CN110882240A

CN110882240A - Polyphenol derivative compound 6-CEPN as therapeutic agent for acute ischemic stroke

Info

Publication number: CN110882240A
Application number: CN201910852701.0A
Authority: CN
Inventors: 沈剑刚; 王明福; 冯靖涵; 邓瑞霞; 杜巧辉
Original assignee: University of Hong Kong HKU
Current assignee: University of Hong Kong HKU
Priority date: 2018-09-10
Filing date: 2019-09-10
Publication date: 2020-03-17

Abstract

The novel flavonoid derivative compound 6-CEPN protects the brain from ischemia/reperfusion (I/R) injury by attenuating peroxynitroso-mediated mitochondrial autophagy. Infarct volume and neurological impairment scores of acute ischemic stroke were significantly improved by 6-CEPN. 6-CEPN was found to inhibit excessive mitochondrial autophagy activation during brain I/R injury, with a concomitant reduction in peroxynitrite production. Specifically, 6-CEPN inhibited the ratio of LC3-II/I and Drp1 expression in the mitochondrial fraction, as well as the protein expression of iNOS and NADPH oxidase subunit p47phox in a dose-dependent manner.

Description

Polyphenol derivative compound 6-CEPN as therapeutic agent for acute ischemic stroke

Technical Field

The subject matter of the present invention provides methods and therapeutic agents for acute ischemic stroke.

Background

Acute Ischemic Stroke (AIS) is a devastating cause of death and disability, depending on the time from the onset of ischemia to treatment, the area of the brain affected and its size. Despite decades of research, no drug therapy is currently approved that stimulates the recovery of neurological function in stroke patients (Endres, Engelhardt et al, 2008). Tissue plasminogen activator (t-PA) is the only approved and widely used drug in clinical therapy.

Many pharmacological drugs, such as eptifibatide (adeye et al, 2015), reteplase (Qureshi et al, 2005), lovastatin (Elkind et al, 2009), granulocyte colony stimulating factor (Shyu et al, 2006), edaravone (Wada et al, 2014) and ethanocaffeine (caffeinol) (Martin-Schild et al, 2009) have been used in clinical trials of AIS by reducing ischemic injury and enhancing functional recovery. Furthermore, the results of pharmacological studies indicate that multiple mechanisms cause different aspects of stroke damage, and therefore several mechanisms must be targeted in the development of stroke medication to provide optimal functional recovery.

Disclosure of Invention

The following presents a simplified summary of the invention in order to provide a basic understanding of some aspects of the invention. This summary is not an extensive overview of the invention. It is intended to neither identify key or critical elements of the invention nor delineate the scope of the invention. Rather, the sole purpose of this summary is to present some concepts of the invention in a simplified form as a prelude to the more detailed description that is presented later.

6-C- (E-phenylvinyl) -naringenin (6-CEPN) is a novel flavonoid compound chemically synthesized from naringenin. Previous studies have shown that 6-CEPN can prevent colorectal cancer by targeting COX-1 (Li et al, 2014). 6-CEPN is also involved in the effects on stroke due to platelet inhibitory activity and antioxidant activity.

Many stroke studies have focused on neuronal damage and mechanisms of neuroprotection, such as inflammatory modulation, blood-brain barrier protection, improvement of immune responses, and the like. However, with the insight into autophagy in stroke, it is believed that drugs targeting autophagy or mitophagy may be effective in reducing injury or maintaining functional recovery after stroke.

AIS is a devastating cause of death and disability, depending on the time from ischemic onset to treatment, the area of the brain affected and the size of the infarct. In AIS pathologies, revascularization often produces large amounts of free radicals, inducing reperfusion injury and exacerbating brain damage. Peroxynitroso (peroxinitrite), a representative Reactive Nitrogen Species (RNS), is an important cytotoxic factor during cerebral ischemia/reperfusion injury. There are several drug candidates entering clinical trials for improving recovery after stroke, such as eptifibatide and reteplase. However, these drug candidates show insufficient protective effects in AIS. Compared to data from existing drug candidates (e.g. eptifibatide, reteplase, lovastatin, edaravone) in the original literature/database, 6-CEPN showed a stronger protective effect on AIS by improving RNS-induced autophagy/mitophagy in animal studies by reducing ischemic injury. Recently, studies have shown that improving autophagy/mitophagy after AIS can reduce brain damage and protect neurological function, emphasizing that autophagy/mitophagy can be a potential therapeutic target for ischemic stroke. The invention discloses that 6-CEPN has stronger protective effect on AIS by reducing ischemic injury in animal research. Here, it was demonstrated that the protective effect of 6-CEPN on cerebral ischemia/reperfusion injury is by modulating peroxynitroso-mediated autophagy/mitochondrial autophagy.

In summary, the subject matter of the present invention provides a novel insight in the treatment of ischemic stroke by targeting peroxynitrite mediated mitophagy, and also provides therapeutic strategies in combination with thrombolytic therapy.

Brief Description of Drawings

Figure 1. 6-CEPN significantly reduced infarct volume and reduced neurological impairment scores following cerebral ischemia-reperfusion injury.

FIG. 2.6-CEPN inhibits the production of peroxynitroso groups and attenuates the level of mitochondrial autophagy in ischemia-reperfused brain.

FIG. 3.6-CEPN reduces SIN-1 induced apoptosis in SH-SY5Y cells.

FIG. 4.6-CEPN directly with ONOO^-Sodium reacts to form new compounds.

FIG. 5 shows the chemical structure of 6-CEPN.

FIG. 6.6-CEPN increased cell viability of HK2 (human kidney) cell line. Panel a and panel b show the cytotoxicity results of 6-CEPN at different concentrations.

Figure 7. effect of naringin on reduction of neurological impairment score and reduction of infarct size in ischemia-reperfused rat brain. Panel a is the neurological impairment score (mNSS) scale results, panel b is the TTC staining results, and panel c is the quantitative analysis of infarct size.

Detailed Description

Herein, 6-CEPN is designed to protect neurons in AIS brain. The AIS-induced oxidative damage was ameliorated by intravenous injection of 10mg/kg of 6-CEPN over 24 hours. The major injury during AIS is caused by an inadequate blood and energy supply during the first 24 hours. For the infarcted area, no nerves were found in the cerebral cortex. Therefore, the neuroprotective activity of drug candidates in AIS shows great potential to help patients recover from AIS, making them more mobile. Recently, RNS-mediated autophagy/mitochondrial autophagy was found to be a potential therapeutic target for ischemic stroke. Overall, 6-CEPN (10mg/kg, i.v.) significantly reduced infarct volume and neurological impairment scores by modulating RNS-mediated autophagy/mitochondrial autophagy early in AIS (0-24 h).

6-CEPN compositions contain 6-CEPN and/or isomers, analogs or prodrugs thereof, or pharmacologically active salts of the compounds. The 6-CEPN composition may be formulated as a pharmaceutical composition containing 6-CEPN and one or more pharmaceutically acceptable excipients.

For the dosage and effective amount, a wide range of amounts of the composition can be used to provide a therapeutic agent for acute ischemic stroke. A specific amount of 6-CEPN is typically used per application depending on a number of factors. As used herein, an effective amount refers to an amount sufficient to induce one or more biological effects, such as an amount that reduces the effects of acute ischemic stroke.

The subject of the present invention provides a new strategy to mitigate acute cerebral ischemia/reperfusion injury and neurological impairment using the flavonoid derivative 6-CEPN. The present invention has great potential for clinical practice in treating and alleviating ischemic stroke. 6-CEPN can be used to treat ischemic stroke through its mitochondrial autophagy regulatory mechanism.

Examples

Example 1

Rat focal cerebral ischemia reperfusion model

Adult male Sprgue-Dawley rats weighing 250-. Animal protocols were approved and supervised by the Committee for use of Live Animals in the university of hong Kong (Committee on the use of Live Animals in Teasching and Research). SD rats were maintained at a controlled temperature (22. + -. 2 ℃) and maintained on a food and water supply. Rats underwent MCAO (middle cerebral artery occlusion) and were then reperfused to mimic cerebral ischemia/reperfusion injury. All rats were randomly assigned to MCAO surgery and sham surgery groups. However, the investigators were aware of the panel assignment. Briefly, rats were anesthetized with 4% isoflurane (Abbott, IL, USA) and maintained with 1.5% isoflurane during induction of anesthesia. During surgery, rats were placed on a heating pad to maintain body temperature at 37 ℃. After shaving, the skin around the neck was disinfected with 75% ethanol and then cut open. Left Common Carotid Artery (CCA), External Carotid Artery (ECA) and Internal Carotid Artery (ICA) were exposed and isolated microscopically by 2cm incision in the middle of the neck. Notably, the nerves (particularly the vagus nerve) were carefully dissected away from CCA and ICA. The ECA was tied with suture and cut as an occlusion (stump). The CCA and ICA are temporarily blocked from blood flow by a kink or a clamp. Then, a monofilament (L3600, jialingco.ltd., china) having a silicon-coated tip and a diameter of 0.36mm was inserted into the ICA and advanced to occlude the source of MCA (middle cerebral artery), inducing blood flow cessation. After 2 hours of occlusion, the artery was again exposed under anesthesia and the monofilament was then removed to achieve reperfusion. Sham rats were subjected to similar surgery except that the MCA was occluded with a monofilament. All rats awakened from anesthesia in recovery cages. For postoperative care, all rats were housed individually until sacrificed. The mortality rate in MCAO-operated rats was about 2%. However, all rats in the sham group survived. These rats were sacrificed 6 hours, 14 hours and 22 hours after reperfusion, respectively.

Example 2

Medical treatment

To clarify ONOO^-Effect in ischemic stroke, FeTMPyP (75854, Cayman, MI, USA) was used as a positive control drug. At the beginning of reperfusion, 3mg/kg of FeTMPyP was administered intravenously once from the femoral vein of rats. Similar to FeTMPyP, 6-CEPN (5, 10mg/kg) was also injected intravenously from the femoral vein, fully dissolved in a mixture of PEG400, ethanol and saline. After injection, the skin was sutured and disinfected with 75% ethanol. The monofilament is then removed for reperfusion. All animals received separate care after surgery.

Example 3

Behavioral testing

The modified neurological impairment score (mNSS) scale is used to assess neurological impairment following ischemic stroke. The mNSS scale scores from 0 to 18, including motor, sensory and reflex tests, to assess neurological dysfunction. Prior to MCAO manipulation, each rat was trained once with reference to the mNSS scale. The higher the score obtained, the more severe the nerve damage is reflected. Behavioral testing was evaluated 24 hours after MCAO.

Example 4

Cerebral infarction volume measurement

After anesthetizing the rats by intraperitoneal injection of a mixture of 200mg/kg ketamine hydrochloride and 20mg/kg xylazine hydrochloride, cardiac perfusion was performed with Phosphate Buffered Saline (PBS). Brain samples were collected and cut into 2mm coronal sections using a rat brain slice chamber (Braintree, MA, USA). The slices obtained are 2-8mm from the frontal pole. Then, the sections were immersed in a 2% TTC (T8877, Sigma) solution and kept at 37 ℃ for 20 minutes in the dark. Stain images were captured by camera and infarct volume was measured and analyzed by ImageJ. To minimize the effects of cerebral edema, the percentage infarct volume was calculated using the following formula: infarct volume percentage ═ volume of right hemisphere-red volume of left hemisphere)/volume of right hemisphere x 100%.

Example 5

Mitochondrial isolation

After cardiac perfusion with PBS, brain tissue was collected and stored at-80 ℃ for further use. Mitochondrial isolation of rat brain was performed using a commercially available kit (89801, Thermo, IL, USA). We have chosen a unique reagent-based approach to achieve simultaneous multiple sample processing. In detail, the rat brain tissue was disrupted by 4 Dounce procedures to obtain a homogeneous suspension. Mitochondria are then isolated from the cytosolic fraction using extraction reagents and differential centrifugation. After isolation of mitochondria, mitochondrial proteins were then extracted with 2% CHAPS (C9426, Sigma) in Tris Buffered Saline (TBS) (300 μ L CHAPS buffer per 200mg brain tissue). In addition, mitochondrial and cytosolic proteins were stored at-80 ℃ for western blot analysis. The integrity and purity of the isolated mitochondria was tested by western blotting using the mitochondrial marker VADC1/Porin (ab15895, Abcam, Cambridge, UK).

Example 6

Western blot analysis

Mitochondrial and cytosolic proteins from rat brain were prepared after mitochondrial isolation. Cell proteins were extracted with RIPA (R0278, Sigma) lysis buffer plus 1% protease inhibitor and phosphatase inhibitor (Sigma). All manipulations were referenced to standard western blotting protocols. After quantitative analysis, equal amounts of protein were added and separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) with 8% to 12% separation gel, and then transferred to a 0.45 μm pore size polyvinylidene fluoride membrane (IPVH00010, EMD Millipore, Germany).After blocking with 5% bovine serum albumin, the membranes were incubated with primary antibody (see table below for details) overnight at 4 ℃ and then washed with TBS-Tween 20 buffer and incubated in secondary antibody (1: 2000) for 2 hours at 4 ℃. Using BIO-RAD ChemiDoc^TMXRS + system (USA), chemiluminescent detection with Enhanced Chemiluminescence (ECL) reagent (RPN2235, ge healthcare, IL, USA).

Example 7

Evaluation of safety

The cytotoxicity of 6-CEPN was tested in the normal renal cell line HK2 cells. Cell viability of HK2 cells was examined by MTT assay 24 hours after treatment with 6-CEPN. a. HK2 cells treated with 6-CEPN (0.0032, 0.016, 0.08, 0.4, 2, 10, 50, 250, 1250. mu.g/ml) were added to the medium. b. HK2 cells treated with 6-CEPN (0.98, 1.95, 3.91, 7.81, 15.63, 31.25, 52.5, 125, 250. mu.g/ml) were added to the medium.

Example 8

Efficacy testing relative to naringin

6-CEPN is modified based on naringenin, a desugared (glycation) product of naringin. Another comparative study was conducted in the present invention on the neuroprotective effect of naringin in experimental rat models of ischemic stroke, as follows.

Rats were subjected to Middle Cerebral Artery Occlusion (MCAO) for 2 hours to allow cerebral ischemia, and then were reperfused for 22 hours. Naringin (80, 120 or 160mg/kg), 6-CEPN (5, 10mg/kg), PDC (peroxynitrite decomposition catalyst) (3mg/kg) or vehicle was administered to rats at the beginning of reperfusion 2 hours after MCAO ischemia.

The results show a better neuroprotective effect than naringenin. Naringin doses ranged from 80-160mg/kg, much higher than 6-CEPN doses, which was shown to reduce infarct volume and improve neurological impairment scores in experimental ischemic stroke models. Importantly, only 10mg/kg of 6-CEPN treatment has shown similar effects to the 160mg/kg dose of naringin.

The results show that 6-CEPN has better neuroprotective effect than naringin and pinocembrin.

Statistics of

Data are expressed as mean ± s.e.m. Differences in measured variables between the two groups were assessed by unpaired two-sided t-test. Multiple comparisons were performed using one-way analysis of variance and Dunnett's multiple comparison test. All data were analyzed using GraphPad Prism (Version 6.0, GraphPad Software inc., CA, USA). Bilateral P <0.05 was considered statistically significant.

Results

The results are shown in FIGS. 1 to 4.

FIG. 1 is the effect of 6-CEPN on infarct volume in stroke rats. Compared to the model group, 6-CEPN significantly reduced infarct volume in a dose-dependent manner. At a concentration of 10mg/kg, 6-CEPN showed better infarct-reducing activity than 3mg/kg pinocembrin and FeTMPyP. Behavioral testing also showed similar results, i.e., 6-CEPN significantly improved balance activity and exercise capacity after two days of treatment.

FIG. 2 shows the results of 6-CEPN, pinocembrin and PDC regulated mitochondrial autophagy in AIS rats. Expression levels of Drp1 and LC3 in mitochondrial proteins were reduced by 6-CEPN, similar to the reduction results for pinocembrin and PDC. Expression levels of proteins associated with apoptosis, e.g. Bax, p47^phoxAnd iNOS, also dose-dependently downregulated by 6-CEPN, while Bcl-2 increased. The results show that 6-CEPN attenuates autophagy/mitochondrial autophagy in ischemic rats.

FIG. 3 shows the effect of 6-CEPN on SIN-1 induced oxidative damage in SH-SY5Y cells. The results show that 6-CEPN improves SIN-1-induced apoptosis of SH-SY5Y cells. Bax and 3-NT are attenuated by 6-CEPN treatment. Furthermore, tissue DNA and HMGB1 regulating transcription increased after SIN-1 treatment, and this condition was disturbed by 6-CEPN treatment.

FIG. 4 reports the direct 6-CEPN connection with ONOO^-Reaction (using HPLC). The 203nm absorption of 6-CEPN showed that 6-CEPN was directly related to ONOO^-Sodium reacts and produces new compounds. The results indicate that 6-CEPN redox on RNS contributes to the protection against ischemic macroangiopathyNeuroprotective effect in mice.

FIG. 5 is the chemical structure of 6-CEPN.

FIG. 6 shows that 6-CEPN is relatively safe and not cytotoxic within 50. mu.g/ml. We found that when the dose reached 250. mu.g/ml, it was significantly toxic to HK2 cells. EC50 for 6-CEPN was 97.75. mu.g/ml (261.09. mu.M). The EC50 concentration was very high. The results indicate that the compounds are safe and valuable for further development.

Data in figure 7 are shown as mean ± s.e.m., where ×) represents P <0.001, compared to the sham-vehicle group; # denotes P <0.05, # denotes P <0.01, # denotes P <0.001, and # denotes P <0.001, compared to the I2/R22-vector group (one-way ANOVA, followed by Dunnett multiple comparison test). Panel a is a behavioral test evaluated by the modified neurological impairment score (mNSS) scale (n-5) following oral administration of naringin, PDC and vehicle. Panel b shows TTC staining of rat cerebral infarct size in sham, I2/R22, I2/R22 plus naringin, and I2/R22 plus PDC (n-5). Panel c is a quantitative analysis of infarct size measured by ImageJ. By way of comparison, the results of the neurological impairment score (mNSS) scale for orally administered 6-CEPN, TTC staining, and quantitative analysis of infarct size by ImageJ are detailed in FIG. 1.

In summary, the present invention relates to the following technical solutions:

use of 6-C- (E-phenylvinyl) -naringenin, or a derivative, isomer or analogue thereof, in the manufacture of a medicament for the alleviation of acute cerebral ischemia/reperfusion injury and impaired neurological function in a patient.

2. The use of claim 1, wherein the unit dose of said medicament contains 60mg to 5g, preferably 300mg to 1g, more preferably 100mg, 200mg, 300mg, 400mg, 500mg or 600mg of 6-C- (E-phenylethenyl) -naringenin, or a derivative, isomer or analogue thereof.

3. The use of

technical scheme

1 or 2, wherein the medicament is used in combination with other medicaments for treating acute cerebral ischemia/reperfusion injury and neurological function damage, preferably the other medicaments for treating acute cerebral ischemia/reperfusion injury and neurological function damage are selected from thrombolytic therapeutic agents, eptifibatide, reteplase, lovastatin and edaravone.

4. The use according to any of claims 1 to 3, wherein the patient is a patient with acute cerebral ischemia/reperfusion injury and impaired neurological function within 24 hours, preferably within 12 hours, preferably within 8 hours, preferably within 4 hours, more preferably within 2 hours of the onset of acute ischemic stroke.

Use of 6-C- (E-phenylvinyl) -naringenin, or a derivative, isomer or analogue thereof, in the manufacture of a medicament for preventing cerebral ischemia/reperfusion injury in a patient.

6. The use of claim 5, wherein the unit dose of the medicament contains 60mg to 5g, preferably 300mg to 1g, more preferably 100mg, 200mg, 300mg, 400mg, 500mg or 600mg of 6-C- (E-phenylethenyl) -naringenin, or a derivative, isomer or analog thereof.

7. The use of

technical scheme

5 or 6, wherein the medicament is used in combination with other medicaments for preventing cerebral ischemia/reperfusion injury, preferably the other medicaments for preventing cerebral ischemia/reperfusion injury are selected from thrombolytic therapeutic drugs, eptifibatide, reteplase, lovastatin, and edaravone.

8. The use according to any of claims 5 to 7, wherein the patient is a patient at risk of an acute ischemic stroke event.

Use of 6-C- (E-phenylvinyl) -naringenin, or a derivative, isomer or analogue thereof, in the manufacture of a medicament for reducing peroxynitroso-mediated autophagy/mitochondrial autophagy.

10. The use of claim 9, wherein the unit dose of said medicament contains 60mg to 5g, preferably 300mg to 1g, more preferably 100mg, 200mg, 300mg, 400mg, 500mg or 600mg of 6-C- (E-phenylethenyl) -naringenin, or a derivative, isomer or analog thereof.

11. The use of claim 9 or 10 wherein the medicament is used in combination with other peroxynitroso-mediated autophagy/mitophagy reducing agents, preferably wherein the other peroxynitroso-mediated autophagy/mitophagy reducing agents are selected from the group consisting of thrombolytic therapy agents, eptifibatide, reteplase, lovastatin, and edaravone.

12. A composition for treating acute ischemic stroke comprising:

6-C- (E-phenylvinyl) -naringenin, or a derivative, isomer, or analog thereof, in an amount effective to treat acute ischemic stroke; and

one or more pharmaceutically acceptable excipients.

13. The composition of claim 12, wherein the composition comprises from 60mg to 5g, preferably from 300mg to 1g, more preferably 100mg, 200mg, 300mg, 400mg, 500mg or 600mg of 6-C- (E-phenylethenyl) -naringenin, or a derivative, isomer or analog thereof.

14. The composition of claim 12 or 13, wherein the composition is used in combination with other drugs for treating acute ischemic stroke, preferably the other drugs for acute ischemic stroke are selected from the group consisting of thrombolytic therapeutic drugs, eptifibatide, reteplase, lovastatin, and edaravone.

15. The composition of any of claims 12-14, wherein the composition is for use in a patient within 24 hours, preferably within 12 hours, preferably within 8 hours, preferably within 4 hours, more preferably within 2 hours of an acute ischemic stroke event.

Unless otherwise indicated in the following examples and elsewhere in the specification and claims, all parts and percentages are by weight, all temperatures are in degrees Celsius, and the pressure is at or near atmospheric.

For any figure or numerical range of a given feature, a number or parameter from one range may be combined with another number or parameter from a different range for the same feature to produce a numerical range.

Other than in the operating examples, all numbers, values and/or expressions referring to parameters, measurements, conditions and the like, used in the specification and claims, if any, are to be understood as modified in all instances by the term "about".

While the invention has been explained in connection with certain embodiments, it is to be understood that various modifications thereof will become apparent to those skilled in the art upon reading the specification. It is, therefore, to be understood that the invention disclosed herein is intended to cover such modifications as fall within the scope of the appended claims.

Claims

Use of 6-C- (E-phenylvinyl) -naringenin, or a derivative, isomer or analogue thereof, in the manufacture of a medicament for the alleviation of acute cerebral ischemia/reperfusion injury and impaired neurological function in a patient.
2. The use of claim 1, wherein the unit dose of the medicament contains 60mg to 5g, preferably 300mg to 1g, more preferably 100mg, 200mg, 300mg, 400mg, 500mg or 600mg of 6-C- (E-phenylvinyl) -naringenin, or a derivative, isomer or analogue thereof.
3. The use according to claim 1, wherein the medicament is used in combination with other medicaments for the treatment of acute cerebral ischemia/reperfusion injury and neurological function damage, preferably the other medicaments for the treatment of acute cerebral ischemia/reperfusion injury and neurological function damage are selected from the group consisting of thrombolytic therapeutic agents, eptifibatide, reteplase, lovastatin, and edaravone.
4. The use of claim 1, wherein the patient is a patient within 24 hours, preferably within 12 hours, preferably within 8 hours, preferably within 4 hours, more preferably within 2 hours of the onset of acute ischemic stroke.
Use of 6-C- (E-phenylvinyl) -naringenin, or a derivative, isomer or analogue thereof, in the manufacture of a medicament for preventing cerebral ischemia/reperfusion injury in a patient.
6. The use of claim 5, wherein the unit dose of the medicament contains 60mg to 5g, preferably 300mg to 1g, more preferably 100mg, 200mg, 300mg, 400mg, 500mg or 600mg of 6-C- (E-phenylethenyl) -naringenin, or a derivative, isomer or analogue thereof.
7. The use of claim 5, wherein the medicament is used in combination with another medicament for preventing cerebral ischemia/reperfusion injury in a patient, preferably the other medicament for preventing cerebral ischemia/reperfusion injury in a patient is selected from the group consisting of thrombolytic therapeutic agents, eptifibatide, reteplase, lovastatin, and edaravone.
8. The use of claim 5, wherein the patient is a patient at risk of an acute ischemic stroke event.
Use of 6-C- (E-phenylvinyl) -naringenin, or a derivative, isomer or analogue thereof, in the manufacture of a medicament for reducing peroxynitroso-mediated autophagy/mitochondrial autophagy.
10. A composition for treating acute ischemic stroke comprising:

6-C- (E-phenylvinyl) -naringenin, or a derivative, isomer, or analog thereof, in an amount effective to treat acute ischemic stroke; and

one or more pharmaceutically acceptable excipients.