Application of lometapate and its compositions in the preparation of drugs for treating liver fibrosis

Abstract

This invention discloses the application of lomestatide and its compositions in the preparation of drugs for treating liver fibrosis. The Jin Zhengjun Q-value method is a classic synergistic discriminant model. Cell experiments based on this model show that lomestatide and obeticholic acid exhibit significant synergistic anti-liver fibrosis effects in a molar ratio of 1:2.5 to 1:10, achieving a synergistic effect greater than the sum of its parts (1+1>2). Therefore, compositions of lomestatide and obeticholic acid in a molar ratio of 1:2.5 to 1:10 show promise for development into drugs for treating liver fibrosis. Furthermore, prior art has not disclosed any therapeutic effect of lomestatide on liver fibrosis; therefore, lomestatide also shows promise for development into a drug for treating liver fibrosis.

Description

Technical Field

[0001] This invention belongs to the pharmaceutical field and relates to the use of lometapate and its compositions in the preparation of drugs for treating liver fibrosis. Background Technology

[0002] Non-alcoholic steatohepatitis (NASH) is a subtype of non-alcoholic fatty liver disease (NAFLD) characterized by hepatocellular damage, innate immune cell-mediated inflammation, and progressive liver fibrosis.

[0003] The progression of liver fibrosis is closely related to the activation of hepatic stellate cells, which are resting, non-parenchymal cells in a normal liver, primarily responsible for storing vitamin A lipid droplets. However, under stimuli such as liver injury, these cells undergo a myofibroblast-like transformation, becoming activated. This transformation is marked by the expression of α-smooth muscle actin (α-SMA) in the matrix and the synthesis and secretion of type I collagen (such as COL1A1 and COL1A2) in the matrix. Human LX-2 cells are commonly used as a vector for in vitro studies of liver fibrosis.

[0004] Liver fibrosis is highly associated with the development of end-stage liver disease. The further development of fibrosis leading to cirrhosis has now become the second most common indication for liver transplantation. However, there are currently no drugs in clinical practice that can effectively treat NASH fibrosis.

[0005] Lomitapide is a highly effective microsomal triglyceride transfer protein inhibitor used to treat homozygous familial hypercholesterolemia, exhibiting good lipid-lowering effects. Currently, there are no reported studies on the use of lomitapide alone or in combination with obeticholic acid for the treatment of NASH and its fibrosis.

[0006] Obeticholic acid (OCA) has been shown to effectively improve various pathological manifestations such as liver fibrosis in NASH patients during clinical trials. However, its efficacy is dose-dependent, and high doses can easily cause many adverse reactions such as itching and abnormal lipid metabolism. Summary of the Invention

[0007] The purpose of this invention is to overcome the shortcomings of the prior art and provide the application of lometapate and its compositions in the preparation of medicaments for treating liver fibrosis.

[0008] The above-mentioned objective of this invention is achieved through the following technical solution:

[0009] A composition for treating liver fibrosis, comprising lometapeptide and obeticholic acid.

[0010] Preferably, the molar ratio of lometasate to obeticholic acid is 1:2.5 to 1:10.

[0011] The use of the above composition in the preparation of drugs for treating liver fibrosis.

[0012] The use of the above composition in the preparation of a medicament for treating liver diseases accompanied by liver fibrosis.

[0013] Preferably, the liver diseases accompanied by liver fibrosis include non-alcoholic fatty liver disease and cirrhosis.

[0014] Preferably, the drug is formulated into a pharmaceutically acceptable dosage form using the above-described composition as the active ingredient and pharmaceutically acceptable excipients as carriers.

[0015] Application of lometabolic acid in the preparation of drugs for treating liver fibrosis.

[0016] Application of lometabolic acid in the preparation of drugs for treating liver diseases with liver fibrosis.

[0017] Preferably, the liver diseases accompanied by liver fibrosis include non-alcoholic fatty liver disease and cirrhosis.

[0018] Preferably, the drug uses lometapetine as the active ingredient and pharmaceutically acceptable excipients as carriers to form a pharmaceutically acceptable dosage form.

[0019] Beneficial effects:

[0020] Jin Zhengjun's Q-value method is a classic synergistic discriminant model. Experimental results based on this model show that lometabine and obeticholic acid exhibit a significant synergistic anti-liver fibrosis effect within a molar ratio range of 1:2.5 to 1:10, achieving a synergistic effect greater than the sum of its parts (1+1>2). Therefore, compositions of lometabine and obeticholic acid in a molar ratio of 1:2.5 to 1:10 show promise for development into drugs for treating liver fibrosis. Furthermore, no prior art has disclosed any therapeutic effect of lometabine on liver fibrosis; therefore, lometabine also holds promise for development into a drug for treating liver fibrosis. Attached Figure Description

[0021] Figure 1 This study compares the levels of fibrosis-related genes in LX-2 cells of different groups 24 hours after drug treatment. Detailed Implementation

[0022] The following describes the substantive content of the present invention in detail with reference to embodiments, but this does not limit the scope of protection of the present invention.

[0023] I. Experimental Materials and Reagents

[0024] Cells: Human hepatic stellate cells LX-2 were purchased from Zhejiang Meisen Cell Technology Co., Ltd.

[0025] Medications: Obeticholic acid (OCA, CAS No.: 459789-99-2) was purchased from GlpBio in the United States; Lomitapide (Lomi, CAS No.: 182431-12-5) was purchased from MedChemExpress.

[0026] Reagents: DMEM high glucose medium (Gibco, USA); fetal bovine serum FBS (TransGold, China); penicillin-streptomycin (Hyclone, USA); 0.25% trypsin-EDTA solution (Boster Biological, China); PBS (Biosharp, China); cell-grade dimethyl sulfoxide (Solarbio, China); RNA isolater Total RNA Extraction Reagent (Nanjing Novizan Biotechnology Co., Ltd.); Evo M-mlv reverse transcription premix kit, SYBR Green Pro Taq HS qPCR kit (Hunan Aikerui Biotechnology Co., Ltd.); DEPC water (Shanghai Beyotime Biotechnology Co., Ltd.); anhydrous ethanol (Nanjing Chemical Reagent Co., Ltd.).

[0027] Consumables: 10cm cell culture dish (SORFA, China); 15mL and 50mL centrifuge tubes (JET BIOFIL, China); cell cryopreservation tubes (Corning, USA); cell counting chamber (Countstar, China); nuclease-free 10, 200, and 1000μL (Axygen, USA); PCR plate and eight-tube strip (Aibotek, China).

[0028] Instruments: 2.5, 10, 100 and 1000 μL pipettes (Eppendorf, Germany); Biosafety cabinet (Thermo, USA); CO2 cell incubator (Thermo, USA); XD202 inverted biological microscope (Nanjing Jiangnan Yongxin Optical Co., Ltd., China); HW-12 electric thermostatic water bath (Shanghai Yiheng Scientific Instrument Co., Ltd., China); IC1000 automated cell counter (Shanghai Ruiyu Biotechnology Co., Ltd., China); TDL-50B low-speed benchtop centrifuge (Shanghai Anting Scientific Instrument Factory, China); Vortex mixer (Haimen Qilinbei Instrument Manufacturing Co., Ltd., China).

[0029] II. Experimental Methods

[0030] 1. Cell Culture

[0031] LX-2 cells were cultured in a high-glucose medium containing 10% FBS, 1% penicillin-streptomycin, and 89% DMEM.

[0032] Cell resuscitation:

[0033] Quickly remove the LX-2 cells frozen in liquid nitrogen and thaw them in a 37°C water bath. Immediately transfer the cell suspension to a 15mL centrifuge tube containing 10mL of complete culture medium and centrifuge at 1000rpm for 5min. Discard the supernatant, resuspend the lower cell layer in 2mL of complete culture medium, and transfer it to a 10cm culture dish pre-filled with 8mL of complete culture medium. Mix well using the cross-hatching method and incubate at 37°C in a 5% CO2 incubator. Wash once with PBS the next day and change the medium.

[0034] Cell culture and passage:

[0035] Observe the cell status daily. When the cell density reaches 90%, perform subculturing: Preheat the complete culture medium, PBS, and trypsin in a 37°C water bath; discard the old culture medium, add 2 mL of PBS to rinse the cell surface, then discard the PBS, repeat twice. Add 1 mL of trypsin and place the cell in the operating table at room temperature for 20-30 seconds to digest. Discard the trypsin to stop digestion. Add 3 mL of complete culture medium and gently pipette. Collect the cell suspension into a 15 mL centrifuge tube and centrifuge at 1000 rpm for 5 minutes. Discard the supernatant, resuspend the lower cell pellet in fresh complete culture medium, and then subculture in a new culture dish at a 1:2 ratio.

[0036] 2. Grouping and Dosing

[0037] Preparation of drug-containing stock solutions: Accurately weigh the powders of OCA and Lomi and dissolve them in a certain volume of dimethyl sulfoxide to prepare drug stock solutions with concentrations of 20mM and 2mM, respectively. After sterile filtration, they can be diluted for cell delivery.

[0038] LX-2 activation control group (Con), 5 μM OCA group, three concentrations of Lomi (0.5, 1, 2 μM) groups, and 5 μM OCA combined with three concentrations of Lomi (0.5, 1, 2 μM) groups were set up, with 3 replicates for each group. LX-2 cells in logarithmic growth phase were collected, digested, centrifuged, resuspended, and then administered at 2 × 10⁻⁶ cells / well. 5 Cells were seeded at a density of cells / well in 6-well plates. After being cultured overnight at 37°C in a 5% CO2 incubator, the control group was replaced with fresh complete medium, while the other wells were replaced with fresh complete medium containing the drug. After incubation at 37°C for 24 hours, cells were collected and mRNA was extracted for the determination of fibrosis-related gene content.

[0039] 3. q-Real-Time PCR Analysis

[0040] RNA primers were designed and synthesized by Nanjing Ruizhen Biotechnology Co., Ltd. The nucleotide sequences of PCR primers for α-SMA, COL1A1, and COL1A2 genes are shown in Table 1. The primers were diluted with nuclease-free water to a working solution of 10 μM for later use.

[0041] Table 1. Nucleotide sequences of RT-PCR primers

[0042]

[0043] Total RNA extraction, reverse transcription, and PCR were performed strictly according to the instructions, using the 2% relative quantification method. －△△ mRNA content was analyzed using the Ct method.

[0044] 4. Data Processing

[0045] Experimental data were processed using GraphpadPrism 8.0 software, and the results are expressed as mean ± SD. Two-tailed unpaired t-tests were used to compare independent samples to determine statistical significance. Compared with group Con, *P < 0.05, **P < 0.01, ***P < 0.001, ns, no statistical significance.

[0046] 5. Evaluation indicators of drug synergistic effects

[0047] The efficacy of each drug used alone or in combination is determined based on the average Q value of the inhibition rate; Q = E a+b / (E a +E b -E a ×E b E a and E b These represent the inhibition rates of the two drugs used alone, E a+b The formula represents the inhibition rate of combined drug therapy; the numerator represents the "measured combined effect," and the denominator is the "expected combined effect"; Q < 0.85 indicates an antagonistic effect, 0.85 ≤ q < 1.15 indicates an additive effect, and q ≥ 1.15 indicates a synergistic effect. The content of fibrosis-related genes used to calculate the inhibition rate is as follows: Figure 1 As shown.

[0048] III. Experimental Results

[0049] 1. Inhibition rate and Q value (mean±SD, n=3) of α-SMA by different concentrations of Lomi combined with OCA

[0050]

[0051] 2. Inhibition rate and Q value (mean ± SD, n = 3) of different concentrations of Lomi combined with OCA on COL1A1

[0052]

[0053] 3. Inhibition rate and Q value (mean ± SD, n = 3) of different concentrations of Lomi combined with OCA on COL1A2

[0054]

[0055] Jin Zhengjun's Q-value method is a classic synergistic discriminant model. Experimental results based on this model show that lometabine and obeticholic acid exhibit a significant synergistic anti-liver fibrosis effect within a molar ratio range of 1:2.5 to 1:10, achieving a synergistic effect greater than the sum of its parts (1+1>2). Therefore, compositions of lometabine and obeticholic acid in a molar ratio of 1:2.5 to 1:10 show promise for development into drugs for treating liver fibrosis. Furthermore, no prior art has disclosed any therapeutic effect of lometabine on liver fibrosis; therefore, lometabine also holds promise for development into a drug for treating liver fibrosis.

[0056] The purpose of the above embodiments is to specifically illustrate the substantive content of the present invention, but those skilled in the art should know that the scope of protection of the present invention should not be limited to the specific embodiments.

Claims

1. A composition for treating liver fibrosis, characterized in that: It is composed of lometasate and obeticholic acid, with a molar ratio of 1:2.5 to 1:

10.

2. The use of the composition according to claim 1 in the preparation of a medicament for treating liver fibrosis.

3. The application according to claim 2, characterized in that: The drug uses the composition of claim 1 as the active ingredient and pharmaceutically acceptable excipients as carriers to form a pharmaceutically acceptable dosage form.

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