TY - GEN A1 - Knauer, Jan Felix A1 - Schulz, Christian A1 - Zemella, Anne A1 - Wüstenhagen, Doreen A. A1 - Walter, Ruben Magnus A1 - Küpper, Jan-Heiner A1 - Kubick, Stefan T1 - Synthesis of mono Cytochrome P450 in a modified CHO-CPR cell-free protein production platform T2 - Scientific Reports N2 - 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. KW - Multidisciplinary Y1 - 2024 U6 - https://doi.org/10.1038/s41598-024-51781-6 SN - 2045-2322 VL - 14 ER - TY - GEN A1 - Schulz, Christian A1 - Herzog, Natalie A1 - Kubick, Stefan A1 - Jung, Friedrich A1 - Küpper, Jan-Heiner T1 - 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 T2 - Cells N2 - 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. KW - Chinese hamster ovary cells KW - CHO-K1 KW - CPR KW - CYP450 KW - cytochrome P450 monooxygenase KW - liver KW - NADPH P450 oxidoreductase KW - phase-1 biotransformation KW - serum-free KW - suspension cells Y1 - 2023 UR - https://www.mdpi.com/2073-4409/12/17/2140#metrics U6 - https://doi.org/10.3390/cells12172140 SN - 2073-4409 VL - 12 IS - 17 ER - TY - GEN A1 - Krüger-Genge, Anne A1 - Köhler, Susanne A1 - Laube, Markus A1 - Haileka, Vanessa A1 - Lemm, Sandy A1 - Majchrzak, Karolina A1 - Kammerer, Sarah A1 - Schulz, Christian A1 - Storsberg, Joachim A1 - Pietzsch, Jens A1 - Küpper, Jan-Heiner A1 - Jung, Friedrich T1 - Anti-Cancer Prodrug Cyclophosphamide Exerts Thrombogenic Effects on Human Venous Endothelial Cells Independent of CYP450 Activation—Relevance to Thrombosis T2 - Cells N2 - 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. KW - cancer KW - cyclophosphamide KW - human umbilical vein endothelial cells KW - HUVEC KW - liver KW - cytochrome P450 enzymes (CYP) KW - thrombosis Y1 - 2023 UR - https://www.mdpi.com/2073-4409/12/15/1965 U6 - https://doi.org/10.3390/cells12151965 SN - 2073-4409 VL - 12 IS - 15 ER - TY - GEN A1 - Schulz, Christian A1 - Stegen, Sarah A1 - Jung, Friedrich A1 - Küpper, Jan-Heiner ED - Lamb, David ED - Syed, Khajamohiddin T1 - Mono-CYP CHO model : a recombinant Chinese hamster ovary cell platform for investigating CYP-specific tamoxifen metabolism T2 - International journal of molecular sciences N2 - The metabolism of drugs and foreign substances in humans typically involves multiple enzymatic steps, particularly in phase-1 biotransformation in the liver, where various cytochrome P450 monooxygenases (CYPs) play crucial roles. This complexity can lead to a wide range of metabolites. Understanding the contributions of individual CYPs and their interactions within these intricate enzyme cascades can be challenging. We recently developed an in vitro biotransformation platform employing various Chinese Hamster Ovarian (CHO) cell clones. These clones express human cytochrome P450 oxidoreductase (CPR), and each is defined by a specific human CYP enzyme expression, thus exhibiting no detectable endogenous CYP enzyme activity (mono-CYP CHO platform). In this study, we investigated whether the mono-CYP CHO platform is a suitable tool for modeling complex drug metabolization reactions in vitro. Tamoxifen (TAM) was selected as a model substance due to its role as a prodrug widely used in breast cancer therapy, where its main active metabolite, endoxifen, arises from a two-step metabolism primarily involving the CYP system. Specifically, the combined activity of CYP3A4 and CYP2D6 is believed to be essential for efficient endoxifen production. However, the physiological metabolization pathway of TAM is more complex and interconnected, and the reasons for TAM’s therapeutic success and variability among patients are not yet fully understood. Analogous to our recently introduced mono-CYP3A4 CHO cells, we generated a CHO cell line expressing human CPR and CYP2D6, including analysis of CYP2D6 expression and specific activity. Comparative studies on the metabolization of TAM were performed with both mono-CYP CHO models individually and in co-culture with intact cells as well as with isolated microsomes. Supernatants were analyzed by HPLC to calculate individual CYP activity for each metabolite. All the picked mono-CYP2D6 clones expressed similar CYP2D6 protein amounts but showed different enzyme activities. Mono-CYP2D6 clone 18 was selected as the most suitable for TAM metabolization based on microsomal activity assays. TAM conversion with mono-CYP2D6 and -3A4 clones, as well as the combination of both, resulted in the formation of the expected main metabolites. Mono-CYP2D6 cells and microsomes produced the highest detected amounts of 4-hydroxytamoxifen and endoxifen, along with N -desmethyltamoxifen and small amounts of N , N -didesmethyltamoxifen. N -desmethyltamoxifen was the only TAM metabolite detected in notable quantities in mono-CYP3A4, while 4-hydroxytamoxifen and endoxifen were present only in trace amounts. In CYP2D6/3A4 co-culture and equal mixtures of both CYP microsomes, all metabolites were detected at concentrations around 50% of those in individual clones, indicating no significant synergistic effects. In conclusion, our mono-CYP CHO model confirmed the essential role of CYP2D6 in synthesizing the active TAM metabolite endoxifen and indicated that CYP2D6 is also involved in producing the by-metabolite N , N -didesmethyltamoxifen. The differences in metabolite spectra between the two mono-CYP models highlight the CYP specificity and sensitivity of our in vitro system. KW - Chinese hamster ovary cells KW - Mono-CYP CHO KW - Liver KW - Phase-1 biotransformation KW - Cytochrome P450 monooxygenase KW - CYP2D6 KW - Tamoxifen KW - Endoxifen KW - DDM-TAM Y1 - 2025 U6 - https://doi.org/10.3390/ijms26093992 SN - 1422-0067 VL - 26 IS - 9 SP - 1 EP - 24 PB - MDPI CY - Basel ER - TY - GEN A1 - Ramm, Franziska A1 - Schulz, Christian A1 - Walter, Ruben M. A1 - Zemella, Anne T1 - Wie CYPs Medikamente verarbeiten : Einblicke durch innovative Labortechniken T2 - BIOspektrum N2 - Cytochrome-P450 enzymes (CYPs) represent a major class of enzymes responsible for the metabolism of xenobiotic substances. To assess the role of CYPs in drug metabolism, model systems such as liver cells are used but they harbor a variety of CYPs. Mono-CYP systems were established with a defined CYP activity to study the underlying mechanisms. We utilize a Mono-CYP system based with cell-free protein synthesis. We have shown that this combination enhances the understanding of CYP reactions. Y1 - 2024 U6 - https://doi.org/10.1007/s12268-024-2307-6 SN - 0947-0867 SN - 1868-6249 VL - 30 IS - 7 SP - 815 EP - 818 PB - Springer Berlin Heidelberg CY - Berlin ; Heidelberg ER -