Multivariate pharmacokinetic/pharmacodynamic (PKPD) analysis with metabolomics shows multiple effects of remoxipride in rats.

The study of central nervous system (CNS) pharmacology is limited by a lack of drug effect biomarkers. Pharmacometabolomics is a promising new tool to identify multiple molecular responses upon drug treatment. However, the pharmacodynamics is typically not evaluated in metabolomics studies, although being important properties of biomarkers. In this study we integrated pharmacometabolomics with pharmacokinetic/pharmacodynamic (PKPD) modeling to identify and quantify the multiple endogenous metabolite dose-response relations for the dopamine D2 antagonist remoxipride. Remoxipride (vehicle, 0.7 or 3.5mg/kg) was administered to rats. Endogenous metabolites were analyzed in plasma using a biogenic amine platform and PKPD models were derived for each single metabolite. These models were clustered on basis of proximity between their PKPD parameter estimates, and PKPD models were subsequently fitted for the individual clusters. Finally, the metabolites were evaluated for being significantly affected by remoxipride. In total 44 metabolites were detected in plasma, many of them showing a dose dependent decrease from baseline. We identified 6 different clusters with different time and dose dependent responses and 18 metabolites were revealed as potential biomarker. The glycine, serine and threonine pathway was associated with remoxipride pharmacology, as well as the brain uptake of the dopamine and serotonin precursors. This is the first time that pharmacometabolomics and PKPD modeling were integrated. The resulting PKPD cluster model described diverse pharmacometabolomics responses and provided a further understanding of remoxipride pharmacodynamics. Future research should focus on the simultaneous pharmacometabolomics analysis in brain and plasma to increase the interpretability of these responses.

Comparison of the agonist-antagonist interaction model and the pool model for the effect of remoxipride on prolactin.

AIMS: The tolerance to the prolactin response following administration of antipsychotic drugs has been modelled as a depletion of a prolactin pool (pool model) and a model where the tolerance is explained by a feedback loop including the dopamine interaction of prolactin release (agonist-antagonist interaction model, (AAI model)). The AAI model was superior to the pool model when analyzing data from clinical trials of risperidone and paliperidone. Here we evaluated the two models using the remoxipride data, designed to challenge the short-term prolactin response, from which the original pool model was built. METHODS: The remoxipride data were collected from a study where eight healthy male subjects received two remoxipride infusions on five occasions. The intervals between the first and second dose on each occasion were 2, 8, 12, 24 and 48 h, respectively. The pool and AAI models were fitted using NONMEM. RESULTS: According to the objective function values the pool model with a circadian rhythm function fitted the data slightly better, while the AAI model was better in describing the circadian rhythm of prolactin. Visual predictive checks revealed that the models predicted the prolactin profiles equally well. CONCLUSIONS: According to the analysis performed here, a previous analysis of several clinical studies and literature reports on prolactin concentrations, it appears that the dopamine feedback mechanism included in the AAI model is better than the storage depletion mechanism in the pool model to estimate the bio-rhythm of prolactin time-course and the tolerance development across different populations, drugs, treatment schedules and time.

Online solid phase extraction with liquid chromatography-tandem mass spectrometry to analyze remoxipride in small plasma-, brain homogenate-, and brain microdialysate samples.

Remoxipride is a selective dopamine D(2) receptor antagonist, and useful as a model compound in mechanism-based pharmacological investigations. To that end, studies in small animals with serial sampling over time are needed. For these small volume samples currently no suitable analytical methods are available. We propose analytical methods for the detection of low concentrations remoxipride in small sample volumes of plasma, brain homogenate, and brain microdialysate, using online solid phase extraction with liquid chromatography-tandem mass spectrometry. Method development, optimization and validation are described in terms of calibration curves, extraction yield, lower limit of quantification (LLOQ), precision, accuracy, inter-day- and intra-day variability. The 20 microl plasma samples showed an extraction yield of 76%, with a LLOQ of 0.5 ng/ml. For 0.6 ml brain homogenate samples the extraction yield was 45%, with a LLOQ of 1.8 ng/ml. The 20 microl brain microdialysate samples, without pre-treatment, had a LLOQ of 0.25 ng/ml. The precision and accuracy were well within the acceptable 15% range. Considering the small sample volumes, the high sensitivity and good reproducibility, the analytical methods are suitable for analyzing small sample volumes with low remoxipride concentrations.

FemtoMolar measurements using accelerator mass spectrometry.

Accelerator mass spectrometry (AMS) is an ultra-sensitive analytical method suitable for the detection of sub-nM concentrations of labeled biological substances such as pharmaceutical drugs in body fluids. A limiting factor in extending the concentration measurements to the sub-pM range is the natural (14)C content in living tissues. This was circumvented by separating the labeled drug from the tissue matrix, using standard high-performance liquid chromatography (HPLC) procedures. As the separated total drug amount is in the few fg range, it is not possible to use a standard AMS sample preparation method, where mg sizes are required. We have utilized a sensitive carbon carrier method where a (14)C-deficient compound is added to the HPLC fractions and the composite sample is prepared and analyzed by AMS. Using 50 microL human blood plasma aliquots, we have demonstrated concentration measurements below 20 fM, containing sub-amol amounts of the labeled drug. The method has the immediate potential of operating in the sub-fM region.

High sensitivity determination of the remoxipride hydroquinone metabolite NCQ-344 in plasma by coupled column reversed-phase liquid chromatography and electrochemical detection.

An HPLC method for the determination of NCQ-344, a remoxipride metabolite with a hydroquinone structure, in human plasma is described. Special precautions for the sampling were necessary as the compound rapidly decomposes. An efficient clean-up of the plasma samples was necessary to make use of the inherent sensitivity of the electrochemical detector. This was accomplished by a fast and simple liquid-liquid extraction at pH 7.05 combined with further cleaning on-line by using a short cyanopropyl column as the first column in a column switching system. A heart-cut from the cyanopropyl column containing the NCQ-344 fraction was then injected onto the analytical octadecyl silica column and NCQ-344 was detected at an oxidation potential of 0.70 V. The absolute recovery was > 95% and concentrations down to 0.10 nM could be determined with acceptable precision. The NCQ-344 levels in a limited number of samples from patients given remoxipride were found to be between 0.10 and 1 nM. The remoxipride concentrations in the same samples were 5,000-20,000 nM.

Stereoselective determination of R-(+)- and S-(-)-remoxipride, a dopamine D2-receptor antagonist, in human plasma by chiral high-performance liquid chromatography.

A stereoselective high-performance liquid chromatographic (HPLC) method is described for the selective and sensitive quantitation in human plasma of R-(+)- and S-(-)-enantiomers of remoxipride. Remoxipride was extracted from basified plasma into hexane-methyl-tert.-butyl ether (20:80, v/v), washed with sodium hydroxide (1.0 M), then back-extracted into phosphoric acid (0.1 M). A structural analog of remoxipride was used as an internal standard. The sample extracts were chromatographed using a silica-based derivatized cellulose chiral column, Chiralcel OD-R, and a reversed-phase eluent containing 30-32% acetonitrile in 0.1 M potassium hexafluorophosphate. Ultraviolet (UV) absorbance detection was performed at 214 nm. Using 0.5-ml plasma aliquots, the method was validated in the concentration range 0.02-2.0 microg/ml and was applied in the investigation of systemic inversion of remoxipride enantiomers in man.

Disposition of remoxipride in Chinese schizophrenic patients.

The disposition of remoxipride was evaluated in 13 male chronic schizophrenic patients. A single 150 mg dose of remoxipride was administered and blood sampling performed over the following 48 hours. The mean (SD) oral clearance and half-life of remoxipride were 74.46 (25.9) ml/min and 5.46 (0.87) hours, respectively. The mean (SD) AUC for remoxipride was 25,320 (9,820) ng.h/ml. A wide interpatient variability was observed. Compared to Caucasian studies there were no significant differences in the disposition of remoxipride.

Determination of risperidone in plasma by high-performance liquid chromatography with electrochemical detection: application to therapeutic drug monitoring in schizophrenic patients.

A highly sensitive high-performance liquid chromatography method with electrochemical detection for the determination of risperidone in plasma has been developed. Remoxipride is used as an internal standard. A simple one-step extraction with 25% methylene dichloride in pentane is used to isolate the drug from the plasma. This is followed by high-performance liquid chromatography analysis on a cyano column with electrochemical detection. Under the experimental conditions described here, commonly coadministered drugs and other antipsychotic drugs did not interfere with the analysis of either risperidone or the internal standard. Also, the available two metabolites of risperidone did not interfere in the assay. This method has sufficient sensitivity to quantitate risperidone accurately at 0.1 ng/mL, when 1 mL of plasma was used for the analysis, with a coefficient of variation of < 9%. This method has been successfully used in the determination of plasma levels of risperidone in schizophrenic patients treated with 4-, 6-, and 8-mg oral doses per day.

Dopamine D2 blocking activity and plasma concentrations of remoxipride and its main metabolites in the rat.

Remoxipride and its active metabolites, the phenolic compounds FLA797(-) and FLA908(-) and the catecholic NCQ436(-) and haloperidol, were examined for their ability to block hypothermia in the rat induced by dopamine (DA) D2 receptor stimulation. In addition, plasma levels of remoxipride and its active metabolites were measured using HPLC methods. Remoxipride (1 mumol/kg), given 30 or 15 min prior to, or 5 and 15 min after, the DA agonists, blocked the hypothermia induced by the DA D2 receptor agonists quinpirole (0.25 mg/kg s.c.) and pergolide (0.1 mg/kg s.c.). Administration of remoxipride by the i.v. or s.c. routes was more effective than by the i.p. route. FLA797(-), FLA908(-), and haloperidol were more effective than remoxipride in preventing the hypothermia caused by quinpirole, while NCQ436(-) was less effective than remoxipride. The variation in time of remoxipride's action and effectiveness in blocking the induced hypothermia followed the variations in plasma concentrations. The plasma concentrations of the active metabolites were below the limit of determination (< 2 nmol/l). Based on estimation of free brain concentrations at effective dose levels together with in vitro affinities for the DA D2 receptor it was concluded that the metabolites FLA797(-), FLA908(-), and NCQ436(-) do not appear to contribute to the antagonism of DA D2 mediated neurotransmission following a low remoxipride dose (1 mumol/kg).

Determination of remoxipride in human plasma and urine by reversed-phase ion-pair high-performance liquid chromatography.

A sensitive method is described for the measurement of remoxipride in human plasma and urine. Remoxipride and its internal standard are extracted from plasma or urine at pH 12 with a mixture of hexane and methyl tert.-butyl ether. After washing the organic phase with base, the compounds are extracted into acid and analyzed on a C18 column with ultraviolet detection at 214 nm. The mobile phase is composed of acetonitrile and aqueous buffer (sodium perchlorate and phosphoric acid, pH 1.7). The limits of reliable quantitation for remoxipride are 12.5 and 50 ng/ml for plasma and urine, respectively. The run times are 6 min for plasma and 3 min for urine. The method has been successfully used to assay remoxipride clinical study samples. This mobile phase has also been successfully applied to the analysis of other basic drugs such as cimetidine, codeine, diltiazem and quinidine with minor modifications.