CN112516285B - Pharmaceutical composition for preventing or treating ischemia/reperfusion injury and application thereof - Google Patents

Abstract

The present invention relates to a pharmaceutical composition and application for preventing or treating ischemia/reperfusion injury, comprising an active ingredient, the active ingredient is composed of an active ingredient A and an active ingredient B, and the active ingredient A is enricasan or A pharmaceutically acceptable salt thereof, the active ingredient B is an echinocandin class drug or a pharmaceutically acceptable salt thereof, and the weight ratio of the active ingredient A and the active ingredient B is 1:0.1-10. The pharmaceutical composition of the present invention can synergistically prevent and/or treat ischemia/reperfusion injury, especially nerve cell injury, more especially the protection of ischemic stroke, and can significantly reduce cerebral ischemia/reperfusion damage.

Description

A kind of pharmaceutical composition and application for preventing or treating ischemia/reperfusion injury

technical field

The invention relates to a pharmaceutical composition and application for preventing or treating ischemia/reperfusion injury, and belongs to the field of biomedicine.

Background technique

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 70-80% of cerebrovascular diseases.

Cerebral ischemic injury is related to various mechanisms, such as excessive formation of free radicals, the toxicity of excitatory amino acids, intracellular calcium overload, and inflammatory reactions. Regardless of the mechanism, the final outcome will lead to nerve cell death, functional destruction, and cerebral infarction. form. There are two main ways of cell death: apoptosis and necrosis. Neuronal apoptosis and necrosis coexist in cerebral ischemic injury. The ischemic core area is dominated by cell necrosis, while the surrounding light and dark zones are dominated by cell apoptosis.

Apoptosis mainly includes endogenous apoptosis pathway (mitochondria-mediated apoptosis pathway) and exogenous apoptosis pathway (death receptor-mediated apoptosis pathway). Caspase family is a large class of regulators of apoptosis, the initiator and final executor 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 caspase enzyme can prevent the development of neuronal apoptosis, reduce the degree of cerebral ischemic injury, and reduce the scope of infarction. The caspase inhibitor, Emricasan, can specifically inhibit caspase-8, caspase-3, and caspase-7, and reduce cerebral ischemia/reperfusion injury.

Studies have shown that necrosis is also tightly regulated by various signaling pathways, called regulated necrosis, among which RIPK1/RIPK3/MLKL-dependent necroptosis has received the most attention. Studies have shown that RIPK1/RIPK3/MLKL-dependent necroptosis is present in a variety of injury-related diseases, including ischemic stroke, myocardial infarction, and renal ischemia/reperfusion injury. Therefore, inhibition of RIPK1/RIPK3-dependent necroptosis has become an effective way to reduce neuronal cell death in ischemic stroke. It has been reported in the literature that the RIPK1 inhibitor necrostatin-1 (Nec-1) can reduce cerebral ischemia injury in mice and improve neurological function, which is closely related to the inhibition of programmed necrosis of nerve cells. However, Nec-1 is only used as a tool drug in animal experiments, and there is currently no clinical drug targeting RIPK1/RIPK3/MLKL for the treatment of ischemic stroke.

In the case of cerebral ischemia/reperfusion injury, if simultaneously inhibiting caspase activity and RIPK1/RIPK3/MLKL pathway can synergistically inhibit neuronal apoptosis and necroptosis, greatly reduce ischemia/reperfusion injury, and at the same time reduce neuronal apoptosis and necroptosis. Reduce the dose of individual drugs and reduce the adverse reactions of light individual drugs.

In our previous study, we found for the first time that the combination of the caspase inhibitor Enricasan and ponatinib, which inhibits the RIPK1/RIPK3/MLKL pathway, has a significant anti-ischemic stroke effect and greatly reduces the lack of Blood/reperfusion injury. However, ponatinib is a tyrosine kinase inhibitor, which has side effects such as vascular thrombosis and cardiotoxicity, which seriously affects the use of ponatinib in stroke. Therefore, the search for cytoprotective drugs that can inhibit the RIPK1/RIPK3/MLKL pathway and The combined application of Enricason has become an urgent problem to be solved.

Echinocandins antifungal drugs can inhibit the formation of fungal cell wall, and through non-competitive inhibition of glucan synthase, resulting in the lack of cell wall glucan during the growth of fungal cells, abnormal osmotic pressure, and ultimately fungal cell lysis. Echinocandins antifungal drugs include caspofungin, micafungin, and anidulafungin, which have good inhibitory activity against Candida and Aspergillus, and are currently mainly used for Treatment of invasive Aspergillus. The applicant has previously found that echinocandins such as micafungin and caspofungin can be used to treat ischemia/reperfusion injury, but it has not been considered to be used in combination with other active ingredients to obtain better effects.

SUMMARY OF THE INVENTION

In view of the deficiencies of the prior art, one of the objects of the present invention is to provide a pharmaceutical composition for preventing or treating ischemia/reperfusion injury; the second object of the present invention is to provide a pharmaceutical composition for preventing or treating ischemia/reperfusion injury. Drug application in reperfusion injury.

The technical scheme of the present invention is as follows:

A pharmaceutical composition for preventing or treating ischemia/reperfusion injury, comprising an active ingredient, the active ingredient is composed of an active ingredient A and an active ingredient B, and the active ingredient A is enricasan or its pharmaceutically acceptable The salt, the active ingredient B is echinocandins or a pharmaceutically acceptable salt thereof, and the weight ratio of the active ingredient A and the active ingredient B is 1:0.1-10.

Further, the weight ratio of the active ingredient A and the active ingredient B is 1:0.2-5.

Further, the weight ratio of the active ingredient A and the active ingredient B is 1:0.4-3.5.

Further, the weight ratio of the active ingredient A and the active ingredient B is one of 1:0.4, 1:1, and 1:3.2.

Further, the echinocandins are one or more of caspofungin, micafungin and anidungin.

Generally, the structural formula of Enricason is shown in formula I

The structural formula of caspofungin is shown in formula II:

The structural formula of micafungin is shown in formula III:

The structural formula of micafungin is shown in formula IV:

Optionally, the pharmaceutically acceptable salt is a pharmaceutically acceptable salt, further, the salt is selected from acetate, benzoate, fumarate, maleate, citrate, tartrate , one of 2,5-dihydroxybenzoate, methanesulfonate, ethanesulfonate, benzenesulfonate, moonsulfonate, hydroquinonesulfonate and p-toluenesulfonate or several.

Further, the pharmaceutical composition can be prepared into any pharmaceutically acceptable dosage form according to known techniques, wherein the preferred dosage form is injection, such as injection, capsule, tablet, granule, spray, powder, lipid One of the body, oral liquid and dripping pills.

Use of the above-mentioned pharmaceutical composition in the preparation of a medicament for preventing or treating ischemia/reperfusion injury.

Further, the mode of administration in the application is one of intramuscular injection, subcutaneous injection, intravenous injection, oral administration, sublingual administration, intralesional or intracerebral or implanted delivery device, and spray administration. Several, preferably intramuscular injection.

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

Further, the ischemia/reperfusion injury is cerebral ischemia/reperfusion injury.

Further, the cerebral ischemia/reperfusion injury includes ischemic stroke.

The inventors of the present invention unexpectedly found that the combined use of Enricason and echinocandins for the prevention or treatment of cerebral ischemia/reperfusion injury is more effective than the single drug in the prevention or treatment. Shows a synergistic effect, which can reduce drug dosage and adverse drug reactions.

The pharmaceutical composition of the present invention can synergistically prevent and/or treat ischemia/reperfusion injury, especially nerve cell injury, more especially the protection of ischemic stroke, and can significantly reduce cerebral ischemia/reperfusion At the same time, it can reduce the dose of the single drug and alleviate the adverse reactions of the single drug, but there is no such treatment method and drug at present. At present, there is no report on the combined application of Enricason and Caspofungin in the prior art, especially no combined application of the two in the prevention and treatment of ischemia/reperfusion injury.

Compared with the prior art, the beneficial effects of the present invention are:

(1) Synergistically prevent or treat ischemia/reperfusion injury, especially cerebral ischemia/reperfusion injury, improve long-term survival rate and reduce disability rate of patients, and can be applied to cerebral ischemia/reperfusion injury;

(2) Reduced adverse drug reactions;

(3) Compared with intravenous injection, the present invention adopts intramuscular injection, which is easy to operate, less irritating, and enhances the patient's compliance.

The research of the present invention finds for the first time that the echinocandin antifungal drug has a new function of inhibiting cell necroptosis and reducing cerebral ischemia/reperfusion injury, and the mechanism is different from its antifungal mechanism. The applicant speculates the mechanism as follows: Echinocandins antifungal drugs can specifically down-regulate RIPK1/RIPK3/MLKL protein and phosphorylation levels and inhibit necroptosis. For the first time, we found that the combination of echinocandin antifungals and enricason can synergistically prevent and treat ischemic stroke, and can significantly reduce cerebral ischemia/reperfusion injury, compared with echinocandins or When used alone, Enricason has better inhibitory effect, can effectively reduce the volume of cerebral infarction in rats, improve neurological function, and has a significant anti-ischemic stroke effect.

Description of drawings

Figure 1: A- rat brain tissue TTC staining and infarct volume measurement, B- rat neurological function score;

Detailed ways

The present invention will be described in detail below with reference to the accompanying drawings and in conjunction with the embodiments. It should be noted that the embodiments of the present invention and the features of the embodiments may be combined with each other under the condition of no conflict.

In the following, the amount of Enricason and the amount of Caspofungin are expressed as a ratio of Enricason and Caspofungin.

Animal experiments: the protective effect of the combination of enricasan and caspofungin on ischemic stroke.

Experimental animals: 48 healthy male SD rats weighing 250-300 g. The experimental animals were raised for a week in an environment with a temperature of 25° C., a relative humidity of 60%, free drinking water, and a fixed amount of time, and then oral administration was performed according to the requirements of each group in the experiment.

Modeling method: Rat cerebral ischemia/reperfusion injury model was established by middle cerebral artery occlusion (MCAO). The steps are as follows: (1) separate the external carotid artery (CCA), separate the right external carotid artery (ECA) and the internal carotid artery (ICA) upward; (2) temporarily clamp the ECA and ICA with ophthalmic forceps, and ligate the proximal end of the CCA (3) Place a knotted spare silk thread at the distal end of the CCA, cut a small cut at the lower end of the thread, insert the suture into the internal carotid artery, tighten the silk thread, release the arterial clips on the ECA and ICA, and follow the The ICA sends the suture to the brain; (4) When it meets resistance, the insertion depth is about 18-20 mm from the CCA bifurcation; (5) After 120 minutes of ischemia, the suture is pulled out and the skin is sutured. , the animals were treated after reperfusion for 24h.

The model's success evaluation standard adopts Longa's "5-point method" to score the neurological deficit of rat cerebral ischemia injury. 0 points: no symptoms of neurological deficit; 1 point: unable to fully straighten the right forelimb; 2 points: right rotation; 3 points: walking to the right; 4 points: unable to walk spontaneously, loss of consciousness. 1 to 4 are classified as valid models.

TTC staining and infarct volume determination in rat brain. After the rats were anesthetized, the brain was quickly taken out, the olfactory bulb and hindbrain were removed, and 4 coronal brain slices, about 2.0 mm thick, were cut from the frontal pole, immediately placed in 1% TTC solution, and incubated at 37°C for 30 min in the dark. Then soak in 10% paraformaldehyde solution for fixation. The infarcted area is white and the non-infarcted area is red. Scanning was performed after each group of brain slices were arranged neatly. ImageJ was then used to measure the infarct area of each brain slice. According to the formula: infarct volume = [(the sum of the infarct area on the front side of each slice + the sum of the infarct area on the back side of each slice)/2]×the thickness of each slice, the same method was used to calculate the whole brain. volume.

Experimental grouping: The experimental animals are randomly divided into 6 groups or 7 groups, namely:

Sham operation group, ischemia/reperfusion group, enricason + ischemia/reperfusion group, caspofungin + ischemia/reperfusion group, different doses of combination therapy + ischemia/reperfusion group, vehicle Group + ischemia/reperfusion group, the rats in each group were given administration after 2 hours of ischemia and 1 hour of reperfusion respectively.

Sham operation group: The left common carotid artery, internal carotid artery and external carotid artery were separated, and no suture was inserted.

Ischemia/reperfusion group (I/R): 2 hours of cerebral ischemia and 24 hours of reperfusion.

Enricason (5mg/kg) + cerebral ischemia/reperfusion group, caspofungin (8mg/kg) + cerebral ischemia/reperfusion group, Enricason (5mg/kg) + caspofungin (2mg/kg) + cerebral ischemia/reperfusion group, Enricason (5mg/kg) + caspofungin (5mg/kg) + cerebral ischemia/reperfusion group, Enricason (2.5mg/kg) kg) + caspofungin (8 mg/kg) + cerebral ischemia/reperfusion group, vehicle + cerebral ischemia/reperfusion group; 1 hour after ischemia 2 h and reperfusion, 6 rats in each group (n = 6), see Figure 1 for the specific experimental results.

Detection indicators: rat neurological function score and infarct volume measurement.

Experimental results:

Effects on the volume of cerebral infarction and neurological function in rats

As shown in A in Figure 1, the I/R group had obvious white infarction, while the cerebral infarction of the rats in the single drug group was significantly reduced, and the cerebral ischemia injury was significantly relieved (**P<0.01vs sham operation group, ^# P <0.05vsI/R group, ^&& P<0.01vsI/R+single drug group), and the effect of combination therapy was significantly better than that of single drug group (**P<0.01vs sham group, ^# P<0.05vsI/R group) , ^& P<0.01 vs I/R + single drug group).

However, the present invention is not limited to cerebral ischemia/reperfusion injury. Since cardiac, hepatic and renal ischemia/reperfusion injury also has up-regulation of Caspase, etc., it can be inferred that the drug is also suitable for the treatment of cardiac, hepatic and renal ischemia/reperfusion injury. perfusion injury.

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

Claims (5)

1. the application of a pharmaceutical composition in the preparation of the medicine for preventing or treating cerebral ischemia/reperfusion injury; it is characterized in that, described pharmaceutical composition comprises active ingredient, and described active ingredient is composed of active ingredient A and active ingredient B composition, the active ingredient A is enricasan or a pharmaceutically acceptable salt thereof, the active ingredient B is caspofungin or a pharmaceutically acceptable salt thereof, the active ingredient A and the active ingredient B are The weight ratio is 1:0.2-5. 2. application according to claim 1 is characterized in that, the mode of administration in described application is intramuscular injection, subcutaneous injection, intravenous injection, oral administration, sublingual oral administration, intralesional or intracerebral or implantation One or more of delivery and spray administration. 3. The use according to claim 1, wherein the cerebral ischemia/reperfusion injury comprises ischemic stroke. 4. The application according to claim 1, wherein the weight ratio of the active ingredient A and the active ingredient B is 1:0.4-3.5. The application according to claim 4, wherein the weight ratio of the active ingredient A and the active ingredient B is one of 1:0.4, 1:1, and 1:3.2.

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