Implications of biological clocks in pharmacology and pharmacokinetics of antitumor drugs.

Mammalians' circadian pacemaker resides in the paired suprachiasmatic nuclei (SCN). SCN control biological rhythms such as the sleep-wake rhythm and homeostatic functions of steroid hormones and their receptors. Alterations in these biological rhythms are implicated in the outcomes of pathogenic conditions such as depression, diabetes, and cancer. Chronotherapy is about optimizing treatment to combat risks and intensity of the disease symptoms that vary depending on the time of day. Thus, conditions/diseases such as allergic rhinitis, arthritis, asthma, myocardial infarction, congestive heart failure, stroke, and peptic ulcer disease, prone to manifest severe symptoms depending on the time of day, would be benefited from chronotherapy. Monitoring rhythm, overcoming rhythm disruption, and manipulating the rhythms from the viewpoints of underlying molecular clocks are essential to enhanced chronopharmacotherapy. New drugs focused on molecular clocks are being developed to improve therapeutics. In this review, we provide a critical summary of literature reports concerning (a) the rationale/mechanisms for time-dependent dosing differences in therapeutic outcomes and safety of antitumor drugs, (b) the molecular pathways underlying biological rhythms, and (c) the possibility of pharmacotherapy based on the intra- and inter-individual variabilities from the viewpoints of the clock genes.

Nuclear Receptors and Clock Components in Cardiovascular Diseases.

Cardiovascular diseases (CVD) are still the first cause of death worldwide. Their main origin is the development of atherosclerotic plaque, which consists in the accumulation of lipids and inflammatory leucocytes within the vascular wall of large vessels. Beyond dyslipidemia, diabetes, obesity, hypertension and smoking, the alteration of circadian rhythms, in shift workers for instance, has recently been recognized as an additional risk factor. Accordingly, targeting a pro-atherogenic pathway at the right time window, namely chronotherapy, has proven its efficiency in reducing plaque progression without affecting healthy tissues in mice, thus providing the rationale of such an approach to treat CVD and to reduce drug side effects. Nuclear receptors are transcriptional factors involved in the control of many physiological processes. Among them, Rev-erbs and RORs control metabolic homeostasis, inflammatory processes and the biological clock. In this review, we discuss the opportunity to dampen atherosclerosis progression by targeting such ligand-activated core clock components in a (chrono-)therapeutic approach in order to treat CVD.

Cell wall remodeling and vesicle trafficking mediate the root clock in Arabidopsis.

In Arabidopsis thaliana, lateral roots initiate in a process preceded by periodic gene expression known as the root clock. We identified the vesicle-trafficking regulator GNOM and its suppressor, ADENOSINE PHOSPHATE RIBOSYLATION FACTOR GTPase ACTIVATION PROTEIN DOMAIN3, as root clock regulators. GNOM is required for the proper distribution of pectin, a mediator of intercellular adhesion, whereas the pectin esterification state is essential for a functional root clock. In sites of lateral root primordia emergence, both esterified and de-esterified pectin variants are differentially distributed. Using a reverse-genetics approach, we show that genes controlling pectin esterification regulate the root clock and lateral root initiation. These results indicate that the balance between esterified and de-esterified pectin states is essential for proper root clock function and the subsequent initiation of lateral root primordia.

Low dose of luteolin activates Nrf2 in the liver of mice at start of the active phase but not that of the inactive phase.

A flavone luteolin has various health-promoting activities. Several studies reported that high dose of luteolin activates the Nrf2/ARE pathway in the liver. However, the effect of the low dose of luteolin that can be taken from a dietary meal on the Nrf2 activation remain unclear. It is expected that the flavonoid metabolism possesses a circadian rhythm, since nutritional metabolism processes daily cycle. In this study we investigated whether an administration affects the Nrf2 activation. ICR mice were orally administered 0.01-10 mg/kg body weight of luteolin once a day for 7 days at two time-points: at the start of active phase (ZT12) or at that of inactive phase (ZT0). Luteolin increased the nuclear translocation of Nrf2, resulting in the increases in its target gene products HO-1 and NQO1 at ZT12 but not at ZT0. The expression level of Nrf2 was lower at ZT12 than at ZT0 in the liver. We also found that the level of luteolin aglycon in the plasma is higher at ZT12 than at ZT0. These results suggest that the low dose of luteolin can activate Nrf2 pathway and the aglycon form of luteolin may mainly contribute to activate the Nrf2 pathway at ZT12 in the liver.

[Evaluation of Naturally Occurring Compounds Regulating Brown/Beige Adipocyte Differentiation].

Brown adipose tissue is a critical regulator of metabolic health, and contributes to thermogenesis by uncoupling oxidative phosphorylation through the action of mitochondrial uncoupling protein 1 (Ucp1). Recent studies have shown that cold exposure and the stimulation of ß3-adrenergic receptors induce the development of brown cell-like "beige" adipocytes in white adipose tissue. Brown and/or beige adipocyte-mediated thermogenesis suppresses high-fat diet-associated obesity. Therefore, the development of brown/beige adipocytes may prevent obesity and metabolic diseases. In the present study, we elucidated whether naturally occurring compounds contribute to regulating the cellular differentiation of brown/beige adipocytes. We screened for the up-regulated expression of Ucp1 during beige adipogenesis using extracts of crude herbal drugs frequently used in Kampo prescriptions (therapeutic drugs in Japanese traditional medicine). This screening revealed that the extract prepared from Citri Unshiu Pericarpium [the peel of Citrus unshiu (Swingle) Marcov.] increased the expression of Ucp1 in beige adipocytes. We also focused on the function of clock genes in regulating brown/beige adipogenesis. Therefore, another aim of the present study was to evaluate naturally occurring compounds that regulate brain and muscle Arnt-like 1 (Bmal1) gene expression. In this review, we focus on naturally occurring compounds that affect regulatory processes in brown/beige adipogenesis, and discuss better preventive strategies for the management of obesity and other metabolic disorders.

Expression of prokineticin 2 and its receptor in the macaque monkey brain.

Prokineticin 2 (PK2) has been indicated as an output signaling molecule for the suprachiasmatic nucleus (SCN) circadian clock. Most of these studies were performed with nocturnal animals, particularly mice and rats. In the current study, the PK2 and its receptor, PKR2, was cloned from a species of diurnal macaque monkey. The macaque monkey PK2 and PKR2 were found to be highly homologous to that of other mammalian species. The mRNA expression of PK2 and PKR2 in the macaque brain was examined by in situ hybridization. The expression patterns of PK2 and PKR2 in the macaque brain were found to be quite similar to that of the mouse brain. Particularly, PK2 mRNA was shown to oscillate in the SCN of the macaque brain in the same phase and with similar amplitude with that of nocturnal mouse brain. PKR2 expression was also detected in known primary SCN targets, including the midline thalamic and hypothalamic nuclei. In addition, we detected the expression of PKR2 mRNA in the dorsal raphe nucleus (DR) of both macaque and mouse brains. As a likely SCN to dorsal raphe projection has previously been indicated, the expression of PKR2 in the raphe nuclei of both macaque and mouse brain signifies a possible role of DR as a previously unrecognized primary SCN projection target.

Chronobiology of bipolar disorder: therapeutic implication.

Multiple lines of evidence suggest that psychopathological symptoms of bipolar disorder arise in part from a malfunction of the circadian system, linking the disease with an abnormal internal timing. Alterations in circadian rhythms and sleep are core elements in the disorders, characterizing both mania and depression and having recently been shown during euthymia. Several human genetic studies have implicated specific genes that make up the genesis of circadian rhythms in the manifestation of mood disorders with polymorphisms in molecular clock genes not only showing an association with the disorder but having also been linked to its phenotypic particularities. Many medications used to treat the disorder, such as antidepressant and mood stabilizers, affect the circadian clock. Finally, circadian rhythms and sleep researches have been the starting point of the developing of chronobiological therapies. These interventions are safe, rapid and effective and they should be considered first-line strategies for bipolar depression.

Thyroid circadian timing: roles in physiology and thyroid malignancies.

The circadian clock represents an anticipatory mechanism, well preserved in evolution. It has a critical impact on most aspects of the physiology of light-sensitive organisms. These rhythmic processes are governed by environmental cues (fluctuations in light intensity and temperature), an internal circadian timing system, and interactions between this timekeeping system and environmental signals. Endocrine body rhythms, including hypothalamic-pituitary-thyroid (HPT) axis rhythms, are tightly regulated by the circadian system. Although the circadian profiles of thyroid-releasing hormone (TRH), thyroid-stimulating hormone (TSH), thyroxine (T4), and triiodothyronine (T3) in blood have been well described, relatively few studies have analyzed molecular mechanisms governing the circadian regulation of HPT axis function. In this review, we will discuss the latest findings in the area of complex regulation of thyroid gland function by the circadian oscillator. We will also highlight the molecular makeup of the human thyroid oscillator as well as the potential link between thyroid malignant transformation and alterations in the clockwork.

Effects of adrenalectomy on daily gene expression rhythms in the rat suprachiasmatic and paraventricular hypothalamic nuclei and in white adipose tissue.

It is assumed that in mammals the circadian rhythms of peripheral clocks are synchronized to the environment via neural, humoral and/or behavioral outputs of the central pacemaker in the suprachiasmatic nucleus of the hypothalamus (SCN). With regard to the humoral outputs, the daily rhythm of the adrenal hormone corticosterone is considered as an important candidate. To examine whether adrenal hormones are necessary for the maintenance of daily rhythms in gene expression in white adipose tissue (WAT), we used RT-PCR to check rhythmic as well as 24 h mean gene expression in WAT from adrenalectomized (ADX) and sham-operated rats. In addition, we investigated the effect of adrenalectomy on gene expression in the hypothalamic SCN and paraventricular nucleus (PVN). Adrenalectomy hardly affected daily rhythms of clock gene expression in WAT. On the other hand, >80% of the rhythmic metabolic/adipokine genes in WAT lost their daily rhythmicity in ADX rats. Likewise, in the hypothalamus adrenalectomy had no major effects on daily rhythms in gene expression, but it did change the expression level of some of the neuropeptide genes. Together, these data indicate that adrenal hormones are important for the maintenance of daily rhythms in metabolic/adipokine gene expression in WAT, without playing a major role in clock gene expression in either WAT or hypothalamus.

The application of multiplex fluorimetric sensor for the analysis of flavonoids content in the medicinal herbs family Asteraceae, Lamiaceae, Rosaceae

BACKGROUND: The aim of our research work was to quantify total flavonoid contents in the leaves of 13 plant species family Asteraceae, 8 representatives of family Lamiaceae and 9 plant species belonging to familyRosaceae, using the multiplex fluorimetric sensor. Fluorescence was measured using optical fluorescence apparatus Multiplex(R) 3 (Force-A, France) for non-destructive flavonoids estimation. The content of total flavonoids was estimated by FLAV index (expressed in relative units), that is deduced from flavonoids UV absorbing properties. RESULTS: Among observed plant species, the highest amount of total flavonoids has been found in leaves ofHelianthus multiflorus (1.65 RU) and Echinops ritro (1.27 RU), Rudbeckia fulgida (1.13 RU) belonging to the family Asteraceae. Lowest flavonoid content has been observed in the leaves of marigold (Calendula officinalis) (0.14 RU) also belonging to family Asteraceae. The highest content of flavonoids among experimental plants of family Rosaceae has been estimated in the leaves of Rosa canina (1.18 RU) and among plant species of family Lamiaceae in the leaves of Coleus blumei (0.90 RU). CONCLUSIONS: This research work was done as pre-screening of flavonoids content in the leaves of plant species belonging to family Asteraceae, Lamiaceae and Rosaceae. Results indicated that statistically significant differences (P > 0.05) in flavonoids content were observed not only between families, but also among individual plant species within one family.

Circadian clock and cancer therapy: an unexpected journey.

Chronotherapy of cancer is one of the oldest examples of the clinical application of biological rhythms. Growing understanding of the importance of biological timing in physiology and pathophysiology of many diseases including cancer reforms our views on the role of biological clocks in cancer and cancer treatment. In the present issue the current progress in circadian clock-dependent mechanisms of cancer, polymorphism and mutations of clock gene in cancer, cancer-associated clock disruptions, and novel chronotherapeutic approaches are discussed.

Time-restricted feeding of rapidly digested starches causes stronger entrainment of the liver clock in PER2::LUCIFERASE knock-in mice.

Restricting feeding to daytime can entrain circadian clocks in peripheral organs of rodents, and nutrients that rapidly increase the blood glucose level are suitable for inducing entrainment. However, dietetic issues, for example, whether or not the diet comprises heated food, have not been fully explored. We therefore hypothesized that rapidly digested starch causes stronger entrainment than slowly digested starch. The entrainment ability of the liver clock in PER2::LUCIFERASE knock-in mice, blood glucose levels, insulin levels, and acute changes in liver clock gene expression were compared between a ß-starch (native)-substituted AIN-93M standard diet and an α-starch (gelatinized)-substituted diet. ß-Corn and ß-rice starch induced larger phase delays of the liver clock, larger blood glucose increases, and higher Per2 gene expression in the liver compared with ß-potato starch. Starch granule size, as examined by electron microscopy, was larger for ß-potato starch than for ß-corn or ß-rice starch. After heating, we obtained gelatinized α-potato, α-corn, and α-rice starch, which showed destruction of the crystal structure and a high level of gelatinization. No difference in the increase of blood glucose or insulin levels was observed between ß-corn and α-corn starch, or between ß-rice and α-rice starch. In contrast, α-potato starch caused higher levels of glucose and insulin compared with ß-potato starch. An α-potato starch-substituted diet induced larger phase delays of the liver clock than did ß-potato starch. Therefore, rapidly digested starch is appropriate for peripheral clock entrainment. Dietetic issues (heated vs unheated) are important when applying basic mouse data to humans.

Biological pacemaker created by percutaneous gene delivery via venous catheters in a porcine model of complete heart block.

BACKGROUND: Pacemaker-dependent patients with device infection require temporary pacing while the infection is treated. External transthoracic pacing is painful and variably effective, while temporary pacing leads are susceptible to superinfection. OBJECTIVE: To create a biological pacemaker delivered via venous catheters in a porcine model of complete heart block, providing a temporary alternative/adjunct to external pacing devices without additional indwelling hardware. METHODS: Complete atrioventricular (AV) nodal block was induced in pigs by radiofrequency ablation after the implantation of a single-chamber electronic pacemaker to maintain a ventricular backup rate of 50 beats/min. An adenoviral vector cocktail (K(AAA) + H2), expressing dominant-negative inward rectifier potassium channel (Kir2.1AAA) and hyperpolarization-activated cation channel (HCN2) genes, was injected into the AV junctional region via a NOGA Myostar catheter advanced through the femoral vein. RESULTS: Animals injected with K(AAA) + H2 maintained a physiologically relevant ventricular rate of 93.5 ± 7 beats/min (n = 4) compared with control animals (average rate, 59.4 ± 4 beats/min; n = 6 at day 7 postinjection; P <.05). Backup electronic pacemaker utilization decreased by almost 4-fold in the K(AAA) + H2 group compared with the control (P <.05), an effect maintained for the entire 14-day window. In contrast to the efficacy of gene delivery into the AV junctional region, open-chest, direct injection of K(AAA) + H2 (or its individual vectors) into the ventricular myocardium failed to elicit significant pacemaker activity. CONCLUSIONS: The right-sided delivery of K(AAA) + H2 to the AV junctional region provided physiologically relevant biological pacing over a 14-day period. Our approach may provide temporary, bridge-to-device pacing for the effective clearance of infection prior to the reimplantation of a definitive electronic pacemaker.

Regulation of circadian behaviour and metabolism by synthetic REV-ERB agonists.

Synchronizing rhythms of behaviour and metabolic processes is important for cardiovascular health and preventing metabolic diseases. The nuclear receptors REV-ERB-α and REV-ERB-ß have an integral role in regulating the expression of core clock proteins driving rhythms in activity and metabolism. Here we describe the identification of potent synthetic REV-ERB agonists with in vivo activity. Administration of synthetic REV-ERB ligands alters circadian behaviour and the circadian pattern of core clock gene expression in the hypothalami of mice. The circadian pattern of expression of an array of metabolic genes in the liver, skeletal muscle and adipose tissue was also altered, resulting in increased energy expenditure. Treatment of diet-induced obese mice with a REV-ERB agonist decreased obesity by reducing fat mass and markedly improving dyslipidaemia and hyperglycaemia. These results indicate that synthetic REV-ERB ligands that pharmacologically target the circadian rhythm may be beneficial in the treatment of sleep disorders as well as metabolic diseases.

REVEILLE8 and PSEUDO-REPONSE REGULATOR5 form a negative feedback loop within the Arabidopsis circadian clock.

Circadian rhythms provide organisms with an adaptive advantage, allowing them to regulate physiological and developmental events so that they occur at the most appropriate time of day. In plants, as in other eukaryotes, multiple transcriptional feedback loops are central to clock function. In one such feedback loop, the Myb-like transcription factors CCA1 and LHY directly repress expression of the pseudoresponse regulator TOC1 by binding to an evening element (EE) in the TOC1 promoter. Another key regulatory circuit involves CCA1 and LHY and the TOC1 homologs PRR5, PRR7, and PRR9. Purification of EE-binding proteins from plant extracts followed by mass spectrometry led to the identification of RVE8, a homolog of CCA1 and LHY. Similar to these well-known clock genes, expression of RVE8 is circadian-regulated with a dawn phase of expression, and RVE8 binds specifically to the EE. However, whereas cca1 and lhy mutants have short period phenotypes and overexpression of either gene causes arrhythmia, rve8 mutants have long-period and RVE8-OX plants have short-period phenotypes. Light input to the clock is normal in rve8, but temperature compensation (a hallmark of circadian rhythms) is perturbed. RVE8 binds to the promoters of both TOC1 and PRR5 in the subjective afternoon, but surprisingly only PRR5 expression is perturbed by overexpression of RVE8. Together, our data indicate that RVE8 promotes expression of a subset of EE-containing clock genes towards the end of the subjective day and forms a negative feedback loop with PRR5. Thus RVE8 and its homologs CCA1 and LHY function close to the circadian oscillator but act via distinct molecular mechanisms.

Sonic hedgehog in temporal control of somite formation.

Vertebrate embryo somite formation is temporally controlled by the cyclic expression of somitogenesis clock genes in the presomitic mesoderm (PSM). The somitogenesis clock is believed to be an intrinsic property of this tissue, operating independently of embryonic midline structures and the signaling molecules produced therein, namely Sonic hedgehog (Shh). This work revisits the notochord signaling contribution to temporal control of PSM segmentation by assessing the rate and number of somites formed and somitogenesis molecular clock gene expression oscillations upon notochord ablation. The absence of the notochord causes a delay in somite formation, accompanied by an increase in the period of molecular clock oscillations. Shh is the notochord-derived signal responsible for this effect, as these alterations are recapitulated by Shh signaling inhibitors and rescued by an external Shh supply. We have characterized chick smoothened expression pattern and have found that the PSM expresses both patched1 and smoothened Shh signal transducers. Upon notochord ablation, patched1, gli1, and fgf8 are down-regulated, whereas gli2 and gli3 are overexpressed. Strikingly, notochord-deprived PSM segmentation rate recovers over time, concomitant with raldh2 overexpression. Accordingly, exogenous RA supplement rescues notochord ablation effects on somite formation. A model is presented in which Shh and RA pathways converge to inhibit PSM Gli activity, ensuring timely somite formation. Altogether, our data provide evidence that a balance between different pathways ensures the robustness of timely somite formation and that notochord-derived Shh is a component of the molecular network regulating the pace of the somitogenesis clock.

Genetic evidence for a role for protein kinase A in the maintenance of sleep and thalamocortical oscillations.

STUDY OBJECTIVES: Genetic manipulation of cAMP-dependent protein kinase A (PKA) in Drosophila has implicated an important role for PKA in sleeplwake state regulation. Here, we characterize the role of this signaling pathway in the regulation of sleep using electroencephalographic (EEG) and electromyographic (EMG) recordings in R(AB) transgenic mice that express a dominant negative form of the regulatory subunit of PKA in neurons within cortex and hippocampus. Previous studies have revealed that these mutant mice have reduced PKA activity that results in the impairment of hippocampus-dependent long-term memory and long-lasting forms of hippocampal synaptic plasticity. DESIGN: PKA assays, in situ hybridization, immunoblots, and sleep studies were performed in R(AB) transgenic mice and wild-type control mice. MEASUREMENTS AND RESULTS: We have found that R(AB) transgenic mice have reduced PKA activity within cortex and reduced Ser845 phosphorylation of the glutamate receptor subunit GluR1. R(AB) transgenic mice exhibit non-rapid eye movement (NREM) sleep fragmentation and increased amounts of rapid eye movement (REM) sleep relative to wild-type mice. Further, R(AB) transgenic mice have more delta power but less sigma power during NREM sleep relative to wild-type mice. After sleep deprivation, the amounts of NREM and REM sleep were comparable between wild-type and R(AB) transgenic mice. However, the homeostatic rebound of sigma power in R(AB) transgenic mice was reduced. CONCLUSIONS: Alterations in cortical synaptic receptors, impairments in sleep continuity, and alterations in sleep oscillations in R(AB) mice imply that PKA is involved not only in synaptic plasticity and memory storage but also in the regulation of sleep/wake states.

Identification, isolation and characterization of HCN4-positive pacemaking cells derived from murine embryonic stem cells during cardiac differentiation.

BACKGROUND: Development of biological pacemaker is a potential treatment for bradyarrhythmias. Pacemaker cells could be extracted from differentiated embryonic stem (ES) cells based on their specific cell marker hyperpolarization-activated cyclic nucleotide-gated (HCN)4. The goal of this study was to develop a method of identification, isolation, and characterization of pacemaking cells derived from differentiated ES cells with GFP driven by HCN4 promoter. METHODS AND RESULTS: Polymerase chain reaction (PCR) screening and southern blot analysis revealed that HCN4p-EGFP trans-gene was stably integrated into the chromosome of mouse AB1 ES cells. RT-PCR and immunostaining results showed similar expression of the specific cardiac pacemaker markers of the HCN4p-EGFP ES cells and its parental AB1 ES cell lines. Although HCN4p-EGFP trans-gene may have slight effect on the general mesodermal differentiation, it had no effect on the pluripotency of ES cells, on the transcription of cardiac specific factors and cardiac contractile proteins, and on the capability of ES cells to differentiate into pacemaker cells. Electrophysiological study indicated that HCN4p-GFP-positive cells revealed the spontaneous action potential, which was slowed by the treatment with 2 mM Cs(+), and expressed the hyperpolarization-activeted cation current I(f) encoded by HCN4 gene. CONCLUSION: By the approach of using stable transfectant of HCN4p-EGFP gene, the identification, isolation, and characterization of ES cell-derived pacemaking cells could be carried out.

[Dosing time based on molecular mechanism of biological clock of hepatic drug metabolic enzyme].

The mammalian circadian pacemaker stays in the paired suprachiasmatic nuclei (SCN). Recent several studies reveal that the circadian rhythms of physiology and behavior are controlled by clock genes. In addition, the effectiveness and toxicity of many drugs vary depending on dosing time associated with 24-h rhythms of biochemical, physiological, and behavioral processes under the control of the circadian clock. Acetaminophen (APAP) is a widely used analgesic drug, and is mainly biotransformed and eliminated as nontoxic conjugates with glucuronic acid and sulfuric acid. Only a small portion of the dose is mainly bioactivated by CYP2E1 to N-acetyl-p-benzoquinone imine (NAPQI), a reactive toxic intermediate. For APAP overdose, glucuronidation and sulfation are saturated and the formation of NAPQI increases. However, the exact mechanisms underlying the chronotoxicity of APAP have not been clarified yet. In the present study, we have clarified that there was a significant dosing time-dependent difference in hepatotoxicity induced by APAP in mice. The mechanism may be related to the rhythmicity of CYP2E1 activity and GSH conjugation. In additon, we investigated whether the liver transcription factor hepatic nuclear factor-1alpha (HNF-1alpha) and clock genes undergoing astriking 24-h rhythm in mouse liver contribute to the 24-h regulation of CYP2E1 activity. A significant 24-h rhythmicity was demonstrated for CYP2E1 activity, protein levels and mRNA levels. HNF-1alpha and clock genes may contribute to produce the 24-h rhythm of CYP2E1 mRNA levels. Metabolism by CYP and GSH conjugation are common metabolic pathways for many drugs such as APAP. These findings support the concept that choosing the most appropriate time of day to administer the drugs associated with metabolic rhythmicity such as CYP and GSH conjugation may reduce hepatotoxicity in experimental and clinical situations. 24-h rhythm of CYP2E1 activity was controlled by HNF-1alpha and clock gene, in a transcriptional level. Identification of rhythmic marker for selecting dosing time will lead improved progress and diffusion of chronopharmacotherapy.