CN118903175A - Application of small molecule drug combination in enhancing metabolism and killing ability of NK cells under hypoxia condition - Google Patents

Abstract

The present invention discloses an application 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 finds for the first time that the combination of honokiol and nicotinamide riboside has a synergistic effect on the regulation of 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

Application of small molecule drugs in combination to enhance the metabolism and killing ability of NK cells under hypoxic conditions

Technical Field

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

Natural killer cells (NK) are a type of innate immune cell that plays a vital role in tumor monitoring. Impaired antitumor 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 of NK cells in liver cancer tumors, thereby inhibiting their antitumor activity. Existing studies have shown that natural killer cells (NK) play a vital role in cancer immune surveillance, but there are currently no reports of targeting the hypoxic environment with small molecule drugs to enhance NK cell metabolism and strengthen its ability to effectively monitor tumors.

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 in vivo and in vitro anti-tumor activity.

Summary of the invention

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, enhances the metabolism and killing ability of NK cells under hypoxic conditions, and thereby improves its effect of inhibiting tumor cell growth and promoting tumor cell apoptosis, and improves its killing effect on tumor cells in vitro and in vivo, by combining two small molecule drugs, magnolia officinalis and magnolia officinalis and nicotinamide riboside, to provide a new strategy for the treatment of tumors.

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

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 honokiol and nicotinamide riboside.

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

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

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

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).

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

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

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

b: Inhibit the growth of tumor cells;

c: Promote apoptosis of tumor cells.

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

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

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

In a further embodiment, the honokiol and nicotinamide riboside can be administered simultaneously, sequentially or at intervals.

The present invention further provides a NK cell obtained by processing using the method described above.

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

The present invention further provides any one or more of the following applications, wherein the application comprises:

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;

b: Application of honokiol and nicotinamide riboside in combination in the preparation of a drug for treating tumors;

c: Application of honokiol in the preparation of drugs for improving the therapeutic effect of nicotinamide riboside on tumors.

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

Beneficial effects of the present invention:

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.

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

Figure 1 is the flow cytometry experimental results of the impaired killing ability of NK cells in the bone marrow of AML patients in Example 1, wherein Figure a is a flow cytometry analysis of the percentage of Annexin V ⁺ K562 cells; Figure b is a statistical graph of the percentage of Annexin V ⁺ K562 cells; Figure c is a flow cytometry analysis of the percentage of Granzyme B ⁺ NK cells, CD107a ⁺ NK cells, and IFN-γ ⁺ NK cells in the total NK cells purified from the bone marrow of AML relapsed and non-relapsed patients; Figure d is a statistical graph of the percentage of Granzyme B ⁺ NK cells, CD107a ⁺ NK cells, and IFN-γ ⁺ 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 of the mean fluorescence intensity MFI of NKG2D, CD38 and CD69 on NK cells isolated and purified from the bone marrow of AML patients.

Figure 2 is the flow cytometry results of the killing function of bone marrow NK (BMNK) cells after in vitro treatment in Example 2, wherein, Figure a is a flow cytometry graph and a statistical graph of the percentage of Annexin V ⁺ primary AML blasts; Figure b is a flow cytometry graph and a statistical graph of the percentage of Granzyme B ⁺ NK cells in the total BMNK cells of AML relapse patients; Figure c is a flow cytometry graph and a statistical graph of the percentage of CD107a ⁺ NK cells in the total BMNK cells of AML relapse patients; Figure d is a flow cytometry graph and a statistical graph of the percentage of IFN-γ ⁺ NK in the total BMNK cells of AML relapse patients; Figure e is a flow cytometry graph of the expression of NKG2D, CD38 and CD160 on BMNK cells of AML relapse patients and a statistical graph of the mean fluorescence intensity MFI; Figure f is a flow cytometry graph and a statistical graph of the percentage of Annexin V ⁺ K562 cells; Figure g is a flow cytometry graph and a statistical graph of the expression of Granzyme B on BMNK cells of AML relapse patients and a statistical graph of the mean fluorescence intensity MFI; Figure h is a flow cytometry graph of the expression of CD107a ⁺ Figure i is a flow cytometry analysis graph and statistical graph of the percentage of NK cells and IFN-γ ⁺ NK cells in the total BMNK cells of AML relapse patients; Figure i is a flow cytometry analysis graph 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 3 is the flow cytometry experimental results of the killing function of NK92MI cells after in vitro treatment in Example 3, wherein Figure a is a flow cytometry analysis graph and a statistical graph of the percentage of Annexin V ⁺ primary AML mother cells; a flow cytometry analysis graph and a statistical graph of the percentage of CD107a ⁺ NK cells and IFN-γ ⁺ NK cells in total NK92MI cells; Figure b is a flow cytometry analysis graph and a statistical graph of the expression of Granzyme B, NKG2D and CD160 on NK92MI cells and a statistical graph of the mean fluorescence intensity MFI; Figure c is a flow cytometry analysis graph and a statistical graph of the percentage of Annexin V ⁺ K562 mother cells; a flow cytometry analysis graph and a statistical graph of the percentage of CD107a ⁺ NK cells and IFN-γ ⁺ NK cells in total NK92MI cells; Figure d is a flow cytometry analysis graph and a statistical graph of the expression of Granzyme B, NKG2D and CD160 on NK92MI cells and a statistical graph of the mean fluorescence intensity MFI.

Figure 4 shows 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

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 should not be construed as limiting the present invention.

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.

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

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

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

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.

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).

In the present invention, Honokiol (HKL) is a bioactive bisphenol phytochemical that can target a variety of signaling molecules and has effective antioxidant, anti-inflammatory, anti-angiogenic and anti-cancer activities. HKL has been shown to inhibit the growth of glioblastoma (GBM) cells and induce apoptosis, inhibiting the growth of breast cancer cells. HKL can effectively inhibit the progression of HCC in in vitro and in vivo models by clearing the effects of hypoxia and lactic acid on HCC cell cycle proteins.

In the present invention, Nicotinamide Riboside (NR) is an endogenous molecule, which is a precursor of nicotinamide adenine dinucleotide (NAD+), has solubility and oral bioavailability, and can increase the level of NAD+ in the body. NAD+ plays an important role in cell metabolism, energy production, DNA repair and gene expression. Studies have shown that nicotinamide riboside may help improve mitochondrial health, stimulate mitochondrial function and induce the generation of new mitochondria, and can also enhance the mitochondrial function of stem cells.

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.

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%.

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

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 both magnolol and nicotinamide riboside.

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

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

The second aspect of the present invention provides a drug, which contains the small molecule drug combination composition described above.

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

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

b: Inhibit the growth of tumor cells;

c: Promote apoptosis of tumor cells.

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 then can inhibit the growth of tumor cells, promote the apoptosis of tumor cells, and ultimately exert the effect of treating tumors.

In the present invention, the tumor includes various types of malignant tumors and benign tumors, wherein the malignant tumor includes but is 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 tumor includes but is not limited to breast fibroma, cyst, lipoma, gallbladder polyp, nodule, etc.

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

In some specific implementation cases of the present invention, the pharmaceutically acceptable auxiliary materials and/or carriers include but are not limited to at least one of diluents, binders, surfactants, adsorption carriers, lubricants, fillers, and disintegrants.

Among them, in some specific implementation cases of the present invention, for example, the diluent may be lactose, sodium chloride, glucose, urea, starch, water, etc., but not limited thereto. For example, the binder may be starch, pregelatinized starch, dextrin, maltodextrin, sucrose, gum arabic, gelatin, methyl cellulose, carboxymethyl cellulose, ethyl cellulose, polyvinyl alcohol, polyethylene glycol, polyvinyl pyrrolidone, alginic acid and alginate, xanthan gum, hydroxypropyl cellulose, hydroxypropyl methyl cellulose, etc., but not limited thereto. For example, the surfactant may be: polyoxyethylene sorbitan fatty acid ester, sodium lauryl sulfate, stearic acid monoglyceride, hexadecanol, etc., but not limited thereto. For example, the adsorption carrier may be starch, lactose, bentonite, silica gel, kaolin, bentonite, etc. For lubricant, for example, zinc stearate, glyceryl monostearate, polyethylene glycol, talcum powder, 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 dodecyl sulfate, etc., but are not limited to this. For filler, for example, mannitol, xylitol, sorbitol, maltose, erythrose, microcrystalline cellulose, polymerized sugar, coupling sugar, glucose, lactose, sucrose, dextrin, starch, sodium alginate, laminarin powder, agar powder, calcium carbonate, sodium bicarbonate, etc., but are not limited to this. For disintegrant, for example, cross-linked vinyl pyrrolidone, sodium carboxymethyl starch, low-substituted hydroxypropyl methyl, cross-linked sodium carboxymethyl cellulose, soybean polysaccharide, etc.

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.

The selection of specific excipients and/or carriers can be carried out according to the dosage form of the drug. When specifically selected, it 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. The term "conducive to administration" as described herein refers to being able to improve the therapeutic effect or improve bioavailability or reduce toxic side effects or improve the adaptability of patients, etc.

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.

Furthermore, the method comprises the following steps:

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

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

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

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.

When the NK cells are CAR-NK cells, an improved CAR-NK cell treatment method can be provided to provide a new treatment plan for cancer patients, that is, before the CAR-NK cells are transmitted into the body of the tumor patient, the CAR-NK cells are pre-treated with the small molecule drug combination composition described above or the drug described above, and then transmitted into the body of the tumor patient to improve the treatment effect of the tumor.

Furthermore, in the present invention, the honokiol and nicotinamide riboside can be administered simultaneously, sequentially or at intervals.

The present invention further provides any one or more of the following applications, wherein the application comprises:

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;

b: Application of honokiol and nicotinamide riboside in combination in the preparation of a drug for treating tumors;

c: Application of honokiol in the preparation of drugs for improving the therapeutic effect of nicotinamide riboside on tumors.

In the present invention, it is verified by 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 implementation cases of the present invention, the concentrations of honokiol and nicotinamide riboside are 0.1mM and 1mM, respectively, and the preferred treatment time is 24h.

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.

The present invention is described below by means of specific examples. It should be noted that the following specific examples are only for illustrative purposes 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 can be obtained from commercial channels.

Example 1 Impaired killing ability of bone marrow NK cells in patients with relapsed AML

1. Experimental Materials

Bone marrow NK cells from patients with acute myeloid leukemia (AML) were derived from bone marrow mononuclear cells (BMMCs), which were isolated by Ficoll density gradient from residual bone marrow samples of AML patients who underwent laboratory testing. These patients included patients with early relapse after allogeneic hematopoietic stem cell transplantation and patients without early relapse, with early relapse defined as relapse within 6 months after complete remission after allogeneic transplantation. NK cells used in in vivo experiments were purified from healthy donor blood: NK cells were purified by magnetic activated cell sorter (MACS) kit (Miltenyi Biotec, Cat. #130-092-657). The purity of NK cells was >93% in each test. All human samples used were approved by the ethics committee (2021-N(H)-120; Hefei, China), and written informed consent was obtained from all patients.

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

2. Experimental methods

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:

Bone marrow NK cells (2×10 ⁶ cells/mL) and K562 target cells (4×10 ⁵ cells/mL) were inoculated in complete RPMI 1640 medium (Thermo Fisher Scientific, 11875119) and co-cultured in a 37°C, 5% CO ₂ incubator for 4 h.

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.

3. Experimental results

The flow cytometry results are shown in Figure 1. Compared with non-relapsed AML patients, the percentage of Annexin V ⁺ (7-AAD, Cat#559925, RRID: AB_2869266; APC-Annexin V, Cat#550474, RRID: AB_2868885) K562 cells in relapsed AML patients was significantly reduced (Figure 1a and Figure 1b). In addition, the proportion of CD107a ⁺ (Cat#560664, RRID: AB_396135), Granzyme B ⁺ (Cat#563389, RRID: AB_2738175) and IFN-γ ⁺ (Cat#506504, RRID: AB_315437) NK cells in relapsed AML patients showed a significant decrease (Figure 1c and Figure 1d). This shows that the effector function of NK cells on tumors is impaired in relapsed AML patients.

Further, please continue to 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 1e and Figure 1f).

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

Example 2 Small molecule drugs combined to enhance the adaptability of BMNK cells to hypoxic environment and improve the killing function of NK cells

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 the activation of NK cells and reverse the damage to the anti-leukemia function of NK cells in relapsed AML patients.

1. Experimental Materials

The NK cells and K562 cells of the AML relapsed patient and the flow cytometry antibodies used are the same as those in Example 1.

Honokiol, HKL (Yuanye, Cat# B20498).

Nicotinamide riboside, NR (TargetMol, Cat# T13795).

2. Experimental methods

Bone marrow NK cells (2×10 ⁶ cells/mL) from relapsed AML patients were inoculated in complete RPMI1640 medium (Thermo Fisher Scientific, 11875119) and divided into the following three groups for drug treatment:

(1) NR alone group, NR dose was 1 mM;

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

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

After drug addition, the cells were cultured at 37°C and 5% CO ₂ to allow the drug to act for 24 hours. Then, the cells in each experimental group (2×10 ⁶ cells/mL) were inoculated with target cells (4×10 ⁵ cells/mL, primary AML blasts or K562 cells) in complete RPMI 1640 medium (Thermo Fisher Scientific, 11875119) and co-cultured in a 37°C, 5% CO ₂ incubator for 4 hours. After the cells in each experimental group were co-cultured, flow cytometry was used to detect the killing function of NK cells.

3. Experimental results

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

The above results indicate 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, in vitro treatment with a combination of NR and HKL restored the degranulation and cytokine secretion capacity of NK cells isolated from these AML patients. In addition, this combined treatment significantly enhanced NK cell activation.

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

1. Experimental Materials

NK92MI cells verified by STR analysis were obtained from Cellcook Biotechnology (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 and 5% _CO2 .

Primary AML blasts were collected from the bone marrow of patients with primary AML in the same manner as in Example 1.

2. Experimental methods

NK92MI cells (2×10 ⁶ cells/mL) were inoculated in Alpha MEM medium and divided into the following 4 experimental groups for drug pretreatment:

(1) Hypoxia group: NK92MI cells were cultured in a hypoxic incubator at 37°C and 5% O ₂ for 24 h;

(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% O ₂ for 24 h;

(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% O ₂ for 24 h;

(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.

After culture, the cells in each experimental group (2×10 ⁶ cells/mL) were inoculated with target cells (4×10 ⁵ cells/mL, primary AML blasts or K562 cells) in Alpha MEM medium and co-cultured for 4 hours in a 37°C, 5% CO ₂ incubator. After the co-cultured cells in each experimental group were labeled with flow cytometry antibodies, the NK cell killing function was detected by flow cytometry.

3. Experimental results

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

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

Example 4 Combination of Nicotinamide Riboside and Honokiol and Combination Index

1. Experimental materials: Same as Example 2.

2. Experimental method: Same as Example 2.

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

4. Experimental results:

The combined drug index evaluation experiment of nicotinamide riboside and honokiol was conducted on primary AML blast cells. The experimental results are shown in Table 1. The results show that the combined treatment of nicotinamide riboside and honokiol has a synergistic effect on the apoptosis of primary AML blast cells (CI < 1). The combined drug index evaluation experiment was conducted on K562 cells. The experimental results are shown in Table 2. The results show that the combined treatment of nicotinamide riboside and honokiol has a synergistic effect on the apoptosis of K562 cells (CI < 1).

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

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

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

1. Experimental Materials

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 (USTCACUC2) Ethics Committee.

HL60 cells were purchased from Shanghai Cell Bank.

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

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

2. Experimental methods (Figure 4a)

a. Animal model construction

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

b. Treatment of NK cells: NK cells (2×10 ⁶ cells/mL) were inoculated in complete RPMI1640 medium (ThermoFisher Scientific, 11875119) and divided into the following three experimental groups for treatment:

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

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

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

All the above experimental groups were cultured in a 37°C, 5% CO ₂ humidified incubator.

c. On the 7th day after mice were loaded with tumors, the NK cells (5.0×10 ⁴ cells/g) treated in each experimental group in step b were adoptively transferred into mice (5 mice in each group). In order to support the survival of NK cells in vivo, IL-2 (50,000 U/mouse) was injected intraperitoneally into the mice once every 2 days.

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

During the entire experiment, the mice were fed with standard full nutrition feeding conditions.

3. Experimental results

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

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, hypoxia-treated NK cells stimulated with NR and HKL showed a significant reduction in tumor burden and prolonged survival compared with NK cells stimulated with NR alone. These results clearly indicate that NK cells effectively restored the impaired anti-leukemia response through the synergistic action of NR and HKL.

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 the metabolism and killing function of NK cells, and further enhancing the killing of tumor cells by NK cells in vivo and in vitro. By targeting NK cells with hypoxia, apoptosis of leukemia cells is promoted and the progression of leukemia is inhibited. It shows that the adaptation of targeted NK cells to hypoxic environments can enhance the metabolism and killing ability of NK cells and promote the treatment of AML.

It should be noted that acute myeloid leukemia is used as an example in this article, 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 be widely used in the treatment of various tumors. Due to limited space, this application will not elaborate on them one by one.

The technical features of the above-described embodiments may be arbitrarily combined. To make the description concise, not all possible combinations of the technical features in the above-described 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.

The above-mentioned embodiments only express several implementation methods of the present invention, and the descriptions thereof are relatively specific and detailed, but they cannot be understood as limiting the scope of the invention patent. It should be pointed out that, for ordinary technicians in this field, several variations and improvements can be made without departing from the concept of the present invention, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be subject to the attached claims.

Claims (10)

1. A small molecule pharmaceutical combination composition for enhancing the metabolic and/or killing capacity of NK cells under hypoxic conditions, comprising honokiol and nicotinamide riboside.

2. The small molecule pharmaceutical combination composition of claim 1, wherein the small molecule pharmaceutical combination composition is a single compound formulation or a combination of two separate single formulations.

3. The small molecule drug combination composition of claim 2, wherein the compound preparation is a compound preparation containing honokiol and nicotinamide riboside;

the combination of the single preparation is the combination of the single preparation containing honokiol and nicotinamide riboside.

4. The small molecule pharmaceutical combination composition of claim 1, wherein the concentration ratio of honokiol to nicotinamide riboside in the small molecule pharmaceutical combination composition is (0.01-1 mM): (0.1-10 mM).

5. A medicament comprising the small molecule pharmaceutical combination composition of any one of claims 1-4, which has any one or more of the following efficacy:

a: enhancing the metabolic and/or killing capacity of NK cells under hypoxic conditions;

b: inhibiting the growth of tumor cells;

c: promoting apoptosis of tumor cells;

preferably, the medicament further comprises pharmaceutically acceptable auxiliary materials and/or carriers.

6. A method for enhancing the metabolic and/or killing capacity of NK cells under hypoxic conditions for non-therapeutic purposes in vitro, comprising the steps of:

NK cells are treated in vitro with a small molecule drug combination composition according to any one of claims 1-4 or a drug according to claim 5.

7. The method of claim 6, wherein honokiol and nicotinamide riboside are administered simultaneously, sequentially or at intervals.

8. An NK cell treated by the method of claim 6 or 7;

preferably, the NK cells are CAR-NK cells.

9. An application as claimed in any one or more of the following, the application comprising:

a: use of a combination of honokiol and nicotinamide riboside in the manufacture of a medicament for enhancing the metabolic and/or killing capacity of NK cells under hypoxic conditions;

b: use of a combination of honokiol and nicotinamide riboside in the manufacture of a medicament for the treatment of a tumor;

c: application of honokiol in preparing medicine for improving tumor treating effect of nicotinamide riboside is provided.

10. The use according to claim 9, wherein the concentration ratio of honokiol to nicotinamide riboside is (0.01-1 mM): (0.1-10 mM).