CN110974904A - Medical application of qi-tonifying and pulse-recovering (freeze-drying) for injection - Google Patents

Abstract

The invention discloses medical application of qi-tonifying pulse-recovering (freeze-drying) for injection, which comprises application of improving the use safety of tissue plasminogen activator (tPA) and application of improving cerebral hemorrhage. The invention uses a mouse cerebral hemorrhage transformation model of tPA thrombolysis after the middle cerebral artery occlusion reperfusion (MCAO/R) induction to verify the improvement effect of qi-tonifying pulse-restoring (freeze-drying) for injection on the hemorrhagic transformation caused by tPA. The improvement effect of the injection for tonifying qi and recovering pulse (freeze-drying) on the cerebral hemorrhage of the mice is verified by using a collagenase-induced cerebral hemorrhage model of the mice.

Description

Medical application of qi-tonifying and pulse-recovering (freeze-drying) for injection

Technical Field

The invention relates to the field of traditional Chinese medicine compound preparations and cerebral apoplexy, in particular to application of qi-tonifying and pulse-restoring injection (freeze-drying) in improving use safety of tissue plasminogen activator (tPA) and application of qi-tonifying and pulse-restoring injection (freeze-drying) in improving cerebral hemorrhage.

Background

Stroke is a frequently encountered Disease in the elderly, characterized by high disability rate, high mortality, and is the leading cause of death in Chinese, severely threatening human health ((1) Zhou M, Wang H, Zeng X, et al, mortality, morbid, and risk factors in China and its services, 1990) -2017: a systematic catalysis for the Global brain of Disease Study 2017[ J ]. Lancet 2019,394(10204): 1-14.). The cerebral apoplexy is mainly divided into cerebral arterial thrombosis and hemorrhagic stroke, wherein the proportion of the cerebral arterial thrombosis accounts for 70 to 80 percent. Ischemic stroke is caused by cerebral ischemia and hypoxia caused by constriction or occlusion of blood vessels, local cerebral necrosis or softening are caused, and finally cerebral dysfunction and death of a stroke patient are caused, and atherosclerosis of carotid artery and vertebral artery is a main cause of ischemic stroke. (2) neurological division of the Chinese medical society, neurological division of the Chinese medical society cerebrovascular disease group, Chinese guidance for acute ischemic stroke 2018J, J of the Chinese neurology, 2018,51(9):666 and 682.). Hemorrhagic stroke, also known as cerebral hemorrhage, refers to hemorrhage caused by vascular rupture in non-traumatic brain parenchyma, and the incidence rate of the hemorrhagic stroke accounts for about 20 percent of the total stroke, but the hemorrhagic stroke is the stroke subtype with the highest clinical mortality rate. Cerebrovascular diseases such as hypertension, anticoagulant drug use, cerebral ischemia-reperfusion injury, cerebral aneurysm, etc. are the main causes of cerebral hemorrhage ((3) van Ash C J, Luitse MJ, Rinkel GJ, et al, index, case failure, and functional outer of the indoor vascular disease over time, recording to, sex, and electronic origin: analytical review and meta-analysis [ J ]. Lancet Neurol,2010.9(2):167 and 176.). The survivors of the cerebral apoplexy are often accompanied with symptoms of dyskinesia, aphasia, blindness and the like, the self-care of life is difficult, and the great burden is brought to families and society of patients ((4) picrorhiza, Gong glory. the economic burden of diseases of the ischemic cerebral apoplexy [ J ]. Chinese sanitary economy, 2003,22(12): 18-20.). Therefore, how to better treat ischemic cerebral apoplexy is a global, especially in china, urgent medical problem to be solved.

Tissue plasminogen activator (tPA) is the only thrombolytic Drug approved by the Food and Drug Administration (FDA) for clinical treatment of ischemic stroke, and can promote thrombolysis and restore normal blood supply to the brain ((5) Zivin JA acid stream therapy with tissue plasminogen activator (tPA) site consumed by the U.S. Food and Drug Administration (FDA) [ J ]. Ann Neurol,2009,66(1): 6-10.). However, because of the narrow treatment time window of tPA (approximately 3-4.5h), thrombolytic treatment of tPA administered to patients beyond the application time window can cause symptoms of cerebral hemorrhage, which greatly limits the clinical use of tPA ((6) Ouk T, Potey C, Maestrini, et al. However, the existing treatments for cerebral hemorrhage mainly include surgical treatment and drug treatment, and surgical excision of hematoma and acute-stage blood pressure reduction treatment can reduce the lump effect and reduce the injury caused by erythrocyte lysis and delayed thrombin release, but the two modes have higher risks of side effects, and clinical results show that the nerve function recovery effect of patients is poor. And some preclinical studies have shown poor results in clinical trials of drugs with improved effects on cerebral hemorrhage ((7) Cordonnier C, Demchuk A, Ziai W, et. intrafibrous haemomorrhage: current applications to access management [ J ] Lancet,2018,392(10154): 1257-) 1268.). Therefore, the medicine capable of effectively improving cerebral hemorrhage and relieving the bleeding side effect of tPA is searched, the medication safety of tPA is improved, and the medicine has important significance for treating cerebral apoplexy clinically. To date, preclinical studies have shown that there are several drugs that can effectively improve cerebral hemorrhage or reduce the hemorrhagic transformation caused by tPA: the blood brain barrier protective agent mainly comprises minocycline, cilostazol, GM6001, fasudil, candesartan, bryodin and the like; the second is an angiogenesis promoting drug which mainly comprises coumarin derivatives IMM-H004, granulocyte colony stimulating factor and the like; the third is other drugs, mainly including vitamin C, DDFPe, erythropoietin and so on ((8) Wang L, Fan W, Cai P, ethyl.recombinant ADAMTS13 process tissue activator-induced magnetic rear linkage in microorganisms [ J ]. Ann neuron, 2013,73(2): 189. 198.(9) Fan X, Jiang Y, Yu Z, et al.combination of alpha. expression peptides for organic thermal reaction in tissues with structural adhesives/diabetes [ J ]. Adv. normal, 2014,71: 410. 10) Tan Z, Li, R, C.C. molecular C, and J.368 phase of polypeptide, molecular cellulose, polysaccharide. The research of the traditional Chinese medicine on the aspects of improving cerebral hemorrhage and relieving hemorrhage transformation caused by tPA is not wide enough, and the traditional Chinese medicine which is published in foreign journals at present mainly comprises a traditional Chinese medicine compound preparation red sage injection and T541 (total astragaloside: total salvianolic acid: panax notoginseng saponins 5: 4: 1) ((11) Chen Q F, Liu Y, Pan C S, et al.

The qi-tonifying pulse-restoring (freeze-drying) (YQFM) for injection is a modern Chinese medicinal compound preparation derived from pulse-activating powder, is composed of three medicines of red ginseng, dwarf lilyturf tuber and Chinese magnoliavine fruit, has the effects of tonifying qi, restoring pulse, nourishing yin and promoting the production of body fluid, is commonly used for treating qi-yin deficiency of exertional angina pectoris of coronary heart disease in clinic, and can be purchased from the market. Meanwhile, the YQFM is clinically found to have obvious curative effect on acute cerebral infarction, improve the neurological deficit of patients and improve the life quality of the patients. (12) whether Cao G, Ye X, Xu Y, et al. YIQIFuMai powder in injection of aqueous solutions of bleeding-woven barrier dynamic function and in vitro of membrane after filtration of blood in injection of bleeding in microorganism [ J ]. DrugDes Dev der 2016,10: 315. (13) Cao G, Chen H, Zhang Y, et al. YIQIMai powder injection of aqueous solutions of the oxidative-glucose depletion-induced fibrous barrier dynamic function and in vitro of bleeding with the same degree of freedom and selectivity of the same degree of freedom and the same degree of freedom, and whether or not the inhibition of cerebral hemorrhage of the same degree of the bleeding is effectively reported by Cao G, Ye X, Xu Y, and the same degree of freedom of NF- κ B1/tPg path of tissue in injection of bleeding [ J ]. Etco-M2016,183).

Disclosure of Invention

The invention discloses a medical application of qi-tonifying pulse-recovering (freeze-drying) for injection in treating cerebral hemorrhage.

The invention also discloses the application of the qi-tonifying pulse-recovering injection in preparing a medicament for treating cerebral hemorrhage.

In some embodiments, the cerebral hemorrhage according to the present invention refers to hemorrhage caused by rupture of blood vessels in non-traumatic brain parenchyma, and in some embodiments, may be intracerebral hemorrhage caused by hypertension, hyperlipidemia, cerebral amyloid angiopathy, cerebral aneurysm, use of anticoagulant drugs, use of thrombolytic drugs, or other bad life habits. In some embodiments, the cerebral hemorrhage described herein is a hemorrhage caused by tissue plasminogen activator (tPA).

The invention also discloses application of the qi-tonifying and pulse-recovering injection in preparing a medicine for treating hemorrhagic stroke.

The invention also provides application of the qi-tonifying pulse-restoring injection in preparing a medicine for relieving hemorrhagic transformation symptoms after the TPA thrombolysis treatment of ischemic stroke.

The invention also provides application of the qi-tonifying and pulse-recovering medicine for injection in improving the safety of the medicine, wherein the medicine is tissue plasminogen activator (tPA).

The invention also provides a method for improving the safety of the tissue plasminogen activator, which is to jointly use the qi-tonifying and pulse-recovering injection.

The invention also provides a pharmaceutical composition, which comprises the qi-tonifying pulse-recovering and tissue plasminogen activator (tPA) for injection. The invention also provides application of the pharmaceutical composition in preparing a medicine for treating cerebral arterial thrombosis.

The invention uses a mouse cerebral hemorrhage transformation model of tPA thrombolysis after the middle cerebral artery occlusion reperfusion (MCAO/R) induction to verify the improvement effect of qi-tonifying pulse-restoring (freeze-drying) for injection on the hemorrhagic transformation caused by tPA. The improvement effect of the injection for tonifying qi and recovering pulse (freeze-drying) on the cerebral hemorrhage of the mice is verified by using a collagenase-induced cerebral hemorrhage model of the mice.

The pharmacological action of qi-tonifying pulse-restoring (freeze-drying) for injection in treating cerebral hemorrhage will be further described with reference to the following examples.

Drawings

FIG. 1 is a schematic diagram of an experimental flow;

figure 2 effect of YQFM on MCAO/R induced brain bleeding volume following tPA thrombolysis in mice (X ± SD, n ═ 6).^##P<0.01vs. sham group; p<0.01vs. vehicle group;^$$P<tPA group at 0.01 vs;

figure 3 effect of YQFM on cerebral hemorrhage area after MCAO/R induced thrombolysis of mouse tPA (X ± SD, n ═ 6).^##P<0.01vs. sham group; p<0.01vs. vehicle group;^$$P<tPA group at 0.01 vs;

figure 4 effect of YQFM on mouse neural function following MCAO/R induced mouse tPA thrombolysis (X ± SD, n ═ 6).^##P<0.01vs. sham group; p<0.01vs. vehicle group;^$$P<tPA group at 0.01 vs;

figure 5 effect of YQFM on mortality within 24h of mice after MCAO/R induced thrombolysis of mouse tPA (n ═ 30).^##P<0.01vs. sham group; p<0.01vs. vehicle group;^$$P<tPA group at 0.01 vs;

FIG. 6 Effect of YQFM on MCAO/R-induced post-thrombolytic mouse tPA blood brain Barrier Permeability ((R))

n＝6)。^##P<0.01vs. sham group; p<0.01vs. vehicle group;^$$P<tPA group at 0.01 vs;

FIG. 7 Effect of YQFM on collagenase induced cerebral hemorrhage volume and neurological function in mice (II)

n＝4)。^##P<0.01vs. sham group;^*P<0.05,**P<model group 0.01vs.

Detailed Description

The present invention will be further described with reference to the following examples, which are intended to illustrate the present invention and not to limit the scope of the present invention, and all simple modifications of the preparation method of the present invention based on the idea of the present invention are within the scope of the present invention. The following examples are experimental methods without specifying specific conditions, and generally follow the methods known in the art. The test materials used in the following examples were purchased from a conventional biochemical reagent store unless otherwise specified.

The embodiment of the invention uses the qi-tonifying pulse-restoring (freeze-drying) for injection as a product of pharmaceutical industry GmbH of Tianjin Tianshi power, and the approval document is a Chinese medicine standard Z20060463.

Example 1

Qi-tonifying and pulse-restoring (freeze-drying) for injection, which is called YQFM for short, has inhibiting effect on hemorrhagic transformation of mice tPA (thrombolysis) induced by middle cerebral artery infarction reperfusion (MCAO/R)

The experimental method comprises the following steps:

a) animal grouping and drug intervention:

male C57BL/6J mice were randomly divided into four groups, sham, vehicle, tPA and tPA + YQFM, 6 animals per group. Preparing a mouse Middle Cerebral Artery Occlusion (MCAO) model by a thread-tying method, slowly pulling out the thread-tying after 45min, loosening a common carotid artery ligature to realize reperfusion, and respectively injecting tPA and YQFM into tail veins at time points of 2h and 2.5h after MCAO operation. The specific grouping situation, mouse surgery and administration operation flow are shown in table 1 and fig. 1:

TABLE 1 animal groups and drug intervention

b) Preparation of mouse middle cerebral artery infarction (MCAO) model by wire embolism method

After fasting for 12 hours, the mice were anesthetized by intraperitoneal injection of 4% chloral hydrate (10mL/kg), and the mice were fixed in the supine position with an adhesive tape. The skin was cut along the midline of the neck, muscle tissue was isolated, and the right common carotid artery and external carotid artery were exposed. The common carotid artery is tied at the proximal end, the external carotid artery is tied at the distal end, and a section of internal carotid artery is dissociated to the deep part. A small opening is punctured at the proximal end of the common carotid artery by a needle, a prepared wire plug is inserted, and the wire plug enters the internal carotid artery through the bifurcation of the common carotid artery. The wire plug is inserted until a slight resistance is met, i.e. the black marked point at the upper end of the wire plug reaches the bifurcation of the common carotid artery. After the insertion of the thread, the preset ligature is tied tightly to prevent the thread plug from sliding out or shifting, and gauze soaked with normal saline is applied to the exposed part of the neck. After MCAO for 45min, loosening the fixing thread, slowly pulling out the thread plug, loosening the common carotid artery ligature to realize reperfusion, and suturing the wound. The whole operation process and the postoperation attention of the mice to keep warm, and the animals are placed in a mouse cage filled with clean padding after the operation to recover water and feed.

c) Preparation of cerebral hemorrhage transformation model

After anesthesia, the mice were injected intraperitoneally with 50% glucose (6 mg/kg). the tPA group mice are injected with tPA by a constant flow injection pump tail vein at the time point of 2h after MCAO operation, the first 1mg/kg of tPA is injected rapidly, the second 9mg/kg of tPA is infused slowly, and the injection is finished in 30 min. Sham and vehicle groups were not injected with tPA.

d) Method for measuring mouse cerebral hemorrhage amount by using hemoglobin kit

Each group of mice was perfused with 1 Xphosphate buffered saline (PBS) from the heart 24h after MCAO surgery until the blood was washed clean, and the brains were taken and photographed. Sucking off the excess liquid and freezing. The olfactory bulb, the brainstem and the cerebellum are cut off, the right hemisphere of the brain is weighed, 1mL of precooled 1 XPBS is added into each 0.1g of brain tissue for homogenization, the brain tissue is transferred into a 1.5mL EP tube for centrifugation (4 ℃, 13000rpm and 30min), 50 mu L of supernatant is taken and added into a 96-well plate, 200 mu L of hemoglobin detection reagent is added, and 3 duplicate wells are formed on each group of samples. After 5min reaction at room temperature, OD was measured at 400nm wavelength and the hemoglobin content in the brain was calculated in μ g/mg according to the protocol in the kit instructions.

e) Hematoxylin-eosin (H & E) staining method for detecting pathological morphological change of mouse brain section

Each group of mice was sacrificed 24h after MCAO surgery, brains were taken and placed in an EP tube containing 4% paraformaldehyde, and were sent to the pathology room of the Jiangsu province drug safety evaluation center for detection.

f) Determination of neuro-behavioral scores

According to the Longa scoring method, each group of mice is judged 24h after MCAO operation, from 0 to 4 points, the higher the score is, the more serious the nerve injury is, and the specific scoring standard is shown in Table 2:

TABLE 2 neurobehavioral Scoring criteria

g) Statistics of mortality within 24h after MCAO in mice

For each group of mice, the total number of mice with successful MCAO was counted (N1), the number of dead mice in each group was counted 24h after MCAO (N2), and the mortality of mice in each group within 24h after MCAO was calculated as follows:

mortality (%) - (N2/N1). times.100%

h) Determination of Evans blue Permeability

22h after MCAO, 2% Evans blue solution (3mL/kg) is injected into tail vein of each group of mice, and after 2h, the mice are perfused with 0.9% physiological saline from heart until blood is washed clean, and brains are taken and photographed. Removing olfactory bulb, brainstem and cerebellum, taking right hemisphere of cerebrum, weighing, adding 1mL of precooled trichloroacetic acid into every 0.1g of brain tissue, fully grinding, transferring into a 1.5mL EP tube, centrifuging (4 ℃, 10000rpm, 30min), taking supernatant, adding into a 96-well plate, measuring OD value at the wavelength of 620nm of each sample in 3 multiple wells, and calculating the content according to a standard curve, wherein the unit is ug/g.

The experimental results are as follows:

a) inhibition effect of YQFM on mouse tPA thrombolysis postcerebral hemorrhage amount induced by MCAO/R

As can be seen from fig. 2, both the mouse brain appearance and the hemoglobin kit test results show that, compared with the sham operation group, the tPA group had significant cerebral hemorrhage, the hemoglobin content was significantly increased, while the bleeding phenomenon of the tPA + YQFM group was alleviated, and the cerebral hemoglobin content was significantly decreased, indicating that YQFM could significantly reduce the amount of cerebral hemorrhage induced by tPA after cerebral ischemia in mice.

b) Improvement effect of YQFM on mouse tPA thrombolysis postcerebral hemorrhage area induced by MCAO/R

As shown by the H & E staining results of the brain sections in fig. 3, the tPA group showed significantly increased cerebral hemorrhage area, and significant vacuolization, infarction, and other injuries of the brain tissue, compared with the sham operation group and the vehicle group; the YQFM and tPA combination group showed a significant reduction in bleeding area compared to the tPA group. The YQFM is suggested to be capable of effectively inhibiting hemorrhagic transformation caused by tPA thrombolysis after cerebral ischemia of mice.

c) Improvement effect of YQFM on MCAO/R-induced mouse tPA postthrombolytic nerve function injury

The results in fig. 4 show that the vehicle group mice had mild to moderate neurological deficit, the tPA group mice had moderate to severe neurological deficit, the degree of neurological deficit was significantly increased in the former two groups, and the YQFM and tPA combined group mice had significantly decreased degree of neurological deficit in the tPA group, as compared to the sham group. The result shows that YQFM can improve mouse tPA thrombolysis aggravated nerve function loss induced by MCAO/R.

d) Effect of YQFM on mouse mortality within 24h after MCAO

As seen in fig. 5, the mortality rate of tPA group was significantly increased and reached more than 50% compared to the sham group and vehicle group; compared with the tPA group, the mice in the YQFM and tPA combined group have obviously reduced mortality. This indicates that YQFM can reduce mortality caused by tPA within 24h after MCAO in mice.

e) Improvement effect of YQFM on blood brain barrier destruction after MCAO/R induced mouse tPA thrombolysis

Figure 6 evans blue leakage results show that evans blue content was significantly increased in tPA group compared to sham group and vehicle group; compared with tPA group, the YQFM + tPA group has significantly reduced Evans blue content. The result shows that the blood brain barrier permeability of the mouse tPA induced by MCAO/R is obviously increased after thrombolysis, and the combination of YQFM and tPA can reduce the blood brain barrier permeability increase caused by tPA.

The research results show that the qi-tonifying pulse-restoring freeze-drying (YQFM) for injection has a remarkable treatment effect on hemorrhagic transformation of mice after thrombolysis of the cerebral arterial thrombosis tPA, effectively improves the nerve function damage and blood brain barrier damage aggravated by the tPA, and inhibits the increase of the death rate of the mice caused by the tPA. Therefore, YQFM can become a candidate drug for clinically treating ischemic stroke by being combined with tPA or singly treating hemorrhagic stroke, and the combination of YQFM and tPA can be an effective means for clinically improving the safety of tPA medication, thereby having great development prospect. Meanwhile, a certain reference basis is provided for expanding the clinical application of the marketed YQFM.

Example 2

Injection for benefiting qi and recovering pulse (freeze-drying) for inhibiting collagenase-induced cerebral hemorrhage of mice

The experimental method comprises the following steps:

male C57BL/6J mice were randomly divided into four groups of 4 animals each, a sham (sham), Model (Model), YQFM prophylactic and YQFM therapeutic dosing groups. Referring to rat brain orientation map, positioning right striatum 2.2mm from the right side of midline and 0.2mm in front of bregma, drilling a round hole with diameter of about 1.0mm, vertically inserting needle with microinjector along the drilling direction, depth of 4mm, slowly injecting collagenase VII-S (0.075U dissolved in 0.5 μ l physiological saline) into brain at uniform speed, retaining needle for 5min, and slowly withdrawing the microinjector. YQFM (0.671g/kg) was injected intraperitoneally 1h before and 1h after molding, respectively. Mice were sacrificed 24h after molding and cerebral hemorrhage volume and neurological function were measured.

The experimental results are as follows:

a) improvement effect of YQFM on collagenase-induced cerebral hemorrhage of mice

The results in fig. 7 show that the cerebral hemorrhage amount and the neurological deficit degree of the mice in the model group are obviously increased, and the cerebral hemorrhage amount and the neurological deficit degree of the mice in the YQFM prevention administration group and the mice in the treatment administration group are obviously reduced.

The research results of the part show that the qi-tonifying pulse-restoring (freeze-drying) for injection can obviously improve the cerebral hemorrhage and nerve function damage of MCAO-induced mice after thrombolytic by tPA, reduce the death rate of the mice and reduce the leakage of blood brain barrier; the qi-tonifying pulse-restoring (freeze-drying) for injection can obviously improve collagenase-induced cerebral hemorrhage and nerve function damage of mice.

Claims (10)

1. The application of qi-tonifying and pulse-recovering injection in preparing a medicine for treating cerebral hemorrhage.

2. The use of claim 1, wherein the cerebral hemorrhage is hemorrhage due to rupture of blood vessels in non-traumatic brain parenchyma.

3. The use according to claim 1, wherein the cerebral hemorrhage is intracerebral hemorrhage caused by hypertension, hyperlipidemia, cerebral amyloid angiopathy, cerebral aneurysm, use of anticoagulant drugs, use of thrombolytic drugs.

4. The use according to claim 1, wherein said cerebral hemorrhage is caused by tissue plasminogen activator.

5. The application of qi-tonifying and pulse-recovering injection in preparing a medicine for treating hemorrhagic stroke.

6. The application of qi-tonifying and pulse-restoring injection in preparing a medicine for relieving hemorrhagic transformation symptoms after ischemic stroke by thrombolysis of tissue plasminogen activator (tPA).

7. The application of qi-tonifying and pulse-recovering injection in improving the safety of a medicament, wherein the medicament is a tissue-type plasminogen activator.

8. A method for improving the safety of a tissue plasminogen activator in medication is to jointly use qi-tonifying and pulse-recovering injection.

9. A pharmaceutical composition comprises qi invigorating, pulse recovering and tissue plasminogen activator for injection.

10. The use of the pharmaceutical composition of claim 9 in the preparation of a medicament for the treatment of cerebral arterial thrombosis.

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