Journal of Pharmacology and Experimental Therapeutics最新文献

Depression is a major global public health challenge, with current treatments often limited by suboptimal efficacy and adverse effects. This study investigated the antidepressant potential of cerebroprotein hydrolysate oral liquid (CHOL), a neuroprotective peptide-based solution derived from porcine brain through enzymatic hydrolysis. In model mice subjected to chronic social defeat stress (CSDS) and chronic restraint stress (CRS), CHOL was found to significantly attenuate depressive-like behaviors. Furthermore, CHOL also effectively reduced corticosterone-induced cell damage in PC12 cells. Metabolomic analysis revealed that depression modeling led to significant disturbances in neurotransmitter-related metabolites, especially norepinephrine, whereas CHOL could restore these metabolites in key brain regions and serum. Mechanism exploration revealed that the elevation of norepinephrine by CHOL was achieved through upregulation of tyrosine hydroxylase expression. Pharmacokinetic studies demonstrated that the 8 peptides in CHOL could rapidly distribute to the brain, with serine proteases, cysteine proteases, and metalloproteases identified as the key enzymes mediating CHOL metabolism. These findings underscored CHOL's preventive and therapeutic potential and provided mechanistic insights for its development as a novel antidepressant strategy. SIGNIFICANCE STATEMENT: This study integrated multiple depression models to confirm the significant antidepressant effects of cerebroprotein hydrolysate oral liquid. For the first time, we elucidated its underlying mechanism by regulating tyrosine hydroxylase expression and promoting norepinephrine synthesis. Furthermore, 8 brain-penetrant peptides were identified, and a targeted protease-inhibition strategy was developed to enhance in vivo exposure, thereby providing novel mechanistic insights and potential translational applications in depression therapeutics.

Since its approval in the early 1960s, 5-fluorouracil (5-FU) has remained an important therapeutic for the treatment of late-stage and metastatic colorectal cancer (CRC). It acts through intracellular conversion to 5-fluoro-2'-deoxyuridine monophosphate (FdUMP) to inhibit thymidylate synthase (TYMS), leading to nucleotide pool imbalance, DNA damage, and disruption of tumor cell proliferation. However, 5-FU is limited by rapid clearance and off-target toxicities, which affects a large proportion of patients with CRC. To address these issues, we developed 5'-(R)-CH₃-FdUMP (Me-FdUMP), a 5'-(R)-CH₃-substituted analog of FdUMP that retains inhibitory activity against purified TYMS. Here, we show that Me-FdUMP is resistant to metabolism by phosphatases and kinases, reduces 5-FU formation, and enhances TYMS inhibition in a human CRC cell line. In mice, Me-FdUMP treatment led to markedly lower 5-FU exposure in the heart and bone marrow, 2 key sites of clinical toxicity. Furthermore, in a mouse xenograft model of human CRC, Me-FdUMP maintained antitumor efficacy comparable to FdUMP. Taken together, these results suggest 5'-(R)-CH₃-substituted FdUMP could be a promising new approach for improving the safety of fluoropyrimidine-based therapeutics. SIGNIFICANCE STATEMENT: Current fluoropyrimidine-based therapeutics for colorectal cancer suffer from metabolic liabilities that can often lead to severe and dose-limiting side-effects. Results reported here highlight a new fluoropyrimidine derivative with enhanced on-target activity in vitro, maintenance of antitumor efficacy in vivo, and impaired metabolism that can reduce exposure of toxic metabolites. This work represents a new strategy to address the shortcomings of current fluoropyrimidine-based therapeutics with the potential to improve patient outcomes.

Background: Arterial medial calcification (AMC) is closely associated with morbidity and mortality in people with chronic kidney disease (CKD). Endogenous bioactive peptides, salusin α and salusin β, are alternative splicing products from preprosalusin encoded by the torsion dystonia-related gene. The present study was designed to explore their roles and mechanisms in AMC under CKD condition. CKD rats with AMC were induced by feeding an adenine (0.75%) with high phosphorus (1.5%) diet for 4 weeks. Calcification in A7r5 cells (rat thoracic aorta smooth muscle cells) was induced with calcifying media. The results showed that in rats with CKD and in the calcifying media-treated A7r5 cells, salusin α protein level was reduced, whereas salusin β was elevated in plasma, in aorta and in A7r5 cells, respectively. Calcification, osteogenic transition, oxidative stress, and extracellular signal-regulated protein kinases (ERK) activation were significantly induced, and these changes were effectively reversed by salusin α application, but notably promoted by salusin β administration. More importantly, salusin α or the ERK activation inhibitor U0126 pretreatment in vitro attenuated the promoting effects of salusin β on calcification, osteogenic transition and oxidative stress and ERK activation which also were alleviated by U0126 treatment in vivo. This study indicates that salusin α can attenuate AMC and counteract the promoting effect of salusin β on AMC by inhibiting oxidative stress and the activation of ERK signaling pathway, suggesting that upregulating the expression of salusin α, but downregulating the expression of salusin β in aorta, may be a good strategy for the treatment of vascular calcification under CKD condition.

Significance statement: The current study found that bioactive peptides, salusin α and salusin β, were important mediators in arterial medial calcification (AMC) under CKD conditions. Salusin α could attenuate AMC and counteract the promoting effect of salusin β on AMC by inhibiting ERK activation and oxidative stress, and the inhibition of ERK activation effectively relieved AMC in CKD. Our findings provide new insights for preventing AMC under CKD condition.

The heterogeneity and treatment resistance of glioblastoma (GBM) can be addressed through multidrug combination therapies that target multiple biological pathways simultaneously. In this study, we explored the repurposing of antiepileptic drugs with potential antitumor effects, combined with the Janus kinase/signal transducer and activator of transcription-3 (JAK/STAT3) inhibitor ruxolitinib (RUX), as an alternative local therapeutic approach for GBM. The cytotoxic effects of valproic acid (VPA), oxcarbazepine (OXC), and gabapentin (GBP) were evaluated on A172 and U251 GBM cells. Both VPA and OXC significantly reduced cell viability, prompting further investigation of their effects in combination with RUX. When tested in 3-dimensional multicellular tumorspheres, the combinations at their IC50 exhibited suboptimal effectiveness compared with single-agent treatment. Using a factorial experimental design based on a minimal combination approach to analyze dose-response data, followed by subsequent Bliss synergy analysis, synergistic interactions were revealed exclusively for RUX + VPA on A172 cells. Although the interaction between RUX and OXC was additive, GBM cells displayed increased sensitivity to this combination, suggesting potential therapeutic value. Ultimately, the most effective drug ratios were assessed using live/dead cell fluorescence staining in 3-dimensional multicellular tumorspheres. The variable treatment response observed among GBM cell lines underscores the need for personalized treatment strategies tailored to the specific molecular profile of individual tumors. SIGNIFICANCE STATEMENT: Given the unmet needs in glioblastoma treatment, the study explores novel combinations of Janus kinase/signal transducer and activator of transcription-3 inhibitor ruxolitinib and antiepileptics for local codelivery, aiming to overcome resistance and heterogeneity through synergistic effects and sustained release via molecularly imprinted reservoirs.

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.

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.

N-lactoyl-phenylalanine (Lac-Phe) has emerged as a signaling metabolite connecting cellular metabolism to systemic physiology. Synthesized through carnosine dipeptidase 2-mediated conjugation of lactate and phenylalanine, Lac-Phe increases acutely in response to exercise and feeding, the primary drivers of its elevation under physiologic conditions. In preclinical models, Lac-Phe acts as a potent regulator of energy balance. Its administration suppresses appetite and reduces body weight in obesity, whereas pharmacologic interventions such as metformin elevate circulating Lac-Phe to produce similar anorexigenic effects. Converging evidence implicates central mechanisms, including inhibition of orexigenic agouti-related peptide neurons, positioning Lac-Phe as a mediator linking peripheral metabolic signals to appetite control. The first human Lac-Phe clinical trial in individuals with obesity began dosing in 2025, evaluating appetite suppression and glucose-lowering effects. Beyond metabolism, Lac-Phe promotes anti-inflammatory macrophage polarization, conferring protection in murine models of colitis and spinal cord injury. Circulating Lac-Phe also rises in conditions such as mitochondrial dysfunction, sepsis, and phenylketonuria, suggesting broader associations with perturbed energy metabolism and systemic stress responses. This review integrates current knowledge spanning molecular mechanisms, physiological regulation, and clinical translation. We examine Lac-Phe biosynthesis, tissue distribution, and regulatory patterns across physiological and disease states, and highlight emerging mechanisms of action in metabolic and inflammatory signaling. Finally, we discuss key knowledge gaps, highlighting the need to define targets, transporters, and tissue sources to shape the next phase of discovery. Collectively, these advances position Lac-Phe at the forefront of exerkine biology and as a promising molecular link between metabolism, immunity, and therapeutic innovation. SIGNIFICANCE STATEMENT: Evidence across molecular, physiological, and translational domains positions Lac-Phe as a promising therapeutic target. This review frames our understanding of Lac-Phe biology-from its biosynthesis to its roles in energy balance and outlines the key questions that will define ongoing discovery.

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.