CHEK1 (CHK1) is a key regulator of the G2/M checkpoint and DNA damage response. Although CHK1 inhibitors (CHK1is) show promise in multiple clinical trials, their further advancement is hampered by the lack of reliable predictive biomarkers. Our previous study demonstrated a nearly 20-fold difference in the sensitivity to a clinical-stage CHK1i SRA737 in prostate cancer (PC) cells. Through bioinformatics analysis, an epigenetic regulator, lysine demethylase 5D (KDM5D), was identified as a potential mediator of differential responses to SRA737. Gain- or loss-of-function studies were performed to investigate how altered KDM5D expression affects CHK1i sensitivity and the underlying mechanisms. Our data demonstrated that higher KDM5D expressions correlated with greater sensitivity to CHK1is in PC cells. In patients with castration-resistant PC (CRPC), a high KDM5D score predicted a better patient response to CHK1i. Knockdown of KDM5D in SRA737-sensitive KDM5D-expressing cells caused resistance to SRA737. Correspondingly, a higher sensitivity to SRA737 was observed in a docetaxel-resistant CRPC cell line with elevated KDM5D, and silencing KDM5D caused resistance to this inhibitor. Mechanistically, depletion of KDM5D activated p38 and induced cyclooxygenase-2 (COX-2) and ATP-binding cassette transporter expression. Inhibition of p38 or COX-2 partially reversed the resistance to CHK1i induced by KDM5D knockdown. Additionally, silencing of p38 increased KDM5D protein expression, indicating a negative feedback loop that may serve to maintain a homeostatic balance between the 2 genes. These data support a key role for KDM5D in modulating CHK1i sensitivity through a novel p38/COX-2 prosurvival pathway in PC cells, with potential predictive value for patients with CRPC receiving these anticancer agents. SIGNIFICANCE STATEMENT: This study demonstrated an important role of an epigenetic regulator KDM5D in regulating CHK1 inhibitor sensitivity via a p38/COX-2-mediated prosurvival pathway in certain castration- or drug-resistant PC cells. Our results indicate that PC cells expressing KDM5D may be more sensitive to targeted inhibition of CHK1 kinase, highlighting the potential predictive value of this gene for CHK1-targeted therapies in PC.

The opioid antagonists, naloxone and nalmefene, are used clinically to rapidly reverse opioid overdose, but often precipitate withdrawal symptoms in opioid-dependent individuals. This study compared 2 medications used for opioid use disorder, buprenorphine and methadone, to naloxone for reversing fentanyl-induced effects in rats. Buprenorphine alone did not produce significant respiratory depression at 0.5-5.0 mg/kg. Rats were challenged with 0.1 mg/kg fentanyl, which resulted in a significant reduction in oxygen saturation (SpO), and naloxone 0.1 mg/kg, buprenorphine 3.0 mg/kg, methadone 2.25 mg/kg, or saline control was given to reverse fentanyl effects. Antinociception and SpO were restored to baseline by 15 minutes after administration of naloxone and buprenorphine. The saline group showed a slow return to baseline SpO within 30 minutes, whereas methadone extended the duration of, but did not enhance, the effects of fentanyl. To determine whether buprenorphine could rapidly (within minutes) reverse fentanyl-induced respiratory depression, rats were given a dose of fentanyl 0.1 mg/kg s.c., followed by saline, naloxone 0.1 mg/kg, or buprenorphine 3.0 mg/kg, and SpO was monitored continuously for 10 minutes. Both naloxone and buprenorphine reversed fentanyl effects within 3.5 minutes, whereas the saline group did not return to baseline levels during the monitoring period. Buprenorphine at 0.3 and 1.0 mg/kg also reversed fentanyl effects, with a slower onset of reversal. In a follow-up study, rats received fentanyl followed by saline, buprenorphine, or methadone for reversal, and blood and brain levels were measured. Fentanyl concentration in the brain was not significantly affected by methadone and buprenorphine treatment, suggesting that differences in SpO were not attributable to pharmacokinetic interactions. These data support repurposing buprenorphine for the treatment of opioid overdose. SIGNIFICANCE STATEMENT: Opioid overdoses cause ∼80,000 annual deaths in the United States. Buprenorphine is an opioid partial agonist used for opioid use disorder. This study used a rat model to compare buprenorphine to naloxone for efficacy in reversing fentanyl-induced respiratory depression.

Pancreatic ductal adenocarcinoma (PDA) is an almost universally fatal disease. Recent advances in the understanding of PDA bioenergetic dynamic equilibrium have illuminated a potential therapeutic target in bromodomain-related protein 4 (BRD4), the most active member of the bromo- and extraterminal domain (BET) protein family of transcription factors. We previously demonstrated that BET inhibitors (BETi) decrease PDA cell proliferation and enhance chemosensitivity. We hypothesized that BETi activates mitophagy and ferroptosis in PDA. Using pharmacological and genetic BRD4 inhibition in PDA patient-derived models, we investigated the effects of BETi on mitochondrial function, mitochondrial protein complex production, ATP production, cellular respiration, autophagy/mitophagy, and murine tumor growth with BMS-986158, a BETi. We determined the role of BRD4 in PDA by evaluating mitophagy and autophagy. In PDA models, we found that BETi decreased cellular respiration (P < .01), decreased ATP production (P < .001), and increased intracellular iron uptake (P < .01) while inducing mitophagy through dysregulated mitochondria complex protein levels. Murine PDA tumors grew slower and were smaller when treated with BETi compared with the control treatment. PDA tumors from experimentally treated mice contained more lipid vacuoles than those from the vehicle control group (P < .01), consistent with ferroptosis. BETi therapy decreased isocitrate dehydrogenase-1 expression, indicating increased chemosensitivity. BETi dysregulate mitochondrial complexes inducing mitophagy. BETi is a promising therapeutic strategy for attacking oncogenic mitochondrial behavior in PDA. We demonstrated a series of mitochondrial-centered events in a temporal sequence leading to cell death. This treatment controls tumors and increases chemosensitivity, offering a novel therapeutic strategy. SIGNIFICANCE STATEMENT: Bromo- and extraterminal domain inhibition is a novel therapeutic strategy for attacking oncogenic mitochondrial behavior in pancreatic ductal adenocarcinoma. Using this strategy in patient-derived models, this study demonstrated a series of mitochondrial-centered events in a temporal sequence leading to cell death and tumor control.

The relationship between neuroinflammatory processes and stress-related disorders is complex with neuroinflammation both resulting from, and contributing to, the stress response. Findings from both preclinical studies and clinical trials suggest that ibudilast (IBUD), a glial cell activation attenuator and phosphodiesterase inhibitor, has shown promise for mitigating the adverse behavioral effects of stress exposure and stress-related neuropsychiatric disorders. The objective of the present study was to determine the effect of IBUD administration on anxiety-like behavioral performance and fear memory expression in rats following stressor exposure. Here, adult male Sprague-Dawley rats were behaviorally tested in the elevated plus maze (EPM) and acoustic startle response tests 2 days before (day 1) and 2 days after (day 5) exposure to a stressor (inescapable footshock). Five days after stressor exposure, IBUD (0-10 mg/kg, i.p.) was administered 1 hour prior to a daily fear expression test session (days 8-11). Behavioral performance in the EPM and acoustic startle test was measured again on the following day (day 12). Following this, multiplex immunoassays were used to determine neuroinflammatory cytokine/chemokine levels in various brain regions. In a separate experiment, the effects of IBUD on locomotor activity and anxiety-like behavior were characterized in an open field test in rats with no history of stressor exposure. Stress exposure significantly (P < .05) reduced open arm exploration in the EPM-effects that were prolonged following IBUD treatment in a dose-dependent manner. Pretest administration of IBUD resulted in significantly (P < .05) greater freezing behavior during the fear expression tests with no evidence of fear extinction, whereas fear extinction was evident in vehicle-treated control rats. In nonstressed rats, IBUD reduced total locomotor activity and center exploration in the open field in a dose-dependent manner. Neuroinflammatory marker levels in the prefrontal cortex and amygdala were positively correlated with anxiety-like behavioral performance outcomes. Together, results suggest IBUD perpetuates stress-induced anxiety-like behavior and stress-associated fear memory expression (ie, hinders fear memory extinction). Further investigations into the interactions between IBUD administration and stressor exposure are needed to understand the implications of administering this drug in the context of stress exposure. SIGNIFICANCE STATEMENT: Ibudilast, a glial cell inhibitor and promising treatment candidate for various psychiatric disorders, was found to prolong fear expression and anxiety-like behavior in stress-exposed rats. Results suggest added characterization and consideration of its interactions with traumatic stress is needed.

Hepatitis B is a severe viral infectious disease, and there is no effective enough way to cure it completely at present. In this study, we first probed into the effect of sirtuin 1 inhibitor III (EX527) on hepatitis B virus (HBV) replication following inhibition of silencing information regulator 1 (SIRT1) and whether EX527 can enhance the efficacy of entecavir (ETV) in anti-HBV therapy. The synergistic interaction of EX527 (10 μM) with ETV (25 μg/mL) was performed in the HBV steady-state replicating cell line HepG2.2.15. The expression profiles of HBV DNA, total RNA, pregenomic RNA, hepatitis B surface antigen, hepatitis B e antigen, HBV X antigen, hepatitis B core antigen, tumor necrosis factor-α, interleukin-6, and nuclear factor-κB were examined by real-time quantitative polymerase chain reaction, ELISA, immunofluorescence, and Western blot. The results show that silenced SIRT1 expression significantly inhibited the replication of HBV. The combination of SIRT1 inhibitor and ETV further reduced HBV DNA levels (24 hours: P = .0012; 95% combination index (CI), 0.1563-0.3164; 48 hours: P = .0025; 95% CI, 0.1990-0.4746; 72 hours: P = .0009; 95% CI, 0.2011-0.3867), total RNA (24 hours: P < .0001; 95% CI, 0.3183-0.4506; 48 hours: P = .0011; 95% CI, 0.2500-0.4964; 72 hours: P = .0011; 95% CI, 0.2978-0.5937) and pregenomic RNA (24 hours: P < .0001; 95% CI, 0.2624-0.3194; 48 hours: P = .0001; 95% CI, 0.3158-0.4554; 72 hours: P = .0022; 95% CI, 0.2586-0.5975) levels compared to ETV monotherapy. Significant reduction in hepatitis B surface antigen (24 hours: P = .0002; 95% CI, 113.3526-173.7049; 48 hours: P < .0001; 95% CI, 128.9962-160.8836; 72 hours: P < .0001; 95% CI, 129.0970-164.7718), hepatitis B e antigen (24 hours: P = .0094; 95% CI, 1.2650-4.9331; 48 hours: P = .0215; 95% CI, 0.7548-5.4610; 72 hours: P = .0003, 95% CI, 4.5457-7.4308), HBV X antigen (24 hours: P = .0009; 95% CI, 0.2263-0.4302; 48 hours: P < .0001; 95% CI, 0.3094-0.3866; 72 hours: P = .0019; 95% CI, 0.2393-0.5325) and hepatitis B core antigen (24 hours: P = .0010; 95% CI, 0.3161-0.6200; 48 hours: P = .0022; 95% CI, 0.2847-0.6571; 72 hours: P = .0012; 95% CI, 0.2571-0.5188) levels were observed in the SIRT1 inhibitor and ETV combination groups. Cotreatment with EX527 and ETV had a more conspicuous effect on the inflammatory factors: tumor necrosis factor-α (mRNA: P < .0001; 95% CI, 0.2207-0.2961; protein: P = .0226; 95% CI, 5.3050-40.7000), interleukin-6 (mRNA: P < .0001; 95% CI, 0.2413-0.3220; protein: P = .0134; 95% CI, 0.5065-1.6998), and nuclear factor-κB (P = .0057; 95% CI, 0.1808-0.4268). These in vitro results indicated that the combination of SIRT1 inhibitor EX527 and ETV can effectively inhibit the replication of HBV. SIGNIFICANCE STATEMENT: This study has important theoretical and practical significance. It explores the factors influencing the replication of the hepatitis B virus from a new perspective and provides new ideas for follow-up research.

Human aging is driven by several interconnected hallmarks, including genomic instability, mitochondrial dysfunction, and cellular senescence, which collectively underlie pathologies such as neurodegeneration and metabolic decline. Despite advances in identifying senescence-associated biomarkers and pathways, conventional antiaging compounds such as resveratrol and fisetin, lack regulatory approval owing to insufficient evidence from large-scale trials. Drug repurposing provides a cost-efficient strategy to target aging pathways by leveraging existing pharmacologic safety profiles. Erythropoietin (EPO) exemplifies this approach, demonstrating pleiotropic antiaging effects through modulation of cell survival pathways and tissue-protective mechanisms. Recent advancements in nonhematopoietic EPO derivatives, such as carbamylated EPO, further unlock its development potential by decoupling therapeutic benefits from erythropoietic activity. This review analyzes EPO molecular antiaging mechanisms and clinical applications in age-related diseases (2015-2025), focusing on multiorgan systemic effects and derivative development beyond anemia. SIGNIFICANCE STATEMENT: This review highlights erythropoietin (EPO) as a promising repurposed drug for combating aging, targeting hallmarks such as oxidative stress and cellular senescence. Crucially, nonhematopoietic EPO derivatives circumvent traditional safety risks while retaining multipathway protective effects in brain, cardiovascular, and metabolic tissues. By leveraging established pharmacology, EPO offers a cost-efficient strategy to advance aging interventions, addressing age-related pathologies beyond anemia.

Metabolic dysfunction-associated steatohepatitis (MASH) poses a significant public health challenge, characterized by liver fat accumulation accompanied with inflammation and cell damage. Patients with MASH commonly exhibit hypercoagulability. Our previous work showed that direct oral anticoagulants exert anti-inflammatory effects in early stages of metabolic dysfunction. Mitigation of thromboinflammation ameliorated the manifestations of cardiometabolic complications. Here, we examine the protective effects of rivaroxaban in a rat model of MASH and compare it to representatives of 2 other antithrombotic drug classes, enoxaparin and clopidogrel, as well as silymarin, a bona fide hepatoprotective agent. Rats were divided into 6 groups: control, MASH (induced by an atherogenic diet), and treatment groups receiving either silymarin (50 mg/kg by mouth), rivaroxaban (20 mg/kg by mouth), enoxaparin (2 mg/kg subcutaneously), or clopidogrel (6.75 mg/kg by mouth) for 8 weeks, starting in the third week of induction. MASH rats showed elevated markers of visceral adipose thromboinflammation, along with liver injury markers (aspartate aminotransferase, alanine aminotransferase, and serum albumin), heightened hepatic levels of inflammatory cytokines (interleukin [IL]-1β and IL-6), reduced antioxidant capacity, and an imbalance in coagulation factors (elevated activated coagulation Factor X and Factor VIII/Protein C) compared with the control group. Anticoagulants and silymarin treatment led to varying degrees of amelioration of these MASH-associated abnormalities. Rivaroxaban demonstrated the most substantial improvement in thromboinflammatory markers, reaching levels comparable to the control group, with percentage improvements of approximately 52%, 49%, and 42% in activated coagulation Factor X, IL-1β, and IL-6, respectively. These findings suggest that direct oral anticoagulants hold promise as therapeutic agents for MASH by targeting the underlying thromboinflammatory state. SIGNIFICANCE STATEMENT: Metabolic dysfunction-associated steatohepatitis is a global health issue. In a rat model of the disease, rivaroxaban improves liver injury markers, outperforming bona fide hepatoprotective substances. This study emphasizes the role of thromboinflammation in metabolic dysfunction-associated steatohepatitis and highlights direct anticoagulants as a potential novel treatment approach.

The pleiotropy of minocycline (MINO), including anti-inflammatory, antioxidant, antimigratory, anti-matrix metalloproteinase (MMP), and neuroprotective effects, has been extensively reported. A novel nonantibiotic MINO derivative, 10-butyl ether minocycline (BEM), was synthesized to retain the pleiotropy of MINO while minimizing side effects such as antibiotic resistance and gut dysbiosis. Previously, we showed that BEM reduced alcohol consumption in dependent murine and porcine models of alcohol use disorder. In this study, we investigated the molecular mechanisms of BEM to determine its potential as a therapeutic agent for neuroimmune and inflammatory conditions such as alcohol use disorder. Here, we report that BEM showed a nearly complete loss of antimicrobial activity against Escherichia coli, Salmonella typhi, and Candida albicans. BEM showed a dose-dependent reduction in cell viability as measured by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, similarly to MINO. BEM also suppressed lipopolysaccharide-induced microglial activation as shown by reduced Iba1 expression in immunohistochemistry and western blot analyses. Inhibition of MMP-9 by BEM (IC50 = 42.2 μM) was improved compared to MINO (IC50 = 60.3 μM), whereas MMP-8 inhibition was moderate (IC50: BEM = 69.4 μM; MINO = 45.4 μM). BEM was found to be effective in inhibiting vascular endothelial growth factor-induced endothelial cell migration and L-glutamine-induced reactive oxygen species levels. Limited inhibition of 15-lipoxygenase activity was observed (IC50: BEM = 92.6 μM; MINO = 65.6 μM). BEM was not toxic to mitochondria, even at high concentrations (200 μM). By eliminating antimicrobial properties while preserving therapeutic pleiotropy, BEM presents an advancement in the development of a promising candidate with multimodal mechanisms to treat neuroimmune-inflammatory pathologies. SIGNIFICANCE STATEMENT: We report mechanisms of action for butyl ether minocycline, a minocycline analog under evaluation for the treatment of alcohol use disorder, which may also show efficacy for other complex disease processes that involve inflammatory or neuroimmune components. We show that butyl ether minocycline had a nearly complete loss of antimicrobial action, yet retained the pleiotropy of minocycline, likely making it a better multimodal therapeutic for long-term treatment of complex diseases with neuroimmune-related components.

The incidence of fatal drug overdoses has increased dramatically over the past decade due to the widespread availability of fentanyl and its analogs. As a complementary strategy to current overdose reversal agents, monoclonal antibodies (mAbs) are in development as therapeutics for prevention and reversal of fentanyl overdose. In the present study, the anti-fentanyl mAb HY6-F9 was tested for reversal of fentanyl-induced respiratory arrest (apnea) in a porcine model. In a first study, following fentanyl-induced apnea, chimeric HY6-F9 and naloxone control were administered as an intravenous bolus. Both chimeric HY6-F9 and naloxone restored spontaneous breathing within 90 seconds. Treatment with mAb increased the concentration of fentanyl in serum by 10-fold within the first minute after mAb bolus administration. In a second study, after induction of apnea, humanized HY6-F9 and naloxone control were administered as a slow intravenous infusion over 10 minutes to determine the ED to restore baseline breathing. In this study, the mean ± SEM ED of humanized HY6-F9 and naloxone to restore baseline respiratory rate were 16.0 ± 1.3 mg/kg and 6.9 ± 1.8 μg/kg, respectively. During mAb infusion, the concentration of fentanyl in serum increased proportionally to the concentration of infused mAb. The anti-fentanyl mAb ablated fentanyl-dependent opioid receptor activation in an in vitro system with concentrations of fentanyl similar to those observed in pigs after mAb treatment. These results demonstrate the efficacy of an anti-fentanyl mAb as a treatment to reverse fentanyl overdose. SIGNIFICANCE STATEMENT: Treatments for opioid use disorder and overdose are urgently needed. Here, we show that an anti-fentanyl monoclonal antibody reversed fentanyl-induced apnea in pigs, and caused rapid (<1 minute) redistribution of fentanyl into serum. Fentanyl was 99% bound by monoclonal antibodies and showed no activity at the opioid receptor.

Cellular senescence, a persistent state of cell cycle arrest, accumulates in aged organisms, contributes to tissue dysfunction, and drives aging-related phenotypes. Clearance of senescent cells decreases chronic, low-grade inflammation and restores tissue repair capacity, thus improving human health and lifespan. Senolytics that selectively eliminate senescent cells have become a promising antiaging strategy. To date, current senolytics are largely developed by repurposing anticancer agents. Therefore, senolytics usually possess various on- and off-target toxicities. These toxicities could preclude their clinical use as antiaging agents, as elderly people are more susceptible to adverse drug effects than young individuals. Proteolysis targeting chimeras as senolytics, termed "SenoTACs," are attractive for more effective treatment of aging-related diseases. In comparison to small molecule inhibitors, SenoTACs can eliminate senescent cells by degrading targeted proteins in a substoichiometric manner, providing better target ability, longer-lasting therapeutic effect, broadened target capability, and decreased drug resistance. Recent efforts have led to the development of several senescence-targeting proteolysis targeting chimeras, including ARV825, PZ15227, 753B, Gal-ARV-771, and Gal-MS99, which exhibit selective senolytic activity and improved safety and efficacy profiles when compared with small molecule inhibitors. In this minireview, we summarize the development of the emerging field. SIGNIFICANCE STATEMENT: The severe toxicities associated with current senolytics may limit their clinical utility as antiaging agents, as older populations are more susceptible to adverse drug effects. PROteolysis TArgeting Chimeras (PROTACs) that induce selective degradation of target proteins, are emerging as a promising therapeutic strategy to address this unmet medical need. Recently, PROTACs have been explored as novel senolytics-termed "SenoTACs," which display improved safety and efficacy in targeting senescent cells for fighting aging-related diseases.

Sulfur is clinically used for treating scabies and pruritus and shows potential in treating atopic dermatitis (AD), though its mechanism remains unclear. The potential targets of sulfur and the differentially expressed genes of AD in the GSE237920 and GSE121212 datasets from the Gene Expression Omnibus database were identified. The intersecting genes were analyzed by Gene Ontology and Kyoto Encyclopedia of Genes and Genomes. A protein-protein interaction network was established using the Search Tool for the Retrieval of Interacting Genes/Proteins database. Through Least Absolute Shrinkage and Selection Operator, Random Forest, and Support Vector Machine-Recursive Feature Elimination algorithms, we screened the core targets. The core genes were validated using receiver operating characteristic analysis, gene set enrichment analysis, and immune cell infiltration analysis. Long noncoding RNA (lncRNA)-microRNA (miRNA)-mRNA bioinformatics networks were predicted using databases. The core lncRNA-miRNA-mRNA axis was identified by intersecting bioinformatics networks with GSE168694. Subsequently, the expression of each node in the axis was verified through animal experiments. Using machine learning and bioinformatics analysis, glucose-6-phosphate isomerase (GPI) was identified as a core target. We then predicted 5 miRNAs and 141 lncRNAs. By validating the results using the GSE168694 dataset, the AC010336.1/miR-3929/GPI axis was identified as the key competing endogenous RNA regulatory axis. Animal experiments demonstrated that sulfur exerted therapeutic effects against AD by restoring epidermal barrier function, reducing immune cell infiltration, and mitigating pruritus. Additionally, animal experiments confirmed that sulfur alleviated AD by modulating the AC010336.1/miR-3929/GPI axis. These novel findings provide valuable insights and guidance for future investigations on the competing endogenous RNA mechanism of traditional Chinese medicine in AD therapy. SIGNIFICANCE STATEMENT: This study investigates the therapeutic mechanisms of sulfur, a traditional Chinese medicine, in the treatment of atopic dermatitis, with elucidate extending to the single-gene regulatory level.

Sleep disturbances are prominent symptoms of opioid withdrawal that contribute to relapse in individuals with opioid use disorder (OUD). Although preclinical studies often examine protracted withdrawal after sustained abstinence, few address the repeated, spontaneous withdrawal cycles that model human patterns of use. Moreover, targeting sleep disruptions provides a novel strategy to reduce relapse risk. The present studies characterized the effects of repeated spontaneous daily withdrawal on sleep and quantitative electroencephalography (qEEG) after once-daily morphine administration in male Sprague-Dawley rats. Daily withdrawal produced persistent sleep disruptions, including reduced rapid eye movement (REM) and non-REM sleep, increased time awake, and sleep fragmentation. Notably, tolerance did not develop to the acute pharmacological effects of morphine or spontaneous daily withdrawal effects on sleep across the 30-day study. Relative spectral analysis (qEEG) revealed progressive shifts from low (δ) to high (γ) frequency power across withdrawal phases, with significant and persistent increases in wake and non-REM relative γ band power during later stages of daily and protracted withdrawal. There were few correlations between somatic behavioral signs and sleep or qEEG supporting our hypothesis that sleep, and brain activity disturbances represent distinct withdrawal phenotypes that should be studied and treated separately from somatic withdrawal behaviors. Together, these findings highlight the chronic and multifaceted nature of opioid withdrawal, particularly its effect on disrupted sleep and brain activity. Collectively, these studies characterized a translationally relevant preclinical model incorporating both sleep and qEEG as potential behavioral and physiological biomarkers for evaluating putative pharmacotherapies for withdrawal-related sleep dysfunction in OUD. SIGNIFICANCE STATEMENT: Sleep disruptions associated with withdrawal in opioid use disorder represent a contributing factor for relapse yet lack targeted treatments. This study reports consistent, spontaneous daily sleep disruptions in rats after once-daily morphine administration, providing a model that can be used for treatment development.

Previous studies show that endothelin-converting enzyme 2 (ECE2), an enzyme involved in nonclassical processing of neuropeptide precursors, regulates the rate and extent of μ and δ opioid receptor recycling, but not internalization following activation by select synthetic and endogenous peptidic agonists. This study focuses on κ opioid receptors (KORs) and examines how prodynorphin and proenkephalin-derived peptides that are substrates of ECE2 modulate the internalization and recycling of both KOR and ECE2; as controls we use peptides that are not ECE2 substrates. First, we use a proximity-based ligation assay to show that KOR and ECE2 are in close proximity to directly interact and cointernalize. Treatment with the peptides induces internalization and recycling of ECE2 at a rate and extent comparable to that of KOR with longer opioid peptides inducing fast and robust internalization and recycling of both ECE2 and KOR compared with shorter peptides. We find that in recombinant cell lines and naïve cells expressing endogenous receptors, a small molecule ECE2 inhibitor attenuates KOR recycling as well as signaling by only peptides that are ECE2 substrates. Taken with the differential expression of ECE2 in the brain (relatively high expression in midbrain and dentate gyrus of the hippocampus and low expression in the striatum), these results highlight a pivotal role for ECE2 in differentially modulating KOR function. SIGNIFICANCE STATEMENT: This study highlights a role for endothelin-converting enzyme 2 in agonist mediated regulation of κ opioid receptor function by select prodynorphin and proenkephalin-derived peptides. Collectively, studies by authors suggest that endothelin-converting enzyme 2 inhibitors could be developed as therapeutics for pathologies involving dysregulations in κ opioid receptor signaling.

Environmental cues that have been associated with drug-taking can evoke drug-craving and drug-seeking and drive relapse. Using the New Response Acquisition procedure, we evaluated the extent to which activation of delta opioid receptors (DORs) changes responding for cocaine-associated stimuli. We hypothesized that activation of DORs, either directly via agonists or indirectly via protected concentrations of endogenous enkephalin peptides, would increase the conditioned reinforcing effects of cues. First, animals undergo Pavlovian conditioning during which rats received 5 infusions of cocaine (0.32 mg/kg/inf) and either paired or unpaired presentations of a stimulus (light + tone) per day for 10 days. Next, nosepokes were added to the operant chamber and rats were allowed to respond for presentations of cocaine-associated stimuli (acquisition). Consistent with previous findings, animals assigned to paired Pavlovian conditioning emitted more responses for cue presentations than animals assigned to the unpaired control. Interestingly, acute administration of SNC80 (DOR agonist; 3.2 mg/kg s.c.) on acquisition session 4 led to robust increases in responding for the cocaine-paired cues in the paired, but also increased responding for cues in rats assigned to unpaired and saline control groups. Further, the enkephalinase inhibitor RB101 (10 mg/kg intravenous), which maintains extracellular concentrations of enkephalins, increased active responding in a DOR-dependent manner. These data suggest that activation at DORs influences behaviors maintained by cues and sheds light on the neurobiology underlying the conditioned reinforcing effects of drug-associated stimuli. SIGNIFICANCE STATEMENT: We used a more rigorous test of conditioned reinforcement to show that activation of delta opioid receptors increases the reinforcing effects of cocaine-paired cues, depending on conditioning history, which implicates the delta opioid receptor system as a target to reduce relapse.

Cocaine-associated stimuli acquire conditioned reinforcing effects and can precipitate relapse. We used the New Response Acquisition procedure to examine factors that influenced the conditioned reinforcing effects of cocaine-associated stimuli in rats. According to this procedure, rats first experienced Pavlovian conditioning, during which they were exposed to intravenous cocaine deliveries and stimulus (light + white noise) presentations. After Pavlovian conditioning, animals learned to respond to the cocaine-paired stimulus alone. The number of responses made for that stimulus reflected the conditioned reinforcing effects of the cocaine-associated stimulus. Across 3 experiments, the extent to which the dose of cocaine during Pavlovian conditioning (experiment 1), food restriction (experiment 2), and the number of cocaine-stimulus pairings and the number of days of Pavlovian conditioning (experiment 3) led to different conditioned reinforcing effects of the cocaine-associated stimulus. Taken together, we found that cocaine-associated cues took on conditioned reinforcing effects dose-dependently, were augmented by food restriction, and were most robust following 10 days of Pavlovian conditioning relative to 5 days of conditioning. These findings advance our understanding of the conditions under which cocaine-associated stimuli can act as a conditioned reinforcer. SIGNIFICANCE STATEMENT: Cocaine-associated stimuli acquire conditioned reinforcing effects via Pavlovian conditioning that drive drug-seeking and relapse. Understanding the conditions under which cocaine-associated stimuli take on conditioned reinforcing effects can be used to inform efforts to curtail cocaine use disorder.

Adiponectin receptor (AdipoR) agonists protect against glomerular inflammation and injury in obesity-related glomerulopathy (ORG), but their molecular mechanisms remain unclear. Given the implication of the ceramide signaling pathway in the pathogenesis of ORG, the present study tested whether AdipoR agonists target acid ceramidase (AC) to inhibit NLRP3 inflammasome activation in podocytes, thereby blocking glomerular inflammation and injury during obesity. Confocal microscopy showed that adiponectin attenuated visfatin-induced NLRP3 inflammasome activation and IL-1β-containing multivesicular body (MVB) formation in podocytes. Nanoparticle tracking analysis revealed that adiponectin suppressed visfatin-induced extracellular vesicle release, an effect dependent on AC activity. Structured illumination microscopy demonstrated that visfatin reduced lysosome-MVB interaction in podocytes, which was restored by adiponectin via enhancement of TRPML1 channel-mediated Ca release. The rescue of lysosome-MVB interaction and TRPML1 channel activity by adiponectin was mimicked by the AC enhancer but interfered with by the AC inhibitor. In vivo, high-fat diet (HFD) treatment induced NLRP3 inflammasome activation and T cell infiltration in glomeruli and increased urinary extracellular vesicle excretion in mice, which were exaggerated by podocyte-specific Smpd1 gene (gene code of acid sphingomyelinase) overexpression in Smpd1/Podo mice compared with WT/WT mice. AdipoRon, a synthetic AdipoR agonist, reduced HFD-induced glomerular inflammation in both WT/WT and Smpd1/Podo mice, but its effect was blocked by AC inhibition. Moreover, podocyte-specific Smpd1 gene overexpression aggravated HFD-induced podocyte injury, proteinuria, and glomerular sclerosis, which were mitigated by AdipoRon in an AC-dependent manner. Additionally, we found that the protective actions of AdipoRon may be mainly attributed to the activation of AdipoR1, but not AdipoR2. Taken together, our findings suggest that AC activation mediates the protective effects of AdipoR agonists against glomerular inflammation and injury in ORG, highlighting AC as a potential therapeutic target. SIGNIFICANCE STATEMENT: This study identifies acid ceramidase as a key mediator of adiponectin receptor agonist action in podocytes, linking its activation to suppression of NLRP3 inflammasome and extracellular vesicle release, and highlighting a novel therapeutic target in obesity-related kidney disease.

The mechanisms underlying L-DOPA-induced dyskinesia (LID) largely arise from maladaptive plasticity in striatal circuits leading to altered neuronal responses to dopamine (DA) signaling. Cyclic nucleotides play a major role in the molecular cascades of DA signaling, and particularly cAMP is known to be associated with LID mechanisms. Cyclic nucleotide levels in striatal neurons are regulated by phosphodiesterases (PDEs), and 1 isoenzyme with selective affinity for cAMP and high expression in the striatum is PDE7. Here, the PDE7 inhibitor OMS-401 was evaluated for antidyskinetic effects in a nonhuman primate (NHP) model of advanced Parkinson's disease. A series of systemic 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine administration followed by chronic L-DOPA treatment were used to induce advanced parkinsonism and reproducible LID in a group of 3 macaques. The effects of the PDE7 inhibitor OMS-401 were analyzed with a dose-response curve design in coadministration trials for 2 doses of L-DOPA (optimal and suboptimal). Motor disability, LID, and drug adverse reactions were assessed using standardized scales for 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-treated NHPs. OMS-401 significantly reduced LIDs in a dose-dependent fashion without interacting with the antiparkinsonian action of L-DOPA or inducing side effects in parkinsonian NHPs. Results confirm that cAMP levels in striatal neurons play a critical role in LID mechanisms, and that PDE7 inhibition may be a strategy to control LID over the long-term DA replacement therapy in Parkinson's disease. SIGNIFICANCE STATEMENT: This study shows that selective phosphodiesterase 7 inhibition with OMS-401 reduces dyskinesia in a Parkinson's primate model without affecting L-DOPA's benefits. Phosphodiesterase 7 inhibition may offer a promising approach for L-DOPA-induced dyskinesia management, providing an alternative to treatments with dose-limiting side effects.

γ-aminobutyric acid type-A receptors associate with auxiliary proteins such as Shisa7 and have allosteric binding sites for benzodiazepines, barbiturates, and alcohols. In this study, Shisa7 was knocked down to assess its involvement in the discriminative-stimulus effects of drugs from each class and in the positive reinforcing effects of alcohol. Twelve male Long-Evans rats were trained to respond in a drug-discrimination procedure with 6 trained to discriminate ethanol (1 g/kg) from saline and 6 trained to discriminate alprazolam (1.8 mg/kg) from cyclodextrin. Following training, cumulative dose-effect curves for ethanol, alprazolam, and pentobarbital were established before and after Shisa7 knockdown was achieved using an intravenous dicer substrate small-interfering ribonucleic acid. A separate group of 6 rats was also trained to consume ethanol orally to assess ethanol intake before and after Shisa7 knockdown. In general, before Shisa7 knockdown, alprazolam and pentobarbital partially substituted for ethanol in ethanol-trained subjects up to doses that decreased response rate, whereas pentobarbital, but not ethanol, partially substituted for alprazolam in alprazolam-trained subjects. After Shisa7 knockdown, the dose-effect curve for ethanol-lever responding in ethanol-trained subjects was shifted downward, the curve for alprazolam-lever responding in alprazolam-trained subjects was shifted rightward, and the curve for pentobarbital was shifted rightward in both ethanol- and alprazolam-trained subjects. Shisa7 knockdown did not affect response rates. In rats orally consuming ethanol, both ethanol intake and dose were significantly decreased after Shisa7 knockdown compared with the dicer substrate small-interfering ribonucleic acid control. These findings demonstrate that reducing Shisa7 levels attenuated the discriminative-stimulus effects of 3 positive allosteric modulators of γ-aminobutyric acid type-A receptors and decreased ethanol's reinforcing effects. SIGNIFICANCE STATEMENT: This study demonstrates that Shisa7, an auxiliary protein associated with γ-aminobutyric acid type-A receptor, plays a crucial role in mediating the discriminative-stimulus effects of ethanol, alprazolam, and pentobarbital, as well as the reinforcing effects of ethanol. By demonstrating that Shisa7 knockdown attenuates the behavioral effects of these drugs, the findings provide new insights into the molecular mechanisms underlying γ-aminobutyric acid type-A receptor-mediated drug effects and potentially identify Shisa7 as a key modulatory mechanism through which these drugs produce their effects.

Polycystic ovary syndrome (PCOS) is a complex endocrine and metabolic disorder characterized by hyperandrogenism and frequently associated with insulin resistance (IR), a key pathogenic factor in PCOS. However, insulin sensitizers commonly used to treat PCOS are often recommended off-label and may cause side effects. This study investigated the therapeutic effects of pancreastatin inhibitor 8 (PSTi8), an insulin sensitizer, in a PCOS rat model. The PCOS rat model was established by daily feeding with a high-fat diet and administering subcutaneous injections of dehydroepiandrosterone at a dose of 60 mg/kg body weight for 21 days and further, followed by 21 days of treatment with PSTi8 (10 mg/kg) and metformin (300 mg/kg). Body weight, estrous cycle, glucose, and insulin tolerance test results were monitored. Ovarian morphology, estrous cycle changes, oxidative stress and inflammatory markers, steroidogenic hormone levels and protein expression, and insulin signaling pathway were assessed to evaluate the therapeutic effectiveness of PSTi8 in PCOS rats. This study found that PSTi8 improved IR and reduced body weight in PCOS rats. PSTi8 lowered serum levels of insulin (27%), testosterone (56%), estradiol (2-fold), progesterone (21%), sex hormone-binding globulin (7.5%), and luteinizing hormone/follicle stimulating hormone ratio (57%). Additionally, PSTi8 helped to restore ovarian morphology, estrous cycle, and improve dyslipidemia. PSTi8 treatment also reduces the oxidative stress level of total superoxide ismutase (16%), glutathione peroxidase (26%), and inflammation in PCOS rats. Furthermore, PSTi8 restores the steroidogenic protein expression and increases PI3K/Akt phosphorylation in PCOS rats. These findings demonstrate PSTi8 exhibited comparable efficacy to metformin in ameliorating IR and ovarian dysfunction in the studied PCOS model. SIGNIFICANCE STATEMENT: Polycystic ovary syndrome (PCOS) increases risk of reproductive and metabolic disorders, partly due to systemic inflammation. This study combined dehydroepiandrosterone with high-fat diet and successfully induced PCOS-like features in rats. PSTi8, a pancreastatin inhibitor known for insulin-sensitizing effects in various disease models, effectively reversed PCOS-associated pathophysiology. PSTi8 improves insulin sensitivity by activating the PI3K/AKT signaling pathway and ameliorates oxidative stress and inflammation in PCOS rats. Additionally, PSTi8 treatment normalized steroidogenesis protein expression and reduced circulating biomarkers linked to cardiovascular risk.

Low-dose aspirin irreversibly acetylates cyclooxygenase (COX)-1 in anucleate platelets and progenitor megakaryocytes, permanently suppressing thromboxane (TX)A-dependent platelet activation. Although aspirin pharmacodynamics is well characterized in platelets, the kinetics of COX inhibition and recovery in human megakaryocytes remains poorly defined, due to ethical issues associated with invasive, bone-marrow trephine sampling, and low megakaryocyte yield. We studied aspirin pharmacodynamics in human megakaryocytic cell lines as a reliable and feasible surrogate model. We characterized COX-1 and COX-2 expression and activity in MEG-01 and CHRF-288-11 megakaryocytic cell lines, treated with a range of aspirin concentrations and exposure duration. COX activity was quantified by the production of TXB from exogenous arachidonic acid. A single 10-μM aspirin exposure suppressed TXB by 90 ± 2% (MEG-01) and 85 ± 4% (CHRF-288-11), with full recovery within 48-72 hours. Both COX-isozymes were detected by western blot and immunohistochemistry; however, selective COX-1 inhibition by SC-560 reduced TXB by >75%, whereas COX-2 inhibition by NS-398 had minimal effect. Repeated aspirin exposure every 24 hours produced concentration- and time-dependent TXB suppression, achieving 89 ± 2% inhibition by day 2 at 1 μM and 73 ± 3% by day 4 at 0.1 μM. TXB biosynthesis recovered by 86 ± 2% and 99 ± 10% at days 2 and 3, respectively. These findings identify COX-1 as the principal source of TXA in megakaryocytes and demonstrate that aspirin inhibits megakaryocyte COX-1 time- and dose-dependently, with delayed recovery likely reflecting de novo synthesis of COX-1 protein, thereby providing mechanistic insight into the sustained antiplatelet effect of low-dose aspirin in humans. SIGNIFICANCE STATEMENT: In human megakaryocyte cell lines, once-daily aspirin treatment at low-concentration range time-dependently inhibits COX-1 with delayed recovery after aspirin withdrawal. This closely mimics the kinetics of platelets, supporting the translational utility of the megakaryocyte-based surrogate model.