Mimicking Drug Metabolism using ROXY EC System ASMS 2018 San Diego, CA, USA

Outline Introduction Drug metabolism – what you should know Case Study 1 – Acetaminophen Case Study 2 – Amodiaquine Examples from Industry Conclusions The lecture will explore on-line EC/MS as a powerful technique to investigate various oxidation and reduction processes in life sciences.

Drug/Xenobiotic Metabolism Many drugs metabolize in the liver by Cytochrome P450 enzymes Drug metabolism consist of two phases: Phase 1: oxidation, reduction, hydrolysis Phase 2: conjugation (adduct formation)

Techniques used in Drug Metabolism 1. Test animals (rodents)  in-vivo 2. Microsomes  in-vitro Rat Liver Microsomes (RLM) Human Liver Microsomes (HLM) 3. Electrochemical mimicking (= simulation)  in-electro

Typical Reactions Catalyzed by Cytochrome P450 Simulated by EC Allylic and aliphatic hydroxylation Possible at high potentials (only with ROXY) Benzylic hydroxylation Possible Desalkylation of amines Desalkylation of ethers and thioethers Ethers: Possible Tioethers: not mimicked Hydroxylation of aromatics Epoxide formation Possible (?) Oxidation of heteroatoms (N, S) R-CH2-CH3 R-CH-CH3 OH N-CH2-R NH + R-CHO R-O-CH2-R R-OH + RCHO O C=C R C C HO R C NH R C+ N R O- H W. Lohmann et al. LC/GC Jan. 2010 5

Case Study 1: Acetaminophen Paracetamol Panadol Tylenol APAP

Metabolism of Acetaminophen Phase 1 Phase 2

Instrumental Set-up Phase 1 Oxidative Metabolism (OxMet) NAPQI

Positive vs. Negative ESI-MS of NAPQI positive ESI D. Tsikas, J.of Chrom. B, 879 (2011) 1476-1484

MS Voltammogram Acetaminophen 800 mV used for oxidation

Instrumental Set-up Phase 2 Conjugation with Glutathione (GSH) NAPQI-GSH NAPQI

Conjugation (Adduct Formation) 0 mV NAPQI-GSH NAPQI 800 mV

Zoom in: Mass Range 445 - 490 0 mV 800 mV

Case Study 2: Amodiaquine Camoquin Flavoquine

Metabolism of Amodiaquine

Mimicking Amodiaquine Metabolism by EC/MS Cell OFF 400mV 1200mV Metabolites 2 & 3 Metabolite 1 Amodiaquine

MS Voltammogram Amodiaquine Met1 Met2 Met3

Conjugation with GSH Met1-GSH Met2-GSH

Examples form Industry

Biotransformation Pathway of Verapamil LC/MS after EC oxidation and via incubation of HLM and RLM

Biotransformation Pathway of Verapamil Molecular formula of Verapamil and it metabolites after EC oxidation and incubation with HLM and RLM

Biotransformation Pathway of Verapamil Conclusions Structure of electrochemically generated metabolites were elucidated on basis of accurate mass data and MS/MS experiments All of the metabolic products of Verapamil incl. Norverapamil can be easily simulated using this purely instrumental technique Furthermore newly reported metabolic reactions products like carbinolamines or imine methides become accessible EC/LC/MS is a powerful tool in drug discovery and development providing faster and often more comprehensive results than in vivo approaches S. Jahn et al., J. of Chromatography A, 1218 (2011) 9210-9220

Generation of Statin Drug Metabolites Simvastatin & Lovastatin (blockbusters)

less than 1 mg of drug = three oxidative metabolites of each parent simvastatin less than 1 mg of drug = three oxidative metabolites of each parent complete NMR characterization was performed. For 3’α hydroxy SV complete 1HNMR first time ever! the electrochemical approach gave higher yields than the enzymatic oxidations lovastatin Bluckbusters > 1 Billion, enzymatic variants Anal. Bioanal. Chem. (2013) 405: 6009-6018

ThP141, 10:30 am - 2:30 pm

> 18 Publications using ROXY™ EC to Mimic Drug Metabolism (last 15 months)  Mechanism of aromatic hydroxylation of lidocaine at a Pt electrode under acidic conditions T Gul, R Bischoff, HP Permentier - Electrochimica Acta, 2017 - Elsevier … 2.2. Electrochemical measurements. Constant potential measurements were performed with an Antec ROXY potentiostat (Antec Leyden, Zoeterwoude, The Netherlands) controlled by Antec Dialogue software. Cyclic voltammetry … Electrochemical simulation of biotransformation reactions of citrinin and dihydroergocristine compared to UV irradiation and Fenton-like reaction J Keller, H Haase, M Koch - Analytical and bioanal. chem, 2017 - Springer … μm spacers. An electrochemical potentiostat ROXY (Antec Leyden) was used to apply a potential ramped between … Electrochemical oxidation of phosphatidylethanolamines studied by mass spectrometry S Colombo, G Coliva, A Kraj, J. of Mass …, 2018 - Wiley Online Library … The EC reactions were conducted in the ROXY EC system (Antec Scientific, Zoeterwoude, NL) consisting of the ROXY potentiostat for the control of the flow-through μ-PrepCell 2.0 … Structural characterization of electrochemically and in vivo generated potential metabolites of selected cardiovascular drugs by EC-UHPLC/ESI-MS using an … M Szultka-Młyńska, S Bajkacz, I Baranowska… - Talanta, 2018 - Elsevier … containing an electrochemical cell ReactorCell™, infusion pump and all the necessary HPLC connections (ROXY™ system Antec Leyden,.. Electrochemistry coupled online to liquid chromatography-mass spectrometry for fast simulation of biotransformation reactions of the insecticide chlorpyrifos TF Mekonnen, U Panne, M Koch - Analytical & bioanal. chem, 2017 - Springer … Mass voltammograms were generated by Roxy ™ potentiostat (Antec Leyden, Zoeterwoude, The Netherlands) equipped with μPrepCell ™…. Prediction of biotransformation products of the fungicide fluopyram by electrochemistry coupled online to liquid chromatography-mass spectrometry and comparison … TF Mekonnen, U Panne, M Koch - Analytical and bioanalytical chemistry, 2018 - Springer … Mass voltammogram of FLP was recorded (continuous scan, 10 mV/s) using a Roxy™ potentiostat (Antec Leyden, Zoeterwoude) ... Investigations of the Copper‐Catalyzed Oxidative Cross‐Coupling of Tetrahydroisoquinolines with Diethylzinc by a Combination of Mass Spectrometric and … JA Willms, H Gleich, M Schrempp;  A European Journal, 2018 - Wiley … Voltage/ion intensity curves were obtained with a commercial electrochemical flow cell [Antec Scientific ROXY potentiostat and … Hydroxylation and dimerization of zearalenone: comparison of chemical, enzymatic and electrochemical oxidation methods J Keller, H Haase, M Koch - World Mycotoxin Journal, 2017 … Oxidation of zearalenone by EC/MS, Fenton-like reaction and Ce(IV) was pumped through the µPrepCell by using a SP2-ROXY dual piston syringe pump (Antec Leyden) and a flow rate of 80 µl/min …   Laccase-and electrochemically mediated conversion of triclosan: Metabolite formation and influence on antibacterial activity E Jahangiri, B Seiwert, T Reemtsma, D Schlosser - Chemosphere, 2017 – Elsevier … 2.4. Electrochemical (EC) conversion of TCS. TCS conversion was performed on a ROXY Potentiostat electrochemical system (Antec,..  Oxidation of 4-Isopropylphenol in Aqueous Solutions as a Model for Oxidation of Flavonoids to Form Dimers IG Zenkevich, TI Pushkareva - Chemistry of Natural Comp., 2018 Springer … Electrochemical oxidation of 4-isopropylphenol (1) (c 50–100 Pg/mL) was conducted in a mixture of aqueous NH 4 OAc (pH ~7.4) and MeCN (3:1) in a ROXY electrochemical cell (Antec, Leyden, … Untargeted screening of phase I metabolism of combretastatin A4 by multi-tool analysis K Jaroch, PZ Goryńska, K Goryński, T Stefański… - Talanta, 2018 – Elsevier … ROXY™ system. All electrochemical experiments mimicking metabolism reactions in liver microsomes were conducted via the ROXY™ EC system (Antec) connected on-line to the high resolution mass … Analysis of Protein-Phenolic Compound Modifications Using Electrochemistry Coupled to Mass Spectrometry C Kallinich, S Schefer, S Rohn - Molecules, 2018 - mdpi.com … Exemplarily, the electrochemical oxidation of chlorogenic acid was optimized by varying the voltage from 0.3 V to 3 V in steps of 0.3 V using a boron-doped diamond electrode and a Roxy ®potentiostat (Antec Scientific, Zoeterwoude, The Netherlands) …

Conclusions EC/MS in Drug Metabolism Fast: minutes vs. weeks No isolation/clean-up Phase I and II metabolism Complementary to in vivo and in vitro Saving rodents (mice, rats) Safety (no exposure to biohazard samples)  EC/MS powerful tool for biomimetic oxidation 27