CN117257782A - Application of melitracin in reversing Oritinib resistance - Google Patents
Application of melitracin in reversing Oritinib resistance Download PDFInfo
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- CN117257782A CN117257782A CN202311478635.8A CN202311478635A CN117257782A CN 117257782 A CN117257782 A CN 117257782A CN 202311478635 A CN202311478635 A CN 202311478635A CN 117257782 A CN117257782 A CN 117257782A
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Classifications
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/13—Amines
- A61K31/135—Amines having aromatic rings, e.g. ketamine, nortriptyline
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/33—Heterocyclic compounds
- A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
- A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
- A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
- A61K31/506—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim not condensed and containing further heterocyclic rings
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
Abstract
The invention discloses an application of melitracin in reversing the drug resistance of octreotide. The invention discovers that the meritroxine or the meritroxine combined with the octreotide can inhibit the proliferation and the clonogenic capacity of lung cancer cells for the first time and can improve the intracellular ROS level of the lung cancer cells. The meretridine and the octreotide have synergistic effect, can obviously enhance the apoptosis capability of the octreotide on PC9/OR cells, and obviously improve the intracellular ROS level of lung cancer cells so as to further promote the apoptosis of the lung cancer cells and enhance the treatment effect of the lung cancer cells (especially non-small cell lung cancer). The invention discovers that the melitracin has a reversing effect on the acquired resistance of the lung cancer of the octreotide for the first time, provides a new method and a new strategy for overcoming the resistance of the octreotide, and simultaneously provides a new application of the melitracin.
Description
Technical Field
The invention belongs to the technical field of medicines, and particularly relates to an application of melitracen in reversing the drug resistance of octreotide.
Background
Lung cancer is a malignant tumor with high morbidity and mortality worldwide. Of these, about 80 to 85% are Non-small cell lung cancer (Non-small cell lung cancer, NSCLC). In recent years, molecular targeted drugs such as EGFR tyrosine kinase inhibitors (Epidermal growth factor receptor-tyrosine kinase inhibitor, EGFR-TKI) have made a breakthrough in the clinical treatment of NSCLC.
Ornitinib (Osi) is the first third generation EGFR-TKI approved by the FDA in the United states, and can selectively irreversibly bind to sensitive mutant EGFR and T790M drug-resistant mutant EGFR to block downstream signaling to achieve the purpose of resisting tumors. In view of its good clinical efficacy and safety, the drug is recommended for first-line treatment of EGFR mutation positive NSCLC patients.
However, also of octenib there is acquired resistance, leading to failure of treatment, disease progression. The reasons for acquired drug resistance are very complex, and the molecular mechanisms that have been found at present mainly comprise two main classes: EGFR-dependent mechanisms, such as EGFRC797S mutations, and the like; EGFR independent mechanisms such as compensatory signaling pathway establishment of c-MET, HER2 and the like, pathological tissue type transformation and the like. In order to overcome the drug resistance of the Ornitinib and enhance the curative effect of the Ornitinib, the present researches show that the panobinostat, the aspirin and the curcumin have a certain reversal effect on the acquired drug resistance of the EGFR-TKI drugs of NSCLC cells, but the drug reversal agent with higher safety, stable curative effect and better effectiveness still needs to be further explored and discovered.
Disclosure of Invention
Based on this, the present invention aims to provide a reversing drug having a reversing effect on the acquired resistance of the lung cancer of octenib.
In order to achieve the above object, the present invention includes the following technical solutions.
In a first aspect, the present invention provides the use of melitracin in the manufacture of a medicament for the prevention and/or treatment of lung cancer.
In some of these embodiments, the lung cancer is an octenib acquired resistant lung cancer.
In some of these embodiments, the lung cancer is human non-small cell lung cancer.
In a second aspect, the invention provides the use of melitracen in combination with octreotide for the manufacture of a medicament for the prevention and/or treatment of lung cancer.
In some of these embodiments, the lung cancer is an octenib acquired resistant lung cancer.
In some of these embodiments, the lung cancer is human non-small cell lung cancer.
In a third aspect, the present invention also provides a combination for the prevention and/or treatment of lung cancer, the active ingredients of which include melitracin and octreotide, which are separate administration units, or which together form a combined administration unit.
In some of these embodiments, the molar ratio of melitracin to octreotide is from 0.5 to 5:1.
in some of these embodiments, the molar ratio of melitracin to octreotide is from 1 to 3:1.
in some of these embodiments, the molar ratio of melitracin to octreotide is from 1.5 to 2.5:1.
in some of these embodiments, the molar ratio of melitracin to octreotide is from 1.8 to 2.2:1.
in some of these embodiments, the molar ratio of melitracin to octreotide is 2:1.
in some of these embodiments, the lung cancer is an octenib acquired resistant lung cancer.
In some of these embodiments, the lung cancer is human non-small cell lung cancer.
In a fourth aspect, the invention also provides the use of melitracin in the manufacture of a reversal medicament for reversing acquired resistance of octreotide in lung cancer.
In some of these embodiments, the lung cancer is human non-small cell lung cancer.
In a fifth aspect, the invention also provides a method for reversing the acquired resistance of the octreotide for lung cancer, which adopts the combination of a reversing drug and the octreotide to realize the reversing of the acquired resistance of the octreotide for lung cancer; the reversing drug is melitracin.
Wherein the melitracin and the octreotide are separate administration units, respectively, or the melitracin and the octreotide together form a combined administration unit, i.e., the melitracin and the octreotide may be administered simultaneously or separately.
In some of these embodiments, the molar ratio of melitracin to octreotide is from 0.5 to 5:1.
in some of these embodiments, the molar ratio of melitracin to octreotide is from 1 to 3:1.
in some of these embodiments, the molar ratio of melitracin to octreotide is from 1.5 to 2.5:1.
in some of these embodiments, the molar ratio of melitracin to octreotide is from 1.8 to 2.8:1.
in some of these embodiments, the molar ratio of melitracin to octreotide is 2:1.
in some of these embodiments, the lung cancer is human non-small cell lung cancer.
The invention discovers that the meretridine or the meretridine combined with the octreotide can inhibit the proliferation and the clone formation of lung cancer cells and simultaneously improve the intracellular ROS level of the lung cancer cells for the first time. The meretridine and the octreotide have synergistic effect, can obviously enhance the apoptosis capability of the octreotide on PC9/OR cells, and obviously improve the intracellular ROS level of lung cancer cells so as to further promote the apoptosis of the lung cancer cells and enhance the treatment effect of the lung cancer cells (especially non-small cell lung cancer).
The invention discovers that the melitracin has a reversing effect on the acquired resistance of the lung cancer of the Ornitanib for the first time, provides a new method and a new strategy for overcoming the resistance of the Ornitanib, and simultaneously provides a new application of the melitracin in preparing a medicine for preventing and/or treating the lung cancer or preparing a medicine for reversing the acquired resistance of the lung cancer of the Ornitanib.
Drawings
FIG. 1 is a validation result of NSCLC octenib acquired drug resistant cell model; wherein,
a is the octenib IC50 value of the parent cells and the drug resistant cells detected by the CCK8 experiment;
b is q-PCR experiment to detect the gene expression condition of ABCB 1;
c is the protein expression condition of the ABCB1 detected by a WB experiment.
FIG. 2 is the effect of melitracin on PC9/OR cell viability; wherein,
a is the structural formula of melitracin;
b is the IC50 value of the melitracin detected by CCK8 experiment;
c is a clone formation experiment to detect the clone inhibition ability of the melitracin on drug-resistant cells.
FIG. 3 is the effect of a combination of melitracin and octreotide on PC9/OR cells; wherein,
a is the result of flow cytometry detection of apoptosis;
b is the result of flow cytometry detection of cellular ROS levels.
Detailed Description
The technical scheme of the invention is further described by the following specific examples. It will be apparent to those skilled in the art that the examples are merely to aid in understanding the invention and are not to be construed as a specific limitation thereof.
Unless defined otherwise, 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 invention belongs. The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention.
The terms "comprising" and "having" and any variations thereof, are intended to cover a non-exclusive inclusion. For example, a process, method, apparatus, article, or device that comprises a list of steps is not limited to the elements or modules listed but may alternatively include additional steps not listed or inherent to such process, method, article, or device.
In the present invention, the term "plurality" means two or more. "and/or", describes an association relationship of an association object, and indicates that there may be three relationships, for example, a and/or B, and may indicate: a exists alone, A and B exist together, and B exists alone. The character "/" generally indicates that the context-dependent object is an "or" relationship.
In order to overcome the drug resistance of the octreotide and enhance the curative effect of the octreotide, the invention discovers a reversing agent (reversing drug) for the acquired drug resistance of the octreotide through a great deal of research and exploration; the research result of the invention shows that: the nervous system medicine-melitracin, a tricyclic diphase antidepressant with oral activity, can obviously enhance the anti-tumor capability of the octreotide and has good anti-tumor effect combined with the octreotide.
In one embodiment of the present invention, the present invention provides the use of melitracen in the manufacture of a medicament for the prevention and/or treatment of lung cancer.
The lung cancer can be common lung cancer or lung cancer with acquired resistance to the drug of the Ornitinib.
Further preferably, the lung cancer is human non-small cell lung cancer.
In another embodiment of the invention, the invention provides the use of melitracen in combination with octreotide for the manufacture of a medicament for the prevention and/or treatment of lung cancer.
The lung cancer can be common lung cancer or lung cancer with acquired resistance to the drug of the Ornitinib.
Further preferably, the lung cancer is human non-small cell lung cancer.
In another embodiment of the present invention, the present invention also provides a combination for preventing and/or treating lung cancer, the active ingredients of which include melitracin and octreotide, which are separate administration units, or which together form a combined administration unit. I.e. the melitracin and the octreotide may be administered simultaneously or separately, the molar ratio of melitracin to octreotide being between 0.5 and 5:1, preferably 1-3:1, further preferably 1.5 to 2.5:1, further preferably 1.8 to 2.2:1, more preferably 2:1.
the lung cancer can be common lung cancer or lung cancer with acquired resistance to the drug of the Ornitinib.
Further preferably, the lung cancer is human non-small cell lung cancer.
The combined medicine of the invention can also be prepared into a pharmaceutical preparation for preventing and/or treating tumors with pharmaceutically acceptable auxiliary materials. The pharmaceutical formulations may be administered to a patient in a variety of routes of administration including, but not limited to, oral, transdermal, intramuscular, subcutaneous, and intravenous injection.
The dosage form of the combined drug of the invention is not limited, and the combined drug can ensure that the active ingredients can reach the dosage form in vivo effectively, and comprises the following components: injection, tablet, film coated tablet, enteric coated tablet, capsule, buccal preparation, granule, powder, unguent, pellet, suspension, pill, powder, spray, drop, suppository, cream, patch, etc.; injection forms are preferred, such as: liquid injection, powder injection, injection tablet, etc.; injection forms include, but are not limited to, crude drug forms, drug-loaded nanoparticles, and the like; injection sites include, but are not limited to, intradermal injection, subcutaneous injection, intramuscular injection, intravenous injection, spinal cavity injection, and the like.
In another embodiment of the invention, the invention also provides the use of melitracin in the manufacture of a reversal medicament for reversing acquired resistance of octreotide in lung cancer.
Wherein the lung cancer is preferably human non-small cell lung cancer.
In another embodiment of the invention, the invention also provides a method for reversing the acquired resistance of the lung cancer of the octenib, which adopts the combination of the reversing drug and the octenib to realize the reversing of the acquired resistance of the lung cancer of the octenib; the reversing drug is melitracin.
Wherein the melitracin and the octreotide are separate administration units, respectively, or the melitracin and the octreotide together form a combined administration unit, i.e., the melitracin and the octreotide may be administered simultaneously or separately. When the medicine is administrated, the molar ratio of the melitracin to the octreotide is 0.5-5:1, preferably 1-3:1, further preferably 1.5 to 2.5:1, further preferably 1.8 to 2.2:1, more preferably 2:1.
wherein the lung cancer is preferably human non-small cell lung cancer.
The invention induces an octreotide drug-resistant cell model by a drug large-dose impact method, utilizes CCK-8 to detect IC50 values of the octreotide in different cells, calculates a drug-resistant coefficient, detects the expression level of a multi-drug-resistant gene ABCB1 by a real-time fluorescent quantitative polymerase chain reaction (Quantitative polymerase chain reaction, qPCR) experiment and a western blotting experiment (WB), and verifies whether the octreotide drug-resistant cell model is successfully constructed. On this basis, the antitumor effect of melitracin and the combination of melitracin and octreotide was evaluated by CCK-8, flow cytometry and cell clone formation experiments, and the synergy was determined by CI values. Meanwhile, the RNA-seq is used for detecting the differential change in drug-resistant strains after the combined drug and the single drug. The invention is proved by the scientific experiments that: the meretridine combined with the octreotide can remarkably inhibit the proliferation capability of an octreotide acquired drug resistant cell model PC9/OR induced by human lung adenocarcinoma PC9 cells, and simultaneously raise the level of active oxygen (reactive oxygen species, ROS) of the cells and promote apoptosis.
The following materials were used in the examples:
RPMI1640 medium, penicillin-streptomycin (100X) was purchased from Gibco corporation, USA;
fetal bovine serum was purchased from the biosciences company of escitalopram, china;
ornitinib (T2490), melitracin (T4274) was purchased from Shanghai Tao Shu Biotechnology Co., ltd;
h2DCFDA (HY-D0940) was purchased from MCE company, USA;
CCK-8 kit (FD 3788) was purchased from Friedel biotechnology Co., ltd;
apoptosis assay kit (KGA 105) was purchased from Jiangsu Kaiki Biotechnology Co., ltd.
The following are specific examples.
Example 1 establishment and verification of NSCLC Ornitinib acquired drug resistant cell model
1. Cell culture
Human non-small cell lung cancer cell line PC9 (EGFR gene exon 19 deletion mutation) was purchased from basic medical institute of China medical sciences. PC9 cells were cultured in RPMI1640 medium containing 10% fetal bovine serum, 1% penicillin and streptomycin at 37℃with 5% CO 2 Culturing in an incubator.
2. Drug resistance model establishment
And establishing an Aoditinib-derived drug-resistant cell strain PC9/OR by using the Aoditinib-sensitive PC9 cells according to a drug large-dose impact method. PC9 cells in the logarithmic phase were treated with octenib at a concentration of 10. Mu.M for 48h, and after withdrawal, the cells recovered to growth to about 80% of the growth density, which was one shock cycle, and subjected to multiple drug shock cycles until the cells could stably grow in the 2. Mu.M octenib-containing medium, i.e., PC9/OR cells were successfully induced.
3. Drug resistance model verification
(1) Cell viability was detected using the CCK-8 assay. The method comprises the following steps:
taking PC9 and PC9/OR cells in logarithmic growth phase, inoculating into 96-well plate (3×10) 3 Individual cells/well), culturing for 24 hr, adding different concentrations of Ornitinib after cell adhesion, and adding 5% CO at 37deg.C 2 After 48 hours of treatment under the condition, adding 10% of CCK-8 detection reagent for further incubation for 1 hour, detecting the absorbance at 450nm by an enzyme-labeled instrument, and calculating the half-maximal inhibitory concentration (halfmaximal inhibitory concentration, IC 50) of the cells; cell viability (%) = (experimental group absorbance-blank group absorbance)/(control group absorbance-blank group absorbance).
The results are shown as a in fig. 1: the IC50 values of octenib in PC9 and PC9/OR were 0.193. Mu.M and 9.493. Mu.M, respectively.
(2) The expression change of the multidrug resistance gene ABCB1 in the cells is detected by adopting a real-time fluorescence quantitative polymerase chain reaction (Quantitativepolymerase chain reaction, qPCR) and a western blotting experiment (WB). The method comprises the following steps:
taking log to growPhase PC9 and PC9/OR cells were seeded in 6-well plates (2X 10) 5 Individual cells/well), at 37 ℃, 5% co 2 Culturing for 48 hours under the condition, extracting cell RNA and total protein, and respectively carrying out qPCR and WB experiments.
The results are shown in FIG. 1 as B (q-PCR assay to detect gene expression of ABCB 1) and C (WB assay to detect protein expression of ABCB 1): ABCB1 expression is elevated in PC9/OR cells.
The above results indicate that: the obtained drug resistant cell strain PC9/OR of the Ornitinib is successfully established and obtained.
Example 2 effect of melitracin on PC9/OR cell viability
Melitracin (Melitracen) is an orally active tricyclic biphasic antidepressant of the formula shown as a in fig. 2. Melitracin inhibits the absorption of Norepinephrine (Norepinephrine) and 5-hydroxytryptamine (5-HT) through the presynaptic membrane, thereby increasing the concentration of single amine transmitters in the synaptic cleft, and is useful for the treatment of depression and anxiety.
1. The effect of melitracin on PC9/OR cell viability was examined using the CCK-8 method. The method comprises the following steps:
PC9/OR cells in logarithmic growth phase were inoculated into 96-well plates (3X 10) 3 Individual cells/well), culturing for 24 hr, and adding different concentrations of meli Qu Xinyu deg.C and 5% CO after the cells adhere to the wall 2 After 24 and 48 hours of treatment under the condition, adding 10% of CCK-8 solution for further incubation for 1 hour, and calculating half-maximal inhibitory concentration (halfmaximal inhibitory concentration, IC 50) of cells after detecting absorbance at 450nm by an enzyme-labeling instrument; cell viability (%) = (experimental group absorbance-blank group absorbance)/(control group absorbance-blank group absorbance).
The results are shown in fig. 2B: IC50 values for PC9/OR cells treated with melitracin for 24h and 48h were 26.72. Mu.M and 17.85. Mu.M, respectively.
2. The effect of melitracin on PC9/OR cell viability was examined using a plate clone formation assay.
The method comprises the following steps:
PC9/OR cells in logarithmic growth phase were seeded in 6-well plates (1X 10) 3 Individual cells/well), culturing for 24 hr, and adding different concentrations after the cells adhere to the wallMildly melitracin, 5% CO at 37 ℃ 2 After 48 hours of treatment under the condition, the culture medium is changed into a culture medium without medicines, and the culture is continued for 7 days. Cells were fixed with 4% paraformaldehyde and then stained with crystal violet.
The results are shown as C in fig. 2: after treatment of PC9/OR cells with melitracin for 48h, the clonogenic potential of the cells was significantly inhibited.
Example 3 effects of combination of melitracin and Ornitinib on PC9/OR cells
1. Apoptosis was detected using flow cytometry. The method comprises the following steps:
the logarithmic phase PC9/OR cells were seeded in 6-well plates (2X 10) 5 Individual cells/well), culturing for 24h, adding 10 μM of melitracin, 5 μM of octreotide, and combination drug (10 μM of melitracin, 5 μM of octreotide) to treat cells after cell adhesion, and treating cells with 5% CO at 37deg.C 2 All cells were collected after 24h of treatment under the conditions. And washing, fixing and staining the cells according to a commercial apoptosis kit, and finally recording fluorescence intensities of an excitation wave FITC channel and a PE channel by using a flow cytometer to analyze apoptosis conditions.
The results are shown in fig. 3 a: the combination regimen significantly enhanced the apoptosis capacity of the oritinib on PC9/OR cells.
2. The ROS levels of the cells were detected using flow cytometry. The method comprises the following steps:
PC9/OR cells in logarithmic growth phase were seeded in 12-well plates (1X 10) 5 Individual cells/well), culturing for 24h, adding 10 μM of melitracin, 5 μM of octreotide, and combination drug (10 μM of melitracin, 5 μM of octreotide) to treat cells after cell adhesion, and treating cells with 5% CO at 37deg.C 2 After 24h of treatment, the original medium was discarded, 10. Mu.M DCFH-DA-containing medium (500. Mu.L/well) was added and stained at 37℃in the absence of light for 30min, and the cells were washed twice with PBS (500. Mu.L/well) to sufficiently remove DCFH-DA not taken into the cells, and the cells were collected for flow cytometry, and fluorescence intensity of FITC channels was measured to analyze the ROS levels in the cells.
The results are shown in fig. 3B: the combined regimen significantly increased ROS levels in PC9/OR cells.
The technical features of the above-described embodiments may be arbitrarily combined, and all possible combinations of the technical features in the above-described embodiments are not described for brevity of description, however, as long as there is no contradiction between the combinations of the technical features, they should be considered as the scope of the description.
The above examples illustrate only a few embodiments of the invention, which are described in detail and are not to be construed as limiting the scope of the invention. It should be noted that it will be apparent to those skilled in the art that several variations and modifications can be made without departing from the spirit of the invention, which are all within the scope of the invention. Accordingly, the scope of protection of the present invention is to be determined by the appended claims.
Claims (10)
1. Use of melitracin in the manufacture of a medicament for the prevention and/or treatment of lung cancer.
2. Use of melitracin in combination with octreotide in the manufacture of a medicament for the prevention and/or treatment of lung cancer.
3. The use according to claim 1 or 2, wherein the lung cancer is of the acquired resistance to octenib.
4. The use according to claim 1 or 2, wherein the lung cancer is human non-small cell lung cancer.
5. A combination for the prophylaxis and/or treatment of lung cancer, characterized in that the active ingredients thereof comprise melitracin and octtinib, which are separate administration units or which together form a combined administration unit.
6. The combination for preventing and/or treating lung cancer according to claim 5, wherein the molar ratio of melitracen to octenib is 1-3:1, a step of; and/or the number of the groups of groups,
the lung cancer is the lung cancer with the acquired drug resistance of the octreotide; and/or the number of the groups of groups,
the lung cancer is human non-small cell lung cancer.
7. The combination for preventing and/or treating lung cancer according to claim 6, wherein the molar ratio of melitracen to octenib is 1.5-2.5:1, preferably 1.8-2.2:1.
8. the use of melitracin in the manufacture of a reversal medicament for reversing acquired resistance of octreotide for lung cancer; preferably, the lung cancer is human non-small cell lung cancer.
9. The method for reversing the acquired resistance of the lung cancer of the octreotide is characterized in that a reversing drug is combined with the octreotide to realize the reversing of the acquired resistance of the lung cancer of the octreotide; the reversing drug is melitracin, and the melitracin and the octreotide are separate administration units or form a combined administration unit together.
10. The method of reversing acquired resistance to lung cancer of claim 9, wherein the lung cancer is human non-small cell lung cancer; and/or the number of the groups of groups,
the molar ratio of the melitracin to the octreotide is 1-3:1, preferably 1.5-2.5:1, more preferably 1.8-2.8:1.
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