CN115869310A - Targeted application of lomitapide in inhibiting FZD10 high-expression tumors - Google Patents
Targeted application of lomitapide in inhibiting FZD10 high-expression tumors Download PDFInfo
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Abstract
The invention discloses targeted application of lomitapide and derivatives thereof in preparing FZD10 high expression resistant tumors and targeted drugs. The lomitapide can inhibit the proliferation, the number and the size of the clone formation of the nasopharyngeal carcinoma cells (CNE 1) at the concentration of 0.1-4 mu M, but the lomitapide cannot inhibit the nasopharyngeal carcinoma cells (CNE 1-CAS-FZD 10) with knockout of FZD10 at the concentration of 0.1-1 mu M. This indicates that binding of lomitapide to FZD10 is specific binding.
Description
Technical Field
The invention relates to application of lomitapide and derivatives thereof in preparation of antitumor drugs, and in particular relates to targeted application of lomitapide and derivatives thereof in preparation of FZD10 high-expression tumor resistant tumors and targeted drugs.
Background
Since the 21 st century, the incidence of cancer has been increasing, which may be related to changes in our lifestyle and exposure to harmful substances, and the current common forms of cancer treatment are chemotherapy, hormone therapy, immunotherapy, radiotherapy and surgery, wherein the research and application of targeted drugs are the focus of cancer treatment. Based on the fact that normal cells are transformed into cancer cells due to abnormal expression of proto-oncogenes and tumor suppressor genes caused by physical, chemical, viral, etc. carcinogenic factors, and that these mutations can result in the change of expression of thousands of genes, there are now various forms of targeted therapies directed to specific single molecular targets or a class of molecular targets in cancer cells. Under targeted therapy, the specific mechanism of action of a drug results in an increase in its therapeutic index.
Nasopharyngeal carcinoma (NPC) is an epitheliogenic tumor that occurs mostly in south China and countries in south east asia, often on the top of the posterior wall of the nasopharynx top, and secondly on the lateral wall. Because of its occult disease, it has a strong tendency to metastasize, with regional lymph nodes and/or distant metastases occurring in about 75% of patients reaching late stage at first visit. Generally, comprehensive treatment mainly based on radiotherapy is very effective on early nasopharyngeal carcinoma, and recurrence or metastasis after treatment is extremely poor in prognosis, which becomes a main reason for failure of nasopharyngeal carcinoma treatment and reduction of survival rate. Therefore, screening the tumor markers of nasopharyngeal carcinoma strives for early detection, reasonable treatment, prognosis prediction and recurrence monitoring, and has very important significance for the clinical diagnosis and treatment of nasopharyngeal carcinoma.
The development of cancer drugs is a multi-step process involving design, synthesis, characterization, efficacy and toxicity testing, and approval. Overall, this process is lengthy and requires a significant capital investment. It is estimated that the development of a new drug typically takes 15 to 18 years and approximately $ 23 million. Most importantly, nearly 90% of drugs fail in clinical trials due to lack of adequate efficacy, unacceptable side effects, or regulatory norms. Therefore, the search for new uses for clinically approved old drugs can accelerate the drug development process. The reuse of old drugs provides a quick transition from the laboratory to the bedside. These drugs have clear pharmacodynamics, pharmacokinetics, dosages, side effects, metabolic characteristics and have been approved by the FDA and other regulatory regulations, passed clinical trials, and are approved for use in humans.
Lomitapide (Lomitapide) is a selective inhibitor of microsomal triglyceride transfer protein (MTP). In 10 months 2007, the FDA awards a lomitamide orphan drug designation for the treatment of Homozygous familial hypercholesterolemia (homozzogous family hyper-cholestilemia, hoFH). The lomitapide and derivatives thereof such as lomitapide mesylate are FDA approved clinical medicines, have high safety, good development and utilization prospects, small toxic and side effects and low price. Therefore, researchers began to develop the use of lomitapide for the treatment of cancer. For example:
the Chinese invention application 202011058670.0 discloses an application of lomitapide in preparing antineoplastic drugs, and the application verifies that N- (2, 2-trifluoroethyl) -9- (4- [4- [4'- (trifluoromethyl) [1,1' -biphenyl ] through cell proliferation experiments]-2-carboxamido]Piperidin-1-yl radical]Butyl) -9H-fluorene-9-carboxamide (lometas, molecular formula C) 39 H 37 F 6 N 3 O 2 Molecular weight of 693.720) in esophageal cancer cell lines (KYSE 150, KYSE 450) and gastric cancer (HGC 27, AGS) cell lines, so as to provide new ideas and bases for clinical research of drugs for preventing and treating tumors.
The Chinese invention application 202110798169.6 also discloses the application of lometaside in preparing antitumor drugs. The application discloses that lometasapine can be dissolved in water and orally taken to assist in the treatment of colon cancer. In vitro and in vivo experiments show that: lomantapide can inhibit intestinal cancer growth.
The results show that the lometasan indeed has a certain effect on resisting tumors. However, lomitapide is not universally applicable to the preparation of antitumor drugs for various occasions.
Therefore, there is a need to identify the targeted use of lomitapide against tumors.
Disclosure of Invention
The invention aims to provide application of lomitapide and derivatives thereof in preparation of tumor-targeted drugs.
The invention also aims to provide an anti-tumor targeted medicament comprising the lomitapide and derivatives thereof.
In one aspect, to achieve the above-mentioned objects of the present invention, the present invention provides the use of lomitapide and derivatives thereof for preparing anti-FZD 10 highly expressed tumor drugs.
Frizzled proteins are a family of Wnt receptors, 10 member proteins in this family, frizzled-10 (FZD 10) being one of the members. FZD10 is a class of proteins with 7-transmembrane structure, similar to G-protein coupled receptors, most frizzled receptors are coupled to the β -catenin canonical signaling pathway, resulting in activation of messengers, inhibition of GSK-3 kinase, nuclear accumulation of β -catenin, and activation of Wnt target genes. The Fzd family has been of interest because of its "switching role" in the Wnt/β -catenin signaling pathway.
The inventors of the present application surprisingly found that in experiments, results of using nasopharyngeal carcinoma Cells (CNEI) and FZD10 knock-out nasopharyngeal carcinoma cells (CNE 1-CAS-FZD 10), lomitapide and derivatives thereof were completely different. For example, lometasapine can inhibit the proliferation, number and size of clonogenic cells of the nasopharyngeal carcinoma (CNE 1) at concentrations of 0.1-4. Mu.M, but lometasapine cannot inhibit the FZD10 knock-out nasopharyngeal carcinoma cells (CNE 1-CAS-FZD 10) at concentrations of 0.1-1. Mu.M. This indicates that the binding of lomitapide to FZD10 is specific, and FZD10 has targeting effect and significance.
Further, the invention provides application of the lomitapide and derivatives thereof in preparing medicaments for resisting nasopharyngeal carcinoma with high expression of FZD 10.
In the invention, the lomitapide and the derivatives thereof can be the lomitapide with the structure shown in the formula I:
formula I is C 39 H 37 F 6 N 3 O 2 Molecular weight is 693.7.
In the present invention, the lomitapide and the derivatives thereof may be lomitapide mesylate having a structure represented by formula II:
formula II is C 40 H 41 F 6 N 3 O 5 S, molecular weight 789.8.
In the invention, the lomitapide and the derivative thereof can also be a lomitapide hydrate with a structure shown in formula III:
the inventor of the invention finds that the lomitapide and the derivative thereof have obvious inhibition effect on cancer cells with high expression of FZD10 when the concentration of the lomitapide and the derivative thereof in animals reaches 2mg/kg-50mg/kg. Preferably, the concentration of the lomitapide and the derivatives thereof is 3mg/kg-30mg/kg; more preferably, the concentration of lomitapide and derivatives thereof is 5mg/kg to 20mg/kg.
For example, in the research of preparing the medicament for inhibiting the growth of the mouse tumor in the human-derived nasopharyngeal carcinoma transplantable tumor model, the inventor finds that the lomitapide can inhibit the growth of the mouse tumor in the human-derived nasopharyngeal carcinoma transplantable tumor model when the concentration of the lomitapide is 10 mg/kg/day.
In another aspect, to achieve the above-mentioned objects of the present invention, the present invention also provides a composition against FZD 10-highly expressed tumors comprising lomitapide and derivatives thereof for achieving the above-mentioned uses of the present invention.
In the composition of the invention, the lomitapide and the derivatives thereof used can be the lomitapide with the structure shown in formula I, the lomitapide mesylate with the structure shown in formula II, the lomitapide hydrate with the structure shown in formula III, and any combination of the lomitapide and the derivatives thereof.
In the above composition of the present invention, at least one pharmaceutically acceptable excipient is further included, the excipient is selected from the group consisting of but not limited to: excipient, disintegrant, carrier, sustained-release agent, adhesive, filler, packaging capsule, various processing aids, etc.
Preferably, the above-mentioned composition of the present invention takes the form of oral preparations such as various oral tablets and capsules.
The invention discovers that the lomitapide and the derivative thereof have a targeted inhibition effect on the cancer cells with high expression of the FZD10, so that the lomitapide and the derivative thereof can be used for preparing a targeted medicament for resisting tumors with high expression of the FZD10, and bring more treatment options for tumor patients.
The present invention will be further described with reference to the accompanying drawings and specific embodiments, which are illustrative of certain specific embodiments of the present invention and are not to be construed as limiting the invention.
Drawings
FIG. 1 shows a schematic Western blot of proteins extracted from nasopharyngeal carcinoma cells (CNE 1) and FZD 10-knocked-out nasopharyngeal carcinoma cells (CNE 1-CAS-FZD 10);
figure 2 shows a schematic of the binding site distribution of FZD10 and lomitapide;
FIG. 3 shows a sequence alignment of the FZD family;
FIG. 4 shows the inhibitory effect of lomethazine at different concentrations on CNE1 cells and CNE1-CAS-FZD 10;
FIG. 5 is a graph comparing the tumor size in mice of the control group and the administered (Lomantapi) group;
FIG. 6 is a graph showing the change in tumor size with time of administration in mice;
FIG. 7 is a graph comparing the size of ex vivo liver of control group and administration (Lomantapi) group mice.
Detailed Description
The materials, equipment and reagents used in this embodiment are as follows:
1.1 nasopharyngeal carcinoma cells
The nasopharyngeal carcinoma cell (CNE 1) and the nasopharyngeal carcinoma cell (CNE 1-CAS-FZD 10) in which FZD10 is knocked out used in the present embodiment are both from the central laboratory of the affiliated tumor hospital of the university school of medicine of shantou.
1.2 Instrument
SWCJCO clean bench Suzhou purification Equipment Co., ltd
BB16 CO2 constant temperature cell culture box: heraeus Japan Ltd
TD5A-WS Large Capacity centrifuge Hunan Saite Xiang apparatus Co., ltd
GS-15R high speed bench refrigerated centrifuge: eppendorf Co, germany
DNP-9052BS electrothermal biochemical incubator: shanghai New Miao medical Instrument manufacturing Co., ltd
MK3 type enzyme labeling instrument: saimer Feishire science and technology Co., ltd
Constant temperature magnetic stirrer: shanghai Si le Instrument factory
LEICA DMI 3000B inverted microscope: olympus corporation of Japan
PVDF membranes (0.45 μm) available from Millipore, USA
Cell culture flasks/dishes from Corning, USA
96/12/6 well plates available from Corning, USA
15/50ml centrifuge tube: purchased from Corning, USA
Pipette (5 ml): purchased from Corning, USA
Rnase-free tip: from Axygen Inc
1.3 reagent
RPMI1640 Medium Gibco Co
Gibco of fetal bovine serum FBS
Penicillin streptomycin mixed solution (100X) Beijing Solaibao Tech Co., ltd
Trypsin Sammermafeishel technologies, inc
Serum-free cell freezing medium Xinsaimei Biotech Co., ltd
Byuntian biotechnology Co Ltd of BCA protein concentration determination kit
SDS-PAGE gel configuration kit Chongqing Bay Biotech Co Ltd
ECL Chemiluminiscence liquid Sammerle Feishell science and technology Co., ltd
Western Blot Marker Saimer Feishale science Co Ltd
Protein (RIPA) lysate Biyunnan Biotech Ltd
Biotech limited of protease inhibitors Biyuntian
Tween20 Amresco Inc. USA
Fuyu Fine chemical Co Ltd of Tianjin sodium chloride
TRIS Beijing Solebao science and technology Co., ltd
Glycine Beijing Solebao science and technology Co., ltd
SDS France Biotech Co
Medical alcohol (75%, 95%) Jiangxi Huikang Kogyo Co., ltd
Guangdong chemical reagent limited of Tianjin city of absolute ethanol
Amresco Inc. of TEMED USA
Biyuntian biotechnology Co Ltd of CCK8 kit
Anti-beta IV Tubulin antibody Guangzhou Zhenzhi Biotechnology Ltd
GAPDH antibody (TA-08) Beijing Zhonghua Jinqiao Co., ltd
Beta-actin antibody (TA-09) Beijing China fir Jinqiao Biotechnology Co., ltd
Cell validation experiment
2.1 Western Blot experiment
Taking CNE1 and CNE1-CAS-FZD10 cells with good growth conditions to extract protein, determining protein concentration according to BCA method, calculating sample amount according to standard curve, and denaturing protein to begin the following steps of electrophoresis, membrane transferring, sealing, TBST washing three times, five minutes each time, primary antibody, TBST washing three times, five minutes each time, secondary antibody, TBST washing three times, five minutes each time and developing
The results obtained are shown in FIG. 1. Wherein, the blot band represents whether the corresponding protein exists in the cell. It can be seen that after cas9 and FZD10 are knocked out, no corresponding band is displayed, and FZD10 is successfully knocked out; among them, GAPDH is a reference, and GAPDH is shallow in fig. 1, indicating that cellular protein is relatively low, but less but more FZD10 is expressed, indicating that the results are accurate.
2.2 sequence comparison
To verify that FZD10 is specific for the Lomitapide (Lomitapide) binding site, an FZD1 family protein sequence alignment will be performed. The sequence alignment procedure was as follows:
a. firstly, obtaining a protein sequence through a Uniprot database (http:// www. Uniprot. Org /);
b. performing multi-sequence comparison on the known FZD family protein sequence by using ClustalX software;
c. the results of ClustalX were beautified using GeneDoc, using different colors to distinguish the conservation of sites (high gray for highly conserved sites, medium gray for medium, low gray for low conserved sites)
Please refer to fig. 2 and fig. 3. The results are shown below, high grey is a highly conserved site, medium grey is medium, low grey is a low conserved site, and the boxed and bold is the binding site of FZD10 and lomitamide, most of which are not conserved sites, thus demonstrating the existence of targeting specificity.
2.3 cytotoxicity assay
CCK8 principle: WST-8 is the main chemical of the CCK8 kit, which can be redox by NAD + to a water-soluble yellow Formazan product (Formazan) in living cells. The more viable cells, the more formazan produced and the darker the color. Therefore, darker staining of the cells indicates a greater number of viable cells, and is often used to test the toxicity of drugs to the cells and to plot cell proliferation-time curves.
(1) Plate paving: taking cells of CNE1 and CNE1-CAS-FZD10 in logarithmic phase, digesting and re-suspending by trypsin, counting the cells under an inverted microscope, respectively adding the cells into two different 96-well plates to make the number of the cells in each well about 4000, and putting the cells into a cell culture box overnight;
(2) Preparing a culture medium: lomitapide was added to the serum-containing medium to a final concentration of 10. Mu.M, and then 9 centrifuge tubes were used to prepare 6 different concentration gradients of medium with 10. Mu.M CPT medium: 1. 10, 100, 1000, 2000, 4000n μm;
(3) The next day, taking out the 96-well plate, sucking out the old culture medium, washing twice with sterilized physiological saline, discarding the waste liquid, adding 200 μ l of prepared culture medium containing medicine into each well, reserving 6 blank wells without adding any cell and medicine as blank contrast, and putting the 96-well plate back to the incubator for continuous culture;
(4) After 48 hours, the 96-well plate was removed, and a medium containing 10% of CCK8 was prepared in advance. Completely sucking out the old culture medium, adding 100 μ l of solution (including the original 6 blank holes) into each hole with a discharging gun, placing into an incubator for continuous culture, taking out after two and a half hours, and measuring absorbance;
(5) Calculating the formula: inhibition rate of drug on cells = (OD value of experimental group-blank control group OD value)/(OD value of control group-blank control group OD value) 100%
The results are shown in FIG. 4. Fig. 4 shows that lomitamide has an inhibitory effect on CNE1, and that this inhibitory effect is reduced after FZD10 is knocked out, and is more pronounced particularly in the low concentration range, e.g., below 3 or 4.
Animal experiments
The 4-week-old BALB/c Nude mice are purchased from Beijing Witongliwa laboratory animal technology Limited and bred in the animal center of Shantou university college of medicine.
When nude mice are 6-8 weeks old, CNE1 cells with good growth condition are inoculated to the right armpit of the animal, the number of the cells is 5 x 10^ 6/mouse, the injected cells are diluted with physiological saline, 200ul of each nude mouse is obtained, when the tumor volume is 200-300mm 3 Grouping is started, i.e. the mice are evenly distributed to each group according to the tumor volume, after 10 groups are grouped, the lometaside is dissolved in a mixture of 10% DMSO +90% normal saline to prepare a corresponding dose, i.e. 10mg/kg of drug solution. Then 2 groups of mice were individually gavaged with normal saline, 10mg/kg lometasa. Tumor volumes were recorded in mice every 2 days. When the tumor volume of the control mice grows to 2000mm fast 3 The experiment was terminated at the left and right.
See fig. 5 and 6 for experimental results. It can be seen that tumor growth in mice was significantly inhibited with the administration.
Observation of side effects of drugs:
liver of ex vivo mice was compared in general, since Lomitapide is a selective inhibitor of microsomal triglyceride transfer protein (MTP) and can lead to the accumulation of large amounts of triglycerides in the liver, leading to the development of fatty liver. From the results in fig. 7, the livers shown in the control group and the administration group were not much different for the ex vivo livers.
Experiment of preparation of composition
50wt% of lometaside and methanesulfonic acid lometaside are taken respectively, and are pressed into oral tablets with 30wt% of anhydrous lactose, 18wt% of microcrystalline cellulose, 1wt% of cross-linked polyvinylpyrrolidone and 1wt% of superfine silica powder.
Claims (10)
1. The application of the lomitapide and the derivative thereof in preparing the anti-FZD 10 high-expression tumor medicine.
2. The use of claim 1, wherein said cancer cell is a nasopharyngeal cancer cell.
3. The use according to claim 1 or 2, wherein the lomitapide and derivatives thereof are lomitapides having the structure shown in formula I:
4. the use as claimed in claim 1 or 2, wherein the lomitapide and derivatives thereof are lomitapide mesylate having the structure shown in formula II:
5. the use of claim 1 or 2, wherein the lomitapide and derivatives thereof are lomitapide hydrates having the structure shown in formula III:
6. the use of claim 1, wherein the concentration of lomitapide and derivatives thereof is 2mg/kg to 50mg/kg.
7. A composition comprising lomitapide and derivatives thereof for use according to any one of claims 1 to 6.
8. The composition of claim 7, wherein the lomitapide and derivatives thereof are lomitapide having a structure shown in formula I, lomitapide mesylate having a structure shown in formula II, and/or lomitapide hydrate having a structure shown in formula III:
9. the composition of claim 8, wherein said composition further comprises at least one pharmaceutically acceptable excipient.
10. The composition of claim 9, wherein the composition is in the form of an oral formulation.
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN107663166A (en) * | 2016-07-28 | 2018-02-06 | 天津汉瑞药业有限公司 | Lome Tapai and its production and use |
| CN111388431A (en) * | 2020-03-02 | 2020-07-10 | 牡丹江医学院 | Oral preparation of lomitapide mesylate and preparation method thereof |
| WO2021167389A1 (en) * | 2020-02-21 | 2021-08-26 | 한국과학기술원 | Pharmaceutical composition for preventing or treating cancer, containing mtor-signaling inhibitor as active ingredient |
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Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107663166A (en) * | 2016-07-28 | 2018-02-06 | 天津汉瑞药业有限公司 | Lome Tapai and its production and use |
| WO2021167389A1 (en) * | 2020-02-21 | 2021-08-26 | 한국과학기술원 | Pharmaceutical composition for preventing or treating cancer, containing mtor-signaling inhibitor as active ingredient |
| CN111388431A (en) * | 2020-03-02 | 2020-07-10 | 牡丹江医学院 | Oral preparation of lomitapide mesylate and preparation method thereof |
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