Piloting the progression of reactive metabolites through

Transcription

1 Piloting the progression of reactive metabolites through drug discovery N Rick Schneider PDM NCE-Biotransformation Groton, CT 06340

2 utline Piloting the progression of reactive metabolites through drug discovery Bioactivation/Reactive Metabolite Review Historical Look at Bioactivation/Reactive Metabolites in Drug Discovery Industry Pfizer Current Strategy/Rationale Integration of multiparametric in vitro assays ( mechanistic ) Putting results into context Summary

3 What are Reactive Metabolites? Chemically Inert Xenobiotic DME Reactive Intermediate Conjugate Macromolecule Adduct Toxicity Necessary, but not sufficient In many cases, adverse events involving the liver, skin, and circulatory system are believed to mediated by chemically highly reactive drug metabolites that covalently modify critical proteins. High dose drugs (> mg/day) that generate reactive metabolites have the poorest safety record (high body burden of reactive metabolites) Idiosyncratic drug toxicity Severe, rare & unpredictable No animal model & often immune-based

4 Bioactivation in Drug Discovery: Why worry? ~31% of pharma attrition is driven by toxicology 2011, 24, Kalgutkar et al. 1 examined 68 drugs recalled or with a black box warning (BBW) due to idiosyncratic adverse drug reactions (IADRs) and the top 200 drugs in the US in 2009 for trends in: daily dose presence of structural alerts evidence for reactive metabolite (RM) formation toxicity mechanism(s) potentially mediated by parent drugs IADR compounds: ~78-86% of drugs associated with toxicity contained structural alerts ~62-69% had evidence indicating that RM formation was a causative factor for toxicity no correlation between physico-chemical properties and IADR occurrence drugs associated with IADRs are frequently the ones with a higher daily dose Drugs withdrawn due to IADR BBW due to IADR > 100 mg (84%) > 100 mg (81%)

5 History Industry Pfizer (2001) In Vitro Reactive Metabolite Detection Assay Based on Conjugate Detection Human Liver Microsomes (HLM) with Glutathione (GSH) with LC-MS detection. Neutral Loss of glutamic acid from (GSH) High throughput Not quantitative Low Sensitivity W.G. Chen, C. Zhang, M.J. Avery, H.G. Fouda, Adv. Exp. Med. Biol., 500 (2001), pp

6 History Industry Pfizer (2001) Merck (2004) In Vitro/In Vivo Assessment Based on Reactive Metabolite/Macromolecule Covalent Binding Principle: Threshold of Covalent Binding Radiolabel synthesis for every candidate Significant FTE Questionable end point D. C. Evans et al., Chem. Res. Toxicol., 17, 3-16 (2004);

7 History Industry Pfizer (2001) Merck (2004) Pfizer (2006) In Vitro Reactive Metabolite Detection Assay Based on Conjugate Detection ti HLM with GSH-ethyl ester (EE) with LC-MS/MS detection. Predicted biotransformation's (with some interpretation) High throughput Too Many False Positives High sensitivity of MS method (MRM) If it wasn t predicted.. Lack of ftrue quantitation (Peak ka Area) Chem. Res. Toxicol. 2006, 19,

8 History Industry Pfizer (2001) Merck (2004) Pfizer (2006) BMS (2007 & 2008) Screening and Identification of GSH-Trapped Reactive Metabolites Using Hybrid Triple Quadruple Linear Ion Trap Mass Spectrometry Joanna Zheng, Li Ma, Baomin Xin, Timothy lah, W. Griffith Humphreys, and Mingshe Zhu * Departments of Bioanalytical Research and Biotransformation, Bristol-Myers Squibb, Princeton, New Jersey Chem. Res. Toxicol., 2007, 20 (5), pp High-Throughput Screening and Characterization of Reactive Metabolites Using Polarity Switching of Hybrid Triple Quadrupole Linear Ion Trap Mass Spectrometry Bo Wen,* Li Ma, Sidney D. Nelson, and Mingshe Zhu Department of Pharmaceutical Candidate ptimization, Bristol-Myers Squibb, Princeton, New Jersey 08543, and Department of Medicinal Chemistry, University of Washington, Seattle, Washington Anal. Chem., 2008, 80 (5), pp

9 History Industry Pfizer (2001) Merck (2004) Pfizer (2006) BMS (2007 & 2008) Pfizer & Sankyo (2009) Astra Zeneca (2011) Chem. Res. Toxicol. 2009, 22, ; DMD 37(2009) ; Chemico-Biological Interactions 192 (2011) 65 71

10 History Industry Pfizer (2001) Merck (2004) Pfizer (2006) BMS (2007 & 2008) Pfizer & Sankyo (2009) Astra Zeneca (2011) Advancement of Previous Covalent Binding Work Dose (Bauman et. al., Nakayama et. al.) Body Burden (Bauman et. al.) Will return to these Reactive Metabolite as a Fraction of Total later.. Metabolic Clearance Matrix of Endpoints (Thompson et. al.) Body Burden, P450 cell tox, mitochondria, BSEP Focused on later stages of discovery with significant investment of time and resources Chem. Res. Toxicol. 2009, 22, ; DMD 37(2009) ; Chemico-Biological Interactions 192 (2011) 65 71

11 History Industry Pfizer (2001) Merck (2004) Pfizer (2006) BMS (2007 & 2008) Pfizer & Sankyo (2009) Astra Zeneca (2011) Pfizer (2015)

12 Strategy Timing Goal: To provide a general perspective on metabolic clearance pathways and bioactivation for a series at Lead Development transition Early Discovery Lead Develop. PreClin Phase I

13 Strategy Timing Goal: To provide a general perspective on metabolic clearance pathways and bioactivation for a series at Lead Development transition Early Discovery Lead Develop. PreClin Phase I Pre-existing Condition? In early discovery, assess RM liability based upon the following triggers: Structural Alert Mech. Based Inhibition -- positive results (eg. CYP3A4) Gene Tox (Ames, IVMN) positive with metabolic activation (S9/NADPH) Unusual metabolites including rearrangements

14 Strategy Experimental In Vitro 1. Human Hepatocyte (HEP) Metabolic Profiling A. Provides a balanced look at all hepatic metabolic pathways 1) In addition, the fraction of the metabolism (f m ) going through the adduct pathway in the context of total metabolic Cl (UV) B. It is not recommended that HLM+GSH be used as an upfront assay. 1) HLM are a very concentrated enzymatic system a) P450 activities can be significantly (1-32x 32x* and 7-23x 23x**) higher in HLM then hhep 2) Do not have full complement of Phase II enzymes or GST s 3) Can provide utility in rank ordering compounds or as a mechanistic tool once the liability has been identified for the series. *B DMD *Brown, DMD 35: , 2007 **Lu, DMD 34: , 2006

15 Strategy Experimental In Vitro 2. HLM + NADPH + dansyl (d)gsh A. The amount of adduct formed (dgsh) can be measured and compared to benchmarked compounds that form reactive metabolites and have known hepatotoxicity Now we know the fraction metabolized (fm fm, HEP) )and relative amount of conjugate (dgsh dgsh)

16 Dansyl GSH Assay Assay Principle N N N H S N H HN HS NH H -HLM incubation -HPLC separation -Establish fluorescence responsivity with dgsh standard H S H P450 S dansylglutathione -Fluorescence does not change with adduct formation raloxifene putative quinoid metaboilite -Quantitate the amount of adduct formed Important: this is an assay that detects BIACTIVATIN, not TXICITY!* *Part of a DSRD risk matrix assay suite possible adduct (m/z 1012) N H N S S N H S H HN H H NH -Compare quantities to (+) controls -Convert to a % of substrate generating adduct(s) -Confirm adduct with accurate m/z (eliminate artifacts) -Developed and published by BMS; we run identically except we now use UPLC to increase throughput

17 Dansyl GSH Assay Possible utcomes for Each Test Compound No adducts detected True adducts detected (can detect 0.1%) MS makes sense MS cannot be readily rationalized for adduct structure Assay artifacts Drug and/or metabolites is fluorescent itself Drug causes degradation of dgsh (e.g. dgssg) Non-metabolic adduct formation (e.g. thiol exchange)

18 Strategy Experimental In Vivo 3. In Vivo A. Use in vivo matrices available (bile, urine, plasma) to examine in vitro relationship. If none exists, no further studies recommended. If so, then 4. Radiolabel A. Teams may choose to invest in labelled studies ( 3 H, 14 C) to provide expansive context of the liability for a given compound Q. How can I be sure I didn t miss something if my series didn t have a trigger? A. at Lead Develop., Drug Induced Liver Injury (DILI) team recommendations put compounds through a matrix of assays including dgsh

19 Return to in vitro Covalent Binding: A Convergence of Findings riginal D.C. Evans, et. al. paper defined the problem and suggested an approach (in vitro covalent binding assay) Three labs have tested the approach in different manners and came to the SAME conclusion: DSE IS IMPRTANT!! Report Drugs Systems bach, et al., (2008) Bauman, et al., (2009) 18 Covalent Binding CL int, Human Liver Microsomes, S-9 Fraction, and Hepatocytes Gan, et al., (2009) 50 Dansyl GSH Adducts in Human Liver Microsomes Nakayama, et al., (2009) 42 Covalent Binding, Human Liver Microsomes, Hepatocytes, and Rat Liver In Vivo **Both known toxics and non-toxics were tested!!! 19

20 Putting risk into context? Title Body burden of covalently bound dose vs. DILI risk SAFE WNG BBW / WDN Nakayama et. al., Drug Metab and Dispos. 2009, 37(9), Bauman et. al., Chem. Res. Toxicol. 2009, 22, Covalent binding and daily dose were significantly correlated with safety category

21 Considering the Integration of Mechanistic in vitro Assays In addition to Covalent Binding Burden in human hepatocytes In vitro panels for Drug induced Liver Injury Bile Salt Export Pump (BSEP) inhibition assess bile acid transport functions (with taurocholate) Mitochondrial dysfunction Freshly isolated rat liver mitochondria HepG2 glu/gal ratios Cytotoxicity: assess cellular lar ATP levels els in THLE cell lines Preview. during retrospective analyses, individual results were not predictive of IADR liability, but matrix assessments have discriminated compounds with high IADR concerns.

22 Association of Liver Injury with Mitochondrial Function and BSEP 72 drugs listed in US National Center for Tox Research Liver Tox Knowledge Base 24 Most DILI, 28 Less DILI, 20 No DILI BSEP & mitochondrial inhibition as function of FDA label categories

23 DILI Severity association with in vitro Panel Assays vs. Covalent Binding Burden Combining in vitro Hazard Matrix scores with CVB burden Multiple Adverse Properties Contribute to Liver Injury R. Thompson et. al., Chem. Res. Toxicol. 2012, 25, 1616 R. Thompson et. al., JPET. 2015, 352,

24 Applying DILI in vitro Matrix Results toward Clinical Exposures Liabilities Daily vs. Dose plasma with ClogP Cmax graph S tti Cli i l l l f f DILI i h i ti i it Setting Clinical exposure levels of concern for DILI using mechanistic in vitro assays F. Shah, et. al. Toxicol. Sciences, 2015, 147(2),

25 REVIEW STRATEGY best approach will include multi-parametric risk assessments HHEP s HLM + dgsh* Bioactivation Has Been bserved (i.e. Hazard has been identified) Stop N RM? Y Estimate f m,rm Bioactivation as Fraction of metabolism (f m ) -- can use HHeps Stop N Measureable fm,rm Y Qualitative X-Species IVIVC In Vivo Relevance? Quantitative Cl % of Dose CB [ 3 H] or [ 14 C] Predict in Covalent Binding Integration of vivo Body Burden Stop N human Y mechanistic Estimate; relevance in vitro assays; assays *r other indication of bioactivation Clinical Dose. Predict t Clinical i l Exposures

26 What is Important? What Should We Be Doing? Dose Pre-LD Post-LD Work early Avoid known structure alerts Drive down dose Potency Intrinsic CL Reduce lipophilicity Check for activation Consider dose Integrate mechanistic assays Predict clinical exposure 26

27 Summary Provide teams with a clear strategy for when and how to assess a series for bioactivation/reactive metabolite formation anticipated / unanticipated scenarios inter species differences in Clint, uncertainty of translating PD effects, uncertainty of clinical dose predictions, etc. Provide a balanced context for the RM results in the scope of metabolic clearance and a path for how to guide the team through potential issues Include assessment of mechanistic in vitro assays & dose predictions Enable teams to make the right staged investments N

28 Acknowledgments Biotransformation colleagues Jon Bauman Anne Hagen Amit Kalgutkar Scott bach Doug Spracklin Alfin Vaz Investigative Toxicology Michael Aleo

29 Backup Slides

30 Bioactivation Assays (Pfizer) GSH Adduct Standard Covalent dgsh (MS) Metabolite ID Binding Throughput Very High (Tier 1) Low 40-80/Week Very, Very Low Quantitative No Semi (from Yes Yes UV) Sensitivity Too High Moderate Moderate Highest Selectivity Too Low Not Tested High High Cost Cheapest Moderate Moderate Luxurious As always, we get what we pay for! Speed Quality Choose Two Cost ther published assays include other types of labeled nucleophile (e.g. [ 35 S]GSH, GSH-quaternary ammonium, etc)

31 Consider Reactive Metabolite Formation AND Dose How high is Detect Reactive Metabolites? high? N YES What defines detectable? Is the Dose High? YES N MAYBE BAD NGD MAYBE 31

32 Putting risk into context? Title Body burden of covalently bound dose vs. DILI risk Now we know the fraction metabolized (fm, HEP) and relative amount of conjugate (dgsh) calculate Daily Body Burden of Covalent Binding: Daily Body Burden of Covalent Binding: D cb = f CL,cb x Daily dose where f CL,cb is the fraction of metabolism comprised by covalent binding: f CL,cb = CL int,cb / (CL int + CL int,cb ) and CL int,cb is the covalent binding intrinsic clearance and CL int is the intrinsic clearance in hepatocytes pmol equiv bound to protein pmol compound in Incubation Projected X Daily Dose = (mg/day) Liver (Body) Burden of Covalent Binding (mg/day) Limitation: This assessment is derived from drugs which generate reactive metabolites in the liver Caveat: Does a hepatocyte assay fully mimic covalent modifier reactivity in other tissues? Bauman et. al., Chem. Res. Toxicol. 2009, 22, Thompson, Chem Res Toxicol 2012

33 Title