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Abstract Context Prior reports of plasma estradiol concentrations and their reference ranges in postmenopausal women identified marked variation, in part due to assay method and body mass index (BMI). Objective To develop an international reference range for plasma estradiol in postmenopausal women, support accurate assessment of concentrations during systemic and local (eg, vaginal) estradiol therapy, and improve risk prediction for diseases, including osteoporosis, breast, and endometrial cancers. Methodology Estradiol concentrations were measured using primarily liquid chromatography-tandem mass spectrometry (LC-MS/MS) assays in 5 international cohorts of community-dwelling, postmenopausal women of primarily European ancestry aged 38-100 years who were not using exogenous estrogens (n = 7206). Measurements were harmonized to the Centers for Disease Control and Prevention (CDC) reference measurement procedure (RMP) by re-assaying a subset of samples for each cohort and generating recalibration equations. Reference intervals were determined for all women, non-obese women, and specific BMI categories. Results Estradiol concentrations from separate LC-MS/MS assays correlated closely with the CDC RMP, and harmonization minimized inter-assay variability. Estradiol concentrations correlated with BMI but not with chronological age. The reference range (2.5th-97.5th percentile) was 1.1-18.2 pg/mL (4.0-66.8 pmol/L) with median of 4.9 pg/mL (18.0 pmol/L) for all postmenopausal women and 1.1-12.5 pg/mL (4.0-45.9 pmol/L) with median of 4.1 pg/mL (15.1 pmol/L) for non-obese women (BMI < 30). Reference ranges increased progressively with higher BMI categories. Conclusion Cross-calibration of estradiol measurements with the CDC RP enabled the development of harmonized plasma estradiol reference ranges for postmenopausal women who were not using exogenous estrogens, suitable for future studies to establish clinical utility. Increasing obesity in postmenopausal women was associated with increasing estradiol concentration and BMI category-specific reference ranges.

Abstract Introduction Narcolepsy type 2 (NT2) is characterized by excessive daytime sleepiness, heterogeneous symptom severity, and typically normal endogenous orexin levels. The Vibrance-2 phase 2 study evaluated alixorexton, a selective orexin 2 receptor agonist, in patients with NT2. Methods Vibrance-2 was a placebo-controlled, dose-ranging phase 2 study. After a 2-week washout from current narcolepsy medications, adults with NT2 meeting ICSD-3-TR diagnostic criteria were randomized 1:1:1:1 to receive placebo or alixorexton once daily at doses of 10, 14, or 18mg for 8 weeks, followed by an optional 5-week open-label extension. Dual primary endpoints were the change from baseline to week 8 in mean sleep latency (MSL) on the Maintenance of Wakefulness Test (MWT) and on the Epworth Sleepiness Scale (ESS). Safety evaluation included treatment-emergent adverse events (TEAEs). P-values were adjusted for multiplicity. Results Ninety-three patients were randomized to placebo (n=24) or alixorexton (10mg, n=23; 14mg, n=22; 18mg, n=24); 90 completed double-blind treatment. At baseline, patients had severe symptoms (MWT: 5.6 minutes; ESS: 17.4). The study met its dual primary endpoints. At week 8, MSL on MWT increased 9.3, 6.7, and 6.7 minutes at 10, 14, and 18mg, respectively, from baseline versus placebo, with statistically significant differences observed for the 14 and 18mg doses (adjusted p< 0.05). ESS improved by -2.1, -3.2, and -3.6 points from baseline versus placebo at week 8 for the 10, 14, and 18mg groups, respectively, with statistical significance observed for the 18mg dose (adjusted p< 0.05), where more than 70% of patients achieved normalization. TEAEs in 10% of all alixorexton-treated patients through week 8 were pollakiuria, insomnia, micturition urgency, dizziness, and headache. Most TEAEs were mild-to-moderate with no serious TEAEs. Conclusion Vibrance-2 was the first large clinical trial in patients with NT2 to demonstrate positive results with an orexin 2 receptor agonist. Alixorexton resulted in statistically significant and clinically meaningful improvements in wakefulness and daytime sleepiness at Week 8 and was generally well tolerated. These results support phase 3 clinical development of alixorexton as a treatment for NT2. Support (if any) Alkermes, Inc.

Abstract Introduction Jazz DUET (Develop hypersomnia Understanding by Evaluating low-sodium oxybate Treatment; NCT05875974) was the first study to evaluate effectiveness/safety of low-sodium oxybate (LXB; Xywav®) >9 g/night (outside the recommended range: 6–9 g/night). This analysis evaluated changes in patient-reported hypersomnolence measures in participants with narcolepsy taking LXB >9 g/night compared with LXB 9 g/night. Methods DUET was a phase 4, prospective, open-label study with screening, 8-day LXB 9 g/night (9g-baseline), titration/optimization, stable-dose, and 8-day end-of-treatment (EOT) optimized LXB >9 g/night (up to 12g; >9g-EOT) periods. Patient-reported measures included Epworth Sleepiness Scale (ESS), electronic sleep diaries (completed daily during 9g-baseline and >9g-EOT; subjective total sleep time [TST], number of awakenings, sleep efficiency [TST divided by time in bed], sleep quality [5-point scale; “very good” to “very poor”], and how rested/refreshed the participant felt upon awakening [5-point scale; “very well” to “not at all”]), Karolinska Sleepiness Scale (KSS; administered 90 minutes post-polysomnography awakening at 9g-baseline and >9g-EOT), and Functional Outcomes of Sleep Questionnaire-10 (FOSQ-10). LSM differences (95% CI) from 9g-baseline to >9g-EOT (nominal P-values) were 9g-baseline-adjusted. Results Forty-eight participants took LXB >9g (mean±SD age: 39.2±10.9 years; 62.5% female). Total stable-dose LXB (mean±SD) was 11.1±1.0 g/night (n=45). From 9g-baseline to >9g-EOT, ESS scores improved (LSM [95% CI]: –3.1 [–4.2, –1.9]; P< 0.0001; n=44), nocturnal TST increased (0.3h [0.1, 0.4]; P=0.0163; n=26), sleep efficiency increased (3.8% [1.7, 5.9]; P=0.0011; n=26), and number of awakenings remained stable (–0.2 [–0.7, 0.3]; P=0.3959; n=26). Percentage of participants rating sleep quality as “very good”/”good” increased from 26.9% (9g-baseline) to 61.5% (>9g-EOT), as did rested sleep (“very well”/”well”/”somewhat” rested) from 57.7% to 69.2%, respectively. From 9g-baseline to >9g-EOT, KSS scores decreased (–0.9 [–1.4, –0.4]; P=0.0004; n=39); FOSQ-10 scores also increased (1.3 [0.5, 2.0]; P=0.0014; n=40). Of 48 participants, 36 (75.0%) experienced ≥1 TEAE (mostly mild/moderate and 1 serious [syncope, unrelated to treatment]). No changes in respiratory polysomnography measures were observed. Conclusion Participants with narcolepsy who optimized to LXB >9 g/night, relative to 9 g/night, reported increased sleep quality and restfulness that paralleled improvements in daytime sleepiness and daytime functioning. TEAEs were consistent with those at lower dosages. Support (if any) Jazz Pharmaceuticals

Abstract Introduction AXS-12 (reboxetine) is a highly selective norepinephrine reuptake inhibitor and cortical dopamine modulator under development for the treatment of narcolepsy. Long-term efficacy and safety of AXS-12 in participants with narcolepsy with cataplexy (NT1) were previously reported from the Phase 3 ENCORE trial. To better understand the impact of AXS-12 on overall functional burden in NT1, we evaluated changes in cognition and functional outcomes during the ENCORE trial. Methods ENCORE included a 24-week open-label extension (OLE) period, followed by a 3-week double-blind randomized-withdrawal (DBRW) period. During the OLE, participants received AXS-12 (5 mg once-daily for 1 week, twice-daily for 23 weeks). Following this, participants were randomized 1:1 to continue twice-daily AXS-12 for 3 weeks or switch to once-daily AXS-12 plus placebo for 1 week, then placebo twice-daily for 2 weeks. P-values reported are nominal. Results ENCORE enrolled 68 participants; 42 completed the OLE and entered the DBRW (AXS-12, n=22; placebo, n=20). In the OLE, participants treated with AXS-12 reported improvement in the NSAQ Ability to Concentrate item at 1 (54.8%) and 6 (58.5%) months. Participants reported improvement in concentration, assessed by PGI-C, at 1 (66.7%) and 6 (70.0%) months. Narcolepsy overall, assessed by the CGI-C, improved at 1 (90.3%) and 6 (90.2%) months; and improved at 1 (77.8%) and 6 (80.0%) months, assessed by the PGI-C. Functional impairment, assessed by the WPAI, was reduced from 53.0% at baseline to 34.0% at 1 month and 24.3% at 6 months. In the DBRW, a greater proportion of participants switched to placebo experienced worsening on the NSAQ Ability to Concentrate item (52.6% vs 14.3%; P=0.011), and PGI-C for ability to concentrate (57.9% vs 22.2%; P=0.029) at 3 weeks compared with those continuing AXS-12. A greater proportion switched to placebo also reported worsening of their narcolepsy overall (PGI-C) at 3 weeks compared with those continuing AXS-12 (52.6% vs 16.7%; P=0.024). Treatment with AXS-12 was generally well tolerated. Conclusion Long-term, open-label treatment with AXS-12 was associated with improvements in cognitive/functional domains and productivity. Randomized withdrawal to placebo led to worsening cognition and narcolepsy overall. These results further support the long-term therapeutic benefit of AXS-12 for narcolepsy. Support (if any) Axsome Therapeutics Inc.

Abstract Human genomic studies link reduced CUB domain-containing protein 1 (CDCP1) expression with myocardial recovery in heart failure. While CDCP1 regulates cardiac fibroblast proliferation in vitro, it’s in vivo role in cardiac fibrosis remains unclear. Using a Cdcp1 -knockout (KO) angiotensin II/phenylephrine mouse model, we show that Cdcp1 deletion reduces echocardiographic left ventricular mass, histologic cardiac fibrosis, and pro-fibrotic gene expression, along with decreased fibroblast activation and inflammatory markers. Spatial transcriptomics identified a pressure overload–expanded fibroblast subpopulation enriched for growth factor and TGF-β signaling (FB5), which was markedly attenuated in Cdcp1 -KO hearts, alongside reduction of a pro-inflammatory cardiomyocyte subtype (CM4). Complementary studies in human ventricular fibroblasts demonstrate that CDCP1 knockdown reduced extracellular matrix gene expression and collagen I deposition. These findings establish CDCP1 as a regulator of cardiac fibrotic remodeling in vivo and open avenues for its further investigation as a potential therapeutic target.

Abstract Prostate adenocarcinomas (PRAD) can acquire resistance to androgen receptor signaling inhibitors through lineage transition to a cell state known as neuroendocrine prostate cancer (NEPC). Using a panel of isogenic PRAD and NEPC mouse tumoroids, we show that NEPC cells acquire new transcription factor (TF) dependencies that function in a previously undefined network. Through selective perturbation of each TF, we identify ASCL1 as a key regulator of NE lineage fate whereas MYCL functions downstream to drive NEPC growth/survival by recruitment of the TIP60/KAT5 acetyltransferase. Interestingly, while dependencies on specific TF family paralogs can vary across NEPC models, all show markedly enhanced dependency on TIP60. Moreover, the H2A.Z-acetyltransferase activity of the TIP60 complex (TIP60-C) is required for NEPC as well as the acetyl-reader BRD8, which is newly incorporated as a TIP60-C subunit with the NEPC transition. Targeted degradation studies in isogenic tumoroids reveal increased dependence on MYCL in NEPC relative to its paralog MYC in PRAD. In addition to a paralog switch (MYC to MYCL), the MYC pathway-addicted NE state is accompanied by a chaperone switch (from TIP60-C to SRCAP) for H2A.Z histone exchange and a coactivator switch (to TIP60) for MYC target gene expression. The NE-specific coupling of MYCL with TIP60 reveals a previously unappreciated opportunity to target MYC-driven NE diseases through pharmacological inhibition of TIP60.

Abstract This work presents a first-of-its-kind artificial intelligence (AI-)integrated MR pulse sequence that detects out-of-voxel (OOV) artifacts in real-time (within-TR) and responds prospectively by updating the crusher gradient scheme. Per Excitation Real-time Execution & Guided Responses with Integrated Neural-network Evaluation (PEREGRINE), allows for deployment of deep learning models and pulse sequence updates. In this study, PEREGRINE operated a time-domain (TD) and frequency-domain (FD) convolutional autoencoder that detect OOV artifacts. Scans without (AI-off) and with (AI-on) updates were collected from the medial prefrontal cortex of healthy volunteers using a MEGA-edited MRS experiment. The degree of OOV contamination (OOV Score) was quantified per transient based upon the prevalence of OOV signals in the TD and FD data. OOV Scores above a user-defined threshold triggered an update of the crusher gradient scheme, iterating through 48 permutations (6 axis transpositions × 8 polarity flips). Within each 2-second TR, PEREGRINE successfully provided single-transient OOV Scores and updated gradients accordingly. No difference was observed between the OOV Scores from the full (“Full” condition) AI-on and AI-off sessions due to the AI-on scan cycling over better and worse gradient permutations relative to the AI-off scan. However, the AI-on scan had significantly lower OOV Scores than the AI-off scan when selecting the transients where PEREGRINE persisted (“Dwell” condition) on a given gradient permutation. Ultimately, Fit Quality Number (FQN) from linear combination modeling improved significantly for the AI-on compared to the AI-off scan. PEREGRINE enabled an adaptive and AI-integrated sequence allowing for real-time evaluation and response to OOV artifacts, identifying gradient modifications that produced less OOV contamination. Graphical Abstract Graphical Abstract Summary PEREGRINE, Per Excitation Real-time Execution & Guided Responses with Integrated Neural-network Evaluation, allows for pulse sequence updates using on-scanner deployed neural networks. To demonstrate the utility, PEREGRINE was used to operate two neural networks (time/frequency domain), trained to detect out-of-voxel artifacts, during a MEGA-edited MRS experiment in the prefrontal cortex. When artifacts were detected, PEREGRINE updated the crusher gradients within the same TR. Real-time, adaptive, and AI-driven MR will provide a long-awaited solution for combatting artifacts and poor data quality. Graphical Abstract Figure

Abstract The genetic contributions of the X chromosome to Alzheimer’s disease (AD) remain poorly understood yet are expected to importantly shape sex differences in AD. We therefore performed large-scale X-chromosome-wide association studies (N=1,240,451), evaluating differential risk due to sex, APOE *4, and escape from X-chromosome inactivation, finding most X-linked loci appear relevant to female-biased AD etiology. In evaluating genetic pleiotropy with hormonal, lipid, and brain imaging traits, we discovered X-linked AD loci converged on white matter traits, particularly in the anterior corona radiata and splenium of the corpus callosum. Through brain-centric functional genomics analyses, we then nominated candidate causal genes, including 5 that appeared highly robust. Notably, we found the escape gene RBBP7 decreases AD risk in APOE *4 carriers likely through higher expression in excitatory neurons to counter tau-related neurodegeneration. Altogether, we provide an atlas of sex and APOE *4-informed candidate X-linked AD risk loci, genes, and mechanisms that will guide future studies.

Abstract Chronic low‐grade inflammation characterizes type 2 diabetes mellitus (T2DM) and underlies the development of peripheral neuropathy, symptoms of which include mechanical allodynia and an exaggerated exercise pressor reflex. The purpose of this study was to determine whether mechanical allodynia and/or specific concentrations of inflammatory mediators coincide with the presence of an exaggerated exercise pressor reflex. Haemoglobin A1c was assessed to detect diabetes onset (threshold ≥5.6%) in male University of California Davis (UCD)‐T2DM rats ( n = 18). Age‐matched, healthy Sprague–Dawley rats ( n = 18) served as controls. Monthly measurements assessed mechanical allodynia (via paw withdrawal threshold), insulin and inflammatory mediators (via ELISA and multiplex kits). Paw withdrawal threshold was not different between groups prior to diabetes onset. However, by 8 weeks post‐onset, T2DM rats demonstrated a significant decrease in pain threshold compared to that before diabetes onset, which persisted for 8 weeks. This is the same period in diabetes progression as when the exercise pressor reflex is exaggerated. Prior to diabetes onset, UCD‑T2DM rats had significantly higher circulating levels of IL‑6 and IL‑1β, and these elevations persisted throughout disease progression, with only CRP showing a significant increase at 8 weeks post‑onset. These findings offer a useful foundation for further exploration into the assessments of mechanical allodynia and inflammation to determine when those with T2DM are at an increased risk for experiencing adverse cardiovascular events.