Pharmaceutical composition for treating drug addiction comprising opioid receptor nanodiscs and method for preparing opioid receptor nanodiscs

Abstract

The present invention relates to a pharmaceutical composition for treating drug addiction comprising opioid receptor nanodiscs and to a method for preparing opioid receptor nanodiscs. Specifically, the opioid receptor nanodiscs, according to the present invention, inhibit the interaction between an opioid receptor and an opioid-based drug without exhibiting toxicity in the body, are delivered to the brain when administered into the body, and significantly restore the delay in a hot-plate response caused by opioid-based drug addiction, thereby being usefully applicable to treatment of opioid-based drug addiction.

Description

Pharmaceutical composition for treating drug addiction comprising opioid receptor nanodiscs and method for manufacturing opioid receptor nanodiscs

[0001] The present invention relates to a pharmaceutical composition for treating drug addiction comprising an opioid receptor nanodisc and a method for manufacturing an opioid receptor nanodisc.

[0002]

[0003] Drug addiction is a seriously recognized public health issue worldwide. Heroin and other opioids, including prescription painkillers, are widely abused and account for a large proportion of illicit drug use. Furthermore, approximately 50% of violent crime in the United States is linked to opioid use, and billions of dollars are spent annually to address this.

[0004] Acute withdrawal symptoms from drug intoxication include sweating, heart palpitations, tachycardia, muscle tension, chest tightness, shortness of breath, tremors, nausea, vomiting, diarrhea, grand mal seizures, cardiac arrest, stroke, hallucinations, and dramatic and traumatic symptoms including delirium tremens (DT). Once acute withdrawal symptoms subside, post-acute withdrawal syndrome develops, which can persist for months or years. Post-acute withdrawal symptoms include fatigue, depression, lack of motivation, and increased pain sensitivity.

[0005] Consequently, many drugs designed to treat drug addiction have been developed in an attempt to alleviate acute and post-acute withdrawal symptoms. However, in most cases, the treatment of withdrawal requires the use of other addictive substances (e.g., morphine or methadone). Treatment also requires addicts to visit the hospital daily for extended periods, resulting in a high relapse rate among patients. Therefore, there is a need for the development of non-addictive treatments capable of effectively curing drug addiction.

[0006] In this regard, Korean Published Patent No. 10-2024-0164255 relates to a pharmaceutical composition for the prevention or treatment of drug addiction containing endocannabinoids, disclosing that endocannabinoids can effectively treat drug addiction by reducing the urge for self-administration of drugs and contribute to the improvement of public health.

[0007]

[0008] The object of the present invention is to provide a pharmaceutical composition for treating drug addiction comprising opioid receptor nanodiscs.

[0009] Another objective of the present invention is to provide a method for manufacturing opioid receptor nanodiscs.

[0010]

[0011] To achieve the above objective, the present invention provides a pharmaceutical composition for the prevention or treatment of opioid-based drug addiction comprising an opioid receptor nanodisc as an active ingredient.

[0012] In addition, the present invention provides a method for manufacturing an opioid receptor nanodisc comprising the step of mixing and reacting an opioid receptor, lipids, and membrane scaffold proteins.

[0013] In addition, the present invention provides an opioid receptor nanodisc produced by the above-described manufacturing method.

[0014] In addition, the present invention provides a method for preventing, improving, or treating opioid-based drug addiction, comprising the step of administering an opioid receptor nanodisc to an individual.

[0015] Furthermore, the present invention provides a use for opioid receptor nanodiscs for use in the manufacture of drugs for the prevention, improvement, or treatment of opioid-based drug addiction.

[0016]

[0017] The opioid receptor nanodisc according to the present invention inhibits the interaction between opioid receptors and opioid-class drugs without exhibiting toxicity in the body, is delivered to the brain upon administration, and significantly restores the delayed hot plate response caused by opioid-class drug intoxication, thereby being useful for the treatment of opioid-class drug intoxication.

[0018]

[0019] Figure 1 is a graph showing the results of confirming the size of an opioid receptor nanodisc manufactured in one embodiment of the present invention.

[0020] Figure 2 is a diagram showing the results of confirming opioid receptors in an opioid receptor nanodisc in one embodiment of the present invention.

[0021] Figure 3 is a graph showing the results of confirming the inhibition of the interaction between the opioid receptor and fentanyl by the opioid receptor nanodisc in one embodiment of the present invention.

[0022] Figure 4 is a diagram (A) showing the results of confirming the distribution of opioid receptor nanodiscs in a mouse body in one embodiment of the present invention, and a graph (B) showing the results of confirming the distribution amount in the body according to the administration time.

[0023] Figure 5 is a diagram showing the results of confirming the distribution of opioid receptor nanodiscs in each mouse tissue in one embodiment of the present invention.

[0024] Figure 6 is a graph showing the results of confirming the distribution of opioid receptor nanodiscs in each mouse tissue according to the administration time (A) and the results of confirming the average distribution amount according to the administration time in all tissues (B) in one embodiment of the present invention.

[0025] Figure 7 is a diagram (A) showing the results of confirming the distribution in brain tissue according to the administration time of opioid receptor nanodiscs in one embodiment of the present invention, and a graph (B) showing the results quantitatively.

[0026] FIG. 8 is a diagram (A) showing the results of confirming the distribution in blood according to the administration time of an opioid receptor nanodisc in one embodiment of the present invention, and a graph (B) showing the results quantitatively.

[0027] Figure 9 is a graph showing the results of confirming the therapeutic effect of opioid receptor nanodiscs on fentanyl addiction according to the dosage in one embodiment of the present invention.

[0028] Figure 10 is a graph showing the results of confirming the therapeutic effect of opioid receptor nanodiscs on fentanyl addiction according to the administration time in one embodiment of the present invention.

[0029] Figure 11 is a diagram showing the result of H&E staining of mouse tissue administered with an opioid receptor nanodisc in one embodiment of the present invention.

[0030] Figure 12 is a graph showing the results of analyzing major factors in the blood of mice administered opioid receptor nanodiscs in one embodiment of the present invention.

[0031]

[0032] The present invention will be described in detail below.

[0033] The present invention provides a pharmaceutical composition for the prevention or treatment of opioid-based drug addiction comprising an opioid receptor nanodisc as an active ingredient.

[0034] As used herein, the term "opioid" refers to a type of narcotic also known as an opioid analgesic. Opioids bind to opioid receptors and exert effects in the brain such as pain relief, alertness, sedation, and anesthesia. Therefore, while opioid-based drugs are used as anesthetics or pain relievers, they can also cause side effects such as itching, sedation, nausea, respiratory depression, constipation, and euphoria; furthermore, long-term use may lead to tolerance, physical dependence, and withdrawal symptoms.

[0035] Meanwhile, the term "opioid receptor" is a protein identified primarily in the central and peripheral nervous systems and the gastrointestinal tract that interacts with opioids, and is one of the Class A G-protein coupled receptors. This opioid receptor exerts an analgesic effect by binding to opioids, inhibiting adenylyl cyclase to reduce intracellular cAMP levels, and simultaneously inhibiting calcium channels in presynaptic neurons while suppressing the opening of potassium channels in postsynaptic neurons.

[0036] The above-mentioned opioid receptor may include all types of opioid receptors known in the ordinary art. In one embodiment of the present invention, the opioid receptor may be a polypeptide composed of the amino acid sequence described in SEQ ID NO. 2. Additionally, the opioid receptor may include a variant in which one or more amino acid residues are substituted, deleted, or inserted from the polypeptide composed of the amino acid sequence described in SEQ ID NO. 2, provided that the protein activity is not altered. Additionally, the opioid receptor may have 80% or more, 90% or more, 95% or more, 98% or more, or 99% or more homology with the polypeptide composed of the amino acid sequence described in SEQ ID NO. 2.

[0037] In addition, the polynucleotide encoding the opioid receptor may also include any sequence known in the ordinary art as a polynucleotide encoding an opioid receptor. The polynucleotide may be composed of the nucleotide sequence described in SEQ ID NO. 1, and may include variants in which one or more nucleotides in the nucleotide sequence are substituted, deleted, or inserted, provided that the activity of the protein produced therefrom is maintained. In addition, the polynucleotide may have 80% or more, 90% or more, 95% or more, 98% or more, or 99% or more homology with the nucleotide sequence described in SEQ ID NO. 1.

[0038] As used herein, the term "nanodisc" refers to a structure in which the sides of a lipid bilayer formed of phospholipids are surrounded by hydrophobic bonds by membrane scaffold proteins. The nanodisc exhibits a flattened disc shape in which the hydrophilic groups of the phospholipids are oriented outward and the hydrophobic groups are oriented inward, and may contain hydrophobic drugs or other substances within the disc shape. That is, the opioid receptor nanodisc according to the present invention may be composed of an opioid receptor, lipids, and a membrane scaffold protein (MSP).

[0039] In this case, the lipid may be a phospholipid. The term "phospholipid" refers to a lipid in which glycerol is bonded to two fatty acids and one phosphate group, or to a compound containing a phosphate group and nitrogen (e.g., choline). The phospholipid comprises a hydrophilic phosphate group and glycerol in the head portion and a hydrophobic long fatty acid chain in the tail portion. The phospholipid may include all types of phospholipids known in the ordinary art, and specifically, the phospholipid may be one or more selected from the group consisting of phosphatidylcholine, phosphatidylglycerol, phosphatidylethanolamine, phosphatidylserine, and phosphatidylinositol.

[0040] 더욱 구체적으로, 상기 포스파티딜콜린은 DOPC(1,2-Dioleoyl-sn-glycero-3-phosphocholine), DLPC(1,2-Dilauroyl-sn-glycero-3-phosphocholine), DMPC(1,2-Dimyristoyl-sn-glycero-3-phosphocholine), DPPC(1,2-Dipalmitoyl-sn-glycero-3-phosphocholine), POPC(1-Palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine), DDPC(1,2-Didecanoyl-sn-glycero-3-phosphocholine), DSPC(1,2-Distearoyl-snglycero-3-phosphocholine), DEPC(1,2-Dierucoyl-sn-glycero-3-phosphocholine), DLOPC(1,2-Dilinoleoyl-snglycero-3-phosphocholine), EPC(Egg phosphatidylcholine), MSPC(1-Myristoyl-2-stearoyl-sn-glycero-3-phosphocholine), PMPC(1-Palmitoyl-2-myristoyl-sn-glycero-3-phosphocholine), PSPC(1-Palmitoyl-2-stearoyl-sn-glycero-3-phosphocholine), SMPC(1-Stearoyl-2-myristoyl-sn-glycero-3-phosphocholine) 및 SPPC(1-Stearoyl-2-palmitoyl-sn-glycero-3-phosphocholine)로 구성된 군으로부터 선택되는 어느 하나 이상일 수 있다.In addition, the phosphatidyl glycerol is DMPG (1,2-Dimyristoyl-sn-glycero-3(Phospho-rac-(1-glycerol))), DPPG (1,2-Dipalmitoyl-sn-glycero-3(Phospho-rac-(1-glycerol))), DSPG(1,2-Distearoyl-sn-glycero3(Phospho-rac-(1-glycerol))), POPG(1-Palmitoyl-2-oleoyl-sn-glycero-3(Phospho-rac-(1-glycerol))), DEPG(1,2-Dierucoyl-sn-glycero-3(Phospho-rac-(1-glycerol))), DLPG (1,2-Dilauroyl-sn-glycero-3(Phosphorac-(1-glycerol))), It may include one or more selected from the group consisting of DOPG (1,2-Dioleoyl-sn-glycero-3(Phospho-rac-(1-glycerol))) and DSPG (1,2-Distearoyl-sn-glycero-3(Phospho-rac-(1-glycerol))). In addition, the phosphatidylethanolamine may include one or more selected from the group consisting of DMPE (1,2-Dimyristoyl-sn-glycero-3-phosphoethanolamine), DPPE (1,2-Dipalmitoyl-sn-glycero-3-phosphoethanolamine), DSPE (1,2-Distearoyl-sn-glycero-3-phosphoethanolamine), DOPE (1,2-Dioleoyl-sn-glycero-3-phosphoethanolamine), DEPE (1,2-Dierucoyl-sn-glycero-3-phosphoethanolamine), DLPE (1,2-Dilauroyl-sn-glycero-3-phosphoethanolamine) and POPE (1-Palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine).In addition, the phosphatidylserine may include one or more selected from the group consisting of DOPS (1,2-Dioleoyl-sn-glycero-3-phosphoserine), DLPS (1,2-Dilauroyl-sn-glycero-3-phosphoserine), DMPS (1,2-Dimyristoyl-sn-glycero-3-phosphoserine), DPPS (1,2-Dipalmitoyl-sn-glycero-3-phosphoserine), DSPS (1,2-Distearoyl-sn-glycero-3-phosphoserine) and POPS (1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-L-serine). Furthermore, the phosphatidylinositol may include one or more selected from the group consisting of phosphatidylinositol-4-phosphate, phosphatidylinositol-4,5-bisphosphate, and phosphatidylinositol-3,4,5-bisphosphate.

[0041] The above term, "membrane scaffold protein," is a protein having a helix structure and exhibiting amphiphilicity, which serves to surround the sides of a lipid bilayer. Specifically, the membrane scaffold protein may be an apolipoprotein (Apo), and more specifically, the apolipoprotein may be one or more selected from the group consisting of apolipoprotein A1 (ApoA-1), apolipoprotein A2 (AproA-2), apolipoprotein B (ApoB), apolipoprotein C (ApoC), apolipoprotein E (ApoE), MSP1 (membrane scaffold protein 1), MSP1D1, MSP1D2, MSP1E1, MSP1E2, MSP1E3, MSP1E3D1, MSP2, MSP2N1, MSP2N2, and MSP2N3.

[0042] The above opioid receptor nanodiscs may be formed by mixing opioid receptors and membrane scaffold proteins in a ratio of 1:1 to 10, 1:1 to 8, 1:1 to 6, 1:1 to 4, 1:2 to 10, 1:2 to 8, 1:2 to 6, 1:2 to 4, 1:4 to 10, 1:4 to 8, or 1:4 to 6. Meanwhile, the above-mentioned opioid receptor nanodisc comprises an opioid receptor and lipids in a ratio of 1:1 to 700, 1:1 to 650, 1:1 to 600, 1:1 to 550, 1:1 to 500, 1:1 to 450, 1:1 to 400, 1:1 to 350, 1:1 to 300, 1:1 to 250, 1:50 to 700, 1:50 to 650, 1:50 to 600, 1:50 to 550, 1:50 to 500, 1:50 to 450, 1:50 to 400, 1:50 to 350, 1:50 to 300, 1:50 to 250, 1:100 to 700, 1:100 to 650, 1:100 to 600, 1:100 to 550, 1:100 to 500, 1:100 to 450, 1:100 to 400, 1:100 to 350, 1:100 to 300, 1:100 to 250, 1:150 to 700, 1:150 to 650, 1:150 to 600, 1:150 to 550, 1:150 to 500, 1:150 to 450, 1:150 to 400, 1:150 to 350, 1:150 to 300, 1:150 to 250, 1:200 to 700, 1:200 to 650, 1:200 to 600, 1:200 to 550, 1:200 to 500, 1:200 to 450, 1:200 to 400, 1:200 to 350, 1:200 to 300, 1:200 to 250, 1:250 to 700, 1:250 to 650, 1:250 to 600, 1:250 to 550, 1:250 to 500, 1:250 to 450, 1:250 to 400, 1:250 to 350,It can be formed by mixing in a ratio of 1:250 to 300, 1:300 to 700, 1:300 to 650, 1:300 to 600, 1:300 to 550, 1:300 to 500, 1:300 to 450, 1:300 to 400, 1:300 to 350, 1:350 to 700, 1:350 to 650, 1:350 to 600, 1:350 to 550, 1:350 to 500, 1:350 to 450, or 1:350 to 400.

[0043] In addition, the opioid receptor nanodisc according to the present invention has a diameter of 50 nm or less, 0.1 to 50 nm, 0.1 to 45 nm, 0.1 to 40 nm, 0.1 to 35 nm, 0.1 to 30 nm, 0.1 to 25 nm, 0.1 to 20 nm, 0.1 to 15 nm, 1 to 50 nm, 1 to 45 nm, 1 to 40 nm, 1 to 35 nm, 1 to 30 nm, 1 to 25 nm, 1 to 20 nm, 1 to 15 nm, 3 to 50 nm, 3 to 45 nm, 3 to 40 nm, 3 to 35 nm, 3 to 30 nm, 3 to 25 nm, 3 to 20 nm, 3 It may have a diameter of up to 15 nm, 5 to 50 nm, 5 to 45 nm, 5 to 40 nm, 5 to 35 nm, 5 to 30 nm, 5 to 25 nm, 5 to 20 nm, 5 to 15 nm, 8 to 50 nm, 8 to 45 nm, 8 to 40 nm, 8 to 35 nm, 8 to 30 nm, 8 to 25 nm, 8 to 20 nm, 8 to 15 nm, 10 to 50 nm, 10 to 45 nm, 10 to 40 nm, 10 to 35 nm, 10 to 30 nm, 10 to 25 nm, 10 to 20 nm, or 10 to 15 nm.

[0044] As used herein, the term "drug intoxication" refers to a condition in which toxic side effects other than the therapeutic effects of the drug are expected to occur in the human body due to the overdose of a therapeutic drug. Such drug intoxication may cause symptoms such as decreased consciousness, respiratory depression, excitement, pupil dilation, cold sweats, increased heart rate, increased blood pressure, tearing, nausea, vomiting, urination, diarrhea, muscle weakness, hyperventilation, and fever. Methods for diagnosing drug intoxication are well known in the ordinary art and can generally be diagnosed through blood or urine tests. Such drug intoxication may be intoxication by opioid-based drugs.

[0045] The above-mentioned opioid class drugs may include all types of drugs known in the ordinary art. For example, the above-mentioned opioid class drugs may be one or more selected from the group consisting of morphine, oxycodone, hydrocodone, hydromorphone, fentanyl, tramadol, and methadone.

[0046] The pharmaceutical composition according to the present invention may contain 0.1 to 95 weight% of an opioid receptor nanodisc, which is an active ingredient, based on the total weight of the composition. In addition, the pharmaceutical composition of the present invention may additionally include one or more active ingredients exhibiting the same or similar functions in addition to the above active ingredient.

[0047] The pharmaceutical composition of the present invention may comprise a carrier, diluent, excipient, or mixture thereof that is commonly used in biological preparations. Any pharmaceutically acceptable carrier suitable for delivering the composition in vivo may be used. Specifically, said carrier may be a compound listed in Merck Index, 13th ed., Merck & Co. Inc., saline solution, sterile water, Ringer's solution, dextrose solution, maltodextrin solution, glycerol, ethanol, or a mixture thereof. Additionally, conventional additives such as antioxidants, buffers, bacteriostatic agents, etc., may be added as needed.

[0048] When formulating the above composition, diluents or excipients such as commonly used fillers, extenders, binders, wetting agents, disintegrants, and surfactants may be added.

[0049] The composition of the present invention may be formulated into an oral preparation or a parenteral preparation. Oral preparations may include solid preparations and liquid preparations. The solid preparation may be a tablet, pill, powder, granule, capsule, or troche, and such solid preparation may be prepared by adding at least one excipient to the composition. The excipient may be starch, calcium carbonate, sucrose, lactose, gelatin, or a mixture thereof. Additionally, the solid preparation may include a lubricant, examples of which include magnesium styrate, talc, etc. Meanwhile, the liquid preparation may be a suspension, an oral liquid, an emulsion, or a syrup. In this case, the liquid preparation may include excipients such as humectants, sweeteners, flavorings, preservatives, etc.

[0050] The above parenteral preparations may include injectables, suppositories, powders for respiratory inhalation, aerosols for spray, powders, and creams. The above injectables may include sterile aqueous solutions, non-aqueous solvents, suspension solvents, emulsions, etc. In this case, as non-aqueous solvents or suspension solvents, vegetable oils such as propylene glycol, polyethylene glycol, or olive oil, or injectable esters such as ethyl oleate may be used.

[0051]

[0052] In addition, the present invention provides a method for manufacturing an opioid receptor nanodisc, comprising the step of mixing and reacting an opioid receptor, a membrane scaffold protein, and a lipid.

[0053] The opioid receptor nanodisc produced by the manufacturing method according to the present invention, and the opioid receptor, membrane scaffold protein, and lipid used in the production of the nanodisc, may have the characteristics described above. At this time, the mixing ratio of the opioid receptor, membrane scaffold protein, and lipid may have the characteristics described above.

[0054] In the above manufacturing method, the reaction may be carried out under conditions known in the ordinary art and, if necessary, under conditions appropriately modified by a person skilled in the art. For example, the reaction may be carried out at a temperature of 4 to 37°C, 4 to 33°C, 4 to 30°C, 4 to 27°C, 4 to 23°C, 8 to 37°C, 8 to 33°C, 8 to 30°C, 8 to 27°C, 8 to 23°C, 12 to 37°C, 12 to 33°C, 12 to 30°C, 12 to 27°C, 12 to 23°C, 16 to 37°C, 16 to 33°C, 16 to 30°C, 16 to 27°C, or 16 to 23°C. In addition, the above reaction may be carried out for 4 to 40 hours, 4 to 35 hours, 4 to 30 hours, 4 to 25 hours, 4 to 20 hours, 8 to 40 hours, 8 to 35 hours, 8 to 30 hours, 8 to 25 hours, 8 to 20 hours, 12 to 40 hours, 12 to 35 hours, 12 to 30 hours, 12 to 25 hours, 12 to 20 hours, 15 to 40 hours, 15 to 35 hours, 15 to 30 hours, 15 to 25 hours, and 15 to 20 hours.

[0055]

[0056] In addition, the present invention provides an opioid receptor nanodisc produced by the above-described manufacturing method.

[0057] The opioid receptor nanodisc according to the present invention may have the characteristics described above. For example, the opioid receptor nanodisc has a diameter of 50 nm or less, 0.1 to 50 nm, 0.1 to 45 nm, 0.1 to 40 nm, 0.1 to 35 nm, 0.1 to 30 nm, 0.1 to 25 nm, 0.1 to 20 nm, 0.1 to 15 nm, 1 to 50 nm, 1 to 45 nm, 1 to 40 nm, 1 to 35 nm, 1 to 30 nm, 1 to 25 nm, 1 to 20 nm, 1 to 15 nm, 3 to 50 nm, 3 to 45 nm, 3 to 40 nm, 3 to 35 nm, 3 to 30 nm, 3 to 25 nm, 3 to 20 nm, 3 It may have a diameter of up to 15 nm, 5 to 50 nm, 5 to 45 nm, 5 to 40 nm, 5 to 35 nm, 5 to 30 nm, 5 to 25 nm, 5 to 20 nm, 5 to 15 nm, 8 to 50 nm, 8 to 45 nm, 8 to 40 nm, 8 to 35 nm, 8 to 30 nm, 8 to 25 nm, 8 to 20 nm, 8 to 15 nm, 10 to 50 nm, 10 to 45 nm, 10 to 40 nm, 10 to 35 nm, 10 to 30 nm, 10 to 25 nm, 10 to 20 nm, or 10 to 15 nm.

[0058]

[0059] In addition, the present invention provides a method for preventing, improving, or treating opioid-based drug addiction, comprising the step of administering an opioid receptor nanodisc to an individual.

[0060] The opioid receptor nanodisc used in the method for preventing, improving, or treating opioid-based drug addiction according to the present invention may have the characteristics described above.

[0061] The above-mentioned individual may be a mammal, and specifically, may be a human.

[0062] The above administration may be administered orally or parenterally depending on the intended method. Parenteral administration may include intravascular, subcutaneous, intramuscular, intraperitoneal, intranasal, rectal, urethral, ​​vaginal, spinal, intracerebral, intraocular, or thoracic injection methods.

[0063] In addition, the above administration may be administered in a pharmaceutically effective amount. This may vary depending on the type and severity of the drug intoxicated, the activity of the drug, the patient's sensitivity to the drug, the time of administration, the route of administration, the duration of treatment, drugs used concurrently, etc. However, for a desirable effect, the amount of the active ingredient included in the pharmaceutical composition according to the present invention may be 0.0001 to 1,000 mg / kg, specifically 0.001 to 500 mg / kg. The above administration may be once or several times a day.

[0064] The composition of the present invention may be administered alone or in combination with other therapeutic agents. When administered in combination, the administration may be sequential or simultaneous.

[0065]

[0066] Furthermore, the present invention provides a use for opioid receptor nanodiscs for use in the manufacture of drugs for the prevention, improvement, or treatment of opioid-based drug addiction.

[0067] The opioid receptor nanodisc used in the manufacture of a drug for the prevention, improvement, or treatment of opioid-based drug addiction according to the present invention may have the characteristics described above.

[0068]

[0069] The present invention is described in detail below by way of the following examples. However, the following examples are merely illustrative of the present invention and do not limit the present invention. Any configuration having substantially the same structure as the technical concept described in the claims of the present invention and achieving the same functional effect is included within the technical scope of the present invention.

[0070]

[0071] Example 1. Fabrication of opioid receptor nanodiscs-(1)

[0072] Nanodisks containing opioid receptors were fabricated using the following method.

[0073] First, the opioid receptor gene fused with the 6×His tag (SEQ No. 1) was cloned into the pET-DEST42 expression vector and transformed into Rosetta strains using conventional methods. The Rosetta strains were cultured overnight at 37°C and 150 rpm using six flasks containing 1 L of culture medium. OD of the culture medium 600 When the value reached 0.45 to 0.5, 1 ml of 1 M IPTG (Isopropyl β-D-1-thiogalactopyranoside) was added, and the culture was continued for 4 more hours under the same conditions. After the culture was finished, the culture medium was centrifuged at 4°C and 7,000 rpm for 10 minutes to obtain a pellet, and lysis buffer was added to lyse the cells. The cell lysate was centrifuged under the conditions described above to obtain the supernatant, and the opioid receptor was purified from the obtained supernatant using a nickel column by a conventional method. A purified opioid receptor, MSP1E3D1 protein, and 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) were mixed in a volume ratio of 1:3:390 at equal molar concentrations and reacted for 16 hours at 20°C and 80 rpm to obtain a nanodisc containing the opioid receptor. SM2 resin (Bio-Rad) was added to the obtained opioid receptor nanodisc to remove detergent substances, and the final opioid receptor nanodisc was produced by purification using size exclusion chromatography.

[0074]

[0075] Example 2. Fabrication of opioid receptor nanodiscs-(2)

[0076] An opioid receptor nanodisc was prepared using the same method and conditions as in Example 1, except that the opioid receptor, MSP1E3D1 protein, and DMPC were mixed in a ratio of 1:3:240.

[0077]

[0078] Example 3. Fabrication of opioid receptor nanodiscs-(3)

[0079] An opioid receptor nanodisc was prepared using the same method and conditions as in Example 1, except that the opioid receptor, MSP1E3D1 protein, and DMPC were mixed in a ratio of 1:5:400.

[0080]

[0081] Example 4. Fabrication of opioid receptor nanodiscs-(4)

[0082] An opioid receptor nanodisc was prepared using the same method and conditions as in Example 1, except that the opioid receptor, MSP1E3D1 protein, and DMPC were mixed in a ratio of 1:5:650.

[0083]

[0084] Experimental Example 1. Verification of Nanodisk Size

[0085] The size of the above-described opioid receptor nanodiscs was confirmed using a particle size analyzer. Specifically, particle size analysis was performed using a zeta sizer, and the results are shown in Figure 1.

[0086] As shown in Figure 1, all nanodiscs prepared in the above examples exhibited a size of 10 to 15 nm, forming nanodiscs of an appropriate size. However, when the opioid receptor, MSP1E3D1 protein, and DMPC were mixed in a ratio of 1:3:390, the nanodisc formation yield was excellent and the formation of aggregates was low.

[0087]

[0088] Experimental Example 2. Confirmation of Opioid Receptors

[0089] The presence of opioid receptors in the fabricated opioid receptor nanodiscs was confirmed as follows using the Western blot method.

[0090] Specifically, an equal amount of 2× sample buffer was added to the opioid receptor nanodiscs prepared in Example 1, and the mixture was placed in boiling water for 10 minutes. Using this as a sample, electrophoresis was performed using a conventional method, and the proteins electrophoresed on the acrylamide gel were transferred to a nitrocellulose membrane. The nitrocellulose membrane was pretreated with 5% skim milk for 1 hour, and an anti-histidine antibody was added as the primary antibody and reacted overnight at 4°C. After the reaction was complete, the cellulose membrane was washed, and an anti-mouse secondary antibody was added and reacted at room temperature for 1 hour. After the reaction was complete, the cellulose membrane was treated with an ECL solution to check for bands, and the results are shown in Figure 2.

[0091] As shown in Figure 2, the manufactured opioid receptor nanodiscs contained opioid receptors of approximately 50 kDa size.

[0092]

[0093] Experimental Example 3. Inhibitory effect of the interaction between opioid narcotics and opioid receptors

[0094] It was confirmed as follows whether the above-mentioned fabricated opioid receptor nanodiscs inhibit the interaction between opioid drugs and opioid receptors.

[0095] First, the HEK-293T cell line was prepared by culturing using conventional methods. The prepared cell line was cultured in 96-well plates at a density of 1 × 10⁶ per well. 4 The cells were divided into groups and cultured overnight. The cultured cell line was transformed with an opioid receptor. At this time, the G protein and the 22F gene were transformed together to confirm the fluorescence signal of the cells. After culturing the cell line for 48 hours, 10 μl of 10 μM forskolin and 10 μl of 100 nM fentanyl were added along with the opioid receptor nanodiscs prepared in Example 1. At this time, the opioid receptor nanodiscs were added at concentrations of 0.0001, 0.001, 0.01, 0.1, 1, or 10 μM, and naloxone was used as a positive control at the same concentration. Afterwards, the cell line was cultured for another 4 hours, and the results confirming competitive interaction by opioid receptor nanodiscs by measuring the fluorescence intensity using a Glo-sensor assay kit and a microplate reader are shown in Figure 3.

[0096] As shown in Figure 3, the interaction between fentanyl and the opioid receptor was inhibited by the opioid receptor nanodisc, and this was dependent on the treatment concentration of the opioid receptor nanodisc.

[0097]

[0098] Experimental Example 4. Distribution in the body-(1)

[0099] The following experiment was performed to confirm the distribution in the body when the above-mentioned opioid receptor nanodiscs were administered.

[0100] First, 200 μl of the opioid receptor nanodisc (1 μM) prepared in Example 1 was intravenously injected into 2-week-old nude mice. At this time, the opioid receptor nanodisc was injected after being stained with cyan 5.5 dye using a conventional method. After 1, 0.5, 1, 3, 6, 9, 12, 24, or 48 hours following the administration of the opioid receptor nanodisc, the whole body of the mice was observed using an IVIS (In-vivooptical imaging system), and the results confirming the distribution of the opioid receptor nanodisc in the body are shown in Fig. 4A. In addition, the results of measuring fluorescence intensity over time after administration are shown in Fig. 4B.

[0101] As shown in Figure 4, 30 minutes after administering the opioid receptor nanodiscs, the nanodiscs were widely distributed in the brain region and migrated from the brain to other tissues as the administration time progressed.

[0102]

[0103] Experimental Example 5. Distribution in the body-(2)

[0104] The distribution of opioid receptor nanodiscs in each organ of the mouse, in which the in vivo distribution of opioid receptor nanodiscs was confirmed above, was confirmed.

[0105] Specifically, mice administered with opioid receptor nanodiscs in Experimental Example 5 and having their distribution in the body confirmed after 3 or 48 hours were sacrificed by conventional means, and major organs (liver, lung, spleen, heart, kidney, brain) were isolated. The distribution of opioid receptor nanodiscs within each tissue was confirmed by performing IVIS analysis using the isolated tissues, and the results of measuring fluorescence intensity over time after administration are shown in Fig. 5. Additionally, the average values ​​for all organs were calculated, and the results of confirming fluorescence intensity over time after administration are shown in Fig. 6B.

[0106] As shown in Figures 5 and 6, the opioid receptor nanodiscs administered into the body were widely distributed in the brain 3 hours after administration and decreased as time passed.

[0107]

[0108] Experimental Example 6. Brain Distribution

[0109] The distribution of opioid receptor nanodiscs in the brain tissue of mice, in which the in vivo distribution of opioid receptor nanodiscs was confirmed above, was confirmed over time after administration. The experiment was performed in the same manner as Experimental Example 5 above, except that brain tissue obtained from mice 1, 3, 6, 24, or 48 hours after administration of opioid receptor nanodiscs was used. As a result, the results of confirming the fluorescence intensity over time after administration are shown in Fig. 7A, and the results of quantitatively calculating the fluorescence intensity from the above results are shown in Fig. 7B.

[0110] As shown in Figure 7, the opioid receptor nanodiscs according to the present invention were mainly distributed in brain tissue at the beginning of administration, and the fluorescence intensity decreased significantly as the administration time progressed.

[0111]

[0112]

[0113] Experimental Example 7. Blood Distribution

[0114] The distribution in the blood of mice in which the in vivo distribution of opioid receptor nanodiscs was confirmed above was confirmed by the following method.

[0115] Specifically, in Experimental Example 5, blood was collected from mice administered opioid receptor nanodiscs at 0, 0.3, 0.6, 1, 3, 6, 9, 12, or 24 hours after administration, and the collected blood was centrifuged at 15,000 g for 20 minutes to separate the plasma. The separated plasma was dispensed into a 96-well plate, and the 96-well plate containing the plasma was analyzed using IVIS. At this time, as a control, a purified opioid receptor was stained with a Cyan 5.5 stained sample and used. As a result, the fluorescence intensity was observed, and the results of quantitatively calculating the fluorescence intensity from the above results are shown in Fig. 8A.

[0116] As shown in Figure 8, unlike when purified opioid receptors were administered, when opioid receptor nanodiscs were administered, high blood distribution and residue levels were observed.

[0117]

[0118] Experimental Example 8. Drug addiction treatment effect-(1)

[0119] The therapeutic effect of the above-prepared opioid receptor nanodiscs on drug addiction was confirmed according to the dosage using the hot-plate latency analysis method.

[0120] First, fentanyl poisoning was induced in 2-week-old albino mice by administering 0.02 mg / kg of fentanyl. At this time, the opioid receptor nanodiscs were intravenously injected at doses of 0.05, 0.1, or 0.2 mg / kg. Thirty minutes after administering the opioid receptor nanodiscs, the mice were placed on a heating plate to measure the time until they exhibited a heat exposure response (lifting forelimbs, lifting hindlimbs, licking forelimbs, etc.), and the results are shown in Fig. 9.

[0121] As shown in Figure 9, the hot plate response delay time decreased in mice administered opioid receptor nanodiscs in a dose-dependent manner.

[0122]

[0123] Experimental Example 9. Drug addiction treatment effect-(2)

[0124] The therapeutic effect of the above-mentioned opioid receptor nanodiscs on drug addiction was confirmed according to the administration time using the hot plate response delay analysis method.

[0125] Specifically, 0.1 mg / kg of opioid receptor nanodiscs were administered to mice administered fentanyl under the same conditions and methods as in Experimental Example 8, and a hot plate response delay analysis was performed 0, 0.1, or 1 hour after administration. At this time, a normal control group with no treatment, a negative control group administered fentanyl, and a positive control group administered 0.1 mg / kg of naloxone were used as control groups. As a result, the time until the mice showed a reaction on the hot plate was measured and is shown in Fig. 10.

[0126] As shown in Fig. 10, the opioid receptor nanodisc according to the present invention significantly shortened the hot plate response delay time increased by fentanyl administration.

[0127]

[0128] Experimental Example 10. Biostability

[0129] The biocompatibility of the opioid receptor nanodisc according to the present invention was confirmed through H&E staining (hematoxylin and eosin staining) and analysis of major factors in blood.

[0130]

[0131] 10-1. H&E Dyeing

[0132] Specifically, H&E staining was performed using the mouse organs obtained in Experimental Example 5 above. At this time, the organs of mice administered opioid receptor nanodiscs and 48 hours later were used, and as a control, the organs of mice administered PBS instead of opioid receptor nanodiscs were used. As a result, the organs stained with H&E dye were observed and are shown in Fig. 11.

[0133] As shown in Figure 11, the tissue staining tendency of mice administered opioid receptor nanodiscs did not differ from that of the control group.

[0134]

[0135] 10-2. Analysis of Major Factors in Blood

[0136] Specifically, using the mouse blood obtained in Experimental Example 5 above, the levels of major blood factors ALB (albumin), ALP (alkaline phosphatase), ALT (alanine aminotransferase), AMY (amylase), TBILI (total bilirubin), BUN (blood urea nitrogen), CA (calcium), PHOS (phosphorus), GLU (glucose), sodium ions (NA+), potassium ions (K+), and T.pro (total protein) were analyzed by conventional methods. At this time, blood from mice administered opioid receptor nanodiscs 48 hours prior was used, and blood from mice administered PBS instead of opioid receptor nanodiscs was used as a control. As a result, the levels of the analyzed major factors are shown in Fig. 12.

[0137] As shown in Figure 12, as the GLU level decreased upon administration of the opioid receptor nanodisc, the ALP, ALT, and TBILI levels decreased; however, based on the fact that the levels of ALP, ALT, etc. generally increase due to drug side effects, this is judged to be a result of nutritional reduction.

[0138] Therefore, from the above results, it was confirmed that the opioid receptor nanodisc according to the present invention can be effectively used to treat opioid-based drug addiction without causing side effects to the human body.

Claims

1. A pharmaceutical composition for the prevention or treatment of opioid-based drug addiction comprising an opioid receptor nanodisc as an active ingredient.

2. A pharmaceutical composition for the prevention or treatment of drug intoxication according to claim 1, wherein the opioid class drug is one or more selected from the group consisting of morphine, oxycodone, hydrocodone, hydromorphone, fentanyl, tramadol, and methadone.

3. A pharmaceutical composition for the prevention or treatment of drug addiction, wherein the opioid receptor of claim 1 is a polypeptide composed of the amino acid sequence described in SEQ ID NO.

2.

4. A pharmaceutical composition for the prevention or treatment of drug addiction, wherein the opioid receptor nanodisc of claim 1 is composed of an opioid receptor, lipids, and a membrane scaffold protein (MSP).

5. A pharmaceutical composition for the prevention or treatment of drug addiction, wherein, in paragraph 4, the lipid is a phospholipid.

6. A pharmaceutical composition for the prevention or treatment of drug intoxication according to claim 5, wherein the phospholipid is one or more selected from the group consisting of phosphatidylcholine, phosphatidylglycerol, phosphatidylethanolamine, phosphatidylserine, and phosphatidylinositol.

7. A pharmaceutical composition for the prevention or treatment of drug intoxication, wherein, in paragraph 4, the membrane scaffold protein is an apolipoprotien.

8. A pharmaceutical composition for the prevention or treatment of drug addiction, wherein the opioid receptor nanodisc according to claim 1 is formed by mixing an opioid receptor and a membrane scaffold protein in a ratio of 1:1 to 10.

9. A pharmaceutical composition for the prevention or treatment of drug addiction, wherein the opioid receptor nanodisc according to claim 1 is formed by mixing an opioid receptor and a lipid in a ratio of 1:1 to 700.

10. A pharmaceutical composition for the prevention or treatment of drug addiction, wherein the opioid receptor nanodisc of claim 1 has a diameter of 50 nm or less.

11. A method for manufacturing an opioid receptor nanodisc comprising the step of mixing and reacting an opioid receptor, a membrane scaffold protein, and a lipid.

12. A method for manufacturing an opioid receptor nanodisc according to claim 11, wherein the reaction is performed at 4 to 37°C.

13. A method for manufacturing an opioid receptor nanodisc according to claim 11, wherein the reaction is performed for 4 to 40 hours.

14. Opioid receptor nanodiscs manufactured by the manufacturing method according to paragraph 11.

15. In claim 14, the opioid receptor nanodisk is an opioid receptor nanodisk having a diameter of 50 nm or less.

16. A method for preventing, improving, or treating opioid-based drug addiction comprising the step of administering an opioid receptor nanodisc to an individual.

17. Use of opioid receptor nanodiscs for use in the manufacture of drugs for the prevention, improvement, or treatment of opioid-based drug addiction.

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