KR100407399B1 - Use of myricetin as an inhibitor for serotonin N-acetyltransferase - Google Patents

Abstract

The present invention, the mycetin as an active ingredient, regulates the amount of serotonin through the inhibition of serotonin N-acetyltransferase activity, and reduces the synthesis of N-acetylserotonin and melatonin acting as a neurotransmitter, thereby insomnia, parallax Useful as a treatment for patients with Jet-lag, night-shift work, delayed-sleep-phase syndrome, and patients with depression and seasonal affective disorders As an activity inhibitor of serotonin N-acetyltransferase that can be used

The myricetin (myricetin, 5, 7, 3 ', 4', 5'-pentahydroxyflavonol),

Characterized in that the formula.

Description

Use inhibitor of serotonin N-acetyltransferase {use of myricetin as an inhibitor for serotonin N-acetyltransferase}

The present invention relates to an activity inhibitor of serotonin N-acetyltransferase (AA-NAT), and more particularly, by using myricetin as an active ingredient, the amount of serotonin is regulated through inhibition of serotonin N-acetyltransferase activity. And reduce N-acetylserotonin and melatonin synthesis, which act as neurotransmitters, thereby reducing insomnia, jet-lag, night-shift work, and delayed-sleep-phase The present invention relates to an inhibitor of serotonin N-acetyltransferase activity, which can be usefully used as a therapeutic agent for patients with syndrome and patients with depression and seasonal affective disorder.

Generally, myricetin (5, 7, 3 ', 4', 5'-pentahydroxyflavonol) is a polyphenol with three rings and belongs to flavonols. It is used as an anti-tumor, anti-inflammatory drug that inhibits the growth of DNA and RNA in cancer, anti-inflammatory drug, and diuretic which shows strong diuretic effect by enhancing kidney and heart function. It is known to be used for such purposes.

The myricetin is the main ingredient of the medicinal herb bark, that is, bark of bark, medicinal bark is used as a stomatitis, blood pressure lowering drug, diuretic, converging branch drug, etc. However, myricetin acts as an inhibitor of serotonin N-acetyltransferase activity to treat various diseases related to serotonin or melatonin, such as insomnia, jet-lag, night-shift work, It has not been found that it can be used as a therapeutic agent for delayed-sleep-phase and premenstrual syndrome.

Melatonin is used to treat sleep disorders caused by jet lag, such as stressful sleep disorders or shift work, and sleep disorders experienced by travelers, that is, jet lag, and is used by night shift workers, including hospital practitioners and long distance drivers. Melatonin is used to alleviate visible sleep disorders, fatigue, digestive disorders, and cardiovascular disorders and to control sleep rhythms. This fact can be seen that by controlling the amount of melatonin can reduce the short-term sleep disorders and control the sleep rhythm. However, in patients with chronic sleep disorders and other psychological disorders such as primary sleep disorders that are seen in the elderly, they are not used for treatment because of the difficulty of taking large amounts of melatonin daily for a certain period of time (Kayumov L, Zhdanova). IV, Shapiro CM, 2000, Seminars in Clinical Neuropsychiatry, 5 (1): 44-55). In this case, if there is a substance that increases the activity of serotonin N-acetyltransferase and induces melatonin synthesis activation, it can be appropriately used.

On the other hand, if the amount of melatonin in the body is out of the normal range and continues at a high level, depression such as seasonal affective disorder occurs. This is because the shorter day length increases the amount of time a person spends in the dark and reduces the light signal to the eye, so that the enzyme activity of serotonin N-acetyltransferase remains high overall, resulting in higher concentrations of melatonin in the body. Turned out. This suggests that melatonin is closely related to emotion regulation, especially depression (Leibenluft E, 1998, Scientific American, 9: 31-35).

Premenstrual syndrome with symptoms such as anxiety, depression, sleep disorders, abdominal pain, anger, seizures, dizziness, fainting, suicidal thoughts, breast discomfort, increased water in tissues, slight weight gain, headache ) Is found in about 40-60% of women of childbearing age (20-30 years). In particular, about 3-5% of all women suffer from serious mental and emotional problems that occur periodically each month. Some cases have been reported with the administration of drugs such as Tricyclic antidepressants or Selective Serotonin Reuptake Inhibitors (Sundblad C, Hedberg MA, Eriksson E. 1993, Neuropsychopharmacology, 9: 133-145). In addition, among women with premenstrual syndrome, melatonin secretion was increased during the menstrual cycle, especially in the luteal phase (Shinohara K, Uchiyama M, Okawa M, Saito K, Kawaguchi M, Funabashi T, Kimura F, 2000). , Neuroscience Letters, 281: 159-162). These results suggest that serotonin and melatonin are closely related to premenstrual syndrome.

Serotonin and melatonin are also important neurotransmitters in the pathogenesis of depression. Low levels of serotonin in the synapses lead to depression, and it is known that serotonin and its metabolites are significantly lower in the brains of patients killed by suicides, and those with low serotonin metabolites in the cerebrospinal fluid are more likely to commit suicide. There is a report. Therefore, if there is a substance that inhibits the activity of serotonin N-acetyltransferase will be able to reduce the depressive effect by melatonin, and maximize the antidepressant effect by increasing the concentration of serotonin at the synapse.

The pineal gland is a neuroendocrine tissue located between two cerebral hemispheres, and serotonin made from tryptophan becomes N-acetylserotonin by serotonin N-acetyltransferase, which is melatonin by hydroxyindole-O-methyltransferase Is formed. In the pineal gland, the amount of serotonin at night is lower than during the day, whereas the amount of melatonin and serotonin N-acetyltransferase activity is more than 100 times higher. Increasing melatonin secretion and serotonin N-acetyltransferase activity activates adenylate cyclase by binding norepinephrine to adrenergic receptors, and increases the amount of cAMP, leading to transcription and translation of the serotonin N-acetyltransferase gene. Activating and maintaining enzyme activity (Klein DC, Coon SL, Roseboom PH, Weller JL, Bernard M, Gastel JA, Zatz M, Iuvone PM, Rodriguez IR, Begay V, Falcon J, Cahill GM, Cassone VM, Baler R, 1997, Recent Prog Horm Res. 52: 307-357). As such, the degree of serotonin and melatonin production in the pineal gland is regulated by serotonin N-acetyltransferase activity. Through these neurotransmitters, several functions of the central and peripheral nervous system, namely serotonin, are depressed, anxious, appetite, and obsessive thought. It is known that melatonin is involved in the regulation of sleep, mood, reproduction, cancer growth, aging, and the like, delusion, mood, arousal, aggressive disposition, pain, sleep, learning, memory, fear and appetite.

Efforts have been made to develop substances to treat various diseases through these neurotransmitters or substances acting on the hormones. For example, in the case of depression indicated by disorders of neurotransmitter systems such as serotonin and noradrenaline, the selection of monovalent amines Reuptake blockers, such as imipramine, fluorcetin, and desipramine, have been developed as antidepressants.

It is an object of the present invention to provide an activity inhibitor of serotonin N-acetyltransferase which specifically acts on serotonin N-acetyltransferase to inhibit its activity and reduce the secretion of melatonin.

Another object of the present invention is to provide an inhibitor of serotonin N-acetyltransferase activity, which can be used as a therapeutic agent for diseases related to serotonin N-acetyltransferase activity, in particular, depression, and has low side effects and excellent stability. It is.

1A is a graph showing that the activity of serotonin N-acetyltransferase is inhibited by the concentration of myricetin,

1B is a graph showing that the activity of serotonin N-acetyltransferase is inhibited according to the mycetin treatment time,

Figure 2a is a graph showing that myricetin acts as a non-competitive inhibitor for tryptamine, a substrate of serotonin N-acetyltransferase,

Figure 2b is a graph showing that myricetin acts as a non-competitive inhibitor for acetyl-CoA, a substrate of serotonin N-acetyltransferase,

3a is a graph showing that the mycetin is shortened in immobility time compared to the control group by the forced swimming test of the white paper,

Figure 3b is a graph showing that the swimming ability is increased in mycetin compared to the control group through the forced swimming test of the white paper.

According to a feature of the present invention for achieving the above object, serotonin N-acetyltransase using mycetin (5,7,3 ', 4', 5'-pentahydroxyflavonol) represented by the following formula as an active ingredient Inhibitors of serotonin N-acetyltransferase, which can be used as therapeutic agents for sleep and depression-related diseases by regulating the amount of serotonin through inhibition of parasease activity and by reducing N-acetylserotonin and melatonin synthesis, which act as neurotransmitters to provide.

Chemical formula.

At this time, according to an additional feature of the present invention, the sleep-related diseases are insomnia, hypersomnia, jet-lag, night-shift work, sleep-sleep- phase, advanced-sleep-phase syndrome, and the depression-related diseases include seasonal affective disorder, premenstrual syndrome, unipolar depression, manic-depressive psychosis, atypical Depression, etc.

In addition, according to another additional feature of the present invention, the 50% inhibition concentration (IC ₅₀ ) of mycetin is about 2.2 μM, and the effective amount of the compound of mycetin represented by the above formula is 0.01 μM to It is preferable that it is 1M.

The above objects and various advantages of the present invention will become more apparent from the preferred embodiments of the present invention described below with reference to the accompanying drawings by those skilled in the art.

Hereinafter, a preferred embodiment according to the present invention will be described in detail with reference to the accompanying drawings.

First, the present inventors have studied to find a new compound that specifically acts on serotonin N-acetyltransferase. As a result, mycetin, which is known only as an anti-tumor, anti-inflammatory drug, and diuretic component, has been applied to serotonin N-acetyltransferase. It has been shown to act specifically to be used as a therapeutic agent for diseases associated with the activity of serotonin N-acetyltransferase, particularly depression. At this time, in the Example, the herbal medicines were searched for compounds that inhibit serotonin N-acetyltransferase activity.

After obtaining a hydrothermal extract using about 300 kinds of herbal medicines, we found a substance that inhibits serotonin N-acetyltransferase activity synthesized in the pineal gland of the white paper using serotonin N-acetyltransferase assay. It was confirmed that myricetin of the constituents most strongly inhibits serotonin N-acetyltransferase activity.

The level of serotonin N-acetyltransferase activity in the pineal gland extract containing serotonin N-acetyltransferase was added [ ³ H] -acetyl CoA and tryptamine, substrates of serotonin N-acetyltransferase, It was measured by treating myricetin and examining the amount of the reaction product [ ³ H] -acetyltrytamine.

As a result of verifying the effect of myricetin on serotonin N-acetyltransferase activity, it effectively inhibited serotonin N-acetyltransferase activity, 50% inhibition effect at 2.2 μM and 98% inhibition effect at 30 μM. Seemed.

<Example 1>

Referring to Figures 1a and 1b, the inhibitory effect of serotonin N-acetyltransferase activity according to the myricetin treatment concentration will be described.

A male white paper (Sprague Dawley, 200-250 gm) was used in the experiment after adapting to a night-day cycle of 12 hours a day or more for a week or more in a constant environment (room temperature 23 ± 1 ° C., relative humidity 60 ± 10%). At 12 o'clock at night, the pineal gland was extracted from the brain of the white paper, and then immersed in 50 mM phosphate buffer (pH 6.8). The transferase was obtained. Serotonin N-acetyltransaction was performed by adding substrates tryptamine, acetyl CoA, [ ³ H] -acetyl CoA, and mycetin for 30 minutes at 37 ° C., and then measuring the radioactivity with a liquid scintillation counter. The degree of furase activity was measured.

Figure 1a is a result showing that serotonin N-acetyltransferase activity is suppressed according to the concentration of myricetin, it can be seen that the serotonin N-acetyltransferase activity is inhibited as the concentration of myricetin increases. When the concentration of myricetin was 2.2 μM, the inhibitory effect was about 50%, and at 30 μM, the inhibitory effect was 98% or more. Figure 1b is a result showing that serotonin N-acetyltransferase activity decreases with treatment time when treated with myricetin, it can be seen that serotonin N-acetyltransferase activity is inhibited with increasing treatment time. After 3 minutes of treatment time, the inhibitory effect was about 30%, and after 15 minutes, the inhibitory effect was about 50%.

<Example 2>

Referring to Figures 2a and 2b, the mechanism of inhibition of myricetin against tryptamine and acetyl CoA, the substrates of serotonin N-acetyltransferase, will be described.

Experiment was carried out in the same manner as in Example 1 above. FIG. 2A shows the degree of inhibition of serotonin N-acetyltransferase activity of myricetin when the concentration of tryptamine was varied from 0.05 mM to 1 mM with a constant concentration of acetyl CoA. It can be seen that myricetin acts as a noncompetitive inhibitor. Figure 2b shows the degree of inhibition of serotonin N-acetyltransferase activity of myricetin when the concentration of acetyl CoA was varied from 1.8μM to 29μM in a state where the concentration of tryptamine was constant. It can be seen that cetin acts as an uncompetitive inhibitor.

<Example 3>

With reference to Figures 3a and 3b, the antidepressant effect in the treatment of myricetin through forced swimming test will be described.

At this time, a forced swimming test method, which is a standardized test called a behavioral despair test, was used. This method is known as a basic experiment to detect antidepressant effects in drug development. In this experiment, a transparent acrylic cylindrical tank 40 cm high and 18 cm in diameter was filled with water at 25 ° C., white paper was left out, and forced to swim for 15 minutes. The water depth is such that the back foot cannot reach the floor when the white paper is swimming or floating in the water.

Secondary forced swimming after the first forced swimming takes place after 24 hours. Secondary forced swimming was maintained in the tank for 5 minutes under the same conditions as the primary forced swimming, and the behavior of the white paper was calculated by dividing it into a floating state, a swimming state, and a climbing state. The floating state was calculated as the total floating time, and the swimming state and the climbing state were divided into 5 units divided by 5 minutes and divided into total 60 units, and at least one movement pattern was recorded within 5 seconds. Mycetin was intraperitoneally injected at a concentration of 20 mg / kg after 15 minutes of forced swimming on the first day, and administered intraperitoneally at a concentration of 20 mg / kg 5 hours before and 1 hour before the 5 minute swimming on the second day.

In the case of secondary forced swimming, the white paper appears to be immobile for much more time as a result of the despair learned, and since the increase in immobilization is somewhat reduced by treatment with antidepressants, this decrease in immobilization acts as an antidepressant. It is thought to be related. Figure 3a shows that the total floating time is reduced by mycetin through the forced swimming test, it can be seen that mycetin has an antidepressant effect in the depression-induced white paper. 3B shows that the swimming state is increased by mycetin through the forced swimming test, but the climbing state is similar to the control group. As a result, the mycetin shows an antidepressant effect by increasing the swimming state.

As a result, the compounds according to the present invention specifically act on only serotonin N-acetyltransferase to inhibit the activity of serotonin N-acetyltransferase. Thus, the compounds may be used for the treatment of central nervous system disorders related to serotonin N-acetyltransferase activity, for treatment of insomnia, jet-lag treatment, night-shift workers and delayed sleep. It can be useful as a treatment for patients with sleep-phase syndrome, depression and seasonal affective disorders, and for patients with premenstrual syndrome.

As a therapeutic agent for the above-mentioned diseases, the compound may be prepared by a general known chemical synthesis method, but preferably obtained by extracting from the bark of the Chinese herb, Myrica rubra Siebold et Zuccarini. When using the bark bark extract containing the compound is more advantageous than the case of the chemical synthesis in terms of stability and cost.

The present invention includes, in addition to non-toxic, inert, pharmaceutically suitable excipients, a pharmaceutical composition containing a compound according to the present invention or consisting of an active compound according to the present invention and a process for preparing the composition. The compounds of the present invention can be administered orally or parenterally during clinical administration and can be used in the form of general pharmaceutical formulations.

It may be administered in a variety of oral and parenteral dosage forms in actual clinical administration, and when formulated, it is prepared by using the usual diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrating agents, and surfactants.

The invention also includes formulations of dosage units. The formulations are in individual dosage forms, for example tablets, coated tablets, capsules, pills, suppositories, and ampoules, and the amount of active compound in the drug corresponds to the fraction or multiple of the individual dosage. The dosage unit may contain, for example, one, two, three, or four times, or 1/2, 1/3, or 1/4 times the individual dosage. Individual dosages preferably contain an amount in which the active compound is administered at one time, which usually corresponds to a total, 1/2, 1/3 or 1/4 of the daily dose.

Pharmaceutically suitable excipients which are nontoxic and inert are solid, semisolid or liquid diluents, fillers and formulation aids of all types. Preferred formulations include tablets, coated tablets, capsules, pills, granules, suppositories, solutions, suspensions and emulsions, pastes, ointments, gels, creams, lotions, powders and sprays and the like.

According to a preferred embodiment of the present invention, the 50% inhibition concentration (IC ₅₀ ) of myricetin is about 2.2 μM, and the effective amount of the compound of the present invention is from 0.01 μM to 1M.

While the invention has been shown and described with respect to particular embodiments, it will be appreciated by those skilled in the art that various modifications and changes can be made without departing from the spirit and scope of the invention as set forth in the appended claims. Anyone can grow up easily.

As described above, myricetin of the present invention acts specifically on serotonin N-acetyltransferase activity, thereby inhibiting serotonin N-acetyltransferase activity, and thus, melatonin and progenitor, which are by-products of serotonin N-acetyltransferase, There is an effect that can be usefully used as a therapeutic agent for diseases related to the serotonin substance.

In particular, it is used as a treatment for insomnia, jet-lag, night-shift work, delayed-sleep-phase syndrome, depression and seasonal affective disorder. There are few side effects and excellent stability.

Claims (5)

(a) mycetin (5,7) represented by the following formula (1) to inhibit the activity of serotonin N-acetyltransferase to regulate the amount of serotonin and to reduce the synthesis of N-acetylserotonin and melatonin that act as neurotransmitters , 3 ', 4', 5'-pentahydroxy flavonol); And (b) a pharmaceutical composition for the treatment of depression or sleep disorders comprising a pharmaceutically acceptable carrier: The method of claim 1, wherein the sleep disorder is insomnia, hypersomnia, Jet-lag, night-shift work, delayed-sleep-phase, or sleep deprivation. Pharmaceutical composition for the treatment of depression or sleep disorders, characterized in that (advanced-sleep-phase). The method of claim 1, wherein the depression is seasonal affective disorder, premenstrual syndrome, unipolar depression, manic-depressive psychosis, or atypical depression. Pharmaceutical compositions for the treatment of sleep disorders. The pharmaceutical composition of claim 1, wherein the 50% inhibition concentration (IC ₅₀ ) of myricetin is 2.2 μM. The pharmaceutical composition for the treatment of depression or sleep disorders according to claim 1, wherein the pharmaceutically effective amount of myricetin represented by Formula 1 is 0.01 μM to 1 M.