阅读说明：本技术 空褐鳞碱类化合物在预防、治疗car-t疗法导致的细胞因子释放综合征中的应用 (Application of anopheline compound in preventing and treating cytokine release syndrome caused by CAR-T therapy ) 是由 刘茂玄 许晨光 黎琴子 曾滢 于 2019-04-08 设计创作，主要内容包括：本发明提供了空褐鳞碱类化合物在制备预防、治疗CAR-T疗法导致的细胞因子释放综合征的制剂中的应用。还提供了一种用于治疗恶性肿瘤、自身免疫性疾病或艾滋病的药物组合物,包括嵌合抗原受体T细胞和空褐鳞碱类化合物。本发明通过将嵌合抗原受体T细胞和空褐鳞碱类化合物联用,可以以较低的经济成本来预防和/或治疗CAR-T细胞疗法导致的CRS副作用,进而发挥出CAR-T细胞疗法的较好治疗效果。(The invention provides application of an alopecuroid compound in preparing a preparation for preventing and treating cytokine release syndrome caused by CAR-T therapy. Also provided is a pharmaceutical composition for treating malignant tumor, autoimmune disease or AIDS, comprising chimeric antigen receptor T cells and an empty limonoid base compound. According to the invention, the chimeric antigen receptor T cell and the anopheline compound are combined, so that CRS side effects caused by CAR-T cell therapy can be prevented and/or treated at a low economic cost, and a good treatment effect of the CAR-T cell therapy is further exerted.)

1. Application of an empty limonoid alkali compound in preparation of a preparation for preventing and/or treating cytokine release syndrome caused by chimeric antigen receptor T cell therapy.

2. The use of claim 1, wherein the null limonoid base compound comprises null limonoid base, a tautomer thereof, a pharmaceutically acceptable salt or prodrug thereof, and a null limonoid base derivative.

3. The use of claim 1, wherein said chimeric antigen receptor T cell therapy comprises treatment of a malignancy, an autoimmune disease, or aids.

4. The use of claim 3, wherein the malignancy comprises one or more of leukemia, lymphoma, multiple myeloma, brain glioma, liver cancer, lung cancer, stomach cancer, esophageal cancer, colon cancer, pancreatic cancer, breast cancer, ovarian cancer, cervical cancer and prostate cancer;

the autoimmune disease comprises one or more of systemic lupus erythematosus, rheumatoid arthritis, chronic active hepatitis, scleroderma, pemphigus, dermatomyositis, ulcerative colitis, and hashimoto's thyroiditis.

5. A pharmaceutical composition comprising a chimeric antigen receptor T cell and an empty limonoid base compound.

6. The pharmaceutical composition of claim 5, wherein the chimeric antigen receptor T cell is a chimeric antigen receptor T cell that targets one or more of CD19, CD20, CD22, CD33, CD38, CD317, BCMA, EGFR, Mesothelin, DR5, c-met, OX40, HER 2.

7. The pharmaceutical composition of claim 5, wherein the T cells used in the chimeric antigen receptor T cells are derived from autologous T cells, allogeneic T cells, or iPSC-induced T cells.

8. The pharmaceutical composition of any one of claims 5-7, wherein said chimeric antigen receptor T cells and said empty limonoid are present alone or in admixture; the administration mode of the chimeric antigen receptor T cells and the empoasca limonoid alkali compound comprises simultaneous administration, sequential administration or administration in different administration modes.

9. The pharmaceutical composition of claim 8, wherein the chimeric antigen receptor T cells are administered intravenously and the administration of the ascochytrine compound is intraperitoneal injection.

10. Use of a pharmaceutical composition according to any one of claims 5-9 for the preparation of a formulation for the treatment of malignant tumors, autoimmune diseases or aids.

Technical Field

The invention relates to the technical field of medicines, in particular to application of an alofuscin compound in preventing and treating cytokine release syndrome caused by CAR-T therapy.

Background

Malignant diseases such as cancer are diseases which seriously harm human health in the global scope and seriously affect life. Currently, the treatment methods for cancer and the like include nonspecific immunotherapy, active immunotherapy, adoptive cellular immunotherapy, and the like. Among them, adoptive cellular immunotherapy is considered as a method in which it is possible to completely eliminate cancer cells in the existing technologies. The chimeric antigen receptor T cell (CAR-T) technology, one of the latest immune cell technologies in current adoptive cell-based therapeutic technologies, is to transfuse genetically modified T lymphocytes (which can express specific chimeric antigen receptors) back into the human body, and activate the T lymphocytes in a manner independent of major histocompatibility complexes, thereby specifically killing malignant target cells such as cancer cells. The CAR-T technology has strong targeting, killing activity and persistence on malignant cells, thereby showing great application prospect in the aspect of treating malignant diseases such as cancer and the like.

Although CAR-T cell therapy has achieved good results in some clinical trials, it has also been associated with serious side effects, such as the most common Cytokine Release Syndrome (CRS) at present, resulting in significant increases in the levels of certain cytokines in the patient's blood, fever, tachycardia, hypotension, etc., and, in severe cases, multiple organ failure and death. Therefore, there is a need to inhibit cytokine release during CAR-T cell immunotherapy and alleviate/eliminate CRS side effects to improve the therapeutic efficacy of CAR-T cells.

Disclosure of Invention

In view of the above, the present invention provides a technical solution for preventing and treating CRS caused by CAR-T cell immunotherapy.

In a first aspect, the invention provides the use of an empty limonoid base compound in the preparation of a formulation for the prevention and/or treatment of Cytokine Release Syndrome (CRS) caused by chimeric antigen receptor T (CAR-T) cell therapy.

In the present invention, the said compound of the empoaspidine class includes empoaspidine (bulbocapline), its tautomer, its pharmaceutically acceptable salt or prodrug, and empoaspidine derivatives.

Wherein the CAR-T cell therapy comprises use in the treatment of a malignancy, an autoimmune disease or AIDS.

Further, the malignant tumor includes one or more of leukemia, lymphoma, multiple myeloma, brain glioma, liver cancer, lung cancer, stomach cancer, esophageal cancer, colon cancer, pancreatic cancer, breast cancer, ovarian cancer, cervical cancer and prostate cancer, but is not limited thereto.

Further, the autoimmune disease includes one or more of systemic lupus erythematosus, rheumatoid arthritis, chronic active hepatitis, scleroderma, pemphigus, dermatomyositis, ulcerative colitis, and hashimoto's thyroiditis, but is not limited thereto.

The compounds of the invention are micromolecular drugs, the price is much lower than that of antibody drugs for inhibiting cell factors after CRS side effects occur, and the purpose of reducing CRS side effects caused by CAR-T cell therapy can be achieved with lower economic cost; meanwhile, the empyrosine can inhibit the activity of various cytokines by inhibiting the release of catecholamine substances, compared with the antibody drug which can only inhibit the activity of one cytokine, so that the prevention and/or treatment effect on CRS is better.

In a second aspect, the invention also provides a pharmaceutical composition comprising a chimeric antigen receptor T (CAR-T) cell and an empty limonoid base compound.

Optionally, the CAR-T cells comprise CAR-T cells that target one or more of CD19, CD20, CD22, CD33, CD38, CD317, BCMA, EGFR, Mesothelin, DR5, c-met, OX40, HER 2.

Wherein the T cells in the CAR-T cells are from autologous T cells, allogeneic T cells, or iPSC-induced T cells.

In the pharmaceutical composition, the CAR-T cells and the anopheline compound can be present independently or in a mixture. When used as a pharmaceutical combination, the two may be administered simultaneously, sequentially or in different modes of administration.

In one embodiment of the invention, the CAR-T cells are administered intravenously and the anopheline compound is administered intraperitoneally.

In a third aspect, the invention also provides an application of the pharmaceutical composition in preparing a preparation for treating malignant tumor, autoimmune disease or AIDS.

According to the invention, the CAR-T cell and the anopheline compound are combined, so that CRS side effects (not only inhibiting the level of a cytokine) caused by CAR-T cell therapy can be prevented and/or treated, and a better treatment effect of CAR-T cell therapy is further exerted.

Detailed Description

While the following is a description of the preferred embodiments of the present invention, it will be understood by those skilled in the art that various changes and modifications may be made without departing from the spirit and scope of the invention.

The embodiment of the invention provides application of an empty limonoid alkali compound in preparing a preparation for preventing and/or treating Cytokine Release Syndrome (CRS) caused by chimeric antigen receptor T (CAR-T) cell therapy.

Wherein the structural formula of the anopheline is shown as the following formula (I):

the "null limonoid compound" of the present invention may include its tautomer, or its pharmaceutically acceptable salt or prodrug, in addition to null limonine (bulbocapline), and include various derivatives that can be modified by simple chemical methods (such as reduction, substitution, etc.) on the side chain group of the skeleton by those skilled in the art, while still retaining the prevention and/or treatment of CRS caused by CAR-T cell therapy, and the synthesis method of these derivatives can be referred to the textbooks in the prior art.

Wherein, "pharmaceutically acceptable salt" refers to a salt of a compound prepared from a pharmaceutically acceptable non-toxic acid (including inorganic acids, organic acids), non-toxic base (including inorganic bases, organic bases), solvate, hydrate or clathrate.

Prodrugs can be prepared in situ during isolation and purification of the compound, or by separately reacting the purified compound with a suitable derivatizing agent. For example, the hydroxy group of the tautomer can be converted to an ester group by treatment with a carboxylic acid in the presence of a catalyst. Examples of cleavable alcohol prodrug moieties include: substituted or unsubstituted, branched or unbranched lower alkyl esters, such as ethyl esters, lower alkenyl esters, di-lower alkylamino lower alkyl esters, such as dimethylaminoethyl ester, acylamino lower alkyl esters, acyloxy lower alkyl esters (such as pivaloyloxymethyl ester), aryl esters, such as phenyl esters, aryl-lower alkyl esters, such as benzyl esters which may be substituted, such as aryl and aryl-lower alkyl esters substituted by methyl, halogen or methoxy, amides, lower alkylamides, di-lower alkylamides, and hydroxyamides.

The empholine is an aporphine (type) alkaloid natural compound derived from the plants of the corydalis genus, can limit the synthesis of catecholamine substances by inhibiting the activity of tyrosine hydroxylase, and further inhibit the release of fine factors, thereby preventing and/or treating CRS caused by CAR-T cell therapy.

The compounds of the invention are micromolecular drugs, the price is much lower than that of antibody drugs for inhibiting cell factors after CRS side effects occur, and the purpose of reducing CRS side effects caused by CAR-T cell therapy can be achieved with lower economic cost; meanwhile, the empyrosine can inhibit the activity of various cytokines by inhibiting the release of catecholamine substances, compared with the antibody drug which can only inhibit the activity of one cytokine, so that the prevention and/or treatment effect on CRS is better.

CAR-T cells of the invention include CAR-T cells that target a variety of different malignant disease targets, such as: targeting one or more of CD19, CD20, CD22, CD33, CD38, CD317, BCMA, EGFR, Mesothelin, DR5, c-met, OX40, HER2, and the like.

Wherein the T cells in the CAR-T cells are from autologous T cells, allogeneic T cells, or iPSC-induced T cells.

Wherein the CAR-T cell therapy comprises use in the treatment of a malignancy, an autoimmune disease or AIDS.

Further, the malignant tumor includes one or more of leukemia, lymphoma, multiple myeloma, brain glioma, liver cancer, lung cancer, stomach cancer, esophageal cancer, colon cancer, pancreatic cancer, breast cancer, ovarian cancer, cervical cancer and prostate cancer, but is not limited thereto.

Further, the autoimmune disease includes one or more of systemic lupus erythematosus, rheumatoid arthritis, chronic active hepatitis, scleroderma, pemphigus, dermatomyositis, ulcerative colitis, and hashimoto's thyroiditis, but is not limited thereto.

Embodiments of the invention also provide a pharmaceutical composition comprising a chimeric antigen receptor T (CAR-T) cell and an empty limonoid base compound. The pharmaceutical composition is used for treating malignant tumor, autoimmune disease or AIDS, and can also be used for preventing and/or treating Cytokine Release Syndrome (CRS) caused by chimeric antigen receptor T (CAR-T) cell therapy.

Optionally, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier. The pharmaceutically acceptable carrier is preferably chemically inert to the alofusrine compounds of the invention and has no deleterious side effects or toxicity under the conditions of use.

Such pharmaceutically acceptable carriers, e.g., excipients, stabilizers, suspending agents or diluents, and the like, are well known to those skilled in the art and are publicly available. Exemplary carriers can include water, physiological saline, aqueous dextrose and related sugar solutions, monohydric alcohols (such as ethanol, isopropanol or cetyl alcohol), glycols (such as propylene glycol or polyethylene glycol), glycerol, ketals (such as 2, 2-dimethyl-1, 3-dioxolane-4-methanol), ethers (such as poly (ethylene glycol) 400), oils, fatty acids, fatty acid esters or glycerides, acetylated fatty acid glycerides with or without pharmaceutically acceptable surfactants, such as soaps or detergents, suspending agents (such as pectin, carbomer, methyl cellulose, hydroxypropyl methyl cellulose or carboxymethyl cellulose), or emulsifying agents and other pharmaceutical adjuvants. The choice of the carrier will depend in part on the particular compound of the anopheline class selected, as well as the particular method used to administer the composition.

In the pharmaceutical composition, the CAR-T cells and the anopheline compound can be present independently or in a mixture. When used as a pharmaceutical combination, the two may be administered simultaneously, sequentially or in different modes of administration.

For example, the CAR-T cells are administered parenterally, e.g., intravenously, intraarterially, subcutaneously, intradermally, intrathecally, or intramuscularly. The CAR-T cells can be dissolved or suspended in a solution suitable for parenteral administration in an acceptable carrier, including isotonic sterile injectable aqueous and non-aqueous solutions. The administration modes of the anopheline compounds comprise oral administration, intravenous injection, subcutaneous injection, intramuscular injection, intraperitoneal administration, sublingual administration, spray nasal administration and the like. When the anopheline compound is administered by intravenous injection, subcutaneous injection, intramuscular injection, etc., it can be made into injection preparation.

In one embodiment of the invention, the CAR-T cells are administered intravenously and the anopheline compound is administered intraperitoneally.

Where the null limonoid is used in conjunction with CAR-T cells, various concentrations of null limonoid may be used in conjunction with various concentrations (or amounts) of CAR-T cells. For example, the CAR-T cells used have a density of 0.5X 10⁶～5×10⁶Individual cells/mL. For example, 1X 10⁶、2×10⁶Or 0.5X 10⁶Individual cells/mL.

In addition, the embodiment of the present invention also provides a method for treating malignant tumor, autoimmune disease or AIDS, which comprises administering the pharmaceutical composition of the present invention to a test subject with malignant target cells.

The concentration of the empty limonoid compound, the concentration (or number) of CAR-T cells can be adjusted depending on the particular subject. Wherein the malignant target cell is a cell targeted by the CAR-T cell. Such as cells corresponding to the above-mentioned malignant tumor, autoimmune disease or AIDS.

Optionally, the CAR-T cell to malignant target cell number ratio is (2-10): 1. for example 2.5:1, 5:1 or 10: 1.

The treatment of CAR-T cell immunotherapy with an empty limonoid resulting in CRS in the treatment of tumors is illustrated below by a specific example. The reagents used in the present invention are all common commercial products, and are all commercially available.

The method comprises the following specific steps:

1. mixing newborn (0-2 days) SGM3(NSG Tg)^{CMV-IL3，CSF2,KITLG}1Eav/MloySzJ) mice were irradiated with sublethal doses (150cGy) and immediately injected intrahepatically with 1X 10⁵Individual human cord blood CD34+ cells, a nhsgm 3 humanized mouse was prepared. Humanized mouse T cells of nhsgm 3 were used to generate CAR-T cells targeted to CD 19.

2. Adult (6-8 weeks) SGM3(NSG Tg)^{CMV-IL3，CSF2,KITLG}1Eav/MloySzJ) mice were irradiated with a sublethal dose (200cGy) and injected immediately via tail vein with 1X 10⁵Individual human cord blood CD34+ cells, HuSGM3 humanized adult mice were prepared.

After 3.4 weeks, the adult mice in step 2 were injected 2X 10 by tail vein injection⁶CD 19-targeted CAR-T cells prepared from nHuSGM3 humanized mice in step 1.

4. Immediately after CAR-T cell injection, 15mg/kg of the orotadenine hydrochloride or an equal volume of PBS was intraperitoneally injected daily for 3 consecutive days.

5. Weighing the body weight of the mice each day after the injection of the CAR-T cells, and measuring the body temperature of the mice; blood was taken every two days and the serum levels of human IL-6, IL-10, TNF- α and mouse serum amyloid A were measured. The CRS-related indexes are continuously detected for 21 days.

As a result, compared with the CAR-T cell therapy only targeting CD19, the CAR-T cell therapy targeting CD19 and the hydrochloride salt of the empty limonine performed on the tumor-bearing adult mice at the same time have lower body temperature and lower contents of cytokines such as human IL-6, IL-10 and TNF-alpha in serum. This indicates that the use of the anopheline compounds has significant therapeutic effects on CRS caused by CAR-T cell immunotherapy in tumor treatment.

The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the present invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. Therefore, the protection scope of the present patent shall be subject to the appended claims.