Application of small molecule drug combination in enhancing the metabolism and killing ability of NK cells under hypoxic conditions

Abstract

The present invention discloses the use of a small molecule drug combination composition in enhancing the metabolism and killing ability of NK cells under hypoxic conditions. The combination composition comprises honokiol and nicotinamide riboside. The present invention discovers for the first time that the combination of honokiol and nicotinamide riboside has a synergistic effect on regulating NK cells under hypoxic conditions, and can significantly enhance the metabolism and killing ability of NK cells under hypoxic conditions, thereby providing a new drug combination strategy for the treatment of tumors.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and specifically relates to a small molecule drug combination composition, and the application of the small molecule drug combination composition in enhancing the metabolism and killing ability of NK cells under hypoxic conditions. Background Art

[0002] Natural killer cells (NK) are a type of innate immune cell that plays a vital role in tumor surveillance. Impaired anti-tumor activity of NK cells is associated with the hypoxic tumor microenvironment (TME) and tumor-derived metabolites (such as lactate) therein. Studies have shown that the hypoxic microenvironment from the tumor impairs the effector function of NK cells and promotes melanoma, pancreatic cancer, and colorectal liver metastasis. In addition, the hypoxic TME changes the metabolism of NK cells by inducing mitochondrial fragmentation in NK cells within liver cancer tumors, thereby inhibiting their anti-tumor activity. Existing studies have shown that natural killer cells (NK) play a vital role in cancer immune surveillance, but there have been no reports of targeting the hypoxic environment with small molecule drugs to enhance NK cell metabolism and strengthen their ability to effectively monitor tumors.

[0003] Therefore, the present invention is dedicated to clarifying a small molecule drug combination composition that can regulate the adaptability of NK cells to hypoxic environment, enhance the metabolism and killing ability of NK cells, and improve the anti-tumor activity in vivo and in vitro. Summary of the Invention

[0004] In view of this, the primary purpose of the present invention is to provide a small molecule drug combination composition, which enhances the adaptability of NK cells to hypoxic environments by combining two small molecule drugs, magnolia officinalis and magnolia officinalis and nicotinamide riboside, to enhance the metabolism and killing ability of NK cells under hypoxic conditions, thereby improving its effect of inhibiting tumor cell growth and promoting tumor cell apoptosis, and improving its killing effect on tumor cells in vitro and in vivo, providing a new strategy for the treatment of tumors.

[0005] In order to achieve the above object, the present invention adopts the following technical solutions:

[0006] The present invention first provides a small molecule drug combination composition for enhancing the metabolism and / or killing ability of NK cells under hypoxic conditions, which comprises magnolol and nicotinamide riboside.

[0007] In a further embodiment, the small molecule drug combination composition is a single compound preparation or a combination of two separate single preparations.

[0008] In a further embodiment, the compound preparation contains honokiol and nicotinamide riboside;

[0009] The single prescription preparation is a combination of a single prescription preparation containing honokiol and a single prescription preparation containing nicotinamide riboside.

[0010] In a further embodiment, in the small molecule drug combination composition, the concentration ratio of honokiol and nicotinamide riboside is (0.01-1 mM): (0.1-10 mM).

[0011] The present invention further provides a drug comprising the aforementioned small molecule drug combination composition.

[0012] The drug has any one or more of the following effects:

[0013] a: Enhance the metabolic capacity and / or killing ability of NK cells under hypoxic conditions;

[0014] b: Inhibit the growth of tumor cells;

[0015] c: Promote apoptosis of tumor cells.

[0016] In a further embodiment, the drug further comprises any pharmaceutically acceptable excipients and / or carriers.

[0017] The present invention further provides an in vitro method for enhancing the metabolic and / or killing ability of NK cells under hypoxic conditions for non-therapeutic purposes, comprising the following steps:

[0018] NK cells are treated in vitro with the small molecule drug combination composition described above or the drug described above.

[0019] In a further embodiment, the honokiol and nicotinamide riboside can be administered simultaneously, sequentially or intermittently.

[0020] The present invention further provides a NK cell obtained by treating with the method described above.

[0021] In a further embodiment, the NK cells are CAR-NK cells.

[0022] The present invention further provides any one or more of the following applications, comprising:

[0023] a: Use of honokiol and nicotinamide riboside in combination in the preparation of a drug for enhancing the metabolic capacity and / or killing capacity of NK cells under hypoxic conditions;

[0024] b: Use of honokiol and nicotinamide riboside in combination in the preparation of a medicament for treating tumors;

[0025] c: Use of honokiol in the preparation of a drug for improving the therapeutic effect of nicotinamide riboside on tumors.

[0026] In a further embodiment, the concentration ratio of honokiol and nicotinamide riboside is (0.01-1 mM): (0.1-10 mM).

[0027] Beneficial effects of the present invention:

[0028] The present invention utilizes two small molecule drugs, honokiol and nicotinamide riboside, in combination to enhance the adaptation of NK cells to a hypoxic environment, thereby improving the metabolism and killing ability of NK cells under hypoxic conditions, thereby improving the effect of inhibiting tumor cell growth and promoting tumor cell apoptosis.

[0029] The small molecule drug combination composition provided in the present invention can enhance NK cell-based cancer treatment by targeted hypoxia regulation, thereby providing a new strategy for the clinical treatment of tumors. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 The results of flow cytometry experiments on the impaired killing ability of NK cells in the bone marrow of AML patients in Example 1 are shown in Figure a. + Flow cytometry analysis of K562 cell percentage; Figure b shows Annexin V + Statistical graph of K562 cell percentage; Figure c shows Granzyme B + NK cells, CD107a + NK cells, IFN-γ + Flow cytometry analysis of the percentage of NK cells in the total NK cells purified from the bone marrow of AML relapsed and non-relapsed patients; Figure d shows Granzyme B + NK cells, CD107a + NK cells, IFN-γ + Figure 5 is a statistical graph showing the percentage of NK cells in the total NK cells purified from the bone marrow of AML relapsed and non-relapsed patients; Figure e is a flow cytometry analysis of the expression of NKG2D, CD38 and CD69 on NK cells isolated and purified from the bone marrow of AML patients; Figure f is a statistical graph showing the mean fluorescence intensity (MFI) of NKG2D, CD38 and CD69 on NK cells isolated and purified from the bone marrow of AML patients.

[0031] Figure 2 Figure 2 shows the flow cytometry results of the killing function of bone marrow NK (BMNK) cells after in vitro treatment in Example 2, wherein Figure a shows Annexin V + Flow cytometry analysis and statistical graph of primary AML blast percentage; Figure b shows Granzyme B + Flow cytometry analysis and statistical graph of the percentage of NK cells in total BMNK cells in patients with relapsed AML; Figure c is CD107a +Flow cytometry analysis and statistical graph of the percentage of NK cells in total BMNK cells in patients with relapsed AML; Figure d shows IFN-γ + Figure 5 is the flow cytometry analysis and statistical graph of the percentage of NK in the total BMNK cells of AML relapse patients; Figure e is the flow cytometry analysis and statistical graph of the mean fluorescence intensity (MFI) of the expression of NKG2D, CD38 and CD160 on BMNK cells of AML relapse patients; Figure f is the Annexin V + Figure g is the flow cytometry analysis and statistical graph of the percentage of K562 cells; Figure g is the flow cytometry analysis and statistical graph of the mean fluorescence intensity (MFI) of Granzyme B expression on BMNK cells of AML relapsed patients; Figure h is the CD107a + NK cells and IFN-γ + Figure i shows the flow cytometry analysis and statistical graph of the percentage of NK cells in the total BMNK cells of AML relapse patients; Figure i shows the flow cytometry analysis and statistical graph of the mean fluorescence intensity (MFI) of the expression of NKG2D, CD38 and CD160 on BMNK cells of AML relapse patients.

[0032] Figure 3 Figure 3 shows the results of the flow cytometry experiment of NK92MI cell killing function after in vitro treatment in Example 3, wherein Figure a shows Annexin V + Flow cytometry analysis and statistical graph of primary AML blast percentage; CD107a + NK cells and IFN-γ + Flow cytometry analysis and statistical graph of the percentage of NK cells in total NK92MI cells; b is the flow cytometry analysis and statistical graph of the expression of Granzyme B, NKG2D and CD160 on NK92MI cells; c is the Annexin V + Flow cytometry analysis and statistical graph of K562 blast percentage; CD107a + NK cells and IFN-γ + Figure a is a flow cytometric analysis graph and statistical graph of the percentage of NK cells in total NK92MI cells; Figure d is a flow cytometric analysis graph and statistical graph of the mean fluorescence intensity (MFI) of Granzyme B, NKG2D, and CD160 expression on NK92MI cells.

[0033] Figure 4 These are the experimental results of studying the effects of the combination of HKL and NR on leukemia cells in a leukemia mouse xenograft model in Example 5, wherein Figure a is a schematic diagram of the experimental process; Figure b is the bioluminescence imaging of AML load; and Figure c is the quantification of AML load as the average value of the total flux (p / s). DETAILED DESCRIPTION

[0034] The embodiments of the present invention are described in detail below. The embodiments described below are exemplary and are only used to explain the present invention, and are not to be construed as limiting the present invention.

[0035] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art of the present invention. The terms used in the specification of the present invention herein are only for the purpose of describing specific embodiments and are not intended to limit the present invention.

[0036] A first aspect of the present invention provides a small molecule drug combination composition, which comprises honokiol and nicotinamide riboside.

[0037] Furthermore, the small molecule drug combination composition is a single compound preparation or a combination of two separate single preparations.

[0038] Furthermore, the compound preparation is a compound preparation containing honokiol and nicotinamide riboside.

[0039] Furthermore, the combination of single-ingredient preparations is a combination of a single-ingredient preparation containing honokiol and a single-ingredient preparation containing nicotinamide riboside.

[0040] Furthermore, in the small molecule drug combination composition, the concentration ratio of honokiol and nicotinamide riboside is (0.01-1 mM): (0.1-10 mM).

[0041] In the present invention, honokiol (HKL) is a bioactive bisphenolic phytochemical that targets multiple signaling molecules and exhibits potent antioxidant, anti-inflammatory, anti-angiogenic, and anti-cancer activities. HKL has been shown to inhibit the growth of glioblastoma (GBM) cells, induce apoptosis, and suppress the growth of breast cancer cells. HKL can effectively inhibit the progression of HCC in both in vitro and in vivo models by eliminating the effects of hypoxia and lactate on HCC cell cycle proteins.

[0042] In the present invention, nicotinamide riboside (NR) is an endogenous molecule that is a precursor of nicotinamide adenine dinucleotide (NAD+). It is soluble and orally bioavailable and can increase NAD+ levels in the body. NAD+ plays an important role in cellular metabolism, energy production, DNA repair, and gene expression. Studies have shown that nicotinamide riboside may help improve mitochondrial health, stimulate mitochondrial function, induce the generation of new mitochondria, and enhance mitochondrial function in stem cells.

[0043] Currently, there are no reports on the combined use of honokiol and nicotinamide riboside to target the ability of NK cells to adapt to hypoxic environments.

[0044] In the present invention, the hypoxia refers to an O2 concentration not exceeding 10%, preferably, between 0.1% and 10%; preferably, the hypoxia refers to an O2 concentration of 5%.

[0045] In the present invention, the small molecule drug combination composition is a single compound preparation or a combination of two separate single preparations.

[0046] Specifically, a compound preparation refers to a preparation made of two or more active pharmaceutical ingredients. For example, when the small molecule drug combination composition of the present invention is a compound preparation, it may contain magnolol and nicotinamide riboside at the same time.

[0047] A single-ingredient preparation refers to a preparation made with a single active pharmaceutical ingredient. For example, when the small molecule drug combination composition of the present invention is a combination of single-ingredient preparations, it can represent a combination of a single-ingredient preparation containing magnolol and a single-ingredient preparation containing nicotinamide riboside.

[0048] In addition, it should be noted that when two single-ingredient preparations are combined, the administration method of the two single-ingredient preparations is not particularly limited and can be administered simultaneously or sequentially. When the administration method is sequential administration, the administration method includes: first administering the single-ingredient preparation containing honokiol, then administering the single-ingredient preparation containing nicotinamide riboside; or first administering the single-ingredient preparation containing nicotinamide riboside, then administering the single-ingredient preparation containing honokiol.

[0049] The second aspect of the present invention provides a drug containing the small molecule drug combination composition described above.

[0050] The drug has any one or more of the following effects:

[0051] a: Enhance the metabolic capacity and / or killing ability of NK cells under hypoxic conditions;

[0052] b: Inhibit the growth of tumor cells;

[0053] c: Promote apoptosis of tumor cells.

[0054] In the present invention, the tumor treatment effect of the drug is achieved mainly because the small molecule drug combination composition enhances the adaptation of NK cells to the hypoxic environment, improves the metabolism and killing ability of NK cells under hypoxic conditions, and thus can inhibit the growth of tumor cells, promote the apoptosis of tumor cells, and ultimately exert the effect of treating tumors.

[0055] In the present invention, the tumor includes various types of malignant tumors and benign tumors, wherein the malignant tumors include but are not limited to lung cancer, breast cancer, colorectal cancer, liver cancer, brain cancer, bone cancer, esophageal cancer, gastric cancer, nasopharyngeal cancer, thyroid cancer, pancreatic cancer, endometrial cancer, ovarian cancer, cervical cancer, renal cell carcinoma, colorectal cancer, prostate cancer, bladder cancer, pancreatic cancer, glioblastoma, melanoma, leukemia, lymphoma, myeloma, etc.; the benign tumors include but are not limited to breast fibroma, cyst, lipoma, gallbladder polyp, nodule, etc.

[0056] Furthermore, it is understood that the drug described in the present invention also includes any pharmaceutically acceptable excipients and / or carriers.

[0057] In some specific embodiments of the present invention, the pharmaceutically acceptable excipients and / or carriers include but are not limited to at least one of diluents, binders, surfactants, adsorption carriers, lubricants, fillers, and disintegrants.

[0058] In some specific embodiments of the present invention, the diluent may be, for example, lactose, sodium chloride, glucose, urea, starch, water, etc., but is not limited thereto. The binder may be, for example, starch, pregelatinized starch, dextrin, maltodextrin, sucrose, gum arabic, gelatin, methylcellulose, carboxymethylcellulose, ethylcellulose, polyvinyl alcohol, polyethylene glycol, polyvinyl pyrrolidone, alginic acid and alginates, xanthan gum, hydroxypropyl cellulose, hydroxypropyl methylcellulose, etc., but is not limited thereto. The surfactant may be, for example, polyoxyethylene sorbitan fatty acid ester, sodium lauryl sulfate, stearic acid monoglyceride, cetyl alcohol, etc., but is not limited thereto. The adsorption carrier may be, for example, starch, lactose, bentonite, silica gel, kaolin, bentonite, etc. For example, lubricants may include zinc stearate, glyceryl monostearate, polyethylene glycol, talc, calcium and magnesium stearate, polyethylene glycol, boric acid powder, hydrogenated vegetable oil, sodium stearyl fumarate, polyoxyethylene monostearate, monolauric sucrose acid ester, sodium lauryl sulfate, magnesium lauryl sulfate, magnesium lauryl sulfate, etc., but are not limited thereto. For fillers, for example, mannitol, xylitol, sorbitol, maltose, erythrose, microcrystalline cellulose, polymerized sugars, coupling sugars, glucose, lactose, sucrose, dextrin, starch, sodium alginate, laminarin powder, agar powder, calcium carbonate, sodium bicarbonate, etc., but are not limited thereto. For disintegrants, for example, cross-linked vinyl pyrrolidone, sodium carboxymethyl starch, low-substituted hydroxypropyl methyl, cross-linked sodium carboxymethyl cellulose, soybean polysaccharides, etc.

[0059] In addition, in the present invention, the auxiliary material and / or carrier may also be at least one of a stabilizer, a buffer, an isotonic agent, a pH regulator or a chelating agent.

[0060] The selection of specific excipients and / or carriers can be carried out according to the dosage form of the drug. When specifically selected, they can be compatible with the active substance, or can effectively improve the stability and solubility of the active ingredient contained in the drug, or can change the release rate and absorption rate of the active substance, thereby ensuring or enhancing the administration effect of the active ingredient. In the present invention, the dosage form of the drug is not particularly limited, and any dosage form known in the art that is conducive to administration can be adopted, for example, it can be: aqueous solution injection, powder injection, pill, powder, tablet, granule, capsule, etc. In some specific implementation cases of the present invention, powder is preferably used. Being conducive to administration as described herein means being able to improve the therapeutic effect or improve bioavailability or reduce toxic and side effects or improve the adaptability of patients, etc.

[0061] The third aspect of the present invention provides an in vitro method for enhancing the metabolic and / or killing ability of NK cells under hypoxic conditions for non-therapeutic purposes.

[0062] Furthermore, the method comprises the following steps:

[0063] NK cells are treated in vitro with the small molecule drug combination composition described above or the drug described above.

[0064] A fourth aspect of the present invention provides a NK cell obtained by the in vitro treatment method described above.

[0065] Through the above-mentioned treatment method, NK cells with excellent hypoxia adaptability, high metabolism and killing effects under hypoxic conditions can be obtained, thereby exerting a better effect of killing tumor cells.

[0066] In the present invention, the NK cells include but are not limited to NK92 cells, NK-92MI cells, KHYG-1 cells, YT cells, CIML-NK cells, NKG cells, NKL cells, NK-YS cells, SNK-6 cells, IMC-1 cells, PB-NK cells, iPSC-NK cells, UCB-NK cells or CAR-NK cells. Preferably, the NK cells are NK-92MI cells or CAR-NK cells.

[0067] When the NK cells are CAR-NK cells, an improved CAR-NK cell treatment method can be provided to provide a new treatment option for cancer patients. That is, before delivering the CAR-NK cells into the body of the tumor patient, the small molecule drug combination composition or the drug described above is used to pre-treat the CAR-NK cells, and then the cells are delivered into the body of the tumor patient to improve the treatment effect of the tumor.

[0068] Furthermore, in the present invention, the honokiol and nicotinamide riboside can be administered simultaneously, sequentially or intermittently.

[0069] The present invention further provides any one or more of the following applications, comprising:

[0070] a: Use of honokiol and nicotinamide riboside in combination in the preparation of a drug for enhancing the metabolic capacity and / or killing capacity of NK cells under hypoxic conditions;

[0071] b: Use of honokiol and nicotinamide riboside in combination in the preparation of a medicament for treating tumors;

[0072] c: Use of honokiol in the preparation of a drug for improving the therapeutic effect of nicotinamide riboside on tumors.

[0073] In the present invention, it was verified through experiments that the concentration ratio of honokiol and nicotinamide riboside is (0.01-1mM): (0.1-10mM), and the treatment time is 18h-30h. The combination of the two has a synergistic effect, which can significantly enhance the metabolism and killing ability of NK cells under hypoxic conditions, thereby better inhibiting the growth of tumor cells and promoting the apoptosis of tumor cells. In some preferred embodiments of the present invention, the concentrations of honokiol and nicotinamide riboside are 0.1mM and 1mM, respectively, and the preferred treatment time is 24h.

[0074] The present invention also provides an improved method for CAR-NK cell therapy, in which CAR-NK cells are treated in vitro with HKL and NR, and then the treated CAR-NK cells are transferred into the patient's body to achieve the purpose of improving the effect of tumor treatment.

[0075] The present invention is described below by means of specific examples. It should be noted that the following specific examples are for illustrative purposes only and do not limit the scope of the present invention in any way. In addition, unless otherwise specified, methods without specific conditions or steps are conventional methods, and the reagents and materials used are all commercially available.

[0076] Example 1 Impaired killing ability of bone marrow NK cells in patients with recurrent AML

[0077] 1. Experimental Materials

[0078] Bone marrow NK cells from patients with acute myeloid leukemia (AML) were isolated from bone marrow mononuclear cells (BMMCs) using Ficoll density gradient separation from residual bone marrow samples from patients with AML who had undergone laboratory testing. These patients included both those who experienced early relapse after allogeneic hematopoietic stem cell transplantation and those who did not experience early relapse, defined as relapse within 6 months of complete remission after allogeneic transplantation. NK cells used in in vivo experiments were purified from healthy donor blood using a magnetic-activated cell sorter (MACS) kit (Miltenyi Biotec, Cat. #130-092-657). The purity of NK cells was >93% in each assay. All human samples used were approved by the institutional ethics committee (2021-N(H)-120; Hefei, China), and written informed consent was obtained from all patients.

[0079] K562 cells were purchased from Shanghai Cell Bank (Chinese Academy of Sciences, Shanghai, China).

[0080] 2. Experimental methods

[0081] Bone marrow NK cells were divided into relapsed AML patients and non-relapsed AML patients, and co-cultured with K562 target cells according to the following steps:

[0082] Bone marrow NK cells (2×10 6 cells / mL) and K562 target cells (4×10 5 The cells were inoculated into complete RPMI 1640 medium (Thermo Fisher Scientific, 11875119) and placed in a 37°C, 5% CO2 incubator for 4 h.

[0083] The co-cultured cells were labeled with flow cytometry antibodies according to the antibody instructions, and the killing function of NK cells was detected by flow cytometry.

[0084] 3. Experimental results

[0085] Flow cytometry results are shown in Figure 1 Annexin V is significantly associated with the relapsed AML patients compared to the non-relapsed AML patients. + (7-AAD, Cat#559925, RRID:AB_2869266; APC-Annexin V, Cat#550474, RRID:AB_2868885) The percentage of K562 cells was significantly reduced ( Figure 1 a and Figure 1 b). In addition, CD107a+ (Cat#560664, RRID: AB_396135), Granzyme B + (Cat#563389, RRID:AB_2738175) and IFN-γ + The proportion of NK cells in relapsed AML patients showed a significant decrease ( Figure 1 c and Figure 1 d). This suggests that the effector function of NK cells against tumors is impaired in relapsed AML patients.

[0086] For further information, please refer to Figure 1 The expression levels of CD38, NKG2D (Cat#562365, RRID: AB_11153309) and CD69 (Cat#555531, RRID: AB_395916) on NK cells of relapsed AML patients were also significantly downregulated compared with those of non-relapsed AML patients ( Figure 1 e and Figure 1 f).

[0087] Based on the above results, it can be seen that the function of NK cells in the hypoxic bone marrow microenvironment is inhibited.

[0088] Example 2: Combination of small molecule drugs enhances the adaptability of BMNK cells to hypoxic environments and improves the killing function of NK cells

[0089] In this example, honokiol and nicotinamide riboside were used to jointly treat NK cells of relapsed AML patients, thereby demonstrating that the combined treatment can significantly enhance NK cell activation and reverse the damage to the anti-leukemia function of NK cells in relapsed AML patients.

[0090] 1. Experimental Materials

[0091] The NK cells and K562 cells from the relapsed AML patient and the flow cytometry antibodies used were the same as those in Example 1.

[0092] Honokiol, HKL (Yuanye, Cat# B20498).

[0093] Nicotinamide riboside, NR (TargetMol, Cat# T13795).

[0094] 2. Experimental methods

[0095] Bone marrow NK cells (2×10 6Cells (cells / mL) were inoculated into complete RPMI1640 medium (Thermo Fisher Scientific, 11875119) and divided into the following three groups for drug treatment:

[0096] (1) NR alone group, NR dose was 1 mM;

[0097] (2) HKL and NR combined use group, the dose of HKL was 100 μ m and the dose of NR was 1 mM;

[0098] (3) The control group did not use HKL and NR, that is, the bone marrow NK cells of relapsed AML patients without drug addition.

[0099] After drug addition, the cells were cultured at 37°C and 5% CO2 for 24 h. 6 cells / mL) and target cells (4×10 5 Primary AML blasts or K562 cells (100 cells / mL) were seeded in complete RPMI 1640 medium (Thermo Fisher Scientific, 11875119) and co-cultured for 4 hours at 37°C in a 5% CO2 incubator. After co-culture, the cells in each experimental group were labeled with flow cytometry antibodies, and the NK cell cytotoxicity was assessed by flow cytometry.

[0100] 3. Experimental results

[0101] Flow cytometry results can be found in Figure 2 It can be seen that after the combined treatment of HKL and NR, Annexin V + The proportions of primary AML cells and K562 cells were significantly increased ( Figure 2 a and Figure 2 f). CD107a + 、Granzyme B + and IFN-γ + The proportion of BMNK cells increased significantly after combined treatment with HKL and NR ( Figure 2 b~2d and Figure 2 g~2h); and the expression levels of CD38, NKG2D and CD160 (Cat#341208, RRID:AB_2561435) in BMNK cells were significantly upregulated ( Figure 2 e and Figure 2 i).

[0102] These results demonstrate that the effector function of BMNK cells from relapsed AML patients on primary AML blasts and AML cell lines was significantly improved after combined treatment with NR and HKL. Specifically, ex vivo treatment with a combination of NR and HKL restored the degranulation and cytokine secretion capacity of NK cells isolated from these AML patients. Furthermore, this combined treatment significantly enhanced NK cell activation.

[0103] Example 3: Combination of small molecule drugs enhances NK92MI's adaptability to hypoxic environments and improves NK cell killing function

[0104] 1. Experimental Materials

[0105] NK92MI cells, verified by STR analysis, were obtained from Cellcook Biotechnology (Guangzhou, China). Cells were cultured in AlphaMEM medium (Cellcook, Cat: CM2003) supplemented with 12.5% ​​horse serum (Cellcook, Cat: CM1001), 12.5% ​​fetal bovine serum (Gibco, Cat: 10099), 0.2 mM inositol, 0.1 mM thiols, and 0.02 mM folic acid. Cells were maintained in a humidified incubator at 37°C with 5% CO2.

[0106] Primary AML blasts were collected from the bone marrow of patients with newly diagnosed AML in the same manner as in Example 1.

[0107] 2. Experimental methods

[0108] NK92MI cells (2×10 6 The cells were inoculated into Alpha MEM medium and divided into the following 4 experimental groups for drug pretreatment:

[0109] (1) Hypoxia group: NK92MI cells were cultured in a hypoxic incubator at 37°C and 5% O2 for 24 h;

[0110] (2) Hypoxia + NR alone group: the NR dose was 1 mM, and NK92MI cells were cultured in a hypoxic incubator at 37°C and 5% O2 for 24 h;

[0111] (3) Hypoxia + HKL and NR combined use group: the dose of HKL was 100 μM, the dose of NR was 1 mM, and NK92MI cells were cultured in a hypoxic incubator at 37°C and 5% O2 for 24 h;

[0112] (4) Control group: HKL and NR were not used and hypoxia culture was not performed. That is, NK92MI cells were cultured in AlphaMEM medium without drug addition in an incubator at 37°C and 5% CO2 for 24 h.

[0113] After culture, cells (2×10 6 cells / mL) and target cells (4×10 5 Primary AML blasts or K562 cells (cells / mL) were inoculated in AlphaMEM medium and co-cultured at 37°C in a 5% CO2 incubator for 4 hours. After co-culture, the cells in each experimental group were labeled with flow cytometry antibodies, and the NK cell cytotoxicity was assessed by flow cytometry.

[0114] 3. Experimental results

[0115] Flow cytometry results can be found in Figure 3 It can be seen that compared with the hypoxia group, after treatment with NR alone and NR and HKL combined, the toxicity of NK cells to primary AML blasts and K562 cells was improved, and the expression of CD107a + and IFN-γ + The proportion of NK cells increased ( Figure 3 a and Figure 3 c). In addition, the expression levels of granzyme B, NKG2D, and CD160 markers in NK cells were upregulated after combined treatment with NR and HKL ( Figure 3 b and Figure 3 d).

[0116] The above results indicate that the combined use of NR and HKL effectively restores the anti-leukemia activity of NK cells by enhancing their adaptation to hypoxic environment.

[0117] Example 4 Combination of Nicotinamide Riboside and Honokiol and Combination Index

[0118] 1. Experimental materials: Same as Example 2.

[0119] 2. Experimental method: Same as Example 2.

[0120] 3. Combination Index Analysis: CalcuSyn analysis software was used to calculate the combination index (CI). The formula for the combination index is CI = D1 / DX1 + D2 / DX2, where D1 and D2 are the concentrations of each drug when used in combination, DX1 and DX2 are the drug concentrations required to achieve fa when the combination achieves the target cell apoptosis rate when either drug is used alone, and fa represents the target cell apoptosis rate achieved by the combination of the two drugs at a given concentration. The software calculates the drug combination index by inputting information such as the dose of each drug, the target cell apoptosis rate of each drug when used alone, the specific ratio of the two drugs, and the target cell apoptosis rate when the drugs are used in combination. A CI < 1 indicates a synergistic effect between the two drugs, while a CI > 1 indicates no synergistic effect between the two drugs.

[0121] 4. Experimental results:

[0122] A combination therapy index evaluation experiment was conducted on primary AML blasts. The results are shown in Table 1, indicating that combined treatment with nicotinamide riboside and honokiol has a synergistic effect on apoptosis in primary AML blasts (CI < 1). A combination therapy index evaluation experiment was conducted on K562 cells. The results are shown in Table 2, indicating that combined treatment with nicotinamide riboside and honokiol has a synergistic effect on apoptosis in K562 cells (CI < 1).

[0123] Table 1 Evaluation of the combined index of nicotinamide riboside and honokiol in primary AML blasts

[0124]

[0125] Table 2 Evaluation of the combined index of nicotinamide riboside and honokiol in K562 cells

[0126]

[0127] Example 5 The combination of HKL and NR can significantly inhibit the growth of leukemia cells in a leukemia mouse xenograft model

[0128] 1. Experimental Materials

[0129] 6-week-old female NOD / ShiLtJGpt-Prkdc em26Cd52 IL-2rg em26Cd22 Gpt (NCG) mice were purchased from GemPharmatech. All animals were housed under specific pathogen-free conditions. All experiments involving mice were performed in accordance with the National Guidelines for Animal Usage in Research and were approved by the U.S. Department of Environmental Health Sciences (USTCACUC2).

[0130] HL60 cells were purchased from Shanghai Cell Bank.

[0131] IL-2 (50000U, Jiangsu Kingsley Pharmaceuticals).

[0132] NK cells were purchased from Miaoshun (Shanghai) Biotechnology Co., Ltd.

[0133] 2. Experimental methods Figure 4 a)

[0134] a. Animal model construction

[0135] Luciferase-labeled HL60 (2.5×10 4 The cells were intravenously injected into NCG mice, and the tumor growth was monitored by bioluminescence imaging using an IVIS Spectral Imaging System (PerkinElmer) to confirm the successful engraftment of leukemia cells.

[0136] b. Treat NK cells and transfer NK cells (2×10 6 Cells (cells / mL) were inoculated in complete RPMI1640 medium (ThermoFisher Scientific, 11875119) and divided into the following three experimental groups:

[0137] (1) Ctrl group, without HKL and NR treatment, i.e., NK cells were cultured in complete RPMI1640 medium without drug addition for 24 h;

[0138] (2) NR alone group, NK cells were pretreated with 1 mM NR for 24 h;

[0139] (3) NR and HKL combined use group: NK cells were treated with 1 mM NR and 100 μM HKL simultaneously for 24 h.

[0140] All the above experimental groups were cultured in a 37°C, 5% CO2 humidified incubator.

[0141] c. On the 7th day after the mice were loaded with tumors, the NK cells (5.0×10 4 To support the survival of NK cells in vivo, IL-2 (50,000 U / mouse) was injected into the peritoneal cavity of the mice once every 2 days.

[0142] d. AML burden was monitored by bioluminescence imaging using an IVIS Spectrum Imaging System (PerkinElmer) at designated time points (7 days, 14 days, 28 days, and 35 days). Quantitative image data were analyzed using Living Image Software (Perkin Elmer).

[0143] Throughout the experiment, the mice were fed with standard full nutrition feeding conditions.

[0144] 3. Experimental results

[0145] The results can be found in Figure 4 Compared with the control group, the growth of IL60 cells and tumors in mice treated with NK cells combined with NR and HKL was significantly inhibited ( Figure 4 b and 4c).

[0146] The above results showed that mice receiving NR and NR and HKL combined-treated NK cells showed significantly lower AML burden and longer survival compared with the control group. Importantly, compared with NK cells stimulated by NR alone, hypoxia-treated NK cells stimulated by NR and HKL showed a significant reduction in tumor burden and prolonged survival. These results clearly indicate that NK cells effectively restore impaired anti-leukemia responses through the synergistic action of NR and HKL.

[0147] Based on the results of the above examples, it can be seen that the combined treatment of HKL and NR can enhance the adaptability of NK cells to hypoxic environments, thereby enhancing NK cell metabolism and killing function, and further enhancing NK cell killing of tumor cells in vivo and in vitro. By targeting NK cells in response to hypoxia, leukemia cell apoptosis is promoted and the progression of leukemia is inhibited. This shows that targeting NK cells to adapt to hypoxic environments can enhance NK cell metabolism and killing ability, promoting the treatment of AML.

[0148] It should be noted that acute myeloid leukemia is used as an example in this article, but this does not mean that the combination of small molecule drugs in this application is only applicable to acute myeloid leukemia. The combination of small molecule drugs in this application can enhance the metabolism and killing ability of NK cells under hypoxic conditions, and can be widely used in the treatment of various tumors. Due to limited space, this application will not elaborate on each one in detail.

[0149] The technical features of the above-mentioned embodiments can be combined arbitrarily. In order to make the description concise, not all possible combinations of the technical features in the above-mentioned embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0150] The above-described embodiments merely illustrate several implementations of the present invention, and while their descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the patent. It should be noted that a person skilled in the art would be able to make numerous variations and improvements without departing from the spirit of the present invention, all of which fall within the scope of protection of the present invention. Therefore, the scope of protection of the patent for this invention shall be determined by the appended claims.

Claims

1. A method for enhancing the metabolism and killing ability of NK cells under hypoxic conditions for non-therapeutic purposes in vitro, characterized in that: The following steps are involved: Treating NK cells in vitro using a small molecule drug combination composition or a drug containing the small molecule drug combination composition; The small molecule drug combination composition includes magnolol and nicotinamide riboside, and the concentration ratio of magnolol and nicotinamide riboside is (0.01~1 mM): (0.1~10 mM).

2. The method according to claim 1, wherein The small molecule drug combination composition is a single compound preparation or a combination of two separate single preparations.

3. The method according to claim 2, wherein The compound preparation is a compound preparation containing honokiol and nicotinamide riboside; The combination of single prescription preparations is a combination of single prescription preparations containing honokiol and containing nicotinamide riboside.

4. The method according to claim 1, wherein The honokiol and nicotinamide riboside can be administered simultaneously, sequentially or intermittently.

5. The method according to claim 1, wherein The drug further includes any pharmaceutically acceptable excipients and / or carriers.

6. A method for preparing NK cells with enhanced metabolism and killing ability under hypoxic conditions, characterized in that: NK cells are treated using the method according to any one of claims 1 to 5.

7. The method according to claim 6, wherein The NK cells are CAR-NK cells.

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