AAPS Journal最新文献

An assessment of parallelism is critical for biomarker assays to confirm whether the assay recognizes the endogenous analyte similarly to the calibrator, the suitability of a surrogate calibrator matrix and the potential need for a minimal required dilution. While the importance of parallelism has been raised in numerous publications there remains a lack of detail on how to conduct and interpret parallelism experiments, as well as some confusion between parallelism, dilution linearity, and spike recovery experiments. This best practice paper provides a detailed discussion of the reasons for conducting parallelism, as well as recommendations for when to conduct parallelism experiments, the number of samples needed, the selection of appropriate surrogate matrices, the interpretation of parallelism data, including graphical and statistical methods, and parallelism results reporting. It emphasizes the need for continuous evaluation of parallelism throughout the assay life cycle to ensure reliable measurement of the desired analyte within the context of use. Finally, a number of short case studies are provided to illustrate the application and interpretation of parallelism.

Innate immune response modulating impurities (IIRMI) with adjuvant potential have emerged as important factors in the immunogenicity risk assessment of protein, peptide, and oligonucleotide therapeutics, particularly for follow-on products where minimal or no clinical studies are available. To assess the impact of differences in impurities on specific cell types, we developed a new IIRMI assay termed multiplexed immunophenotyping for innate activation assessment (MIIAA) that employs spectral flow cytometry to capture single-cell responses to drug products and potential impurities. This technique introduces a new live fluorescent cell barcoding platform that enables sample multiplexing for homogeneous staining with a single fluorescent antibody cocktail composed of identity and activation markers that are acquired simultaneously with a five laser Cytek Aurora. Samples are digitally reassigned to their original testing conditions by positive and negative gating of barcode dyes. Cellular subsets are identified by dimensionality reduction of surface markers with UMAP then gated using cell-specific markers. Here we use trace levels of TLR3, 7/8 and 9 agonists (Poly(I:C), R848, and CpG ODN) to characterize specific responses in B cells, monocytes, cDC and pDC. Importantly, MIIAA captures single-cell responses to nucleic acid impurities in the presence of therapeutic oligonucleotides or monoclonal antibodies with high sensitivity. Taken together, MIIAA offers a powerful immunophenotyping tool to characterize single-cell responses to drug products and potential immunomodulatory impurities that may find utility in drug pipelines to characterize the impact of therapeutics on specific immune cells and to interrogate immunogenic or immunomodulatory risk in comparisons between reference and follow-on products.

Obesity significantly alters drug disposition and contributes to large inter-individual variability in pharmacokinetics (PK). The virtual-twin concept is increasingly used to support model-informed precision dosing in specific populations. In this study, physiologically-based pharmacokinetic models linked with virtual twins (VT-PBPK) have been developed and applied to predict the PK of midazolam and digoxin in patients with obesity (n = 15) and severe obesity (n = 22). The first step of the individualization included basic demographic data with lean liver volume. In the second step, individual serum creatinine, albumin, and hepatic CYP3A4/5, UGT1A4 and P-gp abundance quantified from liver biopsies in the same individuals, were integrated within models. Substrate specific improvements were presented via the stepwise individualization. The final (Step 2) VT-PBPK models predicted midazolam AUC_0-inf,iv within 2-fold for 86% of the individuals (geometric mean fold error, GMFE = 1.5; 95% confidence interval (CI95) = 1.36-1.78), with 36% within the 0.8 to 1.25-fold of the observed values. For digoxin, 97% of C_max and AUC_0-24 values were predicted within 2-fold of the observed data (GMFE = 1.25; CI95 = 1.19-1.33), with 59% of predicted values within the 0.8-1.25-fold range. In the case of digoxin, the prediction accuracy was higher for patients with severe obesity (60% of C_max and AUC_0-24 values within the 1.25-fold range); no clear trends were evident for midazolam. This is the first study that applied the VT-PBPK modelling approach in patients with obesity. It highlights the potential of this approach to predict the PK of other CYP3A and P-gp substrates to support individual dose optimization in this population.

In 2024, 3-Chloromethcathinone (3-CMC) accounted for over 63% of all New Psychoactive Substances seized in Europe, yet its human pharmacology remains poorly understood. This observational, uncontrolled, naturalistic study involved 16 regular psychostimulant users to evaluate and compare 3-CMC metabolism, and distribution in urine and oral fluid (OF) following oral and intranasal administration. Two groups, each consisting of 8 participants (6 males, 2 females) self-administered 3-CMC in two separate sessions: 100-150 mg orally and 60-80 mg intranasally. Urine was collected in two pooled intervals (0-2 h and 2-5 h). Samples were analyzed via four untargeted HPLC-HRMS/MS methods in full MS and ddMS² to characterize the unknown metabolites supported by Compound Discoverer™ software with an established workflow. The data were grouped into four groups concerning the route of administration and the time intervals and the average area were statistically compared with a one-way ANOVA. The parent drug was detected in all the samples at different levels. In total, nine metabolites were observed, of those 4 were phase I and 5 phase II metabolites. Considering the route of administration, distinct metabolic patterns emerged: three metabolites, including two N-acetylated forms and a carboxylated metabolite, were found only after oral intake, suggesting N-acetylation occurs primarily via this route. In contrast, β-OH-3-CMC accumulated more after intranasal use. Furthermore, 3-CMC N- glucuronidation was hypothesized for the first time. These findings indicate that the administration route significantly influences 3-CMC metabolism, highlighting the need for tailored forensic and toxicological assessments.

The anti-drug antibody validation testing and reporting harmonization (ADAH) white paper was published December 2021 and has generated significant interest, highlighting its utility within the scientific community. Following the ADAH white paper, members of the American Association of Pharmaceutical Scientist (AAPS) Therapeutic Product Immunogenicity community published a similar white paper addressing neutralizing antibody validation testing and reporting harmonization (NAbH) in July 2023 which has also been broadly accessed. Given the broad interest in these white papers by the bioanalytical and immunogenicity communities, the authors have solicited user feedback to address noted gaps. This feedback has prompted us to issue an addendum to address these gaps including adding a section on anti drug antibody (ADA) assay cross-validation and ADA data presentation for regulatory submissions, and to update the method sensitivity assessments. In addition, given recent year discussions around the implementation of signal to noise over titer, a section on this topic is included.

The phenomenon of nonlinear pharmacokinetics (PK) mediated by a drug's pharmacological target, also known as target-mediated drug disposition (TMDD), has been increasingly observed in small molecules in the past decade. TMDD class effect with remarkably similar nonlinear PK behaviors has been reported in 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1) inhibitors, monoamine oxidase type B (MAO-B) inhibitors, and soluble epoxide hydrolase (sEH) inhibitors. We anticipated that the occurrence of TMDD class effect might be due to their target capacities falling within a specific range, where nonlinear PK mediated by target binding are more likely to be evident. To test our hypothesis, we employed a mass spectrometry (MS)-based global proteomics approach to quantify the absolute protein concentrations of 11β-HSD1, sEH, and MAO-B in different tissues across species. The estimated total amounts of 11β-HSD1, MAO-B, and sEH in humans were approximately 4994, 4629, and 4137 nmol, respectively. The comparable abundance levels of these proteins suggest that TMDD is more likely to be observed when a drug binds to a target within a specific range, potentially between 1000 nmol and 10000 nmol, which corresponds to nonlinear PK at doses of 1-10 mg for a compound with a molecular weight of 400 g/mol. Our study highlights the importance of early target quantification and provides valuable insights into predicting unusual nonlinear PK caused by TMDD. Additionally, this proteomics-based approach for quantifying absolute target capacity could serve as a valuable tool for both industry and academic researchers in investigating other pharmacological targets.

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.