Pharmaceutical preparations containing lanosterol and its derivatives and cyclodextrin

Abstract

This invention relates to a pharmaceutical formulation containing lanosterol, its derivatives, and cyclodextrin. In one embodiment of the pharmaceutical formulation, water solubility and bioavailability are improved, and bioavailability is increased.

Description

[Technical Field]

[0001] This invention relates to pharmaceutical preparations containing lanosterol and its derivatives, and cyclodextrin. [Background technology]

[0002] Cyclodextrins have pharmaceutical applications and are used to dissolve or stabilize numerous compounds. However, such applications have limitations when applied to therapeutic drugs, as many compounds cannot be complexed with cyclodextrins or become unsuitable for pharmaceutical use [see J. Szejtli, Pharmaceutical Technology, 1991, 24-38; and U.S. Patent No. 5,362,860]. In particular, the bioavailability of drug-cyclodextrin mixtures is often unpredictable, and in practice, the formation of drug-cyclodextrin complexes often reduces the bioavailability of the drug [see T. Loftsson, Pharmaceutical Technology, 1999, 12, 40-50; and Uekama, K, et al., CRC Critical Reviews in therapeutic Drug Carrier Systems, 1987, 3(1), 1-40].

[0003] Meanwhile, research is underway to isolate useful compounds from Chaga mushrooms, and various compounds such as inotodiol, trametenolic acid, lanosterol, and inonotsulide have been isolated (Ma L., et al., 2013). Lanosterol and its derivatives have been shown to have physiological activities such as anticancer, anti-inflammatory, and anti-allergic properties.

[0004] The present inventors have completed an invention for a pharmaceutical formulation to increase the physiological activity of lanosterol and its derivatives. [Overview of the project]

Problems to be Solved by the Invention

[0005] One aspect is to provide a pharmaceutical preparation comprising a compound represented by the following Chemical Formula 1 or a pharmaceutically acceptable salt thereof, and a cyclodextrin derivative represented by the following Chemical Formula 2.

[0006]

Chem.

[0007]

Chem.

[0008] In the above Chemical Formula 1, R1 to R ,

[0010] , 19 , , , 18 , , are independently H, a substituted or unsubstituted alkyl having 1 to 4 carbon atoms, a substituted or unsubstituted alkenyl having 2 to 4 carbon atoms, a substituted or unsubstituted alkynyl having 2 to 4 carbon atoms, a substituted or unsubstituted alkoxy having 1 to 4 carbon atoms, halogen, hydroxy, aldehyde, carboxy, amino, nitro, or cyano, In the above Chemical Formula 1, TIFF2026516040000004.tif22170

[0009] In the above Chemical Formula 2, n is 4, 5 or 6, and R 11 , R 12 , R 13 , R 14 , R 15 , R 16 , R 17 , R 18 and R 19 are independently -OH, a substituted or unsubstituted alkoxy having 1 to 4 carbon atoms, or a substituted or unsubstituted alkylthio having 1 to 4 carbon atoms.

[0010] Another aspect is to provide a method for manufacturing the above preparation.

Means for Solving the Problems

[0011] One aspect provides a pharmaceutical preparation comprising a compound represented by the following Chemical Formula 1 or a pharmaceutically acceptable salt thereof, and a cyclodextrin derivative represented by the following Chemical Formula 2.

[0012]

Chemical Formula

[0013]

Chemical Formula

[0014] In Chemical Formula 1, R1 to R 10 are independently H, a substituted or unsubstituted alkyl having 1 to 4 carbon atoms, a substituted or unsubstituted alkenyl having 2 to 4 carbon atoms, a substituted or unsubstituted alkynyl having 2 to 4 carbon atoms, a substituted or unsubstituted alkoxy having 1 to 4 carbon atoms, halogen, hydroxy, aldehyde, carboxy, amino, nitro, or cyano. In Chemical Formula 1, TIFF2026516T040000007.tif22170

[0015] In Chemical Formula 2, n is 4, 5, or 6, and R[[ID=--33]] 11 , R 12 , R 13 , R 14 , R 15 , R 16 , R 17 , R 18 and R 19 are independently -OH, a substituted or unsubstituted alkoxy having 1 to 4 carbon atoms, or a substituted or unsubstituted alkylthio having 1 to 4 carbon atoms.

[0016] As used herein, the term "comprising" means "including" and is intended not to exclude the presence of additional components, unless otherwise indicated in the context, for example, except when all components total 100%.

[0017] In one embodiment, the compound represented by chemical formula 1 may mean lanosterol and its derivatives.

[0018] The term "lanosterol and its derivatives" as used herein may include one or more selected from the group consisting of lanosterol, inotodiol, trametenolic acid, ganodermadiol, and ganodermatriol.

[0019] The IUPAC name for "Inotodiol" is (3S,5R,10S,13R,14R,17R)-17-[(2S,3R)-3-hydroxy-6-methylhept-5-en-2-yl]-4,4,10,13,14-pentamethyl-2,3,5,6,7,11,12,15,16,17-decahydro-1H-cyclopenta[a]phenanthrene-3-ol The compound is ((3S,5R,10S,13R,14R,17R)-17-[(2S,3R)-3-hydroxy-6-methylhept-5-en-2-yl]-4,4,10,13,14-pentamethyl-2,3,5,6,7,11,12,15,16,17-decahydro-1H-cyclopenta[a]phenanthren-3-ol). The inotodiol is the main physiologically active component of Inonotus obliquus and can be chemically synthesized according to conventional methods, and may be produced as a pharmaceutically acceptable salt, or isolated and purified from Inonotus obliquus extract. The inotodiol compound can exist in the form of a pharmaceutically acceptable salt.

[0020] The term "pharmaceutically acceptable salt" as used herein includes all addition salts of acids or bases and their stereochemical isomers, and may, for example, be addition salts of organic or inorganic acids. The salts include, but are not limited to, any salt that maintains the activity of the parent compound in the target of administration and does not induce undesirable effects.

[0021] Such salts include inorganic and organic salts, for example, acetic acid, nitric acid, aspartic acid, sulfonic acid, sulfuric acid, maleic acid, glutamic acid, formic acid, succinic acid, phosphoric acid, phthalic acid, tannic acid, tartaric acid, hydrobromic acid, propionic acid, benzenesulfonic acid, benzoic acid, stearic acid, lactic acid, bicarbonate, bisulfate, tartaric acid, oxalic acid, butyric acid, calcium edetate, carbonic acid, chlorobenzoic acid, citric acid, edetic acid, toluenesulfonic acid, fumaric acid, glyceptic acid, esylic acid, pamoic acid, gluconic acid, methyl nitrate, malonic acid, hydrochloric acid, hydroiodic acid, hydroxynaphthoic acid, isethionic acid, lactobionic acid, mandelic acid, mucoic acid, These may be naphthalene sulfonic acid, muconic acid, p-nitromethanesulfonic acid, hexamic acid, pantothenic acid, monohydrogen phosphate, dihydrogen phosphate, salicylic acid, sulfamic acid, sulfanilic acid, or methanesulfonic acid.

[0022] Furthermore, the salt forms include alkali and alkaline earth metal salts such as ammonium salts, lithium salts, sodium salts, potassium salts, magnesium salts, and calcium salts; salts containing organic bases such as benzathine, N-methyl-D-glucamine, and hydravamin salts; and salts containing amino acids such as arginine and lysine. Moreover, the salt forms can be converted to free forms by treatment with a suitable base or acid.

[0023] As used herein, the term "inclusion" refers to a stoichiometric molecular phenomenon in which a guest molecule interacts with and is captured by the cavity of a cyclodextrin molecule.

[0024] In this specification, the term "cyclodextrin (CD)" refers to a cyclic compound linked by an α-(1→4)-glycosidic bond, with glucose as the basic unit. The cyclodextrin is a ring-shaped sugar microparticle having a cavity and can be classified into alpha (α), beta (β), and gamma (γ) types depending on its structure. The internal cavities of the cyclodextrin are 5.3 Å for α-cyclodextrin, 6.5 Å for β-cyclodextrin, and 8.3 Å for γ-cyclodextrin. The cyclodextrin has hydrophilic properties because a hydroxyl group is present on the outside of the ring, but exhibits hydrophobicity inside. A substance having a molecular structure suitable for the internal structure of the cyclodextrin can be encapsulated in the hydrophobic internal cavity of the cyclodextrin to form an inclusion compound. In one embodiment, the inotodiol may be encapsulated in the internal cavity of the cyclodextrin.

[0025] In one embodiment, the cyclodextrin derivative represented by chemical formula 2 may be gamma-cyclodextrin (γ-cyclodextrin) of the following chemical formula 3.

[0026] [ka]

[0027] In one embodiment, the pharmaceutical formulation may be a mixture of lanosterol, its derivatives or pharmaceutically acceptable salts thereof, and cyclodextrin in a molar ratio of 1:1 to 10. Specifically, the formulation may be a mixture of lanosterol, its derivatives, and cyclodextrin in molar ratios of 1:1 to 10, 1:1 to 9, 1:1 to 8, 1:1 to 7, 1:1 to 6, 1:1 to 5, 1:1 to 4, 1:1 to 3, or 1:1 to 2. For example, inotodiol and cyclodextrin may be mixed in a molar ratio of 1:3 to form an inclusion complex in which the inotodiol is encapsulated in the cavity of the cyclodextrin.

[0028] Furthermore, the pharmaceutical formulation may have increased water solubility of the hydrophobic lanosterol or its derivatives by encapsulating lanosterol or its derivatives in a cyclodextrin derivative. The pharmaceutical formulation according to one embodiment may increase the bioavailability and anti-inflammatory effect of lanosterol or its derivatives.

[0029] On the other hand, in one embodiment, the formulation may be a solid formulation or a liquid formulation.

[0030] The solid dosage form may be manufactured using any one selected from the group consisting of tablets, capsules, powders, dispersible granules, wafers (cachets), and suppositories, sustained-release formulations, and delayed-release formulations.

[0031] The liquid formulation may be manufactured from any one selected from the group consisting of solutions, suspensions, emulsions, injectable formulations, solutions or sprays for nasal, buccal, or sublingual administration, suitable aerosol formulations for inhalation, transdermal formulations, creams, lotions, and formulations that can be incorporated into transdermal patches.

[0032] In one embodiment, the formulation may further contain pharmaceutically acceptable additives.

[0033] The aforementioned additive may be one or more selected from the group consisting of diluents, binders, disintegrants, and lubricants.

[0034] The diluent may be one or more selected from the group consisting of lactose, cellulose powder, microcrystalline cellulose, silicified microcrystalline cellulose, starch, gelatinized starch, calcium carbonate, cyclodextrin, calcium sulfate, calcium silicate, magnesium carbonate, dicalcium phosphate, tricalcium phosphate, magnesium trisilicate, potassium chloride, sodium chloride, dibasic calcium phosphate dihydrate, tribasic calcium phosphate, kaolin, magnesium carbonate, magnesium oxide, mannitol, maltitol, sorbitol, xylitol, lactose, glucose, maltose, sucrose, glucose, dextrose, fructose, maltodextrin, dextrose, dextrin, and combinations thereof.

[0035] The binder may be one or more selected from the group consisting of povidone, hydroxypropylcellulose, hydroxypropylmethylcellulose, light anhydrous silicic acid, aluminum silicate, calcium silicate, calcium hydrogen phosphate, calcium carbonate, and combinations thereof.

[0036] The disintegrant may be one or more selected from the group consisting of croscarmellose sodium (CrosCMC-Na), carboxymethylcellulose, crospovidone (cross-linked polyvinylpyrrolidone), L-HPC (low-substituted hydroxypropylcellulose), starch, sodium carboxymethyl starch, sodium starch glycolate, partially hydrolyzed starch, and combinations thereof.

[0037] The lubricant may be one or more selected from the group consisting of magnesium stearate, fumaric acid, stearic acid, calcium stearate, sodium stearyl fumarate, polyethylene glycol, starch, talc, highly dispersed (colloidal) silica, magnesium oxide, magnesium carbonate, glyceryl behenate, glyceryl monostearate, silicon dioxide, calcium silicate, magnesium silicate, and combinations thereof.

[0038] The formulation may contain additional excipients commonly used in the art. Such additional excipients may include plasticizers, film-forming agents, colorants, anti-tacking agents, and / or pigments for coating the composition of the present invention.

[0039] Possible additional types of excipients include buffers, flavorings, sweeteners, antioxidants, and / or absorption enhancers.

[0040] The coating base for coating the aforementioned formulation may be one or more selected from the group consisting of polyvinylpyrrolidone (PVP), hydroxypropyl methylcellulose (HPMC), carboxymethylcellulose (sodium salt and calcium salt), ethylcellulose, methylcellulose, hydroxyethylcellulose, ethylhydroxyethylcellulose, hydroxypropylcellulose (HPC), L-HPC (low-substituted HPC), polyvinyl alcohol, polymers of acrylic acid and its salts, vinylpyrrolidone-vinyl acetate copolymer, gelatin, guar gum, partially hydrolyzed starch, alginic acid, xanthan gum, and combinations thereof.

[0041] The compound represented by formula 1 or a pharmaceutically acceptable salt thereof, and the inclusion compound comprising the cyclodextrin derivative represented by formula 2, can be purified as necessary by any method known to those skilled in the art. When such purification methods can be carried out, it should be noted that those skilled in the art are familiar with the purification methods. For example, in a multi-step synthesis that helps to arrive at a particular compound, the purification step can be carried out after all the synthesis steps, after several steps, at various points during the synthesis, and / or at the end of the synthesis. In some methods, one or more purification steps include techniques selected from the group consisting of silica gel column chromatography, C-18 reversed-phase column chromatography, gel filtration chromatography, HPLC (high-performance liquid chromatography), and LC (liquid chromatography).

[0042] Another embodiment provides a method for producing a formulation comprising the step of mixing a compound represented by the following formula 1 or a pharmaceutically acceptable salt thereof with a cyclodextrin derivative represented by the following formula 2.

[0043] [ka]

[0044] [ka]

[0045] In the above formula 1, R1 to R 10 These are independently H, C1-C4 substituted or unsubstituted alkyl, C2-C4 substituted or unsubstituted alkenyl, C2-C4 substituted or unsubstituted alkynyl, C1-C4 substituted or unsubstituted alkoxy, halogen, hydroxy, aldehyde, carboxy, amino, nitro, or cyano. In the above formula 1, TIFF2026516040000011.tif22170

[0046] In the above formula 2, n is 4, 5, or 6, and R 11 , R 12 , R 13 , R 14 , R 15 , R 16 , R 17 , R 18 and R 19 These are independently -OH, a C1-C4 substituted or unsubstituted alkoxy, or a C1-C4 substituted or unsubstituted alkylthio.

[0047] In one embodiment, the compound represented by chemical formula 1 may mean lanosterol and its derivatives. The lanosterol and its derivatives, their pharmaceutically acceptable salts, and cyclodextrins are as described above.

[0048] In one embodiment, the mixing step may involve mixing the compound represented by formula 1 or a pharmaceutically acceptable salt thereof with a cyclodextrin derivative represented by formula 2 in a molar ratio of 1:1 to 10.

[0049] The manufacturing method may include methods such as ultrasonic treatment, high-pressure homogenization, high-temperature treatment, freeze-drying (lyophilization method), kneading method, or co-evaporation method.

[0050] In one embodiment, the step of adding a surfactant to the mixture may further include, but is not limited to, Tween 20, Tween 80, or sucrose ester. The surfactant may be added at a concentration of 0.1 to 5%. [Effects of the Invention]

[0051] According to one embodiment of the pharmaceutical formulation, water solubility, stability in solution, and bioavailability are improved, leading to increased bioavailability and enhanced anti-inflammatory effects. [Brief explanation of the drawing]

[0052] [Figure 1] This diagram illustrates a method for producing a complex in which an inotodiol is encapsulated in a cyclodextrin. [Figure 2] This diagram shows the process of producing a complex in which an inotodiol is encapsulated in a cyclodextrin. [Figure 3] This graph shows the results of differential scanning calorimetry (DSC) analysis, confirming the presence or absence of inclusion complex formation between cyclodextrin and inotodiol. [Figure 3A]This graph shows the results of DSC analysis performed on β-cyclodextrin infused with inotodiol. [Figure 3B] This graph shows the results of DSC analysis performed on gamma-cyclodextrin infused with inotodiol. [Figure 4] This graph shows the formation of inclusion complexes based on the mixing ratio of inotodiol and cyclodextrin. [Figure 5] This graph shows the time-dependent degradation rate of the inotodiol and cyclodextrin inclusion complex. [Figure 6] The results of evaluating the anti-inflammatory efficacy of LPS using a mouse-derived macrophage cell line (Raw264.7) are shown. [Figure 6A] This photograph shows the expression levels of IL-6, IL-1β, and TNF-α in an inclusion complex of an inotodiol that is not encapsulated and an inclusion complex of an inotodiol and a cyclodextrin. [Figure 6B] This graph shows the relative expression levels of IL-6. [Figure 6C] This graph shows the relative expression levels of IL-1β. [Figure 6D] This graph shows the relative expression levels of TNF-α. [Figure 7] This graph shows the EC50 measurement results for unencapsulated inotodiol and inotodiol / γ-cyclodextrin inclusion complexes (inotodiol / γ-CD) in the human mast cell line LUVA cell. [Figure 8] This study confirmed the anti-inflammatory effect of an inotodiol and cyclodextrin inclusion complex (inotodiol / CD) in a systemic mouse model of sepsis. [Figure 8A] This is a schematic diagram illustrating the experimental process for confirming the anti-inflammatory effect in a systemic mouse model of sepsis. [Figure 8B] This graph shows the results of measuring body temperature in sepsis model mice after treating them with an inclusion complex of inotodiol and cyclodextrin following inflammation induction. [Figure 8C]This graph shows the results of examining the expression level of the inflammatory cytokine IL-1β after treating sepsis model mice with an inclusion complex of inotodiol and cyclodextrin following inflammation induction. [Figure 8D] This graph shows the results of examining the expression level of the inflammatory cytokine TNF-α after treating sepsis model mice with an inclusion complex of inotodiol and cyclodextrin following inflammation induction (naive (2 mice), sham (3 mice), Dexa 4 mpk in water (2 mice), G1: inotodiol 0.25 (ino-γCD 0.25 mpk in saline) (4 mice), G2: inotodiol 0.5 (ino-γCD 0.5 mpk in saline) (4 mice), G3: inotodiol 1 (ino 1 mpk in oil) (4 mice), G4: inotodiol 4 (ino 4 mpk in oil) (4 mice)). [Figure 9] This figure shows the results of comparing the in vivo bioavailability of unencapsulated inotodiol and inotodiol and cyclodextrin inclusion complexes according to the dosage. [Figure 9A] This graph shows the results of measuring and comparing the concentration of inotodiol in the serum of mice after oral administration of unencapsulated inotodiol (0.5 mpk) and an inclusion complex of inotodiol and cyclodextrin (0.5 mpk). [Figure 9B] This graph shows the results of measuring and comparing the concentration of inotodiol in the serum of mice after oral administration of unencapsulated inotodiol (1mpk) and an inclusion complex of inotodiol and cyclodextrin (1mpk). [Figure 9C] This graph shows the results of measuring and comparing the concentration of inotodiol in the serum of mice after oral administration of unencapsulated inotodiol (4mpk) and an inclusion complex of inotodiol and cyclodextrin (4mpk). Modes for carrying out the invention

[0053] The invention will be described in more detail through the following examples. However, these examples are for illustrative purposes only, and the scope of the invention is not limited to these examples.

[0054] Example 1. Preparation of an inclusion complex of inotodiol and cyclodextrin

[0055] 1.1 Preparation of inotodiol and γ-cyclodextrin inclusion complex

[0056] A complex in which inotodiol is encapsulated in γ-cyclodextrin was prepared by the method shown in Figures 1 and 2.

[0057] Specifically, γ-cyclodextrin was first dissolved in 9 ml of DDW in proportion to the molar ratio (1:1 to 1:10) of inotodiol. Then, while sonicating (primarily using a microtip: 30% amp, 10 minutes; secondaryly using a flat tip: 50% amp, 5 minutes), 10 mg of inotodiol dissolved in 1 ml of EtOH was gradually injected at 15-second intervals. Next, the solution was incubated in suspension at 25°C for 24 hours and dried in a freeze-dryer (-45°C, 2 to 3 days) to produce the inclusion complex. Furthermore, after sonication, various surfactants were added to the suspension (solution state) as auxiliary agents from 0.1 to 5% to improve the stability of the suspension.

[0058] 1.2 Confirmation of the formation of inotodiol and cyclodextrin inclusion complexes depending on the type of cyclodextrin

[0059] Differential scanning calorimetry (DSC) analysis was used to confirm the presence or absence of inclusion complex (inotodiol / CD) formation produced in Example 1.

[0060] Specifically, 2 mg of each dried sample was placed in a sealed aluminum pan, and the pan was heated under a nitrogen atmosphere at a rate of 10°C / min from 25°C to 310°C. An empty pan was used as a reference. Figure 3 shows a graph confirming the presence or absence of inotodiol / cyclodextrin inclusion complex (inotodiol / CD) formation by DSC analysis.

[0061] Figure 3A is a graph showing the results of DSC analysis performed on β-cyclodextrin (β-CD) with inotodiol encapsulated, and Figure 3B is a graph showing the results of DSC analysis performed on gamma-cyclodextrin (γ-CD) with inotodiol encapsulated.

[0062] As shown in Figures 3A and 3B, the melting peak (192°C) of the inotodiol lipid crystal was detected in the inclusion of inotodiol and β-cyclodextrin (β-CD), but not in the inclusion of inotodiol and γ-cyclodextrin (γ-CD).

[0063] This means that cholesterol, which has a similar structure to inotodiol, forms an inclusion complex with β-cyclodextrin, but inotodiol has two methyl groups on the 4th carbon of the 1st ring and one methyl group on the 14th carbon between the 3rd and 4th rings, and overall occupies a larger volume than cholesterol, and thus forms an inclusion complex only with γ-cyclodextrin, which has a larger internal hydrophobic cavity. In other words, the above result means that inotodiol forms an inclusion complex only with γ-cyclodextrin, which has a larger internal hydrophobic cavity.

[0064] 1.3 Confirmation of inclusion complex formation based on the mixing ratio of inotodiol and cyclodextrin

[0065] The formation of inotodiol / CD inclusion complexes was investigated based on the mixing ratio of inotodiol and cyclodextrin. The results are shown in Table 1 and Figure 4.

[0066] Table 1 shows the results of whether or not inclusion formation occurs when inotodiol and cyclodextrin are mixed in a molar ratio of 1:1 to 5, and Figure 4 is a graph showing the formation of inclusion complexes depending on the mixing ratio (molar ratio) of inotodiol and cyclodextrin.

[0067] As shown in Table 1 and Figure 4, when inotodiol and γ-cyclodextrin were mixed in a 1:3 to 5 molar ratio, it was confirmed that the inotodiol was completely encapsulated by the γ-cyclodextrin.

[0068] [Table 1]

[0069] Experimental Example 1. Confirmation of the degradation rate of the inclusion complex (inotodiol / CD).

[0070] To confirm the efficiency of inotodiol release from the inclusion complex (inotodiol / CD), the degradation rate of the inclusion complex (inotodiol / CD) produced from Example 1 was examined.

[0071] Specifically, γ-cyclodextrin (γ-CD) and the inclusion complex from Example 1 (inotodiol / CD) were each dissolved in PBS (pH 7.5) to a concentration of 0.1 mg / ml. Then, 0.25 U / ml of porcine pancreatic α-amylase (PPA), which has the same drug active site as human and mouse pancreatic α-amylase and exhibits high similarity of 87.1% and 85.5%, respectively, was used to treat the mixture, and the reaction was carried out at 37°C and 100 rpm for 4 hours. The degree of hydrolysis was measured by the Copper bicinchoninate (CBC) method using maltose, a reducing sugar that is a reaction degradation product of α-amylase. The results are shown in Table 2 and Figure 5.

[0072] Table 2 shows the numerical degradation rate of the inclusion complex (inotodiol / CD) over time, and Figure 5 is a graph showing the degradation rate of the inotodiol and cyclodextrin inclusion complex (inotodiol / CD) over time.

[0073] [Table 2]

[0074] As shown in Table 2 and Figure 5, the degradation of the inclusion complex by PPA showed a relatively similar degradation rate to that of γ-cyclodextrin, and both showed a rapid degradation rate from the initial step up to 90 minutes. This means that the ring-open reaction of γ-cyclodextrin occurs early, allowing inotodiol to be released immediately. Furthermore, the inclusion complex of inotodiol and cyclodextrin (inotodiol / CD), which has the same drug active site as human and mouse pancreatic α-amylase (amylase) and exhibits high similarity of 87.1% and 85.5% respectively, showed a high degradation rate under PPA treatment. This means that the inclusion complex (inotodiol / CD) can be applied to humans and mice.

[0075] Experimental Example 2. Comparison of the in-solution stability of unencapsulated inotodiol and inotodiol complex (inotodiol / CD).

[0076] The solution stability of the inclusion complex (inotodiol / CD) produced from Example 1 above was compared with that of uninclusioned inotodiol.

[0077] Specifically, unencapsulated inotodiol or an inclusion complex of inotodiol and cyclodextrin (inotodiol / CD) was mixed with a prepared auxiliary agent. The mixture was prepared at concentrations between 100 and 15,000 ppm by weight of inotodiol and treated at 50°C for 30 minutes using a sonic bath. After that, the mixture was left at room temperature for 24 hours, and stability was determined by visually observing whether precipitation occurred.

[0078] As a result, as shown in Table 3, we confirmed that the inclusion complex (inotodiol / CD) is more stable in solution than the uninclusioned inotodiol.

[0079] [Table 3]

[0080] Experimental Example 3. Comparison of the physiological activity of inotodiol and inotodiol and γ-cyclodextrin inclusion complexes.

[0081] 3.1 Comparison of anti-inflammatory effects in mouse-derived macrophage cell line (Raw264.7)

[0082] The anti-inflammatory effects of the inclusion complex (inotodiol / CD) from Example 1 and uninclusioned inotodiol were compared in a mouse-derived macrophage cell line (Raw264.7).

[0083] Specifically, Raw264.7 is 0.8 × 10 6 Cells were seeded in 96-well plates and cultured overnight. Then, inotodiol and inotodiol-cyclodextrin inclusion complexes were pretreated at different concentrations for 2 hours. After treating with lipopolysaccharide (LPS) at a concentration of 0.1 μg / ml and culturing for 4 hours to induce inflammation, RNA was extracted, and mRNA expression of IL-6, IL-1β, and TNF-α was confirmed by reverse transcription polymerase chain reaction (RT-PCR). The results are shown in Figure 6.

[0084] Figure 6A is an electrophoresis image showing the mRNA expression levels of IL-6, IL-1β, and TNF-α after treatment with unencapsulated inotodiol and inotodiol and cyclodextrin inclusion complexes. Figures 6B to 6D are graphs showing the numerical quantification of the bands in the electrophoresis images using GAPDH. Figure 6B is a graph showing the relative mRNA expression levels of IL-6, Figure 6C is a graph showing the relative mRNA expression levels of IL-1β, and Figure 6D is a graph showing the relative mRNA expression levels of TNF-α.

[0085] As shown in Figures 6A to 6D, in the case of unencapsulated inotodiol, the mRNA expression levels of IL-6 and TNF-α were significantly reduced at a 5 μg / ml treatment. In the case of the inotodiol and γ-cyclodextrin inclusion complex (inotodiol / γ-CD), the mRNA expression levels of IL-1β and TNF-α were significantly reduced at all concentrations. This means that the inotodiol and γ-cyclodextrin inclusion complex (inotodiol / γ-CD) exhibits more effective anti-inflammatory efficacy compared to unencapsulated inotodiol.

[0086] 3.2 Comparison of the degranulation inhibitory effect on mast cells in LUVA cells

[0087] The degranulation inhibitory effect of unencapsulated inotodiol and the inotodiol and γ-cyclodextrin inclusion complex (inotodiol / γ-CD) from Example 1 was compared in human mast cell line (LUVA cell).

[0088] Specifically, LUVA cells are 1 x 10 5Cells were seeded in a 96-well plate and cultured for 1 hour. Then, inotodiol and inotodiol-cyclodextrin inclusion complexes were treated at different concentrations. After treating with IgE at a concentration of 1 μg / ml, the cells were cultured overnight. After removing the supernatant, 50 μl of Tyrode's buffer (130 mM NaCl, 5 mM KCl, 1 mM MgCl2, 1.4 mM CaCl2.2H2O, 10 mM hepes, 5.6 mM glucose, 0.1% BSA) was dispensed into each well. Inotodiol and inotodiol-cyclodextrin inclusion complexes were treated at the same concentrations as before. 20 μl of 2 μM A23187 and 10 μg / ml anti-human IgE antibody complexes were dispensed into each well, and the cells were cultured for 2 hours. Collect 50 μl of the supernatant, mix with 50 μl of 2 mM p-nitrophenyl N-acetyl-bD-glucosamine, and culture for 2 hours. Measure the absorbance at 405 nm. Add 75 μl of 0.1 mM Triton X-100 to the cells remaining on the plate, store at 4°C for 40 minutes to lyse, then collect 50 μl, mix with 50 μl of 2 mM p-nitrophenyl N-acetyl-bD-glucosamine, and culture for 1 hour. Measure the absorbance at 405 nm. Substitute the absorbances of the supernatant and cell lysate into the following formula to calculate the degranulation rate. Compared to the untreated group, determine the drug concentration (EC2) required to reduce the degranulation rate by half. 50 We compared the two, and the results are shown in Figure 7.

[0089]

number

[0090] Figure 7 shows the rate of degranulation and EC2 when LUVA cells are treated with inotodiol that is not encapsulated and with an inclusion complex of inotodiol and γ-cyclodextrin (inotodiol / γ-CD). 50のThis is a graph showing the measurement results.

[0091] As shown in Figure 7, we confirmed that the inclusion complex of inotodiol and γ-cyclodextrin (inotodiol / γ-CD) effectively suppresses mast cell degranulation at relatively lower concentrations compared to uninclusioned inotodiol.

[0092] Furthermore, we confirmed that the required concentration of a drug that can suppress mast cell degranulation by more than 50% when inflammatory stimuli are applied using two types of calcium ionophore + PMA is approximately half as low for the inclusion complex of inotodiol and γ-cyclodextrin (inotodiol / γ-CD) compared to uninclusioned inotodiol.

[0093] 3.3 Comparison of anti-inflammatory effects in a systemic mouse model of sepsis

[0094] The anti-inflammatory effect of the inotodiol and cyclodextrin inclusion complex (inotodiol / CD) described in Example 1 above was confirmed in a systemic mouse model of sepsis.

[0095] Specifically, the effect of the inclusion complex (inotodiol / CD) from Example 1 on clinical symptoms caused by macrophages activated by LPS was confirmed in a systemic mouse model of sepsis. As shown in Figure 8A, mice were pretreated with inotodiol and γ-cyclodextrin mixed in a 1:3 ratio for four consecutive days, and then LPS (5 mg / kg) was injected intraperitoneally one hour after the final treatment.

[0096] Meanwhile, the body temperature of the mice was measured by rectal temperature every two hours for four hours after inflammation was induced with LPS. In addition, the inflammatory cytokines TNF-α and IL-1β produced by macrophages activated by LPS were measured by ELISA in mouse blood samples taken two and six hours, respectively, after inflammation was induced with LPS.

[0097] The anti-inflammatory effects of the inotodiol and cyclodextrin inclusion complex (inotodiol / CD), confirmed during the above experimental process, are shown in Figures 8B to 8D.

[0098] Figure 8B is a graph showing the results of measuring body temperature after treating sepsis model mice with an inclusion complex of inotodiol and cyclodextrin following inflammation induction. Figure 8C is a graph showing the results of confirming the expression level of the inflammatory cytokine IL-1β after treating sepsis model mice with an inclusion complex of inotodiol and cyclodextrin following inflammation induction. Figure 8D is a graph showing the results of confirming the expression level of the inflammatory cytokine TNF-α after treating sepsis model mice with an inclusion complex of inotodiol and cyclodextrin following inflammation induction.

[0099] In Figure 8, the Naive group is the group that received only the same amount of vehicle solution (2mice), the Sham group is the group that received only LPS (3mice), and the Dexa group is the group that received 4.0 mg / kg (mpk) of dexamethasone as a positive control (2mice). Group G1 is the group treated with 0.25 mg / kg (mpk) of inotodiol / γCD dissolved in physiological saline (4mice), Group G2 is the group treated with 0.5 mg / kg (mpk) of inotodiol / γCD dissolved in physiological saline (4mice), Group G3 is the group treated with 1 mg / kg (mpk) of inotodiol dissolved in oil (4mice), and Group G4 is the group treated with 4 mg / kg (mpk) of inotodiol dissolved in oil (4mice).

[0100] Data are presented as the mean ± SD of mice in each group. Statistical significance was confirmed by Bonferroni post-hoc tests according to one-way analysis of variance (ANOVA), and compared to LPS monotherapy, the results were as follows: p<0.005(*), p<0.05(#).

[0101] As shown in Figure 8B, the body temperature of the mice was lowest in the Sham group treated with LPS only and highest in the Naive group administered only the same amount of vehicle solution. The Dexa group and G1-G4 groups showed a rectal temperature distribution between 35 and 36°C.

[0102] This result indicates that, compared to the Naive group which showed normal body temperature, the Sham group experienced a significant decrease in body temperature due to anaphylaxis, but the administration of inotodiol reduced the anaphylactic reaction, preventing or restoring the decrease in body temperature.

[0103] Furthermore, as shown in Figures 8C and 8D, we confirmed that treatment with an inotodiol and cyclodextrin inclusion complex (inotodiol / CD) resulted in a decrease in the expression of inflammatory cytokines (IL-1β, TNF-α).

[0104] In particular, regarding IL-1β expression levels (Figure 8C), we confirmed that not only was the expression level of the inflammatory cytokine (IL-1β) significantly reduced compared to the group treated with LPS alone, but the expression level of IL-β was also reduced more when treated with an inotodiol and cyclodextrin inclusion complex (inotodiol / CD) than when treated with inotodiol dissolved in oil.

[0105] This means that the inclusion complex not only has an anti-inflammatory effect, but also exhibits a more pronounced anti-inflammatory effect than the uninclusioned inotodiol.

[0106] Experimental Example 4. Comparison of Bioavailability of Inotodiol and its Inclusion Complex (inotodiol / CD)

[0107] The bioavailability between the inclusion complex (inotodiol / CD) of Example 1 and inotodiol not encapsulated in cyclodextrin was compared at each concentration.

[0108] Specifically, unencapsulated inotodiol was dissolved in 0.9% physiological saline containing 1% Tween 80, and the inclusion complex (inotodiol / CD) was dissolved only in 0.9% physiological saline. Then, formulations corresponding to 0.5, 1, and 4 mg / kg (mpk) were orally administered to ICR male mice (30 g) at a dose of 200 μl. Blood samples were collected at regular intervals (0.5, 1, 2, 3, and 6 h) after oral administration. The collected blood samples were centrifuged to separate the plasma, and the concentration of inotodiol in the plasma was measured by LC-MS analysis to investigate whether bioavailability had improved. The results are shown in Figure 9.

[0109] Figure 9A is a graph comparing the concentrations of inotodiol in serum after oral administration of unencapsulated inotodiol (0.5 mpk) and an inotodiol and cyclodextrin inclusion complex (0.5 mpk) to mice. Figure 9B is a graph comparing the concentrations of inotodiol in serum after oral administration of unencapsulated inotodiol (1 mpk) and an inotodiol and cyclodextrin inclusion complex (1 mpk) to mice. Figure 9C is a graph comparing the concentrations of inotodiol in serum after oral administration of unencapsulated inotodiol (4 mpk) and an inotodiol and cyclodextrin inclusion complex (4 mpk) to mice.

[0110] As shown in Figures 9A to 9C, the inclusion complex (inotodiol / CD) was demonstrated to have higher bioavailability than uninclusioned inotodiol. Specifically, the inotodiol concentration in mouse serum was generally higher for the inclusion complex (inotodiol / CD) than for uninclusioned inotodiol. This means that the inclusion complex (inotodiol / CD) has higher bioavailability than uninclusioned inotodiol.

Claims

1. The compound represented by the following chemical formula 1 or a pharmaceutically acceptable salt thereof, and Pharmaceutical preparations containing cyclodextrin derivatives represented by the following chemical formula 2: 【Chemistry 1】 【Chemistry 2】 In Chemical Formula 1, R 1 to R 10 are independently H, C 1 to C 4 substituted or unsubstituted alkyl, C 2 to C 4 substituted or unsubstituted alkenyl, C 2 to C 4 substituted or unsubstituted alkynyl, C 1 to C 4 substituted or unsubstituted alkoxy, halogen, hydroxy, aldehyde, carboxy, amino, nitro, or cyano, In the above formula 1, 【change】 In the above formula 2, n is 4, 5, or 6, and R 11 , R 12 , R 13 , R 14 , R 15 , R 16 , R 17 , R 18 , and R 19 These are independently -OH, C 1 ~C 4 Substituted or unsubstituted alkoxy, or C 1 ~C 4 It is a substituted or unsubstituted alkylthio.

2. The pharmaceutical formulation according to claim 1, wherein the compound represented by chemical formula 1 or a pharmaceutically acceptable salt thereof is encapsulated within the cyclodextrin derivative represented by chemical formula 2.

3. The pharmaceutical preparation according to claim 1, wherein the compound represented by chemical formula 1 and the cyclodextrin derivative represented by chemical formula 2 are mixed in a molar ratio of 1:1 to 10.

4. The pharmaceutical preparation according to claim 1, wherein the compound represented by the chemical formula 1 comprises one or more selected from the group consisting of lanosterol, inotodiol, trametenolic acid, ganodermadiol, and ganodermatriol.

5. The pharmaceutical preparation according to claim 1, wherein the cyclodextrin derivative represented by the above chemical formula 2 is gamma-cyclodextrin (γ-cyclodextrin) of the following chemical formula 3. 【Transformation 3】

6. The pharmaceutical preparation according to claim 1, wherein the preparation is a solid preparation or a liquid preparation.

7. The pharmaceutical preparation according to claim 6, wherein the solid preparation is manufactured by any one selected from the group consisting of tablets, capsules, powders, dispersible granules, wafers (cachet), and suppositories, sustained-release preparations and delayed-release preparations.

8. The pharmaceutical formulation according to claim 6, wherein the liquid formulation is manufactured from one selected from the group consisting of a solution, suspension, emulsion, injectable formulation, solution or spray for nasal, buccal, or sublingual administration, suitable aerosol formulation for inhalation, transdermal formulation, cream, lotion, and formulation that can be incorporated into a transdermal patch.

9. The pharmaceutical formulation according to claim 1, further comprising pharmaceutically acceptable additives.

10. The pharmaceutical preparation according to claim 9, wherein the additive is one or more selected from the group consisting of diluents, binders, disintegrants, and lubricants.

11. The pharmaceutical preparation according to claim 10, wherein the diluent is one or more selected from the group consisting of lactose, cellulose powder, microcrystalline cellulose, silicified microcrystalline cellulose, starch, gelatinized starch, calcium carbonate, cyclodextrin, calcium sulfate, calcium silicate, magnesium carbonate, dicalcium phosphate, tricalcium phosphate, magnesium trisilicate, potassium chloride, sodium chloride, dibasic calcium phosphate dihydrate, tribasic calcium phosphate, kaolin, magnesium carbonate, magnesium oxide, mannitol, maltitol, sorbitol, xylitol, lactose, glucose, maltose, sucrose, glucose, dextrose, fructose, maltodextrin, dextrose, dextrin, and combinations thereof.

12. The pharmaceutical formulation according to claim 10, wherein the binder is one or more selected from the group consisting of povidone, hydroxypropylcellulose, hydroxypropylmethylcellulose, light anhydrous silicic acid, aluminum silicate, calcium silicate, calcium hydrogen phosphate, calcium carbonate, and combinations thereof.

13. The pharmaceutical formulation according to claim 10, wherein the disintegrant is one or more selected from the group consisting of croscarmellose sodium (CrosCMC-Na), carboxymethylcellulose, crospovidone (cross-linked polyvinylpyrrolidone), L-HPC (low-substituted hydroxypropylcellulose), starch, sodium carboxymethyl starch, sodium starch glycolate, partially hydrolyzed starch, and combinations thereof.

14. The pharmaceutical formulation according to claim 10, wherein the lubricant is one or more selected from the group consisting of magnesium stearate, fumaric acid, stearic acid, calcium stearate, sodium stearyl fumarate, polyethylene glycol, starch, talc, highly dispersed (colloidal) silica, magnesium oxide, magnesium carbonate, glyceryl behenate, glyceryl monostearate, silicon dioxide, calcium silicate, magnesium silicate, and combinations thereof.

15. The compound represented by the following chemical formula 1 or a pharmaceutically acceptable salt thereof, and A method for producing a pharmaceutical product, comprising the step of mixing a cyclodextrin derivative represented by the following chemical formula 2: 【Chemistry 1】 【Chemistry 2】 In the above formula 1, R 1 ~R 10 H and C are independent of each other. 1 ~C 4 Substituted or unsubstituted alkyl groups, C 2 ~C 4 Substituted or unsubstituted alkenyl, C 2 ~C 4 Substituted or unsubstituted alkynyl, C 1 ~C 4 The substituted or unsubstituted alkoxy, halogen, hydroxy, aldehyde, carboxy, amino, nitro, or cyano are In the above formula 1 【change】 In the above formula 2, n is 4, 5, or 6, and R 11 , R 12 , R 13 , R 14 , R 15 , R 16 , R 17 , R 18 , and R 19 These are independently -OH, C 1 ~C 4 Substituted or unsubstituted alkoxy, or C 1 ~C 4 It is a substituted or unsubstituted alkylthio.

16. The method for producing a formulation according to claim 15, wherein the mixing step involves mixing a compound represented by chemical formula 1 or a pharmaceutically acceptable salt thereof and a cyclodextrin derivative represented by chemical formula 2 in a molar ratio of 1:1 to 10.

17. A method for producing a pharmaceutical product according to claim 16, further comprising the step of adding a surfactant to the mixture.

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