Therapeutic Type I Collagen Peptides

Abstract

The present invention relates to recombinant type I collagen, particularly in undenatured form, for use in a therapeutic method for the oral treatment of inflammatory diseases of the skin and mucous membranes in human or animal patients.

Description

[Technical field]

[0001] The present invention relates to type I collagen, in particular collagen peptides, for use in a therapeutic method for the oral treatment of inflammatory diseases of the skin and mucous membranes of the human or animal body, in particular diseases of the intestinal mucosa. [Background technology]

[0002] Unfortunately, the skin and mucous membranes of humans and animals are frequently and increasingly exposed to inflammatory diseases. In the course of intensive research into the pathogenesis of these diseases, it has been found that such inflammatory diseases are often caused by incorrect immune responses, especially autoimmune diseases. Examples of such inflammatory diseases of the skin and mucous membranes include atopic dermatitis, neurodermatitis, psoriasis, rosacea, acne in the case of the skin, and inflammation of the intestinal mucosa, especially Crohn's disease and ulcerative colitis in the case of the mucous membranes.

[0003] The most common treatments are immunosuppressants such as cortisone and anti-inflammatory drugs, which suppress the body's biological defenses. However, suppressing the immune system's defenses can lead to many side effects, such as skin atrophy and pigmentation disorders, as in the case of topical cortisone therapy for atopic dermatitis. Other treatment options include the use of drugs that act directly on the immune system, stem cell therapy, psychotherapy, occupational therapy, physiotherapy, and nutritional counseling. Treatment-induced immunosuppression protects affected organs, but may increase susceptibility to infections. Stem cell therapy is an ethically and legally controversial option for the treatment of autoimmune diseases and is fraught with health risks.

[0004] From US Patent No. 5,750,144, US Patent No. 5,645,851, US Patent No. 5,529,786 and US Patent No. 5,637,321 it is known to provide an animal tissue-containing composition containing type II collagen for oral administration to treat autoimmune diseases, in particular rheumatoid arthritis. In particular, these documents disclose the use of cartilage tissue-containing materials recovered from natural animal sources and obtaining therefrom a type II collagen-containing composition by various chemical-physical method steps. The type II collagen-containing composition thus obtained is characterized by the presence of non-denatured type II collagen, while it is also characterized by the presence of a large number of secondary components, in particular proteins, antigens and salts, originating from the starting material and the isolation method. No method for treating inflammatory diseases of the skin and mucous membranes, in particular the intestinal mucosa, is disclosed. Summary of the Invention

[0005] There remains a great need for therapeutic options, pharmaceutical compositions and active substances for the treatment of inflammatory diseases of the skin and mucous membranes, in particular diseases of the intestinal mucosa, particularly those that overcome the problems mentioned above, in particular those that identify the cause and eliminate the cause of the disease and / or its symptoms, as well as relieving the suffering and symptoms of the affected patient.

[0006] The technical problem underlying the present invention is therefore to provide a means for overcoming the aforementioned drawbacks, in particular to provide active substances with therapeutic effect and compositions containing them, as well as a method for treating inflammation of the skin and mucous membranes, in particular inflammation of the skin and mucous membranes caused by autoimmune diseases, in particular which can be prepared on a larger industrial scale and cost-effectively in a standardized, reliable and precisely defined form, and which are suitable for use in a method for the oral treatment of inflammatory diseases of the skin and mucous membranes of the human or animal body and / or for maintaining the health of the skin and mucous membranes by virtue of their biological effectiveness.

[0007] The present invention solves the technical problem on which it is based by providing the teaching of the independent claims and also in particular the preferred embodiments and the teaching of the dependent claims herein.

[0008] The present invention relates to type I collagen having a molecular weight of at least 16 kDa for use in a therapeutic method for the oral treatment of inflammatory diseases of the skin and mucosa, in particular immunomodulatory inflammatory diseases of the skin and mucosa, in particular intestinal mucosa diseases, in a human or animal patient.

[0009] The present invention particularly relates to type I collagen for use in a therapeutic method for the oral treatment of inflammatory diseases of the skin and mucosa, in particular diseases of the intestinal mucosa, in a human or animal patient, wherein the type I collagen has a molecular weight of at least 16 kDa and is present in the form of native or recombinant type I collagen.

[0010] The surprising teaching underlying the present invention is that recombinant or natural type I collagen, especially in isolated form, is capable of treating inflammatory diseases of the skin and mucous membranes after oral application, if the molecular weight of this type I collagen is at least 16 kDa. In a particularly preferred embodiment, the recombinant or natural type I collagen with a molecular weight of at least 16 kDa provided according to the present invention is capable of treating inflammatory diseases of the skin and mucous membranes, especially diseases of the intestinal mucosa, especially immunomodulatory diseases.

[0011] In a particularly preferred embodiment, recombinantly prepared type I collagen, in particular type I collagen peptides, are provided for this use according to the invention.

[0012] Surprisingly, the recombinant type I collagen, in particular the recombinant type I collagen peptide, provided in a preferred embodiment of the present invention, can exhibit a biological efficacy at least equivalent to that exhibited by type I collagen recovered from natural sources according to the present invention, i.e. natural type I collagen, and in particular even provides a biological efficacy superior thereto. In a preferred embodiment, the present invention allows the treatment of inflammatory diseases of the skin and mucous membranes in human or animal patients, since very low doses of recombinant type I collagen, in particular type I collagen peptide, i.e. low concentrations of recombinant type I collagen, in particular type I collagen peptide, exhibit high biological efficacy. Thus, the teaching of the present invention has the advantage of providing a reproducible, biotechnologically preparable recombinant type I collagen with a molecular weight of at least 16 kDa for the oral treatment of inflammatory diseases of the skin and mucous membranes, which can be prepared in a standardized manner, which preparation can be carried out on an industrial scale, which is not limited to natural starting materials, which can be prepared with high purity, high yield, contamination-free, and which is characterized in particular by its high biological efficacy allowing its use at low doses.

[0013] The present invention also relates to type I collagen, in particular type I collagen peptides, in particular endogenous collagen, in particular endogenous type I collagen, in particular endogenous type I collagen present in skin and mucosal tissues, having a molecular weight of at least 16 kDa, for inducing oral tolerance, in particular in immunomodulatory inflammatory diseases of the skin and mucosa, in particular in intestinal mucosa diseases.

[0014] The present invention relates to type I collagen (especially recombinant type I collagen peptides), in particular having a molecular weight of at least 16 kDa, and compositions comprising type I collagen peptides (especially recombinant type I collagen peptides), for use in a therapeutic method for the therapeutic treatment or therapeutic prevention of immune intolerance to type I collagen, in particular in immunomodulatory inflammatory diseases of the skin and mucosa, in particular intestinal mucosal diseases, by inducing immune tolerance to type I collagen.

[0015] The present invention also relates to type I collagen having a molecular weight of at least 16 kDa, in particular a recombinant type I collagen peptide, for use in a method for inducing immune tolerance to type I collagen, a composition the administration of which leads to the induction of oral tolerance to type I collagen, in particular in immunomodulatory inflammatory diseases of the skin and mucosa, in particular in intestinal mucosa diseases.

[0016] The natural and recombinant type I collagens, in particular recombinant type I collagen peptides, used according to the invention, having a molecular weight of at least 16 kDa, are characterized by a biological effectiveness that they exert after oral administration in the human or animal body, in particular an immunomodulatory and / or inflammation-modulating biological effectiveness, which in a particularly preferred embodiment is present both in the natural and recombinant (preferably recombinant) type I collagens, in particular in their non-denatured, i.e. native form, and in a preferred embodiment also in the recombinant type I collagen peptides.

[0017] Preferably, the type I collagen, in particular the type I collagen peptides, provided according to the present invention, having a molecular weight of at least 16 kDa, are capable of immunomodulation and / or induction of oral immune tolerance, in particular inducing an immune response and / or oral immune tolerance in the treated human or animal body.

[0018] The natural or recombinant type I collagen with a molecular weight of at least 16 kDa provided according to the invention preferably develops an inhibitory biological efficacy in immunoglobulin synthesis and / or an anti-inflammatory biological efficacy. According to the invention, the reduction of proinflammatory cytokines and the stimulation of anti-inflammatory cytokines can be determined. Without being bound by theory, it is believed that the orally applied natural or recombinant type I collagen with a molecular weight of at least 16 kDa provided according to the invention passes intact or almost intact through the gastrointestinal tract and is recognized in immunomodulatory (particularly immunosuppressive) cells, in particular in the cells of Peyer's patches, and induces immunomodulatory and / or cytokine-modulatory responses and / or signaling cascades that reduce or completely prevent undesirable immune and inflammatory processes in the area of ​​the skin and mucous membranes, in particular the intestinal mucosa.

[0019] Without being bound by theory, oral application of natural or recombinant type I collagen (particularly type I collagen peptides) having a molecular weight of at least 16 kDa provided according to the present invention is believed to induce oral immune tolerance to endogenous type I collagen that induces such undesirable reactions, thereby making it possible to treat inflammatory diseases of the skin and mucosa (particularly intestinal mucosa diseases).

[0020] The recombinant or natural type I collagen (especially recombinant type I collagen peptides) provided according to the present invention, having a molecular weight of at least 16 kDa, preferably has the ability to interact with cells of the treated human or animal patient (especially cells of Peyer's patches) and to bring about, in particular, the stimulation of anti-inflammatory cytokines and the inhibition of pro-inflammatory cytokines, as well as the regeneration of immunosuppressive M2 macrophages and T suppressor cells.

[0021] Inflammatory and immunoregulatory diseases of the skin and mucous membranes which can be particularly preferably treated according to the invention are in particular diseases of the skin (e.g. cutaneous systemic lupus erythematosus, dermatomyositis, lichen sclerosus, neurodermatitis, psoriasis, rosacea, lichen rubra, bullous pemphigoid, pemphigus vulgaris and acne) or diseases of the mucous membranes (in particular the intestinal mucosa) (e.g. Crohn's disease, celiac disease or ulcerative colitis).

[0022] In a particularly preferred embodiment, the present invention relates to type I collagen, wherein the recombinant or natural collagen, in particular the recombinant or natural type I collagen peptide, is present in a homogenous and isolated form with a uniform molecular weight of at least 16 kDa (in particular at least 20 kDa) or as a mixture of isolated collagens (in particular collagen peptide mixture), each collagen (in particular collagen peptide) having a molecular weight of at least 16 kDa, in particular at least 20 kDa.

[0023] Thus, the type I collagen used according to the invention, in particular recombinant or natural type I collagen (in particular type I collagen peptides), can, in one embodiment of the invention, be present in the form of a mixture with type I collagen (in particular type I collagen peptides) of exactly the same size and the same molecular weight, i.e. in a homogenous form with a uniform molecular weight of at least 16 kDa (in particular at least 20 kDa).

[0024] In a further embodiment of the invention, the type I collagen, in particular the recombinant or natural type I collagen (in particular type I collagen peptides) used according to the invention, can be present in the form of a mixture with other type I collagens (in particular type I collagen peptides) which differ from each other in molecular weight, wherein the molecular weight of all type I collagens (in particular type I collagen peptides) of the mixture is at least 16 kDa, in particular at least 20 kDa.

[0025] In a particularly preferred embodiment of the invention, the recombinant or natural type I collagen (in particular type I collagen peptides) used according to the invention having a molecular weight of at least 16 kDa also does not include collagens, in particular collagen peptides, having a molecular weight below 16 kDa.

[0026] In a particularly preferred embodiment of the invention, the recombinant or natural type I collagen (in particular type I collagen peptides) used according to the invention having a molecular weight of at least 16 kDa also does not include type I collagen, in particular type I collagen peptides, having a molecular weight of less than 16 kDa.

[0027] In a further particularly preferred embodiment, the recombinant or natural type I collagen may be present in the form of type I procollagen or mature type I collagen.

[0028] In a further particularly preferred embodiment, the recombinant or natural type I collagen may exist in triple helical form, in particular in the form of a heterotrimer consisting of two I-α1 chains and one I-α2 chain.

[0029] In a particularly preferred embodiment, the recombinant or natural type I collagen is present in its undenatured form, also referred to herein as the native form, ie, having the naturally occurring tertiary and quaternary protein structures.

[0030] In a preferred embodiment, the present invention provides type I collagen (which may be present in the form of type I collagen peptides, i.e. in the form of a single chain, or in the form of multiple chains, e.g. in the form of two or three chains, also referred to herein as triple helix form, in particular in the form of type I procollagen or mature type I collagen, in particular in the form of a heterotrimer consisting of type I-α1 and type I-α2 chains).

[0031] When the natural or recombinant type I collagen according to the present invention does not exist as a single-chain type I collagen peptide, but exists, for example, in a triple helix form, one or all of the individual collagen peptides constituting the triple helix form of collagen I can be realized according to the present invention. In particular, the embodiments disclosed in the present teachings with respect to recombinant or natural type I collagen peptides also apply to type I collagen having one, two or three such single-chain type I collagen peptides, in particular type I collagen consisting only of these, in particular type I collagen consisting of natural (preferably recombinant) type I collagen peptide according to the present invention.

[0032] In a particularly preferred embodiment, the recombinant or natural type I collagen may be bovine type I collagen.

[0033] In a particularly preferred embodiment, the recombinant or natural type I collagen may be type I-α1 or type I-α2 collagen, in particular type I-α1.

[0034] In particularly preferred embodiments, the recombinant or recombinant type I collagen may be present in the form of crosslinked or non-crosslinked fibrils.

[0035] In a particularly preferred embodiment, the recombinant or natural type I collagen (particularly the type I collagen peptide) can be a full-length collagen peptide, ie, have the complete amino acid sequence of a naturally occurring type I collagen peptide.

[0036] In a further particularly preferred embodiment, the recombinant type I collagen is present in the form of recombinant type I collagen peptides, in particular type I-α1.

[0037] In a particularly preferred embodiment, the recombinant type I collagen, in particular the type I collagen peptide, has a molecular weight of 16 to 400 Da, particularly 16 to 390 kDa, particularly 16 to 350 kDa, particularly 16 to 300 kDa, particularly 16 to 110 kDa, particularly 20 to 400 kDa, particularly 20 to 390 kDa, particularly 20 to 350 kDa, particularly 20 to 300 kDa, particularly 20 to 110 kDa, particularly 40 to 110 kDa, particularly 40 to 100 kDa, particularly 21 to 105 kDa, particularly 25 to 100 kDa, particularly 20 to 99 kDa, particularly 25 to 95 kDa, particularly 30 to 95 kDa, particularly 35 to 95 kDa.

[0038] Preferably, the molecular weight of the recombinant type I collagen, in particular the collagen peptide, is 16-25 kDa, in particular 16 kDa.

[0039] Preferably, the molecular weight of the recombinant type I collagen, in particular the collagen peptide, is 20-50 kDa, in particular 20 kDa.

[0040] Preferably, the molecular weight of the recombinant type I collagen, in particular the collagen peptide, is 40-50 kDa, in particular 45 kDa.

[0041] Preferably, the molecular weight of the recombinant type I collagen, in particular the collagen peptide, is 80-100 kDa, in particular 92 kDa.

[0042] In a particularly preferred embodiment, the natural type I collagen, particularly the type I collagen peptide, has a molecular weight of 16 to 400 Da, particularly 16 to 390 kDa, particularly 16 to 350 kDa, particularly 16 to 300 kDa, particularly 16 to 110 kDa, particularly 20 to 400 kDa, particularly 20 to 390 kDa, particularly 20 to 350 kDa, particularly 20 to 300 kDa, particularly 20 to 110 kDa, particularly 40 to 110 kDa, particularly 40 to 100 kDa, particularly 21 to 105 kDa, particularly 25 to 100 kDa, particularly 20 to 99 kDa, particularly 25 to 95 kDa, particularly 30 to 95 kDa, particularly 35 to 95 kDa.

[0043] Preferably, the molecular weight of the natural type I collagen, in particular the collagen peptide, is 16 to 25 kDa, in particular 16 kDa.

[0044] Preferably, the molecular weight of the natural type I collagen, in particular the collagen peptide, is 20 to 50 kDa, in particular 20 kDa.

[0045] Preferably, the molecular weight of the natural type I collagen, in particular the collagen peptide, is 40 to 50 kDa, in particular 45 kDa.

[0046] Preferably, the molecular weight of the natural type I collagen, in particular the collagen peptide, is 80 to 100 kDa, in particular 92 kDa.

[0047] In particularly preferred embodiments, the recombinant or natural type I collagen of the invention, in particular the recombinant or natural type I collagen peptides, are fully or partially hydroxylated, fully or partially glycosylated, or fully or partially hydroxylated and glycosylated.

[0048] In a particularly preferred embodiment, the recombinant or natural type I collagen, in particular the recombinant or natural type I collagen peptide according to the invention, is a non-hydroxylated type I collagen, in particular a type I collagen peptide.

[0049] In a further preferred embodiment, the recombinant or natural type I collagen, in particular the recombinant or natural type I collagen peptide according to the invention, is a hydroxylated type I collagen, in particular a type I collagen peptide.

[0050] Preferably, the recombinant or natural type I collagen, in particular the recombinantly prepared or natural type I collagen peptides, have hydroxylated prolines and / or hydroxylated lysines.

[0051] Preferably, the recombinant or natural type I collagen, in particular the recombinant or natural type I collagen peptide, is non-hydroxylated, partially hydroxylated or fully hydroxylated type I collagen, in particular a type I collagen peptide.

[0052] According to a preferred embodiment of the present invention, the recombinant or natural type I collagen, in particular the recombinant or natural type I collagen peptide, is glycosylated. More preferably, the recombinant or natural type I collagen, in particular the recombinant or natural type I collagen peptide, is glycosylated at at least one hydroxylated lysine. Preferably, each hydroxylated lysine of the recombinant or natural type I collagen, in particular the recombinant or natural type I collagen peptide, is glycosylated.

[0053] In a preferred embodiment of the invention, the recombinant type I collagen, in particular the recombinant type I collagen peptides, are free of amino acid modifications, in particular hydroxylation. Particularly preferably, the recombinant type I collagen, in particular the type I collagen peptides, are free of hydroxylated and / or glycosylated amino acids.

[0054] Preferably, the type I collagen, in particular the type I collagen peptide, according to the present invention has an amino acid sequence present in type I collagen derived from vertebrates (in particular fish, amphibians, reptiles, birds and mammals (in particular pigs, sheep, cows, rodents, kangaroos, horses)) or type I collagen derived from invertebrates (in particular jellyfish), in particular type I collagen derived from cows.

[0055] Particularly preferably, the type I collagen, in particular the type I collagen peptide, comprises the amino acid sequence shown in SEQ ID NO: 2, 4, 6 or 8. More preferably, the type I collagen, in particular the type I collagen peptide, consists of the amino acid sequence shown in SEQ ID NO: 2, 4, 6 or 8.

[0056] In a further preferred embodiment of the invention, the type I collagen, in particular the type I 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 to the amino acid sequence shown in SEQ ID NO: 2, 4, 6 or 8.

[0057] Preferably, the amino acid sequence of the recombinant type I collagen, particularly the type I collagen peptide, is the amino acid sequence of a naturally occurring type I collagen, particularly the type I collagen peptide. Preferably, the amino acid sequence of the recombinant type I collagen, particularly the type I collagen peptide, is the amino acid sequence of a non-naturally occurring type I collagen, particularly the type I collagen peptide. Preferably, the amino acid sequence of the recombinant type I collagen, particularly the type I collagen peptide, is the amino acid sequence of a genetically engineered type I collagen, particularly the type I collagen peptide.

[0058] Particularly preferably, the type I collagen, in particular the type I collagen peptide, according to the present invention has an amino acid sequence present in the α1 chain of non-human collagen, in particular a non-human type I collagen peptide, preferably a non-human type I collagen, in particular an amino acid sequence present in bovine, porcine, equine, ovine, fish or avian collagen, in particular an amino acid sequence present in bovine collagen.

[0059] In a preferred embodiment of the invention, the recombinant type I collagen, in particular the recombinant type I collagen peptides, are collagenase-resistant, in particular resistant to digestion by human collagenase.

[0060] According to a further preferred embodiment of the present invention, recombinant or natural type I collagen, in particular type I collagen peptides, having a molecular weight of at least 16 kDa, are capable of inducing oral immune tolerance, in particular to endogenous collagen, in particular to endogenous type I collagen, in particular to endogenous type I collagen present in the skin and mucous membranes.

[0061] According to a further preferred embodiment of the present invention, recombinant or natural type I collagen, in particular type I collagen peptides, having a molecular weight of at least 16 kDa are capable of inhibiting immunoglobulin synthesis.

[0062] According to a further preferred embodiment of the present invention, recombinant or natural type I collagen, in particular type I collagen peptides, having a molecular weight of at least 16 kDa, are capable of suppressing the synthesis of pro-inflammatory cytokines.

[0063] According to a further preferred embodiment of the invention, recombinant or natural type I collagen, in particular type I collagen peptides, having a molecular weight of at least 16 kDa are capable of stimulating the synthesis of anti-inflammatory cytokines.

[0064] Preferably, the native or recombinant type I collagen provided according to the present invention, in particular the recombinant type I collagen peptide, exhibits the effect of inducing the differentiation of peripheral blood monocytes into immunosuppressive M2 macrophages.

[0065] In a further preferred embodiment, the recombinant or natural type I collagen provided according to the present invention, in particular the recombinant type I collagen peptides, reduce the synthesis of pro-inflammatory cytokines (in particular TNFα and IFNγ) and / or induce the synthesis of anti-inflammatory cytokines (in particular IL-10, IL-4 and TGF-β, in particular IL-10).

[0066] According to a preferred embodiment of the present invention, the recombinant or natural type I collagen, in particular the recombinant type I collagen peptides, provided according to the present invention stimulate / induce the differentiation of naive CD4+ T progenitor cells into T suppressor cells. Particularly preferably, the stimulation / induction of the differentiation of naive CD4+ T progenitor cells into T suppressor cells increases the release of anti-inflammatory cytokines, preferably IL-10, IL-4 and / or TGF-β.

[0067] In a preferred embodiment of the invention, the recombinant or natural type I collagen, in particular the recombinant type I collagen peptides, provided according to the invention reduce the expression of pro-inflammatory cytokines, preferably IL-1β, IFNγ, TNFα and / or IL-6, in particular by dermal fibroblasts.

[0068] According to a further preferred embodiment of the present invention, recombinant or natural type I collagen, in particular type I collagen peptides, having a molecular weight of at least 16 kDa, are capable of inhibiting the synthesis of immunoglobulins, inhibiting the synthesis of pro-inflammatory cytokines, and stimulating the synthesis of anti-inflammatory cytokines.

[0069] According to a preferred embodiment of the present invention, the recombinant or natural type I collagen, in particular type I collagen peptides, for use in a therapeutic method for the oral treatment of inflammatory diseases of the skin and mucous membranes in a human or animal patient, have a molecular weight of 16-400 Da, in particular 16-390 kDa, in particular 16-350 kDa, in particular 16-300 kDa, in particular 16-110 kDa, in particular 20-400 kDa, in particular 20-390 kDa, in particular 20-350 kDa, in particular 20-300 kDa, in particular 20-110 kDa, in particular 40-110 kDa, in particular 40-100 kDa, in particular 21-105 kDa, in particular 25-100 kDa, in particular 20-99 kDa, in particular 25-95 kDa, in particular 30-95 kDa, in particular 35-95 kDa.

[0070] According to a preferred embodiment of the present invention, the recombinant or natural type I collagen, in particular type I collagen peptides, for use in the therapeutic method for the oral treatment of inflammatory diseases of the skin and mucous membranes in human or animal patients have a molecular weight of 35 to 95, in particular 40 to 92 kDa.

[0071] Preferably, the molecular weight of the recombinant or natural type I collagen, especially the collagen peptide, is 16-25 kDa, especially 16 kDa.

[0072] Preferably, the molecular weight of the recombinant or natural type I collagen, especially the collagen peptide, is 20-50 kDa, especially 20 kDa.

[0073] Preferably, the molecular weight of the recombinant or natural type I collagen is 40 to 50 kDa, particularly 45 kDa.

[0074] Preferably, the molecular weight of the recombinant or natural type I collagen is 80 to 100 kDa, particularly 92 kDa.

[0075] In a preferred embodiment of the invention, the recombinant or natural type I collagen according to the invention, in particular the type I collagen peptides, are used for the purposes of the invention alone (i.e. in isolated form), i.e. free from other substances, in particular free from other collagens.

[0076] In a preferred embodiment of the invention, the recombinant or natural type I collagen, in particular the type I collagen peptide, according to the invention is present as a homogenous preparation, in particular as a homogenous preparation of a single recombinant or natural type I collagen (in particular a type I collagen peptide) free of other collagens and having a molecular weight of at least 16 kDa (in particular at least 20 kDa).

[0077] In a preferred embodiment of the invention, the recombinant or natural type I collagen, in particular type I collagen peptides, according to the invention are present as a mixture of type I collagens, in particular type I collagen peptides, all of which have a molecular weight of at least 16 kDa, in particular at least 20 kDa, in particular free of other collagens.

[0078] In a further embodiment of the invention, the recombinant or natural type I collagen according to the invention, in particular the type I collagen peptides, are used as the only biologically effective substance in the applications according to the invention.

[0079] In a particularly preferred embodiment, the present invention relates to a composition comprising at least one type I collagen peptide, in particular at least one type I collagen peptide, having a molecular weight of at least 16 kDa, in particular at least 20 kDa, which composition does not contain other substances, in particular other collagens, other than the at least one recombinant or natural type I collagen, in particular the at least one recombinant or natural type I collagen peptide, and any pharma- ceutically acceptable and / or food-safe carrier.

[0080] In a particularly preferred embodiment, the composition comprising at least one type I collagen peptide, in particular at least one type I collagen peptide, having a molecular weight of at least 16 kDa, in particular at least 20 kDa, is present in a dosage form suitable for oral administration to the human or animal body.

[0081] The present invention also relates to a composition comprising at least one type I collagen peptide, in particular at least one type I collagen peptide according to the invention having a molecular weight of at least 16 kDa, in particular at least 20 kDa, at least one pharma- ceutically acceptable and / or food-safe carrier for use in a therapeutic method for the oral treatment of inflammatory diseases of the skin and mucous membranes, and optionally at least one additive or excipient.

[0082] Thus, the present invention also relates to compositions for use in therapeutic methods for the therapeutic prevention or therapeutic treatment of immune intolerance reactions to type I collagen, particularly endogenous type I collagen, by inducing oral immune tolerance to type I collagen, particularly endogenous type I collagen.

[0083] The present invention also relates to a composition for use in a method for inducing oral tolerance to type I collagen, particularly endogenous type I collagen, which composition induces oral tolerance in a human or animal.

[0084] The present invention also relates to a composition comprising a type I collagen peptide, in particular a recombinant or natural type I collagen peptide, having a molecular weight of at least 16 kDa, in particular at least 20 kDa, for use in inducing oral immune tolerance to type I collagen, in particular endogenous type I collagen, in particular a pharmaceutical composition or a food supplement or a composition contained in a food or luxury food.

[0085] The composition according to the invention for oral administration can in particular be a pharmaceutical composition, a food supplement, or a food or a food treat. In particular, the composition according to the invention is a pharmaceutical composition. In particular, the composition according to the invention is a food supplement.

[0086] The present invention relates in particular to a pharmaceutical composition comprising type I collagen according to the invention, in particular type I collagen peptide, with a molecular weight of at least 16 kDa, in particular at least 20 kDa, and at least one pharma- ceutically acceptable carrier, for use in a therapeutic method for treating inflammatory diseases of the skin and mucous membranes of the human or animal body. Thus, type I collagen according to the invention, in particular type I collagen peptide, can be administered in the form of a pharmaceutical composition. The pharmaceutical composition according to the invention is particularly advantageously administered, for example, in the form of tablets, lozenges, chewable tablets, powders, granules, hard capsules, soft capsules, capsules, bite capsules, dragees, pastilles, extrudates, juices, suspensions, gels or ointments.

[0087] In a particularly preferred embodiment of the invention, the type I collagen, in particular the type I collagen peptides, used according to the invention are present as a dosage form allowing sustained intestinal release, in particular as sustained release capsules.

[0088] In a particularly preferred embodiment, the composition of the invention is present in a form suitable for oral administration of type I collagen, in particular type I collagen peptides, having a molecular weight of at least 16 kDa, in particular at least 20 kDa, in particular in a dose of 1 to 60 mg / day, in particular 5 to 50 mg / day.

[0089] The present invention further relates to a food supplement comprising the type I collagen of the present invention, in particular the type I collagen peptide, with a molecular weight of at least 16 kDa, in particular at least 20 kDa, and at least one food-safe carrier, for use in a therapeutic method for the treatment of inflammatory diseases of the skin and mucous membranes of the human or animal body. Thus, the type I collagen of the present invention, in particular the type I collagen peptide, can be administered in the form of a food supplement. Particularly advantageously, the food supplement of the present invention is present as a hard capsule, soft capsule, capsule, bite-sized capsule, tablet, dragee, pastille, sachet, extrudate, solution, suspension or gel (e.g. in ampoules), granule or powder.

[0090] The present invention also relates to a food or treat comprising the type I collagen of the invention, in particular a type I collagen peptide, with a molecular weight of at least 16 kDa, in particular at least 20 kDa, for use in a therapeutic method for treating inflammatory diseases of the skin and mucous membranes of the human or animal body. According to 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, dairy products (such as yogurt, whey or curd) and milk substitutes (such as soy milk, rice milk, almond milk and coconut milk), a functional food or drink (such as a soft drink or a fitness drink).

[0091] If the recombinant type I collagen, in particular type I collagen peptides, according to preferred embodiments of the present invention are not used as the sole biologically active ingredient of a composition, in particular a pharmaceutical composition, a food supplement, or a food or luxury food, they can be combined with one or more further additives or excipients, in particular those that have a positive effect on general health, in particular on the health of the skin and mucous membranes. Preferred excipients according to the invention are selected from the group consisting of vitamins of the vitamin C, B, D, E and K series, omega-3 fatty acids, omega-6 fatty acids, conjugated linoleic acid, caffeine and its derivatives, guarana extract, rosehip extract, green tea extract, polyphenols, epigallocatechin gallate, creatine, L-carnitine, α-lipoic acid, N-acetylcysteine, NADH, D-ribose, magnesium aspartate, antioxidants (anthocyanins, carotenoids, flavonoids, resveratrol, glutathione, superoxide dismutase, etc.), minerals (iron, magnesium, calcium, zinc, selenium, phosphorus, etc.) and other proteins, hydrolysates and peptides (soy, wheat, whey proteins, etc.).

[0092] In a particularly preferred embodiment, the composition according to the invention, in particular a pharmaceutical composition, a food supplement or a food or a dietary supplement, may comprise a corticosteroid, in particular a glucocorticoid, in particular cortisone.

[0093] In a further preferred embodiment, the composition according to the invention, in particular the pharmaceutical composition, the food supplement, or the food or luxury food, comprises an additive, which may be a recombinantly produced collagen hydrolysate, a collagen hydrolysate from a natural source, a recombinantly produced type I collagen, a type I collagen recovered from a natural source, or a combination thereof, in particular each having a molecular weight of at least 16 kDa, in particular at least 20 kDa.

[0094] In a further preferred embodiment of the invention, the product according to the invention, in particular the pharmaceutical composition, the food supplement or the food or luxury food, does not comprise further proteins or peptides, in particular further collagen peptides, apart from the type I collagen, in particular the type I collagen peptides, according to the invention.

[0095] The present invention also relates to a method for the treatment, in particular for the prevention and / or treatment of inflammatory diseases of the skin and mucous membranes, according to which at least one recombinant or natural type I collagen, in particular type I collagen peptide according to the invention, having a molecular weight of at least 16 kDa, in particular at least 20 kDa, in an amount sufficient for therapeutic purposes is orally administered to the human or animal body, optionally together with a carrier and optionally with excipients or additives.

[0096] The present invention also relates to a method for inducing oral immune tolerance to type I collagen, in particular endogenous type I collagen, in the human or animal body, which comprises administering a sufficient amount for therapeutic purposes of at least one recombinant or natural type I collagen according to the invention, in particular a recombinant or natural type I collagen peptide having a molecular weight of at least 16 kDa, in particular at least 20 kDa, optionally together with a carrier and optionally with excipients or additives, wherein the administration is carried out orally.

[0097] The present invention also relates to a method for the therapeutic treatment or therapeutic prevention of immune intolerance to type I collagen, in particular to type I collagen peptides, which comprises the oral administration of at least one recombinant or natural type I collagen, in particular a recombinant or natural type I collagen peptide according to the invention, having a molecular weight of at least 16 kDa, in particular at least 20 kDa, in an amount sufficient for therapeutic purposes, optionally together with a carrier and optionally with excipients or additives.

[0098] The present invention also relates to the use of recombinant or natural type I collagen, in particular recombinant or natural type I collagen peptides, having a molecular weight of at least 16 kDa, in particular at least 20 kDa, for use in a non-therapeutic method for maintaining the health of the skin and mucous membranes of a human or animal, according to which a sufficient amount of at least one recombinant or natural type I collagen, in particular type I collagen peptide, according to the invention, optionally together with a carrier and optionally with an auxiliary or additive, is orally administered to the human or animal body for maintaining the health of the skin and mucous membranes. In this particularly preferred embodiment of the present invention, the human or animal does not have a skin or mucous membrane disease. Thus, in a particularly preferred embodiment, recombinant or natural type I collagen, in particular recombinant type I collagen peptides, having a molecular weight of at least 16 kDa, in particular at least 20 kDa, is orally administered to a human or animal, which does not have a skin or mucous membrane disease and which maintains the health of its skin and mucous membranes, in particular the health of the intestinal mucosa, by administering natural or recombinant type I collagen, in particular recombinant or natural type I collagen peptides.

[0099] Furthermore, the present invention relates to a method for preparing a recombinant type I collagen, in particular a type I collagen peptide, which can be used according to the present invention and has a molecular weight of at least 16 kDa, in particular at least 20 kDa, comprising: a) providing an expression system comprising at least one expression cassette having at least one nucleotide sequence encoding type I collagen, in particular a type I collagen peptide, having a molecular weight of at least 16 kDa, in particular at least 20 kDa; b) culturing the expression system under conditions allowing expression of type I collagen, in particular type I collagen peptides, and c) recovering type I collagen according to the invention, in particular type I collagen peptides, having a molecular weight of at least 16 kDa, in particular at least 20 kDa; The present invention relates to a method comprising the steps of:

[0100] The process provided according to the invention for preparing recombinant type I collagen, in particular type I collagen peptides, usable according to the invention, with a molecular weight of at least 16 kDa, in particular at least 20 kDa, is particularly characterized by the fact that a precisely defined and recombinantly prepared type I collagen, in particular type I collagen peptides, is recovered, which collagen, in particular due to its biological effectiveness, is suitable for use in methods for the treatment of inflammatory diseases of the skin and mucous membranes of the human or animal body or in methods for maintaining the health of the skin and mucous membranes.

[0101] The recombinant type I collagen, particularly type I collagen peptide, provided according to the present invention has a molecular weight of at least 16 kDa, particularly at least 20 kDa, and has a particularly high purity due to its recombinant production method, compared to type I collagen, particularly type I collagen peptide, recovered from natural sources, for example, by hydrolysis.In addition, the recombinant type I collagen, particularly type I collagen peptide, has the advantage of being biologically effective while being able to be provided on an industrial scale in a wide variety of expression systems without undesirable contamination.

[0102] Type I collagen peptides and their preparation are described, for example, in International Patent Applications WO 2005 / 012356, WO 01 / 34646, WO 01 / 34647, and WO 01 / 34801, which disclose obtaining recombinant type I collagen peptides and their hydroxylation and fibrillation to obtain procollagen in recombinant cell cultures and are fully incorporated into the present disclosure with respect to the preparation of recombinant type I collagen and recombinant type I collagen peptides, particularly in the hydroxylated triple helical form.

[0103] The biological effectiveness of the recombinant type I collagen, in particular type I collagen peptides, found according to the invention, with a molecular weight of at least 16 kDa, in particular at least 20 kDa, and in this connection its (their) suitability when used in a method for the therapeutic treatment of inflammatory diseases of the skin and mucous membranes of the human or animal body, has the advantage that in a preferred embodiment, the type I collagen, in particular type I collagen peptides, directly recovered from the method according to the invention, without the need for further processing steps, are already possessed. Thus, in a preferred embodiment, both the hydroxylated and non-hydroxylated recombinant type I collagen, in particular type I collagen peptides according to the invention, have a biological effectiveness, in particular a biological effectiveness at least equivalent to that of type I collagen recovered from natural sources, and in particular preferably a biological effectiveness superior to that of type I collagen recovered from natural sources.

[0104] What is particularly advantageous here is that the recombinant type I collagen, in particular type I collagen peptides according to the invention, surprisingly have a biological effectiveness even in the non-hydroxylated form, preferably have the same biological effectiveness as type I collagen recovered from natural sources, and particularly preferably have a biological effectiveness superior to that of type I collagen recovered from natural sources.

[0105] Preferably, both the hydroxylated and non-hydroxylated recombinant type I collagen, in particular the type I collagen peptides, according to the present invention exhibit a biological potency, preferably at least equivalent to, and more preferably superior to, type I collagen recovered from natural sources.

[0106] Preferably, the expression system provided in step a) is a host cell, in particular a prokaryotic or eukaryotic cell.

[0107] 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.

[0108] 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.

[0109] In a further preferred embodiment, the expression system, in particular the host cell, is a yeast cell, in particular a Saccharomyces cerevisiae, Komagataella phaffi or Ogataea angusta, Hansenula polymorpha, in particular a Komagataella yeast.

[0110] Preferably, the expression system, in particular the host cell, is a fungal cell, in particular a fungal cell of the species Aspergillus niger.

[0111] In a further 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.

[0112] Preferably, the expression system, in particular the host cell, is an insect cell, in particular an Sf-9, Sf-21 or Tn-5 cell.

[0113] Preferably, the expression system, in particular the host cell, is a plant cell, in particular a corn cell or a tobacco cell.

[0114] In a further preferred embodiment of the invention, 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.Preferably, 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.

[0115] Preferably, the expression system provided in step a) is an expression system having prolyl hydroxylase and / or lysyl hydroxylase activity.Preferably, the expression system provided in step a) is a host cell having prolyl hydroxylase and / or lysyl hydroxylase activity.

[0116] 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, which allows in vivo hydroxylated type I collagen, in particular type I collagen peptides, to be recovered in method step c).

[0117] 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, which allows in vivo hydroxylated type I collagen, in particular type I collagen peptides, to be recovered in method step c).

[0118] In a further preferred embodiment of the 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, 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, whereby in vivo hydroxylated type I collagen, in particular type I collagen peptides, are recovered in method step c).

[0119] The present invention therefore also relates to a method for preparing a recombinant type I collagen, in particular a type I collagen peptide, in particular an in vivo hydroxylated type I collagen, in particular a type I collagen peptide, which can be used according to the invention and has a molecular weight of at least 16 kDa, in particular at least 20 kDa, comprising a) providing an expression system comprising at least one expression cassette, the expression cassette comprising at least one nucleotide sequence encoding a type I collagen, in particular a type I collagen peptide, having a molecular weight of at least 16 kDa, in particular at least 20 kDa, the expression system being capable of hydroxylating proline, lysine or proline and lysine residues of the expressed collagen peptide; b) culturing the expression system under conditions allowing expression and hydroxylation of type I collagen, in particular type I collagen peptides; c) recovering the type I collagen, in particular type I collagen peptide, in particular in vivo hydroxylated type I collagen, in particular type I collagen peptide, according to the invention, having a molecular weight of at least 16 kDa, in particular at least 20 kDa, The present invention relates to a method comprising the steps of:

[0120] Thus, thanks to the above-mentioned method, it is advantageously possible to obtain in vivo hydroxylated recombinantly prepared type I collagen, in particular type I collagen peptides, characterized by a specific pattern of post-translational modifications, in particular hydroxylation and glycosylation, with a molecular weight of at least 16 kDa, in particular at least 20 kDa, depending on the cell-based expression system used. In this way, it is advantageously possible to obtain directly (i.e. without the need for subsequent modification) type I collagen, in particular recombinantly prepared type I collagen peptides, with the desired biological effectiveness, in particular for use in methods for the treatment of inflammatory diseases of the skin and mucous membranes of the human or animal body.

[0121] In a preferred embodiment, the in vivo hydroxylated recombinant collagen peptides prepared according to the invention have biological efficacy. According to a further embodiment of the invention, the expression system provided in step a) is an expression system that 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 have prolyl hydroxylase and lysyl hydroxylase activity.

[0122] The present invention therefore relates to a method for preparing recombinant collagen peptides, in particular non-hydroxylated collagen peptides, which can be used according to the invention and have a molecular weight of at least 16 kDa, in particular at least 20 kDa, comprising: a) providing an expression system comprising at least one expression cassette, the expression cassette comprising at least one nucleotide sequence encoding a type I collagen, in particular a type I collagen peptide, having a molecular weight of at least 16 kDa, in particular at least 20 kDa, the expression system being unable to hydroxylate proline, lysine or proline and lysine residues of the expressed type I collagen, in particular a type I collagen peptide, b) culturing the expression system under conditions allowing expression of type I collagen, in particular type I collagen peptides; c) recovering the type I collagen according to the invention, in particular the type I collagen peptide, in particular the non-hydroxylated type I collagen, in particular the type I collagen peptide, having a molecular weight of at least 16 kDa, in particular at least 20 kDa, The present invention encompasses a method comprising the steps of:

[0123] According to a preferred embodiment of the present invention, at least one nucleotide sequence of at least one expression cassette is codon-optimized, which means that codons in the nucleotide sequence that are not used or preferably not 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 with codons 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.

[0124] In a preferred embodiment of the invention, the type I collagen, in particular the type I collagen peptide, encoded by the nucleotide sequence is a vertebrate type I collagen, in particular a type I collagen peptide, such as a mammal (e.g. a human or a non-human mammal such as a horse, kangaroo, rodent, pig, sheep or cow), a bird (e.g. a chicken), a fish, an amphibian, a reptile, or an invertebrate type I collagen, in particular a type I collagen peptide, such as a jellyfish.

[0125] In a preferred embodiment of the invention, the expression cassette provided in step a) comprises at least one nucleotide sequence as shown in SEQ ID NO: 1, 3, 5 or 7.

[0126] Particularly preferably, the expression cassette provided in step a) comprises at least one nucleotide sequence having 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 to the nucleotide sequence set forth in SEQ ID NO: 1, 3, 5 or 7.

[0127] Particularly preferably, the type I collagen, in particular the type I collagen peptide, encoded by the nucleotide sequence is a type I collagen, in particular a type I collagen peptide, comprising the amino acid sequence shown in SEQ ID NO: 2, 4, 6 or 8. Preferably, the type I collagen, in particular the type I collagen peptide, encoded by the nucleotide sequence comprises the amino acid sequence shown in SEQ ID NO: 2, 4, 6 or 8.

[0128] In a further preferred embodiment of the invention, the type I collagen, in particular the type I collagen peptide, encoded by the nucleotide sequence 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 shown in SEQ ID NO: 2, 4, 6 or 8.

[0129] Preferably, the type I collagen, particularly the type I collagen peptide, encoded by the nucleotide sequence is a naturally occurring type I collagen, particularly the type I collagen peptide. In a further preferred embodiment of the present invention, the type I collagen, particularly the type I collagen peptide, encoded by the nucleotide sequence is not a naturally occurring type I collagen, particularly the type I collagen peptide. Preferably, the type I collagen, particularly the type I collagen peptide, encoded by the nucleotide sequence is a genetically engineered collagen peptide.

[0130] According to a preferred embodiment of the invention, the at least one nucleotide sequence has a molecular weight in the range of 16-400 Da, in particular 16-390 kDa, in particular 16-350 kDa, in particular 16-300 kDa, in particular 16-110 kDa, in particular 20-400 kDa, in particular 20-390 kDa, in particular 20-350 kDa, in particular 20-300 kDa, in particular 20-110 kDa, In particular it encodes a type I collagen peptide which is between 40 and 110 kDa, in particular between 40 and 100 kDa, in particular between 21 and 105 kDa, in particular between 80 and 100 kDa, in particular between 25 and 100 kDa, in particular between 20 and 99 kDa, in particular between 20 kDa, in particular between 25 and 95 kDa, in particular between 30 and 95 kDa, in particular between 35 and 95 kDa, in particular between 92 kDa, in particular between 40 and 50 kDa, in particular between 45 kDa.

[0131] In a particularly preferred embodiment of the invention, the method according to the invention is characterized in that in method step b) conditions are selected which allow the formation of non-denatured (i.e. native) type I collagen, in particular type I collagen peptides.

[0132] In a particularly preferred embodiment of the invention, the method according to the invention is characterized in that in method step b) conditions are selected which allow the formation of the triple helix form of type I collagen, in particular of type I collagen peptides.

[0133] In a particularly preferred embodiment, the method according to the invention allows for the preparation of homogenous and isolated preparations of specific type I collagen peptides that are uniform in molecular weight.

[0134] In particularly preferred embodiments, the present invention also provides a mixture of homogenous and isolated preparations of type I collagen peptides thus prepared, each having a uniform molecular weight.

[0135] The present invention also provides recombinant collagen peptide hydrolysates by dissolution (in particular hydrolysis) of homogeneous and isolated collagen type I peptides of uniform molecular weight prepared by the method according to the invention. In particular, the present invention provides both homogeneous and isolated collagen type I peptides of uniform molecular weight, mixtures thereof or hydrolysates thereof for the oral treatment of inflammatory diseases of the skin and mucous membranes of the human or animal body according to the present invention.

[0136] According to the invention, the method according to the invention is characterized in that following method step b) or c) in method step d), a type I collagen peptide hydrolysate is obtained by dissolution (in particular hydrolysis) of the expressed type I collagen, in particular a type I collagen peptide.

[0137] The type I collagen peptide hydrolysate obtained according to the invention by process step d) can be used as type I collagen peptide according to the invention in the form of this type I collagen peptide hydrolysate or after isolation of one or more type I collagen peptides, preferably in that case in homogeneous and isolated form.

[0138] In a particularly preferred embodiment of the invention, the homogenous and isolated type I collagen peptides present can also be mixed with one another to give a mixture of type I collagen peptides, where all type I collagen peptides have a molecular weight of at least 16 kDa, in particular at least 20 kDa.

[0139] Thus, in a particularly preferred embodiment, the present invention also relates to type I collagen peptides that are present in a homogenous and isolated form, uniform in molecular weight, or that are present in a mixture with recombinant or natural, especially recombinant, type I collagen peptides, or that are present as a hydrolysate of recombinant type I collagen, especially recombinant type I collagen peptides.

[0140] Nucleotide sequences encoding recombinant type I collagen peptides that can be used according to the present invention can be obtained in conventional manner, for example as described in WO 2005 / 012356, WO 01 / 34646, WO 01 / 34647 and WO 01 / 34801.

[0141] In a preferred embodiment of the invention, the type I collagen, in particular type I collagen peptide, usable according to the invention for use in a therapeutic method for the treatment of inflammatory diseases of the skin and mucous membranes of the human or animal body, preferably prepared according to the invention by one of the aforementioned methods, having a molecular weight of at least 16 kDa, in particular at least 20 kDa, is a non-hydroxylated, partially hydroxylated or fully hydroxylated type I collagen peptide, preferably a non-hydroxylated type I collagen peptide, preferably a partially hydroxylated type I collagen peptide, preferably a fully hydroxylated type I collagen peptide.

[0142] In a preferred embodiment of the invention, the type I collagen, in particular the natural or recombinant type I collagen peptide, having a molecular weight of at least 16 kDa, in particular at least 20 kDa, usable according to the invention and preferably prepared by any of the above-mentioned methods for use in a therapeutic method for treating inflammatory diseases of the skin and mucous membranes of the human or animal body, is a glycosylated collagen peptide. Preferably, the type I collagen, in particular the type I collagen peptide, is glycosylated in vivo, more preferably ex vivo.

[0143] In a further preferred embodiment of the invention, the type I collagen, in particular the type I collagen peptide, usable according to the invention, preferably prepared by one of the methods according to the invention, is a non-glycosylated type I collagen, in particular a type I collagen peptide.

[0144] According to the present invention, the term "biological effectiveness" is preferably understood to mean the immunomodulatory capacity of type I collagen, and in particular type I collagen peptides, which can be used according to the present invention, in particular the capacity to regenerate immunosuppressive M2 macrophages from peripheral monocytes and / or the capacity to regenerate immunosuppressive T suppressor cells (in particular from T precursor cells).

[0145] According to the present invention, the term "biological effectiveness" is preferably understood to mean the ability of type I collagen, and in particular type I collagen peptides, which can be used according to the present invention to inhibit the formation and activity of pro-inflammatory cytokines (in particular TNFα, IL-6, IL-1β and IFNγ) or to stimulate the synthesis and activity of anti-inflammatory cytokines (in particular IL-4, IL-10 and TGF-β), in particular both.

[0146] According to the invention, the term "biological effectiveness" is preferably understood to mean that the type I collagen, in particular the type I collagen peptides usable according to the invention, are used for immunomodulation, in particular for the regeneration of immunosuppressive M2 macrophages (in particular from peripheral monocytes) and / or for the regeneration of immunosuppressive T suppressor cells (in particular from T precursor cells) in order to suppress the formation of proinflammatory cytokines (in particular TNFα, IL-6, IL-1β and IFNγ) and for stimulating the synthesis of anti-inflammatory cytokines (in particular IL-4, IL-10 and TGF-β).

[0147] In a particularly preferred embodiment, the biological effectiveness is determined by detection methods known to the skilled artisan, in particular for the immunomodulatory (especially stimulating and inhibiting) activity of the substance, and for anti-inflammatory and pro-inflammatory cytokines. In particular, biological effectiveness in the sense of the present invention is determined by the methods described in Examples 2 to 4. According to the present invention, the term "biological effectiveness" is preferably also understood to mean the ability of type I collagen, in particular type I collagen peptides usable according to the present invention, to induce oral tolerance. In a particularly preferred embodiment, the presence of oral tolerance is determined by detection methods known to the skilled artisan, in particular for the determination of the ability of a substance to induce oral tolerance, in particular by the methods described in Examples 2 to 4.

[0148] In the context of the present invention, proinflammatory cytokines are in particular TNFα, IL-6, IL-1β and IFN-gamma (IFNγ).

[0149] In the context of the present invention, anti-inflammatory cytokines are in particular IL-4, IL-10, TGF-β.

[0150] In the context of the present invention, the term "inhibition" is understood to mean a partial or complete inhibition of the synthesis of a protein, which may in particular be manifested as a reduction or inhibition of protein synthesis or as a reduction or inhibition of the synthesis of mRNA associated with the protein.

[0151] In the context of the present invention, the term "collagen" is understood in its general sense in the art, in particular as defined, for example, in WO 01 / 34646. In a further preferred embodiment, the term "collagen" is understood to mean collagen proteins or peptides with the sequence glycine-proline, glycine-4-hydroxyproline or glycine-X-4-hydroxyproline, preferably with the repeat motif (Gly-XY)n, where X and Y may be any amino acid, preferably proline and 4-hydroxyproline. Particularly preferably, the term "collagen" is understood to mean peptides with the repeat motif (Gly-Pro-Y)n and / or (Gly-X-Hyp)m, where X and Y may be any amino acid.

[0152] "Type I collagen" according to the present invention is collagen in the general sense in the art as understood in accordance with the above, and type I collagen has the amino acid sequence of naturally occurring type I collagen, in particular the amino acid sequence of type I collagen of a vertebrate (in particular pig, sheep, cow, rodent, horse, bird, fish, reptile or amphibian) or an invertebrate (in particular jellyfish).

[0153] Type I collagen may exist as a monomeric collagen peptide, also referred to herein as a single-chain collagen peptide, or as a dimer or trimer, especially a trimer, having at least two, especially three collagen peptides (especially different or the same single-chain collagen peptides).In particular, type I collagen may exist as a triple helical type I collagen peptide, especially native type I collagen.

[0154] In the context of the present invention, the term "type I collagen peptide" is understood to mean a single chain of type I collagen peptide having an amino acid sequence present in type I collagen as defined above, where the peptide is an oligopeptide or a polypeptide. In particular, the type I collagen peptide may be present in a chemically modified form, in particular a hydroxylated and / or glycosylated form, or it may be unmodified.

[0155] Preferably, recombinant type I collagen, particularly type I collagen peptides, used in accordance with the present invention may have sequence variants, particularly function-conserving sequence variants, of naturally occurring type I collagen, particularly type I collagen peptides.

[0156] Thus, "type I collagen" is understood to mean naturally occurring type I collagen, in particular functionally conservative sequence variants of type I collagen peptides, in particular those which have at least 80%, 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 at the amino acid level to the amino acid sequence of naturally occurring type I collagen. According to the present invention, recombinant type I collagen is present when it has either an amino acid sequence that is identical to that present in naturally occurring type I collagen or a function-conserving sequence variant that has 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% amino acid sequence identity compared to naturally occurring type I collagen, particularly from vertebrates (particularly pigs, sheep, cows, rodents, kangaroos, horses, birds, reptiles, amphibians or fish) or from invertebrates (particularly jellyfish), and particularly when this amino acid identity is present compared to the naturally occurring type I collagen amino acid sequence from bovine origin.

[0157] In the context of the present invention, 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.

[0158] According to the present invention, the term "function-conserving sequence variant" is understood to mean a modification of a given, in particular a naturally occurring, amino acid sequence, in particular a substitution, addition and / or deletion of single or multiple amino acids, which results in an amino acid sequence that differs from the given amino acid sequence, but which retains the function characteristic of the given amino acid sequence, in particular its biological effectiveness.

[0159] Preferably, "function-conservative sequence variant" is understood to mean a modification of a given, in particular naturally occurring, amino acid sequence, in which the function, in particular the biological efficacy, characteristic of the given amino acid sequence 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%. Even more preferably, according to the present invention, "function-conservative sequence variant" is understood to mean a modification of a given amino acid sequence, in which the modified amino acid sequence has a sequence homology of 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% to the given amino acid sequence.

[0160] Particularly preferred are sequence variants, in particular "function-conservative sequence variants" in the context of the present invention, which are modifications of a given (particularly naturally occurring) amino acid sequence in which one or more amino acids having specific chemical-physical properties are replaced by one or more amino acids having the same or similar chemical-physical properties, respectively - in particular, for example, an amino acid having a non-polar side chain (Ala, Val, Met, Leu, Ile, Pro, Trp, Phe, etc.) is replaced by another amino acid having a non-polar side chain (Ala, Val, Met, Leu, Ile, Pro, Trp, Phe, etc.). replacement of an amino acid with a polar neutral side chain (such as Tyr, Thr, Gln, Gly, Ser, Cys, Asn) with another amino acid with a polar neutral side chain (such as Tyr, Thr, Gln, Gly, Ser, Cys, Asn), replacement of an amino acid with an acidic side chain (such as Glu, Asp) with another amino acid with an acidic side chain (such as Glu, Asp), and / or replacement of an amino acid with a basic side chain (such as Lys, Arg, His) with another amino acid with a basic side chain (such as Lys, Arg, His). In accordance with this embodiment, the chemical-physical properties of a given amino acid sequence are retained or only slightly altered in the "function-conserving sequence variants".

[0161] In a further embodiment, sequence variants, in particular "function-conserving sequence variants", refer to the fact that 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), such that a function, in particular a biological efficacy, characteristic of the given amino acid sequence, in particular a naturally occurring amino acid sequence, is retained, in particular a biological efficacy according to the invention (demonstration shown in particular in Example 2).

[0162] According to the invention, sequence variants, in particular "function-conserving sequence variants", are understood to mean modifications 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), is added to the given amino acid sequence, in particular a naturally occurring amino acid sequence, and which retains the function characteristic of the given amino acid sequence, in particular a naturally occurring amino acid sequence, and in particular the biological efficacy of the invention, in particular the biological efficacy as demonstrated in Example 2. According to the invention, 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), can be added to the N-terminus, the C-terminus and / or internally of the amino acid sequence.

[0163] In the context of the present invention, the term "amino acid modification" refers to a chemical modification of one or more amino acids that may occur before, after or during the synthesis of recombinant type I collagen, particularly type I collagen peptides, while retaining the original amino acid backbone of type I collagen peptides, particularly one or more proteinogenic amino acids. Thus, this term includes both the use of chemically modified amino acids to synthesize type I collagen, particularly type I collagen peptides, according to the present invention, and the chemical modification of amino acids after or during the synthesis of type I collagen, particularly type I collagen peptides. Typical amino acid modifications of collagen peptides are, in particular, hydroxylation of proline and lysine residues, as well as glycosylation of hydroxylated lysine residues. However, according to the present invention, this term also includes other chemical modifications of amino acids, such as phosphorylation, N-glycosylation, acetylation, methylation or myristoylation.

[0164] In the context of the present invention, recombinant type I collagen (particularly type I collagen peptides) or recombinantly prepared type I collagen (particularly type I collagen peptides) is understood to mean type I collagen, particularly type I collagen peptides, recovered by recombinant preparation by biotechnology using an expression system. According to the present invention, recombinant type I collagen (particularly type I collagen peptides) or recombinantly prepared type I collagen (particularly type I collagen peptides) have in common that they are not recovered from natural sources.

[0165] In a particularly preferred embodiment of the invention, the recombinant type I collagen, in particular the type I collagen peptide, is present in the form of a homogenous preparation consisting of this type I collagen or type I 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 type I collagen or type I collagen peptide. In a preferred embodiment, in the homogenous preparation, only type I collagen or type I collagen peptides of a certain size, i.e. a certain molecular weight, i.e. a single molecular species, in particular having the same amino acid sequence, are present. In a preferred embodiment of the invention, the recombinant type I collagen or type I collagen peptide is present in isolated form. In a particularly preferred embodiment of the invention, the recombinant type I collagen or type I collagen peptide is free from other proteins or peptides, in particular free from other substances (e.g. impurities), in particular free from non-protein substances, free from salts and / or free from other proteins or peptides.

[0166] In a particularly preferred embodiment of the invention, the recombinant type I collagen or type I collagen peptides having a molecular weight of at least 16 kDa also do not comprise collagens, in particular collagen peptides, having a molecular weight below 16 kDa.

[0167] In a particularly preferred embodiment of the present invention, the natural type I collagen, in particular the type I collagen peptide, is present in the form of a homogenous preparation of this type I collagen or type I 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 type I collagen or type I collagen peptide. In a preferred embodiment, in the homogenous preparation, only type I collagen or type I collagen peptides of a certain size, i.e. a certain molecular weight, i.e. a single molecular species, in particular having the same amino acid sequence, are present. In a preferred embodiment of the present invention, the natural type I collagen or type I collagen peptide is present in isolated form. In a particularly preferred embodiment of the present invention, the natural type I collagen or type I collagen peptide is free from other proteins or peptides, in particular free from other substances (e.g. impurities), in particular free from non-protein substances, free from salts and / or free from other proteins or peptides.

[0168] In a particularly preferred embodiment of the invention, the naturally occurring type I collagen or type I collagen peptides having a molecular weight of at least 16 kDa also do not include collagens, in particular collagen peptides, having a molecular weight below 16 kDa.

[0169] In the context of the present invention, the molecular weight is preferably determined by gel permeation chromatography.

[0170] In the context of the present invention, the term "gelatin" is understood in its general sense in the art, in particular as defined, for example, in WO 01 / 34646.

[0171] In the context of the present invention, the term "recombinant DNA" refers to an artificially prepared or engineered DNA molecule prepared in vitro by genetic engineering techniques. In a preferred embodiment, recombinant DNA is composed of components from different organisms of origin.

[0172] In the context of the present invention, the term "expression cassette" is understood to mean a DNA segment that is responsible for the transcription of the information encoded in this segment into RNA (in particular mRNA) and that has at least one promoter and a nucleotide sequence encoding a protein (generally having at least one promoter, at least one nucleotide sequence encoding a protein, and optionally a terminator).

[0173] In the context of the present invention, a "nucleotide sequence" is understood to mean a sequence (particularly a continuous sequence) of nucleotides of a nucleic acid, in particular a nucleic acid strand (particularly a DNA or RNA strand). A "nucleotide sequence" is therefore understood both as a unit of information and as a DNA or RNA strand that physically expresses this information.

[0174] In the context of the present invention, an "expression system" is understood to be a system in which a controlled biosynthesis of a protein of interest can take place. According to the present invention, the term "expression system" includes both cell-free expression systems, in which the components necessary for protein biosynthesis are not present inside the cell, i.e. the protein biosynthesis takes place outside the cell, and cell-based expression systems, in which the protein biosynthesis takes place inside a living cell. In the context of the present invention, a cell-free expression system is preferably a lysate or extract obtained from E. coli, insect cells, wheat germ, tobacco cells or mammalian cells (in particular CHO cells or reticulocytes from rabbits) which contain the components necessary for protein biosynthesis, in particular the translation and transcription systems. When a cell-free expression system is used in one of the methods according to the invention, the term "cultivating" is synonymous with "incubating".

[0175] In the context of the present invention, a "host cell" is understood to be a living cell capable of expressing a peptide or protein coded for by foreign DNA, in particular recombinant DNA.

[0176] According to the present invention, the term "recovering type I collagen, in particular type I collagen peptides" according to method step c) is understood to mean methods known to the skilled artisan for isolating type I collagen or type I collagen peptides from a composition comprising several components, for example by known isolation methods such as centrifugation (in particular differential centrifugation and / or density gradient centrifugation), chromatography (in particular gel filtration chromatography, ion exchange chromatography, affinity chromatography and / or high performance liquid chromatography), electrophoresis, filtration and / or extraction, and the concentration and purification of said components from a composition comprising several components can be achieved by preferably applying several isolation methods in succession. If necessary, collagen can be obtained by cleaving off the C-terminal and / or N-terminal procollagen fragments before, after or during extraction.

[0177] Fibrillation, chemical modification and secretion of the expressed collagen type I peptides can also be carried out preferably within the conditions of method step b).

[0178] According to the present invention, "conditions allowing the expression of type I collagen, in particular type I collagen peptides" is understood to mean conditions that activate or enhance the expression of type I collagen, in particular type I 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 type I collagen, in particular type I collagen peptides, is carried out within the scope of high cell density fermentation, in particular under high pressure, preferably under high atmospheric pressure. The specific conditions that allow the expression of type I collagen, in particular type I 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 type I collagen, in particular type I collagen peptides, may be constitutive or inducible, depending on the structure of the expression cassette.

[0179] In the context of the present invention, a method for the oral treatment of "inflammatory diseases of the skin and mucous membranes" is understood to mean a method for the prevention and / or treatment of inflammatory diseases of the skin and mucous membranes, in particular for the treatment of immunomodulatory inflammatory diseases of the skin and mucous membranes (in particular diseases of the intestinal mucosa), in which the administration of type I collagen is carried out orally.

[0180] In the present invention, inflammatory diseases of the skin and mucosa refer to diseases of the skin and mucosa caused, in particular, by an autoimmune reaction, in particular an excessive immune reaction, and in particular include skin diseases such as cutaneous systemic lupus erythematosus, dermatomyositis, lichen sclerosus, neurodermatitis, psoriasis, rosacea, lichen rubra, bullous pemphigoid, pemphigus vulgaris, and acne, as well as mucosal diseases such as Crohn's disease, celiac disease, and ulcerative colitis, in particular intestinal mucosal diseases.

[0181] According to the present invention, a therapeutic method for the oral treatment of "inflammatory diseases of the skin and mucous membranes" is preferably understood to also mean a method for inducing oral immune tolerance to endogenous collagen, in particular endogenous type I collagen, in particular endogenous type I collagen present in skin and mucosal tissues.

[0182] In the context of the present invention, "immunomodulated" inflammatory diseases of the skin and mucous membranes are understood to mean that the inflammatory diseases of the skin and mucous membranes are caused entirely or at least in part by autoimmune diseases and / or immune intolerance, in particular to type I collagen.

[0183] In the context of the present invention, the terms "comprise" and "have" are understood to mean that in addition to the elements expressly included by these terms, other elements not expressly mentioned can be added. In the context of the present invention, these terms are also understood to mean that only the elements expressly mentioned are included, 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 encompass compositions that, in addition to the elements expressly mentioned, also contain other elements that are not mentioned but have functionally and qualitatively subordinate properties. In this embodiment, the terms "comprise" and "have" are synonymous with the term "consisting essentially of".

[0184] In the context of the present invention, if the first and second decimal places or the second decimal place are not indicated, these digits are to be taken as zero.

[0185] In the context of 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 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).

[0186] Further preferred embodiments are set forth in the dependent claims.

[0187] Without limiting the general concept of the invention, the invention will now be described with reference to exemplary arrangements, embodiments using same and the associated figures.

[0188] SEQ ID NO:1 represents the nucleotide sequence encoding the 16 kDa bovine type I recombinant collagen peptide (CP16) (COL1A1 type;). SEQ ID NO:2 represents the amino acid sequence of the collagen peptide shown in SEQ ID NO:1. SEQ ID NO:3 represents the nucleotide sequence encoding the 20 kDa bovine type I recombinant collagen peptide (CP20) (COL1A1 type). SEQ ID NO:4 represents the amino acid sequence of the collagen peptide shown in SEQ ID NO:3. SEQ ID NO:5 represents the nucleotide sequence encoding the 45 kDa bovine type I recombinant collagen peptide (CP45) (COL1A1 type). SEQ ID NO:6 represents the amino acid sequence of the collagen peptide shown in SEQ ID NO:5. SEQ ID NO:7 represents the nucleotide sequence encoding the 92 kDa bovine type I recombinant collagen peptide (CP90) (COL1A1 type). SEQ ID NO:8 represents the amino acid sequence of the collagen peptide shown in SEQ ID NO:7. SEQ ID NO: 9 represents the amino acid sequence of a hydroxylated bovine control collagen peptide of 8 kDa (7912 Da), hereinafter referred to as CP9. [Brief description of the drawings]

[0189] [Figure 1] Graphs showing stimulation or inhibition of anti-inflammatory cytokines, pro-inflammatory cytokines, and formation of the surface marker CD86 in M2 macrophages (Figures 1 and 2), differentiated macrophages (Figures 3 and 4), T suppressor cells (Figures 5-8), and dermal fibroblasts (Figures 9-11). [Diagram 2] Graphs showing stimulation or inhibition of anti-inflammatory cytokines, pro-inflammatory cytokines, and formation of the surface marker CD86 in M2 macrophages (Figures 1 and 2), differentiated macrophages (Figures 3 and 4), T suppressor cells (Figures 5-8), and dermal fibroblasts (Figures 9-11). [Diagram 3]Graphs showing stimulation or inhibition of anti-inflammatory cytokines, pro-inflammatory cytokines, and formation of the surface marker CD86 in M2 macrophages (Figures 1 and 2), differentiated macrophages (Figures 3 and 4), T suppressor cells (Figures 5-8), and dermal fibroblasts (Figures 9-11). [Figure 4] Graphs showing stimulation or inhibition of anti-inflammatory cytokines, pro-inflammatory cytokines, and formation of the surface marker CD86 in M2 macrophages (Figures 1 and 2), differentiated macrophages (Figures 3 and 4), T suppressor cells (Figures 5-8), and dermal fibroblasts (Figures 9-11). [Diagram 5] Graphs showing stimulation or inhibition of anti-inflammatory cytokines, pro-inflammatory cytokines, and formation of the surface marker CD86 in M2 macrophages (Figures 1 and 2), differentiated macrophages (Figures 3 and 4), T suppressor cells (Figures 5-8), and dermal fibroblasts (Figures 9-11). [Figure 6] Graphs showing stimulation or inhibition of anti-inflammatory cytokines, pro-inflammatory cytokines, and formation of the surface marker CD86 in M2 macrophages (Figures 1 and 2), differentiated macrophages (Figures 3 and 4), T suppressor cells (Figures 5-8), and dermal fibroblasts (Figures 9-11). [Figure 7] Graphs showing stimulation or inhibition of anti-inflammatory cytokines, pro-inflammatory cytokines, and formation of the surface marker CD86 in M2 macrophages (Figures 1 and 2), differentiated macrophages (Figures 3 and 4), T suppressor cells (Figures 5-8), and dermal fibroblasts (Figures 9-11). [Figure 8] Graphs showing stimulation or inhibition of anti-inflammatory cytokines, pro-inflammatory cytokines, and formation of the surface marker CD86 in M2 macrophages (Figures 1 and 2), differentiated macrophages (Figures 3 and 4), T suppressor cells (Figures 5-8), and dermal fibroblasts (Figures 9-11). [Figure 9]Graphs showing stimulation or inhibition of anti-inflammatory cytokines, pro-inflammatory cytokines, and formation of the surface marker CD86 in M2 macrophages (Figures 1 and 2), differentiated macrophages (Figures 3 and 4), T suppressor cells (Figures 5-8), and dermal fibroblasts (Figures 9-11). [Figure 10] Graphs showing stimulation or inhibition of anti-inflammatory cytokines, pro-inflammatory cytokines, and formation of the surface marker CD86 in M2 macrophages (Figures 1 and 2), differentiated macrophages (Figures 3 and 4), T suppressor cells (Figures 5-8), and dermal fibroblasts (Figures 9-11). [Figure 11] Graphs showing stimulation or inhibition of anti-inflammatory cytokines, pro-inflammatory cytokines, and formation of the surface marker CD86 in M2 macrophages (Figures 1 and 2), differentiated macrophages (Figures 3 and 4), T suppressor cells (Figures 5-8), and dermal fibroblasts (Figures 9-11). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS EXAMPLES

[0190] Example 1: Preparation of recombinant type I collagen Recombinantly prepared hydroxylated bovine type I collagen peptides (designated CP16, CP20, CP45 and CP90) having the amino acid sequences shown in SEQ ID NOs: 2, 4, 6 and 8 were recovered by recombinantly expressing, in a Komagataella phaffi yeast strain capable of hydroxylating proline residues, the expression cassettes having the nucleotide sequences shown in SEQ ID NOs: 1, 3, 5 or 7, respectively. Similarly, as an experimental control, the collagen peptide CP9 (SEQ ID NO: 9) known from International Patent Application Publication No. WO2020 / 127929 was used.

[0191] The strains used for recombinant expression of collagen peptides in each case were obtained by integrating into their genome a nucleotide sequence encoding the respective bovine type I collagen peptide or a nucleotide sequence encoding monomeric prolyl-4-hydroxylase from Mimivirus (P4H).

[0192] Example 2: Proof of concept - immune and cytokine modulation by type I collagen peptides of Example 1 The following research was carried out on immune system cells downstream of Peyer's patches.

[0193] The intestinal lining is composed of mucus-secreting enterocytes and an underlying layer of loose connective tissue, the lamina propria. The mucosa is infiltrated by gut-associated lymphoid tissue (GALT), which is part of the immune system and is involved in defending the body against invading pathogens in the intestine.

[0194] The GALT consists of mesenteric lymph nodes of lymphoid tissue, the so-called Peyer's patches. Lymphoid follicles are surrounded by an epithelial layer and specialized M cells, which are characterized by a reduced microvilli border and glycocalyx and are exposed to the inner surface of the intestine.

[0195] M cells are specialized for the uptake of macromolecules such as soluble proteins, peptides, resident and pathogenic microorganisms, and viruses. Peyer's patches recognize and evaluate these macromolecules after transcytosis. Depending on the antigenic compounds, they switch on or off the body's immune response. Pathogens are delivered to mononuclear phagocytes and lymphocytes at the basement membrane of M cells.

[0196] Native type I collagen is recognized by Peyer's patches via its "active" epitope and glycopeptide side chains attached to the collagen.

[0197] Native type I collagen activates macrophages and dendritic cells, and T cell precursors in lymphoid follicles are activated to differentiate into type I collagen-induced regulatory T cells.

[0198] The mature T cells then enter the bloodstream via the thoracic duct and, after reaching the target tissue, release mediators that attenuate the immune response of the tissue by releasing the anti-inflammatory cytokines IL-10, IL-4, and TGF-β, and the pro-inflammatory cytokine TNF-α is downregulated in the target tissue.

[0199] Studies on the direct effects of collagen peptides on Peyer's patches are very difficult to achieve experimentally and are fraught with many methodological problems, so we investigated the effects of the tested peptides on downstream cellular systems of the immune system to demonstrate their effect on specific immune responses.

[0200] The immunomodulatory effect of type I collagen peptides recombinantly prepared according to Example 1 was measured in human peripheral blood monocytes (PBMCs). For this purpose, cells (3H10-25) purchased from 3H-Biomedical AB (Sweden) were used.

[0201] PBMC cells were first cultured in macrophage basal 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 relevant supplement mix (C-28055 supplement, PromoCell, Germany) and 1% amphotericin and 1% penicillin-streptomycin. After 4–24 h of static culture, dead non-adherent monocytes were decanted. Polarization of adherent monocytes into immunosuppressive macrophages of type 2b or type 2c was induced by adding 4 μg / mL of recombinant type I collagen, respectively. The cells were incubated with the samples to be analyzed for 6 days, respectively. Then, 1 μg / mL of lipopolysaccharide from Escherichia coli (LPS, L6529, Merck, Germany) was added to polarize activated macrophages.

[0202] After polarization, the differentiation pattern of the generated macrophages was investigated using specific cell differentiation markers (CD). The differentiation of monocytes into proinflammatory M1 macrophages or into immunosuppressive M2 macrophages was detected using specific markers. For this purpose, the M2 surface markers CD86, CD14, and CD163 were measured using ELISA (enzyme-linked immunosorbent assay). For CD86 (850590096 Diaclone, Holzel Diagnostics, Germany), CD14 (850780096 Diaclone, Hoelzel Diagnostics, Germany), and CD163 (ELH-CD163 RayBiotech, Hoelzel Diagnostics), the respective demonstrations were performed exactly according to the manufacturer's instructions. To exclude differentiation into proinflammatory M1 macrophages, the M1 macrophage markers CD86 (850590096 Diaclone, Hoelzel Diagnostics, Germany) and CD80 (EK0707 Boster PicoKine, Hoelzel Diagnostics, Germany) were also analyzed.

[0203] Furthermore, the effect of type I collagen peptides on the formation of proinflammatory cytokines (TNFα, IFNγ) and anti-inflammatory cytokine (IL-10) in the culture supernatant was examined. TNF (EK0525 Boster PicoKine, Hoelzel Diagnostics, Germany), IL-10 (950060096, Diaclone, Hoelzel Diagnostics, Germany), and IFNγ (EK0373, Boster PicoKine, Hoelzel Diagnostics, Germany) were measured using ELISA according to the manufacturer's instructions.

[0204] The differentiation of monocytes into M1 or M2 macrophages was confirmed using a special differentiation medium (C-28055, PromoCell, Germany) and specific culture additives. Alternatively, monocytes were cultured in macrophage differentiation medium (C-28055, PromoCell, Germany) and supplemented with GM-CSF supplement mix (C60420A, PromoCell, Germany) to indicate M1 differentiation or M-CSF GM-CSF supplement mix (C60442A, PromoCell, Germany) to indicate M2 differentiation.

[0205] The above type I collagen peptides CP90 (molecular weight approximately 92 kDa), CP45 (molecular weight approximately 45 kDa), CP20 (molecular weight approximately 20 kDa), CP16 (molecular weight approximately 16 kDa), and CP9 (molecular weight approximately 8 kDa) were used in the tests.

[0206] The data presented below demonstrate that the 92, 45, 20 and 16 kDa collagen peptides tested demonstrated statistically significant beneficial effects in regenerating immunosuppressive M2 macrophages from peripheral blood monocytes.

[0207] Studies were performed on M2 macrophages. Figure 1 and Table 1 show the mean values ​​(n=6) for the surface marker CD86 relative to untreated control cells. Unless otherwise stated, each measurement was statistically significantly different (p<0.05) from the control group. ns indicates no significant difference from the control group.

[0208] [Table 1]

[0209] Figure 2 and Table 2 show the mean values ​​(n=6) for IL-10 relative to untreated control cells. Unless otherwise noted, each measurement was statistically significantly different (p<0.05) from the control group. ns indicates no significant difference from the control group.

[0210] [Table 2]

[0211] The tested collagen peptides of 92, 45, 20, and 16 kDa showed a statistically significant beneficial effect in regenerating immunosuppressive M2 macrophages from peripheral blood monocytes compared to the control group, as shown by up to a 3.5-fold increase in the M2 macrophage surface marker CD86 (Figure 1) and a 1.8-fold increase in the synthesis of the anti-inflammatory cytokine IL-10 (Figure 2). In contrast, application of the short collagen peptide CP9 (8.0 kDa) did not show a statistically significant effect compared to the untreated control group in any study.

[0212] In differentiated macrophage cells, the cytokine synthesis profile indicated that the investigated collagen type I peptides had an anti-inflammatory effect. Moreover, the synthesis of pro-inflammatory cytokines was suppressed and the synthesis of anti-inflammatory IL-10 was induced.

[0213] The differentiated macrophages were studied as follows. Figure 3 and Table 3 show the mean values ​​(n=6) of IL-10 relative to untreated control cells. Unless otherwise stated, each measured value was statistically significantly different from the control group (p<0.05). ns indicates no significant difference from the control group.

[0214] [Table 3]

[0215] A statistically significant anti-inflammatory effect of the tested collagen type I peptides was demonstrated in differentiated macrophages, as the synthesis of IL-10 cytokine was increased by up to 5.75-fold (Figure 3). In contrast, the CP9 peptide showed only a very weak anti-inflammatory effect.

[0216] The demonstrated anti-inflammatory effects of the test peptides, as evidenced by the IL-10 data above, were confirmed in further experiments.

[0217] The following study was also performed on differentiated macrophages. Figure 4 and Table 4 show the mean values ​​(n=6) for TNF-α relative to untreated control cells. Unless otherwise stated, each measurement was statistically significantly different (p<0.05) from the control group. ns indicates no significant difference from the control group.

[0218] [Table 4]

[0219] Application of the tested collagen peptides resulted in a statistically significant reduction in the proinflammatory cytokine TNFα by up to 0.35-fold (Figure 4). All peptides tested, except for CP9, showed a clear TNFα-reducing effect.

[0220] Example 3: Proof of concept - Naive CD4+ cells stimulated by type I collagen peptides as described in Example 1 + Stimulation of T cell precursors After inducing monocytes into M2 macrophages with CP90, CP45, CP20 and CP16 (Example 2), the macrophage basal medium DXF (C-28057, PromoCell, Germany) was replaced with T cell culture medium (3H800-50-50, 3H Biomedical AB, Sweden). + T cell precursors (3H31-k, 3H Biomedical AB, Sweden) were added. Through direct cell-cell contact between naive T cell precursors and differentiated M2 macrophages and their cytokine cocktail, T cell precursors differentiate into collagen-specific (CP90, CP45, CP20 or CP16) regulatory T suppressor cells.

[0221] Since specific T cell clones are formed at low frequency, mature T suppressor cells were enriched by the ARTE (antigen-reactive T cell enrichment) method. T cells are identified by labeling the cells with cell surface marker (CD) antibodies conjugated to various dyes such as biotin and phycoerythrin, followed by labeling with anti-biotin and anti-PE microbeads. Magnetic microbeads aid in the isolation and collection of specific T cell clonal types. T cells can then be stained with fluorochrome-conjugated antibodies and quantified by flow cytometry. T suppressor cells are identified using forkhead box p3 (FoxP3) and CD25.

[0222] After differentiation, T suppressor cells secrete an anti-inflammatory cytokine cocktail consisting of IL-10, IL-4, and TGF-β.

[0223] The data determined showed that the tested collagen peptides CP90, CP45, CP20, and CP16 exhibited statistically significant beneficial effects in the regeneration of immunosuppressive T suppressor cells.

[0224] Differentiation of T suppressor cells was indicated by expression of forkhead box p3 (FoxP3).The effect of CP90, CP45, CP20, and CP16 collagen peptides on M2 macrophages was indicated by reduced production of proinflammatory cytokines by T suppressor cells.

[0225] The studies shown in Figures 5-8 were performed with T suppressor cells. Figure 5 and Table 5 show the mean values ​​(n=6) relative to untreated control cells for TNF-α. Unless otherwise stated, each measurement was statistically significantly different (p<0.05) from the control group. ns indicates no significant difference from the control group.

[0226] [Table 5]

[0227] Figure 6 and Table 6 show the mean values ​​(n=6) for IL-1β relative to untreated control cells. Unless otherwise noted, each measurement was statistically significantly different (p<0.05) from the control group. ns indicates no significant difference from the control group.

[0228] [Table 6]

[0229] Figure 7 and Table 7 show the mean values ​​(n=6) for IL-6 relative to untreated control cells. Unless otherwise noted, each measurement was statistically significantly different (p<0.05) from the control group. ns indicates no significant difference from the control group.

[0230] [Table 7]

[0231] Figure 8 and Table 8 show the mean values ​​(n=6) for IFNγ relative to untreated control cells. Unless otherwise noted, each measurement was statistically significantly different (p<0.05) from the control group. ns indicates no significant difference from the control group.

[0232] [Table 8]

[0233] The reduction in synthesis rates measured in immunosuppressive T suppressor cells of 0.56-fold for TNFα (Figure 5), 0.75-fold for IL-1β (Figure 6), 0.79-fold for IL-6 (Figure 7) and 0.87-fold for IFNγ (Figure 8) clearly indicated a statistically significant beneficial effect of the tested collagen peptides compared to untreated controls, however no corresponding positive effect was observed after application of CP9 compared to controls.

[0234] The determined data show that the tested type I collagen peptides (CP90, CP45, CP20, CP16) have a clear and statistically significant beneficial effect on the formation of proinflammatory cytokines in immunosuppressive T suppressor cells induced to differentiate from peripheral blood monocytes by M2 macrophages.

[0235] Example 4: Proof of concept - immunosuppression in dermal connective tissue cells Human dermal fibroblasts were cultured in Hams-F12 medium (HAM-12-A, Capricorn, Germany) supplemented with 1% amphotericin, 1% penicillin-streptomycin, and 10% calf serum. After reaching 100% cell confluence, 1 μg / ml lipopolysaccharide (Escherichia coli, L6529, Merck, Germany) was added to induce an inflammatory state in the fibroblasts. Addition of 25 μl / ml of cell supernatant from the T cell differentiation experiment (Example 3) reduced inflammation in the dermal fibroblasts.

[0236] Dermal fibroblasts were studied. Figure 9 and Table 9 show the mean values ​​(n=6) for IL-1β relative to untreated control cells. Unless otherwise stated, each measurement was statistically significantly different (p<0.05) from the control group. ns indicates no significant difference from the control group.

[0237] [Table 9]

[0238] Figure 10 and Table 10 show the mean values ​​(n=6) for TNF-α relative to untreated control cells. Unless otherwise noted, each measurement was statistically significantly different (p<0.05) from the control group. ns indicates no significant difference from the control group.

[0239] [Table 10]

[0240] Figure 11 and Table 11 show the mean values ​​(n=6) based on untreated control cells for IFNγ. Unless otherwise noted, each measurement was statistically significantly different (p<0.05) from the control group. ns indicates no significant difference from the control group.

[0241] [Table 11]

[0242] The anti-inflammatory effect of the tested collagen peptides and native type I collagen in dermal fibroblasts was demonstrated by a maximum 0.2-fold decrease in IL-1β expression (Figure 5), a maximum 0.85-fold decrease in TNFα expression (Figure 6), and a maximum 0.35-fold decrease in IFNγ expression (Figure 7). In contrast, the CP9 peptide did not show a statistically significant anti-inflammatory effect.

[0243] Finally, based on the reduced expression of proinflammatory cytokines (IL-1β, TNFα, IFNγ) in fibroblasts, real-time PCR was used to detect the anti-inflammatory effects of CP90, CP45, CP20, and CP16 in dermal fibroblasts of the target dermal tissues.

Claims

1. 1. Type I collagen for use in a therapeutic method for the oral treatment of inflammatory diseases of the skin and mucous membranes, in particular diseases of the intestinal mucosa, in a human or animal patient, said type I collagen having a molecular weight of at least 16 kDa.

2. 2. Type I collagen for use according to claim 1, wherein the inflammatory disease of the skin and mucosa is an immunomodulatory inflammatory disease of the skin and mucosa, in particular an autoimmune disease, in particular an autoimmune disease of the skin, such as cutaneous systemic lupus erythematosus, dermatomyositis, lichen sclerosus, neurodermatitis, psoriasis, rosacea, lichen rubra, bullous pemphigoid, pemphigus vulgaris and acne, or in particular an autoimmune disease of the mucosa, in particular an autoimmune disease of the intestinal mucosa, such as Crohn's disease, celiac disease or ulcerative colitis.

3. 3. Type I collagen for use according to claim 1 or 2, wherein the recombinant or natural type I collagen, in particular type I collagen peptides, having a molecular weight of at least 16 kDa, also do not include collagens, in particular collagen peptides, having a molecular weight of less than 16 kDa.

4. Type I collagen for use according to any one of claims 1 to 3, wherein the type I collagen is present in triple helical form.

5. 5. Type I collagen for use according to any one of claims 1 to 4, wherein the type I collagen is present in the form of a natural or recombinant type I collagen, in particular a type I collagen peptide, preferably a bovine type I collagen peptide comprising the amino acid sequence as shown in SEQ ID NO: 2, 4, 6 or 8.

6. Recombinant or natural type I collagen, in particular type I collagen peptides, for use according to any one of claims 1 to 5, wherein the molecular weight range of the collagen peptide is 35 to 95, in particular 40 to 92 kDa.

7. Type I collagen for use according to any one of claims 1 to 6, wherein the recombinant or natural type I collagen is in denatured or non-denatured form.

8. Type I collagen for use according to any one of claims 1 to 7, wherein the type I collagen is present fully or partially hydroxylated, fully or partially glycosylated, or fully or partially hydroxylated and glycosylated.

9. Type I collagen for use according to any one of claims 1 to 8, wherein the recombinant type I collagen has been prepared by expression in a eukaryotic host cell, in particular a yeast cell, or in a prokaryotic host cell, in particular E. coli, in particular in the form of a hydroxylated form and / or in the form of a fusion peptide.

10. Type I collagen for use according to any one of claims 1 to 9, wherein the recombinant or natural type I collagen, in particular the recombinant or natural type I collagen peptide, is present in a homogenous isolated form with a uniform molecular weight of at least 16 kDa or as an isolated type I collagen mixture, in particular a type I collagen peptide mixture, in which all type I collagens have a molecular weight of at least 16 kDa.

11. Type I collagen for use according to any one of claims 1 to 10, wherein the type I collagen is type I collagen of a vertebrate animal, in particular a pig, sheep, cow, rodent, kangaroo, horse, bird, reptile, amphibian or fish, or an invertebrate animal, in particular a jellyfish.

12. A composition comprising at least one type I collagen having a molecular weight of at least 16 kDa as described in any one of claims 1 to 11, at least one pharma- ceutically acceptable or food-safe carrier, and optionally at least one additive or excipient, for use in a therapeutic method for the oral treatment of inflammatory diseases of the skin and mucosa, in particular diseases of the intestinal mucosa.

13. 13. The composition for use according to claim 12, wherein at least one excipient is a corticosteroid.

14. 14. The composition for use according to claim 12 or 13, wherein the at least one additive is recombinantly produced collagen hydrolysate, collagen hydrolysate from a natural source, recombinantly produced type I collagen, type I collagen recovered from a natural source, or a combination thereof.

15. 15. The composition for use according to any one of claims 12 to 14, wherein the composition is in the form of a tablet, a troche, a chewable tablet, a powder, a granule, a hard capsule, a soft capsule, a capsule, a bite capsule, a dragee, a pastille, an extrudate, a juice, a suspension or a gel.

16. A composition for use according to any one of claims 12 to 15, suitable for oral administration of type I collagen in a dose of 1 to 60 mg / day.

17. Type I collagen, in particular a recombinant or natural type I collagen peptide, having a molecular weight of at least 16 kDa, for use in the therapeutic treatment or therapeutic prevention of immune intolerance to type I collagen, in particular in immunomodulatory inflammatory diseases of the skin and mucous membranes, in particular in diseases of the intestinal mucosa.

18. Type I collagen, in particular a recombinant or natural type I collagen peptide, having a molecular weight of at least 16 kDa, for use in a method for inducing immune tolerance to type I collagen, in particular in immunomodulatory inflammatory diseases of the skin and mucosa, in particular in intestinal mucosa diseases.

19. The use of recombinant or natural type I collagen, in particular recombinant or natural type I collagen peptides, having a molecular weight of at least 16 kDa, in a dosage form suitable for oral administration for maintaining the health of the skin and mucous membranes, in particular the health of the intestinal mucosa, of humans or animals.

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