Journal of Pharmacology and Experimental Therapeutics最新文献

Low-dose aspirin irreversibly acetylates cyclooxygenase (COX)-1 in anucleate platelets and progenitor megakaryocytes, permanently suppressing thromboxane (TX)A₂-dependent platelet activation. Although aspirin pharmacodynamics is well characterized in platelets, the kinetics of COX inhibition and recovery in human megakaryocytes remains poorly defined, due to ethical issues associated with invasive, bone-marrow trephine sampling, and low megakaryocyte yield. We studied aspirin pharmacodynamics in human megakaryocytic cell lines as a reliable and feasible surrogate model. We characterized COX-1 and COX-2 expression and activity in MEG-01 and CHRF-288-11 megakaryocytic cell lines, treated with a range of aspirin concentrations and exposure duration. COX activity was quantified by the production of TXB₂ from exogenous arachidonic acid. A single 10-μM aspirin exposure suppressed TXB₂ by 90 ± 2% (MEG-01) and 85 ± 4% (CHRF-288-11), with full recovery within 48-72 hours. Both COX-isozymes were detected by western blot and immunohistochemistry; however, selective COX-1 inhibition by SC-560 reduced TXB₂ by >75%, whereas COX-2 inhibition by NS-398 had minimal effect. Repeated aspirin exposure every 24 hours produced concentration- and time-dependent TXB₂ suppression, achieving 89 ± 2% inhibition by day 2 at 1 μM and 73 ± 3% by day 4 at 0.1 μM. TXB₂ biosynthesis recovered by 86 ± 2% and 99 ± 10% at days 2 and 3, respectively. These findings identify COX-1 as the principal source of TXA₂ in megakaryocytes and demonstrate that aspirin inhibits megakaryocyte COX-1 time- and dose-dependently, with delayed recovery likely reflecting de novo synthesis of COX-1 protein, thereby providing mechanistic insight into the sustained antiplatelet effect of low-dose aspirin in humans. SIGNIFICANCE STATEMENT: In human megakaryocyte cell lines, once-daily aspirin treatment at low-concentration range time-dependently inhibits COX-1 with delayed recovery after aspirin withdrawal. This closely mimics the kinetics of platelets, supporting the translational utility of the megakaryocyte-based surrogate model.

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

Despite recent advancements in breast cancer management, it remains the most common malignancy among women worldwide and is the second leading cause of cancer-related deaths in women. Therefore, there is considerable room for improvement in diagnostics, therapeutics, and research related to breast cancer. Although chemotherapy, radiation therapy, surgery, and targeted therapies for estrogen receptor-positive, progesterone receptor-positive, and human epidermal growth factor receptor 2-positive breast cancers remain the standard treatments, numerous new therapeutic targets and treatment modalities are emerging. Since their discovery, nanobodies have attracted significant attention in biomedical science as research tools, diagnostic agents, and therapeutic molecules. While many studies have explored nanobodies, this mini review focuses on recent advancements in their application to breast cancer therapy. Specifically, we examine nanobody-based strategies for targeting uniquely expressed molecules in breast cancer, modulating immune responses, and facilitating nanobody-drug delivery systems, as well as how nanobodies can help overcome the limitations of current treatments. We also discuss the challenges hindering their rapid traslation into clinical practice. SIGNIFICANCE STATEMENT: Breast cancer remains a leading cause of cancer-related deaths in women. Despite substantial therapeutic progress in recent decades, it continues to pose a major global health challenge. Therefore, translating novel therapeutic modalities from the laboratory to clinical settings is critically needed.

Diabetes mellitus is an escalating global health challenge, with current therapies primarily focused on symptom management rather than targeting the root causes of the disease. Emerging mRNA and gene therapies offer a new frontier by targeting disease mechanisms at the molecular level. This review explores the mechanistic landscape, delivery systems, and therapeutic outcomes of mRNA and gene-based interventions in diabetes. mRNA therapies, notably delivered via lipid nanoparticles, have demonstrated potential in restoring insulin production, enhancing angiogenesis, and modulating immune responses. mRNA therapy for both type 1 and type 2 diabetes using mRNAs of vascular endothelial growth factor-A, fibroblast growth factor 21, and glucagon-like peptide-1 linked to the IgG4 Fc region has shown immense potential in preclinical models of diabetes. Gene therapy for long-term correction of insulin signaling and pancreatic cell reprogramming is widely administered using viral (adeno-associated virus, lentivirus) vectors; however nonviral methods such as lipid nanoparticles and chitosan-based nanoparticles are being investigated in preclinical stages. CRISPR/Cas9 based genome-editing tools are another emerging therapeutic option for diabetes that correct metabolic defects and protect β cells. Despite these advances, delivery efficiency, immunogenicity, and off-target effects remain key translational hurdles for these therapies. Ongoing clinical trials for diabetes, including vascular endothelial growth factor A mRNA therapy for diabetic wound healing and fibroblast growth factor 21 gene therapy for type 1 diabetes, highlight the promising applications of these technologies. With careful attention to safety and pharmacokinetics, mRNA and gene therapies hold transformative promise as next-generation treatments for diabetes and its complications. SIGNIFICANCE STATEMENT: This review highlights emerging mRNA and gene therapies targeting the root causes of diabetes. By focusing on molecular mechanisms, delivery systems, and clinical advances, the review outlines next-generation strategies for treating diabetes and its complications.

Monoclonal antibodies are a versatile platform for targeted drug delivery. Their high specificity and favorable pharmacokinetics allow for selective drug delivery to targeted cells. A primary drug delivery application is antibody-drug conjugates (ADCs), which combine monoclonal antibodies with cytotoxic payloads via covalent linkers. While ADCs have shown remarkable clinical success, several limitations remain, including complex conjugation chemistries, heterogeneity in drug-to-antibody ratios, exposed hydrophobic patches, and off-target payload release, which can result in systemic toxicity. To complement existing ADC platforms and mitigate some of these issues, alternative formats referred to as antibody-drug complexes (ADCx) have been developed. ADCx are generated by forming reversible, high-affinity complexes between antibodies and drugs or preformed drug conjugates. This review discusses the ADCx formats reported to date, focusing on the unique advantages and potential limitations of each format. SIGNIFICANCE STATEMENT: Antibody-drug complexes offer a modular, noncovalent alternative to traditional antibody-drug conjugates. This review comprehensively evaluates antibody-drug complex formats, highlighting their potential to expand the utility of antibody-based drug delivery for next-generation therapeutics.

Multidrug resistance of cancer cells is attributed to drug-induced alteration of numerous intracellular processes. Using clinically relevant models of triple-negative breast and non-small lung cancer cells we previously showed that these cells respond to repeated paclitaxel exposure by inter alia lysosome enrichment in ABCC3, ABCC5, and ABCC10, which contribute to drug sequestration in these organelles and reduced drug cytotoxicity. In this study, we provide experimental evidence that transcription of the above-mentioned ABCC genes is enabled by BRG1-based SWI/SNF chromatin remodeling complex. Pharmacological inhibition of SWI/SNF with PFI3 or ACBI1, the PROTAC degrader of SMARCA2/4, substantially reduced transcription of ABCC3, ABCC5, and ABCC10. A similar effect was caused by transient silencing of SMARCA4 (BRG1), but not SMARCA2 (BRM). The deficiency of BRG1 led to extralysosomal distribution of anticancer drugs, their deeper penetration of spheroids, and substantial increase in drug cytotoxicity. Interestingly, in BRG1-deficient cell line paclitaxel triggered mutations, which reverted BRG1 truncating deletion in SMARCA4, thereby restoring SWI/SNF ATPase expression in paclitaxel-resistant cells and increasing transcription of ABCC. Acquisition of drug resistance was associated with BRG1 redistribution in the genome, de novo occurrence at the promoters of genes functionally linked to endolysosomal system, and stronger co-occurrence with EP300. Our study indicates possible target--SWI/SNF complex for anticancer combinatorial interventions in paclitaxel-induced multidrug resistant phenotypes. SIGNIFICANCE STATEMENT: This study provides evidence that BRG1 inhibition with PFI3 and degradation of SMARCA4 mRNA substantially declines lysosomal drug sequestration and potentiate drug toxicity. Therefore, BRG1 targeting can be considered as candidate for combinatorial anticancer therapy with some standard chemotherapy drugs.

Cardiovascular disease (CVD) remains the leading cause of death worldwide despite decades of therapeutic advances. Emerging insights into its etiology have revealed previously unappreciated cellular and molecular drivers beyond traditional risk factors, prompting the development of treatments that target newly identified culprit proteins and cells within cardiovascular tissues. Protein-based biologics-particularly monoclonal antibodies and multispecific proteins-are known for their strength and specificity in targeting and their established use as treatments for other diseases. However, extending biologics to new indications faces challenges: achieving durable effects in diseased tissues and minimizing side effects in healthy tissue. Addressing these long-standing challenges requires fine-tuning biologics' pharmacokinetic properties and pharmacodynamic effects according to target- and disease-specific requirements. In this review, we examine foundational pharmacokinetic and pharmacodynamic principles in the context of cardiovascular-targeted biologics, highlighting the role of protein design in controlling distribution, efficacy, and safety. Additionally, we discuss emerging preclinical and clinical biologics specifically designed for CVDs, as well as emerging opportunities in this landscape. These advances point toward a future where pharmacokinetics guide the rational design of next-generation protein therapeutics for CVD. SIGNIFICANCE STATEMENT: Protein-based biologics hold promise for treating cardiovascular diseases (CVD); however, their successful translation requires understanding how proteins' properties and cardiovascular physiology shape pharmacokinetic and pharmacodynamic behavior. This minireview connects foundational pharmacology principles with strategies in protein engineering suitable for CVD applications. Pharmacokinetic-guided design will accelerate the development of protein therapies that can transform CVD treatment.

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