why and how to determine human metabolites …qin yue, ph.d. metabolism and pharmacokinetics...
TRANSCRIPT
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