AAPS Journal最新文献

The ADA testing strategy for protein therapeutics was established almost two decades ago when assay methodologies were rudimentary, and serious immunogenicity-related safety issues had recently been observed with some biotherapeutics. The current testing paradigm employs multiple tiers and stringent cut points to minimize false negatives, reflecting a conservative stance towards ADA analysis. The development of highly sensitive ADA assay platforms and technologies such as humanized or fully human monoclonal antibody (mAb) drugs has put the traditional, resource-intensive 3-tiered testing approach under scrutiny. ADA data from clinical studies for three different mAb programs were re-assessed to explore the feasibility of a simplified 1-tiered ADA testing strategy with a 1% false positive cut point versus the traditional 3-tiered approach. The analysis demonstrated moderate to strong correlations between screening results (signal-to-noise, S/N) and those of confirmation and titer results, with the vast majority of samples (~ 97%) across all studies having the same ADA positive/negative classification with either testing approach. Furthermore, at the subject level, over 92% had the same ADA category (pre-existing, treatment-emergent, treatment-boosted) under both testing approaches. The re-categorized subjects had low titer ADA responses with no observed clinical implications on pharmacokinetics, efficacy, or safety. Finally, the treatment-emergent ADA incidences were comparable between the 1-tiered and 3-tiered approaches. The results demonstrate that the 1-tiered testing strategy is suitable for ADA assessment in these programs and is likely more widely applicable. Additionally, the 1-tiered approach could expedite data delivery and reduce resource needs in clinical development without compromising data quality or clinical interpretation.

Accurate measurement of plasma protein binding (PPB) is of critical importance in drug discovery. Methodologies for PPB measurement continue to evolve to address the challenges of highly bound compounds. In order to generate high quality PPB data, it is crucial to not only apply state-of-the-art methods and highly sensitive and selective detectors, but also use high-quality plasma. In this study, we found that plasticizers, leaching from polyvinyl chloride (PVC) plasma storage bags, interfered with drug binding to both human α1-acid glycoprotein (AAG) and human serum albumin (HSA). Several AAG and HSA binding drugs were used to probe the differences in PPB using blood/plasma collected and stored in PVC bags or glass tubes through vacutainers. The results showed that plasma collected using vacutainers into the glass tubes has lower plasma fraction unbound (f_u,p) values than those from the PVC bags. The f_u,p differences can be as high as 32-fold. Hence, it is recommended to use vacutainers and glass tubes rather than PVC bags, for blood collection and plasma storage. Plasma from animal species collected using polypropylene syringes into polyethylene tubes showed no differences in f_u,p from plasma collected using vacutainers into glass tubes. Not all compounds are sensitive to plasticizer interference for PPB. It is therefore important to select appropriate positive controls for f_u,p measurement, such as warfarin for HSA and imatinib for AAG, to monitor the quality of plasma and minimize the interference from plasticizers.

Amorphous solid dispersions (ASDs) represent a promising strategy for enhancing the solubility of poorly soluble drugs. However, the mechanisms underlying the physical stability of ASDs remain insufficiently understood. This study aims to investigate these mechanisms and propose quantitative thresholds to predict the maximum stable drug loading using molecular dynamics simulations. Poly(vinylpyrrolidone) (PVP) and poly (vinylpyrrolidone-co-vinyl acetate) (PVPVA64) are selected as polymeric carriers, while naproxen and acetaminophen serve as model drugs, resulting in the formulation of 18 distinct ASDs across four types for comparison with experimental results. Our findings indicate that the molecular mobility of active pharmaceutical ingredients (APIs) is the primary determinant of solid dispersion stability. High polymer concentrations limit drug molecular mobility through spatial structural constraints and ASD viscosity. As drug loading increases, the polymer concentration reaches a critical threshold (C*), beyond which drug-rich regions form, leading to potential aggregation, rearrangement, and recrystallization of drug molecules into more energetically stable forms. Notably, both the interaction energy and diffusion coefficient show sharp fluctuations at the maximum stable drug loading, which can serve as predictive indicators for ASD stability. Additionally, a search strategy is used to identify potential pre-crystalline sites. By integrating kinetic, thermodynamic, and pre-crystalline analyses through molecular dynamics simulations, this study provides a foundation for more accurate predictions of ASD stability, significantly aiding future formulation development.

Recognizing the approach of a dramatic expansion of peptide therapeutics reaching the marketplace in recent years, led by GLP-1 receptor agonists such as semaglutide and liraglutide, the Center for Drug Evaluation and Research (CDER) branch of the US Food and Drug Administration (FDA) issued a final guidance in 2021 that was intended to assist generic drug producers in meeting Abbreviated New Drug Application (ANDA) obligations to establish "sameness" of their active peptide drug relative to that produced by innovator companies. Research and a published report by FDA scientists on best practices followed, which promulgated the use of nuclear magnetic resonance (NMR) and principal component analysis (PCA) and established a quantitative standard by which "sameness" of higher order structure for the applicant's peptide drug could be judged. A key requirement is that drug product samples be analyzed directly and non-invasively, a condition which in practice restricts sample modification to the addition of a small amount of deuterium oxide to allow signal lock and spectral data alignment (as required for NMR analysis). In the study described herein, data are presented to illustrate that 1) relatively small differences in sample pH can cause significant shifting of certain proton resonances, 2) that such resonance shifting is readily reversible and due to the degree of protonation of specific amino acid residues (rather than reflecting differences in higher order structure), and 3) that small differences in pH variability between sample cohorts can frequently cause failure to meet the quantitative benchmark established by the agency. Methodology is presented by which drug sample pHs can be aligned with minimal impact, and a recommendation is made that minor sample pH adjustments be allowed in assessing "sameness" of peptide drug higher order structure.

The development of advanced preclinical models is crucial for the evaluation and validation of novel therapeutic strategies in oncology. Three-dimensional (3D) microtumor models, which incorporate both cancer and stromal cells within biomimetic hydrogels, have emerged as powerful tools that more accurately replicate the complex tumor microenvironment compared to traditional two-dimensional (2D) cell culture systems. In this context, our study aims to develop 3D microtumor models by integrating cancer and stromal cells within an extracellular-matrix-mimetic hydrogel, as a physiologically accurate microtumor model that can serve as an innovative platform for advanced cancer research and drug screening. Microtumors composed of varying ratios of leukemia cells (HL-60) to healthy ovarian stromal cells (SCs) (1:1, 1:10, 1:100, or 1:1000) were encapsulated in PEGylated fibrin hydrogel and cultured for 5 days. The proliferation and dynamics of cancerous and healthy cell populations were evaluated using CD43/Ki67 immunofluorescence double staining. Our findings indicate that tumor development and malignancy progression can be influenced by adjusting cell culture ratios and incubation time. Notably, the HL-60:SCs ratio of 1:100 closely replicated leukemia cell invasion in ovarian tissue, demonstrating detectable malignancy on the third and fifth days without significant changes in total cell density dynamics. This 3D leukemia microtumor model offers superior physiological relevance compared to traditional 2D in vitro assays and shows promising potential for applications in cellular analysis and drug screening.

The use of tacrolimus (FK506) as an immunosuppressant is limited by its low aqueous solubility and bioavailability. The self-microemulsifying drug delivery system (SMEDDS) has successfully improved the solubility of FK506 in our previous study. This study focused on the solidification of liquid SMEDDS to capture the benefits of both liquid SMEDDS and solid dosage forms. Among several porous silica adsorbents evaluated, Aeroperl® 300 Pharma showed the best performance in terms of droplet size, in vitro dissolution, adsorbent-drug compatibility, and tabletabilities. And precoating the adsorbent with polyvinylpyrrolidone K30 resulted in complete drug release. Hydroxypropyl methylcellulose based matrix tablet was developed to achieve a sustained release of FK506. Differential scanning calorimetry and X-ray powder diffraction indicated that FK506 was present in a molecular or amorphous state in the solidified SMEDDS and tablets. In vivo pharmacokinetic studies showed that the self-prepared tablet had improved bioavailability (179.02%) compared to the marketed product Advagraf®. This study provided a promising candidate with improved dissolution and bioavailability for FK506 and a prospective platform for SMEDDS development.

Biotherapeutics are subject to inherent heterogeneity due to the complex biomanufacturing processes. Numerous analytical techniques have been employed to identify, characterize, and monitor critical quality attributes (CQAs) to ensure product safety, and efficacy. Mass spectrometry (MS)-based multi-attribute method (MAM) has become increasingly popular in biopharmaceutical industry due to its potential to replace multiple traditional analytical methods. However, the correlation between MAM and conventional methods remains to be fully understood. Additionally, the complex analytical workflow and limited throughput of MAM restricts its implementation as a quality control (QC) release assay. Herein, we present a simple, robust, and rapid MAM workflow for monitoring CQAs. Our rapid approach allowed us to create a database from ~700 samples, including site-specific post-translational modifications (PTMs) quantitation results using MAM and data from traditional charge variant and oxidation characterization methods. To gain insights from this database, we employ multivariate data analysis (MVDA) to thoroughly exploit the data. By applying partial least squares regression (PLSR) models, we demonstrate the ability to quantitatively predict charge variants in ion exchange chromatography (IEX) assay and oxidation abundances in hydrophobic-interaction chromatography (HIC) assay using MAM data, highlighting the interconnectivity between MAM and traditional product quality assays. These findings help evaluate the suitability of MAM as a replacement for conventional methods for release, and more importantly, contribute to enhanced process and product understanding.

Long non-coding RNAs (lncRNAs) play essential roles as oncogenic factors in cancer progression by influencing cell proliferation, apoptosis, and metastasis pathways. This study aims to investigate the expression changes of LINC01615 in prevalent cancers, explore its correlation with patient mortality rates, and introduce a novel therapeutic approach to reduce LINC01615 expression. Using The Cancer Genome Atlas (TCGA) data, the expression changes of LINC01615 in various cancers were analyzed, and its relationship with patient survival rates through Cox regression analysis weas assessed. Co-expressed pathways related to LINC01615 were identified via network analysis. Potential drugs to decrease LINC01615 expression were identified using the GSE38376 study. Besides, chitosan-coated nanoparticles were fabricated and functionalized with the identified drug, Lapatinib, to examine their effect on lung cancer cell lines and changes in LINC01615 expression. Our results indicated elevated LINC01615 expression in various common cancers, particularly in lung cancer, which was associated with poor prognosis in lung, breast, and kidney cancers. Co-expression network analysis suggested links to metastasis-related genes. Lapatinib, identified through GEO data, was found to modulate LINC01615 expression effectively. Chitosan-gold nanoparticles conjugated with Lapatinib significantly reduced LINC01615 expression in lung cancer cell lines while enhancing apoptosis rates. Therefore, these nanoparticles could be considered a promising therapeutic candidate for treating cancers with overexpression of LINC01615.

A stepwise, data-driven approach for Anti-Drug Antibodies (ADA) singlicate assays has been developed to evaluate the feasibility of singlicate ligand binding assay (LBA) method development, qualification and validation to support our clinical programs. With initial precision runs in method validation and subsequent statistical analysis to directly compare duplicate and singlicate formats, our results indicated no meaningful difference in assay precision between duplicate and singlicate. Consequently, the rest of the ADA method validation proceeded with singlicate analysis yielding acceptable validation results. The validated ADA assay in singlicate has been employed to support a Phase I study. The appropriateness of singlicate analyses is further supported by Signal-to-Noise (S/N) data from the three tiers of the confirmatory positive samples, which showed strong correlation in S/N values.

The article focuses on preparing a nanoformulation based on hydrotalcite and glycyrrhizic acid (GA), seeking a hepatoprotective effect. For this purpose, hydrotalcite-GA formulations were prepared by varying the following conditions to obtain optimal systems in terms of size and PDI (the lowest values), and Z potential (the highest values): (i) type of hydrotalcite (obtained by co-precipitation or calcined hydrotalcite); method used (ultrasound or high shear stirring), and (iii) type of stabilizer (Tween®80 or Pluronic® F-127). The best results were obtained using hydrotalcite obtained by co-precipitation, with high shear stirring and adding a stabilizer, either Tween®80 (HT-T80-GA: mean particle size = 315 nm, PDI = 0.18, Z potential = -20.93) or Pluronic® F-127 (HT-PF127-GA: mean particle size = 307 nm; PDI = 0.27, Z potential = -21.03). After stability studies, the HT-T80-GA formulation was chosen to study antioxidant activity, cytotoxicity, and intracellular penetration capacity. Although the hepatoprotective effect of GA in solution allowed a high viability and antioxidant activity, the fact of including GA in the HT-T80-GA formulation favored its penetration into hepatocytes, with a decrease in Caspase-3/7 expression of C-9 hepatocyte cells treated with H₂O₂, suggesting the capacity to inhibit apoptosis.