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For studying proliferation and determination of survival of cancer cells after irradiation, the multiple MTT assay, based on the reduction of a yellow water soluble tetrazolium salt to a purple water insoluble formazan dye by living cells was modified from a single-point towards a proliferation assay. This assay can be performed with a large number of samples in short time using multi-well-plates, assays can be performed semi-automatically with a microplate reader. Survival, the calculated parameter in this assay, is determined mathematically. Exponential growth in both control and irradiated groups was proven as the underlying basis of the applicability of the multiple MTT assay. The equivalence to a clonogenic survival assay with its disadvantages such as time consumption was proven in two setups including plating of cells before and after irradiation. Three cell lines (A 549, LN 229 and F 98) were included in the experiment to study its principal and general applicability.

We present a generically applicable approach to determine an extensive set of size-dependent critical quality attributes inside nanoparticulate pharmaceutical products. By coupling asymmetrical-flow field-flow fractionation (AF4) measurements directly in-line with solution small angle X-ray scattering (SAXS), vital information such as (i) quantitative, absolute size distribution profiles, (ii) drug loading, (iii) size-dependent internal structures, and (iv) quantitative information on free drug is obtained. Here the validity of the method was demonstrated by characterizing complex mRNA-based lipid nanoparticle products. The approach is particularly applicable to particles in the size range of 100 nm and below, which is highly relevant for pharmaceutical products—both biologics and nanoparticles. The method can be applied as well in other fields, including structural biology and environmental sciences.

Transcutaneous immunization (TCI) via needle-free and non-invasive drug delivery systems is a promising approach for overcoming the current limitations of conventional parenteral vaccination methods. The targeted access to professional antigen-presenting cell (APC) populations within the skin, such as Langerhans cells (LCs), various dermal dendritic cells (dDCs), macrophages, and others makes the skin an ideal vaccination site to specifically shape immune responses as required. The stratum corneum (SC) of the skin is the main penetration barrier that needs to be overcome by the vaccine components in a coordinated way to achieve optimal access to dermal APC populations that induce priming of T-cell or B-cell responses for protective immunity. While there are numerous approaches to penetrating the SC, such as electroporation, sono- or iontophoresis, barrier and ablative methods, jet and powder injectors, and microneedle-mediated transport, we will focus this review on the recent progress made in particle-based systems for TCI. This particular approach delivers vaccine antigens together with adjuvants to perifollicular APCs by diffusion and deposition in hair follicles. Different delivery systems including nanoparticles and lipid-based systems, for example, solid nano-emulsions, and their impact on immune cells and generation of a memory effect are discussed. Moreover, challenges for TCI are addressed, including timely and targeted delivery of antigens and adjuvants to APCs within the skin as well as a deeper understanding of the ill-defined mechanisms leading to the induction of effective memory responses.

Biological small‐angle X‐ray scattering (SAXS) is a versatile and powerful technique for investigating the structural and biophysical properties of biologically and pharmaceutically relevant macromolecules and nanoparticles. SAXS offers detailed insights into macromolecular composition, size, shape and internal structure, while addressing key aspects such as oligomeric state, stability, molecular interactions, and conformational flexibility. Recently, asymmetrical‐flow field‐flow fractionation (AF4) was successfully coupled to SAXS, enabling online size‐based fractionation and analysis of polydisperse samples. This approach allows precise, size‐dependent characterization, offering significant advancements in the study of polydisperse systems. We have integrated an AF4 device at the P12 beamline at the European Molecular Biology Laboratory and implemented technical adaptations allowing full automation to make the system suitable for routine user access. We provide streamlined workflows and troubleshooting resources for both novice and advanced SAXS users thereby equipping them with clear guidance on performing AF4–SAXS measurements. The general principles of our set‐up are easily adaptable to other beamlines which have integrated (or are planning to integrate) a similar system. By coupling asymmetrical‐flow field‐flow fractionation to small‐angle X‐ray scattering (AF4–SAXS), we enable precise, size‐resolved analysis of polydisperse samples. Our automated AF4–SAXS system at the EMBL P12 beamline streamlines workflows, making advanced characterization accessible to both novice and experienced users, with principles adaptable to other facilities.

The present article exemplifies the application of the concept of quality by design (QbD) for the systematic development of a nanoparticulate imiquimod (IMQ) emulsion gel formulation as an investigational medicinal product (IMP) for evaluation in an academic phase-I/II clinical trial for the treatment of actinic keratosis (AK) against the comparator Aldara (EudraCT: 2015-002203-28). The design of the QbD elements of a quality target product profile (QTPP) enables the identification of the critical quality attributes (CQAs) of the drug product as the content of IMQ, the particle-size distribution, the pH, the rheological properties, the permeation rate and the chemical, physical and microbiological stability. Critical material attributes (CMAs) and critical process parameters (CPPs) are identified by using a risk-based approach in an Ishikawa diagram and in a risk-estimation matrix. In this study, the identified CPPs of the wet media ball-milling process’s milling time and milling speed are evaluated in a central composite design of experiments (DoEs) approach, revealing criticality for both factors for the resulting mean particle size, while only the milling time is significantly affecting the polydispersity. To achieve a mean particle size in the range of 300–400 nm with a minimal PdI, the optimal process conditions are found to be 650 rpm for 135 min. Validating the model reveals a good correlation between the predicted and observed values. Adequate control strategies were implemented for intermediate products as in-process controls (IPCs) and quality control (QC) tests of the identified CQAs. The IPC and QC data from 13 “IMI-Gel” batches manufactured in adherence to good manufacturing practice (GMP) reveal consistent quality with minimal batch-to-batch variability.

Several locally acting colon-targeted products to treat colonic diseases have been recently developed and marketed, taking advantage of gastrointestinal physiology to target delivery. Main mechanisms involve pH-dependent, time-controlled and/or enzymatic-triggered release. With site of action located before systemic circulation and troublesome colonic sampling, there is room for the introduction of meaningful in vitro methods for development, quality control (QC) and regulatory applications of these formulations. A one-size-fits-all method seems unrealistic, as the selection of experimental conditions should resemble the physiological features exploited to trigger the release. This article reviews the state of the art for bio-predictive dissolution testing of colon-targeted products. Compendial methods overlook physiological aspects, such as buffer molarity and fluid composition. These are critical for pH-dependent products and time-controlled systems containing ionizable drugs. Moreover, meaningful methods for enzymatic-triggered products including either bacteria or enzymes are completely ignored by pharmacopeias. Bio-predictive testing may accelerate the development of successful products, although this may require complex methodologies. However, for high-throughput routine testing (e.g., QC), simplified methods can be used where balance is struck between simplicity, robustness and transferability on one side and bio-predictivity on the other. Ultimately, bio-predictive methods can occupy a special niche in terms of supplementing plasma concentration data for regulatory approval.

Lipid nanoparticles (LNPs) have gained great attention as carriers for mRNA-based therapeutics, finding applications in various indications, extending beyond their recent use in vaccines for infectious diseases. However, many aspects of LNP structure and their effects on efficacy are not well characterized. To further exploit the potential of mRNA therapeutics, better control of the relationship between LNP formulation composition with internal structure and transfection efficiency in vitro is necessary. We compared two well-established ionizable lipids, namely DODMA and MC3, in combination with two helper lipids, DOPE and DOPC, and two polymer-grafted lipids, either with polysarcosine (pSar) or polyethylene glycol (PEG). In addition to standard physicochemical characterization (size, zeta potential, RNA accessibility), small-angle X-ray scattering (SAXS) was used to analyze the structure of the LNPs. To assess biological activity, we performed transfection and cell-binding assays in human peripheral blood mononuclear cells (hPBMCs) using Thy1.1 reporter mRNA and Cy5-labeled mRNA, respectively. With the SAXS measurements, we were able to clearly reveal the effects of substituting the ionizable and helper lipid on the internal structure of the LNPs. In contrast, pSar as stealth moieties affected the LNPs in a different manner, by changing the surface morphology towards higher roughness. pSar LNPs were generally more active, where the highest transfection efficiency was achieved with the LNP formulation composition of MC3/DOPE/pSar. Our study highlights the utility of pSar for improved mRNA LNP products and the importance of pSar as a novel stealth moiety enhancing efficiency in future LNP formulation development. SAXS can provide valuable information for the rational development of such novel formulations by elucidating structural features in different LNP compositions.

ABSTRACT Purpose The effects of chitosan hydrochloride on the oral absorption of acyclovir in humans were studied to confirm the absorption enhancing effects reported for in vitro and rat studies, respectively. Methods A controlled, open-label, randomized, 3-phase study was conducted in 12 healthy human volunteers. Zovirax 200 mg dispersible tablets co-administered with doses of 400 and 1000 mg chitosan HCl were compared with Zovirax only. Results The expected increased absorption of acyclovir was not observed. On the contrary, mean area under the plasma concentration-time curve (AUC0-12 h) and maximal plasma concentration (C max ) decreased following concomitant chitosan intake (1402 versus 1017 and 982.0 ng∙h/ml and 373 versus 208 and 235 ng/ml, respectively). In addition, T max increased significantly in presence of 1000 mg of chitosan from 1 to 2 h. Conclusions The results of this study in human volunteers did not confirm an absorption enhancing effect of chitosan. Reference values were comparable to literature data, whereas addition of chitosan resulted in significant opposite effects on C max , T max and AUC. Additional studies are needed to investigate the cause of the discrepancy. The observed variability and complex potential interactions may complicate the use of chitosan HCl in oral pharmaceutical formulations.

Hydrogen, as a medical gas, is a promising emerging treatment for many diseases related to inflammation and oxidative stress. Molecular hydrogen can be generated through hydrogen ion reduction by a metal, and magnesium-containing effervescent tablets constitute an attractive formulation strategy for oral delivery. In this regard, saccharide-based excipients represent an important class of potential fillers with high water solubility and sweet taste. In this study, we investigated the effect of different saccharides on the morphological and mechanical properties and the disintegration of hydrogen-generating effervescent tablets prepared by dry granulation. Mannitol was found to be superior to other investigated saccharides and promoted far more rapid hydrogen generation combined with acceptable mechanical properties. In further product optimization involving investigation of lubricant effects, adipic acid was selected for the optimized tablet, due to regulatory considerations.

A comparative study on different enteric-coated hard capsules was performed. The influence of different formulation factors like choice of enteric polymer, triethyl citrate (TEC) concentration (plasticizer), talc concentrations (anti-tacking agent), and different coating process parameters on the sealing performance of the capsule and the disintegration time were investigated. Furthermore, the influence of different disintegration test methods (with disc vs. without disc and 50 mM U.S. Pharmacopoeia (USP) buffer pH 6.8 vs. biopredictive 15 mM phosphate buffer pH 6.5) was evaluated. All formulations showed sufficient but not equivalent acid resistance when tested. Polymer type was the main factor influencing the capsule sealing and disintegration time. In addition, TEC and talc could affect the performance of the formulation. Regarding the choice of the disintegration test method, the presence of a disc had for the most part only limited influence on the results. The choice of disintegration buffer was found to be important in identifying differences between the formulations.