Research projects Publications People

Methods and equipment Cooperation Research funding

The objective is to study liver pharmacodynamics and pharmacokinetics of actual or potential drugs. We focus on executive and regulatory mechanisms of bile production and secretion (enzymes and transporting proteins) and their impairment by various liver diseases. Modulation of these mechanisms exerts significant physiological and pathophysiological consequences including changes in elimination of drugs. Consequent variability in PK/PD of co-administered agents may complicate therapeutic outcomes. Our purpose is therefore to study such changes and to introduce appropriate prophylactic and therapeutic measures. In particular we are working on nonalcoholic fatty liver disease, liver regeneration and cholestatic liver disorders characterized by impaired bile secretion due to extrahepatic biliary obstruction or due to intrahepatic influence of toxic insult like bacterial LPS or ethinylestradiol. Using animals and cellular models of these pathophysiological situations, we study protective effects of drugs modifying activity of PXR, CAR and FXR nuclear receptors, and naturally occurring anti-inflammatory compounds like boldine or EGCG. Furthermore, systemic pharmacokinetics of drugs and xenobiotics is studied in rats including bioavalability, body distribution, metabolism, biliary and urinary excretions.

Research projects

Experimental therapy of Nonalcoholic steatohepatitis, and Intrahepatic cholestasis of Pregnancy

The aim of this branch of research is to study liver effects (pharmacodymacs) of current or potential drugs in common liver disorders. Attention is focused on nonalcoholic steatohepatitis (NASH) as a common disease accompanying numerous metabolic and cardiovascular disorders, and intrahepatic cholestasis (ICP), the most frequent liver disorder during pregnancy. We combine appropriate in vivo models of these situations with detailed analysis of involved regulatory and executive molecules in order to identify dose-response relationship, and responsible mechanisms. Equally important is also description of effects of all such agents in healthy animals because these drugs are frequently used also in clinical situations without NASH and ICP.

Modulation of bile acid homeostasis, and bile production

Bile production is an essential function of the liver and serves as an irreplaceable excretory pathway for elimination of lipophilic endo- and xenobiotics such as cholesterol, BA, bilirubin or drugs. Moreover, as major components of bile, BA are required for micelle formation, intestinal fat digestion, regulation of bacterial growth, and immune response and production of regulatory mediators released to portal circulation such as fibroblast growth factor 19 or glucagon-like peptide 1. In addition, BA as the major metabolites of cholesterol, act as hormones by agonism at several receptors such as farnesoid X receptor (FXR), the G protein-coupled bile acid receptor 1 (TGR5), sphingosine-1-phosphate receptor 2, or pregnane X receptor (PXR), and regulate numerous liver functions including glucose and triglyceride metabolism. Stimulation of these receptors demonstrates promising positive effects in liver diseases such as nonalcoholic steatohepatitis (NASH) or intrahepatic cholestasis. On the other hand, BA accumulated during different forms of cholestasis may have a direct toxic effect on liver cells and tissues. Regulation of bile production and BA homeostasis are therefore key events in liver physiology and pathophysiology. Numerous regulatory events exist in whole process, especially nuclear receptors FXR, PXR, CAR, VDR, as well as feedback and inflammatory signaling triggering mitogen-activated kinases, or directly interacting with promotor sequences as we have recently detected for iron-mediated changes. The aim of this branch of research is therefore the identification of regulatory mechanisms of BA homeostasis and bile production, and characterization of possible pharmacological modulation.

This part is solved in cooperation with the group of prof. PharmDr. Petr Pavek, Ph.D. and prof. PharmD. Petr Nachtigal, Ph.D. from Faculty of Pharmacy in Hradec Kralove.

Drug pharmacokinetics in preclinical studies

Knowledge of pharmacokinetic characteristics of evaluated drugs, i.e. absorption, distribution, metabolism, and excretion is an essential prerequisite for successful pharmacotherapy with minimum of adverse reactions and drug-drug interactions. We therefore focus on evaluation of kinetic parameters including clearance, biliary and urinary secretion, volume of distribution, biological half-life and bioavailability in current or developing drugs. Essential approach is the usage of suitable in vivo and in vitro models together with advanced analytical and mathematical methods.

Recent Publications

• Alaei Faradonbeh F, Lastuvkova H, Cermanova J, Hroch M, Nova Z, Uher M, Hirsova P, Pavek P, Micuda S. Multidrug Resistance-Associated Protein 2 Deficiency Aggravates Estrogen-Induced Impairment of Bile Acid 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, Grøntved L, Braeuning A, Pavek P. Off-target lipid metabolism disruption by the mouse constitutive androstane receptor ligand TCPOBOP in humanized mice. Biochem Pharmacol. 2021 Dec 28;197:114905. doi: 10.1016/j.bcp.2021.114905. IF = 5,858; Q1; AIS 1,144

• Faradonbeh FA, Sa II, Lastuvkova H, Cermanova J, Hroch M, Faistova H, Mokry J, Nova Z, Uher M, Nachtigal P, Pavek P, Micuda S. Metformin impairs bile acid homeostasis in ethinylestradiol-induced cholestasis in mice. Chem Biol Interact. 2021 May 28:109525. doi: 10.1016/j.cbi.2021.109525. IF = 5,192; Q2; AIS 0,728

• Lastuvkova H, Faradonbeh FA, Schreiberova J, Hroch M, Mokry J, Faistova H, Nova Z, Hyspler R, Igreja Sa IC, Nachtigal P, Stefela A, Pavek P, Micuda S. Atorvastatin Modulates Bile Acid Homeostasis in Mice with Diet-Induced Nonalcoholic Steatohepatitis. Int J Mol Sci. 2021 Jun 16;22(12):6468. IF = 5,923; Q2; AIS 1,123

• Vicen M, Igreja Sá IC, Tripská K, Vitverová B, Najmanová I, Eissazadeh S, Micuda S, Nachtigal P. Membrane and soluble endoglin role in cardiovascular and metabolic disorders related to metabolic 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, Kudova E, Pavek P. (E)-7-Ethylidene-lithocholic Acid (7-ELCA) Is a Potent Dual Farnesoid X Receptor (FXR) Antagonist and GPBAR1 Agonist Inhibiting FXR-Induced Gene Expression in Hepatocytes and Stimulating Glucagon-like Peptide-1 Secretion From Enteroendocrine Cells. Front Pharmacol. 2021 Aug 13;12:713149. IF = 5,811; Q1; AIS 1,139

• Igreja Sá IC, Tripska K, Hroch M, Hyspler R, Ticha A, Lastuvkova H, Schreiberova J, Dolezelova E, Eissazadeh S, Vitverova B, Najmanova I, Vasinova M, Pericacho M, Micuda S, Nachtigal P. Soluble Endoglin as a Potential Biomarker of Nonalcoholic Steatohepatitis (NASH) Development, Participating in Aggravation of NASH-Related Changes in Mouse Liver. Int J Mol Sci. 2020 Nov 27;21(23):9021. IF = 5,923; Q1; AIS 1,123

• Smutny T, Dusek J, Hyrsova L, Nekvindova J, Horvatova A, Micuda S, Gerbal-Chaloin S, Pavek P. The 3'-untranslated region contributes to the pregnane X receptor (PXR) expression down-regulation by PXR ligands and up-regulation by glucocorticoids. Acta Pharm Sin B. 2020 Jan;10(1):136-152. doi: 10.1016/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, Gerbal-Chaloin S, Pavek P. Diazepam Promotes Translocation of Human Constitutive Androstane Receptor (CAR) via Direct Interaction with the Ligand-Binding Domain. Cells. 2020 Nov 24;9(12):2532. IF = 6,600; Q2; AIS 1,282

• Stefela A, Kaspar M, Drastik M, Holas O, Hroch M, Smutny T, Skoda J, Hutníková M, Pandey AV, Micuda S, Kudova E, Pavek P. 3β-Isoobeticholic acid efficiently activates the farnesoid X receptor (FXR) due to its epimerization to 3α-epimer by hepatic metabolism. J Steroid Biochem Mol Biol. 2020 Jun 4:105702. IF = 4,292; Q2; AIS 0,905

• Igreja Sá IC, Tripska K, Hroch M, Hyspler R, Ticha A, Lastuvkova H, Schreiberova J, Dolezelova E, Eissazadeh S, Vitverova B, Najmanova I, Vasinova M, Pericacho M, Micuda S, Nachtigal P. Soluble Endoglin as a Potential Biomarker of Nonalcoholic Steatohepatitis (NASH) Development, Participating in Aggravation of NASH-Related Changes in Mouse Liver. Int J Mol Sci. 2020 Nov 27;21(23):9021. IF = 5,923; Q1/2; AIS 1,123

People

Group Leader

prof. MUDr. Stanislav Mičuda, Ph.D.

Postdoctoral researcher

MUDr. Jolana Schreiberová, Ph.D.

Current Ph.D. students

Ing. Hana Laštůvková

Technical assistants

Bc. Kristýna Trubačová

Methods and equipment

The laboratories of LivPharm group at the Department of Pharmacology of Charles University, Faculty of Medicine are well equipped to enable in vivo and in vitrostudies of drug/endobiotic kinetics and dynamics together with detailed analysis of obtained samples by common analytical, biochemical and molecular biology methods.

Instruments

• Quant Studio-7 qPCR (Thermo Fisher Scientific, San Jose, USA) for qRT-PCR nucleic acid analyses;

• Simple WES automatic analyser and/or Bio-Rad electrophoretic and transblot units together with chemiluminiscent analyser Fusion Solo S  (Vilber, France) for Western blot protein analysis;

• Agilent 1260 Infinity II HPLC system (Palo Alto, CA, USA);

• IVIS Spectrum (Perkin Elmer) in vivo imaging system, Vetscan II biochemical analyser, inhalation anesthesia, syringe pumps, heated platforms and temperature controls forin vivo experiments;

• 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 histological evaluation.

• Standard hematological and biochemical analyses of blood and serum samples from rats are performed by routine methods in Central laboratories of University Hospital using Sysmex XE-2100 analyzer (Sysmex, Kobe, Japan) and Modular PP analyzer (Roche, Basel, Switzerland), respectively.

• LC-MS analyses are performed at cooperating Department of Biochemistry on a HPLC system Dionex Ultimate 3000 (Dionex Softron GmbH, Germany) and a triple quadrupole mass spectrometer TSQ Quantum Access Max with H-ESI II probe (Thermo Fisher Scientific, Inc., USA)

Methods

• Animal studies. The kinetics of endogenous or exogenous substances is analyzed using rats or mice by sampling of blood, bile, urine, and stool and by harvesting the tissues at appropriate time points. Liver pharmacodynamics of current or potential therapeutic agents is studied on appropriate animal models of frequently ocuring disorders such as nonalcoholic fatty liver disease or cholestasis during pregnancy. All animals received humane care in accordance with the guidelines set by the institutional Animal Use and Care Committee of Charles University, Faculty of Medicine in Hradec Kralove, Czech 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 histological and immunohistological stainings, detect liver concentrations of reduced and oxidized glutathione, cholesterol and bile acids. BA concentrations in plasma, bile and stool are measured using the LC-MS method. 

• Based on study, we analyze 30-50 genes (by qRT-PCR) and proteins (by Western blot) to uncover changes in enzymes, transporters, and their regulators implemented in lipidomics, and bile production. 

• 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 Holecek, M.D., Ph.D., Department of Physiology, Faculty of Medicine in Hradec Kralove, Charles University in Prague, Simkova 870, Hradec Kralove 50003, Czech Republic

• Prof. Jaroslav Mokrý, M.D. Ph.D.,Department of Histology and Embryology, Faculty of Medicine in Hradec 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, Faculty of Medicine in Hradec Kralove, Charles University in Prague, University Hospital Hradec Kralove, Sokolska 581, Hradec Kralove 50005, Czech Republic

• Jiri Grim, M.D., Ph.D., Department of Oncology, Faculty of Medicine in Hradec Kralove, Charles University in Prague, University Hospital Hradec Kralove, Sokolska 581, Hradec Kralove 50005, Czech Republic

• Prof. Petr Pavek, PharmD., Ph.D.,Department of Pharmacology and Toxicology, Faculty of Pharmacy in Hradec Kralove, Charles University in Prague, Heyrovskeho 1203, Hradec Kralove 50005, Czech Republic

• Prof. Petr Nachtigal, PharmD., Ph.D., Department of Biological and Medical Sciences, Faculty of Pharmacy in Hradec Kralove, Charles University in Prague, Heyrovskeho 1203, Hradec Kralove 50005, Czech Republic

• Prof. Frantisek Staud, PharmD., Ph.D.,Department of Pharmacology and Toxicology, Faculty of Pharmacy in Hradec Kralove, Charles University in Prague, Heyrovskeho 1203, Hradec Kralove 50005, Czech Republic

• Prof. Libor Vítek, M.D., Ph.D., Ústav lékařské biochemie a laboratorní diagnostiky 1. LF UK a VFN

• Petra Hirsova, PharmD., Ph.D., Mayo Clinic, Rochester, USA

• Prof. Mathieu Vinken, Department of Toxicology, Faculty of Medicine and Pharmacy, Vrije Universiteit Brussel, Laarbeeklaan 103, 1090 Brussels, Belgium

• Nathan Cherrington, PhD, ATS, Department of Pharmacology and Toxicology, University of Arizona, Tucson, AZ, 85721, USA.

Research funding