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Xceleron - all rights reserved ©2009 Graham Lappin Chief Scientific Officer Xceleron Inc.

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Presentation on theme: "Xceleron - all rights reserved ©2009 Graham Lappin Chief Scientific Officer Xceleron Inc."— Presentation transcript:

1 Xceleron - all rights reserved ©2009 Graham Lappin Chief Scientific Officer Xceleron Inc

2 Xceleron - all rights reserved ©2009 Summary 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 Xceleron - all rights reserved ©2009 What is MIST? Is the exposure of metabolites in the species used for toxicology studies appropriate to human? How many puns can you think of? Metabolites we have MIST? MIST opportunities The MIST has cleared Metabolites In Safety Testing Gorillas in the MIST! And my favourite ….

4 Xceleron - all rights reserved ©2009 FDA MIST guideline Human metabolites that can raise a safety concern are those formed at greater than 10% of parent drug systemic exposure at steady 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 Regulatory Guidelines (in a nutshell) The ICH guideline should take precedence although a number of pharmaceutical 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 Xceleron - all rights reserved ©2009 Primarily plasma (but also excreta) Structural Elucidation Quantitation Requires the right combination of analytical methods Qualitative and Quantitative Analysis Samples from animal and human studies

6 Xceleron - all rights reserved ©2009 Animal metabolism studies traditionally conducted using 14 C-drug Preclinical Metabolism Studies Mass spectrometry and (perhaps) NMR for structural elucidation Scintillation counting for 14 C for quantitation The need for animal metabolism studies however, are now being questioned eg Obach, Nedderman & Smith Xenobiotica 42(1), 46-56 (2012)

7 Xceleron - all rights reserved ©2009 Human Metabolism Studies Mass spectrometry and (perhaps) NMR for structural elucidation Scintillation counting for 14 C for quantitation The use of 14 C in human studies is more problematic than with animals Ideally First in Man Study

8 Xceleron - all rights reserved ©2009 In vitro 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). Microsome and hepatocyte Incubations with the drug Data used as a guide quantitative predictions in humans generally not good Selection of metabolite standards for synthesis Animal metabolism data

9 Xceleron - all rights reserved ©2009 Non-radiolabelled methods in humans Non-radiolabelled methods in humans Single ascending dose (SAD) Pharmacovigilance Tolerability Pharmacokinetics Pharmacodynamics Samples of plasma and excreta for metabolite analysis Mass spec (eg high resolution mass defect filtering) NMR Sensitive partially-quantitative assay Relatively insensitive quantitative assay Objective is to obtain a human quantitative metabolic pathway to compare with the toxicology species

10 Xceleron - all rights reserved ©2009 Mass spectrometric methods 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 14 C, 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 as isotopic 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 Xceleron - all rights reserved ©2009 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 issue when looking for metabolites 10% of total Use of sensitive NMR cryoprobes * and coupling to LC For fluorine-containing drugs, 18 F-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 Xceleron - all rights reserved ©2009 Human Metabolism Studies Mass spectrometry and (perhaps) NMR for structural elucidation Scintillation counting for 14 C for quantitation Ideally First in Man Study Use of accelerator mass spectrometry AMS for the measurement of 14 C With the use of AMS the burden of radioactivity to human subjects is reduced so low that the study could be classified as non-radioactive

13 Xceleron - all rights reserved ©2009 AMS is exquisitely sensitive to 14 C AMS Atoms separated 12 C, 13 C and 14 C atoms individually counted by differences in mass/charge and energy -Decay of 14 C atom Detected by scintillation counting as photons of light in photomultiplier tube 0.012% of 14 C decays per annum; 2.3 billion 14 C atoms 1 dpm Key Sample containing 12 C 13 C and 14 C atoms 1000 14 C atoms required for valid measurement Scintillation counting

14 Xceleron - all rights reserved ©2009 AMS Instrument 250kV AMS based at Xceleron, Germantown, Maryland

15 Xceleron - all rights reserved ©2009 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 Xceleron - all rights reserved ©2009 Nothing gets MIST (!) In vitro Selection of metabolite standards Monitor in human (add confidence via other MS-screening) Phase-I Human ADME A risk remains of finding unpredicted human metabolites late in development

17 Xceleron - all rights reserved ©2009 Nothing gets MIST (!) In vitro Selection of metabolite standards Phase-I Trace amount of 14 C-drug Full profile and recovery Nothing gets missed

18 Xceleron - all rights reserved ©2009 AMS strategic approach to MIST therapeutic dose of drug containing small amount of 14 C-compound SAD Study Sample collection (plasma & excreta) Mass spectrometry for metabolite identification AMS for metabolite quantification

19 Xceleron - all rights reserved ©2009 AUC Pool approach 1) AUC plasma pool Time (h)Period (h) 00 0.25 0.50.25 10.5 21 42 84 The volume of each plasma sample pooled is adjusted proportional to the collection period 2) 14 C-HPLC Profile Typically one AUC pool is made across all subjects. The pools could however, be made for individual subjects if so desired

20 Xceleron - all rights reserved ©2009 Data interpretation Parent (50% of total) 40% of parent 15% of total 30% of parent 11% total The 14 C makes the peaks directly comparable quantitatively (even if the actual chemical structures are unknown) <10% of parent 3.8% total

21 Xceleron - all rights reserved ©2009 Case study: Lundbeck A/S Lappin, G. Seymour, M. Gross, G. J ø gensen, M. Kall, M and Kv ӕ rnø, L. (2012) Meeting the regulatory requirements in MIST: Human metabolism data in phase-1 using AMS with a tiered bioanalytical approach in metabolite quantification. Bioanalysis in press MIST investigation included in SAD study 10 mg, 500 nCi 14 C-drug administered to the 4 th cohort of 6 subjects Decision on which cohort to include 14 C-drug made last-minute (ie flexible protocols are feasible) Plasma samples taken over time Total 14 C measured in plasma and samples selected for AUC pool AUC pool extracted and analysed by HPLC and AMS

22 Xceleron - all rights reserved ©2009 Total 14 C in plasma AUC for total 14 C (ie total drug-related material) Example of how parent drug (measured with LC-MS) can be compared with total 14 C Metabolites vs parent in plasma

23 Xceleron - all rights reserved ©2009 Metabolite profile by HPLC and AMS Parent drug No metabolite is > 10% of total

24 Xceleron - all rights reserved ©2009 MIST Strategy In vitro metabolism First information on x-species metabolite profiles In vivo metabolism Study in animals with 14 C- labelled compound. First in vitro/in vivo comparison FIM study with 14 C-tracer Information on metabolite profile and systemic exposure in human after SD obtained using AMS Fit for purpose Bioanalytical method Quantify animal exposure of relevant metabolites Validated Bioanalytical method Comparison of systemic exposure in human after MD versus animal species at steady state Synthesize major in vitro metabolites Synthesize major systemic in vivo metabolites Use metabolite standards to identify and quantify key metabolites; potential synthesis of additional metabolites Use metabolite standards for fit for purpose bioanalytical method Use metabolite standards to compare systemic exposure x-species

25 Xceleron - all rights reserved ©2009 MIST data package Direct quantitative comparison, in vitro, animal species and human Confidence everything is accounted for by use of radiolabel Human 14 C & AMS In vitroRatDog 14 C & scintillation counting Metabolite elucidation with MS and NMR Monitoring of late-stage clinical studies with MS Risk mitigation for MIST guidelines

26 Xceleron - all rights reserved ©2009 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 14 C-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 Conclusions

27 Xceleron - all rights reserved ©2009 Mark Seymour and all the folks of Xceleron Gerhard Gross and his co-workers at Lundbeck A/S Celerion and the organisers of ASCPT Acknowledgements Questions gratefully received Graham Lappin Xceleron Inc

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