CN117257803A - Application of lurasidone in preparation of drugs for treating or preventing ischemia/reperfusion injury and cytoprotective drugs - Google Patents

Abstract

The invention relates to application of lurasidone in preparing a medicament for treating or preventing ischemia/reperfusion injury and a cytoprotective medicament. The invention discovers that lurasidone can obviously reduce myocardial infarction dead area and cerebral infarction volume, reduce serum creatine kinase activity, improve neurological functions, reduce myocardial cell and nerve cell death, has myocardial cell and nerve cell protection effect, and can be used for treating or preventing myocardial infarction and ischemic cerebral apoplexy.

Description

Lurasidone in the preparation of drugs and cells for treating or preventing ischemia/reperfusion injury Applications in protective medicine

Technical field

The invention relates to the application of lurasidone in the preparation of drugs and cell protection drugs for treating or preventing ischemia/reperfusion injury, and belongs to the field of biomedicine. The present invention provides new uses and administration methods of lurasidone, including the effect of resisting cardiac and cerebral ischemia/reperfusion injury (especially resisting myocardial infarction and ischemic stroke), and reducing cardiac and cerebral ischemia/reperfusion injury. Reperfusion injury has expanded the indications of lurasidone.

Background technique

Myocardial infarction is mainly caused by cardiac ischemia, which causes ischemia and hypoxia in myocardial cells, which can lead to irreversible damage to myocardial cells. Cerebral infarction, also known as ischemic stroke, is a blood supply disorder in local brain tissue caused by a variety of reasons, leading to brain tissue ischemia, hypoxic necrosis, and loss of neurological function. Ischemic stroke is a common and frequently-occurring disease that seriously endangers human health. It has become the first cause of disability and the third cause of death in the world. The incidence of ischemic stroke accounts for about 80% of cerebrovascular diseases.

One of the main measures in the treatment of infarction is to restore the perfusion and blood supply of the infarcted tissue as soon as possible, but reperfusion treatment is often accompanied by some tissue damage. Injury caused by ischemia and reperfusion is called "ischemia/reperfusion (I/R) injury". I/R injury involves energy metabolism disorders, calcium overload, oxidative stress, inflammatory response and other mechanisms, leading to various death modes such as apoptosis and necrosis of cells. Studies have shown that inhibiting apoptosis and/or necrosis can inhibit the death of myocardial cells and nerve cells caused by ischemia/reperfusion, reduce the degree of cardiac and cerebral ischemia/reperfusion injury, and reduce the scope of infarction.

Necroptosis is a caspase-independent regulated cell necrosis method, mainly composed of receptor-interacting protein kinase (RIPK) 1, RIPK3 and mixed lineage kinase domain-like protein (mixed lineage kinase domain -like, MLKL) mediated. Necroptosis is closely related to a variety of cardiovascular and cerebrovascular diseases, such as cardiocerebral ischemia/reperfusion injury, heart failure, drug-induced myocardial toxicity, atherosclerosis, liver and kidney damage, neurodegenerative diseases, etc. Therefore, inhibiting RIPK1/RIPK3/MLKL-dependent necroptosis can inhibit or reduce cell death and tissue damage caused by the above diseases. It has been reported in the literature that the RIPK1 inhibitor necrostatin-1 (Nec-1) can reduce ischemia/reperfusion injury in mice such as heart, brain, liver, and kidney. However, Nec-1 is only used as a tool drug in animal experiments, and there are currently no drugs targeting RIPK1/RIPK3/MLKL for clinical treatment.

Lurasidone (trade name: Latuda) is an atypical antipsychotic that antagonizes dopamine D2 and 5-hydroxytryptamine 2A (5-HT2A) receptors and is used for the treatment of patients with schizophrenia, but whether it has The anti-ischemia/reperfusion injury effect has not been reported yet.

In the present invention, unless otherwise stated, “lurasidone” shall be understood as “a compound of lurasidone or one of its semi-synthetic derivatives or their salts (salts of compounds or salts of semi-synthetic derivatives). ) or one of their esters (esters of compounds or esters of semi-synthetic derivatives) or one of their ester salts (salts of esters of compounds or esters of semi-synthetic derivatives)”.

Contents of the invention

In view of the shortcomings of the prior art, the object of the present invention is to provide the application of lurasidone in the preparation of drugs for treating or preventing ischemia/reperfusion injury; the second object of the present invention is to provide the application of lurasidone in the preparation of cell protection drugs. Applications in medicine.

In order to solve the above technical problems, the technical solutions of the present invention are as follows:

Application of lurasidone in the preparation of drugs and cytoprotective drugs for treating or preventing ischemia/reperfusion injury.

The structural formula of the lurasidone compound of the present invention is shown in Formula I, and the molecular formula is: C ₂₈ H ₃₆ N ₄ O ₂ S.

Further, the ischemia/reperfusion injury includes myocardial ischemia/reperfusion injury, cerebral ischemia/reperfusion injury, liver ischemia/reperfusion injury, renal ischemia/reperfusion injury, and lung ischemia/reperfusion injury. One or more of injury and intestinal ischemia/reperfusion injury.

Further, the myocardial ischemia/reperfusion injury includes one or more of chronic myocardial ischemia syndrome and myocardial infarction.

Further, the chronic myocardial ischemic syndrome includes one or more of latent coronary heart disease, stable angina pectoris, and ischemic cardiomyopathy.

Further, the myocardial ischemia/reperfusion injury includes myocardial infarction.

Further, the cerebral ischemia/reperfusion injury includes ischemic stroke (also called cerebral infarction).

Based on the same inventive concept, the present invention also provides the application of lurasidone in the preparation of cell protection drugs.

Furthermore, the cytoprotective drugs refer to drugs that have the effect of preventing, inhibiting or treating damage, degeneration or dysfunction of tissues, organs and cells.

Further, the cell protection drugs refer to drugs for preventing, inhibiting or treating cardiovascular system diseases, neurological diseases, ophthalmic diseases, respiratory system and digestive system; preferably, the cells include one of cardiomyocytes and nerve cells. Or several.

Further, the organ includes one or more of the brain, lung, heart, blood vessel, kidney, intestine, pancreas, skin, eye, and cornea.

Further, the cytoprotective drug is a drug used in myocardial ischemia/reperfusion injury and/or cerebral ischemia/reperfusion injury.

The inventor found that lurasidone can reduce the death of myocardial cells and nerve cells caused by myocardial ischemia and cerebral ischemia, significantly reduce the body (area) volume of cardiac and cerebral ischemic infarction, reduce serum creatine kinase activity and improve neurological function. It has scientific functions, reduces the death of cardiomyocytes and nerve cells, has a protective effect on cardiomyocytes and nerve cells, and its effect in reducing cardiac and cerebral ischemia/reperfusion injury is better than currently commonly used cardiovascular and cerebrovascular drugs.

Further, the administration method of the drug is one of intramuscular injection, subcutaneous injection, intravenous injection, intraperitoneal injection, oral administration, sublingual administration, intralesional or intracerebral or implanted delivery, and spray administration. or several, preferably intramuscular, subcutaneous injection, or intravenous injection.

Further, the drug is administered intramuscularly, subcutaneously or intravenously.

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

Further, the dosage form includes one of injections, capsules, tablets, granules, suspensions, emulsions, sprays, powders, liposomes, oral liquids, and dropping pills, among which the preferred dosage form is injection.

Optionally, lurasidone is a pharmaceutically acceptable salt thereof, and the pharmaceutically acceptable salt is a salt commonly used in pharmaceuticals. Further, the salt is selected from 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-dihydroxy One or more of benzoate, methanesulfonate, ethanesulfonate, benzenesulfonate, lunaryl sulfonate, hydroquinone sulfonate and p-toluenesulfonate.

The inventor's research found that lurasidone can specifically downregulate the expression and phosphorylation levels of RIPK1, RIPK3, and MLKL, and can be used as an inhibitor of RIPK1, RIPK3, and MLKL to inhibit cell necroptosis and reduce cell death.

The inventor unexpectedly discovered that lurasidone reduces the death of myocardial cells and nerve cells caused by myocardial ischemia/reperfusion and cerebral ischemia/reperfusion, significantly reduces the volume (area) of cardiac and cerebral ischemic infarction, and reduces serum Creatine kinase activity and improvement of neurological function, can downregulate the expression and phosphorylation levels of RIPK1, RIPK3, and MLKL, and can protect cardiomyocytes and nerve cells. The drug of the present invention is better than currently commonly used cerebrovascular drugs in reducing cardiac and cerebral ischemia/reperfusion injury, can be used to treat myocardial infarction and ischemic stroke, and has good development and application prospects.

The invention expands the indications of lurasidone, and is particularly applicable to myocardial infarction, ischemic stroke and cell protection.

The present invention relates to the application of lurasidone in preparing drugs for treating or preventing ischemia/reperfusion injury, which is disclosed for the first time. It can significantly reduce the area of myocardial infarction and cerebral infarction volume, reduce serum creatine kinase activity and improve neurological function. It is unexpected to reduce the death of cardiomyocytes and nerve cells and to have protective effects on cardiomyocytes and nerve cells. It has nothing to do with the known uses of lurasidone, and there are no other existing compounds that provide relevant enlightenment. It has outstanding The substantive characteristics have made significant progress in the treatment of ischemia/reperfusion injury and cell protection.

Description of the drawings

Figure 1 is a diagram of the effect of lurasidone on myocardial infarction area and serum creatine kinase activity in rats in Example 1; administration was administered for 30 minutes after ischemia, and relevant indicators were detected after 24 hours of reperfusion. A-lurasidone Picture of TTC staining and infarction area measurement of rat myocardial tissue after administration, and serum creatine kinase activity after administration of B-lurasidone.

Figure 2 is a diagram of the effect of lurasidone on cerebral infarction volume and neurological function in rats in Example 2; it was administered after 2 hours of ischemia and 1 hour of reperfusion, and relevant indicators were detected after 24 hours of reperfusion. A-rat brain Picture of tissue TTC staining and infarct volume measurement, picture of B-rat neurological function score.

Figure 3 shows the regulatory effect of lurasidone on MLKL and phosphorylated MLKL (p-MLKL) in rat brain tissue in Example 2.

Detailed ways

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

Application of lurasidone in the preparation of drugs for the treatment of ischemia/reperfusion injury and cell protection drugs.

Materials and methods:

In order to prove the role of lurasidone in resisting ischemia/reperfusion injury and cell protection, the applicant used a rat model of myocardial ischemia/reperfusion injury and an ischemic stroke rat model, and conducted Afterwards, the animals were treated with lurasidone at different time points, and the animals were sacrificed 24 hours after reperfusion. TTC staining and serum creatine kinase activity were used to detect cardiomyocyte damage, and neurobiological scores and TTC staining were used to detect nerve cell damage.

Experimental drug: lurasidone (a compound of lurasidone) was purchased from a reagent company and was dissolved and prepared according to the company's reagent instructions.

Example 1

Animal experiments: Protective effect of lurasidone on myocardial ischemia/reperfusion injury rat model.

Experimental animals:

Several healthy male SD rats weighing 250-300g. The experimental animals were raised for one week in an environment with a temperature of 25°C, a relative humidity of 60%, free access to water, and regular and quantitative administration, and then were administered drugs according to the requirements of each group in the experiment.

Method for establishing the rat myocardial ischemia/reperfusion model: SPF grade Sprague-Dawley rats were anesthetized by intraperitoneal injection of 3% sodium pentobarbital (0.1 mL/kg), fixed on a rat board, and the neck and left side were removed. chest hair. Connect the rat's limbs to the electrocardiograph and observe the changes in the rat's electrocardiogram during the operation. Bluntly separate the neck muscles to expose the trachea, cut a "T"-shaped incision on the trachea, insert the other end of the hose connected to the ventilator into the rat's trachea, and fix it with silk thread. The ventilator parameters were set to a tidal volume of 7 mL/kg and a frequency of 53 times per minute. After the rat's breathing calms down, use a hemostatic forceps to bluntly separate each layer of muscle in the third and fourth intercostal spaces of the left chest, fix the sternum, and gently open the pericardium to expose the entire heart. After the heartbeat returns to regularity, quickly squeeze out the heart. The obvious left coronary vein can be observed between the pulmonary artery cone and the left atrial appendage. Use a 5-0 suture to thread a piece of plastic hose to ligate the left anterior descending branch of the coronary artery, or ligate the left anterior descending coronary artery from the left Insert the needle 2-3mm below the atrial appendage to below the pulmonary artery cone, quickly reset the heart and quickly ligate it. The electrocardiogram changes of rats were monitored, and myocardial infarction was determined by indicators such as ST segment elevation and whitening of the apex of the heart that could be observed with the naked eye. After 1 hour of ischemia, cut the plastic hose at the heart ligation area, the ligation line will be disconnected, and the heart will resume blood flow perfusion. After 3 hours of reperfusion, the sternum at the thoracotomy site was cut, and 2-3 mL of blood was taken from the apex of the heart.

After apical blood sampling, the heart was removed, and all myocardial tissue above the ligation site was cut off, and the right ventricle and interventricular septum were removed. After freezing in a -20°C refrigerator for 30 minutes, take it out, cut the heart into uniformly thick slices, add 1% TTC dye solution (prepared in PBS), and incubate at 37°C for 15 minutes. After staining, remove the dye solution, wash once with PBS, fix with 4% paraformaldehyde for 24 hours, take out the heart sections, observe the staining condition and take photos. The infarct area appears white. The myocardial infarction area and risk area area are measured using ImageJ software, and the percentage of infarct area (Infarct Area, the ratio of the infarct area to the risk area area) of each slice is calculated.

Experimental grouping: The experimental animals were randomly divided into 4 groups, namely:

Sham operation group (Sham group): The rat heart was operated on, but no blood vessel ligation was performed;

Myocardial ischemia/reperfusion group (I/R group): ligation of the left anterior descending coronary artery for 1 hour, suture trimming, and reperfusion for 3 hours;

Lurasidone + myocardial ischemia/reperfusion group (Lurasidone + I/R): After 30 minutes of myocardial ischemia, rats were given intraperitoneal injection of lurasidone (lurasidone dose: 15 mg/kg).

Vehicle+myocardial ischemia/reperfusion group (Vehicle+I/R): The same volume of vehicle was given to rats after myocardial ischemia for 30 minutes.

Blood and myocardial tissue were collected and related indicators were measured: rat myocardial infarction area measurement and serum creatine kinase activity detection.

Serum creatine kinase activity (Creatine Kinase, CK activity) detection

After the ischemia/reperfusion surgery, collect 1-2 mL of blood from the apex of the heart at 3000 rpm, 4°C, centrifuge for 10 min, take the supernatant, and store at -40°C. The steps for measuring serum CK activity according to the instructions of the commercial kit are as follows: Take 10 mL R2 and dissolve a bottle of R1 to prepare a working solution. Take 4 μL of serum and add it to 200 μL of the CK kit working solution. Incubate for 2 min at 37°C and use a microplate reader. Set the wavelength to 340nm, read the absorbance A ₀ , A ₁ , A 2 , and A ₃ at 0, 1, ₂ , and 3 minutes respectively, calculate the change in average absorbance per minute (△A), and calculate the concentration of CK in serum Concentration (U/L).

Experimental results:

Lurasidone has the effect of resisting myocardial ischemia/reperfusion injury in rats, reducing the infarct area of rat myocardial tissue, reducing serum creatine kinase activity, and reducing myocardial cell death.

As shown in A in Figure 1, administration of lurasidone 30 minutes after ischemia can effectively reduce the infarct area of myocardial tissue caused by cardiac ischemia/reperfusion in rats; as shown in B in Figure 1, administration of lurasidone Westerone can significantly reduce serum creatine kinase activity. Data are expressed as mean ± standard error, n=6, **P＜0.01vs Sham, ^## P＜0.01vs I/R.

Experimental results:

Lurasidone can reduce myocardial cell death in rats, alleviate myocardial ischemia/reperfusion injury, has the effect of protecting myocardial cells, and can be used to prepare drugs for the treatment of myocardial ischemia/reperfusion injury.

Example 2

Animal experiments: Protective effect of lurasidone on ischemic stroke rat model.

Experimental animals: healthy male SD rats weighing 250-300g. The experimental animals were raised for one week in an environment with a temperature of 25°C, a relative humidity of 60%, free access to water, and regular and quantitative administration, and then were administered drugs according to the requirements of each group in the experiment.

Modeling method: The middle cerebral artery occlusion (MCAO) method was used to prepare the rat cerebral ischemia/reperfusion injury model. The steps are as follows: (1) Isolate the common carotid artery (CCA), external carotid artery (ECA) and internal carotid artery (ICA) on the left side of the rat; (2) Use ophthalmic tweezers to temporarily clamp the ECA and ICA, and ligate the CCA Proximal end; (3) Place a knotted spare silk thread at the distal end of the CCA, cut a small opening at the lower end of the thread, insert the suture into the internal carotid artery, tighten the silk thread, and release the arteries on the ECA and ICA Clip, and send the suture along the ICA into the skull; (4) Stop when resistance is encountered, and the insertion depth is about 18 to 20 mm from the bifurcation of the CCA; (5) After 120 minutes of ischemia, pull out the suture. The skin was sutured and animals were processed after reperfusion for 24 h.

The model success evaluation criterion uses Longa's "5-point method" to score the neurological deficits of cerebral ischemic injury in rats. 0 points: no neurological deficit symptoms; 1 points: the right forelimb cannot be fully straightened; 2 points: the rat rotates to the right when walking; 3 points: the rat tilts to the right when walking; 4 points: cannot walk spontaneously and loses consciousness. Scores of 1 to 4 are considered effective models.

TTC staining and infarct volume determination of rat brain. After the rat was anesthetized, the brain was quickly taken out, the olfactory bulb and hindbrain were removed, and the brain tissue was cut into coronal brain slices about 2 mm thick starting from the frontal pole, immediately placed in 1% TTC solution, and incubated in the dark at 37°C for 30 min. Then soak and fix with 10% paraformaldehyde solution. The infarcted area appears white and the non-infarcted area appears red. Arrange each group of brain slices neatly before scanning. Then use ImageJ to measure the infarct area of each brain slice. According to the formula: infarct volume = [(front infarction area of each slice + infarct area on the back of each slice)/2] × thickness of each slice. In the same way, the whole brain infarct volume is calculated. The sum of infarct volumes in brain slices.

Western blot (WB) detected the expression levels of MLKL and phosphorylated MLKL. Take an appropriate amount of brain tissue and wash it with pre-cooled PBS. Add magnetic beads to the homogenization tube, place it on ice to pre-cool, and add the washed brain tissue to the homogenization tube for homogenization (70Hz, 180s). Centrifuge at 12,000 rpm for 10 minutes at 4°C and retain the supernatant. After measuring the protein concentration using the BCA method, 20-40ug of protein was separated using 8-10% SDS-PAGE gel and transferred to a PVDF membrane, and used with MLKL and p-MLKL (Abcam, Cambridge, UK) antibodies and β-actin. (Beyotime, Jiangsu) antibody was incubated overnight. The antibody was removed and the membrane was washed. After incubation with the corresponding secondary antibody, it was developed by Molecular Imager ChemiDoc XRS System (Bio-Rad, Philadelphia, PA). β-actin was used as an internal control. The protein gray value was measured by Image J software to evaluate the protein expression level.

Experimental grouping: The experimental animals were randomly divided into 4 groups, namely:

Sham operation group (Sham group): Surgery to separate the internal and external carotid arteries without inserting a thread into the artery.

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

Lurasidone + cerebral ischemia/reperfusion group (lurasidone + I/R): intramuscular injection of lurasidone was given after 2 hours of ischemia, 1 hour, and 6 hours of reperfusion (each administration of lurasidone The amount is 10mg/kg).

Vehicle + cerebral ischemia/reperfusion group (Vehicle+I/R): The same volume of vehicle was given after 2 hours of ischemia, 1 hour, and 6 hours of reperfusion.

Detect neurological function scores and determine cerebral infarct volume in rats.

result:

Lurasidone has the effect of resisting cerebral ischemia/reperfusion injury in rats, reducing cerebral infarction volume, improving neurological deficit symptoms, and reducing nerve cell death.

As shown in A in Figure 2, the I/R group had obvious white infarcts. Rats given lurasidone after 2 hours of ischemia, 1 hour, and 6 hours of reperfusion significantly reduced the cerebral infarct volume and significantly alleviated cerebral ischemia/reperfusion. damage. As shown in B in Figure 2, lurasidone can significantly improve the symptoms of neurological deficit (n=6, **P<0.01vs Sham, ^## P<0.01vs I/R).

As shown in Figure 3, cerebral ischemia/reperfusion induces the upregulation of MLKL and p-MLKL (phosphorylated MLKL) in rat brain tissue, while lurasidone significantly inhibits the upregulation of MLKL and p-MLKL.

Conclusion: Lurasidone has the effect of inhibiting necroptosis, reducing nerve cell death, and alleviating cerebral ischemia/reperfusion injury. It can be used in the preparation of drugs to reduce cerebral ischemia/reperfusion injury and for the treatment of ischemic disease. Stroke.

However, the present invention is not limited to heart and brain ischemia/reperfusion injury. Since the mechanism of liver, kidney, lung and intestinal ischemia/reperfusion injury is similar to that of heart and brain ischemia/reperfusion injury, the drug is also the same. Indicated for the treatment of liver, kidney, lung and intestinal ischemia/reperfusion injury.

The content illustrated in the above embodiments should be understood that these embodiments are only used to illustrate the present invention more clearly, and are not used to limit the scope of the present invention. After reading the present invention, those skilled in the art will be familiar with various equivalent forms of the present invention. All modifications fall within the scope defined by the appended claims of this application.

Claims (10)

1. Use of lurasidone in the manufacture of a medicament for the treatment or prevention of ischemia/reperfusion injury.

2. The use according to claim 1, wherein the ischemia/reperfusion injury comprises one or more of myocardial ischemia/reperfusion injury, cerebral ischemia/reperfusion injury, liver ischemia/reperfusion injury, kidney ischemia/reperfusion injury, lung ischemia/reperfusion injury, and intestinal ischemia/reperfusion injury.

3. The use according to claim 2, wherein the myocardial ischemia/reperfusion injury comprises one or more of chronic myocardial ischemia syndrome, myocardial infarction.

4. The use according to claim 3, wherein the myocardial ischemia/reperfusion injury comprises myocardial infarction.

5. The use according to claim 2, wherein the cerebral ischemia/reperfusion injury comprises ischemic stroke.

6. Use of lurasidone in the preparation of a cytoprotective medicament.

7. The use according to claim 6, wherein the cytoprotective drug is a drug having the effect of preventing, inhibiting or treating injury, degeneration or dysfunction of tissues, organs and cells.

8. The use according to claim 6, wherein the cytoprotective drug is a drug for preventing, inhibiting or treating cardiovascular diseases, nervous system diseases, ophthalmic diseases, respiratory system and digestive system; preferably, the cells include one or more of cardiomyocytes and nerve cells.

9. The use according to claim 7, wherein the organ comprises one or more of brain, lung, heart, blood vessel, kidney, intestine, pancreas, skin, eye, cornea.

10. The use according to claim 6, wherein the cytoprotective drug is a drug for use in myocardial ischemia/reperfusion injury and/or cerebral ischemia/reperfusion injury.