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The research efforts in drug discovery field are based on the knowledge of the molecular aspects of the disease and on the development of new techniques necessary to investigate the biological systems at molecular level. The selection of new leads is therefore a challenging task and involve various essential steps, the first being the identification/validation of new targets, then the selection of molecules able to bind to the target(s), and finally the study of the effects of hitting the target at molecular, cellular and whole animal level. In the case of Alzheimer’s disease (AD), the most common form of dementia in adults, acetylcholinesterase (AChE) has been the first target for the development of new drugs since the discovery of the cholinergic deficit in the central nervous system. However, basic research showed that cognitive deficit could be due not only to a cholinergic deficit but also to a cascade of biochemical events leading to the accumulation in the brain of proteins such as ß-amyloid and phosphorylated tau. Important targets recently emerged are amyloid fibrillogenesis and BACE1, one of the enzymes which cleave APP (amyloid precursor protein). On the other hand, other non cholinergic role of AChE in the AD has been discovered: some evidences suggest that AChE peripheral binding site may play a key role in the development of senile plaques, accelerating -amyloid peptide (A) deposition. Once the disease targets have been selected, the determination of the activity of the new compounds must be carried out quickly and in a way that allows the verification of the design hypothesis. To this aim, in a first instance, high throughput screening methods (HTS) of a large number of compounds for the selection of few lead compounds are examined, based on immobilized enzyme reactor HPLC systems. Secondly, specific methods, which elucidate the selected compound mechanism of action, are described (surface plasmon resonance, circular dichroism), before the ultimate and most advanced tools, transgenic animal models of the disease, can be used to study the effects of single compounds on the disease phenotype.

A previous GC/MS study highlighting the impurity profile of the synthetic pesticide d-allethrin is extended here to validate and confirm the impurities identity through the development of soft ionisation HPLC-MS methods. To accomplish this, we developed a reverse phase LC-MS analysis in gradient elution with two distinct soft ionisation techniques, the atmospheric pressure ionisation with electrospray source (API-ESI) and the chemical ionisation (APCI). A single quadrupole and an ion trap, which allowed the simultaneous determination of the molecular masses and structural information of the impurities by acquisition of collisionally induced (CID) product ions spectrum and in-source fragmentation, were employed as analysers. Single quadrupole and ion trap analysers resulted perfectly matching in the d-allethrin impurity fragmentation patterns. All the main impurities over 0.1% identified by GC/MS were confirmed. Results indicate that the proposed HPLC/MS method was found appropriate to confirm the presence of impurities such as chrysolactone, chloro allethrin derivatives, allethrolone and chrysanthemic acid, excluding their formation under GC/MS strong ionisation condition.

The nucleosome is the basic structural unit of eukariotic chromosomes and consists of a DNA molecule associated with a histone octamer comprised of pairs of the core histones H2A, H2B, H3 and H4. The nucleosomes are joined by linker DNA and histone1 to form chromatin. Histones play an important role in transcription, DNA replication, DNA repair and recombination. Post-translational modification of specific residues in the core histones (acetylation, methylation, phosphorylation, etc.) has been demonstrated to be critical to their regulatory function. In particular acetylation is a very specific phenomenon with various isoforms playing distinct roles. Increased acetylation is generally correlated with transcriptionally active or poised genes. Histone deacetylases’ inhibitors (short chain fatty acids, such as sodium butyrate, hydroxamic acids such as thrichostatin A) have been described as potential cancer therapeutics in a variety of preclinical studies. Inhibitors’ treatment of cells resulted in the increase of highly acetylated isoforms of the histones, which affect gene expression, cell differentiation and apoptosis. It is here described the application of reversed-phase high-pressure liquid chromatography under gradient conditions and mass spectrometry (LC-MS) to analyse global modification levels of core histones.

L-Methotrexate (L-Mtx) belongs to antineoplastic and antireumatic therapeutic categories. It is an antifolate which produced the first striking, although temporary remission in leukemia and the first cure of a solid tumor, choriocarcinoma. D-Mtx can be present as impurity from racemization of the synthetic product or during the shelf life of the pharmaceutical product, due to improper storage conditions. It was found that the pharmacokinetics of D-Mtx is different from that of L-Mtx. Although D-Mtx exhibits similar DHFR inhibitory effect, yet D-Mtx is much less toxic (40-fold lower LD50) and its antitumor effect is proportionately reduced. Many analytical methods have been reported to analyze Mtx in pharmaceutical formulations and in biological fluids by using LC, capillary zone electrophoresis, spectrophotometric and voltammetric techniques. However, there are only few analytical methods which allow the discrimination between L- and D-enantiomers of Mtx. These methods suffer from long time analysis, pre-step derivatization or unsatisfactory resolution for enantiomeric purity estimation in pharmaceutical formulations. The aim of this work was to develop and validate an HPLC method for the enantioselective analysis of Mtx in pharmaceutical formulations, such as tablets and injections. A simple, sensitive and selective HPLC method for the enantioselective analysis of Mtx was developed and validated by using Chirobiotic TTm stationary phase. Acceptable enantioresolution was achieved using a polar organic mobile phase. Essential steps in method validation were studied: linearity, precision, accuracy, suitability and stability. At wavelength 303 nm, the limits of detection and quantification (S/N= 3; S/N= 10) were found to be 0.9 and 3.8 mg/mL . The separation of 0.2% (m/m) D-Mtx (as limit of quantitation) was successfully obtained with resolution 1.72. Enantiopurity of L-Mtx was determined in pharmaceutical formulations (tablets and injections) with inter and intra-days relative standard deviation £ 1.6%.

The electrochemical oxidation and reduction behaviour of adsorbed species of antimetabolic antineoplastic agent Tarabine PFS (Cytosar-U) in Sorensen buffer solution of different pH values at an in situ-mercury film electrode (MFE) is studied using cyclic voltammetry (CV) and Osteryoung square-wave stripping voltammetry (OSWSV). Optimal experimental and operational parameters have been selected for the drug preconcentration and determination in aqueous medium. Based on the adsorption and accumulation of Tarabine PFS using Osteryoung square-wave anodic stripping voltammetry (OSWASV) at MFE, the drug is easily detected as 0.134 ng/ml (5.51 x 10(-10) M). Calibration plots have been constructed at different accumulation times. The standard deviation (n=10) at a concentration level of 6 x 10(-8) M Tarabine PFS is 0.062. The interaction of ssDNA with the drug under the optimal conditions at pH 7.7 has been studied. The formal potentials E degrees and E degrees ' and the equilibrium constants K(1) and K(2) have been calculated for the free form of Tarabine PFS and the bonded form with ssDNA, respectively. It was found that K(2) value for the bonded oxidized form is 298 times than that of K(1) for the bonded reduced form. Therefore, ssDNA has been found to interact strongly with the oxidized form of the drug. The method has been used for the nanogram determination of ssDNA with 1.9% variation coefficient. Detection limit of 3 ng/ml ssDNA has been achieved. Possible interfering organic compounds, cations and anions have been tested. The method has been applied for the drug determination in urine samples, down to 0.23 ng/ml could be easily achieved in such samples.

In the search for new potential drugs for AD treatment, the immobilization of the target enzyme acetylcholinesterase (AChE) and the insertion of the enzyme reactor in a HPLC system have demonstrated to be an excellent tool to rapidly screen for inhibitors. With the purpose of reducing the amount of used enzyme, substrate and inhibitors and of avoiding aspecific interactions between analytes and matrix, in previous studies AChE was covalently immobilized on a silica (1) or monolithic (2) matrix by using an in situ technique. The aim of this work was to prepare a micro-reactor by using a batch-wise technique in the attempt to lower the costs and increase the versatility of the system. Immobilization by batch wise technique involves essentially two steps: immobilization of the target enzyme onto non-packed affinity support followed by packing the stationary phase into an empty column that can be used in a flow system as immobilized enzyme reactor (IMER). Advantages of this technique over the in situ immobilization are: the size of the obtained IMER can be easily varied by using a different amount of bulk material, the optimization of the immobilization procedure can be performed in a parallel on a limited amount of material in a non-flow system, reducing time wasting and costs. In the present study human recombinant AChE was covalently immobilized onto 30 mg of Bakerbond glutaraldehyde-P (Glut-P, J.T. Becker, USA), a wide pore silica based affinity support. Evaluated parameters for the immobilization were: pH, amount of glut-P and contact time. The packing of the AChE-Glut-P into an empty column (Amersham Biosciences, Sweden) gave a micro-IMER of 5 mm ID and 3 mm thickness. After insertion in an HPLC system, chromatographic parameters in terms of mobile phase composition, pH and flow rate were evaluated. Inhibition studies were performed by using well-known inhibitors whose potency was distributed over four orders of magnitude. A particular attention was paid at aspecific interaction between matrix and inhibitors and conditioning time. The results indicate that the small amount of AChE immobilized on a reduced amount of chromatographic support was appropriate for high testing speed.