Composition containing blood-derived growth factors and citric acid, and method for preparing the 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

Technical Field

[0001] The present invention relates to a growth factor-containing composition and a method for preparing the same.

Background Art

[0002] There is a treatment method using plasma enriched with platelets prepared by centrifuging one's own blood, platelet-rich plasma (hereinafter also referred to as "PRP"). This platelet-rich plasma contains a variety of growth factors such as platelet-derived growth factor (hereinafter also referred to as "PDGF"). Since 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 (for example, Patent Document 1).

Prior Art Documents

Patent Documents

[0003]

Patent Document 1

Summary of the Invention

Problems to be Solved by the Invention

[0004] PRP (platelet-rich plasma) having a blood-derived growth factor as an active ingredient and its lyophilized product (PRP-FD) have been widely used in recent years from the viewpoints of promoting tissue repair and anti-inflammatory treatment in the fields of regenerative medicine and orthopedics. In particular, for chronic inflammatory diseases such as joint diseases, attention has been focused on promoting the restoration of homeostasis by administering self-derived growth factors.

[0005] On the other hand, several technical challenges exist in the development and manufacturing of PRP preparations. The first challenge is the desire to recover useful growth factors contained in plasma as efficiently as possible during PRP preparation. However, this process has the problem that undesirable proteolytic enzymes, such as matrix metalloproteinases (MMPs), particularly MMP-3 and MMP-9, are also easily recovered. Since these MMPs have the activity to degrade cartilage matrix, they may exacerbate tissue destruction as a side effect when administered for joint diseases (the second challenge). Furthermore, a third challenge is ensuring the stability of the components during product storage. Growth factors are susceptible to denaturation, inactivation, or degradation under storage conditions, and there is a risk that the practicality and effectiveness of the product will be impaired due to a decrease in physiological activity at the time of use.

[0006] Thus, a key technical challenge in the manufacture of PRP preparations is to simultaneously solve several issues that are independent of each other and potentially conflicting: (1) highly efficient recovery of growth factors, (2) ensuring administration safety by reducing MMP content, and (3) maintaining component stability during storage. The present invention aims to provide a novel preparation method that harmonizes these three elements.

[0007] Furthermore, the present invention also relates to the freeze-dried composition containing blood-derived growth factors itself. Freeze-dried PRP-FD is often used after going through the processes of storage, distribution, and preservation after manufacturing, during which structural deformation of the growth factors and inactivation of their physiological activity may occur. Against this backdrop, one of the objectives of the present invention is to provide a freeze-dried composition containing blood-derived growth factors with excellent storage stability.

[0008] Furthermore, the present invention also relates to blood-derived growth factor-containing compositions themselves. After manufacturing, PRP is often used after going through storage, distribution, and preservation processes, during which structural denaturation or inactivation of physiological activity of growth factors may occur. Against this backdrop, one of the objectives of the present invention is to provide a blood-derived growth factor-containing composition with excellent storage stability. [Means for solving the problem]

[0009] The present invention, which solves the above problems, is as follows. [1] A method for preparing a growth factor-containing composition from blood, A step of separating platelet-rich plasma that does not contain buffy coat components from the aforementioned blood, A step of adding citric acid and / or its salt to the blood or processed product thereof, Methods that include...

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

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

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

[0013] [5] The method according to any one of [1] to [4], wherein the step of separating platelet-rich plasma from the blood is to perform a first centrifugation treatment on the blood at 100 to 1000 G for 1 to 30 minutes to recover the upper layer that does not contain the buffy coat.

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

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

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

[0017] [9] A blood-derived growth factor-containing composition comprising 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 a blood-derived growth factor.

[0019]

[11] A composition containing 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 the salt thereof is 0.001% by mass or more, A blood-derived growth factor-containing composition.

[0020]

[12] wherein the content of the citric acid and / or the salt thereof is 1% by mass or more, [9] to

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

[11] .

[0021]

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

[12] , 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 the citric acid used as an anticoagulant for the blood.

[0023] [[ID=三十一]]

[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 content mass ratio of dextrose when the content mass of the citric acid and / or the salt thereof is 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] A stabilizer for blood-derived growth factor-containing compositions as described in any one of

[16] to

[18] , which is also an anticoagulant. [Effects of the Invention]

[0028] According to the preparation method of the present invention, it is possible to prepare a formulation that improves the recovery efficiency of growth factors contained in plasma, suppresses the content of undesirable proteases such as MMP-3 and MMP-9, and has excellent storage stability. As a result, it is possible to provide a PRP formulation with higher safety and efficacy for joint diseases and other conditions that are the target of treatment.

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

[0030] [Figure 1] This graph shows the results of Test Example 1. It represents the average concentration of MMP-3 in the freeze-dried products of the Example (n=11) and Comparative Example (n=11). The concentration of MMP-3 is shown as a relative amount with the Comparative Example set to 1. [Figure 2] This graph shows the results of Test Example 1. It represents the average MMP-9 content in the freeze-dried products of the Example (n=11) and Comparative Example (n=11). The concentration of MMP-9 is shown as a relative amount with the Comparative Example set to 1. [Figure 3] This graph shows the results of Test Example 2. It represents the citric acid content per gram of freeze-dried material (outlined) and the relative amount of PDGF-BB obtained from 1 mL of blood (filled in black). The relative amount of PDGF-BB is the relative amount when the concentration of PDGF-BB in Example 2 is set to 1. [Figure 4]This graph shows the results of Test Example 3. The vertical axis represents storage stability. Storage stability is shown as the ratio of PDGF-BB content in the 50°C stored product to that in the 22°C stored product. [Figure 5] This graph shows the results of Test Example 4. The vertical axis represents storage stability. Storage stability is shown as the ratio of PDGF-BB content in the 50°C stored product to that in the 22°C stored product. [Figure 6] This graph shows the results of Test Example 5. The vertical axis represents storage stability. Storage stability is shown as the ratio of PDGF-BB in the 50°C stored product to the product stored at 22°C. [Modes for carrying out the invention]

[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 greater and B or less. Furthermore, preferred embodiments and more preferred embodiments illustrated below can be used in appropriate combinations with each other, regardless of expressions such as "for example," "preferred," and "more preferred." In addition, the numerical ranges are illustrative, 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, if A to B and C to D are written, the combination of A to D or C to B can be used). Furthermore, terms such as "contains" or "includes" may be read as "essentially" or "consists only of."

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

[0033] The type of blood used is not particularly limited as long as it contains growth factors. Mammalian blood is a good example of blood to use, and human blood is particularly preferred.

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

[0035] The following details each of the processes mentioned above.

[0036] <Addition process> The method of the present invention includes an addition step of adding citric acid and / or its salt (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, and more preferably sodium citrate. As sodium citrate, one or more selected from monosodium citrate, disodium citrate, and trisodium citrate can be used.

[0037] The timing of the addition step is not particularly limited and can be performed at any stage to adjust the desired citric acid concentration in the final freeze-dried composition. The addition step may also be an embodiment in which citric acid, etc., is added to the blood before the separation step or to the processed blood after the separation step.

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

[0039] When using citric acid or the like as an anticoagulant, embodiments may be used in which heparin or its salts are not used as the anticoagulant. The heparin or its salts not used as a coagulant in this embodiment are not particularly limited. For example, embodiments in which heparin salts such as heparin sodium, heparin potassium, and heparin calcium are not used are preferred.

[0040] When using citric acid or the like as an anticoagulant, it may be used in the form of a citrate-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 process, citric acid and the like may be added in solid form, or they may be added as a solution dissolved in a solvent such as pure water (preferably one that is physiologically acceptable).

[0042] When adding citric acid or the like (more specifically, citric acid and trisodium citrate) as an anticoagulant to the blood before the separation process, the total amount added per 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. It 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 treatment described later can be improved.

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

[0044] It is preferable to carry out the addition step such that the content of citric acid, etc., in the freeze-dried composition after the freeze-drying treatment described later 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, and even more preferably 3% by mass or more. By performing the addition step so that the content of citric acid and other substances in the freeze-dried composition falls within the above range, the efficiency of growth factor extraction and the formulation characteristics of the freeze-dried composition (storage stability, solubility, etc.) can be improved, making it possible to prepare a freeze-dried composition with a higher growth factor content.

[0045] Furthermore, the addition step may be carried out so that the content of citric acid, etc., in the freeze-dried composition after the freeze-drying treatment described later 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, etc., in the freeze-dried composition after the freeze-drying treatment described later 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 treatment described later is within the range described above, by adding citric acid, etc., in the process from before the separation step to the step of removing cells from platelet-rich plasma described later.

[0048] 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 treatment described later falls within the range described above, by adding citric acid, etc., to a target containing plasma-derived cells (specifically platelets).

[0049] From the viewpoint of improving the storage stability of growth factors in freeze-dried compositions, Preferably, prior to the platelet concentration and purification treatment described later, More preferably, prior to the second centrifugal treatment described later, More preferably, prior to the first centrifugation process described later, More preferably, with respect to the blood before the separation process, The addition step is carried out so that the content of citric acid, etc., in the freeze-dried composition after the freeze-drying treatment described later falls within the range described above.

[0050] The total amount of citric acid, etc., added in the addition step is such that the citric acid, etc. content 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, more preferably 0.2 mg or more, more preferably 0.24 mg or more, more preferably 0.27 mg or more, more preferably 0.3 mg or more, more preferably 0.32 mg or more, more preferably 0.34 mg or more, and more preferably 0.36 mg or more. By carrying out the addition process so that the total amount of citric acid and other substances added falls within the above range, the efficiency of growth factor extraction can be improved, and it becomes possible to prepare a freeze-dried composition with a higher growth factor content.

[0051] There is no particular upper limit on 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 carrying out the addition process so that the total amount of citric acid and other substances added falls within the above range, the efficiency of growth factor extraction can be improved, and it becomes possible to prepare a freeze-dried composition with a higher growth factor content.

[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, more preferably 0.3 mg or more, more preferably 0.5 mg or more, more preferably 0.7 mg or more, and more preferably 0.9 mg or more.

[0053] The total amount of citric acid, etc., 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, as a total amount added per whole blood (1 mL). It may also be 6 mg or more, 8 mg or more, or 10 mg or more. By setting the content of citric acid, etc. within the above range, the storage stability of the freeze-dried composition after the freeze-drying treatment described later can be improved.

[0054] As described above, the citric acid, etc., in the addition step may be in the form of an ACD solution. Typically, the mass ratio of dextrose (glucose) to citric acid, etc., in an ACD solution is about 0.7 to 0.8 when the mass of citric acid, etc. is set to 1. From the viewpoint of improving the storage stability of blood-derived growth factors, the addition step may be carried out such that the mass ratio of dextrose (glucose) to citric acid, etc., in the freeze-dried composition after the freeze-drying treatment described later 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 of citric acid, etc. is set to 1.

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

[0056] 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 treatment described later falls within the range described above, by adding citric acid, etc., to a target containing plasma-derived cells (specifically platelets).

[0057] From the viewpoint of improving the storage stability of growth factors in freeze-dried compositions, Preferably, prior to the platelet concentration and purification treatment described later, More preferably, prior to the second centrifugal treatment described later, More preferably, prior to the first centrifugation process described later, More preferably, with respect to the blood before the separation process, The addition step is carried out so that the content of citric acid, etc., in the freeze-dried composition after the freeze-drying treatment described later falls within the range described above. More specifically, it is particularly preferable to carry out the addition step such that the content of citric acid, etc., added as an anticoagulant in the freeze-dried composition after the freeze-drying treatment described later falls within the range described above.

[0058] <Pre-cooling process> When the addition step is performed on blood before the separation step, the process may further include a step of pre-cooling the blood to which citric acid, etc. has been added, before the step of separating platelet-rich plasma from the blood to which citric acid, etc. has been added. The refrigeration temperature in the pre-cooling step of the blood is, for example, 0 to 10°C. The process time for the pre-cooling step of the blood is, for example, more than 0 hours and 120 hours or less. 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] <Separation process> The method of the present invention includes the 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 white blood cells that forms between the red blood cells and the plasma fraction when blood is centrifuged.

[0060] During this separation process, it is preferable to control the temperature of the blood and platelet-rich plasma to between 4 and 25°C.

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

[0062] (First centrifugation process) The first relatively low-speed centrifugal separation process is performed at 100-1200G (corresponding to approximately 200-2000 rpm in a typical centrifuge). The rotational speed is preferably 100-1100G, more preferably 100-500G. The processing time is 1-30 minutes. You can set the time to 5-20 minutes or 5-15 minutes.

[0063] After a first centrifugation process at a relatively low speed, the upper layer containing the plasma fraction (platelet-rich plasma and platelet-poor plasma) is collected, ensuring that it does not contain the buffy coat.

[0064] The step of recovering the upper layer allows for the recovery of any volume such that the fraction containing red blood cells and the buffy coat are (for example, visually) excluded as much as possible. Specifically, for example, the volume ratio of the recovered upper layer to the total volume of 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] Furthermore, a blood-derived growth factor-containing composition prepared by collecting the upper layer to include the buffy coat has a reddish tint. However, a blood-derived growth factor-containing composition prepared by collecting the upper layer to exclude the buffy coat, as in the present invention, does not have a reddish tint. Since a blood-derived growth factor-containing composition prepared by collecting the upper layer to include a buffy coat can be distinguished from the blood-derived growth factor-containing composition of the present invention by the presence or absence of redness, it is possible to prevent medication errors in clinical settings. Furthermore, since the blood-derived growth factor-containing composition of the present invention is not red, it can reduce apprehension when administering it to animals or humans, and can be used to facilitate smooth administration in clinical settings.

[0066] (Second centrifugation process) The platelets, specifically the upper layer recovered after the first centrifugation process, are pelletized by a second, relatively high-speed centrifugation process.

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

[0068] (Platelet purification and concentration process) The platelets can be purified and concentrated by removing the supernatant from the mixture obtained by the second centrifugation process and then suspending it. In this step, any volume can be removed from the supernatant.

[0069] <Activation process> The activation process involves activating platelet-rich plasma by applying physical or chemical stimuli, thereby promoting 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 may be employed in which platelets are brought into contact with a platelet-rich plasma activating agent (generally, calcium ion source compounds such as calcium chloride, calcium phosphate, calcium lactate, and calcium carbonate). Alternatively, a method may be employed in which platelets are brought into contact with a platelet-rich plasma activating agent (generally, platelet activators such as thrombin, adenosine diphosphate (ADP), and collagen). Furthermore, a method may be employed in which platelet-rich plasma is brought into contact with a platelet-rich plasma activating agent.

[0071] Activated platelet-rich plasma may be resuspended by adding a solvent. Any physiologically acceptable solvent can be used for resuspending. Examples include plasma, physiological saline, buffered physiological saline (e.g., phosphate-buffered saline (PBS)), and Ringer's solution (e.g., lactated Ringer's solution, acetate Ringer's solution, bicarbonate Ringer's solution). In this specification, any platelet resuspension obtained by resuspending in any of these solvents will be referred to as "platelet-rich plasma." Furthermore, for purposes such as adjusting the concentration or adding additives, the above solvents may be added to this resuspension, and the resulting solution will also be referred to as "platelet-rich plasma."

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

[0073] During this cell removal process, it is preferable to maintain the platelet-rich plasma temperature at 4-25°C. .

[0074] Platelet-rich plasma contains platelets. Furthermore, according to the method of the present invention, the platelet-rich plasma may also contain leukocytes derived from the buffy coat that could not be completely separated in the separation step, as well as small amounts of erythrocytes.

[0075] Methods for removing blood cells include, for example, heat treatment, acid treatment, and filtering. Of these, filtering is preferred because it carries a low risk of causing denaturation of growth factors. In other words, this step is preferably a filtration step in which blood cells are removed from platelet-rich plasma by filtering.

[0076] There are no restrictions on the filters used for filtering, as long as they can remove blood cells. Since platelets are the smallest blood cells 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 pores increase the risk of clogging. Therefore, pore sizes of 0.2 μm or more, 0.3 μm or more, or 0.4 μm or more are preferable.

[0077] The filtering process may be carried out by centrifugation using a centrifugation tube equipped with the aforementioned filter. In this case, the centrifugation is performed at, for example, 800 to 3000 G (corresponding to approximately 1500 to 5200 rpm in a typical centrifuge). The process times are, for example, 1-30 minutes, 5-15 minutes, and 10 minutes.

[0078] As a result of this process, platelet-rich plasma that has passed through the filter and is substantially free of blood cells can be obtained.

[0079] <Lyophilization process> The method of the present invention may further include the step of freeze-drying 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 performing vacuum freeze-drying.

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

[0083] The freezing process can be performed for as short a time as it takes. Typically, it is carried out at the aforementioned temperature for 6 to 24 hours to ensure that all platelet-rich plasma is frozen. In other words, 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] There are no particular restrictions on the temperature conditions for vacuum freeze-drying. The upper limit of these temperature conditions may be -30°C or lower, -35°C or lower, -40°C or lower, or -45°C or lower. There are no particular restrictions on the lower limit, but -80°C or higher, -75°C or higher, -70°C or higher, -65°C or higher, -55°C or higher, or -5 It may be set to 0°C or higher.

[0085] There are no particular restrictions on the atmospheric pressure conditions for vacuum freeze-drying. The upper limit of these atmospheric 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 higher, and may be 1 Pa or higher, 2 Pa or higher, 3 Pa or higher, 4 Pa ​​or higher, or 5 Pa or higher.

[0086] There are no particular restrictions on the time required for vacuum freeze-drying, and the time can be set according to the liquid volume. The lower limit of the freeze-drying 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 restrictions on the means used to perform the vacuum freeze-drying process; any means can be used. For example, This can also be done using the FDU-1110 (manufactured by Tokyo Rikakikai Co., Ltd.).

[0088] After vacuum freeze-drying, nearly completely dried granular or powdered platelet-rich plasma can be obtained.

[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 form, as long as it contains citric acid and / or a salt thereof. The blood-derived growth factor-containing composition may be, for example, in the form of a platelet-containing fraction (platelet-rich plasma and / or platelet-poor plasma) that has been temporarily separated from whole blood, in the form of a platelet-containing pellet obtained by centrifuging the platelet-containing fraction, in the form of a purified platelet concentrate obtained by suspending the pellet in a pharmacokinetically acceptable aqueous medium such as Ringer's solution or physiological saline, or in the form of cell-free plasma obtained by removing cells from the purified concentrate.

[0091] Furthermore, 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 of the above-described form. This form will be described in detail in the following section.

[0092] The blood-derived growth factor-containing composition of the present invention can be applied to joints such as the shoulders, elbows, limbs, skin, and eyes in 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, and golfer's elbow 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 it can be dissolved or diluted in a pharmacokinetically acceptable aqueous medium such as physiological saline.

[0095] Compositions containing blood-derived growth factors and drug solutions prepared by dissolving them can be used by injection (intravenous, intramuscular, subcutaneous, intrathecal, vaginal, etc.) or topical application (transdermal, ophthalmic, nasal, transdermal absorption, etc.) to the affected area.

[0096] The various conditions and other details regarding the freeze-dried composition containing blood-derived growth factors described below can be applied by substituting them for any blood-derived growth factor-containing composition.

[0097] Freeze-dried composition containing blood-derived growth factors The blood-derived growth factor-containing freeze-dried composition according to the present invention is a freeze-dried product of a blood-derived growth factor-containing composition prepared from blood.

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

[0099] The blood-derived growth factor-containing freeze-dried 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, and golfer's elbow in the fields of orthopedics, dentistry, cosmetic surgery, ophthalmology, and obstetrics and gynecology.

[0100] When used for therapeutic purposes, the lyophilized composition containing blood-derived growth factors can be dissolved in a pharmacokinetically acceptable aqueous medium such as physiological saline before use. It is preferable to dissolve the lyophilized composition containing blood-derived growth factors immediately before use.

[0101] The drug solution prepared by dissolving a freeze-dried composition containing blood-derived growth factors can be used by injection (intravenous, intramuscular, subcutaneous, intrathecal, transvaginal, etc.) or topical application (transdermal, ophthalmic, nasal, transdermal absorption, etc.) to the affected area.

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

[0103] The lower limit of the content of citric acid, etc., 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, and even more preferably 3% by mass or more. A freeze-dried composition containing blood-derived growth factors with a citric acid content within the above range is preferable because it contains more growth factors, and adjusting the citric acid content within the above range can improve formulation properties (storage stability, solubility, etc.).

[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, and even more preferably 6% by mass or more.

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

[0107] The content of citric acid and other elements in the freeze-dried composition prepared from 1 ml of whole blood, which is the raw material, 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 and the like in the freeze-dried composition prepared from 1 ml of whole blood as the raw material is preferably 0.05 mg or more, preferably 0.1 mg or more, more preferably 0.2 mg or more, more preferably 0.3 mg or more, more preferably 0.5 mg or more, more preferably 0.7 mg or more, and still 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 citric acid, etc., that was used as an anticoagulant for blood during the manufacturing process. More specifically, it is preferable that of the citric acid, etc., contained in the freeze-dried composition, 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% is derived from citric acid, etc., that was used as an anticoagulant during the manufacturing 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, 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 of citric acid, etc. is set to 1.

[0111] <Growth factors> The present invention provides a freeze-dried composition containing blood-derived growth factors, platelet-derived growth factors, and epidermal growth factors. It contains anti-inflammatory growth factors, fibroblast growth factors, and vascular endothelial growth factors.

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

[0113] (platelet-derived growth factor) In the freeze-dried composition containing blood-derived growth factors, the content of platelet-derived growth factors is preferably 1000 pg / g or more, preferably 5000 pg / g or more, more preferably 10000 pg / g or more, even more preferably 20000 pg / g or more, even more preferably 50000 pg / g or more, and even more preferably 80000 pg / g or more.

[0114] (epidermal growth factor) In the freeze-dried composition containing blood-derived growth factors, the content of epidermal growth factor 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) In the freeze-dried composition containing blood-derived growth factors, the content of anti-inflammatory growth factors 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) In the freeze-dried composition containing blood-derived growth factors, the content of fibroblast growth factor 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 growth factor) In the freeze-dried composition containing blood-derived growth factors, the content of vascular endothelial growth factor 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 also 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 freeze-dried composition of the present invention may contain the inflammatory cytokines IL-1β, IL-6, and TNF-α. The content of IL-1β and IL-6 is 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, respectively.

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

[0121] In the preparation of blood-derived growth factor-containing compositions, heparin or its salts may be used as anticoagulants. However, heparin-induced thrombocytopenia (HIT) may rarely occur. Furthermore, since heparin or its salts are generally obtained by purification from animals (pig intestines), administering them to a different species (humans) carries an undeniable risk of unexpected infection. Embodiments that substantially do not contain heparin or its salts eliminate the risk of HIT and can be provided without using components derived from a different species. The heparin or its salts that are substantially excluded in these embodiments are not particularly limited. For example, embodiments that substantially do not contain heparin salts such as heparin sodium, heparin potassium, and heparin calcium are preferred.

[0122] In embodiments that substantially do not contain heparin or its salts, as described above, it is preferable to prepare the solution using citric acid or the like instead of heparin or its salts as an anticoagulant.

[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, and even more preferably 200 ng / g or less, more preferably 100 ng / g or less, even more preferably 90 ng It is less than / g.

[0125] MMP-3 and MMP-9 are enzymes that break down collagen and other components that make up cartilage. There are concerns that administering 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 the low levels described above, the therapeutic and preventive effects on joint diseases can be improved.

[0126] In the separation process, platelet-rich plasma free of buffy coat components is separated from the blood, and by using citric acid or the like instead of heparin or its salt as an anticoagulant, the content of MMP-3 and / or MMP-9 can be adjusted to the above-mentioned numerical range.

[0127] In one embodiment, the lyophilized blood-derived growth factor-containing composition of the present invention, and the composition obtained by dissolving it in an aqueous medium such as physiological saline, are not reddish. However, the lyophilized blood-derived growth factor-containing composition prepared by recovering the upper layer containing the buffy coat in the separation process exhibits redness, and can therefore be distinguished from the lyophilized blood-derived growth factor-containing composition of this embodiment by the presence or absence of redness. This helps to prevent drug mix-ups in clinical settings. Furthermore, since the blood-derived growth factor-containing composition of the present invention is not red, it can reduce apprehension when administering it to animals or humans, and can be used to facilitate smooth administration in clinical settings.

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

[0129] Stabilizer for blood-derived growth factor-containing compositions 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 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 of blood-derived growth factors contained in blood-derived growth factor-containing compositions, more specifically, their storage stability and even more specifically, their high-temperature stability.

[0130] The stabilizer of the present invention is used by adding it during the preparation process of a blood-derived growth factor-containing composition. The above-described explanation of the addition process can be directly 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 in the form of an aqueous solution, the content of citric acid, etc., is not particularly limited, but may be, for example, 0.1 to 20% by mass, 0.5 to 10% by mass, and 1 to 5% by mass as a guideline.

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

[0133] Red blood cells use dextrose (glucose) as an energy source to produce ATP within the cell. Because supplying dextrose to red blood cells extends their lifespan, it is sometimes included in anticoagulants. The stabilizer of the present invention may also contain dextrose.

[0134] When the mass content of citric acid and / or its salt in the stabilizer of the present invention is set to 1, the mass ratio of dextrose content 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 the preparation of blood-derived growth factor-containing compositions. The above description can be applied directly to specific embodiments of its use as an anticoagulant.

[0136] The stabilizer of the present invention can be used to stabilize blood-derived growth factors contained in blood-derived growth factor-containing compositions. By adding the stabilizer of the present invention during the preparation process of blood-derived growth factor-containing compositions, 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-freeze-dried compositions such as platelet-containing fractions (platelet-rich plasma and / or platelet-poor plasma) first separated from whole blood, platelet-containing pellets obtained by centrifuging the platelet-containing fraction, purified platelet concentrates obtained by suspending the pellets in a pharmacokinetically acceptable aqueous medium such as Ringer's solution or physiological saline, and cell-free plasma obtained by removing cells from the purified concentrate. Furthermore, the stabilizer of the present invention can be used to improve the stability of blood-derived growth factors in blood-derived growth factor-containing freeze-dried compositions. The above description can be applied directly to blood-derived growth factor-containing compositions and blood-derived growth factor-containing freeze-dried compositions. [Examples]

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

[0139] <Test Example 1> (Preparation of the examples) Whole blood samples were collected from each of the three individuals in blood collection tubes containing citrate-dextrose (ACD) solution. These were refrigerated in a constant temperature bath at 4°C overnight (approximately 16 hours). After storage, a first centrifugation procedure (300G, 10 minutes) was performed to collect the supernatant plasma. Care was taken to ensure that the buffy coat was not included in the plasma collection.

[0140] The plasma, which did not contain the buffy coat component, underwent a second centrifugation (1400G, 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 activated, and 6.5 mL of Ringer's lactate solution was added to stabilize it, followed by resuspension.

[0142] This was transferred to a syringe and filtered using a membrane filter. This filtering separated the platelet-rich plasma into platelets and other cells remaining on the filter and the platelet-rich plasma that passed through the filter. The platelet-rich plasma was transferred to a vial and frozen at -60°C, where it was stored for 24 hours.

[0143] Each vial was subjected to vacuum freeze-drying for approximately 16 hours under conditions of approximately 5 Pa and approximately -45°C. This yielded the sample for the example (n=3).

[0144] Furthermore, sodium citrate was added as appropriate at any stage after obtaining the freeze-dried product from the first centrifugation to adjust the amount of citric acid in the final freeze-dried product.

[0145] (Preparation of comparative examples) Whole blood samples were collected from three individuals using heparin sodium-containing blood collection tubes. These were refrigerated in a constant temperature bath at 4°C overnight (approximately 16 hours). After storage, a first centrifugation (300G, 10 minutes) was performed to collect the supernatant plasma. A second centrifugation (1400G, 10 minutes) was performed on this plasma to remove the supernatant (plasma) (approximately 1 mL of supernatant was left). The pellet was resuspended with the remaining supernatant to obtain a resuspension (platelet-rich plasma). This 1 mL of platelet-rich plasma was activated, and lactated Ringer's solution was added to stabilize the platelet-rich plasma. The mixture was thoroughly mixed to obtain 7.5 mL of platelet-rich plasma.

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

[0147] Each vial was subjected to vacuum freeze-drying for approximately 16 hours under conditions of approximately 5 Pa and approximately -45°C. This yielded comparative samples (n=3).

[0148] (measurement) The citric acid content in the freeze-dried products of the examples and comparative examples was measured using a colorimetric quantitative method. The results were as follows. In the examples, the total amount of citric acid added per 1 mL of blood was 0.1 mg or more, and the citric acid content per 1 g of freeze-dried product was 3.12% by mass. Example: 31.2 mg / g Comparative example: Below the detection limit

[0149] Furthermore, measurements using the ELISA method confirmed that the freeze-dried product of the example contained approximately 20 times more platelet-derived growth factor (PDGF-BB), approximately 13 times more epidermal growth factor (EGF), approximately 11 times more anti-inflammatory growth factor (TGF-β1), approximately 8 times more fibroblast growth factor (bFGF), and approximately 7 times more vascular endothelial growth factor (VEGF) compared to the comparative example.

[0150] Furthermore, measurements using the ELISA method revealed that the MMP-3 and MMP-9 content in the freeze-dried product of the example was less than 75% and less than 14%, respectively, compared to the comparative example.

[0151] Furthermore, the freeze-dried products of the examples containing citric acid showed excellent formulation properties.

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

[0153] The freeze-dried products manufactured under the modified conditions were tested for the content of the various growth factors mentioned above, as well as MMP-3 and MMP-9. The results confirmed that the freeze-dried products of the examples had higher content of these growth factors than the comparative examples. Furthermore, the freeze-dried products of the examples had lower content of these MMPs than the comparative examples. In other words, even when manufactured under modified conditions, the freeze-dried products of the examples showed superior effects compared to the comparative examples, similar to the test examples described above.

[0154] Furthermore, the freeze-dried product of the example containing citric acid, manufactured under modified conditions, also exhibited excellent formulation properties.

[0155] Figures 1 and 2 show the results of measuring the MMP-3 and MMP-9 content of the lyophilized samples of the Examples (n=11) and Comparative Examples (n=11), prepared using the same method as described above, by ELISA. As shown in Figure 1, the MMP-3 content in the lyophilized samples of the Examples was approximately 0.75 times that of the Comparative Examples. Also, as shown in Figure 2, the MMP-9 content in the lyophilized samples of the Examples was approximately 0.14 times that of the Comparative Examples. In other words, a significant improvement in administration safety was confirmed in the lyophilized samples of the Examples due to the reduction in MMP content.

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

[0157] The citric acid content and platelet-derived growth factor (PDGF-BB) content in the lyophilized product were measured using the same method as in Test Example 1. The citric acid content per gram of 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 collected from 1 ml of blood are shown in Table 1 and Figure 3, respectively, based on the measurement results.

[0158] [Table 1]

[0159] As shown in Table 1 and Figure 3, the amount of growth factors contained in the freeze-dried material tends to increase with 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] Furthermore, as shown in Table 1 and Figure 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 extraction.

[0161] Furthermore, similar to the example in Test Example 1 described above, the lyophilized products of Examples 2-7 in Test Example 2 were also measured for growth factors and other components using the ELISA method. As a result, it was confirmed that the lyophilized products of Examples 2-7 had higher levels 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 lower levels of MMP-3 and MMP-9 compared to the lyophilized product of the comparative example in Test Example 1. In other words, it was demonstrated that the lyophilized products of Examples 2-7 were superior to the lyophilized product of the comparative example.

[0162] <Test Example 3> This study aimed to evaluate the effect of differences in citric acid content in lyophilized formulations on the storage stability of growth factors. Specifically, multiple lyophilized formulations were prepared by varying the amount of citric acid added, and after storing them under predetermined temperature conditions, the relationship between citric acid content and the storage stability of growth factors was examined by comparing the remaining amounts of growth factors (PDGF-BB and EGF).

[0163] Blood samples were collected from four healthy individuals, and a total of 10 blood collection tubes were used for each example (3 tubes for 3 individuals, and 1 tube for 1 individual). Blood collection tubes containing ACD solution were used in the tests. In Example 8, the amount of citrate was adjusted by removing the ACD solution from the blood collection tube before blood collection (the final amount of citrate used is shown in Table 2). In Example 9, no manipulation of the ACD solution was performed, and similarly in Example 10, blood collection was 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 to stand overnight at 4°C. In Example 10, after this refrigerated standing period, ACD solution was newly added to each blood collection tube and the amount of citric acid was adjusted by inverting and mixing (the amount of citric acid remaining in the lyophilized product is shown in Table 2).

[0165] Next, all test tubes were centrifuged at 500G for 10 minutes, and the resulting plasma was pooled according to the example. The pooled plasma was dispensed into 15 mL tubes, 4 mL each, and secondary centrifugation was performed. After secondary centrifugation, the supernatant of each tube was removed, leaving 1.0 mL, 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 Example 1.

[0166] Each obtained sample was dispensed into vials of 2 mL or 1.5 mL, frozen at -60°C, and then lyophilized overnight. The lyophilized preparations were sealed and left to stand at 22°C (room temperature) or 50°C for 7 days each. After the end of the storage period, they were frozen in a freezer at -60°C for at least 24 hours.

[0167] For the evaluation of the lyophilized product, it was dissolved in 2.0 mL (or partially 1.5 mL) of sterile 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 in the product stored at 50°C to that in the product stored at 22°C. The results are shown in Table 2 and Figure 4.

[0168] [Table 2]

[0169] As shown in Table 2 and Figure 4, a tendency was observed for storage stability, more specifically high-temperature stability, to improve with increasing citric acid content in the freeze-dried product. In particular, it was found that a remarkable improvement in stability exceeding 90% was achieved when the citric acid content per gram of freeze-dried product was 1.85% by mass or higher.

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

[0171] <Test Example 4> This study aimed to verify whether the citric acid content-dependent effect on improving the storage stability of growth factors in lyophilized formulations, as confirmed in Test Example 3, is maintained even when the timing of citric acid addition is changed.

[0172] In Test Example 4, Examples 11 to 13, which used blood collection tubes containing ACD solution, were tested. The amount of ACD solution pre-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] Aside from using the above-mentioned blood collection tubes, the procedure was the same as in Example 1, up to the removal of the supernatant after secondary centrifugation. Then, 4.5 ml of Ringer's solution was added to the sample in Example 11.

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

[0175] The samples after adding Ringer's solution were processed using the same procedure as in Test Example 3 to obtain the lyophilized products of Examples 11-14. These lyophilized products were subjected to measurement of PDGF-BB and citric acid content, and their storage stability was evaluated, as in Test Example 3. The results are shown in Table 3 and Figure 5.

[0176] [Table 3]

[0177] As shown in Table 3 and Figure 5, similar to Test Example 3, a tendency was observed for storage stability, more specifically high-temperature stability, to improve with increasing citric acid content in the freeze-dried product. This result demonstrates that the citric acid content-dependent effect on improving the storage stability of growth factors in the freeze-dried formulation is maintained regardless of the timing of citric acid addition.

[0178] Furthermore, comparing Test Example 3 and Test Example 4, it is shown that in the example in Test Example 3, a significantly higher stability improvement effect can be obtained even when the citric acid content per gram of freeze-dried material is relatively low. This result indicates that even when achieving the same citric acid content per gram of freeze-dried material, adding it earlier enhances the stabilization effect on growth factors in the freeze-dried material.

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

[0180] <Test Example 5> This study aimed to verify that the citrate-dependent effect on improving the storage stability of growth factors in lyophilized formulations, as confirmed in Test Example 3, is exerted purely in a citrate-dependent manner, without being affected by dextrose (glucose) contained in the ACD solution.

[0181] In Test Example 5, we tested Examples 15 to 18, in which a test tube containing a citrate-dextrose solution (hereinafter referred to as ACD solution) or a citrate solution was used as an anticoagulant, and the final citrate content was changed by adding a dextrose-free citrate solution during the process.

[0182] First, test tubes containing 1.5 ml of ACD solution as an anticoagulant (Examples 15-17) 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 is as follows. Citric acid solution: Aqueous solution of trisodium citrate 22.0 mg / mL and citric acid 8.0 mg / mL (dextrose-free) Furthermore, the only difference in composition between the citric acid solution and the ACD solution is the presence or absence of dextrose.

[0183] Approximately 8.5 mL of blood was collected in each test tube and left to stand overnight at 4°C. Then, the following procedure was 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 (500g, 10 minutes), and plasma was collected from each test tube. The collected plasma was dispensed into 15ml tubes, 4ml each, and after secondary centrifugation, the supernatant was removed, leaving 1.0ml, and lactating Ringer's solution was added to bring the final volume to 5.0ml. Cell-free plasma samples were then prepared using the same procedure as in Test Example 1.

[0185] Each obtained sample was dispensed into a 2 mL vial, frozen at -60°C, and then lyophilized overnight. The lyophilized formulations were sealed and left to stand for 7 days at either 22°C (room temperature) or 50°C.

[0186] The freeze-dried sample, after standing, was redissolved in 2.0 ml of sterile water for injection, and the PDGF-BB content was measured by ELISA. Storage stability was calculated as the ratio of factor amounts in the 50°C stored sample to the 22°C stored sample. The results are shown in Table 4 and Figure 6. Table 4 also shows the amount of ACD solution or citrate solution used as an anticoagulant, the amount of citrate solution added to the collected blood after it had stood overnight, and the amount of citrate added calculated from these total amounts and the concentrations of citrate, etc.

[0187] [Table 4]

[0188] As is clear from the comparison of Examples 15-17, 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 improves as the amount of added citric acid increases. Furthermore, very high stability was observed in Example 18, which did not use an ACD solution containing dextrose. From these results, it is clear 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 showed higher storage stability despite having the same amount of citric acid added as Example 16. This result suggests that the lower the ratio of dextrose to citric acid in the freeze-dried product, the greater the stability of blood-derived growth factors in the freeze-dried product.

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

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

Claims

1. A method for preparing a blood-derived growth factor-containing composition from blood, A separation step is performed on the blood to separate platelet-rich plasma that does not contain buffy coat components from the blood by subjecting it to a first centrifugation process at 100G to 1200G. An addition step of adding citric acid and / or its salt to the blood or the processed product of the blood after the separation step, A purification and concentration step is performed to obtain a platelet pellet by subjecting the platelet-rich plasma, which does not contain the buffy coat component after the separation step, to a second centrifugation treatment at 1000G to 2500G, removing the supernatant, and suspending the pellet in a pharmaceutically acceptable aqueous medium to obtain a purified platelet concentrate. Optionally, a cell removal step to obtain cell-free plasma by removing cells from the purified concentrate, If the cell removal step is included, optionally, a freeze-drying step of freeze-drying the cell-free plasma is further included. Includes, The blood-derived growth factor-containing composition, The non-freeze-dried composition is the purified concentrate or the cell-free plasma, or The lyophilized composition of the cell-free plasma is The aforementioned additive step is, When the blood-derived growth factor-containing composition is the non-freeze-dried composition, the content of citric acid and / or its salt in the freeze-dried composition, assuming that the non-freeze-dried composition has been freeze-dried, shall be 2% by mass or more, or When the blood-derived growth factor-containing composition is the freeze-dried composition, the content of the citric acid and / or its salt in the freeze-dried composition shall be 2% by mass or more. A method comprising adding the citric acid and / or a salt thereof to the blood or the processed product of the blood after the separation step.

2. The method according to claim 1, comprising the cell removal step and the freeze-drying step of freeze-drying the cell-free plasma.

3. The method according to claim 1, further comprising an activation step for activating the purified concentrate.

4. The method according to claim 1, comprising the cell removal step.

5. The method according to claim 1, comprising using the citric acid and / or a salt thereof as an anticoagulant, and not using heparin or a salt thereof as an anticoagulant.

6. A blood-derived growth factor-containing composition prepared by the method according to any one of claims 1 to 5.

7. The blood-derived growth factor-containing composition according to claim 6, which is a freeze-dried composition containing blood-derived growth factors.

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