RU2234323C1 - Preparation eliciting antitumor, antitoxic and radioprotecting effect - Google Patents

Abstract

FIELD: medicine, oncology, chemical-pharmaceutical industry, pharmacy.

SUBSTANCE: invention can be used in clinical practice for treatment of oncological diseases. Invention proposes an allogenic preparation from human placenta based on nucleoproteids complex fragmented to 200-2000 nucleotide pairs and comprising, wt.-%: DNA 60-80; nuclear matrix proteins, 20-40. The advantage of invention involves the presence of complex pharmaceutical activity and provides antitumor, antitoxic and radioprotecting effect of the preparation.

EFFECT: valuable medicinal properties of preparation.

3 cl, 2 tbl, 1 dwg, 3 ex

Description

The invention relates to medicine, in particular pharmacology, and can be used in clinical practice for the treatment of malignant neoplasms.

Modern medicine has a fairly extensive arsenal of drugs for chemotherapy of tumors. Chemotherapeutic agents are mainly represented by a group of cytotoxic drugs, among which the most common and effective are alkylating cytostatics such as cyclophosphamide, chlorobutin and sarcolysin. Due to the lower cytostatic, antiproliferative activity, preparations from the group of antimetabolites (azathioprine, fluorouracil, etc.) are used to a lesser extent. Natural antitumor preparations of plant (colchicine, etc.) and bacterial (rubromycin, etc.) origin are also of limited use due to their high toxicity and narrow therapeutic spectrum of action (treatment of some types of tumors, mainly with exophytic growth). Currently, there are no drugs on the pharmaceutical market of antitumor drugs that have complex pharmacological effects, namely, they have their own antitumor activity in combination with radioprotective and antitoxic effects during complex chemotherapy, mainly using alkylating cytostatics and tumor x-ray therapy.

It is known to use alkylating cytostatics such as cyclophosphamide, which have a wide therapeutic spectrum for the treatment of the most common malignant neoplasms: lung cancer, breast cancer, sarcoma, ovarian cancer, etc. (M.D. Mashkovsky Medicines, Part II, M., Medicine, 1993, p. 498-499; p. 501-502). The disadvantage of these drugs is their high toxicity and inhibitory effect on leukopoiesis. The development of leukopenia is a dangerous complication in the treatment of malignant neoplasms with cytostatics, as it poses a threat of bleeding, a decrease in immunological resistance and the spread of superinfection. Almost any infection with cytostatic therapy, for example, cyclophosphamide or sarcolisin, can cause septic complications, which are practically not treatable with modern antibacterial drugs, since there are no cellular and humoral mechanisms of natural resistance due to suppression of cytostatics.

Currently, the most effective way in the treatment of solid tumors is x-ray therapy (E. Braunwald, Internal Medicine, 2 t., M., Medicine, 1993, p. 50-505), but it also has a significant percentage of complications, primarily associated with immunosuppression, suppression of the white sprout of the blood and the development of superinfection.

Closest to the claimed preparation, the prototype is the preparation “Deoxinate”, which is the sodium salt of double-stranded deoxyribonucleic acid, obtained from sturgeon milk and partially depolymerized by ultrasound to a molecular weight of 2.7-4.0 × 10 ⁵ daltons. The DNA content in the preparation is more than 90%, RNA (from 3 to 5%) and non-histone type proteins associated with DNA are present as impurities in the preparation (PS 42-3922-00). The drug has antitoxic and radioprotective effects and is used as a stimulator of leukopoiesis in chemoradiotherapy conducted by an oncological patient. The drug is available in the form of a 0.5% solution and is intended for subcutaneous and intramuscular administration at the rate of 75 mg of the active substance, regardless of the patient's age.

The disadvantages of “Deoxinate” are

- the DNA contained in the “Deoxinate” has no species specificity, is heterologous and therefore can cause allergic and pyrogenic reactions, which manifest themselves in the form of an increase in temperature. For this reason, the drug cannot be administered intravenously, due to the possible development of anaphylactic shock, often observed with intravenous infusions of species-specific biological substances.

- “Deoxynat” does not have its own antitumor activity and therefore is used only as an auxiliary leukostimulating drug for the prevention of myelodepressions in chemoradiotherapy of cancer patients. At the same time, the leukostimulating effect develops slowly and persistent changes are observed after 4-10 days. The radioprotective effect of “Deoxynat” is indirect in nature and is aimed at correcting leukopenia and stimulating the immune system that develop in acute radiation sickness of the II-III degree of severity.

An object of the present invention is to provide a preparation from species-specific raw materials having antitumor, antitoxic and radioprotective effects.

The technical task is achieved by the proposed drug based on fragmented up to 200-2000 nucleotide pairs of the nucleoprotein complex isolated from human placenta, containing, wt.%:

double-stranded DNA 60-80

nuclear matrix proteins 20-40

The inventive preparation is prepared as follows.

The placenta from healthy women in labor, tested for hepatitis, HIV infection and syphilis, is frozen and stored at -20 ° C in a separate sterile plastic bag, accompanied by a sanitary-hygienic passport, until the preparation is received (conventional name “Panagen”), but no more 1 year from the date of procurement.

After accumulating the required amount, the placenta is thawed for 5-6 hours, washed, homogenized in 10 mM Tris-HCl buffer (pH 7.5) containing 1 mM EDTA. Sodium dodecyl sulfate is added to the resulting homogenate to a final concentration of 2% and incubated at 65 ° C for 6-24 hours. Sodium chloride is added to the resulting lysate to a final concentration of 2 M and incubated for 3-12 hours at 65 ° C. After incubation, the lysate is centrifuged at 9000 g for 10 minutes. An equal volume of acetone cooled to + 10 ° C is added to the obtained supernatant to precipitate the nucleoprotein complex (NPK). The obtained NPK was washed three times with chilled acetone and 96 ° ethanol to remove low molecular weight proteins and peptides unbound from DNA. The purified NPK is dissolved at 65 ° C in 10 mM Tris-HCl buffer (pH 7.5) containing 1 mM EDTA, then the solution is centrifuged at 9000 g for 10 minutes, the supernatant is filtered through a glass filter with a pore diameter of 120 μm and exposed to ultrasound with a frequency of 11-44 kHz for fragmentation of the NPC. The yield of the target product is 0.3-0.5% of the feedstock, the content of the main substance (nucleoprotein complex) is 95.0-99.5%. The molecular weight of the nucleoprotein complex is 1.5x10 ⁶ daltons. The depth of fragmentation is controlled by electrophoresis and upon reaching the required range within 200-2000 nucleotide pairs, the process of exposure to ultrasound is stopped. The solution of the fragmented NPK was centrifuged at 9000 g for 10 minutes, then a twofold volume of chilled 96 ° ethanol was added. The precipitated NPK is washed with 70 ° ethanol and, after drying, the claimed preparation is obtained.

Before use, the drug is dissolved in a pharmaceutically acceptable solvent to a content of the main substance of 0.5-4.0%. The pharmaceutically acceptable solvent used is predominantly phosphate or Tris-HCL buffer mixture, saline.

The determining significant differences of the claimed drug from the drug of the prototype are

- the claimed drug contains a native, species-specific fragmented nucleoprotein complex, consisting of d.s. DNA and related proteins of the nuclear matrix, with the ratio: d.s. DNA 40-80%, proteins 20-60%, which allows to expand the spectrum of therapeutic activity of the drug: the latter has its own antitumor activity in combination with direct radioprotective and antitoxic action (leukostimulating);

- due to a certain depth of fragmentation of the nucleoprotein complex (200-2000 nucleotide pairs), the claimed drug can be attributed to native drugs containing a complete set of fragments of the human genome and, accordingly, in the treatment of cancer in humans, the drug has a replacement, receptor-interrogated, genetically reparative function, as well as anabolic and trophic. In the prototype, the degree of fragmentation is higher (molecular weight distribution is in the range of 2.7-4.0 × 10 ⁵ daltons), which makes the drug more distant from native preparations containing fragments of the genome, moreover, the biological raw materials are non-specific, heterologous and, accordingly, in the treatment of cancer in humans, the function of the drug will be only anabolic or trophic;

- the claimed drug due to the species-specificity and homology of biological material (human placenta) can be administered intravenously, due to which the leukostimulating effect when combined with chemotherapy with cytostatics develops faster and more pronounced.

Known preparations from human placenta, used to stimulate the regeneration of wound surfaces of various etiologies, and also used to treat gastric ulcer, mainly in the form of extracts for intramuscular and subcutaneous injections (Mashkovsky M.D. Medicines. Part I., Part II . - 12th ed., Rev., Rev. and add. - M., 1996). However, from available sources no information was found about a drug similar to the claimed one possessing the above-mentioned complex pharmacological activity.

The antitumor activity of the claimed drug was investigated in an in vivo model. The linear-specific HA-1 tumor maintained in ascites form on A / Sn mice was transplanted intramuscularly (10 tumor cells) to syngeneic A / Sn mice. After 6 days, the same mice were injected intraperitoneally with the claimed preparation obtained from human placenta and NPK obtained from the liver of CBA mice for 11 days, physiological saline was used as a control. Experimental animals were divided into groups:

1st group. Control mice injected with saline.

2nd group. Experimental mice, the introduction of NPK from human placenta (dose of 0.5 mg per mouse daily).

3rd group. Experimental mice, the introduction of NPCs from the liver of CBA mouse mice (dose of 0.5 mg per mouse daily).

As can be seen from the data presented, the claimed drug from human placenta and NPK from the liver of mice have an antitumor effect, however, the antitumor effect of the claimed drug is species-specific and more pronounced.

The radioprotective activity of the claimed drug was investigated in an in vivo model. 4-month-old female CBA mice were irradiated on a gamma installation with an irradiation dose of 980 rad. 4 groups of mice were irradiated simultaneously in one container. Each group consisted of 10 animals:

1. 1st group. Control mice.

2. 2nd group. Mice that received 30 mg of NPA obtained from the liver of CBA mice intraperitoneally 30 minutes before irradiation.

3. 3rd group. Mice administered 30 mg of NPA obtained from the liver of CBA mice intraperitoneally 30 minutes after irradiation, and 0.5 mg at the second and third day after irradiation.

4. 4th group. Mice, which were injected intraperitoneally with 1 mg of NPK obtained from human placenta (claimed composition) 30 minutes after irradiation, and on the second and third day after irradiation, received a dose of 0.5 mg.

The results are presented in table 2.

From table 2 it is seen that the claimed drug has a therapeutic radioprotective effect aimed at reducing the post-radiation biological effects (administration of the drug after irradiation). The radioprotective properties of the claimed drug depend on the species specificity of the NPK and are maximum when used to isolate homologous biological raw materials.

The antitoxic properties of the claimed drug were investigated on a model for the restoration of the number of leukocytes receiving cytostatic cyclophosphamide.

Methodology

A / Sn mice were injected intraperitoneally once with cyclophosphamide (CF) at a dose of 200 mg / kg. Two groups of mice (12 in each) one day before CF and after it daily were injected intraperitoneally with solutions of NPK from human placenta and NPK from the liver of A / Sn mice of 50 μg per mouse. On the 3rd, 5th, and 7th day after the administration of CF, 4 mice from each group were killed by decapitation, blood was taken, and the number of leukocytes in the Goryaev chamber was counted. The control (initial leukocyte count) was intact mice of the same gender and age.

results

2 of 12 mice receiving only CF fell on the 5th day after administration of CF. In groups of mice that received nucleoprotein complexes in addition to CF, there was no death.

The drawing shows that for 3 days. after the introduction of CF, the number of leukocytes in the blood of animals was sharply reduced and amounted to about 6% of the number of leukocytes in intact mice. Moreover, the introduction of NPK did not prevent the decrease in the number of leukocytes, that is, it did not interfere with the action of the cytostatic agent. However, their recovery was significantly accelerated against the background of the administration of NPCs from the liver of mice: the number of leukocytes on day 7 already exceeded the average values of the intact control, whereas in mice that did not receive NPCs, it amounted to about 70% of the initial value. NPK from human placenta had a slightly lesser effect, but significantly exceeded that in control animals that did not receive the claimed drug.

Thus, the claimed drug has an antitoxic effect, namely it stimulates leukopoiesis after its suppression by cytostatics. The antitoxic properties of the claimed preparation also depend on species specificity and are maximum when used to isolate NPK homologous biological raw materials.

The invention is illustrated by the following specific examples of the preparation of the claimed preparation.

Example 1

A placenta (250 g) was thawed for 5 hours, then homogenized in a homogenizer with 250 ml of 10 mM Tris-Hcl buffer (pH 7.5) containing 1 mM EDTA. Sodium dodecyl sulfate is added to the resulting homogenate to a final concentration of 2% by weight and incubated at 65 ° C. for 24 hours. Sodium chloride was added to the resulting lysate to a final concentration of 2 M and incubated for 12 hours at 65 ° C. After incubation, the lysate is centrifuged at 9000 g for 10 minutes. An equal volume of acetone cooled to + 10 ° C is added to the obtained supernatant to precipitate the nucleoprotein complex (NPK). The obtained NPK was washed three times with chilled acetone and 96 ° ethanol to remove low molecular weight proteins and peptides unbound from DNA. The purified NPK is dissolved at 65 ° C in 1 l of 10 mM Tris-Hcl buffer (pH 7.5) containing 1 mM EDTA, then the solution is centrifuged at 9000 g for 10 minutes, the supernatant is filtered through a glass filter with a pore diameter of 120 μm and influence it with ultrasound in the 11 kHz mode for fragmentation of the NPC. The solution of the fragmented NPK was centrifuged at 9000 g for 10 minutes, then a twofold volume of chilled 96 ° ethanol was added. The precipitated NPK is washed with 70 ° ethanol and, after drying, the claimed preparation is obtained on the basis of a nucleoprotein complex with a fragmentation degree of 200 nucleotide pairs of the following composition, wt.%:

DNA 80

nuclear matrix proteins 20

The yield of the target product is 0.37%, the content of the main substance is 98.5%.

Example 2

A placenta (250 g) was thawed for 6 hours, then homogenized in a homogenizer with 250 ml of 10 mM Tris-Hcl buffer (pH 7.5) containing 1 mM EDTA. Sodium dodecyl sulfate is added to the resulting homogenate to a final concentration of 2% by weight and incubated at 65 ° C for 6 hours. Sodium chloride was added to the resulting lysate to a final concentration of 2 M and incubated for 3 hours at 65 ° C. After incubation, the lysate is centrifuged at 9000 g for 10 minutes. An equal volume of acetone cooled to + 10 ° C was added to the obtained supernatant to precipitate NPK. The obtained NPK is washed three times with chilled acetone and 96 ° ethanol to remove low molecular weight proteins and peptides unbound from DNA. The purified NPK is dissolved at 65 ° C in 1 l of 10 mM Tris-Hcl buffer (pH 7.5) containing 1 mM EDTA, then the solution is centrifuged at 9000 g for 10 minutes, the supernatant is filtered through a glass filter with a pore diameter of 120 μm and act on it with ultrasound in the 44 kHz mode to fragment NPK. The solution of the fragmented NPC is centrifuged at 9000 g for 10 minutes, then a twofold volume of chilled 96 ° ethanol is added. The precipitated NPK is washed with 70 ° ethanol and after drying it is obtained the claimed preparation based on NPK with a fragmentation degree of 2000 nucleotide pairs, containing, wt.%:

DNA 40

nuclear matrix proteins 60

The yield of the target product is 0.3%, the content of the main substance is 95%.

Example 3

A placenta (250 g) is thawed for 5 hours, then homogenized in a homogenizer with 250 ml of 10 mM Tris-Hcl buffer (pH 7.5) containing 1 mM EDTA. Sodium dodecyl sulfate is added to the resulting homogenate to a final concentration of 2% by weight and incubated at 65 ° C for 12 hours. Sodium chloride was added to the resulting lysate to a final concentration of 2 M and incubated for 6 hours at 65 ° C. After incubation, the lysate is centrifuged at 9000 g for 10 minutes. An equal volume of acetone cooled to + 10 ° C was added to the obtained supernatant to precipitate NPK. The obtained NPK was washed three times with chilled acetone and 96 ° ethanol to remove low molecular weight proteins and peptides unbound from DNA. The purified NPK is dissolved at 65 ° C in 1 l of 10 mM Tris-Hcl buffer (pH 7.5) containing 1 mM EDTA, then the solution is centrifuged at 9000 g for 10 minutes, the supernatant is filtered through a glass filter with a pore diameter of 120 μm and act on it with ultrasound in the 22 kHz mode to fragment NPK. The solution of the fragmented NPK was centrifuged at 9000 g for 10 minutes, then a twofold volume of chilled 96 ° ethanol was added. The precipitated NPK is washed with 70 ° ethanol and after drying, the inventive preparation is obtained on the basis of NPK with a fragmentation degree of 1000 nucleotide pairs of the following composition, wt.%:

DNA 60

nuclear matrix proteins 40

The yield of the target product is 0.5%, the content of the main substance is 99.5%.

The use of the proposed drug will allow, in comparison with the prototype, to expand the spectrum of pharmaceutical activity, since the claimed drug has its own antitumor activity, as well as reduce the side complications of chemoradiotherapy with a combination of the application, due to the antitoxic and radioprotective properties of the claimed drug.

Claims (3)

1. The drug has antitumor, antitoxic and radioprotective effects, characterized in that it is a fragmented up to 200-2000 pairs of nucleotides nucleoprotein complex with a content, wt.%: Double-stranded DNA 60-80 and nuclear matrix proteins 20-40. 2. The drug according to claim 1, upon receipt of which use species-specific biological raw materials. 3. The drug according to claim 2, upon receipt of which, as a species-specific raw material, a human placenta is used.