RU2738804C1 - Pharmaceutical composition of antiglycating activity in solid dosage form in form of capsules and method for preparation thereof - Google Patents

Abstract

FIELD: medicine.SUBSTANCE: invention refers to medicine, namely to pharmacology and pharmacy and concerns production of an antiglycating agent. Such agent is a pharmaceutical composition in a solid dosage form for oral administration, containing as an active ingredient sodium salt of diethyl ether 4-oxo-1,4-dihydropyrazolo[5,1-c]-1,2,4-triazin-3,8 -dicarboxylic acid monohydrate, with the following ratio of components, wt. %: sodium salt of diethyl ether of 4-oxo-1,4-dihydropyrazolo[5,1-c]-1,2,4-triazine-3,8-dicarboxylic acid monohydrate 95–97.0 %; Plasdone K-29/32 1–5 %; magnesium stearate not more than 1 %.EFFECT: composition provides an antiglycating effect by inhibiting effect on end glycation products of collagen.3 cl, 7 dwg, 2 ex

Description

1. The technical field to which the invention relates

The invention relates to the chemical-pharmaceutical industry, medicine, pharmacology and for the production of antiglycating agents.

2. Technology level

Diabetes mellitus (DM) is a chronic non-infectious disease, the rate of increase in the prevalence of which has reached the scale of the global epidemic [DiabetesAtlas 7th Edition // International Diabetes Federation. 2015.144 p .; Dedov I.I., Shestakova M.V., Vikulova O.K. Epidemiology of diabetes mellitus in Russian Federation: clinical and statistical report according to the federal diabetes registry // Diabetes Mellit. 2017. Vol. 20, no. 1. P. 13-41]. The disease is characterized by a progressive course and the development of a number of acute and late complications, often leading to temporary disability, disability and early death [Dedov, Shestakova. Diabetes mellitus: diagnosis, treatment, prevention. Moscow: LLC "Publishing House" Medical Information Agency ", 2011. 808 rubles]. Therefore, the issue of prevention and treatment of complications of diabetes is one of the most important in modern medicine.

G. Advanced glycation end products cause lipid peroxidation in the human neuronal cell line // J. Alzheimer's Dis. 2003. Vol. 5, №1. P. 25-30.]. Наконец, существует рецептор-опосредованный путь, связанный с индукцией и активацией рецепторов к КПГ (RAGE), запускающих провоспалительные и прокоагулянтные сигнальные пути [Schmidt A.M. et al. The multiligand receptor RAGE as a progression factor amplifying immune and inflammatory responses // J. Clin. Invest. 2001. Vol. 108, №7. P. 949-955]. Активация RAGE участвует не только в патогенезе сахарного диабета, но и в почечной недостаточности [Deane R.J.R. et al. A multimodal RAGE-specific inhibitor reduces amyloid β-mediated brain disorder in a mouse model of Alzheimer disease // J. Clin. Invest. 2012. Vol. 122, №4. P. 1377-1392]. Формирование КПГ в процессе старения и при сахарном диабете обладает выраженным провоспалительным эффектом и приводит к повышению сердечно-сосудистого риска [Hanssen N.M.J, et al. Plasma Advanced Glycation End Products Are Associated With Incident Cardiovascular Events in Individuals With Type 2 Diabetes: A Case-Cohort Study With a Median Follow-up of 10 Years (EPIC-NL) // Diabetes. 2015. Vol. 64, №1. P. 257-265]. Накопление КПГ характерно для всех осложнений диабета, включая нефропатию, ретинопатию, атеросклероз и нейропатию [Vlassara Н., Uribarri J. Advanced Glycation End Products (AGE) and Diabetes: Cause, Effect, or Both? // Curr. Diab. Rep. 2014. Vol. 14, №1 P. 453; Singh V.P. et al. Advanced glycation end products and diabetic complications // Korean J. Physiol. Pharmacol. 2014. Vol. 18, №1. P. 1-14]. Ha основании этого можно утверждать, что, используя ингибиторы образования КПГ, можно существенно уменьшить риск развития осложнений СД.Non-enzymatic interaction between free amino groups of proteins and carbonyl groups of reduced sugars and other carbonyl compounds, leading to the formation of protease-resistant adducts known as advanced glycation end products (AGE) [Ramasamy R. Advanced glycation end products and RAGE: a common thread in aging, diabetes, neurodegeneration, and inflammation // Glycobiology. 2005. Vol. 15, no. 7. P. 16R-28R.]. CNG inhibit the action of nitric oxide [Bierhaus A. etal. AGEs and their interaction with AGE-receptors in vascular disease and diabetes mellitus. I. The AGE concept. // Cardiovasc. Res. 1998. Vol. 37, no. 3. P. 586-600] and are able to induce lipid peroxidation [Gasic-Milenkovic J., Loske S.,

G. Advanced glycation end products cause lipid peroxidation in the human neuronal cell line // J. Alzheimer's Dis. 2003. Vol. 5, no. 1. P. 25-30.]. Finally, there is a receptor-mediated pathway associated with the induction and activation of receptors to AGE (RAGE), which trigger pro-inflammatory and procoagulant signaling pathways [Schmidt AM et al. The multiligand receptor RAGE as a progression factor amplifying immune and inflammatory responses // J. Clin. Invest. 2001. Vol. 108, no. 7. P. 949-955]. RAGE activation is involved not only in the pathogenesis of diabetes mellitus, but also in renal failure [Deane RJR et al. A multimodal RAGE-specific inhibitor reduces amyloid β-mediated brain disorder in a mouse model of Alzheimer disease // J. Clin. Invest. 2012. Vol. 122, no. 4. P. 1377-1392]. The formation of AGE during aging and diabetes mellitus has a pronounced pro-inflammatory effect and leads to an increased cardiovascular risk [Hanssen NMJ, et al. Plasma Advanced Glycation End Products Are Associated With Incident Cardiovascular Events in Individuals With Type 2 Diabetes: A Case-Cohort Study With a Median Follow-up of 10 Years (EPIC-NL) // Diabetes. 2015. Vol. 64, no. 1. P. 257-265]. The accumulation of AGE is characteristic of all complications of diabetes, including nephropathy, retinopathy, atherosclerosis and neuropathy [Vlassara N., Uribarri J. Advanced Glycation End Products (AGE) and Diabetes: Cause, Effect, or Both? // Curr. Diab. Rep. 2014. Vol. 14, No. 1 P. 453; Singh VP et al. Advanced glycation end products and diabetic complications // Korean J. Physiol. Pharmacol. 2014. Vol. 18, no.1. P. 1-14]. Based on this, it can be argued that using inhibitors of the formation of AGEs, it is possible to significantly reduce the risk of developing complications of diabetes.

In the last decade, numerous works have been published devoted to synthetic low molecular weight AGE inhibitors and their therapeutic potential [Sourris K.S., Harcourt V.E., Forbes J.M. A New Perspective on Therapeutic Inhibition of Advanced Glycation in Diabetic Microvascular Complications: Common Downstream Endpoints Achieved Through Disparate Therapeutic Approaches? // Am. J. Nephrol. 2009. Vol. 30, no. 4. P. 323-335; Schalkwijk C.G., Miyata T. Early- and advanced non-enzymatic glycation in diabetic vascular complications: the search for therapeutics // Amino Acids. 2012. Vol. 42, no. 4. P. 1193-1204].

Aminoguanidine (Pimagedine) was the first identified inhibitor of the formation of AGE and studied in most detail [Brownlee M. et al. Aminoguanidine prevents diabetes-induced arterial wall protein cross-linking. // Science. 1986. Vol. 232, # 4758. P. 1629-1632.]. Randomized placebo-controlled trials evaluated the efficacy of aminoguanidine in patients with type 1 and type 2 diabetes. In a phase III clinical study in patients with T1DM, aminoguanidine reduced proteinuria and the progression of retinopathy [Bolton W.K. et al. Randomized Trial of an Inhibitor of Formation of Advanced Glycation End Products in Diabetic Nephropathy // Am. J. Nephrol. 2004. Vol. 24, no. 1. P. 32-40]. This was the first clinical evidence of the effectiveness of inhibition of AGE production in the fight against serious complications of type 1 diabetes. However, due to the unsatisfactory safety profile, revealed prooxidant properties [Suji G., Sivakami S. DNA damage by free radical production by aminoguanidine. // Ann. N.Y. Acad. Sci. 2006. Vol. 1067. P. 191-199] it cannot be used for therapeutic purposes. Nevertheless, aminoguanidine continues to serve as a reference substance in the development of new ANG inhibitors.

Pyridoxamine, a vitamin B6 derivative, prevents the formation of AGE from Amadori intermediates [Ahmad S. et al. Inhibitory Effect of Metformin and Pyridoxamine in the Formation of Early, Intermediate and Advanced Glycation End-Products // PLoS One / ed. Agarwal P.K. 2013. Vol. 8, no. 9. P. e72128]. It slows down the progression of kidney disease, reduces hyperlipidemia and redox imbalance in rats with experimental type 1 diabetes [Degenhardt T.R. et al. Pyridoxamine inhibits early renal disease and dyslipidemia in the streptozotocin-diabetic rat. // Kidney Int. 2002. Vol. 61, no. 3. P. 939-950]. In phase II studies in patients with diabetic nephropathy, pyridoxamine significantly normalized serum creatinine levels, while no differences in urinary albumin excretion were observed [Williams M.E. et al. Effects of pyridoxamine in combined phase 2 studies of patients with type 1 and type 2 diabetes and overt nephropathy. // Am. J. Nephrol. 2007. Vol. 27, no. 6. P. 605-614]. However, it is unlikely that this effect is due to vitamin B6 and thus compromises the use of pyridoxamine [Thornalley P.J., Rabbani N. Therapy: Vitamin B6, B9 and B12 in diabetic nephropathy-beware. // Nat. Rev. Endocrinol. 2010. Vol. 6, no. 9. P. 477-478].

3. The essence of the invention

The objective of the invention is to provide a pharmaceutical composition for oral administration of a drug acting on end products of collagen glycation (AGE) in the form of gelatin capsules containing a pharmacologically acceptable dose of sodium salt of diethyl ether 4-oxo-1,4-dihydropyrazolo [5,1-c ] -1,2,4-triazine-3,8-dicarboxylic acid monohydrate (Sodium 3,8-diethoxycarbonyl-4-oxo-4H-1,2,4-triazolo [5,1-c] [1,2, 4] triazinide monohydrate).

The essence of the invention is illustrated by the following materials: When choosing a dosage form, it was necessary to take into account that the addition of excipients should be minimal, so as not to increase its size, impairing swallowing. It is also undesirable to use carbohydrate derivatives as fillers, because they neutralize the antiglycating effect. The most convenient in this regard are capsules, which, unlike tablets, contain a minimum set of excipients.

Study of the technological properties of the substance of sodium salt of diethyl ether 4-oxo-1,4-dihydropyrazolo [5,1-c] -1,2,4-triazine-3,8-dicarboxylic acid monohydrate (Sodium 3,8-diethoxycarbonyl-4- oxo-4H-1,2,4-triazolo [5,1-c] [1,2,4] triazinide monohydrate) showed that it has a fine-crystalline structure, unsatisfactory flowability, non-uniform particle size, and therefore obtaining the contents of capsules must be carried out using preliminary wet granulation followed by the addition of antifriction substances.

The average therapeutic dose of the substance established in the course of pharmacological studies was 300 mg per dose.

FIG. 1 shows table No. 1, which shows the main technological properties of the sodium salt of diethyl ether 4-oxo-1,4-dihydropyrazolo [5,1-c] -1,2,4-triazine-3,8- dicarboxylic acid monohydrate (Sodium 3,8-diethoxycarbonyl-4-oxo-4H-1,2,4-triazolo [5,1-c] [1,2,4] triazinide monohydrate).

The planned size of the capsules should be within the range of No. 1 (capacity 0.5 cm ³ ). From table No. 1 it follows that with a bulk volume, even with compaction, capsule No. 1 can fit 0.644 × 0.5 = 0.322 g of the substance, i.e. the share of excipients remains no more than 22 mg. At the same time, the main task was to ensure the uniformity of the fractional composition, increase the bulk volume, strength of the granules and their flowability.

FIG. 2 shows table No. 2, which shows 5 model formulations of granulates for the selection of the quantitative and qualitative composition of excipients. The choice of excipients was carried out in a targeted manner: the most frequently used fillers, binders and sliding substances were taken, granulates were obtained, their properties were studied, when unsatisfactory results were obtained, they went on to the next composition, etc. In Table 2, the content of binders is given on a dry basis (to calculate the average weight of the capsule), but the concentrations of the solutions used are shown, so the amount of humectant can be calculated.

The granulates were obtained as follows: the substance and excipients, sifted through a 0.5 mm sieve, were mixed, moistened with a VMV solution. The required amount of humidifier was established empirically. Then the wet mass was granulated through a 3 mm sieve, dried at a temperature of 35-40 ° C, rubbed again through a 1 mm sieve, and powdered with calcium or magnesium stearate. The granulates indicated in table No. 2 were evaluated according to the most important physicochemical and technological indicators according to the methods of the State Pharmacopoeia of the XIV edition [State Pharmacopoeia of the Russian Federation / Ministry of Health of the Russian Federation. - XIV ed. - T. 2. - Moscow, 2018].

Как следует из таблицы №3, по совокупности показателей, находящихся в интервале оптимальных значений, наиболее приемлемыми характеристиками обладал гранулят состава №5, содержащий на одну дозу 300 мг (97%) натриевой соли диэтилового эфира 4-оксо-1,4-дигидропиразоло[5,1-с]-1,2,4-триазин-3,8-дикарбоновой кислоты моногидрата, 6,8 мг (2,2%) пласдона марки К 29/32 и 2,5 мг (0,8%) магния стеарата.FIG. 3 in table No. 3 shows the results of evaluating the technological parameters of model formulations of granulate for capsules of sodium salt of diethyl ether 4-oxo-1,4-dihydropyrazolo [5,1-c] -1,2,4-triazine-3,8-dicarboxylic acid monohydrate (Sodium 3,8-diethoxycarbonyl-4-oxo-4H-1,2,4-triazolo [5,1-c] [1,2,4] triazinide monohydrate)

As follows from table No. 3, according to the totality of indicators that are in the range of optimal values, the most acceptable characteristics were granules of composition No. 5, containing 300 mg (97%) of sodium salt of diethyl ether 4-oxo-1,4-dihydropyrazolo per dose [ 5,1-c] -1,2,4-triazine-3,8-dicarboxylic acid monohydrate, 6.8 mg (2.2%) of plasdon brand K 29/32 and 2.5 mg (0.8%) magnesium stearate.

Technological scheme for obtaining an encapsulated preparation of sodium salt of diethyl ether 4-oxo-1,4-dihydropyrazolo [5,1-c] -1,2,4-triazine-3,8-dicarboxylic acid monohydrate (Sodium 3,8-diethoxycarbonyl-4 -oxo-4H-1,2,4-triazolo [5,1-c] [1,2,4] triazinide monohydrate) included preparation of ingredients (grinding, sieving), moistening of the substance with a solution of VMV from a number of polyvinylpyrrolidone derivatives, granulation of a wet mass , drying, crushing and sieve calibration of dried granules, dusting with antifriction agents selected from a number of stearates, packing the obtained dry mass in the amount of 309.3 mg into gelatin capsules of size No. 1.

The technical result of the present invention is a pharmaceutical composition in solid dosage form (capsules), acting on the end products of collagen glycation (AGE), intended for the prevention and treatment of complications of diabetes mellitus.

Pharmacokinetic properties of the compound in solid dosage form

Methods for studying the pharmacokinetic properties of the compound

The experiments were carried out on male rabbits of the Chinchilla breed, weighing 3-4 kg. The substance of the sodium salt of diethyl ether 4-oxo-1,4-dihydropyrazolo [5,1-c] -1,2,4-triazine-3,8-dicarboxylic acid, monohydrate (Sodium 3,8-diethoxycarbonyl-4-oxo 4H-1,2,4-triazolo [5,1-c] [1,2,4] triazinide monohydrate) and the solid dosage form composition were administered orally using an intragastric tube at a dose of 20.0 mg / kg based on the active substance (experimentally proven therapeutic dose). 12 hours prior to the study, animals were deprived of access to food without limiting water intake.

The HPLC technique with ultraviolet detection was used, which makes it possible to determine the substance under study in a biological material and to carry out pharmacokinetic studies. We used a Shimadzu LC-20 liquid chromatograph with a Shimadzu SPD10-Avp ultraviolet detector (Shimadzu, Japan). Determination of the active substance of the sodium salt of diethyl ester of 4-oxo-1,4-dihydropyrazolo [5,1-c] -1,2,4-triazine-3,8-dicarboxylic acid, monohydrate (Sodium 3,8-diethoxycarbonyl- 4-oxo-4H-1,2,4-triazolo [5,1-c] [1,2,4] triazinide monohydrate) was carried out using an ultraviolet detector on a SupelcoSil LC-18 column 15 cm × 4.6 mm, 5 μm. Optimal conditions for the quantitative determination of the substance: wavelength - 205 nm, temperature - 30 ° С, eluent flow rate - 1 ml / min. The eluent is a mixture of 0.05 M potassium phosphate buffer (pH = 3.0) and acetonitrile in a ratio of 72:28. The lower limit of quantitative determination is 0.5 μg / ml.

To assess the pharmacokinetic properties of the studied medicinal substances, a number of parameters were calculated.

1) The area under the concentration-time pharmacokinetic curve (AUC) is the main pharmacokinetic parameter and characterizes the bioavailability of the drug. This parameter was calculated by the model-independent method of statistical moments in the Microsoft Excel program:

Where

Where

Elimination constant (K _el ), characterizing the decrease in the concentration of the drug in the final (monoexponential) part of the pharmacokinetic curve:

where C _max and T _max are the maximum concentration and the time to reach it, C _last and T _last are the last concentration value and the time of its determination.

2) The half-life (T _1/2 ), reflecting the time during which the drug concentration in the blood is halved:

3) Average retention time, which characterizes the average residence time of a drug molecule (MRT):

where AUMC is the area under the time product concentration-time curve.

4) Total (apparent) clearance (cl), reflecting the rate of release from the drug per unit volume of biofluid, as the ratio of dose (D) to AUC:

5) The total (apparent) volume of distribution (V _d ), which is understood as such a volume, in which the drug would have the same concentration as in the blood plasma, as the ratio of clearance (cl) to the elimination constant (K _el ):

6) Relative bioavailability was calculated as the ratio of the AUC after oral administration of GDF to the AUC after oral administration of the substance of the studied compound to rabbits.

Descriptive statistics were applied to all data using appropriate paired or multiple data comparison criteria consistent with the Gaussian distribution of the data, which are presented in the summary tables.

Test samples:

1) Substance of sodium salt of diethyl ether 4-oxo-1,4-dihydropyrazolo [5,1-c] -1,2,4-triazine-3,8-dicarboxylic acid, monohydrate (Sodium 3,8-diethoxycarbonyl-4- oxo-4H-1,2,4-triazolo [5,1-c] [1,2,4] triazinide monohydrate) (administration to animals orally at a dose of 20.0 mg / kg and blood plasma sampling during 5 half-lives ).

2) Composition in solid dosage form (administration to animals at a dose of 20.0 mg / kg in terms of the active substance) and taking blood plasma samples during 5 half-lives.

Research results:

FIG. Figure 4 shows Figure 1, which shows the pharmacokinetic curve of the content of diethyl ether 4-oxo-1,4-dihydropyrazolo [5,1-c] -1,2,4-triazine-3,8-dicarboxylic acid, monohydrate (Sodium 3 , 8-diethoxycarbonyl-4-oxo-4H-1,2,4-triazolo [5,1-c] [1,2,4] triazinide monohydrate) in the blood of rabbits after intragastric administration of the composition of the finished dosage form (FP) in a dose 20.0 mg / kg (calculated on the active substance) in comparison with the pharmacokinetic curve obtained with the introduction of the pharmaceutical substance (PS), administered at a dose of 20.0 mg / kg, depending on time.

FIG. 5 shows table No. 4, which shows the pharmacokinetic parameters (M ± m) of the active substance in rabbits after oral administration of the substance and composition of a solid dosage form at a dose of 20.0 mg / kg.

Thus, on the basis of the data presented, it has been established that the substance and composition of the finished dosage form are similar in pharmacokinetic profiles. A detailed analysis of the pharmacokinetic parameters of the HDF composition in comparison with the substance did not reveal statistically significant differences.

It has been established that the composition of GLF is characterized by high relative bioavailability (Fig. 5, table No. 4).

Pharmacodynamic properties of the compound in solid dosage form

Modeling experimental streptozotocin diabetes mellitus

The experiments were carried out on 75 sexually mature male Sprague-Dawley rats weighing 250-320 g, which were kept in the vivarium of the Volgograd State Medical University with a natural light regime on a complete nutritional balanced diet for laboratory animals, according to GOST R50258-92. Experimental diabetes mellitus (EDM) was simulated by a single intravenous injection of streptozotocin (Sigma, USA) dissolved in 0.1 M citrate buffer with pH 4.5 at a dose of 45 mg / kg [Kaur, R. Etal. Renoprotective effect of lansoprazole in streptozotocin-induced diabetic nephropathy in wistar rats / R. Kaur, R. Kaur Sodhi, N. Aggarwal, J. Kaur, U.K. Jain // Naunyn-Schmiedeberg's Arch. Pharmacol. 2016. - Vol. 389 (1). - PP. 73-85]. The quantitative determination of glucose in the blood was carried out one day after the administration of cytotoxin and then weekly, in the morning, on an empty stomach, during the entire period of the experiment lasting 12 weeks, using a glucometer "Glucocard Sigma-Mini" (Russia). The experiment took animals with a fasting glucose level of more than 17 mmol / L. One week after streptozotocin administration, the animals were divided into groups of 15 rats. Control groups: intact animals and rats with ESD were injected with distilled water (1 ml / 100 g); experimental groups - the studied substance and the composition of the solid dosage form of the sodium salt of diethyl ether 4-oxo-1,4-dihydropyrazolo [5,1-c] -1,2,4-triazine-3,8-dicarboxylic acid, monohydrate (Sodium 3 , 8-diethoxycarbonyl-4-oxo-4H-1,2,4-triazolo [5,1-c] [1,2,4] triazinide monohydrate) was administered orally at a dose of 20.0 mg / kg based on the active substance and the reference substance aminoguanidine (50.0 mg / kg). Distilled water and solutions of the studied substances were introduced through an intragastric tube daily, once a day. The level of glycosylated hemoglobin was determined using the DIABET-TEST kit (Phosphosorb, Russia). The content of glycation end products (AGEs) in blood serum was determined by the immunochemical method using RatAGEs ELISA Kit (cat. Number CSB-E09413r) (Cusabio, China) on a multifunctional microplate reader Infinite 200 PRO (Tecan, Austria) and expressed in μg / ml serum. Determination of the solubility of collagen in rat tail tendons was determined on a multifunctional microplate reader Infinite 200 PRO (Tecan, Austria) according to the method [Kochakian M., et al. Chronic Dosing With Aminoguanidine and Novel Advanced Glycosylation End Product-Formation Inhibitors Ameliorates Cross-Linking of Tail Tendon Collagen in STZ-Induced Diabetic Rats // M. Kochakian, B.N. Manjula, J.J. Egan / Diabetes. - 1996. - Vol. 45. - PP. 1694-1700.].

Statistical processing of the experimental data was carried out with the calculation of basic statistical indicators (arithmetic mean M, standard error of arithmetic mean m, paired Student's t-test and nonparametric Mann-Whitney test (Mann-Whitney, U-test) with Bonferroni correction [Rebrova O.Yu. Statistical analysis of medical data. Application of the STATISTICA software package. - M .: MediaSfera, 2006. - 312 p.] Using the Statistica 6.0 software packages ("StatSoft", USA), GraphPadPrism 5.0 and MicrosoftExcel (Microsoft, USA).

Test samples:

1. Substance of 4-oxo-1,4-dihydropyrazolo [5,1-c] -1,2,4-triazine-3,8-dicarboxylic acid diethyl ester, monohydrate (Sodium 3,8-diethoxycarbonyl-4-oxo 4H-1,2,4-triazolo [5,1-s] [1,2,4] triazinide monohydrate)) (administration to animals orally at a dose of 20.0 mg / kg for 3 months).

2. Composition in solid dosage form (oral administration to animals at doses of 20.0 mg / kg (based on the active substance) for 3 months).

3. Aminoguanidine (oral administration to animals at a dose of 50.0 mg / kg for 3 months).

Research results:

As a result, it was shown that after the administration of streptozotocin, a significant increase in blood glucose levels above 15 mmol / L was observed in rats with ESD in relation to those of intact animals. The development of diabetes was accompanied by polydipsia, polyuria, the animals were lethargic and apathetic. The blood glucose level in rats of all experimental groups with diabetes mellitus remained significantly high throughout the experiment, exceeding the values of the intact group of animals by an average of 4.9 times in the 1st month, 5.8 times in the 2nd month, in 5.26 times - 3rd month (p <0.05). The results obtained were confirmed by a statistically significant increase in the level of glycated hemoglobin in all experimental groups of rats with streptozotocin diabetes lasting 3 months relative to intact animals by an average of 2.8 times (p <0.05).

FIG. 6 shows table No. 5, which shows the effect of the substance of diethyl ether 4-oxo-1,4-dihydropyrazolo [5,1-c] -1,2,4-triazine-3,8-dicarboxylic acid, monohydrate (Sodium 3, 8-diethoxycarbonyl-4-oxo-4H-1,2,4-triazolo [5,1-c] [1,2,4] triazinide monohydrate), compositions in solid dosage form and the reference drug aminoguanidine, when administered intragastrically on the content of glycosylated hemoglobin (HbA1c) in rats 3 months after the development of experimental diabetes mellitus (M ± m), μg / ml.

Note: * - the differences are statistically significant relative to the Intact control indicators, Student's t-test and nonparametric Mann-Whitney test (Mann-Whitney, U-test) with Bonferroni's correction (p <0.05); # - differences are statistically significant relative to ESD indicators, Student's t-test and nonparametric Mann-Whitney test (Mann-Whitney, U-test) with Bonferroni correction (p <0.05); ESD - experimental diabetes mellitus.

In addition, the formation of end products of glycation (AGE) was confirmed by a statistically significant increase in their concentration in the blood serum of rats with ESD in relation to the intact group by 45.9%, while the solubility of tail collagen in the ESD group decreased by 38.9% (p <0.05) relative to intact control.

FIG. 7 shows table No. 6, which shows the effect of the substance of diethyl ether 4-oxo-1,4-dihydropyrazolo [5,1-c] -1,2,4-triazine-3,8-dicarboxylic acid, monohydrate (Sodium 3, 8-diethoxycarbonyl-4-oxo-4H-1,2,4-triazolo [5,1-c] [1,2,4] triazinide monohydrate), compositions in solid dosage form and the reference drug aminoguanidine, when administered intragastrically on the content of AGE in the blood of rats 3 months after the development of experimental diabetes mellitus (M ± m), μg / ml.

Note: * - the differences are statistically significant relative to the Intact control indicators, Student's t-test and nonparametric Mann-Whitney test (Mann-Whitney, U-test) with Bonferroni's correction (p <0.05); # - differences are statistically significant relative to ESD indicators, Student's t-test and nonparametric Mann-Whitney test (Mann-Whitney, U-test) with Bonferroni correction (p <0.05); ESD - experimental diabetes mellitus.

The introduction of the pharmaceutical substance 4-oxo-1,4-dihydropyrazolo [5,1-c] -1,2,4-triazine-3,8-dicarboxylic acid, monohydrate (Sodium 3,8-diethoxy-carbonyl-4- oxo-4H-1,2,4-triazolo [5,1-c] [1,2,4] triazinide monohydrate) and the composition of the finished dosage form of intragastric drives significantly reduce the content of glycosylated hemoglobin by 51.5% and 49.8% ( p <0.05), the comparison drug aminoguanidine reduced HbA1c by 37.9% (p <0.05) in relation to the ESD group (Fig. 6). The studied substance and composition of the finished dosage form reduced the concentration of CNG in relation to the indicator of animals with ESD by 21.9% and 34.6% (p <0.05), respectively, and the reference drug aminoguanidine reduced CNG by 24.3% relative to rats with ESD (Fig. 7). Under the influence of the substance and composition of the finished dosage form in rats with ESD, the solubility of tail collagen slightly increased by 9.1% and 8.07%, respectively, the reference drug aminoguanidine increased the solubility of tail collagen by 29.8% (p <0.05) relative to the group ESD.

Thus, on the basis of the data presented, it has been established that the substance and composition of the finished dosage form are similar in pharmacodynamic properties.

Therefore, the proposed composition in solid dosage form (capsules), acting on the end products of collagen glycation (AGE), can be used as a means for the prevention and treatment of complications of diabetes mellitus.

The invention is illustrated by the following examples.

Example 1. The substance of the sodium salt of diethyl ether 4-oxo-1,4-dihydropyrazolo [5,1-c] -1,2,4-triazine-3,8-dicarboxylic acid, monohydrate - 970.0 g moistened with 10% aqueous a solution of polyvinylpyrrolidone K30, added in an amount of 220.0 g. Then the wet mass is granulated through a mesh with a mesh size of 3.0 mm, the wet granulate is dried at a temperature not exceeding 40 ° C to a residual moisture content not exceeding 2%. The dried granulate is rubbed through a mesh with a hole diameter of 1 mm and dusted with 8.0 g of magnesium stearate, about 1 kg of the finished encapsulated mass is obtained.

The mass is encapsulated on a manual capsule filling machine "Optima 300" using capsules of size No. 1 with a capacity of 0.5 cm ³ . The average weight of the contents of the capsule is 0.309 ± 7.5% g, the content of the active substance is 300 mg. From 1 kg of mass for encapsulation, 3200 capsules are obtained.

Example 2. The substance of the sodium salt of diethyl ester of 4-oxo-1,4-dihydropyrazolo [5,1-c] -1,2,4-triazine-3,8-dicarboxylic acid, monohydrate - 970.0 g is moistened with 10% aqueous Plasdon solution K 29/32, added in an amount of 220.0 g. Then the wet mass is granulated through a mesh with a mesh size of 3.0 mm, the wet granulate is dried at a temperature not exceeding 40 ° C to a residual moisture content not exceeding 2%. The dried granulate is rubbed through a mesh with a hole diameter of 1 mm and dusted with 8.0 g of magnesium stearate, about 1 kg of the finished encapsulated mass is obtained.

The mass is encapsulated on an Optima 300 manual capsule filling machine using size 3 capsules with a capacity of 0.30 cm ³ . The average mass of the contents of the capsule is 0.206 ± 10% g, the content of the active substance is 200 mg. From 1 kg of mass for encapsulation, 4850 capsules are obtained.

Claims (4)

1. Pharmaceutical composition of antiglycating action in the form of a solid dosage form, characterized in that it contains the substance of sodium salt of diethyl ether 4-oxo-1,4-dihydropyrazolo [5,1-c] -1,2,4- triazine-3,8-dicarboxylic acid monohydrate, with the following ratio of components, wt. %: Sodium salt of diethyl ether 4-oxo-1,4- dihydropyrazolo [5,1-c] -1,2,4-triazine-3,8- 95-97.0 dicarboxylic acid monohydrate PlasdonK 29/32 1-5 Magnesium stearate No more than 1 2. A method of obtaining a pharmaceutical composition of antiglycating action, including 'grinding and sieving the substance and auxiliary substances, moistening the substance of sodium salt of diethyl ether 4-oxo-1,4-dihydropyrazolo [5,1-c] -1,2,4-triazine- 3.8-dicarboxylic acid monohydrate with VMV solution from a number of polyvinylpyrrolidone derivatives, wet mass granulation, drying, crushing and sieve calibration of dried granules, dusting with antifriction agents selected from a number of stearates, and packing the resulting dry mass into gelatin capsules. 3. The method according to claim 2, characterized in that the pharmaceutical composition having antiglycating activity is made in the form of a gelatin capsule for oral administration.

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