CN115212194A - Application of nadolol in preparation of medicine for treating ischemia/reperfusion injury and cell protection medicine - Google Patents

Abstract

The invention relates to application of nadolol in preparation of a medicine for treating ischemia/reperfusion injury and a cell protection medicine. The nadolol is used for preparing a medicine for treating ischemia/reperfusion injury, in particular to a medicine for treating nerve cell injury, and the medicine has a good protection effect on cerebral ischemia/reperfusion (ischemic stroke) and can remarkably relieve the cerebral ischemia/reperfusion injury.

Description

Application of nadolol in preparation of medicine for treating ischemia/reperfusion injury and cell protection medicine

Technical Field

The invention relates to an application of Nadolol (trade name is Corgard) in preparation of a medicine for treating or preventing ischemia/reperfusion injury and a cell protection medicine, belonging to the field of biological medicines. The invention provides new application and administration mode of nadolol, which comprises the protection effect on cerebral ischemia/reperfusion (especially ischemic cerebral apoplexy) and the alleviation of ischemia/reperfusion injury; the cytoprotective medicine is used for protecting organs, tissues or cells against cell damage and dysfunction, and the indication range of the nadolol is expanded; in addition, the invention also covers the application of the nadolol in preparing the medicine for protecting the nerve cell injury.

Background

Cerebral apoplexy is classified into hemorrhagic stroke and ischemic stroke according to different clinical symptoms, wherein the ischemic stroke accounts for about 80%. Ischemic stroke is also called cerebral infarction, is local cerebral tissue blood supply disorder caused by various reasons, leads to cerebral tissue ischemia, hypoxic necrosis and nerve function loss, is a common and frequently encountered disease seriously harming human health, and has become the first cause of disability and the third cause of death in the world.

One of the main measures in the treatment of infarction is to restore the perfusion supply of infarcted tissues as soon as possible, but reperfusion therapy is often accompanied by some tissue damage, causing pathophysiological changes. The damage caused by ischemia and reperfusion is referred to as "ischemia/reperfusion (I/R) damage". I/R injury relates to various mechanisms such as energy metabolism disorder, calcium overload, oxidative stress, inflammatory reaction and the like, and causes various death modes such as apoptosis, necrosis and the like of cells. The research shows that the medicine can inhibit apoptosis and/or necrosis, inhibit myocardial cell and nerve cell death caused by ischemia/reperfusion, relieve the degree of ischemia/reperfusion injury of heart and brain, and reduce infarction range.

Research shows that RIPK1/RIPK3/MLKL dependent necrotic apoptosis exists in various injury related diseases, including ischemic cerebral apoplexy, myocardial infarction, liver and kidney injury, neurodegenerative diseases and the like. Therefore, inhibition of RIPK1/RIPK3/MLKL dependent necrotic apoptosis can inhibit or reduce cell death and tissue damage resulting from the above diseases. The literature reports that the RIPK1 inhibitor, namely, the neostatin-1 (Nec-1), can reduce ischemia/reperfusion injuries of heart, brain, liver, kidney and the like of a mouse. However, nec-1 is only used as a tool medicine in animal experiments, and no RIPK1/RIPK3/MLKL medicine is clinically used for clinical treatment at present.

Nadolol is a beta receptor blocker, has the function of competitively inhibiting catecholamine, is suitable for treating hypertension, and has certain effect on tachyarrhythmia caused by the increase of the concentration of the catecholamine. The heart's contractile force and contractile speed are decreased by decreasing or preventing beta receptor excitation, and the heart's response to exercise or stress is decreased by slowing down conduction through the conduction system. Therefore, can be used for treating angina pectoris. However, whether the composition has the effect of resisting ischemia/reperfusion injury is not reported.

In the present invention, unless otherwise specified, "nadolol" is understood to mean "a compound of nadolol or one of its semisynthetic derivatives or one of their pharmaceutically acceptable salts (a salt of the compound or a salt of the semisynthetic derivative) or one of their esters (an ester of the compound or an ester of the semisynthetic derivative) or one of their ester salts (a salt of the ester of the compound or a salt of the ester of the semisynthetic derivative).

Disclosure of Invention

In view of the defects of the prior art, one of the objects of the present invention is to provide the use of nadolol in the preparation of a medicament for treating or preventing ischemia/reperfusion injury; the invention also aims to provide the application of the nadolol in preparing the cell protection medicament.

The inventor finds that the nadolol can reduce the nerve cell death caused by cerebral ischemia, remarkably reduce the cerebral ischemia infarction (area) volume, improve the neurological function, reduce the nerve cell death and have the nerve cell protection effect.

The structural formula of the nadolol is shown as formula I, and the molecular formula is C ₁₇ H ₂₇ NO ₄ 。

In order to solve the technical problems, the technical scheme of the invention is as follows:

application of nadolol in preparing a medicament for treating or preventing ischemia/reperfusion injury.

Optionally, the ischemia/reperfusion injury is one or more of a brain ischemia/reperfusion injury, a liver ischemia/reperfusion injury, and a kidney ischemia/reperfusion injury.

Optionally, the ischemia/reperfusion injury is a cerebral ischemia/reperfusion injury.

Optionally, the cerebral ischemia/reperfusion injury is ischemic stroke.

Further, the administration mode of the medicine is one or more of intramuscular injection, subcutaneous injection, intravenous injection, oral administration, sublingual buccal administration, delivery in focus or brain or implantation and spray administration, and the preferable mode is oral administration, intramuscular injection, subcutaneous injection or intravenous injection.

Further, the administration mode of the medicine is intramuscular, subcutaneous or intravenous injection.

Furthermore, the medicine can be prepared into any pharmaceutically acceptable dosage form.

Further, the preparation comprises one of injection, oral liquid, dropping pills, capsules, tablets, granules, powder, suspension, emulsion, spray and liposome, wherein the preferable preparation is injection.

Optionally, nadolol is a pharmaceutically acceptable salt thereof, the pharmaceutically acceptable salt is a pharmaceutically commonly used salt, and further, the salt is selected from one or more of acetate, hydrochloric acid, hydrobromic acid, nitric acid, sulfuric acid, phosphoric acid, benzoate, fumarate, maleate, succinic acid, tartaric acid, citrate, oxalic acid, glyoxylic acid, aspartic acid, tartrate, 2, 5-dihydroxybenzoate, methanesulfonate, ethanesulfonate, benzenesulfonate, leucyl sulfonate, hydroquinonesulfonate and p-toluenesulfonate.

Based on the same inventive concept, the invention also provides the application of the nadolol in preparing the cell protection medicament.

Alternatively, the cytoprotective agent refers to an agent having the effect of preventing, inhibiting, or treating damage, degeneration, or dysfunction of tissues, organs, and cells.

Optionally, the cytoprotective agent includes an agent for organs or cells of the nervous system, brain, eye, etc. that resist or cause cell death, such as an agent for treating diseases caused by necrosis and/or pathological apoptosis and/or necroptosis and/or iron death and/or apoptosis and/or autophagy, etc.

Further, the cytoprotective agent is an agent for preventing, inhibiting, or treating a neurological disease or an ophthalmic disease.

Optionally, the organ is one or more of brain, lung, kidney, pancreas, skin, eye, cornea.

Alternatively, the cytoprotective agent refers to an agent that prevents, inhibits, or treats a neurological disease or an ophthalmic disease.

Optionally, the cytoprotective agent is an agent for treating or preventing nerve cell damage.

Optionally, the cell is a neural cell.

Preferably, the organs include the nervous system and the brain. One of the objects of the present invention is to provide a new use of nadolol (either a compound alone or a pharmaceutical composition containing the compound) which can be used for preventing and/or treating cells against or causing cell death processes such as cell death caused by necrosis and/or pathological apoptosis and/or necroptosis and/or iron death and/or apoptosis and/or autophagy, etc.

Further, nadolol can protect, prevent and/or treat cells against cell death or processes that lead to cell death, including inhibition of nerve cell damage, death, such as the treatment and prevention of neurological disorders, glaucoma, retinitis pigmentosa, corneal dystrophy, age-related macular degeneration (AMD), wet or dry AMD-related photoreceptor degeneration, other retinal degenerations, optic neuropathy and optic neuritis, optic nerve drusen, cerebral stroke, alzheimer's disease, parkinson's disease, huntington's disease, parkinson's plus syndrome, ischemia, amyotrophic Lateral Sclerosis (ALS), intracranial hemorrhage, cerebral hemorrhage, trigeminal neuralgia, glossopharyngeal neuralgia, myasthenia gravis, muscular dystrophy, progressive amyotrophic lateral syndrome, bell's palsy, progressive bulbar palsy, spinal muscular atrophy, primary Lateral Sclerosis (PLS), pseudobulbar palsy, invertebrate disc syndrome, cervical spondylosis, hereditary muscular atrophy, plexus disorders, thoracic outlet destruction syndrome, porphyria, peripheral neuropathy, multiple system atrophy, corticobasal degeneration, progressive supranuclear palsy, lewy body dementia, demyelinating disease, frontotemporal dementia, gulfweed-barre syndrome, multiple sclerosis, creutzfeldt-jakob disease, progressive neurotic fibular peroneal disease, myelogenous myelopathy, prion disease, lethal familial disease (FFI), geschwang-schwann-schneiderick syndrome, AIDS, heavy metal-induced dementia, heavy metal chemotherapy induced dementia, and chemotherapy induced dementia; damage to the nervous system caused by mechanical, physical or chemical trauma.

The invention unexpectedly discovers that the nadolol can reduce nerve cell death caused by cerebral ischemia/reperfusion, remarkably reduce the volume of cerebral ischemia infarction, improve neurological function and have the protection effect of nerve cells. The medicine of the invention has the function of relieving cerebral ischemia/reperfusion injury, is possibly used for treating cerebral ischemic stroke, and has good development and application prospects.

The research of the inventor finds that the nadolol can specifically reduce the expression and phosphorylation levels of RIPK1, RIPK3 and MLKL, can be used as RIPK1, RIPK3 and MLKL inhibitors, can inhibit necrosis-like apoptosis, can reduce cell death and improve nerve cell functions.

Nadolol is a beta receptor blocker, has the function of competitively inhibiting catecholamine, and is suitable for treating hypertension. The invention firstly uses nadolol in a rat ischemic stroke model, finds that nadolol can reduce RIPK1, RIPK3 and MLKL expression and phosphorylation levels in brain tissues, and unexpectedly finds that the nadolol can obviously reduce the cerebral infarction (area) volume, improve the neurological function, reduce nerve cell death, has a nerve cell protection effect, is unexpected, is not related to the known application of nadolol, does not have the related revelation given by other existing compounds, has outstanding substantive characteristics, and has obvious progress in treating ischemia/reperfusion injury and protecting cells.

The invention enlarges the indication of nadolol, and can be suitable for cerebral arterial thrombosis and cell protection.

In a word, the nadolol can be used for treating or preventing nerve cell injury, particularly has a protective effect on ischemic stroke, and can obviously relieve cerebral ischemia/reperfusion injury.

Drawings

FIG. 1 is a graph of the effect of nadolol on the cerebral infarction volume and neurological function of rats in example 1: (A) The TTC staining and infarct volume determination condition of the rat brain tissue is shown; and (B) is a graph of the scoring condition of the neurological function of the rat.

FIG. 2 is a graph showing the effect of nadolol on the regulation of MLKL and phosphorylated MLKL (p-MLKL) in rat brain tissue.

Detailed Description

The present invention will be described in detail with reference to examples.

Example 1

Application of nadolol in preparing medicine for treating ischemia/reperfusion injury and medicine with cell protection effect.

Materials and methods:

to demonstrate the role of nadolol in anti-ischemia/reperfusion injury and cytoprotection, applicants used a rat model of ischemic stroke and treated with nadolol at different time points after ischemia/reperfusion, and then sacrificed the animals by reperfusion for 24 hours, and myocardial cell injury was detected by TTC staining and serum creatine kinase activity, and nerve cell injury was detected by neurobiological scoring and TTC staining.

Implementation of the medicine: nadolol (a compound of nadolol) was purchased from reagent companies.

Animal experiments: the protective effect of nadolol on a rat model with cerebral arterial thrombosis is achieved.

Experimental animals: healthy male SD rats weighing 250-300 g. The experimental animals are raised in an environment with the temperature of 25 ℃, the relative humidity of 60%, free drinking water and fixed amount at regular time for one week, and then the medicine is administered according to the requirements of each group of experiments.

The method for establishing the rat model of cerebral arterial thrombosis comprises the following steps: the rat cerebral ischemia/reperfusion model was prepared by the Middle Cerebral Artery Occlusion (MCAO) method. The method comprises the following steps: (1) Isolating the Common Carotid Artery (CCA), external Carotid Artery (ECA) and Internal Carotid Artery (ICA) on the left side of the rat; (2) Temporarily occluding the ECA and ICA with ophthalmic forceps and ligating the CCA proximal; (3) Placing a knotted standby silk thread at the distal end of the CCA, cutting a small opening at the lower end of the thread, inserting the embolus into the internal carotid artery, tightening the silk thread, releasing artery clamps on the ECA and the ICA, and sending the embolus into the cranium along the ICA; (4) Stopping when meeting resistance, and the insertion depth is about 18-20 mm from the bifurcation of the CCA; (5) After 120min of ischemia, the plug thread was pulled out, the skin was sutured, and the animals were treated after 24h of reperfusion.

The model success criterion was used to score neurological deficit in the rat cerebral ischemia/reperfusion injury model using Longa "score 5" method. 0 minute: no symptoms of neurological deficit; 1 minute: the right forelimb cannot be fully straightened; and 2, dividing: rotating to the right; and 3, dividing: the walking is inclined towards the right side; and 4, dividing: the person cannot walk spontaneously and consciousness is lost. 1-4 are classified as valid models.

Rat brain TTC staining and infarct volume determination. After anesthetizing the rat, the brain was quickly removed, the olfactory bulb and hindbrain were removed, 4 pieces of coronal brain were cut from the frontal pole to a thickness of about 2.0mm, immediately placed in 1% TTC solution, and incubated at 37 ℃ in the dark for 30min. Then soaking and fixing the mixture by using 10% paraformaldehyde solution. The infarcted areas appeared white and the non-infarcted areas appeared red. And (5) scanning after the brain slices of each group are arranged in order. And measuring the infarct area of each brain slice by ImageJ, and according to a formula: infarct volume = [ (sum of front infarct size and sum of back infarct size of each sheet)/2 ] × thickness of each sheet, and the whole brain volume was calculated by the same method.

And detecting the expression levels of MLKL and phosphorylated MLKL by Western Blot (WB). A proper amount of brain tissue is taken and washed by adding precooled PBS. The beads were added to the homogenization tube, chilled in ice, and the tissue was homogenized in the tube (70Hz, 180s). Centrifuge at 12000rpm for 10min at 4 ℃ leaving a supernatant. After measuring the protein concentration by BCA method, 20-40 ug of protein was separated by 8-10% SDS-PAGE gel and loaded onto PVDF membrane, and was incubated overnight with MLKL and p-MLKL (Abcam, cambridge, UK) antibody and beta-actin (Beyotime, jiangsu) antibody, and after incubation of the corresponding secondary antibodies, the protein was developed by Molecular Imager ChemiDoc XRS System (Bio-Rad, philadelphia, pa.) and beta-actin was used as reference. Protein expression levels were assessed by measuring protein gray values by Image J software.

Grouping experiments: experimental animals were randomly divided into 4 groups, namely:

sham group (Sham group): the separation operation of the internal artery and the external artery of the neck is carried out without inserting a plug wire into the artery.

Cerebral ischemia/reperfusion group (I/R group): cerebral ischemia for 2h, and reperfusion for 24h.

Nadolol + cerebral ischemia/reperfusion group (Nadolol + I/R): nadolol (5 mg/kg) treatment was given 2h after ischemia and 1h after reperfusion.

Vehicle + cerebral ischemia/reperfusion group (Vehicle + I/R): after 2h of ischemia and 1h of refilling, the solvent is given for treatment.

Detecting the nerve function score of the rat and measuring the cerebral infarction volume.

As a result:

the nadolol has the effects of resisting cerebral ischemia/reperfusion injury of rats, reducing infarct volume, improving neurological deficit symptoms, reducing nerve cell death and protecting nerve cells.

As shown in A in figure 1, the I/R group has obvious white infarction focus, the cerebral infarction volume of rats with nadolol after 2 hours of ischemia and 1 hour of reperfusion is obviously reduced, and cerebral ischemia/reperfusion injury is obviously relieved. As shown in fig. 1, B, nadolol significantly improved neurological deficit symptoms (n =6, # P < 0.01vs Sham, ^# P＜0.05vs I/R， ^## P＜0.01vs I/R)。

FIG. 2 shows that ischemia/reperfusion induces the up-regulation of rat brain tissue MLKL and p-MLKL, while nadolol significantly inhibits the up-regulation of MLKL and p-MLKL.

And (4) conclusion: the nadolol can reduce nerve cell death and relieve cerebral ischemia/reperfusion injury, has the function of protecting nerve cells, can be applied to the preparation of medicaments for relieving the cerebral ischemia/reperfusion injury and the cell protection function, and is used for treating ischemic stroke.

However, the invention is not limited to cerebral ischemia/reperfusion injury, and the medicine is also suitable for treating the hepatic and renal ischemia/reperfusion injury due to the similarity of the mechanism of the hepatic and renal ischemia/reperfusion injury and the cerebral ischemia/reperfusion injury.

The above examples are set forth so that this disclosure will be understood in all instances to be considered illustrative and not restrictive, and that various modifications and equivalent arrangements may be devised by those skilled in the art after reading this disclosure and are intended to be included within the scope of the appended claims.

Claims (9)

1. Application of nadolol in preparing a medicament for treating or preventing ischemia/reperfusion injury.

2. The use of claim 1, wherein the ischemia/reperfusion injury is one or more of cerebral ischemia/reperfusion injury, hepatic ischemia/reperfusion injury and renal ischemia/reperfusion injury.

3. The use of claim 2, wherein the ischemia/reperfusion injury is a cerebral ischemia/reperfusion injury.

4. The use of claim 3, wherein the cerebral ischemia/reperfusion injury is ischemic stroke.

5. Application of nadolol in preparation of cell protection drugs.

6. The use according to claim 5, wherein the cytoprotective agent is an agent having the effect of preventing, inhibiting or treating tissue, organ and cell damage, degeneration or dysfunction.

7. The use of claim 6, wherein the organ is one or more of brain, lung, kidney, pancreas, skin, eye, cornea.

8. The use of claim 5, wherein the cytoprotective agent is an agent for preventing, inhibiting or treating a neurological or ophthalmic disorder.

9. The use according to any one of claims 5 to 8, wherein the cell is a neuronal cell.

Images