Regenerated keratin membrane to match the in vitro drug diffusion through human epidermis

AbstractThis work aimed to develop membranes made of regenerated keratin and ceramides (CERs) to match the barrier property of the human stratum corneum in in vitro percutaneous absorption studies. The membrane composition was optimized on the basis of the in vitro drug diffusion profiles of ibuprofen, propranolol and testosterone chosen as model drugs on the basis of their different diffusion and solubility properties. The data were compared to those obtained using human epidermis.The ATR-FTIR and SEM analyses revealed that CERs were suspended into the regenerated keratin matrix, even if a partial solubilization occurred. It resulted in the membranes being physically stable after exposure to aqueous buffer and/or mineral oil and the fluxes of ibuprofen and propranolol from these vehicles through membranes and human skin were of the same order of magnitude. The best relationship with human epidermis data was obtained with 180μm-thick membrane containing 1% ceramide III and 1% ceramide VI. The data on the testosterone diffusion were affected by the exposure of the membrane to a water/ethanol solution over a prolonged period of time, indicating that such an organic solvent was able to modify the supermolecular organization of keratin and CERs.The keratin/CER membranes can represent a simplified model to assay the in vitro skin permeability study of small molecules

Reciprocal Regulation Between Smad7 and Sirt1 in the Gut

In inflammatory bowel disease (IBD) mucosa, there is over-expression of Smad7, an intracellular inhibitor of the suppressive cytokine transforming growth factor-β1, due to post-transcriptional mechanisms that enhance Smad7 acetylation status thus preventing ubiquitination-mediated proteosomal degradation of the protein. IBD-related inflammation is also marked by defective expression of Sirt1, a class III NAD+-dependent deacetylase, which promotes ubiquitination-mediated proteosomal degradation of various intracellular proteins and triggers anti-inflammatory signals. The aim of our study was to determine whether, in IBD, there is a reciprocal regulation between Smad7 and Sirt1. Smad7 and Sirt1 were examined in mucosal samples of IBD patients and normal controls by Western blotting and immunohistochemistry, and Sirt1 activity was assessed by a fluorimetric assay. To determine whether Smad7 is regulated by Sirt1, normal or IBD lamina propria mononuclear cells (LPMC) were cultured with either Sirt1 inhibitor (Ex527) or activator (Cay10591), respectively. To determine whether Smad7 controls Sirt1 expression, ex vivo organ cultures of IBD mucosal explants were treated with Smad7 sense or antisense oligonucleotide. Moreover, Sirt1 expression was evaluated in LPMC isolated from Smad7-transgenic mice given dextran sulfate sodium (DSS). Upregulation of Smad7 was seen in both the epithelial and lamina propria compartments of IBD patients and this associated with reduced expression and activity of Sirt1. Activation of Sirt1 in IBD LPMC with Cay10591 reduced acetylation and enhanced ubiquitination-driven proteasomal-mediated degradation of Smad7, while inhibition of Sirt1 activation in normal LPMC with Ex527 increased Smad7 expression. Knockdown of Smad7 in IBD mucosal explants enhanced Sirt1 expression, thus suggesting a negative effect of Smad7 on Sirt1 induction. Consistently, mucosal T cells of Smad7-transgenic mice contained reduced levels of Sirt1, a defect that was amplified by induction of DSS colitis. The data suggest the existence of a reciprocal regulatory mechanism between Smad7 and Sirt1, which could contribute to amplify inflammatory signals in the gut

Spotlight on Calcipotriol/Betamethasone Fixed-Dose Combination in Topical Formulations: Is There Still Room for Innovation?

Psoriasis is a lifelong disease which requires treatment adherence for successful management. Considering the complexity of this pathology, the combination of active pharmaceutical ingredients with a synergistic mechanism of action can improve the safety and efficacy of the treatment with respect to the conventional monotherapy. Moreover, a fixed dose of therapeutic agents in a topical formulation offers the possibility to simplify administration, reduce the doses of each active ingredient, and improve patient’s compliance. Among the first-line treatments in mild to moderate psoriasis, the formulation of calcipotriol (Cal) and betamethasone dipropionate (BD) in a single vehicle is challenging due to their chemical incompatibility in an aqueous environment and the formation of degradation products. Based on these considerations, this review aims to provide an overview on the biopharmaceutical properties of Cal/BD fixed-dose combination products available on the market (namely ointment, oleogel, foam, and O/W cream), highlighting also the novel approaches under evaluation. The main differences among topical formulations are discussed considering the different features of the anatomic districts involved in psoriasis and the patient’s adherence. Moreover, since in vitro experiments are fundamental to evaluate the skin permeation profile during the development of an efficacious medicinal product, special emphasis is given to models proposed to mimic psoriatic lesions

Design of Liposomal Lidocaine/Cannabidiol Fixed Combinations for Local Neuropathic Pain Treatment

The administration of drug fixed combinations by nanocarriers is a new attractive approach since it can allow improvements in both the skin penetration of cargo compounds and their synergistic effects. The cutaneous administration of lidocaine (LD) and cannabidiol (CBD) combination can be useful for the local treatment of neuropathic pain. In fact, these drugs might exert a complementary effect on pain acting on sodium and calcium channels. In this study, the feasibility to deliver this combination in the deeper layers of the skin using deformable liposomes was studied. Based on a study of the drug affinity for lipid components performed by DSC, CBD was loaded in the lipid bilayer for limiting the leakage, while LD was loaded in the inner core by a pH gradient method (G-liposomes) or after previous encapsulation in micelle (DiMiL). The effect of the presence of Tween 80 in the liposome membrane was also evaluated. DiMiL increased both the skin permeation and the retention in the dermis of CBD and LD with respect to G-liposomes (R24dermis: 11.52 ± 2.4 against 4.51 ± 0.8 µg/cm2 for CBD; 19.6 ± 2.9 against 3.2 ± 0.1 µg/cm2 for LD). Moreover, both DiMiL and G-liposomes were more efficient than control formulations carrying free drugs in improving drug skin permeation. Interestingly, in the presence of a drug exerting a fluidizing effect such as CBD, the removal of Tween 80 from the composition led to an improved control of drug release and a higher extent of drug retention in the dermis layer

Is PEGylation of Drugs Associated with Hypersensitivity Reactions? An Analysis of the Italian National Spontaneous Adverse Drug Reaction Reporting System

Background and objective: Evidence highlights the allergenic potential of PEGylated drugs because of the production of anti-polyethylene glycol immunoglobulins. We investigated the risk of hypersensitivity reactions of PEGylated drugs using the Italian spontaneous adverse drug reaction reporting system database. Methods: We selected adverse drug reaction reports attributed to medicinal products containing PEGylated active substances and/or PEGylated liposomes from the Italian Spontaneous Reporting System in the period between its inception and March 2021. As comparators, we extracted adverse drug reaction reports of medicinal products containing the same non-PEGylated active substances and/or non-PEGylated liposomes (or compounds belonging to the same mechanistic class). A descriptive analysis of reports of hypersensitivity reactions was performed. Reporting rates and time to onset of hypersensitivity reactions were also calculated in the period between January 2009 and March 2021. As a measure of disproportionality, we calculated the reporting odds ratio. Results: Overall, 3865 adverse drug reaction reports were related to PEGylated medicinal products and 11,961 to their non-PEGylated comparators. Around two-thirds of patients were female and reports mostly concerned patients aged between 46 and 64 years. The frequency of hypersensitivity reactions reporting was higher among PEGylated versus non-PEGylated medicinal products (11.7% vs 9.4%, p < 0.0001). The hypersensitivity reaction reporting rates were higher for PEGylated medicinal products versus non-PEGylated medicinal products, with reporting rate ratios that ranged from 1.4 (95% confidence interval 0.8-2.5) for pegfilgrastim versus filgrastim to 20.0 (95% confidence interval 2.8-143.5) for peginterferon alpha-2a versus interferon alpha-2a. The median time to onset of hypersensitivity reactions was 10 days (interquartile range: 0-61) for PEGylated medicinal products, and 36 days (interquartile range: 3-216) for non-PEGylated comparators. Statistically significant reporting odds ratios were observed when comparing the reporting of hypersensitivity reactions for PEGylated versus non-PEGylated medicinal products (reporting odds ratio: 1.3; 95% confidence interval 1.1-1.4). However, when using all other drugs as comparators, the disproportionality analysis showed no association with hypersensitivity reactions for PEGylated nor non-PEGylated medicinal products, thus suggesting that many other triggers of drug-induced hypersensitivity reactions play a major role. Conclusions: The findings of this analysis of the Italian spontaneous adverse drug reaction database suggest a potential involvement for PEGylation in triggering drug-related hypersensitivity reactions, especially clinically relevant reactions. However, when comparing both PEGylated and non-PEGylated drugs under study to all other drugs no disproportionate reporting of hypersensitivity reactions was observed, probably due to a masking effect owing to the presence in the same database of other medicinal products increasing the threshold required to highlight a safety signal when the entire database is used as a reference

Design and development of plastic antibodies against SARS-CoV-2 RBD based on molecularly imprinted polymers that inhibit in vitro virus infection

The present research study reports the development of plastic antibodies based on Molecularly Imprinted Polymers (MIPs) capable of selectively binding a portion of the novel coronavirus SARS-CoV-2 spike protein. Indeed, molecular imprinting represents a very promising and attractive technology for the synthesis of MIPs characterized by specific recognition abilities for a target molecule. Given these characteristics, MIPs can be considered tailor-made synthetic antibodies obtained by a templating process. After in silico analysis, imprinted nanoparticles were synthesized by inverse microemulsion polymerization and their ability to prevent the interaction between ACE2 and the receptor-binding domain of SARS-CoV-2 was investigated. Of relevance, the developed synthetic antibodies are capable of significantly inhibiting virus replication in Vero cell culture, suggesting their potential application in the treatment, prevention and diagnosis of SARS-CoV-2 infection

Progranulin sustains STAT

Persistent activation of Signal Transducer and Activator of Transcription (STAT)3 occurs in a high percentage of tumors, including colorectal cancer (CRC), thereby contributing to malignant cell proliferation and survival. Although STAT3 is recognized as an attractive therapeutic target in CRC, conventional approaches aimed at inhibiting its functions have met with several limitations. Moreover, the factors that sustain hyper-activation of STAT3 in CRC are not yet fully understood. The identification of tumor-specific STAT3 cofactors may facilitate the development of compounds that interfere exclusively with STAT3 activity in cancer cells. Here, we show that progranulin, a STAT3 cofactor, is upregulated in human CRC as compared to nontumor tissue/cells and its expression correlates with STAT3 activation. Progranulin physically interacts with STAT3 in CRC cells, and its knockdown with a specific antisense oligonucleotide (ASO) inhibits STAT3 activation and restrains the expression of STAT3-related oncogenic proteins, thus causing cell cycle arrest and apoptosis. Moreover, progranulin knockdown reduces STAT3 phosphorylation and cell proliferation induced by tumor-infiltrating leukocyte (TIL)-derived supernatants in CRC cell lines and human CRC explants. These findings indicate that CRC exhibits overexpression of progranulin, and suggest a role for this protein in amplifying the STAT3 pathway in CRC

Hepcidin Upregulation in Colorectal Cancer Associates with Accumulation of Regulatory Macrophages and Epithelial–Mesenchymal Transition and Correlates with Progression of the Disease

Advanced, metastatic colorectal cancer (CRC) is associated with high rate of mortality because of its poor responsiveness to chemotherapy/immunotherapy. Recent studies have shown that hepcidin, a peptide hormone produced mainly by hepatocytes, is expressed by and enhances the growth of tumor cells. We here assessed whether hepcidin expression helps identify subsets of CRC with advanced and aggressive course. By integrating results of in vitro/ex vivo studies with data of bioinformatics databases, we initially showed that hepcidin RNA and protein expression was more pronounced in tissue samples taken from the tumor area, as compared to the macroscopically unaffected, adjacent, colonic mucosa of CRC patients. The induction of hepcidin in the colonic epithelial cell line HCEC-1ct by interleukin (IL)-6, IL-21 and IL-23 occurred via a Stat3-dependent mechanism and, in primary CRC cells, hepcidin co-localized with active Stat3. In CRC tissue, hepcidin content correlated mainly with macrophage accumulation and IL-10 and CD206 expression, two markers of regulatory macrophages. Consistently, both IL-10 and CD206 were up-regulated by hepcidin in blood mononuclear cells. The highest levels of hepcidin were found in metastatic CRC and survival analysis showed that high expression of hepcidin associated with poor prognosis. Moreover, hepcidin expression correlated with markers of epithelial-to-mesenchymal transition and the silencing of hepcidin in CRC cells reduced epithelial-to-mesenchymal transition markers. These findings indicate that hepcidin is markedly induced in the advanced stages of CRC and suggest that it could serve as a prognostic biomarker in CRC

In Vitro Anticancer Activity of Extracellular Vesicles (EVs) Secreted by Gingival Mesenchymal Stromal Cells Primed with Paclitaxel

Interdental papilla are an interesting source of mesenchymal stromal cells (GinPaMSCs), which are easy to isolate and expand in vitro. In our laboratory, GinPaMSCs were isolated, expanded, and characterized by studying their secretome before and after priming with paclitaxel (PTX). The secretome of GinPaMSCs did not affect the growth of cancer cell lines tested in vitro, whereas the secretome of GinPaMSCs primed with paclitaxel (GinPaMSCs/PTX) exerted a significant anticancer effect. GinPaMSCs were able to uptake and then release paclitaxel in amounts pharmacologically effective against cancer cells, as demonstrated in vitro by the direct activity of GinPaMSCs/PTX and their secretome against both human pancreatic carcinoma and squamous carcinoma cells. PTX was associated with extracellular vesicles (EVs) secreted by cells (EVs/PTX), suggesting that PTX is incorporated into exosomes during their biogenesis. The isolation of mesenchymal stromal cells (MSCs) from gingiva is less invasive than that from other tissues (such as bone marrow and fat), and GinPaMSCs provide an optimal substrate for drug-priming to obtain EVs/PTX having anticancer activity. This research may contribute to develop new strategies of cell-mediated drug delivery by EVs that are easy to store without losing function, and could have a superior safety profile in therapy