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Would an h1 Full Agonist Prevent Sleep

Would an h1 Full Agonist Prevent Sleep


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According to Wikipedia the h1 receptors stop firing to effectively manage sleep. Assuming I understand how blocking and non blocking receptors work would a full agonist on the h1 receptors effectively prevent sleep instead of create it.

The histaminergic neurons of the tuberomammillary nucleus become active during the 'wake' cycle, firing at approximately 2 Hz; during slow wave sleep, this firing rate drops to approximately 0.5 Hz. Finally, during REM sleep, histaminergic neurons stop firing altogether. It has been reported that histaminergic neurons have the most wake-selective firing pattern of all known neuronal types.


  • Loratadine is an antihistamine that works selectively on peripheral histamine-1 (H-1) receptors (these are histamine receptors that are located outside of the brain and spinal cord). Because it acts on peripheral histamine receptors, loratadine is much less likely to cause drowsiness compared with some older antihistamines.
  • Histamine is a chemical that is released by mast cells in response to an allergen, and it is responsible for many of the symptoms of an allergic reaction, such as swelling of the mucous membranes, sneezing, and itching. Loratadine binds to histamine receptors and prevents histamine from affecting that receptor, which reduces the symptoms of an allergic reaction.
  • Loratadine belongs to the group of drugs known as antihistamines. Loratadine may also be called an H1-antihistamine, a second-generation antihistamine, or a nonsedating antihistamine.
  • Used to treat allergic-type reactions due to perennial or seasonal allergic rhinitis (hay fever).
  • Effective at controlling symptoms such as sneezing, itching, watery eyes, and runny nose that occur as a result of other respiratory allergens.
  • Can provide relief from the itching that occurs as a result of chronic urticaria (hives). Symptoms include raised, red, itchy bumps, streaks, or blotches on the skin.
  • May be used in the treatment of other allergic skin disorders.
  • Is less likely to cause sedation than older antihistamines.
  • Can be taken once a day.
  • May be given daily regularly when allergens are most prevalent (such as during spring or summer).
  • Although plasma levels of loratadine may be increased by certain drugs (such as ketoconazole, erythromycin, or cimetidine), this has not resulted in any clinically significant changes.
  • Available over-the-counter.
  • Generic loratadine is available.

First-generation Histamine H1-receptor Blockers

In order to alleviate allergy symptoms, the first-generation H1-blockers were developed, beginning with the drug diphenhydramine (Benadryl). Other medications in this class include chlorpheneramine (Chlor-trimeton) and hydroxyzine (Vistaril). H1-receptor blockers work on preventing sinus congestion, seasonal allergies, nausea, itching, and the wheal and flare reaction of the skin.In addition, intravenous or injectable diphenhydramine is often used in the hospital-setting to treat severe allergic reactions such as anaphylaxis. The side effects of these medications include drowsiness, stomach upset, increased heart rate, dry mouth, blurred vision, and confusion. Another class of drugs that block H1-receptors are the tricyclic antidepressants or TCAs, typically used to treat depression. For example, doxepin (Silenor) is a TCA that due to its side effect of sedation is often used to treat insomnia. (Ref 3, 4 and 6)


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Introduction

To understand H1-antihistamines, it is necessary to appreciate the state of science in the 1930s. In his review about his own work,[1] Daniel Bovet wrote “Three naturally occurring amines, acetylcholine, epinephrine, and histamine, may be grouped together because they have a similar chemical structure, are all present in the body fluids, and exert characteristically strong pharmacologic activities. There are alkaloids which interfere with the effects of acetylcholine. Similarly, there are sympatholytic poisons which neutralize or reverse the effects of epinephrine. It seemed possible to me, therefore, that some substance might exist which exerts a specific antagonism toward histamine.” It was against this background that Bovet, who was looking for antagonists of acetylcholine, asked his student, Anne-Marie Staub, to test some of these compounds against histamine. This led to the discovery of the first H1-antihistamine in 1937.[2] Although this compound was too toxic for use in humans, it opened the door for the introduction into the clinic of antergan in 1942,[3] followed by diphenhydramine in 1945[4] and chlorpheniramine, brompheniramine, and promethazine later the same decade.[5]

The histamine H1-receptor

The histamine H1-receptor is a member of the superfamily of G-protein-coupled receptors (GPCRs) [ Figure 1a ]. GPCRs may be viewed as �llular switches” which exist as an equilibrium between the inactive or “off” state and the active or “on” state.[6] In the case of the histamine H1-receptor, histamine cross links sites on transmembrane domains III and V to stabilize the receptor in its active conformation, thus causing the equilibrium to swing to the “on” position[7] [ Figure 1b ]. H1-antihistamines, which are not structurally related to histamine, do not antagonize the binding of histamine but bind to different sites on the receptor to produce the opposite effect. For example, cetirizine cross links sites on transmembrane domains IV and VI to stabilize the receptor in the inactive state and swing the equilibrium to the “off” position[8] [ Figure 1c ]. Thus, H1-antihistamines are not receptor antagonists, but are inverse agonists in that they produce the opposite effect on the receptor to histamine.[6] Consequently, the preferred term to define these drugs is “H1-antihistamines” rather than “histamine antagonists.”

(a) Diagram of a histamine H1-receptor in a membrane showing seven transmembrane domains. Histamine stimulates the receptor following its penetration into the central core of the receptor. (b) A surface view of an activated receptor with histamine linking domains III and V. (c) A surface view of an inactive receptor with cetirizine linking domains IV and VI

The development of H1-antihistamines

Bearing in mind that first-generation H1-antihistamines derive from the same chemical stem from which cholinergic muscarinic antagonists, tranquilizers, antipsychotics, and antihypertensive agents were also developed, it is hardly surprising that they have poor receptor selectivity and often interact with receptors of other biologically active amines causing antimuscarinic, anti-α-adrenergic, and antiserotonin effects. But perhaps their greatest drawback is their ability to cross blood𠄻rain barrier and interfere with histaminergic transmission. Histamine is an important neuromediator in the human brain which contains approximately 64,000 histamine-producing neurons, emanating from the tuberomammillary nucleus.[9] Stimulation of H1-receptors in all of the major parts of the cerebrum, cerebellum, posterior pituitary and spinal where they increase arousal in the circadian sleep/wake cycle, reinforce learning and memory, and have roles in fluid balance, suppression of feeding, control of body temperature, control of cardiovascular system, and mediation of stress-triggered release of adrenocorticotropic hormone (ACTH) and b-endorphin from the pituitary gland.[10] It is not surprising then that antihistamines crossing the blood𠄻rain barrier interfere with all of these processes.

Physiologically, the release of histamine during the day causes arousal, whereas its decreased production at night results in a passive reduction in the arousal response. When taken during the day, first-generation H1-antihistamines, even in the manufacturers’ recommended doses, frequently cause daytime somnolence, sedation, drowsiness, fatigue, and impaired concentration and memory.[11,12] When taken at night, first-generation H1-antihistamines increase the latency to the onset of rapid eye movement (REM) sleep and reduce the duration of REM sleep.[13�] The residual effects of poor sleep, including impairment of attention, vigilance, working memory, and sensory motor performance, are still present in the next morning.[14,16] This is especially problematical with drugs with a long half-life [ Table 1 ]. The detrimental central nervous system (CNS) effects of first-generation H1-antihistamines on learning and examination performance in children and on impairment of the ability of adults to work, drive and fly aircraft have been reviewed in detail in a recent review.[17]

Table 1

Half-lives of first-generation H1-antihistamines

A major advance in antihistamine development occurred in the 1980s with the introduction of second-generation H1-antihistamines,[18] which are minimally or nonsedating because of their limited penetration of the blood𠄻rain barrier. In addition, these drugs are highly selective for the histamine H1-receptor and have no anticholinergic effects. The latest EAACI/GA 2 LEN/EDF/WAO guidelines for the management of urticaria[19] recommend that the first-line treatment for urticaria should be second generation, nonsedating H1-antihistamines. Further, it states “In patients with urticaria and no special indication, we recommend against the routine use of old sedating first-generation antihistamines (strong recommendation, high quality evidence).”

H1-antihistamines in urticaria

Most types of urticaria, including chronic spontaneous urticaria and the majority of inducible urticarias, are mediated primarily by mast cell-derived histamine[20] which reaches very high concentrations due to the poor diffusibility of substances in the dermis.[21,22] They are characterized by short-lived wheals ranging from a few millimeters to several centimeters in diameter which are accompanied by severe itching which is usually worse in the evening or night-time.[23] Standard licensed doses of H1-antihistamines are often ineffective in completely relieving symptoms in many patients for whom increasing the dosage up to four-fold is recommended.[19,24,25] Thus, it is clear that the attributes that dermatologists seek when choosing an H1-antihistamine are: Good efficacy, a rapid onset of action, a long duration of action, and freedom from unwanted effects. Although some of these attributes may be predicted from preclinical and pharmacokinetic studies, it is only in the clinical environment that they may be definitively established.

Efficacy

Two factors determine the efficacy of an H1-antihistamine: The affinity of the drug for H1-receptors (absolute potency) and the concentration of the drug at the sites of the H1-receptors. The affinity of an H1-antihistamine for H1-receptors is determined in vitro in preclinical studies. Comparing the three most recently developed drugs, desloratadine is the most potent antihistamine (Ki: 0.4 nM) followed by levocetirizine (Ki: 3 nM) and fexofenadine (Ki: 10 nM) (the lower the concentration, the higher the potency). The drug concentrations at its site of action could, theoretically, be calculated from its apparent volume of distribution (Vd) which are

5.6 l/kg for desloratadine, levocetirizine, and fexofenadine, respectively.[26] However, Vd does not take into account other factors which influence local tissue concentrations in vivo, such as absorption, metabolism, and plasma binding. In the study of Gillard and colleagues,[27] concentrations of unbound drug in the plasma rather than Vd were used to calculate receptor occupancy, a theoretical indicator of effectiveness in vivo [ Table 2 ]. The validity of these calculations of receptor occupancy is confirmed by the relative inhibition of wheal and flare responses by these drugs.[26,28�]

Table 2

Comparison of receptor occupancy for desloratadine, fexofenadine, and levocetirizine with inhibition of histamine-induced wheal and flare responses 4 and 24 h after drug administration

Speed of onset of action and duration of action

The speed of onset of action of a drug is often equated to the rate of its oral absorption and its duration of action by its plasma concentration. However, this is not strictly correct as seen from Figure 2 . In this study, in children,[31,32] plasma concentrations of drug are near maximum by 30 min and yet it takes a further 1½ h for the drug to diffuse into the extravascular space to produce a maximal clinical effect. In adults, the maximal inhibition of the flare response is

4 h for levocetirizine, fexofenadine, and desloratadine[28,30,33] but may be longer for drugs, such as loratadine and ebastine, which require metabolism to produce their active moiety.[28]

Diagrammatic representation of the pharmacokinetics and pharmacodynamics of levocetirizine for a single oral dose of levocetirizine[31,32]

Figure 2 also shows that the duration of action of levocetirizine in inhibiting the histamine-induced flare response is also much longer than would be predicted from a knowledge of its plasma concentration.[31,32] This is presumably to “trapping” of the drug by its strong and long-lasting binding to histamine H1-receptors.[8] Although less active in the wheal and flare test, desloratadine has a similarly long duration of action.[33] However, the duration of action of fexofenadine, calculated as the time for the wheal to remain inhibited by at least 70%, is less prolonged being 8.5 h for 120 mg fexofenadine compared with 19 h for cetirizine.[34] The primary reason for the shorter duration of action of fexofenadine is that it is actively secreted into the intestine and urine by P-glycoprotein.[35]

Elimination

The metabolism and elimination of H1-antihistamines have been extensively reviewed elsewhere[26,36] and will be only briefly summarized here. Cetirizine and levocetirizine are not metabolized and are excreted primarily unchanged in the urine.[26] Desloratadine undergoes extensive metabolism in liver. Although this gives the potential for drug𠄽rug interactions, no significant interactions have been reported[36] Fexofenadine, which is also minimally metabolized, is excreted primarily in the feces following its active secretion into the intestine under the influence of active drug transporting molecules.[36] This gives the potential for interactions with agents, such as grapefruit juice and St Johns Wort, which inhibit these transporters. Although plasma concentrations of fexofenadine may be increased by these agents, no significant resulting adverse reactions have been reported.[36]

Unwanted effects

Somnolence

A major reason for the reduced penetration of second-generation H1-antihistamines into the brain is because their translocation across the blood𠄻rain barrier is under the control of active transporter proteins, of which the ATP-dependent efflux pump, P-glycoprotein, is the best known.[37,38] It also became apparent that antihistamines differ in their substrate specificity for P-glycoprotein, fexofenadine being a particularly good substrate.[39] In the brain, the H1-receptor occupancy of fexofenadine assessed using positron emission tomography (PET) scanning is negligible, π.1%, and, in psychomotor tests, fexofenadine is not significantly different from placebo.[40] Furthermore, fexofenadine has been shown to be devoid of central nervous effects even at supraclinical doses, up to 360 mg.[41]

Although fexofenadine is devoid of CNS effects, many other second-generation H1-antihistamines still penetrate the brain to a small extent where they have the potential to cause some degree of drowsiness or somnolence, particularly when used in higher doses. For example, PET scanning of the human brain has shown that a single oral doses of 10 mg and 20 mg cetirizine caused 12.5% and 25.2% occupancy of the H1-receptors in prefrontal and cingulate cortices, respectively.[42] These results would explain the repeated clinical findings that the incidence of drowsiness or fatigue is greater with cetirizine than with placebo.[43�] Recent publications have suggested that, at manufacturers’ recommended doses, levocetirizine is less sedative than cetirizine[47] and desloratadine causes negligible somnolence. [36,48] However, it should be pointed out that “mean results” do not reveal everything as some patients may show considerable somnolence, whereas others are unaffected.

Cardiotoxicity

The propensity of astemizole and terfenadine, to block the IKr current, to prolong the QT interval, and to potentially cause serious polymorphic ventricular arrhythmias such as torsades de pointes is well documented.[6,49] These two drugs are no longer approved by regulatory agencies in most countries. In addition, some first-generation H1-antihistamines, such as promethazine,[50] brompheniramine,[51] and diphenhydramine,[52] may also be associated with a prolonged QTc and cardiac arrhythmias when taken in large doses or overdoses. No clinically significant cardiac effects have been reported for the second-generation H1-antihistamines: Loratadine, fexofenadine, mizolastine, ebastine, azelastine, cetirizine, desloratadine, and levocetirizine.[53�]


Author information

Tatsuro Shimamura and Mitsunori Shiroishi: These authors contributed equally to this work.

Affiliations

Human Receptor Crystallography Project, ERATO, Japan Science and Technology Agency, Yoshidakonoe-cho, Sakyo-ku, Kyoto 606-8501, Japan

Tatsuro Shimamura, Mitsunori Shiroishi, Simone Weyand, Hirokazu Tsujimoto, Takuya Kobayashi & So Iwata

Department of Cell Biology, Graduate School of Medicine, Kyoto University, Yoshidakonoe-cho, Sakyo-Ku, Kyoto 606-8501, Japan

Tatsuro Shimamura, Mitsunori Shiroishi, Hirokazu Tsujimoto, Takuya Kobayashi & So Iwata

Department of Molecular Biology, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, California 92037, USA

Tatsuro Shimamura, Vadim Cherezov, Wei Liu, Gye Won Han & Raymond C. Stevens

Graduate School of Pharmaceutical Sciences, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582, Japan

Division of Molecular Biosciences, Membrane Protein Crystallography Group, Imperial College, London SW7 2AZ, UK

Diamond Light Source, Harwell Science and Innovation Campus, Chilton, Didcot, Oxfordshire OX11 0DE, UK

Simone Weyand, Graeme Winter & So Iwata

Skaggs School of Pharmacy and Pharmaceutical Sciences and San Diego Supercomputer Center, University of California, San Diego, La Jolla, California 92093, USA

Vsevolod Katritch & Ruben Abagyan

Systems and Structural Biology Center, RIKEN, 1-7-22 Suehiro-cho Tsurumi-ku, Yokohama 230-0045 Japan


Need To Pull An All-nighter? Reducing Nitric Oxide Gas In The Brain May Help Us Stay Awake

People who must stay awake for long shifts &ndash soldiers, pilots, truckers, students, doctors, parents of newborns &ndash may take comfort from new research showing that preventing the gas nitric oxide from building up in the brain may ward off the sleep urge.

The research, conducted by investigators from Children's Hospital Boston and the University of Helsinki (Helsinki, Finland), ties together previous observations about sleep and finds that nitric oxide production in a specific region of the brain &ndash the basal forebrain &ndash is both necessary and sufficient to produce sleep. The findings appear in two related papers in the August 18 issue of the Journal of Neurochemistry and the September 5 issue of the European Journal of Neuroscience.

"This understanding of sleep physiology should provide a completely new basis for the development of drugs to prevent excessive sleepiness or to promote sleep," says study co-author Paul Rosenberg, MD, PhD, a researcher in the Neurobiology Program at Children's Hospital Boston, and a physician in the Center for Pediatric Sleep Disorders at Children's and in the Sleep Disorders Center at Beth Israel Deaconess Medical Center.

In 1997, senior investigator Tarja Porkka-Heiskanen MD, PhD, now at the University of Helsinki, first showed that when cats are awake for prolonged periods, a compound called adenosine accumulates in their brains, ultimately producing sleep. Once asleep, adenosine levels gradually decline. Rosenberg had been studying how the brain regulates the accumulation of adenosine in the brain for over 10 years, and, in 2000, he and his colleagues demonstrated in brain cells from rats that adenosine's release is stimulated by nitric oxide. The two teams decided to collaborate.

Studying mildly sleep-deprived rats &ndash kept awake for an extra three hours &ndash they found that nitric oxide production in the basal forebrain, but not in other parts of the brain, increased, by 50 to 150 percent. When they injected compounds that inhibit nitric oxide production into this region of the brain, adenosine levels did not increase and sleep was completely abolished (one type of inhibitor abolished dreaming, or REM, sleep, and the other non-REM sleep). The results were identical when the researchers injected a compound that scavenges nitric oxide &ndash mops it up and renders it inactive.

In contrast, when the basal forebrain was infused with a nitric oxide "donor" &ndash an agent that boosts nitric oxide levels &ndash during a normal sleep-wake cycle, adenosine levels increased and the rats fell into a sleep much like the "recovery" sleep that occurs after prolonged wakefulness. Blocking adenosine receptors with caffeine prevented this nitric-oxide-induced slumber.

Rosenberg sees the most pharmaceutical promise in developing drugs that prolong wakefulness by curbing production of nitric oxide or scavenging the gas once it's produced. The opposite &ndash a sleeping pill made from nitric oxide donors -- would be much harder, he says, since these compounds would likely break down before ever reaching the brain. However, one of the ways nitric oxide promotes sleep is by stimulating production of a signaling molecule called cyclic GMP, and it may be possible to achieve the same effect by using drugs that block cyclic GMP's breakdown, Rosenberg says.

One surprise in the research was that the main message telling the brain to go to sleep probably does not come from neurons, but from neighboring glial cells, which appear to produce the greatest amounts of nitric oxide. Until recently, glial cells have been assumed to play only a supportive role in the brain. Rosenberg speculates that the molecules that turn on nitric oxide production in glial cells (as yet undiscovered) might provide additional targets for drug development.

The research was funded by the National Institutes of Health, the Academy of Finland, the European Union, the Finnish Medical Society and the Sigrid Juselius Foundation, and an ESRS Sanofi-Synthelabo Research Award.

Story Source:

Materials provided by Children's Hospital Boston. Note: Content may be edited for style and length.


Results

Breast cancer cell autonomous RIG-I signaling is activated by a synthetic RIG-I mimetic

To assess the potential applicability of a RIG-I agonist in breast cancer, we examined RIG-I/DDX58 expression in a clinical invasive breast cancer data set curated by The Cancer Genome Atlas (TCGA ref. 37). We found genomic DDX58 deletion in only 1 of 817 tumors and mRNA downregulation in only 8 of 817 tumors (Fig. 1A), suggesting that loss of RIG-I expression is a rare event. Similar results were produced upon the analysis of 2509 breast tumors from the METABRIC invasive breast cancer data set (Supplementary Fig. S1A ref. 38). Whole-exome sequencing data identified 3 nonrecurrent missense mutations within DDX58, and no recurrent, truncating, or in-frame mutations (Supplementary Fig. S1B), suggesting that RIG-I/DDX58 is rarely lost or mutated in breast cancers.

RIG-I/DDX58 is expressed in breast cancers and is activated by the RIG-I agonist SLR20. A, TCGA-curated clinical data set of invasive breast cancers (N = 817 ref. 37) was assessed for samples harboring genomic DDX58 loss (solid blue) and/or DDX58 mRNA downregulation (defined as <−1 SD from the mean DDX58 expression among the entire data set and shown in blue outline). Reported scores for IHC analysis of ER and HER2 corresponding to each clinical specimen are shown. B, Whole-cell lysates were assessed by Western analysis using the antibodies indicated to the left of each blot. C, Sixteen hours after transfection with OH-SLR20 and SLR20, cells were fixed, assessed by immunofluorescence to detect RIG-I (green fluorescence), and counterstained with MitoTracker Red (red fluorescence). Left, representative images are shown. The inset shows a high-power magnification of the boxed area within each respective panel. Right, the ratio of RIG-I staining to MitoTracker is shown. MitoTracker staining was quantified as the number of red fluorescent pixels per 40× field using ImageJ. RIG-I immunofluorescent staining was quantified as the number of green fluorescent pixels per 40× field. Each point represents the average value of three random fields per sample, N = 5 samples. Midlines and error bars show average ± SD. P value was calculated using Student unpaired t test. D, Mitochondrial fractions of cells were assessed by Western analysis 18 hours after transfection, using the antibodies shown on the left of each panel. Representative images are shown. N = 3. E, Whole-cell lysates collected 12 hours after transfection were assessed by Western analysis using the antibodies shown on the left of each panel. Representative images are shown. N = 3.

Western analysis confirmed RIG-I expression in two human breast cancer cell lines, MCF7 (ER + ), and BT474 (HER2 amplified Fig. 1B), but not in HER2-amplified, ER + MDA-MB-361 cells (Supplementary Fig. S2A). To determine if RIG-I signaling pathways are functional in breast cancer cells, we used a previously described synthetic minimal RIG-I agonist composed of a double-stranded, triphosphorylated 20-base pair stem-loop RNA, which was then modified with a 5′ triphosphate sequence (SLR20 ref. 39). Previous studies demonstrated that SLRs containing the 5′ ppp motif, but not those lacking the motif, activate type I IFN production via RIG-I/MAVS signaling. We transfected SLR20 (and the nonphosphorylated, but otherwise identical sequence, OH-SLR20) into MCF7 cells and measured RIG-I expression and distribution by immunofluorescence. RIG-I expression was robustly increased in cells transfected with the RIG-I ligand SLR20 as compared with the control ligand OH-SLR20 (Fig. 1C). Counterstaining of mitochondria demonstrated mitochondrial localization of RIG-I in many cells following SLR20 treatment. Further, analysis of mitochondrial cell fractions by Western analysis confirmed mitochondrial RIG-I localization in MCF7 and BT474 cells transfected with SLR20, but not OH-SLR20 (Fig. 1D). Western analysis confirmed RIG-I upregulation following transfection with SLR20 in MCF7, BT474, and mouse 4T1 cells, a mammary tumor line used as a model of aggressive, metastatic, and poorly immunogenic TNBC (Fig. 1E). Importantly, SLR20 increased phosphorylation of the proinflammatory transcription factors p65 (an NF-κB subunit) and STAT1 in MCF7, BT474, and 4T1 cells. Importantly, SLR20 did not affect P-p65 in MDA-MB-361 cells, which lack RIG-I expression (Supplementary Fig. S2B). These data support use of these breast cancer cell lines, and the SLR20 agonist, to model the therapeutic impact of RIG-I signaling in breast cancer.

A nanoparticle-based approach for RIG-I activation in vivo decreases breast tumor growth and metastasis

A recent study examining SLR delivery in vivo confirmed rapid induction of type I IFNs following delivery of a 10-bp SLR sequence (SLR10 ref. 28). However, the impact of RIG-I activation in the complex breast tumor microenvironment has not been explored. We used a nanoparticle-based platform previously optimized for oligonucleotide delivery in vivo for intratumoral (i.t.) treatment of breast tumors with SLR20 (Fig. 2A). These pH-responsive nanoparticles (NP) were composed of amphiphilic diblock copolymers formulated with a hydrophobic core-forming block that is endosomolytic and drives micellar assembly, and a polycationic corona for electrostatic complexation with oligonucleotide (i.e., SLR20), as described previously (36). This formulation has been shown to maximize cytoplasmic delivery of oligonucleotides, an ideal scenario for cytoplasmic RIG-I activation by SLR20. NPs were delivered i.t. to 4T1 mammary tumors grown in WT Balb/c female mice when tumors reached 50 to 100 mm 3 . As an additional control, a third group of tumor-bearing mice were treated by i.t. injection of saline, the vehicle in which NPs were delivered. A total of 3 treatments were administered (days 0, 2, and 4 Fig. 2B). IHC of tumors collected at day 5 (24 hours after final treatment) revealed RIG-I protein upregulation in 4T1 tumors treated with SLR20 NPs over saline-treated or OH-SLR20 NP-treated tumors (Fig. 2C–D and Supplementary Fig. S2C). Further, tumors treated with SLR20 NPs, but not OH-SLR20 NPs, exhibited a 3-fold increase in phosphorylation of STAT1 (Fig. 2C and D), confirming RIG-I signaling in SLR20-treated tumors in vivo.

RIG-I agonist SLR20 induces RIG-I signaling and impairs tumor progression in vivo. A, Schematic representation of nanoparticle formulation used to treat tumor-bearing mice in vivo. B, Schematic of treatment strategy for intratumoral nanoparticle delivery of SLR20 (or OH-SLR20) to WT Balb/c mice harboring 4T1 mammary tumors. Saline was delivered intratumorally as a control. C and D, IHC was used to measure RIG-I and P-STAT1 in tumors harvested at day 14. B, Representative images are shown. N = 5. C, IHC staining for RIG-I and P-STAT1 was quantitated. Each point represents the average of three random fields per sample, N = 5. Midlines show average (±SD). P values were calculated using Student t test. E, Schematic of treatment strategy for intratumoral nanoparticle delivery of SLR20 (or OH-SLR20) to WT Balb/c mice harboring 4T1 mammary tumors. Saline was delivered intratumorally as a control. Tumors were measured throughout treatment (days 0–9) and for 16 days after treatment ceased (days 10–25). Tumors were collected on day 25 (16 days after the final treatment). F, Tumor volume was measured beginning at treatment day 1. N = 10 per group through day 5. N = 5 per group from days 6 to 25. G, Lungs harvested at day 25 were assessed histologically for metastatic lesions. Each point represents the number of metastases per individual mouse. Midlines represent the average (± SD) Student t test. n.s., nonsignificant.

We used a slightly modified treatment scheme to assess the therapeutic impact of SLR20-NP on tumor growth. Tumors were treated on days 0, 3, 6, and 9 with i.t. delivery of SLR20 NPs, at which point treatment stopped and tumor volume was monitored through day 25 (Fig. 2E). Tumors treated with SLR20 NPs did not increase in volume during the treatment window (days 0–9), while tumors treated with saline or with OH-SLR20 NPs increased nearly 4-fold (Fig. 2F). Once treatment was complete, tumors treated with SLR20 resumed volumetric increase, but still grew at a diminished rate as compared with tumors treated with OH-SLR20 NPs or with saline. Lungs harvested from mice on day 25 revealed a decreased number of lung metastases in the SLR20-treated mice as compared with control groups (Fig. 2G). Treatment extended through treatment day 25 (Supplementary Fig. S3A) resulted in sustained tumor growth inhibition in response to SLR20 (Supplementary Fig. S3B).

RIG-I signaling induces breast cancer cell death through tumor cell–intrinsic pathways

We investigated potential mechanisms responsible for decreased tumor growth and metastasis following treatment with SLR20 NPs, first measuring Ki67-positive cells by IHC as a marker of cell proliferation (Fig. 3A Supplementary Fig. S4A). Assessing 4T1 tumors collected on treatment day 5, we found a decreased percentage of Ki67 + tumor cells in samples treated with SLR20 NPs as compared with samples treated with OH-SLR20-NPs or with saline (Fig. 3B). Conversely, tumor cell death, measured by terminal dUTP nick-end labeling (TUNEL) analysis (Fig. 3A), was increased 5-fold in samples treated with SLR20 NPs (Fig. 3B). RIG-I signaling is capable of inducing programmed cell death in many cell types, including some cancer cell types (40), although this possibility remains unclear in breast cancers. Therefore, we transfected MCF7, BT474, and 4T1 cells in culture with SLR20, assessing cells 12 hours after transfection for PARP cleavage, a molecular marker of cell death. Cleaved PARP was increased in cells transfected with SLR20 versus OH-SLR20 (Fig. 3C). Annexin V-FITC staining was used to enumerate apoptotic cells, revealing increased Annexin V + cells following SLR20 treatment in MCF7, BT474, and 4T1 cells (Fig. 3D), but not in MDA-MB-361 cells, which lack RIG-I expression (Supplementary Fig. S4B). Importantly, knockdown of RIG-I in MCF7, BT474, and 4T1 cells using shRNA sequences against RIG-I (Fig. 3E) abrogated the increased Annexin V staining in response to SLR20 (Fig. 3F), while SLR20 remained capable of inducing Annexin V staining in cells expressing nontargeting shRNA sequences. These findings demonstrate that SLR20 activates RIG-I signaling in breast cancer cells, inducing cell death in a tumor cell–intrinsic manner.

RIG-I agonist SLR20 induces tumor cell apoptosis. A and B, histologic analysis of tumor sections using IHC against Ki67 and TUNEL analysis. B, Representative images are shown. N = 5. C, The number of Ki67 + cells and TUNEL + cells per 400× field was quantitated. Each point represents the average of three random fields per sample, N = 5. Midlines show average (± SD). P values were calculated using Student t test. C, Western analysis of whole-cell lysates harvested 12 hours after transfection, using antibodies indicated on the left of each panel. D, Cells were transfected, and after 18 hours, cells were stained with Annexin V-AlexaFluor488 for 4 hours. AlexaFluor488 + cells were imaged by fluorescence microscopy. The number of Annexin V + cells per well was counted. Each point shown represents the average of two experimental replicates, N = 5. Midlines represent the average (± SD). P values were calculated using Student t test. Staurosporine treatment (1 μmol/L) was performed in parallel as a positive control for induction of apoptosis/Annexin V staining. E, Western analysis of whole-cell lysates using antibodies shown on left of each panel. F, Cells were transfected and stained with Annexin V-FITC as shown in D. n.s., nonsignificant.

RIG-I signaling in breast cancer cells induces intrinsic apoptosis and pyroptosis

Because RIG-I signaling is reported to induce apoptosis through several distinct pathways, including intrinsic apoptosis, extrinsic apoptosis, and pyroptosis pathways across a variety of cell types (33), it is unclear by which pathway RIG-I induces cell death in breast cancers. We investigated this using an apoptosis expression array, assessing expression changes in 84 genes associated with the intrinsic and extrinsic apoptosis pathways. RNA harvested from BT474 cells collected 16 hours after transfection harbored changes in 18 of the 84 genes assessed. Genes arranged in order of expression fold change revealed that genes regulating intrinsic apoptosis (e.g., BAD, BAX, CASP9) were downregulated, while expression of genes regulating the extrinsic apoptosis pathway (TNFSF10, FAS, CASP10, CASP8) were upregulated (Fig. 4A), suggesting that the extrinsic apoptosis pathway might be activated in response to RIG-I signaling in breast cancer cells. We confirmed that SLR20 induced expression of the extrinsic apoptotic factor TNFSF10 in MCF7, BT474, and 4T1 cells (Fig. 4B). Additionally, 4T1 tumors treated in vivo with SLR20 NPs were assessed by IHC for expression of the Tnfsf10 gene product, TRAIL. Although TRAIL was expressed at only low levels in 4T1 tumors treated with saline or with OH-SLR20 NPs (Fig. 4C), TRAIL protein levels were markedly upregulated in samples treated with SLR20 NPs.

RIG-I signaling in breast cancer cells induces extrinsic apoptosis and pyroptosis. A, BT474 cells were transfected with SLR20 or OH-SLR20. After 12 hours, RNA was collected and assessed for expression of genes within the intrinsic and extrinsic apoptosis pathway (RT2 Profiler Apoptosis Array). Relative gene-expression values were calculated using the ddCT method, correcting for expression of ACTB and GAPDH, and are shown as expression relative to the average value for each gene in OH-SLR20-transfected cells, as shown in the heat map. Genes (listed at left) were ranked in order of expression fold change, as shown on the right. B, Cells were transfected, and after 12 hours, total RNA was assessed by RT-qPCR to measure expression of the indicated genes involved in pyroptosis. Each point represents the average of three experimental replicates, N = 3. Midlines are average ± SD. Student t test. C, IHC analysis to detect TRAIL in 4T1 tumors harvested on treatment day 5. D and E, Western analysis of whole-cell lysates (D) or membrane fractions (E) harvested 16 hours after transfection using the antibodies shown on left of each panel. F, Cells were transfected, and after 12 hours, total RNA was assessed by RT-qPCR to measure expression of the indicated genes involved in pyroptosis. Each point represents the average of three experimental replicates, N = 3. Midlines are average ±SD. Student t test. G, RNA harvested from 4T1 tumors collected on treatment day 5 was assessed by RT-qPCR for Casp1 gene expression as described in C. H and I, Cells were transfected and immediately treated with caspase-specific inhibitors (each used at 10 μmol/L). After 18 hours, cells were stained with Annexin V-AlexFluor488 for 4 hours (H) or PI for 10 minutes (I). AlexaFluor488 + and PI + cells were imaged by fluorescence microscopy. The number of fluorescent cells per well was counted. Each point shown represents the average of two experimental replicates, N = 5 (H) and N = 4–5 (I). Midlines represent the average (±SD). P values use Student t test.

These data suggest that RIG-I might activate the extrinsic apoptosis pathway in breast cancer cells but do not rule out that RIG-I signaling might also induce breast cancer cells to undergo pyroptosis, an inflammatory type of programmed cell death that requires activation of caspase-1 and oligomerization of gasdermin D on the cell membrane (41). Western analysis of MCF7 and BT474 cells transfected with SLR20 revealed potent activation of caspase-1 (Fig. 4D) and localization of gasdermin D to cell membranes (Fig. 4E). These findings were confirmed in 4T1 cells (Supplementary Fig. S4C). Upregulation of CASP1 and CASP4 (encoding another mediator of pyroptosis, caspase-4) was seen in BT474 cells transfected with SLR20 (Fig. 4F). Importantly, Casp1 levels were increased in 4T1 tumors treated in vivo with SLR20 NPs, but not in tumors treated with OH-SLR20 NPs (Fig. 4G), suggesting the RIG-I–mediated activation of pyroptotic signaling pathway may be maintained even within the complex tumor microenvironment.

Next, we used a selective inhibitor of caspase-10, AEVD-FMK, to block the extrinsic apoptotic pathway, resulting in a moderate, but significant, diminution of Annexin V + cells following treatment with SLR20 (Fig. 4H). In contrast, the caspase-9 inhibitor Z-LEHD-FMK, which blocks activation of the intrinsic apoptotic pathway, had little impact on Annexin V staining in cells transfected with SLR20. We also tested an inhibitor of caspase-1, Z-YVAD-FMK, to block the pyroptosis pathway in SLR20-transfected cells, resulting in partial inhibition of Annexin V staining. These results were confirmed in MCF7 cells (Supplementary Fig. S4D). Interestingly, the combination of the caspase-10 inhibitor with the caspase-1 inhibitor produced a greater reduction in Annexin V staining in BT474 cells as compared with either inhibitor used alone (Fig. 4H), consistent with the idea that these two inhibitors operate through distinct pathways in BT474 cells, and suggesting that RIG-I signaling in breast cancer cells may use both the intrinsic apoptosis pathway and pyroptosis to potently induce programmed cell death. Because pyroptosis produces pores in the plasma membrane (41), making them permeable to PI, we stained MCF7 and BT474 cells with PI at 12 hours after transfection with OH-SLR20 or SLR20, finding a robust increase in PI + staining when cells were transfected with SLR20 (Fig. 4I). However, PI staining was completely abolished in MCF7 and BT474 cells pretreated with the caspase-1 inhibitor, confirming that pyroptosis is induced by SLR20 in breast cancer cells.

RIG-I signaling increases breast tumor-infiltrating leukocytes

In contrast to the intrinsic apoptosis pathway, which is considered an immunologically silent form of programmed cell death, pyroptosis is thought to be an immunogenic form of cell death that may recruit inflammatory leukocytes to the site of a viral infection through cytokine modulation, while increasing immunogenicity of the infected cell through increased expression of the major histocompatibility complex (MHC)-I, the antigen presentation machinery expressed on most nucleated cells. Consistent with this idea, both MCF7 and BT474 breast cancer cells transfected with SLR20 showed upregulation of HLAB (Fig. 5A), encoding a key MHC-I component. The gene B2M, encoding another key MHC-I component, β2 microglobulin, was similarly upregulated in BT474 cells (Fig. 4A).

RIG-I signaling induces immunogenic cell death and increases tumor leukocyte infiltration. A, Cells were transfected, and after 12 hours, total RNA was assessed by RT-qPCR to measure expression of the MHC Class II gene HLAB. Each point represents the average of three experimental replicates, N = 3. Midlines are average ± SD. Student t test. B and C, histologic analysis of tumor sections using IHC against F4/80analysis. B, Representative images are shown. N = 5. C, The number of CD45 + , F4/80 + , CD4 + , and CD8 + cells per 400× field was quantitated. Each point represents the average of three random fields per sample, N = 5. Midlines show average (±SD). P values were calculated using Student t test. D, Schematic of treatment strategy for intraperitoneal delivery of αPD-L1 or IgG, and intratumoral nanoparticle delivery of SLR20 (or OH-SLR20, or saline) to WT Balb/c mice harboring 4T1 mammary tumors. Tumors were measured throughout treatment (days 1–10) and for 8 days after treatment ceased (days 11–18). E, Tumor volume was measured beginning at treatment day 1. N = 7 to 8 per group. n.s., nonsignificant.

We assessed leukocyte recruitment to 4T1 mammary tumors grown in immune-competent Balb/c mice following treatment with SLR20 NPs. IHC analysis for CD45, a pan-leukocyte marker, revealed substantially increased CD45 + cells in tumors treated with SLR20 NPs versus saline or OH-SLR20 NPs (Fig. 5B and Supplementary Fig. S5). Further, IHC analysis using antibodies against F4/80 (a mature macrophage marker), CD4 (a marker of helper T lymphocytes), and CD8, a marker of cytotoxic T lymphocytes (CTL), natural killer T cells (NK-T) and inflammatory dendritic cell (DC) populations, were each increased in tumors treated with SLR20 NPs as compared with saline OH-SLR20 NP-treated tumors (Fig. 5B and C). These data suggest that RIG-I activation results in active recruitment of leukocytes to the TME, consistent with a more immunogenic tumor microenvironment. Consistent with this notion, we found that SLR20 delivery to 4T1 tumors grown in immunocompromised athymic Balb/c (nu/nu) mice displayed more rapid resurgence of tumor growth once the SLR20 treatment was discontinued (Supplementary Fig. S6). This use of the RIG-I ligand to generate a more immunogenic tumor microenvironment was tested more directly using SLR20 in combination with the ICI, αPD-L1. Tumor-bearing WT Balb/c mice were randomized into groups to receive treatment with SLR20, OH-SLR20, or saline and were randomized further into groups receiving αPD-L1 or an isotype-matched control IgG (Fig. 5D). Tumors were treated twice weekly through treatment day 10 and monitored through treatment day 18. We found that tumors treated with SLR20 alone grew at a slower rate than tumors treated with OH-SLR20 or with saline (Fig. 5E). Tumor growth was inhibited by treatment with αPD-L1 alone. However, the combination of SLR20 with αPD-L1 decreased tumor growth to a greater extent than either agent alone, and to a greater extent than αPD-L1 in combination with the control OH-SLR20 NP. These findings are consistent with the idea that SLR20 increases the immunogenicity of the tumor microenvironment in this model of breast cancer.

Cytokine and chemokine modulation by RIG-I signaling in breast cancer cells

Like many PRRs, RIG-I induces expression of inflammatory cytokines required for lymphocyte recruitment (42). Therefore, we measured expression of IFNB1 in MCF7, BT474, and 4T1 cells following transfection with SLR20, revealing IFNB1 upregulation (Fig. 6A). Notably, Ifnb1 upregulation was also seen in 4T1 tumors treated in vivo with SLR20 NPs (Fig. 6B). SLR20-mediated upregulation of IFN1B was impaired in MCF7 and BT474 cells expressing RIG-I-directed shRNA sequences (Fig. 6C Supplementary Fig. S7). This suggests that RIG-I signaling in breast cancer cells might be capable of activating in trans the antigen presenting cells, such as macrophages, within the tumor microenvironment. We tested this hypothesis by harvesting cultured media from 4T1 cells transfected with SLR20 or OH-SLR20, and adding the cultured media to macrophage-derived Raw264.7 cells. After 30 minutes of exposure to cultured media harvested from 4T1 cells treated with SLR20, we found phosphorylation of the proinflammatory transcription factor STAT1 (Supplementary Fig. S8A). Induction of TNF gene expression following transfection with SLR20 also was seen in MCF7, BT474, and 4T1 (Fig. 6D), but not in cells expressing RIG-I shRNA sequences (Supplementary Fig. S8B). To confirm that these gene-expression changes were seen at the protein level, we assessed cultured media harvested from MCF7 cells 48 hours after transfection with SLR20 by cytokine array analysis. Although this array did not carry IFNβ, we observed increased protein levels of TNFα and TNFβ in the cultured media harvested from SLR20 transfected MCF7 cells (Fig. 6E). Additionally, MCF7 cells transfected with SLR20 harbored increased protein expression of several IFNβ-inducible chemokines known to recruit T lymphocytes, including chemokine (C-C) motif ligand (CCL)-3, CCL5, CCL13, C-X-C motif chemokine ligand 11 (CXCL11), lymphotactin/C-motif ligand (XCL1) and interleukin (IL)-8.

RIG-I signaling induces expression of proinflammatory cytokines from breast cancer cells. AD, Cells were transfected, and after 12 hours, total RNA was assessed by RT-qPCR to measure expression of the indicated genes. Each point represents the average of three experimental replicates, N = 3. Midlines are average ± SD. Student t test. E, Cytokine array assessing cultured media harvested from MCF7 cells 48 hours after transfection. Representative images are shown.


Variables that influence severity of effects from mixing ZzzQuil and Alcohol

There are numerous variables that influence the specific effects, total number of effects, and the severity of effects that occur while under the influence of ZzzQuil and alcohol. These variables include: dosages [of each substance] administration details (e.g. co-ingested vs. latency between ingestions) other substances administered (e.g. drugs, supplements, etc.) degree of tolerance to each substance and individual factors (e.g. age, body composition, medical conditions, sex, etc.). When contemplating the effects and/or reactions that you experienced from the ZzzQuil and alcohol combination, it may be helpful to reflect upon these variables.

Dosages

The significance of physiologic effect resulting from the simultaneous ingestion of ZzzQuil and alcohol is principally mediated by dosing. Individuals who ingest lower respective dosages of ZzzQuil and alcohol will exhibit a less significant shift from homeostatic physiology as compared to persons who ingest high doses. For this reason, it is reasonable to suspect that low dose users will experience fewer severe side effects and/or adverse reactions than high dose users. Put simply, someone ingesting a single dose of ZzzQuil after taking a sip of red wine will experience fewer side effects than someone who ingests several doses of ZzzQuil after a few glasses of rum.

  • Low doses: If a combination of ZzzQuil and alcohol is ingested, yet the dosages of each substance within the combination are low, likelihood of serious adverse reactions and/or interaction effects is minimized – compared to higher doses. At low doses, a less significant burden is placed upon enzymes in the liver and kidneys as compared to higher dosages. This means the body can metabolize and excrete the diphenhydramine [within ZzzQuil] and the alcohol more efficiently than if dosages of each were high. More efficient metabolism and elimination decreases likelihood of hepatic and/or renal toxicity and pharmacokinetic-related interactions. Additionally, the pharmacodynamic effect of each substance is less prominent at low dosages, meaning there’s less neurochemical modulation and corresponding CNS depression. For this reason, most healthy adults who ingest a low dose of the combination (ZzzQuil and alcohol) probably won’t experience adverse reactions.
  • High doses: If high doses of ZzzQuil and alcohol are ingested simultaneously, likelihood of serious adverse reactions is amplified. At high doses, a significant burden is placed upon enzymes within the liver and kidneys during metabolism and excretion. High doses of each substance administered simultaneously may prolong hepatic metabolism, induce hepatotoxicity, and/or induce nephrotoxicity. In other words, there may be some serious pharmacokinetic-related interactions that only occur when high doses of diphenhydramine [within ZzzQuil] and alcohol are ingested together. Furthermore, at high doses, we know that the pharmacodynamic effect of each substance is more prominent. For example, large doses of ZzzQuil yields more significant anticholinergic effects than smaller doses, plus exerts relevant noradrenergic and serotonergic effects – neither of which are noticeable at small doses. Not only will intoxication-related side effects be more numerous and severe from high doses of ZzzQuil and alcohol, but the synergistic CNS depression may be significant enough to induce respiratory depression, respiratory failure, and/or death.

ZzzQuil to Alcohol ratio: Because ZzzQuil and alcohol are distinct psychoactive drugs and everyone responds differently to each based on individual factors, it’s difficult to estimate dosing equivalency in regards to potency of physiologic effect. For this reason, it may be challenging to know whether taking 2 ZzzQuil and drinking 1 beer yields a more potent physiologic effect than drinking 2 beers and taking 1 ZzzQuil. When compared, most would suspect that a single dose of ZzzQuil is of greater potency than 1 beer because, on average, a single dose of ZzzQuil is enough to induce extreme sedation and/or sleep, yet this does not usually occur with 1 beer.

Moreover, the sedative/hypnotic effect of diphenhydramine within ZzzQuil persists for a duration of 6 to 8 hours, which is usually longer than the effect of alcohol. If the hypothesis that a single dose of ZzzQuil exerts a more potent physiologic effect than a single standard alcoholic beverage (e.g. 1 beer) is accurate, we’d expect persons who ingest a greater number of ZzzQuil doses relative to standard alcoholic beverages to be most susceptible to severe side effects from the combination of substances relative to others. If forced to estimate dosing equivalency in terms of potency, one might speculate that a single ZzzQuil dose is roughly as potent as the number of drinks it takes to reach a blood-alcohol content reading of 0.04.

Assuming that a single ZzzQuil dose is equal to a BAC of 0.04 in terms of physiologic potency, and you (personally) require 2 beers to reach a BAC of 0.04, we could suggest that a combination of 5 beers and 1 ZzzQuil yields increased susceptibility to severe side effects as compared to a combination of 2 ZzzQuil and 1 beer. In any regard, the greater the amount of alcohol present within the combination, the more severe the alcohol-related side effects will be. The greater the amount of ZzzQuil present within the combination, the more severe the ZzzQuil-related side effects will be. If large amounts of both substances are present within the combination, the more severe the alcohol-related and diphenhydramine-related side effects will be, as well as interaction effects between alcohol and diphenhydramine.

Note: The aforementioned estimations are hypothetical and should not be considered factual. To accurately gauge the physiologic potency of each substance, you’d need to undergo scientific testing. Even after this testing, it would still be difficult to compare ZzzQuil with alcohol due to differences in pharmacokinetics and pharmacodynamics.

Administration details

The details associated with administration of ZzzQuil and alcohol may also influence the specific effects and/or reactions reported by users of this combination. Examples of administration details to consider include: empty vs. full stomach, ingestion latency, rate of administration, and time of administration. Someone who administered ZzzQuil and alcohol rapidly in the morning on an empty stomach may experience totally different reactions than if he/she administered ZzzQuil and alcohol slowly in the afternoon after a large meal.

  • Empty vs. Full stomach: It is known that the consumption of ZzzQuil and alcohol on an empty stomach leads to faster onset of intoxication as compared to drinking with a full stomach (e.g. after a large meal). The presence of food within the stomach prolongs the rate at which ZzzQuil and alcohol are absorbed, metabolized, and distributed. As a result, individuals who ingest ZzzQuil with alcohol on a full stomach will report less rapid onset of intoxication, but a longer duration of intoxicating effect – as compared to those who ingest the combination while fasted. Food within the stomach prior to taking this combination may also help minimize gastric distress by soaking up stomach acid and/or inflammation. To get technical, the macronutrient / micronutrient composition of foods consumed, the total quantity of food consumed, and the time between your last meal and ZzzQuil plus alcohol ingestion (e.g. 2 minutes, 20 minutes, 3 hours, etc.) may influence the physiologic effects that you endure from this combination. Moreover, the extent to which you are hydrated before ingesting ZzzQuil and alcohol can influence your experience on this combination. Dehydration and/or electrolyte imbalances may provoke and/or exacerbate adverse reactions to ZzzQuil and alcohol.
  • Ingestion latency: Another detail to consider is whether ZzzQuil and alcohol were ingested simultaneously, or whether there was a time gap (i.e. latency) between respective ingestions. Someone who co-administers ZzzQuil along with alcohol might experience a different reaction to this substance combination than if he/she had administered ZzzQuil 20 minutes after drinking a beer (or vice-versa). It is reasonable to suspect that the lesser the ingestion latency, the more likely interactions are to occur. Oppositely, the greater the amount of time between respective administrations of ZzzQuil and alcohol, the lower the likelihood of interactions. When time between administration of multiple substances increases, there’s less overlap in respective pharmacokinetics and pharmacodynamics thereby minimizing odds of adverse effects. This is because the first substance administered will have undergone some degree of metabolism and elimination before the second is administered.
  • Time of administration: The time of day at which you administer ZzzQuil and alcohol may also influence the severity of side effects that you experience and your ability to manage them. This is because concentrations of neurochemicals and hormones shift throughout the day in accordance with a person’s circadian rhythm. Most individuals feel alert in the late morning and/or early afternoon (from production of CNS activators like cortisol) and naturally drowsier in the evening (from production of CNS depressants like melatonin). Knowing that ZzzQuil and alcohol induce CNS depression, and that CNS function is more depressed in the evening for most [in accordance with the circadian biology], it’s possible that the nighttime ingestion of these substances may yield [marginally] increased CNS depression as compared to morning or afternoon administration of an equivalent dose. This may increase risk of respiratory depression and/or failure among those who administer the combination in the evening.
  • Rate of administration: The rate at which ZzzQuil and alcohol are administered should also be considered. Rapid administration (and corresponding ingestion) will often provoke a more significant physiologic reaction and side effects than slower administration. Someone who chugs a beer with a dose of ZzzQuil in 2-minute duration may endure more significant physiologic reactions and side effects than if he/she slowly sipped a beer with a ZzzQuil over 20-minute span. Because ZzzQuil doses are not usually divided, rate of administration may apply mostly to alcohol consumption. Nonetheless, a slower rate of administration may allow for more efficient metabolism and/or elimination as compared to rapidly administering a large dose.
  • Mode of administration: The respective modalities of ZzzQuil and alcohol administration can also influence the specific reactions that occur from co-ingestion. Someone who administers ZzzQuil orally and administers alcohol intravenously may react differently than if he/she administered both substances orally. Because ZzzQuil and alcohol are ingested orally by a majority of the populace, it’s not really worth discussing how each particular mode of administration would influence physiologic effect. Just know that various modes of administration such as: insufflation, subcutaneous, intravenous, rectal, etc. – may alter responses to this combination [as compared to other modes of administration].

Additional substances

In addition to the ingestion of ZzzQuil and alcohol, it is necessary to know whether other substances (e.g. dietary supplements, pharmaceutical drugs, illicit drugs) were administered. The administration of substances in addition to ZzzQuil and alcohol may alter pharmacokinetics and/or pharmacodynamics of the combination. Diphenhydramine within ZzzQuil undergoes first-pass metabolism via CYP2D6, but other enzymes such as CYP1A2, CYP2C9, and CYP2C19 also contribute.

Alcohol is metabolized by alcohol dehydrogenase (ADH), aldehyde dehydrogenase (ALDH), cytochrome P450 (2E1), and catalase. Any additional substance that either inhibits or induces activation of the aforestated enzymes can alter metabolism, potency, and duration of effect resulting from ZzzQuil and alcohol. For example, the drug Fomepizole inhibits alcohol dehydrogenase which would interfere with alcohol metabolism and potentiate its intoxicating effect if administered with this combination.

Other substances may alter metabolism in ways that inhibit the physiologic effect of ZzzQuil and alcohol, thereby decreasing odds of adverse pharmacokinetic-related reactions. Additionally, some substances may not interact with the pharmacokinetics of ZzzQuil and alcohol, but might increase hepatic and/or renal burden, whereby hepatotoxicity and/or nephrotoxicity occur. It’s also possible that administration of substances may augment the pharmacodynamic action and/or cumulative CNS effect of ZzzQuil and/or alcohol for enhanced intoxication.

For example, administration of a benzodiazepine along with ZzzQuil and alcohol may potentiate the GABAergic effect of alcohol and synergistically enhance cumulative CNS depression. On the other hand, administration of a psychostimulant like caffeine with ZzzQuil and alcohol may reverse some degree of CNS depression and corresponding effects of drowsiness and fatigue. Also understand that certain substances ingested along with ZzzQuil and alcohol may have no effect upon physiologic responses to the combination.

Note: The greater the number of substances administered along with ZzzQuil and alcohol, the increased likelihood of interaction effects. Furthermore, if another substance is administered that interacts with ZzzQuil and/or alcohol, the dosing may dictate the severity of that interaction.

Degree of Tolerance

The extent to which you’ve developed physiologic tolerance to diphenhydramine (the chief psychoactive substance within ZzzQuil) and/or alcohol will also influence how you respond to the co-ingestion of ZzzQuil and alcohol. Specifically, the lower a person’s tolerance relative to the dosage of ZzzQuil and alcohol administered, the more substantial the physiologic reaction will be, thereby increasing likelihood of serious adverse reactions. On the other hand, the greater a person’s tolerance relative to the dosage of ZzzQuil and alcohol administered, the less substantial the physiologic reaction will be, thereby minimizing likelihood of deleterious effects.

  • Zero tolerance: First-time users of diphenhydramine and alcohol will have zero tolerance prior to an ingestion of the combination. Due to lack of previous exposure to diphenhydramine and alcohol, a first-time user will not have undergone pharmacokinetic-related adaptations (which allow for more efficient metabolism and excretion of each substance) nor pharmacodynamic-related adaptations (which reduce the significance of psychoactive effect derived from each substance). For this reason, a greater burden will be placed upon the liver and kidneys during absorption, metabolism, and excretion following the ingestion of ZzzQuil and alcohol relative to the dosage among persons without tolerance, likely increasing risk of hepatotoxicity and/or nephrotoxicity. Moreover, because zero neuroadaptation will have occurred from previous exposure, a person’s neurochemistry will be especially sensitive to the psychoactive effect exerted by each substance, ultimately increasing likelihood of severe adverse reactions.
  • Moderate tolerance: Individuals who use diphenhydramine and alcohol on a semi-regular basis will likely have some degree of tolerance prior to ingestion of the combination. Because these persons will have ingested diphenhydramine and alcohol before, physiology will have adapted in a few ways to each drug. Enzymes implicated in metabolism and excretion of each substance will have shifted for greater efficiency such that the body breaks down and eliminates each at a faster rate relative to dosage. Additionally, some neuroadaptation will have occurred such that the user will be less sensitive to the psychoactive effect exerted by each substance, thereby reducing likelihood of an adverse reaction relative to dosage – as compared to someone with zero tolerance.
  • High tolerance: Anyone who uses diphenhydramine and alcohol on a regular or frequent basis probably exhibits a high degree of tolerance to each substance. The physiology of persons with high tolerance allows for highly efficient metabolism plus excretion of diphenhydramine and alcohol – compared to infrequent and/or first-time users. Furthermore, more substantial neuroadaptation will have occurred among persons with high tolerance such that neurotransmitter receptor sites will have significantly upregulated and downregulated to maintain homeostasis, which in turn, minimizes the psychoactive effect of each substance. For this reason, it’s logical to suspect that high tolerance [relative to dose of diphenhydramine and alcohol] should minimize odds of adverse reactions from this substance combination.

It should be noted that some individuals may have a high tolerance to alcohol but not diphenhydramine – or vice-versa. Common sense would suggest that, the larger the dose of the substance ingested to which you have low tolerance, the more significant the effect of that particular substance on your physiology from the combination. In other words, someone with a high tolerance to alcohol and a low tolerance to diphenhydramine will probably experience fewer alcohol-related side effects and/or adverse reactions from the combination of ZzzQuil and alcohol with a greater number of diphenhydramine-related side effects – relative to the dosages.

Also worth emphasizing is that the impact of tolerance on responses to ZzzQuil and alcohol is relative to dose. If relativity of dosing weren’t taken into consideration, one might speculate the high tolerance individuals might be more prone to serious adverse reactions from the combination of ZzzQuil and alcohol – possibly as a result of incurring deleterious long-term physiologic adaptations from frequent ingestion of each substance. Moreover, high tolerance users may be at increased risk of exceeding safe dosing thresholds for ZzzQuil and alcohol, some of which may yield severe adverse reactions and/or prove to be fatal.

Individual factors

Individual-specific factors play a significant role in determining how someone reacts to a specific substance or combination of substances (e.g. ZzzQuil and alcohol). Examples of relevant individual-specific factors include: age, body type, genetics, preexisting medical conditions, and sex. It is these factors that explain why two persons with the same degree of tolerance who identically administer equivalent dosages of ZzzQuil and alcohol may respond differently. For example, one individual may report dizziness, hallucinations and vomiting – whereas the other may report fainting with respiratory depression. If attempting to understand a particular reaction to ZzzQuil and alcohol, it should prove useful to account for the individual factors listed below.

    Age: A person’s age may play a substantial role in determining how the body responds to co-ingestion of ZzzQuil and alcohol. Elderly individuals are more likely to: utilize medications for the treatment of age-related medical conditions (some of which may interact with ZzzQuil or alcohol), exhibit impaired organ function (e.g. brain, liver, kidneys), and exhibit decreased water distribution volume, and– ultimately increasing risk of adverse effects from the ingestion of ZzzQuil plus alcohol. Furthermore, research suggests that the metabolism and elimination of both diphenhydramine and alcohol are less efficient with age. Alcohol is also metabolized at a slower rate among older adults due to age-related decreased activation of acetaldehyde dehydrogenase and CYP2E1 enzymes. Additionally, the elimination half-life of diphenhydramine is

13.5 hours in elderly adults compared to

9.2 hours in young adults and


Estradiol Influences Adenosinergic Signaling and NREM Sleep Need in Adult Female Rats

Studies report estradiol (E2) suppresses sleep in females however, the mechanisms of E2 action remain largely undetermined. Our previous findings suggest that the median preoptic nucleus (MnPO) is a key nexus for E2 action on sleep. Here, using behavioral, neurochemical and pharmacological approaches, we investigated whether E2 influenced the sleep homeostat as well as adenosinergic signaling in the MnPO of adult female rats. During the Light Phase, where rats accumulate the majority of sleep, E2 markedly reduced NREM-SWA (a measure of the homeostatic sleep need). Following 6-hours of sleep deprivation, levels of NREM-SWA were significantly increased compared to baseline sleep. However, the NREM-SWA levels were not different between E2 and control treatment despite a significant increase in wake at the expense of NREM sleep. Analysis of NREM-SWA differences between baseline and recovery sleep following sleep deprivation demonstrated that E2 induced a 2-fold increase in delta power compared to controls suggesting that E2 significantly expanded the dynamic range for the sleep homeostat. Correlated with E2-induced changes in physiological markers of homeostatic sleep was a marked increase in extracellular adenosine (a molecular marker of homeostatic sleep need) during unrestricted and recovery sleep following a 6-hour deprivation. Additionally, E2 blocked the ability of an adenosine A2A receptor agonist (CGS-21680) to increase NREM sleep compared to controls. Thus, taken together, the findings that E2 increased extracellular adenosine content, while blocking A2A signaling in the MnPO suggests a potential mechanism for how estrogens impact sleep in the female brain.

Statement of Significance While gonadal steroids and gender are implicated as risk factors for sleep disruptions and insomnia, the relationship between ovarian steroids and sleep is poorly understood. Understanding the mechanisms through which estradiol (E2) is working to influence sleep-wake behavior is a critical first step toward a better understanding of the role of estrogens in sleep pathologies. Using a rodent model, the current study presents novel findings suggesting that estradiol (E2) is influencing adenosinergic actions in the MnPO. The ability of E2 to attenuate the local effects of the A2A receptors in the MnPO suggests that E2 modulation of A2A receptor signaling may underlie estrogenic suppression of sleep behavior as well as changes in homeostatic sleep need.


Watch the video: HYUNDAI H1 2015 (June 2022).