Enzyme assay to study the influence of citalopram on the concentration of psychotropic comedication

Introduction: Citalopram is one of the most frequently prescribed selective serotonin reuptake-inhibitors (SSRI) in the AGATE hospitals (Arbeitsgemeinschaft Arzneimitteltherapie bei psychiatrischen Erkrankungen). It is metabolized by the cytochrome-P₄₅₀ isoenzymes (CYP) 2C19, 2D6 and 3A4. Drug-drug interactions are rarely observed in clinical routine under citalopram and the substance is known not to influence the activity of CYP-isoenzymes. However, when quantifying drug concentrations in psychiatric patients we do see increased concentrations of various drugs that are metabolized via the same CYP isoenzymes. We therefore developed an in vitro method to study the influence of citalopram in comedication on the concentration of those drugs. Methods: Pooled human liver microsomes were incubated in various concentrations of 0.5 to’2.0 mg protein/ml with dipotassiumhydrogenphosphatebuffer (0.1 M, pH7.4), NADPH regenerating system (containing NADP⁺, glucose-6-phosphate, MgCl₂ and glucose-6-phosphatedehydrogenase), drug under study (quetiapine in various concentrations from 250 to 500 ng/ml) and the comedication under investigation (citalopram in various concentrations from 50 to 500 ng/ml) at a temperature of 37°C. Starting at 0 min (baseline) the reaction was stopped by adding acetonitrile to precipitate the protein at multiple incubation times (0, 15, 30, 45, 60, 75, 90, 105, 120, 135, 150, 165, 180, 195, 210, 225, 240, 255 and 270 min). After removing the human liver microsomes by centrifugation the concentrations of the drugs and their metabolites were measured by an HPLC online method with UV-VIS detection. Results: In these experiments, 1thinsp;mg protein/mL of human liver seems likely to to be a good concentration to monitor CYP metabolism. At this concentration, the decrease of the substrate and the increase of the metabolites could be kept in a concentration range above the limit of detection of our HPLC methods. After a time period of 270 min the monitoring of the reaction is stopped, because the enzymes in the human liver microsomes loose their activity. Maximum metabolic turnover was maintained for about 90 min and then decreases to 0 ng/ml/min/mg protein. In presence of an inhibiting comedication the metabolic turnover of the drug under study was reduced. The dimension of inhibition depended on the divergence in affinity between the drugs. Conclusion: With this assay, we could monitor the metabolism of drugs over a time period of about 90 min. In most of the analysis we were quantifying in our TDM measurements there was more than one medication prescribed. Now, we are able to study drug-drug-interactions between substances probably responsible to cause an unexpected plasma concentration.