Use of vitamin b6 for the manufacture of a medicament for the treatment of leukemia

Abstract

This invention belongs to the field of biomedicine and relates to a novel use of vitamin B6. Specifically, it describes the use of vitamin B6 in the preparation of drugs for treating leukemia. This invention demonstrates that vitamin B6 can selectively induce leukemia cell death, induce the death of primary tumor cells in leukemia patients, and delay the death of leukemia model mice, fully illustrating the potential use of vitamin B6 in the preparation of drugs for treating leukemia.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine and relates to new uses of vitamin B6, particularly the use of vitamin B6 in the preparation of drugs for treating leukemia. Background Technology

[0002] Leukemia is a type of malignant clonal disease of hematopoietic stem cells. Clonal leukemia cells proliferate and accumulate in large numbers in the bone marrow and other hematopoietic tissues due to mechanisms such as uncontrolled proliferation, impaired differentiation, and inhibited apoptosis, and infiltrate other non-hematopoietic tissues and organs while suppressing normal hematopoietic function. Based on the degree of differentiation and the length of the natural course of the disease, leukemia can be divided into acute and chronic leukemia. Chemotherapy is a major treatment method for leukemia.

[0003] Because leukemia cells develop resistance to chemotherapy drugs, most patients eventually relapse, leading to low survival rates, especially in elderly patients and those with high-risk genetic traits (Marcucci et al., 2011). Secondly, due to the non-specific cytotoxicity of chemotherapy drugs, while killing leukemia cells, they indiscriminately damage normal hematopoietic stem cells and tissue cells, resulting in significant treatment toxicity. Therefore, efforts are being made to find natural small molecules with lower toxicity for selective targeted therapy of leukemia (Siveen KS et al., 2017).

[0004] Vitamins are a class of trace organic substances that play important roles in human growth, metabolism, and development. Many vitamins are known to be coenzymes or components of enzymes. Currently, numerous studies have shown that vitamin C (Cameronet et al., 1979; Fritz et al., 2014; Doskey et al., 2016; Bonucelli et al., 2017; Marianna et al., 2017; Cimmino et al., 2017; Ngo et al., 2019) and vitamin D (Ng et al., 2019) play important roles in the treatment and prognosis of tumors, including hematological malignancies (Fritz et al., 2014; Cimmino et al., 2017). These results highlight the significant potential of vitamins in cancer treatment. Vitamin B6, also known as pyridoxine, comprises pyridoxine, pyridoxal, and pyridoxamine. It exists in the body as a phosphate ester and is a water-soluble vitamin, easily destroyed by light or alkali, and intolerant of high temperatures. Vitamin B6 is a colorless crystal, readily soluble in water and ethanol, stable in acidic solutions, but easily destroyed in alkaline solutions. Pyridoxine is heat-resistant, while pyridoxal and pyridoxamine are not. Vitamin B6 is abundant in yeast, liver, grains, meat, fish, eggs, beans, and peanuts. Vitamin B6 is a component of certain coenzymes in the body and participates in various metabolic reactions, especially amino acid metabolism. Clinically, vitamin B6 preparations are used to prevent and treat vomiting during pregnancy and radiation sickness. Unlike the pharmacological side effects of vitamin D, vitamin B6 is a water-soluble vitamin and is excreted within 8 hours after digestion. Vitamin B6 produces almost no toxicity when kidney function is normal. Currently, there are no reports of vitamin B6 being used in the treatment of leukemia. Summary of the Invention

[0005] The purpose of this invention is to provide a new use for vitamin B6, specifically the use of vitamin B6 in the preparation of drugs for treating leukemia.

[0006] A further feature of the use described in this invention is that the medicament comprises an effective therapeutic dose of vitamin B6 and a pharmaceutically acceptable carrier.

[0007] At the cellular level, experimental results from this invention show that vitamin B6 selectively kills human and murine leukemia cells THP-1, U937, RAW264.7, Raji, HEL92.1.7, and K562, but has no effect on MEF, MS-1, HCT-8, HCT-116, and HEK293T cells at the same dose.

[0008] In mouse experiments, this invention established a THP-1 acute myeloid leukemia model, and 320 mg / kg of vitamin B6 significantly reduced the tumor burden in mice.

[0009] In a primary leukemia cell experiment, this invention stimulated primary tumor cells from 15 leukemia patients with vitamin B6 and found that all of these primary tumor cells died to some extent. However, at the same dose, it had no effect on peripheral blood mononuclear cells from healthy donors.

[0010] In conclusion, vitamin B6 can selectively induce leukemia cell death, induce the death of primary tumor cells in leukemia patients, and delay the death of leukemia model mice, which fully demonstrates that vitamin B6 may be used to prepare drugs for the treatment of leukemia.

[0011] Currently, due to the high toxicity of chemotherapy and the development of drug resistance in leukemia cells, most patients eventually relapse, resulting in low survival rates. Therefore, the novel use of vitamin B6 provided by this invention has the following beneficial effects: Vitamin B6 is a natural small molecule substance with low toxicity, which can be selectively targeted or used as an adjunct therapy for leukemia. Attached Figure Description

[0012] Figure 1a and Figure 1b This is a graph showing the experimental results of vitamin B6 selectively killing human and murine leukemia cells (THP-1, U937, RAW264.7, Raji, HEL92.1.7, K562). Figure 1a The study showed that vitamin B6 induces leukemia cell death in a dose-dependent manner. Figure 1b The results showed that vitamin B6 had no effect on the activity of MS-1, HCT-8, HEK293T, MEF, and HCT116.

[0013] Figure 2a and Figure 2b This is a diagram showing the experimental results of vitamin B6 delaying the progression of THP-1 cell leukemia in mice and prolonging their survival time. Figure 2a The results showed that vitamin B6 treatment reduced tumor burden in model mice. Figure 2b The results showed that vitamin B6 prolonged the survival time of the model mice.

[0014] Figures 3a to 3c This is a diagram showing the experimental results of vitamin B6 inducing the death of primary tumor cells from leukemia patients. Figure 3a The figure shows that vitamin B6 induces death in primary leukemia cells. Figure 3b show Figure 3a Quantitative statistical results of Annexin V positive staining in the middle Figure 3cThe results showed that vitamin B6 had little effect on peripheral blood mononuclear cells from healthy donors. Detailed Implementation

[0015] The present invention will now be described in detail with reference to specific embodiments and the accompanying drawings.

[0016] Example 1: Cell Experiment

[0017] In this embodiment, THP-1, U937, RAW264.7, Raji, HEL92.1.7, K562, MS-1, HCT-8, HEK293T, MEF, and HCT116 cells were stimulated with different doses of vitamin B6 at the cell line level. After 24 hours, the total ATP level of each cell was measured.

[0018] THP-1: From ATCC cell bank. U937: From ATCC cell bank. RAW264.7: From ATCC cell bank. MS-1: From ATCC cell bank. HCT-8: From ATCC cell bank. HEK293T: From ATCC cell bank. MEF: From ATCC cell bank. HCT116: From ATCC cell bank. Raji: From ATCC cell bank. HEL92.1.7: From ATCC cell bank. K562: From ATCC cell bank. Vitamin B6: Purchased from MedChemExpress, catalog number (HY-B1328).

[0019] The dosages used, from low to high, were 0, 0.1, 0.2, 0.4, 0.8, and 1.6 mg / ml. The experimental procedure was as follows: cultured cells were grouped according to the same cell number and stimulated with different doses of vitamin B6 (0, 0.1, 0.2, 0.4, 0.8, and 1.6 mg / ml). The group with a dose of 0 mg / ml served as the control group. Twenty-four hours after stimulation, the ATP content of the cells was measured using an enhanced ATP assay kit (purchased from Beyotime Biotechnology Co., Ltd., catalog number S0027) to reflect cell viability.

[0020] Data Analysis: After obtaining the ATP content of cells in each group, the survival rate of cells in each group was calculated compared with the control group. Then, an S-shaped dose-dependent curve was fitted with the log value of vitamin B6 dose on the x-axis and the percentage of cell survival on the y-axis (see [link to data]). Figure 1a and Figure 1b ).

[0021] Experimental results are as follows Figure 1a and Figure 1bAs shown, it was surprisingly found that vitamin B6 reduced the survival rate of THP-1, U937, RAW264.7, Raji, HEL92.1.7 and K562 cells in a dose-dependent manner, but had no effect on MS-1, HCT-8, HEK293T, MEF and HCT116 cells.

[0022] Example 2: In vivo experiments in mice

[0023] In this embodiment, THP-1-luciferase cells were injected into severely immunodeficient mice (B-NDG mice) via tail vein to establish an acute leukemia model. The mice were then treated with 320 mg / kg vitamin B6 every other day for 3 weeks. The mice's weight was monitored daily, and bioluminescence was captured by a small animal imaging device at weeks 2, 3, and 4 after treatment. The higher the number of photons in the bioluminescence, the greater the tumor burden.

[0024] Severe immunodeficiency mice (B-NDG mice) were purchased from Beijing Biocytogen Gene Biotechnology Co., Ltd. These mice are mice with the IL2rg gene knocked out in the NOD-scid genetic background. They lack mature T, B and NK cells and are currently recognized internationally as having a high degree of immunodeficiency and are very suitable for the transplantation and growth of human hematopoietic stem cells and peripheral blood mononuclear cells.

[0025] Vitamin B6 was purchased from MedChemExpress, product number (HY-B1328).

[0026] The experimental results are shown in Figure 2. Surprisingly, vitamin B6 significantly reduced bioluminescence in mice and prolonged their survival time, indicating that vitamin B6 delayed the progression of THP-1 cell leukemia in mice and improved the survival of the model mice.

[0027] Example 3: Primary Leukemia Cell Experiment

[0028] This embodiment uses mononuclear cells isolated from the peripheral blood of patients diagnosed with acute leukemia and healthy donors for experiments.

[0029] After stimulating isolated mononuclear cells with vitamin B6, tumor cell viability was detected by flow cytometry after staining with Annexin V-PI apoptosis detection kit (Shanghai Bebo Biotechnology Co., Ltd.).

[0030] The experimental results are shown in Figure 3. Surprisingly, vitamin B6 induced the death of primary tumor cells, while at the same dose, it had little effect on peripheral blood mononuclear cells from healthy donors.

[0031] In summary, this invention demonstrates, both at the cellular and animal model levels, that vitamin B6 effectively kills leukemia cells, significantly delays death in leukemia model mice, improves their survival status, kills primary tumor cells, and has minimal effect on normal cells. This indicates that vitamin B6 has the ability to target and treat leukemia, a discovery that is unprecedented.

[0032] Based on the experimental results of this invention, a treatment drug with fewer side effects can be provided for some patients with refractory acute leukemia or patients who are intolerant to chemotherapy drugs.

[0033] Vitamin B6 is a natural small molecule substance that is metabolized in the liver and excreted through the kidneys, exhibiting low hepatotoxicity and nephrotoxicity. Current clinical data indicates that high doses of vitamin B6 do not have significant toxic side effects on the human body. Furthermore, vitamin B6 is inexpensive, reducing the financial burden on patients, demonstrating its great potential for application in the treatment of leukemia.

[0034] The above descriptions are merely preferred embodiments of the present invention and are not intended to limit the present invention in any way. The present invention is not limited to the above embodiments, and any modifications, alterations, improvements, and variations made by those skilled in the art to the above embodiments without departing from the spirit and concept of the present invention should be considered to fall within the protection scope of the present invention.

Claims

1. Use of pyridoxine as the sole active ingredient for the preparation of a medicament for the treatment of acute myeloid leukemia, characterized in that: The effective therapeutic dose of said pyridoxine is 320 mg / kg; The medicament comprises an effective therapeutic dose of pyridoxine, and a pharmaceutically acceptable carrier.

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