Near-Infrared Transflectance Spectroscopy Discriminates Solutions Containing Two Commercial Formulations of Botulinum Toxin Type A Diluted at Recommended Volumes for Clinical Reconstitution.

Botulinum neurotoxin type A (BoNT-A) is the active substance in pharmaceutical preparations widely used worldwide for the highly effective treatment of various disorders. Among the three commercial formulations of BoNT-A currently available in Italy for neurological indications, abobotulinum A toxin (Dysport®, Ipsen SpA, Milano, Italy) and incobotulinum A toxin (Xeomin®, Merz Pharma Italia srl, Milano, Italy) differ in the content of neurotoxin, non-toxic protein, and excipients. Clinical applications of BoNT-A adopt extremely diluted solutions (10-6 mg/mL) for injection in the target body district. Near-infrared spectroscopy (NIRS) and chemometrics allow rapid, non-invasive, and non-destructive methods for qualitative and quantitative analysis. No data are available to date on the chemometric analysis of the spectral fingerprints acquired from the diluted commercial formulations of BoNT-A. In this proof-of-concept study, we tested whether NIRS can categorize solutions of incobotulinum A toxin (lacking non-toxic proteins) and abobotulinum A toxin (containing non-toxic proteins). Distinct excipients in the two formulations were also analyzed. We acquired transmittance spectra in the visible and short-wave infrared regions (350-2500 nm) by an ASD FieldSpec 4™ Standard-Res Spectrophotoradiometer, using a submerged dip probe designed to read spectra in transflectance mode from liquid samples. After preliminary spectra pre-processing, principal component analysis was applied to characterize the spectral features of the two BoNT-A solutions and those of the various excipients diluted according to clinical standards. Partial least squares-discriminant analysis was used to implement a classification model able to discriminate the BoNT-A solutions and excipients. NIRS distinguished solutions containing distinct BoNT-A commercial formulations (abobotulinum A toxin vs. incobotulinum A toxin) diluted at recommended volumes for clinical reconstitution, distinct proteins (HSA vs. incobotulinum A toxin), very diluted solutions of simple sugars (lactose vs. sucrose), and saline or water. Predictive models of botulinum toxin formulations were also performed with the highest precision and accuracy.

Pharmacokinetic properties of a novel formulation of S-adenosyl-L-methionine phytate.

S-adenosyl-L-methionine (SAM), the main endogenous methyl donor, is the adenosyl derivative of the amino acid methionine, which displays many important roles in cellular metabolism. It is widely used as a food supplement and in some countries is also marketed as a drug. Its interesting nutraceutical and pharmacological properties prompted us to evaluate the pharmacokinetics of a new form of SAM, the phytate salt. The product was administered orally to rats and pharmacokinetic parameters were evaluated by comparing the results with that obtained by administering the SAM tosylated form (SAM PTS). It was found that phytate anion protects SAM from degradation, probably because of steric hindrance exerted by the counterion, and that the SAM phytate displayed significant better pharmacokinetic parameters compared to SAM PTS. These results open to the perspective of the use of new salts of SAM endowed with better pharmacokinetic properties.

Extra-virgin olive oil phenols block cell cycle progression and modulate chemotherapeutic toxicity in bladder cancer cells.

Epidemiological data indicate that the daily consumption of extra­virgin olive oil (EVOO), a common dietary habit of the Mediterranean area, lowers the incidence of certain types of cancer, in particular bladder neoplasm. The aim of the present study was to evaluate the antiproliferative activity of polyphenols extracted from EVOO on bladder cancer (BCa), and to clarify the biological mechanisms that trigger cell death. Furthermore, we also evaluated the ability of low doses of extra­virgin olive oil extract (EVOOE) to modulate the in vitro activity of paclitaxel or mitomycin, two antineoplastic drugs used in the management of different types of cancer. Our results showed that EVOOE significantly inhibited the proliferation and clonogenic ability of T24 and 5637 BCa cells in a dose­dependent manner. Furthermore, cell cycle analysis after EVOOE treatment showed a marked growth arrest prior to mitosis in the G2/M phase for both cell lines, with the subsequent induction of apoptosis only in the T24 cells. Notably, simultaneous treatment of mitomycin C and EVOOE reduced the drug cytotoxicity due to inhibition of ROS production. Conversely, the co­treatment of T24 cells with paclitaxel and the polyphenol extract strongly increased the apoptotic cell death at each tested concentration compared to paclitaxel alone. Our results support the epidemiological evidence indicating that olive oil consumption exerts health benefits and may represent a starting point for the development of new anticancer strategies.

Evaluation of different extraction methods from pomegranate whole fruit or peels and the antioxidant and antiproliferative activity of the polyphenolic fraction.

Pomegranate is a functional food of great interest, due to its multiple beneficial effects on human health. This fruit is rich in anthocyanins and ellagitannins, which exert a protective role towards degenerative diseases. The aim of the present work was to optimize the extraction procedure, from different parts of the fruit, to obtain extracts enriched in selected polyphenols while retaining biological activity. Whole fruits or peels of pomegranate cultivars, with different geographic origin, were subjected to several extraction methods. The obtained extracts were analyzed for polyphenolic content, evaluated for antioxidant capacity and tested for antiproliferative activity on human bladder cancer T24 cells. Two different extraction procedures, employing ethyl acetate as a solvent, were useful in obtaining extracts enriched in ellagic acid and/or punicalagins. Antioxidative and antiproliferative assays demonstrated that the antioxidant capability is directly related to the phenolic content, whereas the antiproliferative activity is to be mainly attributed to ellagic acid.

GMP-grade platelet lysate enhances proliferation and migration of tenon fibroblasts.

Tenon's fibroblasts (TFs), widely employed as in vitro model for many ophthalmological studies, are routinely cultured with FBS. Platelet Lysate (PL), a hemoderivate enriched with growth factors and cytokines has been largely tested in several clinical applications and as substitute of FBS in culture. Here, we investigate whether PL can exert biological effects on TF populations similarly to other cell types. Results show that PL significantly enhances cell proliferation and migration vs. FBS, without influencing cell size/granularity. Upregulation of EGF, VEGF, KDR, MMP2-9, FAK mRNA levels also occurs and phosphorylation of AKT but not of ERK1/2 is significantly enhanced. The inhibition of the PI3kinase/AKT pathway with the specific inhibitor wortmannin, decreases PL-induced cell migration but not proliferation. Condition supernatants containing PL show increased bioavailability of Nitric Oxide and reduced levels of 8-Iso-PGF2-alpha, correlating with cell proliferation and migration. Pro-angiogenic/inflammatory soluble factors (GRO, Angiogenin, EGF, I-309, PARC) are exclusively or greater expressed in media containing PL than FBS. GMP-grade PL preparations positively influence in vitro biological effects of TFs representing a suitable and safer alternative to FBS.

Evaluation of the management of Hr-HPV+/PapTest- women: results at 1-year recall.

With cervical cancer screening the choice of 1-year as a period of follow-up in positive high-risk HPV women without cytological lesions is still under discussion. We evaluated the management of these women and the role of HPV genotyping test. We did a cervical cancer screening study of women aged 35-64 with primary high-risk HPV test. Women positive for high-risk HPV with negative cytology were followed-up after 1 year. In this study we selected women with high-risk HPV+/PapTest- resulted high-risk HPV+ at recall and performed the PapTest and HPV genotyping test. The detection rate of squamous high grade (CIN2+) relative to the total screened cohort was 2.1‰, and it was 0.2‰ at the 1-year recall. The colposcopy performed in women referred at the 1-year recall accounted for 48.8% of the total (baseline + 1-year recall), and 84.3% of these women had no cytological lesions. The most frequent hr-HPV genotype detected was HPV16 and 66.7% of co-infections were due to HPV16 and HPV18. 54.5% of women presented a persistent infection at 1-year recall with the same HPV subtype, 50% of persistent infections was due to HPV16 and 16.7% of these were determined to be CIN2+ histological lesions. Our data show that it may be useful to extend the period of follow-up for women hr-HPV+/PapTest- so as to reduce the number of unnecessary colposcopies due to the transitory infections and that the genotyping test could help to identify the persistent infections in which HPV16 is involved.

Extra virgin olive oil phenols suppress migration and invasion of T24 human bladder cancer cells through modulation of matrix metalloproteinase-2.

The consumption of extra virgin olive oil (EVOO), a common dietary habit of the Mediterranean people, seems to be related to a lower incidence of certain types of cancer including bladder neoplasm. Metastases are the major cause of bladder cancer-related deaths and targeting cell motility has been proposed as a therapeutic strategy to prevent cancer spread. This study aimed to investigate the potential antimetastatic effect of total phenols extracted from EVOO against the human transitional bladder carcinoma cell line T24. We also aimed at verifying that EVOO extract exerts cytotoxic effect on tumor cells without affecting normal urothelial fibroblasts. Our results show that EVOO extract can significantly inhibit the proliferation and motility of T24 bladder cells in a dose-dependent manner. In the same experimental conditions fibroblast proliferation and motility were not significantly modified. Furthermore the enzymatic activity of MMP-2 was inhibited at nontoxic EVOO extract doses only in T24 cells. The qRT-PCR revealed a decrease of the MMP-2 expression and a simultaneous increase of the tissue inhibitors of metalloproteinases expression. Our results may support the epidemiological evidences that link olive oil consumption to health benefits and may represent a starting point for the development of new anticancer strategies.

Chromane derivatives of small aromatic molecules: Chemoenzymatic synthesis and growth inhibitory activity on human tumor cell line LoVo WT.

Aromatic substrates tyrosol (p-hydroxyphenylethanol) and 2,6-dihydroxynaphthalene (2,6-DHN) were converted into chromane derivatives by means of chemoenzymatic reactions catalyzed by the aromatic prenyltransferase of bacterial origin NovQ, using dimethylallyl bromide as allylic substrate instead of the natural isoprenyl pyrophosphate substrate. Stereoselective prenylation occurred in o-position with respect to the phenol hydroxyl in both compounds. Prenylated derivatives were readily converted into chromane products via a selective 6-endo-trig cyclization involving the oxygen atom from the phenol moiety and the double bond of the prenyl substituent, a process catalyzed by FeCl(3). These findings set up the basis of a most convenient two-step, one-pot process which allows for easy recovery of the chromane products in high yields. The chromane derivatives thus obtained were tested for cytotoxicity and pro-apoptotic activity using LoVo WT cells, a line of human colon adenocarcinoma.

Characterization of catechol-thioether-induced apoptosis in human SH-SY5Y neuroblastoma cells.

Recent work has highlighted the involvement of a dopamine derivative, 5-S-cysteinyl-dopamine (CysDA), in neurodegeneration and apoptotic cell death. In this paper we study in further detail the apoptotic process activated by this catechol-thioether derivative of dopamine in SH-SY5Y neuroblastoma cells. CysDA activates a cascade of events by an initial perturbation of Calcium homeostasis in the cell. Cell treatment with the catechol-thioether induces an immediate rise in intracellular Ca(2+) concentration, as demonstrated by a shift in the indo-1 dye emission spectrum, and a sustained high calcium concentration at long times of incubation. Fluorescence microscopy data show that the treatment of cells induces mitochondrial transmembrane potential depolarization, a clear evidence of the onset of apoptotic process. Programmed cell death activation is also demonstrated by cytochrome c release from the mitochondria, by an increased activity of both caspase-8 and -9 and by the poly(ADP-ribose)polymerase (PARP-1) cleavage, yielding the typical 86 kDa fragment due to caspase-3 activity. Overall, our data support the hypothesis that CysDA may induce apoptotic death in neuronal cells, via an initial perturbation of calcium homeostasis in the cytosol.

5-S-Cysteinyl-dopamine effect on the human dopaminergic neuroblastoma cell line SH-SY5Y.

In recent years a catechol-thioether metabolite of dopamine, 5-S-cysteinyl-dopamine, has been identified in certain dopaminergic regions of the brain, notably the Substantia Nigra. 5-S-Cysteinyl-dopamine has received great attention in view of its possible significance as an index of oxidative stress in aging and in neurodegenerative processes, particularly in Parkinson's disease. In the present study the effect of 5-S-cysteinyl-dopamine on human dopaminergic neuroblastoma SH-SY5Y cells is investigated. The substance is highly cytotoxic, even at a concentration as low as 30 microM. Treatment of the cells with 5-S-cysteinyl-dopamine induce the following intracellular responses: a decrease of the mitochondrial transmembrane potential, an increase in reactive oxygen species such as superoxide anion and peroxides, a marked decrease of reduced glutathione and an inhibition of the complex I activity. Caspase-3-like protease activation and oligonucleosomal DNA fragmentation have also been observed. These data are indicative of the onset of apoptotic processes due to 5-S-cysteinyl-dopamine.

P-glycoprotein inserted in planar lipid bilayers formed by liposomes opened on amorphous carbon and Langmuir-Blodgett monolayer.

The insertion of proteins into planar lipid layers is of outstanding interest as the resulting films are suitable for the investigation of protein structure and aggregation in a lipid environment and/or the development of biotechnological applications as biosensors. In this study, purified P-glycoprotein (P-gp), a membrane drug pump, was incorporated in model membranes deposited on solid supports according to the method by Puu and Gustafson, Biochim. Biophys. Acta 1327 (1997) 149-161. The models were formed by a double lipid layer obtained by opening P-gp-containing liposomes onto two hydrophobic supports: amorphous carbon films and Langmuir-Blodgett (L-B) lipid monolayers, which were then observed by transmission electron microscopy and atomic force microscopy, respectively. Before the opening of liposomes, the P-gp structure and functionality were verified by circular dichroism spectroscopy and enzymatic assay. Our micrographs showed that liposomes containing P-gp fuse to the substrates more easily than plain liposomes, which keep their rounded shape. This suggests that the protein plays an essential role in the fusion of liposomes. To localize P-gp, the immunogold labeling of two externally exposed protein epitopes was carried out. Both imaging techniques confirmed that P-gp was successfully incorporated in the model membranes and that the two epitopes preserved the reactivity with specific mAbs, after sample preparation. Model membranes obtained on L-B monolayer incorporated few molecules with respect to those incorporated in the model membrane deposited onto amorphous carbon, probably because of the different mechanism of proteoliposome opening. Finally, all particles appeared as isolated units, suggesting that P-gp molecules were present as monomers.