WO2023048453A1

WO2023048453A1 - Nanoparticles comprising drug dimers, and use thereof

Info

Publication number: WO2023048453A1
Application number: PCT/KR2022/014034
Authority: WO
Inventors: 황도원; 기영욱; 유동혁; 김성한
Original assignee: (주) 테라베스트
Priority date: 2021-09-27
Filing date: 2022-09-20
Publication date: 2023-03-30
Also published as: KR20230044603A

Abstract

The present invention relates to nanoparticles comprising a drug dimer, and a use thereof. In addition, the nanoparticles may further comprise fucoidan. The nanoparticles comprising an obeticholic acid dimer compound can be used for treating a liver disease. The nanoparticles comprising a drug dimer of the present invention can increase a drug content and enhance drug dispersibility. In addition, the nanoparticles have increased in targeting efficiency. Therefore, an effective pharmacological effect can be obtained by using a smaller amount of a drug, and thus commercial applicability is excellent.

Description

Nanoparticles Containing Drug Dimers and Uses Thereof

The present invention relates to nanoparticles comprising drug dimers and uses thereof. More specifically, it relates to nanoparticles comprising an obeticholic acid dimer compound and its use for the treatment of liver diseases.

In the case of hydrophobic drugs, despite excellent pharmacological effects, drug delivery is difficult. Therefore, in order to increase the utilization of hydrophobic drugs, many studies on drug delivery have been conducted. In particular, polymeric nanoparticles have been used for drug delivery systems to increase the low solubility of hydrophobic drugs and the circulation rate in the body, but have limitations in that the manufacturing method is difficult and the drug content is low. To overcome this limitation, a dimeric compound is synthesized by attaching hydrophobic drug monomers to both sides of the linker, and nanoparticles having a high drug content are prepared through self-assembly (Milena Menozzi et al. , J Pharm Sci. , 73(6) 766-770, 1984). However, nanoparticles containing such a dimer compound have disadvantages in that stability and particle size are not homogeneous.

On the other hand, obeticholic acid (OCA, 6-ethylchenodeoxycholic acid, or 6α-ECDCA), a bile acid derivative, shows a strong Farnesoid X Receptor (FXR) activating action, and thus has the potential to treat FXR-mediated diseases. FXR is a nuclear receptor that acts as a bile acid sensor to control bile acid homeostasis, is expressed in various organs, and is involved in biological processes including liver disease, lung disease, kidney disease, intestinal disease, and heart disease, glucose metabolism, insulin metabolism, and lipid metabolism. It was found to be involved in the regulation of the process (Korean Patent Publication No. 2018-0134405).

Accordingly, the present inventors studied to develop a drug dimer having excellent efficacy in treating FXR-mediated diseases, particularly liver diseases, and as a result, prepared a novel obeticholic acid dimer, increased the stability of nanoparticles containing it, A method capable of producing a homogeneous particle size was developed. In addition, the present invention was completed by confirming that when a linker that is degraded in a specific environment such as in the presence of GSH (Glutathione) or hydrogen peroxide is used, a drug monomer is released under a specific condition to exhibit a more effective disease treatment effect.

In order to achieve the above object, one aspect of the present invention provides a novel obeticholic acid dimer compound, a solvate thereof, or a pharmaceutically acceptable salt thereof, and nanoparticles comprising the same.

One aspect of the present invention provides nanoparticles containing the obeticholic acid dimer and fucoidan.

One aspect of the present invention provides a pharmaceutical composition comprising the nanoparticles as an active ingredient.

One aspect of the present invention provides a method for treating liver disease comprising administering the pharmaceutical composition to a subject suffering from liver disease.

One aspect of the present invention provides the use of the obeticholic acid dimer compound, a solvate thereof, or a pharmaceutically acceptable salt for use in preventing or treating liver disease.

One aspect of the present invention provides a use of the obeticholic acid dimer compound, a solvate thereof, or a pharmaceutically acceptable salt for preparing a medicament used for preventing or treating liver disease.

Nanoparticles containing obeticholic acid dimer and fucoidan, which are one embodiment of the present invention, can increase the drug content and improve the dispersibility of the drug. In addition, the nanoparticles have increased targeting efficiency. Therefore, effective pharmacological effects can be obtained using fewer drugs, and drug toxicity can be remarkably reduced. Therefore, the nanoparticles have excellent commercial applicability.

1 shows the ¹ H NMR spectrum of obeticholic acid (OCA).

Figure 2 shows the ¹ H NMR spectrum of obeticholic acid dimer (ssOCA-A) compound.

3a is a graph obtained by analyzing the size and distribution of nanoparticles of obeticholic acid dimer (ssOCA-A) with a particle size analyzer.

Figure 3b is a SEM image of obeticholic acid dimer (ssOCA-A) nanoparticles.

FIG. 4 is an evaluation result of the liver cirrhosis treatment efficacy of obeticholic acid dimer (ssOCA-A) nanoparticles, and confirms the degree of fibrosis by staining the collagen fiber level through Sirius red staining.

5 is an evaluation result of liver cirrhosis treatment efficacy of obeticholic acid dimer (ssOCA-A) nanoparticles, confirming the expression level of TGF-β inducing liver fibrosis.

6 is an evaluation result of the liver cirrhosis treatment efficacy of obeticholic acid dimer (ssOCA-A) nanoparticles, confirming the expression level of α-SMA, an activated hepatic stellate cell marker.

7 shows a ¹ H NMR spectrum of obeticholic acid dimer (ssOCA-E) compound.

8a is a graph obtained by analyzing the size and distribution of nanoparticles of obeticholic acid dimer (ssOCA-E) with a particle size analyzer.

8B is a SEM image of obeticholic acid dimer (ssOCA-E) nanoparticles.

9a is a graph obtained by analyzing the size and distribution of fucoidan-containing obeticholic acid dimer (ssOCA-E) nanoparticles with a particle size analyzer.

Figure 9b is a SEM image of obeticholic acid dimer (ssOCA-E) nanoparticles containing fucoidan.

10a to 10n show the results of hematological analysis as one item of the evaluation of the treatment efficacy of obeticholic acid dimer (ssOCA-E) nanoparticles containing fucoidan. Specifically, the results of measuring AST, triglyceride, GGT, ALT, uric acid, LDL, total protein, cholesterol, HDL, total bilirubin, albumin, total bile acid, glucose, and ALP levels in serum isolated from blood are shown.

Figure 11a is one item of the evaluation of the efficacy of non-alcoholic fatty liver treatment of obeticholic acid dimer (ssOCA-E) nanoparticles containing fucoidan, and the degree of fibrosis of liver tissue through collagen fiber staining by Mason's trichrome staining method It shows the confirmed result. Here, Normal is the normal group, Sham (NASH) group is the non-alcoholic fatty liver (NASH) model, non-drug-untreated positive control group, NASH + OCA is the non-alcoholic fatty liver model, the experimental group in which OCA is administered. , NASH + ssOCA-E Nanogel (P.O.) is an experimental group in which obeticholic acid dimer (ssOCA-E) nanoparticles containing fucoidan are orally administered to a non-alcoholic fatty liver model, NASH + ssOCA-E Nanogel (I.V.) is It shows an experimental group in which obeticholic acid dimer (ssOCA-E) nanoparticles containing fucoidan were intravenously administered to a non-alcoholic fatty liver model.

Figure 11b is one item of the evaluation of the efficacy of non-alcoholic fatty liver treatment of obeticholic acid dimer (ssOCA-E) nanoparticles containing fucoidan, and the degree of fibrosis of liver tissue through collagen fiber staining by Mason's trichrome staining method It is a graph showing the checked result numerically.

Figure 12 shows the result of confirming the degree of expression of TGF-β as one item of evaluation of the efficacy of non-alcoholic fatty liver treatment of obeticholic acid dimer (ssOCA-E) nanoparticles containing fucoidan.

Figure 13 shows the result of confirming the degree of α-SMA expression as one item of the evaluation of the efficacy of non-alcoholic fatty liver treatment of obeticholic acid dimer (ssOCA-E) nanoparticles containing fucoidan.

14 shows a ¹ H NMR spectrum of obeticholic acid dimer (sOCA-E) compound.

15a is a graph obtained by analyzing the size and distribution of fucoidan-containing obeticholic acid dimer (sOCA-E) nanoparticles with a particle size analyzer.

15b is a SEM image of obeticholic acid dimer (sOCA-E) nanoparticles containing fucoidan.

16a to 16n show the results of hematological analysis as one item of the evaluation of the treatment efficacy of obeticholic acid dimer (sOCA-E) nanoparticles containing fucoidan. Specifically, the results of measuring AST, cholesterol, GGT, ALT, ALP, LDL, total protein, triglyceride, HDL, total bilirubin, glucose, total bile acid, uric acid, and albumin levels in serum isolated from blood are shown.

Figure 17a is one item of the evaluation of the efficacy of non-alcoholic fatty liver treatment of obeticholic acid dimer (sOCA-E) nanoparticles containing fucoidan, and the degree of fibrosis of liver tissue through collagen fiber staining by Mason's trichrome staining method It shows the confirmed result. Here, Normal is the normal group, Sham (NASH) group is the non-alcoholic fatty liver (NASH) model, non-drug-untreated positive control group, NASH + OCA is the non-alcoholic fatty liver model, the experimental group in which OCA is administered. , NASH + sOCA-E Nanogel (P.O.) is an experimental group in which obeticholic acid dimer (sOCA-E) nanoparticles containing fucoidan are orally administered to a non-alcoholic fatty liver model, NASH + sOCA-E Nanogel (I.V.) is It shows an experimental group in which obeticholic acid dimer (sOCA-E) nanoparticles containing fucoidan were intravenously administered to a non-alcoholic fatty liver model.

Figure 17b is an item for evaluating the efficacy of non-alcoholic fatty liver treatment of obeticholic acid dimer (sOCA-E) nanoparticles containing fucoidan, and the degree of fibrosis of liver tissue through collagen fiber staining was measured by Mason's trichrome staining method. It is a graph showing the checked result numerically.

Figure 18 shows the result of confirming the degree of expression of TGF-β as one item of evaluation of the efficacy of non-alcoholic fatty liver treatment of fucoidan-containing obeticholic acid dimer (sOCA-E) nanoparticles.

Figure 19 shows the results of confirming the degree of α-SMA expression as one item of the evaluation of the efficacy of non-alcoholic fatty liver treatment of obeticholic acid dimer (sOCA-E) nanoparticles containing fucoidan.

20 shows ^{a 1} H NMR spectrum of BARP ((4-(5-(hydroxymethyl)-5-methyl-1,3,2-dioxaborinan-2-yl)phenyl)methanol) compound.

21 shows a ¹ H NMR spectrum of obeticholic acid dimer (BOCA-E) compound.

22a is a graph obtained by analyzing the size and distribution of fucoidan-containing obeticholic acid dimer (BOCA-E) nanoparticles using a particle size analyzer.

22b is a SEM image of obeticholic acid dimer (BOCA-E) nanoparticles containing fucoidan.

As used herein, the term "solvate" refers to a compound solvated in an organic or inorganic solvent. The solvate is, for example, a hydrate.

As used herein, the term "salt" refers to inorganic and organic acid addition salts of a compound. The pharmaceutically acceptable salt may be a salt that does not cause serious irritation to the organism to which the compound is administered and does not impair the biological activity and physical properties of the compound. The inorganic acid salt may be a hydrochloride, bromate, phosphate, sulfate, or bisulfate salt. The organic acid salt is formate, acetate, propionate, lactate, oxalate, tartrate, malate, maleate, citrate, fumarate, besylate, camsylate, edisyl salt, trichloroacetic acid, trifluoroacetate , benzoate, gluconate, methanesulfonate, glycolate, succinate, 4-toluenesulfonate, galacturonate, embonate, glutamate, ethanesulfonate, benzenesulfonate, p-toluenesulfonate, or aspartate may be an acid salt. The metal salt may be a calcium salt, sodium salt, magnesium salt, strontium salt, or potassium salt.

As used herein, the term "fucoidan" is a sulfated polysaccharide having a sticky viscous structure, and is generally a component contained in brown algae such as wakame and kelp. It is a substance in which a basic sugar and a sulfate group are combined. Fucoidan is known to have various physiological and biological activities such as antioxidants, anticoagulants, anticancer agents, and antibiotics.

As used herein, the term "fucoidan-containing nanoparticles" means that fucoidan is present in the nanoparticles. The term may be expressed as nanoparticles coated with fucoidan. In addition, the term "nanoparticles containing drug dimer and fucoidan" refers to nanoparticles formed by aggregation of fucoidan and drug dimer, and fucoidan may exist inside and/or outside the nanoparticles.

As used herein, the term "obeticholic acid (OCA)" is a bile acid derivative and refers to a compound having the following chemical structure:

Other chemical names for obeticholic acid are 6alpha-ethyl-3alpha,7alpha-dihydroxy-5beta-cholan-24-oic acid, 6α-ethyl-chenodeoxycholic acid, 6-ECDCA, cholan-24-oic acid, 6-ethyl-3,7-dihydroxycholan-24 -Includes 6-ethyl-3,7-dihydroxycholan-24-oic acid, INT-747, Ocaliva, etc. The CAS registry number for obeticholic acid is 459789-99-2, and obeticholic acid refers to all forms of obeticholic acid, eg, non-crystalline, crystalline and substantially pure.

Since obeticholic acid shows strong FXR (Farnesoid X Receptor) activating action, it can be used to treat FXR-mediated diseases. FXR is a nuclear receptor that acts as a bile acid sensor to control bile acid homeostasis, is expressed in various organs, and is involved in biological processes including liver disease, lung disease, kidney disease, intestinal disease, and heart disease, glucose metabolism, insulin metabolism, and lipid metabolism. found to be involved in the regulation of the process.

Hereinafter, the present invention will be described in more detail.

오베티콜린산 이합체 및 이를 포함하는 나노입자Obeticholic acid dimer and nanoparticles containing the same

One aspect of the present invention provides an obeticholic acid dimer compound represented by Formula 1 below, a solvate thereof, or a pharmaceutically acceptable salt thereof:

[Formula 1]

In Formula 1,

A is a C _2-10 linker, which may include -S-, -SS-, -SSS-, -SeSe-, -Se-, -B-, an acetal or a ketal, and X is NH or O can

The linker

,

, and

Is any one selected from the group consisting of; m ₁ ,m ₂ ,p ₁ and p ₂ may each be an integer from 0 to 10.

said m ₁ ,m ₂ ,p ₁ and p ₂ are each an integer of 0 to 10; Specifically, the m ₁ ,m ₂ ,p ₁ and p ₂ may be 0, 1, 2, 3, 4, 5, 6, 7, 8 or 10, respectively.

In this case, the dimer compound may be a compound represented by Formula 1a bound to:

[Formula 1a]

.

An embodiment of the dimer compound may be one represented by any one of the following Chemical Formulas 1b to 1e:

[Formula 1b]

;

[Formula 1c]

;

[Formula 1d]

; and

[Formula 1e]

.

Another aspect of the present invention provides nanoparticles comprising the obeticholic acid dimer compound, a solvate thereof, or a pharmaceutically acceptable salt thereof.

In this case, the size of the nanoparticles may be 250 nm to 500 nm.

Another aspect of the present invention provides nanoparticles containing the obeticholic acid dimer compound and fucoidan.

In this case, the size of the nanoparticles may be 250 nm to 500 nm. In this case, the fucoidan may be included in 10 to 30 parts by weight based on 100 parts by weight of the dimer compound.

Another aspect of the present invention provides a pharmaceutical composition comprising nanoparticles. In this case, the nanoparticles may be nanoparticles containing the obeticholic acid dimer compound or nanoparticles containing the obeticholic acid dimer compound and fucoidan.

The pharmaceutical composition may be used for preventing or treating liver disease.

As used herein, the term "liver disease" refers to a problem in one or more of the various functions performed by the liver, resulting in inability to perform normal metabolism. The liver disease is not limited thereto, but cirrhosis, non-alcoholic fatty liver disease (NAFLD), liver fibrosis, liver cirrhosis, liver cancer, hepatitis, liver ischemia, liver atrophy, acute liver damage, liver toxicity, liver failure, It may be any one or more selected from the group consisting of cholestatic liver disease, primary biliary cirrhosis (PBC), and cholestatic liver disease. The non-alcoholic fatty liver disease (NAFLD) may be non-alcoholic steatohepatitis (NASH).

오베티콜린산 이합체 및 후코이단을 포함하는 나노입자 및 이의 용도Nanoparticles containing obeticholic acid dimer and fucoidan and uses thereof

One aspect of the present invention provides nanoparticles comprising an obeticholic acid dimer compound represented by Formula 1 below:

[Formula 1]

In Formula 1,

The linker

,

, and

The linkage between the drug and the linker, that is, in one embodiment, the linkage between obeticholic acid and the linker may be formed of a hydrolyzable structure such as ester, amide, carbonate, cabamate, or urea. Specifically, the linker may be decomposed by glutathione or active oxygen.

As used herein, the term "nanoparticles comprising obeticholic acid dimers" can be expressed in various ways. For example, when -SS- is included as a linker and the binding site of obeticholic acid and the linker is made of an amide structure, nanoparticles containing ssOCA-A dimer, ssOCA-A dimer nanoparticles, or ssOCA- A can be described as a nanogel. In addition, when -SS- is included as a linker and the bonding site of obeticholic acid and the linker is composed of an ester structure, nanoparticles containing ssOCA-E dimer, ssOCA-E dimer nanoparticles, or ssOCA-E can be described as nanogels. In addition, when -S- is included as a linker and the binding site of obeticholic acid and the linker is made of an ester structure, nanoparticles containing sOCA-E dimer, sOCA-E dimer nanoparticles, or sOCA-E can be described as nanogels. Also, as a linker

Including, and when the bonding site of obeticholic acid and the linker is made of an ester structure, it may be described as a nanoparticle containing a BOCA-E dimer, a BOCA-E dimer nanoparticle, or a BOCA-E nanogel. .

In this case, the nanoparticles may further include fucoidan. In this case, the fucoidan may be 30% or less of the total content of the nanoparticles. Specifically, the drug dimer and fucoidan may have a weight ratio of 70:30 to 95:5. In one embodiment, the weight ratio of the drug dimer and fucoidan may be 70:30, 75:25, 80:20, 85:15, 90:10, or 95:5.

At this time, the fucoidan-containing nanoparticles may have an excellent targeting effect to p-Selectin by fucoidan.

Another aspect of the present invention provides a pharmaceutical composition comprising the nanoparticles.

As used herein, the term "prevention" refers to all activities that suppress or delay the onset of liver disease by administration of the pharmaceutical composition. As used herein, the term "treatment" refers to all activities that improve or beneficially change symptoms of liver disease-related diseases by administration of the pharmaceutical composition.

The liver disease is not limited thereto, but cirrhosis, non-alcoholic fatty liver disease (NAFLD), liver fibrosis, liver cirrhosis, liver cancer, hepatitis, liver ischemia, liver atrophy, acute liver damage, liver toxicity, liver failure, It may be any one or more selected from the group consisting of cholestatic liver disease, primary biliary cirrhosis (PBC), and cholestatic liver disease. The non-alcoholic fatty liver disease (NAFLD) may be non-alcoholic steatohepatitis (NASH).

The pharmaceutical composition may include a pharmaceutically acceptable carrier. The carrier is used as a meaning including an excipient, diluent or auxiliary agent. Such carriers include, for example, lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum acacia, alginates, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, polyvinyl fibre, It may be selected from the group consisting of Rolidone, water, physiological saline, a buffer such as PBS, methylhydroxy benzoate, propylhydroxy benzoate, talc, magnesium stearate, and mineral oil. The composition may include fillers, anti-agglomerating agents, lubricants, wetting agents, flavoring agents, emulsifying agents, preservatives, or combinations thereof.

The pharmaceutical composition may be prepared in any dosage form according to conventional methods. The composition may be formulated as, for example, an oral dosage form (eg, powder, tablet, capsule, syrup, pill, or granule), or a parenteral dosage form (eg, injection). In addition, the composition may be prepared as a systemic formulation or topical formulation.

In the pharmaceutical composition, the solid preparation for oral administration may be tablets, pills, powders, granules, or capsules. The solid formulation may further include an excipient. The excipient may be, for example, starch, calcium carbonate, sucrose, lactose, or gelatin. In addition, the solid preparation may further include a lubricant such as magnesium stearate or talc. In the pharmaceutical composition, the oral liquid formulation may be a suspension, internal solution, emulsion, or syrup. The liquid formulation may include water or liquid paraffin. The liquid formulation may contain excipients such as wetting agents, sweetening agents, flavoring agents, or preservatives. In the pharmaceutical composition, preparations for parenteral administration may be sterilized aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried or suppositories. Non-aqueous solvents or suspensions may include vegetable oils or esters. The vegetable oil may be, for example, propylene glycol, polyethylene glycol, or olive oil. The ester may be, for example, ethyl oleate. The base of the suppository may be witepsol, macrogol, tween 61, cacao butter, laurin fat, or glycerogelatin.

The pharmaceutical composition includes nanoparticles containing an obeticholic acid dimer compound or nanoparticles containing an obeticholic acid dimer compound containing fucoidan according to one aspect as an active ingredient of the pharmaceutical composition. "Active ingredient" refers to a bioactive substance used to achieve pharmacological activity (eg, cancer treatment).

The pharmaceutical composition may include an effective amount of nanoparticles containing an obeticholic acid dimer compound or nanoparticles containing an obeticholic acid dimer compound containing fucoidan according to one aspect. As used herein, the term "effective amount" refers to an amount sufficient to exhibit the effect of preventing or treating a disease when administered to a subject in need of prevention or treatment. The effective amount can be appropriately selected by those skilled in the art depending on the cell or organism to be selected. A preferred dosage of the pharmaceutical composition varies depending on the condition and body weight of the subject, the severity of the disease, the type of drug, the route and period of administration, but can be appropriately selected by those skilled in the art. However, nanoparticles containing the obeticholic acid dimer compound or nanoparticles containing the obeticholic acid dimer compound containing fucoidan are, for example, about 0.0001 mg/kg to about 100 mg/kg, or about 0.001 An amount of mg/kg to about 100 mg/kg can be divided into 1 to 24 times a day, 1 to 7 times every 2 days to 1 week, or 1 to 24 times every 1 month to 12 months. In the pharmaceutical composition, the compound, its solvate, or pharmaceutically acceptable salt may be included in about 0.0001% to about 10% by weight, or about 0.001% to about 1% by weight based on the total weight of the composition. .

The administration method may be oral or parenteral administration. The method of administration can be, for example, oral, transdermal, subcutaneous, rectal, intravenous, intraarterial, intraperitoneal, intramuscular, intrasternal, topical, intranasal, intratracheal, or intradermal routes. . The composition may be administered systemically or topically, and may be administered alone or in combination with other pharmaceutically active compounds.

Specifically, the pharmaceutical composition is not limited thereto, but is cirrhosis, non-alcoholic fatty liver disease (NAFLD; Non-alcoholic fatty liver disease), liver fibrosis, liver cirrhosis, liver cancer, hepatitis, liver ischemia, liver atrophy, acute liver damage, liver toxicity , liver failure, cholestatic liver disease, primary biliary cirrhosis (PBC), and cholestatic liver disease.

An aspect of the present invention is an obeticholic acid dimer compound represented by Formula 1, a solvate thereof, or a pharmaceutically acceptable salt; nanoparticles comprising the obeticholic acid dimer compound, a solvate thereof, or a pharmaceutically acceptable salt thereof; Alternatively, a pharmaceutical composition comprising the nanoparticles is used for preventing or treating diseases related to FXR-mediated diseases, particularly liver diseases.

An aspect of the present invention is an obeticholic acid dimer compound represented by Formula 1, a solvate thereof, or a pharmaceutically acceptable salt; nanoparticles comprising the obeticholic acid dimer compound, a solvate thereof, or a pharmaceutically acceptable salt thereof; Alternatively, a pharmaceutical composition comprising the nanoparticles is used to prepare a medicament used for preventing or treating diseases related to FXR-mediated diseases, particularly liver diseases.

The obeticholic acid dimer compound represented by Formula 1 is as described above. In addition, the contents and diseases of fucoidan are as described above.

나노입자를 이용한 질병 예방 및 치료 방법Disease prevention and treatment method using nanoparticles

An aspect of the present invention is an obeticholic acid dimer compound represented by Formula 1, a solvate thereof, or a pharmaceutically acceptable salt; nanoparticles comprising the obeticholic acid dimer compound, a solvate thereof, or a pharmaceutically acceptable salt thereof; Or it provides a method for preventing or treating liver disease comprising administering to a subject a pharmaceutical composition containing the nanoparticles.

The obeticholic acid dimer compound represented by Formula 1 is as described above. In addition, the subject may be a mammal, for example, a human, mouse, rat, cow, horse, pig, dog, monkey, sheep, goat, ape, or cat. The subject may be an individual suffering from, or highly likely to suffer from, symptoms associated with a disease.

The administration method may be oral or parenteral administration. The method of administration can be, for example, oral, transdermal, subcutaneous, rectal, intravenous, intraarterial, intraperitoneal, intramuscular, intrasternal, topical, intranasal, intratracheal, or intradermal routes. . The pharmaceutical composition may be administered systemically or topically, and may be administered alone or in combination with other pharmaceutically active compounds.

The obeticholic acid dimer compound, a solvate thereof, or a pharmaceutically acceptable salt thereof; nanoparticles comprising the obeticholic acid dimer compound, a solvate thereof, or a pharmaceutically acceptable salt thereof; Alternatively, the preferred dosage of the pharmaceutical composition varies depending on the condition and body weight of the patient, the severity of the disease, the drug type, the route and period of administration, but may be appropriately selected by those skilled in the art. The dosage is, for example, in the range of about 0.001 mg/kg to about 100 mg/kg, about 0.01 mg/kg to about 10 mg/kg, or about 0.1 mg/kg to about 1 mg/kg on an adult basis. can be The administration may be administered once a day, multiple times a day, or once a week, once every 2 weeks, once every 3 weeks, or once every 4 weeks to once a year.

오베티콜린산 이합체 및 후코이단을 포함하는 나노입자 제조 방법Method for preparing nanoparticles containing obeticholic acid dimer and fucoidan

One aspect of the present invention is to obtain nanoparticles containing obeticholic acid dimer and fucoidan, which comprises mixing the obeticholic acid dimer compound represented by Formula 1 below and fucoidan at a weight ratio of 80:20 to 95:5. A manufacturing method is provided:

[Formula 1]

In Formula 1,

The linker

,

, and

Any one selected from the group consisting of; m ₁ ,m ₂ ,p ₁ and p ₂ may each be an integer from 0 to 10.

said m ₁ ,m ₂ ,p ₁ and p ₂ are each an integer of 1 to 10; Specifically, the m ₁ ,m ₂ ,p ₁ and p ₂ may be 0, 1, 2, 3, 4, 5, 6, 7, 8 or 10, respectively.

In the present specification, nanoparticle refers to a compound formed through self-assembly of a dimer compound. In one embodiment of the present invention, the nanoparticles have a nanoscale size, specifically 1 to 999 nm, 100 to 900 nm, 100 to 800 nm, 100 to 700 nm, 100 to 600 nm, 100 to 500 nm, 100 to 400 nm or 100 to 300 nm in size. More specifically, it may be formed in a size of 250 to 500 mm. However, it is not limited thereto.

In the present specification, a linker is a structure connecting drug monomers. For example, there are a sulfide bond-based linker, a thiochital-based linker, a selenide-based linker, and the like. However, it is not limited thereto. In one embodiment of the present invention, the linker based on a sulfide bond may include a sulfide bond or a disulfide bond.

In one embodiment of the present invention, the linker may be cleaved by glutathione or active oxygen. However, it is not limited thereto.

In the present specification, a target cell is a cell having a membrane protein to which fucoidan can bind. In one embodiment of the present invention, target cells may be liver cells. However, it is not limited thereto.

The weight ratio between obeticholic acid dimer and fucoidan may be 80:20 to 95:5. In one embodiment of the present invention, a dispersing agent may be added to the fucoidan solution to increase the dispersibility of the nanoparticles. Specifically, the dispersant includes polyvinyl alcohol (PVA). However, it is not limited thereto.

In one embodiment of the present invention, after preparing the nanoparticles, lyophilization may be further included. However, it is not limited thereto.

Hereinafter, the present invention will be described in more detail by the following examples. However, the following examples are only for exemplifying the present invention, and the scope of the present invention is not limited only to these.

I. 오베티콜린산 이합체 화합물(ssOCA-A)을 포함하는 나노입자I. Nanoparticles Comprising Obeticholic Acid Dimer Compound (ssOCA-A)

Obeticholic acid (OCA; IUPAC name (4R)-4-[(3R,5S,6R,7R,8S,9S,10S,13R,14S,17R)-6-ethyl-3,7-dihydroxy -10,13-dimethyl-2,3,4,5,6,7,8,9,11,12,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-17-yl]pentanoic acid) is as follows, and an obeticholic acid dimer compound was prepared using this.

¹ H NMR (400 MHz, CDCl ₃ ): δ 3.69 (3H, s), 3.4 (3H, s), 2.40-2.18 (6H, m), 1.96-0.84 (m), 0.64 (9H, s).

Molecular Formula: C ₂₆ H ₄₄ O ₄ ; MS/MS (API ⁺ ) Exact Mass: m/z 420.32; MS m/z: 386.03 [M+H] ⁺ .

실시예 1. 오베티콜린산 이합체 화합물(ssOCA-A)의 제조Example 1. Preparation of obeticholic acid dimer compound (ssOCA-A)

Put obeticholic acid (0.5 g), 1-ethyl-3 (dimethylaminophenyl) carbonyldiimide (0.45 g), and 1-hydroxybenzotriazole (0.16 g) in a round flask and add 10 mL of dimethyl sulfoxide. completely dissolved in the side solution. After dissolving cystamine dihydrochloride (0.13g) and triethylamine (0.3g) in 1 mL of dimethyl sulfoxide, they were added dropwise to the reaction solution and reacted at 40°C for 48 hours. After removing triethylamine using a rotary evaporator, it was added dropwise to 150 mL of distilled water, and the resulting precipitate was centrifuged (12000g, 10 minutes) to obtain an obeticholic acid dimer compound (ssOCA-A).

¹ H NMR (400 MHz, DMSO-d6): δ 6.29 (2H, t), 3.71 (4H, s), 3.51 (4H, q), 2.82 (4H, t), 2.34-2.05 (4H, m), 2.00–0.81 (m), 0.68 (6H, s).

Molecular Formula: C ₅₆ H ₉₆ N ₂ O ₆ S ₂ ; MS/MS (API ⁺ ) Exact Mass: m/z 956.67; MS m/z: 462.82 [M+H] ⁺ .

실시예 2. 오베티콜린산 이합체 화합물(ssOCA-A)을 포함하는 나노입자의 제조Example 2. Preparation of nanoparticles containing obeticholic acid dimer compound (ssOCA-A)

10 mg of the obeticholic acid dimer compound (ssOCA-A) prepared in Example 1 was dissolved in 5 mL of tetrahydrofuran, added dropwise to 50 mL of PBS using a syringe, and ultrasonically treated to disperse the nanoparticles. . Thereafter, the organic solvent was removed using a rotary evaporator and freeze-dried using liquid nitrogen. Completely freeze-dried nanoparticles were dispersed in PBS immediately before use. The size, shape and distribution of the nanoparticles as physical properties of the prepared nanoparticles were analyzed using a particle size analyzer and a scanning electron microscope (FIGS. 3a and 3b).

실시예 3. 오베티콜린산 이합체 화합물(ssOCA-A) 및 후코이단을 포함하는 나노입자의 제조Example 3. Preparation of nanoparticles containing obeticholic acid dimer compound (ssOCA-A) and fucoidan

Dissolve 10 mg of ssOCA-A prepared in Example 1 in 5 mL of tetrahydrofuran, and use a syringe to add dropwise to 50 mL of PBS in which 2 mg of Fucoidan (F) is dissolved, and sonicate to obtain nano particles were dispersed. Thereafter, the organic solvent was removed using a rotary evaporator and freeze-dried using liquid nitrogen. Completely freeze-dried nanoparticles were dispersed in PBS immediately before use.

실험예 1. 오베티콜린산 이합체 화합물(ssOCA-A)이 포함된 나노입자의 간경변 치료 효능 평가Experimental Example 1. Evaluation of liver cirrhosis treatment efficacy of nanoparticles containing obeticholic acid dimer compound (ssOCA-A)

Cirrhosis mouse model was constructed by administering CCL4 (carbon tetrachloride, 1.5 mL/kg, 1:3 dilution with olive oil) to mice (BALB/c, 7 weeks old, male) by intraperitoneal injection (i.p.) twice a week for 8 weeks. . At this time, when the mouse was brought in, an external examination of the animal was performed, and after a 7-day acclimatization period, it was moved to the corresponding animal room, and general symptoms were observed every day. General symptoms and body weight changes were confirmed and only healthy animals were used in the test. In addition, during the acclimatization period, the animal's tail was marked with a name pen at the time of acquisition, and the individual identification card during the acclimatization period was attached to the breeding box. During the observation period, the tails of the animals were colored to distinguish the individuals, and the individual identification cards were attached to the breeding boxes. The drinking water was freely consumed by putting purified water filtered using UV sterilization and a filter into a polysulfone drinking water bottle (250 mL).

When creating a liver cirrhosis mouse model, obeticholic acid (OCA; 400 ug/mouse) was administered 15 times (21, 23, 25, 28, 30, 32, 35, 37, 39, 42, 44, 46, 49, 51, 53 days) was orally administered, and the nanoparticles (OCA NP; 400 ug/mouse) prepared in Example 2 were orally administered 15 times to evaluate the efficacy of liver cirrhosis treatment.

Efficacy of liver cirrhosis treatment was evaluated by removing the remaining blood as much as possible by performing perfusion under anesthesia after administering carbon tetrachloride (CCL4) for 8 weeks, and then extracting the liver. The efficacy of liver cirrhosis treatment was evaluated by confirming the degree of fibrosis through collagen fiber staining, the level of expression of TGF-β inducing hepatic fibrosis, and the level of expression of α-SMA, an activated hepatic stellate cell marker.

실험예 1.1. 콜라겐 섬유 염색을 통한 섬유화 정도 확인Experimental Example 1.1. Confirmation of degree of fibrosis through collagen fiber staining

The level of fibrosis in the liver of CCL4-administered mice was confirmed through the Sirius red staining method, which can determine the degree of fibrosis by staining the level of collagen fibers. In liver tissues of CCL4-administered mice, it was observed that collagen was deposited around blood vessels of the liver. Collagen reduction was not observed in the control group in which OCA was administered 15 times through the mouth of a mouse, whereas collagen levels decreased when OCA nanogel, which is a nanoparticle containing an OCA dimer compound, was prepared and administered through the mouth 15 times. Confirmed. Through this, the administration of the OCA nanogel form showed an effect of inhibiting collagen deposition (FIG. 4).

실험예 1.2. 간섬유화를 유도하는 TGF-β 발현 정도 확인Experimental Example 1.2. Confirmation of TGF-β expression level that induces liver fibrosis

TGF-β, which induces liver fibrosis, is a protein secreted by macrophages infiltrating liver tissue and is the main protein responsible for liver fibrosis by stimulating inactive hepatic stellate cells to activate them. When only CCL4 was administered as a positive control group, high TGF-β levels were confirmed in liver tissue. When OCA was administered 15 times orally along with CCL4 administration, a decrease in TGF-β was observed. It was confirmed that it decreased (FIG. 5). It was found that OCA nanogel form was absorbed in the intestine, spread throughout the body, and accumulated in the liver, and OCA was decomposed specifically in the liver inflammatory environment to show a therapeutic effect.

실험예 1.3. 활성화 간성상세포 마커인 α-SMA 발현 정도 확인Experimental Example 1.3. Confirmation of expression level of α-SMA, an activated hepatic stellate cell marker

Liver tissue was cut into 6 μm sections and then fixed with 4% formaldehyde. The fixed liver tissue was treated with an anti-mouse primary antibody (JG-ab124964) for α-SMA and a secondary antibody (Rabbit) for 24 hours. Thereafter, after staining the nucleus with DAPI, expression of α-SMA was evaluated through fluorescence imaging using a confocal laser scanner microscopy. The efficacy of OCA nanogels, that is, nanoparticles containing OCA dimer compounds, was verified by targeting α-SMA, a representative marker of hepatic stellate cells. In general, when hepatic fibrosis is induced by administration of CCL4, α-SMA levels increase through activation of hepatic stellate cells. As shown in FIG. 6, it was confirmed that the α-SMA level around the liver blood vessels was increased in the mouse group administered with CCL4. In addition, when OCA was orally administered 15 times, α-SMA levels did not change. However, α-SMA, a marker for activated hepatic stellate cells, was significantly reduced when nanoparticles (OCA nanogel) containing an OCA dimer compound were administered orally 15 times in a CCL4-administered liver cirrhosis model.

II. 오베티콜린산 이합체 화합물(ssOCA-E)을 포함하는 나노입자II. Nanoparticles comprising obeticholic acid dimer compound (ssOCA-E)

실시예 4. 오베티콜린산 이합체 화합물(ssOCA-E)의 제조Example 4. Preparation of obeticholic acid dimer compound (ssOCA-E)

After dissolving obeticholic acid (1 g), 2,2'-dithiodiethanol (0.18 g), and dimethylaminopyridine (0.014 g) in 50 mL of dichloromethane, the mixture was stirred for 10 minutes. After adding 1-ethyl-3(dimethylaminophenyl)carbonyldiimide (0.9 g) dropwise, the mixture was stirred at room temperature for 16 hours. Reactivity was confirmed using TLC, and when the reaction was all in progress, the reaction solvent was removed and purified through a silica gel column to obtain an obeticholic acid dimer compound (ssOCA-E).

¹ H NMR (400 MHz, CDCl ₃ ): δ 4.34 (2H, t), 4.12 (2H, q), 3.7 (2H, s), 2.91 (4H, m), 2.34-2.25 (4H, m), 2.02 -0.89 (m), 0.66 (6H, s).

Molecular Formula: C ₅₆ H ₉₄ O ₈ S ₂ ; MS/MS (API ⁺ ) Exact Mass: m/z 958.64; MS m/z: 489.88 [M+H] ⁺ .

실시예 5. 오베티콜린산 이합체 화합물(ssOCA-E)을 포함하는 나노입자의 제조Example 5. Preparation of nanoparticles containing obeticholic acid dimer compound (ssOCA-E)

Dissolve 10 mg of the obeticholic acid dimer compound (ssOCA-E) prepared in Example 4 in 5 mL of tetrahydrofuran, add it dropwise to 50 mL of PBS dropwise using a syringe, and sonicate to disperse the nanoparticles. . Thereafter, the organic solvent was removed using a rotary evaporator and freeze-dried using liquid nitrogen. Completely freeze-dried nanoparticles were dispersed in PBS immediately before use. Using a particle size analyzer and a scanning electron microscope, the size, shape and distribution of the nanoparticles were analyzed as physical properties of the prepared nanoparticles (FIGS. 8a and 8b).

실시예 6. 오베티콜린산 이합체 화합물(ssOCA-E) 및 후코이단을 포함하는 나노입자의 제조Example 6. Preparation of nanoparticles containing obeticholic acid dimer compound (ssOCA-E) and fucoidan

Dissolve 10 mg of obeticholic acid dimer compound (ssOCA-E) prepared in Example 4 in 5 mL of tetrahydrofuran, and use a syringe to dissolve 2 mg of fucoidan (F) in 50 mL of PBS. It was added dropwise and ultrasonicated to disperse the nanoparticles. Thereafter, the organic solvent was removed using a rotary evaporator and freeze-dried using liquid nitrogen. Completely freeze-dried nanoparticles were dispersed in PBS immediately before use. The size, shape and distribution of the nanoparticles were analyzed as physical properties of the prepared nanoparticles using a particle size analyzer and a scanning electron microscope (FIGS. 9a and 9b).

실험예 2. 과산화수소수와 글루타치온의 유무에 따른 오베티콜린산 이합체(ssOCA-E) 화합물이 포함된 나노입자의 분해 능력 평가Experimental Example 2. Degradation ability evaluation of nanoparticles containing obeticholic acid dimer (ssOCA-E) compound according to the presence or absence of hydrogen peroxide and glutathione

The linker connection site of the obeticholic acid dimer compound is composed of a hydrolyzable ester bond structure. The linker is decomposed by glutathione or active oxygen, and this was confirmed.

Specifically, 2 mg of the obeticholic acid dimer compound (ssOCA-E) prepared in Example 4 was added to each PBS solution (glutathione 1 mg/mL, hydrogen peroxide solution).50 uM/mL, glutathione 1 mg/mL + hydrogen peroxide solution 50 uM/mL each) into 2 mL and incubated for 48 hours while shaking at 37°C. After 48 hours, in order to evaluate the decomposition ability, organic matter was extracted using ethyl acetate, and after removing water using magnesium sulfate, concentration was performed. The resulting solid was analyzed by MS to confirm the change in molecular weight.

Table 1 below summarizes the results of evaluation of the decomposition ability of nanoparticles containing hydrogen peroxide, glutathione, obeticholic acid dimer (ssOCA-E) compound due to hydrogen peroxide and glutathione.

용매menstruum	Molecular FormulaMolecular Formula	MS/MS (API⁺) Exact MassMS/MS (API ⁺ ) Exact Mass	실측치(MS m/z [M+H]⁺)Actual value (MS m/z [M+H] ⁺ )
PBS 용액PBS solution	C₅₆H₉₄O₈S₂ C ₅₆ H ₉₄ O ₈ S ₂	958.64958.64	489.88489.88
과산화수소수(50 uM/mL) 포함 PBS 용액PBS solution containing hydrogen peroxide (50 uM/mL)	C₂₈H₄₈O₄S₁ C ₂₈ H ₄₈ O ₄ S ₁	480.33480.33	663.48663.48
글루타치온(1 mg/mL) 포함 PBS 용액PBS solution containing glutathione (1 mg/mL)	C₂₈H₄₈O₄S₁ C ₂₈ H ₄₈ O ₄ S ₁	480.33480.33	663.48663.48
과산화수소수(50 uM/mL) 및 글루타치온(1 mg/mL) 포함 PBS 용액PBS solution containing hydrogen peroxide (50 uM/mL) and glutathione (1 mg/mL)	C₂₈H₄₈O₄S₁ C ₂₈ H ₄₈ O ₄ S ₁	480.33480.33	663.37663.37

실험예 3. 오베티콜린산 이합체(ssOCA-E) 화합물이 포함된 나노입자의 비알콜성 지방간 치료 효능 평가Experimental Example 3. Non-alcoholic fatty liver treatment efficacy evaluation of nanoparticles containing obeticholic acid dimer (ssOCA-E) compound

A non-alcoholic fatty liver (NASH) model was constructed in mice (BALB/c, 7 weeks old, male) through an MCD diet (methionine choline-deficient diet).

When making a non-alcoholic fatty liver model, each dose (20 mg/kg) of obeticholic acid (OCA) and the nanoparticles (OCA-Nanogel) prepared in Example 6 was 35 times (daily) from the 2nd week. It was administered orally, and each dose (100 mg/kg) of OCA-Nanogel was intravenously administered 10 times (2 times a week for a total of 5 weeks) to evaluate the efficacy of non-alcoholic fatty liver treatment and other toxicity.

Weight and activity were evaluated daily, and if animals in severe pain or moribund state appeared during the observation period, they were euthanized using carbon dioxide and autopsies were performed to determine the cause of death.

After administration was completed and general symptoms and other specific items were checked for about a week, hematological, histological, protein and other items were evaluated.

실험예 3.1. 혈액학적 분석Experimental Example 3.1. hematological analysis

One week after the end of drug administration, 270 μl of serum was isolated from the blood of each mouse and GOT (AST), triglyceride, GGT (gamma-glutamyl transferase), GPT (ALT), uric acid (LDL), total protein (Total Protein), Cholesterol, HDL, Total Bilirubin, Albumin, Total Bile Acid, Glucose, and ALP were measured.

As a result of the measurement, it was confirmed that AST and ALT, which are liver parameters, decreased compared to the conventional control OCA alone drug (FIGS. 10a and 10b).

실험예 3.2. 콜라겐 섬유 염색을 통한 섬유화 정도 확인Experimental Example 3.2. Confirmation of degree of fibrosis through collagen fiber staining

One week after the end of drug administration, the liver was removed from each mouse and then a block was prepared. Thereafter, fibrosis in the liver tissue of the non-alcoholic fatty liver model was confirmed through the Masson-Tchrome staining method, which can determine the degree of fibrosis by staining collagen, and the degree of fibrosis was identified by digitizing it.

As a result, the fibrosis area compared to the liver area was smaller in the group containing ssOCA dimer than in the OCA alone treatment developed as a treatment for non-alcoholic fatty liver disease. Through this, it was found that less fibrosis of liver tissue occurred, and thus, ssOCA dimer was effective in treating non-alcoholic fatty liver disease (FIGS. 11a and 11b).

실험예 3.3. TGF-β 발현 정도 확인Experimental Example 3.3. Check the level of TGF-β expression

TGF-β, which induces liver fibrosis, is a protein secreted by macrophages infiltrating liver tissue and is the main protein responsible for liver fibrosis by stimulating inactive hepatic stellate cells to activate them. One week after the end of drug administration, livers were removed from each mouse, blocks were prepared, and the differences were compared by staining them.

Recombinant Anti-TGF beta 1 antibody (JG-ab229856) was used as the primary antibody, and TGF-β was detected using Alxexa Fluor ^TM 594 goat anti-rabbit (Invitrogen A11012) as the secondary antibody. dyed. Then, after staining the cell nuclei using DAPI, the degree of TGF-β expression was confirmed.

As a result, a high level of TGF-β was expressed in the non-alcoholic fatty liver model, and when OCA developed as a therapeutic agent was treated alone, the therapeutic effect was not evident. On the other hand, in the case of the nanogel containing the ssOCA dimer, the expression level of TGF-β was significantly decreased. Through this, it was found that the fucoidan-coated nanogel containing the ssOCA dimer was effective in treating non-alcoholic fatty liver. In addition, it was found that the intravascular injection (I.V.) administration method exhibited a higher effect than oral administration (P.O.) of fucoidan nanogels containing ssOCA dimer (FIG. 12).

실험예 3.4. α-SMA 발현 정도 확인Experimental Example 3.4. Confirmation of α-SMA expression level

One week after drug administration was completed, livers were removed from each mouse, and then α-SMA, a marker for activated hepatic stellate cells, was confirmed by preparing a block.

α-SMA using a recombinant anti-alpha smooth muscle antibody (JG-ab124964) as a primary antibody and Alxexa Fluor ^TM 594 goat anti-rabbit (Invitrogen A11012) as a secondary antibody staining proceeded. Then, after staining the nucleus with DAPI, the expression level of α-SMA was confirmed.

As a result, α-SMA was conspicuously expressed in the non-alcoholic fatty liver model, and the therapeutic effect was not evident when OCA developed as a treatment was treated alone. On the other hand, in the experimental group in which the nanogel containing the ssOCA dimer was administered through intravascular injection (I.V), the degree of α-SMA expression was significantly reduced, confirming that the treatment effect of non-alcoholic fatty liver was excellent (FIG. 13).

III. 오베티콜린산 이합체 화합물(sOCA-E)을 포함하는 나노입자III. Nanoparticles comprising obeticholic acid dimer compound (sOCA-E)

실시예 7. 오베티콜린산 이합체 화합물(sOCA-E)의 제조Example 7. Preparation of obeticholic acid dimer compound (sOCA-E)

After dissolving obeticholic acid (0.5 g), thiodiethanol (0.072 g), and dimethylaminopyridine (0.036 g) in 30 mL of dichloromethane, the mixture was stirred for 10 minutes. After adding 1-ethyl-3(dimethylaminophenyl)carbonyldiimide (0.45 g) dropwise, the mixture was stirred at room temperature for 16 hours. Reactivity was confirmed using TLC, and when the reaction was complete, the reaction solvent was removed and purified through a silica gel column to obtain an obeticholic acid dimer compound (sOCA-E).

¹ H NMR (400 MHz, CDCl ₃ ): δ 4.22 (4H, t), 3.78-3.69 (3H, m), 3.4 (2H, m), 2.79 (5H, m), 2.34-2.25 (4H, m) , 1.98–0.85 (m), 0.66 (6H, s).

Molecular Formula: C ₅₆ H ₉₄ O ₈ S; MS/MS (API ⁺ ) Exact Mass: m/z 926.67; MS m/z: 386.03 [M+H] ⁺ .

실시예 8. 오베티콜린산 이합체 화합물(sOCA-E)을 포함하는 나노입자의 제조Example 8. Preparation of nanoparticles containing obeticholic acid dimer compound (sOCA-E)

Dissolve 10 mg of the obeticholic acid dimer compound (sOCA-E) prepared in Example 7 in 5 mL of tetrahydrofuran, add it dropwise to 50 mL of PBS dropwise using a syringe, and ultrasonically treat to disperse the nanoparticles. . Thereafter, the organic solvent was removed using a rotary evaporator and freeze-dried using liquid nitrogen. Completely freeze-dried nanoparticles were dispersed in PBS immediately before use.

실시예 9. 오베티콜린산 이합체 화합물(sOCA-E) 및 후코이단을 포함하는 나노입자의 제조Example 9. Preparation of nanoparticles containing obeticholic acid dimer compound (sOCA-E) and fucoidan

Dissolve 10 mg of obeticholic acid dimer compound (sOCA-E) prepared in Example 7 in 5 mL of tetrahydrofuran, and use a syringe to dissolve 2 mg of fucoidan (F) in 50 mL of PBS. It was added dropwise and ultrasonicated to disperse the nanoparticles. Thereafter, the organic solvent was removed using a rotary evaporator and freeze-dried using liquid nitrogen. Completely freeze-dried nanoparticles were dispersed in PBS immediately before use. The size, shape and distribution of the nanoparticles were analyzed as physical properties of the prepared nanoparticles using a particle size analyzer and a scanning electron microscope (FIGS. 15a and 15b).

실험예 4. 과산화수소수와 글루타치온의 유무에 따른 오베티콜린산 이합체(sOCA-E) 화합물이 포함된 나노입자의 분해 능력 평가Experimental Example 4. Degradation ability evaluation of nanoparticles containing obeticholic acid dimer (sOCA-E) compound according to the presence or absence of hydrogen peroxide and glutathione

Specifically, 2 mg of the obeticholic acid dimer compound (sOCA-E) prepared in Example 7 was added to each PBS solution (glutathione 1 mg/mL, hydrogen peroxide solution).50 uM/mL, glutathione 1 mg/mL + hydrogen peroxide solution 50 uM/mL each) into 2 mL and incubated for 48 hours while shaking at 37°C. After 48 hours, in order to evaluate the decomposition ability, organic matter was extracted using ethyl acetate, and after removing water using magnesium sulfate, concentration was performed. The resulting solid was analyzed by MS to confirm the change in molecular weight.

Table 2 below summarizes the evaluation results of the decomposition ability of nanoparticles containing hydrogen peroxide, glutathione, obeticholic acid dimer (sOCA-E) compound due to hydrogen peroxide and glutathione.

용매menstruum	Molecular FormulaMolecular Formula	MS/MS (API⁺) Exact MassMS/MS (API ⁺ ) Exact Mass	실측치(MS m/z [M+H]⁺)Actual value (MS m/z [M+H] ⁺ )
PBS 용액PBS solution	C₅₆H₉₄O₈SC ₅₆ H ₉₄ O ₈ S	926.67926.67	386.03386.03
과산화수소수(50 uM/mL) 포함 PBS 용액PBS solution containing hydrogen peroxide (50 uM/mL)	C₂₈H₄₈O₄S₁ C ₂₈ H ₄₈ O ₄ S ₁	480.33480.33	663.59663.59
글루타치온(1 mg/mL) 포함 PBS 용액PBS solution containing glutathione (1 mg/mL)	C₂₈H₄₈O₄S₁ C ₂₈ H ₄₈ O ₄ S ₁	480.33480.33	663.48663.48
과산화수소수(50 uM/mL) 및 글루타치온(1 mg/mL) 포함 PBS 용액PBS solution containing hydrogen peroxide (50 uM/mL) and glutathione (1 mg/mL)	C₂₈H₄₈O₄S₁ C ₂₈ H ₄₈ O ₄ S ₁	480.33480.33	663.48663.48

실험예 5. 오베티콜린산 이합체(sOCA-E) 화합물이 포함된 나노입자의 비알콜성 지방간 치료 효능 평가Experimental Example 5. Non-alcoholic fatty liver treatment efficacy evaluation of nanoparticles containing obeticholic acid dimer (sOCA-E) compound

A non-alcoholic fatty liver model was prepared in the same way as in Experimental Example 3, and the nanoparticles (OCA-Nanogel) prepared in Example 9 were orally administered, and the treatment efficacy and Other toxicity evaluations were conducted.

실험예 5.1. 혈액학적 분석Experimental Example 5.1. hematological analysis

One week after the end of drug administration, 270 μl of serum was separated from the blood of each mouse, and GOT (AST), cholesterol (Cholesterol), GGT (Gamma-glutamyl transferase), GPT (ALT), ALP, LDL, total protein (Total Protein) , triglyceride, HDL, total bilirubin, glucose, total bile acid, uric acid, and albumin were measured.

As a result of the measurement, it was confirmed that AST and ALT, which are liver parameters, decreased compared to the existing control OCA alone drug (FIGS. 16a and 16b).

실험예 5.2. 콜라겐 섬유 염색을 통한 섬유화 정도 확인Experimental Example 5.2. Confirmation of degree of fibrosis through collagen fiber staining

Fibrosis in the liver tissue of the non-alcoholic fatty liver model was confirmed through the Masson-Tchrome staining method in the same manner as Experimental Example 3.2, and the degree of fibrosis was identified by digitizing it.

As a result, the fibrosis area compared to the liver area was smaller in the group containing sOCA dimer than in the OCA alone treatment developed as a treatment for non-alcoholic fatty liver disease. Through this, it was found that less fibrosis of liver tissue occurred, and thus, sOCA dimer was effective in treating non-alcoholic fatty liver (FIGS. 17a and 17b).

실험예 5.3. TGF-β 발현 정도 확인Experimental Example 5.3. Check the level of TGF-β expression

In the same manner as in Experimental Example 3.3, after one week after the end of drug administration, the liver was removed from each mouse, and then a block was prepared, and the TGF-β and DAPI staining was performed to confirm the level of TGF-β expression.

As a result, a high level of TGF-β was expressed in the non-alcoholic fatty liver model, and when OCA developed as a therapeutic agent was treated alone, the therapeutic effect was not evident. On the other hand, in the case of the nanogel containing the sOCA dimer, the expression level of TGF-β was significantly decreased. Through this, it was found that the fucoidan-coated nanogel containing the sOCA dimer was effective in treating non-alcoholic fatty liver (FIG. 18).

실험예 5.4. α-SMA 발현 정도 확인Experimental Example 5.4. Confirmation of α-SMA expression level

In the same manner as in Experimental Example 3.4, one week after drug administration was completed, livers were removed from each mouse, and then blocks were prepared, and α-SMA and DAPI were stained to confirm the expression level of α-SMA.

As a result, α-SMA was conspicuously expressed in the non-alcoholic fatty liver model, and the therapeutic effect was not evident when OCA developed as a treatment was treated alone. On the other hand, when the nanogel containing the sOCA dimer was administered, the expression level of α-SMA was significantly decreased. In particular, in the experimental group in which sOCA was administered through intravascular injection (I.V.), a remarkable effect was shown in the treatment of alcoholic fatty liver (FIG. 19).

IV. 오베티콜린산 이합체 화합물(BOCA-E)을 포함하는 나노입자IV. Nanoparticles comprising obeticholic acid dimer compound (BOCA-E)

실시예 10. 오베티콜린산 이합체 화합물(BOCA-E)의 제조Example 10. Preparation of obeticholic acid dimer compound (BOCA-E)

단계 1: BRAP의 합성Step 1: Synthesis of BRAP

After putting 4-hydroxybenzylboronic acid (1 g) and 1,1,1-tris(hydroxymethyl)ethane (0.79 g) in 5 mL of tetrahydrofuran, the mixture was stirred at room temperature. When stirring, 1-2 drops of purified water were added dropwise and stirred until all solutions became clear. The reactivity was confirmed using TLC, and when the reactivity was all progressed, it was purified through a silica gel column, and BRAP ((4-(5-(hydroxymethyl)-5-methyl-1,3,2-dioxaborinan-2-yl)phenyl )methanol) was obtained.

¹ H NMR (400 MHz, CDCl ₃ ): δ 7.79 (2H, d), 7.33 (2H, d), 4.70 (2H, s), 3.80 (6H, m), 0.99 (3H, S).

단계 2: 오베티콜린산 이합체 화합물(BOCA-E)의 합성Step 2: Synthesis of obeticholic acid dimer compound (BOCA-E)

After dissolving obeticholic acid (0.5 g), thiodiethanol (0.072 g), and dimethylaminopyridine (0.036 g) in 30 mL of dichloromethane, the mixture was stirred for 10 minutes. After adding 1-ethyl-3(dimethylaminophenyl)carbonyldiimide (0.45 g) dropwise, the mixture was stirred at room temperature for 16 hours. The reactivity was checked using TLC, and when the reaction was complete, the reaction solvent was removed and purified through a silica gel column to obtain an obeticholic acid dimer compound (BOCA-E).

¹ H NMR (400 MHz, CDCl ₃ ): δ 7.78 (2H, d), 7.33 (2H, d), 4.19 (2H, s), 4.13 (4H, q), 4.03 (4H, m), 3.86 (2H , d), 3.70 (3H, s), 3.54 (3H, m), 3.40 (3H, m), 2.40–2.18 (6H, m), 1.96–0.84 (m), 0.64 (10H, m).

실시예 11. 오베티콜린산 이합체 화합물(BOCA-E)을 포함하는 나노입자의 제조Example 11. Preparation of nanoparticles containing obeticholic acid dimer compound (BOCA-E)

Dissolve 10 mg of the obeticholic acid dimer compound (BOCA-E) prepared in Example 10 in 5 mL of tetrahydrofuran, add it dropwise to 50 mL of PBS dropwise using a syringe, and sonicate to disperse the nanoparticles. . Thereafter, the organic solvent was removed using a rotary evaporator and freeze-dried using liquid nitrogen. Completely freeze-dried nanoparticles were dispersed in PBS immediately before use.

실시예 12. 오베티콜린산 이합체 화합물(BOCA-E) 및 후코이단을 포함하는 나노입자의 제조Example 12. Preparation of nanoparticles containing obeticholic acid dimer compound (BOCA-E) and fucoidan

Dissolve 10 mg of obeticholic acid dimer compound (BOCA-E) prepared in Example 10 in 5 mL of tetrahydrofuran, and use a syringe to dissolve 2 mg of fucoidan (F) in 50 mL of PBS. It was added dropwise and ultrasonicated to disperse the nanoparticles. Thereafter, the organic solvent was removed using a rotary evaporator and freeze-dried using liquid nitrogen. Completely freeze-dried nanoparticles were dispersed in PBS immediately before use. The size, shape and distribution of the nanoparticles as physical properties of the prepared nanoparticles were analyzed using a particle size analyzer and a scanning electron microscope (FIGS. 22a and 22b).

Claims

An obeticholic acid dimer compound represented by Formula 1 below, a solvate thereof, or a pharmaceutically acceptable salt thereof:

[Formula 1]

In Formula 1,

A is a C 2-10 linker, and includes -S-, -SS-, -SSS-, -SeSe-, -Se-, -B-, an acetal or a ketal;

X is NH or O.
According to claim 1,

The A is
,
, and
Any one selected from the group consisting of;

m 1 , m 2 , p 1 and p 2 are each an integer of 0 to 10, an obeticholic acid dimer compound, a solvate thereof, or a pharmaceutically acceptable salt.
According to claim 1,

The dimer compound is an obeticholic acid dimer compound, a solvate thereof, or a pharmaceutically acceptable salt thereof, to which a compound represented by Formula 1a is bound:

[Formula 1a]

.
According to claim 1,

The dimer compound is an obeticholic acid dimer compound represented by any one of the following formulas 1b to 1e, a solvate thereof, or a pharmaceutically acceptable salt thereof:

[Formula 1b]

[Formula 1c]

;

[Formula 1d]

; and

[Formula 1e]

.
A nanoparticle comprising the obeticholic acid dimer compound according to any one of claims 1 to 4, a solvate thereof, or a pharmaceutically acceptable salt thereof.
According to claim 5,

The nanoparticles further comprising fucoidan, the nanoparticles.
According to claim 6,

The nanoparticle size is 250 ㎚ to 500 ㎚ , nanoparticles.
According to claim 6,

The fucoidan is contained in 10 to 30 parts by weight based on 100 parts by weight of the dimer compound, nanoparticles.
A pharmaceutical composition comprising the nanoparticles of claim 5 or 6.
According to claim 9,

The pharmaceutical composition is for the prevention or treatment of liver disease, a pharmaceutical composition.
A method for treating liver disease comprising administering the obeticholic acid dimer compound, a solvate thereof, or a pharmaceutically acceptable salt of any one of claims 1 to 4 to a subject suffering from liver disease.
A method for treating liver disease comprising administering a pharmaceutical composition comprising the nanoparticles of claim 5 or 6 to a subject suffering from liver disease.
Use of the obeticholic acid dimer compound according to any one of claims 1 to 4, a solvate thereof, or a pharmaceutically acceptable salt for use in the prevention or treatment of liver disease.
Use of the obeticholic acid dimer compound according to any one of claims 1 to 4, a solvate thereof, or a pharmaceutically acceptable salt for the manufacture of a medicament used for the prevention or treatment of liver disease.