Waters ASMS Users Meeting May 30 th, Overview

Transcription

1 Application of accurate mass spectrometry to mechanistic studies of metabolismdependent inhibition of cytochrome P45 enzymes Joanna Barbara, Ph.D. XenoTech Waters AM 29 Users Meeting May 3, 29 verview Inhibition of drug metabolizing enzymes Action Implications Cytochrome P45, CYP, enzyme inhibition Alkylamine CYP inhibition application Elucidating mechanism of CYP3A4 inhibition by UPLC/QTF-M metabolite profiling with a novel reversibility sample prep approach 2

2 Reactive metabolites and enzyme inhibition Xenobiotic metabolism usually detoxifying, but sometimes chemically reactive metabolites form e.g. electrophiles, free radicals Metabolism-dependent inhibition can be a specific consequence of formation of some types of reactive metabolites Metabolite binds to metabolizing enzyme and alters it chemically or functionally Clinical implications e.g. drug-drug interactions, mortality Compound development implications e.g. development halt, market withdrawal Mechanistic understanding can help direct compound design to minimize this behavior 3 verview of cytochrome P45 (CYP) enzyme inhibition CYP inhibition Parent drug-protein Metabolite-protein Direct inhibition (reversible) Metabolism-dependent inhibition, MDI (irreversible) More potent direct inhibition e.g. fluoxetine and norfluoxetine Irreversible covalent inhibition e.g. mibefradil Quasi-irreversible noncovalent inhibition e.g. troleandomycin (metabolite-intermediate complex, MIC) 4

3 MDI of CYP3A4 by Posicor (mibefradil), withdrawn 1998 Measure effect of pre-incubation with the test article on the in vitro IC 5 of CYP3A4 marker substrate 1 zero-min preincubation 3 min preincubation Half maximal inhibitory concentration μm 5.5 μm [Mibefradil] (µm) 5 Validated GLP-compliant LC/M/M quantitation (Applied Bio API2) Metabolism-dependent CYP3A4 inhibition by troleandomycin Midazolam 1'-hydroxylation ( Control) 1 Half maximal inhibitory concentration o cofactor.8 μm 3 min preincubation o preincubation 9.5 μm [Troleandomycin] (µm) 6

4 Irreversible MDI of CYP3A4 by mibefradil Reversibility assay incorporating chemical oxidation and ultracentrifugation can distinguish irreversible and quasi-irreversible MDI CYP3A4 activity by formation of 6β-hydroxytestosterone (LC/M/M) Testosterone 6β-Hydroxylation (pmol/mg/min) PI- min PI-3 min Reversibility assay yields no return of enzyme activity 7 o mibefradil Mibefradil Mibefradil + K 3 Fe(C) 6 (resuspended) PI = Preincubation Quasi-irreversible MDI of CYP3A4 by troleandomycin Testosterone 6β-Hydroxylation (pmol/mg/min) Reversibility assay yields return of enzyme activity PI- min PI-3 min o troleandomycin Troleandomycin Troleandomycin + K 3 Fe(C) 6 (resuspended) 8 PI = Preincubation

5 2 and 3 alkylamines and MIC formation MIC formation has been attributed to interaction of a nitroso group introduced to the structure by CYP metabolism nly supported by indirect evidence UV/vis detection of MIC Formaldehyde assays tructural modification & MIC Metabolite profiling and characterization by mass spectrometry can be used to find direct evidence of metabolites and intermediates 9 Dansette et al Adv. Exp. Med. Biol. 197: Macrolide antibiotics Alkylamine test articles C H 3 C H 3 H H H H H CH3 CH3 Troleandomycin Erythromycin 1

6 Diltiazem and metabolites Alkylamine test articles H 3 C H 3 C H 3 C H H CH 3 Diltiazem -Desmethyl diltiazem -Desacetyl diltiazem 11 Metabolism-dependent CYP3A4 inhibition summary All show evidence of metabolism-dependent inhibition of CYP3A4 Test article Troleandomycin Erythromycin Diltiazem -Desmethyl diltiazem -Desacetyl diltiazem IC 5 (μm) o preincubation 3 min preincubation Fold shift ~12 ~5 ~3 ~4 ~3 12

7 Troleandomycin metabolite-intermediate complex (MIC) analysis MIC formation involving the ferrous heme iron can be detected spectrophotometrically 455 nm oret peak HLM Time 1 μm TA 1 mg/ml protein ph 7.4, 37 C 8 min incubation.32 μm MIC formed 13 All test articles showed evidence of MIC involving ferrous heme iron Waters ynapt M metabolite profiling analytical workflow Incubation of drug with in vitro test system Protein precipitation Acquity UPLC separation/ynapt (QTF) detection using M E 1 μm drug;, 3 min; HLM; ADPH 1 min: 1-9 B; water & AC (formic acid); Waters T3 column Positive EI; V & centroid mode; M E and M/M (CE ramp) MetaboLynx X data processing for biotransformation assignment M/M acquisition if necessary Compound-directed mass defect filtering & M E time alignment ± 5 mda; C-heteroatom dealkylation MDF 14 tructural elucidation for biotransformation routes By spectral changes from known parent drug

8 Macrolide antibiotic biotransformation summary Biotransformation dominated by -demethylation and ester hydrolysis -Demethylation -Dealkylation Hydrolysis C H 3 H H H H H CH3 xidation at multiple sites but not on alkylamine 15 o evidence of nitroso or intermediates Diltiazem and metabolite biotransformation summary Biotransformation dominated by -demethylation and ester hydrolysis H 3 C H 3 C H 3 C -Demethylation -Demethylation Hydrolysis H H xidation at multiple sites but not on alkylamine 16 o evidence of nitroso or intermediates

9 ample preparation for metabolite profiling Incubation followed by protein precipitation Drug Aliquot at time-point Add organic stop Transfer supernatent Buffer, ph 7.4 ADPH cofactor Human liver microsomes, HLM PI LC/M 37 C Controls: minute, no cofactor Does inhibitory metabolite precipitate out bound to the protein? 17 Reversibility assay with metabolite profiling Transfer ICE & PI + BUFFER ample should contain proteinbound inhibitory metabolite Incubate 3 min Met ID Treat with FeK 3 (C) 6 Protein precipitation o treatment Protein precipitation AMPLE should contain free inhibitory metabolite CTRL should not contain inhibitory metabolite 18 UPLC/accurate M/M UPLC/accurate M/M

10 ormal diltiazem metabolic profile in human liver microsomes 1 Diltiazem MDF chromatogram minute Desmethyl diltiazem Di--desmethyl diltiazem -Desmethyl--desmethyl diltiazem Diltiazem MDF chromatogram minute Reversibility assay MDF chromatograms for diltiazem 1 -Desmethyl diltiazem 5.4 Di--desmethyl diltiazem -Desmethyl--desmethyl diltiazem 5.1 Diltiazem Unknown: 8 min o treatment Desmethyl diltiazem Di--desmethyl diltiazem -Desmethyl--desmethyl diltiazem 5.12 Diltiazem Unknown: 8 min FeK 3 (C)

11 Low energy M E spectrum for diltiazem inhibitory metabolite 1 Parent diltiazem [M+H] + [X + H] m/z = Δ 413 = amu (2H?) Δ 44 = amu 21 [X - HC + H] [X + a] m/z [X + K] M/M spectrum for diltiazem e L 6 Δ m = [M + H] m/z L = eutral loss

12 M/M spectrum for diltiazem inhibitory metabolite e L 6 Δ m = [M + H] C - source? m/z C - adduct? L = eutral loss Reversibility assay MDF chromatograms for -desmethyl diltiazem 1 p _ ( ) 5.4 -Desmethyl diltiazem TIC 2.27e4 Di--desmethyl diltiazem -Desmethyl--desmethyl diltiazem Unknown: 8 min 4.9 o treatment RD791 Desmeth_Dilt_3Apr9_7_MDF_35 b (1,4. ) 1: TF M E+ 1 TIC e4 -Desmethyl diltiazem Di--desmethyl diltiazem -Desmethyl--desmethyl diltiazem Unknown: 8 min FeK 3 (C) 6 Time

13 Low energy M E spectrum for -desmethyl diltiazem inhibitory metabolite 1 [X + H] Identical to diltiazem inhibitory metabolite 25 [X - HC + H] [X + a] m/z [X + K] M/M spectrum for -desmethyl diltiazem inhibitory metabolite e L 6 Δ m = [M + H] m/z C - adduct?

14 Reversibility assay MDF chromatograms for -desacetyl diltiazem 1 -Desacetyl diltiazem 4.5 TIC 1.93e4 -Desacetyl--desmethyl diltiazem -Desacetyl--desmethyl diltiazem Unknown: 7.5 min o treatment RD791_-Desacet_Dilt_3Apr9_7_MDF_35 b (1,4. ) 1: TF M E+ 1 TIC e4 -Desacetyl diltiazem -Desacetyl--desmethyl diltiazem -Desacetyl--desmethyl diltiazem Unknown: 7.5 min 27 FeK 3 (C) Time Low energy M E spectrum for -desacetyl diltiazem inhibitory metabolite 1 [Y + H] e3-42 from diltiazem inhibitory metabolite Y = X acetyl M/M data indicate modification of alkylamine 28 [Y - HC + H] + [Y + a] + [Y + K] m/z

15 Inhibitory metabolite structure Diltiazem metabolite -Desmethyl diltiazem metabolite Inhibitory metabolite forms by modification of alkylamine Identical metabolite forms with mono and di methylamine 29 Inhibitory metabolite structure C 21 H C [M+H] + m/z th = m/z ex = Δ e = 89.3 ppm Isocyanate metabolite? Mass error is high; unlikely structure Trace inhibitory metabolite in untreated sample: acetonitrile adduct? 3

16 Accurate mass elemental composition data for m/z Compound specific element limits, 5 ppm tolerance, DBE Elemental composition Theoretical m/z Mass error (ppm) i-fit score C 23 H C 24 H C 24 H C 23 H C 21 H C 22 H C 19 H C 2 H C 19 H C 2 H Inhibitory metabolite structure C 23 H H + -[C 2 H 3 ] = C 21 H Change from diltiazem: - CH 4 2 Methyleneamine metabolite Detected species Does this explain why in literature 1 amines do not form MIC? Could blocking this group prevent metabolism-dependent inhibition? 32

17 Conclusions Metabolite profiling of this test group revealed no evidence of nitroso metabolite formation The reversibility assay with LC/M/M metabolite profiling can be used for identification of quasi-irreversible inhibitory metabolites Diltiazem metabolism-dependent inhibition of CYP3A4 may be due to formation of a methyleneamine metabolite Assess di--desmethyl diltiazem for evidence that methyl group is necessary for metabolism-dependent inhibition Revisit the macrolide antibiotics (stronger interaction?) Dr. Andrew Parkinson Dr. David Buckley Brian gilvie Phyllis Yerino Faraz Kazmi Mark Horrigan Dr. Paul Toren eema Muranjan Kammie ettle Tim Blaski Acknowledgments

18 End Joanna Barbara Mass pectrometry pecialist Division of Drug Metabolism and Analytical Chemistry, XenoTech 35