Composition comprising isolated mitochondria for preventing or treating pain and use thereof

Abstract

The present invention relates to a composition comprising isolated mitochondria for preventing or treating pain, and a use thereof. The composition of the present invention can effectively treat or prevent pain and pain-related diseases. Specifically, the isolated mitochondria or the composition comprising same can effectively treat or alleviate pain by inhibiting the expression and / or activity of pain factors, and can specifically treat or alleviate only pain regardless of alleviation of pain-causing etiology.

Description

Composition for preventing or treating pain comprising isolated mitochondria and use thereof

[0001] The present invention relates to a composition for preventing or treating pain comprising isolated mitochondria and its use.

[0002] Pain is a serious condition that reduces both an individual's quality of life and their social productivity. It is defined as an unpleasant sensory and emotional experience associated with actual or potential tissue damage. Pain is a major symptom in a variety of diseases, but its perception is highly subjective, making it one of the most challenging pathologies to effectively diagnose and treat. Pain can severely impair functional abilities and jeopardize a patient's work, social, and family life.

[0003] This pain is very diverse and includes inflammatory pain, neuropathic pain, osteoarthritis pain, post-surgical pain, cancer pain, pain associated with metastatic cancer, pain caused by cancer chemotherapy drugs, trigeminal neuralgia, causalgia, acute herpes and post-herpetic neuralgia, occipital neuralgia, sympathetic dystrophy, fibromyalgia, gout pain, burn pain, phantom limb pain, complex regional pain syndrome, idiopathic pain syndrome, migraine, and chronic pain of unknown etiology.

[0004] Among the aforementioned pains, chronic pain after spinal surgery (CPSS) is a condition characterized by persistent back or lower extremity pain that persists for more than three months after lumbar surgery, with the pain becoming more severe or manifesting differently than before surgery. Existing treatments include exercise, medication, spinal interventions, adhesiolysis, and reoperations. However, the pain experienced by patients with CPSS can be unbearable, and multinational studies have shown that narcotic use rates reach 62%.

[0005] Meanwhile, mitochondria are essential organelles for the survival of eukaryotic cells, involved in the synthesis and regulation of adenosine triphosphate (ATP) as an energy source. Mitochondria are crucial organelles involved in various metabolic pathways within the body, such as cell signaling, cell differentiation, and apoptosis, as well as the control of the cell cycle and growth.

[0006] Accordingly, the inventors of the present invention completed the present invention by confirming that isolated mitochondria can alleviate pain in a CPSS model, thereby discovering that they can be applied as a pain prevention or treatment agent.

[0007] One aspect provides a pharmaceutical composition for preventing or treating pain, comprising isolated mitochondria as an active ingredient.

[0008] Another aspect provides a method for preventing or treating pain, comprising administering to a subject a pharmaceutical composition for preventing or treating pain.

[0009] Another aspect provides a pharmaceutical composition for preventing or treating pain, comprising a hydrogel comprising isolated mitochondria and a biocompatible polymer as active ingredients.

[0010] Another aspect offers a use for mitochondria to prevent or treat pain.

[0011] Another aspect provides the use of mitochondria for manufacturing drugs for preventing or treating pain.

[0012] One aspect is to provide a pharmaceutical composition for preventing or treating pain, comprising isolated mitochondria as an active ingredient.

[0013] As used herein, the term "mitochondria" refers to double-membrane-bound organelles found in most eukaryotic organisms, which produce most of the adenosine triphosphate (ATP) in the cell.

[0014] As used herein, the term "isolated mitochondria" refers to mitochondria obtained from an autologous, allogeneic or xenogeneic source.

[0015] As used herein, the term "autologous mitochondria" refers to mitochondria obtained from the plasma, tissue, bone marrow, or cells of the same individual. Furthermore, the term "allogeneic mitochondria" refers to mitochondria obtained from the plasma, tissue, bone marrow, or cells of an individual of the same species as the individual but having a different genotype for an allele. Furthermore, the term "heterologous mitochondria" refers to mitochondria obtained from the plasma, tissue, bone marrow, or cells of an individual of a different species from the individual.

[0016] At this time, the subject may be a mammal, and preferably a human.

[0017] The mitochondria may be isolated from cells, bone marrow, or plasma of the subject. The mitochondria may be obtained from autologous or allogeneic cells cultured in vitro. In this case, the cells, bone marrow, or plasma may have normal biological activity.

[0018] The term "cell" as used herein refers to a structural or functional unit that constitutes a living organism, consisting of cytoplasm surrounded by a cell membrane, and containing biomolecules such as proteins and nucleic acids. The cell refers to a cell that contains mitochondria within the cell membrane.

[0019] Additionally, the mitochondria may be separated after concentrating and crushing tissue, plasma, bone marrow or cells, or may be separated after crushing from a tissue, plasma, bone marrow or cell sample that has been frozen and then thawed.

[0020] In one specific example, the cell may be any one selected from the group consisting of stem cells, somatic cells, germ cells, and platelets.

[0021] The term "stem cell" as used herein refers to an undifferentiated cell capable of differentiating into various types of tissue cells. The stem cell may be any one selected from the group consisting of mesenchymal stem cells, adult stem cells, induced pluripotent stem cells, embryonic stem cells, bone marrow stem cells, neural stem cells, limbal stem cells, and tissue-derived stem cells.

[0022] At this time, the mesenchymal stem cells may be any one selected from the group consisting of umbilical cord, umbilical cord blood, bone marrow, fat, muscle, nerve, skin, amniotic membrane, and placenta. Preferably, they may be derived from human umbilical cord.

[0023] The term "somatic cell" as used herein refers to a cell excluding germ cells among the cells constituting an organism. The somatic cell may be one selected from the group consisting of muscle cells, hepatocytes, fibroblasts, epithelial cells, nerve cells, adipocytes, osteocytes, periosteal cells, leukocytes, lymphocytes, and mucosal cells. Preferably, the somatic cell may be obtained from muscle cells or hepatocytes with excellent mitochondrial activity. In addition, the somatic cell may be obtained from autologous or allogeneic blood PBMC cells.

[0024] The term "germ cell" as used herein refers to a cell that forms a zygote during reproduction in a sexually reproducing organism. The mitochondria may be obtained from autologous or allogeneic gametes. The gametes may be sperm or eggs.

[0025] As used herein, the term "bone marrow" refers to the semi-solid tissue found in the spongy portion of bone. Human bone marrow produces approximately 500 billion blood cells per day. In particular, bone marrow contains normally active mitochondria.

[0026] As used herein, the term "plasma" refers to the liquid component of blood, excluding blood cells, and refers to the intravascular portion of the extracellular fluid. Plasma comprises up to 95% water and 6 to 8% dissolved proteins or electrolytes. In particular, plasma contains normally active mitochondria.

[0027] The plasma can be obtained by separating it from blood. Specifically, it can be obtained by spinning blood containing an anticoagulant in a centrifuge to separate the supernatant from the blood. Furthermore, plasma can be extracted from the blood through filtration or coagulation. Furthermore, the plasma can be classified based on the blood source. In one specific example, the plasma may be plasma separated from umbilical cord blood or peripheral blood. Preferably, the plasma may be separated from umbilical cord blood.

[0028] In one specific example, the plasma or bone marrow may be obtained and stored from an individual.

[0029] In one specific example, the mitochondria may be isolated from platelets.

[0030] As used herein, the term "platelet" refers to a solid component of blood that plays a crucial role in blood clotting by binding fibrin to form a clot. The mitochondria may be obtained from autologous or allogeneic platelets.

[0031] The isolated mitochondria may have normal biological activity. Specifically, the mitochondria having normal biological activity may have one or more of the following characteristics: (i) having a membrane potential, (ii) generating ATP within the mitochondria, and (iii) removing ROS or reducing the activity of ROS within the mitochondria.

[0032] When isolating mitochondria from specific cells, the separation can be accomplished using various known methods, such as using specific buffer solutions or utilizing potential differences and magnetic fields. Furthermore, the mitochondrial separation may include a step of centrifuging and filtering plasma to remove all cellular components, and a step of centrifuging the filtered plasma.

[0033] The above mitochondrial separation can be obtained by disrupting cells and centrifuging them to maintain mitochondrial activity. Centrifugation may be performed one to three times.

[0034] In one specific example, the method may be performed by culturing cells, centrifuging a composition containing the cells a first time to produce a pellet, resuspending the pellet in a buffer solution and homogenizing it, centrifuging the homogenized solution a second time to produce a supernatant, and centrifuging the supernatant a third time to purify mitochondria. At this time, it is preferable in terms of maintaining cell activity that the time for performing the second centrifugation is controlled to be shorter than the times for performing the first and third centrifugations, and the speed can be increased from the first to the third centrifugation.

[0035] When separating the above mitochondria from plasma, they can be separated using various known methods, such as using a specific buffer solution, ultrasound, a concentration gradient, and a magnetic field.

[0036] The above mitochondrial isolation includes a step of removing cells or organelles from plasma; and a step of purifying mitochondria. In addition, the above mitochondrial isolation may include a step of physically separating endoplasmic reticulum, mitochondrial-associated membrane fragments, and mitochondria.

[0037] In one specific example, the separation may be by centrifugation. Specifically, the separation may be performed through a step of first centrifuging the plasma at low speed to remove cells within the plasma; a step of filtering the plasma to remove cell debris; and a step of second centrifuging the supernatant of the plasma.

[0038] In one specific example, the separation may be by discontinuous concentration gradient and centrifugation. The discontinuous concentration gradient may utilize a sucrose or Percoll concentration gradient. Specifically, the separation may be performed through the steps of: lysing cells using ultrasound; performing a first low-speed centrifugation of plasma to remove cells within the plasma; performing a second centrifugation of the plasma to remove vesicles; loading the supernatant of the plasma onto a discontinuous concentration gradient; and performing a third centrifugation of the separated result.

[0039] The first to third centrifugations may be performed at a temperature of 0 to 10°C, preferably 3 to 5°C. In addition, the time for performing the centrifugation may be 1 to 50 minutes, and may be appropriately adjusted depending on the number of centrifugations and the content of the sample. In addition, the first centrifugation may be performed at a speed of 100 to 1,000 xg, or 200 to 700 xg, or 300 to 450 xg. Additionally, the second or third centrifugation may be performed at a speed of 1 to 2,000 xg, 25 to 1,800 xg, or 500 to 1,600 xg, 100 to 20,000 xg, 500 to 18,000 xg, or 800 to 15,000 xg.

[0040] The membrane proteins of the separated mitochondria can be quantified to quantify the mitochondria. Specifically, the separated mitochondria can be quantified using the bicinchoninic acid assay (BCA) analysis method. At this time, the mitochondria in the pharmaceutical composition can be included at a concentration of 0.1 μg / ml to 1,000 μg / ml, 1 μg / ml to 750 μg / ml, 25 μg / ml to 500 μg / ml, 25 μg / ml to 150 μg / ml, or 25 μg / ml to 100 μg / ml.

[0041] In one embodiment of the present invention, the mitochondria in the pharmaceutical composition are 1 to 100 ㎍ / ㎖, 1 to 80 ㎍ / ㎖, 1 to 60 ㎍ / ㎖, 1 to 50 ㎍ / ㎖, 1 to 40 ㎍ / ㎖, 1 to 30 ㎍ / ㎖, 1 to 25 ㎍ / ㎖, 1 to 20 ㎍ / ㎖, 5 to 100 ㎍ / ㎖, 5 to 80 ㎍ / ㎖, 5 to 60 ㎍ / ㎖, 5 to 50 ㎍ / ㎖, 5 to 40 ㎍ / ㎖, 5 to 30 ㎍ / ㎖, 5 to 25 ㎍ / ㎖, 5 to 20 ㎍ / ㎖, 10 to 100 ㎍ / ㎖, 10 to 80 ㎍ / ㎖, 10 to 60 ㎍ / ㎖, 10 to 50 It may be included at a concentration of ㎍ / ㎖, 10 to 40 ㎍ / ㎖, 10 to 30 ㎍ / ㎖, 10 to 25 ㎍ / ㎖, 10 to 20 ㎍ / ㎖, 15 to 100 ㎍ / ㎖, 15 to 80 ㎍ / ㎖, 15 to 60 ㎍ / ㎖, 15 to 50 ㎍ / ㎖, 15 to 40 ㎍ / ㎖, 15 to 30 ㎍ / ㎖, 15 to 25 ㎍ / ㎖, 15 to 20 ㎍ / ㎖, 20 to 100 ㎍ / ㎖, 20 to 80 ㎍ / ㎖, 20 to 60 ㎍ / ㎖, 20 to 50 ㎍ / ㎖, 20 to 40 ㎍ / ㎖, 20 to 30 ㎍ / ㎖ or 20 to 25 ㎍ / ㎖.

[0042] Additionally, the mitochondria may be in an intact form, a fragmented form, or a combination thereof. In one specific example, the mitochondria may exhibit a pharmacological effect even in a fragmented form if they retain mitochondrial activity.

[0043] Additionally, the number of separated mitochondria can be measured using a particle counter (Multisizer4e, Beckman Coulter).

[0044] In one specific example, the mitochondria in the pharmaceutical composition are 1×10 5 Number of mitochondria / ml to 9×10 9 It can be included in the content of number of mitochondria / ml. Specifically, the mitochondria in the pharmaceutical composition are 1×10 5 / ㎖ to 5×10 9 / ㎖, 2×10 5 / ㎖ to 2×10 9 / ㎖, 5×10 5 / ㎖ to 1×10 9 / ㎖, 1×10 6 / ㎖ to 5×10 8 / ㎖, 2×10 6 / ㎖ to 2×10 8 / ㎖, 5×10 6 / ㎖ to 1×10 8 / ㎖ or 1×10 7 / ㎖ to 5×10 7 / ㎖ content may be included.

[0045] In one embodiment, the mitochondria or composition containing them may be included at a concentration that does not ameliorate or treat the underlying cause of the pain. The mitochondria included in the composition may act specifically on pain without ameliorating or treating the underlying cause of the pain, effectively ameliorating or treating only the pain.

[0046] The term "pain" in the present invention is defined as an unpleasant sensory and emotional experience accompanied by actual or potential tissue damage, and the pain felt by humans can be broadly divided into acute pain and chronic pain. Acute pain is pain caused by nociceptive stimulation resulting from tissue damage due to disease or trauma, and includes pain from childbirth, post-surgical pain, and pain after tissue damage. It generally disappears within 3 to 6 months and is effectively treated with various drugs (narcotic analgesics, non-steroidal anti-inflammatory drugs). Chronic pain is caused by nerve damage and subsequent changes in the nervous system due to various unclear causes, lasts longer than the healing period of the disease or injury that caused it, has unclear boundaries of the painful area, and presents a persistent, dull, deep pain that lasts for more than 3 to 6 months, and the use of narcotic analgesics and non-steroidal anti-inflammatory drugs is limited.

[0047] The pain may include at least one selected from the group consisting of inflammatory pain, neuropathic pain, osteoarthritis pain, post-surgical pain, cancer pain, pain associated with metastatic cancer, pain induced by cancer chemotherapy, trigeminal neuralgia, causalgia, acute herpes and post-herpetic neuralgia, occipital neuralgia, sympathetic dystrophy, fibromyalgia, gouty pain, burn pain, phantom limb pain, complex regional pain syndrome, idiopathic pain syndrome, musculoskeletal pain, musculoskeletal chronic pain, post-spinal surgery chronic pain, migraine, and chronic pain of unknown cause, and specifically, the pain may be at least one selected from the group consisting of musculoskeletal pain, musculoskeletal chronic pain, and post-spinal surgery chronic pain, but is not limited thereto.

[0048] The above musculoskeletal pain (chronic pain) refers to pain that can be caused by diseases of bones, joints, muscles, tendons, ligaments, bursae, or a combination thereof. Accordingly, the above musculoskeletal pain may include one or more selected from the group consisting of bone pain, muscle pain, tendon / ligament pain, bursae pain, and joint pain.

[0049] The above muscle pain may include pain caused by muscle damage, spasm, cramps, stiffness, contraction and / or muscle atrophy.

[0050] The above pain may be caused by increased expression and / or activity of pain factors.

[0051] The pharmaceutical composition may reduce the expression and / or activity of a pain factor. The pain factor may include at least one selected from the group consisting of calcitonin gene-related peptide (CGRP) and substance P, and may specifically reduce the expression and / or activity of substance P.

[0052] The pharmaceutical composition may treat, prevent, or improve pain by reducing the expression and / or activity of pain factors, and specifically, may treat or improve pain by reducing the expression and / or activity of one or more selected from the group consisting of calcitonin gene-related peptide (CGRP) and substance P.

[0053] The pharmaceutical composition may be one that treats, prevents, or improves pain without increasing and / or decreasing an inflammatory response, and specifically, may be one that treats, prevents, or improves pain without affecting the expression / activity level of inflammatory factors such as inflammatory cytokines. The inflammatory cytokines and / or inflammatory factors may include one or more selected from the group consisting of NFkB, IFN-γ, IL-17A, IL-1β, IL-6, and TNF-α.

[0054] The term "treatment" in this specification means any action that improves or beneficially changes the symptoms of pain and pain-related diseases by administering the composition of the present invention.

[0055] The term "prevention" in this specification means any action by which the possibility of the onset of pain and pain-related diseases is suppressed or delayed by administration of the composition of the present invention.

[0056] The pharmaceutical composition may further comprise an ingredient known to be effective in preventing, treating or improving pain, and specifically, may further comprise at least one selected from the group consisting of vitamin D, a nonsteroidal anti-inflammatory drug (NSAID), an antioxidant and an opioid.

[0057] The above components may be in the form of, but are not limited to, compounds, proteins and / or nucleic acids.

[0058] The pharmaceutical composition may include a pharmaceutically acceptable carrier. The term "pharmaceutically acceptable carrier" may refer to a carrier or diluent that does not stimulate a living organism and does not inhibit the biological activity or properties of the injected compound. Here, "pharmaceutically acceptable" means that the carrier does not inhibit the activity of the active ingredient and does not exhibit toxicity exceeding the adaptability of the subject of application (prescription). Any carrier commonly used in the art and pharmaceutically acceptable for the pharmaceutical composition may be used. Non-limiting examples of the above carriers include lactose, dextrose, maltodextrin, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, glycerol, ethanol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, saline solution, sterile water, Ringer's solution, buffered saline, albumin injection solution, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, or mineral oil. These may be used alone or in combination of two or more. The pharmaceutical composition may be prepared as a parenteral formulation according to the administration route by a conventional method known in the art, including a pharmaceutically acceptable carrier in addition to the active ingredient. The above pharmaceutical composition can be formulated and used in the form of an external preparation, suppository, or sterile injection solution according to a conventional method.

[0059] When formulating the above pharmaceutical composition, it may be prepared using a diluent or excipient such as a generally used filler, bulking agent, binder, wetting agent, disintegrant, or surfactant, but may not be limited thereto.

[0060] When the above pharmaceutical composition is prepared as a parenteral dosage form, it can be formulated in the form of injections, transdermal administration, nasal inhalation, and suppositories using a suitable carrier according to a method known in the art. When formulated as an injection, suitable carriers include sterile water, ethanol, polyols such as glycerol or propylene glycol, or mixtures thereof, and preferably, Ringer's solution, phosphate buffered saline (PBS) containing triethanolamine, sterile water for injection, and isotonic solutions such as 5% dextrose can be used. When formulated as a transdermal dosage form, it can be formulated in the form of ointments, creams, lotions, gels, external solutions, pastes, liniments, aerosols, etc. In the case of nasal inhalation, it can be formulated in the form of an aerosol spray using a suitable propellant such as dichlorofluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, or carbon dioxide, and in the case of formulating it as a suppository, the base can be witepsol, tween 61, polyethylene glycol, cacao butter, laurin butter, polyoxyethylene sorbitan fatty acid ester, polyoxyethylene stearate, sorbitan fatty acid ester, etc.

[0061] The pharmaceutical composition may be administered in a pharmaceutically effective amount. The term "pharmaceutically effective amount" refers to an amount sufficient to treat or prevent a disease at a reasonable benefit / risk ratio applicable to medical treatment or prevention, and the effective dosage level may be determined based on the severity of the disease, the activity of the drug, the patient's age, weight, health, sex, the patient's sensitivity to the drug, the time of administration of the composition of the present invention used, the route of administration and the excretion rate, the treatment period, the drug used in combination with or concurrently with the composition of the present invention used, and other factors well known in the medical field. The pharmaceutical composition of the present invention may be administered alone or in combination with a component known to exhibit a therapeutic effect on a known disease. It is important to take all of the above factors into consideration and administer an amount that can achieve the maximum effect with the minimum amount without side effects.

[0062] The dosage of the pharmaceutical composition may be determined by a person skilled in the art in consideration of the intended use, the degree of toxicity of the disease, the patient's age, weight, sex, medical history, or the type of substance used as the active ingredient. For example, the pharmaceutical composition of the present invention may be administered at about 0.1 ng to about 1,000 mg / kg, preferably 1 ng to about 100 mg / kg, per adult, and the frequency of administration of the composition of the present disclosure is not particularly limited thereto, but may be administered once a day or administered in divided doses several times. The dosage or frequency of administration does not limit the scope of the present disclosure in any way.

[0063]

[0064] Another aspect provides a method for preventing or treating pain, comprising administering to a subject a pharmaceutical composition for preventing or treating pain. The same principles as described above apply to the method.

[0065] The term "subject" as used herein may include, without limitation, mammals, birds, reptiles, farmed fish, etc., including dogs, cats, rats, livestock, and humans, which develop or are at risk of developing pain or a pain-related disorder. Furthermore, the subject may exclude humans.

[0066] The pharmaceutical composition described above may be administered in single or multiple doses in a pharmaceutically effective amount. The composition may be formulated and administered in the form of a liquid, powder, aerosol, injection, infusion solution (Ringel), capsule, pill, tablet, suppository, or patch. The pharmaceutical composition for preventing or treating pain may be administered via any conventional route, as long as it can reach the target tissue.

[0067] The above pharmaceutical composition may be administered via a route such as intraperitoneal administration, intravenous administration, intramuscular administration, subcutaneous administration, intradermal administration, transdermal patch administration, oral administration, intranasal administration, intrapulmonary administration, or rectal administration, although it is not particularly limited thereto, depending on the purpose.

[0068]

[0069] Another aspect provides a pharmaceutical composition for preventing or treating pain, comprising a hydrogel containing isolated mitochondria and a biocompatible polymer as an active ingredient. The same principles described above apply to the composition.

[0070] The above hydrogel may have excellent storage convenience because it has the effect of preserving and maintaining the activity of mitochondria for a long period of time.

[0071] The above hydrogel can increase the efficiency of mitochondrial delivery to a target site or target cell because it suppresses the dispersion of mitochondria to other tissues and the mitochondrial delivery effect can be concentrated at the site of administration.

[0072] The above hydrogel can be manufactured by mixing isolated mitochondria and a biocompatible polymer.

[0073] The above biocompatible polymer can be selected appropriately depending on the site of administration.

[0074] The above biocompatible polymer may be a synthetic polymer or a natural polymer.

[0075] The above synthetic polymers are polyethylene (PEA), polyethyleneglycol (PEG), polycaprolactone (PCL), polyalkylcarbonate, polyamino acid, polyhydroxybutyric acid, polyorthoester, polyanhydride, poly(ethylene oxide), poly(propylene oxide), poly(ethylene oxide), Pluronic, polylactide (PLA), polyglycolide (PGA), polybutylene succinate (PBS), poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV), polybutylene terephthalate (PLA). The polymer may be at least one selected from the group consisting of poly(lactic-co-glycolic acid, PLGA), poly(lactic-co-glycolide)-glucose (PLGA-glucose), poly(lactic-co-glycolide)-glucose (PLGA-glucose), and methoxy polyethylene glycol-(polycaprolactone-co-polylactide) (MPEG-(PCL-co-PLLA).

[0076] The above natural polymer may be at least one selected from the group consisting of carboxymethylcellulose (CMC), algin, alginic acid, alginate, hyaluronic acid, polypeptide, protein, gelatin, collagen, albumin, dextran, starch, casein, chitin derivatives, chitosan, and small intestinal submucosa (SIS).

[0077] The above natural polymer may be an extracellular matrix component, for example, hyaluronic acid, collagen, gelatin, albumin, or glycosamidoglycan, and the glycosamidoglycan may be chondroitin sulfate, heparan sulfate, or keratan sulfate.

[0078] The above biocompatible polymer may be crosslinked.

[0079] The biocompatible polymer may have an average molecular weight of 2,000,000 to 4,000,000 Da, 2,300,000 to 4,000,000 Da, 2,000,000 to 3,700,000 Da, 2,200,000 to 3,700,000 Da, or 2,500,000 to 3,500,000 Da.

[0080] The content of the biocompatible polymer may be appropriately increased or decreased as needed, and may be, for example, 1 to 10 wt%, 1 to 5 wt%, or 1 to 3 wt%.

[0081] The content of mitochondria in the hydrogel may be, for example, 0.1 ㎍ / ㎖ to 10 mg / ㎖, 0.1 ㎍ / ㎖ to 5 mg / ㎖, 0.1 ㎍ / ㎖ to 1 mg / ㎖, 1 ㎍ / ㎖ to 10 mg / ㎖, 1 ㎍ / ㎖ to 5 mg / ㎖, 1 ㎍ / ㎖ to 1 mg / ㎖, 5 ㎍ / ㎖ to 10 mg / ㎖, 5 ㎍ / ㎖ to 5 mg / ㎖, 5 ㎍ / ㎖ to 1 mg / ㎖, 10 ㎍ / ㎖ to 10 mg / ㎖, 10 ㎍ / ㎖ to 5 mg / ㎖, 10 ㎍ / ㎖ to 1 mg / ㎖ based on mitochondrial protein.

[0082] The hydrogel may be an injectable formulation. If the hydrogel is an injectable formulation, it is advantageous to have a high storage modulus (G'). The storage modulus of a hydrogel that is easy to inject is, for example, a storage modulus (G') of 2.00 x 10 measured using a rheometer in a frequency range of 0.1 Hz to 10 Hz. 2 Pa to 2.00 x 10 3 Pa, or 2.00 x 10 2 Pa to 4.00 x 10 2 Pa may be, but is not limited to, this.

[0083] The complex viscosity of the hydrogel at 25°C is 3.00 x 10 1 Pa·s to 3.0 x 10 2 Pa·s, or 3.00 x 10 1 Pa·s to 6.0 x 10 1 Pa·s may be, but is not limited to, this.

[0084] The hydrogel may be manufactured into an injectable support (filler) that can be administered by injection. The filler may be a hyaluronic acid filler, for example, a monophasic filler or a piphasic filler.

[0085]

[0086] Another aspect is the use of mitochondria to prevent or treat pain. The same principles described above apply to this use.

[0087]

[0088] Another aspect provides the use of mitochondria for the manufacture of drugs for the prevention or treatment of pain. The same principles described above apply to this use.

[0089] The composition of the present invention can effectively treat or prevent pain and pain-related diseases. Specifically, isolated mitochondria or compositions containing them can effectively treat or alleviate pain by inhibiting the expression and / or activity of pain factors, and can specifically treat or alleviate pain alone, regardless of the improvement of the underlying cause of the pain.

[0090] Figure 1 is a schematic diagram illustrating the process of isolating and obtaining stem cell-derived mitochondria.

[0091] Figure 2 is a schematic diagram illustrating the process of isolating and obtaining platelet-derived mitochondria.

[0092] Figure 3 is a schematic diagram illustrating the process of isolating and obtaining plasma-derived mitochondria.

[0093] Figure 4, A, is a transmission electron microscope observation result of the obtained plasma-derived mitochondria, B is a result of measuring the size and distribution of the obtained plasma-derived mitochondria, and C is a drawing confirming the expression levels of various markers of stem cell-derived, platelet-derived, and plasma-derived mitochondria.

[0094] Figure 5 is a diagram showing the concentration and activity of plasma-derived mitochondria isolated from blood by age and patient (normal or patient group with underlying disease).

[0095] Figure 6 is a schematic diagram illustrating the process of creating a chronic pain after spinal surgery (CPSS) animal model.

[0096] Figure 7 is a drawing showing a spinal magnetic resonance imaging (spine MRI; SIGNA Pioneer 3.0T MR, GE Healthcare Co. USA) photograph taken to confirm paraspinal muscle atrophy that occurred after surgery to create a CPSS animal model.

[0097] Figures 8A and 8B are diagrams showing the results of measuring the cross-sectional area (CSA) of paraspinal muscles on MRI on the day before surgery (pre-surgery), 2 weeks after surgery (post-surgery), and 2 weeks after injection of a treatment (mitochondrion, vitamin D, or a combination of mitochondria / vitamin D) for inducing a CPSS animal model in the control group and CPSS animal model. Meanwhile, in the control group, surgery for inducing a CPSS animal model was not performed, so no surgery was performed post-surgery, and no treatment or PBS was injected post-treat. In addition, in the surgery group, SHE buffer was injected instead of a treatment post-treat.

[0098] Figure 9 shows the results of examining the degree of pain response in the control group and CPSS animal model on the day before surgery (pre-surgery), 2 weeks after surgery (post-surgery), and 2 weeks after injection of a treatment (mitochondrion, vitamin D, or a combination of mitochondria / vitamin D) to induce CPSS animal model.

[0099] Figure 10 is a diagram showing the results of measuring the expression level of pain factors according to mitochondrial treatment in the control group and CPSS animal model.

[0100] Figure 11 is a diagram showing the results of measuring the expression levels of inflammatory cytokines according to the control group, CPSS animal model, and mitochondrial treatment.

[0101] Figure 12 is a diagram showing the results of measuring the expression levels of inflammatory factors according to the control group, CPSS animal model, and mitochondrial treatment.

[0102] The present invention will be described in more detail through the following examples. However, these examples are provided for illustrative purposes only and the scope of the present invention is not limited to these examples.

[0103]

[0104] Example 1: Obtaining mitochondria from umbilical cord-derived mesenchymal stem cells

[0105] The following experiments were performed to isolate / obtain mitochondria from mesenchymal stem cells. A schematic diagram of the isolation / obtaining method is shown in Figure 1.

[0106]

[0107] 1-1: Culturing of umbilical cord-derived stem cells

[0108] Umbilical cord-derived mesenchymal stem cells (IRB number: No.201411-BR-022-02 or No.201806-BR-029-03) were obtained from the Wharton jelly of the umbilical cord and used in the experiment. Specifically, the isolated umbilical cord-derived mesenchymal stem cells were cultured in T-175 culture flasks using Minimum Essential Medium Alpha Modification (MEM Alpha Modification, Hyclone) medium containing 10% fetal bovine serum (FBS; Gibco, Waltham, USA) and 1% penicillin / streptomycin antibiotics (P / S, Hyclone, Logan, USA). The cells were maintained in culture at 37°C and 5% CO2, and the next subculture was performed when the cell density was approximately 80% to 90%.

[0109]

[0110] 1-2: Isolation of mitochondria from umbilical cord-derived mesenchymal stem cells

[0111] Mitochondria were isolated / obtained from umbilical cord-derived mesenchymal stem cells cultured in Example 1-1 above. Specifically, 2 x 10 7 200 ㎕ of SHE buffer [0.25 M sucrose, 20 mM HEPES (pH 7.4), 2 mM EGTA, 10 mM KCl, 1.5 mM MgCl2, 0.1% defatted bovine serum albumin (BSA), pH 7.4] was added to suspend the cells and incubated at 4℃ for 5 minutes. After that, the cell membrane was disrupted using a 1 ㎖ syringe (Koreavaccine, Seoul, South Korea). To remove undisrupted cells and nuclei, centrifugation was performed at 1,500 × g at 4℃ for 5 minutes. The supernatant was collected and centrifuged at 20,000 × g at 4℃ for 10 minutes. To wash the isolated mitochondria, 2 ㎖ of SHE buffer without BSA was added and centrifuged at 20,000 × g at 4℃ for 10 minutes. After removing the supernatant, the mitochondrial pellet was washed twice with Dulbecco's phosphate-buffered saline (DPBS; Welgene, South Korea). The washed pellet was then suspended in 200 μl of DPBS and stored at 4°C. The isolated mitochondria were confirmed to have a pale white suspension.

[0112]

[0113] Example 2: Obtaining platelet- and plasma-derived mitochondria

[0114] The following experiments were conducted to isolate / obtain mitochondria present in platelets and plasma. Schematic diagrams of the isolation / obtaining method are depicted in Figures 2 and 3.

[0115]

[0116] 2-1: Platelet separation

[0117] Donor blood was transported in heparinized tubes and used for the experiment. The donor blood was placed in a 50-mL tube and centrifuged at 3,000 rpm for 5 minutes. After centrifugation, the plasma was separated into a new 50-mL tube and centrifuged at 2,000 x g for 10 minutes. Platelets were obtained from the pellet obtained after centrifugation. The platelet-free plasma was then separated into a new 50-mL tube.

[0118]

[0119] 2-2: Isolation of platelet-derived mitochondria

[0120] Mitochondria were obtained from human platelets in the same manner as in Example 1-2, except that mitochondria were obtained from human platelets obtained in Example 2-1.

[0121]

[0122] 2-3: Isolation of plasma-derived mitochondria

[0123] The plasma from which platelets were separated, obtained in Example 2-1, was centrifuged at 20,000 x g, 4°C for 10 minutes to precipitate mitochondria present in the plasma, and the supernatant was removed. Thereafter, the mitochondria were washed in the same manner using the SHE buffer used in Example 1-2.

[0124]

[0125] Example 3: Characterization of plasma-derived mitochondria

[0126] In order to analyze the characteristics of plasma-derived mitochondria isolated and obtained in Example 2, the following experiments were performed.

[0127] First, the obtained plasma-derived mitochondria were observed using a transmission electron microscope (TEM), and it was confirmed that they formed a double-membrane structure and had a cristae structure (Fig. 4A). In addition, to confirm the size of the obtained plasma-derived mitochondria, dynamic light scattering (DLS) equipment, which measures particle size and distribution, was used, and the result was confirmed to be approximately 383 ± 43.9 nm (Fig. 4B).

[0128] Next, the purity of the umbilical cord-derived mesenchymal stem cell mitochondria, platelet-derived mitochondria, and plasma-derived mitochondria isolated in Examples 1 and 2 was confirmed at the level of specific proteins in each organelle. As a result, mitochondrial-specific proteins, COX IV and TOM20, were detected in all mitochondria. Lysosome- and peroxisome-specific proteins were detected in umbilical cord-derived stem cell mitochondria and platelet-derived mitochondria, whereas they were not detected in plasma-derived mitochondria (Fig. 4C).

[0129]

[0130] Example 4: Evaluation of plasma-derived mitochondrial activity by age and patient

[0131] In order to evaluate the activity of plasma-derived mitochondria isolated in Example 2 above according to the donor's age and presence or absence of underlying disease, the following experiment was performed.

[0132] First, using the bicinchoninic acid assay (BCA) analysis technique, the concentration of plasma-derived mitochondrial proteins obtained from the above-described examples was quantified based on the volume of initially isolated plasma, categorized by donor age and patient type. As a result, no significant differences were observed in the concentration of plasma-derived mitochondria obtained by age or patient type (Figures 5A and 5C).

[0133] Next, to analyze the activity level of plasma-derived mitochondria isolated by age and patient of the donor, 10 μg of mitochondria and 5 mM ADP were used to react for 45 minutes, and then the amount of ATP produced in the mitochondria was measured using the CellTiter-Glo luminescence kit (Promega, Madison, WI, USA), which measures the luminescence signal proportional to the amount of ATP, thereby confirming ATP synthesis activity. As a result, there was no significant difference in the activity of plasma-derived mitochondria obtained according to age and patient status (Figures 5B and 5D).

[0134] Based on the above results, it can be seen that plasma mitochondria exhibit constant activity regardless of the donor's age or disease status.

[0135]

[0136] Example 5: Creation of an animal model of chronic pain after spinal surgery (CPSS).

[0137] To evaluate the pain treatment or alleviation efficacy of the isolated mitochondria of the present invention, a chronic pain after spinal surgery (CPSS) animal model was created using the following method.

[0138] Specifically, to establish an animal model of chronic pain after spinal surgery (CPSS), 12-week-old Sprague-Dawley rats (SD rats, weighing 340-370 g) were divided into a control group and an induction group. In the induction group, the left L5 spinal nerve was transected, and then part of the lamina of the L5-L6 vertebrae, including the facet joint of the left L5-L6 vertebrae, was removed using a rongeur (Fig. 6). The separated muscles were then sutured in layers, and the skin was closed to complete the surgery.

[0139]

[0140] Example 6: Evaluation of improvement in muscle atrophy following mitochondrial treatment

[0141] In order to evaluate whether muscle atrophy caused by spinal surgery could be improved by mitochondrial treatment in the chronic pain syndrome (CPSS) animal model produced in Example 5 above, the following experiments were performed.

[0142] Specifically, mitochondria (Mito), vitamin D (VitD), or a combination thereof (Mito / VitD) were injected as therapeutic agents into the control and experimental groups two weeks after the spinal surgery described in Example 5. Approach was made under a C-type fluoroscopy device for precise injection. The mitochondria injection group was injected with 50 μg of plasma-derived mitochondria isolated and obtained in Example 2 into the left lumbar muscles (L4-L5, L5-L6 level) using a 29 G syringe. Mitochondria were stored in SHE buffer and injected locally in a volume of 100 μl per site. The vitamin D injection group was injected with 150 μg of cholecalciferol into the same sites as above. The combination group was injected with 50 μg of mitochondria and 150 μg of vitamin D into the same sites as above.

[0143] Next, to quantitatively evaluate the changes in paraspinal muscle atrophy associated with CPSS according to surgery and treatment, high-resolution MRI (SIGNA Pioneer 3.0T MR, GE Healthcare Co. USA) was taken for each subject on the day before surgery (Pre-surgery), 2 weeks after surgery (Post-surgery), and 2 weeks after treatment (Post-treat), as shown in Fig. 7. This allowed for a precise evaluation of the anatomical changes and degree of atrophy of the paraspinal muscles due to surgery and / or treatment injection. For the evaluation, the cross-sectional area (CSA) of the multifidus (MF) on both sides at the L5-L6 and L6-S1 levels in the MRI images was measured using the ImageJ program.

[0144] As a result, compared to the control group, in all groups that underwent surgery to induce CPSS (Surgery group, Mito group, VitD group, Mito / VitD group), atrophy of paraspinal muscles was observed at both the L5-L6 level and the L6-S1 level before and after surgery. In addition, it was confirmed that muscle atrophy did not improve within the observation period in most groups, including the mitochondrial injection group, even after treatment (Fig. 8A and B).

[0145] Based on the above results, it can be seen that muscle atrophy in an animal model of chronic pain after spinal surgery is not improved within the observation period even when mitochondria are treated.

[0146]

[0147] Example 7: Evaluation of the pain treatment or relief efficacy of mitochondria.

[0148] To evaluate the pain treatment or relief efficacy of mitochondria, the following experiments were performed.

[0149] Specifically, to evaluate the pain treatment effect of the plasma-derived mitochondria isolated and obtained in Example 2, the von Frey test, a mechanical sensitization method, was performed to quantitatively evaluate the pain threshold. The von Frey test was performed at three specific times: pre-surgery, post-surgery, and post-treatment in the CPSS animal model established in Example 5. Each group was subjected to constant pressure on the left hind paw for 3-5 seconds on a grid floor in a controlled environment until the von Frey filaments were broken. Each animal was evaluated through three independent measurements.

[0150] The von Frey test results above showed that the pain threshold in the left side where surgery was performed was significantly reduced before and after surgery, indicating that the pain was induced by CPSS. Furthermore, in the treatment group, a response evaluation two weeks after the injection of the test drug confirmed that the pain threshold significantly increased in the mitochondria-injected experimental group, indicating that the pain was alleviated or treated by the mitochondrial injection (Fig. 9).

[0151] Based on the above results, it was confirmed that mitochondria did not significantly affect the improvement of muscle atrophy appearing around the surgical area in the CPSS model, but effectively alleviated or treated pain, and thus it can be seen that the isolated mitochondria of the present invention exhibit pain-specific treatment or improvement efficacy.

[0152]

[0153] Example 8: Evaluation of pain-specific factor expression levels by mitochondria

[0154] To evaluate the efficacy of suppressing the expression level of pain factors in mitochondria, the following experiments were performed.

[0155] Specifically, the efficacy of the plasma-derived mitochondria isolated and obtained in Example 2 on suppressing the expression level of pain factors was evaluated, and 1 ml of blood was collected from each of the pre-surgery (0 week), post-surgery (2 weeks), and post-treatment (4 week) groups of the CPSS animal model established in Example 5. The collected blood was collected in an EDTA tube and immediately centrifuged at 1,000xg for 10 minutes to separate the plasma. Next, the change in Substance P, a pain-specific factor in plasma according to the treatment, was confirmed using a Substance P ELISA kit (CUSABIO, Houston, TX, USA). To this end, the plasma of each group was reacted with the reagent included in the kit and then the absorbance was measured.

[0156] As a result, Substance P significantly increased after surgery compared to the control group. However, the mitochondrial injection group (Mito) and the mitochondrial / vitamin D injection group (Mito / VitD) showed a decreased increase, whereas the surgery group and the vitamin D injection group showed a continuous increase in Substance P (Fig. 10).

[0157] Based on the above results, it can be seen that mitochondria can effectively alleviate or treat pain by suppressing the expression level of Substance P, a pain factor.

[0158]

[0159] Example 9: Evaluation of inflammatory cytokine expression levels by mitochondria

[0160] To evaluate the expression levels of inflammatory cytokines following mitochondrial treatment, the following experiments were performed.

[0161] First, 1 ml of blood was collected from the CPSS animal model established in Example 5 before surgery (control), after surgery (surgery), and after treatment. The blood was collected in EDTA tubes and immediately centrifuged at 1,000 × g for 10 minutes to separate the plasma. Next, a multiplex kit (Merck Millipore, Burlington, MA, USA) was used to determine changes in the inflammatory cytokines IFN-γ, IL-17A, IL-1β, IL-6, and TNF-α in the plasma according to treatment. The beads included in the kit were added to each plasma and reacted for 2 hours, and then streptavidin-phycoerythrin was added and reacted for 30 minutes. Thereafter, changes in each inflammatory cytokine were analyzed using the Luminex MAGPIX system (Merck Millipore). As a result of the above experiment, it was confirmed that no inflammatory cytokines increased after surgery, and that no significant changes in the levels of inflammatory cytokines were observed in the mitochondrial injection group (Mito), the vitamin D injection group (VitD), and the mitochondrial / vitamin D injection group (Mito / VitD) (Fig. 11), indicating that no changes were observed in the levels of inflammatory cytokines secreted in the blood following the establishment of the CPSS animal model and treatment with a therapeutic agent.

[0162] Next, tissues were obtained from each individual before surgery (control), after surgery (surgery), and after treatment in the CPSS animal model established in Example 5. Next, Western blotting was performed to confirm the expression pattern of an inflammatory factor (NFkB) at the protein level. Each tissue was finely ground in a mortar and pestle with liquid nitrogen and the concentration was analyzed using the BCA assay technique. Each tissue protein was separated by size through SDS-PAGE and transferred to a PDVF membrane. Thereafter, the membrane was stored in TBS-T containing 5% serum albumin (BSA) to block non-specific reactions, and the primary antibodies NFκB (Santa Cruz; sc-8008), phospho-NFκB (Cell signaling; 3033s), and GAPDH (Cell signaling; 2118s) were added to each membrane and reacted. The next day, secondary antibodies m-IgGκ BP-HRP (Santa Cruz; sc-516102) and mouse anti-rabbit IgG-HRP (Santa Cruz; sc-2357) were added, and after reaction, the reaction was carried out with ECL solution and analyzed using LAS-4000 (Fuji photo film, Tokyo, Japan). As a result of the above experiment, the expression level of inflammatory factors decreased after surgery, and in the mitochondria-injected group (Mito) and the mitochondria / vitamin D-injected group (Mito / VitD), the expression level of inflammatory factors was confirmed to increase (Fig. 12). It can be seen that the above results show a different tendency from Example 8, which evaluated the expression level of pain-specific factors by mitochondria. In addition, based on the fact that NFκB, a short-term inflammatory mediator, was not reduced when mitochondria were injected into the muscle injury site, it can be seen that pain relief by mitochondria is not induced by the relief of inflammation.

[0163] Based on the above results, it can be seen that the mechanism of treating or alleviating pain using mitochondria is different from the inflammatory response and has no effect.

[0164]

[0165] In summary of the results of the above examples, it was confirmed that when mitochondria were treated in the CPSS animal model of the example, there was no significant effect on improving muscle atrophy around the surgical area (Fig. 8), and it was effective in alleviating pain regardless of the inflammatory response (Figs. 11-12) (Figs. 9-10).

[0166]

[0167] The foregoing description of the present invention is provided for illustrative purposes only. Those skilled in the art will readily appreciate that the present invention can be readily modified into other specific forms without altering the technical spirit or essential characteristics of the present invention. Therefore, the embodiments described above should be understood as illustrative in all respects and not restrictive.

Claims

1. A pharmaceutical composition for preventing or treating pain, comprising isolated mitochondria as an active ingredient.

2. A pharmaceutical composition according to claim 1, wherein the mitochondria are isolated from cells or plasma.

3. A pharmaceutical composition according to claim 2, wherein the cell comprises at least one selected from the group consisting of somatic cells, germ cells, stem cells, and blood cells.

4. A pharmaceutical composition according to claim 3, wherein the stem cells include at least one selected from the group consisting of mesenchymal stem cells, induced pluripotent stem cells, and embryonic stem cells.

5. A pharmaceutical composition according to claim 4, wherein the mesenchymal stem cells are derived from at least one selected from the group consisting of umbilical cord, umbilical cord blood, bone marrow, fat, muscle, nerve, skin, amniotic membrane, placenta, synovial fluid, testis, and periosteum.

6. A pharmaceutical composition according to claim 2, wherein the plasma is plasma from bone marrow, umbilical cord blood, or peripheral blood.

7. A pharmaceutical composition according to claim 1, wherein the mitochondria are isolated from platelets.

8. A pharmaceutical composition according to claim 1, wherein the pharmaceutical composition reduces the expression or activity of a pain factor.

9. A pharmaceutical composition according to claim 8, wherein the pain factor comprises at least one selected from the group consisting of calcitonin gene-related peptide (CGRP) and substance P.

10. A pharmaceutical composition according to claim 1, wherein the mitochondria are included at a concentration of 0.1 μg / ml to 1,000 μg / ml.

11. A pharmaceutical composition according to claim 1, wherein the pain comprises at least one selected from the group consisting of inflammatory pain, neuropathic pain, osteoarthritis pain, post-surgical pain, cancer pain, pain associated with metastatic cancer, pain caused by cancer chemotherapy, trigeminal neuralgia, causalgia, acute herpes and post-herpetic neuralgia, occipital neuralgia, sympathetic dystrophy, fibromyalgia, gouty pain, burn pain, phantom limb pain, complex regional pain syndrome, idiopathic pain syndrome, musculoskeletal pain, musculoskeletal chronic pain, chronic pain after spinal surgery, migraine, and chronic pain of unknown cause.

12. A pharmaceutical composition according to claim 1, wherein the composition further comprises at least one selected from the group consisting of vitamin D, a nonsteroidal anti-inflammatory drug (NSAID), an antioxidant, and an opioid.

13. A pharmaceutical composition for preventing or treating pain, comprising a hydrogel containing isolated mitochondria and a biocompatible polymer as an active ingredient.

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