KR20180114860A - Pharmaceutical compositions for preventing or treating intracerebral hemorrhage or conditions arising therefrom comprising rgd-containing elastin-like polypeptide - Google Patents

Abstract

The present invention relates to a pharmaceutical composition for preventing or treating symptoms caused by intracerebral hemorrhage or intracerebral hemorrhage having an RGD contained elastin-like polypeptide. More specifically, the present invention relates to the pharmaceutical composition for preventing or treating intracerebral hemorrhage or symptoms resulting therefrom having the RGD contained elastin-like polypeptide comprising a sequence of the following structural formula, and an elastin-like polypeptide for restoring cerebral vascular rupture. :[Structure] TGPG[VGRGD(VGVPG)6]nWPC.

Description

TECHNICAL FIELD [0001] The present invention relates to a pharmaceutical composition for preventing or treating symptoms caused by intracerebral hemorrhage or intracerebral hemorrhage including an RGD-containing elastin-like polypeptide. [0002] The present invention relates to a pharmaceutical composition for preventing or treating symptoms caused by intracerebral hemorrhage or intracerebral hemorrhage,

The present invention relates to a pharmaceutical composition for preventing or treating symptoms caused by intracerebral hemorrhage or intracerebral hemorrhage comprising an RGD-containing elastin-like polypeptide. More specifically, the present invention relates to a pharmaceutical composition comprising RGD-containing elastin- A pharmaceutical composition for preventing or treating intra-cerebral hemorrhage or symptoms resulting therefrom, and an elastin-like polypeptide for restoring cerebral vascular rupture.

[constitutional formula]

TGPG [VGRGD (VGVPG) ₆ ] _n WPC.

Intracerebral hemorrhage (ICH) is one of the most deadly diseases, accounting for about 15% of the total stroke worldwide. When cerebral blood vessels are ruptured by traumatic injury or aneurysms, the parenchyma around the damaged blood vessels is exacerbated by high pressure due to blood leaks, causing cerebral tissue damage. Neurological dysfunction is directly caused by tissue destruction, increased pressure and cerebral edema. Approximately 75% of all patients with spontaneous ICH deaths or failures occur within one year, and one-month mortality accounts for 40% of all deaths. Although many clinical trials have been conducted for ICH, new therapeutic strategies for treating ICH are still lacking.

Elastin, an extracellular matrix component, provides the elasticity and resilience needed to maintain tissue organization, such as skin and blood vessels. The elastin-like polypeptide (ELP) is derived from human tropoelastin and consists of a poly (Val-Pro-Gly-Xaa-Gly) pentapeptide wherein Xaa can be any amino acid except proline. ELP is non-cytotoxic, readily biodegradable, and can be used in a variety of forms such as hydrogels, microfibers, and cell sheets. The most notable characteristic of ELP is its thermal sensitivity reactivity. At a certain temperature, called the so-called transition temperature ( T t), the ELP changes its physical properties. Exceeding T t, the ELP becomes insoluble and exhibits a collective form.

However, below T t, the ELP becomes soluble and is found in monomeric form. The biocompatibility of ELPs depends on its chemical similarity to human tropoelastin (i.e., elastin precursor) and is therefore well suited for living cells. However, the low cell adhesion of ELP and the lack of its biological function limit the application of ELP to cell growth. To solve this problem, an ELP (V140) fusion protein containing a repeatable integrin-binding ArgGlyAsp (RGD) peptide, TGPG [VGRGD (VGVPG) ₆ ] ₂₀ WPC, REP was developed. REP can promote neuronal adhesion and proliferation on poorly adherent organic scaffolds.

On the other hand, it has been reported that the elastin-like polypeptide (ELP) elastin VGVPG (Valine-Glycine-Valine-Alanine-Proline-Glycine) pentapeptides and the RGD (Arginine-Glycine-Aspartate) (VGRGD (VGVPG) ₆ ] ₂₀ WPC multiblock biopolymer (REP), which has been shown to be effective in tissue regeneration, has been known to be effective in tissue regeneration (Jeon et al, J Biomed Mater Res A, 97: 152, 2011; Patent No. 13500900).

The present inventors assumed that the REP scaffold plays a pivotal role in blocking the ruptured blood vessel and reducing the volume of the hematoma. Thus, the present inventors predicted that the ICH-induced proinflammatory response, such as the activation of immature microglia that change to the M1 phenotype, could be mitigated and predicted that the damaged blood vessel could be repaired.

To confirm this prediction, we measured the volume of the hematoma and analyzed the inflammatory response using immunohistochemistry. The present inventors have found that administration of REP decreases the volume of hematoma, decreases IgG leakage into the substantia nigra, weakens the activity of microglial cell immune response, and induces von Willebrand factor (vWF) expression after ICH And the present invention has been completed.

Accordingly, it is an object of the present invention to provide a pharmaceutical composition for preventing or treating symptoms caused by intracerebral hemorrhage (ICH) or intracerebral hemorrhage including REP.

The present invention also aims to provide a REP for restoring cerebral vascular rupture.

The present invention also provides a method for preventing or treating symptoms caused by intracerebral hemorrhage or intracerebral hemorrhage by administering REP to a patient suffering from intracerebral hemorrhage.

DISCLOSURE OF THE INVENTION The present invention has been made in order to solve the above problems, and it is an object of the present invention to provide a medicament for preventing or treating symptoms caused by intracerebral hemorrhage (ICH) or intracerebral hemorrhage including elastin-like polypeptide (RGD) A pharmaceutical composition is provided.

According to a preferred embodiment of the present invention, the RGD-containing elastin-like polypeptide may be of the sequence of the following structure:

[constitutional formula]

TGPG [VGRGD (VGVPG) ₆ ] _n WPC

In the above formula, n is 10, 12, 15 or 20.

According to another preferred embodiment of the present invention, the symptom caused by intracerebral hemorrhage may be any one selected from the group consisting of hemorrhagic stroke, cerebral hemorrhage, vascular rupture, hematoma expansion, and inflammation induced by intracerebral hemorrhage .

In another preferred embodiment of the present invention, the intracerebral hemorrhage is selected from the group consisting of brain, frontal lobe, parietal lobe, temporal lobe, cerebellum, brainstem, midbrain, larynx, water quality, pituitary, hypothalamus, , And a brain region selected from the group consisting of < RTI ID = 0.0 >

According to another preferred embodiment of the present invention, the cerebral hemorrhage may be any one selected from the group consisting of hypertensive intracerebral hemorrhage, cerebral aneurysm, and subarachnoid hemorrhage.

According to another preferred embodiment of the present invention, the RGD-containing elastin-like polypeptide is useful for reducing hematoma volume, decreasing IgG leakage to the substantia nigra, weakening the activity of microglial cell immune response and von Willebrand factor (vWF) Or a decrease in the expression of the gene of the present invention.

The present invention also provides an elastin-like polypeptide for restoring cerebral vascular rupture consisting of a sequence of the following structure:

[constitutional formula]

TGPG [VGRGD (VGVPG) ₆ ] _n WPC

In the above formula, n is 10, 12, 15 or 20.

According to a preferred embodiment of the present invention, the elastin-like polypeptide can be applied to cerebral vessels ruptured in the form of hydrogel, microfibers and cell sheets.

The present invention also provides a method for preventing or treating symptoms caused by intracerebral hemorrhage or intracerebral hemorrhage by administering REP to a patient suffering from intracerebral hemorrhage.

The REP of the present invention reduces the volume of hematoma caused by ICH, reduces IgG leakage into the substantia nigra, weakens the activity of microglial cell immune response, decreases the expression of von Willebrand factor (vWF) after ICH Thereby preventing the ruptured blood vessels and reducing the volume of the hematomas. Therefore, it can be usefully used for the prevention or treatment of ICH and the symptoms caused therefrom.

FIG. 1A is a graph showing changes in hematoma volume with time, FIG. 1B is an image showing ICH alone, ICH + PBS, ICH + RGD, (N = 7-8 / group, * p <0.05, ** p <0.01, ***) in the ICH + REP group compared to the ICH + ELP p <0.001, Tukey's post-hoc test).
FIG. 2 shows the results of the FAM-labeled REP that was specifically targeted after the cell adhesion assay and intracerebral hemorrhage (ICH). The REP was located in the ipsilateral site target lesion and enhanced cell adhesion after injection. Figure 2A is an image showing that crystal violet stained cells were attached to plates coded with different concentrations of REP, and the images were taken after 1 hour of attachment. FIG. 2B is a graph comparing cell adhesion according to REP dose. FIG. Fig. 2C is an image showing that the fluorescence-tagged REP is specifically observed in the ipsilateral region of the ICH + FAM-REP group (green). Fluorescence was not observed in the ICH alone group (negative control) and in the contralateral region of the ICH + FAM-REP group. PECAM was used as an endothelial cell marker. The nucleus was stained with DAPI (* p <0.05, ** p <0.01, *** p <0.001, scale bar = 100 μm, Dunnett's multiple comparison test).
FIG. 3 shows 2,3,5-triphenyl-2H-tetrazolium chloride (TTC) staining, and TTC staining was performed at 3, 6 and 24 hours after REP administration in a normal control (Cont). There was no ischemic infarction in the Cont + REP group (low magnification = 10 ×, high magnification = 20 ×, scale bar = 2 mm)
FIG. 4 shows the percent of positive-IgG region of the real lesion, FIGS. 4A-4C show immunohistochemistry showing positive regions of IgG in the parenchymal lesion, and FIG. 4D shows IgG expression at all time points (N = 5 / group, * p <0.05, ** p <0.01, *** p <0.001, scale bar = 100 μM, Tukey's post-hoc test).
Figure 5 shows immunohistochemical staining showing vWF expression over time after ICH, and Figures 5A-5C show immunohistochemistry for vWF. Expression of the vWF group was significantly reduced at all time points in the ICH + REP group compared to the ICH alone and the ICH + PBS group. Figure 5D is a graph showing that the vWF expression is significantly reduced in the ICH + REP group compared to ICH alone and ICH + PBS group (n = 6 / group, * p <0.05, ** p <0.01, *** p <0.001, scale bar = 100 μm, Tukey's post-hoc test).
Figure 6 shows the percentage of Iba1-positive microglia cells in real lesions after ICH, Figures 6A-6C are immunohistochemistry showing Iba1-positive microglia cells in substantive lesions, Figure 6D shows the percentage of Iba1- (N = 5 / group, * p &lt; 0.05, ** p &lt; 0.05) in the ICH + PBS group compared to the ICH alone and the ICH + PBS group at all time points <0.01, *** p <0.001, scale bar = 100 μm, Tukey's post-hoc test).

Hereinafter, the present invention will be described in more detail.

As mentioned above, many clinical trials have been conducted regarding ICH, but new therapeutic strategies for treating ICH are still lacking.

Accordingly, the present inventors sought a solution to the above-mentioned problem by providing a REP scaffold that can prevent or treat symptoms caused by intracerebral hemorrhage (ICH) or intracerebral hemorrhage. The REP of the present invention reduces the volume of hematoma caused by ICH, reduces IgG leakage into the substantive brain, weakens the activity of the microglial cell immune response, decreases the expression of von Willebrand factor (vWF) after ICH Thereby preventing the ruptured blood vessels and reducing the volume of the hematomas. Therefore, it can be usefully used for the prevention or treatment of ICH and the symptoms caused therefrom.

Accordingly, the present invention provides a pharmaceutical composition for preventing or treating symptoms caused by intracerebral hemorrhage (ICH) or intracerebral hemorrhage comprising an elastin-like polypeptide (REP) containing RGD (ArgGlyAsp).

According to a preferred embodiment of the present invention, the RGD-containing elastin-like polypeptide may be of the sequence of the following structure:

[constitutional formula]

TGPG [VGRGD (VGVPG) ₆ ] _n WPC

In the above formula, n is 10, 12, 15 or 20.

Intracerebral hemorrhage (ICH) is a form of stroke that causes bleeding into the brain tissue (parenchyma) due to the breakdown of the damaged or injured blood vessels in the brain. Brain damage and consequent neurological damage or death lead to different mechanisms . For example, blood leaks are thought to reduce oxygenation to the brain cells supplied by the affected blood vessels. Also, blood collection can cause edema, and finally, It is also considered to be able to apply pressure. Both edema and hematoma can increase intracranial pressure. Other mechanisms of brain injury caused by ICH are also possible. Specific mechanisms or mechanisms that cause brain damage after ICH in general or in special cases Are not intended to limit the embodiments of the disclosure.

ICH can occur in any part of the brain. Some of the areas of the brain that can be affected by ICH, which can occur in other parts of the brain, include the brain (including frontal lobe, parietal lobe, occipital lobe, temporal lobe), cerebellum, brainstem (including midbrain, , The hypothalamus, the sagittal, the hippocampus, the amygdala, the electric field, or the basal ganglia.

ICH can occur as a result of bleeding from the brain arteries. Representative cerebral arteries include the anterior cerebral artery (ACA), the middle cerebral artery (MCA), the posterior cerebral artery (PCA), the ocular artery, the circle of Willis artery, the posterior subcortical artery (PICA) But is not limited thereto.

Certain symptoms indicative of increased intracranial pressure, such as headache, nausea, vomiting and loss of consciousness may imply the presence of ICH, but definitive diagnosis requires brain imaging. Brain imaging Techniques include computed tomography (CT) scans, angiography, CT angiography, magnetic resonance imaging (MRI), and MR angiography. Other techniques for definitive diagnosis of ICH will be familiar to those of ordinary skill in the art.

Although ICH is less common than ischemic stroke caused by clotting of blood vessels in the brain due to thrombosis, it constitutes about 12% of the total stroke. Unlike ischemic stroke, ICH is particularly associated with hematoma expansion , And are significantly more likely to worsen within the first 24 hours after bleeding begins. Hematoma expansion is associated with worsening patient outcomes.

In the pharmaceutical composition of the present invention, the symptom caused by intracerebral hemorrhage may be any one selected from the group consisting of hemorrhagic stroke, cerebral hemorrhage, vascular rupture, hematoma expansion, and inflammation induced by intracerebral hemorrhage, But is not limited to, and may include all symptoms caused by intracerebral hemorrhage, as is commonly known in the art. The symptoms caused by intracerebral hemorrhage may be any one selected from the group consisting of hemorrhagic stroke, cerebral hemorrhage, vascular rupture, hematoma expansion, and inflammation induced by intracerebral hemorrhage.

In the pharmaceutical composition of the present invention, the RGD-containing elastin-like polypeptide (REP) may be contained in a therapeutically effective amount. In the present invention, the term "therapeutically effective amount" means that when administered to a subject afflicted with ICH, it results in a reduction in the likelihood that the subject will die as a result of ICH, or a decrease in morbidity caused by ICH, Refers to the amount of REP that results in a reduction in neurological damage caused or that results in an improvement in the function, quality of life, or condition of the subject after ICH. The therapeutically effective amount of REP can also be used to prevent or reduce the degree of volume expansion of the hematoma caused by ICH after the onset of bleeding or the diagnosis of ICH, to prevent the expansion of bleeding-posterior edema caused by ICH Reduce its volume, or prevent or reduce an increase in intracranial pressure that may be involved in ICH. A therapeutically effective amount of REP may also delay, or alleviate, alleviate, or attenuate the onset of any of the physical symptoms, symptoms, physiological effects or neurological damage caused by ICH. The therapeutically effective amount of REP may vary depending on such factors as the nature of the specific REP to be used, the disease state of the subject to be treated, age, sex and weight, and the ability of the REP to elicit the desired response in the subject.

In the pharmaceutical composition of the present invention, the REP may also be included in a prophylactically effective amount. In the present invention, a "prophylactically effective amount" is defined as a dose that reduces the likelihood that a subject will become ICH when administered to a subject having one or more ICH risk factors, or reduces the incidence of ICH in a population of subjects having a similar risk profile REP. &Lt; / RTI &gt;

The efficacy and efficacy of REP can be determined based on observations of individual ICH subjects receiving the REP. The efficacy and effective amount of REP can also be determined by performing clinical studies that are regulated in a defined population of ICH subjects according to the knowledge of the ordinary skilled artisan. Based on the results of such clinical studies, the management provider is able to anticipate what is likely to be an effective amount of REP before experiencing a new ICH and administering it to a subject in need of treatment.

Desirably, in the presence of efficacy, a complete prevention or reversal of neurological impairment caused by physical signs or symptoms of ICH (including hematoma expansion, for example) or ICH is not required. Rather, as indicated above, the effective amount of REP only needs to improve the status of the ICH target relative to the status of the target in the absence of treatment with REP.

Non-limiting examples of such carriers, vehicles and other ingredients include, but are not limited to, solvents (e. G., Water, ethanol &lt; RTI ID = 0.0 &gt; (Eg, sucrose, dextrose, lactose), polyalcohols (such as glycerol, mannitol, and the like), saline, phosphate buffered saline), detergents, surfactants, dispersion media, coatings, antibacterial or antifungal agents, isotonic agents, , Sorbitol), salts (such as sodium chloride, potassium chloride), wetting agents, emulsifiers, preservatives, buffers, and agents capable of enhancing the stability or effectiveness of REP.

The pharmaceutical compositions for use in accordance with the disclosure may be in any suitable form for administration to a subject, such as a liquid solution (e.g., injectable and injectable). The pharmaceutical composition may be provided in a pre-mixed format, e.g., ready for administration to a subject in a vial or pre-filled syringe. Such a format does not require reconstitution with a diluent prior to administration. Alternatively, the composition may be presented in lyophilized form requiring reconstitution with a diluent (e. G., Sterile water or saline) prior to administration. In the latter case, the diluent may be provided in a separate container with the lyophilizate. Depending on the knowledge of the ordinarily skilled artisan, the composition may be formulated for storage under refrigeration or at room temperature. The form of the composition will depend, at least in part, on the intended mode of administration. In certain embodiments, the mode of administration is parenteral, including, for example, intravenous, subcutaneous, intraperitoneal, or intramuscular administration.

Other forms as well as parenteral compositions may be formulated in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form refers to physically discrete units adapted as unit dosages for the subject to be treated, each unit being combined with the required pharmaceutical carrier to provide a calculated amount of active compound, calculated to yield the desired therapeutic effect do. Therapeutic pharmaceutical compositions typically must be sterile and stable under the conditions of manufacture and storage.

The pharmaceutical compositions may be formulated as solutions, microemulsions, dispersions, liposomes, or other ordered structures suitable for high drug concentrations. The sterile injectable solution may be formulated with one or a combination of ingredients listed above as appropriate By introducing the required amount of REP into the solvent followed by filtration sterilization. In general, the dispersion is prepared by introducing the active compound into a sterile vehicle containing the basic dispersion medium and other necessary components belonging to those listed above. . In the case of sterile powders for the preparation of sterile injectable solutions, the preferred method of preparation is vacuum drying and freeze-drying, which yields powders of the active ingredient plus any additional desired ingredients from the previously sterile-filtered solution. The proper fluidity of the solution can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants. Prolonged absorption of the injectable composition may be brought about by including in the composition an agent that delays absorption, for example a monostearate salt and gelatin.

The present invention also provides an elastin-like polypeptide for restoring cerebral vascular rupture consisting of a sequence of the following structure:

[constitutional formula]

TGPG [VGRGD (VGVPG) ₆ ] _n WPC

In the above formula, n is 10, 12, 15 or 20.

The elastin-like polypeptides of the present invention can be applied to cerebral blood vessels ruptured in the form of hydrogel, microfibers and cell sheets, but they can be applied without restriction as long as they are suitable for cerebral blood vessel repair.

The present invention also provides a method for preventing or treating symptoms caused by intracerebral hemorrhage or intracerebral hemorrhage by administering REP to a patient suffering from intracerebral hemorrhage.

In the method of the present invention, the REP is the same as the REP included in the pharmaceutical composition, and thus its description of its structure is omitted.

After the initial bleeding caused by ICH, approximately 30% of ICH patients continue bleeding resulting in significant hematoma expansion. Unfortunately, hematoma expansion is associated with worsening patient outcomes. As a method for treating such an ICH patient sub-population, the REP can be administered at a dosage effective to reduce or even prevent hematoma expansion.

Various techniques for measuring hematoma volume when ICH first, and later, as it changes over time, are familiar to those of ordinary skill in the art. Non-limiting examples include brain imaging techniques such as CT, CTA, MRI or MRA. Specific methods include ABC / 2, ABC / 2 with adjusted C values, area measurement and 3D volume rendering. It is common to use computer software to calculate volume based on data collected during a brain scan.

Hematoma volume can be calculated based on data collected using primary brain scans used to establish baseline volume by definitively diagnosing ICH. The hematoma volume may additionally be determined at one or more subsequent scheduled times to monitor whether the hematoma volume is static or swollen, and if so, what rate it is inflated.

In the method of the present invention, the patient refers to a brain capable of undergoing intracerebral hemorrhage, or any animal having a coagulation system that can be activated using the REP of the present invention. In some embodiments, the animal is a mammal such as a human and a non-human primate (e.g., an ape, a monkey, a baboon, a chimpanzee), or another mammal such as a rodent , Dogs, pigs, rabbits, goats, deer, sheep, ferrets, gerbils, guinea pigs, hamsters and bats. Other animals that conform to the knowledge of the ordinary artisan are also possible.

Various human patient risk factors have been identified that contribute to increased risk for ICH. Age is the most important risk factor, and the proportion of ICH increases rapidly in individuals aged over 60 years. Thus, in some methods of the invention, the REP can be administered to patients over 60 years of age to treat or prevent ICH.

On the other hand, hypertension is the most important interchangeable ICH risk factor present in ICH patients in excess of 70%. Untreated hypertension is a greater risk factor than hypertension controlled by therapy, and hypertensive patients under treatment with interrupted medication have a greater risk than those who maintain it. Hypertension is a risk factor for lobar ICH, as well as bleeding in the deep hemisphere and brainstem regions. The relative impact of hypertension as an ICH risk factor is greater in younger patients than in older patients. Thus, in some methods of the invention, the REP can be administered to hypertensive patients to treat or prevent ICH.

The REP should be administered to the patient as soon as possible after the onset of the ICH symptoms or diagnosis of ICH. As mentioned herein, ICH can be suspected at the onset of certain symptoms and can be confirmed by neuroimaging, such as MRI or CT scanning. According to a particular method of the invention, the REP is administered to the patient within 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 hour after the onset of the ICH symptom. In another embodiment, the REP is administered to the patient within 8, 7, 6, 5, 4, 3, 2, 1 hour or less after neurological imaging or other techniques familiar to the ordinarily skilled artisan.

It is well within the knowledge of the ordinary skilled artisan to achieve a target plasma concentration of REP sufficient to treat or prevent ICH. In a non-limiting example, after an estimate of relevant pharmacokinetic parameters, such as the patient's plasma volume or other parameters, is calculated based on the patient's sex, height and weight, or other factors, how much Can be used to calculate whether REP needs to be administered. After administration of REP, plasma concentrations can be monitored according to the knowledge of the ordinary skilled artisan, and such information is used to maintain the concentration in any desired range.

The dosage regimen may be adjusted to provide the optimal desired response (e.g., treatment or prophylactic response). For example, a single transient scan may be administered, or several divided doses may be administered over time, or the dose may be proportionally reduced or increased as indicated by the requirements of the treatment situation.

The dosage value will depend on factors such as the specific REP used and whether it is used alone or in combination with other therapeutic agents, the particular characteristics of the population to be treated, the sub-population or individual subject, and the intended specific therapeutic or prophylactic effect Can vary. Those of ordinary skill in the art will know of other factors that may affect dosage values. In addition, for any particular patient, the specific dosage regimen may need to be adjusted according to the needs of the individual over time and the professional judgment of the management provider administering the REP or supervising its administration. Accordingly, the dosage ranges set forth herein are exemplary only and are not intended to limit the scope or practice of the claimed pharmaceutical composition or method of use.

REP can be administered to a patient, for example, as a composition containing the same, until the appropriate therapeutic or prophylactic effect is achieved with one or more rounds of recovery. Where multiple administrations are used, they may be administered hourly, daily, or at any other suitable interval, including for example a number of daily doses. The REP may also be administered sequentially via, for example, a mini-pump. FXa variants can be administered, for example, via a parenteral route (e.g., intravenously, subcutaneously, intraperitoneally or intramuscularly). REP can generally be administered as part of a pharmaceutical composition as described herein.

In the method of the present invention, when REP is co-administered with one or more second therapeutic agents, it can be administered simultaneously in the same composition, or simultaneously or sequentially in separate compositions. Alternatively, the REP and one or more second therapeutic agents may be administered at different times in separate compositions. The composition comprising REP comprises one species

May be administered before or after the composition comprising the second therapeutic agent. The interval between successive administrations may vary depending on the administration of the REP and the second therapeutic agent, which occurs on the same day in certain embodiments. Combination therapies may be administered hourly, daily or multiple times per week.

In the present invention, the administration of REP reduced hematoma volume, prevented the blood component from leaking into the parenchyma, and weakened the migration of activated microglial cells to the area surrounding the hematoma. In addition, vWF expression levels in the ICH + REP group were significantly reduced compared to the other groups. These results were associated with a reduction in ICH damage, including inflammatory reactions. Previous discoveries have shown that biosynthetic extracellular matrix (ECM) obtained from animal tissue exhibits biological problems such as low attachment, migration, proliferation and differentiation of transplanted cells or surrounding cells. In addition, immune reaction problems such as cytotoxicity were not completely solved. Genetic engineering biopolymers, such as ELP, made by human tropoelastin, are considered new biomaterials for the biomedical field. ELP has several promising properties for biological applications: biocompatibility, biodegradability and non-immunogenicity. Above all, the ELP responds to thermal changes through a soluble-insoluble phase transition. As a result, ELP is at temperatures above T t by the gel (gel) type, is shifting in the sol (sol) type at a temperature of less than T t. Some researchers have used these ELP characteristics for drug and gene delivery in oncology. Although ELPs with low immunogenicity and cytotoxicity can be controlled at the gene level with high affinity for living cells, there is a limit to the ability of these compounds to interact with cells. Indeed, ELP exhibits reduced adhesion properties and limited ability to retain cell growth. In the present invention, administration of ELP (V140) alone after ICH slightly decreased the volume of the hematoma but did not significantly decrease the volume of the hematoma in a time-dependent manner. In particular, the effect of ELP on hematoma volume reduction was not observed at 48 hours after injury.

Recently, TGPG [VGRGD (VGVPG) ₆ ] ₂₀ WPC (REP) in which a biosynthetic extracellular matrix, a cell-adhesive integrin Arg-Gly-Asp (RGD) ligand and an elastin-derived VGVPG pentapeptide are fused has been reported . Previous studies have shown that adhesion and proliferation of neural progenitor cells (NPCs) were significantly up-regulated by REP on poorly adherent organic scaffolds. To improve the effect of ELP on ICH damage, ELP (V140) -based recombinant biomaterial REP was used in the present invention and we investigated the effect of ELP (V140) -based recombinant biomaterial after ICH. REP administration was significantly higher than that of ICH alone (16.7%), ICH + PBS (13.7%), ICH + RGD (15.6%) and ICH + ELP The volume was significantly reduced by 9.1%. These results suggest that REP treatment effectively reduces hematoma volume after collagenase-induced ICH injury. In addition, the amount of IgG leakage after ICH was significantly reduced in ICH + REP group compared to ICH alone and ICH + PBS group. These results indicate that injected heat-sensitive and solubilized REP can form self-assembled coacervate at temperatures above T t and reposition to hemorrhagic lesions to cover ruptured cerebral blood vessels.

In addition, the number of microinvasive cells activated in the parenchyma decreased in the ICH + REP group compared to ICH alone and the ICH + PBS group. The predominant CNS macrophages, or microglia, are known to be immune cells involved in the inflammatory response. These cells are transformed into an activated form upon invasion of microorganisms or other molecular signals. Activated microglial cells migrate to the hemorrhagic region by chemotaxis to remove blood components and other debris. Thus, these results indicate that REP prevents pressure-induced damage of the surrounding tissue caused by bleeding and leakage of blood components from injured blood vessels. As a result, the inflammatory response (i. E., Macrophage entry into hemorrhagic lesions) is reduced.

In addition, self-assembled REP injected into the brain did not block any intact cerebral blood vessels in the normal control group. Inadequate blood supply or occlusion of cerebral blood vessels leads to brain ischemia. The TTC staining results indicate that REP injected into rICA becomes self-assembled gel type at temperatures above T t. However, it did not induce infarction.

As mentioned above, vWF, a 600,000-D to 20,000,000-D glycoprotein secreted by endothelial cells, is an essential co-factor associated with the coagulation pathway and plays an important role in promoting platelet adhesion to subcutaneous collagen. In the coagulation process, vWF acts like a carpet between exposed endothelial cells and activated platelets. In addition, vWF with the RGD sequence binds to the platelet binding site. Indeed, it has been reported that endothelial cells are activated and serum levels of vWF are increased in traumatic brain injury. The present inventors anticipate that REP implanted with rICA will play an important role, such as a carpet, similar to that performed by vWF in injured cerebral vessels, thereby reducing vWF expression as well as reducing bleeding in the early stages of ICH Respectively. Consistent with this expectation, the present invention confirmed that the expression of vWF after ICH was significantly reduced in the REP treatment group compared to ICH alone and the ICH + PBS group.

In addition, the FAM-labeled REP was specifically located in the parenchymal parenchyma surrounding the ipsilateral hematoma. Hyperthermic reactions are spontaneously induced by ischemic or hemorrhagic stroke as well as responses to inflammation that can be caused by microbial infection. The present inventors have demonstrated that the injected FAM-labeled REP molecules accumulate mainly in the ipsilateral ruptured blood vessels, but not in the majority region. Additionally, unlike the prediction that REP will specifically fill the ruptured portion of the blood vessel after ICH, the modified FAM-labeled REP was found to cover cells located in the peri-hematoma lesions in vivo. This phenomenon is presumed to be caused by the adhesiveness of REP. Finally, in the present invention, it was found that REP improves endothelial cell adhesion through cell attachment assays.

Recently, the inventors have found that wounds filled with REP aggregates promote host cell migration from wound edges, while enhancing the survival of transplanted adipose stem cells (ASC) through cell attachment. For a better understanding of the mechanisms underlying the effects of REP, we measured the activity status of kinases that play important roles in Fak, Src, Erk, and Akt signaling kinases, i.e., cell adhesion, migration, and survival . Indeed, this mechanistic investigation suggests that the effect of REP is mediated through the activation of Fak / Src signaling and upregulation of Mek / Erk and PI3K / Akt survival pathways. Therefore, the present inventors predicted that REP would be able to exhibit its positive effects through activation of the same pathway.

In conclusion, the present invention demonstrates that administration of biosynthetic RGD motif-based RE through rICA specifically over ruptured cerebral blood vessels. This positive action reduces hematoma volume, prevents leaking of excess blood components from the blood vessels, weakens the activity of microglial cells, and reduces the expression of vWF. Therefore, REP can be used as a potential therapeutic agent for treating acute phase ICH due to its non-immunogenicity, biocompatibility, biodegradability and various positive effects demonstrated in the present invention.

Hereinafter, the present invention will be described in more detail with reference to Examples. It is to be understood by those skilled in the art that these examples are for illustrative purposes only and that the scope of the present invention is not construed as being limited by these examples.

[ Preparation Example ]

1-1. animal

All animal care and surgical procedures were approved by YLARC's IACUC. 8-week-old male Sprague-Daw (SD) rats (250-260 g) were purchased from Orient (Gyeonggi-do, Korea) and housed in a temperature and humidity controlled sterile (SPF) zone under a 12 hour light period.

1-2. In the brain  Bleeding ( ICH ) Model

All rats were anesthetized with an intraperitoneal (i.p.) injection of a mixture of Zorret (100 mg / kg, Hurst, USA) and xylazine (rumun, 10 mg / kg, Bayer, Germany). The body temperature of the rats was maintained at 36.5 캜 to 37.5 캜 using an exothermic pad and monitored using a rectal probe. The rats were placed in a stereotactic apparatus and a 30 gauge needle was implanted into the striatum (whole: 0.2 mm, side: 3 mm, abdomen: 6 mm). After collagenase type VII (0.5 units, 2 μL) was injected for 10 minutes, the syringe was slowly removed from the skull (ie, 10 minutes). After the operation, the hole was closed with a beeswax (Ethicon, USA).

1-3. REP and 5- Carboxyfluorescein - Preparation and Characterization of Labeled REP (FAM-REP)

Procedures necessary for the synthesis of REP have been reported in the prior art. In summary, ELP was expressed and purified in E. coli BLR (DE3) by reverse cycling. The content of repeatable integrin-bound AspGlyAsp (RGD) peptide and VG (VGVPG) domains was calculated using Compute pI / MW. The purified ELP (V140) -RGD peptide fusion protein (REP) was dissolved in cold PBS (pH 7.2, Gibco, USA). To characterize the thermal changes, the temperature and absorbance change at 350 nm were monitored using Cary Win-UV software. T t is defined as the temperature at which 50% of the maximum turbidity is observed.

The preparation and properties of FAM-REP have been reported in the prior art. To summarize, 5-carboxyfluorescein N-succinimidyl ester (5.0 μmol) dissolved in dimethylsulfoxide (580 μL) to bind 5-carboxyfluorescein (FAM) to the N- Was added to REP (0.97 [mu] mol) dissolved in tetrahydrofuran (20 mL). The mixture was reacted at room temperature for 3 hours to produce FAM-labeled REP. The FAM-REP was purified by repeating the reverse phase transfer of 4 ° C cooling and heating at 45 ° C three times. The extent of FAM labeling was measured according to the protocol of the AnaTag (TM) protein label kit (AnaSpec). T t is the temperature at which the solubilized REP forms highly hydrated hydrogel-like aggregates. The inverse T t of REP was determined by the concentration of REP.

The present inventors have reported that the transition curves of the three REP solutions are characterized by dose-dependent (20, 50, and 100 μM). When the temperature of the solution increased from 15 ℃ to 35 ℃, all three solutions exhibited an increase in absorbance due to the aggregation of REP. T t values were measured at 29.7, 25.9, and 25.3 ° C for REP of 20, 50, and 100 μM, respectively. Because these T t values were lower than the mouse body temperature, the REP solution was self-assembled spontaneously into gel-like aggregates after topical loading into the damaged space.

[ Experimental Example]

1) Experimental method

1-1. medication

All rats were re-anesthetized after ICH and incised neck along the midline. The right external carotid artery (rECA) and the right common carotid artery (rCCA) were permanently tied with a 4-0 canine suture. After arterial ligation, the same amount of REP (ICH + REP group, 100 μM / 100 μL, n = 40) dissolved in 100 μL of cold PBS (ICH + PBS group, n = 30) or cold PBS was injected into the right Were administered via the carotid artery (rICA).

1-2. Measurement of hematoma volume

For histological examination and volume determination of hematomas, the rats were sacrificed at 6, 24 or 48 hours after ICH. The rats were anesthetized by intraperitoneal (ip) injection of a mixture of joretyl (100 mg / kg) and xylazine (lummin, 10 mg / kg) and resuspended in PBS and 4% para And perfused with formaldehyde transcardially. After cardiac perfusion, the brain was removed and sliced with a 2 mm thick coronal section. Percentage of hematoma volume [(hematoma volume / hemispheric volume) × 100] was measured using Image J.

1-3. Immunohistochemistry

Fixed brain tissue was washed with PBS for 6 hours and stored in cryoprotectant (30% sucrose) for 3 days. Tissue was inserted into OCT compound and transferred to -80 ° C. Frozen sections (n = 6, per group) were sliced into sections of 10 μm thickness and air-dried at room temperature. For immunofluorescence analysis, sections (n = 5, per group) were fixed in cold methanol for 15 minutes at -20 &lt; 0 &gt; C. After fixation, they were blocked with donkey serum (1:10) for 1 hour at room temperature to prevent nonspecific binding. The following primary antibodies were used overnight at 4 ° C: polyclonal anti-rat IgG (1:50, Invitrogen, Carlsbad, CA, USA) for detection of IgG leaks in parenchyma, detection of activated microglia PECAM (1:50, Santa Cruz, Dallas, Texas, USA) for labeling endothelial cells (ECs) for anti-Iba1 (1: 200, Abcam, Cambridge, UK) Brant's factor (vWF, 1: 200, Abcam, Cambridge, UK).

The slides were washed 3 times in PBS and incubated with appropriate secondary antibodies conjugated with either rhodamine (1: 200, Millipore) or fluorescein isothiocyanate (FITC, 1: 200, Millipore) Lt; / RTI &gt; The slides were stained with 4,6-diamidino-2-phenylindole (DAPI, Vector, Burlingame, USA) and the nuclei were stained with contrast dye and fixed with a cover slip. All images were obtained with a confocal microscope (LSM700, Carl Zeiss, Thornwood, NY, USA).

1-4. 2,3,5- Triphenyl -2H- Tetrazolium  Chloride ( TTC ) dyeing

The rats were completely anesthetized and injected with REP via the intra-arterial route, the right internal carotid artery (rICA). Rats were euthanized at 3, 6, and 24 hours after REP administration. The brain was removed and cardiac perfusion with PBS was followed by slicing to 2 mm thickness, and the sections were transferred to a 2% TTC (Santa Cruz, Dallas, Texas, USA) solution at 37 ° C for 20 hours.

1-5. Cell adhesion Assay

96-well plates were coded with REP at different concentrations (0, 0.2, 0.6, 1.0 [mu] M) and incubated at 4 [deg.] C. After 12 hours of coating, the excess REP was removed and the wells were washed twice with 200 [mu] L PBS. Brain endothelial cells (b.End3 cells, 2 x 10 ⁵ in 200 μL DMEM) were seeded in each well and cultured for 1 hour. After removing the culture medium, the wells were washed twice rapidly with 200 μL of cold PBS to remove unattached cells. Then, to fix the cells, the wells were treated with ice-cold 200 μL of 100% methanol at 25 ° C for 10 minutes. After fixing, the wells were washed three times with PBS and incubated for 20 min at 25 DEG C with addition of 100 [mu] L of crystal violet solution (0.5% w / v crystal violet in 20% ethanol). After incubation, the wells were washed three times with PBS to remove excess crystal violet. The stained cells were recovered by adding 200 [mu] L of 100% methanol to each well and gently shaking the plate at 25 [deg.] C for 10 minutes. Finally, the extracted crystal violet was measured at 590 nm using a microplate reader (Multiskan, Thermoscienfic, USA).

1-6. Statistical processing

All statistical analyzes were performed using SPSS 18.0 software (IBM Corp., Armonk, NY, USA). Data are expressed as mean ± standard error of the mean (SEM). Differences between the experimental groups were compared using ANOVA. Tukey's post hoc multiple comparison test was used for additional group comparisons. Differences were considered statistically significant at P <0.05 (* p <0.05, ** p <0.01, *** p <0.001).

2) Results

2-1. ICH  Analysis of post-hematoma volume

To assess the volume of the hematoma, different groups of rats were sacrificed at 6, 24 or 48 hours after collagenase-induced intracerebral hemorrhage (Figure 1A). At 6 hours, the ICH + REP group (13.9%), ICH + PBS (13.2%), ICH + RGD (10.1%) and ICH + ELP (V140) 8.1%). The percentage value represents the volume of the hematoma [(hematoma volume / hemispheric volume) x 100]. However, there was no statistical difference between ICH + ELP (V140) group and ICH + REP group at 6 hours. ICH + PBS (13.6%), ICH + RGD (14%), and ICH + ELP (V140) were the only patients with ICH + 10.6%) compared to the control group. In addition, ICH + ICH + RGD (15.6%), and ICH + ELP (V140) (13.9%) compared with ICH alone (16.7%), ICH + PBS And decreased progressively in the REP group (9.1%) (Fig. 1B). Taken together, REP treatment significantly reduces the hematoma volume after collagenase-induced ICH injury.

2-2. Improved brain endothelial cells (b. End3  Cell) attachment and REP location

During hemorrhage following traumatic injury, endothelial cells surrounding the lumen of the blood vessels are exposed. The present inventors hypothesized that REP infusion via rICA would attach to endothelial cells. Thus, the exposed lesion will be covered with REP. Cell attachment assays using crystal violet were performed to investigate whether REP induced adhesion of b.End3 cells (Figs. 2A and 2B). b. End3 cells were inoculated on plates coated dose-dependently with REP (0.2, 0.6, 1 [mu] M). After one hour incubation, the percentage of cells attached to the well containing REP was about 141%, 246%, and 334%, compared to the control (0 [mu] M, PBS). The b.End3 cells cultured on 1 μM REP-coated plates were about 2.3 times higher than the control plates. Next, to confirm that the REP administered via arterial injection reaches the target lesion, FAM-labeled REP is injected into rICA after ICH and the brain slice is stained at a temperature higher than T t (25 ° C) Respectively. Since the main feature of the REP is that it can undergo thermal induced phase transformation, a few variations have been applied to the following procedure. The inventors detected FAM-tagged REP in the ipsilateral but not in the majority of the brain obtained from the FAM-labeled REP group. FAM-like fluorescence (i.e., fictitious-positive signals) was not observed in the ICH single group. FAM-labeled REP changed its physical properties from liquid to gel-like aggregates upon in vivo administration. The gel-like FAM-labeled REP was able to attach to bone marrow cells and specifically blocked ruptured blood vessels (Fig. 2C). Thus, these results indicate that REP promotes endothelial cell adhesion and is specifically located at lesion sites. These data support the results of TCC staining, indicating that REP treatment does not cause infarction of healthy microvessels.

2-3. The relationship between uninjured microvascular occlusion and REP administration

2,3,5-Triphenyl-2H-tetrazolium chloride (TTC) staining is commonly used to assess cellular respiration, allowing differentiation between metabolic activity and inactive tissue. Active tissues are stained dark red, whereas inactive tissues are white. TTC staining was used to determine whether REP aggregates injected into the right internal carotid artery (rICA) specifically targeted lesions without occluding other intact microvascular vessels. Three control rats were sacrificed at 3, 6 or 24 hours after administration of REP without ICH (Cont + REP). No ischemic infarction following REP administration was observed in the group (Fig. 3). These results indicate that heat-sensitive and reversible REPs are biologically stable in vivo by maintaining microvessels intact without infarction despite environmental changes.

2-4. ICH  Peripheral blood component into posterior parenchyma IgG  Protein leakage

On the basis of in vivo results indicating that the volume of the hematoma is reduced after REP administration, the present inventors confirmed the degree of blood vessel leakage of IgG, which is a constituent of plasma, into a lesion in the striatum through the ruptured cerebral blood vessels Immunohistochemistry was performed. In ICH alone and ICH + PBS group, IgG was highly expressed at all time points (Figs. 4A to 4C) compared with ICH + REP group (ICH alone and ICH + PBS treatment 20.3% The relative proportion of IgG-positive areas in the ICH + REP group (10.2%) was less than about half that of the control group. Data also showed that the degree of IgG leakage in the ICH + REP group (8.3%) was significantly reduced compared to ICH alone (29.1%) and ICH + PBS (25.0%) at 24 hours after ICH. In addition, the relative expression of IgG-positive regions in ICH alone, ICH + PBS and ICH + REP groups was approximately 23.5%, 20.5%, and 9.0%, respectively. These values were ~ 2-fold lower than ICH alone and ICH + PBS group at 48 hours after ICH (Figure 4D). This data indicates that REP prevents the blood component from leaking to the brain from ruptured blood vessels after ICH.

2-5. ICH  Of the cerebral blood vessels Integrity  change

To evaluate the warming of cerebral blood vessels, we used anti-von Willebrand factor (vWF) antibodies and anti-platelet-endothelial cell adhesion molecule (PECAM) antibodies as markers for blood vessels to detect damaged cerebral blood vessels And immunohistochemistry was performed. The multiple binding glycoprotein, vWF, plays an important role in the coagulation system and consists of multiple subunits produced by endothelial cells and megakaryocytes. In addition, molecules of vWF are stored in the alpha-granules of endothelial cells and platelets. vWF is necessary for platelets to attach to subcutaneous collagen exposed in the small arteries. Upon bleeding caused by traumatic injury, vWF provides a receptor site for platelets and collagen. The primary receptor site replaces the exposed intravascular subcutaneous collagen known as the innermost vascular lining site and binds to the surface receptors of platelets and glycoproteins Ib / IX / V. Activated platelets then bind and attach vWF. Larger vWF multimers form a flocked carpet that platelets aggregate. vWF expression was significantly increased in ICH alone and in the ICH + PBS group compared to the Cont and ICH + REP groups at 6 hours after ICH (FIGS. 5A and 5D). In addition, vWF was highly expressed in ICH alone and in the ICH + PBS group compared to the Cont and ICH + REP groups at 24 hours after ICH (FIGS. 5B and D). In addition, vWF expression was substantially increased after 48 hours in the ICH alone and in the ICH + PBS group, respectively (Figs. 5C and 5D), as compared to the Cont and ICH + REP groups. These data indicate that REP replaces the role of vWF to fill exposed subcutaneous collagen sites after ICH injury, thereby enhancing vascular integrity and reducing vWF overexpression.

2-6. ICH  Microglia activation after injury

Immature microglia that interact with neurons in the central nervous system (CNS) play an important role similar to macrophages that fight against peripheral viral, bacterial and fungal infections. Endogenous microglia become stimulated by a molecular cue to become an activated form that acts as a macrophage. Surprisingly, microglial activation has been considered detrimental to the treatment of many neurological disorders. To detect activated microglia cells after ICH, we used an anti-ionizing calcium binding adapter molecule 1 (Iba1) antibody that specifically detects activated microglia cells. To test whether REP treatment of traumatic brain injury results in a negative effect such as an immune response, the present inventors performed immunohistochemistry specific to activated microglia. We have found that Iba1-positive cells progressively increase in all groups. However, the relative number of Iba1-positive cells was significantly reduced in the ICH + REP group compared to the other groups. At 6 hours, the percentage of Iba1-positive microglia in the ICH alone, ICH + PBS and ICH + REP groups was 24.6%, 30.0%, and 9.3%, respectively (Figs. 6A and 6D). Also, at 24 hours after ICH, the number of Iba1-positive cells in the ICH + REP group was significantly less than the number of cells observed in the ICH alone and in the ICH + PBS group. The percentage of Iba1-positive cells was 11%, 36.3%, and 37.2% in ICH + REP, ICH alone and ICH + PBS group, respectively (FIGS. 3B and 3D). Furthermore, at 48 hours after ICH, more Iba1-positive cells were detected in the ICH alone (47.9%) and ICH + PBS (37.8%) group than the ICH + REP group (17.6%) (Figs. 6C and 6D). These data suggest that the administration of REP improves the activation of microglial cells resulting from immune responses associated with detrimental effects caused by activated microglia after ICH.

Claims (8)

A pharmaceutical composition for preventing or treating symptoms caused by intracerebral hemorrhage (ICH) or intracerebral hemorrhage comprising RGD (ArgGlyAsp) -containing elastin-like polypeptide (REP). The pharmaceutical composition according to claim 1, wherein the RGD-containing elastin-like polypeptide has a sequence of the following structural formula:
[constitutional formula]
TGPG [VGRGD (VGVPG) ₆ ] _n WPC
In the above formula, n is 10, 12, 15 or 20. The method according to claim 1, wherein the intracerebral hemorrhage is selected from the group consisting of cerebral, frontal, parietal, occipital, temporal, cerebellum, brainstem, midbrain, gait, water, pituitary, hypothalamus, sagittal, hippocampus, amygdala, (ICH) or intracerebral hemorrhage, which is characterized in that it occurs in any one or more brain regions. The method according to claim 1, wherein the symptom caused by intracerebral hemorrhage is selected from the group consisting of hemorrhagic stroke, cerebral hemorrhage, vascular rupture, hematoma expansion, and inflammation induced by intracerebral hemorrhage. A pharmaceutical composition for preventing or treating symptoms caused thereby. The method according to claim 4, wherein the cerebral hemorrhage is one or more selected from the group consisting of hypertensive intracerebral hemorrhage, cerebral aneurysm, and subarachnoid hemorrhage. A pharmaceutical composition for preventing or treating the symptoms caused. 2. The method of claim 1, wherein the RGD-containing elastin-like polypeptide has reduced hematoma volume, decreased IgG leakage to the brain, decreased activity of microglial cell immune response, and decreased expression of von Willebrand factor (vWF) after intracerebral hemorrhage Wherein the compound exhibits at least one of the effects selected from the group consisting of the compound of the formula (I) and the compound of the formula (II). An elastin-like polypeptide for restoring cerebral vascular rupture consisting of a sequence of the following structure:
[constitutional formula]
TGPG [VGRGD (VGVPG) ₆ ] _n WPC
In the above formula, n is 10, 12, 15 or 20. 7. The elastin-like polypeptide according to claim 6, wherein the elastin-like polypeptide is applied to cerebral blood vessels ruptured in the form of hydrogel, microfibers and cell sheets.

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