Compositions containing methylcyclodextrin for the treatment and / or prevention of fatty liver

Abstract

The present invention relates to a novel use of pharmaceutical compositions containing at least one methyl cyclodextrin in the treatment and / or prevention of fatty liver and related diseases. The present invention also relates to the use of methyl cyclodextrins to reduce lipid accumulation in the liver.

Description

Technical Field

[0001] The present invention relates to a pharmaceutical composition for reducing lipid storage in an individual's liver. More specifically, the present invention relates to the use of a pharmaceutical composition in the treatment and / or prevention of fatty liver and diseases or conditions associated with fatty liver.

Background Art

[0002] Non-alcoholic steatohepatitis (non-alcoholic fatty liver disease or NAFLD) is characterized by abnormal accumulation of fat in the liver without excessive alcohol intake.

[0003] NAFLD encompasses a broad range of liver pathologies that can be distinguished into two main entities: fatty liver without or with minimal lobular inflammation (non-alcoholic fatty liver or NAFL) and non-alcoholic steatohepatitis (NASH). NASH is defined by the presence of fatty liver with lobular inflammation and ballooning of hepatocytes. It corresponds to an aggressive form of the disease that promotes the accumulation of fibrosis in the liver parenchyma and progresses to cirrhosis and its complications (liver failure, ascites, rupture of varices, hepatocellular carcinoma).

[0004] The social and medical care impacts of NAFLD and NASH are very significant and are constantly increasing. It is estimated that approximately 30% of the US population suffers from NAFLD. The prevalence of NASH in the US is 8%.

[0005] Several genetic factors have been identified that predispose individuals to NAFLD and its severity. NAFLD occurs in a context of metabolic abnormalities and insulin resistance. The accumulation of metabolic syndrome criteria (waist size, arterial pressure, fasting glucose levels, triglycerides, HDL cholesterol) and the degree of insulin resistance are associated with the prevalence of NAFLD and the exacerbation of its severity (NASH, fibrosis).

[0006] Environmental factors (poor diet, lack of sports activity) are also important risk factors.

[0007] NAFLD is generally a slowly progressing disease, but its natural course remains largely unknown. NASH is an aggressive form of this disease, and compared to patients with NAFL alone, patients with NASH experience faster progression of fibrosis, are more likely to develop cirrhosis, have a higher incidence of liver complications, and have a higher mortality rate.

[0008] The mechanisms involved in the development of this disease include the accumulation of lipids in the liver, followed by inflammation and scarring. In the most advanced stage (cirrhosis), liver tissue is gradually replaced by scar tissue.

[0009] The treatments currently considered are insufficient and, in most cases, act downstream of the pathological process. Examples include caspase or ASK1 inhibitors that act on the cell death process that causes inflammation.

[0010] Finally, there are currently no drugs on the pharmaceutical market that can act upstream to limit lipid accumulation in the liver. Furthermore, there is a constant need for drugs that can control fatty liver and related diseases or conditions. [Overview of the project]

[0011] The applicant's achievement lies in discovering that a methylcyclodextrin-based pharmaceutical composition can reduce lipid storage in the liver and is therefore useful for the treatment and / or prevention of fatty liver disease.

[0012] Accordingly, the present invention relates to a pharmaceutical composition comprising at least one methylcyclodextrin for use in the treatment and / or prevention of fatty liver and related diseases.

[0013] The present invention also relates to the use of methylcyclodextrin for the manufacture of pharmaceuticals for use in the treatment and / or prevention of fatty liver.

[0014] Furthermore, the present invention provides a method for treating and / or preventing fatty liver, comprising administering a therapeutically effective amount of methylcyclodextrin to a patient. [Modes for carrying out the invention]

[0015] The inventors have identified novel uses for pharmaceutical compositions containing at least one methylcyclodextrin in the treatment and / or prevention of fatty liver and related diseases.

[0016] In the context of this invention, the term "fatty liver" encompasses both alcoholic fatty liver and non-alcoholic fatty liver associated with excessive alcohol consumption. Preferably, it includes non-alcoholic fatty liver.

[0017] The term "non-alcoholic fatty liver disease" encompasses all stages of the progression of a condition in which the liver is affected and characterized by excessive lipid accumulation. It may also refer to non-alcoholic fatty liver disease (NAFLD) or non-alcoholic steatohepatitis (NASH).

[0018] The present invention also relates to the treatment and / or prevention of diseases or conditions associated with fatty liver, such as acute or chronic hepatitis, hepatic fibrosis, abdominal obesity, liver failure, and cirrhosis.

[0019] In one embodiment of the present invention, the disease associated with fatty liver is not diabetes.

[0020] Cyclodextrins are cyclic oligosaccharides derived from the enzymatic hydrolysis of starch. The three most common natural cyclodextrins consist of 6, 7, or 8 α-D-glucopyranose units in a chair configuration linked to one another by α-1,4 bonds. They are more commonly referred to as α, β, or γ cyclodextrins, respectively. Their three-dimensional structure is in the form of a frustocone, with the outer part being the hydroxyl group, which is the highly hydrophilic portion of the cyclodextrin. The interior of the cone or cavity of the cyclodextrin consists of hydrogen atoms bonded to the C3 and C5 carbons, as well as oxygen atoms involved in the glycosidic bond, thus giving them nonpolar properties. Cyclodextrins with a hydrophilic outer portion and a hydrophobic cavity are commonly used because of their ability to encapsulate hydrophobic compounds, and therefore for their role as protectors and solubilizers of hydrophobic active ingredients. These are typically found in the food industry, but they can also be found in pharmaceutical forms used as excipients in orally administered pharmaceutical formulations or topically administered cosmetic formulations.

[0021] To improve the water solubility of natural cyclodextrins, many derivatives have been synthesized by grafting various groups onto the hydroxyl functional group. Specifically, the glucopyranose unit of cyclodextrin contains three reactive hydroxyl groups bonded to the C2, C3, and C6 carbon atoms, respectively.

[0022] Examples of derivatives include hydroxypropyl cyclodextrin, methylcyclodextrin, and "sulfated" derivatives of cyclodextrin.

[0023] Surprisingly, the applicant demonstrated that methylcyclodextrin can also be used for the treatment and / or prevention of fatty liver disease. Even more surprisingly, the applicant found that methylcyclodextrin, more specifically methylcyclodextrin with molar substitution values ​​of 0.05 to 1.5, is even more effective than other cyclodextrin derivatives in reducing lipid accumulation in the liver.

[0024] Furthermore, the applicant demonstrated that methylcyclodextrins having molar substitution values ​​of 0.05 to 1.5 can promote increased cholesterol removal.

[0025] The term "molar substitution value (MS)" is understood to mean the number of hydroxyls substituted by methyl groups in each glucopyranose unit. It should be noted that the molar substitution value (MS) corresponds to the number of hydroxyls substituted by methyl groups per cyclodextrin molecule and is therefore different from the degree of molecular substitution (DS), which takes into account the number of glucopyranose units constituting methylcyclodextrin.

[0026] In the present invention, MS can be determined by nuclear magnetic resonance (NMR) or by mass spectrometry (electrospray ionization mass spectrometry (ESI-MS) or matrix-assisted laser desorption / ionization mass spectrometry (MALDI-MS)). While these techniques are known to those skilled in the art, the optimal conditions for determining the methylcyclodextrin of the present invention are particularly well described in Chapter 2, Part B (pp. 59-83) of Romain JACQUET's reference paper, "Hydrophilic cyclodextrins: characterization and study of their enantioselective and complexing properties. Use of liquid chromatography and mass spectrometry," Thesis in Chemistry and Physicochemistry of Compounds of Biological Interest, University of Orleans, 2006, available at http: / / tel.archives-ouvertes.fr / docs / 00 / 18 / 55 / 42 / PDF / jacquet.pdf (accessed November 27, 2013).

[0027] Preferably, MS is determined by NMR according to the following method, i.e., the measurement is performed at 25°C using a DPX 250 MHz Advance instrument (Bruker, Rheinstetten, Germany). Calibration is performed using a D2O signal. Samples of the methylcyclodextrin of the present invention and natural (i.e., unmethylated) cyclodextrin are prepared at a concentration of 5 mg in 0.75 mL of D2O. The solution is evaporated to dryness under a stream of nitrogen and then reconstituted in 0.75 mL of D2O. This operation is repeated twice to ensure the exchange of all protons of the hydroxyl functional group.

[0028] The methylcyclodextrins used in the present invention may correspond to pure products, but it should be noted that generally they correspond to mixtures of methylcyclodextrins with different structures. This is the case, for example, of the product KLEPTOSE® Crysmeb owned by the applicant, which has physical / chemical properties as determined, in particular, in the aforementioned article by Romain JACQUET, particularly in chapter 2, part B (pages 59 - 83).

[0029] As a result, the measured MS in this case is the average of the substitutions occurring at all the glucopyranose units of the whole mixture of methylcyclodextrins.

[0030] This mixture may in particular contain residual natural, i.e. non-methylated cyclodextrins, but generally in negligible amounts, particularly less than 1% by dry weight, preferably less than 0.5% by dry weight, and even more preferably less than 0.1% by dry weight with respect to the total dry weight of the methylcyclodextrins.

[0031] In the context of the present invention, the composition comprises at least one methylcyclodextrin having a molar substitution value of 0.05 to 1.5. Advantageously, the methylcyclodextrin has an MS of 0.1 to 1.4, preferably 0.1 to 1.3, preferably 0.2 to 1.2, preferably 0.3 to 1.1, preferably 0.3 to 1, preferably 0.5 to 0.9, preferably 0.6 to 0.8, for example 0.7, and particularly 0.67. For example, methylcyclodextrin has a range of 0.10-1.40, 0.10-1.30, 0.10-1.20, 0.15-1.40, 0.15-1.30, 0.15-1.20, 0.20-1.40, 0.20-1.30, 0.20-1.20, 0.20-1.10, 0.25-1.40, 0.25-1.30, 0 It may have MS values ​​of 0.25~1.20, 0.25~1.10, 0.15~0.90, 0.15~0.80, 0.25~1.00, 0.25~0.90, 0.25~0.80, 0.30~1.40, 0.30~1.30, 0.30~1.20, 0.30~1.00, 0.50~0.90, and 0.60~0.80.

[0032] Preferably, at least 50%, preferably 60-80%, and typically about 75% of the methyl groups of the methylcyclodextrin used in the context of the present invention are located at hydroxyls bonded to the C2 carbon of the glucopyranose unit.

[0033] At the same time, other methyl groups are generally mainly located on hydroxyls bonded to the C3 and / or C6 carbons of the glucopyranose unit.

[0034] Those skilled in the art know how to determine the distribution of methyl groups on the hydroxyl group of the glucopyranose unit of methylcyclodextrin, for example, by NMR.

[0035] Advantageously, the methylcyclodextrin used in the context of this invention contains seven α-D-glucopyranose units. Therefore, it is methyl-β-cyclodextrin.

[0036] In certain embodiments, the methylcyclodextrin is methyl-β-cyclodextrin and has an MS of 0.05-1.5, preferably 0.1-1.4, preferably 0.1-1.3, preferably 0.2-1.2, preferably 0.3-1.1, preferably 0.4-1, preferably 0.5-0.9, preferably 0.6-0.8, for example 0.7, particularly 0.67. For example, methylcyclodextrin may have MS values ​​of 0.10-1.40, 0.10-1.30, 0.10-1.20, 0.15-1.40, 0.15-1.30, 0.15-1.20, 0.20-1.40, 0.20-1.30, 0.20-1.20, 0.20-1.10, 0.25-1.40, 0.25-1.30, 0.25-1.20, 0.25-1.10, 0.25-1.00, 0.25-0.90, 0.25-0.80, 0.30-1.40, 0.30-1.30, 0.30-1.20, 0.30-1.00, 0.50-0.90, and 0.60-0.80.

[0037] Methylcyclodextrin may be substituted on a hydroxyl bonded to the C2 carbon of the glucopyranose unit, or on a hydroxyl bonded to the C3 and / or C6 carbon of the glucopyranose unit, or on a hydroxyl bonded to the C2, C3 and / or C6 carbon of the glucopyranose unit, preferably a combination of C2 and C6 carbons.

[0038] In another specific embodiment, the methylcyclodextrin is one type of methylcyclodextrin, preferably methyl-β-cyclodextrin, wherein at least 50%, preferably 60-80%, typically about 75%, of its methyl groups are located on a hydroxyl bonded to the C2 carbon of the glucopyranose unit, and has an MS of 0.05-1.5, preferably 0.1-1.4, preferably 0.1-1.3, preferably 0.2-1.2, preferably 0.3-1.1, preferably 0.4-1, preferably 0.5-0.9, preferably 0.6-0.8, for example 0.7, particularly 0.67. For example, methylcyclodextrin may have MS values ​​of 0.10-1.40, 0.10-1.30, 0.10-1.20, 0.15-1.40, 0.15-1.30, 0.15-1.20, 0.20-1.40, 0.20-1.30, 0.20-1.20, 0.20-1.10, 0.25-1.40, 0.25-1.30, 0.25-1.20, 0.25-1.10, 0.25-1.00, 0.25-0.90, 0.25-0.80, 0.30-1.40, 0.30-1.30, 0.30-1.20, 0.30-1.00, 0.50-0.90, and 0.60-0.80.

[0039] In a preferred embodiment, the methylcyclodextrin composition comprises one or more methyl-β-cyclodextrins selected from the group consisting of methyl-β-cyclodextrin substituted on a hydroxyl bonded to the C2 carbon of the glucopyranose unit, methyl-β-cyclodextrin substituted on a hydroxyl bonded to the C3 and / or C6 carbon of the glucopyranose unit, and methyl-β-cyclodextrin substituted on a hydroxyl bonded to the C2, C3 and / or C6 carbon of the glucopyranose unit, preferably the C2 and C6 carbons, and having an MS of 0.05 to 1.5, preferably 0.1 to 1.4, preferably 0.1 to 1.3, preferably 0.2 to 1.2, preferably 0.3 to 1.1, preferably 0.4 to 1, preferably 0.5 to 0.9, preferably 0.6 to 0.8, for example 0.7, particularly 0.67. For example, methylcyclodextrin may have MS values ​​of 0.10-1.40, 0.10-1.30, 0.10-1.20, 0.15-1.40, 0.15-1.30, 0.15-1.20, 0.20-1.40, 0.20-1.30, 0.20-1.20, 0.20-1.10, 0.25-1.40, 0.25-1.30, 0.25-1.20, 0.25-1.10, 0.25-1.00, 0.25-0.90, 0.25-0.80, 0.30-1.40, 0.30-1.30, 0.30-1.20, 0.30-1.00, 0.50-0.90, and 0.60-0.80. Preferably, the methylcyclodextrin composition has at least 50, 60, or 75% methyl molecules substituted on the hydroxyl group bonded to the C2 carbon of the glucopyranose unit.

[0040] As described above, the methylcyclodextrins of the present invention may be mixtures. Mass spectrometry of the product KLEPTOSE® CRYSMEB, which is methyl-β-cyclodextrin, reveals that it is a polydisperse product containing seven major groups of methylcyclodextrins distinguished by their DS. Theoretically, this DS, which can vary from 0 to 21 in methyl-β-cyclodextrin, varies from 2 to 8 in the product KLEPTOSE® CRYSMEB.

[0041] Advantageously, the composition of the present invention comprises a mixture of methylcyclodextrins containing at least 50, 60, 70, 80, or 90% of methylcyclodextrin having an MS of 0.2 to 1.2. Preferably, at least 40, 50, 60, 70, 80, or 90% of methylcyclodextrin have an MS of 0.3 to 1.1. Preferably, at least 30, 40, 50, 60, 70, 80, or 90% of the methylcyclodextrin have an MS of 0.5 to 0.9. Even more preferentially, at least 25, 30, 40, 50, 60, 70, 80, or 90% of the methylcyclodextrin have an MS of 0.6 to 0.8.

[0042] The methylcyclodextrin composition may optionally be prepared by adding various methylcyclodextrins having MS values ​​determined to obtain the composition defined in the present invention, or they may be obtained as a result of such synthesis.

[0043] Therefore, in another specific embodiment, the methylcyclodextrin composition, preferably the methyl-β-cyclodextrin composition, has a substitution profile corresponding to the following, expressed in mole percentages. - 0-5% methyl-β-cyclodextrin containing 2 methyl groups (DS2), - 5-15% methyl-β-cyclodextrin containing 3 methyl groups (DS = 3), - 20-25% methyl-β-cyclodextrin containing 4 methyl groups (DS = 4), - 25-40% methyl-β-cyclodextrin containing 5 methyl groups (DS = 5), - 15-25% methyl-β-cyclodextrin containing 6 methyl groups (DS 6), - 5-15% methyl-β-cyclodextrin containing 7 methyl groups (DS = 7), - It is a methyl-β-cyclodextrin containing 8 methyl groups (DS 8) and making up 0-5% of the total.

[0044] Here, the composition may optionally contain trace amounts of different DS methylcyclodextrins, as well as trace amounts of natural, i.e., unmethylated cyclodextrins, but the total is generally about 100%.

[0045] The substitution profile can be determined by any technique known to those skilled in the art, for example, by ESI-MS or MALDI-TOF-MS. The optimal conditions for determining the substitution profile by these two methods are discussed in detail, in particular, in Chapter 2, Part B, Points II.3 and II.2 (pp. 67-82) and Appendix II of the aforementioned paper by Romain JACQUET.

[0046] In a preferred embodiment, the composition of methylcyclodextrin, preferably methyl-β-cyclodextrin, is such that at least 50%, preferably 60-80%, and typically about 75% of the methyl groups are located at hydroxyls bonded to the C2 carbon of the glucopyranose unit, and has the following substitution profile expressed as a mole percentage. - 0-5% methyl-β-cyclodextrin containing 2 methyl groups (DS2), - 5-15% methyl-β-cyclodextrin containing 3 methyl groups (DS = 3), - 20-25% methyl-β-cyclodextrin containing 4 methyl groups (DS = 4), - 25-40% methyl-β-cyclodextrin containing 5 methyl groups (DS = 5), - 15-25% methyl-β-cyclodextrin containing 6 methyl groups (DS 6), - 5-15% methyl-β-cyclodextrin containing 7 methyl groups (DS = 7), - It is a methyl-β-cyclodextrin containing 8 methyl groups (DS = 8), Here, the composition may optionally contain trace amounts of different DS methylcyclodextrins, as well as trace amounts of natural, i.e., unmethylated cyclodextrins, but the total is generally about 100%.

[0047] Furthermore, depending on the DS in particular, it is possible to explore changing the proportion of methylcyclodextrin molecules or groups, or to separate methylcyclodextrin molecules or groups.

[0048] Therefore, in another specific embodiment, the methylcyclodextrin is a methyl-β-cyclodextrin exhibiting an integer DS in the range of 2 to 8, particularly selected from 2, 3, 4, 5, 6, 7, or 8.

[0049] In another preferred embodiment, the methylcyclodextrin is a methyl-β-cyclodextrin in which at least 50%, preferably 60-80%, typically about 75%, of the methyl groups are located on a hydroxyl bonded to the C2 carbon of the glucopyranose unit, and the DS is an integer in the range of 2-8, particularly selected from 2, 3, 4, 5, 6, 7, or 8.

[0050] In another specific embodiment, methylcyclodextrin, particularly methyl-β-cyclodextrin, has an MS of 0.1-0.3, particularly 0.2-0.3, particularly 0.20-0.30. In another specific embodiment, methylcyclodextrin, particularly methyl-β-cyclodextrin, has an MS of 0.3-0.5, particularly 0.30-0.50. In another specific embodiment, methylcyclodextrin, particularly methyl-β-cyclodextrin, has an MS of 0.5-0.6, particularly 0.50-0.60. In another specific embodiment, methylcyclodextrin, particularly methyl-β-cyclodextrin, has an MS of 0.6-0.7, particularly 0.60-0.70. In another specific embodiment, methylcyclodextrin, particularly methyl-β-cyclodextrin, has an MS of 0.7-0.8, particularly 0.70-0.80. In another specific embodiment, methylcyclodextrin, particularly methyl-β-cyclodextrin, has an MS of 0.8 to 0.9, particularly 0.80 to 0.90. In another specific embodiment, methylcyclodextrin, particularly methyl-β-cyclodextrin, has an MS of 0.9 to 1.1, particularly 0.90 to 1.10. In another specific embodiment, methylcyclodextrin, particularly methyl-β-cyclodextrin, has an MS of 1.1 to 1.2, particularly 1.10 to 1.20.

[0051] Generally, the reducing sugar level of the methylcyclodextrin used in the present invention is less than 1% by dry weight, preferably less than 0.5%.

[0052] The methyl-β-cyclodextrin composition of the present invention can be obtained by the method described in U.S. Patent No. 6,602,860(B1). An example of such a composition is the product KLEPTOSE® CRYSMEB, which is methylated with a molar substitution value of 0.7, more precisely 0.67, per glucose unit.

[0053] Optionally, the compositions of the present invention may further contain an unsubstituted cyclodextrin, particularly β-cyclodextrin, and / or a cyclodextrin, particularly β-cyclodextrin, that is substituted with a sulfobutyl ether (SBE-), hydroxyethyl, hydroxypropyl (HP-), carboxymethyl, carboxyethyl, acetyl, triacetyl, succinyl, ethyl, propyl, butyl, or sulfate group, preferably sulfobutyl and hydroxypropyl, and having a molar substitution value of 0.05 to 1.5.

[0054] However, preferably, the compositions of the present invention do not contain cyclodextrins other than methylcyclodextrin (and optionally, trace amounts of natural cyclodextrin as described above) which are useful for the present invention.

[0055] Optionally, the methylcyclodextrin of the present invention, in particular methyl-β-cyclodextrin, may be substituted with additional groups selected from those listed above. For example, it may be sulfated methyl-β-cyclodextrin.

[0056] However, preferably, the methylcyclodextrin useful in the present invention, particularly methyl-β-cyclodextrin, is not substituted with any group other than a methyl group.

[0057] In another embodiment of the present invention, methylcyclodextrin, as defined in this application and comprising α-D-glucopyranose units linked to one another by α-1,4 bonds, may be partially or completely replaced in the pharmaceutical composition of the present invention by α-D-glucopyranose units linked to one another by α-1,6 bonds.

[0058] In a preferred embodiment of the present invention, methylcyclodextrin is the sole active ingredient of the pharmaceutical composition.

[0059] In another embodiment, the pharmaceutical composition further comprises one or more active ingredients typically selected from those useful for the prevention and / or treatment of symptoms and / or conditions associated with fatty liver.

[0060] The compositions of the present invention may also contain at least one pharmaceutically acceptable excipient. All excipients suitable for galenic forms known to those skilled in the art can be used for systemic administration, preferably orally, parenterally, or cutaneously or via mucous membranes, and especially subcutaneously, intravenously, intramuscularly, intraperitoneally, nasally, pulmonaryly, rectally, cutaneously, intrathecally or spinally, preferably orally.

[0061] Examples include solutions suitable for pharmaceutical use and known to those skilled in the art, such as physiological saline, physiological solutions, isotonic solutions, and buffer solutions. The composition may contain one or more agents or vehicles selected from dispersants, solubilizers, stabilizers, preservatives, etc. Agents or vehicles that can be used in formulations (liquid and / or for injection) include, in particular, methylcellulose, hydroxymethylcellulose, carboxymethylcellulose, polysorbate 80, mannitol, gelatin, lactose, vegetable oils, acacia, and liposomes. The composition can be formulated in the form of injectable suspensions, gels, oils, tablets, suppositories, powders, gel capsules, capsules, aerosols, etc., optionally using galenic forms or devices to ensure sustained and / or delayed release. In this type of formulation, agents such as cellulose, carbonates, or starches are usefully used.

[0062] In the context of the present invention, a composition that can be administered to an individual by injection contains methylcyclodextrin as defined in the present invention in an amount of 1 to 100 mg / kg, preferably 20 to 70 mg / kg, more preferably 30 to 50 mg / kg, and even more preferably 40 mg / kg, relative to the total body weight of the individual. Of course, those skilled in the art can adjust the dose of methylcyclodextrin as defined in this application depending on the body weight of the individual being treated and the mode of administration.

[0063] In a preferred embodiment of the present invention, the pharmaceutical composition can be administered orally.

[0064] When the pharmaceutical composition of the present invention is used in oral form, the amount of methylcyclodextrin administered is such that it can reduce the lipid storage in the patient's liver. The oral dose may be, for example, 10 mg / kg / day to 10,000 mg / kg / day, preferably 20 mg / kg / day to 7,000 mg / kg / day, 50 mg / kg / day to 5,000 mg / kg / day, 75 mg / kg / day to 4,000 mg / kg / day, 100 mg / kg / day to 3,000 mg / kg / day, 200 mg / kg / day to 2,000 mg / kg / day, 300 mg / kg / day to 1,000 mg / kg / day, and more preferably 400 mg / kg / day to 800 mg / kg / day. [Brief explanation of the drawing]

[0065] The following embodiments are used to illustrate and illustrate other aspects and advantages of the present invention and should be considered non-limiting. [Figure 1] Figure 1 shows the changes in body weight over time for the four groups of animals in Example 2. [Figure 2] Figure 2 is a schematic diagram of lipid biosynthesis in de novo and the various genes involved in this biosynthetic pathway. [Figure 3-1] Figure 3 shows the expression levels of various genes involved in de novo lipid biosynthesis in the four animal groups of Example 2. [Figure 3-2] Figure 3 shows the expression levels of various genes involved in de novo lipid biosynthesis in the four animal groups of Example 2. [Figure 3-3] Figure 3 shows the expression levels of various genes involved in de novo lipid biosynthesis in the four animal groups of Example 2. [Figure 3-4] Figure 3 shows the expression levels of various genes involved in de novo lipid biosynthesis in the four animal groups of Example 2. [Figure 4] Figure 4 is a schematic diagram of the various enzymes involved in cholesterol synthesis in de novo settings. [Figure 5-1] Figure 5 shows the expression levels of various genes involved in de novo cholesterol synthesis in the four animal groups of Example 2. [Figure 5-2] Figure 5 shows the expression levels of various genes involved in de novo cholesterol synthesis in the four animal groups of Example 2. [Figure 5-3] Figure 5 shows the expression levels of various genes involved in de novo cholesterol synthesis in the four animal groups of Example 2. [Figure 6-1] Figure 5 shows the expression levels of various genes involved in de novo cholesterol synthesis in the four animal groups of Example 2. [Figure 6-2] Figure 5 shows the expression levels of various genes involved in de novo cholesterol synthesis in the four animal groups of Example 2. [Figure 6-3] Figure 5 shows the expression levels of various genes involved in de novo cholesterol synthesis in the four animal groups of Example 2. [Figure 7] Figure 7 shows the changes in body weight over time for the four groups of animals in Example 3. [Figure 8] Figure 8 shows the liver and adipose tissue weights at the end of the experiment in the four animal groups of Example 3. [Figure 9-1] Figure 9 shows the time course of serum cholesterol levels, triglycerides, unsaturated fatty acids, and LDLc in the four animal groups of Example 3. [Figure 9-2] Figure 9 shows the time course of serum cholesterol levels, triglycerides, unsaturated fatty acids, and LDLc in the four animal groups of Example 3. [Figure 10] Figure 10 shows the measurement results of the percentage of intrahepatic lipids and the amount of cholesterol stored in the liver of the four animal groups in Example 3. [Examples]

[0066] Example 1: Materials and Method Male LVG Golden Syrian hamsters were used in the following experiment. The base diet consisted of AO4C feed sold by SAFE diet. Cholesterol was added to the high-cholesterol diet (obtained from MP Biomedicals).

[0067] Various cyclodextrins were tested. -Firstly, hydroxypropyl-β-cyclodextrin (HPBCD), which is sold by the applicant under the name "KLEPTOSE® HPB" (oral grade), - Secondly, the applicant's proprietary methyl-β-cyclodextrin (MCD) "KLEPTOSE® CRYSMEB".

[0068] Example 2: Preventive effect of methyl-β-cyclodextrin on liver pathology associated with hypercholesterolemia. In this study, 40 Golden Syrian LVG hamsters were divided into four groups of 10 each and fed the following diet for 6 weeks. - "Control group": Normal diet - "Control HC" group: High cholesterol diet (containing 2.5% by weight of cholesterol) - "HC+Crysmeb" group: High cholesterol diet (containing 2.5% by weight cholesterol) + 3% by weight Crysmeb (MCD) - "HC+HPBCD" group: High cholesterol diet (containing 2.5% by weight cholesterol) + 3% by weight HPBCD

[0069] Physiological markers Figure 1 shows the changes in the weight of the animals during the experiment. It can be seen that the high-cholesterol diet did not affect weight. Conversely, the two groups that received cyclodextrin as supplementation showed slower growth.

[0070] At the end of the 42-day experiment, the animals were slaughtered and the weight of various organs was measured.

[0071] Table 1 lists the weights of aorta, brain, liver, small intestine, and epididymal adipose tissue in various treatment groups.

[0072] [Table 1]

[0073] As shown in Table 1, the tissues most affected by cyclodextrin treatment were those involved in oil storage: liver and epididymal adipose tissue (which strongly correlates with the individual's total fat mass). The effect of reducing fat mass was most pronounced with MCD Crysmeb.

[0074] Table 2 shows the plasma triglyceride levels of various groups at different time points: the start of the study (day 0, D0), day 14 (D14), day 28 (D28), and the end of the study (day 43, D43).

[0075] [Table 2]

[0076] The HC diet induced a significant increase in blood triglyceride levels compared to the control group. This increase was normalized by the addition of cyclodextrin, and the effect of MCD Crysmeb was more pronounced.

[0077] Table 3 shows the biochemical data measured in the livers of animals at the end of the 42-day treatment period.

[0078] [Table 3]

[0079] As shown in Table 3, the HC diet causes a significant increase in the amount of lipids stored in the liver, primarily in the form of cholesterol but also in the form of triglycerides. The addition of cyclodextrin to the feed had the effect of reducing this storage. This effect was even more pronounced in MCD, where normal levels of % lipid, cholesterol, and triglyceride storage were measured.

[0080] Biomarker (amount of messenger RNA expressed in the liver) The expression levels of various genes involved in de novo lipid biosynthesis (Figure 3), de novo cholesterol synthesis (Figure 5), and cholesterol removal (Figure 6) were measured in the livers of four groups of animals at the end of the study.

[0081] A cholesterol-rich diet induces increased SCD1 expression, which should lead to increased production of unsaturated fatty acids such as triglycerides. Addition of HPBCD to the feed inhibited the expression of SCD1 and ACC genes compared to the control group, which may be related to decreased synthesis and storage of fatty acids such as triglycerides. Addition of MCD to the feed produced an even more pronounced effect than HPBCD, resulting in overall inhibition of the expression of FAS, ACC, SCD1, and SREBP1 genes (Figure 3).

[0082] As shown in Figure 5, the HC diet resulted in decreased CYP51 gene expression compared to the control group, and therefore decreased cholesterol synthesis. The addition of HPBCD had little effect. In contrast, the addition of Crysmeb MCB increased the expression of SREB2, CYP51a1, and HMGCR genes, and therefore increased cholesterol synthesis instead of fatty acid synthesis.

[0083] As shown in Figure 6, the addition of MCD normalizes the expression of genes involved in cholesterol removal compared to the HC group. This normalization is greater with MCD than with HPBCD.

[0084] In conclusion, various measured parameters indicate that the methylcyclodextrin of the present invention can effectively suppress disorders caused by a high-cholesterol diet (increased fatty acid storage in the liver and increased fatty acid levels).

[0085] The use of methylcyclodextrin according to the present invention promotes a reduction in lipid accumulation and an increase in cholesterol removal in the liver.

[0086] These effects of MCD in the present invention are greater than those of HPBCD, another cyclodextrin.

[0087] Example 3: Therapeutic effect of methyl-β-cyclodextrin on liver pathologies associated with hypercholesterolemia The same protocol as in Example 2 was followed, but in this case, the first stage of inducing hypercholesterolemia was performed for two weeks, followed by the second stage of treatment, during which cyclodextrin was added to the diets of the "HC+Crysmeb" and "HC+HPBCD" groups.

[0088] In this study, 40 Golden Syrian LVG hamsters were divided into four groups of 10 each and fed the following diet for 6 weeks. - "Control group": Normal diet - "Control HC" group: High cholesterol diet (containing 2.5% by weight of cholesterol) - "HC+Crysmeb" group: High cholesterol diet from D1 to D14 (containing 2.5% cholesterol by weight), followed by a high cholesterol diet from D15 to D42 (containing 2.5% cholesterol by weight) + 3% Crysmeb (MCD) - "HC+HPBCD" group: High cholesterol diet from D1 to D14 (containing 2.5% by weight of cholesterol), followed by a high cholesterol diet from D15 to D42 (containing 2.5% by weight of cholesterol) + 3% by weight of HPBCD

[0089] Physiological markers Figure 7 shows the changes in the weight of the animals during the experiment. It can be seen that the high-cholesterol diet did not affect weight. Conversely, the two groups that received cyclodextrin as supplementation showed slower growth.

[0090] At the end of the 42-day experiment, the animals were slaughtered and the weight of various organs was measured.

[0091] Figure 8, which shows the weight of liver and epididymal adipose tissue in various treatment groups, indicates that the tissues most affected by cyclodextrin treatment were those involved in oil storage: liver and epididymal adipose tissue (strongly correlated with the individual's total fat mass). The effect of reducing fat mass was most pronounced with MCD Crysmeb.

[0092] Figure 9 shows the serum concentrations of cholesterol, unsaturated fatty acids, triglycerides, and LDLc in various treatment groups over time.

[0093] The HC diet was found to cause a significant increase in blood triglyceride and cholesterol levels compared to the control group. Addition of MCD Crysmeb 14 days after the HC diet significantly reduced these concentrations, bringing them close to those of the control group.

[0094] As shown in Figure 10, the HC diet causes a significant increase in the amount of lipids stored in the liver, particularly in the form of cholesterol. The addition of cyclodextrin to the diet after the induction period had the effect of reducing this storage.

[0095] In the HC group, histological examination revealed the appearance of microvesicular vacuoles and infiltration by inflammatory cells. Exposure to MCD reduced the severity of these histological signs in the HC + Crysmeb group (data not shown).

[0096] In conclusion, various measured parameters indicate that the methylcyclodextrin of the present invention can effectively suppress disorders caused by a high-cholesterol diet (increased fatty acid storage and increased fatty acid levels in the liver), even when MCD is administered after the induction period of these disorders.

[0097] The use of methylcyclodextrin in the present invention promotes a reduction in lipid accumulation and an increase in cholesterol removal in the liver, and has therapeutic effects in addition to the preventive effects demonstrated in Example 2.

[0098] These effects are greater with MCD of the present invention than with HPBCD, another cyclodextrin.

[0099] Therefore, the applicant has found that the methylcyclodextrin of the present invention allows for the following: - To reduce lipid storage in the body, especially in the liver. - To increase metabolism and cholesterol removal, - To improve the tissue structure of the liver. This demonstrated that it is possible.

[0100] These effects were observed when the methylcyclodextrin of the present invention was administered during the induction of hypercholesterolemia (preventive effect model) or after the onset of hypercholesterolemia induction (therapeutic effect model).

[0101] The observed effect was greater than that observed with another cyclodextrin, hydroxypropyl-β-cyclodextrin.

Claims

1. A pharmaceutical composition for the treatment of fatty liver and related diseases selected from acute or chronic hepatitis, hepatic fibrosis, abdominal obesity, liver failure and cirrhosis, comprising at least one methylcyclodextrin as an active ingredient, wherein the methylcyclodextrin has a molar substitution value of 0.05 to 1.5, for oral administration.

2. A pharmaceutical composition for the treatment of fatty liver and related diseases selected from acute or chronic hepatitis, hepatic fibrosis, abdominal obesity, liver failure and cirrhosis, comprising at least one methylcyclodextrin as an active ingredient, wherein the methylcyclodextrin is the sole active ingredient, and for oral administration.

3. The pharmaceutical composition according to claim 1 or 2, characterized in that the fatty liver is selected from non-alcoholic fatty liver disease ("non-alcoholic fatty liver disease" NAFLD) and non-alcoholic steatohepatitis (NASH).

4. The pharmaceutical composition according to claim 2, characterized in that the methylcyclodextrin has a molar substitution value of 0.05 to 1.

5.

5. The pharmaceutical composition according to any one of claims 1 to 4, characterized in that the methylcyclodextrin is methyl-β-cyclodextrin.

6. The pharmaceutical composition according to any one of claims 1 to 5, characterized in that the methylcyclodextrin is substituted on a hydroxyl bonded to the C2 carbon of the glucopyranose unit, or on a hydroxyl bonded to the C3 and / or C6 carbon of the glucopyranose unit, or on a hydroxyl bonded to the C2, C3 and / or C6 carbon of the glucopyranose unit.

7. The methylcyclodextrin is bonded to the C2 carbon of the glucopyranose unit. A pharmaceutical composition according to any one of claims 1 to 6, comprising one or more methyl-β-cyclodextrins selected from the group consisting of methyl-β-cyclodextrin substituted on a hydroxyl group, methyl-β-cyclodextrin substituted on a hydroxyl group bonded to the C3 and / or C6 carbon of the glucopyranose unit, and methyl-β-cyclodextrin substituted on a hydroxyl group bonded to the C2, C3 and / or C6 carbon of the glucopyranose unit, wherein the methyl-β-cyclodextrin has a molar substitution value of 0.6 to 0.

8.

8. The pharmaceutical composition according to claim 6 or 7, characterized in that the methylcyclodextrin is substituted on hydroxyls bonded to the C2 and C6 carbons of the glucopyranose unit.

9. The pharmaceutical composition according to any one of claims 1 to 8, characterized in that the methylcyclodextrin contains at least 50, 60, or 75% methyl substituted on the hydroxyl bonded to the C2 carbon of the glucopyranose unit.

10. A pharmaceutical composition according to any one of claims 1 to 9, which further promotes the reduction of lipid storage.

11. A pharmaceutical composition according to any one of claims 1 to 10, which further promotes the reduction of lipid accumulation in the liver.

12. A pharmaceutical composition according to any one of claims 1 to 11, which further promotes increased cholesterol removal.

13. The pharmaceutical composition according to any one of claims 1 to 12, characterized in that the methylcyclodextrin has a molar substitution value of 0.2 to 1.

2.

14. The pharmaceutical composition according to any one of claims 1 to 13, characterized in that the methylcyclodextrin has a molar substitution value of 0.4 to 0.

9.

15. The pharmaceutical composition according to any one of claims 1 to 14, characterized in that the methylcyclodextrin has a molar substitution value of 0.6 to 0.8.

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