RU2844426C1 - Use of nitroxysuccinate di(3-hydroxy-4,5-bis(hydroxymethyl)-2-methylpyridinium) as cardioprotective compound in treatment of tumours - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: disclosed is using di(3-hydroxy-4,5-bis(hydroxymethyl)-2-methylpyridinium nitroxysuccinate) as a cardiac tissue Hrf2 transcription factor activator in the therapy of tumours.

EFFECT: invention is aimed at the use of nitroxysuccinate of di(3-hydroxy-4,5-bis (hydroxymethyl)-2-methylpyridinium) as a cardioprotective compound in the treatment of tumours, and also enables to avoid undesirable cytoprotective action for tumour cells.

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Description

The invention relates to the field of medicine, in particular to the development of new cardioprotective compounds, the creation of a new type of drugs based on activators of the transcription factor Nrf2 and the reduction of side effects of antitumor therapy using antioxidant compounds. A promising approach to increasing the resistance of cells and tissues to oxidative damage is the activation of a new therapeutic target - the transcription factor Nrf2 [DOI: 10.1155/2014/260429]. Nrf2 plays a critical role in maintaining cellular homeostasis [DOI: 10.1146/annurev-pharmtox-011112-140320], regulating the basal and inducible expression of antioxidant and cytoprotective detoxifying enzymes of phase II and is the main regulator of oxidative stress signaling in the heart. Nrf2 regulates cardiovascular homeostasis by suppressing oxidative stress and activating the expression of enzymes such as HO-1 and NQO1 [DOI: 10.1152/physiolgenomics.00041.2017]. Thus, targeting Nrf2 can prevent cardiac damage caused by oxidative stress, including the action of anthracycline antibiotics such as doxorubicin [DOI: 10.3892/ijmm.2015.2199, DOI: 10.1093/abbs/gmt082]. An important property of Nrf2 activator compounds for use in oncology studies as cardioprotectors is the ability, on the one hand, to exert some antitumor effect to reduce the doses of cytostatics, and on the other hand, to protect heart cells from oxidative stress. Currently, such drugs are not available for clinical use, although there are examples of Nrf2 activating compounds as antitumor agents or substances that enhance the cytotoxic effect of antitumor drugs in in vitro and in vivo experiments [DOI: 10.3389/fphar.2023.1176819, DOI: 10.1186/s12951-023-02129-1, DOI: 10.1016/j.redox.2023.102625, DOI: 10.3390/cells12172190, DOI: 10.47056/1814-3490-2023-4-283-288].

As a means of preventing cardiotoxicity during tumor chemotherapy, the complexing compound Dexrazoxane is used in the clinic [WO 2007062076 A2, https://patents.google.com/patent/WO2007062076A2/en]. However, this compound does not exhibit antitumor activity.

Thus, the search for new compounds with antioxidant and cardioprotective activity for combination therapy with known antitumor drugs, in particular doxorubicin, remains an important task for medicinal chemistry and oncological research [DOI: 10.3109/03602532.2011.609174].

This problem can be solved by using new antioxidant compounds - activators of the transcription factor Nrf2 based on pyridoxine derivatives. Previously, we synthesized and studied a pyridoxine derivative, di(3-hydroxy-4,5-bis(hydroxymethyl)-2-methylpyridinium) nitroxysuccinate [RU 2712914 C2], which has low cytotoxicity, iron-chelating activity, the ability to reduce lipid peroxidation, increase intracellular nitrogen monoxide levels in vitro [DOI: 10.3390/antiox10091451], as well as activate the expression of the transcription factor Nrf2 and enhance the cytotoxic effect of doxorubicin on tumor cells in vitro [DOI: 10.47056/1814-3490-2023-4-265-275]. Vitamin B6 (pyridoxine) is a natural compound with a wide range of antioxidant activity [DOI: 10.1016/j.freeradbiomed.2003.09.012] and is used in the Russian Federation in the complex treatment and prevention of various cardiovascular diseases, including coronary heart disease, grade II-III angina, myocardial infarction, and ischemic stroke (e.g., Vitabs® cardio). The pyridoxine molecule may be a promising precursor for creating new compounds with antioxidant activity potential [DOI: 10.1079/NRR200125]. Despite the large number of scientific papers devoted to the study of pyridoxine and its derivatives as cardioprotectors (e.g., Mexidol and Emoxipin), the use of such compounds as activators of the Nrf2 transcription factor in heart cells has not been previously considered.

Thus, the objective of the invention is to use di(3-hydroxy-4,5-bis(hydroxymethyl)-2-methylpyridinium) nitroxysuccinate as a cardioprotective compound in tumor therapy.

The stated task is solved by studying the biological activity of di(3-hydroxy-4,5-bis(hydroxymethyl)-2-methylpyridinium nitroxysuccinate), in particular, determining its ability to activate the transcription factor Nrf2 in cardiac tissues in vivo during tumor therapy, activate the expression of cardioprotective genes in cardiac tissues during tumor therapy, and stabilize the activity of cardiomyocyte damage markers during tumor therapy.

Figure 1 shows the effect of compound 1 on the Nrf2 protein content in the heart tissue and tumors of mice with B16 melanoma. The significance of differences from the control is shown as ***P<0.001

Figure 2 shows the effect of compound 1 on the expression level of cardioprotective Nrf2 target genes in the heart tissues and tumors of mice with B16 melanoma. The significance of differences from the control is shown as *P<0.05.

Figure 3 shows the effect of compound 1 on alanine aminotransferase (ALT) activity in the blood of mice with B16 melanoma under normal conditions and under the action of the antitumor drug Doxorubicin.

Figure 4 shows the effect of compound 1 on aspartate aminotransferase (AST) activity in the blood of mice with B16 melanoma under normal conditions and under the action of the antitumor drug Doxorubicin. The significance of differences from the control is shown as **P<0.01, relative to doxorubicin as ##P<0.01

The essence of the proposed invention is characterized by the examples given below. Example 1

The ability of di(3-hydroxy-4,5-bis(hydroxymethyl)-2-methylpyridinium) nitroxysuccinate (1) to increase the content of the transcription factor Nrf2 in the heart tissues of tumor-bearing animals was studied. Experimental melanoma B16 was transplanted subcutaneously into first-generation hybrid mice BDF ₁ (C57Bl/6J×DBA/2). The inoculum was 5×10 ⁶ tumor cells. Male mice were used in the experiment, which were kept in a conventional vivarium with a 12-hour light regime and free access to water and food. Compound 1 was administered intraperitoneally at a dose of 15 mg/kg daily for 10 days. Animals were sacrificed 24 hours after the last administration of 1. The concentration of the transcription factor Nrf2 in heart and tumor tissues was assessed by enzyme-linked immunosorbent assay using the ELISA NFE2L2 mouse Kit (SEL947Mu, Cloud-Clone Corp., USA) according to the manufacturer's protocol.

Figure 1 shows the effect of compound 1 on the Nrf2 protein content in the heart tissues and tumors of mice with B16 melanoma. The significance of differences from the control is shown as ***P<0.001.

It was determined that the introduction of 1 significantly increases the content of the transcription factor Nrf2 in animal heart cells, but does not affect its content in the tumor. The data obtained indicate the selectivity of the action of 1 as an antioxidant and cardioprotective compound, which will avoid an undesirable cytoprotective effect on tumor cells.

Example 2

The ability of di(3-hydroxy-4,5-bis(hydroxymethyl)-2-methylpyridinium) nitroxysuccinate (1) to increase the expression of cardioprotective target genes of the transcription factor Nrf2 in the heart tissues of tumor-bearing animals was studied. Experimental melanoma B16 was transplanted subcutaneously into first-generation hybrid mice BDF ₁ (C57B1/6J×DBA/2). The inoculum was 5*× ^0.6 tumor cells. The experiment used male mice that were kept in a conventional vivarium with a 12-hour light regime and free access to water and food. Compound 1 was administered intraperitoneally at a dose of 15 mg/kg daily for 10 days. The animals were sacrificed 24 hours after the last administration of 1.

Total RNA from the hearts of laboratory animals was isolated using the ExtractRNA reagent (Eurogen, Russia) according to the manufacturer's protocol. The first strand of cDNA was synthesized on the RNA matrix using the MMLV RT kit (Eurogen, Russia). Quantitative real-time PCR was performed using the qPCRmix-HS SYBR reaction mixture (Eurogen, Russia) and the LightCycler® 96 Real-Time PCR System (Roche, Switzerland). The beta-actin gene (ACTB) was used as a reference gene to normalize the expression results. For gene expression analysis, pairs of primers to the following genes were used: GPX1, HMOX1, NQO1, TXN. The level of relative gene expression was calculated using the manufacturer's software based on the results of three independent experiments.

Figure 2 shows the effect of compound 1 on the expression level of cardioprotective Nrf2 target genes in the heart tissues and tumors of mice with B16 melanoma. The significance of differences from the control is shown as *P<0.05.

It was determined that the introduction of 1 significantly increased the expression level of cardioprotective (GPX1, HMOX1, NQO1, TXN) genes of the antioxidant system in animal heart cells, but did not affect their expression level in the tumor. The data obtained indicate the selectivity of the action of 1 as an antioxidant and cardioprotective compound, which will avoid undesirable cytoprotective action for tumor cells.

Example 3

The ability of di(3-hydroxy-4,5-bis(hydroxymethyl)-2-methylpyridinium) nitroxysuccinate (1) to restore the activity level of cardiomyocyte damage markers - alanine aminotransferase and aspartate aminotransferase in the blood of tumor-bearing animals was studied. Experimental melanoma B16 was subcutaneously transplanted into first-generation hybrid mice BDF ₁ (C57B1/6J×DBA/2). The inoculum was 5×10 ⁶ tumor cells. Male mice were used in the experiment, which were kept in a conventional vivarium with a 12-hour light regime and free access to water and food. Compound 1 was administered intraperitoneally at a dose of 15 mg/kg daily for 10 days. Doxorubicin was administered on days 2, 5, and 9 after the start of the experiment at a dose of 6 mg/kg. There were four groups of animals in the experiment: 1 administration for 10 days, three-time administration of doxorubicin, combined administration of doxorubicin and 1, control (administration of saline). Animals were sacrificed 24 hours after the last administration of 1. Biochemical parameters - ALT (alanine aminotransferase) activity; AST (aspartate aminotransferase) activity were determined in blood serum using an automatic biochemical analyzer and BioChem Analette kits (HTI, USA) according to the manufacturer's method. Blood serum was obtained by centrifuging 500 μl of whole blood in test tubes with a coagulator for 10 minutes at 1500 g at room temperature. The de Ritis coefficient was calculated as the ratio of AST/ALT activities.

Figure 3 shows the effect of compound 1 on alanine aminotransferase (ALT) activity in the blood of mice with B16 melanoma under normal conditions and under the action of the antitumor drug Doxorubicin.

Figure 4 shows the effect of compound 1 on the activity of aspartate aminotransferase (AST) in the blood of mice with B16 melanoma under normal conditions and under the action of the antitumor drug Doxorubicin. The significance of differences from the control is shown as **P<0.01, relative to doxorubicin as ^## P<0.01

Table 1 shows the effect of compound 1 on the value of the de Ritis coefficient under normal conditions and under the action of the antitumor drug Doxorubicin for animals with B16 melanoma.

It was determined that the introduction of 1 reduced the activity of the cardiac damage marker ACT in the blood of laboratory animals with B16 melanoma against the background of doxorubicin administration. The use of 1 together with doxorubicin restored the value of the De Ritis coefficient to the normal level. The data obtained indicate that the studied compound, di(3-hydroxy-4,5-bis(hydroxymethyl)-2-methylpyridinium nitroxysuccinate), shows a significant cardioprotective effect when acting together with an anthracycline antibiotic in experimental tumor chemotherapy.

The use of di(3-hydroxy-4,5-bis(hydroxymethyl)-2-methylpyridinium) nitroxysuccinate as a cardioprotective compound in tumor therapy

Claims (1)

The use of di(3-hydroxy-4,5-bis(hydroxymethyl)-2-methylpyridinium) nitroxysuccinate as an activator of the transcription factor Nrf2 in cardiac tissues in tumor therapy.