WO2020114098A1 - Application de polyphénol de thé dans un inhibiteur de points de contrôle immunitaires et dans la préparation de médicaments antitumoraux - Google Patents

Application de polyphénol de thé dans un inhibiteur de points de contrôle immunitaires et dans la préparation de médicaments antitumoraux 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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Chinese (zh)
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李洋
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深圳先进技术研究院
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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

Definitions

  • 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

La présente invention concerne une application de polyphénol de thé dans un inhibiteur de points de contrôle immunitaires et dans la préparation de médicaments antitumoraux.
PCT/CN2019/111656 2018-12-03 2019-10-17 Application de polyphénol de thé dans un inhibiteur de points de contrôle immunitaires et dans la préparation de médicaments antitumoraux WO2020114098A1 (fr)

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