JP5273731B2 - Biorhythm control agent - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for treating circadian rhythm failure by regulating circadian rhythm cycle, and to provide a fundamental therapeutic method of a disease caused by the circadian rhythm failure. <P>SOLUTION: The circadian rhythm failure-ameliorating agent contains lycorins or lycoricidinols of alkaloids of Amaryllidaceae family, or a pharmacologically acceptable salts thereof as an active ingredient. The medicinal composition for the prevention or therapy of the disease caused by the circadian rhythm failure is also provided. <P>COPYRIGHT: (C)2011,JPO&amp;INPIT

Description

  The present invention relates to an agent for improving sleep disorder by controlling circadian rhythm.

In addition to hereditary sleep disorders, various sleep disorders associated with 24-hour society have become social problems. The onset of a series of sleep disorders called circadian rhythm sleep disorders is thought to be related to the biological clock composed of clock genes, but the detailed mechanism is unknown.
As a treatment method for sleep disorders, high-intensity phototherapy requiring a special device, and administration of vitamin B12 (Non-patent Document 1) and a ratonin preparation (Patent Document 1) are generally used.
In addition, benzodiazepine drugs (Non-patent Document 2) are generally used as sleeping drugs, and various half-life drugs have been developed from long-time types to short-time types, intermediate types, and long-term types. . However, treatment methods for sleep disorders using these sleeping drugs are coping therapy, and it is difficult to fundamentally treat sleep disorders. In addition, there are many side effects such as sleepwalking symptoms, and the usage thereof requires careful attention. Recently, a 4-oxoimidazolidine-2-spiropiperidine derivative having an action as an agonist of a nociceptin receptor is expected to have a circadian rhythm sleep disorder treatment effect (Patent Document 2), but is still under research and development. Side effects are also a concern.
Research and development of sleep-improving agents from natural ingredients and foods and drinks is also active, using tea leaves derived teaamine (Patent Document 4), adding whey such as fermented whey with endogenous melatonin secretion effect (patent In addition to literature 5), sleep improvement agents using ginseng extract (non-patent literature 3), phosphatidylcholine (non-patent literature 4) contained in salmon oil have been proposed. These are all derived from materials used for food and drink, so they are safe and can be taken on a daily basis, but their detailed sleep improvement effects and mechanisms of action are fully elucidated. However, there is a great individual difference in the effect.
As described above, most of the conventional treatment methods have an unknown mechanism of action, or are intended to normalize the biological rhythm by adjusting the phase of the biological clock, and the period of the biological clock is abnormal. It is not a fundamental cure for disorders caused by.
The circadian rhythm is controlled by a circadian clock, and in recent years it has become clear that the rhythm of the circadian clock is engraved by a series of genes called clock genes. The biological clock has two functions: biological rhythm phase adjustment and period length adjustment. Regarding circadian rhythm phase regulation, the expression of clock genes is induced by many humoral factors, at least in cell culture systems, and it is assumed that there are many regulatory factors in living organisms. (Non-Patent Document 5).
On the other hand, regarding the regulation of the circadian rhythm cycle length, glycogen synthase kinase 3β (GSK3β) is linked to the biological clock through phosphorylation of clock molecules PERIOD2 (Non-patent Document 11) and REV-ERBα (Non-patent Document 12). A sleep-improving agent using an enzyme activity regulator of c-Jun N-terminal kinase-3 (JNK3) which has an inhibitory effect on BMAL1, one of the transcription factors involved in circadian rhythm. It has been developed (Patent Document 3).
In order to improve sleep disorders fundamentally, it was strongly desired to develop a sleep-improving agent that regulates the cycle length among circadian rhythms. However, until now, detailed circadian rhythm cycles have been detected. Since experimental methods have not been established, circadian rhythm reset (phase adjustment) has mainly been analyzed.
Therefore, it has been desired to provide a fundamental sleep improving agent that regulates the circadian rhythm, particularly the circadian rhythm cycle length.

Japanese National Patent Publication No. 8-502259 WO2003 / 010168 WO2003 / 063907 WO2005 / 097101 WO2005 / 097101 JP-A-61-36221 Japanese Patent Laid-Open No. 11-79992 JP-A-11-255650

“Circadian Rhythm Sleep Clinic” Shigeru Chiba, Kenichi Honma, Emerging Medical Publishers, 2003 “Sleep Disorder Response and Treatment Guidelines”, Makoto Uchiyama, Jiho, 2002 Young Ho Rhee, etal., Psychopharmacology, 101, p. 486-488 (1990) Hidehiko Hibino, FoodStyle21, Vol.7, No.3, p.50-53 (2003) Curr Biol 2000, 10: 1291-4 EMBO Reports 2007, 8: 366-71, Eur J Neurosci 2007, 26: 451-62 Science, 1992, 258, 607-614 Cur. Biol., 2000, 10, 1291-1294 FEBS Lett., 1999, 465, 79-82 Gene to Cells, 2003, 8, 713-720 J. Biol. Chem., 2005, 280, 29397-29402 Science, 2006, 311, 1002-1005 Cell, 2000, 103, 1009-1017 Nature, 2002, 418, 534-539 Mol. Cell. Biol., 2008, 28, 3477-3488 J. Biol. Chem., 1982, 257, 7847-7851 Bioorg. Med. Chem., 2008, 16, 10182-10189 J. Biol. Chem., 1986, 261, 505-507 Biochimica Biophysica et Acta., 1976, 425, 342 Jounalof Natural Products., 1992, 55, 1569 Translational Oncology, 2008, 1, 1-13

  An object of the present invention is to provide a method for treating a sleep disorder by adjusting a circadian rhythm, and to provide a fundamental treatment for a disorder caused by an abnormality of a biological clock.

From the results of recent knockout experiments using circadian rhythm-related genes, it has been almost established that the gene that is the center of biological clock regulation is the Bmal1 gene (Non-patent Document 13).
Traditionally, reporter assays at the cultured cell level using NIH3T3 cells into which a plasmid containing the RORE sequence in the Bmal1 gene promoter, a foreign SV40 promoter, and a luciferase gene have been introduced for transient expression have been performed in mammals. It has been known that the circadian rhythm via Bmal1 gene in the body can be reproduced (Non-patent Document 14). However, in such a conventional reporter assay method using cultured cells, the state of the plasmid in the cell is unstable, and the cell culture is continued in a state where the test substance is added to the medium, and the influence is accurately determined. It was not possible to measure.
The conventional reporter assay method using NIH3T3 cells was intended to search for substances or genes that transiently act on a specific region (RORE sequence) such as the promoter region of the Bmal1 gene. Considered the application of a conventional reporter assay with a completely new concept. In other words, PERIOD was synthesized from the Period gene whose expression was promoted by the heterodimer of transcription factor BMAL1 and transcription factor CLOCK, and PERIOD-CRY complex protein that bound CRY outside the nucleus and translocated into the nucleus was transferred to BMAL1 and CLOCK. The idea was to construct a reporter assay system to search for substances that act over a long period of the circadian rhythm formation cycle itself, which suppresses the activity, and substances that accelerate or slow the cycle.
For this purpose, the inventors first prepared a reporter plasmid in which a luciferase gene serving as a reporter is linked to a region (positions −202 to +27) containing a “RORE” sequence and a transcription initiation site in the Bmal1 gene promoter (FIG. 1). ), The reporter plasmid was introduced into NIH3T3 cells, and a cell line that produced luciferase with stable circadian rhythm was established. Using the established cell line, various substances were allowed to act to monitor luciferase activity. As a result, it has been clarified that ricolin and licoricidinol, alkaloids derived from the Japanese anemone plant, prolong the cycle of the transcriptional rhythm of the Bmal1 gene in a concentration-dependent manner. Regarding not only licholine and licoricidinol but also other alkaloids derived from natural components, there has been no report on the effect of regulating the circadian rhythm cycle length.
Since it is known that ricolin has a protein synthesis inhibitory action, cyclohexamide, a general protein synthesis inhibitor, is used to eliminate the possibility of non-specific action due to protein inhibitory action. However, no effect on the cycle length was observed, and even when the measurement was performed in the presence of ricolin and cyclohexamide, no effect due to the presence or absence of cyclohexamide was observed. These results indicate that the effect of ricolin on the circadian rhythm cycle is apparently due to a mechanism independent of protein synthesis inhibition.
By obtaining the above knowledge, the present invention was completed.

（式中、Ｒ_１及びＲ_２は、それぞれ独立して−Ｈ、−ＣＯＣＨ_３、−ＣＯＣ_２Ｈ_５、−ＣＯＣ_３Ｈ_７又は−ＣＯＣＨ_２ＣＨ（ＯＨ）ＣＨ_３を表す。

＜式（２）＞

（式中、Ｒ_３、Ｒ_４及びＲ_５はそれぞれ独立して−Ｈ又は−ＯＨを表す。）。
〔２〕 下記式（１）もしくは式（２）で表されるヒガンバナ科植物アルカロイド、又はその薬理学的に許容しうる塩もしくはエステルを有効成分として含む、概日リズムの障害に起因した疾患の治療用又は予防用医薬組成物；

＜式（１）＞

（式中、Ｒ_１及びＲ_２は、それぞれ独立して−Ｈ、−ＣＯＣＨ_３、−ＣＯＣ_２Ｈ_５、−ＣＯＣ_３Ｈ_７又は−ＣＯＣＨ_２ＣＨ（ＯＨ）ＣＨ_３を表す。

＜式（２）＞

（式中、Ｒ_３、Ｒ_４及びＲ_５はそれぞれ独立して−Ｈ又は−ＯＨを表す。）。
〔３〕 概日リズムの障害に起因した疾患が、睡眠相前進症候群（ASPS）、睡眠相後退症候群（DSPS）、非24時間睡眠相後退症候群、および季節性うつ病からなる群から選択される疾患である前記〔２〕に記載の医薬組成物。 That is, the present invention includes the following inventions.
[1] An agent for improving circadian rhythm disorder, comprising an Amaryllidaceae alkaloid represented by the following formula (1) or formula (2), or a pharmacologically acceptable salt or ester thereof as an active ingredient;

<Formula (1)>

(Wherein, _{R 1} and _{R 2} are each independently _-H, -COCH 3, represents a _{-COC 2 H 5, -COC 3 H} 7 or _{-COCH 2 CH (OH) CH 3} .

<Formula (2)>

(In the formula, R ₃ , R ₄ and R ₅ each independently represent —H or —OH).
[2] A disease caused by disturbance of circadian rhythm, comprising an Amaryllidaceae alkaloid represented by the following formula (1) or formula (2), or a pharmacologically acceptable salt or ester thereof as an active ingredient: A therapeutic or prophylactic pharmaceutical composition;

<Formula (1)>

(Wherein, _{R 1} and _{R 2} are each independently _-H, -COCH 3, represents a _{-COC 2 H 5, -COC 3 H} 7 or _{-COCH 2 CH (OH) CH 3} .

<Formula (2)>

(In the formula, R ₃ , R ₄ and R ₅ each independently represent —H or —OH).
[3] The disease caused by the circadian rhythm disorder is selected from the group consisting of advanced sleep phase syndrome (ASPS), delayed sleep phase syndrome (DSPS), non-24 hour delayed sleep phase syndrome, and seasonal depression The pharmaceutical composition according to the above [2], which is a disease.

Construction of Bmal1 reporter plasmid The positions of RORE (white) and SAF-A binding sequence (gray) in the genetic map of the Bmal1 promoter region are shown. The region from -202 to +27 containing RORE was inserted upstream of the luciferase gene of plasmid pGL3-dLuc. Circadian rhythm regulation by ricolin The time course of chemiluminescence in the presence of 2.5 μM ricolin (black), the presence of 10 μM ricolin (+), and the control (white) is shown. Effect of protein synthesis inhibitors on circadian rhythm regulation Along with the protein synthesis inhibitor cyclohexamide (CHX: 0.1 nM), 2.5 μM ricolin (black), 10 μM ricolin (+), and control (white) ) Shows the change over time in the amount of chemiluminescence. Circadian rhythm regulation by licoricidinol The time course of chemiluminescence in the presence of 0.1 μM licoricidinol (black), 0.05 μM licoricidinol (+), and control (white) is shown.

1. About Sleep Disorders Sleep disorders are roughly classified into “circadian rhythm sleep disorders” and “sleep breathing disorders”. Circadian rhythm sleep disorders are divided into exogenous acute syndromes and intrinsic chronic syndromes, such as sleep phase advance syndrome and sleep phase regression syndrome, non-24-hour sleep-wake disorder, irregular sleep-wake disorder, Included in intrinsic chronic syndrome. The circadian rhythm sleep disorder is considered to involve the body clock. In particular, the intrinsic chronic syndrome is caused by the abnormal period of the body clock. Conventional treatment methods for sleep disorders, such as light irradiation, melatonin administration, and treatments with various sleeping pills are coping therapy that acts on the reset of the body clock, whereas the present invention aims at The sleep disorder treatment method is a treatment method by improving the period abnormality of the body clock, that is, the “circadian rhythm disorder”.

2. About diseases caused by circadian rhythm disorders Diseases caused by circadian rhythm disorders include advanced sleep phase syndrome (ASPS), late sleep phase syndrome (DSPS), non-24 hour sleep phase regression syndrome, and seasonal depression. It can be considered typical. Other periodic and repetitive disorders such as endogenous manic depression, periodic tension, periodic hypertension, periodic ulcer, irregular ovulation cycle, and diabetes caused by periodic abnormalities of insulin secretion, and cerebral vascular types Circadian rhythm disorder is also considered to be involved in dementia and nighttime hemorrhage in Alzheimer type dementia.

3. About circadian rhythm and Bmal1 gene There are four major factors involved in the regulation of the circadian clock: the Bmal1 and Clock genes that promote the ticking of the circadian clock, and the Cry and Period genes that act as suppressors.
Among these genes, mice that knocked out the Bmal1 gene no longer have any behavioral rhythm, and behave as if the biological rhythm was completely destroyed. In knockout mice such as other clock genes such as Period and Cryptochrome, abnormalities are only observed in the activity cycle, and the Bmal1 gene is the only gene that does not show any behavioral rhythm formation when a single gene is disrupted. Furthermore, in Bmal1 gene knockout mice, expression of clock genes Period1 and Period2 is almost completely suppressed in the suprachiasmatic nucleus (SCN), which is the clock center. From the above, it is considered that the Bmal1 gene is the center of biological clock regulation (Non-patent Document 13).
Recently, the present inventors have elucidated the transcriptional regulation of circadian rhythm via Bmal1 gene by SAF-A binding to “SAF-A binding region (positions −27 to +266)” in the Bmal1 gene promoter region. (Non-patent document 15, Japanese Patent Application No. 2008-25966).

4). Screening for a substance that regulates the circadian rhythm cycle In the present invention, a reporter assay system used for screening a substance that regulates the circadian rhythm cycle is transiently applied to a specific region such as the promoter region of the Bmal1 gene in conventional research. The screening system that has been used to search for substances or genes that act on the drug is modified and used.
First, a reporter plasmid (FIG. 1) prepared by inserting a region of −202 to +27 containing “RORE” of the Bmal1 promoter region upstream of the luciferase gene of the plasmid pGL3-dLuc (Non-patent Document 14) Introduced into NIH3T3 cells according to the technique of Patent Document 14, subcultured, and established a transformed NIH3T3 cell line that produced luciferase with stable circadian rhythm.
From the Period gene whose expression was promoted by the heterodimer of transcription factor BMAL1 and transcription factor CLOCK by adding a test substance to the medium of the established cells and monitoring the luminescence intensity of luciferase over time while culturing. A substance that acts for a long time on a series of circadian rhythm formation cycles in which PERIOD-CRY complex protein, in which PERIOD is synthesized, bound to CRY outside the nucleus and translocated into the nucleus, suppresses BMAL1 and CLOCK transcriptional activity, is the cycle It became possible for the first time to search for substances that speed up or slow down.

（式中、Ｒ_１及びＲ_２は、それぞれ独立して水素であるか、又は水酸基を有しても良い低級アシル基であり、具体的には、−Ｈ、−ＣＯＣＨ_３、−ＣＯＣ_２Ｈ_５、−ＣＯＣ_３Ｈ_７又は−ＣＯＣＨ_２ＣＨ（ＯＨ）ＣＨ_３（Acetyl, Propanoyl, Butanoyl,又はHydroxybutanoyl基）を表す。 5. About the Amaryllidaceae Alkaloids Licolins and Licolicidinols In the present invention, the term "Amphilidae Alkaloids" refers to plant alkaloids derived from Amaryllidaceae plants such as Amaryllidaceae, Narcissus, etc. is there.
The licholines belong to “Amaryllidaceae alkaloids” contained in the Amaryllidaceae plant, and refer to a compound of the following formula (1) having a galanthan skeleton structure.

<Formula (1)>

(In the formula, R ₁ and R ₂ are each independently hydrogen or a lower acyl group which may have a hydroxyl group. Specifically, —H, —COCH ₃ , —COC ₂ H ₅ represents -COC ₃ H ₇ or _{-COCH 2 CH (OH) CH 3} (Acetyl, Propanoyl, Butanoyl, or Hydroxybutanoyl group).

Licolins have been known for a long time to have various biological activities, and include not only protein synthesis inhibitory action (Non-patent Document 18) but also analgesic action, antiviral action, antimalarial action, antitumor action, anti-inflammatory effect. The action has already been reported (Non-patent Document 17). In addition, immunosuppressive action (Patent Document 6) and apoptosis inhibitory action (Patent Document 7) are also known. Although these biological activity mechanisms have not been elucidated in detail, the biochemical mechanism relating to the termination reaction inhibition of the translation process in protein synthesis is best elucidated (Non-patent Document 18).
A typical example of formula (1) is licholine, where R ₁ and R ₂ are both —H. The case where R ₁ is —H and R ₂ is —COCH ₂ CH (OH) CH ₃ (— (3 ′S) -Hydroxybutanoyl) is LT1, and LT1 shares physiological functions with recholine in cell growth inhibitory action and the like. (Non-patent Document 17). In the present invention, ricolin is mainly described as a typical example of the formula (1), but other compounds can be expected to have the same action, and the circadian rhythm cycle length adjustment effect similar to ricolin is effective. I can expect.

（式中、Ｒ_３、Ｒ_４及びＲ_５はそれぞれ独立して−Ｈ又は−ＯＨを表す。） Licolicidinols are compounds of the following formula (2), which are contained in an Amaryllidaceae plant as in the case of lycoline, and are biosynthesized with phenanthridine as a mother skeleton structure.

<Formula (2)>

(In formula, R < ₃ >, R < ₄ > and R < ₅ > represent -H or -OH each independently.)

Like ricolin, licoricidinols are also known to have a protein synthesis inhibitory action (Non-patent Document 19), an antiviral action (Non-patent Document 20), and an apoptosis inhibitory action (Patent Document 8).
A typical example of formula (2) is licoricidinol, where R ₃ , R ₄ and R ₅ are all —OH. When R ₃ is —H and R ₄ and R ₅ are —OH, it is known that lycoricidine has a physiological action common to licoricidinol in antitumor action or the like (non- Patent Document 21). In the present invention, licoricidinol is mainly described as a typical example of the formula (2), but other compounds similar to the formula (2) can be expected to have the same action, and circadian similar to licoricidinol. The effect of adjusting the rhythm cycle length can be expected.

  In the present invention, the above 4. By monitoring the luciferase activity using a transformed NIH3T3 cell line that stably produces circadian rhythm and produced luciferase, ricolin, an alkaloid derived from the Japanese anemone plant, transcribes the Bmal1 gene in a concentration-dependent manner. It became clear that the cycle of the rhythm was extended. Lycoline is known to have a protein synthesis inhibitory effect, but it was confirmed that there was no effect due to the presence or absence of cyclohexamide, a general protein synthesis inhibitor. The possibility of non-specific effects was excluded.

  The target animals to be adjusted for circadian rhythm in the present invention include mammals including primates such as humans, pets such as dogs and cats, domestic animals such as cattle and horses, laboratory animals such as mice and rats, and homology. Birds and fish with high clock genes. Therefore, in the present invention, “Bmal1 gene” refers to a gene or gene product derived from mammals including humans, birds and fish.

6). Circadian rhythm disorder improving agent and pharmaceutical composition for prevention and treatment of diseases caused by circadian rhythm disorder: Amaryllidaceae alkaloids such as ricolins and licoricidinols or pharmacologically acceptable salts thereof, and ingested in vivo Any of the ester-type precursors easily converted in can be used as a circadian rhythm disorder improving agent. Examples of the pharmacologically acceptable salt used in the present invention include acetate, sulfate, hydrochloride and the like.
A pharmaceutical composition for the prevention and treatment of diseases caused by circadian rhythm disorders by containing a circadian rhythm improving agent as an active ingredient and combining with an appropriate pharmacologically acceptable carrier or excipient. be able to.
Such diseases are typically considered to be advanced sleep phase syndrome (ASPS), delayed sleep phase syndrome (DSPS), non-24 hour delayed sleep phase syndrome, and seasonal depression.
Other periodic and repetitive disorders such as endogenous manic depression, periodic tension, periodic hypertension, periodic ulcer, irregular ovulation cycle, and diabetes caused by periodic abnormalities of insulin secretion, and cerebral vascular types Used alone or in combination with other compounds or medicines required for treatment for the treatment of nocturnal epilepsy in dementia or Alzheimer type dementia.
The administration form of the pharmaceutical composition may be systemic administration or local administration, and may be parenteral administration such as injection, salve, or oral administration, and a well-known formulation method can be applied.
The dose can be appropriately adjusted depending on the age, weight, etc. of the patient, and varies depending on the administration form. For example, 0.001 mg to 20 mg, preferably 0.01 mg to 10 mg, per kg of body weight as ricolin hydrochloride, etc. Preferably it is 0.1-1.0 mg, and it is preferable to determine the frequency | count of administration according to the objective.

EXAMPLES Hereinafter, the present invention will be specifically described with reference to examples, but the present invention is not particularly limited to these examples.
Technical terms used in the present invention have meanings commonly understood by those skilled in the art unless otherwise defined. Unless otherwise specified, specific procedures and conditions such as genetic recombination techniques, PCR methods, and other techniques used in the examples of the present invention are as follows: Sambrook and Russell, Molecular Cloning: A Laboratory Manual, 3rd Edition. Harbor Laboratory Press, Plainview, NY (2001), Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York (1989); DM Glover et al. Ed., "DNA Cloning", 2nd ed., Vol. 1 to 4, (The Practical Approach Series), IRL Press, Oxford University Press (1995); Ausubel, FM et al., Current Protocols in Molecular Biology, John Wiley & Sons, New York, NY, 1995; Chemistry Experiment Course 1, Genetic Research Method II ", Tokyo Chemical Doujin (1986); edited by The Japanese Biochemical Society," Shinsei Chemistry Experiment Course 2, Nucleic Acid III (Recombinant DNA Technology) ", Tokyo Chemical Doujin (1992); R. Wu ed., "Methods in Enzymology", Vol. 68 (Recombinant DNA), Academic Press, New York (1980); R. Wu et al. ed., "Methods in Enzymology", Vol. 100 (Recombinant DNA, PartB) & 101 (Recombinant DNA, Part C), Academic Press, New York (1983); R. Wu et al. Ed., "Methods in Enzymology", Vol. 153 (Recombinant DNA, Part D), 154 (Recombinant DNA, Part E) & 155 (Recombinant DNA, Part F), Academic Press, New York (1987), etc., or the method described in the literature cited therein or a method substantially similar thereto.
Moreover, the description content of the prior art literature or the patent application specification cited in the present invention is incorporated as the description of this specification.

(Example 1) Establishment of NIH3T3 cell line stably holding a luciferase gene transcribed by the Bmal1 promoter
As a minimal promoter region necessary for rhythmic expression of the Bmal1 gene, a region of −202 to +27 including “RORE” is selected and inserted upstream of the luciferase gene of the plasmid pGL3-dLuc (Non-patent Document 14). A Bmal1 reporter plasmid was prepared (FIG. 1).
A pTracer-CMV (manufactured by Invitrogen) having an antibiotic (Zeocin) resistance marker gene together with the Bmal1 reporter plasmid is introduced into NIH3T3 cells (by the method described in Non-Patent Document 14), and luciferase transcriptionally regulated by the Bmal1 promoter. Recombinant NIH3T3 cells stably retaining the gene were obtained. Repeated subcultures to establish a transformed NIH3T3 cell line that produces luciferase with a stable circadian rhythm, and the clone in which the circadian rhythm of luciferase activity is most stably observed among the established cells Selected.

(Example 2) Periodic extension of circadian rhythm by ricolin After seeding approximately 5 × 10 ⁵ NIH3T3 cell lines established in Example 1 in a 35 mm culture dish, reset the biological rhythm with 100 nM dexamethasone. It was.
A test substance was added to a medium containing the luminescent substrate luciferin of the established NIH3T3 cell line, and chemiluminescence by the reporter gene was measured in real time for about 120 hours while continuing cell culture. Luminescence was measured for 1 minute every 10 minutes.
Subsequently, 2.5 μM or 10 μM ricolin was added to the medium containing the luminescent substrate luciferin, and chemiluminescence by the reporter gene was measured every 10 minutes for 1 minute in real time while continuing cell culture. When measured over 120 hours, the circadian rhythm period was extended by about 3.8 hours in the presence of 2.5 μM and about 8.6 hours in the presence of 10 μM compared to the control. (Figure 2)

(Example 3) Effect of protein synthesis inhibitor on circadian rhythm regulation As in Example 2, 2.5 μM or 10 μM ricolin was added in the presence of a typical protein synthesis inhibitor cyclohexamide (CHX) 0.1 nM. When added to the culture medium and chemiluminescence by the reporter gene was measured for 120 hours, a decrease in the expression level of the reporter gene was observed by addition of CHX under any culture condition. However, when the circadian rhythm cycle was observed, the result of circadian rhythm cycle extension by ricolin, exactly the same as the result of Example 2, was obtained even in the presence of CHX. (Figure 3)

(Example 4) Period extension of circadian rhythm by licoricidinol
When 0.1 μM licoricidinol was added to the culture medium and chemiluminescence by the reporter gene was measured over 110 hours as in Example 2, the circadian rhythm period was about 1.6 hours in the presence of 0.05 μM compared to the control. For about 3 hours in the presence of 0.1 μM. (Fig. 4)
By obtaining the above results, it was proved that circadian rhythm cycle can be artificially changed by ricolins and licorcidinols, and that this is due to a mechanism independent of protein synthesis inhibition.

Claims (3)

A circadian rhythm cycle extender comprising an Amaryllidaceae alkaloid represented by the following formula (1) or formula (2), or a pharmacologically acceptable salt or ester thereof as an active ingredient;
<Formula (1)>

(Wherein, _{R 1} and _{R 2} are each independently _-H, -COCH 3, represents a _{-COC 2 H 5, -COC 3 H} 7 or _{-COCH 2 CH (OH) CH 3} .
<Formula (2)>

(In the formula, R ₃ , R ₄ and R ₅ each independently represent —H or —OH). See containing Amaryllidaceae plant alkaloid represented by the following formula (1) or formula (2), or a pharmaceutically acceptable salt or ester as an active ingredient, and characterized by extending the period of circadian rhythms A pharmaceutical composition for treating or preventing a disease caused by abnormal circadian rhythm;
<Formula (1)>

(Wherein, _{R 1} and _{R 2} are each independently _-H, -COCH 3, represents a _{-COC 2 H 5, -COC 3 H} 7 or _{-COCH 2 CH (OH) CH 3} .
<Formula (2)>

(In the formula, R ₃ , R ₄ and R ₅ each independently represent —H or —OH). Period caused by abnormal disease of the circadian rhythm, consisting of endogenous manic depression, periodic tension disease, periodic hypertension, cyclic ulcers, irregular ovulation cycle, and periodicity caused by abnormal diabetes of insulin secretion period The pharmaceutical composition according to claim 2, which is a disease selected from the group of sexual / repetitive disorders .

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