Anti-tumor pharmaceutical composition and application thereof

Abstract

The invention discloses an anti-tumor medicine composition and application thereof, the anti-tumor medicine composition has a synergistic effect, the inhibition effect on lung cancer cells PC-9 or NCI-H1975 is remarkably improved, and the anti-tumor medicine composition can be used for inhibiting the growth of source organs of lung adenocarcinoma patients. The anti-tumor pharmaceutical composition comprises an SPTLC1 inhibitor and a targeted drug, and the SPTLC1 inhibitor is a compound for inhibiting the enzyme activity, transcription, translation or protein stability of SPTLC1.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of medicine, in particular to an anti-tumor pharmaceutical composition and application of the pharmaceutical composition. BACKGROUND

[0002] Globally, the number of lung cancer cases and deaths is still increasing year by year, and the GLOBOCAN database shows that there were 1930 million new cancer cases and nearly 1000 million cancer deaths worldwide in 2020, of which lung cancer had the second highest incidence (11.4%) and was the main cause of cancer death. Lung cancer is divided into different histological subtypes, including adenocarcinoma, squamous cell carcinoma, large cell carcinoma (usually referred to as non-small cell lung cancer, NSCLC) and small cell lung cancer. Among them, about 80-85% are NSCLC, and in Asian patients with advanced NSCLC, about 50% of patients have changes in the tyrosine kinase domain of the Epidermal Growth Factor Receptor (EGFR) gene, of which 90% are EGFR exon 19 deletion mutations and exon 21 point mutations. Using small molecule EGFR tyrosine kinase inhibitors (EGFR-Tyrosine Kinase Inhibitors, EGFR-TKIs) combined with the ATP binding pocket of the EGFR tyrosine kinase domain can inhibit the downstream signaling pathway and inhibit tumor cell proliferation.

[0003] Olanzapine is an atypical antipsychotic drug with a molecular formula of C 17 H 20 N4S, mainly used for the treatment of schizophrenia and bipolar disorder, and has fewer adverse reactions. In addition, in improving the quality of life of cancer patients, olanzapine is also used to relieve symptoms such as nausea, vomiting and anorexia caused by chemotherapy. In recent years, its potential application in the field of tumors has attracted attention. Studies have shown that olanzapine can inhibit the proliferation of glioblastoma cells and promote apoptosis. It is currently reported that olanzapine has a certain anti-lung cancer effect, but its specific mechanism of action and target are still unclear.

[0004] Amatinib is the first third-generation EGFR-TKI in China. Through innovative optimization of molecular structure, the introduction of cyclopropyl structure replaces the traditional methyl group, significantly enhancing the selective inhibitory effect of the drug on EGFR sensitive mutations (19 exon deletion / L858R) and T790M drug resistance mutations. Amatinib targets the ATP binding site of the EGFR tyrosine kinase domain through irreversible binding, specifically acting on the Cys797 residue, thereby inhibiting EGFR activity, blocking the signal pathway of tumor cell proliferation and anti-apoptosis, and inhibiting the growth of tumor cells and accelerating cell apoptosis. In terms of pharmacological properties, amatinib has two major advantages: on the one hand, the affinity of amatinib and its metabolites for wild-type EGFR is significantly lower than that of similar drugs (such as osimertinib), and this precise targeting makes the incidence of side effects such as diarrhea and skin rash lower during treatment; on the other hand, the introduction of cyclopropyl in the chemical structure of amatinib increases the lipid solubility of the compound, thereby increasing the permeability of the blood-brain barrier and significantly reducing the risk of intracranial lesion progression. Therefore, amatinib has certain advantages in reducing the risk of brain metastasis progression. Although amatinib shows good efficacy in the treatment of NSCLC patients with EGFR mutations, patients will still develop resistance after a period of use, leading to disease progression. Therefore, it is urgent to find synergists and resistance reversal agents for amatinib to prolong patient survival time.

[0005] The advantages of amatinib compared with osimertinib: 1. In the clinical study of advanced EGFR mutant NSCLC, the short-term efficacy of amatinib and osimertinib in the treatment of advanced EGFR mutant NSCLC patients is similar, and in terms of long-term efficacy, the median progression-free survival and overall survival of amatinib are significantly better than those of osimertinib, and the safety is better. 2. Amatinib is generally well tolerated, no new safety signals are found, and the incidence of skin rash and diarrhea is low, with an incidence of skin rash and diarrhea of ≥3 grade of 0% and 1.4%, respectively, which is lower than that of osimertinib. In addition, the incidence of interstitial pneumonia of osimertinib is about 3%, while no interstitial pneumonia is reported in amatinib. SUMMARY

[0006] To overcome the defects of the prior art, the technical problem to be solved by the present application is to provide an antitumor pharmaceutical composition which has a synergistic effect, significantly improves the growth inhibition effect on lung cancer PC-9 cells and NCI-H1975 cells, and can be used to inhibit the growth of lung adenocarcinoma patient-derived organoids.

[0007] The antitumor pharmaceutical composition comprises an SPTLC1 inhibitor and a targeted drug, wherein the SPTLC1 inhibitor is a compound that inhibits the enzyme activity, transcription, translation or protein stability of SPTLC1.

[0008] The application is a compound preparation composed of an SPTLC1 inhibitor and a targeted drug, both of which are orally administered in clinic, and the compound preparation can improve the drug use compliance of patients and significantly improve the treatment and / or prevention effect of tumors in a synergistic manner.

[0009] Also provided is a use of an anti-tumor pharmaceutical composition in the preparation of an anti-tumor drug.

[0010] Also provided is a use of an anti-tumor pharmaceutical composition in the preparation of an anti-tumor drug, wherein the tumor is a solid tumor with high expression of SPTLC1, and the pharmaceutical composition is used in the preparation of an anti-tumor drug for inhibiting the expression and / or activity of SPTLC1.

[0011] Preferably, the SPTLC1 inhibitor includes a small nucleic acid for inhibiting the transcription of SPTLC1, a small molecule compound for inhibiting the stability of SPTLC1 protein, and a pharmaceutically acceptable carrier or excipient.

[0012] Further preferably, the small molecule compound for inhibiting SPTLC1 is olanzapine, and the molecular formula is: .

[0013] Further preferably, the targeted drug is amatinib, and the molecular formula is: .

[0014] Further preferably, the molar ratio of olanzapine to amatinib is 100-150:0.45-100.

[0015] Further preferably, the pharmaceutically acceptable carrier is a filler, a wetting agent or a binder, a disintegrant, and the preparation form of the pharmaceutical composition is an oral tablet, a granule, an injection or a capsule.

[0016] Further preferably, the anti-tumor pharmaceutical composition is used for inhibiting the proliferation of tumor cells, and the tumor cells are PC-9 cells.

[0017] Further preferably, the anti-tumor pharmaceutical composition is used for inhibiting the growth of patient-derived organoids of lung cancer.

[0018] Compared with the prior art, the application has the following beneficial effects: 1. Through molecular docking, the key binding site of olanzapine binding to SPTLC1 protein is found.

[0019] 2. In PC-9 cells and H1975 cells, it is found that olanzapine can inhibit the expression of SPTLC1 protein.

[0020] 3. Olanzapine combined with amatinib has synergistic effect in PC-9 cells.

[0021] 4. Olanzapine combined with amatinib has good synergistic effect on inhibiting the growth of organoids in human lung adenocarcinoma organoids. BRIEF DESCRIPTION OF DRAWINGS

[0022] Figure 1 is the key binding site of olanzapine on SPTLC1 protein.

[0023] Figure 2 is the Western blotting method for detecting the effect of olanzapine on the expression of SPTLC1 protein in PC-9 cells and NCI-H1975 cells.

[0024] Figure 3 is the CCK-8 experiment for detecting the effect of olanzapine combined with amatinib on the proliferation of PC-9 cells.

[0025] Figure 4 is the human lung adenocarcinoma organoid drug sensitivity detection experiment for exploring the effect of olanzapine combined with amatinib on the growth of organoids. DETAILED DESCRIPTION

[0026] The various illustrative embodiments of the present application will now be described in detail below. The detailed description is made with reference to the accompanying drawings, wherein like reference numerals refer to like elements throughout the various figures. The present application is described with reference to the following examples.

[0027] It should be understood that the terms used in the present application merely describe particular embodiments and are not intended to limit the present application. In addition, for numerical ranges in the present application, it should be understood that each intermediate value between the upper limit and the lower limit of the range is also specifically disclosed. Each smaller range between any stated value or stated range, and any other stated value or stated range, is also encompassed within the scope of the present application. The upper and lower limits of these smaller ranges can be independently included or excluded from the range.

[0028] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present application, preferred methods and materials are described. All publications mentioned in this specification are herein incorporated by reference to disclose and describe the methods and / or materials in connection with which the publications are cited. The citation of any reference in this specification is not intended as an admission that the reference is prior art, but rather that the reference is part of the state of the art. The practice of the application will employ, unless otherwise indicated, conventional methods and techniques within the skill of the art, or as described in the literature.

[0029] Many modifications and variations to the illustrative embodiments described herein will be apparent to those of ordinary skill in the art from the foregoing description. Such modifications and variations are intended to fall within the scope of the present application. Other embodiments will be apparent to those skilled in the art from consideration of the specification and practice of the application disclosed herein. The specification and examples given are exemplary only.

[0030] As used herein, the terms "comprises", "comprising", "includes", "including", "has", "having", "contains", "containing", or variations thereof, are intended to be open-ended terms that mean inclusion, but not limited to, the listed material or list of materials.

[0031] Experimental materials: 1. Cell lines: PC-9 cells and NCI-H1975 cells were purchased from Wuhan Punsun Life Science and Technology Co., Ltd.

[0032] 2. Experimental reagents: Olanzapine and amatinib (catalog number: HY-14541 and HY-112823) were purchased from MCE company; CCK-8 kit (catalog number: CK04) was purchased from DOJINDO company; RIPA (catalog number: P0013B) was purchased from Biyun Tian company; proteinase inhibitor (protein inhibitor cocktail, PIC) (catalog number: P1005) was purchased from Biyun Tian company; PMSF (catalog number: A1060) was purchased from Applygen company. BCA (catalog number: PK10026) was purchased from Proteintech company. Experimental example 1

[0033] The three-dimensional structure of SPTLC1 protein was obtained from PDB protein database, and pretreated, including removing water molecules, adding hydrogen atoms, optimizing atomic coordinates, etc., to ensure the accuracy and integrity of the structure. Based on the molecular docking technology, the binding affinity of small molecule compound olanzapine and SPTLC1 was evaluated, the rationality and stability of the binding mode were evaluated, and the key binding site of olanzapine on SPTLC1 protein was predicted.

[0034] The results are shown in Figure 1 Olanzapine is mainly combined with the 178th, 239th, 323rd, 324th and 327th amino acids of SPTLC1 protein. Experimental example 2

[0035] The effect of olanzapine on the expression of SPTLC1 was detected by Western blotting method.

[0036] First, lung cancer PC-9 cells or NCI-H1975 cells were plated into six-well plates, and after the cells adhered, 0.1% DMSO and 200 μM olanzapine were added to the control group, respectively, and the cells were incubated in a 37°C incubator for 48 hours. After washing twice with pre-cooled PBS, the cells were transferred to 1.5 ml centrifuge tubes, and 200 μl of protein extraction lysis solution (RIPA lysis solution with PIC and PMSF) was added. Lysis for 30 min on ice, centrifugation at 4 °C 12000 rpm for 30 min. Take the supernatant, use the BCA method to quantify the protein and adjust the protein to the same concentration. Add 5x SDS gel loading buffer, mix well and denature at 37 °C for 30 min. Take part of the protein sample for SDS-polyacrylamide gel electrophoresis.

[0037] The results of Western blotting are shown in Figure 2 . The results show that, compared with the control group, olanzapine can significantly inhibit the SPTLC1 protein level in PC-9 cells or NCI-H1975 cells. Experimental Example 3

[0038] The tumor cell proliferation experiment was carried out by CCK-8 experiment to detect the effect of SPTLC1 inhibitor combined with amuvatinib on the proliferation of PC-9 cells.

[0039] Logarithmic growth period PC-9 cells were taken and inoculated in 96-well plates (8000 cells / well), and after 24 h of culture, the cells adhered, different concentrations of amuvatinib single drug or amuvatinib combined with olanzapine were added (solvent was DMSO), and after 24 hours of treatment at 37°C, 5% carbon dioxide, 10% CCK-8 solution was added to each well, and the culture plate was placed in the incubator for 1-4 hours. The absorbance at 450 nm was measured by microplate reader, and the cell survival rate (%) = (experimental group absorbance-blank group absorbance) / (control group absorbance-blank group absorbance) * 100% was calculated; the cell growth inhibition rate (%) = (1- (experimental group absorbance-blank group absorbance) / (control group absorbance-blank group absorbance)) * 100% and IC 50 value.

[0040] The results are shown in Figure 3 and Table 1, and the combination of olanzapine and amuvatinib in PC-9 cells significantly inhibited cell proliferation, which was significantly stronger than amuvatinib alone. When different concentrations of olanzapine and amuvatinib were combined, different combination effects were shown. The specific results are as follows: The IC50 of amitinib monotherapy after 24 hours was 7.218 μM. The IC50 of amitinib combined with 100 μM olanzapine was 1.649 μM, a decrease of approximately 4.37-fold. The IC50 of amitinib combined with 150 μM olanzapine was 0.8931 μM, a decrease of approximately 8.08-fold. Furthermore, the same concentration of amitinib upregulated cell growth inhibition when combined with olanzapine, with a molar ratio of olanzapine to amitinib of 100–150 : 0.45–100.

[0041] Table 1. Cell growth inhibition rate of olanzapine combined with ametinib Olanzapine concentration (μM) Amuvatinib concentration (μM) Cell growth inhibition rate (%) 0 0.45 8.08838644 0 0.9 8.44467285 0 1.8125 18.3418979 0 3.625 22.0794793 0 6.25 47.2147996 0 12.5 66.7488866 0 25 81.8636519 0 50 99.1915039 0 100 98.0781089 100 0.45 34.619035 100 0.9 37.7927945 100 1.8125 43.68475 100 3.625 65.6846741 100 6.25 71.7550587 100 12.5 83.8996242 100 25 93.0868228 100 50 99.7684219 100 100 99.8937018 200 0.45 42.4646163 200 0.9 50.5001453 200 1.8125 58.7681069 200 3.625 67.7707218 200 6.25 80.2598265 200 12.5 99.6214253 200 25 99.7717179 200 50 99.3898643 200 100 99.6092641 .

[0042] The results above suggest that the SPTLC1 inhibitor olanzapine combined with ametinib has a synergistic effect. Experiment Example 4

[0043] The effects of a combination of SPTC1 inhibitor and targeted drug on organoid growth were investigated using a drug sensitivity assay in 3D organoid culture of human lung adenocarcinoma organoids.

[0044] Tumor tissue from EGFR-mutant lung adenocarcinoma patients with high SPTLC1 expression was minced, digested enzymatically to obtain a cell suspension, and further added to Matrigel to form a 3D culture system. The IC50 was measured after adding different gradient concentrations of olanzapine. 50 ( Figure 4 A) Ametinib IC 50 value( Figure 4 B) and IC after olanzapine combined with ametinib 50 value( Figure 4 C). The results show that 0.5 times IC 50 The combination of olanzapine and amitinib significantly increased the sensitivity of human lung adenocarcinoma organoids to amitinib, and the IC50 of amitinib was [missing information]. 50 The value decreased by approximately 2.8 times. These results suggest that reducing SPTLC1 expression with olanzapine may increase the sensitivity of EGFR-mutant lung adenocarcinoma patients to amitinib.

[0045] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any way. Any simple modifications, equivalent changes, and alterations made to the above embodiments based on the technical essence of the present invention shall still fall within the protection scope of the present invention.

Claims

1. An antitumor pharmaceutical composition, characterized by comprising: It comprises an SPTLC1 inhibitor and a targeted drug, wherein the SPTLC1 inhibitor is a compound inhibiting the enzyme activity, transcription, translation or protein stability of SPTLC1. ​ 2. The antitumor pharmaceutical composition according to claim 1, characterized by: The SPTLC1 inhibitor comprises a small nucleic acid inhibiting the transcription of SPTLC1, a small molecule compound inhibiting the protein stability of SPTLC1, and a pharmaceutically acceptable carrier or excipient.

3. The antitumor pharmaceutical composition according to claim 2, characterized by: The small molecule compound inhibiting SPTLC1 is olanzapine, and its molecular formula is: 。 4. The antitumor pharmaceutical composition according to claim 3, characterized by: The targeted drug is amatinib, and its molecular formula is: 。 5. The antitumor pharmaceutical composition according to claim 4, characterized by: The molar ratio of the olanzapine and amatinib is 100-150:0.45-100.

6. The antitumor pharmaceutical composition according to claim 5, characterized by: The pharmaceutically acceptable carrier is a filler, a wetting agent or a binder, a disintegrant; and the preparation form of the pharmaceutical composition is an oral tablet, a granule, an injection or a capsule.

7. The use of an antitumor pharmaceutical composition according to any one of claims 1 to 6, characterized in that: The tumor is non-small cell lung cancer.

8. The use of an antitumor pharmaceutical composition according to any one of claims 1 to 6, characterized in that: The tumor is a solid tumor with high expression of SPTLC1, and the pharmaceutical composition is applied in the preparation of an anti-tumor drug for inhibiting the expression and / or activity of SPTLC1.

9. The use of an antitumor pharmaceutical composition according to claim 8, characterized in that: The anti-tumor pharmaceutical composition is used for inhibiting the proliferation of tumor cells, wherein the tumor cells are PC-9 cells or NCI-H1975 cells.

10. The use of an antitumor pharmaceutical composition according to claim 8, characterized in that: The anti-tumor pharmaceutical composition is used for inhibiting the growth of lung cancer patient-derived organoids.

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