WO2020114098A1 - 茶多酚在作为免疫检查点抑制剂及制备用于抗肿瘤的药物中的应用 - Google Patents

茶多酚在作为免疫检查点抑制剂及制备用于抗肿瘤的药物中的应用 Download PDF

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WO2020114098A1
WO2020114098A1 PCT/CN2019/111656 CN2019111656W WO2020114098A1 WO 2020114098 A1 WO2020114098 A1 WO 2020114098A1 CN 2019111656 W CN2019111656 W CN 2019111656W WO 2020114098 A1 WO2020114098 A1 WO 2020114098A1
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tumor
cancer
epigallocatechin
gallate
catechins
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李洋
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Shenzhen Institute of Advanced Technology of CAS
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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
    • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
    • A61K31/35—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom
    • A61K31/352—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom condensed with carbocyclic rings, e.g. methantheline 
    • A61K31/353—3,4-Dihydrobenzopyrans, e.g. chroman, catechin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/18—Magnoliophyta (angiosperms)
    • A61K36/185—Magnoliopsida (dicotyledons)
    • A61K36/82—Theaceae (Tea family), e.g. camellia
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents

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  • the present disclosure relates to the technical field of biomedicine, and in particular to the application of tea polyphenols as immune checkpoint inhibitors and preparation of anti-tumor drugs.
  • the immune checkpoint blockade represented by PD-1/PD-L1 antibody has a good effect on a variety of tumors.
  • CD8+ T cell activation up-regulates the inhibitory receptor PD-1 to control autoimmune diseases caused by excessive activation of immunity; while in the tumor microenvironment, tumor cells and various inflammatory cells express PD-1
  • the body PD-L1 or PD-L2 depletes T cells, thereby inhibiting the anti-tumor killing function of T cells, so that the tumor can achieve immune escape.
  • the overall objective total response rate is usually less than 30%; the other 25% of patients using PD-1/PD-L1 antibody It will produce an acquired resistance mechanism, which is resistant to tumor immunotherapy, affecting tumor treatment and cure.
  • ORR overall objective total response rate
  • the preparation of therapeutic drugs for immune checkpoints represented by PD-1 antibodies is complicated, expensive, and costly.
  • the purpose of the present disclosure includes, for example, providing the use of tea polyphenols as an immune checkpoint inhibitor to at least alleviate one of the technical problems existing in the prior art.
  • the purpose of the present disclosure also includes, for example, providing the use of tea polyphenols in the preparation of anti-tumor drugs to at least alleviate one of the technical problems existing in the prior art.
  • the present disclosure provides the use of tea polyphenols or pharmaceutically acceptable salts thereof as inhibitors of immune checkpoints including programmed cell death protein 1 (PD-1) and programmed death ligand 1 ( One or more of PD-L1) or Programmed Death Ligand 2 (PD-L2).
  • PD-1 programmed cell death protein 1
  • PD-L1 programmed death ligand 1
  • PD-L2 Programmed Death Ligand 2
  • the present disclosure provides a tea polyphenol or a pharmaceutically acceptable salt thereof for use in treating and/or preventing tumors.
  • the present disclosure provides a pharmaceutical composition for treating and/or preventing tumors.
  • the pharmaceutical composition includes tea polyphenol or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable auxiliary material.
  • the present disclosure provides a method for treating and/or preventing tumors, the method comprising administering to a subject in need thereof a therapeutically effective amount of the tea polyphenol of the present disclosure or a pharmaceutically acceptable salt thereof, or the present The disclosed pharmaceutical composition.
  • the present disclosure provides a method for suppressing the expression of immune checkpoints in a subject, including administering tea polyphenol or a pharmaceutically acceptable salt thereof, or a composition of the present disclosure to the subject.
  • the immune checkpoint includes one or more of PD-1, PD-L1, or PD-L2.
  • the present disclosure provides a method for inhibiting the first cell from expressing PD-1 and/or the second cell from expressing PD-L1 and/or PD-L2, including: making the first cell and/or second cell and tea polyphenol or Contact with pharmaceutically acceptable salts.
  • the contacting is performed in vitro. In one or more embodiments, the contacting occurs in vivo. In one or more embodiments, the first cell is an immune cell. In one or more embodiments, the second cell is a cancer cell or an inflammatory cell.
  • the tea polyphenols include one or more of catechins, flavonoids, flavonols, anthocyanins, phenolic acids, phenolic acids, or polymeric phenols , Preferably catechins;
  • the catechins include one or more of epigallocatechin gallate, epigallocatechin, epicatechin gallate or epicatechin; preferably epigallocatechin Vegetarian gallate (EGCG);
  • EGCG epigallocatechin Vegetarian gallate
  • the epigallocatechin gallate comprises nano-type epigallocatechin gallate.
  • the epigallocatechin gallate inhibits the expression of the immune checkpoint PD-1, PD-L1, or PD-L2 by inhibiting the STAT1 pathway.
  • the present disclosure also provides the use of the above-mentioned immune checkpoint inhibitors in the preparation of anti-tumor drugs, the immune checkpoint inhibitors are tea polyphenols, and the immune checkpoints include PD-1, PD- One or more of L1 or PD-L2, the tea polyphenols achieve anti-tumor by suppressing the expression of immune checkpoints.
  • the tumor includes melanoma, breast cancer, bladder cancer tumor, head and neck cancer tumor, lung cancer tumor, colon cancer tumor, rectal cancer tumor, stomach cancer tumor, prostate cancer tumor, liver cancer tumor, Pancreatic cancer tumor, ovarian cancer tumor or lymphoma.
  • the anti-tumor drug further includes pharmaceutically acceptable excipients.
  • the dosage form of the anti-tumor drug includes an oral preparation or an injection preparation
  • the oral preparations include tablets, capsules, granules, pills, syrups, oral solutions, oral suspensions or oral emulsions;
  • the injection preparation includes an injection solution or a powder injection.
  • the effective dose of the anti-tumor drug is 2-6 mg/kg/day of injection, preferably 2-5 mg/kg/day, more preferably 3-4 mg/ kg/day; or
  • the effective dose of the anti-tumor drug is 5-40 mg/kg/day orally, preferably 10-35 mg/kg/day, more preferably 15-25 mg/kg/day.
  • the tea polyphenols include one or more of catechins, flavonoids, flavonols, anthocyanins, phenolic acids, phenolic acids, or polymeric phenols , Preferably catechins;
  • the catechins include one or more of epigallocatechin gallate, epigallocatechin, epicatechin gallate or epicatechin; preferably epigallocatechin Vegetarian gallate;
  • the epigallocatechin gallate comprises nano-type epigallocatechin gallate.
  • tea polyphenols can be used as inhibitors of immune checkpoints PD-1, PD-L1 and PD-L2 based on a large number of experiments. Tea polyphenols can significantly inhibit immune cell and tumor cell immune checkpoint related signaling pathways Expression, reduce the expression of immune checkpoint receptors and ligands on the surface of immune cells and tumor cells, thereby not only improving the anti-tumor ability of immune cells, but also removing the immune checkpoint ligands that produce tumor immunosuppression from within the tumor Expression, to prevent the occurrence of tumor immune escape. Therefore, tea polyphenols can be used to prepare anti-tumor immune drugs for immune checkpoints, or anti-tumor immune adjuvant drugs for tumor treatment or prevention. In addition, the preparation of tea polyphenols is relatively simple and economical, so the present disclosure not only can more effectively relieve the immunosuppression in the tumor microenvironment, but also can achieve the treatment of tumors at a lower price.
  • 1A is a graph of qPCR detection results of PD-L1 mRNA after treatment with 120 ⁇ M human melanoma cells stimulated with IFN ⁇ by 10 ⁇ M EGCG for 24 hours provided by Example 1 of the present disclosure;
  • 1B is a graph of qPCR detection results of PD-L1 mRNA after treatment with 10 ⁇ M EGCG for IFN ⁇ -stimulated A375 human melanoma cells for 24 hours provided in Example 1 of this disclosure;
  • 1C is a graph showing the results of qPCR detection of PD-L1 mRNA after treatment with 10 ⁇ M EGCG for IFN ⁇ -stimulated human breast cancer cells with MCF7 for 24 hours provided in Example 1 of the present disclosure;
  • 1D is a graph showing qPCR detection results of PD-L1 mRNA after treatment with IFN ⁇ -stimulated MDA-MB-231 human breast cancer cells using 30 ⁇ M EGCG for 24 hours provided by Example 1 of the present disclosure;
  • 2A is a graph of qPCR detection results of PD-L2 mRNA after treatment with 120 ⁇ M human melanoma cells stimulated with IFN ⁇ by 10 ⁇ M EGCG for 24 hours provided by Example 1 of the present disclosure;
  • 2B is a graph of qPCR detection results of PD-L2 mRNA after treatment with IFN ⁇ -stimulated A375 human melanoma cells using 10 ⁇ M EGCG for 24 hours provided by Example 1 of the present disclosure;
  • 2C is a graph showing qPCR detection results of PD-L2 mRNA after treatment with 10 ⁇ M EGCG for IFN ⁇ -stimulated human breast cancer cells with MCF7 for 24 hours provided in Example 1 of the present disclosure;
  • 2D is a graph showing qPCR detection results of PD-L2 mRNA after treatment with IFN ⁇ -stimulated MDA-MB-231 human breast cancer cells using 30 ⁇ M EGCG for 24 hours provided by Example 1 of the present disclosure;
  • 3A is a flow cytometry detection result diagram of PD-L1 protein expression on the cell surface of a 1205Lu human melanoma cell stimulated with IFN ⁇ stimulated with 10 ⁇ M EGCG after 24 hours provided by Example 1 of the present disclosure;
  • 3B is a flow cytometry detection result diagram of PD-L1 protein expression on the cell surface of A375 human melanoma cells stimulated with IFN ⁇ stimulated by 10 ⁇ M EGCG after 24 hours provided by Example 1 of the present disclosure;
  • 3C is a flow cytometry detection result diagram of PD-L1 protein expression on the cell surface after 24 hours of treatment of IFN ⁇ -stimulated human breast cancer cells with 10 ⁇ M EGCG provided by Example 1 of this disclosure;
  • 3D is a flow cytometry detection result diagram of PD-L1 protein expression on the cell surface of MDA-MB-231 human breast cancer cells stimulated with IFN ⁇ stimulated with 30 ⁇ M EGCG after 24 hours provided by Example 1 of the present disclosure;
  • 4A is a flow cytometry detection result diagram of PD-L2 protein expression on the cell surface of a 1205Lu human melanoma cell stimulated with 10 ⁇ M EGCG and treated with IFN ⁇ at 10 ⁇ M for 24 hours;
  • 4B is a flow cytometry detection result diagram of PD-L2 protein expression on the cell surface of A375 human melanoma cells stimulated with IFN ⁇ stimulated with 10 ⁇ M EGCG after 24 hours provided by Example 1 of the present disclosure;
  • 4C is a flow cytometry detection result diagram of PD-L2 protein expression on the cell surface after 24 hours of treatment of IFN ⁇ -stimulated human breast cancer cells with 10 ⁇ M EGCG provided by Example 1 of the present disclosure;
  • 4D is a flow cytometry detection result diagram of PD-L2 protein expression on the cell surface of MDA-MB-231 human breast cancer cells stimulated with IFN ⁇ stimulated with 30 ⁇ M EGCG after 24 hours provided by Example 1 of the present disclosure;
  • 5A is a graph of qPCR detection results of STAT1 mRNA after treatment with 120 ⁇ M human melanoma cells stimulated with IFN ⁇ by 10 ⁇ M EGCG for 24 hours provided by Example 1 of the present disclosure;
  • 5B is a graph of qPCR detection results of IRF1 mRNA after treatment with 120 ⁇ M human melanoma cells stimulated with IFN ⁇ by 10 ⁇ M EGCG for 24 hours provided by Example 1 of the present disclosure;
  • 5C is a Western-blot detection result diagram of phosphorylated STAT1, STAT1, and IRF1 protein expression after treatment with 120 ⁇ M human melanoma cells stimulated with IFN ⁇ by 10 ⁇ M EGCG for 24 hours provided by Example 1 of the present disclosure;
  • 6A is a flow cytometry detection result diagram of PD-1 protein expression on the surface of CD3+ T cells after treating CD3/CD28 activated T lymphocytes with 30 ⁇ M EGCG for 24 hours provided by Example 1 of the present disclosure;
  • 6B is a flow cytometry detection result diagram of PD-1 protein expression on the surface of CD4+ T cells after treating CD3/CD28 activated T lymphocytes with 30 ⁇ M EGCG for 24 hours provided by Example 1 of the present disclosure;
  • 6C is a flow cytometry detection result diagram of PD-1 protein expression on the surface of CD8+ T cells after treating CD3/CD28 activated T lymphocytes with 30 ⁇ M EGCG for 24 hours provided by Example 1 of the present disclosure;
  • 7A is a bar graph showing the average fluorescence intensity detected by flow cytometry of CD3+CD28 surface-activated T lymphocytes treated with 30 ⁇ M EGCG for 24 hours after being treated with 30 ⁇ M EGCG. ;
  • 7B is a bar graph showing the average fluorescence intensity detected by flow cytometry of PD-1 protein expression on the surface of CD4+ T cells after treatment with CD3/CD28 activated T lymphocytes using 30 ⁇ M EGCG for 24 hours provided by Example 1 of the present disclosure ;
  • FIG. 7C is a bar graph showing the average fluorescence intensity detected by flow cytometry of the expression of PD-1 protein on the surface of CD8+T cells after the treatment of CD3/CD28 activated T lymphocytes with 30 ⁇ M EGCG for 24 hours after being treated with 30 ⁇ M EGCG ;
  • 8A is a flow cytometry detection result diagram of PD-L1 protein expression on the cell surface of B16F10 mouse melanoma cells stimulated with 10 ⁇ M EGCG and treated with IFN ⁇ after 24 hours provided in Example 1 of the present disclosure;
  • 8B is a flow cytometry detection result diagram of PD-L2 protein expression on the cell surface of B16F10 mouse melanoma cells stimulated with 10 ⁇ M EGCG and treated with IFN ⁇ after 24 hours provided in Example 1 of the present disclosure;
  • 8C is a graph showing qPCR detection results of PD-L1 mRNA after treatment with 10 ⁇ M EGCG for IFN ⁇ -stimulated B16F10 mouse melanoma cells for 24 hours provided in Example 1 of this disclosure;
  • 8D is a graph showing the results of qPCR detection of PD-L2 mRNA of B16F10 mouse melanoma cells stimulated with IFN ⁇ treated with 10 ⁇ M EGCG after 24 hours provided by Example 1 of the present disclosure;
  • FIG. 9A is a graph showing the detection results of LDH content in the culture medium of spleen CD8+ T cells of C57BL6 mice specifically activated by IFN ⁇ -stimulated B16F10 and B16F10 treated with 10 ⁇ M EGCG after being incubated for 4 hours, as shown in FIG. 9A.
  • 9B is the detection of the activation of tumor cell caspase-3 after co-incubation of B16F10.cOVA.RFP cells treated with 1 ⁇ M EGCG and specifically activated OT-1 mouse spleen CD8+ T cells for 24 hours provided in Example 1 of the present disclosure
  • the result graph, the result is the specific killing of T cells to B16F10.cOVA.RFP tumor cells;
  • Example 10 is a graph of B16F10 melanoma size changes of C57BL6 mice injected intraperitoneally with EGCG provided in Example 1 of the present disclosure
  • 11A is a graph showing the effect of EGCG provided on Example 1 of the present disclosure on the level of PD-L1 transcription in tumor tissue of C57BL6 mice;
  • 11B is a graph showing the effect of EGCG provided on Example 1 of the present disclosure on the transcription level of PD-L2 in tumor tissues of C57BL6 mice;
  • 11C is a graph showing the effect of EGCG provided on Example 1 of the present disclosure on the transcription level of STAT1 in tumor tissue of C57BL6 mice;
  • 11D is a graph showing the effect of EGCG provided on Example 1 of the present disclosure on the transcription level of IRF1 in tumor tissues of C57BL6 mice;
  • 11E is a graph showing the effect of EGCG provided on Example 1 of the present disclosure on the expression of phosphorylated STAT1, STAT1, IRF1 and PD-L1 protein in C57BL6 mouse tumor tissue;
  • FIG. 12 is a graph showing the change in the proportion of T cell population composition of spleen and near-tumor lymph nodes of negative tumor C57BL6 mice provided with Example 1 of the present disclosure after injection of EGCG.
  • Example 13 is a graph showing the change in tumor size in each treatment group after subcutaneous injection of B16F10 tumor in C57BL6 mice provided in Example 1 of the present disclosure.
  • lower limit and upper limit disclosed in the “range” of the present disclosure may be one or more lower limits and one or more upper limits, respectively.
  • preferably and “preferred” refer to embodiments of the disclosure that may provide certain benefits under certain circumstances. However, other embodiments may also be preferred under the same or other circumstances. Furthermore, the recitation of one or more preferred embodiments does not mean that other embodiments are useless, and is not intended to exclude other embodiments from the scope of the present disclosure.
  • the use of tea polyphenols as an immune checkpoint inhibitor is provided, the immune checkpoint including one or more of PD-1, PD-L1, or PD-L2.
  • Tea polyphenols refer to a mixture of polyphenols and their derivatives with a complex composition, large molecular weights and structural differences in tea.
  • the main chemical components are catechins (flavanols), flavonoids and flavonoids Compounds of compounds such as alcohols, anthocyanins, phenolic acids, phenolic acids, and polymerized phenols.
  • Immune checkpoints are a large number of regulatory pathways distributed in the immune system, which play a key role in maintaining the body's own tolerance, the time of immune response and the strength of immune stress. Immune checkpoints begin to participate in the reaction after immune activation, function as a natural suppression feedback loop to reduce inflammation, protect and reduce the immune damage that the immune response may cause to surrounding tissues, and thus avoid incidental involvement of normal tissues.
  • tumors can use the immune checkpoint pathway as the main mechanism of immune evasion, especially the escape of immune killers that can recognize tumor-specific antigen T cells.
  • tea polyphenols can be used as inhibitors of immune checkpoints based on a large number of experiments. Tea polyphenols can significantly inhibit the expression of immune cell and tumor cell immune checkpoint-related signaling pathways and reduce the surface of immune cells and tumor cell cells. The number of immune checkpoint receptors and ligands can not only improve the anti-tumor immune killing of immune cells, but also release the expression of immune checkpoint ligands that produce tumor immunosuppression from the inside of the tumor, preventing tumor immune escape. Therefore, tea polyphenols can be used to prepare anti-tumor immune drugs for immune checkpoints, or anti-tumor immune adjuvant drugs for anti-tumor immunotherapy. In addition, the preparation of tea polyphenols is relatively simple and economical, so the present disclosure not only can more effectively relieve the immunosuppression in the tumor microenvironment, but also can achieve the treatment of tumors at a lower price.
  • the inhibition of immune checkpoints by typical tea polyphenols in the present disclosure can suppress the expression of immune checkpoints by blocking the expression pathway of the immune checkpoints.
  • the tea polyphenols include one or more of catechins, flavonoids, flavonols, anthocyanins, phenolic acids, phenolic acids or polymeric phenols, It is preferably catechin.
  • Catechin has the highest content in tea polyphenols and is easier to extract. It is preferred that catechin as an immune checkpoint inhibitor has stronger efficiency and role in suppressing the expression of immune checkpoints, so as to achieve the purpose of preventing tumor immune escape.
  • the catechins include EGCG (epigallocatechin gallate), EGC (epigallocatechin gallate), ECG (epicatechin gallate) and EC (epicatechin).
  • EGCG epigallocatechin gallate
  • EGC epigallocatechin gallate
  • ECG epicatechin gallate
  • EC epicatechin
  • One or more; EGCG is preferred because the bioavailability of EGCG is relatively high.
  • EGCG can inhibit the expression of PD-1 on the surface of T cells, thereby fully deactivating the immune inactivation of CD8+ T cells, and then activate the anti-tumor immune response to achieve tumor treatment.
  • the EGCG includes nano-like EGCG.
  • Nano EGCG refers to all nano drugs that use nano technology to wrap or package EGCG.
  • the preparation of EGCG into nano-EGCG using nano-drug technology can effectively improve the bioavailability of EGCG and increase the tumor targeting of EGCG, so that EGCG can be effectively enriched and released at the tumor site, thereby increasing the tumor targeting of EGCG And bioavailability.
  • the EGCG suppresses the expression of immune checkpoints by inhibiting the STAT1 pathway.
  • the STAT1 pathway is the STAT1-IRF1 pathway
  • immune checkpoints include PD-1, PD-L1, and PD-L2.
  • the immune checkpoint inhibitor in the preparation of an anti-tumor drug
  • the immune checkpoint inhibitor is tea polyphenol
  • the immune checkpoint includes PD-1 , PD-L1 or PD-L2
  • the tea polyphenols achieve anti-tumor by suppressing the expression of immune checkpoints.
  • the tumor includes melanoma, breast cancer, bladder cancer tumor, head and neck cancer tumor, lung cancer tumor, colon cancer tumor, rectal cancer tumor, gastric cancer tumor, prostate cancer tumor, liver cancer tumor, pancreatic cancer Tumor, ovarian cancer tumor or lymphoma.
  • the anti-tumor drugs further include pharmaceutically acceptable excipients.
  • Pharmaceutically acceptable excipients refer to the excipients and additives used in the production of medicines and formulating prescriptions, which refer to substances that have been reasonably evaluated in terms of safety in addition to the active ingredients and are included in pharmaceutical preparations .
  • the same pharmaceutical adjuvant can be used for pharmaceutical preparations with different administration routes, and have different functions and uses.
  • the pharmaceutically acceptable excipients added to the medicine provided by the present disclosure can play a role of shaping, acting as a carrier or improving stability, and in addition, have important functions such as solubilization, solubilization or sustained and controlled release.
  • Typical but non-limiting pharmaceutically acceptable excipients include: solvents, propellants, solubilizers, co-solvents, emulsifiers, colorants, binders, disintegrants, fillers, lubricants, wetting agents, osmotic pressure adjustment Agents, stabilizers, glidants, flavoring agents, preservatives, suspending agents, coating materials, fragrances, anti-sticking agents, antioxidants, chelating agents, penetration enhancers, pH adjusters, buffers, additives One of plasticizers, surfactants, foaming agents, defoamers, thickeners, clathrates, humectants, absorbents, diluents, flocculants and deflocculants, filter aids or release retardants Or more.
  • the dosage form of the drug includes an oral preparation or an injection preparation.
  • the above-mentioned drugs can be made into any orally acceptable formulation, such as, but not limited to, tablets, capsules, granules, pills, syrups, oral solutions, oral suspensions, or oral emulsions .
  • the carriers used for tablets generally include lactose and corn starch, and lubricants such as magnesium stearate can also be added.
  • Diluents used in capsules generally include lactose and dried corn starch.
  • Oral suspensions are usually used by mixing the active ingredient with suitable emulsifiers and suspending agents.
  • sweeteners can also be added to the above oral formulation forms.
  • the above-mentioned drugs can be made into any formulation acceptable for injection, such as, but not limited to, injections or powder injections.
  • usable carriers and solvents include water, Ringer's solution and isotonic sodium chloride solution.
  • sterilized non-volatile oils can also be used as solvents or suspension media, such as monoglycerides or diglycerides.
  • the effective dose of the anti-tumor drug is intravenous injection 2-6mg/kg/day, for example, it can be, but not limited to, 2mg/kg/day, 3mg/kg/day , 4mg/kg/day, 5mg/kg/day or 6mg/kg/day. or,
  • the effective dose of the anti-tumor drug is 5-40 mg/kg/day orally, for example, but not limited to 5 mg/kg/day, 6.7 mg/kg/day, 10 mg/kg/day, 15 mg /kg/day, 20mg/kg/day, 25mg/kg/day, 30mg/kg/day, 32.4mg/kg/day, 35mg/kg/day or 40mg/kg/day.
  • the administration frequency may be, for example, but not limited to administration twice a day, once a day, once every two days, once a week, or once a month.
  • the drug provided by the present disclosure may be administered in the form of a sustained-release preparation, in which case, less frequent administration is required.
  • the dosage and frequency of administration vary according to the half-life of the preparation in the user's body. In therapeutic applications, it is sometimes necessary to administer relatively high doses at relatively short intervals until the progression of the disease is delayed or stopped, and preferably until the individual manifests After partial or complete improvement of the symptoms of the disease, a preventive regimen can be given to the patient.
  • the preferred injection dosage is 2-5 mg/kg/day, more preferably 3-4 mg/kg/day;
  • the preferred oral administration dose is 10-35 mg/kg/day, more preferably 15-25 mg/kg/day.
  • Typical but non-limiting therapeutic administration is injection, and prophylactic administration is oral.
  • the drug prepared as an immune checkpoint inhibitor can more easily suppress the expression of the immune checkpoint, thereby better achieving the purpose of preventing the immune escape response of the tumor.
  • the cells, drugs, and reagents used in the embodiments of the present disclosure are all from regular and easy-to-purchase channels:
  • cytokines were purchased from Thermo Fisher Scientific
  • EGCG was purchased from Enzo Life Science
  • flow cytometer was Beckman Coulter
  • flow cytometry was purchased from eBioscience
  • real-time PCR instrument was Roche
  • Reverse transcription kit and qPCR reagent are Qiagen company
  • WB antibody is Cell Signaling Technology company
  • WB instrument is Bio-Rad Laboratories company.
  • Example 1 EGCG inhibits immune checkpoint receptor expression
  • the expression of PD-L1 or PD-L2 decreased after 10 hours of treatment with stimulated cells of 10 ⁇ M-30 ⁇ M in EGCG, and the expression of PD-L2 in MCF7 and 231 cells stimulated by IFN ⁇ did not change significantly, so the surface protein inhibition after EGCG treatment Not obvious.
  • 1205Lu tumor cells were seeded in a six-well plate and cultured for 12 hours, after which the treatment group cells were added with a concentration of 10ng/ml IFN ⁇ medium, and 2 hours later, the cells of the EGCG treatment group were added 10 ⁇ M EGCG, and then co-cultured for 24 hours, collecting the cells and performing RNA Extraction and reverse transcription, and use real-time PCR to detect the mRNA expression of STAT1 and IRF1 (GAPDH is the housekeeping gene); perform protein extraction, use Western-blot technology to detect phosphorylation and background STAT1 protein expression and IRF-1 protein Express the situation.
  • GPDH is the housekeeping gene
  • EGCG treatment can significantly inhibit the mRNA and phosphorylation of STAT1 signaling pathway and expression of background protein in IFN ⁇ -stimulated 1205Lu human melanoma cells, and can also significantly inhibit IFN ⁇ -stimulated 1205Lu human melanin.
  • GE Ficoll-Paque
  • CD3+T cells were separated and purified by the method of CD3+Pan Cells Negative selection (MACS Miltenyi Biotec).
  • T cells were activated with CD3/CD28 magnetic beads (Gibco) for 24 hours, the activated magnetic beads were removed, and the EGCG group was treated with 30 ⁇ M EGCG.
  • collect T cells Control group without CD3/CD28 activation, activated group with CD3/CD28 activation, activated+EGCG with EGCG treatment after activation, flow cytometry antibodies CD3+, CD4+, CD8+, PD- 1 Incubate with T cells for 30 minutes. After washing the cells, the flow cytometry was used to detect the expression of PD-1 protein on the surface of each lymphocyte component.
  • continuous administration of EGCG can significantly reduce tumor volume, significantly suppress PD-L1 expression, and significantly increase T cell activation.
  • Mouse melanoma cells B16F10 were inoculated in a six-well plate and cultured for 12 hours. After that, the treatment group cells were added with a medium containing 5ng/ml IFN ⁇ . After 2 hours, the cells in the EGCG treatment group were added with 10 or 30 ⁇ M EGCG, and then cultured for 24 hours. After collecting the cells and incubating with PD-L1 and PD-L2 antibodies for 30 minutes, wash the cells and use flow cytometry to detect the protein expression on the surface of tumor cells.
  • the results are shown in Figures 8A, 8B, 8C, and 8D.
  • the B16F10 mouse melanoma cell line was stimulated with IFN ⁇ and given EGCG treatment, after which the cells were collected for PD-L1 and PD-L2 mRNA detection (GAPDH is a housekeeping gene) and used Flow cytometry detected PD-L1 and PD-L2 proteins on the cell surface, and found that EGCG can significantly inhibit the mRNA and protein expression of PD-L1, but PD-L2 is not expressed in the mouse B16F10 cell line;
  • CTL assay 1 C57BL6 for subcutaneous B16F10 (10 5 cells) in mice, 2 weeks after the extraction of mouse spleen cells, and spleen cells were treated with B16F10 inactivated for 24 hours before using MACS CD8 + T cells; Mouse Melanoma cells B16F10 were seeded in 96-well plates and cultured for 12 hours, then added with 5ng/ml IFN ⁇ medium, 2 hours later added 10 ⁇ M EGCG, and then co-cultured for 24 hours, added CD8+ T cells sorted by the previous magnetic beads sorting , Mix and incubate for 4 hours, and ensure that the ratio of T cells (E) to tumor cells (T) is 40:1.
  • CTL assay 2 Isolate the spleen cells of 6-week-old OT-1 mice (which specifically recognize ovalbumin) and treat them with 2 ⁇ g of SINFEKL polypeptide for 72 hours, wash the spleen cells with PBS, and treat with 20 U/mL IL-2 for 72 After 8 hours, CD8+ T cells were sorted negatively with magnetic beads.
  • B16F10.cOVA.RFP ovalpha-1 (ovalbumin and red fluorescent protein transfection) was seeded in 96-well plates, treated with 1 ⁇ M EGCG for 24 hours, then washed the cells with culture medium, and added OT-1 treated in the previous step CD8+T Cells, the ratio of T cells (E) to tumor cells (T) is 2:1, and then IncuCyte caspase-3 apoptosis reagent (green fluorescence) is added. Apoptosis of B16F10.cOVA.RFP cells was detected in real time within 24 hours with the IncuCyte ZOOM microscope of Essen Bioscience. The T cell only group and the B16F10 cell group only were the standardized numerical control group.
  • Apoptosis results were standardized based on the control values. The results are shown in FIG. 9B.
  • the apoptosis of B16F10.cOVA.RFP cells in the EGCG-treated group was significantly higher than that in the control group without EGCG treatment, indicating that the ability of T cells to kill tumors after EGCG treatment was significantly enhanced.
  • C57BL6 mice were injected subcutaneously with B16F10, and the tumor size was detected.
  • the tumor size was about 50 cubic millimeters
  • EGCG was intraperitoneally injected at a concentration of 1 mg/day/day.
  • the tumor in the control group reached 2000 cubic millimeters, the experiment was terminated, and the tumor tissue was taken for backup , And use flow cytometry to detect the changes of CD3+ T cell components in spleen and near-tumor lymph nodes of mice.
  • the tumor growth is shown in Figure 10.
  • FIGS 11A, 11B, 11C, 11D and 11E The tumor tissues taken in Figure 10 were tested for STAT1, IRF1, PD-L1 and PD-L2 transcription levels and protein levels (beta-Actin is a housekeeping gene ), found that STAT1, IRF1, PD-L1 gene and protein levels were significantly reduced after EGCG treatment, phosphorylated STAT1 protein levels were also significantly reduced after EGCG treatment, PD-L2 no mRNA and protein level expression.
  • a C57BL6 subcutaneous melanoma tumor-bearing mouse model was constructed. When the tumor size reached about 50 mm 3 , it was randomly divided into three groups, and every three days were injected with saline (Control), EGCG aqueous solution (EGCG) and EGCG-loaded nanocarriers through the tail vein. Aqueous dispersion (nanoEGCG), the EGCG dose in the EGCG group and the nanoEGCG group was 0.5 mg/cap.
  • the results are shown in Fig. 13.
  • the statistical analysis is based on 5 mice per group and is analyzed using one-way ANOVA.
  • the results prove that nano-EGCG (nanoEGCG) has better tumor growth inhibition effect than EGCG. *Represents that the EGCG group and the nanoEGCG group are significantly different from the Control group (P ⁇ 0.05), and #represents that the EGCG group and the nanoEGCG group are significantly different (P ⁇ 0.05).
  • the tea polyphenols of the present disclosure can be used as inhibitors of immune checkpoints PD-1, PD-L1 and PD-L2, significantly inhibit the expression of immune cell and tumor cell immune checkpoint related signaling pathways, and reduce the surface of immune cells and tumor cell cells.
  • the expression of immune checkpoint receptors and ligands can not only improve the anti-tumor ability of immune cells, but also release the expression of immune checkpoint ligands that produce tumor immunosuppression from the inside of the tumor, preventing tumor immune escape. Therefore, tea polyphenols can be used to prepare anti-tumor immune drugs for immune checkpoints, or anti-tumor immune adjuvant drugs for tumor treatment or prevention.
  • the preparation of tea polyphenols is relatively simple and economical, so the present disclosure not only can more effectively relieve the immunosuppression in the tumor microenvironment, but also can achieve the treatment of tumors at a lower price.

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Abstract

本公开提供了茶多酚在作为免疫检查点抑制剂及制备用于抗肿瘤的药物中的应用。

Description

茶多酚在作为免疫检查点抑制剂及制备用于抗肿瘤的药物中的应用
相关申请的交叉引用
本公开要求于2018年12月3日提交中国国家知识产权局的申请号为201811478060.9、名称为“茶多酚在作为免疫检查点抑制剂及制备用于抗肿瘤的药物中的应用”的中国专利申请的优先权，其全部内容通过引用结合在本公开中。
技术领域
本公开涉及生物医药技术领域，尤其是涉及一种茶多酚在作为免疫检查点抑制剂及制备用于抗肿瘤的药物中的应用。
背景技术
恶性肿瘤已成为人类面临的最大威胁之一。如何实现对肿瘤的有效治疗成为世界性的难题。经过多年的不断探索，肿瘤免疫治疗终于取得了重要的研究成果，以PD-1/PD-L1抗体为代表的免疫检查点阻断治疗对多种肿瘤具有良好的疗效。CD8+T细胞活化会上调抑制性受体PD-1以控制免疫的过度活化而引起的自身免疫性疾病；而在肿瘤微环境中，肿瘤细胞及多种炎性细胞通过高表达PD-1配体PD-L1或者PD-L2，使T细胞耗竭，进而抑制T细胞的抗肿瘤杀伤功能，使得肿瘤实现免疫逃逸。
尽管PD-1/PD-L1抗体对多种肿瘤均有一定的效应，但整体的客观总反映率(ORR)通常低于30％；另外25％的使用PD-1/PD-L1抗体的病人会产生获得性抵抗机制，对肿瘤免疫治疗产生抗性，影响肿瘤治疗及治愈。目前对于获得性抵抗机制的产生还没有很好的解释，对于具有获得性抵抗机制的病人的肿瘤发展也无有效药物。并且，以PD-1抗体为代表的免疫检查点治疗药物制备复杂、价格昂贵，成本较高。
有鉴于此，特提出本公开。
发明内容
本公开的目的包括例如提供茶多酚在作为免疫检查点抑制剂中的应用，以至少缓解现有技术中存在的技术问题之一。
本公开的目的还包括例如提供茶多酚在制备用于抗肿瘤的药物中的应用，以至少缓解现有技术中存在的技术问题之一。
本公开提供了茶多酚或其药学可接受的盐在作为免疫检查点抑制剂中的应用，所述免疫检查点包括程序性细胞死亡蛋白1(PD-1)、程序性死亡配体1(PD-L1)或程序性死亡配体2(PD-L2)中的一种或多种。
本公开提供了一种茶多酚或其药学可接受的盐，用于治疗和/或预防肿瘤。
本公开提供了一种药物组合物，用于治疗和/或预防肿瘤，药物组合物包括茶多酚或其药学可接受的盐和药学上可接受的辅料。
本公开提供了一种用于治疗和/或预防肿瘤的方法，所述方法包括向有需要的受试者给药治疗有效量的本公开的茶多酚或其药学可接受的盐、或本公开的药物组合物。
本公开提供了一种用于抑制免疫检查点在受试者中表达的方法，包括向受试者施用茶多酚或其药学可接受的盐、或本公开的的组合物。
在一种或多种实施方式中，免疫检查点包括PD-1、PD-L1或PD-L2中的一种或多种。
本公开提供了一种抑制第一细胞表达PD-1和/或第二细胞表达PD-L1和/或PD-L2的方法，包括：使第一细胞和/或第二细胞与茶多酚或其药学可接受的盐进行接触。
在一种或多种实施方式中，接触在体外进行。在一种或多种实施方式中，接触在体内进行。在一种或多种实施方式中，第一细胞为免疫细胞。在一种或多种实施方式中，第二细胞为癌细胞或炎性细胞。
在一种或多种实施方式中，所述茶多酚包括儿茶素类、黄酮、黄酮醇类、花青素类、酚酸、缩酚酸类或聚合酚类中的一种或多种，优选为儿茶素类；
优选地，所述儿茶素包括表没食子儿茶素没食子酸酯、表没食子儿茶素、表儿茶素没食子酸酯或表儿茶素中的一种或多种；优选为表没食子儿茶素没食子酸酯(EGCG)；
优选地，所述表没食子儿茶素没食子酸酯包括纳米类表没食子儿茶素没食子酸酯。
在一种或多种实施方式中，所述表没食子儿茶素没食子酸酯通过抑制STAT1通路实现对免疫检查点PD-1、PD-L1或PD-L2的表达抑制。
另外，本公开还提供了上述的免疫检查点抑制剂在制备用于抗肿瘤的药物中的应用，所述免疫检查点抑制剂为茶多酚，所述免疫检查点包括PD-1、PD-L1或PD-L2中的一种或多种，所述茶多酚通过抑制免疫检查点的表达实现抗肿瘤。
在一种或多种实施方式中，所述肿瘤包括黑色素瘤、乳腺癌肿瘤、膀胱癌肿瘤、头颈癌肿瘤、肺癌肿瘤、结肠癌肿瘤、直肠癌肿瘤、胃癌肿瘤、前列腺癌肿瘤、肝癌肿瘤、胰腺癌肿瘤、卵巢癌肿瘤或淋巴瘤。
在一种或多种实施方式中，所述用于抗肿瘤的药物还包括药学上可接受的辅料。
在一种或多种实施方式中，所述用于抗肿瘤的药物的剂型包括口服制剂或注射制剂；
优选地，所述口服制剂包括片剂、胶囊剂、颗粒剂、丸剂、糖浆剂、口服溶液剂、口服混悬剂或口服乳剂；
优选地，所述注射制剂包括注射液或粉针剂。
在一种或多种实施方式中，所述用于抗肿瘤的药物的有效给药剂量为注射2-6mg/kg/天，优选为2-5mg/kg/天，更优选为3-4mg/kg/天；或
所述用于抗肿瘤的药物的有效给药剂量为口服5-40mg/kg/天，优选为10-35mg/kg/天，更优选为15-25mg/kg/天。
在一种或多种实施方式中，所述茶多酚包括儿茶素类、黄酮、黄酮醇类、花青素类、酚酸、缩酚酸类或聚合酚类中的一种或多种，优选为儿茶素类；
优选地，所述儿茶素包括表没食子儿茶素没食子酸酯、表没食子儿茶素、表儿茶素没食子酸酯或表儿茶素中的一种或多种；优选为表没食子儿茶素没食子酸酯；
优选地，所述表没食子儿茶素没食子酸酯包括纳米类表没食子儿茶素没食子酸酯。
本公开的发明人根据大量实验验证茶多酚可作为免疫检查点PD-1、PD-L1和PD-L2的抑制剂，茶多酚可显著抑制免疫细胞及肿瘤细胞免疫检查点相关信号通路的表达，降低免疫细胞及肿瘤细胞细胞表面的免疫检查点受体和配体的表达，从而既可以提高免疫细胞的抗肿瘤能力，又可以从肿瘤内部解除产生肿瘤免疫抑制的免疫检查点配体的表达，阻止肿瘤免疫逃逸的发生。因此可以利用茶多酚制备针对免疫检查点的抗肿瘤免疫药物，或者抗肿瘤免疫辅助药物进行肿瘤治疗或预防。此外，茶多酚的制备相对简单、经济，因此本公开不仅可以更有效的解除肿瘤微环境中的免疫抑制，同时可以以更低廉的价格实现对肿瘤的治疗。
附图说明
为了更清楚地说明本公开具体实施方式或现有技术中的技术方案，下面将对具体实施方式或现有技术描述中所需要使用的附图作简单地介绍， 显而易见地，下面描述中的附图是本公开的一些实施方式，对于本领域普通技术人员来讲，在不付出创造性劳动的前提下，还可以根据这些附图获得其他的附图。
图1A为本公开实施例1提供的使用10μM EGCG处理IFNγ刺激的1205Lu人黑色素瘤细胞24小时后，PD-L1的mRNA的qPCR检测结果图；
图1B为本公开实施例1提供的使用10μM EGCG处理IFNγ刺激的A375人黑色素瘤细胞24小时后，PD-L1的mRNA的qPCR检测结果图；
图1C为本公开实施例1提供的使用10μM EGCG处理IFNγ刺激的MCF7人乳腺癌细胞24小时后，PD-L1的mRNA的qPCR检测结果图；
图1D为本公开实施例1提供的使用30μM EGCG处理IFNγ刺激的MDA-MB-231人乳腺癌细胞24小时后，PD-L1的mRNA的qPCR检测结果图；
图2A为本公开实施例1提供的使用10μM EGCG处理IFNγ刺激的1205Lu人黑色素瘤细胞24小时后，PD-L2的mRNA的qPCR检测结果图；
图2B为本公开实施例1提供的使用10μM EGCG处理IFNγ刺激的A375人黑色素瘤细胞24小时后，PD-L2的mRNA的qPCR检测结果图；
图2C为本公开实施例1提供的使用10μM EGCG处理IFNγ刺激的MCF7人乳腺癌细胞24小时后，PD-L2的mRNA的qPCR检测结果图；
图2D为本公开实施例1提供的使用30μM EGCG处理IFNγ刺激的MDA-MB-231人乳腺癌细胞24小时后，PD-L2的mRNA的qPCR检测结果图；
图3A为本公开实施例1提供的使用10μM EGCG处理IFNγ刺激的1205Lu人黑色素瘤细胞24小时后，细胞表面PD-L1蛋白表达的流式细胞仪检测结果图；
图3B为本公开实施例1提供的使用10μM EGCG处理IFNγ刺激的A375人黑色素瘤细胞24小时后，细胞表面PD-L1蛋白表达的流式细胞仪检测结果图；
图3C为本公开实施例1提供的使用10μM EGCG处理IFNγ刺激的MCF7人乳腺癌细胞24小时后，细胞表面PD-L1蛋白表达的流式细胞仪检测结果图；
图3D为本公开实施例1提供的使用30μM EGCG处理IFNγ刺激的MDA-MB-231人乳腺癌细胞24小时后，细胞表面PD-L1蛋白表达的流式细胞仪检测结果图；
图4A为本公开实施例1提供的使用10μM EGCG处理IFNγ刺激的1205Lu人黑色素瘤细胞24小时后，细胞表面PD-L2蛋白表达的流式细胞仪检测结果图；
图4B为本公开实施例1提供的使用10μM EGCG处理IFNγ刺激的A375人黑色素瘤细胞24小时后，细胞表面PD-L2蛋白表达的流式细胞仪检测结果图；
图4C为本公开实施例1提供的使用10μM EGCG处理IFNγ刺激的MCF7人乳腺癌细胞24小时后，细胞表面PD-L2蛋白表达的流式细胞仪检测结果图；
图4D为本公开实施例1提供的使用30μM EGCG处理IFNγ刺激的MDA-MB-231人乳腺癌细胞24小时后，细胞表面PD-L2蛋白表达的流式细胞仪检测结果图；
图5A为本公开实施例1提供的使用10μM EGCG处理IFNγ刺激的1205Lu人黑色素瘤细胞24小时后，STAT1的mRNA的qPCR检测结果图；
图5B为本公开实施例1提供的使用10μM EGCG处理IFNγ刺激的1205Lu人黑色素瘤细胞24小时后，IRF1的mRNA的qPCR检测结果图；
图5C为本公开实施例1提供的使用10μM EGCG处理IFNγ刺激的1205Lu人黑色素瘤细胞24小时后，磷酸化STAT1、STAT1、IRF1的蛋白表达的Western-blot检测结果图；
图6A为本公开实施例1提供的使用30μM EGCG处理CD3/CD28激活的T淋巴细胞24小时后，CD3+T细胞表面PD-1蛋白表达的流式细胞仪检测结果图；
图6B为本公开实施例1提供的使用30μM EGCG处理CD3/CD28激活的T淋巴细胞24小时后，CD4+T细胞表面PD-1蛋白表达的流式细胞仪检测结果图；
图6C为本公开实施例1提供的使用30μM EGCG处理CD3/CD28激活的T淋巴细胞24小时后，CD8+T细胞表面PD-1蛋白表达的流式细胞仪检测结果图；
图7A为本公开实施例1提供的使用30μM EGCG处理CD3/CD28激活的T淋巴细胞24小时后，CD3+T细胞表面PD-1蛋白表达的流式细胞仪检测的平均荧光强度柱状统计结果图；
图7B为本公开实施例1提供的使用30μM EGCG处理CD3/CD28激活的T淋巴细胞24小时后，CD4+T细胞表面PD-1蛋白表达的流式细胞仪检测的平均荧光强度柱状统计结果图；
图7C为本公开实施例1提供的使用30μM EGCG处理CD3/CD28激活后T淋巴细胞24小时后，CD8+T细胞表面PD-1蛋白表达的流式细胞仪检测的平均荧光强度柱状统计结果图；
图8A为本公开实施例1提供的使用10μM EGCG处理IFNγ刺激的B16F10小鼠黑色素瘤细胞24小时后，细胞表面PD-L1蛋白表达的流式细胞仪检测结果图；
图8B为本公开实施例1提供的使用10μM EGCG处理IFNγ刺激的B16F10小鼠黑色素瘤细胞24小时后，细胞表面PD-L2蛋白表达的流式细胞仪检测结果图；
图8C为本公开实施例1提供的使用10μM EGCG处理IFNγ刺激的B16F10小鼠黑色素瘤细胞24小时后，PD-L1的mRNA的qPCR检测结果图；
图8D为本公开实施例1提供的使用10μM EGCG处理IFNγ刺激的B16F10小鼠黑色素瘤细胞24小时后，PD-L2的mRNA的qPCR检测结果图；
图9A为本公开实施例1提供的使用10μM EGCG处理IFNγ刺激的B16F10与B16F10特异激活的C57BL6小鼠脾脏CD8+T细胞共孵育4小时后，培养基中LDH含量的检测结果图，其结果为T细胞对B16F10肿瘤细胞的特异杀伤；
图9B为本公开实施例1提供的使用1μM EGCG处理的B16F10.cOVA.RFP细胞与特异激活的OT-1小鼠脾脏CD8+T细胞共孵育24小时后，肿瘤细胞caspase-3的激活情况检测结果图，其结果为T细胞对B16F10.cOVA.RFP肿瘤细胞的特异杀伤；
图10为本公开实施例1提供的腹腔注射EGCG的C57BL6小鼠的B16F10黑色素瘤尺寸变化图；
图11A为本公开实施例1提供的EGCG对C57BL6小鼠肿瘤组织中PD-L1转录水平的影响的结果图；
图11B为本公开实施例1提供的EGCG对C57BL6小鼠肿瘤组织中PD-L2转录水平的影响的结果图；
图11C为本公开实施例1提供的EGCG对C57BL6小鼠肿瘤组织中STAT1转录水平的影响的结果图；
图11D为本公开实施例1提供的EGCG对C57BL6小鼠肿瘤组织中IRF1转录水平的影响的结果图；
图11E为本公开实施例1提供的EGCG对C57BL6小鼠肿瘤组织中磷酸化STAT1、STAT1、IRF1和PD-L1蛋白水平表达影响的结果图；
图12为本公开实施例1提供的负瘤C57BL6小鼠注射EGCG后，其脾脏和近肿瘤淋巴结T细胞种群组成比例变化情况图。
图13为本公开实施例1提供的C57BL6小鼠皮下注射B16F10肿瘤后，各处理组中肿瘤尺寸变化曲线图。
具体实施方式
下面将结合实施例对本公开的技术方案进行清楚、完整地描述，显然，所描述的实施例是本公开一部分实施例，而不是全部的实施例。基于本公开中的实施例，本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其他实施例，都属于本公开保护的范围。
需要说明的是：
本公开中，如果没有特别的说明，本文所提到的所有实施方式以及优选实施方法可以相互组合形成新的技术方案。
本公开中，如果没有特别的说明，本文所提到的所有技术特征以及优选特征可以相互组合形成新的技术方案。
本公开所公开的“范围”以下限和上限的形式，可以分别为一个或多个下限，和一个或多个上限。
本公开中，“优选地”和“优选的”是指在某些情况下可以提供某些益处的本公开的实施例。然而，在相同或其他情况下，其他实施例也可以是优选的。此外，一个或多个优选实施例的叙述并不意味着其他实施例是无用的，并且不旨在将其他实施例排除在本公开的范围之外。
除非另有说明，本文中所用的专业与科学术语与本领域熟练人员所熟悉的意义相同。此外，任何与所记载内容相似或均等的方法或材料也可应用于本公开中。
根据本公开的一个方面，提供了茶多酚在作为免疫检查点抑制剂中的应用，所述免疫检查点包括PD-1、PD-L1或PD-L2中的一种或多种。
茶多酚是指茶叶中一大类组成复杂、分子量及其结构差异很大的多酚类化合物及其衍生物的混合物，主要化学成分为儿茶素类(黄烷醇类)、黄酮及黄酮醇类、花青素类、酚酸及缩酚酸类、聚合酚类等化合物的复合体。
免疫检查点(immune checkpoints)是大量分布在免疫体系中的调节性通路，对维持机体自身的耐受、免疫应答的时间和免疫应激的强弱起到关键的调节作用。免疫检查点在免疫激活后开始参与反应，作为减少炎症的自然抑制反馈环路发挥作用，保障并减少免疫反应对周围组织可能造成的免疫损伤，从而避免正常组织的附带受累。而肿瘤却可以利用免疫检查点通路作为免疫逃避的主要机制，特别是逃避可以识别肿瘤特异性抗原T细胞的免疫杀伤。
本公开的发明人根据大量实验验证茶多酚可作为免疫检查点的抑制剂，茶多酚可以显著抑制免疫细胞及肿瘤细胞免疫检查点相关信号通路的表达，降低免疫细胞及肿瘤细胞细胞表面的免疫检查点受体和配体的数量，既可以提高免疫细胞的抗肿瘤免疫杀伤，又可以从肿瘤内部解除产生肿瘤免疫抑制的免疫检查点配体的表达，阻止肿瘤免疫逃逸的发生。因此可以利用茶多酚制备针对免疫检查点的抗肿瘤免疫药物，或者抗肿瘤免疫辅助药物进行抗肿瘤免疫治疗。此外，茶多酚的制备相对简单、经济，因此本公开不仅可以更有效的解除肿瘤微环境中的免疫抑制，同时可以以更低廉的价格实现对肿瘤的治疗。
本公开中典型的茶多酚对免疫检查点的抑制可以通过阻断免疫检查点的表达通路来抑制免疫检查点的表达。
在一些优选的实施方式中，所述茶多酚包括儿儿茶素类、黄酮、黄酮醇类、花青素类、酚酸、缩酚酸类或聚合酚类中的一种或多种，优选为儿茶素类。
儿茶素在茶多酚中的含量最高，更易于提取，优选儿茶素作为免疫检查点抑制剂在抑制免疫检查点表达上的效率及作用更强，从而达到阻止肿瘤免疫逃逸的目的。
优选地，所述儿茶素包括EGCG(表没食子儿茶素没食子酸酯)、EGC(表没食子儿茶素)、ECG(表儿茶素没食子酸酯)和EC(表儿茶素)中的一种或多种；优选为EGCG，是因为EGCG的生物可利用度相对较高。
EGCG可以抑制T细胞表面PD-1的表达，从而充分解除CD8+T细胞的免疫失活，进而激活抗肿瘤免疫反应，实现肿瘤的治疗。
优选地，所述EGCG包括纳米类EGCG。
纳米类EGCG是指利用纳米技术，对EGCG进行包裹或包装的所有纳米类药物。利用纳米药物的技术将EGCG制备为纳米EGCG，能够有效提高EGCG的生物可利用性，增加EGCG的肿瘤靶向，使EGCG可以特意的在肿瘤部位有效富集及释放，进而增加EGCG的肿瘤靶向和生物可利用性。
在一些优选的实施方式中，所述EGCG通过抑制STAT1通路实现免疫检查点的表达抑制。
其中，STAT1通路为STAT1-IRF1通路，免疫检查点包括PD-1，PD-L1和PD-L2。
根据本公开的另一个方面，提供了上述的免疫检查点抑制剂在制备用于抗肿瘤的药物中的应用，所述免疫检查点抑制剂为茶多酚，所述免疫检查点包括PD-1、PD-L1或PD-L2中的一种或多种，所述茶多酚通过抑制免疫检查点的表达实现抗肿瘤。
在一些优选的实施方式中，所述肿瘤包括黑色素瘤、乳腺癌肿瘤、膀胱癌肿瘤、头颈癌肿瘤、肺癌肿瘤、结肠癌肿瘤、直肠癌肿瘤、胃癌肿瘤、前列腺癌肿瘤、肝癌肿瘤、胰腺癌肿瘤、卵巢癌肿瘤或淋巴瘤。
在一些优选的实施方式中，所述用于抗肿瘤的药物还包括药学上可接受的辅料。
药学上可接受的辅料是指生产药品和调配处方时，使用的赋形剂和附加剂，是指除活性成分外，在安全性方面已进行了合理的评估，并且包含在药物制剂中的物质。同一药用辅料可用于不同给药途径的药物制剂，且有不同的作用和用途。在本公开提供的药物中添加的药学上可接受的辅料，能够起到赋型、充当载体或提高稳定性的作用，此外，还具有增溶、助溶或缓控释等重要功能。
典型但非限制性的药学上可接受的辅料包括：溶剂、抛射剂、增溶剂、助溶剂、乳化剂、着色剂、黏合剂、崩解剂、填充剂、润滑剂、湿润剂、渗透压调节剂、稳定剂、助流剂、矫味剂、防腐剂、助悬剂、包衣材料、芳香剂、抗黏着剂、抗氧剂、螯合剂、渗透促进剂、pH调节剂、缓冲剂、增塑剂、表面活性剂、发泡剂、消泡剂、增稠剂、包合剂、保湿剂、吸收剂、稀释剂、絮凝剂与反絮凝剂、助滤剂或释放阻滞剂中的一种或多种。
在一些优选的实施方式中，药物的剂型包括口服制剂或注射制剂。
当口服用药时，上述药物可制成任意口服可接受的制剂形式，例如可以为，但不限于片剂、胶囊剂、颗粒剂、丸剂、糖浆剂、口服溶液剂、口服混悬剂或口服乳剂。
其中，片剂使用的载体一般包括乳糖和玉米淀粉，另外也可加入润滑剂如硬脂酸镁。胶囊剂使用的稀释剂一般包括乳糖和干燥玉米淀粉。口服混悬剂则通常是将活性成分与适宜的乳化剂和悬浮剂混合使用。
任选地，以上口服制剂形式中还可加入一些甜味剂、芳香剂或着色剂。
当以注射的形式给药时，上述药物可制成任意注射可接受的制剂形式，例如可以为，但不限于注射液或粉针剂。
其中，可使用的载体和溶剂包括水、林格氏溶液和等渗氯化钠溶液。另外，灭菌的非挥发油也可用作溶剂或悬浮介质，如单甘油酯或二甘油酯。
在一些优选的实施方式中，所述用于抗肿瘤的药物的有效给药剂量为静脉注射2-6mg/kg/天，例如可以为，但不限于2mg/kg/天、3mg/kg/天、4mg/kg/天、5mg/kg/天或6mg/kg/天。或，
所述用于抗肿瘤的药物的有效给药剂量为口服5-40mg/kg/天，例如可以为，但不限于5mg/kg/天、6.7mg/kg/天、10mg/kg/天、15mg/kg/天、20mg/kg/天、25mg/kg/天、30mg/kg/天、32.4mg/kg/天、35mg/kg/天或40mg/kg/天。
在一些优选的实施方式中，给药频率例如可以为，但不限于每天两次、每天一次、每两天一次、每周一次或每月一次给药。或者，可以以缓释制剂的形式给予本公开提供的药物，在这种情况下，需要较少的给药频率。
给药剂量和频率根据制剂在用药者体内的半衰期而不同，在治疗性应用中，有时需要以相对短的间隔给予相对高的剂量，直至疾病的进展被延缓或停止，并优选地直至个体表现出疾病症状的部分或完全改善，在此之后，可以给予患者预防方案。
优选的注射给药剂量为2-5mg/kg/天，更优选为3-4mg/kg/天；
优选的口服给药剂量为10-35mg/kg/天，更优选为15-25mg/kg/天。
典型但非限制性的治疗性给药为注射给药，预防性给药为口服给药。
当给药剂量在上述优选范围内时，作为免疫检查点抑制剂制备得到的药物更易抑制免疫检查点的表达，从而更好地达到阻止肿瘤的免疫逃逸反应的目的。
为了有助于更清楚的理解本公开的内容，现结合具体的实施例详细介绍如下。
如无特别说明，本公开实施例中使用的细胞、药品及试剂均来源为正规而易购渠道：
细胞均购买于美国ATCC，细胞因子均购买于Thermo Fisher Scientific公司，EGCG购买于Enzo Life Science公司，流式细胞仪为Beckman Coulter公司，流式抗体购买于eBioscience公司，real-time PCR仪器为Roche公司，反转录试剂盒及qPCR试剂为Qiagen公司，WB抗体为Cell Signaling Technology公司，WB仪器为Bio-Rad Laboratories公司。
实施例1 EGCG抑制免疫检查点受体表达
1205Lu和A375人黑色素瘤细胞或MCF7和MDA-MB-231人乳腺癌细胞被接种于六孔板中培养12小时后，处理组细胞中加入含有10ng/ml IFNγ的培养基以刺激PD-L1和PD-L2的表达，2小时后，EGCG处理组细胞中加入10或30μM EGCG，之后共培养24小时后，收集细胞，进行RNA提取和反转录，并利用real-time PCR检测PD-L1和PD-L2的mRNA表达情况(GAPDH为管家基因)。
结果如图1A、1B、1C、1D、图2A、2B、2C和2D所示。IFNγ刺激1205Lu和A375人黑色素瘤细胞或MCF7和MDA-MB-231人乳腺癌细胞24小时后，收集细胞并qPCR检测PD-L1或PD-L2的基因表达情况，发现其PD-L1或者PD-L2的表达升高。10μM-30μM EGCG处理被刺激细胞24小时候后，PD-L1或者PD-L2的基因表达显著降低。**p<0.01，***p<0.001。
1205Lu和A375人黑色素瘤细胞或MCF7和MDA-MB-231人乳腺癌细胞被接种于六孔板中培养12小时后，处理组细胞中加入含有10ng/ml IFNγ的培养基以刺激PD-L1和PD-L2的表达，2小时后EGCG处理组细胞中加入10或30μM EGCG，之后共培养24小时后，收集细胞，与流式细胞仪PD-L1和PD-L2抗体共孵育30分钟后，清洗细胞，用流式细胞仪检测肿瘤细胞表面蛋白表达水平。
结果如图3A、3B、3C、3D和图4A、4B、4C和4D所示，IFNγ刺激1205Lu和A375人黑色素瘤细胞或MCF7和MDA-MB-231人乳腺癌细胞24小时后，流式细胞仪检测细胞表面PD-L1或PD-L2的表达，发现其PD-L1或者PD-L2的表达显著升高。10μM-30μM EGCG处理被刺激细胞24小时候后，PD-L1或者PD-L2的表达降低，其中，IFNγ刺激的MCF7和231细胞中的PD-L2的表达变化不明显，所以EGCG处理后表面蛋白抑制也不明显。
1205Lu肿瘤细胞被接种于六孔板中培养12小时，之后处理组细胞加入浓度为10ng/ml IFNγ培养基，2小时后EGCG处理组细胞加入10μM EGCG，之后共培养24小时，收集细胞，进行RNA提取及反转录，并利用real-time PCR检测STAT1和IRF1的mRNA表达情况(GAPDH为管家基因)；进行蛋白提取，利用Western-blot技术检测磷酸化和本底STAT1蛋白表达以及IRF-1蛋白表达情况。
结果如图5A、5B和5C所示，EGCG处理能够显著抑制IFNγ刺激的1205Lu人黑色素瘤细胞中STAT1信号通路的mRNA及磷酸化和本底蛋白的表达，也能够显著抑制IFNγ刺激的1205Lu人黑色素瘤细胞中IRF1信号通路的mRNA及蛋白的表达。
利用Ficoll-Paque(GE公司)密度离心的方法，分离人血液中的淋巴细胞，并用CD3+Pan T cells Negative selection(MACS Miltenyi Biotec)的方法分离纯化CD3+T细胞。之后处理组T细胞用CD3/CD28磁珠(Gibco公司)激活24小时后，去除激活磁珠，EGCG组加入30μM EGCG处理。24小时后，收集T细胞(无CD3/CD28激活组为Control,CD3/CD28激活组为activated，激活后加入EGCG处理的为activated+EGCG)，流式细胞仪抗体CD3+，CD4+，CD8+，PD-1与T细胞共孵育30分钟，清洗细胞后，上流式细胞仪检测各淋巴细胞组分细胞表面PD-1蛋白的表达变化。
结果如图6A、6B、6C、7A、7B和7C所示。人血液中分离的人CD3+T细胞，用CD3/CD28磁珠体外激活24小时后，CD3+，CD4+和CD8+T细胞细胞表面PD-1蛋白会有显著的增高；加入30μM EGCG处理T细胞24小时后，收集细胞进行流式细胞仪检测并分别鉴定CD3+，CD4+和CD8+T细胞细胞表面PD-1蛋白表达水平，发现EGCG可以显著抑制激活的CD3+，CD4+和CD8+T细胞表面的PD-1蛋白的表达水平。**p<0.01,***p<0.001，n＝3。
在小鼠黑色素瘤模型中，EGCG持续给药可以使肿瘤体积显著降低，PD-L1表达显著抑制，T细胞活化显著增强。
小鼠黑色素瘤细胞B16F10接种于六孔板中培养12小时，之后处理组细胞中加入含有5ng/ml IFNγ的培养基，2小时后，EGCG处理组细胞加入10或30μM EGCG，之后共培养24小时，收集细胞，与流式细胞仪PD-L1和PD-L2抗体共孵育30分钟后，清洗细胞，并用流式细胞仪检测肿瘤细胞表面蛋白表达水平。
结果如图8A、8B、8C和8D所示，B16F10小鼠黑色素瘤细胞系被IFNγ刺激后给予EGCG处理，之后收集细胞进行PD-L1和PD-L2的mRNA检测(GAPDH为管家基因)，并用流式细胞仪检测细胞表面PD-L1和PD-L2蛋白，发现EGCG可以显著抑制PD-L1的mRNA和蛋白表达，但是PD-L2在小鼠B16F10细胞系中不表达；
结果如图9A和图9B所示，细胞水平检测T细胞特异杀伤肿瘤的能力。
CTL assay 1：对C57BL6小鼠皮下注射B16F10(10 5细胞)，2周后提取小鼠脾脏细胞，并用灭活的B16F10处理脾脏细胞24小时，之后利用磁珠分选CD8+T细胞；小鼠黑色素瘤细胞B16F10接种于96孔板中培养12小时，之后加入含有5ng/ml IFNγ的培养基，2小时后加入10μM EGCG，之后共培养24小时，加入上步磁珠分选的CD8+T细胞，混合孵育4小时，并保证T细胞(E)与肿瘤细胞(T)比例为40:1。4小时后，收集培养基 上清，利用LDH试剂盒检测培养基上清中LDH含量，并以仅有肿瘤和仅有T细胞组中LDH含量为对照，计算处理组中LDH含量，其结果即为T细胞特异杀伤肿瘤细胞的情况(图9A)。
CTL assay 2：分离6周龄的OT-1小鼠(可特异识别卵清蛋白)的脾脏细胞，并用2μg的SINFEKL多肽处理72小时，PBS清洗脾细胞，并用20U/mL的IL-2处理72小时，之后用磁珠negative分选CD8+T细胞。
B16F10.cOVA.RFP(卵清蛋白和红色荧光蛋白转染)接种于96孔板中，加入1μM EGCG处理24小时，之后用培养基清洗细胞，并加入上步骤中处理的OT-1 CD8+T细胞，使T细胞(E)与肿瘤细胞(T)比例为2:1，之后加入IncuCyte caspase-3凋亡试剂(绿色荧光)。用Essen Bioscience公司的IncuCyte ZOOM显微镜24小时内实时检测B16F10.cOVA.RFP细胞的凋亡。仅有T细胞，以及仅有B16F10细胞组为标准化数值对照组。凋亡结果以对照组数值为基础，进行标准化处理。结果如图9B显示，EGCG处理组的B16F10.cOVA.RFP细胞凋亡比无EGCG处理的对照组显著升高，表明EGCG处理后T细胞杀伤肿瘤能力显著增强。
C57BL6小鼠皮下注射B16F10，并检测肿瘤大小，在肿瘤大小为50立方毫米左右时进行EGCG腹腔注射，浓度1mg/只/天，在对照组肿瘤达到2000立方毫米时结束实验，并取肿瘤组织备用，并用流式细胞仪检测小鼠脾脏和近肿瘤淋巴结CD3+T细胞组分的变化。肿瘤生长情况如图10所示。
结果如图11A、11B、11C、11D和11E所示，对图10中所取肿瘤组织进行STAT1、IRF1、PD-L1和PD-L2的转录水平和蛋白水平的检测(beta-Actin为管家基因)，发现STAT1、IRF1、PD-L1基因与蛋白水平在EGCG处理后有显著降低，磷酸化的STAT1蛋白水平在EGCG处理后也有显著降低，PD-L2无mRNA与蛋白水平表达。
结果如图12所示，EGCG处理后的C57BL6负瘤小鼠与对照组比较，其脾脏和近肿瘤淋巴结中的CD8+T细胞种群组成比例明显升高，证明EGCG处理后小鼠的肿瘤免疫杀伤显著增强。
构建C57BL6皮下黑色素瘤荷瘤小鼠模型，肿瘤尺寸达到大约50mm 3时，随机分为三组，每三天分别通过尾静脉注射生理盐水(Control)、EGCG水溶液(EGCG)和负载EGCG的纳米载体水分散液(nanoEGCG)，EGCG组和nanoEGCG组的EGCG剂量为0.5mg/只。
结果如图13所示，C57BL6小鼠皮下B16F10肿瘤生长情况以体积(tumor size)＝长×宽×高表示，统计分析基于每组5只小鼠，并使用one-way ANOVA分析。结果证明纳米EGCG(nanoEGCG)其抑制肿瘤生长的效果比EGCG更好。*代表EGCG组和nanoEGCG组分别与Control组差异显著(P<0.05)，#代表EGCG组和nanoEGCG组差异显著(P<0.05)。
最后应说明的是：以上各实施例仅用以说明本公开的技术方案，而非对其限制；尽管参照前述各实施例对本公开进行了详细的说明，本领域的普通技术人员应当理解：其依然可以对前述各实施例所记载的技术方案进行修改，或者对其中部分或者全部技术特征进行等同替换；而这些修改或者替换，并不使相应技术方案的本质脱离本公开各实施例技术方案的范围。
工业实用性：
本公开的茶多酚可作为免疫检查点PD-1、PD-L1和PD-L2的抑制剂，显著抑制免疫细胞及肿瘤细胞免疫检查点相关信号通路的表达，降低免疫细胞及肿瘤细胞细胞表面的免疫检查点受体和配体的表达，从而既可以提高免疫细胞的抗肿瘤能力，又可以从肿瘤内部解除产生肿瘤免疫抑制的免疫检查点配体的表达，阻止肿瘤免疫逃逸的发生。因此可以利用茶多酚制备针对免疫检查点的抗肿瘤免疫药物，或者抗肿瘤免疫辅助药物进行肿瘤治疗或预防。此外，茶多酚的制备相对简单、经济，因此本公开不仅可以 更有效的解除肿瘤微环境中的免疫抑制，同时可以以更低廉的价格实现对肿瘤的治疗。

Claims (21)

  1. 茶多酚或其药学可接受的盐在作为免疫检查点抑制剂中的应用，其特征在于，所述免疫检查点包括PD-1、PD-L1或PD-L2中的一种或多种。
  2. 根据权利要求1所述的应用，其特征在于，所述茶多酚包括儿茶素类、黄酮、黄酮醇类、花青素类、酚酸、缩酚酸类或聚合酚类中的一种或多种，优选为儿茶素类；
    优选地，所述儿茶素包括表没食子儿茶素没食子酸酯、表没食子儿茶素、表儿茶素没食子酸酯或表儿茶素中的一种或多种；优选为表没食子儿茶素没食子酸酯。
  3. 根据权利要求2所述的应用，其特征在于，所述表没食子儿茶素没食子酸酯包括纳米类表没食子儿茶素没食子酸酯。
  4. 根据权利要求2或3所述的应用，其特征在于，所述表没食子儿茶素没食子酸酯通过抑制STAT1通路实现对免疫检查点PD-1、PD-L1和/或PD-L2的表达抑制。
  5. 如权利要求1-4中任一项所述的免疫检查点抑制剂在制备用于抗肿瘤的药物中的应用，其特征在于，所述免疫检查点抑制剂为茶多酚或其药学可接受的盐，所述免疫检查点包括PD-1、PD-L1或PD-L2中的一种或多种，所述茶多酚通过抑制免疫检查点的表达实现抗肿瘤。
  6. 根据权利要求5所述的应用，其特征在于，所述肿瘤包括黑色素瘤、乳腺癌肿瘤、膀胱癌肿瘤、头颈癌肿瘤、肺癌肿瘤、结肠癌肿瘤、直肠癌肿瘤、胃癌肿瘤、前列腺癌肿瘤、肝癌肿瘤、胰腺癌肿瘤、卵巢癌肿瘤或淋巴瘤中的一种或多种。
  7. 根据权利要求5所述的应用，其特征在于，所述用于抗肿瘤的药物还包括药学上可接受的辅料。
  8. 根据权利要求5-7中任一项所述的应用，其特征在于，所述用于抗肿瘤的药物的剂型包括口服制剂或注射制剂；
    优选地，所述口服制剂包括片剂、胶囊剂、颗粒剂、丸剂、糖浆剂、口服溶液剂、口服混悬剂或口服乳剂；
    优选地，所述注射制剂包括注射液或粉针剂。
  9. 根据权利要求5-8中任一项所述的应用，其特征在于，所述用于抗肿瘤的药物的有效给药剂量为注射2-6mg/kg/天，优选为2-5mg/kg/天，更优选为3-4mg/kg/天；或，
    所述用于抗肿瘤的药物的有效给药剂量为口服5-40mg/kg/天，优选为10-35mg/kg/天，更优选为15-25mg/kg/天。
  10. 根据权利要求5-9任一项所述的应用，其特征在于，所述茶多酚包括儿茶素类、黄酮、黄酮醇类、花青素类、酚酸、缩酚酸类或聚合酚类中的一种或多种，优选为儿茶素类；
    优选地，所述儿茶素包括表没食子儿茶素没食子酸酯、表没食子儿茶素、表儿茶素没食子酸酯或表儿茶素中的一种或多种；优选为表没食子儿茶素没食子酸酯；
    优选地，所述表没食子儿茶素没食子酸酯包括纳米类表没食子儿茶素没食子酸酯。
  11. 一种茶多酚或其药学可接受的盐在治疗和/或预防肿瘤中的应用。
  12. 根据权利要求11所述的应用，其特征在于，所述茶多酚包括儿茶素类、黄酮、黄酮醇类、花青素类、酚酸、缩酚酸类或聚合酚类中的一种或多种，优选为儿茶素类；
    优选地，所述儿茶素包括表没食子儿茶素没食子酸酯、表没食子儿茶素、表儿茶素没食子酸酯或表儿茶素中的一种或多种；优选为表没食子儿茶素没食子酸酯；优选地所述表没食子儿茶素没食子酸酯包括纳米类表没食子儿茶素没食子酸酯。
  13. 根据权利要求11所述的应用，其特征在于，所述肿瘤包括黑色素瘤、乳腺癌肿瘤、膀胱癌肿瘤、头颈癌肿瘤、肺癌肿瘤、结肠癌肿瘤、直 肠癌肿瘤、胃癌肿瘤、前列腺癌肿瘤、肝癌肿瘤、胰腺癌肿瘤、卵巢癌肿瘤或淋巴瘤中的一种或多种。
  14. 一种药物组合物，用于治疗和/或预防肿瘤，其特征在于，所述药物组合物包括茶多酚或其药学可接受的盐、和药学上可接受的辅料，
    优选地所述茶多酚包括儿茶素类、黄酮、黄酮醇类、花青素类、酚酸、缩酚酸类或聚合酚类中的一种或多种，优选为儿茶素类；
    优选地，所述儿茶素包括表没食子儿茶素没食子酸酯、表没食子儿茶素、表儿茶素没食子酸酯或表儿茶素中的一种或多种；优选为表没食子儿茶素没食子酸酯；优选地所述表没食子儿茶素没食子酸酯包括纳米类表没食子儿茶素没食子酸酯；
    优选地，所述辅料包括溶剂、抛射剂、增溶剂、助溶剂、乳化剂、着色剂、黏合剂、崩解剂、填充剂、润滑剂、湿润剂、渗透压调节剂、稳定剂、助流剂、矫味剂、防腐剂、助悬剂、包衣材料、芳香剂、抗黏着剂、抗氧剂、螯合剂、渗透促进剂、pH调节剂、缓冲剂、增塑剂、表面活性剂、发泡剂、消泡剂、增稠剂、包合剂、保湿剂、吸收剂、稀释剂、絮凝剂与反絮凝剂、助滤剂或释放阻滞剂中的一种或多种。
  15. 根据权利要求14所述的药物组合物，其特征在于，所述肿瘤包括黑色素瘤、乳腺癌肿瘤、膀胱癌肿瘤、头颈癌肿瘤、肺癌肿瘤、结肠癌肿瘤、直肠癌肿瘤、胃癌肿瘤、前列腺癌肿瘤、肝癌肿瘤、胰腺癌肿瘤、卵巢癌肿瘤或淋巴瘤中的一种或多种。
  16. 一种用于治疗和/或预防肿瘤的方法，其特征在于，向有需要的受试者给药治疗有效量的根据权利要求11所述的茶多酚或其药学可接受的盐、或根据权利要求13所述的药物组合物。
  17. 根据权利要求16所述的方法，其特征在于，所述肿瘤包括黑色素瘤、乳腺癌肿瘤、膀胱癌肿瘤、头颈癌肿瘤、肺癌肿瘤、结肠癌肿瘤、直 肠癌肿瘤、胃癌肿瘤、前列腺癌肿瘤、肝癌肿瘤、胰腺癌肿瘤、卵巢癌肿瘤或淋巴瘤中的一种或多种。
  18. 根据权利要求16或17所述的方法，其特征在于，所述给药为口服给药或注射给药。
  19. 根据权利要求18中任一项所述的方法，其特征在于，所述给药的剂量为注射2-6mg/kg/天，优选为2-5mg/kg/天，更优选为3-4mg/kg/天；或，
    所述给药的剂量为口服5-40mg/kg/天，优选为10-35mg/kg/天，更优选为15-25mg/kg/天。
  20. 一种用于在受试者中抑制免疫检查点的方法，包括向受试者施用茶多酚或其药学可接受的盐、或根据权利要求13所述的组合物，
    优选地，所述免疫检查点包括PD-1、PD-L1或PD-L2中的一种或多种；
    优选地，所述茶多酚包括儿茶素类、黄酮、黄酮醇类、花青素类、酚酸、缩酚酸类或聚合酚类中的一种或多种，优选为儿茶素类；
    优选地，所述儿茶素包括表没食子儿茶素没食子酸酯、表没食子儿茶素、表儿茶素没食子酸酯或表儿茶素中的一种或多种；优选为表没食子儿茶素没食子酸酯；
    优选地，所述表没食子儿茶素没食子酸酯包括纳米类表没食子儿茶素没食子酸酯。
  21. 一种抑制第一细胞表达PD-1和/或第二细胞表达PD-L1和/或PD-L2的方法，所述方法包括：使所述第一细胞和/或所述第二细胞与茶多酚或其药学可接受的盐进行接触；
    优选地，所述茶多酚包括儿茶素类、黄酮、黄酮醇类、花青素类、酚酸、缩酚酸类或聚合酚类中的一种或多种，优选为儿茶素类；
    优选地，所述儿茶素包括表没食子儿茶素没食子酸酯、表没食子儿茶素、表儿茶素没食子酸酯或表儿茶素中的一种或多种；优选为表没食子儿茶素没食子酸酯；
    优选地，所述表没食子儿茶素没食子酸酯包括纳米类表没食子儿茶素没食子酸酯；
    优选地，所述接触在体外进行；
    优选地，所述接触在体内进行；
    优选地，所述第一细胞为免疫细胞；
    优选地，所述第二细胞为癌细胞或炎性细胞。
PCT/CN2019/111656 2018-12-03 2019-10-17 茶多酚在作为免疫检查点抑制剂及制备用于抗肿瘤的药物中的应用 Ceased WO2020114098A1 (zh)

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