SLEEP

A. Basic and Translational Sleep and Circadian Science I. Mechanisms of Sleep and Circadian Disorders0001Introduction: Narcolepsy is a disorder characterized byhypersomnolence, cataplexy, sleep paralysis, hallucinations andsleep fragmentation. Patients with type 1 narcolepsy have cataplexyand/or hypocretin-1 deficiency. Klotho is a protein expressed bykidneys and choroid plexus, with anti-aging properties. Fibroblastgrowth factor 23 (FGF-23) is a hormone secreted by osteocyteswith actions on mineral metabolism. The purpose of study was toexplore the status of concentration of klotho and FGF23 in thecerebrospinal fluids (CSF) of patients with narcolepsy.Methods: 59 patients with narcolepsy and 17 individuals wereenrolled. We used a radioimmunoassay technique, human klothoenzyme-linked immunosorbent assay (ELISA), human intactFGF23 ELISA and spectrophotometry to measure hypocretin-1,klotho, FGF-23 and phosphorus, respectively. T-Student Testwas used to compare klotho and phosphate concentrations andMann-Whitney U Test was used to compare FGF-23 levels between groups. ANOVA Test was used to compare klotho and phosphate CSF concentrations among narcolepsy patients with CSFhypocretin-1 110pg/ml (HCRT-) and narcolepsy patients withCSF hypocretin-1 110pg/ml (HCRT ) versus control subjects.Results: Klotho and phosphorus CSF levels were lower in narcoleptic patients than in control (908.18 405.51 versus 1265.78 523.26 pg/ml; p 0.004 and 1.34 0.25 versus 1.58 0.23 mg/dl;p 0.001, respectively). We found higher median FGF-23 levels innarcoleptic patients (5.51 versus 4.00 RU/ml; p 0.001). Klothoand phosphorus CSF levels were lower in both HCRT-/HCRT than controls (892.63 388.34/ 925.95 430.76 versus 1265.78 523.26 pg/ml; p 0.014 and 1.35 0.28/ 1.33 0.22 versus 1.58 0.23 mg/dl; p 0.004). Moreover, we found higher median FGF-23levels in both HCRT-/HCRT groups versus controls (5.51/ 6.02versus 4.00 RU/ml in controls), p 0.009.Conclusion: Patients with narcolepsy have decreased CSF concentration of klotho and increased CSF levels of FGF-23. Thesefindings may play a role in understanding the pathogenesis ofnarcolepsy.Support: .ACTIVATION OF NOCICEPTIN/ORPHANIN-FQ PEPTIDE(NOP) RECEPTORS PRODUCES AN INCREASE IN NONREM SLEEP IN RATS AND CONSTITUTES A NOVELAND ATTRACTIVE TARGET FOR THE TREATMENT OFINSOMNIAWhiteside, G. T.1 Hummel, M.2 Knappenberger, T.2 Hiroyama, S.3Itoh, T.3 Takai, N.3 Kyle, D. J.21Imbrium Therapeutics, Stamford, CT, 2Purdue Pharma L.P.,Stamford, CT, 3Shionogi & Co., Ltd., Osaka, JAPAN.Introduction: Treatments for insomnia have targeted GABA, histamine, serotonin, melatonin and orexin receptors. The nociceptin/orphanin-FQ peptide (NOP) receptor is widely expressed in the nervous system. High doses of NOP agonists administered systemicallyor locally into the CNS can result in sedation, however, the utility oftargeting this receptor to treat insomnia has not been fully described.Methods: V117957 is a recently described investigational oral, potent and selective NOP receptor partial agonist. We determinedthe brain Kp in whole brain and multiple sub-regions (50mg/kg)and receptor occupancy in the hypothalamus (30, 300mg/kg) viain vivo displacement using [3H]-NOP-1A. EEG/EMG were determined in rats chronically implanted with electrodes (cortex anddorsal neck muscle) and recorded via telemetry following dosing(3, 30, 300mg/kg); sleep stage was determined from visual analysisof EEG level. Sleep parameters were also assessed in NOP receptorknock-out rats (300mg/kg). The side-effect profile for V117957was determined by functional observation battery, whole-bodyplethysmography, Morris water maze (MWM) (up to 600mg/kg)and conditioned place preference (CPP) assay (up to 300mg/kg).Results: V117957 displayed limited distribution into the CNS butachieved a high level of receptor occupancy (75% at 30mg/kg).Administration of V117957 produced dose-dependent and statisticallysignificant increases in non-REM sleep with a minimally efficaciousdose of 30mg/kg; a coincident dose-dependent and statistically significant decrease in wakefulness and a non-dose-dependent effect on REMsleep occurred. These changes were not seen in knock-out animalsdemonstrating effects are via NOP receptors. At doses higher than thosethat increased non-REM sleep, V117957 had no effects in a functionalobservational battery, did not affect escape latency in MWM or produceCPP; additionally, V117957 did not affect respiratory parameters.Conclusion: We conclude that activation of NOP receptors decreases wakefulness and increases non-REM sleep in rats with animproved preclinical profile compared to historical profiles of current treatments and, therefore, may represent a novel and attractivetarget for the treatment of insomnia.Support: Funded by Shionogi and Imbrium Therapeutics, a subsidiary of Purdue Pharma L.P.0003LGI1 AND CASPR2 AUTOIMMUNITY: SLEEP SYMPTOMS,POLYSOMNOGRAPHY, AND QUANTITATIVE REMSLEEP WITHOUT ATONIADevine, M. F. Feemster, J. C. Lieske, E. A. McCarter, S. J.Sandness, D. J. Steele, T. Boeve, B. F. Silber, M. H. McKeon, A.St. Louis, E. K.Mayo Clinic, Rochester, MN.Introduction: Sleep disturbances, including rapid eye movement(REM) behavior disorder (RBD), are known manifestations ofvoltage-gated-potassium-channel-complex VGKC-IgG seropositivity (VGKC ). Discovery of leucine-rich, glioma inactivatedprotein 1 (LGI1) and contactin-associated protein 2 (CASPR2)have refined our understanding of VGKC . VGKC withoutLGI1/CASPR2-IgG (“double-negative”) has lost its clinical significance. Previous detailed sleep analysis of these subtypes hasbeen limited.Methods: We performed a retrospective study to characterizeclinical and polysomnographic features of LGI1/CASPR2 seropositive (LGI1 /CASPR2 ) and VGKC double-negative patients, including quantitative REM sleep without atonia (RSWA).Quantified RSWA was compared to matched controls and normative RSWA percentiles.0002DECREASED CONCENTRATION OF KLOTHOAND INCREASED CONCENTRATION OF FGF-23 INTHE CEREBROSPINAL FLUID OF PATIENTS WITHNARCOLEPSYOliveira, G. P.1,2 Elias, R. M.3 Fernandes, G. B.1 Moyses, R.3Tufik, S.1 Bichuetti, D. B.1 Coelho, F. M.11Universidade Federal de São Paulo, São Paulo, BRAZIL,2Universidade Federal do Piauí, Teresina, BRAZIL,3Universidade de São Paulo, São Paulo, BRAZIL, 4UniversidadeFederal de São Paulo, São Paulo, BRAZIL.A1SLEEP, Volume 43, Abstract Supplement, 2020

A. Basic and Translational Sleep and Circadian Science I. Mechanisms of Sleep and Circadian DisordersResults: Eleven LGI1 /CASPR2 (LGI1 , 9) and twelve VGKCdouble-negative patients were analyzed. Insomnia was seen in55% of LGI1 /CASPR2 and 8% of VGKC double-negative patients (p 0.05). The LGI1 /CASPR2 group had reduced slowwave sleep compared to the VGKC double-negative group. FiveLGI1 patients had clinical dream enactment behavior (DEB).Eight LGI1 patients met quantitative diagnostic levels of RSWA.Higher RSWA levels were seen in the LGI1 /CASPR2 group.Ten LGI1 /CASPR2 patients received immunotherapy; all tenneurologically benefited with sleep benefits in 6/10.Conclusion: Sleep disorders such as insomnia and RBD are part ofthe LGI1/CASPR2 autoimmune phenotype. Objective sleep manifestations can be seen on polysomnogram in the form of reducedN3 and elevated RSWA as compared to controls. QuantitativeRSWA analysis identified RBD in more LGI1 patients than clinical report or qualitative RSWA. In this study, RBD was only seenwith LGI1 , not CASPR2 . The intermediate RSWA levels ofthe VGKC double-negative patients may suggest a spectrum ofabnormal motor activity in these related antibodies. Additionalstudies are needed to further explore the biomarker potential ofquantitative RSWA in autoimmune neurological conditions.Support: This project was supported by the National CenterforResearch Resources, National Institutes of Health, throughGrant Number 1 UL1 RR024150- 01.insomnia has been completed, which is presented in a separateposter.Support: Taisho Pharmaceutical. Co., Ltd.0005CATAPLEXY TRIGGERED BY SOCIAL CUES: A ROLEFOR OXYTOCIN IN THE AMYGDALAMahoney, C. E.1 Zhao, W.1 Coffey, A.1 Woods, C.1 Kroeger, D.1Scammell, T.11BIDMC/Harvard Medical School, Boston, MA, 2BIDMC/Harvard Medical School, Boston, MA.Introduction: People with narcolepsy type 1 report that cataplexy istriggered most often by positive social experiences such as laughingwith friends, yet the mechanisms through which social interactionpromotes cataplexy are unknown. We hypothesize a subpopulationof central amygdala neurons that are sensitive to the prosocialneuropeptide, oxytocin (CeAOTR), respond to positive valence andtrigger cataplexy.Methods: We have used in vivo calcium imaging, chemogeneticand optogenetic approaches to characterize the activity pattern ofthese neurons and to manipulate their activity state.Results: Cre-dependent anterograde tracing of the CeAOTRneurons of the central amygdala indicate a moderate to dense projection to the REM sleep-regulatory region of the ventral lateralperiaqueductal gray (vlPAG). Additionally, Channel RhodopsinAssisted Circuit Mapping (CRACM) experiments show thatCeAOTR neurons inhibit vlPAG neurons that innervate the REMatonia-promoting region, the sublaterodorsal nucleus. Targetedphotostimulation (15Hz (10ms) for 20sec every hour) of the CeAOTRfibers in the vlPAG doubled the amount of cataplexy. Preliminaryin vivo calcium imaging indicates that the CeAOTR are active justprior to the onset of cataplexy. Chemogenetic and optogenetic activation of CeAOTR neurons increased cataplexy.Conclusion: We conclude that the CeAOTR subpopulation issufficient to promote cataplexy. Our future directions includedetermining the necessity of these oxytocin sensitive neurons incataplexy under different conditions of positive valence.Support: R01 NS106032 and WakeUp Narcolepsy.0004TS-142: A NOVEL AND POTENT DUAL OREXINRECEPTOR ANTAGONIST WITH SLEEP-PROMOTINGEFFECTS IN RATSKambe, D. Hikichi, H. Tokumaru, Y. Ohmichi, M. Konno, Y.Hino, N.Taisho Pharmaceutical Co., LTD., Tokyo, JAPAN.Introduction: The orexin system plays a pivotal role in regulatingsleep and wakefulness, thus, orexin receptors (OX1 and OX2 receptors) have gained much attention as promising therapeutic targetsfor the treatment of insomnia. We synthesized a novel and potentdual orexin receptor antagonist (DORA), ORN0829 (investigationcode name as TS-142), which was designed to have short-actingeffects. Here we report pharmacological and pharmacokinetic profiles of ORN0829 in rats.Methods: The antagonistic activities of ORN0829 were assessedusing calcium mobilization assays. Ala-orexin A-induced [Ca2 ]i response was measured with CHO-K1 cells stably expressinghuman/rat orexin receptor. Rats implanted the EEG/EMG electrodes were orally administrated ORN0829 at doses of 1, 3 or10 mg/kg at the dark onset and sleep-wake stages were inspectedvisually. In addition, pharmacokinetic profiles of ORN0829 wereinvestigated in rats.Results: ORN0829 inhibited Ala-orexin A-increased [Ca2 ]i response with a Kb of 0.67/0.44 nmol/L (for human/rat OX1 receptor), and with a Kb of 0.84/0.80 nmol/L (for human/rat OX2receptor), respectively, indicating that ORN0829 is a potentDORA with no species differences. ORN0829 dose-dependentlyincreased total sleep time and reduced sleep onset latency at dosesof 1, 3 and 10 mg/kg. Importantly, the ORN0829 levels in plasmaand cerebrospinal fluid rapidly reached a maximum concentration,and decreased with an elimination half-life of less than 1 h.Conclusion: The present study indicates that ORN0829 is a noveland potent DORA with sleep-promoting effects, and that it exhibits ideal pharmacokinetic profiles (rapid absorption and shorthalf-life) in rats. A phase 2a study of TS-142 using patients withSLEEP, Volume 43, Abstract Supplement, 20200006THE ROLE OF STRESS IN SLEEP IN NIGHTSHIFT WORKERS: GOING BEYOND CIRCADIANMISALIGNMENTSchaap, E. Sagong, C. Cuamatzi Castelan, A. S. Sayed, J. Roth, T.Drake, C. L. Cheng, P.Henry Ford Health System, Detroit, MI.Introduction: Despite a growing need for nighttime work, fewstudies have characterized the causes of sleep disturbance in nightshift workers beyond circadian misalignment. Recent research suggest that high sleep reactivity to stress (a predisposition for sleepdisturbance due to stress) may also lead to sleep difficulties inshift workers. This study investigated if sleep reactivity is an independent predictor of daytime sleep disturbances after controllingfor circadian phase.Methods: Night shift workers (N 48) completed an 8 hourpolysomnography (PSG) during the daytime following a nightshift (9am - 4pm). Circadian phase was measured using melatoninassays of saliva samples collected over 24 hours under dim light( 10 lux; Dim Light Melatonin Onset [DLMO]). Sleep reactivityA2

A. Basic and Translational Sleep and Circadian Science I. Mechanisms of Sleep and Circadian Disordersshifts required (e.g., with light therapy) for the desired improvement in daytime sleep.Support: Support for this study was provided to PC by the NHLBI(K23HL138166)was measured using the Ford Insomnia Response to Stress Test(FIRST). Linear regressions were conducted with PSG sleepparameters as outcome variables: difficulty falling asleep (SleepOnset Latency [SOL] and Latency to Persistent Sleep [LPS]), difficulty staying asleep (Wake After Sleep Onset [WASO]), and sleepduration (Total Sleep Time [TST]). FIRST was tested as a predictor controlling for DLMO.Results: After controlling for circadian phase, higher FIRSTscores was associated with more difficulty staying asleep(WASO: t[45] 4.059, p 0.001) and shorter sleep duration (TST:t[45] -4.403, p 0.0001), but not predictive of difficulty fallingasleep (SOL: p 0.05). However, higher FIRST scores did predict alonger latency to persistent sleep (LPS: t[45] 2.272, p 0.05).Conclusion: These results suggest that sleep reactivity to stress andcircadian misalignment are independent processes that are both associated with disrupted daytime sleep in night shift workers. Giventhat night shift work can also cause psychosocial stress, treatmentsfocused on circadian misalignment alone may not be sufficient.Our study highlights the need to consider sleep reactivity in theclinical management of shift work disorder.Support: Support for this study was provided to PC by NHLBI(K23HL138166).0008SUVN-G3031, A HISTAMINE H3 RECEPTOR INVERSEAGONIST PRODUCES ROBUST WAKE PROMOTING ANDANTICATAPLECTIC ACTIVITY IN OREXIN KNOCKOUTMICEBenade, V. Daripelli, S. Petlu, S. Subramanian, R. Bhyrapuneni, G.Shinde, A. Rasheed, M. Jayarajan, P. Choudakari, P. Nirogi, R.Suven Life Sciences, Hyderabad, INDIA.Introduction: Narcolepsy is a sleep disorder characterized by excessive daytime sleepiness, sleep paralysis, hallucinations, and insome cases episodes of cataplexy. Results from animal studies indicate the involvement of deficient orexin transmission in narcolepsy which can be circumvented by the activation of histaminergicneurons. SUVN-G3031 is a potent and selective histamine H3 receptor inverse agonist with hKi of 8.7 nM and shows less than 50%inhibition at 1 µM against 70 other targets. SUVN-G3031 exhibited excellent pharmacokinetic properties and brain penetration inpreclinical species. Oral administration of SUVN-G3031 producessignificant increase in histamine, dopamine and norepinephrinelevels in the rat cortex. Long-term safety studies in animals havebeen successfully completed without any concern for further development of SUVN-G3031. In the present study, the effects ofSUVN-G3031 were evaluated in orexin knockout mice, a reliableanimal model of narcolepsy as a proof-of-concept study for thetreatment of narcolepsy with and without cataplexy.Methods: Male orexin knockout mice (10 - 15 weeks old, 25 - 35 gat the time of surgery) were implanted with telemetric device forsimultaneous monitoring of electroencephalography (EEG) andelectromyography. Animals were allowed surgical recovery of 3weeks prior to EEG recording. Effects of SUVN-G3031 (3 and10 mg/kg, p.o.) were evaluated during active period of animals.Results: SUVN-G3031 produced significant increase in wakefulness with concomitant decrease in non-rapid eye movement sleepin orexin knockout mice. SUVN-G3031 also significantly decreasedthe number of cataplectic episodes in orexin knockout mice.Conclusion: Results from the current preclinical study provide astrong basis for the utility of SUVN-G3031 for the treatment ofnarcolepsy with and without cataplexy. SUVN-G3031 is currentlybeing evaluated in a Phase 2 study as monotherapy for the treatment of narcolepsy with and without cataplexy (ClinicalTrials.govIdentifier: NCT04072380).Support: None0007DAYTIME SLEEP IN NIGHT SHIFT WORKERS:QUANTIFYING THE ROLE OF CIRCADIANMISALIGNMENTMann, E. Sagong, C. Cuamatzi Castelan, A. Singh, M. Roth, T.Drake, C. L. Cheng, P.Henry Ford Health System, Detroit, MI.Introduction: Circadian misalignment is commonly cited as a culprit of daytime sleep disturbances in night shift workers; however,the specific impact and magnitude that circadian misalignment hason daytime sleep has not been well-characterized in larger samplesof night shift workers.Methods: Participants included fixed-night shift workers (n 52,ages 18–50) who completed an 8-hour daytime polysomnography(PSG) in the lab following a night shift. Measures of sleep disturbances included: difficulty falling asleep (sleep onset latency [SOL],latency to persistent sleep [LPS]), difficulty staying asleep (sleep efficiency [SE], wake after sleep onset [WASO]), and sleep duration(total sleep time [TST]). Melatonin samples were collected hourlyfor 24 hours under dim light ( 10 lux) and used to determinedim light melatonin offset (DLMOff). Circadian misalignment(CM) was calculated as the time difference between bedtime andDLMOff (higher values represented sleeping after DLMOff), andcorrelated with PSG sleep variables.Results: CM was significantly associated with difficulty stayingasleep (WASO: r 0.48, p 0.001; SE: r -0.45, p 0.001), and sleepduration (TST: r -0.38, p 0.01). Specifically, every 3 hours of CMon average added 19.2 minutes of WASO and reduced TST by 15minutes. In contrast, CM was not significantly correlated withsleep onset difficulties (SOL: r -0.27; LPS: r -0.02).Conclusion: These data suggest that circadian misalignment inshift workers may be a better predictor of difficulties staying asleepand sleep duration during the day relative to difficulties fallingasleep. Because longer work hours (10–12 hours) are common innight shift worker, it may be that sleep initiation difficulties associated with circadian misalignment is masked by elevated fatigue oran increased homeostatic drive from prolonged wakefulness. Theseresults may help guide decisions about the magnitude of phase0009ANTI-STREPTOCOCCAL ANTIBODIES IN CHINESEPATIENTS WITH TYPE -1 NARCOLEPSYDing, Q. Li, J. Xiao, F. Zhang, C. Dong, X. Han, F.Peking University People’s hospital, Beijing, CHINA.Introduction: Narcolepsy type 1 (NT1) is considered to be an autoimmune disease, and streptococcal infection may be an environmental trigger. However, previous studies from Asian narcolepsypatients did not reveal elevated anti-streptolysin O [ASO]. The aimis to investigate whether large sample Chinese patients with NT1have an increase in antistreptococcal antibody titers.A3SLEEP, Volume 43, Abstract Supplement, 2020

A. Basic and Translational Sleep and Circadian Science I. Mechanisms of Sleep and Circadian DisordersMethods: A total of 214 narcolepsy patients and 360 healthy controls were recruited. All patients were DQB1*0602 positive withclear-cut cataplexy or had low CSF hypocretin-1. Participants weretested for ASO and anti DNAse B [ADB]. These patients were divided into five groups according to disease duration, including 29patients less than 3 months; 25 from 3 months to 1 year; 40 from1 to 3 years; 61 from 3 to 10 years and 59 patients over 10 years.Comparison was also made between children and adults with agematched controls, respectively.Results: There were no significant differences between patients andhealthy controls in regard to both ASO 200 IU (19.2% vs. 16.9%,p 0.50) and ADB 480IU (9.8% vs. 10.3%, p 0.86). For childrennarcolepsy patients, ASO positive rates(19.8% vs. 18%, p 0.68)and ADB positive rates(10.4% vs. 12%, p 0.72) had no differences compared to age matched controls. And no difference wasobserved in adult narcolepsy patients either, with ASO positiverates (18.5% vs. 13.8%, p 0.39) and ADB positive rates (9.3% vs.5.3%, p 0.42) compared to age matched controls, respectively.ASO (ADB) positive rates had no significant differences amongdifferent disease duration groups(p 0.55, 0.9).Conclusion: It is indicated that positive rates of ASO and ADBwere not significantly different between Chinese patients with NT1and healthy controls, including recent onset cases and children.Support: The study was supported by the National Natural ScienceFoundation of China (No. 81420108002 and NO. 81570083)frontal regions were significantly increased in patients on dopaminergic medication compared to untreated patients and healthycontrols (P 0.05).Conclusion: Networks with higher intra-network connectivity (i.e.salience, executive, somatomotor, cerebellar) and lower between regions connectivity (i.e. cerebello-frontal, cerebello-parietal) in restless legs syndrome correspond to regions associated with attention,response inhibitory control, and processing of sensory information. Dopaminergic medication normalizes the altered cerebelloparietal communication and increases thalamic connectivity to theprefrontal cortex suggesting that these regions are associated withthe emergence of symptoms in restless legs syndrome.Support: The study was funded by a Grant from TranslationalResearchFund of the government of Tyrol, Austria, and in-kind resources ofthe Medical University of Innsbruck.0011T

Miranda M. Lim, MD, PhD Peter Y. Liu, MBBS, PhD Steven W. Lockley, PhD Faith Luyster, PhD Mark Mahowald, MD Bryce A. Mander, PhD Janna Mantua, PhD George Mashour, MD, PhD . Safwan Badr, MD Michael Grandner, PhD. Table of Contents A