KR102561916B1 - Carbon monoxide-releasing molecule-2-entrapped ultradeformable liposomes and use thereof - Google Patents

Abstract

The present invention relates to the development of hypervariable liposomes containing carbon monoxide-releasing molecule-2, and more specifically, to the topical delivery of carbon monoxide-releasing molecule-2 to acute dermatitis, thereby reducing inflammation mediating cytokines through continuous CO release. It relates to a hypervariable liposome containing carbon monoxide-releasing molecule-2 designed to alleviate inflammation by suppressing its expression.

Description

Carbon monoxide-releasing molecule-2-entrapped ultradeformable liposomes and use thereof {Carbon monoxide-releasing molecule-2-entrapped ultradeformable liposomes and use thereof}

The present invention relates to the development of hypervariable liposomes containing carbon monoxide-releasing molecule-2, and more specifically, to the topical delivery of carbon monoxide-releasing molecule-2 to acute dermatitis, thereby reducing inflammation mediating cytokines through continuous CO release. It relates to a hypervariable liposome containing carbon monoxide-releasing molecule-2 designed to alleviate inflammation by suppressing its expression.

Gas molecules, including nitric oxide (NO), carbon monoxide (CO), and hydrogen sulfide (H ₂ S), have been well-known for the past decades as gaseous transport materials produced in the body and have attracted attention due to their novel therapeutic potential (Ryter SW, Choi AM. Carbon monoxide: present and future indications for a medical gas. Korean J Intern Med 2013;28:123-40) In the case of carbon monoxide, its binding ability to hemoglobin, which is 250 times higher than that of oxygen, is beneficial to living organisms due to toxicity that causes hypoxia. However, carbon monoxide, which is produced naturally in the body, has anti-inflammatory, anti-apoptotic, It exhibits various biological actions such as antiproliferative, antioxidant, angiogenic and cytoprotective actions. (Motterlini R, Otterbein LE. The therapeutic potential of carbon monoxide. Nat Rev Drug Discov 2010;9:728-43, Bauer I, Pannen BH .Bench-to-bedside review: Carbon monoxide-from mitochondrial poisoning to therapeutic use.Crit Care 2009;13:220.) The generation of carbon monoxide in the body is caused by heme by heme oxygenase (HO). It occurs when heme is metabolized into carbon monoxide, biliverdin, and iron ions by the catabolism of Heme oxygenase is divided into heme oxygenase-2, which is a basic structural enzyme, and heme oxygenase-1, which is an inducing enzyme activated by reaction. (Abraham NG, Kappas A. Pharmacological and clinical aspects of heme oxygenase. Pharmacol Rev 2008;60:79-127.) Heme oxygenase-1 is activated by stimuli such as ischemia, hypoxia, inflammation, apoptosis signals, and oxidative/reduction imbalance, and acts as a signal to regulate intracellular, neuronal, and vascular functions. It generates carbon monoxide that acts as a transmitter. (Motterlini R, Mann BE, Foresti R. Therapeutic applications of carbon monoxide-releasing molecules. Expert Opin Investig Drugs 2005;14:1305-18.) It shows anti-inflammatory effect by regulating the mitogen-activated protein kinase (MAPKs) pathway to suppress inflammatory cytokines such as macrophage inflammatory protein-1 beta and interleukin-1 beta.

a-Gallego S, Bernardes GJ. Carbon-monoxide-releasing molecules for the delivery of therapeutic CO in vivo. Angew Chem 2014;53:9712-21.) 일산화탄소방출분자는 빛(CORM-1) 또는 리간드치환반응(CORM-2, CORM-3)에 의해 일산화탄소를 방출하며, 다양한 동물모델에서 일산화탄소의 치료효과들을 입증하였다.(Ismailova A, Kuter D, Bohle DS, Butler IS. An overview of the potential therapeutic applications of CO-releasing molecules. Bioinorg Chem Appl 2018;2018:8547364.) 일산화탄소방출분자-2는 상업적으로 이용가능하며 다양한 연구에서 항염증효과가 입증되었다.(Takagi T, Naito Y, Uchiyama K, Suzuki T, Hirata I, Mizushima K, et al. Carbon monoxide liberated from carbon monoxide-releasing molecule exerts an anti-inflammatory effect on dextran sulfate sodium-induced colitis in mice. Dig Dis Sci 2011;56:1663-71) 그러나 일산화탄소방출분자-2의 일산화탄소 방출 반감기는 1분 내외이며, 지용성이기 때문에 임상적으로 이용하기에는 어려운 단점이 있다.(Motterlini R, Mann BE, Foresti R. Therapeutic applications of carbon monoxide-releasing molecules. Expert Opin Investig Drugs 2005;14:1305-18.) 일산화탄소방출분자-2의 나노기술 기반 전달은 짧은 일산화탄소 방출의 단점을 해결하기 위한 좋은 전달체라고 할 수 있다. 여러 연구에서는 일산화탄소방출분자-2를 함유한 나노입자를 통해 일산화탄소의 방출을 제어방출 하여 치료효과를 개선하였다. 일산화탄소방출분자-2를 함유한 스티렌-말레산 공중합체 미셀은 쥐 대장염 모델에서 일산화탄소방출분자-2 보다 개선된 조직보호효과를 보였다.(Yin H, Fang J, Liao L, Nakamura H, Maeda H. Styrene-maleic acid copolymer-encapsulated CORM2, a water-soluble carbon monoxide (CO) donor with a constant CO-releasing property, exhibits therapeutic potential for inflammatory bowel disease. J Control Release 2014;187:14-21.) 일산화탄소방출분자-2를 함유한 고형지질나노입자는 지속적인 일산화탄소 방출을 통해 카라기난으로 유발된 쥐의 발 염증 모델에서 향상된 항염증 효과를 보였다.(Qureshi OS, Zeb A, Akram M, Kim M-S, Kang J-H, Kim H-S, et al. Enhanced acute anti-inflammatory effects of CORM-2-loaded nanoparticles via sustained carbon monoxide delivery. Eur J Pharm Biopharm 2016;108:187-95.) 본 발명에서는 피부 염증 모델에서 일산화탄소방출분자-2를 함유한 나노운반체의 효과에 대해 입증하고자 한다.As the physiological functions of these endogenous carbon monoxide have been revealed, studies have been conducted on the therapeutic effect through the application of carbon monoxide. Application of carbon monoxide through inhalation has been proven to have strong anti-inflammatory effects in animal models such as inflammation, ischemia, lung and blood vessel damage, sepsis, and graft rejection (Otterbein LE, Mantell LL, Choi AM. Carbon monoxide provides protection against hyperoxic acid). Lung injury. Am J Physiol 1999;276:L688-94, Fujita T, Toda K, Karimova A, Yan SF, Naka Y, Yet SF, et al. Paradoxical rescue from ischemic lung injury by inhaled carbon monoxide driven by derepression of fibrinolysis Nat Med 2001;7:598-604, Foresti R, Bani-Hani MG, Motterlini R. Use of carbon monoxide as a therapeutic agent: promises and challenges. Intensive Care Med 2008;34:649-58.) Inhalation has the disadvantage of requiring strict dose control and special equipment because overdose can be fatal. (Rom. o CC, Bl ttler WA, Seixas JD, Bernardes GJL. Developing drug molecules for therapy with carbon monoxide. Chem Soc Rev 2012;41:3571-83.) To solve this problem, a carbon monoxide-releasing molecule capable of safely delivering carbon monoxide was synthesized. Carbon monoxide-releasing molecules are transition metal carbonyl complexes capable of releasing controlled amounts of carbon monoxide when exposed to an appropriate biological environment. The developed carbon monoxide-releasing molecules include carbon monoxide-releasing molecule-1 (CORM-1, [Mn ₂ (CO) ₁₀ andFe(CO) ₅ ]), carbon monoxide-releasing molecule-2 (CORM-2, [Ru(CO) ₃ Cl ₂ ] ₂ ), carbon monoxide-releasing molecule-3 (CORM-3, [Ru(CO) ₃ Cl-glycinate]) and carbon monoxide-releasing molecule-A1 (CORM-A ₁ [Na ₂ (H ₃ BCO ₂ )] ¹⁴ ). .(Garc

a—Gallego S, Bernardes GJ. Carbon-monoxide-releasing molecules for the delivery of therapeutic CO in vivo. Angew Chem 2014;53:9712-21.) Carbon monoxide-releasing molecules release carbon monoxide by light (CORM-1) or ligand substitution reactions (CORM-2, CORM-3), and the therapeutic effects of carbon monoxide in various animal models have been investigated. (Ismailova A, Kuter D, Bohle DS, Butler IS. An overview of the potential therapeutic applications of CO-releasing molecules. Bioinorg Chem Appl 2018;2018:8547364.) Carbon monoxide-releasing molecule-2 is commercially available and Anti-inflammatory effects have been demonstrated in various studies (Takagi T, Naito Y, Uchiyama K, Suzuki T, Hirata I, Mizushima K, et al. Carbon monoxide liberated from carbon monoxide-releasing molecule exerts an anti-inflammatory effect on dextran sulfate (Motterlini R . can be referred to as a carrier. Several studies have improved the therapeutic effect by controlling the release of carbon monoxide through nanoparticles containing carbon monoxide-releasing molecule-2. Styrene-maleic acid copolymer micelles containing carbon monoxide-releasing molecule-2 showed improved tissue protective effects compared to carbon monoxide-releasing molecule-2 in a rat colitis model (Yin H, Fang J, Liao L, Nakamura H, Maeda H. Styrene-maleic acid copolymer-encapsulated CORM2, a water-soluble carbon monoxide (CO) donor with a constant CO-releasing property, exhibits therapeutic potential for inflammatory bowel disease. J Control Release 2014;187:14-21.) Carbon monoxide-releasing molecule Solid lipid nanoparticles containing -2 showed enhanced anti-inflammatory effects in a rat foot inflammation model induced by carrageenan through sustained carbon monoxide release (Qureshi OS, Zeb A, Akram M, Kim MS, Kang JH, Kim HS , et al. Enhanced acute anti-inflammatory effects of CORM-2-loaded nanoparticles via sustained carbon monoxide delivery. Eur J Pharm Biopharm 2016;108: 187-95.) We want to demonstrate the effectiveness of one nanocarrier.

Next-generation liposomes, known as transfersomes, ultradeformable liposomes (UDLs), and elastic or deformable liposomes, have great potential as delivery vehicles that can improve skin permeation of drugs. (Zeb A, Arif ST, Malik M, Shah FA, Din FU, Qureshi OS, et al. Potential of nanoparticulate carriers for improved drug delivery via skin. J Pharm Investig 2019;49:485-517.) Hypervariable liposomes have phospholipid bilayer and edge activation (Elsayed MMA, Abdallah OY, Naggar VF, Khalafallah NM. Lipid vesicles for skin delivery of drugs: Reviewing three decades of research. Int J Pharm 2007;332:1-16. ) Hypervariable liposomes in which an edge activator is introduced into the phospholipid bilayer have variability and can penetrate deep into the skin past the epidermis. (Cevc G, Blume G. Lipid vesicles penetrate into intact skin owing to the transdermal osmotic gradients and hydration force. Biochim Biophys Acta 1992;1104:226-32.)

Therefore, the hypervariable liposome containing carbon monoxide-releasing molecule-2 can be used as a delivery vehicle that improves skin permeation of carbon monoxide-releasing molecule-2 and improves the half-life of carbon monoxide release to alleviate skin inflammation.

The technical problem to be achieved by the present invention is to provide a transdermal drug delivery system and a pharmaceutical composition for treating dermatitis, which contain hypervariable liposomes as an active ingredient.

However, the technical problem to be achieved by the present invention is not limited to the above-mentioned problems, and other problems not mentioned will be clearly understood by those skilled in the art from the description below.

In order to solve the above problems, the present invention provides a drug delivery system for transdermal penetration comprising a hypervariable liposome as an active ingredient.

In addition, the present invention provides a pharmaceutical composition for the treatment of skin inflammation comprising a hypervariable liposome as an active ingredient.

In addition, the present invention provides a method for treating dermatitis, which includes the step of applying the hypermodifiable liposome to the inflammatory skin area of a subject.

In addition, the present invention provides a hypermodable liposome for the preparation of a drug for treating skin inflammation.

In the present invention, the hypervariable liposome is composed of a double layer composed of a phospholipid and an edge activator, and carbon monoxide-releasing molecule-2 is included in the double layer.

As an embodiment of the present invention, the phospholipids include phosphatidyl choline, phosphatidylethanolamine, phosphatidylserine, phosphatidyl glycerol and phosphatidylinositol ( phosphatidylinositol) may include one or more selected from the group consisting of.

As another embodiment of the present invention, the phospholipid is HPS (hydrogenated phosphatidylcholine), DLPC (Dilauroyl phosphatidylcholine), DMPC (Dimyristoyl phosphatidylcholine), DPPC (Dipalmitoyl phosphatidylcholine), DSPC (Distearoyl phosphatidylcholine), DOPC (Dioleoyl phosphatidylcholine), DEPC (Dierucoyl phosphatidylcholine), POPC (Palmitoyloleoyl phosphatidylcholine), DMPG (Dimyristoyl phosphatidylglycerol, sodium salt), DPPG (Dipalmitoyl phosphatidylglycerol, sodium salt), DSPG (Distearoyl phosphatidylglycerol, sodium salt), DOPG (Dioleoyl phosphatidylglycerol, sodium salt), POPG (Palmitoyloleoyl phosphatidylglycerol, sodium salt), DMPE (Dimyristoyl phosphatidylethanolamine), DPPE (Dipalmitoyl phosphatidylethanolamine), DPPE (Dipalmitoyl phosphatidylethanolamine), DSPE (Distearoyl phosphatidylethanolamine), DOPE (Dioleoyl phosphatidylethanolamine), DMPA (Dimyristoyl phosphatidic acid, sodium salt), DPPA (Dipal mitoylphosphatidic acid, sodium salt), DSPA (Distearoyl phosphatidic acid, sodium salt), and DOPS (Dioleoyl phosphatidylserine, sodium salt).

As another embodiment of the present invention, the edge activator may include at least one selected from the group consisting of polyoxyethylene sorbitan fatty acid esters (Tweens) and sorbitan fatty acid esters (spans). .

As another embodiment of the present invention, the polyoxyethylene sorbitan fatty acid ester (Tweens) is polyoxyethylene (20) sorbitan monolaurate (Tween 20), polyoxyethylene ( 4) Polyoxyethylene (4) sorbitan monolaurate (Tween 21), Polyoxyethylene (20) sorbitan monopalmitate (Tween 40), Polyoxyethylene (20) sorbitan Polyoxyethylene (20) sorbitan monostearate (Tween 60), Polyoxyethylene (4) sorbitan monostearate (Tween 61), Polyoxyethylene (20) sorbitan tristearate ( polyoxyethylene (20) sorbitan tristearate, Tween 65), and polyoxyethylene (4) sorbitan monooleate (Polyoxyethylene (20) sorbitan monooleate, Tween 80).

As another embodiment of the present invention, the sorbitan fatty acid esters (spans) include sorbitan monolaurate (Span 20), sorbitan monopalmitate (Span 40), sorbitan monostea sorbitan monostearate (span 60), sorbitan monooleate (Span 80), sorbitan sesquioleate (Span 83), sorbitan trioleate (Span 85), and It may include one or more selected from the group consisting of sorbitan isostearate (Span 120).

In the present invention, hypervariable liposomes were prepared using formulations of various compositions and concentrations, and as a result of searching for a composition having a size of about 100 nm and satisfying all of the excellent polydispersity index, encapsulation rate, and variability, soybean phosphatidylcholine: Tween80: carbon monoxide release It was confirmed that the size, polydispersity index, encapsulation rate, and variability conditions for the improvement and treatment of acute dermatitis were satisfied in the hypervariable liposome prepared at the ratio of Molecule-2 = 8:2:1, and this was confirmed to improve or treat acute dermatitis. It is intended to be provided for use for treatment.

Therefore, as another embodiment of the present invention, the hypervariable liposome may include 1 to 20% by weight of carbon monoxide-releasing molecule-2, 50 to 80% by weight of phospholipid, and 10 to 40% by weight of an edge activator.

As another embodiment of the present invention, the double layer of the hypervariable liposome may contain a phospholipid and an edge activator in a weight ratio of 8:2.

In addition, the present invention provides a method for preparing an ultra-variable liposome for improving or treating acute dermatitis, comprising mixing soybean phosphatidylcholine, Tween80, and carbon monoxide-releasing molecule-2 in a ratio of 8:2:1 (w/w).

The hypermodable liposome containing carbon monoxide-releasing molecule-2 according to the present invention exhibits improved anti-inflammatory effects in vivo and ex vivo through controlled and efficient carbon monoxide release. In addition, through the controlled carbon monoxide release of carbon monoxide-releasing molecule-2 through the present invention, it is possible to increase not only the anti-inflammatory effect but also the cytoprotective effect, vascular relaxation effect, antiproliferative effect, antioxidant effect, and apoptosis inhibitory effect. .

1 is a schematic diagram of a hypervariable liposome containing carbon monoxide-releasing molecule-2.
FIG. 2 shows a transmission electron microscope (TEM) picture of a hypermodable liposome containing carbon monoxide-releasing molecule-2.
3 is a graph comparing the variability of hypervariable liposomes containing carbon monoxide-releasing molecule-2.
FIG. 4 is a graph comparing carbon monoxide released from a hypervariable liposome containing carbon monoxide-releasing molecule-2 and a carbon monoxide-releasing molecule-2 solution for 180 minutes.
5 is a graph showing the cell viability of RAW 264.7 macrophages treated with carbon monoxide-releasing molecule-2-containing hypervariable liposomes or carbon monoxide-releasing molecule-2 solution.
Figure 6 is a hypervariable liposome containing carbon monoxide-releasing molecule-2 or carbon monoxide-releasing molecule-2 in macrophages inflamed with lipopolysaccharide (LPS) to compare the amount of nitrogen monoxide produced as an indicator of inflammatory response. After processing the solution, it is a graph comparing the amount of nitrite produced by RAW 264.7 macrophages.
Figure 7 shows inflammation through changes in the thickness of the ear when treated with hypermodified liposomes containing carbon monoxide-releasing molecule-2 in the skin inflammation model of mice in which acute skin inflammation was induced by tetradecanoylphorbol acetate (TPA). It is a graph comparing the degree of edema, which is an index of response.
Figure 8 shows the inflammation through the weight change of the ear when treated with hypermodified liposome containing carbon monoxide-releasing molecule-2 in the skin inflammation model of rats in which acute skin inflammation was induced by tetradecanoylphorbol acetate (TPA). It is a graph comparing the degree of edema, which is an index of response.
9 is a comparison of the amount of neutrophils when treated with hypermodified liposomes containing carbon monoxide-releasing molecule-2 in a skin inflammation model of rats in which acute skin inflammation was induced by tetradecanoylphorbol acetate (TPA). It is a graph comparing the degree of reaction.
10 shows the amount of IL-6 mRNA when the hypermodified liposome containing carbon monoxide-releasing molecule-2 was treated in a skin inflammation model of mice in which acute skin inflammation was induced by tetradecanoylphorbol acetate (TPA). It is a graph comparing the degree of inflammatory response by comparing the
Figure 11 shows the amount of IL-1β mRNA when treated with hypermodified liposomes containing carbon monoxide-releasing molecule-2 in a skin inflammation model of mice in which acute skin inflammation was induced by tetradecanoylphorbol acetate (TPA). It is a graph comparing the degree of inflammatory response by comparing the
12 is when the hypermodified liposome containing carbon monoxide-releasing molecule-2 is treated in a skin inflammation model of mice in which acute skin inflammation is induced by tetradecanoylphorbol acetate (TPA), TNF-α¥α mRNA It is a graph comparing the degree of inflammatory response by comparing the amount of

High concentrations of carbon monoxide can cause tissue damage by inducing toxic and poisoning effects in normal tissues by inducing oxygen deficiency. However, low-concentration carbon monoxide has anti-inflammatory effects, cytoprotective effects, vascular relaxation effects, anti-proliferative effects, antioxidant effects, and apoptosis inhibitory effects.

As described above, the present invention aims to reduce toxicity and exhibit anti-inflammatory effects through local skin delivery and controlled release of carbon monoxide by preparing a hypermodable liposome containing carbon monoxide-releasing molecule-2.

Accordingly, the present invention is to develop a hypermodable liposome containing carbon monoxide-releasing molecule-2 for alleviating acute skin inflammation through efficient carbon monoxide delivery, and to provide it for a transdermal drug delivery system and skin inflammation treatment.

In the present invention, the half-life of carbon monoxide release was improved through the invention of hypervariable liposomes containing carbon monoxide-releasing molecule-2, and the anti-inflammatory effect was demonstrated in a skin inflammation model induced by tetradecanoylphorbol acetate (TPA). did

Soybean-derived phosphatidylcholine was used as a component of the phospholipid bilayer and Tween 80 was added as an edge activator to prepare a hypervariable liposome containing carbon monoxide-releasing molecule-2 through lipid membrane hydration and extrusion. After preparation of hypervariable liposomes containing carbon monoxide-releasing molecule-2, physicochemical properties including particle size, polydispersity index, zeta potential, encapsulation rate, shape and variability were evaluated. Carbon monoxide release from hypervariable liposomes containing carbon monoxide-releasing molecule-2 was evaluated by myoglobin analysis. Anti-inflammatory effects in vitro and in vivo were evaluated in lipopolysaccharide (LPS)-induced macrophage and TPA-induced skin inflammation models, respectively.

More specifically, the present inventors, through specific experiments, used soy phosphatidyl choline as a component of the phospholipid bilayer and added Tween80 as an edge activator to obtain lipid film hydration and extrusion methods ( Through extrusion technique), hypervariable liposomes were prepared to contain carbon monoxide-releasing molecule-2. In addition, hypervariable liposomes were prepared by varying the ratio of phosphatidylcholine and Tween80, and their physicochemical properties and variability were evaluated. Confirmed.

In addition, the present inventors confirmed the carbon monoxide release pattern and half-life of carbon monoxide of the hypervariable liposome of Example 2 and the carbon monoxide-releasing molecule-2 solution (Comparative Example 2) through specific experiments, and as a result, the hypervariable liposome of Example 2 had a longer It was confirmed that it released carbon monoxide over time and had a half-life that was increased by about 40 times, and it was confirmed that the storage stability and non-cytotoxic and anti-inflammatory effects of the hypervariable liposome were excellent. Hypermodified liposomes can be provided for the treatment of skin inflammation.

The present invention can apply various transformations and can have various embodiments. Hereinafter, specific embodiments will be illustrated in the drawings and described in detail in the detailed description. However, it should be understood that this is not intended to limit the present invention to specific embodiments, and includes all transformations, equivalents, and substitutes included in the spirit and scope of the present invention. In describing the present invention, if it is determined that a detailed description of related known technologies may obscure the gist of the present invention, the detailed description will be omitted.

[Example]

To prepare a hypervariable liposome containing carbon monoxide-releasing molecule-2, as shown in Table 1, soy phosphatidyl choline was used as a component of the phospholipid bilayer in an appropriate ratio and Tween80 was added as an edge activator to form lipid Hypervariable liposomes containing carbon monoxide-releasing molecule-2 were prepared through a film hydration method and an extrusion technique. More specifically, carbon monoxide-releasing molecule-2, soybean phosphatidylcholine, and Tween80 are dissolved in chloroform in an appropriate ratio in a round bottom flask, and then the solvent is evaporated using a vacuum distillation apparatus to form a thin lipid film. After hydration by adding appropriately warmed 5% glucose solution, the particle size is first reduced through an ultrasonicator and then passed through a 100 nm membrane to uniformly reduce the particle size. Finally, unencapsulated carbon monoxide-releasing molecule-2 was removed by ultrafiltration centrifugation.

Composition (w/w) soybean phosphatidylcholine Tween 80 cholesterol Carbon monoxide-releasing molecule-2 Example 1 9 One - One Example 2 8 2 - One Example 3 7 3 - One Example 4 6 4 - One Comparative Example 1 8 - 2 One Comparative Example 2 - - - One

Experimental Example 1: Physical and chemical properties of hypermodifiable liposomes containing carbon monoxide-releasing molecule-2

After taking a small amount of the composition prepared in the above example and diluting with water, particle size, polydispersity index, and zeta potential were evaluated using a particle sizer (Zeta sizer Nano ZS). The capture efficiency of carbon monoxide-releasing molecule-2 in hypervariable liposomes containing carbon monoxide-releasing molecule-2 was measured by quantifying ruthenium using inductively coupled plasma spectrometry (ICP-AES). The evaluated physical and chemical properties are as summarized in Table 2, and it can be seen that uniform particles of about 100 nm were produced in all examples. The zeta potential is around 40 mV, and it can be seen that the collection efficiency decreases as the ratio of Tween80 increases.

Physical and chemical properties Particle size (nm) polydispersity index Zeta Potential (mV) Capture efficiency (%) Example 1 106.3 ± 1.6 0.070 ± 0.025 43.7 ± 4.9 35.5 ± 0.9 Example 2 100.9 ± 1.5 0.087 ± 0.025 42.7 ± 4.5 31.5±2.0 Example 3 98.6 ± 3.1 0.116 ± 0.005 40.9 ± 4.8 27.1 ± 2.3 Example 4 95.3 ± 1.2 0.141 ± 0.031 38.0 ± 6.8 23.9±2.0

Experimental Example 2: Transmission Electron Microscope (TEM)

The shape of the hypervariable liposome containing carbon monoxide-releasing molecule-2 was confirmed by transmission electron microscopy using Example 2. As shown in FIG. 2, it can be confirmed that round particles having a uniform size are formed.

Experimental Example 3: Evaluation of variability

The variability of the compositions prepared in the above examples was evaluated. After injecting the composition of each example into an extruder equipped with a 50 nm polycarbonate membrane, nitrogen was added at a pressure of 0.1 Mpa to evaluate the amount passing through the membrane for 5 minutes. As a comparative example (Comparative Example 1), liposomes in which cholesterol, generally used for liposome preparation, was added at a ratio of 8:2 were used. Cholesterol is known to play a role in maintaining the rigidity of liposomes. As a result of comparing the variability by evaluating the amount of passing through the membrane for 5 minutes and the particle size after passing through the membrane, as shown in FIG. The highest variability was shown in Example 2, which was composed at a ratio.

Experimental Example 4: Release of carbon monoxide from hypermodified liposomes containing carbon monoxide-releasing molecule-2

Carbon monoxide release from hypervariable liposomes containing carbon monoxide-releasing molecule-2 was evaluated using myoglobin assay. After forcibly reducing myoglobin, when the carbon monoxide-releasing molecule-2 solution or hypervariable liposome containing carbon monoxide-releasing molecule-2 is injected, carbon monoxide is generated and combined with myoglobin to form a carbon monoxide-myoglobin polymer. Carbon monoxide emission over time was evaluated by measuring the concentration of the carbon monoxide-myoglobin polymer through absorbance. As a comparative example (Comparative Example 2), carbon monoxide-releasing molecule-2 solution was used for evaluation. As shown in FIG. 4, Comparative Example 2 showed rapid release at the beginning, whereas Example 2 showed continuous carbon monoxide release. The carbon monoxide emission half-life increased up to 40 times compared to Comparative Example 2.

Experimental Example 5: Evaluation of storage stability of hypervariable liposomes containing carbon monoxide-releasing molecule-2

The storage stability of the hypervariable liposome containing carbon monoxide-releasing molecule-2 was evaluated while storing Example 2 at 4°C (Table 3) and 25°C (Table 4) for 28 days. To evaluate the stability of Example 2, particle size, polydispersity index, zeta potential, and collection efficiency were measured.

Day of the week Particle size (nm) polydispersity index Zeta Potential (mV) Capture efficiency (%) 0 100.9 ± 1.5 0.087 ± 0.025 42.7 ± 4.5 31.5±2.0 One 99.8 ± 2.6 0.059 ± 0.016 45.2 ± 6.8 31.4±2.0 3 98.1 ± 2.3 0.087 ± 0.003 42.2±1.0 30.5±1.8 7 100.3 ± 0.8 0.085 ± 0.019 46.3 ± 3.7 30.1 ± 2.8 14 99.0 ± 5.9 0.077 ± 0.013 45.2 ± 4.4 29.0±2.0 21 97.9 ± 3.2 0.077 ± 0.029 45.1 ± 3.2 28.0 ± 1.1 28 99.2 ± 1.7 0.067 ± 0.020 40.2 ± 6.0 27.2 ± 2.5

Day of the week Particle size (nm) polydispersity index Zeta Potential (mV) Capture efficiency (%) 0 100.9 ± 1.5 0.087 ± 0.025 42.7 ± 4.5 31.5±2.0 One 99.2 ± 2.9 0.085 ± 0.016 41.7 ± 2.6 30.9 ± 2.6 3 96.8 ± 3.6 0.083 ± 0.028 41.8±1.6 29.9 ± 1.9 7 97.6 ± 3.5 0.071 ± 0.007 37.4 ± 4.6 27.4 ± 2.0 14 95.9 ± 2.4 0.067 ± 0.010 22.6 ± 1.7 24.6 ± 3.3 21 89.5 ± 3.2 0.067 ± 0.002 7.7±1.3 23.2 ± 3.9 28 89.5 ± 4.3 0.079 ± 0.021 3.5±1.0 15.5 ± 4.1

Experimental Example 6: Non-cytotoxic and anti-inflammatory effects of hypermodified liposomes containing carbon monoxide-releasing molecule-2 in vitro

The specific cytotoxicity of the hypervariable liposome containing carbon monoxide-releasing molecule-2 was confirmed by evaluating the viability of RAW 264.7 macrophages through the MTT assay. As shown in Figure 5, it was confirmed that there was no cytotoxicity at 10 and 20 μ\μM of Example 2 and Comparative Example 2, and an anti-inflammatory effect test was conducted for both concentrations. For the anti-inflammatory effect, RAW 264.7 macrophages were treated with Experimental Example 2 and Comparative Example 2, respectively, after inducing an inflammatory response using lipopolysaccharide (LPS) to evaluate the production of nitric oxide. Nitric oxide is an indicator of the inflammatory response and can indicate the degree of the inflammatory response. As shown in FIG. 6, it was confirmed that the amount of nitrogen monoxide produced in Example 2 was reduced compared to Comparative Example 2, and through this, the anti-inflammatory effect of Example 2 could be demonstrated.

Experimental Example 7: Anti-inflammatory effect of hypermodable liposomes containing carbon monoxide-releasing molecule-2 in vivo

In order to evaluate the anti-inflammatory effect of hypervariable liposomes containing carbon monoxide-releasing molecule-2 in vivo, a rat acute skin inflammation model was used. After each ear of the rat was treated with Examples and Comparative Examples, acute skin inflammation was induced by treating tetradecanoylphorbol acetate (TPA) in the same ear. At this time, the anti-inflammatory effect according to each Example and Comparative Example was evaluated by the degree of edema through the weight and thickness change of the ear. In addition, the degree of inflammatory response was evaluated by evaluating the number of neutrophils and the expression levels of inflammatory mediating cytokines, IL-6, IL-1β, and TNF-α¥α mRNA. After the experiment was completed, the mouse ear tissue was taken and histopathological evaluation was performed. In this experiment, the anti-inflammatory effect was compared using indomethacin (Comparative Example 3) as a positive control group. As a result, as shown in FIGS. 7 and 8, the change in thickness and weight of the ear was small in Experimental Example 2, and through this, it can be confirmed that the occurrence of edema was suppressed through the anti-inflammatory effect. 9, 10, 11, and 12, when Experimental Example 2 was treated, the number of neutrophils was the lowest, and the expression level of IL-6, IL-1β, and TNF-α¥α mRNA, which are inflammatory mediating cytokines, was the highest. low can be ascertained. In addition, as shown in Table 5, in the histopathological characteristics, when Example 2 was treated, epidermal hyperliferation was most inhibited, and the degree of leucocyte infiltration was also the lowest.

ear edema epidermal hyperkeratinization leukemic infiltrate control group 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 TPA 3.53 ± 0.38 2.47 ± 0.69 3.27 ± 0.43 TPA + Comparative Example 3 2.93 ± 0.43 1.47 ± 0.30 2.60 ± 0.15 TPA + Comparative Example 2 2.87 ± 0.56 1.93 ± 0.72 2.53 ± 0.65 TPA + Example 2 1.13±0.77 ^# 0.93±0.72 ^* 0.73±0.55 ^#

As above, specific parts of the present invention have been described in detail, and for those skilled in the art, it is clear that these specific descriptions are only preferred embodiments, and the scope of the present invention is not limited thereby. something to do. Accordingly, the substantial scope of the present invention will be defined by the appended claims and their equivalents.

Claims (10)

A drug delivery system for transdermal penetration comprising a hypervariable liposome as an active ingredient,
The hypervariable liposome is composed of a double layer consisting of a phospholipid and an edge activator, and a carbon monoxide-releasing molecule-2 is included in the double layer, a transdermal drug delivery system.
According to claim 1,
The phospholipid is selected from the group consisting of phosphatidyl choline, phosphatidylethanolamine, phosphatidylserine, phosphatidyl glycerol and phosphatidylinositol having a fatty acid chain having 12 to 24 carbon atoms. A drug delivery system for transdermal penetration, characterized in that it comprises at least one kind.
According to claim 2,
The phospholipids include hydrogenated phosphatidylcholine (HPS), dilauroyl phosphatidylcholine (DLPC), dimyristoyl phosphatidylcholine (DMPC), dipalmitoyl phosphatidylcholine (DPPC), distearoyl phosphatidylcholine (DSPC), dioleoyl phosphatidylcholine (DOPC), and dierucoylcholine (DEPC). phosphatidylcholine), POPC (Palmitoyloleoyl phosphatidylcholine) , DMPG (Dimyristoyl phosphatidylglycerol, sodium salt), DPPG (Dipalmitoyl phosphatidylglycerol, sodium salt), DSPG (Distearoyl phosphatidylglycerol, sodium salt), DOPG (Dioleoyl phosphatidylglycerol, sodium salt), POPG (Palmitoyloleoyl phosphatidylglycerol, sodium salt), DMPE (Dimy ristoyl phosphatidylethanolamine ); aroyl phosphatidic A transdermal drug delivery system comprising at least one selected from the group consisting of acid, sodium salt) and DOPS (dioleoyl phosphatidylserine, sodium salt). According to claim 1,
The transdermal drug delivery system, characterized in that the edge activator comprises at least one selected from the group consisting of polyoxyethylene sorbitan fatty acid esters (Tweens) and sorbitan fatty acid esters (spans).
According to claim 4,
The polyoxyethylene sorbitan fatty acid ester (Tweens) is polyoxyethylene (20) sorbitan monolaurate (Polyoxyethylene (20) sorbitan monolaurate, Tween 20), polyoxyethylene (4) sorbitan monolaurate (Polyoxyethylene (4) sorbitan monolaurate, Tween 21), Polyoxyethylene (20) sorbitan monopalmitate, Tween 40, Polyoxyethylene (20) sorbitan monostearate monostearate, Tween 60), Polyoxyethylene (4) sorbitan monostearate, Tween 61), Polyoxyethylene (20) sorbitan tristearate, Tween 65 ), and polyoxyethylene (4) sorbitan monooleate (Polyoxyethylene (20) sorbitan monooleate, Tween 80).
According to claim 4,
The sorbitan fatty acid ester (span, spans) is sorbitan monolaurate (Span 20), sorbitan monopalmitate (Span 40), sorbitan monostearate (Span 60), sorbitan monooleate (Span 80), sorbitan sesquioleate (Span 83), sorbitan trioleate (Span 85), and sorbitan isostearate A transdermal drug delivery system comprising at least one selected from the group consisting of Span 120).
According to claim 1,
The hypervariable liposome comprises 1 to 20% by weight of carbon monoxide-releasing molecule-2, 50 to 80% by weight of phospholipid, and 10 to 40% by weight of an edge activator, transdermal drug delivery system.
According to claim 1,
The double layer of the hypervariable liposome is characterized in that the phospholipid and the edge activator are included in a weight ratio of 8: 2, transdermal drug delivery system.
A pharmaceutical composition for the treatment of skin inflammation comprising a hypervariable liposome as an active ingredient,
The hypervariable liposome is composed of a double layer consisting of a phospholipid and an edge activator, and carbon monoxide-releasing molecule-2 is included in the double layer, a pharmaceutical composition.
According to claim 9,
The double layer of the hypervariable liposome is a pharmaceutical composition, characterized in that the phospholipid and the edge activator are included in a weight ratio of 8: 2.