Collagen peptide composition and its use in improving neurological conditions and fibrosis

Abstract

The present invention relates to a cyclic (glycine-proline) enriched peptide composition, and in particular to its use for improving neurological conditions or fibrosis. The present invention further relates to providing a collagen hydrolysate enriched with gly-pro-x tripeptide, and to a method for obtaining a cyclic (glycine-proline) enriched peptide composition, comprising heat-treating the collagen hydrolysate to cyclize at least a portion of the gly-pro-x tripeptide into a cyclic (glycine-proline) structure.

Description

[Technical Field]

[0001] This invention relates to a cyclic (glycine-proline) enriched collagen peptide composition and a method for obtaining a collagen peptide composition. The peptide composition can be used as an active ingredient in food supplements. [Background technology]

[0002] Oxidative stress is increasingly considered a major underlying mechanism in the pathogenesis of various conditions, including brain-related pathologies (e.g., neurological conditions, cognitive decline) and pathological wound healing such as fibrosis (Shroff et al. Curr Pathobiol Rep. 2014;2(4):257-267; Antar et al. Int J Mol Sci. 2023 Feb 16;24(4):4004).

[0003] Cyclic (glycine-proline) compounds (i.e., "cGP") are endogenous diketopiperazines and cyclic dipeptide compounds that may be derived from the cyclization of glycine-proline-glutamate (which is spontaneously cleaved from the IGF-1 N-terminus). Because cGP may exhibit anti-inflammatory and immunomodulatory effects and potentially reduce oxidative stress, it is considered that it may be suitable for improving brain-related conditions or pathological wound healing.

[0004] A drawback is that the synthesis of cyclic peptides such as cGP can be complex and expensive. Conventional methods for producing cGPs include their synthesis from amino acids and subsequent cyclization. These conventional methods are typically complex because they require the removal of organic solvents and resins used in solid-phase synthesis. Production costs can be a limiting factor in the development and availability of cGPs for therapeutic use.

[0005] Another drawback is that isolating CTP is thought to remove most of the dipeptides and free amino acids that typically contribute to the biological effects of collagen peptide compositions. The beneficial effects of cGP, when used alone, appear insufficient to lead to its widespread use (e.g., compared to pharmacological interventions). Therefore, a safer and more effective active ingredient or composition is needed.

[0006] The object of the present invention is to provide novel active ingredients or compositions that can overcome one or more of the above-mentioned limitations, can preferably be obtained from natural materials, and / or can be provided as functional foods or food supplements. A specific object of the present invention is to provide safe and effective active ingredients or compositions suitable for improving the healing of brain-related conditions (e.g., neurological conditions, cognitive impairment), or pathological wounds such as fibrosis. [Overview of the project] [Problems that the invention aims to solve]

[0007] The inventors have discovered a peptide composition that can be obtained from collagen and exhibits strong antioxidant, anti-(neuro)inflammatory, and anti-fibrotic properties. The peptide composition has been found to be particularly effective as an active ingredient for improving neurological disorders, in improving / preventing the decline of normal cognitive functions such as learning ability and memory ability, and / or in preventing and / or treating fibrosis.

[0008] The peptide composition of the present invention is characterized by its particularly high concentration of cyclic (glycine-proline) (i.e., cGP), and further by its content of (cyclic) di- and tripeptides, in particular the presence of glycine-proline-hydroxyproline and glycine-proline-alanine tripeptides (in their relative amounts).

[0009] The present inventors have identified a substance rich in the glycine-proline-x tripeptide sequence, particularly glycine-proline-hydroxyproline (e.g., 1.0 × 10⁻¹⁰). 4 ~1.0×10 5Rich in glycine-proline-alanine tripeptide (e.g., 5 × 10 ppm), 3 ~5×10 4 Heating of intermediate collagen hydrolysates rich in ppm sequences results in approximately 20 times the cGP (e.g., 1.0 × 10⁻¹⁰). 4 ~1.0×10 5 We found that enriching (pp) yields a peptide composition containing a desired amount of (cyclic) di- and tripeptides.

[0010] The inventors have further discovered that cyclization by heating results in a change in the relatively low molecular weight fraction, particularly yielding a peptide composition with a characteristic peak in the molecular weight range of 250-450 Da, which was not evident before cyclization. Since cGP exhibits a typical peak in the size exclusion chromatogram at 250-450 Da due to its shape, it is considered that cGP (Mw=154 Da) contributes to this characteristic peak. Therefore, the molecular weight distribution before and after cyclization is also characteristic of the product disclosed herein.

[0011] It was surprisingly discovered that cGP and further peptides in the peptide composition possess synergistic antioxidant, anti-(neuro)inflammatory, and anti-fibrotic properties. In particular, the combination of cGP with glycine-proline-hydroxyproline and glycine-proline-alanine tripeptide revealed synergistic antioxidant, anti-(neuro)inflammatory, and anti-fibrotic effects. While not bound by theory, the inventors believe that further collagen dipeptides may also contribute to antioxidant properties. In addition, or alternatively, various peptide combinations may together provide the greatest health benefits. [Means for solving the problem]

[0012] In a first aspect, the present invention relates to a peptide composition which can be desired to be obtained by the method disclosed herein, wherein the composition is 1.0 × 10 4 A cyclic (glycine-proline) content of ppm or more, preferably 1.0 × 10⁶. 4 ppm~1.0×105 ppm of cyclic (glycine-proline), 1.0×10 3 ppm or more of glycine-proline-hydroxyproline tripeptide, preferably 1.0×10 3 ~1.0×10 4 ppm of glycine-proline-hydroxyproline tripeptide, and / or 1.0×10 1 ppm or more of glycine-proline-alanine tripeptide, preferably 1.0×10 1 ~1.0×10 3 ppm of glycine-proline-alanine tripeptide. ppm is calculated relative to the total weight of peptides in the peptide composition.

[0013] In a second aspect, the present invention relates to a method for obtaining a peptide composition comprising cyclic (glycine-proline), the method comprising a) providing a collagen hydrolyzate comprising 1.0×10 4 ppm or more of gly-pro-x tripeptide, calculated relative to the total weight of peptides in the collagen hydrolyzate, and having a weight average molecular weight of 500 Da to 3000 Da, and b) preferably comprising heat-treating the collagen hydrolyzate at a temperature of at least 65°C to obtain a peptide composition enriched in cyclic (glycine-proline).

[0014] In a third aspect, the present invention relates to a peptide composition obtainable optionally by the method disclosed herein for use as a medicament.

[0015] In a fourth aspect, the present invention relates to a peptide composition obtainable optionally by the method disclosed herein for use in the prevention and / or treatment of a condition selected from the group consisting of oxidative stress, inflammation, neurological conditions and fibrosis.

[0016] In a fifth aspect, the present invention relates to the non-therapeutic use of the peptide compositions disclosed herein, preferably in the non-therapeutic improvement of cognitive function.

[0017] In a further embodiment, the present invention relates to a topical formulation comprising a peptide composition disclosed herein.

[0018] In a further embodiment, the present invention relates to a cosmetic composition comprising a peptide composition disclosed herein, which is suitable, for example, for the non-therapeutic improvement of skin appearance in fibrous conditions.

[0019] In a further embodiment, the present invention relates to a substrate coated with a peptide composition as defined herein, wherein the substrate is preferably a medical implant. [Modes for carrying out the invention]

[0020] (Peptide composition) Various embodiments of the present invention are exemplified by peptide compositions that can be optionally obtained by the methods disclosed herein.

[0021] When referring to peptide compositions, ppm in this disclosure is calculated relative to the total weight of peptides in the peptide composition unless otherwise specified. When referring to specific collagen hydrolysates, ppm in this disclosure is calculated relative to the total weight of peptides in the collagen hydrolysate unless otherwise specified.

[0022] In one embodiment, the peptide composition of the present invention is • Cyclic (glycine-proline) (e.g., 1.0 × 10⁻⁶) 4 (pp or higher), • Glycine-proline-hydroxyproline tripeptide (e.g., 1.0 × 10⁻⁶) 3 ppm or higher), and / or • Glycine-proline-alanine tripeptide (e.g., 1.0 × 10) 1 Includes (or above ppm)

[0023] In one embodiment, the peptide composition is 1.0 × 10 4 ~2.0×10 5 ppm, for example, 1.0 × 10⁻⁶ 4 ~1.0×10 5 Preferably contains ppm of cyclic (glycine-proline).

[0024] In one embodiment, the peptide composition of the present invention contains at least 1.0 × 10 4 It contains ppm of a cyclic (glycine-proline) molecule. Preferably, the peptide composition contains 1.0 × 10⁻⁶ ppm. 4 ~1.0×10 5 Contains ppm of cyclic (glycine-proline).

[0025] In the context of the present invention, it is preferable to determine the concentration of peptides in the composition by UPLC-MS analysis (ultrahigh-performance liquid chromatography-mass spectrometry).

[0026] In the context of the present invention, the term “cyclic (glycine-proline)” (i.e., cGP) means a cyclic dipeptide containing the amino acids glycine and proline, typically formed by the condensation of a glycine- and (hydroxy)proline-containing linear dipeptide (e.g., Gly-Pro, Pro-Gly, Gly-Hyp, or Hyp-Gly) or a linear tripeptide (e.g., Gly-Pro-Z, Pro-Gly-Z, Gly-Hyp-Z, or Hyp-Gly-Z (where Z is any of the amino acids)). In cGP, the amino acids are linked together in a cyclic or closed-loop configuration, typically via a peptide bond between the carboxyl group of glycine and the amino group of proline. In the context of the present invention, “cyclic glycine-proline” means the same as, and can be used without distinction from, “cyclo(Gly-Pro)”, “cyclo(prolylglycyl)”, “cyclic proline-glycine”, “cyclo(Pro-Gly)”, and “cyclo(glycylprolyl)”.

[0027] In one embodiment, the peptide composition of the present invention contains at least 1.0 × 10 3It contains ppm of glycine-proline-hydroxyproline tripeptide. Preferably, the peptide composition contains 1.0 × 10⁻¹⁶ ppm. 3 ~1.0×10 4 Contains ppm of glycine-proline-hydroxyproline tripeptide.

[0028] In one embodiment, the peptide composition of the present invention contains at least 1.0 × 10 1 It contains ppm of glycine-proline-alanine tripeptide. Preferably, the peptide composition is 1.0 × 10 1 ~1.0×10 3 Contains ppm of glycine-proline-alanine tripeptide.

[0029] 1.0 × 10 used in this specification 1 This means the same as 1.0 × 10 (i.e., 10). 1.0 × 10 as used in this specification 2 This means the same as 1.0 × 100 (i.e., 100). 1.0 × 100 as used in this specification 3 This means the same as 1.0 × 1000 (i.e., 1000). 1.0 × 1000 as used in this specification 4 This means the same as 1.0 × 10000 (i.e., 10000). 1.0 × 10 as used herein 5 This means the same as 1.0 × 100000 (i.e., 100000). 1.0 × 10 as used herein 6 This means the same as 1.0 × 1,000,000 (i.e., 1,000,000).

[0030] In one embodiment, the peptide composition has a weight-average molecular weight of 800 to 2500 Da (Daltons). In another embodiment, the peptide composition has a weight-average molecular weight of 1000 to 2000 Da. In yet another embodiment, the peptide composition has a weight-average molecular weight of 1200 to 1800 Da, preferably 1500 to 1700 Da.

[0031] The peptide composition preferably has a weight-average molecular weight (Mw) in the range of 500 Da to 5000 Da, preferably 750 Da to 3000 Da, more preferably 1000 to 2500 Da, and even more preferably 1200 to 2000 Da. In addition, or alternatively, the peptide composition may have a weight-average molecular weight of at least 500 Da, 600 Da, 700 Da, 800 Da, 900 Da, 1000 Da, 1100 Da, 1200 Da, 1300 Da, 1400 Da, 1500 Da, 1750 Da, 2000 Da, 2250 Da, 2500 Da, 2750 Da, or 3000 Da. In addition, or alternatively, the peptide composition may have a weight-average molecular weight of up to 3500 Da, 3000 Da, or 2500 Da, or 2250 Da, or 2000 Da, or 1750 Da, or 1700 Da, or 1650 Da, or 1600 Da.

[0032] A preferred method for measuring the (average) molecular weight and / or molecular weight distribution of collagen hydrolysates (from which the molecular weight distribution can also be derived) is by high-speed size exclusion chromatography (HPSEC). The following protocol is a preferred HPSEC protocol: Agilent HPLC 1260 Infinity series (G1316A, G1329B, G1311C, G1315D) equipped with TSKgel SWXL pre-columns and G2000SWXL columns (Tosoh Bioscience) is used. Analysis is performed using WinGPC software (PSS). The eluent is 100 mM phosphate buffer pH 5.3. The sample is eluted from the column (e.g., 0.5 mL / min, isocratic) and monitored by UV detection (e.g., 214 nm, analysis time: 40 minutes per injection + 180 minutes equilibrium). Calibration is performed using a Narrow Calibration Standard (Low FILK).

[0033] While not bound by theory, the inventors believe that the relative amounts of cyclic peptides (especially cGP) and other peptides (especially gly-pro-x, gly-pro-hyp, and gly-pro-ala) in a peptide composition contribute to its biological activity, and that further collagen dipeptides may also contribute.

[0034] In one embodiment, the peptide composition is 1.0 × 10 2 ~1.0×10 5 The peptide composition comprises ppm total gly-pro-x total tripeptide and / or total tripeptide. In one embodiment, the peptide composition is 5.0 × 10 2 ~1.0×10 4 The peptide composition contains ppm of gly-pro-x tripeptide and / or total tripeptide. In one embodiment, the peptide composition is 1.0 × 10 3 ~8.0×10 3 The peptide composition contains ppm of gly-pro-x tripeptide and / or total tripeptide. In the embodiment, the peptide composition is 3.0 × 10 3 ~6.0×10 3 The peptide composition contains ppm of gly-pro-x tripeptide and / or total tripeptide. In one embodiment, the peptide composition is 2.0 × 10 3 ~5.0×10 3 Contains ppm of gly-pro-x tripeptide and / or total tripeptide.

[0035] In embodiments of the present invention, the peptide composition is 1.0 × 10 3 ~2.0×10 5 ppm, preferably 1.0 × 10⁻¹⁴ 4 ~1.0×10 5 ppm, more preferably 2.0 × 10 4 ~8.0×10 4 ppm cGP, more preferably 4.0 × 10 4 ~6.0×10 4 Includes ppm cGP

[0036] In embodiments of the present invention, the peptide composition is 5.0 × 10 2 ~1.0×10 4ppm, preferably 2.0 × 10 3 ~8.0×10 3 ppm, more preferably 3.0 × 10 3 ~7.0×10 3 ppm, more preferably 4.0 × 10 3 ~6.0×10 3 Contains ppm of gly-pro-hyp tripeptide.

[0037] In one embodiment, the peptide composition of the present invention contains at least 0.1 ppm of glycine-proline-hydroxyproline tripeptide, or at least 1 ppm of glycine-proline-hydroxyproline tripeptide, or at least 5 ppm of glycine-proline-hydroxyproline, or at least 10 ppm of glycine-proline-hydroxyproline. In addition, or alternatively, in embodiments, the peptide composition of the present invention contains 10,000 ppm or less of glycine-proline-hydroxyproline, or 7,500 ppm or less of glycine-proline-hydroxyproline, or 5,000 ppm or less of glycine-proline-hydroxyproline. In preferred embodiments, the peptide composition of the present invention contains 1 to 1,000 ppm of glycine-proline-hydroxyproline tripeptide, preferably 5 to 500 ppm of glycine-proline-hydroxyproline tripeptide, and more preferably 10 to 100 ppm of glycine-proline-hydroxyproline tripeptide. In preferred embodiments, the peptide composition of the present invention contains 0.1 to 8.0 × 10 3 ppm, preferably 1 to 7.0 × 10 3 Contains ppm of glycine-proline-hydroxyproline tripeptide.

[0038] In embodiments of the present invention, the peptide composition is 1.0 × 10 1 ~1.0×10 4 ppm, preferably 2.0 × 10 1 ~0.5 × 10 4 ppm, more preferably 5.0 × 10⁻¹⁰ 1 ~1.0×10 3 ppm, more preferably 1.0 × 10 2~5.0×10 2 Contains ppm of gly-pro-ala tripeptide.

[0039] In embodiments of the present invention, the peptide composition contains 0.1 to 50 ppm, preferably 0.5 to 20 ppm, and more preferably 1 to 10 ppm of pro-gly-pro tripeptide. In embodiments of the present invention, the peptide composition contains 0.1 to 50 ppm, preferably 0.5 to 20 ppm, and more preferably 1 to 10 ppm of pro-hyp-gly tripeptide.

[0040] In various embodiments, the peptide composition contains 100 to 2500 ppm, preferably 200 to 2000 ppm, of hydroxylysine. The amount of hydroxylysine in the peptide composition may be preferably 300 to 1500 ppm, more preferably 400 to 1000 ppm. In various embodiments, the peptide composition contains 1000 to 50000 ppm, preferably 2000 to 25000 ppm, of hydroxyproline. The amount of hydroxyproline in the peptide composition may be preferably 5000 to 30000 ppm, more preferably 10000 to 20000 ppm, and even more preferably 14000 to 18000 ppm.

[0041] Preferably, the peptide composition is characterized by a cGP:Gly-Pro-Hyp weight ratio of 1:1 to 1000:1, preferably 10:1 to 100:1, calculated with respect to the total weight of peptides in the peptide composition. Preferably, the peptide composition is characterized by a cGP:Gly-Pro-Ala weight ratio of 10:1 to 10000:1, preferably 100:1 to 1000:1. Preferably, the peptide composition is characterized by a cGP:(Gly-Pro-Hyp+gly-pro-ala) weight ratio of 1:1 to 1000:1, preferably 10:1 to 100. Preferably, the peptide composition is characterized by a cGP:Gly-Pro-x weight ratio of 1:1 to 1000:1, preferably 10:1 to 100:1. Preferably, the peptide composition is characterized by a Gly-Pro-Hyp:Gly-Pro-Ala weight ratio of 1:1 to 1000:1, preferably 10:1 to 100:1. Preferably, the peptide composition is characterized by a cGP:total linear dipeptide weight ratio of 1:1 to 10000:1, preferably 1:10 to 1:1000. Preferably, the peptide composition is characterized by a total linear dipeptide:total linear dipeptide weight ratio of 10:1 to 1:10, preferably 5:1 to 1:5, and even more preferably 2:1 to 1:2.

[0042] In embodiments of the present invention, the peptide composition contains 0.1 to 100 ppm, preferably 0.5 to 50 ppm, more preferably 1 to 20 ppm, and even more preferably 2 to 15 ppm of pro-hyp dipeptide. In embodiments of the present invention, the peptide composition contains 0.1 to 200 ppm, preferably 0.5 to 100 ppm, more preferably 1 to 50 ppm, and even more preferably 5 to 30 ppm of hyp-gly dipeptide. In embodiments of the present invention, the peptide composition contains 1.0 × 10 -1 (i.e., 0.1) ~ 1.0 × 10 4 ppm, preferably 1.0 × 10⁻¹⁴ 1 (i.e., 10) ~ 0.5 × 10 4 It contains ppm of pro-gly dipeptide. The amount of pro-gly dipeptide is more preferably 1.0 × 10⁻⁶ 2 ~1.0×10 3 ppm, or 2.0 × 10⁻⁶ 2~8.0×10 2 ppm. In an embodiment of the present invention, the peptide composition is 1.0×10 1 ~5.0×10 4 ppm, preferably 1.0×10 2 ~1.0×10 4 ppm of gly-pro dipeptide. The amount of the gly-pro dipeptide is more preferably 5.0×10 2 ~8.0×10 3 ppm, or 1.0×10 3 ~4.0×10 3 ppm. In an embodiment of the present invention, the peptide composition contains 0.1 to 100 ppm, preferably 0.5 to 50 ppm of ala-pro dipeptide. The amount of the ala-pro dipeptide is more preferably 1 to 20 ppm, or 2 to 40 ppm.

[0043] In an embodiment of the present invention, the peptide composition contains collagen dipeptides in an amount of less than 5.0×10 4 ppm, preferably less than 1.0×10 4 ppm. The amount of the collagen dipeptide may be less than 5.0×10 3 ppm or less than 3.0×10 3 ppm. The amount of the collagen dipeptide may be, for example, 1.0×10 2 ~2.0×10 4 ppm. More preferably, the amount of the collagen dipeptide is 0.5×10 3 ~2.0×10 4 , or 1.0×10 3 ~1.0×10 4 ppm, or 2.0×10 3 ~5.0×10 3 ppm.

[0044] Those skilled in the art know how to convert ppm values ​​to mg / kg, mg / g, or wt.% values ​​for the concentration of a peptide in a peptide composition. In the case of solids, 1 ppm is equal to 1 mg of substance per 1 kg of solid (mg / kg). Therefore, a value of 1000 ppm of peptide X in a peptide composition is calculated relative to the total weight of the peptide in the peptide composition and is equal to 1000 mg / kg, 1 mg / g, or 0.1 wt.%.

[0045] In embodiments of the present invention, the peptide composition contains 0 to 5% by weight, preferably 0.1 to 2% by weight, and more preferably 0.2 to 1% by weight, of peptides having a molecular weight of up to 250 Da.

[0046] In embodiments of the present invention, the peptide composition contains 1 to 25% by weight, preferably 5 to 15% by weight, and more preferably 7 to 13% by weight, of a peptide having a molecular weight of 250 to 450 Da.

[0047] In embodiments of the present invention, the peptide composition contains 1 to 25% by weight, preferably 5 to 15% by weight, and more preferably 7 to 13% by weight, of peptides having a molecular weight of up to 450 Da.

[0048] In embodiments of the present invention, the peptide composition contains 10 to 40% by weight, preferably 15 to 35% by weight, and more preferably 20 to 30% by weight, of peptides having a molecular weight of 450 to 1000 Da.

[0049] In embodiments of the present invention, the peptide composition contains 2 to 30% by weight, preferably 5 to 20% by weight, and more preferably 7 to 15% by weight, of peptides having a molecular weight of 0 to 500 Da or up to 500 Da.

[0050] In embodiments of the present invention, the peptide composition contains 10 to 40% by weight, preferably 15 to 35% by weight, and more preferably 20 to 30% by weight, of peptides having a molecular weight of 500 to 1000 Da.

[0051] In embodiments of the present invention, the peptide composition contains 20 to 70% by weight, preferably 35 to 50% by weight, of peptides having a molecular weight of 1000 to 2000 Da.

[0052] In embodiments of the present invention, the peptide composition contains 10 to 40% by weight, preferably 15 to 30% by weight, of a peptide having a molecular weight of 2000 to 5000 Da.

[0053] In embodiments of the present invention, the peptide composition contains 0.1 to 10% by weight, preferably 0.5 to 5% by weight, of a peptide having a molecular weight of 5000 to 10000 Da.

[0054] In embodiments of the present invention, the peptide composition contains a maximum of 2% by weight, preferably 0.01 to 2% by weight, and more preferably 0.1 to 1% by weight, of peptides having a molecular weight of more than 10,000 Da, calculated relative to the total weight of the total peptides in the peptide composition.

[0055] Preferably, the peptide composition contains 10 to 60% by weight, more preferably 20 to 40% by weight, of peptides having a molecular weight of less than 1000 Da.

[0056] The peptide composition according to the present invention preferably has a polydispersity of 1.3 to 1.9, more preferably 1.4 to 1.8, and even more preferably 1.5 to 1.7.

[0057] The peptide composition obtained by this method may, if desired, be subjected to processes such as filtration, centrifugation, concentration, ultrafiltration, freeze-drying, and / or powdering. The peptide composition is preferably provided as a dry powder.

[0058] The peptide composition of the present invention is preferably a collagen peptide composition. In addition, or alternatively, in one embodiment, the peptide composition of the present invention is obtained entirely from collagen, more preferably from a collagen hydrolysate.

[0059] (Use of peptide composition) Oxidative stress can occur when there is an imbalance between the production of reactive oxygen species (ROS) and the body's ability to neutralize them using antioxidants. ROS are highly reactive molecules that can damage cellular components such as proteins, lipids, and DNA. This damage is generally thought to trigger inflammation, neuroinflammatory responses, and / or responses that activate fibrogenic pathways. Chronic inflammation is also considered a significant driver of fibrosis by promoting fibroblast activation.

[0060] The in vitro 2,2-diphenyl-1-picrylhydrazyl (DPPH) assay measures the ability of a substance or composition to neutralize free radicals, particularly DPPH radicals, and evaluates its antioxidant activity. The DPPH assay is used in the fields of biochemistry, pharmacology, and food science to screen and evaluate the antioxidant capacity of various substance compositions.

[0061] The inventors demonstrated, using a DPPH assay, that cGP and further peptides in the peptide composition of the present invention possess remarkable synergistic antioxidant and anti-inflammatory properties, including anti-neuroinflammatory properties. In particular, synergistic, antioxidant, and anti-inflammatory effects were observed with combinations of cGP with glycine-proline-hydroxyproline and glycine-proline-alanine tripeptides. While not bound by theory, the inventors believe that further collagen dipeptides may also contribute to antioxidant properties. In addition, or alternatively, various peptide combinations may together provide the highest health benefits.

[0062] Various embodiments of the present invention are exemplified by the peptide compositions disclosed herein for use as pharmaceuticals.

[0063] In one embodiment, the peptide composition disclosed herein is the active ingredient.

[0064] In preferred embodiments, the present invention relates to the use of peptide compositions disclosed herein for reducing oxidative stress. In preferred embodiments, the present invention relates to peptide compositions disclosed herein for use in the prevention and / or treatment of oxidative stress. Peptide compositions are also useful in preventing and / or treating conditions or symptoms associated with oxidative stress, for example, but not limited to, inflammatory conditions (e.g., rheumatoid arthritis, inflammatory bowel disease), fibrosis, infections, autoimmune diseases (e.g., rheumatoid arthritis, thyroid disease), neurological conditions, cancer, kidney diseases (e.g., chronic kidney disease, nephropathy), atherosclerosis, or eye diseases (e.g., age-related macular degeneration, cataracts).

[0065] In the context of the present invention, the term “prevent and / or treat” may also be encompassed by the term “improve,” which means to include both “prevent” and “treat” a disease. As used herein, “improve” also encompasses “cure.”

[0066] In the context of this invention, the term “oxidative stress” means a pathological condition characterized by excessive oxidative activity and / or decreased antioxidant levels. Cellular oxidative activity may include, but is not limited to, one or more of the following: oxygen radicals (superoxide anions, hydroxyl radicals, and / or peroxyl radicals); reactive non-radical oxygen species, such as hydrogen peroxide and singlet oxygen; carbon radicals; nitrogen radicals; and sulfur radicals. The pathological conditions of oxidative stress can result, for example, in cell damage, impaired cell function, and / or cell death.

[0067] In preferred embodiments, the present invention relates to the use of peptide compositions disclosed herein for the prevention and / or treatment of diseases characterized by inflammation and / or inflammatory components, for example, but not limited to inflammation (e.g., neuroinflammation), fibrosis, diseases characterized by acute inflammation caused by bacterial or viral infection, such as meningitis, sepsis, malaria, or chronic inflammation, such as rheumatoid arthritis, osteoarthritis, psoriasis, chronic bronchitis, chronic obstructive pulmonary disease, inflammatory bowel disease (ulcerative colitis and Crohn's disease), and multiple sclerosis.

[0068] In preferred embodiments, the present invention relates to the use of peptide compositions disclosed herein for reducing inflammation.

[0069] Antioxidants can play a role in mitigating inflammation by neutralizing reactive oxygen species (ROS) and free radicals, which are often associated with inflammatory processes. The inventors believe that the peptide compositions of the present invention are particularly suitable for reducing inflammation and treating inflammatory conditions. In particular, oxidative stress is involved in various neurological conditions, especially neuroinflammatory conditions and / or neurodegenerative diseases. Accumulating evidence suggests that mitochondrial dysfunction and oxidative stress play a particularly significant role in aging and age-dependent neuropathy. While not bound by theory, compounds with antioxidant and / or anti-inflammatory effects are considered useful in treating these conditions, including by targeting underlying causes.

[0070] In various embodiments, the present invention relates to peptide compositions disclosed herein (which can be obtained by this method) for producing pharmaceuticals, preferably pharmaceuticals for preventing and / or treating the conditions disclosed herein. In preferred embodiments, the present invention relates to peptide compositions for producing pharmaceuticals for preventing and / or treating conditions selected from the group consisting of oxidative stress, inflammation, neurological conditions and fibrosis.

[0071] In various embodiments, the present invention relates to a method for preventing and / or treating a subject, comprising administering to the subject a peptide composition disclosed herein (which can be obtained by this method). The method preferably relates to a method for preventing and / or treating an individual suffering from a pathological condition disclosed herein, for example, a subject suffering from a condition selected from the group consisting of oxidative stress, inflammation, neurological conditions and fibrosis. The method preferably relates to administering an amount of the peptide composition effective in preventing and / or treating the pathological condition.

[0072] (Use in improving fibrosis) Wound healing is a dynamic, multi-stage process necessary for the reconstruction and regeneration of tissue after injury. The wound healing process can be categorized into the following four distinct stages: (1) Hemostasis that stops bleeding, accompanied by vasoconstriction, primary hemostasis and secondary hemostasis, (2) Inflammatory stage in which damaged cells and dead cells are removed along with the pathogen or necrotic tissue fragments, (3) Proliferative stage in which new tissue formation and angiogenesis occur, (4) A remodeling stage in which appropriate tensile strength is achieved through the rearrangement, degradation, and resynthesis of the extracellular matrix (especially collagen).

[0073] Abnormal or excessive responses in one or more stages of wound healing can lead to an undesirable amount of pathological fibrous tissue formation, known as fibrosis or scar tissue formation. Activation of fibroblastic or highly contractile myofibroblasts is considered a major cause of the excessive accumulation of stiff scar tissue seen in pathological fibrotic conditions. While mild fibrosis to a certain extent may be part of the normal tissue replacement process, excessive fibrosis can cause physical problems, as well as significant psychological, emotional, and social problems. Some examples of the adverse effects of excessive fibrosis are illustrated herein.

[0074] Firstly, fibrosis can manifest as an organ-specific pathology. Organ-specific fibrosis can have various underlying causes, but is often associated with inflammation and / or arises as a symptom of an underlying disease. Uncontrolled fibrosis can lead to the destruction of the underlying organ structure and impaired organ function. Organ-specific fibrotic disorders include, for example, pulmonary fibrosis, pulmonary hypertension, asthma, chronic obstructive pulmonary disease, hepatic fibrosis, renal fibrosis, non-alcoholic steatohepatitis (NASH), musculoskeletal fibrosis, atrial fibrosis, Dupuytren's contracture, cellulite, endometriosis, and keloid formation in connective tissue.

[0075] Secondly, fibrosis can occur after tissue manipulation resulting from surgery, i.e., in the form of postoperative fibrosis. For example, postoperative fibrosis is a typical problem seen after abdominal, nerve, spinal, vascular, or thoracic surgery, or other types of surgery using both classical open surgery and arthroscopic / laparoscopic techniques. Postoperative fibrosis can negatively impact surgical and healing outcomes. For example, glaucoma surgery is a difficult procedure and is not widely performed clinically due to the severe complications that can result from excessive fibrosis. Postoperative adhesions (PA) are an example of fibrous tissue, which are the most common driving factors of long-term pathological conditions after abdominal, thoracic, and pelvic surgery, for example.

[0076] Thirdly, fibrosis can occur around medical implants and / or devices, for example, due to trauma, abnormal inflammation / infection, foreign body reaction, and / or (micro) movement of the implant / device. In such cases, the fibrosis may also be called implant-associated fibrosis. For example, breast implants, as well as methods of breast reconstruction and augmentation, are well known and have been used for over 20 years. However, the formation of excessive fibrosis remains a significant problem associated with breast implants. Excessive fibrosis can result in pathological constriction around the implant, also known as capsular contracture. Capsular contracture can result in an implant that is deformed, painful, hard, and can lead to an unnatural appearance and feel. To name a few examples, undesirable fibrosis around implants is also typically seen with implantable sensors, drug delivery devices, pacemakers, cardiovascular stents, and orthopedic implants. Encapsulation of such implants by a fibrous membrane can "shield" the implant from the body, thus hindering proper tissue integration and potentially requiring the removal of the implant.

[0077] Fourth, cutaneous fibrosis can occur as a result of acute injury or skin irritation, and most can be classified as either hypertrophic scars or keloids. The difference between hypertrophic scars and keloids is that hypertrophic scars typically develop within the original wound boundary and tend to regress over time, while keloids typically proliferate unrestricted and rarely regress. Hypertrophic scars or keloids often result from cuts, burns, acne scars, chickenpox scars, perforations, abrasions, surgical cuts, tattoos, tattoo removal, insect bites, and vaccination sites, to name a few. The mechanisms of hypertrophic scar and keloid formation are not yet fully understood, but because the degree of inflammation tends to positively correlate with the final scar size, inflammation is thought to be involved in regulating collagen synthesis.

[0078] Finally, fibrosis is a common symptom resulting from medical conditions such as fatty liver disease, chronic obstructive pulmonary disease, asthma, or systemic sclerosis.

[0079] Several antifibrotic drugs are known that can play a role in alleviating excessive fibrosis. These include the pulmonary fibrosis-approved drug pirfenidone, or the pan-αv integrin inhibitor for pulmonary fibrosis. The limitations of these drugs are that they require approval from federal agencies and a medical prescription, and are associated with several adverse effects. For example, pirfenidone alleviates fibrosis by downregulating the production of growth factors and procollagen I and II. However, pirfenidone is also associated with gastrointestinal, skin, and hepatic side effects. However, as for other non-pharmaceutical antifibrotic products, including both conventional plant-based materials and proteins, there is currently no strong evidence to support their efficacy. Overall, currently known antifibrotic products appear to be inadequately effective, particularly in limiting tissue contraction and activation and / or transdifferentiation in skin cells and / or stromal cells, such as myofibroblasts.

[0080] In particular, the need for active ingredients that can improve fibrosis more effectively and / or safely remains unmet. Specifically, there is a need for active ingredients that can prevent fibrosis as implantable coatings, for example, by improving oxidative stress, and / or improving inflammation, by reducing cell contraction, activation, and / or transdifferentiation in skin cells and / or stromal cells, such as myofibroblasts, and by reducing cell contraction, activation, and / or transdifferentiation. These ingredients have the characteristic of inhibiting myofibroblasts and contraction.

[0081] The inventors have found that the peptide compositions disclosed herein may be particularly effective in improving fibrosis. While not limited by theory, the mechanism of action is thought to involve contraction, activation, redifferentiation, and / or prevention of collagen (type I) overproduction in skin cells and / or stromal cells (e.g., fibroblasts and myofibroblasts).

[0082] This finding by the inventors is surprising because cGP has been suggested in the art as a promoter of type I and type V collagen production (JP 2019206481A), and therefore has been suggested as a suitable anti-aging agent for skin. Considering that fibrosis is characterized by excessive collagen synthesis (mainly type I collagen), this would suggest that cGP would worsen fibrosis. However, the inventors have unexpectedly found that cGP, in combination with other peptides, can enable collagen synthesis while simultaneously inhibiting the excessive collagen synthesis that is a prominent feature of fibrosis. While not bound by theory, providing balanced collagen synthesis may, in part, be a mechanism by which cGP, in combination with other peptides, effectively prevents and / or treats fibrosis.

[0083] Embodiments of the present invention include peptide compositions disclosed herein for use in the prevention and / or treatment of fibrosis. In one of the above examples, the peptide compositions may improve excessive fibrosis and thus provide an alternative to the antifibrotic agents described herein.

[0084] In the context of this invention, the term "fibrosis" refers to a pathological condition characterized by abnormal production, accumulation, and / or deposition of extracellular matrix (proteins) by skin cells and / or stromal cells, particularly fibroblasts and / or myofibroblasts. For example, "abnormal production" means that the production of extracellular matrix (proteins) exceeds the rate of degradation. The term "fibrosis" also encompasses abnormal contraction of the extracellular matrix (proteins). For example, "abnormal contraction" means that the contraction is too high, resulting in excessively stiff tissue and negative functional and / or cosmetic effects. Typically, contraction occurs during the proliferative phase when cell-rich granulation tissue is formed. Those skilled in the art will recognize that contraction is an important part of wound healing because it enables wound closure. However, wound contraction has both positive and negative effects. Wound contraction is beneficial for wound healing by narrowing the wound periphery and leading to wound closure, but excessive contraction can cause undesirable contractures and scarring, resulting in cosmetic and functional problems (Li et al. J Tissue Viability. 2011 Nov;20(4):108-120).

[0085] In the context of the present invention, there are various methods for measuring the presence (and inhibition) of fibrosis, which include in vitro and in vivo methods. Preferred in vitro models include the hydrogel contraction assay and / or myofibroblast transformation assay disclosed herein in the examples. Preferred in vivo models include, for example, the subcutaneous or intraperitoneal implantation model disclosed by Farah et al. (Nat Mater. 2019 August;18(8):892-904) for testing the efficacy of peptide loading and / or peptide-coated implants.

[0086] Suitable models for testing fibrosis include models developed to assess the amount of xenobody reaction (FBR) in vitro or in vivo. For example, Sharifi et al. (Adv Healthc Mater. 2019 Feb;8(4):e1801425) developed an in vitro FBR-on-a-chip model as a physiologically relevant in vitro setting that replicates innate immune cell interactions with implants. In the context of this invention, the FBR-on-a-chip model is a suitable model for testing fibrosis.

[0087] In the context of this invention, the prevention and / or treatment of fibrosis may be therapeutic or non-therapeutic, depending on the severity of the fibrosis and / or the context. A qualified physician can determine on a case-by-case basis whether the fibrosis is pathological or in a pathological subject, or non-pathological or in a healthy subject. Therapeutic use may mean, for example, use in severe fibrosis and / or pathological situations, and / or use when there are symptoms of pain and distress that may lead to (serious) health and psychological risks. Non-therapeutic use may mean, for example, use of fibrosis that is not accompanied by symptoms of pain and distress and / or that resolves spontaneously over time (i.e., is not chronic). Non-therapeutic use particularly includes, for example, cosmetic use to improve the appearance of fibrotic skin, but in non-pathological circumstances and / or when there are no symptoms of pain and distress.

[0088] In preferred embodiments, fibrosis is one or more selected from the group consisting of medical implant-related fibrosis, surgery-related fibrosis, postoperative adhesion formation including adhesion formation, cutaneous fibrosis, hypertrophic scarring, keloids, hepatic fibrosis, pulmonary fibrosis, pancreatic fibrosis, renal fibrosis, vascular fibrosis, endometriosis, atrial fibrosis, and heart fibrosis.

[0089] In preferred embodiments, the fibrosis is organ-specific fibrosis. Organ-specific fibrosis is preferably one or more selected from the group consisting of pulmonary fibrosis, hepatic fibrosis, pancreatic fibrosis, renal fibrosis, musculoskeletal fibrosis, atrial fibrosis, cardiac fibrosis, Dupuytren's contracture, cellulite, endometriosis, and keloid formation in connective tissue.

[0090] In a preferred embodiment, fibrosis is a symptom of a condition selected from the group consisting of pulmonary hypertension, fatty liver disease, Dupuytren's contracture, cellulite, chronic obstructive pulmonary disease, non-alcoholic steatohepatitis (NASH), asthma, and systemic sclerosis.

[0091] In one embodiment, the fibrosis disclosed herein is not the pathophysiology of cystic fibrosis, and / or the peptide composition is not for the prevention and / or treatment of the pathophysiology of cystic fibrosis. Cystic fibrosis is a genetic pathology that typically causes damage and scarring to various organs and may require treatment for thick mucus formation and infection. Nevertheless, the peptide composition may prevent and / or treat fibrosis as a symptom of cystic fibrosis (without targeting the disease itself). Therefore, in one embodiment, fibrosis is fibrosis (as a symptom) in the pathophysiology of cystic fibrosis.

[0092] In one embodiment, the fibrosis disclosed herein is not the pathophysiology of myelofibrosis, and / or the peptide composition is not for the prevention and / or treatment of pathological myelofibrosis. Myelofibrosis is a myelohemorrhagic cancer in which healthy bone marrow is typically replaced by scar tissue. Prevention and / or treatment of myelofibrosis may require treatment targeting the cause and / or underlying myeloproliferative pathology of these diseases. Nevertheless, the peptide composition may prevent and / or treat fibrosis as a symptom of myelofibrosis (without targeting the disease itself). Therefore, in one embodiment, fibrosis is fibrosis (as a symptom) in pathological myelofibrosis.

[0093] The inventors have found a dose-response effect of the peptide composition of the present invention in improving fibrosis. Therefore, the present invention also relates to the optimal amount of the peptide composition for achieving the highest efficacy in the prevention and / or treatment of fibrosis.

[0094] While not bound by theory, various mechanisms may exist that, individually or in combination, produce beneficial antifibrotic effects.

[0095] The inventors have found that peptide compositions are remarkably effective in preventing fibrosis by at least partially preventing contraction, activation, and / or transdifferentiation in skin cells and / or stromal cells, particularly fibroblasts and / or myofibroblasts. In particular, it has been found that peptide compositions can improve the differentiation (transition) of skin cells (e.g., fibroblasts) and / or stromal cells into myofibroblasts.

[0096] Furthermore, the inventors have also found that the peptide composition inhibits the overproduction of collagen and / or provides balanced collagen synthesis, which is most commonly found in type I collagen (e.g., in skin cells and / or stromal cells).

[0097] Excessive collagen production is typically a prominent feature of fibrosis. The cells responsible for collagen production in fibrosis are thought to be fibroblasts and myofibroblasts. For example, pulmonary fibrosis and other types of fibrosis typically promote the dominance of at least type I and / or type III collagen, but type V collagen and other types of collagen may also be overexpressed in fibrosis. In addition, other extracellular matrix proteins such as fibronectin, elastin, laminin, hyaluronan, chondroitin sulfate, and / or heparin sulfate proteoglycans are often increased.

[0098] While not bound by theory, providing balanced collagen synthesis may, in part, be a highly effective mechanism by which the peptide composition of the present invention improves (especially prevents) fibrosis without interfering with normal wound healing (by not completely stopping collagen synthesis).

[0099] In preferred embodiments, fibrosis is prevented and / or treated by improving, preferably reducing, cell activation. In preferred embodiments, fibrosis is prevented and / or treated by improving, preferably reducing, cell differentiation. In preferred embodiments, fibrosis is prevented and / or treated by improving, preferably reducing, collagen overproduction.

[0100] In one embodiment, the peptide composition reduces or increases the migration of cells, preferably skin cells and / or stromal cells. In one embodiment, the peptide composition reduces or increases the proliferation of cells, preferably skin cells and / or stromal cells. In one embodiment, the peptide composition reduces or increases the differentiation (conversion) of cells, preferably skin cells and / or stromal cells. In one embodiment, the peptide composition reduces or increases the activation of cells, preferably skin cells and / or stromal cells. In one embodiment, the peptide composition reduces or increases the contraction of the extracellular matrix, skin cells and / or stromal cells. In one embodiment, the peptide composition reduces or increases wound contraction.

[0101] In preferred embodiments, the peptide composition improves, and preferably reduces, the differentiation (conversion) of skin cells (e.g., fibroblasts) and / or stromal cells into myofibroblasts.

[0102] In one embodiment, a reduction in contraction means, for example, a reduction of at least 5%, preferably at least 10%, more preferably at least 20%, even more preferably at least 30%, and most preferably at least 50%, compared to a negative control group (untreated). The reduction in contraction is preferably measured by the collagen hydrogel contraction assay disclosed herein.

[0103] In one embodiment, a decrease in differentiation and / or transdifferentiation means, for example, a decrease of at least 5%, preferably at least 10%, more preferably at least 20%, even more preferably at least 30%, and most preferably at least 50%, compared to a negative control group (untreated). The decrease in differentiation and / or transdifferentiation is preferably measured by a myofibroblast transformation assay disclosed herein.

[0104] In one embodiment, the peptide composition reduces or inhibits the overexpression of one or more extracellular matrix proteins, preferably one or more collagens, more preferably one or more of type I, type III, and / or type V collagens, (for example, in skin cells and / or stromal cells). In one embodiment, the peptide composition promotes the balanced synthesis of one or more extracellular matrix proteins, preferably one or more collagens, more preferably one or more of type I, type III, and / or type V collagens, (for example, in cells such as skin cells and / or stromal cells).

[0105] In the context of the present invention, the term “skin cell” means any type of cell that is primarily found in the skin (especially the epidermis) and / or any type of cell that provides a specific function in the skin (especially the epidermis), and / or forms the skin (especially the epidermis), and includes keratinocytes, melanocytes, Langerhans cells and Merkel cells. In the context of the present invention, the term “stromal cell” means any multipotent or pluripotent cell that can specifically develop into different types of connective tissue cells. A stromal cell may be a fibroblast, myofibroblast, smooth muscle cell, pericyte or mesenchymal stem / stromal cell. In the context of the present invention, the term “myofibroblast” means a cell type that lies between a fibroblast and a smooth muscle cell during differentiation. "Myofibroblasts" may be formed, for example, by one or more of the following pathways: partial smooth muscle differentiation of fibroblasts, activation of stellate cells, loss of contractile phenotype of smooth muscle cells, direct myofibroblast differentiation of progenitor cells present in interstitial tissue, homing and recruitment of circulating mesenchymal precursors that can differentiate directly as described above or indirectly via other cell types as intermediates, and / or transepithelial-to-mesenchymal differentiation of epithelial cells. In preferred embodiments, myofibroblasts are cells that are positive (e.g., indicated by staining) for markers such as intermediate filament vimentin, α-smooth muscle actin, and paladin.

[0106] (Coating, immobilization) A known strategy for mitigating fibrosis around medical implants is to coat (e.g., immobilize) their surfaces with anti-fibrotic molecules in an attempt to reduce excessive fibrosis. These include coatings based on anti-inflammatory, anti-fibrotic, anti-proliferative, and / or angiogenic agents to prevent excessive cellular fibrosis encapsulation. For example, U.S. Patent No. 6110155 describes a medical catheter carrying the anti-inflammatory corticosteroid dexamethasone as a means of preventing histofibrosis. However, it is preferable to avoid anti-inflammatory agents in order to avoid adverse effects on normal wound healing and to avoid the common side effects of corticosteroids and other anti-inflammatory agents.

[0107] Advantageously, given the limitations described above, the peptide compositions disclosed herein appear particularly well-suited for preventing fibrosis around medical implantable devices, for example, for preventing and / or treating medical implant-associated fibrosis.

[0108] In one embodiment, the present invention relates to a substrate coated with a peptide composition disclosed herein.

[0109] In one embodiment, the peptide composition is provided as a coating on a substrate. The substrate is preferably a medical device such as a medical implant. The medical device is preferably selected from the group consisting of suture materials, drug-eluting beads, cardiovascular implants, stents, catheters, pacemakers, electrical leads, implantable cardiac defibrillators, nerve stimulators, biodegradable implants, interocular lenses, implantable drug delivery devices, implantable pumps, intrauterine devices, surgical mesh implants, breast implants, and prostheses.

[0110] In the context of this invention, the term “medical implant” means a device or tissue placed inside or on the surface of a body. A medical implant may be a prosthesis intended to replace a missing body part. A medical implant may be intended to deliver drugs (e.g., as a drug delivery device), monitor bodily function, or provide support to organs and tissues. The term “medical implant” encompasses implants made from skin, bone, or other body tissue. In the context of this invention, a “medical implant” may be placed permanently or may be removed when it is no longer needed. For example, a stent or hip implant is typically intended to be permanent, while a chemotherapy port or screw for repairing a fracture may be removed when it is no longer needed. In the context of this invention, the term “medical device” means any instrument, machine, or implant intended to treat, cure, prevent, alleviate, or diagnose a disease, and includes any medical device as classified as such by the European Union Medical Devices Regulation and / or the U.S. Food and Drug Administration.

[0111] In various embodiments, the peptide composition is immobilized on a substrate. In the context of the present invention, the term “immobilization” means that a molecule (e.g., a peptide) is chemically linked to a substrate, and the linkage may be direct (e.g., by direct chemical conjugation) or indirect (e.g., via a linker moiety or after chemical functionalization of the substrate). Immobilization in the context of the present invention may be reversible or irreversible. In one embodiment, the peptide composition is preferably not released from the substrate after immobilization. Immobilization in the context of the present invention includes non-specific immobilization (i.e., resulting in randomly oriented immobilized molecules), such as non-covalent adsorption or covalent non-specific adhesion.

[0112] Molecules or compounds may be immobilized directly onto a substrate without the need for a linker. In addition, or alternatively, one or more linker molecules may be provided as an intermediate between the substrate and the molecule to be immobilized. In the context of the present invention, “linker” preferably means a chemical linker that provides one or more chemical bonds as disclosed herein. In one embodiment, the substrate is functionalized during or after the immobilization of the molecule to be immobilized, for example, when the substrate is considered to be chemically inert and / or lacks functionalized moieties for chemical conjugation. In the context of the present invention, the term “functionalization” means altering the physical and / or chemical properties of a surface, preferably providing functional groups that enable improved immobilization of molecules.

[0113] In various embodiments, the peptide composition is provided in an amount of 0.00001 to 10 g, preferably 0.0001 to 1 g, more preferably 0.001 to 0.1 g, and even more preferably 0.01 to 0.1 g, which corresponds to a surface area of ​​1 cm² of the substrate. 2 This is the average amount per unit.

[0114] In various embodiments, the peptide composition is provided on a carrier material (i.e., a carrier) on a substrate. The carrier material may be, for example, a biodegradable material and may allow for more sustained and / or controllable release of the embedded molecule. The carrier may be, for example, one or more of polymer films, microparticles, hydrogels, collagen gels, extracellular matrix gels, or mixtures thereof. Examples of suitable carrier materials in the context of the present invention include carbohydrates and polysaccharides, such as starch, cellulose, dextran, methylcellulose, and hyaluronic acid; proteins or polypeptides, such as albumin, fibrin collagen, and gelatin, or combinations thereof.

[0115] In various embodiments, the substrate and / or carrier material may be fibrous, and the fibers may be within a diameter range of microns and / or nanometers, such as 100 nm to 50 μm, 200 nm to 10 μm, 500 nm to 5 μm, or 1 to 2 μm.

[0116] In various embodiments, the peptide composition is provided in the carrier in an amount calculated relative to the weight of the carrier, for example, 0.00001 to 25 wt.%, or 0.0001 to 10 wt.%, or 0.005 to 1 wt.%. Based on in vitro dose-response curves, the inventors found that preferred concentrations for antifibrosis may be in the range of 0.0001 to 5 wt.%, preferably 0.001 to 1 wt.%, and more preferably 0.01 to 0.1 wt.%, relative to the weight of the carrier.

[0117] Preferably, the carrier conforms to and / or provides an emission profile that lasts for at least 2 days, or 3 days, or 4 days, or 5 days, or 6 days, or 1 week, or 2 weeks, or 3 weeks, or 4 weeks, or 6 weeks, or 8 weeks, or 12 weeks. Preferably, the carrier conforms to and / or provides an emission profile that lasts for up to 12 weeks, or 8 weeks, or 6 weeks, or 4 weeks, or 3 weeks, or 2 weeks, or 1 week.

[0118] Those skilled in the art will know, for example, how to adapt a carrier to a suitable release rate and / or release profile by selecting a suitable polymer for the mixture, the concentration of the polymer, and the thickness of the coating.

[0119] In one embodiment, the peptide composition is releaseable from a substrate and / or coating. In one embodiment, the substrate and / or coating is configured for the release of the peptide composition. In one embodiment, the release is sustained release. In one embodiment, the release follows a sustained release profile.

[0120] As used herein, the terms “sustained release” or “sustained release profile” mean that a compound substance (e.g., a peptide) is released gradually over a long period of time (e.g., several days, several weeks, or several months). In one embodiment, the amount released over the initial period is equal to or less than the amount released over the same period several days or several weeks later. Preferably, the sustained release profile lasts for at least one week, or two weeks, or three weeks, or four weeks, or six weeks, or eight weeks, or twelve weeks.

[0121] In one embodiment, the release exhibits a substantially linear release rate. In the context of the present invention, the term “linear release rate” means that a substance or compound (e.g., a peptide) is released at a rate that does not change by more than about 20%, preferably 10%, over a desired period of time.

[0122] In one embodiment, the peptide composition is released for at least 2 days, or 3 days, or 4 days, or 5 days, or 6 days, or 1 week, or 2 weeks, or 3 weeks, or 4 weeks, or 6 weeks, or 8 weeks, or 12 weeks. In addition, or alternatively, the peptide composition is released for up to 12 weeks, or 8 weeks, or 6 weeks, or 4 weeks, or 3 weeks, or 2 weeks, or 1 week.

[0123] In one embodiment, the peptide composition is released in an average amount of 0.0001g to 10g, 0.001g to 1g, or 0.01g to 0.1g per day.

[0124] The desired release rate, release profile, and / or release amount may vary depending on the body part and / or application.

[0125] In the context of this invention, “release” preferably means in vivo release, i.e., post-implantation release. In vivo release can be determined by implanting a coated sample having a known peptide content, taking samples at various time points, and then determining the amount of residual peptide (i.e., non-released peptide) in the sample. Based on these values, a release profile curve can be plotted. In addition, or alternatively, “release” can preferably be determined in vitro under conditions that approximate or mimic in vivo conditions (temperature, pH, aqueous medium, and / or presence of enzymes, etc.) that may affect the release rate in vivo. A preferred in vitro protocol for measuring release involves incubating the sample in phosphate-buffered saline (PBS) at 37°C and collecting / refreshing the PBS containing the eluted peptide at various time points. The concentration of peptide in the released sample can be measured by any preferred method, such as HPLC. A suitable HPLC protocol for measuring cyclic glycine-proline involves using two eluents, A[0.1%(v / v)TFA / water] and B[0.1%(v / v)TFA / CAN], and linearly increasing eluent B: 0% (0-15 min), 0%-50% (15-45 min), 50%-0% (45-50 min), and 0% (50-60 min). A suitable flow rate is 1.0 mL / min, and a suitable column temperature is 30°C. Elution can be monitored by absorbance at 214 nm. The concentration is normalized against a calibration curve obtained by diluting the stock solution with PBS.

[0126] (Use in improving neurological conditions / brain-related functions) Maintaining or improving neurological and cognitive function is important for general well-being throughout life. A decline in neurological and cognitive function typically results in reduced daily functioning and overall health, often due to adverse effects on several neurological functions, such as neuromotor, neurosensory, or cognitive functions (more specifically, memory, mental flexibility and speed, and learning). This decline is becoming increasingly evident in older adults as part of the aging population and is recognized as a major health and social problem.

[0127] There are several pathological conditions that impair the function of the nervous system and brain, including neurodegenerative and / or neuroinflammatory conditions (e.g., Alzheimer's disease, Parkinson's disease), brain injury (e.g., cerebral ischemia), dementia, depressive disorders, or neuropathy.

[0128] In daily life, the efficiency of individual nerve and brain function can make all sorts of differences, so the need for active ingredients suitable for improving neurological conditions characterized by pathologically impaired function of the nervous system (e.g., resulting from disease or disorder), or for active ingredients that can simply improve nerve function by enhancing and / or preventing the decline of normal cognitive abilities such as learning and memory, is not met.

[0129] Currently, most methods for improving neurological conditions involve pharmacological interventions. For example, cholinesterase inhibitors such as donepezil (Aricept®), galantamine (Razadyne®, Razadyne ER®, Reminyl®, Nivalin®), and rivastigmine tartrate (Exelon®) are known to be effective in treating Alzheimer's disease. Cyclic antidepressants such as amitriptyline and nortriptyline are prescribed for the treatment of neuropathy. Strategies to improve cognitive function typically involve improving neurotransmitter levels and signaling in neurotransmitter pathways thought to be involved in the processes of learning ability and memory formation.

[0130] The main drawback of the aforementioned pharmacological interventions is that they typically have side effects, which limits their widespread use and, in some cases, leads to discontinuation of treatment. In addition, none of the aforementioned pharmacological interventions are considered sufficiently effective, which hinders their widespread use.

[0131] The peptide compositions disclosed herein offer safe and effective alternatives to the pharmacological interventions described above.

[0132] The D-galactose model is a well-recognized animal model that mimics the escalating loss of cognitive and motor abilities in age-dependent neurological disorders (Parameshwaran et al. Rejuvenation Res. 2010 Dec;13(6):729-735). The D-galactose model is used to test diseases such as Alzheimer's disease, Parkinson's disease, cognitive decline, neuroinflammation, ischemic stroke, and age-related changes in the central nervous system. We have shown that the peptide composition of the present invention has the following remarkable therapeutic benefits in the D-galactose model, although the effects do not appear to be limited to this list: • Increased spatial memory and learning, • Reduced neuropathy / Improved neuromotor function (rotarod latency), neuromuscular function (grip strength), and electrophysiological function. • Reduced neuroinflammation.

[0133] Embodiments of the present invention include peptide compositions disclosed herein for use in the prevention and / or treatment of (central) neurological conditions or symptoms resulting therefrom, the neurological conditions encompass one or more selected from inflammation and / or injury of the central nervous system, motor conditions or motor disorders, neuromotor disorders, neuromuscular disorders, neurosensory disorders, psychotic conditions or psychotic disorders, depressive conditions or depressive disorders, anxiety conditions or anxiety disorders, injury of the nervous system and / or brain, cognitive conditions or cognitive disorders, personality conditions or personality disorders, mood conditions or mood disorders, migraines, epilepsy or convulsive disorders, neurodegenerative conditions or neurodegenerative disorders, autistic disorders, neuropathy, neurodevelopmental disorders, and neurotoxicity.

[0134] Inflammation and / or injury of the central nervous system may include concussion injury or traumatic injury, and one or more resulting cognitive impairment, learning impairment, or memory impairment.

[0135] The motor conditions or motor disorders disclosed herein include Parkinson's disease, secondary parkinsonism (e.g., post-encephalitis parkinsonism, Lewy body disease, basal ganglia degenerative diseases, extrapyramidal disorders and motor disorders (e.g., but not limited to tremor, essential tremor and drug-induced tremor, myoclonus, chorea and drug-induced chorea, drug-induced tics and organic tics, drug-induced acute dystonia, drug-induced tardive dyskinesia, L-dopa-induced dyskinesia), and neurological disorders. It may be one or more selected from the group consisting of, for example, neuroleptic-induced movement disorders (including, but not limited to, neuroleptic-induced neuroleptic malignant syndrome (NMS), neuroleptic-induced parkinsonism, neuroleptic-induced early-onset dyskinesia or neuroleptic-induced acute dyskinesia, neuroleptic-induced acute dystonia, neuroleptic-induced acute akathisia, cerebral palsy, neuroleptic-induced tardive dyskinesia, and neuroleptic-induced tremor), restless limb syndrome, and Stiffman syndrome.

[0136] The psychotic conditions or psychotic disorders disclosed herein may be one or more selected from the group consisting of schizophrenia (e.g., paranoid, disorganized, catatonic, undifferentiated, or residual), schizophrenia-like disorders, schizoaffective disorders (e.g., paranoid or depressive), delusional disorders, substance-induced psychotic disorders (e.g., psychotic abnormalities induced by alcohol, amphetamines, cannabis, cocaine, hallucinogens, inhalants, opioids, or phencyclidine), paranoid personality disorders, and schizotypal personality disorders.

[0137] The anxiety conditions or anxiety disorders disclosed herein may be one or more selected from the group consisting of agoraphobia, generalized anxiety disorder (GAD), mixed anxiety-depressive state, obsessive-compulsive disorder (OCD), panic disorder, post-traumatic stress disorder (PTSD), social phobia and other phobias, and panic attacks.

[0138] Examples of injuries to the nervous system and / or brain in the context of the present invention include, but are not limited to, cerebral ischemia, stroke, traumatic brain injury, and spinal cord injury and spinal trauma.

[0139] The cognitive conditions or cognitive impairments disclosed herein may be one or more selected from the group consisting of Alzheimer's disease, Parkinson's disease, delirium, substance-induced persistent delirium, dementia, dementia due to HIV disease, dementia due to Huntington's disease, dementia due to Parkinson's disease, Alzheimer's type dementia, dementia associated with beta-amyloid, behavioral and psychological symptoms of dementia, substance-induced persistent dementia, and mild cognitive impairment.

[0140] The personality disorders disclosed herein may be one or more selected from the group consisting of obsessive-compulsive personality disorder and schizotypal and schizotypal disorders.

[0141] The mood disorders disclosed herein may be one or more selected from the group consisting of bipolar disorders (I and II), cyclothymic disorders, depression, dysthymic disorders, major depressive disorders, treatment-resistant depression, bipolar depression, and substance-induced mood disorders.

[0142] Neurodegenerative disorders may be one or more selected from the group consisting of oxidative stress, for example, Alzheimer's disease, Parkinson's disease, multiple sclerosis, amyotrophic lateral sclerosis (i.e., Lou Gehrig's disease), Huntington's disease, and neurodegenerative lysosomal storage disorders.

[0143] The neuropathy disclosed herein may be one or more selected from the group consisting of diabetic neuropathy, peripheral neuropathy, carpal tunnel syndrome, alcohol neuropathy, idiopathic neuropathy, and Guillain-Barré syndrome.

[0144] In the context of the present invention, neurodevelopmental disorders may be one or more selected from the group consisting of autism spectrum disorder (ASD), intellectual disability (ID), specific learning disability (SLD), communication disorders (e.g., speech disorders, language disorders, and social communication disorders), motor disorders (e.g., developmental coordination disorder (DCD) and Tourette syndrome), and attention deficit hyperactivity disorder (ADHD), preferably ADHD. In preferred embodiments, the neurological condition disclosed herein is ADHD, and / or the use relates to the improvement of ADHD.

[0145] The present invention is further illustrated by peptide compositions disclosed herein for the prevention and / or treatment of neuronal, central nervous system and / or brain damage that causes and / or is associated with increased levels of central nervous system inflammation and / or central nervous system oxidative stress, for example, for use in frailty syndromes, aging, injury, disease, infection and pathology, or for use in promoting the recovery of central nervous system neuronal or brain damage in individuals with injury, disease (e.g., amyotrophic lateral sclerosis and / or multiple sclerosis) and resulting cognitive impairment, learning impairment or memory impairment, disease, infection and pathology that causes and / or is associated with increased levels of central nervous system inflammation and / or central nervous system oxidative stress, and resulting cognitive impairment, learning impairment or memory impairment.

[0146] The use of the present invention as disclosed herein may include administering a therapeutically effective amount of the peptide composition described herein to a subject requiring its use, for example, by administering a therapeutic method.

[0147] In one embodiment, the present invention relates to the use of the peptide compositions described herein for maintaining or improving cognition, particularly for maintaining or improving learning, plasticity and / or long-term memory, and such use may be in therapeutic and / or non-therapeutic situations. In addition, or alternatively, the use of the present invention may relate to maintaining and / or improving neurological and / or brain functions selected from the group consisting of memory, attention, concentration, arousal, mental flexibility and / or speed, learning, intelligence, language skills, problem-solving ability, consciousness, coping with psychological stress or tension, motivation, mobility, decision-making ability, reaction time, and emotional regulation, and such use may be in therapeutic and / or non-therapeutic situations. In embodiments, maintaining and / or improving neurological and / or brain functions may relate to neurological dysfunction, neurological pathologies, and neurological disorders as disclosed herein.

[0148] The terms “cognition,” “cognitive function,” and “cognitive ability” may be used interchangeably and without limitation in the context of this invention, but relate to any mental process or mental state involving learning, memory, image formation, thinking, recognition, reasoning, spatial awareness, speech and language skills, language acquisition, ability to judge and pay attention, arousal, mental flexibility and / or speed, coping with psychological stress or tension, decision-making ability, and reaction time.

[0149] The compositions disclosed herein may have non-therapeutic and / or therapeutic uses in the context of the present invention. Non-therapeutic use is exemplified by administering the compositions of the present invention to healthy subjects who do not receive therapeutic benefits, and non-therapeutic use, in the context of the present invention, typically means enhancing abilities (e.g., cognitive function) starting from a normal state of health. Therapeutic use is exemplified by administering the compositions of the present invention to pathological subjects and / or subjects who receive therapeutic benefits, such as by preventing and / or treating one or more of the following: disease, health risks, psychological risks, or symptoms of pain and distress.

[0150] In one embodiment, the present invention relates to the use of a composition in non-therapeutic improvement of cognitive function.

[0151] In one embodiment, cognitive functions are selected from the group consisting of learning ability, memory, spatial memory, problem-solving ability, recall ability, attention duration, concentration, language processing ability, spatial awareness, speech, language skills, concentration, mental arousal, and reaction time.

[0152] Embodiments of the present invention are exemplified by the non-therapeutic use of the peptide compositions disclosed herein. Such embodiments include enhancing abilities, such as cognitive functions, starting from a normal state of health and / or in otherwise healthy individuals. In preferred embodiments, the present invention relates to the non-therapeutic use of peptide compositions in improving one or more cognitive functions preferably selected from the group consisting of learning, memory, image formation, thinking, recognition, reasoning, spatial awareness, speech skills, language skills, language acquisition, ability to judge and pay attention, arousal, mental flexibility, speed, coping with psychological stress and / or tension, decision-making ability, and reaction time. Non-therapeutic uses of the compositions of the present invention may also be associated with general improvement of muscle control, or improvement of neuromotor or neurosensory function.

[0153] (Administration of peptide composition) In preferred embodiments, the peptide composition (which can be obtained by this method) is provided or present in one or more formulations selected from the group consisting of food formulations, feed formulations, food supplement formulations, feed supplement formulations, or pharmaceutical formulations, preferably as a food supplement formulation. In preferred embodiments, the composition according to the present invention is provided as a food supplement or in a food supplement.

[0154] In a preferred embodiment, the peptide composition (which can be obtained by this method) is provided as a formulation selected from the group consisting of capsules, tablets, or powders, preferably as a powder.

[0155] In preferred embodiments, the peptide composition (which can be obtained by this method) is provided in the form of a formulation selected from the group consisting of a drinkable solution or suspension, a beverage such as beer, a syrup, an artificially flavored beverage, a carbonated beverage, a (water-soluble) powder mixture, a (water-soluble) paste, a (water-soluble) powder, a (water-soluble) tablet, a (water-soluble) pill, a (water-soluble) sugar-coated tablet, a (water-soluble) caplet, a (water-soluble) sachet, or a (water-soluble) capsule. In addition, or alternatively, the peptide composition (which can be obtained by this method) may be present in juices, shakes, dairy beverages, yogurt, yogurt drinks, desserts, functional foods such as energy bars, nutrition bars, slimming bars, or confectionery such as gummies or center-filled gummies.

[0156] In preferred embodiments, the peptide composition (which can be obtained by this method) is administered in a daily dose of 20 mg to 50 g, preferably 50 mg to 40 g, more preferably 0.1 g to 20 g, even more preferably 0.5 g to 10 g, and even more preferably 1 g to 5 g, where the daily dose is the total dry weight of the composition. In preferred embodiments, the peptide composition (which can be obtained by this method) disclosed herein is administered in a unit dose of 10 mg to 25 g, preferably 25 mg to 10 g, more preferably 50 mg to 5 g, and even more preferably 0.1 g to 1 g, where the unit dose is the total dry weight of the composition. In preferred embodiments, the daily dose and / or unit dose is 0.1 g to 20 g, or 0.5 g to 10 g.

[0157] As used herein, the term "daily dose" refers to the total dry weight amount administered to a subject per day. The daily dose of a composition may be administered as a single unit dose, or as two, three, four, or more unit doses. Two or more unit doses may be equal in amount or different. The daily dose of the compositions disclosed herein is preferably administered as two unit doses, more preferably two unit doses corresponding to 30-70%, preferably 40-60%, of the total daily dose. As used herein, the term "unit dose" refers to the total dry weight amount administered to a subject in a single dose. A unit dose is typically a pre-prepared form ready for administration (e.g., a pre-filled dose). A unit dose may be identifiable, for example (similarly), from the product packaging or label.

[0158] In a preferred embodiment, the peptide composition (which can be obtained by this method) is administered to the subject repeatedly, preferably at least once a day, or at least once every other day, and / or to the subject for at least two consecutive weeks, preferably at least four consecutive weeks, more preferably at least six consecutive weeks, and most preferably at least eight consecutive weeks.

[0159] The terms “continuously” or “sequentially” used in relation to administration mean that the administrations occur in a continuous sequence without gaps within a given period. For example, if a composition is administered “for four consecutive days,” this means that the composition is administered at least once a day for four consecutive days (e.g., Monday, Tuesday, Wednesday, and Thursday of the same week), regardless of the number of doses per day or the total number of doses. For example, if a composition is administered “for four consecutive weeks,” this means that the composition is administered at least once a week for four consecutive weeks, regardless of the number of doses per day, the number of doses per week, or the total number of doses.

[0160] In a preferred embodiment, the peptide composition is administered orally, for example, in the form of a food supplement.

[0161] In preferred embodiments, the peptide composition is administered topically, for example, parenterally, preferably subcutaneously and / or transdermally.

[0162] In one embodiment, the peptide composition is administered by injection.

[0163] In preferred embodiments, the peptide composition is provided in the form of a topical formulation. The topical formulation is preferably selected from the group consisting of wound dressings, hydrogels, gels, ointments, plasters, skin substitutes, tissue adhesives, tissue sealants, hemostatic agents, sprays, and bandages. In various embodiments, the present invention relates to topical formulations comprising the peptide compositions disclosed herein.

[0164] The peptide composition may be provided in a topical formulation in an amount calculated relative to the weight of the topical formulation, for example, 0.00001 to 25 wt.%, or 0.0001 to 10 wt.%, or 0.005 to 1 wt.%. Based on in vitro dose-response curves, the inventors found that preferred concentrations for antifibrosis may be in the range of 0.0001 to 5 wt.%, preferably 0.001 to 1 wt.%, and more preferably 0.01 to 0.1 wt.%, relative to the weight of the topical formulation.

[0165] In various embodiments, this embodiment relates to non-therapeutic cosmetic uses of the peptide compositions disclosed herein. Cosmetic uses may be for non-therapeutic improvement of skin appearance, such as scarring, wrinkles, skin discoloration, skin irritation, volume increase, hair loss, post-exfoliation, skin excision, and medical needling. Cosmetic uses may be one or more of intracutaneous or supercutaneous topical administration, intralesional administration, intraepithelial administration, intraepidermal administration, intradermal administration, and subcutaneous administration. In preferred embodiments, administration is topical administration to the skin, for example, on wounded skin.

[0166] In various embodiments, the present invention relates to cosmetic compositions comprising the peptide compositions disclosed herein.

[0167] Peptide compositions can be present in cosmetic compositions and, therefore, may preferably further contain acceptable excipients, lubricants, and additives. Cosmetic compositions may be in any form suitable for topical application, for example, in the form of a solution, gel, solid, paste-like anhydrous product, emulsion obtained by dispersing an oil phase in an aqueous phase, emulsion obtained by dispersing an aqueous phase in an oil phase, multi-emulsion, suspension, microemulsion, microcapsule, fine particles, ionic (liposome) and nonionic vesicle dispersants, foam, and aerosol or patch further containing a compressive propellant. Cosmetic compositions may be selected from the group consisting of soap-type preparations, skin toners, nourishing lotions, essences, nourishing creams, massage creams, packs, gels, makeup bases, foundations, powders, lipsticks, patches, eye creams, eye essences, cleansing creams, cleansing foams, cleansing waters, cleansers, hair shampoos, hair conditioners, hair treatments, hair essences, hair lotions, scalp hair tonics, scalp essences, hair gels, hair sprays, hair packs, body lotions, body creams, body oils, and body essences.

[0168] (Method for obtaining a peptide composition) Conventional methods for producing cGPs include their synthesis from amino acids and subsequent cyclization. These conventional methods are typically complex because they require the removal of organic solvents and resins used in solid-phase synthesis.

[0169] For example, Hayasaka et al. (Food Science and Technology Research. 2016. 22(4):477-483) describe a method for obtaining cGP peptides from natural collagen, involving the following steps: i) Enzymatic hydrolysis of collagen, ii) A step to isolate the collagen tripeptide (CTP) fraction from the hydrolysate (by reverse-phase HPLC), iii) A step of heating the CTP fraction to induce cyclization to a cyclic peptide containing the cGP peptide.

[0170] The method by Hayasaka et al. relies on enriching the cGP (e.g., to >20%) and first isolating the CTP fraction. Isolating CTP is thought to remove most of the dipeptides and free amino acids that typically contribute to the biological effects of the collagen peptide composition. Furthermore, the isolation step involves increased costs and time required for production, i.e., decreased efficiency.

[0171] The inventors have discovered a method for producing cGP starting from natural materials, which has even higher practical values ​​and therefore may be a more promising alternative.

[0172] In particular, it has been found that heat treatment of intermediate collagen hydrolysate products having an appropriate relative amount of peptides enriches cGP and yields a peptide composition further containing desirable amounts of (cyclic) di- and tripeptides. While not bound by theory, the inventors believe that heating promotes the cyclization of linear peptides in intermediate collagen hydrolysate products, especially gly-pro-x tripeptides, and mainly gly-pro-hyp peptides and gly-pro-ala peptides present in intermediate collagen hydrolysate products. It has been further found that heating to induce cyclization significantly increases the amounts of hydroxylysine and hydroxyproline. Therefore, the peptide compositions of the present invention may also have characteristic content of hydroxylysine and / or hydroxyproline.

[0173] In various embodiments, the method of the present invention makes it possible to obtain the peptide compositions disclosed herein.

[0174] In the embodiment, the method of the present invention is a) Calculated relative to the total weight of peptides in collagen hydrolysate, at least 1.0 × 10 4 To provide a collagen hydrolysate preferably containing ppm of gly-pro-x tripeptide, and b) Preferably, the collagen hydrolysate is heated at a temperature of at least 65°C, thereby enabling the acquisition of a cyclic (glycine-proline) enriched peptide composition.

[0175] Preferably, the collagen hydrolysate in the first step has a weight-average molecular weight of 500 Da or more and 3000 Da or less.

[0176] The collagen hydrolysate (provided in step a) is 2.0 × 10 4 ~2.0×10 5 Preferably contains ppm of glycine-proline-x tripeptide. The collagen hydrolysate provided in step a) is 1.0 × 10 4 ~1.0×10 5Preferably contains ppm of glycine-proline-hydroxyproline tripeptide. The collagen hydrolysate (provided in step a) is 5.0 × 10 3 ~5.0×10 4 ppm, preferably 1.0 × 10⁻¹⁴ 4 ~3.0×10 4 Preferably contains ppm of glycine-proline-alanine tripeptide.

[0177] The collagen hydrolysate (provided in step a) preferably contains 10,000 to 100,000 ppm, more preferably 20,000 to 80,000 ppm, of glycine-proline-hydroxyproline tripeptide. For example, the amount of glycine-proline-hydroxyproline tripeptide may be 30,000 to 60,000 ppm.

[0178] In addition, or alternatively, the collagen hydrolysate (provided in step a) may contain at least 1.0 × 10¹⁶ peptides, calculated relative to the total weight of peptides in the collagen hydrolysate. 4 ppm, preferably at least 5.0 × 10⁻¹⁴ 4 ppm, more preferably at least 1.0 × 10⁻¹⁴ 5The collagen hydrolysate (provided in step a) preferably contains a ppm amount of collagen tripeptide, particularly gly-pro-x tripeptide or glycine-proline-alanine tripeptide and / or glycine-proline-hydroxyproline tripeptide. In a preferred embodiment, the collagen hydrolysate (provided in step a) contains 25,000 ppm to 150,000 ppm, preferably 50,000 ppm to 100,000 ppm of collagen tripeptide, particularly gly-pro-x tripeptide, calculated relative to the total weight of peptides in the collagen hydrolysate. In a preferred embodiment, the collagen hydrolysate (provided in step a) contains 10,000 to 100,000 ppm, preferably 20,000 to 80,000 ppm, more preferably 30,000 to 60,000 ppm of glycine-proline-hydroxyproline tripeptide, calculated relative to the total weight of peptides in the collagen hydrolysate. In a preferred embodiment, the collagen hydrolysate (provided in step a) contains 5,000 to 75,000 ppm, preferably 10,000 to 50,000 ppm, and more preferably 20,000 to 40,000 ppm of glycine-proline-alanine tripeptide, calculated relative to the total weight of peptides in the collagen hydrolysate.

[0179] The collagen hydrolysate (provided in step a) is 5.0 × 10 3 Less than ppm, preferably 1.0 × 10⁻¹⁶ 3 Less than ppm, more preferably 5.0 × 10 2 Less than ppm, more preferably 1.0 × 10⁻⁶ 2 Preferably, it contains collagen dipeptides in amounts less than ppm, for example, 10 to 1000 ppm or 50 to 500 ppm.

[0180] The collagen hydrolysate (provided in step a) is 1.0 × 10 5 Less than ppm, preferably 5.0 × 10⁻⁶ 4 Less than ppm, more preferably 1.0 × 10⁻⁶ 4 Less than ppm, more preferably 0.5 × 10 4 Less than ppm, most preferably 1.0 × 10⁻¹⁴ 3Preferably, it contains free amino acids in amounts less than ppm.

[0181] The collagen hydrolysate (provided in step a) preferably features a weight-average molecular weight of at least 800 Da, more preferably at least 1000 Da. The collagen hydrolysate (provided in step a) preferably features a weight-average molecular weight of up to 2500 Da, more preferably up to 2000 Da. In embodiments, the collagen hydrolysate (provided in step a) preferably has a weight-average molecular weight (Mw) in the range of 500 Da to 5000 Da, preferably 750 Da to 3000 Da, more preferably 1000 to 2500 Da, and even more preferably 1200 to 2000 Da. Collagen hydrolysates may have a weight-average molecular weight of at least 500Da, 600Da, 700Da, 800Da, 900Da, 1000Da, 1100Da, 1200Da, 1300Da, 1400Da, 1500Da, 1750Da, 2000Da, 2250Da, 2500Da, 2750Da, or 3000Da. In addition, or alternatively, collagen hydrolysates may have a weight-average molecular weight of up to 3500Da, 3000Da, or 2500Da, or 2250Da, or 2000Da, or 1750Da, or 1700Da, or 1650Da, or 1600Da.

[0182] In preferred embodiments, the collagen hydrolysate has a molecular weight distribution defined by a polydispersity index of 1.0 to 2.0, preferably 1.2 to 1.8, more preferably 1.4 to 1.7, and even more preferably 1.5 to 1.6. In preferred embodiments, the collagen hydrolysate has a molecular weight distribution defined by a polydispersity of 1.1 to 1.7, more preferably 1.2 to 1.6, and even more preferably 1.3 to 1.5.

[0183] In embodiments of the present invention, the collagen hydrolysate contains 0 to 5% by weight, preferably 0.1 to 2% by weight, and more preferably 0.2 to 1% by weight, of peptides having a molecular weight of 0 to 250 Da, or up to 250 Da.

[0184] In embodiments of the present invention, the collagen hydrolysate contains 0 to 5% by weight, preferably 0.1 to 2% by weight, and more preferably 0.2 to 1% by weight, of a peptide having a molecular weight of up to 450 Da.

[0185] In embodiments of the present invention, the collagen hydrolysate contains 0 to 5% by weight, preferably 0.1 to 2% by weight, and more preferably 0.2 to 1% by weight, of a peptide having a molecular weight of 250 to 450 Da.

[0186] In embodiments of the present invention, the collagen hydrolysate contains 25-55% by weight, preferably 30-50% by weight, and more preferably 35-45% by weight, of peptides having a molecular weight of 450-1000 Da.

[0187] In embodiments of the present invention, the collagen hydrolysate contains 0.1 to 10% by weight, preferably 0.5 to 5% by weight, and more preferably 1 to 4% by weight, of peptides having a molecular weight of up to 500 Da.

[0188] In embodiments of the present invention, the collagen hydrolysate contains 20-60% by weight, preferably 25-50% by weight, and more preferably 30-45% by weight, of peptides having a molecular weight of 500-1000 Da. In embodiments of the present invention, the collagen hydrolysate contains 20-60% by weight, preferably 25-50% by weight, and more preferably 30-45% by weight, of peptides having a molecular weight of 1000-2000 Da.

[0189] In embodiments of the present invention, the collagen hydrolysate contains 5 to 40% by weight, preferably 0.5 to 30% by weight, and more preferably 15 to 20% by weight, of peptides having a molecular weight of 2000 to 5000 Da.

[0190] In embodiments of the present invention, the collagen hydrolysate contains 0.1 to 10% by weight, preferably 0.2 to 5% by weight, and more preferably 0.5 to 2% by weight, of a peptide having a molecular weight of 5000 to 10000 Da.

[0191] In embodiments of the present invention, the collagen hydrolysate contains a maximum of 2% by weight, preferably 0.01 to 2% by weight, and more preferably 0.1 to 1% by weight, of peptides having a molecular weight of more than 10,000 Da, calculated relative to the total weight of the total peptides in the peptide composition.

[0192] Preferably, the collagen hydrolysate contains 20-60% by weight, more preferably 30-50% by weight, of peptides having a molecular weight of less than 1000 Da.

[0193] In various embodiments, the collagen hydrolysate (provided in step a) of the present method and / or the peptide composition that can be obtained in step b) comprises one or more cyclic (i.e., cyclo)peptides selected from the group consisting of cyclo(Gly-Ala), cyclo(Gly-Leu), cyclo(Gly-Glu), cyclo(Gly-Ser), cyclo(Gly-Phe), cyclo(Gly-Val), cyclo(Leu-Hyp), and cyclo(Ala-Hyp).

[0194] In various embodiments, the collagen hydrolysate of this method (provided in step a) contains cGP in an amount of 500 to 20,000 ppm, preferably 1,000 to 10,000 ppm. The amount of cGP in the collagen hydrolysate may be preferably 1,000 to 5,000 ppm, more preferably 2,000 to 4,000 ppm.

[0195] In various embodiments, the collagen hydrolysate of this method (provided in step a) contains 1 to 200 ppm, preferably 2 to 100 ppm, of hydroxylysine. The amount of hydroxylysine in the collagen hydrolysate may be preferably 10 to 50 ppm, more preferably 15 to 30 ppm. In various embodiments, the collagen hydrolysate of this method (provided in step a) contains 100 to 2500 ppm, preferably 200 to 1500 ppm, of hydroxyproline. The amount of hydroxyproline in the collagen hydrolysate may be preferably 300 to 1000 ppm, more preferably 400 to 800 ppm.

[0196] In various embodiments, the collagen hydrolysate of the method (step a provided) and / or step b) may be obtained, comprising one or more linear tripeptides selected from the group consisting of Gly-Pro-Hyp, Gly-Pro-Pro, Gly-Pro-Ala, Gly-Ala-Hyp, Gly-Ala-Pro, Gly-Leu-Hyp, Gly-Glu-Hyp, Gly-Glu-Arg, Gly-Pro-Arg, Gly-Ser-Hyp, Gly-Ala-Lys, Gly-Ala-Arg, Gly-Pro-Lys, Gly-Pro-Ser, Gly-Phe-Hyp, Gly-Pro-Gln, Gly-Ala-Ala, Gly-Pro-Val, Gly-Asp-Ala, Gly-Glu-Ala, Gly-Ala-Asp, Gly-Arg-Hyp, and Ala-Hyp-Gly.

[0197] In various embodiments, the collagen hydrolysate (provided in step a) of the present method and / or the peptide composition that can be obtained in step b) comprises one or more linear dipeptides selected from the group consisting of Gly-Pro, Pro-Gly, Gly-Hyp, Pro-Hyp, Hyp-Gly, Ala-Hyp, and Pro-Ala.

[0198] The heat treatment (in step b) may induce dehydration and / or cyclization reactions. The heating time and / or temperature are variable and can be adapted according to the desired yield of the peptide. In addition, or alternatively, the heating time may depend on the hydrolysis step being performed. For example, the inventors have found that the hydrolysis time and / or heat treatment time can be shortened when hydrolysis is performed sequentially using two or more enzymes. The heat treatment may be performed at a temperature of preferably 70°C or higher, for example, 70-120°C, preferably 80-110°C, and more preferably 90-100°C. The temperature is preferably the temperature of the liquid formulation to which the collagen hydrolysate is provided.

[0199] In the context of the present invention, the heat treatment preferably lasts for 8 to 36 hours, more preferably 12 to 24 hours, and even more preferably 16 to 20 hours. In various embodiments, the heating time is at least 0.5, 1, 2, 3, 4, 5, 6, 8, 10, 12, 14, 16, 18, or 20 hours, and in addition, or alternatively, up to 48, 36, 30, 24, 20, 18, 16, 14, 12, 10, 8, 6, 5, or 4 hours.

[0200] (Method for obtaining collagen hydrolysates) The inventors have discovered that digestion of collagen by a microbial collagenase having cleavage activity at the Xaa-|-Gly bond can lead to the extraction of a glycine-proline-x tripeptide sequence (e.g., at least 1.0 × 10⁻¹⁶). 4 ppm, preferably 2.0 × 10 4 ~2.0×10 5 ppm of gly-pro-x tripeptide), especially glycine-proline-hydroxyproline (e.g., 1.0 × 10⁻¹⁰) 4 ~1.0×10 5 ppm) and glycine-proline-alanine tripeptide (e.g., 5 × 10) 3 ~5×10 4 We found that intermediate collagen hydrolysates rich in ppm sequences and further rich in collagen di- and tripeptides can be obtained. In addition, the intermediate collagen hydrolysates are characterized by relatively small amounts of dipeptides and free amino acids, and a more desirable molecular weight distribution.

[0201] In the context of the present invention, the "x" amino acid in the "gly-pro-x" tripeptide may be any of the following amino acid residues: arginine, lysine, histidine, phenylalanine, tyrosine, leucine, isoleucine, methionine, valine, alanine, glycine, proline, glutamic acid, glutamine, serine, threonine, aspartic acid, asparagine, tryptophan, cysteine, hydroxyproline, or hydroxylysine.

[0202] In the context of this invention, unless otherwise specified, the terms “dipeptide” and “tripeptide” refer to linear dipeptides and linear tripeptides (i.e., distinct from cyclic peptides), respectively.

[0203] In embodiments, the present invention relates to a method for obtaining collagen hydrolysates, preferably collagen hydrolysates as disclosed herein.

[0204] A method for obtaining collagen hydrolysates more preferably involves hydrolyzing collagen and / or gelatin starting materials using microbial collagenase.

[0205] In one embodiment, the collagen and / or gelatin starting material is preferably composed of or derived from one or more (animal) collagen-containing tissues.

[0206] In the context of the present invention, the term “collagen” means an amino acid sequence comprising a repeating (Gly-XY) sequence, preferably comprising at least 2, 3, 4, 5, 10, 20, 50, 100, 200, 300, or 400 sequences containing the sequence Gly-XY (where X and Y are amino acid residues independently selected from each other, but X and / or Y are more preferably proline). “Collagen” preferably has sequences found in natural collagen of one or more animal species. In addition, or alternatively, “collagen” may mean a full-length sequence or fragment or subunit thereof of (natural) collagen, preferably one or more of type I to XXVII collagen, more preferably one or more of type I, type II, type III, type IV, type V, or type X collagen, even more preferably one or more of type I, type II, type III, or type IV collagen, or any combination thereof. For example, the term “collagen” may refer to alpha-1(I) chains, alpha-2(I) chains, alpha-1(II) chains, or alpha-1(III) chains, or fragments thereof. The term “collagen” encompasses the triple helix structure formed by the three subunits present in natural collagen. Gelatin in the context of the present invention may be derived from the partial hydrolysis of one or more types of collagen selected from type I to type XXVII collagen, preferably one or more of type I, type II, type III, or type IV collagen, or any combination thereof. In preferred embodiments of the present invention, the collagen may be derived from one or more of skin, cartilage, bone, scales, or sternum, or the collagen may be a mixture of collagens derived from various tissues. The term “skin” as disclosed herein encompasses the meaning of “skin,” i.e., the outer covering of a large animal such as a cow (species), or any other large animal. The terms “skin” and “skin” may be used interchangeably herein and may refer to the outer covering of an animal regardless of size.

[0207] As used herein, the term “collagen hydrolysate” means a mixture of short chains of amino acids derived from a collagen-containing material having natural (full-length) collagen, preferably obtained through a hydrolysis process including enzymatic hydrolysis (also called enzymatic hydrolysis treatment). “Collagen hydrolysate” as used herein may encompass collagen subjected to hydrolysis or partial hydrolysis. The term “collagen hydrolysate” may be used synonymously and without distinction with the terms “collagen hydrolysate,” “hydrolyzed collagen,” or “collagen peptide.” In the context of this invention, the term “gelatin” means a mixture of water-soluble peptides and / or proteins derived from the partial hydrolysis of collagen. The gelatin of this invention may be type A gelatin, type B gelatin, or a combination thereof. Hydrolysis may be carried out by either acidic or alkaline conditions, or by enzymatic hydrolysis, as known in the art. Depending on the physical and chemical methods of partial hydrolysis, the molecular weight of the peptides may be within a wide range (e.g., 10 to 95 kDa). Partial hydrolysis imparts water-retaining and gelling capabilities to gelatin, which typically distinguishes it from "collagen hydrolysates," i.e., products obtained by further hydrolysis of collagen.

[0208] In embodiments, the "collagen hydrolysate" disclosed herein is produced from a collagen-containing material in a single-step process. In embodiments, the "collagen hydrolysate" disclosed herein is produced via an intermediate gelatin step, preferably using, for example, type A and / or type B gelatin (e.g., a mixture of type A and type B gelatin).

[0209] In embodiments, the collagen may be derived from the partial hydrolysis of one or more types of collagen and / or collagen from another animal or animal species, such as bovine, porcine, chicken, and fish, or a combination thereof.

[0210] For hydrolysis, the collagen-containing material (e.g., collagen, gelatin) is preferably provided in a liquid formulation together with one or more enzymes.

[0211] In the context of the present invention, the hydrolysis of collagen and / or gelatin preferably involves enzymatic hydrolysis. Enzymes suitable for enzymatic hydrolysis in the context of the present invention include collagenases, for example, one or more selected from the group consisting of collagenase, acid protease, aminoprotease, serine protease, alkaline protease, neutral protease, flavor protease, complex protease, thiol protease, bromelain, metalloprotease, aspartame, protease, carboxypeptidase, pepsin, chymotrypsin, trypsin, cathepsin K, papain, and subtilisin.

[0212] In a preferred embodiment, the enzyme cleaves Xaa-|-Gly bonds. In one embodiment, the enzyme cleaves Y-Gly bonds in repeating Gly-XY triplets within a collagen sequence, where X or Y represents either amino acid residue. In one embodiment, the enzyme cleaves -Gly bonds within sequences Xaa-Gly-Pro- or -Pro-Xaa-Gly-Pro-. In one embodiment, the enzyme is a collagenase, more preferably a microbial collagenase.

[0213] In the context of the present invention, it is preferable that the enzyme does not produce free amino acids, or produces only a relatively small amount of free amino acids.

[0214] The inventors have discovered that digestion of collagen by a microbial collagenase having cleavage activity at the Xaa-|-Gly bond yields an intermediate collagen hydrolysate product rich in glycine-proline-x tripeptide sequences, particularly glycine-proline-hydroxyproline sequences and glycine-proline-alanine tripeptide sequences, and further containing collagen di- and tripeptides.

[0215] In one embodiment, it is preferable that the enzyme used for hydrolysis is not an enzyme that preferentially cleaves the peptide bond with ginger powder and / or Pro / Hyp at the P2 position. While not bound by theory, the inventors believe that cleavage of the collagen peptide bond with Pro / Hyp at the P2 position results in a collagen hydrolysate containing a considerable amount of X-Hyp-Gly type peptide and not achieving the desired relative amount of cyclic (peptide) upon heating. While not bound by theory, the inventors believe that cleavage of the peptide bond with Pro / Hyp at the P2 position does not result in sufficient enrichment of the glycine-proline-x tripeptide sequence, particularly the glycine-proline-hydroxyproline sequence and the glycine-proline-alanine tripeptide sequence, in the intermediate collagen hydrolysate product.

[0216] In the notation "Xaa-|-Gly," "Xaa" represents any amino acid residue, "|" represents the cleavage site, and "Gly" specifically indicates glycine. Enzymes that have cleavage activity at the Xaa-|-Gly bond cleave peptide bonds where the N-terminal amino acid can be any amino acid "Xaa" and the C-terminal amino acid is glycine.

[0217] In one embodiment, microbial collagenase in the context of the present invention preferentially cleaves Xaa-|-Gly bonds compared to other bonds in collagen.

[0218] In one embodiment, microbial collagenases in the context of the present invention preferentially cleave at the Xaa-|-Gly bond compared to cleavage activity at the peptide bond with Pro / Hyp at the P2 position.

[0219] In the context of the present invention, the cleavage activity of collagenase can be determined by subjecting collagen to collagenase, mapping the peptides produced by enzymatic digestion (e.g., by mass spectrometry), and determining the relative abundance of each peptide to quantify the degree of cleavage at different sites. This method is suitable for evaluating the substrate specificity of the enzyme.

[0220] In the context of the present invention, microbial collagenases are preferably isolated from Bacillus cereus, Clostridium histolyticum, Empedobacter collagenolyticum, Pseudomonas marinoglutinosa, Streptomyces species, such as Streptomyces violaceoruber, Vibrio species, such as Vibrio B-30 (ATCC 21250) or Vibrio alginolyticus (formerly Achromobacter iophagus) or Clostridium histolyticum. In one embodiment, the microbial collagenase is derived from a Streptomyces species. In one embodiment, the microbial collagenase is produced by a self-cloning strain, which is preferably one of the strains disclosed herein.

[0221] In one embodiment, microbial collagenase belongs to the peptidase family M9. In one embodiment, microbial collagenase is classified according to the Enzyme Committee (EC) number as EC 3.4.24 (common name "metalloendopeptidase"), preferably EC 3.4.24.3 (common name "microbial collagenase").

[0222] In a preferred embodiment, the method includes hydrolyzing gelatin with a first enzyme to obtain a gelatin hydrolysate, and hydrolyzing the gelatin hydrolysate with a second enzyme to obtain a collagen hydrolysate.

[0223] In a preferred embodiment, a method for obtaining a collagen hydrolysate is: • A process to obtain gelatin by alkaline hydrolysis or acid hydrolysis of collagen. The step of hydrolyzing gelatin using a first enzyme to obtain a gelatin hydrolysate, wherein the first enzyme is preferably selected from the group consisting of collagenase, acid protease, aminoprotease, serine protease, alkaline protease, neutral protease, flavor protease, complex protease, thiol protease, bromelain, metalloprotease, aspartame, protease, carboxypeptidase, pepsin, chymotrypsin, trypsin, cathepsin K, chymotrypsin, papain and subtilisin, and / or the first enzyme is preferably not a microbial collagenase having cleavage activity at Xaa-|-Gly bonds. Preferably, the process includes a step of hydrolyzing gelatin hydrolysate using a second enzyme, which is a microbial collagenase having cleavage activity at the Xaa-|-Gly bond in collagen, to obtain collagen hydrolysate.

[0224] The inventors have found that sequential hydrolysis by a first enzyme (preferably not cleaved at the Xaa-|-Gly bond) followed by a second enzyme (preferably cleaved at the Xaa-|-Gly bond) can shorten the hydrolysis time and / or the time required for cyclization of the cGP-containing peptide. In addition, or alternatively, sequential hydrolysis by a first enzyme (preferably not cleaved at the Xaa-|-Gly bond) followed by a second enzyme (preferably cleaved at the Xaa-|-Gly bond) can improve the resulting peptide composition and its biological activity (e.g., antioxidant effect, antineurolytic (inflammatory) effect and / or antifibrotic effect).

[0225] In hydrolysis, the material is preferably supplied with an enzyme concentration of 100 to 10,000 ppm, preferably 500 to 5,000 ppm, more preferably 1,000 to 3,000 ppm, and even more preferably 1,500 to 2,500 ppm, calculated relative to the total weight of the collagen-containing material and the liquid formulation. In a preferred embodiment, hydrolysis involves supplying collagen and / or gelatin to a microbial collagenase having cleavage activity at Xaa-|-Gly bonds at a concentration of 500 to 5,000 ppm, preferably 1,000 to 3,000 ppm, and more preferably 1,500 to 2,500 ppm, calculated relative to the total weight of the collagen-containing material and the liquid formulation.

[0226] In embodiments of the present invention, hydrolysis is carried out at a pH of 6.5 to 9.5, preferably 7.0 to 9.0, and more preferably 7.5 to 8.5. In embodiments of the present invention, hydrolysis is carried out at a temperature of 15 to 45°C, preferably 20 to 40°C, and more preferably 25 to 35°C. In embodiments of the present invention, hydrolysis is carried out for 4 to 24 hours, preferably 8 to 20 hours, and more preferably 12 to 16 hours.

[0227] (General definition) The terms "comprising" or "to comprise" and their conjugations are used in the context of this invention in a non-restrictive sense to indicate that the items following the word are included, but that items not specifically mentioned are not excluded.

[0228] The indefinite article "a" or "an" does not rule out the possibility of multiple elements existing unless the context explicitly requires that only one element exists. Therefore, the indefinite article "a" or "an" usually means "at least one."

[0229] In the context of the present invention, a level is considered to have “increased” or “decreased” if it is at least 1% higher or lower than the corresponding level in the control or reference (e.g., at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50%). In addition, or alternatively, a level is considered to have increased or decreased if it is statistically significantly higher or lower than the level in the control or reference (e.g., compared to an earlier point in time), regardless of the magnitude of the change. The term “to reduce” may be used interchangeably with the term “to decrease” in the context of the present invention.

[0230] In the context of this invention, the term "administer" or "administer" means providing a compound, substance, or composition to an object that consumes it. The object that consumes the compound, substance, or composition may administer it to itself. In such cases, the term "administer" can be read as "intake."

[0231] In the context of this invention, the term "subject" means any animal (e.g., a mammal), preferably a human. In the context of this invention, the term "subject" may be used interchangeably with "human" or "individual." A "subject" may be a patient.

[0232] The term “prevent” means ensuring that the subject does not develop a disease. In this specification, an intervention is also considered to be a form of “prevention” if it delays the disease, reduces the severity of the disease, and / or reduces the incidence of the disease, even if it does not completely prevent the disease from occurring. The terms “prevent” or “prevention” may be used for therapeutic and / or non-therapeutic effects. “Prevent” or “prevention” may be defined by a delay, change in severity, and / or change in incidence of at least 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100% compared to a control or reference as measured by any standard technique.

[0233] In the context of this invention, “to treat” means that, if a condition already exists, the intervention reduces and / or cures the condition. The terms “to treat” or “treatment” may be used for therapeutic and / or non-therapeutic effects. Treatment may be a reduction in the severity, incidence, and / or frequency of a condition, for example, by at least 5%, 10%, 20%, 40%, 50%, 60%, 80%, 90%, 95%, or completely (100%) compared to a control or reference as measured by some standard technique.

[0234] In the context of the present invention, the value "0" can also encompass "essentially 0" or "essentially not containing," meaning that the quantity is immeasurable according to standard art and / or below a certain threshold, for example, when referring to a percentage value, so that the quantity can be ignored, preferably less than 0.1, more preferably less than 0.01, even more preferably less than 0.001, and most preferably less than 0.0001. [Brief explanation of the drawing]

[0235] [Figure 1] Radical scavenging activity of cGP, gly-pro-hyp, gly-pro-ala, AP (additive and actual effects), and ascorbic acid (positive control). The additive effect of the cGP + gly-pro-hyp combination is compared with the actual (synergistic) effect of this combination in AP. The radical scavenging activity of cGP, gly-pro-hyp, or gly-pro-ala was normalized to their respective concentrations present in the peptide composition (concentrations according to Table 3). [Figure 2] Effect of AP on hydrogel contraction in fibroblast contraction assays. Fibroblast contraction after 14 days of exposure to various concentrations of AP or TGF-β1 (a positive control that stimulates fibrosis). Each symbol represents an independent experiment and donor (n=3). At least 100 cells were counted per hydrogel. Repeated measures ANOVA by Dunnett's multiple comparison test. *p<0.05;**p<0.01;***p<0.005;****p<0.001 [Figure 3]Effect of AP on hydrogel contraction in adipose stromal cell contraction assay. Adipose stromal cell contraction after 14 days of exposure to various concentrations of AP or TGF-β1 (positive control stimulating fibrosis). Each symbol represents an independent experiment and donor (n=3). At least 100 cells were counted per hydrogel. Repeated measures analysis of variance with Dunnett's multiple comparison test. *p<0.05; **p<0.01; ***p<0.005; ****p<0.001 [Figure 4] Effect of AP on the differentiation of fibroblasts into myofibroblasts. Myofibroblast differentiation in fibroblasts after 14 days of exposure to various concentrations of AP or TGF-β1 (positive control stimulating fibrosis). Each symbol represents an independent experiment and donor (n=3). At least 100 cells were counted per hydrogel. Repeated measures analysis of variance with Dunnett's multiple comparison test. *p<0.05; **p<0.01; ***p<0.005; ****p<0.001 [Figure 5] Effect of AP on the differentiation of adipose stromal cells into myofibroblasts. Myofibroblast differentiation in adipose stromal cells after 14 days of exposure to various concentrations of AP or TGF-β1 (positive control stimulating fibrosis). Each symbol represents an independent experiment and donor (n=3). At least 100 cells were counted per hydrogel. Repeated measures analysis of variance with Dunnett's multiple comparison test. *p<0.05; **p<0.01; ***p<0.005; ****p<0.001 [Figure 6] Effect of treatment on spontaneous alternation behavior in D-galactose-treated mice. * indicates a significant difference (p<0.05) compared to the 60% baseline (dotted line), which is the expected behavior of animals in this model. [Figure 7] Effect of AP on the learning profile of D-galactose-treated mice in the Morris water maze test, p<0.05 (NaCl / H2O vs. D-Gal / H2O), p<0.05 [D-Gal / AP (1722 mg / kg) vs. D-Gal / H2O]. [Figure 8]Interleukin-6 (IL-6) concentrations (mean±SEM) in the cerebral cortex (left), hippocampus (center), and plasma (right). *Significant difference from the negative control group ("NaCl / H2O"), and #Significant difference from the positive control group ("D-Gal / H2O"). [Figure 9] Tumor necrosis factor alpha (TNF-α) concentrations (mean±SEM) in the cerebral cortex (left), hippocampus (center), and plasma (right). *Significant difference from the negative control group ("NaCl / H2O"), and #Significant difference from the positive control group ("D-Gal / H2O"). [Figure 10] Brain-derived neurotrophic factor (BDNF) concentrations (mean±SEM) in the cerebral cortex (left), hippocampus (center), and plasma (right). *Significant difference from the negative control group ("NaCl / H2O"), and #Significant difference from the positive control group ("D-Gal / H2O"). [Figure 11] Interferon gamma (INF-γ) concentrations (mean±SEM) in the cerebral cortex (left) and hippocampus (right). *Significant difference from the negative control group ("NaCl / H2O"), and #Significant difference from the positive control group ("D-Gal / H2O"). [Figure 12] TGF-β concentrations (mean±SEM) in the cerebral cortex. *Significant difference from the negative control group ("NaCl / H2O"), and #Significant difference from the positive control group ("D-Gal / H2O"). [Figure 13] Monocyte chemoattractant protein-1 (MCP-1) concentrations (mean±SEM) in plasma. *Significant difference from the negative control group ("NaCl / H2O"), and #Significant difference from the positive control group ("D-Gal / H2O"). [Figure 14] Neurofilament light chain polypeptide (NfL) concentrations (mean±SEM) in the cerebral cortex (left) and hippocampus (right). *Significant difference from the negative control group ("NaCl / H2O"), and #Significant difference from the positive control group ("D-Gal / H2O").

[0236] Examples

[0237] Example 1: Preparation method and characteristics of cGP-enriched peptide composition

[0238] Enzymatic hydrolysis Porcine gelatin (20% w / w) was enzymatically hydrolyzed using an enzyme (EC 3.4.24.3, derived from Streptomyces species) that has cleavage activity at the Xaa-|-Gly bond in collagen (pH 8, T=30°C, t=16 hours). The enzyme was used at a concentration of 2000 ppm.

[0239] Comparative studies (not shown) demonstrated that this specific enzyme activity leads to a favorable molecular weight distribution and peptide composition, serving as a starting point for further cyclization.

[0240] The peptide was identified by UPLC-MS analysis (ultrahigh-performance liquid chromatography-mass spectrometry).

[0241] Collagen hydrolysates obtained by enzymatic hydrolysis are also called "precursors." Table 1 shows the peptide composition of the precursor. The peptide composition was determined for two distinct replicas produced according to the same protocol. [Table 1]

[0242] Table 2 shows the molecular weight characteristics of the precursor. The peptide composition was determined for two distinct replicas produced according to the same protocol. [Table 2]

[0243] Cyclization by heating Further induction of cGP formation by cyclization was achieved by heating at 90°C for 24 hours. The peptide composition obtained after heating and cyclization is also referred to as "AP" in this specification.

[0244] Table 3 shows the peptide composition of AP. The peptide composition was determined for three distinct replicas generated according to the same protocol. [Table 3]

[0245] Table 4 shows the molecular weight characteristics of AP. The peptide composition was determined for three separate replicates generated according to the same protocol. [Table 4]

[0246] Table 5 shows a comparison of the molecular weight characteristics of the precursor (before heating and cyclization) and AP (after heating and cyclization), based on the measured average values of the individual replicates. [Table 5]

[0247] When comparing the molecular weight data, similar overall Mn and Mw are observed before and after cyclization. However, when comparing the products before and after cyclization, changes in the molecular distribution can be seen.

[0248] As can be seen in Table 5, about 2% of the peptides are below 500 Da before cyclization, and this increases to about 11% after cyclization, whereas about 38% of the peptides are in the range of 500 to 1000 Da before cyclization, and this decreases to about 24% after cyclization.

[0249] Since changes appear to occur in certain relatively low molecular weight fractions due to cyclization, further detailed characterization of the molecular weight distribution of the 0 to 250 Da, 250 to 450 Da, and 450 to 1000 Da fractions was performed (Table 6). [Table 6]

[0250] As can be seen in Table 6, before cyclization, <1% of the peptides have a molecular weight in the range of 250–450 Da, and this increases to about 10% after cyclization, while approximately 39% of the peptides are in the range of 450–1000 Da before cyclization, and this decreases to about 25% after cyclization. Therefore, cyclization appears to change the peptide from the 450–1000 Da range to the 250–450 Da range, resulting in a product with a characteristic peak in the molecular weight range of 250–450 Da. Since cGP shows a typical peak in the size exclusion chromatogram at 250–450 Da due to its shape, it is thought that cGP (Mw=154 Da) contributes to this characteristic peak.

[0251] Table 7 shows a comparison of the peptide compositions of the precursor (before heating and cyclization) and AP (after heating and cyclization) based on the average values ​​of the individual replicates measured. [Table 7]

[0252] We can see that the amount of cGP increases considerably (about 20 times) after cyclization.

[0253] The amounts of hydroxylysine (approximately 35 times) and hydroxyproline (approximately 27 times) also increase considerably after cyclization. Looking at the tripeptide concentrations, the amounts of Gly-pro-ala and Gly-pro-hyp decrease dramatically after cyclization, which can be explained by the formation of cGP.

[0254] Therefore, a comparison of the peptides before and after cyclization shows that cGP was efficiently generated from the Gly-pro-x peptide sequence, particularly from Gly-pro-ala or Gly-pro-hyp.

[0255] Example 2: Radical scavenging activity of cGP-enriched peptide composition

[0256] Example 2 demonstrates the radical scavenging activity of AP. AP was produced as described in Example 1.

[0257] (method) Materials and Reagents • 2,2-diphenyl-1-picrylhydrazyl (DPPH) (R092) (394.32 g / mol) ·methanol distilled water

[0258] DPPH assay protocol The assay was performed in a covered 96-well plate (final volume per well was 200 μL) according to the following protocol: i) Prepare a stock solution of DPPH in methanol. The final concentration per well is 75 μM. ii) Prepare an aqueous stock solution of the test hydrolysate. Prepare a dilution series to obtain a final concentration of 1 mg / mL per well. iii) The final solvent concentration per well is 50 / 50 methanol / water (v / v). iv) Add DPPH stock solution to each test well in a triple line. • Include a positive control (DPPH without the test substance, triple). v) Add the hydrolysate to each test well in a triple sequence. • Include a negative control (test substance without DPPH, triple). vi) Mix well and incubate the mixture in the dark at room temperature for 30 minutes. vii) Measure absorbance at 517 nm under various conditions. viii) Normalize the measured absorbance under the test conditions (sample + DPPH) based on the absorbance of the negative control (test substance without DPPH). ix) Calculate the mean (± standard deviation) absorbance (n=3) for each test sample and the DPPH-positive control (no test substance). x) The following formula: 1 - (average absorbance of test sample / average absorbance of DPPH-positive control) gives the percentage reduction in absorption (compared to the DPPH-positive control).

[0259] (result) Figure 1 shows the radical scavenging activity of cGP, gly-pro-hyp, gly-pro-ala, AP (additive effect and actual effect), and ascorbic acid (positive control). The radical scavenging activity of cGP, gly-pro-hyp, or gly-pro-ala was normalized to their respective concentrations present in AP (concentrations according to Table 3). The additive effect is the theoretical additive effect, which is the sum of the normalized radical scavenging activities of cGP, gly-pro-hyp, and gly-pro-ala.

[0260] It was found that cGP present in AP accounts for 17% of the decrease in DPPH, gly-pro-hyp present in AP accounts for 1% of the decrease in DPPH, and gly-pro-ala present in AP accounts for 0% of the decrease in DPPH.

[0261] Therefore, the additive effect of cGP, gly-pro-hyp, and gly-pro-ala is an 18% reduction in DPPH.

[0262] However, the combination of cGP, gly-pro-hyp, and gly-pro-ala present in AP actually results in a reduction of approximately 30% of DPPH. Therefore, cGP, gly-pro-hyp, and gly-pro-ala have a synergistic effect in reducing DPPH.

[0263] It was further discovered that additional dipeptides (e.g., Pro-Hyp, Gly-pro) and tripeptides in AP can further improve the radical scavenging activity of AP.

[0264] Example 3: Effects of AP on fibrosis

[0265] Example 3 demonstrates the effects of AP in various models of fibrosis.

[0266] (method) material AP was generated as described in Example 1.

[0267] Isolation of fibroblasts and adipocytes (ASCs) Normal skin (including subcutaneous fat) was obtained anonymously from patients who had undergone abdominal fat removal or mastectomy. Fibroblasts and adipocytes (ASCs) were isolated from the skin and subcutaneous adipose tissue, respectively. Fat was removed from the dermis and epidermis and the tissue was cut into fragments. After dispase incubation, the epidermis was removed from the dermis. Both the dermis and adipose tissue were incubated separately at 37°C for 2 hours in a collagenase type II (Gibco, Invitrogen, Paisly, UK) / dispase II (Roche, Mannheim, Germany) solution. The cells were passed through a 40 μm cell strainer and cultured at 37°C in a 5% CO2 atmosphere in DMEM supplemented with 1% penicillin / streptomycin (Invitrogen, Carlsbad, CA, USA) and 1% Ultroser G (BioSepra SA, Cergy-Saint-Christophe, France). Donor-derived cells from n=3 were used in the experiment after passage 2-4.

[0268] Cell culture assay Collagen I was isolated from rat tails and reconstituted in 0.1% acetic acid (4 mg / ml). Fibroblasts were then treated with a 4 mg / ml collagen I solution (cell concentration: 2 × 10⁻⁶). 5 Cells were seeded (at a concentration of cells / ml), and 1 ml of hydrogel per well was poured into a 12-well plate. The hydrogel was polymerized at 37°C for 2 hours. The hydrogel was detached from the well surface and shrunk, and culture medium was added to the wells. The medium containing the peptide was filtered using a 0.2 μm filter, cooled to 37°C, and then added to the culture. Transforming growth factor-β1 (TGF-β1) was used as a control to stimulate fibrosis. The culture medium (containing the peptide) was changed three times a week. Each experimental condition produced intra-experimental replicas. After 2 weeks of culture, the hydrogel was fixed with 4% paraformaldehyde and embedded in paraffin.

[0269] Myofibroblast staining The presence of myofibroblasts was determined by immunohistochemical staining for alpha-smooth muscle actin (α-SMA). Hydrogels were fixed with 4% paraformaldehyde and embedded in paraffin. 5 μm sections were dehydrated and incubated with mouse anti-human α-SMA (1:200, clone 1A4, M0851, DAKO) for 1 hour, followed by blocking with Brightvision Gold for 15 minutes and BrightVision Poly-HRP-Anti Mouse / Rabbit IgG Ruby for 30 minutes. Slides were counterstained with hematoxylin using 3-amino-9-ethylcarbazole as a substrate. The percentage of α-SMA-positive cells was determined by manually counting at least 50 cells per hydrogel (average of 100 cells per hydrogel).

[0270] (result) Figure 2 shows that AP reduces hydrogel contraction in a fibroblast contraction assay. The maximum effect of AP on fibroblasts was observed at the lowest test concentration of 1.7 mg / ml.

[0271] Figure 3 shows that AP reduces hydrogel contraction in an adipocyte (ASC) contraction assay. The maximum effect of AP on ASCs was observed at 1.7 mg / ml. At lower concentrations of AP (i.e., 0.15–0.25 mg / ml), AP activity tended to increase more than that of pure cGP.

[0272] At the end of the 14-day culture period, the hydrogels were treated for paraffin embedding, and the percentage of α-SMA-positive cells was determined by immunohistochemical analysis. According to the shrinkage data, the percentage of α-SMA-positive cells was significantly increased in the TGF-β1-stimulated hydrogel compared to the unexposed control. For both fibroblasts (Figure 4) and ASCs (Figure 5), a clear trend was observed where the percentage of α-SMA-positive cells decreased as the AP concentration increased.

[0273] Figure 4 shows that AP dose-dependently reduces fibroblast differentiation into myofibroblasts, as indicated by the decrease in the percentage of α-SMA-positive cells.

[0274] Figure 5 shows that AP dose-dependently reduces the differentiation of adipocytes into myofibroblasts, as indicated by the decrease in the percentage of α-SMA-positive cells.

[0275] AP was further found to improve the overproduction of collagen I in connective tissue cells such as fibroblasts. AP also appears to improve the production of collagen III and V in connective tissue cells such as fibroblasts.

[0276] AP was found to inhibit macrophage enhancement and contraction in hydrogels containing fibroblasts, suggesting anti-inflammatory properties. AP was also found to stimulate the maturation of monocyte-derived dendritic cells (moDCs) and to stimulate macrophages that lean towards the inflammatory M1 phenotype rather than the fibrous M2 phenotype.

[0277] Overall, though not theoretically bound, blocking or reducing collagen overproduction, and / or regulating the immune response by AP, may be possible mechanisms of action that could lead to (further) the prevention of fibrosis.

[0278] The results show that AP is remarkably effective in preventing fibrosis by at least partially preventing contraction and / or myofibroblast differentiation in connective tissue cells. A dose-dependent effect of cyclic glycine-proline exists in preventing fibrosis.

[0279] Example 4:

[0280] Example 4 demonstrates various neuroprotective properties of AP in a D-galactose mouse model of aging. AP was produced as described in Example 1.

[0281] (method) Test setup Sixty male Swiss mice (3 months old at the time of receipt) were used in the experiment. Table 8 summarizes the test compounds and treatment groups. The test compounds were administered orally once daily for 7 days a week for 69 days. D-galactose (D-GAL) was administered subcutaneously (SC) once daily for 54 days (i.e., starting 15 days after the start of treatment) to induce aging. Therefore, treatment was initiated two weeks before the induction of brain aging. D-Gal was administered one hour after oral administration of the test compound. Three different behavioral tests were used to monitor the effects of the test compounds between days 43 and 54: i) Spontaneous alternation procedure in the Y-maze at D43 (assessment of spatial working memory) ii) Spatial learning in Morris Water Maze (MWM) tests from D46 to D50, and iii) Passive avoidance response (assessment of long-term contextual memory) (trained on D53, retention session on D54).

[0282] The day after the behavioral test (D55), the animals were euthanized for sample collection (plasma, cerebral cortex, and hippocampus) and the following biochemical parameters were determined: • Inflammatory markers: Tumor necrosis factor alpha (TNF-α), Interleukin-6 (IL-6) • Cognitive marker: Brain-derived neurotrophic factor (BDNF) Complementary biomarkers for aging / neurodegeneration: interferon-gamma (IFN-γ), transforming growth factor beta (TGF-β), neurofilamentous light chain polypeptide (NfL), monocyte chemotactic protein-1 (MCP-1)

[0283] Frozen cortical and hippocampal samples were cut into small fragments and homogenized in sterile phosphate-buffered saline (PBS) enriched with a protease inhibitor at a rate of 5 mg of tissue per 100 μl of PBS. The homogenates were then sonicated three times for 10 seconds on ice. The homogenates were then centrifuged at 10,000 rpm at 4°C for 30 minutes, and the supernatant was immediately analyzed by ELISA. For each sample analyzed, the cortical and hippocampal supernatants were diluted to 1 / 5 in sterile PBS enriched with a protease inhibitor cocktail. To avoid proteolysis, all dilutions were performed at 4°C. Standard curve biomarker dilutions were performed at specific concentrations. IL-6, TNF-α, BDNF, INF-γ, TGF-β, and NfL were quantified in double-row ELISA using 100 μl of sample per well in a 96-well plate for each sample. [Table 8]

[0284] (result) General health monitoring and behavioral patterns No adverse effects on weight gain were observed in any of the treatment groups during the study. In the positive control group ("D-Gal / H2O"), loss of spontaneous alternation based on the Y-maze test was observed. Treatment with the lowest dose of AP induced a significant improvement in alternation behavior. Other treatments showed similar trends compared to the 60% baseline (Figure 6). The negative control group ("NaCl / H2O") showed a significantly higher spontaneous alternation rate than the 60% group (p=0.0153), which was considered a characteristic of mouse behavior in the Y-maze. In the positive control group, loss of spontaneous alternation occurred (p=0.0301).

[0285] The MWM test was performed as a tool to test spatial memory and learning (Figure 7). During daily training from day 1 to day 4, control animals were able to locate the hidden platforms, as indicated by a decrease in the swimming time required to find them. The positive control group ("D-Gal / H2O") showed a significant deficit in learning the location of the hidden platforms compared to the negative control group ("NaCl / H2O"), as indicated by a significantly higher swim latency (p=0.0439). Treatment of D-Gal injected animals with AP resulted in improved learning. In the AP 1722 mg / kg group (p=0.0164), the curve was nearly identical to that of the negative control group ("NaCl / H2O").

[0286] Effects on brain biochemical parameters in a D-galactose mouse model Compared to negative control animals ("NaCl / H2O"), positive control animals ("D-Gal / H2O") showed a significant increase in IL-6 concentrations in the cerebral cortex, hippocampus, and plasma (all p ≤ 0.001) (Figure 8). Animals treated with AP 172 mg / kg showed a significant decrease in intracortical and plasma IL-6 concentrations compared to the positive control group. Compared to the positive control group, animals treated with AP 1722 mg / kg showed a potent and significant decrease in IL-6 concentrations in the brain, hippocampus, and plasma. Overall, this suggests a positive effect of this compound on this biomarker.

[0287] Treatment with D-GAL also induced a significant increase in TNF-α concentrations in the cerebral cortex, hippocampus, and plasma of positive control animals ("D-Gal / H2O") compared to negative control animals ("NaCl / H2O") (all p ≤ 0.001) (Figure 9). Compared to animals in the positive control group, decreased TNF-α concentrations were observed in the cerebral cortex, hippocampus, and plasma of animals treated with 172 mg / kg AP and 1722 mg / kg AP. Overall, this suggests a positive effect of AP on the TNF-α biomarker.

[0288] Furthermore, compared to negative control animals ("NaCl / H2O"), a significant decrease in BDNF concentration was observed in the cerebral cortex and hippocampus of positive control animals ("D-Gal / H2O") (p ≤ 0.001) (Figure 10). The same trend was observed in plasma BDNF concentration. Compared to the positive control group, a significant increase in BDNF concentration was observed in the hippocampus of animals treated with AP 172 mg / kg. Compared to the positive control group, a significant increase in BDNF concentration was observed in the brain and hippocampus of animals treated with AP 1722 mg / kg.

[0289] Furthermore, compared to the negative control group ("NaCl / H2O"), a significant increase in INF-γ concentration was observed in the cerebral cortex and hippocampus of the positive control group ("D-Gal / H2O") (p ≤ 0.001) (Figure 11). In this D-Gal mouse model, animals treated with AP 172 mg / kg or 1722 mg / kg showed a decrease in brain INF-γ concentration.

[0290] Compared to negative control animals (Group 1), a significant increase in TGF-β concentration was observed in the cerebral cortex and hippocampus of positive control animals ("D-Gal / H2O") (p ≤ 0.001) (Figure 12). In animals treated with AP 1722 mg / kg, a particularly strong and significant decrease in TGF-β concentration was observed in the cerebral cortex and hippocampus.

[0291] MCP-1 markers were measured in plasma samples from various groups. Compared to negative control animals ("NaCl / H2O"), a significant increase in MCP-1 concentration was observed in the plasma of positive control animals ("D-Gal / H2O"). A particularly strong and significant decrease in MCP-1 concentration was observed in the plasma of animals treated with AP 1722 mg / kg (Figure 13).

[0292] Finally, NfL levels were measured in the cerebral cortex, hippocampus, and plasma of the animals. Compared to negative control animals ("NaCl / H2O"), positive control animals ("D-Gal / H2O") showed a significant increase in NfL concentration in the cerebral cortex and hippocampus. Compared to negative control animals, positive control animals did not show a significant increase in plasma NfL concentration. Animals treated with AP 172 mg / kg showed a decrease in intracerebral cortical NfL concentration compared to the positive control group. Compared to the positive control group, animals treated with AP 1722 mg / kg showed a particularly strong decrease in NfL in the cerebral cortex and hippocampus (Figure 14).

[0293] Based on these results, it is considered that AP administration may be particularly effective in improving cognitive function, such as concentration, attention span, and learning ability, or in improving neurodevelopmental conditions such as ADHD.

Claims

1. 1.0 x 10 ４ cyclic compounds (glycine-proline) in ppm or more, 1.0 x 10 ３ Glycine-proline-hydroxyproline tripeptide in ppm or more, 1.0 x 10 １ Glycine-proline-alanine tripeptide in ppm or more, A peptide composition comprising, A peptide composition in which the ppm is calculated relative to the total weight of the peptides in the peptide composition.

2. 1.0 x 10 ４ ~2.0 x 10 ５ The peptide composition according to claim 1, comprising ppm of a cyclic (glycine-proline) molecule.

3. 1.0 x 10 ３ ~1.0 x 10 ４ A peptide composition according to claim 1 or claim 2, comprising ppm of a glycine-proline-hydroxyproline tripeptide.

4. 1.0×10 １ to 1.0×10 ３ The peptide composition according to any one of claims 1 to 3, comprising glycine-proline-alanine tripeptide in ppm.

5. The peptide composition is calculated as a weight ratio of peptides to the total weight of peptides in the peptide composition, - cGP:Gly-Pro-x tripeptides in a ratio of 10:1 to 100:1, and / or - 10:1 to 100:1 cGP:Gly-Pro-Hyp tripeptides, and / or - cGP:Gly-Pro-Ala tripeptides in a ratio of 100:1 to 1000:1, and / or It has a Gly-Pro-Hyp:Gly-Pro-Ala tripeptide in a ratio of 10:1 to 100:1, A peptide composition according to any one of claims 1 to 4, wherein x is any amino acid.

6. A peptide composition according to any one of claims 1 to 5, having a weight-average molecular weight of 1000 to 2000 Da, preferably 1200 to 1800 Da.

7. Calculated with respect to the total weight of the peptides in the peptide composition, - 0-2% by weight of peptides having a molecular weight of up to 250 Da, and - A peptide having a molecular weight of 250 to 450 Da, in an amount of 5 to 15% by weight, and - A peptide having a molecular weight of 450 to 1000 Da, in a quantity of 15 to 35% by weight. A peptide composition according to any one of claims 1 to 6, comprising:

8. - 35 to 50% by weight of peptides having a molecular weight of 1000 to 2000 Da, calculated relative to the total weight of peptides in the peptide composition, and - 15-30% by weight of peptides having a molecular weight of 2000-5000 Da, and - A peptide having a molecular weight of 5000 to 10000 Da, in an amount of 0.5 to 5% by weight, and - A peptide having a molecular weight exceeding 10,000 Da, in a maximum of 2% by weight, preferably 0.01 to 1% by weight. A peptide composition according to any one of claims 1 to 7, comprising:

9. A peptide composition according to any one of claims 1 to 8, for use as a pharmaceutical product.

10. A peptide composition according to any one of claims 1 to 8, for use in the prevention and / or treatment of a condition selected from the group consisting of neurological conditions and fibrosis.

11. The peptide composition for use according to claim 10, wherein the neurological pathology is one or more selected from the group consisting of central nervous system inflammation, central nervous system inflammatory injury, motor pathology or motor disorder, neuromotor disorder, neuromuscular disorder, neurosensory disorder, psychotic pathology or psychotic disorder, depressive pathology or depressive disorder, anxiety pathology or anxiety disorder, injury to the nervous system and / or brain, cognitive pathology or cognitive impairment, personality pathology or personality disorder, mood pathology or mood disorder, migraine, epilepsy or convulsive disorder, neurodegenerative pathology or neurodegenerative disorder, autism spectrum disorder, neuropathy, neurodevelopmental disorder, and neurotoxicity.

12. The peptide composition for use according to claim 10, wherein the fibrosis is one or more selected from the group consisting of medical implant-related fibrosis, surgery-related fibrosis, organ-specific fibrosis, postoperative adhesion formation, cutaneous fibrosis, cellulite, Dupuytren's contracture, hypertrophic scar, keloid, hepatic fibrosis, pulmonary fibrosis, pancreatic fibrosis, renal fibrosis, vascular fibrosis, endometriosis, atrial fibrosis, and cardiac fibrosis.

13. The peptide composition for use according to claim 10, wherein the fibrosis is a symptom of a condition selected from the group consisting of fatty liver disease, chronic obstructive pulmonary disease, asthma, and systemic sclerosis.

14. A peptide composition for use according to any one of claims 9 to 13, which is administered orally.

15. A peptide composition for use according to any one of claims 9 to 14, administered in a total daily dose of 0.1 to 20 g.

16. Use of the composition according to any one of claims 1 to 8 in non-therapeutic improvement of cognitive function.

17. The use according to claim 16, wherein the cognitive function is selected from the group consisting of learning ability, memory, spatial memory, problem-solving ability, recall ability, attention duration, concentration, language processing ability, spatial awareness, speech, language skills, concentration, mental arousal, and reaction time.

18. A method for obtaining a peptide composition comprising a cyclic (glycine-proline) as described in any one of claims 1 to 8, wherein the method is a) Calculated based on the total weight of peptides in collagen hydrolysate: 1.0 × 10 ４ ~1.0 x 10 ５ ppm of glycine-proline-hydroxyproline tripeptide and 5 × 10 ３ ~5 x 10 ４ To provide a collagen hydrolysate containing ppm glycine-proline-alanine and having a weight-average molecular weight of 500 Da or more and 3000 Da or less, and b) Heat-treating the collagen hydrolysate at a temperature of at least 65°C, A method in which x is any amino acid.

19. The method according to claim 18, wherein the collagen hydrolysate provided in step a) has a weight-average molecular weight of 800 Da or more and 2500 Da or less, preferably 1000 Da or more and 2000 Da or less, and more preferably 1200 Da or more and 1800 Da or less.

20. The collagen hydrolysate provided in step a) - 0-2% by weight of peptides having a molecular weight of up to 250 Da, and - A peptide having a molecular weight of 250 to 450 Da, in an amount of 0 to 2% by weight, and The method according to claim 18 or claim 19, comprising 30 to 50% by weight of a peptide having a molecular weight of 450 to 1000 Da.

21. The collagen hydrolysate provided in step a) - A peptide having a molecular weight of 1000 to 2000 Da, calculated relative to the total weight of the peptides in the peptide composition, in an amount of 25 to 50% by weight, and - A peptide having a molecular weight of 2000 to 5000 Da, in an amount of 0.5 to 30% by weight, and - A peptide having a molecular weight of 5000 to 10000 Da, in an amount of 0.2 to 5% by weight, and The method according to any one of claims 18 to 20, comprising a peptide having a molecular weight of more than 10,000 Da, in a maximum of 2% by weight, preferably 0.01 to 1% by weight.

22. The method according to any one of claims 18 to 21, wherein the heat treatment in step b) is 70 to 120°C, preferably 80 to 100°C.

23. The method according to any one of claims 18 to 22, wherein the heat treatment in step b) lasts for 6 to 72 hours, preferably 12 to 48 hours.

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