RU2320334C1 - Preparation for inhibiting tumor growth and method for inhibiting tumor growth in experiment - Google Patents

Abstract

FIELD: medicine, biology, veterinary practice.

SUBSTANCE: the present innovation deals with preventing malignant neoplasms in domestic animals to be applied in the course of medico-biological trials for developing the methods for preventing and treating oncological diseases. One should introduce daily for animals per os "oligohit aktiv" preparation at the dosage of 90-100 mg/kg body weight before interweaving Pliss' lymphosarcoma at general course of 7 d, not less. The innovation widens the quantity of preparations for inhibiting tumor growth and prolongs life duration in animals due to introducing the above-mentioned preparation.

EFFECT: higher efficiency.

2 cl, 1 ex, 2 tbl

Description

The invention relates to experimental medicine and biology, namely to experimental oncology, and can be used in veterinary practice for the prevention of malignant neoplasms of domestic animals and in biomedical research to develop methods for the prevention and treatment of cancer.

The search for new approaches to the prevention of malignant neoplasms remains an urgent medical and biological task.

Most methods of inhibiting tumor growth include administering chemotherapy drugs [2, 4, 14, 17, 20, 22, 29].

There is a method of preventing tumor disease in an experiment, for which 1 hour after transplanting a tumor cell culture to experimental animals, 1 ml of blood is taken from a subclavian vein in a hermetically sealed vial with an anticoagulant, cyclophosphamide is added therein, the mixture is incubated for 30 minutes, and rats are reinfused intravenously, in a jet, once [22].

A known method of inhibiting tumor growth in an experiment using the cytostatic doxorubicin [17], which was used in ultra-low doses on a model of Lewis lung carcinoma in BDG mice. The drug was administered once intraperitoneally on the next day after inoculation of the tumor in doses of 10 ^-5 , 10 ^-10 , 10 ^-15 and 10 ^-20 M. For comparison, the standard therapeutic dose of doxorubicin 8 mg / kg of animal body weight, which corresponds to 1.4 × 10 ^-3 M. The ability of doxorubicin in ultra-low doses to cause an antitumor effect was found with the specified formulation of the experiment, comparable with the effect obtained with the use of a standard therapeutic dose. On day 12, the tumor size in experimental animals receiving standard and ultra-low doses of the chemotherapy was reduced by an average of 80% compared with the values in the control, i.e. not receiving chemotherapy, animals.

However, the proposed methods, along with the high efficiency of inhibiting tumor growth, cause indirect pathological changes in other organs and systems of the body, which is the main limitation to their use.

A drawback of a number of studies in which an experimental assessment of the effectiveness of the developed methods of tumor inhibition is carried out in vitro [2, 5, 7, 16, 29] is an incomplete understanding of the side effects of the substances used on the body.

In most methods of inhibiting the growth of tumors, the effectiveness of which is shown in vivo, in the experiment the test substances are administered after the induction or transplantation of the tumor [12, 14, 16, 20, 26].

No methods of inhibiting tumor growth with the introduction of substances before the induction or transplantation of the tumor were found.

Therefore, the authors believe that the technical solution is new and meets the criterion of "inventive step".

The objective of the invention is the expansion of the arsenal of tools to inhibit tumor growth, as well as the development of a method of using a new tool.

The task is achieved by the fact that it is proposed:

1. The use of the complex "oligohit asset" as a means of inhibiting tumor growth.

2. A method of inhibiting tumor growth in an experiment, comprising administering a medicament, characterized in that the animals are injected with an oligochit asset daily at a dose of 90-100 mg / kg of body weight before the inoculation of the tumor per os with a total course of at least 7 days.

Oligochit asset (sometimes a synonym is used - chitosan oligosaccharide succinate-ascorbate) (TU 9289-004-57184729-03) is a water-soluble product in which chitosan oligosaccharide (oligochit) (70%), succinate (15%) and ascorbate ( 15%) are in ionic bonds with each other. Under physiological conditions, succinic acid is dissociated, so the name of its anion - succinate, is often used as a synonym for the term "succinic acid", and ascorbate - as a synonym for the term "ascorbic acid." Recent studies have shown that succinate has biological activity with a unique combination of manifestations: it acts as an adaptogen with respect to a healthy organism, and in the presence of pathological manifestations it exhibits an atypically high therapeutic effect for adaptogens [31]. Succinate is a normal cellular metabolite, plays an important role in ensuring the energy balance in the cell [3, 10, 15], has antihypoxic [13, 31, 32], antioxidant [8, 31], radioprotective [11] and detoxifying effects [31] . Several works are known in which the results of the use of succinic acid (UC) for tumor growth are reported: T.Kamata et al. (1970) observed inhibition of growth of M-1 sarcoma in rats after administration of UC [15], A.L. Kovalenko and M.G. Romantsov (1999) showed that as a result of UC administration, a significant decrease in the frequency of spontaneous mouse tumors and an increase in the maximum their life expectancy [8]; EVKozyreva (1997) revealed a decrease in the mortality rate of cancer patients on the background of the complex use of succinic, citric and o-ketoglutaric acids with vitamins and herbs [9]. I. A. Pomytkin and S. P. Soloviev proposed in combination with isotopologists to use UC salt as an additional tool in the treatment of neoplasms [19]. However, in the work of V.N. Anisimov and M.N. Kondrashova it was shown that subcutaneous injections of sodium succinate in animals at a dose of 50 and 250 mg / kg for 10 days, starting from the 3rd day after tumor transplantation, had little effect on the growth of transplanted tumors in mice and rats [1].

Currently, ascorbic acid (AK) is used in the treatment of malignant neoplasms. However, the literature on the effect of AK on tumor growth is controversial. L. S. Trukhanova (1987) on a model of carcinogenesis induced by 1,2-dimethylhydrazine and estradiol dipropionate, it was shown that when mice are injected with CBA line of AK solutions in concentrations of 0.3; 0.75; and 1.5% there is a clear tendency towards a decrease in tumor mass with an increase in AK concentration [24]. In the work of M. Polydock et al. a 2-fold increase in life expectancy was shown in mice with Ehrlich acidic carcinoma against the background of administration of AK at a dose of 10 mg / kg [33], and according to the observations of T. Shingu et al. - acceleration of tumor growth (dose 25 mg / day) [34]. In the experiment of I.A.Kharkovskaya et al. the introduction of AK to guinea pigs at a dose of 0.3 mg / kg of the mass led to complete resorption of the sarcoma, and at a dose of 1 g / kg, accelerated tumor growth [27]. Therefore, the question of the possibilities of using this vitamin in oncology remains open.

In recent years, chitosan and its derivatives have been widely used in various fields of medicine. Chitosan is obtained by processing marine crustaceans, and after enzymatic cleavage of high molecular weight chitosan, a new, water-soluble product is formed - chitosan oligosaccharide (synonym - oligochite). In this form, it is easily absorbed in the intestine and quickly enters the systemic circulation, and then with a blood stream reaches the "target organs". One of the new directions of using it as a carrier for dosage forms [28]. K.A. Treskunov and B.A. Komarov proposed using chitosan in the form of a salt form with L-glutamic acid in combination with special phyto-collections in parallel with radiation therapy and complex chemotherapy before and after surgery in the treatment of cancer [23].

The possibility of using the drug "oligochit asset" as a means to inhibit tumor growth in the experiment was shown on the model of Pliss lymphosarcoma.

The model of an experimental tumor of Plissa lymphosarcoma (LFS) chosen by us, obtained in 1958 after subcutaneous inoculation of a tumor that arose in a rat treated with 3.3 dichlorobenzidine, is characterized by the following features: consists of soft, rounded cells, most of which are occupied by a large nucleus; chromatin is located mainly in the peripheral zone of the nucleus; nucleoli large, hyperchromic; cytoplasm is characterized by moderate basophilia. The tumor has a pronounced infiltrative growth in the border connective and adipose tissue, as well as muscles; metastases [18].

A method of inhibiting tumor growth in an experiment was developed on the model of Pliss lymphosarcoma; it consists in the fact that before the inoculation of the tumor, the animal is given per os daily a solution of the drug "oligochit asset" with a concentration of 90-100 mg / kg body weight, with a total course of at least 7 days. The choice of dose and course of administration is justified by both theoretical data and our own experimental studies. In an article by M.V. Vasin et al. it was shown that the maximum antitoxic and antioxidant effect is achieved at a dose of UC of 100 mg / kg weight [6]; and N.I. Fedotchev et al. revealed that in hypometabolic conditions, effective doses of succinate when administered orally are 50-100 mg / kg of body weight [25]. The term for the introduction of substances is selected as the minimum and corresponding to a single administration for humans [21]. In our studies, it was shown that the use of the drug “oligohit asset” at a concentration of 90-100 mg / kg body weight, a total course of 7 days, is effective for correcting the morphological state of the liver [30] and the free radical balance of the tumor carrier organism.

Studies were conducted and the effectiveness of the use of the drug "oligochit asset" to inhibit tumor growth in the model of Pliss lymphosarcoma was revealed.

The experiment was performed on 20 white outbred rats, which were divided into two groups: control and experimental.

1 group - control (n = 10);

Group 2 - experimental (n = 10).

In the experimental group, healthy animals were daily per os injected with the drug oligochit asset at the rate of 90-100 mg / kg body weight, for a total course of 7 days. Animals of the control group received pure water in the same way.

A day after the drug was discontinued, the animals of the control and experimental groups transplanted subcutaneously the cells of the tumor strain of lymphosarcoma (LFS) Plissa in 0.5 ml of physiological saline into the right thigh. The LFS strain Plissa (acquired at the NN Blokhin ONC RAMS, Moscow) is an experimental tumor obtained in 1958 after subcutaneous inoculation of a tumor that arose in a rat treated with 3,3-dichlorobenzidine [10].

On the 10th, 12th and 19th day after the transplantation of the tumor, linear measurements of the tumor were made with a caliper. And calculated the average tumor volume V (mm ³ ) according to the formula [21]:

V = (a · b ² ) / 2, where a is the length, b is the width.

The degree of inhibition of tumor growth was determined by the inhibition of tumor growth, calculated by the formula [21]:

SRW = (V control-V experience) / V control · 100%,

where V is the average tumor volume (mm ³ ) in the experimental and control groups, respectively, for a specific period.

Significant antitumor effect should be maintained for at least 7 days.

Quantitative criteria for evaluating the inhibitory effect

In the statistical processing of the results, Student's t-test was used to evaluate the significance of digital data using the Bonferonni correction.

The results showed that the use of chitosan succinate-ascorbate oligosaccharide (“oligohit asset”) prior to inoculation of the Plissa LPS tumor strain at the rate of 90-100 mg / kg body weight, with a general course of at least 7 days inhibits the growth of Plissa lymphosarcoma by an average of 63.7% (table .1), which contributes to an increase in the life expectancy of animals of this group by 77.6% (Table 2).

Table 1. Inhibition of the growth of Pliss lymphosarcoma against the background of the introduction of "oligohit asset" before inoculation of the tumor Tumor growth period, days Tumor volume in the control group, cm ³ Tumor volume in the experimental group, cm ³ SRW,% 10 35.5 ± 3.6 12.1 ± 1.5 * 65.9 12 75.6 ± 6.4 30.9 ± 2.6 * 59.1 19 179.9 ± 14.6 61.0 ± 5.46 * 66.1 * - the differences are significant at p≤0.05 compared with the control group. Table 2. Life expectancy of animals - tumor carriers against the background of the introduction of "oligohit asset" before transplantation of Pliss lymphosarcoma. days Rat numbers Control group Experienced group 1/31 17 31 2/32 19 32 3/33 twenty 37 4/34 eighteen 39 5/35 22 38 6/36 23 thirty 7/37 twenty 31 8/38 twenty 37 9/39 eighteen 38 10/40 twenty 37 Days, M ± m 19.7 ± 1.83 35.0 ± 3.52 * * - the differences are significant at p≤0.05 compared with the control group.

Thus, the study of the biological effect of the drug oligohit asset on an experimental model of the transplantable tumor revealed that the use of the drug oligohit asset before inoculation of tumor cells leads to inhibition of tumor growth, which suggests the use of this method for the prevention of malignant neoplasms of domestic animals in veterinary practice and use in biomedical research to develop methods for the prevention and treatment of cancer.

Example No. 1.

1) Rat No. 10, weighing 225 g for 7 days, was administered intragastrically using a probe of pure water 1 ml each. On the 9th day after the beginning of the manipulations, the cells of the Plissa tumor strain of lymphosarcoma (LFS) in 0.5 ml of physiological saline were transplanted subcutaneously into the region of the right thigh. The LFS strain Plissa was acquired at the Research Institute of Experimental Diagnosis and Treatment of Tumors of the Russian Oncology Center. N.N. Blokhina RAMS (Moscow).

On 10, 12 and 19 days after transplantation of LPS cells, Plissa performed linear measurements of the tumor with a caliper and calculated the average tumor volume V (mm ³ ) according to the formula [21]:

V = (a · b ² ) / 2, where a is the length, b is the width.

Tumor volume increased as follows: 10 day tumor growth - 40 mm ³ ; 12 day - 75 mm ³ ; 19 day - 183 mm ³ .

The life span of this animal was 20 days.

2) To rat No. 38, weighing 220 g for 7 days, 1 ml of a solution of chitosan succinate-ascorbate oligosaccharide in a dose of 90-100 mg / kg of animal weight was administered intragastrically with a probe. On the 9th day after the beginning of the manipulations, the cells of the Plissa tumor strain of lymphosarcoma (LFS) in 0.5 ml of physiological saline were transplanted subcutaneously into the region of the right thigh.

On 10, 12 and 19 days after transplantation of the tumor, its volume was 11 mm ³ ; 28 mm ³ and 62 mm ³ , respectively.

The degree of inhibition of tumor growth was determined by the inhibition of tumor growth (SRW) [21]:

SRW = (V control-V experience) / V control · 100%.

The introduction of “chitosan succinate-ascorbate oligosaccharide” prior to the Liss transplantation of Plissa LFS inhibited tumor growth by 72.5%, 62.7% and 63.9%, respectively, according to the selected time frame.

The life expectancy of animal No. 38 was 37 days.

Claims (2)

1. The use of the drug "oligochit asset", which has the property to inhibit Plissa lymphosarcoma. 2. A method of inhibiting the growth of Pliss lymphosarcoma in an experiment, comprising administering an oligochit asset to animals prior to inoculation of Pliss lymphosarcoma per os daily at a dose of 90-100 mg / kg body weight for a total course of at least 7 days.