CN112402613A

CN112402613A - Use of PDE3 inhibitors in combination with cytokines for the treatment of tumors

Info

Publication number: CN112402613A
Application number: CN201910784742.0A
Authority: CN
Inventors: 俞强; 安然
Original assignee: Shanghai Institute of Materia Medica of CAS
Current assignee: Shanghai Institute of Materia Medica of CAS
Priority date: 2019-08-23
Filing date: 2019-08-23
Publication date: 2021-02-26
Also published as: WO2021036937A1

Abstract

The invention relates to the use of PDE3 inhibitors in combination with cytokines for the treatment of tumors. Specifically, provided is a pharmaceutical composition, characterized in that the composition comprises: (a) a therapeutically effective amount of a first active ingredient which is a PDE3 inhibitor; and (b) a therapeutically effective amount of a second active ingredient which is a cytokine. The combined application of the PDE3 inhibitor and the cell factor has obvious synergistic anticancer effect and can be used for treating various cancers.

Description

Use of PDE3 inhibitors in combination with cytokines for the treatment of tumors

Technical Field

The invention relates to the field of medicines, in particular to application of a PDE3 inhibitor and a cytokine in combination therapy of tumors.

Background

Tumors become the first killer of human health, and according to the report of the American cancer society of "2018 Global cancer statistics", there are approximately 1810 ten thousand cancer new cases and 960 ten thousand cancer dead cases globally in 2018. Tumor treatment is always a difficult problem to overcome, and the traditional tumor treatment comprises three treatment modes, namely surgery, chemotherapy and radiotherapy, which achieve great achievement and have great limitations. Traditional chemotherapeutic drugs are mainly cytotoxic drugs such as alkylating agents of DNA (cyclophosphamide, cisplatin, carboplatin), tubulin inhibitors (e.g., paclitaxel), metabolic antagonists of DNA synthesis precursors (methotrexate), topoisomerase II inhibitors (e.g., etoposide), etc., which inhibit normal cell growth while targeting rapidly proliferating and dividing tumor cells, thus causing many serious side effects such as cardiotoxicity, vomiting, alopecia, immunosuppression, etc. Therefore, the search for a treatment mode specifically targeting tumor cells becomes a research and development hotspot of tumor treatment, and the targeted treatment becomes a new generation of tumor treatment mode. The targeted therapy is different from the traditional chemotherapy mode in that a corresponding therapeutic drug is designed aiming at a definite canceration site on the molecular level, so that tumor cells are specifically killed without affecting the surrounding normal tissues.

Therefore, there is a need in the art to develop a drug that provides a therapeutic effect on tumors.

Disclosure of Invention

The invention aims to provide a pharmaceutical composition of a PDE3 inhibitor and a cytokine which have obvious synergistic antitumor effects.

In a first aspect of the present invention, there is provided a pharmaceutical composition comprising:

(a) a therapeutically effective amount of a first active ingredient which is a PDE3 inhibitor; and

(b) a therapeutically effective amount of a second active ingredient which is a cytokine.

In another preferred embodiment, the weight ratio of the first active ingredient to the second active ingredient is 1: (0.1-50), preferably, 1: (0.1-20), more preferably, 1: (0.15-10), most preferably, 1: (0.2-5).

In another preferred embodiment, the PDE3 inhibitor is selected from the group consisting of: a compound of formula I or a pharmaceutically acceptable salt thereof, a compound of formula II or a pharmaceutically acceptable salt thereof, or a combination thereof;

wherein the content of the first and second substances,

r1, R2, R3, R4, R5, R6, R7 and R8 are each independently selected from the group consisting of: a hydrogen atom, a halogen atom, an amino group, a hydroxyl group, a cyano group, an aldehyde group, a nitro group, a substituted or unsubstituted C1-C4 carboxyl group, a substituted or unsubstituted C1-C10 alkyl group, a substituted or unsubstituted C3-C10 cycloalkyl group, a substituted or unsubstituted C2-C10 alkenyl group, a substituted or unsubstituted C2-C10 alkynyl group, a substituted or unsubstituted C6-C10 aryl group, a substituted or unsubstituted C5-C10 heteroaryl group (e.g., a substituted or unsubstituted 5-or 6-membered heterocycle, an 8-to 10-membered heteroaryl group), a substituted or unsubstituted C1-C10 alkoxy group, a substituted or unsubstituted C6-C10 aryl-oxy group, a substituted or unsubstituted C5-C10 heteroaryl-oxy group, a substituted or unsubstituted C1-C10 alkoxy group₂-C₁₀Acyl (, substituted or unsubstituted C)₂-C₁₀An ester group, a substituted or unsubstituted C1-C10 sulfonyl (-SO2-C1-C10 alkyl);

or R1 and R2, R3 and R4 together form a group selected from: substituted or unsubstituted C3-C20 cycloalkyl (preferably C3-C10 cycloalkyl), substituted or unsubstituted C3-C20 heterocycloalkyl (preferably a substituted or unsubstituted 5-or 6-membered heterocycle, 8-to 12-membered heteroaryl bicyclic ring system), carbonyl (═ O);

r9 is selected from the group consisting of: a hydrogen atom, an oxygen atom, a substituted or unsubstituted C1-C10 alkyl group, a substituted or unsubstituted C3-C10 cycloalkyl group, a substituted or unsubstituted C6-C10 aryl group, a substituted or unsubstituted C1-C10 heteroaryl group, a substituted or unsubstituted C1-C10 alkoxy group, a substituted or unsubstituted C6-C10 aryl-oxy group, a substituted or unsubstituted acyl group (preferably-CO-C1-C10 alkyl group), a substituted or unsubstituted C1-C10 sulfonyl group;

wherein, any "substitution" means that one to four (preferably 1,2, 3 or 4) hydrogen atoms on the group are substituted with a substituent selected from the group consisting of: C1-C10 alkyl, C3-C10 cycloalkyl, C1-C10 alkoxy, halogen, hydroxyl, carboxyl (-COOH), C1-C10 aldehyde group, C2-C10 acyl, C2-C10 ester group, amino, phenyl, cyano, -OH, nitro and amino;

wherein the content of the first and second substances,

r11, R12, R13, R14, R15, R17 and R18 are each independently selected from the group consisting of: hydrogen atom, halogen atom, amino group, hydroxyl group, cyano group, aldehyde group, nitro group, substituted or unsubstituted C1-C4 carboxyl group, substituted or unsubstituted C1-C10 alkyl group, substituted or unsubstituted C3-C10 cycloalkyl group, substituted or unsubstituted C2-C10 alkenyl, substituted or unsubstituted C2-C10 alkynyl, substituted or unsubstituted C6-C10 aryl, substituted or unsubstituted C5-C10 heteroaryl (such as substituted or unsubstituted 5-or 6-membered heterocycle, 8-to 10-membered heteroaryl bicyclic ring system), substituted or unsubstituted C1-C10 alkoxy, substituted or unsubstituted C6-C10 aryl-oxy, substituted or unsubstituted C5-C10 heteroaryl-oxy, substituted or unsubstituted C2-C10 acyl, substituted or unsubstituted C2-C10 ester, substituted or unsubstituted C1-C10 sulfonyl (-SO2-C1-C10 alkyl);

r16 is selected from the group consisting of: a hydrogen atom, an oxygen atom, a substituted or unsubstituted C1-C10 alkyl group, a substituted or unsubstituted C3-C10 cycloalkyl group, a substituted or unsubstituted C6-C10 aryl group, a substituted or unsubstituted C5-C10 heteroaryl group, a substituted or unsubstituted C1-C10 alkoxy group, a substituted or unsubstituted C6-C10 aryl-oxy group, a substituted or unsubstituted C1-C10 acyl group, a substituted or unsubstituted C1-C10 sulfonyl group;

represents a single bond or is absent;

wherein, any "substitution" means that one to four (preferably 1,2, 3 or 4) hydrogen atoms on the group are substituted with a substituent selected from the group consisting of: C1-C10 alkyl, C3-C10 cycloalkyl, C1-C10 alkoxy, halogen, hydroxyl, carboxyl (-COOH), C1-C10 aldehyde group, C2-C10 acyl, C2-C10 ester group, amino, phenyl, cyano, OH, nitro and amino.

In another preferred embodiment, R1 is hydrogen.

In another preferred embodiment, R2 is hydrogen.

In another preferred embodiment, R3 is hydrogen.

In another preferred embodiment, R4 is hydrogen.

In another preferred embodiment, R5 is halogen.

In another preferred embodiment, R6 is halogen.

In another preferred embodiment, R7 is hydrogen.

In another preferred embodiment, R8 is hydrogen.

In another preferred embodiment, R9 is hydrogen.

In another preferred embodiment, R11 is hydrogen.

In another preferred embodiment, the halogen is Cl.

In another preferred embodiment, R11 is hydrogen.

In another preferred embodiment, R12 is hydrogen or C1-C6 alkoxy.

In another preferred embodiment, R12 is hydrogen or methoxy.

In another preferred embodiment, R13 is a halogenated C1-C6 alkoxy group.

In another preferred embodiment, R13 is a dihalogenated C1-C6 alkoxy group.

In another preferred embodiment, R13 is halomethoxy.

In another preferred embodiment, R13 is dihalomethoxy.

In another preferred embodiment, R13 is dichloromethoxy.

In another preferred embodiment, R14 is hydrogen or C1-C6 alkoxy.

In another preferred embodiment, R14 is hydrogen or methoxy

In another preferred embodiment, R15 is hydrogen.

In another preferred embodiment, R16 is hydrogen.

In another preferred embodiment, R17 is hydrogen.

In another preferred embodiment, R18 is hydrogen.

In another preferred embodiment, the phenyl group includes an unsubstituted phenyl group or a substituted phenyl group having 1 to 3 substituents.

In another preferred embodiment, the PDE3 inhibitor is selected from the group consisting of: anagrelide (Anagrelide) or a pharmaceutically acceptable salt thereof, zadavirin (Zardaverine) or a pharmaceutically acceptable salt thereof, or a combination thereof.

In another preferred embodiment, the cytokine is selected from the group consisting of: an antiviral cytokine, a tumor necrosis factor, an interferon, a cytokine against DNA replication, or a combination thereof.

In another preferred embodiment, the cytokine is selected from the group consisting of: an interferon, a tumor necrosis factor, or a combination thereof.

In another preferred embodiment, the interferon is selected from the group consisting of: IFN-alpha, IFN-gamma, or a combination thereof.

In another preferred embodiment, the tumor necrosis factor is selected from the group consisting of: tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), TNF- α, or a combination thereof.

In another preferred embodiment, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier.

In a second aspect, the invention provides a kit comprising:

(A) a first formulation comprising a PDE3 inhibitor; and

(B) a second agent comprising a cytokine.

In another preferred embodiment, the kit further comprises instructions.

In another preferred embodiment, the instructions specify that the first agent and the second agent are to be used in combination to prevent and/or treat cancer.

In another preferred embodiment, the first and second formulations are administered simultaneously, separately or sequentially in the prevention and/or treatment of tumors.

In the kit of the present invention, it is to be understood that the first formulation and the second formulation in the kit are independent of each other or can be combined.

In another preferred embodiment, the first formulation and the second formulation are separate from each other in the kit.

In another preferred embodiment, the first preparation is a solid preparation (such as a solid preparation for gastrointestinal tract) or an injection preparation (such as an injection, an infusion solution or a freeze-dried powder injection).

In another preferred embodiment, the second preparation is an injection preparation (such as an injection, an infusion solution or a freeze-dried powder injection).

For example, the first formulation and the second formulation are separate. Typically, the kit of the invention comprises: the preparation comprises a solid preparation (such as a solid preparation for gastrointestinal tract) and an injection preparation (such as an injection, an infusion solution or a freeze-dried powder injection), wherein the solid preparation (such as the solid preparation for gastrointestinal tract) contains a PDE3 inhibitor, and the injection preparation (such as the injection, the infusion solution or the freeze-dried powder injection) contains a cytokine.

In a third aspect of the invention, there is provided an active ingredient combination comprising the following components:

(1) a therapeutically effective amount of a first active ingredient which is a PDE3 inhibitor; and

(2) a therapeutically effective amount of a second active ingredient which is a cytokine.

In a fourth aspect of the present invention, there is provided the use of a pharmaceutical composition according to the first aspect of the present invention, a kit according to the second aspect of the present invention, and/or a combination of active ingredients according to the third aspect of the present invention, for the manufacture of a medicament for the prevention and or treatment of tumours.

In another preferred embodiment, the tumor is selected from the group consisting of: cervical cancer, liver cancer, kidney cancer, colon cancer, brain glioma, melanoma, or combinations thereof.

In a fifth aspect of the present invention, there is provided a method for preventing and/or treating tumors, comprising the steps of: administering to a subject in need thereof a composition according to claim 1.

In another preferred embodiment, the subject is a human or a non-human mammal.

Typically, the non-human mammal includes (but is not limited to): pets (e.g., dogs, cats), livestock (e.g., cattle, sheep, horses, pigs), various zoo animals (pandas, elephants, tigers), and the like.

It is to be understood that within the scope of the present invention, the above-described features of the present invention and those specifically described below (e.g., in the examples) may be combined with each other to form new or preferred embodiments. Not to be reiterated herein, but to the extent of space.

Drawings

FIG. 1A MTT assay A combination of ANA (10nM) and ZARD (20nM) with interferon IFN- α (3000IU/mL), IFN- γ (10ng/mL), apoptosis-related factor TRAIL (100ng/mL) was set up in SMMC7721, Bel7404, A498, FHCC98 cells, respectively. After 72h drug incubation, 30 μ L of MTT was added to each well and incubated for 4-6h, and the OD value was measured at a measurement wavelength of 492nM by a microplate reader (blank: detection medium OD value (no cells spread) control: DMSO (cells spread) was added to the medium in the same volume as the drug to calculate the cell viability ═ 100% (experimental OD-blank OD))/((control OD-blank OD)) x).

FIG. 1B shows MTT assay in combination with ANA (10nM) and ZARD (20nM) with interferon IFN-. alpha. (3000IU/mL), IFN-. gamma. (10ng/mL), apoptosis-related factor TRAIL (100ng/mL) in A2058, Hela, QGY7701, SW1116 cells. After 72h drug incubation, 30. mu.L MTT was added to each well and incubated for 4-6h, and the OD value was measured at a measurement wavelength of 492nM using a microplate reader (blank: detection medium OD value (without plating cells) control group: DMSO (plating cells) was added to the medium in the same volume as the drug to calculate the cell viability ((experiment OD-blank OD))/((control OD-blank OD)) x 100%)

FIG. 2A shows the RTCA assay for IFN- α (3000IU/mL) in combination with ANA (100nM) in Bel7404, A498, SW 1116. And (3) paving 5000/hole plates of the tumor cells, respectively adding a compound to be detected with a specified concentration when the cells grow to a logarithmic growth phase, detecting the growth of the cells in real time by using RTCA, and obtaining a cell growth curve after the experiment is finished.

FIG. 2B shows the RTCA assay for IFN- α (3000IU/mL) in combination with ZARD (100nM) in Bel7404, A498, SW 1116. And (3) paving 5000/hole plates of the tumor cells, respectively adding a compound to be detected with a specified concentration when the cells grow to a logarithmic growth phase, detecting the growth of the cells in real time by using RTCA, and obtaining a cell growth curve after the experiment is finished.

FIG. 2C shows the RTCA detection of IFN-. gamma.10 ng/mL in combination with ANA (100nM) in Bel7404, A498, SW 1116. And (3) paving 5000/hole plates of the tumor cells, respectively adding a compound to be detected with a specified concentration when the cells grow to a logarithmic growth phase, detecting the growth of the cells in real time by using RTCA, and obtaining a cell growth curve after the experiment is finished.

FIG. 2D shows RTCA detection of IFN-. gamma.10 ng/mL in combination with ZARD (100nM) in Bel7404, A498, SW 1116. And (3) paving 5000/hole plates of the tumor cells, respectively adding a compound to be detected with a specified concentration when the cells grow to a logarithmic growth phase, detecting the growth of the cells in real time by using RTCA, and obtaining a cell growth curve after the experiment is finished.

FIG. 2E shows RTCA detection of TRAIL (100ng/mL) in combination with ANA (100nM) in Bel7404, A498, SW 1116. And (3) paving 5000/hole plates of the tumor cells, respectively adding a compound to be detected with a specified concentration when the cells grow to a logarithmic growth phase, detecting the growth of the cells in real time by using RTCA, and obtaining a cell growth curve after the experiment is finished.

FIG. 2F shows RTCA detection of TRAIL (100ng/mL) in combination with ZARD (100nM) in Bel7404, A498, SW 1116. And (3) paving 5000/hole plates of the tumor cells, respectively adding a compound to be detected with a specified concentration when the cells grow to a logarithmic growth phase, detecting the growth of the cells in real time by using RTCA, and obtaining a cell growth curve after the experiment is finished.

FIG. 3A is a combination of TNF- α (10ng/mL) and ANA (100nM) in Bel7404, SW1116 in example 3. And (3) paving 5000/hole plates of the tumor cells, respectively adding a compound to be detected with a specified concentration when the cells grow to a logarithmic growth phase, detecting the growth of the cells in real time by using RTCA, and obtaining a cell growth curve after the experiment is finished.

FIG. 3B shows TNF- α (10ng/mL) in example 3 in combination with ZARD (100nM) in Bel7404, SW 1116. And (3) paving 5000/hole plates of the tumor cells, respectively adding a compound to be detected with a specified concentration when the cells grow to a logarithmic growth phase, detecting the growth of the cells in real time by using RTCA, and obtaining a cell growth curve after the experiment is finished.

Detailed Description

The present inventors have conducted extensive and intensive studies and, as a result, have unexpectedly found that the combined use of a PDE3 inhibitor (such as ANA, or ZARD) and a cytokine (such as TRAIL, IFN-. alpha.or IFN-. gamma.) can significantly improve the therapeutic effect on tumors, and that the synergistic therapeutic effect of the PDE3 inhibitor and the cytokine is significantly superior to that of the PDE3 inhibitor or the cytokine alone. On the basis of this, the present invention has been completed.

Term(s) for

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.

As used herein, the terms "comprises," "comprising," "includes," "including," and "including" are used interchangeably and include not only closed-form definitions, but also semi-closed and open-form definitions. In other words, the term includes "consisting of … …", "consisting essentially of … …".

As used herein, the term "pharmaceutically acceptable carrier" ingredient refers to a substance that is suitable for use in humans and/or animals without undue adverse side effects (such as toxicity, irritation, and allergic response), i.e., at a reasonable benefit/risk ratio.

The term "therapeutically effective amount," as used herein, refers to an amount that produces a function or activity in and is acceptable to humans and/or animals. It will be understood by those skilled in the art that the "therapeutically effective amount" may vary with the form of the pharmaceutical composition, the route of administration, the excipients used, the severity of the disease, and the combination with other drugs.

"prevention" and "treatment" as used herein include delaying and stopping the progression of the disease, or eliminating the disease, and do not require 100% inhibition, elimination, or reversal. In some embodiments, the composition or pharmaceutical composition of the invention prevents, reduces, inhibits, and/or reverses ischemia reperfusion injury, e.g., by at least about 10%, at least about 30%, at least about 50%, or at least about 80%, as compared to levels observed in the absence of the composition or pharmaceutical composition of the invention.

In the present invention, the term "pharmaceutically acceptable salt" refers to a salt formed by the compound of the present invention with an acid or a base, which is suitable for use as a medicament. Pharmaceutically acceptable salts include inorganic and organic salts. One preferred class of salts is that formed by reacting a compound of the present invention with an acid. Suitable acids for forming the salts include, but are not limited to: inorganic acids such as toluenesulfonic acid, hydrochloric acid, hydrobromic acid, hydrofluoric acid, sulfuric acid, nitric acid, phosphoric acid, and the like, organic acids such as formic acid, acetic acid, propionic acid, oxalic acid, malonic acid, succinic acid, fumaric acid, maleic acid, lactic acid, malic acid, tartaric acid, citric acid, picric acid, methanesulfonic acid, benzenesulfonic acid, and the like; and acidic amino acids such as aspartic acid and glutamic acid. One preferred class of salts is that formed from the compounds of the present invention and a base, suitable bases for forming salts include, but are not limited to: inorganic bases such as sodium hydroxide, potassium hydroxide, sodium carbonate, sodium bicarbonate and sodium phosphate, and organic bases such as ammonia, triethylamine and diethylamine.

It is to be understood that substituents and substitution patterns on the compounds of formula I or formula II of the present invention may be selected by one of ordinary skill in the art to produce chemically stable compounds that may be synthesized by techniques known in the art and as set forth below. If substituted with more than one substituent group, it is understood that the multiple groups may be on the same carbon or on different carbons, so long as a stable structure results.

As used herein, the term "substituted" or "substituted" is a radical in which a hydrogen atom on the radical is replaced with a non-hydrogen atom, but which needs to satisfy its valence requirements and which results from the substitution into a chemically stable compound, i.e., a compound which does not spontaneously undergo a transformation such as cyclization, elimination, etc.

Unless otherwise specified, each chiral carbon atom (chiral center) in all compounds of the invention may optionally be in the R configuration or the S configuration, or a mixture of the R configuration and the S configuration.

As used herein, "R1", "R¹"and" R₁"has the same meaning as" and can be substituted for "another, and other similar definitions have the same meaning.

The term "C₁-C₄Carboxy "means C₁-C₃Radicals of the alkyl-COOH structure, in which the alkyl radical may be linear or branched, e.g. CH₃COOH、C₂H₅COOH、C₃H₈COOH、(CH₃)₂CHCOOH, or the like.

As used herein, the term "alkyl", alone or as part of another substituent, means containing onlyStraight chain (i.e., unbranched) or branched saturated hydrocarbon groups of carbon atoms, or a combination of straight and branched groups. When the alkyl group is preceded by a carbon atom number limitation (e.g. C)₁-C₁₀Alkyl) means that the alkyl group contains 1 to 10 carbon atoms, e.g. C₁-C₆Alkyl refers to an alkyl group having 1 to 6 carbon atoms, and representative examples include, but are not limited to, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, or the like.

As used herein, the term "alkenyl", alone or as part of another substituent, refers to a straight or branched chain carbon chain radical having at least one carbon-carbon double bond. When the alkenyl radical is preceded by a carbon atom number limitation (e.g. C)₂-C₁₀) When used, it means that the alkenyl group has 2 to 10 carbon atoms. E.g. C₂-C₆Alkenyl means alkenyl having 2 to 6 carbon atoms and includes ethenyl, propenyl, 1, 2-butenyl, 2, 3-butenyl, butadienyl, or the like.

As used herein, the term "alkynyl", alone or as part of another substituent, refers to an aliphatic hydrocarbon group having at least one carbon-carbon triple bond. The alkynyl group can be linear or branched, or a combination thereof. When alkynyl is preceded by a carbon atom number limitation (e.g. C)₂-C₁₀Alkynyl) means that the alkynyl group contains 2 to 10 carbon atoms. For example, the term "C₂-C₆Alkynyl "refers to straight or branched chain alkynyl groups having 2 to 6 carbon atoms and includes ethynyl, propynyl, isopropynyl, butynyl, isobutynyl, sec-butynyl, tert-butynyl, or the like.

As used herein, the term "cycloalkyl", alone or as part of another substituent, refers to a monocyclic, bicyclic, or polycyclic (fused, bridged, or spiro) ring system group having a saturated or partially saturated unit ring. When a cycloalkyl group is preceded by a carbon atom number limitation (e.g. C)₃-C₁₀) When used, means that the cycloalkyl group has 3 to 10 ring carbon atoms. In some preferred embodiments, the term "C₃-C₁₀Cycloalkyl "refers to a saturated or partially saturated monocyclic or bicyclic alkyl group having 3 to 10 ring carbon atoms and includes cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl and the like,Cyclopentyl, cycloheptyl, or the like.

As used herein, the term "aryl" alone or as part of another substituent refers to an all-carbon monocyclic or fused polycyclic (i.e., rings which share adjacent pairs of carbon atoms) group having a conjugated pi-electron system, when the aryl group is preceded by a definition of the number of carbon atoms, e.g., C₆-C₁₀Aryl means that the aryl group has 6 to 10 ring carbon atoms, such as phenyl and naphthyl. The aryl ring may be fused to other cyclic groups (including saturated or unsaturated rings) but must not contain heteroatoms such as nitrogen, oxygen, or sulfur, while the point of attachment to the parent must be at a carbon atom on the ring with the conjugated pi-electron system. Representative examples of aryl groups include, but are not limited to:

as used herein, the term "heteroaryl", alone or as part of another substituent, refers to an aromatic heterocyclic system having one to more (preferably 1,2, 3, or 4) heteroatoms, which may be monocyclic (monocyclic) or polycyclic (bicyclic, tricyclic, or polycyclic), fused together or covalently linked, where the heteroatoms referred to herein include oxygen, sulfur, and nitrogen. When heteroaryl is previously defined, C5-C10 heteroaryl refers to heteroaryl groups having 5-10 ring atoms. Examples of, for example, 5-membered heteroaryl groups include (but are not limited to): examples of pyrrole, furan, thiophene, imidazole, oxazole, thiazole, 6-membered heteroaryl include, but are not limited to, pyridine, pyrazine, pyridazine, pyrimidine.

As used herein, the term "alkoxy", alone or as part of another substituent, refers to an R-O-group, where R is alkyl, alkyl is as defined herein, and when alkoxy has a carbon atom number limitation, such as C, preceding it₁-C₁₀Alkoxy means that the alkyl group in the alkoxy group has 1 to 10 carbon atoms. Representative examples of alkoxy groups include (but are not limited to): methoxy, ethoxy, n-propoxy, isopropoxy, tert-butoxy, or the like.

The term "acylThe radical "means an R-CO-group in which R is alkyl, alkyl being as defined herein above, e.g." C₂-C₁₀Acyl "means having C₁-C₉Representative examples of alkyl-CO-structural groups, acyl groups include (but are not limited to): CH (CH)₃-CO-、C₂H₅-CO-、C₃H₈-CO-, or the like.

As used herein, the term "ester group", alone or as part of another substituent, refers to a group having the formula R-CO-O-or-CO-O-R, wherein R is alkyl and alkyl is as defined herein, e.g., "C₂-C₁₀The "ester group" means C₁-C₉Radicals of alkyl-CO-O-structure or-CO-O-C₁-C₇Representative examples of alkyl structures, ester groups include (but are not limited to): CH (CH)₃COO-、C₂H₅COO-、C₃H₈COO-、(CH₃)₂CHCOO-、-COOCH₃、-COOC₂H₅、-COOC₃H₈Or the like.

As used herein, the term "C1-C10 sulfonyl", alone or as part of another substituent, refers to-SO 2-C1-C10 alkyl, alkyl being as defined above.

As used herein, the term "C₃-C₂₀Heterocycloalkyl "means having 3 to 20 ring carbon atoms and 1 to 3 heteroatoms (preferably containing 1 nitrogen atom, i.e. with R)¹And R²Common adjacent nitrogen atoms) and polycyclic heterocycles (preferably monocyclic heterocycles), such as a piperidine group, a tetrahydropyrrole group, or the like.

The term "aldehyde group" as used herein refers to — CHO.

As used herein, the term "halogen" refers to F, Cl, Br and I.

As used herein, the term "halo" refers to substitution by halogen.

A first active ingredient

In the present invention, the first active ingredient is a PDE3 inhibitor.

PDE3 belongs to the PDE (phosphodiesterase) family, sharing two subtypes, PDE3A and PDE3B, located on chromosome 12p12 and 11p15, respectively. PDE3 is able to hydrolyze both cAMP and cGMP, secondary messengers of the cell, terminating the conduction of downstream signaling pathways.

In a preferred embodiment, the PDE3 inhibitor is selected from the group consisting of: a compound of formula I or a pharmaceutically acceptable salt thereof, a compound of formula II or a pharmaceutically acceptable salt thereof, or a combination thereof.

Preferably, the PDE3 inhibitor is a compound of formula I, or a pharmaceutically acceptable salt thereof, as follows:

r1, R2, R3, R4, R5, R6, R7 and R8 are as defined above.

Preferably, the PDE3 inhibitor is a compound of formula II or a pharmaceutically acceptable salt thereof, wherein the compound of formula II or the pharmaceutically acceptable salt thereof is as follows:

r11, R12, R13, R14, R15, R17 and R18 are as defined above.

In another preferred embodiment, the PDE3 inhibitor includes (but is not limited to): anagrelide (Anagrelide) or a pharmaceutically acceptable salt thereof, zadavirin (Zardaverine) or a pharmaceutically acceptable salt thereof, or a combination thereof.

In the invention, Anagrelide (Anagrelide) is called ANA for short, and the structural formula is shown as follows:

in the present invention, zadavirin (Zardaverine) is abbreviated as ZARD, and the structural formula is shown as follows:

preferably, the PDE3 inhibitor is Anagrelide (Anagrelide) or a pharmaceutically acceptable salt thereof.

Preferably, the PDE3 inhibitor is zadavalin (Zardaverine) or a pharmaceutically acceptable salt thereof.

A second active ingredient

In the present invention, the second active ingredient is a cytokine.

Preferably, the cytokine includes wild-type and mutant cytokines. The cytokine includes a cytokine derived from a human or non-human mammal. The cytokine may be isolated, recombinantly produced, modified (e.g., PEG-modified) or unmodified.

One preferred class of cytokines are cytokines modified so that the half-life is extended, such as pegylated cytokines, e.g., pegylated TRAIL, pegylated IFN.

In the present invention, representative cytokines include (but are not limited to): an interferon, a tumor necrosis factor, or a combination thereof.

Preferably, the interferon includes (but is not limited to): IFN-alpha, IFN-gamma, or a combination thereof.

Preferably, the tumor necrosis factor includes (but is not limited to): TRAIL, TNF- α, or a combination thereof.

In the present invention, TRAIL is a tumor necrosis factor-related apoptosis-inducing ligand (TNF-related apoptosis-inducing ligand), which is a member of the Tumor Necrosis Factor (TNF) superfamily and can selectively induce cancer cells to undergo apoptosis after being specifically bound to death receptors. TRAIL, which is a selective killing ligand targeting cancer cells, can be highly expressed in cancer cells, plays an important role in the generation, development and treatment of cancer, and is considered to be an anti-tumor cytokine with great development potential.

In the invention, IFN-alpha belongs to type I interferon, and can increase the expression of virus antigen polypeptide by up-regulating the expression of MHC I protein, thereby enhancing the activation of CD8+ T cells and playing an antiviral role. IFN-alpha may exert anti-tumor effects by altering cell differentiation status, interfering with oncogene expression, altering cell surface antigen expression, increasing macrophage phagocytic activity, enhancing lymphocyte cytotoxicity to target cells, and the like. IFN-alpha is clinically used for treating hairy cell leukemia, malignant melanoma and AIDS-related Kaposi sarcoma.

In the invention, IFN-gamma belongs to II type interferon, and IFN-gamma can stimulate the transcriptional expression of immunoglobulin in B cells, up-regulate antigen presenting path, activate macrophage activity and enhance the antiviral and antitumor effects of I type interferon. IFN-gamma is used clinically to treat chronic granulomatous disease and osteonecrosis.

The main role of TNF- α (Tumor necrosis factor- α) is to regulate immune cells. In addition, TNF-alpha can induce apoptosis, induce inflammation, inhibit tumorigenesis and viral replication.

Pharmaceutical composition, kit and active ingredient combination

The present invention provides a pharmaceutical composition comprising:

In another preferred embodiment, the first active ingredient is present in an amount ranging from 0.01% to 99.99% by weight of the total weight of the active ingredients of the composition; preferably 0.1% to 99.9%; more preferably 1% to 99%; preferably 10% to 99%; more preferably from 20% to 99%; more preferably 30 to 99%, still more preferably 40 to 99%.

In another preferred embodiment, the second active ingredient is present in an amount ranging from 0.01% to 99.99% by weight of the total weight of the active ingredients of the composition; preferably 0.1% to 99.9%; more preferably 1% to 99%; more preferably 1% to 90%; more preferably 1% to 80%; more preferably 1 to 70%, still more preferably 1 to 60%.

In a preferred embodiment, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier.

Typically, the pharmaceutical composition comprises:

(a) a therapeutically effective amount of a first active ingredient which is a PDE3 inhibitor;

(b) a therapeutically effective amount of a second active ingredient which is a cytokine; and

(c) a pharmaceutically acceptable carrier.

The pharmaceutical composition containing the first active ingredient and the second active ingredient can be various dosage forms suitable for oral administration, and also can be various external administration preparations or other parenteral administration preparations. For example, the external administration preparation of the present invention may be further prepared by adding adjuvants such as surfactants, transdermal absorption enhancers, preservatives, solvents, antioxidants, humectants, pH regulators, colorants, fragrances, etc. (including but not limited to): liniment, tincture, oil, ointment, plaster, cataplasm, plaster, patch, plastics, membrane, gel, cataplasma, acupoint application agent, spray, aerosol, implant, emulsion, etc. Preferred dosage forms include: various dosage forms for oral administration, implants, and injection preparations (such as injection, infusion solution or lyophilized powder for injection).

It is to be understood that, in the present invention, the carrier is not particularly limited, and is a material commonly used in the art, and the kind, use method, and source thereof are well known to those skilled in the art.

Examples of pharmaceutically acceptable carrier moieties are cellulose and its derivatives (e.g., methylcellulose, ethylcellulose, hydroxypropylmethylcellulose, sodium carboxymethylcellulose, etc.), gelatin, talc, solid lubricants (e.g., stearic acid, magnesium stearate), calcium sulfate, vegetable oils (e.g., soybean oil, sesame oil, peanut oil, olive oil, etc.), polyols (e.g., propylene glycol, glycerin, mannitol, sorbitol, etc.), emulsifiers (e.g., tween), wetting agents (e.g., sodium lauryl sulfate), buffers, chelating agents, thickeners, pH adjusters, transdermal enhancers, colorants, flavors, stabilizers, antioxidants, preservatives, bacteriostats, pyrogen-free water, etc.

The present invention also provides a kit comprising:

(A) a first formulation comprising a PDE3 inhibitor; and

(B) a second agent comprising a cytokine.

In another preferred embodiment, the kit further comprises instructions.

The invention also provides an active ingredient combination, which comprises the following components:

In the active ingredient combination, the first active ingredient and the second active ingredient may be independent of each other, or may be combined together to be present in the form of an active ingredient combination. Preferably, at least one of the active ingredients in the combination of active ingredients is independent.

The pharmaceutical compositions, active ingredient combinations, kits of the invention can be prepared using conventional methods and equipment.

Use and mode of administration

The invention provides an application of the pharmaceutical composition, the kit or the active ingredient combination in preparing a medicament for preventing and/or treating tumors.

Preferably, the tumor includes (but is not limited to): cervical cancer, liver cancer, kidney cancer, colon cancer, brain glioma, melanoma, or combinations thereof.

The first active ingredient and the second active ingredient in the pharmaceutical composition, the kit or the active ingredient combination can generate synergistic effect on the prevention and treatment of tumors and enhance the effect of treating the tumors.

Other anti-tumor agents may be used in combination before, simultaneously with, or after the use of the pharmaceutical composition, kit or active ingredient combination of the present invention.

In the process of drug combination, the interaction of the drugs is divided into additive action, synergistic action and antagonistic action according to the effect of the drugs when used together, the synergistic action means that the effect of the drugs when used together is obviously enhanced compared with the effect when used alone, the additive action means that the effect of the drugs when used together is equivalent to that when used alone, and the antagonistic action means that the effect of the drugs when used together is smaller than that when used alone. In the present invention, it was first found that the combined use of the first active ingredient, the second active ingredient and the third active ingredient has a synergistic effect.

In the process of preventing and/or treating tumors, the administration mode of the invention comprises the sequential administration of the first active ingredient and the second active ingredient, or the simultaneous administration of the first active ingredient and the second active ingredient.

The pharmaceutical formulation is adapted to the mode of administration and, in using the pharmaceutical composition or formulation, a safe and effective amount of the drug is administered to the subject in need thereof (e.g., a human or non-human mammal), wherein the safe and effective daily dosage of the first active ingredient is generally at least about 0.1mg, and in most cases, no more than about 2500 mg. Preferably, the dose is 1mg to 500 mg; a safe and effective amount of the second active ingredient is generally at least about 0.01mg, and in most cases does not exceed 2500 mg. Preferably, the dosage range is from 0.1mg to 2500mg, and a safe and effective amount of the third active ingredient is generally at least about 0.01mg, and in most cases does not exceed 2500 mg. Preferably, the dosage range is 0.1mg to 2500 mg. Of course, the particular dosage will depend upon such factors as the route of administration, the health of the patient, and the like, and is within the skill of the skilled practitioner. When the first active ingredient, the second active ingredient and the third active ingredient are administered sequentially, the interval between administrations is not particularly required. The compositions, active ingredient combinations, pharmaceutical compositions and kits of the present invention comprise a first active ingredient, a second active ingredient and a third active ingredient, each administered simultaneously or sequentially by the same or different routes, including but not limited to: oral administration, injection administration, intratumoral administration, implantation administration, intracavity administration, anal administration, transdermal administration and internal and external application;

preferred administration by injection includes: intravenous injection, intramuscular injection, subcutaneous injection, intracavity injection.

The invention also provides a method for preventing and/or treating tumors, and the pharmaceutical composition, the kit or the active ingredient combination are administered to a needed object.

In another preferred embodiment, the subject is a human or a non-human mammal.

The main advantages of the invention include:

1. in the invention, the combined application of the PDE3 inhibitor (such as ANA or ZARD) and the cell factor (such as TRAIL, TNF-alpha, IFN-alpha or IFN-gamma) obviously enhances the treatment effect on the tumor in a synergistic way, and the synergistic treatment effect of the PDE3 inhibitor and the cell factor is obviously better than that of the PDE3 inhibitor or the cell factor which is used singly.

The invention will be further illustrated with reference to the following specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. The experimental procedures, in which specific conditions are not noted in the following examples, are generally carried out under conventional conditions or conditions recommended by the manufacturers. Unless otherwise indicated, percentages and parts are by weight.

Example 1

Cytotoxicity test

1. Taking the tumor cells in logarithmic growth phase for digestion and counting, inoculating 3000-5000 cells/well into a 96-well plate, wherein each well contains 50ul of culture medium, and performing 5% CO at constant temperature of 37 DEG C₂The cell culture box attached to the wall overnight.

2. After the cells were stabilized, 50ul of the compound (wherein ANA represents Anagrelide (Anagrelide) and ZARD represents Zadavirin (Zardaverine)) at the drug concentration shown in Table 1 was added per well and incubated for 72 h.

3. After the drug incubation time was completed, the response of the cells to the drug (cell number, cell state) was first observed under a microscope, and then 30uL of MTT (5mg/ml in PBS) was added to each well for 3-5 hours.

4. After the MTT incubation is finished, throwing out the liquid of the 96-pore plate to the side of the water tank (throwing out the liquid in the plate as much as possible but not throwing off the blue-violet crystal in the plate), reversely buckling the 96-pore plate on the disposable paper towel, and sucking the residual liquid of the 96-pore plate.

5. Add 150ul DMSO per well and shake horizontally at 600rpm for 3min to allow complete dissolution of crystalline Formazan (Formazan).

6. The cell viability was calculated by the microplate reader at the detection wavelength of 492nM to measure the OD (PS blank: OD of detection medium (without plating cells) control: DMSO (plating cells) in the same volume as the drug was added to the medium)

TABLE 1

Results of the experiment

In the MTT experiment, the treatment results of different tumor cells by each drug group are shown in figure 1A, figure 1B and table 2, and as can be seen from figure 1A and figure 1B, compared with the administration of ANA, ZARD, TRAIL, IFN-alpha and IFN-gamma alone, the combination of ANA, ZARD, interferon IFN-alpha, IFN-gamma and apoptosis-related factor TRAIL can exert synergistic effect, and the synergistic antitumor effect is obviously better than that of the independent use of PDE3 inhibitor or cytokine, especially the combined use of ANA + TRAIL has excellent synergistic antitumor effect.

TABLE 2

Example 2

RTCA experiment

1. Adding 50ul of cell culture medium of cells to be detected into each hole of the E-plate, and detecting the background impedance value

2. 50ul of cell culture medium containing 3000-.

3. Cells were cultured adherent to the wall overnight, when the growth curve entered logarithmic growth phase. And taking out the E-plate from the detection platform, adding 50ul of the compound to be detected into each hole to reach the specified concentration (the final concentration of each group is shown in the table 3), and putting back the E-plate to the detection platform again after the treatment is finished to detect the cell growth in real time.

TABLE 3

Results of the experiment

In the RTCA experiment, the results of the treatment of different tumor cells by each drug group are shown in fig. 2A-2F, and it can be seen from fig. 2A-2F that the combination of ANA, ZARD, interferon IFN- α, IFN- γ and apoptosis-related factor TRAIL can exert synergistic effect compared with the administration of ANA, ZARD, TRAIL, IFN- α and IFN- γ alone, and the synergistic antitumor effect is significantly better than that of the PDE3 inhibitor or cytokine alone, especially the combination of ANA + TRAIL has excellent synergistic antitumor effect.

Example 3

The experimental procedure was substantially the same as in example 2, and the Bel7404, SW1116 cell lines were treated with TNF-. alpha. (10ng/mL), ANA (100nM) and TNF-. alpha. (10ng/mL) + ANA (100 nM); TNF-. alpha. (10ng/mL), ZARD (100nM) and TNF-. alpha. (10ng/mL) + ZARD (100nM) treated Bel7404, SW1116 cell lines.

The experimental results are shown in fig. 3A-3B, and the results show that ANA, ZARD and tumor necrosis factor TNF-alpha can exert synergistic effect, and the synergistic antitumor effect is obviously superior to that of PDE3 inhibitor or tumor necrosis factor TNF-alpha used alone.

All documents referred to herein are incorporated by reference into this application as if each were individually incorporated by reference. Furthermore, it should be understood that various changes and modifications of the present invention can be made by those skilled in the art after reading the above teachings of the present invention, and these equivalents also fall within the scope of the present invention as defined by the appended claims.

Claims

1. A pharmaceutical composition, wherein the composition comprises:

2. The pharmaceutical composition of claim 1, wherein the weight ratio of the first active ingredient to the second active ingredient is 1: (0.1-50), preferably, 1: (0.1-20), more preferably, 1: (0.15-10), most preferably, 1: (0.2-5).

3. The pharmaceutical composition of claim 1, wherein the PDE3 inhibitor is selected from the group consisting of: a compound of formula I or a pharmaceutically acceptable salt thereof, a compound of formula II or a pharmaceutically acceptable salt thereof, or a combination thereof;

wherein the content of the first and second substances,

r1, R2, R3, R4, R5, R6, R7 and R8 are each independently selected from the group consisting of: a hydrogen atom, a halogen atom, an amino group, a hydroxyl group, a cyano group, an aldehyde group, a nitro group, a substituted or unsubstituted C1-C4 carboxyl group, a substituted or unsubstituted C1-C10 alkyl group, a substituted or unsubstituted C3-C10 cycloalkyl group, a substituted or unsubstituted C2-C10 alkenyl group, a substituted or unsubstituted C2-C10 alkynyl group, a substituted or unsubstituted C6-C10 aryl group, a substituted or unsubstituted C5-C10 heteroaryl group (e.g., a substituted or unsubstituted 5-or 6-membered heterocycle, an 8-to 10-membered heteroaryl group), a substituted or unsubstituted C1-C10 alkoxy group, a substituted or unsubstituted C6-C10 aryl-oxy group, a substituted or unsubstituted C5-C10 heteroaryl-oxy groupSubstituted or unsubstituted C₂-C₁₀Acyl (, substituted or unsubstituted C)₂-C₁₀An ester group, a substituted or unsubstituted C1-C10 sulfonyl (-SO2-C1-C10 alkyl);

wherein the content of the first and second substances,

represents a single bond or is absent;

4. The pharmaceutical composition of claim 1, wherein the PDE3 inhibitor is selected from the group consisting of: anagrelide (Anagrelide) or a pharmaceutically acceptable salt thereof, zadavirin (Zardaverine) or a pharmaceutically acceptable salt thereof, or a combination thereof.

5. The pharmaceutical composition of claim 1, wherein the cytokine is selected from the group consisting of: an interferon, a tumor necrosis factor, or a combination thereof.

6. The pharmaceutical composition of claim 1, wherein the pharmaceutical composition further comprises a pharmaceutically acceptable carrier.

7. A kit, comprising:

(A) a first formulation comprising a PDE3 inhibitor; and

(B) a second agent comprising a cytokine.

8. An active ingredient combination, characterized in that the active ingredient combination comprises the following components:

9. Use of a pharmaceutical composition according to claim 1, a kit according to claim 4, and/or an active ingredient combination according to claim 5 for the preparation of a medicament for the prophylaxis and/or treatment of tumors.

10. The use of claim 6, wherein said tumor is selected from the group consisting of: cervical cancer, liver cancer, kidney cancer, colon cancer, brain glioma, melanoma, or combinations thereof.