Pharmaceuticals

18 pages, 1101 KB

Open AccessArticle

A Genetic Algorithm-Based Approach for Quantitative Prediction of Drug-Drug Interactions Caused by Cytochrome P450 3A Inhibition or Induction in Horses

by Veronica Di Paolo, Francesco Maria Ferrari, Italo Poggesi, Mauro Dacasto, Luigi Quintieri and Francesca Capolongo

Pharmaceuticals 2026, 19(6), 815; https://doi.org/10.3390/ph19060815 (registering DOI) - 22 May 2026

Abstract

Introduction: A genetic algorithm (GA)-based approach was designed to predict drug–drug interactions (DDIs) triggered by cytochrome P450 3A (CYP3A) inhibition or induction in horses. Methods: Area under the concentration-time curve ratios (AUCRs), obtained from published in vivo DDI studies in horses, were used [...] Read more.

Introduction: A genetic algorithm (GA)-based approach was designed to predict drug–drug interactions (DDIs) triggered by cytochrome P450 3A (CYP3A) inhibition or induction in horses. Methods: Area under the concentration-time curve ratios (AUCRs), obtained from published in vivo DDI studies in horses, were used to compute the following parameters: (1) the contribution ratio (CR), i.e., the fraction of the substrate dose metabolized via the CYP3A pathway, and (2) the interacting drug’s inhibitory potency or inducing efficacy (IR or IC, respectively). Results: AUCRs for 9 substrates, 12 inhibitors, and 1 inducer of equine CYP3A were predicted and validated with the developed method. More than 96% of predictions fell within the commonly accepted range of 50–200% of observed values. Conclusions: The proposed GA-based method may be a useful tool to estimate possible clinically relevant DDIs when co-administration of a CYP3A substrate and a CYP3A-interacting drug is anticipated. Full article

(This article belongs to the Section Pharmacology)

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28 pages, 7799 KB

Open AccessArticle

Discovery of Benzophenanthridine Alkaloids from Zanthoxylum nitidum That Target the MDM2–p53 Axis in NSCLC

by Nguyen Manh Cuong, Elizaveta Fefilova, Vu Thanh Loc, Natalia Karpova, Nguyen Xuan Ha, Alexandra Daks, Nguyen Viet Ha, Tran Thu Huong, Sergey Parfenyev, Alexander Nazarov, Oleg Semenov, Yulia Gnennaya, Olga Fedorova, Nickolai A. Barlev and Oleg Shuvalov

Pharmaceuticals 2026, 19(6), 814; https://doi.org/10.3390/ph19060814 (registering DOI) - 22 May 2026

Abstract

Background/Objectives: Non-small cell lung cancer (NSCLC) accounts for about 85% of lung cancers and is a leading cause of cancer-related deaths worldwide. Pharmacological targeting of the p53–MDM2 interaction to activate wild-type p53 is a promising strategy for treating NSCLC that retain functional p53 [...] Read more.

Background/Objectives: Non-small cell lung cancer (NSCLC) accounts for about 85% of lung cancers and is a leading cause of cancer-related deaths worldwide. Pharmacological targeting of the p53–MDM2 interaction to activate wild-type p53 is a promising strategy for treating NSCLC that retain functional p53 (approximately 50% of all cases). Methods: We screened 33 ethnomedicinal Vietnamese plant extracts for their anticancer effects using p53-expressing and p53-null NSCLC cell models, as well as two non-cancerous cell lines for control. We used an array of different experimental approaches including NMR spectroscopy; molecular docking; an MTT test; cell cycle analysis; apoptosis analysis; wound healing, migration, and invasion assays; Real-Time PCR; immunoblotting; and Seahorse energy profiling to characterize and study the effects of these bioactive compounds on NSCLC cells. Results: Ethanol extract of Zanthoxylum nitidum stems and twigs demonstrated potent and selective activity by inducing p53-dependent cell cycle arrest and apoptosis. Phytochemical analysis identified several benzophenanthridine alkaloids as active constituents. Molecular docking revealed their strong in silico binding to MDM2. Notably, nitidine was the most promising compound among the molecules tested. Unlike nutlin, but similar to SP141 (two well-known MDM2 inhibitors), nitidine strongly stabilized p53 while concomitantly attenuating MDM2 at the protein level. Surprisingly, this effect was p53-independent. Additionally, nitidine suppressed the EMT master regulator Snail, and hence disrupted cellular bioenergetics and inhibited migration and invasion of NSCLC cells. Conclusions: Our findings identify Z. nitidum and nitidine as promising sources for developing novel MDM2-targeting therapeutics against NSCLC irrespective of the p53 status. Full article

(This article belongs to the Special Issue Medicinal Plants: Exploring Plant-Based Bioactive Compounds for Cancer and Metabolic Diseases Prevention and Intervention)

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19 pages, 789 KB

Open AccessArticle

New Synthetic Strategies Toward DFO*: Enhanced Yield and Purity of a Key Chelator for ⁸⁹Zr Chemistry

by Nils F. Baier, Minqian Miao, Ralf Schirrmacher, Björn Wängler, Gert Fricker and Carmen Wängler

Pharmaceuticals 2026, 19(6), 813; https://doi.org/10.3390/ph19060813 (registering DOI) - 22 May 2026

Abstract

Background: Zirconium-89 (⁸⁹Zr) is a key PET radionuclide and the limited in vivo stability of its clinically used ⁸⁹Zr-DFO complexes has driven the pursuit of improved chelator architectures. Among these, DFO* has attracted particular attention due to its exceptional [...] Read more.

Background: Zirconium-89 (⁸⁹Zr) is a key PET radionuclide and the limited in vivo stability of its clinically used ⁸⁹Zr-DFO complexes has driven the pursuit of improved chelator architectures. Among these, DFO* has attracted particular attention due to its exceptional complex stability with ⁸⁹Zr⁴⁺ and favorable pharmacokinetics of the corresponding bioconjugates in vivo. Despite these advantages, DFO*’s broader application has been hampered by significant synthetic challenges, primarily arising from its pronounced acid sensitivity. Methods: Here, we present a systematic investigation of the acid lability of DFO and DFO*-derived systems, revealing substantial degradation under acidic conditions being commonly applied during preparation and purification. These findings highlight critical limitations of conventional synthetic and purification protocols. To address this, we developed two complementary synthetic routes that consistently avoid fragmentation-inducing conditions. Results: THP/Boc- and TBDPS/Fmoc-based routes provide robust five- and six-step syntheses of DFO*, affording overall yields of 11% and 13%/6.1% and high purity (≥98%) without detectable degradation. Conclusions: By systematically investigating the acid sensitivity of DFO/DFO*-based chelators and providing practical synthetic solutions, this work enables reliable access to DFO* and advances its application in ⁸⁹Zr radiopharmaceutical development. Full article

(This article belongs to the Special Issue Past, Present and Future Radiotracer Techniques: Radiopharmaceuticals in Cancer Theranostics: 2nd Edition)

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16 pages, 967 KB

Open AccessArticle

CYP450 Metabolizer Phenotypes in a Turkish Emergency Cardiac Patient Cohort: A Descriptive Pharmacogenomic Study

by Alten Oskay, Tülay Oskay, Veli Kaan Aydın, Özer Eser, Murat Seyit, Işık Tekin, Mert Özen, Atakan Yılmaz, İbrahim Türkçüer, Gergana Lengerova, Martina Bozhkova, Steliyan Petrov and Aylin Köseler

Pharmaceuticals 2026, 19(6), 812; https://doi.org/10.3390/ph19060812 (registering DOI) - 22 May 2026

Abstract

Background/Objectives: Cytochrome P450 enzymes (CYP2D6, CYP2C19, CYP3A4) play a key role in interindividual variability in cardiovascular drug metabolism. This study aimed to describe metabolizer phenotype distributions in a Turkish emergency cardiac cohort and across diagnostic categories. Methods: This retrospective descriptive pharmacogenomic [...] Read more.

Background/Objectives: Cytochrome P450 enzymes (CYP2D6, CYP2C19, CYP3A4) play a key role in interindividual variability in cardiovascular drug metabolism. This study aimed to describe metabolizer phenotype distributions in a Turkish emergency cardiac cohort and across diagnostic categories. Methods: This retrospective descriptive pharmacogenomic study included 250 patients. Genotyping was performed using TaqMan assays for CYP2D6 (*2, *4, *10, *41), CYP2C19 (*2, *17), and CYP3A4 (*22, *1B). Phenotypes were assigned according to CPIC guidelines. CYP2D6 copy-number variation was not assessed. Results: Non-normal metabolizer phenotypes were observed in 55.6% (CYP2D6), 84.4% (CYP2C19), and 30.4% (CYP3A4) of patients. For CYP2D6, normal (44.4%) and intermediate (42.0%) metabolizers predominated. For CYP2C19, intermediate metabolizers were most frequent (36.0%), followed by normal (22.8%), rapid (17.2%), poor (14.8%), and ultra-rapid metabolizers (9.2%). CYP3A4 showed predominantly normal activity (69.6%). Phenotype distributions varied across diagnoses without clear clustering. Conclusions: A high prevalence of CYP2D6 and CYP2C19 variability with predicted functional relevance based on CPIC was observed, whereas CYP3A4 activity was more stable. These findings provide descriptive pharmacogenomic data to support future genotype-guided cardiovascular therapy studies. Full article

(This article belongs to the Section Pharmacology)

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29 pages, 2543 KB

Open AccessReview

Pharmaceutical Peptides: From Synthesis and Mechanistic Pharmacology to Future Biologic Therapeutics

by Muhammad Yaseen Khan, Touseef Nawaz, Muhammad Sajid Hamid Akash and Adnan Amin

Pharmaceuticals 2026, 19(6), 811; https://doi.org/10.3390/ph19060811 (registering DOI) - 22 May 2026

Abstract

Peptide therapeutics have emerged as a versatile class of biomolecules bridging the gap between small-molecule drugs and large biologics. Advantages of such molecules include high target specificity, potent bioactivity and reduced off-target toxicity. Despite these, broader clinical translation remains constrained by inherent limitations [...] Read more.

Peptide therapeutics have emerged as a versatile class of biomolecules bridging the gap between small-molecule drugs and large biologics. Advantages of such molecules include high target specificity, potent bioactivity and reduced off-target toxicity. Despite these, broader clinical translation remains constrained by inherent limitations like poor metabolic stability, rapid renal clearance, limited membrane permeability and scalable synthesis. This review aims to systematically integrate advances in peptide science across natural discovery, synthetic methodologies, structural engineering, and translational delivery systems, while identifying critical research gaps hindering clinical adoption. We highlight diverse natural sources of bioactive peptides, including plant- (lunasin), animal- (Val-Pro-Pro (VPP) and Ile-Pro-Pro (IPP)), microbial- (nisin and cyclosporine), marine- (dolastatins) and venom-derived (chlorotoxin and ω-conotoxin MVIIA (ziconotide)) agents. Advances in solid-phase peptide synthesis (SPPS), green chemistry, and catalytic strategies are discussed alongside emerging in silico approaches, including artificial intelligence-driven sequence design and molecular modeling. Structural modifications such as cyclization, hydrocarbon stapling, PEGylation, and lipidation are critically evaluated for their role in enhancing pharmacokinetic and pharmacodynamic properties. Furthermore, nanoformulation strategies, including self-assembling peptides and cell-penetrating systems, are examined for their potential to overcome biological barriers. Importantly, this review identifies key unresolved challenges, including the lack of predictive models for peptide delivery systems, safety concerns associated with long-term modifications, and limited in vivo validation of naturally derived peptides. Addressing these gaps through integrated computational and experimental approaches will be essential for advancing next-generation peptide therapeutics. Collectively, this work provides a comprehensive framework for the rational design and translation of peptide-based precision medicines. Full article

(This article belongs to the Special Issue Peptide Synthesis and Drug Development: Exploring Progress and Potential, 2nd Edition)

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32 pages, 2147 KB

Open AccessReview

Harnessing Machine Learning for Accelerated Drug Discovery: Opportunities and Unmet Challenges

by Mohamed El-Tanani, Syed Arman Rabbani, Adil Farooq Wali, Frezah Muhana, Yahia El-Tanani and Rakesh Kumar

Pharmaceuticals 2026, 19(6), 810; https://doi.org/10.3390/ph19060810 (registering DOI) - 22 May 2026

Abstract

The process of drug discovery is one of the most expensive, time-consuming, and high-risk endeavors in modern science. Translating initial scientific insights into safe and effective therapies, supported by genomics, structural biology, and computational chemistry, typically requires more than a decade and substantial [...] Read more.

The process of drug discovery is one of the most expensive, time-consuming, and high-risk endeavors in modern science. Translating initial scientific insights into safe and effective therapies, supported by genomics, structural biology, and computational chemistry, typically requires more than a decade and substantial financial investment. Machine learning (ML) has emerged as a powerful tool for improving efficiency across the drug discovery pipeline. By enabling the analysis of large and complex datasets, ML supports target identification, lead discovery, optimization, and prediction of preclinical and clinical outcomes. Its integration with experimental validation and automation is illustrated by recent advances such as protein structure prediction, AI-driven antifibrotic compound discovery, and antibiotic identification. Despite these advances, significant challenges remain. Model generalizability is limited by data scarcity, heterogeneity, and hidden biases. In addition, the translation of in silico predictions into clinically validated outcomes remains a major bottleneck, and regulatory acceptance is constrained by limited model interpretability. Ethical considerations, including data privacy, equitable representation, and the potential misuse of generative models, further complicate adoption. This review examines the applications of ML across the drug discovery pipeline, with a focus on translational and regulatory considerations. It also discusses emerging directions, including hybrid physics–AI approaches, multimodal foundation models, federated learning, and explainable AI. The effective integration of ML will depend on rigorous validation, interdisciplinary collaboration, responsible data governance, and alignment with regulatory frameworks. Full article

(This article belongs to the Special Issue Artificial Intelligence: New Molecules, Therapeutic Targets and Discovery of New Drugs)

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16 pages, 889 KB

Open AccessArticle

Composite CA15-3, LDH, and Albumin Index as a Predictor of Survival in HER2-Positive Metastatic Breast Cancer Treated with Trastuzumab Emtansine

by Nagihan Kolkıran, Atike Pınar Erdoğan, Mustafa Şahbazlar, Müge Kurul Yeniay, Sinan Ünal, Mehmet Sinan Akarca, Elif Atağ Akyürek, Özge Demirkıran, Bilgin Demir and Ferhat Ekinci

Pharmaceuticals 2026, 19(6), 809; https://doi.org/10.3390/ph19060809 (registering DOI) - 22 May 2026

Abstract

Background/Objectives: Trastuzumab emtansine (T-DM1) is widely used in Human Epidermal Growth Factor Receptor2 (HER2)-positive metastatic breast cancer; however, outcomes vary, and reliable prognostic markers remain limited. We developed the CALA index as a composite biomarker integrating CA15-3, lactate dehydrogenase (LDH), and albumin. [...] Read more.

Background/Objectives: Trastuzumab emtansine (T-DM1) is widely used in Human Epidermal Growth Factor Receptor2 (HER2)-positive metastatic breast cancer; however, outcomes vary, and reliable prognostic markers remain limited. We developed the CALA index as a composite biomarker integrating CA15-3, lactate dehydrogenase (LDH), and albumin. This study aimed to evaluate the prognostic value of the CALA index in metastatic breast cancer treated with T-DM1. Methods: This multicenter retrospective study included 168 patients treated with T-DM1 across four tertiary centers. The CALA index was calculated using pretreatment levels of CA15-3, LDH, and albumin. Receiver operating characteristic (ROC) curve analysis was used to determine the optimal cutoff value, and patients were stratified into groups accordingly. Survival outcomes and independent risk factors were assessed using Kaplan–Meier and Cox regression analyses. Results: The median overall survival (OS) was 26 months (95% CI: 21.3–30.7). ROC analysis identified an optimal CALA cutoff value of 118.3. Patients with CALA ≤ 118.3 demonstrated significantly longer OS compared with those with CALA > 118.3 (log-rank p = 0.006), with 1- and 3-year OS rates of 81.2% and 43.2% versus 69.8% and 22.7%, respectively. In univariate analysis, CALA > 118.3 was associated with worse OS (HR: 1.699; 95% CI: 1.151–2.506; p = 0.008), and this association remained significant in multivariate analysis (HR: 1.671; 95% CI: 1.088–2.565; p = 0.019). Conclusions: The CALA index was associated with overall survival in metastatic breast cancer treated with trastuzumab emtansine and may serve as a practical tool for risk stratification. Full article

(This article belongs to the Section Biopharmaceuticals)

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20 pages, 3316 KB

Open AccessArticle

Lactoferrin Displays Stimulating and Protective Effects on Newly Isolated Phage vB_Sau-E: A New Perspective for Treatment of Staphylococcal Skin Infections

by Urszula Leszczyńska, Małgorzata Stasiłojć, Milena Grzenkowicz, Magdalena Narajczyk, Agnieszka Necel, Lidia Piechowicz, Katarzyna Kosznik-Kwaśnicka and Anna Żywicka

Pharmaceuticals 2026, 19(6), 808; https://doi.org/10.3390/ph19060808 (registering DOI) - 22 May 2026

Abstract

Background/Objectives: Skin and soft tissue infections (SSTIs) represent a significant clinical challenge, largely due to the high prevalence of antibiotic-resistant Staphylococcus aureus, particularly methicillin-resistant S. aureus (MRSA). Treatment is further complicated by biofilm formation, which reduces antibiotic efficacy. The limitations of conventional [...] Read more.

Background/Objectives: Skin and soft tissue infections (SSTIs) represent a significant clinical challenge, largely due to the high prevalence of antibiotic-resistant Staphylococcus aureus, particularly methicillin-resistant S. aureus (MRSA). Treatment is further complicated by biofilm formation, which reduces antibiotic efficacy. The limitations of conventional therapies highlight the need for alternative approaches. Phage therapy has emerged as a promising biological strategy; however, its effectiveness may be constrained by factors such as phage instability and biofilm regrowth. This study aimed to enhance phage-based treatment by combining a newly isolated phage, vB_Sau-E, with lactoferrin (Lf), a multifunctional protein of the innate immune system. Methods: Phage vB_Sau-E was characterized in terms of its infection dynamics and lytic activity. Biocompatibility was further examined using human skin cell lines. The potential effect of Lf was assessed by evaluating its impact on phage infectivity and stability under a range of environmental conditions and by checkerboard assay. Results: Phage vB_Sau-E belongs to the Silviavirus genus in the Herelleviridae family. It was shown to infect 12 out of 22 tested clinical MRSA isolates, with 10 strains identified as good hosts. The phage has a ~30 min life cycle, and ~50 progeny virions are released after bacterial cell lysis. We have also observed that Lf increased plating efficiency and enhanced phage stability at a pH of 5.5 and at −20° C. It also proved to have an additive antibacterial effect, though this was observed to be strain-dependent. Conclusions: Lactoferrin functions as a stabilizing adjuvant for phage vB_Sau-E. Its additive effect supports the development of more effective, biofilm-targeting therapies for staphylococcal SSTIs. Full article

(This article belongs to the Section Biopharmaceuticals)

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21 pages, 8705 KB

Open AccessArticle

Neuroprotective Indole Diterpenoids from the Fungus Tolypocladium album DWS131

by Ai-Lin Liang, Chao Wang, Xing-Yi Chen, Yu-Feng Tan, Wen-Yu Lu, Peng-Ju Xu, Hong-Ping Long, Shao Liu, Jing Li, Wen-Xuan Wang and Xiaobo Xia

Pharmaceuticals 2026, 19(6), 807; https://doi.org/10.3390/ph19060807 (registering DOI) - 22 May 2026

Abstract

Context/Objective: Fungi of the genus Tolypocladium are known for their diverse metabolic capabilities and medicinal potential. Indole diterpenoids (IDTs) represent a structurally unique class of fungal metabolites. Beyond their established roles as mycotoxins, these compounds have recently shown promise for neuroprotective effects. [...] Read more.

Context/Objective: Fungi of the genus Tolypocladium are known for their diverse metabolic capabilities and medicinal potential. Indole diterpenoids (IDTs) represent a structurally unique class of fungal metabolites. Beyond their established roles as mycotoxins, these compounds have recently shown promise for neuroprotective effects. The objective of this study was to isolate and characterize novel IDTs from Tolypocladium album DWS131 and evaluate their neuroprotective activities and underlying mechanisms. Methods: IDTs were isolated through comprehensive chromatographic techniques. Their structures were elucidated using HRESIMS data, 1D/2D NMR spectra, and quantum chemical calculations. Neuroprotective effects were evaluated using glutamate (Glu)-induced R28 cells in vitro and N-methyl-D-aspartic acid-induced mouse models in vivo. A total of 48 mice were utilized for in vivo evaluations, divided into two separate experimental cohorts. In each cohort, mice were randomly assigned to four groups (n = 6 per group). Post-intravitreal injection, retinal survival and visual function were assessed via Brn3a-stained flat-mounts, H&E staining, f-VEP, f-ERG, and OptoDrum. Mechanisms involving the SLC7A11/GPX4/ACSL4 axis were investigated by Western blotting and immunofluorescence. Results: Seven previously undescribed paxilline-type IDTs, tolypindoles A–G (1–7), and two known analogues (8–9) were identified. Compounds 8 and 9 exhibited significant neuroprotection closely associated with the attenuation of oxidative stress and the modulation of ferroptosis-related pathways in Glu-induced R28 cells. In vivo, they preserved retinal ganglion cells, maintained retinal structure, and protected visual function, with compound 8 demonstrating superior efficacy. Mechanistic investigations revealed that both compounds modulate the SLC7A11/GPX4/ACSL4 signaling axis. Conclusions: This study expands the chemical diversity of T. album DWS131. Compounds 8 and 9, characterized by isopentenyl moieties, highlight a promising therapeutic potential for retinal neurodegenerative diseases such as glaucoma. Full article

(This article belongs to the Section Natural Products)

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14 pages, 311 KB

Open AccessReview

Fixed Dose Combinations as an Advantage for the Treatment of Pediatric Tuberculosis: A Narrative Review

by Susanna Esposito, Beatrice Rita Campana, Gaia Giorgia Arnesano and Nicola Principi

Pharmaceuticals 2026, 19(6), 806; https://doi.org/10.3390/ph19060806 (registering DOI) - 22 May 2026

Abstract

Background: Pediatric tuberculosis (TB) remains a major global health concern, accounting for a substantial proportion of TB-related morbidity and mortality worldwide. Treatment in children is particularly challenging due to age-specific pharmacokinetics, difficulties in drug administration, poor palatability, and reliance on caregivers for adherence. [...] Read more.

Background: Pediatric tuberculosis (TB) remains a major global health concern, accounting for a substantial proportion of TB-related morbidity and mortality worldwide. Treatment in children is particularly challenging due to age-specific pharmacokinetics, difficulties in drug administration, poor palatability, and reliance on caregivers for adherence. Objectives: This narrative review aims to evaluate the advantages and limitations of fixed-dose combinations (FDCs) in the treatment of pediatric TB, with a focus on adherence, pharmacological considerations, clinical outcomes, and implementation challenges. Methods: A narrative review of the literature was conducted, including clinical studies, pharmacokinetic analyses, programmatic data, and international guidelines related to the use of FDCs in pediatric TB management. Results: Evidence indicates that pediatric FDCs significantly improve treatment adherence by reducing pill burden and simplifying dosing regimens. They also decrease the risk of medication errors and inadvertent monotherapy, thereby contributing to the prevention of drug resistance. The availability of dispersible, child-friendly formulations has enhanced acceptability and ease of administration. However, limitations persist, including reduced flexibility in dose individualization, challenges in identifying the causative agent in adverse drug reactions, and variable access across settings. Pharmacokinetic concerns, particularly regarding rifampicin exposure, have been addressed in newer WHO-recommended formulations. Conclusions: FDCs represent a critical advancement in pediatric TB management and are strongly supported by international guidelines. Further research is needed to optimize formulations, ensure equitable access, and evaluate long-term clinical outcomes in diverse pediatric populations. Full article

(This article belongs to the Special Issue Advanced Nanosystems and Emerging Therapies: Innovations in Tuberculosis Treatment and Drug Resistance)

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20 pages, 10761 KB

Open AccessArticle

Identification, Determination and Transdermal Behavior Characterization of Nine Components in WenTong HuoXue Cream by UPLC-MS/MS

by Xinran Zhang, Xiaodan Qiu, Xiaolong Kang, Guangzhi Shan and Chenghui He

Pharmaceuticals 2026, 19(6), 805; https://doi.org/10.3390/ph19060805 (registering DOI) - 22 May 2026

Abstract

Background/Objectives: WenTong HuoXue Cream (WTHXC) plays a significant role in the treatment of diabetic peripheral neuropathy (DPN). However, the material basis and quality control methods for this formulation remain largely unexplored. Methods: In this study, UPLC-HRMS/MS combined with standard reference substances was employed [...] Read more.

Background/Objectives: WenTong HuoXue Cream (WTHXC) plays a significant role in the treatment of diabetic peripheral neuropathy (DPN). However, the material basis and quality control methods for this formulation remain largely unexplored. Methods: In this study, UPLC-HRMS/MS combined with standard reference substances was employed to comprehensively identify and confirm the chemical constituents of WTHXC. Furthermore, a rapid and sensitive UPLC-MS/MS method based on multiple reaction monitoring (MRM) mode was developed and validated for the simultaneous quantification of the marker components. Results: Nine compounds were unambiguous characterized, including Di-hydrocapsaicin (DHC), Oxypeucedanin hydrate (OPH), Imperatorin (IMP), Isoimperatorin (IIMP), Xanthotoxin (XAN), Hydroxysafflor yellow A (HSYA), Chlorogenic acid (CGA), Ferulic Acid (FA) and Ligustilide (LIG). The results of method validation denotes that all the analytes showed good linearity between concentration and peak area in the tested ranges, with correlation coefficients (r) not less than 0.9990. The relative standard deviation (RSD) of precision was in the range of 0.57–7.11%. The accuracy of the method, verified by recovery experiments at three concentration levels, ranged from 96.51% to 101.04% for all analytes. Transdermal behavior determination results demonstrate that OPH, HYSA, CGA, FA and LIG exhibited favorable skin permeability and may serve as the key active components of WTHXC. Conclusions: This study elucidates the material basis of WTHXC, providing a scientific foundation for the development of quality control methods and facilitating its broader clinical application. Full article

(This article belongs to the Special Issue Advances in Drug Analysis and Drug Development, 2nd Edition)

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