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A rapid and universal capillary zone electrophoresis (CZE) method was developed to determine the dissociation constants (pK (a)) of the 20 standard proteogenic amino acids. Since some amino acids are poorly detected by UV, capacitively coupled contactless conductivity detection (C(4)D) was used as an additional detection mode. The C(4)D coupling proved to be very successful on a conventional CE-UV instrument, neither inducing supplementary analyses nor instrument modification. In order to reduce the analysis time for pK (a) determination, two strategies were applied: (i) a short-end injection to reduce the effective length, and (ii) a dynamic coating procedure to generate a large electroosmotic flow (EOF), even at pH values as low as 1.5. As a result, the analysis time per amino acid was less than 2 h, using 22 optimized buffers covering a pH range from 1.5 to 12.0 at a constant ionic strength of 50 mM. pK (a) values were calculated using an appropriate mathematical model describing the relationship between effective mobility and pH. The obtained pK (a) values were in accordance with the literature.

The objective was to relate the efficiency of a charged drug to carry current across the skin during iontophoresis to its structural and/or physicochemical properties. The corollary was the establishment of a predictive relationship useful to predict the feasibility of iontophoretic drug delivery, and for the selection and optimization of drug candidates for this route of administration. A dataset of 16 cations, for which iontophoretic fluxes have been measured under identical conditions, with no competition from exogenous co-ions, was compiled. Maximum transport numbers correlated with ion mobilities and decreased with ionic size, the dependence indicating that the electromigration mechanism of iontophoresis would become negligible for drugs of hydrodynamic radius greater than about 8 A. Validation of the model was demonstrated by successfully predicting the transport numbers of three structurally distinct dipeptides, the iontophoretic data for which had been determined under distinctly different experimental conditions. Finally, for the "training" set of cations, a strong linear dependence between their transport numbers in skin and those in aqueous solution was demonstrated; the former were larger by approximately a factor of 1.4 consistent with skin's cation permselectivity. In conclusion, this research offers a practical contribution to the development of a predictive structure-transport model of iontophoresis.

To study the stress-induced effects caused by wounding under a new perspective, a metabolomic strategy based on HPLC-MS has been devised for the model plant Arabidopsis thaliana. To detect induced metabolites and precisely localise these compounds among the numerous constitutive metabolites, HPLC-MS analyses were performed in a two-step strategy. In a first step, rapid direct TOF-MS measurements of the crude leaf extract were performed with a ballistic gradient on a short LC-column. The HPLC-MS data were investigated by multivariate analysis as total mass spectra (TMS). Principal components analysis (PCA) and hierarchical cluster analysis (HCA) on principal coordinates were combined for data treatment. PCA and HCA demonstrated a clear clustering of plant specimens selecting the highest discriminating ions given by the complete data analysis, leading to the specific detection of discrete-induced ions (m/z values). Furthermore, pool constitution with plants of homogeneous behaviour was achieved for confirmatory analysis. In this second step, long high-resolution LC profilings on an UPLC-TOF-MS system were used on pooled samples. This allowed to precisely localise the putative biological marker induced by wounding and by specific extraction of accurate m/z values detected in the screening procedure with the TMS spectra.

Methadone inhibits the cardiac potassium channel hERG and can cause a prolonged QT interval. Methadone is chiral but its therapeutic activity is mainly due to (R)-methadone. Whole-cell patch-clamp experiments using cells expressing hERG showed that (S)-methadone blocked the hERG current 3.5-fold more potently than (R)-methadone (IC50s (half-maximal inhibitory concentrations) at 37 degrees C: 2 and 7 microM). As CYP2B6 slow metabolizer (SM) status results in a reduced ability to metabolize (S)-methadone, electrocardiograms, CYP2B6 genotypes, and (R)- and (S)-methadone plasma concentrations were obtained for 179 patients receiving (R,S)-methadone. The mean heart-rate-corrected QT (QTc) was higher in CYP2B6 SMs (*6/*6 genotype; 439+/-25 ms; n=11) than in extensive metabolizers (non *6/*6; 421+/-25 ms; n=168; P=0.017). CYP2B6 SM status was associated with an increased risk of prolonged QTc (odds ratio=4.5, 95% confidence interval=1.2-17.7; P=0.03). This study reports the first genetic factor implicated in methadone metabolism that may increase the risk of cardiac arrhythmias and sudden death. This risk could be reduced by the administration of (R)-methadone.

A series of NO-donor diarylimidazoles derived from the lead compound Cimicoxib were synthesized and evaluated for their COX-2 inhibitory activity and their stability in whole blood as well as for vasodilator properties. The products are partly transformed into the corresponding alcohols following 24-h incubation in whole blood. All of them display good COX-1/COX-2 selectivity, but are less potent than the lead; a molecular modeling study was carried out to investigate their binding mode. The compounds are also capable of relaxing rat aorta strips precontracted with phenylephrine with a NO-dependent mechanism; this property could confer reduced cardiotoxicity with respect to traditional COX-2 inhibitors.

Numerous studies document that melatonin possesses a broad-spectrum antioxidant activity. It traps a number of reactive oxygen species (ROS) such as hydroxyl and peroxyl radicals, singlet oxygen and hypochlorous acid. It also inhibits peroxynitrite-induced reactions. It is known that atherosclerosis progression involves ROS-induced oxidation of low-density lipoproteins in sub-endothelial space and the depletion of nitric oxide (NO) in blood vessels, as well as a decreased sensitivity of the vessels to the actions of NO. Considering this, a series of new NO-donor antioxidants were designed and synthesized by joining melatonin with NO-donor nitrooxy and furoxan moieties as polyvalent agents potentially useful for the treatment of cardiovascular diseases involving atherosclerotic vascular changes. The in vitro antioxidant properties of the resulting products were assessed in the thiobarbituric acid reactive substances assay (TBARS), the ABTS(+.) as well as in the alkaline phosphatase (ALP) assay. The antioxidant capacities of NO-donor melatonins to inhibit lipoperoxidation (TBARS-IC(50)) was predominantly dependent on their lipophilicity, and therefore on their partitioning process into membranes. On the other hand, their comparable capacity to inhibit protein oxidation (ALP-IC(50)) was independent of their lipophilicity and was consistent with their similar ability to participate in electron transfer reactions. All the NO-donor melatonins were also evaluated for their ability to relax rat aorta strips precontracted with 1 microM phenylephrine. Finally, binding affinities and intrinsic activity studies, carried out at MT(1) and MT(2) receptor subtypes, showed a rather complex picture in need of further investigation.

The natural compound ferulenol, a sesquiterpene prenylated coumarin derivative, was purified from Ferula vesceritensis and its mitochondrial effects were studied. Ferulenol caused inhibition of oxidative phoshorylation. At low concentrations, ferulenol inhibited ATP synthesis by inhibition of the adenine nucleotide translocase without limitation of mitochondrial respiration. At higher concentrations, ferulenol inhibited oxygen consumption. Ferulenol caused specific inhibition of succinate ubiquinone reductase without altering succinate dehydrogenase activity of the complex II. This inhibition results from a limitation of electron transfers initiated by the reduction of ubiquinone to ubiquinol in the ubiquinone cycle. This original mechanism of action makes ferulenol a useful tool to study the physiological role and the mechanism of electron transfer in the complex II. In addition, these data provide an additional mechanism by which ferulenol may alter cell function and demonstrate that mitochondrial dysfunction is an important determinant in Ferula plant toxicity.

The development of in silico and in vitro tools to estimate or predict the passive human skin permeation and distribution of new chemical entities, useful in dermal drug delivery, in absorption studies of toxic compounds, and in the cosmetics industry, is presented. In vitro permeation parameters were measured using the artificial membrane PAMPA-skin. The Volsurf approach was then applied to extract pertinent descriptors from molecular interaction fields characterizing the molecular structure of tested compounds. Two useful three-dimensional solvatochromic models able to predict PAMPA permeation parameters directly from the molecular structure were obtained using the partial least squares analysis. The models also provide valuable information to understand the link between physicochemical and structural properties of tested compounds and their interactions with the artificial membrane PAMPA-skin and can be useful to rapidly estimate their permeation through the human skin.

The impact of species-dependent differences between human and rat MAO B on inhibitor screening was evidenced for two classes of compounds, coumarin and 5H-indeno[1,2-c]pyridazin-5-one derivatives. All examined compounds have shown a greater inhibitor potency toward human MAO B than toward rat MAO B. Moreover, no correlation was found between human and rat pIC(50) values. These divergences have important implications for the design and development of drugs involved in the MAO B metabolic pathway, suggesting that results obtained using rat enzyme cannot be extrapolated to human CNS, a priori. Indeed, the selection of a hit compound for lead generation could be different using human rather than rat enzyme. Moreover, the influence of substituents on the in vitro inhibition of human MAO B was markedly different between homogeneous series of coumarin and 5H-indeno[1,2-c]pyridazin-5-one derivatives, suggesting different binding modes, a hypothesis clearly supported by molecular docking simulations of inhibitors into the active site of human MAO B.

Transdermal iontophoresis enables the controlled, non-invasive administration of peptide therapeutics. The aims of this study were (i) to evaluate the effect of amino acid sequence and the spatial distribution of peptide physicochemical properties on electrotransport, and (ii) to develop a quantitative model to predict peptide transport rates. Experimental results showed that the distribution of molecular properties over the peptide surface significantly affected iontophoretic delivery: different arrangements of the same residues resulted in different transport behavior. Computational studies generated three-dimensional quantitative structure-permeation relationships (3D-QSPR) based on 3D descriptors. The model predicted that iontophoresis was favored by peptide hydrophilicity but hindered by voluminous, localized hydrophobicity. Molecular characteristics that favor electrotransport are the converse of those required for passive diffusion across biological membranes. The data represent the first analysis of peptide electrotransport in terms of the spatial distribution of molecular properties and provide insight into the ab initio prediction of transdermal iontophoretic peptide delivery.

Purpose: The aims of the study are to develop and evaluate an in vitro rat intestine segmental perfusion model for the prediction of the oral fraction absorbed of compounds and to assess the ability of the model to study intestinal metabolism., Methods: The system consisted of a perfusion cell with a rat intestinal segment and three perfusion circulations (donor, receiver, and rinsing circulation). Lucifer yellow (LY) was applied as internal standard together with test compounds in the donor circulation. To validate the model, the permeability of eight noncongeneric passively absorbed drugs was determined. Intestinal N-demethylation of verapamil into norverapamil was followed in the donor and receiver circulations by high-performance liquid chromatography analysis., Results: The in vitro model allowed ranking of the tested compounds according to their in vivo absorption potential. The Spearman's correlation coefficient between the oral fraction absorbed in humans and the ratio of permeation coefficient of test compound to the permeation coefficient of LY within the same experiment was 0.98 (P < 0.01). Moreover, intestinal N-demethylation of verapamil, its permeation, and the permeation of its metabolite norverapamil could be assessed in parallel., Conclusions: Up to six permeation kinetics can be obtained per rat, and the method has shown to be a valuable tool to estimate human oral absorption.

This work was devoted to the search for new artificial membranes allowing a rapid evaluation of passive human skin permeation of compounds with a parallel artificial membrane permeability assay (PAMPA). Effective permeability coefficients (Pe) determined for a set of compounds using the PAMPA technique with isopropyl myristate (IPM) and silicone oil, alone or in mixture, were compared to the corresponding human skin permeability coefficient values (Kp). A good correlation between Pe and Kp was found for compounds tested through a membrane consisting of 70% silicone and 30% IPM. Moreover, positive correlation between the membrane retention of compounds and stratum corneum/water partition coefficients (PSC) was established. These results showed that this new artificial membrane, defined as PAMPA-skin, is able to mimic the main barrier properties of human stratum corneum and can be used for the fast prediction of passive human skin permeability coefficients.

An experimental setup to study the pH dependence of standard ion-transfer potentials at the water/NPOE interface is presented. The system is composed of a microhole generated by laser photoablation in a 12-microm polyethylene terephthalate film, the aqueous phase consisting of a commercial immobilized pH gradient gel reswelled in electrolyte solution and a droplet of organic solution. Two electrodes are used, an Ag/AgCl aqueous electrode and an Ag/AgTPBCl organic electrode. This setup is applied to the study of two ionizable compounds (pyridine, 2,4-dinitrophenol). Thermodynamic parameters such as the standard transfer potential, the Gibbs energy of transfer, and the partition coefficients of the ionized forms as well as the neutral forms of these drug compounds are evaluated by differential pulse voltammetry. The data obtained are summarized in ionic partition diagrams, which are a useful tool for predicting and interpreting the transfer behavior of ionized drugs at the liquid/liquid interfaces mimicking the biological membranes.

Interest in inhibitors of monoamine oxidase type B (MAO B) has grown in recent years, due to their therapeutic potential in aging-related neurodegenerative diseases, such as Parkinson's disease and Alzheimer's disease. This study is devoted to the use of human recombinant MAO B obtained from a Baculovirus expression system (Supersomes MAO B, BD Gentest, MA, USA) as reliable and efficient enzyme source for MAO B inhibitor screening. Comparison of inhibition potencies (pIC50 values) determined with human cloned and human platelet MAO B for the two series of MAO B inhibitors, coumarin and 5H-indeno[1,2-c]pyridazin-5-one derivatives, showed that the difference between pIC50 values obtained with the two enzyme sources was not significant (P>0.05, Student's t-test). Hence, recombinant enzyme is validated as convenient enzyme source for MAO B inhibitor screening.

An RPLC was developed to rapidly determine lipophilicity of neutral and basic compounds using three base deactivated RPLC stationary phases particularly designed for the analysis of basic compounds, namely, Supelcosil ABZ(+)Plus, Discovery RP Amide C16, and Zorbax Extend C18. The work consisted of three sets of experiments. In the first log kw values of neutral compounds were extrapolated using hydroorganic mobile phases at different compositions. Good correlation between log kw and log Poct indicated that the method was appropriate for these supports, without adding a silanol masking agent. In the second set of experiments, isocratic log k values of neutral and basic compounds were measured with three different mobile phases. The best estimation of lipophilicity was obtained for neutral and basic compounds when the secondary interactions were strongly reduced (i. e., when basic compounds were under their neutral form). In the third set of experiments, isocratic retention factors of basic compounds (in their neutral form) were measured with a high-pH mobile phase, on a chemically stable support (Zorbax Extend C18). Under these chromatographic conditions, correlation between the isocratic retention factors and log Poct (log D10.5) for basic compounds was similar to that for neutral compounds.

CZE allows to measure the acidic dissociation constant (pKa) of many drug substances. However, determining the EOF intensity may be time-consuming, especially at a low pH. In order to overcome this drawback, a dynamic coating procedure of the capillary was carried out to increase microEOF, and thus to reduce the analysis time. In addition, this coating procedure enhanced migration time stability. The effective mobilities of 15 compounds were measured at different pH, producing pK'a values dependent on BGE ionic strength. The latter values were corrected with the activity coefficient to obtain a "true" pKa value. The 15 investigated compounds were (i) five acids: namely, salicylic acid, benzoic acid, ketoprofen, phenobarbital, and phenol, (ii) four bases: lidocaine, propafenone, propranolol, and quinine, (iii), five ampholytes: sulfanilamide, sulfabenzamide, sulfadimethoxine, sulfadoxine, and sulfisoxazole, and (iv) one zwitterion: cetirizine. The range of determined pKa values was between 1.2 and 11.2, and close to the pKa values available from the literature.

A liquid chromatography method for the characterization of base deactivated columns was investigated in a collaborative study involving six laboratories. This work was carried out on two chromatographic supports (Xterra RP 18 and Symmetry Shield). Different cooling systems, namely water bath and air oven, were tested and it was shown that column thermoregulation did not significantly influence chromatographic data. In order to control the mobile phase composition, the latter was prepared by weight rather than volume. Thanks to the injection of a set of selected neutral compounds, extra-column effects were evaluated in each of the participating laboratories. The results showed that chromatographic supports tested in different laboratories and following the same test protocol could be effectively compared.

A finite element simulation has been used in order to study the partition chromatography process of one species between an aqueous mobile phase and an organic stationary phase located at the bottom of a rectangular microchannel. The transient model incorporates convection--diffusion of the species in the water phase coupled to the diffusion in the stationary organic phase by the way of the partition kinetics at the interface. The time evolution of the injected species concentration is analyzed versus the velocity of the mobile phase, the detecting position and the thickness of the stationary phase. The comparison of simulation results with both experimental data and analytical model confirm its validity. These simulations show that thin channels can be used to measure log P of molecules from their retention time. Finally, we have shown how the sample velocity can be optimized for a given geometry of the channel and diffusion coefficient of the species.

H3-antagonists possess promising pharmacological effects on awakening, learning and memory, but few data on their access to the central nervous system (CNS) have been reported so far. The purpose of this work was to investigate the relationships between structure and brain penetration of a series of H3-antagonists, using ex vivo binding experiments in rats. H3-antagonists belonging to different chemical classes but all having an imidazole ring, an alkyl spacer, a polar fragment and a lipophilic ending group, were selected among the numerous H3-antagonists recently described by us. Ex vivo binding studies were performed by inhibiting specific [3H]-(R)-alpha-methylhistamine ([3H]-RAMHA) binding to rat cerebral cortical membranes following H3-antagonist peripheral administration. Ionization constants and partition coefficients in n-octanol/water and 1,2-dichloroethane/water were determined by the potentiometric pH-metric method and were compared to the ex vivo binding potencies to analyse structure-property relationships (SPR). In the ex vivo assay, the H3-antagonists showed different potencies (pED50) not correlated to their in vitro H3-receptor binding affinities (pKi). Compound 4a, having a benzothiazol-2-yl-thioethyl chain, showed high ex vivo potency (ED50=1.35 mg kg(-1) i.p.) and a fast brain penetration, eliciting maximal displacement of [3H]-RAMHA already 5 min after i.v. or i.p. administration. Ex vivo binding assays of three compounds, following i.v. and i.p. administration, showed that the observed i.p. ex vivo potencies were not significantly affected by biotransformation. Within the set of compounds, those having a better ability to reach the CNS had a logDoct(7.4) in the range 2-3.5, and a DeltalogPoct-dce < 2. The combined use of two easily measurable physicochemical descriptors, namely logDoct(7.4) (apparent lipophilicity at pH 7.4) and DeltalogPoct-dce (a descriptor of H-bond donor capacity) allowed to model brain permeation of the majority of the compounds examined.

The mitochondrial effects of two fluoride curcumin derivatives were studied. They induced the collapse of mitochondrial membrane potential (DeltaPsi), increased mitochondrial respiration, and decreased O(2)*- production and promoted Ca(2+) release. These effects were reversed by the recoupling agent 6-Ketocholestanol, but not by cyclosporin A, an inhibitor of the permeability transition pore (PTP), suggesting that these compounds act as uncoupling agents. This idea was reinforced by the analysis of the physico-chemical properties of the compounds indicating, that they are mainly in the anionic form in the mitochondrial membrane. Moreover, they are able to induce PTP opening by promoting the oxidation of thiol groups and the release of cytochrome c, making these two molecules potential candidates for induction of apoptosis.

Acetylcholinesterase (AChE) inhibitors are currently the only approved therapy for the treatment of Alzheimer's disease, only a limited number of drugs are commercially available. A library of non-alkaloidal natural compounds was investigated. To this end, a convenient microtitre plate method for assaying AChE inhibition, which allows a complete kinetic analysis of AChE inhibitors, was developed. Seven active compounds with Ki values in the micromolar range were identified, six of which were xanthones. This is the first report that a promising potential for AChE inhibition exists in such non-nitrogenous natural compounds. Furthermore, four xanthones among these xanthones had already been described as monoamine oxidase (MAO) inhibitors, making then dual AChE/MAO inhibitors of great interest.

This review discusses liposome/water lipophilicity in terms of the structure of liposomes, experimental methods, and information content. In a first part, the structural properties of the hydrophobic core and polar surface of liposomes are examined in the light of potential interactions with solute molecules. Particular emphasis is placed on the physicochemical properties of polar headgroups of lipids in liposomes. A second part is dedicated to three useful methods to study liposome/water partitioning, namely potentiometry, equilibrium dialysis, and (1)H-NMR relaxation rates. In each case, the principle and limitations of the method are discussed. The next part presents the structural information encoded in liposome/water lipophilicity, in other words the solutes' structural and physicochemical properties that determine their behavior and hence their partitioning in such systems. This presentation is based on a comparison between isotropic (i.e., solvent/water) and anisotropic (e.g., liposome/water) systems. An important factor to be considered is whether the anisotropic lipid phase is ionized or not. Three examples taken from the authors' laboratories are discussed to illustrate the factors or combinations thereof that govern liposome/water lipophilicity, namely (a) hydrophobic interactions alone, (b) hydrophobic and polar interactions, and (c) conformational effects plus hydrophobic and ionic interactions. The next part presents two studies taken from the field of QSAR to exemplify the use of liposome/water lipophilicity in structure-disposition and structure-activity relationships. In the conclusion, we summarize the interests and limitations of this technology and point to promising developments., (Copyright 2004 Wiley Periodicals, Inc. Med Res Rev, 24, No. 3, 299-324, 2004)

Reversed phase high performance liquid chromatography (RPLC) is currently the method of choice for the analysis of basic compounds. However, with traditional silica materials, secondary interactions between the analyte and residual silanols produce peak tailing which can negatively affect resolution, sensitivity, and reproducibility. In order to reduce these secondary interactions, which comprise ion exchange, hydrogen bonding, and London forces interactions, chromatographic analyses can be carried out at low or high pH values where silanol groups and basic compounds are mostly uncharged. The chromatographic behaviour of a particular bidentate stationary phase, Zorbax Extend C18, was studied with a set of basic and neutral compounds. Thanks to a higher chemical stability than traditional silica based supports, analyses were carried out with a high pH mobile phase, which represents a good alternative to the acidic mobile phases generally used to reduce ion exchange interactions. The performance of this bidentate stationary phase was also compared with that of other supports and it was proved that it is advantageous to work with high pH mobile phases when analyzing basic compounds.

Purpose: Development of reliable mathematical models to predict skin permeability remains a challenging objective. This article examines some of the existing algorithms and critically evaluates their statistical relevance., Methods: Complete statistics were recalculated for a number of published models using a stepwise multiple regression procedure. The predictivity of the models was obtained by cross-validation using a "leave-one-out" deletion pattern. The relative contribution of each independent variable to the models was calculated by a standardization procedure., Results: The heterogeneity of the data in terms of skin origin and experimental conditions has been shown to contribute to the residual variance in existing models. Furthermore, rigorous statistics demonstrate that some published models are based on nonsignificant parameters. As such, they afford misleading mechanistic insight and will lead to over-interpretation of the data., Conclusions: The large number of published models reflects the need for predictive tools in cutaneous drug delivery and toxicology. However, such models are more reliable when confined within well-defined chemical classes, and their applicability is often limited by the narrow property space of the set of permeants under study.

The major hurdle to overcome in the development of 3D-QSAR models using steric, electrostatic, or lipophilic "fields" is related to both conformation selection and subsequent suitable overlay (alignment) of compounds. Therefore, it is of some interest to provide a conformationally sensitive lipophilicity descriptor that is alignment-independent. In this chapter we describe the derivation and parametrization of a new descriptor called 3D-LogP and demonstrate both its conformational sensitivity and its effectiveness in QSAR analysis. The 3D-LogP descriptor provides such a representation in the form of a rapidly computable description of the local lipophilicity at points on a user-defined molecular surface.

The objective of this study was to investigate the reliability of potentiometric measurements of partition coefficients (log P) in the o-NPOE/water system, o-nitrophenyl octyl ether being an organic solvent widely used for the voltammetric determination of the partition coefficient of ions. Three sets of ionizable model compounds were explored in this study. The results showed that the potentiometric technique gave precise and reliable log P(npoe) values in the explored range of 0.1-4.3.

Purpose: A new experimental set-up for studying partitioning of ionizable drugs at the interface between two immiscible electrolyte solutions (ITIES) by amperometry is presented. The method is quite general, as it can be applied to any charged drug molecule., Methods: The procedure is based on 96-well microfilter plates with microporous filters to support 96 organic liquid membranes. The new methodology is first validated using a series of tetra-alkylammonium ions and subsequently used to construct the ion partition diagrams of 3,5-N,N-tetramethylaniline and 2,4-dinitrophenol. The lipophilicity of these drugs was examined by potentiometry and cyclic voltammetry in the NPOE/water system., Results: Cyclic voltammetry resulted in potential-pH profiles of the studied drugs. When the aqueous phase pKa is already known, the logP(NPOE) of lipophilic drugs could be determined using a very little amount of solvents and drugs. The values of the partition coefficients for the neutral forms agree well with those obtained by potentiometry., Conclusions: The procedure based on commercially available 96-well microfilter plates is shown to be useful for determining logP of ionized drugs in a rapid and efficient way.

Three-dimensional quantitative structure-activity relationship (3D-QSAR) analysis was applied to a series of H(3) receptor antagonists characterized by an imidazole ring, an alkyl spacer, and a heterocyclic polar moiety containing an imidazole or a thiazole ring, with a view to investigate the requirements for H(3) receptor affinity on rat cortex membranes. The compounds were aligned based on the hypothesis that the presence of a H-bond donor group in the polar portion of the molecule can increase H(3) receptor affinity. The 3D-QSAR analysis, which was performed using both the CoMFA and CoMSIA protocols, revealed that the presence of a H-bond donor group is not statistically relevant for H(3) receptor affinity. Based on this result, another alignment was adopted that took into consideration the structural features common to all compounds, namely the imidazole ring and the N atom with a free lone pair in the polar portion. The 3D-QSAR models thus obtained showed that H(3) receptor affinity is modulated by the position and direction of the intermolecular interaction elicited by the polar group in the ligands.

Natural geiparvarin 1 and a number of its analogues were prepared and tested as inhibitors of both monoamine oxidase isoforms, MAO-B and MAO-A. The desmethyl congener 6 of geiparvarin, proved potent and selective MAO-B inhibitor (pIC(50)=7.55 vs 4.62). X-ray crystallography and molecular modelling studies helped the understanding of the observed structure-activity relationships.

Purpose: The purpose of this work was to assess the molecular properties that influence solute permeation across siliconemembranes and to compare the results with transport across human skin., Methods: The permeability coefficients (log Kp) of a series of model solutes across silicone membranes were determined from the analysis of simple transport experiments using a pseudosteady-state mathematical model of the diffusion process. Subsequently, structure permeation relationships were constructed and examined, focusing in particular on the difference between solute octanol/water and 1,2 dichloroethane/water partition coefficients (deltalog P(oct-dce)), which re ported upon H-bond donor activity, and the computationally derived molecular hydrogen-bonding potential., Results: The hydrogen-bond donor acidity and the lipophilicity of the compounds examined greatly influenced their permeation across sil cone membranes. Furthermore, for a limited dataset, a significant correlation was identified between solute permeation across silicone membranes and that through human epidermis., Conclusion: The key molecular properties that control solute perme ation across silicone membranes have been identified. For the set of substituted phenols and other unrelated compounds examined here a similar structure-permeation relationship has been derived for their transport through human epidermis, suggesting application of the results to the prediction of flux across biological barriers.

This mini-review begins with a presentation of the hydrogen-bond in the context of other intermolecular recognition forces of significance in molecular biology and molecular pharmacology. This is followed by a survey of the various computational methods available to calculate the hydrogen-bonding capacity of compounds. Such methods use quantum mechanics, molecular mechanics, or algorithms based on experimental fragmental values. A recent and promising advance in the computation of H-bonding capacity is the development of specific molecular interaction fields (MIFs) known as molecular hydrogen-bonding potentials (MHBPs). Their interest in relating molecular properties to pharmacokinetic behaviour is highlighted with two examples, namely oral drug absorption and blood-brain barrier permeation.

This paper first gives a brief review of the main techniques used to measure the lipophilicity of neutral and ionic drugs, namely the shake-flask method, potentiometry, and cyclic voltammetry at liquid-liquid interfaces. The lipophilicity of 28 acidic compounds with various functional groups was studied by potentiometry and cyclic voltammetry in the n-octanol/water and 1,2-dichloroethane/water systems in order to complement our understanding of the lipophilicity of neutral and ionized acids and to clarify the solvation mechanisms responsible for their partition. The parameter diff (log P(N-A)(dce)) (i.e., log P of the neutral acid minus standard log P of the conjugated anion in 1,2-dichloroethane/water) was shown to depend not only on intramolecular interactions and conformational effects in the neutral and anionic forms, but also on the delocalization of the negative charge in the anion, confirming the ability of Born's solvation model to describe qualitatively the effect of the molecular radius on the lipophilicity of ions.

Purpose: This work examines the lipophilic behavior of various zwitterions and shows how distribution profiles in biphasic systems and ionic partition diagrams may improve our understanding of pH-absorption profiles of drugs., Methods: The lipophilicity of various zwitterionic drugs was examined by potentiometry and cyclic voltammetry in the 1,2-dichloroethane/water system to study the intramolecular interactions and conformational effects affecting absorption and activity of zwitterions, as well as to draw their theoretical and experimental ionic partition diagrams., Results: Different theoretical partition diagrams are reported according to the tautomeric constant of the zwitterion. Shifts of apparent PKa are obtained in the ionic partition diagrams of raclopride and eticlopride and compared to the deviations from pH-absorption profile described in the literature for lipophilic drugs. The physicochemical origin of these shifts is discussed., Conclusions: The comparison between pH-absorption profiles and ionic partition diagrams of zwitterions is shown here to be of value for a better mechanistic understanding of absorption processes, thus opening new perspectives in studying pH-absorption profiles of ionizable drugs.

We report a four-component partial least squares discriminant analysis (PLS) model for the prediction of blood-brain barrier (BBB) permeation using descriptors derived from 3D molecular fields. The 3D fields were transformed by VolSurf into suitable 1D descriptors, which were correlated to the ratio of blood-brain partitioning measured at steady state in rats (log C(brain)/C(blood)). The model so obtained sheds light on molecular properties influencing BBB permeation. It can also be used in the virtual screening of new chemicals.

Purpose: To assess the effect of molecular factors influencing retention on immobilized artificial membrane (IAM) high-performance liquid chromatography columns compared to liposomal partitioning and traditional n-octanol/water partition coefficients., Methods: IAM capacity factors were measured at pH 7.0 on an IAM.PC.DD2 stationary phase. Liposomal partitioning at pH 7.0 and n-octanol/water partition coefficients were measured using the pH metric method. Partitioning in egg-phosphatidylcholine (PhC) liposomes was also measured by equilibrium dialysis for a series of beta-blockers., Results: For the ionized beta-blockers, potentiometry and equilibrium dialysis yielded consistent partitioning data. For relatively large bases. IAM retention correlated well with PhC liposome partitioning, hydrophobic forces being mainly involved. For more hydrophilic compounds and for heterogeneous solutes, in contrast, the balance between electrostatic and hydrophobic interactions was not the same in the two systems. Hydrogen bonding, an important factor in liposomes partitioning, played only a minor role in IAM retention., Conclusions: Partitioning in immobilized artificial membranes depends on size, hydrophobicity, and charge. When hydrophobic interactions dominate retention, IAM capacity factors are well correlated with liposomal partitioning. On the contary, for hydrophilic solutes, the two systems do not yield the same information and are not interchangeable.

A number of heterogeneous drugs previously shown to bind to trimetazidine (TMZ) binding sites on mitochondria and to inhibit mitochondrial swelling (Morin et al., Br J Pharmacol 1998;123:1385-94) were investigated here for their physicochemical properties. The molecular parameters measured were the partition coefficients of the neutral and monocationic forms in the n-octanol/water and dichloroethane/water systems, their distribution coefficients at pH 7.4 in these two solvent systems, as well as their distribution coefficients at pH 7.4 in a phosphatidylcholine (PhC) liposomes/water system (log D(lip)(7.4)). Most of these properties were not correlated with affinity to mitochondria or inhibition of mitochondrial swelling. In contrast, log D(lip)(7.4) showed a modest correlation with binding to the low-affinity site (r(2)=0.52) and a better correlation with anti-swelling activity (r(2)=0.69), itself well correlated with binding to the low-affinity sites (r(2)=0.83). Thus, these sites have recognition properties much like those of membranes, as they depend on lipophilicity-hydrophobicity (core binding) and ionic bonds (surface interactions).

A set of 29 3-alkyl 5-arylimidazolidinediones (hydantoins) with affinity for the human cannabinoid CB(1) receptor was studied for their lipophilicity and conformational properties in order to delineate a pharmacophore. These molecules constitute a new template for cannabinoid receptor recognition, since (a) their structure differs from that of classical cannabinoid ligands and (b) antagonism is the mechanism of action of at least three compounds (20, 21, and 23). Indeed, in the [(35)S]-GTP gamma S binding assay using rat cerebellum homogenates, they behave as antagonists without any inverse agonism component. Using a set of selected compounds, experimental lipophilicity was measured by RP-HPLC and calculated by a fragmental method (CLOGP) and a conformation-dependent method (CLIP based on the molecular lipophilicity potential). These approaches revealed two models which differentiate the binding mode of nonpolar and polar hydantoins and which could explain, at least for compounds 20, 21, and 23, the mechanism of action of this new family of cannabinoid ligands.

The objectives of this study were to establish guidelines for the proper measurement of capacity factors (log k(IAMw) on immobilized artificial membrane (IAM) stationary phases. In this context, some aspects related to the extrapolation of log(kIAMw) values, the stability and properties of IAM.PC.DD2 stationary phases and the column-to-column variability are discussed. No significant difference was observed when using either acetonitrile or methanol for the linear extrapolation of log k(IAM) values. However, methanol seems more appropriate when working with ionized compounds. Plotting isocratic capacity factors against the percentage (v/v) of co-solvent instead of the mole fraction leads to more reliable log k(AMW) values. Furthermore, our results with a YMC ODS-AQ and an IAM.PC.DD2 HPLC column indicate that only small differences arise between extrapolated capacity factors when using the (w(w))pH or the (s(w))pH operational scale and correcting or not the ionic strength for dilution caused by the co-solvent. The use of the (s(w))pH scale is recommended when working with ionized compounds in order to avoid parabolic relationships during linear extrapolation. The pH-dependent retention of three ionizable drugs on an IAM.PC.DD2 phase showed that secondary interactions with the charged moieties of the chromatographic surface affect the retention of ionized compounds around physiological pH. Finally, it was shown that column ageing occurs also with IAM.PC.DD2 stationary phases and that it depends on the column as well as on the investigated analyte. The intra-batch variability for IAM.PC.DD2 phases was small, whereas a marked and solute-dependent batch-to-batch variability was apparent.

The rational use of micelles in quantitative structure-activity and quantitative structure-permeation relationships implies a good knowledge of the nature of recognition forces underlying solute-micelle association. The aims of this study were to unravel the intermolecular interaction forces responsible for the association of neutral and ionised compounds with negatively charged sodium dodecyl sulfate (SDS) micelles, using micellar electrokinetic capillary chromatography (MEKC). The MEKC capacity factors (log k(MEKC)) of 36 neutral model solutes were analysed by linear solvation free-energy relationships (LSERs). The results indicate that the size and H-bond acceptor strength of solutes are mainly responsible for their MEKC retention. Compared to n-octanol, the SDS micelles are more cohesive and stronger H-bond donors. Strong attractive electrostatic interactions govern solute-micelle association for positively charged compounds and micelles of the opposite charge, whereas repulsive electrostatic interactions occur between negatively charged solutes and micelles of the same charge. The capacity factors measured for the ionised forms of the acids and bases under study (log k(MEKC)(I)) indeed lie on two distinct plateau, about -1.0 for the former and about 2.0 for the latter and depend on the solute's charge more than on its chemical structure. Thus, the derivation of a diff(log k(MEKC)(N-I)) value, defined as the difference between the log k(MEKC) values of the neutral and charged species, strongly correlates with the respective log k(MEKC)(N) value and does not afford additional information.

The ionic partition diagram methodology has been generalized to address both hydrophilic and lipophilic compounds and to consider biphasic systems with variable phase volume ratios. With this generalized approach electrochemical measurements of ion transfer potentials afford the determination of the standard partition coefficients of all forms of ionizable molecules, including the neutral form, as well as the evaluation of the dissociation constant of monoprotic substances. An interesting consequence of this approach is the definition of an extraction pK(a,ext) which is the apparent pK(a) of neutral acids and bases when dissolved in the organic phase.

Purpose: The "esterase-like activity" of human serum albumin (HSA) is described in the literature, but a contamination of commercially available HSA preparations by plasma cholinesterase is conceivable in some cases. The purpose of the present work was to examine this hypothesis., Methods: The hydrolytic activity of HSA and its inhibition by physostigmine were measured fluorimetrically by monitoring the hydrolysis of the ester substrate moxisylyte. Affinity chromatography was used to separate cholinesterase and HSA. The cholinesterase activity in the eluted fractions was assessed using Ellman's reagent and butyrylthiocholine as substrate., Results: A significant variation in the esterase-like activity of different albumin batches was observed. This activity was strongly inhibited by physostigmine, a well-known inhibitor of cholinesterase. Affinity chromatography led to a complete separation between HSA and the esterase activity, which was found exclusively in the cholinesterase fraction., Conclusions: The apparent esterase-like activity of HSA toward moxisylyte and butyrylthiocholine was due to a contamination by cholinesterase. With these substrates, HSA showed a total lack of esterase-like activity.

A set of 17 coumarin and 2 chromone derivatives with known inhibitory activity toward monoamine oxidase (MAO) A and B were tested as acetylcholinesterase (AChE) inhibitors. All compounds inhibited AChE with values in the micromolar range (3-100 microM). A kinetic study showed that most compounds acted as noncompetitive AChE inhibitors. This finding may be of interest in the context of Alzheimer's disease because recent observations suggest that MAO and AChE inhibition might decrease beta-amyloid deposition.

Literature observations indicate that some psychotropic drugs may have inhibitory activity towards monoamine oxidase (MAO). This study was undertaken to assess the potency, isozyme selectivity and mechanism of inhibition of representative first- and second-generation antidepressant drugs towards rat brain MAO-A and MAO-B. Five tricyclic antidepressants (imipramine, trimipramine, clomipramine, amitriptyline and doxepine) and three selective serotonin reuptake inhibitors (fluoxetine, fluvoxamine and citalopram) were examined. They showed inhibitory activity towards MAO-A and MAO-B, with clear selectivity for MAO-B (Ki in the micromolar range). Their mechanism of inhibition was competitive towards MAO-B and of a mixed competitive type towards MAO-A. The results suggest that some of the drugs examined might also contribute an MAO inhibitory effect in chronically treated patients.

Purpose: The partitioning of cetirizine in a phosphatidylcholine liposomes/water system was compared with that of hydroxyzine and acrivastine to gain insight into the mechanisms of interaction of its various electrical species with membranes., Methods: The lipophilicity profiles of the compounds were obtained from equilibrium dialysis and potentiometry, and compared with changes in NMR relaxation rates., Results: The neutral form of hydroxyzine interacted mainly via hydrophobic interactions with the bilayer lipid core of the membrane, whereas for the cationic form both hydrophobic and electrostatic interactions were involved. Zwitterionic and anionic cetirizine were less lipophilic than its cation, which behaved like the corresponding species of hydroxyzine. Zwitterionic cetirizine interacted more by weak electrostatic interactions with the polar headgroups of phospholipids than by hydrophobic interactions with the membrane interior. The lipophilicity of its anion reflected the balance of repulsive electrostatic interactions between the carboxylate and phosphate groups and the hydrophobic interactions with the lipid core., Conclusion: The study confirms that various mechanisms influence the interaction of solutes with liposomes. Combining experimental techniques and using suitable reference compounds proves useful.

Purpose: This work examines whether ion-pairing contributes to the apparent lipophilicity of cations, which is seen by a shake-flask or titrimetic method to be influenced by the nature and concentration of counter-ions., Methods: To solve this problem, the lipophilicity of several quaternary ammonium drugs was measured by cyclic voltammetry in the 1,2-dichloroethane/water system. The standard ionic partition coefficient values so obtained (log Pdce(o,C)) were correlated with log Poct values calculated by the CLOGP algorithm for the respective neutral molecules., Results: The standard (i.e., intrinsic) lipophilicity values are shown to depend on a, the structure of the ion (nature, volume, charge), and b, on the Galvani potential difference at the ITIES (interface between two immiscible electrolyte solutions)., Conclusions: The standard lipophilicity values were not influenced by counter-ions. In contrast, simulations showed that the increased apparent lipophilicity of cations, as measured by the shake-flask method in the presence of lipophilic anions, is fully accounted for by the resulting increase in the Galvani potential difference.

Hydrogen bonds are major forces of recognition in biochemistry and molecular pharmacology; they are an essential component of intermolecular interactions and determine to a significant extent the 3D-structure of bio-macromolecules. To explore three-dimensional H-bonding properties, a new tool called Molecular Hydrogen-Bonding Potentials (MHBPs) was created. The development of this tool is based on a stepwise procedure similar to the one used successfully to generate the Molecular Lipophilicity Potential (MLP). First, a H-bonding fragmental system was developed starting from published solvatochromic parameters. An atomic H-bonding donor fragmental value (alpha) is associated to each hydrogen atom in a polar moiety. Similarly, an atomic H-bonding acceptor fragmental value (beta) is associated to each polar atom. A distance function and an angle function were defined to take into account variations of the MHBPs in space. The fragmental system and the geometric functions were then combined to generate the MHBPs. These are calculated at each point of an adequate molecular surface or on a three-dimensional grid. The MHBPs were compared with GRID interactions energies and correlated with success to oral drug absorption data. Available examples demonstrate that the MHBPs are a promising computational tool in drug design. Their combination with CoMFA and VolSurf is being studied.

A large series of coumarin derivatives (71 compounds) were tested for their monoamine oxidase A and B (MAO-A and MAO-B) inhibitory activity. Most of the compounds acted preferentially on MAO-B with IC(50) values in the micromolar to low-nanomolar range; high inhibitory activities toward MAO-A were also measured for sulfonic acid esters. The most active compound was 7-[(3, 4-difluorobenzyl)oxy]-3,4-dimethylcoumarin, with an IC(50) value toward MAO-B of 1.14 nM. A QSAR study of 7-X-benzyloxy meta-substituted 3,4-dimethylcoumarin derivatives acting on MAO-B yielded good statistical results (q(2)() = 0.72, r(2)() = 0.86), revealing the importance of lipophilic interactions in modulating the inhibition and excluding any dependence on electronic properties. CoMFA was performed on two data sets of MAO-A and MAO-B inhibitors. The GOLPE procedure, with variable selection criteria, was applied to improve the predictivity of the models and to facilitate the graphical interpretation of results.

Clopidogrel hydrogen sulfate, a thienopyridine derivative, is an ADP receptor antagonist that inhibits platelet aggregation. Clopidogrel is an enantiopure carboxylic ester of S-configuration. The R-enantiomer is devoid of antithrombotic activity and can provoke convulsions at high doses in animals. During preclinical safety evaluation, the possible chiral inversion of clopidogrel has, therefore, been investigated in vivo after repeated oral administration of different dose levels of clopidogrel to male and female rats. Due to rapid metabolism in the liver and low plasma levels of unchanged drug, possible chiral inversion was assessed by monitoring the plasma concentrations of the carboxylic acid metabolites, i.e., the (S)- and (R)-acid, by means of a stereoselective assay. The production of 4 to 8% of (R)-acid was observed. This could be the result of chiral inversion of either clopidogrel or its main metabolite, the (S)-acid. Thus, the possibility of nonenzymatic and enzymatic inversion of clopidogrel and its carboxylic acid metabolite was studied in vitro by chiral HPLC and (1)H NMR. Nonenzymatic chiral inversion of clopidogrel at 37 degrees C in 0.1 M phosphate buffers could be observed but was found to be slow, with estimated half-lives of 7 to 12 days, depending on the pH. The (S)-acid was configurationally fully stable up to 45 days in phosphate buffers. Neither clopidogrel nor its carboxylic acid metabolites were subject to enzymatic chiral inversion in isolated rat hepatocyte suspensions. We conclude that the nonenzymatic inversion of clopidogrel accounts for the 4 to 8% of chiral inversion seen in vivo in the rat.