CN113975378A

CN113975378A - Application of polymerized hemoglobin in preparing medicine for treating cerebral arterial thrombosis

Info

Publication number: CN113975378A
Application number: CN202111376249.9A
Authority: CN
Inventors: 游可为; 李剑波; 张彦鹏; 石松; 王文刚
Original assignee: Runfang Beijing Biotechnology Co ltd; Redpharm Beijing Biomedical Research Institute Co ltd
Current assignee: Redpharm Beijing Biomedical Research Institute Co ltd; Runfang Beijing Biotechnology Co ltd; You Kewei
Priority date: 2021-11-19
Filing date: 2021-11-19
Publication date: 2022-01-28
Also published as: WO2023087421A1

Abstract

The invention relates to an application of polymerized hemoglobin in preparing a medicine for treating cerebral arterial thrombosis, wherein the medicine is a hemoglobin oxygen carrier and is also a colloidal solution, and the particle size of the polymerized hemoglobin is only one thousandth of that of red blood cells. The small-dose perfusion can not bring tissue damage and cell death caused by reperfusion to the brain, but can relieve 'blood shortage' caused by arterial obstruction, and meanwhile, the hemoglobin carries out oxygen loading and unloading from the lung to tissues and organs, carries out targeted blocking on the pathophysiological process of the brain caused by ischemia and anoxia, saves nerve cells in an ischemic penumbra, reduces the death number of the nerve cells, and plays a role in neuroprotection. The medicine can also be added with a nerve protective agent to prepare a compound medicine, has more obvious protective effect on nerve cells of ischemic penumbra, and can prolong the stroke treatment window of rt-PA venous thrombolysis for 6 h.

Description

Application of polymerized hemoglobin in preparing medicine for treating cerebral arterial thrombosis

Technical Field

The invention belongs to the technical field of medicines, and particularly relates to application of polymerized hemoglobin in preparation of a medicine for treating cerebral arterial thrombosis.

Background

The oxygen consumption of normal adult brain tissue accounts for 20-30% of the total oxygen consumption, the energy source of the brain tissue is mainly the aerobic metabolism of sugar, and almost no energy storage exists, so the brain tissue is very sensitive to ischemia and hypoxia injury. Acute ischemic stroke (a IS) refers to a type of clinical syndrome in which ischemia and hypoxic necrosis occur in local brain tissue due to blood supply disorder of the brain caused by various reasons, and corresponding neurological impairment occurs. It has the characteristics of high morbidity, high disability rate and high fatality rate. Devin et al showed that more than 70% of AIS is permanent ischemic stroke (pMCAO).

Cerebral apoplexy is most common in AIS, and 80% -90% of AIS is caused by cerebral artery blockage by thrombus. After the blood flow in the brain is blocked due to the blockage or severe stenosis of the cerebral artery, the blood supply is lost in several minutes in the central part of the brain tissue, and the blood oxygen level is rapidly reduced. The progressive pathophysiological response leads to severe ischemic and hypoxic injury in the infarcted area of the core, and peripheral brain tissue may acquire a quantitative blood flow through the collateral circulation of the blood vessels, maintaining it above the pump level and below the energy required for electrical activity, i.e. the ischemic penumbra. This is partly the prognosis of stroke, which depends on the size of the infarct and the number of ischemic penumbra cells that are rescued after reperfusion. Increasing blood oxygen levels, reducing the metabolic rate of ischemic tissue, and mitigating the oxidative stress injury of reperfusion are emergency measures to rescue the ischemic penumbra. The metabolic reconstruction is particularly important in three nerve cell recovery stages of metabolic reconstruction, structural reconstruction and functional reconstruction. Since brain metabolism relies mainly on aerobic metabolism of glucose oxidation, sufficient oxygen and energy supply are important, and thus all treatments should be applied around how to increase energy and oxygen supply and enable nerve cells to carry out effective metabolism. The key of clinical AIS treatment is to open the blocked cerebral blood vessels as early as possible and recover blood supply in time before the ischemic brain tissue is necrotic, so that an ischemic penumbra is saved, and the fatality rate and the disability rate of ischemic stroke are reduced finally. Although there is no mature treatment scheme to prevent and treat the occurrence of stroke and the loss of nerve function, many experimental drugs can significantly reduce the volume of cerebral infarction in animal models, and double-blind tests are performed clinically to verify the curative effect, which brings hope for human beings to overcome the diseases.

At present, the most effective drug treatment of AIS is still intravenous thrombolysis by administering recombinant tissue plasminogen activator (rt-PA) in an ultra-early stage (less than or equal to 6h), can obviously improve prognosis, and is the only consistent recommendation by home and abroad cerebrovascular disease guidelines. In the face of the severe situation of preventing and controlling the cerebral apoplexy, the early starting of the treatment of the acute cerebral apoplexy is necessary. A well-established treatment modality is thrombolysis. Since the 6h "therapeutic window" of thrombolysis is narrow and the thrombolysis rate is less than 5%, more and more scholars tend to neuroprotective treatment. The neuroprotective treatment is to block the pathological phenomena of energy deficiency, excitatory amino acid toxicity, cell depolarization, necrosis, etc. of nerve cells by influencing the biochemical process of ischemia waterfall. At present, after reticular meta analysis is carried out on the common neuroprotective agents edaravone, ganglioside, butylphthalide, vinpocetine and citicoline, the neurological deficit function can be improved for some in a short time, but the overall curative effect is not obvious, and further verification of the result by high-quality first-to-first research is required.

The present invention has been made in view of the above circumstances.

Disclosure of Invention

In order to solve the problems in the prior art, the invention provides application of polymerized hemoglobin in preparing a medicine for treating cerebral arterial thrombosis, the medicine is a hemoglobin oxygen carrier with a colloid solution property, the small dose of the hemoglobin oxygen carrier can relieve 'blood waste' caused by arterial occlusion, oxygen carrying-oxygen unloading can be carried out from the lung to tissues and organs, tissue damage and cell death caused by ischemia and anoxia are relieved, the number of ischemic penumbra cells is saved, and meanwhile, the blood oxygen level is increased, the metabolic rate of ischemic tissues is reduced, and oxidative stress damage caused by reperfusion is relieved.

The invention provides an application of polymerized hemoglobin in preparing a traditional Chinese medicine for treating cerebral arterial thrombosis.

Furthermore, the medicine is prepared by adding medically acceptable auxiliary materials into the polymerized hemoglobin.

Furthermore, the content of polymerized hemoglobin in each 1L of the medicine is 30.0-130.0 g.

Furthermore, the medicament can also be added with a neuroprotective agent which is commonly used in clinic.

Further, the auxiliary materials are prepared by dissolving the auxiliary materials in 1L of water for injection by weight, and the specific ratio is as follows: 0.87-6.6 g of sodium chloride, 0.3-0.67 g of potassium chloride, 0-0.18 g of alpha-ketoglutarate monopotassium salt, 0-0.81 g of magnesium chloride hexahydrate, 0-3.77 g of histidine monohydrochloride monohydrate, 0-27.92 g of histidine, 0-0.4 g of tryptophan, 0-0.4 g of mannitol, 0.002-0.2 g of calcium chloride dihydrate, 2.0-2.28 g of acetylcysteine, 0-0.48 g of sodium hydroxide and 0-3.1 g of sodium lactate.

Further, the clinical common nerve protective agent comprises one or more of the following components, specifically, the nerve protective agent is dissolved in 1L of water for injection by weight, and the specific proportion is 3.0-60.0 mg of edaravone, 14.0-55.0 mg of ganglioside, 65.0-315.0 mg of butylphthalide, 15.0-125.0 mg of vinpocetine and 65.0-520.0 mg of citicoline.

Further, the medicine is prepared by dissolving the following substances in 1L of water for injection according to the specific ratio: 130.0g of polymerized hemoglobin, 3.2g of sodium chloride, 0.48g of potassium chloride, 0.15g of calcium chloride dihydrate, 0.23g of sodium hydroxide, 1.75g of sodium lactate and 2.14g of acetylcysteine.

Further, the medicine is prepared by dissolving the following substances in 1L of water for injection according to the specific ratio: 65.0g of polymeric hemoglobin, 6.6g of sodium chloride, 0.3g of potassium chloride, 0.2g of calcium chloride dihydrate, 0.48g of sodium hydroxide, 3.0g of sodium lactate, 2.0g of acetylcysteine and 13.70mg of edaravone.

Further, the medicine is prepared by dissolving the following substances in 1L of water for injection according to the specific ratio: 65.0g of polymerized hemoglobin, 6.6g of sodium chloride, 0.3g of potassium chloride, 0.2g of calcium chloride dihydrate, 0.48g of sodium hydroxide, 3.0g of sodium lactate and 2.0g of acetylcysteine.

Further, the medicine is prepared by dissolving the following substances in 1L of water for injection according to the specific ratio: 65.0g of polymerized hemoglobin, 0.87g of sodium chloride, 0.3g of potassium chloride, 0.18g of alpha-ketoglutarate monopotassium salt, 0.81g of magnesium chloride hexahydrate, 3.77g of histidine monohydrochloride monohydrate, 27.92g of histidine, 0.4g of tryptophan, 0.4g of mannitol, 0.002g of calcium chloride dihydrate and 2.28g of acetylcysteine.

Further, the medicine is prepared by dissolving the following substances in 1L of water for injection according to the specific ratio: 30.0g of polymerized hemoglobin, 1.45g of sodium chloride, 0.6g of potassium chloride, 0.12g of alpha-ketoglutarate monopotassium salt, 0.6g of magnesium chloride hexahydrate, 2.2g of histidine monohydrochloride monohydrate, 14.0g of histidine, 0.27g of tryptophan, 0.13g of mannitol, 0.002g of calcium chloride dihydrate and 37.14mg of citicoline.

Further, the pharmaceutical dosage form is an injection.

The polymerized hemoglobin of the present invention is derived from hemoglobin of human or animal origin, which is modified to form a stable molecular polymer, thereby prolonging the half-life of the native hemoglobin in blood and preventing its depolymerization into dimers in vivo. The medicine prepared by the polymerized hemoglobin has the characteristics of no toxicity, no immunogenicity, good rheological property, long circulation retention time, normal physiological metabolic pathway and the like.

The polymerized hemoglobin of the present invention is prepared according to the method for preparing cross-linked hemoglobin disclosed in publication No. CN 110563836A.

The routes of drug administration described in the present invention include, but are not limited to, intravenous injection, and arterial injection routes.

Compared with the prior art, the invention has the beneficial effects that:

(1) the polymerized hemoglobin medicament is a hemoglobin oxygen carrier and is also a colloidal solution, small-dose perfusion cannot bring tissue damage and cell death caused by reperfusion to the brain, but can relieve 'blood waste' caused by artery obstruction, meanwhile, hemoglobin carries out oxygen carrying-oxygen unloading from the lung to tissues and organs, relieves the damage caused by ischemia and anoxia, further saves the number of ischemic penumbra cells, and further prolongs the stroke 'treatment window' of rt-PA venous thrombolysis for 6h, so that more patients who cannot be treated in the limited 'treatment window' can be effectively treated in a wide time period;

(2) the drug is a substitute of red blood cells, is a colloidal solution, the particle size of polymerized hemoglobin is only one thousandth of that of red blood cells, and can reach a capillary bed where the red blood cells cannot reach through a narrow capillary tube, after reperfusion, the reduction of blood flow and partial tissue oxygen partial pressure in a cortical semi-dark area is improved, the number of nerve cells which are abnormal in function but not dead due to ischemic injury around an infarct focus is saved as far as possible, so that the nerve cells are recovered to be normal and the gradual recovery of nerve function is promoted, histopathological analysis of the cortical semi-dark area shows that compared with autologous red blood cells, the perfusion of hemoglobin to the capillary tube is better, the stenosis change is reduced, and the hemoglobin can obviously inhibit the generation of active oxygen in hypoxia/reoxygenation treated cells and also can be used as a neuroprotective agent;

(3) the polymerized hemoglobin prepared by the method has the characteristic of higher oxygen carrying-releasing, can be used for pertinently blocking the pathophysiological process of cerebral ischemia and hypoxia, relieving the death of nerve cells, relieving the damage of the nerve cells and playing a role in protecting nerves, thereby prolonging the treatment window of ischemic stroke, improving the fate, improving the survival efficiency and improving the quality of life.

(4) The animal experiment carried out by the polymerized hemoglobin-based medicament adopts an animal model of permanent ischemic stroke (pMCAO) in the experiment, is different from the animal model applied to transient ischemic stroke (tMCAO) in the prior art, and the pMCAO more practically meets the symptoms of more than AIS70 percent of clinical patients.

(5) The nerve protective agent is added into the medicine, and the nerve tissue can be further protected through mechanisms such as oxygen radical removal and the like, the cerebral edema is relieved, the internal circulation of the brain is further improved, the functions of mitochondria can be promoted, the oxygen radical is inhibited, the apoptosis is inhibited and the like.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, it is obvious that the drawings in the following description are only some embodiments of the present invention, and for those skilled in the art, other drawings can be obtained according to these drawings without any creative effort.

FIG. 1 is a TTC staining pattern of the brain of rats with 1pMCAO in the sham-operated group;

FIG. 2 is a TTC staining pattern of brain of rats with 2pMCAO in the sham operation group;

FIG. 3 is a TTC staining pattern of the brain of 3pMCAO rats in the sham-operated group;

FIG. 4 is a graph of drug group 1pMCAO rat brain TTC staining;

FIG. 5 is a graph of drug group 2pMCAO rat brain TTC staining;

FIG. 6 is a graph of TTC staining of the brain of rats with drug group 3 pMCAO;

FIG. 7 is a graph of TTC staining of rat brain from model 1 pMCAO;

FIG. 8 is a graph of TTC staining of the brain of a model 2pMCAO rat;

FIG. 9 is a graph of TTC staining of rat brain from model 3 pMCAO;

FIG. 10 is a histogram of a protein of interest;

FIG. 11 is a graph showing Caspase-3 content.

Detailed Description

In order to make the objects, technical solutions and advantages of the present invention more apparent, the technical solutions of the present invention will be described in detail below. It is to be understood that the described embodiments are merely exemplary of the invention, and not restrictive of the full scope of the invention. All other embodiments, which can be derived by a person skilled in the art from the examples given herein without making any creative effort, shall fall within the protection scope of the present invention.

The polymerized hemoglobin of the following examples was prepared according to the method of example 1 of CN 110563836A.

Example 1

The present example contains a drug of polymerized hemoglobin, wherein the following substances are dissolved in 1L of water for injection in the following specific proportions: 130.0g of polymerized hemoglobin, 3.2g of sodium chloride, 0.48g of potassium chloride, 0.15g of calcium chloride dihydrate, 0.23g of sodium hydroxide, 1.75g of sodium lactate and 2.14g of acetylcysteine.

Example 2

The compound medicine containing the polymerized hemoglobin and the nerve protective agent is prepared by dissolving the following substances in 1L of water for injection in the following specific proportions: 65.0g of polymeric hemoglobin, 6.6g of sodium chloride, 0.3g of potassium chloride, 0.2g of calcium chloride dihydrate, 0.48g of sodium hydroxide, 3.0g of sodium lactate, 2.0g of acetylcysteine and 13.70mg of edaravone.

Example 3

The present example contains a drug of polymerized hemoglobin, wherein the following substances are dissolved in 1L of water for injection in the following specific proportions: 65.0g of polymerized hemoglobin, 6.6g of sodium chloride, 0.3g of potassium chloride, 0.2g of calcium chloride dihydrate, 0.48g of sodium hydroxide, 3.0g of sodium lactate and 2.0g of acetylcysteine.

Example 4

The present example contains a drug of polymerized hemoglobin, wherein the following substances are dissolved in 1L of water for injection in the following specific proportions: 65.0g of polymerized hemoglobin, 0.87g of sodium chloride, 0.3g of potassium chloride, 0.18g of alpha-ketoglutarate monopotassium salt, 0.81g of magnesium chloride hexahydrate, 3.77g of histidine monohydrochloride monohydrate, 27.92g of histidine, 0.4g of tryptophan, 0.4g of mannitol, 0.002g of calcium chloride dihydrate and 2.28g of acetylcysteine.

Example 5

The compound medicine containing the polymerized hemoglobin and the nerve protective agent is prepared by dissolving the following substances in 1L of water for injection in the following specific proportions: 30.0g of polymerized hemoglobin, 1.45g of sodium chloride, 0.6g of potassium chloride, 0.12g of alpha-ketoglutarate monopotassium salt, 0.6g of magnesium chloride hexahydrate, 2.2g of histidine monohydrochloride monohydrate, 14.0g of histidine, 0.27g of tryptophan, 0.13g of mannitol, 0.002g of calcium chloride dihydrate and 37.14mg of citicoline.

Test example 1 Effect of a drug containing polymerized hemoglobin on intravenous injection of rats with permanent ischemic stroke (pMCAO)

The tested drugs are: a drug comprising polymerized hemoglobin prepared according to example 3, wherein the hemoglobin content is 65.0 g/L;

positive drug: lactated ringer's solution; specification: 20.0 ml/piece;

test animals: SD rats weighing 250-300 g and male animals purchased from Beijing Wittingle laboratory animal technology Limited, wherein the tested animals are raised in a sterile cage independently supplied with air, the padding is 60Co radiation sterilized corncob padding, and the particle size is 4-6 mm. The mice are fed with the special sterilized feed for the mice, and the purified water is freely drunk. The temperature in the animal laboratory is kept at about 25 ℃, the relative humidity is kept at 40-70%, and the illumination is carried out for 12 hours every day.

1. The experimental method comprises the following steps:

1.1 modeling

SD rat is all made ear mark

preparation of pMCAO rat model: the SD rat MCAO model was prepared by a modified Longa wire plug method. A10% chloral hydrate is injected into abdominal cavity to anaesthetize rats, a shaver removes hairs on the neck and abdomen of the rats, the rats are disinfected by 75% ethanol, the rats are fixed on a backing plate of an operating table in a supine position with the abdominal surface facing upwards, a longitudinal incision of about 2cm is made in the middle of the neck by a surgical knife, the cervical superficial cervical mucosa is pulled away, the mucosa in the gap among the digastric muscle, the sternocleidomastoid muscle and the scapular hyoid muscle is pulled away, the Common Carotid Artery (CCA), the External Carotid Artery (ECA) and the Internal Carotid Artery (ICA) are exposed, and the filaments are hung at the far end and near end of the CCA and at the ECA for standby. After ICA is temporarily clamped by the arteriolar clamp, CCA and ECA are tied at the proximal end, a small opening is cut at a position 4mm away from the bifurcation part of the common carotid artery, and a loose knot is tied under the small opening. The plug is inserted through the port along the ECA with ophthalmic forceps, and the free end of the ECA is pulled upward and outward, parallel to the ICA, to straighten the angle with the ICA. The tampon was passed along the ICA and gently pushed slowly until a slight resistance was felt to the insertion depth of about 18mm (i.e., the tampon reached the bifurcation between ICA and CCA), and the loose knot previously tied was tied. After tying, the thread plug in the body is cut short, and the incision is sewed layer by layer and disinfected. After the operation, the rats were kept in lateral decubitus, and the bleeding of the wound and the obstruction of the respiratory tract were noticed.

Surgical sham-operated group rat preparation: SD rats are anesthetized by intraperitoneal injection with 10% chloral hydrate, hairs on the neck and abdomen of the rats are removed by a shaver, the rats are disinfected by 75% ethanol, the rats are fixed on a base plate of an operating table in a supine position with the abdominal surface facing upwards, a longitudinal incision of about 2cm is made in the middle of the neck by a scalpel, the superficial cervical mucosa of the neck is pulled away, the mucosa in the gap between the two abdominal muscles, the sternocleidomastoid muscle and the hyoid muscle is pulled away, the Common Carotid Artery (CCA), the External Carotid Artery (ECA) and the Internal Carotid Artery (ICA) are exposed, and the incisions are sutured layer by layer after the blood vessels are pulled and disinfected. After the operation, the rats are kept in lateral decubitus, and whether bleeding occurs in the wound, whether the respiratory tract is unblocked and the like are noticed.

1.2 grouping and dosing

The grouping and administration dose of SD rats are shown in table 1, and the administration is performed by tail vein injection 3h after the model is built on the SD rats.

TABLE 1 grouping and dosing

Group of	Test drug	Dosage form
			Model set	Lactic acid ringer's solution	12.31ml/kg
Drug group	Polymeric hemoglobin medicine	800mg/kg
			Artificial operation group	Physiological saline	12.31ml/kg

2. Evaluation index

2.1 neurological Scoring

Neurological function was assessed and documented by the Bederson scale (see table 2) by 2 blinded group observers at the onset of pMCAO, 24h post-dose, respectively.

TABLE 2 Bederson scoring method

Grade of rating	Neurological symptoms
		0	No symptoms of nerve damage
1	Injury to the contralateral forelimb during tail lifting does not straighten
		2	Injury to contralateral forelimb flexion without circling behavior
3	Injury to contralateral forelimb flexion with spontaneous circling behavior
		4	Failure to walk spontaneously and unconsciousness
5	Death was caused by death

2.2 blood gas analysis

Blood samples were taken via the femoral artery at 24h after the onset of pMCAO and administration, respectively, and blood gas analysis was immediately performed.

2.3 determination of cerebral infarct volume and evaluation of cerebral edema

After 24h neurological function assessment, each group of (n-4) rats was individually euthanized and stained by TTC. The whole brain tissue of the rat is completely taken out (cerebellum is removed), the rat is rinsed by 0.9 percent of normal saline, the animal brain tissue is placed into a special brain slice mold groove, and the brain tissue is cut into 6 slices by a thin double-sided blade, wherein the thickness of each slice is about 2 mm. The brain slices are sequentially placed into TTC (2, 3, 5-triphenyltetrazolium chloride) staining solution and are stained for 30min at 37 ℃ in a dark place. After 30min, the brain slices were taken out from the TTC staining solution, placed in 10% neutral formalin solution, and fixed in a refrigerator at 4 ℃ for 24 h. After the brain sections were fixed, they were arranged in order and photographed (both anterolateral and caudal). The photos are imported into image analysis software, the areas of the infarct area (white area) and the normal area (red area) are measured, and the formula is shown as follows: v ═ Σ [ (a1+ a2) × d/2] the respective area volumes and the whole brain volume were calculated, where a1, a2 are the areas of the corresponding areas of the anterior and posterior sides of the brain slice, respectively, and d is the slice thickness.

Cerebral infarction (%) - (contralateral hemispheric volume-ipsilateral non-infarcted volume)/contralateral hemispheric volume;

cerebral edema (%) ═ ipsilateral-contralateral/contralateral hemispheric volume.

Wherein, the staining patterns of the TTC of the rat brain of the sham surgery group 1-3pMCAO are shown in figures 1-3, the staining patterns of the TTC of the rat brain of the drug group 1-3pMCAO are shown in figures 4-6, and the staining patterns of the TTC of the rat brain of the model group 1-3pMCAO are shown in figures 7-9.

3. Results of the experiment

3.1 neurological Scoring

After pMCAO attack and administration for 24h, 2 blinded observers in groups respectively adopt a Bederson scoring method (see Table 2) to evaluate the nerve function of experimental rats, the scores of the sham operation group rats are 0 in the result, and the scores of the model group rats and the drug group rats are more than or equal to 3 in the result.

3.2 blood gas analysis

Analysis of the blood gas analysis data results revealed that the oxygen partial pressure (PaO) was higher after the model was created than after the administration of 24 hours₂): the sham operation group and the drug group are increased quickly, while the model group is increased slowly; hemoglobin concentration (ctHb): the sham and drug groups rose faster, while the model group had a slow rise or inhibition; bound oxygen (FO)₂Hb): the sham and drug groups had some elevation, while the model group had a slow elevation or inhibition. The results are shown in table 3.

TABLE 3 blood gas analysis and test results

3.3 determination of cerebral infarct volume

Analysis of the cerebral infarction volume measurement result shows that the cerebral infarction of the sham operation group is almost not cerebral infarction, and the cerebral infarction volume of the drug group is obviously improved compared with that of the model group, and the result is shown in table 4.

TABLE 4 cerebral infarction volume measurement results

3.3 brain edema assessment

Analysis of the results of the cerebral edema determination shows that the brain of the sham operation group has almost no edema, and the cerebral edema of the drug group is obviously improved compared with that of the model group, and the results are shown in table 5.

TABLE 5 cerebral edema volume determination

Experimental example 2 study of inflammatory factor by intravenous injection of drug containing polymerized hemoglobin and reconstituted drug containing polymerized hemoglobin and edaravone in rats with permanent ischemic stroke (pMCAO)

The tested drugs are: the combination prepared in example 2 and containing polymerized hemoglobin and edaravone, wherein the hemoglobin content is 65.0g/L and the edaravone is 13.70 mg/L;

positive drug 1: a drug comprising polymerized hemoglobin prepared according to example 3, wherein the hemoglobin content is 65.0 g/L;

positive drug 2: edaravone injection (specification: 20ml:30 mg; national drug group, national drug industry Limited Co., Ltd.)

Test animals: the same as in test example 1.

1. The experimental method comprises the following steps:

1.1 modeling

The same as in test example 1.

2.2 grouping and dosing

The grouping and administration dose of SD rats are shown in table 6, and the administration is performed by tail vein injection 3h after the model is built on the SD rats.

TABLE 6 grouping and dosing

2. Evaluation index

2.1 neurological Scoring

The same as in test example 1.

2.2 immunoblotting experiments

The samples were fractionated by SDS-PAGE to detect the bands of Caspase-3, HIF-1, IL-6 and GAPDH proteins.

2.3 Caspase-3 detection

Weighing animal part brain tissue, preparing 10% homogenate by PBS in ice bath environment, centrifuging, taking supernatant, measuring protein concentration, and detecting Caspase-3 content according to kit instructions.

3. Results

3.1 neurological Scoring

And (3) performing nerve function evaluation on experimental rats 24h after pMCAO attack and drug administration by 2 blinded observers in a Bederson scoring method, wherein the scores of the rats in the result sham operation group are all 0, and the scores of the rats in the model group, the positive drug 1 group, the positive drug 2 group and the test drug group are all more than or equal to 3.

3.2 immunoblotting experiments

The target protein band is shown in fig. 10, and the results in the target protein band diagram show that, in the ischemic stroke experiment, compared with the model group, the polymerized hemoglobin group and the edaravone group have certain neuroprotective effect, while the tested drug group has better neuroprotective effect, and is close to the sham operation group.

3.3 Caspase-3 content

The content of Caspase-3 is shown in figure 11, Caspase-3 plays an irreplaceable role in apoptosis, and the trend result of the content also proves the result of the target protein band, and the tested drug group is better than the single-component polymerized hemoglobin drug group and the edaravone drug group.

The inventors have also conducted the above experiments on other examples, and the results are substantially consistent and, due to the limited space, are not listed.

The above description is only for the specific embodiment of the present invention, but the scope of the present invention is not limited thereto, and any person skilled in the art can easily conceive of the changes or substitutions within the technical scope of the present invention, and all the changes or substitutions should be covered within the scope of the present invention. Therefore, the protection scope of the present invention shall be subject to the protection scope of the appended claims.

Claims

1. Application of polymerized hemoglobin in preparing medicine for treating cerebral arterial thrombosis is disclosed.

2. The use of claim 1, wherein the medicament is polymerized hemoglobin in combination with a pharmaceutically acceptable excipient.

3. Use according to claim 1 or 2, characterized in that: the medicine can also be added with a neuroprotective agent which is commonly used in clinic.

4. The use according to claim 1 or 2, wherein the amount of polymerized hemoglobin in 1L of the medicament is 30.0-130.0 g.

5. The use of claim 2, wherein the adjuvant is prepared by dissolving in 1L of water for injection by weight, and the specific ratio is as follows: 0.87-6.6 g of sodium chloride, 0.3-0.67 g of potassium chloride, 0-0.18 g of alpha-ketoglutarate monopotassium salt, 0-0.81 g of magnesium chloride hexahydrate, 0-3.77 g of histidine monohydrochloride monohydrate, 0-27.92 g of histidine, 0-0.4 g of tryptophan, 0-0.4 g of mannitol, 0.002-0.2 g of calcium chloride dihydrate, 2.0-2.28 g of acetylcysteine, 0-0.48 g of sodium hydroxide and 0-3.1 g of sodium lactate.

6. Use according to claim 3, characterized in that: the neuroprotective agent commonly used in clinic comprises one or more of the following components, and is specifically dissolved in 1L of water for injection by weight, wherein the specific proportions are 3.0-60.0 mg of edaravone, 14.0-55.0 mg of ganglioside, 65.0-315.0 mg of butylphthalide, 15.0-125.0 mg of vinpocetine and 65.0-520.0 mg of citicoline.

7. The use according to any one of claims 1 to 6, wherein the medicament is prepared by dissolving the following substances in 1L of water for injection in the following specific proportions: 65.0g of polymeric hemoglobin, 6.6g of sodium chloride, 0.3g of potassium chloride, 0.2g of calcium chloride dihydrate, 0.48g of sodium hydroxide, 3.0g of sodium lactate, 2.0g of acetylcysteine and 13.70mg of edaravone.

8. The use according to any one of claims 1 to 6, wherein the medicament is prepared by dissolving the following substances in 1L of water for injection in the following specific proportions: 65.0g of polymerized hemoglobin, 6.6g of sodium chloride, 0.3g of potassium chloride, 0.2g of calcium chloride dihydrate, 0.48g of sodium hydroxide, 3.0g of sodium lactate and 2.0g of acetylcysteine.

9. The use according to any one of claims 1 to 6, wherein the medicament is prepared by dissolving the following substances in 1L of water for injection in the following specific proportions: 65.0g of polymerized hemoglobin, 0.87g of sodium chloride, 0.3g of potassium chloride, 0.18g of alpha-ketoglutarate monopotassium salt, 0.81g of magnesium chloride hexahydrate, 3.77g of histidine monohydrochloride monohydrate, 27.92g of histidine, 0.4g of tryptophan, 0.4g of mannitol, 0.002g of calcium chloride dihydrate and 2.28g of acetylcysteine.

10. The use according to any one of claims 1 to 9, wherein the pharmaceutical dosage form is an injection.