Use of racemates of pinocembrin in preparing medicaments for treating stroke.

The present application claims the priority of Chinese Patent Application No. 200810185559.0 as filed on December 11, 2008 and titled with "Use of pinocembrin in preparing a medicament for treating stroke", and the disclosure of which is incorporated herein by reference.

The present invention relates to the use of pinocembrin racemate in preparing a medicament for treatment and prophylaxis of stroke.

(S)-pinocembrin of Formula II, which chemical name is (S)-2,3-dihydro-5,7-dihydroxy- 2-benyl-4H-1-benzopyran-4-ketone, is a kind of water insoluble flavonone and a natural compound extracted from propolis. In addition, this compound is also found in extractive of a plurality of plants such as Helvetic five-leaved pine, leave of eucalyptus and acacia gum.

Since 1980s, researches all over the world discovered a plurality of pharmacological activities of (S)-pinocembrin, including antibiotic, antivirus, antioxidant and anti-inflammation effects. Chinese Academy of Medical Sciences first disclosed that (S)-pinocembrin has the function of inhibiting vasoconstriction caused by a plurality of factors and protecting damage of many kinds of nerve cells. Details can be found in CN200410037860.9, which disclosed the use of (S)- pinocembrin in treating cerebral ischemia, sequelae of cerebral ischemia, and diseases associated with damage and functional changes of nerve cells, and the mechanism thereof for protecting ischemic brain tissue and nerve cells mainly by inhibiting Calpain, NO and CRP and activating the expression of heat shock protein in the brain. Resumptively, (S)-pinocembrin can treat cerebral infarction. Moreover, Rui Liu etc. in Chinese Academy of Medical Sciences disclosed (S)-pinocembrin had the function of protecting the damaged nerve due to brain ischemic reperfusion or similar situations by the test of ischemia, hypoxia and apoptosis of brain tissue caused by brain ischemic reperfusion model (Pinocembrin protects rat brain against oxidation and apoptosis induced by ischemia-reperfusion both in vivo and in vitro. Brain Res. 2008 Jun 24;1216:104-15.). Yonghao Cheng etc. disclosed synthesis of pinocembrin racemate (Formula □) (Yonghao Cheng, Yabo Duan, Yan Qi etc., Synthesis of 5,7-dihydroxy-flavanone, Chemical Reagents. 2006, 28(7):437). It is known from the prior art that racemate has no direct or certain relationship to efficacy. Overall, their relationship can be reduced to the following several situations: racemate has better effect compared to enantiomers; enantiomers have equal or close activities; enantiomers have activities of different strength; enantiomers have opposite activities; enantiomers have activities of different types. Then we amplify the points by the following examples.

As a α-receptor agonist, levo-isomer of dobutamine affects β-receptor slightly. Similarly, as a β-receptor agonist, dextral-isomer of dobutamine affects α-receptor slightly. Administration by racemate will have an effect of increasing myocardial contractility but not speeding up the heart rate or raising blood pressure;

As an antihistamine, efficacy of isothipendyl racemate by oral administration is 1.4 times better than that of (-)- isomer, and 2.5 times better than that of (+)-isomer by oral administration. The reason why racemate has better efficacy is possibly that one isomer changes absorption of the other so that the bioavailability of the latter is raised, or that one isomer depresses the metabolic rate of the other so that the action time of the latter is extended.

Promethazine as an antihistamine has one chiral molecule. Since its receptors have no selectivity to medicament enantiomers, its two enantiomers have the same pharmacologic actions and show the same strengths ofpharmacological activities.

Propranolol as a β-receptor antagonist mainly depends on its levo-isomer to exhibit antagonistic activity, because its levo-isomer has the same configuration to the β-receptor agonist and can selectively bind to β-receptor, while its dextral- isomer can not.

In another extreme example, L-methyldopa as an antihypertensive drug has pharmacological activities, while D-methyldopa has no activity.

As to Bay k8644, a nifedipine structural analogue, its dextral-enantiomer is a calcium antagonist, whereas its levo-enantiomer is a calcium agonist. The two enantiomers have completely opposite activities.

D-enantiomer of propoxyphene has a strong analgesic activity which is 6 times of the activity of L-enantiomer, but has no antitussive effect. On the contrary, L-enantiomer of propoxyphene has strong antitussive effect. (Medicinal Chemistry, edited by Wensheng Ji, Anliang Li, Higher Education Press: 25-26)

It is further known from the above examples that according to the known pharmacological activity of (S)-pinocembrin, it can not be determined without experiments whether the racemate of pinocembrin or (R)-pinocembrin has the same pharmacological activity and effect of (S)-pinocembrin. The following documents are retrieved in the prior art.

Xiaoming Zhu, etc. disclosed that pinocembrin could expand the vascular of thoracic aorta by endothelium-dependent mechanism and endothelium-independent mechanism (Zhu XM, Fang LH, Li YJ, Du GH. Endothelium-dependent and Endothelium-independent relaxation induced by pinocembrin in rat aortic rings. Vascul Pharmacol. 2007; 46(3): 160).

Mei Gao, etc. disclosed that pinocembrin could improve the glutamate-induced cell injury and apoptosis and also reduce the probability of apoptosis, which provide the evidence of neuro-protective activity of pinocembrin to its effect of anti-cerebral-ischemia (Mei Gao, Wen-cui Zhang, Qing-shan Liu, Juan-juan Hu, Geng-tao Liu, Guan-hua Du. Pinocembrin prevents glutamate-induced apoptosis in SH-S Y5Y neuronal cells via bax/bcl-2 ratio decrease. Eur J Pharmacol. 2008, 591 (1-3):73-9).

Mei Gao, etc. disclosed that pinocembrin could protect cerebral neurovascular of permanent cerebral ischemia in rats (Acute neurovascular unit protective action of pinocembrin against permanent cerebral ischemia in rats. J Asian Nat Prod Res. 2008 May-Jun; 10(5-6): 551-8). It was the first time that the protective action of pinocembrin against permanent focal cerebral ischemia was proved. Its mechanism is still in research.

Hongmei Guang etc. obtained the model of insufficient blood supply to brain by bilateral carotid artery ligation and detected the cognitive function by Morris water maze test. It was disclosed that pinocembrin could bring about improvement in cognitive dysfunction in rats caused by insufficient cerebral blood supply and its mechanism was that pinocembrin could protect the structure and function of mitochondria (Protections of pinocembrin on brain mitochondria contribute to cognitive improvement in chronic cerebral hypoperfused rats. Eur J Pharmacol. 2006 Aug 7; 542 (1-3): 77-83).

The above documents disclose in some extent the pharmacological activities of pinocembrin, but are limited to the action of vascular expansion and neurovascular protection.

It is known in the prior art that acute cerebral ischemic stroke (cerebral ischemic apoplexy) has a high morbidity, a high mortality and a high rate of disability. At present, the most effective treatment is thrombolysis. The earlier the thrombolysis is performed, the better the treatment effect is. The clinical experiences in the past many years showed that the rescue of stroke patients needs to race against time. Once cerebral artery is blocked, the cerebral cells in ischemic region will start up a cascade electrochemical chain reaction quickly, and will produce abundant free radicals, arouse influxing of calcium ions and overloading of intracellular calcium ions and final lead to irreversible damage in brain tissue. Therefore, the time from onset to rescue of stroke should be minimized as much as possible.

In clinical practice, cerebral ischemic stroke can be divided into the following types: ultra-early stage, within 6 hours of onset; early stage, during 6-72 hours of onset; acute later stage, during 72 hours-1 week of onset; recovery stage, 1 week after onset. During the ultra-early stage of ischemic stroke, cerebral infarction has not engendered yet. If normal blood supply can be recovered promptly and harmful metabolites in ischemic tissues can be removed, the patients will have great opportunity to recover completely. So treatment in ultra-early stage will be the best opportunity and will get a good result. Up to early stage of ischemic stroke, persistent ischemia, hypoxia and especially blood-brain barrier being damaged cause that the regions of central infarction tend to form. As compared with ultra-early stage treatment, the treatment in this stage loses a lot of therapeutic value.

In 1996, recombinant tissue plasminogen activator (tPA) passed through the clinical validation of FDA and was approved for use within 3 hours after onset of acute ischemic stroke. This is the only medicament proved effective on treatment of ischemic stroke. Restricted by therapeutic time window, 95% of acute stroke patients abroad can not be treated timely by thrombolysis and in China the number of patients obtaining thrombolysis therapy is less than 1%.

Over the last decade, research for neuroprotective agents becomes a hot spot on stroke treatment. But in 114 stroke trials (up to 49 neuroprotective agents involved) all over the world, few trials were proved successful. This means that still no neuroprotective agent is proved safe and effective on treatment of acute ischemic stroke currently.

From the above analysis, it can be seen that, except tPA, drugs for effectively treating stroke are still in need in clinical practice, and even tPA is restricted by therapeutic time window of only 3 hours.

By experiments, the present invention surprisingly disclosed that pinocembrin racemate can be used to treat stroke. Compared with (S)-pinocembrin, pinocembrin racemate has a longer therapeutic time window (about 6 hours) and will still have therapeutic effect during a longer time period after onset of stroke. The extension of therapeutic time window brings more opportunities for patients of ischemic stroke. Animal experiments show that pinocembrin racemate has significant therapeutic effects.

One object of the present invention is to provide a use of a racemate of pinocembrin, a racemate of pinocembrin salt, a racemate of pinocembrin precursor or a racemate of pinocembrin hydrate in manufacture of a medicament for treating stroke.

The terminology "racemate" means an equimolar mixture of a chiral molecule with optical activity and its enantiomer, which is formed by mixing equivalent amounts of two molecules with opposite rotations and equivalent rotary powers so that their rotations are neutralized due to their intermolecular action.

The salt of pinocembrin is a pharmaceutically acceptable salt of pinocembrin, such as hydrochloride, sulfate and citrate, etc.

The precursor of pinocembrin is a prodrug of pinocembrin, that is, a compound that can produce pharmacological activities only after changed into pinocembrin by conversion in vivo.

Preferably, the stroke is acute ischemic stroke.

It is found by experiments that:

1. 1) Racemate of pinocembrin can be used to treat stroke;
2. 2) Racemate of pinocembrin can improve behavioral changes caused by acute ischemic stroke;
3. 3) Racemate of pinocembrin can improve decrease in cerebral blood flow caused by acute ischemic stroke;
4. 4) Racemate of pinocembrin can decrease volume of cerebral infarct caused by acute ischemic stroke;
5. 5) Racemate of pinocembrin can improve cerebral edema caused by acute ischemic stroke;
6. 6) Racemate of pinocembrin can improve energy metabolism caused by acute ischemic stroke;
7. 7) Racemate of pinocembrin can improve acute inflammation caused by acute ischemic stroke;
8. 8) Racemate of pinocembrin can protect nerve cells from damage caused by acute ischemic stroke.

Therefore, the present invention also relates to a pharmaceutical composition of pinocembrin racemate, which consists of a racemate of pinocembrin, a racemate of pinocembrin salt, a racemate of pinocembrin precursor or a racemate of pinocembrin hydrate and a pharmaceutically acceptable excipient.

The composition of the present invention can be suitable for oral, transdermal, muscular, intravenous or mucosal administration.

The composition of the present invention can be a solid or liquid preparation, which can be prepared by a conventional method.

The composition of the present invention can be a liquid preparation, preferably an injection. The injection is prepared by encapsulating pinocembrin racemate with cyclodextrin or its derivatives, and then adding a suitable excipient.

The present invention is also to provide a use of (R)-pinocembrin, its salts, its precursors or its hydrates in manufacture of a medicament for treatment and prophylaxis of stroke, in which the stroke is ischemic stroke or hemorrhagic stroke.

Pinocembrin racemate used in present invention has the merit of low toxicity and can be used to treat acute ischemic stroke.

• Figure 1 shows the effects of pinocembrin racemate on infarct volume.
• Figure 2 shows the effects of pinocembrin racemate on expression of TNF-α, IL-1β, ICAM-1, VCAM-1, iNOS and AQP-4 in ischemic cerebral tissues after MCAO for 24h; in which

A shows the effects of DL0108 on expression of TNF-α, IL-1β, ICAM-1, VCAM-1, iNOS and AQP-4;

B is a statistic chart that quantifies the expression of TNF-α, IL-1β, ICAM-1, VCAM-1, iNOS and AQP-4 with β-actin as internal standard.

Figure 3 is a Nissl staining figure (200×) of CA1 area in cortex and hippocampus, in which

1. A: sham operation group;
2. B: model group;
3. C: nimodipine 3mg/kg group;
4. D: DL0108 (pinocembrin racemate) 1mg/kg group;
5. E: DL0108 (pinocembrin racemate) 3mg/kg group;
6. F: DL0108 (pinocembrin racemate) 10mg/kg group.

The present invention is illustrated specifically in the following examples.

1. Test drugs: pinocembrin racemate for injection, (R)-pinocembrin for injection and (S)-pinocembrin for injection, which are provided by New Drug Development Laboratory of Drug Research Institute of the Chinese Academy of Medical Sciences (Batch number: 20050601, content: 2.36%), and prepared by the method disclosed in CN200810084682.3, i.e., forming an inclusion complex of pinocembrin racemate or pinocembrin enantiomers with cyclodextrin or its derivatives, and dissolving the complex in a physiological saline when used. Nimodipine as a positive control drug was purchased from Bayer Company (Germany). 2. Experimental animals and grouping:

• Experimental animals: 110 SHRSP rats and 10 normal Wistar rats of 6 weeks.
• Experimental grouping: Wistar rats were normal group; SHRSP rats were grouped as a model group, a nimodipine group (3mg/kg), groups of low, middle and high dose of pinocembrin racemate (3, 10 and 30 mg/kg/d, respectively), groups of low, middle and high dose of (R)-pinocembrin (which doses were identical to those of pinocembrin racemate groups, respectively), and groups of low, middle and high dose of (S)-pinocembrin (which doses were identical to those of pinocembrin racemate groups, respectively).

• Except the rats of the normal group, all SHRSP rats drank 1% sodium chloride solution everyday. The drugs were administered in prescriptive doses respectively for the rats of the nimodipine group (3mg/kg), the groups of low, middle and high dose of pinocembrin racemate (3, 10 and 30 mg/kg/d, respectively), the groups of low, middle and high dose of (R)-pinocembrin (which doses were identical to those of pinocembrin racemate groups, respectively), and the groups of low, middle and high dose of (S)-pinocembrin (which doses were identical to those of pinocembrin racemate groups, respectively). Physiological saline of same volume was administered for the model group. The administration was continued for 14 days after the onset of stroke.

• □ Appearance of twitch on one limb, double forelimbs or whole body; □ appearance of hemiplegia or general paralysis, abdomen attaching to the ground and being unable to stand up; □ to the more serious, appearance of systemic fine tremor, groveling, flaccid paralysis, even death. Besides the symptoms in the above terms, irritation (jumping or leaping up) could be observed in some animals. Once any one of the above symptoms was observed, the onset of stroke was determined, and the time of onset of every animal was recorded.

1. 1) After the last dose, the rats were fasted for 6 hours, and blood samples were collected by blood sampling in eyeground. After decollation, the whole brains of the rats were detached at ice trays for use.
2. 2) Blood of 3mL was dropped rapidly in a cuvette with 1mL 0.074% heparin and mixed. The whole blood viscosity, plasma viscosity, red blood cell (RBC) aggregation index and RBC deformation index were determined with a hemorheology detector according to its specifications.
3. 3) Blood of 2mL was dropped rapidly in a cuvette with 0.2mL 0.13mol/L Sodium citrate) and mixed. The mixture was centrifuged for 10 min at 1000rpm and platelet aggregation ratio was determined with a platelet aggregation meter according to its specifications.
4. 4) Blood of 1mL was dropped rapidly in a cuvette with 0.1mL 0.109mol/L Sodium citrate and mixed. The mixture was centrifuged for 15 min at 3000rpm and plasma fibrinogen in liquid supernatant was determined by euzymelinked immunosorbent assay (ELISA).
5. 5) Blood of 2mL was dropped rapidly in a cuvette with 30µL 7.5% EDTA-2Na and 40µL aprotinin and mixed. After centrifuged for 5 min at 3000rpm, plasma ET-1 in liquid supernatant was determined by isotope radioimmunoassay.
6. 6) The whole brain tissues were weighed one by one, dried in a vacuum over at 110±5 □ for 48 hours, and then weighed again.

Totally 24 rats had died during the experiment. After analysis of pathological autopsy, it was found that 4 of them died of hemorrhagic stroke and other 20 rats died of ischemic stroke. Details can be seen in Table 1.

Compared with the model group, *P<0.05, **P<0.01.

The rats in the normal group were normal in body condition, eating and movement.

From the beginning of the forth week, except the groups of high, middle dose of pinocembrin racemate and the nimodipine group, in other groups there had been constantly some rats attacked by stroke showing symptoms of irritation, paroxysmal convulsions, paralysis, heavy head, eating less, urinary incontinence, hair shaft and becoming thin. According to the above scoring standard of neurological symptoms after stroke onset, beginning with the day of stroke onset, the scores for the groups of high and middle doses of pinocembrin racemate decreased remarkably. Within 2-14 days after onset, their scores were stably below 1.6 and 2.3 respectively. This indicated that the high and middle dose of pinocembrin racemate had significant improvement effects in neurological function. The scores for the group of low dose of pinocembrin racemate and the group of high dose of (R)-pinocembrin not only were relatively high during the beginning several days but also were maintained at 2.5 or above for 14 days. This indicated that the low dose of pinocembrin racemate and the high dose of (R)-pinocembrin had no improvement effect. After onset of stroke, the scores of the nimodipine group were relatively low and were stably below 2.2 after 6 days.

Compared with the normal group,^#P<0.05,^##p<0.01; Compared with the model group, *P<0.05, **P<0.01.

The results show that the model group exhibited significant differences (P<0.01) in the blood hemorheology indexes as compared with the normal group, which illustrated the state of high viscosity and high aggregation of blood of the model group. Compared with the model group, the groups of high, middle and low dose of pinocembrin racemate and the group of high dose of (R)-pinocembrin exhibited a decrease of blood agglomeration in different extents ( P<0.01, P<0.05), especially exhibited a decrease in whole blood viscosity and RBC aggregation index (P<0.01). However, (S)-pinocembrin showed no activity at all. Nimodipine had an effect of decreasing blood viscosity. Results are shown in Table 3.

Suggestion: pinocembrin racemate can decrease blood viscosity in a dose-dependent manner, and (R)-pinocembrin in high dose has the similar effect.

The results of resistance to platelet aggregation in the Table 3 indicated that the platelet aggregation ratio of the model group were higher than that of the normal group (P<0.01). Compared with the model group, the groups of high and middle dose of pinocembrin racemate and the group of high dose of (R)-pinocembrin exhibited a significant decrease in platelet aggregation ratio (P<0.01, P<0.05), while (S)-pinocembrin showed no activity.

Suggestion: pinocembrin racemate can resist platelet aggregation in a dose-dependent manner, and (R)-pinocembrin in high dose also has effect.

The content of plasma fibrinogen in the model group was significantly higher than that in normal group (P<0.01). Compared with the model group, the groups of high, middle and low dose of pinocembrin racemate and the group of high dose of (R)-pinocembrin exhibited a significant decrease in content of plasma fibrinogen (P<0.01) thereby changing the hypercoagulability condition, while (S)-pinocembrin had no activity. Specific results are shown in Table 3.

Compared with the normal group,^#P<0.05,^##p<0.01; Compared with the model group, *P<0.05 , **P<0.01.

As showed in Table 3, the plasma ET-1 in the model group increased rapidly and had significant difference in comparison with the normal group (P<0.01). Compared with the model group, the plasma ET-1 contents in the group of high dose of pinocembrin racemate and the group of high dose of (R)-pinocembrin decreased significantly (P<0.01, P<0.05). The groups of other doses of pinocembrin racemate and (R)-pinocembrin and the groups of (S)-pinocembrin had no activity.

As showed in Table 3, the brain water content in the model group increased markedly and had significant difference in comparison with the normal group (P<0.05). Compared with the model group, the brain water content in the group of high dose of pinocembrin racemate decreased significantly (P<0.05). Other groups had no remarkable activity.

Compared with the model group, pinocembrin racemate can delay the time of stroke onset in dose-dependent manner (P<0.01). Pinocembrin racemate in low dose and (R)-pinocembrin in high dose can also delay the time of stroke onset (P<0.05). Nimodipine can delay the time of stroke onset (P<0.01). It is illustrated that pinocembrin has prophylactic effects on stroke, pinocembrin racemate is superior to (R)-pinocembrin, and (S)-pinocembrin in high, middle and low doses had no activity. The specific results can be seen in Table 1.

Pinocembrin racemate can dose-dependently increase survival ratio after stroke, which indicates pinocembrin racemate has the activity of treating stroke in SHRSP rats.

(R)-pinocembrin has this activity only when administered by high dose, and the activity is similar to that of nimodipine, which indicates that pinocembrin racemate has better activity than (R)-pinocembrin, while (S)-pinocembrin has no significant activity. Suggestions: 1) pinocembrin racemate has effects of prophylaxis and treatment on stroke; 2) (R)-pinocembrin also has effects of prophylaxis and treatment on stroke, although the effects are somewhat less; 3) the treatment effects of pinocembrin racemate are better than that of (R)-pinocembrin generally because (S)-pinocembrin without effects promoted the effects of (R)-pinocembrin.

Test drugs: pinocembrin for injection, provided by New Drug Development Laboratory of Drug Research Institute of the Chinese Academy of Medical Science (Batch number: 20050601, content: 2.36%), and prepared by the method disclosed in CN200810084682.3, i.e., forming an inclusion complex of pinocembrin racemate with cyclodextrin or its derivatives, and dissolving the complex in a physiological saline when used. Nimodipine as the positive control drug was purchased from Bayer Company (Germany).

Experimental animals: 100 male SD rats with a body weight of 250-280g were purchased from the Experimental Animals Institute of the Chinese Academy of Medical Science. The rats were grouped randomly as a sham operation group, a model group, pinocembrin groups and a nimodipine group (3mg/kg).

Experimental model: the model of acute ischemic stroke was made by middle cerebral artery occlusion (MCAO).

The string occlusion method in middle cerebral artery as established by Zea Longa was adopted with proper improvements. 400 mg/kg 10% chloral hydrate was intraperitoneally injected in rats. The external carotid artery (ECA) was separated and ligated at the site where the artery emitted for about 0.8cm. The end of ECA closing to the heart was clipped by an artery clip. A 2mm V-shaped incision was made from ECA ligation to ECA bifurcation. A nylon line was inserted gently into CCA and then into internal carotid artery (ICA) through the bifurcation of ECA and ICA after loosening the artery clip. The nylon line was pushed into the brain through ICA and inserted in depth of 18.5±0.5mm when resistance was felt slightly. The nylon line should reach smaller anterior cerebral artery through the beginning of middle cerebral artery (MCA) and ICA could be ligated and sutured. Stump of the nylon line should be reserved with 1 cm outside the skin.

Rats in the sham operation group were only operated with preoperative anesthesia and vascular dissection without ligation and importing the line.

Statistical method: the results were showed in the format of x̅ ± SD, and the comparison among the groups was performed by one-way analysis of variance (ANOVA).

Neurobehavioral examination was performed before the animals were put to death. The rats were raised off the ground for about 1 chi (1 chi = 1/3 meter) to observe the state of two forelimbs. The rats were placed on the ground and were pushed by their shoulders to observe differences of their resistance. The rats were placed on the ground and were observed by the way of walking. Four-stage scoring method (0-5 scores) was adopted. The higher the score was, the more serious injury existed in their neurobehavior.

1. (1) If the rats acted completely normal, score 0 was recorded.
2. (2) When the rats were raised off the ground, if the rats rotated or retracted forelimb of surgery-contralateral side internally, score 1 was recorded.
3. (3) When the rats were placed on the ground and squeezed with the hand to examine the resistance of both sides, if the resistance of surgery-contralateral side declined, score 2 was recorded.
4. (4) When the rats were placed on the ground to observe the way of walking, if the rats circled around the surgery-contralateral side, score 3 was recorded.
5. (5) If the rats were injured too seriously to move by themselves, score 4 was recorded.

The results were shown in Table 4: the neurobehavioral score of the rats in the sham operation group was 0. The average neurobehavioral score of the rats in the model operation group was 3.4±0.6, in which most animals showed internal rotation or retraction of their forelimbs at surgery-contralateral side, a reduction of muscle stretching force at contralateral side, circling or occasionally circling, and were scored 3; a minority of animals showed only internal rotation of their forelimbs and reduction of resistance, and were scored 2; several animals showed severe symptoms and did move by themselves, and were scored 4.

In the pinocembrin racemate groups (3mg/kg, 10mg/kg, 30mg/kg), symptoms of nerve injury caused by ischemia in animals were improved significantly (P<0.05, P<0.01) showing a dose-effect relationship.

Compared with the sham operation group,^##p<0.01; Compared with the model group, *P<0.05 , **P<0.01.

After neurobehavioral scoring, the rats were put to death by decollation and their brain tissues were removed quickly and put into a refrigerator of -20°C. 10 min later, the brain tissue was moved to an environment of room temperature. After the olfactory bulb, cerebellar and lower brain stem were resected, the brain tissue was cut into five continuous coronal slices with an interval of 2mm and was cut first on the connection midpoint of brain anterior pole and optic decussation, second on optic decussation, third on funnel handle and fourth on midpoint of funnel handle and thick leaves caudate nucleus. Then the brain slices were quickly put into 5ml solution containing 4% TTC and 0.1ml 1mol•L^-1K₂HPO₄ and incubated at a constant temperature of 37°C and in condition of darkness for 30min. During the time period, the brain slices were tipped every 5 min. After TTC staining, normal tissue showed rose red color, while infracted tissue was not stained and showed white color. Every group of brain slices were put in order and photographed. The photographs were processed by an image analysis system and infarct area in every slice was calculated. The infarct volume of every slice was calculated by multiplying the infarct area by the thickness of slice (2mm), and the infarct volume was the sum of infarct volumes of all slices. The infarct volume was showed in percentage in hemisphere so as to eliminate the effects of cerebral edema. Infarct volume%=volume of surgery-contralateral hemisphere-infarct volume of surgery-homolateral hemisphere/volume of surgery-contralateral hemisphere×100%

The results showed that: after injured by cerebral ischemia, no cerebral infarction had been found in the sham operation group, while the infarct volume in the model group was (33.6±4.3)% (P<0.01). Compared with the model group, the infarct volumes in the pinocembrin racemate groups (3, 10, 30 mg/kg) were significantly reduced (P<0.01) and the infarct volumes were respectively (23.1±3.4)%, (21.4±2.1)% and (14.6±1.1)%. The infarct volume in the nimodipine group was (16.7±1.3)% and significantly different from that of the model group (P<0.01). The results were shown in Table 4 and Figure.1.

The results showed that pinocembrin racemate could reduce the volume of cerebral infarction caused by ischemic cerebral stroke.

The rats were fixed pronely on the stereotaxic apparatus and subjected to craniotomy. After cleaning for operation visual field, bregma was used as an origin, and a site situated 2mm behind and 3mm right to the bregma was selected as a measuring point. The region 2-3 cm around the site was thinned by a dental drill. In the process, the integrity of dura was maintained and large vessels were avoided. Probe holder was located and fixed.

The rats were supinely fixed on the operating table to carry out MCAO surgery. When a nylon line was inserted in ICA, it was not inserted into intracranial part until LDF value was stable, and blood flow value in 10 min was recorded, and an average value thereof is used as a baseline value of cerebral blood flow. After the MCAO surgery completed, the nylon line was inserted into the intracranial part. When the blood flow value suddenly declined to 20-30% of the baseline value, it was noted that the blood flow in MCA had been blocked. The blood flow value before MCAO in every group was used as a baseline value (100%) of the group. The blood flow values after surgery were showed in percentages of the baseline value. LDF value was determined at the same site before the animals was put to death.

30 min later after the cerebral ischemia in rats, the rCBF value of the model group was (31.09±5.35)% of baseline value. The rCBF values in the pinocembrin racemate groups (3, 10, 30mg/kg) and the nimodipine group (3mg/kg) were respectively (40.76±6.58)%, (50.09±7.09)%, (53.28±8.03)% and (55.58±6.09)% of their baseline values. It can be seen that cerebral blood flows in all the administration groups recovered rapidly with a significant increase as compared with that of the model group, in which the pinocembrin racemate groups (10, 30mg/kg) and the nimodipine group (3mg/kg) showed significant difference as compared with the model group (P<0.05). Owing to the compensatory of collateral circulation, the extent of regional cerebral blood flow decreased gradually after ischemia occurring, but still kept at a level higher than that in the model group.

Compared with the sham operation group,^##P<0.01; Compared with the model group, *P<0.05.

After surgery, a mixture solution of EB/NF (0.5%, dissolved in normal saline) of 0.25ml was immediately (immediately after administration in the administration groups) injected via tail vein in the animals. 24 hours later, a normal saline was perfused in the hearts of the rats to get rid of unbound dye. The rats were decollated and the brain tissues were promptly removed and separated into ischemic hemisphere and non-ischemic hemisphere. The two hemispheres were weighted respectively and homogenated with 7.5% (w/v) trichloroacetic acid (TCA). The homogenate was divided into two parts, in which 1ml homogenate was adjusted with 52µl NaOH (5N) to reach a neutral pH value, and 200µl of it was taken out to determine the fluorescence intensity (excitation 485nm, emission 535nm), the NF thereof was determined by using a standard curve made with NF solutions of concentration series. Another part of homogenate was centrifuged for 20 min at 12000 g and 4°C. 200µl supernatant was taken out to a microplate and its absorbance was determined at 620nm, and EB thereof was determined by using a standard curve made with EB solutions of concentration series. The results were expressed in form of µg EB (or NF)/g brain wet weight. Percentage of brain edema was expressed as: b⁢r⁢a⁢i⁢n⁢w⁢e⁢i⁢g⁢h⁢t⁢i⁢n⁢i⁢s⁢c⁢h⁢e⁢m⁢i⁢c⁢h⁢e⁢m⁢i⁢s⁢p⁢h⁢e⁢r⁢e-b⁢r⁢a⁢i⁢n⁢w⁢e⁢i⁢g⁢h⁢t⁢i⁢n⁢n⁢o⁢n-i⁢s⁢c⁢h⁢e⁢m⁢i⁢c⁢h⁢e⁢m⁢i⁢s⁢p⁢h⁢e⁢r⁢e/b⁢r⁢a⁢i⁢n⁢w⁢e⁢i⁢g⁢h⁢t⁢i⁢n⁢n⁢o⁢n-i⁢s⁢c⁢h⁢e⁢m⁢i⁢c⁢h⁢e⁢m⁢i⁢s⁢p⁢h⁢e⁢r⁢e×100%

As Table 6 showed, the percentage of brain edema in the model group was (8.3±1.9)%, while the percentages of brain edema in the pinocembrin racemate groups (3mg/kg, 10mg/kg, 30 mg/kg, iv) were respectively (5.5±1.7)%, (4.1±1.5)%, (3.2±2.1)%, which showed significant difference as compared with the model group (p<0.05, p<0.01). Pinocembrin racemate (3mg/kg, 10mg/kg, 30 mg/kg, iv) also had significantly reduced the EB/NF leakage in the tissue. These results showed that pinocembrin could mitigate the tissue edema caused by cerebral ischemia.

Compared with the model group, *P<0.05, **P<0.01.

The results were shown in table 7. After 24 hours of cerebral ischemia, the energy index in the model group decreased to be 42.6% of that before surgery (P<0.05). Compared with the model group, the energy index in pinocembrin racemate groups (3mg/kg, 10mg/kg, 30 mg/kg) increased significantly and increased respectively by 34.6% and 45.8% (P<0.05) relative to the model group, showing a dose-dependence.

Results in recovery test and repeatability test showed that the injection volumes of ATP, ADP and AMP had good linear relationship with peak areas. The obtained r values were respectively r_ATP=0.9897, r_ADP=0.9896, r_AMP=0.9893, r_CrP=0.9981. The recovery rates of 4 kinds of standard materials were respectively (86.6±5.6)%, (94.45±7.5)%, (83.4±6.1)%, (78.69±7.3)%.

Compared with the sham operation group,^#P<0.05; Compared with the model group, *P<0.05.

It was showed in Table 8 that the NO and TNF-α contents in serum in the model group increased significantly, and in the pinocembrin racemate groups (3mg/kg, 10mg/kg, 30 mg/kg), the NO and TNF-α contents in rats' serums at the time 24 hours after ischemia decreased significantly (P < 0.05, P < 0.01).

Compared with the normal group,^#P<0.05,^##P<0.01; Compared with the model group, *P<0.05, **P<0.01.

It can be seen in Figure 2 that the expression of cytokines of TNF-α and IL-1β in tissue increased significantly at the time 24h after ischemia, and the administration of pinocembrin racemate (3mg/kg, 10mg/kg, 30 mg/kg) in early stage of ischemia inhibited the expression of TNF-α, IL-1β in some extents.

It can be seen in Figure 2 that the expression of adhesion molecules of ICAM-1 and VCAM-1 in tissue increased significantly at the time 24h after ischemia, and the administration of pinocembrin racemate (3mg/kg, 10mg/kg, 30 mg/kg) in early stage of ischemia inhibited the expression of TNF-α and IL-1β in some extents.

It can be seen in Figure 2 that the expression of iNOS and AQP-4 protein in tissue increased significantly at the time 24h after ischemia, and the administration of pinocembrin racemate (3mg/kg, 10mg/kg, 30 mg/kg) in early stage of ischemia inhibited the expression of iNOS and AQP-4 protein in some extents.

With β-actin as internal standard, the expressions of TNF-α, IL-1β, ICAM-1, VCAM-1, iNOS, AQP-4 were quantified, and the inhibition effects of pinocembrin racemate on the above proteins were seen intuitively. Pinocembrin racemate in doses of 10mg/kg and 30 mg/kg showed better results.

The above results showed that pinocembrin racemate could decrease acute inflammation caused by acute ischemic stroke.

After cerebral ischemia for 24 hours, the rats were anesthetized by intraperitoneal injection of 10% Chloral hydrate. After perfused via heart with heparinized normal saline for 10 min and 4% paraformaldehyde for 30 min, the brain tissue was taken out and moved in 4% paraformaldehyde to fix, then cut into coronal sections (6µm) by a frozen section machine, and one section was taken out in every 20 sections to stain. The stained sections were expanded on a slide glass treated by polylysine, and then preserved in a refrigerator at -40°C.

Nissel staining: (1) the frozen section was taken out from the refrigerator and dried in room temperature; (2) put into acetone to fix for 30 min and washed by PBS for 3 times, 3 min every time; (3) dipped for 20-30 min in toluidine blue dye and then washed by water for 15min; (4) dehydrated by ethanol in gradient manner and vitrified by xylene and mounted by a neutral gum; (5) observed under a light microscope and photographed for analysis; (6) 4 frozen sections in roughly same position for each rat were taken out and 5 vision fields in hippocampus for each section were observed under 200x light microscope (totally 20 visions per rat). The cells in vision fields were counted. The averages of ratios of the numbers in the administration groups to the number in the model group were calculated for statistical analysis.

The results were shown Figure 3. Hippocampus is an area sensitive to cerebral ischemia. Nissel staining results showed that, after the cerebral tissue was ischemicaly injured, a serious damage in hippocampal neurons, an obvious absence of cells and a loose arrangement of nerve cells were observed. It was showed in quantitative results that nerve cells decreased 79.5±9.7% in comparison with the sham-operation group and the difference was significant (P<0.01). Pinocembrin (3mg/kg, 10mg/kg, 30 mg/kg) could improve the shape of ischemic nerve cells and decrease the loss of nerve cells, in which nerve cells increased by 11.5±8.9%, 36.8±4.9% and 51.7±6.6% in comparison with the model group, indicating significant differences (P<0.05) in comparison with the model group and dose-dependence, which enlightened neuroprotective activities after acute cerebral ischemia.

1. 1) Pinocembrin racemate can relieve behavioral changes caused by ischemic stroke;
2. 2) Pinocembrin racemate can relieve decline of cerebral blood flow caused by ischemic stroke;
3. 3) Pinocembrin racemate can reduce volume of cerebral infarction caused by ischemic stroke;
4. 4) Pinocembrin racemate can relieve cerebral edema caused by ischemic stroke;
5. 5) Pinocembrin racemate can relieve energy metabolism disorder caused by ischemic stroke;
6. 6) Pinocembrin racemate can relieve acute inflammation caused by ischemic stroke;
7. 7) Pinocembrin racemate can relieve nerve cell damage caused by ischemic stroke.

Experimental results showed that, with single intravenous injection in SD rats, LD50 value of pinocembrin racemate was 490.9 (367.6~746.7) mg/kg, LD50 value of (S)-pinocembrin was 375.3 (271.2~538.5) mg/kg, and LD50 value of (R)-pinocembrin was 347.8 (257.4~466.3) mg/kg. The above results showed that pinocembrin racemate had a larger safe range for use. All these drugs had no effect to animal's weight, and their major apparences in term of toxicity were quadriplegic and mild blood stasis in liver and lung.

1. 1. In the test, transient middle cerebral artery occlusion (tMCAO) model of rats was adopted. The reperfusion was performed after focal cerebral ischemia for 2 hours so as to inspect therapeutic time window of pinocembrin after focal cerebral ischemia. The rats were administered by pinocembrin racemate (1 mg/kg, 5 mg/kg, intravenous injection) after reperfusion 1 hour, 4 hours and 6 hours (i.e., ischemia 3 hour, 6 hours and 8 hours). (R)-pinocembrin and (S)-pinocembrin (1 mg/kg, 5 mg/kg, intravenous injection) were administered after reperfusion 1 hour and 4 hours (i.e., ischemia 3 hour and 6 hours). The therapeutic time windows of these drugs in focal cerebral ischemia were assessed by studying their effects to neurological symptom scores, infarct sizes and brain water contents during 24 hours after tMCAO.
2. 2. Materials and methods:

100 male SD rats, 240-280 g, purchased from Beijing Weitong Lihua Experimental Animal Technology Co., Ltd. Certificate of Conformity: SCXK ( JING ) 2007-0001. The animals were raised in a routine way before and after surgery at 23-25°C with free supply of food and water.

Pinocembrin racemate for injection, (R)-pinocembrin for injection and (S)-pinocembrin for injection were provided by the New Drug Development Laboratory in the Drug Research Institute of the Chinese Academy of Medical Sciences (Batch number: 20050601, content: 2.36%), and prepared by the method disclosed in CN200810084682.3, i.e., by forming inclusion complexes of pinocembrin racemate or enantiomers of pinocembrin with cyclodextrin or its derivatives, and dissolving the complexes in physiological saline when used. Nimodipine as positive control drug was purchased from Bayer Company (Germany). Hydroxypropyl β-cyclodextrin was provided by the New Drug Development Laboratory in the Drug Research Institute of the Chinese Academy of Medical Sciences. TTC was purchased from Sigma Company. Other reagents were commercially available reagents of analytical grade.

Rat anesthesia: 400 mg/kg 10% chloral hydrate was intraperitoneally injected in rats and then righting reflex disappeared.

The rats were supinely fixed on an operating table and subjected to anterior neck cut and blunt separation of layers of organization to expose right common carotid artery (CCA). CCA was separated to the segment after bifurcation of internal carotid artery (ICA) and external carotid artery (ECA). Injury to vagus nerve and trachea was avoided. Lines were placed under the CCA and ECA for standby use.

The CCA and ICA were clipped with artery clips. Two No.0 surgery lines were litigated on the ECA distal end with 2~3mm spacing and then the vascular between the two surgery lines was cut. The distal end of ECA was pulled until it formed a straight line with the ICA. An incision was cut on ECA and a nylon line was inserted through ECA into ICA. After the artery clip was loosen, the nylon line was pushed on into the brain through ICA and inserted in depth of 18.5±0.5mm when resistance was felt slightly. The nylon line reached a smaller anterior cerebral artery through the beginning of MCA and then the blood flow obstruction in right MCA was established. the animals were kept in anesthesia during the obstruction. After 2 hours of obstruction, the nylon line was gently pulled out to ECA stub to form reperfusion. During the surgery process, the animals were irradiated by table lamp of 100W to maintain the body temperature. The room temperature was kept in range of 23-25°C.

The rats in the sham operation group were subjected to only preoperative anesthesia and vascular dissection without ligation and importing line.

Sham operation group (intravenous injection of normal saline after 3 hours of ischemia);

Model group (intravenous injection of 50 mg/kg hydroxypropyl β-cyclodextrin after 3 hours of ischemia);

• Pinocembrin racemate group (1 mg/kg, 5 mg/kg, administered after 3, 6 and 8 hours of ischemia);
• (R)- pinocembrin group (1 mg/kg, 5 mg/kg, administered after 3 and 6 hours of ischemia);
• (S)- pinocembrin group (1 mg/kg, 5 mg/kg, administered after 3 and 6 hours of ischemia).

First administration was performed by intravenous injection, and intraperitoneal injection was carried out once 12 hours later with a dose 1.5 times of that for intravenous injection.

Neurobehavioral examination was performed respectively when the animals were awake after surgery and before the animals were put to death. Bederson's score was adopted (see details in Example 2, 1. Neurobehavioral Examination).

After neurobehavioral scoring, the rats were put to death by decollation and their brain tissues were taken out quickly and put into a refrigerator of -20°C. 10 min later, the brain tissues were moved to an environment of room temperature. After the olfactory bulb, cerebellar and lower brain stem were resected, the brain tissue was cut into six continuous coronal slices with an interval of 2mm. Then the brain slices were quickly put into 5ml solution containing 2% TTC and incubated at a constant temperature of 37°C and in a condition of darkness for 30min. During the time, the brain slices were tipped every 5 min. After TTC staining, normal tissue showed rose red color, while the infract tissue was not stained and showed white color. Every group of brain slices were put in order and photographed. The photographs were processed by an image analysis system and the infarct areas in each slice were calculated. The infarct volume of each slice was calculated via multiplying the infarct area by thickness of slice (2mm), and the infarct volume was the sum of infarct volumes of all slices. The infarct volume percentage was expressed as: sick-side infarct volume / total sick-side brain volume.

The wet weight of each animal's brain was weighted before sliced. After staining, the brain slices were dried at 105°C for 24 hours, and the dry weight of each animal's brain was weighted. The brain water contents of animals of various groups were compared. Brain water content = (brain wet weight - brain dry weight) / brain wet weight × 100%.

The data of quantitative measurements were expressed as mean ± SD for t test. The count values were expressed as % for X² test.

The neurobehavioral score in the sham operation group was 0. The average score of rats in the solvent control group was 3.4±0.6, in which most animals showed internal rotation or retraction of their forelimbs of surgery-contralateral side, decrease of muscle stretching force of contralateral side or circling, and were scored 3; a minority of animals only showed internal rotation of their forelimbs and decrease of resistance, and were scored 2; and several animals showed severe symptoms and did not move by themselves, were scored 4.

In the pinocembrin racemate groups (1mg/kg, 5mg/kg), the symptoms of nerve injury after 3 hours and 6 hours of ischemia in animals were improved significantly (P<0.05 ,P<0.01), while the improvement to symptoms after 8 hours of ischemia was not significant, showing dose-dependence in some extent. In the (R)-pinocembrin groups and the (S)-pinocembrin groups (1mg/kg, 5mg/kg), the symptoms of nerve injury after 3 hours of ischemia were improved significantly (P<0.05), while the improvement to symptoms after 6 hours of ischemia was not significant. Furthermore, nimodipine (1 mg/kg) showed no improvement to symptoms after 3 hours of ischemia.

After 24 hours of tMCAO in rats, the consequent brain coronary slices were stained by TTC. There were infarct areas (white) that had not been stained in a certain percentage of animals in each group, indicating that animals automatically restored more or less within 24 hours of reperfusion. In the statistical process, the samples which infarct volume was less than 5% were removed from each group. Pinocembrin racemate (1 mg/kg) reduced infarct volume when initially administered after 3 hours of cerebral ischemia, and its activity was not significant when initially administered after 6 hours of cerebral ischemia. Pinocembrin racemate (5 mg/kg) reduced infarct volume when initially administered after 3 hours and 6 hours of cerebral ischemia (P<0.05, P<0.01), and its activity was not significant when initially administered after 8 hours of cerebral ischemia. These effects showed a dose-dependence in some extents. (R)-pinocembrin and (S)-pinocembrin (1 mg/kg, 5 mg/kg) reduced the infarct volume after 3 hours of cerebral ischemia (P<0.05) and their activities were not significant when initially administered after 6 hours of cerebral ischemia. Furthermore, nimodipine (1 mg/kg) didn't show improvement when initially administered after 3 hours of cerebral ischemia.

In the model group, brain water content of rats increased significantly. Pinocembrin racemate (1 mg/kg, 5 mg/kg) reduced significantly the brain water content after 3 hours and 6 hours of cerebral ischemia (P<0.05, P<0.01) and its activities were not significant when initially administered after 8 hours of cerebral ischemia. These activities were dose-dependent in some extents. (R)-pinocembrin and (S)-pinocembrin (1 mg/kg, 5 mg/kg) reduced brain water content after 3 hours of cerebral ischemia (P<0.05) and their activities were not significant when initially administered after 6 hours of cerebral ischemia. Furthermore, nimodipine (1 mg/kg) didn't show improvement when initially administered after 3 hours of cerebral ischemia. The results are shown in Table 9.

The results showed that the injection of pinocembrin racemate (1 mg/kg, 5 mg/kg) after 3 hours and 6 hours of ischemia decreased neurobehavioral injury and reduced infarct volume and mitigated cerebral edema. The activities were significant and showed a good dose-effect relationship. (R)-pinocembrin and (S)- pinocembrin showed significant effects when initially injected after 3 hours of ischemia in rats but were not effective when initially injected after 6 hours of ischemia. Nimodipine was not significantly effective in the experimental conditions. Pinocembrin racemate (1 mg/kg, 5 mg/kg, iv) had good therapeutic effects to acute ischemic stroke in rats, and the therapeutic time window thereof was about 6 hours.