AAPS Journal最新文献

Generic ophthalmic drug product development is challenging, and innovative methodologies to complement traditional bioequivalence (BE) studies for BE assessment are desirable to promote their evaluation. Ocular physiologically based pharmacokinetic (PBPK) models can provide insight into drug partitioning in eye tissues that are too invasive to access in humans. An approach has been previously validated to translate ocular exposure from rabbits to humans for some ophthalmic solutions and suspensions. This study aims to demonstrate the utility of an ocular PBPK model to predict human exposure following the administration of ophthalmic ointment. Ofloxacin (OFL) ointment is presented as a case study. The Ocular Compartmental Absorption and Transit (OCAT™) model within GastroPlus® v9.9 was used to build a PBPK model for OFL ophthalmic solution and ointment that accounts for OFL release from the formulation into the tears and eye tissues where it is applied, ointment application time, ocular absorption, and distribution in the rabbit eye. The model was subsequently used to predict OFL exposure in humans after topical administration of solution or ointment. Drug and formulation-specific parameters were used as validated in the mode for rabbits. Physiological parameters were adjusted to match human ocular physiology. Simulated human ocular pharmacokinetic (PK) profiles were compared with the observed ocular tissue concentration data to assess the OCAT model's ability to predict human ocular exposure. The OCAT model for rabbits accurately described the observed concentrations in aqueous humor following the topical administration of OFL solution or ointment. For the ointment formulation, the Higuchi release rate and the application time of the formulation were fitted to describe the observed data. After adjustment of physiological parameters to represent the human eye, the predicted aqueous humor concentrations following ocular administration of OFL solution or ointment were within the range of observed values. More case studies for other ophthalmic ointment drug products will be needed to confirm this study's results. Nevertheless, the positive preclinical to clinical extrapolation for OFL represents an important step in the validation process of human exposure using an ocular PBPK model validated in the rabbit.

Minipig models are rapidly gaining popularity in gene therapy preclinical studies. This includes biodistribution studies that are required by regulatory agencies, but are poorly described in the literature. This is particularly true for ddPCR assays with enhanced sensitivity over traditional "real-time" PCR. We, therefore, validated a novel duplex ddPCR assay for quantifying AVGN7, an AAV gene therapeutic featuring a codon-optimized human Smad7 gene and the porcine RPP30 reference gene, in Göttingen minipig tissues. The DNA extraction methods were optimized to ensure maximum recovery of target gene. Validation parameters included accuracy and precision, limit of detection, ruggedness/robustness, selectivity, recovery, dilutional linearity and stability. The assay was sensitive to 10 cp/µg gDNA while intra- and inter-accuracy and precision were demonstrated for the dynamic range of 25,000-5 cp/µL where the inter-assay total error was ≤ 35.5% for AVGN7 and ≤ 4.3% for RPP30. Moreover, ruggedness was assessed over time using multiple analysts, instruments and reagent lots, demonstrating high reliability and reproducibility. Linearity was established across 1,000-fold dilutions and stability after 6 freeze-thaw cycles and 24 h at room temperature. Selectivity was evaluated in unspiked and post-spiked samples using multiple lots of different tissue matrices. High recovery of AVGN7 during nucleic acid extraction was also demonstrated for all tissues. These data not only validate the assay for tracking and quantifying AVGN7 in preclinical studies with Göttingen minipigs, but additionally address challenges of using pig models in general while providing a procedural road map for developing other AAV biodistribution assays.

This study focuses on the development of lyophilized nasal powders of ropinirole hydrochloride (RH), composed of Poloxamer 407 triblock copolymer (P407), surfactant Tween 80 (Tw80) and derivatives of βCD [methyl-β-CD (MβCD) or hydroxy-propyl-β-CD (HPβCD)] for managing Parkinson's disease. The lyophilized compositions were produced by freeze-drying of the corresponding colloidal dispersions. The innovative aspect of this research lies in the integration of these diverse biomaterials to develop advanced drug delivery systems for nasal administration. The optimal lyophilized formulations were blended with spray-dried microparticles of mannitol and lecithin (MLMPs) in various ratios. The blending process resulted in powders with improved morphological characteristics, as evidenced by Scanning Electron Microscopy analysis. Nasal powders were characterized through in vitro diffusion and ex vivo permeation studies. Finally, a comparison between the nasal powders and the corresponding colloidal dispersions was held. Results showed that the lyophilized powders had a superior release profile compared to colloidal dispersions, and blending with MPMPs further enhanced this effect. Ex vivo powders' permeation across rabbit nasal mucosa was found to be more efficient compared to pure RH solution. In particular, the lyophilized formulation of (P407/Tw80/HPβCD)/RH 10:5 blended with MLMPs (25:75) achieved 54.40 ± 3.30% permeation of the loading dose and a mass balance of 98.15 ± 1.61%. This study demonstrates the potential of these formulations for effective nose-to-brain drug delivery. Ongoing in vivo pharmacokinetic studies are being conducted to assess the performance of the optimal formulations in an appropriate animal model.

Human α-1-acid glycoprotein (hAGP) immobilized directly on NHS ester (N-hydroxysuccinimide) activated magnetic beads (hAGP-beads) was developed as a novel tool for studying small molecule hAGP binding. This method offers a straightforward, one-step immobilization process compared to traditional biotin-streptavidin immobilization technique. The hAGP-beads system provides a rapid and convenient alternative to conventional methods like equilibrium dialysis (ED) or ultracentrifugation (UF) for assessing hAGP small molecule interactions. Characterization and evaluation of the hAGP-beads system revealed that equilibrium dissociation constant (K_d) values  of various small molecules obtained by hAGP-beads method correlated well with those determined by ED. This result suggests that the conformation of hAGP binding site is not altered after hAGP covalently binds to magnetic beads through NHS ester  conjugation. Key advantages of the hAGP-beads method include shorter assay incubation times compared to ED (~ 3 min versus 4-6 h) and the ability to quantify both free and hAGP-bound small molecule species, facilitated by simple magnetic separation. Furthermore, long-term storage tests demonstrated that hAGP-beads remain stable and retain its binding functionality at -80°C, significantly reducing the need for frequent preparations. These properties make the hAGP-beads system not only well-suited for general hAGP-related studies, such as small molecule binding, but especially advantageous for high-throughput screening applications.

Lipid nanoparticle-messenger RNA (LNP-mRNA) drug products are a growing class of drug modalities. The unique composition of these drug products requires multiple measurements to account for the different components of these drug modalities. Pharmacokinetic (PK) measurements include measurement of the encapsulated mRNA and components of the LNP in circulation to understand the effectiveness of the therapeutic mRNA. The PK measurements can utilize many different platforms including PCR. Current regulatory guidance documents for bioanalytical method validation are specific to ligand binding and chromatographic assay methods and difficult to interpret for use with molecular workflows. The purpose of this paper is to provide information on considerations for validation of regulated reverse transcription quantitative PCR (RT-qPCR) assays that are used to support the pharmacokinetic analysis of LNP-mRNA drug products.

Increasing the concentration of intravenous (IV) biologic formulations to render them appropriate for subcutaneous (SC) delivery is challenging because it impacts many interrelated variables, including volume, viscosity, and stability. This study gathered drug formulation expert insights regarding these challenges as well as development approach preferences and perceptions concerning formulation volume. Biotechnology and pharmaceutical industry experts familiar with creating high-concentration (≥ 100 mg/mL) biologic drug formulations for SC delivery completed an online survey between 26 April and 7 May 2024. In total, there were 100 respondents included. When asked to rank seven approaches to transitioning a formulation from IV to SC administration, responses showed that increasing drug concentrations to reduce injection volume and/or changing the primary container were considered riskier, more time-consuming, and more costly than maintaining the concentration and using an on-body delivery system (OBDS). The greatest challenges mentioned were solubility issues (75%), viscosity-related challenges (72%), and aggregation issues (68%). Most respondents (69%) reported delays in clinical trials or product launches due to high-concentration SC formulation challenges. Of these, 33.3% experienced delays of 6-9 months (weighted mean: 11.3 months), while 4.3% indicated that trials or launches were canceled entirely due to formulation difficulties. In conclusion, making minimal drug formulation concentration changes to an IV biologic formulation may reduce the risk, time commitment, and cost associated with developing a SC biologic formulation. Further education is needed around the transition of traditional IV formulations to low-concentration, large-volume SC formulations utilizing delivery formats such as an SC infusion pump or OBDS.

As the field of gene therapy advances and as the importance of sex as a biological variable in shaping viral immune responses is recognized, the impact of sex on adeno-associated virus (AAV) vectors mediated gene therapies remain largely unexplored. Here we review current understanding of the immune response against AAV gene therapy as well as the knowledge of sex differences observed in viral responses. We discuss sex differences in innate immune mechanisms such as Toll-like receptor recognition and complement activation, as well as the functional responses of key immune cells such as dendritic cells, macrophages, and T/B cells that are involved in AAV immunogenicity. Variations in pre-existing immunity, including differences in antibody levels and neutralizing activity among sexes, are also described. Additionally, we investigate evidence in the literature of sex differences in AAV transduction in animal and suggest a potential link between the immune responses and higher transductions in males.

Antibody-drug conjugates (ADCs) represent a rapidly expanding class of therapeutics, uniquely combining the specificity of monoclonal antibodies with the potency of cytotoxic small-molecule payloads. Due to their inherent structural complexity and heterogeneous composition, accurate characterization and quantification of ADCs pose significant bioanalytical challenges. This review discusses recent advancements in bioanalytical methodologies, including ligand binding assays (LBAs), liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based approaches, and emerging hybrid LBA-LC-MS/MS platforms. In addition, this review will discuss pharmacokinetic (PK) modeling approaches essential to ADC development, ranging from population PK models to mechanistic frameworks, including physiologically based pharmacokinetic (PBPK) and quantitative systems pharmacology (QSP) models. These modeling strategies allow detailed characterization of ADC absorption, distribution, metabolism, and elimination processes while also accounting for complexities introduced by payload deconjugation and drug-to-antibody ratio variability. By integrating robust bioanalytical methods with advanced modeling techniques, this review provides researchers with essential insights to enhance ADC characterization, inform experimental design, and ultimately facilitate the development of safer, more effective therapeutic candidates.

CAR-T-cells can drive MHC class-I-mediated CD8 + cytotoxic T-cell response towards CAR constructs in addition to an antibody response. Immune response may also develop towards residuals present in the CAR-T cell product such as AAV, CRISPR/CAS9, and expamers. Health authorities recommend developing assays to assess both humoral and cellular immunogenicity towards the CAR-T protein. For the assessment of a humoral response, scientists can leverage the guidance and experience from anti-drug antibody (ADA) assays being developed for biologics. However, measuring CAR-T induced cellular immune responses may be challenging due to factors like cell survival, assay variability, lack of relevant positive controls, reagents, etc. This commentary overviews the strategy for investigating cellular immunogenicity for CAR-T products in development, describing the process for risk assessment, guidance on sample collection, including logistics of cell processing and handling, and design of CAR domain related peptides to elicit the memory response from dosed subjects. The experience gained from cellular immunogenicity assessments implemented for ongoing CAR-T-cell therapies and challenges encountered are presented with concrete recommendations, without disclosure of proprietary data. The clinical relevance/impact of assessing cellular immunogenicity for CAR-T therapies and any association with humoral response will also be delineated.