Mass Spectrometry as the Premier Analytical Tool in

Mass Spectrometry as the Premier Analytical Tool in Drug Discovery and Drug Development Walter Korfmacher Exploratory Drug Metabolism Merck Research Laboratories Kenilworth, NJ USA October 14, 2010 GBMSDG Talk 1

Outline New Drug Discovery Challenges n Mass Spectrometry Basics n Selected In vitro Drug Metabolism Applications n Selected In vivo Drug Metabolism Applications n Metabolite ID Applications n MS Imaging Applications n Conclusions n October 14, 2010 GBMSDG Talk 2

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

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

NEW DRUG DISCOVERY PIPELINE Chemistry Biology--HTS for Receptor Activity DMPK Lead Optimization In-vitro Stability Screen In-vitro Absorption Screen P 450 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 October 14, 2010 GBMSDG Talk 5

The Challenge n How to deal with the multiple compounds at multiple stages in the drug discovery/drug development pipeline. October 14, 2010 GBMSDG Talk 6

The Solution n LC-MS and LC-MS/MS October 14, 2010 GBMSDG Talk 7

Why use Mass Spectrometry? n Specificity! Non-specific techniques, such as UV and fluorescence, are unable to provide proof of the analyte identity n Ease of Use! Modern mass spectrometry software interfaces are easy to use n Versatility! MS is both a qualitative and quantitative technique October 14, 2010 GBMSDG Talk 8

The Challenge n Choosing the right tool for the task : OR Wrench? MS Toolbox Hammer? October 14, 2010 GBMSDG Talk 9

Common MS Tools October 14, 2010 GBMSDG Talk 10

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 October 14, 2010 GBMSDG Talk 11

The Challenge—Compound Synthesis n 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. October 14, 2010 GBMSDG Talk 12

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. October 14, 2010 GBMSDG Talk 13

The Challenge—Lead Optimization In Vitro Screening n At a big Pharma site, one might get 100200 new compounds each week that have to be screened in various in vitro assays. These have to be assayed separately for each screen. October 14, 2010 GBMSDG Talk 14

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. October 14, 2010 GBMSDG Talk 15

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) Q 1 HPLC Column October 14, 2010 Ion source Q 2 Mass analyzer GBMSDG Talk Q 3 Detector 16

Quantitation Gold Standard: MS/MS – Selected Reaction Monitoring (SRM)* Select precursor ion in Q 1 e. g. m/z 216 Fragment precursor ion in Q 2 (Collision Cell) Select product ion in Q 3 e. g. m/z 174 * Often referred to as Multiple Reaction Monitoring (MRM) October 14, 2010 GBMSDG Talk 17

Effects of Stages of Analysis on Signal, Noise, and Signal-to-Noise Important Concept!! S/N Signal Noise 1 October 14, 2010 LC 2 LC-MS GBMSDG Talk 3 LC-MS/MS 4 ? Stages of Analysis 18

LC-TIC LC-MS October 14, 2010 GBMSDG Talk 19

LC-MS/MS 10 ng/ml October 14, 2010 GBMSDG Talk 20

Discovery In Vitro Screening P 450 Assay--Enzyme Inhibition Screen n Caco-2 cells—Absorption Screen n Liver Microsomes/Hepatocytes-Metabolic Stability Screen n Plasma Protein Binding n Each In Vitro Assay Uses LC-MS/MS for the analytical step (typically a triple quadrupole MS system). October 14, 2010 GBMSDG Talk 21

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 October 14, 2010 GBMSDG Talk 22

In Vitro Evaluation of CYP Inhibition Purpose: Evaluate direct and mechanism-based inhibitors for P 450 enzymes (3 A 4, 2 D 6, 2 C 9) to assess potential for drug-drug interactions. Method: P 450 source human liver microsomes incubation cocktail ( substrates testosterone 3 A 4, 2 D 6, 2 C 9) Dextromethorphan 3 A 4 products detection October 14, 2010 2 D 6 6 -hydroxytestosterone dextrophan Tolbutamine 2 C 9 4 -hydroxytobutamide LC-MS-MS GBMSDG Talk 23

In Vitro Evaluation of CYP Inhibition incubation/pre-incubation Stock solution from CDC Serial dilution 1. Coincunation 2. Coincubation • three concentrations/cpd: 20 m. M, 2 m. M and 0. 2 m. M 3. Preincubation • duplicates/each conc. • 30 compounds/set 4. Preincubation October 14, 2010 GBMSDG Talk 24

LC-MS/MS for p 450 Inhibition Screen-Run time is less than 1 minute Substrate for CYP 3 A 4, 6 -hydroxytestosterone Substrate for CYP 2 D 6, dextrophan Internal Standard m/z 258 157 m/z 347 121 Substrate for CYP 2 C 9, 4 -hydroxytolbutamide October 14, 2010 m/z 305 269 GBMSDG Talk m/z 287 171 25

CYP Inhibition Screen 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. October 14, 2010 GBMSDG Talk 26

CYP Inhibition Screen Review October 14, 2010 GBMSDG Talk 27

The Challenge— Metabolic Stability Screening At a big Pharma site, one might get 100200 new compounds each week that have to be screened for metabolic stability. n The challenge is that LC-MS/MS methods have to be developed for each compound. n October 14, 2010 GBMSDG Talk 28

The Solution—LC-MS/MS + Software + Hardware • Use a generic HPLC method. This works for 8090% of the compounds. • Use a vendor-supplied software tool for automated MS/MS method development, e. g. : • • Quick. Quan™ (Thermo-Fisher) • Quan. Optimise™ (Waters-Micromass) • Discovery. Quant™ (AB-Sciex) • Optimizer. TM (Agilent) Use robots for automated sample handling October 14, 2010 GBMSDG Talk 29

Metabolic Stability Assay Example October 14, 2010 GBMSDG Talk 30

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. October 14, 2010 GBMSDG Talk 31

Metabolic Stability Assay Scheme shows how a well organized system is needed to provide high throughput metabolic stability data. October 14, 2010 GBMSDG Talk 32

In Vivo Assays n Various types of in vivo assays are performed as part of new drug discovery and development n The goal may be to understand absorption (A), distribution (D), metabolism (M), or excretion (E) properties of a compound n The goal may be to get pharmacokinetic (PK) information on a compound October 14, 2010 GBMSDG Talk 33

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

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. n The challenge is that LC-MS/MS methods have to be developed for each compound. n October 14, 2010 GBMSDG Talk 35

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. October 14, 2010 GBMSDG Talk 36

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 October 14, 2010 GBMSDG Talk 37

PK Screening Example: CARRS Provide basic pharmacokinetic information for all rapid rat compounds. • AUC (0 -6 hr) • Concentration vs Time Profile (0 -6 hr) Throughput: Up to 96 compounds per week October 14, 2010 GBMSDG Talk 38

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 (Quan. Optimize) October 14, 2010 GBMSDG Talk 39

Preclinical PK Studies n n n 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 GBMSDG the assay. Talk 40 October 14, 2010

Discovery PK Analysis Flowchart The MS/MS instrument is normally a triple quadrupole system October 14, 2010 GBMSDG Talk 41

Rapid MS Method Development ion a Discovery Environment 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 Xu et al. Anal. Chem. --2005 Assay October 14, 2010 Non-routine options GBMSDG Talk

Response ratio Typical Discovery PK Assay 1 – 10, 000 ng/m. L October 14, 2010 GBMSDG Talk 43

Discovery Metabolite ID n Generally this has two components: Lead Optimization Phase--would use unlabelled compounds and in vitro samples to look for major routes of metabolism. n Pre-Recommendation Phase—Look for problem metabolites plus in vitro comparison of human to animal metabolism. n October 14, 2010 GBMSDG Talk 44

Discovery Metabolite ID n Mass Spectrometry serves two purposes: n n 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 October 14, 2010 GBMSDG Talk 45

Triple Quad Scan Functions: to find metabolites n Neutral Loss Scan n n Precursor Ion Scan n n 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) 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 October 14, 2010 GBMSDG Talk 46 a scan set n

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 October 14, 2010 GBMSDG Talk • QTrap MS systems are excellent tools for quantitative analyses as well as for metabolite ID 47 applications

QTrap MS Publication In Vivo PK Samples n 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. October 14, 2010 GBMSDG Talk 48

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

Product Ion Scan Select Precursor Ion Fragmentation Scan Products A key technique for obtaining structural information. Product ion spectrum of a particular compound m 1+ m 2+ October 14, 2010 GBMSDG Talk 50

Discovery Metabolite ID (A) HPLC Radiochromatogram of 14 C-Gemfibrozil at 25 mm Incubated in Human Liver Microsomes Fortified with NADPH and UDPGA; (B) Reconstructed Ion Chromatogram; (C) Full Scan Mass Spectrum of M 1 [M-H]-. Xia, Y. Q. et al. , Use of a quadrupole linear ion trap mass spectrometer in metabolite identification and bioanalysis, Rapid Commun. Mass Spectrom. , 17(11), 1137, 2003. October 14, 2010 GBMSDG Talk MS/MS spectrum of M 1 51

What is MIST? n Mass Spectrometry n Investigators n Security n Trust n MIST will ensure job security for MS metabolite ID experts October 14, 2010 GBMSDG Talk 52

What is MIST? n Metabolites n In n Safety n Testing n MIST will ensure job security for MS metabolite ID experts October 14, 2010 GBMSDG Talk 53

Key MIST Points 1. Human metabolites that can raise a safety concern are those formed at n greater than 10 percent n of parent drug’s systemic exposure n at steady state. 2. Metabolites identified only in human plasma or Metabolites present at disproportionately higher levels in humans than in any of the animal test species should be considered for safety assessment. n Bottom line: Find human metabolites and then be sure they are “covered” in the tox species. October 14, 2010 GBMSDG Talk 54

New MS Tool for Finding Metabolites: HRMS n High Resolution Mass Spectrometry (HRMS) has become the tool of choice for finding metabolites in complex biological matrices. The improved mass resolution can be used to differentiate metabolites from endogenous background n Software tools can use HRMS to find metabolites n The accurate mass of a detected metabolite can help to confirm its identity by leading to its 55 October 14, 2010 empirical formula GBMSDG Talk n

Two HRMS Systems LTQ-Orbitrap The Orbitrap MS provides mass resolution of 10, 000 -100, 000 October 14, 2010 GBMSDG Talk The TOF MS provides mass resolution of 10, 00050, 000 56

Why High Mass Resolution? TOF-MS of Sidenafil Example MS window: 0. 001 Da MS window: 0. 01 Da MS window: 0. 1 Da MS window: 1 Da Scan: 200 - 800 October 14, 2010 GBMSDG Talk 57

Use of Mass Defect Filter for Post. Acquisition Processing of Accurate Mass (High Resolution) LC-MS Data. • “Fish-out” drug-derived peaks from endogenous peaks in a complex biological matrix • Key utility in non-radio-labeled drug-administration • An alternative to triple quadrupole tools: neutral loss scan (NLS) and precursor ion scan (PIS) M. Zhu et al. , Drug Metab. Dispos. 2006, 34, 1722 -1733 October 14, 2010 GBMSDG Talk 58

Mass Defect Filter Reference J. Mass Spectrom. , 2009, 44, 999 -1016. October 14, 2010 GBMSDG Talk 59

Exact Mass and Isotopic Abundance of Common Elements October 14, 2010 GBMSDG Talk 60

Mass Defect Filter Example TIC Processed MS (B) (C) 14 C Source: JMS 2003, 38, 1110 -1112 October 14, 2010 GBMSDG Talk 61

Mass Defect Filter Example (B) (C) Mass spectra of metabolite x at retention time 35. 5 min (A) Full scan spectrum of metabolite x form the unprocessed total ion chromatogram. (B) Detail of (A) in mass range 450 -550 Da. (C) Full scan spectrum of metabolite x (the molecular ion was at m/z 503. 0737 from the MDF processed total ion chromatogram. October 14, 2010 GBMSDG Talk Source: JMS 2003, 38, 1110 -1112 62

Metabolite ID Software Tool: Bg. S-No. RA Published in RCMS, 23, 1563 (2009). October 14, 2010 GBMSDG Talk 63

Metabolite ID Software Tool: Bg. S-No. RA Mouse urine spiked with diclofenac microsomal incubation sample TIC after Background Subtraction TIC after Bg. S-No. RA P = parent October 14, 2010 GBMSDG Talk M 1, M 2, M 3 = metabolites 64

Metabolite ID Software Tool: Bg. S-No. RA Mouse urine spiked with diclofenac microsomal incubation sample—peak at 7. 8 min Unprocessed data Mass spectrum after data processed with Bg. S-No. RA October 14, 2010 GBMSDG Talk 65

Which tool is Best for Metabolite ID? Published in RCMS, 24, 939 (2010). October 14, 2010 GBMSDG Talk 66

Additional Development Stages that use MS Analysis October 14, 2010 GBMSDG Talk 67

TK Study Support n n n Typical study is one compound dosed oral (PO) in a laboratory animal. The goal is to get TK (toxicokinetic) parameters. This is a GLP (Good Laboratory Practices) study. Sample preparation is typically SPE. A multipoint standard curve is prepared for the assay. Analysis is by LC-MS/MS on a triple quadrupole MS/MS system. Usually a SIL (stable isotope label) internal standard is used for the assay. October 14, 2010 GBMSDG Talk 68

Clinical PK Study Support n n n Typical study is one compound dosed oral (PO) in humans. The goal is to get PK parameters. This is a treated as a GLP (Good Laboratory Practices) study. Sample preparation is typically SPE. A multipoint standard curve is prepared for the assay. Analysis is by LC-MS/MS on a triple quadrupole MS/MS system. Usually a SIL (stable isotope label) internal standard is used for the assay. October 14, 2010 GBMSDG Talk 69

Impurities and Degradants These studies are performed to support safety studies or clinical studies. n The goal is to measure any significant impurities or degradants that are in the pharmaceutical test compound batch used for these studies. n Generally, one would use a combination of triple quadrupoles as well as QTOF MS systems as well as the Orbitrap MS system to characterize these compounds. n October 14, 2010 GBMSDG Talk 70

MS Imaging—a Specialty use of MS October 14, 2010 GBMSDG Talk 71

MS Imaging using the MALDI Qq. TOF h Detector + Quadrupole Collision Mass Filter Cell October 14, 2010 GBMSDG Talk Time-of. Flight Analyzer 72

1000 µm Rat Brain Tissue Slice --Rat dosed with clozapine Optical Image Radioautographic Image • MALDI-MS/MS Image is in good agreement with the radioautographic image MALDI-MS/MS Image October 14, 2010 Hsieh Y, et al. , Rapid Commun Mass Spectrom. 2006; 20(6): 965 -72. 73 GBMSDG Talk

Mass Spectral Data Confirms the Presence of Clozapine October 14, 2010 GBMSDG Talk 74

Next Step: Whole Mouse Slice MS Imaging October 14, 2010 GBMSDG Talk 75

Mouse Whole Body Image—Mouse Dosed with Terfenadine Optical image of whole-body mouse slice n 100 mpk p. o. nand sacrificed n 4 h post dosing Source: Chen et al. , Drug Metab. Lett. , 2, 1 -4 (2008) Ion image of terfenadine (parent). stomach GI tract liver Ion image of fexofenadine (metabolite). Result: These MS images allow us to “visualize” first-pass metabolism. October 14, 2010 GBMSDG Talk 76

Advion Nanomate LESA (Liquid Extraction Surface Analysis) System Description of Technology Advion Nanomate ESI Chip Automated liquid extraction-based surface sampling technique utilizing the robotic Advion Nanomate chipbased nanoelectrospray platform. Method invented at Oakridge National Laboratory (ORNL). Developed and commercialized at Advion. Liquid microjunction created between the robotically controlled pipette tip dispensing solvent and surface (e. g. tissue section, blood spots on paper). Liquid microjunction between pipette tip and tissue surface The microfluidics chip contains an array of nanoelectrospray nozzles etched in a silicon wafer eliminating carryover as one tip and one nozzle is used per sample. Can be used for a variety of samples including tissue sections, dried blood spots on paper, samples on MALDI plates for complimentary information by electrospray ionization (ESI), TLC plates, and other planar separation media. Schematic of chip based nano-ESI infusion October 14, 2010 http: //www. advion. com/biosystems/triversa-nanomate/LESA/index. php GBMSDG Talk 77

Whole Body Distribution of a Drug and its Metabolites by QWBA vs LESA 7. 5 mg/kg IV [3 H] Propranolol T T T Male CD-1 Mice QWBA (Quantitative Whole Body Autoradiography): QWBA study with metabolite ID by radioprofiling in conjunction with nanospray MS or accurate mass MS at Merck 7. 5 mg/kg IV ‘Cold’ Propranolol MS Tissue Imaging/Sampling: Male CD-1 Mice Sections transferred to glass slides with UV-activated adhesive or collected on adhesive tape and sent to ORNL for MS tissue 78 imaging/profiling

QWBA Results Confirmed High Levels of [3 H] Propranolol Related Material in Brain, Lung, Liver, and Kidney 40 µm Mouse Whole Body Sagittal Section: 60 min post [3 H]Propranolol IV Dose QWBA Brain Lung 20 µM-eq 40 µM-eq 0 Liver 21 µM-eq Stomach 31µM-eq Kidney 45 µM-eq 100 Autoradioluminograph [3 H]Propranolol Drug Related Material October 14, 2010 GBMSDG Talk 79

Identification of [3 H] Drug Related Material (DRM) in Tissues Unchanged parent detected in lung and brain. Major metabolites in liver and kidney identified as hydroxypropranolol glucuronide metabolites by LCMS/MS-rad. October 14, 2010 GBMSDG Talk 80

Normal Operation using the Advion Nanomate System Mass spectrometer Sampling tip Nozzle Aspirate sample Transfer sample Apply HV, spray voltage Sample October 14, 2010 GBMSDG Talk 81

Operation using the Nanomate System for Surface Sampling – ORNL Invention (Advion LESA) Mass spectrometer Sampling tip Nozzle ~ 1 mm spot size Sample On Surface Solvent Vilmos Kertesz, Gary J. Van Berkel „Fully Automated Liquid Extraction-Based Surface Sampling and Ionization Using a Chip-Based Robotic Nanoelectrospray Platform” J. Mass Spectrom. , 2010 Mar; 45(3): 252 -60. Aspirate solvent Dispense solvent on sample Aspirate sample solution Transfer sample Spray sample 82

LESA: MS for Detection of Propranolol (7. 5 mg/kg IV) and a Major Metabolite in Tissues • Rapid and automated technique to sample tissues including ones on tape used for QWBA. • Successful detection of propranolol and its major glucuronide metabolite not seen by DESI-MS. Lung Brain Kidney Liver Stomach Muscle Dosed tissue Brain Liver Kidney Stomach Muscle Control tissue Propranolol lung liver Propranolol stomach kidney brain muscle brain kidney muscle Hydroxypropranolol glucuronide t, min liver lung stomach Hydroxypropranolol glucuronide t, min

Conclusions n Mass spectrometry is used at multiple stages of new drug discovery and development. Various types of mass spectrometers are utilized including single quadrupoles, triple quadrupoles, Q-Traps, Q-TOFs and Orbitrap MS systems. The need for the multiple types of MS system is due to the variety of assays that are mandated at the different stages in the new drug discovery process. October 14, 2010 GBMSDG Talk 84

MS Reference Books 2009 2008 2005 Available at Amazon. com 2009

Acknowledgments (Thanks to the following for one or more slides) n Waters- n Swapan Micro. Mass n Thermo-Fisher n AB-Sciex n Agilent n Marissa Vavrek n Rick King October 14, 2010 Chowdhury n Joanna Zgoda. Pols n Michelle Reyzer n Yunsheng Hsieh n Fangbiao Li GBMSDG Talk 86

Acknowledgements October 14, 2010 GBMSDG Talk 87

Thank you for your attention! ? October 14, 2010 GBMSDG Talk 88