WO2021037013A1 - 吩噻嗪类或其类似结构的化合物在制药中的新应用 - Google Patents

吩噻嗪类或其类似结构的化合物在制药中的新应用 Download PDF

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WO2021037013A1
WO2021037013A1 PCT/CN2020/110998 CN2020110998W WO2021037013A1 WO 2021037013 A1 WO2021037013 A1 WO 2021037013A1 CN 2020110998 W CN2020110998 W CN 2020110998W WO 2021037013 A1 WO2021037013 A1 WO 2021037013A1
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substituted
unsubstituted
phenothiazine
cells
compound
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PCT/CN2020/110998
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French (fr)
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陈良
范真真
贺嵩敏
陈婕思
桑德尔马克斯
龚守芳
杨晓志
林子青
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广州威溶特医药科技有限公司
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Priority to AU2020338366A priority Critical patent/AU2020338366B2/en
Priority to JP2022513507A priority patent/JP2022547275A/ja
Priority to US17/638,663 priority patent/US20220323454A1/en
Priority to DE112020003610.3T priority patent/DE112020003610T5/de
Publication of WO2021037013A1 publication Critical patent/WO2021037013A1/zh
Priority to JP2024003683A priority patent/JP2024041920A/ja

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/54—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with at least one nitrogen and one sulfur as the ring hetero atoms, e.g. sulthiame
    • A61K31/5415—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with at least one nitrogen and one sulfur as the ring hetero atoms, e.g. sulthiame ortho- or peri-condensed with carbocyclic ring systems, e.g. phenothiazine, chlorpromazine, piroxicam
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/14—Blood; Artificial blood
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    • A—HUMAN NECESSITIES
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    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/0005—Vertebrate antigens
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    • A61K39/00—Medicinal preparations containing antigens or antibodies
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    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A61K39/39533—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals
    • A61K39/3955—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals against proteinaceous materials, e.g. enzymes, hormones, lymphokines
    • A—HUMAN NECESSITIES
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    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/461—Cellular immunotherapy characterised by the cell type used
    • A61K39/4611—T-cells, e.g. tumor infiltrating lymphocytes [TIL], lymphokine-activated killer cells [LAK] or regulatory T cells [Treg]
    • A—HUMAN NECESSITIES
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    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
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    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C07K16/2815—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against CD8
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
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    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C07K16/2818—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against CD28 or CD152
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/515—Animal cells
    • A61K2039/5156—Animal cells expressing foreign proteins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K39/46
    • A61K2239/31—Indexing codes associated with cellular immunotherapy of group A61K39/46 characterized by the route of administration
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K39/46
    • A61K2239/38—Indexing codes associated with cellular immunotherapy of group A61K39/46 characterised by the dose, timing or administration schedule
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K39/46
    • A61K2239/46—Indexing codes associated with cellular immunotherapy of group A61K39/46 characterised by the cancer treated
    • A61K2239/48—Blood cells, e.g. leukemia or lymphoma
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/21—Immunoglobulins specific features characterized by taxonomic origin from primates, e.g. man
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the present invention relates to the technical field of medicines, in particular to a new application of phenothiazines or compounds with similar structures, hydrates, solvates or reduced forms thereof in pharmacy. Another aspect of the present invention relates to the combined application of phenothiazine compounds and adoptive cell therapy. Another aspect of the present invention relates to the combined application of phenothiazine compounds and tumor immunochemotherapeutics, especially PD-1 antibodies.
  • Adoptive cell therapy is a treatment method that includes collecting one or more different types of immune cells from mammals, culturing and/or operating the collected immune cells ex vivo, and combining The cultured and/or manipulated immune cells are returned to the mammal.
  • the in vitro operation of the collected immune cells includes the introduction of recombinant nucleic acid into the immune cells.
  • Adoptive cell therapy includes but is not limited to Tumor Infiltrating Lymphocyte (TIL), Lymphokine Activated Killer (LAK), Cytokine Induced Killer (CIK), Dendritic Dendritic Cell (DC, Dendritic Cell), Natural Killer Cell (NK, Natural Killer), T Cell Receptor Chimeric T Cell (TCR-T, T Cell Receptor TCR-Modified T Cell), Chimeric Antigen Receptor NK Cells (CAR-NK), and Chimeric Antigen Receptor Engineered T Cells (CAR-T, Chimeric Antigen Receptor Engineered Cells (CAR-T, Chimeric Antigen Receptor Engineered Cells (CAR-T, Chimeric Antigen Receptor Engineered Cell), etc.
  • TIL Tumor Infiltrating Lymphocyte
  • LAK Lymphokine Activated Killer
  • CIK Cytokine Induced Killer
  • DC Dendritic Dendritic Cell
  • NK Natural Killer Cell
  • the human immune system can fight against and eliminate invading pathogenic microorganisms or mutated cells.
  • Immune cells are important cells that perform human immune functions.
  • T cell is an important immune cell in the human body. It can recognize the antigen presented by the major histocompatibility complex (MHC) on the target cell through the antigen receptor (TCR) expressed on its cell surface. This is called The first signal.
  • MHC major histocompatibility complex
  • TCR antigen receptor
  • T cells are activated and perform their functions.
  • cytotoxic T cells CTL for short
  • T cells In the process of T cell activation, T cells start to express some molecules that negatively feedback their immune function.
  • PD-1 programmed cell death 1, programmed apoptosis protein 1
  • PD-1 programmed cell death 1, programmed apoptosis protein 1
  • ligand usually PD-L1 on target cells (such as tumor cells)
  • PD-1 initiates its signal transduction, inhibits the function of CTL, and leads to T cells Apoptosis or incompetence.
  • Such signal transduction makes CTL lose its function of killing target cells.
  • PD-1 signal transduction inhibitors of small molecule compounds When drugs are used to block the interaction between PD-L1 and PD-1, the signal transduction of PD-1 is blocked, and the ability of CTL to kill tumor cells is restored.
  • other immune cells can also express PD-1.
  • macrophages, B cells, etc. have all been reported to express PD-1. After suppressing the function of PD-1 with appropriate drugs, the function of these immune cells can be restored to a certain extent.
  • macrophages change from M2 (that is, the type that inhibits CTL function) to M1 (that is, the type that promotes CTL function), and eliminates tumor cells in the body.
  • biological agents such as antibody drugs have been approved by the FDA for clinical treatment. But so far, the drugs used in clinic against PD-1 are all antibodies that belong to the class of biological preparations, and there are no relevant reports about PD-1 signal transduction inhibitors of small molecule compounds.
  • Methylene blue (3,7-bis(dimethylamino)phenothiazine-5-onium chloride) is a phenothiazine salt, which is widely used in chemical indicators, dyes, and biological stains. Recently, some studies have reported its application in medicine. For example, Chinese Patent Publication No. CN 104027338 A discloses the new use of methylene blue against acute cerebral ischemia; Chinese Patent Publication No. CN 103417546 B discloses the post-anesthesia promotion of methylene blue. Sober new application. In addition, some documents disclose methylene blue as a photosensitizer for the preparation of photosensitizing drugs for photodynamic therapy.
  • the present invention provides a pharmaceutical composition
  • a pharmaceutical composition comprising: (a) immune cells suitable for adoptive cell therapy; and (b) phenothiazine compounds, which are selected from compounds represented by formula (I), and Hydrate, solvate or reduced form:
  • Z is selected from: S + , O + , C or N;
  • Y is selected from: N or N + ; and when Z is selected from: S + or O + , Y is N; when Z is selected from C or N, Y is N + ;
  • X - is one or more anions that can form a salt with Z + or N +, so as to achieve electrical neutrality;
  • R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , R 7 , R 8 , R 9 and R 10 are each independently selected from: hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted Cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted alkoxy, substituted or unsubstituted aralkoxy, Sulfur alkoxy, amine, nitro, amino, halogen.
  • the X is selected from inorganic acid anions and organic acid anions.
  • the inorganic anion is preferably Cl -, Br -, I - .
  • the organic acid anion is preferably methanesulfonate anion, ethanesulfonate anion, p-toluenesulfonate anion, benzenesulfonate anion, ethanedisulfonate anion, malonic acid anion and naphthalenedisulfonate anion.
  • R1, R2, R3, R4, R5, R6, R7, R8, R9, and R10 are each independently selected from: hydrogen, substituted or unsubstituted C1-C6 alkyl, substituted or unsubstituted C3 -C8 cycloalkyl, substituted or unsubstituted 3-8 membered heterocycloalkyl, substituted or unsubstituted C5-C10 aryl, substituted or unsubstituted 5-10 membered heteroaryl, substituted or unsubstituted C1 -C6 alkoxy, substituted or unsubstituted arylalkoxy, thioalkoxy, amine, nitro, amino, halogen.
  • the phenothiazine compound is 3,7-bis(dimethylamino)phenothiazine-5-ium chloride.
  • the phenothiazine compound is selected from the 3,7-bis(dimethylamino)phenothiazine-5-onium salt (MTX) of formula II and its hydrate, solvate or reduced form
  • X is one or more anions, so as to achieve electrical neutrality, and is as defined above.
  • the reduced form of the phenothiazine compound is N3, N3, N7, N7-tetramethyl-10H-phenothiazine-3,7-diammonium salt (LMTX) of formula III, its Hydrate or solvate
  • X is one or more anions, so as to achieve electrical neutrality, and is as defined above.
  • the phenothiazine compound is 3,7-bis(dimethylamino)phenothiazine-5-ium chloride or N3,N3,N7,N7-tetramethyl-10H-phene Thiazine-3,7-diammonium bis(methanesulfonate).
  • the immune cells are selected from tumor infiltrating lymphocytes (TIL), chimeric antigen receptor T cells (CAR-T), chimeric antigen receptor NK cells (CAR-NK), and T cell receptors.
  • TIL tumor infiltrating lymphocytes
  • CAR-T chimeric antigen receptor T cells
  • CAR-NK chimeric antigen receptor NK cells
  • T cell receptors T cell receptors.
  • the immune cell is a T cell receptor (TCR) chimeric T cell.
  • TCR T cell receptor
  • the TCR is capable of binding to the SIINFEKL peptide.
  • kits comprising: a) any one of the above-mentioned immune cells, which is formulated in a first dosage form; b) any one of the above-mentioned compounds, which is formulated in a second dosage form .
  • Another aspect of the present invention provides the use of any one of the above-mentioned compounds in the preparation of drugs for the treatment of cancer.
  • the cancer is melanoma, thymic tumor, lung cancer, prostate cancer, breast cancer, ovarian cancer, colorectal cancer, pancreatic cancer, liver cancer, lymphoma, esophageal cancer, bladder cancer, urethral cancer, non-Hodgkin Qijin lymphoma, kidney cancer or brain tumor.
  • Another aspect of the present invention also provides the application of any one of the above-mentioned phenothiazine compounds in the preparation of drugs for improving immune cell function.
  • Another aspect of the present invention provides a method for inhibiting PD-1 signal transduction, including the step of blocking the downstream signal pathway of PD-1.
  • the blocking is achieved by blocking PD-1 from recruiting SHP2 protein.
  • the step of blocking PD-1 from recruiting SHP2 includes contacting the cells with an effective amount of any phenothiazine compound described in the present invention.
  • Another aspect of the present invention provides a method of treating cancer in a subject, the method comprising inhibiting PD-1 signaling in the subject.
  • the inhibition of PD-1 signaling in the subject includes blocking a downstream signaling pathway of PD-1.
  • blocking the downstream signaling pathway of PD-1 includes blocking PD-1 from recruiting SHP2.
  • the step of blocking PD-1 recruitment to SHP2 includes administering to the subject an effective amount of any one of the phenothiazine compounds described in the present invention.
  • Another aspect of the present invention provides a method of treating cancer in a subject, the method comprising administering to the subject an effective amount of any one of the phenothiazine compounds of the present invention, and administering to the subject a second therapy.
  • the second therapy is selected from chemotherapy, radiotherapy, and surgical treatment.
  • the chemotherapy is tumor immunotherapy.
  • the tumor immunotherapy includes administering a PD-1 antibody or a PD-L1 antibody or a functional fragment thereof to the subject.
  • the methods described in this aspect produce a synergistic anti-tumor effect.
  • Another aspect of the present invention provides a pharmaceutical composition for treating cancer, the pharmaceutical composition comprising any one of the phenothiazine compounds described in the present invention, and a second chemotherapeutic agent.
  • the second chemotherapeutic agent is a tumor immunotherapeutic agent.
  • the tumor immunotherapeutic agent is a PD-1 antibody or a PD-L1 antibody or a functional fragment thereof.
  • Another aspect of the present invention provides a method for enhancing the therapeutic effect of a PD-1 antibody or a PD-L1 antibody or a functional fragment thereof in a subject, the method comprising before administering the PD-1 antibody or PD-L1 antibody At the same time or afterwards, an effective amount of any one of the phenothiazine compounds of the present invention is administered to the subject.
  • the method produces a synergistic anti-tumor effect.
  • the inventors of the present invention have discovered through a lot of creative work that the phenothiazines and compounds of similar structure of the present invention can significantly improve the killing of target cells by immune cells when combined with immune cells (especially genetically modified immune cells). Function to achieve a synergistic effect.
  • the phenothiazines and compounds with similar structures of the present invention can inhibit the function of PD-1, block the signal conduction of PD-1, and thus can be used as inhibitors of PD-1 signal conduction.
  • the phenothiazines and compounds with similar structures of the present invention can shrink transplanted tumors or lung cancer in situ in mice to achieve the purpose of treating cancer.
  • small-molecule PD-1 signaling inhibitors have lower cost, simpler preparation processes (such as chemical synthesis), multiple routes of administration, high patient compliance, safety and reliability, etc.
  • the experiment of the present invention also confirmed that the phenothiazine compound of the present invention has an effect equivalent to or superior to PD-1 antibody in terms of tumor suppression.
  • Figure 1 is a graph of the experimental results of Example 1.
  • A shows the expression of PD-1 protein on the surface of untransfected and transfected Jurkat cells;
  • B shows the surface of untransfected and transfected Raji cells.
  • C shows the result of transfected Raji cells inducing the phosphorylation of PD-1 protein Y248 in transfected Jurkat cells;
  • D is MTC promoting Jurkat-PD-1-NFAT-luc cells to secrete IL -2;
  • E shows that MTC or LMT (N3, N3, N7, N7-tetramethyl-10H-phenothiazine-3,7-diammonium bis(methanesulfonate)) promotes Jurkat-PD- Luciferase activity increased in 1-NFAT-luc cells.
  • FIG. 2 shows that MTC promotes the division of OT-1 cells in Example 2; where A is the result of the expression of PD-1 protein on the surface of OT1-CTL cells; B is the result of MTC promotes the division of CTL.
  • FIG. 3 shows that MTC promotes the secretion of effector molecules in OT-1 cells in Example 2, and MTC promotes the secretion of IL-2, IFN ⁇ , Perforin and GranzymeB in CTL cells.
  • FIG. 4 shows that MTC restores the ability of OT-1 T cells to kill target cells;
  • A shows that MTC promotes the killing of EL4-OVA (PD-L1) by CTL;
  • B shows that IFN ⁇ induces B16-F10 to express PD -L1 protein;
  • C and D show that MTC or LMT synergistically increase the killing effect of CTL on B16-OVA cells;
  • E shows that MTC or LMT has no killing effect on B16-WT cells, even if combined with CTL cells No killing effect.
  • Figure 5 is a diagram showing the results of MTC promoting cytotoxic T cells (CTL) to eliminate tumors formed by target cells; wherein, A is a model diagram of transplanted tumors; B, C, and D show that MTC inhibits Rag1-/- mouse transplanted tumors Growth; E shows that MTC inhibits the growth of transplanted tumors in C57BL/6J mice.
  • CTL cytotoxic T cells
  • Figure 6 is a diagram of the results of MTC treatment of primary tumors; wherein, A is a schematic diagram of an experimental model of treatment of tumors in situ; B is the result of MTC clearing tumors through CD8 + T cells.
  • Figure 7 is a diagram showing the results of MTC blocking PD-1 recruiting SHP2; A shows that MTC reduces the binding of PD1 and SHP2; B shows that MTC inhibits the binding of PD1 to recruit SHP2; C shows that MTC inhibits the binding of PD1 and SHP2 Combine.
  • ACT refers to the transfer of immune cells with anti-tumor activity to cancer patients.
  • ACT is a treatment method that involves using lymphocytes with anti-tumor activity, expanding these cells to large numbers in vitro and infusing these cells into a cancer-carrying host.
  • TIL tumor infiltrating lymphocytes
  • CAR-T is an abbreviated form of "chimeric antigen receptor T cell", where the chimeric antigen receptor (CAR) is the core component of CAR-T, giving T cells an HLA-independent way to recognize target cells ( Such as tumor) antigen ability, which enables CAR-modified T cells to recognize a wider range of targets than the natural T cell surface receptor TCR.
  • TCR-T T cell receptor (TCR) chimeric T cell
  • TCR T cell receptor
  • engineered TCR engineered T cell receptor
  • artificial TCR artificial T cell receptor
  • the engineered T cell receptor or the artificial T cell receptor has been genetically modified to have a structure that targets the target antigen, and at the same time retains the structural domains and/or auxiliary molecules in the TCR signal transduction pathway.
  • TCR-T retains all auxiliary molecules in the TCR signal transduction pathway. Therefore, when a small amount of antigen is stimulated, a fully activated state can occur, causing a killing effect on target cells.
  • TCR-T Compared with CAR-T, these TCR-T maintain and apply all the auxiliary molecules on the TCR signal transduction pathway. Therefore, TCR-T is more sensitive to low-concentration, low-copy number antigen recognition than some CAR-T. The therapeutic potential is very large.
  • Tyrosine protein phosphatase non-receptor type 11 is also known as protein tyrosine phosphatase 1D (PTP-1D).
  • Src homology region 2 contains domain phosphatase-2 (SHP-2) or protein tyrosine Amine acid phosphatase 2C (PTP)-2C) is an enzyme encoded by the human PTPN11 gene.
  • SHP-2 is ubiquitously expressed in various tissues and cell types, involving multiple signal transduction pathways, including growth factors such as PDGF, EGF and IGF-1, cytokines such as IL-3, GM-CSF and EPO, etc., and Insulin and interferon.
  • SHP-2 has a compound signal transduction function.
  • SHP-2 is involved in T cell inhibitory signal transduction. Studies have shown that SHP-2 is a downstream molecule of PD-1 signal transduction, which not only inhibits the activation of T cells but also promotes the disability of T cells. Studies have also shown that knocking out SHP2 on T lymphocytes can trigger anti-tumor immunity and inhibit the occurrence of colitis-related cancers in mice.
  • Methionine (MT) is a redox molecule, and depends on environmental conditions (for example, pH, oxygen, reducing agent), in a reduced form of 10H-phenothiazine compound (ie N,N,N',N'- There is an equilibrium state between tetramethyl-10H-phenothiazine-3,7-diamine (LMT) and the oxidized form (MT+).
  • environmental conditions for example, pH, oxygen, reducing agent
  • LMTX salt When LMT exists in its salt form, it is called the LMTX salt, as shown in Formula III.
  • suitable organic acid anions include, but are not limited to, those organic acid anions derived from the following organic acids: 2-acetoxybenzoic acid, acetic acid, ascorbic acid, aspartic acid , Benzoic acid, camphorsulfonic acid, cinnamic acid, citric acid, ethylenediaminetetraacetic acid, ethanedisulfonic acid, ethanesulfonic acid, fumaric acid, glucoheptonic acid, gluconic acid, glutamic acid, glycolic acid, hydroxy Maleic acid, hydroxynaphthoic acid, isethionic acid, lactic acid, lactobionic acid, lauric acid, maleic acid, malic acid, methanesulfonic acid, mucic acid, oleic acid, oxalic acid, palmitic acid, pamoic acid, pantothenic acid (pantothenic), phenylace
  • Suitable polymeric organic anions include but are not limited to those derived from the following polymeric acids: tannic acid, carboxymethyl cellulose.
  • the inorganic acid anion is preferably Cl-, Br-, I-.
  • the organic acid anion is preferably a methanesulfonate anion.
  • MTC methylene blue
  • MT methionine
  • MT+ methionine
  • the chloride salt of is a low molecular weight (319.86) water-soluble tricyclic organic compound with the following structural formula:
  • N3,N3,N7,N7-tetramethyl-10H-phenothiazine-3,7-diammonium bis(methanesulfonate) is shown below.
  • solvate used in this application takes its conventional meaning, which refers to a complex of a solute (such as a compound or a salt of a compound) and a solvent. If the solvent is water, the solvate can conveniently be referred to as a hydrate, such as monohydrate, dihydrate, trihydrate, and the like. Unless otherwise stated, when a specific compound is mentioned, the specific compound also includes its solvate form.
  • Treatment includes administering a compound of the application or a composition of the application to reduce the symptoms or complications of the disease or condition, or to eliminate the disease or condition.
  • the term "alleviation” as used herein is used to describe the process of reducing the severity of the signs or symptoms of a disorder. Symptoms can be alleviated but not eliminated.
  • administration of the composition of the application results in the elimination of signs or symptoms.
  • phenothiazine compounds are selected from the group consisting of compounds represented by formula (I), hydrates, solvates or reduced forms thereof form:
  • Z is selected from: S+, O+, C or N;
  • Y is selected from: N or N+; and when Z is selected from: S+ or O+, Y is selected from N; when Z is selected from C or N, Y is selected from N+; X- is one that can form a salt with Z+ or N+ Or multiple anions to achieve electrical neutrality;
  • R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , R 7 , R 8 , R 9 and R 10 are each independently selected from: hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted Cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted alkoxy, substituted or unsubstituted aralkoxy, Sulfur alkoxy, amine, nitro, amino, halogen.
  • the phenothiazine compound is selected from the 3,7-bis(dimethylamino)phenothiazine-5-onium salt (MTX) of formula II and its hydrate, solvate or reduced form
  • X is one or more anions, so as to achieve electrical neutrality, and is as defined above.
  • the reduced form of the phenothiazine compound is N3, N3, N7, N7-tetramethyl-10H-phenothiazine-3,7-diammonium salt (LMTX) of formula III, its Hydrate or solvate:
  • X is one or more anions, so as to achieve electrical neutrality, and is as defined above.
  • the phenothiazine compound is 3,7-bis(dimethylamino)phenothiazine-5-onium salt (MTX), preferably the phenothiazine compound is 3,7- Bis(dimethylamino)phenothiazin-5-ium chloride.
  • MTX 3,7-bis(dimethylamino)phenothiazine-5-onium salt
  • the phenothiazine compound of formula I is 3,7-bis(dimethylamino)phenothiazine-5-onium salt (MTX) of formula II, its hydrate, solvate or reduced form.
  • MTX 3,7-bis(dimethylamino)phenothiazine-5-onium salt
  • the phenothiazine compound is N3, N3, N7, N7-tetramethyl-10H-phenothiazine-3,7-diammonium salt (LMTX) of formula III.
  • the phenothiazine compound is 3,7-bis(dimethylamino)phenothiazine-5-ium chloride or N3,N3,N7,N7-tetramethyl-10H-phene Thiazine-3,7-diammonium bis(methanesulfonate).
  • the phenothiazine compound of formula I is 3,7-bis(dimethylamino)phenothiazine-5-onium salt (MTX) of formula II, its hydrate, solvate or reduced form.
  • MTX 3,7-bis(dimethylamino)phenothiazine-5-onium salt
  • the phenothiazine compound is N3, N3, N7, N7-tetramethyl-10H-phenothiazine-3,7-diammonium salt (LMTX) of formula III.
  • the phenothiazine compound is 3,7-bis(dimethylamino)phenothiazine-5-ium chloride or N3,N3,N7,N7-tetramethyl-10H-phene Thiazine-3,7-diammonium bis(methanesulfonate).
  • the phenothiazine compound of formula I is 3,7-bis(dimethylamino)phenothiazine-5-onium salt (MTX) of formula II, its hydrate, solvate or reduced form.
  • MTX 3,7-bis(dimethylamino)phenothiazine-5-onium salt
  • the phenothiazine compound is N3, N3, N7, N7-tetramethyl-10H-phenothiazine-3,7-diammonium salt (LMTX) of formula III.
  • the phenothiazine compound is 3,7-bis(dimethylamino)phenothiazine-5-ium chloride or N3,N3,N7,N7-tetramethyl-10H-phene Thiazine-3,7-diammonium bis(methanesulfonate).
  • the phenothiazine compound of formula I is 3,7-bis(dimethylamino)phenothiazine-5-onium salt (MTX) of formula II, its hydrate, solvate or reduced form.
  • MTX 3,7-bis(dimethylamino)phenothiazine-5-onium salt
  • the phenothiazine compound is N3, N3, N7, N7-tetramethyl-10H-phenothiazine-3,7-diammonium salt (LMTX) of formula III.
  • the phenothiazine compound is 3,7-bis(dimethylamino)phenothiazine-5-ium chloride or N3,N3,N7,N7-tetramethyl-10H-phene Thiazine-3,7-diammonium bis(methanesulfonate).
  • the phenothiazine compound of formula I is 3,7-bis(dimethylamino)phenothiazine-5-onium salt (MTX) of formula II, its hydrate, solvate or reduced form.
  • MTX 3,7-bis(dimethylamino)phenothiazine-5-onium salt
  • the phenothiazine compound is N3, N3, N7, N7-tetramethyl-10H-phenothiazine-3,7-diammonium salt (LMTX) of formula III.
  • the phenothiazine compound is 3,7-bis(dimethylamino)phenothiazine-5-ium chloride or N3,N3,N7,N7-tetramethyl-10H-phene Thiazine-3,7-diammonium bis(methanesulfonate).
  • Another aspect of the present invention provides a method for treating cancer, the method comprising administering to a subject suffering from the cancer a therapeutically effective amount of the phenothiazine compound of formula I of the present invention, its hydrate, solvate or further Prototype form.
  • the phenothiazine compound is 3,7-bis(dimethylamino)phenothiazine-5-onium salt (MTX) of formula II, its hydrate, solvate or reduced form.
  • the phenothiazine compound is N3, N3, N7, N7-tetramethyl-10H-phenothiazine-3,7-diammonium salt (LMTX) of formula III.
  • the phenothiazine compound is 3,7-bis(dimethylamino)phenothiazine-5-ium chloride or N3,N3,N7,N7-tetramethyl-10H-phene Thiazine-3,7-diammonium bis(methanesulfonate).
  • the cancer is melanoma, thymic tumor, lung cancer, prostate cancer, breast cancer, ovarian cancer, colorectal cancer, pancreatic cancer, liver cancer, lymphoma, esophageal cancer, bladder cancer, urethral cancer, non-Hodgkin Qijin lymphoma, kidney cancer or brain tumor.
  • the cancer is melanoma.
  • the subject is a mammal, preferably a human.
  • Another aspect of the present invention provides a method for improving immune cell function, the method comprising administering to a subject a therapeutically effective amount of the phenothiazine compound of formula I of the present invention, its hydrate, solvate or reduced form.
  • the phenothiazine compound is 3,7-bis(dimethylamino)phenothiazine-5-onium salt (MTX) of formula II, its hydrate, solvate or reduced form.
  • the phenothiazine compound is N3, N3, N7, N7-tetramethyl-10H-phenothiazine-3,7-diammonium salt (LMTX) of formula III.
  • the phenothiazine compound is 3,7-bis(dimethylamino)phenothiazine-5-ium chloride or N3,N3,N7,N7-tetramethyl-10H-phene Thiazine-3,7-diammonium bis(methanesulfonate).
  • the improvement of immune cell function can achieve an antiviral effect.
  • the subject is a mammal, preferably a human.
  • Another aspect of the present invention provides a method for preventing or treating cancer recurrence, the method comprising administering to a subject suffering from the cancer a therapeutically effective amount of a phenothiazine compound of formula I, its hydrate, solvate or Reduced form.
  • the phenothiazine compound is 3,7-bis(dimethylamino)phenothiazine-5-onium salt (MTX) of formula II, its hydrate, solvate or reduced form.
  • the phenothiazine compound is N3, N3, N7, N7-tetramethyl-10H-phenothiazine-3,7-diammonium salt (LMTX) of formula III.
  • the phenothiazine compound is 3,7-bis(dimethylamino)phenothiazine-5-ium chloride or N3,N3,N7,N7-tetramethyl-10H-phene Thiazine-3,7-diammonium bis(methanesulfonate).
  • the subject has undergone surgery, radiation therapy, or chemotherapy.
  • the method can be used in combination with other cancer therapies.
  • the method can be combined with surgery, radiotherapy or chemotherapy. Therefore, in some embodiments, the method of treating cancer of the present invention further comprises administering a therapeutically effective amount of a second therapeutic agent to a subject suffering from the cancer.
  • the second therapeutic agent is an immune cell suitable for adoptive cell therapy.
  • the second therapeutic agent is administered before, at the same time or after administration of the phenothiazine compound of Formula I, its hydrate, solvate, or reduced form.
  • the second therapeutic agent is a chemotherapeutic agent, preferably, the chemotherapeutic agent is a tumor immunochemotherapeutic agent, more preferably, the chemotherapeutic agent is a PD-1 antibody, a PD-L1 antibody or the like Fragment.
  • the second therapeutic agent is administered before, at the same time, or after administration of the phenothiazine compound of Formula I, its hydrate, solvate, or reduced form.
  • the interval between the two administrations is about 0.1 hour to about 72 hours, for example, the interval is about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 18, 24, 30, 36, 72h administration.
  • the phenothiazine compound, hydrate, solvate, or reduced form of the present invention The form and the second therapeutic agent are provided as separate pharmaceutical compositions. In some embodiments, the separate pharmaceutical compositions are provided in the same kit.
  • the second therapeutic agent is administered simultaneously with the phenothiazine compound of the present invention, its hydrate, solvate or reduced form, in other embodiments, the phenothiazine compound of the present invention, its hydrate, solvent
  • the compound or reduced form and the second therapeutic agent are provided as a single pharmaceutical composition.
  • One aspect of the present invention provides a pharmaceutical composition for the treatment of cancer, which comprises a phenothiazine compound represented by formula I, its hydrate, solvate or reduced form:
  • Z is selected from: S+, O+, C or N;
  • Y is selected from: N or N+; and when Z is selected from: S+ or O+, Y is selected from N; when Z is selected from C or N, Y is selected from N+; X- is one that can form a salt with Z+ or N+ Or multiple anions to achieve electrical neutrality;
  • R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , R 7 , R 8 , R 9 and R 10 are each independently selected from: hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted Cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted alkoxy, substituted or unsubstituted aralkoxy, Sulfur alkoxy, amine, nitro, amino, halogen.
  • the X is selected from inorganic acid anions and organic acid anions.
  • the inorganic anion is preferably Cl -, Br -, I - .
  • the organic acid anion is preferably methanesulfonate anion, ethanesulfonate anion, p-toluenesulfonate anion, benzenesulfonate anion, ethanedisulfonate anion, malonic acid anion and naphthalenedisulfonate anion.
  • the phenothiazine compound is selected from the 3,7-bis(dimethylamino)phenothiazine-5-onium salt (MTX) of formula II and its hydrate, solvate or reduced form:
  • MTX 3,7-bis(dimethylamino)phenothiazine-5-onium salt
  • X is one or more anions, so as to achieve electrical neutrality, and is as defined above.
  • the reduced form of the phenothiazine compound is N3, N3, N7, N7-tetramethyl-10H-phenothiazine-3,7-diammonium salt (LMTX) of formula III, its Hydrate or solvate:
  • X is one or more anions, so as to achieve electrical neutrality, and is as defined above.
  • the phenothiazine compound is 3,7-bis(dimethylamino)phenothiazine-5-onium salt (MTX), preferably the phenothiazine compound is 3,7- Bis(dimethylamino)phenothiazin-5-ium chloride (MTC).
  • MTX 3,7-bis(dimethylamino)phenothiazine-5-onium salt
  • MTC 3,7- Bis(dimethylamino)phenothiazin-5-ium chloride
  • the phenothiazine compound is N3, N3, N7, N7-tetramethyl-10H-phenothiazine-3,7-diammonium bis(methanesulfonate).
  • Another aspect of the present invention provides a pharmaceutical composition for the treatment of cancer, comprising: a phenothiazine compound represented by formula I, its hydrate, solvate or reduced form, and suitable for adoptive cells Immune cells for therapy, wherein formula I and its substituents are as defined above.
  • the immune cells are selected from tumor infiltrating lymphocytes (TIL), chimeric antigen receptor T cells (CAR-T), chimeric antigen receptor NK cells (CAR-NK), and T cell receptors.
  • TIL tumor infiltrating lymphocytes
  • CAR-T chimeric antigen receptor T cells
  • CAR-NK chimeric antigen receptor NK cells
  • T cell receptors T cell receptors.
  • the immune cell is a T cell receptor (TCR) chimeric T cell.
  • TCR T cell receptor
  • the TCR is capable of binding to the SIINFEKL peptide.
  • Another aspect of the present invention provides a pharmaceutical composition for the treatment of cancer, which comprises: a phenothiazine compound represented by formula I, its hydrate, solvate or reduced form, and a pharmaceutical composition for cancer chemotherapy An immunotherapeutic agent, wherein formula I and its substituents are as defined above.
  • the immunotherapeutic agent for cancer chemotherapy is a PD-1 antibody, a PD-L1 antibody or a functional fragment thereof.
  • the pharmaceutical composition of the present invention may include an effective ingredient (for example, any compound of Formula I), which is present in the composition in a mixed form with a pharmaceutically acceptable carrier.
  • the composition of the present invention may also contain two active ingredients (for example, any compound of formula I and adoptive therapy immune cells, or any compound of formula I and PD-1 antibody), which are contained in an appropriate form
  • the pharmaceutical composition when the pharmaceutical composition is administered, the subject is administered the two active ingredients simultaneously or sequentially.
  • the compound of formula I, immune cells suitable for adoptive cell therapy, and a carrier are present in the pharmaceutical composition as a mixture in a predetermined ratio.
  • the compound of formula I and the carrier constitute a part of the pharmaceutical composition in a predetermined ratio
  • immune cells suitable for adoptive cell therapy and the carrier constitute another part of the pharmaceutical composition in a predetermined ratio
  • the combination of the two parts constitutes the pharmaceutical composition
  • a combination of core-shell structure For example, a combination of core-shell structure.
  • Other methods available in pharmacy or pharmaceutical engineering can also be used to combine the two active ingredients without affecting the function of each active ingredient after being administered.
  • kits comprising a first pharmaceutical composition and a second pharmaceutical composition that exist independently, the first pharmaceutical composition comprising a therapeutically effective amount of immune cells suitable for adoptive cell therapy
  • the second pharmaceutical composition comprises a therapeutically effective amount of a phenothiazine compound of formula I or a hydrate or solvate thereof. Therefore, in some embodiments of the kit, the first pharmaceutical composition can be in a separate dosage form, and the second pharmaceutical composition can be in another separate dosage form, and the two dosage forms may be the same or different. In some embodiments, the first pharmaceutical composition and the second pharmaceutical composition in the kit are respectively contained in separate containers.
  • kits comprising a first pharmaceutical composition and a second pharmaceutical composition that exist independently, the first pharmaceutical composition comprising a therapeutically effective amount of a phenothiazine compound of formula I or As a hydrate or solvate thereof, the second pharmaceutical composition contains a therapeutically effective amount of a second chemotherapeutic agent (for example, an immunotherapeutic agent, such as a PD-1 antibody).
  • a second chemotherapeutic agent for example, an immunotherapeutic agent, such as a PD-1 antibody.
  • the first pharmaceutical composition can be in a separate dosage form
  • the second pharmaceutical composition can be in another separate dosage form
  • the two dosage forms may be the same or different.
  • the first pharmaceutical composition and the second pharmaceutical composition in the kit are respectively contained in separate containers.
  • Therapeutically or prophylactically effective amount of the active ingredient can be determined by standard procedures to be administered, considerations may include for example 50, biological half-life, the age, size and weight, and conditions related to the object compound IC. The importance of these and other factors is well known to those of ordinary skill in the art. Generally speaking, the dose is between about 0.01 mg/kg and 50 mg/kg, preferably between 0.1 mg/kg and 20 mg/kg of the subject to be treated.
  • Carriers or excipients can be used to produce pharmaceutical compositions.
  • the carrier or excipient may be selected to facilitate the administration of the compound.
  • carriers include calcium carbonate, calcium phosphate, various sugars (such as lactose, glucose, or sucrose), or starch types, cellulose derivatives, gelatin, vegetable oils, polyethylene glycol, and physiologically compatible solvents.
  • physiologically compatible solvents include sterile water for injection (WFI) solutions, saline solutions, and dextrose.
  • the appropriate dosage form depends in part on the route of administration, such as oral, transdermal, transmucosal, inhalation or by injection (parenteral). Such dosage forms should enable the effective ingredients to reach target cells.
  • the drugs or pharmaceutical compositions of the present invention can be administered by different routes, including intravenous, intraperitoneal, subcutaneous, intramuscular, oral, transmucosal, rectal, transdermal or inhalation.
  • oral administration is preferred.
  • the compound can be formulated into conventional oral dosage forms such as capsules, tablets, and liquid preparations such as syrups, elixirs, and concentrated drops.
  • MTC in the following examples refers to PD-1 signal transduction inhibitor: 3,7-bis(dimethylamino)phenothiazine-5-onium chloride, the structural formula of which is as follows:
  • the LMT in the following examples is N3, N3, N7, N7-tetramethyl-10H-phenothiazine-3,7-diammonium bis(methanesulfonate), the structural formula is as follows:
  • Example 1 MTC improves the function of human T cells
  • Raji cells stably expressing PD-L1 are used to stimulate PD-1 molecules on Jurkat-PD-1.
  • Raji cells are human-derived B lymphocytes that express B7 molecules and can provide the second signal required for T cell activation.
  • Jurkat-PD-1-NFAT-LUC cells were stimulated with 2 ⁇ g/mL CD3 antibody/2 ⁇ g/mL CD28 antibody, and then co-cultured with Raji-PD-L1 cells at a ratio of 1:1 for 6 hours, and then detected by Western blotting Phosphorylation at Y248 of PD-1.
  • IL-2 secretion results show that: Jurkat-PD-1-NFAT-LUC can significantly stimulate IL-2 secretion after PD1 antibody (anti-PD1, Bristol-Myers Squibb Nivolumab) or Raji cells are added; and Compared with the Jurkat-PD-1-NFAT-LUC group without any stimulation, the IL-2 secretion of the experimental group with Raji PDL1 was reduced, while after the addition of MTC or PD1 antibody, the secretion of IL-2 was sharply increased (Figure 1D); And compared with the PD1 antibody group, MTC can more significantly improve the function of Jurkat cells inhibited by PD-1.
  • PD1 antibody anti-PD1, Bristol-Myers Squibb Nivolumab
  • Example 2 MTC can promote the division of OT-1 cells and secrete effector molecules
  • TCR expressed by CD8+ T cells of OT-1 transgenic mice can recognize the SIINFEKL-H-2Kb complex of chicken ovalbumin. Therefore, when the spleen cells of OT-1 mice are cultured in vitro, CD8+ T cells in the spleen can be activated and expanded vigorously under the stimulation of the SIINFEKL peptide, which we call CTL cells.
  • CTL cells and target cells EG7 (EL4 lymphoma cells expressing chicken ovalbumin) were mixed and cultured at a concentration of 1:1, treated with 1 ⁇ M protein transport inhibitor GolgiPlug TM for 6 hours, and then fixed with 4% paraformaldehyde. Cells were punched with 0.1% saponin, and then stained with antibodies against each effector molecule, and flow cytometry was used to detect the effect of MTC on the secretion of CTL effector molecules.
  • EG7 is an EL4 lymphoma cell expressing chicken ovalbumin. Therefore, CTL cells can recognize and perform its killing function.
  • CTL cells When activated OT-1 mouse spleen cells and EG7 cells are mixed in a ratio of 1:1, CTL cells begin to secrete effector molecules, such as IL-2, IFN ⁇ , perforin (Perforin), and Granzyme B (Granzyme B).
  • effector molecules such as IL-2, IFN ⁇ , perforin (Perforin), and Granzyme B (Granzyme B).
  • Add protein transport inhibitor GolgiPlug TM (PTI) and then use FACS for detection.
  • EG7 cells overexpressing PD-L1 on the surface EG7-PD-L1 cell stable strain
  • EG7-PD-L1 cell stable strain can strongly inhibit the secretion of IL-2, IFN ⁇ , Perforin, and Granzyme B by CTL.
  • Example 3 MTC can restore OT-1 T cells to kill target cells
  • EG-7-PD-L1 cells were labeled with 5nM CFSE, then CTL cells and EG-7-PD-L1 were mixed at a ratio of 5:1, and then treated with 1 ⁇ M, 5 ⁇ M, 10 ⁇ M and MTC for 4 hours. Then stained with 10 ⁇ g/mL PI to detect the apoptosis of target cells EG-7-PD-L1 by flow cytometry, and then judge the killing ability of CTL cells to target cells.
  • B16-OVA B16 cells expressing OVA gene
  • CTL cells CTL cells at a ratio of 1:5 for 4 hours
  • MTC MTC (1 ⁇ M) at the same time
  • B16-OVA cells that were not treated with IFN- ⁇ and only treated with DMSO and MTC were used as controls. Observe the apoptosis of B16-OVA under a microscope, and then judge the killing ability of CTL cells to target cells.
  • the melanoma cells B16-OVA (B16 cells expressing OVA gene) were treated with no treatment in the B16-OVA group, and the medium was replaced with water in the Water group; while the other groups were treated with 10 ⁇ g/mL IFN ⁇ , 24 After hours, all groups except the B16-OVA group were mixed and cultured with CTL cells in a ratio of 1:5 for 4 hours, while using MTC (1 ⁇ M), LMT (1 ⁇ M) or anti-PD1 (PD-1 antibody, 10 ⁇ g/ mL, Nivolumab, BMS), observe the death of B16-OVA in each group under a microscope, and then judge the killing ability of CTL cells to target cells.
  • the B16-WT group is not treated with any treatment; the IFN- ⁇ +MTC group and IFN- ⁇ +LMT group are treated with 10 ⁇ g/mL IFN- ⁇ , 24 hours later , And CTL cells were mixed and cultured at a ratio of 1:5 for 4 hours while being treated with MTC (1 ⁇ M) or LMT (1 ⁇ M); while the MTC group and LMT group were only treated with MTC (10 ⁇ M) and LMT (10 ⁇ M) to observe the target The killing ability of cells.
  • Activated OT-1 cells express PD-1 on the surface, so it kills EG-7-PD-L1 cells (OVA gene-modified mouse T lymphoma cells EG7 overexpress mouse PD -L1) has limited capabilities (Figure 4A).
  • MTC can significantly restore the ability of CTL cells to kill EG-7-PD-L1 ( Figure 4A).
  • MTC and LMT have no killing effect on B16-WT cells, and because OT-1 cells cannot recognize B16-WT cells, the combination of MTC/LMT and CTL cells is effective for B16-WT cells. There is no lethal effect. While B16-OVA cells have low expression of MHC-I molecules, and their SIINFEKL peptides are not usually presented, so OT-1 cells cannot recognize and kill B16-OVA cells ( Figure 4C and 4D). When B16-OVA cells are treated with IFN ⁇ , and the SIINFEKL peptide is presented, CTL cells have a killing effect ( Figure 4C and 4D).
  • Example 4 MTC helps cytotoxic T cells eliminate tumors formed by target cells
  • mice After the tumor is fixed for about 5 days after inoculation, we inject activated OT-1 T cells (ie 2 ⁇ 10 6 CTL cells/mouse) through the tail vein, and divide the mice into 3 groups, namely the single CTL cell group , CTL cells+10mg/kg/2days PD-1 antibody group, CTL cells+40mg/kg/day MTC group.
  • activated OT-1 T cells ie 2 ⁇ 10 6 CTL cells/mouse
  • Example 5 MTC can effectively treat tumor in situ
  • transplanted tumor is that the epitope recognized by T cells is very clear, and the system is single, which can clearly explain the problem.
  • the disadvantage is that the model cannot simulate the complex carcinogenesis, tumor formation process and the interaction between tumor and stromal cells of the primary cancer. Therefore, the mouse xenograft model has poor predictability for the effect of the drug in human patients. Therefore, this example uses the transgenic mouse model of lung cancer to further investigate the effect of the MTC of the present invention in treating tumors.
  • mice By feeding EGFR-L858R transgenic mice with food containing tetracycline (DOX), mouse lung epithelial cells can be induced to express human EGFR-L858R mutant (the mutant is common in human lung cancer), within 1-2 months Later, lung cancer in mice can be detected and recorded by Computer Tomography (CT).
  • CT Computer Tomography
  • CT Computer Tomography
  • mice are fed food containing DOX
  • rtTA in the lung epithelial cells undergoes conformational changes after binding to DOX, binding to the sequence of TetO, and starting the expression of EGFR mutants controlled by TetO.
  • These mice developed lung adenocarcinoma in situ after feeding tetracycline for 40 days.
  • These lung adenocarcinomas truly mimic the entire clinical process of lung epithelial cells undergoing cancerous transformation and cancer development under the action of EGFR mutants, and finally the body dies of cancer.
  • Example 6 MTC blocks PD-1 from recruiting SHP2
  • the two fusion genes PD-1-Cluc and Nluc-SHP2 were transferred into liposomes in 293T cells to construct a stable transgenic cell line.
  • the cells were added with different concentrations of MTC and cultured for 6 hours to observe the effect of MTC on luciferase. The effect of reading.

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Abstract

吩噻嗪类化合物在制药中的新应用，其中所述吩噻嗪类化合物选自式(I)所示的化合物、其水合物、溶剂化物或其还原型形式，具体为在制备PD-1信号传导抑制剂中的应用。所述的吩噻嗪类化合物能够抑制PD-1的功能，使PD-1的信号传导受阻，从而可以作为PD-1信号传导抑制剂。在体外实验中发现，该类化合物可以恢复被PD-1抑制的免疫细胞的功能，从而提高CTL等免疫细胞分泌细胞因子和杀伤靶细胞的功能，从而可以提高机体的免疫功能，并且可以有效治疗肿瘤。

Description

吩噻嗪类或其类似结构的化合物在制药中的新应用 技术领域
本发明涉及药物技术领域，特别是涉及一种吩噻嗪类或其类似结构的化合物、其水合物、溶剂化物或还原型形式在制药中的新应用。本发明另一方面涉及吩噻嗪类化合物与过继性细胞疗法的联合应用。本发明另一方面涉及吩噻嗪类化合物与肿瘤免疫化疗剂特别是PD-1抗体的联合应用。
背景技术
过继性细胞疗法(Adoptive cell therapy)是一种治疗方法，其包括从哺乳动物收集一种或多种不同类型的免疫细胞，离体(ex vivo)培养和/或操作收集的免疫细胞，并将经培养和/或操作的免疫细胞返回哺乳动物。对收集的免疫细胞的离体操作包括将重组核酸引入免疫细胞中。过继性细胞疗法包括但不限于肿瘤浸润淋巴细胞(TIL，Tumor Infiltrating Lymphocyte)、淋巴因子激活的杀伤细胞(LAK，Lymphokine Activated Killer)、细胞因子诱导的杀伤细胞(CIK，Cytokine Induced Killer)、树突状细胞(DC，Dendritic Cell)、自然杀伤细胞(NK，Natural Killer)、T细胞受体嵌合型-T细胞(TCR-T，T Cell Receptor TCR-Modified T Cell)、嵌合抗原受体NK细胞(CAR-NK)、及嵌合抗原受体T细胞(CAR-T，Chimeric Antigen Receptor Engineered T Cell)等。
人体免疫系统能对抗并清除外来入侵的病原微生物或变异的细胞，免疫细胞是执行人体免疫功能的重要细胞。其中，T细胞是人体内重要的免疫细胞，它能通过表达在其细胞表面的抗原受体(TCR)识别靶细胞上被主要组织相容性复合物(MHC)递呈的抗原，这称为第一信号。在靶细胞合适的第二信号(如B7分子)协同作用下，T细胞就被激活并执行其功能。如T细胞中的其中一个亚类，细胞毒性T细胞(简称CTL)，被激活后能直接杀伤靶细胞，从而清除被病毒感染的细胞或肿瘤细胞。
在T细胞被激活的过程中，T细胞启动表达负反馈抑制其免疫功能的一些分子，PD-1(即programmed cell death 1，程序性凋亡蛋白1)则是其中最著名的分子之一。当CTL细胞表面的PD-1蛋白受到其配体(通常是靶细胞(如肿瘤细胞)上的PD-L1)的刺激后，PD-1就启动其信号传导，抑制CTL的功能，导致T细胞凋亡或无能。这样的信号传导使得CTL失去了杀伤靶细胞的功能。以人体中的肿瘤细胞为例，这些肿瘤细胞通常在细胞表面表达PD-L1，抑制CTL对其的杀伤，从而能够逃逸免疫监控。而当用药物阻断PD-L1与PD-1相互作用后，PD-1的信号传导受阻，CTL杀伤肿瘤细胞的能力则得以恢复。除了T细胞 之外，其它免疫细胞也能够表达PD-1。例如巨噬细胞、B细胞等都已被报道表达PD-1。用合适的药物抑制PD-1的功能后，这些免疫细胞的功能在一定程度上得以恢复。例如，当阻断PD-1的功能后，巨噬细胞从M2(即抑制CTL功能的类型)转变成为M1(即促进CTL功能的类型)，并清除机体中的肿瘤细胞。目前已经有生物制剂如抗体类药物获得FDA批准以用于临床治疗。但到目前为止，临床使用的针对PD-1的药物都是属于生物类制剂类的抗体，小分子化合物的PD-1信号传导抑制剂却未见相关报道。
亚甲基蓝(3,7-双(二甲氨基)吩噻嗪-5-鎓氯化物)是一种吩噻嗪盐，广泛应用于化学指示剂、染料、生物染色剂等方面。最近有一些研究报道了其在药物方面的应用，比如，中国专利公布号CN 104027338 A公开了亚甲基蓝抗急性脑缺血的新用途；中国专利公告号CN 103417546 B公开了亚甲蓝的麻醉后促清醒的新应用。另外也有一些文献中公开了亚甲基蓝作为光敏剂用于制备光敏药物进行光动力疗法。已有研究人员发现，传统的光动力疗法用于体内治疗时，生物组织对光线的吸收和散射作用造成了激发光强度的衰减，并且恶性肿瘤组织所处的乏氧状态会导致单线态氧的产量较低，由单纯的光敏剂作为光敏药物，其并不具有真正治疗肿瘤的作用(中国专利公告号CN 106668859A)。
因此，尽管现有技术有公开亚甲基蓝在药物方面的一些应用，但并没有相关文献报道将亚甲基蓝应用于PD-1信号传导的抑制，并将其应用于肿瘤的防治或治疗。目前临床上仍然缺乏足够有效的药物来治疗癌症，因此提供一种能够有效肿瘤的药物具有重要的临床意义。
发明内容
本发明的提供了一种药用组合物，包括：(a)适用于过继性细胞疗法的免疫细胞；以及(b)吩噻嗪类化合物，其选自式(I)所示的化合物、其水合物、溶剂化物或还原型形式：
Figure PCTCN2020110998-appb-000001
其中，Z选自：S +、O +、C或N；
Y选自：N或N +；并且当Z选自：S +或O +时，Y为N；当Z选自C或N时，Y为 N +；
X -为能够与Z +或N +成盐的一个或多个阴离子，从而实现电中性；
R 1、R 2、R 3、R 4、R 5、R 6、R 7、R 8、R 9和R 10分别独立地选自：氢、取代或未取代的烷基、取代或未取代的环烷基、取代或未取代的杂环烷基、取代或未取代的芳基、取代或未取代的杂芳基、取代或未取代的烷氧基、取代或未取代的芳烷氧基、硫烷氧基、胺基、硝基、氨基、卤素。
在一些实施方式中，所述X选自无机酸根阴离子和有机酸根阴离子。进一步地，所述无机酸根阴离子优选为Cl -、Br -、I -。所述有机酸根阴离子优选为甲磺酸根阴离子、乙磺酸酸根阴离子、对甲苯磺酸根阴离子、苯磺酸根阴离子、乙二磺酸酸根阴离子、丙二磺酸酸根阴离子和萘二磺酸根阴离子。
在一些实施方式中，R1、R2、R3、R4、R5、R6、R7、R8、R9和R10分别独立地选自：氢、取代或未取代的C1-C6烷基、取代或未取代的C3-C8环烷基、取代或未取代的3-8元杂环烷基、取代或未取代的C5-C10芳基、取代或未取代的5-10元杂芳基、取代或未取代的C1-C6烷氧基、取代或未取代的芳基烷氧基、硫烷氧基、胺基、硝基、氨基、卤素。在一些实施方式中，其中所述吩噻嗪类化合物为3,7-双(二甲氨基)吩噻嗪-5-鎓氯化物。
在一些实施方式中，所述吩噻嗪类化合物选自式II的3,7-双(二甲氨基)吩噻嗪-5-鎓盐(MTX)其水合物、溶剂化物或还原型形式
Figure PCTCN2020110998-appb-000002
其中X为一个或多个阴离子，从而实现电中性，并如以上所定义。
在一些实施方式中，述吩噻嗪类化合物的还原型形式为式III的N3,N3,N7,N7-四甲基-10H-吩噻嗪-3,7-二铵盐(LMTX)、其水合物或溶剂化物
Figure PCTCN2020110998-appb-000003
其中，X为一个或多个阴离子，从而实现电中性，并如以上所定义。
在一些实施方式中，其中所述吩噻嗪类化合物为3,7-双(二甲氨基)吩噻嗪-5-鎓氯化物或N3,N3,N7,N7-四甲基-10H-吩噻嗪-3,7-二铵双(甲磺酸盐)。
在一些实施方式中，其中所述免疫细胞选自肿瘤浸润淋巴细胞(TIL)、嵌合抗原受体T细胞(CAR-T)、嵌合抗原受体NK细胞(CAR-NK)和T细胞受体(TCR)嵌合型T细胞(TCR-T)。
在一些实施方式中，其中所述免疫细胞为T细胞受体(TCR)嵌合型T细胞。
在一些实施方式中，其中所述TCR能够与SIINFEKL肽结合。
本发明再一方面提供一种药盒，包括：a)上述免疫细胞中任一种，其被配制在第一剂型中；b)上述的化合物中的任一个，其被配制在第二剂型中。
本发明再一方面提供上述化合物中的任何一种在制备治疗癌症的药物中的应用。
在一些实施方式中，所述癌症为黑色素瘤、胸腺肿瘤、肺癌、前列腺癌、乳腺癌、卵巢癌、直结肠癌、胰腺癌、肝癌、淋巴癌、食道癌、膀胱癌、尿道癌、非霍奇金淋巴癌、肾癌或脑瘤。
本发明的另一方面还提供上述吩噻嗪类化合物的任何一种在制备提高免疫细胞功能的药物中的应用。
本发明的另一方面提供上述吩噻嗪类化合物的任何一种在制备抑制PD-1信号传导的药物中的应用。本发明的另一方面提供上述吩噻嗪类化合物的任何一种在制备阻断PD-1下游信号通路的药物中的应用。本发明的另一方面提供上述吩噻嗪类化合物的任何一种在制备阻断PD-1招募SHP2蛋白的药物中的应用。
本发明的另一方面提供一种抑制PD-1信号传导的方法，包括阻断PD-1下游信号通路的步骤。在一些实施方式中，所述阻断是通过阻断PD-1招募SHP2蛋白来实现的。在一些实施方式中，所述阻断PD-1招募SHP2的步骤包括将细胞与有效量的本发明所述的任 一种吩噻嗪类化合物接触。
本发明的另一方面提供一种治疗对象中的癌症的方法，所述方法包括抑制对象中的PD-1信号传导。在一些实施方式中，所述抑制对象中的PD-1信号传导包括阻断PD-1下游信号通路。在一些实施方式中，所述阻断PD-1下游信号通路包括阻断PD-1招募SHP2。在一些实施方式中，所述阻断PD-1招募SHP2的步骤包括向对象施用有效量的本发明所述的任一种吩噻嗪类化合物。
本发明的另一方面提供一种治疗对象中的癌症的方法，所述方法包括向所述对象施用有效量的本发明所述的任一种吩噻嗪类化合物，以及向对象施用第二种疗法。在一些实施方式中，所述第二种疗法选自化学疗法、放射性疗法和手术治疗。在一些实施方式中，所述化学疗法是肿瘤免疫疗法。在一些实施方式中，所述肿瘤免疫疗法包括向所述对象施用PD-1抗体或PD-L1抗体或其功能性片段。在一些实施方式中，该方面所述的方法产生协同的抗肿瘤作用。
本发明的另一方面提供一种用于治疗癌症的药物组合物，所述药物组合物包含本发明所述的任一种吩噻嗪类化合物，以及第二种化疗剂。在一些实施方式中，所述第二化疗剂是肿瘤免疫治疗剂。在一些实施方式中，所述肿瘤免疫治疗剂是PD-1抗体或PD-L1抗体或其功能性片段。
本发明的另一方面提供一种增强PD-1抗体或PD-L1抗体或其功能性片段在对象中的疗效的方法，所述方法包括在施用所述PD-1抗体或PD-L1抗体之前、同时或之后，向所述对象施用有效量的本发明所述的任一种吩噻嗪类化合物。在一些实施方式中，该方法产生协同的抗肿瘤作用。
本发明的发明人通过大量创造性劳动发现：本发明的吩噻嗪类及类似结构的化合物能够与免疫细胞(尤其是经过基因修饰过的免疫细胞)合用时能够显著提高免疫细胞对于靶细胞的杀伤作用，达到协同的效果。本发明的吩噻嗪类及类似结构的化合物能够抑制PD-1的功能，使PD-1的信号传导受阻，从而可以作为PD-1信号传导抑制剂。
在体外实验中发现，该类化合物可以恢复被PD-1抑制的免疫细胞的功能，从而提高CTL等免疫细胞分泌细胞因子和杀伤靶细胞的功能，从而可以提高机体的免疫功能。在动物模型中，本发明的吩噻嗪类及类似结构的化合物能够使小鼠体内的移植瘤或原位肺癌萎缩，达到治疗癌症的目的。此外，相对于PD-1抗体等大分子，小分子PD-1信号传导抑制剂具有成本较低、制备工艺较简单(如通过化学合成)、给药途径多、患者顺应性 高、安全可靠等优势。本发明的实验还证实，本发明的吩噻嗪类化合物在肿瘤抑制方面具有与PD-1抗体相当或优于PD-1抗体的效果。
附图说明
图1为实施例1的实验结果图；其中，A示出了未转染和转染的Jurkat细胞表面的PD-1蛋白的表达；B示出了未转染和转染的Raji细胞表面的PD-L1蛋白的表达；C示出了经转染的Raji细胞诱导经转染Jurkat细胞中PD-1蛋白Y248磷酸化的结果；D为MTC促进Jurkat-PD-1-NFAT-luc细胞分泌IL-2的结果；E示出了MTC或LMT(N3,N3,N7,N7-四甲基-10H-吩噻嗪-3,7-二铵双(甲磺酸盐))促进Jurkat-PD-1-NFAT-luc细胞中荧光素酶(luciferase)活力升高。
图2示出了实施例2中MTC促进OT-1细胞的分裂；其中，A为OT1-CTL细胞表面表达PD-1蛋白的结果；B为MTC促进CTL的分裂结果。
图3示出了实施例2中MTC促进OT-1细胞分泌效应分子，MTC促进CTL细胞中IL-2、IFNγ、Perforin和GranzymeB的分泌。
图4示出了MTC恢复OT-1 T细胞杀伤靶细胞的能力；其中，A示出了MTC促进CTL对EL4-OVA(PD-L1)的杀伤；B示出了IFNγ诱导B16-F10表达PD-L1蛋白；C、D示出了MTC或LMT协同地增加了CTL对B16-OVA细胞的杀伤；E示出了MTC或LMT对于B16-WT细胞没有杀伤作用，即使与CTL细胞联合对于细胞仍无杀伤作用。
图5为MTC促进细胞毒性T细胞(CTL)清除靶细胞形成的肿瘤的结果图；其中，A为移植瘤模式图；B、C、D示出了MTC抑制Rag1-/-小鼠移植瘤的生长；E示出了MTC抑制C57BL/6J小鼠移植瘤的生长。
图6为MTC治疗原发肿瘤的结果图；其中，A为治疗原位瘤实验模式图示；B为MTC通过CD8 +T细胞清除肿瘤的结果。
图7为MTC阻断PD-1招募SHP2的结果图；其中，A证明了MTC减少PD1与SHP2的结合；B示出了MTC抑制PD1招募SHP2的结合；C示出了MTC抑制PD1与SHP2的结合。
具体实施方式
术语“过继性细胞疗法”或“adoptive cell therapy”涉及将具有抗肿瘤活性的免疫细胞转移到癌症患者中。在一些实施例中，ACT是一种治疗方法，该方法涉及使用具有抗肿瘤 活性的淋巴细胞，将这些细胞在体外扩增至大量并将这些细胞输注到携带癌症的宿主中。
术语“肿瘤浸润淋巴细胞”或TIL是指已离开血流并迁移到肿瘤中的白细胞。术语“CAR-T”为“嵌合抗原受体T细胞”的简写形式，其中，嵌合抗原受体(CAR)是CAR-T的核心部件，赋予T细胞HLA非依赖的方式识别靶细胞(如肿瘤)抗原的能力，这使得经过CAR改造的T细胞相较于天然T细胞表面受体TCR能够识别更广泛的目标。
术语“TCR-T(T细胞受体(TCR)嵌合型-T细胞)”是指表达有工程化T细胞受体(engineered TCR)或称人造T细胞受体(artificial TCR)的T细胞。所述工程化T细胞受体或人造T细胞受体经过了基因改造，具有靶向目的抗原的结构，同时也保留了TCR信号传导通路中的结构域和/或辅助分子。在某些实施例中，TCR-T保留了TCR信号传导通路中的全部辅助分子因此，在少量抗原刺激时，就可以发生全激活的状态，引起对靶细胞的杀伤效应。相对于CAR-T而言，这些TCR-T保持并应用了TCR信号传导通路上的所有辅助分子，因此TCR-T对低浓度，少拷贝数抗原的识别敏感性高于某些CAR-T，治疗潜力非常大。
酪氨酸蛋白磷酸酶非受体11型(PTPN11)也称为蛋白酪氨酸磷酸酶1D(PTP-1D)，Src同源区2含有结构域磷酸酶-2(SHP-2)或蛋白酪胺酸性磷酸酶2C(PTP)-2C)，是由人类PTPN11基因编码的酶。SHP-2在各种组织和细胞类型中普遍表达，涉及多种信号传导途径，包括由生长因子如PDGF，EGF和IGF-1等，细胞因子如IL-3，GM-CSF和EPO等，以及胰岛素和干扰素。SHP-2具有化合物信号传导功能。它似乎涉及多种信号转导过程，例如Ras-Raf-MAP-ERK途径，Jak-Stat途径和PI3K-Akt途径。它还被证明可与多种信号中间体结合，如Grb2，FRS2，Jak2，PI3激酶的p85亚基，IRS-1和Gab1和Gab2。作为PD-1受体的下游分子，SHP-2参与T细胞抑制性信号的传导。已有研究表明，SHP-2是PD-1信号传导的下游分子，它不仅抑制T细胞活化而且促进T细胞的失能。已有研究还表明，T淋巴细胞上敲除SHP2可引发抗肿瘤免疫，抑制小鼠结肠炎相关癌症的发生。
甲硫堇(MT)是氧化还原分子，并且取决于环境条件(例如，pH、氧、还原剂)，以还原型形式的10H-吩噻嗪化合物(即N,N,N',N'-四甲基-10H-吩噻嗪-3,7-二胺(LMT))和氧化形式(MT+)之间的平衡状态存在。
氧化形式的盐MTX如式II所示，
Figure PCTCN2020110998-appb-000004
当LMT以其盐形式存在时，被称为LMTX盐，如式III所示。
Figure PCTCN2020110998-appb-000005
X -选自无机酸根阴离子和有机酸根阴离子；合适的有机酸阴离子的实例包括但不限于衍生自以下有机酸的那些有机酸阴离子：2-乙酰氧基苯甲酸、乙酸、抗坏血酸、天冬氨酸、苯甲酸、樟脑磺酸、肉桂酸、柠檬酸、乙二胺四乙酸、乙二磺酸、乙磺酸、富马酸、葡庚糖酸、葡糖酸、谷氨酸、羟乙酸、羟马来酸、羟萘甲酸、羟乙磺酸、乳酸、乳糖酸、月桂酸、马来酸、苹果酸、甲磺酸、粘酸、油酸、草酸、棕榈酸、扑酸(pamoic)、泛酸(pantothenic)、苯乙酸、苯磺酸、丙二磺酸、丙酸、丙酮酸、水杨酸、硬脂酸、琥珀酸、对氨基苯磺酸、酒石酸、甲苯磺酸、对甲苯磺酸、萘二磺酸和戊酸。合适的高分子有机阴离子(polymeric organic anion)包括但不限于衍生自以下高分子酸(polymeric acid)的那些高分子有机阴离子：鞣酸、羧甲基纤维素。进一步地，所述无机酸根阴离子优选为Cl-、Br-、I-。所述有机酸根阴离子优选为甲磺酸根阴离子。
其中亚甲蓝(MTC)(本文也称之为3，7-二(二甲基氨基)吩噻嗪-5-鎓氯化物)，它是甲硫堇(MT)的氧化形式(即MT+)的氯化物盐为具有如下结构式的低分子量(319.86)的水溶性的三环有机化合物：
Figure PCTCN2020110998-appb-000006
以及N3,N3,N7,N7-四甲基-10H-吩噻嗪-3,7-二铵双(甲磺酸盐)结构如下所示，
Figure PCTCN2020110998-appb-000007
本申请使用的术语“溶剂化物”取其常规意义，其是指溶质(如化合物、化合物的盐)和溶剂的复合物(complex)。如果溶剂是水，则可方便地将溶剂化物称为水合物，例如一水合物、二水合物、三水合物等。除非另有说明，当提到具体化合物时，该具体化合物也包括其溶剂化物形式。
本文使用的“治疗”包括给予本申请的化合物或本申请的组合物，以减轻疾病或病症的症状或并发症，或消除疾病或病症。本文使用的术语“减轻”用于描述病症的迹象或症状的严重性降低的过程。症状可减轻而没有消除。在一种实施方案中，给予本申请的组合物导致消除迹象或症状。
用途和疗法
本发明的一个方面涉及吩噻嗪类化合物在制备PD-1信号传导抑制剂中的应用，该吩噻嗪类化合物选自式(I)所示的化合物、其水合物、溶剂化物或还原型形式：
Figure PCTCN2020110998-appb-000008
其中，Z选自：S+、O+、C或N；
Y选自：N或N+；并且当Z选自：S+或O+时，Y选自N；当Z选自C或N时，Y选自N+；X-为能够与Z+或N+成盐的一个或多个阴离子，从而实现电中性；
R 1、R 2、R 3、R 4、R 5、R 6、R 7、R 8、R 9和R 10分别独立地选自：氢、取代或未取代的烷基、取代或未取代的环烷基、取代或未取代的杂环烷基、取代或未取代的芳基、取代或未取代的杂芳基、取代或未取代的烷氧基、取代或未取代的芳烷氧基、硫烷氧基、胺基、硝基、氨基、卤素。
在一些实施方式中，所述吩噻嗪类化合物选自式II的3,7-双(二甲氨基)吩噻嗪-5-鎓盐 (MTX)其水合物、溶剂化物或还原型形式
Figure PCTCN2020110998-appb-000009
其中X为一个或多个阴离子，从而实现电中性，并如以上所定义。
在一些实施方式中，述吩噻嗪类化合物的还原型形式为式III的N3,N3,N7,N7-四甲基-10H-吩噻嗪-3,7-二铵盐(LMTX)、其水合物或溶剂化物：
Figure PCTCN2020110998-appb-000010
其中，X为一个或多个阴离子，从而实现电中性，并如以上所定义。
在一些实施方式中，该吩噻嗪类化合物类为3,7-双(二甲氨基)吩噻嗪-5-鎓盐(MTX)，优选地所述吩噻嗪类化合物为3,7-双(二甲氨基)吩噻嗪-5-鎓氯化物。
本发明的另一方面涉及式I的吩噻嗪类化合物、其水合物、溶剂化物或还原型形式在制备治疗癌症的药物中的应用。在一些实施方式中，所述吩噻嗪类化合物为式II的3,7-双(二甲氨基)吩噻嗪-5-鎓盐(MTX)、其水合物、溶剂化物或还原型形式。在一些实施方式中，所述吩噻嗪类化合物为式III的N3,N3,N7,N7-四甲基-10H-吩噻嗪-3,7-二铵盐(LMTX)。在该方面，优选地，所述吩噻嗪类化合物为3,7-双(二甲氨基)吩噻嗪-5-鎓氯化物或N3,N3,N7,N7-四甲基-10H-吩噻嗪-3,7-二铵双(甲磺酸盐)。
本发明的另一个方面涉及式I的吩噻嗪类化合物、其水合物、溶剂化物或还原型形式在制备提高免疫细胞功能的药物中的应用。在一些实施方式中，所述吩噻嗪类化合物为式II的3,7-双(二甲氨基)吩噻嗪-5-鎓盐(MTX)、其水合物、溶剂化物或还原型形式。在一些实施方式中，所述吩噻嗪类化合物为式III的N3,N3,N7,N7-四甲基-10H-吩噻嗪-3,7-二铵盐(LMTX)。在该方面，优选地，所述吩噻嗪类化合物为3,7-双(二甲氨基)吩噻嗪-5-鎓 氯化物或N3,N3,N7,N7-四甲基-10H-吩噻嗪-3,7-二铵双(甲磺酸盐)。
本发明的另一个方面涉及式I的吩噻嗪类化合物、其水合物、溶剂化物或还原型形式在制备预防或治疗癌症复发的药物中的应用。在一些实施方式中，所述吩噻嗪类化合物为式II的3,7-双(二甲氨基)吩噻嗪-5-鎓盐(MTX)、其水合物、溶剂化物或还原型形式。在一些实施方式中，所述吩噻嗪类化合物为式III的N3,N3,N7,N7-四甲基-10H-吩噻嗪-3,7-二铵盐(LMTX)。在该方面，优选地，所述吩噻嗪类化合物为3,7-双(二甲氨基)吩噻嗪-5-鎓氯化物或N3,N3,N7,N7-四甲基-10H-吩噻嗪-3,7-二铵双(甲磺酸盐)。
本发明的另一方面涉及式I的吩噻嗪类化合物、其水合物、溶剂化物或还原型形式在制备阻断PD-1下游信号通路的药物中的应用。在一些实施方式中，所述吩噻嗪类化合物为式II的3,7-双(二甲氨基)吩噻嗪-5-鎓盐(MTX)、其水合物、溶剂化物或还原型形式。在一些实施方式中，所述吩噻嗪类化合物为式III的N3,N3,N7,N7-四甲基-10H-吩噻嗪-3,7-二铵盐(LMTX)。在该方面，优选地，所述吩噻嗪类化合物为3,7-双(二甲氨基)吩噻嗪-5-鎓氯化物或N3,N3,N7,N7-四甲基-10H-吩噻嗪-3,7-二铵双(甲磺酸盐)。
本发明的另一方面涉及式I的吩噻嗪类化合物、其水合物、溶剂化物或还原型形式在制备阻断PD-1招募SHP2蛋白的药物中的应用。在一些实施方式中，所述吩噻嗪类化合物为式II的3,7-双(二甲氨基)吩噻嗪-5-鎓盐(MTX)、其水合物、溶剂化物或还原型形式。在一些实施方式中，所述吩噻嗪类化合物为式III的N3,N3,N7,N7-四甲基-10H-吩噻嗪-3,7-二铵盐(LMTX)。在该方面，优选地，所述吩噻嗪类化合物为3,7-双(二甲氨基)吩噻嗪-5-鎓氯化物或N3,N3,N7,N7-四甲基-10H-吩噻嗪-3,7-二铵双(甲磺酸盐)。
本发明的另一个方面提供一种治疗癌症方法，所述方法包括向患有所述癌症的对象施用治疗有效量的本发明的式I的吩噻嗪类化合物、其水合物、溶剂化物或还原型形式。在一些实施方式中，所述吩噻嗪类化合物为式II的3,7-双(二甲氨基)吩噻嗪-5-鎓盐(MTX)、其水合物、溶剂化物或还原型形式。在一些实施方式中，所述吩噻嗪类化合物为式III的N3,N3,N7,N7-四甲基-10H-吩噻嗪-3,7-二铵盐(LMTX)。在该方面，优选地，所述吩噻嗪类化合物为3,7-双(二甲氨基)吩噻嗪-5-鎓氯化物或N3,N3,N7,N7-四甲基-10H-吩噻嗪-3,7-二铵双(甲磺酸盐)。在一些实施方式中，所述癌症为黑色素瘤、胸腺肿瘤、肺癌、前列腺癌、乳腺癌、卵巢癌、直结肠癌、胰腺癌、肝癌、淋巴癌、食道癌、膀胱癌、尿道癌、非霍奇金淋巴癌、肾癌或脑瘤。在一些实施方式中，所述癌症为黑色素瘤。在一些实施方式中，所述对象为哺乳动物，优选是人。
本发明的另一方面提供一种提高免疫细胞功能的方法，所述方法包括向对象施用治疗有效量的本发明的式I的吩噻嗪类化合物、其水合物、溶剂化物或还原型形式。在一些实施方式中，所述吩噻嗪类化合物为式II的3,7-双(二甲氨基)吩噻嗪-5-鎓盐(MTX)、其水合物、溶剂化物或还原型形式。在一些实施方式中，所述吩噻嗪类化合物为式III的N3,N3,N7,N7-四甲基-10H-吩噻嗪-3,7-二铵盐(LMTX)。在该方面，优选地，所述吩噻嗪类化合物为3,7-双(二甲氨基)吩噻嗪-5-鎓氯化物或N3,N3,N7,N7-四甲基-10H-吩噻嗪-3,7-二铵双(甲磺酸盐)。在一些实施方式中，所述提高免疫细胞功能能够达到抗病毒的效果。在一些实施方式中，所述对象为哺乳动物，优选是人。
本发明的另一方面提供一种预防或治疗癌症复发的方法，所述方法包括向患有所述癌症的对象施用治疗有效量的式I的吩噻嗪类化合物、其水合物、溶剂化物或还原型形式。在一些实施方式中，所述吩噻嗪类化合物为式II的3,7-双(二甲氨基)吩噻嗪-5-鎓盐(MTX)、其水合物、溶剂化物或还原型形式。在一些实施方式中，所述吩噻嗪类化合物为式III的N3,N3,N7,N7-四甲基-10H-吩噻嗪-3,7-二铵盐(LMTX)。在该方面，优选地，所述吩噻嗪类化合物为3,7-双(二甲氨基)吩噻嗪-5-鎓氯化物或N3,N3,N7,N7-四甲基-10H-吩噻嗪-3,7-二铵双(甲磺酸盐)。在一些实施方式中，所述对象经历过手术、放疗或化疗。
在一些实施方式中，所述方法能够与其他癌症的疗法联合应用。例如，所述方法能够与手术、放疗或化疗联合应用。因此，在一些实施方式中，本发明的治疗癌症方法进一步包括向患有所述癌症的对象施用治疗有效量的第二种治疗剂。
在一些实施方式中，所述第二种治疗剂为适用于过继性细胞疗法的免疫细胞。在这些实施方式中，所述第二种治疗剂在施用式I的吩噻嗪类化合物、其水合物、溶剂化物或还原型形式之前、同时或之后施用。
在一些实施方式中，所述第二种治疗剂为化疗剂，优选地，所述化疗剂为肿瘤免疫化疗剂，更优选地，所述化疗剂为PD-1抗体、PD-L1抗体或其片段。在一些实施方式中，所述第二种治疗剂在施用式I的吩噻嗪类化合物、其水合物、溶剂化物或还原型形式之前、同时或之后施用。
当第二种治疗剂与本发明的吩噻嗪类化合物、其水合物或溶剂化物不同时施用时，两者施用间隔约0.1小时至约72小时，例如间隔约0.1、0.2、0.3、0.4、0.5、0.6、0.7、0.8、0.9、1、2、3、4、5、6、7、8、9、10、11、12、18、24、30、36、72h施用。
当第二种治疗剂与本发明的吩噻嗪类化合物、其水合物或溶剂化物同时施用时，在一些实施方式中，本发明的吩噻嗪类化合物、其水合物、溶剂化物或还原型形式与该第二种治疗剂分别以单独的药物组合物形式提供。在一些实施方式中，所述单独的药物组合物被提供在同一个药盒中。当第二种治疗剂与本发明的吩噻嗪类化合物、其水合物、溶剂化物或还原型形式同时施用时，在另一些实施方式中，本发明的吩噻嗪类、其水合物、溶剂化物或还原型形式与该第二种治疗剂以单一的药物组合物形式提供。
药用组合物及药盒
本发明的一个方面提供一种用于治疗癌症的药用组合物，其包含式I所示的吩噻嗪类化合物、其水合物、溶剂化物或还原型形式：
Figure PCTCN2020110998-appb-000011
其中，Z选自：S+、O+、C或N；
Y选自：N或N+；并且当Z选自：S+或O+时，Y选自N；当Z选自C或N时，Y选自N+；X-为能够与Z+或N+成盐的一个或多个阴离子，从而实现电中性；
R 1、R 2、R 3、R 4、R 5、R 6、R 7、R 8、R 9和R 10分别独立地选自：氢、取代或未取代的烷基、取代或未取代的环烷基、取代或未取代的杂环烷基、取代或未取代的芳基、取代或未取代的杂芳基、取代或未取代的烷氧基、取代或未取代的芳烷氧基、硫烷氧基、胺基、硝基、氨基、卤素。
在一些实施方式中，所述X选自无机酸根阴离子和有机酸根阴离子。进一步地，所述无机酸根阴离子优选为Cl -、Br -、I -。所述有机酸根阴离子优选为甲磺酸根阴离子、乙磺酸酸根阴离子、对甲苯磺酸根阴离子、苯磺酸根阴离子、乙二磺酸酸根阴离子、丙二磺酸酸根阴离子和萘二磺酸根阴离子。
在一些实施方式中，所述吩噻嗪类化合物选自式II的3,7-双(二甲氨基)吩噻嗪-5-鎓盐(MTX)其水合物、溶剂化物或还原型形式：
Figure PCTCN2020110998-appb-000012
其中X为一个或多个阴离子，从而实现电中性，并如以上所定义。
在一些实施方式中，述吩噻嗪类化合物的还原型形式为式III的N3,N3,N7,N7-四甲基-10H-吩噻嗪-3,7-二铵盐(LMTX)、其水合物或溶剂化物：
Figure PCTCN2020110998-appb-000013
其中，X为一个或多个阴离子，从而实现电中性，并如以上所定义。
在一些实施方式中，该吩噻嗪类化合物类为3,7-双(二甲氨基)吩噻嗪-5-鎓盐(MTX)，优选地所述吩噻嗪类化合物为3,7-双(二甲氨基)吩噻嗪-5-鎓氯化物(MTC)。
在一些实施方式中，该吩噻嗪类化合物类为N3,N3,N7,N7-四甲基-10H-吩噻嗪-3,7-二铵双(甲磺酸盐)。
本发明的另一个方面提供一种用于治疗癌症的药用组合物，其包含：式I所示的吩噻嗪类化合物、其水合物、溶剂化物或还原型形式，以及适用于过继性细胞疗法的免疫细胞，其中式I及其取代基如以上所定义。
在一些实施方式中，其中所述免疫细胞选自肿瘤浸润淋巴细胞(TIL)、嵌合抗原受体T细胞(CAR-T)、嵌合抗原受体NK细胞(CAR-NK)和T细胞受体(TCR)嵌合型T细胞。
在一些实施方式中，其中所述免疫细胞为T细胞受体(TCR)嵌合型T细胞。
在一些实施方式中，其中所述TCR能够与SIINFEKL肽结合。
本发明的另一个方面提供一种用于治疗癌症的药用组合物，其包含：式I所示的吩噻嗪类化合物、其水合物、溶剂化物或还原型形式，以及用于癌症化疗的免疫治疗剂，其 中式I及其取代基如以上所定义。
在一些实施方式中，所述用于癌症化疗的免疫治疗剂是PD-1抗体、PD-L1抗体或其功能性片段。
因此，本发明的药用组合物可以包含一种有效成分(例如式I的任何一种化合物)，其与药学上可接受的载体以混合形式存在于组合物中。本发明的组合物还可以同时包含两种有效成分(例如式I的任何一种化合物及过继性疗法免疫细胞，或式I的任何一种化合物及PD-1抗体)，它们以适当形式被包含在同一药物组合物中，当施用该药物组合物时，对象同时或按顺序被施用所述两种有效成分。例如，式I化合物、适用于过继性细胞疗法的免疫细胞和载体按预定比例以混合物形式存在于药物组合物中。或者，式I化合物与载体以预定比例组成该药物组合物的一部分，适用于过继性细胞疗法的免疫细胞与载体以预定比例组成该药物组合物的另一部分，两个部分组合构成所述药物组合物，例如以核壳结构组合。药剂学或制药工程上可用的其他方法也可被用来将两种有效成分组合在一起而不影响在被施用后各活性成分的作用发挥。
本发明的另一个方面提供一种药盒，其包含独立存在的第一药物组合物和第二药物组合物，所述第一药物组合物包含治疗有效量的适用于过继性细胞疗法的免疫细胞，所述第二药物组合物包含治疗有效量的式I的吩噻嗪类化合物或其水合物或溶剂化物。因此，在所述药盒的一些实施方式中，所述第一药物组合物能够以单独剂型存在，所述第二药物组合物能够以另一单独剂型存在，两者剂型可以相同或不同。在一些实施方式中，所述药盒中的第一药物组合物和第二药物组合物分别被容纳于单独的容器中。
本发明的另一方面提供一种药盒，其包含独立存在的第一药物组合物和第二药物组合物，所述第一药物组合物包含治疗有效量的式I的吩噻嗪类化合物或其水合物或溶剂化物，所述第二药物组合物包含治疗有效量的第二种化疗剂(例如免疫治疗剂，如PD-1抗体)。因此，在所述药盒的一些实施方式中，所述第一药物组合物能够以单独剂型存在，所述第二药物组合物能够以另一单独剂型存在，两者剂型可以相同或不同。在一些实施方式中，所述药盒中的第一药物组合物和第二药物组合物分别被容纳于单独的容器中。
通过标准程序可以确定待施用的有效成分的治疗或预防有效量，考虑的因素可以包括例如化合物IC 50、生物半衰期、对象的年龄、大小和体重以及与对象有关的病症。这些因素和其它因素的重要性对本领域普通技术人员而言是熟知的。一般而言，剂量为被治疗的对象的大约0.01mg/kg至50mg/kg之间，优选在0.lmg/kg至20mg/kg之间。
载体或赋形剂可以被用于生产药物组合物。所述载体或赋形剂可以被选择为促进化合物的给药。载体的例子包括碳酸钙、磷酸钙、各种糖(例如乳糖、葡萄糖或蔗糖)、或淀粉类型、纤维素衍生物、明胶、植物油、聚乙二醇和生理相容性溶剂。生理上相容性溶剂的例子包括注射用水(WFI)无菌溶液、盐溶液和葡萄糖。
合适的剂型部分地取决于给药途径，例如经口、经皮、经粘膜、吸入或通过注射(肠胃外)。此类剂型应当使有效成分能够到达靶细胞。可以通过不同的路径施用本发明的药物或药物组合物，包括静脉内、腹膜内、皮下、肌内、经口、经粘膜、直肠、经皮或吸入。在一些实施方式中，优选口服。对口服而言，例如，化合物可以被配制为常规口服剂型，例如胶囊、片剂，以及液体制剂，例如糖浆、酏剂和浓缩滴剂。
实施例
以下结合具体实施例对本发明做进一步详细的说明。
以下实施例中的MTC是指PD-1信号传导抑制剂：3,7-双(二甲氨基)吩噻嗪-5-鎓氯化物，其结构式如下：
Figure PCTCN2020110998-appb-000014
以下实施例中的LMT为N3,N3,N7,N7-四甲基-10H-吩噻嗪-3,7-二铵双(甲磺酸盐)，结构式如下：
Figure PCTCN2020110998-appb-000015
实施例1.MTC提高人的T细胞的功能
一、实验方法：
(1)用Jurkat细胞构建高表达PD-1的稳转细胞系。将pCAGin-PD-1质粒电转到Jurkat细胞中，然后用900μg/mL G418抗生素筛选，用流式分选单克隆。然后在Jurkat-PD-1细胞中电转入一个DNA片段，该片段由NFAT结合位点控制荧光素酶(luciferase)的基因(记 作NFAT-LUC)。然后流式分选出单克隆，命名为5号克隆。利用这个细胞，可通过测定luciferase的活力来反映IL-2的表达量。构建稳定表达PD-L1的Raji细胞，将pCDNA-PD-L1质粒电转Raji细胞，用2μg/mL puromycin抗性筛选，然后流式分选单克隆，命名为1号克隆。
(2)稳定表达PD-L1的Raji细胞用来刺激Jurkat-PD-1上的PD-1分子。Raji细胞是人源的B淋巴细胞，表达B7分子，能提供T细胞激活所需的第二信号。将Jurkat-PD-1-NFAT-LUC细胞在2μg/mL CD3抗体/2μg/mL CD28抗体刺激下，然后与Raji-PD-L1细胞以1:1的比例共培养6小时，再通过免疫印迹检测PD-1的Y248位的磷酸化。
(3)在96孔板中，用10μg/mL CD3抗体/10μg/mL CD28抗体包板，4℃过夜，然后用PBS洗掉多余的抗体，将Raji细胞或者Raji-PD-L1细胞以1:1的比例与Jurkat-PD-1-NFAT-LUC细胞共培养；或者在Raji-PD-L1细胞与Jurkat-PD-1-NFAT-LUC细胞以1:1的比例混合的细胞中加入1μM MTC或者10μg/mL的PD1抗体进行共培养；共培养6小时后取上清，用ELISA测IL-2的分泌。
(4)在96孔板中，用10μg/mL CD3抗体/10μg/mL CD28抗体包板，4℃过夜，然后用PBS洗掉多余的抗体，将Raji细胞或者Raji-PD-L1细胞以1:1的比例与Jurkat-PD-1-NFAT-LUC细胞共培养；或者在Raji-PD-L1细胞与Jurkat-PD-1-NFAT-LUC细胞以1:1的比例混合的细胞中加入4.86μM MTC或LMT，共培养6小时后加入luciferase底物，酶标仪测定Luciferase读值。
二、实验结果及分析：
(1)稳定表达PD-1的Jurkat细胞通过FACS测试表明，5号克隆正确表达PD-1分子(图1A)。构建的稳定表达PD-L1的Raji细胞，通过FACS表明1号克隆正确表达PD-L1(图1B)。
(2)将Raji-PD-L1细胞与Jurkat-PD-1细胞共培养6小时后，免疫印迹检测发现PD-1的Y248位被磷酸化(图1C)。由此可知，在表达PD-1的Jurkat与稳定表达PD-L1的Raji细胞的共培养中，PD-1与PD-L1进行了结合。
(3)IL-2的分泌情况结果表明：Jurkat-PD-1-NFAT-LUC在PD1抗体(anti-PD1，百时美施贵宝Nivolumab)或Raji细胞加入后能够显著刺激IL-2的分泌；与未加入任何刺激的Jurkat-PD-1-NFAT-LUC组相比，加入Raji PDL1的实验组的IL-2分泌量降低，而加入MTC或PD1抗体后，IL-2的分泌量急剧增强(图1D)；并且与PD1抗体组相比，MTC能够更 显著地改善被PD-1抑制的Jurkat细胞的功能。
(4)Jurkat-PD-1-NFAT-LUC在CD3抗体/CD28抗体/Raji细胞的刺激下，luciferase的活力升高有限，而加入Raji-PD-L1的细胞luciferase的活力下降。在加入Raji-PD-L1的细胞的同时加入4.86μM MTC或LMT，luciferase的活力急剧升高(图1E)，说明MTC或LMT能改善被PD-1抑制的Jurkat细胞的功能。
实施例2.MTC能够促进OT-1细胞的分裂和分泌效应分子
OT-1转基因小鼠的CD8+T细胞表达的TCR能识别鸡卵清蛋白的SIINFEKL-H-2Kb复合体。所以体外培养OT-1小鼠的脾脏细胞时，在SIINFEKL肽段的刺激下，脾脏中的CD8+T细胞能被激活并剧烈扩增，我们称之为CTL细胞。
一、实验方法：
(1)CTL细胞制备：取OT1小鼠的脾脏，研磨后加入红细胞裂解液裂解红细胞，制成单细胞悬液，培养液为1640+10％FBS+50μMβME+10nM IL-2，调节细胞密度约2-4million/mL,加入10nM OVA257-264，37℃、5％CO 2细胞培养箱进行培养，从CTL制备当天起，每天用FITC标记的PD-1抗体染色，然后流式检测此细胞表面PD-1表达变化。
(2)取上述CTL细胞用5nM CFSE标记，加入10nM SIINFEKL和10nM IL-2的刺激，同时加入10μg/mL小鼠的PD-L1蛋白，实验组还加入100nM的MTC小分子化合物，分别刺激24、48、72小时后，流式观察PD-L1蛋白及MTC对CTL细胞分裂的影响。
(3)将CTL细胞与靶细胞EG7(表达鸡卵清蛋白的EL4淋巴瘤细胞)按1:1的浓度混合培养，用1μM蛋白转运抑制剂GolgiPlug TM处理6小时后，4％多聚甲醛固定细胞，0.1％saponin对细胞进行打孔，然后用各效应分子的抗体进行染色，流式检测MTC对CTL效应分子的分泌情况的影响。
二、实验结果及分析：
(1)在诱导CTL细胞的整个过程中，随着诱导时间增加，其细胞表面PD-1的量逐渐增加(图2A)。
(2)当CTL用SIINFEKL和IL-2刺激，且加入PD-L1蛋白后，PD-L1蛋白能抑制CTL细胞的分裂，而加入100nM的MTC后则能逆转这种抑制(图2B)。
(3)EG7是表达鸡卵清蛋白的EL4淋巴瘤细胞，因此，CTL细胞能识别并执行其杀伤功能。当激活的OT-1小鼠脾细胞和EG7细胞按照1:1的比例混合后，CTL细胞开始分泌 效应分子，如IL-2、IFNγ、穿孔素(Perforin)、颗粒酶B(Granzyme B)。加入蛋白转运抑制剂GolgiPlug TM(PTI)，然后用FACS进行检测。我们发现表面过表达PD-L1的EG7细胞(EG7-PD-L1细胞稳株)能强烈地抑制CTL分泌IL-2、IFNγ、Perforin、Granzyme B。而当我们用MTC处理后，CTL细胞IL-2、IFNγ、Perforin、Granzyme B的分泌量急剧升高(图3)；这说明MTC能够改善PD-1的抑制，促进CTL细胞分泌IL-2、IFNγ、Perforin、Granzyme B。
实施例3.MTC能够恢复OT-1 T细胞杀伤靶细胞
一、实验方法：
(1)CTL细胞的制备见实施例2。
(2)将EG-7-PD-L1细胞用5nM CFSE标记，然后将CTL细胞与EG-7-PD-L1按照5:1的比例混合后，分别用1μM、5μM、10μM MTC处理4小时后再用10μg/mL的PI染色，流式检测靶细胞EG-7-PD-L1的凋亡情况，进而判断CTL细胞对靶细胞的杀伤能力。
(3)将黑色素瘤细胞B16-OVA(表达OVA基因的B16细胞)用10μg/mL IFN-γ处理24小时后，与CTL细胞按照1：5的比例混合培养4小时，同时用MTC(1μM)处理，将未经IFN-γ处理仅分别给予DMSO和MTC处理的B16-OVA细胞作为对照。显微镜观察B16-OVA的凋亡情况，进而判断CTL细胞对靶细胞的杀伤能力。
(4)将黑色素瘤细胞B16-OVA(表达OVA基因的B16细胞)其中B16-OVA组不做任何处理，Water组则将培养基更换为水；而其余各组用10μg/mL IFNγ处理，24小时后，除B16-OVA组之外各组均与CTL细胞按照1：5的比例混合培养4小时，同时用MTC(1μM)、LMT(1μM)或anti-PD1(PD-1抗体，10μg/mL，Nivolumab，BMS)处理，显微镜观察各组B16-OVA的死亡情况，进而判断CTL细胞对靶细胞的杀伤能力。我们还提供未经基因工程改造的黑色素瘤B16-WT，其中B16-WT组不做任何处理；IFN-γ+MTC组与IFN-γ+LMT组用10μg/mL IFN-γ处理，24小时后，与CTL细胞按照1：5的比例混合培养4小时同时用MTC(1μM)或LMT(1μM)处理；而MTC组和LMT组则仅给予MTC(10μM)和LMT(10μM)处理，观察对靶细胞的杀伤能力。
二、实验结果及分析：
(1)激活的OT-1细胞(CTL细胞)表面表达PD-1，因此其杀伤EG-7-PD-L1细胞(OVA基因修饰的小鼠T淋巴瘤细胞EG7中过表达了小鼠的PD-L1)的能力有限(图4A)。在这个系统中，MTC却能显著地恢复CTL细胞对EG-7-PD-L1的杀伤能力(图4A)。
(2)如图4E所示，MTC和LMT对B16-WT细胞并无杀伤作用，而且由于OT-1细胞不能识别B16-WT细胞，因此在MTC/LMT与CTL细胞的联用对于B16-WT并无杀伤作用。而B16-OVA细胞低表达MHC-I分子，其SIINFEKL肽段平时没有被递呈出来，因此OT-1细胞不能识别并杀伤B16-OVA细胞(图4C和4D)。当B16-OVA细胞用IFNγ处理后，SIINFEKL肽段被递呈后，CTL细胞具有杀伤作用(图4C和4D)。但IFNγ处理后，诱导了B16-OVA的PD-L1表达(图4B)。因为CTL对于B16-OVA的这种杀伤作用被PD-1与PD-L1相互作用减弱，所以单独使用CTL时杀伤作用并不显著。我们通过同时加入MTC或LMT则能显著地增强上述条件下CTL细胞对B16-OVA细胞的杀伤量(图4C和4D)；此外，如图4D所示，MTC与CTL细胞的联合杀伤作用与LMT与CTL的联合杀伤作用相当，并且它们的杀伤作用明显强于anti-PD-1组。因此，通过MTC或LMT与CTL细胞联合能够协同地加强对于B16-OVA的杀伤作用。
实施例4.MTC帮助细胞毒性T细胞清除靶细胞形成的肿瘤
上述实施例1-3的实验结果已证实MTC能强烈恢复细胞毒性T细胞杀伤过表达PD-L1的靶细胞。本实施例进一步考察了MTC体内治疗肿瘤的能力。
一、实验方法：
(1)将过表达了鸡的卵清白蛋白OVA和小鼠的PD-L1的淋巴瘤细胞即EL4-OVA-mPDL1细胞重悬于BD Matrigel基质胶中(按照一只小鼠一侧需要2×10 6细胞重悬于100μl BD Matrigel基质胶中)。
(2)将上述细胞和基质胶的混合物接种到Rag1-/-小鼠左右侧腰背部皮下各100μl。
(3)待接种5天左右肿瘤固定后，我们通过尾静脉输入激活的OT-1 T细胞(即2×10 6CTL细胞/只)，同时将小鼠分为3组，即单独CTL细胞组，CTL细胞+10mg/kg/2days PD-1抗体组，CTL细胞+40mg/kg/day MTC组。
(4)从尾静脉注射CTL细胞当天开始测量各组小鼠的移植瘤的长和宽，隔一天测量一次，以长×宽 2/2来计算肿瘤的体积大小(图5A)。
二、实验结果及分析：
(1)与只尾静脉注射CTL细胞的对照组相比，同时腹腔注射PD-1抗体组的小鼠的肿瘤生成受到抑制，肿瘤的体积和重量的增速均受到抑制。由此可知，PD-1抗体组的肿瘤生长受到抑制。
(2)给带有移植瘤的小鼠灌胃给药MTC同样也能强烈地抑制肿瘤的生长(图5B、C、D)。在治疗结束之后，仅输入CTL细胞组无论是肿瘤重量还是肿瘤体积都表明该组小鼠的肿瘤生长迅速，由此可知单独给予CTL不能有效抑制肿瘤的生长。而CTL+PD-1抗体治疗组和CTL+MTC治疗组却都能够显著抑制肿瘤的生长，并且CTL+MTC治疗组治疗效果上是优于CTL+PD-1抗体治疗组。
以与本实施例相似的方法用EG7-mPDL1细胞在C57BL/6J小鼠中得到了相同的结果(图5E)，未给予CTL(CTL free)的小鼠肿瘤生长迅速，而仅给予CTL虽然与CTL free相比能够抑制肿瘤的生长，但其抑制作用仍然弱于给予CTL+PD-1抗体或CTL+MTC联用组的效果。说明MTC能够有效的帮助细胞毒性T细胞清除靶细胞形成的皮下肿瘤。
实施例5.MTC能有效治疗原位瘤
移植瘤的优点是T细胞识别的抗原表位非常清晰，而且体系单一，能清晰地说明问题。缺点是该模型不能模拟原发癌症复杂的癌变、肿瘤形成过程以及肿瘤和基质细胞的相互作用。因此，鼠的移植瘤模型对于药物在人类患者体内的效果的预测性较差。因此，本实施例利用转基因肺癌小鼠模型进一步考察了本发明MTC治疗肿瘤的作用。通过给EGFR-L858R转基因小鼠喂以含四环素(DOX)食物，就能诱导小鼠肺上皮细胞表达人的EGFR-L858R突变体(该突变体常见于人的肺癌)，在1-2个月后，小鼠的肺癌已经能被计算机断层扫描(Computer Tomography,CT)检测并记录。
一、实验方法：
(1)将EGFR-L858R突变的小鼠给予DOX粮食诱导原位肿瘤模型，诱导时间约为40天左右。
(2)计算机断层扫描(Computer Tomography,CT)记录肿瘤的大小及严重程度。
(3)将荷瘤小鼠随机分组，分别给以安慰剂(HKI solution)、MTC(40mg/kg/day in HKI solution)。治疗2周后，CT再次记录肿瘤大小。
(4)为了证明MTC在清除肿瘤的过程中是通过CD8 +T细胞达到目的的，本实施例还进行了以下实验：先用CD8抗体腹腔注射(200mg/只/3天)提前处理一周，将荷瘤小鼠中的CD8 +细胞清除掉，再用MTC和CD8抗体共同治疗2周，CT再次记录肿瘤大小。
二、实验结果及分析：
本实验原理为当给这些小鼠喂养含DOX的食物后，肺上皮细胞中的rtTA结合DOX后发 生构象变化，结合到TetO这个序列上，启动TetO控制的EGFR突变体的表达。这些小鼠在喂养四环素40天后形成原位肺腺癌。这些肺腺癌真实的模拟了肺上皮细胞在EGFR突变体的作用下发生癌变、癌症发展，最后机体死于癌症的整个临床过程。本实施例的实验结果显示安慰剂治疗组的肿瘤快速生长，而通过MTC治疗14天后的小鼠肿瘤的影像学显示肿瘤已基本被清除(图6B)，而在CD8 +T细胞清除情况下的小鼠中的肿瘤在MTC治疗后仍肿瘤的生长并未受到抑制(图6B)。由此可知，MTC能通过激活体内的CTL有效地治疗EGFR-L858R突变的原位肿瘤。
实施例6.MTC阻断PD-1招募SHP2
本实施例初步探究了MTC抑制PD-1传导信号的机理，考察了MTC影响PD-1招募SHP2的能力。PD-1与配体结合可以将SHP2蛋白募集到T细胞受体周围，从而抑制T细胞受体近端激酶的激活，降低Lck所介导的ZAP-70蛋白的磷酸化以及下游信号通路的启动。Luciferase可以拆分为N端和C端两个片段，当这两个片段分别与其他蛋白融合后，如果N端和C端的luciferase片段因为他们的融合蛋白相互作用而靠近后，luciferase的活力就能被检测到，因此，可以通过检测luciferase的活力来间接反映所融合的蛋白的相互作用的强度。
一、实验方法：
(1)首先在293T细胞中脂质体转入PD-1-Cluc和Nluc-SHP2这两个融合基因，构建稳转细胞系，用此细胞加入不同浓度的MTC培养6小时，观察MTC对luciferase读值的影响。
(2)在293T细胞中脂质体转PD-1-GFP和SHP2-mCherry这两个融合基因，构建稳转细胞系，在此细胞中加入不同浓度的MTC培养6小时后再加入1μM PVD(去磷酸化酶抑制剂)处理5分钟，4％多聚甲醛固定后Confocal观察MTC对GFP和mCherry即PD-1和SHP2定位的影响。
(3)在293T细胞中共转染PD-1-Flag质粒和SHP2质粒,然后加入不同浓度的MTC处理，Co-IP检测PD-1招募SHP2的能力。
二、实验结果及分析：
(1)在293T稳转PD-1-Cluc和Nluc-SHP2的稳株中加入不同浓度的MTC，发现MTC能减少luciferase的读值。显示MTC能阻止PD-1招募SHP2(图7A)。
(2)为了进一步证明上步结论，我们用293T稳转PD-1-GFP和SHP2-mCherry的细胞 验证。Confocal结果显示，在磷酸酶的抑制剂PVD的处理下，GFP和mCherry有很好的共定位，显示磷酸化的PD-1招募了SHP2(图7B)。
(3)在上述(2)这个系统中，我们加入MTC后，mCherry从膜上解离下来，呈现细胞质分布(图6B)。
(4)Co-IP结果显示MTC阻止PD-1和SHP2的结合，出人意料地阻断了PD-1的下游信号传导，从而提高了免疫细胞的功能，进而增强了与过继性细胞疗法或其他肿瘤免疫疗法联合应用时的肿瘤细胞杀伤效果(图7C)。
以上所述实施例的各技术特征可以进行任意的组合，为使描述简洁，未对上述实施例中的各个技术特征所有可能的组合都进行描述，然而，只要这些技术特征的组合不存在矛盾，都应当认为是本说明书记载的范围。
以上所述实施例仅表达了本发明的几种实施方式，其描述较为具体和详细，但并不能因此而理解为对发明专利范围的限制。应当指出的是，对于本领域的普通技术人员来说，在不脱离本发明构思的前提下，还可以做出若干变形和改进，这些都属于本发明的保护范围。因此，本发明专利的保护范围应以所附权利要求为准。

Claims (32)

  1. 一种药用组合物，包括：
    (a)适用于过继性细胞疗法的免疫细胞；以及
    (b)吩噻嗪类化合物，其选自式(I)所示的化合物、其水合物、溶剂化物或还原型形式：
    Figure PCTCN2020110998-appb-100001
    其中，Z选自：S +、O +、C或N；
    Y选自：N或N +；并且当Z选自：S +或O +时，Y为N；当Z选自C或N时，Y为N +；
    X -为能够与Z +或N +成盐的一个或多个阴离子，从而实现电中性；
    R 1、R 2、R 3、R 4、R 5、R 6、R 7、R 8、R 9和R 10分别独立地选自：氢、取代或未取代的烷基、取代或未取代的环烷基、取代或未取代的杂环烷基、取代或未取代的芳基、取代或未取代的杂芳基、取代或未取代的烷氧基、取代或未取代的芳烷氧基、硫烷氧基、胺基、硝基、氨基、卤素。
  2. 根据权利要求1所述的药用组合物，其中所述X -选自Cl -、Br -、I -、甲磺酸根阴离子、乙磺酸酸根阴离子、对甲苯磺酸根阴离子、苯磺酸根阴离子、乙二磺酸酸根阴离子、丙二磺酸酸根阴离子和萘二磺酸根阴离子。
  3. 根据权利要求1所述的药用组合物，其中R 1、R 2、R 3、R 4、R 5、R 6、R 7、R 8、R 9和R 10分别独立地选自：氢、取代或未取代的C1-C6烷基、取代或未取代的C3-C8环烷基、取代或未取代的3-8元杂环烷基、取代或未取代的C5-C10芳基、取代或未取代的5-10元杂芳基、取代或未取代的C1-C6烷氧基、取代或未取代的芳基烷氧基、硫烷氧基、胺基、硝基、氨基、卤素。
  4. 根据权利要求1所述的药用组合物，其中所述吩噻嗪类化合物选自式II的3,7-双(二甲氨基)吩噻嗪-5-鎓盐(MTX)、其水合物、溶剂化物或还原型形式：
    Figure PCTCN2020110998-appb-100002
    其中，X -为一个或多个阴离子，从而实现电中性。
  5. 根据权利要求1所述的药用组合物，其中所述吩噻嗪类化合物选自式III的N3,N3,N7,N7-四甲基-10H-吩噻嗪-3,7-二铵盐(LMTX)、其水合物或溶剂化物：
    Figure PCTCN2020110998-appb-100003
    其中，X -为一个或多个阴离子，从而实现电中性。
  6. 根据权利要求1所述的药用组合物，其中所述吩噻嗪类化合物为3,7-双(二甲氨基)吩噻嗪-5-鎓氯化物。
  7. 根据权利要求5所述的药用组合物，其中所述吩噻嗪类化合物为N3,N3,N7,N7-四甲基-10H-吩噻嗪-3,7-二铵双(甲磺酸盐)。
  8. 根据权利要求1-7中任一项所述的药用组合物，其中所述免疫细胞选自肿瘤浸润淋巴细胞(TIL)、嵌合抗原受体T细胞(CAR-T细胞)、嵌合抗原受体NK细胞(CAR-NK细胞)和T细胞受体(TCR)嵌合型T细胞。
  9. 根据权利要求8所述的药用组合物，其中所述免疫细胞为T细胞受体(TCR)嵌合型T细胞。
  10. 根据权利要求9所述的药用组合物，其中所述TCR能够与SIINFEKL肽结合。
  11. 一种药盒，包括：
    a)适用于过继性细胞疗法的免疫细胞，其被配制在第一制剂中；
    b)吩噻嗪类化合物，其被配制在第二制剂中，其选自式(I)所示的化合物、其水合物、 溶剂化物或还原型形式：
    Figure PCTCN2020110998-appb-100004
    其中，Z选自：S +、O +、C或N；
    Y选自：N或N +；并且当Z选自：S +或O +时，Y为N；当Z选自C或N时，Y为N +；
    X -为能够与Z +或N +成盐的一个或多个阴离子，从而实现电中性；
    R 1、R 2、R 3、R 4、R 5、R 6、R 7、R 8、R 9和R 10分别独立地选自：氢、取代或未取代的烷基、取代或未取代的环烷基、取代或未取代的杂环烷基、取代或未取代的芳基、取代或未取代的杂芳基、取代或未取代的烷氧基、取代或未取代的芳烷氧基、硫烷氧基、胺基、硝基、氨基、卤素。
  12. 式(I)所示的化合物、其水合物、溶剂化物或还原型形式在制备治疗癌症的药物中的应用，
    Figure PCTCN2020110998-appb-100005
    其中，Z选自：S +、O +、C或N；
    Y选自：N或N +；并且当Z选自：S +或O +时，Y为N；当Z选自C或N时，Y为N +；
    X -为能够与Z +或N +成盐的一个或多个阴离子，从而实现电中性；
    R 1、R 2、R 3、R 4、R 5、R 6、R 7、R 8、R 9和R 10分别独立地选自：氢、取代或未取代的烷基、取代或未取代的环烷基、取代或未取代的杂环烷基、取代或未取代的芳基、取代或未取代的杂芳基、取代或未取代的烷氧基、取代或未取代的芳烷氧基、硫烷氧基、胺 基、硝基、氨基、卤素。
  13. 根据权利要求12所述的应用，其中所述吩噻嗪类化合物选自式II的3,7-双(二甲氨基)吩噻嗪-5-鎓盐(MTX)、其水合物、溶剂化物或还原型形式：
    Figure PCTCN2020110998-appb-100006
    其中，X -为一个或多个阴离子，从而实现电中性。
  14. 根据权利要求12所述的应用，其中所述吩噻嗪类化合物选自式III的N3,N3,N7,N7-四甲基-10H-吩噻嗪-3,7-二铵盐(LMTX)、其水合物或溶剂化物：
    Figure PCTCN2020110998-appb-100007
    其中，X -为一个或多个阴离子，从而实现电中性。
  15. 根据权利要求12所述的应用，其中所述吩噻嗪类化合物为3,7-双(二甲氨基)吩噻嗪-5-鎓氯化物。
  16. 根据权利要求14所述的应用，其中所述吩噻嗪类化合物为N3,N3,N7,N7-四甲基-10H-吩噻嗪-3,7-二铵双(甲磺酸盐)。
  17. 根据权利要求12所述的应用，其中所述癌症为黑色素瘤、胸腺肿瘤、肺癌、前列腺癌、乳腺癌、卵巢癌、直结肠癌、胰腺癌、肝癌、淋巴癌、食道癌、膀胱癌、尿道癌、非霍奇金淋巴癌、肾癌或脑瘤
  18. 吩噻嗪类化合物在制备PD-1信号传导抑制剂中的应用，其中所述吩噻嗪类化合物选自式(I)所示的化合物、其水合物、溶剂化物或还原型形式：
    Figure PCTCN2020110998-appb-100008
    其中，Z选自：S +、O +、C或N；
    Y选自：N或N +；并且当Z选自：S +或O +时，Y为N；当Z选自C或N时，Y为N +；
    X -为能够与Z +或N +成盐的一个或多个阴离子，从而实现电中性；
    R 1、R 2、R 3、R 4、R 5、R 6、R 7、R 8、R 9和R 10分别独立地选自：氢、取代或未取代的烷基、取代或未取代的环烷基、取代或未取代的杂环烷基、取代或未取代的芳基、取代或未取代的杂芳基、取代或未取代的烷氧基、取代或未取代的芳烷氧基、硫烷氧基、胺基、硝基、氨基、卤素。
  19. 吩噻嗪类化合物在制备提高免疫细胞功能的药物中的应用，其中所述吩噻嗪类化合物选自式(I)所示的化合物、其水合物、溶剂化物或还原型形式：
    Figure PCTCN2020110998-appb-100009
    其中，Z选自：S +、O +、C或N；
    Y选自：N或N +；并且当Z选自：S +或O +时，Y为N；当Z选自C或N时，Y为N +；
    X -为能够与Z +或N +成盐的一个或多个阴离子，从而实现电中性；
    R 1、R 2、R 3、R 4、R 5、R 6、R 7、R 8、R 9和R 10分别独立地选自：氢、取代或未取代的烷基、取代或未取代的环烷基、取代或未取代的杂环烷基、取代或未取代的芳基、取代或未取代的杂芳基、取代或未取代的烷氧基、取代或未取代的芳烷氧基、硫烷氧基、胺基、硝基、氨基、卤素。
  20. 吩噻嗪类化合物在制备阻断PD-1招募SHP2蛋白的药物中的应用，其中所述吩噻嗪类化合物选自式(I)所示的化合物、其水合物、溶剂化物或还原型形式：
    Figure PCTCN2020110998-appb-100010
    其中，Z选自：S +、O +、C或N；
    Y选自：N或N +；并且当Z选自：S +或O +时，Y为N；当Z选自C或N时，Y为N +；
    X -为能够与Z +或N +成盐的一个或多个阴离子，从而实现电中性；
    R 1、R 2、R 3、R 4、R 5、R 6、R 7、R 8、R 9和R 10分别独立地选自：氢、取代或未取代的烷基、取代或未取代的环烷基、取代或未取代的杂环烷基、取代或未取代的芳基、取代或未取代的杂芳基、取代或未取代的烷氧基、取代或未取代的芳烷氧基、硫烷氧基、胺基、硝基、氨基、卤素。
  21. 根据权利要求18至20的任一项所述的应用，其中X -选自Cl -、Br -、I -、甲磺酸根阴离子、乙磺酸酸根阴离子、对甲苯磺酸根阴离子、苯磺酸根阴离子、乙二磺酸酸根阴离子、丙二磺酸酸根阴离子和萘二磺酸根阴离子。
  22. 根据权利要求18至20的任一项所述的应用，其中R 1、R 2、R 3、R 4、R 5、R 6、R 7、R 8、R 9和R 10分别独立地选自：氢、取代或未取代的C1-C6烷基、取代或未取代的C3-C8环烷基、取代或未取代的3-8元杂环烷基、取代或未取代的C5-C10芳基、取代或未取代的5-10元杂芳基、取代或未取代的C1-C6烷氧基、取代或未取代的芳基烷氧基、硫烷氧基、胺基、硝基、氨基、卤素。
  23. 根据权利要求18至20的任一项所述的应用，其中所述吩噻嗪类化合物选自式II的3,7-双(二甲氨基)吩噻嗪-5-鎓盐(MTX)、其水合物、溶剂化物或还原型形式：
    Figure PCTCN2020110998-appb-100011
    其中，X -为一个或多个阴离子，从而实现电中性。
  24. 根据权利要求18至20的任一项所述的应用，其中所述吩噻嗪类化合物选自式III的N3,N3,N7,N7-四甲基-10H-吩噻嗪-3,7-二铵盐(LMTX)、其水合物或溶剂化物：
    Figure PCTCN2020110998-appb-100012
    其中，X -为一个或多个阴离子，从而实现电中性。
  25. 根据权利要求18至20的任一项所述的应用，其中所述吩噻嗪类化合物为3,7-双(二甲氨基)吩噻嗪-5-鎓氯化物或N3,N3,N7,N7-四甲基-10H-吩噻嗪-3,7-二铵双(甲磺酸盐)。
  26. 一种用于治疗癌症的药物组合物，包含：
    (a)吩噻嗪类化合物，其选自式(I)所示的化合物、其水合物、溶剂化物或还原型形式：
    Figure PCTCN2020110998-appb-100013
    其中，Z选自：S +、O +、C或N；
    Y选自：N或N +；并且当Z选自：S +或O +时，Y为N；当Z选自C或N时，Y为N +；
    X -为能够与Z +或N +成盐的一个或多个阴离子，从而实现电中性；
    R 1、R 2、R 3、R 4、R 5、R 6、R 7、R 8、R 9和R 10分别独立地选自：氢、取代或未取代的烷基、取代或未取代的环烷基、取代或未取代的杂环烷基、取代或未取代的芳基、取代或未取代的杂芳基、取代或未取代的烷氧基、取代或未取代的芳烷氧基、硫烷氧基、胺基、硝基、氨基、卤素；以及
    (b)用于癌症治疗的第二种治疗剂。
  27. 根据权利要求26所述的药物组合物，其中所述吩噻嗪类化合物选自式II的3,7-双(二甲氨基)吩噻嗪-5-鎓盐(MTX)、其水合物、溶剂化物或还原型形式：
    Figure PCTCN2020110998-appb-100014
    其中，X -为一个或多个阴离子，从而实现电中性。
  28. 根据权利要求26所述的药物组合物，其中所述吩噻嗪类化合物选自式III的N3,N3,N7,N7-四甲基-10H-吩噻嗪-3,7-二铵盐(LMTX)、其水合物或溶剂化物：
    Figure PCTCN2020110998-appb-100015
    其中，X -为一个或多个阴离子，从而实现电中性。
  29. 根据权利要求26所述的药物组合物，其中所述吩噻嗪类化合物为3,7-双(二甲氨基)吩噻嗪-5-鎓氯化物或N3,N3,N7,N7-四甲基-10H-吩噻嗪-3,7-二铵双(甲磺酸盐)。
  30. 根据权利要求26所述的药物组合物，其中所述第二化疗剂是肿瘤免疫治疗剂。
  31. 根据权利要求26所述的药物组合物，其中所述肿瘤免疫治疗剂是PD-1抗体、PD-L1抗体或其功能性片段。
  32. 一种用于治疗癌症的药盒，包含：
    (a)吩噻嗪类化合物，其被配制在第一制剂中且选自式(I)所示的化合物、其水合物、溶剂化物或还原型形式：
    Figure PCTCN2020110998-appb-100016
    其中，Z选自：S +、O +、C或N；
    Y选自：N或N +；并且当Z选自：S +或O +时，Y为N；当Z选自C或N时，Y为N +；
    X -为能够与Z +或N +成盐的一个或多个阴离子，从而实现电中性；
    R 1、R 2、R 3、R 4、R 5、R 6、R 7、R 8、R 9和R 10分别独立地选自：氢、取代或未取代的烷基、取代或未取代的环烷基、取代或未取代的杂环烷基、取代或未取代的芳基、取代或未取代的杂芳基、取代或未取代的烷氧基、取代或未取代的芳烷氧基、硫烷氧基、胺基、硝基、氨基、卤素；以及
    (b)用于癌症治疗的第二种治疗剂，其被配制在第二制剂中。
PCT/CN2020/110998 2019-08-26 2020-08-25 吩噻嗪类或其类似结构的化合物在制药中的新应用 WO2021037013A1 (zh)

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