Synthetic and recombinantly produced collagen peptides exhibiting biological efficacy

Abstract

The present invention relates to synthetically or recombinantly produced collagen peptides having a molecular weight in the range of 0.18 to 10.0 kDa, in particular 0.18 to 5.0 kDa, in particular 1.1 to 5.0 kDa, for use in methods for therapeutic treatment of the human or animal body, to products comprising the collagen peptides according to the invention, and to methods for producing the collagen peptides according to the invention.

Description

[Technical field]

[0001] The present invention relates to synthetically or recombinantly produced collagen peptides having a molecular weight in the range of 0.18 to 10.0 kDa, in particular 0.18 to 5.0 kDa, in particular 1.1 to 5.0 kDa, for use in methods for the therapeutic treatment of the human or animal body, products comprising the collagen peptides according to the invention, and methods for producing the collagen peptides according to the invention. [Background technology]

[0002] Collagen is an extracellular structural protein found in animals, including mammals, birds and fish. It is usually found in connective tissues, particularly as a component of the extracellular matrix. Tendons, ligaments, cartilage and bones are particularly rich in collagen. Collagen is not present in plants or unicellular organisms.

[0003] Collagen is a type of collagen that is structurally and functionally diverse, and differs in particular in its structure, function, and origin. The polypeptide chains that make up collagen are individually synthesized in the ribosomes of the endoplasmic reticulum in cells in the form of relatively large precursor molecules, and are composed of extensive repeating sequences (Gly-XY). n where X and Y can be any amino acid, but are typically proline and 4-hydroxyproline.

[0004] These precursor polypeptide chains are post-translationally hydroxylated in the endoplasmic reticulum to form hydroxyproline and hydroxylysine residues at proline and lysine residues in the polypeptide chains. Hydroxylation serves to stabilize adjacent collagen polypeptide chains in right-handed triple helices (procollagen) that are formed in cells from three precursor polypeptide chains each.

[0005] The procollagen thus formed is glycosylated within the cell and secreted from the cell in a glycosylated triple-helical form (tropocollagen), which is then formed by peptidase-mediated cleavage of terminal residues to form collagen, which during the fibrillation process assembles into collagen fibrils, which are then covalently cross-linked to form collagen fibers.

[0006] Collagen is also often used in a denatured form, in which case it is called gelatin, or in the form of a hydrolysate.

[0007] When gelatine or collagen is subjected to a hydrolysis process, in particular enzymatic hydrolysis, to obtain collagen peptides, collagen hydrolysates of various compositions and application properties can be produced depending on the type and origin of collagen used and the enzymatic conditions. However, these collagen hydrolysates are mixtures of peptides whose molecular weights are distributed within a certain size range. The use of such collagen hydrolysates, for example as dietary supplements or cosmetic adjuvants, in particular for the prevention and / or treatment of diseases related to bones, joints or connective tissues, has been known for a long time.

[0008] WO 2012 / 065782 describes a collagen hydrolysate obtained from pigskin gelatine, which serves to stimulate the biosynthesis of extracellular matrix proteins by skin cells and is particularly suitable for cosmetic applications.

[0009] WO 2012 / 117012 discloses enzymatically hydrolyzed collagen from bovine leather (Ringerspalt) with an average molecular weight of 1500-8000 Da, which can be used together with prebiotics for the prevention and / or treatment of osteoporosis.

[0010] Although the use of collagen hydrolysates obtained from animal materials is advantageous for many applications and consumer groups, certain consumer groups and application characteristics may make the use of collagen hydrolysates obtained in this way undesirable.For example, certain consumer groups are fundamentally critical or negative towards raw materials obtained from animal materials, whether due to concerns about contamination with microorganisms or drugs harmful to health, such as process aids, or undesirable immune reactions, or due to religious or ethical motivations.Moreover, the manufacturing processes used to obtain collagen hydrolysates obtained from animal materials often involve cumbersome and expensive production, purification and post-treatment steps.

[0011] Against this background, it is not surprising that biotechnology has developed methods for producing gelatine, collagen, collagen hydrolysates and individual collagen peptides using recombinant gene techniques.

[0012] WO 2006 / 052451 discloses the production of recombinant type III collagen in a Pichia pastoris strain that also expresses human prolyl hydroxylase.

[0013] WO 2005 / 012356 discloses the production of gelatin from human type I collagen and individual collagen peptide species of sizes 50 kDa, 65 kDa and 100 kDa in fully hydroxylated, partially hydroxylated and non-hydroxylated forms, respectively.

[0014] Olsen et al. (Protein Expression and Purification, 2005, 40, pg. 346-357) disclose the recombinant production of an 8.5 kDa collagen peptide species from human collagen α1 chain in Pichia pastoris.

[0015] WO 01 / 34646 likewise discloses the production of individual recombinant gelatin species, each with a defined molecular weight of 0-350 kDa generated by a recombinant production route, which may exist in non-hydroxylated, partially hydroxylated or fully hydroxylated form.

[0016] The document discloses the general applicability of individual recombinant gelatin species or mixtures thereof in the food, beverage, cosmetic or pharmaceutical manufacturing industries. Further uses of the disclosed recombinant products are disclosed in the document as photographic compositions and as industrial auxiliaries, e.g. in the semiconductor manufacturing industry and paper manufacturing.

[0017] Thus, recombinant gelatin or collagen peptides of any size, either individually or in mixtures, with or without hydroxylation, are known to be used in a wide range of industrial and non-industrial applications, such as, for example, food production and the treatment of human or animal diseases.

[0018] However, given that many people are increasingly seeking health, an attractive appearance, athletic performance and good physical condition, even as they age, there remains a great demand for foods and preparations for improving and / or maintaining health and treating disease.

[0019] Health, athletic performance, physical attractiveness, and good physical condition are often closely related to the condition of connective tissue. Connective tissue is a tissue that is present in all tissues of the body, for example in muscles, intestines, blood vessels, periodontal tissues, tendons, ligaments, bones, and skin, where it often plays a supporting role. Connective tissue generally has a relatively small number of cells and a large number of cell intermediates, which generally contain extracellular matrix proteins, especially collagen. Connective tissue can occur in the form of loose connective tissue, hard connective tissue, for example skin, tendons, ligaments, and meninges, gelatinous connective tissue, and various other types of connective tissue, especially supportive tissue, i.e. cartilage tissue and bone tissue, which in particular play a decisive role in athletic performance, appearance, the integrity of the body's internal structure, and also in good physical condition. Summary of the Invention

[0020] The technical problem underlying the present invention is therefore to provide means and methods that exhibit biological effectiveness on connective tissues, in particular those of the skin, tendons, ligaments, meninges, cartilage and bone, making it possible, on the one hand, to treat diseases associated with these connective tissues and, on the other hand, to maintain a state of these connective tissues that is considered desirable.

[0021] The present invention is also based on the technical problem of overcoming the above-mentioned drawbacks and providing synthetic or recombinant collagen peptides which can be produced recombinantly, in particular in a standardized, reliable and precisely defined form, also on a larger industrial and low-cost scale, and which, due to their biological effectiveness, are suitable for use in methods for the therapeutic treatment of the human or animal body.

[0022] The present invention solves the technical problem on which it is based by providing the teaching of the independent claims, in particular also the teaching of the preferred embodiments herein and the teaching of the dependent claims.

[0023] The present invention relates to synthetic or recombinant collagen peptides, in particular synthetically or recombinantly produced collagen peptides having a molecular weight in the range of 0.18 to 10.0 kDa, in particular 0.18 to 5.0 kDa, especially 1.1 to 5.0 kDa, for use in methods of therapeutic treatment of the human or animal body.

[0024] The synthetic or recombinant collagen peptides claimed according to the invention, in particular synthetically or recombinantly produced collagen peptides having a molecular weight in the range of 0.18-10.0 kDa, in particular 0.18-5.0 kDa, in particular 1.1-5.0 kDa, preferably exhibit biological activity in connective tissues, in particular in the skin, intestine, in particular the intestinal lining, blood vessels, periodontal tissue and connective tissue of the meninges, connective tissue of tendons and ligaments, as well as in supporting tissues, in particular cartilage tissue, and bone tissue, and are thereby particularly suitable for use in therapeutic or non-therapeutic methods of treatment of the human or animal body.

[0025] According to a preferred embodiment of the present invention, the synthetic or recombinant collagen peptide, in particular the synthetically or recombinantly produced collagen peptide, has the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 6, or a function-conserving sequence variant of one of these amino acid sequences, and preferably the collagen peptide according to the present invention consists of one of these amino acid sequences.

[0026] Preferably, the synthetic or recombinant collagen peptide, in particular the synthetically or recombinantly produced collagen peptide, has the amino acid sequence of SEQ ID NO: 1, and preferably the collagen peptide according to the invention consists of this amino acid sequence.

[0027] Preferably, the synthetic or recombinant collagen peptide, in particular the synthetically or recombinantly produced collagen peptide, has the amino acid sequence of SEQ ID NO: 6, and preferably the collagen peptide according to the invention consists of this amino acid sequence.

[0028] According to another embodiment of the invention, the synthetic or recombinant collagen peptide, in particular the synthetically or recombinantly produced collagen peptide, has a function-conservative sequence variant of one of the amino acid sequences of SEQ ID NO: 1 or SEQ ID NO: 6, preferably the collagen peptide according to the invention consists of a function-conservative sequence variant of one of these amino acid sequences.

[0029] In a preferred embodiment of the present invention, the synthetic or recombinant collagen peptide, in particular the synthetically or recombinantly produced collagen peptide, has an amino acid sequence selected from SEQ ID NOs: 1 to 5, in particular an amino acid sequence selected from SEQ ID NOs: 1 to 4, and preferably the collagen peptide according to the present invention consists of one of these amino acid sequences. Thus, according to this embodiment, the synthetic or recombinant collagen peptide, in particular the synthetically or recombinantly produced collagen peptide, does not have hydroxyproline in its amino acid sequence.

[0030] In a preferred embodiment of the present invention, the synthetic or recombinant collagen peptide, in particular the synthetically or recombinantly produced collagen peptide, has an amino acid sequence selected from SEQ ID NOs: 1 to 5, in particular the amino acid sequence selected from SEQ ID NOs: 1 to 4, and preferably the collagen peptide according to the present invention consists of one of these amino acid sequences, in which at least one prolyl residue and / or at least one lysyl residue is hydroxylated. Thus, according to this embodiment, the synthetic or recombinant collagen peptide, in particular the synthetically or recombinantly produced collagen peptide, has at least one hydroxyproline and / or at least one hydroxylysine in its amino acid sequence.

[0031] In another preferred embodiment of the present invention, the synthetic or recombinant collagen peptide, in particular the synthetically or recombinantly produced collagen peptide, has an amino acid sequence selected from SEQ ID NOs: 6 to 10, in particular an amino acid sequence selected from SEQ ID NOs: 6 to 9, preferably the collagen peptide according to the present invention consists of one of these amino acid sequences.

[0032] According to another preferred embodiment, the synthetic or recombinant collagen peptide, in particular the synthetically or recombinantly produced collagen peptide, has an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-10, in particular an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-4 and 6-9.

[0033] Preferably, the synthetic or recombinant collagen peptide, in particular the synthetically or recombinantly produced collagen peptide, consists of one of the amino acid sequences selected from the group consisting of SEQ ID NOs: 1 to 10, in particular selected from the group consisting of SEQ ID NOs: 1 to 4 and 6 to 9.

[0034] According to another preferred embodiment of the present invention, the synthetic or recombinant collagen peptide, in particular the synthetically or recombinantly produced collagen peptide, has the amino acid sequence of SEQ ID NO: 15, SEQ ID NO: 21, or a function-conserving sequence variant of one of these amino acid sequences, and preferably the collagen peptide according to the present invention consists of one of these amino acid sequences.

[0035] Preferably, the synthetic or recombinant collagen peptide, in particular the synthetically or recombinantly produced collagen peptide, has the amino acid sequence of SEQ ID NO: 15, and preferably the collagen peptide according to the invention consists of this amino acid sequence.

[0036] Preferably, the synthetic or recombinant collagen peptide, in particular the synthetically or recombinantly produced collagen peptide, has the amino acid sequence of SEQ ID NO: 21, and preferably the collagen peptide according to the invention consists of this amino acid sequence.

[0037] According to another embodiment of the invention, the synthetic or recombinant collagen peptide, in particular the synthetically or recombinantly produced collagen peptide, has a function-conservative sequence variant of one of the amino acid sequences of SEQ ID NO: 15 or SEQ ID NO: 21, preferably the collagen peptide according to the invention consists of a function-conservative sequence variant of one of these amino acid sequences.

[0038] In a preferred embodiment of the invention, the synthetic or recombinant collagen peptide, in particular the synthetically or recombinantly produced collagen peptide, has an amino acid sequence selected from SEQ ID NO: 15 and SEQ ID NO: 16, and preferably the collagen peptide according to the invention consists of one of these amino acid sequences. Thus, according to this embodiment, the synthetic or recombinant collagen peptide, in particular the synthetically or recombinantly produced collagen peptide, does not have hydroxyproline in its amino acid sequence.

[0039] In a preferred embodiment of the present invention, the synthetic or recombinant collagen peptide, in particular the synthetically or recombinantly produced collagen peptide, has an amino acid sequence selected from SEQ ID NO: 15 and SEQ ID NO: 16, preferably the collagen peptide according to the present invention consists of one of these amino acid sequences, in which at least one prolyl residue and / or at least one lysyl residue is hydroxylated. Thus, according to this embodiment, the synthetic or recombinant collagen peptide, in particular the synthetically or recombinantly produced collagen peptide, has at least one hydroxyproline and / or at least one hydroxylysine in its amino acid sequence.

[0040] In another preferred embodiment of the invention, the synthetic or recombinant collagen peptide, in particular the synthetically or recombinantly produced collagen peptide, has an amino acid sequence selected from SEQ ID NOs: 21 and 22, preferably the collagen peptide according to the invention consists of one of these amino acid sequences.

[0041] Particularly preferably, the synthetic or recombinant collagen peptide, in particular the synthetically or recombinantly produced collagen peptide, has the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 15, or a function-conserving sequence variant of one of these amino acid sequences, and preferably the collagen peptide according to the invention consists of one of these amino acid sequences.

[0042] According to another preferred embodiment, the synthetic or recombinant collagen peptide, in particular the synthetically or recombinantly produced collagen peptide, has an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-30, in particular selected from the group consisting of SEQ ID NOs: 1-4, 6-9, 12-14, and 18-20.

[0043] Preferably, the synthetic or recombinant collagen peptide, in particular the synthetically or recombinantly produced collagen peptide, consists of one of the amino acid sequences selected from the group consisting of SEQ ID NOs: 1 to 30, in particular selected from the group consisting of SEQ ID NOs: 1 to 4, 6 to 9, 12 to 14, and 18 to 20.

[0044] In another preferred embodiment of the invention, the synthetic or recombinant collagen peptide, in particular the synthetically or recombinantly produced collagen peptide, has an amino acid sequence according to SEQ ID NO:7.

[0045] Preferably, the synthetic or recombinant collagen peptide, in particular the synthetically or recombinantly produced collagen peptide, consists of the amino acid sequence according to SEQ ID NO:7.

[0046] In another preferred embodiment of the invention, the synthetic or recombinant collagen peptide, in particular the synthetically or recombinantly produced collagen peptide, has an amino acid sequence selected from SEQ ID NOs: 17-19, 21 and 22.

[0047] Preferably, the synthetic or recombinant collagen peptide, in particular the synthetically or recombinantly produced collagen peptide, consists of an amino acid sequence selected from SEQ ID NOs: 17-19, 21 and 22.

[0048] In another preferred embodiment of the invention, the synthetic or recombinant collagen peptide, in particular the synthetically or recombinantly produced collagen peptide, has an amino acid sequence selected from SEQ ID NOs: 27-30.

[0049] Preferably, the synthetic or recombinant collagen peptide, in particular the synthetically or recombinantly produced collagen peptide, consists of an amino acid sequence selected from SEQ ID NOs:27-30.

[0050] In a particularly preferred embodiment, the synthetic or recombinant collagen peptides according to the invention, in particular the synthetically or recombinantly produced collagen peptides, are non-hydroxylated collagen peptides.

[0051] In another preferred embodiment, the synthetic or recombinant collagen peptides according to the invention, in particular the synthetically or recombinantly produced collagen peptides, are hydroxylated collagen peptides.

[0052] Preferably, the synthetic or recombinant collagen peptides, in particular the synthetically or recombinantly produced collagen peptides, have hydroxylated prolines and / or hydroxylated lysines.

[0053] Preferably, the synthetic or recombinant collagen peptide, in particular the synthetically or recombinantly produced collagen peptide, is a non-hydroxylated, partially hydroxylated or fully hydroxylated collagen peptide. According to another particularly preferred embodiment, all prolines present in the synthetic or recombinant collagen peptide are hydroxylated.

[0054] According to a preferred embodiment of the present invention, the synthetic or recombinant collagen peptide, particularly the synthetic or recombinantly produced collagen peptide, is glycosylated.Preferably, the synthetic or recombinant collagen peptide, particularly the synthetic or recombinantly produced collagen peptide, is glycosylated on at least one hydroxylated lysine.Preferably, all hydroxylated lysines of the synthetic or recombinant collagen peptide, particularly the synthetic or recombinantly produced collagen peptide, are glycosylated.

[0055] In a preferred embodiment of the invention, the collagen peptide according to the invention has an amino acid sequence present in collagen of type I, type II, type III, type IV, type V, type VI, type VII, type VIII, type IX, type X, type XI, type XII, type XIII, type XIV, type XV, type XVII, type XVIII, type XIX, type XX, type XXI, type XXII, type XXIII, type XXIV, type XXV, type XXVI, type XXVII, preferably has an amino acid sequence present in collagen of type I, type II or type III, preferably type I, preferably type II, preferably type III. Preferably, the collagen peptide according to the invention has an amino acid sequence present in collagen of vertebrates, in particular fish, amphibians, reptiles, birds and mammals, in particular has an amino acid sequence present in collagen of type I, type II or type III, preferably type I, preferably type II, preferably type III of human, bovine, porcine, equine or avian.

[0056] Particularly preferably, the collagen peptide according to the invention has an amino acid sequence which is present in human collagen, in particular in human type I collagen, preferably in the α1 chain of human type I collagen.

[0057] Particularly preferably, the collagen peptide according to the invention has an amino acid sequence present in non-human collagen, in particular an amino acid sequence present in the α1 chain of non-human type I collagen, preferably an amino acid sequence present in bovine, porcine, equine or avian collagen.

[0058] In a preferred embodiment of the invention, the synthetic or recombinant collagen peptides, in particular the synthetically or recombinantly produced collagen peptides, are collagenase resistant, in particular resistant to digestion by human collagenase.

[0059] According to another preferred embodiment of the present invention, synthetic or recombinant collagen peptides, in particular synthetically or recombinantly produced collagen peptides, are capable of inducing the synthesis of extracellular matrix proteins, such as collagen, proteoglycans and / or elastin, in particular collagen, in connective tissue cells, in particular osteoblasts, chondrocytes and / or fibroblasts.

[0060] In a preferred embodiment of the invention, the collagen peptide according to the invention is a synthetically produced collagen peptide, preferably a collagen peptide produced by chemical synthesis, in particular by solid phase synthesis, preferably by Merrifield synthesis, Bailey peptide synthesis or chemoenzymatic synthesis. In another preferred embodiment of the invention, the collagen peptide according to the invention is a recombinantly produced collagen peptide.

[0061] In a preferred embodiment of the present invention, the synthetic or recombinant collagen peptides, in particular the synthetically or recombinantly produced collagen peptides, are free of amino acid modifications, in particular free of hydroxylation. Particularly preferably, the synthetic or recombinant collagen peptides, in particular the synthetically or recombinantly produced collagen peptides, are free of hydroxylated and / or glycosylated amino acids.

[0062] According to a preferred embodiment of the present invention, synthetic or recombinant collagen peptides, in particular synthetically or recombinantly produced collagen peptides, are provided for use in a method for therapeutic treatment of the human or animal body, having a molecular weight of 0.18 to 10.0 kDa, preferably 0.18 to 8.0 kDa, preferably 0.18 to 6.0 kDa, preferably 0.18 to 5.0 kDa, preferably 0.2 to 5.0 kDa, preferably 0.3 to 5.0 kDa, preferably 0.4 to 5.0 kDa, preferably 0.5 to 5.0 kDa, preferably 0.6 to 5.0 kDa, preferably 0.7 to 5.0 kDa, preferably 0.8 to 5.0 kDa, preferably 0.9 to 5.0 kDa, preferably 1.0 to 5.0 kDa, preferably 1.1 to 5.0 kDa. a, preferably 1.2 to 5.0 kDa, preferably 1.3 to 5.0 kDa, preferably 1.4 to 5.0 kDa, preferably 1.5 to 5.0 kDa, preferably 1.6 to 5.0 kDa, preferably 1.7 to 5.0 kDa, preferably 1.8 to 5.0 kDa, preferably 1.9 to 5.0 kDa, preferably 2.0 to 5.0 kDa, preferably 2.1 to 4.9 kDa, preferably 2.2 to 4.8 kDa, preferably 2.3 to 4.7 kDa, preferably 2.4 to 4.6 kDa, preferably 2.5 to 4.5 kDa, preferably 1.2 to 3.2, preferably 1.3 to 3.0 kDa, preferably 1.5 to 3.0 kDa, preferably 1.8 to 3.0 kDa, preferably 2.0 to 3.0 kDa.

[0063] According to another preferred embodiment of the present invention, the synthetic or recombinant collagen peptide, in particular the synthetically or recombinantly produced collagen peptide, for use in a method for therapeutic treatment of the human or animal body, has a molecular weight in the range of 0.18-0.98, preferably 0.19-0.98 kDa, preferably 0.20-0.98 kDa, preferably 0.25-0.98 kDa, preferably 0.30-0.98 kDa, preferably 0.35-0.98 kDa, preferably 0.40-0.98 kDa, preferably 0.45-0.98 kDa, preferably 0.50-0.98 kDa.

[0064] In a preferred embodiment, the present invention further relates to a synthetic or recombinant collagen peptide according to the invention, in particular a synthetically or recombinantly produced collagen peptide, for use in a method for preventing and / or treating bone diseases, in particular osteoporosis.

[0065] In a preferred embodiment, the present invention relates to a synthetic or recombinant collagen peptide according to the invention, in particular a synthetically or recombinantly produced collagen peptide, for use in a method for preventing and / or treating sarcopenia.

[0066] In a preferred embodiment, the present invention relates to a synthetic or recombinant collagen peptide according to the invention, in particular a synthetically or recombinantly produced collagen peptide, for use in a method for preventing and / or treating degenerative loss of muscle mass.

[0067] In a preferred embodiment, the present invention relates to a synthetic or recombinant collagen peptide according to the invention, in particular a synthetically or recombinantly produced collagen peptide, for use in a method for preventing and / or treating cartilage disorders, in particular arthropathy or arthritis.

[0068] In a preferred embodiment, the present invention relates to a synthetic or recombinant collagen peptide according to the invention, in particular a synthetically or recombinantly produced collagen peptide, for use in a method for improving muscle strength.

[0069] In a preferred embodiment, the present invention relates to a synthetic or recombinant collagen peptide according to the invention, in particular a synthetically or recombinantly produced collagen peptide, for use in a method for the prevention and / or treatment of a pathology characterized by reduced mitochondrial activity, in particular a method for the prevention and / or treatment of a pathology characterized by reduced endurance.

[0070] In a preferred embodiment, the present invention relates to a synthetic or recombinant collagen peptide according to the invention, in particular a synthetically or recombinantly produced collagen peptide, for use in a method for stimulating body fat loss.

[0071] In a preferred embodiment, the present invention relates to a synthetic or recombinant collagen peptide according to the invention, in particular a synthetically or recombinantly produced collagen peptide, for use in a method for reducing weight.

[0072] In a preferred embodiment, the present invention relates to a synthetic or recombinant collagen peptide according to the invention, in particular a synthetically or recombinantly produced collagen peptide, for use in a method for the prevention and / or treatment of degenerative joint diseases, in particular osteoarthritis, rheumatoid arthritis, rheumatic diseases, spondylitis and / or connective tissue diseases.

[0073] In a preferred embodiment, the present invention relates to a synthetic or recombinant collagen peptide according to the invention, in particular a synthetically or recombinantly produced collagen peptide, for use in a method for preventing and / or treating tendon or ligament diseases.

[0074] In a preferred embodiment, the present invention relates to a synthetic or recombinant collagen peptide according to the invention, in particular a synthetically or recombinantly produced collagen peptide, for use in a method for the prevention and / or treatment of skin diseases, in particular psoriasis vulgaris, acne, atopic dermatitis, chronic pruritus and / or rosacea.

[0075] In a preferred embodiment, the present invention relates to a synthetic or recombinant collagen peptide according to the invention, in particular a synthetically or recombinantly produced collagen peptide, for use in a method for treating wounds, in particular chronic wounds, acute wounds and / or burns.

[0076] In a preferred embodiment, the present invention relates to a synthetic or recombinant collagen peptide according to the invention, in particular a synthetically or recombinantly produced collagen peptide, for use in a method for the prevention and / or treatment of a neurodegenerative disease.

[0077] In a preferred embodiment, the present invention relates to a synthetic or recombinant collagen peptide according to the invention, in particular a synthetically or recombinantly produced collagen peptide, for use in a method for preventing and / or treating dementia.

[0078] In a preferred embodiment, the present invention relates to a synthetic or recombinant collagen peptide according to the invention, in particular a synthetically or recombinantly produced collagen peptide, for use in a method for preventing and / or treating Alzheimer's disease.

[0079] In a preferred embodiment, the present invention relates to a synthetic or recombinant collagen peptide according to the invention, in particular a synthetically or recombinantly produced collagen peptide, for use in a method for the prevention and / or treatment of a pathology characterized by a decline in mental function.

[0080] In a preferred embodiment, the present invention relates to a synthetic or recombinant collagen peptide according to the invention, in particular a synthetically or recombinantly produced collagen peptide, for use in a method for the prevention and / or treatment of diseases associated with dysfunction of the structure and / or function of the blood-brain barrier, in particular the meninges.

[0081] In a preferred embodiment, the present invention relates to a synthetic or recombinant collagen peptide according to the invention, in particular a synthetically or recombinantly produced collagen peptide, for use in a method for the prevention and / or treatment of intestinal diseases, in particular chronic inflammatory bowel diseases.

[0082] In a preferred embodiment, the present invention relates to a synthetic or recombinant collagen peptide, in particular a synthetically or recombinantly produced collagen peptide, according to the invention for use in a method for preventing and / or treating diseases of the cardiovascular system, in particular diseases of the structure and / or function of blood vessels, in particular diseases of the structure and / or function of the vascular wall, in particular for use in a method for preventing and / or treating hypertension and / or circulatory disorders.

[0083] In a preferred embodiment, the present invention relates to a synthetic or recombinant collagen peptide according to the invention, in particular a synthetically or recombinantly produced collagen peptide, for use in a method for preventing and / or treating periodontal diseases.

[0084] In a preferred embodiment, the present invention further relates to the non-therapeutic use of collagen peptides according to the invention for visually and structurally improving the skin, in particular for reducing the formation of wrinkles, for improving skin elasticity, for increasing skin firmness, for increasing skin hydration, for reducing cellulite and / or for reducing stretch marks, in particular gravitational forces.

[0085] In another preferred embodiment, the present invention relates to the non-therapeutic use of collagen peptides according to the invention for promoting nail growth and / or reducing nail brittleness.

[0086] In a preferred embodiment, the present invention further relates to the non-therapeutic use of collagen peptides according to the invention for visually and structurally improving hair, in particular for improving hair quality, for reducing split ends and / or for reducing or slowing hair loss.

[0087] In another preferred embodiment, the present invention relates to the non-therapeutic use of collagen peptides according to the invention for increasing mitochondrial number and / or mitochondrial activity.

[0088] In another preferred embodiment, the present invention relates to the non-therapeutic use of collagen peptides according to the invention for improving endurance.

[0089] In another preferred embodiment, the present invention relates to the non-therapeutic use of collagen peptides according to the invention for improving mental function.

[0090] In a preferred embodiment of the invention, the synthetic or recombinant collagen peptides according to the invention are used alone, i.e. without other substances, for use in any of the applications provided by the invention.

[0091] In another embodiment of the invention, the synthetic or recombinant collagen peptides according to the invention are used as the only agents exhibiting biological effectiveness in the applications provided by the invention.

[0092] In another preferred embodiment, the synthetic or recombinant collagen peptides according to the invention are used in the applications provided by the invention together with at least one further active agent, in particular another active agent that exhibits biological effectiveness.

[0093] The present invention also relates to a pharmaceutical composition comprising a collagen peptide according to the invention and at least one pharma- ceutically acceptable additive, for use in a method for the therapeutic treatment of the human or animal body, in particular for at least one of the above-mentioned indications. This makes it possible to provide for the administration of the collagen peptide according to the invention in the form of a pharmaceutical composition. Particularly preferably, the pharmaceutical composition according to the invention is administered in the form of, for example, tablets, lozenges, chewable tablets, capsules, bite capsules, dragees, lozenges, liquids, gels or ointments.

[0094] The present invention also relates to a dietary supplement comprising the collagen peptide according to the invention and at least one additive acceptable for food, for use in a method for therapeutic treatment of the human or animal body, in particular for at least one of the aforementioned indications. This makes it possible to provide for the administration of the collagen peptide according to the invention in the form of a dietary supplement. Particularly preferably, the dietary supplement according to the invention is present as a tablet, dragee, pastille, sachet, solution, suspension or gel, for example in an ampoule, as granules or powder. The collagen peptide according to the invention can also be added to various beverages without turbidity due to its good solubility.

[0095] The present invention also relates to a cosmetic product comprising the collagen peptide according to the present invention and at least one skin-compatible additive, for use in a therapeutic treatment method for the human or animal body, in particular for at least one of the above-mentioned indications.This can provide that the collagen peptide according to the present invention is administered in the form of a cosmetic composition.Particularly preferably, the cosmetic composition according to the present invention is administered in the form of, for example, a lotion, an ointment, a cream, a gel, a powder, a splash or a spray.

[0096] The present invention also relates to a food or treat comprising the collagen peptide according to the invention, for use in a method for therapeutic treatment of the human or animal body, in particular for at least one of the above-mentioned indications.In a preferred embodiment, the food or treat is a chocolate bar, a protein bar, a cereal bar, an instant powder for preparing a drink, milk, a dairy product such as yogurt, whey or quark, and dairy substitutes such as soy milk, rice milk, almond milk and coconut milk (so-called functional foods), or a drink, such as a soft drink, a fitness drink.

[0097] Provided that the collagen peptide according to a preferred embodiment of the invention is not used as the sole physiologically active ingredient of a product, in particular a pharmaceutical composition, a dietary supplement, a cosmetic composition, or a food or luxury item, it may be combined with one or more other ingredients that have a positive effect on general health, in particular on endurance. Such ingredients are preferably vitamin C, various vitamins B, D, E and K, omega-3 fatty acids, omega-6 fatty acids, conjugated linoleic acid, caffeine and its derivatives, guarana extract, rosehip extract, green tea extract, epigallocatechin gallate, creatine, L-carnitine, alpha-lipoic acid, N-acetylcysteine, NADH, D-ribose, magnesium aspartate, antioxidants such as anthocyanins, carotenoids, flavonoids, resveratrol, glutathione and superoxide dismutase (SOD), cannabinoids such as cannabidiol (CBD), adaptogens such as Rhodiola rosea, Panax ginseng, Withania somnifera, shiitake mushroom, Ganoderma lucidum, Maca (Lepidium meyenii), mineral substances such as iron, magnesium, calcium, zinc, selenium and phosphorus, and other proteins, hydrolysates and peptides such as soy protein, wheat protein and whey protein.

[0098] In another preferred embodiment of the invention, the product according to the invention, in particular a pharmaceutical composition, a dietary supplement, a cosmetic composition or a food or luxury item, does not contain other proteins or peptides, in particular other collagen peptides, apart from the collagen peptide according to the invention.

[0099] In a preferred embodiment of the present invention, collagen peptide is administered in an amount of 1 to 40 g / day, preferably 1 to 30 g / day, preferably 1 to 20 g / day, preferably 1 to 15 g / day, preferably 2.5 to 30 g / day, preferably 2.5 to 20 g / day, preferably 2.5 to 15 g / day, preferably 2.5 to 10 g / day, preferably 4 to 15 g / day, preferably 4 to 12 g / day, more preferably 5 to 25 g / day, preferably 5 to 15 g / day, preferably 10 to 25 g / day, preferably 12 to 22 g / day, preferably 6 to 15 g / day, particularly 2.5 to 7.5 g / day, preferably 2.5 to 5 g / day.

[0100] The present invention also relates to a method for the prevention and / or treatment of the above-mentioned indications, in particular the above-mentioned therapeutic indications, comprising administering to the human or animal body a sufficient amount for therapeutic purposes of at least one recombinantly or synthetically produced peptide according to the invention, optionally together with additives.

[0101] The present invention also relates to non-therapeutic methods for improving muscle strength, increasing muscle mass, stimulating body fat loss, reducing body weight, maintaining and / or improving bone health, maintaining and / or improving skin health, maintaining and / or improving gut health, maintaining and / or improving vasculature, maintaining and / or improving cardiovascular health, maintaining and / or improving periodontal tissue, maintaining and / or improving nail and hair health of the human or animal body, maintaining and / or increasing mitochondrial number and / or mitochondrial activity, maintaining and / or improving stamina, or maintaining and / or improving mental function, which comprise administering at least one collagen peptide according to the invention to the human or animal body.

[0102] The present invention further provides a method for producing the collagen peptide according to the present invention, a) providing an expression system comprising at least one expression cassette, the expression cassette comprising at least one nucleotide sequence encoding a collagen peptide having a molecular weight in the range of 0.18 to 10 kDa, in particular 0.18 to 5.0 kDa, in particular 1.1 to 5.0 kDa; b) method steps of culturing the expression system under conditions allowing expression of the collagen peptide; c) a method for recovering collagen peptides according to the present invention; The present invention relates to a method comprising the steps of:

[0103] The method for producing collagen peptides according to the invention, in particular synthetic or recombinant collagen peptides having a molecular weight in the range of 0.18-10.0 kDa, in particular 0.18-5.0 kDa, in particular 1.1-5.0 kDa, for the therapeutic treatment of the human or animal body, provided by the present invention, is characterized in that precisely defined and recombinantly produced collagen peptides are obtained, in particular having a molecular weight in the range of 0.18-10.0 kDa, in particular 0.18-5.0 kDa, in particular 1.1-5.0 kDa, which collagen peptides are particularly suitable for use in methods for the therapeutic treatment of the human or animal body due to their biological effectiveness.

[0104] The collagen peptide provided by the present invention exhibits a particularly high purity due to its recombinant production mode, compared to collagen peptide obtained by hydrolysis from natural sources.Moreover, the collagen peptide can be provided in a wide variety of expression systems, even on an industrial scale, without undesirable contamination, and at the same time, the collagen peptide according to the present invention exhibits a favorable biological efficacy, especially for use in therapeutic methods for the treatment of the human or animal body.

[0105] Preferably, the biological effectiveness of the collagen peptides according to the invention found by the present invention, and thus their suitability for use in therapeutic methods for the treatment of the human or animal body, already occurs in the collagen peptides obtained directly from the method according to the present invention, without the need for further processing steps.Thus, both the hydroxylated and non-hydroxylated collagen peptides according to the present invention exhibit biological effectiveness, in particular at least as effective as collagen peptides of the same molecular weight obtained from natural sources and / or mixtures of collagen peptides of the same average molecular weight obtained from natural sources.What is particularly advantageous in this respect is that the collagen peptides according to the present invention surprisingly exhibit biological effectiveness even in the non-hydroxylated form, preferably as effective as hydroxylated collagen peptides of the same molecular weight obtained from natural sources and / or mixtures of hydroxylated collagen peptides of the same average molecular weight obtained from natural sources, and particularly preferably as effective as hydroxylated collagen peptides of the same molecular weight obtained from natural sources and / or mixtures of hydroxylated collagen peptides of the same average molecular weight obtained from natural sources.

[0106] Preferably, both the hydroxylated and non-hydroxylated collagen peptides according to the invention exhibit biological effectiveness in osteoblasts, fibroblasts and / or chondrocytes, preferably in osteoblasts, preferably in fibroblasts, preferably in chondrocytes, preferably in osteoblasts and fibroblasts, preferably in osteoblasts and chondrocytes, preferably in fibroblasts and chondrocytes, preferably exhibit biological effectiveness at least equivalent to that of collagen peptides of the same molecular weight obtained from natural sources and / or mixtures of collagen peptides of equivalent average molecular weight obtained from natural sources, particularly preferably exhibit biological effectiveness superior to that of collagen peptides of the same molecular weight obtained from natural sources and / or mixtures of collagen peptides of equivalent average molecular weight obtained from natural sources.

[0107] The synthetic or recombinant collagen peptides of the invention, in particular the synthetically or recombinantly produced collagen peptides, can show a biological efficacy according to the invention, in particular in connective tissues, in particular in the connective tissues of the skin, intestine, blood vessels and periodontal tissues, brain membranes, tendons, ligaments, cartilage and / or bone, in particular on the basis of in vitro tests for the stimulation of the synthesis of extracellular matrix proteins in osteoblasts, fibroblasts and chondrocytes, as shown in examples 3 to 6.

[0108] In a preferred embodiment, the synthetic or recombinant collagen peptides according to the invention, in particular the synthetically or recombinantly produced collagen peptides of the invention, in particular the synthetically or recombinantly produced hydroxylated collagen peptides, in particular the synthetically or recombinantly produced non-hydroxylated collagen peptides, exhibit biological efficacy, in particular in at least one, preferably at least two, preferably all of the in vitro tests for stimulation of synthesis of extracellular matrix proteins in osteoblasts, fibroblasts and chondrocytes as shown in Examples 3 to 6.

[0109] Preferably, the synthetic or recombinant collagen peptides according to the invention, in particular the synthetically or recombinantly produced collagen peptides of the invention, in particular the synthetically or recombinantly produced hydroxylated collagen peptides, in particular the synthetically or recombinantly produced non-hydroxylated collagen peptides, exhibit biological effectiveness equivalent to that of collagen peptides of the same molecular weight isolated from natural sources, in particular in at least one, preferably at least two, preferably all of the in vitro tests for stimulation of synthesis of extracellular matrix proteins in osteoblasts, fibroblasts and chondrocytes shown in Examples 3 to 6.

[0110] Particularly preferably, the synthetic or recombinant collagen peptides according to the invention, in particular the synthetically or recombinantly produced collagen peptides according to the invention, in particular the synthetically or recombinantly produced hydroxylated collagen peptides, in particular the synthetically or recombinantly produced non-hydroxylated collagen peptides, exhibit a biological effectiveness equivalent to that of a mixture of collagen peptides isolated from a natural source, in particular a mixture of collagen peptides of comparable average molecular weight isolated from a natural source, in particular in at least one, preferably at least two, preferably all of the in vitro tests for stimulation of synthesis of extracellular matrix proteins in osteoblasts, fibroblasts and chondrocytes shown in Examples 3 to 6.

[0111] Particularly preferably, the synthetic or recombinant collagen peptides according to the invention, in particular the synthetically or recombinantly produced collagen peptides of the invention, in particular the synthetically or recombinantly produced hydroxylated collagen peptides, in particular the synthetically or recombinantly produced non-hydroxylated collagen peptides, exhibit greater biological efficacy than collagen peptides of the same molecular weight isolated from natural sources, in particular in at least one, preferably at least two, preferably all of the in vitro tests for stimulation of synthesis of extracellular matrix proteins in osteoblasts, fibroblasts and chondrocytes shown in Examples 3 to 6.

[0112] Particularly preferably, the synthetic or recombinant collagen peptides according to the invention, in particular the synthetically or recombinantly produced collagen peptides according to the invention, in particular the synthetically or recombinantly produced hydroxylated collagen peptides, in particular the synthetically or recombinantly produced non-hydroxylated collagen peptides, exhibit superior biological efficacy to a mixture of collagen peptides isolated from a natural source, in particular a mixture of collagen peptides of comparable average molecular weight isolated from a natural source, and in particular exhibit superior biological efficacy to a mixture of collagen peptides isolated from a natural source, in particular a mixture of collagen peptides of comparable average molecular weight isolated from a natural source, in at least one, preferably at least two, preferably all of the in vitro tests for stimulation of synthesis of extracellular matrix proteins in osteoblasts, fibroblasts and chondrocytes shown in Examples 3 to 6.

[0113] Preferably, the expression system provided in step a) is a host cell, in particular a prokaryotic or eukaryotic cell.

[0114] Preferably, the expression system is a host cell selected from the group consisting of bacterial cells, yeast cells, fungal cells, mammalian cells, insect cells and plant cells.

[0115] Preferably, the expression system, in particular the host cell, is a bacterial cell, in particular a bacterial cell of the species Escherichia coli or Bacillus subtilis.

[0116] In another preferred embodiment, the expression system, in particular the host cell, is a yeast cell, in particular a yeast cell of the species Saccharomyces cerevisiae, Pichia pastoris or Ogataea angusta (Hansenula polymorpha).

[0117] Preferably, the expression system, in particular the host cell, is a fungal cell, in particular a fungal cell of the species Aspergillus niger.

[0118] In another preferred embodiment of the invention, the expression system, in particular the host cell, is a mammalian cell, in particular a CHO cell, a HeLa cell or a HEK293 cell.

[0119] Preferably, the expression system, in particular the host cell, is an insect cell, in particular an Sf-9, Sf-21 or Tn-5 cell.

[0120] Preferably, the expression system, in particular the host cell, is a plant cell, in particular a corn cell or a tobacco cell.

[0121] In another preferred embodiment of the present invention, the expression system provided in step a) is a host cell capable of hydroxylating the proline, lysine or proline and lysine residues of the expressed collagen peptide.Preferably, the expression system provided in step a) is a host cell capable of hydroxylating the proline, lysine or proline and lysine residues of the expressed collagen peptide.

[0122] Preferably, the expression system provided in step a) is an expression system exhibiting prolyl and / or lysyl hydroxylase activity. Preferably, the expression system provided in step a) is a host cell exhibiting prolyl and / or lysyl hydroxylase activity.

[0123] In a preferred embodiment, the expression system provided in step a) is a host cell with at least one expression cassette comprising a polynucleotide sequence encoding prolyl-4 hydroxylase. Particularly preferred, the expression system provided in step a) is a host cell with at least one expression cassette comprising a polynucleotide sequence encoding prolyl-4 hydroxylase, such that in vivo hydroxylated collagen peptides are recovered in method step c).

[0124] In a preferred embodiment, the expression system provided in step a) is a host cell with at least one expression cassette comprising a polynucleotide sequence encoding a lysyl hydroxylase. Particularly preferred, the expression system provided in step a) is a host cell with at least one expression cassette comprising a polynucleotide sequence encoding a lysyl hydroxylase, such that in vivo hydroxylated collagen peptides are recovered in method step c).

[0125] In another preferred embodiment of the present invention, the expression system provided in step a) is a host cell comprising at least one expression cassette comprising a polynucleotide sequence encoding a prolyl-4 hydroxylase and at least one expression cassette comprising a polynucleotide sequence encoding a lysyl hydroxylase. Particularly preferred is that the expression system provided in step a) is a host cell comprising at least one expression cassette comprising a polynucleotide sequence encoding a prolyl-4 hydroxylase and at least one expression cassette comprising a polynucleotide sequence encoding a lysyl hydroxylase, such that in vivo hydroxylated collagen peptides are recovered in method step c).

[0126] The present invention therefore also relates to a method for producing a collagen peptide according to the invention, in particular a method for producing an in vivo hydroxylated collagen peptide, comprising: a) a method step of providing an expression system comprising at least one expression cassette, the expression cassette comprising at least one nucleotide sequence encoding a collagen peptide having a molecular weight in the range of 0.18 to 10 kDa, in particular 0.18 to 5.0 kDa, in particular 1.1 to 5.0 kDa, the expression system being capable of hydroxylating proline, lysine or proline and lysine residues of the expressed collagen peptide; b) the method steps of culturing the expression system under conditions allowing expression and hydroxylation of collagen peptides; c) a method for recovering collagen peptides according to the present invention, in particular collagen peptides hydroxylated in vivo; The present invention includes a method comprising the steps of:

[0127] Thus, the above-mentioned method advantageously allows obtaining in vivo hydroxylated recombinantly produced collagen peptides having a specific molecular weight in the range of 0.18-10.0 kDa, in particular 0.18-5.0 kDa, in particular 1.1-5.0 kDa, characterized by a specific pattern of post-translational modifications, in particular hydroxylation and glycosylation, depending on the cell-based expression system used. In this way, it is advantageously possible to obtain collagen peptides that exhibit the desired biological efficacy, in particular recombinantly produced collagen peptides for use in therapeutic methods for the treatment of the human or animal body, directly, i.e. without the need for subsequent modifications.

[0128] In a preferred embodiment, the in vivo hydroxylated recombinant collagen peptides produced according to the present invention exhibit biological efficacy, in particular in at least one, preferably at least two, preferably all of the in vitro tests for stimulation of synthesis of extracellular matrix proteins in osteoblasts, fibroblasts and chondrocytes shown in Examples 3-6.

[0129] Preferably, the in vivo hydroxylated recombinant collagen peptides produced according to the present invention exhibit biological effectiveness equivalent to that of collagen peptides of the same molecular weight isolated from natural sources, in particular in at least one, preferably at least two, preferably all of the in vitro tests for stimulation of extracellular matrix protein synthesis in osteoblasts, fibroblasts and chondrocytes shown in Examples 3-6.

[0130] Particularly preferably, the in vivo hydroxylated recombinant collagen peptides produced according to the present invention exhibit a biological effectiveness equivalent to that of a mixture of collagen peptides isolated from a natural source, in particular a mixture of collagen peptides of comparable average molecular weight isolated from a natural source, in particular in at least one, preferably at least two, preferably all of the in vitro tests for stimulation of synthesis of extracellular matrix proteins in osteoblasts, fibroblasts and chondrocytes shown in Examples 3 to 6.

[0131] Particularly preferably, the in vivo hydroxylated recombinant collagen peptides produced according to the present invention exhibit greater biological efficacy than collagen peptides of the same molecular weight isolated from natural sources, in particular in at least one, preferably at least two, preferably all of the in vitro tests for stimulation of extracellular matrix protein synthesis in osteoblasts, fibroblasts and chondrocytes shown in Examples 3-6.

[0132] Particularly preferably, the in vivo hydroxylated recombinant collagen peptides produced according to the present invention exhibit greater biological efficacy than mixtures of collagen peptides isolated from natural sources, in particular mixtures of collagen peptides of comparable average molecular weight isolated from natural sources, and in particular exhibit greater biological efficacy than mixtures of collagen peptides isolated from natural sources, in particular mixtures of collagen peptides of comparable average molecular weight isolated from natural sources, in at least one, preferably at least two, preferably all of the in vitro tests for stimulation of synthesis of extracellular matrix proteins in osteoblasts, fibroblasts and chondrocytes shown in Examples 3 to 6.

[0133] According to another embodiment of the invention, the expression system provided in step a) is an expression system which is not capable of causing hydroxylation of proline, lysine or proline and lysine residues of the expressed collagen peptide, in particular the expression system provided in step a) does not exhibit prolyl and lysyl hydroxylase activity.

[0134] The present invention therefore relates to a method for producing collagen peptides according to the invention, in particular for producing non-hydroxylated collagen peptides, comprising the steps of: a) a method step of providing an expression system comprising at least one expression cassette, the expression cassette comprising at least one nucleotide sequence encoding a collagen peptide having a molecular weight in the range of 0.18 to 10 kDa, in particular 0.18 to 5.0 kDa, in particular 1.1 to 5.0 kDa, the expression system being incapable of causing hydroxylation of proline, lysine or proline and lysine residues of the expressed collagen peptide; b) the method steps of culturing the expression system under conditions allowing expression of the collagen peptide; c) a method step of recovering collagen peptides according to the invention, in particular non-hydroxylated collagen peptides; The present invention relates to a method comprising the steps of:

[0135] In a preferred embodiment, the non-hydroxylated recombinant collagen peptides produced according to the present invention exhibit biological efficacy, in particular in at least one, preferably at least two, preferably all of the in vitro tests for stimulation of synthesis of extracellular matrix proteins in osteoblasts, fibroblasts and chondrocytes shown in Examples 3-6.

[0136] Preferably, the non-hydroxylated recombinant collagen peptides produced according to the present invention exhibit biological effectiveness equivalent to that of collagen peptides of the same molecular weight isolated from natural sources, in particular in at least one, preferably at least two, preferably all of the in vitro tests for stimulation of extracellular matrix protein synthesis in osteoblasts, fibroblasts and chondrocytes shown in Examples 3-6.

[0137] Particularly preferably, the non-hydroxylated recombinant collagen peptides produced according to the present invention exhibit a biological effectiveness equivalent to that of a mixture of collagen peptides isolated from a natural source, in particular a mixture of collagen peptides of comparable average molecular weight isolated from a natural source, in particular in at least one, preferably at least two, preferably all of the in vitro tests for stimulation of synthesis of extracellular matrix proteins in osteoblasts, fibroblasts and chondrocytes shown in Examples 3 to 6.

[0138] Particularly preferably, the non-hydroxylated recombinant collagen peptides produced according to the present invention exhibit greater biological efficacy than collagen peptides of the same molecular weight isolated from natural sources, in particular in at least one, preferably at least two, preferably all of the in vitro tests for stimulation of extracellular matrix protein synthesis in osteoblasts, fibroblasts and chondrocytes shown in Examples 3-6.

[0139] Particularly preferably, the non-hydroxylated recombinant collagen peptides produced according to the present invention exhibit superior biological efficacy to mixtures of collagen peptides isolated from natural sources, in particular mixtures of collagen peptides of comparable average molecular weight isolated from natural sources, and in particular exhibit superior biological efficacy to mixtures of collagen peptides isolated from natural sources, in particular mixtures of collagen peptides of comparable average molecular weight isolated from natural sources, in at least one, preferably at least two, preferably all of the in vitro tests for stimulation of synthesis of extracellular matrix proteins in osteoblasts, fibroblasts and chondrocytes shown in Examples 3 to 6.

[0140] According to a preferred embodiment of the present invention, at least one nucleotide sequence of at least one expression cassette is codon-optimized, i.e. codons in the nucleotide sequence that are not used or not preferentially used by the translation system of the provided expression system, in particular the provided cell-based expression system, in particular the provided host cell, are replaced by codons that are preferentially used by the translation system of the provided expression system, in particular the provided cell-based expression system, in particular the provided host cell, without this replacement changing the amino acid sequence of the encoded peptide or protein.

[0141] In a preferred embodiment of the invention, the collagen peptide encoded by the nucleotide sequence is a collagen peptide from a vertebrate, in particular a mammal, such as a human, or a non-human mammal, such as a horse, donkey, kangaroo, sheep, rodent, pig or cow, a bird, such as a chicken, a fish, an amphibian, a reptile or an invertebrate, such as a jellyfish.

[0142] Preferably, the collagen peptide encoded by the nucleotide sequence is selected from type I, type II, type III, type IV, type V, type VI, type VII, type VIII, type IX, type X, type XI, type XII, type XIII, type XIV, type XV, type XVI, type XVII, type XVIII, type XIX, type XX, type XXI, type XXII, type XXIII, type XXIV, type XXV, type XXVI, type XXVII, preferably type I, preferably type II, preferably type III.

[0143] Preferably, the collagen peptide encoded by the nucleotide sequence is a naturally occurring collagen peptide. In another preferred embodiment of the present invention, the collagen peptide encoded by the nucleotide sequence is not a naturally occurring collagen peptide. Preferably, the collagen peptide encoded by the nucleotide sequence is a recombinant collagen peptide.

[0144] According to a preferred embodiment of the present invention, the at least one nucleotide sequence is preferably 0.18 to 10.0 kDa, preferably 0.18 to 8.0 kDa, preferably 0.18 to 6.0 kDa, preferably 0.18 to 5.0 kDa, preferably 0.2 to 5.0 kDa, preferably 0.3 to 5.0 kDa, preferably 0.4 to 5.0 kDa, preferably 0.5 to 5.0 kDa, preferably 0.6 to 5.0 kDa, preferably 0.7 to 5.0 kDa, preferably 0.8 to 5.0 kDa, preferably 0.9 to 5.0 kDa, preferably 1.0 to 5.0 kDa, preferably 1.1 to 5.0 kDa, preferably 1.2 to 5.0 kDa, preferably 1.3 to 5.0 kDa. , preferably 1.4 to 5.0 kDa, preferably 1.5 to 5.0 kDa, preferably 1.6 to 5.0 kDa, preferably 1.7 to 5.0 kDa, preferably 1.8 to 5.0 kDa, preferably 1.9 to 5.0 kDa, preferably 2.0 to 5.0 kDa, preferably 2.1 to 4.9 kDa, preferably 2.2 to 4.8 kDa, preferably 2.3 to 4.7 kDa, preferably 2.4 to 4.6 kDa, preferably 2.5 to 4.5 kDa, preferably 1.2 to 3.2, preferably 1.3 to 3.0 kDa, preferably 1.5 to 3.0 kDa, preferably 1.8 to 3.0 kDa, preferably 2.0 to 3.0 kDa.

[0145] According to another preferred embodiment of the present invention, the at least one nucleotide sequence encodes a collagen peptide having a molecular weight in the range of 0.18-0.98 kDa, preferably 0.19-0.98 kDa, preferably 0.20-0.98 kDa, preferably 0.25-0.98 kDa, preferably 0.30-0.98 kDa, preferably 0.35-0.98 kDa, preferably 0.40-0.98 kDa, preferably 0.45-0.98 kDa, preferably 0.50-0.98 kDa.

[0146] In a preferred embodiment of the invention, the collagen peptide produced by any of the aforementioned methods according to the invention, in particular a synthetic or recombinant collagen peptide having a molecular weight in the range of 0.18 to 10.0 kDa, in particular 0.18 to 5.0 kDa, in particular 1.1 to 5.0 kDa for use in a method for therapeutic treatment of the human or animal body, is a non-hydroxylated, partially hydroxylated or fully hydroxylated collagen peptide, preferably a non-hydroxylated collagen peptide, preferably a partially hydroxylated collagen peptide, preferably a fully hydroxylated collagen peptide.

[0147] In a preferred embodiment of the invention, the collagen peptide produced by any method according to the invention, in particular a synthetic or recombinant collagen peptide having a molecular weight in the range of 0.18-10.0 kDa, in particular 0.18-5.0 kDa, in particular 1.1-5.0 kDa, for use in a method for therapeutic treatment of the human or animal body, is a glycosylated collagen peptide. Preferably, the collagen peptide is glycosylated in vivo, preferably ex vivo.

[0148] In another preferred embodiment of the invention, the collagen peptide produced by any of the methods according to the invention is a non-glycosylated collagen peptide.

[0149] In a particularly preferred embodiment of the invention, in particular any of the methods for producing a collagen peptide according to the invention, at least one expression cassette of the expression system comprises a plurality, preferably at least two, preferably at least three, preferably at least four, preferably at least five contiguous nucleotide sequences, each of which encodes a collagen peptide having a molecular weight in the range of 0.18 to 10.0 kDa, in particular 0.18 to 5.0 kDa, in particular 1.1 to 5.0 kDa.

[0150] According to this embodiment, the expression system first synthesizes precursor collagen peptides, from which the collagen peptides according to the invention can then be obtained by cleavage, in particular by enzymatic cleavage.

[0151] Preferably, the size of the precursor collagen peptide is 2 to 100 kDa, preferably 3 to 80 kDa, preferably 4 to 60 kDa, preferably 5 to 50 kDa, preferably 10 to 45 kDa. In a preferred embodiment, the precursor collagen peptide comprises 2 to 40 collagen peptides according to the present invention, preferably 3 to 30 collagen peptides according to the present invention, preferably 5 to 20 collagen peptides according to the present invention.

[0152] In a preferred embodiment, a plurality of, preferably at least two, preferably at least three, preferably at least four, preferably at least five contiguous nucleotide sequences encoding collagen peptides each having a molecular weight in the range of 0.18 to 10.0 kDa, in particular 0.18 to 5.0 kDa, in particular 1.1 to 5.0 kDa, are separated by sequences capable of cleaving, in particular enzymatically cleaving, the collagen peptide expressed in step b) into a plurality of, preferably at least two, preferably at least three, preferably at least four, preferably at least five collagen peptides according to the present invention.

[0153] According to this embodiment, between step b) and step c), a step is performed in which the collagen peptide expressed in step b) is cleaved to obtain the collagen peptide according to the present invention, particularly a collagen peptide having a molecular weight in the range of 0.18 to 10.0 kDa, particularly 0.18 to 5.0 kDa, particularly 1.1 to 5.0 kDa. Thus, by expressing the collagen peptide in step b) using the expression system provided in step a), and then cleaving this collagen peptide, a plurality of collagen peptides according to the present invention, preferably at least two, preferably at least three, preferably at least four, preferably at least five, are obtained.

[0154] In another preferred embodiment, the plurality, preferably at least two, preferably at least three, preferably at least four, preferably at least five consecutive nucleotide sequences are different nucleotide sequences. Particularly preferred, the plurality, preferably at least two, preferably at least three, preferably at least four, preferably at least five consecutive nucleotide sequences are the same nucleotide sequence.

[0155] In a preferred embodiment of the invention, the cleavage, in particular the enzymatic cleavage, of the collagen peptides expressed in step b), in particular of the precursor collagen peptides, is carried out by means of neutral, alkaline or acidic proteases.

[0156] Preferably, the individual collagen peptides according to the invention of the expressed collagen peptides, in particular precursor collagen peptides, are separated from each other by specific recognition sequences.

[0157] Particularly preferably, the specific recognition sequence is selected from the group consisting of Factor Xa (Ile-(Glu / Asp)-Gly-Arg), TEV (Glu-Asn-Leu-Tyr-Phe-Gln-(Gly / Ser)), thrombin (Leu-Val-Pro-Arg-Gly-Ser), trypsin recognition sequence, and papain recognition sequence.

[0158] In a preferred embodiment, according to the invention, the collagen peptides are administered locally, in particular topically, or systemically, in particular enterally, preferably orally or parenterally.

[0159] According to the present invention, the term "biological effectiveness" preferably means the ability of the collagen peptide according to the invention to stimulate the synthesis of extracellular matrix proteins, in particular collagen, proteoglycan and / or elastin synthesis, in cells, in particular osteoblasts, fibroblasts and / or chondrocytes. According to the present invention, "biological effectiveness" is preferably present when the incubation of cells, in particular osteoblasts, fibroblasts and / or chondrocytes with the collagen peptide according to the present invention stimulates the synthesis of extracellular matrix proteins, in particular collagen, proteoglycan and / or elastin synthesis, and the stimulation of the synthesis of extracellular matrix proteins can be measured in at least one, preferably at least two, preferably all of the in vitro tests for the stimulation of the synthesis of extracellular matrix proteins in osteoblasts, fibroblasts and chondrocytes shown in Examples 3 to 6, compared to untreated cells and / or cells treated with a biologically ineffective agent, in particular untreated osteoblasts, fibroblasts and / or chondrocytes or osteoblasts, fibroblasts and / or chondrocytes treated with a biologically ineffective agent. In a preferred embodiment of the invention, "biologically inactive agent" is understood to mean 160 bloom gelatin from pigskin or 260 bloom gelatin from bovine leather (Ringerspalt).

[0160] According to the present invention, "equivalent biological effectiveness" preferably means that the collagen peptide according to the present invention causes stimulation of the synthesis of extracellular matrix proteins, particularly collagen, proteoglycan and / or elastin, in cells, particularly osteoblasts, fibroblasts and / or chondrocytes, and this stimulation is measured by incubation with a mixture of collagen peptides obtained from natural sources, particularly with a mixture of collagen peptides of equivalent average molecular weight obtained from natural sources, particularly by incubation with Verisol®, manufactured according to European Patent No. 2640352, Fortigel®, manufactured according to International Publication No. 2010 / 149596, or Fortibone®, manufactured according to International Publication No. 2014 / 072235 (European Patent No. 2916855), and is understood to be comparable to the stimulation of cells, particularly osteoblasts, fibroblasts and / or chondrocytes.According to the invention, a "stimulation comparable to" preferably means that the stimulation of the synthesis of extracellular matrix proteins in cells, in particular osteoblasts, fibroblasts and / or chondrocytes, in particular collagen, proteoglycan and / or elastin synthesis, is measurable in at least one, preferably at least two, preferably all of the in vitro tests for the stimulation of the synthesis of extracellular matrix proteins in osteoblasts, fibroblasts and chondrocytes, preferably as shown in Examples 3 to 6, and is achieved by incubation of the cells with a mixture of collagen peptides obtained from a natural source, in particular by incubation of the cells with a mixture of collagen peptides of comparable average molecular weight obtained from a natural source. By incubating the cells with Verisol, in particular produced according to EP 2 640 352, with Fortigel, produced according to WO 2010 / 149596 or with Fortibone, produced according to WO 2014 / 072235 (EP 2 916 855), is understood to mean that the stimulation of the synthesis of extracellular matrix proteins in cells, in particular osteoblasts, fibroblasts and / or chondrocytes, in particular of collagen, proteoglycan and / or elastin, caused by incubation of the cells with Verisol, in particular produced according to EP 2 640 352, with Fortigel, produced according to WO 2010 / 149596 or with Fortibone, produced according to WO 2014 / 072235 (EP 2 916 855), differs by no more than 2%, preferably no more than 1.5%, preferably no more than 1%.

[0161] According to the present invention, the term "better biological effectiveness" is preferably understood as meaning that the collagen peptide according to the invention causes a stimulation of the synthesis of extracellular matrix proteins, in particular collagen, proteoglycan and / or elastin, in cells, in particular osteoblasts, fibroblasts and / or chondrocytes, which stimulation is greater than that of a mixture of collagen peptides obtained from natural sources, in particular a mixture of collagen peptides of comparable average molecular weight obtained from natural sources, in particular Verisol produced according to EP 2 640 352, Fortigel produced according to WO 2010 / 149596 or Fortibone produced according to WO 2014 / 072235 (EP 2 916 855). According to the present invention, the term "better biological effectiveness" preferably refers to a collagen peptide according to the present invention, which is capable of stimulating the synthesis of extracellular matrix proteins, in particular collagen, proteoglycan and / or elastin synthesis, in cells, in particular osteoblasts, fibroblasts and / or chondrocytes, with a mixture of collagen peptides obtained from natural sources, in particular Verisol produced according to EP 2 640 352, Fortigel produced according to WO 2010 / 149596 or mixtures of collagen peptides according to WO 2014 / 072235 (EP 2 916 855). It is understood that stimulation of the synthesis of extracellular matrix proteins, in particular collagen, proteoglycan and / or elastin synthesis in cells, in particular osteoblasts, fibroblasts and / or chondrocytes, by the produced Fortibone is increased by more than 2%, preferably at least 3%, preferably at least 4%, preferably at least 5%, wherein this increase in synthesis is measurable in at least one, preferably at least two, preferably all of the in vitro tests for stimulation of the synthesis of extracellular matrix proteins in osteoblasts, fibroblasts and chondrocytes shown in Examples 3 to 6.

[0162] Thus, according to the present invention, "biological effectiveness", "comparable biological effectiveness" or "superior biological effectiveness" can be present when the collagen peptide according to the present invention is capable of stimulating the synthesis of extracellular matrix proteins, preferably when the synthesis of at least one of the extracellular matrix proteins collagen, proteoglycan or elastin is stimulated, preferably when the synthesis of two or three of these extracellular matrix proteins is stimulated, and this is done in a cell, preferably in at least one of the cell types osteoblasts, fibroblasts or chondrocytes, preferably in two or three of these cell types. Thus, biological effectiveness can also be present in one embodiment of the present invention when the synthesis of only one extracellular matrix protein, in particular one extracellular matrix protein selected from the group consisting of collagen, proteoglycan and elastin, is stimulated, while no stimulation of the synthesis of other extracellular matrix proteins is observed in the cell. Likewise, a biological effect, or an equivalent or better biological effect, may be present if a stimulation of the synthesis of at least one extracellular matrix protein is observed only in one cell type, in particular in one cell type selected from the group consisting of osteoblasts, fibroblasts and chondrocytes, even if such a stimulation does not occur in other cell types. Preferably, it is provided that the synthesis of two or more extracellular matrix proteins, in particular one or more explicitly mentioned extracellular matrix proteins selected from the group consisting of collagens, proteoglycans and elastin, is stimulated in cells, in particular in at least one of the cell types osteoblasts, fibroblasts or chondrocytes, in particular in two or three of these cell types.

[0163] According to the present invention, the "biological efficacy", "comparable biological efficacy" or "superior biological efficacy" of the collagen peptide according to the present invention in osteoblastic cells after incubation of said cells with the collagen peptide according to the present invention can be determined by measuring the expression of the mRNA of the corresponding extracellular matrix protein, in particular selected from the group consisting of collagens, proteoglycans and elastin, in comparison with the expression of the mRNA of the corresponding extracellular matrix protein, in particular selected from the group consisting of collagens, proteoglycans and elastin, in a suitable control, using real-time PCR, in particular by an in vitro test as shown in Example 3.

[0164] According to the present invention, the "biological efficacy", "comparable biological efficacy" or "superior biological efficacy" of the collagen peptide according to the present invention in fibroblasts after incubation of said cells with the collagen peptide according to the present invention can be detected by measuring the expression of the mRNA of the corresponding extracellular matrix protein, in particular selected from the group consisting of collagen, biglycan and versican, in comparison with the expression of the mRNA of the corresponding extracellular matrix protein, in particular selected from the group consisting of collagen, biglycan and versican, in a suitable control, using real-time PCR, in particular by the in vitro test shown in Example 4.

[0165] According to the present invention, the "biological efficacy", "comparable biological efficacy" or "superior biological efficacy" of the collagen peptides according to the present invention in chondrocytes after incubation of said cells with the collagen peptides according to the present invention can be determined by detection of the amount of radiolabeled and synthesized radiolabeled collagen and / or by Alcian blue staining and photometry of the glycosaminoglycans (GAGs) of the synthesized proteoglycans, respectively, in comparison with appropriate controls, in particular by in vitro tests as shown in Example 5.

[0166] According to the invention, the "biological effectiveness", "comparable biological effectiveness" or "superior biological effectiveness" of the collagen peptide according to the invention in osteoblasts, fibroblasts and chondrocytes, in particular fibroblasts and chondrocytes, after incubation of said cells with the collagen peptide according to the invention can be determined by photometric measurement of the amount of dye-labeled synthesized extracellular matrix proteins, in particular selected from the group consisting of collagens, proteoglycans and elastin, particularly preferably selected from the group consisting of collagens and proteoglycans, in comparison with the measurement of the amount of synthesized extracellular matrix proteins, in particular selected from the group consisting of collagens, proteoglycans and elastin, particularly preferably selected from the group consisting of collagens and proteoglycans, in a suitable control, in particular selected from the group consisting of collagens, proteoglycans and elastin, particularly preferably selected from the group consisting of collagens and proteoglycans, in particular by an in vitro test as shown in example 6.

[0167] According to the present invention, the term "biological effectiveness in at least one, preferably at least two, preferably all of the in vitro tests for stimulation of the synthesis of extracellular matrix proteins in osteoblasts, fibroblasts and chondrocytes shown in Examples 3-6" means that the biological effectiveness of the collagen peptide according to the present invention can be demonstrated using any of the assays specified in Examples 3-6. Individual process parameters shown in Examples 3-6 can be varied as appropriate and according to the understanding of the skilled person without affecting the fundamental persuasiveness of the experimental results. In a preferred embodiment of the present invention, the collagen peptide according to the present invention shows biological effectiveness in at least one, preferably at least two, preferably all of the in vitro tests for stimulation of the synthesis of extracellular matrix proteins in osteoblasts, fibroblasts and chondrocytes shown in Examples 3-6 under the process parameters shown in these very examples. Furthermore, the biological effectiveness of the collagen peptide according to the present invention can also be demonstrated in other tests known to the skilled person, in particular in vitro tests, preferably in in vitro tests for stimulation of the synthesis of extracellular matrix proteins in osteoblasts, fibroblasts and chondrocytes.

[0168] According to the present invention, the term "stimulation of the synthesis of extracellular matrix proteins" is understood to mean the promotion of the biosynthesis of at least one protein of the extracellular matrix in cells, preferably osteoblasts, fibroblasts and / or chondrocytes, by exogenous influence, in particular by the collagen peptide according to the present invention, and / or the promotion of the biosynthesis of at least one mRNA encoding an extracellular matrix protein in cells, preferably osteoblasts, fibroblasts and / or chondrocytes. According to the present invention, in particular, the term "stimulation of the synthesis of extracellular matrix proteins" is understood to mean the increase in the amount of at least one extracellular matrix protein secreted by cells, preferably osteoblasts, fibroblasts and / or chondrocytes, by exogenous influence, in particular by the collagen peptide according to the present invention, and / or the increase in the amount of at least one mRNA encoding an extracellular matrix protein synthesized in cells, preferably osteoblasts, fibroblasts and / or chondrocytes.

[0169] In the present invention, the term "collagen" is understood as it is conventionally used in the art, in particular as defined, for example, in WO 01 / 34646. In one preferred embodiment, the term "collagen" relates to collagens of types I to XXVII. In another preferred embodiment, the term "collagen" relates to collagens of the sequence glycine-proline, glycine-4-hydroxyproline or glycine-X-4-hydroxyproline, preferably with the repeating motif (Gly-XY). n where X and Y can be any amino acid, preferably proline and 4-hydroxyproline. Particularly preferably, the term "collagen" refers to a peptide having the repeating motif (Gly-Pro-Y) n and / or (Gly-X-Hyp) m where X and Y may be any amino acid.

[0170] Preferably, in the present invention, the term "gelatin" is understood as it is commonly used in the art, in particular as defined, for example, in WO 01 / 34646.

[0171] In the present invention, the term "collagen peptide" is understood to mean a peptide having an amino acid sequence present in collagen according to the above definition, the peptide being at least a dipeptide, preferably an oligopeptide or a polypeptide. The collagen peptide may be present in particular in a chemically modified form, in particular in a hydroxylated and / or glycosylated form, or it may be unmodified.

[0172] According to the present invention, the term "function-conserving sequence variant" is understood to mean a modification of a given amino acid sequence, in particular a substitution, insertion and / or deletion of single or multiple amino acids, which results in an amino acid sequence which differs from the given amino acid sequence, but which retains the function characteristic of the given amino acid sequence, in particular its biological effectiveness.

[0173] Preferably, a "function-conserving sequence variant" is understood to mean a variant of a given amino acid sequence which retains at least 50%, preferably at least 60%, preferably at least 70%, preferably at least 80%, preferably at least 90%, preferably at least 95%, preferably 100% of the function characteristic of the given amino acid sequence, in particular the biological efficacy, in particular the biological efficacy in connective tissue, in particular the biological efficacy in in vitro tests for stimulation of synthesis of extracellular matrix proteins in osteoblasts, fibroblasts and chondrocytes as shown in Examples 3 to 6. More preferably, according to the present invention, a "function-conserving sequence variant" is understood to mean a variant of a given amino acid sequence which has at least 50%, preferably at least 55%, preferably at least 60%, preferably at least 65%, preferably at least 70%, preferably at least 75%, preferably at least 80%, preferably at least 85%, preferably at least 90% sequence homology to the given amino acid sequence.

[0174] Particularly preferably, "function-conserving sequence variants" according to the invention refer to variants of a given amino acid sequence in which one or more amino acids having given chemical and physical properties are replaced by one or more amino acids having identical or similar chemical and physical properties, in particular, for example, an amino acid having a non-polar side chain (e.g., Ala, Val, Met, Leu, Ile, Pro, Trp, Phe) replaced by another amino acid having a non-polar side chain (e.g., Ala, Val, Met, Leu, Ile, Pro, Trp, Phe), an amino acid having a polar, neutral side chain replaced by another amino acid having a non-polar side chain (e.g., Ala, Val, Met, Leu, Ile, Pro, Trp, Phe), replacement of an amino acid having an acidic side chain (e.g., Glu, Asp) with another amino acid having an acidic side chain (e.g., Glu, Asp); and / or replacement of an amino acid having a basic side chain (e.g., Lys, Arg, His) with another amino acid having a basic side chain (e.g., Lys, Arg, His). In accordance with this embodiment, "function-conserving sequence variants" do not change or only slightly change the chemical and physical properties of a given amino acid sequence.

[0175] In another embodiment, a "function-conserving sequence variant" is a variant in which at least one amino acid of a given amino acid sequence, in particular a naturally occurring amino acid sequence, preferably at least one non-essential amino acid, in particular Ala, Asn, Asp, Glu, Ser, of a given amino acid sequence, in particular a naturally occurring amino acid sequence, is replaced by at least one very specific amino acid, in particular at least one essential amino acid, in particular Ile, Leu, Lys, Met, Phe, Thr, Trp, Val, His, Cys, Tyr, particularly preferably Trp, in which the function characteristic of the given amino acid sequence, in particular a naturally occurring amino acid sequence, in particular the biological efficacy, in particular the biological efficacy in relation to connective tissue, in particular the biological efficacy in an in vitro test for stimulation of the synthesis of extracellular matrix proteins in osteoblasts, fibroblasts and chondrocytes as shown in Examples 3 to 6, is retained by at least 50%, preferably at least 60%, preferably at least 70%, preferably at least 80%, preferably at least 90%, preferably at least 95%, preferably 100%. As an example of this embodiment, the synthetic or recombinant collagen peptides set forth in SEQ ID NOs: 23, 27, and 28, in particular the synthetically or recombinantly produced collagen peptides, are "function-conservative sequence variants" of the synthetic or recombinant collagen peptide set forth in SEQ ID NO: 7, in particular the synthetically or recombinantly produced collagen peptide, in which a non-essential amino acid is replaced with an essential amino acid. Similarly, the synthetic or recombinant collagen peptides set forth in SEQ ID NOs: 24, 29, and 30, in particular the synthetically or recombinantly produced collagen peptides, are "function-conservative sequence variants" of the synthetic or recombinant collagen peptide set forth in SEQ ID NO: 22, in particular the synthetically or recombinantly produced collagen peptide, in which a non-essential amino acid is replaced with an essential amino acid.

[0176] According to the present invention, a "function-conserving sequence variant" is also understood to mean a variant of a given amino acid sequence, in particular a naturally occurring amino acid sequence, in which at least one amino acid, preferably at least one essential amino acid, in particular Ile, Leu, Lys, Met, Phe, Thr, Trp, Val, His, Cys, Tyr, particularly preferably Trp, has been inserted into the given amino acid sequence, in particular a naturally occurring amino acid sequence, in which the function characteristic of the given amino acid sequence, in particular a naturally occurring amino acid sequence, in particular the biological efficacy, in particular the biological efficacy with regard to connective tissue, in particular the biological efficacy in the in vitro tests for stimulation of the synthesis of extracellular matrix proteins in osteoblasts, fibroblasts and chondrocytes as shown in Examples 3 to 6, is retained by at least 50%, preferably at least 60%, preferably at least 70%, preferably at least 80%, preferably at least 90%, preferably at least 95%, preferably 100%. In this case, the invention makes it possible to provide for the insertion of at least one amino acid, preferably at least one essential amino acid, in particular Ile, Leu, Lys, Met, Phe, Thr, Trp, Val, His, Cys, Tyr, particularly preferably Trp, at the N-terminus, C-terminus and / or internally of the amino acid sequence.

[0177] In the present invention, the term "amino acid modification" refers to a chemical change of one or more amino acids that occurs before, after or during the synthesis of a collagen peptide, while retaining the original amino acid backbone of the collagen peptide, in particular one or more proteinogenic amino acids. Thus, the term encompasses both the use of chemically modified amino acids to synthesize collagen peptides according to the invention, and the chemical modification of amino acids after or during the synthesis of collagen peptides. Typical amino acid modifications for collagen peptides are in particular the hydroxylation of proline and lysine residues, and the glycosylation of hydroxylated lysine residues. However, according to the present invention, the term also encompasses other chemical changes of amino acids, such as phosphorylation, N-glycosylation, acetylation, methylation, myristoylation, etc.

[0178] In the present invention, "synthetic or recombinant collagen peptides" or "synthetically or recombinantly produced collagen peptides" are understood to mean collagen peptides obtained by chemical synthesis, in particular solid-phase synthesis, or by recombinant production by biotechnology using expression systems. According to the present invention, "synthetic collagen peptides" or "synthetically produced collagen peptides" and "recombinant collagen peptides" or "recombinantly produced collagen peptides" have in common that they are not obtained from natural sources.

[0179] In the present invention, the term "recombinant DNA" refers to an artificially produced or engineered DNA molecule produced in vitro by genetic engineering techniques. In a preferred embodiment, recombinant DNA is composed of components from different organisms of origin.

[0180] In the present invention, the term "expression cassette" is understood to mean a DNA segment which serves to transcribe the information encoded in said segment into RNA, in particular into mRNA, and which comprises at least one promoter and one nucleotide sequence encoding a protein, usually at least one promoter, at least one nucleotide sequence encoding a protein, and optionally a terminator.

[0181] In the present invention, "nucleotide sequence" is understood to mean the sequence of nucleotides of a nucleic acid, in particular a nucleic acid strand, in particular a DNA or RNA strand. A "nucleotide sequence" should therefore be understood to be both a unit of information and a DNA or RNA strand which physically represents that information.

[0182] In the present invention, the term "expression system" is understood to mean a system capable of carrying out a controlled biosynthesis of a protein of interest. According to the present invention, the term "expression system" encompasses both cell-free expression systems, in which the components necessary for protein biosynthesis are not present in the cell, i.e. the protein biosynthesis takes place outside the cell, as well as cell-based expression systems, in which the protein biosynthesis takes place inside a living cell. In the present invention, a cell-free expression system is preferably a lysate or extract from E. coli, insect cells, wheat germ, tobacco cells or mammalian cells, in particular CHO cells or rabbit reticulocytes, which comprises the components necessary for protein biosynthesis, in particular the translation and transcription systems. When a cell-free expression system is used in any of the methods according to the present invention, the term "culture" is synonymous with "incubate".

[0183] In the present invention, "host cell" is understood to mean a living cell capable of expressing a peptide or protein coded for by a foreign DNA, in particular a recombinant DNA.

[0184] The term "recovery of collagen peptides" in method step c) refers to methods known to the skilled person for isolating collagen peptides from a composition comprising multiple components by known isolation methods, such as centrifugation methods, in particular differential centrifugation and / or density gradient centrifugation, chromatography methods, in particular gel filtration chromatography, ion exchange chromatography, affinity chromatography and / or high performance liquid chromatography, electrophoresis methods, filtration methods and / or extraction methods, whereby enrichment and purification of said components from a composition comprising multiple components can be achieved, preferably by successive application of multiple isolation methods.

[0185] According to the present invention, "conditions allowing expression of collagen peptides" is understood to mean conditions that activate or enhance the expression of collagen peptides, such as in particular temperature, pressure, time, light, and the presence or absence of inducers and / or inhibitors. In a preferred embodiment, the expression of collagen peptides is carried out within the scope of high cell density fermentation, in particular under high pressure, preferably under high pressure. The specific conditions that allow the expression of collagen peptides are known to the skilled artisan and depend on the expression system used and the expression cassette used, in particular the promoter contained therein. The expression of collagen peptides may be constitutive or inducible, depending on the structure of the expression cassette.

[0186] In the present invention, the terms "comprise" and "have" are understood to mean that in addition to the elements explicitly covered by these terms, other elements not explicitly mentioned may be added. In the present invention, these terms are also understood to cover only the elements explicitly mentioned, and no other elements are present. In this particular embodiment, the meaning of the terms "comprise" and "have" is synonymous with the term "consisting of". Furthermore, the terms "comprise" and "have" also cover compositions that, in addition to the elements explicitly mentioned, also contain other elements that are not mentioned but have functionally and qualitatively subordinate properties. In this embodiment, the meaning of the terms "comprise" and "have" is synonymous with the term "consisting essentially of".

[0187] In the present invention, when the first and second decimal places or the second decimal place are not indicated, these digits are assumed to be zero.

[0188] In the present invention, the term "and / or" is understood to mean that all members of the group connected by the term "and / or" are disclosed alternatively to one another and respectively cumulatively to one another in any combination. This means that the expression "A, B and / or C" should be understood as disclosing the following: a) A or B or C, or b) (A and B), or c) (A and C), or d) (B and C), or e) (A and B and C).

[0189] Further preferred embodiments are evident from the dependent claims.

[0190] The invention will now be described with reference to exemplary arrangements, figures and examples without limiting the general inventive concept.

[0191] In the following description: SEQ ID NO:1 is the amino acid sequence Leu-Thr-Gly-Pro-Ile-Gly-Pro-Pro-Gly-Pro-Ala of a non-hydroxylated collagen peptide containing 11 amino acids.

[0192] SEQ ID NO: 2 is the amino acid sequence of a non-hydroxylated collagen peptide containing 33 amino acids: Gly-Ala-Pro-Gly-Lys-Asp-Gly-Val-Arg-Gly-Leu-Thr-Gly-Pro-Ile-Gly-Pro-Pro-Gly-Pro-Ala-Gly-Ala-Pro-Gly-Asp-Lys-Gly-Glu-Ala-Gly-Pro-Ser.

[0193] SEQ ID NO: 3 is the amino acid sequence Gly-Ala-Pro-Gly-Lys-Asp-Gly-Val-Arg-Gly-Leu-Thr-Gly-Pro-Ile-Gly-Pro-Pro-Gly-Pro-Ala-Gly-Ala-Pro of a non-hydroxylated collagen peptide containing 24 amino acids.

[0194] SEQ ID NO: 4 is the amino acid sequence Gly-Ala-Pro-Gly-Lys-Asp-Gly-Val-Arg-Gly-Leu-Thr-Gly-Pro-Ile-Gly-Pro-Pro-Gly-Pro-Ala-Gly-Ala-Arg of a non-hydroxylated collagen peptide containing 24 amino acids.

[0195] SEQ ID NO: 5 is the amino acid sequence of a non-hydroxylated collagen peptide containing 24 amino acids: Gly-Ala-Pro-Gly-Pro-Pro-Gly-Pro-Pro-Gly-Ala-Arg-Gly-Gln-Ala-Gly-Val-Met-Gly-Phe-Pro-Gly-Pro-Lys.

[0196] SEQ ID NO:6 is the amino acid sequence Leu-Thr-Gly-Pro-Ile-Gly-Pro-4Hyp-Gly-Pro-Ala of a hydroxylated collagen peptide containing 11 amino acids.

[0197] SEQ ID NO: 7 is the amino acid sequence of a hydroxylated collagen peptide containing 33 amino acids: Gly-Ala-4Hyp-Gly-Lys-Asp-Gly-Val-Arg-Gly-Leu-Thr-Gly-Pro-Ile-Gly-Pro-4Hyp-Gly-Pro-Ala-Gly-Ala-4Hyp-Gly-Asp-Lys-Gly-Glu-Ala-Gly-Pro-Ser.

[0198] SEQ ID NO: 8 is the amino acid sequence Gly-Ala-4Hyp-Gly-Lys-Asp-Gly-Val-Arg-Gly-Leu-Thr-Gly-Pro-Ile-Gly-Pro-4Hyp-Gly-Pro-Ala-Gly-Ala-4Hyp of a hydroxylated collagen peptide containing 24 amino acids.

[0199] SEQ ID NO: 9 is the amino acid sequence Gly-Ala-4Hyp-Gly-Lys-Asp-Gly-Val-Arg-Gly-Leu-Thr-Gly-Pro-Ile-Gly-Pro-4Hyp-Gly-Pro-Ala-Gly-Ala-Arg of a hydroxylated collagen peptide containing 24 amino acids.

[0200] SEQ ID NO: 10 is the amino acid sequence of a hydroxylated collagen peptide containing 24 amino acids Gly-Ala-4Hyp-Gly-Pro-Pro-Gly-Pro-4Hyp-Gly-Ala-Arg-Gly-Gln-Ala-Gly-Val-Met-Gly-Phe-4Hyp-Gly-Pro-Lys.

[0201] SEQ ID NO: 11 is the amino acid sequence of a non-hydroxylated collagen peptide containing 11 amino acids: Gly-Ala-Pro-Gly-Lys-Asp-Gly-Val-Arg-Gly-Leu.

[0202] SEQ ID NO: 12 is the amino acid sequence Gly-Ala-Pro-Gly-Lys-Asp-Gly-Val-Arg-Gly-Leu-Thr-Gly-Pro-Ile-Gly-Pro-Pro-Gly-Pro-Ala-Gly-Ala of a non-hydroxylated collagen peptide containing 23 amino acids.

[0203] SEQ ID NO: 13 is the amino acid sequence of a non-hydroxylated collagen peptide containing 58 amino acids Gly-Ala-Pro-Gly-Lys-Asp-Gly-Val-Arg-Gly-Leu-Thr-Gly-Pro-Ile-Gly-Pro-Pro-Gly-Pro-Ala-Gly-Ala-Pro-Gly-Ala-Pro-Gly-Asp-Lys-Gly-Glu-Ala-Gly-Pro-Ser-Gly-Pro-Ala-Gly-Pro-Thr-Gly-Ala-Arg-Gly-Ala-Pro-Gly-Asp-Arg-Gly-Glu-Pro-Gly-Pro-Gly-Pro-Gly-Pro-Ala-Gly

[0204] SEQ ID NO: 14 is the amino acid sequence of a non-hydroxylated collagen peptide containing 90 amino acids: Gly-Ala-Pro-Gly-Lys-Asp-Gly-Val-Arg-Gly-Leu-Thr-Gly-Pro-Ile-Gly-Pro-Pro-Gly-Pro-Ala-Gly-Ala-Pro-Gly-Asp-Lys-Gly-Glu-Ala-Gly-Pro-Ser-Gly-Pro-Ala-Gly-Pro-Thr-Gly ly-Ala-Arg-Gly-Ala-Pro-Gly-Asp-Arg-Gly-Glu-Pro-Gly-Pro-Pro-Gly-Pro-Ala-Gly-Phe-Ala-Gly-Pro-Pro-Gly-Ala- Asp-Gly-Gln-Pro-Gly-Ala-Lys-Gly-Glu-Pro-Gly-Asp-Ala-Gly-Ala-Lys-Gly-Asp-Ala-Gly-Pro-Pro-Gly-Pro-Ala.

[0205] SEQ ID NO: 15 is the amino acid sequence of a non-hydroxylated collagen peptide containing 19 amino acids: Lys-Gly-Ala-Pro-Gly-Ala-Asp-Gly-Pro-Ala-Gly-Ala-Pro-Gly-Thr-Pro-Gly-Pro-Gln.

[0206] SEQ ID NO: 16 is the amino acid sequence of the non-hydroxylated collagen peptide containing 35 amino acids Gly-Pro-Pro-Gly-Pro-Ala-Gly-Glu-Lys-Gly-Ala-Pro-Gly-Ala-Asp-Gly-Pro-Ala-Gly-Ala-Pro-Gly-Thr-Pro-Gly-Pro-Gln-Gly-Ile-Ala-Gly-Gln-Arg-Gly-Val.

[0207] SEQ ID NO: 17 is the amino acid sequence of a hydroxylated collagen peptide containing 11 amino acids: Gly-Ala-4Hyp-Gly-Lys-Asp-Gly-Val-Arg-Gly-Leu.

[0208] SEQ ID NO: 18 is the amino acid sequence Gly-Ala-4Hyp-Gly-Lys-Asp-Gly-Val-Arg-Gly-Leu-Thr-Gly-Pro-Ile-Gly-Pro-4Hyp-Gly-Pro-Ala-Gly-Ala of a hydroxylated collagen peptide containing 23 amino acids.

[0209] SEQ ID NO: 19 is the amino acid sequence of a hydroxylated collagen peptide containing 58 amino acids Gly-Ala-4Hyp-Gly-Lys-Asp-Gly-Val-Arg-Gly-Leu-Thr-Gly-Pro-Ile-Gly-Pro-4Hyp-Gly-Pro-Ala-Gly-Ala-4Hyp-Gly-Asp-Lys-Gly-Glu-Ala-Gly-Pro-Ser-Gly-Pro-Ala-Gly-Pro-Thr-Gly-Ala-Arg-Gly-Ala-4Hyp-Gly-Asp-Arg-Gly-Glu-4Hyp-Gly-Pro-4Hyp-Gly-Pro-Ala-Gly.

[0210] SEQ ID NO: 20 is the amino acid sequence of a hydroxylated collagen peptide containing 90 amino acids: Gly-Ala-4Hyp-Gly-Lys-Asp-Gly-Val-Arg-Gly-Leu-Thr-Gly-Pro-Ile-Gly-Pro-4Hyp-Gly-Pro-Ala-Gly-Ala-4Hyp-Gly-Asp-Lys-Gly-Glu-Ala-Gly-Pro-Ser-Gly-Pro-Ala-Gly-Pro-Thr-Gly- Ala-Arg-Gly-Ala-4Hyp-Gly-Asp-Arg-Gly-Glu-4Hyp-Gly-Pro-4Hyp-Gly-Pro-Ala-Gly-Phe-Ala-Gly-Pro-4Hyp-Gly-Ala- Asp-Gly-Gln-4Hyp-Gly-Ala-Lys-Gly-Glu-Pro-Gly-Asp-Ala-Gly-Ala-Lys-Gly-Asp-Ala-Gly-Pro-Pro-Gly-Pro-Ala.

[0211] SEQ ID NO: 21 is the amino acid sequence of a hydroxylated collagen peptide containing 19 amino acids: Lys-Gly-Ala-4Hyp-Gly-Ala-Asp-Gly-Pro-Ala-Gly-Ala-4Hyp-Gly-Thr-Pro-Gly-Pro-Gln.

[0212] SEQ ID NO: 22 is the amino acid sequence of a hydroxylated collagen peptide containing 35 amino acids Gly-Pro-4Hyp-Gly-Pro-Ala-Gly-Glu-Lys-Gly-Ala-4Hyp-Gly-Ala-Asp-Gly-Pro-Ala-Gly-Ala-4Hyp-Gly-Thr-Pro-Gly-Pro-Gln-Gly-Ile-Ala-Gly-Gln-Arg-Gly-Val.

[0213] SEQ ID NO: 23 is the amino acid sequence Gly-Val-4Hyp-Gly-Lys-Tyr-Gly-Val-Arg-Gly-Leu-Thr-Gly-Pro-Ile-Gly-Pro-4Hyp-Gly-Pro-Leu-Gly-Ile-4Hyp-Gly-Trp-Lys-Gly-Phe-Val-Gly-Pro-Thr of a hydroxylated collagen peptide containing 33 amino acids.

[0214] SEQ ID NO: 24 is the amino acid sequence of a hydroxylated collagen peptide containing 36 amino acids Gly-Pro-4Hyp-Gly-Pro-Val-Gly-Thr-Lys-Gly-Ile-4Hyp-Gly-Val-Tyr-Gly-Pro-Leu-Gly-Ile-4Hyp-Gly-Thr-Pro-Gly-Pro-Trp-Gly-Ile-Leu-Gly-Thr-Lys-Arg-Gly-Val.

[0215] SEQ ID NO: 25 is the amino acid sequence of a hydroxylated collagen peptide containing 33 amino acids Gly-Ala-4Hyp-Gly-Lys-Asp-Gly-Val-Arg-Gly-Leu-Thr-Gly-4Hyp-Ile-Gly-4Hyp-4Hyp-Gly-4Hyp-Ala-Gly-Ala-4Hyp-Gly-Asp-Lys-Gly-Glu-Ala-Gly-4Hyp-Ser.

[0216] SEQ ID NO: 26 is the amino acid sequence of hydroxylated collagen peptide containing 39 amino acids: His-His-His-His-His-His-Gly-Ala-4Hyp-Gly-Lys-Asp-Gly-Val-Arg-Gly-Leu-Thr-Gly-Pro-Ile-Gly-Pro-4Hyp-Gly-Pro-Ala-Gly-Ala-4Hyp-Gly-Asp-Lys-Gly-Glu-Ala-Gly-Pro-Ser.

[0217] SEQ ID NO: 27 is the amino acid sequence Gly-Leu-4Hyp-Gly-Lys-Met-Gly-Val-Phe-Gly-Leu-Thr-Gly-Pro-Ile-Gly-Pro-4Hyp-Gly-Pro-Trp-Gly-Val-4Hyp-Gly-His-Lys-Gly-Tyr-Leu-Gly-Pro-Thr of a hydroxylated collagen peptide containing 33 amino acids.

[0218] SEQ ID NO: 28 is the amino acid sequence Gly-Leu-4Hyp-Gly-Lys-Tyr-Gly-Val-His-Gly-Leu-Thr-Gly-Pro-Leu-Gly-Pro-4Hyp-Gly-Pro-Met-Gly-Ile-4Hyp-Gly-Trp-Lys-Gly-Phe-Val-Gly-Pro-Thr of a hydroxylated collagen peptide containing 33 amino acids.

[0219] SEQ ID NO: 29 is the amino acid sequence of a hydroxylated collagen peptide containing 35 amino acids Gly-Pro-4Hyp-Gly-Pro-Leu-Gly-Met-Lys-Gly-Leu-4Hyp-Gly-Val-Trp-Gly-Pro-Phe-Gly-Leu-4Hyp-Gly-Thr-Pro-Gly-Pro-His-Gly-Ile-Thr-Gly-Tyr-Lys-Gly-Val.

[0220] SEQ ID NO: 30 is the amino acid sequence Gly-Pro-4Hyp-Gly-Pro-Val-Gly-Thr-Lys-Gly-Leu-4Hyp-Gly-Phe-Tyr-Gly-Pro-Leu-Gly-Ile-4Hyp-Gly-His-Pro-Gly-Pro-Trp-Gly-Met-Leu-Gly-Thr-Lys-Gly-Val of a hydroxylated collagen peptide containing 35 amino acids. [Brief description of the drawings]

[0221] [Figure 1A] FIG. 1 shows the stimulation of collagen synthesis in human dermal fibroblasts by collagen peptides according to the invention according to SEQ ID NO: 2, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, compared to a mixture of collagen peptides of comparable average molecular weight obtained from natural sources (Verisol) and two different gelatin controls according to Example 6 (160 bloom gelatin from pigskin (Gelatin 160) and 260 bloom gelatin from bovine leather (Rinderspalt) (Gelatin 260)). Quantitative measurement of collagen synthesis fold (Faktor) over untreated samples is shown. Each error bar indicates standard deviation (SEM). [Figure 1B] FIG. 1 shows the stimulation of collagen synthesis in human chondrocytes by collagen peptides of SEQ ID NO:2, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10 according to the present invention compared to a mixture of collagen peptides of comparable average molecular weight obtained from natural sources (Fortigel) according to Example 6. Quantitative measurements of fold collagen synthesis over untreated samples are shown. Each error bar indicates standard deviation (SEM). [Figure 2A] FIG. 1 shows the stimulation of proteoglycan synthesis in human dermal fibroblasts by collagen peptides of SEQ ID NO:2, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10 according to the invention, compared to a mixture of collagen peptides of comparable average molecular weight obtained from natural sources (Verisol) and two different gelatin controls according to Example 6 (160 bloom gelatin from pigskin (Gelatin 160) and 260 bloom gelatin from bovine leather (Gelatin 260)). Quantitative measurements of fold proteoglycan synthesis over untreated samples are shown. Each error bar indicates standard deviation (SEM). [Figure 2B] FIG. 1 shows the stimulation of human chondrocyte proteoglycan synthesis by collagen peptides of SEQ ID NO:2, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10 according to the invention compared to a mixture of collagen peptides of comparable average molecular weight obtained from natural sources (Fortigel) according to Example 6. Quantitative measurements of fold collagen synthesis over untreated samples are shown. Each error bar indicates standard deviation (SEM). [Diagram 3] FIG. 1 shows the stimulation of collagen synthesis in human chondrocytes by collagen peptides of SEQ ID NO: 11 to SEQ ID NO: 26 according to the present invention compared to a mixture of collagen peptides of comparable average molecular weight obtained from natural sources (Fortigel). Quantitative measurements of collagen synthesis fold over untreated samples are shown. Each error bar indicates standard deviation (SEM). [Figure 4] FIG. 1 shows the stimulation of collagen synthesis in human dermal fibroblasts by collagen peptides of SEQ ID NO: 11 to SEQ ID NO: 26 according to the present invention compared to a mixture of collagen peptides of comparable average molecular weight obtained from natural sources (Verisol). Quantitative measurements of collagen synthesis fold over untreated samples are shown. Each error bar indicates standard deviation (SEM). [Diagram 5]FIG. 1 shows the stimulation of proteoglycan synthesis in human chondrocytes by collagen peptides of SEQ ID NO: 11 to SEQ ID NO: 24 according to the present invention compared to a mixture of collagen peptides of comparable average molecular weight obtained from natural sources (Fortigel). Quantitative measurements of collagen synthesis fold over untreated samples are shown. Each error bar indicates standard deviation (SEM). [Figure 6] FIG. 1 shows the stimulation of proteoglycan synthesis in human dermal fibroblasts by collagen peptides of SEQ ID NO: 11 to SEQ ID NO: 26 according to the present invention compared to a mixture of collagen peptides of comparable average molecular weight obtained from natural sources (Verisol). Quantitative measurements of collagen synthesis fold over untreated samples are shown. Each error bar indicates standard deviation (SEM). [Figure 7A] FIG. 2 shows the stimulation of collagen synthesis in human dermal fibroblasts by collagen peptides of SEQ ID NO: 27 to SEQ ID NO: 30 according to the present invention compared to a mixture of collagen peptides of comparable average molecular weight obtained from natural sources (Verisol). Quantitative measurements of fold collagen synthesis over untreated samples are shown. Each error bar indicates standard deviation (SEM). [Figure 7B] FIG. 2 shows the stimulation of proteoglycan synthesis in human dermal fibroblasts by collagen peptides of SEQ ID NO: 27 to SEQ ID NO: 30 according to the present invention compared to a mixture of collagen peptides of comparable average molecular weight obtained from natural sources (Verisol). Quantitative measurements of fold collagen synthesis over untreated samples are shown. Error bars indicate standard deviation (SEM). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS EXAMPLES

[0222] [Example 1] Solid-phase synthesis Collagen peptides having the amino acid sequences of SEQ ID NO:1 to SEQ ID NO:10 and SEQ ID NO:27 to SEQ ID NO:30 according to the present invention were obtained by solid phase synthesis (Merrifield synthesis) on polystyrene resin.

[0223] [Example 2] Recombinant production Furthermore, further collagen peptides, in particular collagen peptides of the amino acid sequences SEQ ID NO: 1 to SEQ ID NO: 10, were obtained by recombinant expression in Pichia pastoris.

[0224] Example 3: Bone health To analyze the biological effectiveness of the collagen peptide according to the invention with regard to the maintenance of bone health and the prevention and treatment of bone diseases, its stimulatory effect on the synthesis of extracellular matrix proteins and enzymes involved in matrix formation and mineralization by osteoblasts was investigated in vitro by measuring the expression levels of the corresponding mRNAs (relative to a control without collagen peptide) by real-time PCR and semi-quantitative evaluation.

[0225] For this, human osteoblasts were isolated by first incubating bone material from knee joints for 1 h at 37 °C under strong agitation in Hank's Balanced Salt Solution supplemented with 7 mg / ml hyaluronidase type I and III-S and 5 mg / ml pronase. Digestion is then continued for 3-5 h at 37 °C in Hank's Balanced Salt Solution supplemented with 16 mg / ml collagenase type CLS IV. After this enzymatic digestion, the obtained primary osteoblasts are cultured in Ham's F12 medium supplemented with 10% fetal bovine serum, 20 U / ml penicillin-streptomycin, 50 μg / ml partricin, 0.05 mg / ml ascorbic acid and 0.15 mg / ml glutamine. Alternatively, primary osteoblasts (product number C-12760 2019) can be purchased from PromoCell GmbH (Heidelberg, Germany) to investigate the biological efficacy. The cells are then cultured in Ham's F12 medium supplemented with 10% fetal bovine serum, 20 U / ml penicillin-streptomycin, 50 μg / ml partlysine and 0.15 mg / ml glutamine.

[0226] To examine the biological efficacy, monolayer cell cultures of isolated human osteoblasts are incubated for 24 hours in medium supplemented with 0.5 mg / ml of each collagen peptide, or in medium without the peptide as a control. Then, the expression levels of each mRNA are measured.

[0227] [Example 4] Skin health The stimulation of collagen (type I) and the synthesis of the proteoglycans biglycan and versican was investigated in human dermal fibroblasts (skin cells) in vitro. For this, cells were incubated for 24 h with 0.5 mg / ml of low molecular weight collagen peptides or collagen peptides according to the invention, respectively, and then the expression levels of collagen RNA, biglycan RNA and versican RNA were measured by real-time PCR and evaluated semi-quantitatively (relative to a control without peptides).

[0228] [Example 5] Cartilage health For cell culture, porcine or human chondrocytes were isolated from cartilage tissue by known methods and cultured at approximately 350,000 cells / cm. 2 The cells are seeded on a culture plate at a density of 1000 x 1000. The culture medium used is Ham's F12 medium containing 10% fetal bovine serum, 10 μg / ml gentamicin, and 5 μg / ml amphoteridine B. Instead of 10 μg / ml gentamicin, 10 μg / ml penicillin-streptomycin can also be used. The culture is maintained in an oxygen-reduced atmosphere (5% O 2 , 5% CO 2 and 90%N 2 ) at 37°C.

[0229] Measurement of collagen biosynthesis Quantification of collagen (basically type II) synthesized by chondrocytes was performed using the method 14 This is done by radiolabeling with C-proline.

[0230] First, radioactive 14C-proline is added and the chondrocytes are incubated under these conditions until the time point of measurement. 14 C-proline and unincorporated 14 To distinguish between C-proline and C-proline, the medium containing the isotope is replaced with pure medium for 3 days. The medium is then removed and distilled water is added to the attached cell layer to disrupt the cell membrane by osmotic stress and release unbound cytoplasmic 14 C-proline is released. The cell debris along with the synthesized extracellular matrix is ​​pelleted by centrifugation. The pellet is resuspended in fresh distilled water and mixed with xylene scintillation cocktail. Then, the cells are counted in a β-counter. 14 By detecting C-proline, the amount of collagen synthesized can be quantified.

[0231] Alternatively, quantification can be performed using the Sircol Collagen Assay Kit (product number 054S5000, 2019, tebu-bio, Offenbach, Germany or Biocolor Ltd., UK) according to the manufacturer's instructions (see Example 6).

[0232] Measurement of proteoglycan biosynthesis Proteoglycan synthesized by chondrocytes is quantified by Alcian blue staining and photometry of glycosaminoglycan (GAG), a component of proteoglycan.

[0233] To measure the GAG ​​content in cell cultures, first remove the culture medium and rinse the attached cell layer with PBS buffer (pH 7). Then fix the cells in a 10% formaldehyde solution in PBS for 2 h at 4 °C. After removing the formaldehyde, add Alcian blue staining reagent (5% Alcian blue in 3% acetic acid) to the cell layer and incubate overnight at 4 °C. Remove unbound Alcian blue and wash it off by carefully rinsing 3-4 times with PBS. Elute the GAG ​​complexes from the cell layer by adding an acidic guanidine solution (8 mol / l). The amount of glycosaminoglycans can then be quantified photometrically at a wavelength of 620 nm.

[0234] Alternatively, quantification can be performed using the Blyscan Glycosaminoglycan Assay Kit (product number 054B3000, 2019, tebu-bio, Offenbach, Germany or Biocolor Ltd., UK) according to the manufacturer's instructions (see Example 6).

[0235] [Example 6] Effect of the synthetic collagen peptide according to the present invention on the biosynthesis of matrix proteins Cell culture: The human cells used were obtained from tebu-bio GmbH (Offenbach, Germany). First, chondrocytes (catalog number 402-05a) or dermal fibroblasts (catalog number 106-05a) were seeded in 12-well culture plates and incubated at 37°C in Ham's F12 medium supplemented with 10% fetal bovine serum, 20 U / ml penicillin-streptomycin, and 50 μg / ml ascorbic acid at 5% CO. 2The medium was replaced with fresh medium every 2 days until the cells were 80% confluent. The following SEQ ID NO:2 (non-hydroxylated), SEQ ID NO:7 (hydroxylated), SEQ ID NO:8 (hydroxylated), SEQ ID NO:9 (hydroxylated), SEQ ID NO:10 (hydroxylated), SEQ ID NO:11 (non-hydroxylated), SEQ ID NO:12 (non-hydroxylated), SEQ ID NO:13 (non-hydroxylated), SEQ ID NO:14 (non-hydroxylated), SEQ ID NO:15 (non-hydroxylated), SEQ ID NO:16 (non-hydroxylated), SEQ ID NO:17 (hydroxylated), SEQ ID NO:18 (hydroxylated), SEQ ID NO:19 (hydroxylated), SEQ ID NO:20 (hydroxylated), SEQ ID NO:21 (hydroxylated), SEQ ID NO:22 (hydroxylated), SEQ ID NO:23 (hydroxylated), SEQ ID NO:24 (hydroxylated), SEQ ID NO:25 (hydroxylated), SEQ ID NO:26 (hydroxylated), SEQ ID NO:27 (hydroxylated), SEQ ID NO:28 (hydroxylated), SEQ ID NO:29 (hydroxylated), SEQ ID NO:30 (hydroxylated), SEQ ID NO:31 (hydroxylated), SEQ ID NO:32 (hydroxylated), SEQ ID NO:33 (hydroxylated), SEQ ID NO:34 (hydroxylated), SEQ ID NO:35 (hydroxylated), SEQ ID NO:36 (hydroxylated), SEQ ID NO:37 (hydroxylated), SEQ ID NO:38 (hydroxylated), SEQ ID NO:39 (hydroxylated), SEQ ID NO:30 (hydroxylated), SEQ ID NO:31 (hydroxylated), SEQ ID NO:32 (hydroxylated), SEQ ID NO:33 (hydroxylated), SEQ ID NO:34 (hydroxylated), SEQ ID NO:35 (hydroxylated), SEQ ID NO:36 (hydroxylated), SEQ ID NO:37 (hydroxylated), SEQ ID NO:38 (hydroxylated), SEQ ID NO To investigate the effect of collagen peptides of SEQ ID NO:20 (hydroxylated), SEQ ID NO:21 (hydroxylated), SEQ ID NO:22 (hydroxylated), SEQ ID NO:23 (hydroxylated), SEQ ID NO:24 (hydroxylated), SEQ ID NO:25 (hydroxylated), SEQ ID NO:26 (hydroxylated), SEQ ID NO:27 (hydroxylated), SEQ ID NO:28 (hydroxylated), SEQ ID NO:29 (hydroxylated), and SEQ ID NO:30 (hydroxylated) on matrix protein biosynthesis, the cell culture medium was replaced with a specific stimulation medium supplemented with 0.5 mg / ml of the specific collagen peptide.

[0236] Collagen Assay: To examine collagen metabolism, cells were stimulated with 0.5 mg / ml of BCP for 3 weeks, and then the amount of newly synthesized collagen was measured. The synthesized collagen was isolated using Sircol-Assay (product number 054S5000, 2019, tebu-bio (Offenbach, Germany) or Biocolor Ltd. (UK)) according to the manufacturer's instructions. Briefly, first, the medium was removed from each, and the attached cell layer was digested at 4 °C overnight using 0.1 mg of pepsin solution in 0.5 M acetic acid. 100 μl of acid-neutralizing reagent was added to neutralize the cell suspension. Next, the synthesized collagen was separated by adding 200 μl of isolation / enrichment solution and shaking vigorously overnight at 4 °C. After centrifugation (12,000 rpm, 10 minutes) to remove the supernatant, the isolated collagen was resuspended in 1 ml of Sircol dye solution. After 30 minutes of shaking and further centrifugation, 750 μl of cold solid acid wash reagent was overlaid on the collagen pellet. After centrifugation again, the supernatant was removed, and the enriched collagen was taken up in 250 μl of alkaline solution. Using 200 μl of each sample solution, the quantification of the synthesized collagen was performed photometrically. The absorbance was measured at a wavelength of 492 nm. The amount of synthesized collagen was determined for each against a standardized collagen solution.

[0237] Compared to untreated controls, an increase in collagen synthesis by dermal fibroblasts can be observed, which were incubated with the collagen peptides of hydroxylated SEQ ID NO: 7 (42% increase), SEQ ID NO: 8 (37% increase), SEQ ID NO: 9 (38% increase), SEQ ID NO: 10 (35% increase), SEQ ID NO: 17 (42% increase), SEQ ID NO: 18 (40% increase), SEQ ID NO: 19 (40% increase), SEQ ID NO: 20 (37% increase), SEQ ID NO: 21 (36% increase), SEQ ID NO: 22 (43% increase), SEQ ID NO: 23 (39% increase), SEQ ID NO: 24 (36% increase), SEQ ID NO: 25 (27% increase), SEQ ID NO: 26 (12% increase), SEQ ID NO: 27 (27% increase), SEQ ID NO: 28 (22% increase), SEQ ID NO: 29 (21% increase) and SEQ ID NO: 30 (21% increase) according to the invention (Figures 1A, 4, 7A). Furthermore, incubation of dermal fibroblasts with non-hydroxylated collagen peptides according to the invention was found to result in at least the same stimulation of collagen synthesis as incubation of the cells with a mixture of hydroxylated collagen peptides of comparable average molecular weight obtained from natural sources (Verisol) (SEQ ID NO:2 (22% increase), SEQ ID NO:11 (19% increase), SEQ ID NO:12 (22% increase), SEQ ID NO:13 (20% increase), SEQ ID NO:14 (22% increase), SEQ ID NO:15 (23% increase) and SEQ ID NO:16 (22% increase)). All collagen peptides according to the invention investigated showed a significant increase in collagen synthesis compared to the gelatin controls (gelatin 160, gelatin 260).

[0238] Also, an increase in collagen synthesis was observed for chondrocytes incubated with collagen peptides of the invention compared to untreated controls. Incubation of chondrocytes with hydroxylated collagen peptides increased collagen synthesis by 28% (SEQ ID NO: 7), 24% (SEQ ID NO: 8, 9), 20% (SEQ ID NO: 10), 33% (SEQ ID NO: 17), 32% (SEQ ID NO: 18), 37% (SEQ ID NO: 19), 30% (SEQ ID NO: 20), 55% (SEQ ID NO: 21), 46% (SEQ ID NO: 22), 19% (SEQ ID NO: 23, 25), 26% (SEQ ID NO: 24) and 12% (SEQ ID NO: 26). Incubation of chondrocytes with non-hydroxylated collagen peptides according to the invention resulted in stimulation of collagen synthesis at least equivalent to that obtained by incubating cells with a mixture of hydroxylated collagen peptides of equivalent average molecular weight from natural sources (Fortigel) (Figure 1B, Figure 3). Here, incubation of chondrocytes with non-hydroxylated collagen peptides increased collagen synthesis by 16% (SEQ ID NO:2, 11), 24% (SEQ ID NO:12), 15% (SEQ ID NO:13), 28% (SEQ ID NO:14), and 31% (SEQ ID NO:15, 16).

[0239] Proteoglycan Assay: For the measurement of proteoglycans, Blyscan-Glycosaminoglycan-Assay (product no. 054B3000, 2019, tebu-bio (Offenbach, Germany) or Biocolor Ltd. (UK)) was used. Here, the biosynthesis of proteoglycans after 2 weeks of stimulation of dermal fibroblasts was investigated. Following the manufacturer's instructions, after removing the medium, the cell layer was overlaid with 1 ml of papain extract and incubated at 65 °C for 3 h with vigorous shaking. The cell suspension was then centrifuged (10000g, 10 min) and the supernatant was collected. After adding 1 ml of Blyscan dye solution and shaking (30 min), the supernatant was centrifuged again (12000 rpm, 10 min) and then removed. The isolated proteoglycan pellet was resuspended in 500 μl of dissociation solution. The amount of synthesized proteoglycan in each 200 μl sample solution was measured photometrically at a wavelength of 656 nm in comparison with an untreated control.

[0240] As is evident from Figures 2A, 6 and 7B, when fibroblasts are incubated with collagen peptides according to the present invention, the synthesis of proteoglycan by dermal fibroblasts is significantly increased compared to untreated controls.For example, incubation of cells with hydroxylated collagen peptides increases proteoglycan synthesis by 41% (SEQ ID NO:7), 23% (SEQ ID NO:8, 25), 27% (SEQ ID NO:9, 21), 25% (SEQ ID NO:10), 24% (SEQ ID NO:17), 31% (SEQ ID NO:18), 29% (SEQ ID NO:19, 20), 28% (SEQ ID NO:22), 22% (SEQ ID NO:23, 28, 30), 26% (SEQ ID NO:24, 27), 6% (SEQ ID NO:26) and 21% (SEQ ID NO:29). Furthermore, it was found that incubation of dermal fibroblasts with non-hydroxylated collagen peptides according to the invention results in a stimulation of proteoglycan synthesis at least equivalent to that obtained by incubating the cells with a mixture of hydroxylated collagen peptides of comparable average molecular weight obtained from natural sources (Verisol). Here, incubation of dermal fibroblasts with non-hydroxylated collagen peptides increased collagen synthesis by 18% (SEQ ID NO: 2, 11, 15, 16), 19% (SEQ ID NO: 12), 16% (SEQ ID NO: 13) and 14% (SEQ ID NO: 14). A significant increase in proteoglycan synthesis could be observed for all collagen peptides according to the invention investigated, compared to the gelatin controls (gelatin 160, gelatin 260).

[0241] Chondrocytes incubated with collagen peptides according to the invention also showed an increase in proteoglycan synthesis compared to untreated controls (Figures 2B and 5). Incubation of chondrocytes with hydroxylated collagen peptides increased collagen synthesis by 30% (SEQ ID NO: 7), 20% (SEQ ID NO: 8), 19% (SEQ ID NO: 9), 29% (SEQ ID NO: 10), 31% (SEQ ID NO: 17), 25% (SEQ ID NO: 18), 18% (SEQ ID NO: 19), 15% (SEQ ID NOs: 20, 24), 22% (SEQ ID NOs: 21), 26% (SEQ ID NOs: 22, 24) and 11% (SEQ ID NO: 23). Incubation of chondrocytes with non-hydroxylated collagen peptides according to the invention resulted in a stimulation of proteoglycan synthesis at least equivalent to that obtained by incubating the cells with a mixture of hydroxylated collagen peptides of equivalent average molecular weight obtained from natural sources (Fortigel) (Figures 5, 2B). Incubation of chondrocytes with non-hydroxylated collagen peptides increased proteoglycan synthesis by 22% (SEQ ID NO:2), 28% (SEQ ID NO:11), 23% (SEQ ID NO:12), 9% (SEQ ID NO:13), 14% (SEQ ID NO:14), 6% (SEQ ID NO:15), and 18% (SEQ ID NO:16).

Claims

1. 1. A synthetic or recombinant collagen peptide for use in a method for the prevention and / or treatment of a bone disease, a cartilage disease, a degenerative joint disease, a tendon or ligament disease, and / or a skin disease, comprising: the collagen peptide has a molecular weight in the range of 0.18 to 10.0 kDa; The collagen peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-30, or an amino acid sequence having at least 90% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-30; The collagen peptides can stimulate the synthesis of extracellular matrix proteins in connective tissue cells. Synthetic or recombinant collagen peptides.

2. 2. The collagen peptide of claim 1, wherein the collagen peptide has the amino acid sequence of SEQ ID NO:1 in which at least one proline residue is hydroxylated.

3. The collagen peptide of claim 1, wherein the collagen peptide has an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-30.

4. Collagen peptide according to any one of claims 1 to 3, wherein the connective tissue cells are osteoblasts, chondrocytes and / or fibroblasts.

5. The collagen peptide of claim 1, wherein the collagen peptide has no amino acid modifications.

6. 5. The collagen peptide of claim 1, wherein the collagen peptide has a hydroxylated proline, a hydroxylated lysine, or a hydroxylated proline and a hydroxylated lysine.

7. 7. The collagen peptide of claim 1, wherein the collagen peptide is glycosylated on at least one hydroxylated lysine.

8. A pharmaceutical composition comprising a collagen peptide according to any one of claims 1 to 7 and at least one pharma- ceutical acceptable additive.

9. A nutritional supplement, food or luxury item comprising a collagen peptide according to any one of claims 1 to 7 and at least one additive acceptable for food.

10. A cosmetic comprising a collagen peptide according to any one of claims 1 to 7 and at least one skin-compatible additive.

11. 1. A composition for use in a non-therapeutic method for maintaining and / or improving bone health, for maintaining and / or improving skin health, for maintaining and / or improving intestinal health, for maintaining and / or improving vasculature, for maintaining and / or improving cardiovascular health, for maintaining and / or improving periodontal tissues, and / or for maintaining and / or improving nail and hair health, comprising The composition comprises at least one synthetic or recombinant collagen peptide having a molecular weight in the range of 0.18 to 10.0 kDa; The recombinant collagen peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-30, or an amino acid sequence having at least 90% sequence identity with an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-30, and is capable of stimulating the synthesis of extracellular matrix proteins in connective tissue cells. composition.

12. 1. A composition for use in a non-therapeutic method for visually and structurally improving skin, comprising: The composition comprises at least one synthetic or recombinant collagen peptide having a molecular weight in the range of 0.18 to 10.0 kDa; The recombinant collagen peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-30, or an amino acid sequence having at least 90% sequence identity with an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-30, and is capable of stimulating the synthesis of extracellular matrix proteins in connective tissue cells. composition.

13. 13. The composition of claim 12, wherein the method is for reducing the formation of wrinkles, for improving skin elasticity, for increasing skin firmness, for increasing skin hydration, for reducing cellulite, and / or for reducing stretch marks.

14. 1. A composition for use in a non-therapeutic method for promoting nail growth and / or reducing nail brittleness, comprising: The composition comprises at least one synthetic or recombinant collagen peptide having a molecular weight in the range of 0.18 to 10.0 kDa; The recombinant collagen peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-30, or an amino acid sequence having at least 90% sequence identity with an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-30, and is capable of stimulating the synthesis of extracellular matrix proteins in connective tissue cells. composition.

15. 1. A composition for use in a non-therapeutic method for visually and structurally improving hair, comprising: The composition comprises at least one synthetic or recombinant collagen peptide having a molecular weight in the range of 0.18 to 10.0 kDa; The recombinant collagen peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-30, or an amino acid sequence having at least 90% sequence identity with an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-30, and is capable of stimulating the synthesis of extracellular matrix proteins in connective tissue cells. composition.

16. 16. The composition of claim 15, wherein the method is for improving hair quality, for reducing split ends, and / or for reducing or slowing hair loss.

17. A method for recombinantly producing a collagen peptide according to any one of claims 1 to 9, comprising the steps of: a) providing an expression system comprising at least one expression cassette, said expression cassette comprising at least one nucleotide sequence encoding a collagen peptide having a molecular weight in the range of 0.18-10 kDa and comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-30, or an amino acid sequence having at least 90% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-30; b) method steps of culturing said expression system under conditions allowing expression of said collagen peptide; c) recovering said collagen peptides; A method comprising:

18. 18. The method of claim 17, wherein the expression system is a host cell selected from the group consisting of a bacterial cell, a yeast cell, a fungal cell, a mammalian cell, an insect cell and a plant cell.

19. 19. The method of claim 17 or 18, wherein the collagen peptide encoded by the nucleotide sequence is a vertebrate collagen peptide.

20. 20. The method of any one of claims 17 to 19, wherein the expression system is a host cell capable of hydroxylating proline, lysine or proline and lysine residues of the expressed collagen peptide.

21. 21. The method of any one of claims 17 to 20, wherein the expression system is a host cell, the host cell comprising at least one expression cassette comprising a polynucleotide sequence encoding prolyl-4 hydroxylase, and recovering in vivo hydroxylated collagen peptides.

22. 22. The method according to any one of claims 17 to 21, wherein the expression system is a host cell, the host cell having at least one expression cassette comprising a polynucleotide sequence encoding a lysyl hydroxylase, and recovering hydroxylated collagen peptides in vivo.

23. 20. The method of any one of claims 17 to 19, wherein the expression system is not capable of causing hydroxylation of proline, lysine or proline and lysine residues of the expressed collagen peptides.

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