Macrophage polarization agent containing organic germanium compound and use thereof

Abstract

To provide novel means for inducing the polarization of macrophages toward the M1 type.SOLUTION: The present invention provides an M1 polarization agent and a SIRPα expression inhibitor for macrophages, which contain at least one selected from among compounds of the general formula (I) in the figure, pharmaceutically acceptable salts and esters thereof, and polymers thereof. The present invention also provides the use of the M1 polarization agent and M1-polarized macrophages in the treatment of diseases or conditions, particularly in the treatment of cancer.SELECTED DRAWING: Figure 1

Description

Technical Field

[0001] The present invention relates to an M1 polarizing agent and SIRPα expression inhibitor for macrophages containing an organogermanium compound, and the use of the M1 polarizing agent and M1-polarized macrophages in the treatment of diseases or conditions, particularly cancer.

Background Art

[0002] Macrophages are one of the important factors in the immune system and have plasticity that can greatly change their form and function in response to local environmental signals. Macrophages are roughly classified into inflammation-promoting M1 type and anti-inflammatory M2 type. M1-type macrophages (M1 macrophages) have high antigen-presenting ability and phagocytic ability, secrete inflammatory cytokines, and contribute to the removal of pathogens such as bacteria and viruses and damaged tissues. M2-type macrophages (M2 macrophages) play roles such as wound healing and immune tolerance by secreting anti-inflammatory cytokines and growth factors. M1 macrophages and M2 macrophages are constantly balancing each other, and when the balance is disrupted, various inflammatory diseases occur.

[0003] Macrophages also play an important role in the cancer area. M1 macrophages exhibit enhanced immune response and phagocytic ability against tumors, and also suppress cancer progression and metastasis. Therefore, various efforts for cancer treatment using M1 macrophages have been underway. However, cancer treatment using M1 macrophages has a problem that phagocytosis of macrophages is suppressed by the binding of SIRPα (signal regulator protein α) on macrophages to CD47 on target cells. Various types of cancer cells highly express CD47 compared to normal cells, and it is considered that they emit a phagocytosis avoidance signal from the CD47-SIRPα immune checkpoint pathway and escape phagocytosis by macrophages. Inhibition of the CD47-SIRPα immune checkpoint using neutralizing antibodies or the like is known to enhance phagocytosis of cancer cells by macrophages and suppress cancer progression.

[0004] In addition, macrophages called TAM (Tumor-Associated Macrophage) present in or near the tumor have M2-like properties, suppress anti-tumor immunity through the secretion of immunosuppressive factors such as anti-inflammatory cytokines, promote cancer metastasis through the secretion of TGF-β, and enhance angiogenesis through the secretion of VEGF. Since TAM provides a microenvironment favorable for the growth of cancer cells in this way, suppression of TAM has also become an important issue in cancer treatment.

[0005] On the other hand, Ge-132 (also called poly-trans-[(2-carboxyethyl)germasesquioxane], repagermanium, and asagermanium) is an organogermanium compound having various physiological effects such as immunostimulatory effect, anti-tumor effect, anti-inflammatory effect, analgesic effect, and synergistic effect with morphine. The anti-tumor effect of Ge-132 is exerted through its hydrolyzate, (3-(trihydroxygermyl)propanoic acid (THGP). In in vivo tests using mice and rats, it has been clarified that it is due to an increase in the secretion of IFN-γ induced by the activation of NK cells and the subsequent activation of macrophages (Non-Patent Documents 1 to 3).

Prior Art Documents

Non-Patent Documents

[0006]

Non-Patent Document 1

Non-Patent Document 2

Non-Patent Document 3

Disclosure of the Invention

Problems to be Solved by the Invention

[0007] An object of the present invention is to provide a new means for inducing the polarization of macrophages into M1 type (M1 polarization).

Means for Solving the Problems

[0008] The present inventors have found that THGP induces the M1 polarization of macrophages under specific conditions, suppresses the expression of SIRPα on macrophages, and further inhibits the epithelial-mesenchymal transition of cancer cells and suppresses the expression of CD47 through the M1 polarization of macrophages, and have completed the following invention.

[0009] (1) A compound of general formula (I)

Chemical Formula

Chemical formula

Chemical formula

Advantages of the Invention

[0010] According to the present invention, M1 polarization of macrophages can be induced, and M1-polarized macrophages can be produced. In addition to having high phagocytic ability, the expression of SIRPα in these macrophages is suppressed, and they can also cause inhibition of epithelial-mesenchymal transition and suppression of CD47 expression in cancer cells. Therefore, they are useful in the treatment of cancer.

Brief Description of the Drawings

[0011]

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Figure 7

Figure 8

Figure 9

Mode for Carrying Out the Invention

[0012] The first aspect of the present invention relates to an M1 polarization agent for macrophages containing at least one of the compounds of general formula (I)

Chemical formula

[0013] Compound The compound used in the present invention is a compound of general formula (I) or a pharmaceutically acceptable salt or ester thereof or a polymer thereof. Here, the polymer is a polymer that gives a compound of general formula (I) or a pharmaceutically acceptable salt or ester thereof by hydrolysis.

[0014] In the compound of general formula (I), R 1 and R 2 are, independently of each other, hydrogen; a halogen such as fluorine, chlorine or bromine; nitro; hydroxy; cyano; C 1-4 alkyl which is linear, branched or cyclic and has 1 to 4 carbon atoms; C 1-4 haloalkyl which is linear, branched or cyclic and has 1 to 4 carbon atoms and is substituted with one or more halogens; C 2-4 alkenyl which is linear, branched or cyclic and has 2 to 4 carbon atoms; C 2-4 haloalkenyl which is linear, branched or cyclic and has 2 to 4 carbon atoms and is substituted with one or more halogens; C 3-4 alkynyl which is linear or branched and has 3 to 4 carbon atoms; C 3-4 haloalkynyl which is linear or branched and has 3 to 4 carbon atoms and is substituted with one or more halogens; -O-C 1-4 alkyl represented by C 1-4 alkoxy; -O-C 1-4 haloalkyl represented by C 1-4 haloalkoxy; -S-C 1-4 alkyl represented by C 1-4 alkylthio; -SO-C 1-4 alkyl represented by C 1-4 alkylsulfinyl; or -SO 2 -C 1-4 alkyl represented by C 1-4 alkylsulfonyl.

[0015] Also, R 1 and R 2 may, together with the two carbon atoms to which they are attached, form a 4- to 10-membered monocyclic or polycyclic saturated ring, a 4- to 10-membered monocyclic or polycyclic partially saturated ring, a 6- to 10-membered monocyclic or polycyclic aromatic ring, or a 5- to 10-membered monocyclic or polycyclic heterocyclic ring containing 1 to 4 atoms selected from nitrogen, oxygen and sulfur.

[0016] A saturated monocyclic or polycyclic ring having 4 to 10 members is a saturated carbon ring having one or more ring structures and having 4 to 10 carbon atoms as ring-constituting atoms. Examples include cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, bicyclooctyl, spirooctyl, and the like.

[0017] A partially saturated monocyclic or polycyclic ring having 4 to 10 members is a partially saturated carbon ring having one or more ring structures and having 4 to 10 carbon atoms as ring-constituting atoms. Examples include cyclobutenyl, cyclopentenyl, cyclohexenyl, cycloheptenyl, cyclooctenyl, bicyclooctenyl, and the like.

[0018] An aromatic monocyclic or polycyclic ring having 6 to 10 members is an aromatic ring having one or more ring structures and having 6 to 10 carbon atoms as ring-constituting atoms. Examples include phenyl, naphthyl, indyl, and the like.

[0019] A heterocyclic ring having 5 to 10 members and containing 1 to 4 atoms selected from the group consisting of nitrogen, oxygen, and sulfur is a saturated ring, a partially saturated ring, or an aromatic ring having one or more ring structures and having 6 to 10 atoms as ring-constituting atoms, wherein 1 to 4 of the ring-constituting atoms are heteroatoms independently selected from the group consisting of nitrogen, oxygen, and sulfur, and the other ring-constituting atoms are carbon atoms. Examples include furanyl, thiophenyl, pyrrolyl, imidazolyl, pyranyl, pyridinyl, pyrimidinyl, pyrazinyl, pyrrolidinyl, piperidinyl, piperazinyl, morpholinyl, indolyl, quinolyl, isoquinolyl, and the like.

[0020] R 1 and R 2 The ring formed by is halogen, nitro, hydroxy, cyano, oxo, C 1-4 alkyl, C 1-4 haloalkyl, C 2-4 alkenyl, C 2-4 haloalkenyl, C 3-4 alkynyl, C 3-4 haloalkynyl, C 1-4Alkoxy, C 1-4 Haloalkoxy, C 1-4 Alkylthio, C 1-4 Alkylsulfinyl and C 1-4 Alkylsulfonyl may be substituted by one or more substituents from the group consisting of. Details of each group are as described in the description of R 1 and R 2 as described in the description.

[0021] In the compound of general formula (I), R 3 is hydrogen, halogen, nitro, hydroxy, cyano, C 1-4 alkyl, C 1-4 haloalkyl, C 2-4 alkenyl, C 2-4 haloalkenyl, C 3-4 alkynyl, C 3-4 haloalkynyl, C 1-4 alkoxy, C 1-4 haloalkoxy, C 1-4 alkylthio, C 1-4 alkylsulfinyl or C 1-4 alkylsulfonyl. Details of each group are as described in the description of R 1 and R 2 as described in the description.

[0022] Compounds preferably used in the present invention are those in which, in general formula (I), R 1 , R 2 and R 3 are, independently of each other, hydrogen, halogen, nitro, hydroxy, cyano, C 1-4 alkyl, C 1-4 haloalkyl, C 2-4 alkenyl, C 2-4 haloalkenyl, C 3-4 alkynyl, C3-4 haloalkynyl, C 1-4 alkoxy, C 1-4 haloalkoxy, C 1-4 alkylthio, C 1-4 alkylsulfinyl or C 1-4It is a compound that is an alkylsulfonyl, or a pharmaceutically acceptable salt or ester thereof, or a polymer of these.

[0023] As the compound of general formula (I) more preferably used in the present invention, R 1 ~R 3 are all hydrogen compounds (THGP); R 1 and R 2 form a 5-membered monocyclic saturated ring together with the two carbon atoms to which they are attached, and R 3 is hydrogen compound (2-(trihydroxygermyl)cyclopentanecarboxylic acid); R 1 and R 2 form a 6-membered monocyclic saturated ring together with the two carbon atoms to which they are attached, and R 3 is hydrogen compound (2-(trihydroxygermyl)cyclohexanecarboxylic acid); R 1 and R 2 form an 8-membered polycyclic saturated ring, especially bicyclo[2.2.2]octane, together with the two carbon atoms to which they are attached, and R 3 is hydrogen compound (for example, 3-(trihydroxygermyl)bicyclo[2.2.2]octane-2-carboxylic acid) can be mentioned.

[0024] In the present invention, particularly preferred compounds are THGP or a pharmaceutically acceptable salt or ester thereof, or a polymer of these.

[0025] The present invention encompasses the use of pharmaceutically acceptable salts or esters of the compounds of general formula (I). Such salts include salts with conventional bases, for example, alkali metal salts (such as sodium salt and potassium salt), alkaline earth metal salts (such as calcium salt and magnesium salt), ammonium salts, or organic amines (such as ethylamine, diethylamine, triethylamine, DIPEA, monoethanolamine, diethanolamine, triethanolamine, dimethylaminoethanol, diethylaminoethanol, tris(hydroxymethyl)aminomethane, choline (2-hydroxy-N,N,N-trimethylethanaminium), procaine, dicyclohexylamine, dibenzylamine, N-methylmorpholine, N-methylpiperidine, arginine, lysine and 1,2-ethylenediamine).

[0026] The pharmaceutically acceptable esters of the compounds of general formula (I) are in vivo hydrolyzable esters of the carboxylic acids of general formula (I). Such esters include, for example, C 1-4 alkyl esters such as methyl, ethyl, tert-butyl esters and the like are preferred.

[0027] The polymers of the compounds of general formula (I) can be represented by general formula (II)

Chemical formula

[0028] The polymer can be obtained by a polymerization reaction in which a compound of the general formula (I) or a pharmaceutically acceptable salt or ester thereof dissolved in an aqueous solution is dried to cause intermolecular dehydration condensation, in the same manner as the production of Ge-132 from THGP. The polymer may have all the constituent units identical or different. The former polymer can be obtained by drying an aqueous solution in which one kind of the compound of the general formula (I) or a pharmaceutically acceptable salt or ester thereof is dissolved, and the latter polymer can be obtained by drying an aqueous solution in which plural kinds of the compound of the general formula (I) or a pharmaceutically acceptable salt or ester thereof are dissolved. Since the polymerization reaction is reversible, by dissolving the polymer in an aqueous medium such as water, the polymer is hydrolyzed to obtain the compound of the general formula (I) or a pharmaceutically acceptable salt or ester thereof.

[0029] Examples of the polymer of the compound of the general formula (I) include Ge-132, a water-soluble organic germanium compound Poly-[(2-carboxyethyl-hydroxygermanium)oxide] which is a linear polymer described in JP-A-57-102895, and a water-soluble organic germanium compound Propagermanium (3-oxygermylpropionic acid polymer) having a ladder structure (a cyclic structure composed of 8 atoms of germanium and oxygen) described in Mizuno et al. (J. Pharm. Sci., 2015, 104 (8), 2482-2488.).

[0030] A further example of the polymer of the compound of the general formula (I) is a compound of the general formula (III)

Chemical formula

[0031] R 4 , R 5 and R 6are independently hydrogen or lower alkyl. Lower alkyl refers to a linear or branched alkyl having 1 to 6 carbon atoms, preferably 1 to 4 carbon atoms, more preferably 1 to 3 carbon atoms. In a preferred embodiment, R 4 , R 5 and R 6 are all hydrogen.

[0032] X in the general formula (III) is an alkali metal cation such as Na + , K + , or an ammonium cation or a quaternary ammonium cation. The quaternary ammonium cation is a cation in which 4 alkyl groups and / or aryl groups are bonded to nitrogen. Preferred quaternary ammonium cations used in the present invention include cations in which 4 linear or branched alkyl groups having 1 to 6 carbon atoms, preferably 1 to 4 carbon atoms, more preferably 1 to 3 carbon atoms are bonded to nitrogen, for example, tetramethylammonium cation, tetraethylammonium cation, tetrapropylammonium cation, ethyltrimethylammonium, triethylmethylammonium, etc. can be mentioned. In a preferred embodiment, X is Na + .

[0033] Suitable examples of the compound of the general formula (III) include those in which R 4 ~R 6 are all hydrogen and X is Na + , 1, 7, 9, 15-tetra(2’-sodium carboxyethylgermanium)-3, 5, 11, 13-tetra-[sodium propanato(2-)-C 3’ , O’]-germanium-2, 4, 6, 8, 10, 12, 14, 16, 17, 18, 19, 20-dodecaoxa-pentacyclo [8.1 1, 5 . 1 7, 11 . 1 9, 13 . 1 3, 15 .] icosane (hereinafter referred to as THGP octamer) can be mentioned.

[0034] The compound of general formula (III) can be produced by crystallizing a compound of general formula (IV) (wherein R 4 ~R 6 is as described in the description of general formula (III)) in a mixed solvent of water and a water-miscible organic solvent.

Chemical formula

[0035] The compound of general formula (IV) can be synthesized as shown in the following scheme using an acrylic acid derivative of general formula (V) and trichlorogermanium.

Chemical formula

[0036] The hydrogermylation of the acrylic acid derivative of general formula (V) and trichlorogermanium can be carried out, for example, at a temperature of about 25 to 40 °C using concentrated hydrochloric acid, diethyl ether or chloroform as a solvent. Then, the compound obtained by hydrogermylation is reacted with an alkali metal hydroxide or an amine in the presence of water, typically in an aqueous solution, to carry out hydrolysis and neutralization, whereby the compound of general formula (IV) can be prepared.

[0037] Alternatively, a known THGP polymer such as Ge-132 is reacted with an alkali metal hydroxide or an amine in the presence of water to prepare a compound of general formula (IV) wherein R 4 ~R 6 is hydrogen.

[0038] The obtained compound of general formula (IV) is then crystallized in a mixed solvent of water and a water-miscible organic solvent. The mixing ratio of water and the organic solvent in the mixed solvent may be 1:3 to 1:20 (v / v), preferably 1:3 to 1:5 (v / v). Examples of the organic solvent used include acetone, methanol, ethanol, 1-propanol, or 2-propanol, etc., and ethanol is particularly preferred.

[0039] By adding the above organic solvent in an amount such that the mixing ratio is within the above range to an aqueous solution of the compound of general formula (IV), a mixed solution for crystallization is prepared. The concentration of the compound of general formula (IV) in this mixed solution may be 2% to 12% (w / v), preferably 8% to 12% (w / v).

[0040] After sufficiently stirring the above mixed solution, the crystals of the compound of general formula (III) can be precipitated by allowing it to stand at room temperature for 12 hours or more, preferably 24 hours or more. Also, to promote crystallization, crystals of the compound of general formula (III) may be added as seed crystals. The obtained crystals can be isolated and purified from the mixed solution by known means such as filtration, washing with an organic solvent such as a lower alcohol, and drying under reduced pressure.

[0041] Polarization of Macrophages The polarization of macrophages means that macrophages greatly change their morphology and function according to local environmental factors. Macrophages that have not been stimulated are usually in a resting state (M0 macrophages), but upon receiving external stimuli, they polarize into two different subsets, broadly classified as M1 type or M2 type (where M2a, M2b, M2c, and M2d exist).

[0042] The M1 polarization of macrophages is induced by IFN-γ alone or by the synergistic action of IFN-γ and other cytokines (such as TNF-α, GM-CSF, etc.) or bacterial-derived components such as LPS. M1 macrophages have high antigen-presenting ability and phagocytic ability, and in addition, produce inflammatory cytokines such as IL-12, IL-1, IL-6, TNF-α, and effector molecules such as nitric oxide and reactive oxygen species. Thereby, M1 macrophages play a role in promoting the elimination of pathogens and cancer cells and promoting the removal of damaged tissues.

[0043] In contrast, the M2 polarization of macrophages is induced by various stimuli such as IL-4, IL-13, TLR agonists, IL-1R, IL-10, TGF-β, and glucocorticoids. M2 macrophages play a role in promoting tissue repair and suppressing the immune response through the production of anti-inflammatory cytokines such as IL-10 and TGF-β. TAM has an M2-like phenotype and is known to promote the proliferation and metastasis of cancer cells, enhance angiogenesis, and reduce the drug sensitivity of cancer cells.

[0044] M1 macrophages and M2 macrophages can be defined using known molecular markers that characterize each of them as indicators. Examples of molecular markers for M1 macrophages include cell surface markers such as CD80 and CD86, cytokines such as TNF-α, IL-1β, and IL-6, and inducible nitric oxide synthase (iNOS). Examples of molecular markers for M2 macrophages include cell surface markers such as CD206 and CD163, cytokines such as TGF-β and IL-10, chemokines such as CXCL2, and arginase, an arginine-degrading enzyme involved in ornithine production.

[0045] The polarization of macrophages refers to the state in which a specific subset becomes dominant in the macrophage cell population. Polarization only requires that a specific subset be dominant over other subsets, and does not necessarily involve a decrease in the number of macrophage cells or a decrease in the expression of molecular markers in other subsets.

[0046] M1 Polarizing Agent The M1 polarizing agent of the present invention (also simply referred to as a polarizing agent) can polarize macrophages into the M1 type. As described above, conventionally, IFN-γ has been considered necessary for M1 polarization. However, the M1 polarizing agent of the present invention can induce M1 polarization of macrophages even under conditions where IFN-γ is absent.

[0047] The M1 polarization of macrophages is carried out by treating macrophage precursor cells with an M1 polarizing agent (polarization treatment). The macrophage precursor cells may be macrophage progenitor cells such as monocytes, or unpolarized macrophages (M0 macrophages). Also, since polarized macrophages can be re-polarized into different subtypes, the macrophage precursor cells may be macrophages polarized into subsets other than the M1 type, typically M2 macrophages.

[0048] When macrophages after M1 polarization are scheduled to be administered to a mammalian individual, the macrophage precursor cells may be derived from the same individual or a different individual as the individual scheduled to receive the administration, that is, the macrophage precursor cells may be autologous or allogeneic. The macrophage precursor cells are preferably derived from the same individual, that is, autologous. The macrophage precursor cells can be prepared by collecting them in advance from the blood, bone marrow, spleen, etc. of a mammalian individual.

[0049] The polarization treatment in vitro can be carried out by culturing macrophage precursor cells in a medium containing a compound of general formula (I) or a pharmaceutically acceptable salt or ester thereof at a concentration of 50 μM to 10 mM for 7 days or more. The medium used in the polarization treatment has no limitation as long as it can culture macrophage precursor cells, and examples thereof include DMEM, MEM, α-MEM, and RPMI1640. The medium may contain additives such as fetal bovine serum (FBS), but does not necessarily contain M1 polarization inducer substances such as IFN-γ.

[0050] The concentration of the compound of general formula (I) or a pharmaceutically acceptable salt or ester thereof in the medium may be 50 μM to 10 mM, for example, 50 μM to 5 mM, preferably 500 μM to 5 mM. When using a polymer of the compound of general formula (I) or a pharmaceutically acceptable salt or ester thereof as a polarizing agent, an amount such that the concentration of the compound of general formula (I) or a pharmaceutically acceptable salt or ester thereof in the medium becomes 50 μM to 10 mM may be added to the medium.

[0051] The culture time for the polarization treatment may be 7 days or more, preferably 10 days or more. The culture time may be long-term. As shown in the examples described below, the inventors have confirmed that M1 polarization continues even when cultured for 120 days. Considering economy, the culture may be terminated when M1 polarization is confirmed. Therefore, the culture time may be, for example, 7 to 120 days. The culture time may be 7 to 90 days, 7 to 45 days, 7 to 40 days, 7 to 30 days, or 7 to 20 days. Preferably, the culture time is 10 to 120 days, 10 to 90 days, 10 to 45 days, 10 to 40 days, 10 to 30 days, or 10 to 20 days.

[0052] When the culture time is less than 10 days, the concentration of the compound of general formula (I) or a pharmaceutically acceptable salt or ester thereof in the medium is preferably 500 μM to 10 mM because there may be variations in the degree of M1 polarization at low concentrations.

[0053] As long as the medium contains the compound of general formula (I) or a pharmaceutically acceptable salt or ester thereof at the above concentration, medium exchange and subculture may be performed during polarized culture.

[0054] The culture temperature and gas concentration may be suitable for individual macrophage raw material cells. For example, the culture can be carried out at 37 °C and 5% CO 2 It can be carried out below.

[0055] Polarization treatment in vivo can be carried out, for example, in the case of oral administration, by administering to a subject a compound of general formula (I) or a pharmaceutically acceptable salt or ester thereof or a polymer thereof in an amount of about 5 to 500 mg / kg body weight per day for about 2 to 30 days.

[0056] M1 polarization of macrophages after polarization treatment can be confirmed by the fact that the number of M1 macrophages in the macrophage cell population is larger than the number of M2 macrophages. Alternatively, M1 polarization can be confirmed by the fact that the degree of increase in the expression of the M1 macrophage molecular marker in the whole macrophage cell population is greater than the degree of increase in the expression of the M2 macrophage molecular marker. For example, the expression of the M1 macrophage molecular marker is increased, and the expression of the M2 macrophage molecular marker has not changed or has decreased.

[0057] As shown in the examples described below, the polarizing agent of the present invention can increase M1 macrophages and decrease M2 macrophages. Therefore, the present invention also provides, in another aspect, an M1 macrophage induction promoter containing a compound of general formula (I) or a pharmaceutically acceptable salt or ester thereof or a polymer thereof, and an M2 macrophage induction inhibitor containing a compound of general formula (I) or a pharmaceutically acceptable salt or ester thereof or a polymer thereof.

[0058] Macrophage SIRPα Expression Inhibitor and CD47-SIRPα Immune Checkpoint Inhibitor The present invention provides, as another aspect, an inhibitor of SIRPα expression in macrophages, which contains a compound of general formula (I), a pharmaceutically acceptable salt or ester thereof, or a polymer thereof.

[0059] SIRPα is a receptor-type membrane protein that is highly expressed on the cell membranes of myeloid cells such as macrophages and nerve cells, and its physiological ligand is CD47. CD47 is highly expressed on the cell membranes of many cancer cells, and it is known to inhibit the phagocytosis of macrophages by binding to SIRPα on macrophages. This cell-cell signal CD47-SIRPα system has attracted attention as an immune checkpoint of the innate immune system, and inhibitors of the CD47-SIRPα immune checkpoint are expected as new cancer molecular target drugs.

[0060] There are two isoforms of human SIRPα and six isoforms of mouse SIRPα. Also, there are three isoforms of human CD47 and five isoforms of mouse CD47. Table 1 shows the amino acid sequences of each isoform registered in the Reference Sequence Database of the National Center for Biotechnology Information (NCBI) and the nucleotide sequences of the cDNA encoding the same. [Table 1]

[0061] The SIRPα expression inhibitor of the present invention can suppress the expression of SIRPα in macrophages and can produce macrophages with reduced expression of SIRPα. The suppression of SIRPα expression can be achieved by culturing macrophage precursor cells in a medium containing a compound of general formula (I) or a pharmaceutically acceptable salt or ester thereof at a concentration of 50 μM to 10 mM for 7 days or more. The macrophage precursor cells, the concentration of the compound of general formula (I) or a pharmaceutically acceptable salt or ester thereof in the medium, the culture time, and other culture conditions are as described in the polarization treatment.

[0062] The suppression of SIRPα expression may be confirmed at the gene level or at the protein level. The suppression of expression at the gene level can be confirmed by hybridization, quantitative PCR, RNA sequencing, or other general methods capable of detecting or quantifying specific gene expression using the nucleotide sequence information of the gene encoding SIRPα. The suppression of expression at the protein level can be confirmed by ELISA, RIA, in situ hybridization, Western blot analysis, or other general methods capable of detecting or quantifying specific protein expression using specific antibodies.

[0063] Macrophages with reduced expression of SIRPα are expected to be able to avoid the CD47-SIRPα immune checkpoint because their binding to CD47 on cancer cells is suppressed. Therefore, the present invention provides, in another aspect, a CD47-SIRPα immune checkpoint inhibitor containing a compound of general formula (I) or a pharmaceutically acceptable salt or ester thereof or a polymer thereof.

[0064] Inhibition of the CD47-SIRPα immune checkpoint can be confirmed by ELISA capable of detecting the binding between CD47 and SIRPα, cell-based reporter assays capable of detecting signal transduction from SIRPα bound with CD47, or other general methods capable of detecting or quantifying the binding or signal transduction between specific proteins.

[0065] Composition As described above, M1-polarized macrophages induced by the compound of general formula (I) or a pharmaceutically acceptable salt or ester thereof or a polymer thereof have high phagocytic ability and can inhibit the CD47-SIRPα immune checkpoint through suppressing the expression of SIRPα. The inventors further found that this M1-polarized macrophage can inhibit the epithelial-mesenchymal transition of cancer cells through its secretions and can suppress the expression of CD47 on cancer cells.

[0066] Epithelial-mesenchymal transition is a phenomenon in which epithelial cells lose their epithelial traits and acquire mesenchymal traits, and is involved in cancer invasion and metastasis in addition to wound healing and accompanying tissue fibrosis. Since cancer cells acquire high metastatic ability, invasiveness, tumorigenicity, and stress resistance through epithelial-mesenchymal transition, inhibition of the epithelial-mesenchymal transition of cancer cells can suppress cancer metastasis and improve drug resistance.

[0067] In addition, suppression of the expression of CD47 on cancer cells, in combination with suppression of the expression of SIRPα on macrophages, is considered to strongly inhibit the CD47-SIRPα immune checkpoint.

[0068] Therefore, by allowing the compound of general formula (I) or a pharmaceutically acceptable salt or ester thereof to coexist with macrophage precursor cells in the body for a long time, M1 polarization of macrophages and suppression of the expression of SIRPα are induced in the body. Furthermore, the induced M1 macrophages inhibit the epithelial-mesenchymal transition of cancer cells and suppress the expression of CD47, so it is considered that cancer can be treated.

[0069] Although the antitumor effect of Ge-132, which is a polymer of THGP, is known, its mechanism is said to be an increase in the secretion of IFN-γ induced by the activation of NK cells and the accompanying induction of M1 macrophages. In patients in whom IFN-γ is not sufficiently produced or in patients with low responsiveness to IFN-γ stimulation in whom M1 polarization of macrophages is less likely to occur due to IFN-γ stimulation, it was considered that Ge-132 could not fully exert its antitumor effect.

[0070] However, according to the present invention, by contacting THGP with macrophage precursor cells for a long time, M1 polarization of macrophages can be induced even in the absence of IFN-γ. This means that THGP can be effective even in cancer patients in whom the antitumor effect of THGP has conventionally been considered not to be exerted.

[0071] Thus, the present invention provides a composition for use in treating a disease or condition in a subject in whom there is no IFN-γ present in an amount capable of inducing M1 polarization of macrophages or in whom M1 polarization of macrophages is not induced by IFN-γ, which composition contains a compound of general formula (I) or a pharmaceutically acceptable salt or ester thereof or a polymer thereof.

[0072] In addition, M1 polarized macrophages prepared by coexisting a compound of general formula (I) or a pharmaceutically acceptable salt or ester thereof in vitro with macrophage precursor cells for a long time are also expected to exert a similar effect by administration to a subject. Therefore, the present invention also provides a composition for use in treating a disease or condition, which composition contains M1 polarized macrophages prepared by a compound of general formula (I) or a pharmaceutically acceptable salt or ester thereof or a polymer thereof.

[0073] In addition, the culture supernatant of M1-polarized macrophages contains various humoral factors produced by macrophages, and these are also considered to be effective in the treatment of diseases or conditions. Therefore, in the present invention, a composition containing the culture supernatant of M1-polarized macrophages prepared by co-existing a compound of general formula (I) or a pharmaceutically acceptable salt or ester thereof with macrophage raw material cells in vitro for a long time can also be used as a composition for the treatment of diseases or symptoms. The culture supernatant may be the culture supernatant during the culture of the polarization treatment or the culture supernatant obtained by newly culturing M1-polarized macrophages.

[0074] The composition containing the compound of general formula (I) or a pharmaceutically acceptable salt or ester thereof or a polymer thereof, the composition containing M1-polarized macrophages, and the composition containing the culture supernatant of M1-polarized macrophages are preferably compositions for cancer treatment used for cancer patients, particularly pharmaceutical compositions for cancer treatment. The composition for cancer treatment can be used to enhance the phagocytosis of cancer cells by macrophages, to suppress the epithelial-mesenchymal transition of cancer cells, and to inhibit the CD47-SIRPα immune checkpoint.

[0075] In addition, the above compositions can be used in the treatment of various diseases or conditions expected to be improved or prevented by M1 macrophages other than cancer, for example, in the treatment of infectious diseases and injuries, and further in diseases or conditions involving chronic inflammation, such as chronic inflammatory diseases, allergic diseases, autoimmune diseases, arteriosclerotic diseases (ischemic heart disease, stroke, etc.), neurodegenerative diseases (Alzheimer's disease), metabolic syndrome and lifestyle-related diseases (obesity, diabetes, dyslipidemia, chronic kidney disease, non-alcoholic fatty liver disease, etc.), urinary disorders, etc.

[0076] The above composition is applicable to mammals such as a subject in need of treatment of a disease or condition, for example, rodents including mice, rats, hamsters, guinea pigs, humans, primates including chimpanzees and rhesus monkeys, livestock including pigs, cows, goats, horses, sheep, and companion animals including dogs and cats. A preferred subject is a human.

[0077] As used herein, the treatment of a disease or condition encompasses all types of interventions permitted in the application of a medicine, quasi-drug, food or drink, or cosmetic for the purpose of curing, temporarily relieving, improving, preventing, etc. a disease or condition, and includes, for example, delaying or stopping the progression of a disease or condition, regression or disappearance of a lesion, prevention of onset or prevention of recurrence, etc.

[0078] The above composition contains an effective amount of a compound of general formula (I) or a pharmaceutically acceptable salt or ester thereof or a polymer thereof, or an effective amount of M1 polarized macrophages, or an effective amount of an M1 polarized macrophage culture supernatant. Here, the "effective amount" means an amount effective for the treatment of a disease or condition, and is appropriately determined by those skilled in the art according to usage, the age of the subject, the nature of the disease or condition, and other conditions.

[0079] In a preferred embodiment, the effective amount of the compound of general formula (I) or a pharmaceutically acceptable salt or ester thereof or a polymer thereof is, for example, 10 μg to 300 mg, preferably 500 μg to 200 mg, more preferably 1 mg to 100 mg per 1 kg of the body weight of the subject administered in the case of oral administration, and the effective amount of the culture supernatant is, for example, 0.001 mg to 100 mg, preferably 0.002 mg to 50 mg, more preferably 0.005 mg to 20 mg per 1 kg of the body weight of the subject administered in the case of intravenous administration. Also, in a preferred embodiment, the effective amount of M1 polarized macrophages is 1×10 4 cells to 1×10 9 cells, preferably 1×10 5 cells to 1×10 8They are cells. These effective amounts can be administered in one or multiple divided doses.

[0080] The above composition can contain pharmaceutically acceptable carriers, buffers, stabilizers, preservatives, excipients and other components, and can be used as a pharmaceutical composition, quasi-drug composition, food or drink composition, or cosmetic composition. Components acceptable in these compositions are well-known to those skilled in the art for each purpose of the composition, and those skilled in the art can appropriately select and use them from the components described in, for example, the 17th revised Japanese Pharmacopoeia and other standards within the scope of their normal implementation ability.

[0081] The above composition can be used in combination with additional means according to its purpose. For example, when the composition is used as a medicine for treating a disease or condition, it can be used in combination with other means effective for treating the disease or condition. When the composition is used as a medicine for treating cancer, combination with chemotherapy, radiotherapy, surgery or immunotherapy is possible.

[0082] The dosage form of the composition containing the compound of general formula (I) or its pharmaceutically acceptable salt or ester or their polymers is arbitrary. For example, oral preparations (tablets, capsules, powders, granules, fine granules, pills, suspensions, emulsions, solutions, syrups, etc.), injections, external preparations (sprays, external solutions, inhalants, ointments, patches, etc.) and the like can be mentioned. The administration route of the composition is not particularly limited and is appropriately determined according to the dosage form. In one of the preferred embodiments, the composition can be administered orally, intravenously, intraperitoneally or transdermally.

[0083] Also, the dosage form of the composition containing M1-polarized macrophages and the composition containing the culture supernatant of M1-polarized macrophages is arbitrary, but it is preferably a parenteral preparation such as an injection. These compositions can be administered, for example, intravenously, intraperitoneally or locally to the affected area.

[0084] The present invention will be described in more detail by the following examples, but the present invention is not limited to these examples.

Example

[0085] Materials and Methods ·Cell Culture The cultured cell line RAW 264.7 derived from mouse macrophages and the cultured cell line B16 4A5 derived from mouse melanoma were provided by the Riken cell bank. The cells were cultured at 37 °C in 5% CO 2 using Dulbecco's modified Eagle's medium (DMEM) (Nissui Pharmaceutical Co., Ltd.) supplemented with 10% fetal bovine serum (FBS). When the cells reached sub-confluence, they were detached with a cell scraper or 0.25% trypsin / 1 mM EDTA and seeded at a ratio of 1:4 to 1:8 every 3 to 4 days. ·THGP Treatment The THGP treatment of RAW 264.7 cells was performed by culturing them in 10% FBS DMEM containing 50, 500, or 5000 μM THGP while subculturing every 3 days. The seeding density of RAW 264.7 cells at the time of subculture was 1×10 6 cells in a 10 cm dish and 5.0×10 5 cells / well in a 6-well plate.

[0086] ·Preparation of Conditional Medium Each of the RAW 264.7 cells cultured in 10% FBS DMEM containing 500 μM THGP for 10 days or more and within 120 days and the RAW 264.7 cells cultured in 10% FBS DMEM without THGP for 10 days or more and within 120 days was seeded at 1×10 6 cells in a 10 cm dish and cultured in 10% FBS DMEM for 3 days. The culture supernatant was collected and stored at -30 °C until use. At the time of use, the culture supernatant was mixed with 10% FBS DMEM at a ratio of 1:1 to obtain a conditional medium.

[0087] ·MTS Assay The MTS assay was performed by measuring the absorbance at 490 nm using the CellTiter 96® AQueous One Solution Cell Proliferation Assay kit (Promega).

[0088] ·Immunofluorescence Staining Cells were fixed with 4% paraformaldehyde in PBS (Wako Pure Chemical Industries), permeabilized with 0.2% Triton X-100 for 10 minutes at room temperature, and then blocked with 1.5% BSA for 30 minutes at room temperature. The primary antibody reaction was carried out overnight at 4°C, and the secondary antibody reaction was carried out for 1 hour at room temperature. As the primary antibodies, anti-B7-2 (CD86) antibody (sc-28347) (Santa Cruz), anti-Mannose Receptor (CD206) antibody (ab64693) (Abcam), and anti-N-Cadherin antibody (Cell signaling) were used. Also, as the secondary antibody, Goat Anti-Mouse IgG H&L (TexasRED) or Goat Anti-Rabbit IgG H&L (FITC) (Abcam) was used. The antibodies were diluted with blocking buffer and used according to the manufacturer's recommended concentration. The nuclei were stained with DAPI (Dojindo Laboratories). Fluorescence observation was performed using a fluorescence microscope Nikon Eclipse TS100, Nikon DS-Fi3 (Nikon), and analysis was carried out using image analysis software NIS-element (Nikon).

[0089] ·Real-Time RT-PCR RNA was extracted from cells using Isogen (Nippon Gene). Using 1 μg of the extracted RNA as a template, reverse transcription reaction was performed at 50 °C for 1 h and 95 °C for 5 min using SuperScript III (Invitrogen). PCR reaction was performed at 95 °C for 5 sec, 60 °C for 30 sec, for 40 cycles using TB Green Premix Ex Taq II (Tli RNaseH Plus) (Takara Bio). Primers used for the PCR reaction are shown in Table 2. RPS18 was used for correction as an internal control. Note that the primer sets for CD47 amplification and SIRPα amplification were designed to amplify sequences common to all isoforms of CD47 and SIRPα, respectively.

Table 2

[0090] ·Western Blot Proteins were extracted from cells using RIPA buffer. Proteins were quantified by the Bradford method (Bio-Rad). SDS-PAGE was performed using 7 μg (for RAW 264.7 cells) or 10 μg (for B16 4A5 cells) of protein. After transfer to a PVDF membrane, blocking was performed using 5% skim milk in TBS-T (Morinaga Milk Industry). The primary antibody reaction was carried out overnight at 4°C, and the secondary antibody reaction was carried out for 1 hour at room temperature. As the primary antibodies, anti-B7-2 (CD86) antibody (sc-28347) (Santa Cruz), anti-Mannose Receptor (CD206) antibody (ab64693) (Abcam), anti-N-Cadherin antibody (Cell signaling), and anti-β-actin antibody (Abcam) were used. Also, as the secondary antibodies, Goat Anti-Mouse IgG H&L (HRP) (ab6789), Goat Anti-Rabbit IgG H&L (HRP) (ab205718), and Donkey Anti-Goat IgG H&L (HRP) (ab205723) (Abcam) were used. The antibodies were diluted with blocking buffer according to the manufacturer's recommended concentration and used. Each band was quantified using the image analysis software Image-Lab (BioRad) and corrected with β-actin.

[0091] ·Phagocytosis of Latex Beads The evaluation of phagocytic ability was performed using a phagocytosis assay kit (Cayman Chemical Company). Rabbit IgG FITC-latex beads were diluted 1:200 and allowed to be phagocytosed by RAW 264.7 cells for 1 hour. The nuclei were stained with Hoechst 33452 (Dojindo Laboratories) and observed under a fluorescence microscope. The captured images were analyzed by NIS-element (Nikon), and the intensity of the fluorescence emitted by FITC was used as an index of phagocytic ability.

[0092] ·Phagocytosis of Cancer Cells (Fluorescent Staining) Raw 264.7 cells stained with Cell tracker Green CMFDA Dye (Thermo fisher) were seeded at 2×10 6 cells / well in a 6-well plate with a cover glass and cultured in DMEM with 10% FBS for 24 hours. Then, B16 4A5 cells stained with Cell tracker Blue CMAC Dye (Thermo fisher) were seeded at 2×10 6 cells / well on the RAW 264.7 cultured cells. After incubation at 37°C for 2 hours, the cells were fixed with 4% paraformaldehyde in PBS (Wako Pure Chemical Industries, Ltd.), observed under a fluorescence microscope, and the images taken were analyzed using NIS-element (Nikon). Cells co-stained with blue and green were regarded as RAW 264.7 cells that had phagocytosed B16 4A5 cells.

[0093] ·Cytotoxicity against Cancer Cells (MTS Assay) RAW 264.7 cells and B16 4A5 cells were seeded at 5×10 3 cells / well and 2.5×10 3 cells / well, respectively, in a 96-well plate and co-cultured in DMEM with 10% FBS for 48 hours. Also, the same number of RAW 264.7 cells and B16 4A5 cells were each cultured alone in the same manner. The cell count was evaluated by MTS assay. The percentage of B16 4A5 cells damaged by RAW 264.7 cells was calculated by subtracting the absorbance in the co-culture from the sum of the absorbances when RAW 264.7 cells and B16 4A5 cells were each cultured alone and then dividing the result by the absorbance when B16 4A5 cells were cultured alone.

[0094] ·ELISA The amount of TGF-β in the culture supernatant was measured using the LEGEND MAX Mouse Latent TGF-β ELISA Kit (BioLegend).

[0095] ·Migration Assay B16 4A5 cells were seeded at 3.0×10 5 cells / well in a 24-well plate and cultured in 10% FBS DMEM for 24 hours. After scraping the cells using a 1000 μl blue tip, the cells were washed twice with PBS (-) and the test medium was added. Cell images were taken at 0 hours and 48 hours after addition, and analyzed using the image analysis software Image J / Fiji (NIH). The area of the cells present in the scraped area after 48 hours was calculated with respect to the area of the scraped area of the cells, and used as an evaluation index for migration ability.

[0096] ·MMP-2 and MMP-9 Activity 30 μl of the culture supernatant was applied to a 10% acrylamide gel containing gelatin, and SDS-PAGE was performed. Using the gel after electrophoresis, the activities of MMP-2 and MMP-9 were evaluated by the Gelatin zymography method. The degree of gelatin degradation by MMP-2 and MMP-9 on the gel was measured by image analysis of the staining intensity of the corresponding bands using Image J / Fiji, and used as the activities of MMP-2 and MMP-9.

[0097] ·Cell Adhesion After culturing B16 4A5 cells in the test medium for 72 hours, the cells were collected. The collected cells were seeded at 5.0×10 4 cells / well in a 96-well plate coated with type I Collagen (Wako Pure Chemical Industries, Ltd.). After one hour, the wells were washed twice with PBS (-) to remove non-adherent cells, and the number of adherent cells was evaluated by MTS assay.

[0098] ·Invasiveness The invasion ability of B16 4A5 cells was evaluated by the Boyden chamber method using 8 μm pore size PET inserts (BD falcon) coated with type I collagen. RAW 264.7 cells were stained with Cell tracker Green CMFDA Dye (Thermo fisher), and B16 4A5 cells were stained with Cell tracker Blue CMAC Dye (Thermo fisher). 1×10 5 cells of RAW 264.7 and 5×10 4 cells of B16 4A5 were seeded on the upper layer of the Boyden chamber. The upper layer was cultured in serum-free medium, and the lower layer was cultured in 10% FBS DMEM for 24 hours. After scraping off the cells that remained without invading using a cotton swab, the cells were fixed with 4% paraformaldehyde in PBS (Wako Pure Chemical Industries, Ltd.), and observations were made with a fluorescence microscope. The cells stained blue were regarded as B16 4A5 cells, and the number of cells visible in one field of view was counted. ·Statistical Analysis Unless otherwise specified, the data were obtained with n = 6 and shown as the mean ± standard deviation. For the test of significant differences between the means, statistical software Excel Statistics was used. For two groups, the Student's t-test was used, and for multiple groups, the Dunnett test was used to verify the significant differences between the groups. A p-value of less than 0.05 was considered a significant difference between the groups (*P < 0.05, **P < 0.01).

[0099] Example 1 M1 Polarization of Macrophages by THGP RAW 264.7 cells were cultured in a medium containing 500 μM THGP for up to 120 days and treated with THGP. As a control, culturing in a THGP-free medium was also performed in parallel. The morphology of RAW264.7 cells changed over time to a spindle shape upon THGP treatment (Figure 1a; the cells on Day 120 are not shown in the figure but showed the same morphological changes as the cells on Day 90). After 10 days of THGP treatment, the proportion of spindle-shaped cells in all cells increased by approximately two-fold compared to the proportion in the control medium (Figure 1b, Figure 1c). Since the spindle shape is the cell morphology of M1 macrophages, it was suggested that RAW 264.7 cells differentiated into M1 macrophages upon THGP treatment.

[0100] The cells on Day 1, Day 10, Day 30, and Day 40 of THGP treatment were passaged, and the cells on the day of passage, 1 day later, and 2 days later were subjected to the MTS assay to measure the cell proliferation rate. A decrease in the cell proliferation rate was observed after THGP treatment for 10 days or more, and the longer the period of THGP treatment, the lower the proliferation rate (Figure 1d). It has been reported that M1 macrophages have slow proliferation, and this decrease in the proliferation rate also suggested that RAW 264.7 cells differentiated into M1 macrophages upon THGP treatment.

[0101] For the cells after THGP treatment for 20 days or more and within 120 days, the expression of CD86, a marker of M1 macrophages, and CD206, a marker of M2 macrophages, was detected by immunofluorescence staining. THGP treatment decreased the proportion of CD206-positive cells in all cells, and at the same time, the M1 / M2 ratio increased (Figure 1e, Figure 1f). This indicated that macrophages were polarized to M1 upon THGP treatment.

[0102] Furthermore, for cells after 20 days of THGP treatment, the gene expressions of markers for M1 macrophages (iNOS, CD80, CD86, TNF-α, IL-1β) and M2 macrophages (arginase, CD206, CD163, TGF-β, CXCL2) were measured by real-time RT-PCR. THGP treatment led to an increase in the gene expression levels of M1 macrophage markers and a decrease in those of M2 macrophage markers (Figs. 2a, 2b), confirming the M1 polarization of macrophages by THGP treatment. Also, the protein expressions of CD86 and CD206 in cells after THGP treatment for 20 days or more and within 120 days showed the same trend as the gene expression (Figs. 2c - 2e).

[0103] Example 2 Influence of THGP Concentration on Macrophage M1 Polarization RAW 264.7 cells were cultured in a medium containing 50, 500, or 5000 μM of THGP for 4, 7, or 10 days for THGP treatment. As a control, culturing in a THGP-free medium was also performed in parallel. The gene expression level of the M1 macrophage marker iNOS in the cultured cells was measured by real-time RT-PCR. The results are shown in Fig. 3. When the THGP concentration was 50 μM, the iNOS expression level in THGP-treated cells exceeded that in control cells at all time points. The expression levels of THGP-treated cells on the 4th and 7th days varied greatly, but the variation became smaller on the 10th day. When the THGP concentration was 500 or 5000 μM, the iNOS expression level in THGP-treated cells on the 4th day was approximately the same as that in the control, but the expression levels on the 7th and 10th days exceeded that in the control, showing a dose-dependent increase. From this, it was shown that induction of M1 macrophages is possible at a THGP concentration of 50 μM or more, but it is preferable to treat for 10 days or more to suppress the variation.

[0104] Example 3 Damage to Cancer Cells by M1 Polarized Macrophages Prepared by THGP Treatment RAW 264.7 cells were cultured in a medium containing 50, 500, or 5000 μM of THGP for 10 days and treated with THGP. As a control, culturing in a THGP-free medium was also performed in parallel. Rabbit IgG FITC-latex beads were added to the cultured cells to evaluate the phagocytic ability against foreign substances. The THGP-treated cells showed enhanced phagocytic ability at any THGP concentration, and the degree of enhancement was dose-dependent (Figs. 4a and 4b).

[0105] In addition, RAW 264.7 cells were cultured in a medium containing 500 μM of THGP for 20 days or more and up to 120 days to obtain RAW T. As a control, RAW 264.7 cells were cultured in a THGP-free medium for 20 days or more and up to 120 days to obtain RAW C. These cells were stained with CMFDA and co-cultured with B16 4A5 cells stained with CMAC to evaluate the phagocytic ability against cancer cells. For comparison, a group (RAW C+THGP) in which RAW C and B16 4A5 cells were co-cultured in the presence of 500 μM of THGP was established.

[0106] The results are shown in Figs. 4c and 4d. In the co-culture with RAW C, almost no B16 4A5 cells were phagocytosed. In the co-culture with RAW C in the presence of THGP, about 10% of B16 4A5 cells were phagocytosed. In contrast, in the co-culture with RAW T, more than 60% of B16 4A5 cells were phagocytosed, and it was confirmed that the M1-polarized macrophages prepared by THGP treatment had a high phagocytic ability.

[0107] When the cytotoxic ability of the M1-polarized macrophages prepared by THGP treatment against B16 4A5 cells was evaluated by an MTS assay under co-culture, results similar to the fluorescence staining results shown in Fig. 4d were obtained (Fig. 5a). For a more detailed elucidation of the mechanism, 5×10 5B16 4A5 cells seeded at cells / well and cultured for 24 hours were further cultured for 48 hours using conditional medium prepared from the culture supernatants of RAW C and RAW T respectively, medium containing 500 μM THGP, or medium without THGP, and cell proliferation was evaluated by the MTS assay. Although the culture supernatants of both RAW C and RAW T inhibited the proliferation of B16 4A5 cells and no difference was observed between the two (Figure 5b), it was shown that the high cytotoxicity of M1-polarized macrophages prepared by THGP treatment against B16 4A5 cells was due to direct phagocytosis rather than through the cytotoxic cytokines they secrete. In addition, B16 4A5 cells cultured in THGP-containing medium showed the same degree of cell proliferation as B16 4A5 cells cultured in THGP-free medium (Figure 5b, Figure 5c), indicating that THGP itself does not affect the proliferation of B16 4A5 cells.

[0108] Furthermore, the cytotoxic ability of M1-polarized macrophages prepared by THGP treatment against B16 4A5 cells was evaluated by MTS assay in the same manner as the test whose results are shown in Fig. 5a, except that 5-FU was added to the medium at a concentration of 1.25 μM during co-culture. Co-culture with RAW C in the presence of 5-FU decreased the viability of B16 4A5 cells more than monoculture in the presence of 5-FU, and further co-culture with RAW T in the presence of 5-FU decreased the viability of B16 4A5 cells more than co-culture with RAW C in the presence of 5-FU (Fig. 5d). For a more detailed elucidation of the mechanism, 5-FU was added to the conditional medium prepared from the culture supernatants of RAW C and RAW T, respectively, or the control THGP-free medium at a concentration of 1.25 μM, and B16 4A5 cells were cultured in these media for 48 hours, and the cell number was evaluated by MTS assay. The culture supernatant of RAW C increased the viability of B16 4A5 cells in the presence of 5-FU, but such an effect was not observed in the culture supernatant of RAW T (Fig. 5e). From this, it was suggested that M1-polarized macrophages prepared by THGP treatment effectively damage cancer cells in combination with anticancer drugs without conferring drug resistance to cancer cells via humoral factors as seen in THGP-untreated macrophages.

[0109] Example 4 Inhibition of the Expression of CD47-SIRPα Immune Checkpoint Molecules by THGP and by M1 Polarized Macrophages Prepared by THGP Treatment In co-culture with B16 4A5 cells, RAW T was observed to accumulate more around B16 4A5 cells than RAW C (Fig. 6a), suggesting that RAW T has a higher recognition ability for B16 4A5 cells than RAW C. Focusing on CD47-SIRPα, which is one of the mechanisms by which macrophages recognize cancer cells, the gene expression of SIRP-α in RAW C and RAW T was measured by real-time RT-PCR, and it was shown that the gene expression of SIRP-α was suppressed in RAW T (Fig. 6b).

[0110] Also, 5×10 5B16 4A5 cells seeded at cells / well and cultured for 24 hours were further cultured for 48 hours using conditional medium prepared from the culture supernatants of RAW C and RAW T, respectively, or medium without THGP. When the CD47 gene expression of B16 4A5 cells after culture was measured by real-time PCR, the culture supernatant of RAW C increased the CD47 gene expression level of B16 4A5 cells, but such an effect was not observed in the culture supernatant of RAW T (Figure 6c). From this, it was shown that THGP suppresses SIRPα gene expression in macrophages and suppresses CD47 gene expression in cancer cells via humoral factors secreted by macrophages.

[0111] Example 5 Inhibition of Epithelial-Mesenchymal Transition of Cancer Cells by M1 Polarized Macrophages Prepared by THGP Treatment As shown in Figure 2b, the gene expression of TGF-β was suppressed in M1-polarized macrophages prepared by THGP treatment. To examine the expression of TGF-β at the protein level, RAW C and RAW T were each seeded at 5.0×10 4 cells / well in a 96-well plate and cultured for 48 hours. The amount of TGF-β in the collected culture supernatant was suppressed, similar to the gene expression (Figure 7a).

[0112] Next, the effect of M1-polarized macrophages prepared by THGP treatment on epithelial-mesenchymal transition was evaluated. B16 4A5 cells seeded at 5×10 5 cells / well in a 6-well plate and cultured for 24 hours were further cultured for 48 hours using conditional medium prepared from the culture supernatants of RAW C and RAW T, respectively, medium containing 500 μM THGP, or medium without THGP. The gene expression of N-cadherin and Vimentin, markers of mesenchymal cells, in B16 4A5 cells after culture was measured by real-time RT-PCR.

[0113] The culture supernatant of RAW C increased the expression levels of N-cadherin and Vimentin genes in B16 4A5 cells, but no such effect was observed in the culture supernatant of RAW T (Fig. 7b, Fig. 7c). N-cadherin showed a similar tendency at the protein level (Fig. 7d, Fig. 7e). This indicates that M1-polarized macrophages prepared by THGP treatment do not induce epithelial-mesenchymal transition of cancer cells through humoral factors, as in the case of non-THGP-treated macrophages. It was speculated that THGP treatment polarized macrophages to M1, which suppressed the production of TGF-β, a major factor in the induction of epithelial-mesenchymal transition, and thus suppressed the induction of epithelial-mesenchymal transition in cancer cells.

[0114] Example 6 Inhibition of the Metastatic Ability of Cancer Cells by M1 Polarized Macrophages Prepared by THGP Treatment Migration assay of B16 4A5 cells was performed using conditional medium prepared from the culture supernatants of RAW C and RAW T, medium containing 500 μM THGP, or medium without THGP as test media. The culture supernatant of RAW C enhanced the migration ability of B16 4A5 cells, but the culture supernatant of RAW T did not show such an effect (Figure 8a and Figure 8b).

[0115] In addition, the MMP-2 and MMP-9 activities of B16 4A5 cells were evaluated using conditional medium prepared from the culture supernatants of RAW C and RAW T, medium containing 500 μM THGP, or medium without THGP as serum-free test media. The culture supernatant of RAW C enhanced the MMP-2 and MMP-9 activities of B16 4A5 cells, but the culture supernatant of RAW T did not show such an effect (Figure 8c).

[0116] Next, the adhesiveness of B16 4A5 cells was evaluated using conditional media prepared from the culture supernatants of RAW C and RAW T as test media. The culture supernatant of RAW T reduced the adhesiveness of B16 4A5 cells more than that of RAW C (Fig. 8d). Furthermore, the invasiveness of B16 4A5 cells was evaluated by co-culture of RAW C or RAW T with B16 4A5 cells using a Boyden chamber. RAW T suppressed the invasiveness of B16 4A5 cells compared to RAW C (Fig. 8e).

[0117] These results indicate that M1-polarized macrophages prepared by THGP treatment do not promote the metastatic potential of cancer cells through humoral factors, as do non-THGP-treated macrophages. It is speculated that THGP treatment polarizes macrophages to M1, suppresses the induction of epithelial-mesenchymal transition in cancer cells, and thus suppresses the promotion of metastasis of cancer cells.

[0118] Comparative Example Confirmation of the Direct Action of THGP on the Epithelial-Mesenchymal Transition and Metastatic Ability of Cancer Cells 5×10 B16 4A5 cells in a 6-well plate 5B16 4A5 cells were treated with TGF-β alone or in combination with TGF-β+THGP by seeding cells / well and culturing them for 1 week in THGP-free medium containing recombinant mouse TGF-β 10 ng / ml (Biolegend) or THGP 500 μM-containing medium with the same concentration of TGF-β. Treatment with TGF-β alone increased mesenchymal-like cells (Figure 9a), and it was confirmed by immunostaining and Western blot analysis of B16 4A5 cells that the protein expression of the mesenchymal cell marker N-cadherin was enhanced (Figures 9b and 9c). The same effect as that of TGF-β alone treatment was also confirmed in the combined treatment of TGF-β+THGP. Furthermore, no difference was observed between TGF-β alone treatment and TGF-β+THGP combined treatment in the invasion assessment of B16 4A5 cells using a Boyden chamber. From this, it was considered that THGP acts on cancer cells not directly but via M1-polarized macrophages prepared by THGP treatment, and suppresses the epithelial-mesenchymal transition and metastatic ability of cancer cells.

Sequence Listing Free-Text

[0119] Base sequence of forward primer for iNOS amplification, SEQ ID NO: 1 Base sequence of reverse primer for iNOS amplification, SEQ ID NO: 2 Base sequence of forward primer for CD80 amplification, SEQ ID NO: 3 Base sequence of reverse primer for CD80 amplification, SEQ ID NO: 4 Base sequence of forward primer for CD86 amplification, SEQ ID NO: 5 Base sequence of reverse primer for CD86 amplification, SEQ ID NO: 6 Base sequence of forward primer for TNF-α amplification, SEQ ID NO: 7 Base sequence of reverse primer for TNF-α amplification, SEQ ID NO: 8 Base sequence of forward primer for Arginase amplification, SEQ ID NO: 9 Base sequence of reverse primer for Arginase amplification, SEQ ID NO: 10 Base sequence of forward primer for amplifying CD163 with SEQ ID NO: 11 Base sequence of reverse primer for amplifying CD163 with SEQ ID NO: 12 Base sequence of forward primer for amplifying CD206 with SEQ ID NO: 13 Base sequence of reverse primer for amplifying CD206 with SEQ ID NO: 14 Base sequence of forward primer for amplifying CD47 with SEQ ID NO: 15 Base sequence of reverse primer for amplifying CD47 with SEQ ID NO: 16 Base sequence of forward primer for amplifying SIRPA with SEQ ID NO: 17 Base sequence of reverse primer for amplifying SIRPA with SEQ ID NO: 18 Base sequence of forward primer for amplifying IL-1β with SEQ ID NO: 19 Base sequence of reverse primer for amplifying IL-1β with SEQ ID NO: 20 Base sequence of forward primer for amplifying TGF-β with SEQ ID NO: 21 Base sequence of reverse primer for amplifying TGF-β with SEQ ID NO: 22 Base sequence of forward primer for amplifying CXCL2 with SEQ ID NO: 23 Base sequence of reverse primer for amplifying CXCL2 with SEQ ID NO: 24 Base sequence of forward primer for amplifying N-cadherin with SEQ ID NO: 25 Base sequence of reverse primer for amplifying N-cadherin with SEQ ID NO: 26 Base sequence of forward primer for amplifying Vimentin with SEQ ID NO: 27 Base sequence of reverse primer for amplifying Vimentin with SEQ ID NO: 28 Base sequence of forward primer for amplifying RPS18 with SEQ ID NO: 29 Base sequence of reverse primer for amplifying RPS18 with SEQ ID NO: 30

Claims

1. General formula (I) 【Chemistry 1】 A compound of the formula: In the formula, R 1 and R 2 are each independently hydrogen, halogen, nitro, hydroxy, cyano, C 1-4 Alkyl, C 1-4 Haloalkyl, C 2-4 Alkenyl, C 2-4 Haloalkenyl, C 3-4 Alkynyl, C 3-4 Haloalkynyl, C 1-4 Alkoxy, C 1-4 Haloalkoxy, C 1-4 Alkylthio, C 1-4 Alkyl sulfinyl or C 1-4 alkylsulfonyl or, together with the two carbon atoms to which they are attached, form a 4- to 10-membered monocyclic or polycyclic saturated ring, a 4- to 10-membered monocyclic or polycyclic partially saturated ring, or a 5- to 10-membered monocyclic or polycyclic saturated or partially unsaturated heterocycle containing 1 to 4 atoms selected from nitrogen, oxygen and sulfur; These rings may be unsubstituted or substituted with halogen, nitro, hydroxy, cyano, oxo, C 1-4 Alkyl, C 1-4 Haloalkyl, C 2-4 Alkenyl, C 2-4 Haloalkenyl, C 3-4 Alkynyl, C 3-4 Haloalkynyl, C 1-4 Alkoxy, C 1-4 Haloalkoxy, C 1-4 Alkylthio, C 1-4 Alkylsulfinyl and C 1-4 substituted by one or more substituents from the group consisting of alkylsulfonyl; R 3 is hydrogen, halogen, nitro, hydroxy, cyano, C 1-4 Alkyl, C 1-4 Haloalkyl, C 2-4 Alkenyl, C 2-4 Haloalkenyl, C 3-4 Alkynyl, C 3-4 Haloalkynyl, C 1-4 Alkoxy, C 1-4 Haloalkoxy, C 1-4 Alkylthio, C 1-4 Alkyl sulfinyl or C 1-4 is alkylsulfonyl, The compound or a pharma- ceutically acceptable salt thereof or C 1-4 An agent for inducing M1 polarization of macrophages in a subject in which IFN-γ is not produced in an amount capable of inducing M1 polarization of macrophages or in which M1 polarization of macrophages is not induced by IFN-γ, comprising an alkyl ester or a polymer thereof.

2. In general formula (I), R 1 , R 2 and R 3 are independently hydrogen, halogen, nitro, hydroxy, cyano, C 1-4 Alkyl, C 1-4 Haloalkyl, C 2-4 Alkenyl, C 2-4 Haloalkenyl, C 3-4 Alkynyl, C3-4 Haloalkynyl, C 1-4 Alkoxy, C 1-4 Haloalkoxy, C 1-4 Alkylthio, C 1-4 Alkyl sulfinyl or C 1-4 A compound which is an alkylsulfonyl, or a pharma- ceutically acceptable salt thereof, or 1-4 The agent according to claim 1 , which contains an alkyl ester or a polymer thereof.

3. In general formula (I), R 1 , R 2 and R 3 or a pharmacy acceptable salt thereof, or 1-4 The agent according to claim 1 or 2, which contains an alkyl ester or a polymer thereof.

4. General formula (I) 【Chemistry 2】 A compound of the formula: In the formula, R 1 and R 2 are each independently hydrogen, halogen, nitro, hydroxy, cyano, C 1-4 Alkyl, C 1-4 Haloalkyl, C 2-4 Alkenyl, C 2-4 Haloalkenyl, C 3-4 Alkynyl, C 3-4 Haloalkynyl, C 1-4 Alkoxy, C 1-4 Haloalkoxy, C 1-4 Alkylthio, C 1-4 Alkyl sulfinyl or C 1-4 alkylsulfonyl or, together with the two carbon atoms to which they are attached, form a 4- to 10-membered monocyclic or polycyclic saturated ring, a 4- to 10-membered monocyclic or polycyclic partially saturated ring, or a 5- to 10-membered monocyclic or polycyclic saturated or partially unsaturated heterocycle containing 1 to 4 atoms selected from nitrogen, oxygen and sulfur; These rings may be unsubstituted or substituted with halogen, nitro, hydroxy, cyano, oxo, C 1-4 Alkyl, C 1-4 Haloalkyl, C 2-4 Alkenyl, C 2-4 Haloalkenyl, C 3-4 Alkynyl, C 3-4 Haloalkynyl, C 1-4 Alkoxy, C 1-4 Haloalkoxy, C 1-4 Alkylthio, C 1-4 Alkylsulfinyl and C 1-4 substituted by one or more substituents from the group consisting of alkylsulfonyl; R 3 is hydrogen, halogen, nitro, hydroxy, cyano, C 1-4 Alkyl, C 1-4 Haloalkyl, C 2-4 Alkenyl, C 2-4 Haloalkenyl, C 3-4 Alkynyl, C 3-4 Haloalkynyl, C 1-4 Alkoxy, C 1-4 Haloalkoxy, C 1-4 Alkylthio, C 1-4 Alkyl sulfinyl or C 1-4 is alkylsulfonyl, The compound or a pharma- ceutically acceptable salt thereof or C 1-4 An agent for suppressing SIRPα expression in macrophages in a subject in which IFN-γ is not produced in an amount capable of inducing M1 polarization of macrophages or in which M1 polarization of macrophages is not induced by IFN-γ, comprising an alkyl ester or a polymer thereof.

5. In general formula (I), R 1 , R 2 and R 3 are independently hydrogen, halogen, nitro, hydroxy, cyano, C 1-4 Alkyl, C 1-4 Haloalkyl, C 2-4 Alkenyl, C 2-4 Haloalkenyl, C 3-4 Alkynyl, C3-4 Haloalkynyl, C 1-4 Alkoxy, C 1-4 Haloalkoxy, C 1-4 Alkylthio, C 1-4 Alkyl sulfinyl or C 1-4 A compound which is an alkylsulfonyl, or a pharma- ceutically acceptable salt thereof, or 1-4 The agent according to claim 4, which contains an alkyl ester or a polymer thereof.

6. In general formula (I), R 1 , R 2 and R 3 or a pharmacy acceptable salt thereof, or 1-4 The agent according to claim 4 or 5, which contains an alkyl ester or a polymer thereof.

7. The agent according to any one of claims 4 to 6, which is used for inhibiting the CD47-SIRPα immune checkpoint.

8. General formula (I) 【Chemistry 3】 A compound of the formula: In the formula, R 1 and R 2 are each independently hydrogen, halogen, nitro, hydroxy, cyano, C 1-4 Alkyl, C 1-4 Haloalkyl, C 2-4 Alkenyl, C 2-4 Haloalkenyl, C 3-4 Alkynyl, C 3-4 Haloalkynyl, C 1-4 Alkoxy, C 1-4 Haloalkoxy, C 1-4 Alkylthio, C 1-4 Alkyl sulfinyl or C 1-4 alkylsulfonyl or, together with the two carbon atoms to which they are attached, form a 4- to 10-membered monocyclic or polycyclic saturated ring, a 4- to 10-membered monocyclic or polycyclic partially saturated ring, or a 5- to 10-membered monocyclic or polycyclic saturated or partially unsaturated heterocycle containing 1 to 4 atoms selected from nitrogen, oxygen and sulfur; These rings may be unsubstituted or substituted with halogen, nitro, hydroxy, cyano, oxo, C 1-4 Alkyl, C 1-4 Haloalkyl, C 2-4 Alkenyl, C 2-4 Haloalkenyl, C 3-4 Alkynyl, C 3-4 Haloalkynyl, C 1-4 Alkoxy, C 1-4 Haloalkoxy, C 1-4 Alkylthio, C 1-4 Alkylsulfinyl and C 1-4 substituted by one or more substituents from the group consisting of alkylsulfonyl; R 3 is hydrogen, halogen, nitro, hydroxy, cyano, C 1-4 Alkyl, C 1-4 Haloalkyl, C 2-4 Alkenyl, C 2-4 Haloalkenyl, C 3-4 Alkynyl, C 3-4 Haloalkynyl, C 1-4 Alkoxy, C 1-4 Haloalkoxy, C 1-4 Alkylthio, C 1-4 Alkyl sulfinyl or C 1-4 is alkylsulfonyl, The compound or a pharma- ceutically acceptable salt thereof or C 1-4 A composition containing an alkyl ester or a polymer thereof for treating a disease or condition that is expected to be ameliorated or prevented by M1 macrophages in a subject in which IFN-γ is not produced in an amount sufficient to induce M1 polarization of macrophages or in which M1 polarization of macrophages is not induced by IFN-γ, wherein the disease or condition is selected from the group consisting of cancer, infectious disease, and trauma.

9. In general formula (I), R 1 , R 2 and R 3 are independently hydrogen, halogen, nitro, hydroxy, cyano, C 1-4 Alkyl, C 1-4 Haloalkyl, C 2-4 Alkenyl, C 2-4 Haloalkenyl, C 3-4 Alkynyl, C3-4 Haloalkynyl, C 1-4 Alkoxy, C 1-4 Haloalkoxy, C 1-4 Alkylthio, C 1-4 Alkyl sulfinyl or C 1-4 A compound which is an alkylsulfonyl, or a pharma- ceutically acceptable salt thereof, or 1-4 The composition according to claim 8, which contains an alkyl ester or a polymer thereof.

10. In general formula (I), R 1 , R 2 and R 3 or a pharmacy acceptable salt thereof, or 1-4 The composition according to claim 8 or 9, which contains an alkyl ester or a polymer thereof.

11. The composition according to any one of claims 8 to 10, which is a pharmaceutical composition for treating cancer.

12. The composition according to any one of claims 8 to 11, for enhancing phagocytosis of cancer cells by macrophages.

13. The composition according to any one of claims 8 to 12, for inhibiting epithelial-mesenchymal transition of cancer cells.

14. A composition according to any one of claims 8 to 13 for inhibiting the CD47-SIRPα immune checkpoint.

15. 50 μM to 10 mM of general formula (I) 【Chemistry 4】 A compound of the formula: In the formula, R 1 and R 2 are each independently hydrogen, halogen, nitro, hydroxy, cyano, C 1-4 Alkyl, C 1-4 Haloalkyl, C 2-4 Alkenyl, C 2-4 Haloalkenyl, C 3-4 Alkynyl, C 3-4 Haloalkynyl, C 1-4 Alkoxy, C 1-4 Haloalkoxy, C 1-4 Alkylthio, C 1-4 Alkyl sulfinyl or C 1-4 alkylsulfonyl or, together with the two carbon atoms to which they are attached, form a 4- to 10-membered monocyclic or polycyclic saturated ring, a 4- to 10-membered monocyclic or polycyclic partially saturated ring, or a 5- to 10-membered monocyclic or polycyclic saturated or partially unsaturated heterocycle containing 1 to 4 atoms selected from nitrogen, oxygen and sulfur; These rings may be unsubstituted or substituted with halogen, nitro, hydroxy, cyano, oxo, C 1-4 Alkyl, C 1-4 Haloalkyl, C 2-4 Alkenyl, C 2-4 Haloalkenyl, C 3-4 Alkynyl, C 3-4 Haloalkynyl, C 1-4 Alkoxy, C 1-4 Haloalkoxy, C 1-4 Alkylthio, C 1-4 Alkylsulfinyl and C 1-4 substituted by one or more substituents from the group consisting of alkylsulfonyl; R 3 is hydrogen, halogen, nitro, hydroxy, cyano, C 1-4 Alkyl, C 1-4 Haloalkyl, C 2-4 Alkenyl, C 2-4 Haloalkenyl, C 3-4 Alkynyl, C 3-4 Haloalkynyl, C 1-4 Alkoxy, C 1-4 Haloalkoxy, C 1-4 Alkylthio, C 1-4 Alkyl sulfinyl or C 1-4 is alkylsulfonyl, The compound or a pharma- ceutically acceptable salt thereof or 1-4 1. A method for producing M1-polarized macrophages, comprising culturing macrophage precursor cells for 7 days or more in a medium containing an alkyl ester, wherein the medium does not contain IFN-γ and the culturing does not include co-culture with T cells.

16. The medium is represented by the general formula (I) R 1 , R 2 and R 3 are independently hydrogen, halogen, nitro, hydroxy, cyano, C 1-4 Alkyl, C 1-4 Haloalkyl, C 2-4 Alkenyl, C 2-4 Haloalkenyl, C 3-4 Alkynyl, C3-4 Haloalkynyl, C 1-4 Alkoxy, C 1-4 Haloalkoxy, C 1-4 Alkylthio, C 1-4 Alkyl sulfinyl or C 1-4 A compound which is an alkylsulfonyl, or a pharma- ceutically acceptable salt thereof, or 1-4 16. The method of claim 15, comprising an alkyl ester.

17. The medium is represented by the general formula (I) R 1 , R 2 and R 3 or a pharma- ceutically acceptable salt thereof, or 1-4 17. The method of claim 15 or 16, comprising an alkyl ester.

18. The method according to any one of claims 15 to 17, wherein the macrophage source cells are macrophage precursor cells, non-polarized macrophages, or macrophages polarized to a subset other than M1 type.

19. The medium contains 500 μM to 5 mM of a compound of formula (I) or a pharma- ceutically acceptable salt thereof or C 1-4 The method of any one of claims 15 to 18, comprising an alkyl ester.

20. The method according to any one of claims 15 to 19, wherein the culture is carried out for 10 days or more.

21. A composition for use in treating a disease or condition that is expected to be ameliorated or prevented by M1 macrophages, comprising M1-polarized macrophages produced by the method of any one of claims 15 to 20 or a culture supernatant thereof, wherein the disease or condition is selected from the group consisting of cancer, infectious diseases, and trauma.

22. 22. The composition of claim 21, which is a pharmaceutical composition for treating cancer.

23. The composition according to claim 21 or 22, for enhancing phagocytosis of cancer cells by macrophages.

24. The composition according to any one of claims 21 to 23, for inhibiting epithelial-mesenchymal transition of cancer cells.

25. A composition according to any one of claims 21 to 24, for inhibiting the CD47-SIRPα immune checkpoint.

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