Dissociation of endocrine responses to the Trier Social Stress Test in Virtual Reality (VR-TSST) by the benzodiazepine alprazolam and the translocator protein 18 kDa (TSPO) ligand etifoxine.

BACKGROUND: Activity of the two major stress systems, the hypothalamic-pituitary-adrenal (HPA) and the sympathetic-adrenal-medullary (SAM) axis, has already been shown to be modulated by different compounds that bind to the central benzodiazepine receptor. Less is known about ligands that modulate the peripheral benzodiazepine receptor - meanwhile known as the translocator protein 18 kDa (TSPO) - which constitute promising candidates in the search of novel anxiolytics. To close this gap, the present study compared the effects of the benzodiazepine alprazolam and the TSPO ligand etifoxine on responses of the HPA and SAM axes to the Trier Social Stress Test, a standardized paradigm to induce acute psychosocial stress in humans, performed in Virtual Reality (VR-TSST). METHODS: Sixty healthy males, aged between 18 and 55 years, were randomly assigned to receive either a daily dose of 1.5 mg alprazolam, 150 mg etifoxine, or placebo over five days. On the last day of intake, they were exposed to the VR-TSST. We assessed changes of salivary cortisol, allopregnanolone, (nor-) epinephrine in serum, TSPO expression in platelets as well as heart rate (HR), skin conductance level (SCL) and self-reports in response to the stress task. Repeated measures ANOVAs were conducted to examine treatment effects on these stress response variables during the course of VR-TSST. RESULTS: The response of salivary cortisol to the VR-TSST was significantly blunted in participants pre-treated with alprazolam but was not affected by etifoxine. While levels of allopregnanolone, epinephrine and norepinephrine increased in response to stress, TSPO expression decreased. None of those endocrine stress markers was affected by the active treatments, whereas TSPO expression increased after etifoxine administration over all study days. There were no effects of the two anxiolytics on HR, SCL or any self-report measurement. CONCLUSION: The current study confirmed the attenuating effects of benzodiazepines on stress-induced HPA axis activity but did not reveal a comparable effect of the TSPO ligand etifoxine. The long-term consequences of a pharmacologically blunted response of the HPA axis to an acute stressor should be further elucidated. Due to the missing effects of etifoxine on stress-related parameters in our sample of healthy subjects, it might be concluded that the therapeutic effects of this TSPO ligand are restricted to stronger or pathological stress responses, respectively.

Implementation of a cost-effective HPLC/UV approach for routine medical quantification of memantine in human serum.

BACKGROUND: In the near future, there will be no innovative drug principle for the treatment of dementia. Therefore, optimizing the efficacy of a drug is at present the most promising way to exploit its full pharmacological potential. METHOD: A high performance liquid chromatography with ultraviolet assay for memantine in serum from demented patients has been developed and validated. The analytical procedure involves offline solid phase extraction cartridges. Because memantine molecules lack chromophoric π-electrons, a derivatization with dansyl chloride was required for detection by ultraviolet (UV) photometry. Analyses were performed on a Dionex high-performance liquid chromatography system with a Phenomenex Luna Phenyl-Hexyl analytical column and 0.02 mol/L potassium dihydrogen phosphate buffer/acetonitrile (40/60 V/V) as mobile phase at a flow rate of 0.4 mL/min. Dansylated memantine was detected at 218 nm; 2 more ultraviolet wavelengths at 254 nm and 336 nm were used as an overlay-control check. RESULTS: The retention time for dansylated memantine was 17.1 ± 0.2 minutes. The calibration curve was linear over a concentration range from 5 to 160 ng/mL (n = 8/r² > 0.999). The method had an accuracy of >90%. Intra-assay and inter-assay coefficients of variation were <5% and <13%, respectively, at 3 different concentrations. The limit of quantification and the limit of detection were 2.9 ng/mL and 0.8 ng/mL, respectively. Among 100 substances prescribed as comedications in the treatment of dementia only fluvoxamine and zuclopenthixole showed retention times close to dansylated memantine (17.8 minutes and 18.1 minutes, respectively). However, these 2 drugs were removed from patients' specimens during solid-phase extraction sample preparation. CONCLUSIONS: The method is applicable under conditions of daily routine as has been demonstrated by application of the method to patient serum samples. The quantification of 29 samples showed that memantine concentrations suggested as "therapeutic" in the literature may only be reached by high doses of memantine.

Implementation of a cost-effective HPLC/UV-approach for medical routine quantification of donepezil in human serum.

A novel, simple, specific and sensitive high performance liquid chromatography (HPLC) assay for the detection and quantification of donepezil in serum of demented patients has been developed and validated. The analytical procedure involves an offline serum preextraction using solid phase extraction (SPE) cartridges (Oasis® HLB, Waters Co). The chromatographic analyses were performed on a Dionex HPLC system with a Phenomenex Luna Phenyl-Hexyl analytical column, and a mobile phase with the two components 0.02 mol/l phosphate buffer and acetonitrile. The flow rate was 0.4 ml/min. For the detection of donepezil three different UV wavelengths were used as an interference-control check. Interference tests between donepezil and 100 of the most commonly used concomitant medications allow quantification of donepezil under the polypharmaceutical conditions of the daily clinical routine. The retention time for donepezil was 12.1 min. The method was validated according to the guidelines of the Society of Toxicology and Forensic Chemistry (GTFCh): The calibration curve was linear over a concentration range from 5 to 160 ng/ml (n=8/r²>0.999). No endogenous compounds were found to interfere with the analyte, which was shown by retention times for the comedication most often prescribed to demented patients. The method had an accuracy of >85%. Intra- and inter-assay coefficients of variation were <6% and <8%, respectively, at three different concentrations. The limit of quantification (LOQ) and the limit of detection (LOD) were found to be 6.1 and 1.7 ng/ml for donepezil. Application of the method to patient serum samples discovered that concentrations suggested as "therapeutic" in the literature may only be reached either by high, off-label dosages or by utilization of inhibitory metabolic effects of the comedication.