A pharmaceutical composition for preventing or treating ischemia / reperfusion injury and use thereof

Abstract

The present application relates to a kind of drug composition and application for preventing or treating ischemia / reperfusion injury, the active ingredient of the drug composition is composed of active ingredient A and active ingredient B, the active ingredient A is telaprevir or its pharmaceutically acceptable salt, the active ingredient B is nadolol or its pharmaceutically acceptable salt, the weight ratio of the active ingredient A and active ingredient B is 1:0.001-1000.The drug composition of the present application can more effectively prevent and / or treat ischemia / reperfusion injury, especially treat nerve cell damage, more especially the protective effect on ischemic stroke, can significantly reduce cerebral ischemia / reperfusion injury.

Description

Technical Field

[0001] This invention relates to a pharmaceutical composition and its application for the prevention or treatment of ischemia / reperfusion injury, belonging to the field of biomedicine. Background Technology

[0002] Stroke is an acute cerebrovascular disease caused by various factors that reduce or interrupt blood supply to the brain, ultimately leading to cell death in the ischemic area. Stroke is characterized by high incidence, high mortality, high teratogenicity, and high recurrence rate, and has become the second leading cause of death and a major cause of disability worldwide. Based on different clinical symptoms, stroke is divided into hemorrhagic stroke and ischemic stroke, with ischemic stroke accounting for approximately 80%.

[0003] Cerebral ischemia-reperfusion injury is related to multiple mechanisms, including excitatory amino acid toxicity, intracellular calcium overload, excessive free radical formation, and inflammatory responses. Regardless of the mechanism, the ultimate outcome is neuronal death, functional impairment, and the formation of cerebral infarction foci. Cell death primarily occurs through two pathways: apoptosis and necrosis. In cerebral ischemia-reperfusion injury, neuronal apoptosis and necrosis coexist. Apoptosis mainly includes the intrinsic apoptosis pathway (mitochondrial-mediated apoptosis pathway) and the extrinsic apoptosis pathway (death receptor-mediated apoptosis pathway). The caspase family is a large class of apoptosis regulators, acting as both initiators and final executors of apoptosis. During cerebral ischemia, the death receptor-mediated apoptosis pathway activates caspase-8, which further activates its downstream effector protease caspase-3 and activates the mitochondrial apoptosis pathway, leading to apoptosis. Studies have shown that inhibiting the activity of caspase8 and caspase3 can prevent the occurrence and development of neuronal apoptosis, reduce the severity of cerebral ischemia-reperfusion injury, and shrink the infarct size.

[0004] Studies have shown that RIPK1 / RIPK3 / MLKL-dependent necroptosis exists in various injury-related diseases, including ischemic stroke, myocardial infarction, and hepatic and renal ischemia / reperfusion injury. Inhibition of RIPK1 / RIPK3-dependent necroptosis, such as the RIPK1 inhibitor necrostatin-1 (Nec-1), can reduce cerebral ischemia injury and improve neurological function in mice. In cases of cerebral ischemia / reperfusion injury, simultaneous inhibition of caspase 8, caspase 3 enzyme activities, and the RIPK1 / RIPK3 / MLKL pathway can synergistically inhibit neuronal apoptosis and necroptosis, significantly alleviating ischemia / reperfusion injury, while also reducing the dosage and adverse reactions of individual drugs.

[0005] Telaprevir is an inhibitor of hepatitis C virus (HCV) NS3 / 4A serine proteases, possessing antiviral activity by inhibiting HCV replication. The applicant's research found that telaprevir inhibits caspase 8 and caspase 3, reducing cardiomyocyte and neuronal cell death caused by myocardial and cerebral ischemia / reperfusion, significantly reducing the infarct size in myocardial and cerebral ischemia, decreasing serum creatine kinase activity, and improving neurological function, thus exhibiting protective effects against cardiomyocytes and neurons.

[0006] Nadolol (trade name Corgard) is a beta-blocker that competitively inhibits catecholamines and is used to treat hypertension. It is also effective against rapid arrhythmias caused by elevated catecholamine levels. The applicant's research found that nadolol can downregulate the expression and phosphorylation levels of RIPK1, RIPK3, and MLKL in brain tissue, significantly reducing infarct volume, improving neurological function, and decreasing neuronal death, thus exhibiting neuroprotective effects.

[0007] However, it remains unclear whether telaprevir and naldolol have a synergistic effect when used together, or whether they can achieve better results in treating ischemia / reperfusion injury. Currently, there are no reports on the combined use of telaprevir and naldolol, especially not on their combined application for the prevention and treatment of ischemia / reperfusion injury. Summary of the Invention

[0008] In view of the shortcomings of the prior art, one objective of the present invention is to provide a pharmaceutical composition for the prevention or treatment of ischemia / reperfusion injury; another objective of the present invention is to provide the application of the pharmaceutical composition in the preparation of drugs for the prevention or treatment of ischemia / reperfusion injury and cell protection.

[0009] To solve the above-mentioned technical problems, the technical solution of the present invention is as follows:

[0010] A pharmaceutical composition for the prevention or treatment of ischemia / reperfusion injury, wherein the active ingredients of the pharmaceutical composition consist of active ingredient A and active ingredient B, wherein active ingredient A is telaprevir or a pharmaceutically acceptable salt thereof, and active ingredient B is naldolol or a pharmaceutically acceptable salt thereof.

[0011] Generally, the structural formula of telaprevir is shown in Formula I, and its molecular formula is C10. 36 H 53 N7O6:

[0012]

[0013] The structural formula of naldolol is shown in Formula II, and its molecular formula is C. 17 H 27NO4:

[0014]

[0015] Furthermore, the weight ratio of active ingredient A to active ingredient B is 1:0.001-1000.

[0016] Furthermore, the weight ratio of active ingredient A to active ingredient B is 1:0.01-100.

[0017] Furthermore, the weight ratio of active ingredient A to active ingredient B is 1:0.03-25.

[0018] Furthermore, the weight ratio of active ingredient A to active ingredient B is 1:0.04-10.

[0019] Furthermore, the weight ratio of active ingredient A to active ingredient B is 1:0.05-0.1.

[0020] Optionally, 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, lecithin sulfonate, hydroquinone sulfonate, and p-toluenesulfonate.

[0021] Furthermore, the pharmaceutical composition can be prepared into any pharmaceutically acceptable dosage form according to known techniques, with the preferred dosage form being an injection, such as an injection, capsule, tablet, granule, powder, spray, liposome, oral liquid, or pellet.

[0022] Based on the same inventive concept, the present invention also provides the use of the pharmaceutical composition described above in the preparation of medicaments for the prevention or treatment of ischemia / reperfusion injury.

[0023] Furthermore, the ischemia / reperfusion injury includes one or more of the following: cerebral ischemia / reperfusion injury, myocardial ischemia / reperfusion injury, hepatic ischemia / reperfusion injury, and renal ischemia / reperfusion injury.

[0024] Furthermore, the ischemia / reperfusion injury is cerebral ischemia / reperfusion injury, preferably, the cerebral ischemia / reperfusion injury is ischemic stroke.

[0025] Furthermore, the administration method in the application is one or more of the following: intramuscular injection, subcutaneous injection, intravenous injection, oral administration, sublingual administration, intralesional or intrabrain delivery device, or spray administration, preferably intramuscular injection.

[0026] Based on the same inventive concept, the present invention also provides the use of the pharmaceutical composition described above in the preparation of cell protection drugs.

[0027] Preferably, the cells are one or more types of cardiomyocytes and nerve cells.

[0028] Optionally, cell-protective drugs include drugs used to resist cell death or processes that cause cell death in organs or cells such as the nervous system, brain, heart, and eyes, for example, drugs used to treat diseases caused by necrosis and / or pathological apoptosis and / or necroptosis and / or pyroptosis and / or autophagy.

[0029] Furthermore, the cell-protective drug refers to a drug that has the effect of preventing, inhibiting or treating damage, degeneration or dysfunction of tissues, organs and cells.

[0030] Furthermore, the organs include one or more of the following: brain, lungs, heart, blood vessels, kidneys, pancreas, skin, eyes, cornea, and joints.

[0031] Furthermore, the cell-protective drug is a drug used to treat or prevent myocardial cell damage or nerve cell damage; or, the cell-protective drug is a drug used to prevent, inhibit, or treat cardiovascular diseases, nervous system diseases, or ophthalmic diseases.

[0032] Optionally, the cell-protective drug is a drug used to treat or prevent myocardial cell damage or nerve cell damage.

[0033] Preferably, the organs targeted by this invention include the nervous system, brain, and heart. One object of this invention is to provide the application of a pharmaceutical composition of terabitvir and naldolol, which can be used to prevent and / or treat cell death caused by processes that resist cell death or lead to cell death, such as necrosis and / or pathological apoptosis and / or necroptosis and / or pyroptosis and / or autophagy.

[0034] Furthermore, the pharmaceutical composition can protect, prevent, and / or treat cells against cell death or processes that lead to cell death, including inhibiting nerve cell damage and death, such as treating and preventing neurological diseases, glaucoma, retinitis pigmentosa, corneal reticular dystrophy, age-related macular degeneration (AMD), wet or dry AMD-related photoreceptor degeneration, other retinal degenerations, optic neuropathy and optic neuritis, optic drusen, stroke, Alzheimer's disease, Parkinson's disease, Huntington's disease, Parkinson's plus syndrome, focal ischemia, amyotrophic lateral sclerosis (ALS), intracranial hemorrhage, cerebral hemorrhage, trigeminal neuralgia, glossopharyngeal neuralgia, myasthenia gravis, muscular atrophy, progressive muscular atrophy, Bell's palsy, progressive bulbar palsy, spinal muscular atrophy, primary muscular atrophy. Lateral sclerosis (PLS), pseudobulbar palsy, invertebral disc syndrome, cervical spondylosis, hereditary muscular atrophy, plexus disorder, thoracic outlet destruction syndrome, porphyria, peripheral neuropathy, multiple system atrophy, corticobasal degeneration, progressive supranuclear palsy, Lewy body dementia, demyelinating diseases, frontotemporal dementia, Gullman-Barré syndrome, multiple sclerosis, Kreutzfeldt-Jacob disease, progressive neurogenic peroneal muscular atrophy, prion disease, fatal familial insomnia (FFI), G.S.-S.S. syndrome (GSS), bovine spongiform encephalopathy, epilepsy, Pick's disease, AIDS dementia syndrome, neurological damage caused by exposure to toxic compounds in groups consisting of industrial solvents, heavy metals, drugs and chemotherapeutic agents; neurological damage caused by mechanical, physical or chemical trauma.

[0035] Furthermore, the pharmaceutical composition can protect, prevent, and / or treat cells against cell death or processes that cause cell death, including inhibiting cardiomyocyte damage and death, such as in the treatment and prevention of cardiovascular diseases, myocardial ischemia and / or vascular ischemia, myocardial infarction, ischemic heart disease, myocardial remodeling, chronic or acute heart failure, hypertrophic cardiomyopathy, and cardiac side effects caused by drug treatment (especially anticancer drugs).

[0036] The application of the drugs described above in the preparation of drugs that reduce cardiovascular toxicity caused by anticancer drugs, wherein the anticancer drugs include, but are not limited to, anthracycline antibiotics, tyrosine kinase inhibitors, fluorouracils, VEGF signaling pathway inhibitors, immune checkpoint inhibitors, platinum-based antitumor drugs, and other antitumor drugs.

[0037] Furthermore, the aforementioned anticancer drugs include anthracycline antibiotics such as doxorubicin, epirubicin, pirarubicin, arubicin, idarubicin, daunorubicin, mitoxantrone, etc.; tyrosine kinase inhibitors such as ninotinib, sunitinib, lapatinib, regorafenib, ponatinib, and dasatinib; fluorouracil derivatives such as fluorouracil, capecitabine, tegafur, and tegafur; VEGF signaling pathway inhibitors such as bevacizumab; immune checkpoint inhibitors such as trastuzumab; platinum-based antitumor drugs such as cisplatin; and other antitumor drugs such as paclitaxel and cyclophosphamide.

[0038] The applicant's research found that naldolol can downregulate the expression and phosphorylation levels of RIPK1, RIPK3, and MLKL in brain tissue, significantly reduce cerebral infarction volume, improve neurological function, reduce neuronal cell death, and has a neuroprotective effect.

[0039] The inventors unexpectedly discovered that the combined use of terabitvir and naldolol for the prevention or treatment of cerebral ischemia / reperfusion injury is more effective than monotherapy, and the two drugs exhibit a synergistic effect, which helps to reduce drug dosage and adverse drug reactions.

[0040] The pharmaceutical composition of this invention synergistically prevents and / or treats ischemia / reperfusion injury, particularly neuronal cell damage, and especially provides protection against ischemic stroke. It significantly reduces cerebral ischemia / reperfusion injury while simultaneously reducing the dosage of individual drugs and mitigating adverse drug reactions. Currently, there are no such treatment methods or drugs. Furthermore, there are no reports in the prior art regarding the combined use of telaprevir and naldolol, especially not regarding their combined application for the prevention and treatment of ischemia / reperfusion injury.

[0041] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0042] (1) The pharmaceutical composition of the present invention can more effectively prevent or treat ischemia / reperfusion injury, especially cerebral ischemia / reperfusion injury, improve the long-term survival rate of patients and reduce the disability rate, and is applicable to cerebral ischemia / reperfusion injury.

[0043] (2) Reduce adverse drug reactions.

[0044] The inventors' research is the first to discover that terabitvir has inhibitory effects on caspase 3 and caspase 8, while naldolol can specifically downregulate RIPK1 / RIPK3 / MLKL protein and phosphorylation levels, inhibiting necrosis-like apoptosis. The combined use of terabitvir and naldolol can synergistically prevent and treat ischemic stroke, significantly reducing cerebral ischemia / reperfusion injury. Compared with naldolol or terabitvir alone, the inhibitory effect is better, effectively reducing the infarct volume in rats, improving neurological function, and exhibiting significant anti-ischemic stroke effects. Attached Figure Description

[0045] Figure 1 Figure A shows the TTC staining and infarct volume measurement of rat brain tissue.

[0046] Figure 1 B shows the rat's neurological function score.

[0047] Figure 2 The diagram shows the regulatory effects of naldolol on MLKL and phosphorylated MLKL (p-MLKL) in rat brain tissue. Detailed Implementation

[0048] The present invention will now be described in detail with reference to the accompanying drawings and embodiments. It should be noted that, unless otherwise specified, the embodiments and features described herein can be combined with each other.

[0049] Example 1

[0050] Animal studies: The protective effect of the combination of telaprevir and naldolol against ischemic stroke.

[0051] Experimental animals: Healthy male SD rats weighing 250–300 g. The experimental animals were housed for one week in an environment with a temperature of 25°C, relative humidity of 60%, free access to water, and regular and quantitative administration. Then, they were administered the drugs orally according to the experimental group requirements.

[0052] Modeling method: A rat model of cerebral ischemia / reperfusion injury was prepared by middle cerebral artery occlusion (MCAO). 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) Temporarily clamp the ECA and ICA with ophthalmic forceps and ligate the proximal end of the CCA; (3) Place a knotted spare suture at the distal end of the CCA, cut a small hole at the lower end of the suture, insert the suture into the internal carotid artery, tighten the suture, release the arterial clamps on the ECA and ICA, and push the suture into the cranium along the ICA; (4) Stop when resistance is encountered. The insertion depth is about 18-20 mm, starting from the bifurcation of the CCA; (5) After 120 min of ischemia, pull out the suture, suture the skin, and reperfuse for 24 h before processing the animal.

[0053] The Longa 5-point scale was used to score the neurological deficits in rats with cerebral ischemia-reperfusion injury. 0 points: no neurological deficit symptoms; 1 point: right forelimb cannot be fully extended; 2 points: rat walks with a rightward rotation; 3 points: walks with a rightward tilt; 4 points: unable to walk spontaneously, loss of consciousness. Scores of 1-4 indicate a valid model.

[0054] TTC staining and infarct volume determination in rat brains. After anesthetizing the rats, the brains were quickly removed, the olfactory bulb and hindbrain were discarded, and five coronal slices, approximately 2.0 mm thick, were cut from the frontal pole. These slices were immediately placed in a 1% TTC solution and incubated at 37°C in the dark for 30 min. Then, they were fixed by soaking in a 10% paraformaldehyde solution. Infarcted areas appeared white, and non-infarcted areas appeared red. Each group of brain slices was neatly arranged and scanned. The infarct area of ​​each slice was measured using ImageJ. The total brain volume was calculated using the formula: Infarct volume = [(Sum of infarct areas on the front of each slice + Sum of infarct areas on the back of each slice) / 2] × thickness of each slice.

[0055] Western blot (WB) was used to detect the expression levels of MLKL and phosphorylated MLKL. A suitable amount of brain tissue was taken and washed with pre-chilled PBS. Magnetic beads were added to the homogenization tube, which was then pre-chilled on ice. The tissue was added to the homogenization tube and homogenized (70 Hz, 180 s). The mixture was centrifuged at 12000 rpm for 10 min at 4 °C, and the supernatant was collected. After determining the protein concentration using the BCA method, 20–40 μg of protein was separated using an 8–10% SDS-PAGE gel and transferred to a PVDF membrane. The membrane was incubated overnight with MLKL and p-MLKL (Abcam, Cambridge, UK) antibodies and β-actin (Beyotime, Jiangsu) antibody. After incubation with the corresponding secondary antibodies, the images were developed using a Molecular Imager ChemiDoc XRS System (Bio-Rad, Philadelphia, PA), with β-actin used as an internal control. The protein grayscale value was measured using ImageJ software to evaluate the protein expression level.

[0056] Experimental grouping: The experimental animals were randomly divided into 6 groups, namely:

[0057] The rats were divided into four groups in order: sham operation group, ischemia / reperfusion group, telaprevir + ischemia / reperfusion group, naldolol + ischemia / reperfusion group, combined drug + ischemia / reperfusion group, and solvent group + ischemia / reperfusion group. Each group of rats was administered the drug 2 hours after ischemia and 1 hour after reperfusion.

[0058] Sham surgery group: The left common carotid artery, internal carotid artery and external carotid artery were separated without inserting a suture.

[0059] Ischemia / reperfusion group (I / R): cerebral ischemia for 2 hours, reperfusion for 24 hours.

[0060] The rat groups included: telaprevir (60 mg / kg) + cerebral ischemia / reperfusion group, nadolol (5 mg / kg) + cerebral ischemia / reperfusion group, telaprevir (60 mg / kg) + nadolol (5 mg / kg) + cerebral ischemia / reperfusion group, and solvent + cerebral ischemia / reperfusion group. Drug administration was performed 1 hour after reperfusion following 2 hours of ischemia. Six rats were in each group (n=6). For detailed experimental results, please refer to [link to relevant documentation]. Figure 1 or Figure 2 .

[0061] Detection indicators: rat neurological function score and infarct volume measurement (see...) Figure 1 Western blot analysis was performed to detect the expression levels of MLKL and phosphorylated MLKL (see...). Figure 2 ).

[0062] Experimental results:

[0063] Effects on cerebral infarction volume and neurological function in rats

[0064] Figure 1 As shown in Figure A, the I / R group had obvious white infarct foci, while the infarct foci in the drug-only group were significantly reduced, significantly alleviating cerebral ischemia-reperfusion injury (**P<0.01 vs sham-operated group). # P<0.05 vs I / R group ## P<0.01 vs I / R group), while the effect of combination therapy was significantly better than that of monotherapy (**P<0.01 vs sham surgery group). ++ P<0.01 vs I / R + drug alone group).

[0065] like Figure 2 As shown, ischemia / reperfusion induces upregulation of MLKL and p-MLKL in rat brain tissue, while naldolol significantly inhibits the upregulation of MLKL and p-MLKL.

[0066] However, this invention is not limited to cerebral ischemia / reperfusion injury. Since ischemia / reperfusion injury of the heart, liver and kidneys also involves Caspase upregulation and necrosis-like apoptosis activation (RIPK1 / RIPK3 / MLKL), it can be inferred that this drug is also suitable for treating ischemia / reperfusion injury of the heart, liver and kidneys.

[0067] The above embodiments should be understood as being used only to illustrate the present invention more clearly, and not to limit the scope of the present invention. After reading the present invention, any modifications of the present invention in various equivalent forms by those skilled in the art fall within the scope defined by the appended claims.

Claims

1. The use of the pharmaceutical composition in the preparation of a medicament for treating cerebral ischemia / reperfusion injury, characterized in that, The active ingredients of the pharmaceutical composition consist of active ingredient A and active ingredient B, wherein active ingredient A is telaprevir or a pharmaceutically acceptable salt thereof, and active ingredient B is naldolol or a pharmaceutically acceptable salt thereof, and the weight ratio of active ingredient A to active ingredient B is 1:0.05-0.

1.

2. The application according to claim 1, characterized in that, The cerebral ischemia / reperfusion injury is referred to as ischemic stroke.

3. The application of the pharmaceutical composition in the preparation of cell-protective drugs; characterized in that, The cells are nerve cells, and the cell-protective drug is a drug used to treat cell damage caused by cerebral ischemia / reperfusion injury. The active ingredients of the pharmaceutical composition consist of active ingredient A and active ingredient B. Active ingredient A is telaprevir or a pharmaceutically acceptable salt thereof, and active ingredient B is naldolol or a pharmaceutically acceptable salt thereof. The weight ratio of active ingredient A to active ingredient B is 1:0.05-0.1.

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