RU2744858C1 - Method of obtaining lyophilisate of sodium aminodihydrophthalazinedione, or tameron medication - Google Patents

Abstract

FIELD: pharmaceuticals.

SUBSTANCE: invention relates to pharmaceuticals and describes a method for producing a drug based on the sodium salt of 5-amino-2,3-dihydrophthalazine-1,4-dione (sodium aminodihydrophthalazinedione) in the form of a lyophilisate. The method includes preparing an aqueous solution of sodium aminodihydrophthalazinedione with a concentration of 03-250 mg / ml; carrying out sterilizing filtration of the resulting solution and dosing the sterile solution into the primary packaging; carrying out lyophilization. Freeze drying is carried out in several stages, while cooling is carried out to minus 75°C - minus 15°C at a rate of 0.5-10 ° C per minute and further drying is carried out until the moisture content in the product is not more than 10%. At the last stage, the freeze-drying chamber is filled with sterile air, sealed and the package is rolled up / sealed.

EFFECT: method according to the invention allows obtaining a stable and highly purified drug in the form of sodium aminodihydrophthalazinedione lyophilisate with increased ergonomics and conversion of the process.

2 cl, 2 tbl, 7 dwg, 6 ex

Description

The invention relates to pharmaceuticals, medicine and veterinary medicine and describes a method for producing a medicinal product based on the sodium salt of 5-amino-2,3-dihydrophthalazine-1,4-dione (sodium aminodihydrophthalazinedione) in the form of a lyophilisate.

A known method of obtaining 2-amino-1,2,3,4-tetrahydrophthalazine-1,4-dione sodium salt of dihydrate (see, for example, Abidov MT "Toxic syndrome in infectious inflammation, pathogenesis and correction", doctoral dissertation , S.-P., 1994), called the drug Galavit (see, for example, "New domestic medicines", Official edition, issue 4, M., 1998, p. 18).

The medicinal preparation Galavit is obtained in the production of the well-known chemiluminescent substance luminol-hydrazide of 3-aminophthalic acid (see, for example, Holzbecker Z. et al. "Organic reagents in inorganic analysis", - M .: Mir, 1979, p. 107) by processing luminol by the action of a base (NaOH) on it, followed by isolation by physicochemical selection from the resulting substance, a by-product - 2-aminophthalhydrazide, which is a luminol isomer and makes up about 2-3% of the total amount of the produced product. When obtaining the drug Galavit, which includes obtaining the starting product and reacting with a base, for example sodium hydroxide (NaOH), a molecular rearrangement reaction of the starting product is carried out and its isomer is obtained, which is then acted upon with sodium hydroxide, and 3-aminophthalhydrazide is obtained as the starting product, after rearrangement - 2-aminophthalhydrazide. The output product is obtained in the form of 2-amino-1,2,3,4-tetrahydrophthalazine-1,4-dione sodium salt of the dihydrate, while the rearrangement is carried out in the presence of a halogen, for example, bromine (Br2), at a temperature of 10-80 ° C ... (Method of obtaining medicinal product Galavit patent RU No. 2138264 with priority from 1999.05.06)

The production of drugs is carried out in repeating cycles, in each of which the initial composition containing 5-amino-2,3-dihydrophthalazine-1,4-dione is exposed to an aqueous solution of an alkali metal hydroxide, and the intermediate composition is isolated in the form of the corresponding 5-amino salt -2,3-dihydrophthalazine-1,4-dione, after which this intermediate composition is dissolved in water or in an appropriate bicarbonate buffer solution and treated to remove impurities to obtain the desired product in the form of 5-amino-2,3-dihydrophthalazine salt -1,4-dione, which is dried after production, and the mother liquors remaining after the isolation of the intermediate composition or the target product are processed, the precipitate and the remaining parts of the mother liquors are either removed from the cycle, or mixed with the original composition, or with the reaction mixture obtained in the subsequent production cycle. Crystallization of the corresponding obtained alkaline salt is carried out and a chemically pure or high-purity product is obtained, the water is preliminarily subjected to boiling for (0.1-2) hours, and when processing the intermediate composition or the target product after their isolation in the form of alkaline salts of 5-amino-2 , 3-dihydrophthalazine-1,4-dione are dried (RF Patent No. 2302863, IPC A61K 31/502, publication date 07.20.2007).

This method of manufacturing a medicinal product is a high-tech, low-waste production process. The method provides for obtaining variants of the drug of different degrees of chemical purity, including pharmaceutical, which significantly expands the possibilities of their use in cases of long-term storage in aqueous solutions. However, this method uses heating of the reaction mixture, which leads to the opening of the cycle and darkening of the reaction mixture and, accordingly, to a decrease in the quality and yield of the final target product.

As the closest analogue, RF Patent No. 2625267 (IPC A61K 31/502, C07D 237/32, publication date 09/22/2016), describing a method for preparing a sterile lyophilized drug based on the sodium salt of 5-amino-2,3-dihydrophthalazine, can be indicated -1,4-dione by processing the anhydrous and / or dihydrous non-sterile substance of the sodium salt of 5-amino-2,3-dihydrophthalazine-1,4-dione.

This method is implemented by processing the intermediate product 5-amino-2,3-dihydrophthalazine-1,4-dione into its sodium salt by dissolving in an aqueous-organic alkaline solution, so that the anhydrous and / or dihydrous substance of the sodium salt 5-amino-2 , 3-dihydrophthalazine-1,4-dione is obtained by crystallization at a temperature of minus 5-7 ° C from an aqueous-organic solution of sodium salt of 5-amino-2,3-dihydrophthalazine-1,4-dione, after which the substance is dissolved in sterile distilled or injection water with a temperature of 50-55 ° C with hydrazine hydrate, acetone with a temperature of 45 ° C is poured in and sterile filtration is performed with a lyophilization step to obtain a sterile drug.

The disadvantage of this method of producing a medicinal product is the use of hydrazine hydrate and acetone in the technology, which are poisonous, chemically hazardous substances that affect the hematopoietic system, liver, and central nervous system. Hydrazine hydrate is a carcinogen. In this patent, the obtained sterile lyophilized preparation has a high humidity, which significantly affects the stability of the preparation during storage and contributes to the degradation of the active substance. Also, the specified method does not describe the process of freeze drying, which significantly affects the final form of the drug, namely the ratio of crystalline forms.

The present invention is a new technological method for the production of the drug "Tameron", namely the lyophilized sodium salt of 5-amino-2,3-dihydrophthalazine-1,4-dione, which has anti-inflammatory, immunomodulatory, antioxidant, regenerating, antiviral, antibacterial, antifungal, neuroprotective, analgesic, antitumor effects.

The task is to ensure increased stability, representativeness of the obtained preparation, reproducibility of the claimed technological process, conditions for its implementation and control, minimization of harmful impurities (isomeric trace impurities such as N-aminoisomer and linear sodium oligomers), environmental harmlessness of the process.

Obtained by the claimed method sodium aminodihydrophthalazinedione (sodium aminodihydrotalazinedione lyophilisate, drug "Tameron") is characterized by a low content of impurities, in particular, residual organic solvents, degradants of sodium aminodihydrophthalazinedione, and rapid recovery of moisture content in an aqueous solution, for example, or in water sodium chloride solution, namely 0.9-1.0% - sodium chloride solution (physiological solution).

The lyophilisate of sodium aminodihydrophthalazinedione obtained according to this invention does not have a local irritating effect upon repeated use, has better absorption and bioavailability in comparison with other monopreparations based on sodium aminodihydrophthalazinedione.

The technical results are achieved due to the fact that a method for obtaining a lyophilized drug "Tameron" has been developed, which is sodium aminodihydrophthalazinedione in the form of stable crystals with a variable molar ratio of substance / water, with an accurate fixation of the amount of water, with a low content of mechanical, chemical and biological impurities, allowing to obtain a stable finished form with increased solubility, with a given ratio of crystalline forms and a low moisture content.

The technical problem posed and the technical result obtained are achieved by the claimed method of obtaining sodium aminodihydrophthalazinedione lyophilisate (5-amino-2,3-dihydrophthalazine-1,4-dione), which includes the following stages:

1) preparation of an aqueous solution of sodium aminodihydrophthalizinedione until it is completely dissolved with a concentration in the solution of 0.3-250 mg / ml for 0.1-1.5 hours;

2) sterilizing filtration of the resulting drug solution under aseptic conditions and dosing it into primary packaging for lyophilized forms;

3) freeze drying of the drug in a freeze drying chamber in several stages, while cooling to a temperature of minus 75 ° C - minus 15 ° C at a rate of 0.5-10 ° C per minute and further drying under vacuum. Drying is carried out until the moisture content in the product is not more than 10%;

4) filling the drying with inert gas or sterile air, sealing the primary packaging and seaming or sealing.

The stage of sterilizing filtration can be carried out several times, depending on the quality of the original pharmaceutical substance.

K., Senker, J. and Breu, J. (2015), The Same at a First Glance: The Diffractogram of a New Polymorph of Anhydrous Sodium Luminolate Almost Perfectly Resembles the Diffraction Trace of an Already Known Polymorph. Z. anorg. allg. Chem., 641: 332-338. https://doi.org/10.1002/zaac.201400604.), LOHKOK02² (² Martin, Т., Greim, D., Milius, W., Niedermaier, M., Ludescher, В., von Wegerer, J., Brysch, W.,

K., Senker, J. and Breu, J. (2015), The Same at a First Glance: The Diffractogram of a New Polymorph of Anhydrous Sodium Luminolate Almost Perfectly Resembles the Diffraction Trace of an Already Known Polymorph. Z. anorg. allg. Chem., 641: 332-338. https://doi.org/10.1002/zaac.201400604.) и код аморфной части степени кристаллизации данного соединения LOHKIE¹ (¹ Rybakov, V.B., Chernyshev, V.V., Paseshnichenko, K.А. et al. Crystallogr. Rep. (2014) 59: 383. https://doi.org/10.1134/S1063774514020187).It is known that sodium aminodihydrophthalazinedione crystals can be in several modifications. Thus, the Cambridge Structural Database (CBSD) contains codes for three modifications of sodium aminodihydrophthalazinedione salts LOHKOK ¹ ( ¹ Rybakov, VB, Chernyshev, VV, Paseshnichenko, K.A. et al. Crystallogr. Rep. (2014) 59: 383. https: //doi.org/10.1134/S1063774514020187.), LOHKOK01 ² ( ² Martin, T., Greim, D., Milius, W., Niedermaier, M., Ludescher, B., von Wegerer, J., Brysch, W .,

K., Senker, J. and Breu, J. (2015), The Same at a First Glance: The Diffractogram of a New Polymorph of Anhydrous Sodium Luminolate Almost Perfectly Resembles the Diffraction Trace of an Already Known Polymorph. Z. anorg. allg. Chem. 641: 332-338. https://doi.org/10.1002/zaac.201400604.), LOHKOK02 ² ( ² Martin, T., Greim, D., Milius, W., Niedermaier, M., Ludescher, B., von Wegerer, J. , Brysch, W.,

K., Senker, J. and Breu, J. (2015), The Same at a First Glance: The Diffractogram of a New Polymorph of Anhydrous Sodium Luminolate Almost Perfectly Resembles the Diffraction Trace of an Already Known Polymorph. Z. anorg. allg. Chem. 641: 332-338. https://doi.org/10.1002/zaac.201400604.) and the code of the amorphous part of the degree of crystallization of this compound LOHKIE ¹ ( ¹ Rybakov, VB, Chernyshev, VV, Paseshnichenko, K.A. et al. Crystallogr. Rep. (2014 ) 59: 383.https: //doi.org/10.1134/S1063774514020187).

Based on the experimental data, it was found that the rate of solution cooling to temperatures of minus 30-40 ° C affects the formation of polymorphic modifications of sodium aminodihydrophthalazinedione in the final product.

For this, we carried out a quantitative phase analysis of the samples obtained at different cooling rates according to the described method, by the Rietveld method using the Bruker Topas5 ³ program ( ³ Bruker TOPAS 5 User Manual. - Karlsruhe, Germany: Bruker AXS GmbH, 2015.).

All diffraction patterns were recorded on a Bruker D8 Advance Vario diffractometer equipped with an X-ray tube with a copper anode and a Ge (111) monochromator (CuKα1) and a LynxEye position-sensitive detector in transmission installations. For samples 1, 2, 3, the shooting interval was 2-60 ° 2θ, step 0.02 ° 2θ. For sample 4, the shooting interval was 2-60 ° 2θ, step 0.02 ° 2θ.

Sample 4 contains a mixture of three polymorphs of the anhydrous sodium salt of luminol; KBSD codes - LOHKOK, LOHKOK01, LOHKOK02. Samples 2 and 3, in addition to the phases of the anhydrous sodium aminodihydrophthalazinedione salt, contain the monohydrate phase of the sodium aminodihydrophthalazinedione salt; code in CBSD LOHKIE2. Sample No. 1, in contrast to the rest of the samples, contains a significant amorphous component. The only crystalline phase in the composition of sample No. 1 is the anhydrous salt of sodium aminodihydrophthalazinedione LOHKOK01. From the ratio of the intensities of the peaks of the crystalline phase and the broadened peaks of the amorphous part, the degree of crystallinity can be estimated at 20%. The quantitative phase compositions of the samples, estimated by the Rietveld method, are presented in table 1 and in figures 1-4.

The following examples are given for illustration, without limiting the claimed invention.

Example 1.

The sodium aminodihydrophthalazinedione substance obtained by the method described in RU patent No. 2673452 C1 is introduced into the water for injection, and stirring is carried out for 30 minutes. Then, sterilizing filtration is carried out through a filter with a pore diameter of 0.22 μm. The sterile solution is dispensed into vials and cooled to a temperature of -45 ° C ± 5 ° C at a rate of 5 ° C per minute. Then, drying is carried out until the moisture content in the preparation is not more than 10%. The drying chamber is filled with sterile air, the vials are sealed and rolled up with aluminum caps.

Example 2.

A microbiologically pure substance of sodium aminodihydrophthalazinedione is added to the water for injection and mixing is carried out for 60 minutes. Then, preliminary filtration is carried out through a filter with a pore size of no more than 0.45 µm and sterilizing filtration through a filter with a nominal pore size of no more than 0.22 µm. The sterile solution is dispensed into ampoules and cooled to a temperature of -20 ° C ± 5 ° C at a rate of 1 ° C per minute. Then, drying is carried out until the moisture content in the preparation is not more than 10%. The drying chamber is filled with an inert gas, the ampoules are sealed.

Example 3.

The results obtained on the stability of the drug by the methods of "accelerated aging" and natural storage showed that the drug is stable and does not lose its properties during storage for up to 4 years (table 2). Stability studies are ongoing.

Example 4.

When comparing the sample obtained by the claimed method, with a standard sample of sodium 5-amino-2,3-dihydrophthalazine-1,4-dione sodium from Sigma by NMR spectroscopy, it was found that there are no foreign impurities in the obtained sample (Fig. 5, 6). H1 NMR data of the sodium aminodihydrophthalazinedione spectrum obtained by the indicated method show the presence of signals of aromatic protons (d, 1H, 6.61 ppm; m, 1H, 7.04 ppm; t, 1H, 7.17 ppm) and NH2 protons groups in the area of 7.34 ppm. The spectrum was recorded by heating the sample in DMSO d6 to 70 ° C until its complete dissolution.

Example 5.

When compared by IR spectroscopy, the sample obtained by the claimed method has a comparable IR spectrum with the spectrum of a standard sample of 5-amino-2,3-dihydrophthalazine-1,4-dione sodium from Sigma (Fig. 7).

Example 6.

Conducted preclinical studies of the drug sodium aminodihydrophthalazinedione obtained according to the claimed method showed:

1) Acute toxicity was assessed on white outbred rats, which were once intramuscularly injected drugs at doses of 40, 200 and 1000 mg / kg by substance, which corresponds to 14-, 70- and 350-fold therapeutic doses for humans. Each dose was tested on 5 males and 5 females. The animals of the control groups were injected with the vehicle - 0.9% saline sodium chloride solution - in the same volume as the test object in all doses. The LD50 of the drug for white outbred rats of both sexes exceeds the maximum tested dose - 1000 mg / kg. The drug does not have a local irritant effect after a single intramuscular injection.

2) The study of chronic toxicity was carried out on white outbred rats, which were injected daily intramuscularly with the drug in doses of 18 and 125 mg / kg by substance for 14 days, which corresponds to 6- and 44-fold therapeutic doses for humans. Each dose of the drug was tested on 10 males and 10 females. The animals of the control groups were injected with the vehicle - 0.9% saline sodium chloride solution - in the same volume as the drug in all doses. According to the results of clinical observations of the general condition of animals, hematological analyzes of peripheral blood, biochemical analyzes of blood serum, clinical analyzes of urine and pathomorphological studies after a 14-day course of administration in the tested doses, no adverse changes caused by the action of the tested drug were revealed. The drug does not have a local irritant effect after repeated use.

3) Assessment of the main pharmacokinetic parameters of the drug was carried out with a single intramuscular injection to rats.

To 50 rats (group I), the formulation was injected intramuscularly in the form of an aqueous solution with an analytical addition of a tritiated drug. When determining the main pharmacokinetic parameters during the simulation, 10 time points were used. There were 5 animals (repetitions) for each time point. Changes in the concentration of the labeled drug in the blood were studied.

As a result of the study, it was found that the drug has a high degree of release and reaches its maximum concentration in the blood in a short period of time.

Thus, the following advantages of the proposed method can be distinguished:

- the use of different methods of freezing minus 45 ° C - minus 15 ° C at a rate of 0.5-10 ° C per minute, followed by freeze drying, contributing to the production in the form of stable crystals of various configurations, which gives various positive biological properties of the drug;

- obtaining a stable medicinal product based on sodium aminodihydrophthalazinedione of high quality with a low moisture content (no more than 10%), mechanical, chemical and biological impurities;

- elimination of the use of toxic hydrazine hydrate and other harmful raw materials;

- increasing conversion, ergonomics and environmental friendliness;

- the formation of resinous substances, linear oligo- and polyimides, isomeric phthalimides is completely suppressed.

Claims (6)

1. A method of obtaining lyophilisate aminodihydrophthalazinedione sodium, including the following stages: 1) preparation of an aqueous solution of sodium aminodihydrophthalizinedione until its complete dissolution with a concentration in the solution of 0.3-250 mg / ml for 0.1-1.5 h; 2) sterilizing filtration of the resulting drug solution under aseptic conditions and dosing it into primary packaging for lyophilized forms; 3) freeze drying of the drug in a freeze drying chamber in several stages, while cooling to a temperature of minus 75 ° C - minus 15 ° C at a rate of 0.5-10 ° C per minute and further drying under vacuum to the moisture content in the product no more than 10%; 4) filling the drying with inert gas or sterile air, sealing the primary packaging and seaming or sealing. 2. A method of obtaining a sodium aminodihydrophthalazinedione lyophilisate according to claim 1, characterized in that the stage of sterilizing filtration can be carried out several times, depending on the quality of the original pharmaceutical substance.

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