LivPharm. ****************************************************************************************** * ****************************************************************************************** Research projects Publications People Methods and equipment Cooperation Research funding The objective is to study liver pharmacodynamics and pharmacokinetics of actual or potenti focus on executive and regulatory mechanisms of bile production and secretion (enzymes and proteins) and their impairment by various liver diseases. Modulation of these mechanisms e physiological and pathophysiological consequences including changes in elimination of drug variability in PK/PD of co-administered agents may complicate therapeutic outcomes. Our pu therefore to study such changes and to introduce appropriate prophylactic and therapeutic particular we are working on nonalcoholic fatty liver disease, liver regeneration and chol disorders characterized by impaired bile secretion due to extrahepatic biliary obstruction intrahepatic influence of toxic insult like bacterial LPS or ethinylestradiol. Using anima models of these pathophysiological situations, we study protective effects of drugs modify of PXR, CAR and FXR nuclear receptors, and naturally occurring anti-inflammatory compounds or EGCG. Furthermore, systemic pharmacokinetics of drugs and xenobiotics is studied in rat bioavalability, body distribution, metabolism, biliary and urinary excretions. ****************************************************************************************** * Research projects ****************************************************************************************** Experimental therapy of Nonalcoholic steatohepatitis, and Intrahepatic cholestasis of Preg The aim of this branch of research is to study liver effects (pharmacodymacs) of current o in common liver disorders. Attention is focused on nonalcoholic steatohepatitis (NASH) as accompanying numerous metabolic and cardiovascular disorders, and intrahepatic cholestasis most frequent liver disorder during pregnancy. We combine appropriate in vivo models of th with detailed analysis of involved regulatory and executive molecules in order to identify relationship, and responsible mechanisms. Equally important is also description of effects agents in healthy animals because these drugs are frequently used also in clinical situati and ICP. Modulation of bile acid homeostasis, and bile production Bile production is an essential function of the liver and serves as an irreplaceable excre elimination of lipophilic endo- and xenobiotics such as cholesterol, BA, bilirubin or drug as major components of bile, BA are required for micelle formation, intestinal fat digesti of bacterial growth, and immune response and production of regulatory mediators released t circulation such as fibroblast growth factor 19 or glucagon-like peptide 1. In addition, B major metabolites of cholesterol, act as hormones by agonism at several receptors such as receptor (FXR), the G protein-coupled bile acid receptor 1 (TGR5), sphingosine-1-phosphate or pregnane X receptor (PXR), and regulate numerous liver functions including glucose and metabolism. Stimulation of these receptors demonstrates promising positive effects in live as nonalcoholic steatohepatitis (NASH) or intrahepatic cholestasis. On the other hand, BA during different forms of cholestasis may have a direct toxic effect on liver cells and ti of bile production and BA homeostasis are therefore key events in liver physiology and pat Numerous regulatory events exist in whole process, especially nuclear receptors FXR, PXR, well as feedback and inflammatory signaling triggering mitogen-activated kinases, or direc with promotor sequences as we have recently detected for iron-mediated changes. The aim of research is therefore the identification of regulatory mechanisms of BA homeostasis and bi and characterization of possible pharmacological modulation. This part is solved in cooperation with the group of prof. PharmDr. Petr Pavek, Ph.D. [ UR portal.faf.cuni.cz/Groups/Group-of-Clinical-and-Molecular-Pharmacotherapy/"] and prof. Pha Nachtigal, Ph.D [ URL "https://portal.faf.cuni.cz/Groups/Pathology-pharmacology-of-cardiov system/"] . from Faculty of Pharmacy in Hradec Kralove. Drug pharmacokinetics in preclinical studies Knowledge of pharmacokinetic characteristics of evaluated drugs, i.e. absorption, distribu and excretion is an essential prerequisite for successful pharmacotherapy with minimum of and drug-drug interactions. We therefore focus on evaluation of kinetic parameters includi biliary and urinary secretion, volume of distribution, biological half-life and bioavailab or developing drugs. Essential approach is the usage of suitable in vivo and in vitro mode advanced analytical and mathematical methods. ****************************************************************************************** * Recent Publications ****************************************************************************************** • Alaei Faradonbeh F, Lastuvkova H, Cermanova J, Hroch M, Nova Z, Uher M, Hirsova P, Pavek Multidrug Resistance-Associated Protein 2 Deficiency Aggravates Estrogen-Induced Impairm Metabolomics in Rats. Front Physiol. 2022 Mar 21;13:859294. IF = 4,3; Q1 dle AIS • Skoda J, Dohnalova K, Chalupsky K, Stahl A, Templin M, Maixnerova J, Micuda S, Gr?ntved A, Pavek P. Off-target lipid metabolism disruption by the mouse constitutive androstane TCPOBOP in humanized mice. Biochem Pharmacol. 2021 Dec 28;197:114905. doi: 10.1016/j.bcp = 5,858; Q1; AIS 1,144 • Faradonbeh FA, Sa II, Lastuvkova H, Cermanova J, Hroch M, Faistova H, Mokry J, Nova Z, U P, Pavek P, Micuda S. Metformin impairs bile acid homeostasis in ethinylestradiol-induce mice. Chem Biol Interact. 2021 May 28:109525. doi: 10.1016/j.cbi.2021.109525. IF = 5,192 • Lastuvkova H, Faradonbeh FA, Schreiberova J, Hroch M, Mokry J, Faistova H, Nova Z, Hyspl IC, Nachtigal P, Stefela A, Pavek P, Micuda S. Atorvastatin Modulates Bile Acid Homeosta Diet-Induced Nonalcoholic Steatohepatitis. Int J Mol Sci. 2021 Jun 16;22(12):6468. IF = 1,123 • Vicen M, Igreja Sá IC, Tripská K, Vitverová B, Najmanová I, Eissazadeh S, Micuda S, Nach Membrane and soluble endoglin role in cardiovascular and metabolic disorders related to syndrome. Cell Mol Life Sci. 2021 Mar;78(6):2405-2418. IF = 9,261; Q1; AIS 2,226 • Stefela A, Kaspar M, Drastik M, Kronenberger T, Micuda S, Dracinsky M, Klepetarova B, Ku P. (E)-7-Ethylidene-lithocholic Acid (7-ELCA) Is a Potent Dual Farnesoid X Receptor (FXR and GPBAR1 Agonist Inhibiting FXR-Induced Gene Expression in Hepatocytes and Stimulating Peptide-1 Secretion From Enteroendocrine Cells. Front Pharmacol. 2021 Aug 13;12:713149. AIS 1,139 • Igreja Sá IC, Tripska K, Hroch M, Hyspler R, Ticha A, Lastuvkova H, Schreiberova J, Dole Eissazadeh S, Vitverova B, Najmanova I, Vasinova M, Pericacho M, Micuda S, Nachtigal P. Endoglin as a Potential Biomarker of Nonalcoholic Steatohepatitis (NASH) Development, Pa Aggravation of NASH-Related Changes in Mouse Liver. Int J Mol Sci. 2020 Nov 27;21(23):90 Q1; AIS 1,123 • Smutny T, Dusek J, Hyrsova L, Nekvindova J, Horvatova A, Micuda S, Gerbal-Chaloin S, Pav untranslated region contributes to the pregnane X receptor (PXR) expression down-regulat ligands and up-regulation by glucocorticoids. Acta Pharm Sin B. 2020 Jan;10(1):136-152. j.apsb.2019.09.010. IF 6,150; Q1; AIS 1,090 • Skoda J, Dusek J, Drastik M, Stefela A, Dohnalova K, Chalupsky K, Smutny T, Micuda S, Ge S, Pavek P. Diazepam Promotes Translocation of Human Constitutive Androstane Receptor (C Interaction with the Ligand-Binding Domain. Cells. 2020 Nov 24;9(12):2532. IF = 6,600; Q • Stefela A, Kaspar M, Drastik M, Holas O, Hroch M, Smutny T, Skoda J, Hutníková M, Pandey Kudova E, Pavek P. 3?-Isoobeticholic acid efficiently activates the farnesoid X receptor its epimerization to 3?-epimer by hepatic metabolism. J Steroid Biochem Mol Biol. 2020 J = 4,292; Q2; AIS 0,905 • Igreja Sá IC, Tripska K, Hroch M, Hyspler R, Ticha A, Lastuvkova H, Schreiberova J, Dole Eissazadeh S, Vitverova B, Najmanova I, Vasinova M, Pericacho M, Micuda S, Nachtigal P. Endoglin as a Potential Biomarker of Nonalcoholic Steatohepatitis (NASH) Development, Pa Aggravation of NASH-Related Changes in Mouse Liver. Int J Mol Sci. 2020 Nov 27;21(23):90 Q1/2; AIS 1,123 ****************************************************************************************** * People ****************************************************************************************** Group Leader prof. MUDr. Stanislav Mičuda, Ph.D. [ URL "https://is.cuni.cz/webapps/whois2/osoba/1577193 lang=cs"]   Postdoctoral researcher MUDr. Jolana Schreiberová, Ph.D. [ URL "https://is.cuni.cz/webapps/whois2/osoba/1915158588 Current Ph.D. students Ing. Hana Laštůvková [ URL "https://is.cuni.cz/webapps/whois2/osoba/1174039587878625/?lang Fatemeh Alaei Faradonbeh, M.Sc. [ MAIL "alaeifaf(zavinac)lfhk.cuni.cz"] Technical assistants Bc. Kristýna Trubačová [ URL "https://is.cuni.cz/webapps/whois2/osoba/1369384408339123/?la ****************************************************************************************** * Methods and equipment ****************************************************************************************** The laboratories of LivPharm group at the Department of Pharmacology of Charles University Medicine are well equipped to enable in vivo and in vitrostudies of drug/endobiotic kineti together with detailed analysis of obtained samples by common analytical, biochemical and methods. Instruments • Quant Studio-7 qPCR (Thermo Fisher Scientific, San Jose, USA) for qRT-PCR nucleic acid a • Simple WES automatic analyser and/or Bio-Rad electrophoretic and transblot units togethe chemiluminiscent analyser Fusion Solo S  (Vilber, France) for Western blot protein analy • Agilent 1260 Infinity II HPLC system (Palo Alto, CA, USA); • IVIS Spectrum (Perkin Elmer) in vivo imaging system, Vetscan II biochemical analyser, in anesthesia, syringe pumps, heated platforms and temperature controls forin vivo experime • Spark microplate reader with CO 2module (Tecan, Grödig, Austria) for biochemical and in vitrocellular analyses; • Microscope BX51 (Olympus) with imageJ (National Institutes of Health, Bethesda, MD; http rsweb.nih.gov/ij/) and NIS software analysis (Laboratory Imaging, Czech Republic) for hi evaluation. • Standard hematological and biochemical analyses of blood and serum samples from rats are routine methods in Central laboratories of University Hospital using Sysmex XE-2100 anal Kobe, Japan) and Modular PP analyzer (Roche, Basel, Switzerland), respectively. • LC-MS analyses are performed at cooperating Department of Biochemistry on a HPLC system 3000 (Dionex Softron GmbH, Germany) and a triple quadrupole mass spectrometer TSQ Quantu with H-ESI II probe (Thermo Fisher Scientific, Inc., USA) Methods • Animal studies. The kinetics of endogenous or exogenous substances is analyzed using rat sampling of blood, bile, urine, and stool and by harvesting the tissues at appropriate t Liver pharmacodynamics of current or potential therapeutic agents is studied on appropri models of frequently ocuring disorders such as nonalcoholic fatty liver disease or chole pregnancy. All animals received humane care in accordance with the guidelines set by the Animal Use and Care Committee of Charles University, Faculty of Medicine in Hradec Kralo Republic. The protocols of experiments are dicussed and approved by the same committee. • To study liver impairment we analyse proper bioindicators in plasma, perform liver histo and immunohistological stainings, detect liver concentrations of reduced and oxidized gl cholesterol and bile acids. BA concentrations in plasma, bile and stool are measured usi method.  • Based on study, we analyze 30-50 genes (by qRT-PCR) and proteins (by Western blot) to un enzymes, transporters, and their regulators implemented in lipidomics, and bile producti • In vitro experiment are performed using HepaRG and HepG2 cells ****************************************************************************************** * Cooperation ****************************************************************************************** • Assoc. prof. Otta Kučera, M.D. Ph.D., Prof. Zuzana Červinkova, M.D., Ph.D., Prof. Milan Ph.D., Department of Physiology, Faculty of Medicine in Hradec Kralove, Charles Universi Simkova 870, Hradec Kralove 50003, Czech Republic • Prof. Jaroslav Mokrý, M.D. Ph.D.,Department of Histology and Embryology, Faculty of Medi Kralove, Charles University in Prague, Simkova 870, Hradec Kralove 50003, Czech Republic • Assoc. Prof. Marian Kacerovsky, M.D., Ph.D., Department of Obstetrics and Gynecology, Fa Medicine in Hradec Kralove, Charles University in Prague, University Hospital Hradec Kra 581, Hradec Kralove 50005, Czech Republic • Jiri Grim, M.D., Ph.D., Department of Oncology, Faculty of Medicine in Hradec Kralove, C University in Prague, University Hospital Hradec Kralove, Sokolska 581, Hradec Kralove 5 Republic • Prof. Petr Pavek, PharmD., Ph.D.,Department of Pharmacology and Toxicology, Faculty of P Hradec Kralove, Charles University in Prague, Heyrovskeho 1203, Hradec Kralove 50005, Cz • Prof. Petr Nachtigal, PharmD., Ph.D., Department of Biological and Medical Sciences, Fac in Hradec Kralove, Charles University in Prague, Heyrovskeho 1203, Hradec Kralove 50005, • Prof. Frantisek Staud, PharmD., Ph.D.,Department of Pharmacology and Toxicology, Faculty Hradec Kralove, Charles University in Prague, Heyrovskeho 1203, Hradec Kralove 50005, Cz • Prof. Libor Vítek, M.D., Ph.D., Ústav lékařské biochemie a laboratorní diagnostiky 1. LF • Petra Hirsova, PharmD., Ph.D., Mayo Clinic, Rochester, USA • Prof. Mathieu Vinken, Department of Toxicology, Faculty of Medicine and Pharmacy, Vrije Brussel, Laarbeeklaan 103, 1090 Brussels, Belgium • Nathan Cherrington, PhD, ATS, Department of Pharmacology and Toxicology, University of A AZ, 85721, USA. ****************************************************************************************** * Research funding ****************************************************************************************** Time period     Title of the grant (number)                          Grant agency              2019-2021 Novel interventions for the treatment of triglycerideGAČRholesterol and bile acids diseases with human CAR ligands (19-14497S) 2018-2020 Role of iron depletion and Mrp2 deficiency for develoGAUKt of estrogen- induced cholestasis (556218) 2018-2020 Effect of cardiovascular drugs on the development of GAUKalcoholic steatohepatitis (346218) 2018-2021 PERSONMED - The centre for personalized medicine deveMŠMT EDRFn age- dependend disorders  (CZ.02.1.01/0.0/0.0/16_048/0007441) 2017-2019 Dynamics of nuclear receptor-mediated gene regulationGAČRlement for the understanding of detoxification functions and optimization of therapy (GA17-06841S) 2015-2017 Tissue and soluble endoglin and their importance in eGAČRhelial dysfunction and atherogenesis in vivo and in vitro (GA15-24015S) 2012-2015 Increasing of the R&D capacity at Charles University MŠMT-OP VVV position for graduates of doctoral studies (Postdoci II UK) - CZ.1.07/2.3.00/30.0061 2012-2014 Breast cancer pathophysiology and its modulation by pIGAt anticancer agents boldine and alpha-tomatine (NT13473) 2009-2011 The study of potential importance of epigallocatechinGAUKlate in the prevention and treatment of the liver injury caused by intrahepatic and extrahepatic cholestasis (116807) 2008-2010 The study of the potential therapeutic importance of GAUKastatin in the treatment of the liver injury caused by acute and chronic extrahepatic cholestasis and biliary cirrhosis in rats. (122408). 2005-2009 COST B.25.10 (1P05OC062): Physiologically based prediCOSTn of pharmacokinetics prior to in vivo studies: Focus on hepatic and renal elimination.  2005-2007 Cytochrome P450 3A4 and multidrug resistance proteinsGAUK1 and MRP2 as a major cause of drug-drug interactions at the level of drug elimination from organism (92/2005/C) 2002-2004 Evaluation of changes in MRP2 expression on the canalGAUKar membrane of hepatocytes and their influence on liver methotrexate elimination (89/2002/C/LFHK) 2000-2005 COST B.15.30. Prediction of metabolic drug-drug interCOSTons based on in vitro experiments 1997-1999 In vivo evaluation of CYP3A4 activity (57/1997/C) GAUK