Substantial Differences in Serum Concentrations of Psychoactive Drugs Measured in Samples Stored for 2 Days or More on Standard Serum Tubes Versus Serum Tubes Containing Gel Separators.

BACKGROUND: Drugs may potentially adsorb to blood collection tubes containing gel separators in the preanalytical phase of therapeutic drug monitoring. The aim of this study was to compare measured concentrations of 28 psychoactive drugs and 13 metabolites in spiked serum samples stored on standard (plain) tubes versus barrier gel tubes during a 2-6-day period at room temperature. METHODS: Drug-free ("blank") serum samples spiked with mixes of antidepressants, antipsychotics, or mood stabilizers (valproic acid and lamotrigine), including relevant metabolites, were transferred to tubes with and without gel, that is, BD Vacutainer SST II Advance gel tubes and BD Vacutainer Glass Serum Tubes (Becton-Dickinson Company, Plymouth, United Kingdom). Mean serum concentrations of the drugs or metabolites measured by ultra-high performance liquid chromatography-tandem mass spectrometry analyses of protein-precipitated samples were compared after storage on plain or gel tubes at 3 time points (day 0, day 2/48 hours, and day 6/144 hours) in room temperature. RESULTS: Mean serum concentrations of all antidepressants, except for one metabolite, and 13 of 18 antipsychotic drugs were significantly lower in gel tubes compared with plain tubes after 2 days of storage (2%-28% lower, P < 0.05). After 6 days of storage, mean serum concentrations of all antipsychotic drugs and antidepressants were significantly lower in gel tubes versus plain tubes (9%-49% lower, P < 0.02), except for amisulpride and O-desmethylvenlafaxine. Serum concentrations of the mood stabilizers were not significantly different in gel tubes compared with plain tubes (P > 0.1). There was a clear relationship between log P (partition coefficient) and residual serum concentrations during gel tube storage (r -0.50 and -0.42 at day 2 and day 6, respectively; P < 0.02). CONCLUSIONS: This study shows that storage on gel for more than 2 days significantly decreases the serum concentrations of antidepressant and antipsychotic drugs as compared to storage in plain tubes. Thus, using tubes with gel separators in the therapeutic drug monitoring of psychoactive drugs should be reconsidered.

Endomyocardial, intralymphocyte, and whole blood concentrations of ciclosporin A in heart transplant recipients.

BACKGROUND: In the early phases following heart transplantation a main challenge is to reduce the impact of acute rejections. Previous studies indicate that intracellular ciclosporin A (CsA) concentration may be a sensitive acute rejection marker in renal transplant recipients. The aims of this study were to evaluate the relationships between CsA concentrations at different target sites as potential therapeutic drug monitoring (TDM) tools in heart transplant recipients. METHODS: Ten heart transplant recipients (8 men, 2 women) on CsA-based immunosuppression were enrolled in this prospective single-center pilot study. Blood samples were obtained once to twice weekly up to 12 weeks post-transplant. One of the routine biopsies was allocated to this study at each sampling time. Whole blood, intralymphocyte, and endomyocardial CsA concentrations were determined with validated HPLC-MS/MS-methods. Mann-Whitney U test was used when evaluating parameters between the two groups of patients. To correlate whole blood, intralymphocyte, and endomyocardial CsA concentrations linear regression analysis was used. RESULTS: Three patients experienced mild rejections. In the study period, the mean (range) intralymphocyte CsA trough concentrations were 10.1 (1.5 to 39) and 8.1 (1.3 to 25) ng/106 cells in the rejection and no-rejection group, respectively (P=0.21). Corresponding whole blood CsA concentrations were 316 (153 to 564) and 301 (152 to 513) ng/mL (P=0.33). There were no correlations between whole blood, intralymphocyte, or endomyocardial concentrations of CsA (P >0.11). CONCLUSIONS: The study did not support an association between decreasing intralymphocyte CsA concentrations and acute rejections. Further, there were no association between blood concentrations and concentrations at sites of action, potentially challenging TDM in these patients.

Metabolism of the active metabolite of quetiapine, N-desalkylquetiapine in vitro.

The antipsychotic drug quetiapine has been approved for the treatment of unipolar and bipolar depression. The antidepressant activity is considered to be mediated by the active metabolite N-desalkylquetiapine, which is mainly formed by CYP3A4. Little is known about the subsequent elimination of this metabolite. Therefore, this study investigated the possible involvement of cytochrome P450 (P450) enzymes in the metabolism of N-desalkylquetiapine. Screening for and interpretation of metabolites were performed by incubating N-desalkylquetiapine in human liver microsomes (HLM) followed by liquid chromatography-tandem mass spectrometry. The possible involvement of P450 enzymes in N-desalkylquetiapine metabolism was evaluated by coincubation of selective P450 inhibitors in HLM and subsequent experiments with recombinant human P450 enzymes. In HLM experiments, three chromatographic peaks were interpreted as possible metabolites of N-desalkylquetiapine, namely, N-desalkylquetiapine sulfoxide, 7-hydroxy-N-desalkylquetiapine, and an unrecognized metabolite (denoted M3). Inhibition of CYP2D6 (by quinidine) reduced formation of 7-hydroxy-N-desalkylquetiapine by 81%, whereas the CYP3A4 inhibitor ketoconazole inhibited formation of N-desalkylquetiapine sulfoxide and M3 by 65 and 34%, respectively. Inhibitors of CYP1A2, CYP2C9, and CYP2C19 showed only limited changes in metabolite formation. In recombinant systems, 7-hydroxy-N-desalkylquetiapine was exclusively formed by CYP2D6, whereas N-desalkylquetiapine sulfoxide and M3 were formed by both CYP3A4 and CYP2D6. Overall, intrinsic clearance of N-desalkylquetiapine was 12-fold higher by recombinant CYP2D6 relative to CYP3A4. In conclusion, N-desalkylquetiapine is metabolized by both CYP2D6 and CYP3A4 in vitro with preference for the former enzyme. The pharmacologically active metabolite, 7-hydroxy-N-desalkylquetiapine, was exclusively formed by CYP2D6, whereas the two other metabolites were mainly formed by CYP3A4.

Olanzapine, but not aripiprazole, weight-independently elevates serum triglycerides and activates lipogenic gene expression in female rats.

Metabolic adverse effects such as weight gain and dyslipidaemia represent a major concern in treatment with several antipsychotic drugs, including olanzapine. It remains unclear whether such metabolic side-effects fully depend on appetite-stimulating actions, or whether some dysmetabolic features induced by antipsychotics may arise through direct perturbation of metabolic pathways in relevant peripheral tissues. Recent clinical and preclinical studies indicate that dyslipidaemia could occur independently of weight gain. Using a rat model, we showed that subchronic treatment with olanzapine induces weight gain and increases adipose tissue mass in rats with free access to food. This effect was also observed for aripiprazole, considered metabolically neutral in the clinical setting. In pair-fed rats with limited food access, neither olanzapine nor aripiprazole induced weight gain. Interestingly, olanzapine, but not aripiprazole, induced weight-independent elevation of serum triglycerides, accompanied by up-regulation of several genes involved in lipid biosynthesis, both in liver and in adipose tissues. Our findings support the existence of tissue-specific, weight-independent direct effects of olanzapine on lipid metabolism.

Acute effects of orexigenic antipsychotic drugs on lipid and carbohydrate metabolism in rat.

OBJECTIVE: This study aims to investigate whether orexigenic antipsychotic drugs may induce dyslipidemia and glucose disturbances in female rats through direct perturbation of metabolically active peripheral tissues, independent of prior weight gain. METHODS: In the current study, we examined whether a single intraperitoneal injection of clozapine or olanzapine induced metabolic disturbances in adult female outbred Sprague-Dawley rats. Serum glucose and lipid parameters were measured during time-course experiments up to 48 h. Real-time quantitative PCR was used to measure specific transcriptional alterations in lipid and carbohydrate metabolism in adipose tissue depots or in the liver. RESULTS: Our results demonstrated that acute administration of clozapine or olanzapine induced a rapid, robust elevation of free fatty acids and glucose in serum, followed by hepatic accumulation of lipids evident after 12-24 h. These metabolic disturbances were associated with biphasic patterns of gluconeogenic and lipid-related gene expression in the liver and in white adipose tissue depots. CONCLUSION: Our results support that clozapine and olanzapine are associated with primary effects on carbohydrate and lipid metabolism associated with transcriptional changes in metabolically active peripheral tissues prior to the development of drug-induced weight gain.

Absolute ProGRP quantification in a clinical relevant concentration range using LC-MS/MS and a comprehensive internal standard.

The objective of this study was to develop a method using liquid chromatography with tandem mass spectrometric detection for the absolute quantification of the small cell lung cancer biomarker ProGRP in human serum, using its tryptic signature peptide NLLGLIEAK. The samples were precipitated for most of its proteins using acetonitrile prior to tryptic digestion. Further sample clean-up and enrichment was achieved by the use of an on-line restricted access media column, followed by separation on a BioBasic C8 column. Detection and quantification was carried out by operating a triple quadrupole MS in the selected reaction monitoring mode. This setup allowed analysis of realistic samples and detections limits in human serum of 150 pg ProGRP on column. Using an internal standard derived from the parent ProGRP after acetylation of the lysine side chain allowed better quantification through variation correction in all sample pretreatment steps, trypsination included.

Acute clozapine exposure in vivo induces lipid accumulation and marked sequential changes in the expression of SREBP, PPAR, and LXR target genes in rat liver.

BACKGROUND: Several antipsychotic drugs (APDs) have high propensity to induce weight gain and dyslipidemia in patients, with clozapine and olanzapine as the most potent drugs. These lipid-related effects have been attributed to drug-mediated blockade or antagonism of histamine H1 and serotonin 5-HT2 receptors as well as activation of hypothalamic AMP-activated protein kinase. We recently showed that APDs activate lipid biosynthesis in cultured liver cells through stimulation of the sterol regulatory element-binding protein (SREBP) transcription factors. OBJECTIVE: The objective of the study was to search for clozapine-related lipogenic effects in peripheral tissues in vivo using rat liver as target organ. MATERIALS AND METHODS: Adult female Sprague-Dawley rats were administered single intraperitoneal injections of clozapine (25 and 50 mg/kg). Hepatic lipid levels were measured during a 48-h time course. Real-time quantitative PCR was used to analyze expression of genes involved in lipid biosynthesis, oxidation, efflux, and lipolysis. RESULTS: We identified an initial up-regulation of central lipogenic SREBP target genes, followed by a marked and sustained down-regulation. We also observed a sequential transcriptional response for fatty acid beta-oxidation and cholesterol efflux genes, normally controlled by the peroxisome proliferator activated receptor alpha and liver X receptor alpha transcription factors, and also down-regulation of genes encoding major lipases. The transcriptional responses were associated with a significant accumulation of triacylglycerol, phospholipids, and cholesterol in the liver. CONCLUSION: These results demonstrate that acute clozapine exposure affects SREBP-regulated lipid biosynthesis as well as other lipid homeostasis pathways. We suggest that such drug-induced effects on lipid metabolism in peripheral tissues are relevant for the metabolic adverse effects associated with clozapine and possibly other APDs.

Pharmacokinetic variability of aripiprazole and the active metabolite dehydroaripiprazole in psychiatric patients.

Aripiprazole is a new atypical antipsychotic drug with a partial agonist activity at dopamine 2 and serotonin 1A receptors. The metabolism of aripiprazole involves both cytochrome P450 2D6 (CYP2D6) and CYP3A4. This study investigated the pharmacokinetic variability of aripiprazole and the active metabolite dehydroaripiprazole on the basis of 155 drug monitoring samples from psychiatric patients treated with therapeutic doses of aripiprazole (10-30 mg/day). Serum concentrations of drug and metabolite were determined by liquid chromatographic and tandem mass spectrometric detection. Pharmacokinetic variability was expressed as the range in concentration/dose (C/D) ratios, and the effect of sex and occasionally coprescribed CYP2D6 or CYP3A4 inhibitors/inducers was studied. In addition, the dose-concentration relationship and combined interquartile range of concentrations obtained at low dose (10-15 mg/day) and high dose (20-30 mg/day) were described. Individual C/D ratios ranged 37-fold for aripiprazole, 78-fold for dehydroaripiprazole, and 27-fold for the active sum of aripiprazole + dehydroaripiprazole. Median C/D ratios in male and female patients differed by less than 15%, and none of the differences were significant (P > 0.14). Cases of concurrent CYP3A4 inducers/inhibitors were not found, but three patients were coprescribed the potent CYP2D6 inhibitors paroxetine or fluoxetine. No consistent difference in C/D ratio was observed in these three patients compared with the rest of the patients. There was a proportional dose-concentration relationship in the population, and the combined interquartile ranges were 230 to 960 nmol/L for aripiprazole and 330 to 1210 nmol/L for aripiprazole + dehydroaripiprazole. In conclusion, pharmacokinetic variability of aripiprazole is extensive in psychiatric patients but apparently not dependent on dose or sex. The variability of the pharmacologic active sum of aripiprazole + dehydroaripiprazole is 25% to 30% less than aripiprazole, suggesting that variability of aripiprazole is partly determined by metabolism to dehydroaripiprazole.

[Dietary supplements--surprise pills?]. / Kosttilskudd--forundringspakke i pilleform?

BACKGROUND: Sales of herbal dietary supplements have increased dramatically. A patient case drew our attention to the problem of incomplete declaration of content. METHODS: Two dietary supplements which the manufacturers claim to be natural, extremely fat-burning and energizing were analysed, as were urine and serum samples from persons taking these supplements. RESULTS: Surprisingly, the herbal dietary supplements contained drugs. Diazepam, clonazepam, ephedrine and metabolites were found when analyzing serum samples after intake of the dietary supplement Thermo-X 650, ephedrine and phenylpropanolamine after intake of the Purple Burn supplement. INTERPRETATION: Use of herbal dietary supplement can have serious consequences, for instance through interactions with drug therapy. Consumers must be given sufficient product information for safe use.