KR102297254B1 - A method for assessing the clinical efficacy of platelet-rich plasma - Google Patents

Abstract

The present application provides a method for evaluating the clinical efficacy of platelet-rich plasma, including measuring the concentration of growth factors, cytokines and cells contained in platelet rich plasma (PRP), clinical efficacy evaluation of platelet-rich plasma It relates to a composition for use, a method for producing platelet-rich plasma, and a pharmaceutical composition for treating or preventing tendinopathy comprising platelet-rich plasma.
According to the clinical efficacy evaluation method of platelet-rich plasma of the present application, by measuring the concentrations of cells, growth factors, and cytokines such as leukocytes, VEGF, and IL-8 contained in platelet-rich plasma, platelets with actual clinically therapeutic effect Since rich plasma can be selected, platelet-rich plasma can be efficiently used for disease treatment.

Description

{A method for assessing the clinical efficacy of platelet-rich plasma}

The present application provides a method for evaluating clinical efficacy of platelet-rich plasma, including measuring the concentrations of growth factors, cytokines and cells contained in platelet rich plasma (PRP), platelet-rich plasma production method, and platelet richness It relates to a pharmaceutical composition for treating or preventing tendinopathy comprising plasma.

Tendon tissue is a highly ordered complex of collagen fibril densely packed with a small number of tenocytes and fibroblasts. Collagen fibers are mainly type I collagen, and there are some other types of collagen and proteoglycans such as decorin, biglycan, and fibromodulin. During the development of tendon tissue, tendon cells are responsible for the synthesis of collagen fibers, and proteoglycans are important components that regulate the assembly of collagen fibers and the acquisition of biomechanical properties of the tendon.

Tendinopathy is a general pathological condition of tendon tissue that appears in clinical practice, and the number of cells increases as the shape of the cells becomes more round and shows the characteristics of apoptotic cell death. In this state, various changes occur in the extracellular matrix, destroying the collagen fibers and losing the unique biomechanical properties used to transmit strong energy.

Therefore, a lot of research on new treatments to treat tendinopathy is ongoing. In the field of regenerative medicine, research is being conducted to restore the function of tendon tissue using growth factors, gene therapy, and tissue engineering technology. treatment methods are being studied.

Platelet-rich plasma (PRP, Patelet Rich Plasma) refers to the centrifugation of blood after special treatment, and refers to the plasma component made to condense platelets more abundantly than normal blood. Platelet-rich plasma shows a higher platelet level compared to whole blood and has various growth factors and cytokines that help wound healing, and these substances include cell proliferation and differentiation, chemotaxis, and angiogenesis. (angiogenesis) plays an important role.

Although many studies have been conducted on the clinical effect of platelet-rich plasma injection, when autologous platelet-rich plasma is obtained after blood collection for therapeutic use, whether it is actually effective platelet-rich plasma has not been studied or reported. there is no state

Accordingly, the present inventors have tried to develop a method for screening platelet-rich plasma having an actual clinically therapeutic effect. As a result, by measuring the concentrations of growth factors, cytokines and cells contained in platelet-rich plasma, there is a specific concentration or higher. In this case, the present invention was completed by developing a method for evaluating whether the platelet-rich plasma is clinically effective.

Republic of Korea Patent Publication No. 10-2019-0063211

A first aspect of the present application is to provide a method for evaluating the clinical efficacy of platelet rich plasma, comprising measuring the concentrations of growth factors, cytokines and cells contained in platelet rich plasma (PRP). .

A second aspect of the present application provides a composition for evaluating the clinical efficacy of platelet rich plasma, comprising a formulation capable of detecting growth factors, cytokines and cells contained in platelet rich plasma (PRP) will be.

A third aspect of the present application is to provide a kit for evaluating the clinical efficacy of platelet-rich plasma, comprising the composition for evaluating the clinical efficacy of platelet-rich plasma.

A fourth aspect of the present application provides a method comprising the steps of: (A) obtaining platelet-rich plasma from a subject; and (B) measuring the concentrations of growth factors, cytokines and cells in the obtained platelet-rich plasma, to provide a method for producing platelet-rich plasma.

A fifth aspect of the present application is to provide a pharmaceutical composition for treating or preventing tendinopathy, comprising platelet-rich plasma.

This will be described in detail as follows. On the other hand, each description and embodiment disclosed herein may also be applied to each other description and embodiment. That is, all combinations of the various elements disclosed in this application fall within the scope of this application. In addition, it cannot be seen that the scope of the present application is limited by the detailed description described below.

A first aspect of the present application provides a method for evaluating clinical efficacy of platelet rich plasma, comprising measuring the concentrations of growth factors, cytokines and cells contained in platelet rich plasma (PRP).

As used throughout this specification, the term "Platelet Rich Plasma (PRP)" is obtained through a centrifugation process and contains a higher concentration of platelets than whole blood. Although platelets have been known to act only in the blood coagulation process, as the understanding of wound healing deepens, it has become increasingly clear that platelets also act in tissue regeneration and healing processes through the action of abundant growth factors and cytokines contained in platelets. It has been found out, and now it is widely used with various indications in various fields such as orthopedics, plastic surgery, neurosurgery, dermatology, dentistry, ophthalmology, and otolaryngology.

According to one embodiment of the present application, the growth factors, cytokines and cells are PDGF (platelet-derived growth factor), TGF-β (transforming growth factor beta), VEGF (vascular endothelial growth factor), IGF-1 (insulin- like growth factor 1), IL-1β (interleukin 1 beta), IL-8 (interleukin 8), and may be one or more of white blood cells (WBC), specifically VEGF, IL-8, and consisting of white blood cells It may include one or more selected from the group, but is not limited thereto.

According to one embodiment of the present application, the method may include measuring the concentration of leukocytes, VEGF and/or IL-8 contained in platelet-rich plasma.

According to one embodiment of the present application, the method may further include the step of determining that the platelet-rich plasma has a clinically therapeutic effect when leukocytes are included in the platelet-rich plasma, specifically, platelet-rich plasma When the concentration of leukocytes contained in the leukocytes is 1.78 x 10 ³ pieces/μL or more, the step of determining that the platelet-rich plasma has a clinically therapeutic effect may be further included.

According to one embodiment of the present application, the concentration of leukocytes contained in platelet-rich plasma is 0.99 x 10 ³ / μL or more, 1.15 x 10 ³ / μL or more, 1.23 x 10 ³ / μL or more, or 1.78 x 10 ³ / μL or more In this case, the platelet-rich plasma may further include the step of determining that it has a clinically therapeutic effect.

According to one embodiment of the present application, the concentration of white blood cells contained in platelet-rich plasma is 0.99 x 10 ³ / μL or more, 1.15 x 10 ³ / μL or more, 10.76 x 10 ³ / μL or more, or 14.79 x 10 ³ / μL or more If, specifically, 10.76 x 10 ³ pieces/μL or more, the platelet-rich plasma may further include the step of determining that there is a clinically therapeutic effect.

According to one embodiment of the present application, the concentration of leukocytes contained in the platelet-rich plasma may be 27.2 x 10 ³ pieces/μL or less, and in general, if it is less than the concentration of leukocytes that may be contained in platelet-rich plasma, it is not limited thereto.

According to one embodiment of the present application, the method may further include the step of determining that the platelet-rich plasma has a clinically therapeutic effect when VEGF is included in the platelet-rich plasma, specifically, platelet-rich plasma If the concentration of VEGF contained in the 37.71 pg / mL or more, the platelet-rich plasma may be one that further comprises the step of determining that there is a clinically therapeutic effect.

According to one embodiment of the present application, when the concentration of VEGF contained in platelet-rich plasma is 8.01 pg/mL or more, 30.06 pg/mL or 37.71 pg/mL or more, it is determined that the platelet-rich plasma has a clinically therapeutic effect. It may be to further include the step of

According to one embodiment of the present application, the concentration of VEGF contained in the platelet-rich plasma may be 145.4 pg/mL or less, and in general, if it is less than the concentration of VEGF that may be contained in platelet-rich plasma, it is not limited thereto.

According to one embodiment of the present application, the method may further include determining that the platelet-rich plasma has a clinically therapeutic effect when IL-8 is included in the platelet-rich plasma.

According to one embodiment of the present application, the clinical effectiveness of platelet-rich plasma in the method is medial and lateral epicondylitis, patellar tendinitis, Achilles tendinitis, rotator cuff tear , plantar fasciitis, chondromalacia, knee ligament injury, acute ligament injury, tendon injury, Elbow epicondylitis, plantar fasciitis fasciitis), Achilles tendon rupture, muscle injury, bone injury, and cartilage injury may be clinically effective for one or more treatments selected from the group consisting of.

According to one embodiment of the present application, the clinical effectiveness of platelet-rich plasma in the method may be clinical effectiveness for the treatment of tendinopathy, specifically, platelet-rich plasma evaluated as clinically effective in the method It may mean that there is a clinically therapeutic effect in the treatment of tendinopathy.

As used throughout this specification, the term “clinical effectiveness” refers to a level of clinically significant therapeutic effect on disease, and in the present specification, whether the therapeutic effect on disease of platelet-rich plasma is clinically significant. Specifically, it may indicate whether the therapeutic effect on tendinopathy is at a clinically significant level, but is not limited thereto.

According to one embodiment of the present application, the clinical effectiveness of the platelet-rich plasma is clinically effective, such as pain reduction, clinical function improvement and/or symptom improvement after 3 months or 6 months after administration of platelet-rich plasma to tendinopathy patients. It may mean that the therapeutic effect is significant, specifically, by administering platelet-rich plasma, pain is reduced by evaluating it with the NRS pain scale score, or the pain level and the degree of daily living are improved through the Constant score. may be, but is not limited thereto.

As used throughout this specification, the term "NRS (Numeral Rating Scale) pain scale" is a quantification of how much the individual patient's pain level is perceived. Specifically, 0 is the patient's no pain and 10 is the patient's unbearable extreme pain. Evaluate after For example, in the case of a patient with pain due to tendinopathy, after conducting several questionnaires such as when the pain began, where the pain was, and what the pain was, 10 would be considered as extreme pain that could not be tolerated. After confirming with the patient how much pain is present at the time of treatment, the effect of treatment such as pain relief can be confirmed by confirming again after treatment.

As used throughout this specification, the term "Constant score" is subdivided into subjective evaluation (35 points) for the degree of pain and ability to live daily life and objective evaluation (65 points) for range of motion and muscle strength. The treatment effect can be confirmed by checking the constant score after.

As used throughout this specification, the term "tendinopathy" refers to a disease in the tendon (tendon), specifically, caused by degenerative changes in the collagen of the tendon itself due to chronic overuse. It occurs when overuse injuries such as repetitive strains continue without giving the tendon time to heal by taking a break when there is a tendon injury. Similarly, Tendinitis refers to inflammation of the tendon itself caused by micro tears of the tendon when a sudden and excessive load (force) is applied to the musculotendinous unit. In clinical practice, tendinitis and tendinopathy are generally used without distinction.

According to one embodiment of the present application, the tendinopathy may include parotitis, tendinitis, tendinitis, tendinitis, proximal hamstring tendinopathy, Achilles tendinopathy, high knee flexor tendinopathy, etc., specifically, rotator cuff tendinopathy (rotator). cuff tendinopathy), but is not limited thereto.

As used throughout this specification, the term “evaluation” refers to determining whether the therapeutic effect of platelet-rich plasma on a disease is clinically effective.

The method of the present application, comprising the step of measuring the concentration of growth factors, cytokines and cells contained in platelet rich plasma (Platelet Rich Plasma, PRP), information providing method for evaluating the clinical efficacy of platelet rich plasma can be

A second aspect of the present application provides a composition for evaluating the clinical efficacy of platelet rich plasma, comprising an agent capable of detecting growth factors, cytokines and cells contained in platelet rich plasma (PRP). The content overlapping with the first aspect is equally applied to the composition of the second aspect.

According to one embodiment of the present application, the growth factors, cytokines and cells are PDGF (platelet-derived growth factor), TGF-β (transforming growth factor beta), VEGF (vascular endothelial growth factor), IGF-1 (insulin- like growth factor 1), IL-1β (interleukin 1 beta), IL-8 (interleukin 8), and may be one or more of white blood cells (WBC), specifically VEGF, IL-8, and consisting of white blood cells It may include one or more selected from the group, but is not limited thereto.

According to one embodiment of the present application, the composition contains 0.99 x 10 ³ / μL or more, 1.15 x 10 ³ / μL or more, 1.23 x 10 ³ / μL or more, or 1.78 x 10 ³ / μL or more of white blood cells contained in platelet-rich plasma. It may include an agent capable of detecting leukocytes, specifically, it may include an agent capable of detecting 1.78 x 10 ³ pieces/μL or more of leukocytes, but is not limited thereto.

According to one embodiment of the present application, the composition contains 0.99 x 10 ³ / μL or more, 1.15 x 10 ³ / μL or more, 10.76 x 10 ³ / μL or more, or 14.79 x 10 ³ / μL or more of white blood cells contained in platelet-rich plasma. It may include an agent capable of detecting

According to one embodiment of the present application, the composition may include an agent capable of detecting VEGF of 8.01 pg/mL or more, 30.06 pg/mL or 37.71 pg/mL or more contained in platelet-rich plasma, specifically 37.71 It may include an agent capable of detecting VEGF of pg/mL or higher, but is not limited thereto.

According to one embodiment of the present application, the clinical effectiveness of the platelet-rich plasma may be clinical effectiveness for the treatment of Tendinopathy, specifically, it may be for the treatment of rotator cuff tendinopathy, However, the present invention is not limited thereto.

A third aspect of the present application provides a kit for evaluating the clinical efficacy of platelet-rich plasma, comprising the composition for evaluating the clinical efficacy of platelet-rich plasma. The contents overlapping with the first aspect and the second aspect are equally applied to the kit of the third aspect.

According to one embodiment of the present application, the kit is PDGF (platelet-derived growth factor), TGF-β (transforming growth factor beta), VEGF (vascular endothelial growth factor), IGF-1 (insulin-like growth factor 1), It may include an agent capable of detecting growth factors, cytokines and cells, such as IL-1β (interleukin 1 beta), IL-8 (interleukin 8), and white blood cells (WBC), specifically It may include an agent capable of detecting one or more selected from the group consisting of VEGF, IL-8, and leukocytes, but is not limited thereto.

According to one embodiment of the present application, the kit may include not only agents capable of detecting various growth factors, cytokines and agents capable of detecting cells, but also other component compositions, solutions or devices suitable for the assay method. .

A fourth aspect of the present disclosure is a method comprising the steps of: (A) obtaining platelet-rich plasma from a subject; and (B) measuring the concentrations of growth factors, cytokines and cells in the obtained platelet-rich plasma, it provides a method for producing platelet-rich plasma. The contents overlapping with the first to third aspects are equally applied to the method of the fourth aspect.

As used throughout this specification, the term "individual" refers to any organism that has or is likely to develop tendinopathy, and specific examples include mammals including mice, monkeys, cattle, pigs, mini-pigs, livestock, humans, and the like. It may include animals, farmed fish, etc., but is not limited thereto.

According to one embodiment of the present application, the method may be a method for producing platelet-rich plasma having clinical effectiveness, specifically, a method for producing platelet-rich plasma having clinical efficacy in treating or preventing tendinopathy.

According to one embodiment of the present application, the platelet-rich plasma may be for the treatment or prevention of tendinopathy, and the tendinopathy is parotiditis, tendinitis, tendinitis, tendinitis, proximal hamstring tendinopathy, Achilles tendinopathy, high flexor flexor tendonopathy. and the like, and specifically, may be for treating or preventing rotator cuff tendinopathy, but is not limited thereto.

According to one embodiment of the present application, the growth factors, cytokines and cells are one or more selected from the group consisting of vascular endothelial growth factor (VEGF), interleukin 8 (IL-8), and white blood cells (WBC). It may include, but is not limited to.

According to one embodiment of the present application, the method may include measuring the concentration of leukocytes, VEGF and/or IL-8 contained in the obtained platelet-rich plasma.

According to one embodiment of the present application, the method may further include the step of selecting platelet-rich plasma containing leukocytes, specifically, selecting platelet-rich plasma having ^{a concentration of 1.78x10 3 cells/μL or more of leukocytes.} It may be to further include a step.

According to one embodiment of the present application, the method comprising the concentration of the white blood cell 0.99 x10 ³ gae / μL or more, 1.15 x10 ³ gae / μL or more, 1.23 x10 ³ gae / μL or more, or 1.78 x10 ³ gae / μL or more platelet-rich plasma It may be to further include the step of selecting the.

According to one embodiment of the present application, the method comprises platelet-rich plasma having ^{a concentration of leukocytes of 0.99 x 10 3} / μL or more, 1.15 x 10 ³ / μL or more, 10.76 x 10 ³ / μL or more, or 14.79 x 10 ^{3 / μL or more} It may be to further include the step of selecting the.

According to one embodiment of the present application, the method may further include the step of selecting platelet-rich plasma containing VEGF, specifically, the step of selecting platelet-rich plasma having a concentration of VEGF of 37.71 pg/mL or more It may be further included.

According to one embodiment of the present application, the method may further include selecting platelet-rich plasma having a VEGF concentration of 8.01 pg/mL or more, 30.06 pg/mL or 37.71 pg/mL or more.

According to one embodiment of the present application, the method may further include selecting platelet-rich plasma containing IL-8.

A fifth aspect of the present application provides a pharmaceutical composition for treating or preventing tendinopathy, comprising platelet-rich plasma. The contents overlapping with the first to fourth aspects are equally applied to the pharmaceutical composition for treating or preventing tendinopathy of the fourth aspect.

According to one embodiment of the present application, the tendinopathy may include parotitis, tendinitis, tendinitis, tendinitis, proximal hamstring tendinopathy, Achilles tendinopathy, high knee flexor tendinopathy, etc., specifically, rotator cuff tendinopathy (rotator). cuff tendinopathy), but is not limited thereto.

According to one embodiment of the present application, the platelet-rich plasma may contain leukocytes, and specifically, 0.99 x10 ³ pieces/μL or more, 1.15 x10 ³ pieces/μL or more, 1.23 x10 ³ pieces/μL or more, or 1.78 x10 ^It may contain 3 pieces/μL or more, but is not limited thereto.

According to one embodiment of the present application, the platelet-rich plasma is 0.99 x 10 ³ / μL or more, 1.15 x 10 ³ / μL or more, 10.76 x 10 ³ / μL or more, or 14.79 x 10 ³ / μL or more of white blood cells. However, it is not limited thereto.

According to one embodiment of the present application, the platelet-rich plasma may contain VEGF, specifically 8.01 pg/mL or more, 30.06 pg/mL or 37.71 pg/mL or more, but is not limited thereto. .

According to one embodiment of the present application, the platelet-rich plasma may contain IL-8.

As used throughout this specification, the term "treatment" refers to any action in which tendinopathy is suppressed or delayed by administration of the pharmaceutical composition of the present application.

As used throughout this specification, the term "prevention" refers to any action that improves or beneficially changes the symptoms of tendinopathy by administration of the pharmaceutical composition of the present application.

According to one embodiment of the present application, the pharmaceutical composition may be formulated and administered as a unit dosage form pharmaceutical preparation suitable for administration in a patient's body according to a conventional method in the pharmaceutical field, and the preparation may be administered once or An effective dosage can be achieved by multiple administrations. As a formulation suitable for this purpose, an injection, an infusion, etc. are preferable as a preparation for parenteral administration. In addition, the pharmaceutical composition may include conventional pharmaceutically acceptable inert carriers and diluents.

Pharmaceutically acceptable carriers and diluents that may be included in the pharmaceutical composition of the present disclosure include excipients such as starch, sugar, and mannitol, fillers and extenders such as calcium phosphate, cellulose derivatives such as carboxymethylcellulose, hydroxypropylcellulose, gelatin, Binders such as alginates and polyvinyl pyrrolidone, lubricants such as talc, calcium stearate, hydrogenated castor oil and polyethylene glycol, disintegrants such as povidone and crospovidone, surfactants such as polysorbate, cetyl alcohol, and glycerol including, but not limited to. The pharmaceutically acceptable carrier and diluent may be biologically and physiologically compatible with the tonsil-derived stem cells and the recipient to which they will be transplanted. Diluents include, but are not limited to, saline, aqueous buffers, solvents, and/or dispersion media. In addition, for example, in the case of injections, preservatives, analgesics, solubilizers or stabilizers, etc., and in the case of formulations for topical administration, a base, excipients, lubricants or preservatives, etc. may be further included. The compositions herein may be used undisturbed or frozen for later use. If frozen, standard cryopreservatives (eg DMSO, glycerol, Epilife® Cell Freezing Medium (Cascade Biologics)) can be added to the cell population prior to freezing.

According to one embodiment of the present application, the pharmaceutical composition for preventing or treating tendinopathy may be administered in a pharmaceutically effective amount to treat tendinopathy or to inhibit the onset of tendinopathy, and the dosage is may vary depending on various factors such as the progress of the disease, the age, weight, characteristics and severity of symptoms, the type of current treatment, the number of treatments, the dosage form and route, and can be easily determined by experts in the field.

The composition of the present application may be administered together or sequentially with the aforementioned pharmacological or physiological components, or may be administered in combination with an additional conventional therapeutic agent, or may be administered sequentially or simultaneously with the conventional therapeutic agent. Such administration may be single or multiple administrations. Taking all of the above factors into consideration, it is important to administer an amount that can obtain the maximum effect with a minimum amount without side effects, and can be easily determined by those skilled in the art.

According to one embodiment of the present application, there is provided a method for preventing or treating tendinopathy, comprising administering to an individual the pharmaceutical composition for preventing or treating tendinopathy. Specifically, the method for preventing or treating tendinopathy of the present application may include administering to a subject other than a human, but is not limited thereto.

As used throughout this specification, the term "individual" refers to a condition in which tendinopathy can be alleviated, suppressed, or treated by administering the pharmaceutical composition of the present application, or a mouse, rat, or hamster, including a human, suffering from or at risk of tendinopathy. , means any animal such as pigs, cattle, and livestock.

As used throughout this specification, the term "administration" means introducing a predetermined substance to an individual in an appropriate way, and the administration route of the pharmaceutical composition of the present application is administered through any general route as long as it can reach the target tissue. can be Intraperitoneal administration, intravenous administration, intramuscular administration, subcutaneous administration, intradermal administration, oral administration, topical administration, intranasal administration, intrapulmonary administration, may be administered intrarectally, but is not limited thereto.

According to the clinical efficacy evaluation method of platelet-rich plasma of the present application, by measuring the concentrations of cells, growth factors, and cytokines such as leukocytes, VEGF, and IL-8 contained in platelet-rich plasma, platelets with actual clinically therapeutic effect Since rich plasma can be selected, platelet-rich plasma can be efficiently used for disease treatment.

Hereinafter, the present application will be described in more detail through Examples and Experimental Examples. However, these Examples and Experimental Examples are for illustrative purposes of the present invention, and the scope of the present application is not limited to these Examples and Experimental Examples.

Example 1: Experimental group and control group setting

To conduct the experiment, 70 patients with rotator cuff tendinopathy were recruited. Among them, 6 patients who underwent surgery and steroid injection were excluded, and the remaining 64 patients were treated with exercise therapy (control group) 34 patients and platelet-rich plasma ( The PRP) treatment group (experimental group) was divided into 30 patients, and the PRP treatment group was further divided into 15 patients in the LP-PRP (Leukocyte-poor platelet-rich plasma) treatment group and 15 patients in the LR-PRP (Leukocyte-rich platelet-rich plasma) treatment group. did.

In addition, among the 64 patients, 1 in the control group and 3 in the experimental group (LP-PRP 2, LR-PRP 1) dropped out, and finally, the experiment was performed with 33 control group and 27 experimental group.

Example 2: Preparation of platelet-rich plasma

For LP-PRP in platelet-rich plasma, 4 ml of LP-PRP was prepared from 15 ml of blood using an ^{ACP TM kit (Arthrex).} Specifically, 15ml of blood was collected from the target patient by connecting one double syringe containing the coagulant and a butterfly needle, and centrifugation was performed at 1500rpm for 5 minutes. Next, while pressing the outer plunger of the double syringe slowly, pulling the inner plunger slowly, 4ml of clear concentration of LP-PRP was separated. Among them, 1.5 ml of LP-PRP was injected into the rotator cuff under ultrasound guidance.

For LR-PRP in platelet-rich plasma, 6 ml of LR-PRP was prepared from 60 ml of blood using a GPSTM III kit (Biomet). Specifically, after injecting 6ml of anticoagulant into the 60ml syringe included in the GPS kit, connecting the 60ml syringe and a butterfly needle to collect 54ml of blood, centrifugation at 3200rpm for about 15 minutes to obtain 6ml of LR-PRP separated. Among them, 1.5 ml of LR-PRP was injected into the rotator cuff under ultrasound guidance.

Example 3: Ultrasound evaluation of rotator cuff tendonopathy and platelet-rich plasma infusion

For ultrasound evaluation of rotator cuff tendinopathy, the thickness of the supraspinatus muscle was measured by placing an ultrasound detector on the supraspinatus muscle in a modified crass position after sitting the experimental group and the control group upright. The longest area to the supraspinatus was measured. The evaluation was performed in both the experimental group and the control group, and was compared before the start of treatment and after the final 6-month follow-up.

For platelet-rich plasma injection, after the first ultrasound evaluation, subjects in the experimental group were seated upright, and the upper arm with degenerative rotator cuff lesion was externally rotated as much as possible. Next, find the cross-section of the biceps iliac ligament through ultrasound, move to the proximal part while maintaining the cross-section, place the ultrasound detector in the supraspinatus muscle, and then use a needle to horizontally place the autologous platelet-rich plasma in the degenerative supraspinatus region under ultrasound guidance 1.5 ml was injected. In the case of subjects injecting platelet-rich plasma into bilateral rotator cuff lesions, 1.5 ml each was injected into both lesions, and platelet-rich plasma was injected into one lesion immediately after injection into other lesions in the same manner.

Example 4: Confirmation of clinical therapeutic effect in patients administered platelet-rich plasma

The degree of clinical function improvement and symptom improvement before, 3 months, and 6 months after administration of platelet-rich plasma of the experimental group set in Example 1 were evaluated by Numeral Rating Scale (NRS) pain scale or Constant score, respectively. Specifically, the NRS pain scale allows the patient to select the level of pain on a scale of 0 to 10 depending on the subject's subjectivity, and the Constant score provides subjective evaluation (35 points) of the pain level and daily living achievability, range of motion and muscle strength. The evaluation was subdivided into objective evaluation (65 points).

Example 5: Confirmation of growth factors, cytokines and cell concentrations in platelet-rich plasma

PDGF (platelet-derived growth factor), TGF-β (transforming growth factor beta), VEGF (vascular endothelial growth factor), IGF-1 (insulin-like) contained in the patient's autologous platelet-rich plasma prepared in Example 2 The concentrations of growth factor 1), IL-1β (interleukin 1 beta), IL-8 (interleukin 8), and white blood cells (WBC) were confirmed.

Multiplex cytokine assay was performed to quantify cytokine concentration levels such as PDGF, IGF-1, TGF-β, IL-1β (interleukin 1 beta), IL-8 (interleukin 8) and VEGF. Specifically, platelet abundance ^{After inoculating 1 Х 10 5} cells per well in a 6-well plate containing plasma cells, culture medium (1 mL) was collected after culturing, and according to the manufacturer's instructions, Milliplex human cytokine/chemokine magnetic panel kit (Millipore , Billerica, MA, USA). In addition, 25 μL of sample and 25 μL of beads were placed in a 96-well plate, and incubated at 4° C. overnight. The next day, the contents of the plate were removed and 25 μL of detection antibody was added to each well, and the plate was incubated for 1 hour at room temperature. After incubation, we added 25 μl of streptavidin-phycoerythrin to each well of the plate containing the detection antibody and 150 μL of sheath fluid to each well. Finally, we read the cytokine and chemokine concentrations in the plate using a cytometer (Luminex 200, Luminex, Austin, TX, USA).

The number of leukocytes was quantified using a blood analyzer (Siemens ADVIA 2120i, Siemens Healthcare Diagnostics, Erlangen, Germany).

Example 6: Statistical Analysis

In order to confirm the correlation between the concentrations of growth factors, cytokines and cells in platelet-rich plasma obtained from a patient with rotator cuff tendonitis confirmed in Example 1, and the clinical therapeutic effect of a patient administered with platelet-rich plasma, correlation analysis (correlation) analysis) and a Generalized Linear Model (GLM) were performed, and an analysis of covariance (ANalysis Of COVariance, ANCOVA) was additionally performed to correct the initial value of the corresponding part. Significant factors were identified through the analysis, and to determine the optimal reference values for these factors (Constant, NRS), a reference value with a significant odds ratio was selected through logistic regression. In addition, when there were a large number of significant reference values, the largest value (max value) was selected as the standard.

Specifically, the data of a total of 60 people (27 people in the experimental group and 33 people in the control group) were used for analysis. During data analysis, continuous variables were expressed as mean ± standard deviation, and categorical variables were expressed as number of times. For categorical data, chi-square test or Fisher exact test was performed. To determine whether there is a difference in blood information for each group, a difference test was performed through an independent two-samples t-test. To determine whether there is a difference in the growth factors for each group, a difference test was performed through the Mann-Whitney test. In order to compare the difference between the PRP group and the control group for clinical scores, the significance of the change according to the intervention was verified through analysis of covariance (ANCOVA). In order to obtain the cut-off value, Spearman correlation analysis was performed to determine significant variables, and then, through regression analysis with initial values corrected, each value was individually compared with other values to calculate the significant value. All statistical analyzes were performed using IBM SPSS version 20.0 (IBM corporation), and statistical significance was set when the P value was less than 0.05.

Example 7: Confirmation of association between growth factors, cytokines and cell concentrations in platelet-rich plasma and clinical therapeutic effect of platelet-rich plasma-NRS pain scale

Based on the clinical therapeutic effect of platelet-rich plasma treatment and the concentration of cytokines and cells in platelet-rich plasma, the concentration of various growth factors, cytokines and cells in platelet-rich plasma and clinical trials of platelet-rich plasma through statistical analysis The correlation of the therapeutic effect was confirmed through the NRS pain scale.

Specifically, the concentration of PDGF, TGF-β, VEGF, IGF-1, IL-1β, IL-8, and leukocytes contained in the initial platelet-rich plasma of the PRP treatment group (experimental group) set in Example 1, the control group and the experimental group in the NRS pain scale at 3 and 6 months after platelet-rich plasma infusion, respectively, based on the cut values of cytokines and cells, values higher and lower than the values were statistically compared, and the corresponding values were not related to the clinical outcome. A cut-off value with a clinically therapeutic effect was confirmed by comparative analysis based on the statistical analysis results to see if there was a significant effect.

As a result, the rotator cuff reconstruction x10 white blood cells in platelet rich plasma as to confirm the clinical benefit of the NRS pain scale at 6 months Variable 0.99 ³ / μL or more, 1.15 x10 ³ after platelet rich plasma injections dialectical patient / μL If it contains more than 1.23 x10 ³ pieces/μL or more or 1.78 x10 ³ pieces/μL or more, it can be determined that a clinically meaningful treatment effect such as relieving the patient's pain can be shown, and the highest value among these In the case of phosphorus 1.78 x 10 ³ pieces/μL or more, it is judged to be able to show the most clinically significant therapeutic effect (Table 1).

* total: the number of tests that were detected in the test

* num: The total number of the cut value value and other values for which regression analysis was performed, the number of values having a value greater than or equal to the cut value

In addition, as a variable that can confirm the clinical effect of the NRS pain scale at 6 months after injection of platelet-rich plasma in patients with rotator cuff tendonitis, VEGF in platelet-rich plasma is 8.01 pg/mL or more, 30.06 pg/mL or 37.71 pg/ If it contains more than mL, it can be judged that a clinically significant therapeutic effect such as relieving the patient's pain can be shown. is considered to be able to show (Table 2).

* total: the number of tests that were detected in the test

* num: The total number of the cut value value and other values for which regression analysis was performed, the number of values having a value greater than or equal to the cut value

In addition, as a variable that can confirm the clinical effect of the NRS pain scale at 6 months after injection of platelet-rich plasma in patients with rotator cuff tendonitis, when IL-8 is contained in platelet-rich plasma of 0 ng/mL or more, the patient's pain It can be determined that it can have a clinically significant therapeutic effect, such as alleviating It can be determined that a significant therapeutic effect can be exhibited (Table 3).

* total: the number of tests that were detected in the test

* num: The total number of the cut value value and other values for which regression analysis was performed, the number of values having a value greater than or equal to the cut value

In addition, the concentration of PDGF, TGF-β, IGF-1 or IL-1β contained in platelet-rich plasma does not seem to show a significant correlation between the clinical therapeutic effect through the actual NRS pain scale.

Example 8: Confirmation of association between growth factors, cytokines and cell concentrations in platelet-rich plasma and clinical therapeutic effect of platelet-rich plasma-Constant score

Based on the clinical therapeutic effect of platelet-rich plasma treatment and the concentration of cytokines and cells in platelet-rich plasma, the concentration of various growth factors, cytokines and cells in platelet-rich plasma and clinical trials of platelet-rich plasma through statistical analysis The correlation of the therapeutic effect was confirmed through the Constant score.

Specifically, the concentration of PDGF, TGF-β, VEGF, IGF-1, IL-1β, IL-8, and leukocytes contained in the initial platelet-rich plasma of the PRP treatment group (experimental group) set in Example 1, the control group and the experimental group Based on the Constant score at 3 and 6 months after platelet-rich plasma injection, and the cut level of each cytokine and cell in A cut-off value with a clinically therapeutic effect was confirmed by comparative analysis based on the results of statistical analysis on the effect.

As a result, the rotator cuff reconstruction dialectical patients after platelet rich plasma injections x10 white blood cells in platelet rich plasma as a variable to determine the clinical effectiveness of the Constant score at 6 months 0.99 ³ / μL or more, 1.15 x10 ³ / μL or more , 10.76 x10 ³ pieces/μL or more or 14.79 x10 ³ pieces/μL or more, it can be judged that a clinically significant treatment effect such as an improvement of the Constant score can be exhibited. It is judged that the case with a high value of 10.78 x 10 ³ pieces/μL or more can show the most clinically significant therapeutic effect (Table 4).

* total: the number of tests that were detected in the test

* num: The total number of the cut value and other values for which regression analysis was performed, the number of values having a value greater than or equal to the cut value

In addition, as a variable that can confirm the clinical effect of the Constant score 6 months after platelet-rich plasma injection in patients with rotator cuff tendonitis, when the platelet-rich plasma contains more than 0 ng/mL of IL-8, the Constant score improves. It can be determined that it can exhibit a clinically significant therapeutic effect such as It can be determined that the effect can be shown (Table 5).

* total: the number of tests that were detected in the test

* num: The total number of the cut value value and other values for which regression analysis was performed, the number of values having a value greater than or equal to the cut value

In addition, the concentration of PDGF, TGF-β, VEGF, IGF-1, or IL-1β contained in platelet-rich plasma does not seem to show a significant correlation between the clinical therapeutic effect through the actual Constant score.

To summarize the above, by checking the concentrations of leukocytes, VEGF and IL-8 contained in the platelet-rich plasma, it is possible to predict in advance that there is a clinical therapeutic effect on the platelet-rich plasma. can be operated efficiently.

From the above description, those skilled in the art to which the present invention pertains will understand that the present invention may be embodied in other specific forms without changing the technical spirit or essential characteristics thereof. In this regard, it should be understood that the embodiments described above are illustrative in all respects and not restrictive. The scope of the present invention should be construed as being included in the scope of the present invention, rather than the above detailed description, all changes or modifications derived from the meaning and scope of the claims described below and their equivalents.

Claims (19)

Measuring the concentration of VEGF (vascular endothelial growth factor) contained in platelet rich plasma (Platelet Rich Plasma, PRP); and
When the concentration of VEGF contained in platelet-rich plasma is 37.71 pg/mL or more, determining that the platelet-rich plasma has a clinically therapeutic effect
As a method for evaluating the clinical efficacy of platelet-rich plasma comprising a,
The method of claim 1, wherein the clinical effectiveness of the platelet-rich plasma is clinical effectiveness for the treatment of Tendinopathy.
According to claim 1,
The method further comprises measuring the concentration of one or more selected from the group consisting of IL-8 (interleukin 8), and white blood cells (WBC, white blood cells), platelet-rich plasma clinical efficacy evaluation method.
3. The method of claim 2, wherein the method
When the concentration of leukocytes contained in platelet-rich plasma is 1.78 x 10 ³ pieces/μL or more, the platelet-rich plasma is clinically effective for evaluating the clinical efficacy of platelet-rich plasma, further comprising the step of determining that it has a therapeutic effect .
delete 3. The method of claim 2,
Wherein the method further comprises the step of determining that the platelet-rich plasma has a clinically therapeutic effect when IL-8 is included in the platelet-rich plasma.
delete As a composition for clinical efficacy evaluation of platelet-rich plasma, comprising a formulation capable of detecting VEGF of 37.71 pg/mL or more contained in platelet rich plasma (PRP),
The clinical effectiveness of the platelet-rich plasma is the clinical efficacy for the treatment of tendinopathy (Tendinopathy), the composition.
8. The method of claim 7,
The composition further comprises an agent capable of detecting one or more selected from the group consisting of IL-8 (interleukin 8), and white blood cells (WBC), for evaluation of clinical efficacy of platelet-rich plasma composition.
The composition for evaluation of clinical efficacy of platelet-rich plasma according to claim 8, wherein the composition comprises ^{a formulation capable of detecting 1.78 x 10 3} pieces/μL or more of leukocytes contained in platelet-rich plasma.
delete delete (A) obtaining platelet-rich plasma from the subject;
(B) measuring the concentration of VEGF in the obtained platelet-rich plasma; and
(C) selecting platelet-rich plasma in which the concentration of VEGF is 37.71 pg/mL or more
As a method for producing platelet-rich plasma, comprising:
The method of claim 1, wherein the platelet-rich plasma is clinically effective for treating Tendinopathy.
13. The method of claim 12, wherein the method
(D) selecting platelet-rich plasma with a concentration of white blood cells of 1.78 x 10 ^{3 cells/μL or more}
Further comprising a, platelet-rich plasma production method.
delete 13. The method of claim 12, wherein the method
(D) selecting platelet-rich plasma containing IL-8
Further comprising a, platelet-rich plasma production method.
A pharmaceutical composition for treating or preventing tendinopathy, comprising platelet-rich plasma,
Wherein the platelet-rich plasma comprises 37.71 pg/mL or more of VEGF.
17. The method of claim 16,
The platelet-rich plasma is 1.78 x 10 ³ pieces / μL or more of a pharmaceutical composition for treating or preventing tendinopathy further comprising white blood cells.
delete 17. The method of claim 16,
The platelet-rich plasma further comprises IL-8, a pharmaceutical composition for treating or preventing tendinopathy.