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Use of AMS in low radioactive dose human
AME studiesGMP
Journées des 04 et 05 Février 2004 Distribution et Transporteurs dans les études de pharmacocinétique
Paris Geert Mannens, Ph.D.Preclinical PharmacokineticsJohnson&Johnson Pharmaceutical Research&Development,A division of Janssen Pharmaceutica N.V.Turnhoutseweg 30B-2340 BeerseBelgium
Outline
• AMS Technology
• Evaluation of AMSPreclinical and clinical experiences
• Conclusions– Advantages– Disadvantages– Perspectives
Outline
• AMS Technology
• Evaluation of AMSPreclinical and clinical experiences
• Conclusions– Advantages– Disadvantages– Perspectives
• Nuclear physics technique• Developed in the USA in the mid 70’s• Primarily used for archaeological (carbon) dating• Mass spectrometer coupled to tandem accelerator• Following graphitization, isotopes are measured
on the basis of their m/z ratio• Highly sensitive determination of 14C
LOQ: 0.005-0.86 dpm/ml (serum extract-faeces)upper limit of detection is 50 dpmideally 10 dpm injected
AMS Accelerator Mass Spectrometry
Sample preparation Graphitisation process
Carbon in biological sample (+ 14C-depleted carbon carrier)
CuO2
900°C Oxidation
Cu
CO2
H2 from
500°C with TiH2 + Zn
Co Catalyst Reduction
H2O
Graphite (0.5-2 mg) (inorganic carbon)
AMSDiagram of an accelerator mass spectrometer (30 m in length !)
Isotopes are accelerated (Tandem Van de Graaff accelerator), electrons are stripped (argon gas) and positive ions are separated using conventional mass spectrometer based on their m/z ratio.
12,13,14C+1to+6
12,13C4+
14C4+
12,13,14C-
• not an absolute value, but 14C/13C or 14C/12C ratio (remember carbon dating)
• AMS data are expressed as the amount of 14C per mg carbon or as pMC (percent modern carbon). Calibration is done with standards with known pMC values.
100 pMC = 1 14C atom/1.18x1012 12C atoms or 97.6 attomole 14C per mg carbon100 pMC = 13.56 dpm 14C/g C
• dpm per g sample is calculated after determining the carbon content with a C,H,N analyser (at 1 g/ml)
(dpm 14C/g C) x (% w/v C in sample) = dpm 14C/ml• 3-9 orders of magnitude more sensitive than LSC (atto- to
zetogram for 14C 10-18 - 10-21 grams).
AMS Output
Comparing AMS to scintillation counting
6 X 105 atoms 14C
ß-
AMS scintillation counter
1 attomole 14C
1000 countsin
2 minutes
1000 countsin
14 years
(t½ = 5730 years)
AMS measures the number of atoms in the sample, while scintillation counters measure the infrequent radioactive decay events in the sample (in any year less than 0.012 % of 14C decays).
Outline
• AMS Technology
• Evaluation of AMSPreclinical and clinical experiences
• Conclusions– Advantages– Disadvantages– Perspectives
Evaluation of AMS [1]Dilution experiment
Analysis of plasma samples, urine samples and methanolic extracts of faeces samples, collected in a clinical study with a convential radioactive dose of a 14C-labelled compound, by LSC (without sample dilution) or by AMS after 600- (< 1.0 µSv) or 5000-fold (< 1 nCi) dilution of the samples.
Evaluation of AMS [1] Dilution experiment
Plasma samples
0
200
400
600
800
0.5 1 2 8 24 48
Time (hours)
Rad
ioac
tivity
(D
PM
/ml)
LSC
AMS 600 x
AMS 5000 x
Evaluation of AMS [1] Dilution experiment
Urine samples
0
10000
20000
30000
40000
0-24 h 24-96 h 168-240 h
Time (hours)
Rad
ioac
tivity
(D
PM/m
l)
LSC
AMS 600 x
AMS 5000 x
Faeces samples (methanolic extracts)
0
200
400
600
800
1000
Stool 5 Stool 7 Stool 9
Stool number
Rad
ioac
tivity
(D
PM/m
l)
LSC
AMS 600 x
AMS 5000 x
Evaluation of AMS [1] Dilution experiment: conclusion
Fairly good to good comparison after both 600- and 5000-fold dilution.
Evaluation study [2]
• Determination of the mass balance and plasma kinetics after single oral administration of a 14C-labelled compound to male and female Wistar rats at a convential radioactive dose (sample analysis by LSC) or at a very low radioactive dose (sample analysis by AMS).
Evaluation study [2]
R115777 (oncology compound) was selected as test item on the basis of the following considerations:
• High radioactive dose preclinical study already performed optimal design for low radioactive dose study.
• Relatively simple metabolism (only 3-5 metabolites).• The mass balance study in healthy volunteers was
scheduled to be performed with AMS.
Evaluation study [2] : Dosing
High radioactive dose Low radioactive doseStudy Number FK2801 FK3463Route Oral gavage Oral gavageTotal dose 10 mg/ kg 10 mg/ kgRadioactive dose 1.48 MBq/ kg (40 µCi/ kg) 14.8 Bq/ kg (0.40 nCi/ kg)Dose/ rat ~ 220 x 105 DPM ~ 220 DPMNumber of rats 5 males and 5 f emales
for urine and f aecescollection;16 males and 16 f emalesfor plasma collection
5 males and 5 f emalesfor urine and f aecescollection;16 males and 16 f emalesfor plasma collection
Evaluation study [2] : sample analysis
• Plasma samples, urine samples and methanolic extracts of faeces samples were analysed by LSC (high radioactive dose study) or AMS (low radioactive dose study).
• Faecal residues were analysed by LSC of the oxidised samples (high radioactive dose study) or AMS (low radioactive dose study).
Evaluation study [2] : results
Faeces (methanolic extracts)
0
10
20
30
40
50
60
70
80
90
100
0-24 h 24-48 h 48-72 h 72-96 h 0-24 h 24-48 h 48-72 h 72-96 h
Male rats Female rats
% o
f dose
LSC
AMS
Evaluation study [2] : results
Urine
0
1
2
3
4
5
6
7
8
9
10
0-4 h 4-8 h 8-24 h 24-48 h 48-72 h 72-96 h 0-4 h 4-8 h 8-24 h 24-48 h 48-72 h 72-96 h
Male rats Female rats
% o
f dos
e
LSC
AMS
Evaluation study [2] : resultsPlasma
0
1000
2000
3000
4000
5000
6000
7000
1 h 3 h 8 h 24 h 1 h 3 h 8 h 24 h
Male rats Female rats
Con
cen
trati
on
of
14
C in
pla
sm
a (
ng
/ml)
AMS - PP
AMS + PP
LSC
Evaluation study [2] : results
Correlation between the high and low radioactive dose was:
• Reasonable to good for faecal excretion.• Poor for urinary excretion (presumably due to
contamination of the urine/faeces collection systems).• Poor for plasma concentrations determined without
protein precipitation (presumably due to high background radioactivity).
• Reasonable for plasma concentrations determined with protein precipitation.
Conclusions for mass balance and plasma kinetics studies in healthy
volunteers
The problems encountered in the rat study are not expected to occur in human AME studies, because:
• It is possible to collect urine and faeces with only minimal risk of contamination with 14C.
• It is possible to collect blank blood/plasma and blank urine from each individual subject, thus allowing for subtraction of the appropriate background.
Conclusions for mass balance and plasma kinetics studies in healthy
volunteersLessons learned:• Laboratories, materials or instruments that have
been previously used in high radioactive dose studies can not be used for low radioactive dose studies involving AMS analysis, not even after extensive cleaning.
• Total radioactivity in plasma should be determined with and without protein precipitation. A gentle protein precipitation method should be used to maximise the recovery.
Human AMS study: study preparations
• Purchase of new glassware, faeces containers, Büchner filters, etc.
• Allocation of laboratory areas that had not been used for activities involving 14C-labelled materials.
• Repeated cleaning of all devices, areas and instruments that were used for activities with 14C-labelled materials.
• AMS analysis of swabs taken from all relevant and potentially contaminated devices, areas and instruments, clinical unit.
• Clinical unit: non-radioactive area
Human AMS study: Dosing
Number of subjects : 4 Route: Oral solution Total dose: 50 mg Radioactive dose: 1.27 kBq = 34.4 nCi = ~76,000 DPM Radiation exposure: 0.9 µSv
For comparison: Thorax X-ray: 100-200 µSv Cosmic radiation: 3 µSv/day
Mass balance and plasma kinetics study in healthy male adult subjects after single oral administration of 14C-R115777Garner R.C. et al. DMD 30:823-830, 2002.
Human AMS study : Sample collection
Urine was collected once before dosing (blank) and in
intervals from 0-4, 4-8, 8-24 , 24-48, 48-72, 72-96, 96-120,
120-144 and 144-168 h after dosing.
Faeces was collected once before dosing (blank) and per
stool up to 168 h after dosing.
Blood (plasma) was collected once before dosing (blank)
and at 1, 2, 3, 4, 6, 8, 24, 32, 48, 72, 96 and 168 h after
dosing.
Human AMS study : Sample analysis
Blood samples were collected and plasma was separated. Faeces samples were homogenised in and extracted with
methanol. The methanolic faeces exctracts and the faecal residues were separated by filtration. The faecal residues were dried and ground.
Radioactivity concentrations in urine samples, methanolic faeces extracts and faecal residues were determined by AMS.
Radioactivity concentrations in plasma samples were determined by AMS with and without protein precipitation.
A pool of urine samples, a pool of methanolic faeces extracts and a pool of plasma samples were injected onto a HPLC column. Eluent fractions were collected. Radioactivity concentrations in individual eluent fractions were determined by AMS.
Human AMS study : Sample analysis
Xceleron (previous CBAMS, Centre for Biomedical Accelerator Mass Spectrometry, York, UK)
Total radioactivity in dosing solution (LSC) (CBAMS)(Packard Tri-Carb TR/SL 2770)4338.59 dpm/ml
Total radioactivity (CBAMS, NEC 15SDH-2 Pelletron) inurine : 45 individual samplesfaeces : 41 individual samples (extract + residue) plasma : 54 individual samples
Human AMS study : Sample analysis
metabolite profiling : AMS after HPLC fractionation, no hyphenated technique (CBAMS) : 2 runs per sample 1 urine sample: before and after enzymatic hydrolysis 1 extract of faeces 1 plasma sample : 3-h overall pool before and after enzymatic
hydrolysis
bioanalysis (Janssen) metabolite identification (Janssen):
comparison of HPLC retention time with known standards confirmation by LC-MSMS
Human AMS study Total excretion in urine and faeces
Total excretion in % of the dose
Sample Subject
1
Subject
2
Subject
3
Subject
4
Mean ± SD
Urine 0-168 h 9.58 22.72 9.27 13.34 13.73 ± 6.27Faeces 0-168 h 87.46 61.16 89.20 81.13 79.74 ± 12.86Total 0-168 h 97.04 83.88 98.47 94.47 93.47 ± 6.60
Garner R.C. et al. DMD 30:823-830, 2002
Human AMS study Total radioactivity and unchanged
R115777 in plasma
0.1
1.0
10.0
100.0
1000.0
10000.0
0 24 48 72 96 120 144 168
Time (h)
Conce
ntr
ati
on (
ng-e
q./
ml) TR in non-pretreated samples
TR in deproteinzed samples
Unchanged R115777
Garner R.C. et al. DMD 30:823-830, 2002
Human AMS study Metabolite profile in urine
0.00
0.20
0.40
0.60
0.80
1.00
1.20
0 10 20 30 40 50 60
Time (min)
dp
m p
er
fract
ion
-glu Urine
+glu Urine
R115777-glucuronide
R115777
R130525
Garner R.C. et al. DMD 30:823-830, 2002
Human AMS study Metabolite profile in faeces
0.00
0.10
0.20
0.30
0.40
0 10 20 30 40 50 60
Time (min)
dpm
per
fra
ctio
n
R130525
R110127
R115777
R101763 R104209
Garner R.C. et al. DMD 30:823-830, 2002
Human AMS study Metabolite profile in plasma
0.00
0.05
0.10
0.15
0.20
0.25
0.30
0.35
0 10 20 30 40 50 60Time (min)
dpm
per
fra
ctio
n - glu Plasma
+ glu PlasmaR115777-glucuronide
R115777
Garner R.C. et al. DMD 30:823-830, 2002
Human AMS study Conclusions
AMS is a suitable method for the determination of the mass balance after giving a very low radioactive dose.
Radioactivity concentrations in non-pretreated plasma samples may be inaccurate, due to high background levels of 14C (~0.5 dpm/ml).
Radioactivity concentrations in deproteinised plasma samples may be underestimated, due to occlusion of radioactivity in precipitated proteins.
AMS analysis of HPLC eluent fractions allows for an evaluation of metabolite profiles, provided the number of metabolites is limited ( 5).
Outline
• AMS Technology
• Evaluation of AMSPreclinical and clinical experiences
• Conclusions– Advantages– Disadvantages– Perspectives
AMS ADVANTAGES OF AMS
• Radioactive dose in humans may be 500-5000 times lower (1 µSv, now ~ 500-1000 µSv)
• Less preclinical data needed for ethics committee
• Opens possibility to conduct studies that were previously impossible
AMS DISADVANTAGES OF AMS
• On-line metabolite profiling is not possible
• Synthesis of high specific activity 14C labelled compound is still necessary
• Contamination !!
• Also senstive for endogenous 14C (importance of predose samples)
AMS Interference from endogenous 14C
0.024 dpm of a plasma extract injected
Future perspectives approach to the application of AMS
in clinical studies Ethical considerations slow elimination : tissue retention / long half-life depot formulationScientific considerations Very potent compounds – extreme low dose inhalation/dermal application : bioavailability studies
with a high radioactive dose evidence for relatively uncomplicated metabolism Limitation with the specific radioactivity (autoradiolysis) micro-dosing concept under evaluation