CN109966275B - Application of quinoid chalcone compound in preparation of antitumor drugs - Google Patents
Application of quinoid chalcone compound in preparation of antitumor drugs Download PDFInfo
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- A61K31/00—Medicinal preparations containing organic active ingredients
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- A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- A61K38/19—Cytokines; Lymphokines; Interferons
- A61K38/21—Interferons [IFN]
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- A61K38/00—Medicinal preparations containing peptides
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Abstract
The invention provides an application of quinoid chalcone compound in preparing antineoplastic drugs, wherein quinoid chalcone is a quinoid chalcone compound with isopentenyl group at ring A, and also provides an application of the quinoid chalcone compound as single drug or combined drug in treating tumor diseases.
Description
Technical Field
The invention relates to an application of quinoid chalcone compound in preparing anti-tumor drugs, and also relates to an application of the quinoid chalcone compound as a combined drug in treating tumor diseases and an anti-tumor drug preparation combination.
Background
Malignant tumor is a disease caused by abnormal cell proliferation, and tumor stem cells play an important role in the survival, proliferation, metastasis and recurrence of tumors. Essentially, tumor stem cells maintain the viability of tumor cell populations through self-renewal and immortalization, the ability to move and migrate conferring the ability of tumor cells to metastasize distally. In recent years, research reports show that in the clinical treatment process, the tumor stem cells can be in a dormant state for a long time and can up-regulate the expression of various drug-resistant molecules, so that the tumor stem cells are insensitive to external physicochemical factors for killing the tumor cells. Tumor dormancy (tumor dormancy) is an occasional clinical state, including cell dormancy, tumor vascular dormancy and immune state dormancy, and is one of the biological characteristics of malignant tumor cells, and is one of the important causes of repeated clinical tumor outbreaks and distant invasion and metastasis. The traditional operation, radiotherapy and chemotherapy means have a certain treatment effect, are accompanied by serious clinical side effects and have the risk of inducing tumor dormancy, and the search for an effective treatment method with small toxic and side effects to improve the life quality of patients is important in medical research.
In vitro killing of cancer cells by using killer T cells, theoretically, the killing effect is more obvious when the number of the killer T cells is larger, however, the inventor of the present invention finds that in the research taking melanoma B16-OVA cells as an example, the killing of the killer T cells to the cancer cells is enhanced along with the increase of the concentration, and even if the number of the T cells is increased after a certain proportion is reached, the B16-OVA cells cannot be completely eliminated. This suggests that there is a group of dormant cancer cells that cannot be killed by specific T cells in clinical treatment, and these dormant cancer cells pose a hidden danger in vivo of tumor patients, and the clinical treatment at present cannot completely eliminate the dormant cancer cells, so that there is a risk of tumor recurrence or metastasis and death in clinical patients.
Although the research results of antitumor drugs continuously appear, the work of searching more effective drugs and means is not stopped, and the method for clearing dormant tumor cells is searched, so that the tumor is really and effectively cleared, the aim of thoroughly treating cancer cells in a clinical tumor patient is fulfilled, and the method is the aim which is sought by the industry and is also expected by the whole society.
Disclosure of Invention
The invention solves the technical problem of providing the application of quinoid chalcone compounds in preparing anti-tumor medicaments, and the micromolecule inhibitor with a special structure can kill tumors, can assist in using interferon and achieve the effect of killing dormant tumor cells.
The invention also provides the application of the quinoid chalcone as an active component of a drug combined with interferon in antitumor treatment, and provides an antitumor preparation combination with active components comprising the quinoid chalcone and the interferon.
One aspect of the present invention provides an application of a quinoid chalcone compound in the preparation of a drug for treating tumor diseases, wherein the quinoid chalcone is a group of quinoid chalcone compounds with an a ring having an isopentenyl group, which has been disclosed and explained in detail in chinese patent application CN201410220017.8, so that the relevant contents are all incorporated into the present application and become a part of the present application.
The quinoid chalcone compound with isopentenyl on the ring A has the following general structure I:
in the formula I, R represents 1 to 4 substituents attached to the ring B, and can be independently selected from: hydrogen, hydroxy, mercapto, methoxy, amino, nitro, halogen (e.g., chlorine, bromine, etc., are relatively common), cyano, mesylate, or formate.
The inventor discovers that the micromolecule inhibitor with the quinoid chalcone structure is used as a single medicine to show a remarkable anti-tumor effect on the basis of a great deal of early exploration, and the micromolecule inhibitor can be used together with clinical anti-tumor medicines to break the dormant state of tumor cells, and shows a better effect of inhibiting and killing the cancer cells compared with the single anti-tumor medicines, so that the micromolecule inhibitor has important significance for thoroughly eliminating the tumor cells in the body of a patient and curing the tumor diseases.
According to the research results of the inventors of the present invention, the quinoid chalcone compound is a kind of small molecule inhibitor (represented by "HB" in the present invention) as a single drug or a combined drug, and is applicable to various clinically diagnosed solid tumors and leukemias, such as malignant melanoma, lung cancer, liver cancer, breast cancer, pancreatic cancer or leukemia.
According to the scheme of the invention, the medicine containing the quinoid chalcone active ingredient for treating the tumor diseases can be a unit preparation, and is more beneficial to use in clinical treatment. The unit preparation of the drug may contain 1-5mg of the quinoid chalcone compound as an active ingredient, depending on the tumor type and the condition of the patient. The dosage form of the pharmaceutical composition can be injection dosage form or oral dosage form, etc.
According to the scheme of the invention, the medicine containing the quinoid chalcone component can be used for single medicine treatment and can also be combined with the current clinical antitumor medicines, for example, the medicine for treating the tumor diseases can also be a preparation combination comprising interferon and the quinoid chalcone compound.
It is also contemplated that the agent for treating neoplastic disease may be in the form of a combination of formulations in a unit formulation. May be a quinoid chalcone compound comprising 1-5mg as an active ingredient and a tumor therapeutically effective amount of interferon. In one embodiment, the unit dosage of the drug in the form of the combination of formulations may comprise 1 × 107～4×107U, or the dose is determined according to the tumor type and the course of the patient.
In still another aspect of the present invention, there is provided a formulation combination of antitumor agents, wherein the therapeutically active ingredients comprise interferon and said quinoid chalcone compound.
The small molecule inhibitor used in the invention is currently used as an anti-inflammatory component, for example, CN201410220017.8 discloses that the small molecule inhibitor can be used for treating vascular and neuroinflammatory diseases and immune inflammatory diseases, but the research of the inventor shows that the small molecule inhibitor has a killing effect on tumor cells and further improves the synergistic effect on the treatment effect of malignant tumor diseases by combining with interferon anti-tumor drugs, which is unexpected.
As to the structure, physicochemical properties and preparation method of the quinoid chalcone compound, refer to Chinese patent application CN 201410220017.8. As an example, some suitable quinoid chalcone compounds are listed below:
the research of the inventor proves that the medicine prepared by taking the quinoid chalcone compound provided by the invention as an active ingredient can be used as a single medicine for treating tumor diseases, can achieve the effect of killing tumors at least partially, can be used together with clinical interferon antitumor medicines, and can effectively kill tumor cells in a dormant state, thereby providing possibility for thoroughly eliminating cancer cells in a patient body.
On the other hand, the quinoid chalcone compound (HB) provided by the invention is used as an active ingredient for preparing a medicine, is used for antitumor treatment, can be combined with other clinical antitumor medicines, for example, is combined with an antitumor vaccine, or is combined with a chemotherapeutic medicine, and the like, and can effectively kill dormant tumor cells, so that the possibility of thoroughly eliminating malignant cancer cells in a patient body is provided.
The quinoid chalcones of the present invention are commercially available or can be prepared synthetically by themselves in accordance with the relevant disclosure (e.g., the aforementioned patent disclosures), and the interferons IFN can be interferon beta and gamma, i.e., IFN-beta, IFN-gamma, currently used clinically.
In order to research and prove that the small-molecule inhibitor kills and inhibits the tumor cells in the dormant state, the inventor cultures the tumor cells by adopting 3D glue to obtain the tumor stem cells, and compared with the traditional method for separating the tumor stem cells, the stem cells obtained by the 3D glue culture technology have the characteristics of strong proliferation capacity, high drug resistance, strong tumor forming capacity, high far-end invasion and metastasis capacity and the like. Moreover, the inventor uses the administration research from human or mouse tumor cell experiments and tumor-bearing mouse models to show that the small molecular inhibitor not only has the effect of inhibiting and killing tumors, but also can remarkably reduce the expression of CD8+ T cell PD-1, has unexpected effect on inhibiting the Kyn-mediated PD-1 expression up-regulation and enhancing the killing of specific CD8+ T cells to corresponding tumor cells, and has reasonable expectation that the small molecular inhibitor can kill differentiated tumor cells and can kill tumor stem cells entering dormancy by combining with the existing clinical antitumor drug IFN or antitumor drug preparations such as antitumor vaccine and chemotherapeutic drug.
As mentioned above, in the anti-tumor single drug and anti-tumor drug preparation combination provided by the invention, the small molecule inhibitor HB single drug and Interferon (IFN) can be unit preparations. The unit preparation is a preparation which meets the requirement of effective components required by one-time administration, such as a unit (needle) injection, and can also be a preparation which can meet the dosage by taking one tablet (granule) or pill and the like as single medicine. The amount of drug required for a single administration to a patient can conveniently be calculated by multiplying the weight of the patient by the unit weight dose required for a single administration to the patient. For example, in the manufacture of a medicament, which is generally considered to be 50-70kg in weight of an adult human, the amount may be initially determined by equivalent dose conversion between the unit weight doses of the experimental animal and the human. For example, the determination can be made by referring to the guidelines proposed by the drug administration such as FDA and SFDA (Huang-Ji Han, et al, "equivalent dose conversion between animals and humans in pharmacological tests", Chinese clinical pharmacology and therapeutics, 2004 Sep; 9(9): 1069-. In the embodiment of the present invention, the dose conversion between human and mouse can be performed by using a conversion coefficient of the body surface area between human and mouse of 0.0026.
In an embodiment of the invention, the small molecule inhibitor HB is administered at 5mg/kg body weight of the mouse, and IFN- β is 5X 10 for a mouse weighing 20g4-1×105U amount is administered. For example, the HB inhibitors are formulated as solutions in water or pharmaceutical solvents and administered to mice at the designed dose or intratumorally, whereas IFN- β is typically administered intratumorally.
Further, based on the results of experiments on mice, which were converted to a design for administration to humans in a normal weight range (e.g., 50-70kg body weight), HB unit formulations containing 1-5mgg as the active ingredient and IFN unit formulations containing 1X 107～4×107U IFN. When the composition is prepared into a preparation combination, the content of HB and interferon also meet the dosage.
The administration mode of the combination of the anti-tumor medicament and the medicinal preparation provided by the invention is as follows: administering a drug comprising the HB inhibitor or the antineoplastic drug formulation combination to a tumor-bearing individual (which may be a human or a mammal) in need of treatment, e.g., an oral (gavage) or an injectable (intravenous, injectable, intraperitoneal, or intratumoral) HB drug at the time of monotherapy; when the combination is used, IFN is injected intravenously, intraperitoneally or intratumorally, and HB medicines are combined for oral administration (intragastrically) or injection (intravenous injection, intraperitoneal injection or intratumoral injection).
The drug containing HB as an active ingredient used in the present invention may be any pharmaceutical composition or preparation prepared according to pharmaceutical techniques, and the content of HB as an active ingredient may be, for example, 0.1 to 95% by weight, and may be formulated into general preparations, sustained-release preparations, controlled-release preparations, targeted preparations and various microparticle drug delivery systems. The administration can be in unit dosage form, and the administration route can be in vivo administration or topical administration, such as intestinal or parenteral administration, e.g., oral, intramuscular, subcutaneous, nasal, oral mucosal, dermal, peritoneal, or rectal administration; can be administered by injection, including intravenous injection, intramuscular injection, subcutaneous injection, intradermal injection, and acupoint injection; can also be liquid dosage form, solid dosage form, such as true solution, colloid, microparticle, emulsion, and suspension; other dosage forms such as tablet, capsule, dripping pill, aerosol, pill, powder, solution, suspension, emulsion, granule, suppository, lyophilized powder for injection, etc.
The present invention also provides a method for treating or preventing tumors, which comprises the process of administering the antitumor drug or the antitumor drug preparation combination to an individual having tumors or an individual having a tendency to develop tumors (which may be a human or a mammal). For example, in monotherapy, HB drugs are administered orally (gavage) or by injection (intravenous, intraperitoneal or intratumoral); in the case of combined administration, administration of IFN (intravenous, intraperitoneal or intratumoral injection), oral administration (intragastric administration) or injection (intravenous, intraperitoneal or intratumoral injection) is combined with administration of HB.
According to the scheme of the invention, the therapeutic administration dosage of the quinoid chalcone compound medicine and the interferon can be controlled according to needs, and recommended unit preparations with different dosages can be prepared, so that the administration to tumor patients at different stages is convenient.
When HB is administered in combination with interferon, the order of administration is not particularly limited, e.g., simultaneous administration, or sequential administration of two agents is possible, with generally not too large an interval between administration, e.g., administration may be completed within 30 minutes to 60 minutes, within 1-12 hours, or within 1-2 days, or administration of each agent according to a separate administration schedule or cycle as prescribed.
Interferon (e.g., IFN- β or IFN- γ) is a cytokine secreted by fibroblasts, has antiviral, innate immune response modulating and antitumor properties, and is a common antitumor biological factor. In the research of the applicant, it is found that interferon has a relatively good anti-tumor effect in clinical treatment application, but a certain amount of treated patients can have tumor recurrence or even metastasis, because IFN-beta or IFN-gamma used alone can only kill part of tumor cells in a differentiated state, and the non-killed tumor cells have stem cell characteristics and enter a dormant state, which is the existence of the dormant tumor cells, so that clinical tumor patients are exposed to the risk of tumor recurrence or metastasis after a period of time (several years). The applicant finds through a large number of experimental researches that the drug taking the quinoid chalcone of which the alpha ring has isoamylene as an active ingredient can kill tumor cells by itself and is combined with IFN-beta or IFN-gamma for administration, so that the aim of killing dormant tumor cells can be fulfilled while differentiated tumor cells are killed, and the dosage of a single drug is reduced while the synergistic effect is remarkably improved.
The antitumor pharmaceutical preparation combination provided by the present invention may further be a combination of the quinoid chalcone compound and a clinical antitumor drug as active ingredients, such as a clinically used antitumor vaccine, various chemotherapeutic drugs, and a drug in which the quinoid chalcone compound of the present invention is given as an active ingredient in combination in the treatment of a diseased individual, and the dosage form, administration dose, administration route and administration mode of the drug are referred to above.
In summary, the scheme of the invention has the following effects:
1. the antitumor drug preparation prepared by utilizing the quinoid chalcone (such as HB-2 and structural analogues thereof in the embodiment) can be used alone to kill tumor cells partially, and can be used together with interferon beta or used together with other clinical antitumor drugs, so that the antitumor drug preparation not only can kill differentiated tumor cells, but also can kill tumor cells entering dormancy, and has the effect of completely eliminating the tumor cells.
2. The anti-tumor medicinal preparation composition can not only realize the effect of killing tumors by enhancing the systemic immune response, but also generate the direct killing effect on the tumors without adjusting the systemic immune response, thereby having the advantages of high safety and no toxic or side effect.
Drawings
FIG. 1 shows the effect of small molecule inhibitor HB on inducing apoptosis in solid tumor and leukemic tumor regenerative cells in 3D culture.
In the figure, A-F show the effect of the quinoid chalcone structural analogues DMF and HB-2 on inducing apoptosis of A375, A549, HepG2, MCF-7, PANC-1 and HL-60 tumor regenerative cells in 3D culture, respectively.
FIG. 2 shows the inhibition of the growth of the small molecule inhibitor HB in solid tumors and leukemia.
In the figure, A-F respectively show the treatment effect of the quinoid chalcone structural analogues such as DMF, HB-2 and the like on A375, A549, HepG2, MCF-7, PANC-1 and HL-60 tumor-bearing mice.
FIG. 3 shows the effect of HB-2 in combination with IFN- β on apoptosis in tumor regenerative cells cultured in 3D.
In the figure:
a is that IFN beta and HB-2 are used together to process human malignant melanoma cells A375 under the 3D culture condition, the apoptosis percentage is detected;
b is human lung cancer cell A549 processed under 3D culture condition by combining IFN beta and HB-2, and the apoptosis percentage is detected;
c, IFN beta and HB-2 are used together to process the human liver cancer cell HepG2 under the 3D culture condition, and the apoptosis percentage is detected;
d is that IFN beta and HB-2 are used together to process human breast cancer cell MCF-7 under the 3D culture condition, and the apoptosis percentage is detected;
e is that IFN beta and HB-2 are used together to process human pancreatic cancer cells PANC-1 under the 3D culture condition, and the apoptosis percentage is detected;
f, adopting IFN beta and HB-2 to process human leukemia cell HL-60 under the 3D culture condition, and detecting the apoptosis percentage.
FIG. 4 is a graph showing the effect of HB-2 in combination with IFN β on the growth of solid tumor bearing mice.
The effect of DMF, HB-2, IFN- β and the combination of IFN- β and HB-2 in reducing the tumor weight of mouse melanoma B16 is shown.
FIG. 5 shows HB-2 vs. in vitro cultured mouse CD8+Influence of expression of PD-1 by T cells.
FIG. 6 shows HB-2 combination specificity CD8+Effect of T cell adoptive on tumor growth in tumor-bearing mice.
Detailed Description
The following examples are provided to further illustrate the embodiments and effects of the present invention, but should not be construed as limiting the scope of the invention.
Tumor cells, drugs and experimental animals used in the following experimental runs:
the OVA-B16 tumor cell line, the B16 mouse melanoma cell line (also called B16 tumor cell line), the A375 human melanoma cell line, the A549 human lung cancer cell line, the HepG2 human liver cancer cell line, the MCF-7 human breast cancer cell line, the PANC-1 human pancreatic cancer cell line and the HL-60 human leukemia cell line can be purchased from American ATCC center or Beijing Cogeneration institute of medicine basic research institute cell center.
The HL-60 cells marked by the luciferase are monoclonal cells which are screened out after the HL-60 cells are transfected with luciferase reporter gene plasmids and stably express the luciferase genes.
Experimental animals: healthy female Balb/C mice, C57BL/C mice, or female NOD-SCID mice, 4-6 weeks old, were purchased from the institute of Chinese medical sciences and institutional animal centers, and the animal experiments were approved by the animal ethics committee of Chinese medical sciences.
DMF (dimethylformamide for short) is purchased from SIGMA company in America and is prepared into solution with corresponding concentration or dosage according to the requirement of experimental design;
human or mouse IFN-beta, cytokine from PeproTech company, make solution of corresponding concentration or dosage according to the experimental design requirement;
the micromolecule inhibitors HB-2, HB-3, HB-7, HB-19, HB-25, HB-31, HB-33 and HB-46 are products synthesized by Chinese medical academy of sciences, can also be synthesized by self according to the method disclosed by Chinese patent application CN201410220017.8, are quinoid chalcone compounds (called small molecule inhibitors HB for short) with isopentenyl group on the A ring, have the following specific structure, and can be prepared into solutions with corresponding concentrations or dosages according to the requirements of experimental design.
3D fibrin soft gel (3D gel), RPMI-1640 medium, PBS are all commercially available.
The statistical results of the experimental data in the following examples are shown in the corresponding figures, where the symbols "+", and "+" represent the statistical differences P <0.05, P <0.01, and P <0.001, respectively, of the results compared to the control results.
A first part: single-use effect detection of small molecule inhibitor HB on solid tumor and leukemia
Example 1: the quinoid chalcone compound can effectively induce apoptosis of solid tumor and leukemia cells under 3D culture conditions by single drug use.
1. Experimental procedure
A375, A549, HepG2, MCF-7, PANC-1 and HL-60 cells are cultured by adopting A3D glue technology, the number of the cells is about 10000 per hole, the cell state is observed after 2 days, and under the good condition, each cultured cell is respectively added with medicine according to the following groups:
control group (control): a common medium (e.g., RPMI-1640 medium) supplemented with glutamine;
experimental groups: glutamine-supplemented normal medium (e.g., RPMI-1640 medium) was supplemented with 10. mu. mol/ml of HB-2, HB-3, HB-7, HB-19, HB-25, HB-31, HB-33, HB-46, and DMF as positive controls.
When the medicine is added, the medicine and the culture medium are uniformly mixed and then added into each group of experimental cells, the beginning of adding the medicine is recorded as day 0, and the apoptosis detection is carried out on the cells in the 3D gel within 96 hours (day 4).
2. Results of the experiment
The statistical results of the groups are shown in fig. 1, and the results show that the drugs of all experimental groups (including positive control DMF) can induce apoptosis of solid tumor and leukemia cells to different degrees, and have significant differences compared with the control group, wherein the effect of inducing apoptosis of solid tumor and leukemia cells by most inhibitors represented by HB-2 is more prominent and is significantly better than that of the positive control DMF.
Example 2: the small molecule inhibitor HB-2 and the structural analogue thereof can inhibit the in vivo growth of solid tumors and leukemia.
1. Experimental procedure
(1) Construction of a375 tumor-bearing mice: subcutaneous inoculation of 1X 10 of NON-SCID mice5A375 cells, when tumor body grows to 5mm x 5mm, dividing tumor-bearing mice into 10 groups with 7 mice in each group randomly, and giving the tumor-bearing mice the following different treatment modes:
control group (control): gavage to PBS;
positive control group: DMF (10mg/kg) was gavaged 1 time every two days.
Experimental group (8 groups): each group is respectively administered with HB-2, HB-3, HB-7, HB-19, HB-25, HB-31, HB-33, HB-46 by intragastric administration, 10mg/kg body weight is administered once every two days;
after mice were sacrificed on day 21, subcutaneous tumors were detached, and the tumor weights of each group were weighed and counted.
(2) Construction of a549 tumor-bearing mice: subcutaneous inoculation of 1X 10 of NON-SCID mice5A549 cells, when tumor body grows to 5mm x 5mm, dividing tumor-bearing mice into 10 groups at random, each group comprises 7, and administering the tumor-bearing mice with the following different treatment modes:
control group (control): gavage to PBS;
positive control group: DMF (10mg/kg body weight) was administered by gavage 1 time every two days.
Experimental group (8 groups): intragastrically administering HB-2, HB-3, HB-7, HB-19, HB-25, HB-31, HB-33, HB-46, 10mg/kg body weight once every two days;
on day 28, the mice were sacrificed and subcutaneous tumors were dissected off, and each group was weighed and counted.
(3) Construction of HepG2 tumor-bearing mice: subcutaneous inoculation of 2X 10 of NON-SCID mice6HepG2 cells, when tumor growth to 5mm x 5mm, tumor-bearing mice equal number of random divided into 10 groups of 7, tumor-bearing mice given the following different treatment:
control group (control): gavage to PBS;
positive control group: DMF (10mg/kg body weight) was administered by gavage 1 time every two days.
Experimental group (8 groups): feeding HB-2, HB-3, HB-7, HB-19, HB-25, HB-31, HB-33 and HB-46 per kg of body weight per intragastric group once every two days;
after mice were sacrificed on day 42, subcutaneous tumors were detached, and the tumor weights of each group were weighed and counted.
(4) Construction of MCF-7 tumor-bearing mice: subcutaneous inoculation of 2X 10 of NON-SCID mice6And (3) dividing the MCF-7 cells into 10 groups with 7 cells in each group in equal amount when tumor bodies grow to 5mm x 5mm, and giving the tumor-bearing mice the following different treatment modes:
control group (control): gavage to PBS;
positive control group: DMF (10mg/kg body weight) was administered by gavage 1 time every two days.
Experimental group (8 groups): intragastrically administering HB-2, HB-3, HB-7, HB-19, HB-25, HB-31, HB-33, HB-46, 10mg/kg body weight once every two days;
after mice were sacrificed on day 56, subcutaneous tumors were detached, and the tumor weights of each group were weighed and counted.
(5) Construction of PANC-1 tumor-bearing mice: subcutaneous inoculation of 5X 10 mice with NON-SCID mice6The PANC-1 cells, when the tumor body grows to 3mm x 3mm, dividing the tumor-bearing mice into 10 groups at random, each group comprises 7 mice, and the tumor-bearing mice are given the following different treatment modes:
control group (control): gavage to PBS;
positive control group: DMF (10mg/kg body weight) was administered by gavage 1 time every two days.
Experimental group (8 groups): intragastrically administering HB-2, HB-3, HB-7, HB-19, HB-25, HB-31, HB-33, HB-46, 10mg/kg body weight once every two days;
after mice were sacrificed on day 56, subcutaneous tumors were detached, and the tumor weights of each group were weighed and counted.
(6) Construction of HL-60 leukemia mouse model: NON-SCID mouse tail vein injection 1X 106The number of mice, equally numbered, was randomly divided into 10 groups of 7 mice each, and 7 days later, the mice were given the following different treatment regimens:
control group (control): gavage to PBS;
positive control group: DMF (10mg/kg body weight) was administered by gavage 1 time every two days.
Experimental group (8 groups): intragastrically administering HB-2, HB-3, HB-7, HB-19, HB-25, HB-31, HB-33, HB-46, 10mg/kg body weight once every two days;
on day 42, the mice were anesthetized and the distribution of leukemia cells in vivo was detected and counted using a small animal in vivo imaging system.
2. Results of the experiment
Statistics of tumor growth for each group of tumor-bearing mice are shown in FIGS. 2A-F.
As can be seen, compared with the control group, all HB single drugs and DMF single drugs can delay and inhibit the growth of solid tumors A375, A549, HepG2, MCF-7, PANC-1 and leukemia cell HL-60 to different degrees, but the effect of HB-2 is more obvious compared with DMF.
The micromolecule inhibitors HB of the invention have different degrees of inhibition effect on the growth of solid tumors and leukemia cells in vivo, and can effectively inhibit the malignant growth of the solid tumors and leukemia cells.
A second part: detection of Effect of Small molecule inhibitors HB-2 and IFN beta on solid tumors and leukemia
Example 3: IFN-beta in combination with HB-2 significantly induced apoptosis in solid tumor cells and leukemia cells under 3D culture conditions.
1. Experimental procedure
Solid tumor cells A375, A549, HepG2, MCF-7, PANC-1 and leukemia cell HL-60 were cultured with 3D gel (24-well plate, cell density 8000/well), cell status was observed the next day, and when cell survival status was good, drug treatment was performed according to the following groups:
control 1 (control): common culture medium and drug solvent;
control group 2(IFN β): adding 6ng/ml mouse IFN beta or 10ng/ml human IFN beta into common culture medium;
experimental group 3 (HB-2): adding 10 mu mol/ml HB-2 into a common culture medium;
experimental group 4(IFN β + DMF): adding 6ng/ml mouse IFN beta or 10ng/ml human IFN beta and 20 mu mol/ml DMF into common culture medium;
experimental group 5 (IFN. beta. + HB-2): adding 6ng/ml mouse IFN beta or 10ng/ml human IFN beta and 10 mu mol/ml HB-2 into the common culture medium;
when the medicine is added, the medicine and the culture medium are uniformly mixed and then added into each group of experimental cells, the cells are cultured for 48 hours, and the apoptosis condition is detected by adopting the flow cytometry.
Note: the experimental use of "murine IFN β" or "human IFN β" is determined by the nature of the tumor cells being cultured, and the following examples are the same.
2. Results of the experiment
The results of the apoptosis rate for each group are shown in a-F in fig. 3 in a control manner.
It can be seen that IFN beta and HB-2 have certain effect on killing solid tumor cells cultured by 3D gel alone, but compared with the combination of IFN beta and small molecule inhibitor, the killing ability of solid tumor cells can be obviously improved by using single drug, and the killing effect of IFN beta and HB-2 on tumors is more obviously higher than that of IFN beta alone, HB-2 alone and IFN beta and DMF.
Example 4: the combined application of IFN-beta and HB-2 can inhibit the tumor growth of the solid tumor-bearing mice and prolong the life cycle of the tumor-bearing mice.
2. Experiment on treatment of tumors in C57BL/C mice with IFN- β and HB-2 in combination
1) Experimental procedure
Constructing tumor-bearing mice: b16 melanoma cells were inoculated into 4-6 week-old C57BL/C mice (inoculum size 1X 10)5Individual cell), 35 mice were randomly divided into 5 groups, the weight of the mice was about 20g, and tumor formation was observed for about 7 days; when the tumor body grows to 5mm x 5mm, the following different treatment modes are respectively given to the tumor-bearing mice according to the group:
control 1 (control): intratumoral injection of PBS only;
experimental group 2 (IFN-. beta.): IFN-beta alone treatment of tumor-bearing mice, each mouse intratumorally injected 1X 105U IFN-beta, once every two days for 10 days;
experimental group 3 (IFN-. beta./DMF): mice were given IFN-. beta.and DMF separately (i.e., in combination with IFN-. beta.and DMF) in the same manner as in experiment group 4, and DMF was administered in the same amount as HB-2.
Experimental group 4 (HB-2): the HB-2 is used for singly treating tumor-bearing mice, the dosage of the HB-2 is every two days, and the administration is carried out according to the weight of the mice of 5mg/kg, the mode is intratumoral injection, and the total injection lasts for 10 days;
experimental group 5 (IFN-. beta. + HB-2): mice were given IFN- β and HB-2, respectively, with the IFN- β being administered as follows: intratumoral administration of 1X 105U IFN-beta is injected intratumorally once every two days for 10 days; the HB-2 administration mode is as follows: injecting HB-2 intratumorally every two days (the dosage is 5mg/kg of the body weight of a mouse), and administering for 10 days; the order of administration of IFN- β and HB-2 is not limited, and each can be administered simultaneously or sequentially;
tumors were weighed after sacrifice on day 28.
2) Results of the experiment
The tumor status of each group of mice is shown in FIG. 4:
it can be seen that, although the treatment of the experimental group has a significant advantage in inhibiting the growth of melanoma, compared to the control group, IFN- β in combination with DMF and IFN- β in combination with HB-2 showed a more significant inhibition of melanoma tumor growth, and HB-2 showed a better effect than DMF.
And a third part: small molecule inhibitor HB-2 to CD8+Effect of T cell PD-1 expression
Example 5: HB-2 can reduce mouse CD8+T cellsExpression of PD-1 enhances the killing function of T cells.
1. Detection of the Effect of HB-2 on the expression of activated mouse CD8+ T cell PD-1
1) Experimental procedure
And (3) obtaining mouse spleen CD8+ T cells by magnetic bead sorting, inoculating the mouse spleen CD8+ T cells in a U-shaped bottom 96-well plate (10 ten thousand/well), adding anti-CD 3/CD28 magnetic beads to activate the T cells, and adding 10ng/mL mouse IL-2 and 50nM beta mercaptoethanol into RPMI-1640 culture medium. Activated T cells were dosed in the following groups:
control 1 (PBS): adding PBS into the culture medium;
experimental group 2 (Kyn): adding Kyn 200 mu mol/ml into the culture medium;
experimental group 3(Kyn + DMF): kyn 200 mu mol/ml and DMF 10 mu mol/ml are added into the culture medium at the same time;
experimental group 4(Kyn + HB-2): kyn 200 mu mol/ml and HB-210 mu mol/ml are added into the culture medium at the same time;
and culturing for 48 hours after adding the medicine, and detecting the PD-1 expression level of each group of T cells by adopting flow cytometry.
2) Results of the experiment
Statistics of the results of detecting the expression level of T cell PD-1 in each group are shown in FIG. 5, wherein the statistics of the control group 1(PBS), the experimental group 2(Kyn), the experimental group 3(Kyn + DMF) and the experimental group 4(Kyn + HB-2) are shown in the column diagram from left to right.
It can be seen that HB-2 can more effectively inhibit Kyn (kynurenine ) mediated CD8+ T cell PD-1 up-regulation than DMF, and the results have significant differences, which indicates that HB-2 can more effectively inhibit small molecule receptors than DMF.
Example 6: the HB-2 combined with the specific CD8+ T cell has obvious effect of adoptively treating the solid tumor of the mouse.
1. Experimental procedure
Constructing tumor-bearing mice: OVA-B16 melanoma cells were inoculated into 4-6 weeks C57BL/6 mice (inoculum size 1X 10)5Individual cell), the mice were randomly divided into 7 groups of 5 mice each, the mice weighed about 20g, and tumor formation was observed for about 7 days; when the tumor body grows to 5mm x 5mm, the following different treatment modes are respectively given to the tumor-bearing mice according to the group:
control 1 (PBS): intratumoral injection of PBS only;
experimental group 2(CD8+ T): OT-1 mouse CD8+ T cell vein adoptive tumor-bearing mouse, once 5 days, 3 times in total, 4x 10 times for adoptive each time6CD8+ T cells;
experimental group 3 (DMF): the DMF single drug is injected into the tumor-bearing mice once every two days for 10 times, and the dosage of each time is 5mg/kg of the body weight of the tumor-bearing mice;
experimental group 4 (HB-2): HB-2 single-drug intratumoral injection tumor-bearing mice is carried out once every two days for 10 times, and the dosage of each time is 5mg/kg of the body weight of the tumor-bearing mice;
experimental group 5(CD8+ T + DMF): OT-1 mouse CD8+ T cell intravenous adoptive combined DMF intratumoral injection, the method and the dosage are the same as those of experimental groups 2 and 3, and the adoptive and administration operation respectively follow the set administration period;
experimental group 6(CD8+ T + HB-2): OT-1 mouse CD8+ T cell intravenous adoptive combined HB-2 intratumoral injection, the method and the dosage are the same as those of experimental groups 2 and 4, and the adoptive and administration operations respectively follow the set administration period;
tumor volume change was calculated by daily measurement of tumor body size at the beginning of the dosing experiment.
2. Results of the experiment
The statistics of tumor body variation of the experimental mice of each group are shown in FIG. 6.
It can be seen that the single CD8+ T cell adoptive therapy or HB-2 single drug has a certain therapeutic effect on the solid tumors of tumor-bearing mice, but the curative effect of the CD8+ T cell adoptive therapy and the small molecule inhibitor therapy is relatively better, and more remarkably, the curative effect of the CD8+ T cell adoptive therapy and the HB-2 combination is obviously better than that of the CD8+ T cell adoptive therapy and the DMF.
The HB-2 can be shown to be more effective than DMF in inhibiting CD8+ T cell small molecule receptor, down regulating the expression of PD-1 inhibitory receptor and enhancing the in vivo killing function of CD8+ T cell.
The above is only a partial experimental result in a large number of studies by the applicant, but it can be stated that:
the small molecule inhibitor (such as HB-2 and the structural analogue thereof used in the experiment) can effectively inhibit the malignant growth of solid tumors and leukemia when used alone;
when the small molecular inhibitor HB (such as HB-2) and IFN beta are jointly used, a better treatment effect can be obtained on the premise of greatly reducing the dosage of a single medicine, which indicates that the small molecular inhibitor HB and the IFN beta are cooperated, so that the effective treatment dosage can be reduced, the curative effect is obviously enhanced, and the effect of enhancing killing effect and delaying the growth of tumor bodies can be achieved in clinical use.
Compared with DMF, the small molecule inhibitor (such as HB-2 and structural analogues thereof used in experiments) can more obviously reduce the expression of CD8+ T cell PD-1, more inhibit Kyn mediated PD-1 up-regulation and enhance the killing of specific CD8+ T cells to corresponding tumor cells.
Claims (12)
1. The application of the quinoid chalcone compound in preparing the medicine for treating tumor diseases is disclosed, wherein the quinoid chalcone compound has the following structure:
the tumor disease is malignant melanoma, lung cancer, liver cancer, breast cancer, pancreatic cancer or leukemia.
2. The use of claim 1, wherein the medicament for treating tumor diseases comprises an injection dosage form and an oral dosage form.
3. The use according to any one of claims 1-2, wherein the medicament for the treatment of a tumor disease is a unit formulation.
4. The use according to claim 3, wherein said unit formulation of the medicament comprises 1 to 5mg of said quinoid chalcone compound as an active ingredient.
5. The use according to claim 1, wherein the medicament for treating tumor diseases is a combination of a formulation comprising interferon and the quinoid chalcone compound.
6. The use according to claim 5, wherein the medicament for the treatment of a tumor disease is a unit formulation.
7. The use according to claim 6, wherein said unit preparation of a medicament comprises 1 to 5mg of said quinoid chalcone compound as an active ingredient and a tumor therapeutically effective amount of interferon.
8. The use of claim 7, wherein the unit dosage form comprises 1 x 10 of the drug7～4×107U interferon.
9. The use according to claim 1 or 2, wherein the medicament for treating tumor diseases is a formulation comprising a clinical antitumor drug in combination with the quinoid chalcone compound.
10. The use according to claim 9, wherein the clinical antineoplastic drug comprises an antineoplastic vaccine or a chemotherapeutic drug.
11. An antitumor agent preparation wherein a therapeutically active ingredient comprises the quinoid chalcone compound according to claim 1 and a clinical antitumor agent.
12. The formulation of claim 11, wherein the clinical antineoplastic drug comprises an antineoplastic vaccine or a chemotherapeutic drug.
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