Stefano Fornasaro

Experimental determination and prediction of bilitranslocase transport activity.

The transport activity of a membrane protein, bilitranslocase (T.C. # 2.A.65.1.1), which acts as a transporter of bilirubin from blood to liver cells, was experimentally determined for a large set of various endogenous compounds, drugs, purine and pyrimidine derivatives. On these grounds, the structure-activity models were developed following the OECD principles of QSAR models and their predictive ability for new chemicals was evaluated. The applicability domain of the models was estimated by Euclidean distances criteria according to the applied modeling method. The selection of the most influential structural variables was an important stage in the adopted modeling methodology. The interpretation of selected variables was performed in order to get an insight into the mechanism of transport through the cell membrane via bilitranslocase. Validation of the optimized models was performed by a previously determined validation set. The classification model was build to separate active from inactive compounds. The resulting accuracy, sensitivity, and specificity were 0.73, 0.89, and 0.64, respectively. Only active compounds were used to develop a predictive model for bilitranslocase inhibition constants. The model showed good predictive ability; Root Mean Squared error of the validation set, RMS(V)=0.29 log units.

Potential of Surface Enhanced Raman Spectroscopy (SERS) in Therapeutic Drug Monitoring (TDM). A Critical Review

Surface-Enhanced Raman Spectroscopy (SERS) is a label-free technique that enables quick monitoring of substances at low concentrations in biological matrices. These advantages make it an attractive tool for the development of point-of-care tests suitable for Therapeutic Drug Monitoring (TDM) of drugs with a narrow therapeutic window, such as chemotherapeutic drugs, immunosuppressants, and various anticonvulsants. In this article, the current applications of SERS in the field of TDM for cancer therapy are discussed in detail and illustrated according to the different strategies and substrates. In particular, future perspectives are provided and special concerns regarding the standardization of self-assembly methods and nanofabrication procedures, quality assurance, and technology readiness are critically evaluated.

Determination of cyanidin 3-glucoside in rat brain, liver and kidneys by UPLC/MS-MS and its application to a short-term pharmacokinetic study.

Anthocyanins exert neuroprotection in various in vitro and in vivo experimental models. However, no details regarding their brain-related pharmacokinetics are so far available to support claims about their direct neuronal bioactivity as well as to design proper formulations of anthocyanin-based products. To gather this missing piece of knowledge, we intravenously administered a bolus of 668 nmol cyanidin 3-glucoside (C3G) in anaesthetized Wistar rats and shortly after (15 s to 20 min) we collected blood, brain, liver, kidneys and urine samples. Extracts thereof were analysed for C3G and its expected metabolites using UPLC/MS-MS. The data enabled to calculate a set of pharmacokinetics parameters. The main finding was the distinctive, rapid distribution of C3G in the brain, with an apparently constant plasma/brain ratio in the physiologically relevant plasma concentration range (19–355 nM). This is the first report that accurately determines the distribution pattern of C3G in the brain, paving the way to the rational design of future tests of neuroprotection by C3G in animal models and humans.

Toward SERS-based point-of-care approaches for therapeutic drug monitoring: the case of methotrexate

To date, in spite of their toxicity, the plasmatic concentration of most chemotherapeutic drugs is difficult to monitor in oncological patients, because their quantitative determination is expensive and time consuming. This contribution reports a first attempt for the direct quantitative determination of a chemotherapeutic drug in human serum samples by means of Surface Enhanced Raman Spectroscopy (SERS). In this study, SERS substrates constituted by Au nanoparticles deposited on paper by a simple dipping method have been used for rapid (few minutes) analysis of diluted human serum spiked with different concentrations of methotrexate, MTX. The drug concentrations were chosen in a range designed to cover typical therapeutic plasmatic values (from nanomolar to millimolar) in oncological patients, and the pertinent calibration was obtained by Partial Least-Squares Regression (PLSR). Stability selection was employed to evaluate the capability of the PLSR model to accurately predict and extract spectral variations correlated to MTX concentration. Such a quantitative determination is crucial for frequent, and hence adherent, therapeutic drug monitoring, TDM, of chemiotherapic drugs, given their heavy side effects. Its low cost, rapid response and the possibility of obtaining spectra with simple and compact instruments, make SERS particularly apt for implementing effective TDM. The promising results obtained in the analytical validation indicate which steps are to be taken on the way toward a clinical validation with real samples from oncological patients, for MTX as well as for other chemotherapeutic drugs.

Bioavailability of Flavonoids: The Role of Cell Membrane Transporters

Abstract Dietary flavonoids play an important role in the prevention of diseases related to oxidative stress in living systems. Although much attention has been focused on studying the protective functions of flavonoids, so far only limited biochemical research was done to tackle the fundamental issue of how flavonoids enter cells. Flavonoids have limited bioavailability, and consequently low plasma concentrations. Thus, in order to enter the intracellular compartment of cells in target tissues, flavonoids must exploit specific cell membrane transporter systems. This interaction between flavonoids and the proteins that mediate their transmembrane transport is of critical importance since it affects both the pharmacokinetic and pharmacodynamic properties of the flavonoids. Extensive knowledge of cell membrane transporters is therefore essential to fully understand the mechanisms of flavonoid-mediated protective activity in the cardiovascular, renal, hepatic, and central nervous systems, and also to elucidate flavonoid-drug interactions.Dietary flavonoids play an important role in the prevention of diseases related to oxidative stress in living systems. Although much attention has been focused on studying the protective functions of flavonoids, so far only limited biochemical research has been done to tackle the fundamental issues of how flavonoids enter cells. Flavonoids have limited bioavailability, and consequently low plasma concentrations. Thus, in order to enter the intracellular compartment of cells in target tissues, flavonoids must exploit specific cell membrane transporter systems. This interaction between flavonoids and the proteins that mediate their transmembrane transport is of critical importance since it affects both the pharmacokinetic and pharmacodynamic properties of flavonoids. Extensive knowledge on cell membrane transporters is, therefore, essential to fully understand the mechanisms of flavonoid-mediated protective activity in the cardiovascular, renal, hepatic and central nervous systems, and also to elucidate flavonoid-drug interactions.

Biofluids and other techniques: general discussion

Richard Dluhy opened a general discussion of the paper by Duncan Graham: In your example of a heterogeneous solution-based assay for multicomponent analysis, what is the concentration of the target fungal ssPCR DNA that is used, and how do you manage the kinetics of the reaction such that the target reaches the probe in a time frame appropriate for a clinical assay?

Long-term stability of an injection-molded zirconia bone-level implant: A testing protocol considering aging kinetics and dynamic fatigue

OBJECTIVE Separately addressing the fatigue resistance (ISO 14801, evaluation of final product) and aging behavior (ISO 13356, standardized sample) of oral implants made from yttria-stabilized zirconia proved to be insufficient in verifying their long-term stability, since (1) implant processing is known to significantly influence transformation kinetics and (2) aging, up from a certain level, is liable to decrease fatigue resistance. Therefore, the aim of this investigation was to apply a new testing protocol considering environmental conditions adequately inducing aging during dynamic fatigue. METHODS Zirconia implants were dynamically loaded (107 cycles), hydrothermally aged (85°, 60 days) or subjected to both treatments simultaneously. Subsequent, monoclinic intensity ratios (Xm) were obtained by locally resolved X-ray microdiffraction (μ-XRD2). Transformation propagation was monitored at cross-sections by μ-Raman spectroscopy and scanning electron microscopy (SEM). Finally, implants were statically loaded to fracture. Linear regression models (fracture load) and mixed models (Xm) were used for statistical analyses. RESULTS All treatments resulted in increased fracture load (p≤0.005), indicating the formation of transformation induced compressive stresses around surface defects during all treatment modalities. However, only hydrothermal and combinational treatment were found to increase Xm (p<0.001). No change in Xm was observed for solely dynamically loaded samples (p≥0.524). Depending on the variable observed, a monoclinic layer thickness of 1-2μm (SEM) or 6-8μm (Raman spectroscopy) was measured at surfaces exposed to water during treatments. SIGNIFICANCE Hydrothermal aging was successfully induced during dynamic fatigue. Therefore, the presented setup might serve as reference protocol for ensuring pre-clinically long-term reliability of zirconia oral implants.

Label-Free Quantification of Anticancer Drug Imatinib in Human Plasma with Surface Enhanced Raman Spectroscopy

Therapeutic drug monitoring (TDM) for anticancer drug imatinib has been suggested as the best way to improve the treatment response and minimize the risk of adverse reactions in chronic myelogenous leukemia (CML) and gastrointestinal stromal tumor (GIST) patients. TDM of oncology treatments with standard analytical methods, such as liquid chromatography-tandem mass spectrometry (LC-MS/MS) is, however, complex and demanding. This paper proposes a new method for quantitation of imatinib in human plasma, based on surface enhanced raman spectroscopy (SERS) and multivariate calibration using partial least-squares regression (PLSR). The best PLSR model was obtained with three latent variables in the range from 123 to 5000 ng/mL of imatinib, providing a standard error of prediction (SEP) of 510 ng/mL. The method was validated in accordance with international guidelines, through the estimate of figures of merit, such as precision, accuracy, systematic error, analytical sensitivity, limits of detection, and quantitation. Moreover, the feasibility and clinical utility of this approach have also been verified using real plasma samples taken from deidentified patients. The results were in good agreement with a clinically validated LC-MS/MS method. The new SERS method presented in this preliminary work showed simplicity, short analysis time, good sensitivity, and could be considered a promising platform for TDM of imatinib treatment in a point-of-care setting.

On the possibility of low cost, adherent therapeutic drug monitoring in oncology

A frequent quantification of drugs concentrations in plasma of patients subject to chemotherapy is seldom performed, mostly because the standard methods (Gas or Liquid Chromatography coupled with Mass Spectroscopy) are expensive and time consuming. In this paper we report the approach pursued in one of the research units of the EU project RAMAN4CLINICS to tackle the problem of a low cost, time adherent quantification of drugs used for oncological patients using a Surface Enhanced Raman Scattering (SERS) spectroscopy. More specifically, the issues concerning the repeatability of the nanostructured substrates will be presented and some promising results to increase the selectivity of the measures toward specific drugs will be discussed, with examples concerning one cytotoxic agent, Irinotecan and one kinase inhibitor, Sunitinib.