Variants of Oxidized Regenerated Cellulose and Their Distinct Effects on Neuronal Tissue

Based on oxidized regenerated cellulose (ORC), several hemostyptic materials, such as Tabotamp^®, Equicel^® and Equitamp^®, have been developed to approach challenging hemostasis in neurosurgery. The present study compares ORC that differ in terms of compositions and properties, regarding their structure, solubility, pH values and effects on neuronal tissue. Cytotoxicity was detected via DNA-binding fluorescence dye in Schwann cells, astrocytes, and neuronal cells. Additionally, organotypic hippocampal slice cultures (OHSC) were analyzed, using propidium iodide, hematoxylin-eosin, and isolectin B₄ staining to investigate the cellular damage, cytoarchitecture, and microglia activation. Whereas Equicel^® led to a neutral pH, Tabotamp^® (pH 2.8) and Equitamp^® (pH 4.8) caused a significant reduction of pH (p < 0.001). Equicel^® and Tabotamp^® increased cytotoxicity significantly in several cell lines (p < 0.01). On OHSC, Tabotamp^® and Equicel^® led to a stronger and deeper damage to the neuronal tissue than Equitamp^® or gauze (p < 0.01). Equicel^® increased strongly the number of microglia cells after 24 h (p < 0.001). Microglia cells were not detectable after Tabotamp^® treatment, presumably due to an artifact caused by strong pH reduction. In summary, our data imply the use of Equicel^®, Tabotamp^® or Equitamp^® for specific applications in distinct clinical settings depending on their localization or tissue properties.     

SCA® Slows the Decline of Functional Parameters Associated with Senescence in Skin Cells

The identification of compounds and natural ingredients that can counteract tissue stress and dysfunction induced by aging in skin cells is warranted. Here, we investigated the activity of the secretion from the snail Cryptomphalus aspersa (SCA^®), an active compound with well-established beneficial effects on skin integrity and aging. To determinate its senescence-regulation mechanisms, we used a model where damage was induced by hydrogen peroxide (H₂O₂). The results showed that SCA^® positively modulated factors involved in cell senescence such as β-galactosidase and cell morphology, secretory efficiency markers (SIRT1/6 and carboxymethyl-lysine), and metabolic and redox homeostasis (mTOR and ROS). This study demonstrated a novel compound that is activity-modulating, reduces cell senescence, and increases longevity to maintain skin homeostasis and functionality.     

Platelets: Still a Therapeutical Target for Haemostatic Disorders

Platelets are cytoplasmatic fragments from bone marrow megakaryocytes present in blood. In this work, we review the basis of platelet mechanisms, their participation in syndromes and in arterial thrombosis, and their potential as a target for designing new antithrombotic agents. The option of new biotechnological sources is also explored.     

Effect of rhBMP-2 Immobilized Anorganic Bovine Bone Matrix on Bone Regeneration

Anorganic bovine bone matrix (Bio-Oss^®) has been used for a long time for bone graft regeneration, but has poor osteoinductive capability. The use of recombinant human bone morphogenetic protein-2 (rhBMP-2) has been suggested to overcome this limitation of Bio-Oss^®. In the present study, heparin-mediated rhBMP-2 was combined with Bio-Oss^® in animal experiments to investigate bone formation performance; heparin was used to control rhBMP-2 release. Two calvarial defects (8 mm diameter) were formed in a white rabbit model and then implanted or not (controls) with Bio-Oss^® or BMP-2/Bio-Oss^®. The Bio-Oss^® and BMP-2/Bio-Oss^® groups had significantly greater new bone areas (expressed as percentages of augmented areas) than the non-implanted controls at four and eight weeks after surgery, and the BMP-2/Bio-Oss^® group (16.50 ± 2.87 (n = 6)) had significantly greater new bone areas than the Bio-Oss^® group (9.43 ± 3.73 (n = 6)) at four weeks. These findings suggest that rhBMP-2 treated heparinized Bio-Oss^® markedly enhances bone regeneration.     

Evidence for Inhibitory Perturbations on the Amplitude,Gating, and Hysteresis of A-Type Potassium Current,Produced by Lacosamide,a Functionalized Amino Acid with Anticonvulsant Properties

Lacosamide (Vimpat^®, LCS) is widely known as a functionalized amino acid with promising anti-convulsant properties; however, adverse events during its use have gradually appeared. Despite its inhibitory effect on voltage-gated Na⁺ current (I_Na), the modifications on varying types of ionic currents caused by this drug remain largely unexplored. In pituitary tumor (GH₃) cells, we found that the presence of LCS concentration-dependently decreased the amplitude of A-type K⁺ current (I_K(A)) elicited in response to membrane depolarization. The I_K(A) amplitude in these cells was sensitive to attenuation by the application of 4-aminopyridine, 4-aminopyridine-3-methanol, or capsaicin but not by that of tetraethylammonium chloride. The effective IC₅₀ value required for its reduction in peak or sustained I_K(A) was calculated to be 102 or 42 µM, respectively, while the value of the dissociation constant (K_D) estimated from the slow component in I_K(A) inactivation at varying LCS concentrations was 52 µM. By use of two-step voltage protocol, the presence of this drug resulted in a rightward shift in the steady-state inactivation curve of I_K(A) as well as in a slowing in the recovery time course of the current block; however, no change in the gating charge of the inactivation curve was detected in its presence. Moreover, the LCS addition led to an attenuation in the degree of voltage-dependent hysteresis for I_K(A) elicitation by long-duration triangular ramp voltage commands. Likewise, the I_K(A) identified in mouse mHippoE-14 neurons was also sensitive to block by LCS, coincident with an elevation in the current inactivation rate. Collectively, apart from its canonical action on I_Na inhibition, LCS was effective at altering the amplitude, gating, and hysteresis of I_K(A) in excitable cells. The modulatory actions on I_K(A), caused by LCS, could interfere with the functional activities of electrically excitable cells (e.g., pituitary tumor cells or hippocampal neurons).     

Tolerability of Oral Supplementation with Microencapsulated Ferric Saccharate Compared to Ferrous Sulphate in Healthy Premenopausal Woman: A Crossover,Randomized, Double-Blind Clinical Trial

A single-center, crossover, randomized, double-blind, and controlled clinical study was conducted to assess the tolerability profile, especially with regard to gastrointestinal complaints, of oral supplementation with AB-Fortis^®, a microencapsulated ferric saccharate (MFS), as compared with conventional ferrous sulphate (FS) in healthy premenopausal women. A dose of 60 mg/day of elemental iron was used. The test products were administered for 14 consecutive days with a washout period of two menstrual episodes and a minimum of one month between the two intervention periods. The subjects completed simple-to-answer questionnaires daily for 14 days during both the intervention and the washout periods, capturing the symptoms associated with oral iron supplementation and overall health aspects. Following product consumption, the incidences of symptoms, numbers of complaints/symptoms, overall intensity, and total days with symptoms were found to be significantly higher for FS consumption as compared to MFS. The better tolerability profile of MFS over FS was further substantiated when both products were compared to a real-life setting (i.e., the washout period). Overall, the administration of both study products was safe with no serious or significant adverse events reported. In summary, the current study shows the better tolerability of the MFS preparation when compared to that of the FS, presenting MFS as a well-tolerated and safe option for improving iron nutrition.     

Treatment with the Probiotic Product Aviguard® Alleviates Inflammatory Responses during Campylobacter jejuni-Induced Acute Enterocolitis in Mice

Prevalences of Campylobacter (C.) jejuni infections are progressively rising globally. Given that probiotic feed additives, such as the commercial product Aviguard^®, have been shown to be effective in reducing enteropathogens, such as Salmonella, in vertebrates, including livestock, we assessed potential anti-pathogenic and immune-modulatory properties of Aviguard^® during acute C. jejuni-induced murine enterocolitis. Therefore, microbiota-depleted IL-10^−/− mice were infected with C. jejuni strain 81-176 by gavage and orally treated with Aviguard^® or placebo from day 2 to 4 post-infection. The applied probiotic bacteria could be rescued from the intestinal tract of treated mice, but with lower obligate anaerobic bacterial counts in C. jejuni-infected as compared to non-infected mice. Whereas comparable gastrointestinal pathogen loads could be detected in both groups until day 6 post-infection, Aviguard^® treatment resulted in improved clinical outcome and attenuated apoptotic cell responses in infected large intestines during acute campylobacteriosis. Furthermore, less distinct pro-inflammatory immune responses could be observed not only in the intestinal tract, but also in extra-intestinal compartments on day 6 post-infection. In conclusion, we show here for the first time that Aviguard^® exerts potent disease-alleviating effects in acute C. jejuni-induced murine enterocolitis and might be a promising probiotic treatment option for severe campylobacteriosis in humans.     

Cultivation of Keratinocytes and Fibroblasts in a Three-Dimensional Bovine Collagen-Elastin Matrix (Matriderm?) and Application for Full Thickness Wound Coverage in Vivo

New skin substitutes for burn medicine or reconstructive surgery pose an important issue in plastic surgery. Matriderm^® is a clinically approved three-dimensional bovine collagen-elastin matrix which is already used as a dermal substitute of full thickness burn wounds. The drawback of an avital matrix is the limited integration in full thickness skin defects, depending on the defect size. To further optimize this process, Matriderm^® has also been studied as a matrix for tissue engineering of skin albeit long-term cultivation of the matrix with cells has been difficult. Cells have generally been seeded onto the matrix with high cell loss and minimal time-consuming migration. Here we developed a cell seeded skin equivalent after microtransfer of cells directly into the matrix. First, cells were cultured, and microinjected into Matriderm^®. Then, cell viability in the matrix was determined by histology in vitro. As a next step, the skin substitute was applied in vivo into a full thickness rodent wound model. The wound coverage and healing was observed over a period of two weeks followed by histological examination assessing cell viability, proliferation and integration into the host. Viable and proliferating cells could be found throughout the entire matrix. The presented skin substitute resembles healthy skin in morphology and integrity. Based on this study, future investigations are planned to examine behaviour of epidermal stem cells injected into a collagen-elastin matrix under the aspects of establishment of stem cell niches and differentiation.     

Bifidobacterium longum subsp. infantis CECT 7210 Reduces Inflammatory Cytokine Secretion in Caco-2 Cells Cultured in the Presence of Escherichia coli CECT 515

Previous works have described the activity of Bifidobacterium longum subsp. infantis CECT 7210 (also commercially named B. infantis IM-1^®) against rotavirus in mice and intestinal pathogens in piglets, as well as its diarrhea-reducing effect on healthy term infants. In the present work, we focused on the intestinal immunomodulatory effects of B. infantis IM-1^® and for this purpose we used the epithelial cell line isolated from colorectal adenocarcinoma Caco-2 and a co-culture system of human dendritic cells (DCs) from peripheral blood together with Caco-2 cells. Single Caco-2 cultures and Caco-2: DC co-cultures were incubated with B. infantis IM-1^® or its supernatant either in the presence or absence of Escherichia coli CECT 515. The B. infantis IM-1^® supernatant exerted a protective effect against the cytotoxicity caused by Escherichia coli CECT 515 on single cultures of Caco-2 cells as viability reached the values of untreated cells. B. infantis IM-1^® and its supernatant also decreased the secretion of pro-inflammatory cytokines by Caco-2 cells and the co-cultures incubated in the presence of E. coli CECT 515, with the response being more modest in the latter, which suggests that DCs modulate the activity of Caco-2 cells. Overall, the results obtained point to the immunomodulatory activity of this probiotic strain, which might underlie its previously reported beneficial effects.     

Sustainable Stabilizer-Free Nanoparticle Formulations of Valsartan Using Eudragit® RLPO

The bioavailability of the antihypertensive drug valsartan can be enhanced by various microencapsulation methods. In the present investigation, valsartan-loaded polymeric nanoparticles were manufactured from Eudragit^® RLPO using an emulsion–solvent evaporation method. Polyvinyl alcohol (PVA) was found to be a suitable stabilizer for the nanoparticles, resulting in a monodisperse colloid system ranging in size between 148 nm and 162 nm. Additionally, a high encapsulation efficiency (96.4%) was observed. However, due to the quaternary ammonium groups of Eudragit^® RLPO, the stabilization of the dispersion could be achieved in the absence of PVA as well. The nanoparticles were reduced in size (by 22%) and exhibited similar encapsulation efficiencies (96.4%). This more cost-effective and sustainable production method reduces the use of excipients and their expected emission into the environment. The drug release from valsartan-loaded nanoparticles was evaluated in a two-stage biorelevant dissolution set-up, leading to the rapid dissolution of valsartan in a simulated intestinal medium. In silico simulations using a model validated previously indicate a potential dose reduction of 60–70% compared to existing drug products. This further reduces the expected emission of the ecotoxic compound into the environment.     

The Effectiveness in Activating M-Type K+ Current Produced by Solifenacin ([(3R)-1-azabicyclo[2.2.2]octan-3-yl] (1S)-1-phenyl-3,4-dihydro-1H-isoquinoline-2-carboxylate): Independent of Its Antimuscarinic Action

Solifenacin (Vesicare^®, SOL), known to be a member of isoquinolines, is a muscarinic antagonist that has anticholinergic effect, and it has been beneficial in treating urinary incontinence and neurogenic detrusor overactivity. However, the information regarding the effects of SOL on membrane ionic currents is largely uncertain, despite its clinically wide use in patients with those disorders. In this study, the whole-cell current recordings revealed that upon membrane depolarization in pituitary GH₃ cells, the exposure to SOL concentration-dependently increased the amplitude of M-type K⁺ current (I_K(M)) with effective EC₅₀ value of 0.34 μM. The activation time constant of I_K(M) was concurrently shortened in the SOL presence, hence yielding the K_D value of 0.55 μM based on minimal reaction scheme. As cells were exposed to SOL, the steady-state activation curve of I_K(M) was shifted along the voltage axis to the left with no change in the gating charge of the current. Upon an isosceles-triangular ramp pulse, the hysteretic area of I_K(M) was increased by adding SOL. As cells were continually exposed to SOL, further application of acetylcholine (1 μM) failed to modify SOL-stimulated I_K(M); however, subsequent addition of thyrotropin releasing hormone (TRH, 1 μM) was able to counteract SOL-induced increase in I_K(M) amplitude. In cell-attached single-channel current recordings, bath addition of SOL led to an increase in the activity of M-type K⁺ (K_M) channels with no change in the single channel conductance; the mean open time of the channel became lengthened. In whole-cell current-clamp recordings, the SOL application reduced the firing of action potentials (APs) in GH₃ cells; however, either subsequent addition of TRH or linopirdine was able to reverse SOL-mediated decrease in AP firing. In hippocampal mHippoE-14 neurons, the I_K(M) was also stimulated by adding SOL. Altogether, findings from this study disclosed for the first time the effectiveness of SOL in interacting with K_M channels and hence in stimulating I_K(M) in electrically excitable cells, and this noticeable action appears to be independent of its antagonistic activity on the canonical binding to muscarinic receptors expressed in GH₃ or mHippoE-14 cells.     

Human Recombinant DNase I (Pulmozyme®) Inhibits Lung Metastases in Murine Metastatic B16 Melanoma Model That Correlates with Restoration of the DNase Activity and the Decrease SINE/LINE and c-Myc Fragments in Blood Cell-Free DNA

Tumor-associated cell-free DNAs (cfDNA) play an important role in the promotion of metastases. Previous studies proved the high antimetastatic potential of bovine pancreatic DNase I and identified short interspersed nuclear elements (SINEs) and long interspersed nuclear elements (LINEs)and fragments of oncogenes in cfDNA as the main molecular targets of enzyme in the bloodstream. Here, recombinant human DNase I (commercial name Pulmozyme^®), which is used for the treatment of cystic fibrosis in humans, was repurposed for the inhibition of lung metastases in the B16 melanoma model in mice. We found that Pulmozyme^® strongly reduced migration and induced apoptosis of B16 cells in vitro and effectively inhibited metastases in lungs and liver in vivo. Pulmozyme^® was shown to be two times more effective when administered intranasally (i.n.) than bovine DNase I, but intramuscular (i.m.) administration forced it to exhibit as high an antimetastatic activity as bovine DNase I. Both DNases administered to mice either i.m. or i.n. enhanced the DNase activity of blood serum to the level of healthy animals, significantly decreased cfDNA concentrations, efficiently degraded SINE and LINE repeats and c-Myc fragments in the bloodstream and induced apoptosis and disintegration of neutrophil extracellular traps in metastatic foci; as a result, this manifested as the inhibition of metastases spread. Thus, Pulmozyme^®, which is already an approved drug, can be recommended for use in the treatment of lung metastases.     

Therapeutic Modulation of the Host Defense by Hemoadsorption with CytoSorb®—Basics,Indications and Perspectives—A Scoping Review

The “normal” immune response to an insult triggers a highly regulated response determined by the interaction of various immunocompetent cells with pro- and anti-inflammatory cytokines. Under pathologic conditions, the massive elevation of cytokine levels (“cytokine storm”) could not be controlled until the recent development of hemoadsorption devices that are able to extract a variety of different DAMPs, PAMPs, and metabolic products from the blood. CytoSorb^® has been approved for adjunctive sepsis therapy since 2011. This review aims to summarize theoretical knowledge, in vitro results, and clinical findings to provide the clinician with pragmatic guidance for daily practice. English-language and peer-reviewed literature identified by a selective literature search in PubMed and published between January 2016 and May 2021 was included. Hemoadsorption can be used successfully as adjunct to a complex therapeutic regimen for various conditions. To the contrary, this nonspecific intervention may potentially worsen patient outcomes in complex immunological processes. CytoSorb^® therapy appears to be safe and useful in various diseases (e.g., rhabdomyolysis, liver failure, or intoxications) as well as in septic shock or cytokine release syndrome, although a conclusive assessment of treatment benefit is not possible and no survival benefit has yet been demonstrated in randomized controlled trials.     

Structural Alterations of Antigens at the Material Interface: An Early Decision Toolbox Facilitating Safe-by-Design Nanovaccine Development

Nanomaterials have found extensive interest in the development of novel vaccines, as adjuvants and/or carriers in vaccination platforms. Conjugation of protein antigens at the particle surface by non-covalent adsorption is the most widely used approach in licensed particulate vaccines. Hence, it is essential to understand proteins’ structural integrity at the material interface in order to develop safe-by-design nanovaccines. In this study, we utilized two model proteins, the wild-type allergen Bet v 1 and its hypoallergenic fold variant (BM4), to compare SiO₂ nanoparticles with Alhydrogel^® as particulate systems. A set of biophysical and functional assays including circular dichroism spectroscopy and proteolytic degradation was used to examine the antigens’ structural integrity at the material interface. Conjugation of both biomolecules to the particulate systems decreased their proteolytic stability. However, we observed qualitative and quantitative differences in antigen processing concomitant with differences in their fold stability. These changes further led to an alteration in IgE epitope recognition. Here, we propose a toolbox of biophysical and functional in vitro assays for the suitability assessment of nanomaterials in the early stages of vaccine development. These tools will aid in safe-by-design innovations and allow fine-tuning the properties of nanoparticle candidates to shape a specific immune response.     

Evaluation of a Gel Containing a Propionibacterium Extract in an In Vivo Model of Wound Healing

Inappropriate wound healing (WH) management can cause significant comorbidities, especially in patients affected by chronic and metabolic diseases, such as diabetes. WH involves several different, partially overlapping processes, including hemostasis, inflammation, cell proliferation, and remodeling. Oxidative stress in WH contributes to WH impairment because of the overexpression of radical oxygen species (ROS) and nitrogen species (RNS). This study aimed to evaluate the in vitro antioxidative action of a gel containing a Propionibacterium extract (Emorsan^® Gel) and assess its skin re-epithelialization properties in a mouse model of WH. The scavenging effects of the bacterial extract were assessed in vitro through the ABTS and DPPH assays and in L-929 murine fibroblasts. The effects of the Emorsan^® Gel were studied in vivo in a murine model of WH. After WH induction, mice were treated daily with vehicle or Emorsan^® Gel for 6 or 12 days. According to the in vitro tests, the Propionibacterium extract exerted an inhibitory effect on ROS and RNS, consequently leading to the reduction in malondialdehyde (MDA) and nitrite levels. Before proceeding with the in vivo study, the Emorsan^® Gel was verified to be unabsorbed. Therefore, the observed effects could be ascribed to a local action. The results obtained in vivo showed that through local reduction of oxidative stress and inflammation (IL-1β, TNF-α), the Emorsan^® Gel significantly reduced the infiltration of mast cells into the injured wound, leading to the amelioration of symptoms such as itch and skin irritation. Therefore, the Emorsan^® Gel improved the speed and percentage of wound area closure by improving the tissue remodeling process, prompting vascular–endothelial growth factor (VEGF) and transforming growth factor (TGF)- β production and reducing the expression of adhesion molecules. Emorsan^® Gel, by its ability to inhibit free radicals, could reduce local inflammation and oxidative stress, thus enhancing the speed of wound healing.     

Simultaneous Monitoring of Monoclonal Antibody Variants by Strong Cation-Exchange Chromatography Hyphenated to Mass Spectrometry to Assess Quality Attributes of Rituximab-Based Biotherapeutics

Different manufacturing processes and storage conditions of biotherapeutics can lead to a significant variability in drug products arising from chemical and enzymatic post-translational modifications (PTMs), resulting in the co-existence of a plethora of proteoforms with different physicochemical properties. To unravel the heterogeneity of these proteoforms, novel approaches employing strong cation-exchange (SCX) high-performance liquid chromatography (HPLC) hyphenated to mass spectrometry (MS) using a pH gradient of volatile salts have been developed in recent years. Here, we apply an established SCX-HPLC-MS method to characterize and compare two rituximab-based biotherapeutics, the originator MabThera^® and its Indian copy product Reditux™. The study assessed molecular differences between the two drug products in terms of C-terminal lysine variants, glycosylation patterns, and other basic and acidic variants. Overall, MabThera^® and Reditux™ displayed differences at the molecular level. MabThera^® showed a higher degree of galactosylated and sialylated glycoforms, while Reditux™ showed increased levels of oligomannose and afucosylated glycoforms. Moreover, the two drug products showed differences in terms of basic variants such as C-terminal lysine and N-terminal truncation, present in Reditux™ but not in MabThera^®. This study demonstrates the capability of this fast SCX-HPLC-MS approach to compare different drug products and simultaneously assess some of their quality attributes.