Use of palmitoylation inhibitors in inducing macrophage apoptosis

Abstract

The application discloses application of a palmitoylation inhibitor in inducing macrophage apoptosis, belongs to the technical field of biological medicine, and particularly relates to a preparation method of a pharmaceutical composition, which comprises the following steps: mixing the palmitoylation inhibitor with a medical reagent to prepare the pharmaceutical composition; the palmitoylation inhibitor is 2-BP, and the use amount of the 2-BP is 0.0015-0.07 wt% of the medical reagent. The application utilizes the 2-BP to specifically up-regulate the expression of CPT1A in the macrophages in a pathological state, thereby selectively inducing the apoptosis of the macrophages, has a smaller influence on normal cells, and significantly improves a safety window of treatment. The pharmaceutical composition can effectively induce the apoptosis of the macrophages, reduce non-specific killing on normal cells, and treat diseases related to abnormal activation or accumulation of the macrophages.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to the application of palmitoylation inhibitors in inducing macrophage apoptosis. Background Technology

[0002] Macrophages are key cells in the innate immune system, playing a central role in inflammatory responses, pathogen clearance, and tissue repair. However, abnormal activation, excessive proliferation, or resistance to apoptosis of macrophages is closely associated with a variety of diseases, such as autoimmune diseases (e.g., rheumatoid arthritis, inflammatory bowel disease), atherosclerosis, fibrotic diseases, and certain types of cancer (e.g., tumor-associated macrophages promote tumor growth and metastasis). Therefore, developing drugs that can specifically induce apoptosis in macrophages under pathological conditions has significant clinical implications.

[0003] Currently, chemotherapy drugs (such as anthracyclines) or radiotherapy methods that induce apoptosis often lack cell selectivity. While killing pathological cells (such as macrophages), they also cause severe damage to normal tissue cells, leading to systemic toxic side effects such as immunosuppression and bone marrow suppression. Furthermore, pathological macrophages (such as tumor-associated macrophages) often acquire apoptosis resistance by upregulating anti-apoptotic proteins (such as Bcl-2 and Mcl-1), making traditional apoptosis-inducing therapies ineffective or even useless.

[0004] Protein palmitoylation is an important post-translational modification that modifies protein membrane localization, stability, protein-protein interactions, and function by adding a 16-carbon palmitic acid chain to cysteine ​​residues. Recent studies have revealed that palmitoylation plays a crucial role in regulating cellular metabolism and immune cell function. Palmitoylation inhibitors (2-bromopalmitate / 2-BP) have been widely used to study the biological functions of this modification, but their application in specifically inducing macrophage apoptosis has not yet been reported. Summary of the Invention

[0005] The purpose of this invention is to provide an application of palmitoylation inhibitors that can effectively induce macrophage apoptosis, reduce non-specific killing of normal cells, and treat diseases related to abnormal macrophage activation or accumulation in inducing macrophage apoptosis.

[0006] Carnitine palmitotransferase 1A (CPT1A) is the rate-limiting enzyme in mitochondrial fatty acid β-oxidation, responsible for converting long-chain acyl-CoA into acylcarnitine, which then enters the mitochondria for oxidative breakdown. CPT1A expression and activity are tightly regulated metabolically. Unexpectedly, this invention reveals that inhibiting macrophage palmitoylation significantly upregulates CPT1A expression, and abnormally high CPT1A expression further irreversibly leads to macrophage apoptosis. Therefore, this discovery provides a novel target and strategy for treating macrophage-related diseases. Inducing macrophage apoptosis by upregulating CPT1A expression, and subsequently using it to treat diseases related to abnormal macrophage activation or accumulation, opens up new pharmaceutical applications for palmitoylation inhibitors.

[0007] The purpose of this invention is to provide a method for preparing a pharmaceutical composition that can specifically induce apoptosis in pathological macrophages. This pharmaceutical composition containing a palmitoylation inhibitor reduces non-specific killing of normal cells, thereby opening up new pharmaceutical applications for palmitoylation inhibitors.

[0008] The technical solution adopted by the present invention to achieve the above objectives is as follows:

[0009] A method for preparing a pharmaceutical composition includes: mixing a palmitoylation inhibitor with a medical reagent to prepare the pharmaceutical composition; wherein the palmitoylation inhibitor is 2-BP, and the amount of 2-BP used is 0.0015-0.07 wt% of the medical reagent. Existing chemotherapy drugs (such as anthracyclines) or radiotherapy lack cell selectivity when inducing apoptosis, causing severe damage to normal tissue cells while killing pathological cells, leading to systemic toxic side effects such as immunosuppression and bone marrow suppression. This invention utilizes the palmitoylation inhibitor (2-BP) to specifically upregulate CPT1A expression in pathological macrophages, thereby selectively inducing apoptosis with minimal impact on normal cells, significantly improving the safety window of treatment. Pathological macrophages (such as tumor-associated macrophages) usually acquire apoptosis resistance by upregulating anti-apoptotic proteins such as Bcl-2 and Mcl-1, leading to the failure of traditional treatment methods. This invention, through a novel pathway of "inhibition of palmitoylation, upregulation of CPT1A, and apoptosis," can effectively bypass or overcome this apoptosis resistance mechanism, providing a new strategy for treating drug-resistant diseases. Palmitoylation inhibitors (2-BP) have previously been used only as research tools to explore the function of protein palmitoylation. This invention reveals for the first time their novel pharmaceutical applications in inducing macrophage apoptosis and treating related diseases. Simultaneously, CPT1A, as a key effector molecule in this pathway, provides a novel therapeutic target for developing new drugs targeting macrophage-related diseases.

[0010] Preferably, the medical reagent includes at least one of water, physiological saline, and PVP.

[0011] Preferably, the medical reagent is water and PVP, wherein PVP and water are mixed in a mass ratio of 1:0.1-10.

[0012] Preferably, the medical reagent is physiological saline and PVP, wherein PVP and physiological saline are mixed in a mass ratio of 1:0.1-10.

[0013] Preferably, the pharmaceutical composition also includes bile acid derivatives.

[0014] More preferably, the bile acid derivative is obtained by reacting bile acid with ethyl tyrosine. In this invention, the bile acid derivative obtained by reacting bile acid with ethyl tyrosine is used in combination with 2-BP to prepare a pharmaceutical composition by mixing with medical reagents. When applied to induce macrophage apoptosis, it can increase the apoptosis rate of macrophages, indicating that the bile acid derivative can be used for new pharmaceutical applications in inducing macrophage apoptosis, especially in the treatment of diseases related to abnormal activation or accumulation of macrophages.

[0015] More preferably, the amount of bile acid derivative used is 0.0003-0.005 wt% of the medical reagent.

[0016] More preferably, in the preparation of the cholic acid derivative, cholic acid and tyrosine ethyl ester are reacted in a solvent containing EDCI, HOBT and DIPEA.

[0017] This invention discloses a pharmaceutical composition prepared by the above method.

[0018] This invention discloses the use of the above-mentioned pharmaceutical composition in the preparation of drugs that induce macrophage apoptosis.

[0019] This invention discloses a pharmaceutical composition comprising 2-BP and a medical reagent.

[0020] Preferably, the medical reagent comprises PVP and water. The PVP and water in the medical reagent are mixed at a mass ratio of 1:0.1-10.

[0021] Preferably, the medical reagent includes PVP and physiological saline. The PVP and physiological saline are mixed in a mass ratio of 1:0.1-10.

[0022] Preferably, the amount of 2-BP used is 0.0015-0.07 wt% of the medical reagent.

[0023] Preferably, the pharmaceutical composition contains a cholic acid derivative, and the amount of the cholic acid derivative used is 0.0003-0.005 wt% of the medical reagent.

[0024] More preferably, in the preparation of the cholic acid derivative, cholic acid is added to DMF, and under an inert gas atmosphere, tyrosine ethyl ester is added, followed by EDCI, HOBT and DIPEA. The mixture is stirred at 20-40°C for 6-24 hours. After the reaction is complete, the mixture is filtered, ethyl acetate is added, and then DMF is removed by washing with saturated brine multiple times. The mixture is dried over anhydrous sodium sulfate, the solvent is removed by rotary evaporation, and then purified by silica gel column chromatography to obtain the cholic acid derivative.

[0025] More preferably, in the preparation of the cholic acid derivative, the amount of cholic acid used is 10-20 wt% of DMF.

[0026] More preferably, nitrogen is used as the inert gas in the preparation of cholic acid derivatives.

[0027] More preferably, in the preparation of the cholic acid derivative, the amount of tyrosine ethyl ester used is 20-40 wt% of the cholic acid.

[0028] More preferably, in the preparation of cholic acid derivatives, the amount of EDCI used is 60-80 wt% of cholic acid.

[0029] More preferably, in the preparation of the cholic acid derivative, the amount of HOBT used is 40-60 wt% of the cholic acid.

[0030] More preferably, in the preparation of the cholic acid derivative, the amount of DIPEA used is 60-70 wt% of the cholic acid.

[0031] More preferably, in the preparation of cholic acid derivatives, the amount of ethyl acetate used is the same as the amount of DMF used, and an appropriate amount of saturated saline solution is used.

[0032] Preferably, the pharmaceutical composition further contains L-tyrosine amide, and the amount of L-tyrosine amide used is 0.00015-0.002 wt% of the medical reagent. In addition to using bile acid derivatives and 2-BP, this invention can further add L-tyrosine amide and mix it with medical reagents to prepare a pharmaceutical composition. When applied to induce macrophage apoptosis, it can further enhance macrophage apoptosis, indicating that bile acid derivatives, 2-BP, and L-tyrosine amide can be used for new pharmaceutical applications in inducing macrophage apoptosis, particularly in the treatment of diseases related to abnormal macrophage activation or accumulation.

[0033] This invention is the first to discover that palmitoylation inhibitors (2-BP) can upregulate the expression of carnitine palmitoyltransferase 1A (CPT1A), thereby inducing macrophage apoptosis. It also reveals a novel pharmaceutical application of palmitoylation inhibitors in inducing macrophage apoptosis, particularly in the treatment of diseases associated with abnormal macrophage activation or accumulation.

[0034] This invention discloses the use of palmitoylation inhibitors in the preparation of drugs for inducing macrophage apoptosis.

[0035] This invention discloses the use of palmitoylation inhibitors in the preparation of medicaments for treating diseases related to abnormal activation or accumulation of macrophages.

[0036] This invention discloses a method for inducing macrophage apoptosis, comprising treating macrophages with an effective dose of a palmitoylation inhibitor.

[0037] This invention discloses a method for treating macrophage-related diseases, comprising administering an effective dose of a palmitoylation inhibitor to a patient in need.

[0038] The 2-BP in this invention can be replaced by other palmitoylation inhibitors: in addition to using 2-BP, other known or newly discovered palmitoylation inhibitors may also achieve similar effects, such as small molecule inhibitors of palmitoyases (such as DHHC family enzymes) and competitive inhibitors based on fatty acid analogs.

[0039] Other methods that can upregulate CPT1A expression can also be used in this invention: in addition to indirectly upregulating CPT1A by inhibiting palmitoylation, the expression or activity of CPT1A can be directly regulated to achieve the same purpose, for example: using CPT1A agonists or expression activators (such as certain nuclear receptor agonists), or directly increasing the level of CPT1A in macrophages through gene therapy (such as viral vectors that overexpress CPT1A).

[0040] This invention utilizes the palmitoylation inhibitor 2-BP to specifically upregulate CPT1A expression in pathological macrophages, thereby selectively inducing apoptosis with minimal impact on normal cells, significantly improving the safety window for treatment. Therefore, it has the following beneficial effects: effectively inducing macrophage apoptosis, reducing non-specific killing of normal cells, and treating diseases related to abnormal macrophage activation or accumulation. Thus, this invention relates to the application of a palmitoylation inhibitor in inducing macrophage apoptosis, which can effectively induce macrophage apoptosis, reduce non-specific killing of normal cells, and treat diseases related to abnormal macrophage activation or accumulation. Attached Figure Description

[0041] Figure 1 The Venn diagram of the intersection of RNA-seq data from two macrophage cell lines and the volcano diagram of differentially expressed genes are shown.

[0042] Figure 2 This image shows the validation of CPT1A, LAMC2, KLF11, and CCR4 expression in two macrophage cell lines.

[0043] Figure 3 The graph shows the cell survival curves for two macrophage cell lines.

[0044] Figure 4This is a diagram showing apoptosis in two macrophage cell lines.

[0045] Figure 5 This is a bar chart showing the apoptosis ratio of the two macrophage cell lines.

[0046] Figure 6 This is a verification diagram of apoptosis proteins in two macrophage cell lines.

[0047] Figure 7 This is a graph showing the apoptosis rate of the RAW264.7 cell line.

[0048] Figure 8 This is a graph showing the apoptosis rate of the J774A.1 cell line. Detailed Implementation

[0049] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0050] The concepts involved in this application will first be described with reference to the accompanying drawings. It should be noted that the following descriptions of various concepts are only for the purpose of making the content of this application easier to understand and do not constitute a limitation on the scope of protection of this application; furthermore, the embodiments and features in the embodiments of this application can be combined with each other unless otherwise specified. This application will now be described in detail with reference to the accompanying drawings and embodiments.

[0051] The research basis of this invention is as follows:

[0052] I. Methods used in the study

[0053] RNA-seq method: Macrophage RNA was isolated and purified using total RNA extraction reagent (TRIzol) according to the instructions. After concentration determination, RNA was denatured. mRNA was enriched using Dynabeads Oligo (dT) magnetic beads (ThermoFisher), and then fragmented under high temperature conditions using a magnesium fragmentation kit (NEBNext® Magnesium RNAFragmentation Module) at 86°C for 7 minutes. A one-strand reaction system was set up to synthesize one-stranded cDNA. A two-strand reaction system was set up to synthesize two-stranded cDNA. End repair, addition of "A" and adapter ligation were performed. The reaction program was set up to add an A base to the 3' end and repair the ends of the double-stranded cDNA to ligate the adapter to the cDNA. PCR was then performed to form a sequencing library. The program was as follows: pre-denaturation at 95°C for 3 minutes, denaturation at 98°C for a total of 8 cycles of 15 seconds each, annealing to 60°C for 15 seconds, extension at 72°C for 30 seconds, and final extension at 72°C for 5 minutes. Finally, paired-end sequencing was performed using an Illumina Novaseq™ 6000 according to standard procedures in PE150 sequencing mode.

[0054] Immunoblot method: After collecting two macrophage cell lines, RIPA lysis buffer was added and the cells were lysed on ice for 30 min. The cells were then centrifuged at 13000 rpm at 4°C for 15 min. The supernatant was collected, and protein content was determined by BCA. Equal amounts of protein from different samples were used for SDS-PAGE to separate proteins. After electrophoresis, the proteins were transferred to PVDF membranes, blocked with 5% skim milk powder, and then blocked overnight with primary antibody. The membranes were then blocked for 2 h with horseradish peroxidase-labeled secondary antibody blocking buffer, and developed with diaminobenzidine (DAB) solution for colorimetric analysis. Primary antibodies included CPT1A protein, GAPDH protein, LAMC2 protein, KLF11 protein, CCR4 protein, Caspase3 protein, cleaved Caspase3 protein, PARP protein, and cleaved PARP protein. The two macrophage cell lines were RAW264.7 and J774A.1.

[0055] Cell survival curves: RAW264.7 cells and J774A.1 cells in logarithmic growth phase were cultured at 6 × 10⁶ cells per well. 3Cells were seeded at a rate of [number] cells / well in 96-well plates and cultured overnight or for 24 hours to ensure cell adhesion and stable growth. Negative and positive control groups were set up, and 2-BP drug solutions were prepared at different concentration gradients, with six replicates for each concentration. The old culture medium was removed from the 96-well plates, and fresh culture medium containing different drug concentrations was added to each well (100 μL). The drug-treated cells were incubated at 37°C in a 5% CO2 incubator for 24 hours. After incubation, 10 μL of CCK-8 reagent was added to each well, the 96-well plate was gently shaken to mix, and then incubated at 37°C for 2 hours. After incubation, the absorbance (OD value) of each well was measured using a microplate reader at 450 nm. The 2-BP drug concentration gradients were 0, 5.86, 11.72, 23.43, 46.88, 93.75, 187.5, 375, 750, and 1500 μM.

[0056] Flow cytometry detection of apoptosis: Log-phase RAW264.7 cells and J774A.1 cells were seeded at 350,000 cells per well in 6-well plates and cultured overnight or for 24 hours to ensure cell adhesion or stable growth. 2-BP drug solutions were prepared at different concentration gradients. After removing the old culture medium, the drug was added, and the cells were incubated for 24 hours. The cell pellet was then collected. After washing twice with 1×PBS, the cells were resuspended in 500 μL of 1×Binding Buffer in each tube, followed by 10 μL of PI and 5 μL of Annexin V-FITC. The mixture was incubated at room temperature in the dark for 5 minutes, and the apoptosis ratio was detected by flow cytometry. The concentration gradients of 2-BP drug were 0, 50, 100, and 150 μM.

[0057] II. Palmitoylation inhibitor (2-BP) upregulates CPT1A expression in macrophages.

[0058] To identify key genes that inhibit palmitoylation in macrophages, this study treated two macrophage cell lines with 100 μM 2-BP for 24 h, followed by RNA-seq data analysis. Figure 1 As shown, in the two macrophage cell lines treated with 2-BP, the genes KLF11, CPT1A, and LAMC2 were upregulated, while the gene CCR4 was downregulated. This was verified by Western blotting, and the results are as follows: Figure 2 As shown, this study found that inhibiting palmitoylation in macrophages led to a significant upregulation of CPT1A expression, while changes in other genes did not conform to the trends observed in RNA-seq. The two macrophage cell lines used were RAW264.7 and J774A.1.

[0059] The cell survival curves in this study are as follows: Figure 3 As shown, the IC50 of the RAW264.7 cell line50 The figure was obtained using the highest concentration of 1.5 mM, with a 2-fold serial dilution.

[0060] III. Palmitoylation inhibitor (2-BP) induces macrophage apoptosis

[0061] To demonstrate that the palmitoylation inhibitor (2-BP) can induce apoptosis in macrophages, this study treated two macrophage cell lines with 50, 100, and 150 μM 2-BP and analyzed the results by flow cytometry. The results are as follows: Figure 4 and Figure 5 As shown, the higher the 2-BP concentration, the more pronounced the apoptosis in both macrophage cell lines. This was subsequently verified using Western blotting experiments. Figure 6 As shown, with increasing 2-BP concentration, the expression of Caspase3 and PARP gradually decreased, while the expression of cleaved Caspase3 and cleaved PARP gradually increased. The two macrophage cell lines used were RAW264.7 and J774A.1.

[0062] Macrophages were treated with 2-BP (2-Bromohexadecanoic acid) and incubated at 37°C for 24 hours.

[0063] Concentration and dosage: Figure 1 The amount of 2-BP used was 100 μM; Figure 2 The dosages of 2-BP used were 0, 50, 100, and 150 μM. Figure 3 IC of China A 50 The figure was obtained using a 2-fold serial dilution of 2-BP at the highest concentration of 1.5 mM. Figure 4 and Figure 5 The dosages of 2-BP used were 0, 50, 100, and 150 μM. Figure 6 The dosages of 2-BP used were 0, 50, 100, and 150 μM.

[0064] Example 1: A pharmaceutical composition

[0065] Pharmaceutical composition: 2-BP and medical reagent. The medical reagent includes water. The amount of 2-BP used is 0.005 wt% of the medical reagent.

[0066] Preparation of the pharmaceutical composition: The pharmaceutical composition was prepared by mixing 2-BP with a medical reagent. The medical reagent included water. The amount of 2-BP used was 0.005 wt% of the medical reagent.

[0067] Example 2: A pharmaceutical composition

[0068] Pharmaceutical composition: 2-BP and medical reagent. The medical reagent includes PVP and water. PVP and water are mixed in a 1:1 mass ratio in the medical reagent, and the amount of 2-BP used is 0.005 wt% of the medical reagent.

[0069] Preparation of the pharmaceutical composition: The pharmaceutical composition was prepared by mixing 2-BP with a medical reagent. The medical reagent included PVP and water. PVP and water were mixed in a 1:1 mass ratio in the medical reagent, and the amount of 2-BP used was 0.005 wt% of the medical reagent.

[0070] Example 3: A pharmaceutical composition

[0071] Pharmaceutical composition: 2-BP, bile acid derivatives, and medical reagents. The medical reagents include PVP and water. PVP and water are mixed in a 1:1 mass ratio in the medical reagents. The amount of 2-BP used is 0.005 wt% of the medical reagents, and the amount of the bile acid derivatives used is 0.001 wt% of the medical reagents.

[0072] Preparation of the pharmaceutical composition: The pharmaceutical composition was prepared by mixing 2-BP, a bile acid derivative, and a medical reagent. The medical reagent included PVP and water. PVP and water were mixed in a 1:1 mass ratio in the medical reagent, and the amount of 2-BP used was 0.005 wt% of the medical reagent, while the amount of the bile acid derivative was 0.001 wt% of the medical reagent.

[0073] Preparation of cholic acid derivatives: Cholic acid was added to DMF. Under an inert gas atmosphere, tyrosine ethyl ester was added, followed by EDCI, HOBT, and DIPEA. The mixture was stirred at 30°C for 12 hours. After the reaction was complete, the mixture was filtered, ethyl acetate was added, and the DMF was removed by repeated washing with saturated brine. The mixture was dried over anhydrous sodium sulfate, and the solvent was removed by rotary evaporation. The cholic acid derivatives were then purified by silica gel column chromatography. The amount of cholic acid used was 15 wt% of DMF, the inert gas was nitrogen, the amount of tyrosine ethyl ester used was 30 wt% of cholic acid, the amount of EDCI used was 70 wt% of cholic acid, the amount of HOBT used was 50 wt% of cholic acid, and the amount of DIPEA used was 65 wt% of cholic acid. The amount of ethyl acetate used was the same as that used for DMF, and an appropriate amount of saturated brine was used.

[0074] Example 4: A pharmaceutical composition

[0075] Pharmaceutical composition: 2-BP, bile acid derivatives, and medical reagents. The medical reagents include PVP and physiological saline. PVP and physiological saline are mixed in a 1:1 mass ratio in the medical reagents. The amount of 2-BP used is 0.005 wt% of the medical reagents, and the amount of bile acid derivatives used is 0.001 wt% of the medical reagents.

[0076] Preparation of the pharmaceutical composition: The pharmaceutical composition was prepared by mixing 2-BP, a bile acid derivative, and a medical reagent. The medical reagent included PVP and physiological saline. PVP and physiological saline were mixed in a 1:1 mass ratio, and the amount of 2-BP used was 0.005 wt% of the medical reagent, while the amount of the bile acid derivative was 0.001 wt% of the medical reagent.

[0077] Preparation of cholic acid derivatives: Cholic acid was added to DMF. Under an inert gas atmosphere, tyrosine ethyl ester was added, followed by EDCI, HOBT, and DIPEA. The mixture was stirred at 30°C for 12 hours. After the reaction was complete, the mixture was filtered, ethyl acetate was added, and the DMF was removed by repeated washing with saturated brine. The mixture was dried over anhydrous sodium sulfate, and the solvent was removed by rotary evaporation. The cholic acid derivatives were then purified by silica gel column chromatography. The amount of cholic acid used was 15 wt% of DMF, the inert gas was nitrogen, the amount of tyrosine ethyl ester used was 30 wt% of cholic acid, the amount of EDCI used was 70 wt% of cholic acid, the amount of HOBT used was 50 wt% of cholic acid, and the amount of DIPEA used was 65 wt% of cholic acid. The amount of ethyl acetate used was the same as that used for DMF, and an appropriate amount of saturated brine was used.

[0078] Example 5: A pharmaceutical composition

[0079] Pharmaceutical composition: 2-BP, bile acid derivative, L-tyrosine amide, and medical reagent. The medical reagent includes PVP and water. PVP and water are mixed in a 1:1 mass ratio in the medical reagent. The amount of 2-BP used is 0.005 wt% of the medical reagent, the amount of L-tyrosine amide used is 0.0005 wt% of the medical reagent, and the amount of bile acid derivative used is 0.001 wt% of the medical reagent.

[0080] Preparation of the pharmaceutical composition: The pharmaceutical composition was prepared by mixing 2-BP, a bile acid derivative, L-tyrosine amide, and a medical reagent. The medical reagent included PVP and water. PVP and water were mixed in a 1:1 mass ratio in the medical reagent. The amount of 2-BP used was 0.005 wt% of the medical reagent, the amount of L-tyrosine amide used was 0.0005 wt% of the medical reagent, and the amount of the bile acid derivative used was 0.001 wt% of the medical reagent.

[0081] Preparation of cholic acid derivatives: Cholic acid was added to DMF. Under an inert gas atmosphere, tyrosine ethyl ester was added, followed by EDCI, HOBT, and DIPEA. The mixture was stirred at 30°C for 12 hours. After the reaction was complete, the mixture was filtered, ethyl acetate was added, and the DMF was removed by repeated washing with saturated brine. The mixture was dried over anhydrous sodium sulfate, and the solvent was removed by rotary evaporation. The cholic acid derivatives were then purified by silica gel column chromatography. The amount of cholic acid used was 15 wt% of DMF, the inert gas was nitrogen, the amount of tyrosine ethyl ester used was 30 wt% of cholic acid, the amount of EDCI used was 70 wt% of cholic acid, the amount of HOBT used was 50 wt% of cholic acid, and the amount of DIPEA used was 65 wt% of cholic acid. The amount of ethyl acetate used was the same as that used for DMF, and an appropriate amount of saturated brine was used.

[0082] Example 6: A pharmaceutical composition

[0083] Pharmaceutical composition: 2-BP, bile acid derivatives, L-tyrosine amide, and medical reagents. The medical reagents include PVP and physiological saline. PVP and physiological saline are mixed in a 1:1 mass ratio in the medical reagents. The amount of 2-BP used is 0.005 wt% of the medical reagents, the amount of L-tyrosine amide used is 0.0005 wt% of the medical reagents, and the amount of bile acid derivatives used is 0.001 wt% of the medical reagents.

[0084] Preparation of the pharmaceutical composition: The pharmaceutical composition was prepared by mixing 2-BP, a bile acid derivative, L-tyrosine amide, and a medical reagent. The medical reagent included PVP and physiological saline. PVP and physiological saline were mixed in a 1:1 mass ratio. The amount of 2-BP used was 0.005 wt% of the medical reagent, the amount of L-tyrosine amide used was 0.0005 wt% of the medical reagent, and the amount of the bile acid derivative used was 0.001 wt% of the medical reagent.

[0085] Preparation of cholic acid derivatives: Cholic acid was added to DMF. Under an inert gas atmosphere, tyrosine ethyl ester was added, followed by EDCI, HOBT, and DIPEA. The mixture was stirred at 30°C for 12 hours. After the reaction was complete, the mixture was filtered, ethyl acetate was added, and the DMF was removed by repeated washing with saturated brine. The mixture was dried over anhydrous sodium sulfate, and the solvent was removed by rotary evaporation. The cholic acid derivatives were then purified by silica gel column chromatography. The amount of cholic acid used was 15 wt% of DMF, the inert gas was nitrogen, the amount of tyrosine ethyl ester used was 30 wt% of cholic acid, the amount of EDCI used was 70 wt% of cholic acid, the amount of HOBT used was 50 wt% of cholic acid, and the amount of DIPEA used was 65 wt% of cholic acid. The amount of ethyl acetate used was the same as that used for DMF, and an appropriate amount of saturated brine was used.

[0086] Comparative Example 1: A pharmaceutical composition

[0087] Pharmaceutical composition: 2-BP, bile acid derivatives, and medical reagents. The medical reagents include PVP and water. PVP and water are mixed in a 1:1 mass ratio in the medical reagents. The amount of 2-BP used is 0.005 wt% of the medical reagents, and the amount of the bile acid derivatives used is 0.0001 wt% of the medical reagents.

[0088] Preparation of the pharmaceutical composition: The pharmaceutical composition was prepared by mixing 2-BP, a bile acid derivative, and a medical reagent. The medical reagent included PVP and water. PVP and water were mixed in a 1:1 mass ratio in the medical reagent, and the amount of 2-BP used was 0.005 wt% of the medical reagent, while the amount of the bile acid derivative was 0.0001 wt% of the medical reagent.

[0089] Preparation of cholic acid derivatives: Cholic acid was added to DMF. Under an inert gas atmosphere, tyrosine ethyl ester was added, followed by EDCI, HOBT, and DIPEA. The mixture was stirred at 30°C for 12 hours. After the reaction was complete, the mixture was filtered, ethyl acetate was added, and the DMF was removed by repeated washing with saturated brine. The mixture was dried over anhydrous sodium sulfate, and the solvent was removed by rotary evaporation. The cholic acid derivatives were then purified by silica gel column chromatography. The amount of cholic acid used was 15 wt% of DMF, the inert gas was nitrogen, the amount of tyrosine ethyl ester used was 30 wt% of cholic acid, the amount of EDCI used was 70 wt% of cholic acid, the amount of HOBT used was 50 wt% of cholic acid, and the amount of DIPEA used was 65 wt% of cholic acid. The amount of ethyl acetate used was the same as that used for DMF, and an appropriate amount of saturated brine was used.

[0090] Experimental example:

[0091] The present invention tested the pharmaceutical compositions in Examples 1-3, 5 and Comparative Example 1 using a flow cytometry to detect apoptosis. The amount of 2-BP used in the pharmaceutical compositions was 0.005 wt% of the medical reagent. The test results on RAW264.7 cells are as follows: Figure 7As shown, S1 is Example 1, S2 is Example 2, S3 is Example 3, S5 is Example 5, and D1 is Comparative Example 1. This invention found that the use of 2-BP can effectively promote apoptosis of RAW264.7 cells, and this was verified in the research foundation section of this invention. Furthermore, when 2-BP is used in combination with a bile acid derivative, the apoptosis rate of RAW264.7 cells can be increased. The medical reagent can be one or more of water, physiological saline, and PVP. The use of water, physiological saline, or PVP will not reduce the effect of 2-BP, nor will it reduce the effect of using 2-BP in combination with the bile acid derivative. The amount of bile acid derivative used needs to meet a certain range; if the amount of bile acid derivative used is too low, it will not significantly improve the apoptosis effect on RAW264.7 cells. In addition to using 2-BP and the bile acid derivative, L-tyrosine amide can also be added to further improve the apoptosis effect on RAW264.7 cells.

[0092] The present invention tested the pharmaceutical compositions in Examples 1-3, 5 and Comparative Example 1 using a flow cytometry to detect apoptosis. The amount of 2-BP used in the pharmaceutical compositions was 0.005 wt% of the medical reagent. The test results on J774A.1 cells are as follows: Figure 8 As shown, S1 is Example 1, S2 is Example 2, S3 is Example 3, S5 is Example 5, and D1 is Comparative Example 1. This invention found that the use of 2-BP can effectively promote apoptosis of J774A.1 cells, and this was verified in the research foundation section of this invention. Furthermore, when 2-BP is used in combination with a bile acid derivative, the apoptosis rate of J774A.1 cells can be increased. The medical reagent can be one or more of water, physiological saline, and PVP. The use of water, physiological saline, or PVP will not reduce the effect of 2-BP, nor will it reduce the effect of using 2-BP in combination with the bile acid derivative. The amount of bile acid derivative used needs to meet a certain range; if the amount of bile acid derivative used is too low, it will not significantly improve the apoptosis effect on J774A.1 cells. In addition to using 2-BP and a bile acid derivative, L-tyrosine amide can also be added to further improve the apoptosis effect on J774A.1 cells.

[0093] The embodiments and / or implementation methods described above are merely preferred embodiments and / or implementation methods for implementing the technology of the present invention, and are not intended to limit the implementation methods of the technology of the present invention in any way. Any person skilled in the art can make some modifications or alterations to other equivalent embodiments without departing from the scope of the technical means disclosed in the content of the present invention, but they should still be regarded as the technology or embodiments that are substantially the same as the present invention.

[0094] This document uses specific examples to illustrate the principles and implementation methods of this application. The descriptions of the above embodiments are only for the purpose of helping to understand the methods and core ideas of this application. The above descriptions are only preferred embodiments of this application. It should be noted that due to the limitations of written expression, while there are objectively infinite specific structures, those skilled in the art can make several improvements, modifications, or changes without departing from the principles of this application, and can also combine the above technical features in an appropriate manner. These improvements, modifications, changes, or combinations, or the direct application of the inventive concept and technical solution to other situations without modification, should all be considered within the scope of protection of this application.

Claims

1. A method for preparing a pharmaceutical composition, comprising: A pharmaceutical composition was prepared by mixing palmitoylation inhibitors, bile acid derivatives, and medical reagents. The palmitoylation inhibitor is 2-BP, and the amount of 2-BP used is 0.0015-0.07 wt% of the medical reagent. The amount of the bile acid derivative used is 0.0003-0.005 wt% of the medical reagent. The preparation method of the cholic acid derivative includes: adding cholic acid to DMF, adding tyrosine ethyl ester under an inert gas atmosphere, then adding EDCI, HOBT and DIPEA, stirring the reaction at 20-40℃ for 6-24 h, filtering after the reaction is completed, adding ethyl acetate, then washing with saturated brine multiple times to remove DMF, drying with anhydrous sodium sulfate, removing the solvent by rotary evaporation, and then purifying with silica gel column chromatography to obtain the cholic acid derivative; The amount of bile acid used is 10-20 wt% of DMF, the amount of tyrosine ethyl ester used is 20-40 wt% of bile acid, the amount of EDCI used is 60-80 wt% of bile acid, the amount of HOBT used is 40-60 wt% of bile acid, and the amount of DIPEA used is 60-70 wt% of bile acid. The medical reagent is selected from at least one of water, physiological saline, and PVP.

2. The method for preparing a pharmaceutical composition according to claim 1, characterized in that: The medical reagent is water and PVP, and PVP and water are mixed in a mass ratio of 1:0.1-10.

3. The method for preparing a pharmaceutical composition according to claim 1, characterized in that: The medical reagent is physiological saline and PVP, and PVP and physiological saline are mixed in a mass ratio of 1:0.1-10.

4. The pharmaceutical composition prepared by any one of the methods described in claims 1-3.

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