RU2375090C1 - Method of tumour growth inhibition - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: invention concerns medicine, namely oncology and can be used for therapy of tumours. The method involves introduction in tumour tissue of octacarboxyphthalocyanine metal - octasodium salts of zinc octacarboxyphthalocyanine in a dose 10-150 mg/kg 1-3 hours prior to ultrasonic irradiation wherein tumour tissue is heated up to temperature 36-42°C.

EFFECT: application of the invention allows inhibiting growth of tumour cells due to solid-state sonic sensitisation with exposure of octacarboxyphthalocyanine metal and ultrasound.

3 tbl, 2 dwg, 22 ex

Description

The invention relates to the field of biology and medicine, and in particular to methods of suppressing the growth of malignant tumors.

Known (international application WO 97103666, A61K 31/40, 1997) is a method of suppressing tumor growth, comprising sequentially introducing into the tumor tissue complexes of cobalt or iron with substituted phthalocyanines or naphthalocyanines and a biogenic reducing agent, in which ascorbic acid was used, in particular . The molar ratio of complex: reducing agent is from 1: 5 to 1:50, and a single dose of cobalt octacarboxyphthalocyanine is 75-100 mg / kg. The considered method allows to suppress the growth of a fairly wide range of malignant neoplasms, as shown by examples both in vitro and in vivo. The disadvantage of this method is its low antitumor activity, which, due to an increase in the dose of the drug, does not allow the presence of ascorbic acid in it, which makes the drug toxic at higher doses.

Closest to the proposed invention is a method of suppressing tumor growth (RF patent 2188054, A61N 7/00, A61K 31/40, 1999). This method includes the introduction of cobalt octacarboxyphthalocyanate (terafthal) and ascorbic acid into the tumor and additional exposure of the tumor tissue by ultrasound at a frequency of 0.5-3 MHz and an intensity of 0.5-5 W / cm ² . In this case, the molar ratio of the complex: reducing agent is from 1: 3 to 1:30, and a single dose of cobalt octacarboxyphthalocyanine is 30-40 mg / kg. In the optimal variant of using the method, the indicator of antitumor activity - inhibition of tumor growth (SRW) is 65-68%. In this technical solution, by reducing the amount of ascorbic acid, the disadvantage of the above-described similar method is partially overcome. However, the antitumor activity of the prototype preparations used remains unsatisfactory and cannot be increased due to the toxicity of the method at higher doses of the preparations used.

An object of the present invention is to find a more effective and less toxic method of suppressing tumor growth.

The problem was solved by introducing octacarboxyphthalocyanine zinc (hereinafter, zinc phthalocyanine) into the tumor tissue with subsequent exposure to ultrasound, which eliminates the toxic effect of ascorbic acid and leads to a significant increase in the therapeutic effectiveness of the method. The used dose of zinc phthalocyanine is 10-150 mg / kg, and ultrasound exposure to the tumor tissue is carried out at a temperature of the tumor tissue 36-42 ° C 1-3 hours after the administration of zinc phthalocyanine.

A distinctive feature of the proposed method in comparison with the prototype is the use of zinc octacarboxyphthalocyanine, which does not exhibit the catalytic activity inherent in the cobalt and iron phthalocyanine complexes, in combination with a biogenic reducing agent to suppress the tumor growth of the octane sodium salt. This indicates a significant difference in the mechanisms of influence of the drug used on the growth of malignant tumors compared with the known method. In the case of the prototype, the introduction of cobalt octacarboxyphthalocyanine and ascorbic acid leads to the formation of superoxide anion radicals, which are an active principle, and which, apparently, is associated with poor tolerance of the known method, due to poor process control. Available experimental data indicate that in the proposed method, zinc phthalocyanine forms a solid phase in the tumor, and therefore it can be assumed that in this case the mechanism of solid-phase sonosensitization is realized and, accordingly, phthalocyanine plays the role of a sonosensitizer, which determines the high therapeutic efficacy of the proposed method.

The range of used doses of octane sodium octacarboxyphthalocyanine zinc not only ensures the achievement of the maximum target effect, but also allows, if necessary, to vary the concentration of the drug in a fairly wide range, which can be significant in the treatment of various types of tumors and individual tolerance of the drug to patients. Moreover, its introduction in an amount less than 10 mg / kg is impractical, since it does not significantly suppress the growth of tumor tissue. The use of doses above 150 mg / kg significantly increases the likelihood of toxic effects.

The choice of temperature and time regimes of ultrasonic irradiation is also due to the need to achieve the maximum therapeutic effect. Moreover, at a temperature of tumor tissue below 36 ° C, the effect is practically not observed, and the use of temperatures above 42 ° C leads to damage to healthy tissues and the loss of the thermosensitizing effect of zinc phthalocyanine.

The need for the administration of zinc phthalocyanine during a certain time interval before ultrasound exposure is also determined by the need to achieve the maximum therapeutic effect associated with the time the drug reaches cellular targets. In this case, ultrasonic irradiation earlier than 1 hour after the administration of zinc phthalocyanine does not provide the necessary therapeutic effect, and later than 3 hours after the administration of zinc phthalocyanine, it worsens the therapeutic effect due to the possible degradation of the drug and its removal from the body.

Determination of the antitumor activity of the proposed method was carried out on mice with transplantable tumors (in vivo). All experiments to evaluate the effectiveness of the proposed invention were performed on tumor strains: melanoma B 16 (examples 1-14 and 22) and highly metastatic Lewis carcinoma into the lungs (examples 15-21).

To evaluate the effect of each exposure according to this invention, the time of doubling the tumor volume "τ" was determined. For this, before and after exposure, the average tumor volume was measured and calculated at different times: V ₀ - tumor volume before exposure; V _t is the tumor volume at the time of measurement. Then V _t / V _{0 was} calculated and “τ” in the compared groups was determined graphically. Efficiency was judged by the coefficient (K), which was calculated by the formula

K = “τ” control / “τ” therapy,

where "τ" of control is the time of doubling the tumor volume in the group without specific effects; "Τ" therapy is the time for doubling the tumor volume in the group exposed to this invention.

The definition of K, as well as the definition of an indicator of inhibition of tumor growth (SRW) in the prototype, is a generally accepted, but more objective indicator of inhibition of tumor growth.

Example 1

The experiment was carried out as follows.

In the experiment, female mice of BDF ₁ hybrids and B16 lines with a body weight of 20-21 grams with v / m transplanted B16 melanoma were used. The method of tumor inoculation of 8 ± 0.05 cm ³ and preparation of animals for the experiment were generally accepted.

Before the experiment, mice were ranked in groups. One group was left control (the number of individuals n = 8) and a physiological solution of sodium chloride was injected intravenously to these mice. The main group received zinc phthalocyanine with ultrasound irradiation (n = 6). Comparison groups received a single ultrasound (n = 6) in optimal conditions of use.

The animal with the B16 tumor was fixed on a special holder and the paw with the tumor was immersed in contact medium (water). Then, zinc phthalocyanine was administered intravenously in an amount of 50 mg / kg in a volume of 0.2 ml of saline. 2 hours after its administration, sonication was performed for 10 minutes. The ultrasound parameters were as follows: frequency 2 MHz, intensity - 4 W / cm ² . The tumor was irradiated uniformly, moving the emitter throughout the tumor. The temperature in the tumor, which was 40 ° C, was fixed with a thermocouple.

Similarly, comparative experiments were conducted on the effects of only ultrasound and terafthal with ascorbic acid and ultrasound (according to the prototype).

The results of the experiment in this example are shown in table 1.

Examples 2-6. The experiments were carried out analogously to example 1, except that the amount of zinc phthalocyanine ranged from 10-150 mg / kg, and the irradiation time from 5 to 20 minutes The experimental results are also shown in table 1.

The observed therapeutic effect, expressed through the coefficient K, for mice treated by the proposed method (examples 2-6), is 150-160% in comparison with the prototype. The death of animals from acute toxicity was not observed.

Table 1.
Dependence of the inhibition of growth of B16 melanoma on the dose of octano-sodium salt of octacarboxyphthalocyanine zinc (zinc phthalocyanine) and the exposure time when exposed to ultrasound (US) Nn example Dose of zinc phthalocyanine (mg / kg) Irradiation time, min TO prototype cobalt phthalocyanine 10 2.5 KM - 10 1,5 Example 1 fifty 10 4.0 Example 2 fifty 5 3.3 Example 3 fifty twenty 4.0 Example 4 10 10 3.8 Example 5 one hundred 10 4.0 Example 6 150 10 4.0

Examples 7-14. The experiments were carried out analogously to example 1, but the temperature of the treatment of tumor tissues varied within 36-43 ° C, and the time from the introduction of zinc phthalocyanine to the start of ultrasonic irradiation from 1 to 3 hours. The dose of zinc phthalocyanine was 50 mg / kg, the ultrasonic irradiation time was 10 minutes, the ultrasonic irradiation parameters were 1.0 MHz, and the power was 4.0 W / cm ² .

The results obtained according to these examples are shown in table 2.

Table 2.
Dependence of inhibition of tumor growth of B16 melanoma on the temperature of tumor tissues and time from the administration of zinc phthalocyanine to ultrasound irradiation. Nn example Temperature ° C Time to irradiation, hour TO 7 36 2 3.2 8 37 2 3.8 9 40 2 4.0 10 42 2 4.0 eleven 43 2 3.9 13 40 one 3.8 fourteen 40 3 4.0

Examples 15-21. The experiments were carried out analogously to example 1, however, Lewis carcinoma was used as the transplanted tumor. The time of ultrasonic irradiation is 10 min, the parameters of ultrasonic irradiation are a frequency of 1.0 MHz, and power is 6.0 W / cm

The results obtained according to these examples are shown in table 3.

Table 3.
Dependence of inhibition of tumor growth of a Lewis carcinoma on the amount of zinc phthalocyanine injected, the temperature of the tumor tissue and the time interval from the administration of zinc phthalocyanine to ultrasound exposure. Nn example The amount of zinc phthalocyanine, mg / kg Tumor tissue temperature, ° C The time interval before exposure, hour TO fifteen fifty 36 2 3.8 16 fifty 40 2 3.9 17 fifty 42 2 4.0 eighteen fifty 40 one 3.8 19 fifty 40 3 3.9 twenty 10 40 2 3,7 21 150 40 2 3.8

Example 22. The experiment was carried out analogously to example 1, except that the ultrasound intensity was 2 W / cm ² .

Ten days after the end of the treatment according to the described example, the animals were killed and a morphological study of tumor tissue was performed.

Electron micrographs of mitochondria of B-16 melanoma tumor cells corresponding to Example 22 are shown in FIG. 1 (control) and 2 (after treatment). In the micrograph of the tumor after treatment (figure 2), mitochondria with destroyed cristae are clearly visible, which indicates cell death.

From the above data it follows that the use of the proposed method can effectively suppress the growth of a number of malignant tumors, namely, to achieve inhibition of tumor growth, as well as reduce the overall toxicity of the method compared to the prototype, in particular, by eliminating the biogenic reducing agent ascorbic acid.

Claims (1)

A method of suppressing tumor growth, including the introduction of metal octacarboxyphthalocyanine into the tumor tissue and subsequent exposure of the tumor tissue to ultrasound, characterized in that the octacarboxyphthalocyanine zinc is introduced as a metal octacarboxyphthalocyanine at a dose of 10-150 mg / kg 1-3 hours before ultrasound and the tumor tissue is heated to a temperature of 36-42 ° C.

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