WO2023068850A1 - Composition for preventing or treating ischemic stroke comprising inhibitor of apoptosis proteins - Google Patents

Abstract

The present invention relates to a composition for preventing or treating ischemic stroke, comprising an inhibitor of apoptosis proteins. More specifically, unlike thrombolytic drugs, which are conventional therapeutic agents for stroke, the present invention can prevent or treat ischemic stroke through a systemic dosage form that penetrates to the blood-brain barrier by using a peptide for inhibiting Fas signaling, which is an inhibitor of apoptosis proteins that is capable of suppressing actual neuronal cell death.

Description

Composition for preventing or treating ischemic stroke containing an apoptosis inhibitory protein

The present invention relates to an injectable composition for preventing or treating ischemic stroke containing an apoptosis inhibitor protein.

Cerebrovascular disease, also called stroke, is one of the three leading causes of death for mankind along with malignant tumors and heart disease. Stroke is a disease that occurs when a blood vessel supplying blood to the brain is blocked or burst, resulting in damage to a local part of the brain, commonly known as 'paralysis'. Symptoms include hemiplegia, sensory disturbances, speech difficulties, speech difficulties, vision and vision disturbances, double vision, headaches, dizziness, impaired consciousness, vegetative state, and dementia. Strokes include “ischemic” stroke (80-85%), which occurs when blood vessels in the brain are completely blocked or severely narrowed and blood flow is not supplied to tissues, and hemorrhagic stroke (15-20%), which is a symptom in which the function of brain cells is damaged by hemorrhage divided into Stroke ranks second in mortality in Korea and third in mortality worldwide, and more than 50% of patients who survive stroke remain with various disabilities, placing a social burden on not only patients but also those who care for them.

Ischemic 　stroke occurs in an overwhelmingly larger proportion than hemorrhagic stroke, and various types of pathological abnormalities appear in the cerebral blood vessels that supply blood to the brain, causing cerebral blood circulation disorders in certain parts of the brain, resulting in reduced brain function or The brain ultimately develops an ischemic infarction. Ischemia refers to a state in which blood supply to a body organ, tissue, or part is reduced, and ultimately leads to irreversible damage, i.e., necrosis of cells and tissues. In particular, the brain and heart are the most sensitive organs of the body to lack of blood flow, and when tissue ischemia occurs, for example, as a result of a stroke or head injury, processes called the ischemic cascade are triggered, resulting in permanent damage to brain tissue. . However, the tissue around it has a penumbra zone that can be recovered, so this area becomes the target of medical treatment.

For ischemic stroke, which accounts for the majority of all strokes, the prognosis of future patients is often determined by the clinical course of the acute phase (within 7 days) or subacute phase (within 4 weeks). In this "ischemic" stroke, recanalization treatment is performed to resupply blood flow so that the brain tissue of the brain-ischemic penumbra, which is a physiological target of acute treatment, functions again. However, until now, it is known that the prognosis of the patient can be improved only when recanalization is performed within 4.5 hours of symptom onset with the intravenous administration method or within 6 hours with the intraarterial method. However, even in Korea and around the world, the rate at which a stroke patient can be found in time and receive recanalization treatment in an appropriate emergency room is extremely low. Therefore, since most patients with acute, ischemic, or stroke do not receive appropriate treatment, a safe and effective new treatment for patients in the acute phase is so urgently needed.

An object of the present invention is to provide a peptide for inhibiting Fas signaling that exhibits a therapeutic effect on ischemic stroke by using a brain-targeting peptide that passes through the blood-brain barrier in the form of an intravenous injection.

In order to achieve the above object, the present invention provides a composition for preventing or treating ischemic stroke comprising a complex represented by the following general formula 1:

[Formula 1]

Lep-PEG-FBP

In the above formula,

Lep is leptin or a leptin-derived brain-targeting peptide comprising the amino acid sequence of 1-33, 12-32, 15-32 or 61-90 of the amino acid sequence of SEQ ID NO: 1,

PEG is polyethylene glycol,

FBP represents a Fas Blocking Peptide consisting of an amino acid sequence represented by Formula 2 below.

[Formula 2]

Xaa ₁ -Cys-Asp-Glu-His-Phe-Xaa ₂ -Xaa ₃

In the above formula,

Xaa ₁ and Xaa ₃ are each independently absent or an arbitrary amino acid;

Xaa ₂ is absent or selected from the group consisting of Ala, Gly, Val, Leu, He, Met, Pro, Ser, Cys, Thr, Asn and Gln.

The present invention also provides a method of treating ischemic stroke comprising administering a therapeutically effective amount of the complex to a subject in need thereof.

The present invention can be applied as a drug delivery platform for various brain diseases by maximizing convenience and fewer side effects through a systemic dosage form that passes through the blood-brain barrier. In addition, unlike conventional thrombolytic drugs, which are conventional stroke treatments, using a peptide for inhibiting Fas signal transmission that can actually inhibit the death of nerve cells can contribute to improving survival rate by delaying cerebral infarction.

Figure 1 shows the drug synthesis and fluorescent substance binding of Leptin-PEG-FBP of the present invention.

Figure 2 shows the result of confirming apoptosis in the hypoxic neuronal cell model of Leptin-PEG-FBP of the present invention through flow cytometry.

Figure 3 shows the results of measuring Fas overexpression in an ischemic stroke mouse model through RT-PCR.

4 illustrates fluorescence images of drug delivery for each organ through photothrombosis-induced stroke modeling.

5 shows fluorescence images of drug delivery through leptin receptor deficient mice.

6 shows fluorescence images of drug delivery for each organ through intravascular middle cerebral artery occlusion modeling.

Figure 7 shows confocal microscopy images of drug delivery through fluorescence pool screening.

8 shows an experimental plan for evaluating drug efficacy in animals according to the present invention.

9 shows the results of cerebral infarct volume measurement using triphenyltetrazolium chloride (TTC).

Figure 10 shows the result of measuring the size of the brain infarct in neural tissue through Nissl staining.

11 shows the results of observation of histological changes through H&E staining.

12 shows the treatment effect of inhibiting neuronal cell death through TUNEL staining.

Figure 13 shows the results of confirming the decrease in the expression of apoptosis proteins through Western blotting.

14 shows the result of observation of cleaved caspase-3 protein through immunohistochemical staining.

During ischemic stroke, cells receive signals of apoptosis due to lack of oxygen and limited nutrient supply. In this regard, the present inventors have reported that ischemic stroke treatment is possible through nasal-to-brain delivery of a Fas Blocking Peptide (FBP) having an apoptosis inhibitory function. In order to treat ischemic stroke by delivering FBP to the brain by intravenous injection, the leptin peptide having the ability to bind to the Leptin receptor expressed in the brain thalamus is bound to FBP-PEG and then intravenously injected, and the complex is blood-brain It was confirmed that it passed through the barrier and was delivered to the part where cerebral ischemia occurred and inhibited the death of brain cells.

Accordingly, the present invention relates to a composition for preventing or treating ischemic stroke comprising a complex represented by the following general formula 1:

[Formula 1]

Lep-PEG-FBP

In the above formula,

Lep is leptin or a leptin-derived brain-targeting peptide comprising the amino acid sequence of 1-33, 12-32, 15-32 or 61-90 of the amino acid sequence of SEQ ID NO: 1,

PEG is polyethylene glycol,

FBP represents a Fas Blocking Peptide consisting of an amino acid sequence represented by Formula 2 below.

[Formula 2]

Xaa ₁ -Cys-Asp-Glu-His-Phe-Xaa ₂ -Xaa ₃

In the above formula,

Xaa ₁ and Xaa ₃ are each independently absent or an arbitrary amino acid;

Xaa ₂ is absent or selected from the group consisting of Ala, Gly, Val, Leu, He, Met, Pro, Ser, Cys, Thr, Asn and Gln.

Fas Blocking Peptide (FBP), an active ingredient of the composition for preventing or treating ischemic stroke of the present invention, has been known as a peptide sequence for inhibiting the interaction between Fas and its ligand, FasL. Fas and its specific ligand, FasL, are members of proteins belonging to the TNF receptor and TNF ligand superfamily (TNFSF), respectively. Interaction between Fas and FasL triggers a cascade of intracellular events leading to cell death in Fas-expressing targets. Fas is a membrane protein expressed in various tissue cells including brain cells, and FasL is mainly expressed in lymphoid organs and immune-related tissues. It has been reported that Fas expression increases during brain damage caused by ischemia (Expression of Fas and Fas Ligand After Experimental Traumatic Brain Injury in the Rat, "J" Cereb "Blood Flow" Metab. "Vol. 20, No. 4, 2000).

The FBP of the present invention is a Fas peptide mimetic, and inhibits Fas activity, particularly Fas-mediated signaling. However, when FBP is administered intravenously or orally, the effective substance does not easily pass through the BBB (Blood-Brain Barrier) and the delivery efficiency to the brain is significantly reduced. Therefore, the present invention uses a Leptin peptide having the ability to pass through the BBB by binding to a Leptin receptor overexpressed in the brain thalamus, and binds FBP using PEG to the Leptin peptide to deliver to the stroke ischemic site through intravenous administration, It is characterized by preventing or treating ischemic stroke by inhibiting brain cell death by blocking the Fas apoptosis pathway.

The term “Lep” or “Leptin” as used herein refers to leptin or a leptin-derived brain-targeting peptide. In one embodiment of the present invention, the leptin-derived brain-targeting peptide of the present invention comprises an amino acid sequence of 1-33, 12-32, 15-32 or 61-90 of the amino acid sequence of SEQ ID NO: 1 , preferably an amino acid sequence of 1-33 or 61-90, more preferably an amino acid sequence of 61-90 (SEQ ID NO: 2).

The polyethylene glycol (or PEG) is a method for stabilizing proteins and inhibiting contact with proteolytic enzymes and kidney loss. PEG non-specifically binds to specific or various sites of the target protein to increase solubility As a result, it is known to be effective in stabilizing proteins and preventing protein hydrolysis and not causing any particular side effects (Sada et al ., J. Fermentation Bioengineering, 71: pp 137-139, 1991). The average molecular weight of PEG usable in the present invention is 500 Da to 2 kDa, or 1 kDa to 2 kDa. The molecular weight of a preferred PEG is about 2 kDa. EG is used to cover both linear and branched polymers. Most PEGs are commercially available. In addition, both ends of PEG may be modified to facilitate binding with Lep, linker peptide, or FBP. For example, it may be modified with a maleimide group, an amide group, or the like.

The Lep and PEG may be connected by a linker peptide serving as a spacer, and may be connected to PEG whose surface is modified by binding to one end of Lep. Examples of such linker peptides include GGGC and RRR. In particular, in the case of GGGC, the -SH group of cysteine and one end may be connected through a bond with a modified PEG moiety.

As used herein, the term "Fas" is also called Fas, Fas receptor (Fas receptor), apoptosis antigen 1 (APO-1), or cluster of differentiation 95 (CD95), a tumor necrosis factor that regulates apoptosis. It is a type of receptor (tumor necrosis factor, TNF). When Fas binds to a ligand, it is activated through multimerization, and as a result, several adapter proteins bind to Fas. The bound adapter proteins activate various apoptosis signal transduction systems, and representative signal transduction regulators include caspase, NF-κB, stress-activated protein kinase (SAPK), and the Bcl-2 family.

As used herein, the term "fas signaling inhibitory peptide (Fas Blocking Peptide, FBP)" binds to the above-described Fas and inhibits the sub-signaling system of Fas as opposed to the conventional Fas ligand, inhibiting activity and apoptosis Means a peptide having an activity.

The FBP is represented by the general formula (2). Specifically, in the above general formula 2,

Xaa ₁ and Xaa ₃ are each independently absent or selected from the group consisting of Tyr, Phe and Trp;

Xaa ₂ is absent or selected from the group consisting of Gly, Ala, Ser, Thr, Met and Cys.

More specifically, in the above general formula 2,

Xaa ₁ and Xaa ₃ are each independently selected from the group consisting of Tyr, Phe and Trp;

Xaa ₂ is selected from the group consisting of Gly, Ala, Ser, Thr, Met and Cys.

In one embodiment of the present invention, in Formula 2, Xaa ₁ and Xaa ₃ may each independently not exist or may be any amino acid, preferably each independently selected from the group consisting of Tyr, Phe and Trp. can

In addition, Xaa ₂ in Formula 1 may not exist or may be selected from the group consisting of Ala, Gly, Val, Leu, Ile, Met, Pro, Ser, Cys, Thr, Asn and Gln, preferably Gly, It may be selected from the group consisting of Ala, Ser, Thr and Cys.

For example, 1) only Xaa ₂ can exist without both Xaa ₁ and Xaa ₃ , 2) Xaa ₂ can exist while either Xaa ₁ or Xaa ₃ exists, 3) Xaa ₁ to Xaa Various combinations are possible, such as none of ₃ .

In one embodiment of the present invention, the amino acid sequence of Formula 2 may be a sequence listed in Table 1 below.

sequence number	Amino acid sequence (5'-3')
3	CDEHF
4	YCDEHF
5	YCDEHFY
6	YCDEHFA
7	YCDEHFC
8	YCDEHFM
9	FCDEHFC
10	YCDEHFCY
11	YCDEHFMY
12	YCDEHFCF

On the other hand, the peptide for inhibiting Fas signaling used in the present invention has improved stability of the peptide, enhanced pharmacological properties (half-life, absorption, potency, efficacy, etc.), altered specificity (eg, broad biological activity spectrum), reduced antigen To obtain properties, the N- and/or C-terminus of the peptide may be modified. The formula may be in the form of an acetyl group, a fluorenyl methoxy carbonyl group, an amide group, a formyl group, a myristyl group, a stearyl group, or polyethylene glycol (PEG) bonded to the N- and/or C-terminus of the peptide. However, any component that can improve peptide modification, particularly stability of the peptide, may be included without limitation. As used herein, the term "stability" refers to storage stability (eg, storage stability at room temperature) as well as in vivo stability that protects the peptides of the present invention from attack by proteolytic enzymes in vivo.

The Lep-PEG-FBP complex of the present invention includes a cross-linking site for complex formation, and the cross-linking site may be one terminal amino acid itself for an amide bond between monomers, a disulfide bond, an imine bond, or an ester bond It may be a specific amino acid for.

For example, as shown in FIG. 1, after blocking the N'-terminus of the Leptin-GGGC peptide (34mer), Mal-PEG-NH2 was used to remove the thiol group present in the cysteine of the linker peptide bound to Leptin and the front end of PEG. A Lep-PEG conjugate can be formed through a reaction between maleimides, and then a Lep-PEG-PBP complex can be formed through an EDC/sulfo-NHS amine covalent bond between an amine group at the PEG terminal and an amine group at the N-terminus of PBP. .

As used herein, the term "treatment" refers to all activities that improve or beneficially change symptoms of ischemic stroke by administration of the pharmaceutical composition according to the present invention.

As used herein, the term "administration" means introducing a predetermined substance, ie, a pharmaceutical composition according to the present invention, into a subject by any suitable method.

In the present invention, the term "therapeutically effective amount" means an amount sufficient to treat a disease with a reasonable benefit/risk ratio applicable to medical treatment, and the effective dose level is dependent on the type, severity, and activity of the drug of the patient's disease. , sensitivity to the drug, time of administration, route of administration and excretion rate, duration of treatment, factors including concomitantly used drugs, and other factors well known in the medical field.

The composition of the present invention includes a pharmaceutically acceptable carrier in addition to the active ingredient. Pharmaceutically acceptable carriers included in the composition of the present invention are those commonly used in formulation, and include lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia gum, calcium phosphate, alginate, gelatin, calcium silicate , microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methyl cellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil, but are not limited thereto no. The composition of the present invention may further include a lubricant, a wetting agent, a sweetening agent, a flavoring agent, an emulsifying agent, a suspending agent, a preservative, and the like in addition to the above components. Suitable pharmaceutically acceptable carriers and agents are described in detail in Remington's Pharmaceutical Sciences (19th ed., 1995).

The composition of the present invention is preferably administered by systemic parenteral administration, and may be administered using, for example, intravenous administration, intraperitoneal administration, intramuscular administration, subcutaneous administration, or local administration.

The suitable dosage of the composition of the present invention varies depending on factors such as formulation method, administration method, patient's age, weight, sex, severity of disease symptoms, food, administration time, administration route, excretion rate and reaction sensitivity, Ordinarily, the skilled practitioner can readily determine and prescribe effective dosages for the desired treatment. According to a preferred embodiment of the present invention, a suitable daily dosage is 0.0001-100 mg/kg (body weight). Administration may be administered once a day, or may be administered in several divided doses.

The composition of the present invention is prepared in unit dosage form by formulating using a pharmaceutically acceptable carrier and/or excipient according to a method that can be easily performed by those skilled in the art, or It can be prepared by placing it in a multi-dose container. In this case, the formulation may be in the form of a solution, suspension or emulsion in an oil or aqueous medium, or may be in the form of an extract, powder, granule, tablet or capsule, and may additionally contain a dispersing agent or stabilizer.

The present invention also relates to a method of treating ischemic stroke comprising administering a therapeutically effective amount of the complex to a subject in need thereof.

The subject may be a human or a non-human animal such as a cow, monkey, bird, cat, mouse, rat, hamster, pig, dog, rabbit, sheep, or horse.

In the treatment method of the present invention, the formulation, administration method, etc. of the complex are the same as those described in the pharmaceutical composition, and detailed descriptions are omitted to avoid redundant description.

Hereinafter, the present invention will be described in detail based on the following examples. However, the following examples are only for exemplifying the present invention, but the scope of the present invention is not limited thereto.

<Example 1> Preparation of Leptin-PEG-FBP

Leptin-PEG-FBP complex was prepared as shown in Figure 1. Briefly, Leptin (61-90, 30mer, SEQ ID NO: 2) peptide, which has the ability to pass through the blood-brain barrier by binding to the Leptin receptor overexpressed in the brain thalamus, is additionally custom-made and used with 4 amino acids GGGC did The LeptinGGGC (34mer) peptide was dissolved in DMSO at a concentration of 100 mg/mL and then diluted 10-fold in PBS (pH 7.4) buffer for use. Leptin peptide at a concentration of 10 mg/mL was blocked at the N-terminus with acetic anhydride for 1 hour at 4°C. Then, 2 kDa Mal-PEG-NH2 was used to react with Leptin's cysteine and PEG's Thiol-Maleimide at a 1:1 molar ratio for 18 hours at 4°C. After the reaction was completed, unbound PEG was removed using a 2kDa MWCO cassette in PBS (pH 7.4) buffer. After purification, the mixture was dissolved in 100 mg/mL DMSO and then covalently bonded to EDC/sulfo-NHS amine at a 1:1 molar ratio of FBP peptide and Leptin-PEG diluted 10-fold in MES (pH 6.5) buffer for 3 hours at room temperature. After the reaction, it was purified in the same way as before and stored at -20 ℃.

<Experimental Example 1> Confirmation of the apoptosis inhibitory effect of the drug through cell experiments

In order to confirm the apoptosis inhibitory effect of Leptin-PEG-FBP, in which Leptin was conjugated to Fas-binding peptide with PEG, fluorescence intensity was observed by flow cytometry after staining with Annexin V (PE). To induce a hypoxic neuron model, Neuro2a cells were seeded in serum-free DMEM medium. Thereafter, FasL (0.1 nM) was pre-treated in a hypoxia (Hypoxia) incubator under 1% O ₂ conditions for 4 hours, and then treated with a peptide at a concentration of 300 μM, and cells in each group were stained with Annexin V fluorescent dye after 12 hours.

As shown in Figure 2, it was confirmed that Leptin-PEG-FBP can inhibit the apoptosis pathway by binding to Fas by observing apoptosis Annexin V fluorescence intensity that is about 30% lower than that of the control group.

<Experimental Example 2> Confirmation of drug delivery difference by organ through intravenous injection of fluorescent peptide in ischemic stroke model

After dissolving the photosensitive dye Rose bengal at 15 mg/mL in sterile saline, intraperitoneally injected at 10 mg/kg per mouse, and after 5 minutes, 561 nm green laser light was applied to the bregma at the intersection of the sagittal and coronal sutures. ) was irradiated to the right skull at 2.5 mm for about 10 minutes to form a blood clot, and ischemic stroke modeling was performed. Brain tissue was collected 24 hours after ischemic stroke induction. After RNA was isolated from the left and right sides of the tissue sample, cDNA was synthesized, and RT-PCR was performed with the Fas primer. there was. After preparing the Lep-PEG-FBP complex as shown in FIG. 1, it was confirmed whether the synthetic peptide to which the fluorescent substance was coupled was selectively delivered to the actual cerebral infarct region. After dissolving the photosensitive dye rose bengal at 15 mg/mL in sterile physiological saline, 10 mg/kg per mouse was intraperitoneally injected. After 5 minutes, 561 nm laser light was applied 2.5 mm right from bregma, the point where the sagittal and coronal sutures intersected. As a result of irradiation on the skull for about 10 minutes, a blood clot was formed, and ischemic stroke modeling was performed. After 12 hours of modeling, the fluorescence-coupled peptide was intravenously injected at 2.5 mg/kg. The number of n for each group was 3. 16 hours after drug injection, each organ of brain, lung, liver, spleen, and kidney was put into a fluorescence imaging device to confirm the degree of drug delivery.

As shown in FIG. 4, most of the group without modeling was filtered in the kidney, and the group using leptin showed strong fluorescence intensity in the right cerebral infarct area where modeling was performed. Through this, it was confirmed that L-P-F, which was synthesized using leptin, remained at the cerebral infarction site and delivered the drug.

Next, receptor-mediated blood-brain barrier penetration drug delivery was confirmed through intravenous injection of a peptide drug combining a fluorescent sample in a leptin receptor deficient animal model (db/db) and a general animal model. After 3 hours from photothrombosis-induced stroke modeling, each peptide drug to which alexa647 fluorescent sample was bound was intravenously injected. Twelve hours after the intravenous injection, the brain, lung, liver, spleen, and kidney organs were sampled and fluorescence images were taken.

As shown in FIG. 5, in the leptin receptor-deficient animal model, it was confirmed that most of the drug was filtered through the kidney without remaining in the brain tissue. On the other hand, in the general animal model, the drug remained bound to the right brain tissue damaged by the ischemic stroke model.

In order to confirm that peptides competing with fluorescent substances are selectively delivered to the actual cerebral infarction area, this is the most commonly used modeling method in stroke rat models. Blood flow to the cerebral artery was blocked, and the filament was removed and reperfused after 60 minutes. After ischemic stroke induction surgery, 2.5 mg/kg of fluorescence-coupled peptide was intravenously injected 6 hours after the study showed that Fas expression was high, and organs were harvested 12 hours later.

As shown in FIG. 6, it was confirmed that the drug was delivered to the right cerebral infarction site most clearly in the Leptin and L-P-F groups compared to PEG-FBP.

After confirming organ-specific drug delivery by fluorescence intensity, the brain tissue was put in an OCT compound and stored at -80 ° C to make a cryoblock and cut into sections to check whether the FBP peptide was bound to Fas expressed on the actual cell membrane. The nucleus was stained using hoechest2000 in a section cut into 5 mm thickness, and as a result of overlapping with the peptide to which the alexa647 fluorescent substance was bound, it was confirmed that the intensity of red peptide fluorescence was strong around the nucleus (FIG. 7).

<Experimental Example 3> Confirmation of the therapeutic effect of inhibiting apoptosis of drugs through ischemic stroke modeling

In order to confirm the therapeutic effect of the drug on inhibition of neuronal cell death through ischemic stroke modeling, as shown in FIG. 8, intravenous injection of FBP at 5 mg/kg twice 3 hours and 6 hours after middle cerebral artery occlusion modeling was performed did The number of n for each group was 5. All experimental animals were sacrificed 48 hours after MCAO, and phosphate-buffered saline was perfused through the left ventricle to remove intravascular blood components, and then brains were removed. The brain tissue excised from the cerebral ischemia induction model is equally divided into sections at 2 mm intervals on a mold. After that, it was reacted at 37 ℃ for 15 minutes in a 2% TTC solution dissolved in sterile physiological saline. After the reaction was completed, it was stored in a 4% paraformaldehyde solution at 4° C. for 24 hours, and then the unstained white brain infarct area was measured through Image J analysis program.

As a result, the L-P-F group showed about 19% less brain infarction (FIG. 9).

Brain tissue slices were stained using 0.5% Nissl staining (cresyl violte), photographed using a full screening microscope, and the size of the infarcted area was measured using an image analysis program. The size of cerebral infarction was measured using an indirect method, which was measured by subtracting the normal area on the cerebral infarct-induced side from the brain area on the opposite side of the cerebral infarct-induced area.

As shown in FIG. 10, the L-P-F group showed 17.2% less brain infarction than the other groups.

Brain tissue made of cryoblock was cut into sections, fixed in 4% paraformaldehyde for 15 minutes, and then H&E staining was performed. As a result of the same observation of ischemic penumbra with an optical microscope, as shown in FIG. 11, a lot of tissue empty space was found due to apoptosis in the other groups compared to the L-P-F drug group, and the shape of the nucleus was triangular and pointed It could be seen that the contraction was

Tissue samples sectioned from cryoblocks made using brain tissue were stained with TUNEL, and the degree of cell death was measured by fluorescence.

As shown in FIG. 12 , significantly less nuclear DAPI fluorescence intensity was measured in the other groups than in the L-P-F drug group, and TUNEL intensity indicating apoptosis in red color was strong. Statistical analysis showed about 28% reduction in apoptosis.

Only the right infarct tissue of the brain tissue was isolated, protein was extracted, and 30 μg of protein per group was applied to each well in a 12% gradient gel to conduct a Western blot experiment.

As shown in FIG. 13, as a result of confirming using the primary antibodies of Fas and Cleaved Caspase-3, the protein expression levels of Fas were reduced by about 2 times and Cleaved Caspase-3 by 0.5 times, indicating that the Fas apoptosis pathway was blocked in the drug-treated group. It was proved molecularly that the treatment effect was shown through

The protein expression level of cleaved Caspase-3, which plays a key role in the apoptosis pathway, was observed in brain tissue sections through immunohistochemical staining.

As shown in FIG. 14, as a result, the cleaved Caspase-3 fluorescence decreased by about 13% in the L-P-F drug group, demonstrating the effect of ischemic stroke treatment by inhibiting apoptosis by FBP binding to Fas.

The present invention can be applied as a systemic dosage form that passes through the blood-brain barrier in the field of treatment of brain diseases such as ischemic stroke.

Claims (14)

1. A composition for preventing or treating ischemic stroke comprising a complex represented by Formula 1 below:

   [Formula 1]

   Lep-PEG-FBP

   In the above formula,

   Lep is leptin or a leptin-derived brain-targeting peptide comprising the amino acid sequence of 1-33, 12-32, 15-32 or 61-90 of the amino acid sequence of SEQ ID NO: 1,

   PEG is polyethylene glycol,

   FBP represents a Fas Blocking Peptide consisting of an amino acid sequence represented by Formula 2 below.

   [Formula 2]

   Xaa ₁ -Cys-Asp-Glu-His-Phe-Xaa ₂ -Xaa ₃

   In the above formula,

   Xaa ₁ and Xaa ₃ are each independently absent or an arbitrary amino acid;

   Xaa ₂ is absent or selected from the group consisting of Ala, Gly, Val, Leu, He, Met, Pro, Ser, Cys, Thr, Asn and Gln.
2. According to claim 1,

   A composition for preventing or treating ischemic stroke, wherein the leptin-derived brain-targeting peptide comprises the amino acid sequence of SEQ ID NO: 2.
3. The method of claim 1, wherein in the general formula 2,

   Xaa ₁ and Xaa ₃ are each independently absent or selected from the group consisting of Tyr, Phe and Trp;

   A composition for preventing or treating ischemic stroke, wherein Xaa ₂ does not exist or is selected from the group consisting of Gly, Ala, Ser, Thr, Met and Cys.
4. The method of claim 1, wherein in the general formula 2,

   Xaa ₁ and Xaa ₃ are each independently selected from the group consisting of Tyr, Phe and Trp;

   Xaa ₂ is selected from the group consisting of Gly, Ala, Ser, Thr, Met and Cys, a composition for preventing or treating ischemic stroke.
5. According to claim 1,

   The amino acid sequence represented by Formula 2 is SEQ ID NOS: any one selected from the group consisting of 3 to 12, a composition for preventing or treating ischemic stroke.
6. According to claim 1,

   The composition is delivered through systemic administration, a composition for preventing or treating ischemic stroke.
7. According to claim 6,

   The systemic administration is intravenous administration, a composition for preventing or treating ischemic stroke.
8. A method for treating ischemic stroke comprising administering a therapeutically effective amount of a complex represented by Formula 1 below to a subject in need thereof:

   [Formula 1]

   Lep-PEG-FBP

   In the above formula,

   Lep is leptin or a leptin-derived brain-targeting peptide comprising the amino acid sequence of 1-33, 12-32, 15-32 or 61-90 of the amino acid sequence of SEQ ID NO: 1,

   PEG is polyethylene glycol,

   FBP represents a Fas Blocking Peptide consisting of an amino acid sequence represented by Formula 2 below.

   [Formula 2]

   Xaa ₁ -Cys-Asp-Glu-His-Phe-Xaa ₂ -Xaa ₃

   In the above formula,

   Xaa ₁ and Xaa ₃ are each independently absent or an arbitrary amino acid;

   Xaa ₂ is absent or selected from the group consisting of Ala, Gly, Val, Leu, He, Met, Pro, Ser, Cys, Thr, Asn and Gln.
9. According to claim 8,

   A method of treating ischemic stroke, wherein the leptin-derived brain-targeting peptide comprises the amino acid sequence of SEQ ID NO: 2.
10. The method of claim 8, wherein in the general formula 2,

    Xaa ₁ and Xaa ₃ are each independently absent or selected from the group consisting of Tyr, Phe and Trp;

    A method for treating ischemic stroke, wherein Xaa ₂ does not exist or is selected from the group consisting of Gly, Ala, Ser, Thr, Met and Cys.
11. The method of claim 8, wherein in the general formula 2,

    Xaa ₁ and Xaa ₃ are each independently selected from the group consisting of Tyr, Phe and Trp;

    Xaa ₂ is selected from the group consisting of Gly, Ala, Ser, Thr, Met and Cys, a method for treating ischemic stroke.
12. According to claim 8,

    The amino acid sequence represented by Formula 2 is any one selected from the group consisting of SEQ ID NOS: 3 to 12, a method for treating ischemic stroke.
13. According to claim 8,

    The method of treating ischemic stroke, wherein the complex is delivered through systemic administration.
14. According to claim 13,

    The systemic administration is an intravenous administration, a method for treating ischemic stroke.

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