Powerful Software Solutions for Drug Metabolite Quantitation and Identification

Powerful Software Solutions for Drug Metabolite Quantitation and Identification

Powerful Software Solutions for Drug Metabolite Quantitation and Identification In this webinar we will discuss LC/MS and NMR software workflows, including new algorithms to add power to your drug metabolite quantitation and identification Today s Presenters from Agilent Technologies: Steve Madden Software Product Manager, MassHunter Frank Delaglio Marketing Manager, Magnetic Resonance Software Doug McIntyre Software Product Manager, MassHunter Dave Russell Applications Scientist, NMR Division Today s Host: Lauren Constable Head of Commissioning, FSG publishers of Bioanalysis

Steve Madden Software Product Manager, MassHunter Agilent Technologies

Doug McIntyre Software Product Manager, MassHunter Agilent Technologies

Powerful Software Solutions for Drug Metabolite Quantitation and Identification Metabolite Identification Quantitative Analysis Qual/Quan June 25, 2013 Steve Madden Doug McIntyre Agilent Technologies Inc. California, USA 5 Customer Presentation July 9, 2013

Metabolite ID in the Drug Discovery & Development Discovery Development Commercialization Target Selection Target Validation Lead Identification Lead Optimization Pre-Clinical Development Clinical Development Regulatory Approval/ Manufacturing Study Aim Optimize metabolic stability of lead compounds Avoid drug-drug interactions Profiling metabolite across species Help select safety species Soft spot analysis modulate metabolic clearance Identify isozyme specific metabolites Identify pharmacologically active metabolites secure IP protection, understand PK/PD and identify new leads Identify reactive metabolites minimize potential IDRs Identify toxic metabolites 6 Customer Presentation July 9, 2013

Quantitation in ADME/PK Discovery Development Commercialization Target Selection Target Validation Lead Identification Lead Optimization Pre-Clinical Development Clinical Development Regulatory Approval/ Manufacturing In vitro ADME In vivo Bioanalysis Metabolic stability Permeability Plasma protein binding Inhibition assay Induction assay Pharmacokinetics Toxicokinetics Pharmacodynamics 7 Customer Presentation July 9, 2013

Agenda Metabolite Identification MassHunter Metabolite ID workflow determining biotransformations Molecular Discovery s Mass-MetaSite workflow for structure prediction and batch processing Quantitative Analysis --Study Manager and Optimizer for automating Acquisition --Compliance features --LIMS connectivity Qual/Quan Application 8 Customer Presentation July 9, 2013

Metabolite Identification Software Tools - Remove the bottleneck of data analysis and interpretation MassHunter Metabolite ID Molecular Discovery s Mass-MetaSite Many Pharma companies use Mass-MetaSite as a global software tool for metabolism studies Goal Increase throughput for metabolite identification Provide rapid confident metabolite identification 9 Customer Presentation July 9, 2013

MassHunter Metabolite ID: Untargeted approach based on MFE comparison Best approach for expected and unexpected metabolites Sample MFE Cmpds Compare Potential Metabolites Confirm + identify Flagged Metabolites Review + conclude Flagged Metabolites Control MFE Cmpds Untargeted approach via comparison of MFE Compound lists Sophisticated Sample-Control Comparison Algorithm based on Molecular Feature Extraction (MFE) allows more comprehensive detection of differences via exploitation of mass and RT resolution. Best suited to find and confirm expected and unexpected metabolites Find all differences and THEN confirm and identify Confirm expected and identify unexpected with multiple and more sophisticated algorithms 10 Customer Presentation July 9, 2013

Mass-MetaSite Software - A desired solution for rapid metabolite Identification Fully automated data analysis and structure elucidation Site of metabolite localization based on MS/MS data Not on in silico predictions as in Metasite Metasite predictions used to designate the most likely SoM within a Markush structure Metabolite structure proposal relies on included reaction mechanism (Phase I & II biotransformations) Batch processing capability for high throughput application 11 Customer Presentation July 9, 2013

Mass-MetaSite Data Analysis Workflow Structure of a substrate Mass spectra raw data DRM search Algorithms Noise suppression Background subtraction Isotope pattern analysis MS/MS fragmentation Mass defect Retention time analysis Metabolite structure assignment Metabolite structure The entire process only takes several minutes 12 Customer Presentation July 9, 2013

LC/MS Quantitation: Two Major ADME/PK Applications Bioanalysis (PK, BA/BE) Measure drug candidate concentration (and metabolites) over time in animals or humans May require compliance option (ADME) Drug Discovery Screening Determine properties of lead molecules (permeability, metabolic stability, ) Often 500 1000 compounds/week Customer Presentation Page 13 July 9, 2013

MassHunter Study Manager for Automating Workflow User selects workflow and submits sample sets (studies) using Excel or text files Coordinates acquisition, compound optimization, quantitation and report generation Uses different Study Creators for each workflow Generic BioAnalysis Watson LIMS connectivity ADME Screening Can be used in compliant mode 14 Customer Presentation July 9, 2013

Fully Automated Quantitation Workflow 1. Specify input File 2. Specify Quant Method 3. Specify Report 4. Submit and go! Customer Presentation 15 July 9, 2013

HT Targeted Quant in Drug Discovery Screening in vitro Import Sample Set Submit Study Optimize Compounds Acquire Samples Analyze Batch Review Results Report Calculate Values Excel or other Electronic Source MassHunte r Study Manager MassHunter Optimizer MassHunter Acquisition MassHunter Quant MassHunter Quant MassHunter Quant Reporting End User Applicati on Software Compound Database Study Manager schedules and automates these steps Which of the synthesized compounds will be viable drug candidates? Customer Presentation Page 16 July 9, 2013

Metabolic Stability Profiles Customer Presentation Page 17 July 9, 2013

Bioanalysis Using Watson LIMS Workflow 1. Create Study Watson TM LIMS 2. Import Study from LIMS 6. Process Study 5. Import Results To LIMS 3. Build Worklist Specify Acq Method & Ref File or Quant Method 4. Auto Quant Batch (Review Results) Create LIMS File Customer Presentation Page 18 July 9, 2013

Targeted Quant in Bioanalysis Watson LIMS Submit Study Run Worklist Analyze Batch Review Results Report Watson LIMS Generate Studies (Sample lists) MassHunter Study Manager MassHunter Acquisition MassHunter Quant MassHunter Quant MassHunter Quant Reporting Upload results (Conc. / Area values) Study Manager schedules and automates these steps LC What are the pharmacokinetic properties of the drug? What does the body do to the drug? Customer Presentation Page 19 July 9, 2013

Software features that support Compliance Customer Presentation Page 20 July 9, 2013

Compliance Key Features Acquisition/Study Manager Quantitative Analysis Mandatory Login Pre-defined Role Based Security UI operations disabled based on Privileges Data file overwrite protection Tamper Detection Capability for Methods and Data files Can require authentication and reason for allowed actions Method versioning Audit Trail on method changes Mandatory Login Configurable Role Based Security Operations can be disallowed based on defined capabilities Tamper Detection Capability for Results Password locked reports Can require authentication and reason for allowed actions Audit Trail on all quant actions Customer Presentation Page 21 July 9, 2013

DATA OpenLAB ECM The ultimate scientific data management solution for the laboratory Centralized data management for Agilent MassHunter Long-term Archival MassHunter Specific Indexing Regulatory Compliance Comprehensive Audit Trails File Revisions Easy to learn & use Extensible to other systems OpenLAB ECM Customer Presentation 22 July 9, 2013

Qual/Quan: on a Single Platform - more information in a single analysis 6550 Q-TOF HRAM MS MS/MS I µm Substrate conc. Metabolic stability Metabolite ID Metabolite Profiling 23 Customer Presentation July 9, 2013

Simultaneous Metabolite Identification 10 min incubation sample Buspirone +O O N N N N N O 1-Pyrimidinylpiperazine -C2H2+O +O +O +2O +2O -C2H2+O +O Buspirone -C2H2 N-oxide +O 24 Customer Presentation July 9, 2013

Normalized Peak Area (%) Metabolic Stability and Metabolite Profiles Buspirone (1.0 µm in RLM) 100 90 80 Buspirone (parent) monohydroxy dihydroxy trihydroxy N-oxide N, N'-desethyl N, N'-desethyl + O 1-PP 70 60 50 40 30 20 10 0 0 10 20 30 40 50 60 Incubation Time (min) 1-PP: pyrimidinylpiperazine 25 Customer Presentation July 9, 2013

Summary and Conclusions For Metabolite ID workflows MassHunter Qual: A great tool for determining specific biotransformations based on MS/MS data. Uses untargeted approach. Mass Metasite: A software provided through Molecular Discovery with the ability for structure prediction and correlating predictions to MS/MS data For Quantitative workflows For Qual/Quan MassHunter Quant Study Manager: fully automate both high throughput ADME screening and BioAnalysis quantitative measurements LIMS integration and Compliance Agilent s 6550 LC/Q-TOF has sufficient quantitative performance to allow performing bioanalytical measurements and providing the accurate mass MS data needed for metabolite identification from one injection 26 Customer Presentation July 9, 2013

27 Thank you!

Frank Delaglio Marketing Manager, Magnetic Resonance Software Agilent Technologies

Dave Russell Applications Scientist, NMR Division Agilent Technologies

Agilent Technologies Magnetic Resonance for ADME/T Studies Making the Complex Simple Frank Delaglio and Dave Russell ADME Webinar 2013

31 Frank Delaglio

What can NMR do in an ADME laboratory? The two primary tasks faced in an ADME study are identification and quantification of metabolites. NMR spectroscopy has been the definitive tool for structural studies over many years. Modern probe technology allows researchers to collect high quality structural data on very small samples, while advances in console design have opened the door to absolute concentration measurements on any sample. While Mass Spectrometry is the predominant technique used for ADME investigations, NMR has a critical role to play in this environment, too. Agilent 600 MHz NMR system Page 32 ADME Webinar 2013

Sample Workflow Agilent NMR systems remove the learning curve The traditional NMR workflow: 1. Insert the sample 2. Load parameters 3. Adjust tuning manually 4. Adjust lock freq. manually 5. Adjust shims manually 6. Run test spectra 7. Adjust experiment parameters manually 8. Collect data 9. Input sample information 10. Save data 11. Process data 12. Adjust processing parameters 13. Save processing parameters The VnmrJ workflow: 1. Insert the sample 2. Select experiments to be collected 3. Input sample information 4. Submit sample o Auto tune o Auto shim o Auto lock o Auto optimization o Auto process o Auto save o Auto archive o Auto email results Operator requires 30 minutes or more for every sample Operator requires 3 minutes for a sample, less for repeat measurements. 33 ADME Webinar 2013

VnmrJ Streamlined Workflows for Sample Submission 34 ADME Webinar 2013

VnmrJ 4.0 NMRPipe SpecView Interactive Principal Component Analysis to Characterize Spectral Differences 35 ADME Webinar 2013

VnmrJ 4.0 Industry-Leading DOSY Solutions 36 ADME Webinar 2013

Page 37 ADME Webinar 2013

38 Dave Russell

NMR as a structural tool Comparison of an SGLT-2 inhibitor and metabolite Sample Courtesy: Mark Grillo, Amgen 39 ADME Webinar 2013

Automated Solvent Suppression 100% water 40 ADME Webinar 2013

Microsample Cold Probe: Excellent Mass Sensitivity for Small Molecule NMR 3 μg Paclitaxel (3 nanomoles) O O O OH 128 transients / 10 minutes O N H O (high-frequency region). OH O OH O H O O O O Paclitaxel Page 41 ADME Webinar 2013

Band-selective Homodecoupled 2D NOESY (bashdnoesy) F1 (ppm) V.V. Krishnamurthy, Magn. Reson Chem. 35, 9 (1997) 1 2 18 N 20 3 4 5 MeO MeO 2 3 1 4 O 6 5 17 7 8 N 10 H 11 16 15 H 13 12 H 14 H 21 O 22 23 6 7 sw=sw1=4006, ni=480, at=0.256 exp. time = 440 min 8 8 7 6 5 4 3 2 1 0 F2 (ppm) Page 42 ADME Webinar 2013

Band-selective Homodecoupled 2D NOESY (bashdnoesy) F1 (ppm) V.V. Krishnamurthy, Magn. Reson Chem. 35, 9 (1997) 1 2 18 N 20 3 4 5 MeO MeO 2 3 1 4 O 6 5 17 7 8 N 10 H 11 16 15 H 13 12 H 14 H 21 O 22 23 6 7 sw=sw1=4006, ni=480, at=0.256 exp. time = 440 min 8 8 7 6 5 4 3 2 1 0 F2 (ppm) Page 43 ADME Webinar 2013

Band-selective Homodecoupled 2D NOESY (bashdnoesy) F1 (ppm) F1 (ppm) A, 3.00 2D NOESY (expansion) 3.10 sw1=4006, ni=480, at=0.256 exp. time = 440 min C, 3.00 2D bashdnoesy 3.10 sw1=92.5, ni=12, at=0.256 Trace I Trace II D, 3.00 3.10 exp. time = 13 min 2D bashdnoesy sw1=92.5, ni=24, at=1-02 7 6 5 4 F2 (ppm) 3 2 1 3.10 3.00 F2 (ppm) exp. time = 30 min Page 44 ADME Webinar 2013

Band-selective Homodecoupled 2D NOESY (bashdnoesy) F1 (ppm) F1 (ppm) A, 3.00 2D NOESY (expansion) 3.10 sw1=4006, ni=480, at=0.256 exp. time = 440 min C, 3.00 2D bashdnoesy 3.10 sw1=92.5, ni=12, at=0.256 Trace I Trace II D, 3.00 3.10 exp. time = 13 min 2D bashdnoesy sw1=92.5, ni=24, at=1-02 7 6 5 4 F2 (ppm) 3 2 1 3.10 3.00 F2 (ppm) exp. time = 30 min Page 45 ADME Webinar 2013

Band-selective Homodecoupled 2D NOESY (bashdnoesy) F1 (ppm) F1 (ppm) A, 3.00 2D NOESY (expansion) 3.10 sw1=4006, ni=480, at=0.256 exp. time = 440 min C, 3.00 2D bashdnoesy 3.10 sw1=92.5, ni=12, at=0.256 Trace I Trace II D, 3.00 3.10 exp. time = 13 min 2D bashdnoesy sw1=92.5, ni=24, at=1-02 7 6 5 4 F2 (ppm) 3 2 1 3.10 3.00 F2 (ppm) exp. time = 30 min Page 46 ADME Webinar 2013

Microsomal Incubation of Bupropion Detecting drug in the protein precipitated sample Blank Incubation sample 2 hours Sample Courtesy: Ron Aoyama, Rigel ADME Webinar 2013

Microsomal Incubation of Bupropion Detecting drug in the protein precipitated sample Band-selective HMBC ADME Webinar 2013

Microsomal Incubation of Bupropion Detecting drug in the protein precipitated sample Band-selective NOESY1D ADME Webinar 2013

Quantitative NMR Spectroscopy - qnmr Why is NMR a valuable tool for quantitation? In contrast to almost all other common quantitative techniques, the NMR experiment is fundamentally quantitative. The response factor for any given nuclei is 1.000, regardless of molecular structure or environment. Historically, the limiting factor for using NMR for routine quantitative studies has been poor reproducibility in the console hardware. Compensating for this required an internal standard or injection of an artificial reference signal. Both of these solutions are suboptimal. Page 50 ADME Webinar 2013

NMR Concentration (mm) Quantitation of Tetracycline in D 2 O 40 μm (19 nanomoles) 450 400 y = 1.0456x + 2.6284 R² = 0.9907 30 μm (14 nanomoles) 350 300 250 20 μm (9 nanomoles) 10 μm (4 nanomoles) 200 150 100 50 0 0 50 100 150 200 250 300 350 400 450 Actual Concentration (mm) LOQ of ~5 μm or 2 nanomoles Page 51 ADME Webinar 2013

Current NMR Analysis Workflow There are various drawbacks to interpretation based on a processed spectrum. Significant issues can be introduced by the Fourier Transform, including baseline issues and phase distortions. FT & phase correct Manual processing. Manual data reduction. Manual everything! Page 52 ADME Webinar 2013

Define regions CRAFT Deconstructing a Spectrum CRAFT Once data have been captured in the spreadsheet, analysis is essentially complete. Simulate Most users want to see how well the data reduction step worked. CRAFT includes a complete set of tools to allow visual inspection of the results. Page 53 ADME Webinar 2013

Spectra of Tetracycline in Plasma Tetracycline is known to bind to endogenous proteins in plasma. NMR can be used to directly measure the concentration of free tetracycline by simple integration of a drug peak. Plasma blank Plasma spiked 20 μm reference Page 54 ADME Webinar 2013

Deconvolution of Tetracycline Resonances Free Concentration = 6.5 μm residual reconstructed experimental Tetracycline is known to bind to endogenous proteins in plasma. NMR can be used to directly measure the concentration of free tetracycline by simple integration of a drug signal. Page 55 ADME Webinar 2013

Summary Agilent NMR systems are easy to use. By providing an array of automated tools and utilities, VnmrJ allows you to focus on learning about your samples rather than learning how to use the software. Sophisticated NMR techniques, such as band-selective experiments, are available to any researcher. A powerful and complete library of over 360 experiments can be used by anyone with just a few mouse clicks. You can use NMR to measure the absolute concentration of practically any sample. Agilent s NMR systems provide the stability and linearity needed to measure absolute concentration without the need for internal standards or artificial reference signals. CRAFT provides automated Spectrum-to-Spreadsheet deconvolution. Deconvolution of NMR spectra has reached reached the 21 st century. Page 56 ADME Webinar 2013

57 Thank you!

info@bioanalysis-zone.com

Look for more webinars brought to you from Bioanalysis and a recording on today s event on www.bioanalysis-zone.com/webinars