KR20210027169A - Pharmaceutical composition for treating or preventing Nonalcoholic Steatohepatitis - Google Patents

Abstract

The present invention relates to a pharmaceutical composition for treating or preventing nonalcoholic steatohepatitis (NASH) comprising: 1) a thiazolidinedione drug, a pharmaceutically acceptable salt, hydrate or crystalline form thereof; 2) carnitine, a pharmaceutically acceptable salt, hydrate or crystalline form thereof; and 3) biphenyl dimethyl dicarboxylate, a pharmaceutically acceptable salt, hydrate or crystalline form thereof. The composition of the present invention has an excellent effect of treating or preventing NASH.

Description

Pharmaceutical composition for treating or preventing Nonalcoholic Steatohepatitis (NASH) {Pharmaceutical composition for treating or preventing Nonalcoholic Steatohepatitis}

The present invention relates to a pharmaceutical composition for treating or preventing non-alcoholic steatohepatitis (NASH), and a method for treating or preventing the same.

Nonalcoholic Fatty Liver Disease (NAFLD) is usually divided into two stages. First, the most common type is simple fatty liver (NAFL, Nonalcoholic Fatty Liver), which is literally a simple fatty liver symptom. The second type of disease is non-alcoholic steatohepatitis (NASH), which is a disease that causes inflammation of hepatocytes by fatty liver.

Non-alcoholic steatohepatitis (NASH) is a common, often "silent" liver disease. It is very histologically similar to alcoholic liver disease, but occurs in people with no alcohol history who drink little or no alcohol. Three main features: abnormal fat accumulation or deposition in the liver (hepatic steatosis), liver inflammation (hepatitis), liver damage or liver tissue damage (liver fibrosis) characterizes NASH and distinguishes it from other liver diseases of metabolic origin. .

Most people with NASH are in good health and are unaware that they have liver problems. Nevertheless, NASH can be severe and can lead to cirrhosis, where the liver is permanently damaged, scarring, and no longer able to function properly. Cirrhosis can even progress further to lead to hepatocellular carcinoma. Approximately 10-15% of patients with histologically proven NASH progressed to cirrhosis and its sequelae such as liver failure and hepatocellular carcinoma (HCC).

NASH can primarily classify suspected patients through commercial blood test panel tests, and further evaluations do not reveal any apparent reason for liver disease (e.g., excessive use of medications, viral hepatitis or alcohol) and x-rays of the liver or When imaging studies reveal fat, NASH is suspected. However, the only means of actually diagnosing NASH and distinguishing between simple fatty liver and NASH is a liver biopsy. For a liver biopsy, a small piece of the liver is removed by inserting a needle through the skin. NASH is diagnosed when damage or fibrosis to inflamed fat and liver cells is confirmed as a result of histologic examination using a microscope. An important part of the information learned from the biopsy is whether scar tissue (fibrotic tissue) has developed in the liver. Currently, no blood test or scan can reliably provide this information. Therefore, diagnosis and treatment effect of NASH can be confirmed only by histopathological method in which the liver tissue is removed and confirmed through a microscope.

As the obese population increases, the incidence of NASH is also increasing, but there are no approved treatments or specific treatment methods for NASH to date. However, there are only suggestions for improving lifestyle habits such as blood sugar control and weight loss (in case of obesity or overweight), diet, exercise, and refraining from alcohol intake through diabetes medications.

Thiazolidinedione (TZD) is a group of drugs having a structure as shown in the following [Chemical Formula 1], and is generally referred to as a glitazone series, which makes the muscle tissue sensitive to insulin so that glucose is absorbed into the muscle. It is known as a type 2 diabetes treatment as a drug that plays a role in lowering blood sugar and blood sugar. Thiazolidinedione-based drugs are currently known such as pioglitazone, robeglitazone and rosiglitazone.

[Formula 1]

Pioglitazone is an analog in which a functional group is attached to the thiazolidinedione, and has the structure of [Chemical Formula 2]. This is a commercially available drug for the treatment of type 2 diabetes and acts to lower blood sugar. Pioglitazone is a potential agonist for peroxisome proliferator-activated receptor-gamma (PPAR-γ), and its specific formula is as follows.

[Formula 2]

Pioglitazone activates the differentiation of adipocytes by acting on the peroxisome proliferator-activated receptor-gamma (PPAR-γ), which is widely distributed in adipose tissue. Through this, it lowers free fatty acids and inhibits the storage of fat and fatty acids in the liver by improving insulin resistance. Recent clinical studies have shown that these actions can prevent abnormal liver fat accumulation or deposition (hepatic steatosis) in NASH patients and further reduce liver inflammation (hepatitis) (Promrat et al., A pilot). study of pioglitazone treatment for nonalcoholic steatohepatitis, HEPATEOLOGY, January 2004).

However, pioglitazone causes unwanted side effects, including drug-drug interactions, cardiovascular effects, edema, weight gain and potential for bladder cancer (e.g. Kus et al., PLoS ONE 6(11): e27126 (2011)). Reference)). Therefore, it is not recommended to chronically administer pioglitazone at high doses, and it is necessary to develop a combination drug including an auxiliary drug that can reduce the side effects of pioglitazone while maintaining the effect.

On the other hand, while the underlying mechanism for liver damage that causes NASH is not known, control over several factors has the potential as a candidate to improve NASH. For example, insulin resistance, release of toxic inflammatory proteins (cytokines, cytokines) by adipocytes, and agents that regulate oxidative stress. Therefore, adjuvant drugs with complex mechanisms capable of simultaneously inhibiting these factors were reviewed.

Carnitine (Carnitine, β-hydroxy-γ acid, 3-hydroxy-4-N,N,N-trimethylaminobutyrate) has a chemical formula as shown in [Chemical Formula 3], and is also known as vitamin BT.

[Formula 3]

Carnitine is an amino acid derivative and acts as an essential cofactor in fatty acid metabolism. It is used for the treatment of primary or secondary carnitine deficiency, and although both L- and DL- isomers are used, only the L-type is recognized for its therapeutic effect. When L-carnitine is administered orally, by injection, or by dialysis, it has the effect of reducing chronic intermittent hemodialysis by increasing the plasma concentration of triglycerides and lowering total cholesterol. It is also used to prevent carnitine deficiency due to hyperlipidemia and toxicity induced by anthra-cyclines and valproate. In addition, in patients with ischemic heart disease, it has been reported that it has a beneficial effect on myocardial function and metabolism, thereby improving exercise resistance in patients with angina [K.L.Goa and R.N.Brogden.Drugs.,1987,34.1; Med. Lett., 1986, 28, 88 (review of carnitine): C.J. Rebouche and A.G. Engel, Mayo Clin. Proc., 1983,58,533 (carnitine metabolism and deficiency syndromes):Lancet, 1987,2,429 (role of carnitine in Reye's syndrome)]. However, no direct therapeutic effect of carnitine against NASH-like hepatitis has been found.

Biphenyl dimethyl dicarboxylate (BDD) is an artificial synthetic material similar to Schizandrin C, an active ingredient of Schisandra chinensis, which has been used as a folk remedy for a long time. It is a compound.

[Formula 4]

This biphenyldimethyldicarboxylate has been proven to have an effect on various chronic and viral hepatitis by animal and clinical experiments, and thus is currently being applied in clinical practice. For example, in animal experiments, biphenyldimethyldicarboxylate has an inhibitory effect on liver damage caused by carbon tetrachloride [Yu HuiQin: Diphenyl-dimethyl-dicarboxylate in treating and preventing hepatitis due drug poisoning, Chinese Medical Journal. , 100(2):122,1987], it has also been reported to significantly lower serum alanine aminotransferase levels in patients with chronic active hepatitis and active liver cirrhosis [Lee Hyo-Seok et al.,'Biphenyldi in patients with chronic active liver disease. A prospective randomized controlled study on the effect of methyldicarboxylate on lowering serum alanine aminotransferase levels', [Journal of Korean Internal Medicine, Vol. 40, No. 2, 1991]. However, the above physiological values are only to confirm the indirect effect on the inhibition of liver damage, and histologically, the actual effect of reducing steatosis or inflammation of liver tissue has not been confirmed. Has not been proven.

The problem to be solved by the present invention is to provide a pharmaceutical composition for the treatment or prevention of non-alcoholic steatohepatitis (NASH).

Another problem to be solved by the present invention is to provide a method for treating or preventing non-alcoholic steatohepatitis comprising administering a pharmaceutical composition to a patient with non-alcoholic steatohepatitis.

The present invention provides a pharmaceutical composition for the treatment or prevention of nonalcoholic steatohepatitis (NASH).

The "Nonalcoholic Steatohepatitis (NASH)" is a type of aggravated disease of nonalcoholic fatty liver disease (NAFLD), and refers to a condition in which inflammation of hepatocytes caused by fatty liver is induced. It is histologically very similar to alcoholic liver disease, but it is defined as NASH when it occurs in people with no alcohol history who drink little or no alcohol, and abnormal fat accumulation or deposition in the liver (hepatic steatosis), and liver inflammation (hepatitis ), liver damage or liver tissue damage (liver fibrosis) characterizes it and distinguishes it from other liver diseases of metabolic origin. When only fat accumulation is observed in the initial stage, it is diagnosed as Nonalcoholic Fatty Liver Disease (NAFLD), but when it develops and accompanies mesenchymal inflammation, liver cell damage, and liver fibrosis, it is classified as NASH. Non-alcoholic steatohepatitis proceeds in the order of mesenchymal inflammation, liver cell damage, and liver fibrosis, and liver fibrosis is also a serious disease in NASH.

In one embodiment of the present invention, the pharmaceutical composition is

One) Thiazolidinedione-based drug, a pharmaceutically acceptable salt, hydrate or crystalline form thereof,

2) Carnitine, a pharmaceutically acceptable salt, hydrate or crystalline form thereof, and

3) Biphenyldimethylcarboxylate, a pharmaceutically acceptable salt, hydrate or crystalline form thereof.

More specifically, in one embodiment of the present invention, the pharmaceutical composition may include pioglitazone, L-carnitine, and biphenyldimethyldicarboxylate.

The “thiazolidinedion drug” is a group of drugs having a basic structure as shown in [Chemical Formula 5], and is generally referred to as a glitazone series, and makes the muscle tissue sensitive to insulin so that it becomes glucose into the muscle. It is known as a type 2 diabetes treatment as a drug that plays a role in lowering blood sugar by helping this absorption. Thiazolidione drugs include pioglitazone, lobeglitazone, and rosiglitazone.

[Formula 5]

In Formula 1, R represents a substituted benzyl group linked with an alkyl group.

In one embodiment of the present invention, R may be a substituted benzene linked with a methyl group, and more specifically, R may be as shown in [Chemical Formula 6].

[Formula 6]

The “carnitine” is 3-hydroxy-4-trimethylammonio-butytric acid and has a structure as shown in [Chemical Formula 3], and is also known as vitamin BT. As an amino acid derivative, it plays a role as an essential coenzyme in fatty acid metabolism. It is used for the treatment of primary or secondary carnitine deficiency, and both L- and DL-isomers can be used.

[Formula 3]

The “pioglitazone” is a thiazolidinedione-based drug having a structure as shown in the following [Chemical Formula 2], and is marketed for the treatment of type 2 diabetes and acts to lower blood sugar. Pioglitazone is a potential agonist for peroxisome proliferator-activated receptor-gamma (PPAR-γ), and its specific formula is as follows. The pioglitazone of the present invention includes all pharmaceutically acceptable salts and crystal forms other than the hydrochloride and potassium salts thereof.

[Formula 2]

The pharmaceutically acceptable salts mentioned in the present invention mean salts commonly used in medicines, benzenesulfonic acid, benzoic acid, citric acid, ethanesulfonic acid, formic acid, fumaric acid, gentisic acid, hydrobromic acid, hydrochloric acid, maleic acid, Salts from malic acid, malonic acid, mandelic acid, methanesulfonic acid, 4-methyl-benzenesulfonic acid, phosphoric acid, salicylic acid, succinic acid, sulfuric acid, tartaric acid, and trifluoroacetic acid.

The “L-Carnitine” is an L isomer of carnitine, an amino acid derivative, and serves as an essential cofactor for fatty acid metabolism, and has the same structure as [Chemical Formula 7].

[Formula 7]

The “biphenyl dimethyl dicarboxylate (BDD)” is also called BDD or DBB, and has the same structure as [Chemical Formula 4].

[Formula 4]

In one embodiment of the present invention, the pharmaceutical composition may include pioglitazone, L-carnitine, and biphenyldimethyldicarboxylate in a weight ratio range of 1:5 to 30:25 to 60, preferably 1:15 to A weight ratio range of 25:35 to 55, more preferably a weight ratio range of 1:15 to 20:40 to 50, more preferably a weight ratio range of 1:15 to 17:40 to 45, most preferably 1: It can be included in the weight ratio range of 16.5:41.

In one embodiment of the present invention, the non-alcoholic steatohepatitis may include liver fibrosis, liver cirrhosis, and hepatocellular carcinoma (HCC), and the pharmaceutical composition of the present invention can treat, stabilize, reduce or Provides a preventive effect.

The “hepatic fibrosis” is formed by the accumulation of fat in the liver and eventually fibrosis as the damaged liver cannot decompose fatty acids, a compound that forms fat, and if it persists, it can develop into cirrhosis.

In one embodiment of the present invention, the pharmaceutical composition may include pioglitazone, L-carnitine, and biphenyldimethyldicarboxylate in a weight ratio range of 1:5 to 30:25 to 60, preferably 1:15 to A weight ratio range of 25:35 to 55, more preferably a weight ratio range of 1:15 to 20:40 to 50, more preferably a weight ratio range of 1:15 to 17:40 to 45, most preferably 1: It can be included in the weight ratio range of 16.5:41.

In one embodiment of the present invention, the non-alcoholic steatohepatitis may include liver fibrosis, liver cirrhosis, and hepatocellular carcinoma (HCC), and the pharmaceutical composition of the present invention can treat, stabilize, reduce or Provides a preventive effect.

The “hepatic fibrosis” is formed by the accumulation of fat in the liver and eventually fibrosis as the damaged liver cannot decompose fatty acids, a compound that forms fat, and if it persists, it can develop into cirrhosis.

The "liver cirrhosis" means that due to chronic inflammation, normal liver tissue is transformed into fibrous tissue such as regenerative nodules (a phenomenon in which small lumps are formed), and the function of the liver is degraded. Scar tissue has occurred throughout the liver, mainly due to irreversible necrosis caused by chronic liver inflammation.

The “hepatocellular carcinoma (HCC)” is the occurrence of cancer in hepatocytes and is the most common cause of death in patients with cirrhosis. It is a primary liver cancer with the highest frequency among liver cancers, and is the fifth most frequent malignant tumor in the world.

In one embodiment of the present invention, the pharmaceutical composition may reduce the NASH score.

The “non-alcoholic steatohepatitis diagnosis score (NASH score)” is intended to quantify histological findings on liver tissue and use it as an index for diagnosing or measuring therapeutic effects of NASH, Microvesicular Vacuolation/Steatosis, It can be obtained by summing all the scores for each item for Lobular Inflammation and Ballooning of Hepatocyte. Specific evaluation methods for this can be confirmed in [Table 2] of the detailed description of the present invention.

When the diagnosis score for non-alcoholic steatohepatitis decreases, it is evaluated as having an effect of treating or preventing NASH.

In one embodiment of the present invention, the pharmaceutical composition is characterized in that it has a therapeutic, stabilizing, reducing or preventing effect on fibrosis or lobular inflammation in liver tissue of non-alcoholic steatohepatitis (NASH).

In one embodiment of the present invention, the pharmaceutical composition is effective in treating, stabilizing, reducing or preventing severe diseases of non-alcoholic steatohepatitis (NASH).

In the present invention, the severe disease of non-alcoholic steatohepatitis (NASH) may be liver fibrosis, liver cirrhosis or hepatocellular carcinoma, and in one embodiment, it means a stage in which fibrosis in liver tissue has progressed.

In one embodiment of the present invention, the pharmaceutical composition has an effect of reducing the non-alcoholic steatohepatitis diagnostic score (NASH score).

In one embodiment of the present invention, the pharmaceutical composition has an effect of treating or preventing fibrosis of non-alcoholic steatohepatitis.

In one embodiment of the present invention, the pharmaceutical composition is used to treat perisinusoidal fibrosis, Chicken-Wire Fibrosis, portal fibrosis, and bridging fibrosis among non-alcoholic steatohepatitis fibrosis. Can be prevented. .

In the present invention, the fibrosis score (FIBROSIS score) is an index for indicating the degree of fibrosis of liver tissue in non-alcoholic steatohepatitis, and is a sum of scores for perivascular fibrosis, periportal fibrosis, and connected fibrosis. Specific evaluation methods can be confirmed in [Table 3] of the detailed description of the present invention. In general, when the FIBROSIS score is greater than 0, it is considered that fibrosis has progressed, and when the FIBROSIS score is no longer increased, it is recognized that there is an effect of preventing, treating or preventing fibrosis in nonalcoholic steatohepatitis. Therefore, if it shows a reduced value compared to the negative control group, it is considered to have a therapeutic or preventive effect.

In one embodiment of the present invention, the pharmaceutical composition has an effect of reducing the fibrosis score (FIBROSIS score) of non-alcoholic steatohepatitis.

In one embodiment of the present invention, the pharmaceutical composition, when administered to a steatohepatitis patient, has the effect of reducing the NASH score by 15% or more compared to the negative control group, and preferably 20 It has the effect of reducing by more than %. In another embodiment of the present invention, the pharmaceutical composition, when administered once daily for 12 weeks to patients with steatohepatitis, reduces the NASH score by 15% or more compared to the negative control group. There is, preferably there is an effect of reducing by 20% or more.

In one embodiment of the present invention, the pharmaceutical composition, when administered to a steatohepatitis patient, has the effect of reducing the fibrosis score by 25% or more compared to the negative control group, preferably 30% or more, more preferably It has the effect of reducing by more than 35%. In another embodiment of the present invention, the pharmaceutical composition, when administered once daily for 12 weeks to patients with steatohepatitis, has the effect of reducing fibrosis score by 25% or more compared to the negative control group, and preferably There is an effect of reducing by 30% or more, more preferably by 35% or more.

The composition for improving liver function according to the present invention may be administered orally (eg, ingestion or inhalation) or parenterally (eg, injection, transdermal absorption, rectal administration), and injection may be performed, for example, It may be intravenous, subcutaneous, intramuscular, or intraperitoneal. The pharmaceutical composition according to the present invention is formulated into tablets, capsules, granules, fine subtilae, powders, sublingual tablets, suppositories, ointments, injections, emulsions, suspensions, syrups, sprays, etc., depending on the route of administration. It can be angry. The various types of pharmaceutical compositions according to the present invention may be prepared by known techniques using a pharmaceutically acceptable carrier commonly used in each formulation. Examples of pharmaceutically acceptable carriers include excipients, binders, disintegrating agents, lubricants, preservatives, antioxidants, isotonic agents, buffering agents, coating agents, sweetening agents, solubilizing agents, bases, dispersing agents, wetting agents. , Suspending agents, stabilizers, colorants, and the like.

The composition for improving liver function according to the present invention may be a pharmaceutical composition. The pharmaceutical composition may additionally contain pharmaceutical adjuvants such as preservatives, stabilizers, wetting agents or emulsification accelerators, salts and/or buffers for controlling osmotic pressure, and other therapeutically useful substances. It can be formulated in the form of a dosage or parenteral administration. In the case of parenteral administration, it can be administered topically to the skin, intravenous injection, subcutaneous injection, intramuscular injection, intraperitoneal injection, transdermal administration, or the like.

The dose and frequency of administration of the pharmaceutical composition of the present invention may vary depending on the method of administration and the age, weight, or disease state of the patient, and when administered orally, the administrable range for each active ingredient is pioglitazone 0.01 to 200 mg/day, BDD 0.01 to 1600 mg/day, L-carnitine 0.01 to 4000 mg/day, preferably 0.1 to 100 mg/day, BDD 1 to 1000 mg/day, L-carnitine 1 to 2000 mg/day, more preferably 1 to 40 mg/day , BDD 10~800mg/day, L-carnitine 10~2000mg/day. In addition, the range of the total administrable content of the composite composition is 0.01 to 6000 mg/day, preferably 0.01 to 4000 mg/day, more preferably 0.01 to 3000 mg/day. The frequency of administration may be administered once a day to four times a day.

The pharmaceutical composition of the present invention may be used alone or in combination with one or more other therapeutic agent(s) (second active ingredient) depending on the severity of the disease to be treated.

In an exemplary embodiment of the present invention, there is provided a method for treating or preventing NASH disease by administering an effective amount of the pharmaceutical composition of the present invention to a NASH patient. Here, the administration may be administered orally (eg, ingestion or inhalation) or parenterally (eg, injection, transdermal absorption, rectal administration).

The pharmaceutical composition of the present invention has excellent therapeutic, stabilizing, reducing or preventing effects on non-alcoholic fatty liver disease (NAFLD), in particular, non-alcoholic steatohepatitis (NASH).

FIG. 1 is a diagram showing and comparing scores and total scores for each item of the NASH score of the negative control, positive control, Example 1, and Comparative Examples 1 to 3. FIG.
FIG. 2 is a diagram illustrating and comparing scores and total scores for each item of the negative control, positive control, Example 1, and FIBROSIS scores of Comparative Examples 1 to 3. FIG.

Hereinafter, the present invention will be described in more detail by examples. However, the Examples and Experimental Examples are only intended to aid understanding of the present invention, and the scope of the present invention is not limited by these Examples in any sense.

Preparation of Experimental Subjects 1. Preparation and identification of NASH-induced mice

The experimental animal was a 7-week-old male rat C57BL6 (Jackson Labs, US), and divided into a control group and an experimental group as shown in Table 1 below. High Fat Diet, HFD) was confirmed to induce NASH.

For the experimental group, 23.1g/L fructose (Sigma, Catalog #F2543) and 17.2g/L glucose (Sigma, Catalog #G8270) were mixed with laboratory tap water at 250ml/weekly, and a high-fat feed (D12079B , Research Diet). On the other hand, laboratory tap water and general feed (Teklad Global Diets-Rodent 2014) were provided for the control group.

The animals are reared in an environment set at a temperature of 22-26℃ and a relative humidity of 30-70%, a dark cycle of 12 hours (lights on at 8 am to light off at 8 pm) and an illuminance of 150 to 300Lux. During the breeding period, the health status was checked twice daily. In addition, 300uL of blood was collected once every 20 weeks after the start of the experiment.

Group name Population Strain Diet term Control 5 C57BL6 Normal diet (standard) 16~24 weeks Experimental group 5 C57BL6 High fat/high fructose diet
(high fat/sucrose) 16~24 weeks

After 16 to 24 weeks, the rats of the control group and the experimental group were euthanized with carbon dioxide, the liver was excised, washed with physiological saline, and stored in 10% Neutral Buffered Formalin (NBF) for a minimum of 24 hours and a maximum of 72 hours.

Preparation of Subjects 1-1. Check whether fatty liver is induced by histological analysis of liver tissue and measurement of the amount of triglyceride in liver tissue

The left lateral lobe section of the liver tissue of the control and experimental mice stored in the formalin was retaken, fixed in Paraffin, hardened, and then sectioned into 5 μm on a slide, and a FFPE (Formalin Fixed Paraffin Embedded) block Was prepared. The section was stained with Sirius Red and then the tissue was analyzed under a microscope (Nikon E600).

Preparation of test subjects 1-2. Non-alcoholic steatohepatitis diagnosis score (NASH score) and fibrosis (FIBROSIS score) check

In order to quantify the effect of non-alcoholic steatohepatitis (NASH) treatment, the severity of major histopathologic factors for diagnosing non-alcoholic steatohepatitis was scored and defined as the NASH score. Specifically, the degree of distribution of microvesicular vacuolation/Steatosis (0-3 points), the number of lobular inflammation (0-3 points), and the degree of ballooning of hepatocytes ( 0-2 points), and the total score of each of these values is called the NAFLD score (0-8 points). It means a cluster (foamy cluster), and is known as an indirect factor that can confirm cellular injury and cytoskeletal damage in liver tissue (Tandra, et al. Journal of Hepatology 2011 vol.55, 654). -659) If the NASH score is 5 or more, it is diagnosed as NASH, if it is less than 2, it is diagnosed as non-NASH, and the value in between is diagnosed as a threshold.

Micro-foaming vacuole can be observed at all magnifications, but in this experimental example, it was carried out at 20 magnification.

Item-1 Item-2 Judgment criteria score NASH score Microfoaming fear
(Microvesicular Vacuolation/Steatosis) <5% 0 5~33% One >33~66% 2 >66% 3 Lobular inflammation
(Lobular Inflammation) No foci 0 <2 foci per 20ⅹfield One 2~4 foci per 20ⅹfield 2 >4 foci per 20ⅹfield 3 Hepatocyte balloon degeneration
(Ballooning of hepatocyte)
H&E stain 400x field None 0 Few balloon cells One Many cells/prominent ballooning 2

In addition, a method for measuring and quantifying the degree of fibrosis, which is the most important for diagnosing NASH and confirming the therapeutic effect, was performed as follows. In general, fibrosis in humans is divided into four stages, and the evaluation items are Perisinusoidal Fibrosis (Chick-Wire Fibrosis), Portal Fibrosis, and Bridging Fibrosis. In this experimental example, the degree of each of the above items was scored, and in particular, perivascular fibrosis (0-3 points) according to the frequency of occurrence of fibrosis in the observed vessels, and context according to the type of fibrosis observed in the observed portal vein. Peripheral fibrosis (0-3 points, 0 points when fibrosis is not confirmed, 1 point when expansion of the portal duct is confirmed due to fibrosis, 2 points when fibrosis is confirmed around the portal vein, 3 points when connected fibrosis is confirmed, In the case of developing cirrhosis, 4 points), connected fibrosis within the observed range, and connected fibrosis according to the number of nodules (0-3 points) were evaluated, and the FIBROSIS score was summed up for each item. Therefore, this FIBROSIS score reflects the principle of fibrosis evaluation in humans, and the higher the score, the higher the degree of fibrosis, so it is evaluated that NASH progressed more, and the lower the score, the better the treatment effect.

Each item of the following FIBROSIS score was performed at all observable magnifications (100x field, 200x field, 400x field, 1000x field).

Item Item-2 Judgment criteria score FIBROSIS score Perivascular fibrosis
(Perisinusoidal Fibrosis, Chicken-Wire Fibrosis) No fibrosis 0 <33% One >33~66% 2 >66% 3 Periportal fibrosis
(Portal Fibrosis) No portal fibrosis
(No portal fibrosis observed) 0 Expanding the perimeter of the context
(Expanded portal areas) One Periportal fibrosis 2 Periportal bridging fibrosis 3 Cirrhosis, widespread nodular remodeling 4 Connected fibrosis
(Bridging Fibrosis, any form) No connected fibrosis
(No bridging fibrosis observed) 0 1 verified
(1 focus) One 1 or more, no nodularity (>1 focus with no nodularity) 2 Bridging fibrosis with some nodular remodeling 3 Cirrhosis, widespred nodular remodeling 4

As a result of confirming the NAFLD score and FIBROSIS score for the liver histological analysis results based on the above criteria, both the experimental group had a NASH score of 5 or more and a FIBROSIS score of 0 or more, and it was confirmed that NASH was induced at weeks 16 to 24 of HFD.

Preparation Example 1. Preparation of a pharmaceutical composition for the treatment of NASH

A pharmaceutical composition was prepared by mixing pioglitazone, BDD, and L-carnitine at a weight ratio of 1:16.5:41 based on the weight ratio of each free salt form. In the preparation of the mixture, pioglitazone is in the form of hydrochloride (manufacturer: Cipla Limited), BDD (manufacturer: Wanbangde pharmaceutical group Co., LTD.) and L-carnitine (manufacturer: Liaoning Nirvana Pharmaceutical co., Ltd.) as a free salt. Was used.

Experimental Example 1. Confirmation of NASH therapeutic effect of pharmaceutical composition

In the preparation of the test subject, for Example 1 in which the diet was proceeded in the same manner as in which it was confirmed that NASH was induced, the pharmaceutical composition prepared in Preparation Example 1 was once daily for 12 weeks at 585 mg/kg (pioglitazone: BDD:L-carnitine=10mg/kg:165mg/kg:410mg/kg) was administered by oral gavage. As a positive control, obeticholic acid (Ocaliva®, OCA) 5.2 mg/kg, as a comparative example, pioglitazone 10 mg/kg, BDD 165 mg/kg, and L-carnitine 410 mg/kg, respectively, at the same time as in the Example. It was administered in the same manner, and 5ml/kg of a placebo composition was administered as a vehicle to the normal group and the negative control group. As a placebo composition, 9% (v/v) Solutol HS-15 (Kolliphor) was used in PBS at a pH of 7.2.

The experimental groups were reared under the same conditions as the experimental examples except for drug administration. Health status was checked twice daily, and blood was collected once a week (0.6ul, twice).

Group name Population Strain Diet drug injection Normal control 10 C57BL6 Normal diet (standard) Vehicle Negative control 10 C57BL6 High fat/high fructose diet
(high fat/sucrose) Vehicle Positive control 10 C57BL6 High fat/sucrose diet OCA Example 1 10 C57BL6 High fat/sucrose diet Manufacturing Example 1 Comparative Example 1 10 C57BL6 High fat/sucrose diet Pioglitazone Comparative Example 2 10 C57BL6 High fat/sucrose diet BDD Comparative Example 3 10 C57BL6 High fat/sucrose diet L-carnitine

After 12 weeks, the mice of the 7 experimental groups were euthanized with carbon dioxide, the liver was excised, washed with physiological saline, and stored in 10% Neutral Buffered Formalin (NBF) for a minimum of 24 hours and a maximum of 72 hours.

Experimental Example 1-1. Confirmation of fatty liver reduction through histological analysis of liver tissue and measurement of triglyceride amount

In the same manner as in Preparation of Experimental Subjects 1-1, livers of the 7 experimental groups were collected and tissue analysis was performed. It was confirmed that the invasion of immune cells around hepatocytes was reduced through high magnification under the microscope, and the degree of fibrosis was reduced by decreasing the bridge due to invasion of satellite cells and immune cells through the low magnification under the microscope.

Specifically, in the negative control group, a red line indicating fibrosis was most often observed, and Comparative Example 2 in which only the BDD composition was administered and the positive control group administered with obeticholic acid also showed a significant degree of fibrosis compared to the negative control group. On the other hand, in Comparative Example 1 in which pioglitazone was administered alone and Comparative Example 3 in which L-carnitine was administered alone, fibrosis was somewhat relieved, and in the micrograph of Example 1 in which the composite composition was administered, a red line indicating connected fibrosis Little was observed. Accordingly, it can be seen that the composite composition administered in Example 1 has the best effect of treating or preventing NASH fibrosis.

Experimental Example 1-2. Non-alcoholic steatohepatitis score (NASH score) and fibrosis (FIBROSIS score) check

Each item of the NASH score and the FIBROSIS score of the 7 experimental groups were evaluated in the same manner as in the preparation of test subjects 1-2, and the results are shown in Tables 5 and 6 and Figs. 1 and 2, respectively.

Group name Administered drugs Microvesicular fear Lobular inflammation Hepatocyte balloon degeneration NASH score Negative control Vehicle 1.3 2.7 1.0 5.0 Positive control OCA 2.0 1.8 1.0 4.8 Example 1 Manufacturing Example 1 1.3 1.7 1.0 4.0 Comparative Example 1 Pioglitazone 1.4 2.8 0.9 5.1 Comparative Example 2 BDD 2.0 2.6 1.0 5.6 Comparative Example 3 L-carnitine 1.5 1.8 1.0 4.3

Group name Administered drugs Perivascular fibrosis Periphery of context
fibrosis Connected fibrosis FIBROSIS score Negative control Vehicle 2.0 3.0 2.0 7.0 Positive control OCA 2.5 2.9 1.8 7.2 Example 1 Manufacturing Example 1 1.8 2.0 0.7 4.5 Comparative Example 1 Pioglitazone 1.6 2.5 1.3 5.4 Comparative Example 2 BDD 2.1 2.8 2.0 6.9 Comparative Example 3 L-carnitine 2.3 2.7 1.7 6.7

As can be seen in Tables 5 and 6 and Figures 1 and 2, in the NASH score and FIBROSIS score evaluation items in Example 1, compared to Comparative Examples 1 to 3 in which each of the single components of the combination formulation of Preparation Example 1 was administered, overall There was a tendency to be excellent, and it was superior to the positive control OCA. In particular, it showed excellent effect on microvesicular vacuole, periportal fibrosis, and connected fibrosis.

In addition, Example 1 showed the lowest value in the overall NASH score, and in particular, in the FIBROSIS score, a significantly lower value compared to other comparative groups and positive controls was confirmed, and the NASH of the composition of Preparation Example 1 administered in Example 1, In particular, it can be seen that there is an excellent treatment or prevention effect for liver fibrosis.

Fibrosis is a symptom that appears in the most severe stage even in NASH, which is an advanced stage of non-alcoholic fatty liver disease (NAFLD), and it can be seen that the composite composition of the present invention has a more excellent effect in the treatment or prevention of severe NASH disease.

Claims (14)

1) Thiazolidinedione-based drug, a pharmaceutically acceptable salt, hydrate or crystalline form thereof,
2) carnitine, a pharmaceutically acceptable salt, hydrate or crystalline form thereof, and
3) Biphenyldimethylcarboxylate, a pharmaceutical composition for the treatment or prevention of non-alcoholic steatohepatitis (NASH) comprising a pharmaceutically acceptable salt, hydrate or crystalline form thereof.
The pharmaceutical composition for the treatment or prevention of non-alcoholic steatohepatitis according to claim 1, wherein the thiazolidinedione-based drug is selected from pioglitazone, robeglitazone, and rosiglitazone.
The pharmaceutical composition for the treatment or prevention of non-alcoholic steatohepatitis according to claim 1, wherein the carnitine is L-carnitine.
The method of claim 1,
The pharmaceutical composition for the treatment or prevention of non-alcoholic steatohepatitis in which the combination ratio of thiazolidinedione-based drug, carnitine and biphenyldimethylcarboxylate of the pharmaceutical composition is 1:10 to 30:30 to 60 based on a weight ratio.
The method of claim 4,
The pharmaceutical composition for the treatment or prevention of non-alcoholic steatohepatitis in which the combination ratio of thiazolidinedione-based drug, carnitine and biphenyldimethylcarboxylate of the pharmaceutical composition is 1:15-20:40-50 based on a weight ratio.
The method of claim 1,
The pharmaceutical composition is a non-alcohol characterized in that it has a therapeutic, stabilizing or reducing effect on hepatic fibrosis, liver cirrhosis, or hepatocellular carcinoma (HCC) of non-alcoholic steatohepatitis. A pharmaceutical composition for the treatment or prevention of sexual steatohepatitis.
The method of claim 1,
The pharmaceutical composition is a pharmaceutical composition for the treatment or prevention of non-alcoholic steatohepatitis, characterized in that it has the effect of improving fibrosis or lobular inflammation in liver tissue of a non-alcoholic steatohepatitis patient.
The method of claim 1,
The pharmaceutical composition is a pharmaceutical composition for the treatment or prevention of non-alcoholic steatohepatitis, characterized in that it has an effect of treating, stabilizing or reducing severe diseases of non-alcoholic steatohepatitis.
The method of claim 1,
The pharmaceutical composition is a pharmaceutical composition for the treatment or prevention of non-alcoholic steatohepatitis, characterized in that it has the effect of reducing the non-alcoholic steatohepatitis diagnostic score (NASH score).
The method of claim 1,
The pharmaceutical composition is a pharmaceutical composition for the treatment or prevention of non-alcoholic steatohepatitis, characterized in that it has the effect of reducing the fibrosis (FIBROSIS score) of non-alcoholic steatohepatitis.
The method of claim 1,
The pharmaceutical composition, when administered to a patient with steatohepatitis, decreases the NASH score by 15% or more compared to the negative control group. A pharmaceutical composition for the treatment or prevention of non-alcoholic steatohepatitis.
The method of claim 1,
The pharmaceutical composition, when administered to a patient with steatohepatitis, reduces fibrosis (FIBROSIS score) by 25% or more compared to the negative control group. A pharmaceutical composition for the treatment or prevention of non-alcoholic steatohepatitis.
A method for treating or preventing nonalcoholic steatohepatitis, comprising administering the composition according to any one of claims 1 to 12 in a therapeutically or prophylactically effective amount.
13. Use of the treatment or prophylaxis of non-alcoholic steatohepatitis, comprising administering a therapeutically or prophylactically effective amount of the composition according to any one of claims 1 to 12.

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