Method for producing gel-like wound healing agent, gel-like wound healing agent, and kit for producing autologous platelet-rich plasma gel for healing wounds

Abstract

The purpose of the present invention is to provide a therapy and a therapeutic agent that have a sufficient effect on wounds or the like in which an improvement tendency cannot be observed in conventional therapies. The present invention relates to a method for producing a gelatinous wound healing agent, the method comprising: mixing (A) an effective amount of platelet-rich plasma, (B) a 150 mg / mL to 300 mg / mL ascorbic acid solution, (C) a 1.0 wt% to 3.0 wt% calcium chloride solution, and (D) thrombin, the mixing ratio of (A) platelet-rich plasma to (B) ascorbic acid solution being 1.0 mL to 3.0 mL with respect to 8.0 mL of (A) platelet-rich plasma, and the mixing ratio of (B) ascorbic acid solution to (A) thrombin being 1.0 mL to 3.0 mL with respect to 8.0 mL of (A) platelet-rich plasma; the mixing ratio of (A) the platelet-rich plasma to (C) the calcium chloride solution is 1.0 mL to 3.0 mL with respect to 8.0 mL of (A) the platelet-rich plasma, and the amount of (D) the thrombin is 500 U to 2000 U with respect to 8.0 mL of (A) the platelet-rich plasma.

Description

Technical Field

[0001] The present invention relates to a method for producing a gelled wound healing agent, a gelled wound healing agent, and a kit for preparing autologous platelet-rich plasma gel for wound healing. Background Art

[0002] It is estimated that there are 10 million diabetic patients in Japan, and if potential patients are included, the number exceeds 20 million. It has also been reported that one of the complications of diabetes is diabetic ulcer, and the prevalence of skin ulcers in diabetic patients reaches about 15%. With the aging of the population, the number of diabetic patients tends to increase, and it is expected that the number of patients with diabetic ulcers, which is one of its complications, will also increase. If diabetic ulcers become severe, amputation of the lower extremities may sometimes be required. Therefore, it is considered important to actively treat diabetic ulcer wounds and prevent the progression of the disease at an early stage. Overseas, the usefulness of autologous platelet-rich plasma (PRP) therapy for such wound treatment has been reported (see Patent Documents 1 and 2).

[0003] Autologous platelet-rich plasma (PRP) therapy is a treatment method in which platelet-rich plasma separated and concentrated from blood collected from a patient is administered to the damaged tissue, and the action of various growth factors contained in the platelet-rich plasma is used to promote tissue regeneration and guide the wound site to healing. Depending on the amount of platelets in the platelet-rich plasma (platelet concentration rate), the treatment method of the platelet-rich plasma, etc., the obtained treatment effect will be different.

[0004] There is a need for a therapy and a therapeutic agent that can improve the problems of the conventionally known autologous platelet-rich plasma (PRP) therapy and can also achieve sufficient effects for wounds and the like for which no improvement tendency is observed in the existing therapy.

[0005] Prior Art Documents

[0006] Patent Documents

[0007] Patent Document 1: Japanese Patent Application Laid-Open No. 2003-524590

[0008] Patent Document 2: Japanese Patent Application Laid-Open No. 2013-507225 Summary of the Invention

[0009] Problems to be Solved by the Invention

[0010] The present invention has been completed based on the above circumstances, and an object thereof is to provide a therapy and a therapeutic agent that can achieve sufficient effects for wounds and the like for which no improvement tendency is observed in the existing therapy.

[0011] Means for Solving the Problems

[0012] The inventors of the present invention conducted in - depth research to solve the above - mentioned problems and found that, in a gel - like wound healing agent using autologous platelet - rich plasma (PRP), by setting the concentrations of ascorbic acid and calcium chloride mixed during production to a specific combination, a gel - like wound healing agent with appropriate hardness as a gel - like wound healing agent, capable of maintaining its form for a long time when applied to skin wounds, and capable of sufficiently protecting the wound site and exerting a high therapeutic effect can be produced. In addition, it was also confirmed that sufficient effects were obtained for wounds and the like for which no improvement tendency was observed in existing therapies to date. The present invention was completed based on these insights.

[0013] That is, the gist of the present invention is as follows.

[0014] [1] A method for producing a gel - like wound healing agent, comprising: mixing

[0015] (A) an effective amount of platelet - rich plasma,

[0016] (B) an ascorbic acid solution at a concentration of 150 mg / mL to 300 mg / mL,

[0017] (C) a calcium chloride solution at a concentration of 1.0 wt% to 3.0 wt% and

[0018] (D) thrombin

[0019] and mixing,

[0020] Regarding the mixing ratio of (A) platelet - rich plasma and (B) ascorbic acid solution, with respect to 8.0 mL of (A) platelet - rich plasma, (B) ascorbic acid solution is 1.0 mL to 3.0 mL,

[0021] Regarding the mixing ratio of (A) platelet - rich plasma and (C) calcium chloride solution, with respect to 8.0 mL of (A) platelet - rich plasma, (C) calcium chloride solution is 1.0 mL to 3.0 mL,

[0022] The amount of (D) thrombin is 500 U to 2000 U with respect to 8.0 mL of (A) platelet - rich plasma.

[0023] [2] The method for producing a gel - like wound healing agent according to [1], wherein (D) thrombin is mixed with (A) platelet - rich plasma and (B) ascorbic acid solution in a state dissolved in (C) calcium chloride solution.

[0024] [3] The method for producing a gel - like wound healing agent according to [2], wherein (B) ascorbic acid solution is mixed in (A) platelet - rich plasma, and then the calcium chloride solution of the aforementioned thrombin is mixed.

[0025] [4] The method for manufacturing the gel-like wound healing agent according to [1] further includes: separating platelet concentrate from other blood components by centrifuging blood, thereby obtaining (A) platelet-rich plasma.

[0026] [5] The method for manufacturing the gel-like wound healing agent according to [4], wherein the aforementioned blood is treated with a calcium chelating agent.

[0027] [6] The method for manufacturing the gel-like wound healing agent according to [4], wherein the conditions for the aforementioned centrifugation are 3000×g to 4500×g, 20 seconds to 1 minute.

[0028] [7] A method for manufacturing a gel sheet wound healing agent, which includes: mixing

[0029] (A) An effective amount of platelet-rich plasma,

[0030] (B) An ascorbic acid solution of 150 mg / mL to 300 mg / mL,

[0031] (C) A calcium chloride solution of 1.0 wt% to 3.0 wt% and

[0032] (D) Thrombin

[0033] mixing,

[0034] Regarding the mixing ratio of (A) platelet-rich plasma and (B) ascorbic acid solution, with respect to 8.0 mL of (A) platelet-rich plasma, (B) ascorbic acid solution is 1.0 mL to 3.0 mL,

[0035] Regarding the mixing ratio of (A) platelet-rich plasma and (C) calcium chloride solution, with respect to 8.0 mL of (A) platelet-rich plasma, (C) calcium chloride solution is 1.0 mL to 3.0 mL,

[0036] (D) The amount of thrombin is 500 U to 2000 U with respect to 8.0 mL of (A) platelet-rich plasma.

[0037] [8] A gel-like wound healing agent, which is obtained by mixing

[0038] (A) An effective amount of platelet-rich plasma,

[0039] (B) An ascorbic acid solution of 150 mg / mL to 300 mg / mL,

[0040] (C) A calcium chloride solution of 1.0 wt% to 3.0 wt% and

[0041] (D) Thrombin

[0042] mixing,

[0043] Regarding the mixing ratio of (A) platelet-rich plasma and (B) ascorbic acid solution, with respect to 8.0 mL of (A) platelet-rich plasma, (B) ascorbic acid solution is 1.0 mL to 3.0 mL.

[0044] Regarding the mixing ratio of (A) platelet-rich plasma and (C) calcium chloride solution, with respect to 8.0 mL of (A) platelet-rich plasma, (C) calcium chloride solution is 1.0 mL to 3.0 mL.

[0045] [9] The gel-like wound healing agent according to [8], wherein (D) thrombin is mixed with (A) platelet-rich plasma and (B) ascorbic acid solution in a state dissolved in (C) calcium chloride solution.

[0046]

[10] The gel-like wound healing agent according to [9], which is obtained by mixing (B) ascorbic acid solution in (A) platelet-rich plasma, and then mixing the calcium chloride solution of the aforementioned thrombin.

[0047]

[11] An autologous platelet-rich plasma gel preparation kit for wound healing, comprising:

[0048] (I) A blood collection needle,

[0049] (II) A blood collection tube and

[0050] (III) A gelation reagent set,

[0051] (III) The gelation reagent set contains an ascorbic acid solution of 150 mg / mL to 300 mg / mL, a calcium chloride solution of 1.0 wt% to 3.0 wt%, and thrombin in a separately independent form.

[0052] Effects of the Invention

[0053] Compared with the conventionally known gel-like wound healing agents using platelet-rich plasma, the gel-like wound healing agent manufactured by the method of the present invention is sufficiently gelated. Therefore, when applied to skin wounds, it can maintain its form for a long time, can sufficiently protect the wound area, and exhibit a high therapeutic effect. In addition, sufficient effects can also be obtained for wounds and the like that have not shown an improvement tendency in existing therapies so far. Furthermore, due to sufficient gelation, it also has the advantage of easy operation. Description of the Drawings

[0054] Figure 1 It is a graph showing the results of comparing the hardness of the gel-like wound healing agent of the present invention with that of the existing product. Detailed Description

[0055] Hereinafter, the manufacturing method of the gel-like wound healing agent of the present invention, the gel-like wound healing agent, and the autologous platelet-rich plasma gel preparation kit for wound healing will be described in detail.

[0056] <Manufacturing Method of Gel-Like Wound Healing Agent>

[0057] The manufacturing method of the gel-like wound healing agent of the present invention is characterized in that it includes: mixing (A) an effective amount of platelet-rich plasma, (B) an ascorbic acid solution of 150 mg / mL to 300 mg / mL, (C) a calcium chloride solution of 1.0 wt% to 3.0 wt%, and (D) thrombin. Regarding the mixing ratio of (A) platelet-rich plasma and (B) ascorbic acid solution, relative to 8.0 mL of (A) platelet-rich plasma, (B) ascorbic acid solution is 1.0 mL to 3.0 mL. Regarding the mixing ratio of (A) platelet-rich plasma and (C) calcium chloride solution, relative to 8.0 mL of (A) platelet-rich plasma, (C) calcium chloride solution is 1.0 mL to 3.0 mL. The amount of (D) thrombin is 500 U to 2000 U relative to 8.0 mL of (A) platelet-rich plasma.

[0058] The manufacturing method of the gel-like wound healing agent of the present invention may also include, before the step of mixing (A) an effective amount of platelet-rich plasma, (B) an ascorbic acid solution of 150 mg / mL to 300 mg / mL, (C) a calcium chloride solution of 1.0 wt% to 3.0 wt%, and (D) thrombin, a step of separating a platelet concentrate from other blood components by centrifuging blood to obtain (A) platelet-rich plasma.

[0059] Compared with the conventionally known gel-like wound healing agent using platelet-rich plasma, the gel-like wound healing agent manufactured by the method of the present invention is sufficiently gelated. Therefore, when applied to skin wounds, it can maintain its shape for a long time, can sufficiently protect the wound area, and exhibit a high therapeutic effect. In addition, sufficient effects can also be obtained for wounds and the like that have not shown an improvement tendency in existing therapies so far. Furthermore, due to sufficient gelation, it also has the advantage of easy operation.

[0060] Hereinafter, each condition constituting the present invention will be specifically described.

[0061] [(A) An Effective Amount of Platelet-Rich Plasma]

[0062] In the method for producing the gel-like wound healing agent of the present invention, the platelet-rich plasma used can be obtained by separating red blood cells and white blood cells from whole blood by methods such as centrifugation. The platelet concentration (platelet concentration ratio) of the obtained platelet-rich plasma can be adjusted by the centrifugation conditions. The platelet-rich plasma used in the method for producing the gel-like wound healing agent of the present invention is preferably a physiological platelet concentration (platelet concentration ratio: 1.0-fold). As the centrifugation conditions for obtaining platelet-rich plasma with a physiological platelet concentration (platelet concentration ratio: 1.0-fold), it is 2000×g to 5000×g, 15 seconds to 3 minutes, preferably 3000×g to 4500×g, 20 seconds to 1 minute, more preferably 4000×g to 4500×g, 20 seconds to 45 seconds, further preferably 4000×g to 4500×g, 30 seconds, and particularly preferably 4236×g, 30 seconds. It is also known that when the platelet concentration of platelet-rich plasma is high, it will have a negative impact on cell proliferation at the wound site and cell migration for tissue regeneration (Reese RJ. Autologous platelet rich plasma (PRP): what do we know? Important concepts relevant to hair restoration surgery. Hair Transplant ForumInt 2010: 14-7). In the present invention, by setting the platelet concentration of platelet-rich plasma to a physiological concentration, the wound healing effect of the gel-like wound healing agent can be improved.

[0063] When collecting blood, an anticoagulant needs to be added to prevent blood coagulation. As such an anticoagulant, a calcium chelating agent is preferably used. As the calcium chelating agent that can be used in the present invention, examples include hydroxycarboxylic acid-based chelating agents, aminopolycarboxylic acid-based chelating agents, aromatic or aliphatic carboxylic acid-based chelating agents, amino acid-based chelating agents, ether carboxylic acid-based chelating agents, phosphonic acid-based chelating agents, polymer electrolyte (including oligomer electrolyte)-based chelating agents, polyols, nitrogen-containing chelating agents such as dimethylglyoxime, and sulfur-containing chelating agents such as mercaptoacetic acid. Among them, a hydroxycarboxylic acid-based chelating agent is preferred.

[0064] Including use.

[0065] Examples of the hydroxycarboxylic acid-based chelating agent include malic acid, citric acid, glycolic acid, gluconic acid, heptonic acid, tartaric acid, lactic acid, and their salts and derivatives. As a commercially available product, ACD-A solution (biological preparation standard blood preservation solution A solution) etc. are available, and it is preferably added to whole blood in an amount that reaches a final concentration of about 10%.

[0066] The method of blood collection can use methods known in the art. For blood collection, materials, equipment, devices, etc. such as blood collection needles, blood collection tubes, and blood collection bags can be used.

[0067] In the platelet-rich plasma of the present invention, blood cell components other than platelets (red blood cells, white blood cells, etc.) may also be mixed in.

[0068] The platelet-rich plasma in the present invention can be autologous (from oneself) obtained from the whole blood of the trauma patient himself / herself, or can be obtained from the whole blood obtained from one or more third parties. In addition, as long as there is no biological incompatibility, the above third party may not be of the same type as the trauma patient. Among them, from the viewpoints of biological compatibility, safety, etc., the platelet-rich plasma is preferably autologous (from oneself) obtained from the whole blood of the trauma patient himself / herself.

[0069] When the platelets contained in the platelet-rich plasma of the present invention are activated by thrombin, collagen, serotonin, adenosine diphosphate (ADP), acetylcholine (ACH), etc., α-granules containing growth factors such as PDGF, coagulation factors, etc. (described in the following paragraphs) are released, and it is considered to promote tissue regeneration at the trauma site, etc.

[0070] The platelets contained in the platelet-rich plasma of the present invention contain platelet-derived growth factor (PDGF), platelet-derived angiogenic factor (PDAF), vascular endothelial growth factor (VEGF), platelet-derived epidermal growth factor (PDEGF), platelet factor 4 (PF-4), transforming growth factor β (TGF-β), acidic fibroblast growth factor (FGF-A), basic fibroblast growth factor (FGF-B), transforming growth factor α (TGF-α), insulin-like growth factors I and II (IGF-1 and IGF-2), β-thromboglobulin-related protein (BTG), thrombospondin (TSP), fibronectin, von Willebrand factor (von Wallinbrand's factor, vWF), fibronectin A (Japanese: フィブロペプチドA), fibrinogen, albumin, plasminogen activator inhibitor I (PAI-1), osteonectin, RANTES (regulated upon The invention relates to factors in the group consisting of cytokines, ...

[0071] The effective amount in "effective amount of platelet-rich plasma" refers to the amount required for wound healing (e.g., reduction of wound volume or surface area; promotion of collagen adhesion, vascular ingrowth (Japanese: endothelial growth), fibroblast growth, or general healing of granular tissue and other biological substances). It should be noted that all embodiments of the invention described in this specification include effective amounts of component substances or combinations thereof in wound treatment.

[0072] [(B) Ascorbic acid solution]

[0073] In the method for producing the gel-like wound healing agent of the present invention, an ascorbic acid solution of 150 mg / mL to 300 mg / mL is mixed into the above-mentioned (A) platelet-rich plasma in an effective amount. At this time, regarding the mixing ratio of (A) platelet-rich plasma and (B) ascorbic acid solution, with respect to 8.0 mL of (A) platelet-rich plasma, (B) ascorbic acid solution is 1.0 mL or more, and (B) ascorbic acid solution is preferably 1.0 mL to 3.0 mL, more preferably 1.0 mL to 2.0 mL, and further preferably 1.0 mL. The concentration of the ascorbic acid solution is 150 mg / mL to 300 mg / mL, preferably 200 mg / mL to 300 mg / mL, more preferably 220 mg / mL to 270 mg / mL, and further preferably 250 mg / mL. By setting the mixing ratio of (A) platelet-rich plasma and (B) ascorbic acid solution and the concentration of the ascorbic acid solution within the above ranges, the hardness and viscosity of the gel-like wound healing agent can be maintained in an appropriate state. Here, the terms "hardness" and "viscosity" are characteristics for determining the degree of gelation of the gel-like wound healing agent, and refer to the degree of hardness, stiffness, softness, and fluidity of the gel-like wound healing agent.

[0074] [(C) Calcium chloride solution]

[0075] In the method for producing the gel-like wound healing agent of the present invention, after mixing an ascorbic acid solution of 150 mg / mL to 300 mg / mL into the above-mentioned (A) platelet-rich plasma in an effective amount, a calcium chloride solution of 1.0 wt% to 3.0 wt% is further mixed. By mixing (C) calcium chloride solution, the action of the calcium chelating agent added during blood collection can be eliminated, and calcium-dependent enzyme reactions can be promoted. The concentration of the calcium chloride solution is preferably 1.0 wt% to 3.0 wt%, more preferably 1.5 wt% to 3.0 wt%, further preferably 1.5 wt% to 2.5 wt%, and particularly preferably 2.0 wt%. Regarding the amount of (C) calcium chloride solution, with respect to the mixing ratio of (A) platelet-rich plasma and (C) calcium chloride solution, with respect to 8.0 mL of (A) platelet-rich plasma, (C) calcium chloride solution is 1.0 mL or more, and (C) calcium chloride solution is preferably 1.0 mL to 3.0 mL, more preferably 1.0 mL to 2.0 mL, and further preferably 1.0 mL. By setting the mixing ratio of (A) platelet-rich plasma and (C) calcium chloride solution and the concentration of the calcium chloride solution within the above ranges, in combination with the above-mentioned (B) ascorbic acid solution, the hardness and viscosity of the gel-like wound healing agent can be in an appropriate state.

[0076] [(D) Thrombin]

[0077] In the present invention, thrombin refers to an endoprotease involved in blood coagulation that converts fibrinogen into fibrin, and in humans, for example, it refers to a substance called activated factor II.

[0078] The thrombin (D) used in the present invention may be thrombin obtained by reacting activated factor X and / or calcium ions, phospholipids, and activated factor V with prothrombin separated from blood or bone marrow by a conventional method, or recombinant thrombin. The source is not particularly limited, and for example, thrombin derived from bovine blood is widely used.

[0079] In the method for producing the gel-like wound healing agent of the present invention, thrombin (D) is mixed in an amount of 500 U to 2000 U with respect to 8.0 mL of platelet-rich plasma (A). Regarding the amount of thrombin (D), with respect to 8.0 mL of platelet-rich plasma (A), it is preferably 500 U to 1500 U, more preferably 750 U to 1250 U, and further preferably about 1000 U. It should be noted that platelet activation and aggregation are promoted by the mixing of thrombin, so it is necessary to mix at a timing immediately before the gel-like wound healing agent of the present invention becomes gel-like.

[0080] In the method for producing the gel-like wound healing agent of the present invention, it is preferable to mix with platelet-rich plasma (A) and ascorbic acid solution (B) in a state where thrombin (D) is dissolved in calcium chloride solution (C). In order to prevent inactivation of thrombin (D), it is preferably dissolved in calcium chloride solution (C) for use during preparation.

[0081] [Preparation steps of gel-like wound healing agent]

[0082] The following specifically describes the steps of the method for producing the gel-like wound healing agent of the present invention. It should be noted that the present invention is not limited to the following specific description.

[0083] (1) Blood (4.5 mL) is collected from the patient himself or a third party. When collecting blood, an anticoagulant such as a calcium chelator (0.5 mL) is added to prevent blood coagulation.

[0084] (2) The blood added with the anticoagulant is centrifuged under the above conditions to obtain platelet-rich plasma (A) with a physiological platelet concentration (platelet concentration ratio: 1.0 times).

[0085] (3) An ascorbic acid solution (B) in an amount approximately 1 / 8 of that of platelet-rich plasma (A) is mixed in 2.5 mL of platelet-rich plasma (A).

[0086] (4) To the mixture obtained in (3), a calcium chloride solution (C) in which thrombin (D) is dissolved and having the same amount as the ascorbic acid solution (B) is added and mixed to gelify it to obtain the gel-like wound healing agent of the present invention.

[0087] The prepared gel-like wound healing agent is used for treatment immediately after preparation.

[0088] <Method for manufacturing gel sheet - type wound healing agent>

[0089] The gel - type wound healing agent obtained by the method for manufacturing a gel - type wound healing agent of the present invention can be made into a sheet form. Therefore, the present invention also includes a method for manufacturing a gel sheet - type wound healing agent, which comprises: mixing (A) an effective amount of platelet - rich plasma, (B) an ascorbic acid solution at a concentration of 150 mg / mL to 300 mg / mL, (C) a calcium chloride solution at a concentration of 1.0 wt% to 3.0 wt%, and (D) thrombin. Regarding the mixing ratio of (A) platelet - rich plasma and (B) ascorbic acid solution, relative to 8.0 mL of (A) platelet - rich plasma, (B) ascorbic acid solution is 1.0 mL to 3.0 mL. Regarding the mixing ratio of (A) platelet - rich plasma and (C) calcium chloride solution, relative to 8.0 mL of (A) platelet - rich plasma, (C) calcium chloride solution is 1.0 mL to 3.0 mL. The amount of (D) thrombin is 500 U to 2000 U relative to 8.0 mL of (A) platelet - rich plasma.

[0090] The specific description of the method for manufacturing the gel sheet - type wound healing agent of the present invention can directly apply the description of the method for manufacturing the gel - type wound healing agent above.

[0091] <Gel - type wound healing agent>

[0092] The gel - type wound healing agent of the present invention is obtained by mixing (A) an effective amount of platelet - rich plasma, (B) an ascorbic acid solution at a concentration of 150 mg / mL to 300 mg / mL, (C) a calcium chloride solution at a concentration of 1.0 wt% to 3.0 wt%, and (D) thrombin. Regarding the mixing ratio of (A) platelet - rich plasma and (B) ascorbic acid solution, relative to 8.0 mL of (A) platelet - rich plasma, (B) ascorbic acid solution is 1.0 mL to 3.0 mL. Regarding the mixing ratio of (A) platelet - rich plasma and (C) calcium chloride solution, relative to 8.0 mL of (A) platelet - rich plasma, (C) calcium chloride solution is 1.0 mL to 3.0 mL. Here, (D) thrombin is preferably mixed with (A) platelet - rich plasma and (B) ascorbic acid solution in a state dissolved in (C) calcium chloride solution. In addition, the gel - type wound healing agent of the present invention is preferably obtained by mixing (B) ascorbic acid solution in (A) platelet - rich plasma, and then mixing the calcium chloride solution of the aforementioned thrombin.

[0093] The gel-like wound healing agent of the present invention is produced by the method for producing the gel-like wound healing agent of the present invention described above. By setting the ascorbic acid concentration and calcium chloride concentration mixed during production to the combination described above, the gel-like wound healing agent of the present invention has an appropriate hardness as a gel-like wound healing agent, and when applied to skin wounds, can maintain its form for a long time, can fully protect the wound area, and exhibit a high therapeutic effect. In addition, sufficient effects are also obtained for wounds and the like for which no improvement tendency has been observed in existing therapies so far. Furthermore, since the gel-like wound healing agent has an appropriate hardness and becomes sheet-like, it also has the advantage of being easy to handle.

[0094] The types of wounds to which the gel-like wound healing agent of the present invention can be applied are not particularly limited. For example, it can be applied to chronic wounds, wounds caused by obesity or diabetes (non-healing diabetic wounds, diabetic ulcers, diabetic neuropathic ulcers, other general diabetic wounds, etc.), burns, pressure ulcers, wounds caused by radiation damage, wounds caused by infection, wounds caused by ischemia, neuropathic ulcers, chronic skin ulcers, etc. In particular, the gel-like wound healing agent of the present invention is suitable for use in wounds for which, even after performing basic wound management (removal of necrotic tissue, etc., infection control, wound debridement, etc.) on the wound and implementing treatment using a wound covering material or a topical agent, etc. for 4 weeks or more in principle, no sufficient effect has been confirmed.

[0095] The gel-like wound healing agent of the present invention may vary depending on the state of the patient's wound. However, from the viewpoint of exhibiting a sufficient therapeutic effect, it is preferably applied (attached) to the wound part based on twice a week, and the treatment is carried out with a maximum of 8 weeks (a maximum of 16 administrations). The amount of the gel-like wound healing agent used once (up to about 6 ml) can be appropriately adjusted according to the size of the wound.

[0096] <Autologous Platelet-Rich Plasma Gel Preparation Kit for Wound Healing>

[0097] The present invention also includes an autologous platelet-rich plasma gel preparation kit for wound healing, which includes: (I) a blood collection needle, (II) a blood collection tube, and (III) a gelation reagent set. The (III) gelation reagent set includes an ascorbic acid solution at 150 mg / mL to 300 mg / mL, a calcium chloride solution at 1.0 wt% to 3.0 wt%, and thrombin in a separately independent form. According to this kit, the gel-like wound healing agent of the present invention can be prepared.

[0098] As (I) a blood collection needle, a conventionally known blood collection needle can be used without particular limitation. In addition, (II) a blood collection tube can be appropriately selected according to the amount of blood collected. (III) The 150 mg / mL to 300 mg / mL ascorbic acid solution, 1.0 wt% to 3.0 wt% calcium chloride solution, and thrombin contained in the gelling reagent set are the same as those used in the method for producing the above-mentioned gel wound healing agent, and as respective descriptions, the description of the item for the method for producing the above-mentioned gel wound healing agent can be directly applied. The above-mentioned thrombin is preferably a freeze-dried product, and is preferably stored frozen or refrigerated to prevent inactivation.

[0099] An example of the procedure for preparing a gel wound healing agent using the autologous platelet-rich plasma gel preparation kit for wound healing is as follows. It should be noted that the procedure for using the autologous platelet-rich plasma gel preparation kit for wound healing is not limited to this.

[0100] (1) Using (I) a blood collection needle, blood is collected from the patient or a third party, and blood (4.5 mL) is collected from (II) a blood collection tube to which an anticoagulant such as a calcium chelator (0.5 mL) has been added in advance to prevent blood clotting.

[0101] (2) The blood and the anticoagulant are thoroughly mixed and centrifuged at 3000×g to 4500×g for 20 seconds to 1 minute to obtain platelet-rich plasma with a physiological platelet concentration (platelet concentration ratio: 1.0 times).

[0102] (3) About 1 / 8 of the amount of 150 mg / mL to 300 mg / mL ascorbic acid solution is mixed with 2.5 mL of platelet-rich plasma.

[0103] (4) An equal amount of a 1.0 wt % to 3.0 wt % calcium chloride solution in which thrombin is dissolved is added to the mixed solution obtained in (3) and mixed to form a gel to prepare an autologous platelet-rich plasma gel for wound healing.

[0104] Example

[0105] Hereinafter, the present invention will be described in detail with reference to Examples and Test Examples, but the present invention is not limited to these Examples and the like.

[0106] 1. Production of gel-like wound healing agent

[0107] 5 mL of blood was collected from a patient with a diabetic ulcer, and ACD-A solution (biological agent standard blood preservative solution A) equivalent to 1 / 9 of the blood volume was mixed in the blood collection tube. Then, centrifugation was performed at 4236×g for 30 seconds to collect the plasma fraction containing platelets (PRP: platelet-rich plasma). 2.5 mL of the collected PRP was added to a 10 mL-sized tube. 313 μL of a 250 mg / mL ascorbic acid solution was added to the PRP and stirred. Lyophilized thrombin at 5000 units per vial was dissolved in 5 mL of 2% calcium chloride solution to prepare a calcium chloride-thrombin solution. 313 μL of the calcium chloride-thrombin solution was added to the PRP after adding ascorbic acid and stirred to induce gelation of the PRP, which was used as the gel-like wound healing agent of the present invention.

[0108] 2. Confirmation of the wound healing effect of the gel-like wound healing agent

[0109] Fifty-four patients with diabetic ulcers (47 cases were the main analysis objects for effectiveness) who did not respond to existing treatments were enrolled in an open-label, single-arm, multi-center trial. Treatment with the above gel-like wound healing agent was performed twice a week for up to 8 weeks. The proportion of cases (effective cases) with a wound radius reduction rate of 50% or more, which was the main evaluation item, was 80.9% (95% confidence interval [66.7, 90.9]), confirming the wound healing effect of the gel-like wound healing agent.

[0110] 3. Comparison of the gel-like wound healing agent of the present invention with existing products

[0111] Blood was collected from the abdominal aorta of rats (SD strain, 8-week-old, male), and ACD-A solution (USP standard) equivalent to 1 / 9 of the blood volume was mixed in the blood collection tube. Then, centrifugation was performed at 4236×g for 30 seconds to collect the plasma fraction containing platelets (PRP: platelet-rich plasma). 1.44 mL of the collected PRP was added to a 2 mL-sized microcentrifuge tube (Eppendorf tube). 180 μL of a 250 mg / mL ascorbic acid solution was added to the PRP and stirred. Lyophilized thrombin at 5000 units per vial was dissolved in 5 mL of 2% calcium chloride solution to prepare a calcium chloride-thrombin solution. 180 μL of the calcium chloride-thrombin solution was added to the PRP after adding ascorbic acid and stirred to induce gelation of the PRP, which was used as the gel-like wound healing agent of Example 1.

[0112] Add 1.44 mL of PRP collected in the same manner as described above to a 2-mL microcentrifuge tube. Add 180 μL of a 500 mg / mL ascorbic acid solution to the PRP and stir. Dissolve 5000 units of freeze-dried thrombin per vial in 5 mL of a 10% calcium chloride solution to prepare a calcium chloride-thrombin solution. Add 180 μL of the calcium chloride-thrombin solution to the PRP after adding ascorbic acid as described above and stir to induce gelation of the PRP, thereby preparing a gel-like wound healing agent of the conventional product (Comparative Example 1).

[0113] Measure the hardness of the gel-like wound healing agents of Example 1 and Comparative Example 1 using a grease consistency meter (GP-DT type, Yoshida Scientific Instruments Co., Ltd.). Specifically, place the gel-like wound healing agents of Example 1 and Comparative Example 1 in containers respectively and let them stand still to make the surfaces flat. After making the height consistent by bringing the tip of a cone of a specified size and weight into contact with the surface of the gel-like wound healing agent, let the cone freely fall for 1 second and measure the distance traveled in the gel. The results are shown in Figure 1 .

[0114] As Figure 1 shown, it can be seen that the gel-like wound healing agent of Example 1 travels a shorter distance in the gel than the gel-like wound healing agent of Comparative Example 1. Based on this result, it is considered that the gel-like wound healing agent of Example 1 is harder than the gel-like wound healing agent of Comparative Example 1 as the conventional product and is sufficiently gelated. Therefore, when the gel-like wound healing agent of Example 1 is applied to skin wounds, it maintains its form for a longer time than existing products, stays at the wound site for a long time, and can exert a sufficient therapeutic effect. In addition, because it is sufficiently gelated, it also has the advantage of being easy to operate. It should be noted that for the gel-like wound healing agent of the present invention, the following unexpected result is obtained: although the ascorbic acid concentration of the ascorbic acid solution and the calcium chloride concentration of the calcium chloride solution used in the preparation are lower than those of the conventional product, the obtained gel is harder than the conventional product.

[0115] Industrial Applicability

[0116] The gel-like wound healing agent produced by the method of the present invention is sufficiently gelated compared with the conventionally known gel-like wound healing agent using platelet-rich plasma. Therefore, when applied to skin wounds, it can maintain its form for a long time, can sufficiently protect the wound site, and exert a high therapeutic effect. In addition, sufficient effects can also be obtained for wounds and the like for which no improvement tendency has been observed in existing therapies so far. Furthermore, because it is sufficiently gelated, it also has the advantage of being easy to operate.

Claims

1. A method for manufacturing a gel-like wound healing agent, comprising: Mix (A) An effective amount of platelet-rich plasma, (B) An ascorbic acid solution of 150 mg / mL to 300 mg / mL, (C) A calcium chloride solution of 1.0 wt% to 3.0 wt%, and (D) Thrombin together. Regarding the mixing ratio of (A) platelet-rich plasma and (B) ascorbic acid solution, relative to 8.0 mL of (A) platelet-rich plasma, (B) ascorbic acid solution is 1.0 mL to 3.0 mL. Regarding the mixing ratio of (A) platelet-rich plasma and (C) calcium chloride solution, relative to 8.0 mL of (A) platelet-rich plasma, (C) calcium chloride solution is 1.0 mL to 3.0 mL. The amount of (D) thrombin is 500 U to 2000 U relative to 8.0 mL of (A) platelet-rich plasma.

2. The method for manufacturing the gel-like wound healing agent according to claim 1, wherein, (D) Thrombin is mixed with (A) platelet-rich plasma and (B) ascorbic acid solution in a state dissolved in (C) calcium chloride solution.

3. The method for manufacturing the gel-like wound healing agent according to claim 2, wherein, Mix (B) ascorbic acid solution in (A) platelet-rich plasma, and then mix the calcium chloride solution of the thrombin.

4. The method for manufacturing the gel-like wound healing agent according to claim 1, further comprising: Platelet concentrate is separated from other blood components by centrifuging blood, thereby obtaining (A) platelet-rich plasma.

5. The method for manufacturing the gel-like wound healing agent according to claim 4, wherein, The blood has been treated with a calcium chelator.

6. The method for manufacturing the gel-like wound healing agent according to claim 4, wherein, The conditions for the centrifugation are 3000×g to 4500×g, 20 seconds to 1 minute.

7. A method for manufacturing a gel sheet wound healing agent, which comprises: Mix (A) An effective amount of platelet-rich plasma, (B) An ascorbic acid solution of 150 mg / mL to 300 mg / mL, (C) A calcium chloride solution of 1.0 wt% to 3.0 wt%, and (D) Thrombin together. Regarding the mixing ratio of (A) platelet-rich plasma and (B) ascorbic acid solution, relative to 8.0 mL of (A) platelet-rich plasma, (B) ascorbic acid solution is 1.0 mL to 3.0 mL. Regarding the mixing ratio of (A) platelet-rich plasma and (C) calcium chloride solution, relative to 8.0 mL of (A) platelet-rich plasma, (C) calcium chloride solution is 1.0 mL to 3.0 mL. The amount of (D) thrombin is 500 U to 2000 U relative to 8.0 mL of (A) platelet-rich plasma.

8. A gel-like wound healing agent, which is obtained by mixing (A) An effective amount of platelet-rich plasma, (B) An ascorbic acid solution of 150 mg / mL to 300 mg / mL, (C) A calcium chloride solution of 1.0 wt% to 3.0 wt%, and (D) Thrombin together, Regarding the mixing ratio of (A) platelet-rich plasma and (B) ascorbic acid solution, relative to 8.0 mL of (A) platelet-rich plasma, (B) ascorbic acid solution is 1.0 mL to 3.0 mL. Regarding the mixing ratio of (A) platelet-rich plasma and (C) calcium chloride solution, relative to 8.0 mL of (A) platelet-rich plasma, (C) calcium chloride solution is 1.0 mL to 3.0 mL.

9. The gelled wound healing agent according to claim 8, wherein, (D) Thrombin is mixed with (A) platelet-rich plasma and (B) ascorbic acid solution in a state dissolved in (C) calcium chloride solution.

10. The gel-like wound healing agent according to claim 9, which is obtained by mixing (B) ascorbic acid solution in (A) platelet-rich plasma, and then mixing the calcium chloride solution of the thrombin.

11. An autologous platelet-rich plasma gel preparation kit for wound healing, comprising: (I) a blood collection needle, (II) a blood collection tube, and (III) a gelation reagent set, (III) The gelation reagent set contains an ascorbic acid solution at 150 mg / mL to 300 mg / mL, a calcium chloride solution at 1.0 wt% to 3.0 wt%, and thrombin in a separately independent form.

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