1. Quantitative Protein Analysis of CYP450 Induction via LC-MRM Analysis

2. 2 © 2010 AB SCIEX Cytochrome P450 Proteins − Cytochrome P450 enzymes are mainly expressed in liver and are responsible for oxidative metabolism of drugs, environmental pollutants, carcinogens, etc − Cytochrome P450 Family of Enzymes –70 p450 protein families in humans –Over 200 different subfamilies / isoforms –Each isoform has different substrate specificities, varied inducibility by different drugs − Important in drug development –Changes in expression of specific isoforms provide information on toxicity of different drugs –Individual patient basal expression levels affect responsiveness to drugs

3. 3 © 2010 AB SCIEX Human CYP Enzymes Drug Metabolism

4. 4 © 2010 AB SCIEX Current Methodologies for Assessing CYP450 Induction − mRNA techniques –Measure the amount of messenger RNA expressed for each enzyme isoform –Assesses only CYP induction from gene transcription changes − Enzymatic activity –Induction by quantifying the metabolite of a CYP-specific probe substrate generated from treated hepatocytes –Relies on the specificity of enzymatic conversion of probe substrates, and a different probe substrate is required for each CYP isozyme which is not always possible − Western Blotting techniques –Measures the actual protein levels of CYP450 enzymes using isoform-specific antibodies. Currently there are only a few good antibodies that are isoform specific Major Challenges in Induction Assay: Assay selectivity, and sensitivity; Different technical expertise and equipment are needed for mRNA or Western Blotting assessment

5. 5 © 2010 AB SCIEX Challenges of Current Approaches − Western Blot Analysis of p450 Expression –Using commercially available antibodies to the various subfamilies of P450 proteins, Western Blot analysis can be used to monitor changes in protein expression –However, commercial antibodies are not available for all protein isoforms –Some antibodies recognize multiple isoforms − An MS-based approach could provide sensitivity and specificity through the detection of individual peptides from specific P450 isoforms Cyp1a1 Cyp1a2 Cyp2e1 Cyp3a4 Control PB induced Changes in expression in response to treatment with phenobarbitol

6. 6 © 2010 AB SCIEX CYP Induction Assay: LC-MS/MS Solution − An LC-MS/MS-based approach can provide sensitivity and specificity through the detection of individual peptides from specific CYP450 isoforms − A fast MS scan speed and the Scheduled MRM™ Algorithm allows for multiplexed protein quantitation − A CYP450 protein assay kit including all reagents, sample preparation procedure and established LC/MS/MS conditions provides easy protein quantitation for human induction studies using current DMPK resources

7. 7 © 2010 AB SCIEX Multiple Reaction Monitoring (MRM) − Highest specificity and sensitivity for detecting components in a complex mixture − Requires QTRAP® System or triple quadrupole MS capability − Largest linear dynamic range for quantitation − Well accepted as the MS technique for quantitation (Pharmaceutical Industry) Fragment peptide Select Peptide Select Fragment Fragmentation Cell Detect Fragment Mass Analyzer Detector

8. 8 © 2010 AB SCIEX Sample Preparation for LC/MS/MS Analysis of Protein Therapeutics − Problem: Protein therapeutics and larger peptide therapeutics are typically too large to directly quantitate using standard MRM assays in mass spectrometry − Solution: Enzymatically digest the protein or large peptide therapeutic into small peptides and monitor one or more peptides as a surrogate –Trypsin is the enzyme of choice for several reasons: –Tryptic peptides are a good size for MRM assays (not too large) –Tryptic peptides tend to fragment well leading to good MRM assays –Trypsin digest quality can be very good when a high grade of trypsin is used Protein Enzyme (Trypsin) Peptides

9. 9 © 2010 AB SCIEX Quantifying Proteins by Multiple Reaction Monitoring Intact protein Enzyme - Trypsin Peptide fragments 200400600800 m/z 0 MS/MS – Q3 m/z * * * * Peptide Q1 m/z Stable Isotope Labeled Internal Standards MRM MethodMRM Results

10. 10 © 2010 AB SCIEX General Strategy for Protein Quant using SIS Peptides Generate a list of peptides that uniquely identify each p450 isoform. Synthesize each of these peptides with a heavy amino acids MRM LC/MS/MS Sequence of Target Protein in sample Design MRM method – monitor heavy and light peptides RQLYSLVGITK* KLQISSDVLAR* RYILNDAVEIR* …KLQISSDVLAR… H2NH2N COOH..RQLYSLVGITK.. …RYILNDAVEIR… Internal Std Target = Concentration of target protein Area of target Area of Internal std * [ Int. Std. ]

11. 11 © 2010 AB SCIEX Control Microsomes Trypsin digest 1D LC-MRM 20 min run Reduce Alkylate Concentration of control P450 Concentration of induced P450 Synthetic heavy peptides -representative of each P450 studied -for internal standard and concentration curve Mix Induced Microsomes 1D LC-MRM 20 min run Trypsin digest Reduce Alkylate [Peak Area Smp/ Peak Area Std] *C Std [Peak Area Smp/ Peak Area Std] *C Std P450 Peptide Assay Workflow

12. 12 © 2010 AB SCIEX Scheduled MRM™ Algorithm Improving MRM Method Efficiency by Maximizing Analyte Utilization − Each MRM monitored only across its expected elution time −  concurrent MRMs − Maintain cycle time and dwell time −  effective duty cycle for every peptide − Maintain analytical precision

13. 13 © 2010 AB SCIEX LC-MRM Assay of CYP Proteins − High assay robustness through monitoring –Multiple MRMs per peptide –Multiple peptides per protein 3A4 -Pep 1 3A4 -Pep 2 3A4 -Pep 3 2B6 -Pep 1 2B6 -Pep 2 2B6 -Pep 3 1A2 -Pep 1 1A2 -Pep 2 1A2 -Pep 3 3A5 -Pep 3 3A5 -Pep 1 3A5 -Pep 2 CYP 1A2 CYP 2B6 CYP 3A4 CYP 3A5

14. 14 © 2010 AB SCIEX How Consistent are MRMs to each Peptide Sample 1 Sample 2 Sample 3

15. 15 © 2010 AB SCIEX Peptide Consistency for CYP 1A2 Sample 1 Sample 2 Sample 3

16. 16 © 2010 AB SCIEX Typical Western Blot Data from Induction Study − The typical results seen in Western blot analysis of protein expression correlates with the observed LC/MS results

17. 17 © 2010 AB SCIEX XIC of (a) control and (b) 3-MC induced microsomes for the one of the peptides from Cyp1A2. (a)(b)Control Sample3-MC Induced Sample Sample Standard

18. 18 © 2010 AB SCIEX LC-MS/MS Protein, mRNA and Activity Assays Control3-MCPBRIF − CYP1A2 –Protein expression changes mirror the changes observed in mRNA assays and enzyme activity changes –3-MC – Prototypical inducer of CYP1A2 by AhR nuclear receptor activation –Phenobarbitol (PB), Rifampicin (RIF), vehicle control – Basal levels of 1A2, no induction

19. 19 © 2010 AB SCIEX − CYP2B6 –Protein expression changes mirror the changes observed in mRNA assays and enzyme activity changes –3-MC, vehicle control – basal levels of 2B6 –Phenobarbitol (PB) – Prototypical inducer of 2B6 by CAR nuclear receptor activation –Rifampicin (RIF) – Also known inducer of 2B6 Control3-MCPBRIF LC-MS/MS Protein, mRNA and Activity Assays

20. 20 © 2010 AB SCIEX − CYP3A4 –Protein expression changes mirror the changes observed in mRNA assays and enzyme activity changes –3-MC – minimal induction of 3A4 –Phenobarbitol (PB) – Significant induction of 3A4 –Rifampicin (RIF) – Prototypical inducer of CYP3A4 by PXR nuclear receptor activation LC-MS/MS Protein, mRNA and Activity Assays Control3-MCPBRIF

21. 21 © 2010 AB SCIEX LC-MS/MS Protein All Cytochrome P450 Proteins − Adding CYP3A5 data relative to other CYPs –Protein expression changes illustrate 3A5 is inducible –3-MC – minimal induction of 3A5 –(PB) – Significant induction of CYP3A5 –(RIF) – Small induction CYP3A5

22. 22 © 2010 AB SCIEX Expandable – 7 CYP isoforms in 10 min run

23. 23 © 2010 AB SCIEX Conclusions - CYP Induction Assay LC-MS/MS Protein Expression Analysis − Highly sensitive, specific, and fast Multiple Reaction Monitoring (MRM) method has been developed: –12 different peptides representing 4 unique P450 proteins (CYP 1A2, 2B6, 3A4 and 3A5) were simultaneously monitored and quantified –2B6, a lower abundant CYP, is easily detected showing good dynamic range of method − Largest protein expression change was observed for microsomes prepared from RIF induced hepatocytes – Cyp3A4 showed an increase in expression upon drug treatment of ~50-fold over control. –S9 or microsomal subcellular fractions can be used − This method was in excellent agreement with existing methods (mRNA, enzyme activity assays)

24. 24 © 2010 AB SCIEX Human Induction Kit (100 Assays) Starter Kit Contents − Heavy peptide mix − Denaturant, Reducing reagent, Alkylating reagent − Digestion buffer − Trypsin − Peptide column − Acquisitions methods for –AB SCIEX Triple Quad™ 5500 and QTRAP® 5500 systems –API 4000™ system, 4000 QTRAP® system, API 5000™ system − Quantitation methods for MultiQuant™ software 1.2 − Microsoft Excel 2007 results template

25. 25 © 2010 AB SCIEX Acknowledgements − AB SCIEX –Sean Seymour –Christie Hunter –Lydia Nuwaysir − CellzDirect –Jeanette Hill –Rob Taylor

26. Thank You for your Attention

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