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WO2025070898A1 - Composition comprenant des mitochondries isolées pour prévenir ou traiter la douleur et son utilisation - Google Patents

Composition comprenant des mitochondries isolées pour prévenir ou traiter la douleur et son utilisation Download PDF

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Publication number
WO2025070898A1
WO2025070898A1 PCT/KR2023/021581 KR2023021581W WO2025070898A1 WO 2025070898 A1 WO2025070898 A1 WO 2025070898A1 KR 2023021581 W KR2023021581 W KR 2023021581W WO 2025070898 A1 WO2025070898 A1 WO 2025070898A1
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Prior art keywords
pain
mitochondria
pharmaceutical composition
plasma
composition according
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English (en)
Korean (ko)
Inventor
최용수
민경훈
김미진
윤창구
김성훈
임익현
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Industry Academic Cooperation Foundation of College of Medicine Pochon CHA University
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Industry Academic Cooperation Foundation of College of Medicine Pochon CHA University
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Priority claimed from KR1020230190348A external-priority patent/KR20250046116A/ko
Application filed by Industry Academic Cooperation Foundation of College of Medicine Pochon CHA University filed Critical Industry Academic Cooperation Foundation of College of Medicine Pochon CHA University
Publication of WO2025070898A1 publication Critical patent/WO2025070898A1/fr
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/14—Blood; Artificial blood
    • A61K35/16—Blood plasma; Blood serum
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/28—Bone marrow; Haematopoietic stem cells; Mesenchymal stem cells of any origin, e.g. adipose-derived stem cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00

Definitions

  • the present invention relates to a composition for preventing or treating pain comprising isolated mitochondria and uses thereof.
  • Pain is a serious condition that reduces both the quality of life of an individual and social productivity. Pain is defined as an unpleasant sensory and emotional experience related to actual or potential tissue damage. Pain is a major symptom in various diseases, but its perception is very subjective, making it one of the most difficult pathologies to effectively diagnose and treat. Pain causes severe impairment of functional ability and jeopardizes the patient's work, social, and family life.
  • This pain is very diverse and includes inflammatory pain, neuropathic pain, osteoarthritis pain, post-surgical pain, cancer pain, pain associated with metastatic cancer, pain induced by cancer chemotherapy drugs, 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, migraine, and chronic pain of unknown etiology.
  • CPSS chronic pain after spinal surgery
  • Existing treatments include exercise, medication, spinal intervention, adhesiolysis, and reoperation, but the pain of patients with CPSS is difficult to endure, and according to a multinational study, the frequency of drug use is known to be as high as 62%.
  • 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 important organelles involved in various metabolic pathways in the body, such as cell signaling, cell differentiation, apoptosis, as well as the control of the cell cycle and cell growth.
  • 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.
  • One aspect provides a pharmaceutical composition for preventing or treating pain, comprising isolated mitochondria as an active ingredient.
  • Another aspect provides a method of preventing or treating pain, comprising administering to a subject a pharmaceutical composition for preventing or treating pain.
  • compositions for preventing or treating pain comprising a hydrogel comprising isolated mitochondria and a biocompatible polymer as active ingredients.
  • Another aspect offers a use for mitochondria to prevent or treat pain.
  • Another aspect provides the use of mitochondria for manufacturing drugs for preventing or treating pain.
  • One aspect is to provide a pharmaceutical composition for preventing or treating pain, comprising isolated mitochondria as an active ingredient.
  • mitochondria refers to double-membrane-bound organelles found in most eukaryotic organisms, which produce most of the adenosine triphosphate (ATP) in the cell.
  • ATP adenosine triphosphate
  • isolated mitochondria refers to mitochondria obtained from an autologous, allogeneic or xenogeneic source.
  • autologous mitochondria refers to mitochondria obtained from the plasma, tissue, bone marrow or cells of the same individual.
  • allogeneic mitochondria refers to mitochondria obtained from the plasma, tissue, bone marrow or cells of an individual belonging to the same species as the individual but having a different genotype for an allele.
  • heterologous mitochondria refers to mitochondria obtained from the plasma, tissue, bone marrow or cells of an individual belonging to a different species from the individual.
  • the subject may be a mammal, and preferably a human.
  • the above mitochondria may be isolated from cells, bone marrow or plasma of the individual.
  • the above 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.
  • cell as used herein means a structural or functional unit constituting a living organism, consisting of cytoplasm surrounded by a cell membrane, and containing biomolecules such as proteins and nucleic acids.
  • the cell means a cell containing mitochondria inside the cell membrane.
  • the mitochondria may be separated and used after concentrating and crushing tissue, plasma, bone marrow or cells, or may be separated and crushed from tissue, plasma, bone marrow or cell samples that have been frozen and then thawed.
  • the cell may be any one selected from the group consisting of stem cells, somatic cells, germ cells, and platelets.
  • stem cell refers to an undifferentiated cell having the ability to differentiate 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.
  • the mesenchymal stem cell 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, it may be derived from human umbilical cord.
  • the term "somatic cell” in this specification refers to a cell excluding germ cells among the cells constituting an individual.
  • the somatic cell may be one selected from the group consisting of muscle cells, hepatocytes, fibroblasts, epithelial cells, nerve cells, adipocytes, bone cells, periosteal cells, leukocytes, lymphocytes, and mucosal cells.
  • muscle cells or hepatocytes with excellent mitochondrial activity.
  • it may be obtained from autologous or allogeneic blood PBMC cells.
  • 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.
  • bone marrow refers to the semi-solid tissue found in the spongy portion of the bone.
  • the bone marrow produces about 500 billion blood cells per day in humans.
  • the bone marrow contains normally active mitochondria.
  • plasma refers to the liquid component of blood excluding blood cells, and refers to the intravascular portion of the extracellular fluid.
  • the plasma contains up to 95% water, 6 to 8% dissolved proteins or electrolytes, etc.
  • the plasma contains normally active mitochondria.
  • the plasma can be obtained by separating from blood.
  • the plasma can be obtained by spinning blood containing an anticoagulant in a centrifuge to separate the supernatant from the blood.
  • the plasma can be extracted from the blood by filtration or coagulation.
  • the plasma can be classified according to the blood from which it is derived.
  • the plasma can be plasma separated from cord blood or peripheral blood. Preferably, it can be separated from cord blood.
  • the plasma or bone marrow may be obtained and stored from an individual.
  • the mitochondria may be isolated from platelets.
  • platelet refers to a solid component that plays an important role in blood clotting by binding fibrin in the blood to form a clot.
  • the mitochondria may be obtained from autologous or allogeneic platelets.
  • the above isolated mitochondria may have normal biological activity.
  • the mitochondria having normal biological activity may have one or more characteristics from the group consisting of (i) having a membrane potential, (ii) generating ATP within the mitochondria, and (iii) removing ROS or reducing the activity of ROS within the mitochondria.
  • the mitochondria When the mitochondria are separated from a specific cell, they can be separated by various known methods, such as using a specific buffer solution or using a potential difference and a magnetic field.
  • the separation of the mitochondria can include a step of centrifuging and filtering the plasma to remove all cellular components, and a step of centrifuging the filtered plasma.
  • the above mitochondrial separation can be obtained by disrupting cells and centrifuging them in order to maintain mitochondrial activity. At this time, centrifugation may be performed once to three times.
  • the method may be performed by the steps of culturing cells, performing a first centrifugation on a composition containing the cells to generate a pellet, resuspending the pellet in a buffer solution and homogenizing it, performing a second centrifugation on the homogenized solution to produce a supernatant, and performing a third centrifugation on the supernatant to purify mitochondria.
  • separation can be done through various known methods, such as using a specific buffer solution, ultrasound, a concentration gradient, and a magnetic field.
  • the above mitochondrial separation includes a step of removing cells or organelles from plasma; a step of purifying mitochondria.
  • the mitochondrial separation may include a step of physically separating endoplasmic reticulum, mitochondrial-associated membrane fragments, and mitochondria.
  • the separation may be by centrifugation. Specifically, the separation may be performed through a step of performing a low-speed primary centrifugation on the plasma to remove cells in the plasma; a step of filtering the plasma to remove cell debris; and a step of performing a secondary centrifugation on the supernatant of the plasma.
  • the separation may be by discontinuous concentration gradient and centrifugation.
  • the discontinuous concentration gradient may utilize a sucrose or Percoll concentration gradient.
  • the separation may be performed through a step of lysing cells using ultrasound; a step of removing cells in plasma by performing a low-speed primary centrifugation of plasma; a step of removing vesicles by performing a secondary centrifugation of plasma; a step of loading the supernatant of plasma onto a discontinuous concentration gradient; and a step of performing a tertiary centrifugation of the separated result.
  • the first to third centrifugations may be performed at a temperature of 0 to 10° C., preferably 3 to 5° C.
  • the time for performing the centrifugation may be 1 to 50 minutes, and may be appropriately adjusted depending on the number of centrifugations, the content of the sample, etc.
  • 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.
  • the second centrifugation or third centrifugation can 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.
  • the membrane protein of the separated mitochondria can be quantified to quantify the mitochondria.
  • the separated mitochondria can be quantified using the BCA (bicinchoninic acid assay) analysis method.
  • the mitochondria in the pharmaceutical composition can be included in a concentration of 0.1 ⁇ g/ml to 1,000 ⁇ g/ml, 1 ⁇ g/ml to 750 ⁇ g/ml, 25 ⁇ g/ml to 500 ⁇ g/ml, 25 ⁇ g/ml to 150 ⁇ g/ml, or 25 ⁇ g/ml to 100 ⁇ g/ml.
  • the mitochondria in the pharmaceutical composition is present in an amount of 1 to 100 ⁇ g/mL, 1 to 80 ⁇ g/mL, 1 to 60 ⁇ g/mL, 1 to 50 ⁇ g/mL, 1 to 40 ⁇ g/mL, 1 to 30 ⁇ g/mL, 1 to 25 ⁇ g/mL, 1 to 20 ⁇ g/mL, 5 to 100 ⁇ g/mL, 5 to 80 ⁇ g/mL, 5 to 60 ⁇ g/mL, 5 to 50 ⁇ g/mL, 5 to 40 ⁇ g/mL, 5 to 30 ⁇ g/mL, 5 to 25 ⁇ g/mL, 5 to 20 ⁇ g/mL, 10 to 100 ⁇ g/mL, 10 to 80 ⁇ g/mL, 10 to 60 ⁇ g/mL, 10 to 50 ⁇ g/ml, 10 to 40 ⁇ g/ml, 10 to 30 ⁇ g/ml, 10 to 25 ⁇ g/ml, 10 to 20 ⁇ g/mL, 10 to
  • the mitochondria may be in an intact form, a fragmented form, or a combination thereof.
  • the mitochondria may exhibit a pharmacological effect even in a fragmented form if they have mitochondrial activity.
  • the number of separated mitochondria can be measured using a particle counter (Multisizer4e, Beckman Coulter).
  • the mitochondria in the pharmaceutical composition may be included in an amount of 1 ⁇ 10 5 mitochondria/mL to 9 ⁇ 10 9 mitochondria/mL.
  • the mitochondria in the pharmaceutical composition may be included in an amount of 1 ⁇ 10 5 /mL to 5 ⁇ 10 9 /mL, 2 ⁇ 10 5 /mL to 2 ⁇ 10 9 /mL, 5 ⁇ 10 5 /mL to 1 ⁇ 10 9 /mL, 1 ⁇ 10 6 /mL to 5 ⁇ 10 8 /mL, 2 ⁇ 10 6 /mL to 2 ⁇ 10 8 /mL, 5 ⁇ 10 6 /mL to 1 ⁇ 10 8 /mL, or 1 ⁇ 10 7 /mL to 5 ⁇ 10 7 /mL.
  • the mitochondria or composition containing them may be included in a concentration that does not improve or treat the cause of the pain itself.
  • the mitochondria included in the composition may act specifically on pain alone, effectively improving or treating only the pain, without treating or improving the cause that initially induced the pain.
  • 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 largely 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 during childbirth, pain after surgery, and pain after tissue damage, and generally disappears within 3 to 6 months, and is effectively treated with various drugs (narcotic analgesics, nonsteroidal anti-inflammatory drugs).
  • Chronic pain is caused by nerve damage and subsequent changes in the nervous system due to various unclear causes, and lasts longer than the healing period of the disease or damage that caused it, the border of the painful area is unclear, and it continuously shows dull and deep pain, and lasts for more than 3 to 6 months, and the use of narcotic analgesics or nonsteroidal anti-inflammatory analgesics is limited.
  • 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 agents, 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, chronic musculoskeletal pain, chronic pain following spinal surgery, migraine, and chronic pain of unknown etiology, and specifically, the pain may be at least one of musculoskeletal pain, chronic musculoskeletal pain, and chronic pain following spinal surgery, but is not limited thereto.
  • the above musculoskeletal pain refers to pain that may be caused by diseases of bones, joints, muscles, tendons, ligaments, bursae, or a combination thereof. Accordingly, the above musculoskeletal pain may include at least one selected from the group consisting of bone pain, muscle pain, tendon/ligament pain, bursae pain, and joint pain.
  • the above muscle pain may include pain caused by muscle damage, contraction, cramping, stiffness, contraction and/or muscle wasting.
  • the above pain may be caused by increased expression and/or activity of pain factors.
  • 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 specifically may reduce the expression and/or activity of substance P.
  • CGRP calcitonin gene-related peptide
  • 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.
  • CGRP calcitonin gene-related peptide
  • the pharmaceutical composition may be for treating, preventing or ameliorating pain without increasing and/or decreasing an inflammatory response, and specifically, may be for treating, preventing or ameliorating pain without affecting the expression/activity level of inflammatory factors such as inflammatory cytokines.
  • the inflammatory cytokines and/or inflammatory factors may include at least one selected from the group consisting of NFkB, IFN- ⁇ , IL-17A, IL-1 ⁇ , IL-6, and TNF- ⁇ .
  • treatment means any action that improves or beneficially changes the symptoms of pain and pain-related diseases by administering the composition of the present invention.
  • prevention 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.
  • 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.
  • NSAID nonsteroidal anti-inflammatory drug
  • the above components may be in the form of, but are not limited to, compounds, proteins and/or nucleic acids.
  • the pharmaceutical composition may include a pharmaceutically acceptable carrier.
  • the "pharmaceutically acceptable carrier” may mean a carrier or diluent that does not stimulate a living organism and does not inhibit the biological activity and properties of the injected compound.
  • the meaning of "pharmaceutically acceptable” means that it does not inhibit the activity of the active ingredient and does not have toxicity that is more than the target of application (prescription) can adapt to.
  • Any type of carrier that can be used in the pharmaceutical composition may be used as long as it is a pharmaceutically acceptable carrier that is commonly used in the relevant technical field.
  • Non-limiting examples of the carrier 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.
  • the pharmaceutical composition may be prepared as a parenteral dosage form 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, respectively, according to a conventional method.
  • the above pharmaceutical composition may be prepared using diluents or excipients such as fillers, bulking agents, binders, wetting agents, disintegrating agents, or surfactants that are commonly used, but may not be limited thereto.
  • diluents or excipients such as fillers, bulking agents, binders, wetting agents, disintegrating agents, or surfactants that are commonly used, but may not be limited thereto.
  • 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.
  • PBS phosphate buffered saline
  • transdermal formulation When formulated as a transdermal formulation, it can be formulated in the form of ointments, creams, lotions, gels, external solutions, pastes, liniments, aerosols, etc.
  • nasal inhalers they 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 suppositories, the bases that can be used include witepsol, Tween 61, polyethylene glycols, cacao butter, laurin butter, polyoxyethylene sorbitan fatty acid esters, polyoxyethylene stearates, and sorbitan fatty acid esters.
  • the pharmaceutical composition may be administered in a pharmaceutically effective amount.
  • pharmaceutically effective amount means 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 according to 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 or simultaneously 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 administer an amount that can obtain the maximum effect with the minimum amount without side effects by considering all of the above factors.
  • 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.
  • the pharmaceutical composition of the present invention may be administered to an adult at about 0.1 ng to about 1,000 mg/kg, preferably 1 ng to about 100 mg/kg, and the frequency of administration of the composition of the present application is not particularly limited thereto, but may be administered once a day or administered several times in divided doses.
  • the dosage or frequency of administration does not limit the scope of the present application in any way.
  • Another aspect is to provide a method for preventing or treating pain, comprising administering to a subject a pharmaceutical composition for preventing or treating pain.
  • a method for preventing or treating pain comprising administering to a subject a pharmaceutical composition for preventing or treating pain.
  • the same parts as described above also apply to the method.
  • subject may include, without limitation, mammals, birds, reptiles, farmed fish, etc., including dogs, cats, rats, livestock, humans, etc., that are suffering from or at risk of suffering from pain or a pain-related disorder. In addition, the subject may exclude humans.
  • the pharmaceutical composition above can be administered in single or multiple doses in a pharmaceutically effective amount.
  • the composition can be formulated and administered in the form of a solution, powder, aerosol, injection, infusion (Ringel), capsule, pill, tablet, suppository or patch.
  • the route of administration of the pharmaceutical composition for preventing or treating pain can be administered through any common route as long as it can reach the target tissue.
  • 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, depending on the intended purpose, but is not particularly limited thereto.
  • 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, depending on the intended purpose, but is not particularly limited thereto.
  • Another aspect is to provide a pharmaceutical composition for preventing or treating pain, comprising a hydrogel comprising isolated mitochondria and a biocompatible polymer as active ingredients.
  • a pharmaceutical composition for preventing or treating pain comprising a hydrogel comprising isolated mitochondria and a biocompatible polymer as active ingredients.
  • the same parts as described above are also applicable to the composition.
  • the above hydrogel may have excellent storage convenience because it has the effect of preserving and maintaining the activity of mitochondria for a long time.
  • the above hydrogel can increase the efficiency of mitochondrial delivery to a target site or target cell because it can suppress the dispersion of mitochondria to other tissues and the mitochondrial delivery effect can be concentrated at the site of administration.
  • the above hydrogel can be manufactured by mixing isolated mitochondria and a biocompatible polymer.
  • the above biocompatible polymer can be selected appropriately depending on the site of administration.
  • the above biocompatible polymer may be a synthetic polymer or a natural polymer.
  • 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 (PCL).
  • PPA polyethylene
  • PEG polyethyleneglycol
  • PCL polycaprolactone
  • PCL polyalkylcarbonate
  • polyamino acid polyhydroxybutyric acid
  • PCL polyorthoester
  • polyanhydride poly(ethylene oxide), poly(propylene oxide), poly(ethylene oxide), Pluronic, polylactide (PLA), polyglycolide (PGA), polybutylene succinate (PBS), poly
  • 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 poly(ethylene glycol)-(polycaprolactone-co-polylactide) (MPEG-(PCL-co-PLLA).
  • 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).
  • CMC carboxymethylcellulose
  • algin alginic acid
  • alginate alginate
  • hyaluronic acid polypeptide
  • protein protein
  • gelatin collagen
  • albumin dextran
  • starch casein
  • casein chitin derivatives
  • chitosan chitosan
  • small intestinal submucosa small intestinal submucosa
  • the above natural polymer may be an extracellular matrix component, and may be, for example, hyaluronic acid, collagen, gelatin, albumin, or glycosamidoglycan, and the glycosamidoglycan may be chondroitin sulfate, heparan sulfate, or keratan sulfate.
  • the above biocompatible polymer may be crosslinked.
  • the biocompatible polymer can 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.
  • the content of the above biocompatible polymer can 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%.
  • the content of mitochondria in the hydrogel may be, for example, 0.1 ⁇ g/ml to 10 mg/ml, 0.1 ⁇ g/ml to 5 mg/ml, 0.1 ⁇ g/ml to 1 mg/ml, 1 ⁇ g/ml to 10 mg/ml, 1 ⁇ g/ml to 5 mg/ml, 1 ⁇ g/ml to 1 mg/ml, 5 ⁇ g/ml to 10 mg/ml, 5 ⁇ g/ml to 5 mg/ml, 5 ⁇ g/ml to 1 mg/ml, 10 ⁇ g/ml to 10 mg/ml, 10 ⁇ g/ml to 5 mg/ml, 10 ⁇ g/ml to 1 mg/ml, based on mitochondrial protein.
  • the hydrogel may be an injectable formulation. If the hydrogel is an injectable formulation, it is advantageous if the hydrogel has a high storage modulus (G').
  • the storage modulus of the hydrogel that is easy to inject may be, for example, a storage modulus (G') of 2.00 x 10 2 Pa to 2.00 x 10 3 Pa, or 2.00 x 10 2 Pa to 4.00 x 10 2 Pa, as measured using a rheometer in a frequency range of 0.1 Hz to 10 Hz, but is not limited thereto.
  • the complex viscosity at 25°C of the easily injectable hydrogel can be, but is not limited to, 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.
  • the above hydrogel can be manufactured into an injectable support (filler) that can be administered by injection.
  • the filler can be a hyaluronic acid filler, and can be, for example, a monophasic filler or a pi-phasic filler.
  • Another aspect is the use of mitochondria to prevent or treat pain.
  • the same parts described above apply to this use as well.
  • Another aspect provides the use of mitochondria for the manufacture of a drug for the prevention or treatment of pain.
  • the same as described above applies to the above use as well.
  • the composition of the present invention can effectively treat or prevent pain and pain-related diseases.
  • the isolated mitochondria or the composition containing them can effectively treat or improve pain by suppressing the expression and/or activity of pain factors, and can specifically treat or improve only pain regardless of the improvement of the cause causing the pain.
  • Figure 1 is a schematic diagram illustrating the process of isolating and obtaining stem cell-derived mitochondria.
  • Figure 2 is a schematic diagram illustrating the process of isolating and obtaining platelet-derived mitochondria.
  • Figure 3 is a schematic diagram illustrating the process of isolating and obtaining plasma-derived mitochondria.
  • Figure 4A is a transmission electron microscope observation result of the obtained plasma-derived mitochondria
  • Figure B is a drawing showing the results of measuring the size and distribution of the obtained plasma-derived mitochondria
  • Figure C is a drawing showing the expression levels of various markers of stem cell-derived, platelet-derived, and plasma-derived mitochondria.
  • 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).
  • Figure 6 is a schematic diagram illustrating the process of creating an animal model of chronic pain after spinal surgery (CPSS).
  • CPSS chronic pain after spinal surgery
  • Figure 7 is a drawing showing a spine magnetic resonance imaging (spine MRI; SIGNA Pioneer 3.0T MR, GE Healthcare Co. USA) image to confirm paraspinal muscle atrophy that occurred after surgery to create a CPSS animal model.
  • spine magnetic resonance imaging spine MRI; SIGNA Pioneer 3.0T MR, GE Healthcare Co. USA
  • Figures 8A and 8B are diagrams showing the results of measuring the cross-sectional area (CSA) of the paraspinal muscles on MRI on the day before surgery for inducing a CPSS animal model (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) in the control group and the CPSS animal model.
  • CSA cross-sectional area
  • Figure 9 shows the results of examining the degree of pain response in the control group and the CPSS animal model on the day before surgery (pre-surgery), 2 weeks after surgery (post-surgery), and 2 weeks after injection of treatment (mitochondrial, vitamin D, or mitochondria/vitamin D combination) to induce the CPSS animal model.
  • Figure 10 is a diagram showing the results of measuring the expression levels of pain factors according to mitochondrial treatment in the control group and CPSS animal model.
  • Figure 11 is a diagram showing the results of measuring the expression levels of inflammatory cytokines in the control group, CPSS animal model, and mitochondrial treatment.
  • 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.
  • Example 1 Obtaining mitochondria from umbilical cord-derived mesenchymal stem cells
  • 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 a T-175 culture flask 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 cultured while maintaining them at 37°C and 5% CO 2 conditions, and the next subculture was performed when the cell density was approximately 80% to 90%.
  • MEM Alpha Modification Minimum Essential Medium Alpha Modification
  • FBS fetal bovine serum
  • P/S penicillin/streptomycin antibiotics
  • Mitochondria were isolated/obtained from the umbilical cord-derived mesenchymal stem cells cultured in the above Example 1-1. Specifically, 200 ⁇ l of SHE buffer [0.25 M Sucrose, 20 mM HEPES (pH 7.4), 2 mM EGTA, 10 mM KCl, 1.5 mM MgCl 2 , 0.1% defatted bovine serum albumin (BSA), pH 7.4] was added to 2 x 10 7 cells, the cells were suspended, and then incubated at 4°C for 5 minutes. After that, the cell membrane was disrupted using a 1 ml syringe (Koreavaccine, Seoul, South Korea).
  • SHE buffer 0.25 M Sucrose, 20 mM HEPES (pH 7.4), 2 mM EGTA, 10 mM KCl, 1.5 mM MgCl 2 , 0.1% defatted bovine serum albumin (BSA), pH 7.4
  • Example 2 Obtaining platelet and plasma derived mitochondria
  • the donor's blood was transported in a heparin tube and used in the experiment.
  • the donor's 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 2,000xg for 10 minutes. Platelets were obtained from the pellet obtained after centrifugation. The plasma from which the platelets were separated was separated into a new 50 ml tube and obtained.
  • 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.
  • Example 2-1 The plasma from which platelets were separated obtained in Example 2-1 was centrifuged at 20,000xg, 4°C for 10 minutes to precipitate the 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.
  • the obtained plasma-derived mitochondria were observed using a transmission electron microscope (TEM) and were found to have a double membrane structure and a cristae structure (Fig. 4A).
  • TEM transmission electron microscope
  • Fig. 4A dynamic light scattering equipment
  • DLS dynamic light scattering
  • Example 2 In order to evaluate the activity of plasma-derived mitochondria isolated in Example 2 above according to the age and underlying disease of the donor, the following experiments were performed.
  • Example 5 Creation of an animal model of chronic pain after spinal surgery (CPSS)
  • CPSS chronic pain after spinal surgery
  • CPSS chronic pain after spinal surgery
  • 12-week-old Sprague-Dawley rats (SD rats, weighing 340-370 g) were divided into a control group and an 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). After that, the separated muscles were sutured in layers, and the skin was closed to complete the surgery.
  • mitochondria mitochondria
  • vitamin D vitamin D
  • Mito/VitD a combination thereof
  • the mitochondria injection group was injected with 50 ⁇ g of the plasma-derived mitochondria isolated and obtained in Example 2 into the left lumbar muscles (L4-L5, L5-L6 levels) using a 29 G syringe.
  • the mitochondria were stored in SHE buffer and locally injected with a volume of 100 ⁇ l per site.
  • the vitamin D injection group was injected with 150 ⁇ g of cholecalciferol into the same sites as above.
  • the combination group was injected with 50 ⁇ g of mitochondria and 150 ⁇ g of vitamin D into the same sites as above.
  • the von Frey test a mechanical sensitization method, was performed for quantitative evaluation of the pain threshold.
  • the von Frey test was performed at three specific times, pre-surgery, post-surgery, and post-treat, in the CPSS animal model established in the above Example 5. Each group applied constant pressure to 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 with three independent measurements.
  • the left side where the surgery was performed showed a significant decrease in the pain threshold before and after the surgery, indicating that the pain was induced by CPSS.
  • the pain threshold was significantly increased in the experimental group that received mitochondria injection in the response evaluation 2 weeks after the injection of the test drug, indicating that the pain was relieved or treated by the injection of mitochondria (Fig. 9).
  • Example 2 the efficacy of the plasma-derived mitochondria isolated and obtained in Example 2 to suppress the expression level of pain factors was evaluated, and 1 ml of blood was collected from the pre-surgery (O week), post-surgery (2 weeks), and post-treatment (4 weeks) groups of the CPSS animal model established in Example 5.
  • the collected blood was collected in an EDTA tube and immediately centrifuged at 1,000 ⁇ g for 10 minutes to separate the plasma.
  • Substance P a pain-specific factor in plasma according to the treatment agent
  • was confirmed using a Substance P ELISA kit (CUSABIO, Houston, TX, USA).
  • the plasma of each group was subjected to a reaction with the reagent included in the kit and then the absorbance was measured.
  • Substance P was significantly increased after surgery compared to the control group.
  • the mitochondria injection group (Mito) and the mitochondria/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).
  • mitochondria can effectively alleviate or treat pain by suppressing the expression level of Substance P, a pain factor.
  • Example 9 Evaluation of inflammatory cytokine expression levels by mitochondria
  • a multiplex kit (Merck Millipore, Burlington, MA, USA) was used to confirm the changes in inflammatory cytokines in the plasma according to treatment with the therapeutic agent, such as IFN- ⁇ , IL-17A, IL-1 ⁇ , IL-6, and TNF- ⁇ .
  • 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.
  • tissue were obtained from each individual before surgery (control), after surgery (surgery), and after treatment in the CPSS animal model established in Example 5.
  • Western blotting was performed to confirm the expression pattern of the inflammatory specific 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 analysis technique.
  • Each tissue protein was separated by size through SDS-PAGE and transferred to a PDVF membrane.

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Abstract

La présente invention concerne une composition comprenant des mitochondries isolées pour prévenir ou traiter la douleur, ainsi que son utilisation. La composition de la présente invention peut traiter ou prévenir efficacement la douleur et les maladies liées à la douleur. Spécifiquement, les mitochondries isolées ou la composition les comprenant peuvent traiter ou soulager efficacement la douleur par inhibition de l'expression et/ou de l'activité de facteurs de douleur, et peuvent spécifiquement traiter ou soulager uniquement la douleur indépendamment du soulagement de l'étiologie provoquant la douleur.
PCT/KR2023/021581 2023-09-26 2023-12-26 Composition comprenant des mitochondries isolées pour prévenir ou traiter la douleur et son utilisation Pending WO2025070898A1 (fr)

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KR10-2023-0129579 2023-09-26
KR20230129579 2023-09-26
KR1020230190348A KR20250046116A (ko) 2023-09-26 2023-12-22 분리된 미토콘드리아를 포함하는 통증 예방 또는 치료용 조성물 및 이의 용도
KR10-2023-0190348 2023-12-22

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Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR20210052365A (ko) * 2019-10-30 2021-05-10 가톨릭대학교 산학협력단 미토콘드리아를 포함하는 골관절염의 예방 또는 치료용 조성물
KR20230015832A (ko) * 2021-07-23 2023-01-31 차의과학대학교 산학협력단 분리된 미토콘드리아를 유효성분으로 포함하는 아셔만 증후군 예방 또는 치료용 약학 조성물
JP2023510230A (ja) * 2020-01-08 2023-03-13 ザ サリー アスター バーディン ブレスト ファウンデーション 筋萎縮性側索硬化症（ａｌｓ）および他の神経変性疾患の処置のための組成物および方法
US20230183380A1 (en) * 2016-01-15 2023-06-15 Children's Medical Center Corporation Therapeutic use of mitochondria and combined mitochondrial agents
KR20230115226A (ko) * 2022-01-25 2023-08-02 차의과학대학교 산학협력단 분리된 미토콘드리아 보존용 조성물, 이를 포함하는 약학적 조성물 및 하이드로겔

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20230183380A1 (en) * 2016-01-15 2023-06-15 Children's Medical Center Corporation Therapeutic use of mitochondria and combined mitochondrial agents
KR20210052365A (ko) * 2019-10-30 2021-05-10 가톨릭대학교 산학협력단 미토콘드리아를 포함하는 골관절염의 예방 또는 치료용 조성물
JP2023510230A (ja) * 2020-01-08 2023-03-13 ザ サリー アスター バーディン ブレスト ファウンデーション 筋萎縮性側索硬化症（ａｌｓ）および他の神経変性疾患の処置のための組成物および方法
KR20230015832A (ko) * 2021-07-23 2023-01-31 차의과학대학교 산학협력단 분리된 미토콘드리아를 유효성분으로 포함하는 아셔만 증후군 예방 또는 치료용 약학 조성물
KR20230115226A (ko) * 2022-01-25 2023-08-02 차의과학대학교 산학협력단 분리된 미토콘드리아 보존용 조성물, 이를 포함하는 약학적 조성물 및 하이드로겔

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