Application of 17 alpha-estradiol in vascular aging related diseases

Abstract

The invention discloses an application of 17 alpha-estradiol in diseases related to vascular aging. The 17alpha-estradiol disclosed by the invention is capable of remarkably improving the vascular endothelial cell activity of the aged male animal and effectively improving the aging function of the aged male animal for the first time. The discovery shows that 17alpha-estradiol can directly act on endothelial dysfunction, which is a core link of vascular senescence, and provides a treatment choice with a brand new mechanism for preventing and treating vascular senescence related diseases such as arteriosclerosis, senile hypertension, coronary heart disease, ischemic cerebral apoplexy, vascular cognitive impairment and peripheral artery disease; due to the effectiveness in an old male animal model, the compound is particularly suitable for blood vessel health maintenance and disease prevention and treatment of middle-aged and old men, and the aim of delaying blood vessel aging is expected to be achieved.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to the application of 17α-estradiol in vascular aging-related diseases. Background Technology

[0002] Vascular aging is a core aspect of overall aging, driven by genetic, lifestyle, and environmental factors, and manifests as a progressive decline in vascular structure and function. Its characteristics include significantly decreased vascular elasticity, exacerbated sclerosis, severe endothelial dysfunction, abnormal luminal remodeling, reduced microcirculation network, and increased risk of thrombosis. The pathological consequences are extensive and far-reaching: reduced elastic fibers in large arteries, abnormal collagen cross-linking, and calcification lead to increased stiffness, causing systolic hypertension and increased pulse pressure (often >60 mmHg); endothelial cell dysfunction results in a sharp decrease in nitric oxide (NO) bioavailability, increased secretion of vasoconstrictors, pro-inflammatory and procoagulant factors, weakening vasodilatory capacity, exacerbating inflammatory responses and coagulation tendency, accelerating the process of atherosclerosis, promoting lipid deposition and vulnerable plaque formation, ultimately leading to serious events such as coronary heart disease and myocardial infarction; and pathological osteoblastic transformation occurs in the smooth muscle cells of the vascular media. Disorders of calcium and phosphorus metabolism further exacerbate calcification, stiffness, and vascular fragility; degeneration of arterioles and capillaries leads to microcirculatory disturbances, clinically manifesting as signs of skin aging, cold extremities, decreased glomerular filtration rate, and an increased risk of cognitive impairment; arteriosclerosis, luminal stenosis, and microcirculatory failure collectively result in insufficient perfusion of critical organs such as the heart, brain, and kidneys, inducing angina pectoris, ischemic stroke, and progressive decline in renal function; weakened endothelial anticoagulation function combined with hemodynamic abnormalities (such as slowed flow velocity) significantly increases the incidence of thromboembolic events such as deep vein thrombosis and pulmonary embolism. Most importantly, vascular aging is a common pathological basis for neurodegenerative diseases such as Alzheimer's disease, cerebral small vessel disease, and Parkinson's disease, accelerating neuropathy through mechanisms such as disrupting the integrity of the blood-brain barrier and inducing chronic endothelial inflammation. Therefore, delaying vascular aging is a key entry point for maintaining overall health and preventing various age-related diseases.

[0003] Current treatment strategies for vascular aging and related conditions mainly rely on traditional drugs and some emerging therapies. The core objectives are to improve vascular structure and function and control cardiovascular risk factors. Traditional drugs include: (1) Renin-angiotensin system inhibitors (ACEIs such as enalapril, ARBs such as valsartan). These drugs reduce vascular remodeling and improve endothelial function by inhibiting the effects of abnormally activated angiotensin, and are suitable for patients with hypertension and coronary heart disease. (2) Calcium channel blockers (such as amlodipine) can dilate blood vessels and improve vascular compliance, and are especially suitable for elderly patients with systolic hypertension. (3) Lipid-lowering drugs such as statins (such as atorvastatin) can lower low-density lipoprotein cholesterol (LDL-C) and stabilize atherosclerotic plaques; PCSK9 inhibitors (such as evolocumab) can provide more effective LDL-C reduction and delay the progression of atherosclerosis. (4) Antiplatelet drugs (such as aspirin and clopidogrel) can inhibit platelet aggregation and reduce the risk of thrombotic events. (5) Vasoactive drugs (such as nitrates and prostacyclin analogs) can improve endothelial function and microcirculation. Emerging drugs such as SGLT2 inhibitors (such as dapagliflozin) and GLP-1 receptor agonists (such as smegglutide and liraglutide) have been shown to have multiple cardiovascular protective effects, including improving vascular endothelium and inhibiting inflammation, while controlling blood sugar, and are particularly suitable for patients with diabetes. Metformin reduces the risk of cardiovascular events by activating the AMPK pathway. In addition, drugs targeting senescent cells (such as 25-hydroxycholesterol), mitochondrial antioxidants (such as MitoQ), and targeted therapies under development (such as UDP-003 and biomimetic nanoparticles) are also being explored.

[0004] However, existing therapies still have significant limitations: (1) Most are symptomatic treatments: Most drugs mainly target a single risk factor (such as hypertension, hyperlipidemia, hyperglycemia) or complication (such as thrombosis), making it difficult to comprehensively intervene in the core biological processes of vascular aging (such as cellular senescence, chronic inflammation, calcification, and microcirculatory degeneration). (2) Insufficient targeting: There is a lack of drugs that can specifically act on key mechanisms of vascular aging (such as improving elasticity, reversing endothelial dysfunction, inhibiting pathological calcification, and promoting microcirculatory regeneration) with controllable side effects. (3) Insufficient consideration of gender differences: Many treatment strategies do not fully consider the gender differences in vascular aging pathology and drug response.

[0005] Against this backdrop, the naturally occurring estrogen isoform 17α-estradiol (17α-E2) has attracted researchers' attention due to its unique biological characteristics. As an isoform of the potent estrogen 17β-estradiol (17β-E2), the unique value of 17α-E2 lies in its "non-feminizing" properties—its binding affinity to classical estrogen receptors (ERα and ERβ) is extremely low (only 1 / 100 to 1 / 200 of that of 17β-E2), thus significantly reducing its stimulatory effect on estrogen-sensitive tissues such as the breast and endometrium, potentially reducing feminizing side effects and the risk of related tissue carcinogenesis. Preclinical studies have revealed a series of remarkable biological effects of 17α-E2: (1) Neuroprotective effects: It can selectively activate the brain-specific ER-X receptor, showing the potential to reduce cerebral ischemia-reperfusion injury and improve cognitive function in animal models. (2) Metabolic regulation: It can activate the liver mTORC2 signaling to improve insulin sensitivity and glucose tolerance; improve muscle mass and function in aged male animals by increasing liver amino acid synthesis; activate the AMPK pathway and inhibit mTORC1 to exert anti-inflammatory and antioxidant effects. (3) Improvement of metabolic diseases: It can reverse obesity-related metabolic disorders and improve non-alcoholic fatty liver in animal models. (4) Endocrine regulation: It can enhance serum testosterone and GnRH levels in aged male animals.

[0006] Although 17α-E2 has shown significant potential in neuroprotection and metabolic regulation, and its mechanism of action involves key pathways closely related to vascular function (such as regulation of inflammation, oxidative stress, and metabolic homeostasis), there is currently no clear research evidence to suggest a direct therapeutic effect of 17α-E2 on vascular aging. Its specific effects on the vascular system, particularly its influence on core aspects of aging such as vascular stiffness, endothelial function, microcirculation, and vascular calcification, remain unknown. Preliminary safety assessments (mainly small-sample trials in postmenopausal women and long-term animal studies) show good tolerability, low carcinogenic risk, and no significant hepatotoxicity or nephrotoxicity reported; however, clinical data to support its safety and efficacy in men are lacking. Summary of the Invention

[0007] To address the shortcomings of existing technologies, this invention provides the application of 17α-estradiol in vascular aging-related diseases, revealing the novel therapeutic value of 17α-estradiol in intervening in the core aspects of vascular aging, and providing an innovative solution for vascular degenerative diseases.

[0008] This invention is achieved through the following technical solution:

[0009] The use of 17α-estradiol or its chemically modified derivatives in the preparation of products for the prevention, delay and / or treatment of vascular aging-related conditions.

[0010] Preferably, the chemical modification is a modification aimed at altering the solubility of the drug molecule and / or its affinity for the target.

[0011] Preferably, the vascular aging-related conditions include decreased vascular elasticity, arteriosclerosis, impaired vascular endothelial function, abnormal vascular lumen, reduced vascular microcirculation, and increased risk of vascular thrombosis.

[0012] Preferably, the target audience for the product is middle-aged and elderly men.

[0013] Preferably, the product is a drug or a laboratory reagent.

[0014] Preferably, the drug further includes a pharmaceutically acceptable carrier.

[0015] Preferably, the dosage form of the drug is any pharmacologically acceptable dosage form.

[0016] The beneficial effects of this invention are as follows:

[0017] (1) This invention is the first unexpected discovery that 17α-estradiol can significantly improve the vitality of vascular endothelial cells in aged male animals and effectively improve their aging-related functional activities. This discovery fills a key scientific gap and establishes for the first time a direct causal relationship between 17α-estradiol and the improvement of aging function of vascular endothelial cells.

[0018] (2) Unlike existing drugs that mainly target downstream risk factors (such as lowering blood pressure, lowering lipids, and antithrombosis), this invention discovers that 17α-estradiol targets the core link of vascular aging—functionally degenerated endothelial cells. By directly enhancing the vitality and function of endothelial cells, it is expected to reverse or delay the initiation and key link of vascular aging—endothelial dysfunction—from its source, thereby improving vascular elasticity, reducing hardening, inhibiting pathological remodeling, and promoting microcirculation health, thus achieving a fundamental intervention in vascular aging.

[0019] (3) Based on the direct effect of 17α-estradiol in improving vascular endothelial aging function, it adds a novel and uniquely mechanistic drug option for the prevention, delay, and treatment of a series of diseases closely related to vascular aging (such as senile isolated systolic hypertension, arteriosclerosis, coronary heart disease, ischemic stroke, vascular cognitive impairment, chronic kidney disease, peripheral artery disease, etc.). Its mechanism of action may be different from or complementary to some existing drugs (such as metformin and SGLT2i), providing a theoretical basis for the future development of combination therapies to more comprehensively combat vascular aging.

[0020] (4) Based on existing small-sample clinical studies (postmenopausal women) and long-term animal experimental data, 17α-estradiol has shown good tolerability, with a significantly lower risk of carcinogenesis than traditional estrogens, and no reports of serious liver or kidney dysfunction. Although its long-term safety in men requires further clinical validation, the existing data provide a positive safety basis for its development for use in elderly men with vascular aging-related diseases. Attached Figure Description

[0021] Figure 1 The effects of Augur perturbation analysis on the major hypothalamic cell groups in aged male rats (OT), control aged male rats (O), and control young male rats (Y) treated with 17α-estradiol in Example 1 are shown in Figure A: 10 major hypothalamic cell types and UMAP dimensionality reduction projection mapping; Figure B: Changes in the number of differences in cell communication among the 10 hypothalamic cell types in the OT vs Y treatment groups; Figure C: Augur perturbation analysis of the 10 major hypothalamic cell groups, divided into 3 groups for comparison (OT vs O, O vs Y, OT vs Y); The 10 cell types are as follows: Neuron, Astro, Oligodendrocyte, OPC, Tany, Ependymal cells, Microglia, Fibroblasts, PTC, and Endothelial cells;

[0022] Figure 2 The results of Augur perturbation analysis and ranking of GOCC pathways related to 70 subcellular components in 10 major hypothalamic cell types in Example 1, performed among different treated samples;

[0023] Figure 3 The results of GOBP enrichment analysis of genes with significant differences before and after 17α-estradiol treatment in aged rat endothelial cells in Example 1 are shown. Detailed Implementation

[0024] The present invention will now be described in further detail with reference to the accompanying drawings and specific embodiments.

[0025] Unless otherwise specified, the technical means used in the following embodiments are all conventional means well known to those skilled in the art, and the experimental methods without specific conditions are all conventional methods in the art.

[0026] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0027] Example 1

[0028] The English name of 17α-estradiol is 17α-estradiol (17α-E2), and its Chinese alternative names are 17a-estradiol, 1,3,5(10)estradiol-3,17α-diol. Its molecular formula is C 18 H 24 O2, molecular weight: 272.38200, CAS number: 57-91-0, structural formula as follows:

[0029]

[0030] 1. Experimental Procedure

[0031] 17α-estradiol was added to the diet of brown Norwegian rats (purchased from Beijing Vital River) at a ratio of 14.4 mg / kg. The rats were allowed free access to food and were divided into 3 groups of 4 rats each. The young group Y was 4 months old, the old group O and the old group OT fed with 17α-estradiol were 30 months old. The OT group was fed with 17α-estradiol diet for 6 consecutive months starting from 24 months of age. The rats were kept in 12-hour light and 12-hour dark environments.

[0032] Intact hypothalamus was removed from euthanized rats and cryopreserved in liquid nitrogen. Two (group O) or three (groups Y and OT) hypothalamus from each group were combined and homogenized using a Durns homogenizer with 500 μL of ice-bath homogenization buffer (containing 0.25 M sucrose, 5 mM calcium chloride, 3 mM magnesium acetate, 10 mM Tris-HCl (pH 8.0), 1 mM DTT, 0.1 mM EDTA, 1× protease inhibitor, and 1 U / μL RiboLock RNase inhibitor). The homogenizer was then washed with 700 μL of ice-bath nuclear wash buffer (containing 0.04% bovine serum albumin, 0.2 U / μL RiboLock RNase inhibitor, 500 mM mannitol, and 0.1 mM benzyl sulfonyl fluoride protease inhibitor in 1× phosphate buffer). The homogenate was filtered through a 70 μm cell filter, and the nuclear fraction was collected. Nuclear fractions were mixed with an equal volume of 50% iodixanol and plated on top of a 30% and 33% iodixanol gradient. The mixture was centrifuged at 10,000 × g for 20 min at 4 °C. After removing the myelin layer from the top layer of the gradient, nuclei were collected from the 30% and 33% iodixanol interface, resuspended in nuclear washing buffer and resuspension buffer, and centrifuged at 500 × g for 5 min at 4 °C to precipitate the nuclei. Nuclei were filtered through a 40 μm cell filter to remove debris and large clumps. Nuclear concentration was manually determined using trypan blue staining and a hemocytometer, and finally adjusted to 700–1200 nuclei / μL. Analysis was performed using the 10X Chromium platform. Reverse transcription, cDNA amplification, and library preparation were performed according to the 10X Genomics ChromiumNext GEM Single Cell 3'Reagent Kits v3.1 protocol. The library was analyzed using Illumina HiSeq. TM Sequencing was performed on 4000.

[0033] The original BCL file was converted to a FASTQ file using 10X Genomics Cell Ranger software (version 3.1.0), and then aligned and quantified. Low-quality barcode and UMI reads were filtered out, and then aligned to a reference genome. Only reads uniquely aligned to the transcriptome and with at least 50% exon overlap were used for UMI counting. UMI sequencing errors were corrected before quantification, and valid barcodes were identified using the EmptyDrops method. A cell × gene matrix was generated based on UMI counting and cell barcode identification. Cells with abnormally high UMI numbers (≥8000) or excessively high mitochondrial gene content (≥15%) were filtered out, and batch effects were corrected using Harmony.

[0034] Three samples were used: a young control (Y), an aged group (O), and an aged group treated with 17α-estradiol (OT). Cell perturbation analysis (O vs Y, OT vs O, OT vs Y) and cell type priority ranking were performed using the R package Augur, with the subsample_size parameter of the calculate_auc function set to 6. Augur analysis was performed on 70 cellular component (CC) pathways from the GO database, and the data were ranked in descending order based on AUC values ​​(data from multiple cell types were integrated before ranking). Differential analysis of intercellular communication was performed using the R package cellchat. Differential analysis used the FindMarkers function of the Seurat package, with the test.use parameter set to wilcox. Significantly differentially regulated genes were divided into upregulated and downregulated sets, and GOBP pathway enrichment analysis was performed separately on the David online website. The two sets were then integrated and plotted.

[0035] 2. Experimental Results

[0036] (1) Augur perturbation analysis of the effects of 17α-E2 treatment on major hypothalamic cell groups in aged male rats (OT), control aged male rats (O), and control young male rats (Y), such as Figure 1 As shown in Figure A, the hypothalamus comprises 10 major cell types. The changes in the number of differentially expressed cell communications among these 10 hypothalamic cell types in the OT vs Y treatment groups (analysis from CellChat) are shown below. Figure 1 As shown in Figure B, the signal changes in endothelial cells (Endo) are relatively concentrated. Augur perturbation analysis was performed on 10 major cell groups of the hypothalamus, divided into 3 groups for comparison (OT vs O, O vs Y, OT vs Y), and the results are shown below. Figure 1 As shown in C, endothelial cells are the most sensitive hypothalamic cell type to 17α-estradiol perturbation.

[0037] (2) Augur perturbation analysis was performed on the GOCC pathway of 70 subcellular components of 10 cell types in the hypothalamus. Based on two-group comparisons (O vs Y, OT vs Y), the results were summarized and sorted in descending order of AUC, showing the top 30 most sensitive subcellular components and their corresponding cell types. The results are as follows: Figure 2 As shown, 27 of the top 30 sensitive components in the OT vs Y group came from endothelial cells (Endo), further confirming the hypersensitivity of endothelial cells to 17α-estradiol.

[0038] (3) Figure 3As shown, enrichment analysis of significantly differentially expressed genes in endothelial cells of aged rats before and after long-term 17α-estradiol feeding revealed that the top 15 David GOBP enriched pathways in endothelial cells were all upregulated, indicating that 17α-estradiol perturbation significantly enhanced various functions of endothelial cells, including angiogenesis, tight junctions between endothelial cells, arterial morphogenesis, and stress fiber assembly, while downregulated GOBP pathways were not significantly enriched.

[0039] In summary, analysis of the major hypothalamic cell groups in aged male rats (OT), control aged male rats (O), and control young male rats (Y) treated with 17α-estradiol showed that endothelial cells exhibited the most concentrated signal changes in the OT vs Y group. Furthermore, in Augur perturbation analysis across the three groups (OT vs O, O vs Y, OT vs Y), endothelial cells were the most sensitive cell type among the 10 major hypothalamic cell types to 17α-estradiol perturbation. GOCC pathway analysis targeting 70 subcellular components of the 10 hypothalamic cell types, and Augur perturbation analysis based on the O vs Y and OT vs Y group comparisons, showed that OT vs Y... Of the top 30 most sensitive subcellular components in group Y, 27 originated from endothelial cells (Endo), further confirming the hypersensitivity of endothelial cells to 17α-estradiol. Simultaneously, enrichment analysis of significantly differentially expressed genes in aged rat endothelial cells before and after long-term 17α-estradiol feeding showed that the top 15 David GOBP enriched pathways in endothelial cells (Endo) were all upregulated, indicating that 17α-estradiol perturbation significantly enhanced multiple endothelial cell functions (such as angiogenesis, tight junctions between endothelial cells, arterial morphology, and stress fiber assembly), while downregulation of GOBP pathways did not show significant enrichment. These experimental results consistently demonstrate that endothelial cells are the most sensitive cell type in the hypothalamus to 17α-estradiol perturbation, and that 17α-estradiol can significantly enhance multiple endothelial cell functions.

[0040] The embodiments described above are only some, not all, of the embodiments of the present invention. The detailed description of the embodiments of the present invention is not intended to limit the scope of the claimed invention, but merely to illustrate selected embodiments. The scope of protection of the present invention is determined by the scope claimed in the claims. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without inventive effort are within the scope of protection of the present invention.

Claims

1. 17 The use of α-estradiol or its chemically modified derivatives in the preparation of products for the prevention, delay and / or treatment of vascular aging-related conditions.

2. The application according to claim 1, characterized in that, The chemical modification is aimed at altering the solubility of the drug molecule and / or its affinity for the target.

3. The application according to claim 1, characterized in that, The vascular aging-related conditions include decreased vascular elasticity, arteriosclerosis, impaired vascular endothelial function, abnormal vascular lumen, reduced vascular microcirculation, and increased risk of vascular thrombosis.

4. The application according to claim 1, characterized in that, The target audience for the product is middle-aged and elderly men.

5. The application according to claim 1, characterized in that, The product is a drug or laboratory reagent.

6. The application according to claim 5, characterized in that, The drug also includes a pharmaceutically acceptable carrier.

7. The application according to claim 5, characterized in that, The dosage form of the drug is any pharmacologically acceptable dosage form.

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