Blood-derived growth-factor-containing composition containing citric acid, and method for preparing same

Abstract

The present invention provides a novel blood-derived growth-factor-containing composition. The present invention for solving the above problem is a method for preparing a growth-factor-containing composition from blood, the method comprising a step for separating platelet-rich plasma that does not contain a buffy coat component from the blood, and a step for adding citric acid and / or a salt thereof to the blood or a processed product thereof.

Description

Blood-derived growth factor-containing composition containing citric acid and method for preparing same

[0001] The present invention relates to growth factor-containing compositions and methods for their preparation.

[0002] There is a treatment method that uses platelet-rich plasma (hereinafter also referred to as "PRP"), which is a plasma enriched with platelets prepared by centrifuging the patient's own blood. This platelet-rich plasma is rich in various growth factors, such as platelet-derived growth factor (hereinafter also referred to as "PDGF"). Because these growth factors play an effective role in wound healing and tissue regeneration, platelet-rich plasma is a promising material in the field of regenerative medicine (see, for example, Patent Document 1).

[0003] JP 2009-195739 A

[0004] Platelet-rich plasma (PRP) and its freeze-dried form (PRP-FD), which contain blood-derived growth factors as active ingredients, have been widely used in recent years for promoting tissue repair and anti-inflammatory treatment in the fields of regenerative medicine and orthopedics. In particular, the administration of autologous growth factors has attracted attention for its ability to promote the restoration of homeostasis in chronic inflammatory diseases such as joint diseases.

[0005] However, the development and manufacture of PRP preparations present several technical challenges. The first challenge is the desire to recover useful growth factors contained in plasma as efficiently as possible during PRP preparation. However, this process also poses the problem of undesirable proteolytic enzymes, such as matrix metalloproteinases (MMPs), particularly MMP-3 and MMP-9. Because these MMPs have the ability to degrade cartilage matrix, administration of these preparations for joint disease may promote tissue destruction as a side effect (a second challenge). A third challenge is ensuring the stability of the product's components during storage. Growth factors are susceptible to denaturation, inactivation, or degradation under storage conditions, which can reduce their physiological activity during use, potentially compromising the product's practicality and efficacy.

[0006] Thus, an important technical challenge in the production of PRP preparations is to simultaneously solve multiple independent and potentially conflicting goals: (1) highly efficient recovery of growth factors, (2) ensuring safe administration by reducing MMP content, and (3) maintaining component stability during storage. The present invention aims to provide a new preparation method that achieves a balance between these three goals.

[0007] The present invention also relates to a freeze-dried composition containing blood-derived growth factors. Freeze-dried PRP-FD is often used after production and undergoes preservation, distribution, and storage, during which time the growth factors may undergo structural denaturation or loss of physiological activity. Against this background, an objective of the present invention is to provide a freeze-dried composition containing blood-derived growth factors that has excellent storage stability.

[0008] The present invention also relates to a blood-derived growth factor-containing composition itself. After production, PRP is often used after going through a process of preservation, distribution, and storage, during which the structure of growth factors may be altered or their physiological activity may be lost. In light of this, an object of the present invention is to provide a blood-derived growth factor-containing composition with excellent storage stability.

[0009] The present invention, which solves the above-mentioned problems, is as follows: [1] A method for preparing a growth factor-containing composition from blood, comprising the steps of: separating platelet-rich plasma not containing buffy coat components from the blood; and adding citric acid and / or a salt thereof to the blood or a processed product thereof.

[0010] [2] The method according to [1], further comprising a step of freeze-drying the platelet-rich plasma.

[0011] [3] The method according to [2], wherein the addition step is carried out so that the content of citric acid or the like in the freeze-dried composition after the freeze-drying is preferably 1% by mass or more.

[0012] [4] The method according to any one of [1] to [3], wherein the total amount of citric acid or the like added in the adding step is 0.1 mg or more per 1 ml of the blood used for preparing the growth factor-containing composition.

[0013] [5] The method according to any one of [1] to [4], wherein the step of separating platelet-rich plasma from the blood comprises subjecting the blood to a first centrifugation treatment at 100 to 1000 G for 1 to 30 minutes, and recovering an upper layer not containing buffy coat.

[0014] [6] The method according to any one of [1] to [5], further comprising a step of activating the platelet-rich plasma.

[0015] [7] The method according to any one of [1] to [6], which comprises a step of removing cells from the platelet-rich plasma.

[0016] [8] The method according to any one of [1] to [7], which comprises using citric acid and / or a salt thereof as an anticoagulant, and does not use heparin or a salt thereof as an anticoagulant.

[0017] [9] A blood-derived growth factor-containing composition containing citric acid and / or a salt thereof, prepared by the method according to any one of [1] to [8].

[0018]

[10] The composition according to [9], which is a freeze-dried composition containing blood-derived growth factors.

[0019]

[11] A blood-derived growth factor-containing composition comprising platelet-derived growth factor, epidermal growth factor, anti-inflammatory growth factor, fibroblast growth factor, vascular endothelial growth factor, and citric acid and / or a salt thereof, wherein the content of the citric acid and / or a salt thereof is 0.001% by mass or more.

[0020]

[12] The blood-derived growth factor-containing composition according to any one of [9] to

[11] , wherein the content of citric acid and / or a salt thereof is 1% by mass or more.

[0021]

[13] The blood-derived growth factor-containing composition according to any one of [9] to

[12] , which is substantially free of heparin or a salt thereof.

[0022]

[14] The blood-derived growth factor-containing composition according to any one of

[11] to

[13] , wherein the citric acid is derived from citric acid used as an anticoagulant for the blood.

[0023]

[15] The blood-derived growth factor-containing composition according to any one of

[11] to

[14] , wherein the blood-derived growth factor-containing composition is a freeze-dried composition.

[0024]

[16] A stabilizer for a blood-derived growth factor-containing composition, comprising citric acid and / or a salt thereof.

[0025]

[17] The stabilizer for a blood-derived growth factor-containing composition according to

[16] , wherein the mass ratio of the dextrose content to the mass of the citric acid and / or a salt thereof is taken as 1 is 0.9 or less.

[0026]

[18] The stabilizer for a blood-derived growth factor-containing composition according to

[16] or

[17] , wherein the blood-derived growth factor-containing composition is a freeze-dried composition.

[0027]

[19] The stabilizer for a blood-derived growth factor-containing composition according to any one of

[16] to

[18] , which is also an anticoagulant.

[0028] The preparation method of the present invention makes it possible to prepare a preparation that improves the recovery efficiency of growth factors contained in plasma, while suppressing the content of undesirable proteases such as MMP-3 and MMP-9, and that has excellent storage stability. This makes it possible to provide a PRP preparation that is safer and more effective for treating joint diseases and other conditions.

[0029] Furthermore, the freeze-dried composition containing blood-derived growth factors of the present invention has excellent storage stability in the amount of growth factors and can retain growth factors for a long period of time, making it extremely useful as a highly reproducible and reliable treatment method in the medical field.

[0030] 1 is a graph showing the results of Test Example 1. The graph shows the average values ​​of MMP-3 concentration in the lyophilized products of Examples (n=11) and Comparative Example (n=11). The MMP-3 concentration is shown as a relative amount when the Comparative Example is set to 1. This graph shows the results of Test Example 1. The graph shows the average values ​​of MMP-9 content in the lyophilized products of Examples (n=11) and Comparative Example (n=11). The MMP-9 concentration is shown as a relative amount when the Comparative Example is set to 1. This graph shows the results of Test Example 2. The citric acid content per 1 g of lyophilized product (open lines) and the relative amount of PDGF-BB extracted from 1 mL of blood (filled lines) are shown. The relative amount of PDGF-BB is the relative amount when the PDGF-BB concentration in Example 2 is set to 1. This graph shows the results of Test Example 3. The vertical axis shows storage stability. Storage stability is shown as the ratio of the amount of PDGF-BB in the product stored at 50°C to that in the product stored at 22°C. This graph shows the results of Test Example 4. The vertical axis represents storage stability. Storage stability is shown as the ratio of the amount of PDGF-BB in the sample stored at 50°C to that in the sample stored at 22°C. This is a graph showing the results of Test Example 5. The vertical axis represents storage stability. Storage stability is shown as the ratio of the amount of PDGF-BB in the sample stored at 50°C to that in the sample stored at 22°C.

[0031] The present invention will be described in detail below. In the embodiments of the present invention, A (numerical value) to B (numerical value) means A or more and B or less. The preferred and more preferred embodiments exemplified below can be used in appropriate combinations, regardless of expressions such as "for example," "preferred," and "more preferred." Numerical ranges are merely examples, and ranges obtained by appropriately combining the upper and lower limits of each range and the numerical values ​​of the examples can also be used (for example, when A to B or C to D is stated, the combinations A to D or C to B can be used). Furthermore, terms such as "contain" or "comprise" may be interpreted as "essentially consisting of" or "consisting only of."

[0032] Method for preparing a composition containing blood-derived growth factors The method for preparing a composition containing blood-derived growth factors of the present invention comprises at least a step of separating platelet-rich plasma that does not contain buffy coat components from blood (hereinafter also referred to as the "separation step"), and a step of adding citric acid and / or a salt thereof to the blood or a processed product thereof (hereinafter also referred to as the "addition step").

[0033] The blood to be used is not particularly limited as long as it contains growth factors. Examples of blood to be used include mammalian blood, and human blood is particularly preferred.

[0034] It is also preferable to use blood collected from an animal to which the blood-derived growth factor-containing composition prepared by the preparation method of the present invention is to be administered.

[0035] Each of the above steps will be described in detail below.

[0036] <Addition Step> The method of the present invention includes an addition step of adding citric acid and / or a salt thereof (hereinafter also referred to as "citric acid, etc.") to blood or a processed product thereof. Suitable examples of citric acid, etc. include metal salts of citric acid, more preferably sodium citrate. As the sodium citrate, one or more types selected from monosodium citrate, disodium citrate, and trisodium citrate can be used.

[0037] The timing of carrying out the addition step is not particularly limited, and it can be carried out at any stage in order to adjust the desired citric acid concentration in the final product, the freeze-dried composition. The addition step may be an embodiment in which citric acid or the like is added to the blood before the separation step or to the treated blood after the separation step.

[0038] In one embodiment, citric acid or the like is used as an anticoagulant. That is, in this embodiment, the addition step is performed on blood before the separation step. In this embodiment, citric acid or the like as an anticoagulant may be added to the collected blood afterwards, but it is preferable to collect the blood in a container such as a blood collection tube or a blood collection bag that contains citric acid or the like in advance.

[0039] When citric acid or the like is used as an anticoagulant, an embodiment can be made in which heparin or a salt thereof is not used as an anticoagulant. In this embodiment, the heparin or a salt thereof that is not used as a coagulant is not particularly limited. For example, an embodiment in which a heparin salt such as heparin sodium, heparin potassium, or heparin calcium is not used is preferred.

[0040] When citric acid or the like is used as an anticoagulant, it may be used in the form of a citric acid-dextrose (ACD) solution, or in the form of an aqueous solution of citric acid or the like that does not contain dextrose (glucose).

[0041] In the addition step, citric acid or the like may be added as a solid, or may be added as a solution dissolved in a solvent such as pure water (preferably a physiologically acceptable solvent).

[0042] When citric acid or the like (more specifically, citric acid and trisodium citrate) is added as an anticoagulant to the blood before the separation step, the total amount added relative to whole blood (1 mL) is preferably 0.1 mg or more, more preferably 1 mg or more, even more preferably 2 mg or more, even more preferably 3 mg or more, and most preferably 5 mg or more, and may also be 6 mg or more, 8 mg or more, or 10 mg or more. By setting the content of citric acid or the like within the above range, the storage stability of the freeze-dried composition after the freeze-drying process described below can be improved.

[0043] In one embodiment, citric acid or the like is added to the upper layer after the first centrifugation treatment described below. In one embodiment, citric acid or the like is added to the precipitate after the supernatant is removed after the second centrifugation treatment described below. In one embodiment, citric acid or the like is added to the freeze-dried composition after the freeze-drying treatment described below.

[0044] The addition step is preferably carried out so that the content of citric acid or the like in the freeze-dried composition after the freeze-drying treatment described below is preferably 0.5% by mass or more, more preferably 1% by mass or more, more preferably 1.5% by mass or more, even more preferably 1.8% by mass or more, even more preferably 2% by mass or more, even more preferably 2.2% by mass or more, even more preferably 2.4% by mass or more, even more preferably 2.6% by mass or more, even more preferably 2.8% by mass or more, even more preferably 3% by mass or more. By carrying out the addition step so that the content of citric acid or the like in the freeze-dried composition falls within the above range, the collection efficiency of the growth factor and the formulation properties (storage stability, solubility, etc.) of the freeze-dried composition can be improved, and a freeze-dried composition with a higher growth factor content can be prepared.

[0045] Furthermore, the addition step may be carried out so that the content of citric acid or the like in the freeze-dried composition after the freeze-drying treatment described below is preferably 10% by mass or less, more preferably 8% by mass or less, even more preferably 6% by mass or less, even more preferably 5% by mass or less, and even more preferably 4.6% by mass or less.

[0046] From the viewpoint of improving the storage stability of growth factors in the freeze-dried composition, it is preferable to carry out the addition step so that the content of citric acid and the like in the freeze-dried composition after the freeze-drying process described below is preferably 0.5% by mass or more, more preferably 1% by mass or more, more preferably 1.5% by mass or more, even more preferably 1.8% by mass or more, even more preferably 2% by mass or more, even more preferably 3% by mass or more, even more preferably 4% by mass or more, even more preferably 5% by mass or more, and even more preferably 6% by mass or more.

[0047] In one embodiment, the addition step is carried out so that the content of citric acid, etc. in the freeze-dried composition after the freeze-drying process described below falls within the above-mentioned range, due to the citric acid, etc. added during the process from before the separation step to the step of removing cells from platelet-rich plasma described below.

[0048] In one embodiment, the addition step is carried out so that the content of citric acid or the like in the freeze-dried composition after the freeze-drying process described below falls within the above-mentioned range, using citric acid or the like added to a subject containing plasma-derived cells (specifically, platelets).

[0049] From the viewpoint of improving the storage stability of growth factors in the freeze-dried composition, the addition step is preferably carried out before the platelet concentration and purification treatment described below, more preferably before the second centrifugation treatment described below, more preferably before the first centrifugation treatment described below, and even more preferably to the blood before the separation step, so that the content of citric acid, etc. in the freeze-dried composition after the freeze-drying treatment described below falls within the above-mentioned range.

[0050] The total amount of citric acid etc. added in the addition step is such that the content of citric acid etc. in the freeze-dried composition prepared from 1 ml of whole blood treated by the method of the present invention is preferably 0.05 mg or more, preferably 0.1 mg or more, more preferably 0.15 mg or more, even more preferably 0.2 mg or more, even more preferably 0.24 mg or more, even more preferably 0.27 mg or more, even more preferably 0.3 mg or more, even more preferably 0.32 mg or more, even more preferably 0.34 mg or more, even more preferably 0.36 mg or more. By performing the addition step so that the total amount of citric acid etc. added falls within the above range, the efficiency of growth factor collection can be improved, and a freeze-dried composition with a higher growth factor content can be prepared.

[0051] There is no particular upper limit to the total amount of citric acid, etc. added in the addition step. The content of citric acid, etc. in the freeze-dried composition prepared from 1 ml of whole blood treated by the method of the present invention is preferably 30 mg or less, more preferably 20 mg or less, even more preferably 10 mg or less, even more preferably 5 mg or less, even more preferably 1 mg or less, and even more preferably 0.8 mg or less. By performing the addition step so that the total amount of citric acid, etc. added falls within the above range, the efficiency of growth factor collection can be improved, and a freeze-dried composition with a higher growth factor content can be prepared.

[0052] From the viewpoint of improving the storage stability of growth factors in the freeze-dried composition, the total amount of citric acid etc. added in the addition step is such that the content of citric acid etc. in the freeze-dried composition prepared from 1 ml of whole blood treated by the method of the present invention is preferably 0.05 mg or more, preferably 0.1 mg or more, more preferably 0.2 mg or more, even more preferably 0.3 mg or more, even more preferably 0.5 mg or more, even more preferably 0.7 mg or more, and even more preferably 0.9 mg or more.

[0053] The total amount of citric acid or the like added in the addition step is preferably 0.1 mg or more, more preferably 1 mg or more, even more preferably 2 mg or more, even more preferably 3 mg or more, and most preferably 5 mg or more relative to whole blood (1 mL), and may also be 6 mg or more, 8 mg or more, or 10 mg or more. By setting the content of citric acid or the like within the above range, the storage stability of the freeze-dried composition after the freeze-drying process described below can be improved.

[0054] As described above, the citric acid or the like in the addition step may be in the form of an ACD solution. Typically, the mass ratio of dextrose (glucose) to citric acid or the like in the ACD solution is about 0.7 to 0.8, when the mass content of citric acid or the like is 1. From the viewpoint of improving the storage stability of blood-derived growth factors, the addition step may be carried out so that the mass ratio of dextrose (glucose) to citric acid or the like in the lyophilized composition after the lyophilization treatment described below is preferably 0.9 or less, more preferably 0.8 or less, even more preferably 0.7 or less, even more preferably 0.5 or less, even more preferably 0.3 or less, even more preferably 0.1 or less, even more preferably 0.01 or less, and even more preferably 0, when the mass content of citric acid or the like is 1.

[0055] In one embodiment, the addition step is carried out so that the total amount of citric acid and the like added during the process from before the separation step to the step of removing cells from platelet-rich plasma described below falls within the above range.

[0056] In one embodiment, the addition step is carried out so that the content of citric acid or the like in the freeze-dried composition after the freeze-drying process described below falls within the above-mentioned range, using citric acid or the like added to a subject containing plasma-derived cells (specifically, platelets).

[0057] From the viewpoint of improving the storage stability of growth factors in the freeze-dried composition, the addition step is preferably carried out before the platelet concentration / purification treatment described below, more preferably before the second centrifugation treatment described below, more preferably before the first centrifugation treatment described below, and even more preferably to the blood before the separation step, so that the content of citric acid, etc. in the freeze-dried composition after the freeze-drying treatment described below falls within the above-mentioned range using citric acid, etc. More specifically, it is particularly preferable to carry out the addition step so that the content of citric acid, etc. in the freeze-dried composition after the freeze-drying treatment described below falls within the above-mentioned range using citric acid, etc. added as an anticoagulant.

[0058] <Pre-cooling Step> When the adding step is performed on blood before the separation step, the method may further include a step of pre-cooling the blood to which citric acid or the like has been added, prior to the step of separating platelet-rich plasma from the blood to which citric acid or the like has been added. The refrigeration temperature in the step of pre-cooling the blood is, for example, 0 to 10°C. The process time for the step of pre-cooling the blood is, for example, more than 0 hours and not more than 120 hours. The lower limit of the process time may be 30 minutes or more, 1 hour or more, 2 hours or more, 4 hours or more, or 8 hours or more. The upper limit of the process time may be 96 hours or less, 72 hours or less, 60 hours or less, 48 ​​hours or less, 36 hours or less, or 24 hours or less.

[0059] The method of the present invention includes a step of separating platelet-rich plasma that does not contain buffy coat components from blood. Here, "buffy coat" refers to a thin, white layer containing many leukocytes that forms between the red blood cells and the plasma fraction when blood is centrifuged.

[0060] During this separation process, the temperature of the blood and platelet-rich plasma is preferably controlled at 4 to 25°C.

[0061] There are no limitations on the means for separating platelet-rich plasma from blood (whole blood), and any known means can be used. Typically, platelet-rich plasma is obtained by subjecting the blood to a first centrifugation process at a relatively low speed to separate the blood into a fraction containing red blood cells, a buffy coat, platelet-rich plasma (PRP), and platelet-poor plasma. Preferably, the separation process includes a first centrifugation process at a relatively low speed and a second centrifugation process at a relatively high speed. The first centrifugation process and the second centrifugation process may each be performed independently multiple times. Alternatively, platelet-rich plasma may be obtained by the first centrifugation process alone at a relatively low speed. The first centrifugation process, the second centrifugation process, and the platelet concentration and purification process are described in detail below.

[0062] (First Centrifugation Treatment) The first centrifugation treatment, which is performed at a relatively low speed, is performed at 100 to 1200 G (corresponding to approximately 200 to 2000 rpm in a typical centrifuge). The rotation conditions are preferably 100 to 1100 G, more preferably 100 to 500 G. The treatment time may be 1 to 30 minutes, 5 to 20 minutes, or 5 to 15 minutes.

[0063] After a first centrifugation step at a relatively low speed, the upper layer containing the plasma fraction (platelet-rich plasma and platelet-poor plasma) is collected without the buffy coat.

[0064] In the step of recovering the upper layer, any volume can be recovered so as to contain as little as possible (e.g., visually) a fraction containing red blood cells and a buffy coat. Specifically, for example, the volume ratio of the upper layer to the total volume of the liquid is preferably 1:0.1 to 0.5, more preferably 1:0.2 to 0.5, and even more preferably 1:0.3 to 0.5.

[0065] Note that blood-derived growth factor-containing compositions prepared by recovering the upper layer so as to contain the buffy coat exhibit a reddish tinge. However, blood-derived growth factor-containing compositions prepared by recovering the upper layer so as not to contain the buffy coat, as in the present invention, do not exhibit a reddish tinge. The blood-derived growth factor-containing compositions prepared by recovering the upper layer so as to contain the buffy coat and the blood-derived growth factor-containing compositions of the present invention can be distinguished by the presence or absence of redness, thereby preventing medication errors in clinical settings. Furthermore, the absence of redness in the blood-derived growth factor-containing compositions of the present invention can reduce caution when administered to animals or humans, making them useful for smooth administration in clinical settings.

[0066] (Second Centrifugation) Platelets are pelleted by a second centrifugation at a relatively high speed from the upper layer collected after the first centrifugation.

[0067] The second centrifugation treatment at a relatively high speed is performed at 1000 to 2500 G (corresponding to approximately 2000 to 5000 rpm in a typical centrifuge). The rotation conditions may be 1100 to 2000 G or 1200 to 1500 G. The treatment time may be 1 to 30 minutes, 5 to 20 minutes, or 5 to 15 minutes.

[0068] (Platelet Purification and Concentration Treatment) The platelets can be purified and concentrated by removing the supernatant from the product obtained by the second centrifugation treatment and then suspending the platelets. Here, any volume of the supernatant can be removed in the step of removing the supernatant.

[0069] <Activation Step> The activation step is a step in which platelet-rich plasma is activated by applying a physical or chemical stimulus to promote the release of numerous growth factors and anti-inflammatory cytokines.

[0070] The method for activating platelet-rich plasma is not particularly limited, and any known method may be employed. For example, a method of contacting platelets with platelets in the presence of an agent that activates platelet-rich plasma (typically, calcium ion source compounds such as calcium chloride, calcium phosphate, calcium lactate, and calcium carbonate) may be employed. Alternatively, a method of contacting platelets with platelets in the presence of an agent that activates platelet-rich plasma (typically, platelet activators such as thrombin, adenosine diphosphate (ADP), and collagen) may be employed. Alternatively, a method of activating platelet-rich plasma by ultrasonic treatment may be employed. Alternatively, a method of activating platelet-rich plasma by freezing and thawing may be employed. Alternatively, a method of activating platelet-rich plasma by a combination of these methods (a method in which the above different agents are added simultaneously, or a method in which ultrasonic treatment and an agent are used in combination) may be employed.

[0071] The activated platelet-rich plasma may be resuspended by adding a solvent. The resuspending solvent may be any physiologically acceptable solvent. Examples include plasma, saline, buffered saline (e.g., phosphate-buffered saline (PBS)), and Ringer's solution (e.g., lactate Ringer's solution, acetate Ringer's solution, bicarbonate Ringer's solution). In this specification, any platelet resuspension resuspended in any solvent is referred to as "platelet-rich plasma." Furthermore, for the purpose of adjusting the concentration or incorporating additives, the above-mentioned solvent may be further added to the resuspension, and the liquid after the addition is also referred to as "platelet-rich plasma."

[0072] <Cell Removal Step> The method of the present invention may further comprise a step of removing blood cells from the platelet-rich plasma.

[0073] During this cell removal step, the platelet-rich plasma is preferably maintained at a temperature of 4 to 25°C.

[0074] Platelet-rich plasma contains platelets. According to the method of the present invention, the platelet-rich plasma may also contain white blood cells derived from the buffy coat that were not completely separated in the separation step, as well as a small amount of red blood cells.

[0075] Examples of means for removing blood cells include heat treatment, acid treatment, and filtering treatment. Among these, filtering treatment is preferred because it has a low risk of causing denaturation of growth factors. That is, this step is preferably a filtration step in which blood cells are removed from the platelet-rich plasma by filtering treatment.

[0076] There are no limitations on the filter used in the filtering process as long as it can remove blood cells. Since platelets have the smallest blood cell size at approximately 2 μm, membrane filters with pore sizes of 1 μm or less, 0.7 μm or less, or 0.5 μm or less can be used. While there is no lower limit on the pore size of the membrane filter, excessively small filters increase the risk of clogging. Therefore, the pore size is preferably 0.2 μm or more, 0.3 μm or more, or 0.4 μm or more.

[0077] The filtering process may be performed by centrifugation using a centrifuge tube equipped with the filter. The centrifugation may be performed at, for example, 800 to 3000 G (corresponding to approximately 1500 to 5200 rpm in a typical centrifuge). The process time may be, for example, 1 to 30 minutes, 5 to 15 minutes, or 10 minutes.

[0078] This treatment results in a filtered platelet-rich plasma that is substantially free of blood cells.

[0079] <Freeze-drying step> The method of the present invention may further comprise a step of freeze-drying the platelet-rich plasma.

[0080] The freeze-drying method is not particularly limited as long as it is a conventionally known freeze-drying method for proteins.

[0081] Freeze-drying involves freezing platelet-rich plasma and then subjecting it to a vacuum freeze-drying process.

[0082] In the freezing process, the temperature of the platelet-rich plasma or the ambient temperature is adjusted to below −60° C., below −70° C., or below −80° C. This may be accomplished by contact with liquid nitrogen or storage in a freezer.

[0083] The freezing process can be performed for just a moment. Typically, the freezing process is performed for 6 to 24 hours at the above-mentioned temperature to ensure that all of the platelet-rich plasma is frozen. That is, the interval between the start of the freezing process and the start of the vacuum freeze-drying process can be 0 to 24 hours or 6 to 24 hours.

[0084] The temperature conditions for the vacuum freeze-drying treatment are not particularly limited. The upper limit of the temperature conditions may be −30° C. or lower, −35° C. or lower, −40° C. or lower, or −45° C. or lower. The lower limit is not particularly limited, but may be −80° C. or higher, −75° C. or higher, −70° C. or higher, −65° C. or higher, −55° C. or higher, or −50° C. or higher.

[0085] The pressure conditions for the vacuum freeze-drying process are not particularly limited. The upper limit of the pressure conditions may be 20 Pa or less, 18 Pa or less, 16 Pa or less, or 15 Pa or less. The lower limit is 0 Pa or more, and may be 1 Pa or more, 2 Pa or more, 3 Pa or more, 4 Pa ​​or more, or 5 Pa or more.

[0086] The time for performing the vacuum freeze-drying process is not particularly limited and can be set according to the amount of liquid. The lower limit of the freeze-drying process time may be, for example, 1 hour or more, 2 hours or more, 3 hours or more, 4 hours or more, 5 hours or more, 6 hours or more, 7 hours or more, or 8 hours or more. On the other hand, the upper limit may be 24 hours or less, 20 hours or less, 15 hours or less, 10 hours or less, or 8 hours or less.

[0087] There are no particular limitations on the means for carrying out the vacuum freeze-drying process, and any means can be used. For example, the process may be carried out using FDU-1110 (manufactured by Tokyo Rikakikai Co., Ltd.).

[0088] After completion of the vacuum freeze-drying process, platelet-rich plasma can be obtained in the form of almost completely dry granules or powder.

[0089] Blood-derived growth factor-containing composition The blood-derived growth factor-containing composition according to the present invention is a blood-derived growth factor-containing composition prepared from blood.

[0090] The blood-derived growth factor-containing composition of the present invention is not limited in its form, so long as it contains citric acid and / or a salt thereof. The blood-derived growth factor-containing composition may be in the form of, for example, a platelet-containing fraction (platelet-rich plasma and / or platelet-poor plasma) primarily separated from whole blood, a platelet-containing pellet obtained by centrifuging the platelet-containing fraction, a purified platelet concentrate obtained by suspending the pellet in a pharmacologically acceptable aqueous medium such as Ringer's solution or physiological saline, or cell-free plasma obtained by removing cells from the purified platelet concentrate.

[0091] The blood-derived growth factor-containing composition of the present invention may also be in the form of a freeze-dried composition obtained by freeze-drying the blood-derived growth factor-containing composition in the form described above. This form will be described in detail in the next section.

[0092] The blood-derived growth factor-containing composition of the present invention can be applied to joints such as shoulders, elbows, limbs, skin, and eyes of humans and animals (including but not limited to mammals such as dogs and cats).

[0093] The blood-derived growth factor-containing composition of the present invention can be used for the treatment and / or prevention of joint diseases such as osteoarthritis, rotator cuff injuries, ligament injuries, tennis elbow, golfer's elbow, etc. in the fields of orthopedics, dentistry, cosmetic surgery, ophthalmology, and obstetrics and gynecology.

[0094] When used for therapeutic purposes, the blood-derived growth factor-containing composition can be used as is, or can be dissolved or diluted in a pharmacologically acceptable aqueous medium such as physiological saline.

[0095] The blood-derived growth factor-containing composition and the medicinal solution prepared by dissolving it can be used by injecting it into the affected area (intravenous administration, intramuscular administration, subcutaneous administration, intrathecal administration, vaginal administration, etc.) or by applying it to the affected area (transdermal administration, ocular instillation, nasal administration, transdermal absorption, etc.).

[0096] The various conditions and the like relating to the freeze-dried composition containing blood-derived growth factors described below can be read and applied to the composition containing blood-derived growth factors.

[0097] Lyophilized Composition Containing Blood-Derived Growth Factors The lyophilized composition containing blood-derived growth factors according to the present invention is a lyophilized product of a composition containing blood-derived growth factors prepared from blood.

[0098] The freeze-dried composition containing blood-derived growth factors of the present invention can be applied to joints such as shoulders, elbows, limbs, skin, and eyes of humans and animals (including but not limited to mammals such as dogs and cats).

[0099] The freeze-dried composition containing blood-derived growth factors of the present invention can be used for the treatment and / or prevention of joint diseases such as osteoarthritis, rotator cuff injuries, ligament injuries, tennis elbow, golfer's elbow, etc. in the fields of orthopedics, dentistry, cosmetic surgery, ophthalmology, and obstetrics and gynecology.

[0100] When used for therapeutic purposes, the freeze-dried composition containing blood-derived growth factors can be dissolved in a pharmacologically acceptable aqueous medium such as physiological saline. The freeze-dried composition containing blood-derived growth factors is preferably dissolved just before use.

[0101] The medicinal solution prepared by dissolving the freeze-dried composition containing blood-derived growth factors can be used by injecting it into the affected area (intravenous administration, intramuscular administration, subcutaneous administration, intrathecal administration, vaginal administration, etc.) or by applying it to the affected area (transdermal administration, ocular instillation, nasal administration, transdermal absorption, etc.).

[0102] <Citric Acid and / or Salts Thereof> The blood-derived growth factor-containing freeze-dried composition of the present invention contains citric acid and / or a salt thereof.

[0103] The lower limit of the content of citric acid or the like is preferably 0.5% by mass or more, 1% by mass or more, more preferably 1.5% by mass or more, even more preferably 1.8% by mass or more, even more preferably 2% by mass or more, even more preferably 2.2% by mass or more, even more preferably 2.4% by mass or more, even more preferably 2.6% by mass or more, even more preferably 2.8% by mass or more, even more preferably 3% by mass or more. A blood-derived growth factor-containing freeze-dried composition having a citric acid or the like content within the above range contains a larger amount of growth factor, and by adjusting the citric acid or the like content within the above range, the formulation properties (storage stability, solubility, etc.) can be improved, which is preferable.

[0104] The upper limit of the content of citric acid etc. is not particularly limited, but is preferably 20% by mass or less, more preferably 10% by mass or less, even more preferably 8% by mass or less, even more preferably 6% by mass or less, even more preferably 5% by mass or less, and even more preferably 4.6% by mass or less.

[0105] From the viewpoint of improving the storage stability of growth factors in the freeze-dried composition, the content of citric acid etc. is preferably 0.5% by mass or more, more preferably 1% by mass or more, more preferably 1.5% by mass or more, even more preferably 1.8% by mass or more, even more preferably 2% by mass or more, even more preferably 3% by mass or more, even more preferably 4% by mass or more, even more preferably 5% by mass or more, even more preferably 6% by mass or more.

[0106] The content of citric acid or the like in a freeze-dried composition prepared from 1 ml of raw whole blood is preferably 0.05 mg or more, preferably 0.1 mg or more, more preferably 0.15 mg or more, even more preferably 0.2 mg or more, even more preferably 0.24 mg or more, even more preferably 0.27 mg or more, even more preferably 0.3 mg or more, even more preferably 0.32 mg or more, even more preferably 0.34 mg or more, even more preferably 0.36 mg or more. A freeze-dried composition containing blood-derived growth factors having a content of citric acid or the like within the above range is preferred because it contains more growth factors and because adjusting the content of citric acid or the like within the above range can improve formulation properties (storage stability, solubility, etc.).

[0107] The content of citric acid and the like in the freeze-dried composition prepared from 1 ml of the raw material whole blood is preferably 30 mg or less, more preferably 10 mg or less, even more preferably 5 mg or less, even more preferably 1 mg or less, even more preferably 0.8 mg or less, and even more preferably 0.6 mg or less.

[0108] From the viewpoint of improving the storage stability of growth factors in the freeze-dried composition, the content of citric acid or the like in the freeze-dried composition prepared from 1 ml of the raw material whole blood is preferably 0.05 mg or more, preferably 0.1 mg or more, more preferably 0.2 mg or more, even more preferably 0.3 mg or more, even more preferably 0.5 mg or more, even more preferably 0.7 mg or more, and even more preferably 0.9 mg or more.

[0109] From the viewpoint of improving the storage stability of growth factors, it is preferable that the citric acid etc. contained in the freeze-dried composition is derived from the citric acid etc. used as a blood anticoagulant in the production process of the freeze-dried composition. More specifically, it is preferable that preferably 10% by mass or more, more preferably 30% by mass or more, even more preferably 50% by mass or more, even more preferably 70% by mass or more, even more preferably 90% by mass or more, and even more preferably 100% of the citric acid etc. contained in the freeze-dried composition is derived from the citric acid etc. used as an anticoagulant in the production process of the freeze-dried composition.

[0110] From the viewpoint of improving the storage stability of growth factors, the mass ratio of dextrose (glucose) to citric acid, etc. in the freeze-dried composition is preferably 0.9 or less, more preferably 0.8 or less, even more preferably 0.7 or less, more preferably 0.5 or less, even more preferably 0.3 or less, even more preferably 0.1 or less, even more preferably 0.01 or less, and even more preferably 0, when the mass ratio of citric acid, etc. is taken as 1.

[0111] <Growth Factors> The blood-derived growth factor-containing freeze-dried composition of the present invention contains platelet-derived growth factor, epidermal growth factor, anti-inflammatory growth factor, fibroblast growth factor, and vascular endothelial growth factor.

[0112] In one embodiment, the platelet-derived growth factor is PDGF-BB, the epidermal growth factor is EGF, the anti-inflammatory growth factor is TGF-β1, the fibroblast growth factor is bFGF, and the vascular endothelial growth factor is VEGF. Each of these growth factors is described below.

[0113] (Platelet-derived growth factor) The content of platelet-derived growth factor in the blood-derived growth factor-containing freeze-dried composition is preferably 1,000 pg / g or more, preferably 5,000 pg / g or more, more preferably 10,000 pg / g or more, even more preferably 20,000 pg / g or more, even more preferably 50,000 pg / g or more, and even more preferably 80,000 pg / g or more.

[0114] (Epidermal growth factor) The content of epidermal growth factor in the blood-derived growth factor-containing freeze-dried composition is preferably 300 pg / g or more, more preferably 1000 pg / g or more, even more preferably 3000 pg / g or more, even more preferably 6000 pg / g or more, even more preferably 8000 pg / g or more, even more preferably 10000 pg / g or more, and even more preferably 12000 pg / g or more.

[0115] (Anti-inflammatory growth factor) The content of anti-inflammatory growth factor in the blood-derived growth factor-containing freeze-dried composition is preferably 20 ng / g or more, more preferably 100 ng / g or more, even more preferably 300 ng / g or more, even more preferably 500 ng / g or more, and even more preferably 700 ng / g or more.

[0116] (Fibroblast growth factor) The content of fibroblast growth factor in the blood-derived growth factor-containing freeze-dried composition is preferably 30 pg / g or more, more preferably 100 pg / g or more, even more preferably 300 pg / g or more, even more preferably 500 pg / g or more, and even more preferably 800 pg / g or more.

[0117] (Vascular endothelial cell growth factor) The content of vascular endothelial cell growth factor in the blood-derived growth factor-containing freeze-dried composition is preferably 30 pg / g or more, more preferably 100 pg / g or more, even more preferably 300 pg / g or more, even more preferably 500 pg / g or more, even more preferably 800 pg / g or more, even more preferably 1000 pg / g or more, even more preferably 2000 pg / g or more, even more preferably 3000 pg / g or more, and even more preferably 4000 pg / g or more.

[0118] (Other Growth Factors) The blood-derived growth factor-containing freeze-dried composition of the present invention may contain other growth factors. The type of growth factor is not particularly limited, but examples include insulin-like growth factor (IGF) and hepatocyte growth factor (HGF).

[0119] <Inflammatory Cytokines> The blood-derived growth factor-containing lyophilized composition of the present invention may contain the inflammatory cytokines IL-1β, IL-6, and TNF-α. The contents of IL-1β and IL-6 are each independently preferably 300 pg / g or less, more preferably 200 pg / g or less, even more preferably 100 pg / g or less, and even more preferably 50 pg / g.

[0120] <Others> The freeze-dried composition containing blood-derived growth factors of the present invention may be embodied as being substantially free of heparin or a salt thereof. Here, "substantially free" means being below the detection limit by ELISA.

[0121] Heparin or a salt thereof is sometimes used as an anticoagulant in the preparation of a blood-derived growth factor-containing composition. However, heparin-induced thrombocytopenia (HIT) can occur in rare cases. Furthermore, because heparin or a salt thereof is generally obtained by purification from animals (pig intestines), administering it to a different species (human) poses an undeniable risk of unexpected infections. In an embodiment that is substantially free of heparin or a salt thereof, there is no risk of HIT and the composition can be provided without using components derived from different species. The heparin or a salt thereof that is substantially free of in this embodiment is not particularly limited. For example, an embodiment that is substantially free of heparin salts such as heparin sodium, heparin potassium, and heparin calcium is preferred.

[0122] In an embodiment that is substantially free of heparin or a salt thereof, as described above, it is preferable to prepare the solution using citric acid or the like as an anticoagulant, without using heparin or a salt thereof.

[0123] In one embodiment, the content of MMP-3 (matrix metalloproteinase-3) in the blood-derived growth factor-containing freeze-dried composition is preferably 110 ng / g or less, more preferably 100 ng / g or less, even more preferably 95 ng / g or less, and even more preferably 90 ng / g or less.

[0124] In one embodiment, the content of MMP-9 (matrix metalloproteinase-9) in the blood-derived growth factor-containing freeze-dried composition is preferably 500 ng / g or less, more preferably 400 ng / g or less, even more preferably 300 ng / g or less, even more preferably 200 ng / g or less, even more preferably 100 ng / g or less, and even more preferably 90 ng / g or less.

[0125] MMP-3 and MMP-9 are enzymes that degrade collagen and other components of cartilage. There is concern that administration of these enzymes to patients with joint diseases may worsen their symptoms. By adjusting the content of MMP-3 and / or MMP-9 in the blood-derived growth factor-containing freeze-dried composition to a low level as described above, the therapeutic and preventive effects of joint diseases can be improved.

[0126] By separating platelet-rich plasma that does not contain buffy coat components from blood in the separation step and using citric acid or the like as an anticoagulant instead of heparin or a salt thereof, the content of MMP-3 and / or MMP-9 can be adjusted to fall within the above-mentioned numerical range.

[0127] In one embodiment, the blood-derived growth factor-containing freeze-dried composition of the present invention and a composition obtained by dissolving the same in an aqueous medium such as physiological saline are not reddish. A blood-derived growth factor-containing freeze-dried composition prepared by recovering the upper layer containing the buffy coat in the separation step exhibits a reddish color, and can be distinguished from the blood-derived growth factor-containing freeze-dried composition of this embodiment by the presence or absence of redness. This prevents drug mix-ups in clinical settings. Furthermore, the absence of redness in the blood-derived growth factor-containing composition of the present invention reduces caution when administered to animals or humans, contributing to smooth administration in clinical settings.

[0128] In one embodiment, the freeze-dried composition containing blood-derived growth factors of the present invention has a lower protein content than a freeze-dried composition containing blood-derived growth factors prepared by recovering the upper layer containing the buffy coat in the separation process.

[0129] Stabilizer for Blood-Derived Growth Factor-Containing Composition The stabilizer for blood-derived growth factor-containing compositions of the present invention is characterized by containing citric acid and / or a salt thereof. As shown in Test Examples 3 to 5 described below, citric acid and the like have the effect of improving the stability of blood-derived growth factor-containing compositions. The stabilizer of the present invention can improve the stability, more specifically storage stability, and even more specifically high-temperature stability, of the blood-derived growth factors contained in the blood-derived growth factor-containing composition.

[0130] The stabilizer of the present invention is added during the preparation of a blood-derived growth factor-containing composition. The above-mentioned explanation regarding the addition step can be applied to specific embodiments of the method for adding the stabilizer of the present invention.

[0131] The dosage form of the stabilizer of the present invention is not particularly limited, and may be in the form of a powder or an aqueous solution. When the stabilizer is in the form of an aqueous solution, the content of citric acid and the like is not particularly limited, but may be, for example, 0.1 to 20% by mass, or 0.5 to 10% by mass, and can be approximately 1 to 5% by mass.

[0132] The stabilizer of the present invention may contain pharmacologically acceptable optional components, such as buffers, metal ions, salts, surfactants, preservatives, and sugar compounds.

[0133] Red blood cells use dextrose (glucose) as an energy source to generate intracellular ATP. By supplying dextrose to red blood cells, the survival time of red blood cells can be extended, and therefore, dextrose is sometimes incorporated into anticoagulants. The stabilizer of the present invention may also contain dextrose.

[0134] When the mass content of citric acid and / or a salt thereof in the stabilizer of the present invention is taken as 1, the mass content ratio of dextrose is preferably 0.9 or less, more preferably 0.7 or less, more preferably 0.5 or less, even more preferably 0.3 or less, even more preferably 0.1 or less, even more preferably 0.01 or less, and even more preferably 0.

[0135] The stabilizer of the present invention can be used as an anticoagulant for blood used in preparing a blood-derived growth factor-containing composition. The above explanation can be applied to specific aspects of its use as an anticoagulant.

[0136] The stabilizer of the present invention can be used to stabilize blood-derived growth factors contained in a blood-derived growth factor-containing composition. By adding and using the stabilizer of the present invention during the preparation process of a blood-derived growth factor-containing composition, the stability of the blood-derived growth factors can be improved.

[0137] The form of the blood-derived growth factor-containing composition to which the stabilizer of the present invention can be applied is not particularly limited. Specifically, the stabilizer of the present invention can be used to improve the stability of blood-derived growth factors in non-lyophilized compositions such as platelet-containing fractions (platelet-rich plasma and / or platelet-poor plasma) primarily separated from whole blood, platelet-containing pellets obtained by centrifuging the platelet-containing fraction, purified platelet concentrates obtained by suspending the pellets in a pharmacologically acceptable aqueous medium such as Ringer's solution or physiological saline, and cell-free plasma obtained by removing cells from the purified concentrate. The stabilizer of the present invention can also be used to improve the stability of blood-derived growth factors in blood-derived growth factor-containing freeze-dried compositions. The above explanations are applicable to blood-derived growth factor-containing compositions and blood-derived growth factor-containing freeze-dried compositions.

[0138] The present invention will be specifically explained below with reference to examples, but the present invention is not limited to these examples.

[0139] <Test Example 1> (Preparation of Example) Whole blood was collected from each of three individuals into blood collection tubes containing an acid citrate-dextrose (ACD) solution. These were refrigerated and stored in a thermostatic chamber at 4°C overnight (approximately 16 hours). After storage, a first centrifugation treatment (300 G, 10 minutes) was performed, and the supernatant, i.e., plasma, was collected. Care was taken to ensure that the buffy coat was not included in the plasma collection.

[0140] This buffy coat component-free plasma was subjected to a second centrifugation treatment (1400 G, 10 minutes) to separate it into a pellet and a supernatant. After separation, the supernatant was retained and the remaining supernatant (plasma) was removed. The pellet was suspended in the remaining supernatant to obtain a suspension (platelet-rich plasma).

[0141] This platelet-rich plasma was subjected to activation treatment, and 6.5 mL of lactated Ringer's solution was added to each sample to stabilize the platelet-rich plasma, followed by resuspension.

[0142] The platelet-rich plasma was transferred to a syringe and filtered through a membrane filter. This filtration separated the platelet-rich plasma into cells such as platelets that remained on the filter and platelet-rich plasma that passed through the filter. The platelet-rich plasma was transferred to a vial, frozen at -60°C, and stored frozen at the same temperature for 24 hours.

[0143] Each vial was subjected to vacuum freeze-drying treatment under conditions of about 5 Pa and about −45° C. for about 16 hours, thereby obtaining samples of the example (n=3).

[0144] Sodium citrate was added as needed at any stage after the first centrifugation to obtain the freeze-dried product, thereby adjusting the amount of citric acid in the freeze-dried product as the final product.

[0145] (Preparation of Comparative Example) Whole blood was collected from three subjects into blood collection tubes containing sodium heparin. These were refrigerated and stored overnight (approximately 16 hours) in a constant temperature bath at 4°C. After storage, a first centrifugation treatment (300G, 10 minutes) was performed to recover the supernatant, i.e., plasma. This plasma was then subjected to a second centrifugation treatment (1400G, 10 minutes) to remove the supernatant (approximately 1 mL of supernatant was left behind). The pellet was resuspended in the remaining supernatant to obtain a resuspension (platelet-rich plasma). 1 mL of this platelet-rich plasma was activated, and lactated Ringer's solution was added to stabilize the platelet-rich plasma. The mixture was then thoroughly stirred to obtain 7.5 mL of platelet-rich plasma.

[0146] After standing for a predetermined time, the platelet-rich plasma was transferred to a syringe and filtered through a membrane filter. This filtering separated the platelet-rich plasma into cells such as platelets that remained on the filter and the liquid that passed through the filter (cell-free plasma). The cell-free plasma was dispensed into vials, frozen at -80°C, and stored frozen at the same temperature for 12 days.

[0147] Each vial was subjected to vacuum freeze-drying treatment under conditions of about 5 Pa and about −45° C. for about 16 hours, thereby obtaining comparative samples (n=3).

[0148] (Measurement) The citric acid content in the lyophilized products of the Examples and Comparative Examples was measured using a colorimetric method. The results were as follows. The total amount of citric acid added per mL of blood used in the Examples was 0.1 mg or more, and the citric acid content per g of the lyophilized product was 3.12 mass%. Example: 31.2 mg / g Comparative Example: below the detection limit

[0149] Furthermore, as a result of measurement by ELISA, it was confirmed that the freeze-dried product of the Example contained about 20 times more platelet-derived growth factor (PDGF-BB), about 13 times more epidermal growth factor (EGF), about 11 times more anti-inflammatory growth factor (TGF-β1), about 8 times more fibroblast growth factor (bFGF), and about 7 times more vascular endothelial growth factor (VEGF) than the freeze-dried product of the Comparative Example.

[0150] Furthermore, as a result of measurement by the same ELISA method, the contents of MMP-3 and MMP-9 in the freeze-dried product of the Example were less than 75% and less than 14%, respectively, of those in the Comparative Example.

[0151] Furthermore, the lyophilized products of the examples containing citric acid exhibited excellent formulation properties.

[0152] In the above-mentioned test example, the conditions for the first centrifugation treatment were "300 G (10 minutes)", but these conditions were changed to "100 G (1 minute, 5 minutes, 10 minutes, 30 minutes), 500 G (1 minute, 5 minutes, 10 minutes, 30 minutes), 700 G (1 minute, 5 minutes, 10 minutes, 30 minutes), and 1000 G (1 minute, 5 minutes, 10 minutes, 30 minutes)", and the freeze-dried products of the examples and comparative examples were similarly produced.

[0153] The contents of the above-mentioned various growth factors, as well as MMP-3 and MMP-9, were measured for the lyophilized products produced under the modified conditions. As a result, it was confirmed that the contents of these growth factors were higher in the lyophilized products of the Examples than in the Comparative Examples. It was also confirmed that the contents of these MMPs were lower in the lyophilized products of the Examples than in the Comparative Examples. In other words, as with the above-mentioned Test Examples, it was confirmed that the lyophilized products of the Examples exhibited superior effects to the Comparative Examples, even when produced under the modified conditions.

[0154] Furthermore, the lyophilized product of the example containing citric acid, which was produced under modified conditions, also showed excellent formulation properties.

[0155] The lyophilized products of the Example (n=11) and Comparative Example (n=11), both prepared by the same method as described above, were analyzed for MMP-3 and MMP-9 contents using ELISA. The results are shown in Figures 1 and 2. As shown in Figure 1, the MMP-3 content in the lyophilized product of the Example was approximately 0.75 times that of the Comparative Example. As shown in Figure 2, the MMP-9 content in the lyophilized product of the Example was approximately 0.14 times that of the Comparative Example. In other words, the lyophilized product of the Example was confirmed to have a significantly improved administration safety due to the reduced MMP content.

[0156] Test Example 2 Lyophilized products were prepared in the same manner as in Test Example 1, except that the total amount of citric acid and other ingredients added in any step from before the separation step to the step of removing cells from platelet-rich plasma was changed to six patterns (Examples 2 to 7) (n=3 for each pattern). The total amount of citric acid added per mL of blood used in the test is as shown in Table 1.

[0157] The citric acid content and platelet-derived growth factor (PDGF-BB) content in the lyophilized product were measured by the same method as in Test Example 1. The citric acid content per 1 g of the lyophilized product, the citric acid content in the lyophilized product prepared from 1 ml of blood used in the test, and the relative amount of PDGF-BB that could be extracted from 1 ml of blood, calculated from the measurement results, are shown in Table 1 and Figure 3.

[0158]

[0159] As shown in Table 1 and Figure 3, the content of growth factors in the freeze-dried product tends to increase depending on the amount of citric acid added. This result indicates that the efficiency of growth factor extraction can be improved by adjusting the amount of citric acid added.

[0160] 3, the growth factor content in the freeze-dried product of Example 6 is significantly higher than that of Example 5. This result indicates that there is a critical point between the amount of citric acid added in Example 5 and the amount of citric acid added in Example 6 that significantly improves the efficiency of growth factor collection.

[0161] Furthermore, similarly to the Example in Test Example 1, the contents of growth factors and the like were also measured by ELISA for the freeze-dried products of Examples 2 to 7 in Test Example 2. As a result, it was confirmed that the freeze-dried products of Examples 2 to 7 had higher contents of platelet-derived growth factor (PDGF-BB), epidermal growth factor (EGF), anti-inflammatory growth factor (TGF-β1), fibroblast growth factor (bFGF), and vascular endothelial growth factor (VEGF), and had lower contents of MMP-3 and MMP-9, compared to the freeze-dried product of the Comparative Example in Test Example 1. In other words, it was demonstrated that the freeze-dried products of Examples 2 to 7 were superior to the freeze-dried product of the Comparative Example.

[0162] Test Example 3: The purpose of this test was to evaluate the effect of different citric acid contents in lyophilized preparations on the storage stability of growth factors. Specifically, several lyophilized preparations were prepared by varying the amount of citric acid added, and after storage at specified temperatures, the remaining amounts of growth factors (PDGF-BB and EGF) were compared to examine the relationship between the citric acid content and the storage stability of growth factors.

[0163] Blood was collected from four healthy volunteers, and a total of 10 blood collection tubes were used for each example (3 tubes for each of 3 volunteers, 1 tube for 1 volunteer). Blood collection tubes containing ACD solution were used for the test. In Example 8, the amount of citric acid was adjusted by removing the ACD solution from the blood collection tube before blood collection (the amount of citric acid finally used is shown in Table 2). In Example 9, no manipulation was performed on the ACD solution, and in Example 10, blood collection was similarly performed without any manipulation. Approximately 8.5 mL of whole blood was collected in each blood collection tube.

[0164] After blood collection, all blood collection tubes were left standing overnight at 4° C. In Example 10, after this refrigerated standing, a new ACD solution was added to each blood collection tube and the tubes were mixed by inversion to adjust the amount of citric acid (the amount of citric acid remaining in the final lyophilized product is shown in Table 2).

[0165] Subsequently, all test tubes were centrifuged at 500 G for 10 minutes, and the resulting plasma was pooled for each experiment. The pooled plasma was dispensed into 15 mL tubes (4 mL each) and subjected to a secondary centrifugation. After the secondary centrifugation, all but 1.0 mL of the supernatant was removed from each tube, and 6.5 mL of Ringer's solution (lactated Ringer's solution) was added to each tube. Cell-free plasma samples were then prepared using the same procedure as in Test Example 1.

[0166] Each sample obtained was dispensed into a vial in an amount of 2 mL or 1.5 mL, frozen at -60°C, and then freeze-dried (overnight). The freeze-dried formulations were sealed and allowed to stand at 22°C (room temperature) or 50°C for 7 days. After storage, the formulations were frozen and stored in a -60°C freezer for 24 hours or more.

[0167] To evaluate the lyophilized product, it was dissolved in 2.0 mL (or 1.5 mL in part) of water for injection, and the PDGF-BB content was measured by ELISA. The citric acid content in the lyophilized product was also quantified. Storage stability was calculated as the ratio of the factor amount of the product stored at 50°C to that of the product stored at 22°C. The results are shown in Table 2 and Figure 4.

[0168]

[0169] As shown in Table 2 and Figure 4, the storage stability, more specifically the high-temperature stability, tended to improve depending on the citric acid content in the lyophilized product. In particular, it was found that a remarkable effect of over 90% stability was obtained when the citric acid content per 1 g of the lyophilized product was 1.85% by mass or more.

[0170] Furthermore, a stability test similar to that in Test Example 3 was also performed on the solution before freeze-drying, i.e., the filter-passed fraction of the platelet resuspension solution, and it was confirmed that results comparable to those of the dry composition were obtained.

[0171] <Test Example 4> The purpose of this test was to verify whether the effect of improving the storage stability of growth factors, which was confirmed in Test Example 3 and depends on the citric acid content in the freeze-dried preparation, is maintained even when the timing of adding citric acid is changed.

[0172] In Test Example 4, tests were conducted on Examples 11 to 13, which used blood collection tubes containing an ACD solution. The amount of ACD solution already contained in the blood collection tubes used in Examples 11 to 14 was the same as that in Example 8 of Test Example 3.

[0173] Except for using the above blood collection tube, the procedure up to removal of the supernatant after the second centrifugation was the same as in Test Example 1. Thereafter, 4.5 ml of Ringer's solution was added to the sample of Example 11.

[0174] On the other hand, different amounts of ACD solution and Ringer's solution were added to the samples of Examples 12 to 14. The amount of ACD solution added was the smallest in Example 12 and the largest in Example 14. The total amount of ACD solution and Ringer's solution added to each sample of Examples 12 to 14 was the same as the amount of Ringer's solution added to the sample of Example 11 (4.5 ml). The total amount of citric acid used in this process is shown in Table 3.

[0175] The samples after addition of Ringer's solution were treated in the same manner as in Test Example 3 to obtain lyophilized products of Examples 11 to 14. The PDGF-BB and citric acid contents of these lyophilized products were measured and their storage stability was evaluated in the same manner as in Test Example 3. The results are shown in Table 3 and FIG.

[0176]

[0177] As shown in Table 3 and Figure 5, there was a tendency for the storage stability, more specifically the high-temperature stability, to improve depending on the citric acid content in the lyophilized product, as in Test Example 3. This result demonstrated that the effect of improving the storage stability of growth factors in the lyophilized preparation, which is dependent on the citric acid content, is maintained regardless of the timing of addition of citric acid.

[0178] Comparing Test Example 3 and Test Example 4, it is shown that even when the citric acid content per gram of lyophilized product is relatively low, a significantly high stability-improving effect can be obtained in the Examples of Test Example 3. This result indicates that even when the same citric acid content per gram of lyophilized product is achieved, the earlier the citric acid is added, the greater the stabilizing effect of the growth factors in the lyophilized product.

[0179] Furthermore, a stability test similar to that in Test Example 4 was also conducted on the solution before freeze-drying, i.e., the filter-passed fraction of the platelet resuspension solution, and it was confirmed that results comparable to those of the dry composition were obtained.

[0180] <Test Example 5> The purpose of this test was to verify that the effect of improving the storage stability of growth factors, which was confirmed in Test Example 3 and is dependent on the citric acid content in the freeze-dried preparation, is exerted purely depending on the amount of citric acid, without being affected by dextrose (glucose) contained in the ACD solution.

[0181] In Test Example 5, test tubes containing a citric acid-dextrose solution (hereinafter referred to as ACD solution) or a citric acid solution were used as an anticoagulant, and dextrose-free citric acid solution was added during the treatment to change the final citric acid content. Tests were conducted on Examples 15 to 18.

[0182] First, test tubes containing 1.5 ml of ACD solution as an anticoagulant (Examples 15 to 17) and test tubes containing 1.5 ml of citric acid solution (Example 18) were prepared. The composition of the citric acid solution used in this test example was as follows: Citric acid solution: an aqueous solution containing 22.0 mg / mL trisodium citrate and 8.0 mg / mL citric acid (dextrose-free). The only difference in composition between the citric acid solution and the ACD solution was the presence or absence of dextrose.

[0183] Approximately 8.5 mL of blood was collected into each test tube and allowed to stand overnight at 4°C. Then, the following operations were performed on each test tube. Example 15: No operation. Example 16: 1.5 mL of citric acid solution was added and mixed by inversion. Example 17: 3.5 mL of citric acid solution was added and mixed by inversion. Example 18: 1.5 mL of citric acid solution was added and mixed by inversion.

[0184] All test tubes were subjected to primary centrifugation (500 g, 10 minutes), and plasma was collected from each tube. The collected plasma was dispensed into 15 ml tubes (4 ml each). After secondary centrifugation, all but 1 ml of the supernatant was removed, and lactated Ringer's solution was added to the tube to a final volume of 5 ml. Cell-free plasma samples were then prepared using the same procedure as in Test Example 1.

[0185] Each sample obtained was dispensed in 2 mL portions into vials, frozen at -60°C, and then freeze-dried (overnight). The freeze-dried formulations were sealed and allowed to stand at 22°C (room temperature) or 50°C for 7 days.

[0186] After standing, the lyophilized material was reconstituted in 2.0 ml of water for injection, and the PDGF-BB content was measured by ELISA. Storage stability was calculated as the ratio of the factor amount in the sample stored at 50°C to that in the sample stored at 22°C. The results are shown in Table 4 and Figure 6. Table 4 also shows the amount of ACD solution or citric acid solution used as an anticoagulant, the amount of citric acid solution added to the collected blood after standing overnight, and the amount of citric acid added, calculated from the total amount and the concentration of citric acid, etc.

[0187]

[0188] As is clear from a comparison of Examples 15 to 17, it was observed that the storage stability improved depending on the citric acid content in the lyophilized product. This result suggests that even in the presence of dextrose used for initial anticoagulation, storage stability improved as the amount of citric acid added increased. Furthermore, very high stability was observed in Example 18, which did not use an ACD solution containing dextrose. These results demonstrate that the effect of improving the storage stability of growth factors in lyophilized formulations is due to the citric acid contained in the lyophilized product.

[0189] Furthermore, as shown in Table 4, Example 18 exhibited higher storage stability despite the same amount of citric acid added as Example 16. This result suggests that the smaller the ratio of dextrose to citric acid, etc., contained in the lyophilized product, the more stable the blood-derived growth factors in the lyophilized product.

[0190] Furthermore, a stability test similar to that in Test Example 5 was also performed on the solution before freeze-drying, i.e., the filter-passed fraction of the platelet resuspension solution, and it was confirmed that results comparable to those of the dry composition were obtained.

[0191] The freeze-dried composition containing blood-derived growth factors of the present invention can be used in regenerative medicine, etc., and in particular, can be used in PRP therapy for humans and animals.

[0192] The blood-derived growth factor-containing composition of the present invention can be used in regenerative medicine, etc., and in particular, can be used in PRP therapy, etc. for humans and animals.

Claims

1. A method for preparing a growth factor-containing composition from blood, comprising the steps of: separating platelet-rich plasma that does not contain buffy coat components from the blood; and adding citric acid and / or a salt thereof to the blood or a processed product thereof.

2. The method of claim 1, further comprising the step of freeze-drying the platelet-rich plasma.

3. The method according to claim 2, wherein the addition step is carried out so that the content of citric acid or the like in the freeze-dried composition after freeze-drying is preferably 1% by mass or more.

4. The method according to claim 1, wherein the total amount of citric acid or the like added in the adding step is 0.1 mg or more per 1 ml of the blood used to prepare the growth factor-containing composition.

5. The method of claim 1, wherein the step of separating platelet-rich plasma from the blood comprises subjecting the blood to a first centrifugation treatment at 100 to 1000 G for 1 to 30 minutes, and recovering an upper layer not containing buffy coat.

6. The method of claim 1, further comprising activating the platelet-rich plasma.

7. The method of claim 1, further comprising removing cells from the platelet-rich plasma.

8. The method of claim 1, comprising using citric acid and / or a salt thereof as an anticoagulant and not using heparin or a salt thereof as an anticoagulant.

9. A freeze-dried composition containing blood-derived growth factors, comprising citric acid and / or a salt thereof, prepared by the method according to any one of claims 1 to 8.

10. A blood-derived growth factor-containing freeze-dried composition comprising platelet-derived growth factor, epidermal growth factor, anti-inflammatory growth factor, fibroblast growth factor, vascular endothelial growth factor, and citric acid and / or a salt thereof, wherein the content of the citric acid and / or a salt thereof is 0.001% by mass or more.

11. The freeze-dried composition containing blood-derived growth factors according to claim 10, wherein the content of citric acid and / or a salt thereof is 1% by mass or more.

12. The blood-derived growth factor-containing freeze-dried composition according to claim 10, which is substantially free of heparin or a salt thereof.

13. A freeze-dried composition containing blood-derived growth factors according to any one of claims 10 to 12, wherein the citric acid is derived from citric acid used as an anticoagulant for the blood.

14. A stabilizer for a blood-derived growth factor-containing composition, comprising citric acid and / or a salt thereof.

15. A stabilizer for a blood-derived growth factor-containing composition according to claim 14, wherein the mass ratio of dextrose to the mass of citric acid and / or its salt is 1 is 0.9 or less.

16. The stabilizer for a blood-derived growth factor-containing composition according to claim 14, wherein the blood-derived growth factor-containing composition is a freeze-dried composition.

17. A stabilizer for a blood-derived growth factor-containing composition according to any one of claims 14 to 16, which is also an anticoagulant.

Images