Analytical Technologies for Metabolite Identification and Quantitation

1. Analytical Technologies for MetaboliteIdentification and Quantitation Graham Lappin Chief Scientific OfficerXceleron Inc

2. Summary Analytical Technologies for MetaboliteIdentification and Quantitation • What methods are available for the identification and quantitation of metabolites to satisfy the MIST guidance? • How a combination of techniques is most powerful • The use of Accelerator Mass Spectrometry as part of an overall strategy • Case studies given as examples

3. What is MIST? Metabolites In Safety Testing How many puns can you think of? Metabolites we have MIST? MIST opportunities The MIST has cleared And my favourite …. Gorillas in the MIST! Is the exposure of metabolites in thespecies used for toxicology studies appropriateto human?

4. The Regulatory Guidelines (in a nutshell) FDA “MIST” guideline “Human metabolites that can raise a safety concern are those formed at greater than 10% of parent drug systemic exposure atsteady state.” ICH M3 guideline “… exposures greater than 10% of total drug-related exposure and at significantly greater levels in humans than the maximum exposure seen in the toxicology studies.” The ICH guideline should take precedence although a number ofpharmaceutical companies still look at steady state The real concern is finding human specific or human disproportionate metabolites at a late stage of drug development

5. Qualitative and Quantitative Analysis Requires the right combination ofanalytical methods Samples fromanimal and human studies Primarily plasma (but also excreta) StructuralElucidation Quantitation

6. Preclinical Metabolism Studies Animal metabolism studiestraditionally conducted using14C-drug Mass spectrometry and (perhaps) NMRfor structural elucidation Scintillation counting for14C for quantitation The need for animalmetabolism studies however, are nowbeing questioned eg Obach, Nedderman & SmithXenobiotica 42(1), 46-56 (2012)

7. Human Metabolism Studies Mass spectrometry and (perhaps) NMRfor structural elucidation Ideally First in ManStudy Scintillation counting for14C for quantitation The use of 14C in human studiesis more problematic than with animals

8. In vitro data Selection ofmetabolitestandards forsynthesis Data used as a guide quantitative predictionsin humans generallynot good Microsome and hepatocyte Incubations with the drug Animal metabolism data Anderson, S, Luffer-Atlas, D and Knadler, MP. Predicting circulating human metabolites: how good are we? Chem Res Toxicol 22(2), 243-56 (2009).

9. Non-radiolabelled methods in humans Samples of plasma and excreta for metabolite analysis Singleascendingdose (SAD) Mass spec(eg high resolutionmass defectfiltering) NMR Relatively insensitive quantitativeassay Pharmacovigilance Tolerability Pharmacokinetics Pharmacodynamics Sensitive partially-quantitativeassay Objective is to obtain a human quantitative metabolic pathway to compare with the toxicology species

10. Mass spectrometric methods • Mass spec excellent for structural elucidation but not quantitative in the absence of standards • Metabolites quantified using standards synthesised based on in vitro and animal data. • Partial-quantification of human metabolites by comparison with same metabolites quantified using 14C, from animal radiolabelled studies. • Use of less structurally-dependent LC-MS ion-sources (eg nanospray*) • Overall, MS methods are excellent at metabolite identification but are not as robustly quantitative asisotopic measurements * - Ramanathan, R, Zhong, R, Blumenkrantz, N, Chowdhury, SK and Alton, KB. Response normalized liquid chromatography nanospray ionization mass spectrometry. J Am Soc Mass Spectrom 18(10), 1891-9 (2007).

11. NMR • NMR provides complete structural elucidation (including chirality). • Quantitative in the absence of standards • Relatively insensitive (typically high ng amounts required) • Assay sensitivity can be an issuewhen looking for metabolites 10% of total • Use of sensitive NMR cryoprobes* and coupling toLC • For fluorine-containing drugs, 18F-NMR is an option • Overall NMR is excellent at metabolite identification and is quantitative but relatively insensitive * - Espina, R, Yu, L, Wang, J, et al. Nuclear magnetic resonance spectroscopy as a quantitative tool to determine the concentrations of biologically produced metabolites: implications in metabolites in safety testing. Chem Res Toxicol 22(2), 299-310 (2009).

12. Human Metabolism Studies Mass spectrometry and (perhaps) NMRfor structural elucidation Ideally First in ManStudy Scintillation counting for14C for quantitation Use of accelerator mass spectrometry AMS for the measurement of14C With the use of AMS the burdenof radioactivity to human subjectsis reduced so low that the study couldbe classified as non-radioactive

13. -Decay of 14C atom Detected by scintillation counting as photons of light in photomultiplier tube Atoms separated 12C,13C and 14C atoms individually counted by differences in mass/charge and energy AMS is exquisitely sensitive to 14C Scintillation counting 0.012% of 14C decays per annum; 2.3 billion 14C atoms ≡ 1 dpm AMS 1000 14C atoms required for valid measurement Key Sample containing12C 13C and 14C atoms

14. AMS Instrument 250kV AMS based at Xceleron, Germantown, Maryland

15. Metabolite profile - Ixabepilone Metabolites in plasma at 4 and 8 hrs Showing complex profiles Comezoglu, S. N., Ly, V. T., Zhang, D., Humphreys, W. G., Bonacorsi, S. J., Everett, D. W., Cohen, M. B., Gan, J., Beumer, J. H., Beijnen, J. H., Schellens, H. M. and Lappin, G., (2009) Drug Metab Pharmacokinet 24(6): 511-522.

16. Nothing gets MIST (!) HumanADME Phase-I Selection of metabolitestandards In vitro Monitor in human (add confidence viaother MS-screening) A risk remains offinding unpredictedhuman metaboliteslate in development

17. Nothing gets MIST (!) Phase-I Trace amount of 14C-drug Selection of metabolitestandards In vitro Full profile and recovery Nothing gets missed

18. AMS strategic approach to MIST “therapeutic” dose of drugcontaining small amount of 14C-compound SADStudy Massspectrometry for metaboliteidentification Sample collection (plasma & excreta) AMS for metabolitequantification

19. AUC Pool approach The volume of each plasmasample pooled is adjusted proportional to the collectionperiod 1) AUC plasma pool Typically one AUC pool is madeacross all subjects. The pools couldhowever, be made for individual subjects if so desired 2) 14C-HPLC Profile

20. Data interpretation Parent (50% of total) 40% of parent 15% of total 30% of parent 11% total <10% of parent 3.8% total The 14C makes the peaks directly comparable quantitatively(even if the actual chemical structures are unknown)

21. Case study: Lundbeck A/S • MIST investigation included in SAD study • 10 mg, 500 nCi 14C-drug administered to the 4th cohort of 6 subjects • Decision on which cohort to include 14C-drug made last-minute (ie flexible protocols are feasible) • Plasma samples taken over time • Total 14C measured in plasma and samples selected for AUC pool • AUC pool extracted and analysed by HPLC and AMS Lappin, G. Seymour, M. Gross, G. Jøgensen, M. Kall, M and Kvӕrnø, L. (2012)Meeting the regulatory requirements in MIST: Human metabolism data in phase-1using AMS with a tiered bioanalytical approach in metabolite quantification.Bioanalysis in press

22. Total 14C in plasma AUC for total 14C (ie total drug-related material) Example of how parent drug (measured with LC-MS) can be compared with total 14C Metabolitesvs parentin plasma

23. Metabolite profile by HPLC and AMS Parent drug No metaboliteis > 10% of total

24. In vitro metabolism First information on x-species metabolite profiles Synthesize major in vitro metabolites MIST Strategy In vivo metabolism Study in animals with 14C-labelled compound. First in vitro/in vivo comparison Synthesize major systemic in vivo metabolites FIM study with 14C-tracer Information on metabolite profile and systemic exposure in human after SD obtained using AMS Use metabolite standards to identify and quantify key metabolites; potential synthesis of additional metabolites “Fit for purpose” Bioanalytical method Quantify animal exposure of relevant metabolites Use metabolite standards for ”fit for purpose” bioanalytical method Validated Bioanalytical method Comparison of systemic exposure in human after MD versus animal species at steady state Use metabolite standards to compare systemic exposure x-species

25. MIST data package In vitro Rat Dog Human 14C & scintillation counting 14C & AMS Direct quantitative comparison, in vitro, animal species and human Confidence everything is accounted for by use of radiolabel Risk mitigation forMIST guidelines Metabolite elucidation with MS and NMRMonitoring of late-stage clinical studies with MS

26. Conclusions • A range of techniques are available for metabolite identification and quantitation to satisfy the MIST requirements • MS is excellent for metabolite identification but only partially-quantitative in absence of authentic standards • NMR is excellent at metabolite identification and quantification but relatively insensitive • AMS enables low levels of 14C-drug to be given in phase-1 studies, it is highly sensitive and fully quantitative but does not provide structural information (other than co-elution with standards) • A combination of methods provides the best strategy to mitigate the risks of finding human disproportionate metabolites late in development

27. Acknowledgements • Mark Seymour and all the folks of Xceleron • Gerhard Gross and his co-workers at Lundbeck A/S • Celerion and the organisers of ASCPT Questions gratefully received Graham Lappin Xceleron Inc graham.lappin@xceleron.com