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Agnieszka Potęga, PhD

Electrochemical simulation: a powerful tool for the elucidation and study of drug metabolism reactions. Agnieszka Potęga, PhD. e-mail: agnieszka.potega@pg.gda.pl. Chemical Faculty. Department of Pharmaceutical Technology and Biochemistry

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Agnieszka Potęga, PhD

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  1. Electrochemical simulation: a powerful tool for the elucidation and study of drug metabolism reactions. Agnieszka Potęga, PhD e-mail: agnieszka.potega@pg.gda.pl ChemicalFaculty Department of Pharmaceutical Technology and Biochemistry Laboratory of Chemistry and Biochemistry of AnticancerDrugs 3rd INTERNATIONAL SUMMIT ON TOXICOLOGY & APPLIED PHARMACOLOGY Chicago, USA, October 20 – 22, 2014

  2. Electrochemical simulation: a powerful tool for the elucidation and study of drug metabolism reactions. Keywords: antitumorcompounds, livercellmicrosomes, cytochrome P450, oxidativedrugmetabolism, electrochemicalsimulation, mass spectrometry 3rd INTERNATIONAL SUMMIT ON TOXICOLOGY & APPLIED PHARMACOLOGY Chicago, USA, October 20 – 22, 2014

  3. Degradationpathway of xenobiotics Phase-I metabolism(activation): oxidationreactions (oftencatalysed by enzymes of the cytochrome P450 superfamily), reduction and hydrolysisreactions(intermediatemetabolites – more polar, less lipophilic) Xenobiotics(nonpolar, lipophilic) Phase-II metabolism(detoxification): enzyme-catalysedconjugationreactions withsmall polarendogenouscompoundsliketripeptideglutathione, glucuronicacid, sulfate, or glycine (metabolites – polar, water-soluble, easilyexcreted from the human body in urineor bile) 3rd INTERNATIONAL SUMMIT ON TOXICOLOGY & APPLIED PHARMACOLOGY Chicago, USA, October 20 – 22, 2014

  4. In vivo and in vitro methods, usinglaboratoryanimals, isolatedlivercells (hepatocytes) ormicrosomes, containingactiveenzymes, i.a. the cytochrome P450 system. laboratoryanimals livercellmicrosomes 3rd INTERNATIONAL SUMMIT ON TOXICOLOGY & APPLIED PHARMACOLOGY Chicago, USA, October 20 – 22, 2014

  5. Cytosol: dehydrogenases oxidases SULTs GSTs NATs Hepatocyte S9 In vivo and in vitro methods, usinglaboratoryanimals, isolatedlivercells (hepatocytes) ormicrosomes, containingactiveenzymes, i.a. the cytochrome P450 system. Microsomes: CYPs FMOs UGTs GSTs Distribution of drugmetabolisingenzymes in livercells. CYP, cytochrome P450 monooxygenase; FMO, flavin-dependent monooxygenase; UGT, UDP-glucuronosyltransferase; GST, glutathionetransferase; SULT, sulfotransferase; NAT, N-acetyltransferase. • Laborious and timeconsuming. • Metabolitesmay form adducts with the cellmatrix, henceisolation and identification of reactivemetabolitesishampered. • Variations in the expression of cytochrome P450 isoforms in eachorganismhave to be takenintoaccount. 3rd INTERNATIONAL SUMMIT ON TOXICOLOGY & APPLIED PHARMACOLOGY Chicago, USA, October 20 – 22, 2014

  6. EC/MS – a powerful platform to simulatevariousoxidation and reductionprocesses. • Electrochemicalcell for the simulation of the oxidative and reductivephase-I metabolism. • High potential for screening of single target compounds in phase-II metabolomicsstudies. • Fast method for directidentificationof reactivemetabolitesbecause of the absence of biologicalmatrices. • Delivers a redoxfingerprint of a (drug) molecule, close to metabolic profile, in a veryshorttime. 3rd INTERNATIONAL SUMMIT ON TOXICOLOGY & APPLIED PHARMACOLOGY Chicago, USA, October 20 – 22, 2014

  7. Instrumental set-up of EC system EC/MS – a powerful platform to simulatevariousoxidation and reductionprocesses. potentiostat • Electrochemicalcell for the simulation of the oxidative and reductivephase-I metabolism. • High potential for screening of single target compounds in phase-II metabolomicsstudies. • Fast method for directidentificationof reactivemetabolitesbecause of the absence of biologicalmatrices. • Delivers a redoxfingerprint of a (drug) molecule, close to metabolic profile, in a veryshorttime. electrochemicalthin-layercell (EC) – reactorcell • high speed • simplicity • ease of automation • low-costinstrumentation 3rd INTERNATIONAL SUMMIT ON TOXICOLOGY & APPLIED PHARMACOLOGY Chicago, USA, October 20 – 22, 2014 dual syringe infusion pump mass spectrometer (MS)

  8. Simulation in anamperometricthin-layercell EC/MS – a powerful platform to simulatevariousoxidation and reductionprocesses. Pd/H2 • Electrochemicalcell for the simulation of the oxidative and reductivephase-I metabolism. • High potential for screening of single target compounds in phase-II metabolomicsstudies. • Fast method for directidentificationof reactivemetabolitesbecause of the absence of biologicalmatrices. • Delivers a redoxfingerprint of a (drug) molecule, close to metabolic profile, in a veryshorttime. • lowadsorption on the electrodesurface • easilyexchangeableworkingelectrode 3rd INTERNATIONAL SUMMIT ON TOXICOLOGY & APPLIED PHARMACOLOGY Chicago, USA, October 20 – 22, 2014

  9. Electrochemicalreaction EC/MS – a powerful platform to simulatevariousoxidation and reductionprocesses. • Electrochemicalcell for the simulation of the oxidative and reductivephase-I metabolism. • High potential for screening of single target compounds in phase-II metabolomicsstudies. • Fast method for directidentificationof reactivemetabolitesbecause of the absence of biologicalmatrices. • Delivers a redoxfingerprint of a (drug) molecule, close to metabolic profile, in a veryshorttime. ΔE 3rd INTERNATIONAL SUMMIT ON TOXICOLOGY & APPLIED PHARMACOLOGY Chicago, USA, October 20 – 22, 2014

  10. Set-upsof the most commonapproaches for the simulation of drugmetabolismreactions EC/MS – a powerful platform to simulatevariousoxidation and reductionprocesses. A EC/MS system Drug • Electrochemicalcell for the simulation of the oxidative and reductivephase-I metabolism. • High potential for screening of single target compounds in phase-II metabolomicsstudies. • Fast method for directidentificationof reactivemetabolitesbecause of the absence of biologicalmatrices. • Delivers a redoxfingerprint of a (drug) molecule, close to metabolic profile, in a veryshorttime. Potentiostat Potentiostat B EC/LC/MS system EC cell EC cell MS detector MS detector Drug LC column Adapted from Baumann et al., Expert Opin DrugMetabToxicol, 2010 3rd INTERNATIONAL SUMMIT ON TOXICOLOGY & APPLIED PHARMACOLOGY Chicago, USA, October 20 – 22, 2014

  11. Typicalphase I reactions and the possibility for theirelectrochemicalsimulation EC/MS – a powerful platform to simulatevariousoxidation and reductionprocesses. • Electrochemicalcell for the simulation of the oxidative and reductivephase-I metabolism. • High potential for screening of single target compounds in phase-II metabolomicsstudies. • Fast method for directidentificationof reactivemetabolitesbecause of the absence of biologicalmatrices. • Delivers a redoxfingerprint of a (drug) molecule, close to metabolic profile, in a veryshorttime. Adapted from Lohmann et al., LC-GC Europe, Jan 2010 3rd INTERNATIONAL SUMMIT ON TOXICOLOGY & APPLIED PHARMACOLOGY Chicago, USA, October 20 – 22, 2014

  12. Typicalphase I reactions and the possibility for theirelectrochemicalsimulation EC/MS – a powerful platform to simulatevariousoxidation and reductionprocesses. • Electrochemicalcell for the simulation of the oxidative and reductivephase-I metabolism. • High potential for screening of single target compounds in phase-II metabolomicsstudies. • Fast method for directidentificationof reactivemetabolitesbecause of the absence of biologicalmatrices. • Delivers a redoxfingerprint of a (drug) molecule, close to metabolic profile, in a veryshorttime. Adapted from Lohmann et al., LC-GC Europe, Jan 2010 3rd INTERNATIONAL SUMMIT ON TOXICOLOGY & APPLIED PHARMACOLOGY Chicago, USA, October 20 – 22, 2014

  13. Financial support by the National Science Centre (Poland) (project SONATA No 2012/07/D/NZ7/03395) • Investigationphase-I metabolism of the model antitumoracridinederivatives, C-1311 and C-1748,under electrochemical conditions coupled to mass spectrometry (EC/MS). • Comparisonthe results obtained by EC/MS with conventional in vitromicrosomalapproach. 3rd INTERNATIONAL SUMMIT ON TOXICOLOGY & APPLIED PHARMACOLOGY Chicago, USA, October 20 – 22, 2014

  14. differentstructures • variousmetabolicpathways • inhibitor of bothtopoisomerases and certain receptor kinases, including FMS-liketyrosinekinase FLT3 (Chau et al., 2006; Skwarska et al., 2010) • activityagainstadvanced solid tumorsunderphase I and II of clinicaltrials, alsotested for the treatment of autoimmunediseases(Capizzi et al., 2008; Isambert et al., 2010) • strongcytotoxicactivityagainst colon cancer cel lines (HCT8 and HT29) (Augustin et al., 2010) and high antitumoractivityagainstseveralprostate(LnCaP, JCA, PC3, TSU) and colon carcinoma xenografts (HCT8) in nude mice (Chen et al., 2002; Tadi et al., 2007) • lowmutagenicpotential and slightmyelosuppressiveproperties(Narayanan et al., 2005) 5-diethyloaminoethyloamino-8-hydroxyimidazoacridinone C-1311 9-(2’-hydroxyethyloamino)-4-methylo-1-nitroacridine C-1748 3rd INTERNATIONAL SUMMIT ON TOXICOLOGY & APPLIED PHARMACOLOGY Chicago, USA, October 20 – 22, 2014

  15. Table:Gradient profile utilized for all HPLC analyses. A:50 mMaqueousammoniumformatebuffer(pH3.4)+5% methanol for HPLC B: Methanol for HPLC+5% H2O • RP-HPLC-DAD/ESI-MS analysis A B Figure:Linearchromatograms of the reactionmicturescontaining0.05 mM(A) C-1311 (B) C-1748, 1 mMNADPH and 2 mg/mLmicrosomal(S9) fractionof livercellsin sodiumphosphatebuffer(pH7.4). 3rd INTERNATIONAL SUMMIT ON TOXICOLOGY & APPLIED PHARMACOLOGY Chicago, USA, October 20 – 22, 2014

  16. Figure: C-1311 abundance vs. electrosynthesistime. 3rd INTERNATIONAL SUMMIT ON TOXICOLOGY & APPLIED PHARMACOLOGY Chicago, USA, October 20 – 22, 2014

  17. Figure: The proposedchemicalstructuresof C-1311metabolitesobtained in enzymatic(—) and electrochemical(—) system([ ]* – intermediateproduct). 3rd INTERNATIONAL SUMMIT ON TOXICOLOGY & APPLIED PHARMACOLOGY Chicago, USA, October 20 – 22, 2014

  18. Figure: C-1748 abundance vs. electrosynthesistime. 3rd INTERNATIONAL SUMMIT ON TOXICOLOGY & APPLIED PHARMACOLOGY Chicago, USA, October 20 – 22, 2014

  19. Figure: The proposedchemicalstructures of C-1748metabolitesobtained in enzymatic(—) and electrochemical(—) system. 3rd INTERNATIONAL SUMMIT ON TOXICOLOGY & APPLIED PHARMACOLOGY Chicago, USA, October 20 – 22, 2014

  20. Electrochemical system is very well-suited for the simulation of the oxidative and reductivemetabolism of antitumoracridinederivatives. • Electrochemical conversion of C-1311 and C-1748 into phase-I metabolites was successfully achieved. • Twomain metabolic products of C-1311 (side chain degradation products) and three products of C-1748 were detected both in the conventional enzymatic approach and in the electrochemical simulation. • Anadditionalproduct of C-1748 (1-aminoacridone) was foundonly in the electrochemicalsimulation. 3rd INTERNATIONAL SUMMIT ON TOXICOLOGY & APPLIED PHARMACOLOGY Chicago, USA, October 20 – 22, 2014

  21. The purelyinstrumentalapproach, based on electrochemicalconversion, is a feasible alternative to the conventionalmicrosomal studies. 3rd INTERNATIONAL SUMMIT ON TOXICOLOGY & APPLIED PHARMACOLOGY Chicago, USA, October 20 – 22, 2014

  22. Application areas of electrochemistry/mass spectrometry reduction of disulfidebridges coupling EC/MS oxidative protein marking cleaving of peptidebonds oxidative DNA demage activesubstance- protein binding MS signalamplification stabilitystudies metabolitesynthesis metabolismstudies 3rd INTERNATIONAL SUMMIT ON TOXICOLOGY & APPLIED PHARMACOLOGY Chicago, USA, October 20 – 22, 2014

  23. Electrochemical simulation: a powerful tool for the elucidation and study of drug metabolism reactions. Zofia Mazerska1, AssocProf Weronika Hewelt-Belka2, MSc Olga Siewruk1 Katarzyna Zapała1 ChemicalFaculty 1Department of Pharmaceutical Technology and Biochemistry 2Department of AnalyticalChemistry 3rd INTERNATIONAL SUMMIT ON TOXICOLOGY & APPLIED PHARMACOLOGY Chicago, USA, October 20 – 22, 2014

  24. Thankyou for yourattention!

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