RU2601406C1 - Agent possessing anti-cachexic, anti-tumour properties and reducing endogenous intoxication level - Google Patents

Abstract

FIELD: pharmaceutics.

SUBSTANCE: invention relates to pharmaceutical industry, particularly to an agent possessing anti-cachexic, anti-tumour properties and reducing endogenous intoxication level. Agent is a dry extract of sandy everlasting leaves and flowers, produced by grinding them, extraction with 96 % ethanol under certain conditions, evaporating, diluting the evaporated residue, first, with distilled water, then, adding chloroform, cooling to the room temperature and centrifuging, followed by separation of water phase and drying it.

EFFECT: described agent possesses the evident anti-cachexic, anti-tumour properties efficiently reduces endogenous intoxication level.

1 cl, 3 tbl, 2 dwg, 2 ex

Description

The invention relates to medicine, the pharmaceutical industry and oncology, and in particular to the creation of drugs based on the newgalenic drug, for the treatment of tumor diseases and cancer cachexia and reduction of endogenous intoxication. The invention allows to significantly reduce the symptoms of cachexia, does not have side effects, contributes to weight gain and makes it possible to continue treatment with antitumor drugs.

Cachexia is a condition accompanied by uncontrolled weight loss, anemia, swelling, loss of appetite, etc. (Uomo et al., J Pancreas (Online) 2006; 7 (2): 157-162). A clearly defined definition of cachexia still does not exist, it is only known that “Patients with developed cachexia are distinguished by anorexia, a feeling of rapid satiety, severe weight loss, weakness, anemia and edema. <...> A poor physical condition during cachexia can be associated with many factors, including weight loss, reduced food intake, and fatigue or depression ”(Fearon et al. Am J Clin Nutr 2006; 83: 1345-50).

Cachexia can develop in some chronic diseases, such as malignant tumors, tuberculosis, diabetes, blood diseases, pathology of the endocrine system, infections and acquired immunodeficiency syndrome. Moreover, malignant tumor depletion progresses with effective treatment due to side effects of anticancer drugs, including myelotoxicity (Dewys WD et al. Eastern Cooperative Oncology Group. Am J Med 1980; 69: 491-7; Steinborn C , Anker SD, Basic Appi Myol 13 (4): 191-201, 2003; Morley JE et al., Am J Clin Nutr 2006, 83: 735-43).

Treatment of cachexia is a very complex process, since as it develops, the patient weakens, metabolic disorders occur, and the mechanisms of nonspecific resistance and immunity are disrupted. In this regard, the continuation of treatment with anticancer drugs, which, as a rule, are characterized by a high level of toxicity, may be impossible.

Thus, cachexia often becomes one of the main obstacles to the treatment of malignant neoplasms.

The use of protein and other nutritional supplements for the treatment of cachexia can provoke the progression of a malignant tumor due to the fact that amino acids are a building material and, first of all, for rapidly dividing tumor cells and shorten the patient's lifespan.

The known drug "Pindolol", which is recommended to be used to stimulate weight gain and prevent weight loss in patients suffering from chronic diseases, including malignant tumors (RU 2485950). The action of this drug, containing β-blocker, is based on a decrease in the activity of the sympathetic nervous system. The disadvantages of this tool is that it only improves the psychological state of patients and increases their appetite, but does not affect the main reason for the development of tumor cachexia - intoxication caused by active metabolism of tumor tissue. Thus, the drug is a symptomatic and not a pathogenetic agent. In addition, Pindolol has a huge list of side effects: insomnia, fatigue, weakness, indigestion, and others, which with prolonged use of the drug provide the opposite effect - progression of cachexia.

It is known that the use of birch fungus (Chaga or Tinder fungus - Inonotus obliquus) in medical practice as part of a symptomatic agent that alleviates the condition of cancer patients is achieved by a painkiller effect and relieves the effects of exhaustion (RU 111909). The infusion is used for malignant tumors of all localizations, but the best effect is observed in cancer of the stomach, lungs and other organs, richly equipped with blood vessels. The medicine is less effective at localizing the tumor in the bones, skin, and brain. The disadvantage of this tool is the fact that this treatment is also symptomatic. Decoctions and infusions of chaga only improve the well-being of patients, reduce pain, but do not relieve the tumor. The drug also has contraindications: it is not recommended to use it for diseases accompanied by fluid retention in the body. In addition, when using this tool, intravenous administration of glucose and the use of penicillin are prohibited, which can lead to undesirable side effects. It was established that with prolonged use of chaga, some patients showed increased excitability of the autonomic nervous system, although with a decrease in the dose of the drug or its complete abolition, this phenomenon gradually disappeared (Saakyan K.R., Vashchenko K.F., Darmogray R.E. Chaga ( black birch mushroom) Overview).

The well-known drug “Celecoxib”, the invention relates to the use of cyclooxygenase-2 inhibitors (“COX-2 inhibitors”) for the treatment and prevention of tumors, disorders associated with tumors and cachexia (RU 2190399). The action is based on the ability of COX-2 to inhibit the production of pro-inflammatory cytokines, especially IL-1 and TNF-α, and leukotrienes, in particular LTB4, as well as to inhibit bone resorption and have analgesic, anti-inflammatory and antipyretic effects. The disadvantages of this tool are its side effects, namely a high risk of gastrointestinal complications, including bleeding, ulceration and perforation of the stomach or intestines. These complications can occur at any time with NSAIDs without warning signs, a fatal outcome is possible. Elderly patients have a higher risk of serious complications from the gastrointestinal tract, the likelihood of these complications also increases with prolonged use. In elderly patients with cachexia with symptoms of heart failure and with impaired liver function, treatment should be started with minimal doses. That is, the use of the drug in people with already developed cachexia has serious limitations.

A known method of treating muscle loss by including in the diet an additional amount of amino acids that stimulate anabolic processes in skeletal muscle. For example, in US Patent Application Publication No. 2004/0122097 Verlaan et al. described nutritional supplements containing leucine and protein that stimulate the formation of muscle tissue. Leucine precursors such as pyruvate and metabolites such as β-hydroxy-β-methylbutyrate and α-ketoisocaproate exhibit properties similar to leucine. It should be noted that β-hydroxy-β-methylbutyrate is not produced by humans in any clinically significant quantities and, therefore, must be replenished. The main disadvantage of this method is that it is contraindicated for the treatment of cachexia, which developed against the background of malignant neoplasms, since the introduction of amino acids into the diet will contribute to the progression of tumor growth.

Thus, currently all existing drugs for the treatment of cachexia have a number of serious disadvantages:

1) a large number of side effects;

2) only symptomatic treatment;

3) prolonged use further exacerbates cachexia.

In addition, not a single antitumor drug not only does not have an anti-cachexic effect due to its own toxicity, but also, on the contrary, exacerbates the depletion of the body.

Sandy immortelle (Helichrysum arendrium (L.) MOENCH) is a perennial herbaceous plant, family Asteraceae. The natural range is almost the entire territory of Europe and Central Asia. It is found in Russia in the European part, Western Siberia, and the Caucasus. In folk medicine, immortelle sand is used for gastritis, constipation, colitis, is used as a liver remedy to regulate bile formation and biliary excretion, the administration of immortelle preparations is justified in the initial stages of gallstone disease. (http://www.rasteniya-lecarstvennie.ru/102-lekarstvennoe-rastenie-bessmertnik-peschanyj.html).

In experiments on rats, the effect of aqueous extracts of Strawflower on the level of dioxidin-induced lipid peroxidation in the blood was established. The ability of immortelle extract to reduce the content of medium-weight molecules by 18% (MSM) and malondialdehyde by 49% (MDA) was revealed only with intramuscular injection. However, these data indicate a decrease in peroxidation under the influence of an external factor. The authors did not study the severity of endogenous intoxication with the introduction of the extract against the background of the tumor process, as well as its effect on the level of lipid hydroperoxides, the effectiveness of the non-invasive oral method of drug administration was not shown (Kurchatova M.A., Durnova N.A., Afanasyeva G. A., Experimental and Clinical Pharmacology, Volume 78, No. 7 (2015) 2015).

Previously, we established the antitumor activity of immortelle extract in intramuscular administration (Skvortsova V.V., Navolokin N.A., Bulletin of Medical Internet Conferences. Volume 3. Issue 2 2013.), but the antichexical effect and the effectiveness of the extract were not oral administration.

The technical task of the present invention is to expand the arsenal of domestic products based on natural plant-based raw materials with complex effects, with the ability to reduce or prevent the development of tumor cachexia, including due to etiotropic (antitumor activity) and pathogenetic effects (reduce endogenous intoxication).

The advantages of the proposed new anti-cachexic drug, created on the basis of the Helichrysum extract, are its low toxicity, the availability of the raw materials from which the product is obtained, as well as a wide range of positive effects.

So, the drug has its own antitumor effect (a decrease in the rate of tumor growth). One of the pathogenetic mechanisms of the therapeutic effect is a decrease in the severity of endogenous intoxication, which is confirmed by a decrease in the levels of malondialdehyde (MDA), lipid hydroperoxides (GPL), and medium-weight molecules (MSM) that accumulate in the blood during tumor growth. The paper demonstrates the possibilities of both oral and intramuscular routes of administration of the drug.

We first established a new anti-cachexic property of the drug. The new anti-cachexic drug is an evaporated extract of the flowers and leaves of the Helichrysum sandwax obtained by grinding them, extraction with 96% alcohol, evaporation of the extract, addition of chloroform, centrifugation and separation into aqueous and chloroform fractions, followed by removal of the chloroform fraction. Extraction with alcohol is carried out in a water bath to a boil and boiled for 14-15 minutes, evaporated at a temperature of 55-60 ° C. The evaporated extract is first diluted with distilled water at 40-50 ° C, then chloroform is added in a proportion of 4/5 parts of water and 1/5 part of chloroform, cooled to room temperature and centrifuged at a speed of 1500 rpm for 15 minutes, then the aqueous fraction is separated and dried it (RU 2482863).

The product is an evaporated viscous transparent extract with a pleasant tart pungent odor, light yellow in color, mixes with water and ethyl alcohol in any proportions and has the following chemical composition: naringin and its soluble aggregate, prunin, quercetin, apigenin and naringenin, as well as 5- Apigenin O-glucoside and isosalipurposide. Using molecular absorption spectroscopy, it was found that the extract contains 73.48 mg of flavonoids in terms of rutin or 17.94 mg in terms of quercetin per 1 g of dry weight of the extract, which is 20.99% and 5.13%, respectively.

The extract obtained in this way is a biologically active composition with new pharmacological properties, namely, antitumor, antioxidant and previously unknown to both the sandy immortelle plant and the anti-cachexic effect described above.

Authenticity and quality parameters of the product.

Collection of material. The material was the raw immortelle sand (flowers) collected in the Lysogorsky district of the Saratov region in the vicinity of s. Ataevka in July 2014, the dried leaves and flowers of the sandwort immortelle were previously ground for a more complete extraction.

Compared to the previously obtained water and alcohol extracts from immortelle grass, the one-off extract of immortelle flowers and leaves obtained by us is non-toxic, which is due to the chemical composition and was previously confirmed in rats [Navolokin N.A., Polukonova N.V., 2013, 2014].

We were the first to carry out experimental work to establish the anti-cachexic and antitumor effect.

In an experiment conducted in accordance with the guidelines for experimental (preclinical) study of new pharmacological substances (Guidelines for preclinical studies of drugs. Part One. / Ed. By A.N. Mironov. - M.: Grif and K, 2012. - 944 pp.], 30 male white laboratory rats weighing 150 ± 50 g were used, which were implanted subcutaneously, in the scapular area, 0.5 ml of a 25% tumor suspension in Hanks's solution of the sarcoma-45 strain obtained from a bank of tumor strains of the State Research Center NN Blokhin Russian Academy of Medical Sciences. my sarcoma was randomly divided into three groups of 10 rats - the first and second experimental, receiving immortelle extract orally and intramuscularly at a dose of 1 g / kg and the third - comparison group, animals with transplanted tumor, but without exposure. the solution was administered orally (in one group) and intramuscularly (in the second), daily, for two weeks, starting 78 hours after tumor transplantation.

The growth dynamics of the tumor was evaluated by changing its volume according to the formula: V = A × B × C / 1000 (mm ³ ), where A is the width, B is the thickness, C is the height of the tumor in mm. Measurements were carried out with an electronic caliper every day from the start of the experiment. The tumor growth inhibition index (ITRO) was calculated - an indicator of the effectiveness of the antitumor drug, calculated by the formula: ITRO [(M _counter- M _experience ) / M _counter ] * 100%, where M _counter is the average tumor mass in the control group, and M _experience is experimental group.

Also, every day the animal was weighed to assess the dynamics of body weight. For a more objective analysis of the results, the true mass of animals was calculated (minus the theoretical mass of the tumor): M _true = M of the _animal -Vp, where M is the mass when weighed, V is the tumor volume, p is the density of the grafted sarcoma (0.83 g / cm ³ ).

Changes in the dynamics of tumor growth.

In the comparison group, the volume of the grafted tumor by the end of the experiment was 25.58 ± 3.72 cm ³ .

With intramuscular administration of immortelle extract, a statistically significant (p <0.005) slowdown in the rate of growth of transplantable sarcoma was noted compared with the control from day 10 of the experiment. At the end of the experiment, the tumor volume in the group with intramuscular injection of the Helichrysum was 7.66 ± 1.37 cm ³ , i.e. was 70% less than in the comparison group - 25.58 ± 3.72 cm ³ (p <0.005). ITRO by weight averaged 47.8% (Fig. 1).

With oral administration of the immortelle extract, a statistically significant (p <0.05) slowdown in tumor growth rates was observed compared with the comparison group, starting from day 9. At the time of the end of the experiment, the tumor volume in the study group was 64.6% less than the volume of the tumor of the comparison group - 9.05 ± 2.96 cm ³ . ITRO by tumor mass was 49.3% (Fig. 1).

Dynamics of the delta of body weight of animals with transplanted tumor.

The measurement was carried out in the following way: first, the theoretical mass of the transplanted sarcoma was calculated (the tumor volume was multiplied by the density), then, to obtain more objective data, the theoretical tumor mass was subtracted from the total rat mass, as a result, the true mass of the animal was obtained. Then, the delta of the body mass of the animals on each day of the experiment was calculated according to the formula: the mass of the animal on the day of the experiment minus the mass of the animal before the experiment.

When assessing the dynamics of the delta of the true body weight of animals in the comparison group, a tendency to the development of tumor cachexia was noted: the animals lost 18% during the experiment.

With the introduction of the immortelle extract both by the oral and intramuscular routes, mass stability and a small but stable increase in it were observed throughout the entire experiment. At the end of the experiment, the following results were obtained:

- the true body weight of animals in the group with the oral route of administration of immortelle extract increased by 11.9% relative to the mass at the time the experiment began (Fig. 2);

- the true body weight of rats in the group with the intramuscular route of administration of immortelle extract increased by 11.8% relative to the weight at the time the experiment began (Fig. 2).

We carried out experimental work to study the effect of immortelle extract on the level of endogenous intoxication (the content of markers of oxidative stress in blood plasma) against the background of tumor growth

The tumor process was modeled by subcutaneous injection of 25% suspension of liver alveolar cancer cells (RS-1) (Bank of tumor strains of the N. N. Blokhin Russian Academy of Medical Sciences RAMS). 1.5 months after transplantation, the tumors of the animals were divided into 3 groups (6 animals per group): the comparison group - with the tumor without exposure and two groups of animals with the tumor with a single intramuscular and oral administration of the sandwort extract of 1 g / kg.

Animals were removed from the experiment one day after the introduction of the extract. The severity of endogenous intoxication and activity of lipid peroxidation processes developing against the background of the tumor process was evaluated by the content of MDA, GPL and MSM in rat serum using spectrophotometric methods.

The following results were obtained.

In the comparison group, MSM indicators were 0.3085 (0.15-0.44) (U / ml); MDA 32.2964 (27.93-35.03) (pmol / mL); GPL - 5.88 (3.07-16.98) (U / ml).

In the blood serum of animals with a tumor process, when the immortelle extract is orally administered, there is a decrease in MSM - by 5%, MDA - by 95%, GPL - by 44% compared with the corresponding parameters of the group of animals with a tumor without administration of the studied drug.

In the group using the intramuscular method of administering the immortelle extract, there is a decrease in MSM - by 29%, MDA - by 95%, GPL - by 40.5% compared with indicators of animals with a tumor without administration of the immortelle drug.

Thus, the introduction of immortelle extract significantly reduces the intensity of lipid peroxidation processes, the level of intermediate products of free radical lipid oxidation in the blood serum of animals, which indicates a decrease in the severity of endogenous intoxication. In addition, it was found that the extract is able to reduce the level of GPL in the blood serum of animals. The effect of preparations based on the immortelle and, in particular, the extract obtained by the described method has not been previously described.

Example 1

For the experiment, we took a white laboratory male rat, which was implanted subcutaneously in the scapular area with 0.5 ml of a 25% tumor suspension in the Hanks solution of the sarcoma-45 strain obtained from a bank of tumor strains of the State Research Center for Oncology, Russian Academy of Sciences. N.N. Blokhina RAMS. 3 days after inoculation, an immortelle extract at a dosage of 1000 mg / kg was orally administered to the experimental animal for 12 days.

The dynamics of tumor growth was evaluated by changing its volume according to the formula: V = A × B × C / 1000, where A is the width, B is the thickness, C is the height of the tumor in mm. Measurements were carried out with an electronic caliper every day from the start of the experiment. Also, every day the animal was weighed to assess the dynamics of body weight. For a more objective analysis of the results, the true mass of animals was calculated (minus the theoretical mass of the tumor): True mass = M-Vp, where M is the mass when weighed, V is the tumor volume, p is the density of the inoculated tumor (0.83 g / cm ³ )

The following data were obtained.

Dynamics of body weight (g): 1 day - 106; 2 day -108; 3 day - 107; 4 day - 108; 5 day - 111; 6 day - 115.5; 7 day - 117; 8 day - 121; 9 day - 125; 10 day - 127; 11 day - 129; 12 day - 131; Day 13 - 133. As a result, there was a tendency toward an increase in rat body weight from the beginning of treatment to the end of the experiment.

The dynamics of the tumor volume (cm ³ ): 1 day - 0.37; 2 day - 0.42; 3 day - 0.51; 4 day - 0.62; 5 day - 0.68; 6 day - 0.79; 7 day - 1.54; 8 day - 2.1; 9 day - 4.21; 10 day - 5.9; 11 day - 9.56; 12 day - 12.2; 13 day - 14.1. Thus, the tumor continued to grow, however, the injected extract slowed the tumor growth rate compared to the standard dynamics of sarcoma growth 45.

The dynamics of body weight minus the theoretical tumor mass (g): 1 day - 105.7; 2 day - 107.6; 3 day - 106.65; 4 day - 107.5; 5 day - 110.4; 6 day - 114.3; 7 day - 115.7; 8 day - 119.2; 9 day - 121.5; 10 day - 122.1; 11 day - 121.6; 12 day - 120.8; 13 day - 121.7. As a result, despite the fact that sarcoma-45 was vaccinated in this animal, and, as you know, malignant neoplasms lead to cachexia, the rat receiving the immortelle extract gained body weight. Moreover, if we subtract the mass of the tumor at the end of the experiment from the mass of the rat, then there remains an increase in body weight relative to the weight of the rat before the experiment. There was also a tendency to slow down the growth rate of the tumor itself in this animal, compared with the standard sarcoma growth 45 described above.

Example 2

For the experiment, we took a white laboratory male rat, which was implanted subcutaneously in the scapular region with 0.5 ml of a 25% tumor suspension in Hanks's solution of a strain of alveolar liver cancer (RS-1). 1.5 months after the tumor was inoculated, the animal was injected intramuscularly with sand immortelle extract at a dose of 1 g / kg. Then it was withdrawn from the experiment one day after the introduction of the extract. The severity of endogenous intoxication and the activity of lipid peroxidation processes developing against the background of the tumor process was evaluated by the content of MDA, GPL and MSM in rat serum using spectrophotometric research methods.

The following results were obtained.

In rats, MSM rates were 0.15 (U / ml); MDA - 1.52 (pmol / mL); GPL -3.5 (U / ml).

As a result, in comparison with the values of LPO products described above in animals with liver cancer, administration of immortelle extract led to a decrease in LPO products (MDA, GPL and MSM), despite the fact that liver cancer was vaccinated in this animal, and, as malignant neoplasms are known to lead to intoxication.

Thus, as can be seen from the description and examples, the agent, which is a dry extract of flowers and leaves of the sandy immortelle, obtained by the above method, has anti-cachexic and antitumor properties and reduces the endogenous level of intoxication. The use of the extract throughout the experiment does not lead to the formation of resistance and contributes to an increase in body weight of animals and reduces the growth rate of the tumor. A feature of this drug is its simultaneous effect on the cause of cachexia and its development mechanism, which can be very promising for the treatment of cachexia in cancer patients, as it is not toxic and has its own antitumor activity.

Claims (1)

An agent with anti-cachexic, antitumor properties and reducing the level of endogenous intoxication, which is a dry extract of leaves and flowers of a sandy immortelle, obtained by grinding them, extraction with 96% alcohol in a water bath until boiling and boiling for 14-15 minutes, evaporation at a temperature of 55 -60 ° C, diluting the evaporated residue first with distilled water at a temperature of 40-50 ° C, then adding chloroform in the proportion of 4/5 parts of water and 1/5 part of chloroform, cooling to room temperature and centrifugation at a speed of 1500 rpm for 15 minutes, followed by separation of the aqueous fraction and drying it.

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