Synthesis of molecular imprinted polymers for selective extraction of domperidone from human serum using high performance liquid chromatography with fluorescence detection.

In this study a novel method is described for selective quantization of domperidone in biological matrices applying molecular imprinted polymers (MIPs) as a sample clean up procedure using high performance liquid chromatography coupled with a fluorescence detector. MIPs were synthesized with chloroform as the porogen, ethylene glycol dimethacrylate as the crosslinker, methacrylic acid as the monomer, and domperidone as the template molecule. The new imprinted polymer was used as a molecular sorbent for separation of domperidone from serum. Molecular recognition properties, binding capacity and selectivity of MIPs were determined. The results demonstrated exceptional affinity for domperidone in biological fluids. The domperidone analytical method using MIPs was verified according to validation parameters, such as selectivity, linearity (5-80ng/mL, r(2)=0.9977), precision and accuracy (10-40ng/mL, intra-day=1.7-5.1%, inter-day=4.5-5.9%, and accuracy 89.07-98.9%).The limit of detection (LOD) and quantization (LOQ) of domperidone was 0.0279 and 0.092ng/mL, respectively. The simplicity and suitable validation parameters makes this a highly valuable selective bioequivalence method for domperidone analysis in human serum.

Oral administration of levo-tetrahydropalmatine attenuates reinstatement of extinguished cocaine seeking by cocaine, stress or drug-associated cues in rats.

Cocaine addiction is characterized by a persistently heightened susceptibility to drug relapse. For this reason, the identification of medications that prevent drug relapse is a critical goal of drug abuse research. Drug re-exposure, the onset of stressful life events, and exposure to cues previously associated with drug use have been identified as determinants of relapse in humans and have been found to reinstate extinguished cocaine seeking in rats. This study examined the effects of acute oral (gavage) administration of levo-tetrahydropalmatine (l-THP), a tetrahydroprotoberberine isoquinoline with a pharmacological profile that includes antagonism of D1, D2 and D3 dopamine receptors, on the reinstatement of extinguished cocaine seeking by a cocaine challenge (10mg/kg, ip), a stressor (uncontrollable electric footshock [EFS]) or response-contingent exposure to a stimulus (tone and light complex) previously associated with drug delivery in male Sprague-Dawley rats. Extinguished drug seeking was reinstated by ip cocaine, EFS, or response-contingent presentation of drug-associated cues in vehicle-pretreated rats following extinction of iv cocaine self-adminisration. Oral administration of either 3.0 or 10.0mg/kg l-THP 1h prior to reinstatement testing significantly attenuated reinstatement by each of the stimuli. Food-reinforced responding and baseline post-extinction responding were significantly attenuated at the 10.0, but not the 3.0mg/kg, l-THP dose, indicating that the effects of 3mg/kg l-THP on reinstatement were likely independent of non-specific motor impairment. These findings further suggest that l-THP may have utility for the treatment of cocaine addiction.

Screening and isolation of a natural dopamine D1 receptor antagonist using cell-based assays.

To develop a cell-based assay to screen for human dopamine D(1) receptor agonists or antagonists from medicinal plant extracts, a stable Chinese hamster ovary (CHO) cell line (CHO-D1R) expressing the human dopamine D(1) receptor was established using an expression vector containing a scaffold attachment region (SAR) element. CHO-D1R cells showed specific binding to [(3)H]-SCH23390 with high affinity (K(d)=1.47+/-0.17 nM) and dose-dependent responses for the dopamine-mediated stimulation of cAMP concentrations (EC(50)=20.6+/-1.44 nM). The screening of medicinal plant extracts using cell-based cAMP assays revealed that an extract of Gleditsia sinensis Lam., which is known to be rich in saponin, had strong antagonist activity for the D(1) receptor. From the activity-guided fractionation and chemical structural analysis of the G. sinensis extract, a compound called gleditsioside F was isolated and was identified to have antagonist activity for the D(1) receptor. Gleditsioside F showed very effective D(1) antagonist activity by inhibiting ligand binding to the D(1) receptor as well as by inhibiting dopamine-mediated increases in cAMP concentration.

Liriodenine inhibits dopamine biosynthesis and L-DOPA-induced dopamine content in PC12 cells.

The inhibitory effects of liriodenine, an aporphine isoquinoline alkaloid, on dopamine biosynthesis and L-DOPA-induced dopamine content increases in PC12 cells were investigated. Treatment of PC12 cells with 5-10 microM liriodenine significantly decreased the intracellular dopamine content in a concentration-dependent manner (IC50 value, 8.4 microM). Liriodenine was not cytotoxic toward PC12 cells at concentrations up to 20 microM. Tyrosine hydroxylase (TH) and aromatic L-amino acid decarboxylase (AADC) activities were inhibited by 10 microM liriodenine to 20-70% and 10-14% of control levels at 3-12 h, respectively; TH activity was more influenced than AADC activity. The levels of TH mRNA, intracellular cyclic AMP and basal Ca2+ concentration were also decreased by 10 microM liriodenine. In addition, 10 microM liriodenine reduced L-DOPA (20-100 microM)-induced increases in dopamine content. However, 10 microM liriodenine resulted in a protective effect against L-DOPA (50-100 microM)-induced cytotoxicity. These results suggest that liriodenine regulates dopamine biosynthesis by partially reducing TH activity and TH gene expression and has protective effects against L-DOPA-induced cytotoxicity in PC12 cells.

Multi-criteria decision making approach and experimental design as chemometric tools to optimize HPLC separation of domperidone and pantoprazole.

This paper deals with multiple response simultaneous optimization using the Derringer's desirability function for the development of a reversed-phase HPLC method for the simultaneous determination of domperidone and pantoprazole in commercial pharmaceutical preparations. Twenty experiments, taking the retention factor of the first peak, the two resolutions, and three retention times as the responses with three important factors, mobile phase composition, buffer molarity and flow rate, were used to design mathematical models. The experimental responses were fitted into a second order polynomial and the six responses simultaneously optimized to predict the optimum conditions for the effective separation of the studied compounds. The optimum assay conditions were: methanol-acetonitrile-dipotassium hydrogen phosphate (pH 7.0; 15.3 mM) (20:33:47, v/v/v) as the mobile phase and at a flow rate of 1.19 ml/min. While using this optimum condition, baseline separation with a minimum resolution of 2.0 and a run time of less than 6 min were achieved. The method showed good agreement between the experimental data and predictive value throughout the studied parameter space. The optimized assay condition was validated according to ICH guidelines to confirm specificity, linearity, accuracy and precision.

Radiotracer synthesis from [(11)C]-iodomethane: a remarkably simple captive solvent method.

A new method of [(11)C]-methylation is described, which attains the goals of simplicity, high radiochemical yields, speed, versatility, and automation. A standard high performance liquid chromatography (HPLC) injection loop on a standard HPLC injection valve is loaded with a solution (80 microL) of precursor (0.3-1.0 mg) in dimethyl formamide (DMF) or dimethyl sulfoxide (DMSO) (+ base if required). At ambient temperature [(11)C]-iodomethane is passed through the loop for 3-4 min with >90% trapping of activity. After a further 1-5 min, the contents of the loop are quantitatively injected onto the HPLC column for purification. Radiochemical yields are equal to or superior to conventional solution methods in all cases, even though no heat is applied. [(11)C]-labeled radiotracers that have been prepared by this method for human or animal studies include Raclopride, N-methylspiperone, Ro 15-1788, FLB 457, RTI-32, Rolipram, SCH 23390, and SKF 82957. Since no vials, transfer lines, cooling, heating, or sealing valves are required, no transfer losses occur, yields are high, and cleanup is minimal, this "loop method" is ideal for most radiopharmaceuticals prepared from [(11)C]-iodomethane.