Loss of tensile strength in roller compacted granules compared to the pregranulation blend is a critical concern when selecting the roller compaction process for drug product manufacturing. While this loss in compactibility is attributed to granule size enlargement and granule hardening by earlier studies, the phenomenon of unified compaction curve (UCC) model to explain this decrease in compactibility has long been overlooked despite of its mechanistic interpretation. The present study offers a profound improvement of the UCC model structure where a proper workflow in predicting granule tabletability was first established from the compaction characteristics of the pregranulation blend only. Following this workflow, the original model though revealed varying extent of overestimation for realistic multicomponent formulations. This shortcoming was addressed by introducing the modified UCC model where different tabletability slopes for pregranulation blend and granules were critically considered that consistently performed better than the UCC model. A correction factor was finally introduced to directly predict granule tabletability of virtual formulations from the compression characteristics of the pregranulation blend. The study demonstrates an integrated digital design-based predictive platform to screen virtual formulations through a minimum 25-fold reduction in batch size compared to typical conventional operations.
Onychomycosis is difficult to treat topically because the keratinized nail plate restricts drug diffusion and solution-based products are readily removed by washing. This study evaluated a fast-drying, polymer-based film-forming nail platform designed to create a wash-resistant surface reservoir and enhance transungual delivery. Terbinafine, efinaconazole, and amorolfine were formulated in the same platform and compared with marketed products (Terclara®, Jublia®, and Loceryl®) using bovine hoof keratin membranes and distal human nail clippings in Franz diffusion cells. In this bovine hoof model, cumulative permeation at 72 h was 9.5-fold higher for terbinafine versus Terclara® and 25.5-fold higher efinaconazole versus Jublia®; although the amorolfine permeation difference was not significant, receptor solutions from all three platform formulations produced significantly larger zones of inhibition against Trichophyton rubrum and Trichophyton mentagrophytes than their comparators, indicating that permeated drug remained biologically active. In a 28-day once-daily distal human nail model, the optimized terbinafine formulation (HLK-5003) maintained cumulative permeation and antifungal activity whereas the solution comparator showed minimal delivery and no detectable inhibition; advantages for the efinaconazole and amorolfine formulations were not established. These in vitro findings indicate that rapid film formation and wash-resistant reservoir behavior can improve sustained transungual delivery in a formulation- and API-dependent manner.
Peptide therapeutics are prone to aggregation, denaturation, and interfacial adsorption, which can compromise product quality, reduce potency, increase immunogenicity, and shorten shelf life. Although surfactants are widely used to stabilize protein biologics, their use in commercial parenteral peptide formulations appears to be more selective. Specifically, the literature does not provide a clear rationale for using surfactants in this context, and their effects on peptides remain poorly understood. Here, we investigate the effects of three common surfactants, polysorbate 80 (PS80), polysorbate 20 (PS20), and poloxamer 188 (PX188), on the conformational stability, oligomerization, and aggregation of liraglutide as the primary model peptide, followed by exploratory evaluation of semaglutide and glucagon, under agitation stress. For liraglutide, which forms dynamic oligomers that are not intrinsically stable under agitation stress, SEC and CD reveal extensive aggregation and structural rearrangement in the presence of both PS80 and PX188, with PS80 driving more rapid aggregation. NMR shows strong, specific interactions between liraglutide and PS80 but not PX188, suggesting different mechanisms: PS80 perturbs the native oligomer via peptide-surfactant interactions, whereas PX188 likely promotes aggregation through interfacial stress. Consistent with this, PS20, with its shorter, less hydrophobic chain, caused less disruption than PS80, underscoring the importance of surfactant-peptide interaction strength. In contrast, semaglutide, which forms more stable oligomers, showed only limited changes in aggregation behavior in the presence of surfactants. For glucagon, which has a strong intrinsic tendency to fibrillate, surfactants modestly changed aggregation rates but did not prevent fibril formation. It is worth noting that, unlike monoclonal antibodies, which primarily adopt monomeric structures stabilized by extensive intramolecular and interdomain interactions, peptides are more flexible and therefore more susceptible to perturbation by surfactants. Overall, surfactant effects on peptide stability are highly molecule-dependent and reflect a balance among peptide-peptide and peptide-surfactant interactions, surfactant-mediated interfacial stress, and the intrinsic stability of peptide oligomers.
Determining the particle size distribution (PSD) of the active pharmaceutical ingredient (API) in nasal spray suspensions is a challenging task that usually requires the use of morphologically-directed Raman spectroscopy (MDRS). A dissolution-based modeling strategy was established to characterize the entire PSD of starting API in mometasone furoate nasal sprays using dissolution data of finished formulations, whereas MDRS measures API PSD in the final products. A dissolution-PSD model was built in-house to derive the API PSD from the dissolution data of target products, with model parameters estimated using test products with known raw API PSD and dissolution profiles. To obtain representative dissolution data that could better reflect the intrinsic API PSD characteristics of the formulation, different sample pretreatment procedures and dissolution approaches were investigated. The results suggested that the dissolution profile acquired by the paddle method following combined enzymatic hydrolysis and ultrasonication pretreatment was preferable for characterizing the starting API particle size features. Model predictions demonstrated that the commercial Nasonex® displayed a bimodal volume-weighted PSD and a relatively high Dv90 of 15.8 μm, which differed from previous reports. A modified MDRS was applied for cross-checking, and the modeled Dv90 was in agreement with the measured results. Cross-validation for the model using five test batches yielded prediction errors mostly <10%, indicating acceptable performance. For nasal suspension products that typically present complex PSD features, the present method may provide a feasible auxiliary tool for guiding formulation screening and optimizing manufacturing parameters in the preliminary development stage.
N-nitrosamine contamination originating from polymer-based packaging materials is an emerging concern, yet amine precursors in these materials or their contribution to nitrosamine formation are not well characterized. In this study, an SPE-LC-MS/MS method was developed to quantify amines in polymer raw materials used for large volume parenteral (LVP) packaging. Amines were detected in these polymer materials with a linear correlation between amine and nitrosamine levels, indicating that amine precursor content in the packaging itself may play an important role for nitrosamine formation. Since such precursors can migrate or react during LVP's shelf life, predictive stability studies were conducted on LVP stored in stability chambers. The results indicated that N-Nitrosodimethylamine levels were markedly higher in stability chamber compared to warehouse storage. These findings indicated that it might be beneficial to control nitrosamine precursors in polymer and conventional stability studies may not fully represent real-world nitrosamine risks.
The feasibility of applying a biowaiver based on Biopharmaceutics Classification System (BCS) to immediate-release oral capsules containing dabigatran etexilate was evaluated according to the ICH M9 guideline. Based on literature data and experimental results, the Dose/Solubility ratio of the drug substance does not meet the criteria for "highly soluble" at pH ≥ 4.5, it is classified as "low solubility" according to the ICH M9 guideline. Likewise, dissolution testing of the comparator product, Prazaxa®, revealed that it fails to satisfy the guideline criteria for "rapidly dissolving" formulations (≥ 85% dissolution within 30 minutes) at either pH 4.5 or 6.8. Mass balance studies indicated that it possesses "low permeability." Together, these findings classify dabigatran etexilate as BCS Class IV. The need to generate an acidic microenvironment using organic acids to achieve adequate absorption suggests a high likelihood that differences in excipient composition could affect bioequivalence (BE). From a clinical perspective, fluctuations in systemic exposure may significantly impact both efficacy and safety. Considering its physicochemical properties, formulation-dependent characteristics and inherent clinical risks, application of a BCS-based biowaiver to solid oral dosage forms of dabigatran etexilate cannot be recommended. Accordingly, demonstration of bioequivalence through in vivo BE studies remains necessary for regulatory approval.
The manufacture of a single biopharmaceutical drug product (DP) batch often requires drug substance (DS) from multiple DS batches. A DS pooling strategy provides operational flexibility, optimizes inventory utilization, and enables production of DP batches of varying sizes to meet fluctuating clinical and commercial demand. This article examines key considerations for designing and implementing an effective DS pooling strategy. These aspects are related to DS, DP, validation of DS pooling, pre- and post-pool DP stability assessment, regulatory considerations, approval and feedback, batch traceability, the timing of introducing DS pooling strategy, age of DS batches at the time of pooling, pooling multiple containers from a single DS batch vs. multiple DS batches, number of DP batches from pooled and non-pooled DS, and post-validation (or post approval) implementation of a DS pooling strategy. Only DS batches that have been successfully released (i.e., meet DS release specifications) should be used for pooling. During DP manufacture using pooled DS, it is important to ensure homogeneity of the pooled material through appropriate mixing techniques and in-process controls (IPCs) or in-process monitoring (IPM) at least until the DP manufacturing process is validated. Before implementing DS pooling for commercial manufacturing, the authors recommend validating this approach by manufacturing at least one process performance qualification (PPQ) DP batch using pooled DS. Manufacturers are expected to demonstrate that DS pooling does not adversely affect the DP stability trajectory. Although no regulatory guidelines explicitly address DS pooling or its implementation requirements, this review summarizes relevant regulatory principles and published regulatory feedback on some aspects of DS pooling for the benefit of manufacturers and readers. Overall, this article aims to support the design and implementation of a scientifically sound DS pooling strategy.
The current work aims to evaluate the efficacy of glycerol-rich vesicular matrices for enhancing the oral bioavailability and antihypertensive capacity of valsartan (VAL), a poorly soluble medication. These vesicular dispersions were generated employing the thin film hydration technique and optimized through the Box-Behnken design to investigate the effect of cholesterol, phospholipid, and glycerol concentrations on the vesicle size (VS), entrapment efficiency (EE), and drug release after 6 h (%Q6h). The optimized formulation (0.46%w/v cholesterol, 4.47% w/v phospholipid, and 10.12% w/v glycerol) exhibited nano-sized vesicles of 172.76 ± 6.47 nm with a narrow size distribution (PDI = 0.185 ± 0.101), a zeta potential of -42.4 ± 0.85 mV, an EE of 80.87 ± 1.58%, and a Q6h of 96.68 ± 1.92%. Additionally, the physical mixture of optimized components in glycerol hydration medium showed a powerful synergistic solubility enhancement, which aligns with carrier-mediated hydration, thereby facilitating the dissolution of over 71% of the target drug load during processing. Morphological characterization using SEM and TEM unveiled spherical, non-aggregated vesicular matrices. FTIR studies confirmed the absence of chemical interactions between the drug and the formulation components, while DSC and XRD analyses evidenced the loss of crystalline character of valsartan upon vesicular incorporation. Ex vivo permeation of the ultra-elastic formulation displayed a flux value (135.07 ± 7.8 µg/cm² h) twice that of the plain drug. Furthermore, the optimized colloidal dispersion remained stable for three months under refrigerated and ambient conditions. Finally, in vivo investigations in dexamethasone-induced hypertensive rabbits showed a significant reduction in mean arterial pressure and a two-fold increase in systemic bioavailability compared to the unprocessed medication. Consequently, the developed vesicular colloidal delivery system holds significant potential for improving valsartan's oral therapeutic delivery.
This study presents the development of bacterial nanocellulose (BNC) membranes derived from "Nata de fique" for local anesthesia applications. The membranes were successfully produced using fique juice as a culture medium and exhibited a characteristic nanofibrillar structure. After a purification process with KOH, they displayed a neutral pH and a high moisture content of 98.12 ± 0.54%. Following mechanical dehydration, their thickness and weight were reduced, but upon loading with articaine hydrochloride and epinephrine, the membranes recovered 87.35 ± 0.32% of their moisture. Electron microscopy confirmed that the structural integrity of the nanofibrillar network remained intact after dehydration and drug loading, allowing for controlled drug release. FTIR analysis validated the successful incorporation of anesthetics without compromising membrane integrity. Adsorption kinetics followed a pseudo-second-order model, with equilibrium reached in 50 minutes, and the membranes absorbed 4.13 ± 0.3 mg of articaine per gram of wet BNC. Desorption studies in simulated physiological fluid revealed a controlled release of up to 88% of the drug within 60 minutes. In vitro studies demonstrated limited cell adhesion, suggesting potential applications as temporary wound dressings that minimize granulation tissue formation. A 48-hour patch test on volunteers confirmed the membranes as "Non-irritating" and dermatologically safe. These findings highlight the potential of BNC derived from "Nata de fique" membranes as a non-invasive alternative for local anesthetic delivery, offering sustained drug release, biocompatibility, and safe skin application.
Human radiolabeled mass-balance studies are central to characterizing drug absorption, distribution, metabolism, and excretion (ADME), but oral-only designs do not directly provide absolute oral bioavailability (F) or systemic clearance (CL). In this study, we evaluated whether F and CL can be estimated from oral pharmacokinetic (PK) data when the fraction absorbed (Fa) and renal clearance (CLr) are known. We derived equations linking oral apparent clearance (CL/F), hepatic extraction, and bioavailability, and tested two assumptions for intestinal first-pass extraction (Fg=1 and Fg=Fh). The approach was validated using 120 approved oral small-molecule drugs with published oral human ADME and intravenous pharmacokinetic data. Predicted CL values were within 2-fold of observed values for 97-99% of drugs and predicted F values were within 2-fold for 98-99%. 64-68% of CL estimates and 74-77% of F estimates were within 80-125% of the observed values. These findings show that oral PK combined with Fa and CLr can provide useful early estimates of F and CL without an intravenous study, supporting drug development and clinical pharmacology study planning.
Activated hepatic stellate cells (HSCs) play a central role in liver fibrosis by promoting extracellular matrix deposition, oxidative stress, and pro-fibrotic signaling. Here, we engineered a membrane-fused hybrid nanoparticle by combining grape-derived exosome-like particles (GELPs) with liposomes to encapsulate JQ1, termed Hybrid@JQ1, and evaluated its anti-fibrotic effects in TGF-β1-activated LX-2 cells. GELPs were successfully isolated and characterized, while the formation of GELP-liposome hybrid nanoparticles were verified by nanoparticle characterization and colocalization analysis. Hybrid@JQ1 exhibited good physicochemical stability and enhanced cellular uptake. In vitro, Hybrid@JQ1 inhibited LX-2 cell proliferation and migration, decreased collagen accumulation, and suppressed α-SMA expression. qPCR gene expression profile further showed that Hybrid@JQ1 downregulated multiple fibrosis-related genes, including α-SMA, COL1A1, COL3A1, and TGF-β, as well as inflammation- and oxidative stress-associated genes such as NOX4, IL6, and TNF-α. These findings indicate that GELP-liposome hybridization is an effective strategy for JQ1 delivery and that Hybrid@JQ1 alleviates fibrotic phenotypes in activated HSCs in vitro. Our study provides a promising plant-derived biomimetic nanoplatform for anti-fibrotic drug delivery.
Monoclonal antibodies (mAbs) frequently encounter fluid-fluid interfaces, such as air-water and oil-water boundaries, during manufacturing, storage, and administration, where adsorp-tion can trigger structural perturbations, aggregation, and loss of efficacy. Despite extensive experimental and computational efforts, a unified understanding that connects molecular-scale mechanisms to macroscopic interfacial behavior remains incomplete. In this review, we provide a comprehensive and up-to-date synthesis of mAb adsorption at fluid-fluid interfaces, with a par-ticular emphasis on the underlying mechanisms, adsorption kinetics, impacts of surfactants and key physicochemical factors governing interfacial behavior. We critically examine experimen-tal approaches spanning macroscopic measurements-such as interfacial tension and interfacial rheology-to microstructural characterization techniques, including spectroscopy, neutron and X-ray reflectometry, that probe interfacial layer structure and organization. we also highlight recent advances in molecular simulations that provide mechanistic insight into adsorption path-ways, orientation, and structural rearrangements of mAbs at hydrophobic interfaces. We further discuss how these insights can inform rational strategies to mitigate interfacial instability and improve the design and formulation of therapeutic proteins.
Aluminum adjuvants sediment during storage and must be resuspended prior to vaccine administration, yet the sediment structures dictating resuspension remain poorly resolved. Here, we used lumogallion-stained aluminum phosphate (AP) adjuvants containing lysozyme or bovine serum albumin (BSA), with and without salt, as model formulations to connect sediment microstructure to resuspension behavior. After 28 days of quiescent settling, confocal laser scanning microscopy was used to image unperturbed sediment networks and residual adjuvant clusters before and after inversion. BSA formulations were hard to resuspend and formed dense, homogeneous sediments with disconnected aqueous pores, while lysozyme and control formulations formed interconnected aqueous networks that were easy to resuspend. Resuspension difficulty increased when aqueous channel connectivity decreased relative to AP connectivity, indicating that the solvent phase topology governs how far inversion-driven fluid stresses stream into sediments. Furthermore, difficult-to-resuspend formulations generated higher numbers of residual ridged clusters after inversion. These results identify sediment topology as an image-based descriptor of adjuvant resuspension that captures outliers from bulk measurements such as sediment packing, because poorly connected aqueous channels and persistent residual clusters are found in formulations resistant to resuspension. Preserving connected solvent pathways may therefore improve the resuspendability of aluminum-adjuvanted vaccine formulations.
Limited penetration and heterogeneous distribution of nanoparticles (NPs) within solid tumors remain major barriers to the clinical efficacy of cancer nanomedicine. Recent reports have shown the transcellular route as the main pathway for NP extravasation in solid tumors. We previously interrogated the NP transfer from one cell to another, termed intercellular exchange, which is a crucial and yet understudied step of transcellular route. Our results identified extracellular vesicles (EVs) as a critical conduit during NP intercellular exchange, and chemical regulation of EV biogenesis may boost up the activity of this process and lead to a more efficient NP delivery into solid tumors. Here, we set out to determine whether a high throughput chemical screening (HTCS) may be used to rapidly identify novel modulators of NP intercellular exchange. To do so, we adapted our spheroid-based intercellular exchange assay into a HTCS-compatible format. A proof-of-the-principal screen was carried out with the NCI Oncology Drug Library (166 FDA-approved anticancer compounds), which identified Venetoclax as a novel enhancer of NP intercellular exchange across multiple cell types and NP formulations. Venetoclax significantly increased NP transfer in both homotypic tumor-cell and heterotypic endothelial-to-tumor transport systems, leading to enhanced NP penetration in tumor spheroids and increased tumor accumulation and intratumoral distribution in vivo. Mechanistically, Venetoclax increased EV-associated protein output without significantly altering EV size distribution or particle concentration, suggesting modulation of EV cargo composition or functional EV subpopulations rather than global stimulation of EV biogenesis. Together, this study establishes a scalable screening platform for identifying pharmacological regulators of EV-mediated NP transport and demonstrates that enhancing intercellular exchange represents a promising strategy for improving nanoparticle delivery into solid tumors.
Conventional chemotherapy with doxorubicin (Dox) is limited by poor selectivity and systemic toxicity. Polymeric micelles based on Pluronic F127 offer a versatile platform for drug delivery, but their stability and responsiveness to tumor-specific stimuli remain critical challenges. To address these issues, two pH-sensitive F127-Dox conjugates were synthesized using cis-aconitic (Cis) and succinic (Suc) linkers. The conjugates were structurally characterized by FTIR and 1H NMR, and their micellization behavior was studied via pyrene fluorescence. Dynamic light scattering was employed to analyze micelle size and pH responsiveness, while UV-Vis spectrophotometry was used to quantify drug loading (DL%) and encapsulation efficiency (EE%). Drug release kinetics were evaluated using the dialysis method at pH 7.4 and 5.5. Conjugation reduced the CMC of F127 from 112 μg/mL to 89 μg/mL (Cis-Dox) and 75 μg/mL (Suc-Dox), enhancing micellar stability. F127-Cis-Dox achieved higher drug loading (5.54%) and encapsulation efficiency (61.8%) compared to F127-Suc-Dox (3.42%, 37.0%) and physically loaded F127-Dox micelles (4.94%, 60.3%). In vitro release showed pronounced pH sensitivity: F127-Cis-Dox released 80.7% of Dox at pH 5.5, compared with 25.2% at pH 7.4, whereas F127-Suc-Dox released 65.0% versus 33.9%, respectively. By contrast, F127-Dox exhibited rapid, nonspecific release (∼85% at pH 7.4, 144 h). These results highlight the importance of linker chemistry in tuning micelle stability and drug release. F127-Cis-Dox, in particular, demonstrates superior pH-responsiveness and drug-loading capacity, making it a promising candidate for tumor-targeted doxorubicin delivery.
To guide optimal dosing, knowledge about target-site drug exposure is essential and is commonly determined using the sampling technique of clinical microdialysis. The extent of target-site pharmacokinetic variability for posaconazole, a highly lipophilic azole antifungal with widely varying plasma pharmacokinetics between patients, is currently unknown. Microdialysis of posaconazole is considered challenging due to high unspecific binding and feasibility should be carefully evaluated in vitro, prior to clinical application. The objective of this study was to foster clinical in vivo applicability through in vitro evaluation of perfusate additives to suppress nonspecific binding of posaconazole. Supporting this aim, a bioanalytical method using mass spectrometry was developed for posaconazole in microdialysate with a lower limit of quantification of 10.0 ng/mL. The investigations addressing compatibility with the microdialysis system were exhausted without accomplishing feasibility of posaconazole for clinical microdialysis, as relative recovery was unfavourably low (<4%) and not congruent with relative delivery values in retrodialysis setting, which were misleadingly high (>82%). An unspecific binding assay suggested that up to 100% of the compound were retained in the tubing material, confirming the hypothesis of unspecific binding. Thus, reliable quantification of unbound posaconazole concentrations at target site was deemed infeasible with currently available catheter materials and clinically acceptable perfusate additives; advances in low-adsorption catheter materials and in-vivo-compatible perfusate formulations are required to enable clinical microdialysis of posaconazole. Utilizing the knowledge gained from exploring microdialysis with posaconazole, an efficient in vitro workflow for feasibility studies on clinical microdialysis with highly lipophilic compounds including early stop criteria was proposed.
Imeglimin (IMG) is a recently developed cyclic biguanide compound used for the management of hyperglycaemia. To comprehend the stability behavior of the polar drug IMG under various degradation environments, an HPLC method was developed, and the major degradation products (DPs) were characterized using mass-spectrometric studies. The mobile phase comprising a 20 mM potassium dihydrogen phosphate (KH2PO4) buffer (pH 3.1) along with ethanol in a simple isocratic mode (98:02, v/v) was employed for optimum resolution of the drug and its DPs using a reverse-phase Luna C18(2) (250 × 4.6 mm, 5 µm) column. Structural characterization of IMG and its DPs was performed using triple-quadrupole LC-MS, employing electrospray ionization (ESI) mode. IMG degradation occurred under acidic, alkaline, peroxide-mediated oxidation, and, to a smaller extent, nitrosation stress conditions, forming twelve DPs. Peak-purity analysis using HPLC-PDA depicted that the IMG peak remained pure under all stressed and stability conditions. Mass-spectrometric analysis enabled the identification of seven previously unreported DPs, thereby expanding the known degradation profile of IMG. The degradation pathways were elucidated based on the plausible structures of the DPs. Consequently, the probable root cause of DP4 formation in the drug product was identified through an experiment involving the enzymatic neutralization of hydrogen peroxide (H2O2). Following ICH Q2(R2) guidelines, the developed method demonstrated suitability for quality control testing of the IMG drug substance and the finished tablet product.
For any pharmaceutical, in development or at the commercial stage, it is critically important that appropriate analytical methodology accompany the product ensuring its safety and efficacy upon release. For vaccines, one of the principal analytical tests is the potency assay, which aims to be predictive of the level of protection seen upon administration. Developing an assay that can measure the potency of a complex drug product formulation can often be challenging due to interference with excipients, most notably adjuvants. Here we present a potency assay developed for a Human Papillomavirus (HPV) vaccine that saw significant interference with the introduction of the adjuvant chitosan. When more traditional means of eliminating interference failed, the enzyme chitosanase was introduced to cleave the adjuvant and rescue the ability to accurately measure potency. When contaminating proteases were observed in a commercial source of chitosanase, an internal effort to supply recombinantly expressed E. coli chitosanase was initiated. This resulted in a pure, active, and consistent supply of chitosanase. These efforts resulted in a robust analytical method that could be used to determine HPV potency in the presence of chitosan. The successful development of this potency assay demonstrates an approach for overcoming interference from adjuvants that may be able to be applied more widely to complex drug product formulations where analytical interference is observed.
