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How to Conduct a Low Energy (Carbon-14)
Radiolabel Human AME study:
Study preparation and planning, design, clinical
procedures, sample collection and mass balance
analysis
如何进行C-14人体AME试验
Dennis Heller, Ph.D. XenoBiotic Laboratories, Inc.
Human Radiolabel “AD ME” Studies
 Absorption, Distribution, Metabolism, Excretion
 Radiolabel (14C - low energy, long half-life)
 “Mass Balance” study (what goes in, must come out)
 Phase I clinical PK study
 Most drugs will require some type of AME study
Other clinical uses of radiolabels
(not covered today)
 Radiopharmaceuticals used in diagnostic Nuclear Medicine (99mTc,
111In, 123I, 67Ga), SPECT, PET imaging (11C, 13N, 18F, 68Ga, 52Fe, 201Tl)
 Gamma Scintigraphy – follow the drug/formulation transit and
dissolution through the body (99mTc, 51Cr, 67Ga, 111In, 123I, 153Sm, 153Gd)
 Radiopharmaceuticals used in Radiotherapy for cancer
(Brachytherapy) (89Sr, 90Y, 131I, , 198Au, 192Ir, 137Cs)
Presentation Outline
 Why we conduct human AME studies
 When we conduct them
 How we conduct them – study design and preparation
 Sample collection and analysis
 Summary
Human 14C-AME Mass Balance Study
Primary Objectives
•
Determine the PK of Total Radioactivity, unchanged drug, and metabolites in
plasma （测定血浆中总放射性、受试物和代谢产物的PK）
•
Determine mass balance and extent of absorption（测定物质平衡和吸收）
•
Determine the clearance pathways (routes of elimination) and rates of elimination
in urine and feces（确定清除途径和从尿粪排除速率）
•
Profile and identify circulating metabolites in plasma, and excreted metabolites in
urine and feces（鉴定血浆中循环代谢产物以及尿粪中排除的代谢产物）
•
Determine the exposure of the parent compound and its major metabolites –
quantify metabolites relative to parent and the total（测定受试物及代谢产物的暴
露量）
Pharmacokinetics of Radioactivity in Plasma
Why is half-life of TRA
longer
than metabolites?
Total Radioactivity (TRA)
Parent
M1
M2
Some drug residues
are covalently bound
to plasma proteins
为什么总放射性的t1/2
长于所有代谢产物
M3
共价键结合
Mass Balance Based on 14C
Total
Urine
Feces
It is straightforward to demonstrate rate and extent of recovery,
in this case ~95% （排泄和回收率结果直观明了）
Human 14C-AME Study Timing
60%
51.9%
50%
40.7%
40%
30%
20%
10%
7.4%
0.0%
0%
Early
Phase I
Early
Phase II
After POC
Phase III
N. Penner, L. Klunk, C. Prakash, Biopharm. Drug Dispos. 30:185-203, 2009.
Typical ADME Knowledge Base Prior to Human
14C-AME Study （进行人体14C-AME试验前需了解的）
• In vitro models for human/preclinical species metabolism
– Microsomes, hepatocytes, S9, liver slices
– Reaction phenotyping for major anticipated pathways
• In vitro models for transport
– P-gp, maybe other uptake or efflux transporters
• Preclinical radiolabeled ADME studies
– May point to a unique biotransformation pathway not detected in vitro
– Could indicate the potential for non-metabolic clearance (biliary, renal)
• PK parameters of parent drug
– Cmax, Tmax, Half-life, etc.
• Plasma profiling from non-radiolabeled human studies
– Can often identify major circulating human metabolites, single, multidose studies
– Preliminary qualitative MIST evaluation
Why Conduct Human 14C-AME Studies As Early As Possible?
（14C-为什么人体越早进行越好）
 Definitive Human metabolic pathway – elucidate structures of
prominent human metabolites – complete picture
 Relative exposure of parent & metabolites, %AUC in plasma
(%Dose in excreta) identify major and long-lived metabolites
 Compare quantitative profiles to pre-clinical Tox species
(MIST guidance*) and in vitro – help to validate Tox species
 Identify any unique human biotransformation pathways?
 Implications for mechanism of action/pharmacology/toxicity –
which metabolites should be monitored in clinical trials? Active
Metabolites?, QT study?
* Metabolites in Safety Testing (MIST): FDA Guidance, 02/2008,
ICH M3 Guidelines, 2009, FDA Guidance M3(R2), 01/2010
Why Conduct Human 14C-AME Studies As Early As Possible?
（14C-为什么人体越早进行越好）
 Plan clinical DDI studies, renal/hepatic impaired （肝肾不正常）,
elderly, pediatric
 Plan future PK and phase II/III study dose levels
 Gender differences
 Polymorphically expressed enzymes, CYP450, Transporters (e.g.
PM, EM subgroups) （多态酶，慢代谢、快代谢）
 Assist with possible BCS class 1 biowaiver
 Plan carcinogenicity study – match metabolites
 Future drug design – protect IP
Preliminary understanding of the
DMPK properties of a drug from
pre-clinical and early clinical work
Human 14C-AME
Study
Mature understanding of DMPK the
properties of a drug – recognition
of remaining knowledge gaps
Human 14C-AME Studies
- Preparation -
Human 14C-AME Study – Preparation
•
Radiolabelling （放射性标记）
 Carbon-14 (14C) preferred isotope.
 Position of Radiolabel – stable incorporation – follows metabolites –
single or multiple positions?
 Radiopurity and Radiochemical stability (potential for radiolysis)
•
Formulating/Dose Preparation
 Solution, suspension, capsule (uniform distribution of hot/cold)
 Hot and cold compound must be chemically identical (salts, free base or
acid, same physical form
 If suspension, dissolve hot and cold together in suitable solvent, then
evaporate solvent
 GMP cold material, quasi-GMP hot material
 UK, Europe – final preparation step of dose formulated to GMP
Radiolabelling
14C
Labeling Replaces 12C within the Molecular Structure
OCH3
O
O
O
N
S
H
NH
O
[14C]Apremilast
M. Hoffman et al. Xenobiotica; 41 (12):
1063-1075, 2011
O
O
* Indicates positions of radiolabel
O
N
O
*
*
O
OH
N
O
O
O
[14C]Peliglitazar
Wang L. et. al. Drug Metab
Dispos.;39:228-238, 2011.
Human 14C-AME Studies
- Dosimetry -
Dosimetry Analysis （辐射剂量分析）
Prediction of the Radiation Absorbed Dose from
Internal Exposure to Low Energy Beta Radiation
Objective
To reasonably (21CFR312.23) predict the radiation
absorbed dose to human volunteers (whole body
and critical organs) from internal exposure to a
radiolabeled drug
Dosimetry - Prediction of Human Radiation
Absorbed Dose from Animal ADME Data
（通过动物ADME试验数据预测人体吸收药物的辐射）
Requires
•
Rodent QWBA or traditional tissue necropsy study
–
Pigmented animals – melanin binding
•
Rodent Excretion balance study
•
Same route of administration as the planned human
study
QWBA Sections of Pigmented Rat
2 hours
28 days
The MIRD System1
D = Ã/m ·  · 
D = absorbed dose (rad or Gy) (1 Gy = 100rad = 1 Joule/kg)
à = cumulative Activity = AUC0- (mCi ·hr)
m = mass of the target organ (g)
 = conversion factor, includes the
MeV/transition info specific to each isotope
(rad ·g/mCi ·hr) = 0.105 for 14C
 = absorbed fraction = 1 for non-penetrating emissions
1. MIRD (Medical Internal Radiation Dose) Primer For Absorbed Dose Calculations,
Revised Edition, 1991, prepared by R. Loevinger, T. F. Budinger, E.E. Watson, Society of
Nuclear Medicine, 136 Madison Ave, New York, NY, 10016
Absorbed Dose per Unit Administered Activity (Ao)
for Non-penetration Radiation
For Carbon-14
D/Ao = Ã/Ao · 0.105/morgan(target)
D/Ao = absorbed dose per unit administered activity (rad/mCi)
Ã/Ao = cumulative activity as a fraction of the administered activity
(%Ao)
= AUC0- of the time activity curve/Ao (mCi·hr/mCi = hr)
• AUC0- values calculated using non-compartmental PK software
• Prior to calculating AUCs, the animal time activity data may be
allometrically scaled by relative organ mass scaling and/or
physiological time scaling
Calculate Effective Dose (ED)1
• Allows non-uniform internal doses to be expressed as an
equivalent whole body dose
• Used for setting dose limits for general public, occupation
workers, fetus
• ED =

T
Abs. Dose Equivalent* (rem or Sv)
·W
T
• ED allows the expression of dose estimates from several
different organs as a single number
– related to overall radiation risk
– allows easier comparison of different procedures in nuclear
medicine as well as diagnostic x-ray, etc.
– Weighting factors are relevant to population averages, thus ED
should not be used to evaluate risk to an individual
*Absorbed Dose Equivalent: rem (rad equivalent man) = rad x quality factor (= 1.0 for b and g)
Sv ‘Sievert’ (1 Sv = 100rem)
1. ICRP Publication 103 (2007), ICRP Publication 60 (1991)
After Dosimetry Calculations
How much Radioactivity (Ci) to Give?
1.
2.
Justify Use: Risk vs. Benefit

Benefit: none for normal healthy volunteers

Risk: no detectable adverse effects from quantity administered
Optimize Exposure:

ALARA (As Low As Reasonably Achievable)

High Enough activity to obtain acceptable signals in biological
samples

Can be an issue if Specific Activity is too low (e.g. for proteins)
Effective Dose ≤ 1 mSv (100 mrem)
= ca. 50-125 Ci Carbon-14 administered activity
Radiation Dose Limits for An Adult
Research Subject - FDA
mrem
mSv
1. Single Dose
3,000
30
2. Annual and Total Dose
Commitment
5,000
50
1. Single Dose
5,000
50
2. Annual and Total Dose
Commitment
15,000
150
21CFR361.1
Whole body, active blood-forming
organs, lens of eye, gonads
Other Organs
Risk Classification:
ICRP Publication 62, 1992
Level of Risk
Risk Category
(Total Risk)
mSv
Level of Social
Benefit Needed
Trivial
(10-6 or less)
<0.1
Minor
Minor
IIa (10-5)
0.1 to 1
Intermediate
1-10
to
Moderate
to
Intermediate
IIb (10-4)
Moderate
III (10-3 or more) > 10
Substantial
Average Effective Dose Equivalent per
Medical X-ray Exam in US
NCRP(93), NCRP (100)
Extremities
Chest
Skull
Cervical Spine
Kidneys, Uterus, Bladder
Pelvis and Hip
CT- Head and Body
Lumbar Spine
IVP (intravenous pyelogram)
Biliary tract
Upper GI
Barium Enema
mrem
mSv
1
6
20
20
55
65
110
130
160
190
245
405
0.01
0.06
0.20
0.20
0.55
0.65
1.10
1.30
1.60
1.90
2.45
4.05
Average Annual Effective Dose
Equivalent for Member of US Population
NCRP(93)
A. Natural Background
1. Cosmic
2. Cosmogenic radionuclides
3. Terrestrial
4. Internal
5. Inhaled
Subtotal Natural
B. Man Made
1. Medical
a. Diagnostic X-rays
b. Nuclear Medicine
2. Consumer Products
3. Other
Subtotal Man Made
Total
mrem
mSv
27
1
28
39
200
295
0.27
0.01
0.28
0.39
2.00
2.95
39
14
11
<1
65
0.39
0.14
0.11
<0.01
0.65
360
3.60
Human 14C-AME Studies
- Study Design -
Human 14C-AME Study – Study Design
•
Considered a “Phase 1” PK Study
•
Route of Administration
 Match intended clinical route – typically oral
•
•
Single Dose


Subjects


•
Drug Dose (mg) – close to predicted efficacious clinical dose
Radioactive “Dose” = activity (Ci) – ca. 50-125 Ci Carbon-14
Normal Healthy volunteers (typically)
4-8 males, sometimes females (non-reproductive status)
Duration of Stay


Depends on drug half-life and clearance
Typically 7-28 days (with exit criteria on mass balance)
Human 14C-AME Studies
Approval and Oversight
Due to administration of radioactivity, there is additional
oversight and approval compared to typical phase I PK study
 Dosimetry – requires rodent ADME study data
 Informed Consent – list Risks from radiation exposure
 Authorized User (FDA 10CFR35.100) – person trained to
administer radioactivity (e.g. Nuclear Medicine Physician)
 Sometimes, additional approval - ARSAC (UK), RDRC (if no
IND)
(ARSAC) Administration of Radioactive Substances Advisory Committee
(RDRC) Radioactive Drug Research Committee (FDA Guidance, 2010)
Human 14C-AME Protocol
Unique Elements
• Subject Exclusion Criteria
 Subject has participated in a radio-labeled clinical trial within the last 12 months
prior to the first dose of study medication
 Subject has had significant radiation exposure (such as serial X-rays, CT scans,
barium studies, occupational exposure) within the last 12 months
• Other restrictions – Female Subjects – non-reproductive status
 Surgically sterile (hysterectomy) or post menopausal
• Screening Tests – If include Female Subjects
 Pregnancy Test A serum pregnancy test will be performed on all female
subjects at Screening. A urine pregnancy test will be performed on all female
subjects at Day –1 (Check-in) and at the end of the study
 Follicle Stimulating Hormone (Female Subjects Only) - A follicle stimulating
hormone test will be performed on female subjects who are less than 1 year
postmenopausal at Screening
Human 14C-AME Protocol
Unique Elements
• Informed Consent
 Includes mention of the use of a small amount of low energy
radioactivity to monitor the metabolism and disposition properties of
the drug and that the risks due to this small exposure are low.
 Typically compare exposure to that obtained when receiving routine
X-rays to the head or abdomen (per dosimetry analysis).
Human 14C-AME Protocol
Unique Elements
This is a confined study!!
•
•
Exit Criteria – Subjects are finished when...

Achieve “mass balance”:  90% of Dose recovered

or ≤1% of Dose in excreta in two consecutive 24 hour
intervals

and < 2 x background in two consecutive PK time points
Requires daily analysis of total radioactivity and
quick turnaround of results
 feedback data to clinical team for decision making
Human 14C-AME Protocol
Sample Collection to Meet Objectives
•
Protocol Objectives - PK Analysis





•
Parent Drug concentration in plasma (LC-MS)
Total Radioactivity (TR) in whole blood, plasma (LSC)
Metabolite profiling/ID and concentrations (AUC) in plasma (whole
blood) (Radioprofiling off-line, on-line)
Total Radioactivity, metabolite profiling/ID and %Dose in excreta
Renal Clearance
Sample Collection

Blood collection – longer time course than for just parent PK


Safety endpoints: typical of a Phase I study
Complete excreta (urine and feces) to achieve mass balance: 7-14
days is typical – subject to exit criteria
 Sometimes expired air
Sample Collection and Processing – Blood/Plasma
Blood Collection – volumes




Whole blood aliquots for TRA analysis – 2 x 1-1.5 mL blood
Plasma for TRA analysis – 2 x 3-5 mL blood
Plasma for cold LC-MS/MS analysis - 2 x 3-5 mL blood
Plasma for metabolite profiling - 2 x 5-10 mL blood
 only for subset of the time points selected for metabolite profiling

Total 24-43 mL per time point (for time points with Radioprofiling sample)
Blood Collection/Processing





Compound specific stabilizer in blood tube? e.g. citric acid, formic acid?
Reserve small aliquot of whole blood for LSC analysis
Standard processing of blood to plasma.
Prepare small aliquot of plasma for immediate LSC analysis for exit
criteria
Prepare and store (-20oC or -70oC) remaining plasma subsamples for
future assay
Sample Collection and Processing – Urine
Urine Collection


Collect each void – record approximate volume, time and date
Add any stabilizer/surfactant if needed
 e.g. 10% 1M phosphate buffer + 2% Tween-20

Address Non-specific binding to collection containers if needed
 e.g. pre-rinse all collection containers with 15% Triton-X 100 solution and let dry

add each void volume to the 24-hour pooling container and store
refrigerated until processing
Urine Processing





Record total volume (or better - weight) from each collection interval
Mix together total volume from each interval thoroughly
Prepare small aliquot for immediate LSC analysis for mass balance/exit
criteria
Prepare and store (-20oC or -70oC) sub-samples (2-4 x 25-50 mL) for future
assay
Do not discard remaining quantities of original samples
Sample Collection and Processing – Feces
Fecal Sample Collection

Collect each void (entire sample) in separate container/bag – time and date
 store refrigerated until processing

Collect toilet paper in separate container/bag
Fecal Sample Processing



Combine stool samples from same 24-hour interval if more than one
Weigh sample – determine net weight of sample (tare weight)
Homogenization
 1 to 3x of 1:1 isopropanol:water or 1:1 methanol:water or just water
 Homogenize in blender/polytron. Add additional aqueous if needed to obtain homogeneous
mixture
 Record all added volumes.



Prepare small aliquot for immediate LSC analysis for mass balance/exit
criteria
Prepare and store (-20oC or -70oC) sub-samples (2 x 20-50 g) for future
assay
Do not discard remaining quantities of original samples
Urine Collection Containers
Individual void collection
tip – use same container for
each subject for duration of
study!
male
female
24-hour collection container
tip – may need more than one
per person per 24 hr interval!
Urine Processing Containers
Beakers to mix and
measure total volume per
interval
Various sizes (2, 4, 6 L)
HDPE or PP, or glass
Sub-sampling –
Tip – do not fill to top!
Fecal Collection Containers
“Hat” configuration for toilet
seat insert – with our
without plastic bag insert
Fecal Homogenization
Wide mouth blending
containers
Various sizes (0.5, 1, 2, 4L)
HDPE or PP
Polytron
homogenizer
Sub-sampling –
Tip – do not fill to top!
Human 14C-AME Study
Preparation at the Clinic
•
Clinical Unit/Subject Training


•
Procedures in place to ensure collection of all urine and feces
Strict procedures and instructions to Subjects to achieve 100%
compliance with sample collection – emphasis on collecting entire
void, not just a portion!
Facilities

Licensed to handle and use radioactivity
 Radiation Safety Officer, Radiation Safety training, Radiation Safety Program



Special areas for radioactive Dose preparation, sample processing
Housing areas for subject confinement for 1- 3 weeks
Strict control of urine and faecal collection
 No ability to flush toilets or discard samples


Sufficient freezer/refrigerator space to stored collected samples
RAD Waste disposal procedures
Human 14C-AME Study
Successful Outcome?
•
Achieve Mass Balance （达到物质平衡）?

90+ % average recovery is considered successful

Typically, individual subject recoveries range from 80-95%.

Typically higher values if Drug mostly excreted in urine
and lower values if Drug mostly excreted in feces

Why?
Human 14C-AME Study
Successful Outcome?
•
Reasons for Low Recovery （回收率低的原因）






Inaccuracies in dose (preparation of delivery, and/or analysis
Incomplete collection of excreta or missed samples
Emesis (non collected), especially after dosing
Inaccuracies in sample processing (mixing, weights and volumes) and
analysis (LSC counting)
Radiolabel lost in expired air – can measure if a possible route of
elimination - based on pre-clinical ADME study
Drug still remaining in body – even if exit criteria met


Long plasma t½
Tissue binding (covalent binding or non-covalent sequestration)
Human AME Studies
- Sample Analysis -
Human 14C-AME Mass Balance Study
Primary Objectives
•
Determine the PK of Total Radioactivity, unchanged drug, and metabolites in
plasma （测定血浆中总放射性、受试物和代谢产物的PK）
•
Determine mass balance and extent of absorption（测定物质平衡和吸收）
•
Determine the clearance pathways (routes of elimination) and rates of elimination
in urine and feces（确定清除途径和从尿粪排除速率）
•
Profile and identify circulating metabolites in plasma, and excreted metabolites in
urine and feces（鉴定血浆中循环代谢产物以及尿粪中排除的代谢产物）
•
Determine the exposure of the parent compound and its major metabolites –
quantify metabolites relative to parent and the total（测定受试物及代谢产物的暴
露量）
Analytical challenges of AME Studies
• Low level radioactivity detection required
– The specific activity(total radioactivity in dose/total mass of dose) used
in AME studies is typically 20-100 times lower than those used for
radiolabeled studies conducted in preclinical species.
• Limited sample availability (plasma/blood)
– AME studies are clinical studies (limited number of subjects, limited
plasma per time point)
• Novel Metabolites
– Some metabolites may be detected for the first time in the course of
conducting a human AME study (human unique/prevalent, unusual
structure, highly polar or non-polar)
– Lack of analytical standards
– Often requires adjustments of bioanalytical methods.
Challenges are overcome with modern instrumentation and analytical techniques
Human 14C-AME: Analytical Activities
Study
Planning
Dose
Preparation
Dosing/ Sample
Collection
Total
Radioactivity
Measurement
Metabolite Profiling /
Met ID/Quantitation
• How much sample is needed to perform various analyses?
• Does the test compound have any properties that require special
consideration (binding to plastic, unstable metabolites, sensitivity to
light, etc.)
Characterization of dose (specific activity, radiochemical purity)
Dose concentration of Delivered dose (pre &post),
sample weights/volumes, interval pooling
stabilizers, additives to containers to prevent NSB
• Liquid scintillation counting (LSC), plasma and urine directly
• Combustion of feces, whole blood
• What sample pooling method is appropriate?
• How should samples be extracted prior to metabolite profiling?
• What radioprofiling method(s) to use?
• Quantitation of parent & metabolites of interest in plasma (and
occasionally excreta) by LC-MS?
• What mass spectrometry method(s) to use?
• What “quality control” measures are appropriate?
Total Radioactivity Analysis (TRA)
•
Sample Processing/Counting


•
Pre-study preparation

•
Plasma and Urine – counted directly by liquid scintillation counting
(LSC)
Blood and fecal homogenate – combusted/oxidized first
Check counting recoveries – spike dosing solution into matrices at
several concentrations and check recoveries – use as future QCs in
the run.
Sample Collection


Stabilizers in blood tube, urine; e.g. citric acid?
Non-specific binding to collection containers for urine?
Procedures to be established & verified prior to study start
Metabolite Profiling（代谢物谱）
• There are two primary objectives of metabolite profiling
1. To obtain estimates of the relative abundance of metabolites
 For excreta - estimate percent of administered dose.
 For plasma - estimate percent of circulating drug-related material
(%AUC of TRA)
2. To structurally characterize metabolites
 The abundance of a metabolite will determine the extent of
characterization required (MIST, institutional guidelines)
• Metabolite profiling steps include:
–
–
–
–
Sample pooling
Sample extraction
Radio-profiling
Metabolite identification
Sample Pooling (Plasma)/AUC Pooling
•
parent
DPM
•
•
Due to issues common to AME studies (low plasma radioactivity, limited
plasma sample availability and large amount of samples) plasma samples are
often pooled to reduce the number of analyses that need to be conducted
A common approach is “AUC pooling” (also known as Hamilton1 pooling)
The pooling method is essentially a mathematical transformation of the
trapezoidal method of calculating AUC
The relative concentrations of drug and metabolites in an AUC pooled sample
should approximate the relative exposure of drug and metabolites within the
time range of the samples pooled
[drug, metabolite]
•
t0 t1 t2
Time
tn
Time Point
Amount of plasma to
add to pool is
proportional to
t0
t1-t0
intermediate
points (tx)
tX+1-tX-1
tn
tn-tn-1
metabolite
Retention Time
1. Hamilton et al., Clin Pharmacol Ther. 29:408-413, 1981
Time Point
001
Plasma Concentration (ng equivalents/mL)
Subject Number
002
003
004
005
Mean
Predose
BLQ
BLQ
0.50 h
75.7
33.3
1h
257
159
1.50 h
245
177
2h
231
172
3h
188
142
4h
153
115
6h
105
76.1
8h
70.2
52.8
10 h
60.5
41.3
12 h
54.5
40.4
24 h
23.6
14.5
48 h
6.99
BLQ
72 h
BLQ
BLQ
96 h
BLQ
BLQ
120 h
BLQ
BLQ
144 h
BLQ
BLQ
168 h
D
BLQ
D
192 h
BLQ
D
D
216 h
D
D
240 h
D
D
264 h
D
D
288 h
BLQ Below the limit of quantitation.
D Subject discharged from clinical unit
BLQ
38.4
140
179
183
141
120
78.8
58.5
50.3
34.8
16.6
BLQ
BLQ
BLQ
BLQ
BLQ
BLQ
BLQ
D
D
D
D
BLQ
138
156
185
188
151
140
87.4
75.2
64.3
41.7
19.4
7.41
BLQ
BLQ
BLQ
BLQ
D
D
D
D
D
D
BLQ
143
180
153
114
102
89.1
54.3
41.8
36.5
31.2
13.7
BLQ
BLQ
BLQ
BLQ
BLQ
BLQ
BLQ
BLQ
BLQ
BLQ
BLQ
0.00
85.6
178
188
177
145
124
80.2
59.7
50.6
40.5
17.6
2.88
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
SD
0.00
52.6
46.1
34.3
41.8
30.5
24.6
18.2
13.4
11.9
8.88
4.01
3.95
0.00
0.00
0.00
0.00
0.00
0.00
N.A.
N.A.
N.A.
N.A.
Pooling Strategies
for Radioprofiling:
Review of Total
Radioactivity Data
PK results
Concentration versus time curves for radioactivity in plasma and blood and parent
drug (cold assay) in plasma
Parent Drug
Cumulative elimination of radioactivity in urine and feces
Majority of the radioactivity (>90%) was recovered within 4 days
Plasma Sample Pooling
Time
points (hr)
Subject 1
Subject 2
Subject 3
...
Subject N
1
2
4
8
12 24 …
A
“AUC Pooling” across time points for
each subject
• Can observe differences between
subjects
• Potential to dilute minor metabolites
• No time course information – only
AUCs
C
“AUC Pooling” across time points and
across subjects
• Cannot observe differences between
subjects
• Potential to dilute minor metabolites
• No time course information – only
global AUC
• Least amount of samples - most cost
efficient, but least amount of
information
B
Pooling across subjects for each time point
• Cannot observe differences between
subjects
• Time course information – Full PK
parameters
*No pooling*
• Observe differences between subjects
• Time course information – Full PK parameters
• Most amount of samples and highest cost
Adapted from: N. Penner, L. Klunk, C. Prakash, Biopharm. Drug Dispos. 30:185-203, 2009
Urine and Feces Sample Pooling
Time
intervals(hr)
0-6
6-12
12-24
Subject 1
Subject 2
24-48
…
A
Equal mass/vol pooling across time
intervals for each subject to 90%
excreted
• Can observe differences between
subjects
• Potential to dilute minor metabolites
• No time course information
Subject 3
...
Subject N
B
Pooling across subjects for each time interval
• Cannot observe differences between
subjects
• Time course information – temporal
appearance of metabolites in excreta
C
Equal mass/vol pooling across time
intervals for each subject to 90%
excreted and across subjects
• Cannot observe differences between
subjects
• Potential to dilute minor metabolites
• No time course information
• Least amount of samples - most cost
efficient, but least amount of
information
*No pooling*
• Observe differences between subjects
• Time course information
• Most amount of samples and highest cost
Sample Pooling – Typical Selection
Plasma –no pooling = full profiling = max number of samples
• 6 subjects x 7 time points = 42 samples
OR
Plasma – some pooling = less samples (13 samples)
• 6 subjects x 1 AUC pooled sample/subject = 6, AND
• 1 pooled sample across all subjects/time point x 7 time points = 7
Urine - pooling across subjects
• 1 pooled sample across all subjects x 5 time intervals = 5 samples
AND/OR
Urine - pooling across time intervals to 90% excretion
• 6 subjects x 1 pooled sample/subject = 6 samples
Feces - pooling across subjects
• 1 pooled sample across all subjects x 4 time intervals = 4 samples
AND/OR
Feces - pooling across time intervals to 90% elimination
• 6 subjects x 1 pooled sample/subject = 6 samples
Metabolite Profiling and Identification
LTQ-Orbitrap XL
FTMS full scan
2
DDA MS (CID or HCD)
1/10
Column
1:10 splitter
9/10 (enough radioactivity
to radio-detectors)
1:1 splitter
Analogue signal
vA RC Dynamic Flow Radio-detector
On-line radio-signal for MS data
Off-line 96-well plate collection for Topcount
Radio-chromatogram HPLC/TopCount
Plasma – 3 hour time point
How Many Radiopeaks to Identify?
•
Each metabolite  10% of total radioactivity (TR) –
based on plasma AUCs


•
Need to identify 80-90% of the TR in plasma


•
Single vs. multiple Dose (steady state)?
MIST guidance
Typically = all radiopeaks >5% of TR (AUCs) including parent
Sometimes includes radiopeaks between 1-5% if critical to the
metabolic pathway
Need to identify 80-90% of TR in excreta

Typically = all radiopeaks >5% of TR (%Dose) including parent
How Many Radiopeaks to Identify?
55.00
In vivo feces sample: 48 % of the dose
50.00
78 metabolites detected in this
sample if >1%
45.00
40.00
35.00
mV
30.00
5% dose
25.00
20.00
15.00
10.00
1% dose
5.00
0.00
12.00
14.00
16 .00
18.00
20.00
22.00
24.00
26.00
28.00
30.00
Minutes
R317573; Faeces Rat m pH=7.5 10%CH3OH 80%CH3CN
32 .00
34.00
36.00
38.00
Metabolite Characterization and
Identification
Propose and Confirm Structures
• LC/MS/MS (product ion scan, neutral loss, parent ions scan,
mass defect, high resolution accurate mass)
•
•
•
•
•
D2O exchange
Chemical derivatization
HPLC mobile phase pH adjustment
Nano- or Micro-spray technology for improved mass data quality
Isotope patterns
• Isolation and purification >10-25 μg
• From in vivo source – urine, feces, plasma, bile
• From in vitro source – microsomes, hepatocytes, microbial, recombinant
• NMR (1H, 13C, 1D, 2D, COSY, NOESY, HMBC, HMQC, etc.)
Human 14C-AME Studies
- Special Cases -
Traditional 14C-AME study
– a single dose study in 4-6 male healthy volunteers –
? What about understanding the AME properties of a drug in…
• Patient populations – oncology
• Renal or hepatic impaired
• Genetic polymorphisms – e.g. study CYP2D6 variants
• Drug interaction studies
• Age specific studies – pediatrics, elderly
• Multi-dose to steady state
In above special cases, may be practical to use ≤ 1 Ci per subject
and use AMS as the detector
 no special dosimetry requirements
 no special facility requirements for use of radioactivity
 Cost considerations
Detecting low levels of radioactivity
 If have very low levels of radioactivity, e.g. at later time points for
drugs with large volume of distribution and slow clearance, or
 If traditional level of activity per subject (50-125 Ci) is prohibitive due
to dosimetry or low SA (with polypeptide/protein for example)
 use Low Background LSC counting
 Use AMS as detector
Not routine, but may be employed as needed
Human 14C-AME Studies
- Summary -
Primary Objectives of a Typical 14C-AME Study
• Determine routes and rates of elimination of drug-related material
– Registration requirement
• Potential effects of organ impairment (renal)
• Pharmacokinetics of drug-related material
– Long-lived metabolites, covalent binding
• Identify metabolites (circulatory, excretory)
– AME studies are particularly well suited to do this
– Information for MIST assessment
– Determine Clearance Pathways
• DDI potential
• Role of polymorphically expressed enzymes
– Identify metabolites that may contribute to pharmacology/toxicology
• Metabolites contribute to the pharmacological effect of >20% of marketed
drugs1.
• Understand PK/PD
• Plan Carcinogenicity study
• Protect IP
1. Fura et. al. Drug Discov Today; 11: 133-142, 2006.
Additional Information Provided by a Typical
Human 14C-AME Study
• Basic knowledge about absorption
– Radioactivity detected in urine following an oral dose must have
been absorbed
– In some cases the extent of metabolism can provide information
about the extent of absorption
– Can sometimes support biowaiver (not discussed)
• Basic knowledge of the relative importance of various
clearance pathways
– Radiolabel allows (rough) quantitation of all excreted metabolites
– In some cases estimates of fraction metabolized (fm) by a given
pathway can be made
– Especially useful when non-P450 routes of clearance are involved
– Can help modeling and simulation of DDI
Human 14C-AME
– Considerations and Limitations
•
Requires synthesis of radiolabel material


•
Time consuming and cost
Regulations
Requires specialized clinical site







Specialized licensing of clinic for use and administration of
radioactivity to humans
Facilities for dose prep (nuclear pharmacist), radioactive dose
verification
Housing area for subject confinement for several weeks with
restricted toilet areas
Collection and storage of excreta (refrigerators and freezers)
Continuous analysis of radioactivity in samples for subject release
criteria
Staff training – collection procedures, radiation safety
Radiation safety program (need RSO), radiation surveys, SOPs
Human 14C-AME
– Considerations and Limitations
•
RAD waste disposal

•
•
Separate waste streams – regulated, cost
Administration of Radioactivity to humans

Potential health risk

Cultural, ethical considerations
Additional oversight and approval of clinical trial

Dosimetry – requires rodent ADME studies

Radiation risks listed in informed consent, Authorized User (FDA
10CFR35.100), ARSAC (UK), RDRC (if no IND)
Thank you