Mass Spectrometry as the Premier Analytical Tool in Drug Discovery and Drug Development - PowerPoint PPT Presentation

colleen-bernard
mass spectrometry as the premier analytical tool in drug discovery and drug development n.
Skip this Video
Loading SlideShow in 5 Seconds..
Mass Spectrometry as the Premier Analytical Tool in Drug Discovery and Drug Development PowerPoint Presentation
Download Presentation
Mass Spectrometry as the Premier Analytical Tool in Drug Discovery and Drug Development

play fullscreen
1 / 88
Download Presentation
Mass Spectrometry as the Premier Analytical Tool in Drug Discovery and Drug Development
557 Views
Download Presentation

Mass Spectrometry as the Premier Analytical Tool in Drug Discovery and Drug Development

- - - - - - - - - - - - - - - - - - - - - - - - - - - E N D - - - - - - - - - - - - - - - - - - - - - - - - - - -
Presentation Transcript

  1. Mass Spectrometry as the Premier Analytical Tool in Drug Discovery and Drug Development Walter Korfmacher Exploratory Drug Metabolism Merck Research Laboratories Kenilworth, NJ USA GBMSDG Talk

  2. Outline • New Drug Discovery Challenges • Mass Spectrometry Basics • Selected In vitro Drug Metabolism Applications • Selected In vivo Drug Metabolism Applications • Metabolite ID Applications • MS Imaging Applications • Conclusions GBMSDG Talk

  3. Topics Not Covered • Proteomics • Biomarker discovery or assay • Metabolomics • High Throughput Screening October 14, 2010 GBMSDG Talk

  4. Drug Discovery:From Library to Market Lead Optimization FDA Approval Clinical Testing Early Discovery Safety Testing Compound Libraries Lead Selection and Optimization Clinical Approved Drug Development Number of compounds Stages of Discovery and Development GBMSDG Talk October 14, 2010

  5. NEW DRUG DISCOVERY PIPELINE Chemistry Biology--HTS for Receptor Activity In-vitro Stability Screen DMPK Lead Optimization In-vitro Absorption Screen P450 Enzyme Inhibition Screen CARRS Oral PK Screen Rat IV / PO PK Dog and Monkey IV / PO PK Metabolite ID Rising Dose and Multiple Dose Studies and Safety Screens Drugs into Development GBMSDG Talk

  6. The Challenge • How to deal with the multiple compounds at multiple stages in the drug discovery/drug development pipeline. GBMSDG Talk

  7. The Solution • LC-MS and LC-MS/MS GBMSDG Talk

  8. Why use Mass Spectrometry? • Specificity! Non-specific techniques, such as UV and fluorescence, are unable to provide proof of the analyte identity • Ease of Use! Modern mass spectrometry software interfaces are easy to use • Versatility! MS is both a qualitative and quantitative technique GBMSDG Talk

  9. The Challenge • Choosing the right tool for the task : OR Wrench? MS Toolbox Hammer? GBMSDG Talk

  10. Common MS Tools GBMSDG Talk

  11. What is LC-MS? Liquid Chromatography Coupled to a Mass Spectrometer (In this case the Mass Spectrometer is a Single Quadrupole instrument) HPLC Column Ion source Mass analyzer Detector APCI or ESI GBMSDG Talk

  12. The Challenge—Compound Synthesis • At a big Pharma site, one might find hundreds of medicinal chemists who might produce 200-1000 new compounds each week. These have to be assayed. GBMSDG Talk

  13. The Solution—LC-MS • The LC-MS system based on a single quadrupole MS is a very useful tool for medicinal chemists who want to know if their synthesis is working correctly—did they make the right compound? Often this is set up as an open access tool. The chemists set up the run and get results within 24 hours. GBMSDG Talk

  14. The Challenge—Lead Optimization In Vitro Screening • At a big Pharma site, one might get 100-200 new compounds each week that have to be screened in various in vitro assays. These have to be assayed separately for each screen. GBMSDG Talk

  15. The Solution—LC-MS/MS • The LC-MS/MS system based on a triple quadrupole MS system is the tool of choice for most quantitative discovery bioanalytical applications. The application of this tool varies with the screen. GBMSDG Talk

  16. What is LC-MS/MS aka Triple Quadrupole Technology? Liquid Chromatography Coupled to a Tandem Mass Spectrometer (In this case the Mass Spectrometer is a Triple Quadrupole instrument) Q1 Q2 Q3 HPLC Column Ion source Mass analyzer Detector GBMSDG Talk

  17. Quantitation Gold Standard:MS/MS – Selected Reaction Monitoring (SRM)* Select Fragment Select precursor ion in Q1 precursor ion in Q2 product ion in Q3 e.g. m/z 216 (Collision Cell) e.g. m/z 174 * Often referred to as Multiple Reaction Monitoring (MRM) GBMSDG Talk

  18. Effects of Stages of Analysis on Signal, Noise, and Signal-to-Noise Important Concept!! S/N Signal Noise 1 2 3 4 LC LC-MS LC-MS/MS ? GBMSDG Talk Stages of Analysis

  19. LC-TIC LC-MS GBMSDG Talk

  20. LC-MS/MS 10 ng/ml GBMSDG Talk

  21. Discovery In Vitro Screening • P450 Assay--Enzyme Inhibition Screen • Caco-2 cells—Absorption Screen • Liver Microsomes/Hepatocytes--Metabolic Stability Screen • Plasma Protein Binding Each In Vitro Assay Uses LC-MS/MS for the analytical step (typically a triple quadrupole MS system). GBMSDG Talk

  22. High Throughput CYP Inhibition Assay Example • Generic LC-MS/MS method • 1 minute gradient • Monitors 3 substrates in a single LC-MS/MS run • Template is used for creating sample list • Automatic results calculation and import into Activity Base GBMSDG Talk

  23. Method: Method: P450 source P450 source P450 source human liver microsomes human liver human liver incubation incubation incubation cocktail ( cocktail 3A4, 2D6, 2C9) substrates substrates substrates testosterone testosterone testosterone Dextromethorphan Dextromethorphan Dextromethorphan Tolbutamine Tolbutamine Tolbutamine 3A4 3A4 3A4 2C9 2C9 2C9 2D6 2D6 2D6 b b b products products products 6 6 6 - - - hydroxytestosterone hydroxytestosterone hydroxytestosterone dextrophan dextrophan dextrophan 4 4 4 - - - hydroxytobutamide hydroxytobutamide hydroxytobutamide detection detection detection LC LC LC - - - MS MS MS - - - MS MS MS In Vitro Evaluation of CYP Inhibition Purpose: Evaluate direct and mechanism-based inhibitors for P450 enzymes (3A4, 2D6, 2C9) to assess potential for drug-drug interactions. GBMSDG Talk

  24. In Vitro Evaluation of CYP Inhibition incubation/pre-incubation Stock solution from CDC Serial dilution 1. Coincunation 2. Coincubation • three concentrations/cpd: 20 mM, 2 mM and 0.2 mM • duplicates/each conc. • 30 compounds/set 3. Preincubation 4. Preincubation GBMSDG Talk

  25. LC-MS/MS for p450 Inhibition Screen-Run time is less than 1 minute Substrate for CYP3A4, 6-hydroxytestosterone m/z 305  269 Substrate for CYP2D6, dextrophan m/z 258  157 Internal Standard m/z 347  121 Substrate for CYP2C9, 4-hydroxytolbutamide m/z 287  171 GBMSDG Talk

  26. CYP Inhibition Screen Throughput •  Throughput: ~ 150 compounds /week, ~ 7000 samples/week •  Analytical cycle-time: 48 hr from delivery to results • NOTE: The advantage for this assay is that it is the same regardless of the test compound—no compound method development needed. GBMSDG Talk

  27. CYP Inhibition Screen Review GBMSDG Talk

  28. The Challenge— Metabolic Stability Screening • At a big Pharma site, one might get 100-200 new compounds each week that have to be screened for metabolic stability. • The challenge is that LC-MS/MS methods have to be developed for each compound. GBMSDG Talk

  29. The Solution—LC-MS/MS + Software + Hardware • Use a generic HPLC method. This works for 80-90% of the compounds. • Use a vendor-supplied software tool for automated MS/MS method development, e.g.: • QuickQuan™ (Thermo-Fisher) • QuanOptimise™ (Waters-Micromass) • DiscoveryQuant™ (AB-Sciex) • OptimizerTM (Agilent) • Use robots for automated sample handling GBMSDG Talk

  30. Metabolic Stability Assay Example GBMSDG Talk

  31. Metabolic Stability Assay Layout shows how a robotic liquid handler can be used to perform the incubation and sample preparation steps in a metabolic stability assay. GBMSDG Talk

  32. Metabolic Stability Assay Scheme shows how a well organized system is needed to provide high throughput metabolic stability data. GBMSDG Talk

  33. In Vivo Assays • Various types of in vivo assays are performed as part of new drug discovery and development • The goal may be to understand absorption (A), distribution (D), metabolism (M), or excretion (E) properties of a compound • The goal may be to get pharmacokinetic (PK) information on a compound GBMSDG Talk

  34. ADME-PK Studies Brain-- D Drug Levels— LC-MS/MS MS Image— MALDI-MS/MS Plasma— A Drug Levels— LC-MS/MS PK Parameters Dose NCE (Drug) PO/IV Liver— D Drug Levels— LC-MS/MS Ref: “Using Mass Spectrometry for Drug Metabolism Studies” W. Korfmacher, ed., CRC Press, 2005. ASMS 2010

  35. The Challenge— In Vivo PK Screening • At a big Pharma site, one might get 50 - 100 new compounds each week that have to be screened for in vivo PK. • The challenge is that LC-MS/MS methods have to be developed for each compound. GBMSDG Talk

  36. The Solution—LC-MS/MS + Software + Planning • Use a generic HPLC method. This works for 80-90% of the compounds. • Use a vendor-supplied software tool for automated MS/MS method development. • Use robots for automated sample handling. • Develop a standard PK screening assay. GBMSDG Talk

  37. In Vivo PK Screening Source: Drug Discovery Today Volume 13, Numbers 7/8 April 2008 Authors: Bo Liu, Jonathan Chang, William P. Gordon, John Isbell, Yingyao Zhou and Tove Tuntland, Department of Pharmacology, Genomics Institute of the Novartis Research Foundation (GNF), San Diego, USA GBMSDG Talk

  38. PK Screening Example: CARRS • Provide basic pharmacokinetic information for all rapid rat compounds. • AUC (0-6hr) • Concentration vs Time Profile (0-6 hr) Throughput: Up to 96 compounds per week GBMSDG Talk

  39. CARRS ASSAY • Protein Precipitation Sample Preparation • Generic UPLC conditions (1-2 min run time) • Triple Quadrupole MS for assay (Two-point standard curve) • Automated MS method development (QuanOptimize) GBMSDG Talk

  40. Preclinical PK Studies • Typical study is one compound dosed oral (PO) and IV (Intravenous) in a laboratory animal. The goal is to get PK parameters in various preclinical species. • Typically this produces 50-60 plasma samples. • Sample preparation is protein precipitation. • A multipoint standard curve is prepared for the assay • Analysis is by LC-MS/MS on a triple quadrupole MS/MS system. Usually a generic internal standard is used for the assay. GBMSDG Talk

  41. Discovery PK Analysis Flowchart The MS/MS instrument is normally a triple quadrupole system GBMSDG Talk

  42. Samples SRM Fail OK LC-MS/MS test run (PP Sample prep.) #1: Matrix effect Revised Chromatography #2: Interference : LLE Enhanced mass resolution SPE #3: standard curve linearity Non-routine options Assay Rapid MS Method Development ion a Discovery Environment Xu et al. Anal. Chem.--2005 GBMSDG Talk October 14, 2010

  43. Typical Discovery PK Assay Response ratio 1 – 10,000 ng/mL GBMSDG Talk

  44. Discovery Metabolite ID • Generally this has two components: • Lead Optimization Phase--would use unlabelled compounds and in vitro samples to look for major routes of metabolism. • Pre-Recommendation Phase—Look for problem metabolites plus in vitro comparison of human to animal metabolism. GBMSDG Talk

  45. Discovery Metabolite ID • Mass Spectrometry serves two purposes: • Finding metabolites-MS systems can be used in various ways to find metabolites in multilple biological matrices (e.g., plasma, bile, urine). • Structure elucidation—MS systems can be used to to obtain partial or complete structural identification for the metabolites GBMSDG Talk

  46. Triple Quad Scan Functions:to find metabolites • Neutral Loss Scan • no prior knowledge of the parent is required—this is used to look for certain classes of metabolites (e.g., glucuronide, sulfate or glutathione conjugates) • Precursor Ion Scan • only fragmentation pattern of parent is required—may find unexpected metabolites • SRM/MRM • the fragmentation pattern of parent is used to predict the fragment ions for likely metabolites—some vendors have software tools that make it easy to build a scan set GBMSDG Talk

  47. MS Tools • Triple Quadrupole MS systems are the premier analytical tool for LC-MS quantitative assays. They are also useful for metabolite ID applications • Q-TOF MS systems are best used for metabolite ID applications and for Imaging MS applications • QTrap MS systems are excellent tools for quantitative analyses as well as for metabolite ID applications GBMSDG Talk

  48. QTrap MS Publication In Vivo PK Samples • Simultaneously quantifying parent drugs and screening for metabolites in plasma pharmacokinetic samples using selected reaction monitoring information-dependent acquisition on a QTrap instrument: Li et al. (Covance), RCMS, 1943, 2005. GBMSDG Talk

  49. Mass Spectrometer Sample from in vivo or in vitro studies 10-50% Injector HPLC 50-90% Radiometric Flow detector 100 100 80 80 60 60 40 40 20 20 0 0 0 0 5 5 10 10 15 15 20 20 25 25 30 30 35 35 40 40 45 45 Time (min) Time (min) Radiocarbon Chromatogram LC/MS Chromatogram Typical Metabolite Profiling Experiments and Instrumentation Radioactivity helps to locate the metabolites in the samples October 14, 2010 GBMSDG Talk

  50. Product ion spectrum of a particular compound m1+ m2+ m2+ m2+ Product Ion Scan Select Precursor Ion Scan Products Fragmentation A key technique for obtaining structural information. GBMSDG Talk