BG110926A - Antitumor medicine, a method for the production of said medicine and a method for its stabilization - Google Patents

Abstract

The invention relates to the field of medicine, in particular, to antitumor medicines based on a platinum compound aqueous solution, to methods for producing the above-mentioned agents and to methodsfor the stabilisation thereof. The method for stabilizing antitumor agents, which are based on the platinum compound aqueous solution, includes the use of a mixture of a fluorouracil base and uracil and urea, as well as the use of a low-proton concentration solution.

Description

The invention relates to the field of medicine, and more specifically to antitumor drugs based on aqueous solution of platinum compounds, which can be used for the treatment of malignant tumors, as well as methods for their preparation and stabilization.

BACKGROUND OF THE INVENTION

Among the antitumor drugs widely used in medical practice, some of the most effective are based on an aqueous solution of the cisplatin monomer complex. Platinum ions act as an active ingredient in these drugs together with ligand carriers that are absorbed by the tumor cells in virtually the same intensity as normal cells. Therefore, drugs containing cisplatin are characterized not only by high antitumor activity but also by high toxicity.

Antitumor drugs based on an aqueous solution of a platinum-deoxyribonucleic acid (Pt-DNA) polymer complex, a product of the reaction of cisplatin and DNA, in which one of the ligand carriers is no less active, but much less toxic platinum ion is a nucleotide embedded in the structure of DNA double-stranded macromolecules that is only intensely absorbed by rapidly developing tumor cells, and therefore antitumor drugs containing a Pt-DNA bond affect tumor tissues with a high degree of selectivity for minor damage to normal tissues [Treatment of inoperable organ tumors in the abdominal cavity, S.A. Shalimov et al.,

-21998, p. 103].

The cisplatin monomer complex and the Pt-DNA polymer complex, which are the main substances in the drugs, are not sufficiently soluble and are chemically unstable in water. Therefore, pharmaceutical additives are added to the drugs based on their aqueous solutions. Pharmacologically acceptable salts, pH regulators and other stabilizing agents are preferably used as additives, preferably those which, at the expense of their own biological activity, which simultaneously with the ability to increase the solubility and chemical stability of the parent substance, provide treatment efficacy and better tolerability.

A known antitumor drug based on an aqueous solution of a platinum derivative with deoxyribonucleic acid polyanion in situ (Pt-DNA) (active ingredient), which includes non-associated Pt-DNA macromolecules of 0.05-0.15 microns in the most its largest dimension, sodium chloride, sodium citrate, ammonium chloride and fluorouracil in the form of the sodium salt (excipient) in the ratio of the components in aqueous solution, in weight%:

Pt-DNA 0.130-0.153 © Sodium chloride 0.080 - 0.090

Sodium citrate is 0.040 - 0.053

Ammonium chloride - 0.015-0.018. Fluorouracil in the form of the sodium salt - 0.025 - 0.370 [see Declaration Patent of Ukraine No. 70 456, published on 15.10.2004, Applicants Shalimov SA, Volchenskova II, Maidanevich N. N.].

As the studies indicate, the aqueous solution of a known drug has a pH of 6.5-7.5 and a density of 1.0012-1.0016 g / cm ³ , in which the unassociated molecules of the Pt-DNA compound have the highest antitumor activity, and fluorouracil has the ability to enhance this activity and to

-3 reduces the toxicity of the platinum derivative if the substance is maintained at a temperature above approximately 35 ° C. Lowering the temperature helps the Pt-DNA molecule bases to enter into cooperative intramolecular interactions more intensely than to intermolecular interactions with the corresponding fluorouracil sodium salt anions. Therefore, the spatial structures of the Pt-DNA molecules condense irreversibly. The intramolecular condensation of structures containing very small platinum atoms with a density of 21.45 g / cm results in the reduction of the spatial structures of the Pt-DNA molecules to an extent where their liquid density exceeds the density of the solution and they precipitate gravitationally in the form of loose sludge.

.Therefore, fluorouracil anions are more likely to interact with protons from solution than with Pt-DNA bases and therefore pass to the state of neutral molecules with their own base, whose solubility in water is limited and which precipitates in the form of crystals. which dissolve as the temperature rises. Sedimentation of active ingredients and excipients causes a decrease in their concentration in the agent solution, thus reducing its antitumor activity and therapeutic power, and also limiting its administration to topical applications and intra-abdominal infusions, but intravenous, intra-arterial infusions and interstitial mode of administration.

Fluorouracil base is known to be used as a structural fragment of tegafur - a precursor of fluorouracil in the body - to produce an antitumor composition containing tegafur and uracil in a 1: 4 molar ratio [patent US5534513, IPC 7 A61K 31IH50 PHARMACEUTICAL CO LTD (JP), published on 09/07/1996 .J.

Urea is known to be used as a diuretic, keratolytic agent, intracranial pressure reducing agent and ophthalmic tonus [M.M.

-4Turkevich, Pharmaceutical Chemistry, 1973, p. 45] and also as an excipient added to solid dosage forms for oral administration.

.High urea concentrations and increasing pH to above 11 are known to be used to denature helix filaments of natural double stranded DNA [A. Leninger. Biochemistry, 1976, p. 737].

Sodium hydroxide is also known to be used in pharmacy to control the pH of solutions of therapeutic agents. Moreover, it is known that, under the influence of high urea concentrations and at pH> 11.0, the helix double strands of natural DNA are denatured [Leninger. Biochemistry, 1976, p. 737].

However, nothing known in the prior art sources for the use of uracil, urea and sodium hydroxide as agents preventing the intramolecular condensation of stranded strands of metal compounds with DNA. Nor is it known to administer urea in dosage forms for parenteral administration. The intervals of effective concentration and the ratio of the active ingredient, fluorouracil and salts in the aqueous solution of the drug claimed are consistent with the ratios indicated in the development of a well-known drug for intraabdominal, intravenous and intraarterial infusions maintained at 35- 40 ° C. Uracil and urea concentration intervals are determined by the fluorouracil, uracil and urea ratio of 1: 1: 1. Sodium hydroxide concentration intervals are limited at the lower end of the pH range, which eliminates the possibility of fluorouracil protonation, and at the upper end of the interval, from pharmaceutical requirements.

There is also another known method of producing an antitumor drug by maintaining the DNA strands for 16-26 hours in a citrate saline solution containing sodium chloride and sodium citrate, dissolving the strands by mechanical stirring and heating the DNA solution to 7090 ° C. , gradual introduction of the cis-dichloro-5diamino-platinum (DDP) citrate-salt solution, heated to the same temperature, into the heated DNA solution as well, addition of fluorouracil sodium salt solution to the mixture and thermostatic control of the mixture of the solution solutions. DNA and DDP at 70-90 ° C for 15-20 minutes [see Declaration Patent of Ukraine No. 70 456, published on 15.10.2004, Applicants Shalimov SA, Volchenskova II, Maidanevich NN ].

The addition of an aqueous solution of the fluorouracil sodium salt to the mixture of DNA and DDP solutions prior to thermostatic control does not help to intensify the intermolecular interactions of the Pt-DNA molecules with the corresponding molecules of the solution and to attenuate the intramolecular interactions with the cooperative eliminates the possibility of fluorouracil anions interacting with protons in solution at temperatures below 35 ° C. Therefore, this well-known method at room temperature does not prevent the irreversible intramolecular condensation of Pt-DNA spatial structures and does not interfere with the protonation of the fluorouracil anions in the drug solution. In order to avoid these phenomena, it is necessary to carry out additional technological operations for the introduction into the drug solution of components that would increase its pH, as well as its density, before thermostatic control. In addition, it is possible to use an antitumor agent prepared in a well-known manner, except that at a temperature of 35-37 ° C, lowering the temperature causes a decrease in the activity of the agent. Also, the high temperature of the agent complicates its storage, transportation and application.

There is a way to stabilize an anti-tumor drug based on an aqueous solution of cisplatin at a concentration of 0.1-1.0 mg / ml using sodium chloride in an amount of 1-20 mg / ml hydrochloric acid to pH 2.0 -3.0 and mannitol (diuretic) in an amount of 2-150 mg / ml [USSR patent 1192596 A, published November 15, 1985].

Aqueous solutions of Pt-DNA compound stabilized with

-6 sodium chloride and sodium citrate (anticoagulation agent) in amounts corresponding to the molar ratio of platinum, DNA phosphorus, sodium chloride and sodium citrate 4: 6: 30: 3 [patent of Ukraine 60597. Published on

15.07.2005; 61543, published August 15, 2005; 66476 A, published at

May 17, 2004].

But the most effective and least toxic antitumor drugs based on aqueous Pt-DNA solution (compounds) are the compounds (derivatives) described above in the patent of Ukraine 70456. They are stabilized with sodium chloride, sodium citrate, ammonium chloride (a drug that causes expectoration and diuretic action) and an additional stabilizing agent, the sodium salt of fluorouracil, in the above ratio of the components of the solution.

The fluorouracil sodium salt potentiates the antitumor and attenuates the toxic effects of the parent substance by being able to act both as a modulator of the pharmacological action of Pt-DNA macromolecules in the body and as a stabilizer of Pt-DNA concentration

macromolecules in medicinal aqueous solution.

However, the aqueous solution of the Pt-DNA compound in known drugs has a pH of 6.5-7.5, whereby the use of the fluorouracil sodium salt allows stabilization of the concentration of Pt-DNA in the solution at a temperature above 35 ° C. Lowering the temperature helps to separate the macromolecules of the Pt-DNA compound from the solution in the form of a loose precipitate and the sodium salt of fluorouracil in the form of basic fluorouracil crystals, which causes a decrease of the parent compound and the concentration of the stabilizing agent in the solution of the detoxifying agent. form of the drug and adversely affect its antitumor activity and therapeutic efficacy. It also limits the storage conditions and administration options to topical administration and intraperitoneal infusions, eliminating intravenous and intraarterial infusions, endolymph injection.

-7and interstitial mode of administration.

SUMMARY OF THE INVENTION

In view of the foregoing, the present invention has to solve a number of problems.

One object of the present invention is to provide an antitumor drug in which the addition of uracil, urea, and sodium hydroxide to a known agent makes it possible to prevent

intramolecular condensation of the parent substance and protonation of the excipient, thereby avoiding their sedimentation and reducing their concentration in the solution over a temperature range of 2515 ° C, thereby increasing the antitumor activity and therapeutic efficacy of the drug and extending the drug its application.

A second object of the present invention is to provide a method for the preparation of an antitumor drug in which the use of aqueous solutions of uracil, urea and sodium hydroxide to a known agent under appropriate technological conditions results in a reduction in the intramolecular interaction rate of a cooperative nature in Pt-DNA molecules on the one hand. On the other hand, an increase in the intensity of interaction of their bases with the corresponding molecules in the solution is achieved, namely with fluorouracil, uracil and urea, which leads to the possibility of creating a drug containing the active ingredient PtDNA, whose molecules do not condense. at the storage temperature of the ready-to-use drug in the range of 25-15 ° C, which in turn prevents the sedimentation of macromolecules from the solution.

The third object of the present invention is to find a method for stabilizing antitumor drugs based on an aqueous solution of a platinum compound with increased selectivity of the specific

-8action and increased therapeutic efficacy.

The present problems are solved by the present invention.

The first problem is solved by an antitumor drug based on an aqueous solution of platinum derivative with deoxyribonucleic acid polyanions in situ (Pt-DNA), which includes unassociated Pt-DNA macromolecules of 0.05-0.15 microns in its largest dimension, sodium chloride, sodium citrate, ammonium chloride and fluorouracil in the form of the sodium salt according to the invention, and additionally contains uracil and urea in a molar ratio of fluorouracil, uracil and urea 1: 1: 1, at a ratio of components in aqueous solution, weight%

• Pt-DNA - 0.130-0.153

Sodium chloride - 0.080 - 0.090

Sodium citrate is 0.040 - 0.053

Ammonium chloride is 0.015-0.018

Fluorouracil in the form of the sodium salt - 0.025 - 0.370

Uracil is 0.018-0.270

Urea - 0.010-0.144, and more sodium hydroxide to reach pH 8.4 - 9.9.

©

At the same time, the density of the solution is 1.0054-1.0058 g / cm ³ .

The second task is solved by obtaining an antitumor drug by maintaining the DNA strands for 16-26 hours in a citrate salt solution containing sodium chloride and sodium citrate, dissolving the strands by stirring mechanically and heating the DNA solution to 7090 ° C. the gradual introduction of the cis-dichlorodiamino-platinum (DDP) citrate-salt solution heated to the same temperature in the heated DNA solution as well, the addition of fluorouracil sodium salt solution to the mixture and thermostatic control of the mixture of D solutions NK and DDP at 70-90 ° C for 15-20 minutes and added to the mixture

-9 solutions of DNA and DDP of an aqueous solution of the sodium salt of fluorouracil before thermostatic control. According to the invention, an aqueous solution of uracil and an aqueous solution of urea in concentrations corresponding to a molar ratio of fluorouracil, uracil and urea 1: 1: 1 must be added before the thermostat control, together with an aqueous solution of the sodium salt of fluorouracil. and also an aqueous sodium hydroxide solution with a pH of 11.0-12.0; the drug solution is then adjusted to pH 8.4-9.9 and to a density of 1.00541,0058 / cm ³ .

The proposed method for the preparation of an antitumor agent allows for increased pH and solution density, which eliminates the possibility of fluorouracil interacting with protons in the solution, which in turn allows the drug to be obtained with non-condensed macromolecules of PtDNA. , with a size of 0.05-0.15 microns in its largest dimension, at the storage temperature of the ready-to-use drug in the range 25-15 ° C. Thus, increasing the density of the solution prevents the sedimentation of macromolecules from the drug solution.

In practice, the antitumor drug is prepared as follows.

The DNA strands were maintained for 16-26 hours in a citrate salt solution containing sodium chloride and sodium citrate. The filaments were dissolved mechanically and the DNA solution heated to 7090 ° C. The citrate-salt solution of cis-dichlorodiamino-platinum (DDP), heated to the same temperature, was gradually introduced into the heated DNA solution as well. An aqueous solution of the fluorouracil sodium salt was then added to the mixture. At the same time, together with the aqueous solution of the sodium salt of fluorouracil, an aqueous solution of uracil and an aqueous solution of urea are added at concentrations corresponding to a molar ratio of fluorouracil, uracil and urea 1: 1: 1, and also an aqueous solution of sodium hydroxide with pH 11.0-12.0. In this way, the drug solution is adjusted to pH 8.4-9.9 and to a density of 1.0054-1.0058 / cm ³ . The mixtures of the DNA and DDP solutions were then maintained at 70-90 ° C for 15-20 minutes.

-10 EXAMPLES FOR CARRYING OUT THE INVENTION

Examples for the preparation of an antitumor drug are presented in Table 1, where Examples 1-3 refer to the drug claimed and Example 4 to a drug already known.

Table 1

Example number Conditions for receiving the medicine DNA storage time, hours heating temperature and thermostat control, ° C Amount of starting components, g / 1 DNA DDP NaCI At ₃ Cyt On ₃ Cy Clock Urea NaOH 1 0.67 0.87 0.80 0.40 3.70 2.70 1.44 0.80 20 90 2 0.68 0.92 0.86 0.48 2.50 1.84 0.99 0.72 26 70 3 0.74 0.96 0.90 0.53 0.25 0.18 0.10 0.60 16 85 4 0.72 0.96 0.90 0.50 2.50 26 85

Symbols in the table: NaCI - sodium chloride, Na ₃ Cyt - sodium citrate, NaFu - fluorouracil in the form of sodium salt, Ura - uracil, Urea.

With the molar weight of uracil 112 g mol, of fluorouracil 153 g mol, of ureas 60 g mol for the first example, the number of moles of uracil is 2.70 / 112 = 0.024; fluorouracil - 3.70 / 153 = 0.024; urea - 1.44 / 60 = 0.024;

For the second example, the number of these substances is 0.016 and for the third example - 0.0016. Therefore, an aqueous solution of uracil and an aqueous solution of urea are added at concentrations corresponding to the molar ratio of fluorouracil, uracil and urea 1: 1: 1.

The characteristics of the drug claimed, depending on the conditions of its preparation, are compared with those of the known drug. The results are presented in Table 2, where

Examples 1-3 relate to the drug claimed and Example 4 to a drug already known.

Table 2

No. of measure Characteristics of the drug obtained Storage temperature, ° C Composition of the drug,% by weight Size of Pt-DNA molecules µm Density of solution g / cnr Pt-DNA NaCl On, Cyt NHtCyt NaFu Clock Urea NaOH Until the pH of the solution is reached 1 15 0,130 0,082 0.053 0.015 0.370 0.272 0,142 9.9 0,05-0,15 1,0056 2 15 0,142 0.080 0.048 0.016 0.250 0,182 0.098 8.4 0.05-0.14 1,0058 20 0,142 0.080 0.048 0.016 0.250 0,182 0.098 8.8 0,05-0,15 1,0054 25 0,142 0.080 0.048 0.016 0.250 0,182 0.098 9.2 0,05-0,15 1,0054 3 20 0.153 0.090 0.040 0.018 0.025 0.016 0.010 8.8 0,05-0,15 1,0056 4 - 25 0,152 0,085 0.046 0.015 0.250 6,9 0.03-0.07 1,0012 15 0,152 0,085 0.046 0.015 0.250 6.6 0.02-0.05 1,0016

The differences in aqueous solutions of the claimed drug and the known drug are evidenced by the different pH values determined potentiometrically and the different density values determined by a pycnometric method. The differences in the spatial structures of the Pt-DNA molecules formed at a temperature below 35 ° C in the known drug and in the claimed drug are confirmed in the examples in the table of their different sizes in the molecular structures, which according to electron microscopic data differ from each other 2-3 times. The size of the molecular structures in the examples of the drug claimed corresponds to the size of the unassociated individual macromolecules of the substance, confirming the lack of intramolecular condensation of Pt-DNA molecules in the drug stored at 25-15 ° C.

• ·

The antitumor activities of the known drug and the drug claimed are tested in a cytotoxicity test on a cultured cell model derived from surgical material from a patient suffering from malignant oligodendroastrocytoma of the brain. Cells were cultured in culture media with standard composition: Eagle medium (40%), fetal calf serum (30-40%), glucose (800 mg%), insulin (0.2 units / ml) at constant gas composition, and temperature of 36.5 ° C in incubator. 4 days later, after inoculation during the period of intensive growth, the control cell culture was treated with normal saline, and the experimental cell cultures were treated with the solutions of the drug claimed and the solution of known drug maintained at temperature 25-15 ° C. The cultures were incubated for 24 hours after treatment. Antitumor activity was assessed by cytotoxicity, which was characterized by the relative amount of cell deaths compared to controls. The results of the calculated relative cell death rate, shown in percentages, are shown in Table 3, where No. 1 is the reference cell culture treated with normal saline; under No. 2-4 the indicators of experimental cell cultures treated with solutions of the drug claimed, and under No. 5 the indicators of experimental cell cultures treated with a solution of a known drug. The data in Table 3 are presented without data on the concentration of salts of sodium chloride, sodium citrate and ammonium chloride.

Table 3

Cell culture no The cure Storage temperature, ° C Concent tation, mkg / ml Percentage of dead cells Pt-DNA NaFu Clock Urea 1 Normal saline 0.7 2 Composition of Example # 1 15 30.0 74,0 54.0 28,0 96.0 3 Composition of Example # 2 15 30.0 50.0 36,0 20,0 86,8 20 30.0 50.0 36,0 20,0 84,3 25 30.0 50.0 36,0 20,0 86,2 4 · Composition of Example # 3 20 30.0 5.0 3.6 2.0 64,7 5 Composition of Example # 4 25 30.0 50.0 52,1 15 30.0 50.0 24.2

The data in Table 3 show that when comparing the quant

evaluation of antitumor activity in the cytotoxicity test, the drug claimed is significantly better than the known drug. Stored at the same temperature of 15 ° C and added to the culture at identical concentrations of Pt-DNA derived from 30 mkg / ml and fluorouracil of 50 mkg / ml, the known drug causes destruction of only 24.2% of the cells, while the drug for which claimed, destroys 86.6%.

Therefore, the proposed drug retains its high antitumor activity and therapeutic efficacy even at temperatures of 25-15 ° C, which greatly facilitates storage, transportation and administration.

The proposed drug can be used in various dosage forms.

The antitumor drug claimed is used in the clinical treatment of primary and metastatic tumors other than the lung, • · • · · · · · · · ·

- · 14 ^{^ ·· ·· ······ · · • χ} '· · · · · · ······ ·· · ······ kidneys, peritoneum and pleura, organs of the digestive system and sarcomatosis of the musculoskeletal system in dosage forms for intravenous, intraarterial and intraabdominal administration; in the treatment of tumor lesions of the head, neck and skin - in dosage forms for topical application in the form of application; for brain tumors - interstitially by intraoperatively implanting an Ommaya reservoir into the bed of the surgically removed tumor. The experience with the clinical administration of the drug claimed has shown that the use of its dosage forms, both intraabdominal and topical, and intravenous, intraarterial and interstitial administration, enhances the effectiveness of chemotherapy, which translates into increased patient life expectancy and improved quality of life during and after treatment courses. However, no significant clinical or laboratory side effects were observed in patients.

The following (third) problem is solved by the invention claimed by proposing a method for stabilizing antitumor agents based on an aqueous solution (compound) of a platinum derivative with deoxyribonucleic acid (Pt-DNA). According to this method, a mixture of basic fluorouracil with uracil and urea is used in a molar ratio of Pt-DNA nucleotide, basic fluorouracil, uracil and urea 1: (6-12: (0.6-1.2: (0.6-1, 2) as an additional stabilizing agent, reducing the proton concentration in the solution to 8.4-9.9 In addition, platinum compounds with deoxyribonucleic acid (Pt-DNA) at a concentration of 1.3 ΟΙ, 53 mg / ml containing sodium chloride in volume 0,80-0,90 mg / ml, sodium citrate in volume 0,40-0,53 mg / ml, ammonium chloride in volume 0,15-0,18 mg / ml and additional stabilizing agent agent according to the invention, a also not used and Pt-DNA complex and whose molecules are not separated from the solution at a temperature below 35 ° C.

As part of the drug, Pt-DNA macromolecules may contain one of the DNA isolated from natural sources or synthesized by genetic engineering methods, without the molecular weight being restricted to

0.3-20 million (million) Dalton.

To increase the pH of the drug solution to 8.4-9.9, either sodium hydroxide or potassium hydroxide or ammonium hydroxide or any other pharmaceutically acceptable pH regulator is used.

The described stabilization method is used in the technology of obtaining the antitumor drugs listed in Table 4, which differ from one another in the nature of the DNA from which the Pt-DNA compound is derived.

Table 4

No. antitumor medicine Characteristics of the DNA from which the Pt-DNA compound is derived Origin Ratio A + T / G + C Molecular Weight, million Daltons 1 Genetically engineered Philadelphia chromosome DNA 1.38 0.3-0.5 2 Salmon cum 1.43 0.3-0.5 3 Rat spit 1.43 3-4 4 Cattle descent, mark B 1.36 4-7 5 Young pig sniff, brand B 1.43 5-6 6 Human liver tumor 1.54 18-20 7 Schvets leukemic tumor in rat 1.04 17-18 8 Cattle descent, mark A 1.36 7-14 9 Young pig sniff, brand A 1.43 12-16

. Symbols in the table: A - adenine, T - thymine, G - guanine, C - cytosine.

In practice, the method of stabilizing antitumor drugs is as follows. The aqueous solution of the additional stabilizing agent containing a mixture of basic fluorouracil and urea is added to the aqueous solution of the Pt-DNA compound whose molecules do not separate from the solution at a temperature below 35 ° C, sodium.

chloride, sodium citrate and ammonium chloride. At the same time, along with the aqueous solution of the stabilizing agent, an aqueous solution of a pH regulator containing sodium hydroxide with a pH of up to 11-12 is added, by which the solution of the targeting agent is adjusted to pH 8.4-9.9.

The specific conditions for the antitumor drug stabilization method presented in Table 4, as well as the amounts of starting components in the solution of the parent compound, in the solution of the additional stabilizing agent and in the solution of the pH regulator, are presented in Table 5, where Examples 1-9 refer to the method claimed, and Examples 10 and 11 to the known method.

Table 5

No. with me Ra No Conditions under which the method is carried out Amount of starting components, g / 1 in the solution of the parent compound in the solution of the additional stabilizing agent in the solution of the narN regulator Pt-DNA NaCl At ₃ Cyt NH4CI NaFu Fu Clock Urea NaOH 1 1 1,30 0.83 0.53 0.17 2.75 0.236 0.127 0.75 2 2 1.53 0.87 0.50 0.15 3.23 0.255 0,149 0.73 3 3 1.48 0.80 0.46 0.18 3.12 0.224 0,132 0.80 4 4 1.50 0.90 0.48 0.15 2.38 0.205 0.122 0.63 5 5 1,30 0.85 0.45 0.16 2.06 0.158 0.106 0.68 6 6 1,30 0.80 0.40 0.15 1.37 0.118 0,063 0.60 7 7 1,40 0.85 0.46 0.16 1.47 0,147 0.080 0.70 8 8 1.53 0.90 0.53 0.18 1.62 0,139 0,099 0.80 9 9 1.48 0.88 0.42 0.17 2,34 0,157 0.108 0.75 10 8 1.50 0.90 0.50 0.18 1.89 11 9 1.48 0.80 0.53 0.15 1.82

Symbols in the table: NaCl - sodium chloride; Na ₃ Cyt - sodium citrate; NH4Cl - ammonium chloride; NaFu - fluorouracil sodium salt; Fu

- basic fluorouracil; Ura - uracil; Urea; NaOH - sodium hydroxide.

The characteristics of the method claimed, depending on the conditions under which it is carried out and the storage temperature of the drug, are compared with the characteristics of the known method using the data presented in Table 6, where Examples 1-9 refer to to the method claimed, and Examples 10 and 11 to the known method.

Table 6

Example number No of an anti-tumor drug Characteristics of the method Storage temperature, ° C Concentration of components in the drug solution, mg / ml Molar ratio of Pt-DNA nucleotide, Fu, Ura and urea Pt- DNA NaCl At ₃ Cyt W1 NaFu Fu NaOH until the pH of the solution is reached 1 1 25 1,30 0.87 0.53 0.17 2.75 9.5 1: 12: 1,2: 1,2 2 2 20 1.53 0.83 0.50 0.15 3.22 9,4 1: 12: 1,1: 1,2. 3 3 20 1.48 0.80 0.46 0.18 3.12 9,8 1: 12: 1: 1,1 4 4 15 1.50 0.90 0.48 0.15 2.37 8.7 1: 9: 0.9: 1.0 5 5 10 1,30 0.85 0.45 0.16 2.05 8.8 1: 9: 0.8: 1.0 6 6 5 1,30 0.80 0.40 0.15 1.37 8.5 1: 6: 0.6: 0.6 7 7 5 1,40 0.84 0.45 0.17 1.47 8.9 1: 6: 0.7: 0.7 8 8 20 1.53 0.90 0.53 0.18 1.62 9.9 1: 6: 0.6: 0.8 8 15 1.53 0.87 0.51 0.16 1.62 9.9 1: 6: 0.6: 0.8 8 10 1.53 0.90 0.52 0.17 1.62 9,8 1: 6: 0.6: 0.8 9 9 15 1.48 0.88 0.42 0.16 2,32 9.2 1: 9: 0.7: 0.9 9 10 1.48 0.87 0.40 0.15 2,32 9.3 1: 9: 0.7: 0.9 9 5 1.48 0.88 0.41 0.16 2,32 9,1 1: 9: 0.7: 0.9 10 8 38 1.50 0.90 0.50 0.18 1.89 7.0 8 15 1.04 0.90 0.50 0.18 1.47 6,8 11 9 38 1.48 0.85 0.53 0.15 1.82 8.0 9 10 0.63 0.85 0.53 0.15 1.19 7,6

Differences in the characteristics of the claimed method and the known method are evidenced by the different pH values determined potentiometrically and by the lack of temperature dependence of the concentrations of the parent compound and of the additional stabilizing agent controlled by quantitative methods. analysis. The differences in concentrations that occur when the temperature drops below 35 ° C by the known method are demonstrated in the examples in the table by reducing the concentration of the Pt-DNA compound from 1.50 mg / ml to 1.04 mg / ml, m .f. by 30.7% and for NaFu from 1.89 mg / ml to 1.47 mg / ml, i. by 22% in the aqueous solution of the drug stored at 15 ° C.

When stored at about 10 ° C the concentration drops to Pt-DNA from 1.48 mg / ml to 0.63 mg / ml, ie. already by as much as 57.4%, and for NaFu from 1.82 mg / ml to 1.19 mg / ml, i. by 34.4%. The concentrations of the parent substance and the additional stabilizing agent in the examples of the claimed method correspond to the concentrations in solutions stored at temperatures above 35 ° C, which confirms the ability of the method to provide conditions under which Pt-DNA macromolecules and molecules of basic fluorouracil should not be separated

from the solution while storing the target product in a temperature range of 25-5 ° C.

The antitumor activity that the claimed method and the known method of drug stabilization can provide is assayed by a cytotoxicity assay on a model of human brain anaplastic astrocytoma cells adapted for cultivation. The cells were kept in Eagle culture medium with added embryonic calf serum, glucose and insulin at a constant gas composition and a temperature of 36.5 ° C in an incubator. During the period of intensive growth, the control cell culture is treated with normal saline, and the experimental cell cultures are treated with the drug solutions stabilized according to the method claimed, and according to the known method, which are previously kept at 10 ° C. The cultures were incubated for 24 hours after treatment. Antitumor activity was assessed by cytotoxicity, which was characterized by the relative amount of cells that died 24 hours after drug treatment compared to controls. The results of the calculated relative cell death rate, shown as a percentage, are shown in Table 7, where under No. 1 are the indicators of the control cell culture treated with normal saline; under No. 2-5 the indicators of experimental cell cultures treated with drug solutions stabilized according to the method claimed, and under No. 6 the indicators of experimental cell cultures treated with drug solution stabilized according to the known method.

Table 7

Cell cultures №№ Storage temperature, ° C The cure Amount in ml of drug per 11 cultivated culture Pt-DNA concentration in cultured culture Percentage of dead cells 1 10 Normal saline 0.02 1.7 2 10 Drug 8 stabilized by the method presented in Example 8 0.02 30.6 86,4 3 10 Drug # 9, stabilized by the method presented in Example 9 0.02 29.6 80.4 4 10 Drug No. 5 stabilized by the method presented in Example 5 0.02 26,0 78,8 5 10 Drug # 3 stabilized by the method presented in Example 3 0.02 29.6 82,1 6 10 Drug # 9, stabilized by the method presented in Example 9 0.02 12.6 32,2

The data in Table 7 indicate that when comparing the quantitative evaluation of the antitumor activity with the cytotoxicity test, the stabilization method claimed is significantly better than the known method. Stored at the same temperature of 10 ° C and applied in equal volumes of 0.02 ml with identical amounts of 1.48 mg / ml Pt-DNA, at a temperature above 35 ° C the aqueous solutions of the drug stabilized by the known method give a concentration of the parent substance only 12.6 mg / l after being added to the culture medium in a volume of 1 l, resulting in destruction of only 32.2%, while aqueous solutions stabilized by the method claimed, after their addition to the culture medium medium, give a Pt-DNA concentration of 29.6 mg / l, resulting in destruction of 80,482.1% of the cell .

Therefore, the proposed stabilization method provides an increased selective specific effect of the Pt-DNA compound and its increased therapeutic efficacy even at temperatures of 25-5 ° C, and also provides facilitated storage conditions and an extended spectrum of

administration by the use of a platinum compound with deoxyribonucleic acid (Pt-DNA), the molecules of which are not separated from the solution at a temperature below 35 ° C by using a mixture of basic fluorouracil with uracil and urea, and also by using the solution with reduced proton concentration.

Antitumor drugs stabilized by the method claimed can be used in different dosage forms.

The experience with the clinical administration of the drugs stabilized by the method claimed shows that their use in dosage forms for both intraabdominal and topical administration, intravenous, intraarterial, endolymph and interstitial administration, enhances the effectiveness of primary and metastatic tumors, localized to the head, neck, lungs, kidneys, organs of the digestive system and musculoskeletal system. Improving efficacy is about increasing the life expectancy of patients and improving quality of life during treatment courses and beyond. However, no significant clinical or laboratory side effects were observed in patients.

Claims (11)

An antitumor drug based on an aqueous solution of a platinum derivative with deoxyribonucleic acid polyanion, characterized in that it comprises unassociated Pt-DNA macromolecules of 0.05-0.15 microns in the largest dimension, sodium chloride, sodium citrate, ammonium chloride and fluorouracil in the form of a sodium salt, characterized in that it further contains uracil and urea in a molar ratio of fluorouracil, uracil and urea 1: 1: 1, in a ratio of components in aqueous solution, by weight %: Pt-DNA - 0,130-0,153 Sodium chloride - 0.080 - 0.090 Sodium citrate is 0.040 - 0.053 Ammonium chloride is 0.015-0.018 Fluorouracil in the form of the sodium salt - 0.025 - 0.370 Uracil is 0.018-0.270 Urea - 0.010-0.144, and more sodium hydroxide to reach pH 8.4 - 9.9. 2. An antitumor drug according to claim 1, characterized in that the density of the solution is 1.0054-1.0058 g / cm. 3. A method for the preparation of an antitumor drug comprising the maintenance of the DNA strands for 16-26 hours in a citrate-saline solution containing sodium chloride and sodium citrate, dissolving the strands by mechanical stirring and heating of the DNA solution to 70-90 ° C, the gradual introduction of the cis-dichloro-diamino-platinum (DDP) citrate-salt solution heated to the same temperature in the same heated DNA solution, adding to the mixture of aqueous sodium salt • · Fluorouracil and thermostatic control of the mixture of DNA and DDP solutions at 70-90 ° C for 15-20 minutes and adding to the mixture of DNA and DDP solutions an aqueous solution of the fluorouracil sodium salt before thermostatic control. This method differs in that before the thermostatic control, together with an aqueous solution of the sodium salt of fluorouracil, an aqueous solution of uracil and an aqueous solution of urea must be added at concentrations corresponding to the molar ratio of fluorouracil, uracil and urea 1: 1: 1 and also an aqueous solution of sodium hydroxide with a pH of 11.0-12.0; the drug solution is then adjusted to pH 8.4-9.9 and to a density of 1.0054-1.0058 / cm ³ . 4. Method for stabilizing antitumor agents based on an aqueous solution of a platinum derivative with deoxyribonucleic acid (PtDNA). This method differs in that it uses a mixture of basic fluorouracil with uracil and urea in a molar ratio of Pt-DNA nucleotide, basic fluorouracil, uracil and urea, 1: (6-12: (0.6-1.2: (0.6-1.2) as an additional stabilizing agent, reducing the proton concentration in the solution to 8.4-9.9. 5. The method of claim 4, wherein the concentration of the Pt-DNA complex is 1.30-1.53 mg / ml. A method according to claim 4, characterized in that the medicament further contains sodium chloride in an amount of 0.80-0.90 mg / ml. 7. The method of claim 4, wherein the medicament further comprises sodium citrate in an amount of 0.40-0.53 mg / ml. A method according to claim 4, characterized in that the medicament contains additional ammonium chloride in an amount of 0.15 - 0.18 mg / ml. A method according to claim 4, characterized in that therapeutically acceptable DNA, recovered from natural sources or synthesized by genetic engineering methods, has a molecular weight of 0.3-20 million Daltons. you * ·> ·· A method according to claim 4, characterized in that either sodium hydroxide or potassium hydroxide or ammonium hydroxide or any other pharmacologically acceptable pH is used to adjust the pH in the drug solutions to 8,4-9,9. regulator. An antitumor drug based on an aqueous solution of a platinum derivative (compound) with deoxyribonucleic acid, stabilized according to claim 4.