Metabolismus und Toxizität von 2-Methylfuran

2-Methylfuran (MF) ist eine Kontaminante, die vor allem in hitzebehandelten Lebensmitteln wie Kaffee oder Lebensmittelkonserven vorkommt. Jedoch ist die Datenlage für eine Risikobewertung lückenhaft hinsichtlich der Exposition und Toxizität (Knutsen et al., 2017). In vivo Studien mit 14C-MF zeigten die Leber als Zielorgan histopathologischer Veränderungen, wie Nekrosen (Ravindranath et al., 1986; Gill et al., 2014). Es wurde die metabolische Aktivierung von MF zu reaktivem Acetylacrolein (AcA) angenommen, welches ein hohes Potential zeigte an Aminosäuren zu binden (Ravindranath et al., 1984a; Ravindranath und Boyd, 1985). In dieser Arbeit wurden die molekularen und chemischen Mechanismen von MF und AcA untersucht, um Rückschlüsse auf die toxikologischen Effekte zu ermöglichen. Zunächst wurde MF in silico hinsichtlich seines toxikologischen Potentials klassifiziert und endpunktspezifisch quantitative Struktur-Wirkungsbeziehungen (QSAR, engl. quantitative structure-activity relationship) analysiert. Positive toxikologische Abschätzungen basierten auf dessen metabolischer Aktivierung zu verschiedenen Metabolite. Diese wurden in zwei Gruppen unterteilt, welche von AcA oder Furfurylalkohol (FFA) abstammten. Im Folgenden wurde AcA synthetisiert und dessen Reaktivität gegenüber N-Acetyl-L-cystein (AcCys), Nα-Acetyl-L-lysin (AcLys), 2′-Desoxyadenosin (dA), 2′-Desoxyguanosin (dG), 2′-Desoxycytosin (dC) sowie 2′-Desoxythymidin (dT) geprüft. Bis auf dT, welches keine Reaktivität zeigte, wurden alle Addukte von AcA charakterisiert, was die Entwicklung sensitiver Quantifizierungsmethoden via (U)HPLC-ESI+/--MS/MS ermöglichte. Anschließend wurde die metabolische Aktivierung von MF zu AcA unter Verwendung humaner (HLM) und Rattenlebermikrosomen (RLM) verifiziert und enzymkinetisch untersucht. Analog wurde die Umsetzung von MF mit CYP 2E1 charakterisiert, welches sich innerhalb der getesteten SupersomesTM CYP 1A2, 2A6, 2C9, 2D6, 2E1 und 3A4 als Schlüsselenzym herausstellte. Zuletzt wurde die Bildung der in chemico identifizierten AcA-Addukte in vitro untersucht. Hierzu wurde die Zytotoxizität von MF in primären Rattenhepatozyten (pRH) mit der in HepG2-Zellen verglichen als auch in Bezug zu der Zytotoxizität von AcA gesetzt. Wurden pRH mit MF und AcA inkubiert, konnte in den Zellüberständen AcLys-AcA zeit- und dosisabhängig nachgewiesen werden. Dies qualifiziert AcLys-AcA als potenzieller Biomarker. Gleichzeitig konnte hiermit gezeigt werden, dass AcA auch auf zellulärer Ebene gebildet wird. Dahingegen wurde weder AcCys-AcA noch die DNA-Addukte dA-AcA, dG-AcA oder dC-AcA in vitro nachgewiesen. Dies deutete auf eine effiziente Detoxifzierung, bzw. Reaktion mit Biomolekülen in der Zelle hin, obgleich die Induktion anderer DNA-Schäden, ggf. auch durch anderen Metabolit, nicht prinzipiell auszuschließen ist. So wurde erstmals in vitro AcA als reaktiver Metabolit von MF nachgewiesen und seine Addukte mit nukleophilen Zellbestandteilen als potenzielle Biomarker untersucht. Diese ermöglicht eine verbesserte Risikobewertung von MF.2-Methylfuran (MF) is a contaminant found primarily in heat-treated foods such as coffee or canned food. However, the data for risk assessment is not only incomplete in terms of exposure but also in terms of toxicity (Knutsen et al., 2017). In vivo studies with 14C-MF indicate that the liver is the target organ for histopathological changes, such as necrosis (Ravindranath et al., 1986; Gill et al., 2014). The metabolic activation of MF to the reactive intermediate acetylacrolein (AcA), which showed high potential to bind to amino acids, was assumed to be the fundamental cause (Ravindranath et al., 1984a; Ravindranath and Boyd, 1985). In this thesis, the molecular and chemical mechanisms of MF and AcA were investigated to draw conclusions about the toxicological effects of MF. To determine the toxicological potential of MF based on its structure, MF was first categorized and its endpoint-specific quantitative structure-activity relationships (QSAR) were analyzed in silico. Positive estimates were based on its metabolic activation to different metabolites, which were then divided into two groups derived from AcA or furfuryl alcohol (FFA). Based on the insights of the in silico analyses and existing literature, AcA was synthesized for further investigation as the most prominent metabolite. That enabled the testing of its reactivity toward N-acetyl-L-cysteine (AcCys), Nα-acetyl-L-lysine (AcLys), 2′-deoxyadenosine (dA), 2′-deoxyguanosine (dG), 2′-deoxycytosine (dC), and 2′-deoxythymidine (dT). Except for dT, which showed no reactivity, all adducts were characterized, allowing the development of sensitive quantification methods via (U)HPLC-MS/MS. In the next step, the metabolic activation of MF to AcA was verified using human (HLM) as well as rat liver microsomes (RLM) and characterized by enzyme kinetics. This was also possible for conversion with CYP 2E1, which was found to be a key enzyme within the tested supersomesTM CYP 1A2, 2A6, 2C9, 2D6, 2E1 and 3A4. The final part of this thesis focused on the formation of the defined AcA adducts in vitro. For this purpose, the cytotoxicity of MF in primary rat hepatocytes (pRH) and HepG2 cells was compared, before it was related to AcA in pRH. When pRH were incubated with MF and AcA, AcLys-AcA was detected in the cell supernatants in a time- and dose-dependent manner. Thus, AcLys-AcA qualifies as a potential biomarker. In addition, the results showed that AcA was indeed formed at the cellular level. Neither AcCys-AcA nor the DNA adducts dA-AcA, dG-AcA or dC-AcA were detected after in vitro incubations. This indicated efficient detoxification, or reaction with biomolecules in cells. Although induction of other DNA damage, possibly by other side metabolites, cannot be generally excluded. In summary AcA was determined as a reactive metabolite of MF in vitro, its adduct formations with nucleophilic cellular components were characterized and investigated as potential biomarker in vitro for the first time. These results improve the risk assessment of MF

Metabolismus und Toxizität von 2-Methylfuran

2-Methylfuran (MF) ist eine Kontaminante, die vor allem in hitzebehandelten Lebensmitteln wie Kaffee oder Lebensmittelkonserven vorkommt. Jedoch ist die Datenlage für eine Risikobewertung lückenhaft hinsichtlich der Exposition und Toxizität (Knutsen et al., 2017). In vivo Studien mit 14C-MF zeigten die Leber als Zielorgan histopathologischer Veränderungen, wie Nekrosen (Ravindranath et al., 1986; Gill et al., 2014). Es wurde die metabolische Aktivierung von MF zu reaktivem Acetylacrolein (AcA) angenommen, welches ein hohes Potential zeigte an Aminosäuren zu binden (Ravindranath et al., 1984a; Ravindranath und Boyd, 1985). In dieser Arbeit wurden die molekularen und chemischen Mechanismen von MF und AcA untersucht, um Rückschlüsse auf die toxikologischen Effekte zu ermöglichen. Zunächst wurde MF in silico hinsichtlich seines toxikologischen Potentials klassifiziert und endpunktspezifisch quantitative Struktur-Wirkungsbeziehungen (QSAR, engl. quantitative structure-activity relationship) analysiert. Positive toxikologische Abschätzungen basierten auf dessen metabolischer Aktivierung zu verschiedenen Metabolite. Diese wurden in zwei Gruppen unterteilt, welche von AcA oder Furfurylalkohol (FFA) abstammten. Im Folgenden wurde AcA synthetisiert und dessen Reaktivität gegenüber N-Acetyl-L-cystein (AcCys), Nα-Acetyl-L-lysin (AcLys), 2′-Desoxyadenosin (dA), 2′-Desoxyguanosin (dG), 2′-Desoxycytosin (dC) sowie 2′-Desoxythymidin (dT) geprüft. Bis auf dT, welches keine Reaktivität zeigte, wurden alle Addukte von AcA charakterisiert, was die Entwicklung sensitiver Quantifizierungsmethoden via (U)HPLC-ESI+/--MS/MS ermöglichte. Anschließend wurde die metabolische Aktivierung von MF zu AcA unter Verwendung humaner (HLM) und Rattenlebermikrosomen (RLM) verifiziert und enzymkinetisch untersucht. Analog wurde die Umsetzung von MF mit CYP 2E1 charakterisiert, welches sich innerhalb der getesteten SupersomesTM CYP 1A2, 2A6, 2C9, 2D6, 2E1 und 3A4 als Schlüsselenzym herausstellte. Zuletzt wurde die Bildung der in chemico identifizierten AcA-Addukte in vitro untersucht. Hierzu wurde die Zytotoxizität von MF in primären Rattenhepatozyten (pRH) mit der in HepG2-Zellen verglichen als auch in Bezug zu der Zytotoxizität von AcA gesetzt. Wurden pRH mit MF und AcA inkubiert, konnte in den Zellüberständen AcLys-AcA zeit- und dosisabhängig nachgewiesen werden. Dies qualifiziert AcLys-AcA als potenzieller Biomarker. Gleichzeitig konnte hiermit gezeigt werden, dass AcA auch auf zellulärer Ebene gebildet wird. Dahingegen wurde weder AcCys-AcA noch die DNA-Addukte dA-AcA, dG-AcA oder dC-AcA in vitro nachgewiesen. Dies deutete auf eine effiziente Detoxifzierung, bzw. Reaktion mit Biomolekülen in der Zelle hin, obgleich die Induktion anderer DNA-Schäden, ggf. auch durch anderen Metabolit, nicht prinzipiell auszuschließen ist. So wurde erstmals in vitro AcA als reaktiver Metabolit von MF nachgewiesen und seine Addukte mit nukleophilen Zellbestandteilen als potenzielle Biomarker untersucht. Diese ermöglicht eine verbesserte Risikobewertung von MF.2-Methylfuran (MF) is a contaminant found primarily in heat-treated foods such as coffee or canned food. However, the data for risk assessment is not only incomplete in terms of exposure but also in terms of toxicity (Knutsen et al., 2017). In vivo studies with 14C-MF indicate that the liver is the target organ for histopathological changes, such as necrosis (Ravindranath et al., 1986; Gill et al., 2014). The metabolic activation of MF to the reactive intermediate acetylacrolein (AcA), which showed high potential to bind to amino acids, was assumed to be the fundamental cause (Ravindranath et al., 1984a; Ravindranath and Boyd, 1985). In this thesis, the molecular and chemical mechanisms of MF and AcA were investigated to draw conclusions about the toxicological effects of MF. To determine the toxicological potential of MF based on its structure, MF was first categorized and its endpoint-specific quantitative structure-activity relationships (QSAR) were analyzed in silico. Positive estimates were based on its metabolic activation to different metabolites, which were then divided into two groups derived from AcA or furfuryl alcohol (FFA). Based on the insights of the in silico analyses and existing literature, AcA was synthesized for further investigation as the most prominent metabolite. That enabled the testing of its reactivity toward N-acetyl-L-cysteine (AcCys), Nα-acetyl-L-lysine (AcLys), 2′-deoxyadenosine (dA), 2′-deoxyguanosine (dG), 2′-deoxycytosine (dC), and 2′-deoxythymidine (dT). Except for dT, which showed no reactivity, all adducts were characterized, allowing the development of sensitive quantification methods via (U)HPLC-MS/MS. In the next step, the metabolic activation of MF to AcA was verified using human (HLM) as well as rat liver microsomes (RLM) and characterized by enzyme kinetics. This was also possible for conversion with CYP 2E1, which was found to be a key enzyme within the tested supersomesTM CYP 1A2, 2A6, 2C9, 2D6, 2E1 and 3A4. The final part of this thesis focused on the formation of the defined AcA adducts in vitro. For this purpose, the cytotoxicity of MF in primary rat hepatocytes (pRH) and HepG2 cells was compared, before it was related to AcA in pRH. When pRH were incubated with MF and AcA, AcLys-AcA was detected in the cell supernatants in a time- and dose-dependent manner. Thus, AcLys-AcA qualifies as a potential biomarker. In addition, the results showed that AcA was indeed formed at the cellular level. Neither AcCys-AcA nor the DNA adducts dA-AcA, dG-AcA or dC-AcA were detected after in vitro incubations. This indicated efficient detoxification, or reaction with biomolecules in cells. Although induction of other DNA damage, possibly by other side metabolites, cannot be generally excluded. In summary AcA was determined as a reactive metabolite of MF in vitro, its adduct formations with nucleophilic cellular components were characterized and investigated as potential biomarker in vitro for the first time. These results improve the risk assessment of MF

Aspects of Medication and Patient participation-an Easy guideLine (AMPEL). A conversation guide increases patients' and physicians' satisfaction with prescription talks

Patients want more information and active participation in medical decisions. Information and active participation correlate with increased adherence. A conversation guide, combining patient-relevant drug information with steps of shared decision-making, was developed to support physicians in effective and efficient prescription talks. Six GP trainees in community-based primary care practices participated in a controlled pilot study in sequential pre-post design. Initially, they conducted 41 prescription talks as usual, i.e., without knowing the guide. Then, they conducted 23 talks considering the guide (post-intervention phase). Immediately after the respective talk, patients filled in a questionnaire on satisfaction with the information on medication and physician-patient interaction, and physicians about their satisfaction with the talk and the application of the guide. Patients felt better informed after guide-based prescription talks (e.g., SIMS-D in median 10 vs. 17, p < 0.05), more actively involved (KPF-A for patient activation 2.9 +/- 0.8 vs. 3.6 +/- 0.8, p < 0.05), and more satisfied with the physician-patient interaction. Physicians rated the guide helpful and feasible. Their satisfaction with the conversation was significantly enhanced during the post-intervention phase. The evaluation of the duration of the talk was not influenced. Enhanced patients' and physicians' satisfaction with prescription talks encourages further examinations of the conversation guide. We invite physicians to try our guide in everyday medical practice

A simulation-based module in pharmacology education reveals and addresses medical students' deficits in leading prescription talks

Although doctor-patient communication is essential for drug prescription, the literature reveals deficits in this area. An educational approach at the Cologne medical faculty aims at identifying and addressing those deficits in medical students. Fifth-year medical students first conducted a simulated prescription talk spontaneously. Subsequently, the conversation was discussed with peer students. A pharmacist moderated the discussion based upon a previously developed conversation guide. Afterwards, the same student had the conversation again, but as if for the first time. Conversations were video-recorded, transcribed and subjected to quantitative content analysis. Four days after the simulation, the students who conducted the talk, those who observed and discussed it, and students who did neither, completed a written test that focused on the content of an effective prescription talk. Content analysis revealed clear deficits in spontaneously led prescription talks. Even essential information as on adverse drug reactions were often lacking. Prescription talks became clearly more informative and comprehensive after the short, guided peer discussion. With regard to a comprehensive, informative prescription talk, the written test showed that both the students who conducted the talk and those who only observed it performed clearly better than the students who did not participate in the educational approach. Deficits regarding prescription talks are present in 5(th) year medical students. We provide an approach to both identify and address these deficits. It thus may be an example for training medical students in simulated and clinical environments like the EACPT recommended to improve pharmacology education

Do the Outcomes of Clinical Efficacy Trials Matter in Regulatory Decision-Making for Biosimilars?

There is an increasing body of evidence supporting a more flexible approach in clinical data requirements for the approval of more complex biosimilar substances such as monoclonal antibodies (mAbs). The aim of this paper is to further analyse the role of quality/chemistry, manufacturing and controls (CMC) and clinical data for the conclusion on biosimilarity and the decision on marketing authorisation (MA). In the present study, we analysed the MA applications (MAAs) of all 33 mAbs and three fusion proteins evaluated by the European Medicines Agency (EMA) between July 2012 and November 2022 with special emphasis on all submitted rituximab (four products) and trastuzumab (seven products) biosimilar candidates, including withdrawn applications. For the two withdrawn applications, the comparative efficacy trials suggested biosimilarity, but the quality/CMC package was not accepted by EMA. We therefore investigated whether a negative MAA outcome could have been predicted based on the evidence generated in the quality/CMC packages, regardless of clinical trial data. For this purpose, we reviewed the respective European Public Assessment Reports (EPARs) or withdrawal assessment reports, and the first regulatory assessments for all these 36 MAAs (i.e. day 120 of the centralized procedure), which are not publicly available. During EMA review, where significant issues are identified which would preclude a marketing authorisation, these issues are raised as questions to the applicant and are classified as major objections (MO). In 67% of cases, the outcome of the quality and clinical assessment was the same, i.e. both the quality and clinical assessments either supported approval or did not support approval. In 11% of cases, MO were identified in the quality part of the submission but not in the clinical data. In 22% of cases, MO were raised on the clinical data package but not on the quality data. However, we found no instance where seemingly negative clinical data, including failed efficacy trials, led to a negative overall decision. In each instance, the failure to confirm similar clinical performance in all investigated aspects was eventually viewed as not being related to the biosimilar per se but as being due to imbalances in the trial arms, immaturity of secondary endpoint results, change in the reference product, or even chance findings. Furthermore, when performing an in-depth analysis of the quality and clinical packages of trastuzumab and rituximab biosimilars, we found that in no case were clinical trial data necessary to resolve residual uncertainties regarding the quality part. The results further support the argument that sufficient evidence for biosimilarity can be obtained from a combination of analytical and functional testing and pharmacokinetic studies which may also generate immunogenicity data. This calls into question the usefulness of comparative efficacy studies for the purposes of regulatory decision-making when approving biosimilar mAbs and fusion proteins. The online version contains supplementary material available at 10.1007/s40259-023-00631-4

Absorption of Anthocyanin Rutinosides after Consumption of a Blackcurrant ( Ribes nigrum L.) Extract.

The dominant anthocyanins in blackcurrant are delphinidin-3-O-rutinoside and cyanidin-3-O-rutinoside. Data on their absorption and distribution in the human body are limited. Therefore, we performed a human pilot study on five healthy male volunteers consuming a blackcurrant (Ribes nigrum L.) extract. The rutinosides and their degradation products gallic acid and protocatechuic acid were determined in plasma and urine. The rutinosides’ concentrations peaked in both plasma and urine samples within 2 h of extract ingestion. The recoveries of delphinidin-3-O-rutinoside and cyanidin-3-O-rutinoside from urine samples were 0.040 ± 0.011% and 0.048 ± 0.016%, respectively, over a 48 h period. Protocatechuic acid concentration increased significantly after ingestion of the blackcurrant extract. Our results show that after ingestion of a blackcurrant extract containing delphinidin-3-O-rutinoside and cyanidin-3-O-rutinoside, significant quantities of biologically active compounds circulated in the plasma and were excreted via urine. Furthermore, these results contribute to the understanding of anthocyanin metabolism in humans

Providing open-label placebos remotely-A randomized controlled trial in allergic rhinitis.

BackgroundPlacebos can reduce physical symptoms even when provided with full honesty and disclosure. Yet, the precise mechanisms underlying the effects of "open-label placebos" (OLPs) have remained subject of debate. Furthermore, it is unclear whether OLPs are similarly effective when provided remotely, as is sometimes required e.g. in the current COVID-19 pandemic.MethodsIn a randomized-controlled trial, we examined the effects of OLP plus treatment as usual (TAU) compared to TAU alone on symptom reduction in people with allergic rhinitis (N = 54) over the course of two weeks. Due to the COVID-19 pandemic, OLP was provided remotely (i.e. sent via postal service). To investigate the potential influence of the clinical encounter on the effects of OLP, we manipulated the perception of the virtual clinical encounter, both with respect to verbal and nonverbal factors (augmented vs. limited encounter).ResultsThe results of the manipulation check confirmed that the augmented clinical encounter was evaluated more positively than the limited encounter, in terms of perceived warmth of the provider. Participants from all treatment groups showed significant symptom reduction from baseline to two weeks later, but OLP had no incremental effect over TAU. Participants benefitted more from OLP when they did not take any other medication against allergic symptoms than when taking medication on demand. When controlling for baseline symptoms, a significant treatment by encounter interaction was found, pointing to greater symptom improvement in the OLP group when the encounter was augmented, whereas the control group improved more when the encounter was limited.DiscussionThe study demonstrates that providing OLP and enhancing the encounter remotely is possible, but their effectiveness might be lower in comparison to previous studies relying on physical patient-provider interaction. The study raises questions for future research about the potential and challenges of remote placebo studies and virtual clinical encounters. The study has been registered as a clinical trial at ISRCTN (record number: 39018)

Correction: Providing open-label placebos remotely-A randomized controlled trial in allergic rhinitis.

[This corrects the article DOI: 10.1371/journal.pone.0248367.]