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WO2023127888A1 - Novel peptides - Google Patents

Novel peptides Download PDF

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Publication number
WO2023127888A1
WO2023127888A1 PCT/JP2022/048245 JP2022048245W WO2023127888A1 WO 2023127888 A1 WO2023127888 A1 WO 2023127888A1 JP 2022048245 W JP2022048245 W JP 2022048245W WO 2023127888 A1 WO2023127888 A1 WO 2023127888A1
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WIPO (PCT)
Prior art keywords
residue
peptide
collagen
gel
gly pro
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PCT/JP2022/048245
Other languages
French (fr)
Inventor
Takaki Koide
Shinichiro ICHISE
Ryo Masuda
Soshi Nakamura
Hiroshi Nose
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Kola-Gen Pharma Inc.
Waseda University
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Application filed by Kola-Gen Pharma Inc., Waseda University filed Critical Kola-Gen Pharma Inc.
Publication of WO2023127888A1 publication Critical patent/WO2023127888A1/en

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/78—Connective tissue peptides, e.g. collagen, elastin, laminin, fibronectin, vitronectin or cold insoluble globulin [CIG]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the present invention relates to a novel peptide, a pharmaceutical composition comprising the peptide, and a gelling agent comprising the peptide, a gel comprising the peptide, and a regenerative medical material comprising the peptide.
  • Collagen is the main structural protein in the extracellular matrix, and it forms supramolecules, such as fibrillar and reticular structures, in the body.
  • the collagen molecule has a right-handed triple-helical structure composed of three polypeptide chains [1].
  • Each polypeptide chain in the triple-helical domain of collagen has Xaa-Yaa-Gly repeats.
  • fibrillar collagen has approximately 340 tandem repeats of Xaa-Yaa-Gly.
  • the amino acids in Xaa and Yaa are often occupied by Pro and 4-hydroxyproline (Hyp/O) residues, respectively, and these imino acid residues increase the thermal stability of the triple helix [2-4].
  • Isolated fibrillar collagen self-assembles into fibrils and then fibers under physiological conditions in vitro, and it then forms a gel.
  • Collagen not only functions as a scaffold for cells, but it also functions as a bio-signaling molecule that regulates cell survival and differentiation by interacting with membrane receptors and soluble proteins.
  • the amino acid sequences recognized by collagen-binding macromolecules have been identified.
  • Collagen-binding integrins ⁇ 1 ⁇ 1, ⁇ 2 ⁇ 1, ⁇ 10 ⁇ 1, and ⁇ 11 ⁇ 1 mediate cell adhesion, survival, and proliferation.
  • the Gly-Phe-Hyp-Gly-Glu-Arg (GFOGER, Hyp/O is 4-hydroxyproline) sequence on the collagen triple helix has been identified as a high-affinity site for integrin ⁇ 2 ⁇ 1, a major collagen-binding integrin [5,6].
  • DDR discoidin domain receptor 1 and 2
  • DDRs recognize Gly-Val-Met-Gly-Phe-Hyp on the collagen triple helix [7].
  • VWF von Willebrand factor
  • SPARC protein acidic and rich in cysteine
  • PEDF pigment epithelium-derived factor
  • heparan sulfate proteoglycans in syndecans competitively bind to Lys-Gly-His-Arg-Gly-Phe-Ser-Gly-Leu on the collagen triple helix and regulate angiogenesis [11-13].
  • Collagen is a useful biomaterial in research and medical applications because of its polymeric properties and biological activities [14]. Recently, development of chemically synthesized artificial collagen as a collagen surrogate has been reported [15,16]. Artificial collagen is a promising biomaterial that can provide matrices with specific biological activities by incorporating bioactive sequences.
  • Peptides with tens of amino acid residues consisting of tandem Xaa-Yaa-Gly repeats form the collagen-like triple helix by self-assembly.
  • collagen-like peptides form the collagen-like triple helix by self-assembly.
  • Several artificial collagens that can form a gel by supramolecular formation of collagen-like peptides have been reported.
  • a trimeric collagen-like peptide in which three peptide strands are staggered and tethered by disulfide bonds forms a gel by elongating the triple helix [17-19].
  • Cell adhesion activity has been introduced into the artificial collagen-like material by incorporating the integrin-binding GFOGER sequence [20].
  • Collagen-like peptides consisting of Pro-Hyp-Gly repeats with Lys and Asp residues in appropriately designed positions form a gel by elongation of the triple helix through sticky-end assembly and further lateral interactions via salt bridges [21,22].
  • rearrangement of the acidic and basic amino acids in the peptide sequence is critical for supramolecular formation. Therefore, the amino acid sequence can hardly rearrange and it is difficult to incorporate bioactive sequences into the peptide sequence.
  • the preparation process requires more than 3 days oxidation in a solution containing 10% (v/v) dimethyl sulfoxide (DMSO) as an oxidant [24], and physical destruction of the gel is irreversible. Therefore, the gel has to be formed into a shape a priori and it is difficult to be applied to a 3D scaffold for cell culture or an injectable gel for medical use. It has been reported that a glycylserine O-acyl isopeptide unit incorporated into a collagen-like peptide sequence prevented triple-helix formation under acidic conditions and induced triple-helix formation by conversion to the amide form at pH 7.4 [25].
  • DMSO dimethyl sulfoxide
  • Pigment epithelium-derived factor shares binding sites in collagen with heparin/heparan sulfate proteoglycans. J. Biol. Chem. 2011, 286, 26364-26374, https://doi.org/10.1074/jbc.m111.252684.
  • Discoidin Domain Receptors Promote ⁇ 1 ⁇ 1- and ⁇ 2 ⁇ 1-Integrin Mediated Cell Adhesion to Collagen by Enhancing Integrin Activation.
  • PLoS One 2012, 7, e52209 https://doi.org/10.1371/journal.pone.0052209.
  • [NPL31] Myllymaki S.M.; Teravainen T.P.; Manninen A. Two distinct integrin-mediated mechanisms contribute to apical lumen formation in epithelial cells.
  • NPL32 Yu W.; Datta A.; Leroy P.; O'Brien L.E.; Mak G.; Jou T.; Matlin K.S.; Mostov K.E.; Zegers M.M.P. ⁇ 1-integrin orients epithelial polarity via Rac1 and laminin. Mol. Biol. Cell 2005, 16, 433-445, https://doi.org/10.1091/mbc.e04-05-0435.
  • NPL33 Chung I.; Enemchukwu N.O.; Khaja S.D.; Murthy N.; Mantalaris A.; Garcia A.J. Bioadhesive hydrogel microenvironments to modulate epithelial morphogenesis.
  • O-acyl isopeptide method efficient synthesis of isopeptide segment and application to racemization-free segment condensation.
  • the present invention provides a novel peptide, a pharmaceutical composition comprising the peptide, and a gelling agent comprising the peptide, a gel comprising the peptide, and a regenerative medical material comprising the peptide.
  • a collagen-like peptide polymer that undergoes a rapid sol-gel transition in situ triggered by a simple pH shift.
  • the transition mechanism is based on O-N acyl migration of an O-acyl isopeptide unit in the collagen-like peptide followed by triple helix formation.
  • the O-acyl isopeptide moiety in the peptide is stable under acidic conditions, and it is converted to the corresponding amide form through O-N acyl migration at physiological pH.
  • Xaa is proline
  • Yaa is hydroxyproline
  • n is 5.
  • the present invention provides a collagen-like peptide polymer that forms a gel within 10 min by triple helix formation triggered by O-N acyl migration under basic conditions by incorporating a glycylserine O-acyl isopeptide unit into the collagen-like peptide sequence.
  • This allowed encapsulation and culturing of cells in the 3D gel, which were not possible using previously developed similar artificial collagen gels [22].
  • the present invention has demonstrated that cyst formation with polarization of epithelial cells can be controlled by incorporating the bioactive sequences of collagen into the artificial collagen gel.
  • This polymer-based 3D cell-culture system is expected to allow investigation of the mechanism by which collagen-binding molecules control the behavior of cells, including proliferation, differentiation, and polarization.
  • injectable gels such as scaffolds for in vitro stem cell culture and carriers for cell transplantation, are also expected.
  • Figure 1 shows a concept of formation of a gel from a collagen-like peptide polymer by O-N acyl migration.
  • the collagen-like peptide which has a glycylserine O-acyl isopeptide at the center of the sequence and two Cys residues at each terminus, is polymerized by cross-linking via disulfide bonds and forms a gel by triple-helix formation indued by O-N acyl migration at physiological pH.
  • Figure 2 shows conformational analysis of the peptides by CD spectroscopy.
  • the peptides were annealed in 0.05% TFA(aq), and the spectra were measured in the presence of 10 mM TCEP.
  • B Monitoring of the CD signal at 225 nm with increasing temperature from 4 to 95 °C. The red line and blue line are C2-ester and C2-amide, respectively.
  • Figure 3 shows evaluation of gel formation and O-N acyl migration of the C2-ester polymer.
  • A Evaluation of gel formation of the C2-ester polymer after addition of 10 ⁇ PBS. Gel formation of the C2-ester polymer was evaluated after addition of 10 ⁇ PBS to the polymer solution at the final concentration of 10 mg/mL followed by incubation at 37 °C for 1 h.
  • a stainless-steel ball was set on the interface of the polymer, and gel formation was judged by whether the ball was held on the interface.
  • the C2-amide polymer polymerized in a solution containing 10% (v/v) DMSO and 0.05% (v/v) TFA at a peptide concentration of 10 mg/mL was also evaluated as a control.
  • the arrowheads indicate the positions of the stainless-steel balls.
  • the C2-ester polymer solution was subjected to addition of 10 ⁇ PBS at a final polymer concentration of 10 mg/mL and incubated at 37 °C for 1 h.
  • FIG. 1 shows morphological analysis of the MDCK-cell spheres in the gels.
  • A Typical morphologies of the MDCK-cell spheres in the gels. The cross-sectional images were obtained by confocal fluorescence microscopy. F-actin and the nuclei were stained in red and blue, respectively. The scale bars indicate 100 ⁇ m.
  • B Quantitative analysis of cyst formation in the gels.
  • the cell spheres with diameter of less than 100 ⁇ m were classified as single-luminal, multi-luminal, or non-luminal spheres.
  • the ellipticity (C) and cross-sectional area (D) of the spheres were measured. The significance of the differences was assessed by non-parametric one-way analysis of variance and the post-hoc Tukey test, and all of the P values ⁇ 0.05 are shown in the figures.
  • Figure 6 shows the kinetics of O-N acyl migration in the C2-ester polymer under each condition (pH 8.95, 7.40, 5.45, and 2.29). The percentage of acyl migration was calculated by the ratio of the amide form in the total peak area of the ester form and the amide form in HPLC analysis.
  • Figure 7 shows the kinetics under the conditions of 0°C and pH 7.40.
  • Figure 8 shows gel formation by copolymerizing C3s-ester with C3s-amide.
  • Figure 9 shows evaluation of gel formation.
  • Figure 10 shows confirmation of gel formation.
  • Figure 11 shows confirmation of gel formation.
  • the present invention discloses a peptide represented by formula (I), wherein m is 2 or 3, n represents an integer of 4 to 10, each of Xaa and Yaa is independently selected from a proline residue, a hydroxyproline residue, an arginine residue, a lysine residue, a valine residue, a leucine residue, an isoleucine residue, a serine residue, a threonine residue, an alanine residue, a glycine residue, an N-isobutyl glycine residue, a phenylalanine residue, a methionine residue, a glutamate residue, an aspartate residue, an asparagine residue, a glutamine residue, a histidine residue, a tryptophan residue or a tyrosine residue, the proline residue may be modified by an amino group or fluorine atom, or pharmaceutically acceptable salts thereof, or a
  • the present invention discloses a peptide represented by formula (II), wherein m is 2 or 3, n represents an integer of 4 to 10, each of Xaa and Yaa is independently selected from a proline residue, a hydroxyproline residue, an arginine residue, a lysine residue, a valine residue, a leucine residue, an isoleucine residue, a serine residue, a threonine residue, an alanine residue, a glycine residue, an N-isobutyl glycine residue, a phenylalanine residue, a methionine residue, a glutamate residue, an aspartate residue, an asparagine residue, a glutamine residue, a histidine residue, a tryptophan residue or a tyrosine residue, the proline residue may be modified by an amino group or fluorine atom, or pharmaceutically acceptable salts thereof, or a solvate of thereof.
  • n is an integer of 4 to
  • the present invention discloses a process for producing the peptide represented by the formula (II), comprising reacting the peptide of formula (I) under physiological pH, wherein m, n, Xaa and Yaa are the same as defined above.
  • the present invention discloses a peptide listed in Table 1,
  • the present invention discloses a peptide listed in Table 2,
  • Another aspect of this invention is directed to a pharmaceutical composition comprising one or more kinds of the peptide represented by formula (I), formula (II), Table 1 or Table 2.
  • Another aspect of this invention is directed to a gelling agent comprising one or more kinds of the peptide represented by formula (I), formula (II), Table 1 or Table 2.
  • Another aspect of this invention is directed to a gel comprising one or more kinds of the peptide represented by formula (I), formula (II), Table 1 or Table 2.
  • Another aspect of this invention is directed to a regenerative medical material comprising one or more kinds of the peptide represented by formula (I), formula (II), Table 1 or Table 2.
  • a peptide thin membrane can, for example, be used as a medical material for adhesion prevention of organs and as a suture material.
  • such medical material may, for example, be placed between an organ and the dermis, or between an organ and another organ, in a surgery, thereby preventing an organ from adhering to the dermis and another organ.
  • the medical material of the present invention thus placed may gradually become soluble and degraded, and then disappear, by peptidase and phagocytes such as macrophage that are present in vivo. Therefore, no further surgery is required to remove such medical material, thus imposing a smaller burden on the prognosis of a patient, and making it possible to improve Quality of life (QOL).
  • QOL Quality of life
  • the peptide of the present invention when using the peptide of the present invention as a research base material having a desired selective activity, it can be used in, for example, researches for unraveling the mechanism of the intracellular information transmission system of the cells that are to bind to such base material.
  • the gel or peptide thin membrane comprising the peptide of the present invention can, for example, be placed on a region of wound, a surgical incision made in a surgery, the cornea and the retina.
  • the gel or peptide thin membrane can, for example, be used as a medical material for promoting wound healing for enhancing the migration and binding of the fibroblasts to a region of wound or a surgical incision, and thus promoting the healing of a wound or surgical incision; or as a medical material for use in cornea and retina regeneration.
  • the abovementioned polymerized peptide is thus produced as a hydrogel of the polymerized peptide of the present invention and water i.e., the polymerized peptide of the present invention can be employed as a gelling agent.
  • the hydrogel of the present invention can be used as a postsurgical adhesion prevention agent for organs, a hemostatic agent, an agent for promoting wound healing, and a regenerative medical material such as a material for cornea or retina regeneration.
  • a polymerized peptide thin membrane as a sheet-shaped membrane.
  • This polymerized peptide thin membrane can, for example, be rehydrated at the time of use so that it can be used as the above material for promoting wound healing, and a regenerative medical material such as an artificial cornea and a cell sheet for an artificial myocardial membrane capable of being transplanted into a body.
  • Conditions that are not specified in the examples will be the common conditions in the art or the recommended conditions of the raw materials by the product manufacturer.
  • the reagents which are not indicated the origin will be the commercially available conventional reagents.
  • MDCK cells were purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA). The cells were cultured in D-MEM (Fujifilm Wako Pure Chemical Industries, Osaka, Japan) containing 10% (v/v) FBS (Thermo Fisher Scientific, Waltham, MA, USA), 100 units/mL penicillin, and 100 ⁇ g/mL streptomycin (Sigma-Aldrich, St. Louis, MO, USA). The cells were maintained at 37 °C in a humidified 5% CO2/air atmosphere. 1.2.
  • the peptides were manually synthesized according to the standard Fmoc-based solid-phase method on 2-chlorotrityl chloride resin (Peptide Institute, Osaka, Japan). The coupling reaction was performed at room temperature for 2 h with 3-5 equivalents of Fmoc-amino acid, DIC, and HOBt in N,N-dimethylformamide. The Fmoc group was deprotected using 20% (v/v) or 30% (v/v) piperidine/DMF for 15 min.
  • Fmoc-Pro-Hyp-Gly-OH (Watanabe Chemical Industries, Hiroshima, Japan) and Boc-Ser(Fmoc-Gly)-OH (Iris Biotech, explanation, Germany) were used for incorporation of Pro-Hyp-Gly repeats and the glycylserine O-acyl isopeptide unit, respectively.
  • the Fmoc group of the amino acid residue next to the glycylserine O-acyl isopeptide unit was deprotected using 25% (v/v) 1-methylpyrrolidine, 2% (v/v) hexamethyleneimine and 3% (w/v) HOBt in N-methylpyrrolidone:DMSO (1:1) [35].
  • the peptides were deprotected and cleaved from the resin by TFA/m-cresol/thioanisole/ethanedithiol/H2O (80:5:5:5:5, v/v) at room temperature for 4 h.
  • the crude peptides were purified by RP-HPLC on a Cosmosil 5C18-AR II column (Nacalai Tesque, Kyoto, Japan) and identified by matrix-assisted laser desorption-ionization time-of-flight mass spectrometry (Autoflex III, Bruker, MA, USA) or electrospray ionization mass spectrometry (AB Sciex, MA, USA).
  • CD analysis The CD spectra were recorded with a J-820 CD spectropolarimeter (JASCO, Tokyo, Japan) equipped with a Peltier thermal controller. The intensity of the spectra was converted into the mean residue weight ellipticity ([ ⁇ ]MRW).
  • the peptides were dissolved in degassed 0.05% (v/v) TFA(aq) containing 10 mM TCEP, followed by annealed at 4 °C overnight after heating at 95 °C for 5 min.
  • the thermal denaturation curve of the triple helix was obtained by monitoring [ ⁇ ]MRW at 225 nm ([ ⁇ ]MRW,225) with increasing temperature from 4 to 95 °C at a rate of 18 °C h-1. 1.4.
  • TCEP was then added to the solution at the final concentration of 50 mM, and the solution was heated at 95 °C for 3 min, followed by RP-HPLC analysis.
  • O-N acyl migration-induced gel formation was similarly assessed by the position of the ball after turning the tube with the ball at the bottom upside down after incubation at 37 °C ( Figure 4D). 1.5.
  • MDCK cells were detached with 0.05% (w/v) trypsin/0.53mM ethylenediaminetetraacetic acid (Fujifilm Wako Pure Chemical Industries) and harvested by centrifugation (120 ⁇ g, 4 min).
  • type I collagen gel 3 mg/mL type I collagen acidic solution (I-AC, Koken, Tokyo, Japan), DMSO, 10 ⁇ D-MEM, and MDCK cell suspension were mixed at a ratio of 15:2:2:1 and incubated at 37 °C. After culturing at 37 °C for 7 days, the cells were fixed with 4% p-formaldehyde/phosphate buffer (Fujifilm Wako Pure Chemical Industries) overnight, permeabilized with 0.1% (v/v) Triton X-100/PBS for 15 min, and blocked with 1% (w/v) skim milk/PBS for 1 h.
  • I-AC type I collagen acidic solution
  • DMSO 10 ⁇ D-MEM
  • MDCK cell suspension 3 mg/mL type I collagen acidic solution (I-AC, Koken, Tokyo, Japan), DMSO, 10 ⁇ D-MEM, and MDCK cell suspension were mixed at a ratio of 15:2:2:1 and incubated at 37 °C. After
  • the nuclei and F-actin were stained by Hoechst 33342 and phalloidin-iFluor conjugate (Cayman Chemical, MI, USA) for 3 h.
  • the cells were observed by confocal fluorescence microscopy (FV-1000, Olympus, Tokyo, Japan) after washing with PBS.
  • FV-1000 confocal fluorescence microscopy
  • the spheres were classified as single-luminal, multi-luminal, or no-luminal spheres using cross-sectional images.
  • the area and ellipticity of the cell spheres were measured by ImageJ software [36].
  • the cross-sectional area was defined as the largest area among the photographs taken of each sphere every 3 ⁇ m in the horizontal direction.
  • the known raw materials of the present invention can be prepared by the conventional synthesis methods in the art, or can be purchased. Peptides 1 to 6 and A196 were synthesized by Life Chemicals Inc. (Ontario, Canada) and purchased from Namiki Shoji Co. Ltd. (Tokyo, Japan).
  • FIG. 1 A strategy to form a gel from a collagen-like peptide polymer by triple helix formation induced by pH-triggered O-N acyl migration at physiological pH has been devised, as shown in Figure 1.
  • This peptide was composed of collagen-like Xaa-Yaa-Gly repeats with two Cys residues at each terminus and a glycylserine O-acyl isopeptide in the center of the sequence.
  • the peptides were cross-linked via disulfide bonds under acidic conditions.
  • the sol-gel transition was expected to be induced by triple helix formation triggered by O-N acyl migration at physiological pH.
  • Boc-Ser(Fmoc-Gly)-OH and Fmoc-Pro-Hyp-Gly-OH were used as building blocks, respectively.
  • the peptides were cleaved from the resin and deprotected by a trifluoroacetic acid (TFA)-based cocktail, and they were purified by reverse-phase high-performance liquid chromatography (RP-HPLC).
  • TFA trifluoroacetic acid
  • RP-HPLC reverse-phase high-performance liquid chromatography
  • the amide-form peptide corresponding to the expected product of O-N acyl migration of C2-ester was also independently prepared by the same procedure (C2-amide, Table 1).
  • Circular dichroism (CD) analysis was performed to confirm that the O-acyl isopeptide unit disturbed triple helix formation, but the amide-form peptide can form the triple helix.
  • C2-ester and C2-amide were dissolved in 0.05% (v/v) TFA(aq) and then annealed at 4°C overnight after heating at 95°C.
  • the CD spectra at 4°C were measured ( Figure 2A). Both C2-ester and C2-amide showed a maximum signal at around 225 nm and a minimum signal at around 200 nm, indicating the polyproline-II-like secondary structure.
  • the signals of C2-ester were smaller than those of C2-amide, indicating that C2-amide had higher helicity.
  • the signal at 225 nm was monitored with increasing temperature (Figure 2B).
  • the signal cooperatively decreased at around 30°C for C2-ester and at around 70°C for C2-amide, and C2-ester showed a more gradual decreasing signal.
  • the cooperative decrease of the signal at 225 nm is a typical property of the collagen-like triple helix, strongly suggesting that both C2-ester and C2-amide formed the triple helix [26].
  • the results also suggested that although C2-ester contained triple-helical population, the triple helix was disturbed and destabilized by the O-acyl isopeptide unit interrupting the continuous Xaa-Yaa-Gly sequence.
  • the signal at 225 nm more gradually decreased with increasing temperature for C2-ester than for C2-amide, suggesting that the triple-helical population of C2-ester was more heterogenous.
  • the Arg residue was introduced to prevent possible lateral aggregation of the Pro-Hyp-Gly repeating peptide [27].
  • the peptide composition and concentration were investigated using the amide-form peptides (C2-amide, C3-amide, and C3l-GPO). The amount of C3l-GPO was fixed at 10% of the total peptide. Copolymers with different ratios of C2-amide and C3-amide were prepared, and the concentration at which the polymer forms a gel was investigated ( Figure 4B).
  • the 5:4:1, 6:3:1, and 7:2:1 C2-ester:C3-amide:C3l-GPO copolymers were in the sol state at concentrations of less than 7, 8, and 10 mg/mL, respectively, while the 8:1:1 and 9:0:1 C2-ester:C3-amide:C3l-GPO copolymers were in the sol state even at 10 mg/mL.
  • the copolymers in the sol state at relatively high concentrations (labeled a-g in Figure 4C) were examined for O-N acyl migration-induced gel formation at pH 7.4.
  • the results of gel formation evaluation after addition of 10 ⁇ PBS to each polymer are shown in Figure 4D.
  • the sol state was maintained even at the highest concentration of 10 mg/ml observed.
  • the Madin-Darby canine kidney (MDCK) cell line a canine renal tubular epithelial cell line, as a typical model for formation of 3D structures in 3D matrices were used.
  • MDCK cells are known to form cysts when cultured in type I collagen gels [28,29].
  • peptides containing each of the three bioactive sequences were prepared (Table 1).
  • the norleucine residue (Nle/X) introduced into C3l-GVXGFO was used as an oxidation-resistant Met analog. This sequence has been reported to function as a ligand for DDR [30].
  • the copolymers composed of C2-ester, C3-amide, and either one of C3l-GPO, C3l-GFOGER, C3l-GVXGFO, or C3l-KGHRGF (called the GPO polymer, GFOGER polymer, GVXGFO polymer, and KGHRGF polymer, respectively) at a ratio of 6:3:1 and a concentration of 7.5 mg/mL were prepared by the same procedure as above. These copolymers were all in the sol state after polymerization.
  • the polymer solution after oxidation was first neutralized to weakly basic pH by adding 10 ⁇ Dulbecco’s modified Eagle’s medium (D-MEM), and the dispersed MDCK cells after trypsinization were mixed at a volume ratio of 17:2:1.
  • the final concentration of the polymer was 6.4 mg/mL.
  • type I collagen acidic solution was mixed with 10 ⁇ D-MEM and MDCK cells, and it formed a gel on a cell-culture dish according to the same procedure.
  • the final collagen concentration of the gel was 2.3 mg/mL.
  • the luminal spheres showed the localization of F-actin along the lumen, indicating formation of apical surfaces inside the sphere.
  • the presence or absence of lumens and the number of lumens of the type I collagen gel and collagen-like peptide polymer gels were compared ( Figures 5B).
  • the type I collagen, GPO polymer, and GFOGER polymer gels were comparable, and fewer spheres formed lumens in the GVXGFO polymer and KGHRGF polymer gels. From comparison of the number of lumens, most of the luminal spheres were single-luminal cysts in the type I collagen gel.
  • the ratios of single-luminal cysts and multi-luminal cysts were similar in the GPO polymer gel without any specific bioactive sequences.
  • the ratio of the single-luminal cyst was higher than that in the GPO polymer gel, and the cyst-forming activity was the highest among the collagen-like peptide polymer gels.
  • the ellipticity and area of the cross-sections of the spheres were measured ( Figure 5C and D). The cross-sections of the spheres in the type I collagen gel were close to perfect circles, whereas those of the spheres formed in all of the collagen-like peptide polymer gels were distorted and their ellipticity was significantly higher.
  • the cross-sectional area of the sphere was not inversely correlated with the single-luminal cyst-forming activity.
  • the cross-sectional areas of the spheres in the type I collagen gel were significantly smaller than those in the collagen-like peptide polymer gels, except for the GFOGER polymer gel. Comparing the introduced bioactive sequences, the cross-sectional areas of the spheres in the GVXGFO polymer gel were significantly larger than those in the other collagen-like peptide polymer gels, and those of the spheres in the KGHRGF polymer gel were significantly larger than those in the GFOGER polymer gel.
  • integrin ⁇ 2 ⁇ 1-binding sequence promotes cyst formation, including polarization of MDCK cells.
  • the contribution of integrin ⁇ 2 ⁇ 1 to cyst formation is supported by previous studies that showed that cyst formation of MDCK cells in type I collagen gels is inhibited by integrin ⁇ 2 ⁇ 1 knockdown and specific inhibitory antibodies [31,32].
  • previous studies using a 3D culture system of synthetic matrices conjugated with the Arg-Gly-Asp peptide, a ligand for integrin ⁇ V ⁇ 3 have also shown that activation of integrin promotes MDCK cell cyst formation [33,34].
  • cyst formation in MDCK cells is suppressed by the Arg-Gly-Gln-Hyp-Gly-Val-Nle-Gly-Phe-Hyp (RGQOGVXGFO) sequence incorporated into C3l-GVXGFO and Lys-Gly-His-Arg-Gly-Phe-Ser-Gly-Leu (KGHRGFSGL) sequences incorporated into C3l-KGHRGF.
  • a polymer of C3s-ester and C3s-amide at a ratio of 4:6 and with peptide concentration of 7.3 mg/ml was neutralized with a buffer solution, and then injected into PBS to investigate the gel formation.
  • a buffer solution 160 mM phosphate buffer (pH 8.0), 2200 mM NaCl, and 42 mM KCl
  • the final concentration was the same salt concentration as PBS
  • the polymer was sucked up with a syringe and injected into the PBS in the tube. After incubating for 20 minutes at 37°C, the tube was turned upside down.
  • the present invention provides a novel peptide, a pharmaceutical composition comprising the peptide, and a gelling agent comprising the peptide, a gel comprising the peptide, and a regenerative medical material comprising the peptide.

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Abstract

A new peptide and a pharmaceutical composition comprising the peptide were provided.

Description

NOVEL PEPTIDES
The present invention relates to a novel peptide, a pharmaceutical composition comprising the peptide, and a gelling agent comprising the peptide, a gel comprising the peptide, and a regenerative medical material comprising the peptide.
Collagen is the main structural protein in the extracellular matrix, and it forms supramolecules, such as fibrillar and reticular structures, in the body. The collagen molecule has a right-handed triple-helical structure composed of three polypeptide chains [1]. Each polypeptide chain in the triple-helical domain of collagen has Xaa-Yaa-Gly repeats. Specifically, fibrillar collagen has approximately 340 tandem repeats of Xaa-Yaa-Gly. The amino acids in Xaa and Yaa are often occupied by Pro and 4-hydroxyproline (Hyp/O) residues, respectively, and these imino acid residues increase the thermal stability of the triple helix [2-4]. Isolated fibrillar collagen self-assembles into fibrils and then fibers under physiological conditions in vitro, and it then forms a gel.　
Collagen not only functions as a scaffold for cells, but it also functions as a bio-signaling molecule that regulates cell survival and differentiation by interacting with membrane receptors and soluble proteins. The amino acid sequences recognized by collagen-binding macromolecules have been identified. Collagen-binding integrins α1β1, α2β1, α10β1, and α11β1 mediate cell adhesion, survival, and proliferation. The Gly-Phe-Hyp-Gly-Glu-Arg (GFOGER, Hyp/O is 4-hydroxyproline) sequence on the collagen triple helix has been identified as a high-affinity site for integrin α2β1, a major collagen-binding integrin [5,6]. The collagen-binding receptor tyrosine kinases, discoidin domain receptor (DDR) 1 and 2, also regulate cell adhesion, survival, and proliferation. DDRs recognize Gly-Val-Met-Gly-Phe-Hyp on the collagen triple helix [7]. Both the von Willebrand factor (VWF) for blood coagulation and secreted protein acidic and rich in cysteine (SPARC) bind to Arg-Gly-Gln-Hyp-Gly-Val-Met-Gly-Phe containing the DDR-binding sequence [8-10]. The pigment epithelium-derived factor (PEDF) and heparan sulfate proteoglycans in syndecans competitively bind to Lys-Gly-His-Arg-Gly-Phe-Ser-Gly-Leu on the collagen triple helix and regulate angiogenesis [11-13].
Collagen is a useful biomaterial in research and medical applications because of its polymeric properties and biological activities [14]. Recently, development of chemically synthesized artificial collagen as a collagen surrogate has been reported [15,16]. Artificial collagen is a promising biomaterial that can provide matrices with specific biological activities by incorporating bioactive sequences.
Peptides with tens of amino acid residues consisting of tandem Xaa-Yaa-Gly repeats (collagen-like peptides) form the collagen-like triple helix by self-assembly. Several artificial collagens that can form a gel by supramolecular formation of collagen-like peptides have been reported. A trimeric collagen-like peptide in which three peptide strands are staggered and tethered by disulfide bonds forms a gel by elongating the triple helix [17-19]. Cell adhesion activity has been introduced into the artificial collagen-like material by incorporating the integrin-binding GFOGER sequence [20]. However, it is not suitable for practical use because the need for stepwise reaction of disulfide bond formation results in high synthetic cost. Collagen-like peptides consisting of Pro-Hyp-Gly repeats with Lys and Asp residues in appropriately designed positions form a gel by elongation of the triple helix through sticky-end assembly and further lateral interactions via salt bridges [21,22]. However, rearrangement of the acidic and basic amino acids in the peptide sequence is critical for supramolecular formation. Therefore, the amino acid sequence can hardly rearrange and it is difficult to incorporate bioactive sequences into the peptide sequence.
Recently, we developed another artificial collagen system by end-to-end cross-linking of collagen-like triple-helical peptides [23]. We found that a peptide with two or more Cys residues at each terminus of the collagen-like sequence formed a gel by triple helix formation and subsequent cross-linking via disulfide bonds. The polymer gel exhibited integrin-dependent cell-attachment activity by incorporating the responsible amino acid sequence into the peptide. The cell-attachment activity was tuned by altering the concentration of the binding sequence in the polymer. The stiffness of the gel could be also tuned by altering the number of Cys residues. However, the preparation process requires more than 3 days oxidation in a solution containing 10% (v/v) dimethyl sulfoxide (DMSO) as an oxidant [24], and physical destruction of the gel is irreversible. Therefore, the gel has to be formed into a shape a priori and it is difficult to be applied to a 3D scaffold for cell culture or an injectable gel for medical use. It has been reported that a glycylserine O-acyl isopeptide unit incorporated into a collagen-like peptide sequence prevented triple-helix formation under acidic conditions and induced triple-helix formation by conversion to the amide form at pH 7.4 [25].
Patent 2003-000000
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The present invention provides a novel peptide, a pharmaceutical composition comprising the peptide, and a gelling agent comprising the peptide, a gel comprising the peptide, and a regenerative medical material comprising the peptide.
After intensive and extensive research, the present inventors have discovered a
collagen-like peptide polymer that undergoes a rapid sol-gel transition in situ triggered by a simple pH shift. The transition mechanism is based on O-N acyl migration of an O-acyl isopeptide unit in the collagen-like peptide followed by triple helix formation. The O-acyl isopeptide moiety in the peptide is stable under acidic conditions, and it is converted to the corresponding amide form through O-N acyl migration at physiological pH.
Specifically, the following inventions are provided.
(1) A peptide represented by formula (I),
Figure JPOXMLDOC01-appb-I000004
wherein m is 2 or 3, n represents an integer of 4 to 10,
each of Xaa and Yaa is independently selected from a proline residue, a hydroxyproline residue, an arginine residue, a lysine residue, a valine residue, a leucine residue, an isoleucine residue, a serine residue, a threonine residue, an alanine residue, a glycine residue, an N-isobutyl glycine residue, a phenylalanine residue, a methionine residue, a glutamate residue, an aspartate residue, an asparagine residue, a glutamine residue, a histidine residue, a tryptophan residue or a tyrosine residue, the proline residue may be modified by an amino group or fluorine atom,
or pharmaceutically acceptable salts thereof, or a solvate of thereof.
(2) The peptide according to (1), wherein, Xaa is proline, Yaa is hydroxyproline, n is 5.
(3) A peptide represented by formula (II),
Figure JPOXMLDOC01-appb-I000005
wherein m is 2 or 3, n represents an integer of 4 to 10,
each of Xaa and Yaa is independently selected from a proline residue, a hydroxyproline residue, an arginine residue, a lysine residue, a valine residue, a leucine residue, an isoleucine residue, a serine residue, a threonine residue, an alanine residue, a glycine residue, an N-isobutyl glycine residue, a phenylalanine residue, a methionine residue, a glutamate residue, an aspartate residue, an asparagine residue, a glutamine residue, a histidine residue, a tryptophan residue or a tyrosine residue, the proline residue may be modified by an amino group or fluorine atom,
or pharmaceutically acceptable salts thereof, or a solvate of thereof.
(4) The peptide according to (3), wherein Xaa is proline, Yaa is hydroxyproline, n is 5.
(5) A process for producing the peptide represented by the formula (II), comprising reacting the peptide of formula (I) under physiological pH, wherein m, n, Xaa and Yaa are the same as defined in (1) and (3).
Figure JPOXMLDOC01-appb-I000006
(6) A pharmaceutical composition comprising the peptides according to each one of (1) to (4).
(7) A gelling agent comprising the peptides according to each one of (1) to (4).
(8) A gel comprising the peptides according to (1) to (4).
(9) A regenerative medical material comprising the peptides according to each one of (1) to (4).
The present invention provides a collagen-like peptide polymer that forms a gel within 10 min by triple helix formation triggered by O-N acyl migration under basic conditions by incorporating a glycylserine O-acyl isopeptide unit into the collagen-like peptide sequence. This allowed encapsulation and culturing of cells in the 3D gel, which were not possible using previously developed similar artificial collagen gels [22]. Furthermore, the present invention has demonstrated that cyst formation with polarization of epithelial cells can be controlled by incorporating the bioactive sequences of collagen into the artificial collagen gel.
This polymer-based 3D cell-culture system is expected to allow investigation of the mechanism by which collagen-binding molecules control the behavior of cells, including proliferation, differentiation, and polarization. In the biomedical field, applications of injectable gels, such as scaffolds for in vitro stem cell culture and carriers for cell transplantation, are also expected.
Figure 1 shows a concept of formation of a gel from a collagen-like peptide polymer by O-N acyl migration. The collagen-like peptide, which has a glycylserine O-acyl isopeptide at the center of the sequence and two Cys residues at each terminus, is polymerized by cross-linking via disulfide bonds and forms a gel by triple-helix formation indued by O-N acyl migration at physiological pH. Figure 2 shows conformational analysis of the peptides by CD spectroscopy. (A) CD spectra of C2-ester and C2-amide at 4 °C. The peptides were annealed in 0.05% TFA(aq), and the spectra were measured in the presence of 10 mM TCEP. (B) Monitoring of the CD signal at 225 nm with increasing temperature from 4 to 95 °C. The red line and blue line are C2-ester and C2-amide, respectively. Figure 3 shows evaluation of gel formation and O-N acyl migration of the C2-ester polymer. (A) Evaluation of gel formation of the C2-ester polymer after addition of 10× PBS. Gel formation of the C2-ester polymer was evaluated after addition of 10× PBS to the polymer solution at the final concentration of 10 mg/mL followed by incubation at 37 °C for 1 h. A stainless-steel ball was set on the interface of the polymer, and gel formation was judged by whether the ball was held on the interface. The C2-amide polymer polymerized in a solution containing 10% (v/v) DMSO and 0.05% (v/v) TFA at a peptide concentration of 10 mg/mL was also evaluated as a control. The arrowheads indicate the positions of the stainless-steel balls. (B) RP-HPLC analysis of the reduced C2-ester polymer before (red) and after (blue) PBS treatment. The C2-ester polymer solution was subjected to addition of 10× PBS at a final polymer concentration of 10 mg/mL and incubated at 37 °C for 1 h. The solution was reduced by TCEP in 0.05% (v/v) TFA(aq), and then analyzed by RP-HPLC with a linear gradient of acetonitrile/water containing 0.05% (v/v) TFA of 10%-20% in 30 min. The peaks at 21 and 22 min were the C2-ester and corresponding amide-form peptide, respectively. Figure 4 shows investigation of the gel-forming conditions of the ester-amide copolymers. (A) Graphical image of the ester-amide copolymer in which the triple-helical content increases after O-N acyl migration at physiological pH. (B) Evaluation of gel formation using copolymers solely composed of the amide-form peptides. C2-amide, C3-amide, and C3l-GPO were copolymerized in 10% (v/v) DMSO for 4 days and gel formation was evaluated by whether a stainless-steel ball was held on the interface of the polymer. (C) Evaluation of gel formation of copolymers composed of the ester and amide-form peptides. C2-ester, C3-amide, and C3l-GPO were copolymerized and gel formation was evaluated by the same procedure as above. (D) Evaluation of gel formation after O-N acyl migration. The copolymers obtained from (C), in which the adopted conditions are labeled a-g, were subjected to addition of 10× PBS and incubated at 37 °C. Gel formation was evaluated by whether the stainless-steel ball was held on top of the tube after turning the tube upside down. The polymer of c’ was 7.5 mg/mL polymer with the same peptide composition as c. The arrowheads indicate the positions of the stainless-steel balls. Figure 5 shows morphological analysis of the MDCK-cell spheres in the gels. (A) Typical morphologies of the MDCK-cell spheres in the gels. The cross-sectional images were obtained by confocal fluorescence microscopy. F-actin and the nuclei were stained in red and blue, respectively. The scale bars indicate 100 μm. (B) Quantitative analysis of cyst formation in the gels. The cell spheres with diameter of less than 100 μm were classified as single-luminal, multi-luminal, or non-luminal spheres. The ellipticity (C) and cross-sectional area (D) of the spheres were measured. The significance of the differences was assessed by non-parametric one-way analysis of variance and the post-hoc Tukey test, and all of the P values <0.05 are shown in the figures. Figure 6 shows the kinetics of O-N acyl migration in the C2-ester polymer under each condition (pH 8.95, 7.40, 5.45, and 2.29). The percentage of acyl migration was calculated by the ratio of the amide form in the total peak area of the ester form and the amide form in HPLC analysis. Figure 7 shows the kinetics under the conditions of 0℃ and pH 7.40. Figure 8 shows gel formation by copolymerizing C3s-ester with C3s-amide. Figure 9 shows evaluation of gel formation. Figure 10 shows confirmation of gel formation. Figure 11 shows confirmation of gel formation.
BEST MODE FOR CARRYING OUT THE INVENTION
In one embodiment, the present invention discloses a peptide represented by formula (I),
Figure JPOXMLDOC01-appb-I000007
wherein m is 2 or 3, n represents an integer of 4 to 10,
each of Xaa and Yaa is independently selected from a proline residue, a hydroxyproline residue, an arginine residue, a lysine residue, a valine residue, a leucine residue, an isoleucine residue, a serine residue, a threonine residue, an alanine residue, a glycine residue, an N-isobutyl glycine residue, a phenylalanine residue, a methionine residue, a glutamate residue, an aspartate residue, an asparagine residue, a glutamine residue, a histidine residue, a tryptophan residue or a tyrosine residue, the proline residue may be modified by an amino group or fluorine atom,
or pharmaceutically acceptable salts thereof, or a solvate of thereof.
Preferably, n is 5, Xaa is proline, and Yaa is hydroxyproline.
In one embodiment, the present invention discloses a peptide represented by formula (II),
Figure JPOXMLDOC01-appb-I000008
wherein m is 2 or 3, n represents an integer of 4 to 10,
each of Xaa and Yaa is independently selected from a proline residue, a hydroxyproline residue, an arginine residue, a lysine residue, a valine residue, a leucine residue, an isoleucine residue, a serine residue, a threonine residue, an alanine residue, a glycine residue, an N-isobutyl glycine residue, a phenylalanine residue, a methionine residue, a glutamate residue, an aspartate residue, an asparagine residue, a glutamine residue, a histidine residue, a tryptophan residue or a tyrosine residue, the proline residue may be modified by an amino group or fluorine atom, or pharmaceutically acceptable salts thereof, or a solvate of thereof.
Preferably, n is 5, Xaa is proline, and Yaa is hydroxyproline.
In one embodiment, the present invention discloses a process for producing the peptide represented by the formula (II), comprising reacting the peptide of formula (I) under physiological pH, wherein m, n, Xaa and Yaa are the same as defined above.
Figure JPOXMLDOC01-appb-I000009
In one embodiment, the present invention discloses a peptide listed in Table 1,
Figure JPOXMLDOC01-appb-I000010
In one embodiment, the present invention discloses a peptide listed in Table 2,
Figure JPOXMLDOC01-appb-I000011
Another aspect of this invention is directed to a pharmaceutical composition comprising one or more kinds of the peptide represented by formula (I), formula (II), Table 1 or Table 2.
Another aspect of this invention is directed to a gelling agent comprising one or more kinds of the peptide represented by formula (I), formula (II), Table 1 or Table 2.
Another aspect of this invention is directed to a gel comprising one or more kinds of the peptide represented by formula (I), formula (II), Table 1 or Table 2.
Another aspect of this invention is directed to a regenerative medical material comprising one or more kinds of the peptide represented by formula (I), formula (II), Table 1 or Table 2.
Since a gel comprising the peptide of the present invention does not impart a specific bioactivity, a peptide thin membrane can, for example, be used as a medical material for adhesion prevention of organs and as a suture material.
If using the gel or peptide thin membrane containing the peptide of the present invention as a medical material for adhesion prevention of organs, such medical material may, for example, be placed between an organ and the dermis, or between an organ and another organ, in a surgery, thereby preventing an organ from adhering to the dermis and another organ. The medical material of the present invention thus placed may gradually become soluble and degraded, and then disappear, by peptidase and phagocytes such as macrophage that are present in vivo. Therefore, no further surgery is required to remove such medical material, thus imposing a smaller burden on the prognosis of a patient, and making it possible to improve Quality of life (QOL).
Further, when using the peptide of the present invention as a research base material having a desired selective activity, it can be used in, for example, researches for unraveling the mechanism of the intracellular information transmission system of the cells that are to bind to such base material.
Furthermore, if used as a medical material, the gel or peptide thin membrane comprising the peptide of the present invention can, for example, be placed on a region of wound, a surgical incision made in a surgery, the cornea and the retina. As a result of allowing receptors present on the cell membranes of the fibroblasts to incorporate the aforementioned binding motifs having binding activities, the gel or peptide thin membrane can, for example, be used as a medical material for promoting wound healing for enhancing the migration and binding of the fibroblasts to a region of wound or a surgical incision, and thus promoting the healing of a wound or surgical incision; or as a medical material for use in cornea and retina regeneration.
If produced in a water solvent, the abovementioned polymerized peptide is thus produced as a hydrogel of the polymerized peptide of the present invention and water i.e., the polymerized peptide of the present invention can be employed as a gelling agent.
Further, the hydrogel of the present invention can be used as a postsurgical adhesion prevention agent for organs, a hemostatic agent, an agent for promoting wound healing, and a regenerative medical material such as a material for cornea or retina regeneration.
In addition, by drying the above hydrogel, there can be prepared a polymerized peptide thin membrane as a sheet-shaped membrane. This polymerized peptide thin membrane can, for example, be rehydrated at the time of use so that it can be used as the above material for promoting wound healing, and a regenerative medical material such as an artificial cornea and a cell sheet for an artificial myocardial membrane capable of being transplanted into a body.
DETAILED DESCRIPTION OF THE INVENTION
Abbreviations used in examples are as follows.
Amino acid residues (all are L forms)
Arg (R): Arginine
Asp (D): Aspartic acid
Cys (C): Cysteine
Gln (Q): Glutamine
Gln (E): Glutamic acid
Gly (G): Glycine
His (H): Histidine
Hyp (O): 4-hydroxyproline
Lys (K): Lysine
Pro (P): Proline
Leu (L): Leucine
Met (M): Methionine
Nle（X）：Norleucine
Phe (F): Phenylalanine
Ser (S): Serine
Val (V): Valine
Protective Groups
Fmoc: 9-fluorenylmethoxycarbonyl
Boc: tert-butoxycarbonyl
Reagents
DIC: N, N′-diisopropylcarbodiimide
DMF: N,N-dimethylformamide
DMSO: Dimethyl sulfoxide
FBS: Fetal bovine serum
HOBt: 1-hydroxybenzotriazole
PBS: Phosphate buffered saline
TCEP: Tris(2-carboxyethyl)phosphine
TFA: Trifluoroacetic acid
Apparatus
RP-HPLC: Reversed-phase high performance liquid chromatography
MALDI-TOF MS: Matrix-assisted laser desorption/ionization time-of-flight mass spectrometer
Others
CD: Circular dichroism
DDR: Discoidin domain receptor
ECM: Extracellular matrix
PEDF: Pigment epithelium-derived factor
vWF: von Willebrand factor
The examples of the invention that are described below are simply provided as examples, and shall not limit the technical scope of the present invention. The technical scope of the present invention is only limited by the descriptions in the scope of claims. The present invention may be modified, for example, elements may be added to the present invention, and the elements of the invention may also be deleted or even substituted without departing from the gist of the present invention.
Conditions that are not specified in the examples will be the common conditions in the art or the recommended conditions of the raw materials by the product manufacturer. The reagents which are not indicated the origin will be the commercially available conventional reagents.
1.Materials and methods
1.1. Cell culture
The MDCK cells were purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA). The cells were cultured in D-MEM (Fujifilm Wako Pure Chemical Industries, Osaka, Japan) containing 10% (v/v) FBS (Thermo Fisher Scientific, Waltham, MA, USA), 100 units/mL penicillin, and 100μg/mL streptomycin (Sigma-Aldrich, St. Louis, MO, USA). The cells were maintained at 37 °C in a humidified 5% CO2/air atmosphere.

1.2. Peptide synthesis

The peptides were manually synthesized according to the standard Fmoc-based solid-phase method on 2-chlorotrityl chloride resin (Peptide Institute, Osaka, Japan). The coupling reaction was performed at room temperature for 2 h with 3-5 equivalents of Fmoc-amino acid, DIC, and HOBt in N,N-dimethylformamide. The Fmoc group was deprotected using 20% (v/v) or 30% (v/v) piperidine/DMF for 15 min. Fmoc-Pro-Hyp-Gly-OH (Watanabe Chemical Industries, Hiroshima, Japan) and Boc-Ser(Fmoc-Gly)-OH (Iris Biotech, Bayern, Germany) were used for incorporation of Pro-Hyp-Gly repeats and the glycylserine O-acyl isopeptide unit, respectively. The Fmoc group of the amino acid residue next to the glycylserine O-acyl isopeptide unit was deprotected using 25% (v/v) 1-methylpyrrolidine, 2% (v/v) hexamethyleneimine and 3% (w/v) HOBt in N-methylpyrrolidone:DMSO (1:1) [35]. The peptides were deprotected and cleaved from the resin by TFA/m-cresol/thioanisole/ethanedithiol/H2O (80:5:5:5:5, v/v) at room temperature for 4 h. The crude peptides were purified by RP-HPLC on a Cosmosil 5C18-AR II column (Nacalai Tesque, Kyoto, Japan) and identified by matrix-assisted laser desorption-ionization time-of-flight mass spectrometry (Autoflex III, Bruker, MA, USA) or electrospray ionization mass spectrometry (AB Sciex, MA, USA).

1.3. CD analysis

The CD spectra were recorded with a J-820 CD spectropolarimeter (JASCO, Tokyo, Japan) equipped with a Peltier thermal controller. The intensity of the spectra was converted into the mean residue weight ellipticity ([θ]MRW). The peptides were dissolved in degassed 0.05% (v/v) TFA(aq) containing 10 mM TCEP, followed by annealed at 4 °C overnight after heating at 95 °C for 5 min. The thermal denaturation curve of the triple helix was obtained by monitoring [θ]MRW at 225 nm ([θ]MRW,225) with increasing temperature from 4 to 95 °C at a rate of 18 °C h-1.

1.4. Preparation of the peptide polymers and evaluation of gel formation
The peptides were dissolved in degassed 0.05% (v/v) TFA(aq) and annealed, as described above. DMSO was added to the peptide solution to a final concentration of 10% (v/v), and the solution was stood at 28 °C for 4 days for polymerization. To induce O-N acyl migration of O-acyl isopeptide in the polymer, 10× PBS [50 mM phosphate buffer (pH 7.4), 100 mM NaCl] was added to the polymer solution, followed by incubation at 37 °C for 5-60 min and dilution with 0.05% (v/v) TFA(aq). TCEP was then added to the solution at the final concentration of 50 mM, and the solution was heated at 95 °C for 3 min, followed by RP-HPLC analysis.

Gel formation was evaluated by gently placing a stainless-steel ball (SUS440C, diameter = 1.5 mm; Funabe Seiko, Hyogo, Japan) on the interfaces (Figure 4B and C). O-N acyl migration-induced gel formation was similarly assessed by the position of the ball after turning the tube with the ball at the bottom upside down after incubation at 37 °C (Figure 4D).

1.5. 3D cell culture
The MDCK cells were detached with 0.05% (w/v) trypsin/0.53mM ethylenediaminetetraacetic acid (Fujifilm Wako Pure Chemical Industries) and harvested by centrifugation (120 ×g, 4 min). The peptide polymer solution (7.5 mg/mL), 10× D-MEM (prepared by dissolving powder mix and NaHCO3, Sigma-Aldrich), and the MDCK cell suspension were mixed at a ratio of 17:2:1, and 30 μL of the mixture was added to a circular silicone mold (diameter = 4 mm, height = 1 mm) set on a glass-based 35-mm dish (Iwaki, Tokyo, Japan) and incubated at 37 °C for gel formation, followed by addition of 2 mL of D-MEM containing 10% (v/v) FBS. To prepare the type I collagen gel, 3 mg/mL type I collagen acidic solution (I-AC, Koken, Tokyo, Japan), DMSO, 10× D-MEM, and MDCK cell suspension were mixed at a ratio of 15:2:2:1 and incubated at 37 °C. After culturing at 37 °C for 7 days, the cells were fixed with 4% p-formaldehyde/phosphate buffer (Fujifilm Wako Pure Chemical Industries) overnight, permeabilized with 0.1% (v/v) Triton X-100/PBS for 15 min, and blocked with 1% (w/v) skim milk/PBS for 1 h. The nuclei and F-actin were stained by Hoechst 33342 and phalloidin-iFluor conjugate (Cayman Chemical, MI, USA) for 3 h. The cells were observed by confocal fluorescence microscopy (FV-1000, Olympus, Tokyo, Japan) after washing with PBS.

For quantitative analysis of cyst formation, the spheres were classified as single-luminal, multi-luminal, or no-luminal spheres using cross-sectional images. The area and ellipticity of the cell spheres were measured by ImageJ software [36]. The cross-sectional area was defined as the largest area among the photographs taken of each sphere every 3 μm in the horizontal direction. The ellipticity was calculated by ellipticity = 1 - (shortest diameter/longest diameter) for the photograph with the largest cross-sectional area. All of analyses were performed blind by a third person.

The known raw materials of the present invention can be prepared by the conventional synthesis methods in the art, or can be purchased. Peptides 1 to 6 and A196 were synthesized by Life Chemicals Inc. (Ontario, Canada) and purchased from Namiki Shoji Co. Ltd. (Tokyo, Japan).
Design of a collagen-like peptide polymer that forms a gel in situ through a sol-gel transition
A strategy to form a gel from a collagen-like peptide polymer by triple helix formation induced by pH-triggered O-N acyl migration at physiological pH has been devised, as shown in Figure 1. This peptide was composed of collagen-like Xaa-Yaa-Gly repeats with two Cys residues at each terminus and a glycylserine O-acyl isopeptide in the center of the sequence. The peptides were cross-linked via disulfide bonds under acidic conditions. The sol-gel transition was expected to be induced by triple helix formation triggered by O-N acyl migration at physiological pH.
Synthesis and structural analysis of the collagen-like peptide containing an O-acyl isopeptide unit
A peptide consisting of twelve repeats of Xaa-Yaa-Gly with two Cys residues at each terminus and a glycylserine O-acyl isopeptide unit at the middle of the sequence (C2-ester, Table 1) has been designed. The peptides were constructed by 9-fluorenylmethoxycarbonyl (Fmoc)-based solid-phase synthesis using N,N’-diisopropylcarbodiimide (DIC) and 1-hydroxybenzotriazole (HOBt) as coupling agents. To incorporate the O-acyl isopeptide unit and Pro-Hyp-Gly repeats, Boc-Ser(Fmoc-Gly)-OH and Fmoc-Pro-Hyp-Gly-OH were used as building blocks, respectively. The peptides were cleaved from the resin and deprotected by a trifluoroacetic acid (TFA)-based cocktail, and they were purified by reverse-phase high-performance liquid chromatography (RP-HPLC). The amide-form peptide corresponding to the expected product of O-N acyl migration of C2-ester was also independently prepared by the same procedure (C2-amide, Table 1).
Figure JPOXMLDOC01-appb-I000012
Circular dichroism (CD) analysis was performed to confirm that the O-acyl isopeptide unit disturbed triple helix formation, but the amide-form peptide can form the triple helix. C2-ester and C2-amide were dissolved in 0.05% (v/v) TFA(aq) and then annealed at 4°C overnight after heating at 95°C. The CD spectra at 4°C were measured (Figure 2A). Both C2-ester and C2-amide showed a maximum signal at around 225 nm and a minimum signal at around 200 nm, indicating the polyproline-II-like secondary structure. The signals of C2-ester were smaller than those of C2-amide, indicating that C2-amide had higher helicity. In addition, the signal at 225 nm was monitored with increasing temperature (Figure 2B). The signal cooperatively decreased at around 30°C for C2-ester and at around 70°C for C2-amide, and C2-ester showed a more gradual decreasing signal. The cooperative decrease of the signal at 225 nm is a typical property of the collagen-like triple helix, strongly suggesting that both C2-ester and C2-amide formed the triple helix [26].

The results also suggested that although C2-ester contained triple-helical population, the triple helix was disturbed and destabilized by the O-acyl isopeptide unit interrupting the continuous Xaa-Yaa-Gly sequence. In addition, the signal at 225 nm more gradually decreased with increasing temperature for C2-ester than for C2-amide, suggesting that the triple-helical population of C2-ester was more heterogenous.
Investigation of gel formation of the C2-ester polymer induced by O-N acyl migration

First, in order to investigate whether C2-ester was in the sol state after oxidative polymerization and whether the polymer sol can be transformed to a gel by increasing the triple helicity by O-N acyl migration at physiological pH, C2-ester was polymerized in a solution containing 10% (v/v) DMSO and 0.05% (v/v) TFA at 28°C for 3 days at a peptide concentration of 11.1 mg/mL after annealing. The polymer was in the sol state after polymerization. Although the polymer was subjected to addition of 10×phosphate-buffered saline (PBS) at pH 7.4 and incubated at 37°C, the polymer, whose final concentration was 10.0 mg/mL, did not form a gel even after 1 h (Figure 3A). Conversely, individually prepared C2-amide at the same concentration formed a gel by polymerization.

To confirm O-N acyl migration in the polymer, the C2-ester polymer was subjected to addition of 10×PBS, incubated at 37°C for 1 h, treated with tris(2-carboxyethyl)phosphine (TCEP) to reduce disulfide bonds, and analyzed by HPLC (Figure 3B). The peak of C2-ester eluted at around 21 min decreased and the peak of the corresponding amide-form peptide eluted at around 22 min increased by PBS treatment. The minor peaks at around 12 and 14 min were N- and C-terminus fragments, which were hydrolyzed products of C2-ester, respectively. However, there was little change in the amounts before and after the reaction, suggesting that they were not involved in O-N acyl migration.

It was revealed that C2-ester could not form a gel by itself even after the ester was converted to the amide. Because the C2-amide polymer formed a gel at the same concentration, prior polymerization of C2-ester is suggested to prevent sufficient triple helix formation to form a gel.

The kinetics under each condition (pH 8.95, 7.40, 5.45, and 2.29）(Fig.6)
The ratio of acyl migration of C2-ester polymer to amide-form and ratio of hydrolyzed products were investigated at each pH. C2-ester polymer was added to 0.5 M borate buffer (pH 8.95), 1 M HEPES (pH 7.40), and 1 M acetate buffer (pH 5.45), respectively, and left at 37℃ for 10, 30, and 180 minutes. Then the samples reduced by TCEP were analyzed by HPLC. From the peak areas of the ester-form raw material, amide-form, and hydrolyzed products, the respective ratios at each time point were determined. As a result, it was observed that the acyl migration proceeded faster as the pH value increased. At pH 8.95, 77% of the amide-form was formed after 10 minutes, 87% of the amide-form was formed after 30 minutes, and 92% of the amide-form was formed after 180 minutes. At pH 7.40, 50% of the amide was formed after 10 minutes, 68% of the amide-form was formed after 30 minutes, and 87% of the amide-form was formed after 180 minutes. At pH 5.45, 44% of the amide-form was formed after 10 minutes, 58% of the amide-form was formed after 30 minutes, and 77% of the amide-form was formed after 180 minutes. On the other hand, when observation was carried out without the addition of buffer (pH 2.29), less than 1% of the amide-form was formed even after 180 minutes. Besides, the ratio of hydrolyzed products was 4% or less at any pH.

The kinetics at pH 7.40 at 0℃(Fig.7)
Furthermore, formation of the amide-form under the conditions of 1 M HEPES (pH 7.40) and 0℃ was verified. As a result, it changed to that 11% of the amide-form was formed after 10 minutes, and 44% of the amide-form was formed after 180 minutes. The migration rate was slower than that at 37℃. Besides, the ratio of hydrolyzed products was 1% or less.
Investigation of the gel-forming conditions of the copolymers of C2-ester and triple-helical peptides

It is considered that the polymer composed solely of C2-ester cannot form sufficient triple helices for gel formation even after O-N acyl migration. Therefore, for the purpose to increase the triple-helical content in the polymer by copolymerizing other triple-helical peptides with C2-ester (Figure 4A), two peptides composed of twelve or fourteen repeats of Pro-Hyp-Gly, two Cys residues at each terminus, and an Arg residue in the middle of the sequence were prepared (C3-amide and C3l-GPO, Table 1). The Arg residue was introduced to prevent possible lateral aggregation of the Pro-Hyp-Gly repeating peptide [27].
First, to determine the conditions under which the polymer forms a gel after O-N acyl migration, the peptide composition and concentration were investigated using the amide-form peptides (C2-amide, C3-amide, and C3l-GPO). The amount of C3l-GPO was fixed at 10% of the total peptide. Copolymers with different ratios of C2-amide and C3-amide were prepared, and the concentration at which the polymer forms a gel was investigated (Figure 4B). It was found that the minimum gel-forming concentrations of the 5:4:1, 6:3:1, 7:2:1, 8:1:1, and 9:0:1 C2-amide:C3-amide:C3l-GPO copolymers were 5, 6, 7, 8, and 9 mg/mL, respectively.

Next, to investigate the compositions and concentrations at which the polymers were in the sol state before O-N acyl migration, copolymers of C2-ester, C3-amide, and C3l-GPO were prepared, and their gel formation was evaluated (Figure 4C). The 5:4:1, 6:3:1, and 7:2:1 C2-ester:C3-amide:C3l-GPO copolymers were in the sol state at concentrations of less than 7, 8, and 10 mg/mL, respectively, while the 8:1:1 and 9:0:1 C2-ester:C3-amide:C3l-GPO copolymers were in the sol state even at 10 mg/mL.

The copolymers in the sol state at relatively high concentrations (labeled a-g in Figure 4C) were examined for O-N acyl migration-induced gel formation at pH 7.4. The results of gel formation evaluation after addition of 10×PBS to each polymer are shown in Figure 4D. The sols transformed to gels within 30 min for the 5:4:1 (b) and 7:2:1 (e) C2-ester:C3-amide:C3l-GPO copolymers at 6 and 9 mg/mL, respectively.

To determine the conditions under which the copolymers rapidly form gels after the pH shift, we also investigated the concentration of the 6:3:1 C2-ester:C3-amide:C3l-GPO copolymer. At a concentration of 7.5 mg/mL, the copolymer was in the sol state after polymerization and formed a gel within 10 min after addition of 10×PBS (labeled c’ in Figure 4D). This revealed a collagen-like peptide polymer that formed a gel within 10 min through a sol-gel transition triggered by O-N acyl migration at pH 7.4.

Gel formation by copolymerizing C3s-ester with C3s-amide (Fig.8)
Figure JPOXMLDOC01-appb-I000013
The composition and concentration of peptides were investigated using a copolymer of C3s-ester and C3s-amide (Table 2) in order to verify the conditions for gel formation by O-N acyl migration. First, the conditions under which the sol state could be maintained even after polymerization were investigated. As a result, the highest concentrations at which the sol state could be maintained were 7 mg/ml for 4:6 and 8 mg/ml for 5:5. For 6:4 and 7:3, the sol state was maintained even at the highest concentration of 10 mg/ml observed.
After that, it was investigated whether the gel would form when the pH of the polymers (a-f) in the sol state was changed to 7.4. As a result, although the gel formed in (d) within 30 minutes, no gel formation was confirmed even after 60 minutes under other conditions. Furthermore, when the same investigation was carried out under the conditions (b') where the ratio of C3s-ester and C3s-amide was 4:6 and the peptide concentration was 7.5 mg/ml, gel formation was observed within 10 minutes.
Application as a 3D scaffold for cell culture
In order to investigate whether the collagen-like peptide polymer optimized above could be applied to 3D cell culture and whether it could regulate specific behaviors of cells by the action of collagen-derived bioactive sequences incorporated into the polymer, the Madin-Darby canine kidney (MDCK) cell line, a canine renal tubular epithelial cell line, as a typical model for formation of 3D structures in 3D matrices were used. MDCK cells are known to form cysts when cultured in type I collagen gels [28,29]. In order to investigate whether MDCK cells also formed cysts in the collagen-like peptide polymer gels containing certain collagen-derived bioactive sequences, peptides containing each of the three bioactive sequences were prepared (Table 1). The norleucine residue (Nle/X) introduced into C3l-GVXGFO was used as an oxidation-resistant Met analog. This sequence has been reported to function as a ligand for DDR [30]. The copolymers composed of C2-ester, C3-amide, and either one of C3l-GPO, C3l-GFOGER, C3l-GVXGFO, or C3l-KGHRGF (called the GPO polymer, GFOGER polymer, GVXGFO polymer, and KGHRGF polymer, respectively) at a ratio of 6:3:1 and a concentration of 7.5 mg/mL were prepared by the same procedure as above. These copolymers were all in the sol state after polymerization.

To embed MDCK cells in the polymer gel, the polymer solution after oxidation was first neutralized to weakly basic pH by adding 10× Dulbecco’s modified Eagle’s medium (D-MEM), and the dispersed MDCK cells after trypsinization were mixed at a volume ratio of 17:2:1. The final concentration of the polymer was 6.4 mg/mL. The mixture was then immediately transferred to molds place in cell-culture dishes and incubated at 37 °C for gel formation. As a control, type I collagen acidic solution was mixed with 10× D-MEM and MDCK cells, and it formed a gel on a cell-culture dish according to the same procedure. The final collagen concentration of the gel was 2.3 mg/mL. After 30 min, D-MEM containing 10% (v/v) fetal bovine serum (FBS) was added onto the gel, and the cells were cultured at 37 °C. After 1 week, the cells were fixed, and the nuclei and F-actin were observed by fluorescent staining. There was little difference in cell proliferation among all of the gels. It was confirmed that the cells of the collagen-like peptide polymer gels were compatible with the 3D cell-culture system.

The cell spheres smaller than 100 μm in diameter were morphologically classified into three categories: cysts with a single lumen, cysts with more than one lumen, and aggregates without a lumen. Typical cross-sectional images of the cell aggregates in this experiment are shown in Figure 5A. The luminal spheres showed the localization of F-actin along the lumen, indicating formation of apical surfaces inside the sphere. The presence or absence of lumens and the number of lumens of the type I collagen gel and collagen-like peptide polymer gels were compared (Figures 5B). In terms of lumen formation, the type I collagen, GPO polymer, and GFOGER polymer gels were comparable, and fewer spheres formed lumens in the GVXGFO polymer and KGHRGF polymer gels. From comparison of the number of lumens, most of the luminal spheres were single-luminal cysts in the type I collagen gel. Conversely, the ratios of single-luminal cysts and multi-luminal cysts were similar in the GPO polymer gel without any specific bioactive sequences. In the GFOGER polymer gel, the ratio of the single-luminal cyst was higher than that in the GPO polymer gel, and the cyst-forming activity was the highest among the collagen-like peptide polymer gels.

The ellipticity and area of the cross-sections of the spheres were measured (Figure 5C and D). The cross-sections of the spheres in the type I collagen gel were close to perfect circles, whereas those of the spheres formed in all of the collagen-like peptide polymer gels were distorted and their ellipticity was significantly higher. The ellipticity seemed to be independent of the bioactive sequences incorporated into the polymers. Overall, the cross-sectional area of the sphere was not inversely correlated with the single-luminal cyst-forming activity. The cross-sectional areas of the spheres in the type I collagen gel were significantly smaller than those in the collagen-like peptide polymer gels, except for the GFOGER polymer gel. Comparing the introduced bioactive sequences, the cross-sectional areas of the spheres in the GVXGFO polymer gel were significantly larger than those in the other collagen-like peptide polymer gels, and those of the spheres in the KGHRGF polymer gel were significantly larger than those in the GFOGER polymer gel.

Overall, these results indicated that the integrin α2β1-binding sequence promotes cyst formation, including polarization of MDCK cells. The contribution of integrin α2β1 to cyst formation is supported by previous studies that showed that cyst formation of MDCK cells in type I collagen gels is inhibited by integrin α2β1 knockdown and specific inhibitory antibodies [31,32]. In addition, previous studies using a 3D culture system of synthetic matrices conjugated with the Arg-Gly-Asp peptide, a ligand for integrin αVβ3, have also shown that activation of integrin promotes MDCK cell cyst formation [33,34].

In contrast to the GFOGER sequence, cyst formation in MDCK cells is suppressed by the Arg-Gly-Gln-Hyp-Gly-Val-Nle-Gly-Phe-Hyp (RGQOGVXGFO) sequence incorporated into C3l-GVXGFO and Lys-Gly-His-Arg-Gly-Phe-Ser-Gly-Leu (KGHRGFSGL) sequences incorporated into C3l-KGHRGF. These sequences bind to cell surface receptors and soluble proteins: DDR [7], VWF [8], and SPARC [9,10] for RGQOGVXGFO, and heparan sulfate of syndecans [11] and PEDF [12,13] for KGHRGFSGL. The mechanisms by which these collagen-binding proteins regulate the polarity and cyst formation need to be further investigated in the future.

In the present invention, three bioactive sequences derived from natural collagen were individually incorporated into artificial collagen-like polymers, but it is also possible to incorporate several bioactive sequences in combinations. The cooperative or competitive effects of multiple collagen receptors could be investigated by taking advantage of the 3D cell-culture system.

Gel formation when a copolymer of C3s-ester and C3s-amide in the sol state was injected into PBS

Peptide polymers were prepared using the method described in Int. J. Mol. Sci., 2022, 23(3), 1584 (Synthetic Collagen-like Polymer That Undergoes a Sol-Gel Transition Triggered by O-N Acyl Migration at Physiological pH, Ichise SF. and Koide T). A polymer of C3s-ester and C3s-amide at a ratio of 4:6 was mixed with 1/500 equivalent of carboxy fluorescein to total peptide and injected into PBS using a syringe. After standing at 26°C for 30 minutes, the tube was turned upside down and fluorescence was observed with a UV illuminator. It was determined as gel formation when fluorescence was observed at the bottom of the tube.

As a result, no gel was formed at 7.0 mg/ml or less, whereas gel formation was confirmed at 7.3 mg/ml or more (Fig. 9).

Gel formation of the polymer of C3s-ester and C3s-amide at a ratio of 4:6 and with peptide concentration of 7.3 mg/ml was investigated over time at 24°C and 37°C. As a result, gel formation was observed within 20 minutes at each temperature (Fig. 10).
A polymer of C3s-ester and C3s-amide at a ratio of 4:6 and with peptide concentration of 7.3 mg/ml was neutralized with a buffer solution, and then injected into PBS to investigate the gel formation. One-fifteenth volume of a buffer solution (160 mM phosphate buffer (pH 8.0), 2200 mM NaCl, and 42 mM KCl) was added (the final concentration was the same salt concentration as PBS) to the polymer containing fluorescein. After certain time of preincubation at 0°C, the polymer was sucked up with a syringe and injected into the PBS in the tube. After incubating for 20 minutes at 37°C, the tube was turned upside down. When fluorescence was observed at the bottom of the tube, it was determined as gel formation (Fig. 11). The pH when the polymer and buffer were mixed was 6.8 to 7.1. The polymer formed a gel, and it could not be sucked up with a syringe at 20 minutes after neutralization.
The present invention provides a novel peptide, a pharmaceutical composition comprising the peptide, and a gelling agent comprising the peptide, a gel comprising the peptide, and a regenerative medical material comprising the peptide.
SEQUENCE LISTING

C2-ester (SEQUENCE 1)
Cys Cys Pro Xaa Gly Pro Xaa Gly Pro Xaa Gly Pro Xaa Gly Pro Xaa Gly Pro Xaa Xaa Xaa Xaa Gly Pro Xaa Gly Pro Xaa Gly Pro Xaa Gly Pro Xaa Gly Pro Xaa Gly Cys Cys

C2-amide (SEQUENCE 2)
Cys Cys Pro Xaa Gly Pro Xaa Gly Pro Xaa Gly Pro Xaa Gly Pro Xaa Gly Pro Xaa Gly
Ser Xaa Gly Pro Xaa Gly Pro Xaa Gly Pro Xaa Gly Pro Xaa Gly Pro Xaa Gly Cys Cys

C3-amide (SEQUENCE 3)
Cys Cys Cys Pro Xaa Gly Pro Xaa Gly Pro Xaa Gly Pro Xaa Gly Pro Xaa Gly Pro Xaa
Gly Pro Arg Gly Pro Xaa Gly Pro Xaa Gly Pro Xaa Gly Pro Xaa Gly Pro Xaa Gly Cys
Cys Cys

C3l-GPO (SEQUENCE 4)
Cys Cys Cys Pro Xaa Gly Pro Xaa Gly Pro Xaa Gly Pro Xaa Gly Pro Xaa Gly Pro Pro
Gly Pro Pro Gly Pro Arg Gly Pro Pro Gly Pro Xaa Gly Pro Xaa Gly Pro Xaa Gly Pro
Xaa Gly Pro Xaa Gly Cys Cys Cys

C3l-GFOGER (SEQUENCE 5)
Cys Cys Cys Pro Xaa Gly Pro Xaa Gly Pro Xaa Gly Pro Xaa Gly Pro Xaa Gly Pro Pro
Gly Phe Xaa Gly Glu Arg Gly Pro Pro Gly Pro Xaa Gly Pro Xaa Gly Pro Xaa Gly Pro
Xaa Gly Pro Xaa Gly Cys Cys Cys

C3l-GVXGFO (SEQUENCE 6)
Cys Cys Cys Pro Xaa Gly Pro Xaa Gly Pro Xaa Gly Pro Xaa Gly Pro Xaa Gly Pro Arg
Gly Gln Xaa Gly Val Xaa Gly Phe Xaa Gly Pro Xaa Gly Pro Xaa Gly Pro Xaa Gly Pro
Xaa Gly Pro Xaa Gly Cys Cys Cys

C3l-KGHRGF (SEQUENCE 7)
Cys Cys Cys Pro Xaa Gly Pro Xaa Gly Pro Xaa Gly Pro Xaa Gly Pro Xaa Gly Pro Lys
Gly His Arg Gly Phe Ser Gly Leu Xaa Gly Pro Xaa Gly Pro Xaa Gly Pro Xaa Gly Pro
Xaa Gly Pro Xaa Gly Cys Cys Cys

C3s-ester (SEQUENCE 8)
Cys Cys Cys Pro Xaa Gly Pro Xaa Gly Pro Xaa Gly Pro Xaa Gly Pro Xaa Gly Pro Xaa Xaa Xaa Xaa Gly Pro Xaa Gly Pro Xaa Gly Pro Xaa Gly Pro Xaa Gly Pro Xaa Gly Cys Cys Cys

C3s-amide (SEQUENCE 9)
Cys Cys Cys Pro Xaa Gly Pro Xaa Gly Pro Xaa Gly Pro Xaa Gly Pro Xaa Gly Pro Xaa
Gly Pro Arg Gly Pro Xaa Gly Pro Xaa Gly Pro Xaa Gly Pro Xaa Gly Pro Xaa Gly Cys
Cys Cys

Claims (9)

  1. A peptide represented by formula (I),
    Figure JPOXMLDOC01-appb-I000001
    wherein m is 2or 3, n represents an integer of 4 to 10,
    each of Xaa and Yaa is independently selected from a proline residue, a hydroxyproline residue, an arginine residue, a lysine residue, a valine residue, a leucine residue, an isoleucine residue, a serine residue, a threonine residue, an alanine residue, a glycine residue, an N-isobutyl glycine residue, a phenylalanine residue, a methionine residue, a glutamate residue, an aspartate residue, an asparagine residue, a glutamine residue, a histidine residue, a tryptophan residue or a tyrosine residue, the proline residue may be modified by an amino group or fluorine atom,
    or pharmaceutically acceptable salts thereof, or a solvate of thereof.
  2. The peptide according to claim 1, wherein, Xaa is proline, Yaa is hydroxyproline, n is 5.
  3. A peptide represented by formula (II),
    Figure JPOXMLDOC01-appb-I000002
    wherein m is 2 or 3, n represents an integer of 4 to 10,
    each of Xaa and Yaa is independently selected from a proline residue, a hydroxyproline residue, an arginine residue, a lysine residue, a valine residue, a leucine residue, an isoleucine residue, a serine residue, a threonine residue, an alanine residue, a glycine residue, an N-isobutyl glycine residue, a phenylalanine residue, a methionine residue, a glutamate residue, an aspartate residue, an asparagine residue, a glutamine residue, a histidine residue, a tryptophan residue or a tyrosine residue, the proline residue may be modified by an amino group or fluorine atom,
    or pharmaceutically acceptable salts thereof, or a solvate of thereof.
  4. The peptide according to claim 3, wherein Xaa is proline, Yaa is hydroxyproline, n is 5.
  5. A process for producing the peptide represented by the formula (II), comprising reacting the peptide of formula (I) under physiological pH, wherein m, n, Xaa and Yaa are the same as defined in claim 1 and 3.
    Figure JPOXMLDOC01-appb-I000003
  6. A pharmaceutical composition comprising the peptides according to each one of the claims 1 to 4.
  7. A gelling agent comprising the peptides according to each one of the claims 1 to 4.
  8. A gel comprising the peptides according to each one of the claims 1 to 4.
  9. A regenerative medical material comprising the peptides according to each one of the claims 1 to 4.
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WO2022050176A1 (en) * 2020-09-02 2022-03-10 コラジェン・ファーマ株式会社 Composition comprising peptide, and use thereof

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