Reactive e Metabolite Trapping

Transcription

1 Reactive e Metabolite Trapping Sylvia Zhao, Ph.D. China R&D and Scientific Affairs June 22, 南京国际药代会议 0 Outline Role of bioactivation in drug toxicity Reactive metabolite trapping methods Application in drug R&D A case study Data interpretation and correlation with drug induced toxicity 1 1

2 Reactive Intermediates/Metabolites and Drug Toxicity Reactive intermediates derived from the metabolism of drugs have been implicated in target organ toxicity and immune-mediated idiosyncratic adverse events For 21 marketed drugs examined, five out of six drugs withdrawn from the market and eight out of 15 drugs given black box warnings provided evidence for the formation of reactive metabolites Properties of reactive intermediate Electrophiles or free radicals Generally unstable with short half-life (msec ~ min), difficult to detect directly Chemically reactive 2 Reactive Metabolite-Induced Toxicity: History and Example Covalent modification of proteins by xenobiotics first reported by Milliers in 1947 The concept of reactive intermediate formation and protein covalent binding contributing to the toxicity of drugs and other chemicals emerged from pioneering academic studies in 1970s First proposed in acetaminophen (APAP)-induced hepatotoxicity (Brodie, BB, 1973) Clinical findings: Safe at therapeutic doses (~15 mg/kg, 4 g/day) Overdose cause hepatotoxicity; 9000 reported cases/year in U.S Centrilobular hepatic necrosis (150 mg/kg and higher, or > 10 g) Potentially fatal or acute liver failure (350 mg/kg ) 2

3 An Example: Acetaminophen Biotransformation Ward C, Sair M Contin Educ Anaesth Crit Care Pain 2010;10: Methodologies to Evaluate Reactive Metabolite for Risk Assessment In silico prediction Structure alert, SAR DEREK, TOPKAT, CASE, OncoLogic These software programs can lead to many false positives Detection of reactive metabolites With the possible exception of acyl glucuronides and certain cyclic iminium ions, most reactive metabolites are short-lived and are not detectable in circulation Their formation can be inferred from the characterization of stable conjugates formed via reaction with in vitro and/or in vivo trapping agents such as reduced glutathione (GSH). Covalent binding 5 3

4 Reactive Metabolite Trapping Yield stable conjugates amenable to structural characterization using LC-MS/MS and/or NMR structure elucidation providing insight into the bioactivation mechanism and hence a rationale on which to base subsequent chemical intervention strategies It does not require radiolabeled material and is amenable to high throughput screening Information provided is purely qualitative in nature yet quantitation may be achieved by radiolabeled material 6 Trapping Agents Soft Nucleophiles Thiol group in GSH Amino groups and aromatic carbons On proteins: N-histidine, S-cysteine, N-valine, In DNA/RNA: N-7-guanine, N-1- and N-3-adenine, O6-guanine Soft electrophilic intermediates include activated double bond such as unsaturated carbonyl compounds, quinones, quinoneimines, quinomethides and diiminoquinones 7 4

5 GSH Adducts The most traditional and common method, using LC-MS/MS analysis by neutral loss (NL) scanning of 129 Da Not very specific, false positives as matrix constituents with the same NL reaction are often detected Poor specificity of positive ion mode NL scanning can be improved by using a 1:1 mixture of stable isotope labeled GSH ( 13 C 2, 15 N- glutathione) and nonlabeled glutathione as a trapping agent, leading to an easily distinguishable molecular ion doublet with three mass units (3 Da) separation for the real GSH conjugates Not all classes of GSH conjugates form the neutral loss of 129 Da in CID (collision-induced i i d dissociation), i leading to false negatives in screening Can be overcome by using precursor ion (PI) scanning for the fragment ion at m/z 272 in negative ion mode, but the detection sensitivity of the PI mode is also relatively poor 8 Trapping Agents-Hard Nucleophiles Amines (e.g., semicarbazide and methoxylamine), amino acids (e.g., lysine), and DNA bases (e.g., guanine and cytosine), For hard electrophiles, including DNA-reactive metabolites (e.g., aldehydes) Cyanide anion For electrophilic iminium species that are generated via metabolism of acyclic and cyclic tertiary amines Hard electrophiles 9 5

6 Case Study (Janssen) Chem. Res. Toxicol. 2011, 24, Case Study: An A 2A /A 1 receptor antagonist for the treatment of Parkinson s disease Positive in Ames (Salmonella typhimurium strain TA1537) and Mouse Lymphoma Assay (L5178Y), but only after the metabolic activation by Aroclor induced rat liver S9 Hypothesis: metabolite(s) but not parent responsible for mutagenic response in TA1537 Lead to intercalation and/or covalent modification of DNA DMPK approaches Reactive metabolite trapping, identification and quantification +S9 -S9 11 6

7 Trapping with CN - 3 cyano conjugates detected by isotopic pattern mass filtering of full scan accurate mass data Reconstructed ion chromatogram (RIC) corresponding to the neutral mass loss of 27 Da from isotopic pattern triggered data-dependent mass analysis China R&D and SA 12 Trapping with Methoxylamine 3 oxime conjugates were detected by interrogation of full scan accurate mass data by isotopic pattern filtering, 2 by a targeted product ion scan RIC corresponding to the base peak at exact m/z from a product ion scan of [M + H]+ of oxime conjugate at m/z 402 following an analysis China R&D and SA 13 7

8 Trapping with GSH 3 GSH conjugates detected from the full scan accurate MS data by isotopic pattern filtering All 3 conjugates displayed neutral mass losses diagnostic of GSH conjugates upon CID of their respective [M +H] +, including losses of pyroglutamic acid (minus 129 Da) and the GSH molecule (minus 307 Da) GSM Reactive Intermediate Structure Elucidation Cyano conjugation Oxime conjugation GSH conjugation Iminium ion Aldehyde Epoxide Unsaturated keto Enamine 15 8

9 Effect on Covalent DNA Binding 16 Impact on Project and Backup Molecule Design 17 9

10 Endogenous Defense Systems Endogenous GSH represents the major cellular scavenger due to the high cellular concentration (5-10 mm) and high polarizability May conjugate hard electrophiles when catalyzed by glutathione S-transferases (GST) Other enzyme systems such as epoxide hydrolyse, quinone reductase, SOD, catalase, GPx Endogenous small molecules such as Vit E and C, uric acid, bilirubine etc Stress leads to the induction of genes and proteins that express chaperone, antioxidant, xenobiotic detoxication, and protein degradation functions 18 Data Interpretation and Application in Drug R&D Detection of conjugates provides evidence for RM formation, However the data needs to be placed in proper context prior to making a decision on discarding a RM positive compound First and foremost, a drug candidate that is devoid of RM formation and/or covalent binding to liver microsomal protein is not a guarantee of its safety Many safe drugs (e.g. propranolol) generate reactive intermediate(s) in in vitro screens Some factors to be considered: Extent of metabolism Target proteins Dose and exposure Indication Disease state and genetics 19 10

11 Extent of Adduct Formation and Drug-Induced Toxicity (BMS) Dansyl-glutathione was used as a trapping agent for the quantitation of adducts Daily Burden of Reactive Metabolites Calculation: Drm=D fa fm frm Gan et al, Chem. Res. Toxicol. 2009, 22, Correlation of GSH Adduct Formation and Hepatotoxicity (GSK) Clinical dose and GSH adduct formation were significantly associated with hepatotoxicity Of the drugs that had dose 100 mg/day, 67% were hepatotoxic in man and 76% of the drugs with dose <100 mg/day were non-hepatotoxic (p < ) Of the drugs positive for GSH adduct formation 65% were hepatotoxic; 58% of the drugs negative in the assay were non-hepatotoxic (p = 0.003) Reese et al, Chemico-Biological Interactions, 2011, 192,

12 Correlation of MBI or CvB and Hepatotoxicity Reese et al, Chemico-Biological Interactions, 2011, 192, Processes for Generation and Characterization of Reactive Intermediates Orhan and Vermeulen, Curr Drug Metab, 2011, 12,

13 Thank you! 13