Use of d-phenylalanine for the preparation of a medicament for the prevention and / or treatment of renal impairment

Abstract

This invention belongs to the field of biomedical technology, specifically relating to the application of dexphosserine in the preparation of drugs for the prevention and / or treatment of kidney injury. The dexphosserine can alleviate glycerol-induced acute kidney injury in mouse models, significantly reduce serum creatinine and urea nitrogen levels in mice with acute kidney injury, and improve kidney pathological damage, thus having a therapeutic effect on acute kidney injury and broad application prospects.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to the application of dexphosserine in the preparation of drugs for the prevention and / or treatment of kidney injury. Background Technology

[0002] Acute kidney injury (AKI) is a clinical syndrome caused by a rapid decline in kidney function due to various etiologies. It can occur in individuals without a prior history of kidney disease as well as in patients with chronic kidney disease. Approximately 5% of hospitalized patients develop AKI, with an incidence rate as high as 30% in intensive care units. Despite increasing attention from the nephrology community, there is currently no specific treatment strategy for AKI, and it has a high mortality rate, making it a critical and acute kidney disease. Therefore, identifying potential drugs for treating AKI is of great clinical significance. Glycerol, also known as glycerol, is a lipid-soluble small molecule. Injection of glycerol can cause muscle breakdown and hemolysis, releasing large amounts of hemoglobin and myoglobin. Myoglobin can lead to renal vasoconstriction, causing constriction of both the afferent arterioles of the glomeruli, resulting in decreased renal blood flow and glomerular filtration rate, leading to renal ischemia and damage to renal tubular function. Because the renal tubules cannot reabsorb the glomeruli, casts form and block the tubules, causing pathological damage to the renal tubules and interstitium. Meanwhile, both myoglobin and hemoglobin can decompose into methemoglobin, which has a direct toxic effect on renal tubules. Various drugs are available for treating acute kidney injury. For example, invention patent CN113663082B discloses that acetylcysteine-stabilized gold nanoclusters can be used to treat acute kidney injury, but the preparation method of gold nanoclusters is complex; invention patent CN109602755A discloses that eleutheroside B has the effect of treating acute kidney injury, but it is used to treat drug-induced acute kidney injury.

[0003] Dexfosfoserine (O-Phospho-L-serine) is a direct precursor in the synthesis of L-serine and an agonist of group III mGluRs (mGluR4, mGluR6, mGluR7, and mGluR8), while also acting as an antagonist of mGluR1 and mGluR2. It is used for nutritional supplementation in cases of excessive fatigue. Currently, there are no reports on the use of dexfosfoserine in treating glycerol-induced acute kidney injury.

[0004] During their research, the inventors unexpectedly discovered that dexphosserine can significantly reduce serum creatinine and blood urea nitrogen levels in mice with acute kidney injury, and can also improve kidney pathological damage, thus having a therapeutic effect on acute kidney injury and broad application prospects. Summary of the Invention

[0005] The purpose of this invention is to provide the use of dexphosphoserine in the preparation of drugs for the prevention and / or treatment of kidney injury, wherein the structural formula of dexphosphoserine is shown in formula (I):

[0006]

[0007] Preferably, the kidney injury is acute kidney injury.

[0008] Preferably, the acute kidney injury is induced by glycerol.

[0009] Preferably, a pharmaceutical formulation is prepared by adding dexphosphoserine as the active compound and other excipients acceptable to pharmaceutical preparations.

[0010] Preferably, the excipients include one or more carriers that function as excipients, stabilizers, antioxidants, colorants, diluents, and sustained-release agents.

[0011] Preferably, the pharmaceutical preparation is any one of the following dosage forms: tablets, capsules, suspensions, emulsions, powder for injection, and injections.

[0012] Preferably, the drug for preventing and / or treating kidney injury has one or more of the following functions:

[0013] (1) Reduce serum creatinine levels in mice with acute kidney injury;

[0014] (2) Reduce the level of blood urea nitrogen in the serum of mice with acute kidney injury.

[0015] The beneficial effects of this invention are: This invention provides the application of dexphosserine in the preparation of drugs for the prevention and / or treatment of kidney injury. The dexphosserine can alleviate glycerol-induced acute kidney injury in mouse models, significantly reduce serum creatinine and urea nitrogen levels in mice with acute kidney injury, and improve kidney pathological damage. It has the effect of treating acute kidney injury and has broad application prospects. Attached Figure Description

[0016] Figure 1 Schematic diagram of serum creatinine levels in mice of each group;

[0017] Figure 2 Schematic diagram of serum urea nitrogen levels in mice of each group;

[0018] Figure 3 HE staining results of mouse kidneys in each group;

[0019] Figure 4 Results of renal tubular injury scores in each group of mice. Detailed Implementation

[0020] In order to make the objectives, technical solutions and advantages of the present invention clearer and more understandable, the following embodiments are used to further elaborate on the present invention. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention. Unless otherwise specified, the experimental methods in the following embodiments are all conventional methods. Unless otherwise specified, the reagent materials used in the following embodiments are all purchased commercially.

[0021] In the present invention, the structural formula of the right phosphoserine is as follows:

[0022]

[0023] Example 1: Effect of right phosphoserine in preparing a drug for treating kidney injury

[0024] 1. Experimental reagents and materials

[0025] Glycerol (99%, G810575, Shanghai Macklin Biochemical Co., Ltd.); 4% paraformaldehyde fixative (P885233, Shanghai Macklin Biochemical Co., Ltd.); ethanol (E809061, Shanghai Macklin Biochemical Co., Ltd.); xylene (X820585, Shanghai Macklin Biochemical Co., Ltd.); hematoxylin-eosin (HE) staining kit (G1120, Beijing Solarbio Science & Technology Co., Ltd.); creatinine assay kit (C011-2-1, Nanjing Bioengineering Institute); urea nitrogen test kit (C013-2-1, Nanjing Bioengineering Institute); 24 male C57BL / 6 mice at 8-12 weeks of age, provided by Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences, with the license number SCXK(Gan)-2020-0002. After being adaptively fed for one week, the animals were given food and free drinking water, and then grouped for experiments.

[0026] Before modeling with glycerol, 99% glycerol was diluted into a 50% glycerol solution with normal saline in a centrifuge tube, and the centrifuge tube was placed on a shaker. It was vibrated for 15 min to mix the solution, and it was prepared and used immediately.

[0027] 2. Experimental grouping and administration methods

[0028] After 1 week of adaptive feeding, 24 male C57BL / 6 mice were randomly divided into a blank control (Control) group, a glycerol model (Glycerol) group, a low-dose right phosphoserine administration (Glycerol+60 mg / kg PS) group, and a high-dose right phosphoserine administration (Glycerol+120 mg / kg PS) group, with 6 mice in each group. The specific grouping and administration methods are as follows:

[0029] 1) Control group: Intraperitoneal injection of physiological saline once a day for 6 days; after the intraperitoneal injection on the 6th day, the mice were deprived of water but allowed to eat for 18 hours. Intraperitoneal injection of physiological saline continued, and 30 minutes after the injection, half of the dose of physiological saline was injected into the muscles of both hind limbs of the mice at a dose of 10 mL / kg. The mice were deprived of water but allowed to eat for 8 hours before being allowed to eat freely.

[0030] 2) Glycerol group: Intraperitoneal injection of physiological saline once a day for 6 days; after the intraperitoneal injection on the 6th day, the mice were deprived of water but not food for 18 hours. Intraperitoneal injection of physiological saline continued, and 30 minutes after the injection, half of the 50% glycerol solution was injected into the muscles of both hind limbs of the mice at a dose of 10 mL / kg. The mice were deprived of water but not food for 8 hours before being allowed to eat freely.

[0031] 3) Glycerol + 60 mg / kg PS group: Mice were injected intraperitoneally with 60 mg / kg dexphosserine (PS) once daily for 6 days. After the intraperitoneal injection on the 6th day, the mice were deprived of water but allowed to eat for 18 hours. Then, the mice were injected intraperitoneally with 60 mg / kg PS. 30 minutes after the injection, half of the 50% glycerol solution was injected into the muscles of both hind limbs at a dose of 10 mL / kg. The mice were then deprived of water but allowed to eat for another 8 hours before being allowed to eat freely.

[0032] 4) Glycerol + 120 mg / kg PS group: Mice were injected intraperitoneally with 120 mg / kg PS once a day for 6 days. After the intraperitoneal injection on the 6th day, the mice were deprived of water but allowed to eat for 18 hours. The intraperitoneal injection of 120 mg / kg PS was continued. 30 minutes after the injection, half of the dose of 50% glycerol solution was injected into the muscles of both hind limbs of the mice at a dose of 10 mL / kg. The mice were deprived of water but allowed to eat for 8 hours before being allowed to eat freely.

[0033] 3. Kidney function test

[0034] After treatment according to the above grouping and administration methods, and continued feeding for 48 hours, blood was collected from the inner canthus of the eye. The blood was collected into centrifuge tubes, left to stand at room temperature for 1 hour, and then centrifuged at 2000g, 4℃ for 10 minutes. The supernatant obtained was the serum sample. The creatinine and urea nitrogen contents of the mouse serum were analyzed using a creatinine assay kit and a blood urea nitrogen test kit.

[0035] After blood collection, the mice were euthanized, and their kidneys were obtained and rinsed 2-3 times with physiological saline to remove blood. The left kidney was fixed in 4% paraformaldehyde for subsequent HE staining, and the right kidney was wrapped in aluminum foil and stored at -80°C.

[0036] 4. HE staining of kidney tissue

[0037] 1) Fixation: The left kidney of the mouse was placed in 4% paraformaldehyde fixative and fixed for 24 hours.

[0038] 2) Washing: Rinse the fixed tissue block with running water for 4-6 hours until there is no formaldehyde odor.

[0039] 3) Dehydration: Place the washed sample in 50%, 70%, and 80% ethanol solutions for 90 min each, then in 95% ethanol solution for 90 min twice, and finally in 100% ethanol solution for 60-90 min three times.

[0040] 4) Transparency: Place in an ethanol:xylene (volume ratio 1:1) solution for 20 min; then dewax with xylene I and II solutions for 20 min each.

[0041] 5) Wax impregnation: This must be carried out in a constant temperature chamber, with the temperature adjusted to be 3°C above the melting point of the paraffin. Paraffin I (45-50°C) 60 min; Paraffin II (56-58°C) 60 min; Paraffin III (56-58°C) 60 min;

[0042] 6) Embedding: Place in a paraffin embedding machine for embedding and use.

[0043] 7) Sectioning: Place the embedded wax block in a -20℃ freezer for 1 hour, then section it to a thickness of about 5μm.

[0044] 8) Display slides: Paraffin sections are heated in a water bath at 45-50℃ and then mounted on glass slides pre-treated with poly-L-lysine.

[0045] 9) Baking the slices: Place the prepared slices in a 45℃ oven and bake for 2-4 hours.

[0046] 10) Staining: Perform staining according to the instructions of the hematoxylin and eosin (HE) staining kit, and observe the HE staining results, as follows: Figure 3 As shown, from left to right, the control group, the Glycerol group, the Glycerol + 60 mg / kg PS group, and the Glycerol + 120 mg / kg PS group are shown. From top to bottom, the HE staining results are magnified 200 and 400 times.

[0047] 11) The scoring rules for renal tubules are as follows: 0 points for no abnormalities; 1 point for obvious dilation and flattened cells; 1 point for brush border damage; 1 point for cell shedding and necrosis in the renal tubule lumen, but without forming casts or cell fragments; 2 points for casts; 2 points for brush border shedding.

[0048] 5. Statistical Analysis

[0049] Experimental data were statistically analyzed using SPSS 23.0 software. Data are expressed as (x±s). One-way ANOVA and LSD-t method were used for pairwise comparisons between groups. P < 0.05 was considered statistically significant. All statistical results were plotted using GraphPad Prism 8 software.

[0050] 6. Experimental Results

[0051] Serum creatinine and blood urea nitrogen are important indicators for evaluating kidney function and are used to detect whether there is kidney damage and the severity of the damage. Figure 1 , Figure 2 The results showed that 60 mg / kg and 120 mg / kg of dexphosserine significantly reduced serum creatinine and blood urea nitrogen levels in mice with acute kidney injury, indicating that 60 mg / kg and 120 mg / kg of dexphosserine can alleviate glycerol-induced acute kidney injury.

[0052] HE staining results of mouse kidneys in each group are as follows: Figure 3 As shown in the figure, the renal tubular injury scores of each group of mice are as follows: Figure 4 As shown, 60 mg / kg of dexphosserine can effectively improve kidney pathological damage, but the effect of 120 mg / kg of dexphosserine is not obvious.

[0053] In summary, this invention provides the application of dexphosphoserine in the treatment of kidney injury. The dexphosphoserine can significantly reduce the serum creatinine and urea nitrogen levels in mice with acute kidney injury, and can also improve kidney pathological damage, thus having a therapeutic effect on acute kidney injury and broad application prospects.

Claims

1. The use of dexphosphoserine in the preparation of drugs for the prevention and / or treatment of kidney injury, wherein the structural formula of dexphosphoserine is shown in Formula (I): ，（Ⅰ） The kidney injury described is acute kidney injury, which is induced by glycerol.

2. The application as described in claim 1, characterized in that, A pharmaceutical formulation is prepared by adding appropriate excipients to a drug formulation, using dexphosphoserine as the active compound.

3. The application as described in claim 2, characterized in that, The excipients include one or more carriers that function as excipients, stabilizers, antioxidants, colorants, diluents, and sustained-release agents.

4. The application as described in claim 3, characterized in that, The pharmaceutical preparation is any one of the following dosage forms: tablets, capsules, suspensions, emulsions, powder for injection, and injections.

5. The application as described in any one of claims 1-4, characterized in that, The aforementioned drugs for the prevention and / or treatment of kidney injury have one or more of the following functions: (1) Reduce serum creatinine levels in mice with acute kidney injury; (2) Reduce the level of blood urea nitrogen in the serum of mice with acute kidney injury.

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