Pharmaceutical compositions and methods of preparing parenteral formulations

Abstract

A pharmaceutical composition in the form of an aqueous solution comprising 1-deoxy-1-N-methylammonio-D-sorbitol succinate, inosine, nicotinamide and methionine, for example as follows: 7.5 to 15.0 g / l of 1-deoxy-1-N-methylammonio-D-sorbitol succinate; 1.50 to 3.0 g / l of inosine; 0.53 to 1.05 g / l of methionine; 0.19 to 0.38 g / l of nicotinamide; water for injection to 1 liter. Also disclosed is a method for the preparation of parenteral formulations containing the claimed pharmaceutical composition. The composition exhibits antidiabetic, renoprotective and wound healing activity and can be used for the long-term treatment of diabetes mellitus and its accompanying complications, in particular diabetic nephropathy, and chronic skin wounds caused by soft tissue damage (diabetic ulcers).

Description

Technical Field

[0001] This invention relates to the pharmaceutical and pharmaceutical industries, and to an aqueous pharmaceutical composition having antidiabetic, nephroprotective and wound-healing activities, as well as a method for preparing a parenteral preparation for the long-term treatment of type II diabetes and its complications, specifically diabetic nephropathy and chronic skin lesions (diabetic ulcers) caused by soft tissue injury. Background Technology

[0002] Diabetes is an endocrine disorder caused by an absolute or relative deficiency of insulin, which impairs the tissue's utilization of glucose. Simultaneously, organs and tissues lose their ability to convert incoming glucose into glycogen, leading to glucose accumulation in the blood and consequently causing endothelial damage in various organs.

[0003] Complications of diabetes include severe damage to the cardiovascular system, eye disease, and kidney disease—impaired excretion, filtration, and synthesis, leading to diabetic nephropathy.

[0004] Diabetic nephropathy occurs due to damage to the nephrons, which are responsible for detoxification and electrolyte balance. Glycosylation occurs in the glomerular filtration membrane, and the distal convoluted tubule epithelium loses its ability to reabsorb salts and other components from the primary urine back into the bloodstream.

[0005] As the disease progresses, the kidneys lose their ability to perform their functions. The concentration of toxins and metabolic products in the blood increases significantly, causing autointoxication in the body. Major complications of diabetic nephropathy include chronic kidney disease, electrolyte disturbances such as potassium, sodium, and phosphorus imbalances, and the development of metabolic acidosis.

[0006] Chronic kidney disease (CKD) is a potentially fatal disease that is often diagnosed at an advanced stage, when vital kidney function has already been lost. CKD is one of the most common complications of diabetes and a risk factor for cardiovascular disease. Nearly 40% of people with type 2 diabetes (T2DM) develop CKD. Despite standard treatments outlined in relevant clinical guidelines, patients with CRD and T2DM face a high risk of CRD progression and cardiovascular complications. Current estimates suggest that over 160 million people with type 2 diabetes worldwide have CRD. In people with type 2 diabetes, CKD is the leading cause of end-stage renal failure, requiring dialysis or kidney transplantation to save lives. Additionally, the condition is associated with chronic wounds on the skin due to soft tissue destruction in the lower extremities. Complications have been observed in people with diabetes for more than 15 to 20 years. In 65% to 85% of cases, this leads to amputation of the affected foot.

[0007] Replacing diabetes with exogenous insulin and medications that lower blood sugar levels is insufficient to fully treat diabetes and its complications. Insulin therapy alone is only effective in the short term and cannot maintain stable blood sugar levels in the future. Therefore, in the early stages of the disease, a combination of oral medications that lower blood sugar and medications that reduce the negative effects of high blood sugar on the kidneys is necessary.

[0008] Therefore, developing combination antidiabetic drugs with a broad spectrum of action is an urgent task.

[0009] There are known compound antidiabetic drugs containing plant-derived ingredients, such as: compositions with a multi-component antidiabetic mechanism of action (RF Patent 2289419), homeopathic antidiabetic agents (RF Patent 2164412), agents with antioxidant, antitumor, immunomodulatory, antidiabetic and antibacterial properties (RF Patent 2598349), antidiabetic compound (RF Patent 2284829), antidiabetic agents (RF Patent 2283659), and compositions with antidiabetic effects (RF Patent 2430735).

[0010] However, due to the multidirectional effects of plant components on the body, such drugs do not have a stable antidiabetic effect. Furthermore, some types of medicinal plant materials may not be available due to their growing region.

[0011] Furthermore, in the treatment of diabetes, combinations of drugs are known to be used that, in addition to relieving disease symptoms, prevent acute complications such as hyperosmolar coma and ketoacidosis, as well as chronic complications such as diabetic neuropathy (RF Patent 2351317). Combinations for treating metabolic disorders (primarily diabetes) can be formulated as combination products or pharmaceutical compositions (RF Patent 2280447). However, these compositions do not possess any nephroprotective activity.

[0012] In addition, oral antidiabetic agents based on succinic acid are known to exist (RF patent 2114617). These products contain a mixture of succinic acid or its salts (such as ammonium succinate and sodium succinate) with sorbitol or xylitol. While the proposed products expand the range of natural antidiabetic agents, they do not address the side effect, specifically renal dysfunction.

[0013] A common drawback of oral dosage forms is that the drugs enter the systemic circulation slowly, they are inactivated in the liver, and they cannot be used when the patient is unconscious.

[0014] A novel quaternary ammonium salt compound, 1-deoxy-1-N-methylammonium-D-sorbitol succinate, derived from succinic acid, is known to have antidiabetic effects, high diuretic activity, and low toxicity (RF Patent No. 2345060, Patent Holder: STPFPOLYSAN Ltd).

[0015] An aqueous solution of 1-deoxy-1-N-methylammonium-D-sorbitol succinate shows promise for development as a parenteral antidiabetic drug and is the closest analogue to the claimed composition.

[0016] However, the effectiveness of this compound is insufficient to comprehensively affect the key pathophysiological processes of diabetes (specifically diabetic nephropathy) because it does not affect the activation of metabolic activity in the glomeruli and tubules of the nephrons, which is necessary to maintain the filtration and reabsorption functions of the kidneys, as well as the healing of diabetic ulcers that form during the long and severe course of diabetes due to poor endothelial function and microcirculation in the skin.

[0017] In addition, long-term, high-dose intravenous administration of this compound carries a high risk because a 10% solution of 1-deoxy-1-N-methylammonium-D-sorbitol succinate has an acidic pH (approximately 4.5), which may cause local irritation and disrupt the body's acid-base balance.

[0018] The basic preparation techniques for parenteral drugs typically include the following main stages: dissolving the active ingredient and excipients in water for injection, mixing until the solution reaches equilibrium, then filtering the solution through a series of filters with different pore sizes, sterilizing the filter, and filling it into a sealed container (Handbook of Pharmaceutical Manufacturing. Sterile Products. Second Edition vol 6, 2009). Summary of the Invention

[0019] The object of the present invention is to produce a pharmaceutical composition in aqueous solution with enhanced antidiabetic activity as well as nephroprotective and wound-healing effects, and to develop a related method for preparing a parenteral preparation for the long-term treatment of diabetes and its complications.

[0020] This objective is achieved by producing a pharmaceutical composition in the form of an aqueous solution containing 1-deoxy-1-N-methylammonium-D-sorbitol succinate, wherein, according to the invention, the pharmaceutical composition further contains inosine, nicotinamide, and methionine as active ingredients, in the following proportions (g / l): 1-Deoxy-1-N-methylammonium-D-sorbitan succinate 7.5-15.0 Inosine 1.50-3.0 Methionine 0.53-1.05 Nicotinamide 0.19-0.38 The maximum amount of water for injection is 1 liter.

[0021] This objective is also achieved by the fact that the pharmaceutical composition in aqueous solution form exhibits antidiabetic activity.

[0022] This objective is also achieved by the fact that the pharmaceutical composition in aqueous form exhibits renal protective activity.

[0023] This objective is also achieved by the fact that the pharmaceutical composition in aqueous solution form exhibits wound healing properties.

[0024] Furthermore, this objective is achieved by a method for preparing a parenteral preparation containing the claimed pharmaceutical composition by dissolving the components in water for injection, mixing, filtering the solution through a sterile filter, and filling it into a sealed container, wherein, according to the invention, compound 1-deoxy-1-N-methylammonium-D-sorbitan succinate is dissolved in water for injection, the solution is adjusted to pH 6.0 to 8.0 with sodium hydroxide, inosine, methionine, nicotinamide are added, and sodium chloride, potassium chloride, and magnesium chloride are also added to achieve an osmotic pressure of 235 to 470 mOsm / l, and then the sealed container containing the resulting solution is terminally sterilized.

[0025] The claimed composition mainly contains 1-deoxy-1-N-methylammonium-D-sorbitol succinate compound with the general formula C. 11 N 22 NО9, and its structure is as follows:

[0026] This compound exerts its anti-diabetic effect by increasing the body's own insulin production and lowering blood glucose and urine glucose levels (RF Patent No. 2345060).

[0027] The claimed pharmaceutical composition also contains active ingredients that activate metabolic processes in the body—inosine, methionine, and nicotinamide. There have been no previous reports of their combined use in antidiabetic compositions.

[0028] Compounds containing metabolites are known to have a general normalizing effect on the body's metabolic processes and are often used as dietary supplements. Combinations containing meglumine succinate, riboxine, methionine, and nicotinamide as active ingredients are known to exhibit hepatoprotective and choleretic activities by stimulating bile acid excretion (RF Patent 2240116).

[0029] Because the nature of diabetes and related diseases or conditions is determined by a variety of factors, the combination of pharmacologically acceptable ingredients with different mechanisms of action does not always yield a combination with preferred properties.

[0030] The present invention demonstrates that the claimed composition comprising 1-deoxy-1-N-methylammonium-D-sorbitol succinate and inosine, nicotinamide and methionine as active ingredients (Example 4) has enhanced antidiabetic activity compared with the prototype (Experiment 2).

[0031] The active ingredients in the composition were selected empirically. Furthermore, their content was not limited to that in Example 4, but was determined by the numerical range of the overall pharmaceutical composition exhibiting the claimed protective effect (Experiment 3).

[0032] The T2DM model in this invention demonstrates for the first time that the claimed composition, in addition to its antidiabetic activity, also exhibits nephroprotective activity by activating metabolic processes in the glomeruli and tubules, which is essential for maintaining the filtration and reabsorption functions of the kidneys (Experiments 2 and 3).

[0033] Furthermore, Experiments 2 and 3 demonstrate that the claimed composition exhibits wound-healing effects by activating metabolism in the skin, which is crucial for the healing of diabetic ulcers formed in severe cases of diabetes due to impaired endothelial function and microcirculation in the skin.

[0034] Therefore, this pharmaceutical composition has a complex antidiabetic mechanism of action designed to alleviate the symptoms of diabetic nephropathy, and is itself a non-toxic composition, so the related medicine can be used safely for a long time.

[0035] According to the present invention, a parenteral formulation is obtained based on the claimed aqueous solution form of the pharmaceutical composition.

[0036] To prepare the drug, 1-deoxy-1-N-methylammonium-D-sorbitan succinate was dissolved in water for injection, and the solution was adjusted to pH 6.0 to 8.0 with sodium hydroxide. This pH range is optimal for both parenteral and intravenous administration. The normal pH of blood plasma is approximately 7.35; therefore, there is a risk of acidosis when using an intravenous solution with a pH of 6.0, and a risk of alkalosis when using a solution with a pH above 8.0, both of which can pose side effects during long-term treatment.

[0037] Then, inosine, methionine, nicotinamide, and additional salts—sodium chloride, potassium chloride, and magnesium chloride—are added to the resulting solution in a certain proportion, and the mixture is stirred until all components are completely dissolved.

[0038] The ratio of sodium chloride, potassium chloride, and magnesium chloride is selected in the following manner: when using therapeutic doses of the drug, the content of sodium, potassium, and magnesium ions is within the physiological range of human blood, and the osmotic pressure of the solution is maintained within 235 to 470 mOsm / l, which is suitable for rapid intravenous administration.

[0039] The solution is then filtered through a sterile filter with a pore size of 0.22 μm and filled into sealed glass or plastic containers, and finally heat-sterilized at 121±1℃ for 15 to 20 minutes.

[0040] In the resulting parenteral solutions, the range of active ingredients and excipients was determined by safety (achieving optimal physicochemical properties of the solution (pH, osmotic pressure, electrolyte content) (Experiment 4)) and by exhibiting antidiabetic, nephroprotective, and wound-healing effects across the entire range of active ingredient content (Experiment 3).

[0041] The claimed invention achieves a broader spectrum of action for pharmaceutical compositions in aqueous form by exhibiting enhanced antidiabetic, nephroprotective, and wound-healing effects.

[0042] Therefore, the resulting parenteral formulation can be used for the long-term treatment of diabetes and its complications (specifically diabetic nephropathy and chronic skin lesions caused by soft tissue damage (diabetic ulcers)). Detailed Implementation

[0043] The quantitative and qualitative content of the claimed composition was determined empirically through studies of the effects of metabolites on the bioenergetics of renal epithelial cells and the antidiabetic activity of 1-deoxy-1-N-methylammonium-D-sorbitan succinate.

[0044] To investigate the effects on renal epithelial cell bioenergetics, solutions of 1-deoxy-1-N-methylammonium-D-sorbitol succinate supplemented with inosine, methionine, and nicotinamide were prepared according to Examples 1 to 4.

[0045] Example 1. Add 900 ml of water to the reactor to dissolve 10.0 g of 1-deoxy-1-N-methylammonium-D-sorbitan succinate, then add 2.0 g of inosine and stir until completely dissolved. Adjust the volume of the solution to 1 liter with water for injection.

[0046] Example 2. Add 900 ml of water to the reactor to dissolve 10.0 g of 1-deoxy-1-N-methylammonium-D-sorbitan succinate, add 0.70 g of methionine and stir until completely dissolved, then bring the volume of the solution to 1 liter with water for injection.

[0047] Example 3. Add 900 ml of water to the reactor to dissolve 10.0 g of 1-deoxy-1-N-methylammonium-D-sorbitan succinate, then add 0.25 g of nicotinamide and stir until completely dissolved. Adjust the volume of the solution to 1 liter with water for injection.

[0048] Example 4. Add 900 ml of water to the reactor to dissolve 10.0 g of 1-deoxy-1-N-methylammonium-D-sorbitan succinate, then add 2.0 g of inosine, 0.70 g of methionine and 0.25 g of nicotinamide, and mix until completely dissolved. Then, bring the volume of the solution to 1 liter with water for injection.

[0049] Using an in vitro renal epithelial cell hypoxia and hypoxia-reperfusion model, the bioactivity of the solutions obtained in Examples 1 to 4 was compared with that of 1-deoxy-1-N-methylammonium-D-sorbitol succinate solution (prototype) – Experiment 1.

[0050] Experiment 1. Studies of compositions and prototypes in an in vitro hypoxia-reperfusion model.

[0051] In this study, the MDCK (Madine-Darby canine kidney cells) renal epithelial cell line was used as the assay system. Cells were cultured in DMEM (Dulbecco modified Eagle medium) at a glucose concentration of 1 g / L, containing 10% FBS, 2 mM L-alanyl-L-glutamine, 100 μg / ml streptomycin, and 100 U / ml penicillin. Cell cultures were maintained in an incubator at 37°C under a humidified atmosphere containing 5% CO2 and 95% air. The cell monolayer density was maintained at at least 70% during the study.

[0052] By adding sodium dithionite solution to cell cultures to simulate reoxygenation after hypoxia, the oxygen present in the culture medium was completely restored (the residual oxygen content in the culture medium was less than 1% of the maximum solubility).

[0053] At the start of the experiment, HBSS (Hanks balanced salt solution) was added to the cells, along with 10% by volume of experimental and prototype samples of compositions 1 to 4. Hypoxia was then simulated by incubating the cells in 5 mM sodium dithionite solution for 180 minutes. Cells were subsequently washed with HBSS and analyzed. The effects of experimental samples (prototype and compositions 1 to 4) under hypoxic and reoxygenation conditions on cell necrosis and apoptosis, changes in cellular bioenergetics (mitochondrial membrane potential (ΔΨm), mitochondrial ATP levels), and cellular antioxidant protection (rate of reactive oxygen species (ROS) generation in the mitochondrial matrix) were assessed.

[0054] The study was conducted using a laser scanning confocal microscope (LSM 900) and employed the fluorescent probes Hoechst 33342 and propidium iodide (PI), as well as annexin V labeled with the fluorescent label AF 488.

[0055] Data were analyzed using dedicated image analysis software ZEN Blue Edition (Zeiss, Germany) and MetaFluor (Molecular Devices, UK). The Kruskal-Wallis test and Dunn post-hoc test were used to determine the statistical significance of differences between data points. Differences with a significance level not exceeding 0.05 were considered statistically significant when describing the results. The obtained results were normalized relative to the median value of normal cells. The results are summarized in Table 1.

[0056] Table 1

[0057] The effects of compositions 1 to 4 on the bioenergetics of renal epithelial cell culture (MDCK cell line (Madine-Darby canine kidney cells)).

[0058] The results were normalized relative to the values ​​of normal cells, Me (Q1; Q3).

[0059]

[0060] Note: – The difference was statistically significant compared to the values ​​of normal cells (p<0.05).

[0061] This indicates that, under hypoxia-reoxygenation conditions, the cell lines treated with the prototype showed statistically significant mitochondrial hyperpolarization (ΔΨm increased by 64%) and a 35% decrease in ATP levels compared to normal cells. There was no difference in ΔΨm levels between normal cells and cells incubated with composition 4, indicating that the membrane potential remained stable within normal limits and mitochondrial ATP was increased.

[0062] A statistically significant increase in ROS synthesis in the mitochondrial matrix was detected in all experimental groups, which is a characteristic of the hypoxia-reoxygenation pathological process [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.].

[0063] When the prototype and composition 2 were added to the cell line, the most significant ROS synthesis in the mitochondrial matrix was observed (increased by 95%), while the lowest (53%) was observed when composition 4 was added.

[0064] Under hypoxia-reoxygenation conditions, the level of cell death in the culture was statistically significantly increased, reaching 9.3% when the original formulation was applied, while it decreased to 2.4% of the total cell number when the formulation 4 was applied.

[0065] Cellular survival under hypoxia-reoxygenation conditions is maintained through a complex protective mechanism associated with global changes in basal metabolic processes. Under low oxygen levels and with reduced role of oxidative phosphorylation in meeting cellular energy requirements, the contribution of alternative sources of ATP increases significantly, with glycolysis being a key factor, leading to a significantly higher rate of cellular glucose consumption.

[0066] The introduction of the prototype and compositions 1 to 4 resulted in higher glucose uptake in cells, with composition 4 showing a statistically significant difference compared to normal cells, where the index was observed to increase by 2.61 times.

[0067] The prototype and compositions 1 through 4 all exhibited cytoprotective effects under hypoxia-reoxygenation conditions. Furthermore, the addition of composition 4 indicated the highest level of activated endogenous defense mechanisms in cell cultures during hypoxia-reoxygenation, due to its role in stabilizing membrane potential, providing adequate levels of ATP and ROS in the mitochondrial matrix, and maintaining bioenergetic mechanisms including glycolysis.

[0068] Therefore, composition 4 is the most promising candidate for development as an antidiabetic drug with nephroprotective activity.

[0069] In addition, a key indicator in diabetes treatment is its impact on wound healing rate.

[0070] Composition 4 (Example 4), containing 10% 1-deoxy-1-N-methylammonium-D-sorbitol succinate, inosine, nicotinamide, and methionine, was used to investigate its effect on the progression of diabetic nephropathy and wound healing rate compared to a solution prepared according to the prototype—a 10% 1-deoxy-1-N-methylammonium-D-sorbitol succinate solution (Experiment 2).

[0071] Experiment 2. Study on anti-diabetic, nephroprotective and wound healing effects in a rat model of diabetic nephropathy.

[0072] A rat model of diabetic nephropathy was established by performing a right nephrectomy with adrenal preservation, administering streptozotocin, and feeding the animals a high-fat diet for 6 weeks. During weeks 7 and 8 of the experiment, streptozotocin was administered intraperitoneally twice weekly at a dose of 25 mg / kg. After confirming diabetes in the rats (blood glucose level ≥11 mmol / L, confirming type 2 diabetes), the animals were randomly assigned to experimental groups to form homogeneous groups based on their blood glucose levels.

[0073] Four groups of animals were formed: 1. Normal animals—those without T2DM progression and without drug administration, n=16; 2. Untreated control group—T2DM + intravenous infusion, n=16; 3. Prototype—T2DM+Application prototype, n=16; 4. Composition 4—T2DM+ application of composition 4, n=16.

[0074] Composition 4 and the solution prepared according to the prototype were administered intravenously to animals with diabetic nephropathy at a dose of 3 ml / kg for 10 consecutive days. An equal volume of intravenous infusion was administered to the untreated control group.

[0075] The animals underwent routine clinical observation daily throughout the experiment.

[0076] On day 10 after the end of the experimental drug administration, the efficacy of the composition claimed to be protected in a diabetic nephropathy model was evaluated.

[0077] Calculate the HOMA-IR index using the following formula:

[0078] To collect urine, rats were placed in metabolic cages for 24 hours. Blood was collected from the tail vein in vivo and placed in test tubes containing a coagulant. Serum creatinine, insulin, glucose, and urea levels were measured using a biochemical analyzer. Animal body weight was measured, and renal excretory function (daily urine volume and concentrations of glucose, protein, and urea in urine) and glomerular filtration rate (GFR) were assessed. GFR was calculated using the following formula:

[0079] The levels of lipid transporter 2 and cystatin C in animal blood and urine were detected using enzyme-linked immunosorbent assay (ELISA) on an EPOCH2 plate spectrophotometer.

[0080] Perform histological examination of the kidney tissue.

[0081] In addition, a full-thickness planar skin wound was created on the back of the rat, and the wound healing was qualitatively and quantitatively assessed. When creating the wound on anesthetized animals, a 1.5 cm diameter skin flap was harvested, and subcutaneous fat was removed. The depth of the injury corresponded to the thickness of the rat skin, including the epidermis, dermis, and subcutaneous tissue. The following parameters were evaluated: Percentage reduction in wound size (A, %) , Where d is the wound diameter on the experimental control days (day 1 and day 10).

[0082] – Linear wound healing rate ( mm 2 / sky): , Where P0 and S0 are the perimeter and area of ​​the wound at the time of the last measurement; Pt and St are the perimeter and area of ​​the wound after a period of time t (days).

[0083] – The wound area was relatively smaller. (ΔS, %) , Where S0 is the wound area at the time of the last measurement; St is the wound area after a period of time t (days).

[0084] Statistical data processing was performed using the Graph Pad Prism 8.0 statistical software package (USA). The Shapiro-Wilk test was used to check the normality of the distribution for each sample. Statistical comparisons between groups were performed using the Tukey test for normal distributions and the Mann-Whitney test for non-normal distributions. Differences with a null hypothesis probability less than 0.05 were considered significant.

[0085] In the untreated control group (Group 2), typical signs of progressive T2DM and diabetic nephropathy were observed—rat weight was statistically significantly reduced by more than 50 g, serum insulin levels decreased by 1.5-fold, and blood glucose, urine glucose, and urine protein levels were elevated compared to the normal animal group. Furthermore, kidney injury markers (lipocalin 2, cystatin C, and creatinine) were also elevated. Additionally, the HOMA-IR index was increased by more than 1.5-fold. The results are summarized in Table 2.

[0086] Table 2

[0087] Effects of intravenous administration of composition 4 and the prototype on blood parameters in diabetic rats, M±SEM, n=16.

[0088]

[0089] Note: – The difference was statistically significant compared with the values ​​of the normal group (p<0.05). – The difference was statistically significant compared with the control group (p<0.05).

[0090] In both the animal group administering the original formulation and the control group, a significant decrease in body weight to 229 ± 10 g was observed, along with the development of glycosuria (urine glucose 4.1 ± 1.5 mmol / L) and proteinuria (urine protein level 23.1 ± 3.3 mg / day). Compared to normal animals, a statistically significant increase in serum creatinine to 69 ± 6 μmol / L, a more than 3-fold increase in glucose, and a 1.3-fold decrease in insulin levels were noted. A significant increase in cystatin C, a marker of kidney injury, was observed: a 1.3-fold increase in serum and a 2-fold increase in urine compared to normal animals. Urinary lipotransferase 2 levels were more than four-fold increased. Compared to the control group, the original formulation exhibited antidiabetic activity.

[0091] Injection of Composition 4 (Group 4) into rats with type 2 diabetes mellitus (T2DM) significantly restored their body weight to 235 ± 11 g, and reduced blood glucose levels by 32% compared to the untreated control group (Group 2). Following injection of Composition 4, a decrease in the HOMA-IR index and levels of renal injury markers was observed—serum creatinine, insulin, and cystatin C concentrations remained at their corresponding levels in normal animals. Urinary lipid transporter 2 levels remained elevated but showed a decreasing trend.

[0092] Therefore, in terms of antidiabetic activity, this composition is superior to the prototype and exhibits significant renal protective activity compared with the prototype and the control group (intravenous infusion).

[0093] Histological images of renal tissue changes in untreated T2DM rats in the control group and rats in the prototype injection group were similar, showing damage to the glomeruli and tubules of the nephrons. In the glomeruli of the nephrons, thickening of the glomerular basement membrane, dilation of the mesangial matrix, and narrowing of the capillary lumen were observed.

[0094] Using composition 4 helps to restore glomeruli in nephrons and reduce lesions in renal structural elements.

[0095] Studies on the wound healing properties of composition 4 showed that, 10 days after intravenous administration of the composition to animals with T2DM, a significant reduction in wound surface area and relative shrinkage of wound area were observed compared to the control group of untreated animals. The results are summarized in Table 3.

[0096] The wound healing effect of administering the original drug to rats with T2DM was not significant.

[0097] Table 3

[0098] The effectiveness of intravenous administration of the compound to be protected, wound healing progress in animals with T2DM, Me(Q1; Q3) n=16.

[0099]

[0100] Note: – The difference was statistically significant compared with the control group (p<0.05).

[0101] Therefore, studies on the efficacy of composition 4 in a diabetic nephropathy model showed that the composition reduced metabolic disturbance levels and had significant anti-diabetic and nephroprotective effects. Furthermore, composition 4 has been reported to have a significant therapeutic effect on wound healing progression in T2DM animals compared to the original formulation.

[0102] To determine the effective range of the active ingredient used in the claimed composition that contributes to anti-diabetic, nephroprotective and wound-healing effects, the composition was diluted with water for injection at different ratios while keeping the proportion of the active ingredient constant.

[0103] Inosine, nicotinamide, and methionine are known to have short half-lives. Therefore, to ensure maximum effect on the target organ (kidney), it is recommended to administer the drug composition at the lowest possible rate over a prolonged period to maintain a constant concentration of the components in the blood and improve therapeutic efficacy.

[0104] Composition 4 (Experiment 2) diluted 10 times with water for injection was used as the base solution, and in order to maintain an effective dose of the active ingredient, the solution was administered at a volume ten times larger (30 ml / kg).

[0105] Experiment 3. Comparative study of the anti-diabetic, nephroprotective and wound-healing effects of drug compositions with different contents of active ingredients in a rat model of diabetic nephropathy.

[0106] A rat model of diabetic nephropathy was established by performing a right nephrectomy with adrenal preservation, administering streptozotocin, and feeding the animals a high-fat diet for 6 weeks. During weeks 7 and 8 of the experiment, streptozotocin was administered intraperitoneally twice weekly at a dose of 25 mg / kg. After confirming diabetes in the rats (blood glucose level ≥11 mmol / L, confirming type 2 diabetes), the animals were randomly assigned to experimental groups to form homogeneous groups based on their blood glucose levels.

[0107] To investigate the effective concentration, the compositions listed in Table 4 were studied: Table 4 Composition.

[0108]

[0109] This study used nine groups of animals: Normal animals – no T2DM and no drugs administered, n=10; Control group – intravenous infusion administered, n=10; Group 1 – Injection Composition 1, n=10; Group 2 – Injection Composition 2, n=10; Group 3 – Injection Composition 3, n=10; Group 4 – Injection Composition 4, n=10; Group 5 – Injection Composition 5, n=10; Group 6 – Injection composition 6, n=10; Group 7 – Injection Composition 7, n=10; Animals with pre-existing diabetic nephropathy were administered the composition (Table 4) via tail vein at a rate of 1 ml / min and a dose of 30 ml / kg using an infusion pump for 10 days. The control group received an equal volume of intravenous infusion.

[0110] The animals underwent routine clinical observation daily throughout the experiment.

[0111] On day 10 after the end of the experimental drug administration, the efficacy of the composition claimed to be protected in a diabetic nephropathy model was evaluated.

[0112] To collect urine, rats were placed in metabolic cages for 24 hours. Blood was collected from the tail vein in vivo and placed in test tubes containing a coagulant. Serum creatinine, insulin, and glucose levels were measured using a biochemical analyzer. Animal body weight was measured, renal excretory function (concentrations of glucose, protein, and urea in urine) was assessed, and glomerular filtration rate (GFR) was estimated. GFR was calculated using the formula from Experiment 2.

[0113] The levels of cystatin C in the blood and urine of animals were detected using an enzyme-linked immunosorbent assay (ELISA) on a plate spectrophotometer.

[0114] In addition, a full-thickness planar skin wound was created on the back of rats, and the wound healing was qualitatively and quantitatively assessed. A 1.5 cm diameter dermal flap was harvested and subcutaneous fat was removed to create the wound in anesthetized animals. The lesion depth corresponded to the thickness of the rat skin, including the epidermis, dermis, and subcutaneous tissue. The percentage reduction in wound area (A, %) and the linear wound healing rate (…) were calculated using the formulas given in Experiment 2. mm 2 / day) and relative reduction in wound area (ΔS, %)

[0115] Statistical data processing was performed using the Graph Pad Prism 8.0 statistical software package (USA). The Shapiro-Wilk test was used to check the normality of the distribution for each sample. Statistical comparisons between groups were performed using the Tukey test for normal distributions and the Mann-Whitney test for non-normal distributions. Differences with a null hypothesis probability less than 0.05 were considered significant.

[0116] Compared to the normal group, in untreated rats with type 2 diabetes mellitus (T2DM), a statistically significant decrease in body weight (over 40 g), a 3-fold increase in glucose levels, a 5.5-fold increase in protein levels, and a 2-fold increase in cystatin C levels were observed in the urine. In the blood, creatinine levels increased to 86 μmol, and glucose levels were more than 5-fold higher than in the normal group. Furthermore, compared to the normal group, the untreated control group showed a significant decrease in bleeding insulin (from 75 pmol / L to 53 pmol / L) and a more than 2-fold decrease in renal function (GFR). The results are summarized in Table 5.

[0117] Table 5

[0118] The efficacy of intravenous administration of solutions with different concentrations of active ingredients in diabetic rats, M±SEM, n=10.

[0119]

[0120] Note: 1. – The difference was statistically significant compared with the values ​​of the normal group (p<0.05). 2. – The difference was statistically significant compared with the control group (p<0.05).

[0121] Administration of solutions with active substance concentrations ranging from 75% to 200% (groups 3 to 7) resulted in a statistically significant increase in rat body weight relative to untreated control animals. When injected with lower concentration solutions (group 1), the animals' body weight remained at the control level.

[0122] Urine analysis showed that when the solution (75% to 150% of the composition) was administered in groups 2 to 5, glucose levels were significantly reduced by 1.8 to 2.4 times compared to the control group, and cystatin C was reduced by 1.7 to 1.9 times. It was also noted that urinary protein levels showed a decreasing trend at all studied concentrations.

[0123] Compared to the control group, administration of all studied concentrations of the solution significantly reduced blood glucose levels by 1.7 to 2.4 times and creatinine levels by 1.2 to 1.7 times in rats. Meanwhile, creatinine concentrations in the blood of rats in groups 1 and 2 remained significantly higher than those in normal animals. In all treatment groups, the concentration of cystatin C, a marker of kidney injury, in the blood of rats was noted to be lower than in normal animals. When the drug was administered at concentrations of 100%, 125%, and 150% (groups 3 to 5, respectively), a significant increase in blood insulin levels was observed relative to the control group.

[0124] After using solutions at concentrations of 100% and 125%, a statistically significant increase in GFR was observed relative to the control group, indicating recovery of renal function in the animals. Furthermore, use of this therapy in groups 2, 5, 6, and 7 helped restore GFR to trend-level values ​​similar to those in the normal group.

[0125] Table 6 summarizes the effects of intravenous administration of solutions at concentrations of 50% to 200% (groups 1 to 7) on wound healing progression in animals with type 2 diabetes mellitus (T2DM). The study showed that when a 100% concentration of the active substance was administered (group 3), a statistically significant increase in wound healing rate and relative shrinkage of wound area was observed compared to the untreated control group.

[0126] In other treatment groups, changes in wound healing progress were observed to be at a trend level.

[0127] Table 6

[0128] Effect of intravenous administration of solutions with different proportions of active substances on wound healing progress in T2DM animals, Me (Q1; Q3), n=10.

[0129]

[0130] Note: – The difference was statistically significant compared with the control group (p<0.05).

[0131] Therefore, solutions with concentrations of 75% to 150%, corresponding to active ingredient contents ranging from 7.5 to 15.0 g / L of 1-deoxy-1-N-methylammonium-D-sorbitan succinate, 1.50 to 3.0 g / L of inosine, 0.53 to 1.05 g / L of methionine, and 0.19 to 0.38 g / L of nicotinamide, exhibit significant antidiabetic activity, as evidenced by the restoration of blood glucose and insulin levels to normal, and also demonstrate nephroprotective activity, as evidenced by the recovery of renal function in diabetic nephropathy and significant wound healing in diabetic animals.

[0132] It should be noted that when the concentration of the active substance decreased to 50% (Group 1) and increased to 175% or above, which is outside the range of the active ingredient value (Groups 6 and 7), a decrease in the effectiveness of the solution on multiple indicators was observed.

[0133] Because the treatment of diabetes and its complications involves long-term medication, to ensure maximum safety, it is recommended that the drug composition be administered as a parenteral drug—in the form of an infusion solution whose ionic composition is physiologically close to that of plasma.

[0134] In this regard, a method has been developed for preparing a parenteral formulation with optimal composition and properties based on the claimed composition, i.e., a solution having physiologically acceptable pH and osmotic pressure, and with the main plasma ion content within physiological standards.

[0135] Therefore, based on the compositions given in Table 4, solutions were prepared by adding sodium hydroxide to adjust the pH to 6.0 to 8.0, and adding sodium chloride, magnesium chloride and potassium chloride salts to achieve physiologically acceptable levels of sodium, potassium, magnesium and chloride ions and osmotic pressure (Table 7).

[0136] Table 7

[0137] Composition and properties.

[0138]

[0139] The presented composition has the following physicochemical properties: osmotic pressure of 309 to 316 mOsmol / L, pH of 6.0 to 8.0, sodium ion content of 130 mOsmol / L, potassium ion content of 4.0 mOsmol / L, magnesium ion content of 1.13 mOsmol / L, and chloride ion content of 104.1 mOsmol / L, corresponding to physiologically acceptable values.

[0140] To conduct safety studies on the composition, solutions with different dilutions were prepared, with the following proportions of all components in the solutions: 50%, 75%, 100%, 125%, 150%, 175%, and 200% of the original values, i.e., Examples 5 to 11.

[0141] Example 5 (50%)

[0142] Add 900 ml of water to the reactor and dissolve 5.0 g of 1-deoxy-1-N-methylammonium-D-sorbitan succinate. Adjust the pH of the solution to 6.0-8.0 with sodium hydroxide. Then add 1.0 g inosine, 0.35 g methionine, and 0.13 g nicotinamide until completely dissolved. Next, add 2.85 g sodium chloride, 0.15 g potassium chloride, and 0.06 g magnesium chloride, and mix until completely dissolved. Finally, adjust the pH of the solution with sodium hydroxide if necessary. Then, bring the solution to a final volume of 1 L with water for injection. Filter the solution through a 0.22 μm sterile filter and fill it into glass vials, sealing each vial with a stopper. Perform final heat sterilization on the prepared vials at 121 ± 1°C for 15-20 minutes. The osmotic pressure of the resulting solution is 157 mOsm / L.

[0143] Example 6 (75%)

[0144] Add 900 ml of water to the reactor and dissolve 7.5 g of 1-deoxy-1-N-methylammonium-D-sorbitan succinate. Adjust the pH of the solution to 6.0-8.0 with sodium hydroxide. Then add 1.5 g inosine, 0.53 g methionine, and 0.19 g nicotinamide until completely dissolved. Next, add 4.28 g sodium chloride, 0.23 g potassium chloride, and 0.09 g magnesium chloride, and mix until completely dissolved. Finally, adjust the pH of the solution with sodium hydroxide if necessary. Then, bring the solution to a final volume of 1 L with water for injection. Filter the solution through a 0.22 μm sterile filter and fill it into glass vials, sealing each vial with a stopper. Perform final heat sterilization on the prepared vials at 121 ± 1°C for 15-20 minutes. The osmotic pressure of the resulting solution is 235 mOsm / L.

[0145] Example 7 (100%)

[0146] Add 900 ml of water to the reactor and dissolve 10.0 g of 1-deoxy-1-N-methylammonium-D-sorbitan succinate. Adjust the pH of the solution to 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. Next, add 5.70 g of sodium chloride, 0.30 g of potassium chloride, and 0.12 g of magnesium chloride, and mix until completely dissolved. Finally, adjust the pH of the solution with sodium hydroxide if necessary. Then, bring the solution to a final volume of 1 L with water for injection. Filter the solution through a 0.22 μm sterile filter and fill it into glass vials, sealing the vials with stoppers. Finally, perform final heat sterilization at 121 ± 1°C for 15-20 minutes. The osmotic pressure of the resulting solution is 313 mOsm / L.

[0147] Example 8 (125%)

[0148] Add 900 ml of water to the reactor and dissolve 12.5 g of 1-deoxy-1-N-methylammonium-D-sorbitan succinate. Adjust the pH of the solution to 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. Next, add 7.13 g of sodium chloride, 0.38 g of potassium chloride, and 0.15 g of magnesium chloride, and mix until completely dissolved. Finally, adjust the pH of the solution with sodium hydroxide if necessary. Then, bring the solution to a final volume of 1 L with water for injection. Filter the solution through a 0.22 μm sterile filter and fill it into glass vials, sealing the vials with stoppers. Finally, perform final heat sterilization at 121 ± 1°C for 15-20 minutes. The osmotic pressure of the resulting solution is 392 mOsm / L.

[0149] Example 9 (150%)

[0150] Add 900 ml of water to the reactor and dissolve 15.0 g of 1-deoxy-1-N-methylammonium-D-sorbitan succinate. Adjust the pH of the solution to 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. Next, add 8.55 g of sodium chloride, 0.45 g of potassium chloride, and 0.18 g of magnesium chloride, and mix until completely dissolved. Finally, adjust the pH of the solution with sodium hydroxide if necessary. Then, bring the solution to a final volume of 1 L with water for injection. Filter the solution through a 0.22 μm sterile filter and fill it into glass vials, sealing each vial with a stopper. Perform final heat sterilization on the prepared vials at 121 ± 1°C for 15-20 minutes. The osmotic pressure of the resulting solution is 470 mOsm / L.

[0151] Example 10 (175%)

[0152] Add 900 ml of water to the reactor and dissolve 17.5 g of 1-deoxy-1-N-methylammonium-D-sorbitan succinate. Adjust the pH of the solution to 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. Next, add 9.98 g of sodium chloride, 0.53 g of potassium chloride, and 0.21 g of magnesium chloride, and mix until completely dissolved. Finally, adjust the pH of the solution with sodium hydroxide if necessary. Then, bring the solution to a final volume of 1 L with water for injection. Filter the solution through a 0.22 μm sterile filter and fill it into glass vials, sealing the vials with stoppers. Finally, perform final heat sterilization at 121 ± 1°C for 15-20 minutes. The osmotic pressure of the resulting solution is 549 mOsm / L.

[0153] Example 11 (200%)

[0154] Add 900 ml of water to the reactor and dissolve 20 g of 1-deoxy-1-N-methylammonium-D-sorbitan succinate. Adjust the pH of the solution to 6.0-8.0 with sodium hydroxide. Then add 4.0 g inosine, 1.40 g methionine, and 0.50 g nicotinamide until completely dissolved. Next, add 11.40 g sodium chloride, 0.60 g potassium chloride, and 0.24 g magnesium chloride, and mix until completely dissolved. Finally, adjust the pH of the solution with sodium hydroxide if necessary. Then, bring the solution to a final volume of 1 L with water for injection. Filter the solution through a 0.22 μm sterile filter and fill it into glass vials, sealing each vial with a stopper. Perform final heat sterilization on the prepared vials at 121 ± 1°C for 15-20 minutes. The osmotic pressure of the resulting solution is 627 mOsm / L.

[0155] Experiment 4. Safety study of combinations with different component contents administered intravenously to rats for 14 days.

[0156] Safety studies were conducted in rats using compositions with different active ingredients (Examples 5 to 11) administered intravenously for 14 days. Based on the results of physiological and clinical laboratory studies, the survival rate of the animals, changes in the clinical manifestations of poisoning, and pathological changes in the animals were assessed.

[0157] Eight groups of animals were formed: - Control group. Intravenous infusion administration, n=10; - Group 1. Compositions with an application concentration of 50% of the active ingredient, n=10; - Group 2. Compositions with an application concentration of 75%, n=10; - Group 3. Compositions with 100% concentration of the applied ingredient, n=10; - Group 4. Compositions with an application ingredient content of 125%, n=10; - Group 5. Compositions with an application ingredient content of 150%, n=10; - Group 6. Compositions with an application ingredient content of 175%, n=10; - Group 7. Compositions with an application ingredient content of 200%, n=10; The various compositions and intravenous infusions were administered into the tail vein of rats at a dose equal to the maximum permissible volume for this species (50 ml / kg). Recommendation of EEC Board No. 33 of November 14, 2023, On Guidelines for Working with Laboratory (Experimental) Animals in Preclinical (Non-Clinical) Studies The drug was administered using an infusion pump at a rate of 1 ml / min. Clinical manifestations of poisoning were assessed (animal appearance, weight, behavioral response, mortality), and blood biochemistry was performed (alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP) activity, total bilirubin, glucose, total cholesterol, total protein), and clinical blood tests were performed (calculation of ESR, red blood cell count, white blood cell count, platelet count, hemoglobin concentration, hematocrit, and white blood cell differential). Cardiovascular parameters (ECG) and respiratory parameters (respiratory rate (RR)) were measured. Blood samples and physiological parameter measurements were performed after administration of the test composition.

[0158] Statistical data processing was performed using the Graph Pad Prism 8.0 statistical software package (USA). The Shapiro-Wilk test was used to check the normality of the distribution for each sample. Statistical comparisons between groups were performed using the Tukey test for normal distributions and the Mann-Whitney test for non-normal distributions. Differences with a null hypothesis probability less than 0.05 were considered significant.

[0159] A 14-day intravenous safety study of compositions with different active ingredients showed the following mortality rates: Group 7 (200% composition) had a mortality rate of 40%; Group 1 (50% composition) had a mortality rate of 30%; Groups 2 and 6 (75% and 175% compositions) had a mortality rate of 20%; and Groups 4 and 5 (125% and 150% compositions) had a mortality rate of 10%. The results are shown in Table 7. No rat deaths were recorded in the group receiving the 100% composition (Group 3).

[0160] At the end of the dosing period, groups 1, 2, 6, and 7 (50%, 75%, 175%, and 200% compositions) showed statistically significant changes in rat motor activity, prickly prickling, and body weight. Compared to the control group, groups 6 and 7 (175% and 200% compositions) showed a significant increase in heart rate (HR) to 582 ± 46 beats / min and a two-fold increase in respiratory rate (RR). Additionally, groups 1, 2, 6, and 7 showed behavioral changes towards depression.

[0161] Studies of blood biochemical parameters showed statistically significant increases in bilirubin and glucose levels in group 1 (50%) and elevated bilirubin levels in group 2 (75%). Significant decreases in white blood cells and red blood cells, as well as elevated ESR, were also recorded in group 1. Compared to the control group, in groups 6 and 7 (175% and 200% compositions), total blood protein significantly increased to 79 ± 5 g / L, ESR increased to 8.0 ± 1.3 mm / h, urinary protein increased 2.5-fold, and urine pH decreased 1.4-fold.

[0162] Table 8

[0163] Physiological and laboratory parameters of rats after intravenous administration of combinations of active ingredients in different proportions 14 days later, M±SEM, n=10.

[0164]

[0165] Based on the data obtained, it can be inferred that repeated intravenous administration of the hypotonic solution (157 mOsm / L) prepared according to Example 5 (Group 1, 50%) for 14 days led to hemolysis of erythrocytes in rats. Meanwhile, when the hypertonic compositions prepared according to Example 10 (175%, 549 mOsm / L) and Example 11 (200%, 627 mOsm / L) were administered, pathological changes indicating impaired electrolyte balance were recorded.

[0166] Considering the animal mortality rate and the results of hematological and biochemical indicators, it can be concluded that the compositions containing 50%, 175%, and 200% of the ingredients have a less than obvious pharmacological safety profile.

[0167] Safety studies have shown that intravenous administration of the composition at a dose of 50 ml / kg for 14 days at concentrations of 75% to 150% of the active ingredient did not cause any changes in the behavior, physiology, or laboratory parameters of the study animals.

[0168] Therefore, in this invention, experiments have confirmed that the solutions containing active ingredients and excipients prepared according to Examples 6 to 9 exhibit significant anti-diabetic effects; in addition, renal function recovery in diabetic nephropathy rats and significant wound healing effects in diabetic animals have also been observed.

[0169] Parenteral preparations obtained according to the methods claimed to be protected can be used for the long-term treatment of diabetes and its complications (specifically diabetic nephropathy and chronic skin lesions caused by soft tissue injury (diabetic ulcers)).

Claims

1. A pharmaceutical composition comprising an aqueous solution of 1-deoxy-1-N-methylammonium-D-sorbitol succinate, characterized in that, The pharmaceutical composition further contains inosine, nicotinamide, and methionine as active ingredients, in the following proportions (g / L): 1-Deoxy-1-N-methylammonium-D-sorbitan succinate 7.5-15.0 Inosine 1.50-3.0 Methionine 0.53-1.05 Nicotinamide 0.19-0.38 The maximum amount of water for injection is 1 liter.

2. The pharmaceutical composition according to claim 1 exhibits antidiabetic activity.

3. The pharmaceutical composition according to claim 1 exhibits renal protective activity.

4. The pharmaceutical composition according to claim 1 exhibits wound healing activity.

5. A method for preparing a parenteral preparation containing the composition according to claim 1 by the following steps: dissolving the components in water for injection, mixing, filtering the solution through a sterile filter, and filling it into a sealed container, characterized in that, 1-Deoxy-1-N-methylammonium-D-sorbitan succinate was dissolved in water for injection, and the solution was adjusted to pH 6.0 to 8.0 with sodium hydroxide. Inosine, methionine, and nicotinamide were added, along with sodium chloride, potassium chloride, and magnesium chloride, to bring the osmotic pressure to an osmotic level of 235 mOsm / l to 470 mOsm / l. The sealed container containing the resulting solution was then terminally sterilized.

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