RU2182482C1 - Method to obtain antitumor preparation - Google Patents

Abstract

FIELD: medicine. SUBSTANCE: solutions of hydroxycarboxylic acids obtained out of natural raw material as humic substances are treated with potassium tetrachloroplatinate by ultrasound impact at irradiation power being 40 W/cm² and 22 kHz frequency for 4-8 min. Humic substances could be obtained by treating humus material with alkaline solutions under normal conditions. As humus material one should use composts, sapropels, high- moor or intermediate-moor peat, humus layers of different soil types, etc. Humic substances could be, also, obtained out of oxidized woody lignin due to treatment with oxygen-bearing gas in alkaline medium at 170 ± 20 C, 1.9-2.5 MPa pressure for 1- 3 h and subsequent cooling the reaction mass, separation of a solid phase against a solution and acidification of the latter up to pH = 2-3. EFFECT: broadened assortment of antitumor preparations obtained out of available material. 8 cl, 7 ex

Description

 The invention relates to the field of production of drugs, in particular to the production of antitumor agents, which are complex compounds of metals, and specifically to a method for producing an antitumor agent containing platinum complexones.

 Antitumor preparations containing platinum complexones, causing a violation of the genetic apparatus of tumor cells, are distinguished by a strong antitumor effect, as a result of which they attract the attention of specialists.

 Platinum drugs have a wide range of antitumor effects. The most studied drug of this class of antitumor drugs - cis-dichlorodiammineplatin (DDP) - is active against tumors of various genesis: spontaneous, transient, induced viruses and chemical carcinogens. The main disadvantage of DDP is its high toxicity. Tests on animals and in the clinic showed that it acts mainly on the kidneys, bone marrow, and the gastrointestinal tract (Biological Aspects of Coordination Chemistry, revised by K. B. Yatsimirsky, Kiev, “Naukova Dumka.” 1979, p. 149-180).

 Another well-known antitumor agent, the so-called oxoplatin, contains a complex compound of tetravalent platinum, cis-diaminodichlorodihydroxyplatin, and has oral activity, lack of nephrotoxicity, inhibition of metastasis growth, high breadth of therapeutic effect (US Pat. RF 2086261, class A 61 K 45 / 05, publ. B.I. 22, 1997).

 The well-known antitumor drug platinum, which belongs to the group of complex compounds of divalent platinum, is effective in the treatment of malignant tumors of the testis and ovaries, breast cancer, disseminated melanoma. Platinum is obtained from platinum-choric acid and hydroxylamine in three stages by the formation of an intermediate alkali metal chloroplatinite, its interaction with hydroxylamine in the presence of alkali metal salts at room temperature in a slightly acidic aqueous medium. The product is purified by recrystallization from dilute hydrochloric acid. (Platinum, leaflet, NGO "Medioeconomics", order 2178).

 There is also known a method of producing an antitumor agent (ed. St. USSR 1701323, A 61 K 45/05, publ. B.I. 48, 1991), in which water containing DNA (high molecular weight natural organic compound) and sodium chloride, the solution is mixed with an aqueous solution of potassium tetrachloroplatinate. Before mixing, ammonium chloride, potassium chloride and potassium acetate are added to the latter, with each solution being heated to the melting temperature of DNA. Sodium citrate is added to the solution containing DNA and sodium chloride. The method allows to obtain a tool that combines high antitumor activity with a pronounced immunomodulating effect.

 Studies of various platinum complexes in animals and clinical trials of DDP and some of its analogues have revealed a variety in the biological properties of platinum complexes: antitumor activity and side effects. An analysis of the available data indicates that minor changes in the molecular structure of the complex can cause radical changes in their biological activity. The existence of a connection between the structure of the complex compound and its antitumor activity stimulates the search for new, platinum-containing drugs - highly active and low toxic.

 The objective of the present invention is to develop a method that allows to obtain from available natural raw materials new effective agents containing platinum and having antitumor activity.

The problem is solved by the fact that a method for producing an antitumor agent based on potassium tetrachloroplatinate is proposed. In accordance with the invention, a solution of potassium tetrachloroplatinate is treated with an aqueous solution containing humic substances, the treatment being carried out under ultrasound with a radiation power of 40 W / cm ² at a frequency of 22 kHz for 4-8 minutes. Humic substances can be obtained by treating the humified material under normal conditions with alkali solutions, to which urea and various complexones, for example Trilon B, can be added. The result is a mixture of salts of humic acids, which is hereinafter called humic substances.

 As humified material, sapropels, humus horizons from various types of soils, composts, peat from upland and transitional bogs can be used.

Humic substances can also be obtained by oxidation of wood lignin, while wood lignin in the form of a water-alkaline suspension is oxidized with an oxygen-containing gas at a temperature of 170 ± 20 ^o C and a pressure of 1.9-2.5 MPa for 1-3 hours. After cooling, the reaction the masses separate the solid phase from the solution, which is unoxidized particles of the original humified material. In solution, salts of humic acids remain, i.e. humic substances. Then, humic acids are isolated from the acidified solution of humic substances, which, after neutralization, are treated with potassium tetrachloroplatinate.

Processing potassium tetrachloroplatinate is advisable to be carried out at a temperature of 40 ± 5 ^o C.

 Ultrasound treatment of potassium tetrachloroplatinate with a radiation power of 40.0 ± 0.5 W and a sound frequency of 22.0 ± 0.5 kHz for 1-25 minutes is possible. The choice of ultrasonic treatment time depends on the composition of humic acids and is chosen experimentally.

It is advisable to water-alkaline suspension containing wood lignin, before oxidation to process with ultrasound with a radiation power of 4-6 W / cm ² and a frequency of 22 kHz for 6-10 minutes In this case, the number of active paramagnetic centers in the substance increases, which leads to an even greater increase in the biological activity of the final product.

 Lignin, which is part of wood, is a valuable medicinal raw material, the reserves of which are practically unlimited. The medicinal properties of lignin are comprehensively considered in the monograph (Levanova VP, Therapeutic lignin. Edited by N. A. Belyakov: Sp (b) 1992, p. 136). Although lignin and preparations based on it in medicine have so far been mainly considered as sorbents, many researchers in addition to sorption properties noted antiseptic, hypocholesterolemic and other properties.

 As wood lignin, a product of acid hydrolysis of wood, lignin-based enterosorbents such as Polyphepan, Lignosorb, etc. can be used.

 "Polyphepan" - non-specific enterosorbent (Reg. 80/1211/3) is a brown powder, odorless and tasteless, with a moisture content of 65%, consists mainly of lignin and contains no more than 20% residual polysaccharides (hydrocellulose).

 Lignosorb (polyphepan paste) VFS 42-2203-93.

 All substances used as feedstock are approved for oral administration.

 Special studies have shown that during the oxidation of lignin under the conditions of the proposed method, the formation of substances harmful to the body, such as polyaromatic hydrocarbons, nitrosoamines and polychlorinated dicyclo-p-dioxins, does not occur.

 The resulting products are concentrated dark brown liquids containing from 0.05 to 1.00% platinum with a specific taste and smell.

 It is well known that the humification process (the process of formation of humic substances) is planetary and the resulting humic substances (otherwise humic acids, which are a mixture of humic acids and fulvic acids) are characterized by similar spectra in the infrared region, regardless of how they were obtained.

 The antitumor activity of the proposed drug is due to its nature. The organic compounds included in its composition are characterized by a large number of covalently active bonds. As a result, it reacts with nucleophilic centers of protein molecules. In this case, the translation and transcription of nucleic acids is disrupted. The presence of platinum ions significantly enhances the antitumor effect of the drug.

 The antitumor mechanism of the proposed substance is currently being studied. The authors suggest that a possible mechanism of antitumor action is its effect on the hypothalamus and thyroid gland and, as a consequence, the activation of the endorphin system.

 The antitumor activity of the obtained product was evaluated by its effect on the growth of tumors in cattle (black and white cows). The latter is due to the fact that the metabolism (metabolism) in cattle is similar in terms of human metabolism and gives representative results.

 The following examples illustrate the proposed method for producing an antitumor agent and illustrate its antitumor activity.

When conducting the experiments described in examples 2-6 below, the treatment of humified materials (sapropel, peat, soil) with an aqueous alkaline solution was carried out under normal conditions, i.e. at a temperature of 20 ^o C and a pressure of 760 mm RT. Art. The result was a suspension containing humic substances in a dissolved state and an insoluble residue.

 Potassium tetrachloroplatinate for the best interaction with humic substances was used in the form of an aqueous solution.

EXAMPLE 1
The initial aqueous-alkaline suspension of lignin (Polyphepan brand, reg. 80/1211/3), a non-specific enteric sorbent, was oxidized with atmospheric oxygen.

Suspension composition
Polyphepan content - 1 kg
Alkali content (sodium hydroxide) - 100 g
Water - 8.1 kg
The oxidation was carried out in a reactor with mechanical stirring at a temperature of 160 ± 5 ^o C at a pressure of 2.5 MPa for 1 h. Air consumption was 5 l / min.

The reaction mass is cooled to room temperature and the precipitate is separated from the solution by filtration. The filtrate, which is an alkaline solution containing humic substances, is acidified with sulfuric acid to pH 2-3. By filtration, a precipitate is isolated in the form of a gel of humic acids, washed with distilled water, and then with a water-alcohol mixture until a pH of 6.0-6.5 is established and dried at 105 ^{° C.} until a homogeneous mass. The obtained humic acids are treated with 5% ammonia solution at the rate of 1 g of humic acids per 80 ml of this solution, thermostatted in a boiling water bath until excess ammonia is removed, filtered through a paper filter and 30 vol.% Distilled water is added. In the resulting solution, which is a salt of humic acids related to humic substances, 0.27 wt.% Potassium tetrachloroplatinate is added per 1 g of humic acid salts and a process of developed acoustic cavitation under the action of ultrasound with a radiation power of 40 W / cm ² and a frequency of 22 kHz for 4 minutes Next, the volume of the solution was adjusted with water to 100 ml.

 The resulting product is an aqueous solution of salts of humic acids related to humic substances, and complex compounds of platinum. This solution was used for animal testing to determine its antitumor activity. The test results are shown below in example 7. To identify the obtained substance, the above solution was analyzed.

The elemental composition of the dry isolated product in terms of organic matter, wt. %: C 54%, H 12%, N 2%, (S + 0) 32% (by difference). The substance decomposes without melting, starting from 168 ^o C.

The results of studying the infrared Fourier spectra show that the product obtained contains bands of close intensity in intensity of 1700 cm ^-1 and 1600 cm ^-1 , characteristic of carboxyl groups, an intense band in the region of 1200 cm ^-1 , a broad band in structure with local maxima of 3400, 3200, 3100 and 2600 cm ^-1 , as well as bands 2926, 2815, 1460 cm ^-1 and bands 2955 and 2870 cm ^-1 , characteristic of methylene groups.

The resulting product has paramagnetism. Symmetrical EPR signals are observed in the product with a q factor of 2,000 ± 0.001 close to the value for a free electron (q = 2.00) and a width of H = 6.1 ± 0.1 Oersted. The concentration of PMC is at the level of (2.5 ± 0.4) x10 ¹⁸ per gram,
The organic part of the product contains 2.9-3.4 mEq of COOH groups in 1 g of product and 6.0-6.4 mEq of phenolic hydroxyls in 1 g of product.

EXAMPLE 2
Lake sapropel with an organic matter content of 32% per dry matter was subjected to alkaline treatment with a 0.1 M potassium hydroxide solution.

For this, 5.6 g of potassium hydroxide was added to 200 g of lake sapropel with a moisture content of 95% and the total volume of the mixture was brought to 1 L, after which the mixture was shaken for 5 minutes and left for 6 hours. The process was carried out under normal conditions. After the indicated period, the precipitate was separated by filtration. The filtrate, which is an alkaline solution containing humic substances, was acidified with phosphoric acid to pH 1-2. The precipitate (gel of humic acids) is separated by filtration, washed with distilled water, and then with a water-alcohol mixture until a pH of 6.0-6.5 is established and dried at 105 ^° C to constant weight. The obtained humic acids are treated with 5% aqueous ammonia at the rate of 1 g of humic acids per 80 ml of this solution, thermostatted in a boiling water bath until the excess ammonia is removed, filtered through a paper filter and 30 vol.% Distilled water is added. In the resulting solution, which is a solution of salts of humic acids related to humic substances, contribute, based on 1 g of humic acids, 0.2 wt. % potassium tetrachloroplatinate and create a process of developed acoustic cavitation under the action of ultrasound with a radiation power of 40 W / cm ² and a frequency of 22 kHz for 8 minutes Next, the volume of the solution was adjusted with water to 100 ml.

The elemental composition of the dry isolated product in terms of organic matter, wt.%: C 60%, H 6%, N 2%, (S + O) 32% (by difference). The substance decomposes without melting, starting from 168 ^o C.

The results of studying the infrared Fourier spectra show that the product obtained contains bands of close intensity in intensity of 1700 cm ^-1 and 1600 cm ^-1 , characteristic of carboxyl groups, an intense band in the region of 1200 cm ^-1 , a broad band in structure with local maxima of 3400, 3200, 3100 and 2600 cm ^-1 , as well as bands 2976, 2815, 1460 cm ^-1 and bands 2955 and 2870 cm ^-1 characteristic of methylene groups.

The resulting product has paramagnetism. Symmetrical EPR signals are observed in the product with a q factor of 2,000 ± 0.001 close to the value for a free electron (q = 2.00) and a width of H = 6.1 ± 0.1 Oersted. The concentration of PMC is at the level of (2.5 ± 0.4) • 10 ¹⁸ per gram.

 The organic part of the product contains 3.0-3.6 mEq of COOH groups in 1 g of the obtained product and 6.0-6.5 mEq of phenolic hydroxyls in 1 g.

EXAMPLE 3
Carried out analogously to example 2, while vermicompost with an organic matter content of 23% per dry matter was subjected to alkaline treatment with a 0.1 M sodium hydroxide solution. 10 g of vermicompost (in terms of a completely dry sample) was poured into 1 l of a 0.1 M sodium hydroxide solution, the mixture was shaken for 1 h, after which the precipitate was separated by filtration. The process was carried out under normal conditions. The filtrate, which is a solution containing humic substances, was acidified with sulfuric acid to pH 1-2 and humic acids were isolated. Further processing was carried out in the same way as shown in examples 1 and 2, except that 0.17 wt.% Potassium tetrachloroplatinate was added to the solution of salts of humic acids based on 1 g of humic acids and heated to 40 ^o C.

 The elemental composition of the dry isolated product in terms of organic matter, wt.%: C 57%, H 7%, N 2%, (O + S) 34% (by difference).

 The organic part of the product contained 3.2-3.8 mEq of COOH groups in 1 g and 6.2 -6.7 mEq of phenolic hydroxyls in 1 g.

 Investigations of infrared spectra and determination of the content of COOH groups and phenolic hydroxyls showed that humic substances were obtained.

EXAMPLE 4
Carried out analogously to example 2, while peat from a high bog with an organic matter content of 73% dry matter was alkaline treated with a 0.2 M potassium hydroxide solution. 5 g of dry peat was poured with 0.2 M potassium hydroxide solution, and then left for 6 hours, after which the precipitate was separated by filtration. The process was carried out under normal conditions. The filtrate, which is a humic substance, was acidified to pH 2-3 with phosphoric acid. Further processing was carried out in the same way as shown in examples 1 and 2, except that the treatment of salts of humic acids with potassium tetrachloroplatinate was carried out at 45 ^o C.

 The elemental composition of the dry isolated product calculated on the organic substance, wt.% C 51%, H 12%, N 2%, (O + S) 35% (by difference).

 The organic part of the product contains 3.4-4.0 mEq of COOH groups in 1 g and 6.3-6.9 mEq of phenolic hydroxyls in 1 g.

 Investigations of infrared spectra and determination of the content of COOH groups and phenolic hydroxyls showed that humic substances were obtained.

EXAMPLE 5
Carried out analogously to example 2, while to 500 ml of lake sapropel with a moisture content of 90% and a solids content of 31% was added 500 ml of distilled water, 5.6 g of potassium hydroxide, 6 g of urea and 37 g of trilon B. After stirring for 1 h The process was carried out under normal conditions. The precipitate was filtered off. The filtrate, which is a solution containing humic substances, was acidified with sulfuric acid to pH 1-2. Further processing was carried out in the same way as shown in examples 1 and 2, except that 0.17 wt.% Potassium tetrachloroplatinate per 1 g of humic acids was added to the humic solution and heated to 40 ^° C.

 The elemental composition of the dry isolated product in terms of organic matter, wt.%: C 52%, H 16%, N 2%, (O + S) 30% (by difference).

 The organic part of the product contains 3.0-3.4 mEq of COOH groups in 1 g and 6.0-6.4 mEq of phenolic hydroxyls in 1 g.

 Investigations of infrared spectra and determination of the content of COOH groups and phenolic hydroxyls showed that humic substances were obtained.

EXAMPLE 6
Carried out analogously to example 2, while to 100 g of the humus soil horizon (horizon A from ordinary black soil) with a total carbon content of 7% was added 500 ml of distilled water, and then 1.12 g of caustic potassium, 6 g of urea and 7.4 g of trilon B.

After stirring for 10 minutes, the mixture was kept under normal conditions for 6 hours and the precipitate was separated by filtration. The filtrate, which is a solution of humic substances, was acidified with sulfuric acid to pH 1-2. Further processing was carried out in the same way as shown in examples 1 and 2, except that 0.2 wt.% Potassium tetrachloroplatinate was added to the solution of salts of humic acids based on 1 g of humic acids and heated to 40 ^° C.

 The elemental composition of the dry isolated product in terms of organic matter, wt.%: C 56%, H 8%, N 3%, (O + S) 33% (by difference).

 The organic part of the product contains 3.4-4.0 mEq of COOH groups in 1 g and 6.4 -7.0 mEq of phenolic hydroxyls in 1 g.

 Investigations of infrared spectra and determination of the content of COOH groups and phenolic hydroxyls showed that humic substances were obtained.

EXAMPLE 7
The preparation obtained in example 1 was tested for non-specific immunotherapy of cattle leukemia.

 In the experiment we used cows of black and motley breed at the age of 5-7 years of one of the enterprises of the Leningrad region. All animals were in standard conditions of loose housing. Diet and caring for animals was similar to the general herd both in experiment and in control.

The drug was stored in glass bottles with a capacity of 10, 20, 25, 30, 50, 100, 200 ml. With the simultaneous processing of a large number of cattle, vials with a preparation of up to 500 ml were taken. The bottles were hermetically sealed with sterile screw caps made of neutral plastic or rubber stoppers. Sterilization of the drug was carried out by autoclaving. They kept the drug in a dark place. The shelf life depends on the ambient temperature, at a temperature of -5 ^o C ...- 10 ^o C up to 2-3 years. Before use, the ampoule with the drug or the bottle was kept in warm water with a temperature of 37 ^o C for 5-10 seconds. Then, subject to sterility, the ampoules were opened and filled into an injection syringe.

 For non-specific immunotherapy of cattle from leukemia, three groups of cows, 12 cows each, were isolated.

 The first group (Experience): 12 cows of black-motley breed with a clinical form of leukemia, reacting positively according to the RID test and hematological studies.

 The second group (Positive control) 12 cows with a clinical form of leukemia. All animals responded positively according to the RID test and hematological studies. This group was not subjected to non-specific immunotherapy.

 The third group (Negative control). 12 cows of a black and motley breed of clinically healthy, negatively reacting according to the RID test and hematological studies.

All three groups of animals were in the same barnyard on loose housing with the same conditions of feeding and care. During the experiment, the animals in the experiment and control were tested in three tests:
establishing the presence of clinical signs of the disease;
serological diagnosis using a RID test;
hematological examination of peripheral blood in all animals.

 Clinical examination of animals was carried out in the presence of veterinarians and employees of the St. Petersburg Veterinary Institute.

 In all animals to the experimental group and in the positive control, an increase in the volume of superficial lymph nodes was observed: inguinal, arrogant and pre-scapular. Especially the inguinal and arterial lymph nodes were enlarged, which were evenly enlarged upon palpation. No signs of tenderness with deep palpation were observed. With percussion of the spleen, an extension of its borders was noted.

 The basis for monitoring the development of the leukemia process was the clinical scheme of the leukemia process according to Riche, 1975. According to this scheme, with the development of metastases in the lymph nodes, proliferative hyperplastic processes occur. At the same time, the lymph nodes increase in volume, which is clearly noticeable during clinical examination. This symptom is the main clinical sign of the manifestation of the disease. Therefore, the process of increasing or decreasing the volume of lymph nodes can be considered, respectively, as a deterioration or improvement in the nature of the disease with leukemia.

 Nonspecific immunotherapy of leukemia was carried out in several cycles according to the following scheme.

 Typically, the test drug was administered intramuscularly every 6 days at 20 ml at a dilution of 1: 100 for 36 days. Then followed a break of 35-40 days.

 In the course of treatment, the size of the inguinal and arrogant lymph nodes of each cow was determined. Measurement of the size of the lymph nodes showed that after the 1st cycle of active nonspecific immunotherapy, a decrease in the volume of lymph nodes to normal sizes was observed in all animals. In a parallel positive experiment in 12 cows with a similar clinical form of leukemia, the enlarged lymph nodes remained unchanged. 35-40 days after the end of the first cycle of active nonspecific immunoteropia in individual animals of the first experimental group, an increase in lymph nodes was again observed.

 After 30-35 days, all animals of the experimental group underwent a second repeated cycle of active non-specific immunotherapy: within 36 days, each animal received every 20 days every 20 days a dilution of 1: 100. After the second course, all animals of the experimental group experienced a stable remission. Clinical symptoms disappeared in all animals, their general condition improved: cows willingly gave in food, milk yield increased. The characteristic clinical signs of the onset of remission of leukemia were noted. Animals became active, the luster of the skin developed, there was no emaciation.

 A control blood test for a RID test study showed a negative result in 60% of the animals.

Claims (9)

1. A method of obtaining an antitumor agent based on potassium tetrachloroplatinate, characterized in that the potassium tetrachloroplatinate solution is treated with an aqueous solution containing humic substances, the treatment being carried out under the action of ultrasound with a radiation power of 40 W / cm ² , with a frequency of 22 kHz for 4- 8 minutes  2. The method according to p. 1, characterized in that the humic substances are obtained by treating the humified material with alkaline solutions under normal conditions.  3. The method according to p. 2, characterized in that the humified material is treated with an alkaline solution in the presence of urea and complexon.  4. The method according to p. 2 or 3, characterized in that as a humified material using peat from bogs or transitional bogs or sapropel, or composts, or humus horizons of different types of soils. 5. The method according to p. 4, characterized in that the treatment with tetrachloroplatinate is carried out at a temperature of 40 ± 5 ^o C.  6. The method according to p. 1, characterized in that the humic substances are obtained from oxidized wood lignin. 7. The method according to p. 7, characterized in that the humic substances are obtained by oxidizing wood lignin in an alkaline environment with an oxygen-containing gas at a temperature of 170 ± 20 ^o C, a pressure of 1.9-2.5 MPa for 1-3 hours and subsequent cooling of the reaction mass, separation of the solid phase from the solution and acidification of the latter to a pH of 2-3. 8. The method according to p. 8, characterized in that before the oxidation of wood lignin, the reaction mass is treated with ultrasound with a radiation power of 4-6 W / cm ² , with a frequency of 22 kHz for 6-10 minutes 9. The method according to p. 8 or 9, characterized in that the treatment with tetrachloroplatinate is carried out at a temperature of 40 ± 5 ^o C.