Composition for treating pain diseases comprising liposomal poloxamer complex

Abstract

The present invention relates to a composition for treating pain diseases and, more specifically, provides: a liposomal complex comprising drug-encapsulated liposomes and poloxamer; and a pharmaceutical composition for treating pain diseases by nerve block, comprising same. The liposomal complex according to the present invention has a hydrophobic / hydrophilic drug encapsulated therein without crystal formation, and can remain at a target site in gel form in the body for a prolonged period, thereby significantly increasing the duration of action, and thus can enhance the efficiency and safety of the drug, and therefore, can be effectively applied to therapeutic fields for a wide range of spinal and pain-related diseases.

Description

Composition for treating pain disorders comprising liposomal poloxamer complex

[0001] The present invention relates to a composition for treating a pain disease, and more particularly, to a pharmaceutical composition for treating a disease that may cause pain, comprising a liposomal poloxamer complex in which a drug is loaded.

[0002] With the increasing human lifespan, interest in and development of treatment technologies for degenerative diseases are inevitably increasing. The prevalence of spinal disorders is also steadily increasing, necessitating the development of diverse treatment technologies.

[0003] Non-invasive treatments are an inevitable trend across all medical fields. In this regard, nerve blocks (e.g., epidural injections, medial branch injections, joint injections) have been used for decades to avoid or delay surgery and manage pain, and their frequency and scope of use are steadily increasing. Furthermore, countless modified forms of these treatments are being developed.

[0004] However, the temporary nature of these effects is a dilemma for all procedures. This is due to the significantly short drug retention time associated with the use of liquid local anesthetics and steroid preparations. Since the development of this technology, the types and forms of these drugs have remained unchanged. Therefore, a new formulation technology is needed that can extend drug retention time and enhance absorption efficiency.

[0005] The purpose of the present invention is to provide a novel liposome poloxamer complex formulation capable of improving the persistence and therapeutic efficiency of a drug and a method for preparing the same.

[0006] Another object of the present invention is to provide a drug delivery system using the above composite formulation.

[0007] Another object of the present invention is to provide a medical use of the above-mentioned composite preparation for a pain-causing disease.

[0008] To achieve the above purpose, the present invention provides a liposome complex comprising a drug-encapsulated liposome and a poloxamer.

[0009] The present invention provides a drug delivery composition comprising the above liposome complex as an active ingredient.

[0010] The present invention provides a pharmaceutical composition for treating pain diseases, comprising the above liposome complex as an active ingredient.

[0011] In addition, the present invention provides a method for preparing a liposome complex, comprising the steps of mixing a hydrophilic drug, a hydrophobic drug, a solvent, and a lipid solution and sonicating the mixture to obtain a drug-encapsulated liposome; and the step of mixing a poloxamer solution into the obtained liposome solution.

[0012] The drugs used in conventional nerve blockade are in liquid form, and their duration of action is short, from a few days to a few weeks, so pain control is limited. However, the liposome poloxamer complex according to the present invention can remain in the target area in the body in a gel form for a long time, significantly extending the duration of action, and by increasing cell penetration, it improves drug absorption efficiency, so that excellent effects can be achieved even with a small amount of drug. Therefore, the frequency of patients' exposure to procedures that carry potential risks (radiation exposure, infection, bleeding, etc.) can be significantly reduced, and potential drug side effects of local anesthetics and steroid preparations can also be reduced.

[0013] Existing drugs have the risk of forming crystals, causing thrombosis, adhesion at the treatment site, etc., but the liposome poloxamer complex according to the present invention can significantly reduce this risk by ensuring that all hydrophobic / hydrophilic drugs are well mixed through the liposome and do not form crystals.

[0014] In addition, when the drug-encapsulated liposome poloxamer complex according to the present invention having the above advantages is provided in the form of a finished product, the efficiency and safety of the treatment can be increased, and it can be effectively applied to a wide range of fields of treatment for spinal and pain-related diseases.

[0015] Figure 1 illustrates a process for manufacturing drug-encapsulated liposomes according to one embodiment of the present invention. (a) illustrates a state when the drugs in Table 1 are mixed, (b) illustrates a step of removing ethanol using a rotary evaporator, and (c) illustrates a step of manufacturing uniform liposomes using ultrasonic treatment.

[0016] Figure 2(a) shows a state in which a drug-encapsulated liposome poloxamer manufactured according to one embodiment of the present invention is loaded into a transwell and gelled, and (b) shows a transwell loaded into a tube containing media.

[0017] Figure 3 is information on the viscosity of honey according to temperature for setting the range of optimal viscosity according to the mixing ratio of the drug according to the present invention.

[0018] Figures 4 and 5 show the viscosity according to the ratio of drug-encapsulated liposomes and poloxamer and temperature according to Experimental Example 1 of the present invention.

[0019] FIG. 6 is an image of the structure of a drug-encapsulated liposome poloxamer manufactured according to one embodiment of the present invention, observed using a transmission electron microscope (TEM).

[0020] Figure 7 shows the results of confirming cytotoxicity according to the concentration of drug-encapsulated liposome poloxamer according to Experimental Example 3 of the present invention.

[0021] Figure 8 shows the results of confirming cytotoxicity according to drug ratio according to Experimental Example 3 of the present invention.

[0022] Figure 9 shows the change in action potential generation in dorsal root ganglion (DRG) neurons of a drug to which a liposomal poloxamer delivery system was applied according to Experimental Example 4 of the present invention.

[0023] Figure 10 shows the number of action potential spikes in DRG neurons according to the release time of a drug to which a liposomal poloxamer delivery vehicle was applied according to Experimental Example 4 of the present invention.

[0024] Figure 11 shows voltage-dependent sodium channel (VGSCs) I of inhibited DRG neurons according to drug release time of drug-encapsulated liposomal poloxamer according to Experimental Example 5 of the present invention. Na It also represents the current representation.

[0025] Figure 12 shows VGSCs I of inhibited DRG neurons according to drug release time of drug-encapsulated liposomal poloxamer according to Experimental Example 5 of the present invention. Na It shows the current-voltage curve.

[0026] Figure 13 shows the current density (nA / pF) of VGSCs generated from DRG neurons under -10 mV stimulation according to the drug release time of drug-encapsulated liposomal poloxamer according to Experimental Example 5 of the present invention.

[0027] Hereinafter, the present invention will be described in detail.

[0028]

[0029] In order to solve the problems of existing drugs, the inventors prepared a liposome complex by mixing poloxamer into drug-encapsulated liposomes, and confirmed that the complex can remain in the body in a gel form, extending the drug retention time and increasing the drug absorption efficiency, thereby completing the present invention.

[0030]

[0031] The present invention provides a liposome complex comprising a drug-encapsulated liposome and a poloxamer.

[0032] The above liposome may have an inner space isolated by a lipid membrane, and a drug may be encapsulated in the inner space.

[0033] The above lipid membrane is formed of at least one selected from the group consisting of phosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol and phosphatidylserine, and may preferably be phosphatidylcholine (lecithin), but is not limited thereto.

[0034] The above liposomes may have an average diameter of 50 to 500 nm, preferably 100 to 300 nm, but is not limited thereto.

[0035] The above drug may include both hydrophobic and hydrophilic drugs, and both hydrophobic and hydrophilic drugs can be effectively bound and dissolved within the liposome, thereby preventing crystal formation of the drug. In other words, both hydrophobic and hydrophilic drugs can be encapsulated within the liposome in a well-dissolved state.

[0036] Specifically, the drug is a drug used in nerve block, and may be selected from local anesthetics or steroid preparations. For example, the hydrophobic drug may be selected from triamcinolone, ropivacaine, bupivacaine, or a pharmaceutically acceptable salt thereof, and the hydrophilic drug may be selected from dexamethasone, lidocaine, hyaluronidase, or a pharmaceutically acceptable salt thereof, but is not limited thereto.

[0037] In this specification, "poloxamer" refers to a triblock polymer of polyethylene oxide (PEO)-polypropylene oxide (PPO)-polyethylene oxide (PEO), which can form a reversible gel in an aqueous solution by controlling temperature. However, single poloxamer gels have limited application as biomaterials due to their low physical stability in aqueous environments.

[0038] The above poloxamer may be selected from the group consisting of pluronic F-68, F-87, F-127, P-105, P-123, L-61 and L-121, and preferably poloxamer 407 (pluronic F-127), but is not limited thereto.

[0039] The above drug-encapsulated liposome and poloxamer may be included in a volume ratio (v / v) of 1: (1 to 3), preferably 1: (1.5 to 2), more preferably 1: (1.75), to form a complex, but is not limited thereto.

[0040] The above complex, when mixed at the above ratio, may have a viscosity (viscosity) in the range of 10,000 to 20,000 cps at body temperature (37°C). The viscosity in the above range is suitable for drug encapsulation, delivery, and maintenance of persistence, and is also an appropriate viscosity that can reduce nerve damage and resulting side effects when injected into the body, and therefore, the above range is preferred.

[0041] The above liposome complex can maintain drug stability and control the degree of drug release by combining drug-encapsulated liposomes and poloxamer.

[0042] The liposome complex may be a thermosensitive complex that contains poloxamer and is liquid at room temperature (20 to 25°C) and changes to a gel at body temperature (35 to 38°C). Accordingly, the liposome complex has the advantage of being able to remain in the desired target area for a long time after being injected into the body in a liquid state and then gelling.

[0043]

[0044] The present invention provides a drug delivery composition comprising the above liposome complex as an active ingredient.

[0045] The above liposome complex can contain both hydrophilic drugs and hydrophobic drugs in dissolved form, and by including poloxamer, it is liquid at room temperature (20 to 25°C) and can change into a gel form at body temperature (35 to 38°C), so that when injected into the body, it can be gelled by body temperature and remain in the desired target site for a long time, thereby improving the drug delivery effect.

[0046]

[0047] The present invention provides a pharmaceutical composition for treating pain diseases, comprising the above liposome complex as an active ingredient.

[0048] The above pain disease may include all diseases that can cause pain, and may be selected from, but is not limited to, degenerative diseases including spinal stenosis, degenerative disc, spondylolisthesis, degenerative facet joint, etc.; fracture, cancer pain, or neuropathic pain disease.

[0049] Preferably, the pain disorder may be treated or alleviated by using nerve block.

[0050] In this specification, “nerve block” means a method of relieving pain or treating a disease by injecting a drug into a nerve or ganglion at the site of pain.

[0051] That is, the liposome complex according to the present invention can be a pharmaceutical composition used for nerve blocking for the treatment of pain diseases or pain relief.

[0052] Specifically, the liposome complex can reduce the firing of action potentials, which play a key role in the excitability and signal transmission of nerve cells.

[0053] The above liposome complex can induce a decrease in the excitability of dorsal root ganglion (DRG) neurons, thereby having enhanced persistence and modulating the transmission of pain signals from the peripheral nervous system to the central nervous system.

[0054] The above liposome complex can increase drug uptake into cells and reduce external release effects, thereby increasing drug persistence and inducing neuroactive effects for a long time.

[0055]

[0056] In this specification, "pharmaceutical composition" means a composition administered for the purpose of preventing or treating a specific disease, and for the purpose of the present invention, means one administered for the treatment of pain diseases, complications caused by the pain, etc.

[0057] The pharmaceutical composition according to the present invention can be prepared according to conventional methods in the pharmaceutical field. The pharmaceutical composition can be combined with an appropriate pharmaceutically acceptable carrier depending on the formulation, and, if necessary, can be prepared by further including excipients, diluents, dispersants, emulsifiers, buffers, stabilizers, binders, disintegrants, solvents, etc. The appropriate carriers, etc., which do not inhibit the activity and properties of the liposome complex according to the present invention, can be selected differently depending on the dosage form and formulation.

[0058] Carriers, excipients, diluents, etc. that may be included in the above pharmaceutical composition include, for example, lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxy benzoate, propylhydroxy benzoate, talc, magnesium stearate, mineral oil, etc.

[0059] The above pharmaceutical composition can be applied in any dosage form, and more specifically, can be selected from the group consisting of oral preparations, injection preparations, mucosal preparations, inhalation preparations, external preparations, and transdermal absorption preparations according to a conventional method, and can preferably be an injection preparation, but is not limited thereto.

[0060] Among the oral dosage forms, the solid dosage forms are in the form of tablets, pills, powders, granules, capsules, etc., and can be prepared by mixing at least one excipient, for example, starch, calcium carbonate, sucrose, lactose, sorbitol, mannitol, cellulose, gelatin, etc., and in addition to simple excipients, lubricants such as magnesium stearate and talc may also be included. In addition, in the case of capsule dosage forms, in addition to the above-mentioned substances, a liquid carrier such as fatty oil may be further included. Among the oral dosage forms, the liquid dosage forms include suspensions, oral solutions, emulsions, syrups, etc., and in addition to commonly used simple diluents such as water and liquid paraffin, various excipients such as wetting agents, sweeteners, fragrances, preservatives, etc. may be included.

[0061] The above parenteral formulations may include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, and suppositories. Non-aqueous solvents and suspensions may include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate. Suppository bases may include witepsol, macrogol, Tween 61, cacao butter, laurin, glycerogelatin, and the like. Without limitation, any suitable formulation known in the art may be used.

[0062]

[0063] The pharmaceutical composition according to the present invention can be administered in a pharmaceutically effective amount.

[0064] As used herein, “pharmaceutically effective amount” means an amount sufficient to treat a disease at a reasonable benefit / risk ratio applicable to medical treatment and not causing adverse effects.

[0065] The effective dosage level of the pharmaceutical composition may vary depending on the intended use, the patient's age, sex, weight, and health condition, the type and severity of the disease, the activity and sensitivity of the drug, the method of administration, the time of administration, the route of administration, and the excretion rate, the duration of treatment, the drugs used in combination or concurrently, and other factors well known in the medical field. For example, although not fixed, it may generally be administered once or several times daily at a dosage of 0.001 to 1000 mg / kg, preferably 0.01 to 100 mg / kg. The above dosage does not limit the scope of the present invention in any way.

[0066] The pharmaceutical composition may be administered to any animal capable of developing a pain disorder, and the animal may include, for example, humans and primates, as well as livestock such as cows, pigs, horses, and dogs.

[0067] The above pharmaceutical composition may be administered via an appropriate route of administration depending on the formulation, and may be administered via various routes, either oral or parenteral, as long as it can reach the target tissue. The method of administration is not particularly limited, and may be administered by conventional methods such as oral, rectal, intravenous, intramuscular, or dermal application, respiratory inhalation, intrauterine epidural, or intracerebroventricular injection.

[0068] The above pharmaceutical composition can be used alone for the prevention or treatment of pain diseases, or can be used in combination with surgery or other drug treatments.

[0069]

[0070] The present invention provides a method for preparing the liposome complex.

[0071] The method for manufacturing a liposome complex according to the present invention may include the steps of mixing a hydrophilic drug, a hydrophobic drug, a solvent, and a lipid solution and sonicating the mixture to obtain a drug-encapsulated liposome; and the step of mixing a poloxamer solution into the obtained liposome solution.

[0072] The above drug may be a local anesthetic or a steroid preparation that can be used for nerve block.

[0073] Specifically, the hydrophilic drug may be selected from, but is not limited to, dexamethasone, lidocaine, hyaluronidase, or a pharmaceutically acceptable salt thereof.

[0074] The hydrophobic drug may be selected from, but is not limited to, triamcinolone, ropivacaine, bupivacaine, or pharmaceutically acceptable salts thereof.

[0075] The solvent may be, but is not limited to, saline solution.

[0076] The above lipid solution is one in which one or more lipid components selected from the group consisting of phosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol, and phosphatidylserine are dissolved in (C1-C4) alcohol or its aqueous solution, and specifically, phosphatidylcholine (lecithin) may be dissolved in ethanol, but is not limited thereto.

[0077] The solvent containing the drug and the lipid solution may be mixed in a ratio of 1:1, but is not limited thereto.

[0078] Liposomes can be obtained by evaporating the solvent of the lipid solution from the above mixture and performing ultrasonic treatment.

[0079] All of the above drugs can be encapsulated inside the liposome in a well-dissolved state.

[0080] Additionally, the solution containing the obtained liposomes can be centrifuged to obtain liposomes having a more uniform size, for example, an average diameter of 50 to 500 nm, preferably 100 to 300 nm, from the supernatant.

[0081] The step of mixing the obtained liposome solution with the poloxamer solution may be performed by mixing in a volume ratio of 1: (1 to 3), preferably 1: (1.5 to 2), more preferably 1: (1.75), and stirring for 20 to 30 hours, but is not limited thereto. When prepared in the above ratio range, it can have an appropriate viscosity.

[0082] By the above mixing, the liposome and poloxamer can be combined to form a uniform dispersion.

[0083] The above liposome and poloxamer can be combined to maintain drug stability and control the degree of drug release.

[0084] The liposome complex comprising the above liposome and poloxamer may be a temperature-sensitive complex that is liquid at room temperature (20 to 25°C) and changes into a gel at body temperature (35 to 38°C). Accordingly, the complex has the advantage of being able to remain in the desired target area for a long time after being injected into the body in a liquid state and then gelling.

[0085] Hereinafter, to aid understanding of the present invention, examples will be given in detail. However, the following examples are intended only to illustrate the scope of the present invention and are not intended to limit its scope. These examples are provided to more fully explain the present invention to those of average skill in the art.

[0086]

[0087] <Example 1> Preparation of drug-encapsulated liposome poloxamer

[0088] 1-1. Drug: DLT [Dexamethasone + Lidocaine + Triamcinolone]

[0089] Dexamethasone (Yuhan Corporation, 5 mg / 1 mL), lidocaine hydrochloride hydrate (Daehan Pharmaceutical Co., Ltd., 400 mg / 20 mL), triamcinolone (Dongkwang Pharmaceutical, 40 mg / 1 mL), and normal saline were prepared.

[0090] The composition ratio of nerve blocking drugs is as shown in Table 1 below.

[0091] Drug concentration volume ratio (v / v) Dexamethasone 5 mg / 1 mL 3 Lidocaine 400 mg / 20 mL 5 Triamcinolone 40 mg / 1 mL 1 Normal saline 0.9 % 5

[0092]

[0093] 1-2. Liposomal Poloxamer (LP)

[0094] Liposomes (lecithin derived from egg yolk) and poloxamer (pluronic F127) were prepared.

[0095]

[0096] 1-3. Preparation of drug-encapsulated liposomes

[0097] When the drug was manufactured with the composition ratio according to Table 1 above, it was confirmed that triamcinolone, a hydrophobic drug, settled down (Fig. 1(a)), so the goal was to first dissolve it.

[0098] 50 mg of phosphatidylcholine (Egg PC, Avanti, cat. 840051P-1g) was dissolved in 125 mL of ethanol and placed in a flask and mixed uniformly. The drug solution listed in Table 1 was then mixed with the phosphatidylcholine dissolved in ethanol in a 1:1 ratio and stirred. Unlike when the drugs listed in Table 1 were mixed (Figure 1(a)), the reaction solution showed no precipitation. The reaction solution was then placed in a reactor and ethanol was evaporated using a rotary evaporator (Figure 1(b)). After evaporation, the mixture was sonicated to form uniform liposomes (Figure 1(c)). The mixture was centrifuged at 4,000 rpm for 10 minutes, and only the supernatant was collected to obtain uniform liposomes of approximately 200 nm, which were ultimately referred to as liposomes. The obtained liposomes were stored refrigerated at 4°C.

[0099]

[0100] 1-4. Preparation of poloxamer solution

[0101] 12 g of poloxamer (pluronic F127) was added to 40 mL of PBS and stirred at 4°C until fully dissolved. Stirring was continued until the solution became clear, thereby preparing a 30% m / w poloxamer solution. The final poloxamer solution was stored refrigerated at 4°C.

[0102]

[0103] 1-5. Preparation of drug-encapsulated liposomal poloxamer

[0104] The obtained liposome solution and poloxamer solution were stirred at 4°C at a liposome:poloxamer ratio of 1:1.75 for 24 hours to prepare a uniformly dispersed liposome-poloxamer composition. During this process, the liposomes and poloxamer stably combined to form a uniform dispersion. The prepared liposome-poloxamer composition was stored in a refrigerator at 4°C.

[0105]

[0106] 1-6. Preparation of drug-releasing media

[0107] 250 μL of the drug-loaded liposome poloxamer prepared in steps 1-5 above was loaded into a 0.3 μm transwell (PET track-etched membrane, FACON). Gelation of the drug-loaded liposome poloxamer was confirmed after incubation at 37°C for 1 hour. After placing the media in a 5 mL tube, the transwell was loaded, and the drug-released media was prepared through incubation for 24, 48, and 72 hours (Fig. 2).

[0108]

[0109] <Experimental Example 1> Viscosity verification of nerve blocking drugs used for spinal diseases

[0110] In order to find the appropriate viscosity of the mixture to maintain the drug's persistence in the spinal nerve block treatment area while having the advantage of dissolving hydrophobic drugs (such as triamcillonone) used in nerve block, we attempted to determine the range of viscosity according to temperature by considering the operating room temperature, body temperature, and the patient's high temperature.

[0111]

[0112] 1-1. Viscosity measurement method

[0113] Viscosity was measured using a rheometer (Anton paar, MCR 702e), and the temperature range was 2℃ to 40℃ (Rate of T: 5℃ / min). Liquids were measured using ① a single-cylinder rotational viscometer, and solid-like gel-like substances were measured using ② a cone-and-plate rotational viscometer.

[0114] ① Single cylinder rotational viscometer: This is a viscometer that measures the torque when a cylinder in a liquid is rotated at a constant angular velocity. The device constant was experimentally determined using a standard solution for viscometer calibration in advance, and the viscosity η of the liquid was calculated according to Equation 1 below.

[0115] <Formula 1>

[0116] [Correction pursuant to Rule 91, May 8, 2025]

[0117] ② Cone-plate rotational viscometer: A viscometer that places a liquid in the gap between a flat disk with the same rotation axis and a cone with a large upper angle, rotates one side, and measures the torque and angular velocity received by the other side. A liquid is placed in the gap between the cone and the flat disk at an angle α, and the cone or flat disk is rotated at a constant angular velocity and constant torque. When the cone or flat disk reaches a steady state, the torque received by the flat disk or cone and the corresponding angular velocity are measured, and the viscosity η of the liquid is calculated according to Equation 2 below.

[0118] <Formula 2>

[0119] [Correction pursuant to Rule 91, May 8, 2025]

[0120]

[0121] 1-2. Setting the viscosity range

[0122] The viscosity range was set by referring to the viscosity of honey among daily products, taking into account the degree to which the spinal nerves are not compressed, and the range of the mixture viscosity was predicted as follows (Fig. 3).

[0123] ① 4℃: Within 500 to 1,000 cps

[0124] ② 20℃: 1,000 to 3,000 cps

[0125] ③ 37℃: 10,000 cps or more

[0126]

[0127] 1-3. Viscosity measurement results

[0128] As shown in Figures 4, 5 and Table 2 below, the ratio (v / v) that falls within the expected viscosity range at a storage temperature of 4°C, an operating room / procedure room temperature of 20°C, and a patient's body temperature of 37°C was set to liposome: poloxamer = 1:1.75.

[0129] Viscostiy (CPS) Temp. liposome + DLT poloxamer L+DLT:P (1:1)L+DLT:P (1:1.25)L+DLT:P (1:1.5)L+DLT:P (1:1.75)L+DLT:P (1:2)L+DLT:P (1:3)L+DLT:P (1:5)L+DLT:P (1:7)4℃4.27126.7723.7726.2628.5641.1543.0172.3498.37143.1920℃3.523395013.7516.1416.7927.4430.35195.161561.23252637℃2.915579573.54150.67233.591497117810381414873955550

[0130]

[0131] <Experimental Example 2> Structural confirmation of drug-encapsulated liposomal poloxamer

[0132] After encapsulating the drug in liposomes, the structure of the complex was confirmed using transmission electron microscopy (TEM) at a concentration ratio of 1.75 poloxamer.

[0133] The morphology of DLT alone (DLT), liposomes loaded with DLT (DLT Liposome), and liposomal poloxamer loaded with DLT (DLT + LP 1:1.75) was confirmed through transmission electron microscopy. As shown in Fig. 6, in the case of DLT alone, an amorphous, undissolved crystal form was observed, and in the group loaded with liposomes, a circular shape was observed. In the case of liposomal poloxamer loaded with liposomes, the circular shape was thicker than that observed in the liposome alone, and the poloxamer was loaded into the liposome shape.

[0134]

[0135] <Experimental Example 3> Confirmation of cytotoxicity of drug-encapsulated liposomal poloxamer

[0136] To determine the cytotoxicity of nerve blocking drugs and drugs encapsulated in LPs in SH-SY5Y (ATCC. Cat. CRL-2266) cells, cell viability was determined using the CCK-8 assay.

[0137] The cytotoxicity according to the concentration of drug-encapsulated liposome-poloxamer (LP) was confirmed as shown in Figure 7.

[0138]

[0139] Additionally, SH-SY5Y cells were seeded at 1.5 × 10 in 96-well plates. 4 After 24 hours of cell / well seeding, the IC of the DLT drug was determined using NS (Normal saline) as a control. 50 Cytotoxicity was confirmed in a total of 7 groups by concentration (DLT (IC50)), concentration of DLT encapsulated in liposomes (DLT (LIPO)), concentration of ratio of liposomes to poloxamer (LP 1:1.5, LP 1:1.75), and concentration of ratio of DLT drug to liposome poloxamer (DLT + LP 1:1.5, DLT + LP 1:1.75).

[0140] As a result, as shown in Fig. 8, cytotoxicity was observed in a time-dependent manner at the concentration of DLT IC50, whereas cytotoxicity was not higher at a concentration of 1.75 poloxamer ratio after encapsulating DLT in liposomes than when treated with LP alone.

[0141]

[0142] <Experimental Example 4> Confirmation of Action Potential Changes in DRG Neurons

[0143] Action potentials play a central role in the excitability and signal transmission of neurons. The generation and propagation of action potentials are precisely regulated by voltage-gated ion channels in the neuronal membrane. In particular, voltage-gated sodium channels (VGSCs) play a crucial role in the generation of action potentials.

[0144] Accordingly, the inventors of the present invention tested whether DLT with liposome + poloxamer delivery system compared to existing DLT reduces the firing of action potentials in dorsal root ganglion neurons (DRG neurons).

[0145]

[0146] The animal experiment protocol included in this experiment was reviewed for ethical and scientific nature by the Institutional Animal Care and Use Committee (IACUC) of Korea University College of Medicine and approved as appropriate (Approval Number: KOREA-2024-0182-C1).

[0147] First, adult mice (15–25 g) 5–7 weeks old were prepared using a known method. The mice were sacrificed with carbon dioxide gas, and the spine was removed. The spine was cut vertically, and the internal spinal cord tissue was removed. The dorsal root ganglion tissue distributed along the dorsal spinal disc was removed using fine forceps, and neurons prepared in 4°C Hanks' balanced salt solution (Hanks' balanced salt solution; Thermo Fisher Scientific, Waltham, Massachusetts, USA) were incubated in 2 mL of Hanks' balanced salt solution containing 0.25% trypsin (Thermo Fisher Scientific, Waltham, Massachusetts, USA) at 37°C for 40 minutes. The tissue was washed with DMEM culture solution and triturated with a flame-polished Pasteur pipette to isolate cells. The isolated cells were centrifuged (1000 RPM, 5 min), resuspended in DMEM, and placed on glass coverslips (diameter 12 mm) coated with 0.1 mg / mL poly-L-ornithine (sigma, St. Louis, MO, USA). DRG neurons were cultured in a 5% carbon dioxide incubator at 37°C.

[0148] Next, we examined whether the spike firing of action potentials in DRG neurons was reduced by inducing repetitive occurrence of action potentials by injecting 100 pA depolarizing current. As shown in Figures 9 and 10, the frequency of occurrence of action potentials decreased in a drug release time-dependent manner (n = 4-5).

[0149] These results indicate that the drug administered via the novel liposome + poloxamer delivery system proposed in the present invention induces a more pronounced reduction in DRG neuron excitability than conventional drug delivery methods. Furthermore, it exhibits enhanced duration of action compared to conventional delivery methods, suggesting its potential to modulate pain signal transmission from the peripheral to the central nervous system.

[0150]

[0151] <Experimental Example 5> Drug Durability Analysis

[0152] Whole-cell recordings were performed using fragment electrodes (3–6 MΩ) pulled from silicate glass using a Brown-Flaming P-97 horizontal micropipette puller (Sutter Instruments, Novato, CA). Voltage- and current-clamp experiments were performed using a Multiclamp 700B amplifier and a Digidata 1550B (both from Molecular Devices, San Jose, CA, USA), and measurements and quantification were performed using pClamp 10.7 and Clampfit 10.7 (Molecular Devices, San Jose, CA, USA). Signals were filtered at 1 kHz and collected at 3 kHz. Electrical recordings were performed at room temperature. To determine drug persistence, residual drug effects were observed at various time points after drug treatment.

[0153] The extracellular solution contained 140 mM NaCl, 2 mM CaCl2, 1 mM MgCl2, 5 mM KCl, 10 mM HEPES, and 10 mM D-glucose, and the pH was adjusted to 7.2–7.4 using NaOH, and the osmolarity was 290–310 mOsm. In the experiment, K + Current (I) K ), Ca 2+ Current (I) ca ) were used to remove tetra-ethylamonium (TEA) and CdCl2.

[0154] The pipette was filled with an intracellular solution containing 130 mM K-gluconate, 5 mM KCl, 0.5 mM EGTA, 10 mM HEPES, 4 mM Mg-ATP, and 0.5 mM Na-GTP, pH adjusted to 7.2–7.4 with KOH, and having an osmolarity of 290–310 mOsm. The extracellular solution was continuously infused into the cells using a gravity-fed infusion system. Recordings were started at least 1 min after obtaining a whole-cell patch.

[0155] In current-clamp experiments, action potential firing was induced by a series of depolarizing current pulses (500 ms duration) increasing in 50 pA steps from -100 pA to +300 pA. I Na is 130 mM CsCl, 1 mM MgCl 2+ Pipettes were used filled with intracellular solution containing 10 mM EGTA, 10 mM HEPES, 4 mM Mg-ATP, 0.5 mM Na-GTP, pH adjusted to 7.2–7.4 with KOH, and having an osmolarity of 290–310 mOsm, induced by pulses of 10 mV steps (100 ms duration) from -60 mV to +80 mV.

[0156]

[0157] I NaAs has already been identified in DRG neurons, especially in small neurons transmitting pain signals, I mainly comes from DRG neurons with a cell diameter of 10 to 30 μm. Na In these small neurons, I was recorded from a rectifying potential of -70 mV to a command potential of +70 mV. Na When analyzing the biophysical characteristics and comparing them with previous reports, I Na The values ​​corresponded to those of voltage-gated sodium channels (VGSCs) (Fig. 11), which is consistent with the properties of VGSCs in DRG neurons.

[0158] Additionally, I of DRG neurons Na As a result of confirming the improved drug effect by the new liposome-poloxamer delivery system, as shown in Figs. 12 and 13, the DLT drug applied with the new delivery system was I Na In the current-clamped mode, hyperpolarizing current injection was clearly eliminated by the novel transmitter and the applied drug (Fig. 13). In addition, I in adult mouse DRG neurons Na The pain suppression effect of the novel delivery system and the applied DLT was release time dependent (Fig. 11).

[0159] To confirm the drug's persistence, the effects of conventional drug administration and the novel delivery system were compared over time (24 h, 48 h, and 72 h) after administration. As shown in Figures 11 to 13, the duration of effect was confirmed to be improved when the novel delivery system and the drug were administered together compared to the conventional delivery system. This indicates that the novel delivery system of the present invention increases drug persistence by increasing intracellular drug uptake and reducing external release, thereby proving that it can induce pain-induced nerve activation for a longer period of time than conventional methods.

[0160]

[0161] While specific aspects of the present invention have been described in detail above, it should be apparent to those skilled in the art that these specific descriptions merely represent preferred embodiments and are not intended to limit the scope of the present invention. In other words, the substantial scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A liposome complex comprising a drug-encapsulated liposome and a poloxamer.

2. In paragraph 1, The above drug, A liposome complex characterized by the dissolution of both hydrophobic and hydrophilic drugs.

3. In paragraph 1, The above poloxamer is, A liposome complex characterized by comprising at least one selected from the group consisting of pluronic F-68, F-87, F-127, P-105, P-123, L-61 and L-121.

4. In paragraph 1, The above drug-encapsulated liposomes and poloxamers are A liposome complex characterized in that it is contained in a volume ratio of 1: (1 to 3).

5. In paragraph 1, The above complex is, A liposome complex characterized by being in a liquid state at room temperature of 20 to 25°C and changing into a gel form at body temperature of 35 to 38°C.

6. A composition for drug delivery comprising a liposome complex according to any one of claims 1 to 5 as an active ingredient.

7. A pharmaceutical composition for treating pain diseases, comprising a liposome complex according to any one of claims 1 to 5 as an active ingredient.

8. In paragraph 7, The above liposome complex is, A pharmaceutical composition characterized by causing a decrease in the excitability of dorsal root ganglion (DRG) neurons.

9. In paragraph 7, The above pain disease is, A pharmaceutical composition characterized in that it is treated using a nerve block.

10. In paragraph 7, The above pain disease is, A pharmaceutical composition characterized by at least one selected from the group consisting of degenerative diseases including spinal stenosis, degenerative disc, spondylolisthesis, or degenerative facet joints; fractures, cancer pain, and neuropathic pain.

11. In paragraph 7, The above pharmaceutical composition, A pharmaceutical composition characterized by comprising at least one selected from the group consisting of oral preparations, injection preparations, mucosal preparations, inhalation preparations, external preparations, and transdermal absorption preparations.

12. A step of mixing a hydrophilic drug, a hydrophobic drug, a solvent, and a lipid solution and sonicating them to obtain drug-encapsulated liposomes; and A method for producing a liposome complex, comprising the step of mixing a poloxamer solution into the liposome solution obtained above.

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