Two Different Quinohemoprotein Amine Dehydrogenases Initiate Anaerobic Degradation of Aromatic Amines in Aromatoleum aromaticum EbN1.

Aromatic amines like 2-phenylethylamine (2-PEA) and benzylamine (BAm) have been identified as novel growth substrates of the betaproteobacterium Aromatoleum aromaticum EbN1, which degrades a wide variety of aromatic compounds in the absence of oxygen under denitrifying growth conditions. The catabolic pathway of these amines was identified, starting with their oxidative deamination to the corresponding aldehydes, which are then further degraded via the enzymes of the phenylalanine or benzyl alcohol metabolic pathways. Two different periplasmic quinohemoprotein amine dehydrogenases involved in 2-PEA or BAm metabolism were identified and characterized. Both enzymes consist of three subunits, contain two heme c cofactors in their α-subunits, and exhibit extensive processing of their γ-subunits, generating four intramolecular thioether bonds and a cysteine tryptophylquinone (CTQ) cofactor. One of the enzymes was present in cells grown with 2-PEA or other substrates, showed an α2ß2γ2 composition, and had a rather broad substrate spectrum, which included 2-PEA, BAm, tyramine, and 1-butylamine. In contrast, the other enzyme was specifically induced in BAm-grown cells, showing an αßγ composition and activity only with BAm and 2-PEA. Since the former enzyme showed the highest catalytic efficiency with 2-PEA and the latter with BAm, they were designated 2-PEADH and benzylamine dehydrogenase (BAmDH). The catalytic properties and inhibition patterns of 2-PEADH and BAmDH showed considerable differences and were compared to previously characterized quinohemoproteins of the same enzyme family.IMPORTANCE The known substrate spectrum of A. aromaticum EbN1 is expanded toward aromatic amines, which are metabolized as sole substrates coupled to denitrification. The characterization of the two quinohemoprotein isoenzymes involved in degrading either 2-PEA or BAm expands the knowledge of this enzyme family and establishes for the first time that the necessary maturation of their quinoid CTQ cofactors does not require the presence of molecular oxygen. Moreover, the study revealed a highly interesting regulatory phenomenon, suggesting that growth with BAm leads to a complete replacement of 2-PEADH by BAmDH, which has considerably different catalytic and inhibition properties.

An innovative investigative approach to characterize the effects observed in a combined fertility study in male and female rats.

This paper describes the characterization of male- and female-mediated effects in a standard ICH rat fertility and early embryonic development study with a discontinued clinical small molecule. In the standard study, the test item had no effect on the number of treated females becoming pregnant, but litter sizes were reduced at the high dose level. In the treated male rats, increased incidences of abnormal sperm, decreases in average sperm path and straight line velocities, and minimal retention of mature sperm in the seminiferous tubules were observed at all dose-levels tested. These findings were unexpected in view of a lack of histopathological changes in the reproductive organs of either gender in 4-week repeat dose studies in rats and monkeys. A follow-up fertility study was conducted using an innovative flexible study design and a single high-dose level. In the first instance, treated male rats were mated with untreated females, followed by necropsy of a subset of males. The intention was then to re-mate the males after an 8-week wash-out period with either treated or untreated females depending on the outcome of the first mating. On this occasion, litter sizes were not affected, but the testicular effects were reproduced. A second mating with treated females reproduced the reduced litter sizes due to increased pre- and post-implantation loss, demonstrating that the effect on fecundity was female-mediated. The testicular changes in males were shown to be reversible after a 12-week recovery period.

An unnatural death by propan-1-ol and propan-2-ol.

A fatality of an inpatient ingesting a disinfectant containing ethanol, propan-1-ol, and propan-2-ol is reported. The alleged survival time was about 1 h. Major findings at autopsy were an extended hemorrhagic lung edema, an edematous brain, and shock kidneys. Concentrations of alcohols and acetone, a major metabolite of propan-2-ol, were determined from body fluids (blood from the heart and the femoral vein, urine, gastric contents) and tissues (brain, muscle, liver, kidneys, lungs) by headspace/gas chromatography using 2-methylpropan-2-ol as the internal standard. All samples investigated were positive for propan-1-ol, propan-2-ol, ethanol, and acetone except stomach contents, where acetone was not detectable. The low concentration of acetone compared to propan-2-ol likely supports the short survival time. The concentration ratios estimated from the results are in accordance with the physico-chemical properties of the particular alcohols, their different affinities towards alcohol dehydrogenase as well as their interdependence during biotransformation. Autopsy did not reveal the cause of death. According to the few published data, blood concentrations of 1.44 and 1.70 mg/g of propan-2-ol and propan-1-ol, respectively, are considered sufficient to have caused the death. This case also points to the need to restrict access to antiseptic solutions containing alcohols in wards with patients at risk.

Structure of the processive rubber oxygenase RoxA from Xanthomonas sp.

Rubber oxygenase A (RoxA) is one of only two known enzymes able to catalyze the oxidative cleavage of latex for biodegradation. RoxA acts as a processive dioxygenase to yield the predominant product 12-oxo-4,8-dimethyl-trideca-4,8-diene-1-al (ODTD), a tri-isoprene unit. Here we present a structural analysis of RoxA from Xanthomonas sp. strain 35Y at a resolution of 1.8 Å. The enzyme is a 75-kDa diheme c-type cytochrome with an unusually low degree of secondary structure. Analysis of the heme group arrangement and peptide chain topology of RoxA confirmed a distant kinship with diheme peroxidases of the CcpA family, but the proteins are functionally distinct, and the extracellular RoxA has evolved to have twice the molecular mass by successively accumulating extensions of peripheral loops. RoxA incorporates both oxygen atoms of its cosubstrate dioxygen into the rubber cleavage product ODTD, and we show that RoxA is isolated with O2 stably bound to the active site heme iron. Activation and cleavage of O2 require binding of polyisoprene, and thus the substrate needs to use hydrophobic access channels to reach the deeply buried active site of RoxA. The location and nature of these channels support a processive mechanism of latex cleavage.

Functional analysis and transcriptional output of the Göttingen minipig genome.

BACKGROUND: In the past decade the Göttingen minipig has gained increasing recognition as animal model in pharmaceutical and safety research because it recapitulates many aspects of human physiology and metabolism. Genome-based comparison of drug targets together with quantitative tissue expression analysis allows rational prediction of pharmacology and cross-reactivity of human drugs in animal models thereby improving drug attrition which is an important challenge in the process of drug development. RESULTS: Here we present a new chromosome level based version of the Göttingen minipig genome together with a comparative transcriptional analysis of tissues with pharmaceutical relevance as basis for translational research. We relied on mapping and assembly of WGS (whole-genome-shotgun sequencing) derived reads to the reference genome of the Duroc pig and predict 19,228 human orthologous protein-coding genes. Genome-based prediction of the sequence of human drug targets enables the prediction of drug cross-reactivity based on conservation of binding sites. We further support the finding that the genome of Sus scrofa contains about ten-times less pseudogenized genes compared to other vertebrates. Among the functional human orthologs of these minipig pseudogenes we found HEPN1, a putative tumor suppressor gene. The genomes of Sus scrofa, the Tibetan boar, the African Bushpig, and the Warthog show sequence conservation of all inactivating HEPN1 mutations suggesting disruption before the evolutionary split of these pig species. We identify 133 Sus scrofa specific, conserved long non-coding RNAs (lncRNAs) in the minipig genome and show that these transcripts are highly conserved in the African pigs and the Tibetan boar suggesting functional significance. Using a new minipig specific microarray we show high conservation of gene expression signatures in 13 tissues with biomedical relevance between humans and adult minipigs. We underline this relationship for minipig and human liver where we could demonstrate similar expression levels for most phase I drug-metabolizing enzymes. Higher expression levels and metabolic activities were found for FMO1, AKR/CRs and for phase II drug metabolizing enzymes in minipig as compared to human. The variability of gene expression in equivalent human and minipig tissues is considerably higher in minipig organs, which is important for study design in case a human target belongs to this variable category in the minipig. The first analysis of gene expression in multiple tissues during development from young to adult shows that the majority of transcriptional programs are concluded four weeks after birth. This finding is in line with the advanced state of human postnatal organ development at comparative age categories and further supports the minipig as model for pediatric drug safety studies. CONCLUSIONS: Genome based assessment of sequence conservation combined with gene expression data in several tissues improves the translational value of the minipig for human drug development. The genome and gene expression data presented here are important resources for researchers using the minipig as model for biomedical research or commercial breeding. Potential impact of our data for comparative genomics, translational research, and experimental medicine are discussed.

Enzymes of anaerobic ethylbenzene and p-ethylphenol catabolism in 'Aromatoleum aromaticum': differentiation and differential induction.

The denitrifying bacterium 'Aromatoleum aromaticum' strain EbN1 is one of the best characterized bacteria regarding anaerobic ethylbenzene degradation. EbN1 also degrades various other aromatic and phenolic compounds in the absence of oxygen, one of them being p-ethylphenol. Despite having similar chemical structures, ethylbenzene and p-ethylphenol have been proposed to be metabolized by completely separate pathways. In this study, we established and applied biochemical and molecular biological methods to show the (almost) exclusive presence and specificity of enzymes involved in the respective degradation pathways by recording enzyme activities, complemented by heme staining, immuno- and biotin-blotting analyses. These combined results substantiated the predicted p-ethylphenol degradation pathway. The identified enzymes include a heme c-containing p-ethylphenol-hydroxylase, both an (R)- and an (S)-specific alcohol dehydrogenase as well as a novel biotin-dependent carboxylase. We also establish an activity assay for benzoylacetate-CoA ligases likely being involved in both metabolic pathways.

[Diuretics and their potential effect on breath-alcohol concentration--a case report]. / Diuretika und ihre Auswirkung auf die Atemalkoholkonzentration--eine Fallvorstellung.

Many objections were raised to breath-alcohol analysis upon its introduction in the field of traffic law enforcement in Germany, but in the meantime this issue has become less relevant in forensic routine work. In the present case, the defending lawyer claimed that the ethanol concentration in the blood and hence in the breath of his client, which was 0.35 mg/l according to the Dräger Alcotest 7110® Evidential and thus above the legal limit of 0.25 mg/l, had been changed by diuretics taken 4 hours before the breath alcohol test, viz. 10 mg of torasemide, a loop diuretic, and 50 mg of spironolactone, a competitive aldosterone antagonist. According to the literature, the maximum urinary output in healthy subjects within the first 4 hours after 10 mg torasemide was 1450 ml. In patients suffering from heart failure, the urinary volume was reduced by a factor of 2.5-3; after chronic intake of torasemide, water loss did not differ from placebo. Spironolactone, which acts on the distal tubule, has little effect on urinary output. In a publication, the loss of water in excess within 24 hours was 90 ml. Co-administration of 100 mg spironolactone and 20 mg furosemide, which roughly compares to 10 mg torasemide, resulted in a mean urinary volume of 1566 ml within the first 4 hours. In terms of the reported case and provided that no compensatory fluid had been taken, a purely theoretical maximum shift of 0.007 mg/ may occur in the breath-alcohol concentration due to the smaller distribution volume even considering maximum urinary excretion values. On the other hand, already mild levels of dehydration may be associated with negative symptoms affecting driving ability.

Simultaneous involvement of a tungsten-containing aldehyde:ferredoxin oxidoreductase and a phenylacetaldehyde dehydrogenase in anaerobic phenylalanine metabolism.

Anaerobic phenylalanine metabolism in the denitrifying betaproteobacterium Aromatoleum aromaticum is initiated by conversion of phenylalanine to phenylacetate, which is further metabolized via benzoyl-coenzyme A (CoA). The formation of phenylacetate is catalyzed by phenylalanine transaminase, phenylpyruvate decarboxylase, and a phenylacetaldehyde-oxidizing enzyme. The presence of these enzymes was detected in extracts of cells grown with phenylalanine and nitrate. We found that two distinct enzymes are involved in the oxidation of phenylacetaldehyde to phenylacetate, an aldehyde:ferredoxin oxidoreductase (AOR) and a phenylacetaldehyde dehydrogenase (PDH). Based on sequence comparison, growth studies with various tungstate concentrations, and metal analysis of the enriched enzyme, AOR was shown to be a tungsten-containing enzyme, necessitating specific cofactor biosynthetic pathways for molybdenum- and tungsten-dependent enzymes simultaneously. We predict from the genome sequence that most enzymes of molybdopterin biosynthesis are shared, while the molybdate/tungstate uptake systems are duplicated and specialized paralogs of the sulfur-inserting MoaD and the metal-inserting MoeA proteins seem to be involved in dedicating biosynthesis toward molybdenum or tungsten cofactors. We also characterized PDH biochemically and identified both NAD(+) and NADP(+) as electron acceptors. We identified the gene coding for the enzyme and purified a recombinant Strep-tagged PDH variant. The homotetrameric enzyme is highly specific for phenylacetaldehyde, has cooperative kinetics toward the substrate, and shows considerable substrate inhibition. Our data suggest that A. aromaticum utilizes PDH as the primary enzyme during anaerobic phenylalanine degradation, whereas AOR is not essential for the metabolic pathway. We hypothesize a function as a detoxifying enzyme if high aldehyde concentrations accumulate in the cytoplasm, which would lead to substrate inhibition of PDH.

Death after smoking of fentanyl, 5F-ADB, 5F-MDMB-P7AICA and other synthetic cannabinoids with a bucket bong.

PURPOSE: We report a case of a polydrug user who consumed various synthetic cannabinoids and fentanyl from a transdermal patch via a bucket bong. Toxicological results from postmortem matrices with special focus on synthetic cannabinoids are discussed in terms of their relevance to the death. METHODS: The samples were analyzed by toxicological screening procedures involving immunoassays and gas chromatography-mass spectrometry (GC-MS) as well as quantitative analyses by means of GC-MS and high-performance liquid chromatography-tandem mass spectrometry (LC-MS/MS). RESULTS: At the autopsy, coronary artery disease and signs of liver congestion were noted, in the absence of acute myocardial ischemic changes. Femoral blood concentrations of fentanyl and pregabalin were 14 ng/mL and 3,200 ng/mL, respectively. In addition, 2.7 ng/mL 5F-ADB and 13 ng/mL 5F-MDMB-P7AICA were detected together with relatively low amounts of 5 other synthetic cannabinoids in cardiac blood. A total number of up to 17 synthetic cannabinoids were detected in kidney, liver, urine and hair. Fentanyl and 5F-ADB were also detected in the water of the bucket bong. CONCLUSIONS: The cause of death could be attributed to an acute mixed intoxication by fentanyl and 5F-ADB (both Toxicological Significance Score (TSS) = 3) with a contribution of pregabalin and 5F-MDMB-P7AICA (TSS = 2), in a subject suffering from pre-existing heart damage. The most plausible mechanism of death consists in a respiratory depression. This case report demonstrates that use of opioids in combination with synthetic cannabinoids might be particularly dangerous.

Functional identification of rubber oxygenase (RoxA) in soil and marine myxobacteria.

The rubber oxygenase (RoxA) of Xanthomonas sp. strain 35Y (RoxA(Xsp)) is so far the only known extracellular c-type diheme cytochrome that is able to cleave poly(cis-1,4-isoprene). All other rubber-degrading bacteria described are Gram positive and employ a nonheme protein (latex-clearing protein [Lcp]) for the postulated primary attack of polyisoprene. Here, we identified RoxA orthologs in the genomes of Haliangium ochraceum, Myxococcus fulvus, Corallococcus coralloides, and Chondromyces apiculatus. The roxA orthologs of H. ochraceum (RoxA(Hoc)), C. coralloides BO35 (RoxA(Cco)), and M. fulvus (RoxA(Mfu)) were functionally expressed in a ΔroxA Xanthomonas sp. 35Y background. All RoxA orthologs oxidatively cleaved polyisoprene, as revealed by restoration of clearing-zone formation and detection of 12-oxo-4,8-dimethyltrideca-4,8-diene-1-al (ODTD) as a cleavage product. RoxA(Xsp), RoxA(Mfu), and RoxA(Cco) were purified and biochemically characterized. The optimal temperature of RoxA(Cco) and RoxA(Mfu) was between 22 and 30°C. All RoxA orthologs as isolated showed an oxidized UV-visible spectrum. Chemical reduction of RoxA(Cco) and RoxA(Mfu) indicated the presence of two slightly different heme centers with absorption maxima between 549 and 553 nm, similar to RoxA(Xsp). Sequence analysis and modeling of the three-dimensional structures of the RoxA orthologs revealed a high degree of similarity to the recently solved RoxA(Xsp) structure and included several conserved residues, notably, W302, F317, and a MauG motif at about H517. Lcp-like sequences were not detected in the genomes of the Xanthomonas sp. 35Y, H. ochraceum, M. fulvus, and C. coralloides. No RoxA orthologs were found in Gram-positive bacteria, and this first description of functional RoxA in Gram-negative bacteria other than Xanthomonas proves that RoxA is more common among rubber degraders than was previously assumed.

Clinical trial considerations on male contraception and collection of pregnancy information from female partners.

BACKGROUND: There is little guidance regarding the risk of exposure of pregnant women/ women of childbearing potential to genotoxic or teratogenic compounds via vaginal dose delivered through seminal fluid during sexual intercourse. METHOD: We summarize current thinking and provide clinical trial considerations for a consistent approach to contraception for males exposed to genotoxic and/or teratogenic compounds or to compounds of unknown teratogenicity, and for collection of pregnancy data from their female partners. RESULTS: Where toxicity testing demonstrates genotoxic potential, condom use is required during exposure and for 5 terminal plasma half-lives plus 74 days (one human spermatogenesis cycle) to avoid conception.For non-genotoxic small molecules and immunoglobulins with unknown teratogenic potential or without a no observed adverse effect level (NOAEL) from embryo-fetal development (EFD) studies and no minimal anticipated biological effect level (MABEL), condom use is recommended for males with pregnant partner/female partner of childbearing potential. For teratogenic small molecules with estimated seminal fluid concentration and a margin between projected maternal area under the curve (AUC) and NOAEL AUC from EFD studies of ≥300 (≥100 for immunoglobulins) or in the absence of a NOAEL with a margin between MABEL plasma concentration and maternal Cmax of ≥300 (≥10 for immunoglobulins), condom use is not required. However, condom use is required for margins below the thresholds previously indicated. For small molecules with available seminal fluid concentrations, condom use is required if margins are <100 instead of <300. Condom use should continue for as long as the projected margin is at or above the defined thresholds. Pregnancy data should be proactively collected if pregnancy occurs during the condom use period required for males exposed to first-in-class molecules or to molecules with a target/class shown to be teratogenic, embryotoxic or fetotoxic in human or preclinical experiments. CONCLUSION: These recommendations, based on a precaution principle, provide a consistent approach for minimizing the risk of embryo-fetal exposure to potentially harmful drugs during pregnancy of female partners of males in clinical trials. Proactive targeted collection of pregnancy information from female partners should help determine the teratogenic potential of a drug and minimize background noise and ethical/logistical issues.

Phe317 is essential for rubber oxygenase RoxA activity.

RoxA is an extracellular c-type diheme cytochrome secreted by Xanthomonas sp. strain 35Y during growth on rubber. RoxA cleaves poly(cis-1,4-isoprene) to 12-oxo-4,8-dimethyltrideca-4,8-diene-1-al (ODTD). Analysis of the RoxA structure revealed that Phe317 is located in close proximity (≈5 Å) to the N-terminal heme that presumably represents the active site. To find evidence of whether Phe317 is important for catalysis, we changed it to tyrosine, tryptophan, leucine, histidine, or alanine. All five RoxA muteins were expressed after integration of the respective gene into the chromosome of a Xanthomonas sp. ΔroxA strain. Residual clearing zone formation on opaque latex agar was found for Xanthomonas sp. strains expressing the Phe317Leu, Phe317Ala, or Phe317His variant (wild type > Leu > Ala > His). Strains in which Phe317 was changed to tyrosine or tryptophan were inactive. Phe317Ala and Phe312Leu RoxA muteins were purified, and polyisoprene cleavage activities were reduced to ≈3% and 10%, respectively. UV-visible spectroscopy of RoxA muteins confirmed that both heme groups were present in an oxidized form, but spectral responses to the addition of low-molecular-weight (inhibitory) ligand molecules such as imidazole and pyridine were different from those of wild-type RoxA. Our results show that residue 317 is involved in interaction with substrates. This is the first report on structure-function analysis of a polyisoprene-cleaving enzyme and on the identification of an amino acid that is essential for polyisoprene cleavage activity.

[Does a positive finding of tetrahydrocannabinol in the blood result from ingestion of Indian frankincense (Boswellia serrata)?]. / Führt die Einnahme von indischem Weihrauch zu einem positiven Tetrahydrocannabinolbefund im Blut?

The exculpatory statement that a positive THC finding in the blood is due to the consumption of hemp products or passive exposure to cannabis smoke has been disproved by the monitoring of hemp products and recent passive inhalation studies conducted in social settings, which showed that these conditions are unlikely to produce a positive result in the blood. The defense that the ingestion of Indian olibanum may result in a positive THC concentration in the blood is unusual; it is based on older publications where authors had speculated on a possible association of the synthetic pathways of THC from terpenoid precursors also being present in olibanum and the biogenesis of THC in hemp. It had further been speculated whether chemical or plant-derived pathways may also occur in humans. A thorough understanding of the different pathways and recently published results have outdated these speculations.

Considerations for and against dosing rodent pups before 7 days of age in juvenile toxicology studies.

This review focuses on preweaning ontogenic and developmental processes that can influence the selection of the appropriate age at which to start dosing rodent pups in juvenile animal studies (JAS). The ICH S11 guideline on 'Nonclinical Safety Testing in Support of Development of Paediatric Medicines' highlights the need to adapt the age from which animals are dosed according to the stage of development in the target organs/tissues of concern in the youngest pediatric patients. Rodents (rat or mouse) are the most common species for JAS. Despite previous practices, based on comparative ontogeny, it is rarely necessary to dose rodents younger than one week of age since postnatal day (PND)7 is appropriate to address concern for the vast majority of organs. In exceptional cases, earlier dosing (e.g., PND4) can be appropriate to address specific concern in preterm neonates and when a tissue of concern has a particularly early developmental trajectory in the rodent compared to humans. The comparative development of the CNS is particularly complex. While exposure of rodents from PND10 covers most CNS development stages relevant to human neonates, a later dosing start (yet, not later than PND14) can sometimes be appropriate to reflect specific aspects (e.g., transformation of GABAergic transmission). An extended study design including subsets of several ages can be helpful to address multiple concerns within a preweaning JAS. Such design can allow for individual assessment of each concern, whilst minimizing (potentially irrelevant) signals from tissues exposed at a developmental stage that do not match the human situation.

Development and validation of an analytical method for the simultaneous determination of the alcohol biomarkers ethyl glucuronide, ethyl sulfate, N-acetyltaurine, and 16:0/18:1-phosphatidylethanol in human blood.

As alcohol is the most common addictive substance worldwide, it is inevitable to advance the established research. New and more substantial analytical methods can be applied to reply to complex questions in legal or forensic contexts. Therefore, an analytical method for the simultaneous determination of four different alcohol biomarkers-ethyl glucuronide, ethyl sulfate, N-acetyltaurine, and 16:0/18:1-phosphatidylethanol-in human blood was developed, validated, and verified. Despite the different chemical properties of the analytes, a specific determination via HPLC-MS/MS was achieved using a novel type of a Phenomenex Luna® Omega Sugar column. Furthermore, all criteria for a successful validation were fulfilled according to forensic guidelines. The method proved to be linear and demonstrates selectivity and sufficient sensitivity for every biomarker. LODs obtained with this method of 2.6 ng/ml (EtG), 4.7 ng/ml (EtS), 12.5 ng/ml (NAcT), and 6.9 ng/ml (PEth) were in an acceptable range for routine applications, and the stability of all analytes over a range of 12 h is given. The verification of the new developed method was performed with authentic samples. Thus, whole blood and postmortem samples were analyzed to obtain information about the drinking behavior, which can answer complex questions regarding alcohol consumption.

Simultaneous Determination of 5- and 6-APB in Blood, Other Body Fluids, Hair and Various Tissues by HPLC--MS-MS.

5-(2-Aminopropyl)benzofuran (5-APB) and 6-(2-aminopropyl)benzofuran (6-APB) are benzofuran analogues of amphetamine and belong to the category of new psychoactive substances. Despite already published fatal 5- and 6-APB intoxication after consumption of both substances in most cases, no sensitive method for the simultaneous detection and quantification of these new psychoactive compounds in human blood samples has yet been developed. Therefore, an easy and fast sample preparation and specific high-performance liquid chromatography and tandem mass spectrometry methods for the determination of both substances in blood were established and validated. In a fatal intoxication in 2017 at the Institute of Forensic and Traffic Medicine in Heidelberg, Germany, concentrations of 850 (5-APB) and 300 ng/mL (6-APB) were determined in peripheral blood. Besides, other body fluids (central blood, urine and bile), hair and various tissues were examined to verify the presence of both compounds and to gain first insights into their distribution. In this publication, we show a method for the simultaneous determination of 5- and 6-APB in human samples by a chromatographic method and to investigate their distribution in the human body.

The benefits, limitations and opportunities of preclinical models for neonatal drug development.

Increased research to improve preclinical models to inform the development of therapeutics for neonatal diseases is an area of great need. This article reviews five common neonatal diseases - bronchopulmonary dysplasia, retinopathy of prematurity, necrotizing enterocolitis, perinatal hypoxic-ischemic encephalopathy and neonatal sepsis - and the available in vivo, in vitro and in silico preclinical models for studying these diseases. Better understanding of the strengths and weaknesses of specialized neonatal disease models will help to improve their utility, may add to the understanding of the mode of action and efficacy of a therapeutic, and/or may improve the understanding of the disease pathology to aid in identification of new therapeutic targets. Although the diseases covered in this article are diverse and require specific approaches, several high-level, overarching key lessons can be learned by evaluating the strengths, weaknesses and gaps in the available models. This Review is intended to help guide current and future researchers toward successful development of therapeutics in these areas of high unmet medical need.

Value of juvenile animal studies.

The Developmental and Reproductive Toxicology Technical Committee of the ILSI Health and Environmental Sciences Institute has undertaken a project to address the impact of juvenile animal studies on pediatric drug development. A workshop, sponsored and organized by the Health and Environmental Sciences Institute Developmental and Reproductive Toxicity Technical Committee, was held on May 5-6, 2010, in Washington, DC, to discuss the outcome of a global survey and the value of juvenile animal studies in the development of drugs intended for use in pediatric patients. During this workshop, summary data from the 2009-2010 survey were presented, and breakout sessions were used to discuss specific case studies to try to assess the impact of juvenile animal studies performed to support specific pediatric drug development. The objectives of the Workshop on The Value of Juvenile Animal Studies were to (1) provide a forum for scientists representing industry, academia, and regulatory agencies to discuss the impact of juvenile animal studies on pediatric drug development, (2) evaluate summary data from the survey to understand how the juvenile study data are being used and their impact in labeling and risk assessment, (3) discuss selected case studies from the survey to highlight key findings, and (4) identify the areas of improvement for the designs of juvenile animal studies. The take home message that resonated from the workshop discussions was that well-designed juvenile animal studies have demonstrated value in support of certain pediatric drug development programs. However, it was also clear that a juvenile animal study is not always warranted.

[Variability of cannabinoid findings in blood]. / Zur Variabilität von Cannabinoidbefunden im Blut.

Cannabis products have been administered for many centuries; today, cannabis is the most widely used illegal drug all over the world. Nevertheless, the interpretation of cannabis findings in blood with regard to consumption behaviour and/or estimating the elapsed time since the last cannabis use is still a very challenging task. A wide variation of pharmacokinetic parameters has been observed even in experimental studies. Different chemical structures of precursors, smoking dynamics, pyrolysis of phytocannabinoids and frequency of drug use affect the amount of THC absorbed. Polymorphic enzymes are involved in phase-I-metabolism of THC. Pharmacological effects of other cannabinoids or medication on the pharmacokinetics of THC have not yet been studied in detail. Hydrolysis of cannabis conjugates may occur during storage of blood samples and processing of specimens for analysis; knowledge on the stability of cannabinoids in forensic specimens is still poor. Whether determination of cannabinoid conjugates may be useful is a matter of further consideration. At present, the broad variation of pharmacokinetic parameters and the limiting factors discussed in the present paper should be taken into account when using data from experimental studies for interpretation of analytical results in forensic case work.

Modified congener analysis: Quantification of cyanide in whole blood, other body fluids, and diverse beverages.

The congener analysis is routinely used for the determination of volatile compounds in body fluids and beverages for forensic investigations. Although intoxications with cyanide via smoke inhalation or ingestion of cyanide salts are frequently encountered in forensic medicine, the inclusion of hydrogen cyanide in this analysis was never studied in detail. In this work, a very simple, fast, and sensitive quantification method with headspace gas chromatography and flame ionization detection for the analysis of cyanide in whole blood-was developed and validated. In contrast to the standard sample preparation of the congener analysis, an acidification step with tartaric acid was added. A limit of detection of 50 ng/ml, good linearity (coefficient of correlation > 0.9997), high accuracy (101.5%-106.4%), and precision (relative standard deviation 1.8%-3.7%) were achieved. Authentic blood samples of 10 forensic cases were investigated with the new method. Furthermore, the method was used for the quantification of cyanide in other body fluids (serum and urine) and diverse beverages. Interferences were investigated, and the addition of aldehydes produced a clear concentration-dependent decrease of the cyanide signal. Besides, the method offers an economical use of limited sample material by the simultaneous determination of cyanide, ethanol, and congener alcohols.