AAPS Journal最新文献

Modified-release (MR) drug products are designed to provide controlled drug delivery over time, offering therapeutic and compliance advantages. However, ensuring consistent in vivo performance requires a thorough understanding of the relation between in vitro dissolution behavior and in vivo drug absorption. In vitro-in vivo correlation (IVIVC) serves as a critical tool in this context, enabling formulation optimization, supporting regulatory decision-making, and streamlining product development. This study aimed to use tofacitinib as a model compound to evaluate three deconvolution methodologies within GPX™ - numerical, compartmental, and mechanistic. Prototype formulations with varying release rates were assessed in a randomized crossover study in healthy volunteers. In vivo fraction absorbed profiles were derived and convoluted to simulate plasma concentration-time profiles, which were then compared to observed clinical data. Prediction errors for key pharmacokinetic parameters (i.e., plasma C_max and AUC) were determined, and 90% confidence intervals for both parameters were calculated to assess bioequivalence between the simulated (convoluted) and observed plasma profiles. The results demonstrate the utility of deconvolution-based IVIVC models for MR product development in a physiologically-based pharmacokinetic (PBPK) framework and offer a strategy for assessing dissolution variability in support of regulatory flexibility and robust formulation lifecycle management.

The number of clinical investigations and approved applications of adeno-associated virus (AAV) based transgene product (TP) delivery has grown steadily. There also has been a growing interest in understanding how anti-AAV and anti-TP immune responses affect the safety and efficacy of these gene therapy treatments. While considerations related to anti-AAV immunity have been discussed in other works, this manuscript focuses on the assessment of anti-TP immune responses, including both humoral and cellular responses. The development of anti-TP antibodies or a cytotoxic cellular response may lead to increased clearance of the TP, elimination of AAV-transduced cells, and consequently, affect the overall durability and efficacy of the treatment. Additionally, the binding and neutralization of residual endogenous protein by anti-TP antibodies might further worsen the clinical condition under treatment. Several topics are explored in this manuscript, including immunogenicity risk factors that can be considered when evaluating the overall risk and impact of anti-TP immunogenicity, potential implications of anti-TP immunogenicity, the importance of assessing anti-TP immunogenicity, and the commonly used analytical methodologies. The manuscript proposes an approach to determining the scope of anti-TP immunogenicity assessment for clinical and non-clinical studies, based on the TP nature, other intrinsic and extrinsic risk factors. Authored by a group of scientists involved in AAV-based therapeutic development from various industry organizations, the manuscript aims to provide recommendations and guidance to industry sponsors, academic laboratories, and regulatory agencies working on AAV-based modalities, with the goal of achieving a more consistent approach to the assessment of anti-TP immune response.

This study aimed to investigate the impact of age on pediatric PK of mAbs and develop a platform PBPK model to support optimal dosing of mAbs in pediatric patients. After extensive literature review 49 mAbs were identified as approved for pediatric use, but only 17 had adequate PK data to support the investigation. It was found that pediatric patients exhibit 20-40% lower initial concentration (i.e. C₀) compared to adults following the same body weight normalized dose. For mAbs demonstrating linear PK, a similar rate of bodyweight normalized clearance was observed between adult and pediatric patients aged 2 years and above, while a faster clearance per kg bodyweight (up to 45% higher) was seen for infants and neonates. The majority of mAbs that demonstrate nonlinear PK were found to have faster bodyweight normalized clearance (up to 350% higher) in pediatric patients. A platform PBPK model was developed to characterize the PK of mAbs in pediatric patients across all age groups. The model was able to adequately (%PE < 35) characterize plasma PK of 11 mAbs with linear PK in pediatric patients aged 0.13 to 17 years following intravenous or subcutaneous administration. The developed model was able to apriori predict antibody PK reasonably well (%PE < 35). The PBPK model was integrated into an interactive web-based R Shiny application ( http://40.67.147.7/ ). The app allows individuals with minimal pharmacometrics expertise to simulate the PK of mAbs in pediatric patients and personalize the dosing of mAbs in patients with sparse PK data from therapeutic drug monitoring.

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.

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%.

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.

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.

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.

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.