Use of inosine in the preparation of a medicine for preventing and / or treating preeclampsia

Abstract

The application belongs to the field of biological medicine, and provides application of inosine in preparation of a drug for preventing and / or treating preeclampsia. The pathological state of preeclampsia is simulated by subcutaneous injection of a solution of nitroso-L-arginine methyl ester (125 mg / kg). From the 9th day of pregnancy, inosine is administered by gavage once a day (100 mg / kg), until the end of pregnancy. Compared with drugs such as labetalol, magnesium sulfate and other drugs with single treatment effect, the application proves that oral inosine can not only significantly improve hypertension and proteinuria caused by preeclampsia, repair placental function, promote intrauterine growth and development of the fetus, correct the imbalance of vascular and anti-angiogenic factors, but also reduce the damage to the kidneys and brain of pregnant women induced by preeclampsia, and exhibits a more comprehensive treatment effect, thereby providing a new therapeutic drug for preventing or treating preeclampsia.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine, specifically relating to the application of inosine in the preparation of drugs for the prevention and / or treatment of preeclampsia. Background Technology

[0002] Preeclampsia (PE) is a pregnancy-specific condition characterized by new-onset hypertension (systolic blood pressure ≥140 mmHg and / or diastolic blood pressure ≥90 mmHg) and proteinuria after 20 weeks of gestation, accompanied by symptoms such as headache, seizures, and visual disturbances. In severe cases, it can cause damage to tissues such as the liver and kidneys, leading to multiple organ failure and even maternal and infant death.

[0003] Currently, clinical treatment strategies for preeclampsia mainly include preventative and symptomatic treatment. Preventative treatment primarily involves oral low-dose aspirin to reduce the incidence of PE. Symptomatic treatment mainly involves oral antihypertensive drugs such as labetalol and nifedipine to lower blood pressure, or intravenous magnesium sulfate to relieve convulsions or spasms. However, existing treatments still have many adverse reactions, including gastrointestinal symptoms such as nausea, vomiting, and upper abdominal pain from oral aspirin, and headache, nausea, and arrhythmia 24 hours after oral labetalol administration. These adverse reactions seriously affect the pregnant woman's pregnancy. Moreover, if the condition cannot be controlled by medication, early termination of pregnancy is the only way to prevent the condition from worsening and save the lives of both mother and baby. However, this increases the risk of infection for the mother, fetal growth retardation, perinatal mortality, and neonatal asphyxia. Therefore, there is an urgent need to find a treatment drug with significant efficacy and good safety.

[0004] Inosine (INO) is a nucleoside composed of adenine and ribose, and is an essential metabolite for purine biosynthesis and degradation. Inosine is also a naturally occurring purine nucleoside formed from the breakdown of adenosine and is considered an inert adenosine metabolite. Inosine function is mediated in a receptor-dependent or receptor-independent manner. The receptor-mediated function of inosine is thought to be related to members of the adenosine receptor family, including G protein-coupled receptors A1, A2A, A2B, and A3. Previous studies have shown that inosine has anti-inflammatory and immunomodulatory effects, and has potential therapeutic effects on systemic inflammation induced by preeclampsia (PE). However, there are currently no studies or reports on the effects of inosine on PE, nor is there evidence that it can regulate placental function or improve symptoms such as PE-related hypertensive renal injury and brain injury. Therefore, this invention aims to protect the important role of inosine in the prevention and treatment of preeclampsia. Summary of the Invention

[0005] To overcome the deficiencies in the prior art, this invention provides the use of inosine in the preparation of drugs for the prevention and / or treatment of preeclampsia. To achieve the above objective, this invention adopts the following technical solution:

[0006] The first aspect of this invention provides the use of inosine in the preparation of medicaments for the prevention and / or treatment of preeclampsia.

[0007] Furthermore, the drug is used to reduce hypertension caused by preeclampsia.

[0008] Furthermore, the drug is used to improve proteinuria caused by preeclampsia.

[0009] Furthermore, the drug is used to improve placental dysfunction caused by preeclampsia.

[0010] Furthermore, the drug is used to improve intrauterine growth restriction in fetuses caused by preeclampsia.

[0011] Furthermore, the drug is used to improve the imbalance between angiogenic factors and anti-angiogenic factors.

[0012] Furthermore, the angiogenic factors include vascular endothelial growth factor (VEGF) and placental growth factor (PlGF), and the anti-angiogenic factors include soluble fms-like tyrosine kinase-1 (sFlt-1).

[0013] Furthermore, the drug is used to improve preeclampsia-induced kidney injury, and to reduce kidney injury including glomerular endothelial cell damage, mesangial cell proliferation and tubulointerstitial inflammation. The drug can reduce the expression of kidney injury molecule-1 (KIM-1) and neutrophil gelatinase-associated lipotransferase (NGAL) in the kidney, and increase the expression of podocyte protein (podocin).

[0014] Furthermore, the drug is used to reverse preeclampsia-induced brain damage, including blood-brain barrier damage. The drug can protect the blood-brain barrier and reduce the expression of central nervous system-specific protein (S100B) and glial fibrillary acidic protein (GFAP) in the circulatory system.

[0015] A second aspect of the present invention provides a medicament for the prevention and / or treatment of preeclampsia, said medicament containing an effective amount of inosine or a pharmaceutically acceptable salt thereof as the main active ingredient.

[0016] Furthermore, the drug also includes pharmaceutically acceptable carriers or excipients.

[0017] Furthermore, the dosage form of the drug is tablets, capsules, oral liquids, granules, powders, pills, powders, suspensions, or injections.

[0018] Furthermore, the drug is administered orally or by injection.

[0019] The present invention has the following beneficial effects:

[0020] This invention simulates the pathological state of preeclampsia by subcutaneously injecting a solution of 125 mg / kg of nitroso-L-arginine methyl ester. Starting from the 9th day of pregnancy, inosine (100 mg / kg) is administered by gavage once daily until the end of pregnancy. Compared to drugs with single therapeutic effects, such as the antihypertensive drug labetalol and the antispasmodic drug magnesium sulfate, this invention demonstrates that oral inosine can significantly reduce hypertension and proteinuria caused by preeclampsia, repair placental function, promote intrauterine fetal growth and development, correct the imbalance of vascular and anti-angiogenic factors, and alleviate kidney and brain damage induced by preeclampsia in pregnant women, exhibiting a more comprehensive therapeutic effect and providing a new therapeutic drug for the prevention or treatment of preeclampsia. Attached Figure Description

[0021] Figure 1 Comparison of blood pressure in each group of mice in Example 1 (n=6);

[0022] Figure 2 For the comparison of urinary protein content in each group of mice in Example 2 (n=6);

[0023] Figure 3 For the comparison of placental weight in each group of mice in Example 3 (n=6);

[0024] Figure 4 For the comparison of placental regions in each group of mice in Example 4 (n=4);

[0025] Figure 5 For the comparison of fetal weights in different groups of mice in Example 5 (n=6);

[0026] Figure 6 For the comparison of the levels of angiogenesis and anti-angiogenic factors in the serum and placenta of mice in each group in Example 6 (n=6);

[0027] Figure 7 For the comparison of kidney injury indicators in each group of mice in Example 7 (n=6);

[0028] Figure 8 This is a comparison of brain injury indicators in the brains of mice in different groups in Example 8 (n=6). Detailed Implementation

[0029] To make the above-mentioned objects, features, and advantages of the present invention more apparent and understandable, specific embodiments of the present invention will be described in detail below with reference to the accompanying drawings, but this should not be construed as limiting the scope of the present invention. Unless otherwise specified, the reagents, methods, and equipment used in the present invention are conventional reagents, methods, and equipment in this technical field.

[0030] Unless otherwise specified, all reagents and materials used in the following examples are commercially available.

[0031] I. Preparation of experimental reagents, materials and animals.

[0032] Drug ingredients:

[0033] Inosine, chemical formula C 10 H 12 N4O5, with a molecular weight of 268.23, is a nucleoside compound formed by the combination of hypoxanthine and ribose.

[0034] Experimental basis:

[0035] 1. Laboratory animals

[0036] Male and female C57BL / 6 mice (8-10 weeks old, weighing 18-20g) were purchased from SPF (Beijing) Biotechnology Co., Ltd. (Beijing, China). Mice were housed under standard light and dark cycles (12h:12h) with free access to food and water. One week after acclimatization, healthy male mice were mated with female mice at a 1:2 ratio overnight. Female mice with sperm plugs were considered pregnant, and this day was defined as the gestation day (GD0). All animal studies were conducted in accordance with protocols and guidelines approved by the Animal Ethics Committee of the Laboratory Animal Center of Southern Medical University.

[0037] 2. Experimental Reagents

[0038]

[0039]

[0040] 3. Drug preparation

[0041] 3.1 N-nitro-L-arginine methyl ester (L-NAME) solution: Weigh 125 mg of L-NAME powder and dilute to 10 mL with physiological saline.

[0042] 3.2 Inosine solution: Weigh 10 mg of inosine powder and dilute to 1 mL with physiological saline.

[0043] 4. Establishment and evaluation of the preeclampsia model

[0044] 4.1 Establishment of the preeclampsia model

[0045] Starting from GD9, pregnant mice were subcutaneously injected daily with 125 mg / kg of nitroso-L-arginine methyl ester (L-NAME) solution for 9 days.

[0046] 4.2 Evaluation of the preeclampsia model

[0047] 4.2.1 Blood Pressure

[0048] The blood pressure of mice in the preeclampsia model group was significantly higher than that of mice in the normal pregnancy group, which indicates that the model was successfully established.

[0049] 4.2.2 Proteinuria

[0050] The urinary protein content in the preeclampsia model group was significantly higher than that in the normal pregnancy group, indicating that the model was successfully established.

[0051] 5. Real-time quantitative PCR

[0052] 5.1 Reverse transcription of RNA

[0053] Prepare a total reaction volume of 20 μL according to the instructions of the reverse transcription kit. Add the required amount of RNA and 4 μL of HiScript IIQ RT SuperMix for qPCR reagent to each 200 μL PCR tube, and then add ddH2O to a final volume of 20 μL. Place the PCR tubes in a PCR amplification instrument and perform the following operations: 50°C for 15 min; 85°C for 5 s; 4°C for ∞. After the reaction, centrifuge, label the time, and store the obtained cDNA at -80°C for later use.

[0054] 5.2 Real-time quantitative PCR (RT-qPCR)

[0055] All reverse transcription products were subjected to real-time quantitative PCR, and the reaction system is shown in Table 1. The primer sequences of relevant genes are shown in Table 2. A two-step PCR amplification system was used, and the method settings are shown in Table 3.

[0056] Table 1. PCR Reaction System

[0057]

[0058]

[0059] Table 2 Primer sequences

[0060]

[0061] Table 3 RT-qPCR reaction procedure

[0062]

[0063] Data processing: Using Gapdh as an internal reference gene, the relative expression level of the target gene was calculated using the 2-ΔΔCt method.

[0064] II. Implementation Examples

[0065] Example 1: Inosine can significantly reduce hypertension caused by preeclampsia.

[0066] Hypertension is the most critical clinical indicator of preeclampsia. To investigate the effect of inosine on blood pressure in preeclampsia mice, this study randomly divided mice into three groups: normal pregnancy group (NP, n=6), preeclampsia model group (PE, n=6), and inosine group (INO, n=6). Mice in the inosine intervention group were administered inosine (100 mg / kg) by gavage starting from day 9 of gestation (GD9), while mice in the normal pregnancy group and preeclampsia model group were administered the same volume of 0.9% saline by gavage. This was done once daily for 9 days. The results are as follows: Figure 1 The results showed that the blood pressure of mice in the preeclampsia model group was significantly higher than that in the normal pregnancy group and the inosine group after GD9, indicating that inosine intervention can significantly reduce hypertension (or increase blood pressure) in preeclampsia mice. (a represents the comparison of systolic blood pressure between the NP group and the PE group on the corresponding date; aaa represents P<0.001; b represents the comparison of systolic blood pressure between the INO group and the PE group on the corresponding date; bbb represents P<0.001; c represents the comparison of systolic blood pressure between the NP group and the INO group on the corresponding date; cc represents P<0.01, ccc represents P<0.001.)

[0067] Example 2: Inosine can significantly improve proteinuria caused by preeclampsia.

[0068] Proteinuria is one of the key clinical indicators of preeclampsia. In this study, subcutaneous injection of nitroso-L-arginine methyl ester (L-NAME) solution was used to simulate the disease state of natural onset of preeclampsia, and the effect of inosine on urinary protein in preeclamptic mice was investigated. Results are shown below. Figure 2 The results showed that the urinary protein content of mice in the preeclampsia model group was significantly higher than that in the normal pregnancy group, and the urinary protein level of mice in the model group was significantly reduced after inosine intervention, indicating that inosine can significantly reduce the proteinuria level of mice in the preeclampsia model group.

[0069] Example 3: Inosine significantly increases placental weight in preeclampsia mice.

[0070] The placenta is rich in blood vessels and nutrients, providing essential substances for fetal growth and development. A comparison of placental weight among different groups of mice yielded the following results: Figure 3 The results showed that, compared with the mice in the normal pregnancy group, the placental weight of the mice in the preeclampsia model group was significantly reduced, and the reduction in placental weight was significantly reversed after inosine intervention, indicating that inosine is beneficial to the growth and development of the placenta in preeclampsia mice.

[0071] Example 4: Inosine can improve placental dysfunction.

[0072] The placenta is mainly divided into three parts: the decidual zone, the junctional zone, and the labyrinthine zone. The placental labyrinthine zone is primarily involved in the synthesis of hormones and cytokines (such as angiogenic factors and anti-angiogenic factors), maintaining normal placental function and fetal growth. The placental junctional zone is mainly responsible for the exchange of oxygen, nutrients, and metabolic waste. A comparison of the placental regions in different groups of mice yielded the following results: Figure 4 As shown, the ratio of the labyrinthine region to the junctional region in the placenta of PE group mice was significantly higher than that in NP group mice, suggesting pathological damage to the placental structure in PE mice. Inosine administration reversed this phenomenon, indicating that inosine can repair the pathological damage to the placental tissue of PE mice and improve placental dysfunction.

[0073] Example 5: Inosine can improve intrauterine growth restriction in preeclampsia mice

[0074] Fetal growth restriction is one of the main complications of preeclampsia. A comparison of fetal weights in different groups of mice yielded the following results: Figure 5 The results showed that compared with mice in the normal pregnancy group, the fetal weight of mice in the preeclampsia model group was significantly reduced, and the intervention of inosine could significantly reverse the reduction in fetal weight, indicating that inosine can promote the growth and development of fetuses in PE mice in utero.

[0075] Example 6: Inosine can improve the imbalance between angiogenic and anti-angiogenic factors.

[0076] During the development of preeclampsia, abnormalities in the vascular system lead to an imbalance between angiogenesis and anti-angiogenic factors, affecting the remodeling of the uterine spiral arteries. A comparison of angiogenesis and anti-angiogenic factors in the serum and placenta of different groups of mice, such as... Figure 6 As shown, compared with the normal pregnancy group, the serum and placenta of mice in the preeclampsia model group showed a significant increase in soluble FMS-like tyrosine kinase-1 (sFlt-1, an anti-angiogenic factor), and a significant decrease in angiogenic factor (VEGF) and placental growth factor (PIGF). Furthermore, the sFlt-1 / PIGF ratio, a disease marker of PE, was significantly increased. Inosine intervention could reverse these phenomena, indicating that inosine can improve the imbalance between angiogenic and anti-angiogenic factors.

[0077] Example 7: Inosine can improve kidney damage induced by preeclampsia.

[0078] Systemic endothelial dysfunction characteristic of preeclampsia manifests in the kidneys as glomerular endothelial hyperplasia and proteinuria. The systemic inflammatory response and oxidative stress triggered by preeclampsia further exacerbate renal pathological damage, leading to a series of pathological changes such as glomerular endothelial cell damage, mesangial cell proliferation, and tubulointerstitial inflammation. Ultimately, this can result in acute kidney injury or even chronic kidney disease, seriously endangering the long-term health of pregnant women. KIM-1 is highly expressed on the surface of renal tubular epithelial cells and released into the urine during renal tubular injury, serving as an early and sensitive biomarker for renal tubular damage. Its elevated levels precede changes in traditional indicators such as serum creatinine, promptly reflecting the degree of renal tubular injury. NGAL, mainly produced by neutrophils, is also synthesized and released in large quantities by renal tubular epithelial cells and glomerular mesangial cells under stimuli such as renal ischemia and toxic injury. Its concentration in serum and urine is significantly increased, making it crucial for the early diagnosis and assessment of acute kidney injury. Podocin is a protein specific to podocytes and plays an important role in maintaining the normal structure and function of podocytes. When podocytes are damaged, podocin expression decreases or becomes abnormal. Comparison of kidney injury markers among different groups of mice yielded the following results: Figure 7 The results showed that, compared with the preeclampsia model group, the expression of KIM-1 and NGAL in preeclampsia pregnant mice was significantly downregulated and the expression of Podocin was significantly upregulated after inosine intervention, indicating that inosine can improve preeclampsia-induced kidney injury.

[0079] Example 8: Inosine can reverse preeclampsia-induced brain damage

[0080] Brain complications arising from preeclampsia (such as eclampsia, cerebral edema, and cerebral hemorrhage) are a leading cause of maternal mortality. In most cases, eclampsia precedes hypertension and is usually asymptomatic. Central nervous system-specific protein (S100B) and glial fibrillary acidic protein (GFAP) are currently being evaluated as biomarkers of brain injury. Comparison of brain injury markers in different groups of mice yielded the following results: Figure 8 As shown, compared with the preeclampsia model group, the expression of S100b and Gfap in the brains of preeclampsia pregnant mice was significantly reduced after inosine intervention, indicating that inosine can reverse preeclampsia-induced brain damage.

[0081] The above embodiments further demonstrate that oral inosine can significantly alleviate hypertension and proteinuria caused by preeclampsia, repair placental pathological tissue structure and functional changes, improve fetal intrauterine growth and development, correct the imbalance between vascular and anti-angiogenic factors, and reverse preeclampsia-induced kidney and brain damage, proving that inosine has a good therapeutic effect on preeclampsia.

[0082] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the invention patent. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the protection scope of the present invention. Therefore, the protection scope of this invention patent should be determined by the appended claims.

Claims

1. The use of inosine in the preparation of drugs for the prevention and / or treatment of preeclampsia.

2. The application according to claim 1, characterized in that, The drug contains an effective amount of inosine or a pharmaceutically acceptable salt thereof as the main active ingredient.

3. The application according to claim 2, characterized in that, The drug also includes pharmaceutically acceptable carriers or excipients.

4. The application according to claim 1, characterized in that, The dosage form of the drug is tablets, capsules, oral liquids, granules, powders, pills, powders, suspensions, or injections.

5. The application according to claim 4, characterized in that, The drug can be administered orally or by injection.

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