Mimicking Oxidative Metabolism “OxMet” using ROXY EC System ASMS 2015 St. Louis, MO, USA

Outline I. Introduction - Drug metabolism – what you should know - Case Study 1 – Acetaminophen - Case Study 2 – Amodiaquine - Examples from Industry II. 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 Adapted from 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 Chroamtography 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

Conclusions EC/MS in Drug Metabolism ionization techniques: ESI (+/-) API, APCI weeks vs. seconds 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 25