CN117838691A

CN117838691A - Application of betrofloxacin or pharmaceutically acceptable salt thereof in preparation of antitumor or interferon activating drugs

Info

Publication number: CN117838691A
Application number: CN202410251613.6A
Authority: CN
Inventors: 刘冰; 游富平; 胡诗雨; 马妮; 龙瑶; 席倩倩; 赵斌强; 谢云飞; 雒钰杰
Original assignee: Beijing Tianyu Hengtai Technology Co ltd; Peking University
Current assignee: Beijing Tianyu Hengtai Technology Co ltd; Peking University
Priority date: 2024-03-06
Filing date: 2024-03-06
Publication date: 2024-04-09
Anticipated expiration: 2044-03-06
Also published as: CN117838691B

Abstract

The invention discloses an application of betrexaban or pharmaceutically acceptable salt thereof in preparing anti-tumor or interferon activating drugs. According to the invention, by screening the small molecular targeted drug capable of activating natural immunity and researching the functions of activating immunity and resisting tumors, the small molecular drug Betraxaban or pharmaceutically acceptable salts, solvates, hydrates, complexes, prodrugs, isomers, analogues, derivatives or metabolites thereof is found to have obvious effect of activating I-type interferon IFN. In addition, the present invention verifies that betrofloxacin or a pharmaceutically acceptable salt, solvate, hydrate, complex, prodrug, isomer, analogue, derivative or metabolite thereof enhances antitumor immunity by activating immune cells.

Description

Application of betrofloxacin or pharmaceutically acceptable salt thereof in preparation of antitumor or interferon activating drugs

Technical Field

The invention belongs to the field of biological medicine, and in particular relates to application of betrexaban or pharmaceutically acceptable salt thereof in preparing an anti-tumor or interferon activating medicine.

Background

Tumor immunotherapy is considered to be one of the most successful approaches in the field of cancer treatment in recent years. Tumor immunotherapy is the first in ten scientific breakthroughs in journal of science 2013. Tumor immunotherapy is largely divided into two categories: cellular immunotherapy and immune checkpoint inhibitor therapy. Among them, the appearance of Immune Checkpoint Inhibitors (ICIs) rapidly alters the therapeutic pattern of a variety of cancers. In the last decade, starting from the first approval of CTLA-4 inhibitors in metastatic melanoma in 2011, PD- (L) 1 inhibitors have now become a routine means of treatment for over 20 different cancer indications. This significant shift is largely driven by the unprecedented antitumor effects of ICIs, which can last for years even if the patient is not subsequently treated continuously.

However, most other cancers respond only 10-25% to single drug PD-1 blockade, except for patients with certain tumors (e.g., melanoma, merck cells, hodgkin's lymphoma, MSI-H tumors) that respond more frequently (40% -70%) to single drug PD-1 blockade. Also, even in patients who initially respond to ICIs, the disease may eventually progress. This has to mention the problem of ICIS resistance. The resistance of ICIs falls into two broad categories: 1) Primary drug resistance: generally refers to patients who do not respond at all, and whose condition is rapid or eventually progressing; 2) Acquired resistance: refers to the patient's response to an initial treatment for a period of time, but eventually also to disease progression.

Immune checkpoint inhibitor therapy is a block against the inhibitory function of T cells in the tumor microenvironment. Patients who do not respond to ICIs treatment are mostly because T cells do not enter the tumor microenvironment, and such tumors are also known as immune privileged tumors or as cold tumors. Natural immunity is effective in activating the immune system, including activating and recruiting T cells, inducing immune cells into the tumor microenvironment, turning a cold tumor into a hot tumor, etc.

Interferon (IFN) is a cytokine protein that activates immune cells (e.g., macrophages and natural killer cells) in humans and enhances host defenses. There are four major pathways that have been found to induce the production of type I interferons: the DNA virus activates the second messenger cGAMP (cyclic GMP-AMP) induction pathway; RNA viruses activate RLRs induction pathways; TLR3 and TLR4 (Toll-ikexepids) activate the adaptor protein tri-induction pathway; TLR7/TLR8 and TLR9 activate the transcription factor IRF 7-induced pathway. After Interferon production, ISGF3 is activated by binding to an Interferon receptor, so that it binds to an Interferon stimulation response element ISRE in the ISG promoter, and then transcription of a large number of Interferon-stimulated genes (ISGs) is induced and expression of the ISGs is regulated. The generated ISG product (ISG) can enhance acquired immunity, disturb tumor growth, and regulate organism immune system. Most cells respond to type I interferons. The induction of expressed ISGs assumes the main biological effects of IFN.

Therefore, how to effectively induce or activate interferon and anti-tumor is a problem to be solved.

Disclosure of Invention

In order to solve at least part of the problems in the prior art, the invention screens the small molecule targeting drugs capable of activating natural immunity and researches the functions of activating immunity and resisting tumor. The inventor discovers that the small molecular medicine betrofloxacin or pharmaceutically acceptable salt, solvate, hydrate, complex, prodrug, isomer, analogue, derivative or metabolite thereof has obvious effect of activating the type I interferon IFN through medicine screening. Specifically, the present invention includes the following.

In a first aspect of the invention there is provided the use of betrexaban or a pharmaceutically acceptable salt, solvate, hydrate, complex, prodrug, isomer, analogue, derivative or metabolite thereof for the manufacture of an antitumor medicament.

In certain embodiments, the use according to the invention, wherein the anti-tumor comprises one of the following:

(1) Inhibit proliferation or growth of tumor cells;

(2) Reducing or lowering tumor volume and/or weight;

(3) Inhibit metastasis of tumors;

(4) Increasing or increasing the number of immune cells, inducing the immune cells to enter the tumor microenvironment;

(5) Promoting or improving the tumor killing function of immune cells;

(6) Activating the production of type I interferon.

In certain embodiments, the use according to the invention, wherein the immune cells comprise one or more of tumor infiltrating lymphocytes, macrophages, natural killer cells, cytokine-induced killer cells, cytotoxic T lymphocytes, dendritic cells in combination with cytokine-induced killer cells, chimeric antigen receptor T cells, and T cell receptor chimeric T cells.

In a second aspect of the invention there is provided the use of betrexaban or a pharmaceutically acceptable salt, solvate, hydrate, complex, prodrug, isomer, analogue, derivative or metabolite thereof in the manufacture of a medicament for activating or promoting a type I interferon.

In certain embodiments, the use according to the invention, wherein said activating or facilitating comprises one of the following:

(a) Activating the target points of the actions of immune and I-type interferon activation signal paths;

(b) Activating or promoting gene expression of ISG family members;

(c) Promote or enhance expression of interferon inducible genes.

In certain embodiments, the use according to the invention, wherein the target comprises a TBK1 target.

In certain embodiments, the use according to the invention, wherein the gene of the ISG family member comprises an ISG15 and/or IFIT gene.

In certain embodiments, the use according to the invention, wherein the interferon inducible gene comprises GO 0006954, WP5095 and/or GO 0032649.

In a third aspect of the invention there is provided the use of betrexaban or a pharmaceutically acceptable salt, solvate, hydrate, complex, prodrug, isomer, analogue, derivative or metabolite thereof in combination with other therapeutic agents.

In certain embodiments, the use according to the invention, wherein the therapeutic agent comprises at least one of a chemotherapeutic agent, an immune checkpoint inhibitor, an anti-tumor drug, and an immunomodulator.

The invention takes the modified eukaryotic expression vector 2fTGH cells (human fibrosarcoma cells) of mammals as a research object, and ISRE-luciferases (Luciferase started by ISRE elements) are inserted into the multiple cloning sites of the eukaryotic expression vector, namely 2fTGH-ISRE-Luci cells, and the expression of Luciferase reporter genes and the activation of type I interferon in the 2f cells are detected and verified by using a microplate reader by using luciferin (luciferin) as a substrate through a chemiluminescent function.

Drawings

FIG. 1 illustrates the results of detection of luciferases after treatment of 2fTGH-ISRE-Luci cells with various drugs.

The four drug treatments 2f and the different times of action of the drugs in FIG. 2 are shown in the graph on the right of FIG. 2, where the left 5 columns represent 6h results and the right 5 columns represent 12h results.

FIG. 3 is a schematic representation of the detection of 2fTGH-ISRE-Luci cell luciferase and RT-qPCR by Betroxaban maleate in combination with TBK1 inhibitor, with the left column of each set being 6h detection and the right column being 24h detection in the left panel of FIG. 3.

FIG. 4 illustrates RT-qPCR detection of ISG15 and IFIT 1-3 mRNA levels after betrexaban maleate has been applied to 2f cells, wherein each column of each set is ISG15, IFIT1, IFIT2 and IFIT3 in order from left to right.

FIG. 5RNA-seq sequencing a list of genes up-regulated by more than 4-fold was selected.

Figure 6 illustrates an exemplary subcutaneous vaccination regimen of betrexaban maleate against antitumor activity of mouse pancreatic cancer cells.

FIG. 7 comparison of body weight and tumor size of two groups of mice 23 days after subcutaneous inoculation.

Fig. 8 illustrates tumor volume and weight data comparison for betrexaban maleate Intraperitoneal Injections group and mock group.

FIG. 9 spleen phenotyping of drug-treated and mock groups.

Detailed Description

Various exemplary embodiments of the invention will now be described in detail, which should not be considered as limiting the invention, but rather as more detailed descriptions of certain aspects, features and embodiments of the invention.

It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. In addition, for numerical ranges in the present invention, it is understood that the upper and lower limits of the ranges and each intermediate value therebetween are specifically disclosed. Every smaller range between any stated value or stated range, and any other stated value or intermediate value within the stated range, is also encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included or excluded in the range.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference for the purpose of disclosing and describing the methods and/or materials associated with the documents. In case of conflict with any incorporated document, the present specification will control.

Use of the same

The invention provides an application of betrexaban or pharmaceutically acceptable salt, solvate, hydrate, complex, prodrug, derivative or metabolite thereof in preparing antitumor or interferon activating drugs.

The term "pharmaceutically acceptable salts" as used herein refers to the organic and inorganic salts of the compound betrofloxacin of the invention. Pharmaceutically acceptable salts are well known in the art, for example, in literature: pharmaceutically acceptable salts are described in detail in S.M. berge et al, describe pharmaceutically acceptable salts in detail in J.pharmaceutical Sciences,66:1-19,1977. Pharmaceutically acceptable salts of non-toxic acids include, but are not limited to: inorganic acid salts such as hydrochloride, hydrobromide, phosphate, sulfate, perchlorate; organic acid salts such as acetates, oxalates, maleates, tartrates, citrates, succinates, malonates; or by other methods described in the book literature, such as ion exchange. Other pharmaceutically acceptable salts include: adipic acid salt, malic acid salt, 2-hydroxypropionate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate, borate, butyrate, camphorate, camphorsulfonate, cyclopentylpropionate, digluconate, dodecylsulfate, ethanesulfonate, formate, fumarate, glucoheptonate, glycerophosphate, gluconate, hemisulfate, heptanoate, caproate, hydroiodidate, 2-hydroxy-ethanesulfonate, lactobionic aldehyde, lactate, laurate, lauryl sulfate, malate, malonate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oleate, palmitate, pamoate, pectate, persulfate, 3-phenylpropionate, picrate, pivalate, propionate, stearate, thiocyanate, p-toluenesulfonate, undecanoate, valerate, and the like. The present invention also contemplates quaternary ammonium salts formed from any compound containing a group of N. The water-soluble or oil-soluble or dispersible product may be obtained by quaternization. Alkali or alkaline earth metals that may form salts include sodium, lithium, potassium, calcium, magnesium, and the like. Pharmaceutically acceptable salts further include suitable, non-toxic ammonium, quaternary ammonium salts and counter-ion forming amine cations such as halides, hydroxides, carboxylates, sulphates, phosphates, nitrates, C1-8 sulphonates, aromatic sulphonates and the like.

It is to be understood that when a compound or embodiment of the invention is shown as a particular salt, the invention includes the corresponding free base and other salts of the corresponding free base (including pharmaceutically acceptable salts of the corresponding free base).

The term "solvate" as used herein refers to an association or complex of one or more solvent molecules with a compound of the invention. Solvents that form solvates include, but are not limited to: water, isopropanol, ethanol, methanol, dimethyl sulfoxide, ethyl acetate, acetic acid, and aminoethanol.

The term "hydrate" as used herein refers to an association or complex of solvent molecules formed from water.

The term "isomer" as used herein includes enantiomeric, diastereomeric, and geometric (or conformational) isomeric forms of a given structure. For example, the invention includes R and S configuration, Z and E double bond isomers, Z and E conformational isomers, single stereochemical isomers and mixtures of enantiomers, diastereomers and geometric (or conformational) isomers of each asymmetric center. Unless otherwise indicated, the present invention includes all tautomeric forms of the disclosed structures of betrixaban.

The term "prodrug" as used herein, means a compound which is converted in vivo to a compound of formula (I) or formula (II):

formula (I);

formula (II).

The term "prodrug" means a pharmacologically inactive derivative of a parent drug molecule that requires spontaneous or enzymatic bioconversion in an organism to release the active drug. Prodrugs are variants or derivatives of the compounds of the invention which have groups that are cleavable under metabolic conditions. Prodrugs become compounds of the present invention that are pharmaceutically active in vivo when they undergo solvent degradation or undergo enzymatic degradation under physiological conditions. Prodrug forms generally offer the advantage of solubility, histocompatibility or delayed release in mammalian organisms (see Bundgard, design of Prodrugs (prodrug design), pp.7-9, 21-24, elsevier, amsterdam 1985 and Silverman, the Organic Chemistry of Drug Design and Drug Action (organic chemistry and pharmaceutical action of pharmaceutical design), pp.352-401, academic Press, san Diego, CA, 1992). Prodrugs known in the art include acid derivatives known to those skilled in the art, such as esters prepared by reacting a parent acid with a suitable alcohol, or amides prepared by reacting a parent acid compound with an amine, or with a basic group that reacts to form an acylated base derivative.

The term "metabolite" as used herein refers to the product of a particular compound or salt thereof obtained in vivo by metabolism. The metabolic products of a compound may be identified by techniques well known in the art, and its activity may be characterized by methods as known in the art. Such products may be obtained by oxidation, reduction, hydrolysis, amidization, deamination, esterification, degreasing, enzymatic cleavage, etc. of the administered compound. Accordingly, the present invention includes metabolites of compounds of formula (I) or (II), including metabolites produced by contacting a compound of formula (I) or (II) of the present invention with a mammal or cell line derived therefrom for a period of time sufficient.

In the present invention, the term "derivative" refers to a product derived from substitution of an atom or a functional group in the compound of formula (I) or (II) with other atoms or functional groups, and its derivative is not particularly limited, including but not limited to an esterified compound, an acylated compound or a metal complex of the compound of formula (I) or (II), and the like.

The invention also provides the use of betrixaban or a pharmaceutically acceptable salt, solvate, hydrate, complex, prodrug, isomer, analogue, derivative or metabolite thereof, in particular betrixaban maleate, in the manufacture of a medicament for the treatment, amelioration or inhibition of a tumour, preferably said tumour is insensitive to an immune checkpoint medicament. The immune checkpoint drug is preferably a CTLA-4 inhibitor, an anti-PD-1 antibody, an anti-PD-L1 antibody or the like.

In the context of the present invention, the insensitivity of a tumor to an immune checkpoint drug means that the tumor has a tumor inhibition rate of less than 50% when treated with a conventional dose of an immune checkpoint drug; preferably, the tumor inhibition rate, expressed as tumor volume inhibition rate TGI (%) and calculated as the length and width of the tumor, is lower than 40%, preferably lower than 30%, preferably lower than 20%, more preferably lower than 10%, when treated with an immune checkpoint drug at the lower dose of the conventional dose range.

In the present invention, examples of the anti-PD-1 antibody include, but are not limited to, for example, CD279, na Wu Liyou mab (nivolumab), pamglizumab (pembrolizumab), terlipressin Li Shan antibody, singal Li Shan antibody, kari Li Zhushan antibody, tirelimumab, and the like. Examples of such anti-PD-L1 antibodies include, but are not limited to, e.g., CD274, durvalumab, and atilizumab (atezolizumab), among others.

The invention also provides the use of the betrixaban or pharmaceutically acceptable salts, solvates, hydrates, complexes, prodrugs, isomers, analogues, derivatives or metabolites thereof, in particular the use of the betrixaban maleate in the manufacture of a medicament for enhancing the anti-tumor efficacy of an immune checkpoint medicament, wherein the immune checkpoint medicament is preferably a CTLA-4 inhibitor, an anti-PD-1 antibody, an anti-PD-L1 antibody or the like.

The invention also provides the use of betrixaban or a pharmaceutically acceptable salt, solvate, hydrate, complex, prodrug, isomer, analogue, derivative or metabolite thereof, in particular betrixaban maleate in combination with other therapeutic agents for the manufacture of a medicament for the anti-tumour and/or activation of type I interferon production. Other therapeutic agents include, but are not limited to, chemotherapeutic agents, immune checkpoint inhibitors, anti-tumor drugs, and immunomodulators. In some embodiments, examples of the additional therapeutic agent are selected from at least one of a TBK1 inhibitor, a PDK1 inhibitor, a CTLA-4 inhibitor, an anti-PD-1 antibody, and an anti-PD-L1 antibody.

The above-mentioned uses of the present invention are achieved by activating or promoting type I interferon, and thus, the present invention also provides the use of betrixaban or a pharmaceutically acceptable salt, solvate, hydrate, complex, prodrug, isomer, analogue, derivative or metabolite thereof, in particular betrixaban maleate, in the manufacture of a medicament for activating or promoting type I interferon.

Further, in the above uses, the medicament contains a therapeutically effective amount of betrixaban or a pharmaceutically acceptable salt, solvate, hydrate, complex, prodrug, isomer, analogue, derivative or metabolite thereof, in particular betrixaban maleate, and optionally a pharmaceutically acceptable excipient or carrier. The term "pharmaceutically acceptable excipient or carrier" as used herein refers to a pharmaceutically acceptable material, composition or vehicle, such as a liquid or solid filler, diluent, excipient, solvent or encapsulating material, and which participates in carrying or transporting an agent from one organ or part of the body to another organ or part of the body. Each excipient or carrier is "acceptable" in the sense of being compatible with the other ingredients of the formulation and not injuring the patient.

The mode of administration of the medicament of the present invention is not particularly limited, and representative modes of administration include, but are not limited to: oral, intratumoral, rectal, parenteral (intravenous, intramuscular or subcutaneous) and topical administration. Correspondingly, the medicine of the invention can be prepared into various clinically acceptable dosage forms, including oral dosage forms, injection dosage forms, local administration dosage forms or external dosage forms, and the like.

Preferably, the betrixaban or pharmaceutically acceptable salts, solvates, hydrates, complexes, prodrugs, isomers, analogues, derivatives or metabolites thereof of the invention may be used clinically alone or in combination with other therapeutic ingredients. The other therapeutic component is selected from at least one of a chemotherapeutic agent, an immune checkpoint inhibitor, an anti-tumor drug, and an immunomodulator. For convenient clinical use, the betrexaban or pharmaceutically acceptable salts, solvates, hydrates, complexes, prodrugs, isomers, analogs, derivatives or metabolites thereof of the invention can be combined with other therapeutic ingredients to prepare a combination medicament.

A therapeutically effective amount of the betrixaban or a pharmaceutically acceptable salt, solvate, hydrate, complex, prodrug, isomer, analogue, derivative or metabolite thereof, particularly betrixaban maleate, of the present invention may be administered to a subject, e.g. a mammal (e.g. a human), in need thereof.

The therapeutically effective amount as defined herein means an amount of the active compound (i.e. betrofloxacin or a pharmaceutically acceptable salt, solvate, hydrate, complex, prodrug, isomer, analogue, derivative or metabolite thereof, particularly betrofloxacin maleate) which is considered pharmaceutically effective for significantly ameliorating the condition without causing serious side effects. For a person of 60kg body weight, the daily dose of betrixaban or a pharmaceutically acceptable salt, solvate, hydrate, complex, prodrug, isomer, analogue, derivative or metabolite thereof is typically from 0.01 to 2000mg, preferably from 0.5 to 500mg or 1 to 500mg, or 0.5 to 250mg, or 0.5 to 200mg, or 0.5 to 150mg, or 0.5 to 100mg, or 0.5 to 50mg, or 0.5 to 40mg, or 0.5 to 30mg, most preferably 0.5 to 25mg. Exemplary effective administration doses are, for example, 0.5mg, 0.75mg, 0.95mg, 1mg, 1.25mg, 1.5mg, 1.75mg, 2mg, 2.5mg, 2.75mg, 3mg, 3.25mg, 3.5mg, 3.75mg, 4mg, 4.25mg, 4.5mg, 4.75mg, 5mg, 5.25mg, 5.5mg, 5.75mg, 6mg, 6.25mg, 6.5mg, 6.75mg, 7mg, 7.25mg, 7.5mg, 7.75mg, 8mg, 8.25mg, 8.5mg, 8.75mg, 9mg, 9.25mg, 9.5mg, 9.75mg, 10mg, 11mg, 12mg, 13mg, 14mg, 15mg, 16mg, 17mg, 18mg, 19mg, 20mg, 21mg, 22mg, 23mg, 24mg, 25mg. Preferably, the daily dose is calculated as betrixaban or a pharmaceutically acceptable salt, solvate, hydrate, complex, prodrug, isomer, analogue, derivative or metabolite thereof, in particular betrixaban maleate. The preparation can be administered once a day in a single dose, can be administered in multiple times per day, or can be used at intervals.

When administered in combination, other therapeutic agents, such as chemotherapeutic agents, immune checkpoint inhibitors, antitumor drugs and immunomodulators, e.g., TBK1 inhibitors, PDK1 inhibitors, CTLA-4 inhibitors, anti-PD-1 antibodies, anti-PD-L1 antibodies, etc., may be administered in amounts of 0.5mg/kg to 30mg/kg, preferably 1 to 20mg/kg, depending on the particular antibody species and type of cancer and stage of development; for example, for a person of 60kg body weight, the dose may generally be 1mg to 1500mg, for example 50mg to 1200mg, or 100mg to 800mg,150mg to 600mg or 200mg to 500mg per administration; exemplary doses per administration are, for example, 50mg, 100mg, 120mg, 160mg, 180mg, 200mg, 240mg, 260mg, 300mg, 320mg, 360mg, 400mg, 500mg, 600mg, 800mg, 1000mg, 1200mg, etc.; the dosing is at intervals, for example 1 time every 3-7 days or 1 time every 1-5 weeks, for example 1 time every 3 days, 1 time every 5 days, 1 time every 1 week, 1 time every 10 days, 1 time every 2 weeks, 1 time every 3 weeks, 1 time every 4 weeks; dosing 1 time every 5 weeks, etc. The particular dosage and frequency of administration should take into account factors such as the route of administration, the health of the patient, etc., which can be determined by a skilled practitioner according to routine skill.

In the present invention, the tumor includes, but is not limited to, a solid tumor or a non-solid tumor, the solid tumor being selected from the group consisting of: breast cancer, prostate cancer, melanoma, osteosarcoma, neuroblastoma, pancreatic cancer, lung cancer, rhabdomyosarcoma, ewing's sarcoma, bladder cancer, colon cancer, liver cancer, ovarian cancer, cervical cancer, nasopharyngeal cancer, laryngeal cancer, gastric cancer, renal cancer, head and neck tumor, esophageal cancer, testicular cancer, merck cell cancer, thyroid cancer or brain cancer. Non-solid tumors include tumors of the blood system such as leukemia, lymphoma (e.g., hodgkin's lymphoma), and tumors of the nervous system.

In the present invention, the term "treatment" refers to both therapeutic treatment and prophylactic or preventative measures, wherein the object is to prevent or slow down (reduce) an undesired physiological change or disorder, such as the progression of a tumor. Beneficial or desired clinical results include, but are not limited to, results that are either detectable or undetectable, including alleviation of symptoms, diminishment of extent of disease, stabilization of disease state (i.e., not worsening), delay or slowing of disease progression, amelioration or palliation of the disease state, and palliation (whether partial or total). Those who have suffered from a cancer or tumor-related disease or those who need to prevent or ameliorate a cancer or tumor-related disease are included in need of treatment.

Unless otherwise indicated, the present invention may detect the expression levels of certain markers, such as genes or proteins, in the tissues and/or cells of cancer or tumor using methods conventional in the art, including but not limited to methods of enzyme-linked immunosorbent, immunohistochemistry, flow cytometry, western blotting, tissue chips, gene detection, and the like.

Example 1

In order to study the immunity activating and anti-tumor effects of the medicine. In this example, after treating 2fTGH-ISRE-Luci cells with different drugs, luciferase activation was compared using a microplate reader.

The experimental steps are as follows: (1) 2fTGH-ISRE-Luci cells were cultured in 96-well plates.

(2) After 18h of cell culture, different drugs were used to act on 2fTGH-ISRE-Luci cells.

(3) And (5) collecting the cells after the medicine is acted for 18 hours, and adding lysis to lyse the cells.

(4) Adding fluorescein excitant, and detecting activation of cytoluciferase treated by different drugs by enzyme-labeled instrument.

(5) The results show that the drug betrofloxacin Ban Malai acid salt and betrofloxacin have stronger effect of activating luciferase.

2. Experimental results

The results are shown in FIG. 1, and FIG. 1 shows the results of detection of luciferases after treatment of 2fTGH-ISRE-Luci cells with different drugs, wherein the drugs Betrexab Ban Malai acid salt, betrexaban have a stronger effect of activating luciferases compared with other drugs, such as small molecule drugs.

Example 2

Based on the experimental results, in this example, after cells were continuously treated with four drugs of betrixaban maleate, betrixaban maleate and rivaroxaban, the comparison was made to detect luciferases, and 6h and 12h drug action time controls were made.

The experimental steps are as follows: (1) 2fTGH-ISRE-Luci cells were cultured in 24-well plates.

(2) After 18h of cell culture, four drugs, namely, betrexaban maleate, betxaban maleate and revaprazaban, were used to act on 2fTGH-ISRE-Luci cells.

(3) Four drugs act on cells, and after different times (6 h and 12 h), the cells are collected, lysis is added to lyse the cells.

(4) Adding fluorescein excitant, and detecting the activation of the four medicaments-treated cytoluciferase by using an enzyme-labeled instrument.

(5) Experiments show that the betrofloxacin and betrofloxacin maleate can activate the reporter gene, and the activation is stronger after the medicine is treated for 12 hours.

2. Experimental results

The results are shown in FIG. 2, and FIG. 2 shows the results of the detection of luciferases after treatment of 2f with four drugs and various times of action of the drugs on 2f cells. The four drugs after 2f cell treatment show that the drug Betraxaban Ban Malai acid salt and Betraxaban have stronger function of activating ISRE-luciferase than the other two drugs according to the luciferase results.

The results of the luciferases after 6h and 12h of the cells with the drug action 2f show that compared with the drug betrofloxacin maleate and betrofloxacin action time, 12 has higher luciferase activity than 6 h. Wherein the betrofloxacin activation fold is about Gao Sanbei in the 12h treatment group compared with the 6h treatment group.

Thus, both betrofloxacin and betrofloxacin maleate were able to activate the reporter gene, and the activation was stronger after 12 hours of drug treatment.

Example 3

Based on the experimental results, in order to study the target of the drug Betroxaban Ban Malai acid salt activated IFN interferon, the embodiment continues to use the drug Betroxaban maleate combined with TANK-binding kinase 1 (TBK 1) inhibitor to act on 2fTGH-ISRE-Luci cells, and then performs luciferase and RT-qPCR detection to verify the signal path of the drug Betroxaban maleate acting target.

The experimental steps are as follows:

(1) 2fTGH-ISRE-Luci cells were cultured in 48-well plates.

(2) After 18h of cell culture, 2fTGH-ISRE-Luci cells were treated with Betraxaban maleate at different concentrations of 20. Mu.M/40. Mu.M in combination with TBK1 inhibitor, respectively.

(3) After the betrofloxacin maleate acts on cells in combination with the TBK1 inhibitor, the cells are collected at different times (6 h and 24 h) respectively. Lyses cells were lysed in 24 wells of a 48 well plate; and collecting samples from the other 24-hole cells, adding lysate to extract RNA, and performing reverse transcription.

(4) Adding a fluorescein excitant into 24 holes of a 48-hole plate, and detecting the activation condition of the four medicaments-treated cell luciferases by using an enzyme-labeled instrument; RNA from 24-well cells was reverse transcribed and then subjected to real-time fluorescent quantitative PCR to relatively quantify the target gene.

(5) Experiments show that the target of the effect of the Betroxas Ban Malai acid salt on activating immunity and the type I interferon activation signal path comprises (or is not limited to) a TBK1 target.

2. Experimental results

The results are shown in FIG. 3, FIG. 3 shows the detection of 2fTGH-ISRE-Luci cell luciferase and RT-qPCR by Betroxaban maleate in combination with TBK1 inhibitor.

Experimental results prove that the activity of luciferase of the betrofloxacin maleate combined with the TBK1 inhibitor is far lower than that of a betrofloxacin Ban Malai acid salt medicament group. This example carries out a comparison of betrixaban maleate at different concentrations of 20 μm/40 μm in combination with TBK1 inhibitor and of the duration of action of the drug for 6h and 24h respectively: wherein the betaxomass Ban Malai acid salt is about 20-fold higher by 40 μm than the 20 μm fold for luciferase activation, indicating that 40 μm is an effective dose; wherein the betrofloxacin maleate has more pronounced 24h than 6h activation; the combination TBK1 shows obvious inhibition effect. Experiments show that the target of the effect of the Betroxas Ban Malai acid salt on activating immunity and the type I interferon activation signal path comprises (or is not limited to) a TBK1 target.

Example 4

Based on the experimental results, the RT-QPCR is used for detecting the gene expression condition of the I type IFN downstream effector signal channel ISG family member after the medicine Betraxaban maleate and the combined TBK1 inhibitor act on 2fTGH-ISRE-Luci cells.

The experimental steps are as follows: (1) 2fTGH-ISRE-Luci cells were cultured in 48-well plates.

(2) After 18h of cell culture, the 2fTGH-ISRE-Luci cells were treated with Betrexaban maleate in combination with TBK1 inhibitor.

(3) And (3) taking out the cells after the Betroxaban maleate and TBK1 inhibitor are combined for 18 hours, and adding a lysate to extract RNA and reverse transcription.

(4) The relative quantification of ISG15 and IFIT genes was performed by real-time fluorescent quantification pcr after reverse transcription of RNA.

(5) Experiments show that the signaling pathway of the Betroxas Ban Malai acid salt activated type I interferon is (but not limited to) TBK1 upstream and ISG downstream.

2. Experimental results

The results are shown in FIG. 4, FIG. 4 shows RT-qPCR detection of ISG15 and IFIT 1-3 mRNA levels after the action of betrexaban maleate on 2f cells.

Experimental results prove that compared with a MOCK group and a DMSO control group, the drug betrofloxaban maleate shows high ISG15 and IFIT gene expression. And the combined TBK1 inhibitor group shows obvious inhibition. Experiments show that the signaling pathway of the Betroxas Ban Malai acid salt activated type I interferon is (but not limited to) TBK1 upstream and ISG downstream.

The results of fig. 3 and 4 together show that betrexed Ban Malai acid salt activates ISG expression in dependence on kinase TBK1.

Example 5

Based on the experimental results, in the embodiment, the medicine betrexaban maleate acts on 2f cells, then RNA is extracted for RNA-seq sequencing, and genes with up-regulation of more than 4 times are selected for analysis.

(2) After 18h of cell culture, the 2fTGH-ISRE-Luci cells were treated with the drug Betraxa Ban Malai acid salt.

(3) And (5) taking the cells after the cells are subjected to the action of the betrixaban maleate for 18 hours, and adding a lysate to extract RNA.

(4) RNA sequencing

(5) Sequencing results showed that the betrexed Ban Malai acid salt treated cells were able to significantly activate up-regulated expression of interferon-induced genes.

2. Experimental results

As a result, as shown in FIG. 5, FIG. 5 shows a list of genes up-regulated by more than 4-fold after the selection of Betroxas Ban Malai acid salt treatment for RNA-seq sequencing, and enrichment of the up-regulated genes in Interferona alpha/beta signaling (R-HAS-909733) was observed. Following the action of the drug betrofloxacin maleate, the up-regulating genes associated with interferon IFN therein were: GO 0006954: inflammatory response; WP5095: overview of proinflammatory and profibrotic mediators; GO 0032649: regulation of type II interferon production.

Taken together, the betrixacum Ban Malai acid salt treated cells were able to significantly activate the type I interferon signaling pathway and up-regulated expression of interferon inducible genes.

Example 6

To verify the antitumor activity of the betrexed Ban Malai acid salt, the present example used Pan02 (mouse pancreatic cancer cells) to vaccinate mice subcutaneously with the vaccination protocol shown in fig. 6. On day 10, mice were randomly selected as mock (control) and betrexed Ban Malai acid salt treatment (Intraperitoneal Injections) and the body weights and tumor sizes of the two groups were compared 23 days after planting.

The experimental steps are as follows: (1) Mice were inoculated subcutaneously with Pan02 (mouse pancreatic cancer cells).

(2) On day 10, mice were randomly selected as mock (control group) and betrexed Ban Malai acid salt treatment group (Intraperitoneal Injections group).

(3) The body weight and tumor size of the two groups of mice were compared 23 days after tumor cell implantation.

2. Experimental results

As shown in fig. 7 and 8, the tumor tissue of the mice was removed 23 days after tumor implantation, and the tumor volume and weight of the betrexed Ban Malai acid salt Intraperitoneal Injections group were significantly smaller than those of the mock group.

Example 7

The validation experiment of example 6 can observe that betrofloxacin maleate can significantly inhibit tumor growth. To explore its molecular mechanism, the number of mouse spleen immune cells was analyzed in this example.

(3) Spleen tissue of two groups of mice was removed 23 days after tumor cell implantation.

(4) Spleen tissue was ground and the spleen tissue was prepared as a single cell suspension using a cell sieve.

(5) Flow cytometry was used to perform phenotypic analysis of spleen cells from both control groups.

(6) The results show that betrofloxaban maleate significantly increases the number of cd8+ T cells in the spleen of mice.

2. Experimental results

As shown in fig. 9, the cd3+cd4+ of the betrexed Ban Malai acid salt Intraperitoneal Injections group was lower than that of the mock group; cd3+cd8+ is higher than mock group.

The application of flow cytometry results shows that betrexaban maleate significantly increases the number of cd8+ T cells in the spleen of mice, as cd8+ T cells have tumor killing function. Thus betrexaban maleate enhances anti-tumor immunity by activating cd8+ T cells. The spleen cell phenotypes of the above two control groups were analyzed.

Finally, it should be noted that the above embodiments are only for illustrating the technical solution of the present invention, and are not limiting; although the invention has been described in detail with reference to the foregoing embodiments, those of ordinary skill in the art will understand that: the technical scheme described in the foregoing embodiments can be modified or some of the technical features thereof can be replaced by equivalents. Such modifications and substitutions do not depart from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. The use of betrofloxacin or a pharmaceutically acceptable salt, solvate, hydrate, complex, prodrug, isomer, analogue, derivative or metabolite thereof for the preparation of an antitumor drug.

2. The use according to claim 1, wherein the anti-tumour comprises at least one of:

(1) Inhibit proliferation or growth of tumor cells;

(2) Reducing or lowering tumor volume and/or weight;

(3) Inhibit metastasis of tumors;

(5) Promoting or improving the tumor killing function of immune cells;

(6) Activating the production of type I interferon.

3. The use according to claim 2, wherein the immune cells comprise one or more of tumor infiltrating lymphocytes, macrophages, natural killer cells, cytokine-induced killer cells, cytotoxic T lymphocytes, dendritic cells in combination with cytokine-induced killer cells, chimeric antigen receptor T cells and T cell receptor chimeric T cells.

4. Use of betrofloxacin or a pharmaceutically acceptable salt, solvate, hydrate, complex, prodrug, isomer, analogue, derivative or metabolite thereof for the manufacture of a medicament for activating or promoting a type I interferon.

5. The use according to claim 4, wherein the activating or facilitating comprises at least one of:

(b) Activating or promoting gene expression of ISG family members;

(c) Promote or enhance expression of interferon inducible genes.

6. The use of claim 5, wherein the target comprises a TBK1 target.

7. The use according to claim 5, wherein the genes of ISG family members comprise one or more of ISG15, IFIT1, IFIT2, IFIT3 and IFIT 5.

8. The use according to claim 5, wherein the interferon inducible genes comprise GO 0006954, WP5095 and/or GO 0032649.

9. The use of betrofloxacin or a pharmaceutically acceptable salt, solvate, hydrate, complex, prodrug, isomer, analogue, derivative or metabolite thereof in combination with other therapeutic agents.

10. The use of claim 9, wherein the therapeutic agent comprises one or more of a chemotherapeutic agent, an immune checkpoint inhibitor, an anti-tumor drug, and an immunomodulatory agent.