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Along with the recent acknowledgement of the World Anti-Doping Agency to use dried blood spot (DBS) samples for routine doping control purposes, there have been propositions to use DBS as a matrix that allows regular proactive remotely supervised self-sampling, providing potential longitudinal monitoring of an athlete's exposure to doping agents. However, several organizational aspects have to be considered before implementation, such as the verification of the sample collections time point. Based on a previous untargeted proteomics workflow utilizing liquid chromatography–high-resolution mass spectrometry (LC–HRMS) to identify protein/peptide markers to define the time since deposition of a bloodstain, the aim of the current study was to develop a targeted LC–HRMS/MS analytical method for promising peptidic target analytes. A long-term DBS storage experiment was carried out over a 3-month period (sample collection time points: 0, 2, 4, 7, 14, 21, 28, 42, 56, 70, 84 and 91 days) with DBS samples of 10 volunteers for longitudinal investigation of signal abundance changes of targeted peptide sequences at different storage temperatures (room temperature [RT], 4°C and −20°C). Prior to experimental analysis, LC–HRMS/MS method characteristics were successfully assessed, including intraday precision, carryover and sample extract stability. For estimation of DBS sample collection time points, ratios of two peptides that originate from the same protein prior to tryptic digestion were created. Two targeted peptide area ratios were found to significantly increase after being stored at RT for 28 days, representing potential markers for future use in routine doping controls that contribute to advancing complementary avenues in anti-doping.

Receptor tyrosine-protein kinase ERBB3 (HER3) is expressed in most EGFR-mutated lung cancers but is not a known mechanism of resistance to EGFR inhibitors. HER3-DXd is an antibody-drug conjugate consisting of a HER3 antibody attached to a topoisomerase I inhibitor payload via a tetrapeptide-based cleavable linker. This phase I, dose escalation/expansion study included patients with locally advanced or metastatic EGFR-mutated non-small cell lung cancer (NSCLC) with prior EGFR tyrosine kinase inhibitor (TKI) therapy. Among 57 patients receiving HER3-DXd 5.6 mg/kg intravenously once every 3 weeks, the confirmed objective response rate by blinded independent central review (Response Evaluation Criteria in Solid Tumors v1.1) was 39% [95% confidence interval (CI), 26.0-52.4], and median progression-free survival was 8.2 (95% CI, 4.4-8.3) months. Responses were observed in patients with known and unknown EGFR TKI resistance mechanisms. Clinical activity was observed across a broad range of HER3 membrane expression. The most common grade ≥3 treatment-emergent adverse events were hematologic toxicities. HER3-DXd has clinical activity in EGFR TKI-resistant cancers independent of resistance mechanisms, providing an approach to treat a broad range of drug-resistant cancers. SIGNIFICANCE: In metastatic EGFR-mutated NSCLC, after disease progression on EGFR TKI therapy, treatment approaches include genotype-directed therapy targeting a known resistance mechanism or chemotherapy. HER3-DXd demonstrated clinical activity spanning known and unknown EGFR TKI resistance mechanisms. HER3-DXd could present a future treatment option agnostic to the EGFR TKI resistance mechanism.See related commentary by Lim et al., p. 16.This article is highlighted in the In This Issue feature, p. 1.

New biomarkers indicating the abuse of drugs and alcohol are still of major interest for clinical and forensic sciences. The endogenous neurotransmitter and approved drug, gamma-hydroxybutyric acid (GHB), is often illegally used for drug-facilitated crimes by spiking GHB into alcoholic beverages. Analytical detection windows of only 6 h in blood and 12 h in urine are often too short to provide reliable proof of GHB ingestion. Therefore, new biomarkers are needed to prove exogenous GHB administration. Previously, amino acid GHB conjugates were discovered in an untargeted metabolomics screening and fatty acid esters with GHB were recently discussed as promising biomarkers to enlarge the analytical detection time windows. However, the development of analytical methods is still slowed down since reference compounds for targeted screenings are still missing. In this paper, we describe simple procedures for the rapid synthesis and purification of amino acid GHB conjugates as well as fatty acid esters, which can be adopted in analytical and clinical/forensic laboratories. Structural characterization data, together with IR, $^1$H-nuclear magnetic resonance (NMR), $^{13}$C-NMR, high-resolution mass spectra (MS), and MS/MS spectra in positive and negative ionization mode are reported for all obtained GHB conjugates and GHB conjugate precursors.

Forensic postmortem case interpretation can be challenging, in particular due to postmortem redistribution (PMR) phenomena. Recent studies have shown that computed tomography (CT)-guided collection of biopsy samples using a robotic arm (virtobot) provides a valuable tool for systematic studies on time-dependent PMR. Utilizing this strategy, several cases involving opioid use such as methadone, fentanyl, tramadol, codeine, oxycodone and hydrocodone were evaluated for time-dependent concentration changes and potential redistribution mechanisms. Upon admission to the institute (t1), blood (femoral and right ventricle heart blood) and tissue biopsy samples (lung, kidney, liver, spleen, thigh muscle and adipose tissue) were collected utilizing CT-guided biopsy. Approximately 24 h later (t2; mean 28 ± 15 h), during the autopsy, samples from the same body regions were collected manually and in addition brain tissue, gastric content, urine and left ventricle heart blood. Analysis was conducted with liquid chromatography tandem mass spectrometry. Significant time-dependent methadone concentration increases in femoral blood (pB) indicate the occurrence of PMR, however, ultimately not relevant for forensic interpretation. The main metabolite of methadone, 2-ethylidene-1,5-dimethyl-3,3-diphenylpyrrolidine (EDDP), showed a less significant trend for PMR. Redistribution by passive diffusion along the muscle-to-pB concentration gradient seems likely for methadone, but not for EDDP. Results for fentanyl suggest extensive PMR. Other opioids such as tramadol, codeine, hydrocodone and oxycodone showed no consistent trend for significant PMR. Overall, CT-guided biopsy sampling proved to be a valuable tool for the investigation of PMR mechanisms.

Zebrafish (Danio rerio) larvae have been suggested as vertebrate model to complement or even replace mammals for rapidly assessing behavioral effects of psychoactive drugs. Yet, divergent responses have been reported in mammals and fish despite the conservation of many drug targets. Cocaine, eg, acts as stimulant in mammals but no such response has been documented for zebrafish larvae. We hypothesized that differences in exposure routes (inhalation or injection in mammals vs waterborne in fish) may be a reason for differences in behavioral responses. We characterized cocaine toxicokinetics by liquid chromatography-mass spectrometry and found its rapid uptake into larvae. We used Matrix-assisted laser desorption ionization-mass spectrometry imaging for the first time to characterize internal distribution of cocaine in zebrafish larvae. Surprisingly, eyes accumulated the highest amount of cocaine and retained most of it even after 48 h depuration. We attribute this to trapping by pigment melanin, a thus far little explored mechanism that may also be relevant for other basic drugs. Cocaine also reached the brain but with levels similar to those in trunk indicating simple passive diffusion as means of distribution which was supported by toxicokinetic models. Although brain levels covered those known to cause hyperactivity in mammals, only hypoactivity (decreased locomotion) was recorded in zebrafish larvae. Our results therefore point to cocaine's anesthetic properties as the dominant mechanism of interaction in the fish: upon entry through the fish skin and gills, it first acts on peripheral nerves rapidly overriding any potential stimulatory response in the brain.

Postmortem redistribution (PMR) can result in artificial drug concentration changes following death and complicate forensic case interpretation. Currently, no accurate methods for PMR prediction exist. Hence, alternative strategies were developed investigating the time-dependent postmortem behavior of diazepam, nordiazepam, morphine, codeine, mirtazapine and citalopram. For 477 authentic postmortem cases, femoral blood samples were collected at two postmortem time-points. All samples were quantified for drugs of abuse (targeted; liquid chromatography-tandem mass spectrometry LC-MS/MS) and characterized for small endogenous molecules (untargeted; gas chromatography-high resolution MS (GC-HRMS). Trends for significant time-dependent concentration decreases (diazepam (n = 137), nordiazepam (n = 126)), increases (mirtazapine (n = 55), citalopram (n = 50)) or minimal median postmortem changes (morphine (n = 122), codeine (n = 92)) could be observed. Robust mathematical mixed effect models were created for the generalized postmortem behavior of diazepam and nordiazepam, which could be used to back-calculate drug concentrations towards a time-point closer to the estimated time of death (caution: inter-individual variability). Significant correlations between time-dependent concentration changes of morphine, mirtazapine and citalopram with individual endogenous molecules could be determined; no correlation was deemed strong enough for successful a posteriori estimation on the occurrence of PMR for specific cases. The current dataset did successfully lead to a significant knowledge gain in further understanding the time-dependent postmortem behavior of the studied drugs (of abuse).

Hair analysis has become a valuable tool in forensic toxicology to assess drug or alcohol abstinence. Yet, hair adulteration by cosmetic products presents a major challenge for forensic hair analysis. Oxidative treatments, e.g. bleaching, may lead to analyte loss and thereby to false negative results. Currently, the eumelanin degradation product 1H‐pyrrole‐2,3,5‐tricarboxylic acid (PTCA) serves as a marker for oxidative hair treatment, but requires the definition of cut‐off values. To investigate further eumelanin degradation products as markers for oxidative hair treatment, hair samples with and without in vitro bleaching (hydrogen peroxide (H2O2) concentrations 1.9 % up to 12 %; incubation times 15 min, 30 min, 60 min) were analyzed by liquid chromatography coupled to high‐resolution time of flight mass spectrometry (HPLC‐HRMS). The distribution of eumelanin degradation products along the hair shaft was investigated for routine applicability after segmentation of cosmetically untreated hair samples and authentically treated hair samples. The signals of the eumelanin degradation products PTCA, 1H‐pyrrole‐2,3,4‐tricarboxylic acid (isoPTCA) and 1H‐pyrrole‐2,3,4,5‐tetracarboxylic acid (PTeCA) were found to be significantly elevated after in vitro bleaching already with low H2O2 concentrations and after short incubation times. In contrast to PTCA and isoPTCA, PTeCA was not detectable in cosmetically untreated segments up to 12 cm from hair root and was only formed through the oxidation process. The results of the study show that the detection of PTeCA within the proximal 3 to 6 cm segment can be applied to reliably detect hair adulteration attempts through hair bleaching.

Background Gamma-hydroxybutyrate (GHB; or sodium oxybate) is an endogenous GHB-/GABAB receptor agonist. It is approved for the application in narcolepsy and has been proposed for potential treatment of Alzheimer’s and Parkinson’s disease, fibromyalgia, and depression, all of which involve neuro-immunological processes. Tryptophan catabolites (TRYCATs), the cortisol awakening response (CAR), and brain derived neurotrophic factor (BDNF) have been suggested as peripheral biomarkers of neuropsychiatric disorders. GHB has been shown to induce a delayed reduction of T helper and natural killer cell counts and alter basal cortisol levels, but GHB’s effects on TRYCATs, CAR and BDNF are unknown. Methods Therefore, TRYCAT and BDNF serum levels as well as CAR and the affective state (Positive and Negative Affect Schedule, PANAS) were measured in the morning after a single nocturnal dose of GHB (50 mg/kg body weight) in 20 healthy male volunteers in a placebo-controlled, balanced, randomized, double-blind, cross-over design. Results In the morning after nocturnal GHB administration, the TRYCATs indolelactic acid, kynurenine, kynurenic acid, 3-hydroxykynurenine, and quinolinic acid, the 3-hydroxykynurenine to kynurenic acid ratio and the CAR were significantly reduced (p<0.05-0.001, Benjamini-Hochberg corrected). The quinolinic acid to kynurenic acid ratio was reduced by trend. Serotonin, tryptophan, and BDNF levels as well as PANAS scores in the morning remained unchanged after nocturnal GHB challenge. Conclusions GHB has postacute effects on peripheral biomarkers of neuropsychiatric disorders, which might be a model to explain some of its therapeutic effects in disorders involving neuro-immunological pathologies (ClinicalTrials.gov: NCT02342366).

Determination of the ethanol concentration in corpses with MRS would allow a reproducible forensic assessment by which evidence is collected in a noninvasive manner. However, although MRS has been successfully used to detect ethanol in vivo, it has not been applied to postmortem ethanol quantification in situ. The present study examined the feasibility of the noninvasive measurement of the ethanol concentration in human corpses with MRS. A total of 15 corpses with suspected alcohol consumption before demise underwent examination in a 3 T whole body scanner. To address the partial overlap of the ethanol and lactate signal in the postmortem spectrum, non‐water‐suppressed single voxel spectra were recorded in the cerebrospinal fluid (CSF) of the left lateral ventricle via the metabolite cycling technique. The ethanol signals were quantified using the internal water as reference standard, as well as based on a reference signal acquired in a phantom. The measured values were compared with biochemically determined concentrations in the blood (BAC) and CSF (CSFAC). In 8 of the 15 corpses a BAC above zero was determined (range 0.03–1.68 g/kg). In all of these 8 corpses, ethanol was measured in CSF with the proposed MRS protocol. The two applied MRS calibration strategies resulted in similar concentrations. However, the MRS measurements generally overestimated the ethanol concentration by 0.09 g/kg (4%) to 0.72 g/kg (45%) as compared with the CSFAC value. The presented MRS protocol allows the measurement of ethanol in the CSF in human corpses and provides an estimation of the ethanol concentration prior to autopsy. Observed deviations from biochemically determined concentrations are mainly explained by the approximate correction of the relaxation attenuation of the ethanol signal.

Gamma-hydroxybutyrate (GHB) is a short-chain fatty acid that occurs naturally in the mammalian brain and that is prescribed as a medication against narcolepsy or used as a drug of abuse. Particularly, its use as a knock-out drug in cases of drug facilitated crimes is of major importance in forensic toxicology. Because of its rapid metabolism and resulting narrow detection windows (<12 h in urine), detection of GHB remains challenging. Thus, there is an urgent call for new markers to improve the reliable detection of GHB use. In the framework of a randomized, placebo-controlled, crossover study in 20 healthy male volunteers, urine samples obtained 4.5 hours post-administration were submitted to untargeted mass spectrometry (MS, QTOF) analysis to identify possible new markers of GHB intake. MS data from four different analytical methods (reversed phase and hydrophilic interaction liquid chromatography; positive and negative electrospray ionization) were filtered for significantly changed features applying uni- and multivariate statistics. From the resulting 42 compounds of interest, eight were finally identified including conjugates of GHB with carnitine, glutamate, and glycine as well as the endogenous compounds glycolate and succinylcarnitine. While GHB conjugates were only detectable in the GHB, but not in the placebo group, glycolate and succinylcarnitine were present in both groups albeit significantly increased through GHB intake. Untargeted metabolomics proved as a suitable tool for the non-hypothesis driven identification of new GHB markers. However, more studies on actual concentrations, detection windows, and stability will be necessary to assess the suitability of these markers for routine application.

Forensic postmortem case interpretation can be challenging, in particular due to postmortem redistribution (PMR) phenomena. Recent studies have shown that computed tomography (CT)-guided collection of biopsy samples using a robotic arm (virtobot) provides a valuable tool for systematic studies on time-dependent PMR. Utilizing this strategy, several cases involving opioid use such as methadone, fentanyl, tramadol, codeine, oxycodone and hydrocodone were evaluated for time-dependent concentration changes and potential redistribution mechanisms. Upon admission to the institute (t1), blood (femoral and right ventricle heart blood) and tissue biopsy samples (lung, kidney, liver, spleen, thigh muscle and adipose tissue) were collected utilizing CT-guided biopsy. Approximately 24 h later (t2; mean 28 ± 15 h), during the autopsy, samples from the same body regions were collected manually and in addition brain tissue, gastric content, urine and left ventricle heart blood. Analysis was conducted with liquid chromatography tandem mass spectrometry. Significant time-dependent methadone concentration increases in femoral blood (pB) indicate the occurrence of PMR, however, ultimately not relevant for forensic interpretation. The main metabolite of methadone, 2-ethylidene-1,5-dimethyl-3,3-diphenylpyrrolidine (EDDP), showed a less significant trend for PMR. Redistribution by passive diffusion along the muscle-to-pB concentration gradient seems likely for methadone, but not for EDDP. Results for fentanyl suggest extensive PMR. Other opioids such as tramadol, codeine, hydrocodone and oxycodone showed no consistent trend for significant PMR. Overall, CT-guided biopsy sampling proved to be a valuable tool for the investigation of PMR mechanisms.

Background and Objective: Lysergic acid diethylamide (LSD) is used recreationally and in clinical research. The aim of the present study was to characterize the pharmacokinetics and exposure–response relationship of oral LSD. Methods: We analyzed pharmacokinetic data from two published placebo-controlled, double-blind, cross-over studies using oral administration of LSD 100 and 200 µg in 24 and 16 subjects, respectively. The pharmacokinetics of the 100-µg dose is shown for the first time and data for the 200-µg dose were reanalyzed and included. Plasma concentrations of LSD, subjective effects, and vital signs were repeatedly assessed. Pharmacokinetic parameters were determined using compartmental modeling. Concentration-effect relationships were described using pharmacokinetic-pharmacodynamic modeling. Results: Geometric mean (95% confidence interval) maximum plasma concentration values of 1.3 (1.2–1.9) and 3.1 (2.6–4.0) ng/mL were reached 1.4 and 1.5 h after administration of 100 and 200 µg LSD, respectively. The plasma half-life was 2.6 h (2.2–3.4 h). The subjective effects lasted (mean ± standard deviation) 8.2 ± 2.1 and 11.6 ± 1.7 h for the 100- and 200-µg LSD doses, respectively. Subjective peak effects were reached 2.8 and 2.5 h after administration of LSD 100 and 200 µg, respectively. A close relationship was observed between the LSD concentration and subjective response within subjects, with moderate counterclockwise hysteresis. Half-maximal effective concentration values were in the range of 1 ng/mL. No correlations were found between plasma LSD concentrations and the effects of LSD across subjects at or near maximum plasma concentration and within dose groups. Conclusions: The present pharmacokinetic data are important for the evaluation of clinical study findings (e.g., functional magnetic resonance imaging studies) and the interpretation of LSD intoxication. Oral LSD presented dose-proportional pharmacokinetics and first-order elimination up to 12 h. Th

Zebrafish (Danio rerio) larvae have been suggested as vertebrate model to complement or even replace mammals for rapidly assessing behavioral effects of psychoactive drugs. Yet, divergent responses have been reported in mammals and fish despite the conservation of many drug targets. Cocaine, eg, acts as stimulant in mammals but no such response has been documented for zebrafish larvae. We hypothesized that differences in exposure routes (inhalation or injection in mammals vs waterborne in fish) may be a reason for differences in behavioral responses. We characterized cocaine toxicokinetics by liquid chromatography-mass spectrometry and found its rapid uptake into larvae. We used Matrix-assisted laser desorption ionization-mass spectrometry imaging for the first time to characterize internal distribution of cocaine in zebrafish larvae. Surprisingly, eyes accumulated the highest amount of cocaine and retained most of it even after 48 h depuration. We attribute this to trapping by pigment melanin, a thus far little explored mechanism that may also be relevant for other basic drugs. Cocaine also reached the brain but with levels similar to those in trunk indicating simple passive diffusion as means of distribution which was supported by toxicokinetic models. Although brain levels covered those known to cause hyperactivity in mammals, only hypoactivity (decreased locomotion) was recorded in zebrafish larvae. Our results therefore point to cocaine’s anesthetic properties as the dominant mechanism of interaction in the fish: upon entry through the fish skin and gills, it first acts on peripheral nerves rapidly overriding any potential stimulatory response in the brain.

3,4-methylenedioxymethamphetamine (MDMA; ecstasy) metabolism is known to be stereoselective, with preference for S-stereoisomers. Its major metabolic step involves CYP2D6-catalyzed demethylenation to 3,4-dihydroxymethamphetamine (DHMA), followed by methylation and conjugation. Alterations in CYP2D6 genotype and/or phenotype have been associated with higher toxicity. Therefore, the impact of CYP2D6 function on the plasma pharmacokinetics of MDMA and its phase I and II metabolites was tested by comparing extensive metabolizers (EMs), intermediate metabolizers (IMs), and EMs that were pretreated with bupropion as a metabolic inhibitor in a controlled MDMA administration study. Blood plasma samples were collected from 16 healthy participants (13 EMs and three IMs) up to 24 h after MDMA administration in a double-blind, placebo-controlled, four-period, cross-over design, with subjects receiving 1 week placebo or bupropion pretreatment followed by a single placebo or MDMA (125 mg) dose. Bupropion pretreatment increased the maximum plasma concentration (Cmax) and area under the plasma concentration-time curve from 0 to 24 h (AUC24) of R-MDMA (9% and 25%, respectively) and S-MDMA (16% and 38%, respectively). Bupropion reduced the Cmax and AUC24 of the CYP2D6-dependently formed metabolite stereoisomers of DHMA 3-sulfate, DHMA 4-sulfate, and 4-hydroxy-3-methoxymethamphetamine (HMMA sulfate and HMMA glucuronide) by approximately 40%. The changes that were observed in IMs were generally comparable to bupropion-pretreated EMs. Although changes in stereoselectivity based on CYP2D6 activity were observed, these likely have low clinical relevance. Bupropion and hydroxybupropion stereoisomer pharmacokinetics were unaltered by MDMA co-administration. The present data might aid further interpretations of toxicity based on CYP2D6-dependent MDMA metabolism.

This paper presents the results of an empirical study concerning conventional and socially responsible mutual funds.We apply a sophisticated operations research algorithm embedded in inverse portfolio optimization on financial market data, ESG-scores and CRSP fund data. Due to our results we cannot find strong evidence of differences between conventional and socially responsible mutual funds. In particular, the calculated risk tolerance parameters describing the real portfolio composition best show that socially responsible mutual funds may be even less concerned about the ESG-scores in the preference functional than conventional funds.

This paper presents the results of an empirical study concerning conventional and socially responsible mutual funds.We apply a sophisticated operations research algorithm embedded in inverse portfolio optimization on financial market data, ESG-scores and CRSP fund data. Due to our results we cannot find strong evidence of differences between conventional and socially responsible mutual funds. In particular, the calculated risk tolerance parameters describing the real portfolio composition best show that socially responsible mutual funds may be even less concerned about the ESG-scores in the preference functional than conventional funds.

From 07.11.04 to 12.11.04, the Dagstuhl Seminar 04461 ``Practical Approaches to Multi-Objective Optimization'' was held in the International Conference and Research Center (IBFI), Schloss Dagstuhl. During the seminar, several participants presented their current research, and ongoing work and open problems were discussed. Abstracts of the presentations given during the seminar as well as abstracts of seminar results and ideas are put together in this paper. The first section describes the seminar topics and goals in general. Links to extended abstracts or full papers are provided, if available.

Summary of the Dagstuhl Seminar 04461. Motivation, proceedings, achievements and feedback, future seminars

Summary of the Dagstuhl Seminar 04461. Motivation, proceedings, achievements and feedback, future seminars

From 07.11.04 to 12.11.04, the Dagstuhl Seminar 04461 ``Practical Approaches to Multi-Objective Optimization'' was held in the International Conference and Research Center (IBFI), Schloss Dagstuhl. During the seminar, several participants presented their current research, and ongoing work and open problems were discussed. Abstracts of the presentations given during the seminar as well as abstracts of seminar results and ideas are put together in this paper. The first section describes the seminar topics and goals in general. Links to extended abstracts or full papers are provided, if available.

Service Test of the Battlefield Illumination System was conducted to determine the suitability of the Battlefield Illumination System for US Army use in a variety of test conditions under simulated field and tactical use. USAIB concludes that the Battlefield Illumination System (Flare, Surface, Parachute, XM183) is capable of providing sufficient illumination to detect targets at a range of 200 meters; is simple to maintain, emplace, and operate and is portable.

Forensic postmortem case interpretation can be challenging, in particular due to postmortem redistribution (PMR) phenomena. Recent studies have shown that computed tomography (CT)-guided collection of biopsy samples using a robotic arm (virtobot) provides a valuable tool for systematic studies on time-dependent PMR. Utilizing this strategy, several cases involving opioid use such as methadone, fentanyl, tramadol, codeine, oxycodone and hydrocodone were evaluated for time-dependent concentration changes and potential redistribution mechanisms. Upon admission to the institute (t1), blood (femoral and right ventricle heart blood) and tissue biopsy samples (lung, kidney, liver, spleen, thigh muscle and adipose tissue) were collected utilizing CT-guided biopsy. Approximately 24 h later (t2; mean 28 ± 15 h), during the autopsy, samples from the same body regions were collected manually and in addition brain tissue, gastric content, urine and left ventricle heart blood. Analysis was conducted with liquid chromatography tandem mass spectrometry. Significant time-dependent methadone concentration increases in femoral blood (pB) indicate the occurrence of PMR, however, ultimately not relevant for forensic interpretation. The main metabolite of methadone, 2-ethylidene-1,5-dimethyl-3,3-diphenylpyrrolidine (EDDP), showed a less significant trend for PMR. Redistribution by passive diffusion along the muscle-to-pB concentration gradient seems likely for methadone, but not for EDDP. Results for fentanyl suggest extensive PMR. Other opioids such as tramadol, codeine, hydrocodone and oxycodone showed no consistent trend for significant PMR. Overall, CT-guided biopsy sampling proved to be a valuable tool for the investigation of PMR mechanisms.

Zebrafish (Danio rerio) larvae have been suggested as vertebrate model to complement or even replace mammals for rapidly assessing behavioral effects of psychoactive drugs. Yet, divergent responses have been reported in mammals and fish despite the conservation of many drug targets. Cocaine, eg, acts as stimulant in mammals but no such response has been documented for zebrafish larvae. We hypothesized that differences in exposure routes (inhalation or injection in mammals vs waterborne in fish) may be a reason for differences in behavioral responses. We characterized cocaine toxicokinetics by liquid chromatography-mass spectrometry and found its rapid uptake into larvae. We used Matrix-assisted laser desorption ionization-mass spectrometry imaging for the first time to characterize internal distribution of cocaine in zebrafish larvae. Surprisingly, eyes accumulated the highest amount of cocaine and retained most of it even after 48 h depuration. We attribute this to trapping by pigment melanin, a thus far little explored mechanism that may also be relevant for other basic drugs. Cocaine also reached the brain but with levels similar to those in trunk indicating simple passive diffusion as means of distribution which was supported by toxicokinetic models. Although brain levels covered those known to cause hyperactivity in mammals, only hypoactivity (decreased locomotion) was recorded in zebrafish larvae. Our results therefore point to cocaine's anesthetic properties as the dominant mechanism of interaction in the fish: upon entry through the fish skin and gills, it first acts on peripheral nerves rapidly overriding any potential stimulatory response in the brain.