Pharmaceutical Research最新文献

{"title":"DeepSeek-LLM with Adaptive RAG for Pharmaceutical Dissolution Prediction.","authors":"Leqi Lin, Xingyu Zhou, Kaiyuan Yang, Yang Liu, Xizhong Chen","doi":"10.1007/s11095-025-03932-1","DOIUrl":"https://doi.org/10.1007/s11095-025-03932-1","url":null,"abstract":"<p><strong>Purpose: </strong>This work aims to accelerate and enhance pharmaceutical drug dissolution prediction by integrating advanced Large Language Models (LLMs) and AI-diffusion models to reduce reliance on time-consuming, costly empirical experiments. The framework sets a foundation for broader adoption of generative AI in drug development.</p><p><strong>Methods: </strong>This work introduces a DeepSeek based LLM framework augmented by prompt engineering (zero-shot, few-shot, chain-of-thought) and adaptive weighted retrieval-augmented generation (RAG) to systematize dissolution profile from basic physical properties. Moreover, a diffusion model synthesizes SEM-derived morphological parameters (e.g., particle size, surface area), circumventing error accumulation from multi-instrument characterization workflows. These parameters feed the RAG database, enabling LLM predictions grounded in structure-performance relationships rather than idealized assumptions.</p><p><strong>Results: </strong>Overall, the LLM generated dissolution profile (few-shot chain-of-thought with RAG) provides a good agreement between experimental and the prediction result among others. Sensitivity analysis is investigated to quantify the reliability and stability of the prompt content. Additionally, diffusion-generated structural data from SEM images combined with the LLM's predictive capabilities are tested to connect macro-scale physical properties with microstructural characteristics, achieving a close profile trend with acceptable RMSE and PCC.</p><p><strong>Conclusions: </strong>This study demonstrates the potential of the DeepSeek-based LLM framework to describe the dissolution of drug powders. Among the different system prompt strategies, few-shot chain-of-thought with RAG performs the best dissolution profile among others. While it may overcomplicate straightforward tasks in certain scenarios. The combination of diffusion models successfully bridges AI-driven insights (e.g., dissolution predictions) with physical and structural drug properties (e.g., particle geometry from SEM images).</p>","PeriodicalId":20027,"journal":{"name":"Pharmaceutical Research","volume":" ","pages":""},"PeriodicalIF":4.3,"publicationDate":"2025-10-08","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145252233","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Potential Transmembrane Proteins-transporters of Chelidonic Acid for its Intracellular Uptake: In Silico Simulation.","authors":"Temur Nasibov, Anna Gorokhova, Konstantin Brazovsky, Alina Ryzhkova, Ekaterina Porokhova, Elena Avdeeva, Mikhail Belousov, Oleg Kokorev, Igor Khlusov","doi":"10.1007/s11095-025-03934-z","DOIUrl":"https://doi.org/10.1007/s11095-025-03934-z","url":null,"abstract":"<p><strong>Introduction: </strong>Small molecules are biologically active organic compounds with molecular weight below 1 kDa. Their small size enables efficient transport across cell membranes and modulation of intracellular signaling, making them promising for drug development. Chelidonic acid (ChA) is a small molecule (184 Da) with a wide range of biological effects, but its transport mechanisms and molecular targets remain unknown.</p><p><strong>Purpose: </strong>The aim of this study is to identify a possibility of ChA uptake by human cells and to search for transporter proteins that may be involved in the intracellular trafficking of ChA using a combination of in silico and in vitro approaches.</p><p><strong>Methods: </strong>Co-culturing of human MCF-7 cells with ChA was conducted in vitro for 4 h and residual (not absorbed by cells) ChA concentration in solution was measured using HPLC. Candidate transporters were screened from databases. Molecular docking was performed with Autodock Vina, and molecular dynamics simulations were run for 50 ns using GROMACS to assess protein-ligand interactions. Statistical analysis used the R language with Newey-West estimator and Welch's t-test. HOLE and VMD were used for 3D-reconstruction and visualization of transport channels.</p><p><strong>Results: </strong>MCF-7 cancer cells uptake ChA through one or several of the common cell transport proteins. Initial screening identified six transmembrane proteins, with further analysis pinpointing three candidates (GLUT3, SVCT1, URAT1) demonstrating structural and functional compatibility for ChA transport.</p><p><strong>Conclusion: </strong>The study contributes to the understanding of the pharmacokinetics and pharmacodynamics of ChA and provides the basis for the rational design of pharmaceutical substances based on it.</p>","PeriodicalId":20027,"journal":{"name":"Pharmaceutical Research","volume":" ","pages":""},"PeriodicalIF":4.3,"publicationDate":"2025-10-08","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145252265","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Metabolic Insights into Drug Absorption: Unveiling Piperine's Transformative Bioenhancing Potential.","authors":"Devika Tripathi, Vivek Kumar Gupta, Prashant Pandey, P S Rajinikanth","doi":"10.1007/s11095-025-03920-5","DOIUrl":"https://doi.org/10.1007/s11095-025-03920-5","url":null,"abstract":"<p><p>The oral bioavailability of drugs is often limited by metabolic barriers, including enzymatic degradation and active efflux processes in the gastrointestinal tract. Piperine, a pungent alkaloid found in black pepper (Piper nigrum), has garnered significant interest as a natural bioenhancer due to its multifaceted ability to inhibit cytochrome P450 enzymes, particularly CYP3A4, and efflux transporters such as P-glycoprotein (P-gp). These actions result in enhanced intestinal absorption and prolonged systemic retention of various therapeutic agents. Additionally, Piperine modulates intestinal permeability and alters the pharmacokinetics of drugs by interfering with first-pass metabolism. Recent developments in nanotechnology have led to innovative formulation strategies, such as nanoemulsions, liposomes, and self-emulsifying drug delivery systems, which further enhance Piperine's solubility, stability, and efficacy. However, despite its promising bioenhancing effects, Piperine exhibits limitations such as poor water solubility, dose-dependent toxicity, reproductive and hepatic concerns, and the potential for significant drug-drug interactions. This review critically examines the mechanistic pathways, formulation advances, pharmacological roles, safety issues, and clinical prospects of Piperine. Furthermore, it emphasizes the need for rigorous clinical trials and regulatory evaluation to validate Piperine's use in pharmaceutical applications. Overall, Piperine represents a potent, yet cautiously applicable, tool in modern drug delivery strategies.</p>","PeriodicalId":20027,"journal":{"name":"Pharmaceutical Research","volume":" ","pages":""},"PeriodicalIF":4.3,"publicationDate":"2025-10-06","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145239493","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Single- and Multiple-dose Pharmacokinetics, Excretion, Metabolism, and Safety of the Antivirulence Agent Fluorothiazinone in Healthy Adults.","authors":"Mark V Savitskii, Natalia E Moskaleva, Svetlana A Appolonova, Alex Brito, Natalia E Bondareva, Nadezhda L Lubenec, Anna B Sheremet, Maria K Ordzhonikidze, Nailya A Zigangirova, Sergey K Zyryanov, Vadim V Tarasov, Alexander L Gintsburg","doi":"10.1007/s11095-025-03919-y","DOIUrl":"https://doi.org/10.1007/s11095-025-03919-y","url":null,"abstract":"<p><strong>Introduction: </strong>Fluorothiazinone (FT) is a novel antivirulence agent formulated as an immediate-release uncoated tablet, with demonstrated efficacy and safety against infections caused by drug-resistant Gram-negative bacteria. In this study, we evaluated the pharmacokinetics, excretion, metabolism, and safety of FT in healthy adults under single- and multiple-dose regimens.</p><p><strong>Methods: </strong>In a randomized study, participants received either a single 1800 mg oral dose of FT or multiple doses according to one of the following regimens: two doses within one day (900 or 1200 mg every 12 h) or 14 doses across 7 days (900 mg every 12 h). Pharmacokinetics, excretion and metabolic profiles were assessed using serial blood and urine sampling.</p><p><strong>Results: </strong>Twenty-nine participants completed the study. Dividing the total daily dose into two equal administrations extended FT's half-life and systemic exposure, representing the optimal dosing strategy. Steady-state pharmacokinetics were observed after 7 days of dosing, indicating extensive tissue distribution. Urinary recovery of unmetabolized FT was low, and identified metabolites are presented. Both single and multiple doses were well tolerated.</p><p><strong>Conclusion: </strong>FT, administered orally, was safe and well tolerated in healthy adults in both single and repeated dosing regimens. The pharmacokinetic data support the continued clinical development of FT, including its use in future phase II and III trials. FT's lack of impact on normal intestinal microflora further substantiates its potential as an antivirulence therapy.</p>","PeriodicalId":20027,"journal":{"name":"Pharmaceutical Research","volume":" ","pages":""},"PeriodicalIF":4.3,"publicationDate":"2025-10-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145207308","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Key Challenges and Optimization Practices for Non-clinical Evaluation of AAV based Gene Therapy for Central Nervous System Disease.","authors":"Dehu Dou, Jing Lu, Xijing Chen, Xuefeng Zhang","doi":"10.1007/s11095-025-03931-2","DOIUrl":"https://doi.org/10.1007/s11095-025-03931-2","url":null,"abstract":"<p><p>Diseases of the central nervous system represent a significant challenge in the field of medical research and clinical practice. The utilization of gene therapy technology represents a promising therapeutic strategy aimed at ameliorating diseases at the genetic level. Nonetheless, the presence of the blood-brain barrier (BBB) and challenges pertaining to delivery efficiency, expression site and level, along with the potential risks linked to overexpression and prolonged expression, raise considerable safety concerns. The design, production, delivery, and expression of adeno-associated virus (AAV) vectors significantly influence both clinical efficacy and safety considerations. This article examines the factors contributing to and potential strategies for addressing the low transduction efficiency associated with the blood-brain barrier, focusing on the optimization of vector design, delivery methods, specific expression, and distribution. The study examines the optimization of promoter synthesis, the application of machine learning algorithms for the enhancement of AAV capsid evolution, and the evaluation of various administration routes. Additionally, it explores innovative delivery methods, including mannitol intra-arterial delivery and focused ultrasound strategies, aimed at improving efficacy and safety. These initiatives offer valuable guidance and insights pertaining to gene therapies aimed at addressing neurodegenerative diseases and various disorders of the central nervous system.</p>","PeriodicalId":20027,"journal":{"name":"Pharmaceutical Research","volume":" ","pages":""},"PeriodicalIF":4.3,"publicationDate":"2025-10-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145207318","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Hydroquinone Hydroxypropyl-β-cyclodextrin Inclusion Complex Based Topical Cream as an Effective Treatment of Melasma by Releasing Hydroquinone in a Controlled Manner.","authors":"Yinling Mu, Hongbing Liu, Xianglin Guo, Yiyu Liu, Wanbing Pan, Fan Zhao, Haibing He, Jingxin Gou, Xing Tang, Tian Yin, Yu Zhang","doi":"10.1007/s11095-025-03925-0","DOIUrl":"https://doi.org/10.1007/s11095-025-03925-0","url":null,"abstract":"<p><strong>Objective: </strong>Hydroquinone (HQ) is globally regarded as the gold-standard topical agent for melasma treatment. However, its clinical utility is limited by several challenges: susceptibility to oxidation induced by light, air, and metal ions, which generates harmful byproducts and triggers adverse effects (e.g., skin irritation, pruritus, and dermatitis). Moreover, excessively high concentration in the skin of hydroquinone due to the burst release of HQ may cause melanocyte cytotoxicity, while systemic penetration raises additional safety concerns. To overcome these limitations and enhance the therapeutic applicability of HQ, this study aims to develop strategies that mitigate its instability and optimize its delivery profile.</p><p><strong>Methods: </strong>In this study, an oil-in-water cream containing hydroquinone cyclodextrin inclusion complex (HQ-HPCD) was designed with PO as the oil phase matrix to improve the treatment of melasma.</p><p><strong>Results: </strong>As expected, the in vitro release profile of HQ-HPCD cream exhibited near-zero-order kinetics, effectively mitigating the irritation associated with burst release-induced peak concentrations. The HQ-HPCD inclusion complex demonstrated enhanced photostability and oxidative resistance compared to free HQ. Then it was confirmed by B16F10 cells that the HQ-HPCD increased the IC₅₀ of HQ by 4.6 times. Moreover, skin condition, tyrosinase activity, SOD (Superoxide Dismutase), MDA (Malondialdehyde) and melanin content in the mice were detected and the results were in line with the in vitro assays, which showed that PO-HQ-HPCD cream has higher resistance to melanin deposition and lower irritation.</p><p><strong>Conclusions: </strong>This study successfully developed a topical formulation that enhances the stability of hydroquinone (HQ) while achieving its sustained release in the epidermal layer, thereby improving its clinical applicability of melasma.</p>","PeriodicalId":20027,"journal":{"name":"Pharmaceutical Research","volume":" ","pages":""},"PeriodicalIF":4.3,"publicationDate":"2025-10-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145207259","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Enhancing Cannabidiol Apparent Solubility and Oral Delivery: Self-assembled Nanomicelles of Amphiphilic Block Copolymer with γ-Polyglutamic Acid-grafted Cholesterol.","authors":"Rui Li, Wenhui Ruan, Liyan Lu, Zhijuan Wu, Rui Hao, Yingli Wang, Jue Chen","doi":"10.1007/s11095-025-03924-1","DOIUrl":"https://doi.org/10.1007/s11095-025-03924-1","url":null,"abstract":"<p><strong>Purpose: </strong>Cannabidiol (CBD) exhibits antiepileptic, anticonvulsant, and anticancer effects. However, its clinical value is limited by poor oral absorption, low water solubility, and poor stability. Therefore, this study aimed to prepare a novel nanomicelles (NMs) to improve the above aspects of CBD.</p><p><strong>Methods: </strong>The amphiphilic polymer was synthesized by selecting γ -polyglutamic acid (γ-PGA) and cholesterol (CHOL) as hydrophilic and hydrophobic materials respectively. The optimal preparation process for CBD/(γ-PGA-g-CHOL) NMs was obtained through single-factor and orthogonal tests. The particle size, potential, stability, morphology, apparent solubility and in vitro drug release behavior of the drug-loaded NMs were characterized. Cytotoxicity, uptake and transport experiments on Caco-2 cells and in vivo pharmacokinetics studies in rats were performed for in vitro and in vivo oral absorption of the drugs.</p><p><strong>Results: </strong>The optimal parameters were a dosage of 2 mg, blank NM concentration of 5 mg/mL, an organic/aqueous ratio of 1:5, and a stirring time of 6 h. The NMs showed pH-sensitive release behavior, with particle size 163.1 ± 2.3 nm, zeta potential -16.5 ± 1.7 mV, encapsulation rate 84.46% ± 0.35%, and drug loading 8.78% ± 0.28%. They were spherical by SEM, stable at 25 ℃ and 37 ℃, and their apparent solubility increased 424-fold. The NMs were biocompatible and improved CBD absorption in uptake/transport tests. Orally administered NMs showed lower apparent clearance value (CL/F), and higher C_max and AUC than those in the free CBD group.</p><p><strong>Conclusions: </strong>These findings reveal potential of the delivery system, γ-PGA-g-CHOL NMs, to improve the stability of CBD and enhance its apparent solubility and systemic exposure.</p>","PeriodicalId":20027,"journal":{"name":"Pharmaceutical Research","volume":" ","pages":""},"PeriodicalIF":4.3,"publicationDate":"2025-10-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145207320","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Establishing PDE4 as a Novel Target of Urolithin-A in Mitigating LPS-induced Inflammation in Retinal Pigmented Epithelium Cells.","authors":"Likhitha Purna Kondapaneni, Meenakshi Arora, Erin M Scott, M N V Ravi Kumar, Raghu Ganugula","doi":"10.1007/s11095-025-03933-0","DOIUrl":"https://doi.org/10.1007/s11095-025-03933-0","url":null,"abstract":"<p><p>Ocular inflammation is a major contributor to vision-threatening disorders, with phosphodiesterase 4 (PDE4), a key regulator of cAMP playing a central role in pro-inflammatory signaling. Although investigational PDE4 inhibitors like Rolipram (RP) show therapeutic promise, their systemic toxicity limits clinical application, underscoring the need for safer, targeted alternatives. Urolithin A (UA), a gut-derived metabolite of ellagic acid with emerging anti-inflammatory properties, was evaluated as a novel PDE4 inhibitor. Molecular docking revealed that UA binds with high affinity to the A-chain of PDE4A (-8.79 kcal/mol), forming unique π-π stacking and multiple hydrogen bonds. In contrast, RP binds preferentially to the B-chain with slightly lower affinity (-8.42 kcal/mol) and fewer stabilizing interactions. While both ligands engage similar catalytic residues, UA exhibited a more extensive binding profile, suggesting enhanced stability and specificity. In lipopolysaccharide (LPS)-stimulated human retinal pigment epithelial cells (ARPE-19), UA significantly inhibited PDE4A activity, elevated intracellular cAMP, and reduced key inflammatory mediators (NF-κB, IL-6, TNF-α), as demonstrated by immunofluorescence, ELISA, and gene expression analysis. These findings support UA's function as an anti-inflammatory agent by inhibiting PDE4A, highlighting its potential as a safer systemic or localized therapy for ocular inflammatory diseases.</p>","PeriodicalId":20027,"journal":{"name":"Pharmaceutical Research","volume":" ","pages":""},"PeriodicalIF":4.3,"publicationDate":"2025-09-22","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145125683","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Investigating the Dissolution of Ibuprofen and Empagliflozin in Aqueous Deep Eutectic Solvent Systems: Experimental and Thermodynamic Modeling Insights.","authors":"Shadi Janfaza, Ali Haghtalab","doi":"10.1007/s11095-025-03921-4","DOIUrl":"10.1007/s11095-025-03921-4","url":null,"abstract":"<p><strong>Purpose: </strong>This study investigated the potential of a deep eutectic solvent (DES) to enhance the dissolution of two poorly water-soluble drugs, ibuprofen (IBU) and empagliflozin (EMPA). The DES was synthesized from tetrabutylphosphonium bromide (TBPB) and diethylene glycol (DEG).</p><p><strong>Methods: </strong>The apparent solubility of IBU and EMPA was measured in aqueous solutions containing eleven different mass fractions of the DES at temperatures ranging from 20 to 40°C. Dissolution kinetics were monitored over 24 h to differentiate between true equilibrium solubility and supersaturated states. The collected experimental data were then analyzed and correlated using three thermodynamic models: Wilson, NRTL, and UNIQUAC.</p><p><strong>Results: </strong>The findings indicated that ibuprofen achieved higher dissolution than empagliflozin in the DES-water system. For both drugs, the dissolution process was endothermic, with solubility increasing as both temperature and DES concentration increased. Among the thermodynamic models tested, the UNIQUAC model provided the most accurate correlation with the experimental dissolution data.</p>","PeriodicalId":20027,"journal":{"name":"Pharmaceutical Research","volume":" ","pages":"1647-1660"},"PeriodicalIF":4.3,"publicationDate":"2025-09-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145001209","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Preclinical Prediction of Resistance Mutations and Proposal of Sequential Treatment Strategies for ALK-positive Lung Cancer Using Next-generation ALK Inhibitors.","authors":"Yuki Takei, Hirotaka Kuroiwa, Chisaki Arai, Yuta Doi, Kentaro Semba","doi":"10.1007/s11095-025-03916-1","DOIUrl":"10.1007/s11095-025-03916-1","url":null,"abstract":"<p><strong>Background: </strong>Anaplastic lymphoma kinase (ALK) gene rearrangements occur in approximately 5% of non-small cell lung cancers (NSCLCs). Although ALK tyrosine kinase inhibitors provide substantial clinical benefits, acquired resistance-conferring mutations frequently emerge, leading to disease progression. Preclinical prediction of these mutations might help guide the development of more effective sequential treatment strategies prior to clinical application.</p><p><strong>Objective: </strong>To predict the emergence of resistance mutations to the investigational ALK inhibitors zotizalkib (TPX-0131), gilteritinib (ASP2215), and neladalkib (NVL-655) following resistance to first-line alectinib and assess the potential of these drugs as second-line therapies.</p><p><strong>Methods: </strong>A polymerase chain reaction (PCR)-based mutagenesis system was used to introduce random mutations into ALK cDNA harboring representative alectinib-resistant mutations. Mutant libraries were expressed in Ba/F3 cells, which were exposed to each inhibitor. Drug-resistant clones were isolated, sequenced, and evaluated for drug sensitivity using viability assays and immunoblotting.</p><p><strong>Results: </strong>Several resistance mutations against zotizalkib, gilteritinib, and neladalkib were identified. Sequential use of these agents effectively suppressed all predicted resistance patterns with G1202R or I1171N.</p><p><strong>Conclusions: </strong>This PCR-based platform provides a valuable approach for anticipating resistance mutations and guiding the design of optimized sequential therapies. Zotizalkib, gilteritinib, and neladalkib might represent promising alternatives to lorlatinib as second-line treatments for ALK-positive NSCLC.</p><p><strong>Key points: </strong>• A PCR-based mutation prediction system was successfully applied to fourth-generation ALK inhibitors. • Neladalkib showed efficacy against G1202R-positive relapses with minimal evidence of secondary resistance mutations. • Sequential combinations of gilteritinib with either neladalkib or ensartinib may sustain efficacy and delay resistance in I1171N-positive relapses.</p>","PeriodicalId":20027,"journal":{"name":"Pharmaceutical Research","volume":" ","pages":"1497-1509"},"PeriodicalIF":4.3,"publicationDate":"2025-09-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145138430","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}