Psychoactive substances as substrates or inhibitors of enzymes in drug metabolism and transport

In contrast to therapeutic drugs, drugs of abuse (DOA) are marketed without (pre)clinical studies. In order to expand the knowledge surrounding toxicokinetics of DOA, the affinity and/or inhibition potential of selected DOA towards human metabolic enzymes such as monooxygenases or monoamine oxidases (MAO) and an efflux transporter, the breast cancer resistance protein (BCRP), was investigated using mass spectrometry techniques. FMO3 was identified as main enzyme involved in the N-oxygenation of N,N-diallyltryptamine and methamphetamine. Furthermore, a broad range of alpha-methyltryptamine-derived new psychoactive substances but also phenethylamines of the 2C-series were identified as MAO inhibitors. Finally, the BCRP ATPase was shown to be potently stimulated or inhibited by 3,4-BDB or WIN 55,212-2, respectively, comparable to model substrates or inhibitors

Reviewing toxicokinetics with a focus on metabolism of new psychoactive substances in the zebrafish (larvae) model

Zebrafish (Danio rerio) share around 70% of their genome with humans and many important enzymes for drug metabolism such as some cytochrome p450s have direct orthologs in zebrafish. To date, several studies showed a similar metabolism to humans in general. Furthermore, although using adult fish as model organism requires approval by an Ethics Committee, using larvae until 120 h postfertilization does not necessarily need any approval at least in the European Union. All these aspects seem to be beneficial for using zebrafish (larvae) in toxicokinetic studies for the so-called new psychoactive substances. These compounds are expected to have similar effects as traditional drugs of abuse but are often not listed as controlled drugs when they first appear on the market. However, no information about their toxicokinetics is available when they appear, which is particularly critical concerning their biotransformation. This knowledge is important for example, for developing urine-based screening procedures or for predicting drug–drug interactions. This focus article aims to briefly introduce into the topic of using zebrafish (larvae) in the context of toxicokinetic studies, particularly metabolism studies, and will highlight some aspects such as the route of administration, which are important to consider when using this model. This article is categorized under: Toxicology > New Psychoactive Substances Toxicology > Analytica

Inhibition and stimulation of the human breast cancer resistance protein as in vitro predictor of drug–drug interactions of drugs of abuse

Transporter-mediated drug–drug interactions (DDI) may induce adverse clinical events. As drugs of abuse (DOA) are marketed without preclinical safety studies, only very limited information about interplay with membrane transporters are available. Therefore, 13 DOA of various classes were tested for their in vitro affinity to the human breast cancer resistance protein (hBCRP), an important efflux transporter. As adenosine 5′-triphosphate (ATP) hydrolysis is crucial for hBCRP activity, adenosine 5′-diphosphate (ADP) formation was measured and used as in vitro marker for hBCRP ATPase activity. ADP quantification was performed by hydrophilic interaction liquid chromatography coupled to high-resolution tandem mass spectrometry and its amount in test compound incubations was compared to that in reference incubations using the hBCRP substrate sulfasalazine or the hBCRP inhibitor orthovanadate. If DOA caused stimulation or inhibition, further investigations such as Michaelis–Menten kinetic modeling or IC50 value determination were conducted. Among the tested DOA, seven compounds showed statistically significant hBCRP ATPase stimulation. The entactogen 3,4-BDB and the plant alkaloid mitragynine were identified as strongest stimulators. Their affinity to the hBCRP ATPase was lower than that of sulfasalazine but comparable to that of rosuvastatin, another hBCRP model substrate. Five DOA showed statistically significant hBCRP ATPase inhibition. Determination of IC50 values identified the synthetic cannabinoid receptor agonists JWH-200 and WIN 55,212-2 as the strongest inhibitors comparable to orthovanadate. The present study clearly demonstrated that tested DOA show in part high affinities to the hBCRP within the range of model substrates or inhibitors. Thus, there is a risk of hBCRP-mediated DDI, which needs to be considered in clinical settings

Altered metabolic pathways elucidated via untargeted in vivo toxicometabolomics in rat urine and plasma samples collected after controlled application of a human equivalent amphetamine dose

Amphetamine is widely consumed as drug of abuse due to its stimulating and cognitive enhancing effects. Since amphetamine has been on the market for quite a long time and it is one of the most commonly used stimulants worldwide, to date there is still limited information on its effects on the metabolome. In recent years, untargeted toxicometabolomics have been increasingly used to study toxicity-related pathways of such drugs of abuse to find and identify important endogenous and exogenous biomarkers. In this study, the acute effects of amphetamine intake on plasma and urinary metabolome in rats were investigated. For this purpose, samples of male Wistar rats after a single dose of amphetamine (5 mg/kg) were compared to a control group using an untargeted metabolomics approach. Analysis was performed using normal and reversed phase liquid chromatography coupled to high-resolution mass spectrometry using positive and negative ionization mode. Statistical evaluation was performed using Welch’s two-sample t test, hierarchical clustering, as well as principal component analysis. The results of this study demonstrate a downregulation of amino acids in plasma samples after amphetamine exposure. Furthermore, four new potential biomarkers N-acetylamphetamine, N-acetyl-4-hydroxyamphetamine, N-acetyl-4-hydroxyamphetamine glucuronide, and amphetamine succinate were identified in urine. The present study complements previous data and shows that several studies are necessary to elucidate altered metabolic pathways associated with acute amphetamine exposure

Toxic plants-Detection of colchicine in a fast systematic clinical toxicology screening using liquid chromatography-mass spectrometry

Colchicum autumnale, which can be mistaken for Allium ursinum, contains the alkaloid colchicine potentially leading to life-threatening up to fatal intoxications. We report two cases of acute intoxications with unexplained circumstances. Using the authors' systematic screening approaches, colchicine could be detected in blood plasma and urine samples using liquid chromatography coupled to linear ion trap mass spectrometry (LC-ITMSn ) and high-resolution tandem mass spectrometry (LC-HRMS/MS). Metabolites of colchicine could be identified in urine for confirmation of screening results. Gas chromatography–mass spectrometry (GC-MS) analysis was also conducted, but colchicine could not be detected. Furthermore, colchicine concentration was estimated via LC-HRMS/MS in plasma samples. Results of the systematic screening indicated the ingestion of colchicine from both subjects. In both cases, the parent compound was detected in blood plasma and urine using the LC-HRMS/MS and LC-ITMSn system. An O-demethylation metabolite was identified in urine samples of both subjects using LC-HRMS/MS; the N-deacetylation product was also found in urine samples of both cases via LC-HRMS/MS and LC-ITMSn . The use of LC-ITMSn resulted only in the detection of the O-demethylation product in case 2. Plasma concentrations were estimated at 2.5 ng/ml and 4.7 ng/ml for cases 1 and 2, respectively. We demonstrated the detection of this highly toxic alkaloid in blood plasma and urine using a time-saving and reliable clinical systematic screening. Furthermore, we identified metabolites of colchicine being rarely discussed in literature, which can be used as additional screening targets

Analytical toxicology of yew constituents in human blood and urine by liquid chromatography-high-resolution tandem mass spectrometry

The active, poisonous constituents in Taxus baccata, the yew plants, are taxine alkaloids whose main action is suggested to be a block of calcium and sodium channels. The main alkaloids are taxine B (30%) and taxine A (1.3%). Symptoms can include bradycardia, bradypnea, diastolic, and cardiac standstill. The current investigation reports the analytical toxicology of human blood and urine to confirm a suspected ingestion of yew needles. This includes the qualitative detection of several yew ingredients, including the main alkaloids, the validated quantification of 3,5-dimethoxyphenol, and the discussion of suitable analytical targets. After analyzing human specimens and yew needle extracts using the developed procedures, the five alkaloids 1-deotaxine B, taxicatin, taxine A, taxine B, and taxine I could be detected and tentatively identified. Finally, taxine A and B can be recommended as analytical targets besides 3,5-dimethoxyphenol

Recent trends in drugs of abuse metabolism studies for mass spectrometry-based analytical screening procedures

The still increasing number of drugs of abuse, particularly the so-called new psychoactive substances (NPS), poses an analytical challenge for clinical and forensic toxicologists but also for doping control. NPS usually belong to various classes such as synthetic cannabinoids, phenethylamines, opioids, or benzodiazepines. Like other xenobiotics, NPS undergo absorption, distribution, metabolism, and excretion processes after consumption, but only very limited data concerning their toxicokinetics and safety properties is available once they appear on the market. The inclusion of metabolites in mass spectral libraries is often crucial for the detection of NPS especially in urine screening approaches. Authentic human samples may represent the gold standard for identification of metabolites but are often not available and clinical studies cannot be performed due to ethical concerns. However, numerous alternative in vitro and in vivo models are available. This trends article will give an overview on selected models, discuss current studies, and highlight recent developments

An easy and fast adenosine 5'-diphosphate quantification procedure based on hydrophilic interaction liquid chromatography-high resolution tandem mass spectrometry for determination of the in vitro adenosine 5'-triphosphatase activity of the human breast cancer resistance protein ABCG2

Interactions with the human breast cancer resistance protein (hBCRP) significantly influence the pharmacokinetic properties of a drug and can even lead to drug-drug interactions. As efflux pump from the ABC superfamily, hBCRP utilized energy gained by adenosine 5′-triphosphate (ATP) hydrolysis for the transmembrane movement of its substrates, while adenosine 5′-diphosphate (ADP) and inorganic phosphate were released. The ADP liberation can be used to detect interactions with the hBCRP ATPase. An ADP quantification method based on hydrophilic interaction liquid chromatography (HILIC) coupled to high resolution tandem mass spectrometry (HR-MS/MS) was developed and successfully validated in accordance to the criteria of the guideline on bioanalytical method validation by the European Medicines Agency. ATP and adenosine 5′-monophosphate were qualitatively included to prevent interferences. Furthermore, a setup consisting of six sample sets was evolved that allowed detection of hBCRP substrate or inhibitor properties of the test compound. The hBCRP substrate sulfasalazine and the hBCRP inhibitor orthovanadate were used as controls. To prove the applicability of the procedure, the effect of amprenavir, indinavir, nelfinavir, ritonavir, and saquinavir on the hBCRP ATPase activity was tested. Nelfinavir, ritonavir, and saquinavir were identified as hBCRP ATPase inhibitors and none of the five HIV protease inhibitors turned out to be an hBCRP substrate. These findings were in line with a pervious publication

Development, validation, and application of a quantitative volumetric absorptive microsampling-based method in finger prick blood by means of LC-HRMS/MS applicable for adherence monitoring of antipsychotics

Volumetric absorptive microsampling (VAMS), an emerging microsampling technique, is expected to overcome some disadvantages of dried blood spots such as volume inaccuracy and influence of hematocrit (HT). This study aimed to develop and evaluate a VAMS-based strategy for quantification of 13 frequently prescribed antipsychotics in finger prick blood within the scope of adherence monitoring to complement already-established qualitative urine analysis. The final workflow consisted of VAMS tip hydration and subsequent precipitation. Samples were analyzed by using reversed-phase ultra-high-performance liquid chromatography and Orbitrap mass spectrometry operated in parallel reaction monitoring mode. The analytical procedure was successfully validated based on international recommendations at three different HT values (20%, 40%, 60%) for most of the analytes. Selectivity and within/between-run accuracy and precision were in accordance with the recommendations in most cases. Internal standard–normalized matrix factor met recommended criteria for all analytes at HT 40%. For the HT values of 20% and 60%, only four substances did not meet the criteria. Dilution integrity was given for all substances, except for olanzapine, allowing a quantification over the whole therapeutic range of selected antipsychotics. Long-term stability in VAMS tips was tested and revealed degradation of five antipsychotic drugs after 1 week of storage at 24 °C. A proof of concept of the applicability of the method was obtained by quantification of a selection of the 13 antipsychotic drugs in VAMS tips and matched plasma samples. Results were coherent between matrices. Thus, VAMS was shown to be a promising alternative for adherence monitoring of at least the investigated antipsychotics

What is the contribution of human FMO3 in the N-oxygenation of selected therapeutic drugs and drugs of abuse?

Little is known about the role of flavin-containing monooxygenases (FMOs) in the metabolism of xenobiotics. FMO3 is the isoform in adult human liver with the highest impact on drug metabolism. The aim of the presented study was to elucidate the contribution of human FMO3 to the N-oxygenation of selected therapeutic drugs and drugs of abuse (DOAs). Its contribution to the in vivo hepatic net clearance of the N-oxygenation products was calculated by application of an extended relative activity factor (RAF) approach to differentiate from contribution of cytochrome P450 (CYP) isoforms. FMO3 and CYP substrates were identified using pooled human liver microsomes after heat inactivation and chemical inhibition, or single enzyme incubations. Kinetic parameters were subsequently determined using recombinant human enzymes and mass spectrometric analysis via authentic reference standards or simple peak areas of the products divided by those of the internal standard. FMO3 was identified as enzyme mainly responsible for the formation of N,N-diallyltryptamine N-oxide and methamphetamine hydroxylamine (>80% contribution for both). A contribution of 50 and 30% was calculated for the formation of N,N-dimethyltryptamine N-oxide and methoxypiperamide N-oxide, respectively. However, FMO3 contributed with less than 5% to the formation of 3-bromomethcathinone hydroxylamine, amitriptyline N-oxide, and clozapine N-oxide. There was no significant difference in the contributions when using calibrations with reference metabolite standards or peak area ratio calculations. The successful application of a modified RAF approach including FMO3 proved the importance of FMO3 in the N-oxygenation of DOAs in human metabolism