The Journal of Pharmacology and Experimental Therapeutics

CNS4 is an agonist concentration-biased NMDAR modulator exhibiting GluN2 subtype-dependent activity. Here we evaluated the translational pharmacology of CNS4 across mice, rats, and client-owned surgical oncology dogs. In mice, CNS4 did not produce conditioned place preference, indicating lack of reward liability. CNS4 produced dose-dependent reductions in locomotor activity without motor impairment on accelerating rotarod testing. CNS4 also maintained body temperature 0.5 to 1 degree Celsius above the vehicle group in mice and dogs, consistent with NMDAR-dependent thermoregulatory activity and contrasting with hypothermic effects reported for NMDAR channel blockers such as ketamine. In fear-conditioning paradigms, CNS4 did not impair fear learning, memory consolidation, or fear expression. CNS4 reduced acute stress-induced sucrose preference, suggesting modulation of stress-responsive neural circuits. In a rat spinal nerve ligation model of neuropathic pain, both single-dose and three-day repeated CNS4 administration significantly reversed mechanical, pressure, and thermal hypersensitivity, supporting analgesic efficacy with no apparent evidence of rapid tolerance development. In client-owned surgical oncology dogs undergoing standard veterinary procedures, preliminary findings from 14 dogs (8 CNS4-treated, 6 vehicle-treated) demonstrated stable intraoperative mean arterial pressure and heart rate. Postoperatively, CNS4-treated dogs exhibited normal physiological recovery, with respiratory rate, heart rate, and body temperature returning toward the normal canine range, and required approximately 8% less propofol for anesthetic induction. Collectively, these findings support CNS4 as a novel NMDAR modulator with thermoregulatory, anti-agitative, analgesic, and autonomic-stabilizing properties without evidence of abuse liability, supporting continued translational development for neuropathic pain and possibly neuropsychiatric disorders.

Ogerin, a positive regulator of GPR68, reduces infarct volume and attenuates blood-brain barrier (BBB) leakage post transient middle cerebral artery occlusion (tMCAO). These data support the potential use of Ogerin for ischemic protection. For therapeutic interventions, tissue distribution, kinetics, and brain penetration are important characteristics for a compound. For these reasons, we performed liquid chromatography-mass spectrometry (LC-MS) analysis of Ogerin in mice. At 3 hours following intraperitoneal (IP) injection, liver exhibited highest level of Ogerin among all major organs assessed. We further analyzed time-dependent changes in Ogerin concentration in serum following intravenous delivery. Brain Ogerin level was ~140 ng/g at 2 hour and ~0.06ng/g at 24 hours. The brain-to-serum ratio of Ogerin was ~0.6 at 5 hr. To determine the effect of Ogerin on signaling, we collected samples 24 hours after tMCAO with or without Ogerin delivered and performed Western blotting. Consistent with the diminished level of Ogerin in brain at 24 hour, tMCAO increased similar changes in phosphorylation of PKA substrates and PKC substrates in vehicle- and Ogerin-treated groups. The pharmacokinetic data suggests that Ogerin is effective mainly during acute hours after injection. This finding generates important insights on the design of effective protocol for Ogerin delivery to potentiate GPR68 function in vivo.

Background and purposeAnthracyclines, such as doxorubicin (DOX), are associated with late-onset kidney dysfunction; however, the mechanisms underlying chronic tubular injury remain poorly understood. We investigated whether chronic low-dose DOX exposure induces persistent mitochondrial dysfunction in renal tubules and evaluated the therapeutic potential of activating the NAD-Sirtuin axis. Experimental ApproachC57BL/6 mice were repeatedly administered low-dose DOX with or without resveratrol (RSV) or nicotinamide mononucleotide (NMN), a sirtuin activator. Renal injury was assessed using neutrophil gelatinase-associated lipocalin (NGAL) staining. Integrated proteomic and RNA sequencing analyses were performed to identify molecular alterations. Mitochondrial morphology and function were evaluated using structured illumination microscopy (SIM) of Massons trichrome-stained paraffin sections and ex vivo Seahorse analysis of freshly isolated renal tubules. Key ResultsChronic DOX administration induced tubular injury, despite preserving serum creatinine levels. Multi-omics analyses consistently demonstrated the suppression of mitochondrial pathways, including oxidative phosphorylation, fatty acid oxidation, and mitochondrial gene expression. SIM revealed mitochondrial fragmentation in tubular epithelial cells, whereas the Seahorse assay showed impaired mitochondrial respiratory capacity in isolated renal tubules. DOX also increased tubular acetylated superoxide dismutase 2 (SOD2) levels and activated inflammatory pathways. Importantly, both RSV and NMN attenuated tubular injury, restored mitochondrial metabolic pathways, reduced SOD2 acetylation, improved mitochondrial morphology, and suppressed inflammatory responses. Conclusions and ImplicationsChronic low-dose DOX exposure induces subclinical renal tubular injury characterized by mitochondrial dysfunction and inflammation. The pharmacological activation of sirtuins confers reno-protective effects by preserving mitochondrial homeostasis. These findings identify mitochondrial dysfunction as a central therapeutic target in DOX-induced nephrotoxicity and support sirtuin modulation as a potential strategy for preventing chemotherapy-related chronic kidney injury. Bullet point summaryO_ST_ABSWhat is already knownC_ST_ABSO_LIDoxorubicin causes cardiotoxicity through mitochondrial dysfunction and oxidative stress. C_LIO_LIDoxorubicin-induced tubular injury and the associated late-onset kidney dysfunction are clinically proven. C_LI What does this study addO_LIChronic low-dose doxorubicin induces tubular mitochondrial dysfunction, as identified by integrated multi-omics analyses. C_LIO_LIResveratrol and NMN preserve mitochondrial integrity and suppress inflammatory responses in renal tubules. C_LI Clinical significanceO_LIMitochondrial dysfunction may represent an early therapeutic target in doxorubicin-associated nephrotoxicity. C_LIO_LIActivation of the NAD-Sirtuin axis could prevent chronic kidney injury in cancer survivors. C_LI

To date, no pharmacotherapy has been approved for cocaine use disorder. Endocannabinoid signaling is closely related to dopamine-dependent reinforcement and may regulate cocaine-related behaviors. In this study, we used common marmosets (Callithrix jacchus) to investigate whether 2-arachidonoylglycerol (2-AG), an endogenous cannabinoid lipid, reduces cocaine reinforcement in a non-restraint oral self-administration model. Marmosets performed oral cocaine self-administration under a fixed-ratio 1 schedule, and cocaine intake was confirmed using plasma benzoylecgonine detection. Dopamine transporter (DAT)-related positron emission tomography (PET) signal, DAT and G protein-coupled receptor 55 (GPR55)-associated fluorescent signals, GPR55/DAT immunofluorescence, and synaptosomal dopamine responses were assessed using 18F-N-(3-fluoropropyl)-2{beta}-carboxymethoxy-3{beta}-(4-iodophenyl) nortropane (18F-FP-CIT) PET imaging, ex vivo fluorescent ligand imaging, confocal microscopy, and dopamine quantification. Marmosets acquired oral cocaine self-administration with preferential active lever responding. Repeated cocaine self-administration reduced striatal DAT-related 18F-FP-CIT PET signal in vivo. In striatal slices, cocaine decreased fluorescent false neurotransmitter (FFN102)-associated DAT signal and increased T1117-associated GPR55 signal. Systemic 2-AG pretreatment reduced cocaine self-administration while restoring diminished circulating 2-AG levels. GPR55 and DAT immunofluorescent signals were colocalized in marmoset brain sections, and 2-AG enhanced cocaine-induced dopamine elevation in synaptosomal preparations. Taken together, these findings suggest that 2-AG attenuates cocaine-taking behavior and may be associated with GPR55/DAT-related dopaminergic responses in common marmosets.

Introduction: Cerebral ischemia induces a reduction in brain pH. GPR4, a proton-sensing G protein-coupled receptor is highly expressed in vascular endothelial cells. Pharmacological inhibition of GPR4 with NE52-QQ57 (NEQQ) reduces ischemia-induced blood-brain barrier (BBB) disruption and brain injury. To facilitate targeting GPR4 in vivo, it is important to better define the effect of NEQQ on post-ischemia signaling and the pharmacokinetics of NEQQ in vivo. Methods: We performed biochemical analysis following transient middle cerebral artery occlusion (tMCAO). We used Liquid Chromatography-Mass spectrometry (LC-MS/MS) to quantify the concentration of NEQQ in serum, brain, and peripheral tissues. Results: NEQQ administration decreased the phosphorylation of PKA substrates, PKC substrates, ERK1/2, and CREB. LC-MS/MS analysis detected NEQQ in both ipsilateral and contralateral hemisphere of the brain at 5 h and 24 h after reperfusion, demonstrating that NEQQ crosses the BBB. In peripheral tissues, NEQQ exhibited highest level in kidney and lowest in the heart. In sham controls, average serum concentrations were ~4 and 0.9 g/g at 5 and 24 h, respectively. Following tMCAO, ipsilateral brain exhibited higher levels as compared to the contralateral side at 5 h while the difference diminished at 24 h. Conclusions: NEQQ attenuated the ischemia-induced activation of multiple signaling pathways. NEQQ exhibited broad distribution in peripheral organs, and its brain level was sufficient for GPR4 inhibition at 24 h after injection. These findings support the use of NEQQ as a pharmacological inhibitor of GPR4-mediated signaling following ischemic stroke.

A growing body of evidence demonstrates that signaling pathways of endogenous lipids (endolipids) modulate the reinforcing properties of opioids including reward and withdrawal. Many of these studies are focused on the endocannabinoid (eCB) system, and its primary eCB ligands, Anandamide (AEA) and 2-arachidonoylglycerol (2-AG). The central hypothesis is that modulation of the eCB system through eCB receptors and enzymes may improve therapeutic outcomes for opioid use disorder (OUD). However, outcomes in preclinical and clinical studies using the low efficacy, CB1/CB2 orthosteric agonist, THC, found limited to no effectiveness, suggesting that targeting this aspect of CB1/CB2 is not a useful therapeutic tool for OUD. Previous studies finding that genetic deletion or pharmacological inhibition of endolipid-regulating enzymes (including eCBs) can alter behavioral sensitivity to opioids provide insight into an alternative approach. If opioid use dysregulates a wide range of endolipid biosynthesis and metabolism, then a clearer understanding of these changes, especially in signaling ligands, will provide a novel avenue to both understand the underlying physiological changes with opioid use as well as providing novel targets for therapeutic interventions. In this study, we tested the hypothesis that if morphine dysregulates multiple endolipid signaling systems in the brain and body, and this dysregulation evolves over time, then these differential effects will be measurable by changes in endolipid levels. Using an Acute (30 minutes post injection 20mg/kg) and a Chronic paradigm (5 days, twice daily, 20mg to 100mg/kg escalating dose) we measured 100 targeted endolipids in 8 brain regions, plasma, liver, and feces in male mice. In the Acute condition, we found that the most screened endolipids were changed in the striatum (42%), while the fewest were changed in the thalamus (19%) and 38% in the plasma. In the Chronic condition, 79% of plasma endolipids were changed. The highest level of change in the CNS was in the cortex (39%). Levels of AEA and 2-AG were largely unchanged; however, levels of the N-acyl GABAs, N-acyl valines, N-acyl taurines, and specific bile acids (e.g. DCA, TCA) showed the most dynamic changes by treatment group. These results provide information on novel endolipid signaling pathways that may contribute to the unwanted side effects of opioids, such as dependence and withdrawal, and provide novel avenues for the development of therapeutic strategies.

Background and PurposeSerotonergic psychedelics affect behavior and physiology, but the relationships among these effects remain poorly understood. In rodents, the head-twitch response is used as a measure of psychedelic-like activity, yet it does not capture changes in physiological state or the performance of learned behaviors. Here, we investigated the acute effects of the 5-HT2A receptor agonist TCB-2 across several behavioral and physiological measures and examined how these effects were modified by pretreatment with the 5-HT2A receptor antagonist volinanserin. Experimental ApproachMice were tested in head-fixed and freely moving conditions. During a learned auditory trace-conditioning task, we measured licking, pupil area, eye position, and blinking. We measured locomotor activity in an open field and quantified head-twitch responses using a DeepLabCut-based method. To examine the contribution of 5-HT2A receptors, mice were pretreated with the 5-HT2A receptor antagonist volinanserin. Key ResultsTCB-2 caused pupil constriction without detectable changes in eye position or blinking when administered alone. TCB-2 also reduced licking at the highest dose, but the cue-locked temporal pattern of licking remained evident. In freely moving mice, TCB-2 reduced locomotor activity and produced a dose-dependent increase in head-twitch responses. Volinanserin partially attenuated TCB-2-induced pupil constriction and reduced head-twitch responses under some conditions, but it did not consistently prevent the other effects of TCB-2. Conclusions and ImplicationsTCB-2 produced distinct effects across physiological and behavioral measures rather than a uniform disruption of behavioral function. Pronounced pupil constriction and head-twitch responses occurred without detectable changes in eye position or blinking, while the temporal organization of conditioned licking was retained despite a reduction in its magnitude. The incomplete and variable effects of volinanserin preclude definitive conclusions about the receptor mechanisms underlying each response. Combining automated head-twitch detection with physiological and task-related measurements provides a broader framework for comparing the pharmacological profiles of serotonergic compounds. What is already knownO_LIClassical psychedelics produce characteristic effects primarily through serotonin 5-HT2A receptor activation. C_LIO_LIHead-twitch responses capture only one dimension of psychedelic-like drug action. C_LI What this study addsO_LITCB-2 reduced locomotion and licking while preserving the cue-locked pattern of conditioned licking. C_LIO_LIPupil constriction occurred without detectable changes in eye position or blinking. C_LI Clinical significanceO_LIMultidimensional phenotyping can distinguish the physiological and behavioral profiles of serotonergic compounds. C_LIO_LIComplementary measures may improve preclinical evaluation of emerging serotonergic therapeutics. C_LI

In pre-clinal studies, VC108, a novel GPR39 inhibitor, increased myocardial O2 tension (mpO2) during different phases of acute myocardial infarction (AMI). The purpose of this work was to investigate the temporal effects of VC108 on mpO2 using a novel population PK/PD model developed from preclinical data using healthy animals and animals subjected to AMI protocols. PK/PD simulations were then performed to explore mpO2 dynamic in untested experimental scenarios. VC108 PK data fitted well to a two-compartment model with linear elimination with body weight, sex, and nominal dose identified as significant covariates influencing clearance and/or central compartment volume. Applying indirect effect model principles for representing mpO2 flux, a structural population PK/PD model was developed to capture mpO2 dynamics during different AMI phases: before and during coronary occlusion and after reperfusion. The model successfully captured basal mpO2 homeostasis, ischemic decline, and reperfusion overshoot, while quantifying the contribution of VC108 to collateral blood flow and functional recovery. Dose response relationships were simulated to measure the effect of different VC108 dose levels on permanent damage, i.e. a reduction in mpO2 at steady state after AMI. The probability of remaining below predefined damage thresholds (mpO2 reduction of 5% to 20%) increased with dose in both sexes, with females consistently achieving greater protection that plateaued around 1 mg/kg of VC108 for higher permanent damage. This model represents a valuable tool to describe, for the first time, VC108 efficacy data and pharmacokinetics with ischemia/reperfusion dynamics and quantify its effect in untested dose levels.

We have examined how ibogaine may enhance synaptic plasticity and neural repair. Extracellular efflux analysis showed that ibogaine (3, 10 or 30 M) produced a rapid (<5 minutes) and concentration-dependent suppression of mitochondrial function and glycolysis in mouse cortical neurons. Cell-based imaging demonstrated this metabolic inhibition was preceded by an elevation of the mitochondrial membrane potential and followed by increased mitochondrial reactive oxygen species levels. Like ibogaine, which blocks potassium channels, mitochondrial Kv1.3 channel (mitoKv1.3) inhibitors produce concordant changes over a similar timeframe. Following treatment with ibogaine (3, 10 or 30 M) for 4 hours, a marked concentration-dependent rebound of energy production occurred 20 hours later suggestive of metabolic preconditioning. Indeed, proteomic profiling showed that oral ibogaine (100 mg/kg) administration to female mice produced changes in spinal cord protein abundance indicative of increasing metabolic stress from 0.25-24 hours that resolved by 72 hours. This was accompanied by activation, inhibition and reactivation of synaptic plasticity and neural repair signaling pathways at 0.25, 4-24 and 72 hours, respectively. Hence, by inducing transient metabolic stress, ibogaine may re-open a neurodevelopmental window characterized by enhanced synaptic plasticity and neural repair. Ibogaine binds with high affinity to the sigma-2 receptor. Like mitoKv1-3 inhibitors and sigma-2 receptor ligands, ibogaine transiently elevated AMP-activated protein kinase (0.25 hours) and temporally suppressed mechanistic target of rapamycin and lipid synthesis pathways (0.25-24 hours) implicated in enhanced synaptic plasticity and neural repair. These findings suggest ibogaine rewires and repairs neural circuits by a metabolic preconditioning mechanism, perhaps involving mitoKv1.3 and the sigma-2 receptor.

Background and Purpose: Among new psychoactive substances, synthetic cannabinoid receptor agonists (SCRAs) encompass considerable structural diversity and show wide variation in cannabinoid receptor 1 (CB1) potency and efficacy, but the molecular and receptor-interaction characteristics underlying these differences remain unclear. We assessed the pharmacological profiles of 16 SCRAs and examined structural features associated with differences in CB1 potency and efficacy. Experimental Approach: CB1 agonist activity was measured in a CB1-G15-based Ca2+ assay, and cataleptic effects were assessed in mice. Ligand-receptor interactions were characterized by molecular docking to an active-state CB1 structure. Principal component analysis (PCA) was applied to residue-level interaction profiles, and the resulting scores were tested for associations with CB1 potency and efficacy. Key Results: Potency and efficacy varied widely across cellular and animal assays, with several SCRAs exhibiting different pharmacological responses across the two experimental systems. PCA identified an interaction pattern whose PC1 scores were significantly associated with Emax but not EC50, with contributions from activation-related CB1 residues including the PHE200-TRP356 toggle switch. Molecular descriptor analysis likewise identified structural features significantly associated with in vitro and in vivo Emax. Conclusion and Implications: Our findings suggest that CB1 efficacy is associated with distinct receptor-interaction patterns among structurally diverse SCRAs. Combining functional pharmacology with receptor-level interaction analysis provides structural insight into SCRA efficacy and may help prioritize emerging compounds for further pharmacological and behavioral evaluation.

Peroxisome proliferator-activated receptor (PPAR) is a nuclear receptor that plays a central role in metabolic adaptation by regulating gene expression programs involved in lipid utilization and energy homeostasis. Although the hepatic functions of PPAR and its target genes are well characterized, less is known about its transcriptional activity in extrahepatic tissues. Pemafibrate (K-877), a selective PPAR modulator (SPPARM), is a high-affinity PPAR ligand with improved potency and selectivity compared with conventional fibrates. Here, we investigated pemafibrate-induced tissue-specific transcriptional responses comparing wild type and whole-body PPAR-deficient littermate mice. Twelve-week-old male PPAR+/+ and PPAR-/- mice were treated daily with pemafibrate (0.1 mg/kg) or vehicle by oral gavage for 14 days. We performed genome-wide transcriptomic analyses in the liver, jejunum, kidney, soleus muscle, and BAT. Pemafibrate-induced transcriptional changes were entirely dependent on PPAR, as no differentially expressed genes were detected in PPAR-/- mice. The magnitude and nature of the response were tissue-specific, with the strongest transcriptional effects observed in the liver and jejunum, whereas the kidney, soleus muscle, and BAT showed limited responses. In the liver, pemafibrate primarily induced the expression of genes involved in fatty acid metabolism, mitochondrial function, and energy production. In the jejunum, pemafibrate activated lipid metabolic programs together with tissue-specific pathways related to cellular homeostasis. Collectively, our findings show that pemafibrate is a highly selective PPAR activator with a preferential transcriptional activity in the liver and intestine after oral administration. This study provides a resource for defining tissue-specific PPAR-responsive gene networks and expands our understanding of PPAR functions beyond the liver.

Alcohol-induced toxicity is driven largely by the accumulation of acetaldehyde and disruption of hepatic redox homeostasis during ethanol metabolism. Oxidation of ethanol by alcohol dehydrogenase (ADH) consumes nicotinamide adenine dinucleotide (NAD) while generating NADH, shifting the intracellular redox state toward a highly reduced environment that impairs mitochondrial function, limits endogenous aldehyde dehydrogenase (ALDH)-mediated acetaldehyde clearance, and promotes oxidative stress and tissue injury. We investigated whether UT-018, a novel metabolic intervention, could support endogenous metabolic resilience during acute ethanol exposure using complementary in vitro and in vivo models. Mechanistic in vitro studies evaluated ADH-dependent NADH generation and NAD add-back experiments, while in vivo investigations assessed serum ALDH-associated activity, circulating acetaldehyde concentrations, and gross gastrointestinal and hepatic morphology following acute ethanol challenge. UT-018 reduced ethanol-associated NADH accumulation in a concentration-dependent manner without evidence of irreversible ADH inhibition. Restoration of NADH generation following supplementation with exogenous NAD demonstrated reversible modulation of ethanol-associated redox biology rather than direct enzymatic inhibition. In vivo, UT-018 enhanced serum ALDH-associated activity, reduced circulating acetaldehyde concentrations by approximately 27 to 33% compared with ethanol-treated controls. Metabolic biomarkers were accompanied by preservation of gross colon and liver morphology following acute ethanol exposure. Collectively, these findings support coordinated biological activity across multiple interconnected stages of alcohol metabolism and support a systems-level mechanism in which restoration of redox homeostasis enhances endogenous aldehyde detoxification, reduces acetaldehyde burden, and preserves tissue integrity. These results identify alcohol metabolism restoration as a promising strategy for enhancing physiological resilience to acute alcohol exposure and provide a rationale for further preclinical and clinical evaluation of UT-018. HighlightsO_LIUT-018 restored ethanol-associated redox homeostasis by reducing excessive NADH accumulation without irreversible inhibition of alcohol dehydrogenase in vitro. C_LIO_LIRestoration of redox balance was associated with enhanced endogenous aldehyde dehydrogenase (ALDH)-associated activity following acute ethanol exposure in vivo. C_LIO_LIUT-018 reduced circulating acetaldehyde concentrations by approximately 30%. C_LIO_LIThe metabolic homeostasis was accompanied by preservation of gross gastrointestinal and hepatic morphology in an acute ethanol challenge model. C_LIO_LIThe collective findings support a systems-level mechanism in which modulation of endogenous alcohol related metabolic pathways enhances physiological resilience to acute alcohol exposure. C_LI

Fentanyl-related overdose deaths now commonly involve non-injection routes, yet preclinical opioid self-administration is modeled predominantly intravenously. Here we establish an oral fentanyl self-administration procedure in male and female inbred C57BL/6 and outbred CD1 mice that measures volitional intake, cue-driven seeking, extinction, and relapse. Mice self-administered oral fentanyl (70 {micro}g/mL) on a fixed-ratio 1 schedule across fifteen 3-hour sessions, followed by ten extinction sessions and a cued reinstatement test. A separate cohort underwent between-session dose thresholding across a quarter-log series from 222 to 22 {micro}g/mL. Seventy-five percent of mice acquired self-administration, with similar rates across genetic background and sex. Responding increased as fentanyl concentration fell, indicating dose-sensitivity toward a preferred drug level. C57BL/6 mice escalated intake and lever pressing across sessions, responded persistently early in extinction before declining, and reinstated pressing to a conditioned cue. CD1 mice consumed high levels from the outset with limited escalation and showed neither extinction nor cued reinstatement of pressing, but shortened their reward-port approach latency when cues returned. This shows that lever presses alone would have misclassified them as weakly conditioned. A composite severity score summing seven components of fentanyl-use risk varied continuously rather than splitting into high and low groups, even among inbred mice. Sex differences were largely confined to C57BL/6 mice, in which females showed stronger cue association and higher severity scores than males. These results reveal separable escalation-prone and relapse-prone phenotypes that track genetic background. Protocols, hardware specifications, and analysis code are openly available, lowering the barrier to adopting oral fentanyl self-administration.

Schizophrenia is a debilitating neuropsychiatric disease that lacks effective treatments for many symptom domains including negative, cognitive and sleep disturbances. Lack of clear disease etiology has hampered the development of new, effective treatments for the unmet needs of people with schizophrenia. Large scale human genetics have identified rare loss of function mutations that substantially increase risk of developing schizophrenia, including in GRIA3, the gene that encodes the GluA3 receptor subunit of the AMPA receptor (AMPAR). Several drug discovery programs have been aimed at developing AMPAR positive allosteric modulators (PAMs) as a novel treatment for schizophrenia. Despite intense drug discovery efforts, there are no FDA approved AMPAR PAMs. We therefore hypothesized that selectively targeting GluA3, the AMPAR subunit implicated by human genetics, could yield a safer and more effective AMPAR PAM for the potential treatment of schizophrenia. Using a combination of medicinal chemistry, in vitro, and in vivo studies, we discovered BRD3290, a GluA3-preferring AMPAR PAM with reasonable potency in heterologous cells, as well as favorable tolerability and brain exposure. Peripheral administration of BRD3290 engaged an established AMPAR PAM target engagement biomarker but did not improve performance of wildtype mice in the novel object recognition task (NOR), in contrast to the nonselective AMPAR PAM PF-4778574, which improved mouse NOR. These findings suggest that the GluA3 selectivity profile of BRD3290 was insufficient to enhance cognitive function in this mouse NOR paradigm. This work highlights the challenges of AMPAR subtype-selective modulation and provides molecular insights into the ability to develop subtype-selective AMPAR PAMs. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=110 SRC="FIGDIR/small/740780v1_ufig1.gif" ALT="Figure 1"> View larger version (28K): org.highwire.dtl.DTLVardef@1c14b74org.highwire.dtl.DTLVardef@140b2b3org.highwire.dtl.DTLVardef@942636org.highwire.dtl.DTLVardef@58bc24_HPS_FORMAT_FIGEXP M_FIG C_FIG

Alkaline ceramidase 2 (ACER2) catalyzes the hydrolysis of ceramides into sphingosine and free fatty acids, playing crucial roles in regulating various biological processes, including but not limited to cell proliferation, survival, and the DNA damage response. However, a pharmacological tool that regulates its catalytic and signaling functions remains to be developed. In this study, N-[(1S,2R)-1-hydroxy-1-phenylpropan-2-yl]dodecane-1-sulfonamide (KPB82) was identified as a potent and selective ACER2 inhibitor. In vitro enzymatic assays showed that KPB82 inhibited ACER2 with an IC50 of 190 nM, while displaying minimal activity against acid and neutral ceramidases (IC50> 100 {micro}M) and no significant inhibition of alkaline ceramidase 1 or alkaline ceramidase 3. Enzymatic kinetic studies revealed that KPB82 is a competitive inhibitor of ACER2. The three-dimensional structure of ACER2 was modeled in the AlphaFold3 server, revealing key Zn2+-coordinating histidine residues and a substrate-binding pocket. Molecular docking studies demonstrated that KPB82 binds ACER2 within this pocket, forming hydrogen bonds with the catalytically important amino residues, Asp93 and His215, and interacting with several hydrophobic residues. Lactate dehydrogenase release assays revealed that KPB82 significantly reduced cytotoxicity in HeLa cells overexpressing ACER2, as well as in HCT116 cells, RT4 cells, and human umbilical vein endothelial cells treated with the DNA-damaging agent doxorubicin. Lipidomic analysis showed that KPB82 inhibited sphingosine production in response to ACER2 overexpression or doxorubicin treatment. Collectively, these findings demonstrate that KPB82 is a potent and selective ACER2 inhibitor capable of suppressing ACER2-dependent biological processes and highlight its potential as a tool for studying ceramide metabolism and ACER2-mediated signaling pathways.

Cinnamaldehyde (CA) is extensively used as flavorant and in traditional medicine. CA is com-monly used in pain research as specific agonist of TRPA1, a polymodal cation channel expressed in nociceptive primary sensory neurons. However, we previously showed that CA inhibits the L-type Ca2+ channel in cardiac and smooth muscle cells, raising the possibility that other ion channels can be modulated by this compound as well. Here, we investigated whether CA exhibits affects the activity on the cardiac NaV1.5 channel. We used the whole-cell patch-clamp technique to record Na+ currents in HEK293T cells expressing the human NaV1.5 channel, as well as in cells expressing hNaV1.5 channels baring mutation in the binding pocket of local anesthetics (LA). Our results show that CA exhibit LA-like actions on hNaV1.5 channels: CA blocks hNaV1.5 currents in a tonic and voltage-dependent fashion. Residues F1760 and Y1767 are important for the blockade of NaV1.5 by CA and a double point mutation F1760A/Y1767A abolishes the blockade of INa by CA. We conclude that CA and LID share common structural determinants for the inhibition of Na+ channels and that CA has LA-like actions.

Histological necrosis is the reference measure of hepatotoxicity but can only be obtained at terminal cull. A time course requires independent cohorts at every timepoint. Circulating alanine aminotransferase (ALT) can be sampled repeatedly in the same animal, and is released in liver injury. We asked whether serial ALT kinetics can be used to estimate histological necrosis in vivo. Forty-five fasted twelve-week-old male C57BL/6J mice received a single intraperitoneal dose of 350 mg/kg acetaminophen (paracetamol). Plasma ALT and microRNA-122 (miR-122) were measured serially from baseline to 48h as cumulative area-under-the-curve (AUC) to cull, against centrilobular necrosis on haematoxylin and eosin sections as reference (range 0 to 59%, mean 32%). A generalised additive model of necrosis on cumulative ALT AUC and time since dosing predicted necrosis with a leave-one-animal-out cross-validated mean absolute error of 7.7% necrotic area (95% CI 5.6 to 10.0; n=49). A panel of traditional regression and machine-learning models all gave equal or larger error, and adding an additional biomarker or regeneration information did not improve prediction. Serial ALT kinetics therefore provide a calibrated, longitudinal measure of hepatic necrosis in vivo and give a more stable estimate of within-group variance for study planning, while supporting reductions in animal use, because one serially-sampled cohort can replace separate cohorts at each timepoint.

Gabapentin (GBP) and pregabalin (PGB) are widely used gabapentinoids. Previously, we have demonstrated, for the first time, that GBP and PGB modulate the nociceptive response to noxious heat in C. elegans at an optimal concentration. In the current study, we use C. elegans and paired thermal nociception assays with direct internal drug concentration measurements to characterize the pharmacokinetic (PK)/pharmacodynamic (PD) relationship of both compounds. Neither drug altered baseline mobility or quadrant preference, confirming that behavioral effects reflected genuine antinociceptive action. Both GBP and PGB produced dose- and time-dependent reductions in thermal avoidance, with 500 uM exposures generating a biphasic, V-shaped time course in which suppression of thermal sensitivity deepened before partially reversing. This partial reversal occurred later with PGB than with GBP. Internal concentrations confirmed dose-dependent absorption and retention for both drugs, yet at 500 uM, internal drug levels remained elevated through 360 min even as behavioral avoidance recovered, indicating that the recovery limb reflects active counter-regulation rather than passive clearance, consistent with previously reported transcriptional and proteomic signatures. Exposure-response profiles were notably flat, suggesting a saturable pharmacodynamic ceiling. Molecular modeling revealed conserved electronic pharmacophores supporting shared alpha-2-delta engagement, alongside shape-descriptor differences that may contribute to divergent absorption kinetics. These findings position C. elegans as a valuable model for dissecting gabapentinoid PK/PD relationships. Beyond mechanistic insight, these findings support the continued investigation of C. elegans as a screening platform whose validation could help address the 3R (Replacement, Reduction, Refinement) principles guiding animal research.

BackgroundAcute kidney injury (AKI) frequently progresses to chronic kidney disease (CKD), yet mechanisms governing this transition remain poorly understood. The endocannabinoid system (ECS), particularly cannabinoid-1 receptor (CB1R), regulates inflammation and metabolism in various organs, but its role in post-AKI maladaptive repair is less established. MethodsWe analyzed CB1R expression in kidney biopsies from pre- and post-transplant recipients and in murine AKI models (ischemia-reperfusion injury [IRI] and folic acid [FA]-induced AKI). Peripheral CB1R blockade was evaluated in FA-AKI model and in human primary kidney proximal tubule cells (hKPTCs). Spatial metabolomics, semi-targeted metabolomic profiling, and gene and protein expression characterized molecular mechanisms. ResultsCB1R expression was increased in kidneys undergoing maladaptive repair in both humans and mice, but remained unchanged during acute injury. In the FA-induced AKI model, the ECS showed stage-specific alterations, with temporal and spatial fluctuations in endocannabinoid levels and their enzymatic regulators. Peripheral CB1R blockade during the repair phase preserved kidney function, reduced injury, and maintained systemic glucose homeostasis. Metabolomic and molecular analyses revealed that CB1R blockade restored dysregulated arginine metabolism and reduced AKT/NF-{kappa}B-p65 pathway in post-AKI kidneys, linking CB1R activation to inflammatory signaling. In hKPTCs, 2-AG-induced activation of CB1R increased VCAM1 expression, a failed-repair marker, while its antagonism reduced TNF/2-AG-induced expression of pro-inflammatory adhesion molecules, chemokines, cytokines, and arginine metabolism enzymes. ConclusionsCB1R overactivation drives AKI-to-CKD progression by promoting inflammatory signaling and metabolic dysregulation. Peripheral CB1R blockade during the repair phase represents a novel therapeutic strategy to prevent maladaptive repair and CKD development after AKI. These findings establish CB1R as a phase-specific therapeutic target for post-AKI intervention. Translational StatementPeripheral CB1R antagonists offer a first-in-class therapeutic strategy to halt progression from acute kidney injury (AKI) to chronic kidney disease (CKD) by selectively targeting maladaptive tubular repair. By blocking CB1R signaling specifically in the kidney, these agents attenuate inflammation, metabolic dysregulation, and fibrogenic pathways that drive failed repair, while sparing central nervous system CB1R and thereby minimizing neuropsychiatric adverse effects. This phase-specific, peripherally restricted approach supports the development of peripheral CB1R antagonists as a viable translational therapy to improve long-term renal outcomes after AKI.

Introduction: Preterm birth is often associated with fetal membrane (FM) inflammation. Opioid-use disorder during pregnancy is a major public health issue as all opioids carry a risk for preterm birth. While maintenance therapy with methadone improves maternal outcomes, it also elevates the risk of preterm birth. We previously reported that the opioid, buprenorphine, induced human FM sterile inflammation. The goal of this study was to investigate if methadone also induced FM inflammation and to determine the mechanisms involved. Methods: Human FM explants were treated with or without methadone in the presence or absence of inhibitors to TLR4/MD2, TLR7, TLR8, NLRP3, the -opioid receptor, p65 NF{kappa}B, p38 MAPK, ERK or JNK. Supernatants were measured for cytokines/chemokines by ELISA. Tissues were analyzed by Western blot for NF{kappa}B and MAPK activity, and by RT-qPCR for the TLR7/8-activating miRs: miR-21a, miR-29a, miR-146a-3p, Let-7b. Pregnant C57BL/6 mice were injected with PBS or methadone on E15.5 and 6hrs later FMs were analyzed by RT-qPCR. Results: Methadone elevated human FM explant secretion of IL-1{beta}, TNF, MIP-1, GM-CSF, and PGE2, and expression of miR-21a. In vivo, methadone elevated mouse FM expression of MIP1A, KC, and COX2. TLR4/MD2 inhibition had no effect on human FM inflammation in response to methadone. NLRP3 inhibition reduced methadone-induced FM IL-1{beta}, TLR7 inhibition reduced methadone-induced FM TNF, and inhibition of either TLR8 or the -opioid receptor reduced methadone-induced secretion of TNF, MIP-1, GM-CSF, and PGE2, but not IL-1{beta} Methadone increased human FM activity of p65 NF{kappa}B, p38 MAPK, JNK, and ERK and inhibition of these signaling pathways collectively reduced methadone-triggered FM inflammation. Finally, naloxone reduced most of the methadone-induced FM inflammatory response. Conclusion: Methadone induces sterile inflammation in human and mouse FMs. In human FMs this inflammation is mediated through activation of innate immune TLR7, TLR8, and NLRP3; the -opioid receptor; and NF{kappa}B/MAPK signaling. These findings provide further insight into the mechanisms underlying the association between opioid use during pregnancy and preterm birth.