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Drug Discovery Programs at BMS

LC-MS in Drug Discovery Timothy V. Olah, Ph.D. Director, Bioanalytical Research Pharmaceutical Research Institute Bristol-Myers Squibb Company timothy.olah@bms.com University of Connecticut 20 February 2007. Drug Discovery Programs at BMS.

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Drug Discovery Programs at BMS

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  1. LC-MS in Drug DiscoveryTimothy V. Olah, Ph.D. Director, Bioanalytical ResearchPharmaceutical Research InstituteBristol-Myers Squibb Companytimothy.olah@bms.comUniversity of Connecticut20 February 2007

  2. Drug Discovery Programs at BMS Wallingford, CT: Virology: HIV (AIDS), Hepatitis C, CNS: Anxiety, Depression, Alzheimer’s, MigraineLawrenceville, NJ:Oncology: CancerImmunology: Rheumatoid Arthritis, AsthmaHopewell, NJ:Cardiovascular: Thrombosis, Atherosclerosis Metabolic Diseases: Diabetes, Obesity

  3. BMS Bioanalytical Research Wallingford:Deborah Barlow, Marc Browning, Mike Donegan, Daniel Morgan, Kim Snow, Sarah Taylor, Kim WidmannLawrenceville:Hollie Booth, Chris Caporuscio, Georgia Cornelius, Celia D'Arienzo, Lorell Discenza, Mohammed Jemal, Zheng Ouyang, Asoka Ranasinghe, Bogdan Sleczka, Yi Tao, Xia Yuan, Jian Wang, Steven WuHopewell:Hong Cai,Cecilia Chi, Jim Smalley, Minjuan Wang, Richard Wong, Baomin Xin, Carrie Xu, Hongwei Zhang, Joanna Zheng

  4. Bioanalytical Research Staffing • Personnel: 30 scientists • Location: WFD (7), LVL (13), HPW (9) • Mass Spectrometers : 30 MS/MS • Full Phase Programs: 34 • Early Phase Programs: 25 • Research Initiatives • Samples 1-4Q 2006: ~184,000

  5. Total Number of Samples Analyzed by BAR (FTE) in Support of Discovery

  6. BAR-Discovery Annual Sample Count

  7. Outline • LC-MS in Drug Discovery • Drug Discovery Process • Strategies and Work Flow Processes • Development and Implementation of Multiple Component LC-MS-based Bioanalytical Methods • Examples, Questions, Comments • What is of interest to you today in regards to the Pharmaceutical Industry ?

  8. Bioanalytical Research’s Goal To generate accurate and precise data in the quantitative analysis of drug discovery candidates in samples from in vivo and in vitro studies that support their biological characterization and optimization as potential development candidates.

  9. The Gilbert Bioanalytical Chronicles“The challenges of bioanalysis stem from the need to accurately and reproducibly measure part per million to part per trillion quantities of analyte in complex biological matrices, full of potentially interfering endogenous or drug-related substances.”John Gilbert et al., “High Performance Liquid Chromatography with Atmospheric Pressure Ionization Tandem Mass Spectrometry as a Tool in Quantitative Bioanalytical Chemistry,” in Biochemical and Biotechnological Applications of Electrospray Ionization Mass Spectrometry, American Chemical Society (1995)

  10. Relative Amounts 1.0 gram 0.001 gram (milligram) 0.000001 gram (microgram) 0.000000001 gram (nanogram) 0.000000000001 gram (picogram)

  11. Stages of Drug Discovery and Development Compound Discovery - Nomination PreClinical - Clinical GLP • Factors for Consideration • Bioanalytical Method Requirements • Application of Technologies • Risk Assessment

  12. Drug Discovery Process A New Chemical Entities from Medicinal Chemistry B H Physical Properties Reporting High Throughput Screening C G Bioassays: Potency / Selectivity Information Management Data Interpretation Automation D Experimental Design ADME Characteristics F Sample Analysis E In Vitro and / or In Vivo Experiments

  13. Drug Discovery Process A New Chemical Entities from Medicinal Chemistry B H Physical Properties Reporting High Throughput Screening C G Bioassays: Potency / Selectivity Information Management Data Interpretation Automation D Experimental Design ADME Characteristics F Sample Analysis E In Vitro and / or In Vivo Experiments

  14. Relationship of Drug Metabolism with Medicinal Chemistry in Early Drug Discovery • Add information to compliment HTS data • Assist in the selection of compounds based upon their ADME characteristics for further evaluation • Provide direction to medicinal chemists for the design of their next compound in a series • Identify trends in the architecture of a class of compounds that correlates with a response in an ADME assay (e.g. Potency with P450 inhibition)

  15. “Absorption, Distribution, Metabolism, Excretion” Duodenum Intestine Stomach (Excretion) (Absorption) Kidney Portal Vein Liver Systemic Circulation (Distribution) (Metabolism)

  16. Desirable ADME Characteristics • Absorption - good solubility and permeability • Distribution - good exposure at the target, minimal elsewhere - acceptable protein binding: estimate “free concentration” • Metabolism - minimal first pass effect - metabolism by two or more CYP (not 2D6) to few metabolites - minimal potential to inhibit or to induce • Excretion - balance between metabolism and excretion of parent drug

  17. Experiments to Assess ADME Characteristics • Absorption - Caco-2 cells, PAMPA, PgP-transport - in vivo PK profiling • Distribution - in vitro protein binding, in vivo tissue distribution studies • Metabolism - Metabolic stability in microsomes, S9 fractions, hepatocytes - P450 Inhibition: microsomes and/or rCYPs, co-administration - P450 Induction: Gene Chips, PXR, multiple dosing studies - Metabolite characterization • Excretion - Quantitation of drugs and metabolites in biological fluids

  18. BAR Responsibilities • Analyze samples from in vivo studies: • Early Exposure (Biology), Coarse PK, Full PK, PK/PD, Tissue Distribution, “Bioequivalence”, TK (CV Telemetry, Pre-ECN, ECN) … • Co-Administration: Screening (N-in-one), P450 Inhibition, Marker Compounds, Stable Label… • All routes of administration (IV, PO, IP, SQ, IN…) • All types of formulations by Pharmaceutics.

  19. BAR Responsibilities • Analyze samples from in vitro studies: • serum protein binding, tissue binding, serum/plasma/chemical stability… • intrinsic clearance, P450 inhibition, pgP, PAMPA, Caco-2… • biological assays, heart/liver perfusion…

  20. BAR Responsibilities • Analyze samples in all types of species and biological matrices: • mice, rats, marmoset, guinea pig, rabbit, dogs, monkeys, chimp, human… • blood, plasma, serum, urine, bile, CSF, SV, brain, lung, heart, liver, GI tract, kidney, muscle, tumor, adipose tissue… • microsomes, hepatocytes, S9 fractions, rCYP, incubation systems, mixed matrices, buffers, dosing solutions…

  21. BAR Responsibilities • Rapidly develop and implement multiple component bioanalytical methods using LC-MS/MS-based detection. • Analyte and Internal Standard • Analyte(s) and Internal Standard(s) • Co-Administration Studies, Pro-Drugs • Parent Compound, Metabolite(s), IS • Parent Compound, Distinct Equilibrium Forms

  22. Keys to Developing Successful Screening Strategies the TIME required to generate data TIME DATA RESULTS the quality of DATA required to generate meaningful results the type of RESULTS that are requested in the screen

  23. “Scaling” Annual BAR Output: Number of Compounds Evaluated • 10,000 - 30,000 evaluated in “Screens” • 1000 - 3000 evaluated in “Early Phase” • 100 - 300 evaluated in detail “Full Phase” • 10 - 30 evaluated for “Nomination” • 1 - 3 compounds “NDA” approved

  24. Bioanalytical Work Flow A Sample Receipt/Tracking B H Method Development Reporting LC-MS/MS C Information Management G Preparation of Analytical Standards & QCs Notebooks and Ancillary Data Programming Automation D Standard Operating Guidelines Sample Preparation F Data Processing E Sample Analysis

  25. What is LC-MS? (LC) Liquid Chromatography: Chromatography is a separations method that relies on differences in partitioning behavior between a flowing mobile phase and a stationary phase to separate the components in a mixture. A column holds the stationary phase and the mobile phase carries the sample through it. Sample components that partition strongly into the stationary phase spend a greater amount of time in the column and are separated from components that stay predominantly in the mobile phase and pass through the column faster. (www.chem.vt.edu/chem-ed/ac-meths.html)

  26. X time meaningful data HPLC Analysis Elution Gradient Recondition 0

  27. What is LC-MS? (MS) Mass Spectrometry: Mass spectrometers use the difference in mass-to-charge ratio (m/z) of ionized compounds to separate them from each other. Compounds have distinctive fragmentation patterns that provide structural information to specifically detect compounds. (www.chem.vt.edu/chem-ed/ac-meths.html)

  28. Types of Mass Spectrometers • LC-MS (single quadrupole) • LC-MS/MS (triple quadrupoles) • LC-Q (ion traps, linear ion traps) • LC-Q-TRAPS (quadrupole linear ion traps) • LC-TOF-MS (time-of-flight) • MALDI-TOF-MS • Q-TOF-MS (quadrupole time-of-flight) • FT-MS (Fourier Transform) • Others

  29. Uses of Mass Spectrometry in Drug Discovery • Qualitative Analysis Elucidation of the structural characteristics of various substances in different matrices: What is in the sample? • Quantitative Analysis Determination of the concentration of various substances in different matrices: How much is in the sample?

  30. Carbamazepine C15H12N2O MW = 236.1 Da. Diltiazem C22H26N2O4S MW = 414.2 Da. Reserpine C33H40N2O9 MW = 608.3 Da. Bumetanide C17H20N2O5S MW = 364.1 Da. Pyrilamine C17H23N3O MW = 285.2 Da. Ketoconazole (ISTD) C26H28N4O4Cl2 MW = 530.1 Da.

  31. Types of MS Detection Methods • Full Scan Spectra • Selected Ion Monitoring • Product Ion Spectra • Neutral Loss Scans • Multiple Reaction Monitoring • Accurate Mass Measurements • Others

  32. Sulfasalazine C18 H14 N4 O5 S MW 398.40

  33. +Q1: 5 MCA scans from sulfasalazine_FinalQ1_Pos.wiff Max. 7.4e6 cps. 399.0 7.4e6 Full Scan Spectrum 7.0e6 6.5e6 6.0e6 5.5e6 5.0e6 4.5e6 4.0e6 Intensity, cps 3.5e6 3.0e6 2.5e6 2.0e6 400.0 1.5e6 1.0e6 400.9 401.1 5.0e5 0.0 396.5 397.0 397.5 398.0 398.5 399.0 399.5 400.0 400.5 401.0 401.5 m/z, amu

  34. m/z 119 m/z 94 m/z 165 m/z 137 m/z 223 Product Ion Spectrum

  35. + ES Ionization + + + + Ar Ar + + Collision gas + + + + + + + + + Q1 – Fixed precursor m/z (m/z “x”) Q2 – Collision chamber Q3 – Fixed product m/z (m/z “y”) + + MRM Transition m/z – m/z Multiple Reaction Monitoring

  36. + ES Ionization + + + + Ar Ar + + Collision gas + + + + + + + + + Q1 – Fixed precursor m/z (m/z 399) Q2 – Collision chamber Q3 – Fixed product m/z (m/z 223) + + MRM Transition m/z 339 – m/z 223 Multiple Reaction Monitoring

  37. Intensity (cps) Time (min) Multiple Reaction Monitoring: specific precursor / product ion pair 399 m/z -> 223 m/z

  38. Plasma Extract MRM: m/z 603-> m/z 483 Plasma Extract SIM: m/z 603 Plasma Extract SIM: m/z 617 Plasma Extract MRM: m/z 617-> m/z 483

  39. Full Scan Spectrum Precursor Ion

  40. Product Ion Spectrum

  41. Multiple Reaction Monitoring: two analytes (m/z 492 -> m/z 336), (m/z 539 -> m/z 332) Multiple Reaction Monitoring (m/z 539 -> m/z 332) Multiple Reaction Monitoring (m/z 492 -> m/z 336)

  42. Examples of the Use of Multiple Component LC-MS/MS-based Bioanalytical Methods in Drug Discovery • Determination of Drugs and Metabolites • Co-Administration Studies

  43. Bioanalytical Method for Determination of BMS-xxxxx and Metabolites in Biological Fluids to Support the Diabetes Program

  44. Structures Parent Compound MW=392.88 “Styrene” Metabolite MW=390.87 “Alcohol” Metabolite MW=408.88 “Ketone” Metabolite MW=406.88

  45. Multiple Component Bioanalytical Method • Mass Spectrometry: Negative Ion Electrospray Multiple Reaction Monitoring Parent Styrene Metabolite • Ketone Metabolite • Alcohol Metabolite • Diol Metabolite • Internal Standard Function Reaction Dwell(secs)* Cone Volt. Col.Energy 2 391.5 > 313.1 0.2 45 15 2 389.4 > 311.1 0.2 45 15 1 405.6 > 327.7 0.1 40 15 1 407.8 > 329.9 0.1 40 15 1 423.3 > 333.5 0.1 45 20 1 379.5 > 289.21 0.1 45 30

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