KR101532211B1 - Pharmaceutical composition and method for treating stoke based on the inhibition of AMPK - Google Patents

Abstract

Provided is a composition for treating stroke which treats stroke by processing a novel compound suppressing zinc neurotoxic AMPK activation which is one of the main causes of stroke. According to an embodiment of the present invention, the composition contains a compound having a chemical formula in the specification as an active ingredient.

Description

[0001] The present invention relates to a pharmaceutical composition and a method for treating a stroke based on AMPK inhibitory function,

The present invention relates to a pharmaceutical composition and method for the treatment of stroke, and more particularly, to a pharmaceutical composition and method for treating a stroke based on AMPK inhibitory function.

Stroke is a term referring to local neurological symptoms caused by sudden onset of cerebral blood flow abnormalities. The brain occupies only 2% of the body weight by weight, but the blood flow to the brain is 15% of the cardiac output and the oxygen consumption is 20% of the total body oxygen consumption. Moreover, since the brain uses only glucose as an energy source, necrosis easily occurs even if the energy supply is interrupted for a while. Thus, cerebral blood flow abnormalities are closely related to brain damage. Brain damage caused by stroke includes various toxic mechanisms such as excitotoxicity, oxidative stress, apoptosis and zinc neurotoxicity, and therefore, it is effective for various neurotoxicity to prevent brain damage Drug discovery is needed.

Currently, many studies have been conducted for the treatment of stroke, but no clear treatment has yet been developed. Cerebral ischemia-reperfusion injury requires an objective evaluation system such as an in vitro model or an animal model for development and evaluation of drugs based on various complicated neuronal cell death mechanisms, but a method for evaluating changes in other vital signs or side effects in drug therapy This preclinical study is very limited and is likely to cause failure due to side effects in clinical trials. In particular, many clinical studies have been conducted centering on NMDA antagonists, but all have failed.

Korean Patent Laid-Open Publication No. 1283416 discloses a neuroprotective method for administering an AMPK inhibitor compound C or an FAS inhibitor C75 to an ischemic mouse to significantly reduce the size of the infarction portion so that the function after stroke or ischemic injury is preserved.

However, the above-described prior arts have only proved the protective effects of neurons against ischemic model animals, and do not guarantee the therapeutic effect against stroke caused by other mechanisms than ischemia. It is an object of the present invention to provide a novel therapeutic agent for stroke, which is based on AMPK inhibitory function, which is effective for various strokes by various mechanisms, to solve various problems including the above problems. However, these problems are exemplary and do not limit the scope of the present invention.

 According to one aspect of the present invention, there is provided a pharmaceutical composition for treating stroke, which comprises, as an active ingredient, a compound having the following structural formula:

.

.

According to another aspect of the present invention there is provided a method of treating a stroke comprising administering to a subject suffering from stroke a compound having the structure:

.

.

According to one embodiment of the present invention as described above, a new compound that inhibits AMPK activity of zinc neurotoxicity, which is a major cause of stroke, can be treated to achieve the therapeutic effect of stroke. Of course, the scope of the present invention is not limited by these effects.

FIG. 1 shows the cytotoxicity of TUNEL staining and post-treatment of Compound C (+ Cpd C), which is an AMPK inhibitor, by TUNEL staining and LDH assay, showing that neurotoxicity after zinc treatment was increased in cultured cortical neurons Which is a quantified graph (B and C).
FIG. 2 is a Western blot photograph (A) and enzyme activity assay (B) graph showing that AMPK activity is increased after zinc treatment in cultured cortical nerve cells.
FIG. 3 shows Western blot photographs (A) showing the increase of Bim expression and caspase-3 activity after zinc treatment in cultured cerebral cortical neurons, and Bim expression after treatment with Compound C (+ Cpd C) And caspase-3 activity were all decreased (Fig.
Figure 4 shows the effect of the enzyme activity on the AMPK activity assay kit (CycLex, Japan) and recombinant AMPK (α2 / β1 / γ1; CycLex, Japan) Of the candidate compounds.
FIG. 5 is a graph showing various neurotoxicities induced in cultured cerebral cortical neurons, followed by treating seven selected compounds, and quantifying the level of apoptosis through LDH assay and observing inhibition of apoptosis.
FIG. 6 is a graph showing the effect of inhibiting brain injury by administering drug # 35 finally selected in an animal model of stroke, and comparing the degree of brain damage in experimental animals with that of the control group.
FIG. 7 is a graph showing the result of acute toxicity test by administering the finally selected drug # 35 to rats and extracting spleen (A), liver (B) and kidney (C).

Definition of Terms:

As used herein, "AMP-activated protein kinase (AMPK)" is a heterotrimeric protein consisting of a catalytic α subunit (α1 or α2) and two regulatory subunits (β and γ). AMPK is phosphorylated and activated when cell energy level is low, and regulates cellular metabolism and regulates gene expression over a long period of time to restore ATP levels. It is known that an increase in AMP / ATP ratio, a change in cell pH and redox state, and an increase in creatine / phosphocreatine ratio activate AMPK.

DETAILED DESCRIPTION OF THE INVENTION [

According to one aspect of the present invention, there is provided a pharmaceutical composition for treating stroke, which comprises, as an active ingredient, a compound having the following structural formula:

.

.

The compound IUPAC designation is N- [2- [1H-indol-3-yl) ethylamino] -2-oxoethyl] -3-phenyl-2,1-benzoxazole-5-carboxamide, AMP-activated protein kinase) enzyme activity to provide a neuroprotective effect. The AMPK enzyme has kinase enzyme activity of the? 2 subunit.

The stroke may be a hemorrhagic stroke, an ischemic stroke, and a metal toxicity stroke.

The metal may be selected from the group consisting of lead, mercury, manganese, arsenic, thallium, iron, zinc, cadmium, bismuth or tin tin).

The ischemic stroke may be a stroke caused by excitatory neuronal cell death or oxidative neuronal cell death. The most widely known mechanisms of neuronal death due to cerebral ischemia are two. One is the accumulation of excess glutamate outside the cell by cerebral ischemia. This glutamate enters the cell and eventually causes excessive accumulation of intracellular calcium, resulting in neuronal death (Kang TC, et al. J. Neurocytol ., 30: 945-955, 2001) and the other is the oxidative neuronal cell cycle that is caused by damage to DNA and cytoplasm due to an increase of in vivo radicals due to abrupt oxygen supply during ischemia-reperfusion Won MH, et al, BrainRes, 836: 70-78, 1999; Sub AY, Chen YM, J. Biomed Sci, 5:....... 401-414, 1998; Flowers F, Zimmerman JJ New Horiz. 6: 169-180,1998).

According to another aspect of the present invention there is provided a method of treating a stroke comprising administering to a subject suffering from stroke a compound having the structure:

.

.

Such administration may be parenteral administration, selected from the group consisting of oral or intravenous infusion, subcutaneous infusion, intracerebroventricular injection, intracerebrospinal fluid injection, intramuscular injection and transdermal absorption.

The effective amount of the compound in the pharmaceutical composition of the present invention may vary depending on the kind of the affected part of the patient, the application site, the number of treatment, the treatment time, the formulation, the condition of the patient, The amount to be used is not particularly limited, but may be 0.01 μg / kg / day to 10 mg / kg / day. The above-mentioned daily dose may be administered once a day or two or three times a day at appropriate intervals, or intermittently administered at intervals of several days.

In the pharmaceutical composition of the present invention, the compound may be contained in an amount of 0.1 to 100% by weight based on the total weight of the composition. The pharmaceutical compositions of the present invention may further comprise suitable carriers, excipients and diluents conventionally used in the manufacture of pharmaceutical compositions. In addition, solid pharmaceutical preparations or liquid pharmaceutical preparations can be used for the preparation of pharmaceutical compositions. The preparation additive may be either organic or inorganic.

Examples of excipients include lactose, sucrose, saccharose, glucose, cornstarch, starch, talc, sorbit, crystalline cellulose, dextrin, kaolin, calcium carbonate and silicon dioxide. Examples of the binder include polyvinyl alcohol, polyvinyl ether, ethylcellulose, methylcellulose, gum arabic, tragacanth, gelatin, shellac, hydroxypropylcellulose, hydroxypropylmethylcellulose, calcium citrate, Dextrin and pectin, and the like. Examples of the lubricant include magnesium stearate, talc, polyethylene glycol, silica, and hydrogenated vegetable oil. Any coloring agent may be used as long as it is usually allowed to be added to pharmaceuticals. These tablets and granules can be suitably coated with sugar (sugar coating), gelatin coating, and others as required. If necessary, preservatives, antioxidants and the like may be added.

The pharmaceutical composition of the present invention can be prepared by any of the formulations conventionally produced in the art (for example, Remington's Pharmaceutical Science (latest edition; Mack Publishing Company, Easton PA), the form of the formulation is not particularly limited . These formulations are generally known prescription Seo literature ^{[Remington's Pharmaceutical Science, 15 th} Edition, 1975, Mack Publishing Company, Easton, Pennsylvania 18042 all Agency: is described in (Chapter 87 Blaug, Seymour).

In the pharmaceutical composition of the present invention, the compound can be administered orally or parenterally. Preferably, the compound is administered by intravenous injection, subcutaneous injection, intracerebroventricular injection, intracerebrospinal fluid injection, , Intramuscular injection, and intraperitoneal injection.

Hereinafter, the present invention will be described in more detail by way of examples. It should be understood, however, that the invention is not limited to the disclosed embodiments, but may be embodied in many different forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete, Is provided to fully inform the user.

General method

Culture of Cerebral Cortex Neurons

The mouse cerebral cortical neurons used in the present invention were extracted from the brain of mouse embryos and cultured in Dulbecco's modified Eagle's medium (DMEM, Gibco, Grand Island, NY) supplemented with 5% fetal bovine serum (FBS) and 5% , US) at 95% humidity, 5% CO ₂ and 37 ° C. The cells were grown at a density of 2 × 10 ⁴ cells in 24-well tissue culture plates for activation and differentiation and cultured in DMEM medium without FBS and HS before zinc and compound treatment.

Example 1: Confirmation of reduction of cytotoxicity by AMPK inhibitor

Zinc toxicity is one of the major causative mechanisms of stroke, and zinc toxicity induced by the cultured cerebral cortical neurons has been confirmed to be reduced by treatment with AMPK inhibitor.

Specifically, the cultured cerebral cortical neurons according to an embodiment of the present invention were treated with 300 μM zinc (ZnCl ₂ ) for 10 minutes and then removed for 10 hours. Cytotoxicity was measured by TUNEL staining or Lactate Dehydrogenase (LDH) ) Analysis. In addition, the cells were treated with 20 μM of Compound C (+ Cpd C, Tocris), which is an AMPK inhibitor, and the decrease of neurotoxicity was observed.

As a result, it was confirmed by TUNEL staining that neurotoxicity after zinc treatment was increased in cerebral cortical neurons (FIG. 1A). The degree of cytotoxicity was quantitated by TUNEL staining and LDH analysis and treated with Compound C (+ Cpd C) Lt; RTI ID = 0.0 > 1B < / RTI > and 1C).

Example 2: AMPK activity after zinc treatment

In order to investigate the relationship between zinc toxicity and AMPK enzyme activity, AMPK activity after zinc treatment in cerebral cortical neurons was examined by western blot analysis and enzyme activity assay.

2-1: Western blot

Cultured cerebral cortical neurons were treated with 300 μM zinc (ZnCl ₂ ) for 10 minutes and then removed. After 0.5, 1, 2, 4, and 6 hours, the cell sample was loaded onto a polyacrylamide gel with protein leather, And were separated according to their size. The antibody was then treated, washed and read.

As a result, phosphorylation of AMPK alpha-1 and alpha-2 threonine residues was observed 30 minutes after zinc treatment, indicating AMPK activity (phosphorylated AMPK). However, no phosphorylation of other serine residues was observed (Figure 2A).

2-2: Enzyme activity assay

Protein extracts were obtained at 0.5, 1, 2, 4, and 6 hours after treatment of zinc on cortical neurons under the same conditions as above, and enzyme activity was measured using the AMPK activity assay kit (CycLex, Japan) .

As a result, it was observed that the activity of AMPK enzyme by zinc treatment increased in a time-dependent manner (Fig. 2B).

Example 3: Inhibition of zinc toxicity by inhibiting AMPK activity

We examined whether the increase of AMPK enzyme activity by treatment of zinc in cerebral cortical neurons is related to apoptosis.

Specifically, 300 μM zinc (ZnCl ₂ ) was treated for 2, 3, 4, 5 and 6 hours in cerebral cortical neurons to induce zinc toxicity. Proteins of each sample were separated and subjected to Western blotting. The relationship between zinc toxicity and apoptosis was confirmed by treating 20 μM of AMPK inhibitor Compound C (+ Cpd C).

As a result, the expression of Bim, a pro-apoptotic protein, which is one of BH3-only Bcl family, was observed and caspase-3 activity was observed 3 hours after zinc treatment (FIG. 3A). However, it was confirmed that the increase of Bim due to zinc toxicity and the increase of caspase-3 activity were all reduced by the treatment with compound C as an AMPK inhibitor (FIG. 3B). Therefore, AMPK enzyme is involved in apoptosis in zinc toxin mechanism, and inhibition of AMPK enzyme activity inhibits zinc poisoning by apoptosis.

Example 4: Screening and inhibitory effect of AMPK activity inhibitor compound

4-1: Structure-based virtual screening

First screening was performed on compounds that might act as inhibitors of AMPK activity.

Specifically, AMPK activity was found to be related to the zinc toxin mechanism through the preceding studies, and other studies also reported increased AMPK activity and Bim expression in the excitotoxic mechanism. The AMPK enzyme is an enzyme in which the α, β and γ subunits function as a complex structure, and the alpha subunit has a kinase enzyme activity. Previous studies have shown that AMPK alpha2 significantly inhibits ischemia-induced neurotoxicity in knock-out mice, unlike alpha1. Accordingly, the present invention selected candidate chemicals capable of inhibiting AMPK enzyme activity targeting alpha2 through structure-based virtual screening, and consequently 208 candidate compounds were selected.

4-2: Analysis of AMPK enzyme activity (AMPK enzyme activity assay)

208 compounds selected in Example 4-1 were obtained from a compound library manufacturer (Interbioscreen, Russia) and subjected to secondary screening by observing their inhibitory effect on AMPK enzyme activity.

Specifically, the AMPK enzyme activity was measured using the AMPK activity assay kit (CycLex, Japan) and recombinant AMPK (α2 / β1 / γ1; CycLex, Japan). As a result of measuring the inhibitory effect of the above-mentioned 208 compounds and the well-known AMPK inhibitor compound C on the AMPK enzyme activity, it was confirmed that 40 drugs showed similar or better inhibitory effects to compound C at 10 μM (Table 1 ). The 40 drugs are shown in Table 1 below.

Compound ID Mean
(% remaining activity) SEM Compound ID Mean
(% remaining activity) SEM #One -1.2626 1.768 # 22 -0.5291 0.000 #2 -1.7934 0.072 # 23 1.0582 0.000 # 3 -0.9684 0.036 # 24 5.0505 5.051 #4 5.5556 0.265 # 25 -0.1793 0.538 # 5 4.2328 0.529 # 26 -1.2554 0.036 # 6 2.3810 0.794 # 27 5.8201 0.000 # 7 3.4392 0.265 # 28 3.9683 1.852 #8 -0.2152 0.215 # 29 0.0000 0.000 # 9 0.5739 0.359 # 30 1.0000 0.667 # 10 -0.3945 0.179 # 31 0.3641 0.850 # 11 5.2910 0.529 # 32 0.1257 0.126 # 12 -1.3245 0.662 # 33 2.9801 2.980 # 13 -0.9684 0.681 # 34 0.3143 0.566 # 14 -0.1435 0.000 # 35 0.1214 0.121 # 15 5.0265 0.265 # 36 0.2514 0.126 # 16 6.3492 0.529 # 37 7.3333 1,000 # 17 2.9101 0.265 # 38 5.6667 4.667 # 18 2.9053 0.681 # 39 1.667 3.333 # 19 4.3400 1.614 # 40 3.6667 0.000 # 20 -1.7575 0.036 Compound C 5.3000 0.760 # 21 0.8967 0.251

4-3: Observation of inhibition effect on zinc toxicity

The tertiary screening was carried out by observing the inhibitory effect on the zinc neurotoxicity of 40 compounds selected in Example 4-2.

Specifically, 400 μM of zinc was cocultured to the cerebral cortical neurons of the cultured mouse for 10 minutes, and after 12 hours, the selected 40 drugs were treated with zinc to observe the neuroprotective effect. The degree of death of the cells was quantitated by LDH analysis. Each effect was averaged and expressed as a numerical value. Separate experiments were performed on each of the selected drugs four times (FIG. 4).

As a result, as shown in FIG. 4, some compounds inhibited cell death due to zinc toxicity. As shown in Table 2, seven compounds (# 6, # 11, # 14, # 17, # 28, # 35 and # 37) significantly inhibited zinc toxicity.

Compound ID valence Compound ID valence #One 0.5487 # 22 0.0200 #2 0.8483 # 23 0.8757 # 3 0.5597 # 24 0.8699 #4 0.0283 # 25 0.4870 # 5 0.9326 # 26 0.6624 # 6 0.0278 # 27 0.3030 # 7 0.0003 # 28 0.0008 #8 0.0001 # 29 0.1137 # 9 0.8649 # 30 0.2142 # 10 0.1253 # 31 0.1777 # 11 0.0311 # 32 0.7276 # 12 0.0000 # 33 0.2386 # 13 0.4384 # 34 0.1043 # 14 0.0220 # 35 0.0016 # 15 0.2378 # 36 0.0381 # 16 0.2488 # 37 0.0290 # 17 0.0048 # 38 0.3225 # 18 0.3961 # 39 0.0027 # 19 0.7590 # 40 0.0390 # 20 0.8889 Compound C 0.0003 # 21 0.6141

4-4: Observation of inhibitory effect on various neurotoxicity

 Four-way screening was carried out by observing the neurotoxic inhibitory effect of the seven compounds selected in Example 4-3.

Specifically, in the cerebral cortical neurons of the cultured mouse, 50 μM of NMDA, 50 μM of FeCl ₂ , 100 μM of H ₂ O ₂ , 2 μM of TPEN (N, N ', N'-tetrakis (2-pyridylmethyl) ethylenediamine, Sigma), 500 nM staurosporine (Abchem) and 10 μM etoposide (Sigma), respectively, to induce cytotoxicity. The 7 selected compounds were treated at a concentration of 20 μM to inhibit apoptosis The degree of apoptosis was quantitated by LDH assay. The neurotoxicity model used includes excitotoxicity, oxidative damage and apoptosis, which are thought to be causative factors of stroke. Of these, NMDA was treated as an excitotoxic model and iron as an oxidative damage model. Toxicity and H ₂ O ₂ toxicity models were used, and TPEN, staurosporine and etoposide toxicity models were used as apoptosis models. The TPEN is a zinc chelator and is known to cause typical apoptosis in neurons. Stressorin is also a kinase inhibitor and is one of the typical cell death inducers. It is a drug known to cause apoptosis due to damage.

As a result, the seven selected drugs were treated together to observe the neuroprotective effect according to the reduction of neurotoxicity. In particular, Compound # 35 and Compound # 28 showed inhibitory effects on all toxicity (FIG. 5). Compound # 35 did not inhibit only the cytosolic toxin in the cell death model, but it was confirmed to be effective in the other two cell death models related to the toxin mechanism induced by the stresporin. Therefore, the compound was selected as the final compound . The compound # 35 was obtained from the place where the library was purchased. The compound # 35 was synthesized from N- [2- [2- (1H-indol-3-yl) ethylamino] -2-oxoethyl] -carboxamide: < / RTI >

.

.

Example 5 Observation of Brain Damage Inhibitory Effect in Stroke Animal Model

To determine whether the compound # 35, which was finally selected in Example 4-4, was also effective in an animal model, the compound was treated with stroke model animals that caused brain damage, .

Specifically, we used a male Sprague-Dawley (SD) rat, 8-9 weeks of age, to construct a permanent middle cerebral artery (MCA) occlusion model. Cerebral blood flow (CBF) was measured using laser-Doppler flowmetry at 30 minutes before and 10 minutes after closure of permanent middle cerebral artery (MCAO) and is shown in Table 3 below. Thereafter, the final selected AMPK inhibitor candidate compound, # 35 (100 ng in 4 μl) or vehicle (10% DMSO) was injected at 0.8 mm back to 3.8 mm depth in 15 minutes before MCAO and 1.2 mm Intracerebroventricular injection was performed. Thereafter, the degree of motor deficit of the rat was assessed. The criteria for evaluation (Longa et al., 1989) were normal (no deficiency), the failure of paw extension was mild mild, and moderate and loss of circling in the opposite direction, or severe deficit in the correct reflex action. Brain was obtained at 24 hours after induction of ischemia in the rat model, and cerebral infarction was measured by staining with 2% 2,3,5-triphenyl tetrazolium chloride (TTC).

As a result, it was finally confirmed that Compound # 35, the final candidate compound discovered through the present invention, markedly inhibited brain damage caused by permanent middle cerebral artery occlusion, which is one of the animal models of stroke (Figure 6 ).

Veh AMPK inhibitor CBF (% of baseline) 36.9 + 2.5 38.4 + 2.4 Weight reduction (g) 48.8 + 2.9 40.0 + 3.5

Example 6: Acute toxicity test < RTI ID = 0.0 >

The final selected compound, # 35, was injected into the rats and the acute toxicity results were observed.

Specifically, Compound # 35, the final selected AMPK inhibitor candidate compound, was injected intraperitoneally (100 μg / kg or vehicle (10% DMSO) per rat in 8-9 week old male Sprague-Dawley intravenous injection) and repeated 4 times.

As a result, no rat was found to die even after lapse of 24 hours, and liver, spleen and kidney organs were sacrificed and their weights were measured. WBC, RBC, BUN, AST, ALT, CREA GLU, etc., but most of the indicators were normal and no noticeable toxicity was observed compared to the control group (FIG. 7). The results for the index are shown in Table 4 below.

Normal range Veh (n = 4) AMPK inhibitor (n = 4) WBCB (x10 ³ cell / uL) 6.6 - 12.6 10.5 ㅁ 3.5 9.9 ㅁ 2.5 RBC (x10 < ³ > cell / uL) 6.8 - 9.8 6.7 ㅁ 0.5 6.4 ㅁ 0.3 BUN (mg / dL) 5 - 21 16.9 ㅁ 2.0 15.2 ㅁ 1.8 AST (U / L) 45.7 - 80.8 64.2 ㅁ 5.0 91.2 ㅁ 15.3 ALT (U / L) 17.5 - 30.2 38.9 ㅁ 0.4 40.2 ㅁ 5.0 CREA (mg / dL) 0.2 - 0.8 0.4 ㅁ 0.1 0.4 ㅁ 0.0 GLU (mg / dL) 50 - 135 175.5 ㅁ 26.5 162.1 ㅁ 13.2

  Taken together, these results indicate that AMPK enzyme plays an important role in zinc toxicity, which is considered to be one of the causative factors of stroke, and has been subjected to screening several times in new candidate compounds having the function of inhibiting AMPK enzyme activity, The compound of the present invention has been shown to be effective as a novel stroke drug based on the AMPK inhibitory function.

While the present invention has been particularly shown and described with reference to exemplary embodiments thereof, it is to be understood that the invention is not limited to the disclosed embodiments, but, on the contrary, is intended to cover various modifications and equivalent arrangements included within the spirit and scope of the appended claims. Accordingly, the true scope of the present invention should be determined by the technical idea of the appended claims.

Claims (6)

A pharmaceutical composition for the treatment of stroke comprising a compound having the following structural formula as an active ingredient:

. The method according to claim 1,
Wherein the stroke is a hemorrhagic stroke, an ischemic stroke or a metal toxicity stroke. 3. The method of claim 2,
The metal may be selected from the group consisting of lead, mercury, manganese, arsenic, thallium, iron, zinc, cadmium, bismuth or tin tin. < / RTI > 3. The method of claim 2,
Wherein said ischemic stroke is caused by excitatory neuronal cell death or oxidative neuronal cell death. A method for treating a stroke in a mammal other than a human, comprising administering a compound having the following structural formula to a mammalian subject other than a human suffering from stroke:

. 6. The method of claim 5,
Such administration may be by parenteral administration selected from the group consisting of oral or intravenous infusion, subcutaneous infusion, intramuscular injection, intracerebroventricular injection, intracerebrospinal fluid injection, intramuscular injection, and percutaneous absorption A method for treating stroke in a mammal other than a human.

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