Paola Palumbo

Clinical Pharmacology of Non-Steroidal Anti-Inflammatory Drugs: A Review

Non-steroidal anti-inflammatory drugs (NSAIDs) are a group of often chemically unrelated compounds with some common therapeutic actions and side effects. They have potent anti-inflammatory, analgesic and antipyretic activity, and are among the most widely used drugs worldwide. It is generally thought that one of their main mechanisms of action is the inhibition of cyclo-oxygenase (COX), the enzyme responsible for biosynthesing the prostaglandins and thromboxane. NSAIDs are also associated with an increased risk of adverse gastrointestinal, renal and cardiovascular effects. This review describes the clinical pharmacology of NSAIDs, their classification, molecular mechanisms of action and adverse effects, including their possible contribution to neuro-inflammation and carcinogenesis, as well as some recent developments aimed at designing effective anti-inflammatory agents with improved safety and tolerability profiles.

Biological effects of low frequency high intensity ultrasound application on ex vivo human adipose tissue.

In the present work the effects of a new low frequency, high intensity ultrasound technology on human adipose tissue ex vivo were studied. In particular, we investigated the effects of both external and surgical ultrasound-irradiation (10 min) by evaluating, other than sample weight loss and fat release, also histological architecture alteration as well apoptosis induction. The influence of saline buffer tissue-infiltration on the effects of ultrasound irradiation was also examined. The results suggest that, in our experimental conditions, both transcutaneous and surgical ultrasound exposure caused a significant weight loss and fat release. This effect was more relevant when the ultrasound intensity was set at 100% (∼ 2.5 W/cm2 for external device; ∼19–21 W/cm2, for surgical device) compared to 70% (∼ 1.8 W/cm2 for external device; ∼13–14 W/cm2 for surgical device). Of note, the effectiveness of ultrasound was much higher when the tissue samples were previously infiltrated with saline buffer, in accordance with the knowledge that ultrasonic waves in aqueous solution better propagate with a consequently more efficient cavitation process. Moreover, the overall effects of ultrasound irradiation did not appear immediately after treatment but persisted over time, being significantly more relevant at 18 h from the end of ultrasound irradiation. Evaluation of histological characteristics of ultrasound-irradiated samples showed a clear alteration of adipose tissue architecture as well a prominent destruction of collagen fibers which were dependent on ultrasound intensity and most relevant in saline buffer-infiltrated samples. The structural changes of collagen bundles present between the lobules of fat cells were confirmed through scanning electron microscopy (SEM) which clearly demonstrated how ultrasound exposure induced a drastic reduction in the compactness of the adipose connective tissue and an irregular arrangement of the fibers with a consequent alteration in the spatial architecture. The analysis of the composition of lipids in the fat released from adipose tissue after ultrasound treatment with surgical device showed, in agreement with the level of adipocyte damage, a significant increase mainly of triglycerides and cholesterol. Finally, ultrasound exposure had been shown to induce apoptosis as shown by the appearance DNA fragmentation. Accordingly, ultrasound treatment led to down-modulation of procaspase-9 expression and an increased level of caspase-3 active form.

Production Conditions Affect the In Vitro Anti-Tumoral Effects of a High Concentration Multi-Strain Probiotic Preparation.

A careful selection of the probiotic agent, standardization of the dose and detailed characterization of the beneficial effects are essential when considering use of a probiotic for the dietary management of serious diseases. However, changes in the manufacturing processes, equipment or facilities can result in differences in the product itself due to the live nature of probiotics. The need to reconfirm safety and/or efficacy for any probiotic product made at a different factory is therefore mandatory. Recently, under the brand VSL#3®, a formulation produced by a manufacturer different from the previous one, has been commercialized in some European countries (the UK and Holland). VSL#3 is a high concentration multi-strain preparation which has been recognized by the main Gastroenterology Associations for the dietary management of pouchitis as well as ulcerative colitis. We have compared the “original” VSL#3 produced in USA with the “newfound” VSL#3 produced in Italy. According to our results, the “newfound” VSL#3 has 130–150% more “dead bacteria” compared to the “original” product, raising concerns for the well-known association between dead microbes with adverse effects. The abilities of bacterial lysates from the two formulations to influence in vitro viability and proliferation of different tumor cell lines also resulted different. The repair of previously scratched monolayers of various adherent tumor cell lines (i.e. HT1080, and Caco-2 cells) was inhibited more significantly by the “original” VSL#3 when compared to the “newfound” VSL#3. Tumor cell cycle profile, in particular cell cycle arrest and apoptotic death of the cancer cells, further confirms that the “original” VSL#3 has a better functional profile than the “newfound” VSL#3, at least in in vitro. Our data stress the importance of the production conditions for the “newfound” VSL#3 considering that this product is intended to be used for the dietary management of patients with very serious diseases, such as chronic inflammatory bowel diseases.

Development of a method for the determination of vardenafil in human plasma by high performance liquid chromatography with UV detection

A simple high-performance liquid chromatographic (HPLC) method with photometric detection is described for the determination of vardenafil hydrochloride, a phosphodiesterase V inhibitor, in human plasma. Chromatographic separation of the analyte and internal standard was achieved on an analytical 250 x 4.6 mm i.d. reversed-phase Kromasil KR 100 C(18) (5 microm particle size) column using a mobile phase of acetonitrile-potassium dihydrogen phosphate (30:70 v/v). The run time was less than 15 min. Column eluate was monitored at 230 nm. The linearity over the concentration range of 10-1500 ng/mL for vardenafil was obtained and the limit of quantification (LOQ) was 10 ng/mL. The method has been applied to analysis of the vardenafil concentrations for application in pharmacokinetic studies.

Bioanalytical method development for quantification of ulifloxacin, fenbufen and felbinac in rat plasma by solid-phase extraction (SPE) and HPLC with PDA detection

A procedure based on solid-phase extraction (SPE) followed by high performance liquid chromatography (HPLC) with PDA detection has been developed for the analysis of multiple drugs in rat plasma. The analytes evaluated were ulifloxacin, fenbufen and felbinac. Eight different solid phase extraction cartridges were tested to evaluate their applicability for the isolation of drugs from rat plasma. Comparison were recovery of different drugs and reproducibility. The samples were analyzed by HPLC using a Kinetex C18 EVO column and acetonitrile-10mM ammonium acetate-methanol as the mobile phase under gradient elution conditions. SPE combined with HPLC-PDA allowed the determination of drugs over a linear range of 0.05-15 μg/mL for ulifloxacin while 0.5-50 μg/mL for felbinac and fenbufen, with limit of detection at 0.05 for ulifloxacin and 0.5 for felbinac and fenbufen. Bond Elut Plexa sorbent was found to provide the most effective clean-up, removing the greatest amount of interfering substance and simultaneously ensuring analyte recoveries higher than 93.54% with relative standard deviation (RSD) <10%. The method was applied with good accuracy and precision in the determination of ulifloxacin, fenbufen and felbinac in rat plasma obtained from rats treated with selected drugs. This method permits its application to pharmacokinetic and pharmacodynamic studies of these analytes and will facilitate detailed investigations on the interactions between new fluoroquinolones and fenbufen.

Immunophenotypic Characterization of Human Glioblastoma Stem Cells: Correlation With Clinical Outcome

Recently, glioma stem cells have been identified as the main cause of glioma propagation and recurrence and a number of several cell markers have been indicated as putative GSC markers. In the present work, a retrospective study to evaluate the prognostic potential of ability to generate GSCs in our series of 15 glioblastoma patients is described. β‐tubulin III, nestin, CD133, GFAP, and SOX‐2 marker expression, both in primary GBM cultures and in respective glioblastoma stem cells (GSCs), was evaluated by flow cytometric analysis. Our results demonstrated various expression levels of these markers in both cell cultures; of note, only those cells expressing SOX‐2 at greater than 30% levels were able to produce in vitro neurospheres. Moreover, statistical analysis revealed that the GSCs generation negatively affected overall survival (OS) (P = 0.000) and progression‐free survival (PFS) (P = 0.001). In addition, a very poor OS (P = 0.000) and PFS (P = 0.000) were observed among patients whose tumors expressed Ki67, evaluated by immunohistochemistry, and showed the ability to generate in vitro GSCs. Overall, the results suggest that in vitro GSCs generation associated to the expression of Ki67 and SOX‐2 may be useful to identify patients at risk of disease progression. J. Cell. Biochem. 116: 864–876, 2015.

Involvement of cPLA2 Inhibition in Dexamethasone-Induced Thymocyte Apoptosis

Various molecular mechanisms have been suggested to be involved in dexamethasone induced thymocyte apoptosis. In this study we show that pharmacological inhibition of cytoplasmic PLA2 in mouse thymocytes for 18 h with arachidonyl trifluoromethyl ketone (AACOCF3) (10 μM) and palmitoyl trifluoromethyl ketone (PACOCF3) (10 μM) induced a drastic increase of thymocyte apoptosis comparable to that observed following Dex (10−7 M) treatment, while inhibition of secretory PLA2 with p-bromophenacyl bromide (pBPB) (20 μM) did not. AACOCF3-induced thymocyte apoptosis, similarly to Dex-induced thymocyte apoptosis, was eliminated by cell pre-treatment with the PI-PLCβ inhibitor, U73122, but not by the PC-PLC inhibitor D609. These observations were corroborated by the ability of AACOCF3, like Dex, to induce a rapid and transient increase in DAG generation. In addition, AACOCF3-induced apoptosis involved the activation of the acidic sphingomyelinase (aSMase) but not of the neutral sphingomyelinase (nSMase), as evaluated by measurements of enzyme activity in cell extracts following thymocyte exposure to AACOCF3 and by the ability of monensin to inhibit AACOCF3-induced thymocyte apoptosis. In addition, the AACOCF3 apoptotic effect resulted in an early increase of ceramide levels. AACOCF3-induced thymocyte apoptosis involved the activation of caspase 3, and cell pre-treatment with a caspase 3 inhibitor prevented AACOCF3-induced apoptosis. These observations suggest that cPLA2 inhibition may have a role in Dex-induced thymocyte apoptosis and highlight the importance of cPLA2 activity in thymocyte survival.

In Vitro Evaluation of Different Methods of Handling Human Liposuction Aspirate and Their Effect on Adipocytes and Adipose Derived Stem Cells

Nowadays, fat tissue transplantation is widely used in regenerative and reconstructive surgery. However, a shared method of lipoaspirate handling for ensuring a good quality fat transplant has not yet been established. The study was to identify a method to recover from the lipoaspirate samples the highest number of human viable adipose tissue‐derived stem cells (hADSCs) included in stromal vascular fraction (SVF) cells and of adipocytes suitable for transplantation, avoiding an extreme handling. We compared the lipoaspirate spontaneous stratification (10‐20‐30 min) with the centrifugation technique at different speeds (90‐400‐1500 × g). After each procedure, lipoaspirate was separated into top oily lipid layer, liquid fraction, “middle layer”, and bottom layer. We assessed the number of both adipocytes in the middle layer and SVF cells in all layers. The histology of middle layer and the surface phenotype of SVF cells by stemness markers (CD105+, CD90+, CD45−) was analyzed as well. The results showed a normal architecture in all conditions except for samples centrifuged at 1500 × g. In both methods, the flow cytometry analysis showed that greater number of ADSCs was in middle layer; in the fluid portion and in bottom layer was not revealed significant expression levels of stemness markers. Our findings indicate that spontaneous stratification at 20 min and centrifugation at 400 × g are efficient approaches to obtain highly viable ADSCs cells and adipocytes, ensuring a good thickness of lipoaspirate for autologous fat transfer. Since an important aspect of surgery practice consists of gain time, the 400 × g centrifugation could be the recommended method when the necessary instrumentation is available. J. Cell. Physiol. 230: 1974–1981, 2015.

Development and validation of a MEPS-UHPLC-PDA method for determination of ulifloxacin in human plasma and urine of patients with peripheral arterial disease.

A novel sensitive analytical method based on the use of a semi-automatic microextraction by packed sorbents (MEPS) techniques combined with ultra high-performance liquid chromatography (UHPLC) with PDA detection has been developed and validate for the analysis of ulifloxacin, the active metabolite of prulifloxacin using danofloxacin as internal standard in human plasma and urine. Different experimental parameters were optimized and validated according to international guidelines. Complete separation of the analytes was achieved with a Waters BEH C18 (50×2.1mm I.D., 1.7μm particle size) analytical column, a mixture of 10mM ammonium acetate (pH 3.0) (A) with and acetonitrile (B) both containing 1% triethylamine were used as mobile phase, at a flow rate of 0.6mL/min in gradient elution, and detection wavelength of 272nm. This method is linear in concentration range of 0.02-10.0μg/mL for plasma and urine, respectively. The limit of quantitation was 20ng/mL for the two fluids. The recoveries of the method were 95% for ulifloxacin in human plasma and urine and 95.5% for the internal standard. Intra- and inter- assay precision and accuracy for ulifloxacin were lower than 10% at all tested concentrations. The proposed method was successfully applied to measure plasma and urine concentrations of ulifloxacin in patients suffering from Peripheral Arterial Disease and for pharmacokinetics study.

VSL#3 Probiotic Differently Influence IEC-6 Intestinal Epithelial Cell Status and Function

The data here reported introduce the wound‐healing assay as a tool for testing probiotics aimed at protecting gastrointestinal mucosal surfaces and to verify the consistency of their manufacturing. At the scope, we compared the in vitro effects of two multi‐strain high concentration formulations both commercialized under the same brand VSL#3 but sourced from different production sites (USA and Italy) on a non‐transformed small‐intestinal epithelial cell line, IEC‐6. The effects on cellular morphology, viability, migration, and H2O2‐induced damage, were assessed before and after the treatment with both VSL#3 formulations. While the USA‐sourced product (“USA‐made”) VSL#3 did not affect monolayer morphology and cellular density, the addition of bacteria from the Italy‐derived product (“Italy‐made”) VSL#3 caused clear morphological cell damage and strongly reduced cellularity. The treatment with “USA‐made” lysate led to a higher rate of wounded monolayer healing, while the addition of “Italy‐made” bacterial lysate did not influence the closure rate as compared to untreated cells. While lysates from “USA‐made” VSL#3 clearly enhanced the formation of elongated and aligned stress fibers, “Italy‐made” lysates had not similar effect. “USA‐made” lysate was able to cause a total inhibition of H2O2‐induced cytotoxic effect whereas “Italy‐made” VSL#3 lysate was unable to protect IEC‐6 cells from H2O2‐induced damage. ROS generation was also differently influenced, thus supporting the hypotesis of a protective action of “USA‐made” VSL#3 lysates, as well as the idea that “Italy‐made” formulation was unable to prevent significantly the H2O2‐induced oxidative stress.