Revisited: Therapeutic and toxic blood concentrations of more than 1100 drugs and other xenobiotics.

In order to assess the significance of drug/substance levels measured in intensive care medicine and clinical and forensic toxicology as well as for therapeutic drug monitoring, it is essential that a comprehensive collection of data is readily available. We revisited and expanded our 2012 compilation of therapeutic and toxic plasma concentration ranges as well as half-lives of now more than 1100 drugs and other xenobiotics.Data have been abstracted from original papers, text books, and previous compilations and have been completed with data collected in our own forensic and clinical toxicology laboratories. We compiled the data presented in the table and the corresponding annotations over the past 30+ years. A previous compilation was completely double-checked, revised, and updated, if necessary. In addition, more than 200 substances, especially drugs who have been introduced since 2012 to the market as well as illegal drugs and other xenobiotics which became known to cause intoxications were added. We carefully referenced all data. Moreover, the annotations providing details were updated and revised, when necessary.For more than 1100 drugs and other xenobiotics, therapeutic ("normal") and, if data was available, toxic, and comatose-fatal plasma/blood concentrations as well as elimination half-lives were compiled in a table.In case of intoxications, the blood concentration of the substance and/or metabolite better predicts the clinical severity of the case when compared to the assumed amount and time of ingestion. Comparing and contrasting the clinical case against the data provided, including the half-life, may support the decision for or against further intensive care. In addition, the data provided are useful for the therapeutic monitoring of pharmacotherapies, to facilitate the diagnostic assessment and monitoring of acute and chronic intoxications as well as to support forensic and clinical expert opinions.

Metabolomics-based parallel discovery of xenobiotics and induced endogenous metabolic dysregulation in clinical toxicology.

Intoxication by xenobiotics triggers the perturbation of metabolic fingerprints in biofluids, including the accumulation of xenobiotic compounds and the dysregulation of endogenous metabolites. In this work, an untargeted metabolomics workflow was developed to simultaneously profile both xenobiotic and endogenous metabolites for the identification of the xenobiotic origin and an in-depth understanding of the intoxication mechanism. This workflow was demonstrated in a real-world clinical case. Plasma samples were collected from four intoxicated children and another three healthy children. Untargeted metabolomics analysis was performed using ultraperformance liquid chromatography (UPLC) coupled to a high-resolution mass spectrometer (HRMS) with data-independent MSE acquisition. LC-MSE data was processed using an untargeted metabolomics data interpretation workflow, in which the identities of xenobiotics and altered endogenous metabolic features were determined via database searching. Five xenobiotic chemicals and 19 endogenous metabolites were found to be dysregulated. Combined with the clinical evidence, penfluridol was confirmed as the xenobiotic toxin. Furthermore, a mechanistic hypothesis was developed to explain the dysregulation of the four endogenous acyl-carnitines. This workflow can be readily applied to a wide range of clinical toxicology cases, offering a powerful and convenient means of simultaneous discovery of intoxication source and the understanding of intoxication mechanisms.

Cardiac biomarkers as sensitive tools to evaluate the impact of xenobiotics on amphibians: the effects of anionic surfactant linear alkylbenzene sulfonate (LAS).

Amphibian populations have been experiencing a drastic decline worldwide. Aquatic contaminants are among the main factors responsible for this decline, especially in the aquatic environment. The linear alkylbenzene sulfonate (LAS) is of particular concern, since it represents 84% of the anionic surfactants' trade. In Brazil, the maximal LAS concentration allowed in fresh waters is 0.5mgL-1, but its potential harmful effects in amphibians remain unknown. Therefore, this study aimed to analyze the effects of a sublethal concentration of LAS (0.5mgL-1) for 96h on sensitive cardiac biomarkers of bullfrog tadpoles, Lithobates catesbeianus (Shaw, 1802). For this, we measured the activity level (AL - % of animals), in situ heart rate (fH - bpm), relative ventricular mass (RVM - % of body mass), in vitro myocardial contractility and cardiac histology of the ventricles. Tadpoles' AL and fH decreased in LAS group. In contrast, the RVM increased, as a result of a hypertrophy of the myocardium, which was corroborated by the enlargement of the nuclear measures and the increase of myocytes' diameters. These cellular effects resulted in an elevation of the in vitro contractile force of ventricle strips. Acceleration in the contraction (TPT - ms) also occurred, although no alterations in the time to relaxation (THR -ms) were observed. Therefore, it can be concluded that even when exposed to an environmentally safe concentration, this surfactant promotes several alterations in the cardiac function of bullfrog tadpoles that can impair their development, making them more susceptible to predators and less competitive in terms of reproduction success. Thus, LAS concentrations that are considered safe by Brazilian by regulatory agencies must be revised in order to minimize a drastic impact over amphibian populations. This study demonstrates the relevance of employing cardiac biomarkers at different levels (e.g., morphological, physiological and cellular) to evaluate effects of xenobiotics in tadpoles.

Mouse Red Blood Cell-Mediated Rare Xenobiotic Phosphorylation of a Drug Molecule Not Intended to Be a Kinase Substrate.

Phosphorylation of xenobiotics is rare, probably owing to a strong evolutionary pressure against it. This rarity may have attracted more attention recently as a result of intentionally designed kinase-substrate analogs that depend on kinase-catalyzed activation to form phosphorylated active drugs. We report a rare phosphorylated metabolite observed unexpectedly in mouse plasma samples after an oral dose of a Tankyrase inhibitor that was not intended to be a kinase substrate, i.e., (S)-2-(4-(6-(3,4-dimethylpiperazin-1-yl)-4-methylpyridin-3-yl)phenyl)-8-(hydroxymethyl)quinazolin-4(3H)-one (AZ2381). The phosphorylated metabolite was not generated in mouse hepatocytes. In vitro experiments showed that the phosphorylation of AZ2381 occurred in mouse whole blood with heparin as anticoagulant but not in mouse plasma. The phosphorylated metabolite was also produced in rat, dog, and human blood, albeit at lower yields than in mouse. Divalent metal ions are required for the phosphorylation since the reaction is inhibited by the metal chelator EDTA. Further investigations with different cellular fractions of mouse blood revealed that the phosphorylation of AZ2381 was mediated by erythrocytes but did not occur with leukocytes. The levels of 18O incorporation into the phosphorylated metabolite when inorganic 18O4-phosphate and γ-18O4-ATP were added to the mouse blood incubations separately suggested that the phosphoryl transfer was from inorganic phosphate rather than ATP. It remains unclear which enzyme present in red blood cells is responsible for this rare phosphorylation.

Generation and Characterization of a Breast Cancer Resistance Protein Humanized Mouse Model.

Breast cancer resistance protein (BCRP) is expressed in various tissues, such as the gut, liver, kidney and blood brain barrier (BBB), where it mediates the unidirectional transport of substrates to the apical/luminal side of polarized cells. Thereby BCRP acts as an efflux pump, mediating the elimination or restricting the entry of endogenous compounds or xenobiotics into tissues and it plays important roles in drug disposition, efficacy and safety. Bcrp knockout mice (Bcrp(-/-)) have been used widely to study the role of this transporter in limiting intestinal absorption and brain penetration of substrate compounds. Here we describe the first generation and characterization of a mouse line humanized for BCRP (hBCRP), in which the mouse coding sequence from the start to stop codon was replaced with the corresponding human genomic region, such that the human transporter is expressed under control of the murineBcrppromoter. We demonstrate robust human and loss of mouse BCRP/Bcrp mRNA and protein expression in the hBCRP mice and the absence of major compensatory changes in the expression of other genes involved in drug metabolism and disposition. Pharmacokinetic and brain distribution studies with several BCRP probe substrates confirmed the functional activity of the human transporter in these mice. Furthermore, we provide practical examples for the use of hBCRP mice to study drug-drug interactions (DDIs). The hBCRP mouse is a promising model to study the in vivo role of human BCRP in limiting absorption and BBB penetration of substrate compounds and to investigate clinically relevant DDIs involving BCRP.

SERUM ANTIOXIDANT PARAMETERS IN PATIENTS POISONED BY DIFFERENT XENOBIOTICS.

There is a great diversity of the acute drugs overdose cases in clinical toxicology. Clinical situation is complicated by the coexistence of factors predisposing to the development of adverse drug reactions (chronic use of drugs, polypharmacy, alcohol or drugs dependence, nutritional disorders) and by the presence of chronic organ damage, especially the liver and the kidney. The aim of this study was to evaluate whether there are sensitive plasma markers belonging to the antioxidant system in patients exposed to various xenobiotics. We measured the activity of antioxidant parameters: catalase (CAT), glutathione peroxidase (GPX3), glutathione (GSH), sulfhydryl groups (-SH), carbonyl groups (=CO) and free radicals (2,2-diphenyl-1-picrylhydrazyl, DPPH, assay) in serum of 49 patients with acute intoxication caused by carbamazepine (CBZ, n = 9), mixed drug intoxication (MDI) (n = 9), alcohol withdrawal syndrome (AWS, n = 9), acetaminophen (APAP, n = 7), tricyclic antidepressants (TCAs) (n = 5), valproic acids (VA, n = 4), narcotics (N, n = 3), and three others (benzodiazepines, BZD, n = 2; barbiturates, n = 1). The results were compared with the parameters of not intoxicated patients (n = 39). All patients had lower catalase activity in comparison to the control group (41.9 ± 16.5 vs. 196.0 ± 82.2 U/mg protein, p = 0.000), while the increase of GSH level was particularly apparent only in patients with AWS (391.3 ± 257.9 µmol/mg protein) compared to the control group (171.4 ± 88.4 µmol/mg protein, p = 0.034) and to patients intoxicated with carbamazepine (152.8 ± 102.5 µmol/mg protein, p = 0.027). Some differences, but without statistical significance, were also observed in GPX3 activity between different groups of poisoned patients.

A PBPK model describing a xenobiotic with a short PK event scale.

Physiologically-based pharmacokinetic (PBPK) modeling has been widely used in human risk assessment and in early drug development to predict human PK from in vitro and/or in vivo animal data. Recently, the application of PBPK modeling has been extended to the evaluation of drug-drug interactions. For most xenobiotic agents, the PK event scale such as elimination is in hours or days. This is much longer than the transit time of the agent in the body, and a PBPK model can be significantly simplified through lumping based on the physiochemical properties, mass transfer, and biotransformation. However, for a xenobiotic agent with a short PK event scale, e.g. in minutes, such an approach is not applicable. In this manuscript, the authors used the observed PK data from an ultrasound contrast agent to illustrate the role of a short PK event scale in the development of a suitable PBPK model. The model development process showed that a PBPK model assuming uniform venous and arterial blood pools, with a static lung model including alveolar and tissue regions, was unable to adequately capture the characteristics of the PK of the agent. Detailed information describing the pulmonary and cardiovascular circulation, and a heterogeneous dynamic lung model became necessary for the model. This exercise once again demonstrates the importance of the principles and methodologies that have been established since the 1960s that need to be followed during PBPK model development.

Polymorphisms in phase I and phase II genes and breast cancer risk and relations to persistent organic pollutant exposure: a case-control study in Inuit women.

BACKGROUND: We have previously reported that chemicals belonging to the persistent organic pollutants (POPs) such as perfluorinated compounds (PFAS) and polychlorinated biphenyls (PCBs) are risk factors in Breast Cancer (BC) development in Greenlandic Inuit women. The present case-control study aimed to investigate the main effect of polymorphisms in genes involved in xenobiotic metabolism and estrogen biosynthesis, CYP1A1, CYP1B1, COMT and CYP17, CYP19 and the BRCA1 founder mutation in relation to BC risk and to explore possible interactions between the gene polymorphisms and serum POP levels on BC risk in Greenlandic Inuit women. METHODS: The study population consisted of 31 BC cases and 115 matched controls, with information on serum levels of POPs. Genotyping was conducted for CYP1A1 (Ile462Val; rs1048943), CYP1B1 (Leu432Val; rs1056836), COMT (Val158Met; rs4680), CYP17A1 (A1> A2; rs743572); CYP19A1 (C> T; rs10046) and CYP19A1 ((TTTA)n repeats) polymorphisms and BRCA1 founder mutation using TaqMan allelic discrimination method and polymerase chain reaction based restriction fragment length polymorphism. The χ2 -test was used to compare categorical variables between cases and controls and the odds ratios were estimated by unconditional logistic regression models. RESULTS: We found an independent association of CYP1A1 (Val) and CYP17 (A1) with BC risk.Furthermore, an increased BC risk was observed for women with high serum levels of perfluorooctane sulfonate (PFOS) and perfluorooctanoic acid (PFOA) and carriers of at least: one CYP1A1 variant Val allele; one variant COMT Met allele; or the common CYP17 A1 allele. No combined effects were seen between PFAS exposure and CYP1B1 and CYP19 polymorphisms. The risk of BC was not found significantly associated with exposure to PCBs and OCPs, regardless of genotype for all investigated SNPs. The frequency of the Greenlandic founder mutation in BRCA1 was as expected higher in cases than in controls. CONCLUSIONS: The BRCA1 founder mutation and polymorphisms in CYP1A1 (Val) and CYP17 (A1) can increase the BC risk among Inuit women and the risk increases with higher serum levels of PFOS and PFOA. Serum PFAS levels were a consistent risk factor of BC, but inter-individual polymorphic differences might cause variations in sensitivity to the PFAS/POP exposure.

Sexual dimorphism in xenobiotic genetic variants-mediated risk for Parkinson's disease.

The vulnerability of dopaminergic neurons to environmental exposures in sporadic Parkinson's disease (PD) has been attributed to altered detoxification by xenobiotic metabolizing genes. Hence, we investigated the influence of genetic polymorphisms in xenobiotic metabolic pathway (CYP1A1 m1, CYP1A1 m2, CYP1A1 m4, COMT p.H108L, GSTT1, and GSTM1) on the susceptibility to PD. We used PCR-RFLP for CYP1A1 and COMT genotyping; multiplex-PCR for GSTT1 and GSTM1 deletion analysis; and spectrophotometric methods to evaluate the oxidative stress markers. Results showed association of CYP1A1 m1 (OR: 2.38, 95 % CI: 1.76-3.22) and COMT p.H108L (OR: 2.08 95 % CI: 1.56-2.77) polymorphisms with risk for PD. Male patients carrying combination of COMT p.H108L and CYP1A1 m1 variant alleles showed an early onset of the disease. There was a significant increase in oxidative stress makers such as malondialdehyde and protein carbonyls; and decrease in glutathione levels in PD cases compared to controls (P < 0.05). To conclude, CYP1A1 m1, COMT p.H108L polymorphisms were associated with PD risk, and sexual dimorphism was observed in these associations.

Comparison of intestinal metabolism of CYP3A substrates between rats and humans: application of portal-systemic concentration difference method.

1. Rats are frequently used in pharmacokinetic studies during drug discovery. However, there is limited information regarding species differences in intestinal availability (Fg) between rats and humans. 2. Here, we directly estimated the fraction of dose absorbed in the portal vein (FaFg) of rats for nine CYP3A substrates using portal-systemic concentration difference method and compared them with human FaFg. No distinct difference in FaFg between the two species was observed, and seven of the nine compounds were within a two-fold difference. Given that their net fraction of dose absorbed (Fa) are expected to be high, this result indicates a moderate correlation in Fg between the two species. 3. In contrast, the in vitro intrinsic clearance (CLint,u) in rat intestinal microsomes tended to be lower than that in humans, and the correlation between intestinal CLint,u and FaFg in rats was poor compared with that in humans. 4. Our finding indicates that rats are appropriate animals for evaluation of the intestinal absorption and metabolism of CYP3A substrates. However, a degree of caution is required when estimating rat Fg from rat intestinal microsomes due to the low metabolic activity and the poor correlation between in vitro and in vivo intestinal metabolism.

Multiscale distribution and bioaccumulation analysis of clofazimine reveals a massive immune system-mediated xenobiotic sequestration response.

Chronic exposure to some well-absorbed but slowly eliminated xenobiotics can lead to their bioaccumulation in living organisms. Here, we studied the bioaccumulation and distribution of clofazimine, a riminophenazine antibiotic used to treat mycobacterial infection. Using mice as a model organism, we performed a multiscale, quantitative analysis to reveal the sites of clofazimine bioaccumulation during chronic, long-term exposure. Remarkably, between 3 and 8 weeks of dietary administration, clofazimine massively redistributed from adipose tissue to liver and spleen. During this time, clofazimine concentration in fat and serum significantly decreased, while the mass of clofazimine in spleen and liver increased by >10-fold. These changes were paralleled by the accumulation of clofazimine in the resident macrophages of the lymphatic organs, with as much as 90% of the clofazimine mass in spleen sequestered in intracellular crystal-like drug inclusions (CLDIs). The amount of clofazimine associated with CLDIs of liver and spleen macrophages disproportionately increased and ultimately accounted for a major fraction of the total clofazimine in the host. After treatment was discontinued, clofazimine was retained in spleen while its concentrations decreased in blood and other organs. Immunologically, clofazimine bioaccumulation induced a local, monocyte-specific upregulation of various chemokines and receptors. However, interleukin-1 receptor antagonist was also upregulated, and the acute-phase response pathways and oxidant capacity decreased or remained unchanged, marking a concomitant activation of an anti-inflammatory response. These experiments indicate an inducible, immune system-dependent, xenobiotic sequestration response affecting the atypical pharmacokinetics of a small molecule chemotherapeutic agent.

Usefulness of the chaotropic effect in sample preparation for chromatographic analysis of acidic xenobiotics in human plasma.

In this study a new RP-HPLC with photo-diode array detection method for the determination of ibuprofen ((RS)-2-(4-isobutylphenyl)propionic acid) in human plasma samples was developed. Samples were prepared by SPE and analyzed by an isocratic elution mode over a C18 column using 80% methanol. A novel sample pretreatment method, based on the addition of ionic liquids possessing chaotropic ions to small human plasma sample (100 µL), was elaborated. 1-Butyl-3-methylimidazolium chloride and 1-butyl-3-methylimidazolium tetrafluoroborate (BMIM BF4 ) were tested from the point of view of extraction yield. Quantification was based on calibration curve applying diclofenac as the internal standard. Owing to dilution of plasma sample by 2 mM aqueous solution of BMIM BF4 before SPE, appropriate sample purification and extraction yields higher than 95% with precision lower than 2% can be achieved. Linear coefficients of correlation (r(2)) were >0.99 in the range of 0.3-5 µg/mL ibuprofen concentration in plasma. The limit of quantification was 65 ng/mL and the detection limit for ibuprofen was 19.5 ng/mL.

The combined effect of persistent organic pollutants in the serum POP mixture in Greenlandic Inuit: xenoestrogenic, xenoandrogenic and dioxin-like transactivities.

Greenlandic Inuit have high body burden of persistent organic pollutants (POPs). We analyzed the combined effect of the actual lipophilic serum POP mixture on estrogen-, androgen- and aryl hydrocarbon-receptor functions as effect biomarkers, and the associations between the effect biomarkers and serum POPs, and lifestyle characteristics. The serum POPs were extracted from 232 Inuit from Ittoqqortoormiit, Narsaq and Qeqertarsuaq. The POP-related receptor transactivities correlated negatively to the POP levels and were associated to the lifestyle characteristics. The POP-related receptor transactivities can be used as effect biomarkers. The serum POPs have hormone disruptive potentials.

Therapeutic and toxic blood concentrations of nearly 1,000 drugs and other xenobiotics.

INTRODUCTION: In order to assess the significance of drug levels measured in intensive care medicine, clinical and forensic toxicology, as well as for therapeutic drug monitoring, it is essential that a comprehensive collection of data is readily available. Therefore, it makes sense to offer a carefully referenced compilation of therapeutic and toxic plasma concentration ranges, as well as half-lives, of a large number of drugs and other xenobiotics for quick and comprehensive information. METHODS: Data have been abstracted from original papers and text books, as well as from previous compilations, and have been completed with data collected in our own forensic and clinical toxicology laboratory. The data presented in the table and corresponding annotations have been developed over the past 20 years and longer. A previous compilation has been completely revised and updated. In addition, more than 170 substances, especially drugs that have been introduced to the market since 2003 as well as illegal drugs, which became known to cause intoxications, were added. All data were carefully referenced and more than 200 new references were included. Moreover, the annotations providing details were completely revised and more than 100 annotations were added. RESULTS: For nearly 1,000 drugs and other xenobiotics, therapeutic ("normal") and, if data were available, toxic and comatose-fatal blood-plasma concentrations and elimination half-lives were compiled in a table. CONCLUSIONS: In case of intoxications, the concentration of the ingested substances and/or metabolites in blood plasma better predicts the clinical severity of the case when compared to the assumed amount and time of ingestion. Comparing and contrasting the clinical case against the data provided, including the half-life, may support the decision for or against further intensive care. In addition, the data provided are useful for the therapeutic monitoring of pharmacotherapies, to facilitate the diagnostic assessment and monitoring of acute and chronic intoxications, and to support forensic and clinical expert opinions.

A quantitative approach to hepatic clearance prediction of metabolism by aldehyde oxidase using custom pooled hepatocytes.

We describe the usability of human pooled hepatocytes for non-CYP metabolism evaluation and an in vivo-in vitro correlation analysis for aldehyde oxidase (AO) substrate compounds using pooled hepatocytes. By comparing intrinsic clearance values of 18 compounds primarily metabolized by AO, UDP-glucuronosyltransferase, carbonyl/aldo-keto reductase, flavin-containing monooxygenase, and monoamineoxidase in individual hepatocytes and pooled hepatocytes from the same individual donors, intrinsic clearance in the pooled hepatocytes was ± 30% of the average clearance value in individuals for 15 of 18 compounds, suggesting that pooled hepatocytes maintained the average activity of the individual hepatocytes. Although the results of an in vivo-in vitro correlation analysis for AO substrate compounds showed a trend toward under-prediction, the underestimation ratios for all AO substrates were nevertheless comparable (7.2- to 14.9-fold), suggesting that hepatic clearance prediction for these compounds can be quantified using empirical scaling. These observations enabled us to obtain specific pooled hepatocytes that showed the expected non-CYP enzyme activities by pre-characterization and to quantify hepatic clearance prediction for AO compounds using an empirical scaling factor.

Inverse associations between long-term weight change and serum concentrations of persistent organic pollutants.

There is emerging evidence that persistent organic pollutants (POPs) can increase the risk of various chronic diseases. As POPs mainly bioaccumulate in adipose tissue, weight change can affect serum concentrations of POPs. However, there are few population-based studies on effects of long-term weight change on serum concentrations of POPs. We examined associations between self-reported weight change over 1 year and 10 years and serum concentrations of seven POPs in 1099 adults aged ≥ 40. Serum concentrations of most POPs were higher in those with long-term weight loss, whereas they were lower in those with long-term weight gain. Adjusted correlation coefficients of each POP with weight change for 10 years were -0.23 (P < 0.01) for trans-nonachlor, -0.16 (P < 0.01) for p,p'-dichlorodiphenyldichloroethylene, and -0.21 (P < 0.01) for ß-hexachlorocyclohexane, -0.16 (P < 0.01) for PCB169, -0.20 (P < 0.01) for PCB180 and -0.17 (P < 0.01) for 1,2,3,4,6,7,8-heptachlorodibenzo-p-dioxin. Weight change for 1 year showed similar but weaker associations, compared with those of long-term weight changes. Although both beneficial health effects after weight loss and harmful health effects after weight gain are generally expected, changes in serum concentrations of POPs in relation to weight change may act on health in directions opposite to what we expect with weight change.

Absorption and disposition of the sphingosine 1-phosphate receptor modulator fingolimod (FTY720) in healthy volunteers: a case of xenobiotic biotransformation following endogenous metabolic pathways.

Fingolimod [(FTY720), Gilenya; 2-amino-2-[2-(4-octylphenyl)ethyl]-1,3-propanediol], a new drug for the treatment of relapsing multiple sclerosis, acts through its phosphate metabolite, which modulates sphingosine 1-phosphate receptors. This represents a novel mechanism of action. In the present work, the absorption and disposition of (14)C-labeled fingolimod were investigated in healthy male volunteers after a single oral dose of 4.5 mg. Total radioactivity was determined in blood, urine, and feces. Fingolimod was quantified in blood. Metabolite profiles were determined in blood and excreta, and metabolite structures were elucidated by mass spectrometry, wet-chemical methods, and comparison with reference compounds. Fingolimod was absorbed slowly but almost completely. The biotransformation of fingolimod involved three main pathways: 1) reversible phosphorylation to fingolimod phosphate [(S)-enantiomer, active principle]; 2) ω-hydroxylation at the octyl chain, catalyzed predominantly by CYP4F enzymes, followed by further oxidation to a carboxylic acid and subsequent ß-oxidation; and 3) formation of ceramide analogs by conjugation with endogenous fatty acids. This metabolism is quite unusual because it follows metabolic pathways of structurally related endogenous compounds rather than biotransformations typical for xenobiotics. The elimination of fingolimod was slow and occurred predominantly by oxidative metabolism whereas fingolimod phosphate was eliminated mainly by dephosphorylation back to fingolimod. Drug-related material was excreted mostly in the urine in the form of oxidation products.

[Determination of blood/serum ratios of different forensically relevant analytes in authentic samples]. / Bestimmung der Blut/Serum-verhältnisse verschiedener forensisch relevanter Analyten in authentischen Proben.

For forensic toxicological investigations only whole blood, but no serum is often available. Pharmacokinetic data are helpful for interpreting the results, but most of these studies indicate serum or plasma concentrations. In order to obtain reliable conversion factors which also take intersubject variability into account, the blood/serum ratios (B/S) of oxycodone, morphine, fentanyl, hydromorphone, zopiclone, MDMA, dexamphetamine, alprazolam, risperidone and 9-hydroxyrisperidone were determined by LC-MS/MS using authentic samples. Blood and corresponding serum samples were obtained from driving studies performed with controlled or known dosages of the above drugs. The analytes were analysed in blood and serum and the following mean B/S ratios (relative standard deviations) were determined: oxycodone 1.48 (8.19 %); morphine 1.03 (3.59 %); fentanyl 0.87 (13.9 %); hydromorphone 1.04 (8.11 %); zopiclone 0.89 (16.1 %); MDMA 1.19 (8.04 %); dexamphetamine 0.89 (10.9 %); alprazolam 0.81 (5.84 %); risperidone 0.65 (7.52 %); 9-hydroxyrisperidone 0.73 (12.3 %). These mean values are largely in line with those reported in the literature. The B/S ratios did not appear to depend on partition coefficients, whereas there was strong evidence that B/S ratios decreased with increasing plasma protein binding.

Biological definition of multiple chemical sensitivity from redox state and cytokine profiling and not from polymorphisms of xenobiotic-metabolizing enzymes.

BACKGROUND: Multiple chemical sensitivity (MCS) is a poorly clinically and biologically defined environment-associated syndrome. Although dysfunctions of phase I/phase II metabolizing enzymes and redox imbalance have been hypothesized, corresponding genetic and metabolic parameters in MCS have not been systematically examined. OBJECTIVES: We sought for genetic, immunological, and metabolic markers in MCS. METHODS: We genotyped patients with diagnosis of MCS, suspected MCS and Italian healthy controls for allelic variants of cytochrome P450 isoforms (CYP2C9, CYP2C19, CYP2D6, and CYP3A5), UDP-glucuronosyl transferase (UGT1A1), and glutathione S-transferases (GSTP1, GSTM1, and GSTT1). Erythrocyte membrane fatty acids, antioxidant (catalase, superoxide dismutase (SOD)) and glutathione metabolizing (GST, glutathione peroxidase (Gpx)) enzymes, whole blood chemiluminescence, total antioxidant capacity, levels of nitrites/nitrates, glutathione, HNE-protein adducts, and a wide spectrum of cytokines in the plasma were determined. RESULTS: Allele and genotype frequencies of CYPs, UGT, GSTM, GSTT, and GSTP were similar in the Italian MCS patients and in the control populations. The activities of erythrocyte catalase and GST were lower, whereas Gpx was higher than normal. Both reduced and oxidised glutathione were decreased, whereas nitrites/nitrates were increased in the MCS groups. The MCS fatty acid profile was shifted to saturated compartment and IFNgamma, IL-8, IL-10, MCP-1, PDGFbb, and VEGF were increased. CONCLUSIONS: Altered redox and cytokine patterns suggest inhibition of expression/activity of metabolizing and antioxidant enzymes in MCS. Metabolic parameters indicating accelerated lipid oxidation, increased nitric oxide production and glutathione depletion in combination with increased plasma inflammatory cytokines should be considered in biological definition and diagnosis of MCS.

Comprehensive plasma-screening for known and unknown substances in doping controls.

Occasionally, doping analysis has been recognized as a competitive challenge between cheating sportsmen and the analytical capabilities of testing laboratories. Both have made immense progress during the last decades, but obviously the athletes have the questionable benefit of frequently being able to switch to new, unknown and untested compounds to enhance their performance. Thus, as analytical counteraction and for effective drug testing, a complementary approach to classical targeted methods is required in order to implement a comprehensive screening procedure for known and unknown xenobiotics. The present study provides a new analytical strategy to circumvent the targeted character of classical doping controls without losing the required sensitivity and specificity. Using 50 microL of plasma only, the method potentially identifies illicit drugs in low ng/mL concentrations. Plasma provides the biological fluid with the circulating, unmodified xenobiotics; thus the identification of unknown compounds is facilitated. After a simple protein precipitation, liquid chromatographic separation and subsequent detection by means of high resolution/high accuracy orbitrap mass spectrometry, the procedure enables the determination of numerous compounds from different classes prohibited by the World Anti-Doping Agency (WADA). A new hyphenated mass spectrometry technology was employed without precursor ion selection for higher collision energy dissociation (HCD) fragmentation experiments. Thus the mass spectra contained all the desired information to identify unknown substances retrospectively. The method was validated for 32 selected model compounds for qualitative purposes considering the parameters specificity, selectivity, limit of detection (<0.1-10 ng/mL), precision (9-28%), robustness, linearity, ion suppression and recovery (80-112%). In addition to the identification of unknown compounds, the plasma samples were simultaneously screened for known prohibited targets.