A Mass Spectrometry Strategy for Protein Quantification Based on the Differential Alkylation of Cysteines Using Iodoacetamide and Acrylamide.

Mass spectrometry has become the most prominent yet evolving technology in quantitative proteomics. Today, a number of label-free and label-based approaches are available for the relative and absolute quantification of proteins and peptides. However, the label-based methods rely solely on the employment of stable isotopes, which are expensive and often limited in availability. Here we propose a label-based quantification strategy, where the mass difference is identified by the differential alkylation of cysteines using iodoacetamide and acrylamide. The alkylation reactions were performed under identical experimental conditions; therefore, the method can be easily integrated into standard proteomic workflows. Using high-resolution mass spectrometry, the feasibility of this approach was assessed with a set of tryptic peptides of human serum albumin. Several critical questions, such as the efficiency of labeling and the effect of the differential alkylation on the peptide retention and fragmentation, were addressed. The concentration of the quality control samples calculated against the calibration curves were within the ±20% acceptance range. It was also demonstrated that heavy labeled peptides exhibit a similar extraction recovery and matrix effect to light ones. Consequently, the approach presented here may be a viable and cost-effective alternative of stable isotope labeling strategies for the quantification of cysteine-containing proteins.

Mathematical modeling of transdermal delivery of topical drug formulations in a dynamic microfluidic diffusion chamber in health and disease.

Mathematical models of epidermal and dermal transport are essential for optimization and development of products for percutaneous delivery both for local and systemic indication and for evaluation of dermal exposure to chemicals for assessing their toxicity. These models often help directly by providing information on the rate of drug penetration through the skin and thus on the dermal or systemic concentration of drugs which is the base of their pharmacological effect. The simulations are also helpful in analyzing experimental data, reducing the number of experiments and translating the in vitro investigations to an in-vivo setting. In this study skin penetration of topically administered caffeine cream was investigated in a skin-on-a-chip microfluidic diffusion chamber at room temperature and at 32°C. Also the transdermal penetration of caffeine in healthy and diseased conditions was compared in mouse skins from intact, psoriatic and allergic animals. In the last experimental setup dexamethasone, indomethacin, piroxicam and diclofenac were examined as a cream formulation for absorption across the dermal barrier. All the measured data were used for making mathematical simulation in a three-compartmental model. The calculated and measured results showed a good match, which findings indicate that our mathematical model might be applied for prediction of drug delivery through the skin under different circumstances and for various drugs in the novel, miniaturized diffusion chamber.

Microparticles and multi-unit systems for advanced drug delivery.

Microparticles have unique benefits in the formulation of multiparticulate and multi-unit type pharmaceutical dosage forms allowing improved drug safety and efficacy with favorable pharmacokinetics and patient centricity. On the other hand, the above advantages are served by high and well reproducible quality attributes of the medicinal product where even flexible design and controlled processability offer success as well as possible longer product life-cycle for the manufacturers. Moreover, the specific demands of patients can be taken into account, including simplified dosing regimens, flexible dosage, drug combinations, palatability, and ease of swallowing. In the more than 70 years since the first modified-release formulation appeared on the market, many new formulations have been marketed and many publications have appeared in the literature. More unique and newer pharmaceutical technologies and excipients have become available for producing tailor-made particles with micrometer dimensions and beyond. All these have contributed to the fact that the sub-units (e.g. minitablets, pellets, microspheres) that make up a multiparticulate system can vary widely in composition and properties. Some units have mucoadhesive properties and others can float to contribute to a suitable release profile that can be designed for the multiparticulate formula as a whole. Nowadays, there are some available formulations on the market, which are able to release the active substance even for several months (3 or 6 months depending on the type of treatment). In this review, the latest developments in technologies that have been used for a long time are presented, as well as innovative solutions such as the applicability of 3D printing to produce subunits of multiparticulate systems. Furthermore, the diversity of multiparticulate systems, different routes of administration are also presented, touching the ones which are capable of carrying the active substance as well as the relevant, commercially available multiparticle-based medical devices. The versatility in size from 1 µm and multiplicity of formulation technologies promise a solid foundation for the future applications of dosage form design and development.

Inhalation Dosage Forms: A Focus on Dry Powder Inhalers and Their Advancements.

In this review, an extensive analysis of dry powder inhalers (DPIs) is offered, focusing on their characteristics, formulation, stability, and manufacturing. The advantages of pulmonary delivery were investigated, as well as the significance of the particle size in drug deposition. The preparation of DPI formulations was also comprehensively explored, including physico-chemical characterization of powders, powder processing techniques, and formulation considerations. In addition to manufacturing procedures, testing methods were also discussed, providing insights into the development and evaluation of DPI formulations. This review also explores the design basics and critical attributes specific to DPIs, highlighting the significance of their optimization to achieve an effective inhalation therapy. Additionally, the morphology and stability of 3 DPI capsules (Spiriva, Braltus, and Onbrez) were investigated, offering valuable insights into the properties of these formulations. Altogether, these findings contribute to a deeper understanding of DPIs and their development, performance, and optimization of inhalation dosage forms.

Viscoelasticity of Liposomal Dispersions.

Janus-faced viscoelastic gelling agents-possessing both elastic and viscous characteristics-provide materials with unique features including strengthening ability under stress and a liquid-like character with lower viscosities under relaxed conditions. The mentioned multifunctional character is manifested in several body fluids such as human tears, synovial liquids, skin tissues and mucins, endowing the fluids with a special physical resistance property that can be analyzed by dynamic oscillatory rheology. Therefore, during the development of pharmaceutical or cosmetical formulations-with the intention of mimicking the physiological conditions-rheological studies on viscoelasticity are strongly recommended and the selection of viscoelastic preparations is highlighted. In our study, we aimed to determine the viscoelasticity of various liposomal dispersions. We intended to evaluate the impact of lipid concentration, the presence of cholesterol or 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) and the gelling agents polyvinyl alcohol (PVA) and hydroxyethylcellulose (HEC) on the viscoelasticity of vesicular systems. Furthermore, the effect of two model drugs (phenyl salicylate and caffeine) on the viscoelastic behavior of liposomal systems was studied. Based on our measurements, the oscillation rheological properties of the liposomal formulations were influenced both by the composition and the lamellarity/size of the lipid vesicles.

Determination of the Main Phase Transition Temperature of Phospholipids by Oscillatory Rheology.

Knowledge of the physical and chemical properties of phospholipids, such as phase transition temperatures (Tc), is of great importance in order to reveal the functionalities of biological and artificial membranes. Our research group developed an oscillatory rheological method for the simple and rapid determination of phase transition temperatures (Tc). The phospholipids constructing the membranes undergo conformational changes at their Tc, which cause alterations of viscoelastic properties of the molecules. The oscillatory technique recommended by us proved to be appropriate to reveal the altered molecular properties of phospholipids as tracking the slightest changes in the viscoelasticity. Our study demonstrates the abrupt changes in rheological properties at Tc for the following phospholipids: 1,2-Dimyristoyl-sn-glycero-3-Phosphocholine (DMPC), 1,2-Dipalmitoyl-sn-glycero-3-Phosphatidylcholine (DPPC), and 1,2-Distearoyl-sn-glycero-3-Phosphocholine (DSPC), proving that the applied methodology is adequate for determining the Tc of phospholipids.

Rheological Considerations of Pharmaceutical Formulations: Focus on Viscoelasticity.

Controlling rheological properties offers the opportunity to gain insight into the physical characteristics, structure, stability and drug release rate of formulations. To better understand the physical properties of hydrogels, not only rotational but also oscillatory experiments should be performed. Viscoelastic properties, including elastic and viscous properties, are measured using oscillatory rheology. The gel strength and elasticity of hydrogels are of great importance for pharmaceutical development as the application of viscoelastic preparations has considerably expanded in recent decades. Viscosupplementation, ophthalmic surgery and tissue engineering are just a few examples from the wide range of possible applications of viscoelastic hydrogels. Hyaluronic acid, alginate, gellan gum, pectin and chitosan are remarkable representatives of gelling agents that attract great attention applied in biomedical fields. This review provides a brief summary of rheological properties, highlighting the viscoelasticity of hydrogels with great potential in biomedicine.

Specific nasopharyngeal Corynebacterium strains serve as gatekeepers against SARS-CoV-2 infection.

The SARS-CoV-2 virus is still causing a worldwide problem. The virus settles primarily on the nasal mucosa, and the infection and its course depend on individual susceptibility. Our aim was to investigate the nasopharynx composition's role in the individual susceptibility. During the first phase of SARS-CoV-2 pandemic, nasopharyngeal microbiome samples of close contact unvaccinated patients were investigated by 16S rRNA analysis and by culturing. The whole genome of cultured Corynebacteria was sequenced. The relative expression of ACE2, TMPRSS2, and cathepsin L on Caco-2 cells and the strength of S1-ACE2 binding were determined in the presence of Corynebacteria. From 55 close contacts exposed to identical SARS-CoV-2 exposure, 26 patients became infected and 29 remained uninfected. The nasopharyngeal microbiome analysis showed significantly higher abundance of Corynebacteria in uninfected group. Corynebacterium accolens could be cultivated only from uninfected individuals and Corynebacterium propinquum from both infected and uninfected. Corynebacteria from uninfected patient significantly reduced the ACE2 and cathepsin L expression. C. accolens significantly reduced the TMPRSS2 expression compared to other Corynebacteria. Furthermore, Corynebacterium spp. weakened the binding of the S1-ACE2. Most C. accolens isolates harbored the TAG lipase LipS1 gene. Based on these results, the presence of Corynebacterium spp. in the nasopharyngeal microbiota, especially C. accolens strains, could reduce the individual susceptibility to SARS-CoV-2 infection by several mechanisms: by downregulation the ACE2, the TMPRSS2 receptors, and cathepsin L in the host; through the inhibition of S1-ACE2 binding; and lipase production. These results suggest the use of C. accolens strains as probiotics in the nasopharynx in the future.

Transdermal Delivery of α-Aminophosphonates as Semisolid Formulations-An In Vitro-Ex Vivo Study.

α-Aminophosphonates are organophosphorus compounds with an obvious similarity with α-amino acids. Owing to their biological and pharmacological characteristics, they have attracted the attention of many medicinal chemists. α-Aminophosphonates are known to exhibit antiviral, antitumor, antimicrobial, antioxidant and antibacterial activities, which can all be important in pathological dermatological conditions. However, their ADMET properties are not well studied. The aim of the current study was to provide preliminary information about the skin penetration of three preselected α-aminophosphonates when applying them as topical cream formulations in static and dynamic diffusion chambers. The results indicate that aminophosphonate 1a, without any substituent in the para position, shows the best release from the formulation and the highest absorption through the excised skin. However, based on our previous study, the in vitro pharmacological potency was higher in the case of para-substituted molecules 1b and 1c. The particle size and rheological studies revealed that the 2% cream of aminophosphonate 1a was the most homogenous formulation. In conclusion, the most promising molecule was 1a, but further experiments are proposed to uncover the possible transporter interactions in the skin, optimize the topical formulations and improve PK/PD profiles in case of transdermal delivery.

The Effect of the Particle Size Reduction on the Biorelevant Solubility and Dissolution of Poorly Soluble Drugs with Different Acid-Base Character.

Particle size reduction is a commonly used process to improve the solubility and the dissolution of drug formulations. The solubility of a drug in the gastrointestinal tract is a crucial parameter, because it can greatly influence the bioavailability. This work provides a comprehensive investigation of the effect of the particle size, pH, biorelevant media and polymers (PVA and PVPK-25) on the solubility and dissolution of drug formulations using three model compounds with different acid-base characteristics (papaverine hydrochloride, furosemide and niflumic acid). It was demonstrated that micronization does not change the equilibrium solubility of a drug, but it results in a faster dissolution. In contrast, nanonization can improve the equilibrium solubility of a drug, but the selection of the appropriate excipient used for nanonization is essential, because out of the two used polymers, only the PVPK-25 had an increasing effect on the solubility. This phenomenon can be explained by the molecular structure of the excipients. Based on laser diffraction measurements, PVPK-25 could also inhibit the aggregation of the particles more effectively than PVA, but none of the polymers could hold the nanonized samples in the submicron range until the end of the measurements.

Structural and Functional Analysis of Excised Skins and Human Reconstructed Epidermis with Confocal Raman Spectroscopy and in Microfluidic Diffusion Chambers.

Several ex vivo and in vitro skin models are available in the toolbox of dermatological and cosmetic research. Some of them are widely used in drug penetration testing. The excised skins show higher variability, while the in vitro skins provide more reproducible data. The aim of the current study was to compare the chemical composition of different skin models (excised rat skin, excised human skin and human-reconstructed epidermis) by measurement of ceramides, cholesterol, lactate, urea, protein and water at different depths of the tissues. The second goal was to compile a testing system, which includes a skin-on-a-chip diffusion setup and a confocal Raman spectroscopy for testing drug diffusion across the skin barrier and accumulation in the tissue models. A hydrophilic drug caffeine and the P-glycoprotein substrate quinidine were used in the study as topical cream formulations. The results indicate that although the transdermal diffusion of quinidine is lower, the skin accumulation was comparable for the two drugs. The various skin models showed different chemical compositions. The human skin was abundant in ceramides and cholesterol, while the reconstructed skin contained less water and more urea and protein. Based on these results, it can be concluded that skin-on-a-chip and confocal Raman microspectroscopy are suitable for testing drug penetration and distribution at different skin layers within an exposition window. Furthermore, obese human skin should be treated with caution for skin absorption testing due to its unbalanced composition.

Review on Starter Pellets: Inert and Functional Cores.

A significant proportion of pharmaceuticals are now considered multiparticulate systems. Modified-release drug delivery formulations can be designed with engineering precision, and patient-centric dosing can be accomplished relatively easily using multi-unit systems. In many cases, Multiple-Unit Pellet Systems (MUPS) are formulated on the basis of a neutral excipient core which may carry the layered drug surrounded also by functional coating. In the present summary, commonly used starter pellets are presented. The manuscript describes the main properties of the various nuclei related to their micro- and macrostructure. In the case of layered pellets formed based on different inert pellet cores, the drug release mechanism can be expected in detail. Finally, the authors would like to prove the industrial significance of inert cores by presenting some of the commercially available formulations.

Real-time coating thickness measurement and defect recognition of film coated tablets with machine vision and deep learning.

This paper presents a system, where images acquired with a digital camera are coupled with image analysis and deep learning to identify and categorize film coating defects and to measure the film coating thickness of tablets. There were 5 different classes of defective tablets, and the YOLOv5 algorithm was utilized to recognize defects, the accuracy of the classification was 98.2%. In order to characterize coating thickness, the diameter of the tablets in pixels was measured, which was used to measure the coating thickness of the tablets. The proposed system can be easily scaled up to match the production capability of continuous film coaters. With the developed technique, the complete screening of the produced tablets can be achieved in real-time resulting in the improvement of quality control.

Development and Dissolution Study of a ß-Galactosidase Containing Drinking Straw.

Today, in addition to many different physicochemical and pharmacological properties of the active ingredients and excipients, the developer of a pharmaceutical formulation must take into account several factors during the formulation process in order for the patient to cooperate to use the formulation accurately. One of the innovative solutions in paediatrics may be the use of medicated drinking straws. For our studies, we successfully prepared lactase-containing, rapid disintegration particles by two techniques commonly used in the pharmaceutical industry. The simulation of the usage of the filled straws was presented from a new perspective for the patient by an in vitro method. The effect of the temperature of the liquid used during the administration of the straw and the effect of the frequency during the application on the dissolution rate were investigated. According to our results, in the case of a straw containing rapidly dissolving particles, the temperature of the used liquid and the mode of administration (frequency) play a significant role in the release rate from the composition.

Altered Glycosylation of Human Alpha-1-Acid Glycoprotein as a Biomarker for Malignant Melanoma.

A high-resolution HILIC-MS/MS method was developed to analyze anthranilic acid derivatives of N-glycans released from human serum alpha-1-acid glycoprotein (AGP). The method was applied to samples obtained from 18 patients suffering from high-risk malignant melanoma as well as 19 healthy individuals. It enabled the identification of 102 glycan isomers separating isomers that differ only in sialic acid linkage (α-2,3, α-2,6) or in fucose positions (core, antenna). Comparative assessment of the samples revealed that upregulation of certain fucosylated glycans and downregulation of their nonfucosylated counterparts occurred in cancer patients. An increased ratio of isomers with more α-2,6-linked sialic acids was also observed. Linear discriminant analysis (LDA) combining 10 variables with the highest discriminatory power was employed to categorize the samples based on their glycosylation pattern. The performance of the method was tested by cross-validation, resulting in an overall classification success rate of 96.7%. The approach presented here is significantly superior to serological marker S100B protein in terms of sensitivity and negative predictive power in the population studied. Therefore, it may effectively support the diagnosis of malignant melanoma as a biomarker.

Optimization and Development of Albumin-Biopolymer Bioconjugates with Solubility-Improving Properties.

Bioconjugation is an emerging field in the food and pharmaceutical industry. Due to its biocompatibility and high ligand binding capacity, albumin is widely used in modern drug delivery systems. However, the protein is sensitive to environmental stresses; albumin conjugates, on the other hand, have improved functional properties. Biopolymers are gaining interest due to their biodegradability and safety, compared to synthetic polymers. In this study, albumin-biopolymer bioconjugates were prepared by nonenzymatic Maillard reaction at 60 °C and 80% relative humidity. This nonenzymatic conjugation takes place between reducing sugars and available amino groups of a protein in certain conditions. The optimal molar ratio and time for the conjugation were studied by several investigation methods, including circular dichroism and fluorescence spectroscopy, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and determination of available amino groups with ortho-phthaldialdehyde (OPA) assay. All of the measurements provided evidence for the covalent bonding of albumin and biopolymers, resulting in bioconjugates. Based on the results, a higher molar ratio and longer time are necessary to complete the reaction with the available amino groups. However, the optimal parameters are specific to each given biopolymer. The rheological behavior of the conjugates is characteristic of the initial biopolymer, which can be useful in drug development. Moreover, both the physical characteristics of albumin and the solubility-improving capacity were enhanced. Therefore, the potential use of albumin-biopolymer bioconjugates in the pharmaceutical industry could be considered.

Image Analysis: A Versatile Tool in the Manufacturing and Quality Control of Pharmaceutical Dosage Forms.

In pharmaceutical sciences, visual inspection is one of the oldest methods used for description in pharmacopeias and is still an important part of the characterization and qualification of active ingredients, excipients, and dosage forms. With the development of technology, it is now also possible to take images of various pharmaceutical dosage forms with different imaging methods in a size range that is hardly visible or completely invisible to the human eye. By analyzing high-quality designs, physicochemical processes can be understood, and the results can be used even in the optimization of the composition of the dosage form and in the development of its production. The present study aims to show some of the countless ways image analysis can be used in the manufacturing and quality assessment of different dosage forms. This summary also includes measurements and an evaluation of, amongst others, a less studied dosage form, medicated foams.

Investigating thermal stability based on the structural changes of lactase enzyme by several orthogonal methods.

Thermal stability of lactase (ß-galactosidase) enzyme has been studied by a variety of physico-chemical methods. ß-galactosidase is the main active ingredient of medications for lactose intolerance. It is typically produced industrially by the Aspergillus oryzae filamentous fungus. Lactase was used as a model to help understand thermal stability of enzyme-type biopharmaceuticals. Enzyme activity (hydrolyzation of lactose) of ß-galactosidase was determined after storing the solid enzyme substance at various temperatures. For a better understanding of the relationship between structure and activity changes we determined the mass and size of the molecules with gel electrophoresis and dynamic light scattering and detected aggregation processes. A bottom-up proteomic procedure was used to determine the primary amino acid sequence and to investigate changes in the N-glycosylation pattern of the protein. NMR and CD spectroscopic methods were used to observe changes in higher order structures and to reveal relationships between structural and functional changes.

Evaluation of Biodegradable PVA-Based 3D Printed Carriers during Dissolution.

The presence of additive manufacturing, especially 3D printing, has the potential to revolutionize pharmaceutical manufacturing owing to the distinctive capabilities of personalized pharmaceutical manufacturing. This study's aim was to examine the behavior of commonly used polyvinyl alcohol (PVA) under in vitro dissolution conditions. Polylactic acid (PLA) was also used as a comparator. The carriers were designed and fabricated using computer-aided design (CAD). After printing the containers, the behavior of PVA under in vitro simulated biorelevant conditions was monitored by gravimetry and dynamic light scattering (DLS) methods. The results show that in all the dissolution media PVA carriers were dissolved; the particle size was under 300 nm. However, the dissolution rate was different in various dissolution media. In addition to studying the PVA, as drug delivery carriers, the kinetics of drug release were investigated. These dissolution test results accompanied with UV spectrophotometry tracking indirectly determine the possibilities for modifying the output of quality by computer design.

Investigation of propellant-free aqueous foams as pharmaceutical carrier systems.

Due to their light consistency and good spreadability, aqueous foams are considered as convenient and highly accepted drug carrier systems that are of great importance in the field of topical drug delivery. The production of a stable, easy to dose, preferably environmentally harmless foam formulation is challenging. Therefore, foam characterisation requires a complex approach: several tests are to be performed throughout the formulation. Our study primarily aims to investigate the quality attributes of propellant-free foam-forming additives. Throughout the research, we focused on acquiring knowledge about the properties of pharmaceutical excipients suitable for foam formulations and their effect on foam characteristics. Not only were the relative foam density, actuated foam weight and the foam collapse tendencies studied, but also the initial liquid properties. Along with surface tension determination, bubble-forming experiments were carried out. The bubble size and rate of formation, standardised by using a texture analyser, were followed by image analysis. Analysing the bubble-forming properties of dilute surfactant solutions allows assumptions on the properties of foam formed from the more concentrated solutions. The size and number of bubbles in the produced foams are related to the kinetics of single bubble formation. For comparison, commercially available medicated foams were studied.