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Computational and Regression Modeling Methodologies for Investigating Adrenal Steroid Metabolism in in Vitro and Clinical Studies

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Computational and Regression Modeling Methodologies for Investigating Adrenal Steroid Metabolism in in Vitro and Clinical Studies Aus der Klinik für Direktor: Frau/ Herr Prof. Dr. …………………………. Computational and regression modeling methodologies for investigating adrenal steroid metabolism in in vitro and clinical studies D i s s e r t a t i o n s s c h r i f t zur Erlangung des akademischen Grades Doctor of Philosophy (Ph.D.) vorgelegt der Medizinischen Fakultät Carl Gustav Carus der Technischen Universität Dresden von Anastasios Mangelis aus Heraklion Griechenland Dresden 2018 2. Blatt (2. Seite) 1. Gutachter: 2. Gutachter: Tag der mündlichen Prüfung: (Verteidigungstermin) gez.: ---------------------------------------- -----------------Vorsitzender der Promotionskommission Anmerkung: Die Eintragung der Gutachter und Tag der mündlichen Prüfung (Verteidigung) erfolgt nach Festlegung von Seiten der Medizinischen Fakultät Carl Gustav Carus der TU Dresden. Sie wird durch die Promovenden nach der Verteidigung zwecks Übergabe der fünf Pflichtexemplare an die Zweigbibliothek Medizin in gedruckter Form oder handschriftlich vorgenommen. 2 “In Praise of Learning” Study from bottom up, for it’s you who will take charge; it is not too late! Study the ABC; it is not enough. but study it! Do not become discouraged, begin! You have to know everything! You must prepare to take command, now! Study, man in exile! Study, man in prison! Study, wife in your kitchen! Study, old-age pensioner! You must prepare to take command now! Find yourself a school, homeless! Look for knowledge, you who freeze! You who starve, reach out for a book: it will be a weapon. You must prepare to take command now. Don’t be afraid to question, comrades! Never believe in good faith; see for yourself! What you yourself don’t learn, you don’t know. Question the reckoning, you yourself must pay it; Set down your finger on each small item, asking: where do you get this? You must prepare to take charge now! Bertolt Brecht, Germany, 1931 3 Acknowledgements Firstly, I would like to thank my advisor Dr. Eisenhofer for the support of my PhD study and related research and for always having his office door open for me. His guidance helped me during research and writing of this thesis. Besides my advisor, I would like to express my sincere gratitude to the rest of my PhD committee members, Prof. Dr. Deussen and Dr. Dieterich, for the endless patience and for sharing knowledge as true teachers. I would also like to thank Prof. Dr. Morawietz for kindly reviewing this thesis and for his valuable comments. I would also like to thank Prof. Dr. Angela Huebner, Dr. Katrin Koehler and Dr. Ramona Juehlen for their kindness, support and for sharing their lab facilities with me. Without their precious support, it would not have been possible for me to conduct this research. I would also like to thank Dr. Mirko Pietzsch and Denise Kaden for sharing their knowledge and expertise on mass spectrometry. Also, I would like to thank Isabel Poser for translating my summary into German. I also want to thank present and past members of the Eisenhofer lab in Dresden: Dr. Susan Richter, who supported me throughout my thesis, discussed scientific issues, helped me with organizing my experiments and writing this thesis and shared the stress and enthusiasm necessary for completing this difficult task. Dr. Nan Qin, Dr. Christina Pamporaki and Dr. Nicole Bechmann for sharing their knowledge and for giving me scientific support without hesitation. Also Denise, Isabel, Tina, Karsten and Romi for making the lab a nicer working environment. I would also like to thank Elena kai Maria for their friendship, their true support and for making Haus 28 a friend-meeting point. I would like to thank my grandmother Maria and my mother Katerina for their patience raising a difficult child and for teaching me how to love. My father Kostas, who taught me to think and to never stop fighting. Special thanks to Peter, Ilektra and my sister Maria for being my solid rock in every windy day. I feel privileged to be the only one in this world to call her sister and friend. I owe to thank my friends Michalis, Maria, Sisi, Maria and Leuteris, Vasia and Nikos, for being present despite the distance and for supporting me for many long and hard years. Above all, my wife, Vivian, for helping me with her brilliant scientific thinking, sharing her knowledge like a true scientist and for giving me a hand every time I was down. Her bright mind and loving soul always make me better myself. She has offered me the great gift of feeling complete. 4 Publication Note The present dissertation is mostly based on work previously published in peer-reviewed journals. The papers containing part of this work are listed below: 1. Mangelis A, Dieterich P, Peitzsch M, Richter S, Jühlen R, Hübner A, Willenberg HS, Deussen A, Lenders JW, Eisenhofer G., Computational analysis of liquid chromatography-tandem mass spectrometric steroid profiling in NCI H295R cells following angiotensin II, forskolin and abiraterone treatment. J Steroid Biochem Mol Biol. 2016 Jan;155(Pt A):67-75. doi: 10.1016/j.jsbmb.2015.09.038. Epub 2015 Oct 3. 2. Mangelis A, Jühlen R, Dieterich P, Peitzsch M, Lenders JWM, Hahner S, Schirbel A, Eisenhofer G., A steady state system for in vitro evaluation of steroidogenic pathway dynamics: Application for CYP11B1, CYP11B2 and CYP17 inhibitors. J Steroid Biochem Mol Biol. 2018 Dec 6. pii: S0960-0760(18)30464-3. doi: 10.1016/j.jsbmb.2018.12.003. [Epub ahead of print] 3. Eisenhofer G, Peitzsch M, Kaden D, Langton K, Pamporaki C, Masjkur J, Tsatsaronis G, Mangelis A, Williams TA, Reincke M, Lenders JWM, Bornstein SR., Reference intervals for plasma concentrations of adrenal steroids measured by LC-MS/MS: Impact of gender, age, oral contraceptives, body mass index and blood pressure status. Clin Chim Acta. 2017 Jul;470:115-124. doi: 10.1016/j.cca.2017.05.002. Epub 2017 May 4. 5 Contents Acknowledgements ....................................................................................................................... 4 Publication Note ............................................................................................................................ 5 Contents ........................................................................................................................................ 6 Abbreviations ............................................................................................................................... 9 1. Introduction ........................................................................................................................ 10 1.1. The adrenal gland ..................................................................................................... 10 1.1.1. Adrenal cortex steroids regulation ................................................................. 10 1.1.2. Physiological metabolic processes of steroidogenesis ............................... 13 1.2. Adrenal cortical dysfunction and therapeutic challenges ................................... 15 1.3. The adrenocortical cell line NCI –H295R .............................................................. 16 1.4. Chemical regulators of adrenal steroidogenesis ................................................. 17 1.5. Computational mechanistic modelling of adrenal metabolism .......................... 18 1.5.1. Static cell culture models for characterizing pathway dynamics .............. 18 1.5.2. Steady state as a tool for intracellular fluxes estimation ........................... 19 2. Hypothesis and aims ........................................................................................................ 20 3. Materials and methods .................................................................................................... 22 3.1. Experimental overview ............................................................................................. 22 3.1.1. Computational mechanistic modeling of steroid metabolism .......................... 22 3.1.2 A steady state system for in vitro evaluation of steroidogenic pathway dynamics ............................................................................................................................ 24 3.1.3 Calculation of age and gender adjusted reference intervals for plasma adrenal steroids for healthy population ........................................................................ 27 3.2 Liquid chromatography – tandem mass spectrometry ........................................ 29 3.3. Static cell culture mechanistic model..................................................................... 29 3.3.1. Cell culture conditions ...................................................................................... 29 3.3.2. Computational model representation for steroid metabolism and cell proliferation using ODE systems .................................................................................... 29 3.3.3. Metabolic pathways .......................................................................................... 30 3.3.4. Transport processes ........................................................................................ 32 3.3.5. Parameter estimation ....................................................................................... 34 3.4. Steady state model system for computation of pathway kinetics ...................... 35 3.4.1. Cell culture conditions for continuous flow culture system ......................... 35 6 3.4.2 Steroid secretion rates and intracellular fluxes calculation under steady state conditions ................................................................................................................
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