Medicinal composition and method of producing a parenteral preparation

Abstract

A medicinal composition in the form of an aqueous solution contains 1-deoxy-1-N-methylammonium-D-glucitol succinate, inosine, nicotinamide and methionine in the following proportions: 7.5-15.0 g / l 1-deoxy-1-N-methylammonium-D-glucitol succinate; 1.50-3.0 g / l inosine; 0.53-1.05 g / l methionine; 0.19-0.38 g / l nicotinamide; water for injection to 1 litre. Also disclosed is a method of producing a parenteral preparation containing the claimed medicinal composition. The composition exhibits anti-diabetic, nephroprotective and wound-healing activity and can be used in the long-term treatment of diabetes mellitus and attendant complications, particularly diabetic nephropathy and chronic skin wounds caused by soft tissue damage (diabetic ulcers).

Description

[0001]

[0002] A medicinal composition in the form of an aqueous solution and a method for producing a parenteral drug based on it.

[0003] Field of technology.

[0004] The invention relates to medicine and the pharmaceutical industry, and concerns a medicinal composition in the form of an aqueous solution that has antidiabetic, nephroprotective and wound-healing activity, as well as a method for obtaining a parenteral drug based on it, which can be used in the long-term treatment of diabetes mellitus and its complications, in particular diabetic nephropathy and chronic wounds on the skin due to the destruction of soft tissues (diabetic ulcers).

[0005] Prior art.

[0006] Diabetes mellitus is an endocrine disorder that impairs tissue glucose utilization due to an absolute or relative deficiency of the hormone insulin. This causes organs and tissues to lose the ability to convert incoming glucose into glycogen, leading to glucose accumulation in the blood and, consequently, damage to the endothelium of various organs.

[0007] Complications of diabetes mellitus include significant damage to the cardiovascular system, visual organs, as well as kidney damage - a disruption of their excretory, filtering and synthesizing capacity, which leads to diabetic nephropathy.

[0008] Diabetic nephropathy develops due to damage to the nephrons, which detoxify the body and regulate water and salt balance. Glycosylation of the protein filtering membranes of the glomeruli occurs, and the epithelium of the distal tubules loses the ability to reabsorb salts and other components from the primary urine back into the blood.

[0009] As the disease progresses, the kidneys lose their ability to function. The concentration of toxins and metabolic waste products in the blood increases significantly, leading to self-poisoning of the body. The main complications of diabetic nephropathy include chronic kidney disease, electrolyte imbalances such as potassium, sodium, and phosphorus, and the development of metabolic acidosis.

[0010] Chronic kidney disease (CKD) is a potentially fatal condition typically diagnosed in its late stages, when significant renal function has already been exhausted. CKD is one of the most common complications of diabetes and is also a risk factor for cardiovascular disease. Chronic kidney disease develops in nearly 40% of patients with type 2 diabetes mellitus (T2DM). Despite standard therapy, the principles of which are established in relevant clinical guidelines, patients with CKD and T2DM remain at high risk of CKD progression and cardiovascular complications. It is estimated that over 160 million patients with type 2 diabetes worldwide suffer from CKD. In patients with type 2 diabetes, chronic kidney disease is the leading cause of end-stage renal failure, requiring life-saving dialysis or kidney transplantation.Furthermore, this pathology is accompanied by the development of chronic skin wounds due to the destruction of soft tissue in the lower extremities. This complication is observed in patients with diabetes who have had the condition for more than 15-20 years. In 65-85% of cases, this leads to amputation of the affected foot.

[0011] Replacement therapy with exogenous insulin and glucose-lowering medications is insufficient for the comprehensive treatment of diabetes mellitus and its complications. Insulin-only treatment is effective only for a short period of time and subsequently fails to maintain stable glucose levels, necessitating combination oral therapy with medications that lower blood glucose and mitigate the negative effects on the kidneys caused by elevated blood glucose levels, even in the early stages of the disease.

[0012] Thus, the creation of complex antidiabetic drugs with a broad spectrum of action is a pressing task.

[0013] There are known complex antidiabetic medicinal products containing components of plant origin, for example: “Composition having a multicomponent mechanism of antidiabetic action” (RU Patent 2289419), “Homeopathic antidiabetic agent” (RU Patent 2164412), “A product having antioxidant, antitumor, immunomodulatory, antidiabetic and antibacterial action” (RU Patent 2598349), “Antidiabetic collection” (RU Patent 2284829), “Antidiabetic agent” (RU Patent 2283659), “Composition having antidiabetic action” (RU Patent 2430735).

[0014] However, due to the varied effects of plant components on the body, such medications do not provide a consistent antidiabetic effect. Furthermore, some types of medicinal plant materials may be unavailable due to their growing areas.

[0015] Combinations of medications are also used in the treatment of diabetes. These combinations, while alleviating symptoms, prevent both acute complications, such as hyperosmotic coma and ketoacidosis, and chronic complications, such as diabetic neuropathy (Russian Patent 2351317). Combinations for the treatment of metabolic disorders, primarily diabetes, can be formulated as a single drug or pharmaceutical composition (Russian Patent 2280447). However, these combinations do not possess nephroprotective activity.

[0016] Also known is an "Antidiabetic Agent Based on Succinic Acid" for oral use (RU Patent 2114617). This agent contains succinic acid or its salts, such as ammonium succinate and sodium succinate, mixed with sorbitol or xylitol. This proposed agent expands the arsenal of natural antidiabetic agents, but does not address side effects, particularly kidney dysfunction.

[0017] Common disadvantages of oral dosage forms of drugs are the slow entry of the drug into the systemic circulation, they are inactivated in the liver and cannot be used in an unconscious patient.

[0018] A new chemical compound from the group of quaternary ammonium salts of succinic acid, 1-Deoxy-1-M-methylammonium-O-glucitol succinate, is known, which has an antidiabetic effect, high diuretic activity, and low toxicity (RU Patent No. 2345060, patent holder LLC NTFF Polisan).

[0019] An aqueous solution of the compound 1-Deoxy-1-N-methylammonium-O-glucitol succinate is promising for the creation of antidiabetic drugs for parenteral use and is the closest analogue of the claimed composition. PCI7RU2025 / 000240

[0020] However, the effectiveness of this compound is insufficient to comprehensively influence the key pathophysiological processes of diabetes, in particular, diabetic nephropathy, since it does not activate metabolic activity in the glomeruli and tubules of the nephron, which is required to maintain the filtering and reabsorbing function of the kidneys, as well as the healing of diabetic ulcers that form during the long-term severe course of diabetic disease as a result of impaired endothelial function and microcirculation in the skin.

[0021] In addition, long-term intravenous administration of the compound in large volumes poses a high risk, since a 10% solution of 1-deoxy-1-N-methylammonium-B-glucitol succinate has an acidic pH (about 4.5), which can lead to local irritation and disruption of the acid-base balance of the body.

[0022] The basic technology for the production of drugs for parenteral administration usually includes the following main steps: dissolution of active and auxiliary components in water for injection, mixing until the solution reaches equilibrium, subsequent filtration of the solution through a series of filters with different porosity, sterilizing filtration and filling into sealed containers (Handbook of Pharmaceutical Manufacturing. Sterile Products. Second Edition vol 6, 2009).

[0023] Brief description of the invention.

[0024] The objective of the present invention is to create a medicinal composition in the form of an aqueous solution having increased antidiabetic activity, as well as nephroprotective and wound-healing activity, and to develop a method for obtaining a parenteral drug based on it, which can be used in the long-term treatment of diabetes mellitus and its complications.

[0025] The stated problem is solved by creating a medicinal composition in the form of an aqueous solution containing 1-Deoxy-1-H-methylammonium-E-glucitol succinate, which, according to the invention, additionally contains inosine, nicotinamide, methionine as active components, in the following ratio of components, g / l:

[0026] 1-Deoxy- 1-Ь1-methylammonium-B-glucitol succinate 7.5-15.0

[0027] Inosine 1.50-3.0

[0028] Methionine 0.53-1.05

[0029] Nicotinamide 0.19-0.38

[0030] Water for injection up to 1 liter. This problem is solved by the fact that the medicinal composition in the form of an aqueous solution exhibits antidiabetic activity.

[0031] The stated problem is also solved by the fact that the medicinal composition in the form of an aqueous solution exhibits nephroprotective activity.

[0032] The stated problem is also solved by the fact that the medicinal composition in the form of an aqueous solution exhibits wound-healing activity.

[0033] In addition, the stated problem is solved by a method for obtaining a parenteral preparation containing the claimed medicinal composition by dissolving the components in water for injection, mixing, filtering the solution through a sterilizing filter and pouring it into sealed containers, in which, according to the invention, the compound 1-Deoxy-1-I-methylammonium-B-glucitol succinate is dissolved in water for injection, the solution is adjusted to pH 6.0-8.0 with sodium hydroxide, inosine, methionine, nicotinamide are added, and sodium chloride, potassium chloride and magnesium chloride are additionally added to an osmolarity of 235-470 mOsm / l, then the sealed containers with the resulting solution are subjected to final sterilization.

[0034] The basis of the claimed composition contains the compound 1-Deoxy-1-N-methylammonium-B-glucitol succinate, general formula C11H22NO9, structural formula:

[0035] This compound has an antidiabetic effect by increasing the body's production of insulin and reducing glucose levels in the blood and urine (RU Patent No. 2345060).

[0036] The claimed medicinal composition additionally contains the active ingredients inosine, methionine, and nicotinamide, which activate metabolic processes in the body. Their combined use in antidiabetic compositions is previously unknown.

[0037] Well-known compounds containing metabolites have a general normalizing effect on metabolic processes in the body and are often used as dietary supplements. A well-known combination containing sodium glucamine succinate, riboxin, methionine, and nicotinamide as active ingredients exhibits hepatoprotective and choleretic activity by stimulating bile acid flow (Russian Patent 2240116). Since the nature of diabetes and related diseases or conditions is determined by many factors, combining pharmacologically acceptable components with different mechanisms of action does not always result in combinations with favorable properties.

[0038] The invention shows that the claimed composition, containing 1-Deoxy-1-I-methylammonium-N-glucitol succinate and inosine, nicotinamide and methionine (example 4) as active components, has increased antidiabetic activity compared to the prototype (experiment 2).

[0039] The content of active components in the composition was selected empirically. However, their content is not limited by Example 4 and is determined by the range of values ​​at which the medicinal composition as a whole exhibits the stated activity (Experiment 3).

[0040] Using a model of type 2 diabetes, the invention demonstrated for the first time that the claimed composition, along with antidiabetic activity, also exhibits nephroprotective activity due to the activation of metabolic processes in the renal glomeruli and tubules, which is required to maintain the filtering and reabsorbing function of the kidneys (experiments 2, 3).

[0041] In addition, in experiments 2 and 3 it was shown that the claimed composition exhibits wound-healing activity due to the activation of metabolism in the skin pores, which is an important property for the healing of diabetic ulcers that form in severe cases of diabetic disease as a result of impaired endothelial function and impaired microcirculation in the skin.

[0042] Thus, the medicinal composition has a complex mechanism of antidiabetic action, aimed at reducing the symptoms of diabetic nephropathy and is non-toxic, which determines the safety of long-term use of the drug prepared on its basis.

[0043] According to the invention, a preparation for parenteral administration is obtained on the basis of the claimed medicinal composition in the form of an aqueous solution.

[0044] To prepare the drug, 1-Deoxy-1-N-methylammonium-O-glucitol succinate is dissolved in water for injection, and the solution is adjusted to a pH of 6.0-8.0 with sodium hydroxide. This pH range is most suitable for parenteral solutions, particularly intravenous solutions. The normal pH of blood plasma is approximately 7.35, so using intravenous solutions with a pH of 6.0 carries a risk of acidosis, while solutions with a pH above 8.0 carry a risk of alkalosis, which poses a risk of side effects during long-term treatment. Inosine, methionine, and nicotinamide are then added to the resulting solution, along with salts (sodium chloride, potassium chloride, and magnesium chloride) in a specific ratio, and the mixture is stirred until the components are completely dissolved.

[0045] The content of sodium, potassium and magnesium chlorides is selected in such a way that the content of sodium, potassium and magnesium ions, when using therapeutic doses of the drug, is within the physiological norm for human blood, and the osmolarity of the solution remains within the range of 235-470 mOsm / l, acceptable for rapid intravenous administration.

[0046] The solution is then filtered through a sterilizing filter with a pore diameter of 0.22 µm, and the solution is poured into hermetically sealed glass or plastic containers, which are then subjected to final thermal sterilization at a temperature of 121±1°C for 15-20 minutes.

[0047] In the resulting solution for parenteral administration, the range of content of active and auxiliary components is determined by both safety due to the achievement of optimal physicochemical properties of the solution (pH, osmolarity, electrolyte content) of the solution (experiment 4), and the manifestation of antidiabetic, nephroprotective and wound-healing activities in the entire range of content of active components (experiment 3).

[0048] The implementation of the claimed invention achieves an expansion of the spectrum of action of the medicinal composition in the form of an aqueous solution due to the manifestation of increased antidiabetic, nephroprotective and wound-healing activity.

[0049] Thus, the resulting drug for parenteral administration can be used in the long-term treatment of diabetes mellitus and its complications, in particular diabetic nephropathy and chronic skin wounds due to the destruction of soft tissue (diabetic ulcers).

[0050] Detailed description of the invention.

[0051] The quantitative and qualitative composition of the claimed composition was selected empirically during the study of the influence of metabolic components on the bioenergetic indices of the renal epithelium and the antidiabetic activity of the compound 1-Deoxy-1-M-methylammonium-O-glucitol succinate.

[0052] To study the effect on the bioenergetic parameters of the renal epithelium, solutions of 1-Deoxy-1-N-methylammonium-O-glucitol succinate were prepared with the addition of inosine, methionine and nicotinamide, prepared in accordance with examples 1^. Example 1. 900 ml of water are placed in a reactor, 10.0 g of 1-Deoxy-1-N-methylammonium-B-glucitol succinate are dissolved, 2.0 g of inosine are then added and mixed until completely dissolved, the volume of the solution is brought to 1 l with water for injection.

[0053] Example 2. 900 ml of water are placed in a reactor, 10.0 g of 1-Deoxy-1-N-methylammonium-E-glucitol succinate are dissolved, 0.70 g of methionine are added and stirred until completely dissolved, the volume of the solution is brought to 1 l with water for injection.

[0054] Example 3. 900 ml of water are placed in a reactor, 10.0 g of 1-Deoxy-1-N-methylammonium-B-glucitol succinate are dissolved, then 0.25 g of nicotinamide is added and stirred until completely dissolved, the volume of the solution is brought to 1 l with water for injection.

[0055] Example 4. 900 ml of water are placed in a reactor, 10.0 g of 1-Deoxy-1-N-methylammonium-B-glucitol succinate are dissolved, then 2.0 g of inosine, 0.70 g of methionine and 0.25 g of nicotinamide are added and mixed until completely dissolved, the volume of the solution is brought to 1 l with water for injection.

[0056] The biological activity of the solutions obtained in Examples 1-4, compared with a solution of compound 1-Deoxy- lN-methylammonium-B-glucitol succinate (prototype), was studied using a model of hypoxia and hypoxia-reperfusion of renal epithelium in vitro - Experiment 1.

[0057] Experiment 1 Study of compositions and prototype on the in vitro hypoxia and reperfusion model.

[0058] The Madine-Darby Canine kidney cell (MBSC) renal epithelial cell line was used as a test system in the study. Cells were cultivated in Bulbecco's Modified Eagle's Medium (BMEM) with a glucose concentration of 1 g / L, containing 10% FBS, 2 mM β-alanyl-β-glutamine, 100 μg / mL streptomycin, and 100 U / mL penicillin. Cell cultures were maintained in an incubator at 37°C in a humidified atmosphere containing 5% CO2 and 95% air. The cell monolayer density throughout the study was at least 70%.

[0059] Hypoxia followed by reoxygenation was modeled by adding a sodium dithionite solution to the cell culture, which resulted in complete restoration of the oxygen present in the medium (the residual oxygen content in the medium was less than 1% of the maximum solubility).

[0060] At the beginning of the experiment, HBSS (Hanks' balanced salt solution) was added to the cells, with 10% of the volume of the experimental samples with compositions 1-4 and prototype added to it, and then hypoxia was modeled by incubating the cells in a 5 mM sodium dithionite solution for 180 minutes. Then, the cells were washed with HBSS and analyzed. The effect of the experimental samples (prototype and compositions 1-4) under hypoxic and reoxygenation conditions on the development of necrosis and PCI7RU2025 / 000240 apoptosis in cells, on changes in cell bioenergetics indicators (assessment of the mitochondrial membrane potential (L t), ATP level in mitochondria), the level of activation of antioxidant protection of cells (assessment of the rate of formation of reactive oxygen species (ROS) in the mitochondrial matrix) was assessed.

[0061] The study was performed using a laser scanning confocal microscope LSM 900 using fluorescent probes - Hoechst 33342 and propidium iodide (PI), as well as annexin V labeled with the fluorescent label AF 488.

[0062] Data analysis was performed using specialized image analysis software: ZEN Blue Edition (Zeiss, Germany) and MetaFluor (Molecular Devices, UK). The Kruskal-Wallis test with Dunn's post hoc test was used to determine the statistical significance of differences between data. Differences with a significance level of no more than 0.05 were considered statistically significant. The results were normalized to the median value relative to the values ​​of intact cells. The study results are presented in Table 1.

[0063] Table 1

[0064] Study of the influence of compositions 1-4 on the bioenergetic parameters of kidney epithelial cell culture (MDCK cell line (Madine-Darby Canine kidney cells).

[0065] The results are normalized to the values ​​of intact cells, Me (Q1;Q3). Note: * - differences are statistically significant (p<0.05) compared to the values ​​of intact cells.

[0066] It was shown that under hypoxia-reoxygenation conditions, the cell line treated with the prototype exhibited statistically significant mitochondrial hyperpolarization (DHT increased by 64%) and a 35% decrease in ATP levels relative to intact cells. The absence of differences in DHT levels between intact cells and cells incubated in the presence of composition 4 indicated stabilization of the membrane potential within the normal range and an increase in mitochondrial ATP.

[0067] A statistically significant increase in ROS synthesis in the mitochondrial matrix was recorded in all experimental groups, which is characteristic of the pathological process of hypoxia-reoxygenation [Thomas LW, Ashcroft M. Exploring the molecular interface between hypoxia-inducible factor signaling and mitochondria / / Cellular and Molecular Life Sciences. 2019. No. 9 (76). P. 1759-1777.].

[0068] The most pronounced synthesis of ROS in the mitochondrial matrix was observed when adding the prototype and composition 2 to the cell line (95%) and the lowest when adding composition 4 - 53%.

[0069] The level of cell death in the culture against the background of hypoxia-reoxygenation statistically significantly increased and reached 9.3% upon the introduction of the prototype and decreased upon the addition of composition 4 to 2.4% of the total number of cells.

[0070] Maintaining cell viability under hypoxia-reoxygenation conditions is ensured by complex protective mechanisms associated with global changes in basic metabolic processes. Under conditions of low oxygen levels and a reduced role of oxidative phosphorylation in meeting cellular energy needs, the contribution of alternative ATP sources, a key one being glycolysis, significantly increases, contributing to a significant increase in the rate of cellular glucose consumption.

[0071] The introduction of the prototype and compositions 1-4 contributed to an increase in glucose absorption by cells, with statistically significant differences relative to intact cells being recorded in composition 4, where an increase in this indicator by 2.61 times was noted.

[0072] The prototype and compositions 1-4 exhibited cytoprotective activity under hypoxia-reoxygenation conditions. Moreover, the addition of composition 4 demonstrated the highest level of activation of endogenous protective mechanisms in the cell culture during hypoxia-reoxygenation by stabilizing membrane potential, maintaining ATP and ROS levels in the mitochondrial matrix, and activating glycolysis processes to support bioenergetic mechanisms.

[0073] Thus, composition 4 is the most promising for the development of an antidiabetic drug with nephroprotective activity.

[0074] In addition, an important indicator in the treatment of diabetes is the effect on the rate of wound healing.

[0075] Composition 4 (Example 4) containing 10% 1-Deoxy- lN-methylammonium-D-glucitol succinate, inosine, nicotinamide and methionine was used to study the effect on the development of diabetic nephropathy and the rate of wound healing in comparison with a solution prepared according to the prototype - 10% solution of 1-Deoxy- lN-methylammonium-D-glucitol succinate (Experiment 2).

[0076] Experiment 2. Study of antidiabetic, nephroprotective and wound-healing activity in a model of diabetic nephropathy in rats.

[0077] Diabetic nephropathy in rats was modeled by performing right-sided nephrectomy with adrenal preservation, administering streptozotocin, and placing the animals on a high-fat diet for 6 weeks. Streptozotocin was administered intraperitoneally at a dose of 25 mg / kg twice with a weekly interval on weeks 7 and 8 of the experiment. After verifying diabetes mellitus in the rats (blood glucose levels > 11 mmol / L, confirming the development of T2DM), the animals were randomized into experimental groups based on blood glucose levels to form homogeneous groups.

[0078] Four groups of animals were formed:

[0079] 1. Intact animals - without the development of type 2 diabetes and without the administration of drugs, n = 16;

[0080] 2. Control without treatment - type 2 diabetes + administration of saline solution, n = 16;

[0081] 3. Prototype - SD2 + introduction of prototype, n = 16;

[0082] 4. Composition 4 - SD2 + introduction of composition 4, n = 16.

[0083] Animals with developed diabetic nephropathy were administered composition 4 and a prototype solution intravenously at doses of 3 ml / kg for 10 days. The untreated control group received an equivalent volume of saline solution.

[0084] General clinical observation of the animals was carried out daily throughout the experiment. The efficacy of the proposed composition in the diabetic nephropathy model was assessed 10 days after the end of administration of the study drugs.

[0085] The insulin resistance index (HOMA-IR) was calculated using the formula: fasting glucose x fasting insulin

[0086] NOMA — 1R = - - - - - - —

[0087] 22.5

[0088] To collect urine, rats were placed in metabolic cages for 24 hours. Blood was collected from the tail vein during life into tubes containing a clot activator. A biochemical analyzer was used to determine serum creatinine, insulin, glucose, and urea. The animals' body weights were measured, and renal excretory function (daily urine output and urinary glucose, protein, and urea concentrations) and glomerular filtration rate (GFR) were assessed. GFR was calculated using the formula: ml / mmol / h.

[0089] - creatinine in urine - x urine volume in 24 hours (ml)

[0090] SCF _ _ V l 7 _ ,MKM0LY > serum creatinine (-) x 0.001

[0091] - - x 1400 (min) rat weight (kg) v 7

[0092] The level of lipocalin 2 and cystatin C in the blood and urine of animals was studied using the enzyme immunoassay method on an EPOCH2 plate spectrophotometer.

[0093] A histological examination of kidney tissue was performed.

[0094] Additionally, full-thickness planar skin wounds were created on the rats' backs, and a qualitative and quantitative assessment of wound healing was performed. When creating wounds in anesthetized animals, a 1.5-cm-diameter skin flap was excised, also removing subcutaneous fat. The depth of the lesion corresponded to the thickness of the rat's skin, including the epidermis, dermis, and hypodermis. The following parameters were calculated:

[0095] Percentage of wound reduction (A, %): where d is the diameter of the wounds on the control days of the experiment: 1st and 10th.

[0096] - Linear rate of wound healing (m, mm 2 / day):

[0097] _ 2 x(S0-S t )

[0098] V = - , tx(P0-P t ) ' where Po and So are the perimeter and area of ​​the wounds during the previous measurement; Pt and St are the perimeter and area of ​​the wounds after a time interval t (day).

[0099] - Relative decrease in wound area (AS, %):

[0100] AS = x 100%,

[0101] S o xt PC17RU2025 / 000240 where So is the wound area at the previous measurement; St is the wound area after a time interval t (day).

[0102] Statistical data processing was performed using the GraphPad Prism 8.0 statistical software package (USA). The distribution of each sample was tested for normality using the Shapiro-Wilk test. Between-group statistical comparisons were performed using the Tukey test for normal distributions and the Mann-Whitney test for non-normal distributions. Differences with a null hypothesis probability of less than 0.05 were considered significant.

[0103] In the untreated control group of animals (Group 2), typical signs of developing type 2 diabetes and diabetic nephropathy developed: a statistically significant decrease in body weight of more than 50 g and a 1.5-fold drop in blood insulin levels were recorded, as well as increased glucose and protein levels in the blood and urine, as well as increased markers of kidney damage (lipocalin 2, nystatin C, and creatinine) compared to values ​​in the intact group of animals. Furthermore, the HOMA-IR index increased by more than 1.5 times. The results are presented in Table 2.

[0104] Table 2

[0105] The effectiveness of intravenous administration of composition 4 and Prototype on blood parameters in rats with diabetes mellitus, M±SEM, n=16.

[0106]

[0107] Notes:

[0108] * - differences are statistically significant (p<0.05) compared with the values ​​of the intact group;

[0109] # - differences are statistically significant (p<0.05) compared with the values ​​of the control group.

[0110] In the group of animals administered the prototype, as well as in the control group, a significant decrease in body weight to 229±10 g, the development of glucosuria (urine glucose 4.1±1.5 mmol / l) and proteinuria (urine protein level 23.1±3.3 mg / day) were noted. In the blood of animals, a statistically significant increase in creatinine to 69±6 μmol / l, glucose by more than 3 times, as well as a decrease in the insulin level by 1.3 times compared to the values ​​​​in intact animals were recorded. A significant increase in the kidney damage marker - cystatin C by 1.3 times in the blood and 2 times in the urine was recorded in relation to the indicator in intact animals. The level of lipocalin 2 in the urine increased by more than 4 times. The prototype had antidiabetic activity compared to the control group.

[0111] Administration of Composition 4 (Group 4) to rats with T2DM resulted in a statistically significant restoration of body weight to 235±11 g and a 32% reduction in blood glucose levels compared to the untreated control group (Group 2). Administration of Composition 4 resulted in a reduction in the HOMA-IR index and renal damage markers—creatinine, insulin, and cystatin C blood levels remained at the same level as those in intact animals. Urinary lipocalin 2 levels remained elevated but showed a downward trend.

[0112] Thus, the composition surpasses the prototype in antidiabetic activity and also exhibits significant nephroprotective activity compared to the prototype and the control group (administration of saline). The histological changes in the kidney tissue of rats with type 2 diabetes in the untreated control group and in the prototype group were similar and revealed damage to the glomeruli and tubules of the nephrons. Thickening of the glomerular basement membrane and expansion of the mesangial matrix, as well as narrowing of the capillary lumens, were observed in the glomeruli of the nephrons.

[0113] The use of composition 4 contributed to the restoration of nephron glomeruli and a reduction in the level of changes in the structural elements of the kidneys.

[0114] A study of the wound-healing properties of composition 4 revealed that 10 days of intravenous administration to animals with type 2 diabetes resulted in a significant reduction in wound surface area and relative decrease in wound size compared to the untreated control group. The results are presented in Table 3.

[0115] Administration of the prototype to rats with type 2 diabetes caused a less pronounced wound healing effect.

[0116] Table 3

[0117] Efficiency of intravenous administration of the claimed compound, dynamics of the wound healing process in animals with type 2 diabetes, Me (Q1;Q3) n=16.

[0118] Note: # - differences are statistically significant (p<0.05) compared to the values ​​of the control group.

[0119] Thus, a study of the efficacy of Composition 4 in a diabetic nephropathy model demonstrated that this composition reduces metabolic disturbances and exhibits pronounced antidiabetic and nephroprotective effects. Furthermore, it was demonstrated that Composition 4 has a significant therapeutic effect on wound healing dynamics in animals with type 2 diabetes compared to the prototype.

[0120] To establish the range of effective values ​​of the active components of the claimed composition, providing antidiabetic, nephroprotective and wound-healing activity, various dilutions of the composition in water for injection were prepared while maintaining the ratio of active components.

[0121] Inosine, nicotinamide, and methionine are known to have short half-lives. Therefore, to ensure maximum effect on the target organ (the kidneys), it is advisable to administer the medicinal composition over a long period at a minimal rate. This will maintain a constant concentration of the components in the blood over an extended period and increase the effectiveness of treatment.

[0122] Composition 4 (experiment 2), diluted with water for injection 10 times, was taken as the base solution, and to maintain the effective dose of active components, the solutions were administered in a tenfold increased volume - 30 ml / kg.

[0123] Experiment 3. Comparative study of the antidiabetic, nephroprotective and wound-healing activity of compositions with different contents of active components in a model of diabetic nephropathy in rats.

[0124] Diabetic nephropathy in rats was modeled by performing right-sided nephrectomy with preservation of the adrenal gland, administering streptozotocin, and placing the animals on a high-fat diet for 6 weeks. Streptozotocin was administered intraperitoneally at a dose of 25 mg / kg twice with a weekly interval on the 7th and 8th weeks of the experiment. After verification of diabetes mellitus in rats (blood glucose levels > 11 mmol / L, confirming the development of T2DM), the animals were randomized into experimental groups based on blood glucose levels to form homogeneous groups.

[0125] To study the effective concentrations, the compositions given in Table 4 were examined:

[0126] Table 4

[0127] Compositions of compositions.

[0128] To conduct the study, 9 groups of animals were formed:

[0129] • Intact – animals without the development of type 2 diabetes and without the administration of drugs, n = 10;

[0130] • Control – saline solution was administered, n = 10;

[0131] • Group 1 – composition 1 was administered, n = 10;

[0132] • Group 2 – composition 2 was administered, n = 10;

[0133] • Group 3 – composition 3 was administered, n = 10;

[0134] • Group 4 – composition 4 was administered, n = 10;

[0135] • Group 5 – composition 5 was administered, n = 10;

[0136] • Group 6 – composition 6 was introduced, n = 10;

[0137] • Group 7 – composition 7 was administered, n = 10.

[0138] Animals with developed diabetic nephropathy were administered the compositions (Table 4) via the tail vein using infusion pumps at a rate of 1 ml / min at a dose of 30 ml / kg for 10 days. The control group received an equivalent volume of saline solution.

[0139] General clinical observation of animals was carried out daily throughout the experiment.

[0140] The effectiveness of the claimed composition on the diabetic nephropathy model was assessed 10 days after the end of administration of the study drugs.

[0141] To collect urine, rats were placed in metabolic cages for 24 hours. Blood was collected from the tail vein during life into tubes containing clot activator. Creatinine, insulin, and glucose were determined in the serum using a biochemical analyzer. The animals' body weights were measured, and renal excretory function (urine glucose, protein, and urea concentrations) and SCF were assessed. SCF was calculated using the formula given in Experiment 2.

[0142] Cystatin C levels in the animals' blood and urine were measured using an enzyme-linked immunosorbent assay (ELISA) on a plate spectrophotometer. Additionally, full-thickness planar skin wounds were created on the rats' backs, and wound healing was assessed qualitatively and quantitatively. Wounds were created in anesthetized animals by excising a 1.5 cm diameter skin flap and removing subcutaneous fat. The depth of the lesion corresponded to the thickness of the rat's skin, including the epidermis, dermis, and hypodermis. The percentage of wound reduction (A, %), the linear rate of wound healing (u, mm2 / day), and the relative decrease in wound area (AS, %) were calculated using the formulas given in Experiment 2.

[0143] Statistical data processing was performed using the GraphPad Prism 8.0 statistical software package (USA). The distribution of each sample was tested for normality using the Shapiro-Wilk test. Between-group statistical comparisons were performed using the Tukey test for normal distributions and the Mann-Whitney test for non-normal distributions. Differences with a null hypothesis probability of less than 0.05 were considered significant.

[0144] In the urine of untreated rats with type 2 diabetes (the control group), a statistically significant decrease in body weight of more than 40 g was observed, as well as a threefold increase in glucose, a 5.5-fold increase in protein, and a twofold increase in cystatin C compared to the intact group. In the blood, creatinine levels increased to 86 μmol and glucose levels increased more than fivefold compared to the intact group. Furthermore, the untreated control group showed a significant decrease in blood insulin (from 75 to 53 pmol / L) and a more than twofold decrease in the renal function index (SCF) compared to the intact group. The study results are presented in Table 5.

[0145] Table 5

[0146] Efficiency of intravenous administration of a solution with different contents of active components in rats with diabetes mellitus, M±SEM, n=10.

[0147] Notes:

[0148] 1. * - differences are statistically significant (p<0.05) compared with the values ​​of the intact group;

[0149] 2. # - differences are statistically significant (p<0.05) compared with the values ​​of the control group.

[0150] Administration of a solution with a concentration of 75-200% active ingredients (Groups 3-7) resulted in a statistically significant increase in body weight in rats relative to the untreated control group. Following administration of a lower concentration (Group 1), body weight remained at the control group level. Urine analysis revealed that administration of the solution in Groups 2-5 (75-150%) resulted in a significant decrease in glucose levels by 1.8-2.4 times and cystatin C by 1.7-1.9 times compared to the control group. Furthermore, a trend-like decrease in urine protein levels was recorded across all concentrations studied.

[0151] Administration of the solution at all studied concentrations significantly reduced glucose levels by 1.7-2.4 times and creatinine by 1.2-1.7 times in the blood of rats relative to control group values. However, creatinine concentrations in the blood of rats from groups 1 and 2 remained significantly higher relative to intact animals. A decrease in the concentration of cystatin C, a marker of renal impairment, was recorded in the blood of rats in all treatment groups relative to intact animals. A significant increase in blood insulin levels relative to control group values ​​was observed with administration of the drug at concentrations of 100, 125, and 150% (groups 3-5, respectively).

[0152] A statistically significant increase in SCF relative to the control group was observed following administration of the solution at concentrations of 100% and 125%, indicating restoration of renal function in the animals. Meanwhile, treatment in groups 2, 5, 6, and 7 resulted in a trend toward restoration of SCF to levels similar to those in the intact group.

[0153] The study of the effectiveness of intravenous administration of the solution in concentrations of 50-200% (groups 1-7) on the dynamics of the wound healing process in animals with type 2 diabetes is presented in Table 6. It was shown that when administering a solution with a concentration of active ingredients of 100% (group 3), a statistically significant increase in the rate of healing of the wound surface and the relative decrease in the wound area were recorded compared to the values ​​​​in the control group without treatment.

[0154] In the remaining treatment groups, a change in the indicators of wound healing dynamics was noted at the trend level.

[0155] Table 6. The effectiveness of intravenous administration of a solution with different ratios of active substances on the dynamics of the wound healing process in animals with type 2 diabetes, Me (Q1;

[0156] Q3), n=10.

[0157]

[0158] Note: # - differences are statistically significant (p<0.05) compared to the values ​​of the control group.

[0159] Thus, solutions with a concentration in the range from 75% to 150%, which corresponds to the content of active components in the range of values ​​​​1-Deoxy-1-N-methylammonium-B-glucitol succinate 7.5-15.0 g / l, inosine 1.50-3.0 g / l, methionine 0.53- 1.05 g / l, nicotinamide 0.19-0.38 g / l, have a pronounced antidiabetic activity, as evidenced by the restoration of normal levels of glucose and insulin in the blood, as well as nephroprotective activity, which is confirmed by the restoration of indicators of the functional state of the kidneys in diabetic nephropathy and a significant wound healing effect in animals with diabetes mellitus.

[0160] It should be noted that with a decrease in the concentration of active substances to 50% (group 1), and an increase to 175% and higher, i.e. outside the range of values ​​of active components (groups 6 and 7), a decrease in the effectiveness of the solution was noted for a number of indicators.

[0161] Since the treatment of diabetes and its complications involves long-term drug therapy, to ensure maximum safety, it is advisable to administer the medicinal composition as a parenteral drug - in the form of an infusion solution, the ionic composition of which is close in physiological properties to blood plasma.

[0162] In this regard, a method has been developed for obtaining, based on the claimed composition, a parenteral drug with an optimal composition and properties, namely, a solution with a physiologically acceptable pH value and osmolarity, and a content of the main ions of blood plasma within the physiological norm.

[0163] For this purpose, based on the compositions given in Table 4, a solution is prepared with the addition of sodium hydroxide to bring the pH to 6.0-8.0, as well as sodium chloride, magnesium chloride, and potassium chloride salts to achieve physiologically acceptable contents of sodium, potassium, magnesium, and chloride ions and osmolarity (Table 7).

[0164] Table 7

[0165] Composition and properties of the composition.

[0166] The presented composition has the following physicochemical properties: osmolarity 309-316 mOsmol / l, pH=6.0-8.0, sodium ion content 130 mmol / l, potassium 4.0 mmol / l, magnesium 1.13 mmol / l, chlorides 104.1 mmol / l, which corresponds to physiologically acceptable values.

[0167] To conduct safety studies of this composition, various dilutions were prepared with the content of all components in the solution - 50%, 75%, 100%, 125%, 150%, 175% and 200% of the original values, examples 5-11.

[0168] Example 5 (50%)

[0169] Place 900 ml of water in a reactor, dissolve 5.0 g of 1-Deoxy- lN-methylammonium-D-glucitol succinate, bring the solution to pH 6.0-8.0 with sodium hydroxide, then add 1.0 g of inosine, 0.35 g of methionine and 0.13 g of nicotinamide until completely dissolved, then add 2.85 g of sodium chloride, 0.15 g of potassium chloride and 0.06 g of magnesium chloride and stir until completely dissolved and finally bring the pH of the solution to pH with sodium hydroxide if necessary. Then, bring the volume of the solution to 1 liter with water for injection. Filter the solution through a sterilizing filter with a pore diameter of 0.22 μm and pour into glass vials, seal with a stopper. The resulting vials are subjected to terminal heat sterilization at 121±1°C for 15-20 minutes. The resulting solution has an osmolarity of 157 mOsm / L.

[0170] Example 6 (75%)

[0171] Place 900 ml of water in a reactor, dissolve 7.5 g of 1-Deoxy- lN-methylammonium-D-glucitol succinate, bring the solution to pH 6.0-8.0 with sodium hydroxide, then add 1.5 g of inosine, 0.53 g of methionine and 0.19 g of nicotinamide until completely dissolved, then add 4.28 g of sodium chloride, 0.23 g of potassium chloride and 0.09 g of magnesium chloride and stir until completely dissolved and finally bring the pH of the solution to pH with sodium hydroxide if necessary. Then, bring the volume of the solution to 1 liter with water for injection. Filter the solution through a sterilizing filter with a pore diameter of 0.22 μm and pour into glass vials, seal with a stopper. The resulting vials are subjected to terminal heat sterilization at 121±1°C for 15-20 minutes. The resulting solution has an osmolarity of 235 mOsm / L.

[0172] Example 7 (100%)

[0173] Place 900 ml of water in a reactor, dissolve 10.0 g of 1-Deoxy- lN-methylammonium-D-glucitol succinate, bring the solution to pH 6.0-8.0 with sodium hydroxide, then add 2.0 g of inosine, 0.70 g of methionine and 0.25 g of nicotinamide until completely dissolved, then add 5.70 g of sodium chloride, 0.30 g of potassium chloride and 0.12 g of magnesium chloride and stir until completely dissolved and finally bring the solution to pH with sodium hydroxide if necessary. Then, bring the volume of the solution to 1 liter with water for injection. Filter the solution through a sterilizing filter with a pore diameter of 0.22 μm and pour into glass vials, seal with a stopper. The resulting vials are subjected to terminal heat sterilization at 121±1°C for 15-20 minutes. The resulting solution has an osmolarity of 313 mOsm / L.

[0174] Example 8 (125%)

[0175] Place 900 ml of water in a reactor, dissolve 12.5 g of 1-Deoxy- lN-methylammonium-D-glucitol succinate, bring the solution to pH 6.0-8.0 with sodium hydroxide, then add 2.5 g of inosine, 0.88 g of methionine and 0.31 g of nicotinamide until completely dissolved, then add 7.13 g of sodium chloride, 0.38 g of potassium chloride and 0.15 g of magnesium chloride and stir until completely dissolved and finally bring the pH of the solution to pH with sodium hydroxide if necessary. Then, bring the volume of the solution to 1 liter with water for injection. Filter the solution through a sterilizing filter with a pore diameter of 0.22 μm and pour into glass vials, seal with a stopper. The resulting vials are subjected to terminal heat sterilization at 121±1°C for 15-20 minutes. The resulting solution has an osmolarity of 392 mOsm / L.

[0176] Example 9 (150%)

[0177] Place 900 ml of water in a reactor, dissolve 15.0 g of 1-Deoxy- lN-methylammonium-D-glucitol succinate, bring the solution to pH 6.0-8.0 with sodium hydroxide, then add 3.0 g of inosine, 1.05 g of methionine and 0.38 g of nicotinamide until completely dissolved, then add 8.55 g of sodium chloride, 0.45 g of potassium chloride and 0.18 g of magnesium chloride and stir until completely dissolved and finally bring the solution to pH with sodium hydroxide if necessary. Then, bring the volume of the solution to 1 liter with water for injection. Filter the solution through a sterilizing filter with a pore diameter of 0.22 μm and pour into glass vials, seal with a stopper. The resulting vials are subjected to terminal heat sterilization at 121±1°C for 15-20 minutes. The resulting solution has an osmolarity of 470 mOsm / L.

[0178] Example 10 (175%)

[0179] Place 900 ml of water in a reactor, dissolve 17.5 g of 1-Deoxy- lN-methyl ammonium-D-glucitol succinate, bring the solution to pH 6.0-8.0 with sodium hydroxide, then add 3.5 g of inosine, 1.23 g of methionine and 0.44 g of nicotinamide until completely dissolved, then add 9.98 g of sodium chloride, 0.53 g of potassium chloride and 0.21 g of magnesium chloride and stir until completely dissolved and finally bring the pH of the solution to pH with sodium hydroxide if necessary. Then, bring the volume of the solution to 1 liter with water for injection. Filter the solution through a sterilizing filter with a pore diameter of 0.22 μm and pour into glass vials, seal with a stopper. The resulting vials are subjected to terminal heat sterilization at 121±1°C for 15-20 minutes. The resulting solution has an osmolarity of 549 mOsm / L.

[0180] Example 11 (200%)

[0181] Place 900 ml of water in a reactor, dissolve 20 g of 1-Deoxy- lN-methylammonium-B-glucitol succinate, bring the solution to pH 6.0-8.0 with sodium hydroxide, then add 4.0 g of inosine, 1.40 g of methionine and 0.50 g of nicotinamide until completely dissolved, then add 11.40 g of sodium chloride, 0.60 g of potassium chloride and 0.24 g of magnesium chloride and stir until completely dissolved and finally bring the solution to pH with sodium hydroxide if necessary. Then, bring the volume of the solution to 1 liter with water for injection. Filter the solution through a sterilizing filter with a pore diameter of 0.22 μm and pour into glass vials, seal with a stopper. The resulting vials are subjected to terminal heat sterilization at 121±1°C for 15-20 minutes. The resulting solution has an osmolarity of 627 mOsm / L.

[0182] Experiment 4. Study of the safety of a composition with different component contents upon repeated intravenous administration to rats over 14 days.

[0183] A safety study was conducted using different active ingredient compositions (examples 5-11) with repeated intravenous administration to rats over 14 days. Animal survival and changes in the clinical picture of intoxication were assessed, and pathological changes in the animals were studied based on the results of physiological and clinical laboratory studies.

[0184] 8 groups of animals were formed:

[0185] - Control. Introduction of physiological solution, n = 10;

[0186] Group 1. Introduction of a composition with a component content of 50%, n = 10;

[0187] - Group 2. Introduction of a composition with a component content of 75%, n = 10;

[0188] - Group 3. Introduction of a composition with a component content of 100%, n = 10;

[0189] - Group 4. Introduction of a composition with a component content of 125%, n = 10;

[0190] - Group 5. Introduction of a composition with a component content of 150%, n = 10;

[0191] - Group 6. Introduction of a composition with a component content of 175%, n = 10;

[0192] - Group 7. Introduction of a composition with a component content of 200%, n = 10.

[0193] The compositions and saline solution were administered to rats via the tail vein in the maximum permitted volume for this animal species (50 ml / kg) [Recommendation of the Board of the Eurasian Economic Commission of November 14, 2023, No. 33 "On the Guidelines for Working with Laboratory (Experimental) Animals During Preclinical (Non-Clinical) Studies"]. Administration was carried out using infusion pumps at an administration rate of 1 ml / min. The clinical picture of intoxication was assessed (animal appearance, body weight, behavioral reactions, mortality), a biochemical blood test was performed (alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP) activity, total bilirubin, glucose, total cholesterol, total protein), a clinical blood test (ESR, number of erythrocytes, leukocytes, platelets, hemoglobin concentration, hematocrit, leukocyte formula was calculated), cardiovascular system parameters were measured (ECG, respiratory system (respiratory rate (RR)).Blood sampling and measurement of physiological parameters were performed after administration of the study compositions. Statistical data processing was performed using the GraphPad Prism 8.0 statistical software package (USA). The distribution of each sample was tested for normality using the Shapiro-Wilk test. Between-group statistical comparisons were performed using the Tukey test for normal distributions and the Mann-Whitney test for abnormal distributions. Differences with a null hypothesis probability of less than 0.05 were considered significant.

[0194] A safety study of intravenous administration of a composition with different active ingredient compositions over 14 days showed a mortality rate of 40% in group 7 with a 200% composition, a mortality rate of 30% in group 1 (50% composition), a mortality rate of 20% in groups 2 and 6 (75 and 175% compositions), and a mortality rate of 10% in groups 4 and 5 (125 and 150% compositions). The study results are presented in Table 7. No mortality was recorded in the group of animals administered the composition with a 100% composition (group 3).

[0195] At the end of the administration period, a decrease in motor activity, piloerection, and a statistically significant change in body weight were observed in groups 1, 2, 6, and 7 (50, 75, 175, and 200% formulations). In groups 6 and 7 (175 and 200% formulations), a significant increase in heart rate to 582±46 bpm and a twofold increase in respiratory rate were observed compared to the control group. Furthermore, behavioral changes toward depression were observed in groups 1, 2, 6, and 7.

[0196] A study of blood biochemistry parameters revealed a statistically significant increase in bilirubin and glucose levels in the blood in Group 1 (50% composition) and an increase in bilirubin in Group 2 (75%). A significant decrease in leukocytes and erythrocytes and an increase in ESR were also recorded in Group 1. In Groups 6 and 7 (175 and 200% composition), a significant increase in total protein in the blood to 79±5 g / L, ESR to 8.0±1.3 mm / h, urine protein by 2.5 times, and a decrease in urine pH by 1.4 times relative to the control group values ​​were noted.

[0197] Table 8

[0198] Study of physiological and laboratory parameters of rats following intravenous administration of compositions with different contents of active substances for 14 days, M±SEM, n=10.

[0199] Physiological indicators PC17RU2025 / 000240

[0200] Based on the data obtained, it can be assumed that repeated intravenous administration over 14 days of a hypo-osmolar solution (157 mOsm / L), prepared according to Example 5 (Group 1, 50%), led to the development of erythrocyte hemolysis in rats. Moreover, upon administration of compositions with hyperosmolar formulations prepared according to Examples 10 (175%, 549 mOsm / L) and 11 (200%, 627 mOsm / L), pathological changes indicating a disturbance of the water-salt balance were recorded.

[0201] Taking into account the results of animal mortality, as well as hematological and biochemical parameters, it can be concluded that compositions with a component content of 50%, 175% and 200% had less pronounced pharmacological safety.

[0202] A safety study demonstrated that intravenous administration of compositions containing active ingredients ranging from 75% to 150% at a dose of 50 ml / kg for 14 days did not result in changes in behavioral, physiological, or laboratory parameters in experimental animals. Thus, the present invention experimentally confirmed that the resulting solutions containing active and auxiliary components according to Examples 6-9 exhibit a pronounced antidiabetic effect. Furthermore, renal function in diabetic nephropathy is restored, and a significant wound-healing effect is observed in animals with diabetes mellitus.

[0203] The parenteral drug obtained according to the claimed method can be used in the long-term treatment of diabetes mellitus and its complications, in particular diabetic nephropathy and chronic skin wounds due to the destruction of soft tissues (diabetic ulcers).

Claims

CLAUSES OF THE INVENTION 1. A medicinal composition in the form of an aqueous solution containing the compound 1-Deoxy-1-T-methylammonium-O-glucitol succinate, characterized in that it additionally contains inosine, nicotinamide, and methionine as active components, in the following ratio of components, g / l: 1-Deoxy- 1-N-methylammonium-O-glucitol succinate 7.5-15.0 Inosine 1.50-3.0 Methionine 0.53-1.05 Nicotinamide 0.19-0.38 Water for injection up to 1 liter 2. A medicinal composition according to claim 1, exhibiting antidiabetic activity.

3. A medicinal composition according to claim 1, exhibiting nephroprotective activity.

4. A medicinal composition according to claim 1, exhibiting wound healing activity.

5. A method for producing a parenteral preparation containing the composition according to claim 1 by dissolving the components in water for injection, mixing, filtering the solution through a sterilizing filter and pouring it into sealed containers, characterized in that the compound 1-Deoxy-1-N-methylammonium-N-glucitol succinate is dissolved in water for injection, the solution is adjusted to pH 6.0-8.0 with sodium hydroxide, inosine, methionine, nicotinamide are added, and sodium chloride, potassium chloride and magnesium chloride are additionally added to an osmolarity of 235-470 mOsm / l, then the sealed containers with the resulting solution are subjected to final sterilization.

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