Use of small-molecule drug combination in enhancing metabolic and killing abilities of NK cells under hypoxic conditions

Abstract

Use of a small-molecule drug combination composition in enhancing the metabolic and killing abilities of NK cells under hypoxic conditions. The combination composition comprises honokiol and nicotinamide nucleoside. A combination of honokiol and nicotinamide nucleoside has a synergistic effect on the regulation of NK cells under hypoxic conditions, and can significantly enhance the metabolic and killing abilities of NK cells under hypoxic conditions. Thus, the present invention provides a new drug combination strategy for the treatment of tumors.

Description

Application of small molecule drug combination therapy in enhancing NK cell metabolism and killing ability under hypoxic conditions Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a small molecule drug combination composition and its application in enhancing the metabolism and killing ability of NK cells under hypoxic conditions. Background Technology

[0002] Natural killer (NK) cells are innate immune cells that play a crucial role in tumor surveillance. Impaired antitumor activity of NK cells is associated with the hypoxic tumor microenvironment (TME) and its tumor-derived metabolites (such as lactate). Studies have shown that the hypoxic tumor microenvironment impairs the effector function of NK cells and promotes melanoma, pancreatic cancer, and colorectal liver metastases. Furthermore, the hypoxic TME alters NK cell metabolism by inducing mitochondrial fragmentation in NK cells within liver cancer tumors, thereby inhibiting their antitumor activity. While existing research indicates that NK cells play a vital role in cancer immune surveillance, there are currently no reports of using small molecule drugs to target the hypoxic environment to enhance NK cell metabolism and strengthen their ability to effectively monitor tumors.

[0003] Therefore, this invention aims to elucidate a small molecule drug combination composition that can regulate the adaptation of NK cells to hypoxic environments, enhance the metabolism and killing ability of NK cells, and improve in vivo and in vitro antitumor activity. Summary of the Invention

[0004] In view of this, the primary objective of the present invention is to provide a combination composition of small molecule drugs that enhances the adaptability of NK cells to hypoxic environments by combining small molecule drugs with honokiol and honokiol with nicotinamide nucleoside, thereby enhancing the metabolic and killing ability of NK cells under hypoxic conditions, improving their effect of inhibiting tumor cell growth and promoting tumor cell apoptosis, and enhancing their killing effect on tumor cells in vivo and in vitro, thus providing a new strategy for the treatment of tumors.

[0005] To achieve the above objectives, the present invention adopts the following technical solution:

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

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

[0008] In a further embodiment, the compound preparation contains magnolol and nicotinamide nucleoside;

[0009] The single-ingredient preparation is a combination of single-ingredient preparations containing magnolol and nicotinamide nucleoside.

[0010] In a further embodiment, the concentration ratio of magnolol and nicotinamide nucleoside in the small molecule drug combination composition is 0.01~1:0.1~10.

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

[0012] The drug has 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: Inhibits the growth of tumor cells;

[0015] c: Promotes apoptosis of tumor cells.

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

[0017] This invention further provides a method for enhancing the metabolic and / or cytotoxic capacity of NK cells under hypoxic conditions in vitro for non-therapeutic purposes, comprising the following steps:

[0018] NK cells were treated in vitro using the small molecule drug combination composition or the drugs described above.

[0019] In a further embodiment, magnolol and nicotinamide nucleoside can be administered simultaneously, sequentially, or at intervals.

[0020] The present invention further provides an NK cell, which is obtained by processing using the method described above.

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

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

[0023] a: Application of magnolol and nicotinamide nucleoside in the preparation of drugs for enhancing the metabolic and / or killing capacity of NK cells under hypoxic conditions;

[0024] b: Application of magnolol and nicotinamide nucleoside in the preparation of drugs for treating tumors;

[0025] c: The application of magnolol in the preparation of drugs for improving the therapeutic effect of nicotinamide nucleoside on tumors.

[0026] In a further embodiment, the concentration ratio of magnolol and nicotinamide nucleoside is 0.01~1:0.1~10.

[0027] In this invention, the combination of two small molecule drugs, magnolol and nicotinamide nucleoside, enhances the adaptation of NK cells to a hypoxic environment, improves the metabolism and killing ability of NK cells under hypoxic conditions, and thus enhances the effect of inhibiting tumor cell growth and promoting tumor cell apoptosis.

[0028] The small molecule drug combination composition provided in this invention can enhance NK cell-based cancer therapy by targeting hypoxia regulation, thereby providing a new strategy for the clinical treatment of tumors. Attached Figure Description

[0029] Figure 1 shows the flow cytometry results of impaired NK cell killing ability in the bone marrow of AML patients in Example 1. Figure 1a shows Annexin V... + Flow cytometry plot of K562 cell percentage; Figure 1b shows Annexin V. + A statistical graph of the percentage of K562 cells; Figure 1c shows Granzyme B. + NK cells, CD107a + NK cells, IFN-γ + Flow cytometry plot of the percentage of total NK cells purified from bone marrow in AML relapsed and non-relapsed patients; Figure 1d shows Granzyme B. + NK cells, CD107a + NK cells, IFN-γ + Figure 1e shows the flow cytometry analysis of the expression of NK cells in total NK cells purified from bone marrow of AML relapsed and non-relapsed patients; Figure 1f shows the statistical graph of the mean fluorescence intensity (MFI) of NK cells purified from bone marrow of AML patients.

[0030] Figure 2 shows the flow cytometry results of bone marrow NK (BMNK) cell killing function after in vitro treatment in Example 2. Figure 2a shows the Annexin V... + Flow cytometry and statistical plots of the percentage of primary AML blast cells; Figure 2b shows Granzyme B. + Flow cytometry plots and statistical graphs showing the percentage of NK cells in total BMNK cells in AML relapsed patients; Figure 2c shows CD107a. +Flow cytometry plots and statistical graphs showing the percentage of NK cells in total BMNK cells in AML relapsed patients; Figure 2d shows IFN-γ. + Figure 2e shows the flow cytometry analysis and statistical graph of the percentage of NK cells in total BMNK cells of AML relapsed patients; Figure 2f shows the flow cytometry analysis and statistical graph of the mean fluorescence intensity (MFI) of NKG2D, CD38, and CD160 expression in BMNK cells of AML relapsed patients; + Flow cytometry plots and statistical graphs of K562 cell percentages; Figure 2g shows flow cytometry plots and statistical graphs of mean fluorescence intensity (MFI) of Granzyme B expression in BMNK cells of AML relapsed patients; Figure 2h shows CD107a... + NK cells and IFN-γ + Flow cytometry plots and statistical graphs showing the percentage of NK cells in total BMNK cells of AML relapsed patients; Figure 2i shows flow cytometry plots and statistical graphs of mean fluorescence intensity (MFI) of NKG2D, CD38, and CD160 expression in BMNK cells of AML relapsed patients.

[0031] Figure 3 shows the flow cytometry results of the NK92MI cell killing function after in vitro treatment in Example 3. Among them, Figure 3a shows the Annexin V... + Flow cytometry plots and statistical graphs of the percentage of primary AML blast cells; CD107a + NK cells and IFN-γ + Figure 3b shows the flow cytometry analysis and statistical graph of the percentage of NK cells in total NK92MI cells; Figure 3c shows the flow cytometry analysis and statistical graph of the mean fluorescence intensity (MFI) of Granzyme B, NKG2D, and CD160 expression in NK92MI cells; + Flow cytometry plots and statistical graphs of K562 blastocyte percentage; CD107a + NK cells and IFN-γ + Flow cytometry plots and statistical graphs showing the percentage of NK cells in total NK92MI cells; Figure 3d shows flow cytometry plots and statistical graphs of mean fluorescence intensity (MFI) of Granzyme B, NKG2D, and CD160 expression in NK92MI cells.

[0032] Figure 4 shows the experimental results of studying the effect of HKL and NR combined on leukemia cells in a mouse xenograft model of leukemia in Example 5. Figure 4a is a schematic diagram of the experimental procedure; Figure 4b is a bioluminescent imaging of AML load; and Figure 4c is the average value (p / s) of AML load quantified as total throughput. Embodiments of the present invention

[0033] The first aspect of the present invention provides a small molecule drug combination composition comprising magnolol and nicotinamide nucleoside.

[0034] Furthermore, the small molecule drug combination is a single compound preparation or a combination of two separate single preparations.

[0035] Furthermore, the compound preparation is a compound preparation containing magnolol and nicotinamide nucleoside.

[0036] Furthermore, the combination of the single-ingredient preparations is a combination of a single-ingredient preparation containing magnolol and a single-ingredient preparation containing nicotinamide nucleoside.

[0037] Furthermore, in the small molecule drug combination composition, the concentration ratio of magnolol and nicotinamide nucleoside is 0.01~1:0.1~10.

[0038] In this invention, honokiol (HKL) is a bioactive bisphenol phytochemical that targets multiple signaling molecules and possesses effective antioxidant, anti-inflammatory, anti-angiogenic, and anticancer activities. HKL has been shown to inhibit the growth of glioblastoma (GBM) cells and induce apoptosis, as well as inhibit the growth of breast cancer cells. HKL can effectively inhibit the progression of HCC in in vitro and in vivo models by scavenging the effects of hypoxia and lactate on HCC cell cyclins.

[0039] In this invention, nicotinamide riboside (NR) is an endogenous molecule that is a precursor to nicotinamide adenine dinucleotide (NAD+). It is soluble and orally bioavailable, and can increase NAD+ levels in the body. NAD+ plays a crucial role in cellular metabolism, energy production, DNA repair, and gene expression. Previous studies have shown that nicotinamide riboside may help improve mitochondrial health, stimulate mitochondrial function, and induce the generation of new mitochondria, and may also enhance mitochondrial function in stem cells.

[0040] There are currently no research reports on the combined use of magnolol and nicotinamide nucleoside to target the hypoxic adaptation ability of NK cells.

[0041] In this invention, low oxygen means an O2 concentration of no more than 10%, preferably 0.1% to 10%; more preferably, low oxygen means an O2 concentration of 5%.

[0042] In this invention, the small molecule drug combination composition is a single compound preparation or a combination of two separate single preparations.

[0043] 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 in this invention is a compound preparation, it can mean that it simultaneously contains magnolol and nicotinamide nucleoside.

[0044] For single-component formulations, it refers to a formulation made with a single active pharmaceutical ingredient. For example, when the combination of small molecule drugs in this invention is a combination of single-component formulations, it can be represented as a combination of single-component formulations containing magnolol and nicotinamide nucleoside, respectively.

[0045] Furthermore, it should be noted that when a combination of two single-component preparations is used, there are no particular restrictions on the administration method of these two preparations; they can be administered simultaneously or sequentially. When administered sequentially, the administration methods include: first administering the single-component preparation containing magnolol, and then administering the single-component preparation containing nicotinamide nucleoside; or first administering the single-component preparation containing nicotinamide nucleoside, and then administering the single-component preparation containing magnolol.

[0046] A second aspect of the present invention provides a medicament comprising the small molecule drug combination composition described above.

[0047] The drug has one or more of the following effects:

[0048] a: Enhance the metabolic capacity and / or killing ability of NK cells under hypoxic conditions;

[0049] b: Inhibits the growth of tumor cells;

[0050] c: Promotes apoptosis of tumor cells.

[0051] In this invention, the therapeutic effect of the drug on tumors is mainly due to the fact that the combination of small molecule drugs enhances the adaptation of NK cells to the hypoxic environment, improves the metabolism and killing ability of NK cells under hypoxic conditions, thereby inhibiting the growth of tumor cells, promoting the apoptosis of tumor cells, and ultimately exerting the therapeutic effect on tumors.

[0052] In this invention, the tumors include various types of malignant and benign tumors. 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 carcinoma, 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 fibroadenomas, cysts, lipomas, gallbladder polyps, nodules, etc.

[0053] Furthermore, it is understood that the medicaments described in this invention also include pharmaceutically acceptable excipients and / or carriers.

[0054] 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, adsorbents, lubricants, fillers, and disintegrants.

[0055] 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, polyvinylpyrrolidone, alginate and alginates, xanthan gum, hydroxypropylcellulose, hydroxypropyl methylcellulose, etc., but is not limited thereto. The surfactant may be, for example, polyethylene oxide sorbitan fatty acid ester, sodium lauryl sulfate, glyceryl monostearate, hexadecyl alcohol, etc., but is not limited thereto. The adsorbent carrier may be, for example, starch, lactose, bentonite, silica gel, kaolin, soap clay, etc. Examples of lubricants include zinc stearate, glyceryl monostearate, polyethylene glycol, talc, calcium and magnesium stearate, polyethylene glycol, boric acid powder, hydrogenated vegetable oil, sodium stearate fumarate, polyoxyethylene monostearate, monolauric sucrose, sodium lauryl sulfate, magnesium lauryl sulfate, magnesium dodecyl sulfate, etc., but are not limited to these. Examples of fillers include mannitol, xylitol, sorbitol, maltose, erythrose, microcrystalline cellulose, polysaccharides, coupled sugars, glucose, lactose, sucrose, dextrin, starch, sodium alginate, kelp polysaccharide powder, agar powder, calcium carbonate, sodium bicarbonate, etc., but are not limited to these. Examples of disintegrants include crosylvinylpyrrolidone, sodium carboxymethyl starch, low-substituted hydroxypropylmethyl, crosylcarboxymethyl cellulose sodium, soybean polysaccharides, etc.

[0056] Furthermore, in this invention, the excipients and / or carriers may also be at least one of stabilizers, buffers, isotonic agents, pH adjusters, or chelating agents.

[0057] The selection of specific excipients and / or carriers can be based on the dosage form of the drug. Specifically, the selection should be compatible with the active substance, or effectively improve the stability and solubility of the active ingredient contained in the drug, or alter the release and absorption rate of the active substance, thereby ensuring or enhancing the drug delivery effect. In this invention, the dosage form of the drug is not particularly limited, and any dosage form known in the art that is beneficial for drug delivery can be used, such as: aqueous injection, powder for injection, pills, powders, tablets, granules, capsules, etc. In some specific embodiments of this invention, powder is preferred. The term "beneficial for drug delivery" as used herein refers to improving therapeutic effects, increasing bioavailability, reducing toxic side effects, or improving patient adaptability, etc.

[0058] The third aspect of the present invention provides a method for enhancing the metabolic and / or killing ability of NK cells under hypoxic conditions in vitro for non-therapeutic purposes.

[0059] Furthermore, the method includes the following steps:

[0060] NK cells were treated in vitro using the small molecule drug combination composition or the drugs described above.

[0061] A fourth aspect of the present invention provides an NK cell obtained by the in vitro processing method described above.

[0062] The above treatment methods can be used to obtain NK cells with excellent hypoxia adaptability and high metabolism and killing effect under hypoxia conditions, thereby achieving better tumor cell killing effect.

[0063] In this 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.

[0064] When the NK cells are CAR-NK cells, a modified CAR-NK cell therapy can be provided to offer a new treatment option for cancer patients. This involves pretreating the CAR-NK cells with the small molecule drug combination or the drugs described above before delivering them to the cancer patient, thereby improving the treatment effect.

[0065] Furthermore, in this invention, magnolol and nicotinamide nucleoside can be administered simultaneously, sequentially, or at intervals.

[0066] The present invention further provides one or more of the following applications, said applications including:

[0067] a: Application of magnolol and nicotinamide nucleoside in the preparation of drugs for enhancing the metabolic and / or killing capacity of NK cells under hypoxic conditions;

[0068] b: Application of magnolol and nicotinamide nucleoside in the preparation of drugs for treating tumors;

[0069] c: The application of magnolol in the preparation of drugs for improving the therapeutic effect of nicotinamide nucleoside on tumors.

[0070] In this invention, experiments verified that the concentration ratio of magnolol and nicotinamide nucleoside was (0.01~1 mM):(0.1~10 mM), and the treatment time was 18 h-30 h. The combined use of these two drugs has a synergistic effect, significantly enhancing the metabolic and killing capacity of NK cells under hypoxic conditions, thereby better inhibiting tumor cell growth and promoting tumor cell apoptosis. In some preferred embodiments of this invention, the concentrations of magnolol and nicotinamide nucleoside are 0.1 mM and 1 mM, respectively, and the preferred treatment time is 24 h.

[0071] This invention also provides an improved method for CAR-NK cell therapy, in which CAR-NK cells are treated in vitro with a combination of HKL and NR, and then the treated CAR-NK cells are delivered into the patient to improve the therapeutic effect on tumors.

[0072] The present invention will be described below through specific embodiments. It should be noted that the specific embodiments below are for illustrative purposes only and do not limit the scope of the present invention in any way. In addition, unless otherwise specified, methods that do not specifically describe conditions or steps are conventional methods, and the reagents and materials used can be obtained commercially.

[0073] Example 1: Impaired NK cell killing ability in bone marrow of AML relapsed patients

[0074] 1. Experimental Materials

[0075] Bone marrow NK cells from patients with acute myeloid leukemia (AML) were derived from bone marrow mononuclear cells (BMMCs), which were isolated from residual bone marrow samples of AML patients who underwent laboratory testing using the Ficoll density gradient method. These patients included those who relapsed early after allogeneic hematopoietic stem cell transplantation and those who did not relapse early; early relapse was 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: NK cells were purified using a magnetically activated cell sorter (MACS) kit (Miltenyi Biotec, Cat. #130-092-657). NK cell purity was >93% in each assay. All human samples used were approved by the Ethics Committee of the First Affiliated Hospital of the University of Science and Technology of China (2021-N(H)-120; Hefei, China), and written informed consent was obtained from all patients.

[0076] K562 cells were purchased from Shanghai Cell Bank (Chinese Academy of Sciences, Shanghai, China).

[0077] 2. Experimental Methods

[0078] Bone marrow NK cells were divided into those from relapsed AML patients and those from non-relapsed AML patients, and were co-cultured with K562 target cells separately according to the following steps:

[0079] Bone marrow NK cells (2×10) 6 (cells / mL) and K562 target cells (4 × 10⁻⁶) 5 (Numbers / mL) were inoculated into complete RPMI 1640 medium (Thermo Fisher Scientific, 11875119) and co-cultured at 37°C in a 5% CO2 incubator for 4 hours.

[0080] Following the antibody instructions, the co-cultured cells were labeled with the antibody by flow cytometry, and the NK cell killing function was detected by flow cytometry.

[0081] 3. Experimental Results

[0082] The flow cytometry results are shown in Figure 1. Annexin V was observed in relapsed AML patients compared to 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 (Fig. 1a and Fig. 1b). Furthermore, CD107a... +(Cat# 560664, RRID: AB_396135), Granzyme B + (Cat# 563389, RRID: AB_2738175) and IFN-γ + The proportion of NK cells (Cat# 506504, RRID: AB_315437) was significantly reduced in relapsed AML patients (Figures 1c and 1d). This indicates that the effector function of NK cells against tumors is impaired in relapsed AML patients.

[0083] Furthermore, referring to Figure 1, the expression levels of CD38, NKG2D (Cat# 562365, RRID: AB_11153309), and CD69 (Cat# 555531, RRID: AB_395916) on NK cells were significantly downregulated in relapsed AML patients compared to non-relapsed AML patients (Figure 1e and Figure 1f).

[0084] The results above indicate that NK cells in a hypoxic bone marrow microenvironment have suppressed function.

[0085] Example 2: Combination of small molecule drugs enhances the adaptability of BMNK cells to hypoxic environments and improves NK cell killing function.

[0086] In this embodiment, NK cells from patients with relapsed AML were treated with a combination of magnolol and nicotinamide nucleoside, 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 patients with relapsed AML.

[0087] 1. Experimental Materials

[0088] The NK cells and K562 cells used in AML relapse patients, as well as the flow cytometry antibody used, were the same as in Example 1.

[0089] And magnolol, HKL (Yuanye, Cat#B20498), nicotinamide nucleoside, NR (TargetMol, Cat#T13795).

[0090] 2. Experimental Methods

[0091] Bone marrow NK cells (2×10⁻⁶) from relapsed AML patients 6 (number / mL) were inoculated into complete RPMI 1640 medium (Thermo Fisher Scientific, 11875119) and treated in the following 3 groups:

[0092] (1) NR group used alone, NR dose is 1 mM;

[0093] (2) The group using HKL and NR in combination, with HKL dose of 100 μm and NR dose of 1 mM;

[0094] (3) Control group, without HKL and NR, i.e. bone marrow NK cells of patients with relapsed AML who did not receive any medication.

[0095] After drug administration, cells were cultured at 37°C and 5% CO2 for 24 hours to allow the drug to take effect. Then, cells from each experimental group (2 × 10⁻⁶ cells) were... 6 (cells / mL) and target cells (4×10) respectively 5 Cells (or primary AML blast cells or K562 cells) were seeded in complete RPMI 1640 medium (Thermo Fisher Scientific, 11875119) and co-cultured at 37°C in a 5% CO2 incubator for 4 hours. After co-culturing, the cells from each experimental group were labeled with antibodies by flow cytometry, and the NK cell killing function was detected by flow cytometry.

[0096] 3. Experimental Results

[0097] Please refer to Figure 2 for the flow cytometry results. It can be seen that after combined treatment with HKL and NR, Annexin V... + The proportions of primary AML cells and K562 cells were significantly increased (Fig. 2a and Fig. 2f). CD107a + Granzyme B + and IFN-γ + The proportion of BMNK cells increased significantly after combined treatment with HKL and NR (Fig. 2b-2d and Fig. 2g-2h); and the expression levels of CD38, NKG2D and CD160 (Cat# 341208, RRID: AB_ 2561435) in BMNK cells were significantly upregulated (Fig. 2e and Fig. 2i).

[0098] These results indicate that the effector function of BMNK cells from relapsed AML patients on primary AML blast cells and AML cell lines was significantly improved after combined treatment with NR and HKL. Specifically, in vitro treatment with combined NR and HKL restored the degranulation and cytokine secretion capabilities of NK cells isolated from these AML patients. Furthermore, this combined treatment significantly enhanced NK cell activation.

[0099] Example 3: Combination of small molecule drugs enhances the adaptability of NK92MI to hypoxic environments and improves NK cell killing function.

[0100] 1. Experimental Materials

[0101] NK92MI cells, validated by STR analysis, were obtained from Cellcook Biotechnology Co., Ltd. (Guangzhou, China). Cells were cultured in Alpha MEM 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 thiol, and 0.02 mM folic acid. Cells were stored in a humidified incubator at 37°C with 5% CO2.

[0102] Primary AML blast cells were collected from the bone marrow of newly diagnosed AML patients using the same method as in Example 1.

[0103] 2. Experimental Methods

[0104] NK92MI cells (2×10) 6 (Number of cells / mL) were inoculated into Alpha MEM medium and pretreated with the drug in the following 4 experimental groups:

[0105] (1) Hypoxia group: NK92MI cells were cultured in a hypoxia incubator at 37℃ and 5% O2 for 24h;

[0106] (2) Hypoxia + NR alone group, NR dose is 1 mM, NK92MI cells are cultured in a hypoxia incubator at 37℃ and 5% O2 for 24h;

[0107] (3) Hypoxia + HKL and NR combined use group, HKL dose is 100μm, NR dose is 1mM, NK92MI cells are cultured in a hypoxia incubator at 37℃ and 5% O2 for 24h.

[0108] (4) Control group: without HKL and NR and without hypoxia culture, NK92MI cells were cultured in Alpha MEM medium without drugs in an incubator at 37°C and 5% CO2 for 24 h.

[0109] After culturing, cells from each experimental group (2×10⁻⁶) were... 6 (cells / mL) and target cells (4×10) respectively 5 Cells (or primary AML blast cells or K562 cells) were seeded in Alpha MEM medium and co-cultured at 37°C in a 5% CO2 incubator for 4 hours. After co-culturing, the cells from each experimental group were labeled with antibodies by flow cytometry, and the NK cell killing function was detected by flow cytometry.

[0110] 3. Experimental Results

[0111] Please refer to Figure 3 for the flow cytometry results. It can be seen that, compared to the hypoxia group, NK cell cytotoxicity against primary AML blast cells and K562 cells was improved after treatment with NR alone and in combination with NR and HKL, while also increasing CD107a expression. + and IFN-γ + The proportion of NK cells increased (Fig. 3a and Fig. 3c). In addition, after combined treatment with NR and HKL, the expression levels of granzyme B, NKG2D and CD160 markers in NK cells were upregulated (Fig. 3b and Fig. 3d).

[0112] 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 environments.

[0113] Example 4: Combined use of nicotinamide nucleoside and magnolol and their combination index

[0114] 1. Experimental materials: Same as in Example 2.

[0115] 2. Experimental method: Same as Example 2.

[0116] 3. Combination Index Analysis: The combination index (CI) was calculated using CalcuSyn analysis software. The formula for the combination index is CI = D1 / DX1 + D2 / DX2, where D1 and D2 are the individual concentrations of the two drugs when used in combination, DX1 and DX2 are the drug concentrations required to achieve the target cell apoptosis rate of fa when the combination drug reaches fa, and fa represents the target cell apoptosis rate achieved by using the two drugs in combination at a certain concentration. By inputting information such as the dosage of the single drug, the target cell apoptosis rate of the drug when used alone, the specific ratio of the two drugs, and the target cell apoptosis rate when the drugs are used in combination, the software can calculate the combination index. CI < 1 indicates that the two drugs have a synergistic effect, while CI > 1 indicates that the two drugs have no synergistic effect.

[0117] 4. Experimental Results:

[0118] A combined treatment index assessment experiment was conducted on primary AML blast cells using nicotinamide nucleoside and magnolol. The results are shown in Table 1. The results indicate that the combined treatment of nicotinamide nucleoside and magnolol has a synergistic effect on apoptosis in primary AML blast cells (CI < 1). A combined treatment index assessment experiment was also conducted on K562 cells. The results are shown in Table 2. The results indicate that the combined treatment of nicotinamide nucleoside and magnolol has a synergistic effect on apoptosis in K562 cells (CI < 1).

[0119] Table 1 Assessment of the combined index of nicotinamide nucleoside and magnolol in primary AML blast cells

[0120] NR(mM)HKL(mM)CI0.50.050.3570.10.010.588

[0121] Table 2 Assessment of the combined index of nicotinamide nucleoside and magnolol in K562 cells

[0122] NR(mM)HKL(mM)CI0.50.050.2380.10.010.454

[0123] Example 5: The combined use of HKL and NR significantly inhibited the growth of leukemia cells in a mouse xenograft model of leukemia.

[0124] 1. Experimental Materials

[0125] 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 conducted in accordance with the National Guidelines for Animal Usage in Research and were approved by the Ethics Committee of the University of Science and Technology of China (USTCACUC2).

[0126] HL60 cells were purchased from Shanghai Cell Bank.

[0127] IL-2 (50000 U, Jiangsu Kingsley Pharmaceuticals).

[0128] NK cells were purchased from Miaoshun (Shanghai) Biotechnology Co., Ltd.

[0129] 2. Experimental method (as shown in Figure 4a)

[0130] a. Animal model construction

[0131] HL60 labeled with luciferase (2.5 × 10⁻⁶) 4 Leukemia cells (number per g) were intravenously injected into NCG mice, and tumor growth was monitored using bioluminescence imaging via the IVIS spectral imaging system (PerkinElmer) to confirm successful leukemia cell transplantation.

[0132] b. Treat NK cells, add NK cells (2×10⁻⁶) 6 (Number of cells / mL) were inoculated into complete RPMI 1640 medium (Thermo Fisher Scientific, 11875119) and treated in the following 3 experimental groups:

[0133] (1) Ctrl group, without HKL and NR treatment, i.e. NK cells were cultured in untreated complete RPMI 1640 medium for 24 h;

[0134] (2) In the NR group alone, NK cells were pretreated with 1 mM NR for 24 h;

[0135] (3) In the group using NR and HKL in combination, NK cells were treated with 1mM NR and 100µM HKL simultaneously for 24h.

[0136] All experimental groups were cultured in an incubator at 37℃ and 5% CO2 humidification.

[0137] c. On day 7 after tumor implantation in mice, NK cells (5.0 × 10⁻⁶) from each experimental group treated in step b were... 4 IL-2 (50,000 U / mouse) was adopted into mice (5 mice per group) to support the survival of NK cells in vivo. The mice were injected intraperitoneally with IL-2 every 2 days.

[0138] d. AML load was monitored using the IVIS spectral imaging system (PerkinElmer) via bioluminescence imaging at specified time points (7d, 14d, 28d, and 35d). Quantitative image data were analyzed using Living Image Software (PerkinElmer).

[0139] Throughout the experiment, the mice were fed a standard complete diet.

[0140] 3. Experimental Results

[0141] The results are shown in Figure 4. Compared with the control group, the growth of IL60 cells and tumors in mice treated with NR and HKL were significantly inhibited (Figures 4b and 4c).

[0142] These results indicate that, compared with the control group, mice receiving NK cells treated with NR and the combined treatment of NR and HKL exhibited significantly lower AML burden and longer survival. Importantly, compared with NK cells stimulated by NR alone, NK cells subjected to hypoxia treatment with the combined stimulation of NR and HKL showed a significant reduction in tumor burden and prolonged survival. These results clearly demonstrate that NK cells effectively restore the impaired antileukemic response through the synergistic effect of NR and HKL.

[0143] The results of the above examples demonstrate that combined treatment with HKL and NR enhances the adaptability of NK cells to hypoxic environments, thereby enhancing NK cell metabolism and killing function, and ultimately strengthening the killing of tumor cells by NK cells both in vivo and in vitro. Targeting NK cells under hypoxia promotes apoptosis of leukemia cells and inhibits leukemia progression. This indicates that targeting NK cell adaptation to hypoxic environments can enhance NK cell metabolism and killing ability, promoting the treatment of AML.

[0144] It should be noted that this article uses acute myeloid leukemia as an example, but it 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 thus be widely used in the treatment of various tumors. Due to space limitations, this application will not elaborate on each one in detail.

Claims

1. A small molecule drug combination composition for enhancing the metabolism and / or killing capacity of NK cells under hypoxic conditions, characterized in that, The small molecule drug combination composition comprises honokiol and nicotinamide riboside.

2. The small-molecule drug combination composition of claim 1, wherein The small molecule drug combination composition is a single complex preparation or a combination of two separate single preparations.

3. The small-molecule drug combination composition of claim 2, wherein The complex preparation comprises honokiol and nicotinamide riboside. The combination of the single preparations comprises honokiol and a single preparation containing nicotinamide riboside.

4. The small-molecule drug combination composition of claim 1, wherein In the small molecule drug combination composition, the concentration ratio of honokiol and nicotinamide riboside is 0.01-1:0.1-10.

5. A medicament, characterized by comprising a compound of the formula (I) or a pharmaceutically acceptable salt thereof. The small molecule drug combination composition comprises honokiol and nicotinamide riboside. The drug has any one or more of the following effects: a: enhancing the metabolic capacity and / or killing capacity of NK cells under hypoxic conditions; b: inhibiting the growth of tumor cells; c: promoting the apoptosis of tumor cells.

6. A method of enhancing the metabolism and / or killing capacity of NK cells under hypoxic conditions in vitro for non-therapeutic purposes, characterized in that, Preferably, the drug further comprises any pharmaceutically acceptable excipient and / or carrier. The method comprises the following steps:

7. The method of claim 6, wherein, The NK cells are treated in vitro using the small molecule drug combination composition according to any one of claims 1-4 or the drug according to claim 5.

8. An NK cell, characterized in that, The honokiol and nicotinamide riboside can be administered simultaneously, sequentially or at intervals. The NK cells are obtained by the method according to claim 6 or 7. Preferably, the NK cells are CAR-NK cells.

9. The use of any one or more of the following: a: honokiol and nicotinamide riboside in the preparation of a drug for enhancing the metabolic capacity and / or killing capacity of NK cells under hypoxic conditions; b: honokiol and nicotinamide riboside in the preparation of a drug for treating tumors; 10. The use according to claim 9, wherein the compound is ###0002### c: honokiol in the preparation of a drug for improving the effect of nicotinamide riboside on treating tumors. The concentration ratio of honokiol and nicotinamide riboside is 0.01-1:0.1-10.

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