Journal of Pharmacology and Experimental Therapeutics最新文献

This study aimed to investigate the effects of soluble epoxide hydrolase (sEH) inhibition on osteoclast differentiation and activity in vitro and in vivo, as well as to elucidate the signaling pathways associated with osteoclastogenesis. Primary murine bone marrow monocytes were stimulated with macrophage colony-stimulating factor and receptor activator of nuclear factor kappa B ligand to induce osteoclastogenesis and treated with the sEH inhibitor 1-(1-propanoylpiperidin-4-yl)-3-[4-(trifluoromethoxy)phenyl]urea (TPPU) (0.1-10 μM). Tartrate-resistant acid phosphatase staining, gene expression analyses, and immunofluorescence were used to evaluate osteoclast formation, transcriptional regulation, and cell fusion. A murine model of ligature-induced periodontitis was used to assess in vivo effects of sEH inhibition (TPPU 10 mg/kg). Alveolar bone loss was quantified by histomorphometry, and gingival gene expression was analyzed. In vitro, sEH inhibition significantly reduced tartrate-resistant acid phosphatase-positive multinucleated osteoclast formation, downregulated the expression of key transcription factors and osteoclast activity-related genes. Immunofluorescence analysis revealed attenuation of mitogen-activated protein kinase signaling and reduced dendritic cell-specific transmembrane protein expression, indicating impaired cell fusion. In vivo, TPPU treatment preserved alveolar bone structure, reduced osteoclast-like cell numbers, and decreased the expression of osteoclastic markers in gingival tissues during experimental periodontitis. sEH acts as a crucial regulator of osteoclast differentiation and function. Pharmacological inhibition of sEH suppresses osteoclastogenesis and protects against inflammatory bone loss. Therefore, targeting sEH may represent a novel therapeutic approach to modulate osteoclast activity and prevent bone destruction in periodontitis and other bone-resorptive diseases. SIGNIFICANCE STATEMENT: This study provides direct evidence that soluble epoxide hydrolase inhibition modulates osteoclast differentiation and fusion, contributing to reduced inflammatory bone loss. By demonstrating effects on osteoclast-intrinsic pathways while also influencing the inflammatory microenvironment, our findings support soluble epoxide hydrolase as a pharmacological target for chronic inflammatory bone-resorptive diseases.

Neurolysin (Nln) is a peptidase recognized for its cerebroprotective function in acute ischemic stroke. This study aimed to identify small molecule activators of Nln as research tools to further explore the role of this enzyme in stroke and other neurological disorders. Building on our previous computational screen of ∼140,000 compounds from the National Cancer Institute Developmental Therapeutics Program database, we extended experimental testing to the top 100 candidates using an Nln enzymatic assay. A pyridine-piperazine derivative (Py-Pip) was identified as a hit molecule and was characterized in detail. Py-Pip concentration-dependently enhanced the hydrolysis of both synthetic and natural substrates (neurotensin, angiotensin I, and bradykinin) by rat Nln, and displayed comparable activating effects on human and mouse orthologs. Importantly, Py-Pip exhibited a favorable selectivity profile, showing no potentiation of homologous metallopeptidases or unrelated enzymes. Kinetic analysis revealed that Py-Pip increases the catalytic efficiency (V_max/K_m) of Nln via a nonessential activation mechanism, whereas competition assays with inhibitor dynorphin A(1-13) confirmed that Py-Pip acts at a distinct, nonoverlapping site. Direct binding was further validated by orthogonal biophysical techniques, including differential scanning fluorimetry, microscale thermophoresis, and biolayer interferometry, whereas circular dichroism spectroscopy indicated activator-induced secondary structural changes. These findings validate that Nln activity can be enhanced by small molecules and establish Py-Pip as a novel, nonpeptide scaffold for developing potent, "drug-like" activators to investigate Nln biology and therapeutic potential. SIGNIFICANCE STATEMENT: This study reports the discovery of a novel nonpeptide small molecule that selectively enhances the activity of neurolysin (Nln), a peptidase implicated in cerebroprotection. Unlike previous peptide-based activators, this molecule provides a stable, "drug-like" scaffold and a structural foundation for the development of potent Nln activators to probe Nln biology and therapeutic potential in ischemic stroke.

Nicotine dependence and obesity are global health issues. Emerging studies suggest that obese individuals are more likely to develop nicotine dependence. Despite this knowledge, there have been few investigations into whether nicotine dose and/or chemical flavorants commonly found in nicotine-containing products modulate nicotine dependence in obese models. Therefore, our objective was to examine whether low (6 mg/mL) or high (60 mg/mL) dose nicotine, with or without flavor impact vaping-related behaviors in a diet-induced obesity (DIO) mouse model. Briefly, adult male and female DIO mice were trained to self-administer 6 or 60 mg/mL nicotine, with or without menthol and green apple (GA) flavors using an e-Vape self-administration assay. Overall, DIO female mice assigned to flavored e-liquids self-administered more nicotine than their male counterparts. Additionally, DIO female mice assigned to 6 mg/mL nicotine + menthol exhibited higher nicotine reinforcement-related behavior than female mice assigned 6 mg/mL nicotine (unflavored). Reinforcement-related behavior was not changed in DIO male mice assigned to e-liquids containing 60 mg/mL nicotine. However, DIO female mice exhibited menthol- and GA-induced enhancements in reinforcement-related behavior with 60 mg/mL nicotine. By combining a noncontingent vapor exposure assay with fast-scan cyclic voltammetry, we observed that menthol and GA flavorants, when combined with 6 or 60 mg/mL nicotine, increased tonic- and phasic-stimulated dopamine signaling in the nucleus accumbens core. Together, these preclinical findings provide additional evidence that nicotine plus popular chemical flavorants such as menthol and GA could alter dopamine signaling in the reward pathway and promote vaping-related behaviors in a DIO mouse model. SIGNIFICANCE STATEMENT: This study highlights the effects of flavorants on vaping-related behaviors in a diet-induced obese mouse model. Collectively, this study revealed that flavorants commonly found in vaping products alter dopamine release in the nucleus accumbens core-a phenomenon linked with nicotine addiction.

Small interfering RNA (siRNA) therapeutics are an emerging modality for treating genetic and metabolic diseases, with 8 approved drugs now in clinical use. Despite substantial advances in delivery technologies, including lipid nanoparticles and N-acetylgalactosamine conjugates, inefficient intracellular trafficking, particularly endosomal escape, remains a critical limitation. Here, we identify cellular cholesterol as a key regulator of siRNA intracellular trafficking, endosomal escape, and pharmacologic efficacy. Using a 2D hepatocyte cell culture model and cationic-lipid-mediated delivery, we show that pharmacologic cholesterol reduction via statin treatment significantly impairs siRNA-mediated gene silencing with minimal effects on cellular uptake, indicating a post-internalization trafficking defect. Cholesterol supplementation restores silencing, confirming its essential role in functional siRNA activity. Confocal imaging reveals increased siRNA entrapment in late endosomes following statin treatment, consistent with impaired endosomal escape. Notably, chloroquine, an endosomal escape enhancer, rescues gene silencing under cholesterol-reduced conditions. Mechanistically, we identify annexin A2 (ANXA2) as a critical mediator of this cholesterol-sensitive trafficking pathway, as ANXA2 knockdown abrogates the restorative effect of cholesterol supplementation. Together, these findings uncover a previously unrecognized cholesterol- and ANXA2-dependent mechanism regulating siRNA efficacy. While these mechanistic insights are specific to cationic-lipid-based delivery, they highlight intracellular cholesterol as an important determinant of siRNA endosomal escape. Future studies using microphysiological systems or in vivo models will be essential to validate and extend these findings beyond this 2D cell culture model. SIGNIFICANCE STATEMENT: This study uncovers cholesterol as an essential and previously unrecognized determinant of small interfering RNA therapeutic efficacy, acting through annexin A2 to enable endosomal escape, a critical bottleneck in RNA drug delivery. The findings position cholesterol modulation as a viable approach to improve the intracellular delivery and therapeutic effectiveness of RNA-based drugs.

Methyl (1-{[6-{[(1S)-1-cyclopropylethyl]amino}-2-(pyrazolo[5,1-b][1,3]thiazol-7-yl)pyrimidin-4-yl]carbonyl}piperidin-4-yl)carbamate mono(4-methylbenzenesulfonate) monohydrate (NS-229) is a novel Janus kinase 1 inhibitor currently being evaluated in a phase 2 global study (NCT06046222) for the treatment of eosinophilic granulomatosis with polyangiitis (EGPA). We investigated the nonclinical efficacy of NS-229 to support its therapeutic use in treating EGPA. Its effects were investigated in human peripheral blood eosinophils, human peripheral blood mononuclear cells, and a mouse model of eosinophilic vasculitis induced by ovalbumin. In human peripheral blood eosinophils, NS-229 and an anti-interleukin (IL)-5 antibody, but not prednisolone, significantly decreased the expression of CD69 induced by IL-5. In human peripheral blood mononuclear cells, NS-229 and prednisolone, but not the anti-IL-5 antibody, significantly decreased the production of cytokines such as interferon gamma, IL-5, and IL-13, induced by anti-CD3/CD28 antibody. NS-229 inhibited the development of vascular lesions, decreased eosinophil counts in the blood and bronchoalveolar lavage fluid, and lowered bronchoalveolar lavage fluid lymphocyte counts in the ovalbumin-induced eosinophilic vasculitis mouse model. The effects of NS-229 in the mouse model were comparable to those of prednisolone and tofacitinib, a pan-Janus kinase inhibitor. Regarding safety, NS-229 did not influence the platelet or red blood cell counts, which were significantly elevated with tofacitinib and prednisolone, respectively. NS-229 did not affect body weight, which was significantly increased with tofacitinib and significantly decreased with prednisolone. Collectively, the nonclinical investigation of NS-229 showed a suppression of multiple cytokine signals and inhibition of vascular lesion formation without impacting the relevant side-effect parameters, suggesting its potential as an additional treatment option for EGPA. SIGNIFICANCE STATEMENT: NS-229 inhibited the formation of vascular lesions in a mouse model of ovalbumin-induced eosinophilic vasculitis without affecting certain side-effect parameters. The underlying mechanism of action is suggested to be the selective inhibition of multiple cytokine signals via JAK1.

Although chemotherapy remains a life-saving intervention for numerous cancer patients, it is often accompanied by depressive symptoms and cognitive impairments, "chemobrain." Noteworthy, multiple studies emphasize the role of glycogen synthase kinase 3β (GSK-3β) in depression and chemobrain; nevertheless, no available data relate GSK-3β inhibitors to chemobrain. Herein, this study aims to investigate the effect of the GSK-3β inhibitor, lithium, on behavioral and neurobiological abnormalities in a doxorubicin (DOX)-induced rat model of chemobrain. The chemobrain model was established through weekly intraperitoneal injections of doxorubicin (2 mg/kg/wk) for a duration of 4 weeks, whereas lithium (100 mg/kg/d, i.p.) was administered concomitantly over the same period. Behavioral, neurochemical, and histopathological evaluations were performed after the experimental protocol. DOX-induced depressive-like behaviors and cognitive impairments, with reduction in prefrontal cortex tropomyosin receptor kinase B receptors, brain-derived neurotrophic factor protein kinase B (BDNF), and phosphorylated protein kinase B, elevating the levels of the active form of GSK-3β, which lessened phosphorylated mammalian target of rapamycin/nuclear factor-erythroid 2-related factor 2/heme oxygenase-1 and BDNF/synapsin-1 pathways, while triggering overexpression of NF-κB, proinflammatory cytokines, oxidative stress, apoptosis, tau hyperphosphorylation, and neurodegeneration. Lithium ameliorated DOX-induced behavioral, neurochemical, and histological abnormalities. To the best of our knowledge, this study presents the first evidence that lithium treatment can modulate DOX-induced depression and cognitive deficits, potentially through revamping the BDNF/tropomyosin-related kinase receptor B/protein kinase B/GSK-3β/mammalian target of rapamycin/nuclear factor-erythroid 2-related factor 2/heme oxygenase-1 signaling cascade, thereby attenuating oxidative stress, neuroinflammation, apoptosis, neurofibrillary tangles, and subsequent neurodegeneration. SIGNIFICANCE STATEMENT: To the best of our knowledge, this study is the first to detect antidepressant and procognitive effects of lithium in DOX-induced chemobrain via GSK-3β inhibition. Accordingly, lithium offers a promising therapeutic target for the management of chemotherapy-induced depression and chemobrain.

Cisplatin remains a cornerstone of chemotherapy, but its clinical use is often limited by cisplatin-induced acute kidney injury, a condition driven by oxidative stress, inflammation, and mitochondrial dysfunction. Here, we developed naringenin-functionalized polyester nanoparticles (P2Ns-NAR) to enhance the oral delivery and therapeutic efficacy of urolithin A (UA), a mitochondrial-targeting metabolite with cytoprotective properties. The resulting formulation, P2Ns-NAR-UA, conferred kidney protection in vitro and in vivo, outperforming the nontargeted nanoparticle formulation (P2Ns-UA). Notably, in vivo efficacy was achieved at a 50% lower dose. Molecular docking studies suggest UA exhibits a favorable heme oxygenase-1 binding energy of -7.43 kcal/mol, supporting its potential as a promising drug candidate. Mechanistic studies demonstrated that P2Ns-NAR-UA upregulate heme oxygenase-1 and activate PTEN-induced putative kinase 1/Parkin-mediated mitophagy, promoting mitochondrial quality control and preserving dynamics by increasing mitofusin-1/2 and reducing dynamin-related protein 1 and mitochondrial fission protein 1 expression. Treatment also attenuated inflammatory cytokines (interleukin 6, interleukin 8, and tumor necrosis factor-α), immune activation markers (cluster of differentiation 80 and 45), and kidney injury biomarkers (neutrophil gelatinase-associated lipocalin, cystatin C, and osteopontin). Histological analysis confirmed reduced tubular damage and fibrosis. These findings establish P2Ns-NAR-UA as a promising oral therapeutic platform to mitigate cisplatin-induced acute kidney injury through coordinated modulation of inflammation, oxidative stress, and mitochondrial homeostasis. Further investigation in cisplatin-resistant cancer models is warranted to establish this platform's dual therapeutic potential and translational value. SIGNIFICANCE STATEMENT: This study shows that naringenin-functionalized polyester nanoparticles improves intestinal uptake of encapsulated agents through intestinal folate receptors. Naringenin-functionalized polyester nanoparticles loaded with urolithin A (P2Ns-NAR-UA) doubles the efficacy of polyester nanoparticles loaded with urolithin A, achieving comparable results at half the dose. The formulation enhances cell health, reduces inflammation, and restores kidney function, making it a promising adjuvant to cisplatin therapy by improving outcomes while minimizing toxicity.

TMEM16A forms a Ca²⁺-activated Cl^- (Cl_Ca) channel that plays essential roles in the cardiovascular, gastrointestinal, and central nervous systems. Dysregulation of TMEM16A expression has been implicated in the development of several diseases, making selective TMEM16A modulators attractive therapeutic candidates. Here, the effects of lidocaine, a voltage-gated Na⁺ (Na_V) channel blocker widely used as a local anesthetic and antiarrhythmic drug, on TMEM16A-mediated Cl_Ca currents were investigated using whole-cell patch-clamp recordings in human embryonic kidney 293 cells stably expressing human TMEM16A. Lidocaine, an amide-type local anesthetic, inhibited TMEM16A Cl_Ca currents in a concentration-dependent manner (IC₅₀ = 0.69 mM). Similarly, tetracaine, an ester-type local anesthetic, suppressed TMEM16A Cl_Ca currents. Lidocaine produced weaker inhibition of human TMEM16B Cl_Ca currents (IC₅₀ = 1.50 mM). Among Na_V channel blockers, the antiarrhythmic drugs, mexiletine and quinidine, inhibited TMEM16A currents, whereas the anticonvulsants, phenytoin and carbamazepine, showed no effect. In monocrotaline-induced pulmonary arterial hypertension (PAH) rats, in which TMEM16A expression is upregulated, lidocaine exerted stronger inhibitory effects on Cl_Ca currents in pulmonary arterial smooth muscle cells compared with those in control rats. Daily administration of lidocaine (30 mg/kg for 14 days) improved in vivo PAH parameters, including right ventricular systolic pressure, Fulton index, and pulmonary vascular remodeling, in monocrotaline-induced PAH rats. In conclusion, lidocaine inhibits TMEM16A Cl_Ca channels independently of Na_V channel blockade and attenuates PAH progression, supporting its potential as a repositioned therapeutic candidate for PAH. SIGNIFICANCE STATEMENT: Lidocaine, a voltage-gated Na⁺ channel blocker widely used as a local anesthetic and antiarrhythmic drug, significantly inhibited TMEM16A Ca²⁺-activated Cl^- channels. Lidocaine also ameliorated pulmonary arterial hypertension (PAH) progression in experimental PAH rats, suggesting that it directly targets TMEM16A Cl_Ca channels and represents a promising repositioned therapeutic option for PAH.