CN116421605A

CN116421605A - Use of ISX-9 in the treatment of circadian amplitude decline and sleep disorders associated with aging

Info

Publication number: CN116421605A
Application number: CN202210001616.5A
Authority: CN
Inventors: 张洪钧; 唐芸棋
Original assignee: Shanghai Institute of Nutrition and Health of CAS; Center for Excellence in Brain Science and Intelligence Technology Chinese Academy of Sciences
Current assignee: Shanghai Institute of Nutrition and Health of CAS; Center for Excellence in Brain Science and Intelligence Technology Chinese Academy of Sciences
Priority date: 2022-01-04
Filing date: 2022-01-04
Publication date: 2023-07-14
Anticipated expiration: 2042-01-04
Also published as: WO2023130675A1; CN116421605B

Abstract

The invention provides an application of ISX-9. In particular, ISX-9 can be used to prepare a composition for one or more uses selected from the group consisting of: (i) preventing and/or treating circadian rhythm disorders; (ii) preventing and/or treating sleep disorders; (iii) increasing circadian amplitude; and/or (iv) increasing the activity or expression level of the CaMKII delta protein. The active ingredient ISX-9 of the invention can be stable and continuous, and can effectively treat circadian rhythm disorder and related diseases of elderly subjects in-vivo, ex-vivo and in-vitro environments.

Description

Use of ISX-9 in the treatment of circadian amplitude decline and sleep disorders associated with aging

Technical Field

The invention relates to the field of medicaments, in particular to the use of ISX-9 for the treatment of circadian amplitude decline and sleep disorders associated with aging.

Background

Biological clocks are an internal timer that orderly coordinates a variety of physiological activities, including circadian hormone cycles, feeding, sleep, body temperature, and metabolic activities. Disruption of circadian rhythms in model organisms mimics a variety of conditions associated with human diseases such as cardiovascular dysfunction, diabetes, cancer, sleep disorders, depression, and neurodegenerative diseases. Notably, all of the above diseases are highly correlated with the aging process, and thus the decline in biological clock intensity is also considered as one of the indicators of aging. Given that biological rhythm amplitude degradation is a common feature of health impairment, interventions aimed at preventing biological rhythm amplitude decay as well as shrinking are expected to be applied in the treatment of circadian rhythm disorders and related diseases. Through screening means, previous studies have found that a variety of compounds can be used to modulate the amplitude or phase of biological rhythms, while elucidating the biological clock protein targets and related mechanisms of their action. However, since long-term phase dislocation is harmful to health, small molecules that cause significant phase changes should be carefully used; in addition to specific uses, such as modulation of jet lag, the examination of how to correctly and safely use these small molecules for chronic disease treatment for long periods of time still requires a large amount of clinical data.

Disclosure of Invention

The invention aims to provide an application of ISX-9 which can be stable and continuous and can effectively treat circadian rhythm disorder and related diseases of elderly subjects in-vivo, ex-vivo and in-vitro environments.

In a first aspect of the invention there is provided the use of an ISX-9, said ISX-9 having the structure according to the formula:

the ISX-9 is used for preparing a composition for one or more uses selected from the group consisting of: (i) preventing and/or treating circadian rhythm disorders; (ii) preventing and/or treating sleep disorders; (iii) increasing circadian amplitude; and/or (iv) increasing the activity or expression level of the CaMKII delta protein.

In another preferred embodiment, the composition is a pharmaceutical composition or a nutraceutical composition.

In another preferred embodiment, the composition is used for (i) preventing and/or treating circadian rhythm disorders in a middle aged and elderly subject; (ii) preventing and/or treating sleep disorders; (iii) increasing circadian amplitude; and/or (iv) increasing the activity or expression level of the CaMKII delta protein.

In another preferred embodiment, the middle-aged and elderly subject is a subject that has an age of 2/3 over the average life span, more preferably 3/4 over the average life span.

In another preferred example, when the subject is a human, the middle-aged and elderly subject is a subject with an age of 45 years or more.

In another preferred example, when the subject is a mouse, the middle-aged and elderly subject is a subject having a month age of 12 months or more, more preferably 14 months or more.

In another preferred embodiment, the circadian rhythm disorder comprises circadian rhythm disorder due to aging.

In another preferred embodiment, the prevention and/or treatment of circadian rhythm disorder means that one or more indicators selected from the group consisting of:

(i) Improving circadian amplitude;

(ii) Improving metabolic circadian rhythmicity fluctuations;

(iii) Preventing and/or treating circadian rhythm sleep disorder.

In another preferred embodiment, the increasing circadian amplitude means that administration of ISX-9 increases circadian amplitude by at least 1.2-fold, preferably by at least 1.5-fold, for example by at least 2-fold, at least 3-fold, at least 5-fold, compared to a control to which ISX-9 was not administered.

In another preferred embodiment, the composition increases circadian amplitude without altering circadian cycle.

In another preferred embodiment, the composition is sustained for more than 3 days, preferably within 4-14 days, more preferably within 7-14 days.

In another preferred embodiment, the preventing and/or treating circadian rhythm disorder comprises increasing activity of a biological clock gene, and the biological clock gene is selected from the group consisting of: bmal1, per2, dbp, clock, cry1, cry2, npas2, nr1d1, rorα, or combinations thereof.

In another preferred embodiment, the prevention and/or treatment of circadian rhythm disorder may be performed in cells, in vitro tissues and/or in vivo.

In another preferred embodiment, the cells comprise mouse embryonic fibroblasts.

In another preferred embodiment, the in vitro tissue comprises the suprachiasmatic nucleus (SCN) and/or the pituitary.

In another preferred embodiment, the composition does not affect the circadian rhythm of the young subject.

In another preferred embodiment, the young subject is a subject having an age of not more than 2/3 of the average life, more preferably more than 1/2 of the average life.

In another preferred embodiment, when the subject is a human, the young subject is a subject having an age of 30 years or less.

In another preferred example, when the subject is a mouse, the young subject is a subject having a month age of 8 months or less, more preferably 4 months or less, still more preferably 2 months or less.

In another preferred embodiment, the preventing and/or treating circadian rhythm disorder comprises increasing circadian rhythm amplitude by increasing BMAL1 activity.

In another preferred embodiment, the increasing BMAL1 activity comprises promoting phosphorylation of BMAL 1.

In another preferred embodiment, the promotion of BMAL1 phosphorylation is mediated by CaMKII delta.

In another preferred embodiment, the increased BMAL1 activity further promotes CaMKII delta self-expression.

In another preferred embodiment, the composition is used for promoting Ca ²⁺ Inflow into the body.

In another preferred embodiment, caMKII delta responds to Ca ²⁺ Following signaling, BMAL1 transcriptional activity is upregulated by phosphorylating BMAL 1.

In another preferred embodiment, the preventing and/or treating circadian rhythm disorder comprises enhancing BMAL1 activity and enhancing CaMKII delta expression itself.

In another preferred embodiment, the preventing and/or treating circadian rhythm disorder comprises increasing circadian rhythm amplitude by a positive feedback cycle of CaMKII delta, BMAL1, wherein the positive feedback cycle comprises ISX-9 enhancing BMAL1 activity by CaMKII delta-mediated phosphorylation of BMAL1, and the increase in BMAL1 activity further increases expression of CaMKII delta itself.

In another preferred embodiment, the metabolic circadian fluctuation-enhancing feature is one or more of the following:

(a-1) increasing the circadian amplitude of the respiratory exchange ratio;

(a-2) enhancing the object O ₂ Circadian amplitude of consumption;

(a-3) enhancing the object CO ₂ A generated circadian amplitude;

(a-4) correcting the period of the respiratory exchange ratio;

(a-5) does not affect the food intake and/or body weight of the subject.

In another preferred embodiment, the prevention and/or treatment of sleep disorders has one or more of the following characteristics:

(b-1) increasing the wakefulness of the subject during the active period;

(b-2) reducing rapid eye movement REM and slow wave NREM sleep of said subject during an active period;

(b-3) increasing the circadian amplitude of the subject's body temperature;

(b-4) increasing the fast wave gamma power of the subject during an active period;

(b-5) increasing slow wave delta power of the subject during a rest period;

(b-6) improving sleep-awake homeostasis in said subject.

In another preferred embodiment, the composition contains ISX-9 or a pharmaceutically acceptable salt thereof at a concentration of 0.1. Mu.M or more, preferably 0.25. Mu.M or more, more preferably 10. Mu.M or more.

In another preferred embodiment, the composition contains 0.1 to 99wt%, preferably 0.5 to 95wt%, more preferably 1 to 90wt% of ISX-9 or a pharmaceutically acceptable salt thereof.

In another preferred embodiment, the composition further comprises a pharmaceutically acceptable excipient and/or carrier.

In another preferred embodiment, the dosage form of the composition is selected from the group consisting of: solid, liquid or semi-solid formulations.

In another preferred embodiment, the dosage form of the composition is selected from the group consisting of: gel, patch, tablet, capsule, powder, ointment, powder, injection, aqua, enteric sustained-release preparation or injection.

In another preferred embodiment, the composition is in the form of an injection.

In another preferred embodiment, the composition is administered to a subject by: oral, transdermal, intravenous, intramuscular or anorectal administration.

In a second aspect of the invention there is provided the use of deuterated species of ISX-9, said ISX-9 having the structure according to the formula:

the deuterated of ISX-9 is used to prepare a composition for one or more uses selected from the group consisting of: (i) preventing and/or treating circadian rhythm disorders; (ii) preventing and/or treating sleep disorders; (iii) increasing circadian amplitude; and/or (iv) increasing the activity or expression level of the CaMKII delta protein.

In another preferred embodiment, the composition is as described in the first aspect of the invention.

In a third aspect of the invention there is provided the use of a CaMKII delta high expression modulator for the preparation of a composition for one or more uses selected from the group consisting of: (i) preventing and/or treating circadian rhythm disorders; and/or (ii) modulating BMAL1 activity.

In another preferred embodiment, the CaMKII delta is rhythmically expressed by Camk2 d.

In another preferred embodiment, the composition is used to increase the expression level of CaMKII delta in subcortical regions and hypothalamus.

In another preferred embodiment, the preventing and/or treating circadian rhythm disorder comprises: by promoting the phosphorylation of BMAL1, the activity of BMAL1 and the expression of CaMKII delta are improved.

In another preferred embodiment, the prevention and/or treatment of circadian rhythm disorder is as described in the first aspect of the invention.

In a fourth aspect of the present invention, there is provided a pharmaceutical composition comprising:

ISX-9 or a deuterated or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.

In another preferred example, the pharmaceutical composition may comprise other circadian rhythm regulators, wherein the circadian rhythm regulators are selected from the group consisting of: atrasentan (atastant), hhAntag, OSI-930, GW4064, 2-NP, or combinations thereof.

In another preferred embodiment, the dosage form of the pharmaceutical composition is selected from the group consisting of: solid, liquid or semi-solid formulations.

In another preferred embodiment, the dosage form of the pharmaceutical composition is selected from the group consisting of: gel, patch, tablet, capsule, powder, ointment, powder, injection, aqua, enteric sustained-release preparation or injection.

In another preferred embodiment, the pharmaceutical composition is in the form of an injection.

In another preferred embodiment, the dosage form of the pharmaceutical composition is selected from the group consisting of: oral dosage forms, transdermal injection dosage forms, intravenous injection dosage forms, intramuscular injection dosage forms.

In another preferred example, the injection solution of the injection contains one or more of physiological saline, glucose, a stabilizer, a preservative, a suspending agent or an emulsifying agent.

In a fifth aspect of the invention, there is provided (i) a method of preventing and/or treating circadian rhythm disorders; (ii) preventing and/or treating sleep disorders; and/or (iii) a method of increasing circadian amplitude, comprising the steps of: administering to a subject in need thereof a safe and effective amount of ISX-9 or a pharmaceutically acceptable salt thereof.

In another preferred embodiment, the subject is a middle aged or elderly subject.

In another preferred embodiment, the middle aged and elderly subjects are as described in the first aspect of the invention.

In another preferred embodiment, the subject is a mammal, such as a human, mouse, rat, hamster, rabbit, cat, dog, cow, sheep, monkey, and the like.

In another preferred embodiment, ISX-9 or a pharmaceutically acceptable salt thereof is used at an effective concentration of 0.1 to 50. Mu.M, more preferably 0.25 to 10. Mu.M.

It is understood that within the scope of the present invention, the above-described technical features of the present invention and technical features specifically described below (e.g., in the examples) may be combined with each other to constitute new or preferred technical solutions. And are limited to a space, and are not described in detail herein.

Drawings

FIG. 1 shows the identification of ISX-9 as a biorhythmic amplitude enhancer by small molecule screening.

(A) Schematic of compound screening procedure and validation. (B) Heat map of the results of the preliminary screening of 6777 small molecules (left). The points represent PER 2::: LUC bioluminescence relative values at 24 and 30 hour time points, normalized to vehicle control. (C) mPer2 after each small molecule (red curve) treatment ^Luc Real-time bioluminescence recordings of MEF cells showed an amplitude enhancing effect of small molecules. Control is the rhythmic fluctuation of DMSO treatment (black curve display). (D) Small molecule post-treatment cycle analysis associated with FIG. 1C. 9: ISX-9; a: atrasentan (atastan); h: hhANtag; o: o (O) SI-930; g: GW6064 and 2:2-NP. (E) Amplitude evaluation results of 4-day bioluminescence peaks of small molecule treatment. The amplitude of each compound was normalized to its respective day 1 peak. (grey represents DMSO and orange represents dosing group) (F) Dbp: luc and Rev-erb. Alpha. Luc expression changes in response to small molecule treatment; DMSO group was set to 1. Data were checked using unpaired Student's t (ns, not significant, # p<0.05，**p<0.01 Data are shown as mean ± SD.

FIG. 2 shows ISX-9 enhanced mPer2 ^Luc The biorhythmic amplitude effects of MEFs are dose dependent.

(A) Chemical structure of ISX-9. (B) ISX-9 at mPer2 ^Luc Dose-dependent effects in MEFs. (C) Cell viability assay 72 hours after MEF cell treatment with ISX-9. Data are shown as mean ± SD. Unpaired Student's t was used for verification (ns, not significant).

FIG. 3 shows that ISX-9 enhances biological clock gene expression in MEF cells.

Quantitative PCR analysis of biological clock gene expression in MEFs treated with DMSO (black) or ISX-9 (red) at various time points. Data are shown as mean ± SD.

FIG. 4 shows that the biorhythmic amplitude enhancement effect of ISX-9 is Zhong Danbai BMAL1 dependent.

(A) Protein levels of BMAL1, REV-ERBα and DBP were detected by immunoblotting after increasing the therapeutic dose of ISX-9. (B) MEF cells of Bmal1 Wild Type (WT), heterozygous (HET) or Knocked Out (KO) were treated with DMSO (D) or 10. Mu.M ISX-9 (9), and then cells were harvested at 36 hours or 48 hours post-treatment for immunoblotting of BMAL1 and DBP. (C) LUC real-time bioluminescence recordings of Bmal1 wild-type (WT), heterozygous (HET) or knock-out (KO) MEF cells after treatment with DMSO (D) or 10. Mu.M ISX-9 (9) PER 2. (D) ISX-9 (10. Mu.M) treatment of mPer2 ^Luc In vitro PER2 in SCN and pituitary of mice:: LUC rhythmic recordings. Data are shown as mean ± SEM. (E) Immunohistochemical analysis of PER2 levels in SCN of 6 month old mice treated with DMSO or ISX-9 (20 mg/Kg) for 7 days. The right panel shows the quantification of PER2, shown as mean.+ -. SEM. Unpaired Student's t test was used (ns, not significant, # p<0.05，**p<0.01，***p<0.001)。

FIG. 5 shows that ISX-9 younger the circadian metabolism of middle aged and elderly mice.

(A) Respiratory Exchange Ratio (RER), oxygen consumption (VO) of 2 month old (blue curve, n=5) versus 14 month old (gray curve, n=6) mice ₂ ) Carbon dioxide production (VCO) ₂ ). (B) RER, VO after 14 month old mice were treated with DMSO (gray curve, n=6) or ISX-9 (orange curve, n=6) ₂ And VCO (Voltage controlled Oscillator) ₂ As a result. (C) RER, VO after 2 month old mice were treated with DMSO (blue, n=5) or ISX-9 (purple, n=5) ₂ And VCO (Voltage controlled Oscillator) ₂ As a result. (D) The RER fluctuation cycle analysis of the metabolic cages was performed using the software ClockLab. (E) Daily water intake, food consumption and body weight changes in ISX-9 treated, as well as untreated groups of mice. Daily (F) intake and (G) food consumption records in ISX-9 treated, as well as untreated, young mice groups. Using unpaired Student's t test (ns, not significant, # p <0.05，**p<0.01，***p<0.001 Data are shown as mean ± SEM.

Figure 6 shows the sleep, activity, body temperature and diurnal brain electrical differences in young versus middle aged and elderly mice.

(A) Awake, NREM sleep and REM sleep time percentages for 2 months of age (blue, n=4) and 14 months of age mice (gray, n=8). The bottom represents the on period (white portion, 07:00-19:00) and the off period (black portion, 19:00-07:00), respectively. (B) Circadian activity and body temperature changes over a 24-hour period in 2 month old (blue, n=4) and 14 month old mice (gray, n=8). (C) EEG power analysis of 2 month old (blue, n=4) and 14 month old mice (gray, n=8). The ratio of the total power in circadian rhythms for delta (0.5-4 Hz), theta (4-8 Hz), alpha (8-12 Hz), sigma (12-16 Hz), beta (16-32 Hz) and gamma (> 32 Hz) powers, respectively, is shown. Data are shown as mean ± SEM using unpaired Student's t test (ns, not significant, < p 0.05, < p <0.01, < p < 0.001).

FIG. 7 shows the difference in sleep, activity, body temperature and day and night brain electricity of the middle-aged and elderly mice before and after ISX-9 treatment.

(A) Awake, NREM sleep and REM sleep time percentages of 14 month old mice treated with DMSO (gray curve, n=8) or ISX-9 (orange curve, n=8). The bottom represents the on period (white portion, 07:00-19:00) and the off period (black portion, 19:00-07:00), respectively. (B) Day and night activity and body temperature change over a 24 hour period in DMSO treated 2 month old mice (blue curve, n=4), DMSO (gray curve, n=8), or ISX-9 (orange curve, n=8) treated 14 month old mice. (C) Electroencephalographic power analysis of 14 month old mice treated with DMSO (gray curve, n=8) or ISX-9 (orange curve, n=8). The proportion of total power in the circadian rhythm is shown for delta (0.5-4 Hz), theta (4-8 Hz), alpha (8-12 Hz), sigma (12-16 Hz), beta (16-32 Hz) and gamma (> 32 Hz), respectively. (D) Number of state transitions between awake and sleep phases during the first hour of switching on (ZT 0-1) or off (ZT 12-13). Data are shown as mean ± SEM using unpaired Student's t test (ns, not significant, < p 0.05, < p <0.01, < p < 0.001).

FIG. 8 shows ISX-9 triggered Ca ²⁺ Inflow and increased CaMKII delta-related phosphorylation of BMAL 1.

(A) After DMSO (black curve) or 10. Mu.M ISX-9 (orange curve) treatment in MEF cells, ca ²⁺ Inflow measurement and measurement of Fluo-4AM fluorescence change (left). The right graph shows a representative image at a specified point in time; scale bar, 50 μm. Data are shown as mean ± SD using unpaired Student's t test. (B) PER 2-LUC fluctuation under DMSO (black curve), ISX-9 (red curve) and ISX-9 and CaMKII inhibitor KN93 (magenta curve) treatment conditions. (C) LUC fluctuations in PER2 at DMSO, ISX-9, and treatment with ISX-9 and knock down of Camk2a, camk2b, camk2d, or Camk2g alone. (D) The levels of BMAL1 phosphorylation in MEF cells following DMSO (D) or 10. Mu.M ISX-9 (9) treatment were analyzed using a Phos-tag gel. (E) In vitro kinase assays were performed on purified CaMKII α, β, δ and γ using purified BMAL1 as substrate. The highest activity was set to 1 to facilitate comparison between subtypes. Data represent mean ± SD. Assay using unpaired Student's t (.p)<0.05，**p<0.01，***p<0.001)。

FIG. 9 shows single molecule RNA fluorescence in situ hybridization of Camk2 subtype in adult mouse brain.

Single molecule RNA fluorescence in situ hybridization analysis of CamK2a, b, d and g in coronal brain sections at time points ZT2 or ZT14 from 2 month wild type mice. Camk2d showed higher expression in subcortical areas and hypothalamus. Camk2a and Camk2b showed significant, higher expression in striatum and cortex. Scale bar, 1000 μm.

FIG. 10 shows the analysis of the rhythmic oscillation of Camk2 subtype in the supraoptic nucleus by single molecule RNA fluorescence in situ hybridization.

SmFISH of CamK2a, b, d and g at the indicated times in SCN of 2 month old wild type mice. Expression levels in SCN were normalized after quantification and set to 1 at the first time point, data shown as mean ± SEM.

FIG. 11 shows that ISX-9 reactivates Camk2d expression in aged mouse SCN.

(A) The expression of CamK2a, b, d and g in SCN of 2 month old and 18 month old mice was aligned at ZT10 time point (SmFISH assay). Expression levels were quantified from SCN regions, and then the intensity of each Camk2 subtype in 2 month old mice was set to 1 (n=3/group). (B) Expression of CamK2a, b, d and g of SCN from 16 month old mice treated with DMSO or ISX-9 (SmFISH). Expression levels were quantified and the average brightness of DMSO control group was set to 1 (n=4/group). Data are shown as mean ± SEM using unpaired Student's t test (ns, not significant, ×p <0.05, ×p < 0.01).

FIG. 12 shows that Camk2d, as a well-defined biological clock control gene, induces and promotes expression of the clock gene Dbp: luc in response to ISX-9.

(A) E-box schematic of the mCamk2 promoter and construction of luciferase reporter plasmid. (B) Camk2: luc bioluminescence assay in N2a cells treated with DMSO or 10. Mu.M ISX-9. The value of the luminescence of the Luc in Camk2d treated with DMSO was set to 1. (C) In vitro kinase assays of purified CaMKII delta WT and K43M mutants, using BMAL1 as substrate. (D) Bioluminescence detection of Dbp: luc under conditions of expression of CaMKII delta WT or K43M mutant. Unpaired Student's t test (< p 0.05, < p 0.01, < p < 0.001) was used and the data are shown as mean ± SD.

FIG. 13 shows a schematic of the mechanism by which ISX-9 enhances circadian amplitude by activating BMAL1 phosphorylation function of CaMKII delta.

Detailed Description

The present inventors have made extensive and intensive studies, and as a result, have provided a use of ISX-9 for treating circadian rhythm disorders by a large number of screening and testing. The inventors have surprisingly found for the first time that ISX-9 can continuously and effectively increase circadian amplitude, treat sleep disorders, improve metabolic fluctuations in circadian rhythm, and treat circadian rhythm disturbances in elderly subjects in vivo, ex vivo, and in vitro environments without affecting circadian cycle.

Furthermore, the present invention further demonstrates the sensitization of Ca by ISX-9 ²⁺ Inflow, thereby enhancing CaMKII delta signal, and promotion of BMAL1 phosphorylation mediated by CaMKII delta, thereby increasing circadian amplitude in senescent cells. Thus, designs employing ISX-9 and targeting CaMKII delta are expected to be an important tool for intervention in circadian rhythm decay, even in aging-related diseases.

Terminology

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

As used herein, when used in reference to a specifically recited value, the term "about" means that the value can vary no more than 1% from the recited value. For example, as used herein, the expression "about 100" includes 99 and 101 and all values therebetween (e.g., 99.1, 99.2, 99.3, 99.4, etc.).

As used herein, the term "comprising" or "including" can be open, semi-closed, and closed. In other words, the term also includes "consisting essentially of …," or "consisting of ….

As used herein, the term "room temperature" or "normal temperature" refers to a temperature of 4-40 ℃, preferably 25±5 ℃.

As used herein, the term "plurality" or "plurality" refers to 2 or more, such as 2, 3, 4, 5 or 6, per species.

Active ingredient

The active ingredient of the invention is ISX-9 or pharmaceutically acceptable salt or deuterated product thereof.

According to one embodiment, the small molecule compound for treating circadian rhythm disorder is ISX-9, and the application concentration is 1-50 mu M.

The acidic fragments which may be included in the active ingredients of the present invention may form physiologically acceptable salts with various organic or inorganic bases. Typical base-forming salts include ammonium salts, alkali metal salts such as sodium, lithium, potassium salts, alkaline earth metal salts such as calcium, magnesium salts, and salts with organic bases (e.g., organic amines), such as benzathine, dicyclohexylamine, hydrabamine (salts with N, N-bis (dehydroabietyl) ethylenediamine), N-methyl-D-glucamine, N-methyl-D-glucamide, t-butylamine, and salts with amino acids such as arginine, lysine, and the like.

As used herein, the term "pharmaceutically acceptable" ingredient refers to a substance that is suitable for use in humans and/or animals without undue adverse side effects (such as toxicity, irritation, and allergic response), commensurate with a reasonable benefit/risk ratio.

As used herein, the term "deuterated" refers to the substitution of one or more hydrogens of the active ingredient of the present invention with deuterium. Deuteration may be mono-, di-, poly-or full-substituted.

As used herein, the term "effective amount" refers to an amount that is functional or active in and acceptable to a human and/or animal. It will be appreciated by those of ordinary skill in the art that the "effective amount" may vary depending on the form of the pharmaceutical composition, nutraceutical composition, the adjuvant used, the severity of the disease, and the combination with other drugs, nutraceuticals, or foods.

"promoting", "accelerating" as used herein includes improving circadian rhythm disorders or its associated sleep disorders, and does not necessarily require 100% cure. In some embodiments, the active ingredients of the present invention improve circadian rhythms by, for example, at least about 10%, at least about 30%, at least about 50%, or at least about 80% compared to when the active ingredients of the present invention are not present.

Circadian rhythm disorder

Biodysrhythmias (Circadian dysregulation) are accompanied by the occurrence of a variety of diseases, including metabolic disorders and aging.

The active ingredients of the invention are particularly suitable for middle-aged and elderly subjects.

Compositions and methods of administration

The composition of the present invention comprises ISX-9 or a pharmaceutically acceptable salt thereof as an active ingredient.

The compositions of the present invention comprise a safe and effective amount of ISX-9 or a pharmaceutically acceptable salt thereof, in combination with a pharmaceutically acceptable excipient or carrier.

Typically, the compositions of the present invention contain 1-2000mg of the active ingredient/agent of the present invention, more preferably 10-500mg of the active ingredient/agent of the present invention. Preferably, the "one dose" is a capsule or tablet.

"pharmaceutically acceptable carrier" means: one or more compatible solid or liquid filler or gel materials which are suitable for human use and must be of sufficient purity and sufficiently low toxicity. "compatibility" as used herein means that the components of the composition are capable of blending with and between the active ingredients of the present invention without significantly reducing the efficacy of the active ingredients. Examples of pharmaceutically acceptable carrier moieties are cellulose and its derivatives (e.g., sodium carboxymethylcellulose, sodium ethylcellulose, cellulose acetate, and the like), gelatin, talc, solid lubricants (e.g., stearic acid, magnesium stearate), calcium sulfate, vegetable oils (e.g., soybean oil, sesame oil, peanut oil, olive oil, and the like), polyols (e.g., propylene glycol, glycerol, mannitol, sorbitol, and the like), emulsifiers (e.g.

) Wetting agents (e.g. sodium lauryl sulphate), colouring agents, flavouring agents, stabilisers, antioxidants, anti-oxidantsA decay agent, pyrogen-free water, and the like.

The mode of administration of the active ingredients or compositions of the present invention is not particularly limited, and representative modes of administration include (but are not limited to): oral, rectal, parenteral (intravenous, intramuscular or subcutaneous).

Solid dosage forms for oral administration include capsules, tablets, pills, powders and granules. In these solid dosage forms, the active ingredient is admixed with at least one conventional inert excipient (or carrier), such as sodium citrate or dicalcium phosphate, or with the following ingredients: (a) Fillers or compatibilizers, for example, starch, lactose, sucrose, glucose, mannitol and silicic acid; (b) Binders, for example, hydroxymethyl cellulose, alginate, gelatin, polyvinylpyrrolidone, sucrose and acacia; (c) humectants, e.g., glycerin; (d) Disintegrants, for example, agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain complex silicates, and sodium carbonate; (e) a slow solvent, such as paraffin; (f) an absorption accelerator, e.g., a quaternary amine compound; (g) Wetting agents, such as cetyl alcohol and glycerol monostearate; (h) an adsorbent, for example, kaolin; and (i) a lubricant, for example, talc, calcium stearate, magnesium stearate, solid polyethylene glycol, sodium lauryl sulfate, or mixtures thereof. In capsules, tablets and pills, the dosage forms may also comprise buffering agents.

Solid dosage forms such as tablets, dragees, capsules, pills and granules can be prepared with coatings and shells, such as enteric coatings and other materials well known in the art. They may contain opacifying agents and the release of the active ingredient in such a composition may be released in a delayed manner in a certain part of the digestive tract. Examples of embedding components that can be used are polymeric substances and waxes. The active ingredient may also be in the form of microcapsules with one or more of the above excipients, if desired.

Liquid dosage forms for oral administration include pharmaceutically or nutraceutically acceptable emulsions, solutions, suspensions, syrups or tinctures. In addition to the active ingredient, the liquid dosage forms may contain inert diluents commonly used in the art such as, for example, water or other solvents, solubilizing agents and emulsifiers such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, propylene glycol, 1, 3-butylene glycol, dimethylformamide and oils, in particular, cottonseed, groundnut, corn germ, olive, castor and sesame oils or mixtures of these substances and the like.

In addition to these inert diluents, the compositions can also include adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, and perfuming agents.

Suspensions, in addition to the active ingredient, may contain suspending agents as, for example, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum methoxide and agar-agar or mixtures of these substances, and the like.

Compositions for parenteral injection may comprise physiologically acceptable sterile aqueous or anhydrous solutions, dispersions, suspensions or emulsions, and sterile powders for reconstitution into sterile injectable solutions or dispersions. Suitable aqueous and nonaqueous carriers, diluents, solvents or excipients include water, ethanol, polyols and suitable mixtures thereof.

Dosage forms of the active ingredients of the present invention for topical application include gels (e.g., injectable hydrogels), patches, ointments, powders, sprays and inhalants. The active ingredient is mixed under sterile conditions with a physiologically acceptable carrier and any preservatives, buffers, or propellants which may be required if necessary.

The active ingredients of the present invention may be administered alone or in combination with other therapeutic agents. In another preferred embodiment, the composition further comprises one or more additional therapeutic agents. Preferably, the therapeutic agent is selected from the group consisting of: atrasentan (atastant), hhAntag, OSI-930, GW4064, 2-NP, or combinations thereof.

In certain embodiments, the active ingredients of the present invention are used simultaneously with, or sequentially with, other agents that are part of a combination therapeutic regimen in the same or separate formulations.

The general range of therapeutically effective doses for the compositions of the active ingredients of the present invention will be: about 1 to 2000 mg/day, about 10 to about 1000 mg/day, about 10 to about 500 mg/day, about 10 to about 250 mg/day, about 10 to about 100 mg/day, or about 10 to about 50 mg/day. A therapeutically effective dose will be administered in one or more doses. However, it will be appreciated that the particular dosage of a compound of the invention for any particular patient will depend on a variety of factors, such as the age, sex, weight, general health, diet, individual response of the patient to be treated, the time of administration, the severity of the disease to be treated, the activity of the particular compound administered, the dosage form, the mode of application and concomitant medication. The therapeutically effective amount for a given situation can be determined by routine experimentation and is within the ability and judgment of a clinician or physician. In any event, the active ingredient or composition will be administered in multiple doses based on the individual circumstances of the subject to be administered and in a manner that allows for the delivery of a therapeutically effective amount.

Examples of subjects to which the active ingredients or compositions of the present invention are administered include mammals (e.g., humans, mice, rats, hamsters, rabbits, cats, dogs, cattle, sheep, monkeys, etc.).

The main advantages of the invention include:

1) ISX-9 can treat circadian rhythm disorder, and has excellent effects in enhancing amplitude, persistence and the like, and has dose-dependence, low cytotoxicity and no influence on circadian cycle;

2) The present invention demonstrates that Camk2d acts as a true clocked gene, regulating BMAL1 activity and forming a positive feedback cycle that, if enhanced, reverses the circadian decay caused by aging.

The invention will be further illustrated with reference to specific examples. It is to be understood that these examples are illustrative of the present invention and are not intended to limit the scope of the present invention. The experimental methods, in which specific conditions are not noted in the following examples, are generally conducted under conventional conditions or under conditions recommended by the manufacturer. Percentages and parts are weight percentages and parts unless otherwise indicated.

Materials and methods

Cell lines

HEK293T, NIH-3T3 and N2a cell lines were obtained from ATCC. Mouse embryoFetal fibroblast MEF from C57 wild type, bmal1 ^-/- Or mPer2 ^Luc/+ The mice were isolated from E13.5d embryos. Cells were cultured in DMEM (Invitrogen) containing 10% FBS and 100U/ml penicillin/streptomycin and 5% CO at 37 ℃C ₂ Is cultured in a humidified environment. The present invention uses a PCR-based method (MP 0035, sigma) to examine all cell lines, ensuring that all cell lines are mycoplasma free.

Animal strain

Reporting mouse strain mPer2 ^Luc (006852) and Bmal1-floxed condition line (007668) were obtained from Jackson Laboratory. Mice were housed in Specific Pathogen Free (SPF) animal housing facilities at a 12:12 hour light-dark cycle. Animal experiments were performed according to the protocols approved by the biomedical research ethics committee of scientific research, shanghai nutrition and health institute and the brain science center and the intelligent technical institute of the national academy of sciences.

Plasmid(s)

For luciferase-based expression assays, the mouse Camk2a/b/d/g, dbp and Rev-erba promoter fragments were PCR amplified from mouse genomic DNA and then cloned into pGL3-Basic-luc vector (E1751, promega) via MluI and BglII sites. Constructs for over-expression of Bmal1, FLAG-Camk2a, b, d and g were cloned into the pCAGGS vector with FLAG tag fusion via NotI and MluI sites. The Camk2d K43M mutant was generated using PCR-based site-directed mutagenesis and then subcloned into the same pCAGGS backbone.

Reagent(s)

Small molecules for biorhythmic testing were purchased from the following companies: ISX-9 is from Selleck; GW4064, OSI-930, atrasentan (atlas), forskolin and KN93 from Med Chem Express;2-NP and HhANtag were from TargetMol. Antibodies were purchased from the following sources: anti-BMAL 1 (14020,Cell Signaling Technology), anti-DBP (12662-1-AP, proteinTech), anti-PER 2 (13168, ABclonal), anti-REV-ERBα (13418,Cell Signaling Technology), anti-GAPDH (60004, proteinTech), HRP-conjugated goat anti-rabbit secondary antibody (1706515, bio-Rad) and HRP-conjugated goat anti-mouse secondary antibody (1706516, bio-Rad).

Small molecule screening

Primary small molecule screening was performed in 384 well plates (Natural Products library, NIH clinical series, LOPAC series and Spectrum series from Sigma-Aldrich; new compound library, epigenetic library and GPCR and G protein library from Selleck; approved drug screening library and inhibitor library from TargetMol; stem cell regulator library from Merck; neurotransmitter library from Tocris Bioscience; unique New compound library from MCE; and nuclear receptor ligand agonist or antagonist library from Enzo Life Sciences). The screening procedure used about 3000 mPer2 ^Luc MEF cells were inoculated for 48 hours and treated with 10 μm small molecule drug. After small molecule drug treatment, ONE-Glo was passed at two time points of 24 hours and 30 hours, respectively ^TM Luciferase assay System (E6120, promega) measures luciferase activity. After chemiluminescent measurement, cell viability was determined by alamarBlue assay (Invitrogen).

Real-time bioluminescence recording

To record PER 2:LUC bioluminescence in real time, approximately 2.5x10 was inoculated ⁵ mPer2 ^Luc MEF cells were then cultured in DMEM in 3.5cm dishes for 48 hours until the cells were full. Cells were then treated with 200nM dexamethasone, synchronized for 1 hour, and finally 200. Mu.M fluorescein (A5030, tokyo chemical industry) was added to serum-free DMEM medium and the specified small molecules at 10. Mu.M or specified concentrations were incubated and bioluminescence recorded for 4 days prior to bioluminescence recording. Recording conditions were continuous recording with the dishes in an opaque box (LumiCycle 32, actetics) equipped with a photon multiplier tube recording (PMT) detector assembly. The data were processed using LumiCycle analysis software (actetics), including results of amplitude and period.

Tissue culture for bioluminescence recording

Slice and view of upper core SCNReal-time bioluminescenceThe recording was performed as described by savely ev et al (savely ev et al, 2011). The basic steps are that mPer2 with the size of 6 months is firstly collected ^Luc Whole brain of mouseThe SCN fraction was then cut to 300 μm thickness by McIlwain Tissue Chopper (TC 752, cavey Laboratory Engineering co.ltd). SCN sections were placed on a culture membrane (PICM 0RG50, millipore) and 1.5ml recording medium (1 XDMEM, 1 XB 27 supplement, 4.2mM NaHCO) was added to the peripheral dishes ₃ 10mM HEPES, 1 Xpenicillin/streptomycin and 200. Mu.M fluorescein) were used for SCN tissue culture; the control group contained DMSO and the experimental group contained ISX-9 (10. Mu.M). The amplitude enhancement effect of ISX-9 was detected by continuous PMT recording (LumiCycle 32, actimetrics). The recording conditions of the pituitary are the same as those of SCN.

Quantitative RT-PCR analysis

Total RNA was extracted using the RNeasy Mini Kit (Qiagen) according to the manufacturer's instructions. Approximately 3. Mu.g of RNA was used for cDNA generation using Omniscript RT Kit (205111, qiagen). Real-time PCR reactions were assembled using SYBR Green PCR Kit (208054, qiagen) and then analyzed in ABI viiiatm 7. Quantum real-time PCR software was used to determine gene expression levels and normalize to the ribosomal reference gene, rpl19. The primer sequences are listed in Table 1.

TABLE 1 primer sequences

Immunoblotting

After cell harvest, cells were lysed in a frozen lysis buffer (50 mM Tris, 150mM NaCl, 5mM MgCl pH 7.5) supplemented with protease inhibitor (118735800, sigma-Aldrich) and phosphatase inhibitor (4906837001, roche) ₂ 0.5% Nonidet P-40 and 1% Triton X-100). The protein concentration of the cell lysates was measured by Bradford assay, after which the protein content of each sample was adjusted to be equal for quantitative immunoblot analysis. Phos-tag gel electrophoresis was performed by incubating cell lysates with 40. Mu.M acrylamide Phos-tag ligand (F4002, APExBIO) and 80. Mu.M MnCl ₂ Electrophoresis analysis was performed in a 4% acrylamide gel of (2).

Immunohistochemistry

Will be specialImmediately after euthanasia of age-fixed mice, the whole brain was removed, dehydrated and cryoprotected in 30% sucrose, and finally brain tissue was embedded in OCT embedding medium (4583, sakura). Coronal brain sections 30 μm thick were harvested using a frozen microtome (CM 1850, leica) and then passed through 3%H ₂ O ₂ The treatment quenches residual peroxidase activity in the sample. The antigen retrieval step was performed by 0.05% trypsin treatment for 5 min at room temperature, followed by blocking the brain sections with TNB blocking buffer (100 mM Tris-HCl, pH7.5, 150mM NaCl,10%FBS), followed by covering the brain sections with anti-PER 2 antibody dilution (1:300) and incubation overnight at 4 ℃. After 3 washes with TNT buffer (100 mM Tris-HCl, pH7.5, 150mM NaCl,0.05%Tween 20) at intervals, HRP-conjugated anti-rabbit secondary antibody (1:1000) was overlaid on brain sections and incubation was continued for 1.5 hours at room temperature. After 3 washes in TNT buffer, brain sections were incubated with fluorophore-labeled tyramine substrate (TSA plus Cyanine3 kit, perkin Elmer) and DAPI (10 ng/ml, sigma-Aldrich), and fluorescent labeling was observed by confocal microscopy (Cell bserver, ZEISS). Fluorescence histochemical staining results were analyzed and quantified by ImageJ.

Metabolism cage

Indirect calorimetric experiments in mice were performed by the CLAMS-16 metabolism cage system (Columbus Instruments). Groups of

mice

2 and 14 months old were treated with ISX-9 (20 mg/kg) or DMSO for 7 days, and the mice were individually housed in metabolic cages for 2 days for metabolic data collection. Sufficient conventional feed and drinking water was provided throughout the experiment. The VO of the mice was automatically measured every 10 minutes under 12:12 hours of light and shade cycle and 25 ℃ of temperature control ₂ 、VCO ₂ Food intake, water intake and activity, data were collected for 3 consecutive days. Calculation of physical activity and respiratory exchange ratio by companion software CLAX (Columbus Instruments) (rer=vco) ₂ /VO ₂ )。

Electroencephalogram recording and electroencephalogram data analysis

Electroencephalogram recording of mice was performed by HD-X02 telemetry of implants (DSI PhysioTel) ^TM ) Feeding inAnd (3) row. The implant implantation operation is firstly performed with deep anesthesia by single intramuscular injection of 5mg/kg pentobarbital (pentobarbital), and then two EEG electrodes are respectively fixed on the skull by small screws, and recording sites are 1mm outside the skull Bregma line, 1mm before the Bregma line and 3mm. Post-operative recovery was promoted by intraperitoneal injection of 5% dextrose solution (40 μl/g body weight) for 7 days in mice after surgery. Activity, body temperature, EEG, and EMG signals were collected by MX2 hub and RPC-1 receiver connected to a computer. First, 2 days were recorded under normal light and dark conditions as a pretreatment baseline. Mice of each age group were given a continuous record of EEG and EMG as control data for 3 days after 7 days of intraperitoneal injection of DMSO and 7 days of cessation of treatment. Finally, the same group of mice was injected with ISX-9 (20 mg/kg) as an experimental group for 7 days, stopped for 7 days, and then subjected to EEG and EMG recordings for 3 consecutive days. Mouse EEG sleep analysis awake, fast eye movement sleep REM and slow wave NREM sleep stages were analyzed and summarized using the matched software NeuroScore 3.2.0 (DSI) and the switching transitions of circadian sleep-wake were counted by slow wave, fast wave power over a 10 second time window. EEG power in each band (delta: 0.5-4Hz, theta: 4-8Hz, alpha: 8-12Hz, sigma: 12-16Hz, beta: 16-32Hz, gamma: >32 Hz) is shown expressed as an average data point per hour.

²⁺ In vitro Ca imaging

MEF cells (2 x10 ⁵ ) Inoculated onto a glass bottom petri dish (80002, NEST), and after 24 hours Fluo-4AM (2. Mu.M, molecular Probes) was incubated in serum-free DMEM for 45 minutes at 37 ℃. After washing unbound dye twice with PBS, ca was measured on cells ²⁺ Baseline (excitation 488nm, emission 520 nm) for 15 minutes. Recording Ca was started at the same time as DMSO or 10. Mu.M ISX-9 addition (time point 0) ²⁺ -inflow. Images were collected by fluorescence microscopy (Cell o server, ZEISS) at 1 minute intervals for 15 minutes to observe calcium influx.

siRNA knockdown

Silencing oligonucleotides for mouse Camk2a/b/d/g and scramble controls were purchased from GenePharma. MEF cells were transiently transfected with siRNA oligonucleotides using Lipofectamine 3000 (Invitrogen) and then subjected to real-time bioluminescence recording as described above under experimental conditions.

Protein purification and kinase assay

The pCAGGS plasmid (5. Mu.g) encoding FLAG-Bmal1, FLAG-Camk2 subtype and mutant was transfected to 2X10, respectively ⁶ In HEK293T cells, after 48 hours of expression, cells were harvested and lysed in 400 μl lysis buffer containing protease inhibitor (Roche). Approximately 3mg of cell lysate was incubated with 20. Mu.l of anti-FLAG-M2 magnetic beads (Sigma) for 4 hours at 4℃and then the impurities were washed 3 times with TBS buffer (50 mM Tris, pH7.5, 150mM NaCl) and finally the BMAL1 or CaMKII purified proteins were eluted through 100. Mu.l of 3 XFLAG short peptide (150 ng/. Mu.l, sigma). Use of kinase Activity kit (EA 004, R) &DSystems) in CaMKII: the kinase assay was performed at a ratio of BMAL 1:4, and finally the absorbance of the kinase reaction was measured at 620 nm.

Single molecule RNA in situ hybridization

Single molecule in situ hybridization was performed using an RNAscope multiplex fluorescent v 2-kit (Advanced Cell Diagnostics). The basic procedure was euthanized, perfused with 4% paraformaldehyde, carefully removed from the brain, fixed overnight in 4% paraformaldehyde, and then frozen for brain tissue protection by soaking in 30% sucrose-1 XPBS at 4deg.C. After embedding with OCT embedding solution, 30 μm coronal brain sections were collected, then dehydrated, quenched for peroxidase activity, and finally target RNA was exposed by Protease Plus solution (322381,Advanced Cell Diagnostics). After washing the sections with water, they were incubated with Camk2a, 2b, 2d, 2g of target probes (product catalog numbers 445231, 453601, 508941, 522071,Advanced Cell Diagnostics,1:300 dilution) in a HybEZ oven at 40℃for 2 hours. Amplification, HRP peroxidase incorporation and tyramine staining steps were performed as described in the user manual (323100-USM, advanced Cell Diagnostics), followed by DAPI counterstaining and fluorescent microscopy photographs (VS 120, OLYMPUS). Images were analyzed and quantified by ImageJ.

Luciferase assay

N2a cells were plated at 8X10 per well 24 hours prior to transfection ⁴ The density of individual cells was seeded into 24-well dishes.Bmal1 reporter plasmid and Camk2 overexpression plasmid were transfected with Lipofectamine 3000 (Invitrogen) at a ratio of 200:100ng per well. After 24 hours, cells were treated with 100nM dexamethasone (dexamethasone) for 15 minutes for synchronization and treated with DMSO (control) or 10. Mu.M ISX-9 (experimental) in DMEM. Cells were harvested 24 hours later and lysed in 100. Mu.l 1 Xglo lysis buffer (E266A, promega) and incubated with substrate (ONE-Glo ^TM Luciferase detection system, E6120, promega) was mixed for luciferase measurement.

Quantification and statistical analysis

Individual in vitro experiments were performed at least 3 times unless noted otherwise. Animal experiments were repeated at least twice independently and consistent dosing effect was determined. Results are expressed as mean ± SD or mean ± SEM, as shown in the legend. The statistical significance is marked as insignificant, ns; * p <0.05; * P <0.01; * P <0.001.

Example 1. Small molecule screening ISX-9 was identified as a circadian amplitude enhancer.

In order to find new biological rhythm regulation small molecules for more durable enhancement of biological clock amplitude and to minimally interfere with 24 hour circadian cycles, the present invention is directed to a method of enhancing biological clock amplitude by mPer2 ^Luc Fibroblasts were screened for 8060 small molecule compounds, which covered major targets such as kinases, epigenetic factors, and GPCRs (fig. 1A).

To verify amplitude and phase changes in high throughput primary screening, the inventors measured mPer2 at 24h and 30h, respectively, after drug treatment ^Luc Chemiluminescence levels of fibroblasts (fig. 1A and B). A compound may be described as an amplitude modulator if it is found that the effect of the compound on the up-or down-regulation of the expression of LUC at two time points is consistent compared to the untreated control; if the effects on the two time points are opposite, it can be stated that the compound is a phase modifier.

After the elimination of compounds with significant cytotoxicity, the invention focuses on the remaining 6777 compounds and gives 270 amplitude enhancers capable of increasing PER 2:LUC amplitude by a factor of 1.2. Among them, ISX-9, atrasentan (atasten), hhANtag, OSI-930, GW4064 and 2-NP are the main candidates for increasing PER2:: LUC level (FIG. 1C).

Real-time recordings of PER 2:LUC luminescence demonstrated that all six drug candidates were able to increase PER 2:LUC amplitude with a rhythmic period maintained near 24 hours without affecting circadian phase (FIG. 1D). Notably, most drug candidates significantly enhanced PER 2:LUC amplitude only on day 1, and then significantly decreased in effect for the remaining 3 days (FIG. 1E).

ISX-9 is the only amplitude enhancer that continuously maintains rhythmic oscillations in the 4-day test. The amplitude enhancement of ISX-9 is broad and can be used to boost the expression of other important rhythmic genes, such as Dbp and Rev-Erbα (FIG. 1F). In these cases, ISX-9 again showed its optimal ability to modulate biological clocks among the six drug candidates.

ISX-9 is an isoxazole compound and has neurogenic activity (FIG. 2A). In the test of the present invention it was found that ISX-9 had a dose dependent effect on increasing PER 2:LUC amplitude (FIG. 2B) and still showed low cytotoxicity at the highest concentration tested of 10. Mu.M (FIG. 2C).

In summary, ISX-9 has excellent effects in enhancing amplitude, persistence, etc., and has dose-dependence, low cytotoxicity, and does not affect circadian cycle, so ISX-9 can be used as a circadian rhythm amplitude enhancer.

Example 2. Relationship of ISX-9 to biological clock genes in cultured cells.

As can be seen from FIG. 3, in agreement with the luciferase assay, ISX-9 increased the expression of biological clock genes including Bmal1, per2, dbp, and other core biological clock genes in Mouse Embryo Fibroblasts (MEFs), and the like.

DBP protein levels are highly sensitive to ISX-9 doses compared to BMAL1 and REV-ERBα (FIG. 4A) and thus can be used as a biological clock protein marker for reporting ISX-9 effects in the peripheral system.

Interestingly, the inventors found that ISX-9 induced DBP and PER2 to LUC at Bmal1 ^+/- Attenuation in mouse embryonic fibroblasts, bmal1 ^-/- Mouse embryo fibroblast middle nodeThe rhythmicity disappeared, indicating that the amplitude enhancement effect of ISX-9 was dependent on BMAL1 activity (fig. 4B and C).

Example 3 in vitro, ex vivo and in vivo amplitude enhancement effects of ISX-9

As can be seen from FIG. 4, ISX-9 is also capable of stimulating higher PER2:: LUC rhythm amplitude in the visual cross upper core (SCN) and pituitary equivalent external culture recordings (FIG. 4D); and mice seven days after administration of ISX-9 showed higher PER2 rhythmin levels in SCN (fig. 4E), demonstrating overall the amplitude enhancing effect of ISX-9 in cells, in vitro tissues, and in vivo.

EXAMPLE 4 in vivo modulation of the metabolism of ISX-9 in senior mice

To explore the metabolic regulation of ISX-9 in vivo, the present invention further tested the metabolic rate of young (2 months of age) and middle aged and elderly (14 months of age) mice.

Firstly, establishing a steady-state environment of a circadian rhythm, waking up a mouse in a dark environment under a light-dark cycle of 12:12 hours, and enabling the mouse to enter a rest state in a bright environment; circadian rhythm disorder experimental group is 14 month-old mice.

Young mice exhibited significant circadian fluctuations in Respiratory Exchange Ratio (RER) and showed lower RER in the resting/fasted state of the day (fig. 5A). Middle-aged and elderly groups showed poor ren rhythmicity in the metabolic cages, with only a brief increase in ren in the last half of the night, representing the carbohydrate metabolism after eating (fig. 5A).

In vivo injection of ISX-9 in the middle aged and elderly mice increased the magnitude of RER (FIG. 5B), and did not alter the magnitude of RER in the young group (FIG. 5C), indicating the applicability of ISX-9 in rhythm regulation during aging. In addition, the present invention found that ISX-9 was able to correct the RER cycle from 26 hours to 24 hours in the middle aged and elderly group of mice (fig. 5D), without affecting food intake and body weight in the middle aged and elderly group, except for slightly promoting night time water intake (fig. 5E).

Since ISX-9 treatment did not affect the young population (FIGS. 5F and 5G), the results indicate that ISX-9 selectively ameliorates the metabolic disorders associated with aging. Since treatment of ISX-9 was performed prior to the metabolic cage experiment, the experimental results further demonstrate that ISX-9 is sustainable in promoting circadian amplitude in vivo.

EXAMPLE 5 ISX-9 improvement of sleep disorders in middle aged and elderly mice

ISX-9 experimental group showed lower O during daytime ₂ Consumption and CO ₂ This compound was produced, indicating that it can make rest of middle-aged and elderly mice more stable (fig. 5B). To investigate whether ISX-9 can indeed improve sleep disorders, the present invention performed telemetry recording of electroencephalograms (EEG) and analysis of sleep status in young and middle aged mice.

By comparison, the invention finds that the difference between wakefulness and slow wave sleep (NREM) of two groups of mice at night is most remarkable. The middle aged and elderly group exhibited higher NREM sleep during the night's active period, showing lower wakefulness (fig. 6A). The middle aged and elderly mice also exhibited a circadian rhythm disorder phenotype with greatly reduced activity, reduced body temperature fluctuations (FIG. 6B), reduced beta/gamma fast waves and insignificant delta/theta slow wave fluctuations (FIG. 6C) compared to the young mice, which indicated an overall decrease in intensity of circadian rhythms in the brain during aging.

To test whether ISX-9 can induce circadian rhythm and enhance sleep homeostasis, the inventors administered 20mg/Kg of ISX-9 to the same group of mice for 7 consecutive days, and began EEG recording 7 days after stopping the treatment.

The present invention found that pretreatment with ISX-9 greatly improved the mid-night wakefulness of middle-aged and old mice while reducing fast eye REM and slow wave NREM sleep (fig. 7A). The treated middle-aged and elderly mice also exhibited higher activity at night, while at rest at lower body temperature during the day (fig. 7B), which results indicate that ISX-9 assisted in adjusting the middle-aged and elderly mice to a more alert, active state while awake, and maintained a better, lower body temperature resting state during sleep. The present invention also notes that treated middle aged and elderly mice have higher slow wave delta power during rest and higher fast wave gamma power during wakefulness, both of which show an improvement in brain electrical circadian rhythm homeostasis (fig. 7C).

By calculating the transition from awake-sleep phase upon switching on (ZT 0-1) and off (ZT 12-13), the present invention found that ISX-9 significantly inhibited frequent awake-sleep transitions in the middle aged and elderly group, causing the middle aged and elderly group to approach a more stable sleep-awake steady state observed in healthy young mice (fig. 7D).

EXAMPLE 6 ISX-9 triggering Ca ²⁺ Inflow and associated with Camk2d Activity

Previous studies demonstrated that ISX-9 can pass through Ca ²⁺ Signaling promotes neuronal differentiation. The invention first tracks intracellular Ca in MEF by Fluo-4 AM fluorochromes ²⁺ Level, and confirm that ISX-9 can trigger Ca rapidly in the test ²⁺ Inflow (fig. 8A).

Consistent with previous reports, the present invention also found that ISX-9 effects were mediated by CaMKII activity, as the CaMKII specific antagonist KN93 completely abrogated the ISX-9 enhancement of PER 2:LUC (FIG. 8B). Of the four CaMKII subtypes, the inventors noted that Camk2b and Camk2d knockdown significantly reduced the ISX-9 effect in real-time PER 2:LUC recordings, but not in Camk2a and Camk2g knockdown (FIG. 8C), showing that the effect of ISX-9 was more dependent on both the CaMKII beta and CaMKII delta subtypes.

Whereas ISX-9 showed a high degree of BMAL1 dependence at elevated DBP and PER 2:LUC levels (FIGS. 4B and C) and triggered phosphorylation of BMAL1 (FIG. 8D), the present invention further examined the activity of CaMKII on phosphorylation of BMAL1 by biochemical methods and compared differences between CaMKII subtypes.

CaMKII delta showed the highest activity on purified BMAL1 in the kinase assay (FIG. 8E), along with the results of Camk2d knockdown affecting PER2:: LUC expression, which data demonstrate that CaMKII delta is critical for mediating BMAL1 dependent ISX-9 effects. These results suggest a junction ISX-9, ca ²⁺ Signal and BMAL1 phosphorylate to regulate the forward feedback loop of biorhythmic amplitude.

Single molecule RNA fluorescence in situ hybridization (smFISH) analysis showed that Camk2d was more abundantly expressed in subcortical and hypothalamic areas in the mouse brain (fig. 9) and showed clear rhythmic expression that peaked at ZT18 time point in the biological Zhong Zhongshu visual crossing upper core (SCN) (fig. 10), well in agreement with the potential function expected to modulate circadian rhythms. Other CaMKII subtype transcripts, such as Camk2a and Camk2b, showed slight fluctuations and low expression in SCN, with expression more enriched in striatum and cortex (fig. 9). Further, the circadian rhythm effect of ISX-9 on SCN is related to Camk2 d.

EXAMPLE 7 reactivation of Camk2d expression in aged mouse SCN by ISX-9

The significant decrease in Camk2d in SCN in aged mice (FIG. 11A), while ISX-9 increased Camk2d expression (FIG. 11B), suggested that ISX-9 was beneficial in re-increasing CamKII delta levels during aging. The addition of CaMKII delta can up-regulate BMAL1 transcriptional activity by phosphorylating BMAL1, further reversing the circadian rhythm amplitude decline caused by aging. The expression levels of Camk2a and Camk2B were unchanged in the older SCN and did not respond to ISX-9 treatment (FIGS. 11, A and B).

Example 8 Camk2d appears to be a true clock control gene that induces and facilitates control of biological clocks in response to ISX-9.

Whereas Camk2d expression is rhythmic and can be triggered by ISX-9, the present invention searches for specific E-box sequences in the Camk2 promoter. The present invention found that all of the Camk2 subtypes contained E-box elements, including classical E-box sequences (CACGTG) in both the Camk2d and Camk2g promoters (FIG. 12A). After analysis using promoter reporting in N2a cells, the present invention found that the Camk2d promoter was most sensitive to CLOCK: BMAL1 activation, verifying that Camk2d is a well-defined biological CLOCK control gene (FIG. 12B).

In addition, ISX-9 may support the transcription factor CLOCK BMAL1 further induced Camk2 and showed the best effect on Camk2d expression (FIG. 12B), consistent with the effect in aged SCN (FIG. 11B). To demonstrate the direct effect of CaMKII delta on BMAL1 phosphorylation, the inventors purified the CaMKII delta kinase inactivated K43M mutant and detected a significant decrease in BMAL1 phosphorylation by biochemical methods (fig. 12C). In addition, caMKII delta K43M mutants had significantly reduced effect on activation of Dbp: luc expression (FIG. 12D), again confirming that CaMKII delta can up-regulate biorhythmic amplitude by phosphorylating BMAL 1.

Taken together, the results indicate that CaMKII delta acts as a biological clock-regulated clock protein, modulating BMAL1 activity and forming a positive feedback cycle, enhancing this positive feedback cycle, e.g., using CaMKII agonist ISX-9, reversing circadian decay caused by aging, rejuvenating aging guests (fig. 13).

Discussion of the invention

The invention provides an intervention measure based on a small molecular medicine, which is used for effectively and continuously amplifying circadian amplitude and solving the problem of gradual attenuation of the circadian amplitude in the elderly. Part of the reason for this attenuation is that the amount of CaMKII delta expressed decreases during aging, thus limiting the ability to regulate biological clocks by CaMKII delta phosphorylating and initiating BMAL 1.

Previous work has shown that calcium fluctuations are the fundamental signal for circadian rhythm generation in the supranuclear of the visual junction, the present invention further demonstrates sensitization of Ca by ISX-9 ²⁺ Inflow is a convenient way to boost the CaMKII delta signal and thereby increase the circadian amplitude of senescent cells. In addition to the neurogenic activity of ISX-9, and the improvement of neurodegenerative diseases, the application of ISX-9 is expected to have potential in anti-aging, especially in the treatment of nervous system diseases.

In summary, designs employing ISX-9 and targeting CaMKII delta are expected to be important tools for intervention in circadian rhythm decay, even in aging-related diseases.

All documents mentioned in this application are incorporated by reference as if each were individually incorporated by reference. Further, it will be appreciated that various changes and modifications may be made by those skilled in the art after reading the above teachings, and such equivalents are intended to fall within the scope of the claims appended hereto.

Claims

1. Use of ISX-9, said ISX-9 having the structure according to the formula:

characterized in that said ISX-9 is used for preparing a composition for one or more uses selected from the group consisting of: (i) preventing and/or treating circadian rhythm disorders; (ii) preventing and/or treating sleep disorders; (iii) increasing circadian amplitude; and/or (iv) increasing the activity or expression level of the CaMKII delta protein.

2. The use according to claim 1, wherein the composition is for (i) preventing and/or treating circadian rhythm disorders in a middle aged and elderly subject; (ii) preventing and/or treating sleep disorders; (iii) increasing circadian amplitude; and/or (iv) increasing the activity or expression level of the CaMKII delta protein.

3. Use according to claim 1, wherein the prevention and/or treatment of circadian rhythm disorders refers to an improvement in one or more indicators selected from the group consisting of:

(i) Improving circadian amplitude;

(ii) Improving metabolic circadian rhythmicity fluctuations;

(iii) Preventing and/or treating circadian rhythm sleep disorder.

4. The use according to claim 1, wherein the prevention and/or treatment of circadian rhythm disorder comprises increasing activity of a biological clock gene, and wherein the biological clock gene is selected from the group consisting of: bmal1, per2, dbp, clock, cry1, cry2, npas2, nr1d1, rorα, or combinations thereof.

5. The use according to claim 1, wherein the prevention and/or treatment of circadian rhythm disorders comprises increasing circadian amplitude by increasing BMAL1 activity.

6. The use according to claim 1, wherein the prevention and/or treatment of circadian rhythm disorders comprises enhancing BMAL1 activity and increasing expression of CaMKII δ itself.

7. Use of deuterated species of ISX-9, said ISX-9 having a structure according to the formula:

characterized in that said deuterated forms of ISX-9 are used for preparing a composition for one or more uses selected from the group consisting of: (i) preventing and/or treating circadian rhythm disorders; (ii) preventing and/or treating sleep disorders; (iii) increasing circadian amplitude; and/or (iv) increasing the activity or expression level of the CaMKII delta protein.

8. Use of a CaMKII delta high expression modulator for the preparation of a composition for one or more uses selected from the group consisting of: (i) preventing and/or treating circadian rhythm disorders; and/or (ii) modulating BMAL1 activity.

9. The use according to claim 8, wherein the prevention and/or treatment of circadian rhythm disorders comprises: by promoting the phosphorylation of BMAL1, the activity of BMAL1 and the expression of CaMKII delta are improved.

10. A pharmaceutical composition, the pharmaceutical composition comprising: ISX-9 or a deuterated or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.