CB1 receptor-mediated control of the release of endocannabinoids (as assessed by microdialysis coupled with LC/MS) in the rat hypothalamus.

In the present study, we examined the occurrence and potential regulation of endocannabinoid release by cannabinoid CB1 receptors in the rat brain. To this end, we developed a highly sensitive (limit of sensitivity 30-300 amol) new analytical method, combining online brain microdialysis with solid-phase extraction-liquid chromatography-tandem mass spectrometry, which allowed the detection in real time of trace amounts of endocannabinoids in the extracellular fluid. In the hypothalamus, anandamide and 2-arachidonoyl-glycerol release was stimulated following depolarization via local administration of K(+), with or without addition of Ca(2+), or glutamate application. Inhibition of fatty acid amide hydrolase by systemic administration of intraperitoneal (i.p.) URB597 (0.5 mg/kg) induced an increase of anandamide, but not 2-arachidonoyl-glycerol, outflow. The CB1 receptor antagonist rimonabant (10 mg/kg i.p.) increased, whereas the CB1 agonist WIN55,212-2 (2.5 mg/kg i.p.) decreased, anandamide release. Interestingly, the same treatments induced opposite changes in 2-arachidonoyl-glycerol release. At a dose of 3 mg/kg i.p., which by itself did not affect endocannabinoid release, rimonabant fully antagonized the effect of WIN55,212-2 (2.5 mg/kg i.p.). Taken together, these results suggest that CB1 receptors are able to control the local release of endocannabinoids in the hypothalamus via a feedback mechanism and strengthen the view that anandamide and 2-arachidonoyl-glycerol have distinct physiological roles.

Chiral separation strategy in polar organic solvent chromatography and performance comparison with normal-phase liquid and supercritical-fluid chromatography.

A strategy, including a rapid screening and several optimisation steps, for the separation of chiral molecules of pharmaceutical interest by polar organic solvent chromatography (POSC), using four polysaccharide-based stationary phases, is proposed and compared with previously reported strategies in normal-phase (NPLC) and supercritical fluid chromatography (SFC). In a first part of this paper, different examples demonstrate the effectiveness of the POSC strategy for fast method development. Optimisation is based on the use of experimental design to map the experimental domain in an efficient way. In the second part, the best screening results, obtained after performance of earlier defined chromatographic screening strategies in NPLC and SFC, are compared to those obtained in POSC. The three techniques show complementary separation results and allowed baseline separation of 23 of 25 compounds. POSC is found to be a very interesting separation mode compared to NPLC, because of the many fast (< 10 min) baseline separations obtained.

Screening approach for chiral separation of pharmaceuticals IV. Polar organic solvent chromatography.

The aim of this work is to determine generic screening conditions and an initial simple separation strategy allowing the rapid separation of drug enantiomers in polar organic solvent chromatography (POSC). Four cellulose/amylose-based stationary phases were investigated in detail using two mobile phase basis solvents commonly applied in this mode, i.e. acetonitrile and methanol. Polar mode is interesting for use in purification of enantiomers. In a first step, the parameters potentially influencing the separation, such as addition of an alcohol to the polar organic solvent or the type of mobile phase additive(s), were examined by means of experimental designs. Afterwards, the factors found most important are investigated in more detail. Results showed that the cellulose- and amylose-based stationary phases have very broad and complementary enantiorecognition abilities in the POSC mode. The type of organic solvent for the mobile phase appeared to have a dramatic influence on the quality of the separation. Based on the results, a screening strategy was proposed. Enantioseparation was observed in more than 85% of the tested compounds and analysis times of last eluted peak were usually below 10 min.

Strategy for the chiral separation of non-acidic pharmaceuticals using capillary electrochromatography.

In completion of an earlier defined generic chiral screening approach, a generic separation strategy for basic, bifunctional, and neutral compounds was proposed and evaluated. This strategy adds to a previously defined strategy for acidic compounds. The screening experiment of the actual strategy used a mobile phase of 5 mM phosphate buffer pH 11.5/ACN (30/70 v/v), a temperature of 25 degrees C, and a voltage of 15 kV. The selected chiral stationary phases were Chiralpak AD-RH, Chiralcel OD-RH, Chiralcel OJ-RH, and Chiralpak AS-RH, all based on polysaccharide selectors. It was seen that 31 out of 48 test compounds were partially or baseline-resolved under screening conditions. After execution of the optimization steps of the strategy, this number increased to 41, with a total of 21 baseline-separated compounds. Combined with the results obtained from the acidic test set examined in the earlier defined strategy, of all tested compounds 82.5% showed enantioselectivity and 49.2% could be baseline-separated.

Screening approach for chiral separation of pharmaceuticals. Part III. Supercritical fluid chromatography for analysis and purification in drug discovery.

High-throughput and performance analysis and purification of enantiomers are important parts of drug discovery and provide high-quality compounds for pharmacological testing. We have previously reported two parts describing chiral chromatographic screens using normal-phase (NPLC) and reversed-phase (RPLC) liquid chromatography, in order to cope with increasing numbers of new compounds generated by chemistry programs. We present in this part the development and implementation of a third faster screen using supercritical fluid chromatography (SFC) to maximize chance in achieving rapid enantiomer resolution of large numbers of compounds in a minimum of time. The SFC screen utilizes a narrow combination of only four columns (Chirlapak AD and AS, and Chiralcel OD and OJ) and two solvent modifiers (methanol and isopropanol). A modifier and column-switching setup was employed to allow the entire screening process to be serially run in the order AD> OD > OJ > AS and methanol > isopropanol, so that the screening for a given molecule can be stopped when separation is achieved. The switching system was fully automated for unattended operation of multiple compounds. An optimization procedure was also defined, which can be performed if needed for unsuccessful separations in the screening step. The chiral SFC strategy proved its performance and robustness in resolution of hundreds proprietary chiral molecules generated by drug discovery programs, with a success rate exceeding 95%. In addition, the generic capability of the strategy was evaluated by applying the screen and optimization methodology to a test set comprising 40 marketed drugs differing from proprietary compounds in terms of chemical diversity, revealing a similar high success rate of 98%. Chiral separations developed at the analytical scale work easily and equally well at the semi-preparative level, as illustrated with an example. The SFC screen allows resolution of compounds that were partially separated by NPLC or not separated at all by RPLC, demonstrating the utility of implementing complementary chromatographic techniques. The SFC screen is currently an integral part of our analytical support to discovery chemical programs and is considered the first try for chiral separations of new compounds, because it offers a higher success rate, performance and throughput.

Capillary electrochromatographic chiral separations with potential for pharmaceutical analysis.

The use of capillary electrochromatography as a chiral separation technique for pharmaceutical applications is reviewed. Publications of the past 10 years that provide a potential practical application in pharmaceutical analysis are considered. Method development or validation, separation strategies, and potential routine analysis by the methods/applications cited are the main subjects on which we focused our attention. The indirect chiral separation method was only used once in CEC mode. In the direct chiral separations, the use of chiral stationary phases was obviously preferred over the use of chiral mobile phases with non-chiral stationary phases. Amongst the chiral stationary phases, those based on macrocyclic antibiotics and polysaccharide selectors were the most frequently used. Monolithic stationary phases also have several applications, but not so extended as those with packed capillary electrochromatography. The considered papers not only describe the applicability of the technique for relatively large sets of chiral analytes, they also showed that various types of stationary phases can be produced in-house in a simple manner. However, to survive as a mature separation technique, considerable time and effort are still needed to solve some disadvantages currently characterizing capillary electrochromatography.

Separation strategy for acidic chiral pharmaceuticals with capillary electrochromatography on polysaccharide stationary phases.

The effect of five factors on the capillary electrochromatographic enantioseparation of acidic compounds was studied using an experimental design. The studied factors were pH, acetonitrile content in the mobile phase, temperature, buffer concentration, and applied voltage. These experiments allowed defining a generic separation strategy applicable on acidic compounds with chemical and structural diversity. The starting screening conditions consist of a 45 mM ammonium formate electrolyte at pH 2.9 mixed with 65% acetonitrile, an applied voltage of 15 kV, and a temperature of 25 degrees C. The screening phase occasionally can be followed by an optimization procedure. Evaluation of the proposed strategy pointed out that it allows achieving baseline resolution within a relatively short time when a beginning of separation is obtained at the starting conditions. This strategy revealed enantioselectivity for 11 compounds out of 15, of which 10 could be baseline-separated after the proposed optimization steps.

Enantioseparations by capillary electro-chromatography: differences exhibited by normal- and reversed-phase versions of polysaccharide stationary phases.

The influence of using normal-phase and reversed-phase versions of four commercial polysaccharide stationary phases on chiral separations was investigated with capillary electrochromatography (CEC). Both versions of the stationary phases, Chiralcel OD, OJ, and Chiralpak AD, AS were tested for the separation of two basic, two acidic, a bifunctional, and a neutral compound. Different background electrolytes were used, two at low pH for the acid, bifunctional and neutral substances, and three at high pH for the basic, bifunctional and neutral ones. This setup allowed evaluating differences between both stationary-phase versions and between mobile-phase compositions on a chiral separation. Duplicate CEC columns of each stationary phase were in-house prepared and tested, giving information about the intercolumn reproducibility. In general, reversed-phase versions of the current commercial polysaccharide stationary phases are found to be best for reversed-phase CEC, even though at high pH no significant differences were seen between both versions. Most differences were observed at low pH. For acidic compounds, it was seen that an ammonium formate electrolyte performed best, which is also an excellent electrolyte if coupling with mass spectrometry is desired. For basic, bifunctional and neutral compounds, no significant differences between the three tested electrolytes were observed at high pH. Here, a phosphate buffer is preferred as electrolyte because of its buffering capacities. However, if coupling to mass spectrometry is wanted, the more volatile ammonium bicarbonate electrolyte can be used as an alternative.

Enantioseparations of basic and bifunctional pharmaceuticals by capillary electrochromatography using polysaccharide stationary phases.

A fast screening strategy was developed in capillary electrochromatography (CEC) for the chiral separation of basic and bifunctional compounds. The screening conditions were determined on polysaccharide chiral stationary phases using 15 pharmaceutical compounds. The content and type of organic modifier, as well as the pH of the mobile phase appeared to have the largest influence on the chiral resolution. It was seen that for acidic compounds, our approach was not suitable. A generic mobile phase for basic and bifunctional compounds was determined. The testing on 20 additional compounds showed that the proposed mobile phase performed well since enantioselectivity was observed for 86% of the investigated compounds. A comparison of CEC and reversed-phase liquid chromatography (RPLC) results was attempted to demonstrate the potential of the used technique for chiral method development.

Robustness testing of chiral separations by capillary electrophoresis using highly-sulfated cyclodextrins.

The robustness of a generic method for chiral separation in capillary electrophoresis using highly-sulfated cyclodextrins in a low pH phosphate buffer and the "short-end injection technique" was studied. In this study, we focused on the robustness of the separations and not of the quantitative analysis of the enantiomers. The robustness was evaluated for the enantiomeric separation of a basic (propranolol), a neutral (praziquantel) and an acidic (warfarin) compound. The influence of eight factors which were believed to affect significantly the separations was studied using a 11-factor, 12-experiment Plackett-design. Statistical interpretation of the factor effects on different analytical responses (selectivity and resolution) was performed. The separations of the three compounds could be considered as rather robust as the factor effects were generally not significant (alpha = 0.05) and small.

Structure and stereochemistry of the active metabolite of clopidogrel.

Clopidogrel (SR25990C, PLAVIX) is a potent antiplatelet drug, which has been recently launched and is indicated for the prevention of vascular thrombotic events in patients at risk. Clopidogrel is inactive in vitro, and a hepatic biotransformation is necessary to express the full antiaggregating activity of the drug. Moreover, 2-oxo-clopidogrel has been previously suggested to be the essential key intermediate metabolite from which the active metabolite is formed. In the present paper, we give the evidence of the occurrence of an in vitro active metabolite after incubation of 2-oxo-clopidogrel with human liver microsomes. This metabolite was purified by liquid chromatography, and its structure was studied by a combination of mass spectometry (MS) and NMR experiments. MS results suggested that the active metabolite belongs to a family of eight stereoisomers with the following primary chemical structure: 2-[1-[1-(2-chlorophenyl)-2-methoxy-2-oxoethyl]-4-sulfanyl-3-piperidinylidene]acetic acid. Chiral supercritical fluid chromatography resolved these isomers. However, only one of the eight metabolites retained the biological activity, thus underlining the critical importance of associated absolute configuration. Because of its highly labile character, probably due to a very reactive thiol function, structural elucidation of the active metabolite was performed on the stabilized acrylonitrile derivative. Conjunction of all our results suggested that the active metabolite is of S configuration at C 7 and Z configuration at C 3-C 16 double bound.