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• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K33/00—Medicinal preparations containing inorganic active ingredients
  • A61K33/24—Heavy metals; Compounds thereof
  • A61K33/243—Platinum; Compounds thereof
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
  • A61K31/7088—Compounds having three or more nucleosides or nucleotides
  • A61K31/713—Double-stranded nucleic acids or oligonucleotides
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/28—Compounds containing heavy metals
  • A61K31/282—Platinum compounds
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/33—Heterocyclic compounds
  • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
  • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
  • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
  • A61K31/513—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim having oxo groups directly attached to the heterocyclic ring, e.g. cytosine
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
  • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
  • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
  • A61K47/16—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing nitrogen, e.g. nitro-, nitroso-, azo-compounds, nitriles, cyanates
  • A61K47/18—Amines; Amides; Ureas; Quaternary ammonium compounds; Amino acids; Oligopeptides having up to five amino acids
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K9/00—Medicinal preparations characterised by special physical form
  • A61K9/0012—Galenical forms characterised by the site of application
  • A61K9/0019—Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K9/00—Medicinal preparations characterised by special physical form
  • A61K9/08—Solutions
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P35/00—Antineoplastic agents

Definitions

• the invention relates to medicine, namely to antitumor agents based on an aqueous solution of platinum compounds, which can be used to treat malignant tumors, to methods for their preparation and to methods for their stabilization.
• antitumor agents widely used in medical practice, some of the most effective are based on an aqueous solution of the cisplatin monomeric complex.
• the active agent in these agents is platinum ion, coordinated by carrier ligands that are absorbed by tumor cells almost as intensively as normal ones, and therefore drugs containing cisplatin are characterized not only by high antitumor activity, but also by high toxicity.
• PT-DPC platinum with deoxyribonucleic acid
• one of the ligands-carriers of the platinum ion is a nucleotide embedded in the double-stranded structure spirals of DNA macromolecules, intensively absorbed only by rapidly multiplying tumor cells, and therefore antitumor agents containing the Pt-DNA compound are highly selected elnosti affect tumor tissue insignificant damage normal [Treatment of inoperable tumors of the abdominal cavity. CA. Shalimov et al. 1998, p. 103].
• the cisplatin monomeric complex and the Pt-DNA polymer complex which are the main substances in the products, are not sufficiently soluble and chemically unstable in water, therefore pharmaceutical additives are introduced into the products based on their aqueous solutions.
• Pharmacologically acceptable salts, pH regulators and other stabilizing agents are used as additives, preferably those which, due to their own biological activity, which is manifested simultaneously with the ability to increase the solubility and chemical stability of the basic substance, make it possible to ensure the effectiveness of treatment with better tolerance.
• a known antitumor agent based on an aqueous solution of a compound of platinum with a polyanion of deoxyribonucleic acid (Pt DNA) (active substance) includes the 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 sodium salt (excipient) at a ratio of components in an aqueous solution, mass. %:
• an aqueous solution of the known drug has a pH of 6.5-7.5 and a density of 1.0012 - 1.0016 g / cm 3 , in which unassociated molecules of the Pt-DNA compound have the highest antitumor activity, and fluorouracil has the ability to enhance this activity and reduce the toxicity of the platinum compound upon aging of the obtained agent in the temperature range above 35 ° C.
• fluorouracil anions tend to interact with protons of the solution to a greater extent than with Pt DNA bases, and therefore turn into a state of neutral molecules of their own base, the solubility of which is limited in water and which settles in the form of crystals that are soluble with increasing temperature.
• the precipitation of the active and auxiliary substances causes a decrease in their concentration in the solution of the drug and, thereby, reduces its antitumor activity and therapeutic effectiveness, limits its use only to local applications and intraperitoneal infusions, excluding intravenous, intraarterial infusions and interstitial route of administration.
• urea as a diuretic, keratolytic, a means of reducing intracranial and intraocular pressure is known [M.M. Turkevich. Pharmaceutical Chemistry, 1973, p. 45], and also as an excipient added to solid dosage forms for oral administration.
• uracil, urea and sodium hydroxide as a means of preventing the intramolecular condensation of spiralized strands of metal compounds with DNA.
• the use of urea in dosage forms for parenteral administration is also not known.
• the effective concentration ranges and the ratio of the active substance, fluorouracil and salts in the aqueous solution of the agent, which is claimed, correspond to the ratios established during the development of the known means for intraperitoneal, intravenous and intra-arterial infusions, which are maintained at a temperature of 35-40 ° C.
• the ranges of concentrations of uracil and urea determined by the molar ratio of fluorouracil, uracil and urea 1: 1: 1.
• the ranges of sodium hydroxide concentrations are limited from below by a pH value, which excludes the possibility of protonation of fluorouracil, and from above by pharmaceutical requirements.
• citrate-saline solution which contains sodium chloride and sodium citrate
• the antitumor agent obtained in a known manner it is possible to use only at a temperature of 35-37 0 C, as with decreasing temperature the activity of the agent decreases.
• the high temperature of the product complicates its storage, transportation and use.
• a known method of stabilizing an antitumor agent based on an aqueous solution of cisplatin with a concentration of 0.1 to 1.0 mg / ml which uses sodium chloride in an amount of 1 to 20 mg / ml, hydrochloric acid to pH 2.0-3.0 and mannitol (diuretic) in an amount of 2-150 mg / ml [US Pat. USSR 1192596 A publ. 11/15/85].
• the most effective and least toxic antitumor agent based on an aqueous solution of the Pt-DNA compound is the agent described above in US Pat. Ukraine 70456, which is stabilized with sodium chloride, sodium citrate, ammonium chloride (an agent that causes an expectorant effect and enhances the action of diurethins) and an additional stabilizing agent with the fluorouracil sodium salt in the ratio of components in the solution mentioned above.
• Sodium salt of fluorouracil enhances the antitumor and weakens the toxic effects of the main substance, as it can act both as a modulator of the pharmacological action of Pt-DNA macromolecules in the body, and as a stabilizer of the concentration of Pt-DNA macromolecules in an aqueous solution of the drug.
• an aqueous solution of the Pt-DNA compound in a known agent has a pH of 6.5-7.5, at which the use of the sodium salt of fluorouracil can stabilize the concentration of Pt-DNA in the solution at a temperature above 35 ° C.
• this invention poses a number of tasks that need to be addressed.
• One of the challenges facing the present invention is to provide an antitumor agent in which, by adding uracil, urea and sodium hydroxide to the known agent, it is possible to prevent intramolecular condensation of the basic substance and protonate the excipient and thereby avoid their precipitation and decrease their concentration in solution in the temperature range of 25-15 ° C, which achieve an increase in antitumor activity and therapeutic efficacy of the drug and expanded renie its application.
• the second challenge facing the invention is to develop a method for producing an antitumor agent, in which, by using aqueous solutions of uracil, urea and sodium hydroxide under appropriate technological conditions, on the one hand, weakening the intensity of intramolecular interactions of a cooperative nature in Pt-DNA molecules and, on the other hand, increasing the intensity of the interaction of their bases with related solution molecules, namely fluorouracil, uracil and urea, which e enable creation means which contains the active substance Pt-DNA molecules which do not condense at the temperature keeping ready means in the range 25-15 ° C, which in turn prevents and prevents precipitation of macromolecules from a solution.
• the third task facing the invention is to create a method for stabilizing antitumor agents based on an aqueous solution of a platinum compound with increased selectivity of a specific action and increased therapeutic efficacy. The tasks are solved by the proposed invention.
• Pt-DNA deoxyribonucleic acid
• the second task is solved due to the fact that the proposed method for producing an antitumor agent by keeping DNA fibers in a citrate-salt solution containing sodium chloride and sodium citrate for 16-26 hours, dissolving the fibers with mechanical stirring and heating the DNA solution to 70-90 0 C, the gradual introduction into a heated solution of DNA heated to that the temperature of the citrate-salt solution of cis-dichlorodiamineplatinum (DDP), thermostating of a mixture of DNA and DDP solutions at 70-90 0 C for 15-20 minutes and adding to the mixture before thermostating an aqueous solution of sodium fluorouracil, in which, according to the invention, before thermostating simultaneously with an aqueous solution of sodium salt of fluorouracil additionally introduce an aqueous solution of uracil and an aqueous solution of urea with concentrations that correspond to a molar ratio of fluorouracil, uracil and urea of 1: 1: 1, and water th solution of sodium hydroxide with a
• the proposed method for producing an antitumor agent allows one to achieve increased pH and density of the solution, which excludes the possibility of interaction of fluorouracil with protons of the solution, and this, in turn, provides means with Pt-DNA macromolecules that do not condense, 0.05-0, 15 microns in the largest dimension at a temperature of keeping the finished product in the range of 25-15 0 C. In this case, an increase in the density of the solution prevents the precipitation of macromolecules from the solution of the product.
• the use of urea in parenteral dosage forms for parenteral administration is also not known.
• the effective ranges of concentrations and ratios of the basic substance and salts in an aqueous solution stabilized by the method that is claimed correspond to the concentrations and ratios established during the development of a known method for producing a Pt-DNA compound in an aqueous solution for intraperitoneal, intravenous and intraarterial infusions that have a pH of 6.5 - 7.5 and are maintained at a temperature of 35-40 ° C.
• the concentration ranges of the fluorouracil, uracil and urea base are determined by the molar ratio of the nucleotide PT-DNA, the base of the fungoracil, uracil and urea, which is l :( 6-12) :( 0.6-l, 2) :( 0.6-1.2).
• the ranges of concentration of the pH regulator are limited from below to values that exclude the possibility of crystallization of the fluorouracil base, and from above to pharmaceutical requirements.
• the proposed stabilization method does not allow Pt-DNA macromolecules and fluorouracil base molecules to be isolated from the solution, which in turn provides antitumor agents with such concentrations of components that do not change in their dosage forms at a temperature of keeping the finished product in the range of 25-5 0 C with prolonged storage.
• an antitumor agent was prepared as follows.
• DNA fibers were kept for 16-26 hours in a citrate-salt solution, which contained sodium chloride and sodium citrate. They were dissolved with mechanical stirring and heating the DNA solution to 70-90 ° C. A citrate-salt solution of cis-dichlorodiamineplatinum (DDP) heated to the same temperature was gradually introduced into the heated DNA solution. Then, an aqueous solution of the fluorouracil sodium salt was added to the mixture.
• DDP cis-dichlorodiamineplatinum
• the number of moles for these substances is 0,
• an aqueous solution of uracil and an aqueous solution of urea were introduced at concentrations that corresponded to a molar ratio of 1: 1: 1 of fluorouracil, uracil and urea.
• the differences in aqueous solutions of the claimed agent and the known are confirmed by different pH values, which are determined potentiometrically, and different density values found pycnometrically.
• the differences in the spatial structures of Pt-DNA molecules, which are formed at temperatures below 35 0 C in a known tool and in the inventive tool are confirmed in the table examples by their different sizes in molecular structures, which according to electron microscopy are 2-3 times different from each other.
• the size of the molecular structures in the examples of the claimed agent corresponds to the size of the unassociated individual macromolecules of the substance, which confirms the absence of intramolecular condensation of Pt-DNA molecules in the agent, kept at a temperature of 25 - 15 0 C.
• the antitumor activity of the known agent and the claimed agent was investigated in a cytotoxicity test on a model of cultured cells obtained from surgical material a patient with malignant oligodendroastrocytoma of the brain.
• the cells were cultured in a nutrient mixture of standard composition: medium needle (40%), fetal calf serum (30-40%), glucose (800 milligrams%), insulin (0.2 units / ml) with a constant regime of gas composition and temperature 36 , 5 ° C in an incubator. 4 days after seeding, during the period of intensive growth, the cells of the control culture were treated with physiological saline, and the cells of the experimental cultures were treated with solutions of the inventive agent and a solution of a known agent aged at temperatures of 25-15 ° C.
• the proposed tool retains its high antitumor activity and therapeutic efficacy even at temperatures of 25-15 ° C, which greatly simplifies its storage, transportation and use.
• the proposed tool can be used in different dosage forms.
• the antitumor agent of the invention was used in the clinical treatment of primary and metastatic tumors of the lungs, kidneys, peritoneum and pleura, digestive organs and musculoskeletal system in dosage forms for intravenous, intraarterial and intraperitoneal administration; in the treatment of tumor lesions of the head, neck and skin - in dosage forms for topical application in the form of applications; in brain tumors - interstitially with intraoperative implantation of an Ommua reservoir in the bed of a surgically removed tumor.
• the clinical experience of the claimed drug showed that the use of its dosage forms for both intraperitoneal and topical administration, and dosage forms for intravenous, intraarterial and interstitial administration, increases the effectiveness of chemotherapy, which is expressed in increasing the life expectancy of patients and improving the quality of life during the course of treatment and in a long period of time. In this case, no important clinical or laboratory manifestations of side effects in patients were detected.
• the next (third) problem is solved by this invention due to the fact that a method for stabilizing antitumor agents based on an aqueous solution of a compound of platinum with deoxyribonucleic acid (Pt-DNA) is proposed, in accordance with which, as an additional stabilizing agent, a mixture of fluorouracil base with uracil and urea in a molar ratio of PT DNA nucleotide, fluorouracil, uracil and urea base l: (6-12) :( 0.6-l, 2) :( 0.6-l, 2) with a decrease in the proton concentration in the solution to 8 4 - 9.9.
• Pt-DNA platinum with deoxyribonucleic acid
• platinum compounds with deoxyribonucleic acid are used at a concentration of 1.30-1.53 mg / ml, containing sodium chloride in an amount of 0.80-0.90 mg / ml, sodium citrate in an amount of 0.40 - 0.53 mg / ml, ammonium chloride in an amount of 0.15 - 0.18 mg / ml and an additional stabilizing agent, according to the invention, use the Pt-DNA complex, whose molecules below 35 0 C are not isolated from the solution.
• Pt-DNA macromolecules may contain one of the DNA isolated from natural sources or synthesized by genetic engineering with a molecular weight, not limited to, 0.3 - 20 million daltons.
• Either sodium hydroxide, or potassium hydroxide, or ammonium hydroxide, or some other pharmacologically acceptable pH regulator is used to adjust the pH of the solution in the media to 8.4 - 9.9.
• the inventive method of stabilization was applied in the technology for producing antitumor agents listed in Table 4, which differed from each other by the nature of DNA preceding the Pt-DNA compound.
• the method of stabilizing antitumor agents was carried out as follows.
• an aqueous solution containing a compound of Pt-DNA, macromolecules whose lower than 35 0 C are not released from solution sodium chloride, sodium citrate and ammonium chloride
• an aqueous solution of an additional stabilizing agent containing a mixture of a base of fluorouracil, uracil and urea.
• an additional aqueous solution of a pH regulator was added, containing sodium hydroxide to a pH of 11.0-12.0, with which a solution of the target agent was adjusted to a pH of 8.4 - 9.9.
• the differences in the characteristics of the claimed and known method are confirmed by different ranges of pH values, measured potentiometrically, and the absence of a temperature dependence of the concentrations of the main substance and the additional stabilizing agent, controlled by quantitative analysis methods.
• the differences in concentrations that occur at temperatures below 35 0 C in the known method are confirmed in the examples of the table by a decrease in the concentration of the Pt-DNA compound from 1.50 mg / ml to 1.04 mg / ml, i.e. 30.7%, and NaFu from 1.89 mg / ml to 1.47 mg / ml, i.e. 22% in an aqueous solution of the product, aged at 15 ° C.
• concentrations of the basic substance and the additional stabilizing agent in the examples of the method that is claimed correspond to the concentrations in solutions maintained at temperatures above 35 ° C, which confirms the possibility of the method to ensure conditions under which Pt-DNA macromolecules and fluorouracil base molecules do not stand out from the solution while maintaining the target product in the temperature range 25-5 ° C.
• the antitumor activity which the claimed and known methods of stabilizing means can provide, was investigated in a cytotoxicity test on a model of human brain malignant astrocytoma adapted for cell culture.
• Cells were maintained in Eagle's nutrient medium with the addition of fetal calf serum, glucose, and insulin under a constant regime of gas composition and a temperature of 36.5 ° C in an incubator.
• the cells of the control culture were treated with physiological saline, and the cells of the experimental cultures with solutions of means stabilized by the claimed and known method, which were previously kept at a temperature of 10 0 C.
• the antitumor activity was evaluated by cytotoxicity, characterized by the relative number of cells killed in relation to the control 24 hours after processing means.
• the proposed method provides increased stabilization of the selective action of a specific DNA complex Pt- and enhanced therapeutic efficacy even at temperatures of 25 to 5 0 C, and provides enhanced means storage conditions and extended the range of their applications by using the platinum complex with deoxyribonucleic acid (PT DNA), whose macromolecules below 35 0 C are not isolated from the solution, due to the use of a mixture of fluorouracil base with uracil and urea, and also due to the use of solution with a low concentration of protons.
• PT DNA deoxyribonucleic acid
• Antitumor agents stabilized by the present method can be used in different dosage forms.

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• 2009
  • 2009-11-23 PL PL394863A patent/PL219734B1/pl unknown
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