Qin Yue, Ph.D.

Metabolism and Pharmacokinetics

Novartis Institutes for Biomedical Sciences

Emeryville, CA, USA

Why and How to Determine Human

Metabolites Early in Drug Development

早期对人体体内代谢产物的分析策略和意义

Metabolism is the major elimination pathway of

xenobiotics

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Metabolism converts a drug to new chemicals via metabolic reactions catalyzed by drug metabolizing enzymes

Metabolic reactions

Phase I, functionalization

Oxidation, reduction, hydrolysis, etc

Phase II, conjugation

Glucuronidation, sulfation, actylation, GSH conjugation, etc

Metabolism, in most cases, leads to form more

polar metabolites and loss of pharmacological

activity, but in some cases

Pharmacological activation and alternation;

Toxicological activation

Metabolism>Renal>Bile

Williams et al DMD 2004

CYP>UGT>Esterase

Importance to assess metabolite(s) exposure in

clinical studies

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Assist to interpret and understand efficacy and safety data

On-target pharmacology related to efficacy, and potentially important for establishing PK/PD relationship in the PoC study and beyond

On- or off-target pharmacology related to safety

Exposure of both parent and metabolites may change under various intrinsic (such as renal or hepatic impairment) and extrinsic conditions (such as DDI)

Human circulating metabolite exposures are not always predictable from pre-clinical studies or in vitro studies.

Safety coverage of human metabolites needs to be demonstrated in preclinical toxicological species.

Safety Testing of Drug Metabolites (MIST) Guidance for industry

Finalized in 2008

Applies to small molecule nonbiologic drug products

doesn’t apply to some cancer therapies (risk vs benefit)

Nonclinical safety evaluation of a human metabolite(s) is only warranted when

Its exposure is >10% of the parent at steady state

Disproportionately higher in human than in animal

Phase II metabolites are excluded except for acylglucuronide

Encourage to identify differences in drug metabolism between animals and human as early as possible

Guidance on Nonclinical Safety Studies M3 (R2) ICH Guideline

Approved by the steering committee of M3 (R1) revision under step 4 in 2009

Nonclinical characterization of a human metabolite(s) is only warranted when that metabolite(s) is observed at exposures greater than 10% of total drug-related exposure and at significantly greater levels in humans than the maximum exposure seen in the toxicity studies.

ICH guidance supersedes FDA guidance.

Qualitative or semi-quantitative

LC-MS/MS based assay

In vitro across species metabolite ID

LM, S9, hepatocyte, etc

In vivo pre-clinical metabolite ID

Plasma, urine, bile, feces

Metabolite ID in preclinical TK and First-in-human SAD and MAD studies

Quantitative

Radio-labelled Metabolite ID

In vitro

(Non)rodent ADME

human ADME

LC-RAD-MS/MS

High cost and late development

Quantitation by validated BA method

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From discovery to late development

Metabolite ID studies

Discovery Pre-

phase 1

Phase

1

Phase

2a/2b

Phase

3

Phase

4

Early opportunity to identify human unique or disproportionate

metabolites

Metabolite analysis in First-in-Human study

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Objective

Early opportunity to explore human unique or disproportionate metabolites to address MIST concern

• Profiling human circulating metabolites to learn any surprised or new metabolites;

• Semi-quantitatively assess relative exposures of metabolites in human and preclinical species

Relatively quantitate or semi-quantitate metabolites using 14C-metabolites as calibrators if available;

Quantitation of known active metabolites with qualified or validated method when standards of metabolites are available;

A tiered approach to metabolite quantification

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Fit for purpose

Aubry AF, et al. Bioanalysis (2014), 6(5), 651-664

Discovery Pre-

phase 1

Phase

1

Phase

2a/2b

Phase

3

Phase

4

Tier I or II exploratory (screening or research) approaches

Tier III or IV qualified or validated approaches - Active and significant metabolites

Stabilization of study sample is important

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From collection to analysis

General approaches

• Consideration of anticoagulants

• Suitable temperature for storage and sample extraction work out

• Light protection

Special cases

• Adjustment of matrix pH

- Acylglucuronide is unstable under neutral and basic condition

• 2% lactic acid or 20-100mM citric acid

- N-glucuronide is unstable under acid condition

• Use of enzyme inhibitors

- Hydrolysis of ester-containing drugs

- Catechol autoxidation

- Lactone and acid interconversion

Stabilization of study sample is important

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Acidify to stabilize acyl glucuronide

Gao, H and Obach, S, DMD. 2012, 40, 1290-96

Metabolite profiling in human plasma from First-in-

Human study

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Plasma samples are generated from Cmax pool and Hamilton plasma pooling AUC0-t

LC-HRMS technologies coupled with intelligent data acquisition and processing are typically used to detect and identify metabolites

Estimate metabolite levels in plasma by applying corrected MS response factors

Constructed for individual metabolites from the preclinical in vitro and in vivo

radiolabeled studies - Generated metabolites in in vitro system with radiolabeled material

- Precipitated and the supernatant was dried and reconstituted

- Spike metabolite mixture into blank human plasma

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Ionization is not equal molar response

Challenges to quantify metabolites without standard by LC-MS

Timmerman P, et al, Bioanalysis (2010), 2, 1185-1194

Rapid Commun. Mass Spectrom. 21, 497–502 (2007).

Estimation of biological [14C] metabolite standard concentrations

Measurement of metabolite MS response in target matrix to get response ratio R

R=MSUNK / MSREF

Estimation of the metabolites in an unknown sample

Bioanalysis (2010) 2(7), 1195–1210

Selection of pseudo internal standard Pseudo internal standard confers some of the advantages

of a traditional internal standard but is already a component of the reference and unknown samples. The pseudo internal standard (PIS) would most often be the parent drug itself.

Determination of reference correction factor Reference correction factor establishes the direct

relationship between metabolite-to-PIS response ratios for radiodetection versus MS peak area.

Determination of relative abundance ratio & relative molar ratio Relative abundance ratio for metabolite-to-PIS based on

MS peak area in the nonradioactive sample is multiplied by the reference correction factor to convert it into the molar-based relative molar ratio.

Estimation of metabolite concentrations in unknown samples Estimating metabolite exposure is accomplished by taking

measured pharmacokinetic data for the PIS (usually parent drug) and multiplying it by the relative molar ratio and a molecular weight correction for metabolite and parent.

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Estimate metabolite level in human plasma with 14C-metabolites as calibrators

Comparable between a validated method and an exploratory radioactivity-MS response approach

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Huskey, SE, et al, Bioanalysis, 2014, 6. 617-628

Subject NVS2 M2 M3 M8 Total

A 9585 1900 31980 11811 55276

B 23332 1756 41637 16775 83500

C 3979 1745 72586 19353 97663

D 4868 1492 88555 51910 146825

E 48237 4176 81036 18331 151780

F 46851 24818 102541 14989 189199

mean % 16.3 3.6 52.5 17.5 89.9

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Early metabolite profiling in FIH discovered major human metabolites in human

Lot1

Lot2

Exposure of NVS2 and its major metabolites

in human plasma (ng*Eq*hr/ml)

Metabolite profile of NVS2 in human

Hepatocyte

Huskey, SE, et al, Bioanalysis, 2014, 6. 617-628

1. An unexpected major metabolite (M3)

detected in human plasma.

2. The M3 is the secondary metabolite of M8,

which is limitation of in vitro system.

3. Would it be covered in pre-clinical tox

species.

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Can we quantitatively compare metabolite exposure in human and preclinical species as early as possible?

Timmerman P, et al, Bioanalysis (2010), 2, 1185-1194

Challenges to quantify metabolites without standard by LC-MS

Matrix could significantly suppress on metabolite MS response

Metabolite exposure in human and preclinical species can be relatively assessed by MS peak area ratios

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When radiolabeled parent and metabolite standards are not available

Samples: TK samples and FIH plasma samples (SAD or MAD)

Sample pool: Hamilton plasma AUC pooling

Mixing matrices to reduce matrix effect

TKmix = TK AUCpool + human plasma blank

Humanmix = FIH AUCpool+ TK plasma blank

Sample processing

LC-MS/MS analysis (HRMS or MRM)

Report exposure multiples of metabolites on MS peak area ratios:

TKmix / Humanmix

If the ratio>2, higher exposure in animal can be confidently claimed

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Animal to human ratio measurement error and confidence limits. The error bar is 95% confidence interval.

(A) Fresh animal and human samples measured side by side on day 1.

(B) Animal and human samples measured side by side on the same day (n = 5, e.g., day 1, 30, 105, 254,

and 314).

Gao, H, et al, Analytical Chemistry, 2015, 87, 11771-11776

When the ratio <2...

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May prioritize the human ADME study to determine if metabolite exposure is 10% of total drug-related exposure

If so, standards of metabolites may be synthesized and prepare for qualified or validated methods

Species Rata Doga Rabbitb Humanb

Oral Dose 10 mpk 3 mpk 50 mpk 200 mg

Plasma AUC

(ng*Eq*h/mL) Feces (% Dose)

Plasma AUC

(ng*Eq*h/mL) Feces (% Dose)

Plasma AUC

(ng*Eq*h/mL)

Plasma AUC

(ng*Eq*h/mL)

Compound A 12078 11.4 14200 ND 12864 22298

M18 3072 4.6 2800 ND 414 1243

M23 ND 5.6 ND 30.3 34 1460

aQuantification of metabolites in plasma from rats (n=3) and dogs (n=3) by radioactivity

bExploratory quantification of metabolites in plasma from rabbits (n=5) and Humans (n=6)

Wenkui Li, 2016, 10th WRIB in Orlando, FL

M23 >10% of the total radioactivity AUC0-120h

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All required studies according to guidance:

• Genotoxicity

• Repeated dose toxicity

• Other preclinical safety evaluation

• Bioanalytical method development and validation for M23 in rat and dog plasma in support GLP tox studies

Exposures of major metabolites in human plasma

Summary

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Identify differences in drug metabolism between animals and human as early as possible

Tiered approach is encouraged to be implemented for metabolite characterization and quantitation to address regulatory requirements

Only assays for confirmed important human metabolites (i.e., unique/disproportionate or contributing significantly to the activity) are validated for routine use in late development.

Acknowledgements

Patrick Rudewicz, Novartis

Wenkui Li, Novartis

Huskey Su-Er, Novartis

MAP and DMPK teams