Application of dehydroequol in treatment of neonatal hypoxic ischemic encephalopathy

Abstract

The invention belongs to the technical field of medical treatment, and discloses application of dehydroequol to treatment of neonatal hypoxic ischemic encephalopathy. The dehydroequol is used for treating the neonatal hypoxic-ischemic encephalopathy, and it is proved that the dehydroequol can reduce the area of cerebral infarction induced by hypoxia and ischemia, relieve encephaledema and inhibit oxidative stress and copper death, and has a treatment effect on neonatal hypoxic-ischemic brain injury. Dehydroequol is a medicine for treating hypoxic ischemic encephalopathy of newborns and has a development prospect.

Description

Technical Field

[0001] This invention belongs to, but is not limited to, the field of medical technology, and particularly relates to the application of dehydroestrol in the treatment of neonatal hypoxic-ischemic encephalopathy. Background Technology

[0002] Hypoxic-ischemic encephalopathy (HIE) is a brain injury syndrome caused by reduced cerebral blood flow (ischemia) and oxygen supply (hypoxia) during the perinatal period. It is a leading cause of death and disability in children worldwide, and there is currently no effective treatment. Epidemiological surveys show that more than one million people die from HIE globally each year. Children with HIE often experience long-term neurological dysfunctions such as epilepsy, cerebral palsy, intellectual disability, learning and behavioral disorders, severely impacting their quality of life. The pathophysiological process of neonatal HIE brain injury is complex, involving multiple aspects including energy metabolism disorders, oxidative stress, excitatory amino acid toxicity, inflammatory cytokine damage, mitochondrial dysfunction, and neuronal cell death.

[0003] Phenoxodiol (PXD) is an isoflavone derivative with a low metabolic rate and high biological activity. Domestic and international studies have shown that PXD has anti-tumor, anti-neuroinflammatory, and neuroprotective effects. PXD (0.3 mg / kg, 1 mg / kg) can reduce the infarct area and alleviate cerebral edema in neonatal rats with hypoxic-ischemic encephalopathy (HIE) after 24 hours, indicating that PXD has an inhibitory effect on HIE brain injury. PXD reduces MDA content and increases SOD and GSH activity in the cortical injury area, indicating that PXD can protect against HIE brain injury by inhibiting oxidative stress. PXD also reduces Cu in the ischemic cerebral cortex tissue of neonatal rats with HIE. 2+ Levels, reducing Cu in brain tissue 2+ Furthermore, PXD can reduce the expression of copper death-related proteins FDX1, LIAS, and HSP70 in the cerebral cortex tissue of the HIE ischemic injury area in newborn rats, and reduce HI-induced DLAT oligomerization, suggesting that PXD can reduce Cu... 2+ Accumulation inhibits copper death, thereby alleviating HIE brain damage in newborn rats.

[0004] (1) Close to related patent 1: EP4255459A1 - ω-3 fatty acid emulsion in combination or replacement therapy for HIE

[0005] Patent content: This patent discloses a neuroprotective composition and method for neonatal hypoxic-ischemic encephalopathy (HIE). The main component is an injectable emulsion of omega-3 fatty acid diesters or triesters (such as DHA and EPA), which can be used alone or in combination with therapeutic hypothermia (HT) to reduce the risk of brain damage caused by HIE.

[0006] Existing technical problems:

[0007] *These emulsions need to be prepared and injected urgently within a very short time after infection (such as within hours after birth), which is difficult to do for premature or low birth weight infants;

[0008] *The stability, emulsifying properties, in vivo pharmacokinetics and efficacy of the emulsion in combination with low-temperature treatment are still unclear;

[0009] *Insufficient research on lipid metabolic burden and safety in extremely preterm infants may increase metabolic or immune risks.

[0010] (2) Closely related patent 2: US20160362443A1 - Preparation of DHEA esters to improve drug delivery performance (not for HIE)

[0011] Patent content: This patent relates to a method for converting untreated DHEA into fat-soluble esters such as DHEA enanthate, mainly for prolonging the metabolic persistence of DHEA in the human body, improving physical performance and immune function. It is a chronic supplement or enhancement purpose, which is different from the treatment of HIE.

[0012] Existing technical problems:

[0013] *This technology is designed for long-term sustained-release formulations and exercise enhancement applications, and has not been developed for dosing regimens for low birth weight newborns or HIE acute conditions;

[0014] The permeability and safety of DHEA-ester metabolic pathways in the neonatal brain are not yet clear.

[0015] *It does not take into account the pathological state of HIE, such as changes in the blood-brain barrier after hypoxia, the emergency window period, and other clinical emergency characteristics, and lacks applicability for emergency treatment.

[0016] (3) Summary and existing technological gaps: Why is "dehydroestrol" still a technological gap as a small molecule treatment for HIE?

[0017] *Limitations of current technology: The only standard treatment for HIE is hypothermia therapy, which has limited effectiveness; large molecular formulations such as ω-3 lipid emulsions present challenges in preparation and metabolism; and existing DHEA ester technology is geared towards chronic use and is out of step with emergency treatment procedures.

[0018] *Technological gaps and application potential: Dehydroestradiol (Phenoxodiol, PXD and other small molecules) has the potential to be rapidly formulated, easily penetrate the blood-brain barrier, and have antioxidant, anti-inflammatory and neuroprotective effects.

[0019] *Key innovation: Developing PXD-type small molecule drug compositions (possibly including acceptable carriers or diluents) suitable for neonatal HIE, which can fill the technological gaps in rapid onset of action, small volume, safe entry into brain tissue, and easy synergy with standard treatments such as hypothermia therapy. Summary of the Invention

[0020] To address the problems existing in the prior art, this invention provides an application of dehydroestrone in the treatment of neonatal hypoxic-ischemic encephalopathy.

[0021] This invention is achieved by providing a dehydroestrol, the specific structural formula of which is as follows:

[0022]

[0023] Another objective of this invention is to provide an application of the aforementioned dehydroestrol in the treatment of neonatal hypoxic-ischemic encephalopathy.

[0024] Furthermore, neonatal hypoxic-ischemic encephalopathy refers to a brain injury syndrome caused by reduced cerebral blood flow (ischemia) and oxygen (hypoxia) supply during the perinatal period.

[0025] Another object of the present invention is to provide a pharmaceutical composition comprising an acceptable carrier and / or diluent on the dehydroestradiol for the treatment of neonatal hypoxic-ischemic encephalopathy.

[0026] Furthermore, neonatal hypoxic-ischemic encephalopathy refers to a brain injury syndrome caused by reduced cerebral blood flow (ischemia) and oxygen (hypoxia) supply during the perinatal period.

[0027] Based on the above technical solutions and the technical problems solved, the advantages and positive effects of the technical solution to be protected by this invention are as follows:

[0028] This invention utilizes dehydroestrol to treat neonatal hypoxic-ischemic encephalopathy, demonstrating significant technical advantages and positive effects. Firstly, from a mechanism of action perspective, dehydroestrol can alleviate brain damage caused by ischemia and hypoxia through a multi-target approach. On one hand, it can effectively reduce the area of ​​cerebral infarction; on the other hand, it can also alleviate cerebral edema, demonstrating its advantage in protecting the structural integrity of brain tissue during the acute phase. This dual protective effect provides a new approach for drug intervention in neonatal hypoxic-ischemic encephalopathy.

[0029] Secondly, this invention reveals the unique therapeutic effects of dehydroephrine in inhibiting oxidative stress and copper death. In hypoxic-ischemic brain injury, excessive production of reactive oxygen species and copper-related cell death are crucial factors leading to irreversible neuronal damage. Dehydroephrine, by significantly reducing oxidative stress levels and blocking the copper death pathway, inhibits neuronal death at its source, demonstrating a more comprehensive and deeper protective effect compared to conventional antioxidant drugs.

[0030] Furthermore, compared to existing conventional hypothermia therapy or symptomatic supportive treatment, the dehydroephrine intervention proposed in this invention has significant pharmacological innovations. Cooling or symptomatic treatment is mostly limited to improving the local metabolic and circulatory environment, while dehydroephrine works through multiple pathways and mechanisms, not only improving acute nerve damage but also potentially providing long-term protection for subsequent neurological function recovery. This differentiated advantage makes it highly substitutable and complementary in clinical applications.

[0031] Furthermore, this invention utilizes dehydroephrine with pharmaceutically acceptable carriers and / or diluents to prepare a pharmaceutical composition, demonstrating good formulation feasibility and clinical application potential. This composition enables stable release and safe delivery of the active ingredient, providing a solid foundation for subsequent drug development and clinical application. Compared to some structurally complex or difficult-to-prepare candidate drugs, dehydroephrine formulations are simple, can be produced on an industrial scale, and possess significant industrialization advantages.

[0032] Finally, from a social and medical perspective, this invention provides a promising new drug candidate that could overcome existing treatment limitations. Neonatal hypoxic-ischemic encephalopathy is a serious disease prevalent during the perinatal period, lacking specific and effective drug treatments, and there is an urgent clinical need for safe and efficient intervention programs. This invention demonstrates the therapeutic potential of dehydroestrol through experimental data and pharmacological verification, filling a technological gap in this field and providing a new approach to reducing neonatal disability and mortality rates, possessing significant clinical application prospects and social significance. Attached Figure Description

[0033] Figure 1 This is a graph showing the effect of dehydroestrol on the cerebral infarction area of ​​a newborn rat HIE model, provided in an embodiment of the present invention.

[0034] Figure 2 This is a graph showing the effect of dehydroestrol on brain edema in a newborn rat HIE model, provided in an embodiment of the present invention.

[0035] Figure 3 This is a graph showing the effect of dehydroestrol on oxidative stress-related indicators in the cerebral cortex of the ischemic injury area of ​​newborn rats in HIE, as provided in the embodiments of the present invention.

[0036] Figure 4The dehydroestrone provided in this embodiment of the invention has an effect on the Cu in the cerebral cortex of the HIE ischemic injury area in newborn rats. 2+ Horizontal influence diagram;

[0037] Figure 5 This is a diagram showing the effect of dehydroestrone on the expression of FDX1, LIAS and HSP70 proteins in the cerebral cortex of the HIE ischemic injury area in newborn rats, as provided in the embodiments of the present invention.

[0038] Figure 6 This is a diagram illustrating the effect of dehydroestrol on DLAT oligomerization in the cerebral cortex of neonatal rats with HIE ischemic injury, as provided in this embodiment of the invention.

[0039] Figure 7 This is a diagram showing how dehydroestrone, as provided in this embodiment of the invention, can reduce neuronal loss in the cortical region on the side of HIE injury in newborn rats.

[0040] Figure 8 This invention provides a diagram showing how dehydroestrone can improve the behavioral function of newborn rats during the acute phase of HIE. Detailed Implementation

[0041] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention.

[0042] Example 1

[0043] In an animal experiment, a hypoxic-ischemic encephalopathy model was established in 7-day-old neonatal SD rats by ligating the right common carotid artery and placing them in a hypoxic chamber. After successful modeling, the experimental animals were randomly divided into four groups: a control group, a model group, and treatment groups (0.3 mg / kg and 1 mg / kg). The treatment groups were administered dehydroestradiol (0.3 mg / kg and 1 mg / kg) via intraperitoneal injection, while the control and model groups were given an equal volume of organic solvent. Twenty-four hours after treatment, brain tissue sections were analyzed using TTC staining. The results showed that the infarct area in the treatment group was significantly reduced, the degree of cerebral edema was significantly alleviated, and the neurological function scores were better than those in the model group, suggesting that dehydroestradiol has a clear neuroprotective effect on hypoxic-ischemic brain injury.

[0044] This invention provides a dehydroestrol, the specific structural formula of which is as follows:

[0045]

[0046] This invention provides an application of the dehydroestrol in the treatment of neonatal hypoxic-ischemic encephalopathy.

[0047] Neonatal hypoxic-ischemic encephalopathy refers to a brain injury syndrome caused by reduced cerebral blood flow (ischemia) and oxygen (hypoxia) supply during the perinatal period.

[0048] This invention provides an application of a pharmaceutical composition consisting of an acceptable carrier and / or diluent on the dehydroestradiol in the treatment of neonatal hypoxic-ischemic encephalopathy.

[0049] Neonatal hypoxic-ischemic encephalopathy refers to a brain injury syndrome caused by reduced cerebral blood flow (ischemia) and oxygen (hypoxia) supply during the perinatal period.

[0050] The following section uses specific experiments to illustrate the role of dehydroestrone in the treatment of neonatal hypoxic-ischemic encephalopathy.

[0051] (1) Preparation of a neonatal rat model of hypoxic-ischemic encephalopathy (HIE): Neonatal SD rats, 7 days old, of any sex, weighing >12g. Anesthetized with 2-3% isoflurane (induction to awakening time not exceeding 10 min), fixed in a supine position, with neck skin disinfected, and a midline incision made under a microscope. The right common carotid artery was isolated, electrocoagulated, and the incision was glued together with tissue adhesive, and the skin was disinfected again. After the neonatal rats recovered from anesthesia, they were returned to their mothers for feeding. 120 minutes post-surgery, the neonatal rats were placed in a 37℃ hypoxic chamber for 1 hour (7% O2), then normal oxygen conditions were restored, and they were returned to their mothers for feeding. The PXD treatment group received a subcutaneous injection 30 min before surgery. In the HIE group, the right common carotid artery was isolated, electrocoagulated, and an equal volume of solvent was injected subcutaneously. In the sham-operated group, the right common carotid artery was isolated, but electrocoagulation and hypoxia were not performed; an equal volume of solvent was injected subcutaneously. The room temperature was strictly controlled at 23℃~25℃ during surgery.

[0052] (2) Determination of the extent of cerebral infarction: The brain was removed by decapitation under anesthesia, and the coronal plane was cut into 5 slices. The slices were stained with 0.5% TTC at 37°C in the dark for 10 min, fixed with 4% paraformaldehyde, and photographed.

[0053] (3) Detection of oxidative stress indicators: The brain tissue was homogenized and the SOD, GSH activity and MDA content in the brain tissue were detected according to the kit instructions.

[0054] (4) Intracellular copper detection: Follow the instructions of the copper assay kit (ab272528, Abcam, Boston, MA, USA).

[0055] (5) Western blot method: Brain tissue was fully lysed with cell lysis buffer, centrifuged at 12000g for 5 min at 4℃, and protein was quantified using the BCA method. 50 μg of total protein was added to the loading buffer and boiled for denaturation. The protein was then subjected to SDS / PAGE gel electrophoresis and electrotransfer to an NC membrane. After blocking, the protein was incubated with primary antibody at 4℃ overnight, and with secondary antibody at room temperature for 1 h. After incubation with chemiluminescent reagent for 1 min, the protein was photographed and analyzed using a chemiluminescent immunoassay system to detect the content of the target protein.

[0056] (6) Statistics: SPSS 20.0 software was used to analyze the experimental data. All data are expressed as mean ± standard deviation. One-way ANOVA was used for overall comparisons between groups, and SNK test was used for multiple comparisons within groups. P < 0.05 was considered statistically significant.

[0057] (7) Experimental results showed that dehydroestrone (0.3 mg / kg and 1 mg / kg) could reduce the cerebral infarction area and alleviate cerebral edema in neonatal rat HIE models 24 h later, indicating that PXD has an inhibitory effect on HIE brain injury; PXD reduced the MDA content in the cortical injury area and increased the SOD and GSH activities, indicating that PXD can protect against HIE brain injury by inhibiting oxidative stress; PXD reduced Cu in the cerebral cortex tissue of the ischemic injury area of ​​neonatal rats with HIE. 2+ Levels, reducing Cu in brain tissue 2+ Simultaneously, PXD reduced the expression of copper death-related proteins FDX1, LIAS, and HSP70 in the ischemic cerebral cortex of neonatal rats with HIE, and decreased HI-induced DLAT oligomerization, suggesting that PXD can reduce Cu... 2+ Accumulation inhibits copper death, thereby alleviating HIE brain damage in newborn rats.

[0058] Figure 1 The effect of dehydroestrol on the infarct area in a newborn rat HIE model;

[0059] Figure 2 The effect of dehydroestrol on brain edema in a neonatal rat HIE model;

[0060] Figure 3 The effect of dehydroestrone on oxidative stress-related indicators in the cerebral cortex of the ischemic injury area of ​​newborn rats with HIE;

[0061] Figure 4 Dehydroestrone affects the Cu in the ischemic cerebral cortex of the HIE (hypoxic-ischemic encephalopathy) area in newborn rats. 2+ The influence of level;

[0062] Figure 5 The effect of dehydroestrone on the expression of FDX1, LIAS and HSP70 proteins in the cerebral cortex of the HIE ischemic injury area in newborn rats.

[0063] Figure 6 Effects of dehydroestrol on DLAT oligomerization in the ischemic cerebral cortex of neonatal rats with HIE.

[0064] Dehydroephedrine can reduce the area of ​​cerebral infarction induced by hypoxia-ischemia, alleviate cerebral edema, inhibit oxidative stress and copper death, and play a therapeutic role in neonatal hypoxic-ischemic brain injury. Dehydroephedrine is a promising drug for the treatment of neonatal hypoxic-ischemic encephalopathy.

[0065] Figure 7 Dehydroestrone can reduce neuronal loss in the cortical region on the side of HIE injury in newborn rats.

[0066] Corresponding detection methods:

[0067] Immunofluorescence detection of NeuN was used to evaluate neuronal loss: After anesthesia, animals were internally fixed by cardiac perfusion with 4% paraformaldehyde. Brain tissue was harvested, refixed, and dehydrated using a 20% and 30% sucrose gradient. Frozen sections were then prepared. Sections were washed with PBS, blocked with 3% BSA + 0.5% Triton X-100 at room temperature for 1 hour, then incubated with NeuN primary antibody for 30 minutes at room temperature, and overnight at 4°C. The next day, the sections were warmed for 30 minutes, washed with PBS, incubated with secondary fluorescent antibody, protected from light, and incubated at room temperature for 1 hour. After washing with PBS, the sections were mounted, dried, and mounted with a DAPI-containing fluorescent anti-quenching agent. The results were observed and analyzed under a fluorescence microscope.

[0068] Figure 8 Dehydroestrone can improve the acute phase behavior of HIE in newborn rats.

[0069] The corresponding behavioral testing method is the righting reflex test: Place the newborn rat on a clean, flat wooden board with its head, neck, and back pressed against the board, then release it. Record the time it takes for the newborn rat to roll from its supine position to its limbs on the ground, accurate to 0.01 seconds.

[0070] Cliff avoidance reflex: Place two-thirds of a newborn rat's body on the edge of a high platform, hold its tail, and use a stopwatch to record the time it takes to move backward or sideways away from the edge of the platform, accurate to 0.01s.

[0071] Negative geotaxis test: A rat is placed head down on a rough wooden board at a 45° angle to a horizontal table. A newborn rat is placed near the top of the board to prevent it from seeing or touching objects on the table and thus moving. After the newborn rat is placed, a stopwatch is used to record the time it takes for it to rotate its body 180° and start climbing upwards with its head up, accurate to 0.01s.

[0072] Forelimb suspension test: The rat's forelimbs were placed on a thin rope 45 cm high, with a diameter of approximately 2 mm. Cotton was placed underneath to prevent injury from falls. The time it took for the newborn rat to fall from the rope was recorded using a stopwatch, accurate to 0.01 s.

[0073] Example 1

[0074] In this embodiment, dehydroephrine is used as the active ingredient to prepare an injection solution. Specifically, dehydroephrine is dissolved in an organic solvent, and pharmaceutically acceptable stabilizers and diluents are added to formulate a pharmaceutical composition suitable for intravenous administration. The prepared drug exhibits good stability in physicochemical property tests and is suitable for clinical infusion use.

[0075] The therapeutic effect of this drug on hypoxic-ischemic encephalopathy in newborn rats was verified using animal models. Results showed that animals treated with dehydroestrol experienced a significant reduction in cerebral infarction volume, relief of cerebral edema, and better neurological function scores than the control group, demonstrating the drug formulation's good clinical application value.

[0076] Example 2

[0077] In this embodiment, a modified Rice-Vannucci neonatal mouse hypoxic-ischemic model was used, and brain injury was induced by ligating the carotid artery and exposing the mouse to a hypoxic environment. After modeling, dehydroestrol solution was administered via intraperitoneal injection, and the pathological changes in brain tissue were observed.

[0078] Experimental results showed that the area of ​​cerebral cortex necrosis was significantly reduced in the dehydroestrone treatment group, and TTC staining results showed a reduction in the infarct area of ​​the cerebral cortex. This indicates that dehydroestrone can exert a protective effect against brain injury caused by hypoxia and ischemia by reducing the infarct area and alleviating cerebral edema.

[0079] Example 3

[0080] In this embodiment, the inhibitory effect of dehydroephrine on oxidative stress in a neonatal hypoxic-ischemic encephalopathy model was investigated. After the model was established, indicators such as malondialdehyde (MDA) and superoxide dismutase (SOD) in brain tissue were measured. After intervention with dehydroephrine, compared with the control group, MDA levels decreased and SOD activity significantly increased.

[0081] Further examination of biomarkers related to the copper-induced cell death pathway revealed that dehydroequadol could reduce the accumulation of copper ions in neurons and decrease the expression of copper death-related proteins. This indicates that dehydroequadol not only inhibits oxidative stress but also effectively blocks the copper death pathway, thereby enhancing neuroprotective effects.

[0082] Example 5

[0083] In this embodiment, neonatal hypoxic-ischemic encephalopathy model animals were treated with intravenous injection of dehydroestrol. The drug was administered within 30 minutes after model establishment, once daily for 3 consecutive days. The treated group animals showed reduced neurobehavioral deficits and improved motor dysfunction.

[0084] TTC staining of brain tissue showed that the infarct area in the treatment group was significantly smaller than that in the control group. Electron microscopy revealed that dehydroestrol treatment maintained the structural integrity of neuronal mitochondria and reduced cell apoptosis. This indicates that this treatment method can effectively alleviate brain damage caused by hypoxia and ischemia, and has clear feasibility and application value.

[0085] The above description is merely a specific embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any modifications, equivalent substitutions, and improvements made by those skilled in the art within the scope of the technology disclosed in the present invention, and within the spirit and principles of the present invention, should be covered within the scope of protection of the present invention.

Claims

1. A drug for treating hypoxic-ischemic encephalopathy in newborns, characterized in that, The drug contains dehydroestrol as its active ingredient.

2. The drug according to claim 1, characterized in that, The drug also contains a pharmaceutically acceptable carrier and / or diluent.

3. The use of dehydroestrol in the preparation of a drug for treating hypoxic-ischemic encephalopathy in newborns, characterized in that, The neonatal hypoxic-ischemic encephalopathy is caused by reduced cerebral blood flow and insufficient oxygen supply during the perinatal period.

4. The use according to claim 3, characterized in that, The drug achieves its neuroprotective effect by reducing the area of ​​cerebral infarction and alleviating cerebral edema.

5. The use of dehydroestrol in the preparation of a drug for inhibiting oxidative stress-related brain injury in neonatal hypoxic-ischemic encephalopathy, characterized in that, The dehydroestradiol inhibits oxidative stress by reducing the level of reactive oxygen species.

6. The use according to claim 5, characterized in that, The dehydroestrone further reduces neuronal death by inhibiting copper death.

7. The use of a pharmaceutical composition comprising dehydroestrol in the preparation of a drug for improving the prognosis of hypoxic-ischemic encephalopathy in newborns, characterized in that, The pharmaceutical composition can promote the recovery of neurological function in newborns.

8. The use according to claim 7, characterized in that, The pharmaceutical composition reduces the disability rate of neonatal hypoxic-ischemic encephalopathy by protecting the structural integrity of neurons.

9. A method for treating hypoxic-ischemic encephalopathy in newborns, characterized in that, Administering drugs containing dehydroestrol to patients can help reduce brain damage caused by ischemia and hypoxia.

10. The method according to claim 9, characterized in that, The administration method is oral, injection, or intravenous infusion.

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