Biologically active collagen hybridizing peptides

Abstract

The modified collagen hybridizing peptide (CHP) may include one or more binding partners crosslinked to the CHP. CHP has the formula (I): (Gly-XY) a~b wherein Gly is glycine, at least one of X and Y is proline, modified proline and / or hydroxyproline, a is 3, and b is 20. The one or more binding partners are selected from the group consisting of an integrin site, an integrin binding site, a cross-linking site, von Willebrand factor (VWF), discoidin domain receptor (DDR) 1, DDR2, a SPARC binding peptide, a fibronectin binding peptide, a modified integrin binding peptide, a matrix metalloproteinase (MMP) cleavage site, a cathepsin K (CATK) site, and an osteoclast-associated receptor (OSCAR).

Description

[Background technology]

[0001] Collagen homeostasis is a complex process involving both collagen synthesis and degradation. Collagen homeostasis is tightly controlled primarily by a specialized group of cells called fibroblasts. Fibroblasts produce both collagen and matrix metalloproteinases (MMPs) in response to environmental conditions such as chemical signals, cell-to-extracellular matrix (ECM) interactions, UV light, and force. The most abundant ECM protein is collagen. Collagen has the existing binding sequences necessary to assume a triple-helical form for recognition by fibroblasts, MMPs, cathepsin K, or other cell types including integrins, discoidin domain receptors, osteoclast-associated receptors (OSCARs), and many others. Importantly, fibroblasts respond to mechanical signals through integrin-mediated interactions with collagen.

[0002]

[0002] Many events can cause fibroblasts to reduce collagen production, including diseases such as osteoarthritis; natural events such as aging (senescent fibroblast senescence); reduced mechanical stimulation; and environmental factors such as UV damage or chemical signals such as reactive oxygen species. Reduced collagen synthesis often leads to reduced mechanical stimulation, as fibroblasts have less surrounding collagen to interact with. This reduced mechanical stimulation can lead to increased matrix metalloproteinase (MMP) production, essentially creating a negative feedback loop in which reduced mechanical stimulation leads to increased MMP production and reduced collagen production, further reducing mechanical stimulation. This can occur in dermal fibroblasts in aged skin, whether due to increased UV exposure or natural aging. A similar process can occur in tissues such as tendons or cartilage, which contain mechanically damaged collagen.

[0003]

[0003] Fibroblasts can escape this negative feedback loop by providing additional binding sites for fibroblasts to attach. Providing new mechanical cues in the microenvironment increases collagen production, thereby increasing the mechanical stimulus for cells to create a positive feedback loop and promote tissue regeneration and repair. Therefore, methods and compositions that can stimulate fibroblasts to increase collagen production to restore the structural integrity of the ECM and repair damage to collagen-containing tissues are desirable. Summary of the Invention

[0004] In one aspect, to solve the problem of stimulating fibroblasts to regulate the production of ECM components, such as collagen and MMPs, the present disclosure provides a modified collagen hybridizing peptide (CHP), comprising one or more binding partners crosslinked to the CHP. CHP has the formula I: (Gly-XY) a~b wherein Gly is glycine, at least one of X and Y is proline, modified proline and / or hydroxyproline, a is 3, and b is 20. The one or more binding partners are selected from the group consisting of an integrin site, an integrin binding site, a cross-linking site, von Willebrand factor (VWF), discoidin domain receptor (DDR) 1, DDR2, a SPARC binding peptide, a fibronectin binding peptide, a modified integrin binding peptide, a matrix metalloproteinase (MMP) cleavage site, a cathepsin K (CATK) site, and an osteoclast-associated receptor (OSCAR).

[0005]

[0005] In one aspect, to solve the problem of increasing collagen production in a subject, the present disclosure provides a composition comprising a modified collagen hybridizing peptide (CHP) disclosed herein.

[0006]

[0006] In one aspect, to solve the problem of increasing collagen degradation in a subject, the present disclosure provides a composition comprising a modified collagen hybridizing peptide (CHP) disclosed herein.

[0007]

[0007] In one aspect, to solve the problem of increasing MMP production in a subject, the present disclosure provides a composition comprising a modified collagen hybridizing peptide (CHP) disclosed herein.

[0008]

[0008] In one aspect, to solve the problem of decreasing collagen production in a subject, the present disclosure provides a composition comprising a modified collagen hybridizing peptide (CHP) disclosed herein.

[0009]

[0009] In one aspect, to solve the problem of reducing MMP production in a subject, the present disclosure provides a composition comprising a modified collagen hybridizing peptide (CHP) disclosed herein.

[0010]

[0010] In another aspect, the present disclosure further provides a method for increasing collagen production in a subject, the method comprising administering to the subject a cosmetic composition comprising a modified collagen hybridizing peptide (CHP) disclosed herein.

[0011]

[0011] In one aspect, to solve the problem of inducing skin repair in a subject, the present disclosure provides a composition comprising a modified collagen hybridizing peptide (CHP) disclosed herein.

[0012]

[0012] In another aspect, the present disclosure further provides a method for inducing skin repair in a subject, the method comprising the step of administering to the subject a cosmetic composition comprising a modified collagen hybridizing peptide (CHP) disclosed herein.

[0013]

[0013] In one aspect, to solve the problem of inducing tissue repair in a subject, the present disclosure provides a composition comprising a modified collagen hybridizing peptide (CHP) disclosed herein.

[0014]

[0014] In another aspect, the present disclosure further provides a method for inducing tissue repair in a subject, the method comprising administering to the subject a medical device composition comprising a modified collagen hybridizing peptide (CHP) disclosed herein. [Brief description of the drawings]

[0015] [Figure 1A] FIG. 1 illustrates a biologically active CHP that contains a triple-helix forming moiety covalently linked to a biologically active sequence. [Figure 1B] Diagram of the unique binding mechanism of bioactive CHP, which renders it inactive in a single-chain conformation but activates it upon triple-helix formation with damaged collagen, allowing collagen-producing cells to bind to and mechanically sense the newly formed triple helices. [Figure 2A] FIG. 1 shows CHP targeting and binding to damaged collagen induced by collagenase activity in an in vivo mouse model of tendonitis. [Figure 2B] 1 is an image showing the striking difference in CHP binding between normal and osteoarthritic (OA) knee joints in stained human tissue sections. [Figure 2C] 1 is an image showing increased CHP binding in aged human tissue sections of skin compared to human tissue sections of young tissue. [Figure 3A]1 is a graph showing the first derivative from the thermal melting curve (Tm) of the bioactive CHP disclosed herein. Tm was measured from 4°C to 80°C at a rate of 0.5°C per minute and indicates the thermal transition temperature as CHP goes from an ordered triple helical structure to a more disordered monomeric structure. The first derivative shows the inflection point of the Tm curve as the minimum of the graph. This point corresponds to the melting temperature of CHP, i.e., the temperature required to break down any homotrimeric triple helices that have formed. This indicates that the bioactive CHP has a Tm close to body temperature (37°C), making the bioactive CHP an ideal candidate for binding to damaged collagen in the body. Each CHP was dissolved in DI water at a concentration of 150 μM. Before measuring Tm, each CHP was allowed to fully fold by placing it in a refrigerator (4°C) for 48 hours. [Figure 3B] 1 is a graph showing the average amount of CHP binding at each bioactive binding site in B-G3-(GfO)5-binding site-(GfO)5. A 10% gelatin solution (w / v) from porcine skin (Sigma-Aldrich 48724-100G-F) was prepared in 1×PBS. The 10% gelatin solution was kept under constant heating (65°C) to prevent it from gelling. Next, 25 μL of the heated 10% gelatin solution was added to coat the bottom of a 96-well plate, and the excess was then immediately removed to leave a thin layer of gelatin. The plate was cooled at 4°C for 15 minutes. Then, 100 μL of EDC crosslinking solution consisting of 1×MES buffer, EDC (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride), and NHS (N-hydroxysuccinimide) was added to the surface of each well and allowed to crosslink overnight at 4°C. The cross-linking solution was removed and each well was then washed 3 times with 200 μL of 1×PBS. Next, 50 μL of CHP was added at a concentration of 100 μM and allowed to bind overnight at 37° C. After binding, the wells were washed 3 times with 200 μL of 1×PBS and 20 μL of FITC-streptavidin fluorophore (0.01 mg / ml) was added to each well and allowed to bind for 1 hour at room temperature to visualize the amount of CHP binding. [Figure 4A]Images of human dermal fibroblast (HDF) cells from healthy (Figure 4A), injured (Figure 4B) and bioactive CHP-treated injured (Figure 4C) models in 2.5D gelatin experiments. For the 2.5D gelatin experiments, 10% gelatin solution was made from pig skin (Sigma-Aldrich 48724-100G-F) in 1x PBS and kept at 65°C to prevent gelation before use. Then, 250 μL of the 10% gelatin solution was added to each corresponding well of a 24-well plate. The plate was cooled at 4°C for 15 minutes. Then, 400 μL of EDC crosslinking solution. The crosslinking solution was removed and the wells were washed three times using 400 μL of 1x PBS. Then, 70% EtOH was added to each well and the plate was placed under UV light for 1 hour for sterilization. The wells were washed five times with 1 mL of 1x PBS. The CHP was pre-heated at 80°C for 15 minutes. A master mix of 15,000 human dermal fibroblasts (ATCC), 50 μL of 20 μM CHP, and 950 μL of fibroblast growth medium with 10% FBS was made for each well in each treatment. 1000 μL of this mix was added to each corresponding well. Wells were imaged at 24 hours at 4x magnification on an EVOS Brightfield. Images were analyzed with ImageJ software. [Figure 4B]Images of human dermal fibroblast (HDF) cells from healthy (Figure 4A), injured (Figure 4B) and bioactive CHP-treated injured (Figure 4C) models in 2.5D gelatin experiments. For the 2.5D gelatin experiments, 10% gelatin solution was made from pig skin (Sigma-Aldrich 48724-100G-F) in 1x PBS and kept at 65°C to prevent gelation before use. Then, 250 μL of the 10% gelatin solution was added to each corresponding well of a 24-well plate. The plate was cooled at 4°C for 15 minutes. Then, 400 μL of EDC crosslinking solution. The crosslinking solution was removed and the wells were washed three times using 400 μL of 1x PBS. Then, 70% EtOH was added to each well and the plate was placed under UV light for 1 hour for sterilization. The wells were washed five times with 1 mL of 1x PBS. The CHP was pre-heated at 80°C for 15 minutes. A master mix of 15,000 human dermal fibroblasts (ATCC), 50 μL of 20 μM CHP, and 950 μL of fibroblast growth medium with 10% FBS was made for each well in each treatment. 1000 μL of this mix was added to each corresponding well. Wells were imaged at 24 hours at 4x magnification on an EVOS Brightfield. Images were analyzed with ImageJ software. [Figure 4C]Images of human dermal fibroblast (HDF) cells from healthy (Figure 4A), injured (Figure 4B) and bioactive CHP-treated injured (Figure 4C) models in 2.5D gelatin experiments. For the 2.5D gelatin experiments, 10% gelatin solution was made from pig skin (Sigma-Aldrich 48724-100G-F) in 1x PBS and kept at 65°C to prevent gelation before use. Then, 250 μL of the 10% gelatin solution was added to each corresponding well of a 24-well plate. The plate was cooled at 4°C for 15 minutes. Then, 400 μL of EDC crosslinking solution. The crosslinking solution was removed and the wells were washed three times using 400 μL of 1x PBS. Then, 70% EtOH was added to each well and the plate was placed under UV light for 1 hour for sterilization. The wells were washed five times with 1 mL of 1x PBS. The CHP was pre-heated at 80°C for 15 minutes. A master mix of 15,000 human dermal fibroblasts (ATCC), 50 μL of 20 μM CHP, and 950 μL of fibroblast growth medium with 10% FBS was made for each well in each treatment. 1000 μL of this mix was added to each corresponding well. Wells were imaged at 24 hours at 4x magnification on an EVOS Brightfield. Images were analyzed with ImageJ software. [Figure 4D] Figure 1 shows the results of HDF cell length in a 2.5D gelatin experiment by creating 12 unit cells and collecting images after 24 hours of incubation at 37°C. To reduce subjectivity in analyzing cell length, cells were randomly selected and counted until 100 cells were analyzed per image. To determine cell length, individual cells were measured with the internal ImageJ measurement tool. [Figure 4E] Figure 1 is a graph showing the average cell number after 24 hours of incubation in 250 μL of 10% gelatin. To count the number of cells, images were analyzed using ImageJ Maxima Finder. [Figure 5A]Figure 1 shows the delta CT fold change values ​​from PBS by ex vivo native models purchased from GenoSkin. The models passed all OC testing by GenoSkin. The specimens were surgically removed from the abdomen of a 69 year old Caucasian female with type 1 phototype skin (light skin). Upon receiving the ex vivo models, 1mL of the supplied GenoSkin medium was added to each model and allowed to calibrate in situ for 1 hour at 37°C. The CHP was heated to 80°C for 15 minutes and then rapidly quenched on ice (approximately 30-90 seconds). Next, 100μL of 100μM CHP was added to 900μL of medium. For each treatment, the medium was removed from each model and 1000μL of fresh medium+CHP was added to each well. The CHP was allowed to absorb from the bottom of the model to simulate an in situ injection. The medium+CHP mixture was removed and replenished daily. On day 7 after surgery (day 6 of treatment), models were homogenized and lysed for RNA extraction according to the Qiagen RNeasy Micro Kit protocol. RNA was quantified in nanodroplets. Reverse transcription was performed according to the Qiagen QuantiTect Reverse Transcription Kit. RT-PCR was performed using Taqman reagents with Col1A1 as the gene of interest and 18S as the housekeeping gene. Values ​​reported are delta CT fold changes from PBS wells. [Figure 5B]Figure 1 shows the delta CT fold change values ​​from PBS by ex vivo native models purchased from GenoSkin. The models passed all OC testing by GenoSkin. The specimens were surgically removed from the abdomen of a 69 year old Caucasian female with type 1 phototype skin (light skin). Upon receiving the ex vivo models, 1mL of the supplied GenoSkin medium was added to each model and allowed to calibrate in situ for 1 hour at 37°C. The CHP was heated to 80°C for 15 minutes and then rapidly quenched on ice (approximately 30-90 seconds). Next, 100μL of 100μM CHP was added to 900μL of medium. For each treatment, the medium was removed from each model and 1000μL of fresh medium+CHP was added to each well. The CHP was allowed to absorb from the bottom of the model to simulate an in situ injection. The medium+CHP mixture was removed and replenished daily. On day 7 after surgery (day 6 of treatment), models were homogenized and lysed for RNA extraction according to the Qiagen RNeasy Micro Kit protocol. RNA was quantified in nanodroplets. Reverse transcription was performed according to the Qiagen QuantiTect Reverse Transcription Kit. RT-PCR was performed using Taqman reagents with Col1A1 as the gene of interest and 18S as the housekeeping gene. Values ​​reported are delta CT fold changes from PBS wells. [Figure 5C]1 is a graph showing ELISA results for procollagen I in an ex vivo hole punch wound healing model. The ex vivo hole punch models were purchased from GenoSkin. The specimens were surgically removed from the abdomen of a female, 63 years old, African American, with type 5 phototype skin (dark skin). Upon receiving the ex vivo models, 1 mL of the supplied GenoSkin medium was added to each model and allowed to calibrate undisturbed at 37°C for 1 hour. Each day, the GenoSkin medium was removed from each model and replaced with 1000 μL of fresh medium. The CHP was heated at 80°C for 15 minutes and then rapidly quenched on ice (approximately 30-90 seconds). Next, 100 μL of a 100 μM CHP solution was administered on top of the model and replaced every 24 hours. The medium was collected daily, then centrifuged and aliquoted into 100 μl samples. These samples were stored at -80°C. Samples were then processed using the manufacturer's protocol for Abeam's Procollagen I ELISA Kits (AB120966). [Figure 6] 1 is a graph showing changes in gene expression caused by the bioactive CHP disclosed herein. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0016] Detailed Description

[0028] In the following, exemplary embodiments of the present disclosure are described in detail. However, the present disclosure is not limited to the embodiments disclosed below, but may be implemented in various forms. The following embodiments are described to enable those skilled in the art to embody and implement the embodiments of the present disclosure.

[0017]

[0029] Disclosed are substances, compositions, and components that can be used in, with, or in preparation for the disclosed methods and compositions, or that are products of the disclosed methods and compositions. Where these and other substances are disclosed herein, and combinations, subsets, interactions, groups, etc. of these substances are disclosed, it is understood that although specific reference to the various individual and collective combinations and permutations of these compounds may not be expressly disclosed, each is specifically contemplated and described herein. For example, where peptide conjugates are disclosed and discussed, and multiple modifications that can be made to multiple molecules that comprise the peptide conjugates are discussed, each combination and permutation of the peptide conjugates and possible modifications are specifically contemplated unless specifically indicated to the contrary. Thus, where classes of molecules A, B, and C are disclosed, and further classes of molecules D, E, and F, as well as examples of combined molecules A-D, each is individually and collectively contemplated, even if each is not individually described. Thus, in this example, each of the combinations A-E, A-F, B-D, B-E, B-F, C-D, C-E, and C-F is specifically contemplated and should be considered disclosed from the disclosure of A, B, and C; D, E, and F; and example combinations A-D. Likewise, any subset or combination of these is specifically contemplated and disclosed. Thus, for example, the subgroups A-E, B-F, and C-E are specifically contemplated and should be considered disclosed from the disclosure of A, B, and C; D, E, and F; and example combinations A-D. This concept applies to all aspects of this application, including, but not limited to, steps in methods of making and using the disclosed compositions. Thus, where there are multiple possible additional steps, it is understood that each of these additional steps can be performed by any particular embodiment or combination of embodiments of the disclosed methods, and each such combination should be considered specifically contemplated and disclosed.

[0018] definition

[0030] The terms first, second, etc. may be used to describe various elements, but these elements are not limited by these terms. These terms are only used to distinguish one element from another. For example, a first element may be referred to as a second element, and similarly, a second element may be referred to as a first element, without departing from the scope of the example embodiments. The term "and / or" includes any and all combinations of one or more of the associated listed items.

[0019]

[0031] It must be noted that, as used herein, in the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, a reference to "a peptide" includes a plurality of such peptides, a reference to "the peptide" is a reference to one or more peptides and equivalents thereof known to those skilled in the art, and so forth. Similarly, the word "or" is intended to include "and" unless the context clearly dictates otherwise.

[0020]

[0032] As used herein in the specification and claims, the phrase "at least one" in reference to a list of one or more elements should be understood to mean at least one element selected from any one or more of the elements in the list of elements, but not necessarily including at least one of each element specifically listed in the list of elements, and not excluding any combination of elements in the list of elements. This definition also allows for elements other than those specifically identified in the list of elements to which the phrase "at least one" refers, whether related or not to those elements specifically identified, may optionally be present. Thus, as a non-limiting example, "at least one of A and B" (or, equivalently, "at least one of A or B," or, equivalently, "at least one of A and / or B") can refer to, in one embodiment, at least one A (optionally including elements other than B), with no B present, and optionally including more than one; in another embodiment, at least one B (optionally including elements other than A), with no A present, and optionally including more than one; in yet another embodiment, at least one A, optionally including more than one, and at least one B, optionally including more than one, (optionally including other elements); etc.

[0021]

[0033] The terms "comprising," "including," "having," and the like are used interchangeably and have the same meaning. Similarly, "comprises," "includes," "has," and the like are used interchangeably and have the same meaning. In particular, each of these terms is defined consistent with the general U.S. patent law definition of "comprising," and is therefore interpreted as the open term meaning "at least the following," and not excluding additional features, limitations, aspects, and the like. Thus, for example, "an apparatus having components a, b, and c" means that the apparatus includes at least components a, b, and c. Similarly, the phrase: "a method involving steps a, b, and c" means that the method includes at least steps a, b, and c. Additionally, although steps and processes may be outlined in a particular order herein, one of ordinary skill in the art will recognize that designating the steps and processes may vary unless a particular order is clearly indicated by the context.

[0022]

[0034] As used herein, the term "about" refers to numerical values, including, for example, integers, fractions, and percentages, whether or not expressly indicated. The term "about" generally refers to a range of numerical values ​​(e.g., + / - 5, 6, 7, 8, 9, or 10% of the stated value) that one of ordinary skill in the art would consider equivalent to the stated value (e.g., having the same function or result). In some instances, the term "about" may include numerical values ​​that are rounded to the nearest significant figure. In general, the term "about" includes values ​​for a given quantity that fall within the acceptable range of manufacture, formulation, and / or measurement, at least.

[0023]

[0035] As used herein, "collagen" may be from any tissue type (e.g., bone, dermis, tendon, ligament, etc.). Collagen may refer to a molecule in which three alpha chains of a polyproline II-like structure are folded together into a triple helix. In addition, collagen may apply to any protein that contains a triple helix region, including collagen types I-XXVIII and bacterial collagen. The term "collagen" as used herein may refer to all forms of collagen, including artificial collagen and collagen that has been processed or otherwise modified. In some embodiments, the collagen is selected from type I collagen, type II collagen, type III collagen, type IV collagen, type V collagen, type VI collagen, type VII collagen, type VIII collagen, type IX collagen, type X collagen, type XI collagen, type XII collagen, type XIII collagen, type XIV collagen, type XV collagen, type XVI collagen, type XVII collagen, type XVIII collagen, type XIX collagen, type XX collagen, type XXI collagen, type XXII collagen, type XXIII collagen, type XXIV collagen, type XXV collagen, type XXVI collagen, type XXVII collagen, type XXVIII collagen, and combinations thereof.

[0024]

[0036] As used herein, the term "proline or modified proline" refers to the amino acid proline and its various isomers, analogs and variants, including both natural and non-natural isomers. In one example, the modified proline contains an electron-withdrawing group. Examples of modified prolines include, but are not limited to, hydroxyproline, methylated proline, 4-fluoroproline, and 4-chloroproline.

[0025]

[0037] In some embodiments, the method excludes collecting a sample from a subject. In some embodiments, "subject" as used herein may refer to a human or animal or bacteria or cell cultures from any of the aforementioned groups. Non-limiting examples of animals include vertebrates, such as primates, rodents, livestock animals, or game animals. Primates include chimpanzees, cynomolgus monkeys, spider monkeys, and macaques (e.g., rhesus monkeys). Rodents include mice, rats, marmots, ferrets, rabbits, and hamsters. Livestock and game animals include cows, horses, pigs, deer, bison, buffalo, elk, feline species (e.g., domestic cats), and canine species (e.g., dogs, foxes, wolves). Fish include chondrichthyes (cartilaginous fish) and bony fish (bony fish). The subject may be a mammal. The mammal may be, but is not limited to, a human, a non-human primate, a mouse, a rat, a dog, a cat, a horse, or a cow. In addition, the methods described herein can be used to diagnose and / or treat livestock or pet animals. The term does not refer to a particular age or sex. Thus, adult and neonatal subjects, as well as fetuses, whether male or female, are intended to be included within the scope of the term.

[0026]

[0038] The term "treating" refers to partially or completely alleviating, ameliorating, alleviating, delaying, inhibiting progression, reducing the severity, and / or reducing the incidence of one or more symptoms or characteristics of a particular disease, disorder, and / or condition. For example, "treating" a disease or injury involving collagen damage can refer to reducing or eliminating the amount of damaged / denatured collagen. Treatment may be administered to subjects who do not exhibit symptoms of the disease, disorder, and / or condition and / or to subjects who exhibit only early signs of the disease, disorder, and / or condition for the purpose of reducing the risk of developing pathology associated with the disease, disorder, and / or condition.

[0027] I. introduction

[0039] Exemplary embodiments of the present disclosure are described in detail below with reference to the accompanying drawings. To facilitate understanding of the present disclosure, like numerals refer to like elements throughout the description of the figures and descriptions of the same elements are not repeated.

[0028] II. skin aging

[0040] As we age, we have decreased collagen production and increased MMP production in the skin, which causes wrinkled (and thin) skin. Figure 1 illustrates the pathway for aging-induced production of reactive oxygen species (ROS) and MMP in fibroblasts. First, ROS are generated by exposure to external factors, such as UV and pollution. Excess ROS further exacerbates the damage to tissues by pushing young fibroblasts into a pathway of increased ROS and MMP production (negative feedback loop).

[0029]

[0041] Loss of collagen and increased ROS levels in the ECM cause skin fibroblasts to round and become senescent. This leads to further ROS production and MMP production, further reducing collagen production, thereby destroying collagen in the ECM that supports fibroblasts in an elongated "young" morphology. Loss of collagen further pushes fibroblasts to a rounded "senescent" morphology, creating an accelerating negative feedback loop. A similar phenomenon occurs when collagen tissues such as tendons are damaged, for example, by trauma (i.e., tearing). High ROS levels and collagen matrix destruction resulting from trauma accelerate fibroblast rounding.

[0030]

[0042] To reverse the effects of aging on the ECM or to repair damaged collagen tissue, the problem of providing mechanical stimulation to rounded fibroblasts needs to be solved.

[0031] III. Stimulating fibroblasts to increase collagen production

[0043] In one aspect, to solve the problem of stimulating fibroblasts to increase collagen production, the present disclosure provides a modified collagen hybridizing peptide (CHP), comprising one or more binding partners crosslinked to the CHP. CHP has the formula I: (Gly-XY) a~b wherein Gly is glycine, at least one of X and Y is proline, modified proline and / or hydroxyproline, a is 3, and b is 20. The one or more binding partners are selected from the group consisting of an integrin site, an integrin binding site, a cross-linking site, von Willebrand factor (VWF), discoidin domain receptor (DDR) 1 and 2, a SPARC binding peptide, a fibronectin binding peptide, a modified integrin binding peptide, a matrix metalloproteinase (MMP) cleavage site, a cathepsin K (CATK) site, and an osteoclast-associated receptor (OSCAR).

[0032] IIIa. Collagen Hybridizing Peptide (CHP)

[0044] Collagen is the most abundant protein in the human body and is an essential component of nearly all organs and tissues, providing structure for cell attachment and growth. All types of collagen from all species share a triple helical protein structure that is found almost exclusively in collagen.

[0033]

[0045] In certain embodiments, CHP comprises a short repeating tripeptide that can specifically target damaged collagen in tissues by recognizing structural motifs not available in intact collagen molecules (e.g., polyproline II-like helices in alpha chains).By modifying CHP to induce specific biological activities, CHP can be used to improve the microenvironment around fibroblasts, and thus CHP can regulate collagen production by adhering to ECM and increasing mechanical stimuli and feedback within the ECM.

[0034]

[0046] In certain embodiments, CHP comprises a specific sequence (also referred to herein as "active sequence or bioactive sequence"), which can crosslink to triple helix forming moieties on both sides of the bioactive sequence. When fibroblasts recognize the bioactive sequence and they are in triple helix form, CHP targets damaged collagen and forms triple helix, thus preventing fibroblasts from binding to the bioactive sequence unless the bioactive sequence is in triple helix structure. Therefore, the modified CHP of the present disclosure does not bind to fibroblasts in solution. Instead, the binding between fibroblasts and modified CHP occurs only when fibroblasts are localized to CHP bound to damaged collagen.

[0035] IV. Modified collagen hybridizing peptide (CHP)

[0047] FIG. 6 shows the results of a bioactive CHP with the sequence CF-Ahx-(GPO)3-GFOGER-(GPO)3. GFOGER is the binding site for α1β1 and α2β1 integrins. The modified CHP also contains sequences with a high propensity to form triple helices, with GPO repeats on either side of the integrin binding site GFOGER. These sequences ensure that the CHP can stably bind to damaged collagen. Thus, in one example, the modified CHP has the sequence CF-Ahx-(GPO)3-GFOGER-(GPO)3. The modified CHP is incorporated into gelatin (coated on a slide) and forms a collagen triple helix such that the integrin binding site GFOGER is only present in the CHP. The α2β1 integrin mainly interacts only with a single alpha chain, with a minority of interactions occurring with the second alpha chain and not with the third alpha chain of the collagen triple helix. As a result, binding between fibroblasts and the integrin binding domain occurs when CHP binds to degraded collagen.

[0036]

[0048] In certain embodiments, the active sequence is selected to be a binding site for a molecule and / or marker specific for fibroblasts to recruit and / or bind to fibroblasts at sites of collagen degradation or damage, thereby stimulating fibroblasts to either increase collagen production, clear damaged collagen faster, reduce MMP production, or some combination of the above to induce tissue repair. Thus, the one or more binding partners may include, but are not limited to, MMP cleavage sites, integrin sites, integrin binding sites, fibronectin binding sites, cathepsin K (CATK) binding sites, cross-linking sites, von Willebrand factor (VWF), discoidin domain receptor (DDR) 1, DDR2, SPARC binding peptides, modified integrin binding peptides, and osteoclast-associated receptor (OSCAR).

[0037]

[0049] In certain embodiments, the modified CHP disclosed herein comprises one or more binding sites cross-linked to the CHP. The CHP has the formula I: (Gly-XY) a~b wherein Gly is glycine, at least one of X and Y is proline, hydroxyproline and / or modified proline, a is 3 and b is 20. The one or more binding partners are selected from the group consisting of an integrin site, an integrin binding site, a cross-linking site, von Willebrand factor (VWF), discoidin domain receptor (DDR) 1, DDR2, a SPARC binding peptide, a fibronectin binding peptide, a modified integrin binding peptide, a matrix metalloproteinase (MMP) cleavage site, a cathepsin K (CATK) site, and an osteoclast-associated receptor (OSCAR).

[0038]

[0050] In some embodiments, the modified CHP has the formula I: (Gly-XY) a~bwherein Gly is glycine, at least one of X and Y is proline, modified proline, and / or hydroxyproline, a is 3, and b is 20.

[0039]

[0051] In an exemplary embodiment, a is 3 and b is 4. In an exemplary embodiment, a is 3 and b is 5. In an exemplary embodiment, a is 3 and b is 6. In an exemplary embodiment, a is 3 and b is 7. In an exemplary embodiment, a is 3 and b is 8. In an exemplary embodiment, a is 3 and b is 9. In an exemplary embodiment, a is 3 and b is 10. In an exemplary embodiment, a is 3 and b is 11. In an exemplary embodiment, a is 3 and b is 12. In an exemplary embodiment, a is 3 and b is 13. In an exemplary embodiment, a is 3 and b is 14. In an exemplary embodiment, a is 3 and b is 15. In an exemplary embodiment, a is 3 and b is 16. In an exemplary embodiment, a is 3 and b is 17. In an exemplary embodiment, a is 3 and b is 18. In an exemplary embodiment, a is 3 and b is 19. In an exemplary embodiment, a is 3 and b is 20.

[0040]

[0052] In an exemplary embodiment, a is 4 and b is 5. In an exemplary embodiment, a is 4 and b is 6. In an exemplary embodiment, a is 4 and b is 7. In an exemplary embodiment, a is 4 and b is 8. In an exemplary embodiment, a is 4 and b is 9. In an exemplary embodiment, a is 4 and b is 10. In an exemplary embodiment, a is 4 and b is 11. In an exemplary embodiment, a is 4 and b is 12. In an exemplary embodiment, a is 4 and b is 13. In an exemplary embodiment, a is 4 and b is 14. In an exemplary embodiment, a is 4 and b is 15. In an exemplary embodiment, a is 4 and b is 16. In an exemplary embodiment, a is 4 and b is 17. In an exemplary embodiment, a is 4 and b is 18. In an exemplary embodiment, a is 4 and b is 19. In one exemplary embodiment, a is 4 and b is 20.

[0041]

[0053] In an exemplary embodiment, a is 5 and b is 6. In an exemplary embodiment, a is 5 and b is 7. In an exemplary embodiment, a is 5 and b is 8. In an exemplary embodiment, a is 5 and b is 9. In an exemplary embodiment, a is 5 and b is 10. In an exemplary embodiment, a is 5 and b is 11. In an exemplary embodiment, a is 5 and b is 12. In an exemplary embodiment, a is 5 and b is 13. In an exemplary embodiment, a is 5 and b is 14. In an exemplary embodiment, a is 5 and b is 15. In an exemplary embodiment, a is 5 and b is 16. In an exemplary embodiment, a is 5 and b is 17. In an exemplary embodiment, a is 5 and b is 18. In an exemplary embodiment, a is 5 and b is 19. In an exemplary embodiment, a is 5 and b is 20.

[0042]

[0054] In an exemplary embodiment, a is 6 and b is 7. In an exemplary embodiment, a is 6 and b is 8. In an exemplary embodiment, a is 6 and b is 9. In an exemplary embodiment, a is 6 and b is 10. In an exemplary embodiment, a is 6 and b is 11. In an exemplary embodiment, a is 6 and b is 12. In an exemplary embodiment, a is 6 and b is 13. In an exemplary embodiment, a is 6 and b is 14. In an exemplary embodiment, a is 6 and b is 15. In an exemplary embodiment, a is 6 and b is 16. In an exemplary embodiment, a is 6 and b is 17. In an exemplary embodiment, a is 6 and b is 18. In an exemplary embodiment, a is 6 and b is 19. In an exemplary embodiment, a is 6 and b is 20.

[0043]

[0055] In an exemplary embodiment, a is 7 and b is 8. In an exemplary embodiment, a is 7 and b is 9. In an exemplary embodiment, a is 7 and b is 10. In an exemplary embodiment, a is 7 and b is 11. In an exemplary embodiment, a is 7 and b is 12. In an exemplary embodiment, a is 7 and b is 13. In an exemplary embodiment, a is 7 and b is 14. In an exemplary embodiment, a is 7 and b is 15. In an exemplary embodiment, a is 7 and b is 17. In an exemplary embodiment, a is 7 and b is 17. In an exemplary embodiment, a is 7 and b is 18. In an exemplary embodiment, a is 7 and b is 19. In an exemplary embodiment, a is 7 and b is 20.

[0044]

[0056] In an exemplary embodiment, a is 8 and b is 9. In an exemplary embodiment, a is 8 and b is 10. In an exemplary embodiment, a is 8 and b is 11. In an exemplary embodiment, a is 8 and b is 12. In an exemplary embodiment, a is 8 and b is 13. In an exemplary embodiment, a is 8 and b is 14. In an exemplary embodiment, a is 8 and b is 15. In an exemplary embodiment, a is 8 and b is 16. In an exemplary embodiment, a is 8 and b is 17. In an exemplary embodiment, a is 8 and b is 18. In an exemplary embodiment, a is 8 and b is 19. In an exemplary embodiment, a is 8 and b is 20.

[0045]

[0057] In an exemplary embodiment, a is 9 and b is 10. In an exemplary embodiment, a is 9 and b is 11. In an exemplary embodiment, a is 9 and b is 12. In an exemplary embodiment, a is 9 and b is 13. In an exemplary embodiment, a is 9 and b is 14. In an exemplary embodiment, a is 9 and b is 15. In an exemplary embodiment, a is 9 and b is 16. In an exemplary embodiment, a is 9 and b is 17. In an exemplary embodiment, a is 9 and b is 18. In an exemplary embodiment, a is 9 and b is 19. In an exemplary embodiment, a is 9 and b is 20.

[0046]

[0058] In an exemplary embodiment, a is 10 and b is 11. In an exemplary embodiment, a is 10 and b is 12. In an exemplary embodiment, a is 10 and b is 13. In an exemplary embodiment, a is 10 and b is 14. In an exemplary embodiment, a is 10 and b is 15. In an exemplary embodiment, a is 10 and b is 16. In an exemplary embodiment, a is 10 and b is 17. In an exemplary embodiment, a is 10 and b is 18. In an exemplary embodiment, a is 10 and b is 19. In an exemplary embodiment, a is 10 and b is 20.

[0047]

[0059] In an exemplary embodiment, a is 11 and b is 12. In an exemplary embodiment, a is 11 and b is 13. In an exemplary embodiment, a is 11 and b is 14. In an exemplary embodiment, a is 11 and b is 15. In an exemplary embodiment, a is 11 and b is 16. In an exemplary embodiment, a is 11 and b is 17. In an exemplary embodiment, a is 11 and b is 18. In an exemplary embodiment, a is 11 and b is 19. In an exemplary embodiment, a is 11 and b is 20.

[0048]

[0060] In an exemplary embodiment, a is 12 and b is 13. In an exemplary embodiment, a is 12 and b is 14. In an exemplary embodiment, a is 12 and b is 15. In an exemplary embodiment, a is 12 and b is 16. In an exemplary embodiment, a is 12 and b is 17. In an exemplary embodiment, a is 12 and b is 18. In an exemplary embodiment, a is 12 and b is 19. In an exemplary embodiment, a is 12 and b is 20.

[0049]

[0061] In an exemplary embodiment, a is 13 and b is 14. In an exemplary embodiment, a is 13 and b is 15. In an exemplary embodiment, a is 13 and b is 16. In an exemplary embodiment, a is 13 and b is 17. In an exemplary embodiment, a is 13 and b is 18. In an exemplary embodiment, a is 13 and b is 19. In an exemplary embodiment, a is 13 and b is 20.

[0050]

[0062] In an exemplary embodiment, a is 14 and b is 15. In an exemplary embodiment, a is 14 and b is 16. In an exemplary embodiment, a is 14 and b is 17. In an exemplary embodiment, a is 14 and b is 18. In an exemplary embodiment, a is 14 and b is 19. In an exemplary embodiment, a is 14 and b is 20.

[0051]

[0063] In an exemplary embodiment, a is 15 and b is 16. In an exemplary embodiment, a is 15 and b is 17. In an exemplary embodiment, a is 15 and b is 18. In an exemplary embodiment, a is 15 and b is 19. In an exemplary embodiment, a is 15 and b is 20.

[0052]

[0064] In an exemplary embodiment, a is 16 and b is 17. In an exemplary embodiment, a is 16 and b is 18. In an exemplary embodiment, a is 16 and b is 19. In an exemplary embodiment, a is 16 and b is 20.

[0053]

[0065] In an exemplary embodiment, a is 17 and b is 18. In an exemplary embodiment, a is 17 and b is 19. In an exemplary embodiment, a is 17 and b is 20.

[0054]

[0066] In an exemplary embodiment, a is 18 and b is 19. In an exemplary embodiment, a is 18 and b is 20. In an exemplary embodiment, a is 19 and b is 20.

[0055]

[0067] In some embodiments, the modified CHP has the formula I: (Gly-XY) a~b wherein Gly is glycine, at least one of X and Y is proline, modified proline, and / or hydroxyproline, a is 3, and b is 20.

[0056]

[0068] In some embodiments, the CHP described herein comprises any one of SEQ ID NOs: 1-169 as shown in Table 1 below.

[0069]

[0057] [Table 1] TIFF2024537778000002.tif225149 TIFF2024537778000003.tif225149 TIFF2024537778000004.tif225149 TIFF2024537778000005.tif35149

[0058]

[0070] In certain sequences given in Table 1 above, "NH2" represents the amidated C-terminus. In certain sequences given in Table 1 above, "f" in the "GfO" sequence represents 2S,4S-4-fluoroproline (cis structure). In certain sequences given in Table 1 above, "c" in the "GcO" sequence represents c=cis-chloroproline (2S,4S-4-chloroproline).

[0059]

[0071] In some embodiments, the CHP has a sequence selected from Table 2 below.

[0060] [Table 2]

[0061]

[0072] In some embodiments, the modified CHP has the formula I: (Gly-XY) a~b wherein Gly is glycine, at least one of X and Y is proline, modified proline, and / or hydroxyproline, a is 3, and b is 20. The integrin binding site comprises integrin α1β1 and has a sequence selected from the sequences listed in Table 3.

[0062] [Table 3]

[0063]

[0073] In some embodiments, the modified CHP has the formula I: (Gly-XY) a~b wherein Gly is glycine, at least one of X and Y is proline, modified proline, and / or hydroxyproline, a is 3, and b is 20. The integrin binding site comprises integrin α2β1 and has a sequence selected from the sequences listed in Table 4.

[0064] [Table 4]

[0065]

[0074] In some embodiments, the modified CHP has the formula I: (Gly-XY) a~bwherein Gly is glycine, at least one of X and Y is proline, modified proline, and / or hydroxyproline, a is 3, and b is 20. The integrin binding site comprises integrin α10β1 and has a sequence selected from the sequences listed in Table 5.

[0066] [Table 5]

[0067]

[0075] In some embodiments, the modified CHP has the formula I: (Gly-XY) a~b wherein Gly is glycine, at least one of X and Y is proline, modified proline, and / or hydroxyproline, a is 3, and b is 20. The integrin binding site comprises integrin α11β1 and has a sequence selected from the sequences listed in Table 6.

[0068] [Table 6]

[0069]

[0076] In some embodiments, the modified CHP comprises a crosslinking moiety crosslinked to a CHP having a sequence represented by formula I: (Gly-XY)a-b, where Gly is glycine, at least one of X and Y is proline, modified proline, and / or hydroxyproline, a is 3, and b is 20. The crosslinking moiety has a sequence selected from the sequences listed in Table 7.

[0070] [Table 7]

[0071]

[0077] In Table 6, "x" refers to an amino acid residue, particularly a naturally occurring amino acid residue.

[0072]

[0078] In some embodiments, the cross-linking site is a sugar cross-linking site.

[0073]

[0079] In some embodiments, one or more binding partners comprise a cross-linking site that comprises a sequence having at least 85% sequence identity to SEQ ID NO:17.

[0074]

[0080] In some embodiments, the modified CHP comprises a VWF, DDR1, DDR2, and SPARC binding peptide crosslinked to a CHP having a sequence represented by formula I: (Gly-XY)a-b, where Gly is glycine, at least one of X and Y is proline, modified proline, and / or hydroxyproline, a is 3, and b is 20. The crosslinking site has a sequence selected from the sequences listed in Table 8.

[0075] [Table 8]

[0081] In Table 8, "x" refers to an amino acid residue, particularly a naturally occurring amino acid residue.

[0076]

[0082] In some embodiments, the one or more binding partners include VWF, DDR1, DDR2, and SPARC binding peptides comprising sequences having at least 85% sequence identity to SEQ ID NOs: 18-21.

[0077]

[0083] In some embodiments, the modified CHP comprises a fibronectin binding motif crosslinked to a CHP having a sequence represented by formula I: (Gly-XY)a-b, where Gly is glycine, at least one of X and Y is proline, modified proline, and / or hydroxyproline, a is 3, and b is 20. The crosslinking site has a sequence selected from the sequences listed in Table 9.

[0078] [Table 9]

[0084] In some embodiments, one or more binding partners comprises a fibronectin binding motif comprising a sequence having at least 85% sequence identity to SEQ ID NO:22 or SEQ ID NO:23.

[0079]

[0085] In some embodiments, the modified CHP comprises an engineered integrin-binding motif crosslinked to a CHP having a sequence represented by formula I: (Gly-XY)a-b, where Gly is glycine, at least one of X and Y is proline, modified proline, and / or hydroxyproline, a is 3, and b is 20.

[0080]

[0086] In some embodiments, the one or more binding partners comprises a modified integrin binding motif comprising a sequence having at least 85% sequence identity to SEQ ID NO:24 or 25.

[0081]

[0087] In some embodiments, the modified CHP comprises an MMP cleavage site crosslinked to a CHP having a sequence represented by formula I: (Gly-XY)a-b, where Gly is glycine, at least one of X and Y is proline, modified proline, and / or hydroxyproline, a is 3, and b is 20. The crosslinked site has a sequence selected from the sequences listed in Table 10:

[0082] [Table 10]

[0083]

[0088] In some embodiments, the one or more binding partners comprises an MMP cleavage site comprising a sequence having at least 85% sequence identity to SEQ ID NO:26 or 27.

[0084]

[0089] In some embodiments, the CHP has a sequence selected from the group consisting of SEQ ID NO:XXX.

[0085]

[0090] In some embodiments, the modified CHP has one binding partner cross-linked to the C-terminus of the CHP. In some embodiments, the modified CHP has one binding partner cross-linked to the N-terminus of the CHP.

[0086]

[0091] In some embodiments, the modified CHP has two binding partners, the first of which is cross-linked to the C-terminus of the CHP and the second of which is cross-linked to the N-terminus of the CHP.

[0087]

[0092] In some embodiments, the one or more binding partners are attached to the C-terminus of CHP and the N-terminus of CHP.

[0088]

[0093] In some embodiments, the modified CHP and one or more binding partners are directly crosslinked to the CHP. In some embodiments, the one or more binding partners are crosslinked to the CHP by zero or more spacers. Examples of spacers include, but are not limited to, amino acid(s) such as GGG, and chemical spacer(s) such as 6-aminohexanoic acid (Ahx).

[0089]

[0094] In some embodiments, the modified CHP comprises a cap at the N-terminus of the CHP. In some embodiments, the cap comprises an acetyl group. In some embodiments, the cap is directly attached to the N-terminus of the CHP. In some embodiments, the modified CHP comprises a spacer between the N-terminus of the CHP and the cap.

[0090]

[0095] In some embodiments, the one or more binding partners of the modified CHP are directly attached to the N-terminus of the CHP, such that the one or more binding partners are attached between the cap and the N-terminus of the CHP. In some embodiments, the modified CHP comprises one or more binding partners attached to the N-terminus of the CHP, one or more spacers attached to the one or more binding partners, and a cap attached to the one or more spacers, such that the one or more spacers are between the cap and the one or more binding partners. In some embodiments, the modified CHP has the structure: cap-spacer-CHP-binding partner-CHP. In some embodiments, the cap and CHP are directly attached. In some embodiments, the cap and one or more binding partners are directly attached.

[0091] V. A composition comprising a modified CHP.

[0096] In one aspect of the disclosure, any of the modified CHPs described herein may be included in a therapeutic, medical device, therapeutic medical device, or cosmetic composition. In some embodiments, the composition is formulated such that the modified CHPs included in the composition do not form triple helices with other modified CHPs.

[0092]

[0097] In some embodiments, the composition includes a carrier, for example, for carrying the modified CHP through an epithelial layer. Examples of carriers include, but are not limited to, micelles, dendrites, lipids, microemulsions, nanoemulsions, solid lipid nanoparticles, nanostructured lipid carriers, liposomes, transfersomes, ethosomes, niosomes, collagen, extracellular matrix, and artificial extracellular matrix.

[0093]

[0098] In some embodiments, the composition is a cosmetic composition formulated for administration by injection or topical application, hi some embodiments, the cosmetic composition may be formulated for administration by microdermal injection, for example, using an array of microneedles containing the cosmetic composition.

[0094]

[0099] In some embodiments, a composition comprising a modified CHP is formulated for administration by local injection, topical application, or physical application.

[0095]

[0100] In some embodiments, compositions comprising modified CHPs are formulated as putties, meshes, patches, adhesives, creams, sutures, eye drops, and / or other formulations that can be administered topically or by physical application.

[0096]

[0101] In some embodiments, compositions comprising modified CHP are formulated for systemic administration by injection, oral administration, drops, spray, intravenous injection, intramuscular injection, suppository, enema, or vaporizer.

[0097] VI. Uses of modified CHP and compositions containing modified CHP

[0102] As discussed herein, the modified CHP and / or compositions comprising the modified CHP described herein can be used to modulate fibroblast morphology and induce fibroblast adhesion, thereby inducing fibroblasts to produce more collagen.

[0098]

[0103] Therefore, the modified CHP and / or composition comprising modified CHP described herein can be used to regulate fibroblast production of collagen or MMP in a subject.In one embodiment, increasing collagen production leads to a reduction in the appearance of wrinkles, thereby reducing the effect of aging on the skin of a subject.Thus, in one embodiment of the present disclosure, a method for increasing collagen production in a subject can include administering to a subject the modified CHP and / or composition comprising modified CHP described herein.The administration of the modified CHP and / or composition comprising modified CHP can be performed by injection, microdermal injection or topical application.

[0099]

[0104] Changing fibroblast morphology and inducing fibroblasts to produce more collagen can further reduce fibroblast-induced production of MMP and enhance skin repair.Therefore, in one aspect of the present disclosure, a method for reducing MMP production in a subject can include administering to a subject the modified CHP and / or a composition comprising modified CHP described herein.Administering modified CHP and / or a composition comprising modified CHP can be performed by injection, microdermal injection or topical application.

[0100]

[0105] In another embodiment, by applying MMP sites as bioactive sites in CHP, we can increase the collagen degradation rate. By removing partially damaged collagen faster and more efficiently, newly generated collagen can bind to existing fibrils faster. Therefore, by removing damaged collagen, we can effectively restart the collagen fibril formation process, ultimately resulting in a more dense and aligned collagen matrix.

[0101]

[0106] Increased collagen production in fibroblasts can aid in skin repair, for example, in skin damaged by trauma or aging. Thus, the modified CHP and / or compositions comprising modified CHP described herein can be used to induce skin repair in a subject in need thereof. Thus, in one aspect of the present disclosure, a method for inducing skin repair in a subject in need thereof can include administering to a subject the modified CHP and / or compositions comprising modified CHP described herein. The composition comprising modified CHP can be a cosmetic composition or a therapeutic composition. In some embodiments, the administration of the modified CHP and / or compositions comprising modified CHP can be performed by injection, microdermal injection, or topical application. In some embodiments, the administration of the modified CHP and / or compositions comprising modified CHP can be performed by application as a putty, mesh, patch, adhesive, cream, suture, or eye drop.

[0102]

[0107] The modified CHP can contain a variety of binding partners, including various cross-linking motifs, and thus can be used to regulate cell expression of various cells by modifying their microenvironment, inducing specific cell binding, or cytokine and enzymatic responses in the extracellular matrix. The term "cell expression" refers to the cell expression of one or more proteins, such as collagen, MMP, and one or more extracellular matrix proteins. Thus, in one embodiment of the present disclosure, a method for regulating the cell expression of a particular cell type may include contacting the modified CHP with the corresponding cell type. Examples of cells whose cell expression can be regulated using the modified CHP described herein include, but are not limited to, bone cells, tendon cells, chondrocytes, fibroblasts, osteoblasts, or mesenchymal stem cells (MSCs).

[0103]

[0108] In some embodiments, modulating cell expression may include decreasing non-triple helical collagen concentration in the microenvironment surrounding the cell, e.g., bone cells, tenocytes, chondrocytes, fibroblasts, osteoblasts, or mesenchymal stem cells (MSCs). In some embodiments, modulating cell expression may include increasing collagen expression in the cell, e.g., bone cells, tenocytes, chondrocytes, fibroblasts, osteoblasts, or mesenchymal stem cells (MSCs).

[0104]

[0109] In some embodiments, the modified CHP may be contacted with the corresponding cell type by administering the modified CHP by local injection, intravenous injection, topical application, or physical application. In some embodiments, administration of the modified CHP may be performed by application as a putty, mesh, patch, glue, cream, suture, or eye drops. In some embodiments, the modified CHP is administered with a carrier, such as collagen, extracellular matrix, artificial extracellular matrix, polymer carrier, protein carrier, mineral, glycosaminoglycan (GAG), bioactive glass, liposome, and mixtures thereof. EXAMPLES

[0105] Example 1:

[0110] FIG. 2A shows CHP targeting and binding to damaged collagen induced by collagenase activity in an in vivo mouse model of tendonitis.

[0106]

[0111] A C57BL / 6J mouse model was used to test CHP on mouse tendons. Bacterial collagenase was used to create a tendonitis model.

[0107]

[0112] Mice were injected subcutaneously with a 10 μl volume of 1 nmol CHP solution. Joints were imaged at specific time points (2 h, 5 h, 24 h, and 72 h) to compare sham (PBS-injected knees) versus collagenase-injected knees.

[0108] Example 2

[0113] FIG. 2B shows targeting and binding by CHP to damaged collagen in human samples obtained from osteoarthritic knee joints compared to normal knee joints.

[0109]

[0114] Formalin-fixed paraffin-embedded (FFPE) sections from knee joints (osteoarthritic and normal knee joints) were deparaffinized according to standard protocols, followed by the addition of CHP preservative. CHP preservative was adjusted to 20 μM, heated to 80°C for 5 min, snap-chilled on ice for approximately 60 s, and then added to the tissue sections. The volume was sufficient to cover the entire tissue (100-200 μl). CHP was allowed to stain overnight at 4°C. It was then washed off with 1xPBS for 3x5 min, followed by counterstaining with DAPI.

[0110] Example 3

[0115] FIG. 2C shows targeting and binding by CHP to damaged collagen in aged compared to young human skin tissue.

[0111]

[0116] For frozen sections, OCT was washed off with 3x5 min washes in 1x PBS. CHP stock solution was adjusted to 20 μM, heated to 80°C for 5 min, then rapidly quenched in ice water for approximately 60 s before being added to the tissue sections. The volume was sufficient to cover the entire tissue (100-200 μl). CHP was allowed to stain overnight at 4°C. It was then washed off with 1x PBS for 3x5 min before being counterstained with DAPI.

[0112] Example 4

[0117] FIG. 6 shows the changes in gene expression caused by the bioactive CHP disclosed herein.

[0113]

[0118] IMR-90 human lung fibroblast cells were purchased from ITCC. The cells were thawed and cultured in EMEM supplemented with 10% FBS and 1% penicillin-streptomycin. The cells were cultured to 80-95% confluence and then subcultured for three passages before being used in the experiments.

[0114]

[0119] Experimental wells were prepared by adding 100 μL of cell culture medium to the wells of a 96-well gelatin-coated plate (corning) or 300 μL of medium to the wells of a 48-well uncoated cell culture plate. The peptides listed below were added to each well (5 μL for 96-well plates, 10 μL for 48-well plates).

[0120] CF-Ahx-(GPO)3-GFOGER-(GPO)3-heating, 2.5μM

[0121] CF-Ahx-(GPO)3-GFOGER-(GPO)3-non-heated (triple helix), 2.5 μM

[0122] CF-Ahx-(GPO)3-EGORFG-(GPO)3-2.5μM

[0123] Gelatin derived from pig skin (30μg / mL)

[0124] Bovine skin collagen (30μg / mL)

[0125] During the passage, cells were quantified and 2100 IMR-90 cells were added to each well of a 96-well gelatin-coated plate. For uncoated plates, IMR-90 cells were added to each well. Additional medium was added to bring the volume to 200 μL (96-well) or 500 μL (48-well). Cells were cultured at 37° C., 5% CO2 for 3 days and then imaged. IMR-90 cells were nearly confluent, and wells that had gelatin or collagen added to the medium appeared to have greater confluence and more directionality in cell growth. Medium was added after 1 day (day 4) and then cultured for another 2 days, after which cells were imaged and lysed. qPCR was performed on cell lysates after cDNA synthesis (cell-to-CT kit, Thermo Fisher scientific).

[0115]

[0126] Unless otherwise indicated, all numbers expressing quantities of ingredients, properties, such as molecular weights, reaction conditions, and other things used in the specification and claims should be understood in all instances to be modified by the term "about." Thus, unless indicated to the contrary, the numerical variables set forth in the specification and the appended claims are approximations that may vary depending on the desired properties sought to be obtained by the present disclosure. At the very least, and without any attempt to limit the application of the doctrine of equivalents to the scope of the claims, each numerical variable should at least be interpreted in light of the number of reported significant digits and by applying ordinary rounding techniques.

[0116]

[0127] Notwithstanding that the numerical ranges and variables setting forth the broad scope of the disclosure are approximations, the numerical values ​​set forth in the specific examples are reported as precisely as possible, however, any numerical value inherently contains certain errors necessarily resulting from the standard deviation found in their respective testing measurements.

[0117]

[0128] Groupings of alternative elements or embodiments of the disclosure disclosed herein should not be construed as limiting. Each group member may be referred to and claimed individually or in any combination with other members of the group or other elements found herein. It is anticipated that one or more members of a group may be included in or deleted from a group for reasons of convenience and / or patentability. When any such inclusion or deletion occurs, the specification is considered to contain the group as modified, thus fulfilling the written description of all Markush groups used in the appended claims.

[0118]

[0129] Certain embodiments of the present disclosure are described herein, including the best mode known to the inventors for carrying out the disclosure. Naturally, variations on these described embodiments will become apparent to those skilled in the art upon reading the foregoing specification. The inventors anticipate that those skilled in the art will utilize such variations as necessary, and the inventors intend for the present disclosure to be practiced otherwise than as specifically described herein. Accordingly, this disclosure includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, any combination of the above-described elements in all possible variations thereof is encompassed by the present disclosure unless otherwise indicated herein or otherwise clearly contradicted by context.

[0119]

[0130] Certain embodiments disclosed herein may be further limited in the claims using the phrases "consisting of" or "consisting essentially of." The transition term "consisting of," when used in a claim, excludes any element, step, or ingredient not specified in the claim, whether originally filed or added by amendment. The transition term "consisting essentially of" limits the claim to the materials or steps specified and to those materials or steps that do not materially affect the basic and novel characteristic(s). Embodiments of the present disclosure so claimed are essentially or specifically described and enabled herein.

[0120]

[0131] It should be understood that the embodiments of the present disclosure disclosed herein are illustrative of the principles of the present disclosure. Other variations that can be utilized are within the scope of the present disclosure. Thus, by way of example, but not of limitation, alternative forms of the present disclosure can be utilized in accordance with the teachings herein. Thus, the present disclosure is not limited to that precisely as shown and described.

[0121]

[0132] Although the present disclosure has been described and illustrated herein by reference to various specific materials, procedures and examples, it is understood that the disclosure is not limited to the specific combination of materials and procedures selected for its purpose. Numerous variations on such details can be implied as would be understood by one of ordinary skill in the art. It is intended that the specification and examples be considered as exemplary only, with a true scope and spirit of the present disclosure being indicated by the following claims. All references, patents, and patent applications mentioned in this application are incorporated herein by reference in their entirety.

Claims

**Claim 1** A modified collagen hybridizing peptide (CHP) comprising one or more binding partners crosslinked to the CHP and having the formula I: (Gly-X-Y) ａ～ｂ (Formula I) (wherein Gly is glycine, at least one of X and Y is proline, modified proline, and / or hydroxyproline, a is 3, and b is 20) having the sequence represented by wherein the one or more binding partners are selected from the group consisting of an integrin site, an integrin binding site, a crosslinking site, von Willebrand factor (VWF), discoidin domain receptor (DDR) 1, DDR2, a SPARC binding peptide, a fibronectin binding peptide, a modified integrin binding peptide, a matrix metalloproteinase (MMP) cleavage site, a cathepsin K (CATK) site, and an osteoclast-associated receptor (OSCAR); the modified CHP. **Claim 2** The modified CHP according to claim 1, wherein the one or more binding partners comprise an integrin site. **Claim 3** The modified CHP according to claim 1, wherein the one or more binding partners comprise an integrin binding site. **Claim 4** The modified CHP according to claim 1, wherein the one or more binding partners comprise an integrin binding site comprising a sequence having at least 85% sequence identity to any one of SEQ ID NOs: 1 to 16. **Claim 5** The modified CHP according to claim 3, wherein the integrin binding site comprises integrin α1β1. **Claim 6** The modified CHP according to claim 3, wherein the integrin binding site comprises integrin α2β1. **Claim 7** The modified CHP according to claim 3, wherein the integrin binding site comprises integrin α10β1. **Claim 8** The modified CHP according to claim 3, wherein the integrin binding site comprises integrin α11β1. **Claim 9** The modified CHP according to claim 1, wherein the one or more binding partners comprise a crosslinking site. **Claim 10** The modified CHP according to claim 1, wherein the one or more binding partners comprise a sugar crosslinking site. **Claim 11** The modified CHP according to claim 1, wherein the one or more binding partners comprise a crosslinking site comprising a sequence having at least 85% sequence identity to SEQ ID NO:

17. **Claim 12** The modified CHP according to claim 1, wherein the one or more binding partners comprise VWF.

13. The modified CHP according to claim 1, wherein the one or more binding partners comprise a DDR1, DDR2, or SPARC binding peptide.

14. The modified CHP according to claim 1, wherein the one or more binding partners comprise a VWF binding peptide comprising a sequence having at least 85% sequence identity to any one of SEQ ID NOs: 18-21.

15. The modified CHP according to claim 1, wherein the one or more binding partners comprise a DDR1, DDR2, or SPARC binding peptide comprising a sequence having at least 85% sequence identity to any one of SEQ ID NOs: 18-21.

16. The modified CHP according to claim 1, wherein the one or more binding partners comprise a fibronectin binding motif or sequence.

17. The modified CHP according to claim 1, wherein the one or more binding partners comprise a fibronectin binding motif comprising a sequence having at least 85% sequence identity to SEQ ID NO: 22 or 23.

18. The modified CHP according to claim 1, wherein the one or more binding partners comprise a modified integrin binding motif.

19. The modified CHP according to claim 1, wherein the one or more binding partners comprise a modified integrin binding motif comprising a sequence having at least 85% sequence identity to SEQ ID NO: 24 or 25.

20. The modified CHP according to claim 1, wherein the one or more binding partners comprise an MMP cleavage site.

21. The modified CHP according to claim 1, wherein the one or more binding partners comprise an MMP cleavage site comprising a sequence having at least 85% sequence identity to SEQ ID NO: 26 or 27.

22. The modified CHP according to claim 1, wherein the CHP has a sequence selected from the group consisting of SEQ ID NOs: 1-169.

23. The modified CHP according to claim 1, having one binding partner, wherein the one binding partner is cross-linked to the C-terminus of the CHP.

24. The modified CHP according to claim 1, having one binding partner, wherein the one binding partner is cross-linked to the N-terminus of the CHP.

25. The modified CHP according to claim 1, having two binding partners, wherein one of the two binding partners is cross-linked to the C-terminus of the CHP and the other of the two binding partners is cross-linked to the N-terminus of the CHP.

26. The modified CHP according to claim 1, wherein the one or more binding partners are bound to the CHP at the N-terminus and are bound to another CHP at the C-terminus.

27. The modified CHP according to claim 1, wherein the one or more binding partners are directly cross-linked to the CHP.

28. The modified CHP according to claim 1, wherein the one or more binding partners are cross-linked to the CHP via one or more spacers.

29. The modified CHP according to claim 1, comprising a cap at the N-terminus of the modified CHP.

30. The modified CHP according to claim 29, wherein the cap comprises an acetyl group (Ac).

31. The modified CHP according to claim 29 or 30, wherein the CHP is directly cross-linked to the cap.

32. The modified CHP according to claim 29 or 30, comprising one or more spacers between the cap and the CHP.

33. The modified CHP according to claim 29 or 30, comprising one or more spacers between the cap and the one or more binding partners.

34. The modified CHP according to claim 29 or 30, wherein the one or more binding partners are directly cross-linked to the cap.

35. The modified CHP according to claim 29 or 30, wherein the CHP comprises the cap-spacer-CHP-binding partner-CHP.

36. A composition comprising the modified CHP according to any one of claims 1 to 30.

37. The composition according to claim 36, wherein the modified CHP does not form a triple helix with another modified CHP.

38. The composition according to claim 36, further comprising a carrier.

39. A cosmetic composition, wherein the carrier comprises at least one selected from the group consisting of micelles, dendritic protrusions, lipids, microemulsions, nanoemulsions, solid lipid nanoparticles, nanostructured lipid carriers, liposomes, transferosomes, ethosomes, niosomes, collagen matrices, extracellular matrices, and artificial extracellular matrices, the composition according to claim 38.

40. The cosmetic composition according to claim 39, which is for increasing collagen production in a subject by administration to the subject.

41. The composition according to claim 40, wherein the administration is carried out by injection, microdermal injection, or topical application.

42. The cosmetic composition according to claim 39, which is for reducing MMP production in a subject by administration to the subject.

43. The composition according to claim 42, wherein the administration is carried out by injection, microdermal injection, or topical application.

44. The cosmetic composition according to claim 39, which is for inducing skin repair in a subject by administration to the subject.

45. The composition according to claim 44, wherein the administration is carried out by injection, microdermal injection, or topical application.

46. By contacting the modified CHP according to any one of claims 1 to 30 with any one of osteocytes, tenocytes, chondrocytes, fibroblasts, osteoblasts, and MSCs, for regulating cell expression of osteocytes, tenocytes, chondrocytes, fibroblasts, osteoblasts, or mesenchymal stem cells (MSCs), a composition comprising the modified CHP according to any one of claims 1 to 30.

47. The composition according to claim 46, wherein the cell expression comprises the expression of one or more of collagen, MMP, and extracellular matrix proteins.

48. The composition according to claim 46, which is for reducing the concentration of non-triple helix collagen in the microenvironment surrounding the osteocytes, tenocytes, chondrocytes, fibroblasts, osteoblasts, or MSCs.

49. The composition according to claim 46, which is for increasing collagen expression in the osteocytes, tenocytes, chondrocytes, fibroblasts, osteoblasts, or MSCs.

50. The composition according to claim 46, wherein the administration is carried out by local injection, intravenous injection, topical application, or physical application.

51. The composition according to claim 46, which is for administration as a paste, mesh, patch, adhesive, cream, suture, or eye drop.

52. The composition according to claim 46, which is for administering the modified CHP together with a carrier.

53. The composition according to claim 52, wherein the carrier comprises at least one selected from the group consisting of collagen, extracellular matrix, artificial extracellular matrix, polymer carrier, protein carrier, mineral, glycosaminoglycan (GAGs), bioactive glass, liposome, and mixtures thereof.

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