JP5747493B2 - Platelet release promoter and method for promoting platelet release - Google Patents

Description

  The present invention relates to a platelet release promoter useful for preparing a platelet release factor used for the purpose of wound healing and the like, and a method for promoting platelet release.

  Platelets include platelet-derived growth factor (PDGF), transforming growth factor-β (TGF-β), epidermal growth factor (EGF), vascular endothelial growth factor (VEGF), platelet factor 4, β-thromboglobulin. For example, a factor having biological activity is included. These factors are released into the plasma in a platelet release reaction that follows platelet aggregation, such as during vascular injury. These factors (hereinafter also referred to as platelet-releasing factors) promote wound healing by attracting macrophages and fibroblasts to the injured part and promoting synthesis of extracellular matrix. In the medical field, attempts have been made to apply the effects of these platelet-releasing factors to wound treatment. For example, in cases where blood circulation in the wound area is insufficient, the platelet-releasing factor is concentrated externally to the wound area. The therapeutic effect is obtained by supplying.

  As factors that activate platelets, thrombin, collagen, thromboxane A2, ADP, adrenaline and the like are known. Among these, thrombin is most commonly used as a platelet activator used in the preparation of platelet gels and concentrated platelet wound healing agents in vitro, and as a base material for hemostatic and wound dressings, it is biocompatible. Many excellent collagens are used.

  Collagen is a representative extracellular matrix most abundant in the human body, and platelets start to aggregate when blood is exposed to collagen when blood vessels are damaged. The adhesion of platelets to collagen follows the indirect adhesion via von Willebrand factor in blood, followed by collagen receptors present on the platelet surface in the -Arg-Gly-Asp-sequence (RGD sequence) of collagen. This occurs when integrin α2β1 (GPIA / IIa) is directly bound, or glycoprotein VI (GPVI), which is a collagen receptor present on the platelet surface, binds to the triple helical structure of collagen. It is clear that the triple helix structure of collagen plays an important role in the interaction between collagen and platelets, and that denatured collagen (gelatin), which has collapsed the triple helix structure by heating collagen, does not cause platelet activation. It has become.

By the way, it is known that a continuous motif of XY-Gly tripeptide contributes to the triple helical structure of collagen, and X is often Pro and Y is often Pro or Hyp. Hyp is usually a 4Hyp (for example, trans-4-hydroxy-L-proline) residue. Non-patent Documents 1 and 2, a polypeptide having a triple helical structure - (Gly-Pro-Hyp) 8 ~ 10 - crosslinked material is disclosed, further, that these substances have a platelet aggregation-promoting activity Is disclosed.

  Furthermore, in Patent Document 1 and Non-Patent Document 3, a high molecular weight polymer of a polypeptide having a triple helical structure (Pro-Hyp-Gly)-has platelet aggregation promoting activity without requiring crosslinking. It is disclosed.

However, when trying to release an effective factor for wound healing such as PDGF from platelets by platelet release reaction for the purpose of wound healing, natural collagen is derived from animals and the risk of infectious diseases has not been eliminated. In addition,-(Gly-Pro-Hyp) ₁₀ -described above lacks heat stability, and in order to maintain high activity, it needs to be chemically crosslinked to form a quaternary structure. We couldn't wipe out the safety concerns caused by the residue. Furthermore, the polymer of-(Gly-Pro-Hyp)-has high thermostable platelet aggregation-promoting activity when polymerized, but lacks the RGD sequence that is the binding site of platelet GPIa / IIa. No platelet release activity has been confirmed so far.

WO2008 / 077559

The JOURNAL OF BIOLOGICAL CHEMISTRY 1994; 269 (19): 13899-13903 The Biochemical Journal 1995; 306; 337-344. FEBS Letter 2009; 583; 81-87

  It is an object of the present invention to provide a platelet release promoter that has high safety and thermal stability, can be used at low cost, and exhibits a high platelet release promoting effect.

  As a result of intensive studies to solve the above-mentioned problems, the present inventors have found that a polymer peptide containing-(Pro-Hyp-Gly)-or-(Pro-Pro-Gly)-as a constituent unit promotes platelet release. It has been found that the polymer peptide has activity and that the polymer peptide exhibits platelet release promoting activity even at a low concentration as compared with natural collagen, and the present invention has been completed.

That is, the following [1] to [7] can be exemplified as the present invention.
[1] A platelet release accelerator containing a polymer having a structural unit represented by the following formula (1) and having an average molecular weight of 500,000 to 10,000,000.
-(Pro-X-Gly)-(1)
(In the formula, X represents Pro or Hyp.)
[2] When the molecular weight of the polymer is measured by gel permeation chromatography, 80% or more of the total peak area is included in the molecular weight range of 20,000 to 20 million. Platelet release promoter.
[3] The platelet release promoter according to [1] or [2], wherein the polymer has a triple helical structure.
[4] The platelet release promoter according to any one of [1] to [3], wherein the polymer exhibits platelet release promoting activity at a concentration of 0.05 μg / mL.
[5] The platelet release promoter according to any one of [1] to [4], wherein the polymer is insolubilized by thermal crosslinking.
[6] A method for promoting platelet release, comprising a step of exposing the platelet release promoter according to any one of [1] to [5] to platelets in vitro.
[7] A wound healing material that retains the platelet release promoter according to any one of [1] to [5].

  According to the present invention, it is possible to provide a platelet release promoter that exhibits a high platelet release promoting effect. In addition, the present invention can effectively promote the platelet release reaction as compared with natural collagen.

The figure which shows PDGF release promotion activity (Example 1) of a platelet release promotion peptide. The figure which shows PDGF release promotion activity (comparative example 1) of commercially available porcine type I collagen. The figure which shows the PDGF release promotion activity (Example 2) of the platelet release promotion peptide heat-crosslinked. The figure which shows PDGF release acceleration | stimulation activity (comparative example 2) of the commercially available bovine dermal collagen cross-linked.

  Hereinafter, preferred embodiments of the present invention will be described, and the present invention will be described in more detail.

<Platelet release promoter of the present invention>
The platelet release promoting agent of the present invention contains a polymer containing a structural unit represented by the following formula (1) (also referred to as “platelet release promoting peptide of the present invention” or “platelet release promoting peptide”). -(Pro-X-Gly)-(1)
(In the formula, X represents Pro or Hyp.)
In the platelet release-promoting peptide, all Xs may be Pro, all Xs may be Hyp, and Pro and Hyp may be mixed as X in any ratio. Regarding X, the ratio of Pro to Hyp is not particularly limited, but Hyp: Pro (molar ratio) = 100: 0 to 5:95 is preferable in terms of thermal stability of the triple helix structure, and 100: 0 to 50: 50 is more preferable, and 100: 0 to 90:10 is more preferable. Hyp is usually a 4Hyp (for example, trans-4-hydroxy-L-proline) residue.

  Regarding the molecular weight of the platelet release-promoting peptide, the weight average molecular weight measured by gel permeation chromatography is preferably 500,000 or more, more preferably 1,000,000 or more, from the viewpoint of the thermal stability of the triple helix. Preferably, it is 2 million or more, more preferably 3 million or more. The weight average molecular weight is preferably 10 million or less, and more preferably 8 million or less. Further, when the molecular weight of the platelet release promoting peptide is measured by gel permeation chromatography, the total peak area is preferably 80% or more, more preferably 90% or more, still more preferably 95% or more, particularly preferably. 98% or more is included in the molecular weight range of 20,000 to 20 million. Further, when the molecular weight of the platelet release promoting peptide is measured by gel permeation chromatography, the detected peak may be single or plural. In addition, the molecular weight of the platelet release promoting peptide measured by gel permeation chromatography is assumed to be a pullulan equivalent molecular weight.

  In addition, the platelet release-promoting peptide may contain other amino acid residues or peptide residues as long as it does not interfere with the thermal stability of the triple helix. The content of other amino acid residues and peptide residues is, for example, 20% or less, preferably 10% or less, more preferably 5% or less, even more preferably, as the number of amino acid residues, relative to the whole platelet release promoting peptide. Is 2% or less, particularly preferably 1% or less. The platelet release promoting peptide may be linear or may have one or more branches. Further, the platelet release-promoting peptide may be modified such as glycosylation and sulfation.

  At least a part of the platelet release-promoting peptide forms a triple-helical structure with high heat stability. The platelet release-promoting peptide preferably exhibits a positive cotton effect at a wavelength of 220 to 230 nm and a negative cotton effect at a wavelength of 195 to 205 nm in a circular dichroism spectrum in a liquid state of 90 ° C. or lower. That is, it is preferable that at least a part of the platelet release-promoting peptide forms a triple helical structure in a liquid state at 90 ° C. or lower.

  Platelet release promoting peptides have platelet release promoting activity. Therefore, the platelet release promoting agent of the present invention containing the platelet release promoting peptide of the present invention exhibits a platelet release promoting effect.

  “Promoting platelet release” means that “platelet release” is promoted. “Platelet release” refers to release of a biologically active factor contained in platelets. Examples of the biologically active factor contained in platelets include platelet-derived growth factor (PDGF), transforming growth factor-β (TGF-β), epidermal growth factor (EGF), and vascular endothelial growth factor (VEGF). , Platelet factor 4 and β-thromboglobulin. These factors are also collectively referred to as platelet release factors.

  In the present invention, “promoting platelet release” means, for example, that the release amount of platelet releasing factor is increased as compared with the case where platelets are not exposed to the platelet release promoting peptide. In the present invention, “platelet release promotion” specifically means that the amount of platelet release factor released is preferably 20% or more, more preferably 50%, compared to the case where platelets are not exposed to the platelet release-promoting peptide. More preferably, it means 100% or more, more preferably 150% or more. In the present invention, it is preferable that at least the release of PDGF is promoted. The platelet release-promoting peptide of the present invention preferably exhibits a platelet release-promoting effect at a lower concentration than natural collagen. For example, it preferably exhibits a platelet release-promoting effect at a concentration of 5 μg / mL or less. It is more preferable to show a platelet release promoting effect at a concentration of 5 μg / mL or less. The platelet release promoting peptide of the present invention preferably exhibits a platelet release promoting effect at a concentration of 0.05 μg / mL, for example.

  The platelet release promoting peptide can be used as it is as the platelet release promoting agent of the present invention. That is, the platelet release promoting agent of the present invention may contain only a platelet release promoting peptide.

  Moreover, the platelet release promoter of the present invention may contain components other than the platelet release promoting peptide as long as the platelet release promoting effect is achieved. Components other than the platelet release-promoting peptide may be selected in consideration of storage stability, ease of handling, stability of activity, etc., and are not particularly limited. Examples thereof include polyvinyl alcohol, polylactic acid, and cellulose derivatives that are generally used for medical purposes.

  In addition, the platelet release promoter of the present invention may be formulated in any form such as solid, liquid, gel. In formulation, pharmacological agents such as excipients, binders, disintegrants, lubricants, stabilizers, flavoring agents, diluents, surfactants, or injection solvents that are usually used as pharmaceutical carriers. Any acceptable additive can be used. The platelet release promoter of the present invention can be used for promoting platelet release in vitro or in vivo, as it is, or diluted or dissolved with water, physiological saline, buffer solution or the like. It goes without saying that even when diluted or dissolved in this way, it is included in the range of the platelet release accelerator of the present invention.

  The platelet release promoter of the present invention can be used for wound treatment, for example, by directly administering to the wound site. The dose of the platelet release promoter of the present invention is not particularly limited, and may be appropriately set according to various conditions such as the degree of platelet release promoting activity of the platelet release promoter and the degree of wound.

  The platelet release promoter of the present invention can be used for wound treatment, for example, by holding it on an arbitrary carrier and bringing the carrier holding such a platelet release promoter into contact with the wound site. That is, the present invention provides a wound healing material that retains the platelet release promoter of the present invention. Although it does not restrict | limit especially as a support | carrier, For example, the wound dressing material normally used can be used. Although it does not restrict | limit especially as a wound dressing material, For example, the sheet | seat containing a bandage, gauze, a hydrocolloid, etc. are mentioned. That is, for example, a solution of a platelet release promoting peptide can be used in a state of being soaked in gauze or the like. The amount of the platelet release promoter of the present invention held in the carrier is not particularly limited, and may be appropriately set according to various conditions such as the degree of platelet release promoting activity of the platelet release promoter and the degree of wound.

<Method for producing platelet release-promoting peptide>
Hereinafter, a method for producing a platelet release-promoting peptide will be described in detail.
The method for producing a platelet release promoting peptide is not particularly limited, and any platelet release promoting peptide obtained by any method can be used as a platelet release promoting agent.

  The platelet release-promoting peptide can be produced, for example, by polycondensation of one or two constituent tripeptides, ie, Pro-Hyp-Gly and / or Pro-Pro-Gly, in a solvent. For example, when producing a polymer having-(Pro-Hyp-Gly)-as a structural unit, the tripeptide used for polycondensation may be Hyp-Gly-Pro or Gly-Pro-Hyp. Needless to say. A method of performing a polypeptide polycondensation reaction in a solvent is preferable in that it is not necessary to repeat deprotection and amino acid bonding, and a platelet release-promoting peptide can be produced at a low cost. In addition, if it manufactures by chemical synthesis, it is preferable at the point which does not mix the pathogenic factor derived from the animal which was a problem when using the conventional collagen. Tripeptides can be produced, for example, using known solid phase synthesis methods or liquid phase synthesis methods.

  The polycondensation reaction of a tripeptide is usually performed in a solvent. A solvent will not be restrict | limited especially if the tripeptide used as a raw material can be melt | dissolved or suspended, For example, water or an organic solvent can be used. Specific examples of the solvent include water, amides (such as dimethylformamide, dimethylacetamide, and hexamethylphosphoramide), sulfoxides (such as dimethylsulfoxide), nitrogen-containing cyclic compounds (such as N-methylpyrrolidone and pyridine), and nitriles. (Such as acetonitrile), ethers (such as dioxane and tetrahydrofuran), alcohols (such as methyl alcohol, ethyl alcohol, and propyl alcohol), and mixed solvents thereof. Of these solvents, water, dimethylformamide, or dimethyl sulfoxide is preferably used.

  The polycondensation reaction of the tripeptide is preferably performed in the presence of a dehydrating agent (dehydrating condensing agent). When the reaction is carried out in the presence of a dehydration condensation agent and a condensation aid, the condensation reaction proceeds smoothly while suppressing dimerization and cyclization.

  The dehydrating condensing agent is not particularly limited as long as dehydrating condensation can be efficiently performed in the solvent, and examples thereof include a carbodiimide condensing agent, a fluorophosphate condensing agent, and diphenyl phosphoryl azide (DPPA). Examples of the carbodiimide condensing agent include diisopropylcarbodiimide (DIPC), 1-ethyl-3- (3-dimethylaminopropyl) -carbodiimide (EDC = WSCI), 1-ethyl-3- (3-dimethylaminopropyl) -carbodiimide hydrochloride Salt (WSCI · HCl), dicyclohexylcarbodiimide (DCC), and the like. Fluorophosphate condensing agents include O- (7-azabenzotriazol-1-yl) -1,1,3,3-tetramethyluronium hexafluorophosphate, O-benzotriazol-1-yl-N, N , N ′, N′-tetramethyluronium hexafluorophosphate, benzotriazol-1-yl-oxy-tris-pyrrolidinophosphonium hexafluorophosphate, benzotriazol-1-yl-tris (dimethylamino) phosphonium hexafluorophosphate Salt (BOP) etc. are mentioned. Of these, carbodiimide condensing agents such as WSCI and WSCI · HCl are preferred. These dehydrating condensation agents can be used alone or in combination of two or more.

  The condensation aid is not particularly limited as long as it accelerates the condensation reaction. For example, N-hydroxypolycarboxylic imides, N-hydroxytriazoles, N-hydroxytriazines, 2-hydroxyimino-2-cyanoacetate ethyl Examples include esters. N-hydroxypolycarboxylic imides include N-hydroxydicarboxylic imides, and N-hydroxydicarboxylic imides include N-hydroxysuccinimide (HONSu), N-hydroxy-5-norbornene. -2,3-dicarboxylic acid imide (HONB) and the like. Examples of N-hydroxytriazoles include 1-hydroxybenzotriazole (HOBt). Examples of N-hydroxytriazines include 3-hydroxy-4-oxo-3,4-dihydro-1,2,3-benzotriazine (HOOBt). Among these, N-hydroxydicarboxylic imides such as HONSu, N-hydroxybenzotriazoles such as HOBt and HOBt, and N-hydroxybenzotriazines are preferable. These condensation aids can be used alone or in combination of two or more.

  It is preferable to use a dehydrating condensing agent and a condensing aid in an appropriate combination. For example, DCC or WSCI can be used as a dehydrating condensing agent, and HONSu, HOBt, or HOOBt can be used in combination as a condensing aid.

  The amount of the dehydrating condensing agent used is usually 0.7 to 5 mol, preferably 0.8 to 2.5 mol, more preferably, when a non-aqueous solvent not containing water is used per 1 mol of the total amount of tripeptide. Is in the range of 0.9 to 2.3 moles, for example 1-2 moles. In a solvent containing water (aqueous solvent), since the dehydration condensing agent is deactivated by water, the amount of the dehydrating condensing agent used is usually 2 to 500 mol with respect to 1 mol of the total tripeptide, preferably It is 5-250 mol, More preferably, it is the range of 10-125 mol.

  The amount of the condensation aid is usually 0.5 to 5 mol, preferably 0.7 to 2 mol, more preferably 0.8 to 0.1 mol with respect to 1 mol of the total tripeptide regardless of the type of solvent. The range is 1.5 mol.

  In the polycondensation reaction, the pH of the reaction system may be adjusted. When adjusting the pH of the reaction system, the pH of the reaction solution is usually adjusted to around neutral (pH = about 6 to 8). The pH can be adjusted using an inorganic base (sodium hydroxide, potassium hydroxide, sodium carbonate, sodium hydrogen carbonate, etc.), an organic base, an inorganic acid (hydrochloric acid, etc.) or an organic acid. You may carry out using the base which is not concerned. Examples of the base not involved in the condensation reaction include tertiary amines, for example, trialkylamines such as trimethylamine, triethylamine, and diisopropylethylamine, and heterocyclic tertiary amines such as N-methylmorpholine and pyridine. . The amount of such a base used can usually be selected from a range of about 1 to 2 times the total number of moles of the tripeptide.

  Since the reagent used for the reaction remains in the peptide obtained by polycondensation of the tripeptide as described above, it is preferable to remove the remaining reagent and replace it with a storage solvent. The remaining reagent can be removed using a known method such as a dialysis method, a column method, or an ultrafiltration method.

  The preservation solvent is not particularly limited as long as it can suppress changes in physical properties and biological properties of the obtained peptide. Examples thereof include water, physiological saline, a buffer having a buffer capacity from weak acid to weak alkali, and an aqueous alcohol solution. In addition, the manufacturing method of these peptides is explained in full detail in Unexamined-Japanese-Patent No. 2003-321500.

  The usage form of the peptide of the present invention is not particularly limited, and may be, for example, liquid, gel, or dry.

  The dried product may be in any form such as powder, two-dimensional form (film, sheet, etc.), and three-dimensional form. The drying method is not particularly limited, and a known method such as a freeze drying method, a spray drying method, or a reduced pressure drying method can be used. For example, a sponge-like dried product can be obtained by freeze drying, and a powdery dried product can be obtained by spray drying. In addition, a peptide sheet or film can be obtained by casting and drying a peptide solution or suspension on a peelable base (for example, a fluororesin container such as polytetrafluoroethylene).

  In drying, other components may be mixed. Other components are not particularly limited as long as the platelet release promoting effect is achieved, and for example, polyvinyl alcohol, cellulose derivatives, polylactic acid, and the like can be used.

  The dried platelet release-promoting peptide can be sufficiently high enough to keep its shape even when used alone, but the strength can be further increased by thermal crosslinking. The heat-crosslinked platelet release-promoting peptide is insoluble in water. The degree of thermal crosslinking depends on the temperature and time of heating, and the crosslinking proceeds as the temperature and / or time increases. Specifically, by heating at 180 ° C. or higher for 2 hours or longer, crosslinking proceeds to such an extent that the dried platelet release-promoting peptide is insoluble in water. In order to suppress carbonization and side reactions of the peptide, heating at 230 ° C. or lower and 10 hours or shorter is preferable. Thermal crosslinking can be performed in the air or in a nitrogen atmosphere, but it is preferably performed in a nitrogen atmosphere in order to suppress side reactions.

<Promoting method of platelet release>
Platelet release can be promoted by exposing the platelets to the platelet release promoter of the present invention containing a platelet release-promoting peptide. That is, the present invention provides a method for promoting platelet release (hereinafter also referred to as the method of the present invention), which comprises the step of exposing platelets to the platelet release promoter of the present invention. In the method of the present invention, purified platelets may be exposed or any sample containing platelets may be exposed. Samples containing platelets include whole blood and platelet-rich plasma. As long as the sample contains platelets, the step of exposing the sample to the platelet release-promoting agent is included within the range of exposing the platelet to the platelet release-promoting agent.

  In the method of the present invention, the concentration of the platelet release promoting peptide in the reaction system is not particularly limited as long as the platelet release promoting effect is achieved, and can be set as appropriate. The concentration of the platelet release-promoting peptide is, for example, preferably usually 0.01 μg / mL or more, more preferably 0.025 μg / mL or more, as the final concentration when exposing blood, and 0.05 μg / mL. More preferably, it is more than mL. The concentration of the platelet release promoting peptide may be, for example, 100 μg / mL or less, or 50 μg / mL or less. The concentration of the platelet release-promoting peptide can be, for example, about 0.05 μg / mL.

  In the method of the present invention, the reaction temperature is not particularly limited as long as the platelet release promoting effect is achieved, and can be set as appropriate. For example, the reaction temperature is usually preferably from 10 ° C to 50 ° C, and more preferably from 20 ° C to 40 ° C. The reaction temperature may be, for example, about 37 ° C.

  In the method of the present invention, the reaction time is not particularly limited as long as the platelet release promoting effect is achieved, and can be set as appropriate. For example, the reaction time is usually preferably 10 seconds or longer, more preferably 30 seconds or longer, and even more preferably 1 minute or longer. The reaction time may be, for example, 10 hours or less, or 1 hour or less. The reaction time can be, for example, about 5 minutes.

  In the method of the present invention, the reaction system may contain any component other than the platelet release promoter of the present invention and platelets as long as the platelet release promoting effect is achieved.

  In addition, platelet release factor can be efficiently produced by promoting platelet release by the method of the present invention. That is, this invention provides the manufacturing method of the platelet release factor including the process of exposing platelets to the platelet release promoter of this invention. The released platelet releasing factor may be purified as necessary. Purification can be performed, for example, by a known method. The produced platelet releasing factor is similar to the platelet release promoting agent of the present invention, for example, by directly administering it to the wound site, or holding it on an arbitrary carrier, and a carrier that holds such a platelet releasing factor. By contacting the wound site, it can be used for wound treatment.

  Hereinafter, the present invention will be described more specifically with reference to examples. However, the present invention is not limited to these examples. The “water” used below means ultrapure water unless otherwise specified.

Experimental Example 1: Synthesis of platelet release-promoting peptide 1 g of tripeptide represented by Pro-Hyp-Gly synthesized by the liquid phase method was dissolved in 20 mL of 10 mM phosphate buffer (pH 7.4) and cooled to 4 ° C. . To this was added 473 mg of 1-hydroxybenzotriazole (HOBt) at 4.degree. C. and 3.35 g of 1-ethyl-3- (3-dimethylaminopropyl) -carbodiimide hydrochloride (WSCI.HCl) for 2 hours at 4.degree. Stir, then warm to 20 ° C. and stir for 46 hours. After completion of the reaction, water was added to the reaction solution to dilute it twice, and this solution was put into a dialysis cellulose tube (UC27-32-100, manufactured by Biscay) and dialyzed against 2 L of water for 48 hours. At that time, the water was changed four times at intervals of 6 hours or more. The polypeptide aqueous solution thus obtained was used as a platelet release promoting peptide solution in the following experiments.

  After dialysis, the platelet release-promoting peptide solution was diluted 50 times (volume ratio) with water, and gel permeation chromatography (high performance liquid chromatography: Agilent 1100 series, column: GM6000PWXL-CP (Tosoh), flow rate: 0 .5 ml / min., Mobile phase: 20 mM potassium phosphate buffer (pH 3.0) / methanol = 8/2 (volume ratio)), peak of polypeptide in the range of 20,000 to 20 million molecular weight The weight average molecular weight was about 6 million. As a molecular weight standard, pullulan having a known molecular weight (SHODEX STANDARD P-82 (manufactured by Showa Denko KK) (molecular weight range 5,900 to 788,000)) was used.

  After dialysis, the platelet release-promoting peptide solution is diluted 60 times (volume ratio) with water, and a circular dichroism spectrum (Joscope-made circular dichroism dispersometer J-820, optical path length 1 mm) is obtained at 25 ° C. When measured, a positive cotton effect was confirmed at 225 nm, and a negative cotton effect was confirmed at 198 nm. Therefore, it was confirmed that at least a part of the platelet release-promoting peptide obtained as described above formed a triple helical structure in a liquid state at 25 ° C.

Experimental Example 2: Thermal Crosslinking of Platelet Release-Promoting Peptide The platelet release-promoting peptide solution prepared in Experimental Example 1 was made into an aqueous solution having a peptide concentration of 0.5%, placed in a Teflon (registered trademark) container, and lyophilized. The resulting dried product was sponge-like, immediately wetted by water and dissolved in water in a few minutes. On the other hand, when a part of the obtained sponge-like dried product was heated at 180 ° C. for 2 hours under a nitrogen atmosphere, the dried product after heating did not dissolve in water even after 1 day had passed after addition.

Example 1: Platelet-release-promoting peptide platelet-derived growth factor (PDGF) release-promoting activity 0.8 ml of 1/10 volume of 3.8% sodium citrate from the abdominal aorta of female ICR mice (body weight 25-35 g, Slc) Blood was collected and mixed well for anticoagulation treatment. To this, the platelet release promoting peptide solution prepared in Experimental Example 1 was added so that the final concentration of the peptide was 0.005 to 5 μg / ml, and the mixture was shaken at 37 ° C. for 5 minutes. Thereafter, the cells were centrifuged at 800 G to remove blood cells, and then PDGF contained in the serum was measured using R & D System's Quantikine Mouse / Rat PDGF-BB Immunoassay (Cat. No. MBB00). The results are shown in FIG. The platelet release-promoting peptide prepared in Experimental Example 1 showed a remarkable PDGF release-promoting activity at 0.05 μg / mL.

Comparative Example 1: PDGF Release Promoting Activity of Porcine Type I Collagen PDGF releasing activity was measured in the same manner as in Example 1 except that commercially available porcine type I collagen was used instead of the platelet release promoting peptide. The results are shown in FIG. Porcine type I collagen did not show significant PDGF release promoting activity even at 5 μg / mL.

  Therefore, it was revealed that the platelet release-promoting peptide prepared in Experimental Example 1 has a significantly higher PDGF release-promoting activity as compared with natural collagen.

Example 2: PDGF release promoting activity of heat-crosslinked platelet release-promoting peptide The heat-crosslinked peptide prepared in Experimental Example 2 was wetted in 20 mM acetic acid and homogenized for 5 minutes. This suspension was appropriately diluted with a phosphate buffer having a pH of 6.8, and PDGF releasing activity was evaluated according to the method described in Example 1. The results are shown in FIG. The thermally cross-linked peptide prepared in Experimental Example 2 started to exhibit PDGF release promoting activity from 0.025 μg / mL, and PDGF release promoting activity was almost saturated at 0.05 μg / mL.

Comparative Example 2: PDGF release-promoting activity of heat-crosslinked bovine dermis-derived collagen As in Example 2 except that a commercially available heat-crosslinked bovine dermis-derived collagen was used instead of the heat-crosslinked peptide prepared in Experimental Example 2. The release activity of PDGF was measured by the method described above. The results are shown in FIG. Bovine dermis-derived collagen began to exhibit PDGF release promoting activity from 5 μg / mL, and PDGF release promoting activity almost saturated at 25 μg / mL.

  Therefore, the thermally cross-linked peptide prepared in Experimental Example 2 has a PDGF release promoting activity at a concentration of about 1/100 that of the heat-crosslinked bovine dermis-derived collagen, which is remarkable as compared with the heat-crosslinked natural collagen. It was revealed that it has a high PDGF release promoting activity.

  From the above, it was shown that the platelet release promoting peptide of the present invention has platelet release promoting activity. In addition, the platelet release-promoting peptide of the present invention has a platelet release-promoting activity at a lower concentration than that of natural collagen, and clearly has a significantly higher PDGF release-promoting activity compared with natural collagen. became.

  According to the present invention, platelet release can be effectively promoted as compared with natural collagen. Therefore, the present invention can be used for the preparation of platelet-releasing factor from platelets in vitro and rapid platelet activation at the wound site, and is therefore useful for wound treatment.

Claims (6)

Platelet-derived growth factor (PDGF) release promoter (platelet aggregation ) containing a polymer represented by the following formula (1) and having a weight average molecular weight of 500,000 to 10,000,000 Excluding those used for triggering).
-(Pro-X-Gly)-(1)
(In the formula, X represents Pro or Hyp.) The platelet-derived platelet according to claim 1, wherein when the molecular weight of the polymer is measured by gel permeation chromatography, 80% or more of the total peak area is included in the molecular weight range of 20,000 to 20 million. Growth factor (PDGF) release enhancer. 3. The platelet-derived growth factor (PDGF) release promoter according to claim 1, wherein the polymer has a triple helical structure. The release promotion of platelet-derived growth factor (PDGF) according to any one of claims 1 to 3, wherein the polymer exhibits a platelet release factor release promoting activity at a concentration of 0.05 µg / mL. Agent. The release promoter for platelet-derived growth factor (PDGF) according to any one of claims 1 to 4, wherein the polymer is insolubilized by thermal crosslinking. A method for promoting the release of platelet-derived growth factor (PDGF) , comprising the step of exposing the platelet in vitro to the platelet-derived growth factor (PDGF) release promoter according to any one of claims 1 to 5. .

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