Effects of feed intake and water hardness on fluralaner pharmacokinetics in layer chickens.

BACKGROUND: Fluralaner is a novel drug belonging to the isoxazoline class that acts on external parasites of domestic animals. It is used systemically via drinking water, especially against red poultry mite in layer chickens. Fluralaner is frequently used in layers infected with D. gallinae. However, no study to date has investigated the effects of feed intake and water hardness. OBJECTIVES: This study aimed to investigate the effects of variable water hardness and feed intake on the pharmacokinetic profile of fluralaner. METHODS: Layer chickens were divided into four groups (n = 8): fed + purified water (Group 1), feed restricted + purified water (Group 2), feed restricted + hard water (Group 3), and feed restricted + soft water (Group 4). After administering a single dose of the drug with drinking water, the blood samples were collected for 21 days. Fluralaner concentrations in plasma samples were determined by liquid chromatography/tandem mass spectrometry. The maximum plasma concentration (Cmax), time to reach maximum plasma concentration (tmax), area under the concentration-time curve values (AUC0-21d), half-life (t1/2), and other pharmacokinetic parameters were calculated. RESULTS: Although the highest maximum plasma concentration (Cmax) was determined in Group 1 (fed + purified water), no statistically significant difference was found in the Cmax, tmax, t1/2, MRT0-inf_obs, Vz/Fobs, and Cl/F_obs parameters between the experimental groups. CONCLUSIONS: It was concluded that the feed intake or water hardness did not change the pharmacokinetic profile of fluralaner in layer chickens. Therefore, fluralaner could be used before or after feeding with the varying water hardness in poultry industry.

Characterization, optimization, and evaluation of preservative efficacy of carboxymethyl cellulose/hydromagnesite stromatolite bio-nanocomposite.

Currently, researchers are focusing on the development of nano-additive preservatives during the worldwide COVID-19 pandemic. This research aimed to constitute a small sized preservative nano-formulation which emerges from the biopolymer carboxymethyl cellulose (a green stabilizing agent) and hydromagnesite stromatolite (a fossilized natural additive). In this study, we investigated the optimization of the experimental design of carboxymethyl cellulose/hydromagnesite stromatolite (CMC/HS) bio-nanocomposites using a green and one-step sonochemical method at room temperature. In addition, we constructed a mathematical model which relates the intrinsic viscosity with all operating variables, and we carried out statistical error analysis to assess the validity of the proposed model. The characterization and chemical functional groups of CMC/HS bio-nanocomposites were determined by different advanced techniques such as SEM, HRTEM, DLS, FTIR, XRD, and BET. The challenge test was used to show the preservative efficacy of CMC/HS bio-nanocomposites against Staphylococcus aureus, Pseudomonas aeruginosa, Escherichia coli, Candida albicans, and Aspergillus brasiliensis. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltrazolium bromide (MTT) assay was performed on L929 cells to evaluate the in vitro cytotoxicity of CMC/HS bio-nanocomposites. According to the results, we showed that the synthesized CMC/HS bio-nanocomposites have no cytototoxic effects on L929 fibroblast cells and could be considered to be an alternative green nano-additive preservative against pathogenic microorganisms.

Effects of Tyrosine Kinase Inhibitor-masitinib Mesylate on Canine Mammary Tumour Cell Lines.

INTRODUCTION: Masitinib mesylate, a selective tyrosine kinase inhibitor of the c-KIT receptor, is used for the treatment of mast cell tumours in dogs. Masitinib has previously been investigated in various cancers; however, its potential anticancer effect in canine mammary tumours (CMTs) is unknown. In the present paper, we investigated the antiproliferative effect of masitinib in CMT cells and its possible mechanisms of action. MATERIAL AND METHODS: The effect of masitinib on the proliferation of CMT-U27 and CMT-U309 cells was assessed by MTT assay and DNA fragmentation. Flow cytometric analysis was used to measure the effect of masitinib on apoptosis and the cell cycle. Additionally, vascular endothelial growth factor levels (VEGF) were measured, and the proliferation marker Ki-67 was visualised in immunocytochemical stainings in CMT cells. RESULTS: Treatment with masitinib inhibited the proliferation of CMT cells in a concentration-dependent manner. Maximal apoptotic activity and DNA fragmentation were observed at approximately IC50 of masitinib in both cell lines. In addition, cell cycle distribution was altered and VEGF levels and Ki-67 proliferation indices were decreased in masitinib-treated cells in comparison with control cells. CONCLUSION: In this study, masitinib suppressed cell proliferation concomitantly via induction of apoptosis and cell cycle arrest by decreasing VEGF levels and the Ki-67 proliferation index in CMT-U27 and CMT-U309 cells in vitro, suggesting its potential as a therapeutic tool in the clinical setting of mammary cancer treatment in dogs.

The screening of the safety profile of polymeric based amoxicillin nanoparticles in various test systems.

Nanotechnology-based drugs show superiority over conventional medicines because of increased bioavailability, lower accumulation in non-target tissues, and improved therapeutic index with increased accumulation at target sites. However, it is important to be aware of possible problems related to the toxicity of these products, which have therapeutically superior properties. Accordingly, the present study was designed to investigate the safety profile of amoxicillin nanoparticles (AmxNPs) that we developed to increase the oral bioavailability of amoxicillin (Amx) in poultry. In the first part of the study, the genotoxicity potential of AmxNPs was evaluated using the Ames test and the in vitro comet assay. The results of Ames test showed that none of the tested concentrations of Amx and AmxNPs cause a significant increase in the revertant number of Salmonella typhimurium strains TA98, and TA100, either with or without metabolic activation. Similarly, the comet assay revealed that AmxNPs did not induce DNA damage at any of the concentrations used, whereas high-dose (200 µg/mL) of Amx caused a significant increase in the percentage of DNA in the tail. In the second part of the study, the toxicity potential of AmxNPs on broilers was investigated by measuring biochemical parameters. In vivo results demonstrated that AmxNps did not cause a significant change in biochemical parameters, whereas Amx increased ALT, glucose, and cholesterol levels at certain sampling times. The obtained findings suggest that AmxNPs could be a safe promising potential drug in drug delivery systems.

In silico and in vitro studies of two non-imidazole multiple targeting agents at histamine H3 receptors and cholinesterase enzymes.

Recently, multi-target directed ligands have been of research interest for multifactorial disorders such as Alzheimer's disease (AD). Since H3 receptors (H3 Rs) and cholinesterases are involved in pathophysiology of AD, identification of dual-acting compounds capable of improving cholinergic neurotransmission is of importance in AD pharmacotherapy. In the present study, H3 R antagonistic activity combined with anticholinesterase properties of two previously computationally identified lead compounds, that is, compound 3 (6-chloro-N-methyl-N-[3-(4-methylpiperazin-1-yl)propyl]-1H-indole-2-carboxamide) and compound 4 (7-chloro-N-[(1-methylpiperidin-3-yl)methyl]-1,2,3,4-tetrahydroisoquinoline-2-carboxamide), was tested. Moreover, molecular docking and binding free energy calculations were conducted for binding mode and affinity prediction of studied ligands toward cholinesterases. Biological evaluations revealed inhibitory activity of ligands in nanomolar (compound 3: H3 R EC50  = 0.73 nM; compound 4: H3 R EC50  = 31 nM) and micromolar values (compound 3: AChE IC50  = 9.09 µM, BuChE IC50  = 21.10 µM; compound 4: AChE IC50  = 8.40 µM, BuChE IC50  = 4.93 µM) for H3 R antagonism and cholinesterase inhibition, respectively. Binding free energies yielded good consistency with cholinesterase inhibitory profiles. The results of this study can be used for lead optimization where dual inhibitory activity on H3 R and cholinesterases is needed. Such ligands can exert their biological activity in a synergistic manner resulting in higher potency and efficacy.

In Vitro Evaluation of Antioxidant, Anti-Inflammatory, Antimicrobial and Wound Healing Potential of Thymus Sipyleus Boiss. Subsp. Rosulans (Borbas) Jalas.

Thymus sipyleus Boiss. subsp. rosulans (Borbas) Jalas (TS) is a commonly used plant in the treatment of various complaints, including skin wounds in Turkish folk medicine. Despite the widespread traditional use of TS, there is not any scientific report confirming the effectiveness of this plant on the healing process. This research aimed to investigate the effects of different extracts obtained from TS on biological events during wound healing, on a cellular basis. In this context, proliferative activities of the extracts, as well as the effects on wound closure and hydroxyproline synthesis, were determined. In addition to wound healing properties, the antioxidant, antibacterial and anti-inflammatory activities of the extracts were evaluated. Decoction (D) and infusion (I) extracts contained the highest amount of phenolic content and showed the most potent activity against DPPH radical. All extracts exhibited complete protection against the damage induced by hydrogen peroxide (H2O2) by increasing cell viability compared to only H2O2-treated groups, both in co-treatment and pre-treatment protocols. None of the extracts exhibited cytotoxic activity, and most of the extracts from the TS stimulated fibroblast proliferation and migration. All TS extracts exert anti-inflammatory activity by suppressing the overproduction of tumor necrosis factor-alpha (TNF-α) and nitric oxide (NO). The most pronounced activity on hydroxyproline synthesis was observed in D extract. In summary, it was observed that TS extracts can promote the healing process by enhancing fibroblast migration, proliferation and collagen synthesis as well as suppressing pro-inflammatory cytokines. The obtained data in this work support the traditional use of TS as a valuable plant-based compound for the treatment of wounds.

Investigation of the in vitro cytotoxic effects and wound healing activity of ternary composite substance (hollow silica sphere/gum arabic/methylene blue).

We aimed to test the adsorption of the methylene blue (MB) on HSEPCGUM as a dye and also to test the obtained ternary biocomposite substance (HSEPCGUM-MB) on wound healing. Hollow silica spheres (HSS) are used in the pharmaceutical and biochemical field, because of low toxic, highly biocompatible and mechanically stable by large surface areas. HSS was obtained by mechanochemistry method. The obtained HSS was treated with epichlorohydrin to carry out an epoxidation process (HSEPC). Then, HSEPC was functionalized by treatment with gum arabic (HSEPCGUM). MB was adsorbed onto HSEPCGUM and the adsorption maximum capacity of HSEPCGUM (Xmax) was obtained 333 mg g-1. For in vitro studies, according to the cytotoxicity test results, ternary biocomposite substance (HSEPCGUM-MB) was studied at non-cytotoxic concentrations 10, 50 and 100 µg/ml and wound closure was found as 55% (100 µg/ml) as compared to control.

Response of cultured normal canine mammary epithelial cells to deracoxib-doxorubicin combination.

Currently, there is a growing interest in combining anticancer drugs with the aim to improve outcome in patients suffering from tumours and reduce the long-term toxicity associated with the current standard of treatment. In this study, we evaluated the possible role of deracoxib against the toxicity of doxorubicin on normal canine mammary epithelial cells. The effect of deracoxib and doxorubicin combination on cell viability was determined by MTT assay. Apoptosis was characterised by flow cytometry. Cell nitrite concentrations were measured with the Griess reaction. Deracoxib (50 and 100 µM) treatment decreased the cytotoxic action of doxorubicin at 0.9 µM in the cells, from 33.63% to 13.4% and 25.82%, respectively. Our results also showed that the reverse effect of deracoxib on doxorubicin-induced cytotoxic activity in the cells was associated with a marked (3.04- to 3.57-fold) decrease in apoptosis. In additional studies identifying the mechanism of the observed effect, deracoxib exhibited an activity to prevent doxorubicin-mediated overproduction of nitric oxide in the cells. Our in vitro study results indicate that deracoxib (50 and 100 µM) can be beneficial in protecting normal cells from the toxic effect of doxorubicin in conjunction with apoptosis by the modulation of nitric oxide production.

Identification of a RON tyrosine kinase receptor binding peptide using phage display technique and computational modeling of its binding mode.

RON (Recepteur d'Origine Nantais) tyrosine kinase receptor is a promising target for therapeutic intervention in cancer therapy. The aim of this work was identification of RON-binding peptides using phage display and computational modeling their mode of binding. A 12-mer peptide phage library was utilized to perform biopanning against RON. The RON-binding ability of the selected peptide-displaying phage and their possible binding sites were examined by ELISA. Binding modes and affinities were also predicted by docking and molecular dynamics (MD) simulation. The results of ELISA experiment showed that P6 peptide displaying phage has higher affinity for RON compared to others and its binding site is located out of ligand binding site. Docking and MD simulation results also indicated higher affinity of P6 to RON as well as its exosite-binding feature. Taken together, our data suggest a capacity for P6 peptide (FEHSLYKEMTHL) to be utilized as RON binding agent, and hence be used for various purposes, including design of drug delivery systems for transferring cytotoxic agents to RON-positive cancer cells, interfering with RON signaling, peptidomimetics design, and diagnostic imaging.

Characterizing the Hot Spots Involved in RON-MSPß Complex Formation Using In Silico Alanine Scanning Mutagenesis and Molecular Dynamics Simulation.

Purpose: Implication of protein-protein interactions (PPIs) in development of many diseases such as cancer makes them attractive for therapeutic intervention and rational drug design. RON (Recepteur d'Origine Nantais) tyrosine kinase receptor has gained considerable attention as promising target in cancer therapy. The activation of RON via its ligand, macrophage stimulation protein (MSP) is the most common mechanism of activation for this receptor. The aim of the current study was to perform in silico alanine scanning mutagenesis and to calculate binding energy for prediction of hot spots in protein-protein interface between RON and MSPß chain (MSPß). Methods: In this work the residues at the interface of RON-MSPß complex were mutated to alanine and then molecular dynamics simulation was used to calculate binding free energy. Results: The results revealed that Gln193, Arg220, Glu287, Pro288, Glu289, and His424 residues from RON and Arg521, His528, Ser565, Glu658, and Arg683 from MSPß may play important roles in protein-protein interaction between RON and MSP. Conclusion: Identification of these RON hot spots is important in designing anti-RON drugs when the aim is to disrupt RON-MSP interaction. In the same way, the acquired information regarding the critical amino acids of MSPß can be used in the process of rational drug design for developing MSP antagonizing agents, the development of novel MSP mimicking peptides where inhibition of RON activation is required, and the design of experimental site directed mutagenesis studies.

Strategies of targeting the extracellular domain of RON tyrosine kinase receptor for cancer therapy and drug delivery.

PURPOSE: Cancer is one of the most important life-threatening diseases in the world. The current efforts to combat cancer are being focused on molecular-targeted therapies. The main purpose of such approaches is based on targeting cancer cell-specific molecules to minimize toxicity for the normal cells. RON (Recepteur d'Origine Nantais) tyrosine kinase receptor is one of the promising targets in cancer-targeted therapy and drug delivery. METHODS: In this review, we will summarize the available agents against extracellular domain of RON with potential antitumor activities. RESULTS: The presented antibodies and antibody drug conjugates against RON in this review showed wide spectrum of in vitro and in vivo antitumor activities promising the hope for them entering the clinical trials. CONCLUSION: Due to critical role of extracellular domain of RON in receptor activation, the development of therapeutic agents against this region could lead to fruitful outcome in cancer therapy.

In vitro effects of doxorubicin and deracoxib on oxidative-stress-related parameters in canine mammary carcinoma cells.

The present study evaluated the effects of doxorubicin (DOX) and deracoxib (DER), as single agents and in combination treatments, on antioxidant parameters in the canine mammary carcinoma cell line CMT-U27. The cells were exposed to DOX and DER for 24, 48 and 72 h. The viability and malondialdehyde (MDA), nitric oxide (NO), catalase (CAT), superoxide dismutase (SOD), glutathione peroxidase (GSHPx) and total glutathione (GSH) activities of CMT-U27 cells were determined. The half inhibition concentration (IC50) of DOX was found to be ∼0.9 µM in the 72-h period. IC50 and 1/10 IC50 concentrations of DOX were combined with all concentrations of DER (50-1000 µM) in the combination experiments. The results showed increased oxidative status associated with significant decreases of CAT and GSH levels in CMT-U27 cells exposed to 10-µM and higher concentrations of DOX compared to control cells. In contrast, there were no significant changes in the groups tested with any of the concentrations of DER (50-1000 µM). In combination treatments, DER attenuated DOX-induced oxidative damage by modulating the enzymatic and non-enzymatic components in CMT-U27 cells. We suggest that the combination of DOX and DER can be beneficial in the treatment of cancer cells by increasing cellular responses to oxidative stress. In conclusion, the use of COX inhibitor in conjunction with a chemotherapeutic agent may provide a basis for new concepts of cancer treatment through systematic modulation of the antioxidant defence systems in mammary cancers of animals.

The effects of piroxicam and deracoxib on canine mammary tumour cell line.

Cyclooxygenase (COX) inhibitors, already widely used for the treatment of pain and inflammation, are considered as promising compounds for the prevention and treatment of neoplasia. The aim of our study was to determine the direct antiproliferative effects of nonsteroidal anti-inflammatory drugs (NSAIDs), piroxicam and deracoxib, at a variety of concentrations as both single and combined treatments on canine mammary carcinoma cell line CMT-U27 and to understand the mechanisms of cell death. MTT assay was performed to determine cell viability, and flow cytometric analyses were performed to evaluate apoptosis and cell cycle alterations. Significant decrease in cell viability was observed at high concentrations of piroxicam and deracoxib in both single and combined treatments after 72 h incubation. Combined treatment produced a significantly greater inhibition than that caused by either agent alone. Also apoptotic cell number was increased by both drugs at the cytotoxic concentrations. However, concomitant treatment of cells with piroxicam and deracoxib resulted in significant induction of apoptosis at lower concentrations and accumulation of cells in the G0/G1 phase. Significant cytotoxic effects exhibited by the combination of piroxicam and deracoxib against canine mammary carcinoma cells in vitro suggest an attractive approach for the treatment of canine mammary carcinoma.