The Journal of Pharmacology and Experimental Therapeutics

PurposeWe previously showed that mice expressing a phosphorylation-deficient kappa opioid receptor mutant (K4A) exhibited reduced U50,488H-induced anti-scratching tolerance in males and reduced conditioned place aversion in females, without changes in acute anti-scratching or hypo-locomotor effects. Here, we examined whether K4A mutations, which markedly diminish {beta}-arrestin-mediated signaling, alter U50,488H-induced increases in serum corticosterone and urine output. MethodsK4A and wildtype mice received U50,488H (5 mg/kg, s.c.) or saline. Serum corticosterone was measured by ELISA 1 h later. Urine was collected for 1 h beginning 10 min after injection. ResultsU50,488H increased serum corticosterone to similar levels in wildtype and K4A mice of both sexes. Basal corticosterone levels were higher in females than males regardless of genotype. U50,488H also significantly increased urine output in both sexes, with no genotype differences. However, the increase in urine output was greater in males than females. ConclusionsKOR phosphorylation and associated {beta}-arrestin-mediated signaling are not required for U50,488H-induced increases in serum corticosterone or diuresis in either sex. These findings also demonstrate, for the first time, that KOR activation produces greater diuresis in male than female mice.

Acetaminophen (APAP) overdose is the leading cause of acute liver failure (ALF), with acute kidney injury (AKI) contributing substantially to morbidity and mortality in those patients. To determine whether APAP-induced AKI depends on hepatic CYP2E1-mediated bioactivation, we used CYP2E1^flox/flox^ mice treated with AAV8-TBG-Cre to selectively delete hepatic CYP2E1 while preserving renal metabolism. Male and female mice received APAP (600 mg/kg) and were evaluated up to 48 hours for liver and kidney injury. Liver-specific CYP2E1 deletion reduced APAP hepatotoxicity, confirming the absence of hepatic NAPQI formation. Despite this protection, both male and female mice treated with AAV8-TBG-Cre and APAP developed progressive renal injury, with marked increases in blood urea nitrogen (BUN) and creatinine, tubular vacuolation, and strong induction of KIM-1 and osteopontin, along with apoptotic cell death at 48 hours. Notably, female mice, lacking renal CYP2E1 and displaying no detectable renal protein adducts, still progressed to AKI, demonstrating that kidney injury can occur through CYP-independent mechanisms. Given that APAP-induced AKI is a delayed injury, we further considered p-aminophenol (PAP), a deacetylation product of APAP, as a potential CYP-independent contributor. These findings support the concept that non-CYP pathways, including PAP formation, may contribute to kidney injury during the later phase of toxicity, although this pathway likely represents only one component of a multifactorial injury process. Together, these results demonstrate that APAP-induced AKI is a kidney-intrinsic process that can develop independently of both hepatic and renal CYP2E1 activity, emphasizing the need for kidney-specific therapeutic strategies for preventing APAP-induced renal injury.

Background & Aims: Activation of apoptosis signal-regulating kinase 1 (ASK1), a ubiquitous redox-sensitive kinase, results in inflammation, apoptosis, and fibrosis, key common pathways in human liver disease. SRT-015 is a novel, small molecule inhibitor of ASK1. This study evaluated the in vitro efficacy of SRT-015, compared it to other ASK1 inhibitors, and determined the in vivo efficacy of SRT-015 across multiple acute and chronic liver disease models. Methods: In vitro studies determined the kinase potency and selectivity of SRT-015, and cellular studies were used to demonstrate direct mechanisms of action. The cardiac hERG channel inhibition was assessed and PK determined in rodents and nonhuman primates. In vivo studies evaluated SRT-015 efficacy in rodent models of drug-induced hepatotoxicity (acetaminophen (APAP) overdose), alcohol-associated liver disease (ALD), metabolic-disease associated steatohepatitis (MASH) and cholestatic disease (bile duct ligation, BDL). Results: SRT-015, was demonstrated a selective ASK1 kinase, and SRT-015 treatment directly inhibited fibrosis, apoptosis and inflammation in activated human fibroblasts, hepatocytes and PBMCs, respectively without safety signals or hERG inhibition. Other ASK1 inhibitors had safety concerns or limited functional activity. Liver and kidney selective PK were observed for SRT-015 in all species evaluated. In vivo, SRT-015 treatment was efficacious in the acute mouse APAP overdose and ALD model significantly (P<0.05) decreasing serum ALT. Using a therapeutic diet-induced obesity (DIO)-MASH model with biopsy-verified fibrosis, SRT-015 treatment significantly (P<0.05) inhibited DIO-induced liver enzymes, hepatomegaly, fibrosis, inflammation, and apoptosis independent of body weight loss whereas treatment with selonsertib was ineffective. In a rat cholestatic model, SRT-015 treatment significantly (P<0.05) decreased fibrosis and stellate cell activation. Conclusions: These findings support SRT-015 as a potential therapeutic for human liver diseases of any etiology.

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

Repeated low-dose psilocybin is being developed as a scalable outpatient treatment for mood and anxiety disorders, but chronic exposure raises concern because psilocin binds the cardiac serotonin 5-HT2B receptor, whose sustained agonism causes drug-induced valvular heart disease (VHD). We evaluated this risk using an exposure-response model that incorporates functional efficacy and exposure duration rather than binding affinity alone. Plasma psilocin concentrations were converted into the time-integrated increment in 5-HT2B Gq signaling above endogenous serotonergic tone ({Delta}TIA) and calibrated against drugs and conditions with known valvular outcomes. All modeled exposures known to cause human VHD scored {Delta}TIA [&ge;] +172 %{middle dot}h/day, whereas exposures not associated with VHD scored [&le;] +28. A candidate 3 mg daily psilocybin regimen scored {Delta}TIA +3, roughly two orders of magnitude below the weakest valvulopathic exposure. This safety margin arises from psilocins low-efficacy partial agonism at 5-HT2B (Emax {approx}51.8% of serotonin, compared with 96% for norfenfluramine) and its short half-life ({approx} 2.5 h), which prevents accumulation and produces brief daily receptor engagement. In support of the model, rats receiving continuous psilocin for 12 days at plasma concentrations {approx}2.4-fold above the projected human peak for 3 mg daily psilocybin showed no valvular lesions by blinded histopathology. This exposure duration however cannot exclude slowly developing fibrosis. Emerging human data, including serial echocardiography in repeated LSD microdosing and a large observational cohort, are also agreement with the model. Collectively, these findings suggest a favorable safety margin for daily, sub-hallucinogenic psilocybin use in clinical indications. Nevertheless, continued pharmacological and clinical investigations should include prospective echocardiographic monitoring to advance the clinical safety profile of sub-hallucinogenic psilocybin and support its evaluation across a broad array of therapeutic programs. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=54 SRC="FIGDIR/small/739440v1_ufig1.gif" ALT="Figure 1"> View larger version (10K): org.highwire.dtl.DTLVardef@1ef0508org.highwire.dtl.DTLVardef@1337d52org.highwire.dtl.DTLVardef@1689943org.highwire.dtl.DTLVardef@261605_HPS_FORMAT_FIGEXP M_FIG C_FIG Three key determinants of cardiac safety margins for repeated low-dose psilocybin are shown. Psilocin is a low-efficacy partial agonist at 5-HT2B (ceiling {approx}52% vs 96% for norfenfluramine; left). Its short half-life yields a brief daily pulse of receptor engagement rather than a sustained plateau (center). The resulting integrated 5-HT2B signal ({Delta}TIA) at 3 mg daily lies roughly two orders of magnitude below valvulopathic exposures, and continuous in vivo exposure produced no valvulopathy (right).

The need for improved treatments for chronic pain has driven increased interest in cannabis-based therapeutics. Peripheral dorsal root ganglion (DRG) neurons, including nociceptors, express cannabinoid receptors (CB1 and CB2), suggesting that modulation of DRG excitability may provide an effective strategy for peripheral analgesia. Here, we investigated the effects of cannabidiol (CBD), {Delta}9-tetrahydrocannabinol (THC), terpene mixtures as well as cannabis plant extracts on neuronal excitability in small-diameter mouse DRG neurons using whole-cell current-clamp electrophysiology and assessed potential synergistic interactions. Both CBD and THC produced a concentration- and time-dependent inhibition of rheobase-evoked action potential firing, which were reversible in the presence of bovine serum albumin (BSA), both with similar estimated IC50 values of 5 M (. Terpene mixtures, as well as individual terpenes (linalool, {beta}-pinene, and myrcene), similarly reduced neuronal firing. Co-application of CBD with THC or terpenes enhanced inhibition, consistent with synergistic interactions and the known "entourage effect." Application of WIN55,212-2 (WIN), a non-selective cannabinoid receptor agonist, in the presence of CBD also accelerated the time-dependent inhibition of neuronal firing. The inhibition of firing by the CB2-selective inverse agonist JTE-907 indicated the presence of CB2 receptors on DRG neurons. Plant extracts from the Cannabis sativa leaves also reversibly inhibited neuronal firing. CBD and a terpenes mixture produced modest effects on hERG channels, whereas plants extracts had negligible effects. Collectively, these findings demonstrate that phytocannabinoids and terpenes suppress peripheral sensory neuron excitability via receptor-dependent and indirect mechanisms, supporting their potential as non-opioid analgesics. Their synergistic interactions suggest that multi-component formulations may enhance analgesic effects.

Malignant brain tumors are among the most aggressive and difficult to treat human cancers. Glioblastomas (World Health Organization grade IV gliomas) are particularly lethal and refractory to treatment. Few drugs exist that are even somewhat effective. Our investigation of the physiologic role of fatty acid (FA) activating enzymes (acyl-CoA synthetase; ACS) identified an ACS that was widely expressed in gliomas but not in normal glial cells. Depletion of this enzyme, ACSVL3 (very long-chain ACS3), by knockdown or knockout decreased the malignant behavior of several glioma cell models including U87MG and Mayo-22 cells both in culture and when grown as xenografts. Hypothesizing that ACSVL3 is a potential therapeutic target in glioma, we conducted a search for inhibitors of this enzyme and found that CB5 (grassofermata) was a promising candidate. Treating U87MG glioma cells with CB5 slowed growth in monolayer culture; the growth rate was similar to that seen in cells in which ACSVL3 was either knocked down or knocked out. CB5 inhibited growth in a dose-dependent manner over a narrow range, and concentrations above 10 M were toxic. Treatment at the lower dose of 3 M inhibited growth of U87MG cells but was reversible, suggesting that this dose was not toxic. CB5- treated U87MG cells exhibited an altered morphology with a larger size and longer projections. In contrast, normal human fibroblasts treated with 10 M CB5, a concentration that was toxic to U87MG cells, showed no effect on either growth rate or morphology. Treating U87MG cells with 3 M CB5 induced differentiation as shown by increased expression of the astrocyte-specific marker glial fibrillary acidic protein (GFAP). In contrast, GFAP levels remained low in ACSVL3 knockdown cells. CB5- treated U87MG cells were less invasive, and thus less malignant, than either untreated cells or ACSVL3 knockout cells when assessed by a scratch wound healing assay. Acute treatment of U87MG cells with 3 M CB5 decreased the ability of these cells to degrade FA of differing chain lengths from 16-24 carbons by {beta}-oxidation, suggesting that decreased ACS enzyme activity contributes at least in part to the drugs mechanism of action. NOD/SCID mice receiving up to 32 mg/kg/day CB5 by intraperitoneal injection showed no obvious side effects, suggesting that the drug was well-tolerated. Xenografts induced by subcutaneous injection of U87MG cells in the flanks of NOD/SCID mice were allowed to grow for 8 days after which half of the mice were treated with 2 mg/kg/day CB5. After 7 days of treatment, xenograft growth slowed in the treated mice and by 12 days tumor size had begun to decrease, suggesting therapeutic efficacy. When a similar study was done using xenografts induced by subcutaneous injection of Mayo-22 cells, which are maintained as subcutaneous tumors in mice rather than in cell culture, the effect of CB5 on tumor growth or weight at sacrifice was not statistically significant. The results of these studies suggest that CB5 may have therapeutic value in malignant glioma. Additional studies using other glioma models and other drugs chemically related to CB5 seem warranted.

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

Acute kidney injury (AKI) affects millions of patients annually and is associated with high morbidity and mortality, to date no curative treatment exists. Drug-induced nephrotoxicity accounts for up to 25% of AKI cases, but individual susceptibility remains hard to predict. While genetic factors are suspected to play a part in this variability, the pharmacogenomics of nephrotoxin-induced kidney injury remain largely unknown. To investigate genetically determined susceptibility, we used precision-cut kidney slices (PCKS) from the two founder strains of the BXD mouse consortium, C57BL/6J and DBA/2J. PCKS preserves tissue architecture and cell-cell interaction, allowing close experimental control while maintaining the renal microenvironment. Slices were exposed to cyclosporine A (80 nM for 6h, 20nM for 24h and 48h) and Tunicamycin (1 {micro}M for 6h and for 24h) as well as normoxia (4{degrees}C for 20h) and hyperoxia (4{degrees}C for 20h and 4h in incubator). Slices were then analysed using histopathological scoring, TUNEL staining, ATP quantification and bulk RNA sequencing. We found that the main source of variation was experimental duration. Nevertheless, a subtle difference between the strains could be observed for both cyclosporine A and Tunicamycin, with DBA/2J showing a stronger response to nephrotoxic stress, including lower ATP levels, higher proportion of apoptotic cells and a more pronounced transcriptomic response. For both strains, normoxia was the least harmful condition. These findings support the hypothesis that the BXD founder strains differ in their susceptibility to nephrotoxic kidney injury and support the use of PCKS as a relevant ex vivo model for studying early renal stress response. This provides the foundation to extend this approach to a broader spectrum of the BXD population to identify genetic loci and candidate genes involved in genetic susceptibility to nephrotoxins.

An acetaminophen (APAP) overdose is the leading cause of drug-induced hepatotoxicity and acute liver failure (ALF) in the United States. While N-acetylcysteine (NAC), is highly effective when administered early after an overdose, its efficacy decreases with delayed administration. Since most patients present late to the clinic, there is an urgent need for novel late-acting therapeutic options to prevent progression to ALF. We previously demonstrated the benefit of delayed activation of the Adenosine A2B Receptor (A2BAR) in attenuating APAP-induced hepatotoxicity and this study focuses on its effects on liver recovery after injury. Fasted male C57BL/6J mice were treated with 300 mg/kg APAP, followed by activation of A2BAR 6 or 9 h later and sacrifice 24, 48 or 72 h post-APAP with evaluation of liver injury, the innate immune response and liver regeneration. Delayed activation of A2BAR significantly enhanced liver recovery, with accelerated repopulation of the liver by Kupffer cells, increased macrophage migration to the necrotic areas and their faster resolution. A2BAR activation also upregulated lipid metabolism related genes in non-parenchymal cells and cell proliferation and metabolism genes in hepatocytes. Remarkably, genes such as Cidec and Plin2, crucial for lipid droplet formation, were upregulated, indicating that A2ABR activation enhances lipid metabolism which plays a key role in providing energy for liver regeneration. Overall, these findings highlight the potential of A2BAR activation not only in protecting against liver injury, but also in promoting and accelerating liver regeneration by modulating the innate immune responses and metabolic pathways.

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.

Cannabidiol (CBD) is a non-psychoactive component of cannabis that has been studied as a potential therapy for chronic pain. CBD attenuates behavioral hypersensitivities in models of neuropathic pain, and promotes production of bioactive lipids (e.g., anandamide), altering lipid signaling. However, a lack of understanding of the mechanisms underlying the therapeutic effects of CBD has hindered development and application of CBD to mechanism-based therapies for pain in people. We asked whether the analgesics effects of CBD were dependent upon the enzyme NAPE-PLD. We used a mouse model of chemotherapy-induced peripheral neuropathy (CIPN) to evaluate the acute and chronic antinociceptive effects of CBD and investigate its mechanisms. Pharmacological specificity was tested with antagonists targeting CB1, CB2, PPAR{gamma}, and PPAR receptors. Mechanisms were further examined using NAPE-PLD and GPR55 knockout mice. We also assessed repeated CBD dosing during both the development and maintenance of paclitaxel-induced CIPN in wild-type, GPR55 KO, and NAPE-PLD KO mice. CBD suppressed paclitaxel-induced behavioral hypersensitivities; these effects were attenuated by a PPAR and PPAR{gamma} antagonists, but not CB1 or CB2 antagonists. CBD reduced both the development and maintenance of neuropathic nociception in a model CIPN in wild-type mice, but these effects were absent in NAPE-PLD KO mice. By contrast, anti-allodynic efficacy of CBD was fully preserved in GPR55 KO mice. Pharmacological blockade of the PPAR receptor and genetic deletion of NAPE-PLD abolished the antinociceptive effects of CBD in a model of CIPN, suggesting a pivotal role for NAPE-PLD and PPAR receptors in CBD-mediated analgesia in chemotherapy-induced neuropathic pain.

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

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.

Hypoxia-inducible factor prolyl hydroxylase (HIF-PH) inhibitors are widely used for the treatment of renal anemia; however, their effects on intraocular vascular endothelial growth factor (VEGF) expression remain unclear. In this study, we examined the effects of all five HIF-PH inhibitors --roxadustat, daprodustat, vadadustat, enarodustat, and molidustat--on Vegfa expression in the retina in mice. C57BL/6J mice were orally administered each inhibitor. Six hours after administration, the kidney, retina, and liver were collected, and transcription levels were quantified by real-time quantitative reverse transcription PCR. Renal Epo transcription was significantly increased by molidustat (P < 0.01), roxadustat (P < 0.01), and enarodustat (P < 0.05). Retinal Vegfa transcription was significantly increased by four inhibitors (P < 0.01), with molidustat showing no significant effect. In the liver, Vegfa transcription was increased by daprodustat (P < 0.05) and vadadustat (P < 0.01). Furthermore, renal Epo and retinal Vegfa transcription levels showed a moderate positive correlation with a marginal trend toward statistical significance (r = 0.37, P = 0.08). These findings indicate that HIF-PH inhibitors differentially regulate hypoxia-responsive genes across tissues and suggest that retinal VEGF upregulation should be considered when evaluating the safety of these agents.

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.

Parkinsons disease (PD) is characterised by progressive dopaminergic neurodegeneration in the substantia nigra, leading to debilitating motor dysfunction. Current treatments remain largely symptomatic, highlighting the need for disease-modifying therapies. {beta}1 integrin, implicated in neuroinflammation and trophic signalling, represents a candidate therapeutic target. We investigated whether allosteric {beta}1 integrin modulation could attenuate motor asymmetry in the unilateral 6-hydroxydopamine (6-OHDA) mouse model of PD. Adult male C57BL/6 mice received intracerebral 6-OHDA into the substantia nigra. The anti-{beta}1 integrin antibody JB1a (50 {micro}g) was administered prophylactically (3 days pre-lesion) or therapeutically (3 or 7 days post-lesion). Motor asymmetry was assessed through spontaneous circling (5 min) and apomorphine-induced (0.5 mg/kg s.c.) circling (30 min). 6-OHDA induced dose-dependent contralateral circling, confirming nigrostriatal lesion. Pre-treatment with JB1a (3 days before 6-OHDA) reduced apomorphine-induced circling, although this did not reach statistical significance (28.5 {+/-} 12.8, n = 4 versus 38.6 {+/-} 7.5, n = 8; p>0.05). Post-treatment at 3 days post-lesion produced no statistically significant change in either spontaneous or apomorphine-induced circling (p>0.05). Post-treatment at 7 days post-lesion reduced apomorphine-induced circling by approximately 50%, with values returning to those of sham-operated controls (n =8-9; p<0.01). These findings, obtained in a murine 6-OHDA model, indicate that allosteric {beta}1 integrin modulation attenuates lesion-induced motor asymmetry with apparent temporal specificity. As apomorphine-induced rotation reflects post-synaptic dopamine receptor supersensitivity rather than direct neuronal preservation, and as histological confirmation of dopaminergic integrity was not obtainable in this study, the present data should be interpreted as proof-of-concept behavioural evidence requiring further mechanistic and translational validation in models incorporating -synuclein pathology. The findings are not directly generalizable to human Parkinsons disease. The histological confirmation of lesion extent was not available and as such the behavioural findings are correspondingly interpreted as a proof-of-concept observation requiring histological replication.

Volume-regulated anion channels (VRACs), formed by leucine-rich repeat-containing 8 (LRRC8) proteins, are ubiquitously expressed chloride channels essential for cell volume regulation and implicated in diverse physiological and pathological processes. Small-molecule VRAC inhibitors have been reported to modulate paracrine signaling, proliferation, differentiation, migration, and apoptosis, and have been patented for potential therapeutic applications in stroke, cardiovascular and metabolic diseases, and cancer. However, growing evidence indicates that many commonly used VRAC blockers exert substantial off-target effects and frequently fail to reproduce phenotypes observed after deletion of the essential VRAC subunit LRRC8A. Here, we systematically compared effects of several widely used pharmacological VRAC inhibitors with outcomes of molecular downregulation of LRRC8A in limiting proliferation of malignant glioblastoma cells derived from surgical specimens. NIH/3T3 fibroblasts served as a non-malignant control. In serum-containing media, structurally diverse VRAC blockers (DCPIB, DIDS, carbenoxolone, phloretin, and bromadiolone) reduced proliferation in a non-uniform manner, with potencies that did not correlate with reported VRAC affinities and varied markedly among cell lines. Radiotracer-based measurements of VRAC activity indicated that these discrepancies were largely attributable to binding of inhibitors to serum albumin. When experiments were repeated under serum-free conditions, all inhibitors except DIDS and phloretin induced extensive death of both malignant and non-malignant cells, confirmed by microscopy and LDH release assays. This cytotoxicity was accompanied by a marked reduction in intracellular ATP levels, consistent with previously reported mitochondrial uncoupling effects. In contrast, LRRC8A knockdown reduced proliferation without substantial cell death. Together, these findings demonstrate that most commercially available VRAC blockers limit proliferation and viability predominantly through VRAC-independent mechanisms. Under standard culture conditions, serum albumin masks much of their intrinsic cytotoxicity. These results underscore the need for rigorous molecular controls in pharmacological studies and provide basis for developing more selective and less toxic VRAC-targeting agents.

Obesity-driven metabolic syndrome poses a critical global threat, yet standard therapies like GLP-1 receptor agonists trigger substantial lean mass wasting, with muscle loss accounting for up to 40% of reduced weight. Here we identify a non-canonical metabolic application for dronedarone hydrochloride, an anti-arrhythmic benzofuran derivative. In diet-induced and ob/ob obese mice, short-term dronedarone hydrochloride administration dose-dependently reduces food intake, clears visceral and subcutaneous adiposity, and reverses steatohepatitis. Head-to-head trials show that dronedarone hydrochloride achieves glycemic control and fat clearance non-inferior to semaglutide, tirzepatide, and empagliflozin, but uniquely and completely preserves skeletal muscle mass. Mechanistically, dronedarone hydrochloride operates independently of central hypothalamic appetite-regulating neuropeptides and the peripheral leptin pathway. By decoupling fat reduction from sarcopenia, our findings establish dronedarone hydrochloride as a muscle-sparing therapeutic candidate for metabolic syndrome.