CN117897058A

CN117897058A - Collagen compositions and methods of use thereof

Info

Publication number: CN117897058A
Application number: CN202280031834.1A
Authority: CN
Inventors: L·戴; D·L·小汉德林; S·路; M·德莱乌; D·杜尔彭; D·威廉姆森; I·汉森-塞万提斯; B·拉乌
Original assignee: Modern Meadow Inc
Current assignee: Modern Meadow Inc
Priority date: 2021-04-30
Filing date: 2022-04-29
Publication date: 2024-04-16

Abstract

The present disclosure relates to recombinant collagen, peptide fragments thereof, and sequence variants thereof. Also provided are novel pichia pastoris strains for use in the production of the recombinant collagen, fragments thereof, and sequence variants thereof, and methods of using the recombinant collagen, fragments thereof, and sequence variants thereof.

Description

Collagen compositions and methods of use thereof

Reference to an electronically submitted sequence Listing

The contents of the sequence listing submitted electronically in the ASCII text file (designation: 4431_0825c04_seqliping_ST25; size: 841,608; and date of creation: 2022, 4, 27 days) submitted with the present application are incorporated herein by reference in their entirety.

Background

Collagen is one of the most important proteins in the human body, and is present in connective tissue such as cartilage, bone, tendon, ligament, and skin, and it is a major protein in the extracellular matrix of human cells. "recombinant collagen" refers to a family of at least 28 different naturally occurring collagen types prepared using recombinant techniques.

Collagen has many known uses. In the cosmetic and skin care industries, for example, skin care compositions comprising collagen are useful to combat the effects of aging and environmental stresses on the appearance, elasticity and thickness of skin. For example, aging and environmental factors can lead to skin conditions including, but not limited to, fine lines, wrinkles, dry skin, enlarged pores, skin discoloration, reduced elasticity, unwanted hair, thinning of the skin, purpura, actinic keratosis, itching, eczema, acne, rosacea, erythema, telangiectasias, actinic telangiectasias, skin cancer, and hypertrophic rosacea. Although there are many skin care products on the market that improve the appearance of skin, many consumers are hesitant to use chemically synthesized products, which they consider to be environmentally unfriendly or unsafe.

Disclosure of Invention

In some embodiments, the present disclosure provides a recombinant collagen fragment having a molecular weight of about 50kDa and a sequence identity of at least about 85% to the amino acid sequence shown in SEQ ID NO. 986. In some embodiments, the recombinant collagen fragment is non-hydroxylated. In some embodiments, the recombinant collagen fragment is hydroxylated. In some embodiments, the collagen fragment has the amino acid sequence shown in SEQ ID NO. 986.

In some embodiments, the present disclosure provides a sequence variant of a recombinant collagen fragment having a molecular weight of about 50kDa and a sequence identity of at least about 85% to the amino acid sequence shown in SEQ ID NO. 986, wherein the sequence variant comprises the amino acid sequence shown in any one of SEQ ID NO. 987 through SEQ ID NO. 1015. In some embodiments, the sequence variant is not hydroxylated. In some embodiments, the sequence variant is hydroxylated.

In some embodiments, the present disclosure provides a composition comprising a recombinant collagen fragment having a molecular weight of about 50kDa and a sequence identity of at least about 85% to the amino acid sequence shown in SEQ ID NO. 986. In some embodiments, the present disclosure provides a composition comprising a sequence variant of a recombinant collagen fragment having a molecular weight of about 50kDa and a sequence identity of at least about 85% to the amino acid sequence shown in SEQ ID NO. 986, wherein the sequence variant comprises the amino acid sequence shown in any one of SEQ ID NO. 987 through SEQ ID NO. 1015. In some embodiments, the composition further comprises one or more peptides formed from the hydrolysis of a collagen fragment having the amino acid sequence shown in SEQ ID NO. 986. In some embodiments, at least one of the one or more peptides formed from hydrolysis of a collagen fragment having the amino acid sequence shown in SEQ ID NO. 986 has an amino acid sequence according to one of SEQ ID NO. 2 through SEQ ID NO. 972. In some embodiments, the composition further comprises a pharmaceutically acceptable excipient or cosmetically acceptable excipient.

In some embodiments, the present disclosure provides a method of producing a composition comprising a recombinant collagen fragment having a molecular weight of about 50kDa and a sequence identity of at least about 85% to the amino acid sequence shown in SEQ ID NO. 986, comprising producing the recombinant collagen fragment in a genetically engineered yeast strain. In some embodiments, the yeast is Pichia pastoris (Pichia pastoris). In some embodiments, the yeast has been transformed with a plasmid comprising the nucleic acid sequence shown in SEQ ID NO. 973. In some embodiments, the yeast is a yeast that has been transformed with a plasmid comprising the nucleic acid sequence set forth in SEQ ID NO. 974.

In some embodiments, the method comprises: (i) fermenting the genetically engineered yeast in a fermentation broth; (ii) Recovering from the fermentation broth a recombinant collagen fragment secreted by the genetically engineered yeast; and (iii) optionally purifying the recombinant collagen fragment. In some embodiments, the method further comprises hydroxylating the recombinant collagen fragment ex vivo.

In some embodiments, the disclosure also provides a method of producing a sequence variant disclosed herein, the method comprising producing the recombinant collagen fragment in a genetically engineered yeast strain. In some embodiments, the yeast is Pichia pastoris (Pichia pastoris). In some embodiments, the yeast has been transformed with a plasmid comprising the nucleic acid sequence set forth in any one of SEQ ID NOS: 1045 to 1073.

In some embodiments, the method comprises: (i) fermenting the genetically engineered yeast in a fermentation broth; (ii) Recovering from the fermentation broth a sequence variant of the recombinant collagen fragment secreted by the genetically engineered yeast; and (iii) optionally purifying the recombinant collagen fragment.

In some embodiments, the method further comprises hydroxylating the recombinant collagen sequence variant ex vivo.

In some embodiments, the present disclosure provides a yeast strain genetically engineered to produce a recombinant collagen fragment described herein, wherein the yeast strain comprises a vector comprising a DNA sequence encoding the recombinant collagen.

In some embodiments, the present disclosure provides a yeast strain genetically engineered to produce a sequence variant described herein, wherein the yeast strain comprises a vector comprising a DNA sequence encoding the variant.

The yeast strain of claim 24, wherein the vector comprises a nucleic acid sequence comprising the DNA sequence set forth in SEQ ID No. 973. In some embodiments, the yeast strain further comprises a second vector comprising a nucleic acid sequence comprising the DNA sequence set forth in SEQ ID NO. 974. In some embodiments, the vector comprises a nucleic acid sequence comprising a DNA sequence set forth in any one of SEQ ID NOS: 1045 to SEQ ID NO: 1073. In some embodiments, the yeast strain described herein is pichia pastoris.

In some embodiments, the present disclosure provides a method of treating a skin condition, the method comprising administering to a subject in need thereof an effective amount of a recombinant collagen fragment described herein, or sequence variant thereof, having a molecular weight of about 50kDa and a sequence identity of at least about 85% with the amino acid sequence set forth in SEQ ID No. 986.

In some embodiments, the present disclosure provides a method of treating a skin condition, the method comprising administering to a subject in need thereof an effective amount of a composition described herein. In some embodiments, the skin condition includes fine lines, wrinkles, dry skin, enlarged pores, skin discoloration, reduced elasticity, unwanted hair, skin thinning, purpura, actinic keratosis, itching, eczema, acne, rosacea, erythema, telangiectasia, actinic telangiectasia, skin cancer, or hypertrophic rosacea. In some embodiments, the composition is topically applied to an area of skin. In some embodiments, the skin region is selected from the group consisting of: facial surfaces, scalp, neck, ears, shoulders, chest (including breast and/or collar), arms, hands, legs, stomach, buttocks, groin, back, feet, and combinations thereof.

In some embodiments, the present disclosure provides a method of increasing collagen production in a cell, the method comprising administering to the cell an effective amount of a recombinant collagen fragment described herein, or sequence variant thereof, having a molecular weight of about 50kDa and a sequence identity of at least about 85% with the amino acid sequence shown in SEQ ID NO. 986. In some embodiments, the method increases the production of type I collagen. In some embodiments, the cells are fibroblasts. In some embodiments, the cells are cultured cells. In some embodiments, the fragment or variant is formulated in a composition. In some embodiments, the fragment has the amino acid sequence shown in SEQ ID NO. 986. In some embodiments, the sequence variant has the amino acid sequence set forth in any one of SEQ ID NOs 987 to 1015.

In some embodiments, the present disclosure provides a skin care product comprising a composition as described herein for reducing appearance of wrinkles, evening skin tone, providing moisture, reducing appearance of dark under-eye circles, increasing collagen content of skin, increasing skin density, improving skin firmness and elasticity, improving appearance of fine lines and wrinkles, smoothing skin texture, increasing skin shine and brightness, improving appearance of sagging skin, whitening skin, or any combination thereof.

In some embodiments, the present disclosure provides a method of treating a wound in a human subject in need thereof, the method comprising administering a composition described herein to the wound of the subject, wherein administration of the recombinant collagen fragment induces production of human type I collagen, human type III collagen, or a combination thereof. In some embodiments, the collagen fragments are topically applied to the wound.

In some embodiments, the present disclosure provides a recombinant collagen fragment having a molecular weight of about 50kDa and a sequence identity of at least about 85% to the amino acid sequence shown in SEQ ID NO. 1 or SEQ ID NO. 986. In some embodiments, the recombinant collagen fragment may be non-hydroxylated. In some embodiments, the recombinant collagen fragment may be hydroxylated. In some embodiments, the collagen fragment may have the amino acid sequence shown in SEQ ID NO. 1 or SEQ ID NO. 986. In some embodiments, the present disclosure provides a recombinant collagen fragment comprising an amino acid sequence according to any one of SEQ ID NOs 2 to 972.

In some embodiments, the present disclosure provides a composition comprising a recombinant collagen fragment described herein. In some embodiments, the composition further comprises one or more peptides formed from the hydrolysis of a collagen fragment having the amino acid sequence shown in SEQ ID NO. 1 or from the hydrolysis of a collagen fragment having the amino acid sequence shown in SEQ ID NO. 986.

In some embodiments of the present disclosure, at least one of the one or more peptides formed from hydrolysis of a collagen fragment having the amino acid sequence shown in SEQ ID No. 1 has an amino acid sequence according to one of SEQ ID No. 2 to SEQ ID No. 972. In some embodiments of the present disclosure, at least one of the one or more peptides formed from hydrolysis of a collagen fragment having the amino acid sequence shown in SEQ ID NO. 986 has an amino acid sequence according to one of SEQ ID NO. 2 through SEQ ID NO. 972. In some embodiments, the composition further comprises a pharmaceutically acceptable excipient or cosmetically acceptable excipient.

In some embodiments, the disclosure also provides a method of producing a recombinant collagen fragment, the method comprising producing the recombinant collagen fragment in a genetically engineered yeast strain. In some embodiments, the yeast may be pichia pastoris. In some embodiments, the yeast may be transformed with a plasmid comprising the nucleic acid sequence shown in SEQ ID NO. 973. In some embodiments, the yeast may be a yeast that has been further transformed with a plasmid comprising the nucleic acid sequence set forth in SEQ ID NO. 974. In some embodiments, the method comprises: (i) fermenting the genetically engineered yeast in a fermentation broth; (ii) Recovering from the fermentation broth a recombinant collagen fragment secreted by the genetically engineered yeast; and (iii) optionally purifying the recombinant collagen fragment. In some embodiments, the method further comprises hydroxylating the recombinant collagen fragment ex vivo.

In some embodiments, the present disclosure provides a yeast strain genetically engineered to produce a recombinant collagen fragment described herein, wherein the yeast strain comprises a vector comprising a DNA sequence encoding the recombinant collagen. In some embodiments, the vector comprises a nucleic acid sequence comprising the DNA sequence set forth in SEQ ID NO 973. In some embodiments, the yeast strains described herein further comprise a second vector comprising a nucleic acid sequence comprising the DNA sequence set forth in SEQ ID NO. 974. In some embodiments, the yeast strain may be pichia pastoris.

In some embodiments, the present disclosure provides a method of treating a skin condition, the method comprising administering to a subject in need thereof an effective amount of a recombinant collagen fragment described herein. In some embodiments, the present disclosure provides a method of treating a skin condition, the method comprising administering to a subject in need thereof an effective amount of a composition disclosed herein. In some embodiments, the skin condition may be fine lines, wrinkles, dry skin, enlarged pores, skin discoloration, reduced elasticity, unwanted hair, thinning skin, purpura, actinic keratosis, itching, eczema, acne, rosacea, erythema, telangiectasia, actinic telangiectasia, skin cancer, or hypertrophic rosacea. In some embodiments, the composition may be topically applied to an area of skin. In some embodiments, the skin region may be selected from the group consisting of: facial surfaces, scalp, neck, ears, shoulders, chest (including breast and/or collar), arms, hands, legs, stomach, buttocks, groin, back, feet, and combinations thereof.

In some embodiments, the present disclosure provides a method of increasing collagen production in a cell, the method comprising administering to the cell an effective amount of a recombinant collagen fragment described herein. In some embodiments, the method may increase the production of type I collagen. In some embodiments, the method may increase the production of type III collagen. In some embodiments, the cells may be fibroblasts. In some embodiments, the cells may be cultured cells.

In some embodiments of the methods disclosed herein, the fragments can be formulated in a composition. In some embodiments, the fragment may have an amino acid sequence according to SEQ ID NO. 1 or SEQ ID NO. 986.

In some embodiments, the present disclosure provides a method of treating a wound in a human subject in need thereof, the method comprising administering to the wound of the subject a composition comprising a recombinant collagen fragment disclosed herein, wherein administration of the recombinant collagen fragment induces production of human type I collagen, human type III collagen, or a combination thereof. In certain embodiments of these methods, the collagen fragments are topically applied to the wound.

Drawings

FIG. 1 shows a vector diagram of a bleomycin resistant vector A, a plasmid encoding a 50kDa fragment of human collagen III.

FIG. 2 shows a vector diagram of vector B, which is a plasmid encoding a 50kDa fragment of human collagen III, N-acetyltransferase and beta-lactamase.

FIG. 3A shows biomass density curves for yeast cultures expressing a 50kDa fragment of human collagen as detected by optical density measurement at 600 nm. The X-axis is the fermentation time in hours. The Y-axis is biomass density.

FIG. 3B shows biomass density curves for yeast cultures expressing a 50kDa fragment of human collagen as detected by wet cell weight measurement. The X-axis is the fermentation time in hours. The Y-axis is wet cell weight.

FIG. 3C shows glycerol concentration of a yeast culture relative to the fermentation time of a 50kDa fragment of human collagen expressed. The X-axis is the fermentation time in hours. The Y-axis is glycerol concentration.

FIG. 3D shows the carbon dioxide release rate (CER) of a yeast culture relative to the fermentation time of a 50kDa fragment of human collagen expressed. The X-axis is the fermentation time in hours. The Y-axis is CER.

FIG. 4 shows the abundance of peptides of various sizes generated by hydrolysis of a 50kDa fragment of human collagen as detected by mass spectrometry. Analysis showed that a series of peptides appeared after one week of incubation at room temperature and that the peptides present after one and three weeks of incubation were identical.

FIG. 5 shows a flow chart of a method for purifying an unhydroxylated 50kDa fragment of human collagen.

FIG. 6 shows a flow chart of a method for purifying a hydroxylated 50kDa fragment of human collagen.

FIG. 7 shows the percent recovery of a 50kDa fragment of human collagen during individual steps of its purification process, as well as the percent overall recovery during all steps up to those individual steps.

FIG. 8 shows the percent hydroxylation of the 50kDa fragment of human collagen achieved over time during an ex vivo hydroxylation reaction.

Fig. 9A and 9B show thermograms of 50kDa collagen before and after hydroxylation, respectively. The hydroxylated 50kDa collagen establishes rheology and has improved thermal stability after hydroxylation.

Fig. 10 shows a circular dichroism spectrum of hydroxylated 50kDa collagen showing the expected profile for spiral collagen.

Fig. 11A and 11B show the results of MTT assays for measuring the effect of various concentrations of different collagen preparations on the viability of fibroblasts. No collagen was observed to affect cell viability.

Fig. 12A and 12B show the results of type I collagen assays for detecting the effect of different collagen and collagen fragment preparations on type I collagen synthesis in treated primary human skin fibroblasts.

Fig. 13A and 13B show the results of a type III collagen assay for detecting the effect of different collagen and collagen fragment preparations on type III collagen synthesis in treated primary human skin fibroblasts.

Fig. 14 shows the measured solubility and properties of collagen and collagen fragment solutions.

Fig. 15A and 15B show the results of EpiOcular tests performed on human recombinant collagen III and a 50kDa fragment of human collagen.

Fig. 16A provides statistics for each attribute objectively graded for formulation 1 and formulation 2.

Fig. 16B and 16C provide the self-assessment problems and results for formulation 1 and formulation 2.

Figure 16D shows statistics of collagen levels in skin before and after administration of formulation 1 or formulation 2 for 6 weeks.

Fig. 17A-17H show the results of MTT assays for measuring the effect of various concentrations of different collagen variant preparations on the viability of fibroblasts.

Fig. 18A-18H show the results of a type I collagen assay for detecting the effect of different collagen variant preparations on type I collagen synthesis in treated primary human skin fibroblasts.

Fig. 19A-19H show the results of a type III collagen assay for detecting the effect of different collagen variant preparations on type III collagen synthesis in treated primary human skin fibroblasts.

Detailed Description

Definition of the definition

The indefinite articles "a" and "an" describe an element or component mean that there is one or at least one of the element or component. Although the articles are generally employed to refer to a modified noun as a singular noun, the articles "a" and "an" as used herein also include plural referents unless otherwise specified in the context of a specific situation. Similarly, the definite article "the" as used herein also means that the modified noun may be singular or plural unless otherwise specified in the context.

As used herein, the term "about" as used with a numerical value means "within 10% of the specified value" unless explicitly stated otherwise. For example, "about 5 wt%" means 4.5 wt% to 5.5 wt%.

The term "modified" when applied to the collagen fragments disclosed herein refers to collagen fragments comprising an amino acid sequence that is at least 70%, 80%, 90%, 95% or 99% identical or similar to the amino acid sequence of a biologically active molecule. In some embodiments, the modified collagen fragment comprises an amino acid sequence that is at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of the native sequence or a previously engineered sequence. The modified sequence may comprise additions, deletions, substitutions or combinations thereof to the amino acid sequence of the native molecule or a previously engineered molecule. For example, a modified collagen fragment may bind to or lack 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more amino acid residues as compared to the native collagen sequence. Such selections may be made to alter the looseness or tightness of the recombinant collagen. The degree of hydroxylation of collagen is related to the looseness or tightness of the triple helix of collagen. The modified collagen fragments may also comprise chemical modifications to the polypeptide, such as cross-linking between cysteine residues, or hydroxylated or glycosylated residues. As used herein, the terms "variant" and "sequence variant" refer to polypeptide sequences that are about 75% to about 99% identical to the amino acid sequence set forth in SEQ ID No. 986.

The terms "pharmaceutically acceptable" and "cosmetically acceptable" when applied to a carrier, excipient, or stabilizer useful in the compositions described herein refer to a carrier, excipient, or stabilizer that is non-toxic to the recipient at the dosage and concentration employed.

As used herein, the term "tissue repair" refers to the restoration of tissue architecture and function after injury in the context of the healing of damaged tissue. Tissue regeneration refers to the type of healing in which new growth restores portions of damaged tissue to a normal state.

A.Collagen

The term "collagen" refers to any known type of collagen, including type I to type XX collagen, described below, as well as any other collagen, whether natural, synthetic, semi-synthetic, or recombinant. The term collagen includes collagen, collagen fragments, collagen-like proteins, triple helix collagen, alpha chains, monomers, gelatin, trimers, and combinations thereof. Collagen includes all of the collagens, modified collagens and collagen-like proteins described herein. The term also encompasses procollagen and collagen-like or collagenous proteins comprising the motif (Gly-X-Y) n (where n is an integer). The term encompasses molecules of collagen and collagen-like proteins, trimers of collagen molecules, collagen fibrils, and fibers of collagen fibrils. The term also refers to chemically, enzymatically or recombinantly modified collagen or collagen-like molecules that may be fibrillated, as well as collagen, collagen-like molecules and fragments of collagenous molecules that are capable of assembling into nanofibers. The native or engineered recombinant collagen molecule will typically comprise a repetitive- (Gly-X-Y) n-sequence.

As used herein, collagen is a generic term for a family of at least 28 different collagen types. A variety of different collagen types have been identified in a range of species, including bovine, ovine, porcine, chicken, marine, plant and human collagens. Animal skin is typically type I collagen, but other types of collagen may be used to form leather, including type III collagen. The term "collagen" encompasses unprocessed (e.g., procollagen), as well as post-translationally modified and proteolytically processed collagens having triple helical structures. Type I collagen is the major fibrous collagen of bone and skin, accounting for about 80-90% of the total collagen of an organism. Type I collagen is the major structural macromolecule present in the extracellular matrix of multicellular organisms and accounts for about 20% of the total protein mass. Type I collagen is a heterotrimeric molecule comprising two α1 (I) chains and one α2 (I) chain, encoded by COL1A1 and COL1A2 genes, respectively. In vivo assembly of type I collagen fibrils, fibers and fiber bundles occurs during development and provides mechanical support to tissue while allowing cell movement and nutrient transport. Other collagen types are less abundant than type I collagen and exhibit a different distribution pattern. Type III collagen is the predominant fibrous collagen present in skin and vascular tissue. Type III collagen is homotrimeric collagen comprising three identical α1 (III) chains encoded by the COL3A1 gene.

B.Recombinant collagen and recombinant collagen fragments

As used herein, the term "recombinant collagen" refers to a family of at least 28 different naturally occurring collagen types prepared using recombinant techniques, including but not limited to collagen types I through XXVIII.

In some embodiments, the recombinant collagen described herein is a recombinant collagen fragment. The recombinant collagen fragment may be a fragment of the complete amino acid sequence of a native collagen molecule capable of forming procollagen (trimeric collagen), or the fragment may be a modified collagen molecule or a fragment of a truncated collagen molecule having at least 70%, 80%, 90%, 95%, 96%, 97%, 98% or 99% identity or similarity to the native collagen amino acid sequence (or to a fibril forming region thereof or to a segment essentially comprising [ Gly-X-Y ] n).

Exemplary collagen sequences from which fragments may be derived include the amino acid sequences of Col1A1, col1A2, and Col3A1, such as those described in the following accession numbers: p02461.4 (SEQ ID NO:982; human Col3A 1) (www.ncbi.nlm.nih.gov/protein/124056490), NP-001029211.1 (SEQ ID NO:978; bovine Col1A 1) (www.ncbi.nlm.nih.gov/protein/77404252), NP-776945.1 (SEQ ID NO:979; bovine Col1A 2) (www.ncbi.nlm.nih.gov/protein/27806257), and NP-001070299.1 (SEQ ID NO:980; bovine Col3A 1) (www.ncbi.nlm.nih.gov/protein/116003881), which are incorporated herein by reference.

The gene encoding collagen may be truncated or otherwise modified to add or remove sequences, for example, to encode a collagen fragment as disclosed herein. In addition, genetic modifications may be made to customize the size of the polynucleotide or vector, target the expressed protein to the endoplasmic reticulum or other cellular or extracellular compartment, or control the length of the encoded protein. Polynucleotides encoding collagen fragments may be modified. For example, the polynucleotide coding sequence of a collagen or collagen fragment can be modified to encode a protein that is at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 100% identical or similar to a known amino acid sequence. Such modifications may include codon modification or codon optimization of the polynucleotide encoding the collagen fragment.

In some embodiments, the collagen fragments disclosed herein can have a molecular weight of about 40kDa to about 60 kDa. In some embodiments, the collagen fragment may have a molecular weight of about 40kDa, about 41kDa, about 42kDa, about 43kDa, about 44kDa, about 45kDa, about 46kDa, about 47kDa, about 48kDa, about 49kDa, about 50kDa, about 51kDa, about 52kDa, about 53kDa, about 54kDa, about 55kDa, about 56kDa, about 57kDa, about 58kDa, about 59kDa, or about 60 kDa. In particular embodiments, the collagen fragment may have a molecular weight of about 50 kDa.

In some embodiments, the collagen fragments described herein can have an amino acid chain length of about 350 amino acids to about 600 amino acids, and can overlap with the amino acid sequence of SEQ ID NO. 1 or SEQ ID NO. 986. In some embodiments, the overlapping collagen fragments described herein can have a length of about 350 amino acids, about 370 amino acids, about 390 amino acids, about 400 amino acids, about 420 amino acids, about 440 amino acids, about 460 amino acids, about 480 amino acids, about 500 amino acids, about 510 amino acids, about 520 amino acids, about 530 amino acids, about 540 amino acids, about 550 amino acids, about 560 amino acids, about 570 amino acids, about 580 amino acids, about 590 amino acids, or about 600 amino acids.

In some embodiments, the collagen fragments described herein can have an amino acid sequence according to SEQ ID NO. 1 or SEQ ID NO. 986. In some embodiments, the collagen fragment may have at least about 70%, at least about 75%, at least about 80%, about 85%, at least about 87.5%, at least about 90%, at least about 92.5%, at least about 95%, at least about 97.5%, at least about 98%, at least about 99%, or 100% sequence identity or similarity to SEQ ID No. 1. In some embodiments, the collagen fragment may have at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 87.5%, at least about 90%, at least about 92.5%, at least about 95%, at least about 97.5%, at least about 98%, at least about 99%, or 100% sequence identity or similarity to SEQ ID NO. 986.

The amino acid sequence of SEQ ID NO. 1 is: DVKSGVAVGGLAGYPGPAGPPGPPGPPGTSGHPGSPGSPGYQGPPGEPGQAGPSGPPGPPGAIGPSGPAGKDGESGRPGRPGERGLPGPPGIKGPAGIPGFPGMKGHRGFDGRNGEKGETGAPGLKGENGLPGENGAPGPMGPRGAPGERGRPGLPGAAGARGNDGARGSDGQPGPPGPPGTAGFPGSPGAKGEVGPAGSPGSNGAPGQRGEPGPQGHAGAQGPPGPPGINGSPGGKGEMGPAGIPGAPGLMGARGPPGPAGANGAPGLRGGAGEPGKNGAKGEPGPRGERGEAGIPGVPGAKGEDGKDGSPGEPGANGLPGAAGERGAPGFRGPAGPNGIPGEKGPAGERGAPGPAGPRGAAGEPGRDGVPGGPGMRGMPGSPGGPGSDGKPGPPGSQGESGRPGPPGPSGPRGQPGVMGFPGPKGNDGAPGKNGERGGPGG PGPQGPPGKNGETGPQGPPGPTGPGGDKGDTGPPGPQGLQGLPGTGGPPGENGKPGEPGPKGDAGAPGAPGGKGDAGAPGERGPP.

The amino acid sequence of SEQ ID NO. 986 is: DVKSGVAVGGLAGYPGPAGPPGPPGPPGTSGHPGSPGSPGYQGPPGEPGQAGPSGPPGPPGAIGPSGPAGKDGESGRPGRPGERGLPGPPGIKGPAGIPGFPGMKGHRGFDGRNGEKGETGAPGLKGENGLPGENGAPGPMGPRGAPGERGRPGLPGAAGARGNDGARGSDGQPGPPGPPGTAGFPGSPGAKGEVGPAGSPGSNGAPGQRGEPGPQGHAGAQGPPGPPGINGSPGGKGEMGPAGIPGAPGLMGARGPPGPAGANGAPGLRGGAGEPGKNGAKGEPGPRGERGEAGIPGVPGAKGEDGKDGSPGEPGANGLPGAAGERGAPGFRGPAGPNGIPGEKGPAGERGAPGPAGPRGAAGEPGRDGVPGGPGMRGMPGSPGGPGSDGKPGPPGSQGESGRPGPPGPSGPRGQPGVMGFPGPKGNDGAPGKNGERGGPGGPGPQGPPGKNGETGPQGPPGPTGPGGDKGDTGPPGPQGLQGLPGTGGPPGENGKPGEPGPKGDAGAPGAPGGKGDAGAPGERGPPAIAGIGGEKAGGFAPYYG.

In some embodiments, collagen fragments described herein may have an amino acid chain length of about 350 amino acids to about 600 amino acids, and may have at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 87.5%, at least about 90%, at least about 92.5%, at least about 95%, at least about 97.5%, at least about 98%, at least about 99%, or 100% sequence identity or similarity to SEQ ID NO 1 or SEQ ID NO 986. In some embodiments, such collagen fragments described herein can have a length of about 350 amino acids, about 370 amino acids, about 390 amino acids, about 400 amino acids, about 420 amino acids, about 440 amino acids, about 460 amino acids, about 480 amino acids, about 500 amino acids, about 510 amino acids, about 520 amino acids, about 530 amino acids, about 540 amino acids, about 550 amino acids, about 560 amino acids, about 570 amino acids, about 580 amino acids, about 590 amino acids, or about 600 amino acids.

In some embodiments, the recombinant collagen may comprise a hydrolysate of a collagen fragment, wherein the hydrolysate may have a sequence that is part of SEQ ID NO. 1 or SEQ ID NO. 986. In some embodiments, the hydrolysate can have a sequence according to one of SEQ ID NO. 2 through SEQ ID NO. 972.

In some embodiments, the lysine, proline or lysine residues and proline residues present in the collagen fragment are non-hydroxylated. In other embodiments, the collagen fragments described herein can be hydroxylated. In some embodiments, at least 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or 100% (or any intermediate value or subrange) of the lysine, proline or lysine residues and proline residues in the recombinant collagen fragment can be hydroxylated. Hydroxylated collagen can establish rheology and improve the thermal stability of collagen molecules or fragments. The hydroxylated collagen and hydroxylated collagen fragments are also resistant to high concentration pepsin digestion, e.g., pepsin to total protein ratio of 1:25 to 1:1.

The degree of hydroxylation of proline, lysine or proline and lysine residues in a collagen fragment can be estimated by determining the melting temperature of hydrated collagen (such as a hydrogel) and comparing the melting point of the hydrogel to a "control" collagen fragment having a known content of hydroxylated amino acid residues. The collagen melting temperature may be in the range of 25 ℃ to 40 ℃, with more highly hydroxylated collagen generally having a higher melting temperature.

In some embodiments, the collagen fragments described herein can have amino acid sequences as set forth in table 1 or table 2 below. In some embodiments, the collagen fragment may be a collagen fragment sequence variant having the amino acid sequence set forth in table 3.

Table 1.

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Table 2.

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In some embodiments, the collagen fragment sequence variants described herein can have the amino acid sequences provided in table 4 below. In some embodiments, the collagen fragment sequence variants described herein can have an amino acid sequence encoded by a nucleic acid sequence set forth in table 5 below. The relationship between the amino acid sequence, the nucleic acid sequence encoding the amino acid sequence and the sequence similarity to SEQ ID NO. 986 is provided in Table 3 below.

Table 3.

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Table 4.

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In some embodiments, the collagen fragment sequence variants described herein can have an amino acid sequence encoded by a nucleic acid sequence set forth in table 5 below.

Table 5.

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C.Compositions comprising collagen fragments and sequence variants thereof

In some embodiments, the present disclosure provides a composition comprising one or more recombinant collagen fragments disclosed herein and at least one pharmaceutically or cosmetically acceptable excipient. In particular embodiments, the composition may comprise a recombinant collagen fragment according to SEQ ID NO. 1 or SEQ ID NO. 986, and at least one pharmaceutically acceptable excipient or cosmetically acceptable excipient. In further embodiments, the composition may comprise a recombinant collagen fragment having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 87.5%, at least about 90%, at least about 92.5%, at least about 95%, at least about 97.5%, at least about 98%, at least about 99% or 100% sequence identity or similarity to SEQ ID No. 1 or SEQ ID No. 986, and at least one pharmaceutically or cosmetically acceptable excipient. In still further embodiments, the composition may comprise a recombinant collagen fragment having about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or 100% identity or similarity to SEQ ID NO. 1 or SEQ ID NO. 986, and at least one pharmaceutically or cosmetically acceptable excipient. In still further embodiments, the composition may comprise a recombinant collagen fragment having about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or 100% sequence identity or similarity to SEQ ID No. 1 or SEQ ID No. 986, and at least one excipient suitable for use in a dietary supplement (e.g., a nutritional supplement).

In some embodiments, the composition may comprise a hydrolysate of collagen fragments and at least one pharmaceutically or cosmetically acceptable excipient, wherein the hydrolysate may have a sequence as part of SEQ ID NO. 1 or SEQ ID NO. 986. In some embodiments, the composition comprises a hydrolysate having a sequence according to one or more of SEQ ID NOs 2 to 972, and at least one pharmaceutically acceptable excipient or cosmetically acceptable excipient. In some embodiments, the composition may comprise any one of the hydrolysates shown in SEQ ID NO. 2 through SEQ ID NO. 972, and at least one pharmaceutically acceptable excipient or cosmetically acceptable excipient. In some embodiments, the composition may comprise a recombinant collagen fragment having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 87.5%, at least about 90%, at least about 92.5%, at least about 95%, at least about 97.5%, at least about 98%, at least about 99%, or 100% sequence identity or similarity to one of SEQ ID nos. 2 to 972, and at least one pharmaceutically or cosmetically acceptable excipient.

In yet another embodiment, the composition may comprise a recombinant collagen fragment according to SEQ ID NO. 1 or SEQ ID NO. 986, a hydrolysate having a sequence according to one of SEQ ID NO. 2 to SEQ ID NO. 972, and at least one pharmaceutically or cosmetically acceptable excipient. In yet another embodiment, the composition may comprise a recombinant collagen fragment according to SEQ ID NO. 1 or SEQ ID NO. 986, a plurality of hydrolysates having sequences according to any of SEQ ID NO. 2 to SEQ ID NO. 972 that may be the same or different, and at least one pharmaceutically or cosmetically acceptable excipient. In certain embodiments, the number of hydrolysates present in the various hydrolysates in the composition may increase over time, temperature, pH, or due to other conditions that generally result in the hydrolysis or otherwise decomposition of recombinant collagen fragments (such as recombinant collagen fragments according to SEQ ID NO:1 or SEQ ID NO: 986). In other embodiments, the composition may be stabilized with one or more stabilizers such that the concentration of recombinant collagen fragments according to SEQ ID NO:1 or SEQ ID NO:986 and the concentration of each fragment of the plurality of fragments remain substantially constant (i.e., do not vary by more than.+ -. 5% as measured by HPLC over a given period of time) or remain constant. In certain embodiments, the recombinant collagen fragment, such as the recombinant collagen fragment according to SEQ ID No. 1 or SEQ ID No. 986, is hydrolyzable such that less than about 10%, about 10% to about 20%, about 20% to about 30%, about 30% to about 40%, about 40% to about 50%, about 50% to about 60%, about 60% to about 70%, about 70% to about 80%, about 80% to about 90%, or about 90% to about 100% of the unhydrolyzed recombinant fragment remains in the composition as measured by HPLC. In other embodiments, the composition can comprise a mixture of recombinant collagen fragments (e.g., recombinant collagen fragments according to SEQ ID NO:1 or SEQ ID NO: 986) and hydrolysates of the recombinant collagen fragments (e.g., collagen fragments according to any one of SEQ ID NO:2 to SEQ ID NO: 972) such that the weight of hydrolysates in the composition is less than about 10%, about 10% to about 20%, about 20% to about 30%, about 30% to about 40%, about 40% to about 50%, about 50% to about 60%, about 60% to about 70%, about 70% to about 80%, about 80% to about 90%, or about 90% to about 100% of the weight of collagen-related protein in the composition.

In some embodiments, the present disclosure provides a composition comprising one or more recombinant collagen fragments disclosed herein, and at least one pharmaceutically or cosmetically acceptable excipient. In particular embodiments, the composition may comprise a recombinant collagen fragment sequence variant according to any one of SEQ ID NO. 987 to SEQ ID NO. 1015, and at least one pharmaceutically acceptable excipient or cosmetically acceptable excipient. In further embodiments, the composition may comprise a recombinant collagen fragment sequence variant having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 87.5%, at least about 90%, at least about 92.5%, at least about 95%, at least about 97.5%, at least about 98%, or at least about 99% sequence identity or similarity to SEQ ID No. 986, and at least one pharmaceutically or cosmetically acceptable excipient. In still further embodiments, the composition may comprise a recombinant collagen fragment sequence variant having about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identity or similarity to SEQ ID NO. 986, and at least one pharmaceutically or cosmetically acceptable excipient. In still further embodiments, the composition may comprise a recombinant collagen fragment sequence variant having about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% sequence identity or similarity to SEQ ID NO. 986, and at least one pharmaceutically or cosmetically acceptable excipient.

In some embodiments, the composition may comprise a recombinant collagen fragment having an amino acid chain length of about 350 amino acids to about 600 amino acids that overlaps with the amino acid sequence in SEQ ID NO. 1 or SEQ ID NO. 986. In some embodiments, the composition comprises a recombinant collagen fragment having a length of about 350 amino acids, about 370 amino acids, about 390 amino acids, about 400 amino acids, about 420 amino acids, about 440 amino acids, about 460 amino acids, about 480 amino acids, about 500 amino acids, about 510 amino acids, about 520 amino acids, about 530 amino acids, about 540 amino acids, about 550 amino acids, about 560 amino acids, about 570 amino acids, about 580 amino acids, about 590 amino acids, or about 600 amino acids. In a specific embodiment, the composition comprises a recombinant collagen fragment having an amino acid chain length of 528 amino acids. In a specific embodiment, the composition comprises a recombinant collagen fragment having an amino acid chain length of 546 amino acids.

In some embodiments, the composition comprises a recombinant collagen fragment sequence variant according to any one of SEQ ID nos. 987 to 1015, the recombinant collagen fragment sequence variant having a length of about 350 amino acids, about 370 amino acids, about 390 amino acids, about 400 amino acids, about 420 amino acids, about 440 amino acids, about 460 amino acids, about 480 amino acids, about 500 amino acids, about 510 amino acids, about 520 amino acids, about 530 amino acids, about 540 amino acids, about 550 amino acids, about 560 amino acids, about 570 amino acids, about 580 amino acids, about 590 amino acids, or about 600 amino acids. In a specific embodiment, the composition comprises a recombinant collagen fragment having an amino acid chain length of 528 amino acids. In a specific embodiment, the composition comprises a recombinant collagen fragment having an amino acid chain length of 546 amino acids.

In some embodiments, the composition may comprise a recombinant collagen fragment or sequence variant thereof having a chain length of about 350 amino acids to about 600 amino acids, and may have at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 87.5%, at least about 90%, at least about 92.5%, at least about 95%, at least about 97.5%, at least about 98%, at least about 99% or 100% sequence identity or similarity to SEQ ID No. 1 or SEQ ID No. 986. In some embodiments, such collagen fragments described herein can have a length of about 350 amino acids, about 370 amino acids, about 390 amino acids, about 400 amino acids, about 420 amino acids, about 440 amino acids, about 460 amino acids, about 480 amino acids, about 500 amino acids, about 510 amino acids, about 520 amino acids, about 530 amino acids, about 540 amino acids, about 550 amino acids, about 560 amino acids, about 570 amino acids, about 580 amino acids, about 590 amino acids, or about 600 amino acids.

In some embodiments, the composition may comprise a hydrolysate of collagen fragments, wherein the hydrolysate may have an amino acid chain length of about 10 amino acids to about 75 amino acids. In some embodiments, the composition may comprise a hydrolysate of collagen fragments having an amino acid chain length of about 20 amino acids to about 50 amino acids. In some embodiments, the hydrolysates described herein may have a length of about 10 amino acids, about 15 amino acids, about 20 amino acids, about 25 amino acids, about 30 amino acids, about 35 amino acids, about 40 amino acids, about 45 amino acids, about 50 amino acids, about 55 amino acids, about 60 amino acids, about 65 amino acids, about 70 amino acids, or about 75 amino acids.

In some embodiments, the composition may comprise about 5ppm to about 500ppm of recombinant collagen fragments or sequence variants thereof. In some embodiments, the composition can comprise about 5ppm, about 10ppm, about 25ppm, about 50ppm, about 100ppm, about 150ppm, about 200ppm, about 250ppm, about 300ppm, about 350ppm, about 400ppm, about 450ppm, or about 500ppm of the recombinant collagen fragment. In some embodiments, the composition can comprise about 5ppm, about 10ppm, about 25ppm, about 50ppm, about 100ppm, about 150ppm, about 200ppm, about 250ppm, about 300ppm, about 350ppm, about 400ppm, about 450ppm, or about 500ppm of the recombinant collagen fragment sequence variant. In some embodiments, the composition may comprise about 5ppm, about 10ppm, about 25ppm, about 50ppm, about 100ppm, about 150ppm, about 200ppm, about 250ppm, about 300ppm, about 350ppm, about 400ppm, about 450ppm, or about 500ppm of the recombinant collagen fragment having a sequence according to SEQ ID NO:1 or SEQ ID NO: 986. In some embodiments, the composition may comprise about 5ppm, about 10ppm, about 25ppm, about 50ppm, about 100ppm, about 150ppm, about 200ppm, about 250ppm, about 300ppm, about 350ppm, about 400ppm, about 450ppm, or about 500ppm of one or more hydrolysates of recombinant collagen fragments. In some embodiments, the composition may comprise about 5ppm, about 10ppm, about 25ppm, about 50ppm, about 100ppm, about 150ppm, about 200ppm, about 250ppm, about 300ppm, about 350ppm, about 400ppm, about 450ppm, or about 500ppm of one or more hydrolysates of recombinant collagen fragments having a sequence according to SEQ ID NO:1 or SEQ ID NO: 986. In some embodiments, the composition can comprise about 5ppm, about 10ppm, about 25ppm, about 50ppm, about 100ppm, about 150ppm, about 200ppm, about 250ppm, about 300ppm, about 350ppm, about 400ppm, about 450ppm, or about 500ppm of a recombinant collagen fragment sequence variant according to any one of SEQ ID NOs 987 to 1015. In some embodiments, the composition may comprise a mixture of recombinant collagen fragments or variants thereof at a concentration of about 5ppm to about 500ppm and one or more hydrolysates of the recombinant collagen fragments at a concentration of about 5ppm to about 500 ppm. In some embodiments, the composition may comprise a mixture of a recombinant collagen fragment having a sequence according to SEQ ID NO:1 or SEQ ID NO:986, or a variant thereof, at a concentration of about 5ppm to about 500ppm and one or more hydrolysates of the recombinant collagen fragment at a concentration of about 5ppm to about 500 ppm.

In some embodiments, the composition can be prepared with about 0.1% to about 20% by volume of a solution of about 0.5% to about 25% by weight of the recombinant collagen fragment. In some embodiments, the composition can be prepared with about 0.1% to about 20% by volume of about 0.5% to about 25% by weight of the recombinant collagen fragment sequence variant solution. In some of these embodiments, the composition can comprise about 0.1 vol%, about 0.2 vol%, about 0.3 vol%, about 0.4 vol%, about 0.5 vol%, about 0.6 vol%, about 0.7 vol%, about 0.8 vol%, about 0.9 vol%, about 1 vol%, about 2 vol%, about 3 vol%, about 4 vol%, about 5 vol%, about 6 vol%, about 7 vol%, about 8 vol%, about 9 vol%, about 10 vol%, about 11 vol%, about 12 vol%, about 13 vol%, about 14 vol%, about 15 vol%, about 16 vol%, about 17 vol%, about 18 vol%, about 19 vol%, or about 20 vol% of the recombinant collagen fragment solution or the recombinant collagen fragment sequence variant solution. In some of these embodiments, the recombinant collagen fragment solution comprises about 0.5 wt%, about 0.6 wt%, about 0.7 wt%, about 0.8 wt%, about 0.9 wt%, about 1 wt%, about 2 wt%, about 3 wt%, about 4 wt%, about 5 wt%, about 6 wt%, about 7 wt%, about 8 wt%, about 9 wt%, about 10 wt%, about 11 wt%, about 12 wt%, about 13 wt%, about 14 wt%, about 15 wt%, about 16 wt%, about 17 wt%, about 18 wt%, about 19 wt%, about 20 wt%, about 21 wt%, about 22 wt%, about 23 wt%, about 24 wt%, or about 25 wt% of the recombinant collagen fragment, or about 0.5 wt%, about 0.6 wt%, about 0.7 wt%, about 0.8 wt%, about 0.9 wt%, about 1 wt%, about 2 wt%, about 3 wt%, about 4 wt%, about 5 wt%, about 6 wt%, about 7 wt%, about 8 wt%, about 9 wt%, about 10 wt%, about 11 wt%, about 12 wt%, about 13 wt%, about 14 wt%, about 15 wt%, about 16 wt%, about 17 wt%, about 18 wt%, about 19 wt%, about 20 wt%, about 21 wt%, about 22 wt%, about 23 wt%, about 24 wt% or about 25 wt% of the recombinant collagen fragment sequence variant. In some of these embodiments, the recombinant collagen fragment solution comprises about 0.1 wt%, about 0.2 wt%, about 0.3 wt%, about 0.4 wt%, about 0.5 wt%, about 0.6 wt%, about 0.7 wt%, about 0.8 wt%, about 0.9 wt%, about 1 wt%, about 2 wt%, about 3 wt%, about 4 wt%, about 5 wt%, about 6 wt%, about 7 wt%, about 8 wt%, about 9 wt%, about 10 wt%, about 11 wt%, about 12 wt%, about 13 wt%, about 14 wt%, about 15 wt%, about 16 wt%, about 17 wt%, about 18 wt%, about 19 wt%, about 20 wt%, about 21 wt%, about 22 wt%, about 23 wt%, about 24 wt%, or about 25 wt% of a collagen fragment having a sequence according to SEQ ID NO 1 or SEQ ID NO 986. In some of these embodiments, the recombinant collagen fragment solution comprises about 0.1 wt%, about 0.2 wt%, about 0.3 wt%, about 0.4 wt%, about 0.5 wt%, about 0.6 wt%, about 0.7 wt%, about 0.8 wt%, about 0.9 wt%, about 1 wt%, about 2 wt%, about 3 wt%, about 4 wt%, about 5 wt%, about 6 wt%, about 7 wt%, about 8 wt%, about 9 wt%, about 10 wt%, about 11 wt%, about 12 wt%, about 13 wt%, about 14 wt%, about 15 wt%, about 16 wt%, about 17 wt%, about 18 wt%, about 19 wt%, about 20 wt%, about 21 wt%, about 22 wt%, about 23 wt%, about 24 wt%, or about 25 wt% of one or more hydrolysates of the recombinant collagen fragments. In some of these embodiments, the recombinant collagen fragment solution comprises about 0.1 wt%, about 0.2 wt%, about 0.3 wt%, about 0.4 wt%, about 0.5 wt%, about 0.6 wt%, about 0.7 wt%, about 0.8 wt%, about 0.9 wt%, about 1 wt%, about 2 wt%, about 3 wt%, about 4 wt%, about 5 wt%, about 6 wt%, about 7 wt%, about 8 wt%, about 9 wt%, about 10 wt%, about 11 wt%, about 12 wt%, about 13 wt%, about 14 wt%, about 15 wt%, about 16 wt%, about 17 wt%, about 18 wt%, about 19 wt%, about 20 wt%, about 21 wt%, about 22 wt%, about 23 wt%, about 24 wt%, or about 25 wt% of a hydrolysate of one or more recombinant collagen fragments having a sequence according to one or more of SEQ ID NOs 2 to 972. In some of these embodiments, the recombinant collagen fragment sequence variant solution comprises about 0.1 wt%, about 0.2 wt%, about 0.3 wt%, about 0.4 wt%, about 0.5 wt%, about 0.6 wt%, about 0.7 wt%, about 0.8 wt%, about 0.9 wt%, about 1 wt%, about 2 wt%, about 3 wt%, about 4 wt%, about 5 wt%, about 6 wt%, about 7 wt%, about 8 wt%, about 9 wt%, about 10 wt%, about 11 wt%, about 12 wt%, about 13 wt%, about 14 wt%, about 15 wt%, about 16 wt%, about 17 wt%, about 18 wt%, about 19 wt%, about 20 wt%, about 21 wt%, about 22 wt%, about 23 wt%, about 24 wt%, or about 25 wt% of a recombinant collagen fragment sequence variant or variant having a sequence according to one or more of SEQ ID nos. 987 to 1015. In some of these embodiments, the composition can be prepared using a mixture of about 0.1% to about 20% by volume of about 0.5% to about 25% by weight of the recombinant collagen fragment and one or more hydrolysates of the recombinant collagen fragment. In some of these embodiments, the composition can be prepared using a mixture of about 0.1% to about 20% by volume of about 0.5% to about 25% by weight of a recombinant collagen fragment having a sequence according to SEQ ID NO. 1 or SEQ ID NO. 986 and one or more hydrolysates of the recombinant collagen fragment.

In some embodiments, the composition can be prepared using about 0.1% to about 20% by volume of a solution of about 0.5% to about 25% by weight comprising a mixture of recombinant collagen fragments having a sequence according to SEQ ID NO. 1 or SEQ ID NO. 986 and one or more hydrolysates of any of the recombinant collagen fragments. In some embodiments, the composition can be prepared using about 0.1% to about 20% by volume of a solution of about 0.5% to about 25% by weight comprising a mixture of recombinant collagen fragment sequence variants having a sequence according to any one of SEQ ID NOs 987 to 1015. In some embodiments, the composition can be prepared with about 0.1% to about 20% by volume of a solution comprising a mixture of one or more hydrolysates of any recombinant collagen fragment having a sequence according to SEQ ID No. 1 or SEQ ID No. 986 and about 0.5% to about 25% by weight of a recombinant collagen fragment sequence variant solution comprising a sequence variant having a sequence according to any one of SEQ ID No. 987 to SEQ ID No. 1015.

In some of these embodiments, the recombinant collagen fragment and recombinant collagen fragment sequence variant solution comprises about 0.1 wt%, about 0.2 wt%, about 0.3 wt%, about 0.4 wt%, about 0.5 wt%, about 0.6 wt%, about 0.7 wt%, about 0.8 wt%, about 0.9 wt%, about 1 wt%, about 2 wt%, about 3 wt%, about 4 wt%, about 5 wt%, about 6 wt%, about 7 wt%, about 8 wt%, about 9 wt%, about 10 wt%, about 11 wt%, about 12 wt%, about 13 wt%, about 14 wt%, about 15 wt%, about 16 wt%, about 17 wt%, about 18 wt%, about 19 wt%, about 20 wt%, about 21 wt%, about 22 wt%, about 23 wt%, about 24 wt%, or about 25 wt% of a recombinant collagen fragment or more hydrolysate having a sequence according to one or more of SEQ ID NOs 2 to 972.

In some embodiments, the composition can comprise about 0.0005 wt.% to about 25 wt.% of the recombinant collagen fragment, about 0.001 wt.% to about 25 wt.% of the recombinant collagen fragment, about 0.01 wt.% to about 25 wt.% of the recombinant collagen fragment, about 0.1 wt.% to about 25 wt.% of the recombinant collagen fragment, about 0.5 wt.% to about 20 wt.% of the recombinant collagen fragment, about 0.7 wt.% to about 17 wt.% of the recombinant collagen fragment, about 1 wt.% to about 15 wt.% of the recombinant collagen fragment, about 2 wt.% to about 12 wt.% of the recombinant collagen fragment, about 2 wt.% to about 10 wt.% of the recombinant collagen fragment, about 3 wt.% to about 9 wt.% of the recombinant collagen fragment, about 4 wt.% to about 8 wt.% of the recombinant collagen fragment, or about 5 wt.% to about 7 wt.% of the recombinant collagen fragment.

In some embodiments, the composition can comprise about 0.0005 wt%, about 0.001 wt%, about 0.01 wt%, about 0.1 wt%, about 0.2 wt%, about 0.3 wt%, about 0.4 wt%, about 0.5 wt%, about 0.6 wt%, about 0.7 wt%, about 0.8 wt%, about 0.9 wt%, about 1 wt%, about 2 wt%, about 3 wt%, about 4 wt%, about 5 wt%, about 6 wt%, about 7 wt%, about 8 wt%, about 9 wt%, about 10 wt%, about 11 wt%, about 12 wt%, about 13 wt%, about 14 wt%, about 15 wt%, about 16 wt%, about 17 wt%, about 18 wt%, about 19 wt%, about 20 wt%, about 21 wt%, about 22 wt%, about 23 wt%, about 24 wt% or about 25 wt% of the recombinant collagen fragment. In some embodiments, the composition may comprise about 0.0005 wt%, about 0.001 wt%, about 0.01 wt%, about 0.1 wt%, about 0.2 wt%, about 0.3 wt%, about 0.4 wt%, about 0.5 wt%, about 0.6 wt%, about 0.7 wt%, about 0.8 wt%, about 0.9 wt%, about 1 wt%, about 2 wt%, about 3 wt%, about 4 wt%, about 5 wt%, about 6 wt%, about 7 wt%, about 8 wt%, about 9 wt%, about 10 wt%, about 11 wt%, about 12 wt%, about 13 wt%, about 14 wt%, about 15 wt%, about 16 wt%, about 17 wt%, about 18 wt%, about 19 wt%, about 20 wt%, about 21 wt%, about 22 wt%, about 23 wt%, about 24 wt%, or about 25 wt% of a collagen fragment having a sequence according to SEQ ID NO. 1 or SEQ ID NO 986. In some embodiments, the composition may comprise about 0.0005 wt%, about 0.001 wt%, about 0.01 wt%, about 0.1 wt%, about 0.2 wt%, about 0.3 wt%, about 0.4 wt%, about 0.5 wt%, about 0.6 wt%, about 0.7 wt%, about 0.8 wt%, about 0.9 wt%, about 1 wt%, about 2 wt%, about 3 wt%, about 4 wt%, about 5 wt%, about 6 wt%, about 7 wt%, about 8 wt%, about 9 wt%, about 10 wt%, about 11 wt%, about 12 wt%, about 13 wt%, about 14 wt%, about 15 wt%, about 16 wt%, about 17 wt%, about 18 wt%, about 19 wt%, about 20 wt%, about 21 wt%, about 22 wt%, about 23 wt%, about 24 wt% or about 25 wt% of the one or more hydrolysates of the recombinant collagen fragments. In some embodiments, the composition may comprise about 0.0005 wt%, about 0.001 wt%, about 0.01 wt%, about 0.1 wt%, about 0.2 wt%, about 0.3 wt%, about 0.4 wt%, about 0.5 wt%, about 0.6 wt%, about 0.7 wt%, about 0.8 wt%, about 0.9 wt%, about 1 wt%, about 2 wt%, about 3 wt%, about 4 wt%, about 5 wt%, about 6 wt%, about 7 wt%, about 8 wt%, about 9 wt%, about 10 wt%, about 11 wt%, about 12 wt%, about 13 wt%, about 14 wt%, about 15 wt%, about 16 wt%, about 17 wt%, about 18 wt%, about 19 wt%, about 20 wt%, about 21 wt%, about 22 wt%, about 23 wt%, about 24 wt%, or about 25 wt% of the recombinant collagen fragment having one or more sequences according to one or more of SEQ ID NOs 2 to SEQ ID NOs 972. In some embodiments, the composition can comprise about 0.0005 wt%, about 0.001 wt%, about 0.01 wt%, about 0.1 wt%, about 0.2 wt%, about 0.3 wt%, about 0.4 wt%, about 0.5 wt%, about 0.6 wt%, about 0.7 wt%, about 0.8 wt%, about 0.9 wt%, about 1 wt%, about 2 wt%, about 3 wt%, about 4 wt%, about 5 wt%, about 6 wt%, about 7 wt%, about 8 wt%, about 9 wt%, about 10 wt%, about 11 wt%, about 12 wt%, about 13 wt%, about 14 wt%, about 15 wt%, about 16 wt%, about 17 wt%, about 18 wt%, about 19 wt%, about 20 wt%, about 21 wt%, about 22 wt%, about 23 wt%, about 24 wt% or about 25 wt% of the recombinant collagen fragment and a mixture of one or more hydrolysates of the recombinant collagen fragments. In some embodiments, the cosmetic composition may comprise about 0.0005 wt%, about 0.001 wt%, about 0.01 wt%, about 0.1 wt%, about 0.2 wt%, about 0.3 wt%, about 0.4 wt%, about 0.5 wt%, about 0.6 wt%, about 0.7 wt%, about 0.8 wt%, about 0.9 wt%, about 1 wt%, about 2 wt%, about 3 wt%, about 4 wt%, about 5 wt%, about 6 wt%, about 7 wt%, about 8 wt%, about 9 wt%, about 10 wt%, about 11 wt%, about 12 wt%, about 13 wt%, about 14 wt%, about 15 wt%, about 16 wt%, about 17 wt%, about 18 wt%, about 19 wt%, about 20 wt%, about 21 wt%, about 22 wt%, about 23 wt%, about 24 wt%, or about 25 wt% of the recombinant collagen fragment having a sequence according to SEQ ID NO:1 or SEQ ID NO 986, or a mixture of the recombinant collagen fragments.

In some embodiments, the composition can comprise about 0.0005 wt.% to about 25 wt.% of the recombinant collagen fragment sequence variant, about 0.001 wt.% to about 25 wt.% of the recombinant collagen fragment sequence variant, about 0.01 wt.% to about 25 wt.% of the recombinant collagen fragment sequence variant, about 0.1 wt.% to about 25 wt.% of the recombinant collagen fragment sequence variant, about 0.5 wt.% to about 20 wt.% of the recombinant collagen fragment sequence variant, about 0.7 wt.% to about 17 wt.% of the recombinant collagen fragment sequence variant, about 1 wt.% to about 15 wt.% of the recombinant collagen fragment sequence variant, about 2 wt.% to about 12 wt.% of the recombinant collagen fragment sequence variant, about 2 wt.% to about 10 wt.% of the recombinant collagen fragment sequence variant, about 3 wt.% to about 9 wt.% of the recombinant collagen fragment sequence variant, about 4 wt.% to about 8 wt.% of the recombinant collagen fragment sequence variant, or about 5 wt.% to about 7 wt.% of the recombinant collagen fragment sequence variant.

In some embodiments, the composition can comprise about 0.0005 wt%, about 0.001 wt%, about 0.01 wt%, about 0.1 wt%, about 0.2 wt%, about 0.3 wt%, about 0.4 wt%, about 0.5 wt%, about 0.6 wt%, about 0.7 wt%, about 0.8 wt%, about 0.9 wt%, about 1 wt%, about 2 wt%, about 3 wt%, about 4 wt%, about 5 wt%, about 6 wt%, about 7 wt%, about 8 wt%, about 9 wt%, about 10 wt%, about 11 wt%, about 12 wt%, about 13 wt%, about 14 wt%, about 15 wt%, about 16 wt%, about 17 wt%, about 18 wt%, about 19 wt%, about 20 wt%, about 21 wt%, about 22 wt%, about 23 wt%, about 24 wt% or about 25 wt% of the recombinant collagen fragment sequence variant. In some embodiments, the composition may comprise about 0.0005 wt%, about 0.001 wt%, about 0.01 wt%, about 0.1 wt%, about 0.2 wt%, about 0.3 wt%, about 0.4 wt%, about 0.5 wt%, about 0.6 wt%, about 0.7 wt%, about 0.8 wt%, about 0.9 wt%, about 1 wt%, about 2 wt%, about 3 wt%, about 4 wt%, about 5 wt%, about 6 wt%, about 7 wt%, about 8 wt%, about 9 wt%, about 10 wt%, about 11 wt%, about 12 wt%, about 13 wt%, about 14 wt%, about 15 wt%, about 16 wt%, about 17 wt%, about 18 wt%, about 19 wt%, about 20 wt%, about 21 wt%, about 22 wt%, about 23 wt%, about 24 wt%, or about 25 wt% of the collagen fragment having a sequence according to any one of SEQ ID NOs 1015 to SEQ ID NOs 987 to SEQ ID NOs. In some embodiments, the composition can comprise about 0.0005 wt%, about 0.001 wt%, about 0.01 wt%, about 0.1 wt%, about 0.2 wt%, about 0.3 wt%, about 0.4 wt%, about 0.5 wt%, about 0.6 wt%, about 0.7 wt%, about 0.8 wt%, about 0.9 wt%, about 1 wt%, about 2 wt%, about 3 wt%, about 4 wt%, about 5 wt%, about 6 wt%, about 7 wt%, about 8 wt%, about 9 wt%, about 10 wt%, about 11 wt%, about 12 wt%, about 13 wt%, about 14 wt%, about 15 wt%, about 16 wt%, about 17 wt%, about 18 wt%, about 19 wt%, about 20 wt%, about 21 wt%, about 22 wt%, about 23 wt%, about 24 wt% or about 25 wt% of the recombinant collagen fragment and a mixed variant of one or more recombinant collagen fragment sequences. In some embodiments, the cosmetic composition may comprise about 0.0005 wt%, about 0.001 wt%, about 0.01 wt%, about 0.1 wt%, about 0.2 wt%, about 0.3 wt%, about 0.4 wt%, about 0.5 wt%, about 0.6 wt%, about 0.7 wt%, about 0.8 wt%, about 0.9 wt%, about 1 wt%, about 2 wt%, about 3 wt%, about 4 wt%, about 5 wt%, about 6 wt%, about 7 wt%, about 8 wt%, about 9 wt%, about 10 wt%, about 11 wt%, about 12 wt%, about 13 wt%, about 14 wt%, about 15 wt%, about 16 wt%, about 17 wt%, about 18 wt%, about 19 wt%, about 20 wt%, about 21 wt%, about 22 wt%, about 23 wt%, about 24 wt%, or about 25 wt% of a collagen fragment having a sequence according to SEQ ID NO. 1 or SEQ ID NO 986 and a mixture of collagen fragments having a sequence according to SEQ ID NO 987 to any one of the variant or more of SEQ ID NO 987 to 98.

Skin care

In some embodiments, the compositions described herein can be used to improve the aesthetic appearance of skin and/or its appendages, such as the surface appearance and/or texture of skin. In some embodiments, the compositions described herein may be formulated for use in the body and face, hands and feet, including use as treatments for the eye area, nails and hair. The term "surface appearance" means visual and/or tactile irregularities in the skin and/or scalp, including wrinkles and fine lines, expression lines in the space above the forehead and between the eyebrows, wrinkles and/or fine lines around the mouth and/or sagging of the area around the lips and upper lip (the area between the upper lip and nose), uneven skin tone (freckles, actinic lentigo), appearance and/or visibility of pores, papery appearance of skin, imperfections in skin micro-relief such as varicella or acne scars, imperfections of oily skin (shiny appearance, etc.). The term "skin texture" may refer to loose, sagging, less firm, less elastic skin, and/or skin that has sagging.

In some embodiments, the compositions described herein can be used to improve the aesthetic appearance of skin, including improving the appearance of expression lines. The expression lines are created by the action of stress exerted on the skin by the underlying muscles. Age and environmental factors (such as exposure to sunlight) can deepen and persist expression lines. Expression lines are characterized by furrows in the area of the skin muscles around the holes formed by the nose (nasal sulcus), mouth (perioral lines and so-called picric lines) and eyes (fish tail lines), as well as between the eyebrows (glabella wrinkles or lions lines) and on the forehead.

In some embodiments, the compositions described herein can be used to improve the aesthetic appearance of skin and/or the visibility of pores. The visibility of pores may be due to excess sebum, aging, loss of firmness, relaxation, stress, fatigue, improper hygiene, climatic factors, or any combination thereof. The compositions described herein can tighten pores making them less visible.

In some embodiments, the compositions described herein can be used to improve the paper appearance and skin touch behavior of skin. In particular, older skin may visually present the appearance of cigarette paper, giving it an appearance similar to paper sedge. The paper-like appearance of the skin can be seen on the face and back of the elderly.

In some embodiments, the compositions described herein may be compositions for facial, hand, foot, or body protection, treatment, or care, for example, day creams, night creams, make-up removal creams, sun protection compositions, body creams for skin protection or care, post-sun creams, skin lotions, gels, foams, artificial tanning compositions, and post-shave compositions. In some embodiments, the compositions described herein may be formulated, for example, as solutions, suspensions, lotions, creams, essences, gels, balms, gels, oils, oil-in-creams, micellar water, facial sprays, facial essences, concealers or skin tone correction formulations, toners (based on water and/or alcohol), paints, polishes, sticks, pencils, sprays, aerosols, ointments, cleaning liquid detergents, solid sticks, shampoos, hair conditioners, hair styling products, pastes, foams, powders, mousses, balms, shave creams, wipes, strips, patches, wound dressings, adhesive bandages, hydrogels, film forming products, facial and skin masks, cosmetics (e.g., foundations, eye liners, eye shadows), exfoliants, deodorants, and antiperspirants, and the like. Exemplary formulations are provided herein.

In some embodiments, the compositions described herein may be cosmetic compositions, and the at least one excipient may be a cosmetically acceptable excipient. Cosmetically acceptable excipients are excipients suitable for use in cosmetic products. Exemplary cosmetically acceptable excipients are described below.

In some embodiments, the cosmetic compositions described herein may comprise a hydrolysate of recombinant collagen fragments, and at least one excipient, such as a cosmetically acceptable excipient. In some embodiments, the cosmetic compositions described herein may comprise a recombinant collagen fragment, one or more hydrolysates of the recombinant collagen fragment, and at least one excipient, such as a cosmetically acceptable excipient.

In some embodiments, the cosmetic composition may comprise ingredients commonly used in cosmetic products such as skin care, eye care, nail care, and hair care products. These ingredients may include, but are not limited to, soaps, antimicrobial agents, anti-inflammatory agents, moisturizers, waxy alcohols, hydration agents, moisturizers, permeation enhancers, emulsifiers, natural or synthetic oils, solvents, fats, surfactants, detergents, gelling agents, emollients, antioxidants, fragrances, paints, polishes, fillers, thickeners, waxes, odor absorbers, dyes, colorants, powders, viscosity control agents, anesthetics, antipruritics, plant extracts, conditioning agents, darkening or whitening agents, humectants, mica, minerals, polyphenols, silicones or silicone derivatives (such as dimethicones), sunscreens, vitamins, botanicals, alcohols (such as denatured alcohols and ethanol), polyols, polyol ethers, and other ingredients listed in international cosmetic ingredient dictionary and handbook 13 (2009), the entire contents of which are incorporated herein by reference. In certain embodiments, a given component may perform more than one function and may belong to more than one category.

In some embodiments, the compositions described herein may be therapeutic compositions, and the at least one excipient may be a therapeutically acceptable excipient. The therapeutic compositions may be used to treat one or more conditions, such as reducing or preventing scar tissue formation, promoting healing, promoting tissue regeneration, minimizing local inflammation, minimizing tissue rejection, and/or enhancing skin and/or hair graft integration. Therapeutically acceptable excipients are excipients that can be used as vehicles or mediums for the active substance and include excipients commonly used in therapeutic compositions (i.e., compositions useful for treating one or more conditions). Exemplary therapeutically acceptable excipients are described below.

In some embodiments, the compositions described herein may be a dietary composition or dietary supplement, and the at least one excipient may be, for example, a food or beverage additive. A "dietary supplement" is a preparation intended to supplement a diet and can be used to provide nutrients or additives that may be absent from a person's diet or may not be consumed in sufficient amounts. In some embodiments, the dietary supplement may be provided in any of the usual solid or liquid dosage forms for oral administration including, but not limited to, capsules, tablets, pills, powders, granules or powders, soft and hard gelatin capsules and/or as soft gelatin capsules. Suitable excipients include, but are not limited to lactose or milk sugar, high molecular weight polyethylene glycols and the like. Exemplary dietary compositions are described below.

The compositions described herein may also include one or more of the following additional components. Exemplary contemplated additional components are as follows; however, the present disclosure is not limited to these exemplary additional components.

In some embodiments, the compositions described herein may further comprise one or more anti-wrinkling agents. Anti-wrinkle agents are compounds that cause an increase in the synthesis and/or activity of certain enzymes of the skin that reduce the appearance of wrinkles and/or fine lines when the composition is contacted with an area of wrinkled skin (e.g., the body or face, including the eye area). Exemplary anti-wrinkle agents include, but are not limited to, desquamation agents, anti-glycation agents, nitric oxide synthase inhibitors, muscle relaxants and/or anti-skin shrink agents, anti-radical agents, and mixtures thereof.

Additional exemplary anti-wrinkling agents that may be included in the compositions described herein include, but are not limited to, adenosine and its derivatives, retinol and its derivatives (e.g., retinol palmitate), ascorbic acid and its derivatives (e.g., magnesium ascorbyl phosphate and ascorbyl glucoside), tocopherols and its derivatives (e.g., tocopheryl acetate), niacin and its precursors (e.g., niacinamide), ubiquinone, glutathione and its precursors (e.g., L-2-oxothiazolidine-4-carboxylic acid), C-glycoside compounds (also known as C-glycosyl compounds) and its derivatives (e.g., beta-C-xylosyl derivatives under the trade name PRO-XYLANE, plant extracts (e.g., sea fennel extract and olive leaf extract), vegetable proteins and hydrolysates thereof (e.g., rice or soy protein hydrolysates), algae extracts (e.g., kelp algae extract), bacterial extracts, sapogenins (e.g., diosgenin), yam extracts (e.g., wild yam extract), alpha-hydroxy acids, beta-hydroxy acids (e.g., salicylic acid and 5- (n-octanoyl) salicylic acid), oligopeptides and pseudodipeptides and acylated derivatives thereof (e.g., {2- [ acetyl (3- (trifluoromethyl) phenyl) amino ] -3-methylbutyrylamino } acetic acid), lipopeptides (e.g., MATRIXYL 3000 available from Croda)), lycopene, manganese salts, magnesium salts (e.g., gluconate) and combinations of any of the foregoing.

Exemplary adenosine derivatives include, but are not limited to, 2' -deoxyadenosine; 2',3' -isopropylidene adenosine; toyocamycin, 1-methyladenosine; n-6-methyladenosine, adenosine N-oxide, 6-methyl-mercaptopurine nucleoside and 6-chloropurine nucleoside. Other adenosine derivatives include adenosine receptor agonists, including phenylisopropyladenosine ("PIA")/1-methylisoguanosine, ns-Cyclohexyladenosine (CHA), N6-Cyclopentyladenosine (CPA), 2-chloro-Ns-cyclopentyl-adenosine, 2-chloroadenosine, N6-phenyladenosine, 2-phenylaminoadenosine, MECA, ne-phenethyladenosine, 2-p- (2-carboxyethyl) phenethylamino-5 '-N-ethylcarboxamido-adenosine (CGS-21680), (N-ethylcarboxamido) adenosine-S- (NECA), 5' - (N-cyclopropylcarboxamido) adenosine, DPMA (PD 129,944) and adendil (metrifudil).

In some embodiments, the compositions described herein may comprise one or more adenosine derivatives that increase the intracellular concentration of adenosine, such as erythro-9- (2-hydroxy-3-nonyl) adenine ("EHNA"), tubercidin iodide, or a combination thereof. Additional adenosine derivatives contemplated herein include adenosine salts and alkyl esters of adenosine.

In some embodiments, the composition may further comprise one or more pearlescing agents. Pearlizing agents are rainbow particles of any shape, especially produced by certain shellfish in their shells. Alternatively, the pearlescing agent may be synthesized, i.e., made manually. The pearlescent agent may be selected from white pearlescent agents such as, but not limited to, mica covered with titanium oxide or with bismuth oxychloride; colored pearlescing agents such as, but not limited to, bismuth oxychloride-based pearlescing agents; titanium oxide coated mica covered with iron oxide; in particular titanium oxide coated mica covered with ferric blue or chromium oxide; or titanium oxide coated mica covered with an organic pigment.

In some embodiments, the composition may further comprise one or more hydroxy acids. Examples of hydroxy acids include beta hydroxy acids such as salicylic acid, acetylsalicylic acid, and the like. Other exemplary hydroxy acids suitable for use in the composition include citric acid, glycolic acid, hydroxycaproic acid, hydroxycaprylic acid, lactic acid, malic acid, tartaric acid, polyhydroxy acids (including gluconolactone), and any combination thereof.

In some embodiments, the composition may further comprise one or more emulsifiers. The emulsifier prevents the separation of different components (such as oil and water) in the emulsion. Suitable emulsifiers include, but are not limited to: polysorbate, laureth-4, potassium cetyl sulfate, glyceryl caprylate, and any combination thereof.

In some embodiments, the composition may further comprise one or more chelating agents. Chelating agents bind to metal ions and prevent them from chemically reacting with other substances in the formulation. Suitable chelating agents include, but are not limited to: sodium phytate, disodium edetate, tetrasodium glutamate diacetate and trisodium ethylenediamine disuccinate.

In addition to the chelating agent, tetrasodium glutamate diacetate can also be used as a stabilizer in any of the compositions described herein.

In some embodiments, the composition may further comprise one or more antimicrobial agents. Suitable antimicrobial agents include, but are not limited to: octanoyl glyceryl ether, benzalkonium chloride, benzethonium chloride, and chloroxylenol (PCMX), tea tree oil, witch hazel oil, rosemary oil, lemon oil, and any combination thereof.

In some embodiments, the composition may further comprise one or more humectants (water retaining agents) to improve moisture levels in the skin. Non-limiting examples of humectants suitable for use in the compositions described herein are described in WO 98/22085, WO 98/1844 and WO 97/01326 and include: amino acids and derivatives thereof such as proline and arginine aspartate, 1, 3-butanediol, propylene glycol, pentanediol, water, soft-dried algae extract, creatinine, diglycerol, biogel-1, glucosamine salts, glucuronates, glutamate, polyethylene glycol ethers of glycerol (e.g., glycerol polyether 20), glycerol monopropoxylate, glycogen, hexylene glycol, honey, hydrogenated starch hydrolysates, hydrolyzed glycosaminoglycans (such as xanthan gum and biogel-1), inositol, keratin amino acids, glycosaminoglycans, methoxyppeg 10, methyl gluceth-10 and methyl gluceth-20, methyl glucose, 3-methyl-1, 3-butanediol, N-acetylglucosamine salts, polyethylene glycols and derivatives thereof (such as PEG 15 butanediol, PEG 4, PEG 5 pentaerythritol, PEG 6, PEG 8, PEG 9), propylene glycol, pentaerythritol, 1, 2-pentanediol, glycerol ether, 2-pyrrolidone-5-carboxylic acid (including salts and esters thereof), saccharide isomers, sericin, sodium ethyl uric acid, sodium hyaluronate, sodium polygalactote, sodium glutamate, sorbitol, 6, sorbitol, and trisaccharide, and derivatives thereof (such as PEG 15 butanediol, PEG 4, PEG 5 pentaerythritol, PEG 6, PEG 9), and derivatives thereof, such as methyl glucitol, and derivatives thereof.

Other humectants suitable for use herein include polyols selected from the group consisting of: glycerol, diglycerol, glycerol, erythritol, arabitol, xylitol, ribitol, mannitol, sorbitol, galactitol, fucitol, maltitol, mannose, inositol, triethylene glycol, sodium Pyrrolidone Carboxylate (PCA), zinc PCA, and derivatives and mixtures thereof.

In some embodiments, the composition may further comprise a gellant comprising a polyacrylamide-based polymer. The polyacrylamide-based polymer may be a derivative thereof other than polyacrylamide itself, and may be a mixture of various types of polymers, and may also be a copolymer having acrylamide and its derivative as monomers. The gellant is useful for providing a good appearance to the skin, providing a cooling sensation to the skin, and providing a fresh, non-tacky sensation to the skin. In some embodiments, the gelling agent may include one or more pigments, or one or more fillers, including: inorganic pigments (including extender pigments, coloring pigments and whitening pigments), organic pigments, pearlescent pigments, macromolecular powders, functional pigments, talc, mica, kaolin, calcium carbonate, magnesium carbonate, silicic anhydride, aluminum silicate, magnesium silicate, calcium silicate, aluminum oxide, barium sulfate, red iron oxide, yellow iron oxide, black iron oxide, chromium oxide, ultramarine blue, prussian blue, carbon black, zinc oxide, mica titanium, fish scales, bismuth oxychloride, boron nitride, nylon powder, silk powder, carbomers, tar pigments, natural pigments and titanium oxides such as amorphous or rutile and/or anatase crystals.

In some embodiments, the composition may include water and oil resistant pigments and/or fillers, and may also include any conventionally used water and/or oil repellent agents to impart water and oil repellency to the pigment, such as fluorochemicals. Representative fluorine compounds that are conventionally used and that can serve as water and oil repellents include compounds having perfluoroalkyl groups such as perfluoroalkyl phosphates, perfluoroalkyl silanes, perfluoroalkyl silazanes, polyhexafluoropropanes, perfluoroalkyl-containing organosiloxanes, perfluoropolyethers, perfluoroalkyl alcohols, perfluoroalkyl acrylate polymers, and derivatives thereof. The perfluoroalkyl phosphate esters can provide uniform and stable pigment dispersion within the formulation of the gel composition, and the perfluoroalkyl silanes can have excellent compatibility with other cosmetic ingredients. In addition, the perfluoroalkyl phosphate-diethanolamine salts produced by Asahi Glass as Asahi guard AG530 on the market, and perfluoroalkyl silane coupling agents such as LP-IT and LP-4T of Shin-Etsu Silicone, can be used.

Representative gelling agents include, but are not limited to, those marketed by Seppic under the trade names Sepigel 305, sepigel501, and Sepigel 600. Sepigel 305 is a composition containing about 40% polyacrylamide, about 24% of-C ₁₃ -C ₁₄ Isoparaffin and about 6% laureth-7 (wherein laureth-7 is of formula C ₁₂ H ₂₅ --(OCH ₂ CH ₂ ) _n - -OH, wherein n has an average value of 7). Sepigel 600 is a mixture of acrylamide/acrylamide-2-propanesulfonate copolymer, isohexadecane, and polysorbate 80 (polyoxyethylene sorbitan monooleate (20 EO)). Suitable gelling agents comprising polyacrylamide-based polymers are disclosed in EP 0503 853 (Scott Bader Company ltd.), the disclosure of which is incorporated herein by reference.

In some embodiments, the composition may further comprise Hyaluronic Acid (HA). In some embodiments, HA may be in a non-crosslinked state. In some embodiments, the HA may be in a cross-linked state. Like collagen, HA is an important structural component of human tissue. Hyaluronan, also known as Hyaluronic Acid (HA), is a non-sulfated glycosaminoglycan that is widely distributed in connective, epithelial and nervous tissues of the human body. Hyaluronan is abundant in different layers of the skin, where it has a variety of functions, e.g. ensuring good hydration, assisting the organization of the extracellular matrix, acting as a filling material; and participates in the tissue repair mechanism. However, with age, the amount of hyaluronan present in the skin decreases.

In some embodiments, the compositions described herein can be dermal filler compositions, such as injectable dermal filler compositions. Dermal fillers are typically made of collagen and may optionally comprise HA. The dermal filler composition can be suitable for use on the face and body, including, for example, periocular, on or around the cheek, on or around the collar, on or around the hand, on or around the nail, on or around the ear (including on the earlobe), on or around the leg, and on or around the foot.

In some embodiments, the compositions described herein may be used with microneedle arrays, such as arrays included in a patch or patch. The microneedle array may include a plurality of microneedles of sufficient length to penetrate the skin through the stratum corneum and into the active epidermis. In some embodiments, it may be desirable to deliver the polypeptide to the epidermis/dermis junction area for cosmetic or therapeutic purposes.

In some embodiments, the compositions described herein may be used with a microneedle patch or patch. Microneedles and microneedle patches are suitable for delivering collagen into the epidermis and dermis of human skin on the face and body, including, for example, the eyes, cheeks, lips, neckline, and hands. In some embodiments, the microneedles used to deliver the composition in a targeted manner into the epidermis and dermis are injectable microneedles, drug-coated metallic microneedles, or microneedles with dissolvable tips. Exemplary methods and disclosures regarding microneedles can be found in, for example, aditya et al, kinetics of collagen microneedledrug delivery system, journal of Drug Delivery Science and Technology, volume 52, pages 618-623 (month 8 of 2019) and Sun et al, transdermal Delivery of Functional Collagen Via Polyvinylpyrrolidone Microneedles, ann.biomed.eng.,43 (12): 2978-2990 (2015), each of which is incorporated by reference.

In some embodiments, the composition may further comprise a waxy lipid, such as a ceramide. Ceramides help create a barrier against permeability, which helps to prevent dryness and irritation, and may also protect the epidermis from environmental damage.

In some embodiments, the composition may further comprise vitamin a or a vitamin a derivative. Examples of vitamin derivatives include, but are not limited to, retinoids such as retinaldehyde, retinoic acid esters, retinol, retinoic acid, isotretinoin, adapalene, tazarotene, and the like. The term "retinoid" includes both cis and trans derivatives of retinoids (e.g., all-trans retinoic acid, 13-cis retinoic acid, 13-trans retinoic acid, and 9-cis retinoic acid).

In some embodiments, the composition may further comprise vitamin C or a derivative thereof, such as ascorbic acid, ascorbates (e.g., tetrahexyldecyl ascorbate), and the like.

In some embodiments, the composition may further comprise vitamin B, such as biotin (i.e., vitamin B7), nicotinamide, and the like.

In some embodiments, the composition may further comprise vitamin E, such as alpha-tocopherol, beta-tocopherol, gamma-tocopherol, and sigma-tocopherol and their associated corresponding tocotrienols, and the like.

In some embodiments, the composition may further comprise vitamin K and derivatives thereof.

Any vitamin, vitamin analog, or derivative thereof that can be suitably formulated as a topical composition is contemplated for the present disclosure.

In some embodiments, the compositions disclosed herein may further comprise one or more thickening agents. Thickening agents, i.e., structure enhancers, can suspend pigments and/or increase viscosity in the composition. Thickeners and/or structure builders suitable for the compositions of the present invention include, but are not limited to, organically modified clays, fumed silica, trihydroxystearin, silicone gels or elastomers, ammonium acrylodimethyltaurate/VP copolymer, C10-30 alkyl acrylate/crosslinked polymers, and mixtures thereof.

Suitable organically modified clays include, but are not limited to, hectorite, bentonite, smectite and montmorilloniteOrganomodified versions of stone clays (such as those sold under the trade nameThose sold under the trade name Elementis SpecialtiesThose sold from Sud-Chemie and under the trade name +.>Those sold from Southern Clay Products). Hydrophilic modified fumed silica includes, but is not limited to, WACKER- >N20 and T30 grades (Wacker-Chemie AG) under the trade name +.>(Evonik) hydrophilic stage. Silicone gels or silicone elastomers include, but are not limited to, "KSG" thickening series (KSG-15, KSG-16, KSG-18, KSG-41, KSG-42, KSG-43, KSG-44) from Shin-Etsu Silicones, DOW>9040. 9041, 9045 and 9546 silicone elastomer blends, SFE839 from Momentive Performance Materials ^TM And Velvesil ^TM Silicone gel, from Wacker-Chemie AG +.>RG-100。

In some embodiments, the compositions disclosed herein may further comprise one or more liposoluble/lipodispersible film-forming agents. Liposoluble/lipid-dispersible film formers suitable for use herein include, but are not limited to, organosilicone resins (e.g., trimethylsiloxysilicate, such as SRI 000 from GE Silicones) and copolymers of organosilicone resins (e.g., diisostearyl trimethylol propane monosilane)Oxysilicates such as SF1318 from GE Silicones); a fluorinated silicone resin; acrylic and/or vinyl-based polymers or copolymers, including Silicones and/or fluorinated versions (e.g., silicone acrylates of the "KP" series from Shin-Etsu Silicones, and 3M ^TM Silicone "Plus" polymers VS70 and SA 70); polyurethanes (e.g., hydroxy ester triglyceride derived Poly from Alzo International)A series); polyesters (e.g.. From Inolex Chemical Company +.>A series of polymeric polyesters); and mixtures thereof.

In some embodiments, the compositions disclosed herein may further comprise one or more colorants. Colorants suitable for use herein include all inorganic and organic pigments/dyes, including mineral or pearlescent pigments suitable for use in cosmetic compositions. Such colorants include those with or without a surface coating or treatment. The colorant may enhance the coloring and/or light scattering and/or light reflecting effects of the composition.

In some embodiments, the compositions disclosed herein may further comprise one or more sunscreens, such as mineral and/or physical sunscreens. Sunscreens may block UVA and/or UVB radiation. Exemplary UVA sunscreens include, but are not limited to, avobenzone (avobenzone), terephthalidenediocamphorsulfonic acid, disodium bissulfenyl, disodium bezimidazole tetrasulfonate, diethylamino hydroxybenzoyl hexyl benzoate, bisdiethylamino hydroxybenzoyl benzoate, bisbenzoxazolyl phenyl ethyl hexyl amino triazine, and combinations thereof.

Exemplary UVB sunscreens include, but are not limited to, octocrylene, oxacinoate, xin Shuiyang ester, homosalate, enrolment Li, ethylhexyl triazone, enzacamine, al Mi Luozhi (amioxate), diethylhexyl butyramidotriazine, benzylidene malonate polysiloxane, pamoate-O, triethanolamine salicylate, cinoxate (cinoxate), para-aminobenzoic acid, and derivatives thereof, and combinations thereof.

Exemplary sunscreens that absorb UVA and UVB radiation are, for example, oxybenzone (oxybenzone), meradite, titanium dioxide, zinc oxide, bis-octotriazole, bei Moqu octyl (bemtrilinol), cresol trisiloxane (drometrizole trisiloxane), shu Liben ketone (sulibenzone), dihydroxybenzone (dioxybenzone), or combinations thereof.

Specific suitable sunscreens include, but are not limited to, p-aminobenzoic acid, salts and derivatives thereof (ethyl, isobutyl, glyceride, p-dimethylaminobenzoic acid, anthranilate (i.e., O-aminobenzoate, methyl ester, menthyl ester, phenyl ester, benzyl ester, phenethyl ester, linalyl ester, terpinyl ester and cyclohexenyl ester), salicylates (amyl ester, phenyl ester, benzyl ester, menthyl ester, glyceride and dipropylene glycol ester), cinnamic acid derivatives (methyl ester and benzyl ester, alpha-phenylcinnamonitrile, butylcinnamoyl pyruvate), dihydroxycinnamic acid derivatives (umbelliferone, methylumbelliferone, methylacetyl-umbelliferone), trihydroxycinnamic acid derivatives (esculetin, methylesculetin, daphnetin and glucoside, esculin and daphnoside), hydrocarbons (diphenylbutadiene, stilbene), and the like dibenzylideneacetone and benzylidene acetophenone, naphthol sulfonates (sodium salts of 2-naphthol-3, 3-disulfonic acid and 2-naphthol-6, 8-disulfonic acid), ci hydroxynaphthoic acid and salts thereof, o-hydroxybiphenyl disulfonate and p-hydroxybiphenyl disulfonate, coumarin derivatives (7-hydroxycoumarin, 7-methylcoumarin, 3-phenylcoumarin), diazoles (2-acetyl-3-bromoindazole, phenylbenzoxazole, methylnaphthoxazole, various arylbenzothiazoles), quinines (bisulfate, sulfate, chloride, oleate and tannate), quinoline derivatives (8-hydroxyquinoline salt, 2-phenylquinoline), benzoquinone derivatives (2-acetyl-3-bromoindazole), hydroxy or methoxy substituted benzophenones, uric acid and valicacid (e.g., hexaethyl ether), tannic acid and derivatives thereof (e.g., hexaethyl ether), (butyl carbitol) (6-propylpiperonyl) ether, hydroquinone, benzophenone (hydroxyphenyl, sulfoisophenone, dihydroxybenzophenone, benzoylresorcinol, 2, 4' -tetrahydroxybenzophenone, 2' -dihydroxy-4, 4' -dimethoxybenzophenone, ostaphenone, 4-isopropyldibenzoylmethane, butyl methoxydibenzoylmethane, etoricene (etocerene) and 4-isopropyl-dibenzoylmethane, titanium dioxide, iron oxide, zinc oxide and mixtures thereof other cosmetically acceptable sunscreens and concentrations (weight percent of total cosmetic sunscreens compositions) include diethanolamine methoxycinnamate (10% or less), ethyl-bis (hydroxypropyl) aminobenzoate (5% or less), aminobenzoate (3% or less), 4-isopropyldibenzoylmethane (5% or less), 4-methylbenzylidene (6% or less), camphor, p-dibenzenesulfonate (10% or less) and optionally also referred to herein as salts of D-aspartic acid, 10% or less, and combinations thereof, the term "derivatives" of D-aspartic acid and D-alanine refers to D-aspartic acid and D-alanine molecules covalently bound to any organic group through their amino, carboxyl or side chains, provided that the effect of D-aspartic acid and D-alanine on promotion of collagen production is not impaired. Exemplary organic groups include, but are not limited to, protecting groups such as N-phenylacetyl and 4,4' -Dimethoxytrityl (DMT) groups; biopolymers such as proteins, peptides, sugars, lipids and nucleic acids; synthetic polymers such as polystyrene, polyethylene (polyethylene), polyethylene (polyvinyl), polypropylene, and polyester; and functional groups such as ester groups. The ester groups may include, for example, aliphatic esters such as methyl and ethyl esters; and aromatic esters.

In some embodiments, the composition may further comprise one or more general purpose skin care additives, for example conditioning agents such as silicones. In some embodiments, the composition may further comprise one or more shark liver oils, such as squalane and/or squalene. In some embodiments, the composition may further comprise one or more polysaccharides produced by microalgae, e.g., alginic acid.

In some embodiments, the composition may further comprise at least one preservative. In some embodiments, the at least one preservative may be a quaternary ammonium compound, a halogenated phenol, sorbic acid, potassium sorbate, benzoic acid, sodium benzoate, sodium citrate, sodium anisole, octanoyl hydroxamic acid, sodium levulinate, phenoxyethanol, or a combination thereof.

The pH of the compositions described herein may range from about 4 to about 8, about 4.7 to about 5.5, about 5 to about 7, about 6 to about 7, about 6.1 to about 6.8, or about 6.4 to about 6.6.

Soap

In some embodiments, the cosmetic compositions described herein may be cleansing compositions or soaps, including conventional soaps and cleansers in the form of solid bars, cleansing bars, body washes, emulsions, creams, foam cream gels, or liquid soaps in the form of gels that may be packaged in tubes, bottles, pump bottles, aerosol shower foam, or foam pump bottles. Soaps are useful in cosmetic methods for cleaning the residue of dirt of human keratinous materials in the presence of water, collecting to form foam, and the foam and residue of dirt formed are removed by rinsing with water, and are useful on any part of the body and face disclosed herein, including, for example, body skin, face, hands, lips, eyelids, nails, hair, eyelashes, and/or eyebrows.

Traditionally, solid soaps include alkali metal fatty acid salts and fatty acid potassium soaps, and liquid soaps include detergent formulations of four major families: (1) a lauryl sulfate-based detergent formulation; detergent formulations based on alpha-olefin sulfonates; (3) Detergent formulations based on a mixture of synthetic anionic, amphoteric and/or nonionic surfactants; (4) Based on a mixed formulation of soap and synthetic surfactant. Liquid soaps typically contain a thickening system selected from, for example, electrolytes such as sodium chloride, potassium chloride or potassium sulfate; alkanolamides such as cocamide DEA or cocamide MEA; esters of polyethylene glycol with a monobasic acid or stearic acid, such as polyethylene glycol distearate 6000 or mixtures thereof, and the liquid soap is contained in a cosmetically acceptable aqueous medium. However, both solid and liquid soaps may contain any suitable additional ingredients, such as those listed below, in any combination.

In some embodiments, the cleaning compositions or soaps described herein may further comprise: one or more cellulose compounds, or polysaccharide compounds having in their structure chains of glucose residues linked by β -1,4 bonds; one or more fatty acids comprising a linear or branched, saturated or unsaturated alkyl chain having from 6 to 30 carbon atoms or from 12 to 22 carbon atoms; one or more fatty acids including lauric, myristic, palmitic and stearic acid, linolenic acid and mixtures thereof; and one or more inorganic bases including alkali metal hydroxides (sodium hydroxide and potassium hydroxide), metal hydroxides, or ammonia, or organic bases such as triethanolamine, monoethanolamine, monoisopropanolamine, N-methylglucamine, lysine, and arginine.

In some embodiments, the compositions described herein may further comprise one or more anionic surfactants or salts, including alkali metal salts such as sodium salts, ammonium salts, amine salts, amino alkoxides, or salts of alkaline earth metals (e.g., magnesium) of the following types: alkyl sulfate, alkyl ether sulfate, alkyl amido ether sulfate, alkylaryl polyether sulfate, monoglyceride sulfate; alkyl sulfonates, alkylamide sulfonates, alkylaryl sulfonates, alpha-olefin-sulfonates, paraffin-sulfonates; alkyl sulfosuccinate, alkyl ether sulfosuccinate, alkyl amide sulfosuccinate; alkyl sulfoacetates; acyl sarcosinates; and acyl glutamates, the alkyl and acyl groups of all these compounds having 6 to 24 carbon atoms and the aryl group representing phenyl or benzyl; c of polyglycoside Carboxylic acids ₆ -C ₂₄ Alkyl esters such as alkyl glucosides citrate, alkyl polyglucosides tartrate and alkyl polyglucosides sulfosuccinate, alkyl sulfosuccinates, acyl isethionates and N-acyl taurates, the alkyl or acyl groups of all of these compounds having from 12 to 20 carbon atoms; and/or acyl lactylates having 8 to 20 carbon atoms in the acyl group and mixtures thereof. In some embodiments, alkyl-D-galactoside uronic acid, polyoxyalkylene (C ₆ -C ₂₄ ) Ether carboxylic acid, polyoxyalkylene (C) ₆ -C ₂₄ ) Aryl (C) ₆ -C ₂₄ ) Polyoxyalkylene ether carboxylic acid, polyoxyalkylene (C) ₆ -C ₂₄ ) Alkylamidoether carboxylic acids, especially those containing from 2 to 50 ethylene oxide groups; and their alkali metal, ammonium, amine, amino alkoxide or alkaline earth metal salts may also be suitable.

Suitable C containing 1 to 30 ethylene oxide groups ₆ -C ₂₄ Alkyl ether sulfates include alkali metal or alkaline earth metal salts, ammonium salts, amine salts or amino alkoxides, sodium salts and oxyethylenated (C) ₁₂ -C ₁₄ ) Alkyl ether sulfates having an average number of ethylene oxide groups of 1 to 4 and comprising sodium laureth sulfate (CTFA name), such as the commercial product sold by cog produced under the name texpon AOS225UP texpon N702 texpon NSW or the commercial product sold by Huntsman company under the names emepicol ESB3/FL2, emepicol ESB3/FL3, emepicol ESB70/FL 2.

Suitable amphoteric surfactants include, but are not limited to, derivatives of aliphatic secondary or tertiary amines in which the aliphatic radical is a straight or branched chain containing from 8 to 22 carbon atoms. The amphoteric surfactant may contain at least one water-soluble anionic group such as carboxylate, sulfonate, sulfate, phosphate or phosphonate groups, (C) ₈ -C ₂₀ ) Alkyl betaines, sulfobetaines, (C) ₈ -C ₂₀ ) Alkylamido (C) ₆ -C ₈ ) Alkyl betaines or (C) ₈ -C ₂₀ ) Alkylamidoalkyl (C) ₆ -C ₈ ) Sulfobetaines, and mixtures thereof.

Suitable amine derivatives include those described by the namesThe products sold, as described in U.S. Pat. No. 2,528,378 and U.S. Pat. No. 2,781,354, are filed in the CTFA dictionary, 1982, 3 rd edition, under the nomenclature amphoglycinate and amphoglycinate. Other suitable amine derivatives include those concentrated by Rhodia under the trade nameThose amines sold by C2M classified under the following names in CTFA dictionary, 5 th edition 1993Derivatives: cocoyl amphodiacetate disodium salt, lauroyl amphodiacetate disodium salt, decanoyl amphodiacetate disodium salt, octanoyl amphodiacetate disodium salt, cocoyl amphodipropionic acid disodium salt, lauroyl amphodipropionic acid disodium salt, decanoyl amphodipropionic acid disodium salt, octanoyl amphodipropionic acid disodium salt, lauroyl amphodipropionic acid, cocoyl amphodipropionic acid and cocoyl amphodiacetate. Suitable alkyl (C) ₈ -C ₂₀ ) Betaines include cocoamidopropyl betaine and coco betaine such as commercial MIRATAINE BB/FLA from RHODIA or commercial EMPIGEN BB/FL from Huntsman.

In some embodiments, the cleaning compositions or soaps described herein may be sulfate-free and may include a sulfate-free surfactant system.

In some embodiments, the cleaning compositions or soaps described herein may also contain one or more thickeners of the nonionic cellulose compound type. Suitable cellulose compounds include, but are not limited to, nonionic cellulose ethers, including methyl cellulose and ethyl cellulose; hydroxyalkyl celluloses such as hydroxymethyl cellulose, hydroxyethyl cellulose, and hydroxypropyl cellulose; mixed hydroxyalkyl-alkyl cellulose such as hydroxypropyl-methyl cellulose, hydroxyethyl ethyl cellulose and hydroxybutyl-methyl cellulose, and alkyl chain modified hydroxyalkyl cellulose. Suitable hydroxypropyl methylcellulose includes commercial METHOCEL E, F, J and K sold by Dow corning, even more particularly METHOCEL E4 MQG or METHOCEL F4M. Suitable cellulosic components may be in crystalline form, microcrystalline form or mixtures thereof.

In some embodiments, the cleaning compositions or soaps described herein may further comprise one or more additional thickeners, including electrolytes such as sodium chloride, potassium chloride, or potassium sulfate; alkanolamides such as cocamide DEA or cocamide MEA; esters of polyethylene glycol and mono-or stearic acid such as polyethylene glycol distearate 6000 or mixtures thereof; polysaccharide biopolymers such as xanthan gum, guar gum, alginates; synthetic polymers such as polyacrylic acids such as CARBOPOL 980, CARBOPOL 1382, commercially available from NOVEON, acrylate/acrylonitrile copolymers such as HYPAN SS201, commercially available from KINGSTON; clays such as smectites, modified or unmodified hectorites such as Rheox produce the marketed BENTONE product, southern Clay Products produce the marketed LAPONITE product, RT Vanderbilt produces the marketed VEEGUM HS product, and mixtures thereof.

In some embodiments, the cleaning compositions or soaps described herein may also contain one or more nonionic surfactants. These are known compounds (see MR Porter "Handbook of Surfactants", blackie)&Son edit (Glasgow and London), 1991, pages 116-178) and may be selected from alcohols, alpha-diols, (C) ₁ -C ₂₀ ) Alkylphenols or polyethoxylated, polypropoxylated or polyglycerolated fatty acids having a fatty chain comprising, for example, 8 to 18 atoms, the number of ethylene oxide groups or propylene oxide may be in the range of 2 to 50 and the number of glycerol groups may be in the range of 2 to 30; copolymers of ethylene oxide and propylene; condensates of ethylene oxide and propylene oxide on fatty alcohols; a polyethoxylated fatty amide having from 2 to 30 moles of ethylene oxide, a polyglycerolated fatty amide comprising an average of from 1 to 5 glycerol groups; a polyethoxylated fatty amine having from 2 to 30 moles of ethylene oxide, an ethoxylated sorbitan fatty acid ester having from 2 to 30 moles of ethylene oxide; sucrose fatty acid ester, polyethylene glycol fatty acid ester, (C6-C24) alkyl polyglycoside, N-alkyl (C ₆ -C ₂₄ ) Glucosamine derivatives, amine oxides, such as alkyl (C) ₁₀ -C ₁₄ ) Oxides or N-acyl groups of amines (C ₁₀ -C ₁₄ ) -aminopropylmorpholine oxides, and mixtures thereof.

Other suitable nonionic surfactants include, but are not limited to, alkyl Polyglucosides (APGs), maltitol esters, polyglycerolated fatty alcohols, glucosamine derivatives (e.g., 2-ethylhexyloxycarbonyl-N-methylglucamine), and mixtures thereof. Suitable alkyl polyglucosides include those containing an alkyl group containing 6 to 30 carbon atoms and a hydrophilic group (glucoside). Exemplary alkyl polyglucoside packagesIncludes decyl glucoside (alkyl-C9/C11-polyglucoside (1.4)), including Mydol, which is known by the name of Kao ChemicalsProducts sold under the name Plantaren2000>Products sold and under the name Oramix NS +.>A product for sale; and octyl/decyl glucoside, including those sold under the name Oramix CG by SEPPIC Corp->A product for sale; plant 1200 +.>And plant->Sold lauryl glucoside, and coco glucoside, e.g. by Cognis under the name plant +.>And (5) selling the product.

Suitable maltose derivatives include those described in document EP-A-566 438, such as 0-octanoyl-6 '-D-maltose or O-dodecanoyl-6' -D-maltose described in document FR-2 739 556.

In some embodiments, the cleansing composition or soap may be formulated in a cosmetically acceptable aqueous medium. In addition to water, suitable cosmetically acceptable aqueous media may include one or more solvents such as lower alcohols containing 1 to 6 carbon atoms, such as ethanol; polyols such as glycerol; diols such as butanediol, isopentyl glycol, propylene glycol, polyethylene glycols (such as PEG-8), sorbitol; sugars such as glucose, fructose, maltose, lactose, sucrose, and mixtures thereof. The amount of solvent in the compositions disclosed herein may range from 0.1 wt% to 95 wt%.

In some embodiments, the cleaning compositions or soaps described herein may also comprise one or more cationic polymers of the polyquaternium type, which may provide softness and lubricity to the foaming composition. Suitable cationic polymers include polyquaternium 5 such as the product MERQUAT 5 produced by CALGON, polyquaternium 6 such as the product SALCARE SC produced by CIBA and the product MERQUAT 100 produced by CALGON, polyquaternium 7 such as the product MERQUAT S, MERQUAT 2200 and MERQUAT 550 produced by CALGON, and the product SALCARE SC 10 produced by CIBA, polyquaternium 10 such as the product Polymer JR400 produced by Amerchol, polyquaternium 11 such as the product GAFQUAT 755, GAFQUAT 755N and GAFQUAT 734 produced by ISP, polyquaternium 15 such as the product ROHM product ROHAGIT KF 720F produced by CALGON, polyquaternium 16 such as the product LUVIQUAT 905 produced by BASF, LUQUAT 370, LUFC 552 and VIQUAT 550 produced by CALGON, polyquaternium 22 such as the product 280 produced by CAGON, polyquaternium 10 such as the product 60 produced by CAROHM, and polyquaternium 40 such as the product 60 produced by BAOCR 46 such as the product 60 produced by BAOCR, and polyquaternium 39 such as the product 60 produced by BASION, and polyquaternium 15 such as the product 60 such as the product RQUAT produced by CAQUS.

In some embodiments, the cleansing compositions or soaps described herein may further comprise one or more adjuvants or additives for use in cosmetic compositions. Suitable adjuvants or additives include, but are not limited to: oils, actives, fragrances, preservatives, chelating agents, pearlescers or sunscreens, pigments, pearlescers, inorganic or organic fillers such as talc, kaolin, silica powder or polyethylene, soluble dyes or any combination thereof.

Examples of oils include vegetable oils (jojoba, avocado, sesame, sunflower, corn, soybean, safflower, grape seed oils), mineral oils (petrolatum, optionally hydrogenated isoparaffins), synthetic oils (isopropyl myristate, cetostearyl octanoate, polyisobutylene, ethylhexyl palmitate, alkyl benzoate), volatile or non-volatile silicone oils such as Polydimethylsiloxane (PDMS) and cyclomethicone or cyclomethicone, and fluorinated or fluorosilicone oils and mixtures thereof.

Exemplary active agents include sunscreens, desquamation agents, moisturizers, depigmenting agents, pro-coloring agents, alpha-hydroxy acids, antibacterial agents, anti-radical agents, anti-fouling agents, anti-inflammatory agents, retinoids, extracts of algae, mushrooms, vegetables, yeasts, bacteria, hydrolyzed proteins, partially hydrolyzed or unhydrolyzed proteins, enzymes, hormones, vitamins and their derivatives, flavonoids and isoflavones, and mixtures thereof.

The cleaning compositions or soaps described herein may have a pH in the range of 6 to 10, depending on the application chosen. The pH may be adjusted to the desired value conventionally by adding a base (organic or inorganic), for example ammonia or a primary, secondary or tertiary (poly) amine, such as monoethanolamine, diethanolamine, triethanolamine, isopropanolamine or 1, 3-propanediamine, to the composition, or by adding an inorganic or organic acid, such as a carboxylic acid, for example citric acid. In the case of shower gels, the pH can vary from 8 to 10.

In some embodiments, the cleaning compositions described herein may further comprise optional additives such as colorants, fragrances, antibacterial agents, preservatives, antioxidants, beads (fragrance, exfoliating or moisturizing beads), mica, glitter, shea butter beads, opacifiers, pearlescers, and other such ingredients. In some embodiments, the composition has a high transparency (about 2NTU to about 25 NTU), a target viscosity in the range of about 1/4 to about 1/8 that is easily dispensed from the orifice (about 4,000 centipoise to about 10,000 centipoise), and a yield value (about 3 pascals to about 15 pascals) that allows the composition to suspend multiple additives with uniform distribution and enhanced stability (e.g., about 8 months at 120°f (49 ℃)), and any mixtures thereof.

In some embodiments, the cleansing composition or soap may further comprise one or more moisturizers/emollients. Moisturizers may be included in bar or liquid soap compositions to provide conditioning benefits to the skin. The term "moisturizer" describes a material that imparts a smooth and soft feel to the skin surface.

There are two ways to reduce the loss of water from the stratum corneum. One is to deposit an occlusive layer on the skin surface that reduces the evaporation rate. The second approach is to add a non-occlusive hygroscopic substance to the stratum corneum which will retain water and make it available to the stratum corneum to alter its physical properties and produce the cosmetically desirable effect. Non-occlusive moisturizers also work by improving the lubricity of the skin.

Both occlusive and non-occlusive moisturizers are contemplated for use in the compositions described herein. Exemplary moisturizers include long chain fatty acids, liquid water-soluble polyols, glycerin, propylene glycol, sorbitol, polyethylene glycol, ethoxylated/propoxylated ethers of methyl glucose (e.g., methyl glucitol polyether-20), ethoxylated/propoxylated ethers of lanolin alcohol (e.g., available from Amerchol co.) ) Coconut and tallow fatty acids, liquid water-soluble polyols (e.g., glycerol, propylene glycol, butylene glycol, hexylene glycol, polypropylene glycol, and polyethylene glycol).

Non-occlusive moisturizers can naturally occur in the stratum corneum of the skin, such as sodium pyrrolidone carboxylate, lactic acid, urea, L-proline, guanidine, and pyrrolidone. Examples of other non-occlusive moisturizers include cetyl esters, myristyl, isodecyl or isopropyl esters of adipic, lactic, oleic, stearic, isostearic, myristic or linoleic acid, as well as many of their corresponding alcohol esters (sodium isostearyl-2-lactate, sodium octyllactate), hydrolyzed and other collagen-derived proteins, aloe vera gel and acetamide MEA (N-acetyl ethanolamine). Other examples of occlusive and non-occlusive types of moisturizers are disclosed in "Emollients-A Critical Evaluation", cosmetics & tools, j.mausner, 1981, incorporated herein by reference.

Exemplary occlusive moisturizers include petrolatum, mineral oil, beeswax, silicone, lanolin and oil-soluble lanolin derivatives, saturated and unsaturated fatty alcohols such as behenyl alcohol, squalene and squalane, and various animal and vegetable oils such as almond oil, peanut oil, wheat germ oil, linseed oil, jojoba oil, apricot kernel oil, walnut oil, palm nut oil, pistachio oil, sesame oil, rapeseed oil, juniper oil, corn oil, peach kernel oil, poppy seed oil, pine oil, castor oil, soybean oil, avocado oil, sunflower oil, coconut oil, hazelnut oil, olive oil, grape seed oil and sunflower seed oil.

Hair care

In some embodiments, the composition is a hair care composition comprising ingredients commonly used in hair care products. These ingredients may include, but are not limited to, cleaners, lathering agents, hydration agents, surfactants, detergents, gelling agents, fragrances, plant extracts, conditioning agents, humectants, silicones or silicone derivatives, thickening agents, sunscreens, vitamins, alcohols, polyols, polyol ethers, and other commonly used ingredients in shampoos, conditioners, and styling agents. Hair care products typically include one or more surfactants, one or more viscosity modifiers, one or more preservatives, and one or more fragrances, as well as any of the ingredients listed below or combinations thereof.

In general, hair can be damaged and friable by external atmospheric factors such as the effects of light and bad weather, as well as by mechanical or chemical treatments such as brushing, combing, dyeing, bleaching, perming, and/or relaxing, and over time, hair can become dry, rough, dull, and/or friable. To overcome these drawbacks, it is common practice to use a care composition that properly conditions the hair, thereby imparting satisfactory cosmetic properties thereto, in particular in terms of smoothness, gloss, softness, flexibility, brightness, natural feel and good detangling properties. These hair care compositions may be, for example, shampoos, conditioning shampoos, hair conditioners, hair films, lotions, gels, shampoos and creams, which may be rinsed off or left in the composition. In various embodiments, these compositions generally comprise a combination of cationic conditioning agents such as cationic surfactants, cationic polymers, silicones, and/or fatty substances such as fatty alcohols in order to impart satisfactory properties to the hair in terms of softness, smoothness and flexibility. Exemplary compositions may comprise silicones, which are known to improve the cosmetic properties of hair in terms of smoothness and flexibility (as shown in U.S. patent No. 5,374,421, each incorporated herein by reference).

In some embodiments, the hair care composition may further comprise one or more non-amino polyalkylsiloxanes, one or more oxyethylenated polymers in the presence of fatty alcohols, one or more non-amino polyalkylsiloxanes comprising at least one alkyl chain having at least 12 carbon atoms, one or more oxyethylenated polymers, and/or one or more fatty alcohols. Exemplary oxyethylenated polymers may have a molecular weight of greater than or equal to 10 ⁶ Is a weight average molecular weight of (2). In some embodiments, the oxyethylenated polymer may be selected from those having the formula H (OCH) ₂ CH ₂ ) _z OH, wherein z is an integer greater than or equal to 30,000. In certain embodiments, z may range from 30,000 to 120,000, or from 40,000 to 95,000. In some embodiments, the oxyethylenated polymer may be PEG-45M (z=45,000), such as the product sold by Amerchol corporation under the name Polyox WSR N60K, and PEG-90M (z=90 000), and mixtures thereof.

In some embodiments, the hair care composition may further comprise one or more fatty alcohols. The term "fatty alcohol" means any saturated or unsaturated, straight or branched chain alcohol containing at least 8 carbon atoms and not being oxyalkylated. Exemplary fatty alcohols are at room temperature (25 ℃) and atmospheric pressure (1.013 x 10 ₅ Pa) is solid. Exemplary fatty alcohols include cetyl alcohol, stearyl alcohol, oleyl alcohol, behenyl alcohol, linoleyl alcohol, palmitoleic alcohol, arachidonic alcohol, erucyl alcohol, cetostearyl alcohol (or cetostearyl alcohol)Alcohols) and mixtures thereof.

In some embodiments, the hair care composition may further comprise one or more conditioning agents, including cationic surfactants, cationic polymers, and mixtures thereof. The term "cationic surfactant" means a surfactant that is positively charged when included in the compositions described herein. Suitable cationic surfactants may be selected from the group consisting of optionally polyoxyalkylated primary, secondary or tertiary fatty amines or salts and quaternary ammonium salts thereof, and mixtures thereof. An exemplary fatty amine is stearamidopropyl dimethylamine. Exemplary quaternary ammonium salts include tetraalkylammonium salts including dialkyldimethylammonium salts or alkyltrimethylammonium salts wherein the alkyl group contains about 16 to 22 carbon atoms, particularly behenyl trimethylammonium, distearyl dimethylammonium, cetyl trimethylammonium, or benzyl dimethyl stearyl ammonium salt, or alternatively, palmitoylamide propyltrimethylammonium salt, stearamidopropyl trimethylammonium salt, stearamidopropyl dimethylcetylstearyl ammonium salt, or stearamidopropyl dimethyl (myristylacetic acid) ammonium salt sold under the name CERAPHOL 70 by Van Dyk.

The term "cationic polymer" means any polymer containing cationic groups and/or groups that can be ionized into cationic groups, which may be non-siliceous. Exemplary cationic polymers include any known polymer for styling hair, such as those described in patent application EP-A-0337 354 and French patent applications FR-A-2 270 846, 2 383 660, 2 598 611, 2 470 596 and 2 519 863, each of which is incorporated by reference in its entirety. Additional exemplary cationic polymers include those containing units comprising primary, secondary, tertiary and/or quaternary amine groups, which may form part of the polymer backbone or may be carried by pendant substituents directly attached thereto. Suitable cationic polymers may have a cationic polymer chain length of greater than 10 ⁵ Polymers of the type including polyamines, polyaminoamides and polyquaterniums, including those described in French patent 2 505 348 and 2 542 997, each of which is incorporated by referenceThe manner in which these are incorporated herein in their entirety.

In some embodiments, the hair care composition may further comprise a "non-sulfate" cleanser, a foaming agent, or a surfactant. Suitable "non-sulfate" agents include, but are not limited to: sodium lauroyl methyl isethionate propylene glycol, sodium methyl oleoyl taurate and sodium cocoyl isethionate.

In some embodiments, the hair care composition may further comprise any of the following ingredients or mixtures thereof: quaternary ammonium polymers of quaternary ammonium compounds, vinylpyrrolidone and/or vinylimidazole synthesized from rapeseed, such as the products sold by BASF company under the names LUVIQUAT FC 905, FC 550 and FC 370 and LUVIQUAT Excellence; cationic polysaccharides, including cationic celluloses, including cellulose ether derivatives containing quaternary ammonium groups, cationic cellulose copolymers or cellulose derivatives grafted with water-soluble quaternary ammonium monomers, and cationic galactomannan gums. An exemplary cellulose ether derivative comprising quaternary ammonium groups is described in french patent 1 492 597. These polymers are also defined in the CTFA dictionary as quaternary amines of hydroxyethyl cellulose that have been reacted with epoxides substituted with trimethylammonium groups. Cationic cellulose copolymers or cellulose derivatives grafted with water-soluble quaternary ammonium monomers are described in U.S. patent No. 4,131,576, such as hydroxyalkyl cellulose, e.g. hydroxymethyl cellulose, hydroxyethyl cellulose or hydroxypropyl cellulose, grafted with, inter alia, methacryloylethyl trimethyl ammonium, methacrylamidopropyl trimethyl ammonium or dimethyldiallyl ammonium salts. Suitable related celluloses are e.g. with C ₈ -C ₃₀ Fatty chain quaternized alkylhydroxyethyl cellulose such as the product QUATRISOFT LM 200 (INCI name Polyquaternium-24) sold by Amerchol/Dow Chemical company and the product CRODACEL QM (INCI name PG-hydroxyethyl cellulose cocodimethyl ammonium chloride), CRODACEL QL (C) ₁₂ Alkyl) (INCI name PG-hydroxyethyl cellulose lauryl dimethyl ammonium chloride) and CRODACEL QS (C) ₁₈ Alkyl) (INCI name PG-hydroxyethyl cellulose stearyl dimethyl ammonium chloride). Other suitable fatty chain hydroxyethylcellulose derivatives include those from Amerchol +.Commercial products SOFTCAT Polymer SL from Dow chemical company, such as SL-100, SL-60, SL-30 and SL-5 under INCI name Polyquaternium-67. Suitable cationic galactomannan gums are described in U.S. patent nos. 3,589,578 and 4 031 307. Suitable cellulosic components may be in crystalline form, microcrystalline form or mixtures thereof.

In some embodiments, the hair care composition may further comprise one or more cationic proteins or cationic protein hydrolysates, polyalkyleneimines (including polyethyleneimines), polymers containing vinylpyridine or vinylpyridinium units, polyamine and epichlorohydrin condensates, ji Duoya urea and chitin derivatives, animal protein hydrolysates with trimethylbenzyl ammonium groups (such as the product sold by Croda company under the name Crotein BTA and known as benzyltrimethyl ammonium hydrolyses animal proteins in CTFA dictionaries), protein hydrolysates with quaternary ammonium groups on the polypeptide chain, said ammonium groups comprising at least one alkyl group having from 1 to 18 carbon atoms.

In some embodiments, the hair care composition may further comprise one or more quaternized vegetable proteins, such as those from wheat, corn or soy proteins, for example quaternized wheat proteins, including those sold by Croda corporation under the designation hydroritum WQ or QM known as coco dimethyl ammonium hydrolyzed wheat proteins in the CTFA dictionary under the designation hydroritum QL known as lauryl dimethyl ammonium hydrolyzed wheat proteins in the CTFA dictionary or other sold under the designation hydroritum QS known as stearyl dimethyl ammonium hydrolyzed wheat proteins in the CTFA dictionary.

In some embodiments, the hair care composition may further comprise one or more polyamines, such as POLYQUART R H, sold by Cognis, under the name polyethylene glycol tallow based polyamine in the CTFA dictionary. Other suitable polymers include those sold by BASF under the name Lupamin, as well as products sold under the names Lupamin 9095, lupamin 5095, lupamin 1095, lupamin 9030, and Lupamin 9010.

In some embodiments, the hair care composition may further comprise one or more ofSeed was grown at room temperature (25 ℃) and atmospheric pressure (1.013.10 ⁵ Pa) a fatty substance that is liquid. The term "fatty substance" means a fatty substance at normal temperature (25 ℃) and atmospheric pressure (1.013 x 10 ⁵ Pa) (solubility less than 5%, less than 1% or less than 0.1%). The fatty material is typically soluble in an organic solvent, such as chloroform, methylene chloride, carbon tetrachloride, ethanol, benzene, toluene, tetrahydrofuran (THF), liquid petrolatum or decamethyl cyclopentasiloxane, under the same conditions of temperature and pressure. The liquid fatty substances of the present disclosure may be non-polyoxyethylenated and non-polyglycerolated. The term "oil" means a mixture of oil at room temperature (25 ℃) and atmospheric pressure (1.013 x 10 ⁵ Pa) is a liquid "fatty substance". The term "non-silicone oil" means an oil that does not contain any silicon atoms (Si) and the term "silicone oil" means an oil that contains at least one silicon atom. The liquid fatty substance may be selected from non-silicone oils such as, in particular, C ₆ -C ₁₆ Liquid hydrocarbons, liquid hydrocarbons containing more than 16 carbon atoms, non-silicone oils of animal origin, triglycerides of vegetable or synthetic origin, fluorooils, liquid fatty acids and/or fatty alcohol esters other than triglycerides, and mixtures thereof. The liquid hydrocarbon may be linear, branched or optionally cyclic and comprises hexane, cyclohexane, undecane, dodecane, tridecane or isoparaffin, such as isohexadecane, isodecane or isododecane, and mixtures thereof. Suitable straight or branched chain liquid hydrocarbons of mineral or synthetic origin containing more than 16 carbon atoms may be selected from liquid paraffin, petrolatum, liquid petrolatum, mineral oil, polydecene and hydrogenated polyisobutene (such as PARLEAM) and mixtures thereof. Hydrocarbon-based oils of animal origin, such as perhydro squalene, may be used.

Exemplary triglycerides of vegetable or synthetic origin may be selected from liquid fatty acid triglycerides containing 6 to 30 carbon atoms, such as heptanoic or octanoic acid triglycerides, or alternatively, more particularly from those present in vegetable oils, such as coconut oil, sunflower oil, corn oil, soybean oil, marrow oil, grape seed oil, sesame oil, hazelnut oil, almond oil, macadamia nut oil, alara oil (arara oil), castor oil, avocado oil, jojoba oil, shea butter, or synthetic caprylic/capric triglycerides, such as those sold by Stearineries Dubois company or those sold under the names MIGLYOL 810, 812 and 818 by the company Dynamit Nobel, and mixtures thereof. Suitable fluoro oils include perfluoromethyl cyclopentane and perfluoro-1, 3-dimethylcyclohexane, such as flute esc PC1 and flute esc PC3 from BNFL Fluorochemicals company; perfluoro-1, 2-dimethylcyclobutane; perfluoroalkanes such as dodecafluoropentane and tetradecane sold by 3M under the names PF 5050 and PF 5060, or bromoperfluorooctane sold by Atochem under the name foralky; nonafluoromethoxybutane and nonafluoroethoxyisobutane; perfluoromorpholine derivatives such as 4-trifluoromethyl perfluoromorpholine sold by 3M company under the name PF 5052.

Suitable monoesters include dihydroabietyl behenate; octyl dodecanol behenate; isocetyl behenate; cetyl lactate; c (C) ₁₂ -C ₁₅ Alkyl lactate; isostearyl lactate; lauryl lactate; linolic acid ester; oleyl lactate; (iso) stearyl octanoate; isocetyl octanoate; octyl octanoate; cetyl octanoate; decyl oleate; isocetyl isostearate; isocetyl laurate; isocetyl stearate; isodecyl octanoate; isodecyl oleate; isononyl isononanoate; isostearyl palmitate; acetyl methyl ricinoleate; myristyl alcohol stearate; octyl isononanoate; 2-ethylhexyl isononanoate; octyl palmitate; octyl nonanoate; octyl stearate; octyl dodecyl erucate; erucic acid oil ester; ethyl palmitate and isopropyl palmitate; 2-ethylhexyl palmitate, 2-octyldecyl palmitate, alkyl myristates (such as isopropyl myristate, butyl myristate, cetyl myristate, 2-octyldodecyl myristate, myristyl myristate, stearic acid ester), hexyl stearate, butyl stearate, isobutyl stearate; dioctyl malate, hexyl laurate, 2-hexyl decyl laurate, and mixtures thereof.

In some embodiments, the hair care composition may further comprise diethyl sebacate, diisopropyl adipate, di-n-propyl adipate, dioctyl adipate, diisostearyl adipate, dioctyl maleate, glyceryl undecylenate, octyl dodecyl stearoyl stearate, pentaerythritol monoricinoleate, pentaerythritol tetraisononanoate, pentaerythritol tetranonanoate, pentaerythritol tetraisostearate, pentaerythritol tetraoctanoate, propylene dioctate, propylene glycol dicaprate, tridecyl erucate, triisopropyl citrate, triisostearyl citrate, glyceryl trilactate, glyceryl trioctate, trioctyldodecyl citrate, trioctyl citrate, propylene glycol dioctate, neopentyl glycol diheptanoate, diethylene glycol diisononanoate, and polyethylene glycol distearate, and mixtures thereof.

In some embodiments, the hair care composition may further comprise one or more fatty acid esters, one or more sugar esters, and/or one or more C ₆ -C ₃₀ (such as C ₁₂ -C ₂₂ ) Diesters of fatty acids. The term "sugar" means an oxygenated hydrocarbon-based compound containing several alcohol functions, with or without aldehyde or ketone functions, and containing at least 4 carbon atoms. Suitable sugars may include monosaccharides, oligosaccharides or polysaccharides such as sucrose (or cane sugar), glucose, galactose, ribose, fucose, maltose, fructose, mannose, arabinose, xylose and lactose, and derivatives thereof such as alkyl derivatives such as methyl derivatives, e.g., methyl glucose. Suitable esters may include oleic, lauric, palmitic, myristic, behenic, cocoic, stearic, linoleic, linolenic, capric, arachidonic or mixtures thereof, such as oleic/palmitate, oleic/stearate or palmitate/stearate mixed esters. Suitable mono-and diesters also include mono-or di-oleate, mono-or di-stearate, mono-or di-behenate, mono-or di-oil palmitate, mono-or di-linoleate, mono-or di-linolenate or mono-or di-oil stearate of sucrose, glucose or methyl glucose, including the products sold under the name glucose DO by Amerchol corporation, which are methyl glucose dioleate . Other exemplary esters or ester mixtures of sugars and fatty acids that may also be mentioned include: products sold by Crodesta under the designations F160, F140, F110, F90, F70 and SL40 represent sucrose palmitate/stearate formed from 73% mono-and 27% di-and tri-esters, from 61% mono-and 39% di-, tri-and tetra-esters, from 52% mono-and 48% di-, tri-and tetra-esters, from 45% mono-and 55% di-, tri-and tetra-esters, from 39% mono-and 61% di-, tri-and tetra-esters and sucrose monolaurate, respectively; a product sold under the name Ryoto Sugar Esters, for example reference B370 and corresponding to sucrose behenate formed from 20% monoester and 80% diester-triester-polyester; sucrose mono-dipalmitate/stearate sold by Goldschmidt under the name TEGOSOFT PSE.

In some embodiments, the hair care composition may further comprise a pH adjuster such as citric acid and/or sodium hydroxide. Any commonly used pH adjusting agent for hair care compositions is contemplated for use herein.

In various embodiments, the hair care composition may further comprise any of the following ingredients and/or mixtures thereof: sodium laurylmethylisethionate (detergents and foaming agents), liquid fatty substances (including silicone oils other than the non-amino polyalkylsiloxanes discussed hereinabove) and organomodified polysiloxanes comprising at least one functional group selected from amino, aryl and alkoxy groups. Organopolysiloxane is defined in greater detail in Chemistry and Technology of Silicones (1968) by Walter Noll, academic Press, the entire contents of which are hereby incorporated by reference. They may be volatile or non-volatile. Suitable cyclic polydialkylsiloxanes include octamethyl cyclopentasiloxane sold by Union Carbide under the name VOLATILE SILICONE 7207 or by Rhodia under the name SILVILONE 70045V2, decamethyl cyclopentasiloxane sold by Union Carbide under the name VOLATILE SILICONE 7158 and by Rhodia under the name SILVILBIONE 70045V5, and mixtures thereof. Ring copolymers of the dimethylsiloxane/methylalkylsiloxane type, such as VOLATILE SILICONE FZ 3109 sold by Union Carbide, are also suitable. Exemplary cyclic polydialkylsiloxanes having organosilicon compounds include octamethyl cyclotetrasiloxane and tetra (trimethylsilyl) pentaerythritol (50/50) as well as mixtures of octamethyl cyclotetrasiloxane with oxy-1, 1' -bis (2, 2', 3' -hexa-trimethylsiloxy) neopentane, linear volatile polydialkylsiloxanes such as those sold under the name SH 200 by Toray Silicones Inc. Silicones belonging to this category are also described in Cosmetics and Toiletries, volume 91, 1 month 76, pages 27-32, todd & byrs, "Volatile Silicone Fluids for Cosmetics", which is incorporated herein by reference.

Exemplary suitable nonvolatile polydialkylsiloxanes include polydimethylsiloxanes having trimethylsilyl end groups such as the 47 and 70 047 series of silbin oils or MIRASIL oils sold by Rhodia, for example 70 047v 500 000 oils; MIRASIL series of oils sold by Rhodia; 200 series of oils from Dow Corning, inc., such as having a viscosity of 60,000 mm ² DC200 of/s; vissil oil from General Electric and certain oils of SF series from General Electric (SF 96, SF 18), polydimethylsiloxanes with dimethylsilanol end groups known under the designation dimethiconol (CTFA), such as the oils of series 48 from Rhodia corporation.

Exemplary organomodified silicones include polyalkylarylsiloxanes, and products sold under the following names: SILBIONE oil from Rhodia series 70 641; oils from the rhodiola series 70 633 and 763; oil from Dow Corning556 cosmetic grade fluid; silicones from the Bayer PK series, such as product PK20; silicones from the PN and PH series of Bayer, such as products PN1000 and PH1000; certain oils from the SF series of General Electric, such as SF 1023, SF 1154, SF 1250, and SF 1265; products sold by genese under the names GP 4Silicone Fluid and GP 7100 or by Dow Corning company under the names Q2 8220 and Dow Corning 929 or 939.

In various embodiments, the hair care composition may further comprise one or more additional surfactants other than the cationic surfactants described above, including anionic surfactants, amphoteric surfactantsSurfactants or zwitterionic surfactants, nonionic surfactants, and mixtures thereof. Exemplary anionic surfactants include alkyl sulfates, alkyl ether sulfates, alkyl amide ether sulfates, alkylaryl polyether sulfates, monoglyceride sulfates, alkyl sulfonates, alkyl amide sulfonates, alkylaryl sulfonates, alpha-olefin sulfonates, paraffin sulfonates, alkyl sulfosuccinates, alkyl ether sulfosuccinates, alkyl amide sulfosuccinates, alkyl sulfoacetates, acyl sarcosinates, acyl glutamates, alkyl sulfosuccinamates, acyl isethionates, and N- - (C) s ₁ -C ₄ ) Alkyl N-acyl taurates, salts of alkyl mono-and poly-glycoside-polycarboxylic acids, acyl lactates, D-galactoside aldonates, alkyl ether carboxylates, alkyl aryl ether carboxylates, alkyl amide ether carboxylates; and the corresponding non-salified forms of all of these compounds; the alkyl and acyl groups (unless otherwise mentioned) of all these compounds typically contain from 6 to 24 carbon atoms and aryl groups typically represent phenyl groups. The anionic surfactants in salt form may include alkali metal salts such as sodium or potassium salts, sodium salts, ammonium salts, amine salts and aminoalkoxides, or alkaline earth metal salts such as magnesium salts. Exemplary amino alkoxides include monoethanolamine, diethanolamine and triethanolamine salts, monoisopropanolamine, diisopropanolamine or triisopropanolamine salts, 2-amino-2-methyl-1-propanolate, 2-amino-2-methyl-1, 3-propanediol salts, and tris (hydroxymethyl) aminomethane salts.

Suitable anionic surfactants also include mild anionic surfactants, i.e., anionic surfactants having no sulfate functionality, including polyoxyalkylene alkyl ether carboxylic acids; polyoxyalkylene alkylated alkylaryl ether carboxylic acids; polyoxyalkylene alkylated alkylamide ether carboxylic acids, especially those containing from 2 to 50 ethylene oxide groups; alkyl-D-galactoside uronic acid; acyl sarcosinates, acyl glutamates; and alkyl polyglycoside carboxylates such as those sold under the name AKYPO RLM 45CA from Kao.

Exemplary suitable amphoteric or zwitterionic surfactants can be aliphatic secondary or tertiary amine derivatives, optionally quaternizedWherein the aliphatic group is a straight or branched chain containing from 8 to 22 carbon atoms, wherein the amine derivative contains at least one anionic group, e.g. a carboxylate, sulfonate, sulfate, phosphate or phosphonate group, such as (C) ₈ -C ₂₀ ) Alkyl betaines, sulfobetaines, (C) ₈ -C ₂₀ ) Alkylamides (C) ₃ -C ₈ ) Alkyl betaines or (C) ₈ -C ₂₀ ) Alkylamides (C) ₆ -C ₈ ) Alkyl sulfobetaines. Any suitable aliphatic secondary or tertiary amine derivative may be present in coconut oil or hydrolyzed linseed oil and the like. Representative compounds were classified under the following names in CTFA dictionary, 5 th edition 1993: disodium cocoyl amphodiacetate, disodium lauroyl amphodiacetate, disodium caproyl amphodiacetate, disodium capryloyl amphodiacetate, disodium cocoyl amphodipropionate, disodium lauroyl amphodipropionate, disodium caproyl amphodipropionate, disodium capryloyl amphodipropionate, lauroyl amphodipropionic acid, and cocoyl amphodipropionic acid.

For example, cocoyl amphodiacetate sold by Rhodia under the trade name SODIUM C2M Concentrate and diethylaminopropyl cocoyl asparagine sold by Chimex under the name Chimexane HB are suitable for use in the disclosed compositions.

Suitable nonionic surfactants are described in Blackie&Son publication M.R.Porter Handbook of Surfactants (Glasgow and London), 1991, pages 116-178, and includes fatty alcohols, fatty alpha-diols, fats (C) ₁ -C ₂₀ ) Alkylphenols and fatty acids, which may be ethoxylated, propoxylated or glycerinated and contain at least one fatty chain containing from 8 to 18 carbon atoms, the number of ethylene oxide or propylene oxide groups ranging from 1 to 200 and the number of glycerol groups ranging from 1 to 30. Condensates of ethylene oxide and propylene oxide with fatty alcohols, ethoxylated fatty amides having 1 to 30 ethylene oxide units, polyglycerolated fatty amides containing on average 1 to 5 and especially 1.5 to 4 glycerol groups, ethoxylated fatty esters of sorbitan containing 1 to 30 ethylene oxide units, fats of sucroseFatty acid esters, fatty acid esters of polyethylene glycol, (C) ₆ -C ₂₄ ) Alkyl polyglycoside, oxyethylenated vegetable oil, N- (C) ₆ -C ₂₄ ) Alkyl glucosamine derivatives, amine oxides such as (C) ₁₀ -C ₁₄ ) Alkylamine oxides or N- (C) ₁₀ -C ₁₄ ) Acylaminopropyl morpholine oxide is also suitable for use in the compositions disclosed herein.

Other suitable nonionic surfactants include Alkyl Polyglucosides (APGs), maltitol esters, polyglycerolated fatty alcohols, glucosamine derivatives (e.g., 2-ethylhexyloxycarbonyl-N-methylglucamine), and mixtures thereof. Alkyl polyglucosides are those containing an alkyl group containing from 6 to 30 carbon atoms and containing a hydrophilic group (glucoside). Exemplary alkyl polyglucosides include decyl glucoside (alkyl-C9/C11-polyglucoside (1.4)), including those sold under the name Mydol by Kao ChemicalsProducts sold under the name Plantaren 2000>Products sold and under the name Oramix NS +.>A product for sale; and octyl/decyl glucoside, including those sold under the name Oramix CG by SEPPIC Corp->A product for sale; plant 1200 +.>And plant->Sold lauryl glucoside, and coco glucoside, e.g. by Cognis under the name plant +.>And (5) selling the product.

Suitable maltose derivatives include those described in document EP-A-566 438, such as 0-octanoyl-6 '-D-maltose or O-dodecanoyl-6' -D-maltose described in document FR-2 739 556. Each of these files is incorporated by reference in its entirety.

In some embodiments, the hair care composition may be formulated in a cosmetically acceptable medium. The term "cosmetically acceptable medium" means a medium compatible with human keratin fibers such as hair. The cosmetically acceptable medium may be formed from water or from a mixture of water and one or more cosmetically acceptable solvents selected from lower alcohols such as ethanol and isopropanol; polyols and polyol ethers including 2-butoxyethanol, propylene glycol monomethyl ether, diethylene glycol monoethyl ether and monomethyl ether and mixtures thereof.

In some embodiments, the hair care composition may further comprise any of the following additives or mixtures thereof: solid fatty substances other than fatty alcohols such as waxes, anionic polymers, nonionic polymers or amphoteric polymers or mixtures thereof, antidandruff agents, anti-seborrheic agents, agents for preventing hair loss and/or for promoting hair regrowth, vitamins and provitamins (including panthenol), sunscreens, inorganic or organic pigments, chelating agents, plasticizers, solubilisers, acidifying agents, inorganic or organic thickeners (in particular polymeric thickeners other than oxyethylenated polymers), opacifiers or pearlescers, antioxidants, hydroxy acids, fragrances and/or preservatives.

Nail care

In some embodiments, the cosmetic composition may comprise ingredients commonly used in nail care products. Nail care products include, but are not limited to, nail treatments (including nail enhancers, topcoats, and basecoats), nail polish remover, hand skin care, foot skin care, desiccants, and correction pens (including nail polish remover).

In some embodiments, the nail care composition may be a nail treatment composition. The nail treatment composition may include a composition for treating an ingrown nail or nail deformity, a composition for topical treatment of nail infections (including fungal infections), nail enhancers, topcoats, basecoats, polish removers, or any combination thereof. In some embodiments, the nail treatment composition may be formulated as a topical nail lacquer or nail polish, cream, solution, suspension, lotion, serum, gel, balsam, gel, oil-in-cream, and/or wipe for treating hands and/or feet.

In some embodiments, the nail treatment composition may be a nail enhancer. Nail enhancers can treat the fingernails and toenails to harden, strengthen, and promote the growth of the nails to prevent or minimize cracking, crazing, splitting, and peeling, and can include any known composition for preventing and healing sidewall hoof cracks (quater crack) while increasing the growth of the horseshoe, having a protein consistency similar to that of a fingernail and toenail, including: lanolin, beef tallow, beeswax, rosin, copper acetate and turpentine. In some embodiments, the nail treatment composition may further comprise titanium dioxide, such as TI-PURE R900 from e.i. dupont.

In some embodiments, the nail treatment composition may be a primer coating. In some embodiments, the primer layer may be a liquid composition comprising at least one adhesion-providing polymer, for example, the polymer is copolymerized from Methyl Methacrylate (MMA) and methacrylic acid (MAA) to form a polymer composed of polymethyl methacrylate (PMMA) and polymethacrylic acid (PMAA), where the MAA monomer fraction may vary from 0 to 100%. In some embodiments, suitable polymers for use as the primer layer include hydroxyethyl methacrylate (HEMA), hydroxypropyl methacrylate (HPMA), ethyl Methacrylate (EMA), tetrahydrofurfuryl methacrylate (THFMA), pyromellitic dianhydride di (meth) acrylate, pyromellitic dianhydride glycerol dimethacrylate, pyromellitic dimethacrylate, methacryloxyethyl maleate, 2-hydroxyethyl methacrylate/succinate, 1, 3-glycerol dimethacrylate/succinate adducts, phthalic acid monoethyl methacrylate, and mixtures thereof. In some aspects, the basecoat layer may also comprise a non-reactive, solvent-soluble film-forming polymer, such as a cellulose ester, for example, cellulose acetate alkylate, cellulose acetate butyrate, or cellulose acetate propionate. The exemplary ingredients described above are not limiting.

In some embodiments, the nail treatment composition may be a topcoat, such as a quick-drying topcoat. The topcoat may include a base resin that is cellulose acetate butyrate, a film former that is a methacrylate polymer, a thermally curable or photocurable monomer that is a monofunctional methacrylate and a crosslinker (i.e., difunctional methacrylate and trifunctional methacrylate), a thermal initiator or photoinitiator in solution with an aliphatic ester and an alcohol, and a photoreactive coating. Suitable solvents include acetates and alcohols, particularly ethyl acetate, butyl acetate and isopropyl alcohol. The photoreactive coating can include photoreactive monomers including methacrylate monomers such as: cyclohexyl methacrylate, n-decyl methacrylate, 2-ethylhexyl methacrylate, ethyl methacrylate, hydroxypropyl methacrylate, isobornyl methacrylate, 2-methoxyethyl methacrylate; difunctional and trifunctional methacrylate monomers, crosslinkers such as dicarbamate dimethacrylate, ethylene glycol dimethacrylate, 1, 10-decanediol dimethacrylate, 1, 6-hexanediol dimethacrylate and trimethylolpropane trimethacrylate. Commercially available photoinitiators suitable for use include, but are not limited to: benzoin methyl ether, 2-hydroxy-2-methyl-1-phenyl-1-propanone ("Darocur 1173"), diethoxyacetophenone, and benzyl diketal. The photoinitiator is an oligomeric mixture of phenylacetone, such as a mixture of 2,4, 6-trimethylbenzophenone and 4-methylbenzophenone, and a mixture of oligomeric- [ 2-hydroxy-2-methyl-1 [4- (1-methylvinyl) phenyl ] propanone ] and 2-hydroxy-2-methyl-phenylpropanone sold by Sartomer under the names "Esacure KIP 100F" and "Esacure TZT photoinitiator".

In some embodiments, the nail care composition may be a nail polish or a nail polish composition. In some embodiments, the nail polish or nail polish composition may further comprise a nitrocellulose film former, a latex film former, a polycarbodiimide film former, a low Volatile Organic Compound (VOC), and a polycarbodiimide film former. The polycarbodiimide may include a polymer having a plurality of carbodiimide groups attached to a polymer backbone. For example, U.S. patent No. 5,352,400 (the disclosure of which is incorporated herein by reference) discloses polymers and copolymers derived from alpha-methylstyrene-isocyanate. Suitable polycarbodiimide compounds include, but are not limited to, those commercially available from suppliers Nisshinbo (including those known under the names CARBODILITE series V-02, V02-L2, SV-02, E-02, V-10, SW-12G, E-03A), picassian, and 3M.

In some embodiments, the nail polish or nail polish composition may further comprise one or more latex polymers, including carboxyl functional acrylate latex polymers, carboxyl functional polyurethane latex polymers, carboxyl functional silicone latex polymers, carboxyl functional non-acrylate latex polymers, and mixtures thereof. In various embodiments, a suitable latex polymer may be a film-forming latex polymer or a non-film-forming latex polymer. In some embodiments, the latex polymer may be a carboxyl functional acrylate latex polymer, such as those resulting from homo-or copolymerization of ethylenically unsaturated monomers selected from the group consisting of: vinyl monomers, (meth) acrylic monomers, (meth) acrylamide monomers, unsaturated monocarboxylic and dicarboxylic acids, esters of (meth) acrylic monomers, and amides of (meth) acrylic monomers. As used herein, the term "(meth) acryl" and variants thereof means acryl or methacryl. The (meth) acrylic monomer may be selected from, for example, acrylic acid, methacrylic acid, citraconic acid, itaconic acid, maleic acid, fumaric acid, crotonic acid, and maleic anhydride. As non-limiting examples, esters of (meth) acrylic acid monomers may be C1-C8 alkyl (meth) acrylates such as methyl (meth) acrylate, ethyl (meth) acrylate, propyl (meth) acrylate, isopropyl (meth) acrylate, butyl (meth) acrylate, t-butyl (meth) acrylate, pentyl (meth) acrylate, isopentyl (meth) acrylate, neopentyl (meth) acrylate, hexyl (meth) acrylate, isohexyl (meth) acrylate, 2-ethylhexyl (meth) acrylate, cyclohexyl (meth) acrylate, isohexyl (meth) acrylate, heptyl (meth) acrylate, isoheptyl (meth) acrylate, octyl (meth) acrylate, isooctyl (meth) acrylate, allyl (meth) acrylate, and combinations thereof. Amides of (meth) acrylic monomers may be made, for example, from (meth) acrylamides, especially N-alkyl (meth) acrylamides, especially N- (C1-C12) alkyl (meth) acrylates such as N-ethyl (meth) acrylamide, N-t-butyl (meth) acrylamide, N-t-octyl (meth) acrylamide, N-hydroxymethyl (meth) acrylamide and N-diacetone (meth) acrylamide, and combinations thereof.

Vinyl monomers may include, but are not limited to, vinyl cyanide compounds such as acrylonitrile and methacrylonitrile; vinyl esters such as vinyl formate, vinyl acetate, vinyl propionate, vinyl neodecanoate, vinyl pivalate, vinyl benzoate and vinyl-tert-butyl benzoate, triallyl cyanurate; vinyl halides such as vinyl chloride and vinylidene chloride; aromatic monovinyl or divinyl compounds such as styrene,. Alpha. -methylstyrene, chlorostyrene, alkylstyrene, divinylbenzene and diallyl phthalate, as well as p-styrenesulfonic acid, vinylsulfonic acid, 2- (meth) acryloxyethylsulfonic acid, 2- (meth) acrylamide-2-methylpropanesulfonic acid and mixtures thereof.

The list of monomers herein is not limiting and it is understood that any monomer known to those skilled in the art may be used, including acrylic monomers and/or vinyl monomers (including monomers modified with silicone chains).

In some non-limiting exemplary embodiments, the carboxyl functional acrylate latex polymer may be selected from the following aqueous dispersions: methacrylic acid/ethyl acrylate copolymer (INCI: acrylate copolymers such as the LUVIFLEX SOFT of BASF), PEG/PPG-23/6 dimethicone citraconate/C10-30 alkyl PEG-25 methacrylate/acrylic acid/methacrylic acid/ethyl acrylate/trimethylolpropane PEG-15 triacrylate copolymers (INCI: polyacrylate-2 cross-linked polymers such as the Fixate super hold. Tm.) of Lubrizol, styrene/acrylic acid copolymers such as the Acudyne Shine of Dow Chemical, ethyl-hexyl acrylate/methyl methacrylate/butyl acrylate/acrylic acid/methacrylic acid copolymers (INCI: acrylate ethyl-hexyl acrylate copolymers such as daiosol 5000SJ,Daito Kasei Kogyo), acrylic acid/acrylate copolymers (INCI name: acrylate copolymers such as daiosol 5000AD,Daito Kasei Kogyo), acrylate copolymers such as those known under the trade name decryl AQF (Akzo Nobel), under the trade name lulamer CR (Akzo), under the trade name of lulamer CR (Akzo), acrylic acid/hydroxy acrylate copolymers such as the trade name of lulamer CR (Akzo) 180, and styrene/acrylic acid copolymers such as the trade name of ethyl acrylate/acrylic acid ester (window) from law of law Chemical, styrene/acrylic acid copolymer (20, acrylic acid copolymer (window) and acrylic acid ester copolymer (window) of acrylic acid/acrylic acid ester/acrylic acid copolymer (window) of styrene/acrylic acid ester/acrylic acid copolymer (window) 20).

In some embodiments, the nail care composition may comprise a nail polish remover. Nail polish removers may include polyol compounds including glycerin, glycols, polyglycerols, esters of polyols, and mixtures thereof. The diols may contain 2 to 12 carbon atoms, for example glycerol, propylene glycol, butylene glycol, propylene glycol, hexylene glycol, polyglycerol, dipropylene glycol and diethylene glycol. Suitable esters of polyols include liquid esters of saturated or unsaturated, linear or branched C1-C26 polyols. Examples of esters of suitable polyols include, but are not limited to, esters of dihydric, trihydric, tetrahydroxy or penta-hydroxy alcohols. The ester of the polyhydric alcohol may be a glyceride such as glyceryl triglycolate, glyceryl tricitrate, glyceryl trilactate, glyceryl tributyrate, glyceryl triheptanoate, glyceryl trioctanoate, and the like.

The nail polish remover may also comprise a lower alcohol, containing from 1 to 8 carbon atoms. The lower alcohols may contain 2 to 6 carbon atoms, such as 2 to 5 carbon atoms. Examples of lower alcohols include, but are not limited to, ethanol, propanol, butanol, pentanol, isopropanol, isobutanol, and isopentanol. Nail polish removers may also include high boiling ester compounds including, but not limited to, carbonates, adipates, sebacates, and succinates. Exemplary high boiling ester compounds include, but are not limited to, alkylene carbonates such as propylene carbonate, dimethyl succinate, diethyl succinate, dimethyl glutarate, diethyl glutarate, dimethyl sebacate, diethyl sebacate, diisopropyl sebacate, bis (2-ethylhexyl) sebacate, dimethyl adipate, diisopropyl adipate, di-n-propyl adipate, dioctyl adipate, bis (2-ethylhexyl) adipate, diisostearyl adipate, ethyl maleate, bis (2-ethylhexyl) maleate, triisopropyl citrate, triisocetyl citrate, triisostearyl citrate, trioctyldodecyl citrate, and triisostearyl citrate.

Nail polish removers may also include thickening agents including, but not limited to: nonionic, anionic, cationic, amphiphilic and amphoteric polymers, as well as other known rheology modifiers, such as cellulose-based thickeners, such as hydroxyethyl cellulose, hydroxypropyl cellulose, methyl cellulose and ethyl hydroxyethyl cellulose. Some notable cellulose derivatives include hydroxy-modified cellulose polymers such as hydroxyethyl cellulose, e.g., those having a molecular weight in excess of 500,000 daltons, such as NATROSOL 250HHR and hydroxypropyl cellulose, e.g., KLUCEL MF, both available from Ashland, covington, ky. The thickening agent may be a polysaccharide such as levan, dextran, galactan and mannan, or a heteropolysaccharide such as hemicellulose, pullulan (pullulan) or a branched polysaccharide such as gum arabic and pullulan, or a mixed polysaccharide such as starch. The thickening agent may be an acrylic thickening agent (acrylic thickener) or an acrylamide thickening agent (acrylamide thickener). The thickening agent may include at least one monomer that performs a weak acid function, such as acrylic acid, methacrylic acid, itaconic acid, crotonic acid, maleic acid, and/or fumaric acid. The thickening agent may include a monomer that performs a strong acid function, such as a monomer having a sulfonic acid type or phosphonic acid type function, such as 2-acrylamide-2-methylpropanesulfonic Acid (AMPS). The thickening agent may include a cross-linking agent such as Methylene Bisacrylamide (MBA), ethylene glycol diacrylate, polyethylene glycol dimethacrylate, diacrylamides, cyanomethacrylates, ethyleneoxyethyl acrylate or methacrylate, formaldehyde, glyoxal, and glycidyl ether type compositions such as ethylene glycol diglycidyl ether or epoxides. Suitable acrylic thickeners are disclosed in U.S. patent application publication nos. 2004/0028637 and 2008/0196174, both of which are incorporated herein by reference. In some embodiments, the thickening agent may include an organoclay (hydrophobically treated clay) or a hydrophilic clay.

In some embodiments, the thickening agent may include an abrasive compound (abrasive system). An "abrasive compound" is a compound capable of providing abrading or mechanical exfoliation. Abrasive particles may include perlite, pumice, zeolite, hydrated silica, calcium carbonate, dicalcium phosphate dihydrate, calcium pyrophosphate, alumina, sodium bicarbonate, polylactic acid, and synthetic polymeric materials such as polyethylene, polypropylene, polyethylene terephthalate, polymethyl methacrylate, or nylon. In certain embodiments, the medium hardness abrasive comprises perlite, such as cosmetic grade perlite available under the name IMERCARE 270P-Scub from Imerrys. In certain embodiments, the soft grind is a sugar, ground kernel or shell powder, such as almond, coconut shell, or spherical wax (e.g., carnauba jojoba oil); the powder of the shell of the jatropha curcas, etc.

In some embodiments, the nail care composition may further comprise additives commonly used in cosmetic compositions and known to the skilled artisan, including solvents, preservatives, fragrances, oils, waxes, surfactants, antioxidants, anti-free radical agents, humectants, dispersants, defoamers, neutralizing agents, stabilizers, active ingredients selected from the group consisting of essential oils, UV masking agents, sunscreens, moisturizers, vitamins, proteins, ceramides, plant extracts, fibers, and the like, and mixtures thereof.

Therapeutic compositions

In some embodiments, the compositions described herein may be therapeutic compositions for treating one or more conditions. For example, in some embodiments, administration of the compositions described herein may promote wound healing, reduce or prevent scar tissue formation, promote tissue regeneration, minimize local inflammation, minimize tissue rejection, and/or enhance graft integration. In some embodiments, the composition may be formulated as an injectable material, such as a hydrogel. Collagen hydrogels exhibit a large uniform surface area and can be used as a delivery system for collagen and optionally one or more additional therapeutic agents. The injectable collagen material may also form a scaffold or network capable of replacing tissue functions and supporting tissue regeneration. In certain embodiments, the composition may be administered topically. In certain embodiments, the composition may be injected transdermally, intradermally, or subcutaneously.

In some embodiments, the therapeutic compositions described herein may comprise one or more additional therapeutic and/or prophylactic agents in addition to the collagen fragments described elsewhere herein. The one or more additional therapeutic and/or prophylactic agents may be small molecule active agents or biomolecules, such as enzymes or proteins, polypeptides or nucleic acids.

Non-limiting examples of additional therapeutic and/or prophylactic agents include anti-cancer agents, antimicrobial agents (including antiviral, antibacterial, antifungal, and antiparasitic agents), antioxidants, analgesics, local anesthetics, anti-inflammatory agents, cytokines, immunosuppressants, antiallergic agents, essential nutrients, growth factors (such as fibroblast growth factor, hepatocyte growth factor, platelet-derived growth factor, vascular endothelial growth factor, and insulin-like growth factor), and combinations thereof. The specific dosages of additional therapeutic and/or prophylactic agents can be readily determined by those skilled in the art. See Ansel, howard C. Et al Pharmaceutical Dosage Forms and Drug Delivery Systems (6 th edition) Williams and Wilkins, malvern, pa (1995).

In other embodiments, the recombinant collagen fragment compositions described herein can be used in combination with cellular delivery, e.g., delivery of stem cells, pluripotent cells, somatic cells, and combinations thereof.

While the present disclosure contemplates that the therapeutic and/or prophylactic agent will be an integral part of the compositions described herein, in some embodiments, the therapeutic and/or prophylactic agent may be administered prior to, concurrently with, or after administration of the therapeutic compositions described herein. In other words, in some embodiments, the one or more additional therapeutic and/or prophylactic agents may not be incorporated entirely into the present compositions, but rather may be provided separately from the present compositions.

Non-limiting examples of suitable local anesthetics that can be included in the compositions of the present invention include but are not limited to, ambukaine (ambukaine), aciclone (amolanone), acicloline (amolanine), procaine (benoxinate), benzocaine (benzocaine), ding Yangka (betaxocaine), benzocaine (biphenamine), bupivacaine (bupivacaine), bupivacaine (butacaine), aminobutyric acid (butamben), bupivacaine (butanilidine), butylaminocarine (butthamine), ding Yangka (butaxycaine), carbocaine (carbocaine), chloroprocaine (chloroprocaine), cocaethylene (cocaine), cocaine (cocaine), cyclomethicaine (cyclomethiocarine), dibucaine (dibucaine), dimethylprocaine (dimethylquinone) dimethyl-cocaine, diperoxide, dyclonine, ecgonidine, ethyl chloride, etidocaine, beta-eucaine, you Puluo (euprocin), phenamine, furacarine, hexycaine, oxybutynin (hydroxy tetracaine), isobutyl para-aminobenzoate, leucaine mesylate, levo Sha Quer (levoxadiol), lidocaine (lidocaine), carbocaine (mepivacaine), mepropbicaine, mebucaine, methyl chloride, meltecaine, mycetin, melnucaine, naepaine, otacaine, orthocarpine, oxcarbaine, parathocarine, phenacaine, phenol, pirocaine, pidocaine, polidocaine, pramoxine, prilocaine, procaine, and propidium-carpaine (procaine), procaine (procaine), propidium-carpaine (procaine), procalcitonin (procaine), pseudococaine (psuedococaine), pyrrolcaine (pyrrocaine), ropivacaine (ropivacaine), salicylalcohol, tetracaine (tetracaine), tolica (tolycaine), trimecaine (trimecaine), zolamine (zolamine), and combinations thereof.

Non-limiting examples of suitable antiviral agents include ganciclovir (ganciclovir) and acyclovir (acyclovir). Non-limiting examples of suitable antibiotic agents include aminoglycosides such as streptomycin, amikacin, gentamicin, and tobramycin; ansamycins such as geldanamycin (geldanamycin) and herbimycin (herbimycin); carbacephem; carbapenems; cephalosporins; glycopeptides such as vancomycin, teicoplanin (teicoplanin), and telavancin Mo Xin; lincomamides; lipopeptides such as daptomycin (daptomycin); macrolides such as azithromycin (azithromycin), clarithromycin (clarithromycin), dirithromycin (dirithromycin) and erythromycin (erythromycin); monoamines; nitrofurans; penicillins; polypeptides such as bacitracin (colistin), colistin (colistin) and polymyxin B; quinolones; sulfonamides; and tetracyclines. Additional exemplary antimicrobial agents include iodine, silver compounds, moxifloxacin (moxifloxacin), ciprofloxacin (ciprofloxacin), levofloxacin (levofloxacin), cefazolin (cefazolin), tigecycline (tigecycline), gentamicin, ceftazidime, ofloxacin (ofloxacin), gatifloxacin (gatifloxacin), amphotericin (amphotericin), voriconazole (voriconazole), natamycin (natamycin).

Non-limiting examples of suitable anti-inflammatory agents include steroidal active agents including glucocorticoids, progestins, mineralocorticoids, and corticosteroids. Exemplary non-steroidal anti-inflammatory drugs include ketorolac (ketorolac), ibuprofen (ibuprofen), nepafenac (nepafenac), diclofenac (dichlorophenofenac), aspirin (aspirin), and naproxen (naproxen). Other exemplary anti-inflammatory agents include triamcinolone acetonide (triamcinolone acetonide), fluocinolone acetonide (fluocinolone acetonide), prednisolone (prednisolone), dexamethasone (dexamethasone), loteprednol, fluorometholone (fluoromertholone), and dipotassium glycyrrhizinate (dipotassium glycyrrhizate).

In some embodiments, the composition may further comprise one or more additional pharmaceutically active agents. Exemplary agents may include non-steroidal anti-inflammatory agents (NSAIDs), such as flurbiprofen (flurbiprofen), ibuprofen, naproxen, indomethacin (indomethacin), and related compounds. In some embodiments, the composition may further comprise one or more antimitotic agents, including colchicine (colchicine), paclitaxel (taxol), and related compounds. In some embodiments, the composition may further comprise one or more topical disinfectants, such as benzoyl peroxide. In some embodiments, the composition may further comprise one or more polysaccharides produced by microalgae, e.g., alginic acid.

In some embodiments, the composition may further comprise one or more immunomodulatory drugs. Exemplary immunomodulatory drugs include imiquimod (imiquimod), cyclosporin (cycloporine), tacrolimus (tacrolimus), and rapamycin (rapamycin).

In some embodiments, the composition may further comprise one or more cytokines. Exemplary suitable cytokines include, but are not limited to, IL-10, TGF-beta, IL-25, and IL-35. In certain embodiments, the cytokine may induce Treg activation (e.g., IL-25) and inhibit Thl7 activation (e.g., IL-10) to minimize rejection.

In some embodiments, the compositions described herein further comprise at least one eukaryotic cell type. Some exemplary eukaryotic cell types include stem cells, mesenchymal stem cells, keratinocytes, fibroblasts, melanocytes, adipocytes, immune cells such as T lymphocytes, B lymphocytes, natural killer cells, and dendritic cells, or combinations thereof. In some embodiments, the stem cells may be adipose-derived mesenchymal stem cells. Functional features of mesenchymal Stem Cells that may be beneficial for wound healing include their ability to migrate to the site of injury or inflammation, participate in regeneration of damaged tissue, stimulate proliferation and differentiation of resident progenitor Cells, promote recovery of damaged Cells by growth factor secretion and matrix remodeling, and exert unique immunomodulatory and anti-inflammatory effects (see, e.g., phinney OG et al, stem Cells,25:2896-2902 (2007); chamberlain G et al, stem Cells,25:2739-2749 (2007); dazzi F et al, curr Opin Oncol.19:650-655 (2007)). Each of these references is incorporated by reference in its entirety.

In some embodiments, eukaryotic cells may be responsible for increasing the structural integrity of connective tissue and/or promoting healing. In some embodiments, eukaryotic cells (such as fibroblasts) may be responsible for enhancing or promoting growth or attachment of cells or tissues.

In some embodiments, the recombinant collagen composition may be contacted with 1) an implanted hair graft and 2) tissue into which the hair graft is implanted. The recipient site for hair transplantation includes the scalp, facial area, armpit or chest area, or pubic area. Specific areas of the facial area include eyebrows, eyelids, beard, sideburns, chin, and cheeks. The recipient site may be any area of skin where hair is desired to appear to the subject. In some embodiments, contact between the compositions described herein and the implanted hair and surrounding tissue of the recipient site promotes nutrient perfusion from the surrounding tissue into the graft and increases the survival rate of the graft as compared to a graft implanted without the compositions described herein. In certain embodiments, contact between the compositions described herein and the implanted hair and contact between the compositions described herein and surrounding tissue at the recipient site promote vascularization around the implanted graft such that the survival rate of the graft is increased compared to a graft implanted without the compositions described herein.

Dietary compositions

In some embodiments, the compositions described herein may be a dietary composition useful for providing collagen to a subject in need thereof. For example, in some embodiments, consumption of the dietary compositions described herein may provide health and/or skin benefits, such as increased collagen uptake, relief of joint pain, and improvement of skin health. In certain embodiments, the composition may be in powder, capsule, liquid form, or any other suitable form.

In some embodiments, the dietary composition may comprise one or more nutritional ingredients, such as: ascorbic acid, biotin, chromium nicotinate, cupric citrate, D-calcium pantothenate, cyanocobalamin, linseed, flax, folic acid, fructooligosaccharides (fiber), magnesium oxide, manganese citrate, maltodextrin, medium chain triglycerides, flavoring agents, nicotinamide, potassium citrate, potassium iodide, riboflavin, sugarcane (saccharum officinarum), sodium molybdate dihydrate, sodium selenate (selenium), soy protein isolate, stevia leaf extract/stevia, thiamine HCl, tricalcium phosphate, vitamin a palmitate, vitamin D3, xanthan gum, zinc citrate, cellulose gum, guar gum, pyridoxine hydrochloride, salts, tocopherols, antioxidants (e.g., resveratrol, coQ10, brazil, lycopene and pomegranate), natural or artificial sweeteners (e.g., glucose, sucrose, fructose, sugar, cyclohexasulfamate, aspartame), sucralose, aspartame, acesulfame K or sorbitol), flavoring agents (such as extracts of stevia, volatile oils, chocolate (e.g., chocolate), chocolate, other than any of the combination thereof, and the like.

In some embodiments, as described above, the compositions described herein may be in the form of an alcohol-based or water-based toner. Exemplary toner formulations are described below.

Alcohol-based toners

The components are as follows:	% weight/weight
		Water (Water)	Residual components
Recombinant collagen fragments disclosed herein	About 0.0005% to about 25%
		Alcohol denaturant	10％-20％
Pentanediol	5％-10％
		Glycerol	1％-5％
Gluconolactone	0.1％-1％
		Dipotassium glycyrrhizinate	0.1％-1％
Sodium citrate	0.1％-1％
		Sodium benzoate	0.1％-1％

Water-based toner

In some embodiments as described above, the compositions described herein may be in the form of a cream, gel, or serum. Exemplary cream, gel or concentrate formulations are described below.

Cream

The components are as follows:	% weight/weight
		Water (Water)	Residual components
Recombinant collagen fragments disclosed herein	About 0.0005% to about 25%
		Cetostearyl alcohol	5％-10％
Glycerol	1％-5％
		Squalane (Squalene)	1％-5％
Butyrospermum parkii (shea butter)	1％-5％
		Caprylic acid glyceride	1％-5％
Microcrystalline cellulose	1％-5％
		Glyceryl stearate	0.1％-1％
Tocopheryl acetate	0.1％-1％
		Cetostearyl glucoside	0.1％-1％
Stearoyl sodium glutamate	0.1％-1％
		Cellulose gum	0.1％-1％
Xanthan gum	0.1％-1％
		Octanoyl hydroxamic acid	0.1％-1％
Sodium phytate	0.01％-0.1％

Gel

The components are as follows:	% weight/weight
		Water (Water)	Residual components
Recombinant collagen fragments disclosed herein	About 0.0005% to about 25%
		Sodium phytate	0.1％-1％
Sodium hydroxide	0.1％-1％
		Carbomer (carbomer)	0.1％-1％
Phenoxyethanol	0.1％-1％

Essence liquid

In some embodiments as described above, the compositions described herein may be shampoos or conditioners. Exemplary shampoo or conditioner formulations are described below.

Shampoo

The components are as follows:	% weight/weight
		Water (Water)	Residual components
Recombinant collagen fragments disclosed herein	About 0.0005% to about 25%
		Cocamidopropyl betaine	5％-10％
Lauroyl methyl hydroxyethyl sodium sulfonate	5％-10％
		Propylene glycol	1％-5％
Sodium methyl Betayl taurate	1％-5％
		Sodium cocoyl isethionate	1％-5％
Ethylenediamine disuccinic acid trisodium salt	0.1％-1％
		Octanoyl hydroxamic acid	0.1％-1％
Panthenol	0.1％-1％
		Citric acid	0.1％-1％
Octanediol	0.1％-1％
		Sodium benzoate	0.1％-1％

Conditioning agent

D.Yeast strains

The present disclosure can use yeast to produce the collagen fragments described herein. In some embodiments, modified yeast may be used to produce collagen fragments. Suitable yeasts include Pichia (Pichia), candida (Candida), fabry (Komatagaella), hansenula (Hansenula), cryptococcus (Cryptococcus), saccharomyces (Saccharomyces), and combinations thereof. In some embodiments, the yeast may be from the genus pichia. The yeast may be modified or hybridized. Hybrid yeasts can be prepared by cultivating different strains of the same species, different species of the same genus or strains of different genus. Examples of yeast strains suitable for producing collagen fragments disclosed herein include Pichia pastoris (Pichia pastoris), pichia membranaefaciens (Pichia membranifaciens), pichia desertification (Pichia deserticola), pichia cephalocereana, pichia pastoris (Pichia eremophila), pichia pastoris (Pichia myanmarensis), pichia anomala (Pichia anomala), pichia pastoris (Pichia nakasei), pichia siamensis, pichia heel, pichia pastoris (Pichia barker), pichia norway (Pichia norvegensis), pichia Gao Wenbi red yeast (Pichia thermomethanolica), pichia stipitis, pichia sub-film (Pichia subpelliculosa), pichia beet (Pichia exigua), pichia western (Pichia occidentalis), cactus (Pichia cactophila), and the like.

In one embodiment, a pichia pastoris strain can be engineered to express a codon optimized polynucleotide encoding a collagen fragment.

In some embodiments, a collagen fragment encoded by a yeast host cell is fused to a polypeptide sequence that facilitates secretion of the collagen fragment from the yeast. For example, the vector may encode a chimeric gene comprising a coding sequence for a collagen fragment fused to a sequence encoding a secretory peptide. Secretion sequences that may be used for this purpose include Saccharomyces (Saccharomyces) alpha mating factor Prepro sequence, saccharomyces alpha mating factor Pre sequence, PHO1 secretion signal, alpha-amylase signal sequence from Aspergillus niger (Aspergillus niger), protein with internal repeat 1 signal sequence, glucoamylase signal sequence from Aspergillus awamori (Aspergillus awamori), serum albumin signal sequence from Homo sapiens (Homo sapiens), inulinase signal sequence from Kluyveromyces marxianus (Kluyveromcyes maxianus), invertase signal sequence from Saccharomyces cerevisiae (Saccharomyces cerevisiae), killer protein signal sequence from Saccharomyces cerevisiae and lysozyme signal sequence from chicken (Gallus galius). Other secretion sequences known in the art may also be used.

In some embodiments, one or more of the following yeast promoters may be incorporated into the vector to facilitate transcription of mRNA encoding a protein of interest (e.g., a collagen fragment). Promoters are known in the art and include pAOX1, pDas2, pPMP20, pCAT, pDF, pGAP, pFDH1, pFLD1, pTAL1, pFBA2, pAOX2, pRKI1, pRPE2, pPEX5, pDAK1, pFGH1, pADH2, pTPI1, pFBP1, pTAL1, pPFK1, pGPM1, and pGCW14.

In some embodiments, a yeast termination sequence is incorporated into the vector to terminate transcription of mRNA encoding a protein of interest (e.g., a collagen fragment). Terminators include, but are not limited to, AOX1 TT, das2 TT, AOD TT, PMP TT, cat1 TT, TPI TT, FDH1 TT, TEF1 TT, FLD1 TT, GCW14 TT, FBA2 TT, ADH2 TT, FBP1 TT, and GAP TT.

In some embodiments, the recombinant collagen fragments described herein are produced in genetically engineered yeast strains. In some embodiments, the yeast is pichia pastoris.

In some embodiments, the yeast is transformed with a plasmid (vector) comprising the nucleic acid sequence set forth in SEQ ID NO. 973. In some embodiments, to improve recombinant protein expression, the yeast is re-transformed (i.e., double transformed) with a second plasmid. In some embodiments, the yeast is doubly transformed with a plasmid comprising the nucleic acid sequence set forth in SEQ ID NO. 974.

In some embodiments, the recombinant collagen fragments described herein are produced by a method comprising the steps of:

(i) Fermenting a yeast strain in a fermentation broth;

(ii) Recovering from the fermentation broth a recombinant collagen fragment secreted by the yeast strain; and

(iii) Optionally, these recombinant collagen fragments are purified.

In some embodiments, the recombinant collagen fragment may then undergo ex vivo hydroxylation.

In some embodiments, the recombinant collagen fragment may be produced in a genetically engineered yeast strain. In some embodiments, the yeast may be pichia pastoris. In some embodiments, the yeast may be transformed with a plasmid comprising the nucleic acid sequence shown in SEQ ID NO. 973. In some embodiments, the yeast may be doubly transformed with a plasmid comprising the nucleic acid sequence set forth in SEQ ID NO. 974.

In some embodiments, the recombinant collagen fragment sequence variants described herein can be produced by a method comprising the steps of:

(i) Fermenting a yeast strain in a fermentation broth;

(ii) Recovering from the fermentation broth the recombinant collagen fragment sequence variant secreted by the yeast strain;

and

(iii) Optionally, these recombinant collagen fragment sequence variants are purified.

In some embodiments, these recombinant collagen fragment sequence variants may then undergo ex vivo hydroxylation.

In some embodiments, the recombinant collagen fragment sequence variants described herein can be produced in genetically engineered yeast strains. In some embodiments, the yeast may be pichia pastoris. In some embodiments, the yeast may be transformed with a plasmid comprising the nucleic acid sequence shown in any one of SEQ ID No. 1045 to SEQ ID No. 1073. In some embodiments, the yeast may be doubly transformed with a nucleic acid sequence as set forth in any one of SEQ ID NOS: 1045 to 1073.

DNA may be introduced into the yeast strain by electroporation. Transformants can be selected, for example, using leucine, tryptophan, uracil or histidine auxotrophic host yeast cells in conjunction with a selectable marker gene such as LEU2, TRP1, URA3, HIS3 or LEU 2-D. The DNA sequence of the collagen fragment may be introduced into the yeast via a vector, for example by electroporation. The DNA may be inserted into a vector. Suitable vectors include, but are not limited to, pHTX1-BiDi-P4HA-Pre-P4HB Hygro, pHTX1-BiDi-P4HA-PHO1-P4HB Hygro, pGCW14-pGAP1-BiDi-P4HA-Prepro-P4HB G418, pGCW14-pGAP1-BiDi-P4HA-PHO1-P4HB Hygro, pDF-Col3A1 modified bleomycin, pCAT-Col3A1 modified bleomycin, pDF-Col3A1 modified bleomycin with AOX1 landing pad, pHTX1-BiDi-P4HA-Pre-Pro-P4HB Hygro. These vectors typically contain at least one restriction site for DNA linearization. Once within the yeast strain, the DNA can be inserted into the yeast genome and used to produce collagen fragments.

In some embodiments, the disclosure provides a yeast strain (e.g., pichia pastoris) genetically engineered to produce a recombinant collagen fragment described herein, wherein the yeast strain comprises a vector comprising a DNA sequence encoding the recombinant collagen fragment. In some embodiments, the vector may comprise a nucleic acid sequence comprising the DNA sequence set forth in SEQ ID NO 973. In some embodiments, the vector may comprise a nucleic acid sequence comprising the DNA sequence set forth in SEQ ID NO 974. In some embodiments, the vector is inserted into the yeast by electroporation.

In some embodiments, the present disclosure provides a vector comprising a DNA sequence for producing a collagen fragment, wherein the vector comprises the nucleic acid sequence set forth in SEQ ID NO 973.

In some embodiments, the present disclosure provides a vector comprising a DNA sequence for producing a collagen fragment, wherein the vector comprises the nucleic acid sequence set forth in SEQ ID NO 974.

In some embodiments, the present disclosure provides a vector comprising a DNA sequence for producing a collagen fragment, wherein the vector comprises a nucleic acid sequence set forth in any one of SEQ ID NO:1045 to SEQ ID NO: 1073.

In some embodiments, the present disclosure provides a yeast strain that produces a collagen fragment, the yeast strain comprising a vector comprising a DNA sequence for producing a recombinant collagen fragment described herein.

Promoters may improve the production of recombinant proteins, and may be included in vectors comprising sequences encoding a protein of interest (e.g., a collagen fragment as described herein). Suitable promoters for use in preparing the collagen fragments disclosed herein include, but are not limited to, AOX1 methanol inducible promoters, pDF de-repressed promoters, pCAT de-repressed promoters, das1-Das2 methanol inducible bi-directional promoters, pHTX1 constitutive bi-directional promoters, pGCW14-pGAP1 constitutive bi-directional promoters, and combinations thereof. Suitable methanol inducible promoters include, but are not limited to, AOX2, das1, das2, pDF, pCAT, pPMP, pFDH1, pFLD1, pTAL2, pFBA2, pPEX5, pDAK1, pFGH1, pRKI1, prsp 2, and combinations thereof.

A terminator may be placed at the end of each open reading frame utilized in the vector incorporated into the yeast. The DNA sequence of the terminator may be inserted into the vector. For replication vectors, an origin of replication is necessary to initiate replication. The DNA sequence of the origin of replication is inserted into a vector. One or more DNA sequences containing homology to the yeast genome may be incorporated into the vector to facilitate recombination and incorporation into the yeast genome or to stabilize the vector once transformed into a yeast cell.

The vector may also typically comprise at least one selectable marker for selecting for yeast cells that have been successfully transformed. Markers are sometimes associated with antibiotic resistance, and markers may also be associated with the ability to grow with or without certain amino acids (auxotrophic markers). Suitable auxotrophic markers include, but are not limited to ADE, HIS, URA, LEU, LYS, TRP and combinations thereof. To provide for selection of yeast cells containing the recombinant vector, at least one DNA sequence of a selectable marker may be incorporated into the vector.

The engineered yeast cells described above can be used as hosts to produce the collagen fragments described herein. To this end, the cells may be placed in a medium within a fermentation chamber and supplied with dissolved oxygen and a carbon source (e.g., glycerol) under controlled pH conditions for a period ranging from 12 hours to 1 week. Suitable media include, but are not limited to, buffered glycerol complex media (BMGY), buffered methanol complex media (BMMY), and yeast extract peptone glucose (YPD).

E.Application method

Increasing collagen production in cells

The inventors have surprisingly found that the collagen fragments described in the present disclosure can induce the production of type I collagen and type III collagen in cells such as fibroblasts. Thus, when administered to a cell, such as a fibroblast or other suitable cell, the collagen fragments described herein (i.e., collagen fragments having the amino acid sequence of SEQ ID NO:1 or SEQ ID NO:986, or compositions comprising such fragments) can advantageously induce collagen formation in the cell to which they are administered.

Thus, in some embodiments, the present disclosure provides methods of increasing collagen production in a cell comprising administering to the cell a collagen fragment described herein. Such administration may consist of directly exposing the cells to an amount of collagen fragments sufficient to induce the cells to produce greater amounts of collagen. In some embodiments, the method comprises administering the collagen fragment as part of a composition comprising the collagen fragment and at least one pharmaceutically acceptable excipient. In some embodiments, the collagen fragments can be administered to cells in their natural context, such as cells in an organism or tissue. In some embodiments, the collagen fragments may be topically applied to the skin. In some embodiments, the collagen fragments may be topically applied to human skin. In some embodiments, the collagen fragments may be applied to the cultured cells as part of the culture medium.

In some embodiments, the cells may be fibroblasts. In some embodiments, these cells may be fibroblasts in their natural context (e.g., within human skin). In some embodiments, the cells may be primary human fibroblasts. In some embodiments, the cells may be muscle cells, transformed human cells, cardiac muscle cells, endothelial cells, stem cells, or induced pluripotent stem cells.

In some embodiments, the recombinant collagen fragments or compositions comprising one or more fragments disclosed herein can be applied to a wound, such as a incision, laceration, split, tear, scratch, abrasion, or laceration, in order to increase collagen production at the wound site and/or surrounding tissue thereof. As part of the wound healing process, fibroblasts migrate to the wound site and produce collagen necessary for wound repair. These cells eventually fill the wound cavity with a network of interwoven collagen wires that duly align themselves into a firm band and form a permanent new tissue. Thus, in some embodiments, a recombinant collagen fragment according to SEQ ID NO. 1 or SEQ ID NO. 986, or a composition comprising the fragment, may be applied to a wound, such as a incision, laceration, split, laceration, scratch, abrasion, or laceration, in order to increase collagen production at the wound site and/or surrounding tissue thereof. In some embodiments, one or more hydrolysates according to the recombinant collagen fragment of SEQ ID NO. 1 or SEQ ID NO. 986 or compositions comprising such hydrolysates may be applied to a wound, such as a incision, laceration, split, tear, scratch, abrasion or laceration, in order to increase collagen production at the wound site and/or surrounding tissue thereof. In some embodiments, a composition comprising a recombinant collagen fragment according to SEQ ID No. 1 or SEQ ID No. 986 and a mixture of one or more hydrolysates of the recombinant collagen fragment may be applied to a wound, such as a incision, laceration, split, laceration, scratch, abrasion or laceration, in order to increase collagen production at the wound site and/or surrounding tissue thereof.

Thus, in certain embodiments, the present disclosure provides methods of applying a composition comprising a recombinant collagen fragment, a hydrolysate of a recombinant collagen fragment, or a combination thereof to a wound, such as a cut, laceration, split, tear, scratch, abrasion, or laceration. In certain embodiments, the wound may be a wound in a human subject. In some embodiments, the method comprises providing a composition for promoting collagen production and wound healing in the skin of a subject in need thereof, wherein the composition comprises a recombinant collagen fragment described herein (e.g., a fragment comprising SEQ ID NO:1 or SEQ ID NO: 986). In some embodiments, the methods comprise providing a composition for promoting collagen production and wound healing in the skin of a subject in need thereof, wherein the composition comprises one or more hydrolysates of the recombinant collagen fragments described herein (e.g., hydrolysates comprising one or more of SEQ ID NOs: 2 to 972). In some embodiments, the method comprises providing a composition for promoting collagen production and wound healing in the skin of a subject in need thereof as described herein, wherein the composition comprises a recombinant collagen fragment and a mixture of one or more hydrolysates of the recombinant collagen fragment (e.g., a mixture comprising the fragment of SEQ ID NO:1 or SEQ ID NO:986 and the hydrolysate comprising SEQ ID NO:2 to SEQ ID NO: 972).

In some embodiments, the method can include administering to the cell a collagen fragment having the amino acid sequence of SEQ ID NO. 1 or SEQ ID NO. 986. In some embodiments, the method can include administering to the cell a hydrolysate resulting from hydrolysis of a collagen fragment having the amino acid sequence of SEQ ID NO:1 or SEQ ID NO:986 (e.g., administering one or more hydrolysates having sequences according to SEQ ID NO:2 through SEQ ID NO: 972). In some embodiments, the method comprises administering to the cell a recombinant collagen fragment described herein, and one or more hydrolysates of the recombinant collagen fragment. In some embodiments, the method can include administering to the cell a collagen fragment having the amino acid sequence of SEQ ID NO. 1 or SEQ ID NO. 986, and one or more hydrolysates of the fragment having sequences according to SEQ ID NO. 2 through SEQ ID NO. 972.

In some embodiments, the method may increase the production of type I collagen. In some embodiments, the method may increase the production of type III collagen. In some embodiments, the method can increase production of both type I collagen and type III collagen.

Skin care

In some embodiments, the compositions described herein may be skin care compositions that are useful for treating an area of skin by topically applying the skin care composition to the area of skin.

In some embodiments, the compositions described herein may be skin care compositions that are useful for treating skin, hair, and nails by consuming the composition as a dietary supplement.

In some embodiments, applying the compositions described herein to skin can improve or maintain skin quality and reduce or eliminate signs of aging. Signs of aging include, but are not limited to, all externally visually and tactilely perceptible manifestations, any other macroscopic or microscopic effects due to skin aging. Such signs may be induced or caused by intrinsic or extrinsic factors, such as chronological age and/or environmental damage. These signs may be caused by processes that include, but are not limited to, development of tissue discontinuities such as wrinkles and deep wrinkles, fine lines, skin lines, fissures, ridges, macropores (e.g., associated with accessory structures such as sweat gland ducts, sebaceous glands, or hair follicles) or non-uniformities or roughness, loss of skin elasticity (loss and/or inactivation of functional skin elastin), sagging (including edema of the eye area and lower jaw), loss of skin tightness, loss of skin deformation recoil, discoloration (including eye circles), appearance of spots, sallowness, hyperpigmented skin areas such as age spots and freckles, keratoses, abnormal differentiation, hyperkeratosis, elastosis, collagen breakdown, and other histological changes in the dermis, epidermis, the skin vasculature (e.g., telangiectasia or spider vessels), and underlying tissues (e.g., fat and/or muscle), especially those that are close to the skin.

In some embodiments, the compositions as described herein may be suitable for use as dermal fillers. The dermal filler composition may replace the lost endogenous matrix polymer, or enhance/promote the function of existing matrix polymers, in order to treat skin conditions caused by aging or damage. The dermal filler composition can fill wrinkles, lines, folds, scars, and strengthen skin tissue, such as plumping the lips, or fill the depressed eyes or cheeks. Early dermal filler products were typically made from collagen. One common matrix polymer used in modern dermal filler compositions is hyaluronan. Because hyaluronan is natural to the human body, it is generally well tolerated and the risk of treatment for a variety of skin conditions is quite low.

In some embodiments, the compositions as described herein may be suitable for use with a microneedle sheet or patch. Microneedles may provide the compositions described herein to any area of the face and body to achieve cosmetic benefits or wound healing benefits.

In some embodiments, the compositions described herein may be therapeutic compositions useful for reducing or preventing scar tissue formation, promoting healing, promoting tissue regeneration, minimizing local inflammation, minimizing tissue rejection, and/or enhancing skin and/or hair graft integration.

In some embodiments, the present disclosure provides a method of treating a skin condition, the method comprising administering to a subject in need thereof an effective amount of a composition comprising a recombinant collagen fragment. In some embodiments, the skin condition may be fine lines, wrinkles, dry skin, enlarged pores, skin discoloration, reduced elasticity, unwanted hair, thinning skin, purpura, actinic keratosis, itching, eczema, acne, rosacea, erythema, telangiectasia, actinic telangiectasia, skin cancer, or hypertrophic rosacea.

In some embodiments, the composition may be topically applied to an area of skin. In some embodiments, the skin region may be selected from the group consisting of: facial surfaces, scalp, neck, ears, shoulders, chest (including breast and/or collar), arms, hands, legs, stomach, buttocks, groin, back, feet, and combinations thereof.

In some embodiments, the composition may be topically applied to the facial surface. In some embodiments, the facial surface may be selected from the group consisting of: forehead, eye, perioral surface, chin surface, orbit surface, nasal surface, cheek skin surface, and combinations thereof.

In some embodiments, the present disclosure provides a method for improving collagen production in skin, the method comprising administering an effective amount of a composition comprising a recombinant collagen fragment.

Description of the embodiments

E1. A recombinant collagen fragment having a molecular weight of about 50kDa and a sequence identity of at least about 85% with the amino acid sequence shown in SEQ ID No. 986.

E2. The recombinant collagen fragment according to embodiment 1, wherein the recombinant collagen fragment is non-hydroxylated.

E3. The recombinant collagen fragment according to embodiment 1, wherein the recombinant collagen fragment is hydroxylated.

E4. The recombinant collagen fragment according to embodiment 1, wherein the collagen fragment has the amino acid sequence shown in SEQ ID NO. 986.

E5. A sequence variant of the recombinant collagen fragment according to embodiment 1, wherein the sequence variant comprises an amino acid sequence shown in any one of SEQ ID NOs 987 to 1015.

E6. The sequence variant according to embodiment 5, wherein the sequence variant is non-hydroxylated.

E7. The sequence variant according to embodiment 5, wherein the sequence variant is hydroxylated.

E8. A composition comprising the recombinant collagen fragment according to any one of embodiments 1 to 4.

E9. A composition comprising a sequence variant according to any one of embodiments 5 to 7.

E10. The composition of embodiment 8, wherein the composition further comprises one or more peptides formed from the hydrolysis of a collagen fragment having the amino acid sequence shown in SEQ ID No. 986.

E11. The composition according to embodiment 10, wherein at least one of the one or more peptides formed from the hydrolysis of the collagen fragment having the amino acid sequence shown in SEQ ID No. 986 has an amino acid sequence according to one of SEQ ID No. 2 to SEQ ID No. 972.

E12. The composition according to any one of embodiments 8 to 11, further comprising a pharmaceutically acceptable excipient or a cosmetically acceptable excipient.

E13. A method of producing a recombinant collagen fragment according to any one of embodiments 1 to 4, the method comprising producing the recombinant collagen fragment in a genetically engineered yeast strain.

E14. The method according to embodiment 13, wherein the yeast is pichia pastoris.

E15. The method according to embodiment 13 or 14, wherein the yeast has been transformed with a plasmid comprising the nucleic acid sequence shown in SEQ ID NO. 973.

E16. The method according to any one of embodiments 13 to 15, wherein the yeast is a yeast which has been transformed with a plasmid comprising the nucleic acid sequence shown in SEQ ID NO 974.

E17. The method according to any one of embodiments 13 to 16, wherein the method comprises:

(i) Fermenting genetically engineered yeast in a fermentation broth;

(ii) Recovering from the fermentation broth a recombinant collagen fragment secreted by the genetically engineered yeast; and

(iii) Optionally, these recombinant collagen fragments are purified.

E18. The method of embodiment 17, further comprising hydroxylating the recombinant collagen fragment ex vivo.

E19. A method of producing a sequence variant according to any one of embodiments 5 to 7, comprising producing the recombinant collagen fragment in a genetically engineered yeast strain.

E20. The method according to embodiment 19, wherein the yeast is pichia pastoris.

E21. The method according to embodiment 19 or 20, wherein the yeast has been transformed with a plasmid comprising the nucleic acid sequence shown in any one of SEQ ID NOS: 1045 to 1073.

E22. The method according to any one of embodiments 19 to 21, wherein the method comprises:

(i) Fermenting genetically engineered yeast in a fermentation broth;

(ii) Recovering from the fermentation broth a sequence variant of the recombinant collagen fragment secreted by the genetically engineered yeast; and

(iii) Optionally, these recombinant collagen fragments are purified.

E23. The method according to embodiment 22, further comprising hydroxylating the recombinant collagen sequence variant ex vivo.

E24. A yeast strain genetically engineered to produce a recombinant collagen fragment according to any one of embodiments 1 to 4, wherein the yeast strain comprises a vector comprising a DNA sequence encoding the recombinant collagen.

E25. A yeast strain genetically engineered to produce a sequence variant according to any one of embodiments 5 to 7, wherein the yeast strain comprises a vector comprising a DNA sequence encoding the variant.

E26. The yeast strain of embodiment 24, wherein the vector comprises a nucleic acid sequence comprising the DNA sequence shown in SEQ ID NO. 973.

E27. The yeast strain of embodiment 26, further comprising a second vector comprising a nucleic acid sequence comprising the DNA sequence set forth in SEQ ID No. 974.

E28. The yeast strain of embodiment 25, wherein the vector comprises a nucleic acid sequence comprising a DNA sequence set forth in any one of SEQ ID NOS: 1045 to 1073.

E29. The yeast strain according to any one of embodiments 24 to 28, wherein the yeast strain is pichia pastoris.

E30. A method of treating a skin condition, the method comprising administering to a subject in need thereof an effective amount of the recombinant collagen fragment according to any one of embodiments 1 to 4 or the sequence variant according to any one of embodiments 5 to 7.

E31. A method of treating a skin condition, the method comprising administering to a subject in need thereof an effective amount of the composition according to any one of embodiments 8-12.

E32. The method of embodiment 31, wherein the skin condition comprises fine lines, wrinkles, dry skin, enlarged pores, skin discoloration, reduced elasticity, unwanted hair, thinning skin, purpura, actinic keratosis, itching, eczema, acne, rosacea, erythema, telangiectasia, actinic telangiectasia, skin cancer, or hypertrophic rosacea.

E33. The method according to embodiment 31 or 32, wherein the composition is topically applied to an area of skin.

E34. The method according to embodiment 33, wherein the skin area is selected from the group consisting of: facial surfaces, scalp, neck, ears, shoulders, chest (including breast and/or collar), arms, hands, legs, stomach, buttocks, groin, back, feet, and combinations thereof.

E35. A method of increasing collagen production in a cell, the method comprising administering to the cell an effective amount of a recombinant collagen fragment according to any one of embodiments 1 to 4 or a sequence variant according to any one of embodiments 5 to 7.

E36. The method of embodiment 35, wherein the method increases type I collagen production.

E37. The method of embodiment 34 or 35, wherein the method increases type III collagen production.

E38. The method according to any one of embodiments 35 to 37, wherein the cells are fibroblasts.

E39. The method according to any one of embodiments 35 to 38, wherein the cells are cultured cells.

E40. The method according to any one of embodiments 35 to 39, wherein the fragment or variant is formulated in a composition.

E41. The method according to any one of embodiments 35 to 41, wherein the fragment has the amino acid sequence shown in SEQ ID NO. 986.

E42. The method according to any one of embodiments 35 to 41, wherein the sequence variant has an amino acid sequence as set forth in any one of SEQ ID NO:987 to SEQ ID NO: 1015.

E43. A skin care product comprising the composition according to any one of embodiments 8 to 12 for reducing appearance of wrinkles, evening skin color, providing moisture, reducing appearance of dark under-eye circles, increasing collagen content of skin, increasing skin density, improving skin firmness and elasticity, improving appearance of fine lines and wrinkles, smoothing skin texture, increasing skin luster and brightness, improving appearance of sagging skin, whitening skin, or any combination thereof.

E44. A method of treating a wound in a human subject in need thereof, the method comprising administering to the wound of the subject a composition according to any one of embodiments 8 to 12, wherein administration of the recombinant collagen fragment induces production of human type I collagen, human type III collagen, or a combination thereof.

E45. The method of embodiment 44, wherein the collagen fragment is topically applied to the wound.

E46. A recombinant collagen fragment having a molecular weight of about 50kDa and a sequence identity of at least about 85% to the amino acid sequence set forth in SEQ ID No. 1.

E47. The recombinant collagen fragment according to embodiment 46, wherein the recombinant collagen fragment is non-hydroxylated.

E48. The recombinant collagen fragment according to embodiment 46, wherein the recombinant collagen fragment is hydroxylated.

E49. The recombinant collagen fragment according to embodiment 48, wherein the collagen fragment has the amino acid sequence shown in SEQ ID NO. 1.

E50. A recombinant collagen fragment comprising an amino acid sequence according to any one of SEQ ID NOs 2 to 972.

E51. A composition comprising a recombinant collagen fragment according to any one of embodiments 46 to 50.

E52. The composition of embodiment 51, wherein the composition further comprises one or more peptides formed from the hydrolysis of a collagen fragment having the amino acid sequence shown in SEQ ID No. 1.

E53. The composition according to embodiment 52, wherein at least one of the one or more peptides formed from the hydrolysis of the collagen fragment having the amino acid sequence shown in SEQ ID No. 1 has an amino acid sequence according to one of SEQ ID No. 2 to SEQ ID No. 972.

E54. The composition according to any one of embodiments 51 to 53, further comprising a pharmaceutically acceptable excipient or a cosmetically acceptable excipient.

E55. A method of producing a recombinant collagen fragment according to any one of embodiments 46 to 50, the method comprising producing the recombinant collagen fragment in a genetically engineered yeast strain.

E56. The method of embodiment 55, wherein the yeast is pichia pastoris.

E57. The method according to embodiment 55 or 56, wherein the yeast has been transformed with a plasmid comprising the nucleic acid sequence shown in SEQ ID NO. 973.

E58. The method according to any one of embodiments 55 to 57, wherein the yeast is a yeast that has been transformed with a plasmid comprising the nucleic acid sequence shown in SEQ ID NO 974.

E59. The method according to any one of embodiments 55 to 58, wherein the method comprises:

(i) Fermenting genetically engineered yeast in a fermentation broth;

(iii) Optionally, these recombinant collagen fragments are purified.

E60. The method of embodiment 59, further comprising hydroxylating the recombinant collagen fragment ex vivo.

E61. A yeast strain genetically engineered to produce a recombinant collagen fragment according to any one of embodiments 46 to 50, wherein the yeast strain comprises a vector comprising a DNA sequence encoding the recombinant collagen.

E62. The yeast strain of embodiment 61, wherein the vector comprises a nucleic acid sequence comprising the DNA sequence shown in SEQ ID NO. 973.

E63. The yeast strain of embodiment 62, further comprising a second vector comprising a nucleic acid sequence comprising the DNA sequence set forth in SEQ ID NO 974.

E64. The yeast strain of any one of embodiments 61 to 63, wherein the yeast strain is pichia pastoris.

E65. A method of treating a skin condition, the method comprising administering an effective amount of the recombinant collagen fragment according to any one of embodiments 46 to 50 to a subject in need thereof.

E66. A method of treating a skin condition, the method comprising administering to a subject in need thereof an effective amount of the composition according to any one of embodiments 51-54.

E67. The method of embodiment 66, wherein the skin condition comprises fine lines, wrinkles, dry skin, enlarged pores, skin discoloration, reduced elasticity, unwanted hair, thinning skin, purpura, actinic keratosis, itching, eczema, acne, rosacea, erythema, telangiectasia, actinic telangiectasia, skin cancer, or hypertrophic rosacea.

E68. The method of embodiment 66 or 67, wherein the composition is topically applied to an area of skin.

E69. The method of embodiment 68, wherein the skin region is selected from the group consisting of: facial surfaces, scalp, neck, ears, shoulders, chest (including breast and/or collar), arms, hands, legs, stomach, buttocks, groin, back, feet, and combinations thereof.

E70. A method of increasing collagen production in a cell, the method comprising administering to the cell an effective amount of a recombinant collagen fragment according to any one of embodiments 46 to 50.

E71. The method of embodiment 70, wherein the method increases type I collagen production.

E72. The method of embodiment 70 or 71, wherein the method increases production of type III collagen.

E73. The method according to any one of embodiments 70-72, wherein the cells are fibroblasts.

E74. The method according to any one of embodiments 70 to 73, wherein the cells are cultured cells.

E75. The method according to any one of embodiments 70-74, wherein the fragment is formulated in a composition.

76. The method according to any one of embodiments 70 to 75, wherein the fragment has the amino acid sequence shown in SEQ ID NO. 1.

E77. A skin care product comprising the composition according to any one of embodiments 51 to 54 for reducing appearance of wrinkles, evening skin color, providing moisture, reducing appearance of dark under-eye circles, increasing collagen content of skin, increasing skin density, improving skin firmness and elasticity, improving appearance of fine lines and wrinkles, smoothing skin texture, increasing skin shine and brightness, improving appearance of sagging skin, whitening skin, or any combination thereof.

E78. A method of treating a wound in a human subject in need thereof, the method comprising administering to the wound of the subject a composition according to any one of embodiments 51 to 54, wherein administration of the recombinant collagen fragment induces production of human type I collagen, human type III collagen, or a combination thereof.

E79. The method of embodiment 78, wherein the collagen fragment is topically applied to the wound.

Examples

Example 1: production of Pichia strains producing truncated human collagen III fragments

A plasmid encoding the secreted 50kDa human collagen of secretion signal having the amino acid sequence according to SEQ ID NO. 986 ("50 kDa human collagen fragment") and having bleomycin resistance was constructed as follows, designated vector A, and is shown in FIG. 1. To generate the secretion signal fragment, the vector of SEQ ID NO. 984 was amplified by Polymerase Chain Reaction (PCR) with primer 1 (SEQ ID NO. 975) and primer 2 (SEQ ID NO. 976). PCR was performed with PHUSION PLUSDNA POLYMERASE using an initial denaturation at 95 ℃ for 2 min, followed by 25 cycles of 30 sec at 95 ℃, 30 sec at 60 ℃ and 15 sec at 72 ℃ and a final extension at 72 ℃ for 5 min. To generate a 50kDa gel comprising the amino acid according to SEQ ID NO:986 The original fragment was amplified by PCR with primer 3 (SEQ ID NO: 981) and primer 4 (SEQ ID NO: 977) for the vector of SEQ ID NO:985, respectively. PCR was performed using an initial denaturation at 95 ℃ for 2 min, followed by 25 cycles of 30 seconds at 95 ℃, 30 seconds at 60 ℃ and 1 min at 72 ℃, and a final extension at 72 ℃ for 5 min. The MMV132 vector (see U.S.2019/0040400, incorporated herein by reference in its entirety) backbone was digested with an Mly I restriction endonuclease. All DNA fragments were purified by agarose gel electrophoresis. Using GibsonThe master mix (New England Biolabs) was assembled into pieces.

The resulting circular plasmid DNA was transformed into DH 5. Alpha. E.coli (E.coli). Transformants resistant to bleomycin were obtained after overnight growth at 37 ℃. The 50kDa collagen fragment construct was verified by Sanger sequencing. By PureLink ^TM Plasmid DNA from positive clones was purified using the HiPure plasmid extraction kit (Invitrogen). DNA was linearized by swai digestion.

For expression of the 50kDa collagen fragment, linearized plasmids were transformed into a strain of Pichia pastoris PP97 (French colt (Komagataella phaffii)) by electroporation with the BIO-RAD GENE PULSER XCELL total system. Transformants were grown on YPD agar containing 1M sorbitol and 500. Mu.g/mL bleomycin at 30 ℃. The resulting strain containing a 50kDa human collagen fragment under bleomycin resistance was designated Pichia pastoris strain 1.

To further improve recombinant protein expression, a second transformation of the 50kDa human collagen fragment construct was performed with a nociceptin N-acetyltransferase (NAT) tag. First, molecular cloning is performed to generate the necessary plasmid DNA. A novel vector encoding the signal sequence, the 50kDa human collagen fragment, NAT and beta-lactamase was produced as follows, and this vector was designated vector B (see FIG. 2). Vector a (fig. 1) was digested with Xba I and Eco RV restriction enzymes. The vector containing NAT resistant SEQ ID NO. 983 was digested with Nde I and Bsa I restriction enzymes. The vector fragments containing the 50kDa human collagen fragment and NAT resistant vector fragment, respectively, were purified from agarose gel. Fragments were ligated via Gibson assembly and transformed into DH 5. Alpha. Competent E.coli (E.coli). For the anti-carbenicillin transformants, the 50kDa human collagen fragment construct was verified by Sanger sequencing. Plasmid DNA was purified via medium extraction, linearized with Swa I restriction enzyme, and transformed into pichia pastoris strain 1. The resulting doubly transformed strain was designated pichia pastoris strain 2.

To identify pichia clones with high expression levels, small-scale cultures were grown in 96 deep-well blocks for 40 hours. The cells were pelleted by centrifugation and the supernatant harvested. Recombinant protein expression was assessed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Custom primary antibodies using C-terminal peptides against collagen (Abmart) Western blotting was performed with an 800CW goat anti-mouse IgG secondary antibody (LI-COR).

Screening for collagen production was performed by a sandwich enzyme-linked immunosorbent assay (ELISA). This assay uses the custom-made C-terminal peptide antibody (Abmart) as a capture antibody described above, as well as a commercially available detection antibody, anti-COL 3A horseradish peroxidase conjugate (sc-271249,Santa Cruz). The signal was generated with 3,3', 5' -Tetramethylbenzidine (TMB) and the reaction was quenched with 2M sulfuric acid. The signal was detected by measuring the absorbance at 450nm with a spectrophotometer. Using these methods, pichia pastoris strain 3 was selected as the best clone.

Example 2: fermentation of selected clones

The three-step fermentation process was started by propagating Pichia pastoris strain 3 clones in shake flasks, followed by seed fermentation for about 23-27 hours, which seeds were then used to seed the production fermentor. The production fermentor was run for about 72 hours, with a 24 hour batch phase followed by a 48 hour fed-batch phase. Glycerol was used as a carbon source and glycerol feed was started at the end of the batch phase to maintain a constant maximum OTR value of 150 mmol/L/hr. The OTR of 150 mmol/L/hr was maintained by a constant glycerol feed and by adjusting the initial pressure to an overpressure of 800mbar and a aeration of 1 vvm. By automatic addition of The pH was controlled to 6.+ -. 0.05 with 25% ammonium hydroxide and the temperature and DO were controlled to 32 ℃ and 25% (saturated at 800mbar overpressure), respectively. Sampling during the whole fermentation process and measuring OD ₆₀₀ Wet cell weight, dry weight, residual nitrogen and residual glycerol. (FIGS. 3A-3D).

Example 3: purification of truncated human collagen III fragments

Fermentation supernatant recovery

The fermentation broth comprises a supernatant containing secreted 50kDa human collagen having an amino acid sequence according to SEQ ID NO. 986 ("50 kDa human collagen fragment"), which is first recovered by centrifugation. Depending on the process scale, the supernatant was recovered by batch centrifugation using a Sorvall centrifuge (10 min,10 ℃,17568 Xg) or using a disc stack centrifuge. To recover the supernatant by disc stack centrifugation, the broth was diluted with deionized water to a Wet Cell Weight (WCW) of 200 g/kg. The disc stack centrifuge (GEA HFC-15) is operated at a maximum bowl speed and a feed rate of 350L/h. After the feed was passed through the centrifuge once, the discharged solids were diluted back to 200g/kg WCW before the second pass. The supernatants from the two passes were combined and the secondary clarification was continued. Flowcharts showing the purification process of the resulting 50kDa human collagen fragment are shown in FIGS. 5 and 6. Fig. 5 shows a purification method for preparing non-hydroxylated 50kDa human collagen, and fig. 6 shows a purification method for preparing hydroxylated 50kDa human collagen.

Filter cake filtration

As a secondary clarification step, cake filtration was performed using a metachem 470mm filter press. For each plate used, 0.379kg of Celite 512 diatomaceous earth was mixed with 20kg of water and applied as a precoat on the filter press. 0.5 wt% Celite 512 diatomaceous earth and 0.5 wt% Celite SuperCel Fine DE were added to the supernatant and material and passed through a filter press to remove residual particulate matter.

UF concentration

The pH was adjusted to 4 by adding 50% sulfuric acid before concentrating the collagen solution. The volume of the resulting solution was reduced to a minimum of 1/10 by cross-flow filtration using a spiral wound PES membrane of 10kDa cut-off.

Percolation

Diafiltration is performed to remove small impurities such as salts from the product. Prior to diafiltration, the pH was adjusted to 8 by the addition of acid (50% sulfuric acid) or base (10N sodium hydroxide) as necessary. In a 10kDa mPES hollow fiber moduleDiafiltration was performed in either a KR2i filtration carriage or an Alfa Laval M20 filtration carriage equipped with a 10kDa PES spiral wound module. Deionized water was used as the diafiltration medium and diafiltration was continued until the conductivity of the solution reached equilibrium.

Preservative agent

The diafiltered product was diluted by the addition of Deionized (DI) water to achieve 2% dissolved solids concentration, and during this dilution 1, 2-hexanediol and butanediol were added to achieve effective concentrations of 3% and 5%, respectively.

A graph depicting the overall recovery of a 50kDa human collagen fragment during various steps of its purification process and during all steps up to those various steps is shown in fig. 7.

Example 4: characterization of truncated human collagen III fragments

Mass Spectrometry (MS) and sequence analysis

The digestion in solution was performed using an S-Trap microcentrifuge column (ProtiFi, huntington, N.Y., USA) following the slightly modified Strap protocol as previously described. A25. Mu.L sample of protein (30. Mu.g) in 50mM TEAB pH 8.5, 6M urea, 2M thiourea, 1% SDS, 10mM DTT was reduced at 34℃for 1h, followed by alkylation with 50mM iodoacetamide in the dark at room temperature for 45min and then quenching with Dithiothreitol (DTT) at a final concentration of 40 mM. After quenching, 12% phosphoric acid was added to a final concentration of 1.2%. Thereafter, a 1:7 dilution (v/v) was performed with 90% methanol, 0.1M Ambic pH 7.9. The sample was then placed in an S-Trap spin column and centrifuged at 4000Xg for 30 seconds. Then washed three times with 150. Mu.L of 90% methanol, 0.1M Atmic 7.9. Digestion was performed with 125. Mu.L of LysC in 120 ng/. Mu.L (1:10 w/w) solution in 0.1M Ambic added to the top of the column. LysC solution was absorbed into a highly hydrophilic matrix, spin column and incubated overnight (16 hours) at 37 ℃. After incubation, the digested peptides were eluted from the S-trap column with 40. Mu.L of each 50mM TEAB pH 8.5 followed by 0.2% formic acid and finally 50% acetonitrile-0.2% formic acid. The three eluted peptide washes were pooled together and evaporated to dryness by Speedvac SC110 (Thermo Savant, milford, MA). Half (15. Mu.g) of the Lys digested peptide was reconstituted and digested with 33. Mu.L of chymotrypsin in 60 ng/. Mu.L solution in 50mM Ambic (1:7.5 w/w) for 16 hours at 37 ℃. Digestion was stopped by adding 10 μl of 5% fa and evaporated to dryness.

The doubly digested peptide was reconstituted in 150 μl of 0.5% formic acid ("FA") and 2.5 μl was injected into the nano LC-MS/MS system. LysC-chymotrypsin peptide was eluted at 300nL/min in a 60min gradient from 5% to 33% acn-0.1% FA. The operation of Orbitrap fusion is essentially the same as described above, switching the additional EThcD MS2 scan for 3-7 charged ions in the ion trap to the CID MS2 scan for 2-3 charged ions in a 3 second "highest speed" workflow. The calibrated ETD parameters (reaction time and maximum reagent injection time) were applied to ETD supplemented with additional 20% HCD activation energy to generate an EThcD MS2 spectrum.

By combining the results of both LysC and chymotrypsin sequential digestion and trypsin single digestion, 97.1% sequence coverage of the target sequence was obtained. The nucleotides in the reconstructed sequence were confirmed to be 100% identical to the relevant part of human collagen III.

Example 5: ex vivo hydroxylation reaction and analysis

Reaction

The fermentation supernatant containing non-hydroxylated 50kDa human collagen having the amino acid sequence according to SEQ ID NO. 986 (non-hydroxylated "50kDa human collagen fragment") was partially purified using salt precipitation. Ammonium sulfate was added as a salt to the precipitate to a final concentration of 800mM, the pH was adjusted to 4.+ -. 0.05 with 50% sulfuric acid, and then the supernatant was mixed and incubated at room temperature for 30 minutes. The 50kDa human collagen fragment was recovered from the supernatant by centrifugation at 17000Xg and the resulting pellet was resuspended in half volume of deionized water.

PP547 cell pellet was individually resuspended in lysis buffer (50 mM sodium phosphate) to a concentration of 30% w/w to 45% w/w. The pH of the cleavage feed was adjusted to 9.+ -. 0.05 with 2M citric acid and/or 2M sodium hydroxide. This feed was then split in bead powder according to standard splitting time, volume and cleaning methods.

The lysate was mixed together with the purified supernatant containing the 50kDa human collagen fragment on a magnetic stirrer under gentle agitation, and then 25mM to 65mM AKG, 25mM to 65mM DTT and 2mM to 5mM ascorbic acid were added as dry powders. During the addition of AKG, DTT and ascorbate, the pH was measured constantly and adjusted to avoid being lower than pH 6.5±0.05 (pH <6.5 inactivated P4H enzyme). Then 0.1mM to 0.25mM ferrous sulfate was added from the freshly prepared stock solution at 0.5M. This reaction feed was then transferred to a reaction vessel equipped with pH controls, temperature controls, mixing controls, and aeration controls. Once the reaction feed was added to the vessel, aeration was set at 1 to 2vvm, mixed to 150 to 500rpm depending on the tank OTR configuration, temperature was controlled to 26 to 32 ℃ and pH was controlled to 7.5±0.05. After 5 hours of reaction, the vessel pH was adjusted to 4±0.05 by automatic addition of 2M citric acid, the temperature of the reaction vessel was adjusted to 20 ℃ to 26 ℃, and nitrogen gas was bubbled through the system to maintain zero Dissolved Oxygen (DO). After 16 hours of incubation under the above conditions, clarification was performed once by centrifuging the reaction product at 17000 xg. After primary clarification, secondary clarification was performed in a cake filtration system using diatomaceous earth. The clarified reaction product was cooled to 12 ℃ and concentrated in an ultrafiltration system with a cut-off of 10kDa to reduce the volume to 1/10 to 1/15. The hydroxylated 50kDa collagen fragment was then precipitated from the solution using 800mM sodium sulfate. In some cases, further precipitation may be performed using 1M sodium chloride. The precipitated protein was then resuspended in deionized water and, depending on the end use of the 50kDa human collagen fragment, one of 0.01N HCl, 0.001N HCl or deionized water was used to remove residual salts by diafiltration using a 10kDa cut-off. As a final step, the diafiltration product was clarified by further centrifugation at 17,000Xg for 10 minutes to remove all particulate matter. Figure 8 shows the percent hydroxylation of the 50kDa human collagen fragment achieved over time during this ex vivo hydroxylation reaction. Fig. 9A and 9B show the thermograms of the 50kDa human collagen fragment before and after hydroxylation, respectively.

Analysis of hydrolyzed peptides

The final purified product contained the native protease co-purified with the 50kDa human collagen fragment. Incubation at room temperature (t=25℃) allowed the protease to further hydrolyze the 50kDa human collagen fragment to produce short collagen peptides. The final product was incubated at room temperature for three weeks to investigate the rate and extent of hydrolysis. After incubation for one and three weeks at room temperature (t=25℃), samples were analyzed by mass spectrometry. (FIG. 4). To examine the relative abundance and sequence of short collagen peptides, no enzymatic digestion was performed. MS spectra showed that the peptide distribution was unchanged after the first week, with nearly 70% of the short peptides (1 kDa to 6 kDa) being 1kDa to 1.5kDa in length. No peptides greater than 6kDa were measured in this assay. The peptide mapping showed that the detected peptide covered 97% of the sequence of the original recombinant collagen amino acid sequence (SEQ ID NO: 986). Peptides identified by mass spectrometry after one week of incubation are provided in table 1 above. Peptides identified by mass spectrometry after three weeks of incubation are provided in table 2 above.

Example 6: stimulation of collagen I and III synthesis by truncated human collagen III in fibroblasts

A fibroblast culture model was used to assess the ability of the test material to exert an effect on collagen synthesis. The study also assessed the viability of cells after exposure to the test material.

MTT assay

The change in cell number can be assessed by an MTT assay. MTT assay is a colorimetric assay of cellular metabolic activity reflecting the number of living cells. Mitochondrial reduction of MTT resulted in the formation of insoluble purple fomamzine crystals, which were extracted from cells with isopropanol and quantified spectrophotometrically. The intensity of the purple color is proportional to the metabolic activity of the cell and inversely proportional to the toxicity of the test material.

Fibroblast cells were inoculated into 0.5ml of Fibroblast Growth Medium (FGM) in individual wells of a 24-well plate at 37.+ -. 2 ℃ and 5.+ -. 1% CO ₂ Incubate overnight. The next day, the medium was removed by aspiration to eliminate any non-adherent cells and replaced with 0.5ml fresh FGM. Cells were grown until confluent, with medium changed every 48 to 72 hours. After fusion was reached, the cells were treated with DMEM supplemented with 1.5% fbs for 24 hours to wash out any effects of growth factors contained in normal medium. After this 24-hour washout period, the cells were treated with the test material at the indicated concentration in FGM with 1.5% FBS (see fig. 11A and 11B). TGF-B (50 ng/ml) was used as a positive control for inducing collagen expression. Untreated cells (negative control) received DMEM with only 1.5% FBS. As a negative control, cells were treated with 100 μm of bDcAMP in FGM with 1.5% FBS. Cells were incubated for 48 hours and cell culture medium was collected at the end of the incubation period and either stored frozen (-75 ℃) or assayed immediately. Materials were tested in triplicate.

The samples tested were the unhydroxylated 50kDa fragment of human type III collagen (SEQ ID NO: 986) ("hcol III 50kDa unhydroxylated"), the hydroxylated (28%) 50kDa fragment of human type III collagen (also SEQ ID NO: 986) ("hcol III 50kDa;28% hydroxylated), full length bovine collagen 3 with hydroxylation (7%), marine collagen (Ashland), arabinogen (Lipoid Kosmetik AG), recombinant human collagen 21 (Geltor) (" HumColl21 "), BIOLLAGEN (Jland Biotech) and full length bovine collagen with hydroxylation (45%) type III (" full length bcol3 "). Each collagen sample was diluted in a range of concentration percentages in tissue culture medium FGM containing 1.5% FBS (see fig. 11A and 11B). The non-hydroxylated 50kDa fragment of type III human collagen (SEQ ID NO: 986) was tested at 0.1 wt%, 0.05 wt%, 0.01 wt%, 0.005 wt% and 0.001 wt% in culture medium. Hydroxylated 50kDa fragment of type III human collagen (SEQ ID NO: 986) was tested at 0.01 wt%, 0.005 wt%, 0.001 wt%, 0.0005 wt% and 0.0001 wt% in culture medium. Full length type III bovine collagen with 7% hydroxylation was tested at 0.05 wt%, 0.01 wt%, 0.005 wt%, 0.001 wt% and 0.0005 wt% in medium. Marine collagen was tested at 1 wt%, 0.5 wt%, 0.01 wt%, 0.05 wt% and 0.01 wt% in culture medium. The arabinoxylans were tested at 1 wt%, 0.5 wt%, 0.01 wt%, 0.05 wt% and 0.01 wt% in the medium. Human collagen 21 was tested at 0.1 wt%, 0.05 wt%, 0.01 wt%, 0.005 wt% and 0.001 wt% in the medium. Human collagen 21 was tested at 0.1 wt%, 0.05 wt%, 0.01 wt%, 0.005 wt% and 0.001 wt% in the medium. Human collagen 21 was tested at 0.1 wt%, 0.05 wt%, 0.01 wt%, 0.005 wt% and 0.001 wt% in the medium. Full length bovine type III collagen with 45% hydroxylation was tested at 0.015 wt%, 0.003 wt%, 0.0015 wt%, 0.0003 wt% and 0.00015 wt% in culture medium.

After 2 days incubation, the cell culture medium was removed (see above) and the fibroblasts were washed twice with PBS to remove all remaining test material. After the last wash, 500. Mu.l of DMEM supplemented with 0.5mg/ml MTT was added to each well and the cells were incubated at 37.+ -. 2 ℃ and 5.+ -. 1% CO ₂ Incubate for 1 hour. After incubation, DMEM/MTT solution was removed and cells were washed once more with PBS, then 0.5ml isopropanol was added to the wells to extract purple fomamzine crystals. 200 microliters of isopropyl extract was transferred to a 96-well plate and the plate was read at 540nm using isopropyl alcohol as a blank. The mean MTT uptake value of the negative control cells was calculated and used to represent 100% cell viability. Individual MTT values from cells undergoing various treatments were then divided by the average of negative control cells and expressed as a percentage to determine the change in cell viability caused by each treatment.

The results of the MTT assay are shown in fig. 11A and 11B. As shown, no effect of the tested collagen on cell viability was observed. The viability of each treated cell sample was at least as great as untreated control cells.

Type I collagen assay

Fibroblasts are the primary source of extracellular matrix peptides, including collagen. Procollagen (Procollagen) is a large peptide synthesized by fibroblasts in the dermis layer of the skin and is a precursor of collagen. When the pro-collagen is processed to form mature collagen, the propeptide portion is cleaved off as a type I C-peptide. The mature collagen and type I C-peptide fragments are then released into the extracellular environment. With collagen synthesis, the type I C-peptide fragments accumulate in the tissue culture medium. Because of the 1:1 stoichiometric ratio between the two parts of the procollagen peptide, the determination of type I C-peptide reflects the amount of collagen synthesized. To measure the effect of different forms of collagen on collagen synthesis and secretion, type 1C-peptide was determined via ELISA-based methods.

Fibroblast cells were inoculated into 0.5ml of Fibroblast Growth Medium (FGM) in individual wells of a 24-well plate at 37.+ -. 2 ℃ and 5.+ -. 1% CO ₂ Incubate overnight. The next day, the medium was removed by aspiration to eliminate any non-adherent cells and replaced with 0.5ml fresh FGM. Cells were grown until confluent, with medium changed every 48 to 72 hours. After fusion was reached, the cells were treated with DMEM supplemented with 1.5% fbs for 24 hours to wash out any effects of growth factors contained in normal medium. After this 24-hour washout period, cells were treated with the following test materials at the indicated concentrations (see fig. 12A and 12B) in FGM with 1.5% FBS. TGF-B (50 ng/ml) was used as a positive control for inducing collagen expression. Untreated cells (negative control) received DMEM with only 1.5% fbs. Cells were incubated for 48 hours and cell culture medium was collected at the end of the incubation period and either stored frozen (-75 ℃) or assayed immediately. Materials were tested in triplicate.

The samples tested were the non-hydroxylated 50kDa fragment of human type III collagen (SEQ ID NO: 986), the hydroxylated (28%) 50kDa fragment of human type III collagen (also SEQ ID NO: 986), full length bovine collagen 3 with hydroxylation (7%), marine collagen (Ashland), arabinogen (Lipoid Kosmetik AG), recombinant human collagen 21 (Geltor), BIOLLAGEN (Jland Biotech) and full length bovine type III collagen with hydroxylation (45%). Each collagen sample was diluted in a range of concentration percentages in tissue culture medium FGM containing 1.5% FBS (see fig. 11A and 11B). The non-hydroxylated 50kDa fragment of type III human collagen (SEQ ID NO: 986) was tested at 0.1 wt%, 0.05 wt%, 0.01 wt%, 0.005 wt% and 0.001 wt% in culture medium. Hydroxylated 50kDa fragment of type III human collagen (SEQ ID NO: 986) was tested at 0.01 wt%, 0.005 wt%, 0.001 wt%, 0.0005 wt% and 0.0001 wt% in culture medium. Full length type III bovine collagen with 7% hydroxylation was tested at 0.05 wt%, 0.01 wt%, 0.005 wt%, 0.001 wt% and 0.0005 wt% in medium. Marine collagen was tested at 1 wt%, 0.5 wt%, 0.01 wt%, 0.05 wt% and 0.01 wt% in culture medium. The arabinoxylans were tested at 1 wt%, 0.5 wt%, 0.01 wt%, 0.05 wt% and 0.01 wt% in the medium. Human collagen 21 was tested at 0.1 wt%, 0.05 wt%, 0.01 wt%, 0.005 wt% and 0.001 wt% in the medium. Human collagen 21 was tested at 0.1 wt%, 0.05 wt%, 0.01 wt%, 0.005 wt% and 0.001 wt% in the medium. Human collagen 21 was tested at 0.1 wt%, 0.05 wt%, 0.01 wt%, 0.005 wt% and 0.001 wt% in the medium. Full length bovine type III collagen with 45% hydroxylation was tested at 0.015 wt%, 0.003 wt%, 0.0015 wt%, 0.0003 wt% and 0.00015 wt% in culture medium.

For ELISA assays, a series of type I C-peptide standards ranging from 0ng/ml to 640ng/ml were prepared. Next, ELISA microplates were prepared by removing any unwanted strips from the plate frame, followed by adding 100. Mu.l of peroxidase-labeled anti-procollagen type I-C peptide antibody to each well used in the assay. Twenty (20) μl of the sample (collected tissue culture medium) or standard was then added to the appropriate wells and the microwells were covered and allowed to incubate at 37 ℃ for 3±0.25 hours.

After incubation, wells were aspirated and washed three times with 400 μl wash buffer. After the last wash was removed, 100 μl of peroxidase substrate solution (hydrogen peroxide+tetramethylbenzidine as chromophore) was added to each well and the plate was incubated for 15±5 minutes at room temperature. After incubation, 100 μl of stop solution (1N sulfuric acid) was added to each well and the plates were read at 450nm using a microplate reader. To quantify the amount of each species present, a standard curve is generated using known concentrations of each species. Regression analysis is performed to establish a line that best fits the data points. The absorbance values of the test material and untreated samples were used to estimate the amount of each species present in each sample. The mean values were compared using ANOVA, with n=3 for each treatment. Statistical significance was set to p <0.05.

The results of type I collagen assay are shown in fig. 12A and 12B. These results were obtained after treating cells with a range of weight percent concentrations of each collagen solution. These data show that 0.1% of the 50kDa human collagen fragment (SEQ ID NO: 986) unexpectedly increases the amount of type I collagen secreted by the treated fibroblasts. Treatment with 0.1 wt% (final concentration) of the 50kDa human collagen fragment increased collagen I expression by more than 200% relative to untreated cells. Although effects were observed with the non-hydroxylated 50kDa human collagen fragment (SEQ ID NO: 986) rather than hydroxylated collagen, this difference could be attributed to the fact that hydroxylated collagen was not tested at the highest concentration (0.1%). Cells were treated separately with a series of different collagens at equal or even higher concentrations. Cells were treated with a series of collagens of similar size or smaller to the 50kDa human collagen fragment, in particular hydrolysed marine collagen, acacia source, human collagen 21 and BIOLLAGEN. These similarly sized collagens had no effect on the production of type I collagens, even when those collagens were tested at a 10-fold percent concentration of the 50kDa fragment. Cells were treated with full-length bovine collagen III at about 7% hydroxylation and about 45% hydroxylation. Full-length bovine collagen III is capable of inducing collagen I expression, but only when it is highly hydroxylated (about 45%). Thus, these data indicate that compositions comprising a 50kDa human collagen fragment are useful when increased production of type I collagen is desired.

Type III collagen assay

Type III collagen is synthesized as a large propeptide by dermal fibroblasts. When the peptide is processed to form mature type III collagen, the propeptide portion is cleaved off (type III N-peptide). The mature collagen and type III N-peptide fragments are then released into the extracellular environment. With collagen synthesis, the type III N-peptide fragments accumulate in the tissue culture medium. Because of the 1:1 stoichiometric ratio between the two parts of the procollagen peptide, the determination of type III N-peptide reflects the amount of collagen synthesized. To measure the effect of different forms of collagen on collagen synthesis and secretion, type III N-peptides were determined via ELISA-based methods.

Fibroblast cells were inoculated into 0.5ml of Fibroblast Growth Medium (FGM) in individual wells of a 24-well plate at 37.+ -. 2 ℃ and 5.+ -. 1% CO ₂ Incubate overnight. The next day, the medium was removed by aspiration to eliminate any non-adherent cells and replaced with 0.5ml fresh FGM. Cells were grown until confluent, with medium changed every 48 to 72 hours. After fusion was reached, the cells were treated with DMEM supplemented with 1.5% fbs for 24 hours to wash out any effects of growth factors contained in normal medium. After this 24-hour washout period, cells were treated with the following test materials at the indicated concentrations (see fig. 13A and 13B) in FGM with 1.5% FBS. TGF-B (50 ng/ml) was used as a positive control for inducing collagen expression. Untreated cells (negative control) received DMEM with only 1.5% fbs. Cells were incubated for 48 hours and cell culture medium was collected at the end of the incubation period and either stored frozen (-75 ℃) or assayed immediately. Materials were tested in triplicate.

The samples tested were the non-hydroxylated 50kDa fragment of human type III collagen (SEQ ID NO: 986), the hydroxylated (28%) 50kDa fragment of human type III collagen (SEQ ID NO: 986), full length bovine collagen 3 with hydroxylation (7%), marine collagen (Ashland), acacia senegal (Lipoid Kosmetik AG), recombinant human collagen 21 (Geltor), BIOLLAGEN (Jland Biotech) and full length bovine type III collagen with hydroxylation (45%). Each collagen sample was diluted in a range of concentration percentages in tissue culture medium FGM containing 1.5% FBS (see fig. 11A and 11B). The non-hydroxylated 50kDa fragment of type III human collagen (SEQ ID NO: 986) was tested at 0.1 wt%, 0.05 wt%, 0.01 wt%, 0.005 wt% and 0.001 wt% in culture medium. Hydroxylated 50kDa fragment of type III human collagen (SEQ ID NO: 986) was tested at 0.01 wt%, 0.005 wt%, 0.001 wt%, 0.0005 wt% and 0.0001 wt% in culture medium. Full length type III bovine collagen with 7% hydroxylation was tested at 0.05 vol%, 0.01 vol%, 0.005 vol%, 0.001 vol% and 0.0005 vol% in medium. Marine collagen was tested at 1%, 0.5%, 0.01%, 0.05% and 0.01% by volume in the medium. The arabinoxylans were tested at 1%, 0.5%, 0.01%, 0.05% and 0.01% by volume in the medium. Human collagen 21 was tested at 0.1%, 0.05%, 0.01%, 0.005% and 0.001% by volume in the medium. Human collagen 21 was tested at 0.1%, 0.05%, 0.01%, 0.005% and 0.001% by volume in the medium. Human collagen 21 was tested at 0.1%, 0.05%, 0.01%, 0.005% and 0.001% by volume in the medium. Full length bovine type III collagen with 45% hydroxylation was tested at 0.015%, 0.003%, 0.0015%, 0.0003% and 0.00015% by volume in culture medium.

For ELISA assays, a series of standards were prepared and 100. Mu.l of these standards or samples were added to the wells of a type III collagen ELISA plate. Plates were then incubated at 37℃for 1.5 hours. After incubation, the ELISA plates were then washed twice with wash buffer, followed by 100 μl of detection solution. The ELISA plates were then incubated at 37℃for 1 hour. After incubation, all ELISA plates were washed with wash solution, followed by addition of 100 μl HRP conjugate solution and incubation at 37 ℃ for 30 minutes. After incubation, the ELISA plate was again washed and 100 μl of substrate solution was added to each well, and the well plate was incubated at room temperature for 10-30 minutes to allow the chromogenic reaction to occur. At the end of the chromogenic reaction, 100 μl of stop solution was added to each well and the plate was read at 460nm using a plate reader. To quantify the amount of each species present, a standard curve is generated using known concentrations of each species. Regression analysis is performed to establish a line that best fits the data points. The absorbance values of the test material and untreated samples were used to estimate the amount of each species present in each sample. The mean values were compared using ANOVA, with n=3 for each treatment. Statistical significance was set to p <0.05.

The results of type III collagen assay are shown in fig. 13A and 13B. These results were obtained after treating cells with a range of weight percent concentrations of each collagen solution. These results show that treatment with 0.1% of the 50kDa human collagen fragment (SEQ ID NO: 986) unexpectedly increased the amount of type III collagen secreted by the treated fibroblasts by more than 200% relative to untreated cells. A smaller but still significant effect was also observed after treatment with 0.01% of the 50kDa human collagen fragment (SEQ ID NO:986; both hydroxylated and unhydroxylated). No effect on collagen III was observed after treatment with bovine collagen III (7% hydroxylated), arabinogen, human collagen 21 or biolagen, even when these collagens were tested at a percentage concentration up to 10-fold higher than the 50kDa fragment. Full-length bovine collagen III is capable of inducing collagen I expression, but only when it is highly hydroxylated (about 45%). Marine collagen induced effects on collagen III expression only when tested at a 10-fold higher percentage concentration of the 50kDa human collagen fragment. Thus, these data indicate that compositions comprising a 50kDa human collagen fragment are useful when increased production of type III collagen is desired.

Example 7: additional characterization of truncated human collagen III fragments

An additional measurement was performed on the 50kDa human collagen fragment (SEQ ID NO: 986) to further characterize the fragment.

Physical Properties

The solubility and properties of the purified non-hydroxylated preparation and the purified hydroxylated preparation of the 50kDa human collagen fragment (SEQ ID NO: 986) in various solutions were evaluated. These properties were compared to those of full-length bovine collagen 3. The following samples were each dissolved in deionized water at a concentration of 1 wt%, and stored with 1% phenoxyethanol:

1. non-hydroxylated 50kDa fragment of human collagen 3

2. Hydroxylated (28%) 50kDa fragment of human collagen 3

3. Full-length bovine collagen 3 with 7% hydroxylation

4. Full-length bovine collagen 3 with 45% hydroxylation

The solubilized collagen solution was compared with COLLUME (2%; geltor), human collagen 21 (2%; geltor) and marine collagen NPNF (Tri-K), each of which was treated in the same manner. The results of the analysis are shown in table 14. The 50kDa collagen fragment tested is soluble and non-adhesive with little or no color or odor.

Four collagen samples were also added at 3 wt% to seven base formulations to test compatibility and performance. These base solutions are gels (carbomer system), essences (acrylamide/VP copolymer and acrylate/C10-30 alkyl acrylate cross-linked polymer system), cream emulsions (oil/water), hair conditioners (containing cetyltrimethylammonium chloride and behenyl trimethylammonium methyl sulfate), shampoos/cleaners (sulfate-free), water-based toners and alcohol-based toners. The non-hydroxylated collagen was compatible with all the base formulations tested, while the hydroxylated collagen was compatible with all the base formulations except the alcohol-based toner. Furthermore, within the scope of the base formulation tested, the 50kDa human collagen fragments exhibited greater compatibility than full-length bovine collagen.

These results demonstrate that the 50kDa human collagen fragment is a viable material for potential use in personal care formulations, provided that it is properly incorporated into a suitable formulation. Typically, it exhibits good color and odor and can be easily incorporated into the tested formulation. The properties of the collagen fragments provide possible benefits in skin care formulations in terms of enhancing elasticity, reducing wrinkles, smoothing, preventing moisture loss, tightening and film formation. No upper level of use of these materials in certain formulation types is observed in terms of aesthetics or compatibility. No negative skin feel was observed for any 1% solution at any of the inclusion levels.

HRI for skin irritationPT test

To determine the stimulatory and allergenic (contact allergy) potential of the 50kDa human collagen fragment (SEQ ID NO: 986), a volunteer study was performed in which collagen solutions were repeatedly applied to human skin. The study involved testing of hydroxylated and non-hydroxylated collagen fragment solutions.

A patch containing a 7.5mm paper tray was immersed in a solution containing 2 wt% of a 50kDa human collagen fragment (SEQ ID NO: 986). The patch was then secured directly to the skin of the intrascapular region of the back, either to the right or left of the midline, and the subject was excluded using either a non-wetting test area or a direct sunlight exposure of the test area. One patch was administered to each patient for each collagen solution tested. After the first application, the patch was left in place for 48 hours. Subjects were instructed not to remove the patch until their planned 48 hour visit. Thereafter, the subjects were instructed to remove the patch 24 hours after administration for the remainder of the study. The procedure was repeated until a series of nine consecutive 24 hour exposures three times a week had been made for three consecutive weeks. The test sites were assessed by trained laboratory personnel prior to each reapplication. A retest/challenge dose was administered once to the previously unexposed test site after a 10-14 day rest period. Retested sites were assessed by trained laboratory personnel 48 hours and 96 hours after administration.

For any collagen solution, no adverse events of any kind were reported during the course of the study in any of the 55 test subjects. In contrast, administration of the positive control (2% sodium lauryl sulfate solution) resulted in one subject experiencing a grade 4 response (erythema, induration, and bulla) and three subjects experiencing a grade 1 response (erythema at least three quarters of the patch area). Thus, there is no indication that there is a possibility of noticing skin irritation or sensitization (contact allergy) to the 50kDa human collagen fragment.

Example 8: cosmetic composition

To characterize the application of a 50kDa human collagen tablet on human skinThe effect of the paragraph (SEQ ID NO: 986), a volunteer study was performed in which collagen solutions were repeatedly applied to human skin. Two different formulations were administered, each of these formulations comprising a 50kDa human collagen fragment in a composition comprising 1.5% xanthan @CG-BT), 0.2% potassium sorbate, 0.3% sodium benzoate, and 0.05% citric acid, pH 5.2. Formulation 1 contained a 50kDa human collagen fragment at a final concentration of 0.05%, while formulation 2 contained a 50kDa human collagen fragment solution at a final concentration of 0.002%. Twenty-five subjects were administered formulation 1 daily for 12 weeks, while 24 subjects were administered formulation 2 daily for 12 weeks.

Objective clinical efficacy assessment was performed by clinical grading specialists before the start of administration, 6 weeks after administration and 12 weeks after administration. The grader checked the skin using a 10-point sequence scale (10-Point Ordinal Scales on the Face) on the face: texture and smoothness (vision), skin uniformity, firmness (vision), gloss, sagging, and fine lines/wrinkles. With respect to the degree of improvement, the results of these evaluations are shown in fig. 16A. Statistically significant improvements in skin texture/smoothness (vision), skin tone uniformity, firmness (vision), sagging, and fine lines/wrinkles were observed after 6 and 12 weeks of use of the dose 1 and dose 2 products. For gloss, statistically significant improvement was observed after 6 and 12 weeks of use of the dose of product, but only after 12 weeks of use of the dose of 2.

Subjects also performed subjective self-assessment 6 weeks and 12 weeks after administration. These assessments take the form of a series of questions presented to the subject, as listed in fig. 16B and 16C. Broadly, formulation 1 and formulation 2 were uniformly characterized as resulting in an improvement in skin condition, with greater than 50% of patients noticing an improvement (i.e., consent or strong consent) with respect to almost all problems.

Using(Astron clinical Ltd.) spectrophotometric Pi Nafen analysis (SIA) was performed to determine collagen levels in the skin before and after 6 weeks and 12 weeks of application of either formulation 1 or formulation 2. As shown in fig. 16D, administration of both formulation 1 and formulation 2 resulted in 18% increase in collagen levels after 6 weeks of administration and 25% increase in collagen after 12 weeks of administration, with almost all patients having some improvement in collagen.

The viscoelastic properties (firmness and elasticity) of the skin were measured by applying suction to the skin surface using skin elasticity tester MPA 580 (courage+khazaka, germany). A statistically significant reduction in RO was observed after 12 weeks of use of the product compared to T0 for dose 1, indicating an improvement in skin firmness. A statistically significant increase in elasticity was observed after 6 and 12 weeks of use of the product compared to T0 for dose 1 and dose 2.

Example 9: generation of truncated human collagen III fragment sequence variants

A plasmid encoding a human collagen variant secreted by a secretion signal having an amino acid sequence according to SEQ ID NO. 987 to SEQ ID NO. 1015 was constructed. The plasmid DNA is shown as the nucleic acid sequence according to SEQ ID NO. 1045 to SEQ ID NO. 1073.

As in example 1, each plasmid was transformed into a strain of Pichia pastoris PP97 by electroporation using the BIO-RAD GENE PULSER XCELL total system. The resulting strain was used to express the variants. In some cases, the strain is doubly transformed to improve recombinant protein expression. Then, the three-step fermentation method of example 2 was used.

Example 10: preparation and analysis of collagen variant samples

Preparation of samples

Fermentation harvest

The broth is removed from the fermentor and mixed at about 200RPM to 400RPM to ensure homogeneity of the solution. Then, the cells were removed from the fermentation supernatant. The fermentation broth was transferred to a 1L Sorvall flask and centrifuged at maximum speed (17,218 g) for 20min at 20 ℃. The supernatant was poured into a 1L to 5L container and the pellet was discarded. The fermentation supernatant was stored in a-20 ℃ refrigerator until ready for use.

pH and ammonium sulfate precipitation and recovery

The fermentation supernatant was thawed in a room temperature water bath. 1M ammonium sulfate was added to the fermentation supernatant and then mixed at about 200RPM to 400RPM to ensure that the solution was homogeneous. The amount of ammonium sulfate mass required to target the concentration of 1M was calculated and added according to the following equation. Density=1.07 kg/L

Density=1.07 kg/L

The pH was adjusted to 4.+ -. 0.05 with 50% sulfuric acid. And the solution was incubated for 30 minutes while mixing at about 200RPM to 400RPM to ensure homogeneity of the solution. The pH was checked again and adjusted back to pH 4.+ -. 0.05 by adding 50% sulfuric acid or 10N NaOH if necessary. The pH/salt precipitated pellets were recovered. The solution was transferred to a 1L Sorvall flask and centrifuged at maximum speed (17,218 g) for 20min at 4 ℃. The supernatant was then decanted into a beaker and the waste discarded. The precipitated protein pellets were resuspended in water and the fermentation supernatant stored in a-20 ℃ freezer until ready for use.

Ultra-filtration

The resuspended protein solution was thawed in a room temperature water bath. The system was provided with a 10kD hollow fiber filter and a water flux test was performed. The 0.1N NaOH storage solution was drained from the membrane housing and disposed of, and the system was rinsed with water. Clean water flux at transmembrane pressures (TMP) of about 3psi, 5psi and 10psi was performed. Then, 10x phosphate salt aqueous solution was added to the resuspended pellets and pH was adjusted. The solution was mixed at about 200RPM to 400RPM until the solution was homogeneous. The amount of 10X PBS was calculated according to the following equation and added to achieve a final concentration of 1X. * Let density=1 g/mL

If necessary, the pH was adjusted to 7.2 by adding 10N NaOH. Any insoluble particles were removed and the solution was transferred to a 1L Sorvall flask and centrifuged at maximum speed (17,618 g) for 20min at 4 ℃. The supernatant was decanted into a beaker for further processing and the pellet waste was discarded. The resuspended protein solution was concentrated by: the pump was set at 1020mL/min, back pressure was applied to achieve a transmembrane pressure (TMP) of 20psi to 25psi, and concentrated up to 200mL.

The concentrated protein solution was diafiltered with 3 diafiltration volumes of water by: the pump was set at 1020mL/min, back pressure was applied to achieve a 20psi to 25psi TMP, and water was used to diafiltration until 3 diafiltration volumes were achieved. An inspection was performed to confirm that total solids >1.5%. If the total solids is <1.5%, the protein solution is concentrated to a minimum volume (about 75 mL). The product was removed from the system and stored in a-20 ℃ freezer until ready for use. A water rinse was performed, the system was subjected to a clean-in-place solution (CIP) containing a 1% CIP 100 solution, and the membrane was placed in a 0.1M sodium hydroxide storage solution.

Formulations

The diafiltered protein solution was thawed in a room temperature water bath and 10x Phosphate Buffered Saline (PBS) was added. The solution was mixed at about 200RPM to 400RPM until the solution was homogeneous. The amount of 10x Phosphate Buffered Saline (PBS) volume required to target the 1x final concentration was calculated using an excel calculator.

* Let density=1 g/mL

The pH was adjusted to 7.2.+ -. 0.1 (pH 7.1-7.3) using concentrated hydrochloric acid (HCl) or 10N sodium hydroxide (NaOH). The final formulated product was vacuum sterile filtered in a biosafety cabinet with a 0.2 μm sterile bottle cap filter and evaluated.

Data collection method

SDS-PAGE of proteins

Protein quantification, monitoring and detection are provided by electrophoretic separation via sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Standards were prepared gravimetrically at concentrations of 0.25g/L, 0.125g/L and 0.0625g/L to create standard curves. The working solution for each standard was stored at 4 ℃. Based on the estimated concentration, the samples were diluted with deionized water to meet the standard curve. Lithium Dodecyl Sulfate (LDS) running buffer was added as a 1:1 solution to each individual standard and sample. It is heated to break down disulfide bonds and denature proteins so that they can pass through the pores of the Tris acetate gel. Tris acetate Gel was placed in Midi Gel pot. Tris-acetate buffer (1X) was added to the gel cassette. In SDS-PAGE, each gel contained the following: molecular weight markers, standard curve points, samples and positive controls. Once standards and samples were loaded into the gel wells, the gel cassettes were covered with power cables connected to electrophoresis at 125 volts and 300 watts for one hour, twenty minutes.

The gel was removed from the plastic box and placed in a Licor incubation box for a two hour staining procedure using Page-Blue protein staining solution and placed on an orbital shaker under gentle agitation.

The Page-Blue solution was washed away and the gel-stripping process was started using 10% methanol. The gel was incubated on an orbital shaker with gentle agitation for 60 minutes. Then, methanol was washed away and the gel was further destained with Millipore water. The gel was incubated on an orbital shaker with gentle agitation for 60 minutes.

The gel was Image scanned using an Image lab densitometer. Samples positive for collagen expression have strong band migration at the appropriate molecular weight corresponding to the positive control. These bands were analyzed using the application "app v2.01 TT". A CSV excel file is created and added to the corresponding "qSDS template".

Using a statistical control chart, each gel was subjected to a quality control check to ensure that all system applicability parameters were met before reporting the data.

Sequence verification of proteins (LC/MS analysis)：

All protein samples were subjected to sequence verification by LC/MS as in section above: enzymatic digestion of proteins (for LC/MS analysis) digestion is performed as described in the following. After enzymatic digestion, the identified peptide sequences of the digested peptides were matched to the theoretical protein sequence to calculate sequence coverage (calculated by dividing the number of amino acids in all peptides found by the total number of amino acids in the entire protein sequence). Sequence coverage and detection maps of the target variant proteins identified in each sample were generated for visualization using Byonic software (Protein Metrics inc., san Carlos, CA).

In addition to the% total sequence coverage, additional sequence validation calculations (% potential sequence coverage) were calculated to address the identification limitations of LC/MS instruments. This calculation omits peptides present in the digested sample that are too small to be detected by the LC/MS instrument in its current setting. Thus, the% potential sequence coverage is calculated as the total number of amino acids in all peptides found divided by the total number of amino acids in the peptide that might be detected by LC/MS in its current acquisition mode. The% unexplained sequence is calculated as the complementary sequence with% potential sequence coverage, representing the portion of the protein sequence that should be seen after digestion based on the trypsin digestion rules but not with the matching peptide.

Glycosylation analysis of proteins (LC/MS analysis)：

All protein samples were subjected to sequence verification by LC/MS as in section above: enzymatic digestion of proteins (for LC/MS analysis) digestion is performed as described in the following. After enzymatic digestion, mass spectrometry raw data, agilent x.d files, were processed using Byonic and byogic software (Protein Metrics inc., san Carlos, CA) and searched for the corresponding homemade variant Protein sequences. The Byonic search parameters were set to include digestion of peptides cleaved at the C-terminus to Arg residues and Lys residues using fully specific trypsin, allowing up to two missed cleaves. The precursor mass tolerance was set to 10ppm and the fragment mass tolerance was 0.02Da, with QTOF/HCD fragmentation pattern. Post-translational modifications considered within the search parameters include ureido methylation (Cys; +57.021464 Da), deamidation (Asn, gln; +0.984016 Da), gln- > pyro-Glu (N-terminal Gln; -17.026549 Da), oxidation (Met; + 15.994915), and acetylation (protein N-terminal; +42.010565 Da). All modifications were considered variable except for ureido methylation set to fix the modified Cys. The glycopeptides in the samples were identified after a separate, more focused search using a database of 20 fungal N-glycans and 14 fungal O-glycans. The data were filtered at 1% protein false discovery rate (FDR or 20% counter). The Byonic results file is entered into Byogic for further detailed analysis of the results report. After peptide mapping, the peak area of the matching peptide was used to calculate the ratio of glycosylated to non-glycosylated Ser (S) or Thr (T) residues in the peptide sequence. For each S/T detected, its single% glycosylation is calculated as: (sum of XIC areas of glycosylated peptides)/(sum of XIC areas of glycosylated+unmodified peptides). The total average% glycosylation of the samples was then calculated by averaging the% of all detected (non-zero) Ser/Thr modified individual S/T glycosylation.

Example 11: stimulation of collagen I and III synthesis by truncated human collagen III variants in fibroblasts

Fibroblast culture models were used to assess the ability of human collagen III variants ("second set of test materials") having the amino acid sequences shown in SEQ ID NO:987 through SEQ ID NO:1015 to exert an effect on collagen synthesis. The study also assessed viability of the cells after exposure to the second set of test materials.

Overview of the test methods

Fibroblasts are the major source of extracellular matrix peptides, including structural proteins type I collagen and type III collagen. In this study, human skin fibroblasts were treated with a second set of test materials for 48 hours, after which changes in extracellular matrix (ECM) components in cell culture medium were measured using an ELISA-based method.

MTT assay

In addition to variations in ECM component production, the effect of the second set of test materials on cell viability was assessed using the MTT assay. MTT assays for the second set of test materials were performed as described for the test materials discussed in example 6.

Samples were divided into batches 1 to 3 for testing. Batch 1 to batch 3 include each of the truncated human collagen III variants provided herein having the amino acid sequence shown in SEQ ID nos. 987 to 1015. The relationship between variants (identified by the "variant letter"), their amino acid sequences, their nucleic acid sequences, the plasmid sequences used to prepare the pichia pastoris strain for collagen expression, and identity to SEQ ID No. 986 is provided in table 6 below.

Table 6.

Variants in the second set of test materials of lot 1 ("lot 1 test materials") include variants H, O, G, Q, R, V, T, P and Z. Batch 1 test materials were prepared as active substances dissolved in solution. Each variant collagen sample was diluted in a range of concentration percentages in tissue culture medium FGM containing 1.5% FBS (see fig. 17A-17B). The concentrations reported in the results represent the final concentration (v/v) of the solution in the tissue culture medium.

Variant H (SEQ ID No: 994) was tested at 5%, 1%, 0.5%, 0.01% and 0.005% of the starting concentration, which correspond to actual test concentrations of 0.068%, 0.014%, 0.007%, 0.000% and 0.000%, respectively.

Variants O (SEQ ID No: 1001), R (SEQ ID No: 1004) and P (SEQ ID No: 1002) were tested at 20%, 10%, 5%, 1% and 0.01% of the original concentration, which correspond to the following actual test concentrations: 0.127%, 0.063%, 0.032%, 0.006% and 0.000% (variant O); 0.103%, 0.052%, 0.026%, 0.005% and 0.000% (variant R); and 0.081%, 0.041%, 0.020%, 0.004% and 0.000% (variant P).

Variants G (SEQ ID NO: 993), Q (SEQ ID NO: 1003), V (SEQ ID NO: 1008), T (SEQ ID NO: 1006) and Z (SEQ ID NO: 1012) were tested at 50%, 25%, 10%, 1% and 0.01% of the original concentration, which correspond to the following actual test concentrations: 0.614%, 0.307%, 0.123%, 0.012%, and 0.000% (variant G); 0.369%, 0.185%, 0.074%, 0.007% and 0.000% (variant Q); 0.307%, 0.153%, 0.061%, 0.006% and 0.000% (variant V); 0.019%, 0.009%, 0.004%, 0.000% (variant 23); and 0.767%, 0.383%, 0.153%, 0.015% and 0.000% (variant Z).

Variants in the second set of test materials of lot 2 ("lot 2 test materials") include variants A, L, N, B, D, S, U, C, W, Z, AA, E and F. Batch 2 test materials were prepared as active substances dissolved in solution. Each variant collagen sample was diluted in a range of concentration percentages in tissue culture medium FGM containing 1.5% FBS (see fig. 17C-17F). The concentrations reported in the results represent the final concentration (v/v) of the solution in the tissue culture medium.

Variants A (SEQ ID NO: 987), B (SEQ ID NO: 988), D (SEQ ID NO: 990), C (SEQ ID NO: 989) and E (SEQ ID NO: 991) were tested at 5%, 1%, 0.5%, 0.01% and 0.005% of the starting concentration, which correspond to the following actual test concentrations: 0.040%, 0.008%, 0.004% and 0.000% (variant a); 0.043%, 0.009%, 0.004% and 0.000% (variant B); 0.025%, 0.005%, 0.0025%, 0.00005% and 0.000025% (variant B); .0.109%, 0.022%, 0.011%, 0.000% (variant C); and 0.060%, 0.012%, 0.006%, 0.000% (variant E).

Variant L (SEQ ID NO: 998) and variant Z (SEQ ID NO: 1012) were tested at 50%, 25%, 10%, 1% and 0.01% of the original concentration, which correspond to the following actual test concentrations: 0.563%, 0.281%, 0.113%, 0.011% and 0.000% (variant L); and 0.767%, 0.383%, 0.153%, 0.015% and 0.000% (variant Z).

Variants N (SEQ ID NO: 1000), S (SEQ ID NO: 1005), U (SEQ ID NO: 1007) and F (SEQ ID NO: 992) were tested at 20%, 10%, 5%, 1%, 0.01% of the starting concentration, which correspond to the following actual test concentrations: 0.125%, 0.063%, 0.031%, 0.006% and 0.000%, respectively (variant N); 0.146%, 0.073%, 0.036%, 0.007% and 0.000%, respectively (variant S); 0.026%, 0.013%, 0.006%, 0.001% and 0.000%, respectively (variant U); and 0.155%, 0.078%, 0.039%, 0.008% and 0.000%, respectively (variant F).

Variants W (SEQ ID NO: 1009) and AA (SEQ ID NO: 1013) were tested at 25%, 10%, 5%, 1% and 0.01% of the original concentration, which correspond to the following actual test concentrations: 0.126%, 0.051%, 0.025%, 0.005% and 0.000%, respectively (variant W); 0.608%, 0.243%, 0.122%, 0.024% and 0.000%, respectively (variant AA).

Variants in the second set of test materials of lot 3 ("lot 3 test materials") include variants I, J, K, M, X, Y, BB and CC. Batch 3 test materials were prepared as active substances dissolved in solution. Each variant collagen sample was diluted in a range of concentration percentages in tissue culture medium FGM containing 1.5% FBS (see fig. 17C-17H). The concentrations reported in the results represent the final concentration (v/v) of the solution in the tissue culture medium.

Variants J (SEQ ID NO: 996), M (SEQ ID NO: 999), Y (SEQ ID NO: 1011) and I (SEQ ID NO: 995) were tested at 30%, 20%, 10%, 1% and 0.01% of the original concentration, which correspond to the following actual test concentrations: 0.193%, 0.129%, 0.064%, 0.006% and 0.000%, respectively (variant J); 0.180%, 0.120%, 0.060%, 0.006%, 0.000%, respectively (variant M); 0.080%, 0.053%, 0.027%, 0.003% and 0.000%, respectively (variant Y); and 0.204%, 0.136%, 0.068%, 0.007% and 0.000%, respectively (variant I).

Variant X (SEQ ID No: 1010) was tested at 50%, 25%, 10%, 1% and 0.01% of the starting concentration, which corresponds to the actual test concentrations of 0.103%, 0.051%, 0.021%, 0.002% and 0.000%.

Variants BB (SEQ ID NO: 1014) and CC (SEQ ID NO: 1015) were tested at 25%, 10%, 5%, 1%, 0.01% of the original concentration, which correspond to the following actual test concentrations: 0.414%, 0.165%, 0.083%, 0.017% and 0.000%, respectively (variant 41); 0.471%, 0.188%, 0.094%, 0.019% and 0.000%, respectively (variant 42).

Variant K (SEQ ID No: 1009) was tested at 20%, 10%, 5%, 1% and 0.01% of the starting concentration, which corresponds to actual test concentrations of 0.270%, 0.135%, 0.068%, 0.014% and 0.000%, respectively.

The results of the MTT assay are shown in fig. 17A to 17H, and the results are summarized in tables 7A to 7C below. In the table, "increase" indicates that the treatment significantly increased the endpoint, while "decrease" indicates that the treatment significantly decreased the endpoint.

Type I collagen assay

Fibroblasts are the primary source of extracellular matrix peptides, including collagen. Procollagen (Procollagen) is a large peptide synthesized by fibroblasts in the dermis layer of the skin and is a precursor of collagen. When the pro-collagen is processed to form mature collagen, the propeptide portion is cleaved off as a type I C-peptide. The mature collagen and type I C-peptide fragments are then released into the extracellular environment. With collagen synthesis, the type I C-peptide fragments accumulate in the tissue culture medium. Because of the 1:1 stoichiometric ratio between the two parts of the procollagen peptide, the determination of type I C-peptide reflects the amount of collagen synthesized. To measure the effect of different forms of collagen on collagen synthesis and secretion, type 1C-peptide was determined via ELISA-based methods. Type I collagen assays for the second set of test materials were performed as described for the test materials discussed in example 6.

Samples were divided into batches 1 to 3 for testing as described above. Batch 1 to batch 3 include each of the truncated human collagen III variants provided herein having the amino acid sequence shown in SEQ ID nos. 987 to 1015. Variants (identified by the "variant letter"), their amino acid sequences and their identity to SEQ ID No. 986 are provided in Table 6 above.

Variants in the second set of test materials were tested for each lot at the concentrations described in the MTT assay section above.

The results of type I collagen assay are shown in fig. 18A-18H, and the results are summarized in tables 7A-7C below. In the table, "increase" indicates that the treatment significantly increased the endpoint, while "decrease" indicates that the treatment significantly decreased the endpoint.

Type III collagen assay

Type III collagen is synthesized as a large propeptide by dermal fibroblasts. When the peptide is processed to form mature type III collagen, the propeptide portion is cleaved off (type III N-peptide). The mature collagen and type III N-peptide fragments are then released into the extracellular environment. With collagen synthesis, the type III N-peptide fragments accumulate in the tissue culture medium. Because of the 1:1 stoichiometric ratio between the two parts of the procollagen peptide, the determination of type III N-peptide reflects the amount of collagen synthesized. To measure the effect of different forms of collagen on collagen synthesis and secretion, type III N-peptides were determined via ELISA-based methods. Type III collagen assays for the second set of test materials were performed as described in example 6.

Samples were divided into batches 1 to 3 for testing as described above. Batch 1 to batch 3 include each of the truncated human collagen III variants provided herein having the amino acid sequence shown in SEQ ID nos. 987 to 1015. Variants (identified by "variant number"), their amino acid sequences, and their identity to SEQ ID No. 986 are provided in Table 6 above.

Variants in the second set of test materials were tested for each lot at the concentrations described in the MTT assay section above. Each variant collagen sample was diluted in a range of concentration percentages in tissue culture medium FGM containing 1.5% FBS (see fig. 19A-19B). The concentrations reported in the results represent the final concentration (v/v) of the solution in the tissue culture medium.

The results of the collagen III assay are shown in fig. 19A-19H, and the results are summarized in tables 7A-7C below. In the table, "increase" indicates that the treatment significantly increased the endpoint, while "decrease" indicates that the treatment significantly decreased the endpoint.

Table 7A to table 7C effect summary-batch 1 to batch 3

TABLE 7A

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TABLE 7B

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TABLE 7C

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The results of the MTT assay showed that at certain concentrations, variants B, C, E, F, G, H, I, J, K, L, M, N, P, Q, R, S, T, U, V, W, X, Y, Z, AA, BB and CC had at least as much viability or increased cell viability as untreated control cells.

The results of the collagen I assay show that at certain concentrations, the increase in variants C, D, G, J, L, N, Q, U, W, Z and AA increases the amount of type I collagen secreted by the treated fibroblasts. These data indicate that compositions comprising these recombinant collagen variants are useful when increased production of type I collagen is desired.

The results of the collagen III assay show that at certain concentrations, variant G, H, L, N, O, P, Q, R, S, T, U, V, W, Z, AA increased the amount of type III collagen secreted by the treated fibroblasts. These data indicate that compositions comprising these recombinant collagen variants are useful when increased production of type III collagen is desired.

The collagen variants described in this example were produced and purified by the same protocol. However, the impurity levels can vary from sample to sample, which results in different toxicity level results in the MTT assay. Generally, lower titers are associated with higher impurity levels. Thus, some variants with low titers were not amenable to collagen stimulation assessment testing at relatively high concentrations.

The breadth and scope of the present disclosure should not be limited by any of the above-described exemplary embodiments, but should be defined only in accordance with the following claims and their equivalents.

Claims

1. A recombinant collagen fragment having a molecular weight of about 50kDa and a sequence identity of at least about 85% with the amino acid sequence shown in SEQ ID No. 986.

2. The recombinant collagen fragment according to claim 1, wherein the recombinant collagen fragment is non-hydroxylated.

3. The recombinant collagen fragment according to claim 1, wherein the recombinant collagen fragment is hydroxylated.

4. The recombinant collagen fragment according to claim 1, wherein the collagen fragment has the amino acid sequence shown in seq id No. 986.

5. A sequence variant of a recombinant collagen fragment according to claim 1, wherein said sequence variant comprises the amino acid sequence shown in any one of SEQ ID NOs 987 to 1015.

6. The sequence variant of claim 5, wherein the sequence variant is non-hydroxylated.

7. The sequence variant of claim 5, wherein the sequence variant is hydroxylated.

8. A composition comprising the recombinant collagen fragment according to any one of claims 1 to 4.

9. A composition comprising the sequence variant according to any one of claims 5 to 7.

10. The composition of claim 8, wherein the composition further comprises one or more peptides formed from the hydrolysis of a collagen fragment having the amino acid sequence shown in SEQ ID No. 986.

11. The composition of claim 10, wherein at least one of the one or more peptides formed from the hydrolysis of the collagen fragment having the amino acid sequence shown in SEQ ID No. 986 has an amino acid sequence according to one of SEQ ID No. 2 to SEQ ID No. 972.

12. The composition of any one of claims 8 to 11, further comprising a pharmaceutically acceptable excipient or a cosmetically acceptable excipient.

13. A method of producing the recombinant collagen fragment according to any one of claims 1 to 4, the method comprising producing the recombinant collagen fragment in a genetically engineered yeast strain.

14. The method of claim 13, wherein the yeast is pichia pastoris.

15. The method according to claim 13 or 14, wherein the yeast has been transformed with a plasmid comprising the nucleic acid sequence shown in seq id No. 973.

16. The method according to any one of claims 13 to 15, wherein the yeast is a yeast which has been transformed with a plasmid comprising the nucleic acid sequence shown in SEQ ID NO 974.

17. The method according to any one of claims 13 to 16, wherein the method comprises:

(i) Fermenting genetically engineered yeast in a fermentation broth;

(iii) Optionally, purifying the recombinant collagen fragment.

18. The method of claim 17, further comprising hydroxylating the recombinant collagen fragment ex vivo.

19. A method of producing a sequence variant according to any one of claims 5 to 7, the method comprising producing the recombinant collagen fragment in a genetically engineered yeast strain.

20. The method of claim 19, wherein the yeast is pichia pastoris.

21. The method according to claim 19 or 20, wherein the yeast has been transformed with a plasmid comprising the nucleic acid sequence shown in any one of SEQ ID NOs 1045 to 1073.

22. The method according to any one of claims 19 to 21, wherein the method comprises:

(i) Fermenting genetically engineered yeast in a fermentation broth;

(ii) Recovering from the fermentation broth a recombinant collagen fragment sequence variant secreted by the genetically engineered yeast; and

(iii) Optionally, purifying the recombinant collagen fragment.

23. The method of claim 22, further comprising hydroxylating the recombinant collagen sequence variant ex vivo.

24. A yeast strain genetically engineered to produce a recombinant collagen fragment according to any one of claims 1 to 4, wherein the yeast strain comprises a vector comprising a DNA sequence encoding the recombinant collagen.

25. A yeast strain genetically engineered to produce a sequence variant according to any one of claims 5 to 7, wherein the yeast strain comprises a vector comprising a DNA sequence encoding the variant.

26. The yeast strain of claim 24, wherein the vector comprises a nucleic acid sequence comprising the DNA sequence set forth in SEQ ID No. 973.

27. The yeast strain of claim 26, further comprising a second vector comprising a nucleic acid sequence comprising the DNA sequence set forth in SEQ ID No. 974.

28. The yeast strain of claim 25, wherein the vector comprises a nucleic acid sequence comprising a DNA sequence set forth in any one of SEQ ID NOs 1045 to 1073.

29. The yeast strain of any one of claims 24 to 28, wherein the yeast strain is pichia pastoris.

30. A method of treating a skin condition, the method comprising administering to a subject in need thereof an effective amount of the recombinant collagen fragment according to any one of claims 1 to 4 or the sequence variant according to any one of claims 5 to 7.

31. A method of treating a skin condition, the method comprising administering to a subject in need thereof an effective amount of the composition of any one of claims 8 to 12.

32. The method of claim 31, wherein the skin condition comprises fine lines, wrinkles, dry skin, enlarged pores, skin discoloration, reduced elasticity, unwanted hair, skin thinning, purpura, actinic keratosis, itching, eczema, acne, rosacea, erythema, telangiectasia, actinic telangiectasia, skin cancer, or hypertrophic rosacea.

33. The method of claim 31 or 32, wherein the composition is topically applied to an area of skin.

34. The method of claim 33, wherein the skin region is selected from the group consisting of: facial surfaces, scalp, neck, ears, shoulders, chest (including breast and/or collar), arms, hands, legs, stomach, buttocks, groin, back, feet, and combinations thereof.

35. A method of increasing collagen production in a cell, the method comprising administering to the cell an effective amount of a recombinant collagen fragment according to any one of claims 1 to 4 or a sequence variant according to any one of claims 5 to 7.

36. The method of claim 35, wherein the method increases type I collagen production.

37. The method of claim 34 or 35, wherein the method increases type III collagen production.

38. The method of any one of claims 35 to 37, wherein the cells are fibroblasts.

39. The method of any one of claims 35 to 38, wherein the cell is a cultured cell.

40. The method of any one of claims 35 to 39, wherein the fragment or variant is formulated in a composition.

41. The method according to any one of claims 35 to 41, wherein the fragment has the amino acid sequence shown in seq id No. 986.

42. The method of any one of claims 35 to 41, wherein the sequence variant has an amino acid sequence set forth in any one of SEQ ID NOs 987 to 1015.

43. A skin care product comprising the composition according to any one of claims 8 to 12 for reducing appearance of wrinkles, evening skin tone, providing moisture, reducing appearance of dark under-eye circles, increasing collagen content of skin, increasing skin density, improving skin firmness and elasticity, improving appearance of fine lines and wrinkles, smoothing skin texture, increasing skin luster and brightness, improving appearance of sagging skin, whitening skin, or any combination thereof.

44. A method of treating a wound in a human subject in need thereof, the method comprising administering the composition of any one of claims 8 to 12 to the wound of the subject, wherein administration of the recombinant collagen fragment induces production of human type I collagen, human type III collagen, or a combination thereof.

45. The method of claim 44, wherein the collagen fragment is topically applied to the wound.

46. A recombinant collagen fragment having a molecular weight of about 50kDa and a sequence identity of at least about 85% to the amino acid sequence set forth in SEQ ID No. 1.

47. The recombinant collagen fragment according to claim 46, wherein the recombinant collagen fragment is non-hydroxylated.

48. The recombinant collagen fragment according to claim 46, wherein the recombinant collagen fragment is hydroxylated.

49. The recombinant collagen fragment according to claim 48, wherein the collagen fragment has the amino acid sequence shown in SEQ ID NO. 1.

50. A recombinant collagen fragment comprising an amino acid sequence according to any one of SEQ ID NOs 2 to 972.

51. A composition comprising the recombinant collagen fragment according to any one of claims 46 to 50.

52. The composition of claim 51, wherein the composition further comprises one or more peptides formed from the hydrolysis of a collagen fragment having the amino acid sequence shown in SEQ ID NO. 1.

53. The composition of claim 52, wherein at least one of the one or more peptides formed from the hydrolysis of the collagen fragment having the amino acid sequence shown in SEQ ID No. 1 has an amino acid sequence according to one of SEQ ID No. 2 to SEQ ID No. 972.

54. The composition of any one of claims 51 to 53, further comprising a pharmaceutically acceptable excipient or a cosmetically acceptable excipient.

55. A method of producing a recombinant collagen fragment according to any one of claims 46 to 50, said method comprising producing said recombinant collagen fragment in a genetically engineered yeast strain.

56. The method of claim 55, wherein the yeast is Pichia pastoris.

57. The method according to claim 55 or 56, wherein said yeast has been transformed with a plasmid comprising the nucleic acid sequence shown in SEQ ID NO. 973.

58. The method of any one of claims 55 to 57, wherein the yeast is a yeast that has been transformed with a plasmid comprising the nucleic acid sequence set forth in SEQ ID NO 974.

59. The method of any one of claims 55 to 58, wherein the method comprises:

(i) Fermenting genetically engineered yeast in a fermentation broth;

(iii) Optionally, purifying the recombinant collagen fragment.

60. The method of claim 59, further comprising hydroxylating the recombinant collagen fragment ex vivo.

61. A yeast strain genetically engineered to produce a recombinant collagen fragment according to any one of claims 46 to 50, wherein the yeast strain comprises a vector comprising a DNA sequence encoding the recombinant collagen.

62. The yeast strain of claim 61, wherein the vector comprises a nucleic acid sequence comprising the DNA sequence set forth in SEQ ID NO 973.

63. The yeast strain of claim 62, further comprising a second vector comprising a nucleic acid sequence comprising the DNA sequence set forth in SEQ ID NO. 974.

64. The yeast strain of any one of claims 61 to 63, wherein the yeast strain is pichia pastoris.

65. A method of treating a skin condition, the method comprising administering an effective amount of the recombinant collagen fragment according to any one of claims 46 to 50 to a subject in need thereof.

66. A method of treating a skin condition, the method comprising administering to a subject in need thereof an effective amount of the composition of any one of claims 51-54.

67. The method of claim 66, wherein the skin condition comprises fine lines, wrinkles, dry skin, enlarged pores, skin discoloration, reduced elasticity, unwanted hair, skin thinning, purpura, actinic keratosis, itching, eczema, acne, rosacea, erythema, telangiectasia, actinic telangiectasia, skin cancer, or hypertrophic rosacea.

68. The method of claim 66 or 67, wherein the composition is topically applied to an area of skin.

69. The method of claim 68, wherein the skin region is selected from the group consisting of: facial surfaces, scalp, neck, ears, shoulders, chest (including breast and/or collar), arms, hands, legs, stomach, buttocks, groin, back, feet, and combinations thereof.

70. A method of increasing collagen production in a cell, the method comprising administering to the cell an effective amount of the recombinant collagen fragment according to any one of claims 46 to 50.

71. The method of claim 70, wherein the method increases type I collagen production.

72. The method of claim 70 or 71, wherein the method increases type III collagen production.

73. The method of any one of claims 70-72, wherein the cell is a fibroblast.

74. The method of any one of claims 70-73, wherein the cell is a cultured cell.

75. The method of any one of claims 70-74, wherein the fragments are formulated in a composition.

76. The method according to any one of claims 70 to 75, wherein said fragment has the amino acid sequence shown in seq id No. 1.

77. A skin care product comprising the composition according to any one of claims 51 to 54 for reducing appearance of wrinkles, evening skin tone, providing moisture, reducing appearance of dark under-eye circles, increasing collagen content of skin, increasing skin density, improving skin firmness and elasticity, improving appearance of fine lines and wrinkles, smoothing skin texture, increasing skin luster and brightness, improving appearance of sagging skin, whitening skin, or any combination thereof.

78. A method of treating a wound in a human subject in need thereof, the method comprising administering the composition of any one of claims 51 to 54 to the wound of the subject, wherein administration of the recombinant collagen fragment induces production of human type I collagen, human type III collagen, or a combination thereof.

79. The method of claim 78, wherein the collagen fragment is topically applied to the wound.