Ionic self-assembling peptides

Abstract

Provided herein are ionic self-assembling peptides, pharmaceutical compositions comprising the peptides, and methods of using and making the same.

Description

[0001] Priority claim

[0002] This application claims the benefit of U.S. Provisional Application Serial No. 62 / 693,877, filed on July 3, 2018. The entire contents of the foregoing are incorporated herein by reference.

[0003] Sequence Listing

[0004] This application contains a sequence listing, which has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. The ASCII copy was created on July 1, 2019, is named 46406-0029WO1 SEQ.txt and is 24,576 bytes in size. Technical Field

[0005] The present disclosure generally relates to self-assembling peptides, pharmaceutical compositions comprising the same, and methods of use thereof. Background Art

[0006] Self-assembling peptides have been developed for a variety of purposes, including scaffolds for tissue engineering and regenerative medicine, drug delivery, three-dimensional tissue culture, and hemostasis. Examples of such self-assembling peptides include β-sheet peptides with alternating positive and negative charges and hydrophobic residues (1) (e.g., RADA 16 (SEQ ID NO: 91), IEIK13 (SEQ ID NO: 92), and KLDL12 (SEQ ID NO: 93)), β-sheet peptides with alternating sequences of nonionic, polar residues and hydrophobic residues (2), and β-sheet peptides with repeated nonionic, polar residues (3). However, such self-assembling peptides have practical limitations. For example, pharmaceutical compositions including these self-assembling peptides with alternating positive and negative charges and hydrophobic residues (e.g., RADA 16 (SEQ ID NO: 91)) must be formulated at an acidic pH to dissolve the peptide, which may result in cell and / or tissue damage when administered to a subject. Therefore, there remains a need for improved self-assembling peptides that can be used for therapeutic applications. Summary of the invention

[0007] The present disclosure is based at least in part on the development of ionic self-assembling peptides (ionic self-assembling peptides), which have a specific combination of ionic polar amino acids (ionic polar amino acid, ionic polar amino acids), hydrophobic amino acids and non-ionic polar amino acids (non-ionic polar amino acid, non-ionic polar amino acids). The self-assembling peptides described previously generally include an equal amount of amino acid residues with a net negative charge and a net positive charge at neutral pH. These peptides are prepared at acidic or alkaline pH to charge the peptides so that the peptides are retained in the solution, and the composition comprising the peptides is flowable and injectable. If prepared at neutral pH, phase separation and precipitation of the peptides will be observed. For example, RADA16 (SEQ ID NO:91) has four positive charges and four negative charges at neutral pH. As shown in Figure 20A, when prepared at pH 7.5, RADA 16 (SEQ ID NO:91) precipitates.

[0008] In contrast, the inclusion of ionic polar amino acids in the self-assembling peptides provided herein causes the peptides to have a non-zero net charge at neutral pH, thereby allowing the peptides to be formulated at neutral pH. Advantageously, the ionic self-assembling peptides provided herein can be formulated at neutral pH and remain soluble and stable in solution. Without wishing to be bound by any particular theory, pharmaceutical compositions comprising the ionic self-assembling peptides described herein can be formulated at neutral pH and applied to subjects without causing tissue damage that may be associated with pharmaceutical compositions with acidic or alkaline pH. In addition, ionic self-assembling peptides can be used to prepare improved hydrogels with favorable physical properties under physiological conditions. For example, an aqueous solution of self-assembling peptides can be produced that remains stable, flowable and injectable under physiological conditions. After gelation, the self-assembling peptides can form a hydrogel with shear thinning, thixotropy and rheological properties, which can be used for clinical, industrial and / or research applications.

[0009] In some aspects, the present disclosure provides self-assembling peptides and pharmaceutical compositions comprising the self-assembling peptides, wherein the self-assembling peptides comprise or consist of an amino acid sequence listed in the following formula: [(X)i(Y)j(Z)k(Y)l]m(X)n (Formula I), [(Y)i(X)j(Y)k(Z)l]m(Y)n (Formula II), [(Z)i(Y)j(X)k(Y)l]m(Z)n (Formula III), or [(Y)i(Z)j(Y)k(X)l]m(Y)n (Formula IV), wherein each (X) is independently an ionic, polar amino acid, wherein each (Y) is independently a hydrophobic amino acid, wherein each (Z) is independently a non-ionic, polar amino acid, wherein each i, j, k and l are independently an integer ≥1, wherein m is an integer ≥2, and wherein n=0 or an integer ≥1. In some embodiments, at least one of i, j, k, and l is independently an integer of 1. In some embodiments, at least one of i, j, k, and l is independently an integer of 2. In some embodiments, each of i, j, k, and l is 1. In some embodiments, m is independently an integer of 2, 3, or 4.

[0010] In some embodiments, the self-assembling peptide comprises an amino acid sequence listed in formula I or consists of an amino acid sequence listed in formula I. In some embodiments, the self-assembling peptide comprises an amino acid sequence listed in formula II or consists of an amino acid sequence listed in formula II. In some embodiments, the self-assembling peptide comprises an amino acid sequence listed in formula III or consists of an amino acid sequence listed in formula III. In some embodiments, the self-assembling peptide comprises an amino acid sequence listed in formula IV or consists of an amino acid sequence listed in formula IV.

[0011] In some embodiments, each (X) is a basic amino acid (eg, arginine, lysine, histidine, or ornithine). In some embodiments, each (X) is an acidic amino acid (eg, aspartic acid or glutamic acid).

[0012] In some embodiments, each (Y) is alanine, valine, leucine, isoleucine, methionine, phenylalanine, tryptophan, or glycine.

[0013] In some embodiments, each (Z) is serine, threonine, tyrosine, cysteine, glutamine, asparagine, or methionine.

[0014] In some embodiments, the self-assembling peptide comprises or consists of the amino acid sequence set forth in SEQ ID NOs: 1-20 and 94-96.

[0015] In some embodiments, the self-assembling peptide comprises an N-terminal functional group, a C-terminal functional group, or both. In some embodiments, the N-terminal functional group is an acetyl, a formyl, a pyroglutamyl (pGlu), biotin, a polyethylene glycol (PEG), urea, an alkylamine, a carbamate, a sulfonamide, a dansyl, a 2,4-dinitrophenyl, a fluorescein, a 7-methoxycoumarinacetic acid, a 9-fluorenylmethoxycarbonyl, a palmitic acid, a succinyl, a chloroacetyl, a maleimide, a benzyloxycarbonyl, a bromoacetyl, a nitrilotriacetyl, a tert-butyloxycarbonyl, a 4-hydroxyphenylpropionic acid, an allyloxycarbonyl, a butyric acid, a fatty acid, or a trityl. In some embodiments, the C-terminal functional group is an amide, N-alkylamide, aldehyde, ester, alcohol, para-nitroanilide (pNA), 7-amino-4-methylcoumarin (Amc), hydrazide, hydroxamic acid, chloromethyl ketone, p-nitroaniline, p-nitrophenol, hydroxysuccinimide ester (hydroxysucinimide), fluoromethyl ketone, cysteamide (cysteamide, mercaptoacetylation), 9-fluorenylmethyl (Fm) ester, allyl ester, 2,4-dimethoxybenzyl ester, 2-phenylisopropyl ester, p-nitrobenzyl ester and 2-chlorotrityl ester. In some embodiments, the self-assembling peptide comprises or consists of the amino acid sequences listed in SEQ ID NOs: 21-40 and 97-99.

[0016] In some embodiments, the self-assembling peptide comprises at least one (e.g., one, two, three, four or more) bioactive peptide motif. In some embodiments, at least one bioactive peptide motif is present at the N-terminus of the self-assembling peptide. In some embodiments, at least one bioactive peptide motif is present at the C-terminus of the self-assembling peptide. In some embodiments, at least one bioactive peptide motif is derived from laminin-1, collagen IV, fibronectin, elastin, bone marrow homing peptide 1 (bone marrow homing peptide 1, bone marrow guiding peptide 1), bone marrow homing peptide 2 or myelopeptide (myelopeptide, bone marrow peptide, osteogenic peptide). In some embodiments, at least one bioactive peptide motif comprises an amino acid sequence set forth in any one of SEQ ID NO: 41-70 or is composed of an amino acid sequence set forth in any one of SEQ ID NO: 41-70. In some embodiments, the self-assembling peptide comprises an amino acid sequence set forth in SEQ ID NO: 71-90 or is composed of an amino acid sequence set forth in SEQ ID NO: 71-90.

[0017] In some embodiments, the pharmaceutical composition comprises a tonicity agent. In some embodiments, the tonicity agent comprises one or more salts selected from the group consisting of NaCl, KCl, MgCl2, CaCl2, NH4Cl, Na2HPO4, KH2PO4 and CaSO4. In some embodiments, the tonicity agent further comprises one or more sugars selected from the group consisting of dextrose, mannitol, glycerol, sucrose and trehalose. In some embodiments (e.g., when the tonicity agent is one or more salts), the concentration of the tonicity agent is about 0.01M to about 0.3M. In some embodiments (e.g., when the tonicity agent is one or more salts), the concentration of the tonicity agent is about 0.15M. In some embodiments (e.g., when the tonicity agent is one or more sugars), the concentration of the tonicity agent is about 0.1 to 10% (w / v). In some embodiments (e.g., when the tonicity agent is one or more sugars), the concentration of the tonicity agent is about 10% (w / v). In some embodiments, the tonicity agent increases the rheological properties of the composition or hydrogel peptide comprising the self-assembling peptide described herein.

[0018] In some embodiments, the pH of the pharmaceutical composition is from about 6 to about 8. In some embodiments, the pH of the pharmaceutical composition is from about 7 to about 7.5. In some embodiments, the net charge of the self-assembling peptide in the pharmaceutical composition is greater than or equal to +1 or less than or equal to -1. In some embodiments, the net charge of the self-assembling peptide in the pharmaceutical composition is from about +1 to about +6 (e.g., +1, +2, +3, +4, +5, or +6). In some embodiments, the net charge of the self-assembling peptide in the pharmaceutical composition is from about -1 to about -6 (e.g., -1, -2, -3, -4, -5, or -6).

[0019] In some embodiments, the concentration of the self-assembling peptides in the pharmaceutical composition is about 0.01% (w / v) to about 10% (w / v). In some embodiments, the concentration of the self-assembling peptides in the pharmaceutical composition is about 0.1% (w / v) to about 5% (w / v). In some embodiments, the concentration of the self-assembling peptides in the pharmaceutical composition is about 0.5% (w / v) to about 1.5% (w / v). In some embodiments, the concentration of the self-assembling peptides in the pharmaceutical composition is about 1% (w / v).

[0020] In some embodiments, the pharmaceutical composition comprises separated cells (e.g., stem cells). In some embodiments, the separated cells are mammalian cells. In some embodiments, mammalian cells are immune cells, stem cells, chondrocyte progenitor cells (chondrocyte progenitor cell), pancreatic progenitor cells, myoblasts, fibroblasts, keratinocytes, neurons, glial cells, astrocytes, preadipocytes, adipocytes, vascular endothelial cells, endothelial progenitor cells, mesenchymal cells, neural stem cells, immune cells (e.g., B cells and T cells), smooth muscle progenitor cells, cardiomyocytes, fetal dermal fibroblasts, epidermal keratinocytes, myoblasts, and capillary endothelial cells.

[0021] In some embodiments, the pharmaceutical composition comprises at least one (e.g., one, two, three, four, five or more) bioactive agent. In some embodiments, the bioactive agent is a hormone, a growth factor, insulin, an enzyme, siRNA, shRNA, antisense RNA, antisense DNA, mRNA, antibiotics, antibodies, or anti-inflammatory agents.

[0022] In some embodiments, the pharmaceutical composition is an aqueous solution. In some embodiments, the pharmaceutical composition is a hydrogel.

[0023] In some embodiments, the pharmaceutical composition is a hydrogel having a storage modulus of at least about 10 Pascals (Pa) (e.g., about 25 Pa, about 50 Pa, about 100 Pa, about 150 Pa, about 250 Pa, about 500 Pa, about 750 Pa, about 1000 Pa, or more).

[0024] In another aspect, the present disclosure provides an article of manufacture comprising the self-assembling peptides or pharmaceutical compositions described herein. In some embodiments, the article of manufacture is a syringe, a vial, an autoinjector, a tube, or a catheter.

[0025] In another aspect, the present disclosure provides a method of treating a subject in need thereof, comprising administering to the subject an effective amount of a self-assembling peptide or pharmaceutical composition described herein.

[0026] On the other hand, the present disclosure provides a method for promoting tissue repair or regeneration in a subject in need thereof, including contacting the subject's tissue with a self-assembling peptide or pharmaceutical composition as described herein, thereby promoting tissue repair or tissue regeneration. In some embodiments, the tissue is skin, bone, cartilage, neural tissue, ligament, tendon, vascular tissue or muscle. In some embodiments, the tissue is optic nerve tissue. In some embodiments, the tissue is cardiac tissue. In some embodiments, the subject suffers from a congenital disease or illness, resulting in the need for tissue repair or regeneration. In some embodiments, the subject suffers from damage (e.g., surgery, trauma, stroke, tumor or disease or illness), resulting in the need for tissue repair or regeneration.

[0027] On the other hand, the present disclosure provides a method for promoting wound healing in a subject in need thereof, comprising contacting a wound of the subject (or alternatively applying to the subject's wound) an effective amount of a self-assembling peptide or pharmaceutical composition as described herein, thereby promoting wound healing and / or antibacterial activity. In some embodiments, the wound comprises or consists of an abrasion, burn, chapped, worn, cut, ulcer, laceration, incision or scratch.

[0028] In another aspect, the present disclosure provides a method for stopping or preventing (or alternatively reducing) bleeding at a site in a subject's body, comprising contacting the site with (or alternatively applying to a wound of a subject) a self-assembling peptide or pharmaceutical composition described herein, thereby creating a physical barrier, thereby stopping or preventing (or reducing) bleeding at the site in the subject's body.

[0029] On the other hand, the present disclosure provides a method for removing a lesion from a site in the gastrointestinal tract of a subject, comprising contacting the submucosal layer below the lesion (or, alternatively, applying to the submucosal layer below the lesion) the self-assembling peptide or pharmaceutical composition described herein in an amount sufficient to lift the lesion; and removing the lesion from the gastrointestinal site of the subject. In some embodiments, the lesion comprises a polyp, an ulcer, or a tumor. In some embodiments, the lesion is present in a gastrointestinal region of the subject selected from the oral cavity, throat, esophagus, stomach, small intestine, large intestine, colon, and rectum.

[0030] In yet another aspect, the present disclosure provides a method of culturing a cell, comprising contacting the cell with a pharmaceutical composition described herein.

[0031] In another aspect, provided herein is a method of treating pulmonary bullae in a subject, comprising:

[0032] introducing a delivery device into a target area of ​​a bulla of a subject;

[0033] positioning an end of the delivery device at a target area where bullae need to be treated;

[0034] administering the self-assembling peptide or pharmaceutical composition described herein to a target area via a delivery device in an effective amount and concentration to form a barrier under physiological conditions of the target area to treat pulmonary bullae;

[0035] removing the delivery device from the target area; and

[0036] The bullae are collapsed before or after administration of the solution.

[0037] In another aspect, provided herein is a method for reducing adhesion to biological tissue, the method comprising administering to the biological tissue an effective amount of the self-assembling peptide or pharmaceutical composition described herein, thereby reducing adhesion to the biological tissue.

[0038] In another aspect, provided herein is a method of filling a bone void in a subject, comprising

[0039] introducing a delivery device into a bone of a subject;

[0040] positioning an end of the delivery device adjacent a void in the bone where bone growth is desired;

[0041] administering the self-assembling peptide or pharmaceutical composition described herein via a delivery device at a concentration sufficient to form a hydrogel scaffold under physiological conditions; and

[0042] The delivery device is removed.

[0043] In another aspect, provided herein is a method of treating dry eye in a subject, comprising administering to the subject's eye an effective amount of a self-assembling peptide or pharmaceutical composition described herein.

[0044] The present invention further provides a self-assembling peptide for use in any of the methods described herein, which comprises or consists of an amino acid sequence as set forth in the following formula: [(X)i(Y)j(Z)k(Y)l]m(X)n(Formula I), [(Y)i(X)j(Y)k(Z)l]m(Y)n(Formula II), [(Z)i(Y)j(X)k(Y)l]m(Z)n(Formula III) or [(Y)i(Z)j(Y)k(X)l]m(Y)n(Formula IV).

[0045] Other features and advantages of the invention will be apparent from the following detailed description and drawings, and from the claims. BRIEF DESCRIPTION OF THE DRAWINGS

[0046] Figure 1 Shown are net charge measurements of KLNL12 (SEQ ID NO: 21) at various pHs.

[0047] Figure 2 Shown are net charge measurements of NLEL12 (SEQ ID NO: 33) at various pHs.

[0048] Figure 3 Shown are net charge measurements of RADA16 (SEQ ID NO:91) at various pHs.

[0049] Figure 4 Schematic representation of the molecular structure and electronic charge of RANA16 (SEQ ID NO: 39) at pH 7.5 is shown.

[0050] Figure 5 Schematic diagram showing the molecular structure and electronic charge of RADA16 (SEQ ID NO:91) at pH 7.5.

[0051] Figure 6-8 Shear thinning properties of exemplary aqueous pharmaceutical compositions including the self-assembling peptides KLNL12 (SEQ ID NO:21), IQIK13 (SEQ ID NO:28), or NLEL12 (SEQ ID NO:33) are shown.

[0052] Figure 6 The shear-thinning properties of an aqueous pharmaceutical composition including 1% (w / v) KLNL12 (SEQ ID NO: 21) at pH 7.5 are shown, as reflected by changes in viscosity in response to various shear rates.

[0053] Figure 7 The shear-thinning properties of an aqueous pharmaceutical composition including 1% (w / v) IQIK13 (SEQ ID NO: 28) at pH 7.5 are shown, as reflected by changes in viscosity in response to various shear rates.

[0054] Figure 8 The shear-thinning properties of an aqueous pharmaceutical composition including 1% (w / v) NLEL12 (SEQ ID NO: 33) at pH 7.5 are shown, as reflected by changes in viscosity in response to various shear rates.

[0055] Figure 9-10 The shear thinning properties of exemplary aqueous pharmaceutical compositions including the self-assembling peptides KLNL12 (SEQ ID NO:21) or NLEL12 (SEQ ID NO:33) are shown.

[0056] Fig. 9 The shear thinning properties of an aqueous pharmaceutical composition including 1% (w / v) KLNL12 (SEQ ID NO: 21) containing 0.9% NaCl at pH 7.5 are shown, as reflected by changes in viscosity in response to various shear rates.

[0057] Fig.10 The shear thinning properties of an aqueous pharmaceutical composition including 1% NLEL12 (SEQ ID NO: 33) containing 0.9% NaCl at pH 7.5 are shown, as reflected by changes in viscosity in response to various shear rates.

[0058] Figure 11-12 The thixotropic properties of exemplary aqueous pharmaceutical compositions including the self-assembling peptides KLNL12 (SEQ ID NO:21) or NLEL12 (SEQ ID NO:33) are shown.

[0059] Fig.11 The thixotropic properties of an aqueous pharmaceutical composition including 1% (w / v) KLNL12 (SEQ ID NO: 21) containing 0.9% NaCl at pH 7.5 are shown, as reflected by the change in mechanical strength after removal of shear stress.

[0060] Fig.12 The thixotropic properties of an aqueous pharmaceutical composition including 1% NLEL12 (SEQ ID NO: 33) containing 0.9% NaCl at pH 7.5 are shown, as reflected by the change in mechanical strength after removal of shear stress.

[0061] Figure 13-15 Shown are changes in rheological properties of exemplary aqueous pharmaceutical compositions including the self-assembling peptides KLNL12 (SEQ ID NO:21), KIQI13 (SEQ ID NO:29), or NLEL12 (SEQ ID NO:33) in response to exposure to DMEM.

[0062] Fig.13 Shown are changes in the rheological properties of aqueous pharmaceutical compositions including 1% (w / v) KLNL12 (SEQ ID NO: 21) at pH 7.5, as reflected by increased mechanical strength after exposure to DMEM.

[0063] Fig.14 Shown are changes in the rheological properties of aqueous pharmaceutical compositions including 1% (w / v) KIQI13 (SEQ ID NO: 29) at pH 7.5, as reflected by increased mechanical strength after exposure to DMEM.

[0064] Fig.15 Shown are changes in the rheological properties of an aqueous pharmaceutical composition including 1% (w / v) NLEL12 (SEQ ID NO: 33) at pH 7.5, as reflected by increased mechanical strength after exposure to DMEM.

[0065] Figure 16-18Shown are changes in rheological properties of exemplary aqueous pharmaceutical compositions including the self-assembling peptides KLNL12 (SEQ ID NO:21), KIQI13 (SEQ ID NO:29), or NLEL12 (SEQ ID NO:33) in response to exposure to DMEM.

[0066] Fig.16 Shown are changes in the rheological properties of an aqueous pharmaceutical composition including 1% (w / v) KLNL12 (SEQ ID NO: 21) containing 0.9% NaCl at pH 7.5, as reflected by increased mechanical strength after exposure to DMEM.

[0067] Fig.17 Shown are changes in the rheological properties of an aqueous pharmaceutical composition including 1% (w / v) KIQI13 (SEQ ID NO: 29) containing 0.9% NaCl at pH 7.5, as reflected by increased mechanical strength after exposure to DMEM.

[0068] Fig.18 Shown are changes in the rheological properties of an aqueous pharmaceutical composition including 1% NLEL12 (SEQ ID NO: 33) containing 0.9% NaCl at pH 7.5, as reflected by increased mechanical strength after exposure to DMEM.

[0069] Fig.19 is a schematic diagram of a macromolecular structure that can be formed by the self-assembling peptides of the present invention.

[0070] Figures 20A-20C below are photographs showing the appearance of aqueous pharmaceutical compositions including the specified self-assembling peptides at pH 7.5. Figure 20A is a photograph showing the appearance of an aqueous pharmaceutical composition including RADA 16 (SEQ ID NO: 91) at pH 7.5.

[0071] FIG. 20B is a photograph showing the appearance of an aqueous pharmaceutical composition including KLNL12 (SEQ ID NO: 21) at pH 7.5.

[0072] FIG. 20C is a photograph showing the appearance of an aqueous pharmaceutical composition including NLEL12 (SEQ ID NO: 33) at pH 7.5.

[0073] Figures 21A and 21B show the appearance of the submucosa of the pig stomach 0-5 minutes after injection of the indicated pharmaceutical compositions (Figure 21A) and 15-20 minutes after injection (Figure 21B). The injection sites are indicated by dashed black ovals: (1) 2 mL of 0.1% (w / v) KLNL12 (SEQ ID NO:21), 0.9% (w / v) NaCl, pH 7.5; (2) 2 mL of 0.1% (w / v) NLKL12 (SEQ ID NO:23), 0.9% (w / v) NaCl, pH 7.5; (3) 2 mL of 0.1% (w / v) KIQI13 (SEQ ID NO:29), 0.9% (w / v) NaCl, pH 7.5; (4) 2 mL of 0.2% (w / v) RADA16 (SEQ ID NO:91), pH 2.5; (5) 2 mL of (6) 2 mL of saline, pH 7.5.

[0074] Figures 22A-22C The appearance of the dissected porcine stomach submucosa after injection of 2 mL saline pH 7.5 ( FIG. 22A ), or 2 mL aqueous pharmaceutical composition pH 2.5 containing 0.2% (w / v) RADA16 (SEQ ID NO: 91) ( FIG. 22B ), or 2 ml aqueous pharmaceutical composition pH 2.5 including 0.2% (w / v) RADA16 (SEQ ID NO: 91), 0.9% NaCl (w / v) ( FIG. 22C ) is shown. The black oval represents the white and fuzzy-looking area.

[0075] Figures 23A, 23B, 23C and 23D show the appearance of the submucosal layer of pig stomach after injection of 2 mL of a pharmaceutical composition comprising 0.1% KLNL12 (SEQ ID NO:21), 0.9% NaCl, pH 7.5 (Figure 23A); 2 mL of a pharmaceutical composition comprising 0.2% NLEL12 (SEQ ID NO:33), 0.9% NaCl, pH 7.5 (Figure 23B); 2 mL of a pharmaceutical composition comprising 0.2% QLEL12 (SEQ ID NO:35), 0.9% NaCl, pH 7.5 (Figure 23C); or 2 mL of a pharmaceutical composition comprising 0.2% LELQ12 (SEQ ID NO:36), 0.9% NaCl, pH 7.5.

[0076] Fig.24A Shown are changes in the rheological properties of an aqueous pharmaceutical composition comprising 0.15% (w / v) QLEL12 (SEQ ID NO: 35) and 0.9% NaCl (w / v) at pH 7.5, as reflected by increased mechanical strength after exposure to DMEM.

[0077] Fig. 24BRheological properties of aqueous pharmaceutical compositions comprising various concentrations of QLEL12 (SEQ ID NO: 35) between 0.1% (w / v) and 0.3% (w / v) and 0.9% NaCl (w / v) at pH 7.5 are shown; a linear regression was performed.

[0078] Figures 25A-25D Shown are the elevation height and lifting capability of the submucosal layer of the canine stomach and colon following injection of 0.5 mL of QLEL12 (SEQ ID NO:35) at various concentrations between 0.1% (w / v) and 0.3% (w / v) at pH 7.5 and 0.9% NaCl (w / v).

[0079] These and other advantages of the present technology will be apparent when reference is made to the following description. DETAILED DESCRIPTION

[0080] definition

[0081] Unless otherwise defined herein, scientific and technical terms used in this disclosure shall have the meanings commonly understood by those of ordinary skill in the art. In general, the terms and techniques related to chemistry, cell and tissue culture, molecular biology, cell and cancer biology, neurobiology, neurochemistry, virology, immunology, microbiology, pharmacology, genetics, and protein and nucleic acid chemistry described herein are well-known and commonly used in the art.

[0082] The methods and techniques of the present disclosure are generally performed according to conventional methods well known in the art and as described in various general and more specific references that are cited and discussed throughout the specification unless otherwise indicated.

[0083] Chemical terms used herein are used according to conventional usage in the art, as exemplified by “The McGraw-Hill Dictionary of Chemical Terms,” Parker S., Ed., McGraw-Hill, San Francisco, CA (1985).

[0084] All publications, patents, and published patent disclosures cited in this disclosure are expressly incorporated herein by reference. In case of conflict, the present specification, including its specific definitions, will control.

[0085] As used herein, the term "self-assembly" refers to the ability of certain peptides to spontaneously self-associate into higher order structures (e.g., beta sheets). For example, a pharmaceutical composition comprising self-assembling peptides in solution form transforms into a gel state when the self-assembling peptides self-associate. In some embodiments, the interactions between the individual self-assembling peptides are reversible, allowing the composition to reversibly transform between a gel state and a solution state. The interactions between the individual self-assembling peptides can be non-covalent interactions, including hydrogen bonds, ionic interactions, electrostatic interactions (e.g., through van der Waals forces), and hydrophobic interactions. In various embodiments, the self-assembled peptide nanostructure comprises a beta sheet peptide. The nanostructure can be a nanofiber or a network of nanofibers. For illustrative purposes, Fig.19 Self-assembling peptides are shown assembling into beta sheets 1901. These beta sheets can self-assemble into nanofibers 1902. Multiple nanofibers 1902 can self-assemble into a membrane network 1903. In some embodiments, the self-assembly of peptides described herein into higher order structures is responsive to one or more environmental triggers (e.g., changes in one or more of pH, temperature, ionic strength, osmolarity, osmolarity, applied pressure, applied shear stress, etc.).

[0086] As used herein, the term "administering" is intended to include, but is not limited to, applying, introducing, or injecting the self-assembling peptides described herein and / or pharmaceutical compositions comprising the self-assembling peptides.

[0087] As used herein, the term "amino acid residue" or "amino acid" includes natural and synthetic amino acid residues, including D- and L-amino acids; α-, β-, and γ-amino acids; chemically modified amino acids; naturally occurring non-protein amino acids; rare amino acids; and chemically synthesized compounds having the properties known in the art as amino acids.

[0088] As used herein, the phrases "therapeutically effective amount," "effective amount," or "effective dose" refer to an amount of a pharmaceutical composition that, when delivered to a subject, provides a therapeutic or aesthetic benefit in the treatment, prevention, or management of a tissue, disease, and / or condition. The determination of a therapeutically effective amount is well within the capabilities of one skilled in the art. In general, a therapeutically effective amount may vary with the subject's medical history, age, condition, sex, severity and type of medical condition in the subject, and the administration of other pharmaceutically active agents.

[0089] As used herein, the term "hydrogel" refers to a composition comprising a three-dimensional network of self-assembling peptides. The term hydrogel can be used to refer to a self-assembling peptide network in a dry state (xerogel) or a wet state. In the wet state, the hydrogel can contain a high water content (e.g., about 90% to about 99.9% water). Hydrogels have many properties required for biomedical applications. For example, hydrogels can be made non-toxic and compatible with tissues. In addition, hydrogels are generally highly permeable to water, ions, and small molecules.

[0090] As used herein, the term "isolated" with respect to a cell refers to a cell that has been mechanically separated from the environment in which it is naturally found.

[0091] As used herein, the term "tonicity agent" refers to an agent that can be used to adjust the osmotic pressure of the pharmaceutical compositions described herein. Exemplary tonicity agents include, but are not limited to, sodium chloride, calcium chloride, potassium chloride, potassium phosphate, and sugars (e.g., dextrose and sucrose). In some embodiments, the tonicity agent is sodium chloride. The tonicity agent can increase the rheological properties of a solution or composition (e.g., a hydrogel) comprising the self-assembling peptides disclosed herein.

[0092] As used herein, "pharmaceutical composition" refers to a composition comprising a self-assembling peptide and other components (eg, physiologically suitable carriers and / or excipients).

[0093] As used herein, the terms "protein" and "peptide" are used interchangeably and refer to a polymer of amino acid residues interconnected by peptide bonds between the α-amino and carboxyl groups of adjacent residues. The terms "protein" and "peptide" include polymers with modified amino acid residues (e.g., phosphorylated, amidated or glycosylated amino acid residues) and amino acid analogs.

[0094] As used herein, the term "subject" refers to a human or non-human animal. The term includes, but is not limited to, mammals (e.g., humans, other primates, pigs, rodents (e.g., mice and rats or hamsters), rabbits, guinea pigs, cattle, horses, cats, dogs, sheep, and goats). In some embodiments, the subject is a human. In some embodiments, the subject is an adult subject. In some embodiments, the subject is a pediatric human subject.

[0095] As used herein, the term "wound" refers to a trauma in the tissue of a subject (eg, a human subject), such as an abrasion, burn, chapter, abrasion, cut, ulcer, laceration, incision, or scratch.

[0096] Unless otherwise defined, all technical and scientific terms used herein have the same meanings as those of ordinary skill in the art to which the present invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described below. All publications, patent applications, patents and other references mentioned herein are incorporated herein by reference in their entirety. In the event of a conflict, this specification (including definitions) shall prevail. In addition, materials, methods and examples are illustrative only and are not intended to be limiting. Reference or identification to any file in this application does not mean that the document can be used as the prior art of the present invention.

[0097] Other features and advantages of the invention will be apparent from the following detailed description, and from the claims.

[0098] Pharmaceutical compositions and self-assembling peptides

[0099] In one aspect, the present disclosure provides self-assembling peptides and pharmaceutical compositions comprising at least one self-assembling peptide described herein. When present in an aqueous solution, the self-assembling peptides spontaneously assemble into higher order structures through intermolecular and intramolecular electrostatic interactions. These higher order structures include beta sheets, nanofiber structures, and three-dimensional networks or mesh structures. When these higher order structures form aqueous solutions, they may become hydrogels with various desired properties. In addition to the self-assembling peptides, the pharmaceutical composition may also include other additives, such as tonicity agents, buffers, pharmaceutically acceptable excipients, biomolecules, therapeutic agents, and cells.

[0100] In some embodiments, the self-assembling peptide is biodegradable. As used herein, "biodegradable" refers to a material that is degraded or decomposed into a component that can be metabolized or excreted by a subject in a few minutes to a few years (for example, one day, two days, three days, four days, five days, six days, seven days, two weeks, three weeks, four weeks, two months, three months, six months, one year, two years or longer) when interacting with a physiological environment. In some embodiments, the self-assembling peptide can be degraded by cracking the peptide chain, for example, by hydrolysis or enzymatic cleavage. Although the self-assembling peptide (and the higher order structure (such as support) formed by the self-assembling peptide) can be biodegradable, these higher order structures preferably maintain their structural integrity within a period of time required for their intended use.

[0101] The self-assembling peptides provided herein comprise or consist of an amino acid sequence as set forth in the following formula:

[0102] [(X)i(Y)j(Z)k(Y)l]m(X)n (Formula I),

[0103] [(Y)i(X)j(Y)k(Z)l]m(Y)n (Formula II),

[0104] [(Z)i(Y)j(X)k(Y)l]m(Z)n (Formula III) or

[0105] [(Y)i(Z)j(Y)k(X)l]m(Y)n (Formula IV),

[0106] Wherein each (X) is independently an ionic, polar amino acid, wherein each (Y) is independently a hydrophobic amino acid, wherein each (Z) is independently a nonionic, polar amino acid, wherein each i, j, k and l is independently an integer ≥1, wherein m is an integer ≥2, and wherein n=0 or an integer ≥1. In some embodiments, each i, j, k and l is independently an integer 1. In some embodiments, each i, j, k and l is independently an integer 2. In some embodiments, each i, j, k and l is independently an integer 3. In some embodiments, n is 0. In some embodiments, n is an integer ≥1 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or greater). In some embodiments, m is an integer 1. In some embodiments, m is an integer ≥2 (e.g., 2, 3, 4, 6, 7, 8, 9, 10, 11, 12 or greater). In some embodiments, m is an integer 2, 3 or 4. In some embodiments, each (X) is the same amino acid, each (Y) is the same amino acid and / or each (Z) is the same type of amino acid residue.

[0107] In some embodiments, the self-assembling peptide comprises or consists of an amino acid sequence set forth in [(X)i(Y)j(Z)k(Y)l]m(X)n (Formula I), wherein each (X) is independently an ionic, polar amino acid, wherein each (Y) is independently a hydrophobic amino acid, wherein each (Z) is independently a nonionic, polar amino acid, wherein each i, j, k, and l is independently an integer ≥1, wherein m is an integer ≥2, and wherein n=0 or an integer ≥1. In some embodiments, each i, j, k, and l is independently an integer 1. In some embodiments, each i, j, k, and l is independently an integer 2. In some embodiments, each i, j, k, and l is independently an integer 3. In some embodiments, n is 0. In some embodiments, n is an integer ≥1 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or greater). In some embodiments, m is an integer 1. In some embodiments, m is an integer ≥ 2 (e.g., 2, 3, 4, 6, 7, 8, 9, 10, 11, 12 or more). In some embodiments, m is an integer 2, 3 or 4.

[0108] In some embodiments, the self-assembling peptide comprises or consists of an amino acid sequence set forth in [(Y)i(X)j(Y)k(Z)l]m(Y)n (Formula II), wherein each (X) is independently an ionic, polar amino acid, wherein each (Y) is independently a hydrophobic amino acid, wherein each (Z) is independently a nonionic, polar amino acid, wherein each i, j, k, and l is independently an integer ≥1, wherein m is an integer ≥2, and wherein n=0 or an integer ≥1. In some embodiments, each i, j, k, and l is independently an integer 1. In some embodiments, each i, j, k, and l is independently an integer 2. In some embodiments, each i, j, k, and l is independently an integer 3. In some embodiments, n is 0. In some embodiments, n is an integer ≥1 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or greater). In some embodiments, m is an integer 1. In some embodiments, m is an integer ≥ 2 (e.g., 2, 3, 4, 6, 7, 8, 9, 10, 11, 12 or more). In some embodiments, m is an integer 2, 3 or 4.

[0109] In some embodiments, the self-assembling peptide comprises or consists of an amino acid sequence set forth in [(Z)i(Y)j(X)k(Y)l]m(Z)n (Formula III), wherein each (X) is independently an ionic, polar amino acid, wherein each (Y) is independently a hydrophobic amino acid, wherein each (Z) is independently a nonionic, polar amino acid, wherein each i, j, k, and l is independently an integer ≥1, wherein m is an integer ≥2, and wherein n=0 or an integer ≥1. In some embodiments, each i, j, k, and l is independently an integer 1. In some embodiments, each i, j, k, and l is independently an integer 2. In some embodiments, each i, j, k, and l is independently an integer 3. In some embodiments, n is 0. In some embodiments, n is an integer ≥1 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or greater). In some embodiments, m is an integer 1. In some embodiments, m is an integer ≥ 2 (e.g., 2, 3, 4, 6, 7, 8, 9, 10, 11, 12 or more). In some embodiments, m is an integer 2, 3 or 4.

[0110] In some embodiments, the self-assembling peptide comprises or consists of an amino acid sequence set forth in [(Y)i(Z)j(Y)k(X)l]m(Y)n (Formula IV), wherein each (X) is independently an ionic, polar amino acid, wherein each (Y) is independently a hydrophobic amino acid, wherein each (Z) is independently a nonionic, polar amino acid, wherein each i, j, k, and l is independently an integer ≥1, wherein m is an integer ≥2, and wherein n=0 or an integer ≥1. In some embodiments, each i, j, k, and l is independently an integer 1. In some embodiments, each i, j, k, and l is independently an integer 2. In some embodiments, each i, j, k, and l is independently an integer 3. In some embodiments, n is 0. In some embodiments, n is an integer ≥1 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or greater). In some embodiments, m is an integer 1. In some embodiments, m is an integer ≥ 2 (e.g., 2, 3, 4, 6, 7, 8, 9, 10, 11, 12 or more). In some embodiments, m is an integer 2, 3 or 4.

[0111] In some embodiments, the self-assembling peptide comprises or consists of an amino acid sequence set forth in [(X)i(Y)j(Z)k(Y)l]m (Formula V), wherein each (X) is independently an ionic, polar amino acid, wherein each (Y) is independently a hydrophobic amino acid, wherein each (Z) is independently a nonionic, polar amino acid, wherein each i, j, k, and l is independently an integer ≥1, wherein m is an integer ≥2, and wherein n=0 or an integer ≥1. In some embodiments, each i, j, k, and l is independently an integer 1. In some embodiments, each i, j, k, and l is independently an integer 2. In some embodiments, each i, j, k, and l is independently an integer 3. In some embodiments, n is 0. In some embodiments, n is an integer ≥1 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or greater). In some embodiments, m is an integer 1. In some embodiments, m is an integer ≥ 2 (e.g., 2, 3, 4, 6, 7, 8, 9, 10, 11, 12 or more). In some embodiments, m is an integer 2, 3 or 4.

[0112] In some embodiments, the self-assembling peptide comprises or consists of an amino acid sequence set forth in [(Y)i(X)j(Y)k(Z)l]m (Formula VI), wherein each (X) is independently an ionic, polar amino acid, wherein each (Y) is independently a hydrophobic amino acid, wherein each (Z) is independently a nonionic, polar amino acid, wherein each i, j, k, and l is independently an integer ≥1, wherein m is an integer ≥2, and wherein n=0 or an integer ≥1. In some embodiments, each i, j, k, and l is independently an integer 1. In some embodiments, each i, j, k, and l is independently an integer 2. In some embodiments, each i, j, k, and l is independently an integer 3. In some embodiments, n is 0. In some embodiments, n is an integer ≥1 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or greater). In some embodiments, m is an integer 1. In some embodiments, m is an integer ≥ 2 (e.g., 2, 3, 4, 6, 7, 8, 9, 10, 11, 12 or more). In some embodiments, m is an integer 2, 3 or 4.

[0113] In some embodiments, the self-assembling peptide comprises or consists of an amino acid sequence set forth in [(Z)i(Y)j(X)k(Y)l]m (Formula VIII), wherein each (X) is independently an ionic, polar amino acid, wherein each (Y) is independently a hydrophobic amino acid, wherein each (Z) is independently a nonionic, polar amino acid, wherein each i, j, k, and l is independently an integer ≥1, wherein m is an integer ≥2, and wherein n=0 or an integer ≥1. In some embodiments, each i, j, k, and l is independently an integer 1. In some embodiments, each i, j, k, and l is independently an integer 2. In some embodiments, each i, j, k, and l is independently an integer 3. In some embodiments, n is 0. In some embodiments, n is an integer ≥1 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or greater). In some embodiments, m is an integer 1. In some embodiments, m is an integer ≥ 2 (e.g., 2, 3, 4, 6, 7, 8, 9, 10, 11, 12 or more). In some embodiments, m is an integer 2, 3 or 4.

[0114] In some embodiments, the self-assembling peptide comprises or consists of an amino acid sequence set forth in [(Y)i(Z)j(Y)k(X)l]m (Formula VIII), wherein each (X) is independently an ionic, polar amino acid, wherein each (Y) is independently a hydrophobic amino acid, wherein each (Z) is independently a nonionic, polar amino acid, wherein each i, j, k, and l is independently an integer ≥1, wherein m is an integer ≥2, and wherein n=0 or an integer ≥1. In some embodiments, each i, j, k, and l is independently an integer 1. In some embodiments, each i, j, k, and l is independently an integer 2. In some embodiments, each i, j, k, and l is independently an integer 3. In some embodiments, n is 0. In some embodiments, n is an integer ≥1 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or greater). In some embodiments, m is an integer 1. In some embodiments, m is an integer ≥ 2 (e.g., 2, 3, 4, 6, 7, 8, 9, 10, 11, 12 or more). In some embodiments, m is an integer 2, 3 or 4.

[0115] Each (X) in the self-assembling peptide of formula I-IV can be an acidic amino acid or a basic amino acid. For example, in some embodiments, each (X) is a basic amino acid, including but not limited to arginine, lysine, histidine and ornithine. In some embodiments, each (X) is an acidic amino acid, including but not limited to aspartic acid and glutamic acid.

[0116] Each (Y) in the self-assembling peptides of Formulas I-IV can be alanine, valine, leucine, isoleucine, methionine, phenylalanine, tryptophan, or glycine.

[0117] Each (Z) is selected from the group consisting of serine, threonine, tyrosine, cysteine, glutamine, asparagine and methionine.

[0118] The pharmaceutical compositions described herein may include one type of self-assembling peptide or a variety of different types of self-assembling peptides. For example, in some embodiments, the pharmaceutical compositions provided herein may include one type of self-assembling peptide (e.g., a self-assembling peptide comprising an amino acid sequence described in Formula I or consisting thereof). In some embodiments, the pharmaceutical compositions described herein include two or more types of self-assembling peptides (e.g., two, three, four, five, six, seven, eight, nine, ten or more types). When the pharmaceutical compositions provided herein include more than one type of self-assembling peptide, the self-assembling peptides may include an amino acid sequence described in a single formula (i.e., one of Formulas I-IV), or may include an amino acid sequence described in different formulas. When there are multiple types of self-assembling peptides in the pharmaceutical compositions provided herein, different types of peptides may interact and form a higher order structure (e.g., a β sheet).

[0119] In some embodiments, self-assembling peptide comprises about 8 amino acid residues to about 50 amino acid residues or is made up of about 8 amino acid residues to about 50 amino acid residues.In some embodiments, self-assembling peptide comprises about 8 amino acid residues to 18 amino acid residues or is made up of about 8 amino acid residues to 18 amino acid residues.For example, self-assembling peptide can comprise 8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29,30,31,32,33,34,35,36,37,38,39,40,41,42,43,44,45,46,47,48,49 or 40 amino acid residues.The size of self-assembling peptide can make the tertiary structure of self-assembling peptide not destroy the ability of this peptide and other self-assembling peptides to form higher order structure (for example nanofiber or β sheet). In some embodiments, the self-assembling peptide comprises or consists of about 12 amino acid residues. In some embodiments, the self-assembling peptide comprises or consists of about 13 amino acid residues. In some embodiments, the self-assembling peptide comprises or consists of about 14 amino acid residues. In some embodiments, the self-assembling peptide comprises or consists of about 15 amino acid residues. In some embodiments, the self-assembling peptide comprises or consists of about 16 amino acid residues. In some embodiments, the self-assembling peptide comprises or consists of about 17 amino acid residues. In some embodiments, the self-assembling peptide comprises or consists of about 18 amino acid residues. In some embodiments, the self-assembling peptide comprises or consists of about 19 amino acid residues. In some embodiments, the self-assembling peptide comprises or consists of about 20 amino acid residues. In some embodiments, the self-assembling peptide comprises or consists of about 21 amino acid residues. In some embodiments, the self-assembling peptide comprises or consists of about 22 amino acid residues. In some embodiments, the self-assembling peptide comprises or consists of about 23 amino acid residues. In some embodiments, the self-assembling peptide comprises or consists of about 24 amino acid residues. In some embodiments, the self-assembling peptide comprises or consists of about 25 amino acid residues. In some embodiments, the self-assembling peptide comprises or consists of about 26 amino acid residues. In some embodiments, the self-assembling peptide comprises or consists of about 27 amino acid residues. In some embodiments, the self-assembling peptide comprises or consists of about 28 amino acid residues. In some embodiments, the self-assembling peptide comprises or consists of about 29 amino acid residues. In some embodiments, the self-assembling peptide comprises or consists of about 30 amino acid residues.

[0120] Table 1 below provides exemplary self-assembling peptides. The exemplary self-assembling peptides of Table 1 include alternating ionic, polar amino acid residues, hydrophobic amino acid residues and non-ionic, polar amino acid residues. In some embodiments, the pharmaceutical compositions provided herein comprise a self-assembling peptide comprising or consisting of an amino acid sequence set forth in SEQ ID NOs: 1-20 and 94-96.

[0121] Table 1. Exemplary self-assembling peptides

[0122]

[0123]

[0124] In some embodiments, the self-assembling peptide may include one or more functional groups. In some embodiments, the functional group can prevent or delay the degradation of the self-assembling peptide. In some embodiments, the self-assembling peptide includes an N-terminal functional group. In some embodiments, the self-assembling peptide includes a C-terminal functional group. In some embodiments, the self-assembling peptide includes an N-terminal functional group and a C-terminal functional group. For example, in some embodiments, the functional group prevents or delays the degradation of the self-assembling peptide by an enzyme (e.g., in vivo) (e.g., acetyl, formyl, chloroacetyl, benzyloxycarbonyl, bromoacetyl, tert-butyloxycarbonyl, 9-fluorenylmethoxycarbonyl, allyloxycarbonyl, methyl ester, benzyl ester or tert-butyl ester). Exemplary N-terminal functional groups include, but are not limited to, acetyl, formyl, pyroglutamyl (pGlu), biotin, polyethylene glycol (PEG), urea, alkylamine, carbamate, sulfonamide (e.g., 4-toluenesulfonyl, 4-nitrobenzenesulfonyl), dansyl, 2,4-dinitrophenyl, fluorescein, 7-methoxycoumarinacetic acid, 9-fluorenylmethoxycarbonyl, palmitic acid, succinyl, chloroacetyl, maleimide, benzyloxycarbonyl, bromoacetyl, nitrilotriacetyl, tert-butyloxycarbonyl, 4-hydroxyphenylpropionic acid, allyloxycarbonyl, butyric acid, fatty acids (e.g., caproic acid, octanoic acid, capric acid, palmitic acid, stearic acid, myristic acid, and lauric acid), and trityl. Exemplary C-terminal functional groups include, but are not limited to, amide, N-alkylamide, aldehyde, ester (e.g., methyl, benzyl or tert-butyl), alcohol, p-nitroanilide (pNA), 7-amino-4-methylcoumarin (Amc), hydrazide, hydroxamic acid, chloromethyl ketone, p-nitroaniline, p-nitrophenol, hydroxysuccinimide ester, fluoromethyl ketone, cysteamide, 9-fluorenylmethyl (Fm) ester, allyl ester, 2,4-dimethoxybenzyl ester, 2-phenylisopropyl ester, p-nitrobenzyl ester, and 2-chlorotrityl ester.

[0125] In some embodiments, the self-assembling peptides described herein may include an N-terminal acetyl group and / or a C-terminal amine group. For example, Table 2 below provides exemplary self-assembling peptides that simultaneously include these functional groups. In some embodiments, the pharmaceutical compositions provided herein include self-assembling peptides that include or consist of the amino acid sequences set forth in SEQ ID NOs: 21-40 and 97-99.

[0126] Table 2. Exemplary self-assembling peptides containing an N-terminal acetyl group and a C-terminal amine group

[0127]

[0128] In some embodiments, the self-assembling peptides described herein include at least one bioactive peptide motif. Without wishing to be bound by any particular theory, the bioactive peptide motif can promote one or more biological processes in, above or around the pharmaceutical composition (e.g., hydrogel) described herein, including, for example, cell adhesion, differentiation, proliferation, recruitment and / or homing; neurite outgrowth; bioactive molecule recruitment (e.g., as a binding site for bioactive molecules), retention and / or recombination. The bioactive peptide motif can be present anywhere in the amino acid sequence of the self-assembling peptide and preferably does not interfere with the ability of the self-assembling peptide to form a higher-order structure (e.g., nanofiber or β sheet) via intramolecular interactions or intermolecular interactions with other self-assembling peptides. For example, in some embodiments, the bioactive peptide motif is present at the N-terminus of the self-assembling peptide. In some embodiments, the bioactive peptide motif is present at the C-terminus of the self-assembling peptide. The self-assembling peptide can include one or more (e.g., one, two, three, four, five, six, seven, eight or more) bioactive peptide motifs. When the self-assembling peptide includes more than one bioactive peptide motif, each motif can be of the same type, or each motif can be different. Exemplary bioactive peptide motifs can be derived from proteins such as laminin-1, collagen IV, fibronectin, elastin, bone marrow homing peptide 1, bone marrow homing peptide 2, or myelin. Non-limiting examples of bioactive motifs are provided in Table 3 below. In some embodiments, the self-assembling peptides provided herein comprise a bioactive peptide motif comprising an amino acid sequence set forth in SEQ ID NO: 41-70.

[0129] Table 3. Exemplary bioactive peptide motifs

[0130]

[0131]

[0132] In some embodiments, the self-assembling peptide comprises the bioactive peptide motifs shown below in Table 4. In some embodiments, the self-assembling peptide comprises or consists of the amino acid sequence set forth in SEQ ID NOs: 71-90.

[0133] Table 4. Exemplary self-assembling peptides containing bioactive peptide motifs

[0134]

[0135] The concentration of the self-assembling peptide present in the pharmaceutical composition provided herein can be varied to change the rheological properties of the composition. In some embodiments, the pharmaceutical composition may include about 0.01% (w / v) to about 10% (w / v) self-assembling peptides described herein. In some embodiments, the pharmaceutical composition may include about 0.1% (w / v) to about 5% (w / v) self-assembling peptides described herein. In some embodiments, the pharmaceutical composition may include about 0.1% (w / v) to about 2% (w / v) self-assembling peptides described herein. In some embodiments, the pharmaceutical composition may include about 0.5% (w / v) to about 3% (w / v) self-assembling peptides described herein. In some embodiments, the pharmaceutical composition may include about 0.5% (w / v) to about 1.5% (w / v) self-assembling peptides described herein. In some embodiments, the pharmaceutical composition may include about 1% (w / v) to about 3% (w / v) self-assembling peptides described herein. In some embodiments, the pharmaceutical composition comprises about 0.5%, about 1% (w / v), about 1.5% (w / v), about 2% (w / v), about 2.5% (w / v), about 3% (w / v), about 3.5% (w / v), about 4% (w / v), about 4.5% (w / v) or about 5% (w / v) of a self-assembling peptide described herein.

[0136] In some embodiments, the pharmaceutical composition can be an aqueous solution or a hydrogel, or it can be dehydrated (e.g., a powder). The hydrogel formed by the self-assembling peptides described herein can be porous or solid. In some embodiments, the hydrogel comprises pores having an average diameter of about 1 nm to about 2,000 μm, about 10 nm to about 1,000 μm, about 10 nm to about 500 μm, about 10 nm to about 100 μm, about 10 nm to about 1 μm, about 5 nm to about 500 nm, or about 5 nm to about 250 nm.

[0137] In some embodiments, the pharmaceutical composition provided herein can be a hydrogel of different sizes and geometries, including films and particles, such as nanoparticles or microparticles. In some embodiments, the pharmaceutical composition can be layered on a surface (e.g., a hydrogel). The pharmaceutical composition particle size can be changed according to the expected specific use of such particles. Typically, the particle can have at least one dimension in the range of about 1000 μm to about 2000 μm (e.g., about 1,000 μm, about 1,100 μm, 1,200 μm, 1,300 μm, 1,400 μm, 1,500 μm, 1,600 μm, 1,700 μm, 1,800 μm, 1,900 μm, or 2,000 μm).

[0138] The self-assembling peptides described herein can be advantageously formulated at physiological pH and the self-assembling peptides remain dissolved and stable therein. In some embodiments, the pH of the pharmaceutical composition described herein is about 5 to about 8. In some embodiments, the pH of the pharmaceutical composition is about 6 to about 8. In some embodiments, the pH of the pharmaceutical composition is about 5.5 to about 7.5. In some embodiments, the pH of the pharmaceutical composition is about 6 to about 7.4. In some embodiments, the pH of the pharmaceutical composition is about 6.5 to about 7.5. In some embodiments, the pH of the pharmaceutical composition is about 7 to about 7.5. In some embodiments, the pH of the pharmaceutical composition is about 6.5, about 6.6, about 6.7, about 6.8, about 6.9, about 7.0, about 7.1, about 7.2, about 7.3, about 7.4, about 7.5, about 7.6, about 7.7, about 7.8, about 7.9 or about 8.0. The pH of the pharmaceutical composition described herein can be adjusted by including a buffer system. Alternatively, the self-assembling peptide can be used as a buffer in the pharmaceutical composition. The pH of the pharmaceutical composition may be adjusted using one or more acids or bases, such as sodium hydroxide, potassium hydroxide, hydrochloric acid, phosphoric acid, sodium carbonate, and sodium bicarbonate.

[0139] In some embodiments, the pharmaceutical composition may include a buffer such as phosphate buffered saline (PBS). In some embodiments, the pharmaceutical composition may include a culture medium for cell culture, including but not limited to Dulbecco's modified Eagle's medium (DMEM), DME / F12, minimal essential medium (MEM), basal medium Eagle (BME), RPMI1640, F-10, F-12, α-minimal essential medium (α-MEM), Glasgow's minimal essential medium (G-MEM), PF CHO (SAFC Biosciences) and Iscove's modified Dulbecco's medium.

[0140] The pharmaceutical compositions described herein can be formulated so that the pH of the composition is neutral or physiological (e.g., pH is about 6 to about 8) and the net charge of the self-assembling peptides therein is non-zero. Without wishing to be bound by any particular theory, when formulated at neutral or physiological pH, the non-zero net charge of the self-assembling peptides makes the peptides prone to forming electrostatic interactions with molecules of opposite charge. This property can be advantageously used to promote the adhesion of the pharmaceutical composition to the desired surface. This is particularly advantageous in clinical applications in which the pharmaceutical composition is applied to tissues rich in biomolecules (e.g., glycoproteins) with non-zero net charge.

[0141] In addition, without wishing to be bound by any particular theory, the self-assembling peptides described herein having a net positive charge when formulated at physiological pH are particularly advantageous in therapeutic applications because they may have bactericidal and / or bacteriostatic effects. Therefore, the self-assembling peptides can be readily used in a variety of applications where it is desired to reduce bacterial burden.

[0142] In some embodiments, when the pH of the pharmaceutical composition is about 5 to about 8, the net charge of the self-assembling peptide is greater than or equal to +1 or less than or equal to -1. In some embodiments, when the pH of the pharmaceutical composition is about 5 to about 8 (e.g., 7.4), the net charge of the self-assembling peptide is about +1 to about +6 (e.g., +1, +2, +3, +4, +5, or +6). In some embodiments, when the pH of the pharmaceutical composition is about 5 to about 8 (e.g., 7.4), the net charge of the self-assembling peptide is about -1 to about -6 (e.g., -1, -2, -3, -4, -5, or -6).

[0143] In some embodiments, the elastic modulus of the pharmaceutical composition provided herein is 10 Pascals (Pa) to about 10,000 Pa. As used herein, the term "elastic modulus" refers to the tendency of a composition to deform elastically (i.e., non-permanently) when a force is applied. Typically, the elastic modulus of an object is defined as the slope of its stress-strain curve in the elastic deformation region. Specifying how to measure stress and strain (including direction), multiple types of elastic moduli can be defined. Young's modulus (E) describes tensile elasticity, or the tendency of an object to deform along an axis when opposite forces are applied to the object along the axis; it is defined as the ratio of tensile stress to tensile strain. It is often referred to as the elastic modulus. The shear modulus or stiffness modulus (G or μ) describes the shear tendency (shape deformation under constant volume) when an object is subjected to opposite forces; it defines shear stress under shear strain. The shear modulus is part of the derivation of viscosity. The bulk modulus (K) describes the bulk elasticity, or the tendency of an object to deform in all directions when uniformly loaded in all directions; it is defined as the volume stress under volume strain and is the inverse of the compressibility. Bulk modulus is the extension of Young's modulus to three dimensions. In some embodiments, the storage modulus of a pharmaceutical composition (e.g., hydrogel) described herein is at least about 10Pa. In some embodiments, the storage modulus of a pharmaceutical composition described herein is at least about 50Pa. In some embodiments, the storage modulus of a pharmaceutical composition described herein is at least about 100Pa. In some embodiments, the storage modulus of a pharmaceutical composition described herein is at least about 500Pa. In some embodiments, the storage modulus of a pharmaceutical composition described herein is at least about 1,000Pa. In some embodiments, the storage modulus of a pharmaceutical composition described herein is about 10Pa to about 5,000Pa. In some embodiments, the storage modulus of a pharmaceutical composition described herein is about 50Pa to about 500Pa. In some embodiments, the storage modulus of a pharmaceutical composition described herein is about 10Pa to about 1,000Pa. In some embodiments, the storage modulus of a pharmaceutical composition described herein is about 500Pa to about 5,000Pa.

[0144] In some embodiments, the increased ionic strength can increase the rigidity and / or gelation kinetics of the self-assembling peptides present in the pharmaceutical compositions described herein. In some embodiments, the increased ionic strength can be a physiological ionic strength. When the composition is applied to and / or on a subject (e.g., a human subject), the ionic strength of the pharmaceutical composition can be increased. Alternatively, the ionic strength of the pharmaceutical composition described herein can be increased by mixing the composition with one or more tonicity agents. Exemplary tonicity agents include, but are not limited to, sodium chloride, calcium chloride, potassium chloride, and potassium phosphate. In some embodiments, the pharmaceutical compositions described herein do not include tonicity agents. In some embodiments, the pharmaceutical compositions described herein include about 0.01M to about 0.3M tonicity agents. In some embodiments, the pharmaceutical compositions described herein include about 0.1M to about 0.3M tonicity agents. In some embodiments, the pharmaceutical compositions described herein include about 0.1M to about 0.2M tonicity agents. In some embodiments, the pharmaceutical compositions described herein comprise 0.01 M, 0.02 M, 0.03 M, 0.04 M, 0.05 M, 0.06 M, 0.07 M, 0.08 M, 0.09 M, 0.1 M, 0.15 M, 0.2 M, 0.25 M, or 0.3 M tonicity agent.

[0145] In some embodiments, the pharmaceutical compositions described herein are formulated so that they are hypotonic relative to the site of administration to the subject. In some embodiments, the pharmaceutical compositions described herein are formulated so that they are isotonic relative to the site of administration to the subject. In some embodiments, the pharmaceutical compositions described herein are formulated so that they are hypertonic relative to the site of administration to the subject.

[0146] In some embodiments, the pharmaceutical compositions described herein comprise cells (e.g., isolated cells). In some embodiments, the pharmaceutical compositions (e.g., hydrogels) can enhance the viability of the cells therein, thereby facilitating the delivery of living cells to a desired site (e.g., injured or defective body tissue). In some embodiments, the pharmaceutical composition is an aqueous solution comprising about 10 4 to about 10 8 cells / mL (e.g. 10 4 , 10 5 , 10 6 , 10 7 In some embodiments, the pharmaceutical composition is an aqueous solution comprising about 10 4 to about 10 6 In some embodiments, the pharmaceutical composition comprises one cell type. In some embodiments, the pharmaceutical composition comprises more than one cell type (e.g., two, three, four, five or more cell types).

[0147] Depending on the desired application, any suitable cell may be included in the pharmaceutical composition. For example, in some embodiments the pharmaceutical composition (e.g., hydrogel) is inoculated with animal cells or plant cells. When it is a hydrogel, the cell can be encapsulated in the hydrogel. In some embodiments, the pharmaceutical composition comprises mammalian cells. Exemplary mammalian cells include, but are not limited to, stem cells (embryonic stem cells, mesenchymal stem cells, stem cells derived from bone marrow, hematopoietic stem cells, neural stem cells and hair follicle stem cells), chondroprogenitor cells, pancreatic progenitor cells, myoblasts, fibroblasts, keratinocytes, neurons, glial cells, astrocytes, preadipocytes, adipocytes, vascular endothelial cells, endothelial progenitor cells, mesenchymal cells, neural stem cells, immune cells (e.g., B cells and T cells), smooth muscle progenitor cells, cardiomyocytes, fetal dermal fibroblasts, epidermal keratinocytes, myoblasts and capillary endothelial cells. In some embodiments, the cell is a genetically modified cell (e.g., a cell modified to express and secrete a desired compound, such as a growth factor, a differentiation factor, a cytokine, a chimeric antigen receptor, an antibody or a fragment thereof). In some embodiments, the cell is a tissue culture cell line cell. Exemplary tissue culture cell line cells include, but are not limited to, C166 cells, C6 glioma cell line, AML12, HeLa cells, and Chinese hamster ovary cells (CHO cells).

[0148] In some embodiments, the pharmaceutical compositions provided herein comprise biologically active molecules, including but not limited to extracellular matrix proteins (e.g., fibronectin, vitronectin and laminin), cytokines, growth factors, differentiation factors, insulin, nucleic acids, vitamins, fatty acids and therapeutic agents.

[0149] Exemplary growth factors and cytokines include, but are not limited to, stem cell factor (SCF), granulocyte colony stimulating factor (G-CSF), granulocyte-macrophage stimulating factor (GM-CSF), stromal cell-derived factor-1, vascular endothelial growth factor (VEGF), platelet-derived growth factor (PDGF), angiogenin, epidermal growth factor (EGF), basic fibroblast growth factor (bFGF), hepatocyte nuclear factor-1 (HNF-1), nerve growth factor (NGF), bone morphogenetic protein (BMP), fibroblast growth factor (FGF), hepatocyte growth factor, insulin-like growth factor (IGF-1), interleukin (IL)-3, IL-1a, IL-1β, IL-6, IL-7, IL-8, IL-11 and IL-13, colony stimulating factor, thrombopoietin, erythropoietin, fit3 ligand, and tumor necrosis factor alpha (TNFα).

[0150] In some embodiments, the bioactive molecule is a therapeutic agent. As used herein, the term "therapeutic agent" refers to a compound used in a subject for diagnosis, treatment or prevention of a disease or condition. Any therapeutic agent known to be useful in the diagnosis, treatment or prevention of a disease or condition may be included in the pharmaceutical composition provided herein. Therapeutic agents include pharmacologically active compounds (e.g., antibiotics and anti-inflammatory agents), hormones, DNA (e.g., plasmid DNA), RNA, siRNA, shRNA, antisense RNA, proteins (e.g., antibodies and fragments thereof and enzymes), lipids, pro-inflammatory molecules and combinations thereof. Any of the therapeutic agents may be combined to the extent that such combinations are biocompatible. The amount of the therapeutic agent in the pharmaceutical composition will depend on a variety of factors, including, for example, the effective dose of the therapeutic agent required for a specific course of action and the time required to release the therapeutic agent. For example, other exemplary therapeutic agents and appropriate dosages and regimens are described in Harrison's Principles of Internal Medicine, 19th Edition, Eds. DL Kasper et al., McGraw-Hill, New York, NY (2015); Physician's Desk Reference, 71st Edition, Montvale, NJ, Physician's Desk Reference Inc. (2016); and Goodman and Gilman's: The Pharmacological Basis of Therapeutics, 13th Edition, Eds. LL Brunton et al., McGraw-Hill, New York, NY (2018), each of which is incorporated herein by reference.

[0151] Uses of self-assembling peptides and pharmaceutical compositions

[0152] The self-assembling peptides and pharmaceutical compositions described herein can be used in a variety of in vitro and in vivo applications, including cell culture applications and clinical applications.

[0153] Cell culture

[0154] In some embodiments, the self-assembling peptides and pharmaceutical compositions described herein can be used for cultured cells (e.g., isolated human cells). For example, the hydrogel comprising the self-assembling peptides described herein can be a substrate for cell culture. As mentioned above, the hydrogel formed by the self-assembling peptides described herein can include a first-order or higher-order structure (e.g., nanofibers and three-dimensional nets), which provide a space-time substrate for cells. Advantageously, since the hydrogels can be prepared at physiological pH, they can provide a suitable environment for cell growth and proliferation.

[0155] Any cell type described in detail above can be cultured in the hydrogel described herein. Cells can be directly seeded onto a preformed hydrogel, or they can be mixed with an aqueous pharmaceutical composition to form a hydrogel after gelation. The liquid phase of the hydrogel can be supplemented with one or more culture medium components (e.g., growth factors and serum) to provide the necessary factors to promote cell survival and proliferation. The appropriate conditions and factors necessary to maintain cell survival and growth are known in the art (see, e.g., Freshney, Culture of Animal Cells: A Manual of Basic Technique, Wiley-Liss, New York, NY (2000); and Cells: A Laboratory Manual (Spector, DL, Goldman, RD and Leinwand, LA, eds.), Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1998).

[0156] The self-assembling peptides and pharmaceutical compositions provided herein can be used in a variety of applications, including, for example, for cell culture, in cosmetics (e.g., skin care products and hair care products), in tissue engineering, as coating agents (e.g., for medical devices, such as contact lenses), as lubricants (e.g., in eye drops or as joint lubricants), as hemostats, as desiccants, as bone fillers, as artificial vitreous, as artificial lenses, as drug delivery devices, as water-retaining materials, and in biomaterial applications.

[0157] Clinical Application

[0158] In some embodiments, the self-assembling peptides and pharmaceutical compositions described herein can be used for a variety of clinical applications. Therefore, various methods for treating a subject are provided herein, which are administered to a specific site in the subject or on the subject in an effective amount of the pharmaceutical composition described herein. As described above, the specific advantage of the pharmaceutical compositions described herein is that they can be formulated at physiological pH (e.g., about pH 6 to about pH 8), and therefore, compared to pharmaceutical compositions of self-assembling peptides formulated at acidic or alkaline pH, pain caused by contact with the subject can be alleviated and tissue damage can be reduced.

[0159] Depending on the form of the pharmaceutical composition (e.g., aqueous solution or hydrogel) and the route of administration used, the amount of the pharmaceutical composition applied (e.g., applied or injected) to the subject, such as volume or concentration, can be changed. The exact formula, route of administration, volume and amount of the pharmaceutical composition can be selected according to the subject's condition and according to the specific target area or position where the pharmaceutical composition is applied. The specific dosage and treatment regimen for any particular subject may depend on a variety of factors, including a specific self-assembling peptide, the size of the treated area, the desired thickness of the resulting hydrogel (when the aqueous solution is applied) and the length of treatment time. Other factors that may affect the specific dosage and treatment regimen include age, weight, health status, sex, time of administration, degradation rate, severity and course of the disease, condition or symptoms. In some embodiments, the pharmaceutical composition can be administered in a single dose. In other embodiments, the pharmaceutical composition can be administered in more than one dose (e.g., two, three, four, five, six or more doses).

[0160] In some embodiments, the pharmaceutical composition is administered to a subject in the form of a pre-polymerized hydrogel. In some embodiments, the pharmaceutical composition is a hydrogel formed in vitro and administered to a desired location in a subject.

[0161] In some embodiments, the pharmaceutical composition is administered to a subject in the form of an aqueous solution. The aqueous solution can form a hydrogel in vivo after administration. Since the self-assembling peptides polymerize in response to changes in tonicity, a hydrogel can be formed when in contact with a subject during administration or shortly thereafter.

[0162] The pharmaceutical composition can be administered by introducing a delivery device at or near a predetermined or desired target area of ​​the experimenter. The delivery device can be a conventional device, or is designed to achieve at least one of the following: reach a specific target area, achieve a specific application scheme, deliver a specific target volume, amount or concentration, and accurately deliver to the target area. Suitable delivery devices include but are not limited to syringes, pipettes, tubes, catheters, syringe catheters, automatic injectors and other devices based on needles that arrive at the predetermined or desired target area of ​​the experimenter. The specification of the syringe needle can be selected to provide a pharmaceutical composition from a syringe to the sufficient flow rate of the target area.

[0163] The effective amount may comprise a pharmaceutical composition having a volume of about 0.1 milliliters (mL) to about 100 mL (if administered in the form of an aqueous solution). In some embodiments, the effective amount may be a pharmaceutical composition of about 0.1 mL to about 10 mL. In some embodiments, the effective amount may be a pharmaceutical composition of about 0.5 mL to about 5 mL. In some embodiments, the effective amount may be a pharmaceutical composition of about 1 mL to about 5 mL. In some embodiments, the effective amount may be a pharmaceutical composition of about 0.5 mL. In some embodiments, the effective amount may be a pharmaceutical composition of about 1.0 mL. In some embodiments, the effective amount may be a pharmaceutical composition of about 1.5 mL. In some embodiments, the effective amount may be a pharmaceutical composition of about 2.0 mL. In some embodiments, the effective amount may be a pharmaceutical composition of about 2.5 mL. In some embodiments, the effective amount may be about 0.1 mL to about 5 mL per cm 2 In some embodiments, the effective amount can be about 1 mL per cm 2 In some embodiments, the effective amount can be about 2 mL per cm 2 Target area.

[0164] The pharmaceutical composition can be administered to a subject by any suitable route known in the art, including but not limited to injection, implantation, microinjection, and direct application. Administration by injection includes but is not limited to cutaneous, intramuscular, intradermal, subdermal, and subcutaneous injections.

[0165] In some embodiments, the pharmaceutical composition is administered (e.g., injected or implanted) to a location of the subject in a single dose to produce the desired result. In some embodiments, the pharmaceutical composition is administered in multiple doses to produce the desired result. In some embodiments, when multiple doses of the pharmaceutical composition are administered, each administration is performed after a specific time period, such as 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 2 weeks, 1 month, 2 months, 3 months, or longer.

[0166] In some embodiments, the hydrogel formed or present at the site of the subject can be removed by contacting the hydrogel with a solution, the solution comprising a tonicity agent with a low osmotic concentration compared to the hydrogel. In some embodiments, the hydrogel formed or present at the site of the subject can be removed by contacting the hydrogel with water. In some embodiments, the hydrogel formed or present at the site of the subject can be removed by contacting the hydrogel with a solution, the solution being low osmotic relative to the site of the subject. In some embodiments, the hydrogel formed or present at the site of the subject can be removed by contacting the hydrogel with a solution, the solution being isotonic with respect to the site of the subject, with sugar (e.g., dextrose and sucrose). In some embodiments, the hydrogel formed or present at the site of the subject can be removed by contacting the hydrogel with a solution, the solution being isotonic, containing salts of low ionic strength (e.g., NaCl-"normal saline") compared to salts in body fluids.

[0167] The hydrogel can be in contact with the solution for a sufficient time to destroy the hydrogel (e.g., destroy the structure or morphology) and / or to detach the hydrogel from the site of the subject. In some embodiments, the hydrogel is in contact with the solution for about 5 minutes, about 10 minutes, about 20 minutes, about 30 minutes, about 1 hour, about 2 hours, about 3 hours or more.

[0168] In some embodiments, pharmaceutical compositions as described herein can be used as delivery devices for releasing bioactive molecules or therapeutic agents to specific targets. In some embodiments, pharmaceutical compositions can be formulated so that the composition allows spontaneous release of bioactive molecules or therapeutic agents (e.g., after being administered to a subject). In some embodiments, pharmaceutical compositions can be formulated so that pharmaceutical compositions allow controlled release of bioactive molecules or therapeutic agents. In some embodiments, bioactive molecules or therapeutic agents are released from pharmaceutical compositions (e.g., hydrogels) for a long time (e.g., from about 12 hours to about 2 months). In some embodiments, bioactive molecules or therapeutic agents are released from pharmaceutical compositions for about 12 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 2 weeks, 3 weeks, 1 month, 2 months, 3 months or longer. According to the amino acid sequence of the self-assembling peptides present in the pharmaceutical composition, the concentration of the self-assembling peptides in the pharmaceutical composition, and the biochemical and physical properties of the bioactive molecules or therapeutic agents, release kinetics can be changed.

[0169] 1. Gastrointestinal obstruction

[0170] The self-assembling peptides and pharmaceutical compositions described herein can be used to prevent gastrointestinal obstruction in a subject. Methods for preventing gastrointestinal obstruction using pharmaceutical compositions comprising self-assembling peptides are known in the art, and can be suitable for delivering pharmaceutical compositions and self-assembling peptides as described herein (see, for example, U.S. Patent No. 9,724,448, the entire contents of which are expressly incorporated herein by reference). For example, endoscopic mucosal resection (EMR) and endoscopic submucosal dissection (ESD) are the main surgical options for resection of lesions (such as polyps, ulcers, and gastrointestinal cancer) (see, for example, Wallace (2017) Gastroenterol. Hepatol. (NY) 13 (6): 371-4). Although EMR and ESD are minimally invasive surgeries, during the healing process, the tightening / contraction of scars may cause gastrointestinal obstruction. One form of gastrointestinal obstruction may be stenosis, tubular organs or structures (such as the gastrointestinal tract) stenosis, which may cause partial or complete obstruction of the gastrointestinal tract. A method for preventing stenosis in the gastrointestinal tract of a subject (e.g., in one or more of the oral cavity, throat, esophagus, stomach, small intestine, large intestine, colon, or rectum) is provided. In some embodiments, an effective amount of a pharmaceutical composition as described herein (e.g., an aqueous solution) is administered to a site in the gastrointestinal tract of a subject using a medical device (e.g., a syringe, a pipette, a tube, a syringe catheter, a catheter, or an endoscope). In some embodiments, the aqueous solution forms a hydrogel at the site of application, thereby allowing prevention of stenosis at the site of application. In some embodiments, the self-assembling peptide can promote mucosal epithelial formation to prevent or reduce postoperative scar formation, which can help prevent or reduce gastrointestinal obstruction or stenosis. In some embodiments, the hydrogel provides a scaffold for cell infiltration, promoting healing of the hydrogel application site.

[0171] In some embodiments, before lesion excision (e.g., using EMR or ESD), a pharmaceutical composition provided herein (e.g., aqueous solution) can be applied (e.g., by injection) to the submucosal layer of the gastrointestinal tract of a subject. The submucosal layer is a thinner connective tissue layer with a loose structure. In some embodiments, a pharmaceutical composition is injected into the submucosal layer to form a bulla and the lesion is lifted, thereby helping to remove the lesion. In some embodiments, the pharmaceutical composition raises the submucosal layer for about 20 minutes to about 1 hour. In some embodiments, the pharmaceutical composition raises the submucosal layer for about 20 minutes, about 30 minutes, about 40 minutes, about 50 minutes, about 1 hour, about 2 hours, about 3 hours, about 4 hours, about 5 hours or longer.

[0172] 2. Tissue repair or regeneration

[0173] The self-assembling peptides and pharmaceutical compositions described herein can be used to promote or enhance the repair or regeneration of tissues in subjects. Self-assembling peptides and pharmaceutical compositions can promote or enhance different types of tissues, including but not limited to skin, bone, cartilage, neural tissue, ligament, tendon, vascular tissue and muscle (e.g., cardiac tissue). In some embodiments, the subject suffers from a congenital disease or condition, resulting in the need for tissue repair or regeneration. In some embodiments, the subject has been injured, resulting in the need for tissue regeneration. Injury may be caused by surgery, trauma, stroke, tumor, disease or condition (e.g., neurodegenerative disease or condition). The methods and compositions described herein can restore the structural and / or functional integrity of tissues (e.g., the structural and / or functional state of tissues before injury). Methods for promoting tissue regeneration using a hydrogel comprising a self-assembling peptide are known in the art, and may be suitable for use with the self-assembling peptides and hydrogels described herein (see, e.g., U.S. Patent No. 7,846,891 and U.S. Publication No. 2016 / 0362451 and 2017 / 0128172, the entire contents of each of which are expressly incorporated herein by reference).

[0174] In some embodiments, the self-assembling peptides and pharmaceutical compositions described herein can be used to improve or treat the effects of organ tissue degeneration, repair damage to an organ or other body structure, or form an organ or other body structure. Such organs or body structures include, but are not necessarily limited to, vascular tissue, brain, neural tissue, esophagus, fallopian tubes, heart, intestine, gallbladder, kidney, liver, lung, ovary, pancreas, prostate, bladder, bone, spinal cord, spleen, stomach, testicles, thymus, thyroid, trachea, ureter, urethra, uterus, and skin.

[0175] By providing self-assembling peptides and pharmaceutical compositions to bioactive molecules (e.g., growth factors, cell adhesion molecules, integrins, etc.), tissue repair and regeneration can be enhanced. In some embodiments, the bioactive molecules are present in the pharmaceutical composition. In some embodiments, cells that produce one or more bioactive molecules are present in the pharmaceutical composition. For example, genetically modified cells that produce and / or secrete one or more bioactive molecules can be present in the pharmaceutical composition.

[0176] The self-assembling peptides and pharmaceutical compositions described herein can be used to promote the regeneration or repair of ocular tissues (e.g., optic nerve, corneal stroma, and lens cortex). For example, the self-assembling peptides and pharmaceutical compositions described herein can be used to treat subjects suffering from retinopathy or retinal / macular disorders.

[0177] In some embodiments, the self-assembling peptides and pharmaceutical compositions described herein can be used to promote or enhance the repair or regeneration of nerve tissue at an injured site in a subject. For example, when administered to an injured site, the pharmaceutical compositions described herein provide an environment that allows the repair or regeneration of nerve tissue and axonal growth at the injured site.

[0178] 3. Wound healing and antimicrobial dressings

[0179] The self-assembling peptides and pharmaceutical compositions described herein can be used to promote wound healing or skin reconstruction or treat wounds (e.g., burns) in a subject in need thereof. For example, the pharmaceutical compositions described herein can be directly applied to wounds or can be suitable for use with gauze or tulle to promote wound healing, promote skin reconstruction or treat wounds. For example, in some embodiments, the pharmaceutical compositions described herein can be injected into a biopsy site or wound site produced by surgical intervention (e.g., tumor resection). The pharmaceutical compositions described herein can also be used to promote the healing of chronic wounds, such as skin lesions and diabetic ulcers. The method of using self-assembling peptides to promote wound healing or skin reconstruction and treat wounds is known in the art, and can be suitable for use with self-assembling peptides and pharmaceutical compositions described herein (see, e.g., U.S. Publication No. 2011 / 0002880 and International Publication No. WO 2017 / 210416, the entire contents of each of which are expressly incorporated herein by reference).

[0180] In some embodiments, the self-assembling peptides and pharmaceutical compositions described herein can be used as antimicrobial agents. In some embodiments, the self-assembling peptides and pharmaceutical compositions described herein can be used as antimicrobial dressings.

[0181] 4. Stop bleeding

[0182] The self-assembling peptides and pharmaceutical compositions described herein can be used to promote hemostasis in a subject. The self-assembling peptides and pharmaceutical compositions described herein can be used to prevent or control blood loss (e.g., during surgery or after trauma) of blood vessels (e.g., arteries, veins, aortas) and subject body organs. Methods for promoting hemostasis in a subject are known in the art and may be suitable for use with the self-assembling peptides and pharmaceutical compositions described herein (see, e.g., International Publication No. WO2017 / 210421, the entire contents of which are expressly incorporated herein by reference).

[0183] In some embodiments, self-assembling peptides and pharmaceutical compositions as described herein can be used to establish physical barriers to prevent or prevent bleeding during surgery or endoscopy (e.g., in hepatectomy, splenectomy, vaginoplasty, cholecystectomy, coronary artery bypass grafting or femoral bypass grafting). In some embodiments, self-assembling peptides and pharmaceutical compositions as described herein can be used to prevent or prevent exudative bleeding of blood vessels and solid organ substance. In some embodiments, self-assembling peptides and pharmaceutical compositions as described herein can be used to prevent or prevent exudative bleeding caused by vascular anastomosis (e.g., anastomosis with natural or artificial blood vessels). In some embodiments, self-assembling peptides and pharmaceutical compositions as described herein can be used to prevent or prevent exudative bleeding of small blood vessels or capillaries in the gastrointestinal tract of a subject (e.g., during endoscopic submucosal dissection (EMD) of the gastrointestinal tract, laparoscopic resection of the gastrointestinal tract, or endoscopic mucosal resection (EMR) of the gastrointestinal tract.

[0184] 5. Local drug / therapeutic agent delivery

[0185] In one aspect, the present disclosure provides a combination composition comprising: (i) a self-assembling peptide, including a self-assembling peptide or a pharmaceutical composition described herein, and (ii) one or more payload agents (e.g., therapeutic agents), wherein the storage modulus of the combination composition is from about 0.1 to about 100 Pa (e.g., at a frequency of 5 radians / second and 0.1 Pa oscillatory stress), and / or the viscosity of the combination composition at room temperature is from about 0.5 Pa to about 50,000 Pa. In some embodiments, the one or more payload agents (e.g., therapeutic agents) of the combination composition are substantially uniformly distributed in the combination composition.

[0186] On the other hand, the self-assembling peptides and pharmaceutical compositions described herein can be used to deliver drugs and / or therapeutic agents locally to diseased and / or defective sites in the body. The drug / therapeutic agent can be mixed with a peptide solution and injected using a needle, nozzle or catheter. The drug can be delivered and positioned in the tissue and then slowly released before the hydrogel is absorbed into the body.

[0187] The term "agent" as used herein refers to a compound or entity of any chemical class, including, for example, polypeptides, nucleic acids, sugars, lipids, small molecules, metals, or combinations thereof. In some embodiments, the agent is or comprises a natural product because it is found in nature and / or obtained from nature. In some embodiments, the agent is or comprises one or more artificial entities because it is designed, engineered, and / or produced by human hands, and / or cannot be found in nature. In some embodiments, the agent can be used in an isolated or pure form; in some embodiments, the agent can be used in a crude form. In some embodiments, potential agents can be provided as a collection or library, for example, screening can be performed to identify or characterize active agents therein. Some specific embodiments of agents that can be used according to the present invention include small molecules, antibodies, antibody fragments, aptamers, nucleic acids (e.g., siRNA, shRNA, DNA / RNA hybrids, antisense oligonucleotides, mRNA, CRISPR systems, and ribozymes), peptides, peptide mimetics, etc. In some embodiments, the agent is or comprises a polymer. In some embodiments, the agent is not a polymer and / or is substantially free of any polymer. In some embodiments, the agent comprises at least one polymer portion. In some embodiments, the agent lacks or is substantially free of any polymer moiety. In some embodiments, the agent is a cell and / or tissue. In some embodiments, the agent is a cell lysate or comprises a cell lysate. In some embodiments, the agent is or comprises a cellular material and / or a multicellular material (e.g., a micropillar graft and / or a micrograft).

[0188] As used herein, the phrase "therapeutic agent" generally refers to any agent that can cause a desired pharmacological effect when applied to an organism. In some embodiments, if a statistically significant effect is shown in an appropriate population, the agent is considered a therapeutic agent. In some embodiments, the appropriate population can be a model organism population. In some embodiments, the appropriate population can be defined by various criteria, such as a specific age group, gender, genetic background, previous clinical conditions, etc. In some embodiments, a therapeutic agent is a substance that can be used to alleviate, improve, alleviate, inhibit, prevent, delay the onset of one or more symptoms or characteristics of a disease, illness and / or condition, reduce its severity and / or reduce its incidence. In some embodiments, a "therapeutic agent" refers to an agent that has been or needs to be approved by a government agency before being listed for administration to humans. In some embodiments, a "therapeutic agent" is an agent that requires a medical prescription for human administration.

[0189] In some specific embodiments, the combination composition of the present invention comprises one or more payload agents, such as therapeutic agents or detection agents. Such agents include, for example, compounds or entities of any chemical class, including, for example, polypeptides, nucleic acids, sugars, lipids, small molecules, metals, or combinations thereof. It is clear from the context that in some embodiments, the agent can be or include a cell or organism, or a part, extract, or component thereof. In some embodiments, the agent is or includes a natural product found in nature and / or obtained from nature. In some embodiments, the agent is or includes one or more artificial entities because it is designed, engineered, and / or produced by human hands, and / or cannot be found in nature. In some embodiments, the agent can be used in an isolated or pure form; in some embodiments, the agent can be used in a crude form. In some embodiments, potential agents are provided as a collection or library, which can, for example, be screened to identify or characterize the active agent therein. Some specific forms of agents that can be utilized according to the present invention include small molecules (e.g., antibiotics, anticancer drugs, analgesics, anti-inflammatory drugs, steroids, antipsychotics), antibodies, antibody fragments, aptamers, nucleic acids (e.g., siRNA, shRNA, DNA / RNA hybrids, antisense oligonucleotides, ribozymes), peptides, peptide mimetics, proteins, fusion proteins, vaccines, anticoagulants, cytokines, hormones, enzymes, blood factors, extracellular matrix components, etc. In some embodiments, the agent can be selected from microtransplanted tissues, drugs (e.g., antibiotics), and biologics (e.g., growth factors and / or other molecules / proteins). In some embodiments, the agent is a cytokine (e.g., epidermal growth factor, nerve growth factor, transforming growth factor α and β, platelet-derived growth factor, insulin-like growth factor, vascular endothelial growth factor).

[0190] In some embodiments, the combination of the self-assembling peptide or pharmaceutical composition described herein and one or more payload agents (e.g., therapeutic agents) is used to treat a disease or condition, such as a disease or condition known or suspected to be treatable by a therapeutic agent described herein (e.g., infection, cancer, cardiovascular disease, nervous system disease), and / or for wound healing, bone / cartilage repair / regeneration, soft tissue regeneration. The combination of the self-assembling peptide or pharmaceutical composition described herein and one or more payload agents (e.g., therapeutic agents) can be administered to a subject in a variety of ways, and administration is not limited to any particular method. In some embodiments, the combination of the self-assembling peptide or pharmaceutical composition described herein and one or more payload agents (e.g., therapeutic agents) is administered to a subject by a device, a medical device, an implant, a dental implant, a breast implant, a prosthesis, a needle, a stent, or a catheter, or applied to a device, a medical device, an implant, a dental implant, a breast implant, a prosthesis, a needle, a stent, or a catheter. Other methods of administration are described, for example, in patent application numbers US2011-0002880, WO / 2008 / 073395, US2011 / 0201541, US 2014-0329914, US2015-0105336, WO / 2014 / 136081, WO / 2014 / 141143, and U.S. Pat. No. US7846891, the entire contents of each of which are incorporated herein by reference in their entirety for any and all purposes.

[0191] Other methods and embodiments may be found in WO 2017 / 120092, the entire contents of which are incorporated herein by reference for any and all purposes.

[0192] 6. Leak occlusion

[0193] In some embodiments, the self-assembling peptides and pharmaceutical compositions described herein can be used for air leak occlusion. In some embodiments, the self-assembling peptides and pharmaceutical compositions described herein can be used to treat bullae. In multiple embodiments, a method for treating bullae and / or air leaks in a subject is provided. The method may include introducing a delivery device into a target area of ​​bullae in the subject. The method may also include positioning the end of the delivery device in a target area where bullae need to be treated. The method may also include administering a solution containing the self-assembling peptides or pharmaceutical compositions described herein to the target area in an effective amount and at an effective concentration by means of a delivery device to form a barrier under physiological conditions in the target area, thereby treating bullae. The method may also include removing the delivery device from the target area.

[0194] Other methods and embodiments may be found in WO 2013 / 030673, WO 2015 / 138473, and U.S. Patent 10,245,299, the entire contents of each of which are hereby incorporated by reference in their entirety for any and all purposes.

[0195] 7. Prevent and reduce adhesion

[0196] In some embodiments, the self-assembling peptides and pharmaceutical compositions described herein can be used to prevent or reduce biological tissue adhesion. In various embodiments, the invention provides a method for reducing adhesion to biological tissue, the method comprising administering an effective amount of the self-assembling peptides and pharmaceutical compositions described herein to the biological tissue, wherein the self-assembling peptides and pharmaceutical compositions described herein reduce adhesion to the biological tissue.

[0197] In various aspects, the present invention provides a method for promoting anti-adhesion, comprising: introducing a delivery device into a target area; positioning the end of the delivery device in the target area where anti-adhesion is required; applying a solution containing the self-assembling peptide or pharmaceutical composition described herein to the target area in an effective amount and at an effective concentration through the delivery device to promote anti-adhesion; and removing the delivery device from the target area.

[0198] In some embodiments, the biological tissue comprises the epicardium, the peritoneum, the cecum, the intestine, preferably the large intestine and / or the colon. In some embodiments, the target region comprises the epicardium, the peritoneum, the cecum, the intestine, preferably the large intestine and / or the colon.

[0199] Other methods and embodiments may be found in US 2019-0091376 Al, the entire contents of which are incorporated herein by reference for any and all purposes.

[0200] 8. In vitro cell culture

[0201] In some embodiments, the self-assembling peptides and pharmaceutical compositions described herein can be used in 2D cell culture and / or 3D cell culture.

[0202] 9. Bone void filler

[0203] In some embodiments, the self-assembling peptides and pharmaceutical compositions described herein can be used to fill bone voids.

[0204] In some embodiments, a method for filling a bone void in a subject is provided. In some embodiments, the method may include introducing a delivery device into a subject's bone, positioning the end of the delivery device near a void in the bone where bone growth needs to be promoted, administering a solution containing a self-assembling peptide or pharmaceutical composition of the present disclosure through the delivery device in an effective amount and at a concentration of about 0.1 w / v percentage to about 5 w / v percentage peptide to form a hydrogel scaffold under physiological conditions, thereby promoting bone growth at the target site, and removing the delivery device from the subject.

[0205] In one or more embodiments, the self-assembling peptides and pharmaceutical compositions described herein can be used as bone void fillers (BVFs), which are absorbed and replaced by bones during the healing process after application to the target site. Peptide hydrogels can be placed in bony voids or gaps in the skeletal system. In certain embodiments, the self-assembling peptides and their self-assembling structures can be used as cell culture supports to repair and replace various tissues, and as scaffolds to encapsulate living cells. The self-assembling peptides and pharmaceutical compositions described herein can promote tissue regeneration and the production of related extracellular matrix proteins. In at least some embodiments, the self-assembling peptides and pharmaceutical compositions described herein are non-immunogenic and represent improvements over existing materials for this indication, including desalted freeze-dried allogeneic bone (DFDBA) preparations.

[0206] Other methods and embodiments may be found in US 2017-0128622 Al, the entire contents of which are incorporated herein by reference for any and all purposes.

[0207] 10. Artificial tears for dry eyes

[0208] In some embodiments, the self-assembling peptides and pharmaceutical compositions described herein can be used to treat dry eye. In some embodiments, the self-assembling peptides and pharmaceutical compositions described herein can be used in artificial tears.

[0209] 11. Repair of joint cartilage

[0210] In some embodiments, the self-assembling peptides and pharmaceutical compositions described herein can be used to repair articular cartilage. In some embodiments, the self-assembling peptides and pharmaceutical compositions described herein can be used as injectable compositions. Without being bound by theory, injectable hydrogels containing the self-assembling peptides and pharmaceutical compositions described herein can enhance the therapeutic effect and improve the ease of application. Ideal injectable scaffolds for cartilage regeneration should generally meet the following criteria: (i) easy to apply under physiological conditions, (ii) ensure injectability (gelation by chemical or physical cross-linking after injection), (iii) excellent biocompatibility and potential biodegradability, (iv) can mimic cartilage ECM characteristics and promote cell chondrogenesis potential, (v) can easily fill the internal defect site of the joint and integrate with the surrounding natural cartilage tissue, rather than easily displaced, and (vi) if associated with local drug delivery, have a sustained release curve.

[0211] 12. Cosmetic uses (dermal fillers in cosmetic surgery)

[0212] In some embodiments, the self-assembling peptides and pharmaceutical compositions described herein can be used as dermal fillers. In some embodiments, the self-assembling peptides and pharmaceutical compositions described herein can be used as injectable compositions. Without being bound by theory, injectable hydrogels containing the self-assembling peptides and pharmaceutical compositions described herein can enhance their therapeutic effects and improve their ease of application. Ideal injectable scaffolds for cartilage regeneration should generally meet the following criteria: (i) easy to apply under physiological conditions, (ii) ensure injectability (gelation by chemical or physical cross-linking after injection), (iii) excellent biocompatibility and potential biodegradability, (iv) able to mimic cartilage ECM characteristics and promote cell chondrogenesis potential, (v) able to easily fill the defect site inside the joint and integrate with the surrounding natural cartilage tissue, rather than easily displaced, and (vi) if associated with local drug delivery, have a sustained release curve.

[0213] In some embodiments, the self-assembling peptides and pharmaceutical compositions described herein can be used to plump up the lips. In some embodiments, the self-assembling peptides and pharmaceutical compositions described herein can be used to fill or smooth wrinkles. In some embodiments, the self-assembling peptides and pharmaceutical compositions described herein can be used to soften facial wrinkles. In some embodiments, the self-assembling peptides and pharmaceutical compositions described herein can be used to enhance shallow contours. In some embodiments, the self-assembling peptides and pharmaceutical compositions described herein can be used to reconstruct contour deformations of a subject's face. In some embodiments, the self-assembling peptides and pharmaceutical compositions described herein can be used to improve the appearance of sunken scars. In some embodiments, the self-assembling peptides and pharmaceutical compositions described herein can be used to reduce or eliminate shadows under the lower eyelid.

[0214] Reagent test kit

[0215] On the other hand, the present disclosure provides kits and articles comprising pharmaceutical compositions and / or self-assembling peptides as described herein. The kit may include instructions for the appropriate preparation and / or use of the components thereof. As described herein, the kit further includes tools that can be used to prepare and / or deliver pharmaceutical compositions or self-assembling peptides to a subject (e.g., a human subject).

[0216] In some embodiments, the kit contains separate containers, dividers or compartments for the pharmaceutical composition and the informational material. For example, the pharmaceutical composition can be contained in a bottle, vial or syringe, and the informational material can be contained in a plastic sleeve or packet. In some embodiments, the separate elements of the kit are contained in a single undivided container. For example, the pharmaceutical composition or the self-assembling peptide can be contained in a bottle, vial or syringe with the informational material attached in the form of a label.

[0217] In some embodiments, the test kit comprises a plurality of (e.g., a pack) of separate containers, each containing one or more unit dosage forms of a pharmaceutical composition or self-assembling peptide. For example, the test kit may include a plurality of syringes, ampoules, foil packages, or blister packages, each containing a single unit dose of a pharmaceutical composition or self-assembling peptide. In some embodiments, the components of the test kit are stored in sealed vials, such as with rubber or silicone caps (e.g., polybutadiene or polyisoprene caps). In some embodiments, the components of the test kit are stored under inert conditions (e.g., under nitrogen or other inert gases such as argon). In some embodiments, the components of the test kit are stored under anhydrous conditions (e.g., with a desiccant). In some embodiments, the components of the test kit are stored in light-shielding containers (e.g., amber vials). In some embodiments, the container of the test kit may be airtight and / or waterproof.

[0218] The kit may further comprise a solution comprising a tonicity agent. These solutions may be packaged alone or in combination with a pharmaceutical composition comprising a self-assembling peptide provided herein.

[0219] Also provided are articles, including, for example, syringes, automatic injectors, tubes and catheters (e.g., with or without guidewires). In some embodiments, the articles can be pre-filled with self-assembling peptides or pharmaceutical compositions as described herein. These articles can be separated from or included in a kit as described herein.

[0220] Further aspects and embodiments of the invention are set forth in the following numbered paragraphs:

[0221] 1. A pharmaceutical composition comprising a self-assembling peptide, wherein the self-assembling peptide comprises an amino acid sequence as set forth in the following formula:

[0222] [(X)i(Y)j(Z)k(Y)l]m(X)n Formula I,

[0223] [(Y)i(X)j(Y)k(Z)l]m(Y)n Formula II,

[0224] [(Z)i(Y)j(X)k(Y)l]m(Z)n Formula III, or

[0225] [(Y)i(Z)j(Y)k(X)l]m(Y)n Formula IV,

[0226] wherein each (X) is independently an ionic, polar amino acid, wherein each (Y) is independently a hydrophobic amino acid, wherein each (Z) is independently a nonionic, polar amino acid, wherein each i, j, k and l is independently an integer ≥1, wherein m is an integer ≥2, and wherein n=0 or an integer ≥1.

[0227] 2. The pharmaceutical composition of paragraph 1, wherein the self-assembling peptide comprises the amino acid sequence set forth in Formula I.

[0228] 3. The pharmaceutical composition of paragraph 1, wherein the self-assembling peptide comprises the amino acid sequence set forth in Formula II.

[0229] 4. The pharmaceutical composition of paragraph 1, wherein the self-assembling peptide comprises the amino acid sequence set forth in Formula III.

[0230] 5. The pharmaceutical composition of paragraph 1, wherein the self-assembling peptide comprises the amino acid sequence set forth in Formula IV.

[0231] 6. The pharmaceutical composition of any one of paragraphs 1-5, wherein each (X) is a basic amino acid.

[0232] 7. The pharmaceutical composition of paragraph 6, wherein the basic amino acid is selected from the group consisting of arginine, lysine, histidine and ornithine.

[0233] 8. The peptide of any of paragraphs 1-5, wherein each (X) is an acidic amino acid.

[0234] 9. The peptide of paragraph 8, wherein the acidic amino acid is selected from the group consisting of aspartic acid and glutamic acid.

[0235] 10. The pharmaceutical composition of any one of paragraphs 1-9, wherein each (Y) is selected from the group consisting of alanine, valine, leucine, isoleucine, methionine, phenylalanine, tryptophan and glycine.

[0236] 11. The pharmaceutical composition of any of paragraphs 1-10, wherein each (Z) is selected from the group consisting of serine, threonine, tyrosine, cysteine, glutamine, asparagine and methionine.

[0237] 12. The pharmaceutical composition of any of paragraphs 1-11, wherein at least one of i, j, k and l is independently an integer of 1.

[0238] 13. The pharmaceutical composition of any one of paragraphs 1-12, wherein at least one of i, j, k and l is independently an integer of 2.

[0239] 14. The pharmaceutical composition of any one of paragraphs 1-13, wherein m is independently an integer of 2 or 3.

[0240] 15. The pharmaceutical composition of any one of paragraphs 1-14, wherein the self-assembling peptide comprises the amino acid sequence set forth in SEQ ID NO: 1-20.

[0241] 16. The pharmaceutical composition of any one of paragraphs 1-15, wherein the self-assembling peptide comprises an N-terminal functional group, a C-terminal functional group, or both.

[0242] 17. The pharmaceutical composition of paragraph 16, wherein the N-terminal functional group is selected from the group consisting of acetyl, formyl, pyroglutamyl (pGlu), biotin, polyethylene glycol (PEG), urea, alkylamine, carbamate, sulfonamide, dansyl, 2,4-dinitrophenyl, fluorescein, 7-methoxycoumarinacetic acid, 9-fluorenylmethoxycarbonyl, palmitic acid, succinyl, chloroacetyl, maleimide, benzyloxycarbonyl, bromoacetyl, nitrilotriacetyl, tert-butyloxycarbonyl, 4-hydroxyphenylpropionic acid, allyloxycarbonyl, butyric acid, fatty acid and trityl.

[0243] 18. The pharmaceutical composition of paragraph 16, wherein the C-terminal functional group is selected from the group consisting of: amide, N-alkylamide, aldehyde, ester, alcohol, p-nitroanilide (pNA), 7-amino-4-methylcoumarin (Amc), hydrazide, hydroxamic acid, chloromethyl ketone, p-nitroaniline, p-nitrophenol, hydroxysuccinimide ester, fluoromethyl ketone, cysteamide, 9-fluorenylmethyl (Fm) ester, allyl ester, 2,4-dimethoxybenzyl ester, 2-phenylisopropyl ester, p-nitrobenzyl ester and 2-chlorotrityl ester.

[0244] 19. The pharmaceutical composition of any one of paragraphs 1-16, wherein the self-assembling peptide comprises the amino acid sequence set forth in SEQ ID NO: 21-40.

[0245] 20. The pharmaceutical composition of any one of paragraphs 1-19, wherein the self-assembling peptide further comprises at least one biologically active peptide motif.

[0246] 21. The pharmaceutical composition of any one of paragraphs 1-20, wherein at least one biologically active peptide motif is present at the N-terminus of the self-assembling peptide.

[0247] 22. The pharmaceutical composition of any one of paragraphs 1-20, wherein at least one biologically active peptide motif is present at the C-terminus of the self-assembling peptide.

[0248] 23. The pharmaceutical composition of any of paragraphs 20-22, wherein at least one bioactive peptide motif is derived from laminin-1, collagen IV, fibronectin, elastin, bone marrow homing peptide 1, bone marrow homing peptide 2 or myelin.

[0249] 24. The pharmaceutical composition of any one of paragraphs 20-22, wherein at least one biologically active peptide motif comprises the amino acid sequence set forth in any one of SEQ ID NOs: 41-70.

[0250] 25. The pharmaceutical composition of any one of paragraphs 1-20, wherein the self-assembling peptide comprises the amino acid sequence set forth in SEQ ID NO: 71-90.

[0251] 26. The pharmaceutical composition of any one of paragraphs 1-25, further comprising a tonicity agent.

[0252] 27. The pharmaceutical composition of paragraph 26, wherein the tonicity agent is present at a concentration of about 0.01M to about 0.3M.

[0253] 28. The pharmaceutical composition of paragraph 26, wherein the tonicity agent is present at a concentration of about 0.15M.

[0254] 29. The pharmaceutical composition of any of paragraphs 1-28, having a pH of about 6 to about 8.

[0255] 30. The pharmaceutical composition of any of paragraphs 1-28, having a pH of about 7 to about 7.5.

[0256] 31. The pharmaceutical composition of paragraph 29 or paragraph 30, wherein the net charge of the self-assembling peptide is greater than or equal to +1 or less than or equal to -1.

[0257] 32. The pharmaceutical composition of paragraph 29 or paragraph 30, wherein the net charge of the self-assembling peptide is from about +1 to about +6.

[0258] 33. The pharmaceutical composition of paragraph 29 or paragraph 30, wherein the net charge of the self-assembling peptide is from about -1 to about -6.

[0259] 34. The pharmaceutical composition of any of paragraphs 1-33, wherein the concentration of the self-assembling peptide is from about 0.01% (w / v) to about 10% (w / v).

[0260] 35. The pharmaceutical composition of any of paragraphs 1-33, wherein the concentration of the self-assembling peptide is from about 0.1% (w / v) to about 5% (w / v).

[0261] 36. The pharmaceutical composition of any of paragraphs 1-33, wherein the concentration of the self-assembling peptide is from about 0.5% (w / v) to about 1.5% (w / v).

[0262] 37. The pharmaceutical composition of any of paragraphs 1-33, wherein the concentration of the self-assembling peptide is about 1% (w / v).

[0263] 38. The pharmaceutical composition of any of paragraphs 1-37, further comprising isolated cells.

[0264] 39. The pharmaceutical composition of paragraph 38, wherein the isolated cells are mammalian cells.

[0265] 40. The pharmaceutical composition of paragraph 38 or paragraph 39, wherein the mammalian cells are immune cells, stem cells, chondrocyte progenitor cells, pancreatic progenitor cells, myoblasts, fibroblasts, keratinocytes, neuronal cells, glial cells, astrocytes, preadipocytes, adipocytes, vascular endothelial cells, endothelial progenitor cells, mesenchymal cells, neural stem cells, immune cells (such as B cells and T cells), smooth muscle progenitor cells, cardiomyocytes, fetal dermal fibroblasts, epidermal keratinocytes, myoblasts and capillary endothelial cells.

[0266] 41. The pharmaceutical composition of any one of paragraphs 1-40, wherein the pharmaceutical composition further comprises a biologically active agent.

[0267] 42. The pharmaceutical composition of paragraph 41, wherein the bioactive agent is selected from the group consisting of hormones, growth factors, insulin, enzymes, siRNA, shRNA, antisense-RNA, antibiotics, antibodies and anti-inflammatory agents.

[0268] 43. The pharmaceutical composition of any one of paragraphs 1-42, wherein the pharmaceutical composition is an aqueous solution.

[0269] 44. The pharmaceutical composition of any of paragraphs 1-42, wherein the pharmaceutical composition is a hydrogel.

[0270] 45. The pharmaceutical composition of paragraph 44, wherein the pharmaceutical composition is a hydrogel, and wherein the hydrogel comprises a storage modulus of at least about 10 Pascals (Pa).

[0271] 46. ​​An article of manufacture comprising the pharmaceutical composition of any one of paragraphs 1-45.

[0272] 47. The article of manufacture of paragraph 46, wherein the article is a syringe, vial, autoinjector, tube or catheter.

[0273] 48. A method of treating a subject in need thereof, comprising administering to the subject an effective amount of the pharmaceutical composition of any of paragraphs 1-45.

[0274] 49. A method of promoting tissue repair or regeneration in a subject in need thereof, comprising contacting a tissue of the subject with the pharmaceutical composition of any of paragraphs 1-45, thereby promoting tissue repair or regeneration of the tissue.

[0275] 50. The method of paragraph 49, wherein the tissue is skin, bone, cartilage, neural tissue, ligament, tendon, vascular tissue or muscle.

[0276] 51. The method of paragraph 49, wherein the tissue is optic nerve tissue.

[0277] 52. The method of paragraph 49, wherein the tissue is cardiac tissue.

[0278] 53. The method of any of paragraphs 49-52, wherein the subject suffers from a congenital disease or condition resulting in a need for tissue repair or regeneration.

[0279] 54. The method of any of paragraphs 49-52, wherein the subject suffers from an injury resulting in a need for tissue repair or regeneration.

[0280] 55. The method of paragraph 54, wherein the injury is the result of surgery, trauma, stroke, tumor, or disease or condition.

[0281] 56. A method of promoting wound healing in a subject in need thereof, comprising contacting a wound of the subject with the pharmaceutical composition of any of paragraphs 1-45, thereby promoting wound healing.

[0282] 57. The method of paragraph 56, wherein the wound comprises an abrasion, burn, chapped skin, abrasion, cut, ulcer, laceration, incision or scratch.

[0283] 58. A method of stopping or preventing bleeding at a site within a subject, comprising contacting the site with the pharmaceutical composition of any of paragraphs 1-45, wherein the pharmaceutical composition creates a physical barrier, thereby stopping or preventing bleeding at the site within the subject.

[0284] 59. A method of resecting a lesion from a gastrointestinal tract site of a subject, comprising:

[0285] a. contacting the submucosal layer beneath the lesion with a pharmaceutical composition according to any one of paragraphs 1-45, thereby lifting the lesion; and

[0286] b. Resection of the lesion from the subject's gastrointestinal tract.

[0287] 60. The method of paragraph 59, wherein the lesion comprises a polyp, an ulcer, or a tumor.

[0288] 61. The method of paragraph 59 or paragraph 60, wherein the lesion is present in a region of the gastrointestinal tract of the subject selected from the group consisting of the mouth, throat, esophagus, stomach, small intestine, large intestine, colon and rectum.

[0289] 62. A method of culturing cells comprising contacting the cells with a pharmaceutical composition according to any one of paragraphs 1-45.

[0290] The following examples are illustrative and non-restrictive. By reading this disclosure, many changes in the technology will become apparent to those skilled in the art. Therefore, the technical scope should not be determined with reference to the embodiments, but should be determined with reference to the full scope of the attached claims and their equivalents.

[0291] Example

[0292] Materials and methods

[0293] The experiments described in the following examples were performed using the following materials and methods.

[0294] Peptide synthesis

[0295] All peptides used in the examples herein were synthesized by conventional solid peptide synthesis using an automated peptide synthesizer.

[0296] Viscosity measurement

[0297] The peptide (200 μL) prepared as an aqueous solution was placed between the plates of a rheometer (DHR-1, TA Instruments, 20 mm plate, measuring geometry 500 μm) and the flow rate was adjusted from 0.001 s -1 Up to 100s -1 Shear rate measurement of viscosity. Thixotropy measurement

[0298] The peptide (200 μL) prepared as an aqueous solution was placed between the plates of a rheometer (DHR-1, TA Instruments, 20 mm plate, measuring geometry 500 μm), and the sample was subjected to a shear rate of 1000 s -1 The plate was rapidly rotated for 1 min at 1 rad s -1 The storage modulus was measured at angular frequency for 10 minutes.

[0299] Sweep frequency test

[0300] The peptides formulated as aqueous solutions were placed between the plates of a rheometer (DHR-1, TA Instruments, 20 mm plate, measuring geometry of 500 μm or 40 mm cone and plate, measuring geometry of 2.0° cone angle) and frequency stress sweep tests were performed at 0.1 Hz to 10 Hz with a strain of 0.1%; measurements were performed at 37° C. after a relaxation time of 2 minutes. For frequency sweep tests after exposure to Dulbecco's Modified Eagle Medium (DMEM), 10 mL of DMEM was added to the chamber around the plate, and the samples in the plate were then immersed in DMEM and the frequency test was performed as indicated after 20 minutes.

[0301] Endoscopic mucosal resection and endoscopic submucosal dissection

[0302] As described in Uraoka et al. (2009) Drug Des. Devel. Ther. 2: 131-8, which is incorporated herein by reference, an in vivo porcine animal model was used for endoscopic mucosal and submucosal dissection. Briefly, 2 mL of the selected peptide formulation was injected between the muscle and submucosal layers of the porcine stomach, and the submucosal layer was dissected with an electrosurgical knife. The injection site and the overall appearance of the submucosal tissue dissected at the injection site were analyzed.

[0303] Example 1. Determination of the net charge of exemplary self-assembling peptides in formulations at different pH ranges

[0304] A plurality of exemplary self-assembling peptides described herein (Table 5; SEQ ID NOs: 21-40) were designed to have a net positive or negative charge when formulated at pH 7.5. In contrast, previously described self-assembling peptides had a net charge of zero at the same pH. The net charge of the exemplary self-assembling peptides in pH 7.5 solution was determined and is shown in Table 5. Although the previously described self-assembling peptides RADA 16, IEIK13, and KLD12 (SEQ ID NOs: 91-93, respectively) had a net charge of about zero at pH 7.5, the newly developed self-assembling peptides had a net positive charge or a net negative charge at the same pH.

[0305] like Figure 1-3 As shown, this net charge behavior extends to around the pH 7.5 test point shown in Table 5. For example, Figure 1 The net charge of KLNL12 (SEQ ID NO:21, about +3 at about neutral pH) is shown as a function of pH. Figure 2 The net charge of NLEL12 (SEQ ID NO: 33, about -3 at about neutral pH) is shown as a function of pH. Figure 3The net charge of RADA16 (SEQ ID NO:91, about 0 at about neutral pH) is shown as a function of pH.

[0306] Table 5. Net charge of exemplary self-assembling peptides at pH 7.5

[0307]

[0308]

[0309] *Exemplary conventional self-assembling peptide for comparison

[0310] Figure 4-5 Molecular models of the atomic structure and net charge of exemplary self-assembling peptides are provided. Figure 4 It is shown that RANA16 (SEQ ID NO: 39) has a net charge of +4 at pH 7.5. In contrast, Figure 5 The net charge of RADA16 (SEQ ID NO: 91) at pH 7.5 was shown to be 0. The structures were computationally simulated using MarvinSketch and both showed a β-sheet conformation. Although RANA16 (SEQ ID NO: 39) and RADA 16 (SEQ ID NO: 91) exhibited similar structural conformations at pH 7.5, the net charges of the two peptides were significantly different, with RANA16 (SEQ ID NO: 39) having a +4 net positive charge and RADA16 (SEQ ID NO: 91) having a zero net charge.

[0311] Example 2. Characterization of the solubility of exemplary self-assembling peptides in aqueous pharmaceutical compositions formulated at pH 2.5 and 7.5

[0312] A variety of exemplary self-assembling peptides described herein (see Table 5; SEQ ID NOs: 21-40) are designed to have a non-zero net charge when formulated in a solution at pH 7.5, have high solubility, and transform from a liquid state to a hydrogel after administration (e.g., after injection into a subject or mixing with a solution containing a specific concentration of a tonicity agent (e.g., salt). In contrast, the previously described self-assembling peptides RADA16, IEIK13, and KLDL12 (SEQ ID NOs: 91-93, respectively) have a zero net charge and must be formulated in a solution at an acidic pH to maintain their ability to conditionally transform from a liquid state to a hydrogel after administration.

[0313] To determine the solubility of the exemplary self-assembling peptides in aqueous pharmaceutical compositions formulated at acidic and neutral pH, the appearance of the pharmaceutical compositions was evaluated. A homogeneous and transparent appearance reflects adequate solubility of the peptide in solution and minimal hydrogel formation. Phase separation and turbid appearance reflect poor solubility of the peptide in solution and / or formation of a hydrogel.

[0314] Table 6 below and Figure 20 show the appearance of exemplary self-assembling peptides at pH 2.5 and pH 7.5. The aqueous pharmaceutical compositions containing the newly described self-assembling peptides were transparent and homogeneous at pH 7.5, while solutions of the previously disclosed self-assembling peptides RADA16 (SEQ ID NO: 91), IEIK13 (SEQ ID NO: 92), and KLDL12 (SEQ ID NO: 93) exhibited phase separation and turbid appearance at pH 7.5 (see Table 6 and Figure 20). Figure 20A-C ).

[0315] Table 6. Appearance of exemplary self-assembling peptides at pH 2.5 and pH 7.5

[0316]

[0317]

[0318] Example 3. Characterization of the solubility of aqueous pharmaceutical compositions formulated at pH 7.5 and having different ionic strengths

[0319] The self-assembling peptides described herein (Table 5; SEQ ID NO: 21-40) are configured to have a non-zero net charge, high solubility, and transition from a liquid state to a hydrogel after administration when formulated as an aqueous solution with isotonic ionic strength (e.g., 0.15M salt ions) at pH 7.5. In order to determine the solubility of aqueous pharmaceutical compositions formulated at pH 7.5 at different ionic strengths, aqueous pharmaceutical compositions with different concentrations of sodium chloride were prepared and their appearance was evaluated. A homogeneous and transparent appearance reflects the full solubility of the peptide in solution and minimal hydrogel formation. Phase separation and turbid appearance reflect poor solubility of the peptide in solution and / or the formation of a hydrogel.

[0320] In general, an increase in the amount of hydrophobic residues correlates with a decrease in solubility at higher ionic strengths, as shown in Table 7. Thus, the amino acid composition of the self-assembling peptides can be manipulated to achieve a desired solubility at a desired ionic strength.

[0321] Table 7. Appearance of exemplary self-assembling peptides at different ionic strengths

[0322]

[0323]

[0324] *Peptides precipitate at about pH 7.5, so prepare at about pH 2.2-2.3

[0325] Example 4. Determination of shear-thinning and thixotropic behavior of aqueous pharmaceutical compositions containing exemplary self-assembling peptides formulated at various ionic strengths at pH 7.5

[0326] The self-assembling peptides disclosed herein (Table 5 SEQ ID NO: 21-40) are designed to have a non-zero net charge and high solubility when formulated in a solution at pH 7.5 in the presence of isotonic ionic strength (defined as 0.15M salt ions), and are converted from a liquid state to a hydrogel after administration to a subject. In some clinical applications, the administration of a pharmaceutical composition comprising a self-assembling peptide requires that the composition be subjected to pressure and / or shear thinning (e.g., injection by a syringe needle or during transfer by a pump-based system). Ideally, a pharmaceutical composition comprising a self-assembling peptide as described herein shows a reduced viscosity under pressure or shear thinning forces, and returns to a base viscosity after removing these forces. In order to determine the shear-thinning properties of a pharmaceutical composition of a self-assembling peptide formulated at pH 7.5, a formulation comprising different ionic strengths was prepared using sodium chloride of different concentrations, and viscosity was measured at an increased shear rate. The thixotropic properties of the formulation and the storage modulus that varies over time at a specific frequency and pressure were also determined.

[0327] Figure 6-8 Shown are experimentally determined viscosities of aqueous pharmaceutical compositions containing 1% (w / v) KLNL12 (SEQ ID NO:21), 1% (w / v) IQIK13 (SEQ ID NO:28), or 1% (w / v) NLEL12 (SEQ ID NO:33) at pH 7.5. Fig. 9 and 10 The experimentally determined viscosity of aqueous pharmaceutical compositions containing 1% KLNL12 (SEQ ID NO: 21) or 1% NLEL12 (SEQ ID NO: 33) and 0.9% NaCl (simulating physiological conditions) at pH 7.5 is shown. The data show that the aqueous pharmaceutical composition exhibits shear thinning. That is, as the shear rate increases, the viscosity decreases. This property can advantageously facilitate the administration and use of the pharmaceutical composition (e.g., due to reduced viscosity when pumping) while maintaining a desired viscosity at the site of administration (e.g., in the absence of applied shear forces).

[0328] Fig.11 and 12The thixotropic properties of pharmaceutical compositions comprising 1% KLNL12 (SEQ ID NO: 21) or 1% NLEL12 (SEQ ID NO: 33) and 0.9% NaCl at pH 7.5 are shown after removal of shear stress. The aqueous pharmaceutical compositions were flowed for 1 minute at a shear rate of 1000 1 / s, and the storage modulus at a frequency of 10 rad / s and a stress of 0.1 Pa was tested over time. The aqueous pharmaceutical compositions tested exhibited thixotropic properties. This property can advantageously facilitate the administration and use of pharmaceutical compositions because they exhibit predictable and reliable properties after application of mechanical stress.

[0329] Example 5. Determination of the rheological properties of pharmaceutical compositions containing self-assembling peptides

[0330] The rheological properties of aqueous pharmaceutical compositions comprising exemplary self-assembling peptides KLNL12 (SEQ ID NO:21), KIQI13 (SEQ ID NO:29), or NLEL12 (SEQ ID NO:33) were determined by performing a frequency sweep test on the storage modulus.

[0331] Figure 13-15 Shown are the results of a 1% (w / v) KLNL12 (SEQ ID NO: 21) ( Fig.13 )、KIQI13 (SEQ ID NO: 29) ( Fig.14 ) or NLEL12 (SEQ ID NO:33) ( Fig.15 ) at pH 7.5. In each case, the rheological properties (e.g., mechanical strength) of the peptide solutions increased after DMEM treatment.

[0332] Figure 16-18 Shown are the results of DMEM containing 1% (w / v) KLNL12 (SEQ ID NO: 21) before and after exposure to Fig.16 )、KIQI13 (SEQ ID NO: 29) ( Fig.17 ) or NLEL12 (SEQ ID NO:33) ( Fig.18 ) Rheological properties of aqueous pharmaceutical compositions with isotonic salinity (0.9% NaCl) at pH 7.5. The rheological properties (e.g. mechanical strength) of peptide solutions increased after DMEM treatment.

[0333] The above data demonstrate that aqueous pharmaceutical compositions comprising KLNL12 (SEQ ID NO:21), KIQI13 (SEQ ID NO:29) or NLEL12 (SEQ ID NO:33) exhibit enhanced mechanical strength in the presence of physiological fluids and are therefore suitable for in vivo therapeutic applications.

[0334] Example 6. Pharmaceutical compositions comprising exemplary self-assembling peptides exhibit reduced tissue damage compared to pharmaceutical compositions of RADA16

[0335] Considering that the self-assembling peptides disclosed herein (Table 5; SEQ ID NO: 21-40) have a non-zero net charge when formulated at neutral pH (e.g., pH 7.5), it can be inferred that these compositions exhibit reduced side effects when administered or contacted with mammalian tissues compared to compositions comprising previously described self-assembling peptides (e.g., RADA16) formulated at acidic pH. Therefore, an in vivo porcine model system was used to evaluate the ability of these compositions to induce submucosal elevation after injection into the gastrointestinal submucosal layer, as well as the extent of tissue damage.

[0336] In this model, a submucosal injection is performed with a liquid composition, and the morphology of the injection site is analyzed. During the removal of lesions from the gastrointestinal tract, an aqueous solution is used for submucosal injection to facilitate the removal of lesions by providing a safety cushion during the removal process (e.g., Kim et al. (2013) World J. Gastroenterol. 19(20): 3069-76, incorporated herein by reference). Here, an aqueous pharmaceutical composition comprising three exemplary self-assembling peptides (KLNL12 (SEQ ID NO: 21), NLKL12 (SEQ ID NO: 23), or KIQI13 (SEQ ID NO: 29)) formulated at pH 7.5 is mixed with an aqueous pharmaceutical composition comprising the previously described RADA 16 self-assembling peptide (formulated at pH 2.5), a 0.4% sodium hyaluronate solution ( Boston Scientific Japan KK, Tokyo, Japan) and saline pH 7.5 were compared. As shown in Figures 21A and 21B, injection of a composition containing KLNL12 (SEQ ID NO: 21), NLKL12 (SEQ ID NO: 23), or KIQI13 (SEQ ID NO: 29) resulted in submucosal elevation. Over time, these compositions showed a significant improvement over saline and Better submucosal elevation.

[0337] After dissecting the tissue at the injection site using an electrosurgical knife, the effect of injecting an aqueous pharmaceutical composition pH 2.5 containing RADA 16 (SEQ ID NO: 91) with and without 0.9% (w / v) NaCl on the submucosal tissue morphology was analyzed. As shown in Figures 22A and 22B, injection of a composition containing RADA16 (SEQ ID NO: 91) with and without 0.9% NaCl resulted in tissue damage, including mucosal aggregation, which appeared as a white precipitate near the injection site. In contrast, no tissue damage was observed when saline was injected (Figure 22A).

[0338] The effect of injection of an aqueous pharmaceutical composition containing 0.9% (w / v) NaCl at pH 7.5 and comprising the exemplary self-assembling peptides KLNL12 (SEQ ID NO: 21), NLEL12 (SEQ ID NO: 33), QLEL12 (SEQ ID NO: 35), or LELQ12 (SEQ ID NO: 36) on the morphology of the submucosal tissue was analyzed after dissection of the tissue at the injection site using an electrosurgical unit. Figures 23A-23D As shown, no mucosal aggregation was observed at the injection sites, and the morphology of these sites was easily visible.

[0339] As an additional control, the effect of injection of phosphate buffered saline (PBS) at pH 7.4 or pH 2.5 on the morphology of the submucosal tissue was analyzed after dissecting the tissue at the injection site using an electrosurgical knife. Mucosal aggregation was observed only at the site of injection of acidic PBS (i.e., pH 2.5), showing as a white precipitate near the injection site (data not shown), confirming that the change in mucosal morphology appears to be due to the pH of the composition.

[0340] Table 8 below provides a summary of the appearance of the gastric mucosa following injection of each of the compositions used in this example.

[0341] Table 8. Appearance of the pig gastric mucosa at the injection site

[0342]

[0343]

[0344] The data provided in this example show that injection of a pH 2.5 aqueous pharmaceutical composition of a RADA16 formulation (SEQ ID NO: 91) into the submucosal layer of the pig stomach results in mucosal aggregation after about 10-15 minutes. In contrast, injection of a pH 7.5 aqueous pharmaceutical composition comprising exemplary self-assembling peptides KLNL12 (SEQ ID NO: 21), KIQI12 (SEQ ID NO: 99), NLEL12 (SEQ ID NO: 33), QLEL12 (SEQ ID NO: 35) or LELQ12 (SEQ ID NO: 36) does not appear to change the submucosal tissue morphology. This result is consistent with the view that an acidic composition (e.g., a composition containing RADA16) can damage mucosal tissue, while injection of a composition of neutral pH can reduce tissue damage.

[0345] In another embodiment, mucosal aggregation near the injection site, visualized as a white precipitate, was assessed in porcine esophagus in vivo.

[0346] Table 9 below provides a summary of the appearance of the esophageal mucosa following injection of each of the compositions used in this example.

[0347] Mucosal aggregation was observed at the site of injection of KIQI12 (SEQ ID NO: 99) as well as acidic PBS (pH 2) and RADA16 (SEQ ID NO: 91). KIQI12 (SEQ ID NO: 99) is a representative peptide among peptides that are positively charged at physiological pH. However, no mucosal aggregation was observed using NLEL12 (SEQ ID NO: 33), QLEL12 (SEQ ID NO: 35), and LELQ12 (SEQ ID NO: 36), which are negatively charged at physiological pH. This result suggests that in some submucosal tissues comprising the esophagus, changes in mucosal morphology appear to be attributed to pH and the positive or negative charge of the composition at physiological pH.

[0348] Table 9. Appearance of the porcine esophageal mucosa at the injection site

[0349]

[0350]

[0351] Example 7. Pharmaceutical composition with increased mechanical strength

[0352] In some embodiments, a volume of 700 μl of the peptide formulated as an aqueous solution was placed between the plates of a rheometer (DHR-1, TA Instruments, 40 mm cone and plate, measuring geometry with a cone angle of 2.0°) and a frequency sweep test was performed at a frequency of 0.1 Hz to 10 Hz using a strain of 0.1%; the measurement was performed after a 2-minute relaxation time at 37° C. In some embodiments, the frequency sweep test was performed after exposure to Dulbecco's Modified Eagle Medium (DMEM), wherein 10 mL of DMEM was added to the chamber around the plate, the sample in the plate was immersed in DMEM, and the frequency test was performed as indicated after 20 minutes.

[0353] Fig.24A The changes in the rheological properties of aqueous pharmaceutical compositions containing 0.15% (w / v) QLEL12 (SEQ ID NO: 35) and 0.9% NaCl (w / v) at pH 7.5 are shown, as reflected by the increased mechanical strength after exposure to DMEM. At a frequency of 0.1 Hz, the mechanical strength of the composition containing the self-assembling peptide increased by 23 times. At a frequency of 1 Hz, the mechanical strength of the composition containing the self-assembling peptide increased by 20 times. At a frequency of 10 Hz, the mechanical strength of the composition containing the self-assembling peptide increased by 9 times.

[0354] Fig. 24B The rheological properties of aqueous pharmaceutical compositions containing various concentrations of QLEL12 (SEQ ID NO: 35) between 0.1% (w / v) and 0.3% (w / v) and 0.9% NaCl (w / v) at pH 7.5 are shown. There is a linear relationship between the rheological properties and the concentration of the self-assembling peptide. Increasing the concentration of the self-assembling peptide in the composition leads to increased mechanical strength.

[0355] Example 8. Pharmaceutical composition with elevated submucosal layer and enhanced lifting capacity

[0356] In some embodiments, the ability of peptide solutions to elevate the submucosal layer was evaluated using an ex vivo canine model. For this test, QLEL12 (SEQ ID NO: 35) was selected because it showed good rheological properties and no unclear / turbid sites in the submucosal layer. 0.1%, 0.15%, 0.2%, 0.3% (w / v) QLEL12 (SEQ ID NO: 35) solutions were prepared using 0.9% NaCl at pH 7.5. Normal saline was used as a reference solution. In the ex vivo canine model, 0.5 mL of each sample was injected into the submucosal layer of the stomach or colon. After the initial injection, the height of the elevation was measured using an electronic caliper at time points of 0 min, 10 min, 20 min, 30 min, 40 min, 50 min, and 60 min. After each sample solution was injected with a syringe needle under the submucosal layer of the stomach and colon, the height of the elevation was measured with an electronic caliper. The elevation height is the distance between the bottom of the flat surface of the skin and the top of the swollen bulla after injection under the submucosal layer.

[0357] The lifting ability of the injection solution to lift the submucosal layer from the muscle layer after injection of the solution was measured. In some embodiments, the muscle layer is the gastrointestinal tract. If the initial lift height and the overall lift height over time are high enough to perform a submucosal resection procedure, it is considered to have good lifting ability.

[0358] Fig.25A Shown is the increased height of the submucosa in canine stomach following injection of 0.5 mL of various concentrations of QLEL12 (SEQ ID NO: 35) between 0.1% (w / v) and 0.3% (w / v) at pH 7.5 and 0.9% NaCl (w / v).

[0359] Fig.25B Shown are the relative elevations to the initial height in the canine colon following injection of 0.5 mL of various concentrations of QLEL12 (SEQ ID NO: 35) between 0.1% (w / v) and 0.3% (w / v) at pH 7.5 and 0.9% NaCl (w / v).

[0360] Fig.25C Shown is the increased height of the submucosa in the canine colon following injection of 0.5 mL of various concentrations of QLEL12 (SEQ ID NO: 35) between 0.1% (w / v) and 0.3% (w / v) at pH 7.5 and 0.9% NaCl (w / v).

[0361] Fig.25DShown are the relative elevations to the initial height in the canine colon following injection of 0.5 mL of various concentrations of QLEL12 (SEQ ID NO: 35) between 0.1% (w / v) and 0.3% (w / v) at pH 7.5 and 0.9% NaCl (w / v).

[0362] For all tested concentrations, treatment with a composition comprising a representative self-assembling peptide resulted in increased retention of the elevated height of the submucosal layer in the colon and stomach. For all tested concentrations, treatment with a composition comprising a representative self-assembling peptide also resulted in increased retention of the relative elevation of the submucosal layer in the colon and stomach. The lifting capacity of the composition comprising a representative self-assembling peptide also showed good lifting capacity, which helped to easily remove certain parts of the submucosal layer.

[0363] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention belongs. Methods and materials for use in the present invention are described herein; other suitable methods and materials known in the art may also be used. The materials, methods, and examples are illustrative only and are not intended to be limiting. All publications, patent applications, patents, sequences, database entries, and other references mentioned herein are incorporated herein by reference in their entirety. In the event of a conflict, the present specification (including definitions) shall prevail.

Claims

1. A pharmaceutical composition comprising a self-assembling peptide, wherein: The self-assembling peptide consists of the amino acid sequence listed in SEQ ID NO:

15.

2. The pharmaceutical composition according to claim 1, wherein The self-assembling peptide comprises one or two modifications of the following: a) an N-terminal functional group selected from the group consisting of acetyl, formyl, pyroglutamyl (pGlu), biotin, urea, carbamate, sulfonamide, dansyl, 2,4-dinitrophenyl, fluorescein, 7-methoxycoumarinacetic acid, 9-fluorenylmethoxycarbonyl, palmitic acid, succinyl, maleimide, benzyloxycarbonyl, tert-butyloxycarbonyl, 4-hydroxyphenylpropionic acid, allyloxycarbonyl, butyric acid, hexanoic acid, caprylic acid, capric acid, stearic acid, myristic acid, lauric acid, and trityl, and b) a C-terminal functional group selected from the group consisting of amino, N-alkylamide, cysteamide, methyl ester, benzyl ester, tert-butyl ester, 7-amino-4-methylcoumarin (Amc), hydrazide, hydroxamic acid, chloromethyl ketone, p-nitrophenol and fluoromethyl ketone.

3. The pharmaceutical composition according to claim 1, wherein The self-assembling peptide comprises one or two modifications of the following: a) an N-terminal functional group selected from the group consisting of chloroacetyl, bromoacetyl and nitrilotriacetyl, and b) a C-terminal functional group selected from the group consisting of p-nitroanilide, p-nitroaniline, hydroxysuccinimide ester, 9-fluorenylmethyl ester, allyl ester, 2,4-dimethoxybenzyl ester, 2-phenylisopropyl ester, p-nitrobenzyl ester and 2-chlorotrityl ester.

4. The pharmaceutical composition according to claim 1, further comprising a tonicity agent, wherein The tonicity agent comprises: One or more salts selected from the group consisting of NaCl, KCl, MgCl2, CaCl2, NH4Cl, Na2HPO4, KH2PO4 and CaSO4; or One or more sugars selected from the group consisting of dextrose, mannitol, glycerol, sucrose and trehalose.

5. The pharmaceutical composition according to claim 4, wherein The tonicity agent comprises one or more salts and is present in a concentration of 0.01M to 0.3M.

6. The pharmaceutical composition according to claim 4, wherein The tonicity agent comprises one or more salts and is present at a concentration of 0.15M.

7. The pharmaceutical composition according to claim 4, wherein The tonicity agent comprises one or more sugars and is present in a concentration of 0.1-10% w / v.

8. The pharmaceutical composition according to claim 4, wherein The tonicity agent comprises one or more sugars and is present at a concentration of 10% w / v.

9. The pharmaceutical composition according to claim 1, having a pH of 6 to 8.

10. The pharmaceutical composition according to claim 9, wherein The net charge of the self-assembling peptide is greater than or equal to +1 or less than or equal to -1.

11. The pharmaceutical composition according to claim 1, wherein The concentration of the self-assembling peptide is 0.01% w / v to 10% w / v.

12. The pharmaceutical composition according to claim 1, wherein The concentration of the self-assembling peptide is 0.1% w / v to 5% w / v.

13. The pharmaceutical composition according to claim 1, wherein The concentration of the self-assembling peptide is 0.5% w / v to 1.5% w / v.

14. The pharmaceutical composition according to claim 1, wherein The concentration of the self-assembling peptide was 1% w / v.

15. The pharmaceutical composition of claim 1, further comprising isolated cells.

16. The pharmaceutical composition according to claim 15, wherein The isolated cells are mammalian cells selected from the group consisting of immune cells, stem cells, myoblasts, fibroblasts, keratinocytes, neurons, glial cells, astrocytes, preadipocytes, adipocytes, endothelial cells, mesenchymal cells and cardiomyocytes.

17. The pharmaceutical composition according to claim 15, wherein The isolated cells are mammalian cells selected from the group consisting of chondrocyte progenitor cells, pancreatic progenitor cells, endothelial progenitor cells, and smooth muscle progenitor cells.

18. The pharmaceutical composition according to claim 1, wherein The pharmaceutical composition also comprises a bioactive agent.

19. The pharmaceutical composition according to claim 18, wherein The bioactive agent is selected from the group consisting of hormones, growth factors, enzymes, siRNA, shRNA, antisense RNA, antibiotics, antibodies and anti-inflammatory agents.

20. The pharmaceutical composition according to claim 1, wherein The pharmaceutical composition is an aqueous solution.

21. The pharmaceutical composition according to claim 1, wherein The pharmaceutical composition is a hydrogel.

22. The pharmaceutical composition according to claim 21, wherein The hydrogel comprises a storage modulus of at least 10 Pascals (Pa).

23. A prepared product comprising the pharmaceutical composition of claim 1, wherein: The article of manufacture is a syringe, a vial or a catheter.

24. Use of the pharmaceutical composition according to claim 1 in the preparation of a medicament for use in the process of resecting a lesion from a gastrointestinal tract site of a subject, wherein the resecting lesion comprises: administering the pharmaceutical composition to the submucosal layer beneath the lesion in an amount sufficient to lift the lesion; and The lesion is resected from the gastrointestinal tract site of the subject.

25. The use according to claim 24, wherein The lesions include polyps, ulcers or tumors.

26. The use according to claim 24, wherein The lesion is present in a region of the gastrointestinal tract selected from the group consisting of: mouth, throat, esophagus, stomach, small intestine, large intestine, colon and rectum.

27. A method for culturing cells in vitro, comprising contacting the cells with the pharmaceutical composition according to claim 1.

28. A self-assembling peptide consisting of the amino acid sequence listed in SEQ ID NO:35.

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