Method for manufacturing eye drops

Abstract

To provide a convenient production method for a PRP eye drop with a sufficiently large amount of bioactive substance contained in PRP.SOLUTION: The present invention discloses a method for producing a PRP eye drop with increased TGFβ1 and EGF levels, where the PRP prepared from blood is stored at low temperatures for 7 days to 35 days.SELECTED DRAWING: None

Description

Technical Field

[0001] The present invention relates to a method for producing a platelet-rich plasma eye drop.

Background Art

[0002] In graft-versus-host disease, corneal transplantation, Sjögren's syndrome, severe dry eye, etc., intractable corneal and conjunctival epithelial disorders occur, which not only cause deterioration of subjective symptoms but also problems such as decreased visual acuity, increased susceptibility to infection, and perforation. Treatment for these corneal and conjunctival epithelial disorders mainly focuses on eye drop therapy. In Japan, mucin production promoters such as diclofenac sodium and rebamipide, and eye drops containing sodium hyaluronate for treating corneal and conjunctival epithelial disorders have been approved and are used clinically. However, there are intractable corneal and conjunctival epithelial disorders that are not effective in these eye drop therapies for treating corneal and conjunctival epithelial disorders, and there is a need to develop new therapeutic agents.

[0003] From this perspective, serum (hereinafter referred to as AS), which has components similar to tears and contains bioactive substances necessary for wound healing, has been used as an eye drop (Non-Patent Documents 1 to 4). On the other hand, there are intractable corneal and conjunctival epithelial disorders that are not effective even with AS eye drops. Recently, platelet-rich plasma (hereinafter referred to as PRP), which contains many bioactive substances, has been expected as a new eye drop for treating corneal and conjunctival epithelial disorders (Non-Patent Document 5). However, the blood cell components in PRP eye drops, the growth factors that contribute to the promotion of corneal epithelial disorder healing, the behavior during the storage period from the preparation of the active ingredient to its use, and its sterility have not been clarified.

Prior Art Documents

Non-Patent Documents

[0004]

Non-Patent Document 1

Outdoor Tool2

Outdoor Tools3

Outdoor Tools 4

Direct Environment 5

[0005] Since PRP eye drops are autologous eye drops prepared by collecting the patient's blood and centrifuging it, it is unclear whether the concentrations of TGFβ1 and EGF, which are physiologically active substances necessary for wound healing of the corneal epithelium contained in PRP, are sufficient. Furthermore, the dynamics of physiologically active substances during the storage period from preparation to use, and the sterility of the eye drops, have not been clarified. Therefore, the object of the present invention is to provide a simple method for producing PRP eye drops with a sufficiently high amount of physiologically active substances. [Means for solving the problem]

[0006] Therefore, the inventors refrigerated PRP prepared from collected blood and measured the content of physiologically active substances over time, while also checking for the growth of bacteria and fungi in the eye drops. They found that the concentrations of TGFβ1 and EGF increased significantly from 7 days after storage compared to immediately after preparation, and that no bacteria or other organisms grew until about 35 days later. Thus, they discovered that PRP eye drops with increased concentrations of physiologically active substances could be easily obtained, and thus completed the present invention.

[0007] In other words, the present invention provides the following inventions [1] to [6]. [1] A method for producing PRP eye drops with elevated TGFβ1 and EGF concentrations, characterized by refrigerating PRP prepared from blood for 7 to 35 days. [2] A method for manufacturing PRP eye drops according to [1], wherein the TGFβ1 concentration in the PRP is 35 pg / mL or higher and the EGF concentration is 1000 pg / mL or higher. [3] A method for manufacturing PRP eye drops according to [1] or [2], wherein the PRP is PRP eye drops for autologous use. [4] A method for manufacturing PRP eye drops as described in any of [1] to [3], wherein refrigeration is performed at a temperature of 0°C to 8°C. [5] A method for manufacturing a PRP eye drop solution according to any one of [1] to [4], wherein the PRP eye drop solution is a PRP eye drop solution for the treatment of autologous corneal and conjunctival epithelial disorders. [6] A method for manufacturing PRP eye drops as described in any of [1] to [5], wherein the refrigerated storage period is 10 to 35 days. [Effects of the Invention]

[0008] According to the present invention, a PRP eye drop solution with sufficiently elevated TGFβ1 and EGF concentrations, useful for treating corneal and conjunctival epithelial disorders, can be obtained through simple procedures. Furthermore, the obtained eye drop solution can be safely used as an autologous eye drop solution without the development of bacteria or other contaminants. In particular, it is useful for intractable corneal and conjunctival disorders such as chronic graft-versus-host disease, Stevens-Johnson syndrome, Sjögren's syndrome, and severe dry eye. This method is useful for manufacturing autologous PRP eye drops for the treatment of epithelial damage. [Brief explanation of the drawing]

[0009] [Figure 1] This shows the change in platelet count in PRP over time. [Figure 2] This shows the time course of fibronectin concentration in PRP. [Figure 3] This shows the time course of TGFβ1 concentration in PRP. [Figure 4] This shows the change in EGF concentration in PRP over time. [Figure 5] The results of bacterial and fungal culture tests 28 days after PRP preparation are shown. [Modes for carrying out the invention]

[0010] The present invention relates to a method for producing PRP eye drops with elevated TGFβ1 and EGF concentrations, characterized by refrigerating PRP prepared from blood for 7 to 35 days. The raw material, blood, is the blood of the patient to whom the eye drops will be administered. PRP is typically prepared by centrifugation immediately after blood collection. Several centrifugation methods exist, including centrifugation at 800g for 10 minutes, centrifugation at 2000g for 7 minutes, and a two-stage centrifugation method involving centrifugation at 400g for 10 minutes followed by centrifugation at 2000g for 3 minutes. Of these, the centrifugation method at 2000g for 7 minutes and the two-stage method (centrifugation at 400g for 10 minutes followed by centrifugation at 2000g for 3 minutes) are preferred because refrigerated storage significantly increases TGFβ1 and EGF concentrations. Several platelet-rich plasma (PRP) preparation kits have also been developed, and PRP can be prepared using these kits. It is preferable that the PRP eye drops contain 300,000 to 900,000 platelets / μL.

[0011] Next, the PRP is refrigerated for 7 to 35 days. The refrigeration temperature is preferably 0°C to 8°C, and more preferably 4°C ± 2°C. It is also preferable to store the PRP in a sealed container such as a syringe or a bottle for storing injection solutions. After refrigeration, PRP showed an increase in TGFβ1 and EGF concentrations starting around 7 days after preparation, compared to immediately after preparation, and this increase continued until about 35 days later. Fibronectin concentration remained unchanged from immediately after preparation until 28 days later. Furthermore, platelet count gradually decreased from immediately after preparation.

[0012] The refrigerated storage period is preferably a period during which the TGFβ1 concentration and EGF concentration have risen sufficiently and no bacteria or fungi have grown. Preferably, the PRP is stored for a period during which the TGFβ1 concentration is 35 pg / mL or higher and the EGF concentration is 1000 pg / mL or higher. Furthermore, it is preferable to store the PRP for a period during which the TGFβ1 concentration is 40 pg / mL or higher and the EGF concentration is 1500 pg / mL or higher. Such a storage period is preferably 10 to 35 days, more preferably 14 to 35 days, and even more preferably 14 to 28 days.

[0013] The PRP refrigerated and stored during the above period has sufficiently increased TGFβ1 concentration and EGF concentration, ensured platelet count, and has no bacteria or fungi generated, and thus is useful as a PRP eye drop, particularly a PRP eye drop for self - instillation. Further, it is useful as a PRP eye drop for treating autologous corneal epithelial disorders. Particularly, it is useful as an autologous PRP eye drop for treating intractable corneal epithelial disorders such as chronic graft - versus - host disease, Stevens - Johnson syndrome, Sjögren's syndrome, and severe dry eye.

Examples

[0014] Next, the present invention will be further described in detail with reference to examples, but the present invention is not limited to these examples.

[0015] Example 1 (1) Preparation of PRP PRP was prepared using a PRP preparation kit from Mycells. First, 11 mL of the patient's venous blood was collected in each of two blood collection tubes of the preparation kit, and the collected blood was quickly transported to the PRP preparation room in Juntendo University and centrifuged at 2000 g for 7 minutes. After centrifugation, it was transferred to a clean bench in the PRP preparation room, and the plasma supernatant was removed so that 2 mL of plasma remained in each blood collection tube. By thoroughly stirring the remaining plasma, 2 mL of PRP each, a total of 4 mL, was prepared. After filtering this PRP through a filter, it was transferred to a sterilized light - shielding eye drop bottle from M & I Chemical Co., Ltd. (M & I Chemical Co., Ltd. product code 4731 Poly Point No. 3 10 cc, main body color brown, cap color white) to prepare a PRP eye drop.

[0016] (2) Refrigerated storage and time - dependent changes of each component The PRP was placed in a sterilized light - shielding eye drop bottle and refrigerated at 4°C. Platelet count, TGFβ1, EGF, and fibronectin were measured on the production day, 7 days later, and 28 days later. Also, it was inspected whether bacteria or fungi had occurred during the storage period. Platelet count: Measured by an automatic blood cell analyzer TGFβ1, EGF, fibronectin: Measured by an ELISA kit Bacteria, fungi: Measured by a culture test

[0017] (3) Measurement results The changes in platelet count, fibronectin, TGFβ1, and EGF over time are shown in Figures 1, 2, 3, and 4. Platelet counts were highest on the day of preparation and gradually decreased until 28 days later (Figure 1). Fibronectin concentration remained almost unchanged from the day of preparation to 28 days later, at 200 pg / mL to 250 pg / mL (Figure 2). TGFβ1 concentration was around 15 pg / mL on the day of preparation, but increased to 35 pg / mL after 7 days and to 60 pg / mL after 28 days (Figure 3). EGF concentration was 165 pg / mL on the day of preparation, but increased to 1000 pg / mL after 7 days and to 3000 pg / mL after 28 days (Figure 4). Furthermore, no bacterial or fungal growth was observed even after 28 days (Figure 5).

Claims

1. A method for producing PRP eye drops, comprising a step of increasing the TGFβ1 and EGF concentrations by refrigerating PRP prepared from blood for 14 to 35 days.

2. A method for producing PRP eye drops according to claim 1, wherein the TGFβ1 concentration in the PRP is 35 pg / mL or higher and the EGF concentration is 1000 pg / mL or higher.

3. A method for producing PRP eye drops according to claim 1 or 2, wherein the PRP is PRP eye drops for autologous use.

4. A method for producing PRP eye drops according to any one of claims 1 to 3, wherein refrigeration is performed at a temperature of 0°C to 8°C.

5. A method for producing a PRP eye drop according to any one of claims 1 to 4, wherein the PRP eye drop is a PRP eye drop for the treatment of autologous corneal and conjunctival epithelial disorders.

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