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Heterodimers 4 and 5 were effective inhibitors of acetylcholinesterase (AChE) activity and AChE-induced amyloid-beta (A beta) aggregation. The peculiar biological profile of 4 can be relevant in studying the molecular basis underlying the nonclassical action of AChE and in addressing the question whether AChE inhibitors can affect the neurotoxic cascade leading to Alzheimer's disease. Compound 4 emerged as the most potent heterodimer so far available to inhibit AChE-induced A beta aggregation.

Polymeric micelles based on polyvinyl alcohol substituted with oleic acid were used as vehicles for progesterone and folic acid. The ability of this amphiphilic polymer to entrap lipophilic drugs and to generate stable micelles in aqueous neutral medium makes it a good candidate for drug delivery. The release of the loaded drugs in acidic environments represents another important property of these systems. Size of micelles, their stability, and their drug-loading capacity were evaluated, as well as the in vitro controlled-release profiles at pH 7.4 and 5.5.

In the present paper we expanded SAR studies of 3, the ethyl analogue of the AChE inhibitor caproctamine (2), by investigating the role of its octamethylene spacer separating the two amide functions through the replacement with dipiperidine and dianiline moieties. Compounds 4 and 8 were the most interesting of the two series of compounds. Compound 4 was the most potent AChE inhibitor with a pIC50 value of 8.48 +/- 0.02, while displaying also significant muscarinic M2 antagonistic activity (pKb value of 6.18 +/- 0.20). The availability of a suitable assay allowed us to verify whether 2, 3, 4, and 8 inhibit AChE-induced Abeta aggregation. Although all four derivatives caused a mixed type of AChE inhibition (active site and PAS), only 4 and 8, which bear an inner constrained spacer, were able to inhibit AChE-induced Abeta aggregation to a greater extent than donepezil. Clearly, the ability of an AChE inhibitor, based on a linear polyamine backbone, to bind both AChE sites may not be a sufficient condition to inhibit also AChE-induced Abeta aggregation. Dipiperidine derivative 4 emerged as a valuable pharmacological tool and a promising lead compound for new ligands to investigate and, hopefully, treat Alzheimer's disease.

Rivastigmine (1), an acetylcholinesterase (AChE) inhibitor approved in 2000 for the treatment of Alzheimer disease, bears a carbamate moiety in its structure, which is able to react covalently with the active site of the enzyme. Kinetic and structural studies on the interaction of 1 with different cholinesterases have been published, giving deeper, but not definitive, insights on the catalysis mechanism. On the basis of these findings and in connection with our previous studies on a series of benzopyrano[4,3-b]pyrrole carbamates as AChE inhibitors, we designed a series of conformationally restricted analogues of 1 by including the dimethylamino-alpha-methylbenzyl moiety in different tricyclic systems. A superimposition between the conformation of 1 and the carbon derivative 4, as obtained from Monte Carlo simulations, supported the idea that the tricyclic derivatives might act as rigid analogues of 1. The biological profile of 4-9, assessed in vitro against human AChE and BChE, validated our rational design. Compound 5, bearing a sulfur-containing system, showed the highest inhibitory activity, being 192-fold more potent than 1. In the present study, the most potent inhibitors were always methyl derivatives 3-5, endowed with a nanomolar range potency, whereas the ethyl ones were 40 times less potent. A reasonable explanation for this finding might be a steric hindrance effect between the ethyl group of 1 and His440 in the active site, as already suggested by the crystal structure of the complex AChE/1. The unfavorable influence of the carbamic N-alkyl chain on AChE inhibition is less striking when considering BChE inhibition, since BChE is characterized by a bigger acyl binding pocket than AChE. In fact, methyl carbamates 3-5 did not show AChE/BChE selectivity, whereas compounds 6-9 were significantly more potent in inhibiting BChE than AChE activity. At 100 microM, 5 was found to inhibit the AChE-induced aggregation only by 19% likely because it is not able to strongly interact with the peripheral anionic site of AChE, which plays an essential role in the Abeta aggregation mediated by the enzyme but is lacking in BChE structure.

A two-dimensional achiral/chiral HPLC method with circular dichroism (CD) detection was optimized for the stereochemical resolution and determination of the elution order of the eight stereoisomers of synthetic allethrin. A monolithic silica HPLC column (Chromolith, Merck, 100 mm x 4.6 mm i.d.) was put orthogonally to an enantioselective OJ Daicel column (250 mm x 4.6 mm i.d.) by means of a switching valve. The resolution of cis and trans diastereoisomers on the silica column was obtained by using a mobile phase consisting of n-hexane:tert-butyl methyl ether (96:4) (v/v) at a flow rate of 1 ml min(-1). The cis and trans peaks were then switched to the enantioselective OJ column separately in two subsequent injections. The resolution of the four trans stereoisomers was accomplished by using n-hexane:tert-butyl methyl ether (90:10) (v/v), while the mobile phase composition for the four cis stereoisomers consisted of n-hexane:isopropanol (99.3:0.7) (v/v). The CD based detection system allowed the determination of the elution order on the basis of the CD signals of the single stereoisomers, together with the injection of pure stereoisomers. Under the final conditions, the validated method was applied to the determination of stereoisomeric composition and absolute configuration of the prevailing stereoisomers of real samples, i.e. commercial batches of different sources of d-allethrin.

The development and characterization of a human recombinant acetylcholinesterase (hrAChE) micro-immobilized-enzyme reactor (IMER), prepared by using an in situ immobilization procedure is reported. hrAChE was covalently immobilized on an ethylenediamine (EDA) monolithic convective interaction media (CIM) disk (12 mm x 3 mm i.d.), previously derivatized with glutaraldehyde. The optimal conditions for the immobilization were: 12 microg of enzyme dissolved in 800 microl of phosphate buffer (50 mM, pH 6.0). The mixture was gently agitated overnight at 4 degrees C. The resulting Schiff bases were reduced by cyanoborohydride and the remaining aldehydic groups were condensed with monoethanolamine. Under these conditions, 0.22 U of hrAChE were immobilized with retention of 3.0% of the initial enzymatic activity. The activity of the immobilized hrAChE was stable for over 60 days. The activity and kinetic parameters of the hrAChE micro-IMER were investigated by inserting the micro-IMER in a HPLC system and it was demonstrated that the enzyme retained its activity. The micro-IMER was characterized in terms of units of immobilized enzyme and best conditions for immobilization yield. IMERs were compared for their relative enzyme stability, immobilized units, yield and aspecific matrix interactions. The effect of AChE inhibitors was evaluated by the simultaneous injection of each inhibitor with the substrate. The relative IC50 values were found in agreement with those derived by the conventional kinetic spectrophotometric method. In comparison with previously developed AChE-based IMERs, AChE monolithic micro-IMER showed advantages in terms of reduction of analysis time (2 min), lower aspecific matrix interactions and lower backpressure. Included in a HPLC system, it can be used for the rapid screening of new compounds' inhibitory potency. The advantages over the conventional methods are the increased enzyme stability and system automation which allows a large number of compounds to be analyzed in continuous.

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.

A GC/FID/MS method was developed for the identification and quantification of d-allethrin (DA) and its major impurities in commercial samples. Optimisation of the experimental conditions was carried out considering such important requirements as resolution, reproducibility, detection limits of 0.1% (m/m) for the impurities, and short analysis time. Under the optimised final conditions the method was validated for specificity, precision (CV% = 0.133 at 2.10 mg/mL and CV% = 0.035 at 3.00 mg/mL), linearity (0-3.00 microg injected), limits of detection (0.09 ng injected) and quantitation (0.28 ng injected), and robustness. The DA related impurities were identified by using a GC/MS method with ion trap mass detection and also by comparison with synthesised standards. The most abundant impurities were crysolactone, allethrolone, chrysanthemic acid, and chloro-derivatives of DA.

This review addresses the use of high-performance liquid chromatography (HPLC) and capillary electrophoresis (CE) as affinity separation methods to characterise drugs or potential drugs-bio-polymer interactions. Targets for the development of new drugs such as enzymes (IMERs), receptors, and membrane proteins were immobilized on solid supports. After the insertion in the HPLC system, these immobilized bio-polymers were used for the determination of binding constants of specific ligands, substrates and inhibitors of pharmaceutical interest, by frontal analyses and zonal elution methods. The most used bio-polymer immobilization techniques and methods for assessing the amount of active immobilized protein are reported. Examples of increased stability of immobilized enzymes with reduced amount of used protein were shown and the advantages in terms of recovery for reuse, reproducibility and on-line high-throughput screening for potential ligands are evidenced. Dealing with the acquisition of relevant pharmacokinetic data, examples concerning human serum albumin binding studies are reviewed. In particular, papers are reported in which the serum carrier has been studied to monitor the enantioselective binding of chiral drugs and the mutual interaction between co-administered drugs by CE and HPLC. Finally CE, as merging techniques with very promising and interesting application of microscale analysis of drugs' binding parameters to immobilized bio-polymers is examined.

The interaction between human serum albumin (HSA) and the acetylcholinesterase inhibitor donepezil, has been studied by means of capillary electrophoresis frontal analysis (CE/FA) and circular dichroism. CE/FA enabled rapid and direct estimation of the quantity of free donepezil present at equilibrium with a physiological level of serum albumin (600 micromol L(-1)). Application of Scatchard analysis enabled estimation of the binding parameters of HSA towards donepezil, such as association constant and number of binding sites on one protein molecule. Furthermore, due to enantioseparation ability shown by HSA on donepezil in CE mode, displacement experiments were carried out using ketoprofen and warfarin as coadditives to the HSA based running buffer. The addition of these compounds reduced the enantioresolution of donepezil by HSA only when used at high concentration. These data were confirmed and corroborated by circular dichroism (CD) experiments. Using CD, bilirubin was also applied as a ligand specific to site III of HSA. The observed behaviour suggested that donepezil could be considered a ligand with independent binding to sites I and II; although site III is not the highest affinity site, indirect interaction (i.e. cooperative binding) can be assumed.

The photophysical and photochemical properties of bumetanide, a sulfonamide diuretic drug, have been investigated and the photodegradation of the drug on exposure to UV-B and UV-A radiation monitored by fluorimetric and chromatographic (HPLC) methods. The main photoproducts were isolated by solid-phase extraction and liquid chromatographic procedures, and their structure elucidated by 1H-NMR and mass spectrometry. Bumetanide generates only extremely small quantities of singlet oxygen (1O2) upon irradiation in the presence of oxygen. The in vitro 3T3 N RU phototoxicity test yielded no evidence of phototoxic action.

Ambenonium (1), an old AChE inhibitor, is endowed with an outstanding affinity and a peculiar mechanism of action that, taken together, make it a very promising pharmacological tool for the treatment of Alzheimer's disease (AD). Unfortunately, the bisquaternary structure of 1 prevents its passage through the blood brain barrier. In a search of centrally active ambenonium derivatives, we planned to synthesize tertiary amines of 1, such as 2 and 3. In addition, to add new insights into the binding mechanism of the inhibitor, we designed constrained analogues of ambenonium by incorporating the diamine functions into cyclic moieties (4-12). The biological evaluation of the new compounds has been assessed in vitro against human AChE and BChE. All tertiary amine derivatives resulted more than 1000-fold less potent than 1 and, unlike prototype, did not show any selectivity between the two enzymes. This result, because of recent findings concerning the role of BChE in AD, makes our compounds, endowed with a well-balanced profile of AChE/BChE inhibition, valuable candidates for further development. To better clarify the interactions that account for the high affinity of 1, docking simulations and molecular dynamics studies on the AChE-1 complex were also carried out.

The photostability of the diuretic drugs triamterene and furosemide, individually and combined, was evaluated. Spectrophotometric, spectrofluorimetric and chromatographic (HPLC) methods were applied to monitor the drug photodegradation. Furosemide was confirmed to be highly photolable in both pH 7.4 solution and methanol. Differently triamterene proved to be highly fluorescent (emission quantum yield: 0.9 in methanol and 0.8 in pH 7.4 solution), but essentially photostable (photochemical reaction quantum yield: congruent with 5 x 10(-4)) under exposure at 365 and 313 nm radiations. When the combined drugs in pH 7.4 solutions were exposed to 365 nm radiations a significant photoprotective effect of triamterene on furosemide was observed. The photoreactivity of the drugs was exploited to develop an HPLC method involving a post-column on-line photochemical derivatization useful to confirm the analyte identity in a commercial dosage form (tablets). The commercial product, containing the combined drugs, proved to be photostable also after long (65 h) light exposure.

Amphiphilic copolymers have been the object of growing scientific interest due to their ability to form polymeric micelles in aqueous environments entrapping lipophilic drugs in their inner core. In this study, polyvinylalcohol substituted with oleic acid was employed as an amphiphilic micellar carrier for folic acid (FA), a model drug similar for its chemical-physical characteristics to methotrexate. In order to investigate the stability of the polymeric micelles, the drug incorporation and the kinetic aspects of drug release from these systems, selective analytical methods are required. The development of three analytical methods suitable for selectively identifying and reliably determining FA contained in the micelles and in the delivery systems is reported. UV derivative (first and second order) spectrophotometry was first applied to the aqueous solution of the FA containing micelles obtained at pH 9.0 and provided a characteristic spectral profiling with sharp peaks, related to the analyte, whose amplitude was used for quantitative application. A second approach involved a solid phase extraction (strong anion exchanger), which provided an effective clean up of the FA micelles solution, allowing accurate analysis to be performed also by a conventional spectrophotometric method. A RP-HPLC method, selectively supplying the FA separation from the micelles' components, was then used as a reference method to determine the accuracy of the spectrophotometric methods. These methods were applied to various micelle composition and to the delivery system study.

In recent years, the investigation of acetylcholinesterase (AChE) inhibitors has gained further interest, because the involvement of the peripheral site of the enzyme in the beta-amyloid (Abeta) aggregation process has been disclosed. We present here, for the first time, a direct evidence of the Abeta antiaggregating action of an AChE inhibitor (AP2238) purposely designed to bind at both the catalytic and the peripheral sites of the human enzyme.

Administration of the second-generation antihistamine, terfenadine, is sometimes associated with photosensitivity and other skin reactions. To obtain information on its photoreactivity, we used a stepwise experimental approach involving tests for photostability, phototoxicity (PT) (mouse fibroblast cell line [3T3] neutral red uptake [NRU] test) and photomutagenicity (with standard Ames salmonella tester strains TA98, TA100 and TA102). Terfenadine was not phototoxic to cultured mammalian cells under the conditions used (i.e. 5000/161 mJ cm(-2) UVA-UVB). Natural sunlight and UV radiations caused considerable drug decomposition and formation of several photoproducts. Addition of the irradiated terfenadine solution (i.e. a mixture of photoproducts) to the tester did not significantly increase background mutation frequency. Irradiation of terfenadine coplated with the TA102 strain induced a clear-cut photomutagenic response, the magnitude of which was dependent upon the precursor compound concentration and the UV dose (212/7 to 339/11 mJ cm(-2) UVA-UVB). These findings demonstrate that in vitro terfenadine is photomutagenic in absence of PT. Further in vitro and in vivo studies are therefore needed to provide an adequate safety assessment of the photochemical genotoxicity--carcinogenicity potential of terfenadine. In the meantime, patients should be advised to avoid excessive exposure to sunlight.

Continuing our studies on polyamine-based compounds of potential interest in the field of Alzheimer's disease therapeutics, we investigated the structure-activity relationships (SAR) of a lead compound (caproctamine, 3) identified in a previous work. In particular, we varied the substituents on the phenyl ring and on the nitrogen functions (both the amine and the amide), and studied the effects of such modifications on the inhibitory potency against isolated acetyl- and butyryl-cholinesterase (AChE and BChE). Moreover, the ability of selected compounds to reverse the d-tubocurarine-induced neuromuscular blockade and their antagonism toward muscarinic M(2) receptors in guinea pig left atrium were assayed. The most interesting SAR result was the identification of a relationship between the electronic characteristics of 2-substituents (measured by pK(a)) and the AChE inhibitory potency (pIC(50)) of tertiary amine compounds 6-12, which was confirmed by the invariance of the pIC(50) values of the corresponding methiodide derivatives 14-20. With regard to the biological profile, the most interesting compound was the N-ethyl-analogue of caproctamine (9), that showed pIC(50) values of 7.73 (+/-0.02) and 5.65 (+/-0.03) against AChE and BChE, respectively. The ability to increase the acetylcholine level was maintained in the functional assay (pAI(50) for reversing the neuromuscular blockade was 6.45 (+/-0.07)), as well as the ability to antagonize the M(2) receptors (pK(b) = 5.65 (+/-0.06)). Moreover, 9 showed a long duration of action as AChE inhibitor, an useful property in view of a possible development of this compound as a therapeutic agent.

The glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH) has been extensively studied as a target for new drugs to be used in the treatment of various parasitic diseases. The standard approach to the determination of GAPDH activity utilizes solubilized free enzyme and is limited by the enzyme's low stability. In the current study the stability of GAPDH was significantly increased through the covalent immobilization of the enzyme on a wide-pore silica support containing glutaraldehyde (Glut-P). The optimal conditions for the immobilization were: 100 mg Glut-P stationary phase, approximately 150 microg of enzyme dissolved in pyrophosphate buffer (15 mM, pH 8.5). The mixture was gently agitated for 6 h at 4 degrees C. Under these conditions 91.3% of protein was immobilized on 100 mg of Glut-P support with retention of 2.97% of the initial enzymatic activity. The activity of the immobilized GAPDH was stable for over 30 days. The GAPDH-Glut-P stationary phase was packed into a glass column to produce a GAPDH immobilized enzyme reactor (GAPDH-IMER). The activity and kinetic parameters of the GAPDH-IMER were investigated and the results demonstrated that the enzyme retained its activity and sensitivity to the competitive inhibitor agaric acid.

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 Alzheimer's 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.

An RP-HPLC study for the pKa determination of a series of basic compounds related to caproctamine, a dibenzylaminediamide reversible inhibitor of acetylcholinesterase, is reported. The 2-substituted analogues, bearing substituents with different electronegativity, were analysed by RP-HPLC by using C18 C4 stationary phases with a mobile phase consisting of mixture of acetonitrile and triethylamine phosphate buffer (pH range comprised between 4 and 10). Typical sigmoidal curves were obtained, showing the dependence of the capacity factors upon pH. In general, the retention of the investigated basic analytes increased with increasing of the pH. The inflection point of the pH sigmoidal dependence was used for the dissociation constant determination at a fixed acetonitrile percentage. When plotting pKa vs. percent of acetonitrile in the mobile phase for two representative compounds, linear regression were obtained: the y intercept gave the aqueous pKa(w). The pKa estimation by HPLC method was found to be useful to underline the difference of benzylamine basicity produced by the ortho aromatic substituents. The variation of pKa values (6.15-7.80) within the series of compounds was correlated with the electronic properties of the ortho-substituents through the Hammett sigma parameter, whereas the ability of substituents to accept H-bond was found to play a role in determining the conformational behavior of the molecules.

An isocratic reversed-phase liquid chromatographic (HPLC) method is proposed for the practical and reliable determination of triclosan, an antimicrobic agent incorporated into a variety of personal heath care products. Chromatographic separations were performed on a C-18 column using acetonitrile-TEA phosphate (70 mM; pH 3.5) 55:45 (v/v) as mobile phase and UV detection at 230 and 280 nm. The selectivity of the method was assured by the on-line photodiode array detector. The identity of the triclosan peak was also confirmed by HPLC MS. The method was successfully applied to the determination of triclosan in commercially available health care products (deodorant stick, dentifrice gel, mouthrinse, toothpaste and handwash). All the products displayed triclosan concentrations in compliance with the EEC directive (< or = 0.3%,).

A series of 2-(4-biphenylyl)-3,3'-hydroxy-substituted phenyl propionic acid, with anti-inflammatory properties, bearing two chiral centres, were studied by HPLC upon HSA-CSP (human serum albumin-based chiral stationary phase). The compounds were analysed in their stereoisomeric erythro and threo forms. The study involved the enantioselective analysis on HSA-CSP, the determination of the racemate lipophilicity (log k'(w)), a QSRR (quantitative structure-retention relationship) analysis and CD study for the assessment of the absolute configuration of the most retained enantiomer. Lipophilicity was found to be an important factor affecting the affinity of the compounds for the HSA stationary phase, but electronic properties seemed to play a role. The position of the substituent of the phenyl group on carbon 3 was found important to modulate stereoselective interaction, the highest value of enantioselectivities being found for the erythro ortho-substituted phenyl derivatives. The previously proposed two steps mechanism of enantiodiscrimination for cyclohexylphenyl substituted derivatives was confirmed for this series of derivatives bearing the biphenylyl moiety.

The reversible binding of valproate to human serum albumin determines a decrease of the binding of ligands that selectively bind to site I, site II, and bilirubin binding site. The binding inhibition was followed by displacement chromatography methodology using increasing concentrations of the competitor, i.e. valproate, in the mobile phase. Significant binding inhibition was observed for drugs binding at site I and site II. The greater displacement was observed for the more retained enantiomer of benzodiazepines and profens. A reduction of the affinity was observed also in the case of phenol red, this compound being selected as representative of bilirubin binding site. Difference circular dichroism spectroscopy was also used to characterise the binding of valproate to human serum albumin. This antiepilectic drug was proved to affect the binding at site I, II, and bilirubin binding site. The data have physiological relevance because significant inhibition of the binding resulted at clinic concentrations of valproate.

In this work, we further investigated a class of carbamic cholinesterase inhibitors introduced in a previous paper (Rampa et al. J. Med. Chem. 1998, 41, 3976). Some new omega-[N-methyl-N-(3-alkylcarbamoyloxyphenyl)methyl]aminoalkoxyaryl analogues were designed, synthesized, and evaluated for their inhibitory activity against both acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE). The structure of the lead compound (xanthostigmine) was systematically varied with the aim to optimize the different parts of the molecule. Moreover, such a structure-activity relationships (SAR) study was integrated with a kinetic analysis of the mechanism of AChE inhibition for two representative compounds. The structural modifications lead to a compound (12b) showing an IC(50) value for the AChE inhibition of 0.32 +/- 0.09 nM and to a group of BuChE inhibitors also active at the nanomolar level, the most potent of which (15d) was characterized by an IC(50) value of 3.3 +/- 0.4 nM. The kinetic analysis allowed for clarification of the role played by different molecular moieties with regard to the rate of AChE carbamoylation and the duration of inhibition. On the basis of the results presented here, it was concluded that the cholinesterase inhibitors of this class possess promising characteristics in view of a potential development as drugs for the treatment of Alzheimer's disease.

The photostability of diltiazem was studied in aqueous solutions at different pH values. Firstly, the hydrolysis of the drug to desacetyldiltiazem in alkaline medium was evaluated and then the drug photodegradation under exposure to UVA-UVB radiation (solar simulator) was monitored by HPLC methods. The main photoproduct was isolated and characterized as diltiazem-S-oxide on the basis of the NMR and mass spectra. The HPLC method was also applied to the selective analysis of diltiazem in commercial formulations. Tests on mutagenicity and photomutagenicity of the drug were also carried out using Salmonella typhimurium TA 102 strain. In this testing the drug neither was mutagenic nor toxic.

An immobilised acetylcholinesterase (AChE) stationary phase was prepared by using an in situ AChE immobilisation procedure. A stainless steel column packed with epoxide silica was connected to the HPLC system and the enzyme solution at pH 5.8 was recycled through the column at a flow-rate of 0.5 ml/min for 24 h. The activity of the immobilised AChE was determined by injecting the substrate acetylthiocholine, using as mobile phase 0.1 M phosphate buffer (pH 7.4) containing Ellman's reagent [5,5'-dithio-bis(2-nitrobenzoic acid)] and measuring the area of the obtained peak with UV detection at 412 nm. The effect of AChE inhibitors tacrine, edrophonium and donepezil were evaluated by the simultaneous injection of each inhibitor with the substrate. The resulting decrease in the AChE activity, as expressed by the decrease of the peak area detected at 412 nm, was related to the concentration and potency of the solutes. The obtained IC50 values were compared with those derived by the conventional spectrophotometric method. This immobilized enzyme reactor, included in a chromatographic system, can be used for the rapid screening for new inhibitors allowing for the on-line determination of a compound's inhibitory potency. The advantages over the conventional methods are the increased enzyme stability and system automation which allows a large number of compounds to be analysed continuously.

The analysis of donepezil, a centrally acting acetylcholine esterase inhibitor, is described by a CZE method suitable for applications in pharmaceutical field. A rapid migration of the analyte was obtained under acidic conditions (pH 3.0); with detection wavelength of 320 nm a LOD of 0.8 x 10(-3) mg/ml was provided. Applications on real sample (pharmaceuticals) were carried out using two different instruments with comparable results in terms of reproducibility and accuracy. The use of chiral selectors in the running buffer allowed the enantioseparation of donepezil; charged cyclodextrins (carboxymethyl-beta-cyclodextrin and sulfated-beta-cyclodextrin) were suitable for the chiral resolution of the analyte. Interesting results were also obtained using human serum albumin. The protein-based CE enantioseparation was carried out at pH 7.4 avoiding the partial filling technique due to the good absorptivity of donepezil at 320 nm. Interestingly, the use of bicine as BGE provided a significative improvement in the enantioresolution compared to that obtained by phosphate buffer.

The purpose of this study was to obtain information on the photochemical and phototoxic properties of Labetalol, a beta-blocker drug. Preliminary information on the drug photoreactivity was achieved using a flow system with a photochemical reactor on-line with a diode array detection system. Photophysical and photochemical investigations on the drug were performed in aqueous solutions at different pH values using spectrophotometric and fluorimetric methods; the photodegradation quantum yield was found to be 2.7 x 10(-3) at pH 5.8 and 1.5 x 10(-2) at pH 11.5. Forced photodegradation of labetalol solutions under exposure to UVA--UVB radiations (xenon arc lamp) was monitored by reversed-phase liquid chromatography. The main photodegradation products were isolated and characterized by NMR and mass spectrometry; labetalol was found to give 3-amino-1-phenylbutane and salicylamide-4-carboxaldehyde as the main photoproducts. Preliminary phototoxic testings on human keratinocyte cultures were performed evaluating the viability of the cells by the neutral-red uptake assay; mutagenic and photomutagenicity tests were also carried out based on Salmonella typhimurium strains. As a result, labetalol was found to be photolabile,mainly in alkaline medium, but evidences of significant phototoxic and photomutagenic effects by the drug were not observed.

In a search for less flexible analogues of caproctamine (1), a diamine diamide endowed with an interesting AChE affinity profile, we discovered compound 2, in which the terminal 2-methoxybenzyl groups of 1 have been incorporated into a tricyclic system. Since this compound retains good AChE inhibitory activity and its hexahydrochromeno[4,3-b]pyrrole moiety is reminiscent of the hexahydropyrrolo[2,3-b]indole of physostigmine (3), we have designed and synthesized carbamates 4-6, and their biological evaluation has been assessed in vitro against human AChE and BChE. The 6-carbamate 4 was almost as potent as physostigmine and was 60- and 550-fold more potent than the 7-carbamate 5 and the 8-carbamate 6, respectively. The two enantiomers of 4, (-)-4 and (+)-4, did not show a marked enantioselectivity. Finally, a similar time-dependent pattern of inhibition of AChE was observed for 3 and 4.

The photostability of melatonin, a hormone used as supplementary drug in the alleviation of jet-lag and other sleep disorders, was studied. The drug photodegradation at different pH values was monitored by HPLC methods. The main photoproduct was isolated and characterised on the basis of the NMR, FTIR, and mass spectra. A HPLC method, in combination with a post-column on-line photochemical derivatisation was developed for the selective and reliable quality control of commercially available melatonin containing products.

In this study, we attempted to derive a comprehensive SAR picture for the class of acetylcholinesterase (AChE) inhibitors related to tacrine, a drug currently in use for the treatment of the Alzheimer's disease. To this aim, we synthesized and tested a series of 9-amino-1,2,3,4-tetrahydroacridine derivatives substituted in the positions 6 and 7 of the acridine nucleus and bearing selected groups on the 9-amino function. By means of the Hansch approach, QSAR equations were obtained, quantitatively accounting for both the detrimental steric effect of substituents in position 7 and the favorable electron-attracting effect exerted by substituents in positions 6 and 7 of the 9-amino-1,2,3,4-tetrahydroacridine derivatives. The three-dimensional (3D) properties of the inhibitors were taken into consideration by performing a CoMFA analysis on the series of AChE inhibitors made by 12 9-amino-1,2,3, 4-tetrahydroacridines and 13 11H-indeno[1,2-b]quinolin-10-ylamines previously developed in our laboratory. The alignment of the molecules to be submitted to the CoMFA procedure was carried out by taking advantage of docking models calculated for the interactions of both the unsubstituted 9-amino-1,2,3,4-tetrahydroacridine and 11H-indeno[1,2-b]quinolin-10-ylamine with the target enzyme. A highly significant CoMFA model was obtained using the steric field alone, and the features of such a 3D QSAR model were compared with the classical QSAR equations previously calculated. The two models appeared consistent, the main aspects they had in common being (a) the individuation of the strongly negative contribution of the substituents in position 7 of tacrine and (b) a tentative assignment of the hydrophobic character to the favorable effect exerted by the substituents in position 6. Finally, a new previously unreported tacrine derivative designed on the basis of both the classical and the 3D QSAR equations was synthesized and kinetically evaluated, to test the predictive ability of the QSAR models. The 6-bromo-9-amino-1,2,3,4-tetrahydroacridine was predicted to have a pIC(50) value of 7.31 by the classical QSAR model and 7.40 by the CoMFA model, while its experimental IC(50) value was equal to 0.066 (+/-0.009) microM, corresponding to a pIC(50) of 7.18, showing a reasonable agreement between predicted and observed AChE inhibition data.

The binding characteristics of a series of 2,3-substituted 3-hydroxypropionic acids, with anti-inflammatory properties, bearing two chiral centres, were studied by HPLC upon HSA (human serum albumin)-based stationary phase. The compounds were analysed in their stereoisomeric erythro and threo forms and the chromatographic conditions for enantioseparation of the erythro and threo forms were studied on human serum albumin stationary phase. The enantiomer elution order was determined by injection of the enriched samples or by carrying out the CD spectra of each enantiomeric fraction. The absolute configuration of the single enantiomers was assigned on the basis of their CD spectra. A QSRR study was performed by subjecting the chromatographic data of the compounds to multiparameter regression analysis against various molecular descriptors to have insight into the chiral recognition mechanism. The lipophilicity appeared to be the most important parameter in determining the affinity to the protein, the compounds' capacity factors being linearly correlated to the experimental RP-HPLC partition coefficients (log k'w). The enantioselectivity factors (alpha) related to the enantiomers of the erythro and threo forms were studied taking into consideration both the physico-chemical parameters and the conformational behaviour of the compounds.

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.

Capillary electrophoresis (CE) was applied to the determination of angiotensin-converting enzyme (ACE) inhibitors in pharmaceuticals (tablets). Since a free solution CE system failed to reach a complete separation of closely related compounds (lisinopril, ramipril, benazepril, quinapril), alkylsulfonic additives (sodium heptansulfonate and (+)-10-camphorsulphonic acid) were added to the running buffer: improved separations were obtained suggesting a favourable effect of ion-pairing interactions between analytes and additives. The separations were carried out in acidic medium and a systematic investigation of electrophoretic parameters was made to evaluate the performance of the selected additives. Under the optimized conditions, ramipril and benazepril in their commercial dosage forms were determined confirming the applicability of the developed CE approach to the analysis of pharmaceutical samples; the results were also compared with those obtained applying a previously described and validated HPLC method.

A series of derivatives of the known M1 selective muscarinic receptor agonist McN-A-343 (1) was designed with the aim of investigating the effects of structural variations on both the butynyl chain and the phenyl ring of 1. The butynyl chain was replaced with an aromatic spacer, and the effects of such a modification on the stereoelectronic properties of the molecules were theoretically studied and considered compatible with muscarinic receptor affinity. Substituents on the phenyl ring of 1 were selected so as to vary their electronic and hydrophobic properties. This design strategy did not produce muscarinic M1 receptor agonists more potent than the prototype 1, even if some analogues displayed functional selectivity for different muscarinic receptor subtypes. Compounds 3 and 7 were selective agonists towards muscarinic M3 receptors, while compounds 14, 16 and 18 were selective muscarinic M2 receptor agonists. The most interesting derivative was 8, a full agonist at muscarinic M3 receptors devoid of activity at both muscarinic M1 and M2 subtypes. The pharmacological profile of the series was further characterized by studying the anticholinesterase and miotic activities of some representative compounds. Compounds 3-8 turned out to be weak acetylcholinesterase inhibitors, while derivatives 4, 6, 8 and 11 were able to significantly reduce the pupillary diameter in rabbit, indicating 8 as an effective miotic agent.

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 Alzheimer's 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).

A new sensitive, selective, and versatile circular dichroism (CD)-based HPLC detection system was used for the validation of the enantiomeric purity assay in the quality control of chiral drugs upon nonchiral stationary phases. The precision and the accuracy of the method were checked for selected samples showing values of the anisotropy factor on the order of 10(-1) to 10(-4). Very high accuracy has been obtained also in the case of extreme enantiomeric purity values (/=99% e.p.) and of a low anisotropy factor (g = 2 x 10(-4)) compound. The high selectivity of this detection system allows a selective monitoring of analytes in complex mixtures and makes the baseline stable., (Copyright 2000 Wiley-Liss, Inc.)

The photostability of the beta-blocker drug Atenolol was evaluated at pH 9, 7.4 and 4.0. The drug was exposed to UVA-UVB radiations and the photoproducts were detected by reversed phase LC methods. The photodegradation was found to increase with the pH value decreasing. The major photodegradation product at pH 7.4 was identified as 2-(4-hydroxyphenyl)acetamide. The LC method developed for routine analyses (column: C-18 Alltima; mobile phase: TEA acetate (pH 4; 0.01 M)-acetonitrile 96:4) was found to be suitable for the stability indicating determination of Atenolol in pharmaceutical dosage forms.

The transfer of potassium salts of dinitrophenyl amino acids from water to chloroform by the lipophilic guanosine derivative 1 takes place enantioselectively. Depending on the K(+):1 ratio, G-quartets of 1 self-assemble into octamers (O) or polymers.

Chemically oversulfated galactosaminoglycans with potential as therapeutic agents (inhibitors of human leukocyte elastase) were tested as chiral selectors in capillary electrophoresis of basic racemates. The high anionic character of these compounds provides them with anodic mobility in acidic buffer; using uncoated capillaries, the enantioresolution of racemic basic drugs was obtained at pH 2.5. Dimethindene, chloroquine and chlorpheniramine were enantioresolved applying negative voltage (-15 kV) while the other analytes (propranolol, pindolol, tetrahydrozoline and cloperastine) exhibited catodic migration. The addition of organic solvents to the running buffer was evaluated in order to increase the resolution; methanol provides the best results and in general, baseline separation of the analytes was reached. The studied oversulfated mucopolysaccharide, shows the same ionic character of heparin but presents different stereochemistry and sites of sulfation. A comparison with heparin, used in the same acidic conditions, may underline the role of ionic, spatial and steric features of glycosaminoglycans in the enantiorecognition.

The ability of capillary zone electrophoresis in the development of analytical methods devoted to the quality control of the thiol drug penicillamine is shown. Using 50 mM phosphate running buffer (pH 2.5), good quantitations of underivatized penicillamine and its disulfide were achieved; detection at 200 nm allowed checking the presence of the disulfide impurity in pharmaceuticals. The use of 1,1-[ethenylidenebis(sulfonyl)]bis-benzene as a thiol specific reagent resulted in an increased sensitivity for the quantitation of D-penicillamine (limit of detection at 200 nm wavelength was 1.5 microM). Introducing beta-cyclodextrin as chiral selector in the running buffer, enantioseparation of D-L-penicillamine was obtained; for this purpose (+)-camphor-10-sulfonic acid, a chiral ion-pairing reagent, was found to be an essential additive in obtaining a baseline separation. The resulting enantioseparative system was validated in order to evaluate the presence of the toxic L-penicillamine enantiomer in pharmaceutical samples.

In an effort to learn more about the general structure-activity relationships of cocaine with the aim to elucidate those structural features that might confer antagonistic properties to such analogues, we describe herein our synthetic efforts to prepare two-carbon bridge functionalized (methoxylated and hydroxylated) analogues. Our approach makes use of a modification of the classical Willstatter synthesis of cocaine: Mannich type cyclization of acetonedicarboxylic acid monomethyl ester with methylamine hydrochloride and 2-methoxysuccindialdehyde in a citrate buffer solution afforded the 6- and 7-substituted 2-carbomethoxy-3-tropinones 3a,b and 4a,b in approximate yields of 64%. Reduction of the (+/-)-tropinone derivatives was performed with sodium amalgam in a sulfuric acid solution to afford a mixture of (+/-)-methoxyecgonine and (+/-)-methoxypseudoecgonine derivatives 5, 11 and 6, 7, 12, 13. Benzoylation of these alcohols yielded the desired cocaine and pseudococaine-like compounds 8, 14 and 9, 10, 15, 16. Additionally, we show that enzymatic hydrolysis of these cocaine analogues using pig liver esterase (PLE) affords a practical means for achieving their chemical resolution. The enantiomers of the methoxycocaine analogues were also prepared starting from chiral (+)- and (-)-6-methoxytropinone. All new analogues were examined for their ability to displace [3H]mazindol binding and to inhibit high-affinity uptake of [3H]dopamine into striatal nerve ending (synaptosomes). It appeared evident that methoxylation of the cocaine two-carbon bridge provides compounds of particular interest: the Ki for the binding of the methoxypseudococaines is about two to four times smaller than the Ki for inhibition of dopamine uptake, thus enabling these compounds capable of countering the effects of cocaine to some extent.

Acetylcholinesterase (AChE) inhibitors are one of the most actively investigated classes of compounds in the search for an effective treatment of Alzheimer's disease. This work describes the synthesis, AChE inhibitory activity, and structure-activity relationships of some compounds related to a recently discovered series of AChE inhibitors: the omega-[N-methyl-N-(3-alkylcarbamoyloxyphenyl)methyl]aminoalkoxy xanthen-9-ones. The influence of structural variations on the inhibitory potency was carefully investigated by modifying different parts of the parent molecule, and a theoretical model of the binding of one representative compound to the enzyme was developed. The biological properties of the series were investigated in some detail by considering not only the activity on isolated enzyme but the selectivity with respect to butyrylcholinesterase (BuChE) and the in vitro inhibitory activity on rat cerebral cortex as well. Some of the newly synthesized derivatives, when tested on isolated and/or AChE-enriched rat brain cortex fraction, displayed a selective inhibitory activity and were more active than physostigmine. In particular, compound 13, an azaxanthone derivative, displayed the best rat cortex AChE inhibition (190-fold higher than physostigmine), as well as a high degree of enzyme selectivity (over 60-fold more selective for AChE than for BuChE). When tested in the isolated enzyme, compound 13 was less active, suggesting some differences either in drug availability/biotransformation or in the inhibitor-sensitive residues of the enzyme when biologically positioned in rat brain membranes.

Purpose: Vasoactive cocktails are widely used in diagnosing and treating erectile dysfunction, especially in poor responders to prostaglandin E1 (PGE1). However, very little information as to their chemical interactions and stability is available, despite the huge amount of published work regarding their clinical efficacy. Obviously, medical and legal problems are involved., Materials and Methods: We analyzed four kinds of vasoactive cocktails, composed of papaverine, phentolamine and PGE1 in different combinations, using High Performance Liquid Chromatography analysis after 5 to 60 days of storage at temperatures between 2 and 8C. SPSS MANOVA analysis and a t-test for paired samples were used for statistical purposes., Results: Papaverine and phentolamine concentrations showed no significant variations during the 2 month study, ranging from a minimum of 96.75+/-1.20 to a maximum of 103.00+/-0.20% of the starting values. In the same period, PGE1 showed an accelerated degradation profile, reaching concentration values, after 60 days, of 76.00+/-2.28% and 70.20+/-2.02% when added to phentolamine or papaverine respectively and 70.00+/-2.40% with both., Conclusions: Papaverine and phentolamine are characterized by chemical stability when blended together or with PGE1. Papaverine and/or phentolamine increase the naturally occurring degradation of PGE1 in physiological solution. This effect is most evident in the first 10 days. Papaverine has the greatest deteriorating effect on PGE1. A safe and proper use of these cocktails should take into account the variations of PGE1 concentration.

Dermatan sulfate (DS), a complex, polydispersed, sulfate polysaccharide was investigated as a useful chiral selector in capillary electrophoresis for the enantioresolution of a variety of drugs. Analysis was carried out in a fused-silica capillary column of 48.5 cm length (40 cm to detector window) x 50 microns I.D., and the separation buffer consisted of citric acid-Tris containing DS; the applied voltage was 15 kV and the detection wavelength was 220 nm. The effects of buffer pH, the dermatan concentration and run temperature on the enantioseparation and migration were examined. The method was applied to the enantioresolution of a representative set of twenty basic drugs. At all pH values used (3.0, 4.5 and 6.5) the addition of DS resulted in an increased migration time due to analyte-DS interaction. Using DS concentration of 2% (w/W), at pH 4.5, enantiomeric separations could be obtained for more than 50% of the examined drugs; resorcinic drugs; resorcinic moiety was found to be a very favourable structural feature for obtaining high enantioresolution values.

A Gas chromatographic method with mass detector was developed to identify and determine nitromusks in incense sticks of different origin (India, China, Tibet). The proposed method was found useful to correlate dermatological allergic reactions with the use and composition of commercial incense sticks. The incense sticks were powdered, extracted with methanol and after the addition of 1-eicosanol as internal standard, injected into the GC-MS, using 25 m bonded phase fused capillary column methyl, 5% phenyl silicone (0.32 mm I.D., 0.25 microns film thickness). Musk ambrette was identified and determined in one kind of chinese incense together with musk ketone and musk xylene. The latter compound was also found alone in another kind of chinese incense.

Capillary electrophoresis (CE) and chiral stationary phase (CSP) HPLC methods were investigated for the determination of enantiomeric purity of alpha1-adrenoreceptor antagonists related to WB 4101. In the CE study, the enantioseparation of the analytes was performed by studying the effect of different types of cyclodextrin in the buffer, namely heptakis (2,6-di-O-methyl)-beta-cyclodextrin (DMCD), hydroxypropyl-beta-cyclodextrin (HPCD) and beta-cyclodextrin (beta-CD). HPCD was found to be the most effective chiral selector in the enantioseparation of all the compounds, with high resolution values. A HPLC method, using immobilised serum protein columns, human serum albumin (HSA) and alpha1-acid glycoprotein (AGP), was also investigated. Two benzodioxane racemates were well resolved on a mixed tpe (50% HSA and 50% AGP) column, with enantioselective binding on AGP column.

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.

A series of 12 chiral arylcarboxylic acids were chromatographed on an immobilized human serum albumin chiral stationary phase (HSA-CSP). The effects of solute structure on chromatographic retentions and enantioselective separations were examined by linear regression analysis and the construction of quantitative structure-enantioselective retention relationships. Competitive displacement studies were also conducted using R-ibuprofen as the displacing agent. The results indicate that the enantioselective retention of the solutes takes place at the indole-benzodiazepine site (site II) on the HSA molecule and that chiral recognition is affected by the hydrophobicity and steric volume of the solutes. The displacement studies also identified a cooperative allosteric interaction induced by the binding of R-ibuprofen to site II.