Technical field

The invention belongs to biomedicine technical fields, specifically, being related to a kind of for preventing and/or treating cerebral ischemia Combination product and its application.

Background technology

Cerebral arterial thrombosis has high incidence, high mortality, high disability rate, high relapse rate, the several spies of high health care costs Point is the great chronic disease for endangering China's residents ' health.Clinical data statistics display, the global cerebral apoplexy death rate already exceed cancer Disease becomes the highest disease of the Single diseases death rate.It is well known that inflammatory reaction is the important set during cerebral ischemia pathology damage At part, and aggravate the latency of cerebral ischemia.In addition to this, cerebral arterial thrombosis is often accompanied by brain edema and hemorrhagic Conversion, wherein brain edema is the complication of cerebral arterial thrombosis most serious, and 80% dies of the palsy later stage in apoplexy patient death Pernicious brain edema.Therefore the prevention of inflammation and brain edema is significant to the treatment of cerebral arterial thrombosis.

Sulfonylureas receptor 1 (sulfonylurea receptor 1, Sur1)-transient receptor potential channel M4 types The channel (transient receptor potential melastatin 4, Trpm4) plays the formation of brain edema important Effect.Pertinent literature reports, cerebral arterial thrombosis occur be within 4-8 hour brain edema formation critical period, in this period ischemic The Sur1 receptors in the position channels cerebrovascular tapetum cell film Sur1-Trpm4 will appear the expression of irritability height, the Sur1 of great expression Receptor causes the channels Sur1-Trpm4 to be activated.The activation in the channels Sur1-Trpm4 increases cationic especially sodium ion and enters cell It is interior, since the reason driving water of osmotic pressure pours in into the cell, causes ishemic part cell or even tissue that oedema occurs, be soldier There is the key pathological mechanism of brain edema in middle patient.Sur1 inhibitor is combined the opening for inhibiting this channel with Sur1 receptors, from And effectively inhibit the formation of brain edema.Representative Sur1 inhibitor glibenclamide is directed to the treatment of palsy associated with hydrocephalus in U.S. State enters the clinical III phases.

Qinghaosu and its derivative are a kind of new antimalarial agents of China scientific worker research and development, are now widely used for The treatment of malaria.With the continuous deepening of research, other than its Antimalarial, scientist is also found that qinghaosu and its spreads out in succession Biology pharmacological action in other respects, including antitumor action, the effect to immunocyte, anti-fibrosis effect, it is anti-it is fat, Anti-diabetic and antifungic action etc., wherein Antimalarial and antitumor action are the most noticeable.Qinghaosu and its derivative Research of the object in terms of inflammatory is based primarily upon its therapeutic effect to autoimmune disease, such as lupus erythematosus and rheumatoid arthrosis Inflammation, researches show that it includes NF-Kb and cell factor, such as IL-1, IL-6 and IL-8 etc. equal to multiple inflammatory relevant target proteins There is regulating and controlling effect.But the application of qinghaosu and its derivative in terms for the treatment of cerebral ischemia diseases so far there are no research report.

In view of this special to propose the present invention.

Invention content

The technical problem to be solved in the present invention is to overcome the deficiencies of the prior art and provide a kind of for preventing and/or controlling Combination product and its application of cerebral ischemia are treated, has synergistic effect between the ingredient of the combination product.

In order to solve the above technical problems, the present invention adopts the following technical scheme that：

A kind of combination product for preventing and/or treating cerebral ischemia, wherein the combination product is to press down containing Sur1 The pharmaceutical composition of preparation and qinghaosu Sesquiterpene lactones compound；

Or it is the first medicament containing Sur1 inhibitor and the second medicament containing qinghaosu Sesquiterpene lactones compound Combination.

Specifically, on the one hand, it is provided by the present invention for preventing and/or treating the combination product of cerebral ischemia, it can be with It is that Sur1 inhibitor and qinghaosu Sesquiterpene lactones compound are put together and to form a kind of pharmaceutical composition, the pharmaceutical composition Can a kind of compound preparation further be made with pharmaceutically acceptable auxiliary material in object.

On the other hand, provided by the present invention for preventing and/or treat the combination product of cerebral ischemia, can also be by containing There are the first medicament of Sur1 inhibitor and the second medicament containing qinghaosu Sesquiterpene lactones compound to carry out drug combination. When drug combination, the medication of the first medicament and second medicament sequence in no particular order, can be first with the first medicament, can also be first with the Two medicaments can simultaneously be used with two medicaments.

Further, in the pharmaceutical composition Sur1 inhibitor and qinghaosu Sesquiterpene lactones compound quality Than for (1~3):(2500~5000)；

Qinghaosu Sesquiterpene lactones compound in Sur1 inhibitor and second medicament in first medicament in the combination Mass ratio be (1~3):(2500~5000).

Further, the pharmaceutical composition is Sur1 inhibitor, qinghaosu Sesquiterpene lactones compound and pharmacy Pharmaceutically acceptable dosage form, optimizing injection, more preferable injection or freeze-dried powder is made in upper acceptable auxiliary material.

Its supplementary product kind and preparation method can refer to the prior art.

Further, first medicament is made with pharmaceutically acceptable auxiliary material for Sur1 inhibitor and can pharmaceutically connect The dosage form received；

The second medicament is that pharmacy is made with pharmaceutically acceptable auxiliary material in qinghaosu Sesquiterpene lactones compound Upper acceptable dosage form, optimizing injection, more preferable injection or freeze-dried powder.

In the present invention, by the first medicament containing Sur1 inhibitor and qinghaosu Sesquiterpene lactones compound can be contained Second medicament carry out drug combination.First medicament or second medicament can be existing preparations in the prior art, Can be that Sur1 inhibitor or qinghaosu Sesquiterpene lactones compound are added pharmaceutically acceptable auxiliary material respectively to make respectively At pharmaceutically acceptable dosage form.The preparation method of supplementary product kind and each dosage form can refer to the prior art.

Further, the Sur1 inhibitor is selected from glibenclamide, orinase, Repaglinide, meglitinide, miaow One or more of lattice row azoles or Glimepiride.

Further, the qinghaosu Sesquiterpene lactones compound be the sesquiterpene lactone extracted from artemisia annua and The derivative of qinghaosu.

Further, the qinghaosu Sesquiterpene lactones compound is Artesunate, dihydroartemisinine or Artemether.

The present invention also provides the combination products to prepare the application in treating Imaging in Patients with Cerebral Ischemia Disease drug.

The cerebral ischemia is cerebral apoplexy.

After adopting the above technical scheme, the present invention has the advantages that compared with prior art：

The present invention provides a kind of containing Sur1 inhibitor and qinghaosu Sesquiterpene lactones compound, wherein Sur1 inhibitor Can be by inhibiting sur1 to mitigate brain edema, and qinghaosu Sesquiterpene lactones compound can be by inhibiting inflammatory factor and related egg White hair waves anti-inflammatory effect.The two has the function of synergy when being used cooperatively in appropriate proportions.By Sur1 inhibitor and sweet wormwood Plain Sesquiterpene lactones compound drug combination in appropriate proportions, can be than being applied alone in Sur1 inhibitor or qinghaosu sequiterpene Ester type compound has better therapeutic effect to cerebral ischemia diseases.

The specific implementation mode of the present invention is described in further detail below in conjunction with the accompanying drawings.

Description of the drawings

Fig. 1 shows cerebral infarction volume percentage, sham-operation group vs model groups,^###P ＜ 0.001；Administration group vs model groups,^**P ＜ 0.01,^*P ＜ 0.05；

Fig. 2 shows edema volume percentage, sham-operation group vs model groups,^###p<0.001；Administration group vs model groups,^**P ＜ 0.01；

Fig. 3 shows Serum TNF-α and IL-6 expressions, sham-operation group vs model groups,^###P ＜ 0.001；Administration group vs models Group,^***P ＜ 0.001,^*P ＜ 0.05；

It should be noted that these attached drawings and verbal description are not intended to the design model limiting the invention in any way It encloses, but is that those skilled in the art illustrate idea of the invention by referring to specific embodiments.

Specific implementation mode

In order to make the object, technical scheme and advantages of the embodiment of the invention clearer, below in conjunction with the embodiment of the present invention In attached drawing, the technical solution in embodiment is clearly and completely described, following embodiment for illustrating the present invention, but It is not limited to the scope of the present invention.

It is below embodiment 1 to embodiment 7, the composition of pharmaceutical composition is shown in Table 1 in each embodiment, preparation method To weigh each substance by the quality, then it is mixed uniformly to get pharmaceutical composition.

Table 1

 

Aforementioned pharmaceutical compositions can further add pharmaceutically acceptable auxiliary material and be prepared into pharmaceutically acceptable dosage form, Optimizing injection, including but not limited to injection or freeze-dried powder.

The research of test example 1, glibenclamide and Artesunate to cerebral ischemia reperfusion mouse brain injury protective effect

It prepares Kunming mouse MCAO ischemia-reperfusion models and causes cerebral ischemia and Brain edema, give glibenclamide and sweet wormwood amber Ester injection treatment, does positive control, after the treatment by infarction of brain volume ratio, edema volume with Edaravone Injection Than, the evaluation of measuring Sur1 inhibitor glibenclamide and sweet wormwood of Neuroscore and brain tissue inflammation's factor TNF-α and IL-6 Plain Sesquiterpene lactones compound is combined the therapeutic effect to cerebral apoplexy.

1, experimental animal and grouping

Kunming mouse 90 (is purchased from Kunming Medical University's SPF Animal Lab), male, 25-28g.It is randomly divided into 9 Experimental group, every group 10, including：

1. sham-operation group：Arteria carotis communis, external carotid artery and internal carotid, but the not embolism cerebrovascular are isolated, iv approach is given Give the solvent (0.9% physiological saline) for the treatment of group's equivalent；

2. model control group：It prepares cerebral ischemia (MCAO) and fills model again, intravenous injection (iv) approach is given with treatment group etc. The solvent (0.9% physiological saline) of amount；

3. 10 μ g/kg groups of glibenclamide：Cerebral ischemic model is prepared, iv approach gives glibenclamide；

4. 30 μ g/kg groups of glibenclamide：Cerebral ischemic model is prepared, iv approach gives glibenclamide；

5. Artesunate 25mg/kg groups：Cerebral ischemic model is prepared, iv approach gives Artesunate；

6. Artesunate 50mg/kg groups：Cerebral ischemic model is prepared, iv approach gives Artesunate；

7. 10 μ g/kg+ Artesunate 25mg/kg groups of glibenclamide：Cerebral ischemic model is prepared, iv approach gives glibenclamide And Artesunate

8. 30 μ g/kg+ Artesunate 50mg/kg groups of glibenclamide：Cerebral ischemic model is prepared, iv approach gives glibenclamide And Artesunate

Positive drug 9. (Edaravone 3mg/kg) control group：Cerebral ischemic model is prepared, iv approach gives Edaravone；

2, prepared by model：

Arterial occlusion (MCAO) model in transience mouse brain is prepared using line brush.Its operating procedure is as follows：First will Record of weighing after healthy Kunming mouse number is lain on the back and is fixed on operating table with 4% chloraldurate intraperitoneal injection of anesthesia, 60 DEG C of incandescent lamp irradiations are being placed at operating table 30cm, and mouse temperature is made to maintain 37 DEG C or so.It cuts neck and hits exactly skin Skin detaches subcutaneous tissue and is bubbled body of gland, exposes ectotrachea muscle, and separating mouse right muscles, to isolate right side neck total Artery (CCA), external carotid artery (ECA) and internal carotid (ICA), in CCA proximal parts and ECA distal ends, (distance CCA crotches are about 5mm) place's hanging wire is spare.Then distal end ligatures CCA and ECA.With No. 2 syringe needles from CCA away from slotting from the about 0.5cm of the forks ECA and ICA Enter line bolt.Insertion depth touches bolt line to when about 1cm (at mark point) with ophthalmic tweezers, feels that the when of having resistance indicates to have been inserted into Position, at this moment gently fastens bolt line, skin suture with filament.Nylon embolus line is extracted after 2h, so that its blood flow is led to again, is divided cage to put mouse Raising, pays attention to warming in steaming again.

3, it is administered

Glibenclamide is administered successively in three times for 0h after ischemia-reperfusion (being administered immediately after pulling out Outlet bolt), 3h and 6h, Administering mode is tail vein injection (iv).Artesunate is 0h Bolos intravenous administrations after ischemia-reperfusion.Artesunate is with 5% NaHCO₃Dissolving, glibenclamide are dissolved by heating with 1% meglumine.

4, experimental endpoints sample collection

It before putting to death rat, detaches abdominal aorta and takes blood 4mL, after standing 1h, 3000r/min centrifuges 10min, draws blood Clearly.- 20 DEG C of refrigerators are put into, TNF-α and IL-6 concentration are measured using Double antibody sandwich-ELISA (ELISA).Strictly It is operated by ELISA kit operation instructions.

5, evaluation index

5.1 Neuroscore：After rat operation for 24 hours, neurological deficit score is carried out to experimental animal and recorded, methods of marking For Longa methods, standards of grading are as follows：

0 point：Impassivity defective symptom；

1 point：Offside forelimb cannot stretch completely；

2 points：It turn-takes to offside；

3 points：It walks and is tilted to offside；

4 points：It spontaneous cannot walk, the loss of consciousness.

5.2 cerebral infarction volumes detect

5.3 brain water contents measure：Swelling volume and swelling percentage are calculated using swelling volumetric method.

6, statistical analysis

Each level area of brain section, infarct size etc. are measured using Imagepro plus6.0.Pass through Graphpad 5 softwares of Prism are for statistical analysis to data, one-way or two-way repeat measure ANOVA post-hoc Dunnett ' test carry out comparison among groups, and the standard of significant difference is 0.05.

7, experimental result

7.1 cerebral infarction volumes detect：By ischemia-reperfusion for 24 hours after mouse row excessively anaesthetize, directly take brain after broken end, set In in mouse brain mold, then it is sliced from front to back by brain is coronal, is divided into 4, is put in 1.0%TTC solution, 37 DEG C are protected from light 10min dyeing is incubated, infarcted region is not colored, and normal cerebral tissue dyes red.Then it takes pictures, passes through to every a piece of brain piece Imagepro plus6.0 image analysis systems measure each level area, ischemic areas, half brain area of ischemic side (homonymy area), Half brain area (to lateral area) of non-ischemic side calculates respective volume (mm in conjunction with slice thickness³), and calculate Infarction volume and account for offside The percentage of cerebral hemisphere volume.The results are shown in Figure 1, model group, positive controls (Edaravone -3mg/kg), Ge Lieben Urea 10ug/kg, glibenclamide 30ug/kg, Artesunate 25mg/kg groups, Artesunate 50mg/kg groups, glibenclamide 10ug/kg + Artesunate 25mg/kg combinations group, glibenclamide 30ug/kg+ Artesunate 50mg/kg combination groups are compared with sham-operation group, all There is different degrees of infarct, shows that MCAO ischemia-reperfusion models are successfully established；Wherein, compared with model group, Artesunate 25mg/kg groups, Artesunate 50mg/kg groups, glibenclamide 10ug/kg+ Artesunate 25mg/kg combination group glibenclamides 30ug/kg+ Artesunate 50mg/kg combinations groups can be substantially reduced infarct size, and be better than positive controls.

7.2 experiments calculate swelling volume and swelling percentage using swelling volumetric method.As shown in Fig. 2, compared with model group, Glibenclamide 30ug/kg and glibenclamide 30ug/kg+ Artesunate 50mg/kg combinations groups can significantly mitigate Brain edema, and be better than Positive controls.

7.3 Neurological deficits

Neurological deficits reflect the case where drug improves prognosis to mouse cerebral apoplexy, and the results are shown in Table 1：

Table 1, embodiment MACO mouse Nerves neurological deficit score (n=6)

Group	Neurological deficits (average value)
		Sham-operation group	0
Model group	3.2
		Glibenclamide 10ug/kg	2.1
Glibenclamide 30ug/kg	2.0
		Artesunate 25mg/kg	2.4
Artesunate 50mg/kg	2.1
		Glibenclamide 10ug/kg+ Artesunates 25mg/kg	1.6
Glibenclamide 30ug/kg+ Artesunates 50mg/kg	1.2
		Edaravone 3mg/kg	1.8

TNF-α and IL-6 are horizontal in 7.4 serum

Using enzyme-linked immunization, detects separate groups of mice serum cytokines TNF-α and IL-6 is horizontal.As a result it shows：With Normal group mouse is compared, model group TNF-α and IL-6 contents significantly increase (^###p<0.001)；Compared with model group mouse, Artesunate group, glibenclamide 10ug/kg+ Artesunates 25mg/kg combinations group and glibenclamide 30ug/kg+ Artesunates 50mg/kg combinations group can significantly reduce TNF-α and IL-6 contents in serum (^***p<0.001,^*p<0.05).As a result see Fig. 3：

8, conclusion

Show from the mouse MCAO ischemia-reperfusion models treatment drug effect evaluation result carried out in animal level green in the present invention Artemisic succinate, glibenclamide and Artesunate combination can significantly reduce Serum TNF-α and IL-6 contents, reduce brain edema and infraction, Especially combination group can improve prognosis, improve life in patients, have good development and application values.

Above-mentioned experiment is also carried out to the other combination products of the present invention, the result obtained is similar to above-mentioned test result.

The above is only presently preferred embodiments of the present invention, is not intended to limit the present invention in any form, though So the present invention has been disclosed as a preferred embodiment, and however, it is not intended to limit the invention, the technology people of any familiar present invention Member without departing from the scope of the present invention, when the technology contents using above-mentioned prompt make it is a little variation or be modified to The equivalent embodiment of equivalent variations, it is right according to the technical essence of the invention as long as being the content without departing from technical solution of the present invention Any simple modification, equivalent change and modification made by above example, in the range of still falling within the present invention program.

技术领域

本发明属于生物医药技术领域，具体地说，涉及一种用于预防和/或治疗脑缺血的组合产品及其应用。

背景技术

缺血性脑卒中具有高发病率、高死亡率、高致残率、高复发率、高医疗花费几个特点，是危害我国居民健康的重大慢性病。临床数据统计显示，全球脑卒中死亡率已经超过癌症成为单病种死亡率最高的疾病。众所周知，炎症反应是脑缺血病理损伤过程中的重要组成部分，也是加重脑缺血损伤的潜在因素。除此之外，缺血性脑卒中常伴有脑水肿和出血性转化，其中脑水肿是缺血性脑卒中最严重的并发症，卒中患者死亡中80％死于卒中后期的恶性脑水肿。因此炎症及脑水肿的防治对缺血性脑卒中的治疗有重要意义。

磺脲类受体1(sulfonylurea receptor 1，Sur1)-瞬时受体电位通道M4型(transient receptor potential melastatin 4，Trpm4)通道对于脑水肿的形成起着重要作用。相关文献报道，缺血性脑卒中发生4-8小时是脑水肿形成的关键时期，在这时期缺血部位脑血管内壁细胞膜Sur1-Trpm4通道的Sur1受体会出现应激性高表达，大量表达的Sur1受体导致Sur1-Trpm4通道激活。Sur1-Trpm4通道的激活增加阳离子特别是钠离子进入细胞内，由于渗透压的缘故驱动水大量涌入细胞内，引起缺血部位细胞乃至组织发生水肿，是卒中患者出现脑水肿的关键病理机制。Sur1抑制剂与Sur1受体结合抑制这个通道的开放，从而有效地抑制脑水肿的形成。代表性Sur1抑制剂格列本脲针对卒中后脑水肿的治疗已在美国进入临床III期。

青蒿素及其衍生物是我国科学工作者研究开发的一类抗疟新药，目前广泛应用于疟疾的治疗。随着研究的不断深入，除了其抗疟作用外，科学家还相继发现了青蒿素及其衍生物在其他方面的药理作用，包括抗肿瘤作用、对免疫细胞的作用、抗纤维化作用、抗肥胖、抗糖尿病以及抗真菌作用等，其中抗疟作用和抗肿瘤作用最为引人关注。青蒿素及其衍生物在炎性方面的研究主要基于其对自身免疫性疾病的治疗作用，如红斑狼疮和类风湿关节炎，研究显示其对多个炎性相关靶蛋白包括NF-Kb和细胞因子，例如IL-1、IL-6和IL-8等均有调控作用。但青蒿素及其衍生物在治疗脑缺血性疾病方面的应用至今未见研究报道。

有鉴于此特提出本发明。

发明内容

本发明要解决的技术问题在于克服现有技术的不足，提供一种用于预防和/或治疗脑缺血的组合产品及其应用，该组合产品的成分之间具备协同增效的作用。

为解决上述技术问题，本发明采用如下技术方案：

一种用于预防和/或治疗脑缺血的组合产品，其中，所述的组合产品为含有Sur1抑制剂和青蒿素倍半萜内酯类化合物的药物组合物；

或为含有Sur1抑制剂的第一药剂和含有青蒿素倍半萜内酯类化合物的第二药剂的组合。

具体地说，一方面，本发明所提供的用于预防和/或治疗脑缺血的组合产品，可以是将Sur1抑制剂和青蒿素倍半萜内酯类化合物放在一起形成一种药物组合物，该药物组合物可进一步与药学上可接受的辅料制成一种复方制剂。

另一方面，本发明所提供的用于预防和/或治疗脑缺血的组合产品，也可以是将含有Sur1抑制剂的第一药剂和含有青蒿素倍半萜内酯类化合物的第二药剂进行联合用药。在联合用药时，第一药剂与第二药剂的用药顺序不分先后，可以先用第一药剂，也可以先用第二药剂，还可以两个药剂同时使用。

进一步的，所述的药物组合物中Sur1抑制剂和青蒿素倍半萜内酯类化合物的质量比为(1～3):(2500～5000)；

所述的组合中第一药剂中Sur1抑制剂和第二药剂中青蒿素倍半萜内酯类化合物的质量比为(1～3):(2500～5000)。

进一步的，所述的药物组合物为Sur1抑制剂、青蒿素倍半萜内酯类化合物与药学上可接受的辅料制成药学上可接受的剂型，优选注射剂，更优选注射液或冻干粉针。

其辅料种类和制备方法可参照现有技术。

进一步的，所述的第一药剂为Sur1抑制剂与药学上可接受的辅料制成药学上可接受的剂型；

所述的第二药剂为青蒿素倍半萜内酯类化合物与药学上可接受的辅料制成药学上可接受的剂型，优选注射剂，更优选注射液或冻干粉针。

本发明中，可以将含有Sur1抑制剂的第一药剂和含有青蒿素倍半萜内酯类化合物的第二药剂进行联合用药。所述的第一药剂或第二药剂可以是现有技术中已有的制剂，也可以是将Sur1抑制剂或青蒿素倍半萜内酯类化合物分别添加药学上可接受的辅料分别制成药学上可接受的剂型。辅料种类和各剂型的制备方法可参照现有技术。

进一步的，所述的Sur1抑制剂选自格列本脲、甲苯磺丁脲、瑞格列奈、美格列奈、咪格列唑或格列美脲中的一种或几种。

进一步的，所述的青蒿素倍半萜内酯类化合物是从黄花蒿中提取的倍半萜内酯及青蒿素的衍生物。

进一步的，所述的青蒿素倍半萜内酯类化合物为青蒿琥酯、双氢青蒿素或蒿甲醚。

本发明还提供所述的组合产品在制备治疗脑缺血疾病药物中的应用。

所述的脑缺血为脑卒中。

采用上述技术方案后，本发明与现有技术相比具有以下有益效果：

本发明提供一种含有Sur1抑制剂和青蒿素倍半萜内酯类化合物，其中Sur1抑制剂可通过抑制sur1减轻脑水肿，而青蒿素倍半萜内酯类化合物可通过抑制炎症因子及相关蛋白发挥抗炎作用。两者以适当比例配合使用时具有协同增效的作用。将Sur1抑制剂和青蒿素倍半萜内酯类化合物以适当比例联合用药，可以比单用Sur1抑制剂或者青蒿素倍半萜内酯类化合物对脑缺血性疾病有更好的治疗效果。

下面结合附图对本发明的具体实施方式作进一步详细的描述。

附图说明

图1示脑梗死体积百分比，假手术组vs模型组，^###p＜0.001；给药组vs模型组，^**p＜0.01，^*p＜0.05；

图2示脑水肿体积百分比，假手术组vs模型组，^###p<0.001；给药组vs模型组，^**p＜0.01；

图3示血清TNF-α和IL-6表达水平，假手术组vs模型组，^###p＜0.001；给药组vs模型组，^***p＜0.001，^*p＜0.05；

需要说明的是，这些附图和文字描述并不旨在以任何方式限制本发明的构思范围，而是通过参考特定实施例为本领域技术人员说明本发明的概念。

具体实施方式

为使本发明实施例的目的、技术方案和优点更加清楚，下面将结合本发明实施例中的附图，对实施例中的技术方案进行清楚、完整地描述，以下实施例用于说明本发明，但不用来限制本发明的范围。

以下为实施例1至实施例7，各实施例中药物组合物的组成见表1所示，其制备方法为按所述质量称取各物质，然后将其混合均匀，即得药物组合物。

表1

 

上述药物组合物可进一步添加药学上可接受的辅料制备成药学上可接受的剂型，优选注射剂，包括但不限于注射液或冻干粉针。

试验例1、格列本脲与青蒿琥酯对脑缺血再灌小鼠脑损伤保护作用的研究

制备昆明种小鼠MCAO缺血再灌模型造成脑缺血及脑肿胀，给予格列本脲及青蒿琥酯注射治疗，用依达拉奉注射液做阳性对照，在治疗后通过对大脑梗死体积比、水肿体积比、神经功能评分、及脑组织炎症因子TNF-α和IL-6的测定评价Sur1抑制剂格列本脲及青蒿素倍半萜内酯类化合物联用对脑卒中的治疗作用。

1、实验动物及分组

昆明种小鼠90只(购自昆明医科大学SPF动物实验室)，雄性，25-28g。随机分为9个实验组，每组10只，包括：

①假手术组：分离出颈总动脉、颈外动脉及颈内动脉，但不栓塞脑血管，iv途径给予治疗组等量的溶剂(0.9％生理盐水)；

②模型对照组：制备脑缺血(MCAO)再灌模型，静脉注射(iv)途径给予与治疗组等量的溶剂(0.9％生理盐水)；

③格列本脲10μg/kg组：制备脑缺血模型，iv途径给予格列本脲；

④格列本脲30μg/kg组：制备脑缺血模型，iv途径给予格列本脲；

⑤青蒿琥酯25mg/kg组：制备脑缺血模型，iv途径给予青蒿琥酯；

⑥青蒿琥酯50mg/kg组：制备脑缺血模型，iv途径给予青蒿琥酯；

⑦格列本脲10μg/kg+青蒿琥酯25mg/kg组：制备脑缺血模型，iv途径给予格列本脲及青蒿琥酯

⑧格列本脲30μg/kg+青蒿琥酯50mg/kg组：制备脑缺血模型，iv途径给予格列本脲及青蒿琥酯

⑨阳性药(依达拉奉3mg/kg)对照组：制备脑缺血模型，iv途径给予依达拉奉；

2、模型制备：

采用线栓法制备短暂性小鼠大脑中动脉闭塞(MCAO)模型。其操作步骤如下：先将健康昆明种小鼠编号后称重记录，以4％的水合氯醛腹腔注射麻醉，仰卧固定于手术台上，在距离手术台30cm处放置60℃白炽灯照射，使小鼠体温维持在37℃左右。切开颈部正中皮肤，分离皮下组织及鼓泡腺体，暴露出气管外层肌肉，分离小鼠右侧肌肉、分离出右侧颈总动脉(CCA)、颈外动脉(ECA)和颈内动脉(ICA)，在CCA近心端及ECA远心端(距离CCA分叉处约5mm)处挂线备用。然后远心端结扎CCA和ECA。用2号针头从CCA距ECA和ICA岔口约0.5cm处插入线栓。插入深度到约1cm(标记点处)时用眼科镊轻推栓线，感觉到有阻力时即表示已插到位，这时用细线轻轻系牢栓线，缝合皮肤。2h后拔出尼龙栓线，使其血流再通，将小鼠分笼放回笼内饲养，注意保暖。

3、给药

格列本脲为缺血再灌注后0h(即拔出线栓后立即给药)、3h和6h分三次依次给药，给药方式为尾静脉注射(iv)。青蒿琥酯为缺血再灌注后0h静注给药。青蒿琥酯用5％的NaHCO₃溶解，格列本脲均用1％的葡甲胺加热溶解。

4、实验终点样品采集

在处死大鼠前，分离腹主动脉并取血4mL，静置1h后，3000r/min离心10min，吸取血清。放入-20℃冰箱，采用双抗体夹心酶联免疫吸附法(ELISA)测定TNF-α和IL-6浓度。严格按ELISA试剂盒使用说明书操作。

5、评价指标

5.1神经功能评分：大鼠手术后24h，对实验动物进行行为学评分并记录，评分方法为Longa法，评分标准如下：

0分：无神经缺损症状；

1分：对侧前肢不能完全伸直；

2分：向对侧转圈；

3分：行走向对侧倾斜；

4分：不能自发行走，意识丧失。

5.2脑梗死体积检测

5.3脑含水量测定：采用肿胀体积法计算肿胀体积及肿胀百分比。

6、统计学分析

采用Imagepro plus6.0测定脑切片各层面面积、梗死面积等。通过GraphpadPrism 5软件对数据进行统计分析，one-way或two-way repeat measure ANOVA post-hocDunnett’test进行组间比较，显著性差异的标准为0.05。

7、实验结果

7.1脑梗死体积检测：将缺血再灌注24h后的小鼠行过量麻醉，直接断头后取脑，置于小鼠脑模具内，然后将大脑冠状从前向后切片，分为4片，放于1.0％TTC溶液中，37℃避光温育10min染色，梗死区不着色，正常脑组织染成红色。然后对每一片脑片进行拍照，经Imagepro plus6.0图像分析系统测量各层面面积、缺血面积、缺血侧半脑面积(同侧面积)、未缺血侧半脑面积(对侧面积)，结合切片厚度计算相应体积(mm³),并计算梗死体积占对侧大脑半球体积的百分比。结果如图1所示，模型组、阳性对照组(依达拉奉-3mg/kg)、格列本脲10ug/kg、格列本脲30ug/kg、青蒿琥酯25mg/kg组、青蒿琥酯50mg/kg组、格列本脲10ug/kg+青蒿琥酯25mg/kg联用组、格列本脲30ug/kg+青蒿琥酯50mg/kg联用组与假手术组相比，都出现了不同程度梗死，表明MCAO缺血再灌模型成功建立；其中，与模型组相比，青蒿琥酯25mg/kg组、青蒿琥酯50mg/kg组、格列本脲10ug/kg+青蒿琥酯25mg/kg联用组格列本脲30ug/kg+青蒿琥酯50mg/kg联用组能显著减小梗死面积，且优于阳性对照组。

7.2实验采用肿胀体积法计算肿胀体积及肿胀百分比。如图2所示，与模型组比较，格列本脲30ug/kg和格列本脲30ug/kg+青蒿琥酯50mg/kg联用组能显著减轻脑肿胀，且优于阳性对照组。

7.3神经行为学评分

神经行为学评分反映药物对小鼠脑卒中改善预后的情况，结果见表1所示：

表1、实施例MACO小鼠神经行为学评分(n＝6)

组别	神经行为学评分(平均值)
		假手术组	0
模型组	3.2
		格列苯脲10ug/kg	2.1
格列苯脲30ug/kg	2.0
		青蒿琥酯25mg/kg	2.4
青蒿琥酯50mg/kg	2.1
		格列苯脲10ug/kg+青蒿琥酯25mg/kg	1.6
格列苯脲30ug/kg+青蒿琥酯50mg/kg	1.2
		依达拉奉3mg/kg	1.8

7.4血清中TNF-α和IL-6水平

采用酶联免疫法，检测不同组小鼠血清细胞因子TNF-α和IL-6水平。结果显示：与正常对照组小鼠比较，模型组TNF-α和IL-6含量显著增高(^###p<0.001)；与模型组小鼠比较，青蒿琥酯组、格列本脲10ug/kg+青蒿琥酯25mg/kg联用组及格列本脲30ug/kg+青蒿琥酯50mg/kg联用组可显著降低血清中TNF-α和IL-6含量(^***p<0.001，^*p<0.05)。结果见图3：

8、结论

从在动物水平进行的小鼠MCAO缺血再灌模型治疗药效评价结果表明，本发明中青蒿琥酯、格列本脲与青蒿琥酯联用能显著降低血清TNF-α和IL-6含量，减少脑水肿及梗塞，特别是联用组能改善预后、提高患者生存质量，具有很好的开发应用价值。

对本发明其它组合产品也进行了上述试验，其获得的结果与上述试验结果相似。

以上所述仅是本发明的较佳实施例而已，并非对本发明作任何形式上的限制，虽然本发明已以较佳实施例揭露如上，然而并非用以限定本发明，任何熟悉本发明的技术人员在不脱离本发明技术方案范围内，当可利用上述提示的技术内容作出些许变动或修饰为等同变化的等效实施例，但凡是未脱离本发明技术方案的内容，依据本发明的技术实质对以上实施例所作的任何简单修改、等同变化与修饰，均仍属于本发明方案的范围内。