CN110876751B

CN110876751B - Application of trametinib in preparation of medicines for preventing and/or treating nonalcoholic hepatitis and/or nonalcoholic fatty liver disease

Info

Publication number: CN110876751B
Application number: CN201911033194.4A
Authority: CN
Inventors: 王靖翔; 刘祖瑞; 高明景
Original assignee: Beijing Yiyao Technology Co ltd
Current assignee: Beijing Yiyao Technology Co ltd
Priority date: 2019-10-28
Filing date: 2019-10-28
Publication date: 2023-03-14
Anticipated expiration: 2039-10-28
Also published as: CN110876751A; WO2021082683A1

Abstract

The invention provides an application of trametinib in preparation of drugs for preventing and/or treating nonalcoholic hepatitis, and also provides an application of trametinib in preparation of drugs for preventing and/or treating nonalcoholic fatty liver diseases. The invention provides application of trametinib in preparation of drugs for preventing and/or treating non-alcoholic hepatitis and non-alcoholic fatty liver diseases, and results show that the trametinib can effectively reduce ALT, AST, CHO and TG values in an MCD model and reduce liver indexes, and a trametinib treatment group has small staining surface value, no fat granules and no inflammatory necrosis; trametinib can also effectively reduce fat and inflammatory reaction in a high-fat/fructose model, and a trametinib treatment group is small in dyeing surface value, obviously reduced in fat particles and small in damage.

Description

Application of trametinib in preparation of medicines for preventing and/or treating nonalcoholic hepatitis and/or nonalcoholic fatty liver disease

Technical Field

The invention belongs to the field of pharmacy, and particularly relates to application of trametinib in preparation of medicines for preventing and/or treating non-alcoholic hepatitis and/or non-alcoholic fatty liver diseases.

Background

As the standard of living increases, non-alcoholic fatty liver disease (NAFLD) affects the health of more and more people. Non-alcoholic fatty liver disease (NAFLD) refers to the condition of excess fat in the liver of a human who has little or no alcohol consumption. The most common NAFLD is a non-severe disease known as hepatic steatosis (fatty liver), where fat accumulates in hepatocytes: although this is not normal, it may not itself damage the liver. NAFLD is most commonly found in individuals with a series of risk factors known as metabolic syndrome, characterized by elevated fasting blood glucose, or postprandial glucose intolerance, overweight or obesity, hyperlipidemia (e.g., cholesterol and triglycerides and low high density lipids, protein cholesterol (HDL-C) levels, and hypertension); not all patients have all manifestations of the metabolic syndrome. Obesity is considered to be the most common cause of NAFLD; some experts estimate that approximately two-thirds of obese adults and half of obese children may have fatty liver. Most patients with NAFLD are asymptomatic, with a normal physical examination (although the liver may be slightly enlarged); children may develop symptoms such as abdominal pain and fatigue, and may develop dark skin discoloration (acanthosis nigricans) in the form of patches. Diagnosis of NAFLD is often the first suspected overweight or obese person who has been found to have a slight elevation in their liver blood test in routine examinations, although NAFLD may be present in normal liver blood tests or accidentally found in imaging examinations such as abdominal ultrasound or CT scans. The most common diagnostic modalities are confirmed by imaging studies, such as liver ultrasound or Magnetic Resonance Imaging (MRI).

Some nonalcoholic steatohepatitis (NAFLD) patients may develop a more serious disease-nonalcoholic steatohepatitis (NASH): about 2-5% of adults and up to 20% of obese people are likely to suffer from NASH. In NASH, fat accumulation in the liver is associated with inflammation and scarring to varying degrees. NASH is a potentially serious disease with a great risk of developing end-stage liver disease, cirrhosis and hepatocellular carcinoma. Some patients with cirrhosis are at risk of liver failure and may eventually require liver transplantation.

NAFLD can be distinguished from NASH by NAFLD Activity Score (NAS): steatosis liver biopsy histopathology score (0-3), lobular inflammation (0-2), and hepatocyte swelling (0-2). NAS less than 3 corresponds to NAFLD,3-4 corresponds to critical NASH, NAS greater than 5 corresponds to NASH. Biopsies were also scored as fibrosis (0 to 4).

NASH is the leading cause of end-stage liver disease; NAFLD and to a greater extent NASH are, however, closely related to the state of metabolic syndrome, including insulin resistance (pre-diabetes) and type 2 diabetes (T2 DM) as well as abdominal obesity. T2DM is the most significant predictor of poor prognosis for NAFLD, while elevated liver enzymes are considered unreliable. The incidence of NASH is higher in patients with long-standing T2DM, and the majority of patients with cryptogenic cirrhosis are obese and/or diabetic. Studies have shown that 60% of patients with T2DM and NAFLD are biopsy-confirmed to be NASH, and 75% of patients with diabetes and hypertension develop advanced liver fibrosis, while only 7% of patients without these two diseases. Haukeland, "abnormal glucose tolerance is a predictor of nonalcoholic steatohepatitis and fibrosis in nonalcoholic steatohepatitis patients," Scand j. Gastenstrol, 401469-1477 (2005), reporting that Impaired Glucose Tolerance (IGT) and T2DM are the only independent risk factors for severe NAFLD and NASH, increasing the chance of morbidity. Mofrad, "the clinical and histological spectrum of nonalcoholic fatty liver disease is normally associated with alanine transaminase levels", liver disease, 371286-1292 (2003), reports a study demonstrating the predictive value of elevated liver transaminase for NASH diagnosis in NAFLD patients and finding that T2DM is the only independently relevant factor. Tide increases the risk of late stage fibrosis. NASH is therefore an overlooked complication of T2DM, often associated with fibrosis, with approximately 10% of patients leading to cirrhosis; the risk of hepatocellular carcinoma is also increased in patients with T2DM and NASH. Patients with NAFLD and NASH often exhibit mixed dyslipidemia and the other metabolic disorders described above, including the atherosclerotic Low Density Lipoprotein (LDL) phenotype, consisting primarily of small, dense particles. The metabolic syndrome and NAFLD/NASH are characterized by increased cardiovascular inflammation, measured by elevation of high sensitivity C-reactive protein (hscrp) and other inflammatory cytokines.

The incidence of global obesity, metabolic syndrome, pre-diabetes and diabetes is high, and is expected to double to 3.66 billion by 2030. It is estimated that 2540 million (11.5%) of 2011 american diabetics develop diabetes, 3770 million (14.5%) by 2031, and 20.2% of hispanic adults suffer from diabetes. Since about 70% of patients with T2DM have fatty liver and necrosis and fibrosis (i.e., NASH) are more severe in diabetic patients, diabetes epidemiology indicates a significant increase in the incidence of NASH and chronic liver disease patients. Noninvasive evaluation of hepatic steatosis using nuclear magnetic resonance imaging, NAFLD prevalence was estimated to be 34% in the united states or about 8000 ten thousand, with greater than 5% in two thirds of obese subjects. However, this prevalence is considered to be much higher in T2 DM. MRI showed a prevalence of NAFLD of 76% in 107 unselected T2DM patients, similar to recent studies in italy and brazil. Recent studies have shown that the prevalence of NAFLD is rapidly increasing in obese children and adolescents, particularly in children of hispanic lineage.

There is currently no approved drug for the prevention or treatment of NAFLD or NASH. Many pharmaceutical interventions have been tried in NAFLD/NASH, but the overall effect is limited. Antioxidants can prevent lipid peroxidation and cytoprotective agents can stabilize phospholipid membranes, but attempts to date have been unsuccessful or only moderately effective with antioxidants including ursodeoxycholic acid, vitamin E (alpha-tocopherol) and C, and pentoxifylline. Compared to diet and exercise alone to achieve weight loss ("weight loss alone"), orlistat and other weight loss drugs do not have significant benefits. Most of the weight loss studies in NAFLD/NASH were short-term and limited success pioneering studies, reporting only a slight improvement in necrosis or fibrosis. A randomized, double-blind, placebo-controlled 6-month trial (Belfort, "placebo-controlled trial of pioglitazone on non-alcoholic steatohepatitis patients", n.engl.j.med.,355, 2297-2307 (2006)) with pioglitazone alone (thiazolidinedione peroxisome proliferator-activated receptor-gamma (ppary γ) agonist and insulin sensitizer) reduced weight, failed to demonstrate any improvement in weight loss with pioglitazone alone, but treatment with pioglitazone improved glycemic control, insulin sensitivity, systemic inflammatory indicators. LAMMation (including hscrp, tumor necrosis factor alpha and transforming growth factor beta) and liver histology in NASH and IGT or T2DM patients. Pioglitazone treatment also improved the infrared spectrum of fat, liver and muscle and was associated with a reduction of necrotic plaques by about 50% (p < 0.002) and a reduction of fibrosis by 37% (p = 0.08). Improvement in hepatocyte injury and fibrosis was recently reported in another 12-month pioglitazone control trial. In contrast, while the first randomized clinical study of rosiglitazone, another thiazolidinedione approved for diabetes treatment, showed that rosiglitazone treatment had no significant effect on necrosis, inflammation or fibrosis in NASH, it was able to reduce insulin, plasma alanine Aminotransferase (ALT) levels and steatosis. Preliminary reports of a 2 year open follow-up for this trial were also disappointing, with no significant benefit from rosiglitazone treatment. Therefore, the most effective drug for NASH is pioglitazone. Unfortunately, pioglitazone is also associated with a significant increase in the risk of weight gain, edema, congestive heart failure and osteoporotic fractures in both women and men.

GW510516, a potent peroxisome proliferator-activated receptor-delta (ppar delta) agonist, improved diet-induced obesity and insulin resistance in normal mice, with increased metabolic rate and fatty acid beta-oxidation. It also significantly improved diabetes, demonstrating that both glucose and insulin levels are greatly reduced in genetically obese db/db mice. GW510516 has been demonstrated to lower TGS, low density lipoprotein cholesterol (LDL-C), apolipoprotein B (APOB) and insulin levels, increase HDL-C and insulin sensitivity in two first phase studies in healthy subjects, one of which also showed a 20% reduction in liver fat; and a later study demonstrated the role of these effects in subchronic brain damage due to dyslipidemia. CTS for abdominal obesity (20% reduction in liver fat, 30% reduction in fasting TGS, 26% reduction in APOB, 23% reduction in LDL-C, 40% reduction in fasting non-esterified fatty acids, 11% reduction in fasting insulin). However, after GW510516 was observed to be associated with rapid development of various organ cancers in animal studies, the development of GW510516 was stopped.

The peroxisome proliferator-activated receptor- α/peroxisome proliferator-activated receptor- δ (PPAR α/δ) agonist GFT505 has preferential α (EC 50=6 nm) and secondary δ (EC 50= 47nm) agonist activity. The lipid-regulating effect of gft505 has been demonstrated in two healthy subjects and in t2dm patients, combined abdominal obesity and mixed dyslipidemia, combined abdominal obesity and pre-diabetes, atherosclerotic dyslipidemia and insulin resistance. These effects include a reduction in TGS, non-HDL-C and total cholesterol, LDL-C and APOB; increase of HDL-C. Preclinical studies in rodent models of NAFLD/NASH showed that GFT505 treatment can reduce hepatic steatosis, inflammation and fibrosis, and reduce hepatic dysfunction markers; in clinical studies, however, GFT505 has been reported. Reducing a range of markers of liver dysfunction, including alanine aminotransferase, alkaline phosphatase (alp) and gamma-glutamyl transferase (ggt). One NASH liver biopsy basal phase 2 trial with a period of 1 year patients initially enrolled to receive 80 mg/day of GFT505 or placebo treatment, a 6 month interim safety analysis showed no safety issues affecting ongoing studies; the second enrollment phase selected 120 mg/day of GFT505 or placebo-treated patients.

In a phase 2 study for NASH sponsored by the national diabetes and digestive and renal disease institute, a semi-synthetic bile acid analog, obeticholic acid (OCA, 6 α -ethylchenodeoxycholic acid), a potent farnesoid X receptor agonist, was studied. This study was discontinued at the beginning of 1 month 2014 because about half of the 283 subjects had completed the study, when interim analysis in a program showed that the primary endpoint had been reached. Treatment (OCA 25 mg/day, 72 weeks) resulted in a highly statistically significant improvement in the primary histological endpoint (p =0.0024 on an intention treatment basis compared to placebo), defined as a reduction in NAS at least two points, with no fibrotic exacerbations.

Despite the reports of the primary benefits of GFT505 and OCA, there remains an important unmet clinical need for an effective and well-tolerated drug that can prevent or slow the progression of NAFLD and NASH.

Trametinib is an MEK inhibitor with an IC50 of 2nM for MEK1 and MEK2 enzymes, the mesocultural name of trametinib: n- [3- [ 3-cyclopropyl-5- [ (2-fluoro-4-iodophenyl) amino ] -3,4,6, 7-tetrahydro-6, 8-dimethyl-2, 4, 7-trioxopyridino [4,3-d ] pyrimidin-1 (2H) -yl ] phenyl ] acetamide; the molecular formula is as follows: C26H23FIN5O4; molecular weight: 615.39; CAS accession number: 871700-17-3. Trametinib has poor solubility and solvate has good solubility. Trametinib (0.1-100 nM) blocks TNF α and IL-6 in peripheral blood cells. Trametinib can inhibit colon cancer cells of 9/10 of human beings, and after the trametinib is administrated, the cell cycle can be blocked in a G1 phase. 0.1mg/kg trametinib or 10mg/kg leflunomide was effectively inhibited in adjuvant-induced arthritis (AIA) and type II collagen-induced arthritis (CIA) models. Trametinib and GSK2118436 are used together to effectively prevent cell growth, reduce Erk phosphorylation, reduce cyclin D1 and increase P27 protein. Trametinib (0.3 mg/kg,1mg/kg, oral) was effective in inhibiting HS-29 tumor transplantation in a nude mouse tumor transplantation model. However, trametinib has not been reported to prevent and treat NAFLD and NASH.

Disclosure of Invention

The purpose of the invention is as follows: the purpose of the present invention is to provide an effective and well-tolerated drug that can prevent or slow down the progression of NAFLD and NASH.

The technical scheme is as follows: in a first aspect of the invention, the application of trametinib in preparation of medicines for preventing and/or treating non-alcoholic hepatitis is provided.

Preferably, trametinib is used in an amount of 0.001mg/kg to 0.10mg/kg, preferably 0.03mg/kg to 0.10mg/kg.

In a second aspect of the invention, the application of trametinib in preparation of medicines for preventing and/or treating non-alcoholic fatty liver diseases is provided.

In a third aspect of the present invention, there is provided a composition for the prevention and/or treatment of non-alcoholic hepatitis, said composition comprising trametinib, a pharmaceutically acceptable salt thereof, and at least one pharmaceutically acceptable carrier, diluent, excipient or adjuvant.

Preferably, the pharmaceutically acceptable carrier comprises a filler, a binder, a disintegrant, a lubricant, a glidant.

Has the beneficial effects that: the invention provides application of trametinib in preparation of drugs for preventing and/or treating nonalcoholic hepatitis and nonalcoholic steatohepatitis, and results show that the trametinib can effectively reduce ALT and AST values in an MCD model, and a trametinib treatment group has small dyeing surface value, disappears fat particles and is free of inflammatory necrosis; trametinib can also effectively reduce fat and inflammatory reaction in a high-fat/fructose model, and a trametinib treatment group is small in dyeing surface value, obviously reduced in fat particles and small in damage.

Drawings

FIG. 1a is a graph of ALT levels in a mouse MCD model activity assay experiment;

FIG. 1b is a graph showing AST levels in the mouse MCD model activity assay experiment;

FIG. 1c is a graph of CHO levels in a mouse MCD model activity test experiment;

FIG. 1d is a graph of TG levels in a mouse MCD model activity assay experiment;

FIG. 2a is a schematic sectional view of a liver of a set of MCD models stained with oil Red O;

FIG. 2b is a schematic section of liver treated with a low dose post-treatment of MCD model group stained with oil red O;

FIG. 2c is a schematic section of liver treated after the MCD model group administered with oil red O staining;

FIG. 2d is a graph showing the result of an oil red O staining MCD model set experiment;

FIG. 3a is a schematic slice of a liver of the MCD model set;

FIG. 3b is a liver pathological section of the MCD model group at low dose;

FIG. 3c is a schematic slice of liver disease in the high dose group of MCD model group;

the above figures are the results of MCD model tests, wherein N is the normal group, M is the model group, T-0.03mg/kg is the low dose group, and T-0.1mg/kg is the high dose group.

FIG. 4a is a schematic sectional view of liver treated with sirius red staining in normal group;

FIG. 4b is a schematic slice of a liver treated with sirius red staining for the HDF + HF model group;

FIG. 4c is a schematic sectional view of a liver treated with sirius red staining after administration to the HDF + HF model group;

FIG. 4d is a graph showing the effect of HDF + HF model group experiments by sirius red staining method;

FIG. 5a is a photograph of a pathological section treated by oil-red-O staining in a HDF + HF normal group;

FIG. 5b is a schematic sectional view of liver treated by HDF + HF model group oil red O staining method;

FIG. 5c is a schematic section of liver stained with oil Red O treatment after administration of the HDF + HF model group;

FIG. 5d is a graph showing the experimental results of the HDF + HF model group by oil red O staining method;

FIG. 6a is a schematic sectional view of liver in the normal group;

FIG. 6b is a schematic section of the liver of the HDF + HF model set;

FIG. 6c is a schematic section of liver after administration of the HDF + HF model group;

FIG. 7a is a graph comparing the liver morphology after administration of the normal group, HDF + HF model and group;

FIG. 7b is a graph comparing liver indices in the normal group, HDF + HF model, and group after dosing;

FIG. 8a is a graph of ALT levels in a mouse HDF + HF model activity test experiment;

FIG. 8b is a graph of AST levels in a mouse HDF + HF model activity test experiment;

FIG. 8c is a graph of CHO levels in a mouse HDF + HF model activity test experiment;

FIG. 8d is a graph of TG levels in a mouse HDF + HF model activity assay experiment;

FIG. 9a is a graph of serum glucose levels in a mouse HDF + HF model;

FIG. 9b is a graph of mouse HDF + HF model serum insulin levels;

FIG. 9c is a graph of serum glucose concentration versus time for the mouse HDF + HF model;

FIG. 9c is a graph of a mouse HDF + HF model glucose antagonism test;

FIG. 9d is a graph of mouse HDF + HF model insulin resistance index;

FIG. 10a is a graph of HDF + HF model food consumption in mice after dosing;

FIG. 10b is a graph of the change in body weight of the mouse HDF + HF model after administration.

The above figures are all results of high-sugar and high-fat (HDF + HF) model tests, wherein N is normal group, HDF + HF/G is model group, and T-0.1mg/kg is administration group.

Detailed Description

The following detailed description of embodiments of the present invention is provided in connection with the accompanying drawings and examples. The following examples are intended to illustrate the invention, but are not intended to limit the scope of the invention.

Example 1

Trametinib was tested for activity on a mouse MCD model (NASH model):

the experimental method comprises the following steps: (1) The 8-week old C57BL/6N was kept under environmental control and free access to standard feed and drinking water. After one week of acclimation, the mice were divided into four groups, normal group mice were fed with normal feed, model group mice and administration group mice (low dose, high dose) were fed with methionine choline deficient feed (MCD, a 02082002B).

Two weeks after MCD feeding, mice in the administration group were injected intraperitoneally with vehicle (0.5% sodium carboxymethylcellulose (CMC-Na, sigma)) containing trametinib once daily for 14 consecutive days, mice in the model group and normal group were injected with vehicle at the same frequency.mice were sacrificed after 14 days, and mouse body weight and liver tissue were weighed.

Portions of each liver were fixed with 4% paraformaldehyde and analyzed for liver histology by Hematoxylin and Eosin (HE) staining and oil red staining. Other tissues were collected and frozen in liquid nitrogen for use, and serum was collected to measure metabolite parameters.

The experimental results are as follows:

(1) The activity test result shows that: 0.03mg/kg and 0.10mg/kg of trametinib can reduce ALT by 30.2% and 67.5%, and AST by 25.4% and 73.5%. CHO, TGs and HDL are similar to the MCD group, see FIGS. 1a, 1b, 1c and 1d;

(2) Liver pathological sections by oil red O staining showed: the MCD group has large staining value, a large amount of fat granules in the liver and severe inflammatory necrosis, while the trametinib treatment group has small staining value, no fat granules and no inflammatory necrosis, which is shown in figures 2a, 2b, 2c and 2d;

(3) Liver pathological section shows that the liver of MCD group contains a large amount of fat particles and has serious inflammatory necrosis, and the fatty particles of the trimetinib group disappear and have no inflammatory necrosis, so that the liver index can be effectively reduced. See fig. 3a, 3b and 3c.

Example 2

Trametinib was tested for activity on a mouse high fat diet/fructose model (NASH model):

the experimental method comprises the following steps: after one week of adaptation to the 8-week old C57BL/6N new environment, the mice were divided into three groups, and the normal group was fed with normal diet, and the model group mice and the administration group mice were fed with High Fat Diet (HFD) for eight weeks.

After normal control group and administration group mice were given normal drinking water and normal diet, and model group mice and administration group mice were given high fructose and high glucose drinking water (23.1 g fructose and 18.9g glucose added to 1L water) and continued to be given high fat diet for 8 weeks, the administration group mice were intraperitoneally injected with vehicle containing trametinib (0.5% sodium carboxymethylcellulose (CMC-Na, sigma) (trametinib amount of 0.1 mg/kg) per body weight once a day for 14 consecutive days.14 days, fasting blood glucose was measured after 14 days, and orbital blood was centrifuged to take serum for measuring fasting insulin.the mice were given 2g/kg of glucose solution intraperitoneally injected and blood glucose of 0, 30,60,90,120min was measured by tail tip blood using a Roche glucometer, then the mice were sacrificed, and mouse weights and liver tissues were weighed.

Portions of each liver were fixed with 4% paraformaldehyde and analyzed for liver histology by Hematoxylin and Eosin (HE) staining, oil red staining, and sirius red staining. Other tissues were collected and frozen in liquid nitrogen for use, and serum was collected to measure metabolite parameters.

The experimental results are as follows:

(1) Sirius red stained liver pathology sections showed: the staining surface value of the model group is larger, the liver contains a large amount of fat granules, and the inflammatory necrosis is serious, while the staining surface value of the administration group is smaller, the fat granules are fewer, the inflammatory necrosis is less, and the staining area is obviously reduced, which is shown in fig. 4a, 4b, 4c and 4d;

(2) Liver pathological sections by oil red O staining showed: the HFD + HF group has larger staining value, a large amount of fat granules in the liver and serious inflammatory necrosis, while the administration group has smaller staining value, less fat granules and lighter inflammation, and the staining area is obviously reduced, as shown in figures 5a, 5b, 5c and 5d;

(3) Liver pathological section shows that the liver of the model group contains a large amount of fat particles and has severe inflammatory necrosis, while the administration group has fewer fat particles and has lighter inflammation, which is shown in figures 6a, 6b and 6c.

(4) Trametinib can significantly improve liver morphology of high-fat and high-sugar dieters, and reduce liver index, as shown in fig. 7a and 7b.

(5) The activity test result shows that: trametinib can significantly reduce ALT, AST, CHO, and TGs, see fig. 8a, 8b, 8c, and 8d;

(6) The activity test result shows that: trametinib can significantly improve serum glucose, serum insulin levels, insulin antagonism, see fig. 9a, 9b, 9c and 9d;

(7) Trametinib at therapeutic doses, mice fed and weighed no significant difference from the control group, see fig. 10a, 10b.

The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the invention. It should be noted that various changes and modifications can be made by those skilled in the art without departing from the spirit of the invention, and these changes and modifications are all within the scope of the invention. Therefore, the protection scope of the present patent shall be subject to the appended claims.

Claims

1. The application of trametinib in preparing the medicine for preventing and/or treating nonalcoholic hepatitis is that the dosage of the trametinib is 0.001-0.10 mg/kg.

2. Use according to claim 1, characterized in that: the dosage of trametinib is 0.005-0.1mg/kg.

3. The application of trametinib in preparing the medicine for preventing and/or treating non-alcoholic fatty liver disease, wherein the dosage of the trametinib is 0.001-0.10 mg/kg.

4. Use according to claim 3, characterized in that: the amount of trametinib is 0.005-0.1mg/kg.