Current Pharmaceutical Biotechnology - Volume 22, Issue 2, 2021

COVID-19 is a pandemic, caused by the novel coronavirus 2 (SARS-CoV-2) which is a severe acute respiratory syndrome. The devastating impact of this novel coronavirus outbreak has necessitated the need for rapid and effective antiviral therapies against SARS-CoV-2 to control the spread of the disease and importantly, alleviate the severe life-threatening symptoms and disorders. Drug repurposing strategy offers an attractive, immediate and realistic approach to tackle this growing pandemic of COVID-19. Due to the similarities with the SARS-CoV-1 virus and phylogenetic relation to the MERS-CoV virus, accelerated screening of approved drugs and the development of repositioning strategies have proved to be critical for the survival of many COVID-19 patients. Numerous scientific investigations from the initial years of the coronavirus outbreak along with upcoming advances of immunotherapy and vaccines, may prove to be beneficial. Currently, several repurposing strategies are under different phases of clinical trials and provide a definitive framework for the development of future therapies for the effective treatment of COVID-19. This review article summarizes the latest developments and trends in drug repurposing strategy for COVID-19 treatment.

Mesenchymal Stem Cells (MSCs), a form of adult stem cells, are known to have a selfrenewing property and the potential to specialize into a multitude of cells and tissues such as adipocytes, cartilage cells, and fibroblasts. MSCs can migrate and home to the desired target zone where inflammation is present. The unique characteristics of MSCs in repairing, differentiation, regeneration, and the high capacity of immune modulation have attracted tremendous attention for exerting them in clinical purposes, as they contribute to the tissue regeneration process and anti-tumor activity. The MSCs-based treatment has demonstrated remarkable applicability towards various diseases such as heart and bone malignancies, and cancer cells. Importantly, genetically engineered MSCs, as a stateof- the-art therapeutic approach, could address some clinical hurdles by systemic secretion of cytokines and other agents with a short half-life and high toxicity. Therefore, understanding the biological aspects and the characteristics of MSCs is an imperative issue of concern. Herein, we provide an overview of the therapeutic application and the biological features of MSCs against different inflammatory diseases and cancer cells. We further shed light on MSCs' physiological interaction, such as migration, homing, and tissue repairing mechanisms in different healthy and inflamed tissues.

Nowadays, consumers have become increasingly attentive to human health and the use of more natural products. Consequently, the demand for natural preservatives in the food industry is more frequent. This has led to intense research to discover new antimicrobial compounds of natural origin that could effectively fight foodborne pathogens. This research aims to safeguard the health of consumers and, above all, to avoid potentially harmful chemical compounds. Lactobacillus is a bacterial genus belonging to the Lactic Acid Bacteria and many strains are defined GRAS, generally recognized as safe. These strains are able to produce substances with antibacterial activity against food spoilage bacteria and contaminating pathogens: the bacteriocins. The aim of this review was to focus on this genus and its capability to produce antibacterial peptides. The review collected all the information from the last few years about bacteriocins produced by Lactobacillus strains, isolated from clinical or food samples, with remarkable antimicrobial activities useful for being exploited in the food field. In addition, the advantages and disadvantages of their use and the possible ways of improvement for industrial applications were described.

Background: Several human diseases like Parkinson’s, Alzheimer’s disease, and systemic amyloidosis are associated with the misfolding and aggregation of protein molecules. Objective: The present study demonstrated the comparison of 4-methyl coumarin and 4-methylthiocoumarin derivative for their anti-amyloidogenic and disaggregation activities. The hen egg-white lysozyme is used as a model system to study protein aggregation and disaggregation under in vitro conditions. Methods: Techniques used in the study were Thioflavin T fluorescence assay, intrinsic fluorescence assay, circular dichroism, transmission electron microscopy, and molecular dynamics. Results: Fifteen compounds were screened for their anti-amyloidogenic and disaggregation potential. Six compounds significantly inhibited the fibril formation, whereas ten compounds showed disaggregation property of pre-formed fibrils. Under in vitro conditions, the compound C3 and C7 showed significant inhibition of fibril formation in a concentration-dependent manner as compared to control. C3 and C7 demonstrated 93% and 76% inhibition of fibril formation, respectively. Furthermore, C3 and C7 exhibited 83% and 76% disaggregation activity, respectively, of pre-formed HEWL fibrils at their highest concentration. These anti-amyloidogenic and disaggregation potential of C3 and C7 were validated by intrinsic fluorescence, CD, molecular dynamics, and TEM study. Discussion: 4-methylthiocoumarins derivatives have shown better anti-amyloidogenic activity as compared to 4-methylcoumarin derivatives for both amyloid formation as well as disaggregation of preformed amyloid fibrils. Structurally, the derivatives of 4-methylthiocoumarins (C3 and C7) contain thio group on 2nd position that might be responsible for anti-amyloidogenic activity as compared to 4- methylcoumarin derivatives (C2 and C4). Conclusion: C3 and C7 are novel 4-methylthiocoumarin derivatives that can be used as a lead for alleviation and symptoms associated with protein aggregation disorders.

Background: The present limitations related to the ocular administration of antifungal drugs for the treatment of fungal keratitis include poor ocular bioavailability, limited retention time, and low ocular tissue penetration. Methods: This study aimed to prepare a novel ophthalmic voriconazole-loaded nanosuspension based on Eudragit RS 100. Pharmasolve® was explored as a corneal permeation enhancer in voriconazole ophthalmic formulation using in vitro and in vivo experiments. Briefly, 1% voriconazole-loaded nanosuspension was prepared using the quasi-emulsion solvent evaporation process. Results: Characterizations of the voriconazole-loaded nanosuspension by Zetasizer Nano ZS and Transmission Electron Microscope (TEM) showed a uniform spherical shape without any agglomeration. The well-discreted nanoparticle with a size of 138 ± 1.3 nm was achieved with high entrapment efficiency (98.6 ± 2.5%) and positive zeta potential in the range of 22.5-31.2mV, indicating excellent physical stability. Discussion: Voriconazole-loaded nanosuspension containing the penetration enhancer displayed good permeability both in vitro and in vivo compared with the commercial voriconazole injection. The voriconazole-loaded nanosuspension exhibited good antifungal activity, significantly inhibiting the growth of Candida albicans at a lower concentration of voriconazole (2.5μg/mL, p < 0.05). Conclusion: In conclusion, the voriconazole-loaded nanosuspension containing Pharmasolve® can be used as an effective ophthalmic formulation for the topical ocular delivery of voriconazole.

Background: Oleamide is an essential substance for human health. So, the plants with high oleamide content are great sources for health care products. Objective: This study is conducted to investigate the quality of oleamide in plants and test the bioactivity in the selected two studied species. Methods: The three Ipomoea and five Dillenia species including Ipomoea alba, Ipomoea aquatica and Ipomoea pes-caprae, and Dillenia indica, Dillenia obovata, Dillenia ovata, Dillenia parviflora and Dillenia pentagyna were investigated for the quantity of oleamide by high-performance liquid chromatography. The biological activity test was conducted on the powder formulation of the chosen plants, Dillenia ovata and Dillenia parviflora at a ratio of 30:70, for anti-inflammatory activity ex vivo on a panel of molecular targets through ion channel inhibition including voltage-gated sodium channel, voltage-gated potassium channel, and the cardiac ion as human ether-a-go-go related gene. Results: The results showed that the leaf extracts of I. aquatica and D. ovata gave the highest and subsequent oleamide quantity i.e. 7.52 and 5.17 mg/g, respectively. Out of the Dillenia formulation which contained various compounds, oleamide showed the highest percentages of inhibition at 8.0-20.0%, and 6.2-14.2% in voltage-gated sodium channel, and voltage-gated potassium channel which had slightly lower values than the oleamide standard, and no effect as 0.0% value inhibition in the cardiac ion channel. Conclusion: The Dillenia formulation exhibits anti-inflammatory activity without affecting the heart. Accordingly, the three studied Ipomoea and three studied Dillenia species may be used for the same activity as a single component or formulation with effective solvent for disease treatments.

Background: The anticancer effects of Phyllanthus amarus extract on various cancer cells have been investigated, however, the effects of its major constituents on HCT116 human colorectal cancer cells have not been reported. Objective: In the present study, we investigated the cytotoxic effect of 80% ethanol extract of P. amarus and its marker constituents (phyllanthin, hypophyllanthin, gallic acid, niranthin, greraniin, phyltetralin, isolintetralin, corilagin and ellagic acid) on HCT116 and their underlying mechanisms of action. Methods: Their antiproliferative and apoptotic effects on HCT 116 were performed using MTT assay and flow cytometric analysis, respectively, while caspases 3/7, 8 and 9 activities were examined using the colorimetric method. The expression of cleaved poly ADP ribose polymerase enzyme (PARP) and cytochrome c proteins was investigated by the immune-blot technique. Results and Discussion: HPLC and LC-MS/MS analyses demonstrated that the extract contained mainly lignans and polyphenols. The plant samples markedly suppressed the growth and expansion of HCT116 cells in a concentration- and time-dependent manner with no toxicity against normal human fibroblast CCD18 Co. P. amarus extract, phyllanthin and gallic acid induced mode of cell death primarily through apoptosis as confirmed by the exteriorization of phosphatidylserine. Caspases 3/7, 8, and 9 activities increased in a concentration-dependent manner following 24h treatment. The expressions of cleaved PARP (Asp 214) and cytochrome c were markedly upregulated. Conclusion: P. amarus extract, phyllanthin and gallic acid exhibited an apoptotic effect on HCT116 cells through the caspases-dependent pathway.

Background: Apigenin, a natural plant flavone, has been shown to possess a variety of biological properties. Objective: In this report, a highly selective and sensitive LC-MS/MS method was developed and validated for the determination of apigenin in rat plasma. Methods: Analysts were separated on the HSS T3 column (1.8 μm 2.1×100 mm) using acetonitrile and 0.1% formic acid in 2mM ammonium acetate buffer at a supply rate of 0.200 mL/min as eluent in gradient model. Results: Plasma samples were treated by protein precipitation using acetonitrile for the recovery ranging from 86.5% to 90.1% for apigenin. The calibration curves followed linearity in the concentration range of 0.50-500ng/mL. The inter-day and intra-day precisions at different QC levels within 13.1% and the accuracies ranged from -10.6% to 8.6%. Conclusion: The assay has been successfully applied to the pharmacokinetic study of apigenin in rats.

Objectives: In order to prevent infections through dummies used during Cardiopulmonary Resuscitation (CPR) training, we analyzed the microbiological contamination on dummies used in CPR institutions. Methods: A total of 31 dummy samples were collected from 13 different institutions in Korea, and were evaluated for the number of contaminating bacteria and fungi on the surface. PCR and biochemical tests were performed to identify pathogenic bacteria and fungi, including Methicillin-Resistant Staphylococcus aureus (MRSA). Moreover, we further assessed the survival rate of microorganisms on the surface of the dummies. Results: We assessed the total number of microorganisms on the surface to be 77,752CFU/cm² (±50,047CFU), which is up to 188 times higher than the required surface contamination level. Grampositive cocci such as Micrococcus spp. and Staphylococcus spp. accounted for the highest proportion (55.3%). Especially, we detected three MRSA strains. Considering the isolated fungi and yeast, Aspergillus spp. and Candidia spp. accounted for the highest proportion. Assessing the contamination level simulation and survival rate on the humanoid surface showed that within two weeks of training, the level of contamination on the dummy’s surface exceeded the standard, and artificially contaminated pathogenic strains on the surface of the dummy survived for at least 40 days. Conclusion: To minimize the possibility of secondary infections during CPR training, there is a requirement for a standardized protocol for proper microbiological management of dummies.

Background: Diabetes Mellitus (DM) is characterized by hyperglycemia (high blood glucose levels) which is due to the destruction of insulin-producing β-cells in the islets of Langerhans in the pancreas. It is associated with oxidative and endoplasmic reticulum stress. The plant alkaloid Palmatine has been previously reported to possess antidiabetic and antioxidant properties as well as other protective properties against kidney and liver tissue damage. Objective: Here, we investigated the ability of Palmatine to reduce the up-regulation of chaperone proteins Glucose Regulatory Protein 78 (GRP78), and Calreticulin (CALR) protein in a Streptozotocin (STZ)-induced diabetic rat model. Methods: Streptozotocin (STZ) induced diabetes in Sprague Dawley rats treated with 2mg/kg of Palmatine for 12 weeks after the elevation of plasma glucose levels above 11mmol/L post-STZ administration. Proteins were extracted from the pancreas after treatment and Two-Dimensional gel electrophoresis (2-DE), PDQuest 2-D analysis software genomic solutions and mass spectrometer were used to analyze differentially expressed protein. Mass Spectrometry (MS/MS), Multidimensional Protein Identification Technology (MudPIT) was used for protein identification. Results: There was an up-regulation of the expression of chaperone proteins CALR and GRP78 and down-regulation of the expression of antioxidant and protection proteins peroxidoxin 4 (Prdx4), protein disulfide isomerase (PDIA2/3), Glutathione-S-Transferase (GSTs), and Serum Albumin (ALB) in non-diabetic rats. Palmatine treatment down-regulated the expression of chaperone proteins CALR and GRP78 and up-regulated the expression of Prdx4, PDIA2/3, GST, and ALB. Conclusion: Palmatine may have activated antioxidant proteins, which protected the cells against reactive oxygen species and endoplasmic stress. The result is in consonance with our previous report on Palmatine.

Background: The chicken eggshells and their subcrustal membranes are a valuable source of calcium, but they are not further processed but disposed of as waste from the food industry. Chicken eggshells have high content (>95%) of calcium carbonate. Some properties suggest that eggshells may be a promising alternative to the present calcium sources used in the pharmaceutical industry. Methods: The effect of roasting chicken eggshells with a selected organic acid (citric or fumaric or lactic acid) on microbiological purity, including the presence of fungi and bacteria Salmonella spp., Staphylococcus aureus, Escherichia coli of obtained calcium salts, was investigated. In this study, chicken eggshells were subjected to chemical reactions with organic acids (citric, fumaric or lactic acid) at two different calcium-acid molar ratios (1:1 or 1:3) and the mixture was heat-treated for 1 or 3 hours at a temperature of 100°C or 120°C. Results and Discussion: It was found that lactic acid was 100% effective against fungi, and the remaining citric and fumaric acids were -50% (regardless of the other examined conditions). The type of acid used has a significant effect on fungal growth inhibition (p<0.05). Fumaric acid and lactic acid will be nearly 100% effective against bacteria (100% fumaric acid and 97% lactic acid effectiveness), regardless of other factors. Conclusion: Lactic acid is the most effective against pathogenic flora - fungi and bacteria. The transformation of chicken eggshells into calcium lactate can provide us with sterile calcium salt, free of 100% fungi and 97% of all bacteria.

Background: Clinical studies indicate that recombinant tumor necrosis factor receptor:Fc fusion protein (rhTNFR:Fc) quickly alleviates symptoms and physical signs of active Ankylosing Spondylitis (AS), improving the manifestation of spinal inflammation on radiological imaging. However, the regulatory mechanism of rhTNFR:Fc in the chemokine pathway is unclear. Thus we study the mechanism of phlogogenic activity of CXCL16/CXCR6 in AS and the related mechanism of rhTNFR: Fc treatment. Methods: Thirty-two cases of active AS were treated with rhTNFR:Fc for 3 consecutive months. Clinical response was evaluated at baseline and after treatment. CXCL16/CXCR6 expression as well as Receptor Activator Of Nuclear Factor-b Ligand (RANKL)/Osteoprotegerin (OPG), essential molecules for osteoclast differentiation, were studied in AS before and after treatment. Further, the proliferation of lymphocytes and the RANKL level stimulated by recombinant human CXCL16 (rhCXCL16) were measured in vitro. Results: Thirty cases responded to rhTNFR:Fc treatment. The RANKL level, RANKL/OPG ratio, CXCLl6 level in serum, and CXCLl6 and CXCR6 mRNA levels in active AS were higher than those in controls and treated patients (P<0.001). rhCXCL16 treatment increased lymphocyte proliferation and RANKL level in active AS (P<0.001), but not in controls or treated patients (P>0.05). A positive linear correlation was noted between CXCL16 serum levels and RANKL/OPG ratio and between CXCL16 levels and C-reactive protein results (P<0.001). Conclusions: Our findings suggest that rhTNFR:Fc suppresses inflammation and bone destruction of AS by reducing the RANKL/OPG ratio through inhibition of the CXCL16/CXCR6 pathway.

Background: Salinomycin, an ionophore antibiotic, has a strong anti-cancer effect, inducing the apoptosis of cancer cells and cancer stem cells. Objective: The aim of the study was to assess the influence of salinomycin on the expression profile of genes related to stemness and miRNA regulating their expression in endometrial cancer cells. Methods: Endometrial cancer cells of cell line Ishikawa were exposed to salinomycin at concentrations in the range of 0.1-100 μM, with the aim of determining its pro-apoptotic potential and the concentration which would cause the death of 50% of the cells (Sulforhodamine B test). In the following stages, the cells were incubated with the drug at a concentration of 1μM for 12,24 and 48 hour periods and compared to the control. Determining the changes in the expression of the genes related to stemness and regulating their miRNA was done using the microarray technique and RTqPCR. ELISA assay was performed in order to determine the level of TGFβ2, COL14A1, CDH2, WNT5A in cell culture under salinomycin treatment in comparison to the control. Results: Salinomycin caused the apoptosis of cells. For the concentration of 0.1 μM, a decrease in the population of living cells by 11.9% was determined. For 1 μM, it was 49.8%, for 10 μM -69.4%, and for a concentration of 100 μM - 87.9%. The most noticeable changes in the expression caused by the addition of salinomycin into the culture were noted for mRNA: TGFβ2; WNT5A (up-regulated); COL14A1; CDH2 (down-regulated), as well as miRNA: hsa-miR-411 (up-regulated); hsa-miR-200a; hsa-miR-33a; hsa-miR-199a; hsa-miR-371-5p; hsa-miR-374; hsa-miR-374b (down-regulated). Conclusion: It was confirmed that salinomycin has an influence on the stemness process. The most noticeable changes in the expression were noted for mRNA: TGFβ2; COL14A1; CDH2; WNT5A, as well as for miRNA: hsa-miR-200a; hsa-miR-33a; hsa-miR-199a; hsa-miR-371-5p; hsa-miR-411; hsa-miR- 374a; hsa-miR-374b.