Vincenza Andrisano

β-Amyloid aggregation induced by human acetylcholinesterase: inhibition studies

The aggregation of beta-amyloid (1-40) (Abeta) induced by human recombinant acetylcholinesterase (HuAChE) was studied by means of circular dichroism (CD) and by thioflavin T fluorescence spectroscopy. Abeta was incubated alone and with HuAChE. The kinetic of fibrils formation was followed for 48 hr. The increasing beta-conformation content induced by HuAChE, preliminary to the formation of Abeta fibrils, was determined by circular dichroism. This phenomenon was found to be related to the thioflavin T emission of fluorescence at 490 nm. Incubation experiments were performed in the presence of known AChE inhibitors (physostigmine, edrophonium, decamethonium, propidium) and drugs used for Alzheimers disease (AD) (tacrine, donepezil), to test their capability of preventing the HuAChE-induced Abeta aggregation. The non-competitive or mixed mode of AChE inhibition was confirmed to be an essential feature. At 100 microM propidium, decamethonium, donepezil and physostigmine were found to inhibit the HuAChE-induced Abeta aggregation by 82, 25, 22 and 30%, respectively.

Inhibition of Acetylcholinesterase, β-Amyloid Aggregation, and NMDA Receptors in Alzheimer's Disease : A Promising Direction for the Multi-target-Directed Ligands Gold Rush

Alzheimers disease (AD) is a multifactorial syndrome with several target proteins contributing to its etiology. To confront AD, an innovative strategy is to design single chemical entities able to simultaneously modulate more than one target. Here, we present compounds that inhibit acetylcholinesterase and NMDA receptor activity. Furthermore, these compounds inhibit AChE-induced Abeta aggregation and display antioxidant properties, emerging as lead candidates for treating AD.

Pyrano(3,2-c)quinoline-6-Chlorotacrine Hybrids as a Novel Family of Acetylcholinesterase- and β-Amyloid-Directed Anti-Alzheimer Compounds

Two isomeric series of dual binding site acetylcholinesterase (AChE) inhibitors have been designed, synthesized, and tested for their ability to inhibit AChE, butyrylcholinesterase, AChE-induced and self-induced beta-amyloid (Abeta) aggregation, and beta-secretase (BACE-1) and to cross blood-brain barrier. The new hybrids consist of a unit of 6-chlorotacrine and a multicomponent reaction-derived pyrano[3,2-c]quinoline scaffold as the active-site and peripheral-site interacting moieties, respectively, connected through an oligomethylene linker containing an amido group at variable position. Indeed, molecular modeling and kinetic studies have confirmed the dual site binding of these compounds. The new hybrids, and particularly 27, retain the potent and selective human AChE inhibitory activity of the parent 6-chlorotacrine while exhibiting a significant in vitro inhibitory activity toward the AChE-induced and self-induced Abeta aggregation and toward BACE-1, as well as ability to enter the central nervous system, which makes them promising anti-Alzheimer lead compounds.

Benzofuran-Based Hybrid Compounds for the Inhibition of Cholinesterase Activity, β Amyloid Aggregation, and Aβ Neurotoxicity

The complex etiology of Alzheimers disease (AD) prompts scientists to develop multitarget strategies to combat causes and symptoms. We therefore designed, synthesized, and tested new hybrid molecules linking a benzofuran ring to a N-methyl- N-benzylamine through a heptyloxy chain, affording a series of potential multifunctional drugs for AD. The cholinesterase inhibitory activity was extended to the inhibition of Abeta fibril formation for 1, 3, and 5. Compound 3 showed an additional neuroprotective effect.

Analysis of ACE inhibitors in pharmaceutical dosage forms by derivative UV spectroscopy and liquid chromatography (HPLC)

Derivative UV spectroscopy and high performance liquid chromatography (HPLC) were applied to the determination of angiotensin-converting enzyme (ACE) inhibitors in their pharmaceutical dosage forms. For spectrophotometric determinations, the more appropriate derivative order was selected for each drug: ramipril (third-order), benazepril (second-order), enalapril maleate (second-order), lisinopril (first- and second-order) and quinapril (first-order). Reverse phase HPLC procedures (ODS column) were developed able to provide a single, symmetric peak for each drug; mixtures A-B, where A is 20 mM sodium heptansulphonate (pH 2.5) and B is acetonitrile-THF (95:5 v/v), proved to be suitable mobile phases to obtain selective separations of the cited ACE inhibitors. At ambient temperature, a low pH value (2.5) was found to be critical to avoid peak splitting and band broadening.

Acetylcholinesterase inhibitors for potential use in Alzheimer's disease: molecular modeling, synthesis and kinetic evaluation of 11H-indeno-[1,2-b]-quinolin-10-ylamine derivatives.

Continuing our work on tetracyclic tacrine analogues, we synthesized a series of acetylcholinesterase (AChE) inhibitors of 11H-indeno-[1,2-b]-quinolin-10-ylaminic structure. Selected substituents were placed in synthetically accessible positions of the tetracyclic nucleus, in order to explore the structure-activity relationships (SAR) and the mode of action of this class of anticholinesterases. A molecular modeling investigation of the binding interaction of the lead compound (1a) with the AChE active site was performed, from which it resulted that, despite the rather wide and rigid structure of 1a, there may still be the possibility to introduce some small substituent in some positions of the tetracycle. However, from the examination of the experimental IC50 values, it derived that the indenoquinoline nucleus probably represents the maximum allowable molecular size for rigid compounds binding to AChE. In fact, only a fluorine atom in position 2 maintains the AChE inhibitory potency of the parent compound, and, actually, increases the AChE-selectivity with respect to the butyrylcholinesterase inhibition. By studying the kinetics of AChE inhibition for two representative compounds of the series, it resulted that the lead compound (1a) shows an inhibition of mixed type, binding to both the active and the peripheral sites, while the more sterically hindered analogue 2n seems to interact only at the external binding site of the enzyme. This finding seems particularly important in the context of Alzheimers disease research in the light of recent observations showing that peripheral AChE inhibitors might decrease the aggregating effects of the enzyme on the beta-amyloid peptide (betaA).

Application of high-performance liquid chromatography with diode-array detection and on-line post-column photochemical derivatization to the determination of analgesics

HPLC analyses of pharmaceutical dosage forms containing analgesics and related compounds (acetylsalicyclic acid, paracetamol, propyphenazone, caffeine and chlorpheniramine) were performed on C18 and cyano columns under reversed-phase conditions. The performance of the methods was enhanced by introducing postcolumn on-line photochemical derivatization in combination with a diode-array detection. The column effluents were subjected on-line to UV irradiation (254 nm) and the characteristic photo-induced spectral modifications were useful for the unambiguous identification of the various analgesic compounds. The proposed HPLC methods were successfully applied to the analysis of commercially available analgesic dosage forms.

Structure-Activity Relationships of Acetylcholinesterase Noncovalent Inhibitors Based on a Polyamine Backbone. 4. Further Investigation on the Inner Spacer

Novel multi-target-directed ligands were designed by replacing the inner dipiperidino function of 3 with less flexible or completely rigid moieties to obtain compounds endowed with multiple biological properties that might be relevant to Alzheimers disease. 15 was the most interesting, inhibiting AChE in the nanomolar range and inhibiting AChE-induced and self-promoted beta-amyloid aggregation in the micromolar range.

Design of experiments for capillary electrophoretic enantioresolution of salbutamol using dermatan sulfate.

Statistical experimental design was used for the optimization and for robustness evaluation of a capillary electrophoretic method developed for the enantioresolution of salbutamol. Dermatan sulfate was used as chiral selector. The goal of the study was to obtain an efficient and fast separation. An eight-run Plackett-Burman matrix was used during the optimization process for the screening of the factors and to adjust the experimental domain under study. Response surface methodology was adopted after the screening phase to obtain information about how the factors percentage of chiral selector, pH and voltage affected the considered responses resolution and analysis time. The Derringer desirability function, which makes it possible to combine results obtained for properties measured on different scales, was used to simultaneously optimize the two responses. Robustness testing was carried out using a Plackett-Burman matrix. The method was found robust as regards the response resolution while voltage and chiral selector were found to be critical factors for the robustness of analysis time response. The proposed CE method permitted the complete enantioseparation of racemic salbutamol and was applied to its chiral resolution in spiked urine samples.

On-line post-column photochemical derivatization in liquid chromatographic—diode-array detection analysis of binary drug mixtures☆

HPLC methods were developed for the analysis of pharmaceutical creams containing binary drug mixtures (betamethasone valerate-chlorocresol; hydrocortisone-miconazole nitrate; desonide pivalate-chlorhexidine; dexamethasone-clotrimazole; triamcinolone acetonide-econazole nitrate). The chromatographic separations were performed on C-18 and cyano columns under reversed-phase conditions. A post-column on-line photochemical reactor (irradiation at 254 nm) was arranged between the analytical column and the diode-array detector to enhance the performance of the method. Two UV spectra (photoreactor on and off) were obtained for each analyte and these additional sources of information proved to be useful for the unambiguous identification of the various analytes. The method was applied to the quality control of commercial creams using a solid-phase extraction procedure for the sample clean-up.