Current practices and future vision on metabolite profiling and

quantification in GSK

Graeme Young – September EBF Workshop, Brussels 2015

Property of GlaxoSmithKline

Overview

• GSK strategy – Human First ! [“Human is the best model for human”]

• Cold metabolism – the “free data” concept

• Cold metabolism + 14C tracer

• Specific assays……absolute quantification with authentic standards

• Long winded approach – use of 14C for multiple species metabolite profiling

• Future vision

Presentation title 3

Steady state Quantification

Definitive structures

In silico prediction

Better contextualise

after FTIH

Mechanism? Did it leave? How much? Where is it? What is it?

Early human focus

Human ADME – human is the best model for human

NMR

AMS MS

Tracer approaches – novel designs

Human first approach – FTIH: first opportunity to understand human ADME

Differentiated development – reduced animal work, less up front cost

Integrated tools – complementary analytical tools dedicated to metabolism

Bile string Urine Blood/Plasma Phase I opportunity

Less reliance on conventional 14C

study Routes of metabolism – the missing piece of the jigsaw

Need to quantify Need to identify Need to detect Analytical foundation

Early patient focus

Problems with historical approach

in silico Too many options; how

predictable for human

especially circulation

in vitro “artificial” system;

good for routes

~70% predictable

Not routinely used to select

toxicology species

Pre-clinical in vivo

Resource intensive

ID, quant. on non-human mets.

What are likely to be relevant?

Conventional Human Radiolabel Study

HRS Circulating metabolites

Routes of excretion

% urine % faeces

complex ethics Phase 2b/3

Body Burden %dose/metabolite & % unextracted

Single dose Human dose

What can we do in humans earlier? – the “free data” concept

Human Non-radiolabel Circulating metabolites

Routes of excretion

% urine

Single & Repeat dose

Faeces!

duodenal Bile*

*Entero-Test® ; GSK has used this approach in > 12 clinical studies (most in early clinical)

Guiney WJ, Beaumont C, Thomas SR, Robertson DC, McHugh SM, Koch A, Richards D. Use of Entero-Test, a simple approach for non-invasive clinical evaluation of the biliary disposition of drugs. Br J Clin Pharmacol 2011; 72: 133-42.

Cold metabolism….”free data”

NMR based cold metabolism approach

DRUG

30

PLASMA

70

Re

spo

nse

HPLC fractionation

Pool or bulk sample

Concentrate/extract sample

NMR NMR of fractions

% Quantify

Time

MET. M1

Dear GJ, Roberts AD, Beaumont C, North SE. Evaluation of preparative high performance liquid chromatography and cryoprobe-nuclear magnetic resonance spectroscopy for the early quantitative estimation of drug metabolites in human plasma. J. Chromatogr. B 2008; 876: 182–90.

Cold metabolism : healthy subject example

Glucuronidation is major route of elimination

• Contribution of oxidative metabolism (CYP3A4) unclear from in vitro data

• Human bile was analysed in early clinical development (non-invasive bile string)

• The primary pathway of metabolism was identified as O-glucuronidation

• Data used to inform on DDI risk strategy

Bloomer JC, Nash M, Webb A, Miller BE, Lazaar AL, Beaumont C, Guiney WJ. Assessment of potential drug interactions by characterization of human drug metabolism pathways using non-invasive bile sampling. Br J Clin Pharmacol 2013; 75: 488–96.

Cold metabolism example : patient focussed

• Sitamaquine is an orally active 8-aminoquinoline - shown potential for treatment

of Visceral leishmaniasis (VL) in Africa and India

• Despite potential for sitamaquine in treating VL, notable toxicity has been demonstrated in non-clinical species and clinically relevant exposure is only achieved in one species (the rat)

N

NH

O

N

• Animal welfare/ethical issues preclude further animal studies with sitamaquine

and development has been via carefully conducted clinical studies in patients with

VL, with intensive safety monitoring

• Also precludes a Human Radiolabel Study (HRS) in healthy volunteers

• In vivo human metabolism data generated via cold metabolism approach

urine and plasma examined by NMR and MS to determine the nature and

amounts of metabolite

Cold metabolism example : patient focussed

Minimum Body Burden

Major circulating

Major metabolic route

Renal Clearance

Cold metabolism example : Sitamaquine – Patient focussed

Cold metabolism + 14C tracer

Cold metabolism + 14C tracer

• Design using tracers of 13C- or 14C-labelled drug

tracer choice dictated by assay sensitivity and study endpoints IV 13C- or 14C-tracer dosed concomittant to Oral therapeutic dose

• Repeat dose 14C study designs are now feasible

often low ionising radiation exposure; <1µCi dose

• IV microtracer study design* adopted by many Pharma supplementary benefit of 14C over the “cold metabolism” approach provides the “what is it?” and the “how much?” providing valuable PK/ADME information particularly for early stage assets

* Approaches to intravenous clinical pharmacokinetics: Recent developments with isotopic microtracers, Lappin G., The Journal of Clinical Pharmacology 2015, on-line advance version, 1–13.

Cold metabolism + 14C tracer: example

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Time after oral dose (hours)

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nce

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atio

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f SR

T2

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4 in

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sma

(n

g/m

L)

Oral (dose of 250mg)

IV data from 14C tracer of 100ug

(Normalised to 250mg)

Provided by AMS (parent isolation by UPLC)

Provided by LC/MS

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dpm

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uL H

PLC

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ctio

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M17

M16

M21,M22

M27

M26

M19 M18

M8

F%

Human PK

Metabolism

• combining MS, NMR, AMS

Cold metabolism + 14C tracer - Metabolism

• Data from metabolic profiling “informs” for further development strategies

– provide useful data for safety assessment and clinical study designs

assessment of total observed drug related material in urine from PO therapeutic route dose (NMR)

• Each technology (AMS, LC/MS & NMR ) provides different insights

14C use allows assessment of eg. extraction recoveries

more comprehensive comparison of metabolism in animals and human

definition of full structural ID and amounts

specific assay for isolated metabolite (by “LC+AMS”) also possible

Synthetic standards – specific assays

Frequency of occurrence……quantitative metabolite assays (within bioanalytical group)

• Proportion of [in-house] clinical assays which included metabolites

In 2015 (Jan to August) it was ~5%

Interestingly - in 2008 (Jan to August ) it was ~15% [larger data set than 2015]

on the decrease? ; pragmatism on the rise?

possible impact of “differential development” ?

Specific assays by LC/MS

The longwinded approach….

The longwinded approach….

• Human ADME support produced evidence of two “major” metabolites – known instability issue - dilute and shoot approach and AMS applied

0

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0 10 20 30

dp

m

Time (mins)

.

Parent

M2/others

M4

M2/others ~8% M4 = 11%

Human ADME data Plasma Pool

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0 20 40 60

dp

m

Time (mins)

.

M2

M2 resolved and no longer considered a major metabolite.

Human ADME data Plasma Pool

• Synthesis of metabolite standards not viable

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0.1

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0 10 20 30

dp

m

Time (mins)

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Parent

M2/others

M4

M2/others ~8% M4 = 11%

Human ADME data Plasma Pool

0

0.1

0.2

0 20 40 60

dp

m

Time (mins)

.

M2

M2 resolved and no longer considered a major metabolite.

Human ADME data Plasma Pool

Elapsed Time > 1 Year

The longwinded approach….

• Follow-on dosing of 14C-drug to rodent and non-rodent

• Cover estimates made based on quantification via profiling

Future Vision

• Drug to target – human tissue sampling

leaning towards imaging for more intact tissue assessment

moving from qualitative to more quantitative [mimetic model approach]*

• Increased use of microtracer approaches ; combined power of AMS, MS, NMR

access to key matrix – bile sampling [particularly useful with IV admin.]

• Greater focus on patients rather than healthy subjects…….

Future vision….

* A Mimetic Tissue Model for the Quantification of Drug Distributions by MALDI Imaging Mass Spectrometry, Groseclose M.R. and Castellino S., Anal.Chem., 2013 , 85 (21):10099-106.

GSK Clinical studies (incl. patients) – drug to site of action/tissue of interest

Skin cores…. healthy skin and lesions

Important Note – even when we have shown that drug appears to be close to the target site or site of toxicity (real or inferred) ; has not necessarily lead to efficacy or provided clear link to toxicity …but at least one step (maybe several) closer to supporting the “pillars of success” than eg. analysis of drug in blood plasma……

* Hair sampling…

drug therapy

adherence

monitoring?

Urine bladder…..

Dermal

distbn. of

Drug XX

*Brain tumour sampling…

Cancer drug to

target tissue

*Lung lavage…

Pulmonary PK of

drug and active

metabolite(s)

Lung lavage…

Seminal fluid analysis…

* Potential/planned

Drug Z risk

assessment

Colon and rectum analysis..

Drug X to target

tissue

Skeletal muscle analysis…

Drug Y to target

tissue

*Polydendritic cells…

Delivery to

target cells in

systemic

Pulmonary PK for IV

antibiotic & multiple

inhaled cpds.

Cerebrospinal fluid5…

Fosdevirine safety

“investigation”

5. Central nervous system distribution and metabolism of fosdevirine (GSK2248761), a non-nucleoside reverse transcriptase inhibitor : an LC-MS and matrix-assisted laser desorption/ionization imaging MS investigation into central nervous system toxicity, Catellino S. et al., Chem. Res. Toxicol., 2013, 26, 241-251.

6. Safety, Pharmacokinetic, and Functional Effects of the Nogo-A Monoclonal Antibody in Amyotrophic Lateral Sclerosis: A Randomized, First-In-Human Clinical Trial, Meininger V et al., www.plosone.org, 2014, 9(5).

Skeletal muscle6 analysis…

Ozanezumab to

target tissue

safety

“investigation”

Summary • Human in vivo data as first intent – metabolism in animals later

• What is the metabolite structure and how much ?

access to human bile and urine

circulating metabolites are only one part of the story [assessing body/tissue burden]

mechanism of metabolite formation is important

IV µtracer design with ADME endpoints provides opportunities for early human IV PK and Abs. Bio. + Met ID/Quant

Drug to target – human tissue sampling

Greater focus on patients rather than healthy subjects…….

Summary

Acknowledgements • GSK DMPK colleagues present and past including -

Stephanie North, Gordon Dear, Andy Roberts, Claire Beaumont, Billy Guiney,

Steve Thomas, Steve Castellino, David Wagner, Jill Pirhalla, Ernie Schubert, Igor

Goljer, Steve Corless, Clive Felgate, Adrian Pereira, Mike Tucker

• Xceleron (AMS CRO), Vitalea Science (AMS CRO), PRA, SIMBEC, Quotient and Comprehensive Development for support of the IV µtracer studies • The patients and healthy volunteer subjects who participated in the clinical studies*

* The human biological samples were sourced ethically and their research use was in accord with the terms of the informed consents

Questions?

Back-ups

1.52.02.53.03.54.04.55.05.56.06.57.07.58.08.5 ppm

Metabolite signal masked by noise

0.00 2.50 5.00 7.50 10.00 12.50 15.00 17.50 20.00 22.50 25.00 27.50 30.00 32.50 35.00 37.50 40.00 42.50Time1

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%

14.61

12.55

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16.17

12.5311.19

10.79

10.28

9.69

7.274.173.84 8.55

12.70

12.94

13.14

600/700 NMR - cryoprobes

UPLC/MS Mass defect filtering

NMR Approach Oral dose > 50 mg

~ 5 μg drug in plasma pool

Mass Spectrometry Approach Oral dose 1-50 mg

Technology improvements - Entero-Test ®

• FTIH metabolism focus Provides steady state systemic metabolites

Urinary metabolites

Biliary excretion is often a major route of elimination

Not easy to get bile out of humans

• Entero-Test® (absorbent string) provides easy non-invasive mechanism to collect human bile (used on several FTIH studies); ~ 1mL sampled

• Bile ; extremely dirty matrix – reliant on technology platforms to maximise data value

Assess data from SD human plasma

Are there any metabolites >5% oDRM*

Assess data from residual rodent TK plasma

Are human metabolites of concern present?

Plan RD rodent study to coincide with RD human

(if insufficient TK plasma available )

Assess data from residual norodent TK plasma

Are human metabolites of concern present?

Consider options for RD non-rodent study to

coincide with RD human (if insufficient TK plasma

available )

Await RD human data

Alternative options may be considered

Await RD human data

No concerns at this time

YES

NO

YES

YES

NO

NO

Step 1: LC/MS of residual rodent & non-rodent TK plasma (no data processing)

Step 2: Quantitative & qualitative assessment of SD human plasma

Assess data from RD human plasma

Are there any metabolites >5% oDRM*

Assess data from residual rodent & non-rodent TK

plasma

Are human metabolites of concern present ?

Generate & use appropriate non-clinical plasma to

assess exposure multiples with human

(LC/MS peak area ratios)

Discuss alternative options with SA

eg. alternative non-clinical species; dosing of

metabolite No further action required

at this stage

YES

NO

YES#

NO

Step 3: Quantitative & qualitative assessment of RD Human Plasma

* Or metabolites of safety concern based on chemical structure

If insufficient cover

#Stability of metabolites assessed first if residual TK plasma to be used

Additional References

• The use of isotopes in the determination of absolute bioavailability of drugs in humans, Lappin G., Rowland M., and Garner R.C., Expert Opin. Drug Metab. Toxicol., 2006, 2(3), 419-426

[Provides a good comparison of the pros and cons to use of stable label versus radioisotopes for absolute bioavailability studies]

• Accelerator MS: its role as a frontline bioanalytical technique, Seymour M., Bioanalysis, 2011, 3(24), 2817-2823. [Mentions use of IV tracer following repeated oral administration to provide kinetics at steady state, and clearly outlines the advantages

of concomitant dosing versus the traditional cross-over design]

• When opportunity met aspirational goals: accelerator MS, microdosing and absolute bioavailability studies , Arnold ME and LaCreta

F., Bioanalysis, 2012, 4(15), 1831-1834. [Acknowledges “renewed requirement” for defining absolute bioavailability in humans and provides support for the technology that now makes this more facile for many molecules]

• Microdosing: A Critical Assessment of Human Data, Rowland M., J. Pharm. Sci., 2012, Published online in WileyOnline Library

(wileyonlinelibrary.com). DOI 10.1002/jps.23290. [Recent position piece on the general microdosing approach , including concomitant dosing by PO and IV routes]

• Human ADME Properties of Drug Molecules: a Plethora of Approaches, Beaumont C., Young GC, Cavalier T. and Young M, Br. J.Clin.

Pharmacol., 78:6, 1185-1200. [Variety of cold, tracer and alternative approaches discussed]

‘Human first’ strategy to assess victim DDI risk

Acknowledgements: Aarti Patel and Claire Beaumont

Determining Absolute Bioavailability (F%) in Humans

Variable PK in human?

Early assessment of F%

- permeability limited?

- first pass metabolism?

F> 20 (?)%

Yes

No Yes

Consider

Termination of

Development #

Consider addition of

other ADME endpoints

No

High % (>85%ǂ)

of parent drug

excreted

unchanged in

human urine?

No Yes

Conduct Abs. Bio.

study prior to

submission of

regulatory file?

Investigative Regulatory Requirement

Defer to later stage of

development

(prior to submission)

Develop

recommendation for

waiver of Abs. Bio.

study ? High clearance

(>70% LBF)?

No further

formulation effort Worth pursuing formulation development to decrease

variability

- address solubility and dissolution rate issues

- reduce dose burden; cost of goods improvement

No Yes

# Unless drug targetted to have low bioavailability eg. local action in GI tract, or pro-drug ǂ Per FDA BCS guidance draft 2015

Pooling

• Urine

– Equal volumes from all subjects (0-24h) combined to generate a 500 mL pool

• Plasma

– Combined across subjects and time points in a time adjusted manner to create a sample representative of the AUC 0-24h (70 mL)

NMR

Time

Resp

on

se

30 METABOLITE

70 DRUG

% PLASMA/ URINE

(1) Pooling

(2) Extraction

(3) Fractionation

(4) Identification

(5) Quantification

UPLC MS

Extraction

• Urine

– 500 mL lyophilised

– Reconstituted in 50 mL of 10% aqueous methanol

• Plasma

– Protein precipitated with acetonitrile. Supernatants combined, dried under N2 and reconstituted in 50 mL of 10% aqueous methanol

NMR

Time

Resp

on

se

30 METABOLITE

70 DRUG

% PLASMA/ URINE

(1) Pooling

(2) Extraction

(3) Fractionation

(4) Identification

(5) Quantification

UPLC MS

HPLC

• HPLC scaled from analytical where possible

• Typically 10 mm – 21.2mm i.d. columns

• Column eluent is fractionated at 15 s intervals to give a reasonable compromise between maintaining chromatographic resolution and limiting fraction numbers

• On-line MS to aid structural elucidation

NMR

Time

Resp

on

se

30 METABOLITE

70 DRUG

% PLASMA/ URINE

(1) Pooling

(2) Extraction

(3) Fractionation

(4) Identification

(5) Quantification

UPLC MS

NMR

• HPLC fraction are dried and reconstituted or analysed directly if using D2O mobile phase

• 10 - 100 μg of metabolite may be isolated from urine allowing for the usual selection of NMR experiments to determine structure

• Metabolites isolated from plasma are typically lower (<10μg) NMR will be limited to 1D proton only

• To reach acceptable detection limits in plasma cryoprobes are essential

NMR

Time

Resp

on

se

30 METABOLITE

70 DRUG

% PLASMA/ URINE

(1) Pooling

(2) Extraction

(3) Fractionation

(4) Identification

(5) Quantification

UPLC MS