The Use of the QbD Approach to Optimize the Co-Loading of Simvastatin and Doxorubicin in Liposomes for a Synergistic Anticancer Effect.

The present study aimed to optimize a liposomal formulation co-encapsulating simvastatin (SIM) and doxorubicin (DOX) that has future perspectives in anticancer therapy. The optimization process was performed by implementing the Quality by Design concept and by considering the results of a previous screening study. Failure Mode and Effects Analysis was used for the identification of the potential critical factors, i.e., phospholipid, SIM and DOX concentration, which were assessed in an optimization experimental design with the purpose of designing an optimal formulation. The optimal formulation, meeting the established quality profile, was additionally characterized in terms of the release profile and antiproliferative effects. During dissolution studies, a novel chronoamperometric method was used for the simultaneous quantification of SIM and DOX. The obtained data confirmed the similarity of this method with a validated HPLC method. The anticancer potential of the optimal formulation was tested against two human cancerous cell lines, namely T47D-KBluc human mammary ductal carcinoma cell line and A549 human pulmonary cancer cell line. The results highlighted that the antiproliferative effect of the optimal formulation is concentration dependent and favors a synergistic effect of the two drugs.

Enhanced Recovery of Phenolic and Tocopherolic Compounds from Walnut (Juglans Regia L.) Male Flowers Based on Process Optimization of Ultrasonic Assisted-Extraction: Phytochemical Profile and Biological Activities.

The extraction of bioactive compounds present in walnut (Juglans regia L.) male flowers (WMFs) was performed based on an experimental design using ultrasonic-assisted extraction. Solvent nature, extraction time, and water content were selected as experimental variables, and phenolic, flavonoidic, and condensed tannins contents and antioxidant properties were evaluated. Acetone was the solvent with the highest extraction performance, with the extracts obtained using this solvent displaying an increased concentration of bioactive compounds and increased antioxidant activities. For several extracts with high bioactive content, individual polyphenolic and tocopherolic compounds were evaluated by means of LC-MS and LC-MS/MS. The best extraction conditions for polyphenolic (2.86 mg gallic acid equivalents/g WMF) and tocopherolic compounds (29.4 µg/g WMF) were acetone with 40% water content (N20) and acetone with 20% water content (N15), respectively. Although the total tocopherol concentrations were lower than in other Juglans regia parts, most of the total tocopherol quantity was provided by the highly biologically active δ-tocopherol (84%). Significant quantities of quercetin (101.9 µg/g), hyperoside (2662.9 µg/g), quercitrin (405.7 µg/g), and isoquercitrin (1293.7 µg/g) were determined in WMF (N20). Both extracts inhibited the enzymatic activity of α-glucosidase and tyrosinase; however, an increased inhibition was observed for N20, the extract with the higher polyphenolic content. Conversely, N15 had higher anticancerous activity on the cell lines used, with a moderate selectivity towards the cancerous phenotype being observed for both extracts. At non-cytotoxic concentrations, both extracts displayed good antioxidant activities in cellular cultures, decreasing basal and H2O2-induced oxidative stress. This is the first characterization of both hydrophilic and lipophilic phytochemicals in WMF extracts. The outcomes of our study reveal that walnut male flowers have strong biological activities, thus justifying further research to demonstrate their usefulness in the food, pharmaceutical, and/or cosmetic industries.

Beneficial effects of the novel marine oxygen carrier M101 during cold preservation of rat and human pancreas.

Ischaemia impairs organ quality during preservation in a time-dependent manner, due to a lack of oxygen supply. Its impact on pancreas and islet transplantation outcome has been demonstrated by a correlation between cold ischaemia time and poor islet isolation efficiency. Our goal in the present study was to improve pancreas and islet quality using a novel natural oxygen carrier (M101, 2 g/L), which has been proven safe and efficient in other clinical applications, including kidney transplantation, and for several pre-clinical transplantation models. When M101 was added to the preservation solution of rat pancreas during ischaemia, a decrease in oxidative stress (ROS), necrosis (HMGB1), and cellular stress pathway (p38 MAPK)activity was observed. Freshly isolated islets had improved function when M101 was injected in the pancreas. Additionally, human pancreases exposed to M101 for 3 hours had an increase in complex 1 mitochondrial activity, as well as activation of AKT activity, a cell survival marker. Insulin secretion was also up-regulated for isolated islets. In summary, these results demonstrate a positive effect of the oxygen carrier M101 on rat and human pancreas during preservation, with an overall improvement in post-isolation islet quality.

Insights on the Role of Putative Muscle-Derived Factors on Pancreatic Beta Cell Function.

Skeletal muscle is a main target of insulin action that plays a pivotal role in postprandial glucose disposal. Importantly, skeletal muscle insulin sensitivity relates inversely with pancreatic insulin secretion, which prompted the hypothesis of the existence of a skeletal muscle-pancreas crosstalk mediated through an endocrine factor. The observation that changes in skeletal muscle glucose metabolism are accompanied by altered insulin secretion supports this hypothesis. Meanwhile, a muscle-derived circulating factor affecting in vivo insulin secretion remains elusive. This factor may correspond to peptides/proteins (so called myokines), exosomes and their cargo, and metabolites. We hereby review the most remarkable evidence encouraging the possibility of such inter-organ communication, with special focus on muscle-derived factors that may potentially mediate such skeletal muscle-pancreas crosstalk.

Estrogenic and anti-estrogenic activity of butylparaben, butylated hydroxyanisole, butylated hydroxytoluene and propyl gallate and their binary mixtures on two estrogen responsive cell lines (T47D-Kbluc, MCF-7).

The estrogenic and anti-estrogenic effects of butylparaben (BuPB), butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT) and propyl gallate (PG) were evaluated for individual compounds as well as for binary mixtures, using an estrogen-dependent reporter gene assay in T47D-Kbluc breast cancer cells and an estrogen-dependent proliferation assay in MCF-7 breast cancer cells. In terms of estrogenicity the potency of the selected compounds increased from BHA < PG < BuPB in the luciferase assay (with BHT showing no significant estrogenic activity), while in the proliferation assay the following order was observed: BHT < BHA < BuPB (with PG showing no significant estrogenic activity). Non-monotonic dose-response curves were obtained for BuPB (in both assays) and PG (in the luciferase assay), respectively. In the presence of estradiol, a significant anti-estrogenic activity was observed in both cell lines for PG, BuPB and BHA, while BHT showed weak anti-estrogenic activity only in T47D-Kbluc cells. The evaluation of binary mixtures confirmed the endocrine disruptive potential of the compounds, their individual potency being correlated with that of the mixtures. All mixtures were able to reduce the estradiol-induced luminescence or cell proliferation, an effect that was accurately predicted by the dose addition mathematical model, suggesting the same (or at least partially overlapping) modes of action for the tested compounds. The results of the present study emphasize the importance of a cumulative risk assessment of endocrine disruptors.

Individual and combined in vitro (anti)androgenic effects of certain food additives and cosmetic preservatives.

The individual and combined (binary mixtures) (anti)androgenic effect of butylparaben (BuPB), butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT) and propyl gallate (PG) was evaluated using the MDA-kb2 cell line. Exposing these cells to AR agonists results in the expression of the reporter gene (encoding for luciferase) and luminescence can be measured in order to monitor the activity of the reporter protein. In case of the evaluation of the anti-androgenic effect, the individual test compounds or binary mixtures were tested in the presence of a fixed concentration of a strong AR agonist (1000 pM 5-alpha-dihydrotestosterone; DHT). Cell viability was assessed using a resazurin based assay. For PG, this is the first report in the literature concerning its (anti)androgenic activity. In case of both individual and mixture testing none of the compounds or binary combinations showed androgenic activity. When tested in the presence of DHT, BuPB, BHA and BHT proved to be weak anti-androgens and this was confirmed during the evaluation of binary mixtures (BuPB+BHA, BuPB+BHT and BHA+BHT). Besides performing the in vitro testing of the binary combinations, two mathematical models (dose addition and response addition) were evaluated in terms of accuracy of prediction of the anti-androgenic effect of the selected binary mixtures. The dose addition model guaranteed a good correlation between the experimental and predicted data. However, no estimation was possible in case of mixtures containing PG, due to the lack of effect of the compound in case of the individual testing.

Estrogenic/antiestrogenic activity of selected selective serotonin reuptake inhibitors.

BACKGROUND AND AIMS: Selective serotonin reuptake inhibitors (SSRIs) are one of the most prescribed classes of psychotropics. Even though the SSRI class consists of 6 molecules (citalopram, escitalopram, fluoxetine, fluvoxamine, paroxetine and sertraline), only fluoxetine was intensively studied for endocrine disruptive effects, while the other SSRIs received less attention. This study was designed to evaluate the estrogenic/antiestrogenic effect of fluoxetine, sertraline and paroxetine. METHODS: The in vitro (anti)estrogenic activity was assessed using a firefly luciferase reporter construct in the T47D-KBluc breast cancer cell line. These cells express nuclear estrogen receptors that can activate the transcription of the luciferase reporter gene upon binding of estrogen receptor agonists. RESULTS: All three compounds were found to interact with the estrogen receptor. Fluoxetine had dual properties, weak estrogenic at lower concentrations and antiestrogenic effect at higher concentrations. Sertraline shared the same properties with fluoxetine, but also increased the estradiol-mediated transcriptional activity. Paroxetine presented only one type of effect, the ability to increase the estradiol-mediated transcriptional activity. CONCLUSIONS: Overall, our results indicate a possible interaction of SSRIs with the estrogen receptor. As SSRIs are being used by all categories of population, including pregnant women or children, establishing whether they can affect the endocrine mediated mechanisms should be a priority.

In vitro modulation of estrogen receptor activity by norfluoxetine.

BACKGROUND AND AIMS: Selective serotonin reuptake inhibitors (SSRIs) are antidepressants increasingly prescribed for pregnancy and postpartum depression. However, these compounds can cross the placenta and also pass into breast milk, thus reaching the fetus and infant during critical developmental stages, potentially causing adverse effects. Fluoxetine, a widely used SSRI, has been shown to affect (neuro)endocrine signaling in various organisms, including humans. This compound can also interact with estrogen receptors in vitro and cause an estrogen-dependent uterotrophic response in rodents. Consequently, the aim of the present study was to assess if the active metabolite of fluoxetine, namely norfluoxetine (NFLX), shares the same capacity for estrogen receptor interaction. METHODS: The in vitro (anti)estrogenic activity of norfluoxetine was assessed using a firefly luciferase reporter construct in the T47D-Kbluc breast cancer cell line. These cells express nuclear estrogen receptors (ERs) that can activate the transcription of the luciferase reporter gene upon binding of ER agonists. Light emission was monitored in case of cells exposed to norfluoxetine or mixtures of norfluoxetine-estradiol. Cell viability was assessed using a resazurin-based assay. RESULTS: During individual testing, NFLX was able to induce a significant increase in luciferase activity compared to control, but only at the highest concentration tested (10 µM). In binary mixtures with estradiol (30 pM constant concentration) a significant increase in luminescence was observed at low submicromolar norfluoxetine concentrations compared to estradiol alone. CONCLUSION: Norfluoxetine can induce estrogenic effects in vitro and can potentiate the activity of estradiol. However, further studies are needed to clarify if these observed estrogenic effects may have detrimental consequences for human exposure.