NMR in Drug Metabolism Extraction NMR - Keeping up with the Pace of Drug Discovery. Dr Ute Gerhard

Transcription

1 F NMR in Drug Metabolism Extraction NMR - Keeping up with the Pace of Drug Discovery Dr Ute Gerhard

2 The Early Days Eat plant Feel Worse Feel Better Try again Drug!

3 The Golden Age Make molecule Feed to rats Rats OK Rats not OK Drug!

4 Today Lots of money and commitment to basic research? No Target known? Yes Do we have a lead? Add to screening set and try again It s OK Drug! Yes Physiology, biochemistry, pharmacology: Work out new drug target It doesn t work Assay How on Earth did you get here? Clinical trials Spend lots of It s not OK Drat. Is the problem fixable? No Wait for research to be done It works well It s OK Yes No Make more compounds Pharmacokinetics Chewed up by liver Excreted by kidneys Yes It s not OK Not absorbed Why not? Try and fix this Found something Not brain penetrant Found something No Test screening set Combichem! Kills rats on contact Tastes horrible Found nothing Found nothing You lose. Shareholders unhappy. Start again!

5 DMPK in Drug Discovery Lead identification Preliminary PK & major metabolic routes Optimize leads for affinity/efficacy Provide feedback on DMPK parameters Select best compound Profile compound: in vivo profile in higher species P450 induction/inhibition etc.etc. Identify metabolite structures whenever possible by LC-MS (QToF for highest sensitivity) Scale up remaining metabolites in best species (in vivo/vitro) & identify structures (NMR/MS)

6 NMR in Metabolism studies Why? Detailed structural details Ideal for metabolite structures NMR signal is directly proportional to concentration Ideal for quantitative studies But Relatively poor sensitivity compared to mass spectrometry Slow Needs a lot of clean sample

7 NMR in Metabolism studies Metabolite structure identification Isolation-NMR, LC-MS-NMR, Extraction-NMR All standard 1D and 2D experiments Metabolic routes, improve microsomal stability etc. Excretion studies ( 19 F NMR): Metabolite tracking in biofluids (in the absence of a radiolabel) Kinetic studies (time course of 1D or 2D spectra) Glucuronide migration rates

8 A typical DM NMR sample Samples are typically obtained from in vitro incubations (liver microsomes, CYP450) or in vivo experiments (bile, urine, faeces etc.) The compound of interest may be contained in a complex biomatrix -> extraction procedures Compared with chemistry samples, we may not have much sample sample sizes of less than 1 μg are not unusual -> scale up for NMR experiments is required

9 The good news NMR spectrum of parent is usually available, and fully assigned for both 13 C and 1 H We know the mass of the metabolite Potentially other relevant information from LC-MS data (e.g. polarity, fragmentation) What do we want to learn? Where has oxidation, conjugation, or rearrangement taken place? Put another way, how does the metabolite s NMR spectrum differ from that of parent? For many drug metabolites, a clean 1D proton NMR spectrum is sufficient to identify the structure

10 The challenge NMR sensitivity Advances in NMR technologies allows rapid 1D & 2D data acquisition on much smaller sample sizes Higher fields Cryo probes and improved probe design Improved electronics Speed of analysis Structural results provided in a time window relevant to the lead optimisation

11 Extraction-NMR

12 Protocol for in vitro incubation and extraction Systems: hepatocytes, S9, microsomes, cytosol 50 or 100 µm incubation (+ NADPH) LC-MS profiling: are the significant metabolites present? Extraction: Liquid/liquid or SPE Solvent and ph Evaporate solvent and re-dissolve LC-NMR or prep HPLC NMR

13 Protocol for in vitro incubation and extraction Systems: hepatocytes, S9, microsomes, cytosol 50 or 100 µm incubation (+ NADPH) LC-MS profiling: are the significant metabolites present? Extraction: Liquid/liquid or SPE Solvent and ph Evaporate solvent and re-dissolve in d 4 -methanol NMR of crude mixture: Extraction-NMR If structure can be determined - no further work If not - continue with conventional isolation techniques LC-NMR or prep HPLC NMR

14 Proton spectrum: sample after extraction of incubate The region below 5.5 ppm is obstructed by the residual biological matrix Surprisingly clear in the aromatic region Predominantly useful where aromatic regions of a molecule are metabolised

15 Extraction-NMR: 1 H with 19 F decoupling A5 A6 A4 N O A2 NH 2 A2 A6 A4 B2 A5 B5 B6 B6 B5 B4 B4 R N H B2 F C2 C6 C5 C6 C5 OH R N H C2 F

16 NADPH O N N O P OH OH OH HO OH O NH 2 H 2 N N N O O OH P O O - P O O N O O

17 Extraction-NMR - 19 F spectrum The 19 F spectrum can be a diagnostic tool provided hydroxylation occurs in the vicinity of the fluorine atom -> strong influences on the 19 F chemical shift Proton couplings are difficult to resolve due to the nature of the sample Parent Metabolite

18 Extraction-NMR - 1D Me R Parent R' METABOLITE O Microsomal extract? N NH 2

19 Extraction-NMR COSY Me R R' HO R R'

20 Extraction-NMR TOCSY Cresset s European User Group meeting

21 Extraction-NMR - NOESY R N R O N + Cresset s European User Group meeting

22 Proton NMR of a rat bile sample extract * ethyl acetate: extraction solvent * * *

23 19 F NMR of extracted bile sample * Parent Major metabolite # Minor metabolites F2 F2 # # # # #

24 1 H- 19 F COSY of extracted bile sample F' 3' 4' F to H3 R 6 5' F F F to H3 4 3 F R' F' 3' 4' F to H6 F to H6 R HO 6 3 F R' 5'

25 Why determine metabolite structures? Potentially toxic metabolites

26 The power of accurate mass measurement M+16 [M+O?] % M+34 M+14 [M+O-2H?] parent endogenous Time 10 μm incubation in Rhesus monkey liver microsomes: LC-MS trace

27 The power of accurate mass measurement M M+O % M M+ H 2 O 2 M M+HO 2 -F parent endogenous Time 10 μm incubation in Rhesus monkey liver microsomes: LC-MS trace

28 Extraction NMR: 19 F NMR Scale up to from 100 µm incubation in Rhesus monkey liver microsomes: Extraction NMR: 1 H NMR spectrum very complex Parent R F Initial conclusions: Two fluorine containing metabolites One is probably hydroxylated next to fluorine

29 The structures Analytical scale HPLC separation 1D proton and 2D NMR data on isolated peaks R F OH OH R OH R OH! M+34 M+14 M+16 F OH

30 Oxidative stress and drug metabolism Reactive metabolites commonly formed under oxidative (P450) or reductive processes Biological systems are rich in nucleophiles Covalent binding to proteins critical for function Formation of protein-protein crosslinks Immunological response via antigen formation Br Br CYP450 O Covalent binding to tissue macromolecules Hepatic necrosis

31 Phase II metabolism Defense mechanism exists for dealing with electrophiles Reaction with the thiol nucleophile glutathione (GSH) Tripeptide -Cys-Glu-Gly - found at 10 mm intracellularly Mediated by the enzyme glutathione-s-transferase (GST) Detoxication step Br Br Br epoxide glutathione OH OH hydrolase O S-transferase OH Glutathione Glutathione conjugation (Phase II metabolism) can be saturated

32 Summary NMR does provides invaluable information through precise metabolite structures The real challenge in determining the structures of metabolites is the nature of the samples Technologies such as cryo probes, LC-MS-NMR, caplc-nmr etc. are key tools to make NMR an invaluable tool in drug metabolism The detection limits for state-of-the-art NMR systems are now where LC-MS was a decade ago and things will only get better