in particular GL-0001 and GL-0007, and three peptides from patent application WO2018 / 069442, namely peptide Co-3 of sequence HADGTFISDYSTILDNLAARDFINWLIQTKITD (SEQ ID NO: 14), peptide Co-7 of sequence HAEGTFISDYSIAMDKLAARDFINWLIQTKITD (SEQ ID NO: 15) and peptide Co-19 of sequence HADGTFISDYSTILDNLAARDFINWLIQTKGKK (SEQ ID NO: 16).

[0260] Peptides Co-3, Co-7 and Co-19 are considered as dual GIP / GLP-2 analogs in WO2018 / 069442. However, no data on the deposition and maturation of bone matrix of osteoblasts (i.e., collagen maturity) are provided. This experimental scheme enables the present invention to evaluate the effects of dual GIP / GLP-2 analogs as synergists, additive agents or antagonists.

[0261] Materials and methods

[0262] 1. Peptide sequence

[0263] All peptides were prepared by GeneCust, Boynes, France by Fmoc synthesis. Purity and peptide composition were verified by HPLC and mass spectrometry. Peptide sequences, batch numbers and purity are shown in Table 5 below.

[0264] Table 5: Peptides used

[0265]

[0266] 2. Sequence homology

[0267] To determine the percentage of homology between the peptides of the present invention and the peptides disclosed in WO2018 / 069442, the peptide sequences were aligned using the Needleman-Wunsch algorithm in Matlab R2016b (nwalign function).

[0268] 3. Data

[0269] Briefly, MC3T3-E1 subclone 4 cells were grown and propagated in α-MEM supplemented with 5% fetal bovine serum (FBS), 5% calf serum (BCS), 100 U / mL penicillin and 100 μg / mL streptomycin at 37° C. in a humidified atmosphere enriched with 5% CO 2 .

[0270] For the collagen maturation assay, cells were dissociated with 0.05% trypsin-EDTA and cultured at 1.5 × 10 4 cells / cm 2 Cells were plated at a density of 100 μg / mL and grown to confluence in α-MEM supplemented with 5% FBS, 5% BCS, 100 U / mL penicillin and 100 μg / mL streptomycin. At confluence, the medium was replaced with differentiation medium containing αMEM supplemented with 5% FBS, 5% BCS, 100 U / mL penicillin, 100 μg / mL streptomycin, 50 μg / ml ascorbic acid and various concentrations of GIP, GLP-2 or dual GIP / GLP-2 analogs. This day was considered day 1. The differentiation medium was changed every two days. On day 14, osteoblast cultures were decellularized by incubation in 0.2 M sodium cacodylate buffer (pH 7.4) containing 0.1% triton X100 for 4 hours on an orbital shaker. The cultures were rinsed at least 6 times with milliQ water, fixed in anhydrous ethanol, scraped from the culture dish, and transferred to a BaF2 window, where the cultures were air-dried. The integrity of the collagen extracellular matrix was verified by comparing the obtained Fourier transform infrared spectra with those of commercially available collagen. Spectral analysis was performed using a Bruker Vertex 70 spectrometer (Bruker optics, Ettlingen, Germany) interfaced with a Bruker Hyperion 3000 infrared microscope equipped with a standard single element mercury cadmium telluride (MCT) detector. Calibration of the infrared spectrometer was performed once a week using polystyrene film standards (Bruker optics). The 4 cm -1The resolution of 100 nm was 240 nm, and the infrared spectra were recorded in transmission mode with an average of 32 scans. Background spectral images were collected from the same BaF2 window at the beginning and end of each experiment under the same conditions to ensure instrument stability. At least 5 spectra were acquired for each condition and analyzed in Matlab R2016b (The Mathworks, Natick, MA) using a laboratory-made routine script (version 2.0). The collagen maturity index was determined as the relative ratio of mature trivalent to immature divalent collagen crosslinks, using their respective 1660 cm-1 peaks at amide I. -1 and 1690cm -1 The sub-band at is determined.

[0271] For the GIP and GLP-2 datasets, the present invention used previously acquired and analyzed collagen maturation data.

[0272] 4. Assessment of synergistic modes of action

[0273] First, the collagen maturity obtained in control cultures (i.e., lacking the dual GIP / GLP-2 analog) was averaged and subtracted for all data sets. To determine the effect of administering the dual GIP / GLP-2 analog, the transformed data set was divided by the mean of the untreated cultures. For each peptide concentration, the mean, SD, and number of events were calculated. These data were then exported to GraphPad Prism (version 8.0) for further analysis. The GraphPad Prism analysis consisted of the following steps: (1) transforming drug concentrations to log(concentration), (2) curve fitting using a Gaussian function, stimulated dose-response (four parameters), the sum of two Gaussian functions, or a 6th order polynomial function, and (3) estimating EC50 and E for each drug. max .

[0274] In addition, to study synergistic effects, the inventors estimated the effects of dual GIP / GLP-2 analogs at fixed doses of 0.2, 0.4, 0.8, 0.9, 1.0, 1.2, and 1.5 times the EC50 of GIP and GLP-2 and the EC50 of GIP / GLP-2. At each concentration, the combined effect index was calculated using the Chou & Talalay model (Chou & Talalay (1983) Trends Pharmacol Sci 4: 450-454) as follows:

[0275] CI=(E GIP +E GLP2 -(E GIP ×E GLP2 )) / E DA

[0276] Where EGIP 、E GLP2 and E DA Represents the effects observed with GIP alone, GLP-2 alone or the dual agonist, respectively. The inventors have previously observed that although GIP and GLP-2 exhibit different maximal effects on collagen maturation, their respective EC50s are roughly similar.

[0277] 5. Statistical Analysis

[0278] The difference in the combination index at EC50 and 1.5xEC50 was compared with additivity (defined as a value of 1.0 ± 0.15) using a two-tailed t-test. P values ​​< 0.05 were considered significant. Data are expressed as mean ± SD.

[0279] result

[0280] 1. Sequence homology between the peptides of the present invention and the peptides disclosed in WO2018 / 069442

[0281] The percentage of sequence homology was calculated by computer and is given in Table 6 below. The sequence alignment between GL-0001 and GL-0007, and the peptides disclosed in WO2018 / 069442 is also given in Table 6 below.

[0282] Peptide Co-7 from WO2018 / 069442 showed the greatest sequence homology with both GL-0001 and GL-0007. Because data on peptides Co-3 and Co-19 against alkaline phosphatase were provided in WO2018 / 069442, the inventors also selected these two peptides for further comparison.

[0283] Table 6: Percentage of sequence homology between the peptides of the present invention and the peptides from WO2018 / 069442

[0284]

[0285] The differences in amino acid composition (same or of the same type) at the same position between the peptides of the present invention and the peptides of WO2018 / 069442 are summarized in Fig.19 middle.

[0286] For GL-0001, the major differences from peptides Co-3, Co-7 and Co-19 are at positions 7, 12, 13, 31, 32 and 33. For GL-0007, the major differences from peptides Co-3, Co-7 and Co-19 are at positions 7, 12, 13, 31, 32 and 33.

[0287] 2. Collagen Maturation as a Function of Peptide Concentration

[0288] The collagen maturity index obtained as the ratio of trivalent mature / bivalent immature collagen cross-links was measured in the extracellular matrix. Fig. 20 The effects of the peptides of the present invention and the peptides of WO2018 / 069442 on this parameter are represented.

[0289] As expected, and confirming previous data, GL-0001 and GL-0007 increased collagen maturity above the index observed in the absence of the peptides (dashed line). Interestingly, the effects of peptides Co-3, Co-7 and Co-19 in increasing the collagen maturity index were very modest.

[0290] 3. Dose-effect curve of dual GIP / GLP-2 analogs on collagen maturation

[0291] Dose response curves were generated for each bis-GIP / GLP-2 analog. The best fitting model for each bis-GIP / GLP-2 analog is given in Table 7.

[0292] Table 7: Estimates of the best-fit model for the dose-response curves

[0293] Dual GIP / GLP-2 analogs Fitting the model R2 GL-0001 Gaussian function 0.647 GL-0007 Gaussian function 0.590 Co-3 6th order polynomial 0.210 Co-7 6th order polynomial 0.404 Co-19 The sum of two Gaussian functions 0.248

[0294] 4. EC50 of dual GIP / GLP-2 analogs on collagen maturation

[0295] The EC50 of each di-GIP / GLP-2 analog was identified from the dose-effect curve. The relevant EC50 is given in Table 8.

[0296] Table 8: EC50 and E of dose-effect curves max Data represent mean ± SD of 5 individual experiments.

[0297]

[0298] As shown previously, [D-Ala2]-GIP 1-30 [Gly2]-GLP-2 showed similar EC50s, so the average EC50 of 69 pM was used to determine the combined effect index. Interestingly, all dual GIP / GLP-2 analogs showed EC50s in the same range, suggesting that the same concentrations can be used to achieve a half-maximal effect. However, for the maximum effect (E max ) as GL-0001 and GL-0007 were clearly almost twice the maximal effect observed with the WO2018 / 069442 peptide.

[0299] 5. Combined effect index of dual GIP / GLP-2 analogs on collagen maturation

[0300] Based on the Chou & Talalay model, the combined effect index was calculated at 0.2, 0.4, 0.8, 0.9, 1.0, 1.2 and 1.5 times the average EC50 of GIP and GLP-2 (i.e., 14, 27, 54, 61, 69, 82 and 100 pM). Fig.21 The combined effect index is plotted against concentration to show the evolution of CI with varying doses.

[0301] Tables 9 and 10 present the respective combination effect indices for each dual GIP / GLP-2 analog at EC50 and 1.5xEC50.

[0302] Table 9: Combined effect indices on collagen maturation at EC50 and their interpretation

[0303] Compound CI at EC50 P-value Interpretation GL-0001 0.16±0.02 <0.0001 Strong synergy GL-0007 0.14±0.02 <0.0001 Strong synergy Co-3 1.29±1.65 0.704 Add Co-7 1.53±1.10 0.315 Add Co-19 277.7±617.4 0.346 Add

[0304] Table 10: Combined effect indices on collagen maturation at 1.5x EC50 and their interpretation

[0305] Compound CI at 1.5xEC50 P-value Interpretation GL-0001 0.20±0.03 <0.0001 Strong synergy GL-0007 0.17±0.02 <0.0001 Strong synergy Co-3 1.79±2.20 0.448 Add Co-7 2.30±1.01 0.021 Antagonism Co-19 389.0±864 0.345 Add

[0306] CI>10: very strong antagonism; CI 3.3-10: strong antagonism; CI 1.45-3.3: antagonism; CI1.15-1.45: moderate antagonism; CI 0.85-1.15: additive effect; CI0.85-0.65: moderate synergistic effect; CI 0.3-0.65: synergistic effect; CI 0.1-0.3 strong synergistic effect; CI<0.1: very strong synergistic effect.

[0307] Data are given as mean ± SD of 5-13 experiments. A two-tailed t-test was used to assess whether the CI was significantly different from additivity (1.0 ± 0.15).

[0308] GL-0001 and GL-0007 showed a combined action index at 69pM and 100pM suggesting strong synergy. Previously, the inventors have shown that the combined action index of GL-0001 and GL-0007 has similar behavior supporting synergy. This study improves the previously obtained data.

[0309] Interestingly, none of the peptides Co-3, Co-7 and Co-19 displayed synergistic properties, but rather exhibited additive or antagonistic effects depending on the concentration.

[0310] Based on the combined effect index at EC50 and 1.5xEC50, only GL-0001 and GL-0007 appear to be good candidates for synergy.

[0311] in conclusion

[0312] This study compared the effects of the dual GIP / GLP-2 analogs of the present invention and the peptides disclosed in the WO2018 / 069442 patent application.

[0313] GL-0001 and GL-0007 are peptides of the present invention, and they are selected because they represent the best compromise in terms of the effect on collagen maturity and osteoclastogenesis. Sequence alignment with WO2018 / 069442 peptides shows that peptide Co-7 is the closest peptide to GL-0001 and GL-0007. Since data on alkaline phosphatase are provided using peptide Co-3 and peptide Co-19 in WO2018 / 069442, the inventors decided to compare the effects of GL-0001 and GL-0007 head to head on the one hand, and on the other hand, compare the effects of peptides Co-3, Co-7 and Co-19. Although all peptides show similar EC50, there are obvious differences in the amplitude of the maximum effect. This suggests that for the same dose of peptides, the effect of the peptides of the present invention on collagen maturity is greater. In addition, the pharmacological mechanism behind the dual GIP / GLP-2 analogs is solved, and the peptides of the present invention show strong synergy. The WO2018 / 069442 peptides showed either additive or antagonistic modes of action.

[0314] Example 4 : Comparison of the peptides of the present invention with the peptides of the prior art

[0315] The aim of this study was to compare the potential of the dual GIP / GLP-2 analogs of the invention, in particular GL-0001 and GL-0007, and three peptides from patent application WO2018 / 069442 (i.e., peptide Co-3, peptide Co-7 and peptide Co-19) on osteoclastogenesis in vitro.

[0316] Materials and methods

[0317] 1. Peptide sequence

[0318] The peptides are disclosed in Example 3.

[0319] 2. Cells and propagation methods

[0320] Murine Raw 264.7 cells were grown and expanded in a propagation medium containing Dulbecco's modified Eagle's medium (DMEM-Gibco) supplemented with 10% fetal bovine serum (FBS-Lonza), 100 U / mL penicillin, and 100 μg / mL streptomycin (Gibco) at 37°C in a humidified atmosphere enriched with 5% CO2. The first passage after thawing Raw 264.7 cells was considered to be passage 1. Cells up to passage 8 were used.

[0321] 3. Osteoclastogenesis Assay

[0322] Rat Raw 264.7 cells were scraped from plastic dishes and cultured at 1.25 × 10 4 cells / cm 2 Cells were plated at a density of 1:1 and grown in propagation medium enriched with 10 ng / ml soluble murine RANKL (Bio-Techne, ref 462-TEC-010). After 110 hours, cells were fixed with formalin (10% in PBS buffer) for 10 minutes and rinsed in distilled water before staining with tartrate-resistant acid phosphatase (TRAcP).

[0323] TRAcP was histochemically visualized by synchronous coupled reaction at 37°C in the dark for 90 minutes using naphthol AS-BI-phosphate (Sigma Aldrich) as substrate and Fast Violet B (Sigma-Aldrich) as diazonium salt. The cultures were rinsed three times in distilled water and residual activity was inhibited by 4% NaF (Sigma-Aldrich) for 30 minutes. The cells were then rinsed in distilled water and allowed to dry. TRAcP-positive cells with more than three nuclei were identified as osteoclasts. The number of newly generated osteoclasts was assessed by histologists who were blinded to the treatment intervention using optical microscopy. In this example, the cells previously identified using (D-Ala 2 )GIP 1-30 and (Gly 2 ) The data set obtained with GLP-2 was used for comparison and calculation of the combined effect index.

[0324] 4. Data Evaluation

[0325] The osteoclast counts obtained using the dual GIP / GLP-2 analogs were first reported as a percentage of the osteoclast counts observed in the absence of the peptide (RANKL alone). For each concentration of peptide, the mean, SD, and number of events were calculated. These data were then exported to GraphPad Prism (version 8.0) for further analysis. The GraphPad Prism analysis consisted of the following steps: (1) transforming the drug concentrations to log(concentration), (2) performing curve fitting using the three-parameter Log(inhibitor) versus the response model (constrained to an upper limit of 100), and (3) estimating the IC50 for each drug.

[0326] In addition, to investigate synergistic effects, the inventors converted osteoclastogenesis data to inhibitory effects by subtracting each osteoclastogenesis percentage from 100. These data were then exported to GraphPad Prism. The analysis consisted of the following steps: (1) transforming drug concentrations to log(concentration), (2) performing curve fitting using a three-parameter Log(agonist) versus response model (with a lower constraint of 0), and (3) estimating EC50 and E for each drug. max Effect of Dual GIP / GLP-2 Analogs at EC50 of GIP and GLP-2 (36.8 pM) Using data from previously performed experiments, the combined action index was calculated according to Chou & Talalay (Chou & Talalay (1983) Trends Pharmacol Sci 4:450-454) as follows:

[0327] CI=(E GIP +E GLP2 -(E GIP ×E GLP2 )) / E DA

[0328] Where E GIP 、E GLP2 and E DA Represents the effects observed with GIP alone, GLP-2 alone or the dual agonist, respectively.

[0329] result

[0330] 1. Osteoclastogenesis

[0331] All peptides reduced the amount of newly generated osteoclasts at the highest concentration. For all peptides, the curve fits of osteoclast responses were Fig. 22 IC50 was derived from the curve fit and is given in Table 11.

[0332] Table 11: IC50 calculated from curve fitting of osteoclastogenesis versus peptide concentration.

[0333]

[0334] ND: Not determined because the maximal effect did not reach 50% inhibition. *: Determined from previous experiments.

[0335] Interestingly, (D-Ala 2 )GIP 1-30 and (Gly 2 ) GLP-2 presented a similar IC50. A two-tailed t-test confirmed this finding. Thus, the intermediate IC50 of 36.8 pM was used to investigate the "synergistic" properties of the dual GIP / GLP-2 analogs.

[0336] 2. Dose-response inhibitory effect and pharmacological mechanism of dual GIP / GLP-2 analogs on osteoclastogenesis

[0337] To evaluate the response of each dual GIP / GLP-2 analog, the inventors transformed the data into the percentage of inhibition of osteoclastogenesis. The data were then curve fitted and the R2 values ​​for each curve fit are presented in Table 12.

[0338] Table 12: Estimates of the best-fit model for the dose-response curves

[0339]

[0340] Dose-effect curves were generated for each dual GIP / GLP-2 analog and presented in Fig.23 For each dual GIP / GLP-2 analog, the EC50 and maximum inhibitory effect (E max ) and are shown in Table 13.

[0341] Table 13: EC50 and E50 of determination of dose-effect curves max .

[0342]

[0343] Data are presented as mean ± SD of 5 separate experiments. *: Data obtained in previous experiments.

[0344] It will be interesting to determine whether the osteoclast response observed with the dual GIP / GLP-2 analogs is equal to, greater than, or less than that observed with D-Ala 2 )GIP 1-30 and (Gly 2 ) GLP-2 combined administration. To answer this question, the combined effect index was calculated according to Chou & Talalay and reported in Table 14. In addition, Fig.24 Middle pair in (D-Ala2 )GIP 1-30 and (Gly 2 )EC of GLP-2 50 The combined effect index was plotted.

[0345] Table 14: Combination effect index and pharmacological interpretation at EC50 of GIP and GLP-2

[0346] Compound CI at EC50 Interpretation GL-0001 1.10 Add GL-0007 0.82 Moderate collaboration Co-3 48.00 Very strong antagonism Co-7 271.28 Very strong antagonism Co-19 1.94 Antagonism

[0347] CI>10: very strong antagonism; CI 3.3-10: strong antagonism; CI 1.45-3.3: antagonism; CI1.15-1.45: moderate antagonism; CI 0.85-1.15: additive effect; CI 0.85-0.65: moderate synergism; CI 0.3-0.65: synergism; CI 0.1-0.3 strong synergism; CI<0.1: very strong synergism.

[0348] A two-tailed t-test was used to assess whether the CI was significantly different from additivity (1.0 ± 0.15).

[0349] in conclusion

[0350] This study shows that the peptides of the present invention, especially GL-0001 and GL-0007, exhibit lower EC50 than the peptides of the prior art. This suggests that lower doses of GL-0001 and GL-0007 can be used to achieve 50% inhibition of osteoclast formation. In addition, the E max The values ​​showed that both peptides achieved more than 85% inhibition of osteoclast formation, which was higher than that of (Gly 2 )GLP-2. The pharmacological mechanisms behind the effects of GL-0001 and GL-0007 range from moderate synergism to additive effects. Interestingly, compared with (D-Ala 2 )GIP 1-30 or (Gly 2 )GLP-2, peptides Co-3, Co-7 and Co-19 disclosed in WO2018 / 069442 showed equal or higher EC50, suggesting that the same dose or even higher dose is required to inhibit osteoclast formation by 50%. In addition, the highest inhibitory effect of these three peptides is at most ~62.4%, which is lower than that of using (Gly 2 )GLP-2 or (D-Ala 2 )GIP 1-30 Furthermore, the pharmacological modes of action of these three peptides range from antagonism to very strong antagonism.

Claims

1. An isolated peptide, wherein the peptide is selected from the group consisting of: - GL-0001 of sequence HGEGSFGSDMSIALDKLAARDFVNWLLQTKITD (SEQ ID NO: 3), - GL-0007 of sequence HGEGSFGSDFSIALDKLAARDFVNWLLQTK-NH2 (SEQ ID NO: 4), - GL-0001-tag of the sequence HGEGSFGSDMSIALDKLAARDFVNWLLQTKITDGAADDDD DD (SEQ ID NO: 5), - GL-0002 of sequence HGEGSFVSDMSIVLDKLAARDFVNWLLQTK-NH2 (SEQ ID NO: 6), - GL-0003 of sequence HGEGSFVSEMSIVLDKLAARDFVNWLLQTK-NH2 (SEQ ID NO: 7), - GL-0004 of sequence HGEGSFVSDMSVVLDKLAARDFVNWLLQTK-NH2 (SEQ ID NO: 8), - GL-0005 of sequence HGEGSFVSDLSVVLDKLAARDFVNWLLQTK-NH2 (SEQ ID NO: 9), - GL-0006 of sequence HGEGSFVSDFSVVLDKLAARDFVNWLLQTK-NH2 (SEQ ID NO: 10), - GL-0008 of sequence HGEGSFTSDFSIALDKLAARDFVNWLLQTK-NH2 (SEQ ID NO: 11), and - GL-0009 of sequence HGEGSFVSDFSIALDKLAARDFVNWLLQTK-NH2 (SEQ ID NO: 12). 2 . The peptide according to claim 1 , which is peptide GL-0001 of the sequence HGEGSFGSDMSIALDKLAARDFVNWLLQTKITD (SEQ ID NO: 3).

3. A pharmaceutical composition comprising the peptide defined in claim 1 or 2.

4. An implantable medical device coated with a peptide as defined in claim 1 or 2.

5. Use of a peptide as defined in claim 1 or a pharmaceutical composition as defined in claim 3 for the preparation of a medicament for treating and / or preventing a bone disease in a subject.

6. The method according to claim 5, wherein the bone disease is selected from primary or secondary osteoporosis, osteopenia, diabetic bone disease, osteomalacia and rickets, osteodystrophy, hypercalcemia of malignancy, osteopenia due to bone metastasis, osteosarcoma, Ewing's osteoma, osteochondroma, bone abnormalities caused by cancer treatment, osteogenesis imperfecta, osteomyelitis, achondroplasia, avascular necrosis, osteopetrosis, myositis ossificans, periodontal disease, hyperparathyroidism, periarticular erosions in rheumatoid arthritis, bone erosions in ankylosing spondylitis and bone loss in anorexia nervosa.

7. The use according to claim 6, wherein the bone disease is selected from Paget's disease of bone, postmenopausal osteoporosis, glucocorticoid-induced osteoporosis, disuse osteoporosis and senile osteoporosis.

8. The use according to claim 5, wherein the bone disease is a manifestation of metabolic disorder or hormonal disorder in the bone.

9. A bone filling biomaterial comprising the peptide defined in claim 1.

10. Use of the peptide defined in claim 1 in the preparation of a bone filling biomaterial, wherein the bone filling biomaterial is used for bone regeneration.

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