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The interpretation of analytical results in forensic postmortem (PM) cases often poses a great challenge, in particular, due to possible PM redistribution (PMR) phenomena. In terms of new synthetic opioids, such data are usually not available and, if so, they are from case reports without the exact knowledge of dose, user habits, time of consumption or PM interval (PMI). Hence, a controlled toxicokinetic pig study was performed allowing the examination of PM tissue distribution and possible PMR of U-47700, tramadol and the main metabolites N-desmethyl-U-47700 and O-desmethyltramadol (ODT). For this purpose, 12 domestic pigs received an intravenous dose of 100 µg/kg body weight (BW) U-47700 or 1,000 µg/kg BW tramadol, respectively. The animals were put to death with T61 8 h after administration, and relevant organs, tissues and body fluids were sampled. Subsequently, the animals were stored at room temperature (RT), and the samples were taken again after 24, 48, and 72 h PM. Following homogenization and solid-phase extraction, quantification was performed applying a standard addition approach and liquid chromatography–tandem mass spectrometry. Only low-to-moderate concentration changes of U-47700, tramadol and their main metabolites were found in the analyzed tissue specimens and body fluids during storage at RT depending on the chosen PMI. On the contrary, a remarkable concentration increase of tramadol was observed in the liver tissue. These findings indicate that both synthetic opioids and their main metabolites are only slightly prone to PMR and central blood might be the matrix of choice for quantification of these substances.

In spite of a decreasing number of new releases, new synthetic opioids (NSOs) are gaining increasing importance in postmortem (PM) forensic toxicology. For the interpretation of analytical results, toxicokinetic (TK) data, e.g., on tissue distribution, are helpful. Concerning NSOs, such data are usually not available due to the lack of controlled human studies. Hence, a controlled TK study using pigs was carried out, and the tissue distribution of U-47700 and tramadol as reference was examined. Twelve pigs received an intravenous dose of 100 µg/kg body weight (BW) U-47700 or 1,000 µg/kg BW tramadol. Eight hours after administration, the animals were put to death with T61. Relevant organs, body fluids and tissues were sampled. After homogenization and solid-phase extraction, quantification was performed applying standard addition and liquid chromatography--tandem mass spectrometry. At the time of death, the two parent compounds were determined in all analyzed specimens. Regarding U-47700, concentrations were highest in duodenum content, bile fluid and adipose tissue (AT). Concerning tramadol, next to bile fluid and duodenum content, highest concentrations were determined in the lung. Regarding the metabolites, N-desmethyl-U-47700 and O-desmethyltramadol (ODT) were detected in all analyzed specimens except for AT (ODT). Higher metabolite concentrations were found in specimens involved in metabolism. N-desmethyl-U-47700 showed much higher concentrations in routinely analyzed organs (lung, liver and kidney) than U-47700. To conclude, besides the routinely analyzed specimens in PM toxicology such as blood, urine or standard specimens like kidney or liver, AT, bile fluid and duodenum content could serve as alternative matrices. In case of U-47700, quantification of the main metabolite N-desmethyl-U-47700 is highly recommendable.

Metabolism studies have shown that the synthetic cannabinoid (SC) 5F-MDMB-P7AICA is predominantly degraded by ester hydrolysis to 5F-MDMB-P7AICA dimethyl butanoic acid. To investigate the stability of 5F-MDMB-P7AICA during storage for a certain period of time or smoking, in vitro stability tests were performed. Blood and serum samples were collected repeatedly during a toxicokinetic study using a pig model and were retested after a 5- and 12-month storage at different temperatures (−20°C, 4°C or room temperature (RT)). Analysis was performed using fully validated liquid chromatography tandem mass spectrometry methods following liquid–liquid extraction and protein precipitation. One set of samples was analyzed immediately following the experiment (without storage (WS)). In the WS samples, 5F-MDMB-P7AICA and 5F-MDMB-P7AICA dimethyl butanoic acid were present in every sample collected throughout the whole experiment. Analysis of the blood and serum samples stored for 5 and 12 months at −20°C and 4°C revealed relatively stable concentrations of the parent substance and the dimethyl butanoic acid metabolite. Regarding the samples stored at RT, the concentrations of 5F-MDMB-P7AICA decreased, while the concentrations of the hydrolysis product increased. This change could particularly be observed in samples with a high initial concentration of the analytes. A further screening of the samples stored at RT revealed no other degradation products. In conclusion, the SC 5F-MDMB-P7AICA could be detected even after 12 months of storage at RT and therefore seems to be more stable than its isomer, 5F-ADB. Regarding the smoke condensate, besides the parent compound, only trace amounts of dimethyl butanoic acid were found.

New psychoactive substances (NPS), especially synthetic cannabinoids (SC) remain a public health concern. Due to ethical reasons, systematic controlled human studies to elucidate their toxicodynamics and/or toxicokinetics are usually not possible. However, such knowledge is necessary, for example, for determination of screening targets and interpretation of clinical and forensic toxicological data. In the present study, the feasibility of the pig model as an alternative for human in vivo metabolism studies of SC was investigated. For this purpose, the metabolic pattern of the SC methyl-2-{[1-(5-fluoropentyl)-1H-pyrrolo[2,3-b]pyridine-3-carbonyl]amino}-3,3-dimethylbutanoate (5F-MDMB-P7AICA) was elucidated in pig urine following inhalative administration (dosage: 200 µg/kg of body weight). The results were compared with human and pig liver microsomal assays and literature. In addition, different incubations with isolated cytochrome-P450 (CYP) monooxygenases were conducted to identify the involved isozymes. In total, nine phase I and three phase II metabolites were identified in pig urine. The most abundant reactions were ester hydrolysis, ester hydrolysis combined with glucuronidation and ester hydrolysis combined with hydroxylation at the tert-butyl moiety. The parent compound was only found up to 1 h after administration in pig urine. The metabolite formed after hydroxylation and glucuronidation was detectable for 2 h, the one formed after ester hydrolyzation and defluorination for 4 h after administration. All other metabolites were detected during the whole sampling time. The most abundant metabolites were also detected using both microsomal incubations and monooxygenase screenings revealed that CYP3A4 catalyzed most reactions. Finally, pig data showed to be in line with published human data. To conclude, the main metabolites recommended in previous studies as urinary targets were confirmed by using pig urine. The used pig model seems therefore to be a suitable alternative for in vivo metabolism studies of 7-azaindole-derived SC.

Background Vascularization is a major hallmark in the pathogenesis of endometriosis. An increasing number of studies suggests that multiple mechanisms contribute to the vascularization of endometriotic lesions, including angiogenesis, vasculogenesis and inosculation. Objective and rationale In this review, we provide an overview of the basic mechanisms of vascularization in endometriosis and give special emphasis on their future clinical implications in the diagnosis and therapy of the disease. Search methods Literature searches were performed in PubMed for English articles with the key words 'endometriosis', 'endometriotic lesions', 'angiogenesis', 'vascularization', 'vasculogenesis', 'endothelial progenitor cells' and 'inosculation'. The searches included both animal and human studies. No restriction was set for the publication date. Outcomes The engraftment of endometriotic lesions is typically associated with angiogenesis, i.e. the formation of new blood vessels from pre-existing ones. This angiogenic process underlies the complex regulation by angiogenic growth factors and hormones, which activate intracellular pathways and associated signaling molecules. In addition, circulating endothelial progenitor cells (EPCs) are mobilized from the bone marrow and recruited into endometriotic lesions, where they are incorporated into the endothelium of newly developing microvessels, referred to as vasculogenesis. Finally, preformed microvessels in shed endometrial fragments inosculate with the surrounding host microvasculature, resulting in a rapid blood supply to the ectopic tissue. These vascularization modes offer different possibilities for the establishment of novel diagnostic and therapeutic approaches. Angiogenic growth factors and EPCs may serve as biomarkers for the diagnosis and classification of endometriosis. Blood vessel formation and mature microvessels in endometriotic lesions may be targeted by means of anti-angiogenic compounds and vascular-disrupting agents. Wider implications The establishment of vascularization-based approaches in the management of endometriosis still represents a major challenge. For diagnostic purposes, reliable angiogenic and vasculogenic biomarker panels exhibiting a high sensitivity and specificity must be identified. For therapeutic purposes, novel compounds selectively targeting the vascularization of endometriotic lesions without inducing severe side effects are required. Recent progress in the field of endometriosis research indicates that these goals may be achieved in the near future.

Nerve/glial antigen (NG)2 enhances cell proliferation, migration as well as chemoresistance and is associated with a poor clinical outcome in glioblastoma. Since regulatory mechanisms of NG2 expression are still largely unknown, we herein investigated the impact of protein kinase CK2 inhibition on NG2 expression in human glioblastoma cells. CK2 was inhibited in NG2-positive human glioblastoma cell lines (A1207, U87) and primary human glioblastoma cells by pharmacological treatment (CX-4945, TBB) or siRNA. NG2 expression was analyzed by flow cytometry, Western Blot and qRT-PCR. Cytotoxicity and viability were assessed by WST-1, LDH and BrdU assays. Scratch, sprouting and BrdU assays as well as growth curves were performed to examine cell migration and proliferation. Truncated fragments of the human NG2 promotor were generated and their transcriptional activity was assessed using reporter gene assays. The effect of CK2 inhibition on tumor growth was investigated in xenografts within the flanks of NOD/SCID mice. Finally, effects of CK2 inhibition were analyzed in primary human glioblastoma cells. We found that inhibition of CK2 significantly reduces NG2 protein levels in A1207 (19%±6.2; Mean±SD), U87 (35%±11.3) and primary human glioblastoma cells (41%) when compared to controls. Further analyses revealed that this is due to a decreased NG2 mRNA level. Of note, we identified a 200 base pair fragment, including a binding site for the CK2-dependent transcription factor SP-1. Functional assays showed no cytotoxic effects of the decreased NG2 expression after CK2 inhibition, whereas cell migration and proliferation were markedly reduced. Moreover, we found that CX-4945 treatment decreases tumor growth, which is associated with a diminished NG2 expression (56%±13.0) when compared to controls. In conclusion, we identified CK2 as a novel regulator of NG2 gene expression in human glioblastoma cells. Hence, pharmacological inhibition of CK2 may represent a novel strategy in the therapy of NG2-positive glioblastoma.

background: Angiogenesis, i.e. the development of new blood vessels from pre-existing ones, represents an integral part in the patho- genesis of endometriosis. During the last decade, an increasing number of studies have therefore focused on the anti-angiogenic treatment of the disease. The present review provides a systematic overview of these studies and critically discusses the future role of anti-angiogenic therapy in the multimodal management of endometriosis. methods: Literature searches were performed in PubMed, MEDLINE and ISI Web of Knowledge for original articles published before the end of March 2012, written in the English language and focusing on anti-angiogenic approaches for the therapy of endometriosis. The searches included both animal and human studies. results: Numerous compounds of different substance groups have been shown to exert anti-angiogenic effects on endometriotic lesions under experimental in vitro and in vivo conditions. These include growth factor inhibitors, endogenous angiogenesis inhibitors, fumagillin analogues, statins, cyclo-oxygenase-2 inhibitors, phytochemical compounds, immunomodulators, dopamine agonists, peroxisome prolifer- ator-activated receptor agonists, progestins, danazol and gonadotropin-releasing hormone (GnRH) agonists. However, clinical evidence for their efficacy in anti-angiogenic endometriosis therapy is still lacking. conclusions: Anti-angiogenic compounds hold great promise for the future treatment of endometriosis because they may inhibit the establishment of new endometriotic lesions in early stages of the disease or after surgical treatment. Further experimental studies, controlled clinical trials in particular, are required now to clarify which compounds fulfil these expectations without inducing severe side effects in patients with endometriosis.

Notwithstanding non-robotic, thoracoscopic preparation of the internal mammary artery (IMA) is a difficult surgical task, an appropriate experimental training model is lacking. We evaluated the young domestic pig for this purpose. Four domestic female pigs (30-40 kg body weight) were used for this study. Bilateral thoracoscopic preparation of the IMA was carried out under continuous, pressure controlled CO(2) insufflation. A 30 degrees rigid thoracoscope was inserted through a 10-mm port in the 5th/6th intercostal space (ICS) dorsally to the posterior axillary line. The dissection instrument (Ultracision Harmonic Scalpel) was inserted (5-mm port) in the 7th ICS at the posterior axillary line and the endo-forceps (5-mm port) in the 5th ICS at the posterior axillary line. Thoracoscopic IMA preparation in pig resulted more difficult than in man. A total of seven IMAs were prepared in their full intrathoracic length. A change in the preparation technique (lateral detachment of the endothoracic muscle) improved the safety of the procedure, allowing all four respective IMAs to be prepared safely, while the initial technique ensued an injury for 2 out of 3 vessels. The described young domestic pig model is suitable for experimental training of bilateral thoracoscopic IMA preparation.

BACKGROUND: Epigallocatechin-3-gallate (EGCG), the major component of green tea, is a pleiotropic substance, which may inhibit tumor growth via multiple intracellular signaling pathways. Herein, we studied whether EGCG may also be effective in the treatment of endometriosis. METHODS: We investigated the effect of EGCG on activation by estradiol (E 2 ), proliferation and vascular endothelial growth factor (VEGF) expression of isolated hamster endometrial stromal cells and glandular cells in vitro using the water-soluble tetrazolium (WST)-1 colorimetric assay and western blot analysis. In the dorsal skinfold chamber model of Syrian golden hamsters, which were treated for 14 days with EGCG or vehicle, we further analyzed angiogenesis, blood perfusion and tissue integrity of both endometriotic lesions and ovarian follicles by intravital fluorescence microscopy and histology. RESULTS: We found that EGCG suppresses E 2 -stimulated activation, proliferation and VEGF expression of endometrial cells in vitro (all P < 0.05). Furthermore, EGCG selectively inhibited angiogenesis and blood perfusion (P < 0.05) of endometriotic lesions in vivo without affecting blood vessel development in ovarian follicles. Histology confirmed that EGCG-treatment induces regression of the endometriotic lesions. CONCLUSIONS: Our data indicate that EGCG might be a promising therapeutic agent in the treatment of endometriosis, preventing the establishment of new endometriotic lesions.

Background Excessive loss of functional liver mass results in hepatic dysfunction and is associated with an increased sensitivity to infection. This experimental study investigated the role of Rho-kinase in hepatectomy-induced sensitization to endotoxin. Methods Male C57BL/6J mice underwent 68 per cent hepatectomy and were injected 24 h later with 100 µg Escherichia coli lipopolysaccharide (LPS). Simultaneously, animals received either fasudil or Y-27632 for Rho-kinase inhibition, or phosphate-buffered saline. Untreated hepatectomized animals served as positive controls and sham-operated animals as negative controls. Liver injury and inflammatory parameters were assessed 6 h after LPS challenge by serum alanine aminotransferase (ALT) levels, histomorphology and enzyme-linked immunosorbent assay. Results Hepatectomy resulted in a significant susceptibility to LPS, as indicated by inflammatory leucocyte recruitment (mean(s.e.m.) 10(1) leucocytes per high-power field), hepatocellular disintegration (ALT 22·4(3·1) µkat/l) and apoptotic cell death (3·8(0·2) per cent). Rho-kinase inhibition reduced leucocytic infiltration by more than 33 per cent, abolished hepatocellular apoptosis entirely, and reduced tumour necrosis factor α expression by more than 48 per cent and CXC chemokine expression by more than 36 per cent. Conclusion Hepatectomy increased susceptibility to LPS by Rho-kinase-dependent mechanisms. Blocking Rho-kinase signalling decreased LPS-induced liver injury in hepatectomized mice.

Endometriosis represents one of the most common gynaecological disorders. According to the implantation theory, angiogenesis is a major prerequisite for the initiation and progression of the disease. Thus, during the last decade, many studies have focused on the mechanisms regulating angiogenesis in endometriotic lesions. For this purpose, sophisticated in vitro and in vivo approaches have been established, which are highlighted in this review. Enzyme-linked immunosorbent assays demonstrate the imbalance of pro- and anti-angiogenic growth factors in isolated peritoneal fluid from endometriosis patients. Histological, immunohistochemical and gene expression analyses of endometriotic tissue provide detailed information on the angio-architecture of endometriotic lesions and the different growth factor expression by various cell populations. Moreover, cell culture systems are useful tools for the identification of hormonal and immunological factors involved in the angiogenic process. Finally, sophisticated in vivo models, such as rodent models of peritoneal endometriosis as well as the chorioallantoic membrane assay and the dorsal skinfold chamber, allow for the detailed analysis of blood vessel development in ectopic endometrium and the efficacy of angiogenesis inhibitors. The findings resulting from all these approaches will help to provide better insights into the pathophysiology of endometriosis and to establish new anti-angiogenic treatment strategies for the future.