KR101936836B1 - Pharmaceutical composition for prevention and treatment of ischemic brain disease - Google Patents

Abstract

The present invention relates to a therapeutic use of ischemic brain disease of zatebradine as a hyperpolarization-activated cyclic nucleotide-gated channel (HCN) channel inhibitor. According to the present invention, zatebradine as HCN channel inhibitor, can effectively inhibit nerve cell damage caused by ischemia reperfusion, by blocking hyperexcitability of a nerve cell during reperfusion after ischemic stroke. Therefore, the present invention can be useful for preventing, alleviating and treating ischemic brain disease including ischemic stroke.

Description

[0001] The present invention relates to a pharmaceutical composition for prevention and treatment of ischemic brain diseases,

The present invention relates to the use of Zatebradine as an HCN channel inhibitor for treating ischemic brain diseases.

Stroke is the third most common cause of death with cancer and cardiovascular disease. It is the most frequent among the diseases that invade the nervous system, and its incidence is gradually increasing.

Stroke is usually divided into two types: ischemic stroke caused by inability of blood to pass through the cerebral blood vessels and hemorrhagic stroke due to cerebral hemorrhage. Among them, the incidence of ischemic stroke is higher. Especially, acute ischemic stroke is a dangerous disease that can die within a few hours.

Ischemic stroke, which is caused by blockage of blood flow by thrombus or embolization, is more common in about 80% of stroke patients. When ischemic stroke causes blood circulation to cease and blood circulation resumes, cell and tissue damage occurs, which is called 'ischemic reperfusion injury'.

The cause of ischemic reperfusion injury is sudden increase of blood flow in the oxygen deficiency state and sudden increase of intracellular calcium concentration. Increased intracellular calcium causes damage to the mitochondria, resulting in active oxygen, resulting in inflammatory reactions leading to cell damage. Excitotoxicity is one of the causes of increased calcium in the cells. The excitotoxicity is that nerve cells are excessively excited, and the nerve cell toxicity is increased by the increase of calcium influx of nerve cells due to hyperactivity of nerve cells.

Methods for controlling such reperfusion injury include regulating the rate of reperfusion, hypothermia, and drug therapy. Ischemic reperfusion injury appears to be greater with faster blood flow. Therefore, in order to prevent sudden re-supply of blood, blood is slowly taken in. The heart also undergoes ischemic postconditioning, which protects against reperfusion injury by repeated preperfusion and reperfusion for a short time before reperfusion, but the effect on the prognosis of the patient is still insufficient. The clinical efficacy of hypothermic treatment was also confirmed in patients who were initially treated with acute ischemic stroke by Ajou University Hospital and Chungnam University Hospital. Hypothermia was addressed in the hypothermic treatment of patients with cardiac arrest. In patients with severe stroke, hypothermia was performed after intravascular reoprosthesion to reduce reperfusion injury, thus confirming intravascular therapy and neuroprotective effects. In addition, studies have shown that when anticipated ischemic reperfusion injury, antioxidants or immunosuppressants can be administered in advance to prevent them. However, antioxidants are limited due to their toxicity, and some drugs such as cyclosporine, a type of immunosuppressant, are undergoing clinical studies.

In addition, it is known that neuroprotective agents administered within 6 hours after the stroke can reduce neuronal cell death due to hypoxia induced by stroke. However, within 3 hours immediately after the stroke, t-PA (tissuetype plasminogen activator has been used to restore blood flow, there has been no clinically effective neuroprotective agent for treating cerebral ischemia.

As a result of repeated studies to develop a therapeutic agent for a new ischemic brain disease, it has been found that Zatebradine, which is used as an inhibitor of hyperpolarization-activated cyclic nucleotide-gated channel (HCN) It is possible to inhibit neuronal cell damage due to reperfusion, thereby completing the present invention.

Korean Patent Publication No. 10-2016-0053027 Korean Patent No. 10-1645654

Accordingly, it is an object of the present invention to provide a pharmaceutical composition for preventing or treating ischemic brain diseases that prevents neuronal damage due to reperfusion by preventing overexcitation of neurons in reperfusion.

In order to achieve the above object,

The present invention provides a pharmaceutical composition for preventing or treating ischemic brain diseases containing an HCN (hyperpolarization-activated cyclic nucleotide gated) channel inhibitor as an active ingredient.

In one embodiment of the present invention, the HCN channel inhibitor may be Zatebradine.

In one embodiment of the present invention, the Zatebradine may be contained in the composition at a concentration of 0.1 to 1000 μM.

In one embodiment of the present invention, the Zatebradine may have an effect of preventing or treating ischemic brain disease through a mechanism of inhibiting neuronal cell damage due to reperfusion after ischemic stroke.

In one embodiment of the present invention, the disorder is selected from the group consisting of stroke, cerebral infarction, cerebral ischemia, thrombosis, em-bolism, transient ischemic attack, lacune, head trauma, cerebral circulatory metabolic disorder, brain functional coma, traumatic brain injury, and hypoxic brain injury.

As an HCN channel inhibitor according to the present invention, Zatebradine can effectively inhibit neuronal cell damage due to ischemic reperfusion by preventing hyperactivity of nerve cells generated during reperfusion after ischemic stroke. As a result, ischemic stroke And can be usefully used for prevention, improvement and treatment of diseases.

FIG. 1 is a graph showing the inhibitory effect of necrotic cells and excitations induced by reperfusion of the HCN channel inhibitor, Zatebradine. To induce reperfusion after ischemic stroke, hypoxic and reoxygenation were induced, As shown in FIG. '(-) Zatebradine' is the group not treated with zatebradine before induction of hypoxia, and '(+) Zatebradine' is treated with zatebradine before hypoxic induction.
FIG. 2 is a graph showing the inhibitory effects of the HCN channel inhibitor, ZD7288, on neuronal cells and excitability. To induce reperfusion after ischemic stroke, hypoxic and reoxygenation were induced, . '(-) ZD7288' is a group not treated with ZD7288 before induction of hypoxia, and '(+) ZD7288' is a group treated with ZD7288 before induction of hypoxic induction.
FIG. 3 shows the measurement of the activation of the HCN channel by reperfusion using the magnitude of the voltage sag when a negative current (-200 to -50 pA) is injected.
FIG. 4 is a schematic diagram showing the position of Zatebradine, which is an HCN channel inhibitor, in the process of neuronal damage during reperfusion after ischemic stroke. When ischemic stroke occurs and oxygen deficiency appears, reperfusion results in the damage of cells and tissues due to the increase of intracellular calcium concentration, mitochondria damage, increase of active oxygen and inflammation reaction sequentially. At this time, treatment of Zatebradine before reperfusion inhibits increase of intracellular calcium concentration by inhibiting the increase of excitability of neuron, thereby restricting reperfusion injury.

The present invention provides a pharmaceutical composition for the prevention or treatment of ischemic brain diseases containing an HCN (hyperpolarization-activated cyclic nucleotide gated) channel inhibitor as an active ingredient.

In the present invention, the 'HCN channel' is a hyperpolarization-activated cyclic nucleotide gated (HCN) channel, and is an ion channel distributed in the heart and brain nerve cells. This ion channel is generally activated when the membrane of the neuron is hyperpolarized, and the cation is opened into the cell, and the membrane is depolarized.

In one embodiment of the present invention, the HCN channel inhibitor may be Zatebradine.

Accordingly, the present invention may be a pharmaceutical composition for preventing or treating ischemic brain disease containing, as an active ingredient, a Zatebradine compound represented by the following formula (1) or a pharmaceutically acceptable salt thereof:

≪ Formula 1 >

Zatebradine of the present invention has the molecular formula C ₂₆ H ₃₆ N ₂ O ₅ and molecular weight of 456.579 as a known compound as a conventional HCN channel inhibitor.

In order to examine the possibility of zatebradine compound as a therapeutic agent for ischemic brain diseases, the present inventors have investigated the possibility of treating zygomycin-induced neurodegenerative diseases by using CA1 neurons in hippocampus in rat brain slices, As a result, it has the effect of inhibiting neurons and excitement by reperfusion after ischemic stroke, and thus it is useful as a drug, such as a pharmacological composition, as a substance useful for prevention, improvement or treatment of ischemic brain diseases For the first time.

Ischemic brain diseases that can be prevented or treated with the composition of the present invention include stroke, cerebral infarction, cerebral ischemia, thrombosis, em-bolism, transient ischemic attack, lacune, head trauma, brain circulatory metabolic disorders, brain function coma, traumatic brain injury, and hypoxic brain injury.

The compounds according to the invention can be used in the form of salts, preferably pharmaceutically acceptable salts. The salt is preferably an acid addition salt formed by a pharmaceutically acceptable free acid, and the free acid may be an organic acid or an inorganic acid. The organic acids include, but are not limited to, citric, acetic, lactic, tartaric, maleic, fumaric, formic, propionic, oxalic, trifluroacetic, benzoic, gluconic, methosulfonic, glycolic, succinic, Glutamic acid and aspartic acid. The inorganic acid includes, but is not limited to, hydrochloric acid, bromic acid, sulfuric acid, and phosphoric acid.

The Zatebradine according to the present invention may be a commercially available product or may be prepared by a chemical synthesis method known in the art.

The composition of the present invention is a pharmaceutical composition comprising the above-mentioned Zatebradine as an active ingredient. The composition of the present invention may be prepared by using pharmaceutically acceptable and physiologically acceptable adjuvants in addition to the active ingredients. , A disintegrating agent, a sweetening agent, a binder, a coating agent, a swelling agent, a lubricant, a lubricant or a flavoring agent.

The pharmaceutical composition may be formulated into a pharmaceutical composition containing at least one pharmaceutically acceptable carrier in addition to the above-described active ingredients for administration.

The pharmaceutical composition may be in the form of granules, powders, tablets, coated tablets, capsules, suppositories, liquids, syrups, juices, suspensions, emulsions, drops or injectable solutions. For example, for formulation into tablets or capsules, the active ingredient may be combined with an oral, non-toxic pharmaceutically acceptable inert carrier such as ethanol, glycerol, water, and the like. Also, if desired or necessary, suitable binders, lubricants, disintegrants and coloring agents may also be included as a mixture. Suitable binders include, but are not limited to, natural sugars such as starch, gelatin, glucose or beta-lactose, natural and synthetic gums such as corn sweeteners, acacia, tracker candles or sodium oleate, sodium stearate, magnesium stearate, sodium Benzoate, sodium acetate, sodium chloride, and the like. Disintegrants include, but are not limited to, starch, methyl cellulose, agar, bentonite, xanthan gum and the like. Acceptable pharmaceutical carriers for compositions that are formulated into a liquid solution include sterile solutions suitable for the living body such as saline, sterile water, Ringer's solution, buffered saline, albumin injection solution, dextrose solution, maltodextrin solution, glycerol, One or more of these components may be mixed and used. If necessary, other conventional additives such as an antioxidant, a buffer, and a bacteriostatic agent may be added. In addition, diluents, dispersants, surfactants, binders, and lubricants may be additionally added to formulate into injectable solutions, pills, capsules, granules or tablets such as aqueous solutions, suspensions, emulsions and the like. Further, it can be suitably formulated according to each disease or ingredient, using the method disclosed in Remington's Pharmaceutical Science, Mack Publishing Company, Easton PA as an appropriate method in the field.

In one embodiment of the present invention, the Zatebradine of the present invention may be contained in the composition at a concentration of 0.1 to 1000 μM, and the Zatebradine of the present invention may be contained at a concentration of 0.1 to 95 % ≪ / RTI > by weight.

The present invention also provides the use of a composition comprising zatebradine as an active ingredient for the manufacture of a medicament for the prevention and treatment of ischemic stroke. The composition of the present invention comprising Zatebradine as an active ingredient can be used for the preparation of a medicament for the prevention and treatment of ischemic stroke.

The present invention also provides a method of preventing and treating ischemic stroke comprising administering to a mammal Zatebradine. The administration can be done prior to reperfusion after ischemic stroke. For example, administration of Zatebradine within 6 hours after stroke can reduce the death of neurons by hypoxia induced by stroke.

The term "mammal " as used herein refers to a mammal that is the subject of treatment, observation or experimentation, preferably a human.

The term "therapeutically effective amount " as used herein refers to the amount of active ingredient or pharmaceutical composition that elicits a biological or medical response in a tissue system, animal or human, as contemplated by a researcher, veterinarian, The amount that induces the relief of the symptoms of the disease or disorder being treated. It will be apparent to those skilled in the art that the therapeutically effective dose and the number of administrations of the active ingredient of the present invention will vary depending on the desired effect. The optimal dosage to be administered can therefore be readily determined by those skilled in the art and will depend upon the nature of the disease, the severity of the disease, the amount of active and other ingredients contained in the composition, the type of formulation, and the age, , Sex and diet, time of administration, route of administration and rate of administration of the composition, duration of treatment, concurrent administration of the drug, and the like.

In the therapeutic method of the present invention, the composition comprising the zatebradine of the present invention as an active ingredient can be administered orally, rectally, intravenously, intraarterally, intraperitoneally, intramuscularly, intrastrally, transdermally, topically, Lt; RTI ID = 0.0 > route. ≪ / RTI >

The present invention also provides a neuroprotective agent comprising zatebradine as an active ingredient.

Hereinafter, the present invention will be described in more detail with reference to Examples. It will be apparent to those skilled in the art that these embodiments are merely illustrative of the present invention and that the scope of the present invention is not limited to these embodiments.

≪ Example 1 >

Inhibition of nerve cell damage by ischemic reperfusion of Zatebradine

In the present invention, in order to examine the inhibitory effects of Zetabradine and ZD7288, which are known as hyperpolarization-activated cyclic nucleotide gated (HCN) channel inhibitors, on ischemic reperfusion injury, Zdebradine 'and' ZD7288 'were pretreated, respectively, and the degree of nerve cell damage following induction of reoxygenation was measured. For reference, hypoxic and reoxygenation were induced to experimentally induce reperfusion after ischemic stroke.

In detail, electrophysiological experiments using hippocampal CA1 neurons were performed in rat brain slices. SD rats (12-21 days old) were anesthetized with carbon dioxide, and then brain slices were prepared at a thickness of 300 μm and cultured at 32 ° C for 30 minutes. The brain slices were placed in artificial cerebrospinal fluid bubbled in 95% O2, 5% CO ₂ and maintained in physiological response. To confirm excitability of neurons, whole-cell patch clamps were performed at 200 pA and 200 pA The electrical response was measured every 5 minutes by current injection for 600 ms while increasing the pA by 50 pA. The Axopatch 700B amplifier was used for the measurement. The patch electrode to be measured had a resistance value of 3-5 MΩ and filled with a solution having a composition similar to that of the intracellular solution. In order to experimentally induce ischemic reperfusion injury, brain sections were sequentially hypoxicized (using 8% O ₂ , 5% CO ₂ , 87% N ₂ ) and reoxygenation (95% O ₂ , 5% CO ₂ ) Nerve inflammation was induced together to produce a similar effect to the pathological response. At this time, before hypoxia induction, zeta bradine hydrochloride (Sigma, catalog #: Z0127) and ZD7288 (Tocris, catalog #: 1000) were treated with 10 μM and 20 μM concentration for 20 minutes or 10 minutes before induction of hypoxia , But not at the time of induction of hypoxia. The results were analyzed using IGOR Pro and excel software. The significance was confirmed by Student's t-test.

First, in order to confirm the excitability change of the neuron, the frequency of the action potential generated when the current of 100 pA was injected after the ischemic reperfusion induction was confirmed.

As a result, hypoxia decreased the excitability of nerve cells and increased excitability by re-digestion. It was confirmed that excitability was significantly higher than before hypoxia, and when Zatebradine was pretreated, It was confirmed that the excitability of the cells did not increase (see Fig. 1).

On the other hand, when ZD7288, a commonly used HCN channel inhibitor, was pretreated, it was found that unlike zatebradine, it did not inhibit excitability of neurons that were increased by reperfusion (see FIG. 2). In other words, it was confirmed that ZD7288 does not affect the increase of the activity voltage frequency of nerve cell unlike zetabradine.

These results demonstrate the usefulness of zatebradine as an inhibitor of ischemic reperfusion injury because it inhibits excessive excitement of nerve cells and inhibits cellular damage among HCN channel inhibitors.

On the other hand, as shown in FIG. 3, it was confirmed that the increase in excitability of nerve cells during reoxygenation was accompanied by an increase in excitatory current through the HCN channel. That is, it was confirmed that the voltage sag indicated by the HCN channel was decreased by hypoxia, while it was significantly increased by the reoxygenation. The activity of the HCN channel was measured using the magnitude of the voltage sag when a negative current (-200 to -50 pA) was injected.

The present invention has been described with reference to the preferred embodiments. It will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention as defined by the appended claims. Therefore, the disclosed embodiments should be considered in an illustrative rather than a restrictive sense. The scope of the present invention is defined by the appended claims rather than by the foregoing description, and all differences within the scope of equivalents thereof should be construed as being included in the present invention.

Claims (5)

A pharmaceutical composition for preventing or treating ischemic brain diseases containing zatebradine as an active ingredient. delete The method according to claim 1,
Wherein the Zatebradine is contained in the composition at a concentration of 0.1 to 1000 μM. The method according to claim 1,
Wherein the Zatebradine has an effect of preventing or treating ischemic brain disease through a mechanism of inhibiting neuronal damage caused by reperfusion after ischemic stroke. The method according to any one of claims 1 and 3 to 4,
The disorder is characterized by at least one of cerebral ischemia, stroke, cerebral ischemia, thrombosis, em-bolism, transient ischemic attack, lacune, head trauma, A cerebral function coma, a traumatic brain injury, and a hypoxic brain injury.

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