阅读说明：本技术 多甲氧基黄酮在改善昼夜节律失调的用途 (Use of polymethoxyflavone for improving circadian rhythm disorder ) 是由 陈铮 S-H·柳 J·S·高桥 于 2018-03-05 设计创作，主要内容包括：公开了包括向患有或有风险患上时钟受控失调(例如代谢综合症或其组成症状)的哺乳动物给予包含至少一种多甲氧基黄酮的组合物的方法。公开了包括向患有或有风险患上睡眠障碍；老化；和/或患有或有风险患上心境障碍的哺乳动物给予包含至少一种多甲氧基黄酮的组合物的方法。多甲氧基黄酮可以是川陈皮素和/或橘皮素。所述组合物还可以包含其他化合物,例如烟酰胺核苷和/或紫檀芪,和/或预期改善代谢综合征、睡眠障碍、心境障碍、老化、心血管疾病、免疫失调、神经退行性疾病和/或癌症中的一种或多种症状的其他化合物。(Methods are disclosed that include administering a composition comprising at least polymethoxylated flavones to a mammal having or at risk of developing a clockwork-controlled disorder (e.g., metabolic syndrome or a constituent thereof). methods are disclosed that include administering a composition comprising at least polymethoxylated flavones to a mammal having or at risk of developing a sleep disorder, aging, and/or having or at risk of developing a mood disorder.)

1, a method comprising:

administering to a mammal suffering from or at risk of developing metabolic syndrome or a constituent symptom thereof a composition comprising at least polymethoxylated flavones.

2. The method of claim 1, wherein at least polymethoxylated flavones have formula I:

wherein R is¹、R²、R³、R⁴、R⁵And R⁶Each independently is-H or-OCH₃Provided that R is¹、R²、R³、R⁴、R⁵And R⁶At least two of which are-OCH₃。

3. The method of claim 2, wherein at least polymethoxylated flavones are selected from the group consisting of nobiletin, hesperetin and both.

4. The method of claim 1 wherein the administration is at a dose of 20mg polymethoxyflavone/kg body weight of the mammal to 2000mg polymethoxyflavone/kg body weight of the mammal and at a rate of from twice daily to times weekly for at least weeks.

5. The method of claim 4 wherein the administration is at a dose of 200mg of polymethoxylated flavone per kg of body weight of the mammal at a rate of times every two days for at least ten days.

6. The method of claim 1, wherein the administration is by the oral route.

7. The method of claim 6, wherein the composition is in a form selected from the group consisting of a liquid, a tablet, a capsule, a caplet, and a soluble powder.

The method of claim 8, , comprising:

administering to a mammal suffering from or at risk of developing a sleep disorder a composition comprising at least polymethoxylated flavones.

9. The method of claim 8, wherein at least polymethoxylated flavones have formula I:

wherein R is¹、R²、R³、R⁴、R⁵And R⁶Each independently is-H or-OCH₃Provided that R is¹、R²、R³、R⁴、R⁵And R⁶At least two of which are-OCH₃。

10. The method of claim 9, wherein at least polymethoxylated flavones are selected from the group consisting of nobiletin, hesperetin and both.

11. The method of claim 8 wherein the administration is at a dose of 20mg polymethoxylated flavone/kg body weight of the mammal to 2000mg polymethoxylated flavone/kg body weight of the mammal and at a rate of from twice daily to times weekly.

12. The method of claim 11 wherein the administration is at a dose of 200mg of polymethoxylated flavone per kg of body weight of the mammal and at a rate of times every two days.

13. The method of claim 8, wherein the administration is by the oral route.

14. The method of claim 13, wherein the composition is in a form selected from the group consisting of a liquid, a tablet, a capsule, a caplet, and a soluble powder.

15. The method of claim 8, wherein the sleep disorder is selected from the group consisting of insomnia, somnolence, narcolepsy, Delayed Sleep Phase Syndrome (DSPS), advanced sleep phase syndrome (ASPD), non-24 hour sleep arousal disorder, irregular sleep arousal rhythm, and shift work sleep disorder.

16, a method, comprising:

administering to a mammal suffering from aging a composition comprising at least polymethoxylated flavones.

17. The method of claim 16, wherein at least polymethoxylated flavones have formula I:

wherein R is¹、R²、R³、R⁴、R⁵And R⁶Each independently is-H or-OCH₃Provided that R is¹、R²、R³、R⁴、R⁵And R⁶At least two of which are-OCH₃。

18. The method of claim 17, wherein at least polymethoxylated flavones are selected from the group consisting of nobiletin, hesperetin and both.

19. The method of claim 16 wherein the administration is at a dose of 20mg polymethoxylated flavone/kg body weight of the mammal to 2000mg polymethoxylated flavone/kg body weight of the mammal and at a rate of from twice daily to times weekly.

20. The method of claim 19 wherein the administration is at a dose of 200mg of polymethoxylated flavone per kg of body weight of the mammal and at a rate of times every two days.

21. The method of claim 16, wherein the administration is by the oral route.

22. The method of claim 21, wherein the composition is in a form selected from the group consisting of a liquid, a tablet, a capsule, a caplet, and a soluble powder.

Technical Field

The present disclosure relates generally to the field of mammalian health, more specifically, it relates to the use of polymethoxylated flavones to ameliorate circadian rhythm disturbances.

Background

In mammals, the CLOCK system is organized hierarchically, and the central pacemaker coordinates the peripheral tissue CLOCK in the hypothalamic suprachiasmatic nucleus (SCN) to perform physiological functions (Takahashi et al, 2008). cell autonomous molecular oscillators are the basic components of the CLOCK system, consisting of interlocking feedback loops (Dibner et al, 2010). the core loop consisting of positive (transcription activator CLOCK/BMAL1 or NPAS2/BMAL1) and negative (PER1/2 and CRY1/2) arms is responsible for generating molecular rhythms, while the competitive nuclear receptors REV-ERB and ROR 2012 regulate BMAL1 expression to confer rhythm stability and robustness (Zhang and Kay, 2010. the molecular oscillator drives gene-specific expression throughout the tissue cycle by transcription and post-transcription mechanisms (Zhang et al; Zhang et al, 2014).

The basic process strictly regulated by the clock system is metabolism (Asher and Schibler, 2011; Bass and Takahashi, 2010; Gerhart-Hines and Lazar, 2015; Green et al, 2008; Rutter et al, 2002), as metabolites (Eckel-Mahan et al, 2012) and metabolic gene expression (Yang et al, 2006; Zhang et al, 2014) broadly display circadian rhythm oscillationsOverlapping metabolic defects in clock-disrupted mice are shown (Green et al, 2008). For example, a diurnal mouse mutant Clock with a dominant negative allele has been found^Δ19/Δ19(Viterterna et al 1994; King et al 1997) exhibit -series metabolic disorders including obesity, hyperlipidemia, hepatic steatosis, hyperglycemia, hypoinsulinemia, and respiratory uncoupling (Marcheva et al 2010; Shi et al 2013; Turek et al 2005).

Recent studies explored strategies to directly manipulate circadian rhythms to improve metabolic syndrome (Antoch and Kondratov, 2013; Chen et al, 2013; Farrow et al, 2012; Schroeder and Colwell, 2013). For example, the time-limited intake of a High Fat Diet (HFD) was shown to protect mice from metabolic diseases (Hatori et al, 2012). In the nocturnal specific HFD protocol, the oscillation amplitude of the clock and metabolic gene expression are significantly enhanced, indicating that during activity, the body can best consume the incoming nutrients through the synergistic effects of clock-related pathways. To circumvent compliance issues inherent in behavioral interventions, pharmacological approaches involving clock-adjusted small molecules have also been investigated (Chang et al, 2015; Chen et al, 2012, 2013; Hirota et al, 2012; Isojima et al, 2009; Meng et al, 2010; Solt et al, 2012; Wallach and Kramer, 2015). For example, small molecule agonists acting on REV-ERB nuclear receptors show beneficial metabolic effects (Solt et al, 2012), suggesting that regulatory compounds may improve metabolism through clock components or clock-related mechanisms.

The present inventors previously identified several clock amplitude enhanced small molecules (CEMs) in high-throughput chemical screening using reporter cells with highly robust rhythms (Chen et al, 2012,2013). Hybrid Clock when applied to culture^Δ19/+PER 2:Lucreporter cells, in which the reporter rhythm oscillates at a weaker amplitude (approximately one third ) relative to Wild Type (WT) Clock +/+ cells, these CEMs are able to restore the reporter rhythm amplitude to near normal levels.

However, there remains a need for clock amplitude enhancing small molecules (CEMs), including or more symptoms of metabolic disorders that may be demonstrated to be ameliorated in an in vivo mammalian model.

Disclosure of Invention

In embodiments, the present disclosure relates to methods comprising administering to a mammal having or at risk of developing metabolic syndrome or its constituent symptoms, a composition comprising at least polymethoxylated flavones.

In embodiments, the present disclosure relates to methods comprising administering to a mammal having or at risk of having a sleep disorder, a composition comprising at least polymethoxylated flavones.

In embodiments, the disclosure relates to methods comprising administering to a mammal with aging a composition comprising at least polymethoxylated flavones.

In embodiments, the present disclosure relates to methods comprising administering to a mammal having or at risk of developing a mood disorder, a composition comprising at least polymethoxylated flavones.

Polymethoxylated flavones, such as nobiletin (nobiletin) and tangeretin (tandetin), are small clock amplitude enhancing molecules (CEM) which, as demonstrated by the present inventors, improve or more symptoms of metabolic syndrome in vivo mammalian models.

Polymethoxylated flavones, such as nobiletin and hesperetin, can ameliorate or more symptoms of chronic diseases which can be caused or exacerbated by disruption of the circadian rhythm of a mammal, including but not limited to cardiovascular disease, immune disorders, neurodegenerative diseases and cancer.

Drawings

The following figures form part of the specification and are included to provide a further illustration of certain aspects of the disclosure may be better understood by reference to or more of these drawings in combination with the detailed description of specific embodiments presented herein.

Fig. 1A presents the chemical structure of nobiletin.

FIG. 1B shows the nobiletin versus PER2:: Luc Clock in the NIH clinical library (left) and the Microsource Spectrum library (right)^Δ19/+An increase in the rhythm of the reporter gene.

Fig. 1C shows the dose-dependent effect of nobiletin on reporter gene rhythm from PER2rLucSV cells (left) and quantification of amplitude response to nobiletin dose (right).

FIG. 1D shows that nobiletin cannot save PER 2:Lucclock^Δ19/Δ19Reporter gene rhythm in fibroblasts.

FIG. 1E shows nobiletin from PER2:: Luc WT (left) and PER2:: Luc Clock^Δ19/+(right) clock enhancement in pituitary explants of mice was reported.

Figure 1F shows a graph of activity changes (n-5) (left) illustrating the effect of vehicle or nobiletin on day-night behavior in WT mice fed RC. The indicated genotypes are summarized at L: d (12 hours light, 12 hours dark) or D: d (constant darkness) average wave pattern of roller motion during 12-14 days (n-5) (middle); and the indicated genotypes are at L: d or D: total wheel activity per day (n ═ 5) under condition D (right).

Fig. 2A shows the structure of hesperetin.

Fig. 2B shows the identification of hesperetin as a clock enhancing molecule. First, fully confluent PER2 was stimulated with Fsk:LUCClock^Δ19/+The cells were reported for 1 hour, and then the compound was added to the plate. Reporter gene luminescence was recorded in an EnVision microplate reader over 4 days.

FIG. 2C shows clock enhancement of NOB in peripheral tissues including lung (apical) and liver (basal) explants from PER2:: Luc WT report mice. The graph represents at least three experiments.

FIG. 2D shows that NOB did not enhance reporter rhythm in SCN explants from PER2:: Luc WT reporter mice. The medium was changed 7 days after the culture (day 0). DMSO or NOB were administered at the indicated times (dashed lines). Compared with the rhythm before and after NOB administration, no increase in amplitude was observed with NOB.

FIG. 2E shows PER2 pretreated with Forskolin (Forskolin) for 1 hour by real-time qPCR analysis the mRNA level expression of the clock gene in LucSV cells.

FIG. 2F shows a representative Western blot of circadian clock proteins in cells treated as in (E).

Fig. 2G shows Western blot analysis quantification of clock protein in (F) (n-2-3). Data are presented as mean ± SEM. P <0.05, p <0.01 and p < 0.001. NOB compared to DMSO.

Figure 3A shows the determination of the NOB levels in the plasma, brain and liver of mice by oral gavage at a dose of 200mg/kg body weight followed by LC-MS/MS (n-3).

Figure 3B shows weight gain at the end of the 10-week treatment period (n-8-15). In FIGS. 3B-3J, for WT or Clock^Δ19/Δ19Mutant mice were fed a High Fat (HF) diet and treated with vehicle or NOB every other day (n-8-15).

Fig. 3C shows food intake during the dark (solid) and light (open) phases corresponding to the subjective night and day. The Y-axis represents the cumulative food intake during the dark or light phase (n-8-15).

Figure 3D shows visceral WAT cell size with reduced NOB. Images of three regions 200 μm apart, representing >500 cells, were analyzed.

Fig. 3E shows the quantification of VO2 (oxygen consumption volume) (n-8) for each group for the dark (solid bars) and light (open bars) phases.

Fig. 3F shows that the energy expenditure of the circadian cycle is recorded (n-8).

Fig. 3G shows the quantification of the respiratory quotient for the dark and light phases (n-8).

FIG. 3H shows WT and Clock fed with HFD treated with vehicle or NOB^Δ19/Δ19Total liver weight of mutant mice (n ═ 8-15).

FIG. 3I shows WT and Clock fed with HFD treated with vehicle or NOB^Δ19/Δ19Representative image of whole liver of mutant mice (I) (n ═ 8-15). Figure 3J shows histological analysis of the liver after 10 weeks of treatment. The liver was stained with oil red O to visualize lipid droplets.

FIG. 4A shows WT or Clock^Δ19/Δ19Mean body weight of mutant mice fed with HFD and treated with vehicle (wt. hf. veh and clk. hf. veh) or nobiletin (wt. hf. nobiletin and clk.hf. nobiletin) for 10 weeks (n ═ 8-15).

Fig. 4B shows the daily food intake (n-8-15) of the four groups of mice involved in fig. 4A.

Figure 4C shows the body weight composition (n-3) of the four groups of mice involved in figure 4A as analyzed by nuclear magnetic resonance.

Figure 4D shows histological analysis of white fat (WAT) after 10 weeks of treatment of the four groups of mice referred to in figure 4A. WAT was stained for H & E.

Fig. 4E shows the circadian rhythm (oxygen consumption) of VO2 in the four groups of mice involved in fig. 4A (n-8).

Figure 4F shows a graph of activity changes (n-7) (left) illustrating the effect of vehicle or nobiletin on circadian behavior in WT mice fed with HFD; the indicated genotypes are summarized at L: d or D: average wave pattern of roller motion during 12-14 days of D (n-7); and the indicated genotypes are at L: d or D: total wheel activity per day (n ═ 7) under condition D (right).

FIG. 4G shows Clock illustrating vehicle or nobiletin in HFD feeding^Δ19/Δ19(ii) a pattern of activity changes in the effect on circadian behavior in mutant mice (n-3) (left); the indicated genotypes are summarized at L: d or D: average wave pattern (n-3) of roller motion during day 10-12 of D (middle); the indicated genotypes are indicated at L: d or D: total wheel activity per day (n ═ 3) under condition D (right).

FIG. 5A shows WT (left) and Clock fed with HFD treated with vehicle or nobiletin at two opposite time points^Δ19/Δ19Fasting blood glucose levels (n-8-15) in the mutant mice (right).

FIG. 5B shows nobiletin versus WT and Clock fed with HFD as measured by the Glucose Tolerance Test (GTT) (n 8-15)^Δ19/Δ19Influence of glucose tolerance of mutant mice (left) and area under the curve (AUC) (right).

FIG. 5C shows nobiletin versus WT and Clock fed with HFD as measured by the insulin resistance test (ITT) (n-8-15)^Δ19/Δ19Effect of insulin resistance in mutant mice (left) and area under the curve (AUC) (right).

FIG. 5D showsHFD-fed WT mice treated with vehicle or nobiletin and Clock^Δ19/Δ19Blood insulin levels in mutant mice (n-8-15).

Fig. 5E shows total Triglyceride (TG) levels and cholesterol (TC) levels (n-8-15) in blood after 10 weeks of treatment.

Fig. 5F shows total TG levels and TC levels in the liver 10 weeks after treatment (n-8-15).

FIG. 5G shows WT and Clock fed with HFD after 10 weeks of treatment^Δ19/Δ19Total liver H of mutant mice&And E, dyeing.

Fig. 6A shows body weight at the end of the 9-week treatment period (n-12). NOB did not affect metabolic homeostasis in mice fed regular food.

Figure 6B shows the weight gain at the end of the 9-week treatment period (n-12). NOB did not affect metabolic homeostasis in mice fed regular food.

Fig. 6C shows fasting blood glucose levels (n-12). NOB did not affect metabolic homeostasis in mice fed regular food.

Fig. 6D shows the Glucose Tolerance Test (GTT) (n-12). NOB did not affect metabolic homeostasis in mice fed regular food.

Fig. 6E shows the insulin resistance test (ITT) (n-12). NOB did not affect metabolic homeostasis in mice fed regular food.

Fig. 7A shows the structures of Naringin (NAR) and naringenin (naringenin).

FIG. 7B shows that naringin did not enhance PER 2:LucSVor PER 2:LUCclock^Δ19/+The reporter gene in the reporter cell emits light.

FIG. 7C shows that naringin did not enhance PER 2:LucSVor PER 2:LUCclock^Δ19/+The reporter gene in the reporter cell emits light.

FIG. 7D shows naringenin does not enhance PER 2:LucSVor PER 2:LUCclock^Δ19/+The reporter gene in the reporter cell emits light.

FIG. 7E shows Western blot analysis of PER 2:per 2 protein in LucSV cells treated with 5 μ M MAR or DMSO at time 0, collected times to 32 hours every 4 hours, and then immunoblotted with antibody.

FIG. 7F shows the real-time qPCR analysis of Per2mRNA levels in LucSV cells treated as discussed with respect to FIG. 7E for PER 2.

Figure 8A shows the mean body weight (left) and body weight gain (right) (n-8-15) of HFD-fed mice treated with vehicle or NAR (wt. hf. veh, wt. hf. NAR, clk. hf. veh, and clk. hf. NAR) after 10 weeks of treatment.

FIG. 8B shows that NAR reduced WT mice but not Clock during day and night^Δ19/Δ19Fasting blood glucose levels in the mutant mice (n-8-15).

FIG. 8C shows that NAR shows a mild effect on glucose tolerance as measured by the Glucose Tolerance Test (GTT), at WT and Clock^Δ19/Δ19The mutant mice were indistinguishable from each other (n-8-15). Quantification of the area under the curve (AUC) is also shown.

FIG. 8D shows that NAR shows a mild effect on insulin sensitivity as measured by the insulin resistance test (ITT), at WT and Clock^Δ19/Δ19The mutant mice were indistinguishable from each other (n-8-15). Quantification of the area under the curve (AUC) by ITT is also shown.

FIG. 8E shows a block diagram at WT and Clock^Δ19/Δ19In mutant mice, NAR reduced blood insulin levels to a similar extent (n-8-15).

Fig. 8F shows blood total Triglyceride (TG) levels (n-8-15) after 10 weeks of treatment.

Fig. 8G shows cholesterol (TC) levels (n-8-15) after 10 weeks of treatment.

FIG. 9A shows db/db or db/db Clock fed with RC^Δ19/Δ19Average body weight of double mutant mice, which were treated with vehicle (db.veh and db.clk.veh) or nobiletin (Db. nobiletin and db.clk.nobiletin) for 10 weeks (n ═ 6-8) (left). At the beginning of treatment, mice were 6-8 weeks old. And (3) right: the average body weight of these four groups of mice increased.

FIG. 9B shows db/db (left) and db/db Clock at two opposite time points^Δ19/Δ19Fasting blood glucose levels in double mutant mice (right) (n-6-8).

FIG. 9C shows nobiletin versus db/db and db/db Clock as measured by GTT^Δ19/Δ19Effect of glucose tolerance in double mutant mice (n ═ 6-8). And (3) right: AUC.

FIG. 9D shows nobiletin vs db/db and db/db Clock as measured by ITT^Δ19/Δ19Effect of insulin resistance in double mutant mice (n ═ 6-8). And (3) right: AUC.

FIG. 9E shows db/db and db/db Clock^Δ19/Δ19Total TG levels in mice (n-6-8).

FIG. 9F shows db/db and db/db Clock^Δ19/Δ19Total blood TC levels in mice (n-6-8).

FIG. 9G shows nobiletin vs db/db and db/db Clock^Δ19/Δ19Effect of circulating insulin levels in mice (n-6-8).

Fig. 10A shows the determination of protein and mRNA expression of clock genes in liver samples collected from WT mice fed with HFD by Western blotting, the mice were treated with vehicle (WT. hf.veh) or nobiletin (WT. hf. nobiletin). WT mice fed with RC (WT. RC. veh) were used as controls in the comparison (n-3 or 4).

Figure 10B shows determination of protein and mRNA expression of clock genes in liver samples collected from WT mice fed HFD by real-time qPCR, mice treated with vehicle (WT. hf. veh) or nobiletin (WT. hf. nobiletin). WT mice fed with RC (WT. RC. veh) were used as controls in the comparison (n-3 or 4).

FIG. 10C shows a heat map of microarray gene expression data showing that the expression pattern of 56 genes was altered by HFD, nobiletin reversed their expression to varying degrees at the time points of ZT2 and ZT14 to approximate RC levels in the liver of WT mice.

Fig. 10D shows the functional classification of 56 genes in (C) by the GO program. The percentage of genes sharing GO biological processes is shown.

Figure 10E shows real-time qPCR analysis of mRNA expression of clock-controlled metabolic output genes in the liver from mice treated as above.

Fig. 11A shows a ratio heat map. The expression data in figure 10C were calculated to derive fold-changes, indicating that NOB reversed the expression pattern of 56 genes altered by HFD in mouse liver at ZT2 and ZT14 time points. The scale shows the fold change (ratio). NOB/HF represents the hf.nob to hf.veh expression ratio; HF/RC represents the expression ratio of hf.veh to rc.veh.

Fig. 11B shows that thirty genes showed clock protein binding, while the remaining genes did not (blank region) analysis is based on published ChIP-Seq results (Koike et al, 2012; Cho et al, 2012) scale expressing the magnitude of DNA occupancy, as quantified by enrichment of immunoprecipitated DNA fragments that bind each transcription factor.

Fig. 11C shows the expression of ROR α (left) and ROR γ (right) mRNA in Hepa1-6 cells treated with control (Ctrl) or mouse ROR α/γ siRNA and vehicle (DMSO) or NOB (3 μ M, 12h) (n-4).

FIG. 11D shows that highlighted within the network is ROR α/γ, which acts as a node for genes regulated by NOB indicating genes that are down-and up-regulated by HFD (left) or NOB (right), whose intensities correspond to fold-changes.

Fig. 12A shows a saturation curve (n-3) for ROR α -LBD and ROR γ -LBD generated with 100ng ROR α -LBD (top) and 200ng ROR γ -LBD (bottom) combined with 25- [3H ] -OHC filtration assay.

FIG. 12B shows a Scatchard plot of the saturation curve results for ROR α -LBD (top) and ROR γ -LBD (bottom) for 25- [3H ] -OHC obtained from FIG. 12A, which corresponds to n ═ 3. this analysis gives a dissociation constant (Kd) of 6.10nM, total number of binding sites (Bmax) of 100fmol/mg protein for ROR α, 6.67nM and 410fmol/mg protein for ROR γ.

FIG. 12C shows an in vitro competitive radioligand binding assay demonstrating that Nobiletin (NOB) (C) binds directly to ROR α -LBD and ROR γ -LBD, rather than Naringin (NAR), within the indicated dose range.

Figure 12D shows an in vitro competitive radioligand binding assay that showed that naringenin did not bind directly to ROR α -LBD and ROR γ -LBD over the indicated dose range.

FIG. 12E shows a mammalian single hybrid assay showing the interaction of nobiletin with ROR-LBD human embryonic kidney 293T cells were co-transfected with a GAL4 reporter construct with expression vectors for GAL 4DBD-ROR α LBD or GAL 4DBD-ROR γ LBD.

FIG. 12F shows a mammalian single-hybrid assay showing a lack of naringin interaction with ROR-LBD A GAL4 reporter construct with expression vectors for GAL 4DBD-ROR α LBD or GAL 4DBD-ROR γ LBD was co-transfected with human embryonic kidney 293T cells SR1001 was used as a positive control).

FIG.12G shows that in the presence of ROR α or ROR γ in Hepa1-6 cells, dose-dependent increase in the expression of luciferase reporter driven by Bmal1 promoter by nobiletin with WT, but not mutant RORE ectopic expression of REV-ERB α abrogates reporter activation.

Figure 12H shows abrogation of nobiletin induction by ROR α/γ expression by siRNA knock-out of luciferase reporter gene expression driven by Bmal1 promoter in Hepa1-6 and U2OS cells.

Figure 12I shows real-time qPCR analysis of ROR α/γ target gene in the same mouse liver sample as figure 10A.

In all figures, data are presented as mean ± SEM. P <0.05, p <0.01, p <0.001, # p <0.05 and # # p < 0.001.

Detailed Description

In embodiments, the present disclosure relates to methods comprising administering to a mammal having or at risk of developing metabolic syndrome or its constituent symptoms a composition comprising at least polymethoxylated flavones.

"Metabolic syndrome" is a collection of at least three of the following medical symptoms in patients, abdominal obesity, elevated blood pressure, elevated fasting glucose, high serum triglycerides and low density lipoproteins (HDL). if the mammal's diet is high in carbohydrate (especially sugars), sedentary lifestyle, and/or experiences chronic stress, as well as other experiences and behaviors known to those skilled in the art, the mammal may be at risk for developing metabolic syndrome.

Flavonoids have the -like structure known in the art, which includes a pentadecane skeleton containing two benzene rings and heterocycles a subset of flavonoids includes flavones, isoflavones and neoflavones polymethoxyflavones are flavones containing at least two methoxy moieties.

In embodiments, at least of the polymethoxylated flavones have formula I:

wherein R is¹、R²、R³、R⁴、R⁵And R⁶Each independently is-H or-OCH₃Provided that R is¹、R²、R³、R⁴、R⁵And R⁶At least two of which are-OCH₃。

In an example of step , the at least polymethoxylated flavones may be selected from the group consisting of nobiletin (formula II), hesperetin (formula III) and both.

The composition may also comprise at least carriers the at least carriers may be any or more materials with which the at least polymethoxylated flavones may be mixed or combined in the desired form and suitable for human or animal consumption the composition may be in a form selected from the group consisting of liquid, tablet, capsule, caplet and soluble powder, and one of skill in the art will be able to routinely select or more suitable carriers depending on the particular form desired for a given embodiment of the composition.

In embodiments, at least carriers can be or include water, gelatin, or cellulose.

In embodiments, at least carriers can be or include microcrystalline cellulose, hypromellose, vegetable magnesium stearate, or silicon dioxide.

Alternatively or additionally, the composition may also comprise flavouring agents, such as citrus flavour, non-citrus fruit flavour, herbal flavour, vanilla flavour or chocolate flavour and other suitable flavouring agents.

Alternatively or additionally, the composition can comprise or more ingredients in addition to the polymethoxylated flavone that is expected to reduce or more symptoms of metabolic syndrome for example, in embodiments, the composition can further comprise steps of cinnamon, used alone or in conjunction with other ingredients that are expected to reduce or more symptoms of metabolic syndrome.

Alternatively or additionally, the composition may comprise or more ingredients intended to reduce symptoms other than metabolic syndrome and/or or more ingredients intended to improve or more aspects of the overall health and well-being of the mammal.

The compositions can be administered to a mammal at any dose and rate that provides at least polymethoxylated flavones at a concentration below a detrimental level for any duration in blood or other body tissue or fluid, it is desirable that the duration can be sufficiently long to reduce the severity of or risk of developing metabolic syndrome in a patient.

In embodiments, the dosage of 20mg polymethoxyflavone/kg mammal body weight to 2000mg polymethoxyflavone/kg mammal body weight, and the dosage is from twice daily to times weekly for at least weeks, in a specific embodiment, the dosage of 200mg polymethoxyflavone/kg mammal body weight is times every two days for at least ten weeks.

In an example of step , where the composition is further steps comprising nicotinamide riboside and pterostilbene, the dosage of nicotinamide riboside can be from 1mg nicotinamide riboside/kg mammal body weight to 10mg nicotinamide riboside/kg mammal body weight, the dosage of pterostilbene can be from 0.2mg pterostilbene/kg mammal body weight to 2.5 pterostilbene/kg mammal body weight, and the administration rate can be times per day.

In the mammal may be a homo sapiens other mammals for which reduction of metabolic syndrome or risk thereof may be desired include, but are not limited to work animals, pack animals, animals for transportation (e.g., horses), racing animals (e.g., horses or lions), meat animals, fur or skin producing animals, dairy animals, work dogs, companion animals, and the like.

In another embodiment, the composition may be provided in the form of a tablet or capsule, and in this form, the composition may be provided in the form of a tablet or lozenge that dissolves when placed in the mouth of a user.

In another embodiments, the disclosure relates to methods comprising administering to a mammal having or at risk of having a sleep disorder a composition comprising at least polymethoxylated flavones.

As used herein, "sleep disorder" refers to a non-transitory impairment of a mammal's ability to enter, exit, or maintain sleep over or during a desired time or duration, while not being bound by theory, disruption of the mammal's circadian rhythm may cause or exacerbate or more sleep disorders in embodiments, the sleep disorder may be selected from the group consisting of insomnia, somnolence, narcolepsy, Delayed Sleep Phase Syndrome (DSPS), advanced sleep phase syndrome (ASPD), non-24 hour sleep arousal disorder, irregular sleep arousal rhythm, and shift work sleep disorder.

In the method of this example, a composition comprising at least polymethoxylated flavones and any other components (e.g., or more carriers (nicotinamide riboside and/or pterostilbene)) and any formulations thereof may be as described above in 0 in examples, the composition for use in the method may comprise or more ingredients other than polymethoxylated flavones that are expected to reduce or more symptoms of sleep disorders, for example, in examples, the composition may further comprise steps of melatonin, valerian, or more valerianic acids, slips, or more kavalactones, kavain, chamomile, apigenin, passion flower, lemon balm, plant of genus comfrey (skullcap), hops, lavender, l-tryptophan, St John's wort, acerola, or more phenolic acids, glycosides and or more cannabinoids.

Similarly, administration of the compositions, either alone or in combination, including dosage, rate, duration and route, may also be as described above.

In yet another embodiments, the disclosure relates to methods comprising administering to a mammal with aging a composition comprising at least polymethoxylated flavones.

As used herein, "aging" refers to a sustained increase in aging in a mammal. While not being bound by theory, disruption of the mammalian circadian rhythm may exacerbate aging. Exemplary symptoms of aging include, but are not limited to, decreased muscle mass, decreased bone density, decreased immune system function, decreased memory, decreased cognitive function, increased wrinkles, increased liver spots, increased gray and white hair, hair loss, and decreased overall vitality.

In the method of this embodiment, a composition comprising at least polymethoxylated flavones and any other ingredients (e.g., or more carriers (nicotinamide riboside and/or pterostilbene)) and any formulations thereof may be as described aboveIn examples, the composition may further comprise steps of at least 0 of resveratrol, β -carotene, vitamin A, vitamin B6, vitamin B9, vitamin B12, vitamin C, vitamin E, 1 or more curcumins, turmeric, 2 or more green tea polyphenols, or more catechins, epigallocatechin 3-gallate, grape seed extract, or more carotenoids, lutein, zeaxanthin, cryptoxanthin, astaxanthin, canthaxanthin, lycopene, or more xanthophylls, or more phytosterols, sitosterol, stigmasterol, campesterol, calcium, or more omega-3 fatty acids, eicosapentaenoic acid, docosahexaenoic acid, glucosamine, chondroitin, collagen, quercetin, dietary fiber, or more probiotics, Lactobacillus, Bifidobacterium, or more zinc, prebiotics, calcium, vitamin Q, prebiotics, calcium, vitamin Q, and vitamin Q₁₀Ginkgo, blueberry, cranberry, oregano, nectarine, assai, rosa damascena, cocoa, green tea, olive oil, HSP-12.6, tannic acid, caffeic acid, rosmarinic acid, spermidine, or thioflavin T, and the like.

Similarly, administration of the compositions, either alone or in combination, including dosage, rate, duration and route, may also be as described above.

In still another embodiments, the present disclosure relates to methods comprising administering to a mammal having or at risk of developing a mood disorder a composition comprising at least polymethoxylated flavones.

As used herein, "mood disorder" refers to a non-transitory change in a baseline emotional state of a mammal. Examples of mood disorders include, but are not limited to, mania, hypomania, unipolar depression, bipolar disorder, and the like. While not being bound by theory, mood disorders can be caused or exacerbated by disruption of a mammalian circadian rhythm.

In the methods of this example, compositions comprising at least polymethoxylated flavones and any other ingredients (e.g., or more carriers (nicotinamide riboside and/or pterostilbene)) and any formulations thereof may be as described above in examples, compositions for use in the methods may comprise or more ingredients other than polymethoxylated flavones that are expected to reduce or more symptoms of mood disorders.

Similarly, administration of the compositions, either alone or in combination, including dosage, rate, duration and route, may also be as described above.

In additional embodiments, the present disclosure relates to methods comprising administering to a mammal having or at risk of having a cardiovascular disease, an immune disorder, a neurodegenerative disease, cancer, or two or more thereof, a composition comprising at least polymethoxylated flavones.

In the method of this embodiment, a composition comprising at least polymethoxylated flavones and any other ingredients (e.g., carrier(s) (nicotinamide riboside and/or pterostilbene)) as well as any formulations thereof may be as described above in embodiments, the composition for use in the method may comprise ingredients in addition to polymethoxylated flavones that are expected to reduce symptoms of one or more diseases or disorders.

Similarly, administration of the compositions, either alone or in combination, including dosage, rate, duration and route, may also be as described above.

The following examples are included to illustrate specific embodiments of the invention. It should be appreciated by those of skill in the art that the techniques disclosed in the examples which follow represent techniques discovered by the inventor to function well in the practice of the invention. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention.

Examples of the invention

Example 1 identification of nobiletin as a clock regulator

To identify Clock amplitude enhanced small molecules (CEMs), a hybrid Clock is used^Δ19/+PER2 Luc reporter cells screened an internal compound library with 5,300 small molecules that exhibited a sustained reporter rhythm with attenuated amplitude relative to Wild Type (WT) cells. In particular, chemical screening for circadian clock modulators was performed in the chemogenomics core facility of the houston health science center, texas university (UTHSC-H). The internal chemical library screened consisted of compounds from the National Institute of Health (NIH) clinical library, the national cancer institute library and the Microsource Spectrum library. Screening was performed mainly based on the previously described protocol (Chen et al, 2012). Briefly, Clock from Luc bioluminescent reporter gene expressing PER2^Δ19/+Immortalized fibroblasts from heterozygous mice were plated into 96-well plates after confluence, the cells were incubated with 5 μ M forskolin for 1-2 hours, then chemical compounds were added to the plates using a robotic arm (Beckman), then monitored continuously for several days in a temperature controlled EnVision plate reader (Perkin Elmer) data analysis was performed using MultiCycle software (Actimetrics) to measure cycle, phase and amplitude.

Other materials and methods are as follows:

animals and cell lines.Animal feeding for all studies, except for tissue explant experiments, was performed under IACUC guidelines and operated as described in the protocols of animals approved by the houston health science center (UTHSC-H), university of texas. Male Wild Type (WT) Clock^Δ19/Δ19Db/db and db/db Clock^Δ19/Δ19The mice were all in the genetic background of C57BL/6J, using Clock obtained from Takahashi and Jackson laboratories (#000697), respectively^Δ19/+(Antoch et al, 1997; King et al, 1997) and db/+ breeders were obtained from heterozygote-bred littermates. In a standard animal facility at 12 hours: 12 hours of light irradiation: mice were housed in groups (2-4/cage) on the dark cycle. Mice that showed aggressive behavior towards cage partners were removed. For day-night exercise and metabolic laboratory studies, mice were housed in single cages in satellite facilities approved by the UTHSC-H animal welfare committee. According to the southwest university of TexasIACUC guidelines of the medical center (UTSW) maintained PER2:: Luc reporter knock-in mice for tissue explant experiments. Adult mouse ear fibroblasts and Mouse Embryonic Fibroblasts (MEFs) have been previously described (Chen et al, 2012).

High-throughput chemical screening and validation.Chemical screening for circadian clock modulators is performed in the chemogenomics core facility of UTHSC-H. The screened internal chemical library consisted of compounds from the NIH clinical library, NCI library and MicrosourceSpectrum library. Screening was performed mainly based on the previously described protocol (Chen et al, 2012). Briefly, 15,000 Clocks from expressing PER2:: Luc bioluminescent reporter genes were prepared^Δ19/+Immortalized fibroblasts from heterozygous mice were plated in each well of a 96-well plate and incubated for 3-4d to grow to confluence. The cells were then incubated with 5 μ M forskolin for 1-2 h, followed by addition of chemical compounds to the plate using a robotic arm (Beckman), and then monitored continuously for several days in a temperature-controlled EnVision microplate reader (Perkin Elmer). Data analysis was performed using MultiCycle software (Actimetrics) to measure period, phase and amplitude. Among compounds that showed greater than 2X SD effect on circadian amplitude and/or cycle, NOB was independently identified from two sub-libraries as significantly enhancing circadian amplitude. NOB and the structurally related analogue Naringin (NAR) were purchased again from commercial sources including Sigma and GenDEPOT and dose response validated using PER2:: LucSV reported fibroblasts showing stronger bioluminescent signal, allowing accurate measurement of circadian clock effects of compounds (Chen et al, 2012). Tissue explant experiments were performed as previously described (Chen et al, 2012).

Circadian motor activity.NOB-or vehicle-treated WT fed with RC, WT fed with HFD and Clock^Δ19/+Mice were used for day-night motor activity experiments. Briefly, mice were first treated at 12 hr: illumination for 12 hr: the dark (LD) cycle is maintained for at least 2 weeks and then released to a constant dark, free-motion state. The mice were then kept in constant darkness for an additional 2 weeks. The roller data is downloaded as VitalView data file and passes ActiView and Actogram J programAssays were performed (Schmid et al, 2011; Zheng et al, 1999).

Mouse treatment, body weight and mass composition measurements.For diet-induced obesity, male mice of 6 weeks of age were fed HFD (D12492, study diet) until the end of the experimental protocol, during the entire experiment, mice were treated with vehicle (DMSO) or NOB (200mg/kg body weight) by oral gavage within the time window ZT8-10 every days for several reasons, the dosage protocol was chosen at the indicated level every days first, previous in vivo studies had used similar total amounts for mouse treatment (100 + 125 mg/kg/day) (Lee et al, 2013; Li et al, 2006) oral gavage steps were performed later in the afternoon (ZT8-10) before their start of active period, as this may result in from previous studies, 2012, no daily dose was chosen so as to avoid taking the step itself as an artificial timer to drive experimental mice in the middle of a single dose condition (trial pharmacokinetics) was performed (PK 29 hours) and thus the results of a significant daily dose was not observed in the study despite the observation of the non-usual observation of the results of the observation of the non-daily dose profile of the study, especially after No. 5 h observation of the study (S) and no observation of the course of the study.

Weekly body weights were monitored for the different treated mice over 10 weeks. Body weight composition was measured at the end of the experiment using a minispec mqNMR spectrometer (Bruker Optics, texas) (Garcia et al, 2013). Mice in the control group were fed regular diet (Purina 5001) while both treatment groups were in HFD. For db/db and db/db Clock^Δ19/⁺Mice, male mice of 6-8 weeks of age were group housed (2-3 mice/cage) and maintained on a regular diet. Mice were orally gavaged with DMSO or NOB as described above.

Pharmacokinetic studies in mice.NOB were administered orally at a dose of 200mg/kg in a 0.25% sodium carboxymethylcellulose (CMC) suspension at ZT 8. 0.0, 1.0, 2.0, 4.0, 8.0 and 24.0 hours after oral gavageThree mice were sacrificed at each time point. NOB in plasma, brain and liver were determined by LC-MS/MS (API 4000: EQ-RS-MS-006).

Energy expenditure and food intake measurements.Energy expenditure was examined by measuring oxygen consumption by indirect calorimetry as described (Chutkow et al, 2010; Daniels et al, 2010). After 8 weeks of the above treatment, each group of mice was placed in a room of a comprehensive laboratory animal monitoring system (CLAMS, Columbus Instruments, Columbus, Ohio) at room temperature (22 ℃ -24 ℃). After adaptation of the mice to the metabolic compartment, O2 consumption and CO2 production were continuously recorded over a 24 hour period. The average O2 consumption between different treatments was calculated and compared. Food and water were provided ad libitum. To measure food intake, in mice treated as above, food particles were weighed every three hours over 24 hours. Daily food intake was calculated from the average food intake of 3 independent experiments.

Serum and liver lipid assays.Serum samples were obtained from treated mice at ZT2 as previously described (Jeong et al, 2015). Hepatic triglycerides and cholesterol were extracted as previously described (Liu et al, 2012). Triglyceride and cholesterol levels in liver and serum were assessed by serum triglyceride assay kit (Sigma) and cholesterol assay kit (Cayman), respectively. Assay plates were read using a TECAN M200 instrument (TECAN) according to the manufacturer's instructions.

Glucose tolerance and insulin resistance tests (GTT and ITT).Glucose Tolerance Tests (GTT) and Insulin Tolerance Tests (ITT) are performed substantially as described above (He et al, 2015; Jeong et al, 2015). Briefly, GTT and ITT were performed at ZT2 and ZT8, respectively, after overnight and 5-hour fasting. Glucose levels were measured from tail blood using the ONETOUCH UltraMini blood glucose monitoring system (LifeScan) 15, 30, 60 or 120 minutes before and after injection of 1g/kg glucose or 0.75U/kg insulin (Sigma) at ZT2 and ZT8, respectively. Serum insulin levels were measured using a rat/mouse insulin Elisa kit (Millipore) according to the manufacturer's instructions. Plasma samples were collected at ZT2 for lipid determination as described above.

Histological analysis of liver and adipose tissue.For microscopic analysis of lipid accumulation in the liver, Tissue samples were collected and immediately embedded in Tissue-Tek OCT cryostat moulds (Leica) and then frozen at-80 ℃. Tissue sections were stained in 0.5% oil red O and counterstained with Mayer hematoxylin for 1 minute. In addition, liver, brown adipose and white adipose tissues were embedded in paraffin and treated with hematoxylin and eosin (H)&E) And (6) dyeing. Microscope images were obtained on an Olympus BX60 microscope.

Real-time qPCR and Western blot analysis.For qPCR analysis, cells were plated in 6-well plates at an initial density of 3X105 cells and incubated for 2-4 days, followed by synchronization treatment (5 μ M Fsk or 100nM Dex) followed by compound treatment RNA samples were prepared using purex rnaract rnanol for cDNA synthesis and real-time pcr using MaxPro3000Thermocycler (Agilent) (GenDEPOT) the qPCR primers used are listed in the table shown in example 4 antibodies were prepared as described (Yoo et al, 2013) from similarly grown and treated cells in 60mm dishes and tissue extracts (Yoo et al, 2013), Western blot analysis (GenDEPOT) using REV-ERB α (Pierce and Cell signalling), PER1(Lee et al, 2001) and other clock proteins (Yoo et al, 2013).

And (4) carrying out microarray analysis.Total RNA prepared from liver tissue of WT mice treated with RC feed, vehicle treatment, and HFD feed, vehicle, or NOB for 10 weeks was reverse transcribed into cDNA, then labeled with biotin-UTP and hybridized with Illumina mouse WG-6v2.0Expression BeadChip genes with statistically significant fold change differences were clustered using center correlation (Cluster 3.0) and then visualized as heatmaps on Tree View. furthermore, genes with statistically significant differences derived from microarray Analysis were introduced into Ingenity Pathway Analysis (IPA, http:// www.ingenuity.com). in IPA, the differentially expressed genes were mapped to the genetic network in Ingenity Knowledge Database to generate group networks, then Cluster 3.0 was used to generate heatmaps as described previously (Koike et al, Chip-seq.) data stored in published BI data series published by the International publication, Ebus 2002, published by the same publication, Inc. published by general AnalysisAccession number GSE 78848.

A plasmid.In our laboratory, plasmids containing retinoic acid-related orphan receptor response elements (RORE) from the mouse Bmal1 promoter (pcDNA3.1B-G4DBD-ROR α LBD and pcDNA3.1BG4DBD-ROR γ LBD) were constructed. specifically, 1960bp sequence (-0/+ 130) of the mouse Bmal1 promoter with WT (AAAGTAGGTCA and AAAGTAGGTTA) or mutation (AAAGTACACGA) RORE was PCR-amplified from mouse genomic DNA and cloned into pGL 3-promoter luciferase vector in order to express ROR α or ROR γ -GAL4 protein, mouse ROR α -LBD (aa 261-523) or ROR γ -LBD (aa 250-516) was PCR-amplified from mouse genomic DNA and cloned into GAL 4-DBD-containing vector pcDNA3.1B.

Single hybrid reporter gene assay.For mammalian single-hybrid assays, HEK293T cells were co-transfected with pcDNA3.1B-G4DBD-ROR α/γ LBD, pGL4.31 and TK promoter Renilla (Renilla) Luciferase constructs (tK.pRL.) to explore the regulation of the Bmal1 Reporter, Hepa1-6 cells were co-transfected with Bmal1-WT or mutant RORE Reporter plasmids (ROR α, ROR γ or Rev-erb α expression constructs) and tK.pRL. mice and human siRNAs targeting R α and ROR γ were purchased from Santa Cruz. transfected with Lipofectamine 2000 reagent (Invitrogen), 24 hours after transfection, cells were treated with vehicle or NOB, 24 hours after treatment lysates were collected and the agonist activity of firefly and the agonist and renal Luciferase agonist activities were measured using the Dual-Luciferase Reper System (Promefer), regardless of the nature of the mutual agonist activity of these chimeric ligands, the chimeric ligands, 2010, chimeric ligands, etc.

Radioligand receptor binding assays.A few modifications were made to the previously described protocol (Kumar et al, 2010; Wang et al, 2010 b.) for the saturation binding experiments, 100ng ROR α -LBD or 200ng ROR γ -LBD were combined with 25- [3H ]]Hydroxycholesterol (OHC) in assay buffer [50mM HEPES, pH 7.4, 0.05% Bovine Serum Albumin (BSA), 150mM NaCl and 5mM MgCl2]And (4) carrying out incubation. Ligand binding was determined by a filter binding assay to calculate Kd values. For the competitive binding assay, 25- [3H ] was measured at 4.5nM]100ng ROR α -LBD or200ng ROR gamma-LBD with various concentrations of nobiletin, naringin or naringenin incubation. Ki was determined using the Cheng-Prusoff equation.

RNA-mediated interference.Hepa1-6 cells on 24-well plates were transfected with control siRNA and siRNA versus mouse ROR α and ROR γ (Santa Cruz) twenty-four hours post transfection, cells were treated with DMSO or NOB (3 μ M) 12 hours post treatment, cells were harvested and total rna was isolated.

And (5) carrying out statistical analysis.Data are presented as mean ± SEM. Statistical significance was determined by one-way or two-way analysis of variance with multiple sets of comparisons according to the Turkey and Dunnett tests. Consider P<0.05 has statistical significance.

In all examples, data are presented as mean ± SEM. Statistical significance was determined by one-way or two-way analysis of variance with multiple sets of comparisons according to either the Turkey or Dunnett test. A P value <0.05 was considered to indicate statistical significance.

Finds out natural polymethoxyflavone and nobiletin rich in orange peel from two sublibraries respectively, and can enhance Clock^Δ19/⁺Reporter Gene rhythm of cells (FIGS. 1A and 1B.) hesperetin ( analogs similar to nobiletin) similarly enhanced Clock^Δ19/⁺PER2 in cells Luc reporter Gene rhythm (FIGS. 2A and 2B). Nobiletin robustly enhances PER2:: rhythmic amplitude of LucSV reporter gene (Chen et al, 2012) and extends the cycle in a dose-dependent manner, estimating half the maximum effective concentration<5.0mM (FIG. 1C). Similar to the previously reported CEM (Chen et al, 2012,2013), nobiletin is a homozygous Clock with disrupted recovered Clock^Δ19/⁺The rhythmic aspect of the reporter cell was ineffective (fig. 1D). Importantly, nobiletin enhances Clock^Δ19/⁺And PER2 in the peripheral tissue explants of WT reporter knock-in mice Luc reporter rhythm (fig. 1E and 2C), but not in SCN, the latter being resistant to external perturbations due to robust inter-neuron coupling (fig. 2D) (Buhr et al, 2010; Chen et al, 2012; Liu et al, 2007). anti-SCN-mediated anti-nobiletinIn nature, normal roller activity and periodicity were observed in WT C57BL/6J mice treated with nobiletin (FIG. 1F).

Nobiletin has shown various beneficial effects (Ben-Aziz, 1967; Cui et al, 2010; mullvihill et al, 2011; Nagase et al, 2005; Walle, 2007). however, its effect as a regulator of circadian clock was previously unknown. although nobiletin slightly changes and reduces PER2:: the transcription level of LucSV cells at PER2 of CT20 (fig. 2E), PER2 protein was found to accumulate to a higher level (fig. 2F-2G), which is -dependent on increased bioluminescence and indicates a post-transcriptional mechanism of PER2 enrichment, nobiletin also changes the expression of other core clock genes (fig. 2E-2G). in particular, CRY1 (heterodimeric partner of PER in negative arm of shaker), although the transcription level significantly decreased, it shows a slight trend of greater abundance of protein (fig. 2E and 2F): simultaneously with y 8295. the idea of yay 1 and PER 58387, which may lead to a stable circadian amplitude increase of rat fibroblast and like (2011-5: pei 58387 and/ha) as well as a stable circadian rhythm for rat.

Example 2 robust clock-dependent metabolic protection of nobiletin against diet-induced obesity (DIO) mice

Recent studies have shown that nobiletin has a protective effect on metabolic syndrome (Kurowska and Manthey, 2004; Lee et al, 2010, 2013; Mulvihill et al, 2011; Roza et al, 2007). Thus, pharmacokinetic studies revealed that nobiletin had significant brain and systemic exposure (fig. 3A). To solve the problem that the metabolic protection of nobiletin depends on the Clock function, the use of WT and Clock-destroyed Clock is selected^Δ19/+Diet-induced obesity (DIO) mouse model of mice.

Animal feeding for all studies, except for tissue explant experiments, was performed under Institutional Animal Care and Use Committee (IACUC) guidelines and operated as described in the animal protocol approved by the houston health science center of texas university (UTHSC-H). PER2:: Luc reporter knock-in mice for tissue explant experiments were maintained according to the guidelines of the southwest medical center IACUC, university of Texas. Adult mouse ear fibroblasts and mouse embryo fibroblasts have been previously described (Chen et al, 2012).

For diet-induced obesity, male mice of 6 weeks of age were fed HFD (D12492; study diet) until the end of the experimental protocol throughout the entire experimental period, mice were treated with vehicle (DMSO) or nobiletin (200mg/kg body weight) by oral gavage within the time window ZT8-ZT10 every days.

In WT C57BL/6J mice fed HFD, treatment with nobiletin significantly reduced body weight gain for 10 weeks relative to vehicle control (fig. 4A and 3B). Nobiletin did not significantly alter food intake relative to vehicle control (fig. 4B and 3C). Body weight composition analysis revealed that weight loss was mainly due to fat mass loss (fig. 4C) and white adipocyte cell size (fig. 4D and 3D). Notably, the Clock is being fed with HFD^Δ19/Δ19In mutant mice (fig. 4A and 3B), nobiletin treatment resulted in only a very mild decrease in weight gain, whereas nobiletin treatment did not substantially alter fat mass and adipocyte size (fig. 4C, 4D and 3D). although mutant mice consumed more food during the light phase than WT mice (Turek et al, 2005), nobiletin did not alter the food intake of mutant mice (fig. 4B and 3C). indicating increased energy consumption, the WT mice treated with nobiletin had greatly increased oxygen consumption compared to controls throughout the day-night cycle, the greatest increase was found during the early dark phase (fig. 4E and 3E). in WT mice treated with nobiletin, the respiratory quotient also increased (fig. 3F), as opposed to which shifted from lipid-biased metabolism to a more balanced contribution of all major macronutrients^Δ19/Δ19Energy expenditure or respiratory quotient in mice did not increase (FIGS. 4E and 3F). with its effect on body weight and energy expenditure of , nobiletin significantly increased the roller activity level in WT mice fed with HFD relative to control treatment (FIG. 4F), in contrast to the control treatmentClock^Δ19/Δ19No significant difference in activity levels was detected between the treatments of the mice (fig. 4G).

Nobiletin also improved glucose and lipid homeostasis in WT mice, but not Clock^Δ19/Δ19A mouse. Nobiletin reduced fasting blood glucose levels in WT mice (fig. 5A) and significantly improved glucose tolerance and insulin sensitivity (fig. 5B and 5C). Interestingly, blood insulin levels were greatly reduced in WT mice treated with nobiletin relative to vehicle control (fig. 5D). Nobiletin also significantly reduced total Triglyceride (TG) and Total Cholesterol (TC) levels in WT serum and liver (fig. 5E and 5F). Hematoxylin and eosin (H)&E) And oil red O staining revealed that nobiletin significantly improved the steatosis in DIO WT liver and substantially eliminated the formation of lipid droplets (fig. 5G and 3H-3J). In contrast, nobiletin did not significantly improve Clock compared to WT mice^Δ19/Δ19Glucose and lipid homeostasis in mice (FIG. 5), on Clock^Δ19/Δ19The beneficial effects of hepatic steatosis were significantly diminished (FIGS. 5G and 3H-3J). In contrast to HFD, regular diet (RC) feeding did not result in metabolic dysregulation, and nobiletin treatment did not show significant beneficial effects on metabolic homeostasis (fig. 6). Taken together, these results demonstrate the clock-dependent efficacy of nobiletin on metabolic syndrome.

Non-methoxyflavanones, such as naringin and its aglycone derivative naringenin, are also naturally occurring flavonoids (fig. 7A) (Assini et al, 2013), but they fail to enhance cellular circadian rhythms in our primary screen (fig. 7B-7F.) naringin showed a significant attenuation of weight gain and lipid and glucose homeostasis compared to nobiletin (fig. 8) compared to Mulvihill et al, 2009, 2011) ^Δ19/Δ19There was essentially no difference between the C57BL/6J mice.

Example 3 clock-dependent Metabolic protection of nobiletin on db/db diabetic mice

Considering the improvement in glucose homeostasis in DIO mice treated with nobiletin, we next investigated the effect of nobiletin on db/db mice (which are established fatness)Genetic mouse models of obesity and diabetes, which lack functional leptin receptors) and clock action. Nobiletin treatment severely hampered weight gain in db/db mice (fig. 9A), reduced fasting blood glucose levels (fig. 9B), improved glucose tolerance and insulin sensitivity (fig. 9C and 9D), and reduced serum total Triglyceride (TG) and Total Cholesterol (TC) levels in db/db mice (fig. 9E and 9F). In contrast, db/db Clock^Δ19/Δ19Double mutant mice showed significantly reduced response to nobiletin (figure 9). Db/db Clock under vehicle treatment^Δ19/Δ19Mutant mice showed lower insulin levels than db/db, as compared to what was previously done in Clock^Δ19/Δ19β cell deficiency and hypoinsulinemia results reported in mice (Marcheva et al, 2010.) Chuanchenpi treatment in db/db and db/db Clock^Δ19/Δ19Circulating insulin levels were greatly reduced in both mutant mice, with the former showing a more pronounced reduction (fig. 9G). These results are correlated with db/db Clock versus db/db^Δ19/Δ19 for moderate to low insulin sensitivity, consider Clock^Δ19/Δ19Age-dependent defects in cell proliferation and insulin secretion of the mesopancreas β (Marcheva et al, 2010), future studies will explore the mechanistic relationship between the effects of nobiletin on insulin levels and β cell function and/or insulin sensitivity.

Example 4 identification of nobiletin responsive genes to liver by microarray

To characterize the molecular basis of the role of nobiletin in WT mice fed with HFD, studies focused on the major metabolic organ liver, where we observed that nobiletin had a strong protective effect. Specifically, total RNA prepared from liver tissue from rc.veh and hf.veh or HF nobiletin WT mice, which lasted 10 weeks, was reverse transcribed into cDNA, then labeled with biotin-UTP and hybridized to Illumina mouse WG-6v2.0Expression bearchip. The data discussed in this publication have been stored in the gene expression corpus (GEO) of the national center for biotechnology information, available under GEO series accession number GSE 78848. Real-time qPCR and western blotting analysis of circadian gene expression was performed as described previously (Yoo et al, 2013). The primers used were as follows:

for mammalian single-hybrid assays, ligand interaction with these chimeric receptors has been shown to reduce the transcriptional activity of these chimeric receptors, regardless of the nature of the ligand (agonist or inverse agonist) (Wang et al, 2010a, 2010 b). For radioligand receptor binding assays, the protocol previously described was followed with minor modifications (Kumar et al, 2010; Wang et al, 2010 b).

Compared to previous studies (Kohsaka et al, 2007) , mice fed with HFD, vehicle treated (HF. Veh) had generally lower amplitude of oscillation of clock gene expression in the liver relative to mice lacking RC feed, vehicle treated (RC.Veh) (FIGS. 10A and 10B). Tochenodermadin improved circadian clock transcriptional oscillation and largely restored clock rhythm (FIGS. 10A and 10B) (mice [ HF. nobiletin ] treated with HFD feed, nobiletin compared to HF.Veh mice) and had robust physiological efficacy in order to characterize metabolic output gene expression, microarray analysis was performed to analyze gene expression changes at time points ZT2 and ZT14 in a pairwise comparison analysis (comparison expressed as HF/RC) and HF. velvetoene and HF. vel and HF. velet (comparison expressed as HF/RC) and HF/VEH/phne and HF/phne (expressed as dermoepide/493 544, phne/493) and showed no changes in response to baseline expression of heparin expression (mK) and PCR) of the observed in response to the basal expression of hepatic gene expression of extracellular expression of heparin, e.phne, e.g, E, e.g, E, e.g. showed no change in response to the basal expression of hepatic transcriptional activity (7, E, e.g, E, e.g. observed in a transcriptional regulation, e.g. 5, e.g. observed in a basal expression of hepatic, E, e.g. 5, e.g. observed in a basal expression of hepatic transcriptional regulation, e.g, e.g. 5, E, e.g, e.g. 5, e.g. observed in a basal expression of hepatic transcriptional regulation, e.g. indicative of hepatic transcriptional regulation, e.g. a basal expression, e.g. a basal expression of hepatic transcriptional regulation, E, e.g. a, e.g. observed in a, e.g. a basal expression of basal expression, and in a, E, e.g. a, E.

Example 5 retinoic acid receptor-like orphan receptor (ROR) as a direct protein target for nobiletin.

A cross-examination of previous circadian chromatin immunoprecipitation sequencing studies (Cho et al, 2012; Koike et al, 2012) revealed that 63% of the nobiletin response genes showed promoter occupancy of the core clock protein (fig. 11B), particularly REV-ERB (protein encoded by the reverse DNA chain of c-erbA), REV-ERB and ROR, as negative and positive transcription factors, respectively, competed for binding to the RORE promoter element, playing an important role in circadian rhythm, metabolism and inflammation (Gerhart-hins et al, 2013; Jetten et al, 2013; Kojetin and Burris, 2014.) among previous ROR γ t inhibitor screens, nobiletin hit many of the main screens, but instead activated ROR γ t, and thus was not validated in step (Huh et al, 2011.) the ROR family receptors consist of α, β and γ isoforms and the ROR-r α and the ROR γ domain binding to tissue and more similar structures (rohl et al, 2011 β; soller et al, 2010).

To characterize the direct interaction between nobiletin and ROR proteins, a ROR competitive radioligand binding assay (Kumar et al, 2010; Wang et al, 2010B) with 25- [3H ] -hydroxycholesterol (25- [3H ] -OHC) was performed (Kumar et al, 2010; Wang et al, 2010B) saturation curve and Scatchard plot validating the assay using Kd values similar to those previously reported (Kumar et al, 2010; Wang et al, 2010) importantly, nobiletin showed robust competitive binding to LBD of ROR α and ROR γ with higher affinity to ROR γ (fig. 12C; see Ki comparison) as opposed to naringin or its aglycone derivative naringenin showing significantly reduced binding within the same concentration range (fig. 12C and 12D) with these binding assay results , nobiletin showed mammalian activity as did in the single hybrid reporter gene assay GAL 82-56 and ROR 56 (fig. 12C and 12D) and no evidence of the activity of these chimeric receptors as had been shown by mutual agonist activity of the chimeric receptor ligands (GAL 3H-r 34, GAL et al, 2010) and the inverse agonist activity of these chimeric receptors was shown to be of the receptor agonist (r 20 a) and the receptor agonist (r 20 a).

Characterization of the effect of nobiletin on ROR α/γ transcriptional activity using functional assays it was found that in the presence of ROR α or ROR γ in Hepa1-6 cells, nobiletin dose-dependently increased the activity of the Bmal1 promoter-driven luciferase reporter gene (Preitner et al, 2002) (fig.12g) in contrast to WT rather than mutant RORE element , abrogation of the Rora/C gene encoding ROR α/γ abolished the nobiletin-mediated induction of Bmal1 promoter-driven reporter gene activity in Hepa1-6 and U2OS cells (fig. 12H and 11C) by small interfering rna (sirna) and relative to control treatment, several target genes (e.g. Cyp7b1, IkBa and 4) were induced in DIO mouse liver treated with nobiletin (fig. 12I) relative to control treatment, and the ingenio rease gene also showed an important role in the curculin-mediated luciferase activity in the overall robinally expressed response to robinar α, overall robinary activity.

Example summary

In summary, our unbiased chemical screening identified clock-enhanced polymethoxylated flavones, particularly nobiletin. Strong evidence from genetic and pharmacological studies suggests that nobiletin has Clock gene-dependent efficacy in preventing metabolic syndrome in mice, providing evidence for mammals that circadian rhythm amplitude enhancement is a pharmacological intervention strategy for metabolic diseases and other Clock-related conditions (e.g., age-related decline). Beneficial results of circadian amplitude enhancement may include enhanced efficacy of physiology, expanded stimulation range and sensitized response, suggesting that circadian amplitude enhancement may enhance energy metabolism, time-limited feeding, and thus energy expenditure, playing a major role in determining the degree of obesity caused by HFD feeding.

Polymethoxylated flavones elicit a variety of benefits in mice and humans, including alleviation of the effects on cancer, inflammation, atherosclerosis and recently metabolic disorders and neurodegenerative diseases (Cui et al, 2010; Evans et al, 2012; Kurowska and Manthey, 2004; Lee et al, 2013; mullvihill et al, 2009; Nohara et al, 2015 a). Polymethoxylated flavones generally showed good pharmacokinetic profiles (Evans et al, 2012; Saigusa et al, 2011), and no significant toxicity was observed in long-term treated mice in this and previous studies (Lee et al, 2013; mullvihill et al, 2011). The inventors group showed the role of nobiletin in ammonia treatment regulated by urea cycle, and nobiletin in Clock^Δ19/Δ19The transcriptional induction of the rate-limiting Cps1 gene was impaired in mutant mice (Nohara et al, 2015 a.) this study demonstrates a direct role for nobiletin in the circadian clock enhancement, particularly in the activation of the ROR receptor.

In summary, nobiletin is clock-enhanced natural compounds that activate ROR and prevent metabolic syndrome in a clock-dependent manner, suggesting that such clock-enhanced compounds may be applicable to other diseases (e.g., mood and sleep disorders) and aging.

All of the compositions and methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the compositions and methods of this invention have been described in terms of preferred embodiments, it will be apparent to those of skill in the art that variations may be applied to the compositions and methods and in the steps or in the sequence of steps of the methods described herein without departing from the concept, spirit and scope of the invention. More specifically, it will be apparent that certain agents which are both chemically and physiologically related may be substituted for the agents described herein while the same or similar results would be achieved. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the invention as defined by the appended claims.

Reference to the literature

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