EP4313299A1 - Rekombinantes typ ii-kollagen zur therapeutischen verwendung - Google Patents

Rekombinantes typ ii-kollagen zur therapeutischen verwendung

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Publication number
EP4313299A1
EP4313299A1 EP22719203.6A EP22719203A EP4313299A1 EP 4313299 A1 EP4313299 A1 EP 4313299A1 EP 22719203 A EP22719203 A EP 22719203A EP 4313299 A1 EP4313299 A1 EP 4313299A1
Authority
EP
European Patent Office
Prior art keywords
collagen
type
recombinant
recombinant type
peptide
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
EP22719203.6A
Other languages
German (de)
English (en)
French (fr)
Inventor
Stephan Hausmanns
Hans-Ulrich Frech
Steffen Oesser
Martin Hahn
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Gelita AG
Original Assignee
Gelita AG
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Gelita AG filed Critical Gelita AG
Publication of EP4313299A1 publication Critical patent/EP4313299A1/de
Pending legal-status Critical Current

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/78—Connective tissue peptides, e.g. collagen, elastin, laminin, fibronectin, vitronectin or cold insoluble globulin [CIG]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A61P19/02—Drugs for skeletal disorders for joint disorders, e.g. arthritis, arthrosis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/39—Connective tissue peptides, e.g. collagen, elastin, laminin, fibronectin, vitronectin, cold insoluble globulin [CIG]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0053—Mouth and digestive tract, i.e. intraoral and peroral administration
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/80—Vectors or expression systems specially adapted for eukaryotic hosts for fungi
    • C12N15/81—Vectors or expression systems specially adapted for eukaryotic hosts for fungi for yeasts
    • C12N15/815—Vectors or expression systems specially adapted for eukaryotic hosts for fungi for yeasts for yeasts other than Saccharomyces

Definitions

  • the present invention relates to recombinant type II collagen for use in a therapeutic method for the oral therapy of cartilage diseases in a human or animal body.
  • Collagen is an extracellular structural protein found in animals such as mammals, birds and fish. It is usually found there in the connective tissue, particularly as a component of the extracellular matrix. Tendons, ligaments, cartilage and bones are particularly rich in collagen. In contrast, collagens are not naturally found in plants and unicellular organisms.
  • Collagens occur in different, structurally and functionally different types and differ in terms of their structure, function and origin, among other things.
  • the polypeptide chains building up the collagen are synthesized individually in the cell on the ribosomes of the endoplasmic reticulum in the form of larger precursor molecules and have extensive repetitive (Gly-X-Y)n sequences, where X and Y can be any amino acid, but mostly proline and 4- are hydroxyproline.
  • precursor polypeptide chains are post-translationally hydroxylated to proline and fysine residues of the polypeptide chain in the endoplasmic reticulum to form hydroxyproline and hydroxylysine residues.
  • the hydroxylation serves to stabilize neighboring collagen polypeptide chains of the right-hand triple helix formed in the cell, each consisting of three of the precursor polypeptide chains (procollagen).
  • the procollagen formed in this way is glycosylated intracellularly, secreted from the cell in the glycosylated triple-helical form (tropocollagen) and then mature collagen is formed by peptidase-mediated cleavage of the terminal residues. As part of a fibrillogenesis process, this accumulates to form collagen fibrils, which are then covalently cross-linked and form collagen fibers.
  • Collagen is also often used in denatured form, sometimes also referred to as gelatine, or in the form of its hydrolyzates. If gelatine or collagen is subjected to hydrolytic processes, in particular an enzymatic hydrolysis, to obtain collagen peptides, collagen hydrolysates with a wide variety of compositions and application profiles can be produced depending on the collagen type used and origin as well as the enzymatic conditions. However, these collagen hydrolysates represent a mixture of peptides whose molecular weights are distributed over certain size ranges. The use of such collagen hydrolyzates, for example as a dietary supplement or as a cosmetic aid, has been known for a long time, among other things for the prevention and/or treatment of complaints associated with the bones, the joints or the connective tissue.
  • WO 2012/065782 describes collagen hydrolyzates obtained from pigskin gelatine, which are used to stimulate the biosynthesis of extracellular matrix proteins by skin cells and are particularly suitable for cosmetic purposes.
  • WO 2012/117012 discloses enzymatically hydrolyzed collagen from bovine splits with an average molecular weight of 1500 to 8000 Da, which can be used together with a prebiotic to prevent and/or treat osteoporosis.
  • collagen hydrolysates obtained from animal materials has advantages for many applications and consumer groups, the use of collagen hydrolysates obtained in this way can also be less desirable with regard to certain consumer groups and application profiles.
  • Certain consumer groups are fundamentally critical or hostile to raw materials derived from animal materials, whether they fear contamination with harmful microorganisms or agents, such as processing aids, or unwanted immune reactions, or for religious or ethical reasons.
  • the manufacturing processes used to obtain collagen hydrolyzates obtained from animal materials often include complex and expensive disruption, purification and further processing steps.
  • WO 2006/052451 A2 discloses the production of recombinant type III collagen in Pichia pastoris strains which also express human prolyl hydroxylases.
  • WO 2005/012356 A2 discloses the production of gelatin from human collagen type I and individual 50 kDa, 65 kDa and 100 kDa collagen peptide species in fully hydroxylated, partially and non-hydroxylated forms, respectively.
  • Olsen et al. Provides the recombinant production of an 8.5 kDa collagen peptide species from the a1 chain of human collagen in Pichia pastoris.
  • WO 01/34646 A2 also discloses the production of individual recombinant gelatin species with a defined molecular weight of up to 350 kDa resulting from the recombinant production route, which can be present in non-hydroxylated, partially or completely hydroxylated form.
  • Type II collagen is a collagen that occurs specifically in cartilage tissue and is usually present there in the form of a homotrimer of a1 chains.
  • the production of recombinant type II collagen peptides, their hydroxylation by means of a prolyl-4-hydroxylase and their hydrogenation and formation into procollagen and the associated triple helix formation is described, for example, in US Pat. No. 5,593,859.
  • US Pat. No. 5,399,347 describes the oral administration of water-soluble, highly purified type II collagen from natural sources for the treatment of arthritis.
  • the production of this collagen is extremely difficult, expensive and also susceptible to contamination, in particular from microbes.
  • US 7,083,820 and EP 1 435 906 disclose the use of methods for obtaining animal tissue-containing compositions containing non-denatured type II collagen, with special process steps eliminating microbial contaminants, but at the same time the original, non-denatured form of the Type II collagen should be preserved.
  • the present invention is therefore based on the technical problem of providing type II collagen that overcomes the aforementioned disadvantages, in particular that can be produced in a standardized, reliable and precisely defined form, even on a larger industrial and cost-effective scale, and because of its biological effectiveness for use in a method for the oral therapy of cartilage diseases in the human or animal body and/or for maintaining cartilage health.
  • the present invention solves the technical problem on which it is based by providing the teachings of the independent patent claims, in particular also the teachings of the preferred embodiments in the description and the dependent patent claims.
  • the present invention relates to a recombinant type II collagen, in particular a non-denatured recombinant type II collagen, for use in a therapeutic method for the oral therapy of cartilage diseases in a human or animal patient.
  • the present invention is based on the surprising teaching that recombinantly produced type II collagen, in particular also in isolated form, is able to treat cartilage diseases after oral administration.
  • recombinantly produced type II collagen in particular also in isolated form
  • the materials and process steps required in the prior art for the production of therapeutically effective type II collagen compositions in particular the use of natural animal cartilage tissue and the use of certain process steps to maintain the nativity of the collagen present in this starting tissue, are not used or not carried out and in addition the secondary components present in the starting material in the according to the invention provided recombinantly produced type II collagen are not present, a biological effectiveness, in particular a surprisingly high biological effectiveness of the recombinantly produced type II collagen according to the present procedure could be shown.
  • the recombinant type II collagen provided according to the invention in particular recombinant type II collagen peptides, is able to exhibit a biological effectiveness, in particular one that is at least the same as that of type II collagen obtained from natural sources, in particular it is even provided improved biological effectiveness.
  • the present invention enables cartilage diseases in human or animal patients due to the high biological effectiveness of the recombinant type II collagen, in particular type II collagen peptides, in very low doses, ie low concentrations of type II collagen, in particular type II collagen peptide to treat.
  • the recombinant type II collagens provided according to the invention are capable of treating immune-modulated cartilage diseases, in particular an autoimmune disease, in particular polychondritis or rheumatoid arthritis.
  • an immunomodulated cartilage disease is a disease characterized by immune intolerance.
  • the present invention also relates to recombinant type II collagen, in particular recombinant type II collagen peptides, for the treatment of cartilage diseases, which can be inflammatory and/or degenerative cartilage diseases, in particular rheumatoid arthritis or arthrosis.
  • the present invention also relates to recombinant type II collagen, in particular recombinant type II collagen peptides for inducing oral tolerance, in particular for inducing oral tolerance to endogenous collagen, in particular endogenous type II collagen, in particular endogenous type II collagen present in cartilage tissue .
  • the present invention relates to recombinant type II collagen, particularly recombinant type II collagen peptides, and compositions comprising recombinant type II collagen, particularly recombinant type II collagen peptides, for use in a method of therapeutically treating or therapeutically preventing immune intolerance to collagen type II, particularly by inducing immune tolerance to type II collagen.
  • the present invention also relates to recombinant type II collagen, in particular recombinant type II collagen peptides for use in a method of inducing immune tolerance to type II collagen, in particular a composition the administration of which results in the induction of oral tolerance to type II collagen.
  • the teaching of the present invention therefore advantageously provides a reproducible, biotechnologically producible recombinant type II collagen for the oral therapy of cartilage diseases, which can be produced in a standardized manner, can be produced on an industrial scale, and which is free of contamination in high purity and yield without the limitation can be produced by natural starting materials and is characterized in particular due to its high biological effectiveness in that it can be used in small doses.
  • the recombinant type II collagens used according to the invention are characterized by a biological activity which unfolds after oral administration in human or animal bodies, in particular an immune-modulating and/or inflammation-modulating biological activity.
  • this biological activity is present in particular for full-length recombinant type II collagens present in non-denatured, i.e. native, form, but in a preferred embodiment also for collagen peptides with a shortened length present in the form of recombinant type II collagen peptides.
  • type II collagens provided according to the invention are preferably capable of immunomodulation and/or induction of oral tolerance, in particular they bring about an immune response and/or induction of oral tolerance in the treated human or animal body.
  • the recombinant type II collagens provided according to the invention preferably develop a biological activity that suppresses the synthesis of immunoglobulins and/or an anti-inflammatory biological activity.
  • the reduction per inflammatory and the stimulation of anti-inflammatory cytokines could be determined.
  • the orally administered recombinant type II collagen provided according to the invention in particular recombinant type II collagen peptides, survives the gastrointestinal passage completely or largely undamaged and triggers immune-modulating and/or cytokine-regulating reactions and/or signaling cascades in immunomodulating, in particular immunosuppressor cells, in particular cells of Peyer's plaque, which trigger undesirable immune reactions and inflammatory processes in the area of cartilage , especially articular cartilage, reduce or completely prevent.
  • endogenous type II collagen or fragments thereof which is present in or on damaged or degenerated cartilage tissue, can induce an autoimmune reaction which leads to inflammation, immunoglobulin formation and degenerative and destructive processes in the cartilage, which lead to further cartilage destruction and degeneration contribute and ultimately contribute to the appearance of osteoarthritis, rheumatoid arthritis and similar diseases.
  • the oral administration of recombinant type II collagen provided according to the invention, in particular type II collagen peptides appears to bring about oral tolerance to endogenously present type II collagen which triggers such undesirable reactions, so that treatment of cartilage diseases is made possible.
  • the recombinant type II collagens provided according to the invention are preferably equipped with the ability to interact with cells of the treated human or animal patient, in particular with cells of Peyer's plaque, and in particular to stimulate anti-inflammatory and lead to inhibition of pro-inflammatory cytokines and inhibition of immunoglobulins.
  • the recombinant type II collagens provided according to the invention in particular recombinant type II collagen peptides, preferably show an inducing effect on the differentiation of peripheral blood monocytes into immunosuppressive M2 macrophages.
  • the recombinant type II collagens provided according to the invention lead to a reduction in the synthesis of inflammatory cytokines, in particular TNFa and IFNY, and/or to an induction of the synthesis of anti-inflammatory cytokines, especially IL IO.
  • the recombinant type II collagens provided according to the invention lead to a stimulation/induction of the differentiation of na ⁇ ve CD4 + T Progenitor cells to T suppressor cells.
  • the stimulation/induction of the differentiation of na ⁇ ve CD4 + T progenitor cells into T suppressor cells particularly preferably leads to an increased release of anti-inflammatory cytokines, preferably IL-10, IL-4 and/or TGF- ⁇ .
  • the recombinant type II collagens provided according to the invention bring about reduced expression of pro-inflammatory cytokines, preferably of IL-lß, TNFa and/or IL-6, by chondrocytes, particularly by articular chondrocytes.
  • the recombinant type II collagen is present in a non-denatured, ie native, form.
  • the recombinant type II collagen is in triple-helical form.
  • the present invention provides a recombinant type II collagen, which can be present in the form of a type II collagen peptide, i.e. in single-stranded form, or in multi-stranded, for example two- or three-stranded form, here also as triple helical form, in particular in the form of a type II procollagen or mature type II collagen, in particular in the form of a homotrimer of type II al chains.
  • the recombinant type II collagen according to the invention is not present as a single-stranded type II collagen peptide, but in a triple-helical form, for example, one or all of the individual collagen peptides that build the triple-helical form of the recombinant type II collagen can, according to of the present invention.
  • the statements on recombinant type II collagen peptides disclosed in the present teaching also apply to type II collagens which have one, two or three such single-stranded type II collagen peptides, in particular are completely composed of them, in particular of the recombinant type according to the invention II collagen peptides.
  • the recombinant type II collagen can be bovine type II collagen (type IIB collagen). In a further particularly preferred embodiment, the recombinant type II collagen can be present in the form of type II procollagen or mature type II collagen.
  • the recombinant type II collagen can be in triple-helical form, in particular in the form of a homotrimer of three type II-al chains.
  • the recombinant type II collagen is in non-denatured form, also referred to here as native form, ie it has the naturally occurring tertiary and quaternary protein structure.
  • the recombinant type II collagen can be present in the form of crosslinked or non-crosslinked fibrils.
  • the recombinant type II collagen in particular type II collagen peptide, can be a full-length collagen peptide, ie it can have the complete amino acid sequence of a naturally occurring type II collagen peptide.
  • the recombinant type II collagen is in the form of a type II collagen peptide.
  • the recombinant type II collagen, in particular type II collagen peptide can be a type II collagen peptide with a molecular weight in the range from 5 to 400 kDa, in particular 10 to 390 kDa, in particular 10 to 350 kDa, in particular 10 to 300 kDa, in particular 10 to 110 kDa, in particular 40 to 110 kDa, in particular 40 to 100 kDa, in particular 11 to 105 kDa, in particular 15 to 100 kDa, in particular 20 to 99 kDa, in particular 25 to 95 kDa, in particular 30 to 95 kDa, especially 35 to 95 kDa.
  • the recombinant type II collagen, in particular collagen peptide preferably has a molecular weight in a range from 40 to 50 kDa, in particular 45 kDa.
  • the recombinant type II collagen peptide can be present in a triple-helical form.
  • the recombinant type II collagen, in particular the recombinant type II collagen peptide, of the present invention is complete or partial hydroxylated, fully or partially glycolized, or fully or partially hydroxylated and glycolized.
  • the recombinant type II collagen, in particular the recombinant type II collagen peptide, according to the present invention is a non-hydroxylated type II collagen, in particular type II collagen peptide.
  • the recombinant type II collagen, in particular the recombinant type II collagen peptide, according to the present invention is a hydroxylated type II collagen, in particular type II collagen peptide.
  • the recombinant type II collagen in particular the recombinantly produced type II collagen peptide, preferably has hydroxylated proline and/or hydroxylated lysine.
  • the recombinant type II collagen in particular the recombinant type II collagen peptide, is a non-hydroxylated, partially hydroxylated or fully hydroxylated type II collagen, in particular type II collagen peptide.
  • the recombinant type II collagen in particular the recombinant type II collagen peptide
  • the recombinant type II collagen, in particular the recombinant type II collagen peptide is preferably glycosylated on at least one hydroxylated lysine.
  • any hydroxylated lysine of the recombinant type II collagen, particularly the recombinant type II collagen peptide is glycosylated.
  • the recombinant type II collagen in particular recombinant type II collagen peptide, has no amino acid modification, in particular no hydroxylation.
  • the recombinant type II collagen, in particular type II collagen peptide particularly preferably has no hydroxylated and/or glycosylated amino acids.
  • the type II collagen according to the invention in particular type II collagen peptide, preferably has a type II collagen from vertebrates, in particular from fish, amphibians, reptiles, birds and mammals, in particular in pigs, sheep, cattle, rodents, kangaroos, horses or from invertebrates, in particular jellyfish, occurring amino acid sequence, in particular an amino acid sequence occurring in type II collagen from bovine.
  • the type II collagen, in particular type II collagen peptide particularly preferably comprises the amino acid sequence according to SEQ ID No. 2.
  • the type II collagen, in particular type II collagen peptide preferably consists of the amino acid sequence according to SEQ ID No. 2.
  • the type II collagen in particular type II collagen peptide, has at least 80%, preferably at least 85%, preferably at least 90%, preferably at least 95%, preferably at least 96%, preferably at least 97% preferably at least 98%, preferably at least 99%, sequence identity with the amino acid sequence according to SEQ ID No. 2.
  • the type II collagen, in particular type II collagen peptide comprises the amino acid sequence as shown in SEQ ID No. 4.
  • the type II collagen, in particular type II collagen peptide preferably consists of the amino acid sequence as shown in SEQ ID #4
  • the type II collagen in particular type II collagen peptide, has at least 80%, preferably at least 85%, preferably at least 90%, preferably at least 95%, preferably at least 96%, preferably at least 97% preferably at least 98%, preferably at least 99%, sequence identity with the amino acid sequence according to SEQ ID No. 4.
  • the amino acid sequence of the recombinant type II collagen, in particular type II collagen peptide is preferably the amino acid sequence of a naturally occurring type II collagen, in particular type II collagen peptide.
  • the amino acid sequence of the recombinant type II collagen, especially type II collagen peptide is the amino acid sequence of a naturally non-occurring type II collagen, especially type II collagen peptide.
  • the amino acid sequence of the recombinant type II collagen, in particular type II collagen peptide is preferably the amino acid sequence of a genetically modified type II collagen, in particular type II collagen peptide.
  • the type II collagen according to the invention in particular type II collagen peptide, particularly preferably has an amino acid sequence occurring in non-human collagen, in particular in non-human type II collagen peptides, preferably in the a1 chain of non-human type II collagen , in particular an amino acid sequence occurring in bovine, porcine, equine, ovine, piscine or avian collagen, in particular an amino acid sequence occurring in bovine collagen.
  • the recombinant type II collagen, in particular the recombinant type II collagen peptide is collagenase-resistant, in particular resistant to digestion by human collagenases.
  • the recombinant type II collagen in particular type II collagen peptide, is capable of inducing oral tolerance, in particular against endogenously present collagen, in particular endogenously present type II collagen, in particular in cartilage tissue endogenous type II collagen.
  • the recombinant type II collagen in particular type II collagen peptide, is capable of suppressing the synthesis of immunoglobulins.
  • the recombinant type II collagen in particular type II collagen peptide, is capable of suppressing the synthesis of pro-inflammatory cytokines.
  • the recombinant type II collagen in particular type II collagen peptide, is capable of stimulating the synthesis of anti-inflammatory cytokines.
  • the recombinant type II collagen in particular type II collagen peptide, is capable of suppressing the synthesis of immunoglobulins, suppressing the synthesis of pro-inflammatory cytokines and synthesis of anti-inflammatory ones to stimulate cytokines.
  • the recombinant type II collagen, in particular type II collagen peptide, for use in a therapeutic method for the oral therapy of cartilage diseases in a human or animal patient has a molecular weight in the range from 5 to 400 kDa, in particular 10 to 390 kDa, in particular 10 to 350 kDa, in particular 10 to 300 kDa, in particular 10 to 110 kDa, in particular 40 to 110 kDa, in particular 40 to 100 kDa, in particular 11 to 105 kDa, in particular 15 to 100 kDa, in particular 20 to 99 kDa, in particular 25 to 95 kDa, in particular 30 to 95 kDa, in particular 35 to 95 kDa.
  • the recombinant type II collagen preferably has a molecular weight in a range from 40 to 50 kDa, in particular 45 kDa.
  • the recombinant type II collagen according to the invention in particular type II collagen peptide, is used alone, ie in isolated form, ie without other substances, in the use provided according to the invention.
  • the recombinant type II collagen according to the invention in particular type II collagen peptide, is present as a homogeneous preparation, in particular as a homogeneous preparation of a single recombinant type II collagen, in particular type II collagen peptide, with a uniform molecular weight.
  • the recombinant type II collagen according to the invention in particular type II collagen peptide, is used as the only substance having biological activity in the use provided according to the invention.
  • the present invention relates to at least one recombinant type II collagen, in particular at least one recombinant type II collagen peptide, having compositions which, in addition to the at least one recombinant type II collagen, in particular the at least one recombinant type II collagen peptide, and optionally a pharmaceutically acceptable and/or food-compatible carrier, contain no other substances.
  • the composition containing at least one recombinant type II collagen, in particular at least one recombinant type II collagen peptide is present in a dosage form suitable for oral administration in a human or animal body.
  • the present invention also relates to a composition
  • a composition comprising at least one recombinant type II collagen, in particular at least one recombinant type II collagen peptide, according to the present invention and at least one pharmaceutically acceptable and/or food-compatible carrier and, optionally, at least one additive or excipient for Use in a therapeutic method for the oral therapy of cartilage diseases.
  • the present invention therefore also relates to a composition for use in a method for the therapeutic prophylaxis or therapeutic treatment of Immune intolerance responses to type II collagen, particularly endogenous type II collagen, through the induction of oral tolerance to type II collagen, particularly endogenous type II collagen.
  • the present invention also relates to a composition for use in a method of inducing oral tolerance to type II collagen, in particular endogenous type II collagen, which composition results in the induction of oral tolerance in the human or animal body.
  • the present invention also relates to a composition
  • a composition comprising recombinant type II collagen, in particular recombinant type II collagen peptides, in particular pharmaceutical compositions or dietary supplements, or food or beverages, for use in inducing oral tolerance to type II collagen, in particular endogenous type II -Collagen.
  • compositions according to the invention for oral administration can in particular be pharmaceutical compositions, dietary supplements or foodstuffs and semi-luxury foods.
  • the compositions according to the invention are pharmaceutical compositions.
  • the compositions according to the invention are food supplements.
  • the present invention relates in particular to a pharmaceutical composition
  • a pharmaceutical composition comprising a type II collagen according to the invention, in particular type II collagen peptide, and at least one pharmaceutically acceptable carrier, and the pharmaceutical composition for use in a method for the therapeutic treatment of cartilage diseases in the human or animal body.
  • the pharmaceutical composition according to the invention is particularly advantageously administered, for example, in the form of tablets, lozenges, chewable tablets, powder, granules, hard capsules, soft capsules, capsules, bite capsules, coated tablets, lozenges, extrudates, juices, suspensions, gels or ointments.
  • the type II collagen used according to the invention in particular type II collagen peptide, is present in a dosage form which enables delayed intestinal release, in particular sustained-release capsules.
  • the composition according to the invention is in a form suitable for oral administration, in particular with a dose of 1 to 60 mg/day, in particular 5 to 50 mg/day, of recombinant type II collagen.
  • the present invention further relates to a dietary supplement comprising a type II collagen according to the invention, in particular type II collagen peptide, and at least one food-acceptable carrier, and the dietary supplement for use in a method for the therapeutic treatment of cartilage diseases in the human or animal body.
  • a dietary supplement comprising a type II collagen according to the invention, in particular type II collagen peptide, and at least one food-acceptable carrier, and the dietary supplement for use in a method for the therapeutic treatment of cartilage diseases in the human or animal body.
  • the food supplement according to the invention is particularly advantageously in the form of a hard capsule, soft capsule, capsule, bite capsule, tablet, coated tablet, lozenge, sachet, extrudate, solution, suspension or gel, for example in an ampoule, as granules or powder.
  • the invention also relates to a foodstuff or luxury foodstuff comprising a type II collagen according to the invention, in particular type II collagen peptide, and the foodstuff or luxury foodstuff for use in a method for the therapeutic treatment of cartilage diseases in the human or animal body.
  • the food or luxury food is a chocolate bar, protein bar, cereal bar, instant powder for preparing drinks, milk, milk products, for example yoghurt, whey or quark and milk substitutes, for example soy milk, rice milk, almond milk and coconut milk, functional food or a drink, for example a refreshing or fitness drink.
  • the recombinant type II collagen, in particular type II collagen peptide, according to a preferred embodiment of the invention is not to be used as the sole biologically active component of a composition, in particular a pharmaceutical composition, a dietary supplement, or a foodstuff or luxury food, it can are combined with one or more other additives or auxiliaries, in particular those that have a positive effect on general health, in particular on cartilage health and/or endurance capacity.
  • preferred excipients are selected from the group consisting of vitamin C, vitamins from the B, D, E and K series, omega-3 fatty acids, omega-6 fatty acids, conjugated linolenic acids, caffeine and its derivatives, guarana extract, Rosehip Extract, Green Tea Extract, Epigallocatechin Gallate, Creatine, L-Carnitine, ⁇ -Lipoic Acid, N- Acetylcysteine, NADH, D-ribose, magnesium aspartate, antioxidants such as anthocyanins, carotenoids, flavonoids, resveratrol, glutathione and superoxide dismutase, minerals such as iron, magnesium, calcium, zinc, selenium and phosphorus, as well as other proteins, hydrolysates and peptides such as soy and wheat - and whey protein.
  • composition according to the invention in particular the pharmaceutical composition, the dietary supplement or the food, or semi-luxury food
  • Excipient for example chondrotin, chondrotin sulfate, hyaluronic acid, aflapin, univestin 5-glocsin, glucosamine, glucosamine sulfate and/or methylsulfonylmethane (MSM).
  • MSM methylsulfonylmethane
  • composition according to the invention in particular the pharmaceutical composition, the dietary supplement or a foodstuff or semi-luxury food
  • Additive wherein the additive may be a recombinantly produced collagen hydrolyzate, a naturally sourced collagen hydrolyzate, a recombinantly produced Type I collagen, a naturally sourced Type I collagen, or a combination thereof.
  • the products according to the invention in particular the pharmaceutical composition, the dietary supplement, or the food or luxury food, contain no other proteins or peptides, in particular no other collagen peptides, in addition to the type II collagen according to the invention, in particular type II collagen peptide .
  • the present invention also relates to methods for therapy, in particular for the prevention and / or treatment of cartilage diseases, according to which the human or animal body is given an amount sufficient for the therapeutic purpose of at least one of the recombinant type II collagens according to the invention, in particular type II collagen peptides, if necessary administered orally with a carrier and, optionally, an adjuvant or additive.
  • the present invention also relates to methods for inducing oral tolerance to type II collagen, in particular endogenous type II collagen, in a human or animal body, comprising the administration of an amount sufficient for a therapeutic purpose of at least one of the recombinant type II collagens according to the invention, especially recombinant type II collagen peptides, optionally by means of a carrier and, optionally, an adjuvant or additive, the administration being oral.
  • the present invention also relates to methods for the therapeutic treatment or therapeutic prophylaxis of immune intolerance to type II collagen, in particular type II collagen peptide, comprising the oral administration of an amount sufficient for a therapeutic purpose of at least one of the recombinant type II collagens according to the invention, in particular recombinant type II collagen peptides, optionally by means of a carrier and, optionally, an adjuvant or additive.
  • the present invention also relates to the use of recombinant type II collagen, in particular recombinant type II collagen peptides, in non-therapeutic methods of maintaining cartilage health in a human or animal, according to which the human or animal body receives an amount sufficient to maintain cartilage health at least one of the recombinant type II collagens according to the invention, in particular type II collagen peptides, optionally with a carrier and, optionally, an adjuvant or additive, is administered orally.
  • the human or animal does not have a cartilage disease.
  • oral administration of recombinant type II collagen, in particular recombinant type II collagen peptides, to a human or animal which does not have a cartilage disease and by administering the recombinant type II collagen, in particular, can accordingly be provided recombinant type II collagen peptide, maintains its cartilage health.
  • the present invention relates to a method for producing a recombinant type II collagen which can be used according to the invention, in particular a type II collagen peptide, comprising the method steps: a) providing an expression system which has at least one expression cassette, the expression cassette having at least one nucleotide sequence which is a type II collagen, in particular type II collagen peptide, b) culturing the expression system under conditions which allow the expression of the type II collagen, in particular type II collagen peptide, and c) Obtaining the type II collagen according to the invention, in particular type II collagen peptide.
  • the method provided according to the invention for the production of a recombinant type II collagen that can be used according to the invention, in particular type II collagen peptide is characterized in particular by the fact that a precisely defined, recombinantly produced type II collagen, in particular type II collagen peptide, is obtained which , In particular because of its biological effectiveness, for use in a method for the therapeutic treatment of cartilage diseases of the human or animal body or for maintaining cartilage health.
  • the type II collagen provided according to the invention in particular type II collagen peptide, has a particularly high purity due to its recombinant production method compared to type II collagen obtained hydrolytically from natural sources, in particular type II collagen peptides. It can be provided in a wide variety of expression systems, even on an industrial scale, without undesired contamination, with the type II collagen according to the invention, in particular type II collagen peptide, advantageously also having biological activity at the same time.
  • Recombinant type II collagen and its production is described for example in US 5,593,859.
  • This document discloses the production of recombinant type II collagen peptides and the hydroxylation and fibrillogenesis for production of procollagen in recombinant cell culture and is related to the production of recombinant type II collagen and recombinant type II collagen peptides, especially in hydroxylated and triple-helical form , fully included in the present disclosure content.
  • type II collagen found according to the invention in particular of type II collagen peptides, and associated with it or their suitability for use in a method for the therapeutic treatment of cartilage diseases of the human or animal body comes in a preferred embodiment advantageously already directly from the Type II collagen obtained according to the invention, in particular type II collagen peptides, without the need for further processing steps.
  • both the hydroxylated and the non-hydroxylated type II collagens, in particular type II collagen peptides, according to the present invention have a biological activity in a preferred embodiment, in particular at least the same biological activity as type II collagen obtained from natural sources, especially prefers better biological effectiveness than type II collagen derived from natural sources.
  • type II collagens according to the invention in particular type II collagen peptides, surprisingly have a biological activity even in non-hydroxylated form, preferably the same biological activity as type II collagen obtained from natural sources, particularly preferably a better one biological effectiveness as type II collagen derived from natural sources.
  • Both the hydroxylated and the non-hydroxylated type II collagens, in particular type II collagen peptides, according to the present invention preferably show a biological activity, preferably at least the same biological activity as type II collagen obtained from natural sources, particularly preferably a better one biological effectiveness such as type II collagen derived from natural sources.
  • the expression system provided in step a) is preferably a host cell, in particular a prokaryotic or eukaryotic cell.
  • the expression system is preferably a host cell selected from the group consisting of bacterial cell, yeast cell, fungal cell, mammalian cell, insect cell and plant cell.
  • the expression system in particular the host cell, is preferably a bacterial cell, in particular of the Escherichia coli or Bacillus subtilis species.
  • the expression system in particular the host cell, is a yeast cell, in particular of the species Saccharomyces cerevisiae, Pichia pastoris or Ogataea angusta (Hansenula polymorpha), in particular Pichia pastoris.
  • the expression system in particular the host cell, is preferably a fungal cell, in particular of the species Aspergillus niger.
  • the expression system in particular the host cell, is a mammalian cell, in particular a CHO cell, a HeLa cell or a HEK293 cell.
  • the expression system in particular the host cell, is preferably an insect cell, in particular an Sf-9, Sf-21 or Tn-5 cell.
  • the expression system, in particular the host cell is preferably a plant cell, in particular a maize or tobacco cell.
  • the expression system provided in step a) is a host cell capable of hydroxylating proline, lysine or proline and lysine residues of the expressed collagen peptide.
  • the expression system provided in step a) is preferably a host cell which is capable of hydroxylating proline, lysine or proline and lysine residues of the expressed collagen peptide.
  • the expression system provided in step a) is preferably an expression system which has prolyl hydroxylase and/or lysyl hydroxylase activity.
  • the expression system provided in step a) is preferably a host cell which has prolyl hydroxylase and/or lysyl hydroxylase activity.
  • the expression system provided in step a) is a host cell which has at least one expression cassette which comprises a prolyl-4-hydroxylase-encoding polynucleotide sequence.
  • the expression system provided in step a) is particularly preferably a host cell which has at least one expression cassette which comprises a prolyl-4-hydroxylase-encoding polynucleotide sequence, so that in method step c) an in vivo hydroxylated type II collagen, in particular type II collagen collagen peptide is obtained.
  • the expression system provided in step a) is a host cell which has at least one expression cassette which comprises a polynucleotide sequence coding for lysylhydroxylase.
  • the expression system provided in step a) is particularly preferably a host cell which has at least one expression cassette which comprises a lysyl hydroxylase-encoding polynucleotide sequence, so that in method step c) an in vivo hydroxylated type II collagen, in particular type II collagen peptide, is obtained becomes.
  • the expression system provided in step a) is a host cell which has at least one expression cassette which comprises a prolyl-4-hydroxylase-encoding polynucleotide sequence and at least one expression cassette which comprises a lysyl-hydroxylase-encoding polynucleotide sequence.
  • the expression system provided in step a) is particularly preferably a host cell which has at least one expression cassette which comprises a prolyl-4-hydroxylase-encoding polynucleotide sequence and at least one expression cassette which comprises a lysyl-hydroxylase-encoding polynucleotide sequence that in method step c) an in vivo hydroxylated type II collagen, in particular type II collagen peptide, is obtained.
  • the prolyl-4-hydroxylase-encoding polynucleotide sequence comprises the nucleotide sequence according to SEQ ID No. 5.
  • the prolyl-4-hydroxylase-encoding polynucleotide sequence particularly preferably codes for a monomeric prolyl-4-hydroxylase, in particular for a Prolyl-4-hydroxylase which has an amino acid sequence according to SEQ ID No. 6, preferably consists of an amino acid sequence according to SEQ ID No. 6.
  • the present invention thus also relates to a method for producing a recombinant type II collagen which can be used according to the invention, in particular a type II collagen peptide, in particular an in vivo hydroxylated type II collagen, in particular a type II collagen peptide, comprising the method steps a) providing an expression system, which has at least one expression cassette, wherein the expression cassette has at least one nucleotide sequence which encodes a type II collagen, in particular type II collagen peptide, and wherein the expression system is capable of hydroxylating proline, lysine or proline and lysine residues of the expressed collagen peptide , b) culturing the expression system under conditions which allow the expression and hydroxylation of type II collagen, in particular type II collagen peptide, c) obtaining the type II collagen according to the invention, in particular type II collagen peptide, in particular of the in vivo hydroxylated type II colla genes, particularly type II collagen peptides.
  • the recombinant in vivo hydroxylated collagen peptide produced according to the invention has a biological activity.
  • the expression system provided in step a) is an expression system which is incapable of causing hydroxylation of proline, lysine or proline and lysine residues of the expressed collagen peptide, in particular that has in step a) provided expression system no prolyl hydroxylase and lysyl hydroxylase activity.
  • the present invention thus comprises a method for producing a recombinant collagen peptide which can be used according to the invention, in particular a non-hydroxylated collagen peptide, comprising the method steps a) providing an expression system which has at least one expression cassette, the expression cassette having at least one nucleotide sequence which is a type II - encoding collagen, in particular type II collagen peptide, and wherein the expression system is not capable of hydroxylating proline, lysine or proline and lysine residues of the expressed type II collagen, in particular type II collagen peptide, b) culturing the expression system under Conditions enabling the expression of type II collagen, in particular type II collagen peptide, c) obtaining the type II collagen according to the invention, in particular type II collagen peptide, in particular non-hydroxylated type II collagen, in particular type II collagen peptide .
  • the at least one nucleotide sequence of the at least one expression cassette is codon-optimized, which means that those codons in the nucleotide sequence that are not or are not preferably used are replaced by those that are preferably 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 thereby changing the amino acid sequence of the encoded peptide or protein.
  • the type II collagen encoded by the nucleotide sequence, in particular type II collagen peptide is a type II Collagen, in particular type II collagen peptide, of a vertebrate, in particular a mammal, for example a human or a non-human mammal, for example a horse, kangaroo, rodent, pig, sheep or cattle, a bird, for example a chicken, a fish, an amphibian, a reptile or an invertebrate such as a jellyfish.
  • the expression cassette provided in step a) comprises at least one nucleotide sequence according to SEQ ID No. 1.
  • the expression cassette provided in step a) comprises at least one nucleotide sequence with a sequence identity of at least 90%, preferably at least 95%, preferably at least 96%, preferably at least 97%, preferably at least 98%, preferably at least 99%, to the nucleotide sequence according to SEQ ID No. 1.
  • the type II collagen encoded by the nucleotide sequence in particular type II collagen peptide
  • the type II encoded by the nucleotide sequence is preferred -Collagen, in particular type II collagen peptide, from the amino acid sequence according to SEQ ID No. 2.
  • the type II collagen encoded by the nucleotide sequence in particular type II collagen peptide, has at least 80%, preferably at least 85%, preferably at least 90%, preferably at least 95%, preferably at least 96% at least 97%, preferably at least 98%, preferably at least 99% sequence identity with the amino acid sequence according to SEQ ID No. 2.
  • the expression cassette provided in step a) comprises at least one nucleotide sequence according to SEQ ID No. 3.
  • the expression cassette provided in step a) comprises at least one nucleotide sequence with a sequence identity of at least 90%, preferably at least 95%, preferably at least 96%, preferably at least 97%, preferably at least 98%, preferably at least 99%, to the nucleotide sequence according to SEQ ID No. 3.
  • the type II collagen encoded by the nucleotide sequence, in particular type II collagen peptide is a type II collagen, in particular type II collagen peptide, comprising the amino acid sequence according to SEQ ID No. 4.
  • the type II collagen encoded by the nucleotide sequence in particular type II collagen peptide, has at least 80%, preferably at least 85%, preferably at least 90%, preferably at least 95%, preferably at least 96% at least 97%, preferably at least 98%, preferably at least 99% sequence identity with the amino acid sequence according to SEQ ID No. 4.
  • the type II collagen, in particular type II collagen peptide, encoded by the nucleotide sequence is a naturally occurring type II collagen, in particular type II collagen peptide.
  • the type II collagen, in particular type II collagen peptide, encoded by the nucleotide sequence is not a naturally occurring type II collagen, in particular type II collagen peptide.
  • the type II collagen encoded by the nucleotide sequence, in particular type II collagen peptide is preferably a genetically modified collagen peptide.
  • the at least one nucleotide sequence encodes a type II collagen peptide having a molecular weight in a range from 5 to 400 kDa, in particular 10 to 390 kDa, in particular 10 to 350 kDa, in particular 10 to 300 kDa, in particular 10 to 110 kDa, in particular 40 to 110 kDa, in particular 40 to 100 kDa, in particular 11 to 105 kDa, in particular 15 to 100 kDa, in particular 20 to 99 kDa, in particular 25 to 95 kDa, in particular 30 to 95 kDa, in particular 35 to 95 kDa , especially from 40 to 50 kDa, especially 45 kDa.
  • the methods according to the invention are distinguished in that in method step b) conditions are selected which enable the formation of a non-denatured, ie native, type II collagen, in particular type II collagen peptide.
  • the methods according to the invention are distinguished in that in method step b) conditions are selected which enable the formation of a triple-helical form of type II collagen, in particular type II collagen peptides.
  • the methods according to the invention can lead to the production of homogeneous and isolated preparations of specific type II collagen peptides with a uniform molecular weight.
  • the invention also provides mixtures of isolated and homogeneous preparations of type II collagen peptides produced in this way, each with a uniform molecular weight.
  • the invention also envisages providing recombinant collagen peptide hydrolyzates by lysis, in particular hydrolysis, from type II collagen peptides which are produced by means of the method according to the invention and are optionally present homogeneously and in isolation and have a uniform molecular weight.
  • the present invention provides in particular for providing both the homogeneously isolated type II collagen peptides with a uniform molecular weight, their mixtures or their hydrolyzates for the oral therapy of cartilage diseases of a human or animal body provided according to the invention.
  • the methods of the invention are characterized in that following method step b) or c) in a method step d) in a by lysis, in particular hydrolysis, of the expressed type II collagen, in particular type II collagen peptide, a recombinant Type II collagen peptide hydrolyzate is obtained.
  • the type II collagen peptide hydrolyzate obtained according to the invention by process step d) can be used as the recombinant type II collagen peptide according to the invention either in the form of this type II collagen hydrolyzate or after isolation of one or more type II collagen peptides, preferably present homogeneously and isolated.
  • type II collagen peptides present homogeneously and in isolation are mixed with one another and thus represent a mixture of recombinant type II collagen peptides.
  • the invention therefore also relates to a recombinant type II collagen peptide which is present in an isolated, homogeneous form with a uniform molecular weight, or as a recombinant type II collagen peptide which is in a mixture with recombinant or natural, in particular recombinant type II collagen peptides is present, or is present in a hydrolyzate of a recombinant type II collagen, in particular a recombinant type II collagen peptide.
  • nucleotide sequences coding for the recombinant type II collagen peptide which can be replaced according to the invention can be obtained in a manner customary in the art, for example as described in WO 93/07889, US 2006/0147501, US 5,593,859 or US 2008/0081353.
  • the type II collagen in particular type II collagen peptide, which can be used according to the invention and is preferably produced using one of the aforementioned methods according to the invention, is a non- hydroxylated, partially hydroxylated or fully hydroxylated type II collagen peptide, preferably a non-hydroxylated type II collagen peptide, preferably a partially hydroxylated type II collagen peptide, preferably a fully hydroxylated type II collagen peptide.
  • the type II collagen that can be used according to the invention and is preferably produced using one of the above methods, in particular recombinant type II collagen peptide is a glycosylated collagen peptide for use in a method for the therapeutic treatment of cartilage diseases in the human or animal body .
  • the type II collagen, in particular type II collagen peptide is preferably glycosylated in vivo, preferably glycosylated ex vivo.
  • the type II collagen, in particular type II collagen peptide which can be used according to the invention and is preferably produced using one of the methods according to the invention is a non-glycosylated type II collagen, in particular type II collagen peptide.
  • biological activity preferably means the ability of the type II collagens that can be used according to the invention, in particular type II collagen peptides, to immunomodulate, in particular to suppress the synthesis of immunoglobulins, in particular IgE, IgA, IgM and/or IgG.
  • the term “biological effectiveness” preferably also means the ability of the type II collagens that can be used according to the invention, in particular type II collagen peptides, to suppress the formation and activity of pro-inflammatory cytokines, in particular TNFa, IL-6 and IENg or to stimulate the synthesis and activity of anti-inflammatory cytokines, particularly IL-4, IL-10 and TGF- ⁇ 1, particularly both.
  • the term “biological activity” is preferably understood to mean that the type II collagens that can be used according to the invention, in particular type II collagen peptides, for immunomodulation, in particular for suppressing the synthesis of immunoglobulins, in particular IgE, IgA, IgM and/or IgG, for suppression of the production of pro-inflammatory cytokines, in particular TNFa, IL-6 and IENg, and are capable of stimulating the synthesis of anti-inflammatory cytokines, in particular IL-4, IL-10 and TGF- ⁇ 1.
  • the biological effectiveness is determined in particular by means of detection methods familiar to the person skilled in the art for immunomodulating, in particular stimulating and suppressing, activities of substances and for anti-inflammatory cytokines and pro-inflammatory cytokines.
  • the biological activity within the meaning of the present invention is determined using the procedure according to Example 2, Example 3, Example 4 and/or Example 5.
  • the term “biological effectiveness” is preferably used according to the invention to also include the ability of the type that can be used according to the invention II collagens, in particular type II collagen peptides, understood to induce oral tolerance.
  • the presence of oral tolerance is determined by means of detection methods familiar to those skilled in the art for determining the ability of a substance to induce oral tolerance, in particular by means of the procedure according to Example 2, Example 3, Example 4 and/or Example 5 .
  • pro-inflammatory cytokines are in particular TNF ⁇ , IF-6 and IFN-gamma.
  • anti-inflammatory cytokines are in particular IF-4, IF-10 and TGF- ⁇ 1.
  • suppression is understood to mean the partial or complete suppression of a synthesis of proteins, which can manifest itself in particular as a reduction or inhibition of protein synthesis or as a reduction or inhibition of mRNA synthesis affecting the proteins.
  • the term “collagen” is understood in a manner customary in the art, in particular as defined, for example, in WO 01/34646.
  • the term “collagen” refers to a collagen protein having the sequence glycine-proline, glycine-4-hydroxyproline or glycine-X-4-hydroxyproline, preferably the repetitive motif (Gly-X-Y)n Peptide understood, where X and Y are any amino acid may be, preferably proline and 4-hydroxyproline.
  • the term “collagen” particularly preferably means a peptide having the repetitive motif (Gly-Pro-Y)n and/or (Gly-X-Hyp)m, where X and Y can be any amino acid.
  • a “type II collagen” according to the present invention is a collagen as is understood in the art according to the above statements, wherein the type II collagen has the amino acid sequence of a naturally occurring type II collagen, in particular the amino acid sequence of a type II collagen of a vertebrate, in particular pig, sheep, bovine, rodent, horse, bird, fish, reptile or amphibian or an invertebrate, in particular jellyfish.
  • the type II collagen can be present as a monomeric collagen peptide, also referred to here as a single-stranded collagen peptide, or as a dimer or trimer, in particular a trimer, having at least two, in particular three, collagen peptides, in particular from the same single-stranded collagen peptides.
  • the type II collagen can be present as a triple-helical type II collagen peptide, in particular native type II collagen.
  • type II collagen peptide means a single-stranded type II collagen peptide which has an amino acid sequence occurring in type II collagen as defined above, the peptide being an oligopeptide or polypeptide .
  • the type II collagen peptide can be present in particular in chemically modified form, in particular hydroxylated and/or glycosylated form, or it can be unmodified.
  • the recombinant type II collagen used according to the invention in particular type II collagen peptide, can preferably have a sequence modification, in particular a function-preserving sequence modification of a naturally occurring type II collagen, in particular type II collagen peptide.
  • a “type II collagen” is also understood as a function-preserving sequence modification of a naturally occurring type II collagen, in particular a type II collagen peptide, in particular if this has an amino acid sequence identity of at least 80% to the amino acid sequence of the naturally occurring type II collagen at the amino acid level occurring type II collagen have, in particular at least 85%, in particular at least 90%, in particular at least 95%, in particular at least 96%, in particular at least 97%, in particular at least 98%, in particular at least 99% amino acid sequence identity.
  • a type II collagen is present when if the recombinant type II collagen either has exactly the amino acid sequence that occurs in a naturally occurring type II collagen or if a functional sequence modification with an amino acid sequence identity of at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% over a naturally occurring type II collagen, in particular over naturally occurring type II collagen from a Vertebrate, in particular pig, sheep, bovine, rodent, kangaroo, horse, a bird, a reptile, an amphibian or a fish or an invertebrate, in particular jellyfish, is present, in particular when this amino acid identity is compared to a naturally occurring type II bovine collagen amino acid sequence.
  • amino acid sequence identity is determined using the Smith-Waterman algorithm (SSE2, Michael Farrar, 2006, 7.2 November 2010) with the parameters BL50 matrix (15:-5), Open/ext: -12 /-2, determined.
  • the term "function-preserving sequence modification” is understood to mean the modification of a given, in particular naturally occurring, amino acid sequence, in particular the replacement, addition and / or deletion of one or more amino acids, which leads to an amino acid sequence that differs from the given amino acid sequence, which However, the modified amino acid sequence retains the function characteristic of the given amino acid sequence, in particular its biological effectiveness.
  • a “function-preserving sequence modification” is preferably understood as meaning a modification of a given, in particular naturally occurring, amino acid sequence in which the function characteristic of the given amino acid sequence, in particular a biological effectiveness, is 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% is maintained.
  • a “function-preserving sequence modification” is understood as meaning a modification of a given amino acid sequence in which the modified amino acid sequence accounts for 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.
  • sequence modification in particular a "function-preserving sequence modification" in connection with the present invention, a modification of a given, in particular naturally occurring amino acid sequence, in which one or more amino acids with certain chemical-physical properties are replaced by one or more amino acids with the same or similar ones chemical-physical properties have been replaced, in particular, for example, an amino acid with a non-polar side chain (e.g. Ala, Val, Met, Leu, Ile, Pro, Trp, Phe) by another amino acid with a non-polar side chain (e.g. Ala, Val, Met, Leu , Ile, Pro, Trp, Phe), an amino acid with a polar neural side chain (e.g.
  • a non-polar side chain e.g. Ala, Val, Met, Leu, Ile, Pro, Trp, Phe
  • an amino acid with a polar neural side chain e.g.
  • sequence modification in particular a "function-preserving sequence modification”
  • a sequence modification in particular a "function-preserving sequence modification” is also understood to mean the modification of a given amino acid sequence, in particular a naturally occurring amino acid sequence, which consists in the given amino acid sequence, in particular the naturally occurring amino acid sequence, containing at least one amino acid, preferably at least one essential one Amino acid, in particular Ile, Leu, Lys, Met, Phe, Thr, Trp, Val, His, Cys, Tyr, particularly preferably Trp, was added, the function characteristic of the given amino acid sequence, in particular the naturally occurring amino acid sequence, in particular the biological effectiveness of the present invention, in particular the biological effectiveness according to the evidence presented in example 2, example 3, example 4 and/or example 5, is retained.
  • the at least one amino acid preferably the at least one essential amino acid, in particular Ile, Leu, Lys, Met, Phe, Thr, Trp, Val, His, Cys, Tyr, particularly preferably Trp, N-terminal, C -add terminally and/or within the amino acid sequence.
  • amino acid modification refers to a chemical modification of one or more amino acids that may have taken place before, after or during the synthesis of the recombinant type II collagen, in particular type II collagen peptide, while retaining the original amino acid basic structure, in particular one or more proteinogenic amino acids, the type II collagen peptide.
  • the term thus includes both the use of chemically modified amino acids for the synthesis of the type II collagen according to the invention, in particular type II collagen peptide, and the chemical modification of the amino acids after or during the synthesis of the type II collagen, in particular type II collagen peptide.
  • Amino acid modifications typical of collagen peptides are, in particular, hydroxylations on proline and lysine residues and glycosylation of hydroxylated lysine residues. According to the invention, however, the term also includes other chemical changes in amino acids, such as phosphorylation, N-glycosylation, acetylation, methylation or myristoylation.
  • a recombinant type II collagen, in particular type II collagen peptide, or a recombinantly produced type II collagen, in particular type II collagen peptide is a type II collagen obtained by biotechnological recombinant production using an expression system , especially type II collagen peptide understood.
  • the recombinant type II collagen, in particular type II collagen peptide, or the recombinant produced type II collagen, in particular type II collagen peptide in common that these are not obtained from natural sources.
  • the recombinant type II collagen, in particular type II collagen peptide is in the form of a homogeneous preparation of this type II collagen or type II collagen peptide, in particular such a preparation comprises at least 90% by weight, preferably at least 95% by weight, in particular at least 98% by weight, in particular at least 99% by weight, preferably 100% by weight of the type II collagen or the type II collagen peptide.
  • a certain specific size ie a certain molecular weight, ie a single molecular species, are present in a homogeneous preparation.
  • the recombinant type II collagen or the type II collagen peptide is present in isolated form.
  • the recombinant type II collagen or the type II collagen peptide is free from other proteins or peptides, in particular free from other substances, for example impurities, in particular free from non-protein material, free from salts and /or free from other proteins or peptides.
  • the term “gelatin” is understood in a manner customary in the art, in particular as defined, for example, in WO 01/34646.
  • the term “recombinant DNA” refers to an artificially produced or manipulated DNA molecule that was produced in vitro using genetic engineering methods.
  • the recombinant DNA is composed of components from different organisms of origin.
  • expression cassette means a DNA segment that is responsible for the transcription of the information encoded in this segment into an RNA, in particular into an mRNA, and at least one promoter and one protein-coding nucleotide sequence, in usually has at least one promoter, at least one protein-coding nucleotide sequence and optionally a terminator.
  • nucleotide sequence is the sequence of the nucleotides of a nucleic acid, in particular a nucleic acid strand, in particular a DNA or RNA strand understood.
  • a “nucleotide sequence” is therefore to be understood both as an informational unit and as the DNA or RNA strand that physically manifests this information.
  • an “expression system” means a system in which a targeted and controlled protein biosynthesis can take place.
  • the term "expression system” according to the invention includes both cell-free expression systems in which the components required for protein biosynthesis are not present within a cell, i.e. protein biosynthesis takes place outside of a cell, and cell-based expression systems in which protein biosynthesis takes place within a living cell.
  • a cell-free expression system is preferably a lysate or an extract from E. coli, insect cells, wheat germs, tobacco cells or mammalian cells, in particular CHO cells or reticulocytes from rabbits, which contains the components necessary for protein biosynthesis, in particular a translation and a transcription system.
  • the term “culturing” is synonymous with "incubating”.
  • a “host cell” is understood to mean a living cell which is capable of expressing peptides or proteins encoded in foreign DNA, in particular in recombinant DNA.
  • the term "obtaining the type II collagen, in particular type II collagen peptide", according to method step c) according to the invention is a method known to those skilled in the art for isolating type II collagen or type II collagen peptide from a composition containing several components by means of known Isolation methods, such as centrifugation methods, in particular differential centrifugation and/or density gradient centrifugation, chromatographic methods, in particular gel filtration, ion exchange, affinity and/or high-performance liquid chromatography, electrophoresis methods, filtration methods and/or extraction methods, understood, with an enrichment and purification of the component in question of the multi-component composition can preferably be achieved by sequential application of multiple isolation methods.
  • Isolation methods such as centrifugation methods, in particular differential centrifugation and/or density gradient centrifugation, chromatographic methods, in particular gel filtration, ion exchange, affinity and/or high-performance liquid chromatography, electrophoresis methods, filtration methods and/or extraction methods, understood, with an
  • C- and/or N-terminal procollagen fragments can be cleaved off before, after or during the extraction in order to obtain collagen. Fibrillogenesis, chemical modifications and secretion of the expressed type II collagen peptide can preferably also take place under the conditions of method step b).
  • condition that enable the expression of type II collagen, in particular type II collagen peptides allow” conditions such as, in particular, temperature, pressure, time, light and the presence or absence of inducers and/or repressors that Activate or enhance expression of type II collagen, in particular type II collagen peptides.
  • the type II collagen, in particular type II collagen peptide is expressed in the context of a high cell density fermentation, in particular under high pressure, preferably high air pressure.
  • the specific conditions which allow expression of the type II collagen, in particular type II collagen peptide are known to the person skilled in the art and depend on the expression system used and the expression cassette used, in particular the promoter contained therein. Depending on the structure of the expression cassette, expression of type II collagen, in particular type II collagen peptide, can be constitutive or inducible.
  • a therapeutic method for the oral therapy of “cartilage diseases” is understood to mean a method for the prevention and/or treatment of cartilage diseases, in particular for the treatment of cartilage diseases, with the recombinant type II collagen being administered orally.
  • Cartilage diseases within the meaning of the present invention are in particular inflammatory, degenerative cartilage diseases and/or cartilage diseases caused by autoimmune actions, in particular by excessive immune reactions, in particular arthrosis and/or rheumatoid arthritis.
  • cartilage diseases are in particular articular cartilage diseases, in particular of the joints in the feet, knees, fingers, wrists, hips and spine.
  • a therapeutic method for the oral therapy of “cartilage diseases” is preferably also understood to mean a method for inducing oral tolerance to endogenous collagen, in particular endogenous type II collagen, in particular endogenous type II collagen present in or on cartilage tissue.
  • the terms “comprising” and “having” mean that in addition to the elements explicitly covered by these terms other elements that are not explicitly mentioned can also be added. In connection with the present invention, these terms also mean that only the elements explicitly mentioned are covered and no further elements are present. In this particular embodiment, the meaning of the terms “comprising” and “comprising” is synonymous with the term “consisting of”. In addition, the terms “comprising” and “having” also include compositions that, in addition to the elements explicitly mentioned, also contain other elements that are not mentioned, but which are of a functionally and qualitatively subordinate nature. In this embodiment, the terms “comprising” and “comprising” are synonymous with the term “consisting essentially of”.
  • the first and second decimal place or the second decimal place are/is not specified, they are/is to be set as 0.
  • SEQ ID NO: 1 The nucleotide coding sequence of bovine type II collagen (CP90)
  • SEQ ID No. 2 The amino acid sequence of bovine type II collagen (CP90) (1012
  • SEQ ID NO:3 The nucleotide coding sequence of a bovine type II collagen
  • SEQ ID NO: 4 The amino acid sequence of a bovine type II collagen-derived collagen
  • Collagen peptide (CP45) (500 amino acids).
  • SEQ ID No. 5 The nucleotide coding sequence of a monomeric prolyl-4-hydroxylase
  • SEQ ID NO:6 The amino acid sequence of a monomer encoded by SEQ ID NO:5
  • SEQ ID No. 7 The nucleotide sequence of the plasmid pAOXsec-ColII-1.
  • SEQ ID No. 8 The nucleotide sequence of the plasmid pAOXsec-ColII-1 s.a.
  • SEQ ID No. 9 The nucleotide sequence of the plasmid pAOX_Mimi-int 3.0.
  • Figure 1 A plasmid map of pAOXsec-ColII-1.
  • Figure 2 A plasmid map of pAOXsec-ColII-1 s.a.
  • Figure 3 A plasmid map of pAOX_Mimi-int 3.0.
  • Recombinantly produced hydroxylated full-length type II collagen with the amino acid sequence according to SEQ ID No. 2 (CP90) was obtained by recombinant expression of an expression cassette having the nucleotide sequence according to SEQ ID No. 1 in a Pichia pastoris strain capable of hydroxylating proline residues.
  • a type II collagen-based, recombinantly produced hydroxylated collagen peptide with the amino acid sequence according to SEQ ID No. 4 (CP45) was produced by recombinant expression of an expression cassette having the nucleotide sequence according to SEQ ID No. 3 in a Pichia pastoris capable of hydroxylating proline residues. tribe won.
  • the Pichia pastoris strains used for the recombinant expression of CP90 or CP45 were derived from genomic integration of the coding nucleotide sequence of bovine type II collagen (CP90) or the coding nucleotide sequence of a collagen peptide (CP45) derived from bovine type II collagen ( col2al ). by means of the Integration plasmids pAOXsec-ColII-1 ( Figure 1) or pAOXsec-ColII- 1sa ( Figure 2) and the coding nucleotide sequence of a monomeric prolyl-4-hydroxylase from mimi virus (PH4) using the integration plasmid pAOX_Mimi-int 3.0 ( Figure 3). .
  • the immunomodulatory effect of the native recombinantly produced type II collagen (recombinantly produced full-length collagen type II) according to example 1 was determined in commercial healthy murine Peyer's Patch M cells (SCC142M, Sigma Aldrich, Germany).
  • the M cells were cultured in ITES-ERDF medium, which promotes the production of immunoglobulins.
  • the culture medium was supplemented with 10% fetal calf serum, 10 pg/mL insulin, 20 pg/mL transferrin, 20 pM ethanolamine and 25 nM selenite (ITES).
  • the synthesis of immunoglobulins in the cell culture supernatant was determined by specific enzyme-linked immunosorbent assays against IgE, IgA, IgM and IgG.
  • RNA expression of pro-inflammatory and anti-inflammatory cytokines was tested after a cultivation period of 1-5 days.
  • the collagen stock solution was dissolved in 0.01N acetic acid in a 1:1 (v/v) ratio to prepare a working solution of type II collagen.
  • Complete Freund's Adjuvant (CFA) was added to the collagen working solution at a 1:1 (v/v) ratio.
  • mice 8-week-old male DB A/1J mice were housed at 20 ⁇ 2 °C under a 12/12 h light-dark cycle and 55 ⁇ 10% humidity, standard laboratory baby diet and water ad libitum. Oral tolerance was induced by feeding 2 mg/mL native recombinant type II collagen (100 pg dissolved in 0.05 N acetic acid) every other day for a 2-week intervention and an equal amount of phosphate-buffered saline (PBS) as a placebo control compared. Mice received six consecutive administrations of collagen prior to subsequent induction of rheumatoid arthritis.
  • PBS phosphate-buffered saline
  • mice were anesthetized by intraperitoneal injection of 100 pL ketamine-xylazine solution in phosphate-buffered saline (1:100 v/v; 62.5 mg ketamine, 0.625 mg xylazine in 10 mL PBS). Each mouse received 100 pL of the final CFA-collagen solution subcutaneously in the tail 2 cm in front of the base of the tail without penetrating blood vessels so as to induce rheumatoid arthritis (RA).
  • RA rheumatoid arthritis
  • mice against the type II collagen were boosted by repeating the injection after three weeks of maintenance.
  • the boost injection was given closer to the base of the tail to increase the development of rheumatoid arthritis.
  • the degree of arthritis was assessed three times a week for up to 12 weeks.
  • the severity of the arthritis was rated on a scale from 0 to 4:
  • 0 no edema or swelling
  • 1 mild edema and erythema limited to foot and/or ankle
  • 2 slight edema and erythema from ankle to tarsal bone
  • 3 moderate edema and erythema from ankle to tarsal bone
  • 4 edema and erythema from ankle to entire leg.
  • mice were sacrificed, Peyer's patch M cells were isolated and cultured in ITES-ERDF medium as described above.
  • the RNA expression of cytokines and the synthesis of immunoglobulins in type II collagen immunized murine M cells and the PBS controls was determined as described above.
  • the data obtained showed a beneficial effect of recombinantly produced type II collagen in murine Peyer's plaque cells.
  • RNA expression profile of the cytokines showed an anti-inflammatory effect of recombinantly produced type II collagen.
  • the synthesis of immunoglobulins was suppressed in healthy M cells.
  • PBMC peripheral blood monocytes
  • the PBMC cells were cultivated in macrophage base medium DXF (C-28057, PromoCell, Germany) in cell culture flasks coated with human fibronectin (C-43060, PromoCell, Germany).
  • the culture medium was supplemented with the associated supplement mix (supplement to C-28055, PromoCell, Germany) and with 1% amphotericin and 1% penicillin-streptomycin.
  • a redifferentiation of the adherent monocytes to immunosuppressive type 2b or 2c macrophages was induced by the addition of 4 pg/mF recombinant type II collagen.
  • the cells were each incubated with the recombinant collagen peptides for 6 days.
  • the macrophages activated in this way were then polarized by adding 1 pg/mF fipopolysaccharides from Escherichia coli (FPS, F6529, Merck, Germany).
  • the differentiation pattern of the generated macrophages was then examined using specific cell differentiation markers (CDs).
  • CDs specific cell differentiation markers
  • the differentiation of the monocytes into inflammation-inducing M1 macrophages or into immunosuppressive M2 macrophages was demonstrated using specific markers.
  • the M2 surface markers CD86, CD14 and CD163 were determined using EFISA (“Enzyme-Finked Immunosorbent Assay”).
  • CD86 850590096 Diaclone, Hölzel Diagnostics, Germany
  • CD14 850780096 Diaclone, Hölzel Diagnostics, Germany
  • CD 163 EH-CD163 RayBiotech, Hölzel Diagnostics
  • CD86 850590096 Diaclone, Hölzel Diagnostics, Germany
  • CD80 EK0707 Boster PicoKine, Hölzel Diagnostics, Germany
  • TNFa pro-inflammatory
  • IFNY anti-inflammatory cytokines
  • IE-10 anti-inflammatory cytokines
  • the differentiation of the monocytes into M1 and M2 macrophages was checked using a special differentiation medium (C-28055, PromoCell, Germany) and specific cultivation additives.
  • the data obtained showed a statistically significant (p ⁇ 0.05), advantageous effect of the type II collagen peptides CP90 and CP45 used on the differentiation to immunosuppressive M2 macrophages from peripheral blood monocytes.
  • Example 4 Stimulation of na ⁇ ve CD4 + T progenitor cells
  • the macrophage base medium DXF (C-28057, PromoCell, Germany) was inoculated against T cell culture medium (3H800-50-50, 3H Biomedical AB, Sweden).
  • Na ⁇ ve CD4 + T progenitor cells (3H31-k, 3H Biomedical AB, Sweden) were added to the differentiated M2 macrophages.
  • T progenitor cells differentiate into regulatory T suppressor cells.
  • the mature T-suppressor cells were enriched using the ARTE (antigen-reactive T-cell enrichment) method.
  • the specificity of the T cells is determined by labeling the cells with cell surface marker (CD) antibodies that are coupled to various dyes such as biotin or phycoerythrin. To enrich for the specific T cell clone types were these are then separated using anti-biotin and anti-PE-coupled magnetic MicroBeads.
  • CD cell surface marker
  • T cells could be stained with fluorochrome-conjugated antibodies and quantified by flow cytometry.
  • T suppressor cells are identified by Forkhead Box p3 (FoxP3) and CD25.
  • the data obtained also showed a significant beneficial effect of the recombinantly produced type II collagen peptides CP90 and CP45 on the formation of immunosuppressive T-suppressor cells.
  • the data obtained show an advantageous effect of the recombinantly produced type II collagen peptides CP90 and CP45 through the statistically significant (p ⁇ 0.05) increased formation of anti-inflammatory cytokines in immunosuppressive T-suppressor cells.
  • the results support the underlying principle of action in terms of oral tolerance induction through oral application of type II collagen or type II collagen peptides (CP90 and CP45).

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DE102010060564A1 (de) 2010-11-15 2012-05-16 Gelita Ag Verwendung von Kollagenhydrolysat zur Verbesserung der Gesundheit der menschlichen Haut, Haare und/oder Nägel
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