CN114762696A

CN114762696A - Use of deuterium-depleted water and related products

Info

Publication number: CN114762696A
Application number: CN202110037564.2A
Authority: CN
Inventors: 姜标; 杨异凤; 张缘园; 路文文
Original assignee: ShanghaiTech University
Current assignee: ShanghaiTech University
Priority date: 2021-01-12
Filing date: 2021-01-12
Publication date: 2022-07-19
Anticipated expiration: 2041-01-12
Also published as: CN114762696B

Abstract

The invention belongs to the field of biomedical research, and particularly relates to application of deuterium-depleted water in any one or more aspects of the following aspects: preparing a biological enzyme regulating agent; preparing a cell glycolysis inhibitor; preparing a tumor treatment product; preparing a tumor proliferation inhibitor; preparing a tumor cell clone inhibitor; preparing angiogenesis promoting ability inhibitor of tumor cells; preparing a tumor growth inhibitor; preparing an antitumor drug synergist; preparing a synergist of the enzyme inhibitor; preparing a cardiovascular drug synergist; preparing a drug synergist for the basic metabolic diseases; the deuterium depleted water has a deuterium content higher than 0.1ppm and lower than 135 ppm. The product of the invention regulates and controls the expression and enzyme activity of key biological enzymes in cells of a living body, changes the metabolic pathway and metabolic level of the biological enzymes, thereby inhibiting the proliferation of tumor cells, limiting the development of cardiovascular and respiratory disease focuses, delaying and controlling the progress of other basic metabolic diseases, and can be applied to the tumor inhibition related to the cell metabolic pathway.

Description

Use of deuterium-depleted water and related products

Technical Field

The invention belongs to the field of biomedical research, and particularly relates to application of deuterium-depleted water and a related product.

Background

Water is the source of life and plays a fundamental role in the life metabolic process of organisms. In 1934 scientists in the United states of America particularly used spectroscopic methods to find deuterium (D, stable isotope of hydrogen), so that water in nature was essentially protium water (H)₂O, customarily known as "light water") and deuterium water (D)₂O, HDO, customarily known as "heavy", "semi-heavy") and very little tritium water (T)₂O) and mixtures of several isotopic compounds. In ordinary water, protium water is the most, while deuterium content is about 150ppm, and tritium water is negligible. As shown in Table 1, heavy water (D) was found by separation measurement₂O), semi-heavy water (HDO), light water (H)₂O) the three isotopic compounds are significantly different from each other in many physicochemical properties, such as deuterium oxide (D)₂O) viscosity ratio H₂O is about 20 percent higher, and the pH value D at normal temperature₂O is 7.43 and H₂O is 7.0. Accordingly, heavy water (D)₂O) lower ionization capacity, and the deuterium ion (D +) compound has stronger chemical bond, and the compound formed by it is more stable, and its chemical activity will be lower than that of the same configuration protium (H +) compound. Initially, the scientific community began to research deuterium as a raw material of hydrogen bombs and atomic bombs, and gradually expanded to the fields of life sciences and medical health as the research goes deep, and in particular, the research on the application of Deuterium Depleted Water (DDW) became a current hotspot.

Since various kinds of amino acids constituting a protein need to be bound by means of hydrogen bonds (protium or deuterium bonds), and water, which simultaneously provides the hydrogen bonds, is a basic condition for all biochemical reactions of a living body, the hydrogen bonds play a very important role in the structure and function of the protein. Although the proportion of deuterium (D) in ordinary water is not high, it has been found that deuterium (D) can play a significant role in biological metabolism, and the strength of this role is closely related to the content or concentration of deuterium (D) in water. Experiments show that deuterium in biological body fluid can inhibit or stimulate metabolic processes of a living system, and influences the growth speed of cells, the activity of enzyme and the influence of cell energetics. Experiments show that under high deuterium concentration, the population doubling time of human adipose-derived stem cells (ADSCs) is prolonged, which indicates that the cell cycle is slowed down and the cell proliferation rate is reduced. Deuterated and deuterium depleted media exhibit acute and chronic cytotoxicity, respectively. Migration is minimal in deuterated media and higher in media with deuterium content close to natural. After 3 days of culture in deuterated media, the metabolic activity of the cells decreased. In contrast, human adipose stem cells (ADSCs) metabolic activity was increased in deuterium depleted medium. Also, the data indicate that Deuterium Depleted Water (DDW) has antidotal properties that accelerate metabolism. It follows that deuterium concentrations in biological fluids have a regulatory effect on cellular metabolic pathways and levels. However, it is not clear what mechanism deuterium-depleted water specifically affects the metabolic pathways and levels of cells in the previous studies, and the application of deuterium-depleted water has been in an empirical level and has not been applied effectively in the related biomedical field in a large scale.

Disclosure of Invention

In order to overcome the problems in the prior art, the invention aims to provide the application of the deuterium-depleted water and related products.

In order to achieve the above objects and other related objects, the present invention adopts the following technical solutions:

in a first aspect of the invention, there is provided the use of deuterium-depleted water in any one or more of the following:

preparing a biological enzyme regulator;

preparing a cell glycolysis inhibitor;

preparing a tumor treatment product;

preparing a tumor proliferation inhibitor;

preparing a tumor cell clone inhibitor;

preparing an angiogenesis promoting ability inhibitor of tumor cells;

preparing a tumor growth inhibitor;

preparing an antitumor drug synergist;

preparing a synergist of the enzyme inhibitor;

preparing a cardiovascular drug synergist;

preparing a drug synergist for the basic metabolic diseases;

the deuterium content of the deuterium water is higher than 0.1ppm and lower than 135 ppm.

In a second aspect, the invention provides a biological enzyme modulator comprising deuterium-depleted water having a deuterium content of greater than 0.1ppm and less than 135 ppm.

The third aspect of the invention provides a deuterium-depleted composition, which comprises the above biological enzyme regulator and any one of the following drugs:

antineoplastic drugs, cardiovascular drugs, drugs for respiratory diseases, drugs for basic metabolic diseases or enzyme inhibitors.

In a fourth aspect, the invention provides a cell culture medium comprising the aforementioned biological enzyme modulator and a basal cell culture medium selected from BME cell culture medium, MEM cell culture medium, DMEM cell culture medium, IMDM cell culture medium, RPMI-1640 cell culture medium, HamF10 cell culture medium, DMEM/F12 cell culture medium, M199 cell culture medium, McCoy5A cell culture medium, L15 cell culture medium, primary cell culture medium or stem cell culture medium.

In a fifth aspect, the invention provides the use of a biological enzyme modulator, or a deuterium-depleted composition, or a cell culture medium as hereinbefore described for the modulation of cellular metabolism.

Compared with the prior art, the invention has the following beneficial effects:

compared with the prior art which has been searched at present, the invention has the advantages that: the deuterium-depleted biological enzyme regulating agent based on the main component of the deuterium-depleted water has no toxic or side effect on a living body, the preparation process is simpler than that of an enzyme inhibitor in the prior art, and compared with other inhibitors which need to be used in a mode of injection, instillation and the like, the deuterium-depleted biological enzyme regulating agent can be directly taken orally, and the safety is high after long-term use; the deuterium-depleted composition based on the deuterium-depleted water and other compounds has a synergistic gain effect, can obviously reduce the dosage of other combined compounds on the premise of ensuring better effect or no reduction of the effect, not only obviously reduces the total medication cost, but also obviously reduces the toxic and side effects of other combined compounds on a living body; the deuterium-depleted biological enzyme modulators of the present invention also have modulating utility for a variety of metabolic key enzymes, and thus are more applicable to a variety of disease types and have a larger field of application than prior art biological enzyme inhibitors.

Another advantage of the present invention is that the deuterium depleted biological enzyme modulator of the present invention has biochemical functions other than enzyme inhibitors, including but not limited to improving cellular microenvironment, enhancing immune cell activity, promoting the rate of metabolism of the organism itself, improving pharmacokinetic profile of prior art combination compounds, etc.

Drawings

FIG. 1 shows that glioma cells were cultured directly after culture medium prepared by using the bio-enzyme control agent of the present invention (in this case, 25pppm deuterium-depleted water) and distilled water. Glioma cells with a well A and a well B of U251 were cultured in a medium prepared directly with 150ppm of normal distilled water, and glioma cells with a well C and a well D of U251 were cultured in a medium prepared directly with the biological enzyme regulator of the present invention (which in this case consists of 25pppm deuterium-depleted water).

FIG. 2 shows that glioma cells were cultured 14 days after the culture medium prepared from the biological enzyme control agent of the present invention (in this case, 25pppm deuterium-depleted water) and normal distilled water was left to stand. The glioma cells with the hole A and the hole B are cultured by a culture medium (after being placed at low temperature for 14 days) prepared by common distilled water with the concentration of 150ppm, and the glioma cells with the hole C and the hole D are cultured by a culture medium (after being placed at low temperature for 14 days) prepared by the biological enzyme regulator.

FIG. 3 shows the inhibition of the proliferation of brain glioma cells U87, U251 and T98G by the bioenzyme modulators of the invention (which in this case consist of 25pppm deuterium-depleted water).

FIG. 4 shows the inhibition of the monoclonal formation of mouse glioma cells (ALTS1C1) and human glial cells (U251) by the bioenzyme modulators of the invention (which in this case consist of 25pppm deuterium-depleted water).

FIG. 5 tumor cell supernatants (U87, U251, T98G) cultured with the biological enzyme modulators of the present invention (this example consisting of 25pppm deuterium-depleted water) inhibited angiogenesis of endothelial cells EVC 304.

FIG. 6, the biological enzyme modulator of the present invention (this example consists of 25pppm deuterium-depleted water) significantly inhibited the growth of subcutaneous transplantable tumors in NOD-SCID immunodeficient mice and C57BL/6 immune healthy mice.

Figure 7, immunohistochemistry detected Ki67 and CD31 expression in mice subcutaneous tumors.

FIG. 8, cell cloning experiments to examine the effect of PFK15 on glioma proliferation.

FIG. 9, Western blot to examine the expression of PFKFB3, HKI, PKM2 and GAPDH of glioma cells cultured in medium formulated with double distilled water and a biological enzyme modulator according to the present invention (in this case consisting of 25pppm deuterium depleted water).

FIG. 10 shows Western blot analysis of the expression of phosphorylated ERK and phosphorylated RSK signaling pathway molecules in glioma cells cultured in medium containing deuterium-depleted biological enzyme modulators (25 ppm deuterium-depleted water in this case) and double distilled water.

Figure 11, qPCR detects mRNA levels of PFKFB3 of glioma cells.

FIG. 12, Western blot to examine the expression of PFKFB3 in ALTS1C1 cells cultured in medium with double distilled water and inhibited protein synthesis with CHX, in accordance with the present invention low deuterium bioenzyme modulators (in this case consisting of 25ppm low deuterium water).

FIG. 13, combination of deuterium-depleted water (DDW) with rapamycin (rapamycin)

FIG. 14, combination of deuterium-depleted water (DDW) and everolimus (everolimus)

FIG. 15, deuterium-depleted water (DDW) in combination with sapercetin (INK-128)

FIG. 16, Deuterium Depleted Water (DDW) in combination with Temozolomide (TMZ)

FIG. 17, CDX model experiment (ALTS 1C1 followed by temozolomide tumor inhibitor) in C57BL/6 mice.

FIG. 18, CDX model experiment in C57BL/6 mice (ALTS 1C1 inoculated with atuzumab for tumor suppression).

FIG. 19 shows that the deuterium content of the low-deuterium biological enzyme regulator of the invention is reduced to greatly inhibit the expression level of the low-deuterium glycolysis key enzyme PFKFB 3. 150ppm refers to normal distilled water. 50ppm, 25ppm are deuterium-depleted biological enzyme modulators of the invention with different levels of deuterium content.

Figure 20, the deuterium depleted biological enzyme modulators (composed of deuterium depleted water with different deuterium content) of the present invention compared with PFK15 enzyme inhibitor effect.

Detailed Description

TABLE 1 comparison of physical Properties of three isotopic oxide portions of Hydrogen

Cellular metabolic pathways and levels are directly related to the health of cells and, in turn, affect the health of the organism. In the case of eukaryotic cells, there are generally two pathways for the metabolism of intracellular sugars, glycolysis and mitochondrial oxidative phosphorylation. Glycolysis is an oxygen-independent metabolic pathway; whereas mitochondrial oxidative phosphorylation needs to be carried out under aerobic conditions. Under normal aerobic conditions, the major energy source of most cells is the metabolic pathway by mitochondrial oxidative phosphorylation, and correspondingly glycolysis is inhibited (Pasteur Effect). However, in pathological conditions, such as tumor cells, one can supply itself by glycolysis without relying on mitochondria and oxygen, and at the same time, it can increase the glycolysis rate to 200 times that of normal cells, so that even under oxygen-rich conditions, tumor cells continue to select this metabolic mode. However, this metabolic mode, which relies primarily on glycolysis for energy, produces excess lactate and a range of tumor metabolites, known as the Warburg effect. These excess lactate enters the extracellular environment through monocarboxylic acid transporters (MCTs), particularly monocarboxylic acid-transporter 4(MCT4), and thereby acidifies the Tumor Microenvironment (TME). The acidic extracellular environment is favorable for competitive uptake of glucose by tumor cells and effector T cells, inhibits proliferation of the effector T cells which also take anaerobic glycolysis as a main metabolic mode, and provides help for tumor immune escape. Glycolysis replaces oxidative phosphorylation to supply energy, inhibits immune T cells from starting external apoptosis commands, and mitochondria cannot normally start apoptosis programs, so that the life cycle of tumor cells is prolonged, otherwise, glycolysis is inhibited, lactic acid is reduced, and normal recognition of T cells and the apoptosis programs are started. Thus, aerobic glycolysis and the resultant acidification of TME have a strong influence on the T-cell mediated anti-tumor immune response and the activity of tumor-infiltrating myeloid cells.

When the energy metabolism of cells is mainly changed to a glycolysis-dependent mode, the occurrence and development of various metabolic-related diseases can be caused, including malignant tumors, cardiovascular diseases, diabetes and other metabolic diseases. Accordingly, if the glycolytic metabolic pathway of the above pathological cells can be reversed or the metabolic level of the pathological cells can be reduced, it is possible to delay the development of metabolic-related diseases and to achieve a good therapeutic effect. Specifically, glycolysis uses glucose as a substrate, and converts glucose into pyruvate through a series of cascade enzymatic reactions, and releases free energy to form high-energy compounds, ATP and NADH. In the glycolysis process, three rate-limiting enzymes regulate the rate of glucose decomposition, one of which is Phosphofructokinase (PFK), which catalyzes the conversion of fructose-6-phosphate into fructose-1, 6-diphosphate. Phosphofructokinase-2/fructose-2, 6-diphosphatase 3 (PFKFB 3) has dual activities of kinase and phosphatase, and fructose-2, 6-bisphosphate catalytically synthesized by the phosphofructokinase-2/fructose-2, 6-bisphosphate is an allosteric activator of PFK, and is helpful for improving the activity of PFK, increasing the generation of fructose-1, 6-bisphosphate and promoting the glycolysis process. Therefore, by inhibiting the activity of PFKFB3, the production of fructose-2, 6-diphosphate is reduced, and further the activity of PFK is reduced, the glycolysis process can be inhibited, and finally the energy production of tumor cells is reduced.

It has been shown that overexpression of PFKFB3 leads to exacerbation of respiratory disease. The pulmonary hypertension model is replicated by constructing endothelial cell and smooth muscle cell specific PFKFB3 knockout mice and simultaneously utilizing chronic hypoxia induced mice. Research results show that after PFKFB3 is knocked out specifically by endothelial cells, glycolysis level of the endothelial cells is reduced remarkably, and generation of pyruvate serving as a glycolytic metabolite is inhibited. The reduction of the pyruvate level leads to the weakening of the inhibition capability on PHDs, leads HIF2A in cells to be degraded and increased, and finally leads the expression of growth factors, proinflammatory cytokines and cell adhesion factors to be reduced, thereby inhibiting the abnormal proliferation of pulmonary vascular smooth muscle cells and the infiltration of pulmonary perivascular inflammatory cells and inhibiting the development of hypoxia-induced pulmonary hypertension. In another study on smooth muscle glycolysis, it is found that after PFKFB3 is knocked out specifically by smooth muscle cells, the lactic acid content of glycolytic metabolites is reduced, so that phosphorylation activation of calpain-2 dependent on ERK1/2 is reduced, collagen synthesis in pulmonary vascular smooth muscle cells is reduced, abnormal proliferation of smooth muscle cells is weakened, and pulmonary vascular remodeling in the development process of pulmonary hypertension is inhibited. Research also finds that the endothelial cell specific PFKFB3 knockout can protect mice from septicemia and acute lung injury caused by LPS, and the endothelial cell PFKFB3 can regulate the expression of adhesion factors ICAM-1 and VCAM-1 and the release of inflammatory factors IL-1 beta and TNF-alpha through an NF-KB signal channel, thereby promoting tissue damage caused by excessive inflammatory reaction. Therefore, inhibition of PFKFB3 in vascular cells has a very positive and effective effect on the treatment of the above-mentioned several serious lung diseases.

Further studies have shown that SRC-3 protein, steroid hormone receptor co-activator 3(SRC-3), induces tumor production, PFKFB4 acts as the most prominent kinase regulating cell proliferation, inhibiting its expression reduces SRC-3 activity in tumor cells, while its overexpression promotes SRC-3 activity. The most important function of PFKFB4 metabolic kinases is indeed to add phosphate groups to sugar molecules in the pathway of the Warburg effect; meanwhile, when PFKFB4 adds a phosphate group to SRC-3, it activates SRC-3, converting it into a potent driver of breast and other cancers. In a mouse model, researchers successfully achieved growth inhibition and volume reduction of breast cancer tumors by removing PFKFB4 from the tumor, inhibiting phosphorylation of SRC-3 rendering SRC-3 unable to accept phosphate groups, having a tumor control effect, and being able to almost completely eliminate recurrence and metastasis of breast cancer. Although the energy provided by sugar metabolism is not high, the PFKFB4 kinase in the process can induce the production of cancer cells. Thus, PFKFB4 is linked to the interaction between the glycolytic pathway and the oncogenic activation of the transcriptional coactivator SRC-3. Phosphorylation of SRC-3 rapidly increases its transcriptional activity, promoting gene synthesis that drives glucose flux. Thus, PFKFB4 is also an important enzyme for tumor inhibition. In conjunction with the current research progress, all PFKFB enzymes (PFKFB1-4) are enzymes that activate fructose 2, 6-diphosphate (F2,6P2), of which PFKFB3 is the enzyme with the highest expression, so its effect on glycolysis regulation is most pronounced.

PFK15(PFK-015) can be used in laboratories to inhibit the activity of PFKFB3 in cells, and experiments can confirm that the activity of PFKFB3 in cells is reduced remarkably after PFK15 inhibitor is used. Patent application No. CN104520274A "PFKFB 3 inhibitor and method for use as anticancer therapeutic agent" proposes a PFKFB3 inhibitor PFK158 which is superior to PFK15, has a significant improvement in inhibition efficiency, pharmacokinetic properties, in vivo tolerance and safety, and is claimed to be applicable to most tumor diseases. Patent application No. CN111228265A "application of p38 γ inhibitor in preparing medicine for treating pancreatic cancer", another PFKFB3 inhibitor, 3PO (C13H10N2O), is mentioned; patent application No. CN109134434A "quinoline or quinazoline compound, and preparation method and application thereof" mention a quinoline or quinazoline compound, which can effectively inhibit activity of PFKFB in tumor cells, especially can effectively inhibit kinase activity of PFKFB3, thereby reducing level of fructose 2, 6-diphosphate (F2,6P 2). The patent application number CN109134336A 'diaryl ether compound, and a preparation method and application thereof' provide a diaryl ether compound which can inhibit the activity of PFKFB3 in tumor cells and effectively block the activation of key enzymes in the glycolysis process. Patent CN107875150A "application of AZ67 small molecule in preparation of PFKFB3 activity inhibitor" discloses application of AZ67 small molecule in preparation of PFKFB3 activity inhibitor, and inhibits activity of PFKFB3 through AZ67 small molecule. Based on the various compound PFKFB3 inhibitors disclosed by the invention, the inhibitors generally have the defects of complex synthesis process, high medication cost and large toxic and side effects.

By combining the regulation and control effects of the PFKFB1-4 on cell glycolysis and other protein synthesis and the regulation and control effects of deuterium concentration in the biological fluid on cell metabolism, in order to find out the exact regulation and control mechanism of deuterium-depleted water on cell metabolism, the research on the cell metabolism process by deuterium-depleted water is selected, and the regulation and control effects on the expression of key enzymes such as PFKFB3 and the like can be exactly realized by means of cell in-vitro culture modeling, biological model modeling, metabonomics analysis and the like, and the effect of degrading the enzymes is also realized. Therefore, in order to overcome the defects of the biological enzyme inhibitor in the prior art, the invention provides a deuterium-depleted biological enzyme regulator taking deuterium-depleted water as a main component, and a deuterium-depleted composition combining deuterium-depleted water and a specific compound. The research shows that the biological enzyme inhibitor taking the deuterium-depleted water as the main component can inhibit the expression of a key enzyme PFKFB3 in cells and has a degradation effect on the expressed PFKFB3, so that a more comprehensive inhibition effect on PFKFB3 superior to the prior art can be obtained, and simultaneously, the biological enzyme inhibitor has an inhibition effect on PFKFB1-4 enzymes, so that the biological enzyme inhibitor can participate in the regulation and control of metabolic pathways related to PFKFB1, PFKFB2 and PFKFB 4. Through the inhibition of PFKFB enzyme, the cell glycolytic metabolism is regulated, the SRC protein expression is inhibited, and other deuterium sensitive biological enzyme related metabolic pathways are inhibited, so that the functional polypeptide can independently play a role in the control and treatment of tumor inhibition, cardiovascular diseases, respiratory diseases and other metabolic basic diseases. Meanwhile, compared with the single use of the deuterium-depleted water, the deuterium-depleted composition can improve the effect of controlling cell metabolism or the curative effect of diseases; compared with the single use of the specific compound, the deuterium-depleted composition can reduce the conventional use amount of the specific compound, reduce the toxic and side effects of the specific compound on a living body and reduce the use economic cost. Meanwhile, the low-deuterium biological enzyme regulator can also be used for regulating and controlling specific cell metabolic pathways and metabolic levels in the fields of biological engineering and life science research.

Before the present embodiments are further described, it is to be understood that the scope of the invention is not to be limited to the specific embodiments described below; it is also to be understood that the terminology used in the examples herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention. Test methods in which specific conditions are not specified in the following examples are generally carried out under conventional conditions or under conditions recommended by the respective manufacturers.

When numerical ranges are given in the examples, it is understood that both endpoints of each of the numerical ranges and any value therebetween can be selected unless the invention otherwise indicated. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In addition to the specific methods, devices, and materials used in the examples, any methods, devices, and materials similar or equivalent to those described in the examples may be used in the practice of the invention in addition to the specific methods, devices, and materials used in the examples, in keeping with the knowledge of one skilled in the art and with the description of the invention.

Unless otherwise indicated, the experimental methods, detection methods, and preparation methods disclosed herein all employ techniques conventional in the art of molecular biology, biochemistry, chromatin structure and analysis, analytical chemistry, cell culture, recombinant DNA technology, and related arts.

The invention provides a low-deuterium biological enzyme regulating agent and a low-deuterium composition thereof. The deuterium-depleted biological enzyme regulator regulates and controls the expression and enzyme activity of key biological enzymes in cells of a living body, changes the metabolic pathway and metabolic level of the biological enzymes, thereby inhibiting the proliferation of tumor cells, limiting the development of focuses of cardiovascular and respiratory diseases, delaying and controlling the progress of other basic metabolic diseases, and can be applied to the fields of tumor inhibition, inflammatory reaction, disease control and adjuvant therapy of cardiovascular diseases and respiratory diseases related to cell metabolic pathways, and the like. Meanwhile, the low-deuterium biological enzyme regulating agent can be combined with a cell culture medium to form a low-deuterium composition, and is applied to the process links or test flows related to cell metabolism regulation in bioengineering and life science research. In addition, the deuterium-depleted water and the specific compound are combined to form the deuterium-depleted composition, and compared with the deuterium-depleted water which is used alone, the deuterium-depleted composition can improve the effect of controlling cell metabolism or the curative effect of diseases; compared with the single use of the specific compound, the deuterium-depleted composition can reduce the conventional use amount of the specific compound, relieve the toxic and side effects of the specific compound on a living body and reduce the use economic cost. Compared with the prior art, the invention has the advantages of relatively simple preparation and configuration process, low application cost and small toxic and side effects on life bodies including cells on the whole.

Use of deuterium depleted water in any one or more of:

preparing a biological enzyme regulator;

preparing a cell glycolysis inhibitor;

preparing a tumor treatment product;

preparing a tumor proliferation inhibitor;

preparing a tumor cell clone inhibitor;

preparing an angiogenesis promoting ability inhibitor of tumor cells;

preparing a tumor growth inhibitor;

preparing an antitumor drug synergist;

preparing a synergist of the enzyme inhibitor;

preparing a cardiovascular drug synergist;

preparing a drug synergist for the basic metabolic diseases;

the deuterium-depleted water has a deuterium content higher than 0.1ppm and lower than 135 ppm.

In a preferred embodiment, the deuterium depleted water has a deuterium content of 15ppm to 100 ppm.

Specifically, the biological enzyme regulating agent is a preparation which has an inhibiting and/or degrading effect on biological enzymes.

Having inhibitory effects on biological enzymes include, but are not limited to: inhibiting the expression or activity of a biological enzyme.

Inhibiting the activity of a biological enzyme means reducing the activity of the biological enzyme. Preferably, the activity of the biological enzyme is reduced by at least 10%, preferably by at least 30%, more preferably by at least 50%, even more preferably by at least 70%, and most preferably by at least 90% compared to the activity prior to inhibition.

The inhibition of the expression of the biological enzyme may specifically be inhibition of transcription or translation of the biological enzyme gene, and specifically may refer to: making the gene of the biological enzyme non-transcribed or reducing the transcriptional activity of the gene of the biological enzyme, or making the gene of the biological enzyme non-translated or reducing the translation level of the gene of the biological enzyme.

The degrading effect is to break down the biological enzymes already present.

The deuterium-depleted biological enzyme modulators modulate objects including, but not limited to, fructose-2-phosphate kinase/fructose-2, 6-bisphosphatase (PFKFB enzyme), SRC proteins, and other metabolic key enzymes.

The method is particularly suitable for the regulation of PFKFB3 enzyme, and can realize the function of regulating glycolytic metabolism; particularly suitable for the regulation of PFKFB4 enzyme, can inhibit the expression of SRC protein.

More generally, the biological enzyme regulator of the present invention has a regulating effect on various biological enzymes affecting basic metabolism.

In one embodiment, the tumor is selected from a glioma.

In one embodiment, the anti-neoplastic agent is selected from the group consisting of cytotoxic agents, hormonal agents, biological response modifiers, monoclonal antibody agents, cell differentiation inducers (e.g., retinoids, arsenite, etc.), apoptosis inducers, angiogenesis inhibitors or epidermal growth factor receptor inhibitors (e.g., gefitinib, erlotinib, etc.).

In a further embodiment, the anti-neoplastic drug is selected from rapamycin, everolimus, temozolomide, sapercetin, or a PD-L1 antibody.

In one embodiment, the enzyme inhibitor is selected from PFKFB inhibitors.

In a further embodiment, the PFKFB inhibitor is selected from PFK15, PFK158, 3PO, quinoline or quinazoline, diaryl ether or AZ 67.

In one embodiment, the cardiovascular agent is selected from the group consisting of anti-anginal agents (nitrates, nitroglycerols, nifedipine, diltiazem, etc.), anti-arrhythmic agents, anti-hypertensive agents, anti-cardiac insufficiency agents, peripheral vasodilators.

In one embodiment, the basal metabolic disease agent is selected from agents whose primary indication is diabetes, diabetic ketoacidosis, hyperglycemic hyperosmolar syndrome, hypoglycemia, gout, protein-energy dystrophy, vitamin a deficiency, scurvy, vitamin D deficiency, or osteoporosis disease.

The deuterium-depleted water inhibits tumor cell proliferation, inflammatory responses, limits cardiovascular or respiratory disease focus development, or retards and controls other underlying metabolic disease processes by altering cellular or other living body metabolic pathways and levels.

The biological enzyme regulator comprises deuterium-depleted water with deuterium content higher than 0.1ppm and lower than 135 ppm.

Furthermore, the deuterium-depleted water is an effective component or one of the effective components of the biological regulator.

Preferably, the deuterium-depleted water has a deuterium content of 15ppm to 100 ppm.

The biological enzyme regulating agent is a preparation which has inhibition and/or degradation effects on biological enzymes.

The biological enzyme is selected from PFKFB enzyme or SRC protein.

The deuterium-depleted composition of an embodiment of the present invention includes the above biological enzyme regulator, and any one of the following drugs:

In one embodiment, the tumor is selected from a glioma.

The dosage of the combined medicine in the deuterium-depleted composition can be reduced to 50-80% of the dosage of the combined medicine under the condition of single medicine application, and experiments show that the inhibition effect of the deuterium-depleted composition on the target biological enzyme is better than or not lower than that of the single conventional dosage medicine (single deuterium-depleted water or single medicine or single biological enzyme inhibitor of the prior art), and simultaneously, the toxic and side effects of the medicine combined with the deuterium-depleted water on a living body are reduced, and the synergistic gain effect of adding one to two is achieved.

In one embodiment, the enzyme inhibitor is selected from PFKFB inhibitors.

In a further embodiment, the PFKFB inhibitor is selected from PFK15, PFK158, 3PO, quinoline or quinazoline compounds, diaryl ethers or AZ 67.

In one embodiment, the cardiovascular agent is selected from the group consisting of anti-anginal agents (nitrates, nitroglycerols, nifedipine, diltiazem, etc.), antiarrhythmic agents, antihypertensive agents, anti-cardiac insufficiency agents, peripheral vasodilators.

In one embodiment, the basal metabolic disease agent is selected from agents whose primary indication is diabetes, diabetic ketoacidosis, hyperglycemic hyperosmolar syndrome, hypoglycemia, gout, protein-energy dystrophy, vitamin a deficiency, scurvy, vitamin D deficiency, or osteoporosis;

the cell culture medium of an embodiment of the invention comprises the above biological enzyme regulator and a basal cell culture medium selected from BME cell culture medium, MEM cell culture medium, DMEM cell culture medium, IMDM cell culture medium, RPMI-1640 cell culture medium, HamF10 cell culture medium, DMEM/F12 cell culture medium, M199 cell culture medium, McCoy5A culture medium, L15 cell culture medium, primary cell culture medium or stem cell culture medium.

The aforementioned biological enzyme modulators, or deuterium-depleted compositions, or cell culture media may be used in the regulation of cellular metabolism.

Further, the use is of non-disease therapeutic or diagnostic purpose;

the application belongs to the field of control bioengineering or life science research; before application, the biological enzyme regulator, the deuterium-depleted composition or the cell culture medium is subjected to preset treatment, or the deuterium concentration of deuterium-depleted water or the combination ratio of deuterium-depleted water and a combined compound is adjusted; or adjusting the dosage, time frequency of application of a biological enzyme modulator, or said deuterium depleted composition, or said cell culture medium.

The pre-treatment satisfies the condition that water molecular groups in the deuterium-depleted water and other components of the deuterium-depleted composition or components of the culture medium form a uniform distribution and an effective combination state.

Further, the pre-treatment methods for the deuterium depleted composition of the present invention include, but are not limited to, uniformly stirring the deuterium depleted composition, filtering with a filter, shielding from light, storing at a suitable temperature, standing to eliminate vibration, packaging as necessary, and proper sterilization. Meanwhile, the corresponding preset treatment needs to meet the requirement of a certain time length, the time length is usually between 0 day and 14 days, and meanwhile, a certain time can be properly prolonged according to the requirement until the uniform distribution and the effective combination state of the water molecular groups in the deuterium-depleted water and the combined compound or the culture medium components are realized after the preset treatment.

Further, the above-mentioned necessary pre-treatment method is a necessary condition for effectively exerting the effect of the bio-enzyme regulator of the present invention, based on the case of combining deuterium-depleted water with other compounds. In particular, without the above-mentioned pre-treatment, it is possible to reduce or substantially lose the effect of a composition simply made up according to the constitution of the ingredients of the present invention.

As described above, although the biological enzyme regulator of the present invention can be administered and exert a positive effect at any stage of disease development or cell development, in order to exert the regulation effect of the biological enzyme regulator of the present invention sufficiently, it should be administered as early as possible at a known disease process node or a development stage of a cell line, and appropriate dosage, administration time frequency and administration duration are ensured.

In the present invention, the biological enzyme regulator and the deuterium-depleted biological enzyme regulator have the same meaning.

Example 1, the inhibition of cellular glycolysis by the deuterium-depleted biological enzyme modulators of the present invention was confirmed using metabolomic analysis.

In order to explore the influence of deuterium-depleted water on the metabolism in animals, the serum of CDX model nude mice fed by the deuterium-depleted biological enzyme regulator with the deuterium-depleted water as the main component and the double distilled water is collected and subjected to metabonomic analysis. The experimental procedure was as follows:

(1) Collecting serum: collecting blood of fed nude mice by orbital hemospasia, centrifuging each nude mouse at 3000 rpm for 15min to obtain supernatant, transferring into 1.5mL centrifuge tube, and storing in refrigerator at-80 deg.C for use.

(2) Sample preparation: placing the frozen serum sample at room temperature for natural thawing, taking 100 μ l of serum, adding 400 μ l of methanol (containing 10 μ g/ml of ribitol internal standard), vortexing for 1min, standing for 10min at 4 ℃, centrifuging for 15min at 13800 Xg, taking 200 μ l of supernatant, placing in a 1.5ml centrifuge tube, and drying by nitrogen.

(3) Derivatization of the sample: adding 15mg/ml methoxylamine pyridine solution 50 μ l into the sample residue dried by nitrogen blow, vortexing for 30s, placing in an oven at 70 ℃ for reacting for 60min, adding MSTFA (containing 1% TMCS)50 μ l, vortexing for 1min, reacting at room temperature for 30min, adding 100 μ l n-heptane, vortexing for 30s, centrifuging for 5min at 1500 Xg, and taking 100 μ l of supernatant to a sample injection vial.

(4) GC-MS analysis: chromatographic conditions, namely, an Agilen DB-5MS gas chromatographic column (30m multiplied by 0.25mm, 0.25 mu m) is adopted for chromatographic separation, the temperature is programmed to be raised to 70 ℃ at the initial temperature, kept for 3min, raised to 220 ℃ at the speed of 4 ℃/min, raised to 300 ℃ at the speed of 10 ℃/min, kept for 6min, the temperature of a sample inlet is 250 ℃, and the split ratio is 10: 1, the temperature of a transmission line is 260 ℃, the carrier gas is high-purity helium, the flow rate is 1ml/min, and the sample injection amount is 1 mu l. Mass spectrum conditions: EI ion source is adopted, the electron energy is 70ev, and the ion source temperature is 200 ℃; mass range (m/z): 50-650 amu; the solvent delay time is 6min, and the mass spectrum acquisition time is 50 min.

(5) And (3) data analysis: the raw data were imported into SIMCA-P V11.0.0 (Umetrics, Sweden) software for partial least squares-discriminant analysis (PLS-DA), and the model was evaluated based on the correlation R2, Q2 values. The differential metabolites were further analyzed by a two-sided t-test (model vs control) and differences were considered statistically significant when P < 0.05.

As shown in table 2, metabolomics results show that deuterium-depleted water reduces the expression of some acidic substances in the blood of mice, wherein glucosamine-6-phosphate is involved in the glycolysis pathway, and the reduction thereof indicates that the glycolysis metabolic pathway can be inhibited by using the deuterium-depleted enzyme modulator of the present invention.

Table 2 deuterium-depleted bio-enzyme modulator (composed of deuterium-depleted water) of the present invention and double distilled water-fed nude mouse hemometabonomics differential molecules

Example 2, the low deuterium bio-enzyme modulator of the present invention is combined with the medium powder to involve the necessary pre-treatment.

It should be noted that the cell culture medium (low deuterium combined culture medium) prepared by combining the low deuterium biological enzyme modulator and the cell culture medium powder according to the present invention as described above and below requires a pre-set treatment as necessary. Specifically, DMEM medium powder (containing glucose and various amino acids) is prepared by adding deuterium-depleted water, sterile filtration is carried out by using a 0.22-micron filter membrane, the mixture is kept stand at the low temperature of 4-8 ℃ for 2 weeks, and after the deuterium-depleted water small molecular groups fully permeate into medium components, the mixture is used for cell culture.

FIG. 1 shows a deuterium-depleted culture medium prepared by combining the deuterium-depleted biological enzyme modulator (in this case, 25ppm of deuterium-depleted water) of the present invention with culture medium powder without pre-treatment, and culturing glioma cells directly using a culture medium prepared with normal distilled water. Among them, the glioma cells in the A and B wells are cultured in a medium directly prepared by using 150ppm of common distilled water, and the glioma cells in the C and D wells are cultured in a medium directly prepared by using 25ppm of deuterium-depleted water.

FIG. 2 shows a deuterium-depleted medium prepared by combining a deuterium-depleted biological enzyme modulator (in this case, 25ppm of deuterium-depleted water) and medium powder according to the present invention after a pre-treatment for 14 days, and culturing glioma cells using a medium prepared by combining normal distilled water and medium powder. Among them, the A and B wells showed the result of culturing the U251 glioma cells in a medium prepared with 150ppm of normal distilled water (after 14 days of low-temperature standing), and the C and D wells showed the result of culturing the U251 glioma cells in a medium prepared with 25ppm of deuterium-depleted water (after 14 days of low-temperature standing).

Comparing fig. 1 and fig. 2, we can find that: and (3) the deuterium-depleted water small molecular group is fully mixed with nutrient components of the culture medium through low-temperature presetting for at least 14 days, and compared with a culture result of the culture medium prepared by using double distilled water, the deuterium-depleted water small molecular group has an obvious inhibition effect on the growth of tumor cells, so that the cell cloning formation is greatly reduced. Therefore, the capacity of directly preparing the culture medium by using the deuterium-depleted water to inhibit the proliferation of the tumor cells is weak without adopting necessary preset measures; the biological enzyme regulating agent of the invention adopting necessary preset measures has obvious inhibition capability on tumor cell proliferation.

Example 3 the effectiveness of the deuterium-depleted biological enzyme modulator of the present invention in inhibiting the proliferation of tumor cells was confirmed using the CCK8 method.

This example compares the difference in tumor cell proliferation rates in two media using the CCK8 method. One DMEM high-sugar medium prepared based on the deuterium-depleted biological enzyme regulator (consisting of 25ppm deuterium-depleted water in this example) and the other DMEM high-sugar medium prepared based on double distilled water (150 ppm).

Cell Counting Kit-8(CCK-8) experiment:

1) taking U87/T98G/U251 glioma cells in logarithmic growth phase, adjusting the cell density to 5 × 10⁴mL, 100. mu.L of cells per well (5000 per well) were plated in 96-well plates,

2) each cell is divided into two groups, one group is paved with 6 holes, the DMEM high-sugar culture medium prepared by the deuterium-depleted biological enzyme regulator DDW (the example is composed of 25ppm deuterium-depleted water) or the CTL double distilled water (150ppm) of the control group is used for treating for 1 to 3 days,

3) the absorbance values at 450nm were measured on

days

1, 2 and 3 using CCK8 reagent, and the values of each group of cells were counted using graphpad software.

Cell Counting Kit-8(CCK-8) can count various cells. The reagent contains WST-8, namely 2- (2-methoxy-4-nitrophenyl) -3- (4-nitrophenyl) -5- (2, 4-disulfonic acid benzene) -2H-tetrazole monosodium salt. It is reduced by dehydrogenase in the cell to a yellow formazan product with high water solubility under the action of an electron carrier. The number of formazan produced was proportional to the number of living cells, and thus viable cells were counted.

As shown in fig. 3, the test group using the bio-enzyme regulator of the present invention (which is composed of 25ppm deuterium-depleted water in this example) showed a statistically significant difference from the control group using double distilled water, that is, the bio-enzyme regulator of the present invention (which is composed of 25ppm deuterium-depleted water in this example) significantly inhibited the proliferation of brain glioma cells U87, U251 and T98G, and the inhibitory effect thereof was relatively enhanced with time.

Example 4, the inhibition effect of the biological enzyme regulator on tumor cells was verified by using a monoclonal formation test.

This example utilizes a monoclonal formation experiment. The specific scheme is as follows:

1) taking glioma cells and umbilical vein endothelial cells (EVC304) in logarithmic growth phase, and adjusting cell density to 1 × 10⁵mL, 10. mu.l of cells per well (1000 per well) were plated in 6-well plates, each cell in two groups, one group in 3 wells,

2) the DMEM high-sugar culture medium prepared by the biological enzyme regulator (composed of 25ppm deuterium-depleted water DDW in this example) or double distilled water (150ppm) is used for treatment,

3) then placing at 37 ℃ and 5% CO₂The constant temperature incubator is used for 1-2 weeks, 200 mu L of culture medium is supplemented every two days,

4) after 1-2 weeks, the six-well plate was removed, the culture medium was poured off, and fixed with methanol. Staining with giemsa for 10 min. Cell clones with a diameter of more than 75 μm (or containing more than 50 cells) were counted under the microscope.

As shown in FIG. 4, the cell clone number of the low deuterium combined culture medium (ALTS1C1-DDW group, U251-DDW group) containing the biological enzyme regulator of the present invention is significantly lower than that of the control group. The experimental group and the control group use the same culture medium powder, and the difference is only whether the biological enzyme regulator is used, which shows that the biological enzyme regulator DDW of the invention can obviously inhibit the clonogenic capacity of mouse glioma cells (ALTS1C1) and human glial cells (U251).

Example 5, the ability of the biological enzyme regulator to inhibit angiogenesis of tumor cells was verified by tube-forming experiments.

This example was performed using an angio-tube experiment. The specific scheme is as follows: taking three glioma cells in logarithmic growth phase, adjusting cell density to 1 × 10⁵5mL of cells were plated in 6-well plates, each of which was divided into two groups, and cultured in a 10% FBS-containing DMEM high-sugar medium at 37 ℃ in 5% CO₂Culturing in a constant temperature incubator, and culturing for 24 hours in a serum-free DMEM high-sugar medium prepared by replacing the deuterium-depleted biological enzyme regulator (consisting of 25ppm deuterium-depleted water in this example) or double distilled water (150ppm) on the next day, transferring two groups of cell supernatants into 15mL centrifuge tubes respectively, centrifuging at 2000rpm for 5min, collecting the supernatants, and storing in a refrigerator at-40 ℃. Then, human umbilical vein vascular endothelial cells (EVC304) in the logarithmic growth phase were collected, the cells were digested with 0.25% trypsin and counted, an equal amount of cell suspension was transferred to 6 15mL centrifuge tubes, centrifuged at 1000rpm for 5min, the EVC304 cells were resuspended in the collected glioma cell supernatant with the density adjusted to 1.5 × 105/mL, a matrigel diluent (matrigel and EBM medium) from BD was first applied to a 96-well plate, and after half an hour, the cell suspension was added to 100 μ L of cells per well (15000 cells/well), incubated in a constant temperature incubator at 37 ℃ and 5% CO2 for 4-6 hours, and observed under a microscope and photographed and recorded.

As shown in FIG. 5, the EVC304-U87-DDW group, EVC304-U251-DDW group and EVC304-T98G-DDW group cultured by the biological enzyme regulator of the present invention had no obvious tube formation, while the control group had obvious tube formation. The experimental group and the control group use the same culture medium powder, and the difference is only whether the biological enzyme regulator DDW is used, which shows that the biological enzyme regulator DDW can inhibit the angiogenesis promoting capacity of the tumor cells.

Example 6 validation of inhibition of tumor growth in mice by the Low deuterium Bio-enzyme modulators of the invention

Animal experiments:

(1) pretreatment of C57BL/6 mice or nude mice: mice were randomly divided into two groups of 6-8 mice each, one group was fed with double distilled water, and one group was fed with the deuterium-depleted bio-enzyme modulator of the present invention (consisting of 25ppm deuterium-depleted water in this example), and placed in an SPF-class animal house for 3-4 weeks.

(2) And (3) processing the cells: culturing ALTS1C1 mouse glioma cell to logarithmic growth phase, subculturing, taking out when culturing to 60-80% fusion rate, digesting with 0.25% pancreatin, centrifuging at 1000rpm for 5min, diluting with PBS to 2.5 × 10⁷Cell suspension in ml.

(3) Injection of C57BL/6 mice or nude mice: the C57BL/6 mouse nude mouse was grasped from the back by the right hand, and subcutaneously injected from one side of the back by a 1ml insulin injection needle to 5X 10 ⁶And (0.2mL) cells.

(4) Animal feeding: placing C57BL/6 mouse or nude mouse into SPF animal room, and keeping feeding with double distilled water or deuterium-depleted water for 3-4 weeks.

(5) Collecting and processing a specimen: the mice were removed, sacrificed by cervical dislocation, and tumors on the backs of C57BL/6 mice or nude mice were removed with scissors and forceps and photographed side by side. The tumor specimen is taken out and placed in liquid nitrogen for protein extraction in the later period. The sections were placed in formalin and fixed embedded followed by immunohistochemical staining.

As shown in FIG. 6, the tumor size of ALTS1C1-DDW group to which the deuterium-depleted bioenzyme regulator of the present invention (consisting of 25ppm deuterium-depleted water in this example) was applied was significantly smaller than that of ALTS1C1-H2O group using double distilled water, both in immunized C57BL/6 mice and in immunodeficient nude mice, and thus the deuterium-depleted bioenzyme regulator of the present invention had a significant inhibitory effect on the growth of ALTS1C1 glioma cells in mice.

To explore the mechanism of deuterium depleted water action in the CDX model, we analyzed tissue sections of ALTS1C1 subcutaneous tumors from C57BL/6 mice in the double-distilled water-fed group and the deuterium-depleted bio-enzyme modulator (consisting of 25ppm deuterium depleted water in this example) fed group of the invention using immunohistochemical staining. As shown in fig. 7, the deuterium-depleted biological enzyme modulator (in this case consisting of 25ppm deuterium-depleted water) according to the present invention was found to reduce the expression of the tumor cell proliferation-associated molecule Ki67 and the expression of the angiogenesis marker CD 31.

Example 7 validation of the mechanism of inhibition of glioma cell proliferation by controlling PFKFB3 to alter glycolysis

To explore the effect of the key enzyme PFKFB3 related to glycolysis on brain glioma proliferation, we validated the difference in clonogenic capacity of PFKFB3 inhibitor PFK15(1 μ M) on double distilled water medium, and glioma cells cultured in medium formulated with the deuterium-depleted bioenzyme modulators described herein (this example consists of 25ppm deuterium-depleted water) and PFK15(1 μ M) compositions using a clonogenic assay. As shown in fig. 8, experiments prove that the proliferation capacity of glioma cells can be remarkably inhibited by inhibiting PFKFB3, and meanwhile, if the deuterium-depleted composition consisting of the deuterium-depleted bio-enzyme regulator (in this case, 25ppm of deuterium-depleted water) and PFK15 is adopted, a clear synergistic gain effect is obtained, and the control capacity of the proliferation of glioma cells is increased by one and more than two.

Example 8, the regulatory effect of the deuterium-depleted biological enzyme modulators of the present invention on key enzymes in cells was validated and analyzed.

To explore whether the deuterium depleted biological enzyme modulators of the present invention (consisting of 25ppm deuterium depleted water in this example) affect the glycolysis related key enzymes PFKFB3, HKI and PFKM2, we first tested ALTS1C1/U87/T98G/U251 cells cultured in double distilled water or media formulated with deuterium depleted biological enzyme modulators of the present invention (consisting of 25ppm deuterium depleted water in this example) using the western blot method. As shown in fig. 9, we found that the deuterium depleted biological enzyme modulators of the present invention (which in this case consisted of 25ppm deuterium depleted water) significantly inhibited the expression of PFKFB3 in glioma cells without significantly altering the expression of HKI and PFKM 2.

To explore whether the deuterium depleted biological enzyme modulators of the present invention (which in this case consist of 25ppm deuterium depleted water) affect the modulation of signal pathway molecules upstream of PFKFB3, we first performed the validation of phosphorylated RSK and ERK signal pathway molecules using western blot in ALTS1C1/U251/T98G cells cultured in double distilled water or media formulated with the deuterium depleted biological enzyme modulators of the present invention. As shown in fig. 10, we found that the deuterium-depleted biological enzyme modulators of the present invention significantly inhibited the expression of phosphorylated RSK1 in glioma cells, but did not significantly alter the expression of phosphorylated ERK, phosphorylated RSK2, and RSK 3.

To explore whether the deuterium-depleted bio-enzyme modulators of the present invention (consisting of 25ppm deuterium-depleted water in this example) affected the expression of mRNA levels of the glycolysis-related key enzyme PFKFB3, we performed a fluorescent quantitative PCR method in U87/T98G cells cultured in media formulated in double distilled water or deuterium-depleted water. As shown in fig. 11, we found that the deuterium depleted biological enzyme modulators of the present invention (this example consists of 25ppm deuterium depleted water) did not significantly alter the expression of the transcription level of PFKFB 3.

To explore whether the deuterium depleted biological enzyme modulators of the present invention (which in this case consist of 25ppm deuterium depleted water) affect the rate of post-translational degradation of proteins of the glycolysis-related key enzyme PFKFB3, we treated ALTS1C1 cells cultured in media formulated in double distilled water or deuterium depleted water with the protein synthesis inhibitor cycloheximide CHX (20 μ g/ml) for 0-2-4-6 hours, and then collected the cellular proteins for detection by the western blot method. As shown in fig. 12, we found that the deuterium depleted biological enzyme modulators of the present invention (which in this case consist of 25ppm deuterium depleted water) accelerate the degradation of PFKFB3 in ALTS1C1 glioma cells.

Example 9 the deuterium-depleted biological enzyme modulator of the present invention is combined with a specific drug compound to form a deuterium-depleted composition with tumor inhibition effect.

We performed the combination of deuterium depleted water and rapamycin in the glioma cell line U251, T98G, A172 (FIG. 13), using the method of example 3, except that the DDW, CTL in step 2 was replaced by DDW, CTL, DDW + rapamycin (1nM), CTL + rapamycin (1nM), using the method of example 3.

Similarly, using the method of example 3 in combination with deuterium depleted water and everolimus in glioma cell line U251, T98G, a172 (fig. 14), with the exception that DDW, CTL in step 2 were replaced with DDW, CTL, DDW + everolimus (4nM), CTL + everolimus (4 nM).

Similarly, the procedure of example 3 was used in conjunction with deuterowater and saperchroman in glioma cell line U251, T98G, A172 (FIG. 15), except that DDW and CTL in step 2 were replaced by DDW, CTL, DDW + INK-128(5nM), CTL + INK-128(5 nM).

And in mouse glioma cell line ALTS1C1 and human glioma cell line T98G, using the method of example 4, except that the six groups were used, replacing 25ppm deuterium depleted water and 150ppm double distilled water in step 2 with 25ppm + DMSO,25ppm +100 μ M TMZ,25ppm +200 μ M TMZ,150ppm + DMSO,150ppm +100 μ M TMZ,150ppm +200 μ M TMZ.

The experimental result shows that the combined group of the deuterium-depleted water and the medicines has the best tumor inhibition effect.

In order to maximize the applicability of the deuterium depleted compositions of the present invention to clinical trials, we used CDX animal experiments to investigate whether the deuterium depleted biological enzyme modulators of the present invention (which in this case consist of 25ppm deuterium depleted water) in combination with temozolomide could inhibit the growth of subcutaneous tumours formed by glioma cells. Mice were fed with double distilled water and 25ppm deuterium depleted water for 3 weeks, then ALTS1C1 was inoculated subcutaneously into C57BL/6 mice to form subcutaneous tumors, and then the mice were further fed with double distilled water and 25ppm deuterium depleted water, and 5 days after subcutaneous tumor inoculation, temozolomide (double distilled water group with 10mg/kg or 20mg/kg doses; deuterium depleted water group with 10mg/kg dose) in a diluted configuration with double distilled water or 25ppm deuterium depleted water was injected intraperitoneally, once a day, and the control group was injected with the same amount of solvent. This was until the subcutaneous tumor grew for 3 weeks. The experiment shows that: 1) the deuterium-depleted biological enzyme regulator DDW can inhibit the growth of tumors in mice. 2) Under the condition of reducing the drug amount of TMZ by 50%, the tumor inhibition effect of DDW and TMZ combined DDW + TMZ (10mg/kg) is still better than that of H2O + TMZ (20mg/kg), namely, the deuterium-depleted composition has a synergistic gain effect of increasing one to more than two on tumor inhibition, as shown in figure 17.

To further investigate whether the use of deuterium-depleted water in combination with an immunomodulator could enhance the anti-glioma effect, we used a CDX animal experiment to observe the effect of deuterium-depleted water in combination with the PD-L1 antibody, atuzumab. Mice were fed with double distilled water and 25ppm deuterium-depleted water for 3 weeks, then ALTS1C1 was inoculated subcutaneously into C57BL/6 mice to form subcutaneous tumors, and then the mice were further fed with double distilled water and 25ppm deuterium-depleted water, 5 days after subcutaneous tumor inoculation, along with dilution of the prepared alemtuzumab (at a dose of 10 mg/kg) with double distilled water or 25ppm deuterium-depleted water, were injected intraperitoneally once every 3 days for a total of 3 needles, and the control group was injected with equal amount of solvent, and thus subcutaneous tumors were grown for 3 weeks. Experiments prove that the deuterium-depleted water can obviously enhance the anti-glioma effect of tializumab (figure 18).

Example 10, the bio-enzyme regulator of the present invention inhibits the key enzyme PFKFB3 strongly as the deuterium content decreases.

T98G-150PPM refers to common distilled water group, T98G-50PPM, T98G-25PPM refers to the deuterium-depleted biological enzyme regulator of the invention, the deuterium content of which is respectively 50PPM and 25PPM deuterium-depleted water. As shown in FIG. 19, the deuterium content of the biological enzyme regulator with low deuterium content increases the inhibition and degradation capability of the biological enzyme regulator with low deuterium content on the expression level of the key enzyme PFKFB3 in low sugar glycolysis. That is, the extent of this degradation change is related to the decrease in deuterium content from the point of view of the concentration gradient.

As shown in fig. 20, 25ppm of deuterium-depleted water constituted the deuterium-depleted biological enzyme modulator of the present invention had more significant inhibition and degradation of the expression of PFKFB3 compared to the PFK15 inhibitor group.

The regulation of metabolic processes of other diseases or other cell culture processes in the present invention is analogous to this logic and method, and is not described here.

While the invention has been described with respect to a preferred embodiment, it will be understood by those skilled in the art that the foregoing and other changes, omissions and deviations in the form and detail thereof may be made without departing from the scope of this invention. Those skilled in the art can make various changes, modifications and equivalents to the disclosed technology without departing from the spirit and scope of the present invention, and all such changes, modifications and equivalents are intended to be included therein as equivalents of the present invention; meanwhile, any equivalent changes, modifications and evolutions of the above embodiments according to the essential technology of the present invention are still within the scope of the technical solution of the present invention.

Claims

1. Use of deuterium depleted water in any one or more of:

preparing a biological enzyme regulating agent;

preparing a cell glycolysis inhibitor;

preparing a tumor treatment product;

preparing a tumor proliferation inhibitor;

preparing a tumor cell clone inhibitor;

preparing angiogenesis promoting ability inhibitor of tumor cells;

preparing a tumor growth inhibitor;

preparing an antitumor drug synergist;

preparing a synergist of the enzyme inhibitor;

preparing a cardiovascular drug synergist;

preparing a drug synergist for the basic metabolic diseases;

2. The use of claim 1, further comprising one or more of the following features:

1) the deuterium-depleted water has a deuterium content of 15ppm to 100 ppm.

2) The biological enzyme regulating agent is a preparation with inhibition and/or degradation effects on biological enzymes;

3) the tumor is selected from glioma;

4) the anti-tumor drug is selected from cytotoxic drugs, hormone drugs, biological response regulators, monoclonal antibody drugs, cell differentiation inducers, apoptosis inducers, angiogenesis inhibitors or epidermal growth factor receptor inhibitors;

5) the enzyme inhibitor is selected from PFKFB inhibitor;

6) The cardiovascular medicine is selected from anti-angina, anti-arrhythmia, anti-hypertension, anti-cardiac insufficiency or peripheral vasodilator;

7) the basal metabolic disease drug is selected from drugs with main indications of diabetes, diabetic ketoacidosis, hyperglycemia and hyperosmolar syndrome, hypoglycemia, gout, protein-energy malnutrition, vitamin A deficiency disease, scurvy, vitamin D deficiency disease or osteoporosis disease;

8) the deuterium-depleted water inhibits tumor cell proliferation, inflammatory responses, limits cardiovascular or respiratory disease focus development, or retards and controls other underlying metabolic disease processes by altering cellular or other living body metabolic pathways and levels.

3. Use according to claim 2, further comprising one or more of the following features:

a. in feature 2), the biological enzyme is selected from a PFKFB enzyme or a SRC protein;

b. the anti-tumor drug is selected from rapamycin, everolimus, temozolomide, sapercetin or PD-L1 antibody;

c. in feature 5), the PFKFB inhibitor is selected from PFK15, PFK158, 3PO, quinoline or quinazoline compounds, diaryl ether compounds or AZ 67.

4. A biological enzyme modulator, wherein the biological enzyme modulator comprises deuterium-depleted water having a deuterium content of greater than 0.1ppm and less than 135 ppm.

5. The biological enzyme modulator according to claim 4, further comprising one or more of the following features:

1) the deuterium-depleted water is an active ingredient or one of the active ingredients of the biological regulating agent;

2) the deuterium content of the deuterium-depleted water is 15ppm to 100 ppm;

3) the biological enzyme regulating agent is a preparation with inhibition and/or degradation effects on biological enzymes;

4) the biological enzyme is selected from PFKFB enzyme or SRC protein.

6. A deuterium depleted composition comprising a biological enzyme modulator according to any one of claims 4 to 5, and a drug selected from the group consisting of: antineoplastic drugs, cardiovascular drugs, drugs for respiratory diseases, drugs for basic metabolic diseases or enzyme inhibitors.

7. The deuterium depleted composition of claim 6, further comprising any one or more of the following features:

1) the tumor is selected from glioma;

2) the anti-tumor drug is selected from cytotoxic drugs, hormone drugs, biological response regulators, monoclonal antibody drugs, cell differentiation inducers, apoptosis inducers, angiogenesis inhibitors or epidermal growth factor receptor inhibitors;

3) The enzyme inhibitor is selected from PFKFB inhibitors;

4) the cardiovascular medicine is selected from anti-angina, anti-arrhythmia, anti-hypertension, anti-cardiac insufficiency or peripheral vasodilator;

5) the drug for the basal metabolic disease is selected from drugs with main indications of diabetes, diabetic ketoacidosis, hyperglycemia and hyperosmolar syndrome, hypoglycemia, gout, protein-energy malnutrition, vitamin A deficiency disease, scurvy, vitamin D deficiency disease or osteoporosis disease.

8. The deuterium depleted composition of claim 7, further comprising any one or more of the following features:

a. the anti-tumor drug is selected from rapamycin, everolimus, temozolomide, sapercetin or PD-L1 antibody;

b. the PFKFB inhibitor is selected from PFK15, PFK158, 3PO, quinoline or quinazoline compounds, diaryl ether compounds or AZ 67.

9. A cell culture medium comprising the biological enzyme modulator of any one of claims 4-5 and a basal cell culture medium selected from BME cell culture medium, MEM cell culture medium, DMEM cell culture medium, IMDM cell culture medium, RPMI-1640 cell culture medium, HamF10 cell culture medium, DMEM/F12 cell culture medium, M199 cell culture medium, McCoy5A culture medium, L15 cell culture medium, primary cell culture medium, or stem cell culture medium.

10. Use of a biological enzyme modulator according to any one of claims 4 to 5, or a deuterium depleted composition according to any one of claims 6 to 8, or a cell culture medium according to claim 9, for the modulation of cellular metabolism.

11. The use of claim 10, further comprising one or more of the following features:

1) said use belongs to a non-disease therapeutic or diagnostic purpose;

2) the application belongs to the field of control bioengineering or life science research;

3) before application, the biological enzyme regulator, the deuterium-depleted composition or the cell culture medium is subjected to preset treatment, or the deuterium concentration of deuterium-depleted water or the combination ratio of deuterium-depleted water and a combined compound is adjusted; or adjusting the dosage, time frequency of application of a biological enzyme modulator, or said deuterium depleted composition, or said cell culture medium.

12. The use of claim 11, wherein said pre-treatment is such that water clusters in the deuterium depleted water are uniformly distributed and effectively bound to other components of the deuterium depleted composition or to components of the culture medium.