@misc{SchulzKammererKuepper, author = {Schulz, Christian and Kammerer, Sarah and K{\"u}pper, Jan-Heiner}, title = {NADPH-cytochrome P450 reductase expression and enzymatic activity in primary-like human hepatocytes and HepG2 cells for in vitro biotransformation studies}, series = {Clinical Hemorheology and Microcirculation}, volume = {73}, journal = {Clinical Hemorheology and Microcirculation}, number = {1}, issn = {1386-0291}, doi = {10.3233/CH-199226}, pages = {249 -- 260}, language = {en} } @misc{UhligGehreKammereretal., author = {Uhlig, Katja and Gehre, Christian P. and Kammerer, Sarah and K{\"u}pper, Jan-Heiner and Coleman, D. and P{\"u}schel, G. and Duschl, Claus}, title = {Real-time monitoring of oxygen consumption of hepatocytes in a microbioreactor}, series = {Toxicology Letters}, volume = {295}, journal = {Toxicology Letters}, number = {Supplement 1}, issn = {0378-4274}, doi = {10.1016/j.toxlet.2018.06.652}, pages = {S115}, language = {en} } @misc{SteinbrechtPfeiferHerzogetal., author = {Steinbrecht, Susanne and Pfeifer, Nadine and Herzog, Natalie and Katzenberger, Nadine and Schulz, Christian and Kammerer, Sarah and K{\"u}pper, Jan-Heiner}, title = {HepG2-1A2 C2 and C7: Lentivirus vector-mediated stable and functional overexpression of cytochrome P450 1A2 in human hepatoblastoma cells}, series = {Toxicology Letters}, volume = {319}, journal = {Toxicology Letters}, issn = {0378-4274}, doi = {10.1016/j.toxlet.2019.11.006}, pages = {155 -- 159}, abstract = {Novel HepG2 cell clones 1A2 C2 and 1A2 C7 were independently generated by lentiviral transduction to functionally overexpress cytochrome P450 1A2 (CYP1A2). We found similar and stable CYP1A2 transcript and protein levels in both cell clones leading to specific enzyme activities of about 370 pmol paracetamol x min-1 x mg-1 protein analyzed by phenacetin conversion. Both clones showed dramatically increased sensitivity to the hepatotoxic compound aflatoxin B1 (EC50<100 nM) when compared to parental HepG2 cells (EC50 ∼5 μM). Thus, newly established cell lines are an appropriate tool to study metabolism and toxicity of substances depending on conversion by CYP1A2.}, 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{MartinaKammererPrantletal., author = {Martina, Georgieva and Kammerer, Sarah and Prantl, Lukas and Jung, Friedrich and Stroszczynski, Christian and Jung, Ernst Michael}, title = {Imaging of breast implant and implant-associated complications: Capsular contracture and intraor extracapsular rupture}, series = {Clinical Hemorheology and Microcirculation}, volume = {76}, journal = {Clinical Hemorheology and Microcirculation}, number = {2}, issn = {1875-8622}, doi = {10.3233/CH-209218}, pages = {221 -- 231}, abstract = {BACKGROUND: In recent years, follow-up after breast reconstruction with silicone implants and the detection of complications have been relieved by the possibility of improved diagnostic methods. METHODS: Between January 2015 and December 2019 a total of 40 patients (29-84 years) with silicone implants were included in this retrospective study. The implants were examined clinically and with modern imaging: general ultrasound imaging (US), magnetic resonance imaging (MRI), high resolution computed tomography (CT) and positron emission tomography -computed tomography (PET-CT). If necessary, a histological/cytological sample was taken. The breast implants were assessed by three radiologists specialized in breast imaging. The grade of capsular contracture was classified according to the Baker classification. RESULTS: All 40 women obtained a clinical examination and an US diagnostic to identify early and more common complications such as implant folding and capsular fibrosis. Depending on the clinical examination and ultrasound findings additional MRI (n = 10), CT (n = 9) and/or PET-CT (n = 2) were performed. 16 patients had implants folding proven with US (n = 16), MRI (n = 6) and CT (n = 1). The grade of capsular fibrosis was determined according to the Baker classification. The following results were obtained in our study: 25 breast implants with Baker grade I and eleven breast implants with Baker grade II, both proven with US; one breast implants with Baker grade III and one breast implant with Baker grade IV, proven with US (n = 2), MRI (n = 1) and CT (n = 1). One patient had intracapsular rupture and one patient had extracapsular rupture, both detected on CT and surgically proven. No patient had a silicone accumulation in the lymph nodes. One patient had pathologically enlarged axillary lymph nodes, which were evaluated as inflammatory changes in PET-CT. Long-term complications such as the development of malignant breast tumors could not be observed. CONCLUSION: To detect early complications after breast implant surgery, a regular clinical examination is indispensable. Imaging methods complement each other and if they are used multimodal, it is easier to identify early complications. Modern diagnostic modalities like ultrasound and magnetic resonance imaging expand the spectrum and improve diagnostic safety.}, language = {en} } @misc{JungWertheimerPutzetal., author = {Jung, Ernst Michael and Wertheimer, T. and Putz, F. J. and Jung, Friedrich and Kammerer, Sarah and Pregler, B. and Luerken, L. and Stroszczynski, Christian and Beyer, L.}, title = {Contrast enhanced ultrasound (CEUS) with parametric imaging and time intensity curve analysis (TIC) for evaluation of the success of prostate arterial embolization (PAE) in cases of prostate hyperplasia}, series = {Clinical Hemorheology and Microcirculation}, volume = {76}, journal = {Clinical Hemorheology and Microcirculation}, number = {2}, issn = {1875-8622}, doi = {10.3233/CH-209202}, pages = {143 -- 153}, abstract = {AIM: To evaluate the use of dynamic contrast enhanced ultrasound (CEUS) with parametric color-coded imaging and time intensity curve analysis (TIC) for planning and follow-up after prostate arterial embolization (PAE). MATERIAL/METHOD: Before and after selective iliacal embolization by PAE with a follow up of 6 months 18 male patients (43-78 years, mean 63±3.5 years) with histopathological proven benign prostate hyperplasia were examined by one experienced examiner. A multifrequency high resolution probe (1-6 MHz) was used for transabdominal ultrasound and CEUS with bolus injections of 2.4 ml sulphur-hexafluoride microbubbles. Independent evaluation of color-coded parametric imaging before and after PAE by in PACS stored DICOM loops from arterial phase (10-15 s) up to 1min were performed. Criteria for successful treatment were reduction of early arterial enhancement by changes of time to peak (TTP) and area under the curve (AUC) by measurements in 8 regions of interest (ROI) of 5 mm in diameter at the margin and in the center and changes from hyperenhancement in parametric imaging (perfusion evaluation of arterial enhancement over 15 s) from red and yellow to blue and green by partial infarctions. Reference imaging method was the contrast high resolution 3 tesla magnetic resonance tomography (MRI) using 3D vibe sequences before and after PAE and for the follow up after 3 and 6 months. RESULTS: PAE was technically and clinically successful in all 18 patients with less clinical symptoms and reduction of the gland volume. In all cases color-coded CEUS parametric imaging was able to evaluate partial infarction after embolization with changes from red and yellow to green and blue colors in the embolization areas. Relevant changes could be evaluated for TIC-analysis of CEUS with reduced arterial enhancement in the arterial phase and prolonged enhancement of up to 1 min with significant changes (p = 0.0024). The area under the curve (AUC) decreased from 676±255.04 rU (160 rU-1049 rU) before PAE to 370.43±255.19 rU (45 rU-858 rU) after PAE. Time to peak (TTP) did not change significantly (p = 0.6877); TTP before PAE was 25.82±9.04 s (12.3 s-42.5 s) and after PAE 24.43±9.10 s (12-39 s). Prostate volume decreased significantly (p = 0.0045) from 86.93±34.98 ml (30-139 ml) before PAE to 50.57±26.26 ml (19-117 ml) after PAE. There were no major complications and, in most cases (14/18) a volume reduction of the benign prostate hyperplasia occurred. CONCLUSION: Performed by an experienced examiner CEUS with parametric imaging and TIC-analysis is highly useful to further establish prostatic artery embolization (PAE) as a successful minimal invasive treatment of benign prostatic hyperplasia.}, language = {en} } @misc{GehreFlechnerKammereretal., author = {Gehre, Christian P. and Flechner, Marie and Kammerer, Sarah and K{\"u}pper, Jan-Heiner and Coleman, Charles Dominic and P{\"u}schel, Gerhard Paul and Uhlig, Katja and Duschl, Claus}, title = {Real time monitoring of oxygen uptake of hepatocytes in a microreactor using optical microsensors}, series = {Scientific Reports}, volume = {10}, journal = {Scientific Reports}, issn = {2045-2322}, doi = {10.1038/s41598-020-70785-6}, abstract = {Most in vitro test systems for the assessment of toxicity are based on endpoint measurements and cannot contribute much to the establishment of mechanistic models, which are crucially important for further progress in this field. Hence, in recent years, much effort has been put into the development of methods that generate kinetic data. Real time measurements of the metabolic activity of cells based on the use of oxygen sensitive microsensor beads have been shown to provide access to the mode of action of compounds in hepatocytes. However, for fully exploiting this approach a detailed knowledge of the microenvironment of the cells is required. In this work, we investigate the cellular behaviour of three types of hepatocytes, HepG2 cells, HepG2-3A4 cells and primary mouse hepatocytes, towards their exposure to acetaminophen when the availability of oxygen for the cell is systematically varied. We show that the relative emergence of two modes of action, one NAPQI dependent and the other one transient and NAPQI independent, scale with expression level of CYP3A4. The transient cellular response associated to mitochondrial respiration is used to characterise the influence of the initial oxygen concentration in the wells before exposure to acetaminophen on the cell behaviour. A simple model is presented to describe the behaviour of the cells in this scenario. It demonstrates the level of control over the role of oxygen supply in these experiments. This is crucial for establishing this approach into a reliable and powerful method for the assessment of toxicity.}, language = {en} } @misc{SteinbrechtKiebistKoenigetal., author = {Steinbrecht, Susanne and Kiebist, Jan and K{\"o}nig, Rosalie and Thiessen, Markus and Schmidtke, Kai-Uwe and Kammerer, Sarah and K{\"u}pper, Jan-Heiner and Scheibner, Katrin}, title = {Synthesis of cyclophosphamide metabolites by a peroxygenase from Marasmius rotula for toxicological studies on human cancer cells}, series = {AMB Express}, volume = {10}, journal = {AMB Express}, issn = {2191-0855}, doi = {10.1186/s13568-020-01064-w}, pages = {13}, abstract = {Cyclophosphamide (CPA) represents a widely used anti-cancer prodrug that is converted by liver cytochrome P450 (CYP) enzymes into the primary metabolite 4-hydroxycyclophosphamide (4-OH-CPA), followed by non-enzymatic generation of the bioactive metabolites phosphoramide mustard and acrolein. The use of human drug metabolites as authentic standards to evaluate their toxicity is essential for drug development. However, the chemical synthesis of 4-OH-CPA is complex and leads to only low yields and undesired side products. In past years, fungal unspecific peroxygenases (UPOs) have raised to powerful biocatalysts. They can exert the identical selective oxyfunctionalization of organic compounds and drugs as known for CYP enzymes with hydrogen peroxide being used as sole cosubstrate. Herein, we report the efficient enzymatic hydroxylation of CPA using the unspecific peroxygenase from Marasmius rotula (MroUPO) in a simple reaction design. Depending on the conditions used the primary liver metabolite 4-OH-CPA, its tautomer aldophosphamide (APA) and the overoxidized product 4-ketocyclophosphamide (4-keto-CPA) could be obtained. Using a kinetically controlled approach 4-OH-CPA was isolated with a yield of 32\% (purity > 97.6\%). Two human cancer cell lines (HepG2 and MCF-7) were treated with purified 4-OH-CPA produced by MroUPO (4-OH-CPAUPO). 4-OH-CPAUPO-induced cytotoxicity as measured by a luminescent cell viability assay and its genotoxicity as measured by γH2AX foci formation was not significantly different to the commercially available standard. The high yield of 4-OH-CPAUPO and its biological activity demonstrate that UPOs can be efficiently used to produce CYP-specific drug metabolites for pharmacological assessment.}, 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{BrauneKruegerGengeKammereretal., author = {Braune, Steffen and Kr{\"u}ger-Genge, Anne and Kammerer, Sarah and Jung, Friedrich and K{\"u}pper, Jan-Heiner}, title = {Phycocyanin from Arthrospira platensis as Potential Anti-Cancer Drug: Review of In Vitro and In Vivo Studies}, series = {Life}, volume = {11}, journal = {Life}, number = {2}, doi = {10.3390/life11020091}, pages = {91}, language = {en} } @misc{Kammerer, author = {Kammerer, Sarah}, title = {Three-Dimensional Liver Culture Systems to Maintain Primary Hepatic Properties for Toxicological Analysis In Vitro}, series = {International Journal of Molecular Sciences}, volume = {22}, journal = {International Journal of Molecular Sciences}, number = {19}, issn = {1422-0067}, doi = {10.3390/ijms221910214}, language = {en} } @misc{JungKammererBrandensteinetal., author = {Jung, Ernst Michael and Kammerer, Sarah and Brandenstein, M. and Putz, F. J. and Stroszczynski, Christian and Jung, Friedrich}, title = {High resolution flow (HR Flow) and Glazing Flow in cases of hepatic flow changes: Comparison to color-coded doppler sonography (CCDS)}, series = {Clinical Hemorheology and Microcirculation}, volume = {79}, journal = {Clinical Hemorheology and Microcirculation}, number = {1}, issn = {1875-8622}, doi = {10.3233/CH-219102}, pages = {3 -- 17}, abstract = {AIM: To evaluate the possibilities of flow detection using high resolution flow (HR Flow) and Glazing Flow technique in patients with liver parenchymal changes and flow changes in comparison to color-coded Doppler sonography (CCDS). MATERIAL AND METHODS: All examinations were performed using a multi frequency matrix convex probe with high resolution technique (SC6-1U/Resona7, Mindray, Shenzhen, China) by one experienced examiner to evaluate the venous, portal venous and arterial liver flow with digital documentation of the dynamic flow parameters like peak systolic flow, end-diastolic flow and resistance index. For liver parenchymal stiffness changes shear wave elastography was performed with at least 10 measurements. By two independent readers an elevation was performed to evaluate the image quality and the degree of flow artefacts, from 0 = not available to 5 points with excellent image quality without flow artefacts. RESULTS: All 40 patients (24 men, 16 women, age 27-83 years, mean 56±5 years) showed morphology changes from B-Mode of the liver parenchyma to inhomogeneous tissue with higher stiffness evaluated by the shear wave elastography (1.45 m/s up to 2.79 m/s±1.79 m/s, considering F1 up to F4 fibrosis) and in 15 cases histopathologically proven liver cirrhosis. In 9 cases after non-acute thrombosis flow reduction of the portal vein was the reason for the diameter less than 5 mm. Flow parameters for the venous flow were between 8 cm/s up to 29 cm/s, mean 14±4 cm/s, for the hepatic portal vein 5 cm/s up to 57 cm/s, mean 17±5 cm/s, for the hepatic artery systolic flow between 50 cm/s up to 127±33 cm/s, end-diastolic flow from 22 cm/s up to 47±8 cm/s. Resistance index for the hepatic artery was between 0.41 up to 0.73, mean 0.67±0.25. The image quality for CCDS over all cases was evaluated for CCDS between 1 up to 4. The mean quality was 2.5±0.5, for HR Flow in combination with Glazing Flow 3±0.5, with significant differences for the 2 readers (P < 0.01). CONCLUSION: Combination of HR Flow with Glazing Flow could be helpful to evaluate morphological und hemodynamic changes of liver arterial flow, portal venous and venous flow. Reduction of flow artefacts in combination with a higher image quality could be helpful for optimizing the digital measurements also for follow up examinations.}, language = {en} } @misc{VagiannisZhangBudagagaetal., author = {Vagiannis, Dimitrios and Zhang, Yu and Budagaga, Youssif and Novotna, Eva and Skarka, Adam and Kammerer, Sarah and K{\"u}pper, Jan-Heiner and Hofman, Jakub}, title = {Alisertib shows negligible potential for perpetrating pharmacokinetic drug-drug interactions on ABCB1, ABCG2 and cytochromes P450, but acts as dual-activity resistance modulator through the inhibition of ABCC1 transporter}, series = {Toxicology and Applied Pharmacology}, volume = {Vol. 434}, journal = {Toxicology and Applied Pharmacology}, issn = {0041-008X}, doi = {10.1016/j.taap.2021.115823}, pages = {13}, language = {en} } @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{KruegerGengeKoehlerLaubeetal., author = {Kr{\"u}ger-Genge, Anne and K{\"o}hler, Susanne and Laube, Markus and Haileka, Vanessa and Lemm, Sandy and Majchrzak, Karolina and Kammerer, Sarah and Schulz, Christian and Storsberg, Joachim and Pietzsch, Jens and K{\"u}pper, Jan-Heiner and Jung, Friedrich}, title = {Anti-Cancer Prodrug Cyclophosphamide Exerts Thrombogenic Effects on Human Venous Endothelial Cells Independent of CYP450 Activation—Relevance to Thrombosis}, series = {Cells}, volume = {12}, journal = {Cells}, number = {15}, issn = {2073-4409}, doi = {10.3390/cells12151965}, abstract = {Cancer patients are at a very high risk of serious thrombotic events, often fatal. The causes discussed include the detachment of thrombogenic particles from tumor cells or the adverse effects of chemotherapeutic agents. Cytostatic agents can either act directly on their targets or, in the case of a prodrug approach, require metabolization for their action. Cyclophosphamide (CPA) is a widely used cytostatic drug that requires prodrug activation by cytochrome P450 enzymes (CYP) in the liver. We hypothesize that CPA could induce thrombosis in one of the following ways: (1) damage to endothelial cells (EC) after intra-endothelial metabolization; or (2) direct damage to EC without prior metabolization. In order to investigate this hypothesis, endothelial cells (HUVEC) were treated with CPA in clinically relevant concentrations for up to 8 days. HUVECs were chosen as a model representing the first place of action after intravenous CPA administration. No expression of CYP2B6, CYP3A4, CYP2C9 and CYP2C19 was found in HUVEC, but a weak expression of CYP2C18 was observed. CPA treatment of HUVEC induced DNA damage and a reduced formation of an EC monolayer and caused an increased release of prostacyclin (PGI2) and thromboxane (TXA) associated with a shift of the PGI2/TXA balance to a prothrombotic state. In an in vivo scenario, such processes would promote the risk of thrombus formation.}, 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{StockHoferLochmannetal., author = {Stock, Valentina and Hofer, Rebecca and Lochmann, Franziska and Spanke, Vera and Liedl, Klaus R. and Troppmair, Jakob and Langer, Thierry and Gstach, Hubert and Dank, Christian and Mayhew, Chris A. and Kammerer, Sarah and Ruzsanyi, Veronika}, title = {Tolterodine is a novel candidate for assessing CYP3A4 activity through metabolic volatiles to predict drug responses}, series = {Scientific reports}, volume = {15}, journal = {Scientific reports}, number = {1}, publisher = {Nature Publishing Group UK}, address = {London}, issn = {2045-2322}, doi = {10.1038/s41598-025-86450-9}, pages = {1 -- 12}, abstract = {Cytochrome P450 (CYP) 3A4 plays a major role in drug metabolism. Its activity could be determined by non-invasive and cost-effective assays, such as breath analysis, for the personalised monitoring of drug response. For the first time, we identify an isotopically unlabelled CYP3A4 substrate, tolterodine that leads to the formation of a non-toxic volatile metabolite, acetone, which could potentially be applied to monitor CYP3A4 activity in humans. In vitro biotransformation of tolterodine by HepG2 cells overexpressing CYP3A4, CYP2D6 or CYP2C9 was investigated by LC-MS analysis of cell culture supernatant for the non-volatile metabolite, N-dealkylated tolterodine, and PTR-ToF-MS analysis of the headspace for acetone. The highest level of the N-dealkylated metabolite was produced by HepG2-CYP3A4. Concentration dependent effects of tolterodine were analysed, resulting in TC50 values of 414 µM and 375 µM for HepG2-CYP3A4 and reference cells, respectively. Acetone and N-dealkylated tolterodine levels increased continuously over 24 h in HepG2-CYP3A4. Treatment with either a pan-CYP inhibitor, 1-aminobenzotriazole, or a CYP3A4 inhibitor, ketoconazole, considerably reduced the production of both metabolites in HepG2-CYP3A4 cells. These findings pave the way for the further development of non-invasive breath tests using unlabelled precursors to determine CYP enzyme activity in individuals.}, language = {en} } @misc{StockHoferSpankeetal., author = {Stock, Valentina and Hofer, Rebecca and Spanke, Vera A. and Liedl, Klaus R. and Troppmair, Jakob and Langer, Thierry and Dank, Christian and Gstach, Hubert and Kammerer, Sarah and Ruzsanyi, Veronika}, title = {Diisopromine as a non-labelled CYP3A4 substrate : implications for breath test development}, series = {Bioorganic chemistry}, volume = {163}, journal = {Bioorganic chemistry}, number = {6}, publisher = {Elsevier BV}, address = {Amsterdam}, issn = {0045-2068}, doi = {10.1016/j.bioorg.2025.108772}, pages = {1134 -- 1146}, abstract = {Breath tests for the prediction of patient-specific drug response require suitable substrates that when metabolised in the human body yield specific, unlabelled volatile metabolites. This study explores diisopromine as a potential substrate due to its structural similarity to tolterodine, the first identified non-labelled CYP3A4 substrate producing a volatile metabolite. In contrast to tolterodine, diisopromine contains a phenyl-group instead of p-cresol. During biotransformation, both compounds undergo N-dealkylation, forming acetone, though their non-volatile metabolites differ. Mass spectrometric techniques such as PTR-ToF-MS and LC-MS were used to determine volatile and non-volatile metabolites, respectively. A design of experiments was used to determine the optimal reaction time and substrate concentration of diisopromine to maximise the production of the non-volatile metabolite while maintaining high cell viability. The specificity for diisopromine was investigated with three different CYP isoforms using HepG2 clones that overexpress the respective isoform. As expected, highest levels of N-dealkylated diisopromine were obtained by CYP3A4, with minor contributions from CYP2D6 and CYP2C9. The absence of a stereocenter in diisopromine (i) enhances its biotransformation efficiency (yielding 1.5 times more N-dealkylated metabolite and 1.4 times more acetone than tolterodine), (ii) shifts its metabolism more towards N-dealkylation and (iii) decreases the toxicity to the cells (with TC50 values of almost 1000 μM). The production of both metabolites in HepG2-CYP3A4 cells was significantly inhibited following treatment with either 1-aminobenzotriazole or ketoconazole. These findings highlight that the minor structural modification in diisopromine enhances the biotransformation efficiency, paving the way for its potential development for a (non-) invasive CYP assay.}, language = {en} } @misc{StockHoferLiedletal., author = {Stock, Valentina and Hofer, Rebecca and Liedl, Klaus R. and Troppmair, Jakob and Langer, Thierry and Gstach, Hubert and Dank, Christian and Kammerer, Sarah and Ruzsanyi, Veronika}, title = {Towards the use of metabolic volatiles in breath for determining drug response : gstachamine as an unlabeled substrate to measure CYP3A4 activity}, series = {ChemMedChem : an official journal of the EFMC}, volume = {20}, journal = {ChemMedChem : an official journal of the EFMC}, number = {18}, publisher = {Wiley}, address = {Weinheim}, issn = {1860-7179}, doi = {10.1002/cmdc.202500492}, pages = {1134 -- 1146}, abstract = {Breath analysis is a promising noninvasive diagnostic tool, but the clinical applicability of breath tests depends on several factors. A salient criterion pertains to the presence of substrates with the ability to produce detectable volatile metabolites during the metabolism. In this work, we evaluated the potential of two candidate compounds, namely gstachidine and gstachamine, for their use in metabolic breath analysis. Both substrates were evaluated for their toxicity and metabolic conversion in HepG2 cell clones overexpressing CYP3A4. Gstachidine was found to be toxic and did not produce any volatile metabolite. In contrast, gstachamine successfully generated butanone as a volatile metabolite, making it the first substrate to yield a stable VOC detectable exclusively at low ppbV levels in breath. To characterize its biotransformation, we conducted time-dependent analyses, as well as CYP specificity, toxicity, and inhibition investigations regarding the production of N-dealkylated gstachamine or butanone. The results demonstrated that gstachamine had a high metabolic turnover and a strong CYP3A4-dependency in the production of the specific N-dealkylated metabolite. Furthermore, a substantial reduction in the production of both metabolites was observed in HepG2-CYP3A4 cells following treatment with CYP inhibitors 1-aminobenzotriazole or ketoconazole. The results suggest that gstachamine has potential for noninvasive CYP3A4 metabolism monitoring.}, language = {en} } @misc{JansenAlAssarafetal., author = {Jansen, Gerrit and Al, Marjon and Assaraf, Yehuda G. and Kammerer, Sarah and van Meerloo, Johan and Ossenkoppele, Gert J. and Cloos, Jacqueline and Peters, Godefridus J.}, title = {Statins markedly potentiate aminopeptidase inhibitor activity against (drug-resistant) human acute myeloid leukemia cells}, series = {Cancer drug resistance}, volume = {6}, journal = {Cancer drug resistance}, number = {2}, publisher = {OAE Publishing Inc.}, issn = {2578-532X}, doi = {10.20517/cdr.2023.20}, pages = {430 -- 446}, abstract = {Aim: This study aimed to decipher the molecular mechanism underlying the synergistic effect of inhibitors of the mevalonate-cholesterol pathway (i.e., statins) and aminopeptidase inhibitors (APis) on APi-sensitive and -resistant acute myeloid leukemia (AML) cells. Methods: U937 cells and their sublines with low and high levels of acquired resistance to (6S)-[(R)-2-((S)-Hydroxy-hydroxycarbamoyl-methoxy-methyl)-4-methyl-pentanoylamino]-3,3 dimethyl-butyric acid cyclopentyl ester (CHR2863), an APi prodrug, served as main AML cell line models. Drug combination effects were assessed with CHR2863 and in vitro non-toxic concentrations of various statins upon cell growth inhibition, cell cycle effects, and apoptosis induction. Mechanistic studies involved analysis of Rheb prenylation required for mTOR activation. Results: A strong synergy of CHR2863 with the statins simvastatin, fluvastatin, lovastatin, and pravastatin was demonstrated in U937 cells and two CHR2863-resistant sublines. This potent synergy between simvastatin and CHR2863 was also observed with a series of other human AML cell lines (e.g., THP1, MV4-11, and KG1), but not with acute lymphocytic leukemia or multiple solid tumor cell lines. This synergistic activity was: (i) specific for APis (e.g., CHR2863 and Bestatin), rather than for other cytotoxic agents; and (ii) corroborated by enhanced induction of apoptosis and cell cycle arrest which increased the sub-G1 fraction. Consistently, statin potentiation of CHR2863 activity was abrogated by co-administration of mevalonate and/or farnesyl pyrophosphate, suggesting the involvement of protein prenylation; this was experimentally confirmed by impaired Rheb prenylation by simvastatin. Conclusion: These novel findings suggest that the combined inhibitory effect of impaired Rheb prenylation and CHR2863-dependent mTOR inhibition instigates a potent synergistic inhibition of statins and APis on human AML cells.}, language = {en} }