Analytical Technologies for Metabolite Identification and Quantitation

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Analytical Technologies for MetaboliteIdentification and Quantitation

Graham LappinChief Scientific Officer

Xceleron Inc


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

Analytical Technologies for MetaboliteIdentification and Quantitation


What is MIST?

Is the exposure of metabolites in thespecies used for toxicology studies appropriateto human?

How many puns can you think of?Metabolites we have MIST?MIST opportunitiesThe MIST has cleared

MetabolitesInSafetyTesting

Gorillas in the MIST!And my favourite ….


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 Regulatory Guidelines (in a nutshell)

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


Primarily plasma (but also excreta)

StructuralElucidation Quantitation

Requires the right combination ofanalytical methods

Qualitative and Quantitative Analysis

Samples fromanimal and human studies


Animal metabolism studiestraditionally conducted using14C-drug

Preclinical Metabolism Studies

Mass spectrometryand (perhaps) NMRfor structural elucidation

Scintillation counting for14C for quantitation

The need for animalmetabolism studies however, are nowbeing questionedeg Obach, Nedderman & Smith Xenobiotica 42(1), 46-56 (2012)


Human Metabolism Studies



The use of 14C in human studiesis more problematic than with animals

Ideally First in ManStudy


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 hepatocyteIncubations with the drug

Data used as a guidequantitative predictionsin humans generallynot good

Selection ofmetabolitestandards forsynthesis

Animal metabolism data


Non-radiolabelled methods in humans

Singleascendingdose (SAD)

PharmacovigilanceTolerabilityPharmacokineticsPharmacodynamics

Samples of plasma and excreta for metabolite analysis

Mass spec(eg high resolutionmass defectfiltering)

NMR

Sensitive partially-quantitativeassay

Relatively insensitive quantitativeassay

Objective is to obtain a human quantitative metabolic pathway to compare with the toxicology species


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).


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).


Human Metabolism Studies



Ideally First in ManStudy

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


AMS is exquisitely sensitive to 14C

AMS

Atoms separated 12C,13C and 14C atomsindividually countedby differences in mass/charge

and energy

-Decay of 14C atomDetected by scintillation counting as photons

of light in photomultiplier tube

0.012% of 14C decays per annum; 2.3 billion 14C atoms ≡ 1 dpm

Key

Sample containing 12C 13C and 14C atoms

1000 14C atoms required for valid measurement

Scintillation counting


AMS Instrument

250kV AMS based at Xceleron, Germantown, Maryland


Metabolite profile - Ixabepilone

Metabolites in plasma at 4 and 8 hrsShowing 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.


Nothing gets MIST (!)

In vitro

Selection of metabolitestandards

Monitor in human(add confidence viaother MS-screening)

Phase-IHumanADME

A risk remains offinding unpredictedhuman metaboliteslate in development


Nothing gets MIST (!)

In vitro

Selection of metabolitestandards

Phase-ITrace amount of 14C-drug

Full profile and recoveryNothing gets missed


AMS strategic approach to MIST

“therapeutic” dose of drugcontaining small amount of 14C-compoundSAD

Study

Sample collection (plasma & excreta)

Massspectrometryfor metaboliteidentification

AMS for metabolitequantification


AUC Pool approach

1) AUC plasma pool

Time (h) Period (h)

0 0

0.25 0.25

0.5 0.25

1 0.5

2 1

4 2

8 4

The volume of each plasmasample pooled is adjusted proportional to the collectionperiod

2) 14C-HPLC Profile

Typically one AUC pool is madeacross all subjects. The pools couldhowever, be made for individual subjects if so desired


Data interpretation

Parent(50% of total)40% of parent

15% of total

30% of parent11% total

The 14C makes the peaks directly comparable quantitatively(even if the actual chemical structures are unknown)

<10% of parent3.8% total


Case study: Lundbeck A/S

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

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


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


Metabolite profile by HPLC and AMS

Parent drug

No metaboliteis > 10% of total


MIST StrategyIn vitro metabolismFirst information on x-species metabolite profiles

In vivo metabolism Study in animals with 14C-labelled compound. First in vitro/in vivo comparison

FIM study with 14C-tracerInformation 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


MIST data package

Direct quantitative comparison, in vitro, animal species and humanConfidence everything is accounted for by use of radiolabel

Human

14C & AMS

In vitro Rat Dog

14C & scintillation counting

Metabolite elucidation with MS and NMRMonitoring of late-stage clinical studies with MS

Risk mitigation forMIST guidelines


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

Conclusions


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 LappinXceleron [email protected]

Analytical Technologies for Metabolite Identification and Quantitation

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