WO2023130675A1 - Isx-9在治疗衰老相关的昼夜节律幅度下降和睡眠障碍方面的应用 - Google Patents

Isx-9在治疗衰老相关的昼夜节律幅度下降和睡眠障碍方面的应用 Download PDF

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WO2023130675A1
WO2023130675A1 PCT/CN2022/099218 CN2022099218W WO2023130675A1 WO 2023130675 A1 WO2023130675 A1 WO 2023130675A1 CN 2022099218 W CN2022099218 W CN 2022099218W WO 2023130675 A1 WO2023130675 A1 WO 2023130675A1
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isx
circadian rhythm
composition
increasing
bmal1
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PCT/CN2022/099218
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English (en)
French (fr)
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张洪钧
唐芸棋
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中国科学院脑科学与智能技术卓越创新中心
中国科学院上海营养与健康研究所
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Publication of WO2023130675A1 publication Critical patent/WO2023130675A1/zh

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/41—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with two or more ring hetero atoms, at least one of which being nitrogen, e.g. tetrazole
    • A61K31/42—Oxazoles
    • A61K31/422—Oxazoles not condensed and containing further heterocyclic rings
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/20—Hypnotics; Sedatives
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00

Definitions

  • the invention relates to the field of medicine, in particular to the application of ISX-9 in the treatment of aging-related circadian rhythm amplitude decline and sleep disorders.
  • the circadian clock is an intrinsic timekeeper that coordinates a variety of physiological activities in an orderly manner, including circadian hormone cycles, eating, sleeping, body temperature, and metabolic activity. Disruption of circadian rhythms in model organisms can mimic a variety of symptoms associated with human diseases, such as cardiovascular dysfunction, diabetes, cancer, sleep disorders, depression, and neurodegenerative diseases. It is worth noting that all the above-mentioned diseases are highly related to the aging process, so the decline in the strength of the biological clock is also considered as one of the indicators of aging. Given that decreased circadian rhythm amplitude is a common feature of impaired health, interventions aimed at preventing circadian rhythm amplitude decline and shrinkage are expected to have applications in the treatment of circadian rhythm disorders and their associated diseases.
  • the purpose of the present invention is to provide an application of ISX-9 that can be stable, sustained, and effective in treating circadian rhythm disorders and related diseases of elderly subjects in vivo, in vitro, and in vitro.
  • ISX-9 a kind of purposes of ISX-9 is provided, and described ISX-9 has the structure shown in the following formula:
  • the ISX-9 is used to prepare a composition, and the composition is used for one or more purposes selected from the following group: (i) preventing and/or treating circadian rhythm disorders; (ii) preventing and/or treating Sleep disturbance; (iii) increasing the amplitude of circadian rhythm; and/or (iv) increasing the activity or expression of CaMKII ⁇ protein.
  • the composition is a pharmaceutical composition or a health product composition.
  • the composition is used to (i) prevent and/or treat circadian rhythm disorders; (ii) prevent and/or treat sleep disorders; (iii) increase the amplitude of circadian rhythm in middle-aged and elderly subjects; And/or (iv) increasing the activity or expression of CaMKII ⁇ protein.
  • the middle-aged and elderly subjects refer to subjects whose age exceeds 2/3 of the average life expectancy, and more preferably exceeds 3/4 of the average life expectancy.
  • the middle-aged and elderly subjects are subjects aged ⁇ 45 years old.
  • the middle-aged and elderly subject is a subject with a monthly age ⁇ 12 months, more preferably a monthly age ⁇ 14 months.
  • the circadian rhythm disorder includes circadian rhythm disorder caused by aging.
  • the prevention and/or treatment of circadian rhythm disorders refers to the improvement of one or more indicators selected from the following group:
  • the increase of the circadian rhythm amplitude means that compared with the control without ISX-9 administration, the administration of ISX-9 increases the circadian rhythm amplitude by at least 1.2 times, preferably at least 1.5 times, such as at least 2 times, at least 3 times, at least 5 times.
  • the composition increases the amplitude of circadian rhythm without changing the cycle of circadian rhythm.
  • the composition is persistent for more than 3 days, preferably within 4-14 days, more preferably within 7-14 days.
  • the prevention and/or treatment of circadian rhythm disorders includes increasing the activity of biological clock genes, and the biological clock genes are selected from the group consisting of Bmal1, Per2, Dbp, Clock, Cry1, Cry2, Npas2, Nr1d1, Ror ⁇ , or a combination thereof.
  • the prevention and/or treatment of circadian rhythm disorders can be performed in cells, tissues in vitro and/or in vivo.
  • the cells include mouse embryonic fibroblasts.
  • the extracorporeal tissues include suprachiasmatic nucleus (SCN) and/or pituitary gland.
  • the composition does not affect the circadian rhythm of the young subjects.
  • the young subject refers to a subject whose age does not exceed 2/3 of the average life expectancy, and more preferably exceeds 1/2 of the average life expectancy.
  • the young subject when the subject is a human, is a subject whose age is ⁇ 30 years old.
  • the young subject when the subject is a mouse, is a subject whose age is ⁇ 8 months, more preferably ⁇ 4 months, more preferably ⁇ 2 months.
  • the prevention and/or treatment of circadian rhythm disorder includes increasing the circadian rhythm amplitude by increasing the activity of BMAL1.
  • said increasing BMAL1 activity includes promoting BMAL1 phosphorylation.
  • the promotion of BMAL1 phosphorylation is mediated by CaMKII ⁇ .
  • increasing the activity of BMAL1 can further promote the expression of CaMKII ⁇ itself.
  • the composition is used to promote Ca 2+ influx.
  • CaMKII ⁇ up-regulates BMAL1 transcriptional activity by phosphorylating BMAL1 after responding to Ca 2+ signals.
  • the prevention and/or treatment of circadian rhythm disorders includes enhancing BMAL1 activity and increasing the expression of CaMKII ⁇ itself.
  • the prevention and/or treatment of circadian rhythm disorders includes increasing the amplitude of circadian rhythm through the positive feedback loop of CaMKII ⁇ and BMAL1, wherein the positive feedback loop includes the phosphorylation of BMAL1 mediated by ISX-9 through CaMKII ⁇ Enhanced BMAL1 activity, and increased BMAL1 activity further increased the expression of CaMKII ⁇ itself.
  • the improved circadian fluctuation of metabolism has one or more of the following characteristics:
  • (a-5) does not affect food intake and/or body weight of the subject.
  • the prevention and/or treatment of sleep disorders has one or more of the following characteristics:
  • the composition contains ISX-9 or a pharmaceutically acceptable salt thereof at a concentration ⁇ 0.1 ⁇ M, preferably ⁇ 0.25 ⁇ M, more preferably ⁇ 10 ⁇ M.
  • the composition contains 0.1-99wt%, preferably 0.5-95wt%, more preferably 1-90wt% of ISX-9 or a pharmaceutically acceptable salt thereof.
  • composition further includes pharmaceutically acceptable excipients and/or carriers.
  • the dosage form of the composition is selected from the group consisting of solid preparation, liquid preparation or semi-solid preparation.
  • the dosage form of the composition is selected from the group consisting of gel, patch, tablet, capsule, powder, ointment, powder, injection, water, enteric sustained-release preparation or injection.
  • the dosage form of the composition is injection.
  • composition is administered to the subject by oral administration, transdermal injection, intravenous injection, intramuscular injection or anorectal administration.
  • the ISX-9 has a structure as shown in the following formula:
  • the deuterated substance of ISX-9 is used to prepare a composition, and the composition is used for one or more purposes selected from the following group: (i) preventing and/or treating circadian rhythm disorders; (ii) preventing and /or treating sleep disorders; (iii) increasing the amplitude of circadian rhythm; and/or (iv) increasing the activity or expression of CaMKII ⁇ protein.
  • composition is as described in the first aspect of the present invention.
  • a use of a CaMKII ⁇ high expression regulator is provided for preparing a composition, and the composition is used for one or more purposes selected from the following group: (i) preventing and/or or treating circadian rhythm disorders; and/or (ii) modulating BMAL1 activity.
  • the CaMKII ⁇ is rhythmically expressed by Camk2d.
  • the composition is used to increase the expression level of CaMKII ⁇ in the subcortical area and hypothalamus.
  • the prevention and/or treatment of circadian rhythm disorders includes: increasing BMAL1 activity and increasing the expression of CaMKII ⁇ itself by promoting BMAL1 phosphorylation.
  • the prevention and/or treatment of circadian rhythm disorder is as described in the first aspect of the present invention.
  • composition further includes pharmaceutically acceptable excipients and/or carriers.
  • the dosage form of the composition is selected from the group consisting of solid preparation, liquid preparation or semi-solid preparation.
  • the dosage form of the composition is selected from the group consisting of gel, patch, tablet, capsule, powder, ointment, powder, injection, water, enteric sustained-release preparation or injection.
  • the dosage form of the composition is injection.
  • composition is administered to the subject by oral administration, transdermal injection, intravenous injection, intramuscular injection or anorectal administration.
  • a pharmaceutical composition comprising:
  • ISX-9 or its deuterated product or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier are examples of pharmaceutically acceptable carriers.
  • the pharmaceutical composition may include other circadian rhythm regulators, wherein the circadian rhythm regulators are selected from the group consisting of Atrasentan, HhAntag, OSI-930, GW4064, 2- NP, or a combination thereof.
  • the dosage form of the pharmaceutical composition is selected from the group consisting of solid preparation, liquid preparation or semi-solid preparation.
  • the dosage form of the pharmaceutical composition is selected from the following group: gel, patch, tablet, capsule, powder, ointment, powder, injection, water, enteric sustained-release preparation or injection .
  • the dosage form of the pharmaceutical composition is injection.
  • the dosage form of the pharmaceutical composition is selected from the group consisting of oral dosage form, transdermal injection dosage form, intravenous injection dosage form, and intramuscular injection dosage form.
  • the injection of the injection contains one or more of physiological saline, glucose, stabilizer, preservative, suspending agent or emulsifying agent.
  • a method for (i) preventing and/or treating circadian rhythm disorders; (ii) preventing and/or treating sleep disorders; and/or (iii) increasing the amplitude of circadian rhythm comprising the steps : administering a safe and effective amount of ISX-9 or a pharmaceutically acceptable salt thereof to a subject in need thereof.
  • the subject is a middle-aged and elderly subject.
  • the middle-aged and elderly subjects are as described in the first aspect of the present invention.
  • the subject is a mammal, such as human, mouse, rat, hamster, rabbit, cat, dog, cow, sheep, monkey, etc.
  • the effective concentration of ISX-9 or a pharmaceutically acceptable salt thereof is 0.1-50 ⁇ M, more preferably 0.25-10 ⁇ M.
  • Figure 1 shows the identification of ISX-9 as a biorhythm amplitude enhancer by small molecule screening.
  • FIG. 1C (A) Schematic of the compound screening procedure and validation.
  • B Heatmap of primary screening results for 6777 small molecules (left). Points represent relative values of PER2::LUC bioluminescence at 24 hr and 30 hr time points, normalized to vehicle control.
  • C Real-time bioluminescence recordings of mPer2 Luc MEF cells treated with each small molecule (red curve), showing the amplitude-enhancing effect of the small molecule. Controls are rhythmic fluctuations treated with DMSO (shown in black curve).
  • D Cycle analysis after small molecule treatment associated with Figure 1C. 9: ISX-9; A: Atrasentan; H: HhAntag; O: OSI-930; G: GW6064 and 2: 2-NP.
  • Figure 2 shows that ISX-9 enhances the circadian rhythm amplitude of mPer2 Luc MEFs in a dose-dependent manner.
  • FIG. 3 shows that ISX-9 enhances circadian clock gene expression in MEF cells.
  • FIG. 4 shows that the circadian rhythm amplitude enhancement effect of ISX-9 is dependent on the clock protein BMAL1.
  • A Protein levels of BMAL1, REV-ERB ⁇ , and DBP were detected by immunoblotting after increasing doses of ISX-9 treatment.
  • B Bmal1 wild-type (WT), heterozygous (HET) or knockout (KO) MEF cells were treated with DMSO (D) or 10 ⁇ M ISX-9 (9), and then 36 hours or 48 hours after treatment Cells were harvested at time points for immunoblotting detection of BMAL1 and DBP.
  • C PER2::LUC real-time bioluminescence recordings of Bmal1 wild-type (WT), heterozygous (HET) or knockout (KO) MEF cells treated with DMSO (D) or 10 ⁇ M ISX-9 (9).
  • Figure 5 shows that ISX-9 rejuvenates the circadian metabolism of middle-aged and aged mice.
  • Figure 6 shows the differences in sleep, activity, body temperature, and diurnal EEG of young versus middle-aged and aged mice.
  • Figure 7 shows the differences in sleep, activity, body temperature and diurnal EEG of middle-aged and aged mice before and after ISX-9 treatment.
  • Figure 8 shows that ISX-9 triggers Ca 2+ influx and increases CaMKII-associated BMAL1 phosphorylation.
  • A Schematic representation of Ca influx measurements and Fluo-4AM fluorescence changes following DMSO (black curve) or 10 ⁇ M ISX-9 (orange curve) treatment in MEF cells (left). Right panels show representative images at indicated time points; scale bar, 50 ⁇ m. Data are shown as mean ⁇ SD using unpaired Student's t-test.
  • B Fluctuation of PER2::LUC under the treatment conditions of DMSO (black curve), ISX-9 (red curve) and ISX-9 with CaMKII inhibitor KN93 (magenta curve).
  • C PER2::LUC fluctuations in DMSO, ISX-9, and ISX-9 treatment with knockdown of Camk2a, Camk2b, Camk2d, or Camk2g alone.
  • Figure 9 shows single molecule RNA fluorescence in situ hybridization of Camk2 isoforms in adult mouse brain.
  • Camk2a, b, d, and g Single-molecule RNA fluorescence in situ hybridization analysis of CamK2a, b, d, and g in coronal brain sections from 2-month-old wild-type mice at time point ZT2 or ZT14.
  • Camk2d showed higher expression in subcortical areas and hypothalamus.
  • Camk2a and Camk2b showed marked, higher expression in the striatum and cortex.
  • Scale bar 1000 ⁇ m.
  • Figure 10 shows the rhythmic oscillations of Camk2 isoforms in the suprachiasmatic nucleus analyzed by single-molecule RNA fluorescence in situ hybridization.
  • Figure 11 shows that ISX-9 reactivates Camk2d expression in the SCN of aged mice.
  • (A) Comparison of the expression of CamK2a, b, d and g in the SCN of 2-month-old and 18-month-old mice at ZT10 time point (SmFISH detection). Expression levels were quantified from SCN regions, then the intensity of each Camk2 isoform in 2-month-old mice was set to 1 (n 3/group).
  • (B) Expression of CamK2a, b, d, and g in the SCN of 16-month-old mice treated with DMSO or ISX-9 (SmFISH). Expression levels were quantified and the mean brightness of the DMSO control group was set at 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 clear circadian clock control gene, can induce and promote the expression of the clock gene Dbp:Luc in response to ISX-9.
  • Figure 13 shows a schematic diagram of the mechanism by which ISX-9 enhances the amplitude of circadian rhythm by activating the BMAL1 phosphorylation function of CaMKII ⁇ .
  • the present inventor provides a use of ISX-9 in the treatment of circadian rhythm disorders.
  • the inventors unexpectedly found for the first time that ISX-9 can continuously and effectively increase the amplitude of circadian rhythm without affecting the circadian rhythm cycle, treat sleep disorders, improve metabolic fluctuations in circadian rhythm, and can be used in vivo, in vitro, and in vitro. Both can treat circadian rhythm disorders in middle-aged and elderly subjects.
  • the present invention further proves that ISX-9 sensitizes Ca 2+ influx, thereby strengthening CaMKII ⁇ signal, and promotes BMAL1 phosphorylation mediated by CaMKII ⁇ , thereby increasing the circadian rhythm amplitude of aging cells. Therefore, the design of applying ISX-9 and targeting CaMKII ⁇ is expected to be an important means to intervene in circadian rhythm decline, and even aging-related diseases.
  • the term "about” when used in reference to a specifically recited value means that the value may vary by no more than 1% from the recited value.
  • the expression “about 100” includes all values between 99 and 101 and in between (eg, 99.1, 99.2, 99.3, 99.4, etc.).
  • the term “comprises” or “includes (comprising)” can be open, semi-closed and closed. In other words, the term also includes “consisting essentially of”, or “consisting of”.
  • room temperature or "normal temperature” refers to a temperature of 4-40°C, preferably 25 ⁇ 5°C.
  • the term “plurality” or “multiple” refers to 2/type or more, such as 2, 3, 4, 5 or 6/type.
  • the active ingredient of the present invention is ISX-9 or its pharmaceutically acceptable salt or its deuterated product.
  • the small molecule compound proposed by the technical solution of the present invention for treating circadian rhythm disorders is ISX-9, and its administration concentration is 1-50 ⁇ M.
  • the acidic moieties that may be contained in the active ingredients of the present invention may form physiologically acceptable salts with various organic or inorganic bases.
  • Typical salts formed with bases include ammonium salts, alkali metal salts such as sodium, lithium, potassium salts, alkaline earth metal salts such as calcium, magnesium salts and salts formed with organic bases (such as organic amines), such as benzathine, dicyclohexylamine , Hipamine (salt with N,N-di(dehydroabietyl)ethylenediamine), N-methyl-D-glucamine, N-methyl-D-glucamide, tert-butyl Amines, and salts with amino acids such as arginine, lysine, etc.
  • the term "pharmaceutically acceptable” ingredient refers to a substance suitable for use in humans and/or animals without undue adverse side effects (such as toxicity, irritation and allergic reactions), i.e. having a reasonable benefit/risk ratio.
  • deuterated refers to the replacement of one or more hydrogens of the active ingredients of the present invention with deuterium.
  • Deuterium can be monosubstituted, disubstituted, polysubstituted or fully substituted.
  • the term "effective amount” refers to an amount that produces functions or activities on humans and/or animals and is acceptable to humans and/or animals. Those of ordinary skill in the art should understand that the "effective amount” can vary depending on the form of the pharmaceutical composition, health product composition, excipients used, disease severity, and combination with other drugs, health products or food. vary with each other.
  • the "promoting" and “accelerating” in the present invention include improving the circadian rhythm disorder or its related sleep disorders, and do not necessarily require 100% cure.
  • the active ingredient of the invention improves circadian rhythm, eg, by at least about 10%, at least about 30%, at least about 50%, or at least about 80%, compared to the absence of the active ingredient of the invention.
  • Circadian dysregulation is associated with the occurrence of many diseases, including metabolic disorders and aging.
  • the active ingredients of the present invention are especially suitable for middle-aged and elderly subjects.
  • composition of the present invention comprises ISX-9 or a pharmaceutically acceptable salt thereof as an active ingredient.
  • composition of the present invention comprises ISX-9 or a pharmaceutically acceptable salt thereof within a safe and effective amount range, and a pharmaceutically acceptable excipient or carrier.
  • the composition of the present invention contains 1-2000 mg of the active ingredient/dose of the present invention, more preferably, 10-500 mg of the active ingredient/dose of the present invention.
  • the "one dose” is a capsule or tablet.
  • “Pharmaceutically acceptable carrier” refers to: one or more compatible solid or liquid fillers or gel substances, which are suitable for human use, and must have sufficient purity and low toxicity. "Compatibility” here means that each component in the composition can be blended with the active ingredient of the present invention and with each other without significantly reducing the efficacy of the active ingredient.
  • Examples of pharmaceutically acceptable carrier parts include cellulose and derivatives thereof (such as sodium carboxymethylcellulose, sodium ethylcellulose, cellulose acetate, etc.), gelatin, talc, solid lubricants (such as stearic acid , magnesium stearate), calcium sulfate, vegetable oil (such as soybean oil, sesame oil, peanut oil, olive oil, etc.), polyols (such as propylene glycol, glycerin, mannitol, sorbitol, etc.), emulsifiers (such as ), wetting agent (such as sodium lauryl sulfate), coloring agent, flavoring agent, stabilizer, antioxidant, preservative, pyrogen-free water, etc.
  • cellulose and derivatives thereof such as sodium carboxymethylcellulose, sodium ethylcellulose, cellulose acetate, etc.
  • gelatin such as talc
  • solid lubricants such as stearic acid , magnesium stearate
  • calcium sulfate such
  • the mode of administration of the active ingredient or composition of the present invention is not particularly limited, and representative modes of administration include (but not limited to): oral, rectal, parenteral (intravenous, intramuscular or subcutaneous).
  • Solid dosage forms for oral administration include capsules, tablets, pills, powders and granules.
  • the active ingredient is mixed with at least one conventional inert excipient (or carrier), such as sodium citrate or dicalcium phosphate, or with: (a) fillers or extenders such as , starch, lactose, sucrose, glucose, mannitol, and silicic acid; (b) binders, such as hydroxymethylcellulose, alginate, gelatin, polyvinylpyrrolidone, sucrose, and acacia; (c) humectants , for example, glycerol; (d) disintegrants, for example, agar, calcium carbonate, potato starch or tapioca starch, alginic acid, certain complex silicates, and sodium carbonate; (e) slow agents, for example paraffin; (f ) absorption accelerators such as quaternary ammonium compounds; (g) wetting agents such as cetyl alcohol and glycerol,
  • Solid dosage forms such as tablets, dragees, capsules, pills, and granules can be prepared with coatings and shell materials, such as enteric coatings and others well known in the art. They may contain opacifying agents, and the release of the active ingredient from such compositions may be in a certain part of the digestive tract in a delayed manner.
  • coatings and shell materials such as enteric coatings and others well known in the art. They may contain opacifying agents, and the release of the active ingredient from such compositions may be in a certain part of the digestive tract in a delayed manner.
  • examples of usable embedding components are polymeric substances and waxy substances.
  • the active ingredient can also be in the form of microcapsules with one or more of the above-mentioned excipients, if necessary.
  • Liquid dosage forms for oral administration include pharmaceutically or nutraceutical acceptable emulsions, solutions, suspensions, syrups or tinctures.
  • liquid dosage forms may contain inert diluents conventionally used in the art, such as water or other solvents, solubilizers and emulsifiers, for example, ethanol, isopropanol, ethyl carbonate, ethyl acetate, propylene glycol, 1,3-Butanediol, dimethylformamide and oils, especially cottonseed oil, peanut oil, corn germ oil, olive oil, castor oil and sesame oil or mixtures of these substances, etc.
  • inert diluents conventionally used in the art, such as water or other solvents, solubilizers and emulsifiers, for example, ethanol, isopropanol, ethyl carbonate, ethyl acetate, propylene glycol, 1,3-Butanediol, dimethyl
  • compositions can also contain adjuvants, such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, and perfuming agents.
  • adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, and perfuming agents.
  • Suspensions in addition to the active ingredients, may contain suspending agents, for example, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum methoxide and agar, or mixtures of these substances, etc. .
  • suspending agents for example, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum methoxide and agar, or mixtures of these substances, etc.
  • 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 non-aqueous carriers, diluents, solvents or vehicles include water, ethanol, polyols and suitable mixtures thereof.
  • Dosage forms for topical administration of the active ingredient of this invention include gels (eg, 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.
  • composition further includes one or more other therapeutic agents.
  • the therapeutic agent is selected from the group consisting of Atrasentan, HhAntag, OSI-930, GW4064, 2-NP, or a combination thereof.
  • the active ingredients of the invention are administered concurrently with, or sequentially with, other agents that are part of a combined therapeutic regimen, in the same or separate formulations.
  • compositions of active ingredients of the present invention will be: about 1-2000 mg/day, about 10-about 1000 mg/day, about 10-about 500 mg/day, about 10-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. It is to be understood, however, that the particular dosage of a compound of the invention for any particular patient will depend on a variety of factors, for example, the age, sex, weight, general health, diet, individual response, time of administration, time to be treated, and other factors of the patient to be treated. The severity of the disease, the activity of the specific compound administered, the dosage form, mode of application and concomitant drugs.
  • the therapeutically effective amount for a given situation can be determined by routine experimentation and is within the ability and judgment of the clinician or physician.
  • the active ingredient or composition will be administered in multiple doses based on the individual condition of the subject to which it will be administered and in such a manner as to allow delivery of a therapeutically effective amount.
  • subjects to whom the active ingredient or composition of the present invention is administered include mammals (for example, humans, mice, rats, hamsters, rabbits, cats, dogs, cows, sheep, monkeys, etc.).
  • ISX-9 can treat circadian rhythm disorders, and has excellent effects in terms of enhancing amplitude and persistence, and has dose-dependent, low cytotoxicity and does not affect the circadian rhythm cycle;
  • Camk2d behaves as a real clock control gene, which can regulate BMAL1 activity and form a positive feedback loop. If this positive feedback loop is enhanced, the circadian rhythm decay caused by aging can be reversed.
  • HEK293T, NIH-3T3 and N2a cell lines were obtained from ATCC.
  • Mouse embryonic fibroblast MEFs were isolated from E13.5d embryos of C57 wild-type, Bmal1 -/- or mPer2 Luc/+ mice.
  • Cells were cultured in DMEM (Invitrogen) containing 10% FBS and 100 U/ml penicillin/streptomycin at 37°C in a humidified environment with 5% CO2 .
  • the inventors checked all cell lines using a PCR-based method (MP0035, Sigma) to ensure that all cell lines were free of Mycoplasma.
  • the reporter mouse strain mPer2 Luc (006852) and the Bmal1-floxed conditional strain (007668) were obtained from Jackson Laboratory. Mice were housed in a Specific Pathogen Free (SPF) animal house facility on a 12:12 hr light-dark cycle. Animal experiments were carried out in accordance with the protocols approved by the Biomedical Research Ethics Committee of the scientific research institution, the Shanghai Institute of Nutrition and Health and Center of Excellence in Brain Science, and the Institute of Intelligent Technology, Chinese Academy of Sciences.
  • SPF Specific Pathogen Free
  • 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).
  • Constructs for overexpression of Bmal1, FLAG-Bmal1, FLAG-Camk2a, b, d and g were cloned through NotI and MluI sites into pCAGGS vector with FLAG tag fusion.
  • the Camk2d K43M mutant was generated using PCR-based site-directed mutagenesis and then subcloned into the same pCAGGS backbone.
  • Small molecules for circadian rhythm testing were purchased from the following companies: ISX-9 from Selleck; GW4064, OSI-930, Atrasentan, Forskolin and KN93 from Med Chem Express; 2-NP and HhAntag from TargetMol.
  • Antibodies were purchased from the following resources: anti-BMAL1 (14020, Cell Signaling Technology), anti-DBP (12662-1-AP, ProteinTech), anti-PER2 (13168, ABclonal), anti-REV-ERB ⁇ (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).
  • luciferase activity was measured by ONE-Glo TM luciferase assay system (E6120, Promega) at two time points of 24 hours and 30 hours, respectively. Following chemiluminescence measurements, cell survival was determined by the alamarBlue assay (Invitrogen).
  • PER2::LUC bioluminescence For real-time recording of PER2::LUC bioluminescence, approximately 2.5x10 5 mPer2 Luc MEF cells were seeded and cultured in DMEM for 48 hours in 3.5 cm dishes until the cells became confluent. Afterwards, the cells were treated with 200 nM dexamethasone and synchronized for 1 hour. Finally, 200 ⁇ M fluorescein (A5030, Tokyo Chemical Industries, Ltd.) and 10 ⁇ M or specified concentrations of the specified small molecules were added to the serum-free DMEM medium before bioluminescence recording and incubated and recorded. Bioluminescence for 4 days.
  • the recording conditions were continuous recording by placing the petri dish in a light-tight box (LumiCycle32, Actimetrics) equipped with a photon multiplier tube recording (PMT) detector assembly. Data were processed using LumiCycle analysis software (Actimetrics), including results such as amplitude and period.
  • Sectioning and real-time bioluminescent recording of the suprachiasmatic nucleus SCN was performed as described by Savelyev et al. (Savelyev et al., 2011). The basic steps are first to harvest the whole brain of 6-month-old mPer2 Luc mice, and then cut the SCN part with a thickness of 300 ⁇ m by McIlwain Tissue Chopper (TC752, Cavey Laboratory Engineering Co.Ltd).
  • SCN slices were placed on culture membranes (PICMORG50, Millipore), and 1.5 ml recording medium (1x DMEM, 1x B27 supplement, 4.2 mM NaHCO 3 , 10 mM HEPES, 1X penicillin/streptomycin, and 200 ⁇ M luciferin) was added to the peripheral Petri dish SCN tissue culture was performed; the control group contained DMSO and the experimental group contained ISX-9 (10 ⁇ M). The amplitude-enhancing effect of ISX-9 was detected by continuous PMT recordings (LumiCycle32, Actimetrics). The recording conditions for the pituitary were the same as for the SCN.
  • Cells were harvested and lysed in frozen lysis buffer (50 mM Tris, 150 mM NaCl, 5 mM MgCl 2 , 0.5% Nonidet P-40 and 1% TritonX-100). The protein concentration of cell lysates was measured by Bradford assay, after which the protein content of each sample was adjusted to equal for quantitative immunoblot analysis. Phos-tag gel electrophoresis was carried out by electrophoresis analysis of cell lysate in 4% acrylamide gel containing 40 ⁇ M acrylamide Phos-tag ligand (F4002, APExBIO) and 80 ⁇ M MnCl 2 .
  • frozen lysis buffer 50 mM Tris, 150 mM NaCl, 5 mM MgCl 2 , 0.5% Nonidet P-40 and 1% TritonX-100.
  • the protein concentration of cell lysates was measured by Bradford assay, after which the protein content of each sample was adjusted to equal for quantitative immunoblot analysis.
  • mice After euthanizing mice of a specific age, they were immediately perfused with 4% paraformaldehyde, then the whole brain was removed, dehydrated and cryoprotected in 30% sucrose, and finally the brain tissue was embedded in OCT embedding medium (4583 , SAKURA).
  • OCT embedding medium 4583 , SAKURA.
  • a 30 ⁇ m-thick coronal brain section was harvested using a cryostat (CM1850, Leica), and then treated with 3% H 2 O 2 to quench the residual peroxidase activity in the sample.
  • the antigen retrieval step was performed by 0.05% trypsinization for 5 min at room temperature, and then the brain sections were blocked with TNB blocking buffer (100 mM Tris-HCl, pH 7.5, 150 mM NaCl, 10% FBS), followed by addition of anti-PER2 antibody dilution (1:300) cover the brain slices and incubate overnight at 4°C. After washing 3 times with TNT buffer (100mM Tris-HCl, pH 7.5, 150mM NaCl, 0.05% Tween 20) every other day, cover the brain slices with HRP-conjugated anti-rabbit secondary antibody (1:1000), and continue at room temperature Incubate for 1.5 hours.
  • TNB blocking buffer 100 mM Tris-HCl, pH 7.5, 150 mM NaCl, 10% FBS
  • brain slices were incubated with fluorophore-labeled tyramide substrate (TSA plus Cyanine3 kit, Perkin Elmer) and DAPI (10 ng/ml, Sigma-Aldrich), and then examined by confocal microscopy (Cell Observer, ZEISS) to observe fluorescent labels.
  • TSA plus Cyanine3 kit, Perkin Elmer fluorophore-labeled tyramide substrate
  • DAPI 10 ng/ml, Sigma-Aldrich
  • Mouse EEG recordings were performed with the HD-X02 telemetry implant (DSI PhysioTel TM ).
  • the implant implantation operation was first deeply anesthetized by a single intramuscular injection of 5 mg/kg pentobarbital (pentobarbital), and then two EEG electrodes were respectively fixed on the skull with small screws, and the recording site was 1 mm lateral to the Bregma line of the skull , 1mm and 3mm before the Bregma line.
  • 5% glucose solution 40 ⁇ l/g body weight
  • Activity, body temperature, EEG and EMG signals were collected by the MX2 hub and RPC-1 receiver connected to the computer.
  • Mouse EEG sleep analysis uses the supporting software NeuroScore 3.2.0 (DSI) to analyze and summarize wakefulness, rapid eye movement sleep REM and slow wave NREM sleep stages, and count the power of slow waves and fast waves in a 10-second time window for circadian sleep- Awakening Toggle Shift.
  • the EEG power of each frequency band (:0.5-4Hz,:4-8Hz,:8-12Hz,:12-16Hz,:16-32Hz,:>32Hz) is displayed as an hourly average data point.
  • MEF cells (2 ⁇ 10 5 ) were seeded on glass-bottom culture dishes (801002, NEST), and after 24 hours, Fluo-4AM (2 ⁇ M, Molecular Probes) was incubated in serum-free DMEM at 37° C. for 45 minutes. After two washes with PBS to wash away unbound dye, the cells' baseline Ca2 + (excitation 488 nm, emission 520 nm) was measured for 15 minutes. Ca 2+ -influx was recorded starting at the same time as DMSO or 10 ⁇ M ISX-9 addition (time point 0). Images were collected at 1 min intervals by a fluorescence microscope (Cell Observer, ZEISS) for 15 min to observe calcium influx.
  • Fluo-4AM 2 ⁇ M, Molecular Probes
  • 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), followed by real-time bioluminescent recordings as described above for the experimental conditions.
  • the pCAGGS plasmids (5 ⁇ g) encoding FLAG-Bmal1, FLAG-Camk2 isoforms and mutants were transfected into 2 ⁇ 10 6 HEK293T cells respectively, and after 48 hours of expression, the cells were collected and incubated in 400 ⁇ l lysis buffer containing protease inhibitors (Roche) Cells were lysed in solution.
  • kinase assays were performed using a kinase activity kit (EA004, R&D Systems) at a ratio of CaMKII:BMAL1 1:4, and finally the absorbance of the kinase reaction was measured at 620 nm.
  • RNAscope Multiplex Fluorescent v2-Kit Advanced Cell Diagnostics
  • the basic procedure is to euthanize the mouse, perfuse it with 4% paraformaldehyde, carefully remove the brain and post-fix overnight in 4% paraformaldehyde, then freeze the brain by soaking in 30% sucrose-1X PBS at 4°C Protect. After embedding with OCT embedding solution, 30 ⁇ m coronal brain slices were collected, followed by dehydration, quenching of peroxidase activity, and finally exposure of target RNA by Protease Plus solution (322381, Advanced Cell Diagnostics).
  • N2a cells were seeded into 24-well dishes at a density of 8x104 cells per well.
  • the Bmal1 reporter plasmid and the Camk2 overexpression plasmid were transfected with Lipofectamine 3000 (Invitrogen) at a ratio of 200:100ng per well.
  • Lipofectamine 3000 Invitrogen
  • cells were treated with 100 nM dexamethasone (dexamethasone) for 15 minutes after synchronization in DMEM with the addition of DMSO (control group) or 10 ⁇ M ISX-9 (experimental group).
  • Example 1 Small molecule screen identified ISX-9 as a circadian rhythm amplitude enhancer.
  • the present inventors conducted a compound screen of 8060 small molecules covering kinases in mPer2 Luc fibroblasts , epigenetic factors, and GPCRs (Fig. 1A).
  • the inventors measured the chemiluminescence levels of mPer2 Luc fibroblasts at 24h and 30h after drug treatment, respectively (Fig. 1A and B). If a compound is found to have consistent upregulation or downregulation of PER2::LUC expression at two time points compared to an untreated control, the compound can be described as an amplitude modulator; If the effect is opposite, it can be explained that the compound is a phase regulator.
  • the present invention focused on the remaining 6777 compounds and resulted in 270 amplitude enhancers capable of increasing the amplitude of PER2::LUC by 1.2-fold.
  • ISX-9, Atrasentan, HhAntag, OSI-930, GW4064, and 2-NP were the main candidates for increasing PER2::LUC levels (Fig. 1C).
  • ISX-9 was the only amplitude enhancer that consistently maintained rhythmic oscillations during the 4-day test.
  • the amplitude enhancement effect of ISX-9 is broad and can be used to increase the expression of other important rhythm genes, such as Dbp and Rev-Erb ⁇ (Fig. 1F).
  • ISX-9 again showed its best ability to regulate the biological clock among the six drug candidates.
  • ISX-9 is an isoxazole compound and has neurogenic activity (Fig. 2A). ISX-9 was found to have a dose-dependent effect on increasing the PER2::LUC amplitude in the assay of the present invention (Fig. 2B) and still showed low cytotoxicity at the highest concentration tested, 10 ⁇ (Fig. 2C).
  • ISX-9 has excellent effects in terms of enhancing amplitude and persistence, and has dose-dependent, low cytotoxicity and does not affect the circadian rhythm cycle, so ISX-9 can be used as a circadian rhythm amplitude enhancer.
  • ISX-9 increased the expression of circadian clock genes in mouse embryonic fibroblasts (MEFs), including Bmal1, Per2, Dbp and other core circadian clock genes.
  • DBP protein levels were highly sensitive to ISX-9 dose (Fig. 4A), and thus could serve as a clock protein marker reporting the effects of ISX-9 in the peripheral system.
  • ISX-9 can also stimulate higher PER2::LUC rhythm amplitude in in vitro culture recordings such as suprachiasmatic nucleus (SCN) and pituitary (Figure 4D); Mice showed higher levels of PER2 rhythmic protein in the SCN (Fig. 4E), overall illustrating the amplitude-enhancing effect of ISX-9 in cells, tissues in vitro, and in vivo.
  • SCN suprachiasmatic nucleus
  • Figure 4D pituitary
  • Mice showed higher levels of PER2 rhythmic protein in the SCN (Fig. 4E), overall illustrating the amplitude-enhancing effect of ISX-9 in cells, tissues in vitro, and in vivo.
  • the present invention further tested the metabolic rate of young (2-month-old) and middle-aged (14-month-old) mice.
  • mice were awake in the dark environment, and the mice were put into a resting state in the bright environment; the circadian rhythm disorder experimental group was 14-month-old mice .
  • the ISX-9 experimental group showed lower O 2 consumption and CO 2 production during the day, indicating that the compound could stabilize the rest of middle-aged and aged mice (Fig. 5B).
  • the present invention performed electroencephalogram (EEG) telemetry recordings and analyzed sleep states in young and middle-aged mice.
  • EEG electroencephalogram
  • the present invention finds that the difference between wakefulness and slow-wave sleep (NREM) of the two groups of mice at night is the most significant.
  • the middle-aged and elderly groups showed higher NREM sleep during the nocturnal active period, indicating their lower level of wakefulness (Fig. 6A).
  • the mice in the middle-aged and elderly groups also showed a significant reduction in activity, reduced body temperature fluctuations (Figure 6B), decreased ⁇ / ⁇ fast waves, and insignificant delta/ ⁇ slow wave fluctuations (Figure 6C) and other rhythm disturbances phenotype, these results indicate an overall decrease in the strength of circadian rhythms in the brain during aging.
  • the inventor administered 20 mg/Kg ISX-9 to the same group of mice for 7 consecutive days, and started EEG recording 7 days after stopping the treatment.
  • the present invention found that the pretreatment of ISX-9 greatly improved the sobriety of middle-aged and aged mice in the first half of the night, while reducing rapid eye movement REM and slow wave NREM sleep ( FIG. 7A ).
  • Treated middle-aged and aged mice also showed higher activity at night and lower body temperature during daytime rest (Figure 7B), the above results suggest that ISX-9 assists middle-aged and aged mice to adjust to wakefulness more effectively. Alertness, activeness, and better, cooler rest during sleep.
  • the inventors also noted that the treated middle-aged and aged mice had higher slow-wave delta power during rest and higher fast-wave gamma power during wakefulness, both of which showed improvements in brain EEG circadian rhythm homeostasis ( Figure 7C).
  • the present invention found that ISX-9 significantly inhibited the frequent waking-sleep transitions of the middle-aged and elderly groups, making the middle-aged and elderly groups This approached a more stable sleep-wake homeostasis observed in healthy young mice (Fig. 7D).
  • the present invention first tracked intracellular Ca 2+ levels in MEFs by Fluo-4AM fluorescent dye, and confirmed that ISX-9 can rapidly trigger Ca 2+ influx in the test ( FIG. 8A ).
  • the present inventors also found that the ISX-9 effect was mediated by CaMKII activity, as the CaMKII-specific antagonist KN93 completely abolished ISX-9's PER2::LUC enhancing effect (Fig. 8B).
  • the inventors noticed that Camk2b and Camk2d knockdown significantly reduced the ISX-9 effect in real-time PER2::LUC recordings, but not Camk2a and Camk2g knockdown (Fig. 8C), showing that ISX- The effect of 9 is more dependent on the two subtypes of CaMKII ⁇ and CaMKII ⁇ .
  • CaMKII ⁇ exhibited the highest activity against purified BMAL1 in the kinase assay (Fig. 8E), and together with Camk2d knockdown affecting PER2::LUC expression, these data suggest that CaMKII ⁇ is critical for mediating BMAL1-dependent ISX-9 effects . These results suggest a positive feedback loop linking ISX-9, Ca signaling , and BMAL1 phosphorylation to regulate circadian rhythm amplitude.
  • RNA fluorescence in situ hybridization smFISH analysis showed that Camk2d in the mouse brain was more abundantly expressed in the subcortical area and hypothalamus (Fig. The time point ZT18 peaked in the suprachiasmatic nucleus (SCN) (Fig. 10), in good agreement with its expected potential function in regulating circadian rhythms.
  • Transcripts of other CaMKII isoforms, such as Camk2a and Camk2b showed slight fluctuations and low expression in the SCN, and their expression was more enriched in the striatum and cortex (Fig. 9). It is further illustrated that the circadian effect of ISX-9 on SCN is related to Camk2d.
  • Camk2d appears to be a bona fide clock-controlled gene that induces and promotes control of the circadian clock in response to ISX-9.
  • Camk2d expression is rhythmic and can be triggered by ISX-9
  • the present inventors searched for specific E-box sequences in the Camk2 promoter.
  • the present inventors found that all Camk2 subtypes contain E-box elements, including the canonical E-box sequence (CACGTG) in the Camk2d and Camk2g promoters ( FIG. 12A ).
  • the present invention found that the Camk2d promoter is the most sensitive to CLOCK:BMAL1 activation, verifying that Camk2d is a clear circadian clock control gene ( FIG. 12B ).
  • ISX-9 could support the transcription factor CLOCK:BMAL1 to further induce Camk2 and showed the best effect on Camk2d expression (Fig. 12B), consistent with the effect in aged SCN (Fig. 11B).
  • the inventors purified the CaMKII ⁇ kinase-inactivated K43M mutant, and detected a significant decrease in the phosphorylation of BMAL1 by biochemical methods ( FIG. 12C ).
  • the effect of the CaMKII ⁇ K43M mutant on activating the expression of Dbp:Luc was significantly reduced ( FIG. 12D ), which again confirmed that CaMKII ⁇ could up-regulate the amplitude of biological rhythm by phosphorylating BMAL1.
  • the present invention proposes an intervention measure based on small molecule drugs, which is used to effectively and continuously amplify the amplitude of the circadian rhythm and solve the problem of gradual attenuation of the amplitude of the rhythm during old age.
  • This attenuation is partly due to reduced expression of CaMKII ⁇ during aging, thereby limiting the ability to phosphorylate and activate BMAL1 to regulate the circadian clock through CaMKII ⁇ .

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Abstract

本发明提供了一种ISX-9的用途。具体地，ISX-9可用于制备组合物，所述组合物用于选自下组的一种或多种用途：(i)预防和/或治疗昼夜节律失调；(ii)预防和/或治疗睡眠障碍；(iii)提高昼夜节律振幅；和/或(iv)提高CaMKIIδ蛋白的活性或表达量。本发明的活性成分ISX-9能够稳定，持续，在体内、离体、体外环境均能有效治疗老年对象的昼夜节律失调及其相关疾病。

Description

ISX-9在治疗衰老相关的昼夜节律幅度下降和睡眠障碍方面的应用 技术领域
本发明涉及药物领域，具体地涉及ISX-9在治疗衰老相关的昼夜节律幅度下降和睡眠障碍方面的应用。
背景技术
生物钟是一种内在的计时器，它有序地协调多种生理活动，包括昼夜激素周期、进食、睡眠、体温和代谢活动。在模式生物中破坏昼夜节律能模拟多种与人类疾病相关的症状，如心血管功能障碍、糖尿病、癌症、睡眠障碍、抑郁症和神经退行性疾病等。值得注意的是，所有上述疾病都与衰老过程高度相关，因此生物钟强度的衰退也被认为是衰老的指标之一。鉴于生物节律振幅下降是健康受损的常见特征，因此旨在防止生物节律振幅衰退以及缩小的干预措施可望应用于昼夜节律失调及其相关疾病的治疗。通过筛选手段，前人研究已发现多种化合物可以用于调节生物节律的振幅或相位，同时阐明了其作用的生物钟蛋白靶点及相关机制。但由于长期的相位错位对健康有害，因此导致相位明显改变的小分子应谨慎使用；除了特定用途，例如调节时差之外，如何长期地正确并安全使用这些小分子用于慢性疾病治疗仍需大量临床数据的检验。
发明内容
本发明的目的就是提供一种ISX-9能够稳定，持续，在体内、离体、体外环境均能有效治疗老年对象的昼夜节律失调及其相关疾病的应用。
在本发明的第一方面，提供了一种ISX-9的用途，所述的ISX-9具有如下式所示的结构：
Figure PCTCN2022099218-appb-000001
所述的ISX-9用于制备组合物，所述组合物用于选自下组的一种或多种用途：(i)预防和/或治疗昼夜节律失调；(ii)预防和/或治疗睡眠障碍；(iii)提高昼夜 节律振幅；和/或(iv)提高CaMKIIδ蛋白的活性或表达量。
在另一优选例中，所述的组合物为药物组合物或保健品组合物。
在另一优选例中，所述的组合物用于在中老年对象中(i)预防和/或治疗昼夜节律失调；(ii)预防和/或治疗睡眠障碍；(iii)提高昼夜节律振幅；和/或(iv)提高CaMKIIδ蛋白的活性或表达量。
在另一优选例中，所述中老年对象是指年龄超过平均寿命2/3的对象，更佳地超过平均寿命3/4。
在另一优选例中，所述对象为人时，所述中老年对象为年龄≥45周岁的对象。
在另一优选例中，所述对象为小鼠时，所述中老年对象为月龄≥12月龄，更佳地月龄≥14月龄的对象。
在另一优选例中，所述昼夜节律失调包括由于年龄增长而导致的昼夜节律失调。
在另一优选例中，所述预防和/或治疗昼夜节律失调指选自下组的一个或多个指标得到改善：
(i)提高昼夜节律振幅；
(ii)提高代谢昼夜节律性波动；
(iii)预防和/或治疗昼夜节律性的睡眠障碍。
在另一优选例中，所述提高昼夜节律幅度是指相比未施用ISX-9的对照，施用ISX-9将昼夜节律幅度提高了至少1.2倍，优选至少1.5倍，例如至少2倍、至少3倍、至少5倍。
在另一优选例中，所述的组合物在提高昼夜节律幅度同时不改变昼夜节律周期。
在另一优选例中，所述的组合物在3天以上具有持续性，优选在4-14天内，更优选在7-14天内。
在另一优选例中，所述预防和/或治疗昼夜节律失调包括提高生物钟基因活性，且所述生物钟基因选自下组：Bmal1、Per2、Dbp、Clock、Cry1、Cry2、Npas2、Nr1d1、Rorα，或其组合。
在另一优选例中，所述预防和/或治疗昼夜节律失调可在细胞、体外组织和/或体内进行。
在另一优选例中，所述细胞包括小鼠胚胎成纤维细胞。
在另一优选例中，所述体外组织包括视交叉上核(SCN)和/或垂体。
在另一优选例中，所述组合物不影响年青对象的昼夜节律。
在另一优选例中，所述年青对象是指年龄不超过平均寿命2/3的对象，更佳地超过平均寿命1/2。
在另一优选例中，所述对象为人时，所述年青对象为年龄≤30周岁的对象。
在另一优选例中，所述对象为小鼠时，所述年青对象为月龄≤8月龄的对象，更佳地月龄≤4月龄，更佳地月龄≤2月龄。
在另一优选例中，所述预防和/或治疗昼夜节律失调包括通过提高BMAL1活性从而提高昼夜节律振幅。
在另一优选例中，所述提高BMAL1活性包括促进BMAL1磷酸化。
在另一优选例中，所述促进BMAL1磷酸化由CaMKIIδ介导。
在另一优选例中，所述提高BMAL1活性可进一步促进CaMKIIδ自身表达。
在另一优选例中，所述组合物用于促进Ca 2+流入。
在另一优选例中，CaMKIIδ响应Ca 2+信号后，通过磷酸化BMAL1上调BMAL1转录活性。
在另一优选例中，所述预防和/或治疗昼夜节律失调包括增强BMAL1活性和提高CaMKIIδ自身的表达。
在另一优选例中，所述预防和/或治疗昼夜节律失调包括通过CaMKIIδ、BMAL1的正向反馈循环提高昼夜节律振幅，其中所述正向反馈循环包括ISX-9通过CaMKIIδ介导的BMAL1磷酸化增强BMAL1活性，且BMAL1活性提高进一步提高CaMKIIδ自身的表达。
在另一优选例中，所述提高代谢昼夜节律性波动具有以下的一个或多个特征：
(a-1)提高呼吸交换比的昼夜节律性幅度；
(a-2)提高所述对象O 2消耗的昼夜节律性幅度；
(a-3)提高所述对象CO 2产生的昼夜节律性幅度；
(a-4)校正呼吸交换比的周期；
(a-5)不影响所述对象的食物摄入量和/或体重。
在另一优选例中，所述预防和/或治疗睡眠障碍具有以下的一个或多个特征：
(b-1)提高所述对象在活动周期的清醒程度；
(b-2)降低所述对象在活动周期的快速动眼REM和慢波NREM睡眠；
(b-3)提高所述对象的体温的昼夜节律性幅度；
(b-4)提高所述对象在活动周期的快波γ功率；
(b-5)提高所述对象在休息周期的慢波δ功率；
(b-6)改善所述对象的睡眠-清醒稳态。
在另一优选例中，所述组合物中含有浓度≥0.1μM，较佳地浓度≥0.25μM，更佳地浓度≥10μM的ISX-9或其药学上可接受的盐。
在另一优选例中，所述组合物含有0.1-99wt％，较佳地0.5-95wt％，更佳地1-90wt％的ISX-9或其药学上可接受的盐。
在另一优选例中，所述组合物还包括药学上可接受的赋形剂和/或载体。
在另一优选例中，所述组合物的剂型选自下组：固体制剂、液体制剂或半固体制剂。
在另一优选例中，所述组合物的剂型选自下组：凝胶剂、贴剂、片剂、胶囊剂、散剂、膏剂、粉剂、针剂、水剂、肠溶缓释制剂或注射剂。
在另一优选例中，所述组合物的剂型为注射剂。
在另一优选例中，所述组合物通过以下方式施用于受试者：口服、经皮注射、静脉注射、肌肉注射或肛肠给药。
在本发明的第二方面，提供了一种ISX-9的氘代物的用途，所述的ISX-9具有如下式所示的结构：
Figure PCTCN2022099218-appb-000002
所述的ISX-9的氘代物用于制备组合物，所述组合物用于选自下组的一种或多种用途：(i)预防和/或治疗昼夜节律失调；(ii)预防和/或治疗睡眠障碍；(iii)提高昼夜节律振幅；和/或(iv)提高CaMKIIδ蛋白的活性或表达量。
在另一优选例中，所述组合物如本发明第一方面所述。
在本发明的第三方面，提供了一种CaMKIIδ高表达调节剂的用途，用于制备组合物，所述组合物用于选自下组的一种或多种用途：(i)预防和/或治疗昼夜节律失调；和/或(ii)调节BMAL1活性。
在另一优选例中，所述CaMKIIδ由Camk2d节律性表达。
在另一优选例中，所述的组合物用于提高CaMKIIδ在皮层下区域和下丘脑中的表达水平。
在另一优选例中，所述预防和/或治疗昼夜节律失调包括：通过促进BMAL1磷酸化从而提高BMAL1活性和提高CaMKIIδ自身的表达。
在另一优选例中，所述预防和/或治疗昼夜节律失调如本发明第一方面所述。
在另一优选例中，所述组合物还包括药学上可接受的赋形剂和/或载体。
在另一优选例中，所述组合物的剂型选自下组：固体制剂、液体制剂或半固体制剂。
在另一优选例中，所述组合物的剂型选自下组：凝胶剂、贴剂、片剂、胶囊剂、散剂、膏剂、粉剂、针剂、水剂、肠溶缓释制剂或注射剂。
在另一优选例中，所述组合物的剂型为注射剂。
在另一优选例中，所述组合物通过以下方式施用于受试者：口服、经皮注射、静脉注射、肌肉注射或肛肠给药。
在本发明的第四方面，提供了一种药物组合物，所述药物组合物包括：
ISX-9或其氘代物或其药学上可接受的盐，以及药学上可接受的载体。
在另一优选例中，所述药物组合物可包括其他昼夜节律调节剂，其中所述昼夜节律调节剂选自下组：阿曲生坦(Atrasentan)、HhAntag、OSI-930、GW4064、2-NP，或其组合。
在另一优选例中，所述药物组合物的剂型选自下组：固体制剂、液体制剂或半固体制剂。
在另一优选例中，所述药物组合物的剂型选自下组：凝胶剂、贴剂、片剂、胶囊剂、散剂、膏剂、粉剂、针剂、水剂、肠溶缓释制剂或注射剂。
在另一优选例中，所述药物组合物的剂型为注射剂。
在另一优选例中，所述药物组合物的剂型选自下组：口服剂型、经皮注射剂型、 静脉注射剂型、肌肉内注射剂型。
在另一优选例中，所述注射剂的注射液中含有生理盐水、葡萄糖、稳定剂、防腐剂、悬浮剂或乳化剂的一种或多种。
在本发明的第五方面，提供了一种(i)预防和/或治疗昼夜节律失调；(ii)预防和/或治疗睡眠障碍；和/或(iii)提高昼夜节律振幅的方法，包括步骤：向有此需要的对象施用安全有效量的ISX-9或其药学上可接受的盐。
在另一优选例中，所述对象为中老年对象。
在另一优选例中，所述中老年对象如本发明第一方面所述。
在另一优选例中，所述对象为哺乳动物，如人、小鼠、大鼠、仓鼠、兔、猫、狗、牛、绵羊、猴等。
在另一优选例中，ISX-9或其药学上可接受的盐的有效使用浓度为0.1-50μM，更佳地为0.25-10μM。
应理解，在本发明范围内中，本发明的上述各技术特征和在下文(如实施例)中具体描述的各技术特征之间都可以互相组合，从而构成新的或优选的技术方案。限于篇幅，在此不再一一累述。
附图说明
图1显示了通过小分子筛选鉴定出ISX-9为生物节律振幅增强剂。
(A)化合物筛选程序和验证的示意图。(B)6777个小分子(左)的初步筛选结果热图。点代表24小时和30小时时间点的PER2::LUC生物发光相对值，以载体对照进行归一化展示。(C)各个小分子(红色曲线)处理后的mPer2 LucMEF细胞的实时生物发光记录，显示小分子的振幅增强效果。对照为DMSO处理的节律波动(黑色曲线显示)。(D)与图1C相关的小分子处理后周期分析。9：ISX-9；A：阿曲生坦(Atrasentan)；H：HhAntag；O：OSI-930；G：GW6064和2：2-NP。(E)小分子治疗的4天生物发光峰的振幅评估结果。将每种化合物的振幅归一化为其各自的第1天峰值。(灰色代表DMSO，橙色代表加药组)(F)Dbp:Luc和Rev-erbα:Luc表达的变化对小分子治疗的响应；DMSO组设为1。数据使用未配对的Student’s t检验(ns，不显著，*p<0.05，**p<0.01)，数据显示为平均值±SD。
图2显示了ISX-9增强mPer2 Luc MEFs的生物节律振幅效果具有剂量依赖性。
(A)ISX-9的化学结构。(B)ISX-9在mPer2 Luc MEFs中的剂量依赖性效应。(C)在MEF细胞处理ISX-9后72小时的细胞存活测试。数据显示为平均值±SD。使用未配对的Student’s t检验(ns，不显著)。
图3显示了ISX-9增强了MEF细胞的生物钟基因表达。
在不同时间点用DMSO(黑色)或ISX-9(红色)处理的MEF中生物钟基因表达的定量PCR分析。数据显示为平均值±SD。
图4显示了ISX-9的生物节律振幅增强效果依赖于钟蛋白BMAL1。
(A)在增加ISX-9治疗剂量后，通过免疫印迹检测BMAL1、REV-ERBα和DBP的蛋白水平。(B)Bmal1野生型(WT)、杂合型(HET)或敲除(KO)的MEF细胞用DMSO(D)或10μM ISX-9(9)处理，然后在处理后36小时或48小时的时间点收取细胞进行BMAL1和DBP的免疫印迹检测。(C)Bmal1野生型(WT)、杂合型(HET)或敲除(KO)的MEF细胞用DMSO(D)或10μM ISX-9(9)处理后的PER2::LUC实时生物发光记录。(D)ISX-9(10μM)处理mPer2 Luc小鼠的SCN和垂体中的体外PER2::LUC节律记录。数据显示为平均值±SEM。(E)用DMSO或ISX-9(20mg/Kg)处理7天的6月龄小鼠的SCN中PER2水平的免疫组织化学分析。右图显示PER2的量化结果，显示为平均值±SEM。使用了未配对的Student’s t检验(ns，不显著，*p<0.05，**p<0.01，***p<0.001)。
图5显示了ISX-9使中老年小鼠的昼夜代谢年轻化。
(A)2个月大(蓝色曲线，n＝5)与14个月大(灰色曲线，n＝6)小鼠的呼吸交换比(RER)、耗氧量(VO 2)、二氧化碳产生量(VCO 2)。(B)用DMSO(灰色曲线，n＝6)或ISX-9(橙色曲线，n＝6)处理14月龄小鼠后的RER、VO 2和VCO 2结果。(C)用DMSO(蓝色，n＝5)或ISX-9(紫色，n＝5)处理2月龄小鼠后的RER、VO 2和VCO 2结果。(D)代谢笼的RER波动周期分析，使用软件ClockLab处理。(E)在ISX-9处理，以及未处理的各小鼠组的每日饮水量、食物消耗及体重变化。在ISX-9处理，以及未处理的的年轻小鼠组的(F)每日饮水量和(G)食物消耗记录。使用未配对的Student’s t检验(ns，不显著，*p<0.05，**p<0.01，***p<0.001)，数据显示为平均值±SEM。
图6显示了年轻对比中老年小鼠的睡眠、活动量、体温和昼夜脑电差异。
(A)2月龄(蓝色，n＝4)与14月龄的小鼠(灰色，n＝8)的清醒、NREM睡眠和REM睡眠时间百分比。底部分别表示亮灯时段(白色部分，07:00-19:00)和 熄灯时段(黑色部分，19:00-07:00)。(B)2个月龄(蓝色，n＝4)与14个月龄的小鼠(灰色，n＝8)在24小时周期内的昼夜活动和体温变化。(C)2个月龄(蓝色，n＝4)与14个月龄的小鼠(灰色，n＝8)EEG功率分析。分别显示δ(0.5-4Hz)、θ(4-8Hz)、α(8-12Hz)、σ(12-16Hz)、β(16-32Hz)和γ(>32Hz)功率在昼夜节律中总功率的比例。使用未配对的Student’s t检验(ns，不显著，*p<0.05，**p<0.01，***p<0.001)，数据显示为平均值±SEM。
图7显示了中老年小鼠在ISX-9处理前后的睡眠、活动量、体温和昼夜脑电差异。
(A)用DMSO(灰色曲线，n＝8)或ISX-9(橙色曲线，n＝8)处理的14月龄小鼠的清醒、NREM睡眠和REM睡眠时间百分比。底部分别表示亮灯时段(白色部分，07:00-19:00)和熄灯时段(黑色部分，19:00-07:00)。(B)DMSO治疗的2个月龄小鼠(蓝色曲线，n＝4)、DMSO(灰色曲线，n＝8)或ISX-9(橙色曲线，n＝8)处理的14个月龄小鼠在24小时周期内的昼夜活动和体温变化。(C)用DMSO(灰色曲线，n＝8)或ISX-9(橙色曲线，n＝8)处理的14个月龄小鼠的脑电图功率分析。分别显示了δ(0.5-4Hz)、θ(4-8Hz)、α(8-12Hz)、σ(12-16Hz)、β(16-32Hz)和γ(>32Hz)在昼夜节律中总功率的比例。(D)在开灯(ZT0-1)或关灯(ZT12-13)的第一个小时内清醒和睡眠阶段之间的状态转换数。使用未配对的Student’s t检验(ns，不显著，*p<0.05，**p<0.01，***p<0.001)，数据显示为平均值±SEM。
图8显示了ISX-9触发Ca 2+内流并提高CaMKII相关的BMAL1磷酸化。
(A)在MEF细胞中进行DMSO(黑色曲线)或10μM ISX-9(橙色曲线)处理后，Ca 2+流入测量和Fluo-4AM荧光变化的测定示意图(左)。右图显示在指定时间点的代表性图像；比例尺，50μm。使用未配对的Student’s t检验，数据显示为平均值±SD。(B)PER2::LUC在DMSO(黑色曲线)、ISX-9(红色曲线)和ISX-9与CaMKII抑制剂KN93(品红色曲线)处理条件下的波动。(C)DMSO、ISX-9以及用ISX-9处理并单独敲低Camk2a、Camk2b、Camk2d或Camk2g下的PER2::LUC波动。(D)使用Phos-tag凝胶分析DMSO(D)或10μM ISX-9(9)处理后的MEF细胞内BMAL1磷酸化水平。(E)使用纯化的BMAL1作为底物对纯化的CaMKIIα、β、 和γ进行体外激酶测定。最高活性设置为1，以便于亚型之间的比较。数据代表平均值±SD。使用未配对的Student’s t检验(*p<0.05，**p<0.01，***p<0.001)。
图9显示了Camk2亚型在成年小鼠脑中的单分子RNA荧光原位杂交。
来自2个月野生型小鼠在时间点ZT2或ZT14的冠状面脑切片中CamK2a、b、d和 g的单分子RNA荧光原位杂交分析。Camk2d在皮层下区域和下丘脑中表现出更高的表达。Camk2a和Camk2b在纹状体和皮质中表现出明显的、更高的表达。比例尺，1000μm。
图10显示了单分子RNA荧光原位杂交分析Camk2亚型在视交叉上核中的节律震荡。
2月龄的野生型小鼠的SCN中在指定的时间的CamK2a、b、d和g的SmFISH。SCN中的表达水平在量化后归一化并设定第一个时间点为1，数据显示为平均值±SEM。
图11显示了ISX-9重新激活老年小鼠SCN中的Camk2d表达。
(A)在ZT10时间点比对2月龄和18月龄小鼠SCN中的CamK2a、b、d和g的表达(SmFISH检测)。从SCN区域量化表达水平，然后将2个月龄小鼠中每个Camk2亚型的强度设置为1(n＝3/组)。(B)用DMSO或ISX-9处理的16个月龄小鼠SCN的CamK2a、b、d和g的表达(SmFISH)。表达水平被量化并设定DMSO对照组的平均亮度为1(n＝4/组)。使用未配对的Student’s t检验(ns，不显著，*p<0.05，**p<0.01)，数据显示为平均值±SEM。
图12显示了Camk2d作为明确的生物钟控制基因，可以响应ISX-9诱导并促进钟基因Dbp:Luc的表达。
(A)mCamk2启动子的E-box示意图及荧光素酶报导质粒构建。(B)用DMSO或10μM ISX-9处理的N2a细胞中的Camk2:Luc生物发光测定。DMSO处理的Camk2d:Luc生物发光值设置为1。(C)纯化的CaMKIIδWT和K43M突变体的体外激酶测定，使用BMAL1作为底物。(D)表达CaMKIIδWT或K43M突变体条件下Dbp:Luc的生物发光检测，。使用未配对的Student’s t检验(*p<0.05，**p<0.01，***p<0.001)，数据显示为平均值±SD。
图13显示了ISX-9通过激活CaMKIIδ的BMAL1磷酸化功能增强昼夜节律振幅的机制示意图。
具体实施方式
本发明人经过广泛而深入的研究，通过大量筛选和测试，提供了一种ISX-9在治疗昼夜节律失调方面的用途。本发明人首次意外地发现，ISX-9可以在不影响昼夜节律周期的情况下持续有效地提高昼夜节律振幅，治疗睡眠障碍，改善昼夜节律中的代谢波动，并且在体内、离体、体外环境均可以治疗中老年对 象的昼夜节律失调。
此外，本发明还进一步证明通过ISX-9敏化Ca 2+流入，从而加强CaMKIIδ信号，并由CaMKIIδ介导促进BMAL1磷酸化，从而提高衰老细胞昼夜节律振幅。因此，应用ISX-9和靶向CaMKIIδ的设计可望作为干预昼夜节律衰退，甚至是衰老相关的疾病的重要手段。
术语
除非另有定义，否则本文中所用的全部技术术语和科学术语均具有如本发明所属领域普通技术人员通常理解的相同含义。
如本文所用，在提到具体列举的数值中使用时，术语“约”意指该值可以从列举的值变动不多于1％。例如，如本文所用，表述“约100”包括99和101和之间的全部值(例如，99.1、99.2、99.3、99.4等)。
如本文所用，术语“含有”或“包括(包含)”可以是开放式、半封闭式和封闭式的。换言之，所述术语也包括“基本上由…构成”、或“由…构成”。
如本文所用，术语“室温”或“常温”是指温度为4-40℃，较佳地，25±5℃。
如本文所用，术语“多个”或“多种”指2个/种或以上，如2、3、4、5或6个/种。
活性成分
本发明的活性成分为ISX-9或其药学上可接受的盐或其氘代物。
Figure PCTCN2022099218-appb-000003
根据一个实施方式，本发明技术方案提出的一种治疗昼夜节律失调的小分子化合物，为ISX-9，其施用浓度为1-50μM。
本发明的活性成分可能含有的酸性片段可能会和各种有机或无机碱形成生理学上可接受的盐。典型的碱形成的盐包括铵盐、碱金属盐如钠、锂、钾盐，碱土金属盐如钙、镁盐和有机碱形成的盐(如有机胺)，如苄星、二环已基胺、海巴胺(与N,N-二(去氢枞基)乙二胺形成的盐)、N-甲基-D-葡糖胺、N-甲基-D-葡糖酰胺、叔丁基胺，以及和氨基酸如精氨酸、赖氨酸等等形成的盐。
如本文所用，术语“药学上可接受的”成分是指适用于人和/或动物而无过度 不良副反应(如毒性、刺激和变态反应)，即有合理的效益/风险比的物质。
如本文所用，术语“氘代”指本发明的活性成分的一个或多个氢被氘所取代。氘代可以是一取代、二取代、多取代或全取代。
如本文所用，术语“有效量”，是指对人和/或动物产生功能或活性的且可被人和/或动物所接受的量。本领域的普通技术人员应该理解，所述的“有效量”可随着药物组合物、保健品组合物的形式、所用的辅料、疾病的严重程度以及与其他药物、保健物或食物联合等情况的不同而有所不同。
本发明所述的“促进”、“加速”包括改善昼夜节律失调或其相关睡眠障碍，并不必需要100％治愈。在一些实施方案中，与不存在本发明的活性成分时相比，本发明的活性成分将昼夜节律改善了例如至少约10％、至少约30％、至少约50％、或至少约80％。
昼夜节律失调
生物节律失调(Circadian dysregulation)伴随着多种疾病的发生，包括代谢紊乱和老化等。
本发明的活性成分尤其适用于中老年对象。
组合物和施用方法
本发明的组合物包含ISX-9或其药学上可接受的盐作为活性成分。
本发明的组合物包含安全有效量范围内的ISX-9或其药学上可接受的盐，以及药学上可接受的赋形剂或载体。
通常，本发明的组合物含有1-2000mg本发明活性成分/剂，更佳地，含有10-500mg本发明活性成分/剂。较佳地，所述的“一剂”为一个胶囊或药片。
“药学上可接受的载体”指的是：一种或多种相容性固体或液体填料或凝胶物质，它们适合于人使用，而且必须有足够的纯度和足够低的毒性。“相容性”在此指的是组合物中各组份能和本发明的活性成分以及它们之间相互掺和，而不明显降低活性成分的药效。药学上可以接受的载体部分例子有纤维素及其衍生物(如羧甲基纤维素钠、乙基纤维素钠、纤维素乙酸酯等)、明胶、滑石、固体润滑剂(如硬脂酸、硬脂酸镁)、硫酸钙、植物油(如豆油、芝麻油、花生油、橄榄油等)、多元醇(如丙二醇、甘油、甘露醇、山梨醇等)、乳化剂(如
Figure PCTCN2022099218-appb-000004
)、润湿剂(如十二烷基硫酸钠)、着色剂、调味剂、稳定剂、抗氧化剂、防腐剂、无热 原水等。
本发明活性成分或组合物的施用方式没有特别限制，代表性的施用方式包括(但并不限于)：口服、直肠、肠胃外(静脉内、肌肉内或皮下)。
用于口服施用的固体剂型包括胶囊剂、片剂、丸剂、散剂和颗粒剂。在这些固体剂型中，活性活性成分与至少一种常规惰性赋形剂(或载体)混合，如柠檬酸钠或磷酸二钙，或与下述成分混合：(a)填料或增容剂，例如，淀粉、乳糖、蔗糖、葡萄糖、甘露醇和硅酸；(b)粘合剂，例如，羟甲基纤维素、藻酸盐、明胶、聚乙烯基吡咯烷酮、蔗糖和阿拉伯胶；(c)保湿剂，例如，甘油；(d)崩解剂，例如，琼脂、碳酸钙、马铃薯淀粉或木薯淀粉、藻酸、某些复合硅酸盐、和碳酸钠；(e)缓溶剂，例如石蜡；(f)吸收加速剂，例如，季胺化合物；(g)润湿剂，例如鲸蜡醇和单硬脂酸甘油酯；(h)吸附剂，例如，高岭土；和(i)润滑剂，例如，滑石、硬脂酸钙、硬脂酸镁、固体聚乙二醇、十二烷基硫酸钠，或其混合物。胶囊剂、片剂和丸剂中，剂型也可包含缓冲剂。
固体剂型如片剂、糖丸、胶囊剂、丸剂和颗粒剂可采用包衣和壳材制备，如肠衣和其它本领域公知的材料。它们可包含不透明剂，并且，这种组合物中活性活性成分的释放可以延迟的方式在消化道内的某一部分中释放。可采用的包埋组分的实例是聚合物质和蜡类物质。必要时，活性活性成分也可与上述赋形剂中的一种或多种形成微胶囊形式。
用于口服施用的液体剂型包括药学上或保健品学上可接受的乳液、溶液、悬浮液、糖浆或酊剂。除了活性活性成分外，液体剂型可包含本领域中常规采用的惰性稀释剂，如水或其它溶剂，增溶剂和乳化剂，例知，乙醇、异丙醇、碳酸乙酯、乙酸乙酯、丙二醇、1,3-丁二醇、二甲基甲酰胺以及油，特别是棉籽油、花生油、玉米胚油、橄榄油、蓖麻油和芝麻油或这些物质的混合物等。
除了这些惰性稀释剂外，组合物也可包含助剂，如润湿剂、乳化剂和悬浮剂、甜味剂、矫味剂和香料。
除了活性活性成分外，悬浮液可包含悬浮剂，例如，乙氧基化异十八烷醇、聚氧乙烯山梨醇和脱水山梨醇酯、微晶纤维素、甲醇铝和琼脂或这些物质的混合物等。
用于肠胃外注射的组合物可包含生理上可接受的无菌含水或无水溶液、分散液、悬浮液或乳液，和用于重新溶解成无菌的可注射溶液或分散液的无菌粉末。适宜的含水和非水载体、稀释剂、溶剂或赋形剂包括水、乙醇、多元醇及 其适宜的混合物。
用于局部施用的本发明活性成分的剂型包括凝胶剂(如可注射水凝胶)、贴剂、软膏剂、散剂、喷射剂和吸入剂。活性成分在无菌条件下与生理上可接受的载体及任何防腐剂、缓冲剂，或必要时可能需要的推进剂一起混合。
本发明活性成分可以单独施用，或者与其他治疗剂联合给药。在另一优选例中，所述组合物还包含一种或多种其他的治疗剂。优选地，所述治疗剂选自下组：阿曲生坦(Atrasentan)、HhAntag、OSI-930、GW4064、2-NP，或其组合。
在某些实施方式中，本发明的活性成分在相同或分开的制剂中与作为联合治疗方案的部分的其它试剂同时使用，或与所述其它试剂依次使用。
本发明的活性成分的组合物的治疗有效剂量的一般范围将是：约1-2000mg/天、约10-约1000mg/天、约10-约500mg/天、约10-约250mg/天、约10-约100mg/天，或约10-约50mg/天。治疗有效剂量将以一个或多个剂量给予。然而，应理解，对于任何特定患者的本发明化合物的特定剂量将取决于多种因素，例如，待治疗的患者的年龄、性别、体重、一般健康状况、饮食、个体响应，给予时间、待治疗的疾病的严重性、施用的具体化合物的活性、剂型、应用模式和伴用药物。给定情况的治疗有效量能用常规实验测定，并在临床医生或医师能力和判断范围内。在任何情况中，所述活性成分或组合物将基于施用对象的个体情况以多个剂量给予并以允许递送治疗有效量的方式给予。
本发明的活性成分或组合物的施用对象的实例包括哺乳动物(例如，人、小鼠、大鼠、仓鼠、兔、猫、狗、牛、绵羊、猴等)。
本发明的主要优点包括：
1)ISX-9可治疗昼夜节律失调，且从增强振幅、持续性等方面均具有优异效果，并且具有剂量依赖性、低细胞毒性且不影响昼夜节律周期；
2)本发明证明Camk2d表现为真正的钟控制基因，可调节BMAL1活性并形成正反馈循环，若增强此正反馈循环，则可逆转衰老引起的昼夜节律衰变。
下面结合具体实施例，进一步阐述本发明。应理解，这些实施例仅用于说明本发明而不用于限制本发明的范围。下列实施例中未注明具体条件的实验方法，通常按照常规条件，或按照制造厂商所建议的条件。除非另外说明，否则百分比和份数是重量百分比和重量份数。
材料和方法
细胞系
HEK293T、NIH-3T3和N2a细胞系获自ATCC。小鼠胚胎成纤维细胞MEF从C57野生型、Bmal1 -/-或mPer2 Luc/+小鼠的E13.5d胚胎中分离。细胞在含有10％FBS和100U/ml青霉素/链霉素的DMEM(Invitrogen)中培养，并在37℃有5％CO 2的加湿环境中培养。本发明使用基于PCR的方法(MP0035,Sigma)检查所有细胞系，确保所有细胞系不含支原体。
动物品系
报告小鼠品系mPer2 Luc(006852)和Bmal1-floxed条件品系(007668)获自Jackson Laboratory。小鼠在12:12小时光暗循环下饲养在无特定病原体(SPF)动物房设施中。动物实验按照科研单位的生物医学研究伦理委员会、上海营养与健康研究所和脑科学卓越中心与中国科学院智能技术院批准的方案进行。
质粒
对于基于荧光素酶的表达测定，小鼠Camk2a/b/d/g、Dbp和Rev-erba启动子片段从小鼠基因组DNA中进行PCR扩增，然后通过MluI和BglII位点克隆到pGL3-Basic-luc载体(E1751，Promega)中。用于过表达Bmal1、FLAG-Bmal1、FLAG-Camk2a、b、d和g的构建体通过NotI和MluI位点克隆到具有FLAG标签融合的pCAGGS载体中。Camk2d K43M突变体是使用基于PCR的定点诱变产生的，然后亚克隆到相同的pCAGGS骨架中。
试剂
用于生物节律测试的小分子购自以下公司：ISX-9来自Selleck；GW4064、OSI-930、阿曲生坦(Atrasentan)、Forskolin和KN93来自Med Chem Express；2-NP和HhAntag来自TargetMol。抗体购自以下资源：抗BMAL1(14020，Cell Signaling Technology)，抗DBP(12662-1-AP，ProteinTech)，抗PER2(13168，ABclonal)，抗REV-ERBα(13418，Cell Signaling Technology)、抗GAPDH(60004，ProteinTech)、HRP偶联的山羊抗兔二抗(1706515，Bio-Rad)和HRP偶联的山羊抗小鼠二抗(1706516，Bio-Rad)。
小分子筛选
在384孔板中进行初级小分子筛选(Natural Products库、NIH临床系列、LOPAC系列和Spectrum系列来自Sigma-Aldrich；新化合物库、表观遗传学库和GPCR和G蛋白库来自Selleck；批准药物筛选库和抑制剂库来自TargetMol；干细胞调节器库来自Merck；神经递质库来自Tocris Bioscience；独家新化合物库来自MCE；以及核受体配体激动剂或拮抗剂库来自Enzo Life Sciences)。筛选流程使用大约3000个mPer2 Luc MEF细胞，接种48小时后加入10μM小分子药物处理。在小分子药物处理后，分别于24小时和30小时两个时间点通过ONE-Glo TM荧光素酶测定系统(E6120，Promega)测量荧光素酶活性。在化学发光测量之后，通过alamarBlue测定法(Invitrogen)确定细胞存活。
实时生物发光记录
为了实时记录PER2::LUC生物发光，接种约2.5x10 5mPer2 Luc MEF细胞，然后在3.5cm培养皿中用DMEM培养48小时直至细胞长满。之后使用200nM地塞米松处理细胞，同步化1小时，最后在生物发光记录前在无血清DMEM培养基中添加200μM荧光素(A5030，东京化学工业)以及10μM或指定浓度的指定小分子培养并记录生物发光4天。记录条件为将培养皿置于配置了光子倍增管记录(PMT)检测器组件的不透光箱体中(LumiCycle32,Actimetrics)连续记录。数据使用LumiCycle分析软件(Actimetrics)处理，包括振幅和周期等结果。
用于生物发光记录的组织培养
视交叉上核SCN的切片和 实时生物发光记录按照Savelyev等人(Savelyev等人，2011)的描述进行。基本步骤为首先收取6个月大的mPer2 Luc小鼠全脑，然后通过McIlwain Tissue Chopper(TC752，Cavey Laboratory Engineering Co.Ltd)将SCN部分切成300μm厚。SCN切片置于培养膜(PICM0RG50,Millipore)上，外围培养皿加入1.5ml记录培养基(1x DMEM、1x B27补充剂、4.2mM NaHCO 3、10mM HEPES、1X青霉素/链霉素和200μM荧光素)进行SCN组织培养；对照组包含DMSO，实验组包含ISX-9(10μM)。通过连续PMT记录(LumiCycle32，Actimetrics)检测ISX-9的振幅增强效果。垂体的记录条件与SCN相同。
定量RT-PCR分析
根据制造商的说明，使用RNeasy Mini Kit(Qiagen)提取总RNA。使用Omniscript RT Kit(205111,Qiagen)将大约3μg RNA用于cDNA生成。使用SYBR Green PCR Kit(208054，Qiagen)组装实时PCR反应，然后在ABI ViiATM 7中进行分析。QuantStudioTM实时PCR软件用于确定基因表达水平并归一化为核糖体参考基因,Rpl19。引物序列列于表1。
表1引物序列
Figure PCTCN2022099218-appb-000005
免疫印迹
细胞收取后裂解于补充了蛋白酶抑制剂(118735800，Sigma-Aldrich)和磷酸酶抑制剂(4906837001，Roche)的冷冻裂解缓冲液(pH7.5的50mM Tris、150mM NaCl、5mM MgCl 2、0.5％Nonidet P-40和1％TritonX-100)中。通过Bradford测定法测量细胞裂解液的蛋白质浓度，之后调整各样品的蛋白质含量至相等，用于定量免疫印迹分析。Phos-tag凝胶电泳做法为将细胞裂解物在含有40μM丙烯酰胺Phos-tag配体(F4002，APExBIO)和80μM MnCl 2的4％丙烯酰胺凝胶中进行电泳分析。
免疫组化
将特定年龄的小鼠安乐死后，立即用4％多聚甲醛灌注，随后将全脑取出，在30％蔗糖中进行脱水与冷冻保护，最后将脑组织置于OCT包埋剂中包埋(4583，SAKURA)。使用冰冻切片机(CM1850，Leica)收取30μm厚的冠状面脑切片，然后通过3％H 2O 2处理淬灭样品中残留的过氧化酶活性。在室温下通过0.05％胰蛋白酶处理5分钟进行抗原修复步骤，然后用TNB封闭缓冲液(100mM Tris-HCl，pH7.5，150mM NaCl，10％FBS)封闭脑切片，接着加入抗PER2抗体稀释液(1:300) 覆盖脑切片，4℃孵育过夜。隔日用TNT缓冲液(100mM Tris-HCl，pH 7.5，150mM NaCl，0.05％Tween 20)洗涤3次后，将HRP偶联的抗兔二抗(1:1000)覆盖于脑切片上，室温下继续孵育1.5小时。在TNT缓冲液洗涤3次后，将脑切片与荧光团标记的酪胺底物(TSA plus Cyanine3试剂盒，Perkin Elmer)和DAPI(10ng/ml，Sigma-Aldrich)一起孵育，然后通过共聚焦显微镜(Cell Observer，ZEISS)观察荧光标记。荧光组化染色结果由ImageJ分析和量化。
代谢笼
小鼠的间接热量测定实验通过CLAMS-16代谢笼系统(Columbus Instruments)进行。2个月大和14个月大的小鼠群体在ISX-9(20mg/kg)或DMSO处理7天后，将小鼠单独饲养于代谢笼中适应2天后进行代谢数据采集。整个实验过程中提供充足的常规饲料与饮水。在12:12小时的明暗循环以及25℃的温控条件下，每隔10分钟自动测量小鼠的VO 2、VCO 2、食物摄入量、饮水量和活动量，采集连续3天数据。通过配套软件CLAX(Columbus Instruments)计算体力活动和呼吸交换比(RER＝VCO 2/VO 2)。
脑电图记录和脑电数据分析
小鼠脑电图记录是通过HD-X02遥测植入子(DSI PhysioTel TM)进行。植入子埋植手术首先通过单次肌肉注射5mg/kg戊巴比妥(pentobarbital)进行深度麻醉，之后将两个EEG电极分别通过小螺丝钉固定于颅骨上，记录位点为颅骨Bregma线外侧1mm、Bregma线前1mm与3mm。手术后，在小鼠腹腔注射5％葡萄糖溶液(40μl/g体重)7天促进术后恢复。活动量、体温、EEG和EMG信号由连接到计算机的MX2集线器和RPC-1接收器收集。首先在正常光暗条件下记录2天作为预处理基线。各年龄段的小鼠在腹腔注射DMSO 7天，停止处理7天后，连续记录3天的EEG和EMG作为对照组数据。最后，同一组小鼠作为实验组注射ISX-9(20mg/kg)7天，停药7天，然后进行连续3天的EEG和EMG记录。小鼠EEG睡眠分析使用配套软件NeuroScore 3.2.0(DSI)，分析并总结清醒、快速动眼睡眠REM和慢波NREM睡眠阶段，并通过10秒时间窗口的慢波、快波功率统计昼夜睡眠-觉醒的切换转变。各频段的EEG功率(:0.5-4Hz,:4-8Hz,:8-12Hz,:12-16Hz,:16-32Hz,:>32Hz)显示以每小时平均数据点表达。
体外Ca 2+成像
将MEF细胞(2x10 5)接种在玻璃底培养皿(801002，NEST)上，24小时后将Fluo-4AM(2μM，Molecular Probes)在无血清DMEM中于37℃孵育45分钟。用PBS冲洗两次清洗未结合的染料后，测量细胞的Ca 2+基线(激发488nm，发射520nm)15分钟。在DMSO或10μM ISX-9添加同时(时间点0)开始记录Ca 2+-流入。通过荧光显微镜(Cell Observer，ZEISS)以1分钟的间隔收集图像，持续15分钟观察钙内流。
siRNA敲低
用于小鼠Camk2a/b/d/g和scramble对照的沉默寡核苷酸购自GenePharma。使用Lipofectamine 3000(Invitrogen)用siRNA寡核苷酸瞬时转染MEF细胞，然后如上面实验条件所述进行实时生物发光记录。
蛋白质纯化和激酶测定
将编码FLAG-Bmal1、FLAG-Camk2亚型和突变体的pCAGGS质粒(5μg)分别转染到2x10 6HEK293T细胞中，表达48小时后，收集细胞并在含有蛋白酶抑制剂(Roche)的400μl裂解缓冲液中裂解细胞。将大约3mg细胞裂解液与20μl抗FLAG-M2磁珠(Sigma)在4℃下孵育4小时，然后通过TBS缓冲液(50mM Tris，pH7.5，150mM NaCl)清洗杂质3次，最后通过100μl的3X FLAG短肽(150ng/μl，Sigma)洗脱BMAL1或CaMKII纯化蛋白。使用激酶活性试剂盒(EA004，R&DSystems)以CaMKII：BMAL1 1:4的比例进行激酶测定，最后在620nm测量激酶反应的吸光度。
单分子RNA原位杂交
使用RNAscope多重荧光v2-试剂盒(Advanced Cell Diagnostics)进行单分子原位杂交。基本步骤为，将小鼠安乐死，用4％多聚甲醛灌注，小心取出大脑后在4％多聚甲醛中后固定过夜，然后在4℃下浸泡于30％蔗糖-1X PBS中进行脑组织冷冻保护。用OCT包埋液包埋后，收集30μm的冠状面脑切片，然后进行脱水、过氧化物酶活性淬灭，最后通过Protease Plus溶液(322381，Advanced Cell Diagnostics)暴露目标RNA。切片用水洗涤后，与Camk2a、2b、2d、2g目标探针(产品目录号码445231、453601、508941、522071，Advanced Cell Diagnostics， 1:300稀释)在HybEZ烘箱中40℃孵育2小时。依照用户手册(323100-USM，Advanced Cell Diagnostics)中的描述进行扩增、掺入HRP过氧化物酶和酪胺染色等步骤，然后进行DAPI复染和荧光显微镜观察拍照(VS120，OLYMPUS)。图像通过ImageJ分析和量化。
荧光素酶测定
在转染前24小时，将N2a细胞以每孔8x10 4个细胞的密度接种到24孔培养皿中。用Lipofectamine 3000(Invitrogen)以200:100ng每孔的比例转染Bmal1报告质粒及Camk2过表达质粒。24小时后，使用100nM地塞米松(dexamethasone)处理细胞15分钟同步化后，在DMEM中用加入DMSO(对照组)或10μM ISX-9(实验组)处理。24小时后收取细胞并在100μl 1X Glo裂解缓冲液(E266A，Promega)中裂解，并与底物(ONE-Glo TM荧光素酶检测系统，E6120，Promega)混合用于荧光素酶测量。
量化和统计分析
除非另有标注，个别体外实验至少进行3次。动物实验至少独立重复两次，并确定给药效果一致。结果均以平均值±SD或平均值±SEM表示，如图例所示。统计显著性标记为不显著，ns；*p<0.05；**p<0.01；***p<0.001。
实施例1.小分子筛选将ISX-9鉴定为昼夜节律幅度增强剂。
为了发现新的生物节律调节小分子用于更持久地增强生物钟振幅，并且最小地干扰24小时昼夜周期，本发明通过mPer2 Luc成纤维细胞进行了8060个小分子的化合物筛查，其中覆盖了激酶、表观遗传因子和GPCR等主要标靶(图1A)。
为了验证高通量初筛中的振幅与相位改变，发明人分别在药物处理后24h和30h测量mPer2 Luc成纤维细胞的化学发光水平(图1A和B)。与未处理的对照相比，如果发现某化合物对PER2::LUC的表达在两个时间点的上调或下调影响是一致的，则该化合物可以说明为振幅调节剂；如果对两个时间点的作用是相反的，则可以说明该化合物为相位调节剂。
在排除了具有明显细胞毒性的化合物后，本发明专注于剩余的6777种化合物，并得到270种能够将PER2::LUC振幅增加1.2倍的振幅增强剂。其中，ISX-9、阿曲生坦(Atrasentan)、HhAntag、OSI-930、GW4064和2-NP是提高PER2::LUC水 平的主要候选(图1C)。
PER2::LUC发光的实时记录证实了六个候选药物都能够在不影响昼夜节律相位的情况下增加PER2::LUC振幅，节律周期都维持在接近24小时(图1D)。值得注意的是，多数候选药物仅在第1天明显增强了PER2::LUC幅度，然后在剩余的3天内效果显著下降(图1E)。
ISX-9是唯一在4天的测试中持续维持节律震荡的振幅增强剂。ISX-9的振幅增强作用是广效的，可用于提升其他重要节律基因，例如Dbp和Rev-Erbα的表达(图1F)。在这些情况下，ISX-9在六种候选药物中再次显示出其调节生物钟的最佳能力。
ISX-9是一种异恶唑化合物，并具有促神经发生的活性(图2A)。在本发明的测试中发现ISX-9在提高PER2::LUC振幅上具有剂量依赖效应(图2B)，并且在检验的最高浓度10μM条件下时仍显示低细胞毒性(图2C)。
综上所述，ISX-9从增强振幅、持续性等方面均具有优异效果，并且具有剂量依赖性、低细胞毒性且不影响昼夜节律周期，因此ISX-9可用作昼夜节律幅度增强剂。
实施例2.ISX-9与培养细胞中的生物钟基因的关系。
由图3可知，与荧光素酶分析一致，ISX-9增加了小鼠胚胎成纤维细胞(MEF)中生物钟基因的表达，包括Bmal1、Per2、Dbp以及其他核心生物钟基因等。
与BMAL1和REV-ERBα相比，DBP蛋白水平对ISX-9剂量高度敏感(图4A)，因此可以作为在外周系统中报告ISX-9效果的生物钟蛋白标记物。
有趣的是，发明人发现ISX-9对DBP以及PER2::LUC的诱导效果在Bmal1 +/-小鼠胚胎成纤维细胞中减弱，在Bmal1 -/-小鼠胚胎成纤维细胞中节律性消失，表明ISX-9的振幅增强效果依赖于BMAL1活性(图4B和C)。
实施例3.ISX-9的体外、离体和体内的振幅增强效果
由图4可知，ISX-9还能在视交叉上核(SCN)和垂体等体外培养记录中激发更高的PER2::LUC节律振幅(图4D)；并且施打ISX-9七天后的小鼠在SCN中显示出更高的PER2节律蛋白水平(图4E)，整体说明了ISX-9在细胞、体外组织，以及体内的振幅增强效果。
实施例4.ISX-9在体内调节中老年小鼠的代谢作用
为了探索ISX-9在体内的代谢调节作用，本发明进一步测试了年轻(2个月龄)和中老年(14个月龄)小鼠的代谢率。
首先建立昼夜节律稳态环境，在12:12小时的明暗循环下，在黑暗环境下使小鼠清醒，在明亮环境下使小鼠进入休息状态；昼夜节律失调实验组为14个月龄小鼠。
年轻小鼠表现出明显的呼吸交换比(RER)的昼夜节律性波动，并且在白天的休息/禁食状态下显示较低的RER(图5A)。中老年组在代谢笼中表现出较差的RER节律性，仅在上半夜表现出RER的短暂升高，代表进食后的碳水化合物代谢结果(图5A)。
在中老年组小鼠体内注射ISX-9可增加RER的幅度(图5B)，并且没有改变年轻组的RER幅度(图5C)，显示ISX-9在衰老过程中节律调节的应用性。此外，本发明发现ISX-9能将中老年组小鼠的RER周期从26小时校正到24小时(图5D)，除了略微促进了夜间的水分摄取外，不影响中老年组的食物摄入量以及体重(图5E)。
由于ISX-9治疗不影响年轻群体(图5F和5G)，结果表明ISX-9选择性地改善与衰老相关的代谢失调。由于ISX-9的处理在代谢笼实验之前进行，实验结果进一步证实了ISX-9在促进体内昼夜节律幅度上是可持续的。
实施例5.ISX-9改善中老年小鼠的睡眠障碍
ISX-9实验组在白天表现出较低的O 2耗用以及CO 2产生，表明该化合物可以使中老年小鼠的休息更加稳定(图5B)。为了研究ISX-9是否确实可以改善睡眠障碍，本发明在年轻与中老年组的小鼠进行了脑电图(EEG)的遥测记录并分析睡眠状态。
通过比对，本发明发现两组小鼠在夜间的清醒和慢波睡眠(NREM)差异最为显著。中老年组表现出夜间活动期时较高的NREM睡眠，显示其较低的清醒程度(图6A)。与年轻组相比，中老年组小鼠还表现出活动量大幅减少、体温波动减小(图6B)、β/γ快波降低和δ/θ慢波波动不明显(图6C)等节律紊乱表型，这些结果表明了衰老过程中脑内昼夜节律强度的整体下降。
为了测试ISX-9是否可以引导昼夜节律，强化睡眠稳态，发明人给同一组小鼠连续7天施打了20mg/Kg的ISX-9，停止处理7天后开始脑电EEG记录。
本发明发现ISX-9的预处理大幅提升了中老龄小鼠上半夜的清醒程度，同时降低了快速动眼REM和慢波NREM睡眠(图7A)。接受治疗的中老年小鼠在夜间也表现出较高的活动力，同时在白天休息时表现出较低的体温(图7B)，上述结果表明ISX-9协助中老年小鼠调整至清醒时更加警觉、活跃的状态，并在睡觉期间保持更好的、体温更低的休息状态。本发明也注意到接受治疗的中老年小鼠在休息期间具有更高的慢波delta功率，在清醒期间具有更高的快波gamma功率，两者都显示大脑脑电昼夜节律稳态的改善(图7C)。
通过计算刚开灯(ZT0-1)和刚关灯(ZT12-13)时清醒-睡眠阶段的转变，本发明发现ISX-9显著抑制了中老年组频繁的清醒-睡眠转换，使中老年组趋近于在健康年轻小鼠中观察到的更稳定的睡眠-清醒稳态(图7D)。
实施例6.ISX-9触发Ca 2+流入且与Camk2d活性相关
前人研究证实ISX-9可通过Ca 2+信号传导促进神经元分化。本发明首先通过Fluo-4AM荧光染料在MEF中跟踪细胞内的Ca 2+水平，并证实了ISX-9能够在测试中快速触发Ca 2+流入(图8A)。
与之前的报告一致，本发明还发现ISX-9效应是由CaMKII活性介导的，因为CaMKII特异性拮抗剂KN93完全消除了ISX-9对PER2::LUC增强作用(图8B)。在四种CaMKII亚型中，发明人注意到Camk2b和Camk2d敲低在实时PER2::LUC记录中明显降低了ISX-9效应，但在Camk2a和Camk2g敲低中没有(图8C)，显示ISX-9的效果更加依赖于CaMKIIβ和CaMKIIδ两个亚型。
鉴于ISX-9在提高DBP和PER2::LUC水平上显示出高度的BMAL1依赖性(图4B和C)，并能触发BMAL1的磷酸化(图8D)，本发明进一步采用生化方法检验了CaMKII对BMAL1磷酸化的活性，并比较CaMKII亚型之间的差异。
CaMKIIδ在激酶测定中对纯化的BMAL1表现出最高活性(图8E)，连同Camk2d敲低后影响PER2::LUC表达的结果，上述数据说明了CaMKIIδ对介导BMAL1依赖的ISX-9效应至关重要。这些结果提出了一个连接ISX-9、Ca 2+信号和BMAL1磷酸化以调节生物节律振幅的正向反馈循环。
单分子RNA荧光原位杂交(smFISH)分析表明，小鼠大脑中的Camk2d在皮层下区域和下丘脑中表达更为丰富(图9)，并显示出清晰的节律性表达，其在生物钟中枢视交叉上核(SCN)中的ZT18时间点达到峰值(图10)，与预期其调节昼夜节律的潜在功能非常吻合。其他CaMKII亚型转录产物，如Camk2a和Camk2b 在SCN中表现出轻微的波动和低表达，其表达更加富集于纹状体和皮质中(图9)。进一步说明，ISX-9对SCN中的昼夜节律作用与Camk2d有关。
实施例7.ISX-9重新激活老年小鼠SCN中的Camk2d表达
老年小鼠的SCN中Camk2d显著降低(图11A)，而ISX-9能提高Camk2d表达(图11B)，这些结果提示ISX-9有益于在衰老过程中重新提高CaMKIIδ水平。加上CaMKIIδ可通过磷酸化BMAL1上调BMAL1转录活性，进一步逆转了衰老导致的昼夜节律幅度下降。Camk2a和Camk2b的表达水平在年老的SCN中没有改变，对ISX-9治疗也没有反应(图11，A和B)。
实施例8.Camk2d表现为真正的时钟控制基因，可以响应ISX-9诱导并促进生物钟的控制。
鉴于Camk2d表达是节律性的并且可以由ISX-9触发，本发明在Camk2启动子中搜索了特定E-box序列。本发明发现所有Camk2亚型都包含E-box元件，包括在Camk2d和Camk2g启动子中的经典E-box序列(CACGTG)(图12A)。在N2a细胞中使用启动子报告分析后，本发明发现Camk2d启动子对CLOCK:BMAL1激活最敏感，验证了Camk2d是一个明确的生物钟控制基因(图12B)。
此外，ISX-9可支持转录因子CLOCK:BMAL1进一步诱导Camk2，并且在Camk2d表达上显示最佳效果(图12B)，与老年SCN中的效果一致(图11B)。为证明CaMKIIδ对BMAL1磷酸化的直接作用，发明人纯化了CaMKIIδ激酶失活K43M突变体，并通过生化方法检测出BMAL1的磷酸化明显减少(图12C)。另外，CaMKIIδK43M突变体对激活Dbp:Luc表达的效果明显降低(图12D)，上述再次证实CaMKIIδ可通过磷酸化BMAL1上调生物节律振幅。
综上所述，结果表明CaMKIIδ作为生物钟调控的钟蛋白，可调节BMAL1活性并形成正反馈循环，增强此正反馈循环，例如使用CaMKII激动剂ISX-9，可逆转衰老引起的昼夜节律衰变，使衰老客体年轻化并恢复活力(图13)。
讨论
本发明提出了一种基于小分子药物的干预措施，用于有效并持续地放大昼夜节律振幅，解决老年过程中节律幅度逐渐衰减的问题。这种衰减的部分原因是衰老过程中CaMKIIδ表达量减少，因而限制了通过CaMKIIδ磷酸化并启动BMAL1调 节生物钟的能力。
前人工作已表明钙波动是视交叉上核中生成昼夜节律的基本信号，本发明进一步证明通过ISX-9敏化Ca 2+流入是加强CaMKIIδ信号从而提高衰老细胞昼夜节律振幅的便捷方法。加上ISX-9的促神经发生活性，以及其改善神经退行性疾病等前人发现，使用ISX-9可望在抗衰老，尤其是在治疗神经系统疾病方面具有潜力。
综上所述，应用ISX-9和靶向CaMKIIδ的设计可望作为干预昼夜节律衰退，甚至是衰老相关的疾病的重要手段。
在本发明提及的所有文献都在本申请中引用作为参考，就如同每一篇文献被单独引用作为参考那样。此外应理解，在阅读了本发明的上述讲授内容之后，本领域技术人员可以对本发明作各种改动或修改，这些等价形式同样落于本申请所附权利要求书所限定的范围。

Claims (16)

  1. 一种ISX-9的用途，所述的ISX-9具有如下式所示的结构：
    Figure PCTCN2022099218-appb-100001
    其特征在于，所述的ISX-9用于制备组合物，所述组合物用于选自下组的一种或多种用途：(i)预防和/或治疗昼夜节律失调；(ii)预防和/或治疗睡眠障碍；(iii)提高昼夜节律振幅；和/或(iv)提高CaMKIIδ蛋白的活性或表达量。
  2. 如权利要求1所述的用途，其特征在于，所述的组合物用于在中老年对象中(i)预防和/或治疗昼夜节律失调；(ii)预防和/或治疗睡眠障碍；(iii)提高昼夜节律振幅；和/或(iv)提高CaMKIIδ蛋白的活性或表达量。
  3. 如权利要求1所述的用途，其特征在于，所述昼夜节律失调包括由于年龄增长而导致的昼夜节律失调。
  4. 如权利要求1所述的用途，其特征在于，所述预防和/或治疗昼夜节律失调指选自下组的一个或多个指标得到改善：
    (i)提高昼夜节律振幅；
    (ii)提高代谢昼夜节律性波动；
    (iii)预防和/或治疗昼夜节律性的睡眠障碍。
  5. 如权利要求1所述的用途，其特征在于，所述预防和/或治疗昼夜节律失调包括提高生物钟基因活性，且所述生物钟基因选自下组：Bmal1、Per2、Dbp、Clock、Cry1、Cry2、Npas2、Nr1d1、Rorα，或其组合。
  6. 如权利要求1所述的用途，其特征在于，所述预防和/或治疗昼夜节律失调包括通过提高BMAL1活性从而提高昼夜节律振幅。
  7. 如权利要求1所述的用途，其特征在于，所述预防和/或治疗昼夜节律失调包括增强BMAL1活性和提高CaMKIIδ自身的表达。
  8. 如权利要求1所述的用途，其特征在于，所述组合物中含有浓度≥0.1μM，较佳地浓度≥0.25μM，更佳地浓度≥10μM的ISX-9或其药学上可接受的盐。
  9. 如权利要求1所述的用途，其特征在于，所述组合物的剂型选自下组：固体 制剂、液体制剂或半固体制剂。
  10. 如权利要求1所述的用途，其特征在于，所述组合物通过以下方式施用于受试者：口服、经皮注射、静脉注射、肌肉注射或肛肠给药。
  11. 一种ISX-9的氘代物的用途，所述的ISX-9具有如下式所示的结构：
    Figure PCTCN2022099218-appb-100002
    其特征在于，所述的ISX-9的氘代物用于制备组合物，所述组合物用于选自下组的一种或多种用途：(i)预防和/或治疗昼夜节律失调；(ii)预防和/或治疗睡眠障碍；(iii)提高昼夜节律振幅；和/或(iv)提高CaMKIIδ蛋白的活性或表达量。
  12. 一种CaMKIIδ高表达调节剂的用途，其特征在于，用于制备组合物，所述组合物用于选自下组的一种或多种用途：(i)预防和/或治疗昼夜节律失调；和/或(ii)调节BMAL1活性。
  13. 如权利要求12所述的用途，其特征在于，所述预防和/或治疗昼夜节律失调包括：通过促进BMAL1磷酸化从而提高BMAL1活性和提高CaMKIIδ自身的表达。
  14. 如权利要求12所述的用途，其特征在于，所述的组合物用于提高CaMKIIδ在皮层下区域和下丘脑中的表达水平。
  15. 一种药物组合物，其特征在于，所述药物组合物包括：ISX-9或其氘代物或其药学上可接受的盐，以及药学上可接受的载体。
  16. 一种(i)预防和/或治疗昼夜节律失调；(ii)预防和/或治疗睡眠障碍；和/或(iii)提高昼夜节律振幅的方法，其特征在于，包括步骤：向有此需要的对象施用安全有效量的ISX-9或其药学上可接受的盐。
PCT/CN2022/099218 2022-01-04 2022-06-16 Isx-9在治疗衰老相关的昼夜节律幅度下降和睡眠障碍方面的应用 WO2023130675A1 (zh)

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