AAPS Journal最新文献

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.

Intravenous dosing of L- and D-eflornithine in a racemic mixture is a currently recommended late-stage gambiense human African trypanosomiasis (g-HAT) treatment, either as 14-day monotherapy or in combination with oral nifurtimox for seven days. However, an oral eflornithine treatment against late-stage g-HAT would be preferable. Pharmacokinetics of eflornithine are enantioselective with different oral absorption of the enantiomers. L-eflornithine has a greater in vitro antitrypanosomal potency than D-eflornithine. This study aimed to integrate knowledge about in vitro potency and literature data from the only clinical study with enantiospecific pharmacokinetic oral data to predict L-eflornithine concentrations in plasma and cerebrospinal fluid to estimate the probability of target attainment. L- and D-eflornithine concentrations in cerebrospinal fluid from the clinical study with enantiospecific data were described with a compartment model that was validated using external data with total eflornithine concentrations. Simulations were performed with oral L-eflornithine doses ranging between 125 to 1000 mg/kg/day administered twice to twelve times daily. The probability of target attainment analysis showed that oral L-eflornithine doses of 750 mg/kg/day administered four or twelve times daily (i.e., drinking a solution every 2nd hour) as monotherapy would be needed to acquire efficacious exposures. In combination with nifurtimox, L-eflornithine dosed at 375 mg/kg/day four or twelve times daily would give exposures over the threshold concentration in cerebrospinal fluid. The presented simulation framework may serve as a starting point to find a suitable oral dose regimen to assess the clinical potential for an oral L-eflornithine-based combination treatments against late-stage g-HAT.

A virtual bioequivalence (VBE) approach utilizing physiologically based pharmacokinetic (PBPK) modeling presents a compelling alternative for pharmaceutical industries. This method can significantly reduce the time and cost associated with clinical bioequivalence (BE) trials while minimizing the risk of detecting a type II error (a false negative), as well as a type I error (a false positive). Additionally, it aligns with ethical considerations by obviating the need to expose healthy volunteers to investigational drugs. To secure a biowaiver through the VBE approach, it is essential to translate compendial in vitro dissolution data into pharmacokinetic (PK) parameters using PBPK modeling to evaluate BE. This article reflects three concepts: compendial dissolution test, PBPK modeling, and BE, reviewing their historical development and current research. It highlights the gaps and challenges and discusses potential future opportunities to strengthen clear linkage among three concepts. A deeper understanding of the mechanisms and mathematical translation of dosage form disintegration and API particle dissolution can enhance the prediction of dosage form performance in both in vivo and in vitro settings. Future advancement of PBPK modeling should incorporate these developments to improve confidence in correlating compendial dissolution data with quantitative risk assessments of BE outcomes.

Accurate quantitation of circulating messenger RNA (mRNA) is critical for the quantitation of lipid nanoparticle-formulated mRNA (mRNA-LNP) drug products. This study evaluated the concordance between branched DNA (bDNA) and reverse transcription quantitative polymerase chain reaction (RT-qPCR) assays for quantifying mRNA in human serum from a phase 1 clinical trial. We compared analytical performance across bDNA and two RT-qPCR workflows-RNA purification and a simplified NP-40 detergent-based treatment. A total of 77 clinical serum samples were analyzed. Method performance was assessed using assay precision, accuracy, and total error, along with linear regression and Bland-Altman analyses to evaluate inter-platform concordance. Noncompartmental PK analysis was performed on a subset of samples from four subjects. Results showed that RT-qPCR methods yielded lower mRNA concentrations than bDNA, with a consistent negative bias more pronounced in NP-40-treated samples. The purification RT-qPCR method showed closer agreement with bDNA across the quantitative range (R² = 0.878) than NP-40 treated RT-qPCR (R² = 0.736). Despite quantitative differences, PK parameters derived from all methods were comparable, supporting RT-qPCR's suitability for clinical mRNA quantification. NP-40 treatment offered workflow efficiency and lower sample volume requirements, whereas mRNA purification had improved concordance with bDNA. These findings support the feasibility of adopting RT-qPCR as a viable alternative to bDNA method for mRNA quantification, with method selection guided by study phase, throughput needs, and available matrix volume. Cross-platform comparability ensures robust bioanalytical support for clinical development of mRNA drug candidates.