The AAPS Journal[JOURNAL]
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200 papers
Revisiting the Anti-Drug Antibody Assay Drug Tolerance of a Commercial Biological Product.
Zanghi J, Yu N, Andrews BT … +6 more , Chan P, Usdin M, Chiu C, Lin W, Lin A, Song Y
AAPS J · 2025 Dec · PMID 41436902 · Publisher ↗
Drug tolerance (DT) is a critical attribute of anti-drug antibody (ADA) assays for assessing clinical immunogenicity. We present a unique situation where a previously approved commercial product, atezolizumab, required r...
Drug tolerance (DT) is a critical attribute of anti-drug antibody (ADA) assays for assessing clinical immunogenicity. We present a unique situation where a previously approved commercial product, atezolizumab, required re-assessment of the assay DT to meet an increased drug exposure demand arising from a new route of administration (subcutaneous) and align with updated health authority (HA) regulations. Rather than redevelop the existing ADA assay, which could disrupt ongoing clinical trials, we identified a new anti-idiotype (anti-ID) antibody surrogate that demonstrated that the assay maintained adequate DT for the new route of administration. This streamlined approach addressed concerns regarding higher serum trough concentrations with subcutaneous administration and stricter sensitivity expectations. We established a target DT concentration based on population pharmacokinetic modeling to ensure adequate ADA characterization at steady state. This case study highlights the value of having alternative surrogate ADAs and demonstrates that achieving stringent DT requirements can be accomplished without extensive method redevelopment. We also introduce the broader implications of surrogate ADA selection, binding kinetics, and the clinical relevance of achieving high DT in the context of atezolizumab's efficacy and safety profile. This work also emphasizes the importance of considering bioanalytical assay characteristics, such as DT, throughout a product's lifecycle.
Quantifying the Full-to-Empty Adeno-Associated Virus (AAV) Capsid Ratios and Their Impact on Transduction Efficiency in vitro.
Hoyle B, Hill J, Bello D
AAPS J · 2025 Dec · PMID 41408431 · Publisher ↗
Adeno-associated virus (AAV) vectors are made up of a protein shell that typically encapsidates a single stranded viral deoxyribonucleic acid (DNA) genome up to a total of ~ 4700 nucleotides. Classical AAV Manufacturing...
Adeno-associated virus (AAV) vectors are made up of a protein shell that typically encapsidates a single stranded viral deoxyribonucleic acid (DNA) genome up to a total of ~ 4700 nucleotides. Classical AAV Manufacturing processes yield a mixture of capsids that either contain no payload, partial DNA, or the gene of interest (GOI). Several studies have generated variable data indicating that empty capsids (1) affect transduction efficiency by engaging the immune system's neutralizing antibodies, (2) do not improve full capsid uptake, (3) cause liver toxicity at high capsid loads, or (4) alter biodistribution. To study the effect of different empty capsid ratios on transduction efficiency, multiple AAV serotypes and full-to-empty methods were utilized in conjunction with a 5-day in vitro potency method. Total protein expression levels for each AAV were measured by a luciferase reporter after differentiation of C2C12 mouse myoblast cells into myotubes. Empty capsid load regardless of serotype identity did yield a statistically significant change in total protein expression levels. This data suggests that limiting the number of empty capsids regardless of the serotype is advised to ensure the highest dose of GOI containing AAVs reach target cells. Manufacturing practices should continue to reduce the overall empty capsid load in each batch to ensure most AAV particles contain the GOI. The findings of this project could aid in process optimization and method development strategies for cell-based assays to quantify the level of protein expression in AAVs containing other GOIs.
Substantial Comparability of Singlicate Versus Duplicate Analysis-A Retrospective Evaluation of Clinical Immunogenicity in a Biosimilar PK Study.
Lichtenfels M, Fan J, Guerrieri D … +4 more , Hackl MW, Izquierdo AV, Cen L, Pötzl J
AAPS J · 2025 Dec · PMID 41408420 · Publisher ↗
To investigate the feasibility of a singlicate-based approach for immunogenicity assays in the biosimilar setting by comparing singlicate and duplicate data of an established Anti-Drug Antibodies (ADA) assay and from a b...
To investigate the feasibility of a singlicate-based approach for immunogenicity assays in the biosimilar setting by comparing singlicate and duplicate data of an established Anti-Drug Antibodies (ADA) assay and from a biosimilar study. We re-calculated the screening, confirmatory, and titer cut-points using singlicate values. The ADA method validation initially performed in duplicates was re-evaluated based on singlicate data. We performed variance component analysis to investigate the contribution of well-well variance on overall data variability. We re-assessed the clinical immunogenicity study data based on singlicate values. The ADA assay validation parameters were comparable between duplicate and singlicate-based evaluation. The variance component analysis confirmed the negligible influence of well-to-well variability. The use of singlicates would not have impacted the immunogenicity outcome of the clinical study. The singlicate-based analysis in our study would have reduced the analytical workload by about ~ 40%.
Impacts of Antibody Structure and Mixtures on Receptor Signaling for Antibody-Dependent Cellular Cytotoxicity.
Guan J, Wang T, Tessier PM … +1 more , Thurber GM
AAPS J · 2025 Dec · PMID 41408018 · Full text
Antibody-Dependent Cellular Cytotoxicity (ADCC) is a key mechanism of action for humoral immune response, which is important for clinical antibodies such as trastuzumab and cetuximab. The level of ADCC is dependent on mu...
Antibody-Dependent Cellular Cytotoxicity (ADCC) is a key mechanism of action for humoral immune response, which is important for clinical antibodies such as trastuzumab and cetuximab. The level of ADCC is dependent on multiple properties such as antibody isotype, Fab affinity, epitope, and geometry in the immune synapse. Here, we integrated computational simulations with experiments to analyze the impacts of several key factors on ADCC, including binding affinity, target expression, hinge flexibility, and antibody valency. The kinetic model was adapted to simulate antibody cross-linking between tumor and immune-reporter cells, followed by signal activation. Given the complexity of the interactions between cells and the formation of the immunological synapse, we fitted the effective on-rates within the synapse that are hard to determine a priori. With minimal fitting, the model successfully replicated the trends of immune activation for a series of trastuzumab structural mutants. The simulations demonstrated that antibody variants with a higher likelihood of monovalent target binding, such as single-arm antibodies, as well as those with low Fab affinity and reduced hinge flexibility, increased signaling. The model was able to capture the efficacy of mixtures of antibodies with different Fc domains, which are relevant for combination treatments such as trastuzumab and pertuzumab. Interestingly, the fraction of receptors blocked with antibody combinations was more important than total receptor expression, implying restrictions on diffusion of free receptors in the synapse. Overall, the simulations showed close agreement with experimental observations, providing a tool to interpret the ADCC results and guide the design of antibody therapeutics.
Correction: Randomized-Exposure Mixture-Model Analysis (REMIX) allowing Type-1 Error Controlled Exposure-Response Modelling.
Wojtyniak D, Guk J, Wicha SG
AAPS J · 2025 Dec · PMID 41408014 · Publisher ↗
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Implementing Synthetic Multi-Transmembrane Receptor Protein in a Duplex Immunoassay for Assessment of Neutralizing Antibody to a Bispecific Biotherapeutic.
Wu B, Devlin J, Schnarr M … +4 more , Ji Y, Zhao B, Jiang Y, Yang TY
AAPS J · 2025 Dec · PMID 41407979 · Publisher ↗
The development of multi-specific biotherapeutics has revolutionized targeted therapy by simultaneously engaging multiple receptors or pathways, thereby enhancing therapeutic efficacy and specificity. However, evaluating...
The development of multi-specific biotherapeutics has revolutionized targeted therapy by simultaneously engaging multiple receptors or pathways, thereby enhancing therapeutic efficacy and specificity. However, evaluating the immunogenic potential of these complex molecules remains a significant challenge, particularly in the reliable detection of neutralizing antibodies (NAbs). To support the development of a bispecific biotherapeutic, we initially established a cell-based bioassay utilizing a cytotoxicity assay platform for NAb assessment. This traditional approach faced significant limitations due to severely limited drug tolerance which prevented accurate NAb classification. To address this hurdle, we developed a non-cell based competitive ligand binding (CLB) assay. The primary obstacle was the multi-transmembrane receptor target, which lacks a soluble form suitable for conventional immunoassays. We successfully addressed this challenge by leveraging a novel synthetic version of the multi-transmembrane receptor as the surrogate target. This enabled the development of a duplex competitive ligand binding assay utilizing a homogeneous bead-based AlphaLISA™ assay platform. This innovative duplex NAb assay significantly increased drug tolerance by at least 170-fold when compared to the cell-based assay, enabling sensitive and specific detection of NAb against each drug target binding domain. The AlphaLISA™ based CLB NAb exhibited negligible matrix interference and generated excellent intra-assay and inter-assay precision, with data concordant across different reagent lots and plate readers. Our results demonstrate that the AlphaLISA™ assay platform offers a robust, sensitive, and drug-tolerant alternative to traditional cell-based NAb assays. This approach provides a superior solution for assessment of NAb against multi-specific biotherapeutics targeting multi-transmembrane receptors.
Sodium Alginate Hydrogels Loaded with Mesenchymal Stem Cells-Derived Extracellular Vesicles: Safety and Cell Migration Potential.
Henrique Domingos da Silva L, Paris Junior JA, Lopes Francisco RC … +8 more , Trovatti E, Tavares Junior AG, Chorilli M, Falcão Haddad F, Scarim CB, Ferreira Floriano J, Fardim P, Resende FA
AAPS J · 2025 Dec · PMID 41407940 · Publisher ↗
Wound healing is a complex process often impaired in severe injuries, requiring innovative therapeutic strategies. Mesenchymal stem cell-derived extracellular vesicles (MSC-EVs) modulate key cellular pathways, but their...
Wound healing is a complex process often impaired in severe injuries, requiring innovative therapeutic strategies. Mesenchymal stem cell-derived extracellular vesicles (MSC-EVs) modulate key cellular pathways, but their clinical application is limited by low stability and bioavailability. This study aimed to evaluate the safety and potential of sodium alginate hydrogels (SAH-EVs) loaded with mesenchymal stem cell-derived extracellular vesicles, focusing on cell migration, cytotoxicity, genotoxicity, and irritation potential. MSC-EVs from Sprague-Dawley rat bone marrow were isolated from conditioned medium collected at 24, 36, 48 and 60 h using size exclusion chromatography and characterized by Nanoparticle Tracking Analysis. The highest EV concentration was obtained from the conditioned medium collected at 36 h, with a main peak at 123 nm. The heterogeneous particle population suggests the presence of EV subtypes. Scanning Electron Microscopy confirmed successful MSC-EVs incorporation into hydrogels with desirable viscoelastic properties. SAH-EVs stimulated HaCaT keratinocyte migration while exhibiting low cytotoxicity in 2D and 3D models, with no genotoxic or mutagenic effects. HET-CAM assays confirmed the absence of irritation potential. These findings highlight the potential of SAH-EVs as a safe biomaterial and lay the groundwork for further investigations into their role in wound healing, reinforcing their relevance in regenerative medicine and tissue engineering.
New Frontiers in ADC Analysis: Advances in Chromatography, Electrophoresis, and Mass Spectrometry.
Beck A, D'Atri V, Verhoev O … +9 more , Alez-Martin L, Letissier L, Delobel A, Fleury-Souverain S, François YN, Cianférani S, Gahoual R, Guillarme D, Hernandez-Alba O
AAPS J · 2025 Dec · PMID 41372718 · Publisher ↗
Antibody-drug conjugates (ADCs) are composed of a tumor-targeting mAb conjugated to a cytotoxic payload to enable the selective delivery of the cytotoxic moiety while reducing the side effects and immunogenicity to the p...
Antibody-drug conjugates (ADCs) are composed of a tumor-targeting mAb conjugated to a cytotoxic payload to enable the selective delivery of the cytotoxic moiety while reducing the side effects and immunogenicity to the patient. Since the first ADC approval by the FDA, the design of new generation of ADC products has been extensively developed to improve the therapeutic efficiency of first-generation ADCs. Concomitantly, different analytical methods have been improved to enable critical quality attributes (CQA) assessment and thus support ADCs development and production at different stages. In this context, liquid chromatography (LC), capillary electrophoresis (CE), and mass spectrometry (MS) have played a predominant role in ADCs characterization, showcasing the advantages of these methods to identify and potentially quantify the different ADCs populations resulting from the bioconjugation process. This review provides a detailed overview about cutting-edge analytical methods with a particular focus on studies reported during the last five years related to LC, CE, and MS, allowing not only a deeper insight into ADCs structure, but also to provide further evidence about their in vitro and in vivo biotransformation products. The maturity, robustness, and high throughput associated to these methods allow their progressive introduction in regulatory and clinical environments, ensuring the structural integrity of ADCs prior to their administration to the patients. The examples reported in this review article clearly highlight the relevance of using tailored analytical strategies for a more comprehensive ADC characterization and thus continue the contribution to early developability assessment and bring next-generation ADCs into the market.
A Novel Oral Disintegrating Tablet Containing Aprepitant Nanocrystal Developed by Freeze-Drying Technology: An Effective Strategy to Enhance Aprepitant Bioavailability.
Li Y, Yi M, Ma K … +5 more , Gou J, Yin T, Zhang Y, Tang X, He H
AAPS J · 2025 Dec · PMID 41372558 · Publisher ↗
This study developed a novel aprepitant (APT) nanocrystal-containing oral disintegrating tablet (ODT) to enhance the oral bioavailability of APT through "rapid disintegration". APT nanocrystal suspension (APT-NS) was pre...
This study developed a novel aprepitant (APT) nanocrystal-containing oral disintegrating tablet (ODT) to enhance the oral bioavailability of APT through "rapid disintegration". APT nanocrystal suspension (APT-NS) was prepared via miniaturized media milling, with a particle size of 199.25 ± 12.88 nm, a polydispersity index (PDI) of 0.148 ± 0.026 and zeta potential of -24.82 ± 1.28 mV. The APT-NS was spray-dried into dry powders (APT-NCS) and further processed into ODTs via freeze-drying. Through central composite design-response surface methodology (CCD-RSM) optimization, the final APT-ODT formulation demonstrated rapid disintegration (< 5 s) and excellent dissolution (> 95% within 2 min). Results of X-ray diffraction (XRD), differential scanning calorimetry (DSC), and Fourier transform infrared spectroscopy (FTIR) confirmed the absence of crystalline transformation or chemical degradation during processing. Pharmacokinetic results demonstrated that both APT-NS and APT-ODTs exhibited approximately twofold higher AUC (28.51 ± 1.62 μg·h/mL and 32.61 ± 2.19 μg·h/mL, respectively) compared to free APT. In conclusion, the optimized APT-ODT successfully improved the oral bioavailability of APT, representing a promising new strategy for clinical application.
Meta-Analysis of the Placebo Response in Chronic and Episodic Migraine: Insights from Migraine Preventive Drug Trials.
Nookala AR, Panse N, Gottipati G … +4 more , Fitter HD, Uppoor R, Mehta M, Sabarinath S
AAPS J · 2025 Dec · PMID 41366588 · Full text
Randomized, well-controlled trials are the gold-standard for evaluating novel therapies, but they often fail to fully account for the placebo response, particularly in pain-related conditions such as migraine, where subj...
Randomized, well-controlled trials are the gold-standard for evaluating novel therapies, but they often fail to fully account for the placebo response, particularly in pain-related conditions such as migraine, where subjective improvement is common in placebo groups. This placebo response is influenced by individual-level factors, such as prior treatment experience, expectations of treatment benefit, and demographic characteristics such as age, race, sex, and clinical trial location. As clinical trials grow increasingly global and diverse, ensuring balanced demographic distribution across treatment arms is essential to accurately assess efficacy. We conducted a meta-analysis of placebo arm data from 14 pivotal Phase 2 and 3 trials that supported approval of six drugs for the preventive treatment of episodic and chronic migraine. Data were stratified by migraine type and analyzed by demographic variables including age, sex, race, menopause status, trial region, prior prophylactic medication use, route of administration, and frequency. Changes from baseline were evaluated for monthly migraine days (primary endpoint) and migraine-related symptoms (e.g., headache days, headache days of moderate/severe intensity, photophobia/phonophobia days, and nausea/vomiting days). Placebo responses were consistently higher in subjects who had not used prior preventive medications, African Americans, and participants enrolled in North America. These findings suggest that placebo response in migraine trials may be modulated by demographic and treatment-related factors, especially when subjective endpoints are used. Accounting for these variables during trial design and subject allocation could help minimize bias, reduce the risk of false negative outcomes, and enhance the likelihood of accurately demonstrating treatment efficacy.
Simultaneous Target-Mediated Drug Disposition-Pharmacodynamic (TMDD-PD) Modeling of Finasteride and Dutasteride: Impact of Target Binding and Turnover on Non-linear Pharmacokinetics.
Iwaki Y, Lee W, Aoki Y … +1 more , Sugiyama Y
AAPS J · 2025 Dec · PMID 41366580 · Publisher ↗
Target-mediated drug disposition (TMDD) refers to non-linear pharmacokinetic (PK) profiles arising from the saturable interaction between a drug and its pharmacological target. Recently, our group revisited the TMDD case...
Target-mediated drug disposition (TMDD) refers to non-linear pharmacokinetic (PK) profiles arising from the saturable interaction between a drug and its pharmacological target. Recently, our group revisited the TMDD cases observed in small-molecule drugs interacting with high-specificity targets, obtaining quantitative insights into in vivo target binding. In this study, we developed a physiologically-based PK (PBPK) model incorporating TMDD and pharmacodynamic (PD) responses (TMDD-PD) for finasteride and dutasteride, two time-dependent inhibitors of 5α-reductase (5αR). In addition to the tissue- and subtype-dependent 5αR inhibition, the model incorporated irreversible inactivation of 5αR and its turnover to account for the mechanism of time-dependent inhibition. We simultaneously analyzed the non-linear PK and PD (dihydrotestosterone level decline and recovery) data for both finasteride and dutasteride. Our model effectively captured the observed PK/PD profiles of both drugs, and the model-derived 5αR inhibition parameters were comparable to those obtained from in vitro 5αR inhibition data. Sensitivity analysis revealed that saturation of target binding is the primary driver of the non-linear PK and corresponding PD profiles, while slow turnover of 5αR contributes to the prolonged PD effect. Our results further suggest that the distinct PD profiles of finasteride and dutasteride are attributable to their differing inhibition characteristics against 5αR subtypes (selectivity and affinity). These findings extend our previous work and further support the utility of TMDD-PD modeling for optimizing clinical dose and improving therapeutic outcomes for small-molecule drugs exhibiting TMDD with time-dependent target inhibition.
Drug Dissolution Enhancement Using 3D-Printed Silica-Based Oral Films.
Blaháčková D, Elbl J, Lammerding LC … +4 more , Mašková E, Muselík J, Kašlík J, Gajdziok J
AAPS J · 2025 Dec · PMID 41361598 · Publisher ↗
Orodispersible films (ODFs) are increasingly employed for individualized drug delivery due to their ease of administration and precise dosing. However, their drug loading capacity is often limited by the need to maintain...
Orodispersible films (ODFs) are increasingly employed for individualized drug delivery due to their ease of administration and precise dosing. However, their drug loading capacity is often limited by the need to maintain thin, flexible structures, posing a particular challenge for incorporating poorly soluble drugs. This study aimed to develop and characterize porous ODF matrices optimized for 3D printing of medicated inks. The primary objective was to investigate the impact of macroporosity on the dissolution kinetics of both poorly soluble and readily soluble drugs, with a focus on enhancing the release of the poorly soluble dexamethasone. Porous ODFs were fabricated via solvent casting using silica- and silicate-based porogens, then loaded with caffeine or dexamethasone through 3D printing. The films were comprehensively characterized using structural (micro-CT, BET), mechanical, and solid-state techniques (SEM, Raman microscopy, FTIR, XRD) to assess porosity, drug crystallization behavior, and drug-matrix compatibility. Drug release was evaluated through dissolution studies. Silica-based porogens yielded films with tunable macroporosity, supporting high drug loads (up to 3-5 times the ink volume). Dexamethasone printed on the SY2 substrate exhibited markedly enhanced dissolution (79.2 ± 1.8%) compared to its powdered form (29.9 ± 11.5%), achieving 61.5% release within 20 min. In contrast, caffeine (readily soluble) showed a transient reduction in dissolution rate during the initial two minutes, attributed to increased particle size and delayed film disintegration. Overall, integrating porous matrix design with 3D printing significantly improved the dissolution of poorly soluble dexamethasone without inducing drug-matrix interactions, confirming that structural modifications drive the enhanced release.
Reference Standard Calibration Challenges in the Case of Erythropoietin: Impact on Potency and Biosimilarity Determination.
TerWee J, Wilson-Landy K, Wang Y … +1 more , Hellman N
AAPS J · 2025 Dec · PMID 41350961 · Publisher ↗
International reference standards have been established as gold standards for several biological products. Use of international units can drive consistency and standardization for biosimilar products across manufacturers...
International reference standards have been established as gold standards for several biological products. Use of international units can drive consistency and standardization for biosimilar products across manufacturers. However, our research and development for Retacrit® (epoetin alfa-epbx), the first and only biosimilar FDA approved for all indications of Epogen®/Procrit® (epoetin alfa), showed that use of a compendial reference standard overestimated the potency of both the biosimilar and the originator product. Possible causes are or imprecision in the methods used to assign potency, or differences in the isoform composition between the compendial reference standard and product. Tracing the history of international reference standard potency assignment for erythropoietin provided insight into this issue. An internal product specific reference standard and process for qualifying and maintaining future replacements are recommended. Use of specific activity as compared to percent potency or units/mL was a useful tool and can provide a means to normalize data from multiple methods and samples with differences in labelled activity from various manufacturers.
Utilizing Physiologically Based Pharmacokinetic Modeling and Virtual Simulation for Simvastatin Tablets to Evaluate the Sensitivity of Using Parent vs Metabolite as Analyte on Bioequivalence Assessment.
Cheng YH, Wu F, Yoon M … +2 more , Zhao L, Fang L
AAPS J · 2025 Dec · PMID 41350955 · Publisher ↗
Typically, parent drug is measured for bioequivalence (BE) assessment because it's more sensitive to detect formulation differences, compared to its metabolite(s). For simvastatin immediate release (IR) tablets, current...
Typically, parent drug is measured for bioequivalence (BE) assessment because it's more sensitive to detect formulation differences, compared to its metabolite(s). For simvastatin immediate release (IR) tablets, current product-specific guidance (PSG) recommends measuring both parent and metabolite but taking metabolite as supportive data. This study aims to utilize physiologically based pharmacokinetic (PBPK) modeling and virtual BE (VBE) simulation to evaluate the sensitivity of parent vs metabolite as analyte on BE assessment, using simvastatin case and explore relevant mechanism. PBPK model was developed to describe drug exposures of parent drug simvastatin (SV) and metabolite simvastatin acid (SVA) in healthy individuals administered with 20-80 mg IR tablets under fasting condition. VBE simulations were conducted to evaluate the sensitivity of SV and SVA as analytes to assess BE between test product and reference listed drug. PBPK model incorporating enzyme- and transporter-mediated kinetics reasonably captures fasting PK profiles for SV and SVA. VBE simulations indicate that parent drug, in general, is more sensitive to demonstrate BE as compared to metabolite. However, this study highlighted the importance of conducting BE analysis using PK data for both SV and SVA when the test product contains certain excipients in the formulation that may impact transporter activity for changing clearance and subsequent drug exposure of metabolite. The VBE simulation results further implied that in some cases, SVA as analyte is more sensitive to show drug exposure differences and may enhance the assessment of formulation effect, as compared to SV. This aligns with current PSG recommendations.
A Design of Experiments and Risk Management-Driven Analytical Platform for Charge Variant Analysis of Therapeutic Antibodies by Imaged Capillary Isoelectric Focusing.
Ghizzani V, Orlandini S, Ascione A … +7 more , Pasquini B, Tengattini S, Temporini C, Gotti R, Massolini G, Furlanetto S, Luciani F
AAPS J · 2025 Dec · PMID 41350510 · Publisher ↗
The evaluation of monoclonal antibodies (mAbs) charge variants during their entire life cycle is crucial, as their profiles represent a critical quality attribute of biotherapeutics. While the current scenario still show...
The evaluation of monoclonal antibodies (mAbs) charge variants during their entire life cycle is crucial, as their profiles represent a critical quality attribute of biotherapeutics. While the current scenario still shows marked analytical non-uniformity in the evaluation of charge variants with imaged capillary isoelectric focusing (icIEF) with many "product-specific" methods, regulatory authorities are increasingly encouraging the utilization of horizontal standards, such as Platform Analytical Procedures (PAPs). A practical Analytical Quality by Design (AQbD) workflow is provided, emphasizing the Design of Experiments as a Quality Risk Management tool to develop a PAP based on icIEF, able to accurately measure charge variants pI values. Infliximab was chosen as the leading molecule. The cause-effect matrix, combined with an asymmetric screening design, identified key parameters exerting a critical impact on the Analytical Procedure Attributes. PAP quality measurements were ensured by a 10% risk acceptance level, employing response surface methodology and Monte Carlo simulation. The developed PAP was validated using three independent System Suitability samples and exhibited a low bias in the pI measurement (less than 2%), while maintaining satisfactory separation performance. Good intra-day and inter-day repeatability, combined with a robustness test and an exploratory application to real samples of three different therapeutic mAbs, confirmed its versatility. The study supports regulatory trends by demonstrating the successful application of AQbD in PAP development. This icIEF platform would ensure a systematically consistent analysis of charge variants, where pI is promoted to an objective tool, to be used as an additional reliable parameter in the Quality Control context.
Improvements in Data Quality Can Boost Efficiency and Reduce Development Costs: A Pharmacometric CRO's Perspective.
de la Peña A, Fiedler-Kelly J, Humphrey RL … +1 more , Barrett JS
AAPS J · 2025 Dec · PMID 41350485 · Publisher ↗
Drug development can take up to 15 years, costing as much as $11 billion USD, and relies heavily on high-quality data. The goal of this investigation of contract research organizations (CROs) was to assess the impact of...
Drug development can take up to 15 years, costing as much as $11 billion USD, and relies heavily on high-quality data. The goal of this investigation of contract research organizations (CROs) was to assess the impact of data management activities (such as curation, quality assessment and integration) on model-informed drug development (MIDD) deliverables. A survey was sent to a diverse sample of CROs, to evaluate their baseline experience with assessing the data quality of sponsor-provided data and the time required to create analysis-ready datasets. It was distributed to 44 colleagues from 32 companies offering pharmacometrics services, including data management. The survey included 11 questions; 9 were multiple choice and 2 open-ended. Responses were gathered anonymously to ensure confidentiality and intellectual property protection and later shared with all participants. Of the 17 survey respondents, most develop data specifications and create analysis-ready datasets. The majority (65%) said the data they received from sponsors was rarely (< 10%) immediately usable due to improper formatting and quality issues like missing data and inconsistencies. Over 50% cited lack of definition/specifications as the primary reason. Assuming an average programming cost of $250/hour, cleaning client data takes CROs 3 to 24 h, costing between $750 and $6000 per dataset. Significant time is spent on rectifying poor-quality data. Automated data quality assessments can improve efficiency checks, though automation alone cannot resolve all quality issues. Better communication, collaboration, and systematic approaches to address data quality issues involving automation and AI are essential to further improve data quality.
Metronidazole Pharmacokinetics Across Species: Meta-Analysis Integrating Allometric Scaling and Minimal Physiologically-Based Pharmacokinetic Modeling.
Kir F, Jusko WJ
AAPS J · 2025 Dec · PMID 41345821 · Publisher ↗
Metronidazole (MTZ) is frequently used in both human and veterinary medicine to treat infections caused by certain protozoa and anaerobic bacteria. This study investigates the pharmacokinetic (PK) profiles of MTZ for ava...
Metronidazole (MTZ) is frequently used in both human and veterinary medicine to treat infections caused by certain protozoa and anaerobic bacteria. This study investigates the pharmacokinetic (PK) profiles of MTZ for available species in the literature by utilizing a linear, allometric, minimal physiologically-based PK (mPBPK) model. High quality PK data for intravenous (IV, n = 13) and oral (PO, n = 10) single doses were collected. Reported clearances (CL) and volumes of distribution (V) were highly correlated (R = 0.957, 0.969) with body weights (BW) with allometric power coefficients of 0.97 and 0.87. A mPBPK model with one perfusion-limited tissue compartment was used to evaluate MTZ PK using anatomical and physiological parameters for each species. The mPBPK model adequately captured the IV PK profiles when using species-specific CL values and a generalized tissue:plasma partition coefficient (K = 0.792 (CV 2.76%)) except for sheep and goats that had very low K values. The IV and PO profiles were best fitted jointly with shared physiological parameters and species-specific clearances, K values (range 0.55 to 1.44, mean 0.86), and bioavailability (F 0.32 - 1.00, mean 0.73). Overall, successfully integrating allometric scaling into a mPBPK model for diverse species revealed very consistent disposition of MTZ with generally BW-proportional CL values, reasonably conserved K values, and a moderate range of absorption rates and high bioavailability.
The Impact of Cognate Antigen Binding on the FcRn-mediated Transcytosis and Recycling of Monoclonal Antibodies.
Vijayanand S, Lagasse HAD, Tegenge MA … +2 more , Sauna ZE, Golding B
AAPS J · 2025 Dec · PMID 41339598 · Publisher ↗
Immunoglobulin G (IgG) antibodies rely on neonatal Fc receptor (FcRn)-mediated recycling and transcytosis for prolonged serum half-life and tissue distribution. However, high antigen loads during infection may alter FcRn...
Immunoglobulin G (IgG) antibodies rely on neonatal Fc receptor (FcRn)-mediated recycling and transcytosis for prolonged serum half-life and tissue distribution. However, high antigen loads during infection may alter FcRn-mediated trafficking, impacting therapeutic efficacy. This study investigates how cognate antigen binding influences FcRn-mediated transport of two SARS-CoV-2-specific (SCoV-2) monoclonal antibodies: Sotrovimab, an Fc-engineered antibody with enhanced FcRn affinity, and B38, a non-engineered comparator. We evaluated antibody binding using ELISA and bio-layer interferometry (BLI) and assessed FcRn-mediated transport through transcytosis and recycling assays in MDCK cells expressing human FcRn. Experiments were conducted with and without SCoV-2 wild-type (WT) spike protein (SP) at a 1:1 molar ratio. Sotrovimab demonstrated superior binding affinity to both SCoV-2 WT SP and FcRn, exhibiting greater baseline transcytosis and recycling efficiency. However, antigen presence significantly reduced transcytosis for both antibodies, with Sotrovimab showing a more pronounced decrease (46.7% vs. 23% for B38). Recycling responses also diverged: Sotrovimab showed a modest, non-significant decrease while recycling of B38 significantly increased. Kinetic analysis revealed antigen binding altered FcRn interactions differently. The higher binding affinity of Sotrovimab was due to reduced dissociation at neutral pH, potentially trapping complexes intracellularly. B38 showed faster association at pH 6.0 without compromised dissociation. These data suggest that cognate antigen binding and interaction of immune complexes (ICs) with FcRn, play a major role in influencing transcytosis and recycling of mAb. These findings emphasize the complex interplay between antigen binding and FcRn function, with implications for antibody dosing strategies during infection to optimize tissue distribution and efficacy.
Preclinical Development of GT-14, a Novel Inhibitor of Gα2 Protein: Comprehensive Evaluation of Physicochemical, Metabolic Characteristics and Tissue Distribution.
Sarkar M, Du T, Chen Y … +7 more , Lawrence YVM, Ma J, Khan SA, Oyelere AK, Liang D, Gao S, Xie H
AAPS J · 2025 Dec · PMID 41324835 · Full text
GT-14, identified as [(E)-4-((1-(1-methyl-1H-indol-2-yl) ethylidene)amino)phenol], is a novel inhibitor targeting the Gα2 protein, which is crucial in facilitating cell migration and invasion in prostate, ovarian, and br...
GT-14, identified as [(E)-4-((1-(1-methyl-1H-indol-2-yl) ethylidene)amino)phenol], is a novel inhibitor targeting the Gα2 protein, which is crucial in facilitating cell migration and invasion in prostate, ovarian, and breast cancer cells. therefore a valuable target for treating metastatic castration-resistant prostate cancer (mCRPC). In this study, GT-14's physicochemical properties, permeability, metabolic behavior, and tissue distribution were assessed. The results showed that GT-14 exhibited very slight aqueous solubility at room temperature (0.11 mg/mL) but was soluble in solvents including dimethyl sulfoxide and dimethyl acetamide, and sparingly or slightly soluble in several cosolvents. GT-14 exhibited a distinct pH-dependent solubility profile, being stable across a broad pH range (1.2-7.4) but degrading in strongly basic conditions. It exhibited high permeability (1.3 x 10 cm/s) in Caco-2 cell culture models and therefore identified as a BCS II compound. Hepatic microsomal studies revealed that GT-14 underwent Phase I metabolism, with more than 90% remaining in 60 min incubation in rat liver microsomes. A stable co-solvent formulation was developed to enable intravenous administration for pharmacokinetic studies. Previous pharmacokinetic studies showed that GT-14 exhibited biphasic disposition with a terminal plasma elimination half-life of 268.07 minutes (> 4 hours). Tissue distribution analysis indicated the highest concentration of GT-14 in the prostate, followed by the kidneys, lungs, heart, and liver. Our study presents an early-stage preclinical drug development roadmap that integrates modern technologies for efficiency and success, using GT-14 as a model compound. It showed promising characteristics, reinforcing its potential as a new therapeutic agent for mCRPC.
A Machine Learning-Empowered Quantitative Structure-Activity Relationship Model for Predicting the Plasma Half-life of Drugs in Dogs.
Wu X, Wu PY, Chou WC … +2 more , Tell LA, Lin Z
AAPS J · 2025 Nov · PMID 41254440 · Publisher ↗
Understanding a drug's plasma half-life is essential in guiding dosage regimens and optimizing therapeutic outcomes, particularly in the early stages of drug development. By using published pharmacokinetic data from Food...
Understanding a drug's plasma half-life is essential in guiding dosage regimens and optimizing therapeutic outcomes, particularly in the early stages of drug development. By using published pharmacokinetic data from Food Animal Residue Avoidance Databank, we collected 560 data points of plasma half-lives for different drugs in dogs following intravenous administration. The dataset was then preprocessed and the mean elimination half-life for each drug was selected in the final clean dataset for model training and testing. Five types of chemical descriptors and four types of supervised machine learning (ML) algorithms were employed to build ML-empowered Quantitative Structure-Activity Relationship (QSAR) models. Model performances were assessed by determination coefficient (R) and root mean square error values. The results showed that the Deep Neural Networks model with all-combined descriptor type had the best performance with R = 0.80 for the fivefold cross-validation set and R = 0.57 for the testing set. Furthermore, the applicability domains of the well-trained models are shown via Williams plots. This study reports an ML-based QSAR tool in predicting the elimination half-lives of drugs in dogs based on only chemical structures. This approach can be used to support drug development in dogs and provides a basis for potential interspecies extrapolation.
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