The AAPS Journal最新文献

To evaluate the artificial intelligence (AI)-guided AlphaFold algorithm for studying the binding interactions of human huntingtin and the aggregation of huntingtin peptides. Variants of huntingtin protein implicated in Huntington’s disease were used as a model system to evaluate AlphaFold. Variants of huntingtin and huntingtin peptides with polyglutamine tracts (PQT) containing 21, 31, 51, or 78 glutamines were studied. The 3-dimensional structures of huntingtin variants and their interactions with huntingtin-associated protein-40 (HAP40) were obtained. Aggregation experiments were conducted with peptide sequences corresponding to variants of PQT, amino terminal sequence (NTS) plus PQT, NTS plus PQT plus proline rich region (PRR), and the 300 amino acid sequence from the NTS through HEAT3 of huntingtin. Oligomerization experiments with 1, 3, 6, or 12 peptide sequences were used to assess the quaternary structures of aggregates. The PQT and PQT plus NTS peptides formed a helical secondary structure that formed a central core in the quaternary structure of the aggregates The PRR formed an extended type II polyproline helix that did not participate in central core the aggregates. The distance between the amino and carboxyl termini of disease-linked 31Q, 51Q, and 78Q variants of full-length huntingtin was prominently decreased compared to the 21Q huntingtin. The interaction of HAP40 with the 78Q variant increased the distance between the amino and carboxyl termini. AlphaFold identified key tertiary structure changes in human huntingtin that have been independently corroborated in experimental models. The results highlight the utility of AlphaFold for hypothesis generation in pharmaceutical research.

Pre-existing anti-AAV antibodies can be detected using ligand binding-based assay formats. One such format is the MSD-based bridging assay, which uses sulfo-tag-labeled AAV vectors as detection reagents. However, no method has been developed to accurately measure the degree of sulfo-tag labeling on AAV vectors. To fill this gap, we developed a liquid chromatography-high resolution mass spectrometry (LC-HRMS) method to assess the degree of labeling (DoL) of sulfo-tag on AAV5 vectors, enabling the measurement of the DoL on AAV5 at six increasing levels of sulfo-tag challenge ratio. In addition, a Biacore-based assay was used to evaluate the binding affinity between an anti-AAV5 monoclonal antibody and various sulfo-tag labeled AAV5 vectors. The results indicated that increased DoL of sulfo-tag labeling on AAV5 did not compromise the binding affinity.

Our study further employed the MSD-bridging assay to detect the binding Signal/Noise (S/N) ratios of four anti-AAV5 monoclonal antibodies (mAbs) to various sulfo-tag-labeled AAV5 vectors. The findings revealed a strong correlation between the degree of sulfo-tag labeling and both the S/N ratios and the sensitivity of MSD bridging assays. This result underscores the importance of optimizing the labeling of detection reagents to enhance assay sensitivity for detecting anti-AAV5 antibodies.

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A recent FDA draft guidance discusses statistical considerations for demonstrating comparability of cell and gene therapy products and processes. One experimental study described in the guidance is the split-apheresis design. The FDA draft guidance recommends a paired data analysis for such a design. This paper demonstrates that the paired analysis is under powered for some quality attributes for practical sample sizes of three to five donors unless a significant portion of variability is attributed to donor. Addition of historic lots from the pre-change process can increase the power for these attributes. This paper provides appropriate statistical methods for including this information.

The International Council for Harmonization of Technical Requirements for Pharmaceuticals for Human Use (ICH) adopted Guideline M10 entitled "Bioanalytical Method Validation and Study Sample Analysis" in May 2022. In October 2023, approximately one year after the adoption of the ICH M10 guideline, a "Hot Topic" session was held during the AAPS PharmSci 360 meeting to discuss the implementation of the guideline. The session focused on items the bioanalytical community felt were challenging to implement or ambiguous within the guideline. These topics included cross-validation, parallelism, comparative bioavailability studies, combination drug stability, endogenous analyte bioanalysis, and dilution QCs. In addition, the regulatory perspective on the guideline was presented. This report provides a summary of the Hot Topic session.

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Oral administration of peptide represents a promising delivery route, however, it is hindered by the harsh gastrointestinal environment, leading to low in vivo absorption. In this study, auto-adaptive protein corona-AT 1002-cationic liposomes (Pc-AT-CLs) are constructed with the characteristic of hydrophilic and electrically neutral surface properties for the encapsulation of liraglutide. BSA protein corona is used to coat AT-CLs reducing the adherence of mucus, and may fall off after penetrating the mucus layer. Transmucus transport experiment demonstrated that the mucus penetration amount of Pc-AT-CLs are 1.45 times that of AT-CLs. After penetrating the mucus layer, AT-CLs complete transmembrane transport by the dual action of AT and cationic surface properties. Transmembrane transport experiment demonstrated that the apparent permeability coefficient (P_app) of AT-CLs is 2.03 times that of CLs. In vivo tests demonstrated that Pc-AT-CLs exhibited a significant hypoglycemic effect and enhanced the relative bioavailability comparing to free liraglutide. Pc-AT-CLs protect liraglutide from degradation, facilitate its absorption, and ultimately improve its oral bioavailability.

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In this study, AT-CLs (AT-1002 peptide attached to cationic liposomes) and coated with BSA (bovine serum albumin) together form Pc-AT-CLs. The purpose of simultaneously improved mucus permeating ability and transepithelial absorption. When the carrier penetrates the mucus, the BSA protein corona coating may fall off. Then, AT-CLs carriers possessing cationic and hydrophobic surfaces, which will facilitate uptake of intracellular and paracellular.

Fig: The schematic diagram of Pc-AT-CLs administration and the transport process through the mucus layer and epithelial barrier.

The aim of this study was to develop a model to predict individual subject disease trajectories including parameter uncertainty and accounting for missing data in rare neurological diseases, showcased by the ultra-rare disease Autosomal-Recessive Spastic Ataxia Charlevoix Saguenay (ARSACS). We modelled the change in SARA (Scale for Assessment and Rating of Ataxia) score versus Time Since Onset of symptoms using non-linear mixed effect models for a population of 173 patients with ARSACS included in the prospective real-world multicenter Autosomal Recessive Cerebellar Ataxia (ARCA) registry. We used the Multivariate Imputation Chained Equation (MICE) algorithm to impute missing covariates, and a covariate selection procedure with a pooled p-value to account for the multiply imputed data sets. We then investigated the impact of covariates and population parameter uncertainty on the prediction of the individual trajectories up to 5 years after their last visit. A four-parameter logistic function was selected. Men were estimated to have a 25% lower SARA score at disease onset and a moderately higher maximum SARA score, and time to progression (T50) was estimated to be 35% lower in patients with age of onset over 15 years. The population disease progression rate started slowly at 0.1 points per year peaking to a maximum of 0.8 points per year (at 36.8 years since onset of symptoms). The prediction intervals for SARA scores 5 years after the last visit were large (median 7.4 points, Q1-Q3: 6.4–8.5); their size was mostly driven by individual parameter uncertainty and individual disease progression rate at that time.

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Advair Diskus is an essential treatment for asthma and chronic obstructive pulmonary disease. It is a dry powder inhaler with a combination of fluticasone propionate (FP) and salmeterol xinafoate (SX). However, the pharmacokinetics (PK) batch-to-batch variability of the reference-listed drug (RLD) hindered its generic product development. This work developed the PK models for inhaled FP and SX that could represent potential batch variability. Two batches each of the reference and the test product (R₁, R₂, T₁, T₂) of Advair Diskus (100 μg FP/50 μg SX inhalation) were administered to 60 healthy subjects in a 4-period, 4-sequence crossover study. The failure of the bioequivalence (BE) between R₁ and R₂ confirmed the high between-batch variability of the RLD. Non-linear mixed effect modeling was used to estimate the population mean PK parameters for each batch. For FP, a 2-compartment model with a sequential dual zero-order absorption best described the PK profile. For SX, a 2-compartment model with a first-order absorption model best fit the data. Both models were able to capture the plasma concentration, the maximum concentration, and the total exposure (AUC_inf) adequately for each batch, which could be used to simulate the BE study in the future. In vitro properties were also measured for each batch, and the batch with a higher fraction of the fine particle (diameter < 1 µm, < 2 µm) had a higher AUC_inf. This positive correlation for both FP and SX could potentially assist the batch selection for the PK BE study.

Immunogenicity evaluation is a critical part of drug development. Regulatory guidelines from multiple health agencies provide recommendations for the development and validation of anti-drug antibody (ADA) assays to assess immunogenicity in clinical trials. These recommendations primarily describe an ADA method run in one bioanalytical laboratory supporting a biotherapeutic molecule; however, there are increasing instances that may necessitate the support of the ADA method being run in more than one laboratory. A program can rapidly expand into multiple clinical studies within one or multiple countries, where the most appropriate way to support the program is by having multiple laboratories perform ADA sample analysis. In addition, there may be certain country-specific challenges that may make it infeasible to transport samples outside of the country for analysis. China for example has a lengthy sample exportation process that has potential to negatively impact study timelines. If multiple laboratories analyze samples using the same ADA method, comparable method performance should be established. Here, we describe a three-way assessment of ADA assay comparability between two US-based bioanalytical laboratories and one based in China.

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Comparative bioavailability studies often involve multiple groups of subjects for a variety of reasons, such as clinical capacity limitations. This raises questions about the validity of pooling data from these groups in the statistical analysis and whether a group-by-treatment interaction should be evaluated. We investigated the presence or absence of group-by-treatment interactions through both simulation techniques and a meta-study of well-controlled trials. Our findings reveal that the test falsely detects an interaction when no true group-by-treatment interaction exists. Conversely, when a true group-by-treatment interaction does exist, it often goes undetected. In our meta-study, the detected group-by-treatment interactions were observed at approximately the level of the test and, thus, can be considered false positives. Testing for a group-by-treatment interaction is both misleading and uninformative. It often falsely identifies an interaction when none exists and fails to detect a real one. This occurs because the test is performed between subjects in crossover designs, and studies are powered to compare treatments within subjects. This work demonstrates a lack of utility for including a group-by-treatment interaction in the model when assessing single-site comparative bioavailability studies, and the clinical trial study structure is divided into groups.

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