RU2707554C1 - Composition inhibiting tumor growth and survival rate - Google Patents

Abstract

FIELD: biology.

SUBSTANCE: invention relates to molecular biology and is a composition for growth inhibition and tumor cell survival comprising 2-amino-2-deoxy-D-glucose (glucosamine D) and 2-deoxy-D-glucose (2-DG), wherein concentration of 2-amino-2-deoxy-D-glucose (glucosamine D) and 2-deoxy-D-glucose (2-DG) is equal to 3–10 mM for each component, respectively, and the ratio of components is equal to 1:1.

EFFECT: use of the declared invention enables higher stability of the composition, provides high selectivity with respect to tumor cells due to combined effect on tumor cells and higher inhibition of proliferation.

1 cl, 7 dwg, 1 ex

Description

The invention relates to the field of molecular biology, exploring new possibilities for increasing the selective death of tumor cells without damaging normal ones.

Tumor chemotherapy has traditionally been aimed at inhibiting the proliferation of cancer cells. A decrease in proliferative activity is accompanied by a blocking of progression along the cycle and subsequent death of the tumor cell. A unique metabolic feature of tumor cells is aerobic glycolysis, which leads to their increased dependence on glucose. Glucose analogues glucosamine D, and 2-deoxy-D-glucose (2-DG) are glycolysis inhibitors, namely the first stage of glycolysis, inhibiting the activity of hexokinase. In the works: Journal of Zhejiang University SCIENCE In 2006, 7, 608-614 [1]; Nature 1953, 171, 252-254 [2] shows the toxicity of D-glucosamine for several malignant cell lines and in vivo tumors at concentrations having little effect on normal cells. Its antioxidant and anti-inflammatory ability has been shown, which makes it an attractive multifunctional therapeutic agent: Life Science, 2016, 152, 21-29 [3]. It is also known that 2-deoxy-D-glucose is toxic to various types of cancer cells. In addition, it increases the effectiveness of radiation therapy and chemotherapy. 2-deoxy-D-glucose is widely used in research as a metabolic inhibitor: Nat. Rev. Cancer 2014, 14 (4), 263-276 [4]. It has been shown that, on different cell lines of cancer cells, the ability of 2-DG to slow down proliferation, stop the cell cycle, accompanied by the induction of moderate apoptosis, up to a complete stop of the cell cycle and massive apoptosis: Anticancer Research 2006, 26, 3561-3566 [5]. The selective efficacy of 2-deoxy-D-glucose in concentrations from 20 to 80 mM was shown to induce apoptotic death of tumor cells and to block the cell cycle in G2 / M phases, including, depending on the level of glucose in the nutrient medium: Pathological physiology and experimental therapy: Pathological physiology and experimental therapy 2014, 58 (4), 78-85 [6]. It is known that D-glucosamine can both induce and inhibit apoptosis depending on the type of cells and the activity of a specific biochemical pathway.

It is also known the use of glucosamine derivatives in combination with other substances to increase toxicity to tumor cells:

Known patent RU 2138257, IPC A61K 31/375, A61K 31/19 "Pharmaceutical composition for the prevention and treatment of cancer", which proposed D-glucosamine in combination with other substances [7]. The composition includes at least three active compounds: at least one amino acid, at least one vitamin, and at least one component selected from the group consisting of adenine, 2-deoxy-D-ribose, D-mannose, D-glucosamine , malic acid, oxalic acetic acid, adenosine triphosphate and / or their pharmaceutically acceptable salts, given that if the composition contains, in addition to amino acids, only malic acid and a vitamin, then the vitamin cannot be ascorbic acid. That is, D-glucosamine was used as the third component and in approximately one of the 10 possible formulations. We used concentrations that reduced cell proliferation precisely with combined exposure.

In this composition, only D-glucosamine has selectivity for tumor cells, the remaining components can change the metabolism of both normal and tumor cells, which leads to an uncertainty of the expected result.

Known patent WO 2005025581, IPC A61K 31/375, A61K 31/19 "Use of n-acetyl-d-aminog lycosamine in preparation of drugs for the treatment of cacer and metastasis" [8], where it is proposed to use a derivative of D-glucosamine - - N-acetyl-D-aminoglycosamine for the treatment of cancer and metastases. The authors suggest that N-acetyl-D-aminoglycosamine can integrate into the cell membrane, be included in the process of cancer metabolism and stop the transfer of energy to cells, which leads to their death. The authors claim that this drug showed a very significant effect both in laboratory experiments and in animal models and in human tests.

The lack of analogue. Glucosamine is unstable, therefore, its salts, hydrochloride or sulfate or N-acetyl, are mainly used in therapy.

N-acetylglucosamine is not soluble in water, soluble in concentrated organic and inorganic acids, which reduces the effectiveness and selectivity of exposure to tumor cells.

Patent CA 26663398, IPC A61K 31/7028 "A caner sensitizer containing glucosamine, glucosamine derivatives or salts thereof (priority from 2006-06-16) [9] is considered as a prototype. The composition consists of D-glucosamine and its sulfate salts, which are considered as additives to increase the sensitivity of cancer cells to anticancer agents without causing side effects.The second component is chemotherapeutic drugs: mechlorethamine, chlorambucil, phenylalanine, mustard, cyclophosphamide, ifosfamide, carmustine (BCNU), lomustine (CCNU), streptozoto, thioplatin, thiot Otepaa, dactinomycin (actinomycin D), doxorubicin (adriamycin), daunorubicin, idarubicin, mitoxantrone, plicamycin, mitomycin C, bleomycin, vincristine, vinblastine, paclitaxel, docetaxel, etoposide, tenipozekan, topototekan, tramp.

The used concentration of D-glucosamine or its salt of -10 mM is non-toxic to normal cells, i.e. selective for tumor cells.

The disadvantages of the prototype are that the second component of the composition is chemotherapeutic agents, which are toxic for both healthy and tumor cells, i.e. do not have selectivity for tumor cells. In addition, the sulfate form of glucosamine is not stable enough. Assessment of cell viability was not adequately assessed only using MTT, which is based on the restoration of formazan using intracellular NAD (P) H-oxidoreductases, i.e. by metabolic activity (viability). Despite the huge number of works using this simple method, conclusions about the inhibition of proliferation based on analyzes of metabolic viability may be erroneous, because they may not correlate with growth inhibition and / or loss of clonogenic survival, this is stated in the works: Sci Rep . 2018. v.8.n.1: 1531 [10]; J Cell Mol Med. 2010 [11].

Technical effect: the creation of a stable composition and having a higher selectivity with respect to tumor cells.

An object of the present invention is to provide a composition that acts in combination with tumor cells by increasing inhibition of proliferation.

The technical effect is achieved by the fact that in the known composition that inhibits the growth and survival of tumor cells, which includes two components, one of which is a glucosamine derivative as an inhibitor of cell metabolism, a new one is that 2-amino-2-deoxy-D is used as a glucosamine derivative -glucose, and the second component is a second inhibitor of cellular metabolism of 2-deoxy-D-glucose.

The concentration of the claimed components 3-10 mm for each component and their ratio is 1: 1.

The claimed combination of features is based on experimental data showing inhibition of tumor cell proliferation in various phases of the cycle: glucosamine inhibits the progression of cells from G1 to S-phase, and 2-DG blocks cells in G2 / M, which provides a synergistic effect of the combined action in this composition components of the composition and leads to apoptotic death of tumor cells.

The claimed combination of features is not known from open sources of information.

To clarify the invention, the following graphic materials.

In FIG. 1 shows the structural formulas of the composition: 2-amino-2-deoxy-D-glucose (D-glucosamine) on the left, 2-deoxy-D-glucose (2-DG) on the right

In FIG. Figure 2 presents the results of experiments to assess the effect of various concentrations of D-glucosamine and 2-deoxy-D-glucose (2-DG) on the cell viability of two lines, carried out using the MTT method.

In FIG. Figure 3 presents the results of a cytometric analysis of the effect of 20 mM D-glucosamine on cycle progression and apoptotic death of HeLa G63 and ECV 304.

In FIG. 3 (A) presents the results of a cytometric analysis during the treatment of HeLa G63 cells with glucosamine at a concentration of 0.1 mM and without treatment.

In FIG. Figure 4 presents data on the assessment of changes in cell concentration in samples after 48-hour incubation with D-glucosamine at the same initial concentration when sieving them in vials (effect on cell growth).

In FIG. 5 in the form of diagrams reflecting the level of apoptotically dying cells (cells with DNA content <2s), the results of separate and combined treatment of cells with glucosamine D and 2-deoxy-D-glucose in various concentrations are presented, followed by their cultivation in a medium with normal (full medium: 4 g / l) and low glucose (starvation: 1 g / l).

In FIG. Figure 6 presents the results of experiments evaluating the influence of glucose level in a nutrient medium on cell progression in a cycle in the form of cell distribution by DNA content (2c-G1, 3c-S, 4c-G2 / M phase of the cell cycle).

In FIG. 7 presents the results of a cytometric analysis of the influence of glycolysis inhibitors of D-glucosamine and 2-DG on the distribution of ECV line cells by DNA content when they are cultured in a nutrient medium with different glucose levels. (<2s apoptosis, 2s - G1, 3s -S, 4s-G2 / M phases of the cell cycle).

Examples of specific implementation of the method.

Objects of study: cells of the cervical epithelioid carcinoma HeLa G 63 line, human endothelial cells ECV 304 line. These cell lines are cultured in the Needle medium (Biolot company) with the addition of 10% cattle serum (Biolot company) and 100 units ./ml penicillin-streptomycin (Gibco BRL) at 37 ° C. Subcultured every three days.

As proliferation inhibitors, 2-amino-2-deoxy-D-glucose was used as described above, and the second component is a second glycolysis inhibitor of 2-deoxy-D-glucose. Both components are hexokinase inhibitors. In contrast to the considered analogues (where only glucosamine is used as an inhibitor of hexokinase.)

It is important that glucosamine hydrochloride is proposed as a glucosamine salt, glucosamine HCl is non-toxic, has high hydrophilicity and is more stable than glucosamine sulfate. Considering the fact that glucosamine hydrochloride does not require stabilization and is used practically in its pure form, a number of scientists believe that glucosamine hydrochloride: Cartilage is better to use in therapy. 2016 Jan; 7 (1): 70-81 [12]

The following is an analysis of cytotoxicity using the MTT method: Biotechnol Annu Rev 11 127-152 (2005) [13]. Colorimetric analysis during the restoration of the yellow tetrazolium salt to purple formazan in the mitochondria of viable cells. Cells are seeded at a concentration of 1 × 10 ⁵ cells / well in 24-well plates. After incubation for 24 hours in a nutrient medium, either glucosamine D (Sigma), or 2-deoxy-D-glucose, or both glucosamine + 2-decose-D-glucose (Sigma) in doses of 3 to 10 mm, in the ratio 1: 1. Control without processing. On the day of analysis, after (24-48 hours) the culture medium in each well was replaced with fresh culture medium containing MTT (3- (4,5-dimethylthiazol-2-yl) -2,5-diphenyltetrazolium) (Sigma) at a concentration of 0.5 mg per ml, followed by incubation in it for an additional 4 hours. After removing the medium, 500 μl of DMSO was added to each well, and the formazan crystals were dissolved using an orbital shaker and analyzed on a Thermoficher reader. Cell survival was presented as a percentage of the test group relative to the control. These experiments were performed for three samples and were repeated at least at least three times, for each experiment the average of three independent experiments and standard deviations are given.

It was found (Fig. 2) that treatment of cells with glucosamine and 2-deoxy-D-glucose leads to a dose-dependent decrease in metabolic activity in the studied cell lines. At the same time, tumor cells (HeLa G63) were approximately 3 times more sensitive to glucosamine than non-tumor ones (ECV 304). An approximately equal effect was recorded at glucosamine doses of 3 mM for HeLa G63 and 10 mM for ECV 304. When treating cells with 2-deoxy-D-glucose, an equal effect was recorded for HeLa G63 10 mM, for ECV 304 - 20 mM, i.e. HeLa G 63 cells were twice as sensitive to 2-deoxy-D-glucose as ECV 304. The obtained data confirm the presence of selective cytotoxic dose-dependent effects in each of the preparations. With increasing concentration, differences between cell lines become more pronounced.

In contrast to the prototype, a study of the effect of this composition on cell growth and survival was also carried out using a cytometric analysis of the composition of the cell population (Fig. 3). For this, the cells were incubated with preparations in the indicated concentration range, washed and incubated with an ethidium bromide fluorescent probe. 1 hour before the analysis on a flow cytometer, the cells were incubated for 10 minutes in a hypotonic solution of ethidium bromide, which quantitatively binds to the DNA of the cells; therefore, the distribution of cells by DNA content makes it possible to follow the cell progression in a cycle: 2c-G1 phase; 3c-S phase; 4c-G2 / M phase. The cytotoxicity of the composition was assessed by the level of cells with a DNA content less than diploid (<2 s or sub-G1 population), which appear as a result of nuclear fragmentation during apoptotic cell death: Flow Cytometry Data Analysis: Basic Concepts and Statistics [14] and in parallel morphological analysis of the population on cells by microscopy of drugs. It was found that after 48 hours of treatment of cells of both lines with D-glucosamine at a concentration of 20 mM (for some cell lines, a concentration of 20 mM D-glucosamine is considered toxic), an increase in the proportion of cells in G _{1 is} recorded, which indicates a violation of the cell progression along the cycle. A 20-fold decrease in the concentration of D-glucosamine to 0.1 mM (Fig. 3A) also leads to cell accumulation in the G1 phase, but without a visible cytotoxic effect. For tumor cells, the appearance of cells with a DNA content of <2s cells (apoptotically dying cells) is recorded. Compared with the untreated control (2%), the level of cells with a content of <2s increased by almost 8 times (15%). ECV 304 cells only slowed the progression of the cell cycle, the proportion of cells in the G1 phase increased from 46% in the control to 60% after treatment with D-glucosamine at a concentration of 20 mM. The proportion of cells with DNA content <2s did not change, which suggests the absence of cell death of the ECV 304 line. In morphological analysis, cells with fragmented nuclei (apoptotically dying cells) were also not recorded.

It was found (Fig. 4) that after 48-hour incubation of cells with D-glucosamine at concentrations of 3 mM and 10 mM, the growth of HeLa cells sharply decreases to 55 and 40% of the control. Moreover, the concentration of ECV cells after similar treatment is 95-85% of the control, which indicates the selective toxicity of D-glucosamine in relation to tumor cells. These results correlated with the results of cytometric and morphological analysis of the composition of the cell population and no correlation with the data obtained using the MTT method.

It was found (Fig. 5) that treating cells with a glucosamine D + 2-deoxy-D-glucose composition in various concentrations, followed by culturing them in a medium with normal (full medium) (4 g / l) and low glucose (hungry medium - 1 g / l) led to an increase in the death of tumor cells compared with the separate treatment of each of the agents of the composition. For example, 5 mM glucosamine D + 5 mM 2-deoxy-D-glucose induced greater cell death than 10 mM glucosamine D or 10 mM 2-deoxy-D-glucose, separately, although the total concentration of inhibitors in the medium was the same (10 mm). When cells are cultured in an environment with a low glucose content (1 g / l), the effectiveness of the combined treatment increases. For example, the combined treatment of 5 mM glucosamine D + 5 mM 2-deoxy-D-glucose in complete medium and 3 mM glucosamine + 3 mM 2-deoxy-D-glucose in a hungry medium induced approximately the same number of apoptotically dying cells (20%), and 10 mM glucosamine + 10 mM 2-deoxy-D-glucose in complete medium, as 5 mM glucosamine + 5 mM 2-deoxy-D-glucose in a hungry medium, i.e. the level of glucose in the nutrient medium changed the cytotoxic effect twice and even more. Thus, the synergistic effect of the claimed composition and its amplification while lowering the level of glucose in the medium is proved.

It was found (Fig. 6) that treatment of HeLa G63 cells with glucosamine leads to an increase in the level of cells with a DNA content of 2c compared to the untreated control, i.e. glucosamine inhibits the progression of cells from G1 to S-phase in a dose-dependent manner; a decrease in the level of glucose in the nutrient medium somewhat eliminates the dose dependence. Treatment of 2-DG cells at the same concentrations and with the same treatment mode leads to an increase in the level of cells with 4c DNA content (G2 / M phase), this is especially evident when cells are incubated in a medium with a low glucose content and when combined with the composition. Inhibition of cell progression at two points of the cell cycle with combined exposure leads to an increase in the cytotoxicity of even low concentrations of D-glucosamine and 2-DG. Using this composition, therapeutic doses can be significantly reduced, selectivity of drugs can be increased, and possible side effects can be avoided.

It was found (Fig. 7) that treatment of ECV 304 D-glucosamine, 2-DG cells at a concentration of 3 mM did not violate the cycle progression, because the level of cells in the contents of 2c (G1 phase) and 4c (G2 / M phase) did not differ significantly from the control. The level of cells with DNA content <2s (apoptotically dying cells) is very low (5-7%) and practically does not differ from the control. At a concentration of 10 mM, an insignificant increase in the proportion of 2c cells after treatment with glucosamine and an increase in the proportion of 4c after treatment with 2-DG are recorded, when cultured in a nutrient medium with both normal (4 g / l) and low (1 g / l) glucose . Thus, we prove the selectivity of this composition in relation only to tumor cells.

Thus, FIG. 2, FIG. 2a, FIG. 4, FIG. 5 and FIG. 6, that the claimed composition has a combined effect on tumor cells by increasing inhibition of proliferation by blocking cells in the G1 / S phases, and in G2 / M, phases of the cell cycle, which leads to apoptotic death of tumor cells.

Thus, a composition is proposed that contains two hexokinase inhibitors, namely 2-amino-2-deoxy-D-glucose (D-glucosamine) and 2-deoxy-D-glucose (2-DG) (the structural formulas of which are shown in FIG. . 1), acting on tumor cells in combination: glucosamine D in the studied concentrations inhibits the progression of cells from G1 to S-phase, and 2-DG blocks cells in G2 / M. Violation of the control over cell progression at a cycle at two points of the cell cycle leads to an increase in cytotoxicity even at low concentrations of D-glucosamine and 2-DG, which can significantly reduce the therapeutic concentration of drugs to a level that does not have any noticeable negative effect on normal cells.

The invention provides high antitumor activity and selectivity of action of D-glucosamine and 2-deoxy-D-glucose (2-DG) while significantly reducing the possible toxic effects on normal cells.

The inventive composition can be used in biological research and in preclinical trials. D-glucosamine is a pharmacopeia drug and is widely used in medicine as a drug substance to inhibit the development of degenerative processes in the joints, restore their function and reduce joint pain. The fact that it is a well-known pharmacopeia drug with a fairly long history of its use in medical practice greatly facilitates its possible use, including for the treatment of cancer, since this drug not only does not require any preclinical studies on its toxicity , safety and possible side effects, but is also widely available in the form of dosage forms in the pharmacy network, as well as nutritional supplements.

List of references

1. Zhang L, Liu W-S, Han B-Q, et. al. Antitumor activities of D-glucosamine and its derivatives. Journal of Zhejiang University SCIENCE B 2006, 7, 608-614;

2. J.H. Quastel, A. Cantero, Inhibition of tumor growth by D-glucosamine, Nature 1953, 171, 252-254;

3. Dalirfardouei R., Rfrimi G, Jamialahmadi K. Molecular mechanisms and biomedical applications of glucosamine as a potential multifunctional therapeutic agent. Life Science, 2016, 152, 21-29.

4. Lee A.M. Glucose regulated proteins in cancer: molecular mechanisms and therapeutic potential. Nat. Rev. Cancer 2014, 14 (4), 263-276.

5. Zhang X.D., Deslandes E., Villedieu M., Poulain L., Duval M., Gauduchon P., Schwartz L., Card P. Effect of 2-Deoxy-D-glucose on Various Malignant Cell Lines In Vitro. Anticancer Research 2006, 26, 3561-3566

6. Gigliano N.Ya., Bondarev GN, Konevega LV, et al. Possible mechanisms of the selective action of glycolysis inhibitors on endotheliocytes and human carcinoma cells in culture. Pathological physiology and experimental therapy. 2014, 58 (4), 78-85.

7. RU 2138257 C1 "Pharmaceutical composition for the prevention and treatment of cancer" priority 08/08/1994, IPC A61K 31/375, A61K 31/19

8. WO 2005025581 A1 "Use of n-acetyl-d-aminog lycosamine in preparation of drugs for the treatment of cacer and metastasis" priority 2003.09.17, IPC A61K 31/7008

9. CA 2663398 C "A cancer sensitizer comprising glucosamine, glucosamine derivatives or salts thereof 2012-01-31, priority 2006.06.16, IPC A61K 31/7028, IPC prototype.

10. Rai Y., Pathak R., Kumari N. et. al. Mitochondrial biogenesis and metabolic hyperactivation limits the application of MTT assay in the assessment of radiation induced growth inhibition. Sci rep. 2018.v.8.n.1: 1531

11. Quent VM, Loessner D, Friis T, et. al. DW. Discrepancies between metabolic activity and DNA content as tool to assess cell proliferation in cancer research. J Cell Mol Med. 2010

12. Bascoul-Colombo C, Garaiova I, Plummer SF, Harwood JL, Caterson B, Hughes CE. Glucosamine Hydrochloride but Not Chondroitin Sulfate Prevents Cartilage Degradation and Inflammation Induced by Interleukin-la in Bovine Cartilage Explants. Cartilage 2016 Jan; 7 (1): 70-81.

13. Berridge, M.V., Tan, A.S., Herst, P. M. .. Tetrazolium dyes as tools in cell biology: New insights into their cellular reduction. Biotechnol Annu Rev 11 127-152 (2005).

14. Watson J V 2005. Flow Cytometry Data Analysis: Basic Concepts and Statistics).

Claims (1)

Composition for inhibiting the growth and survival of tumor cells, including 2-amino-2-deoxy-D-glucose (glucosamine D) and 2-deoxy-D-glucose (2-DG), while the concentration for 2-amino-2-deoxy Β-D-glucose (glucosamine D) and 2-deoxy-D-glucose (2DG) are 3 to 10 mM for each component, respectively, and the component ratio is 1: 1.

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