Claude Vaccher

Study of the complexation of risperidone and 9-hydroxyrisperidone with cyclodextrin hosts using affinity capillary electrophoresis and 1H NMR spectroscopy

The complexation of risperidone (Risp) and 9-hydroxyrisperidone (9-OH-Risp), atypical antipsychotics, with seven cyclodextrins (CDs) of pharmaceutical interest (native and hydroxypropylated (HP) alpha-, beta-, gamma-CDs and methyl (Me)-beta-CD) was studied by affinity capillary electrophoresis (ACE) and nuclear magnetic resonance spectroscopy (NMR) for acidic pH 2.5 and physiological pH 7.4. The 1:1 stoichiometry of the complexes was established by (1)H NMR spectroscopy using the continuous variation method developed by Job. The apparent binding constants of the 14 complexes at both pH were determined by ACE through the linear Scotts plots. The NMR spectroscopy investigation of the binding constants was achieved for the two CDs allowing the highest complexation: the beta-CD and Me-beta-CD. Both ACE and NMR spectroscopy studies provide similar conclusions by considering the influence of the 9-hydroxylation, the influence of the CD substitution and the influence of the pH. Moreover, the NMR spectroscopy results have allowed to suppose a pH-dependent inclusion mechanism. A thermodynamic study was then performed by ACE at both pH for the Risp.Me-beta-CD and 9-OH-Risp.Me-beta-CD complexes: the opposite signs of the entropic change (DeltaS degrees <0 at pH 2.5 and DeltaS degrees >0 at pH 7.4) confirms the influence of the pH on the complexation mechanism and the possible difference in the depth of the analyte inclusion in the hydrophobic cavity of the CD. Last, the two-dimensional ROESY (rotating-frame Overhauser spectroscopy) ((1)H-(1)H) and HOESY (heteronuclear Overhauser effect spectroscopy) ((19)F-(1)H) experiments have proved the inclusion of the aromatic part of the Risp and 9-OH-Risp in the hydrophobic CD cavity and lead us to propose a model of complexation.

Comparative study of the complex forming ability and enantioselectivity of cyclodextrin polymers by CE and 1H NMR.

The interactions between nine drugs (baclofen, bupivacaine, chlorpheniramine, ketoconazole, paliperidone, promethazine, propranolol, risperidone and verapamil) and six cyclodextrins (α-CD, β-CD, γ-CD, HP-β-CD, HP-γ-CD and Me-β-CD) or six polymers of cyclodextrins (polyα-CD, polyβ-CD, polyγ-CD, polyHP-β-CD, polyHP-γ-CD and polyMe-β-CD) were studied by affinity capillary electrophoresis and/or (1)H NMR at pH 2.5. An exhaustive qualitative study was performed through the determination of the retardation factor. Then, four compounds and both β-CD and polyβ-CD were selected for the quantitative study of the interactions at pH 2.5 and 7.0. By comparing the results obtained with the β-CD and polyβ-CD, it appears that the apparent binding constants are up to five times higher with the polymer. The 2D-NMR results seem to indicate that the structure of the polymeric network favours the inclusion of the guest in the hydrophobic cavity of the CD units. Moreover, the poly-CDs have shown very high enantioselective abilities at both pH.

Benzofuran analogues of baclofen: a new class of central and peripheral GABAB-receptor antagonists.

Two novel beta-(benzo[b]furan) analogues of baclofen (4-amino-3-benzo[b]furan-2-ylbutanoic acid, 9G, and 4-amino-3-(5-methoxybenzo[b]furan-2-yl)butanoic acid, 9H) antagonised the baclofen-induced depression of twitch contractions in the guinea-pig isolated ileum (estimated apparent pA2 3.9 and 4.1 respectively); both 9G and 9H also antagonised in a dose-dependent manner the baclofen-induced reduction of repetitive paroxysmal discharges in rat neo-cortical slice preparations maintained in Mg2+-free Krebs solution. These benzofurans evidently represents a new class of GABAB-receptor antagonist.

P-glycoprotein and cytochrome P450 3A4 involvement in risperidone transport using an in vitro Caco-2/TC7 model and an in vivo model.

The possible involvement of P-glycoprotein (P-gp) and cytochrome P450 (CYP) 3A4 in risperidone transport was investigated using in vitro and in vivo models. Firstly, uptake studies were performed on a Caco-2/TC7 cell monolayer; the effects of 1 microg ml(-1) risperidone on apparent permeability were determined for secretory and absorptive directions, in the presence or absence of various P-gp and CYP3A4 inhibitors (verapamil, ketoconazole, erythromycin), and of an associated multidrug-resistant protein inhibitor (indomethacin). Secondly, on a conscious rat model, risperidone pharmacokinetic parameters, notably absorption parameters, were determined using compartmental and deconvolution methods. Three groups of seven rats received respectively an IV risperidone dose, an oral risperidone dose (PO group) and the same oral risperidone dose after verapamil administration (POV group). No formation of 9-hydroxyrisperidone was observed on Caco-2 cells after risperidone administration; there was no evidence that intestinal CYP3A4 is involved in risperidone metabolising. Risperidone secretory permeation was higher than absorptive permeation. Verapamil increased risperidone absorption permeation and decreased its secretory permeation. Indomethacin did not modify these permeation values. In rats, verapamil led to an increase in both risperidone and 9-hydroxyrisperidone plasmatic concentrations. The fraction absorbed in the verapamil group was 3.18 times higher than in the oral group (65.9% and 20.7% for POV group and PO group). The absorption rate constant was lower in the verapamil group. Our results indicate that P-gp decreases the intestinal absorption of risperidone and that intestinal CYP3A4 is not involved in risperidone metabolism.

Diastereomeric resolution of nucleoside analogues, new potential antiviral agents, using high-performance liquid chromatography on polysaccharide-type chiral stationary phases.

This paper describes the separation of the four sets of stereoisomers of nucleoside analogs, new potential antiviral agents by direct analytical HPLC methods using derivatized cellulose and amylose chiral stationary phases. The resolution was made using normal-phase methodology with a mobile phase consisting of n-hexane-alcohol (ethanol or 2-propanol) in various percentages, and a silica-based cellulose tris-3,5-dimethylphenylcarbamate (Chiralcel OD-H), or tris-methylbenzoate (Chiralcel OJ) and a silica-based amylose tris-3,5-dimethylphenylcarbamate (Chiralpak AD) or tris-(S)-1-phenylethylcarbamate (Chiralpak AS). The effects of structural features on the extent of discrimination between the stereoisomers were examined through the retention, the selectivity and the resolution factors as well as the elution order. Baseline separation (Rs>1.5) was easily obtained in many cases. The resolution results were complementary between the different columns.

A chemometric approach to elucidate the parameter impact in the hyphenation of evaporative light scattering detector to supercritical fluid chromatography.

The aim of this work was to elucidate the effects of parameters influencing the evaporative light scattering detector (ELSD) response when it was coupled to supercritical fluid chromatography (SFC). Phthalates, currently used as plasticizers in medical devices, were selected as model compounds. The configuration of the hyphenation setup was firstly optimized and shown that both peak efficiency and sensitivity were improved by connecting the ELSD to the SFC before the back pressure regulator (BPR). By using a tee-junction which splits the flow after the PDA towards the collect fraction (or waste) and the ELSD, this instrument configuration has the advantage to be applicable for small-scale preparative SFC. The impacts of other parameters such as mobile phase composition and flow rate, outlet pressure, column oven temperature and ELSD drift tube temperature on the ELSD signal were evaluated using a chemometric approach. First, it was demonstrated that a classical mobile phase composed of CO2-methanol 90:10 (v/v) was suitable to obtain great nebulization efficiency. The flow rate of the eluent was the second main effect factor. The setting must be as low as possible to avoid the loss of large particle size in the drift tube resulting in a loss of signal intensity. Concerning the outlet pressure, the configuration of the setup between SFC and ELSD requires a setting as high as possible to limit the partial liquid-vapor separation of the mobile phase in the restrictor tube. Finally, due to the low quantity of solvent which must be evaporated in the detector, a drift tube temperature of 25 °C is suitable for the hyphenation of ELSD to SFC. In the optimized conditions, the proposed SFC/ELSD method could be suitable to quantify plasticizers in medical devices.

Dual CD system in capillary electrophoresis for direct separation of the four stereoisomers of agonist and antagonist melatoninergic ligands

In this study, baseline separation of the stereoisomers of six tetrahydronaphthalenic derivatives (agonists and antagonists for the melatonin (N‐acetyl‐5‐methoxytryptamin) binding sites) was successfully achieved using CE and CDs as chiral selectors. The method for the simultaneous chiral separation of the four stereoisomers uses a capillary dynamically coated with polyethylene oxide and a dual CD system. Optimisation was performed first upon the constituents of the CD system, by varying neutral and anionic CD type, size and concentration, at first in mono‐CD systems and subsequently in dual neutral/anionic CD systems. Once these characteristics of the dual CD system were established, operational parameters such as voltage and temperature were then optimised. Under the optimal conditions (i.e. 1.5% w/v of highly S‐β‐CD and 10 mM of γ‐CD in 25 mM phosphate buffer (pH 2.5) as the BGE, separation voltage 20 kV and a temperature of 25°C), complete resolution of the six molecules was accomplished. Preliminary results for repeatability and the migration order of the optimised method are described.

Chiral capillary electrophoretic determination of the enantiomeric purity of tetrahydronaphthalenic derivatives, melatoninergic ligands, using highly sulfated β‐cyclodextrins

Using cyclodextrin capillary zone electrophoresis (CD‐CZE), baseline separation of synthetic tetrahydronaphthalenic derivatives, potential melatoninergic compounds, was achieved. A method for the enantioresolution of these tetralins and determination of their enantiomeric purity was developped using anionic CDs (highly sulfated‐CD or highly S‐CD) as chiral selectors and capillaries dynamically coated with polyethylene oxide (PEO). Operational parameters such as the nature and concentration of the chiral selectors, buffer pH, organic modifiers, temperature and applied voltage were investigated. The use of charged CDs provides a driving force for our neutral compounds in the running buffer and enantiomeric resolution by inclusion of compounds in the CD cavity. The highly S‐β‐CD was found to be the most effective complexing agent, allowing good enantiomeric resolution. The complete resolution of three tetralin compounds was obtained using 25 mM phosphate buffer at pH 2.5 containing 2.5% w/v of highly S‐β‐CD at 25°C with an applied field of 0.25 kV/cm. The apparent association constants of the inclusion complexes were calculated. This optimized method was validated in terms of linearity, sensitivity, accuracy and recovery. The enantiomeric purity for the three molecules was determined and the detection limit of enantiomer impurities is about 0.3–0.6%.

Synthesis, pharmacology and X-ray studies of baclofen analogues

Baclofen (β-p-chlorophenyl-GABA) is the reference selective agonist for the bicuculline-insensitive GABAB receptor. The search for new compounds having a high affinity for the GABAB receptor is very important to clarify structural requirements. In that sense, we report the synthesis, binding studies and X-ray determinations of various 3-heteroaromatic γ-aminobutyric acids. Biochemical investigations concerning their abilities to displace [3H] muscimol (GABAA) and [3H] baclofen (GABAB) in binding studies showed that the 4-amino-3-(5-methoxybenzo[b]furan-2-yl)butanoic acid 6a (IC50 = 22.16 μM/R (−) [3H] baclofen; IC50 = 5.6 μM/RS [3H] baclofen) has a specific affinity for the GABAB receptor. The crystal structure of compounds 6a and 6b associated with computer graphics molecular superimpositions allows some structural requirements for GABAB receptor ligands to be proposed.

Direct separation of 4-amino-3-(4-chlorophenyl)butyric acid and analogues, GABAB ligands, using a chiral crown ether stationary phase

Abstract The direct resolution of baclofen (β- p -chlorophenyl-γ-aminobutyric acid) and a series of four analogues was achieved by HPLC on an enantioselective, crown ether column, CR(+). Chromatography was carried out with perchloric acid as mobile phase and methanol as organic modifier. The effects of temperature, pH, eluent composition and substituents are discussed. The absolute configuration is attributed. The best results were obtained with baclofen (α = 1.48; R s = 8.07).