CN112206229A - Composition for treating fatty liver, hepatitis and hepatic fibrosis and its preparation method - Google Patents

Composition for treating fatty liver, hepatitis and hepatic fibrosis and its preparation method Download PDF

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CN112206229A
CN112206229A CN201910622895.5A CN201910622895A CN112206229A CN 112206229 A CN112206229 A CN 112206229A CN 201910622895 A CN201910622895 A CN 201910622895A CN 112206229 A CN112206229 A CN 112206229A
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borneol
composition
edaravone
group
phenyl
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安文吉
张正平
王磊
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Yantai Yenepharma Co ltd
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Yantai Yenepharma Co ltd
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/41—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with two or more ring hetero atoms, at least one of which being nitrogen, e.g. tetrazole
    • A61K31/415—1,2-Diazoles
    • A61K31/4152—1,2-Diazoles having oxo groups directly attached to the heterocyclic ring, e.g. antipyrine, phenylbutazone, sulfinpyrazone
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/045—Hydroxy compounds, e.g. alcohols; Salts thereof, e.g. alcoholates
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/16—Drugs for disorders of the alimentary tract or the digestive system for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics

Abstract

The invention relates to application of a composition in preparing a medicine for treating non-alcoholic fatty liver disease (NAFL), non-alcoholic steatohepatitis (NASH) and hepatic fibrosis and application of the composition in preparing the medicine for treating the related disorders, wherein the composition contains 3-methyl-1-phenyl-2-pyrazoline-5-ketone or pharmaceutically acceptable salt thereof and borneol, and the medicine is a medicine for improving the non-alcoholic fatty liver disease (NAFL), the non-alcoholic steatohepatitis (NASH) and the hepatic fibrosis.

Description

Composition for treating fatty liver, hepatitis and hepatic fibrosis and its preparation method
Technical Field
The invention belongs to the field of pharmacy, and relates to application of a composition of 3-methyl-1-phenyl-2-pyrazoline-5-ketone and borneol in treating non-alcoholic fatty liver disease (NAFL), non-alcoholic steatohepatitis (NASH), hepatic fibrosis and related disorders.
Background
NAFLD (nonalcoholic fatty Liver Disease) is a Liver Disease characterized by hepatocyte lipid deposition induced by glycolipid metabolic disorders, and the Disease spectrum includes simple fatty Liver (NAFL), NASH (nonalcoholic steatohepatitis, NASH), NASH-associated cirrhosis and hepatocellular carcinoma (Clinical Liver Disease, 2018, 11 (4): 81-81).
NAFLD has increased worldwide in prevalence and incidence over the last 20 years, with NAFLD being 25.24% worldwide (hepatology, 2016;64: 73-84). The prevalence rate of NAFLD is greatly increased in the last decade in China, and reaches 26-45%, which has replaced the first chronic liver disease of China with chronic viral liver diseases (World JGastroenterenol.2013; 19: 5334-.
Traditional drug therapies have very limited efficacy in NAFLD, and the development of new targeted drugs is receiving attention and expectations from researchers in the field of NAFLD. In 2019, the FDA granted obeticholic acid a breakthrough medication eligibility to treat NASH with liver fibrosis, a fast-lane position to treat primary biliary cirrhosis, and an orphan position to treat primary biliary cirrhosis and primary sclerosing cholangitis. Other new drugs include glucagon-like peptide-1 receptor agonists, peroxisome proliferator activated receptor- α/γ dual receptor agonists, mitochondrially targeted insulin sensitizers, agonists targeting bile acid signaling pathways, acetyl-coa carboxylase inhibitors, thyroxine analogs, drugs that block apoptosis, drugs that inhibit inflammatory responses, lysyl oxidase-like protein-2 blockers, Rho kinase-2 inhibitors, and the like, which have been studied in small samples and even phase 2 clinical trial data show that the drug may effectively act on multiple signaling pathways for insulin resistance, lipid metabolism, apoptosis, inflammatory responses, and fibrotic processes, is expected to become a new treatment option, but needs to be studied in the future to further define the curative effect and the safety (Nat Rev Drug Discov. 2016;15: 249-.
In recent years, a plurality of main molecular signal pathways and related regulation mechanisms which are involved in metabolic disorder, inflammatory reaction and fibrosis development in the process of NAFLD formation are advanced in a successive way, (1) the insulin resistance of NAFLD patients is proved to be a signal transduction disorder behind an insulin receptor on a molecular level (cell. 2012;148: 852) 871), namely, the combination of insulin and the receptor thereof starts the autophosphorylation activation of the receptor, and further, the disorder in the process of activating phosphatidylinositol 3 kinase, serine/threonine and downstream effector molecules is transmitted and activated hierarchically. Wherein the active product components of fatty acid metabolism (such as sphingolipids and diacylglycerol) activated protein kinase C (Nature 2014;510:84-91; Prog Lipid Res.2013; 52:175-, Cholesterol crystallization, etc.) activates proinflammatory pathway of caspase-1, and inflammatory factors activates apoptosis pathway initiated by nuclear factor NF-kB and JNK 1; (3) the key link for starting the generation of NAFLD fibrosis, namely related pathways leading to hepatic stellate cell activation, are clear, and comprise a Hedgehog pathway (hepatology. 2012;55: 1711-.
Free radicals (radicals) refer to ions, atoms, radicals or molecules containing unpaired electrons. Normally, the generation and elimination of free radicals in the human body are in dynamic equilibrium, however, when the free radical balance in the human body is disrupted due to the injury of the body, the inflammation reaction, the pathological changes, or the influence of external environmental factors, the excess free radicals attack the biological membrane, initiate a series of free radical chain reactions, cause the oxidative damage of proteins, enzymes, lipids, and nucleic acids in the body, and affect the normal metabolism in the body, thereby causing diseases (Physiological Reviews, 2002, 82(1): 47-95). Numerous human and animal studies have observed associations between the disease state of NAFL/NASH and biomarkers of oxidative stress or lipid oxidation (Clin Sci (Lond); 2004;106: 261-. The major Lipid peroxidation products produced using linoleic acid are reported to be significantly elevated in NASH patients compared to steatosis patients, and there is a strong correlation between these oxidation products and liver histopathology, such as inflammation, fibrosis and steatosis (J Lipid Res 2010;51: 3046-.
3-methyl-1-phenyl-2-pyrazolin-5-one is capable of scavenging free hydroxyl radicals and peroxynitrite (Experimental & Clinical diagnostics, 2004, 9 (3): 177). It limits the degree of lipid peroxidation through free radical generation and cell membrane damage caused by oxidative stress, exerts protective and antioxidant effects and delays disease progression. It has been reported that 3-methyl-1-phenyl-2-pyrazolin-5-one protects brain, heart (Life Sci.71 (2002) 2249) and liver (Eur. J. Pharmacol. 465 (2003) 163; Journal of pharmacological and Experimental Therapeutics, 2003, 307(1): 74-82.) from free radical mediated damage in various Experimental models.
The borneol is divided into synthetic borneol and natural borneol. The synthetic borneol contains isoborneol, while the natural borneol does not contain isoborneol. It has been shown that borneol improves intestinal absorption (AAPS PharmSciTech.2011 Dec;12 (4): 1044-9), blood brain barrier (J Asian Nat Prod Res.2014, 16 (6): 648-57; J Ethnopharmacol. 2015, 162: 270-7) and corneal permeability (Pharmazie. 2006, 61 (9): 783-8), neuroprotection (Neuroscience 2011, 176: 408-19; Eur J Pharmacol. 2014, 740: 522-31), anti-inflammatory (Infiammation. 2014, 37 (4): 1148-57), antioxidant and protective DNA damage (J Agric Food Chem. 2014, 62 (28): 6632-9), enhances 1101 GABA receptor function (Biocheamacol. 2014, 2005 (7): 11), and also improves rat function, antithrombotic activity (Am J2008. 719; Chimed 4; 719-27).
Disclosure of Invention
According to the prior art, whether the composition of 3-methyl-1-phenyl-2-pyrazoline-5-ketone and borneol has a treatment effect on non-alcoholic fatty liver (NAFL), non-alcoholic steatohepatitis (NASH), hepatic fibrosis and related disorders cannot be presumed, and whether the composition in the existing mass ratio of 10:1 to 1:10 shows a synergistic effect cannot be judged.
The invention unexpectedly discovers the remarkable effect of the composition of the 3-methyl-1-phenyl-2-pyrazoline-5-ketone and the borneol on preparing drugs for treating non-alcoholic fatty liver disease (NAFL), non-alcoholic steatohepatitis (NASH) and hepatic fibrosis and related disorders.
Furthermore, the pharmaceutical composition can synergistically enhance the efficacy of treating non-alcoholic fatty liver disease (NAFL), non-alcoholic steatohepatitis (NASH) and hepatic fibrosis; compared with the single use of 3-methyl-1-phenyl-2-pyrazoline-5-ketone or pharmaceutically acceptable salt thereof or the single use of borneol, the compound has stronger drug effect.
Furthermore, the weight ratio of the pharmaceutical composition 3-methyl-1-phenyl-2-pyrazoline-5-one or the pharmaceutically acceptable salt thereof to borneol is 1: 10-10: 1.
Furthermore, the weight ratio of the pharmaceutical composition 3-methyl-1-phenyl-2-pyrazoline-5-one or the pharmaceutically acceptable salt thereof to borneol is 1: 5-5: 1.
The borneol of the invention comprises natural borneol.
Wherein the drug is a drug for improving non-alcoholic fatty liver disease (NAFL), non-alcoholic steatohepatitis (NASH) and hepatic fibrosis.
Drawings
FIG. 1 relative cell viability values for each group
FIG. 2 protective Effect of edaravone and borneol compositions on lipotoxicity in HepG2 cells induced by free fatty acids
FIG. 3 median Effect plot of synergistic analysis of protective Effect of edaravone and borneol compositions on lipotoxicity in HepG2 cells
FIG. 4 protective Effect of edaravone and borneol compositions on lipotoxicity in HepG2 cells induced by free fatty acids
FIG. 5 median Effect plot of synergistic analysis of protective Effect of edaravone and borneol compositions on lipotoxicity in HepG2 cells
FIG. 6 the degree of phosphorylation of NF-. kappa. B p65 in each administration group
FIG. 7 relative phosphorylation of JNK in each administration group
FIG. 8 shows the relative expression amounts of a-SMA in each administration group
Detailed Description
The following examples illustrate the invention and should not be construed as limiting it. The edaravone mentioned in the examples is 3-methyl-1-phenyl-2-pyrazolin-5-one and the composition is a combination of 3-methyl-1-phenyl-2-pyrazolin-5-one and borneol.
Example 1: protective effect of edaravone and borneol composition on lipotoxicity in HepG2 cells induced by free fatty acid
1 materials and methods
1.1 cells
HepG2 was purchased from ATCC and cultured in DMEM medium containing 10% fetal bovine serum, 1% penicillin and streptomycin at 37 ℃ in 5% CO2 incubator.
1.2 drugs and reagents
The mass ratio of edaravone to borneol is 100: 1-1: 100
Edaravone (Sigma-Aldrich, M70800-100G)
Borneol (Sigma-Aldrich, 420247-1G)
FFAs (oleic acid/palmitic acid, 2:1)
Oleic acid (Sigma-Aldrich, O1008-1G)
Palmitic acid (Sigma-Aldrich, P5585-10G)
Cell titer Glo Cell viability assay kit (Promega, G7571)
1.3 test methods
1.3.1 cell models
All cells were seeded in cell culture flasks at least 24 hours before treatment. To induce FFA overload, 70% confluent HepG2 cells were exposed to a mixture of different concentrations of long chain FFA (oleic acid/palmitic acid, 2:1) in medium containing 1% FFA-free BSA. Stock solutions of 30mM FFA were diluted in medium containing 1% BSA to obtain the desired final concentration.
HepG2 cells at 8X 103Initial density of individual cells/well was seeded in 96-well plates, incubated for 2h with varying concentrations of edaravone and borneol composition and cells, followed by addition of 3 mffa (BLANK control with 0.1% DMSO),after 24 hours of incubation, Cell viability (%) of each group was measured using Cell titer Glo Cell viability assay kit.
Grouping is as follows (table 1):
TABLE 1 Edaravone and borneol combination administration schematic
Administration group E0 E1 E2 E3 E4 E5
B0 E0B0 E1B0 E2B0 E3B0 E4B0 E5B0
B1 E0B1 E1B1 E2B1 E3B1 E4B1 E5B1
B2 E0B2 E1B2 E2B2 E3B2 E4B2 E5B2
B3 E0B3 E1B3 E2B3 E3B3 E4B3 E5B3
B4 E0B4 E1B4 E2B4 E3B4 E4B4 E5B4
B5 E0B5 E1B5 E2B5 E3B5 E4B5 E5B5
BLANK
Wherein:
edaravone concentrations were 0, 0.1, 0.3, 1, 3, 10 (μ g/ml), n =4 for columns E0 to E5, respectively;
borneol concentrations of 0, 0.1, 0.3, 1, 3, 10 (μ g/ml), n =4, administered from row B0 to row B5, respectively;
EB is the combination of edaravone and borneol;
BLANK is BLANK control, and FFAs and the composition of borneol and edaravone are not added during molding.
1.3.2 detection index
Cell viability
And detecting the chemiluminescence intensity of each well by using a microplate reader, taking the average value of the BLANK control group as 100% of cell viability, and comparing the chemiluminescence intensity values of other groups with the chemiluminescence intensity value of the BLANK control group to calculate the relative cell viability.
1.4 statistical methods
The statistical software of Graph Pad Prism7 is used for analysis, the measured data is expressed by X +/-s, the mean comparison among multiple groups adopts one-factor variance analysis, the two-by-two comparison adopts Dunnett test, and P <0.05 indicates that the difference has statistical significance.
2 results of the test
As shown in figure 1, FFAs induced HepG2 free fatty acid overload resulting in HepG2 cytotoxicity, the relative cell viability of the modeling group E0B0 was significantly reduced compared to the BLANK group. The protective effect of the administration combination of E1B1 on the cell protective effect of E1B0, the administration combination of E2B1 on the cell protective effect of E2B0, the administration combination of E3B1 on the cell protective effect of E3B0, the administration combination of E4B1 on the cell protective effect of E4B0 and the administration combination of E5B1 on the cell protective effect of E5B0 caused by free fatty acid overload is not significantly different (P > 0.05), which indicates that the borneol in the composition has no synergistic effect on the cell protective effect of edaravone at the concentration of 0.1 mu g/ml. The protective effects of the E1B2 group, the E1B3 group, the E1B4 group and the E1B5 group are remarkably different relative to the protective effects of the E1B0 group (P is less than 0.05); the protective effects of the E2B2 group, the E2B3 group, the E2B4 group and the E2B5 group are remarkably different relative to the protective effects of the E2B0 group (P < 0.05); the protective effects of the E3B2 group, the E3B3 group, the E3B4 group and the E3B5 group are remarkably different relative to the protective effects of the E3B0 group (P < 0.05); the protective effects of the E4B2 group, the E4B3 group, the E4B4 group and the E4B5 group are remarkably different relative to the protective effects of the E4B0 group (P < 0.05); the protective effects of the E5B2 group, the E5B3 group, the E5B4 group, the E5B5 group and the E5B0 group are remarkably different (P is less than 0.05); these show that the borneol in the composition has synergistic effect on the cell protection effect of edaravone at the concentration of 0.3, 1, 3 and 10 mu g/ml.
3 conclusion
Compared with an E0B0 control group, the edaravone and borneol combined administration group can reduce FFAs-induced liver cell damage in a dose-dependent manner, and meanwhile, the edaravone and borneol combined administration group is combined within a mass ratio of 1: 10-10: 1, and has a significant difference compared with the control group.
Example 2: synergistic analysis of protective effects of edaravone and borneol compositions on lipotoxicity in HepG2 cells induced by free fatty acid
1.1 cells
HepG2 was purchased from ATCC and cultured in DMEM medium containing 10% fetal bovine serum, 1% penicillin and streptomycin at 37 ℃ in 5% CO2 incubator.
1.2 drugs and reagents
The edaravone and borneol composition is in a mass ratio of 10: 1-1: 10
Edaravone (Sigma-Aldrich, M70800-100G)
Borneol (Sigma-Aldrich, 420247-1G)
FFAs (oleic acid/palmitic acid, 2:1)
Oleic acid (Sigma-Aldrich, O1008-1G)
Palmitic acid (Sigma-Aldrich, P5585-10G)
Cell titer Glo Cell viability assay kit (Promega, G7571)
1.3 test methods
1.3.1: cell model
All cells were seeded in cell culture flasks at least 24 hours before treatment. To induce FFA overload, 70% confluent HepG2 cells were exposed to a mixture of different concentrations of long chain FFA (oleic acid/palmitic acid, 2:1) in medium containing 1% FFA-free BSA. Stock solutions of 30mM FFA were diluted in medium containing 1% BSA to obtain the desired final concentration.
HepG2 cells at 8X 103The initial density of each Cell/well is inoculated in a 96-well plate, the edaravone and borneol composition with different concentrations and the cells are incubated for 2h, then 3mMFFA (0.1% DMSO is added in a BLANK control group) is added, the incubation is carried out for 24 h, the Cell viability (%) of each group is detected by using a Cell titer Glo Cell viability detection kit, and the inhibition rate of the composition on the lipotoxicity in the cells is calculated according to the following formula: inhibition (%) = (administration group cell viability-model group cell viability)/(100% cell viability-model group cell viability) × 100.
Grouping is as follows (table 2, table 3, table 4, table 5):
TABLE 2 Edaravone and borneol concentrations administered alone (Eda. Edaravone; bor. borneol)
Eda. (ng/mL) Bor. (ng/mL)
13200 13200
4400 4400
1467 1467
488.9 488.9
163.0 163.0
54.32 54.32
18.11 18.11
6.036 6.036
Table 3 edaravone and borneol in a 10:1 proportion of the combination administration concentration (Eda. edaravone; bor. borneol)
Total concentration of composition (ng/mL) Eda. (ng/mL) Bor. (ng/mL)
13200 12000 1200
4400 4000 400
1467 1333 133.3
488.9 444.4 44.44
163.0 148.1 14.81
54.32 49.38 4.938
18.11 16.46 1.646
6.036 5.487 0.5487
Table 4 edaravone and borneol in a 1:1 proportion of the combination administration concentration (Eda. edaravone; bor. borneol)
Total concentration of composition (ng/mL) Eda. (ng/mL) Bor. (ng/mL)
13200 6600 6600
4400 2200 2200
1467 733.3 733.3
488.9 244.4 244.4
163.0 81.48 81.48
54.32 27.16 27.16
18.11 9.053 9.053
6.036 3.018 3.018
Table 5 edaravone and borneol in a 1:10 proportion combination administration concentration (Eda. edaravone; bor. borneol)
Total concentration of composition (ng/mL) Eda. (ng/mL) Bor. (ng/mL)
13200 1200 12000
4400 400 4000
1467 133.3 1333
488.9 44.44 444.4
163.0 14.81 148.1
54.32 4.938 49.38
18.11 1.646 16.46
6.036 0.5487 5.487
1.4 statistical methods
Analysis was performed using Graph Pad Prism7 statistical software and dose-dependent curves were fitted using the log (inhibitor) vs. normalized response-Variable slope equation. The combination index was calculated by calcusyn2.0 software using the multi-drug effect equation.
The experimental results are as follows:
2.1 protective Effect of different proportions of edaravone and borneol on lipotoxicity in HepG2 cells induced by free fatty acid the fitted curve is shown in FIG. 2. the different proportions of the compositions (edaravone: borneol =10:1, 1:1 and 1: 10) can reduce toxicity of free fatty acid overload on HepG2 cells dose-dependently in the total concentration range of 6.0-13200 ng/mL.
2.2 synergistic analysis (Table 6, FIG. 3)
By using the calcusyn2.0 software and calculating the combination drug index by using the multi-drug effect equation (table 3), it can be known that when the edaravone and borneol are administered at the combination ratio of 10:1, 1:1 and 1:10, the protective effect of the combination of the edaravone and borneol on the lipid toxicity in HepG2 cells induced by free fatty acid has a synergistic effect (CI value is less than 0.9), and fig. 3 is a median effect diagram of the synergistic analysis of the protective effect of the edaravone and borneol combination on the lipid toxicity in HepG2 cells.
TABLE 6 synergistic analysis of protective Effect of edaravone and borneol compositions on lipotoxicity in HepG2 cells (Eda. is edaravone; bor. is borneol; CI is combination Effect index)
Administration combination CI value (ED 50) CI value (ED 75) CI value (ED 90)
Eda. N/A N/A N/A
Bor. N/A N/A N/A
Eda.+Bor.(10:1) 0.45206 0.39205 0.3405
Eda.+Bor.(1:1) 0.02561 0.01303 0.00673
Eda.+Bor.(1:10) 0.53312 0.6857 0.97336
2.3 conclusion
The synergistic analysis shows that when the mass ratio of the edaravone to the borneol is 1: 10-10: 1, the combination index CI is less than 0.9, and the edaravone and borneol composition has the effect of synergistically reducing the lipotoxicity of HepG2 cells caused by free fatty acid.
Example 3: synergistic analysis of protective effects of edaravone and borneol compositions on lipotoxicity in HepG2 cells induced by free fatty acid
1.1 cells
HepG2 was purchased from ATCC and cultured in DMEM medium containing 10% fetal bovine serum, 1% penicillin and streptomycin at 37 ℃ in 5% CO2 incubator.
1.2 drugs and reagents
The edaravone and borneol composition is prepared from 5: 1-1: 5 by mass
Edaravone (Sigma-Aldrich, M70800-100G)
Borneol (Sigma-Aldrich, 420247-1G)
FFAs (oleic acid/palmitic acid, 2:1)
Oleic acid (Sigma-Aldrich, O1008-1G)
Palmitic acid (Sigma-Aldrich, P5585-10G)
Cell titer Glo Cell viability assay kit (Promega, G7571)
1.3 test methods
1.3.1: cell model
All cells were seeded in cell culture flasks at least 24 hours before treatment. To induce FFA overload, 70% confluent HepG2 cells were exposed to a mixture of different concentrations of long chain FFA (oleic acid/palmitic acid, 2:1) in medium containing 1% FFA-free BSA. Stock solutions of 30mM FFA were diluted in medium containing 1% BSA to obtain the desired final concentration.
HepG2 cells at 8X 103The initial density of each Cell/well is inoculated in a 96-well plate, the edaravone and borneol composition with different concentrations and the cells are incubated for 2h, then 3mMFFA (0.1% DMSO is added in a BLANK control group) is added, the incubation is carried out for 24 h, the Cell viability (%) of each group is detected by using a Cell titer Glo Cell viability detection kit, and the inhibition rate of the composition on the lipotoxicity in the cells is calculated according to the following formula: inhibition (%) = (administration group cell viability-model group cell viability)/(100% cell viability-model group cell viability) × 100.
Grouping is as follows (table 7, table 8, table 9, table 10):
TABLE 7 Edaravone and borneol concentrations for single administration (Eda. Edaravone; bor. borneol)
Eda.(ng/mL) Bor.(ng/mL)
13200 13200
4400 4400
1467 1467
488.9 488.9
163.0 163.0
54.32 54.32
18.11 18.11
6.036 6.036
Table 8 edaravone and borneol in 5:1 proportion of the combination administration concentration (Eda. edaravone; bor. borneol)
Total concentration of composition (ng/mL) Eda. (ng/mL) Bor. (ng/mL)
13200 11000 2200
4400 3667 733.3
1467 1222 244.4
488.9 407.4 81.48
163.0 135.8 27.16
54.32 45.27 9.053
18.11 15.09 3.018
6.036 5.030 1.006
Table 9 edaravone and borneol in a ratio of 1:1 proportion of the combination administration concentration (Eda. edaravone; bor. borneol)
Total concentration of composition (ng/mL) Eda. (ng/mL) Bor. (ng/mL)
13200 6600 6600
4400 2200 2200
1467 733.3 733.3
488.9 244.4 244.4
163.0 81.48 81.48
54.32 27.16 27.16
18.11 9.053 9.053
6.036 3.018 3.018
Table 10 edaravone and borneol in a ratio of 1:5 proportion combination administration concentration (Eda. edaravone; bor. borneol)
Total concentration of composition (ng/mL) Eda. (ng/mL) Bor. (ng/mL)
13200 2200 11000
4400 733.3 3667
1467 244.4 1222
488.9 81.48 407.4
163.0 27.16 135.8
54.32 9.053 45.27
18.11 3.018 15.09
6.036 1.006 5.030
1.4 statistical methods
Analysis was performed using Graph Pad Prism7 statistical software and dose-dependent curves were fitted using the log (inhibitor) vs. normalized response-Variable slope equation. The combination index was calculated by calcusyn2.0 software using the multi-drug effect equation.
The experimental results are as follows:
2.1 protective Effect of different proportions of edaravone and borneol on lipotoxicity in HepG2 cells induced by free fatty acid the fitted curve is shown in FIG. 4. the different proportions of the compositions (edaravone: borneol =5:1, 1:1 and 1: 5) can reduce toxicity of free fatty acid overload on HepG2 cells dose-dependently in the total concentration range of 6.0-13200 ng/mL.
2.2 synergistic analysis (Table 11, FIG. 5)
By using the calcusyn2.0 software and calculating the combination drug index by using the multi-drug effect equation (table 8), it can be known that when the edaravone and borneol are administered at the combination ratio of 5:1, 1:1 and 1:5, the protective effect of the combination of the edaravone and borneol on the lipid toxicity in HepG2 cells induced by free fatty acid has a synergistic effect (CI value is less than 0.9), and fig. 5 is a median effect diagram of the synergistic analysis of the protective effect of the edaravone and borneol combination on the lipid toxicity in HepG2 cells.
TABLE 11 synergistic analysis of protective Effect of edaravone and borneol compositions on lipotoxicity in HepG2 cells (Eda. is edaravone; bor. is borneol; CI is combination Effect index)
Administration combination CI value (ED 50) CI value (ED 75) CI value (ED 90)
Eda. N/A N/A N/A
Bor. N/A N/A N/A
Eda.+Bor.(5:1) 0.02524 0.01345 0.00718
Eda.+Bor.(1:1) 0.02561 0.01303 0.00673
Eda.+Bor.(1:5) 0.01952 0.01097 0.00654
2.3 conclusion
According to synergistic analysis, when the mass ratio of the edaravone to the borneol is 1: 5-5: 1, the combination index CI is less than 0.9, and the edaravone and borneol composition has the effect of synergistically reducing the lipotoxicity of HepG2 cells caused by free fatty acid.
Example 4: research on effect of edaravone and borneol composition on JNK, NF-kB signaling pathway in TNF-alpha activated HepG2 cells
1 materials and methods
1.1 cells
HepG2 was purchased from ATCC and cultured in DMEM medium containing 10% fetal bovine serum, 1% penicillin and streptomycin at 37 ℃ in 5% CO2 incubator.
1.2 drugs and reagents
The edaravone and borneol composition is in a mass ratio of 10: 1-1: 10
Edaravone (Sigma-Aldrich, M70800-100G)
Borneol (Sigma-Aldrich, 420247-1G)
Human recombinant TNF-alpha (Abcam, ab 9642)
The antibody Phospho-NF-. kappa. B p65 (Ser 536) (CST, # 3031)
Antibody NF- κ B p65 (CST, # 8242)
Antibody phophor c-Jun NH2-terminal kinase (JNK) (CST, # 3270)
Antibody c-Jun (JNK) (CST, # 9165)
1.3 test methods
1.3.1: cell model
Cells were preincubated with the indicated concentrations of reagents for 2 hours, followed by incubation with 10 ng/mL TNF-. alpha.for 4 hours. After 4 hours, cell lysates containing antibody XL-665 labeled Phospho-NF-. kappa. B p65 (Ser 536), antibody EulabeledNF-. kappa. B p65, antibody XL-665 labeled Phospho c-Jun NH2-terminal kinase (JNK), and antibody Eulabeled c-Jun (JNK) were added, and after 1 hour of incubation, the degree of phosphorylation of NF-. kappa. B p65 and JNK was examined using the HTRF method.
Grouping is as follows (table 12):
TABLE 12 Edaravone and borneol combination administration schematic diagram
Administration group E0 E1 E2 E3
B0 E0B0 E1B0 E2B0 E3B0
B1 E0B1 E1B1 E2B1 E3B1
B2 E0B2 E1B2 E2B2 E3B2
B3 E0B3 E1B3 E2B3 E3B3
BLANK
Wherein:
edaravone concentrations were 0, 1, 5, 10 (μ g/ml), n =4 for columns E0 to E3, respectively.
Borneol concentrations were 0, 1, 5, 10 (μ g/ml), n =4 for administration in rows B0 to B3, respectively.
BLANK is a BLANK control group, and the composition of TNF-alpha, borneol and edaravone is not added during molding.
1.3.2 detection index
1.3.2.1 degree of phosphorylation of NF-. kappa. B p65
The phosphorylation intensity relative to NF-. kappa. B p65 was calculated by lysing the cells with a lysate containing the antibody XL-665 labeled Phospho-NF-. kappa. B p65 (Ser 536) and the antibody EulabeledNF-. kappa. B p65, detecting the 665nm and 620nm wavelengths in each set of cell lysates by the HTRF method, and dividing the intensity of fluorescence at 665nm by the intensity at 620 nm.
1.3.2.2 degree of JNK phosphorylation
The phosphorylation intensity relative to JNK can be calculated by lysing the cells using cell lysates containing the antibody XL-665 labeled phosphor c-Jun NH2-terminal kinase (JNK) and the antibody Eulabeledc-Jun (JNK), detecting 665nm and 620nm wavelengths in the cell lysates of each group using the HTRF method, and dividing the fluorescence intensity at 665nm wavelength by the intensity at 620nm wavelength.
1.4 statistical methods
The statistical software of Graph Pad Prism7 is used for analysis, the measured data is expressed by X +/-s, the mean comparison among multiple groups adopts one-factor variance analysis, the two-by-two comparison adopts Dunnett test, and P <0.05 indicates that the difference has statistical significance.
2 results of the test
2.1 Effect of Edaravone borneol composition on NF- κ B phosphorylation
Data were counted using one-way ANOVA (one-way ANOVA) with the degree of phosphorylation of NF- κ B p65 of group E0B0 as a control. The results are shown in FIG. 6. The results show that the phosphorylation degrees of JNF- κ B P65 of groups E0B2, E0B3, E1B1, E1B2, E1B3, E2B0, E2B1, E2B2, E2B3, E3B0, E3B1, E3B2 and E3B3 are significantly different relative to those of groups E0B0 (P < 0.05).
2.2 Effect of Edaravone borneol composition JNK phosphorylation
Data were statistically analyzed by one-way ANOVA (one-way ANOVA) using JNK phosphorylation of group E0B0 as a control, and the results are shown in FIG. 7. The results show that the JNK phosphorylation degrees of groups E0B2, E0B3, E1B1, E1B2, E1B3, E2B0, E2B1, E2B2, E2B3, E3B0, E3B1, E3B2, and E3B3 are significantly different relative to group E0B0 (P < 0.05).
3 conclusion
From the above results, it is known that the edaravone and borneol combined administration group can significantly reduce the phosphorylation degree of NF- κ B and the phosphorylation degree of JNK in TNF- α activated hepatocytes compared with the E0B0 control group, and has great advantages compared with the administration of two drugs alone.
Example 5: study on effect of edaravone and borneol composition on alpha-SMA signal pathway in TGF-beta activated LX-2 cells
1 materials and methods
1.1 cells
LX-2 was purchased from ATCC and cultured in DMEM medium containing 10% fetal bovine serum, 1% penicillin and streptomycin at 37 ℃ in 5% CO2 incubator.
1.2 drugs and reagents
The edaravone and borneol composition is in a mass ratio of 10: 1-1: 10
Edaravone (Sigma-Aldrich, M70800-100G)
Borneol (Sigma-Aldrich, 420247-1G)
Human recombinant TGF-beta (Abcam, ab 50036)
ELISA kit for detecting alpha-SMA (Abcam, ab 240678)
ELISA kit for detecting beta-actin (LSBio, LS-F10737-1)
1.3 test methods
1.3.1: cell model
Cells were preincubated with the indicated concentrations of reagents for 3.5 hours, followed by another 48 hour incubation with 5 ng/mL TGF- β.
Cells were lysed after 48 hours and the extent of α -SMA and β -actin expression was measured using ELISA.
The orthogonal groupings are as follows (table 13):
TABLE 13 Edaravone and borneol combination administration schematic
Administration group E0 E1 E2 E3
B0 E0B0 E1B0 E2B0 E3B0
B1 E0B1 E1B1 E2B1 E3B1
B2 E0B2 E1B2 E2B2 E3B2
B3 E0B3 E1B3 E2B3 E3B3
BLANK
Wherein:
edaravone concentrations were 0, 1, 5, 10 (μ g/ml), n =4 for columns E0 to E3, respectively.
Borneol concentrations were 0, 1, 5, 10 (μ g/ml), n =4 for administration in rows B0 to B3, respectively.
BLANK is a BLANK control group, and no TGF-beta, borneol and edaravone composition is added during molding.
1.3.2 detection index
ELISA detects the expression conditions of alpha-SMA and beta-actin, and the relative alpha-SMA expression quantity can be obtained by dividing the absolute expression quantity of alpha-SMA by the absolute expression quantity of beta-actin.
1.4 statistical methods
The statistical software of Graph Pad Prism7 is used for analysis, the measured data is expressed by X +/-s, the mean comparison among multiple groups adopts one-factor variance analysis, the two-by-two comparison adopts Dunnett test, and P <0.05 indicates that the difference has statistical significance.
2 results of the test
The relative alpha-SMA expression level can be obtained by dividing the absolute alpha-SMA expression level by the absolute beta-actin expression level. The relative expression level of the alpha-SMA in the E0B0 group is taken as a control, and single-way ANOVA (one-way ANOVA) is adopted to count the data. The results are shown in FIG. 8. The results show that the relative expression amounts of alpha-SMA in groups E0B2, E0B3, E1B1, E1B2, E1B3, E2B0, E2B1, E2B2, E2B3, E3B0, E3B1, E3B2 and E3B3 are significantly different relative to that in group E0B0 (P < 0.05).
2.2 conclusion
From the results, the edaravone and borneol combined administration group can obviously reduce the expression degree of alpha-SMA in the hepatic stellate cells activated by TGF-beta relative to a control group, and has great advantages compared with the single administration of two medicines.

Claims (10)

1. The application of a composition in preparing a medicament for treating non-alcoholic fatty liver disease (NAFL), non-alcoholic steatohepatitis (NASH) and hepatic fibrosis is disclosed, wherein the composition contains 3-methyl-1-phenyl-2-pyrazoline-5-ketone or pharmaceutically acceptable salt thereof and borneol.
2. A composition containing 3-methyl-1-phenyl-2-pyrazolin-5-one or its pharmaceutically acceptable salt and Borneolum Syntheticum has synergistic effect in treating non-alcoholic fatty liver disease (NAFL), non-alcoholic steatohepatitis (NASH) and hepatic fibrosis.
3. The composition according to any one of claims 1 to 2, wherein the weight ratio of the 3-methyl-1-phenyl-2-pyrazolin-5-one or the pharmaceutically acceptable salt thereof to borneol is 1: 10-10: 1.
4. The composition according to any one of claims 1 to 3, wherein the weight ratio of the 3-methyl-1-phenyl-2-pyrazolin-5-one or the pharmaceutically acceptable salt thereof to borneol is 1: 5-5: 1.
5. The composition according to any one of claims 1 to 4, wherein the weight ratio of the 3-methyl-1-phenyl-2-pyrazolin-5-one or the pharmaceutically acceptable salt thereof to borneol is 1: 1-5: 1.
6. The composition according to any one of claims 1 to 4, wherein the weight ratio of the 3-methyl-1-phenyl-2-pyrazolin-5-one or the pharmaceutically acceptable salt thereof to borneol is 1: 5-1: 1.
7. Use according to claims 1 to 6, characterized in that said composition is a medicament for the treatment of non-alcoholic fatty liver disease (NAFL).
8. Use according to claims 1 to 6, characterized in that said composition is a medicament for the treatment of non-alcoholic steatohepatitis (NASH).
9. Use according to claims 1 to 6, characterized in that said composition is a medicament for the treatment of hepatic fibrosis.
10. The use according to claims 1 to 6, characterized in that the borneol is natural borneol.
CN201910622895.5A 2019-07-11 2019-07-11 Composition for treating fatty liver, hepatitis and hepatic fibrosis and its preparation method Pending CN112206229A (en)

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