@misc{VagiannisBudagagaMorelletal., author = {Vagiannis, Dimitrios and Budagaga, Youssif and Morell, Anselm and Zhang, Yu and Novotna, Eva and Skarka, Adam and Kammerer, Sarah and K{\"u}pper, Jan-Heiner and Hanke, Ivo and Rozkoš, Tom{\´a}š and Hofman, Jakub}, title = {Tepotinib Inhibits Several Drug Efflux Transporters and Biotransformation Enzymes: The Role in Drug-Drug Interactions and Targeting Cytostatic Resistance In Vitro and Ex Vivo}, series = {International Journal of Molecular Sciences}, volume = {22}, journal = {International Journal of Molecular Sciences}, number = {21}, issn = {1422-0067}, doi = {10.3390/ijms222111936}, language = {en} } @misc{KuenzelHoffmannWeberetal., author = {K{\"u}nzel, Stephan R. and Hoffmann, Maximilian and Weber, Silvio and K{\"u}nzel, Karolina and K{\"a}mmerer, Susanne and G{\"u}nscht, Mario and Klapproth, Erik and Rausch, Johanna S. E. and Sadek, Mirna S. and Kolanowski, Tomasz and Meyer-Roxlau, Stefanie and Piorkowski, Christopher and Tugtekin, Sems M. and Rose-John, Stefan and Yin, Xiaoke and Mayr, Manuel and Kuhlmann, Jan Dominik and Wimberger, Pauline and Gr{\"u}tzmann, Konrad and Herzog, Natalie and K{\"u}pper, Jan-Heiner and O'Reilly, Molly and Kabir, S. Nashitha and Sommerfeld, Laura C. and Guan, Kaomei and Wielockx, Ben and Fabritz, Larissa and Nattel, Stanley and Ravens, Ursula and Dobrev, Dobromir and Wagner, Michael and El-Armouche, Ali}, title = {Diminished PLK2 Induces Cardiac Fibrosis and Promotes Atrial Fibrillation}, series = {Circulation Research}, volume = {129}, journal = {Circulation Research}, number = {8}, issn = {1524-4571}, doi = {10.1161/CIRCRESAHA.121.319425}, pages = {804 -- 820}, abstract = {Rationale: Fibrosis promotes the maintenance of atrial fibrillation (AF), making it resistant to therapy. Improved understanding of the molecular mechanisms leading to atrial fibrosis will open new pathways toward effective antifibrotic therapies. Objective: This study aims to decipher the mechanistic interplay between PLK2 (polo-like kinase 2) and the profibrotic cytokine OPN (osteopontin) in the pathogenesis of atrial fibrosis and AF. Methods and Results: Atrial PLK2 mRNA expression was 10-fold higher in human fibroblasts than in cardiomyocytes. Compared with sinus rhythm, right atrial appendages and isolated right atrial fibroblasts from patients with AF showed downregulation of PLK2 mRNA and protein, along with increased PLK2 promotor methylation. Genetic deletion as well as pharmacological inhibition of PLK2 induced profibrotic phenotype conversion in cardiac fibroblasts and led to a striking de novo secretion of OPN. Accordingly, PLK2-deficient (PLK2 knockout) mice showed cardiac fibrosis and were prone to experimentally induced AF. In line with these findings, OPN plasma levels were significantly higher only in patients with AF with atrial low-voltage zones (surrogates of fibrosis) compared with sinus rhythm controls. Mechanistically, we identified ERK1/2 as the relevant downstream mediator of PLK2 leading to increased OPN expression. Finally, oral treatment with the clinically available drug mesalazine, known to inhibit ERK1/2, prevented cardiac OPN overexpression and reversed the pathological PLK2 knockout phenotype in PLK2 knockout mice. Conclusions: Abnormal PLK2/ERK1/2/OPN axis function critically contributes to AF-related atrial fibrosis, suggesting reinforcing PLK2 activity and/or OPN inhibition as innovative targets to prevent fibrosis progression in AF. Mesalazine derivatives may be used as lead compounds for the development of novel anti-AF agents targeting fibrosis.}, language = {en} } @misc{LauRangarajanKruegerGengeetal., author = {Lau, S. and Rangarajan, R. and Kr{\"u}ger-Genge, Anne and Braune, Steffen and Kammerer, Sarah and K{\"u}pper, Jan-Heiner and Lendlein, Andreas and Jung, Friedrich}, title = {Effects of acrolein in comparison to its prodrug cyclophosphamide on human primary endothelial cells in vitro}, series = {Toxicology in Vitro}, volume = {62}, journal = {Toxicology in Vitro}, issn = {0887-2333}, doi = {10.1016/j.tiv.2019.104685}, pages = {8}, abstract = {Cyclophosphamide (CPA) is one of the most successful anticancer prodrugs that becomes effective after biotransformation in the liver resulting in the toxic metabolite acrolein. Cancer is often accompanied by thromboembolic events, which might be a result of dysfunctional endothelial cells due to CPA treatment. Here, the effect of 1 mM CPA or acrolein (10/50/100/500 μM) on human umbilical vein endothelial cells (HUVECs) was analyzed after two days of treatment. The addition of CPA or 10 μM acrolein did not affect HUVECs. However, concentrations of 100 μM and 500 μM acrolein significantly reduced the number of adherent cells by 86 ± 13\% and 99 ± 1\% and cell viability by 51 ± 29\% and 93 ± 8\% compared to the control. Moreover, pronounced stress fibers as well as multiple nuclei were observed and von Willebrand factor (vWF) was completely released. Lactate dehydrogenase was 8.5 ± 7.0-fold and 252.9 ± 42.9-fold increased showing a loss of cell membrane integrity. The prostacyclin and thromboxane secretion was significantly increased by the addition of 500 μM acrolein (43.1 ± 17.6-fold and 246.4 ± 106.3-fold) indicating cell activation/pertubation. High doses of acrolein led to HUVEC death and loss of vWF production. This effect might be associated with the increased incidence of thromboembolic events in cancer patients treated with high doses of CPA.}, language = {en} } @misc{KuepperJungKriegeretal., author = {K{\"u}pper, Jan-Heiner and Jung, Friedrich and Krieger, Volker and Hufert, Frank}, title = {A comparison of mortality rates between European and Asian States}, series = {Clinical Hemorheology and Microcirculation}, volume = {75}, journal = {Clinical Hemorheology and Microcirculation}, number = {1}, issn = {1875-8622}, doi = {10.3233/CH-209008}, pages = {3 -- 5}, language = {en} } @misc{VagiannisNovotnaSkarkaetal., author = {Vagiannis, Dimitrios and Novotna, Eva and Skarka, Adam and Kammerer, Sarah and K{\"u}pper, Jan-Heiner and Chen, Si and Guo, Lei and Staud, Frantisek and Hofman, Jakub}, title = {Ensartinib (X-396) Effectively Modulates Pharmacokinetic Resistance Mediated by ABCB1 and ABCG2 Drug Efflux Transporters and CYP3A4 Biotransformation Enzyme}, series = {Cancers}, volume = {12}, journal = {Cancers}, number = {4}, issn = {2072-6694}, doi = {10.3390/cancers12040813}, pages = {27}, abstract = {Ensartinib (X-396) is a promising tyrosine kinase inhibitor currently undergoing advanced clinical evaluation for the treatment of non-small cell lung cancer. In this work, we investigate possible interactions of this promising drug candidate with ATP-binding cassette (ABC) drug efflux transporters and cytochrome P450 biotransformation enzymes (CYPs), which play major roles in multidrug resistance (MDR) and pharmacokinetic drug-drug interactions (DDIs). Accumulation studies showed that ensartinib is a potent inhibitor of ABCB1 and ABCG2 transporters. Additionally, incubation experiments with recombinant CYPs showed that ensartinib significantly inhibits CYP3A4 and CYP2C9. Subsequent molecular docking studies confirmed these findings. Drug combination experiments demonstrated that ensartinib synergistically potentiates the antiproliferative effects of daunorubicin, mitoxantrone, and docetaxel in ABCB1, ABCG2, and CYP3A4-overexpressing cellular models, respectively. Advantageously, ensartinib's antitumor efficiency was not compromised by the presence of MDR-associated ABC transporters, although it acted as a substrate of ABCB1 in Madin-Darby Canine Kidney II (MDCKII) monolayer transport assays. Finally, we demonstrated that ensartinib had no significant effect on the mRNA-level expression of examined transporters and enzymes in physiological and lung tumor cellular models. In conclusion, ensartinib may perpetrate clinically relevant pharmacokinetic DDIs and modulate ABCB1-, ABCG2-, and CYP3A4-mediated MDR. The in vitro findings presented here will provide a valuable foundation for future in vivo investigations.}, language = {en} } @misc{JungKriegerHufertetal., author = {Jung, Friedrich and Krieger, Volker and Hufert, Frank and K{\"u}pper, Jan-Heiner}, title = {How we should respond to the Coronavirus SARS-CoV-2 outbreak: A German perspective}, series = {Clinical Hemorheology and Microcirculation}, volume = {74}, journal = {Clinical Hemorheology and Microcirculation}, number = {4}, issn = {1875-8622}, doi = {10.3233/CH-209004}, pages = {363 -- 372}, abstract = {BACKGROUND: In the early phase of the COVID-19 pandemic Germany missed to set up efficient containment measures. Consequently, the number of cases increased exponentially until a lockdown was implemented to suppress the spread of SARS-CoV-2. Fortunately, Germany has a high capability for coronavirus lab testing and more than 30,000 ICU beds. These capabilities and the lockdown turned out to be an advantage to combat the pandemic and to prevent a health-system overload. AIM: The aim was to predict the plateau day of SARS-CoV-2 infections or deaths. RESULTS: The effect on the viral spread of the German measures taken and the impact on the peak of new infection cases is shown. By normalizing daily case numbers, the plateau day of the current outbreak in Germany could be calculated to be reached at April 12, 2020 (day 103 of 2020). CONCLUSION: Normalized case number curves are helpful to predict the time point at which no further new infections will occur if the epidemic situation remains stable. Upon reaching the plateau day during a lockdown phase, a residual time-period of about 2-3 weeks can be utilized to prepare a safe unlocking period. As can be learned from Asian countries such as South Korea and Taiwan there must be strict rules to keep the risk of infection low. Those include social distancing, face mask wearing in combination with digital contact tracing and serosurveillance studies. Following those rules, a safe dance around the infection curve allows to keep the population at a reduced infection rate.}, language = {en} } @misc{KammererNowakMantkeetal., author = {Kammerer, Sarah and Nowak, Elisabeth and Mantke, Ren{\´e} and Jung, Friedrich and K{\"u}pper, Jan-Heiner}, title = {In vitro simulation of the liver first-pass effect with biotransformation-competent HepG2 cells to study effects of MG-132 on liver and cancer cells}, series = {Clinical Hemorheology and Microcirculation}, volume = {86}, journal = {Clinical Hemorheology and Microcirculation}, number = {1-2}, issn = {1386-0291}, doi = {10.3233/CH-238108}, pages = {159 -- 168}, abstract = {BACKGROUND: Liver biotransformation is the major route for drug metabolism in humans, often catalysed by cytochrome P450 (CYP) enzymes. This first-pass effect can lead to hepatotoxicity and influences the bioavailability of drugs. OBJECTIVE: We aimed to establish in vitro culture systems simulating the liver first-pass to study effects of the proteasome inhibitor MG-132 simultaneously on hepatocytes and cancer cells. METHODS: The first-pass effect was simulated by conditioned medium transfer (CMT) from pre-treated HepG2 CYP3A4-overexpressing cells to either pancreatic cancer cell line PANC-1 or primary colon cancer cells, and by indirect co-culture (CC) of liver and cancer cells in a shared medium compartment. Experimental proteasome inhibitor MG-132 was used as test substance as it is detoxified by CYP3A4. RESULTS: Cancer cells showed higher viabilities in the first-pass simulation by CMT and CC formats when compared to monocultures indicating effective detoxification of MG-132 by HepG2 CYP3A4-overexpressing cells. HepG2-CYP3A4 cells showed reduced viabilites after treatment with MG-132. CONCLUSIONS: We successfully established two different culture systems to simulate the liver first-pass effect in vitro. Such systems easily allow to study drug effects simultaneously on liver and on target cancer cells. They are of great value in pre-clinical cancer research, pharmaceutical research and drug development.}, language = {en} } @misc{SchulzHerzogKubicketal., author = {Schulz, Christian and Herzog, Natalie and Kubick, Stefan and Jung, Friedrich and K{\"u}pper, Jan-Heiner}, title = {Stable Chinese Hamster Ovary Suspension Cell Lines Harboring Recombinant Human Cytochrome P450 Oxidoreductase and Human Cytochrome P450 Monooxygenases as Platform for In Vitro Biotransformation Studies}, series = {Cells}, volume = {12}, journal = {Cells}, number = {17}, issn = {2073-4409}, doi = {10.3390/cells12172140}, abstract = {In the liver, phase-1 biotransformation of drugs and other xenobiotics is largely facilitated by enzyme complexes consisting of cytochrome P450 oxidoreductase (CPR) and cytochrome P450 monooxygenases (CYPs). Generated from human liver-derived cell lines, recombinant in vitro cell systems with overexpression of defined phase-1 enzymes are widely used for pharmacological and toxicological drug assessment and laboratory-scale production of drug-specific reference metabolites. Most, if not all, of these cell lines, however, display some background activity of several CYPs, making it difficult to attribute effects to defined CYPs. The aim of this study was to generate cell lines with stable overexpression of human phase-1 enzymes based on Chinese hamster ovary (CHO) suspension cells. Cells were sequentially modified with cDNAs for human CPR in combination with CYP1A2, CYP2B6, or CYP3A4, using lentiviral gene transfer. In parallel, CYP-overexpressing cell lines without recombinant CPR were generated. Successful recombinant expression was demonstrated by mRNA and protein analyses. Using prototypical CYP-substrates, generated cell lines proved to display specific enzyme activities of each overexpressed CYP while we did not find any endogenous activity of those CYPs in parental CHO cells. Interestingly, cell lines revealed some evidence that the dependence of CYP activity on CPR could vary between CYPs. This needs to be confirmed in further studies. Recombinant expression of CPR was also shown to enhance CYP3A4-independent metabolisation of testosterone to androstenedione in CHO cells. We propose the novel serum-free CHO suspension cell lines with enhanced CPR and/or defined CYP activity as a promising "humanised" in vitro model to study the specific effects of those human CYPs. This could be relevant for toxicology and/or pharmacology studies in the pharmaceutical industry or medicine.}, language = {en} } @misc{KnauerSchulzZemellaetal., author = {Knauer, Jan Felix and Schulz, Christian and Zemella, Anne and W{\"u}stenhagen, Doreen A. and Walter, Ruben Magnus and K{\"u}pper, Jan-Heiner and Kubick, Stefan}, title = {Synthesis of mono Cytochrome P450 in a modified CHO-CPR cell-free protein production platform}, series = {Scientific Reports}, volume = {14}, journal = {Scientific Reports}, issn = {2045-2322}, doi = {10.1038/s41598-024-51781-6}, pages = {14}, abstract = {AbstractCytochromes P450 (CYPs) are a group of monooxygenases that can be found in almost all kinds of organisms. For CYPs to receive electrons from co-substrate NADPH, the activity of NADPH-Cytochrome-P450-oxidoreductase (CPR) is required as well. In humans, CYPs are an integral part of liver-based phase-1 biotransformation, which is essential for the metabolization of multiple xenobiotics and drugs. Consequently, CYPs are important players during drug development and therefore these enzymes are implemented in diverse screening applications. For these applications it is usually advantageous to use mono CYP microsomes containing only the CYP of interest. The generation of mono-CYP containing mammalian cells and vesicles is difficult since endogenous CYPs are present in many cell types that contain the necessary co-factors. By obtaining translationally active lysates from a modified CHO-CPR cell line, it is now possible to generate mono CYPs in a cell-free protein synthesis process in a straightforward manner. As a proof of principle, the synthesis of active human CYPs from three different CYP450 gene families (CYP1A2, CYP2B6 and CYP3A4), which are of outstanding interest in industry and academia was demonstrated. Luciferase based activity assays confirm the activity of the produced CYPs and enable the individual adaptation of the synthesis process for efficient cell-free enzyme production. Furthermore, they allow for substrate and inhibitor screenings not only for wild-type CYPs but also for mutants and further CYP isoforms and variants. As an example, the turnover of selected CYP substrates by cell-free synthesized CYPs was demonstrated via an indirect luciferase assay-based screening setup.}, language = {en} }