Profiling withanolide A for therapeutic targets in neurodegenerative diseases.

To identify new potential therapeutic targets for neurodegenerative diseases, we initiated activity-based protein profiling studies with withanolide A (WitA), a known neuritogenic constituent of Withania somnifera root with unknown mechanism of action. Molecular probes were designed and synthesized, and led to the discovery of the glucocorticoid receptor (GR) as potential target. Molecular modeling calculations using the VirtualToxLab predicted a weak binding affinity of WitA for GR. Neurite outgrowth experiments in human neuroblastoma SH-SY5Y cells further supported a glucocorticoid-dependent mechanism, finding that WitA was able to reverse the outgrowth inhibition mediated by dexamethasone (Dex). However, further GR binding and transactivation assays found no direct interference of WitA. Further molecular modeling analysis suggested that WitA, although forming several contacts with residues in the GR binding pocket, is lacking key stabilizing interactions as observed for Dex. Taken together, the data suggest that WitA-dependent induction of neurite outgrowth is not through a direct effect on GR, but might be mediated through a closely related pathway. Further experiments should evaluate a possible role of GR modulators and/or related signaling pathways such as ERK, Akt, NF-κB, TRα, or Hsp90 as potential targets in the WitA-mediated neuromodulatory effects.

Removal of batch effects using stratified subsampling of metabolomic data for in vitro endocrine disruptors screening.

The human adrenal cell line H295R constitutes a well-established model to evaluate potential alterations of steroidogenic pathways as a result of chemical exposure. However, to date most assays are based on the targeted investigation of a limited number of steroid hormones, thus preventing in-depth mechanistic interpretation with respect to steroidogenesis. In that context, analytical strategies coupling liquid chromatography and high-resolution mass spectrometry (LC-HRMS) have been reported as promising methods for an extended monitoring of steroid metabolites. However, unwanted sources of variability occurring during the acquisition process, including batch effects, may prevent relevant biochemical information to be properly highlighted. Dedicated data mining strategies are therefore needed to overcome these limitations, and extract relevant extended steroidomic profiles. The present study combines an untargeted LC-HRMS acquisition strategy with automated steroid metabolite annotation based on accurate mass and isotopic patterns, and a chemometric tool allowing the different sources of variability to be decomposed based on experimental design. This workflow was applied to the extended monitoring of steroidogenic dysregulations due to endocrine disrupting chemicals (EDCs) exposure in H295R cell cultures. A series of six chemicals, including acetyl tributylcitrate, octyl methoxycinnamate, torcetrapib, forskolin, linuron, and octocrylene, and dimethylsulfoxide as solvent control, were investigated through the simultaneous monitoring of 130 potential steroid metabolites, repeating the whole experiment independently three times. A stratified subsampling strategy was carried out to remove efficiently systematic batch variations and highlight subgroups of chemicals with similar steroid patterns. The proposed approach was reported as a potent screening strategy, as it allowed specific alterations of the steroid hormone biosynthesis and metabolism related to distinct mechanisms of action to be distinguished.

Effects of lisdexamfetamine on plasma steroid concentrations compared with d-amphetamine in healthy subjects: A randomized, double-blind, placebo-controlled study.

The novel d-amphetamine prodrug lisdexamfetamine is applied to treat attention-deficit/hyperactivity disorder (ADHD). d-Amphetamine releases dopamine and norepinephrine and stimulates the hypothalamic-pituitary-adrenal (HPA) axis, which may contribute to its reinforcing effects and risk of abuse. However, no data is currently available on the effects of lisdexamfetamine on circulating steroids. This randomized, double-blind, placebo-controlled, cross-over study evaluated the effects of equimolar doses of d-amphetamine (40 mg) and lisdexamfetamine (100 mg) and placebo on circulating steroids in 24 healthy subjects. Plasma steroid and d-amphetamine levels were determined up to 24 h. Delayed increase and peak levels of plasma d-amphetamine concentrations were observed following lisdexamfetamine treatment compared with d-amphetamine administration, however the maximal concentrations and total exposure (area under the curve [AUC]) were similar. Lisdexamfetamine and d-amphetamine significantly enhanced plasma levels of adrenocorticotropic hormone, glucocorticoids (cortisol, cortisone, corticosterone, 11-dehydrocorticosterone, and 11-deoxycortisol), androgens (dehydroepiandrosterone, dehydroepiandrosterone sulfate, and Δ4-androstene-3,17-dione [androstenedione]), and progesterone (only in men) compared with placebo. Steroid concentration-time curves were shifted to later time points due to a non-significantly later onset following lisdexamfetamine administration than after d-amphetamine, however maximal plasma steroid concentrations and AUCs did not differ between the active treatments. None of the active treatments altered plasma levels of the mineralocorticoids aldosterone and 11-deoxycorticosterone or the androgen testosterone compared with placebo. The effects of the amphetamines on glucocorticoid production were similar to those that were previously reported for methylphenidate (60 mg) but weaker than those for the serotonin releaser 3,4-methylenedioxymethamphetamine (MDMA; 125 mg) or direct serotonin receptor agonist lysergic acid diethylamide (LSD; 0.2 mg). Lisdexamfetamine produced comparable HPA axis activation and had similar pharmacokinetics than d-amphetamine, except for a delayed time of onset. Thus, serotonin (MDMA, LSD) may more effectively stimulate the HPA axis than dopamine and norepinephrine (D-amphetamine).

Acute effects of lisdexamfetamine and D-amphetamine on social cognition and cognitive performance in a placebo-controlled study in healthy subjects.

RATIONALE: Amphetamines are used as medications but are also misused as cognitive enhancers by healthy subjects and may have additional effects on social cognition. METHODS: We investigated the acute effects of single, high, equimolar doses of D-amphetamine (40 mg) and lisdexamfetamine (100 mg) on social cognition and cognitive performance using a randomized, placebo-controlled, double-blind, cross-over design in 24 healthy volunteers. Effects on social cognition were assessed using the Facial Emotion Recognition Task (FERT), Multifaceted Empathy Test (MET), and Sexual Arousal Task (SAT). Cognitive performance was measured using the Digit Symbol Substitution Test (DSST), Digit Span (DS), Stop-Signal Task (SST), and Mackworth Clock Test (MCT). RESULTS: D-Amphetamine and lisdexamfetamine had small effects on measures of social cognition. There were no effects on emotion recognition on the FERT. D-Amphetamine increased direct empathy on the MET, but only for positive stimuli. Both amphetamines increased ratings of pleasantness and attractiveness on the SAT in response to sexual but also to neutral stimuli. D-Amphetamine and lisdexamfetamine increased cognitive performance (go-accuracy and vigilance on the SST and MCT, respectively). Lisdexamfetamine increased processing speed on the DSST. Neither drug had an effect on the DS. CONCLUSION: Single, high, equimolar doses of D-amphetamine and lisdexamfetamine enhanced certain aspects of cognitive performance in healthy non-sleep-deprived subjects. Both amphetamines also slightly altered aspects of social cognition. Whether these small effects also influence social interaction behavior in amphetamine users remains to be investigated. TRIAL REGISTRATION: The study was registered at ClinicalTrials.gov (NCT02668926).

Chiral analysis of amphetamines in hair by liquid chromatography-tandem mass spectrometry: compliance-monitoring of attention deficit hyperactivity disorder (ADHD) patients under Elvanse® therapy and identification after controlled low-dose application.

Amphetamine (AMP) is used as an illicit drug and also for the treatment of attention deficit hyperactivity disorder (ADHD). Respective drugs most often contain the enantiomer (S)-AMP as active compound or (S)-AMP is formed from the prodrug lisdexamfetamine (Elvanse®) whereas the illicit drug is usually traded as racemate ((R/S)-AMP). A differentiation between the use of the medically prescribed drug and the abuse of illicit street amphetamine is of great importance, for example in retrospective consumption monitoring by hair analysis. A liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the chiral separation and quantitation of (S)- and (R)-AMP in hair was developed. For this purpose, 20 mg hair was extracted and derivatized with N-(2,4-dinitro-5-fluorophenyl)-L(S)-valinamide L(S)-(DNPV) to yield amphetamine diastereomers. Baseline separation of the resulting diastereomers was achieved on a high-pressure liquid-chromatography system (HPLC) coupled to a Sciex QTRAP® 5500 linear ion trap quadrupole mass spectrometer. The method was successfully validated. Analysis of hair samples from nine Elvanse® patients revealed only (S)-AMP in eight cases; one subject showed both enantiomers indicating a (side-) consumption of street amphetamine. The analysis of the 16 amphetamine users' samples showed only racemic amphetamine. Furthermore, it could be shown in a controlled study that (S)-AMP can be detected after administration of even very low doses of lisdexamfetamine and dexamphetamine, which can be of interest in forensic toxicology and especially in drug-facilitated crime (DFC). The method now enables the retrospective compliance-monitoring of ADHD patients and the differentiation between medically prescribed intake of (S)-amphetamine and abuse of illicit street amphetamine.

Pharmacokinetics and Pharmacodynamics of Lisdexamfetamine Compared with D-Amphetamine in Healthy Subjects.

Rationale: Lisdexamfetamine is a prodrug of D-amphetamine used for the treatment of attention-deficit/hyperactivity disorder (ADHD). Lisdexamfetamine is thought to have a prolonged pharmacokinetic profile compared with oral D-amphetamine, possibly associated with lower drug liking and a lower risk of oral misuse. However, differences in the pharmacokinetics and pharmacodynamics of lisdexamfetamine and D-amphetamine have not been directly compared. Methods: Equimolar doses of D-amphetamine (40 mg) and lisdexamfetamine (100 mg), and placebo were administered in 24 healthy subjects in a randomized, double-blind, placebo-controlled, cross-over study. Plasma concentrations of amphetamine, subjective effects, and vital signs were repeatedly assessed. The pharmacokinetic parameters were determined using compartmental modeling. Results: The increase in plasma concentrations of amphetamine had a 0.6 ± 0.6 h (mean ± SD) longer lag time and reached peak levels 1.1 ± 1.5 h later after lisdexamfetamine administration compared with D-amphetamine administration, but no differences in maximal concentrations or total exposure (AUC) were found between the two treatments. Consistent with the pharmacokinetics, the subjective and cardiovascular stimulant effects of lisdexamfetamine also occurred later compared with D-amphetamine. However, no differences in peak ratings of potentially abuse-related subjective drug effects (e.g., drug liking, drug high, stimulation, happy, well-being, and self-confidence) were observed after lisdexamfetamine administration compared with D-amphetamine administration. Lisdexamfetamine and D-amphetamine also produced similar peak increases in mean arterial blood pressure, heart rate, body temperature, pupil size, and adverse effects. Conclusion: The pharmacokinetics and pharmacodynamics of lisdexamfetamine are similar to D-amphetamine administered 1h later. Lisdexamfetamine is likely associated with a similar risk of oral abuse as D-amphetamine. The study was registered at ClinicalTrials.gov (NCT02668926).

Enhanced metabolite annotation via dynamic retention time prediction: Steroidogenesis alterations as a case study.

The development of metabolomics based on ultra-high pressure liquid chromatography coupled to high-resolution mass spectrometry (UHPLC-HRMS) now allows hundreds to thousands of metabolites to be simultaneously monitored in biological matrices. In that context, bioinformatics and multivariate data analysis (MVA) play a crucial role in the detection of relevant alteration patterns. However, sound biological interpretations must necessarily be supported by metabolite identifications to be definitive or at least have a high degree of confidence. Each compound, should be characterised by unique molecular properties. Among them, the exact mass and the chromatographic retention time are recognised as major and complementary criteria for compound identification. While the former is easily derived from the molecular structure, building generic and accurate retention time open databases still constitutes a critical issue because of the vast diversity of instruments, stationary phases and operating conditions in UHPLC-HRMS. Because several hits matching a molecular formula obtained from an exact mass and an isotopic pattern are often generated for each analyte, this methodology rarely allows a unique and unambiguous molecular identity to be gained. This work aims to provide a flexible solution to facilitate reliable compound annotation based on retention time in reversed-phase liquid chromatography (RPLC). It proposes an innovative approach based on the chromatographic linear solvent strength (LSS) theory, allowing retention times under any gradient conditions at fixed temperature, stationary phase and mobile phase type to be predicted. Starting from a subset of the Human Metabolite Database (HMDB), a new dynamic database involving LSS parameters was developed. A real case study involving steroidogenesis alterations due to forskolin exposure was conducted using the adrenal H295R OECD reference cell model for endocrine disruptor screening. The prediction of retention times was successfully achieved, facilitating steroid identification. An automated procedure which implements the compound annotation levels encouraged by the Metabolite Standard Initiative (MSI) and the Coordination of Standards in Metabolomics (COSMOS) was also developed to speed up the process and enhance the data reusability.

Steroid profiling in H295R cells to identify chemicals potentially disrupting the production of adrenal steroids.

The validated OECD test guideline 456 based on human adrenal H295R cells promotes measurement of testosterone and estradiol production as read-out to identify potential endocrine disrupting chemicals. This study aimed to establish optimal conditions for using H295R cells to detect chemicals interfering with the production of key adrenal steroids. H295R cells' supernatants were characterized by liquid chromatography-mass spectrometry (LC-MS)-based steroid profiling, and the influence of experimental conditions including time and serum content was assessed. Steroid profiles were determined before and after incubation with reference compounds and chemicals to be tested for potential disruption of adrenal steroidogenesis. The H295R cells cultivated according to the OECD test guideline produced progestins, glucocorticoids, mineralocorticoids and adrenal androgens but only very low amounts of testosterone. However, testosterone contained in Nu-serum was metabolized during the 48h incubation. Thus, inclusion of positive and negative controls and a steroid profile of the complete medium prior to the experiment (t=0h) was necessary to characterize H295R cells' steroid production and indicate alterations caused by exposure to chemicals. Among the tested chemicals, octyl methoxycinnamate and acetyl tributylcitrate resembled the corticosteroid induction pattern of the positive control torcetrapib. Gene expression analysis revealed that octyl methoxycinnamate and acetyl tributylcitrate enhanced CYP11B2 expression, although less pronounced than torcetrapib. Further experiments need to assess the toxicological relevance of octyl methoxycinnamate- and acetyl tributylcitrate-induced corticosteroid production. In conclusion, the extended profiling and appropriate controls allow detecting chemicals that act on steroidogenesis and provide initial mechanistic evidence for prioritizing chemicals for further investigations.

Disruption of steroidogenesis: Cell models for mechanistic investigations and as screening tools.

In the modern world, humans are exposed during their whole life to a large number of synthetic chemicals. Some of these chemicals have the potential to disrupt endocrine functions and contribute to the development and/or progression of major diseases. Every year approximately 1000 novel chemicals, used in industrial production, agriculture, consumer products or as pharmaceuticals, are reaching the market, often with limited safety assessment regarding potential endocrine activities. Steroids are essential endocrine hormones, and the importance of the steroidogenesis pathway as a target for endocrine disrupting chemicals (EDCs) has been recognized by leading scientists and authorities. Cell lines have a prominent role in the initial stages of toxicity assessment, i.e. for mechanistic investigations and for the medium to high throughput analysis of chemicals for potential steroidogenesis disrupting activities. Nevertheless, the users have to be aware of the limitations of the existing cell models in order to apply them properly, and there is a great demand for improved cell-based testing systems and protocols. This review intends to provide an overview of the available cell lines for studying effects of chemicals on gonadal and adrenal steroidogenesis, their use and limitations, as well as the need for future improvements of cell-based testing systems and protocols.

Formation of threohydrobupropion from bupropion is dependent on 11ß-hydroxysteroid dehydrogenase 1.

Bupropion is widely used for treatment of depression and as a smoking-cessation drug. Despite more than 20 years of therapeutic use, its metabolism is not fully understood. While CYP2B6 is known to form hydroxybupropion, the enzyme(s) generating erythro- and threohydrobupropion have long remained unclear. Previous experiments using microsomal preparations and the nonspecific inhibitor glycyrrhetinic acid suggested a role for 11ß-hydroxysteroid dehydrogenase 1 (11ß-HSD1) in the formation of both erythro- and threohydrobupropion. 11ß-HSD1 catalyzes the conversion of inactive glucocorticoids (cortisone, prednisone) to their active forms (cortisol, prednisolone). Moreover, it accepts several other substrates. Here, we used for the first time recombinant 11ß-HSD1 to assess its role in the carbonyl reduction of bupropion. Furthermore, we applied human, rat, and mouse liver microsomes and a selective inhibitor to characterize species-specific differences and to estimate the relative contribution of 11ß-HSD1 to bupropion metabolism. The results revealed 11ß-HSD1 as the major enzyme responsible for threohydrobupropion formation. The reaction was stereoselective and no erythrohydrobupropion was formed. Human liver microsomes showed 10 and 80 times higher activity than rat and mouse liver microsomes, respectively. The formation of erythrohydrobupropion was not altered in experiments with microsomes from 11ß-HSD1-deficient mice or upon incubation with 11ß-HSD1 inhibitor, indicating the existence of another carbonyl reductase that generates erythrohydrobupropion. Molecular docking supported the experimental findings and suggested that 11ß-HSD1 selectively converts R-bupropion to threohydrobupropion. Enzyme inhibition experiments suggested that exposure to bupropion is not likely to impair 11ß-HSD1-dependent glucocorticoid activation but that pharmacological administration of cortisone or prednisone may inhibit 11ß-HSD1-dependent bupropion metabolism.

Carbonyl reduction of triadimefon by human and rodent 11ß-hydroxysteroid dehydrogenase 1.

11ß-Hydroxysteroid dehydrogenase 1 (11ß-HSD1) catalyzes the conversion of inactive 11-oxo glucocorticoids (endogenous cortisone, 11-dehydrocorticosterone and synthetic prednisone) to their potent 11ß-hydroxyl forms (cortisol, corticosterone and prednisolone). Besides, 11ß-HSD1 accepts several other substrates. Using rodent liver microsomes and the unspecific inhibitor glycyrrhetinic acid, it has been proposed earlier that 11ß-HSD1 catalyzes the reversible conversion of the fungicide triadimefon to triadimenol. In the present study, recombinant human, rat and mouse enzymes together with a highly selective 11ß-HSD1 inhibitor were applied to assess the role of 11ß-HSD1 in the reduction of triadimefon and to uncover species-specific differences. To further demonstrate the role of 11ß-HSD1 in the carbonyl reduction of triadimefon, microsomes from liver-specific 11ß-HSD1-deficient mice were employed. Molecular docking was applied to investigate substrate binding. The results revealed important species differences and demonstrated the irreversible 11ß-HSD1-dependent reduction of triadimefon. Human liver microsomes showed 4 and 8 times higher activity than rat and mouse liver microsomes. The apparent Vmax/Km of recombinant human 11ß-HSD1 was 5 and 15 times higher than that of mouse and rat 11ß-HSD1, respectively, indicating isoform-specific differences and different expression levels for the three species. Experiments using inhibitors and microsomes from 11ß-HSD1-deficient mice indicated that 11ß-HSD1 is the major if not only enzyme responsible for triadimenol formation. The IC50 values of triadimefon and triadimenol for cortisone reduction suggested that exposure to these xenobiotica unlikely impairs the 11ß-HSD1-dependent glucocorticoid activation. However, elevated glucocorticoids during stress or upon pharmacological administration likely inhibit 11ß-HSD1-dependent metabolism of triadimefon in humans.

Species-specific differences in the inhibition of human and zebrafish 11ß-hydroxysteroid dehydrogenase 2 by thiram and organotins.

Dithiocarbamates and organotins can inhibit enzymes by interacting with functionally essential sulfhydryl groups. Both classes of chemicals were shown to inhibit human 11ß-hydroxysteroid dehydrogenase 2 (11ß-HSD2), which converts active cortisol into inactive cortisone and has a role in renal and intestinal electrolyte regulation and in the feto-placental barrier to maternal glucocorticoids. In fish, 11ß-HSD2 has a dual role by inactivating glucocorticoids and generating the major androgen 11-ketotestosterone. Inhibition of this enzyme may enhance glucocorticoid and diminish androgen effects in fish. Here, we characterized 11ß-HSD2 activity of the model species zebrafish. A comparison with human and mouse 11ß-HSD2 revealed species-specific substrate preference. Unexpectedly, assessment of the effects of thiram and several organotins on the activity of zebrafish 11ß-HSD2 showed weak inhibition by thiram and no inhibition by any of the organotins tested. Sequence comparison revealed the presence of an alanine at position 253 on zebrafish 11ß-HSD2, corresponding to cysteine-264 in the substrate-binding pocket of the human enzyme. Substitution of alanine-253 by cysteine resulted in a more than 10-fold increased sensitivity of zebrafish 11ß-HSD2 to thiram. Mutating cysteine-264 on human 11ß-HSD2 to serine resulted in 100-fold lower inhibitory activity. Our results demonstrate significant species differences in the sensitivity of human and zebrafish 11ß-HSD2 to inhibition by thiram and organotins. Site-directed mutagenesis revealed a key role of cysteine-264 in the substrate-binding pocket of human 11ß-HSD2 for sensitivity to sulfhydryl modifying agents.