RU2494744C1 - Anticancer therapeutic agent of acadesine - Google Patents

Abstract

FIELD: medicine, pharmaceutics.

SUBSTANCE: what is presented is a therapeutic agent of acadesine with high anti-cancer activity, containing acadesine, as well as a non-steroid anti-inflammatory preparation: ibuprofen, or indomethacin, or aspirin.

EFFECT: what is shown is synergetic anti-cancer action of the claimed agent on a B-cell leukaemia model.

3 tbl, 3 ex

Description

The invention relates to medicine, in particular oncology and hematology, in particular to a therapeutic agent for the treatment of B-cell leukemia based on 5-aminoimidazole-4-carboxamide-1-ribofuranoside (AICAR, acadezin) in combination with one of the non-steroidal anti-inflammatory drugs (NSAIDs) )

Among the common types of malignant tumors are hemoblastoses (tumors of the hematopoietic system), in particular malignancy of B-lymphocytes (B-cell leukemia). At present, basic polychemotherapy (anthracycline antibiotics, alkylating agents, antimitotic drugs, etc.) is practically the only way to save life and maintain its relatively high quality in patients with B-cell leukemia. In relapses of the disease, new generation drugs are used - protein kinase inhibitors dasatinib and ponatinib. These treatment regimens are carried out as multi-month and multi-year courses and, therefore, are expensive [Tre AR, Gonzalez-Angulo AM. Breast cancer and metastasis: on the way toward individualized therapy. Cancer Genomics Proteomics. 2012 9 (5): 297-310; Rodrigues G, Sanatani M. Age and comorbidity considerations related to radiotherapy and chemotherapy administration. Semin Radiat Oncol. 2012, 22 (4): 277-283)].

A feature of tumor cells is the acquisition of resistance to almost any drug. This is due to a violation of the mechanisms of death in tumor cells and the survival of variants with non-functioning signaling pathways of programmed death (apoptosis). This most important difficulty of antitumor therapy makes it necessary to search for substances that can cause non-apoptotic types of tumor cell death. These substances include acadezin [Van Den Neste E, Van den Berghe G, Bontemps F. AICA-riboside (acadesine), an activator of AMP-activated protein kinase with potential for application in hematologic malignancies. Expert Opin. Investig. Drugs 2010, 19 (4): 571-578; Drew BG, Kingwell BA. Acadesine, an adenosine-regulating agent with the potential for widespread indications. Expert Opin. Pharmacother 2008, 9 (12): 2137-2144].

Akadezin is important for antitumor therapy due to the unique mechanism of antitumor action: this compound causes complex death with the participation of the necrosis mechanism [Glazunova et al., AICAR is a broad-spectrum antitumor drug. Oncological journal of the public association "Belarusian Oncological Society", 2012, volume 6, p.16]. The practical use of acadesine is constrained by the extremely high cost of the substance obtained by organic synthesis. Recently, an effective method for producing acadesine using microbiological synthesis using a recombinant producer (RU 2405833) has been developed. The cytotoxicity of the natural substance for the culture of B-leukemia cells does not differ from the activity of synthetic acadezin [Glazunova et al., AICAR is a broad-spectrum antitumor drug. Oncological journal of the public association "Belarusian Oncological Society", 2012, volume 6, p.16].

The molecular mechanisms of the antitumor effect of acadesine are far from clear, however, it is currently considered as a promising anti-leukemia drug [Leukemia PLoS ONE, 2009.4 (11), e2889, p.1-11; Expert Opin.Invest. Drugs 2010.19 (4), p. 571-578; Blood. 2010., 116 (16) 3023-3032; Oncotarget. 2011, 2 (12), p.1322-1328; Mol. Cancer Ther. 2011, 10 (3), p. 437-447].

The closest analogue of the claimed invention is acadezin - a therapeutic agent with antitumor activity, characterized by selective suppression of b-lymphocytes, but not T-lymphocytes [WO 203/080076, Blood. 2003, 101, p. 3674-3680; Leukemia. 2005, 19, p. 292-294]. The disadvantage of acadesine as an antitumor agent is its rapid conversion into purine nucleotides, which reduces its intracellular content. However, it is known that some NSAIDs (ibuprofen, indomethacin, aspirin) inhibit the in vitro activity of the transformylase cyclohydrolase enzyme, which is responsible for the conversion of acadesine to purine nucleotides [Baggott JE, Morgan SL, T, Vaughn WH, Hine RJ. Inhibition of folate-dependent enzymes by non-steroidal anti-inflammatory drugs. Biochem J. 1992, 15; 282 (Pt l): 197-202].

The objective of the present invention is to develop a therapeutic agent based on acadesine with a more pronounced antitumor activity.

The problem was solved by developing a therapeutic agent based on acadesine, which also includes NSAIDs: ibuprofen, or indomethacin, or aspirin.

The invention is confirmed by the following examples.

Example 1. Cytotoxicity of acadesine and its combinations with NSAIDs for mouse B-lymphocytic leukemia cells (line P388).

Cells of the B-cell leukemia line (P388 line) were cultured in Dulbecco's modified Eagle medium (DMEM medium) supplemented with 2 mM L-glutamine, 100 U / ml penicillin and 100 μg / ml streptomycin (culture medium) at 37 ° C, 5% CO ₂ in a humidified atmosphere. On the day of the experiment, cells were counted in a Goryaev chamber and suspended in culture medium to a concentration of 50,000 cells / ml. 190 μl of cell suspension (-10 thousand cells) were added to the wells of a 96-well plate (Nunc, Denmark) in culture medium and incubated for 16 h at 37 ° C, 5% CO ₂ . Serial dilutions of acadezin, ibuprofen, indomethacin, aspirin and piroxicam from stock solutions (10 mM in dimethyl sulfoxide for each test compound except aspirin; 100 mg / ml for aspirin) were prepared. From each dilution, the required final concentration of the drug in the well was obtained in a volume of 5-10 μl. A solution of dimethyl sulfoxide was added to control wells to a final concentration of 0.5%. Cells were incubated with each compound individually or with combinations of acadesine with the remaining compounds for 72 hours. Each concentration was examined in triplicate. After incubation, 20 μl of an aqueous solution (5 mg / ml) of 3- (4,5-dimethylthiazol-2-yl) -2,5-diphenyltetrazolium bromide was added to the wells. Cells were incubated for 2 h until the development of dark purple color of the cells (formazan), the culture medium was removed. 100 μl of dimethyl sulfoxide was added to the cells and suspended. The optical density was measured on a spectrophotometer at a wavelength of 570 nm, subtracting the optical density at 690 nm. The percentage of surviving cells was calculated as the ratio of optical density (OD _{570 nm} -OD _{690 nm} ) of the control (without preparations) wells to the corresponding indicator in wells with a particular concentration of the test compound or a combination of the test compounds.

Table 1 Cytotoxicity of acadesine and NSAIDs for P388 cell line. Compound Concentration Acadezine 0 1.5 3 7 fifteen 30 μm Ibuprofen 0 mm one hundred* one hundred one hundred 94 62 29th 0.2 mm one hundred one hundred 98 65 32 12 0.4 mm one hundred 60 39 eighteen 8 5 Indomethacin 0 mm one hundred one hundred one hundred 94 62 29th 0.1 mm 96 fifteen 12 5 four 3 Aspirin 0 mm one hundred one hundred one hundred 94 62 29th 0.5 mg / ml 98 64 51 32 21 fifteen 1 mg / ml 95 46 32 twenty 16 8

Notes: *% of surviving cells (average of 3 independent measurements). The most pronounced results of synergism of acadesine with other compounds are emphasized.

From the above data it follows that the effect of only acadezin on B-leukemia cells was toxic in the concentration range of 15-30 μM. The compounds with which acadezin combinations were tested are used in non-toxic or weakly toxic concentrations. A synergistic effect was observed for ibuprofen, or indomethacin, or aspirin. Thus, acadezin (3 μM) and ibuprofen (0.4 mmol) separately did not cause pronounced cell death, and only 39% of the cells survived with the combination of the two compounds at those concentrations. Pronounced synergism is seen with the combined action of 1.5-3 μM acadezin and 0.1 mm indomethacin.

Thus, non-steroidal anti-inflammatory agents: ibuprofen, or indomethacin, or aspirin, enhance the cytotoxicity of acadezin for cultured cells of B-lymphocytic leukemia.

Example 2. Antitumor activity of acadesine in combination with ibuprofen in a model of B-cell leukemia P388 in laboratory mice.

Cells of P388 lymphocytic leukemia induced in DBA / 2 mice by smearing the skin with methylcholanthrene were transplanted into first generation BDF ₁ hybrid mice (C ₅₇ Bl / _6j × DBA / 2) subcutaneously in the right axillary region. Cells were obtained from ascites fluid of tumor-bearing mice. The number of cells for vaccination is 10 ⁶ cells in a nutrient medium 199. Treatment was started 24 hours after inoculation of tumor cells.

Groups of animals were formed to obtain statistically significant results: the control group consisted of 12 mice, experimental - from 8 animals.

The criterion for evaluating the antitumor effect was the inhibition of tumor growth (SRW,%). To calculate the SRW, we measured the three maximum mutually perpendicular sizes of the tumor (length, width, height) in each animal and calculated its volume, and then the average tumor volume in the group. Tumor volume was measured every 3 days, starting from the 7th day of the experiment, when the tumors in untreated animals become measurable.

Inhibition of tumor growth is calculated by the formula:

TPO (%) = (Vk-Vo) / Vk × 100, where

Vk is the average volume of tumors in the control group (mm ³ );

Vo is the average volume of tumors in the experimental group (mm ³ ).

Akadezin was administered intraperitoneally at a therapeutic dose of 80 mg / kg, as well as at an ineffective dose of 50 mg / kg intraperitoneally for 10 days. Ibuprofen was administered orally at a dose of 100 mg / kg for 10 days.

With the combined use of acadesine in two doses and ibuprofen, a synergistic antitumor effect was observed with the interaction of the two drugs. The combined use of acadesine at a dose of 80 mg / kg and an ineffective dose of ibuprofen of 100 mg / kg the antitumor effect is higher: TPO = 87% -84% -68% than when using acadesine at an ineffective dose of 50 mg / kg and the same dose of ibuprofen: TPO = 64% -77% -58%. This difference in the antitumor effect of the two groups is not statistically significant (p> 0.05). However, with the combined use of acadesine (80 mg / kg) and ibuprofen (100 m / kg), the death of 3 out of 8 mice receiving this combination was observed (37.5% of cases).

table 2 The antitumor effect of acadesine in combination with ibuprofen on B-cell leukemia P388. Group Dose mg / kg Days of introduction Route of administration SRW,% (days from the start of the experiment) Death from toxicity *** 8 10 13 Acadezine 80 1-10 in / br 55 * 46 37 0/8 - “- fifty 1-10 - “- 17 49 38 0/8 Ibuprofen one hundred 1-10 per os 35 twenty four 0/8 Akadezin + 80 1-10 b / w per os 87 ** 84 ** 68 ** 3/8 Ibuprofen one hundred Akadezin + fifty 1-10 b / w per os 54 ** 77 ** 58 ** 0/8 Ibuprofen one hundred

Notes: * p <0.05 with respect to control; ** p> 0.05 between the group of combined administration of acadesine at a dose of 80 mg / kg with ibuprofen at a dose of 100 mg / kg and the group of combined administration of acadesine at a dose of 50 mg / kg with ibuprofen at a dose of 100 mg / kg; *** in the numerator: the number of mice that died from toxicity in the first 3 days after treatment, in the denominator: the total number of animals in the group.

Thus, acadezin in combination with NSAIDs ibuprofen exhibits a synergistic antitumor effect on B-cell leukemia P388.

Example 3. The antitumor activity of acadesine in combination with aspirin in a model of B-cell leukemia P388 in laboratory mice.

The antitumor activity of acadesine in combination with aspirin was determined according to the scheme shown in example 2, but acadesine was administered intraperitoneally at a therapeutic dose of 60 mg / kg, and aspirin at a dose of 200 mg / kg orally for 10 days.

Table 3 The antitumor effect of acadesine in combination with aspirin on B-cell leukemia P388. Group Dose, Days Way SRW,% (days from the beginning Death from mg / kg introduce- introduce- experience) toxic niya niya 8 10 13 nosti *** Acadezine 60 1-10 in / br 58 * 62 * 37 ** 0/8 Aspirin 200 1-10 per os 43 19 44 0/8 Akadezin + 60 1-10 in / br 58 * 63 * 73 * ^, ** 1/8 Aspirin 200 per os

Notes: * p <0.05 with respect to control; ** p <0.05 between the group of combined administration of acadezin at a dose of 60 mg / kg with aspirin at a dose of 200 mg / kg and the group of acadezin at a dose of 50 mg / kg in monotherapy; *** in the numerator: the number of mice that died from toxicity in the first 3 days after treatment, in the denominator: the total number of animals in the group.

From the data shown in table 3, it follows that acadezin in the monotherapy mode caused inhibition of tumor growth (SRW) 8-10 days after the start of administration (58% and 62%). The value of TPO did not differ from that with a combination of acadesine and aspirin at the indicated times (58% and 63%). However, subsequently, the therapeutic effect of acadesine weakened: by the 13th day after the start of acadezine monotherapy, TPO was only 37%), while the combined effect of acadesine and aspirin showed a pronounced effect:% TPO = 73 (table 3). Therefore, the combination of acadesine with aspirin leads to a persistent and pronounced antitumor effect, while the effectiveness of acadesine alone is short-lived. Aspirin in the monotherapy regimen also exerted an antitumor effect, and its effect in combination with acadezin by the 13th day of treatment should be considered additive (TPO 37% acadezin, 44% aspirin, 73% combination of acadezine and aspirin).

Thus, from the results presented in tables 2 and 3, it follows that the claimed therapeutic agent in appropriate doses exhibits a more pronounced antitumor activity than acadesine.

Claims (1)

The drug with anticancer activity based on acadesine, characterized in that it further includes a non-steroidal anti-inflammatory drug: ibuprofen, or indomethacin, or aspirin.