40
Use of AMS in low radioactive dose human AME studies GMP Journées des 04 et 05 Février 2004 Distribution et Transporteurs dans les études de pharmacocinétique Paris Geert Mannens, Ph.D. Preclinical Pharmacokinetics Johnson&Johnson Pharmaceutical Research&Development, A division of Janssen Pharmaceutica N.V. Turnhoutseweg 30 B-2340 Beerse Belgium

Use of AMS in low radioactive dose human AME studies GMP Journées des 04 et 05 Février 2004 Distribution et Transporteurs dans les études de pharmacocinétique

Embed Size (px)

Citation preview

Page 1: Use of AMS in low radioactive dose human AME studies GMP Journées des 04 et 05 Février 2004 Distribution et Transporteurs dans les études de pharmacocinétique

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

Page 2: Use of AMS in low radioactive dose human AME studies GMP Journées des 04 et 05 Février 2004 Distribution et Transporteurs dans les études de pharmacocinétique

Outline

• AMS Technology

• Evaluation of AMSPreclinical and clinical experiences

• Conclusions– Advantages– Disadvantages– Perspectives

Page 3: Use of AMS in low radioactive dose human AME studies GMP Journées des 04 et 05 Février 2004 Distribution et Transporteurs dans les études de pharmacocinétique

Outline

• AMS Technology

• Evaluation of AMSPreclinical and clinical experiences

• Conclusions– Advantages– Disadvantages– Perspectives

Page 4: Use of AMS in low radioactive dose human AME studies GMP Journées des 04 et 05 Février 2004 Distribution et Transporteurs dans les études de pharmacocinétique

• 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

Page 5: Use of AMS in low radioactive dose human AME studies GMP Journées des 04 et 05 Février 2004 Distribution et Transporteurs dans les études de pharmacocinétique

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)

Page 6: Use of AMS in low radioactive dose human AME studies GMP Journées des 04 et 05 Février 2004 Distribution et Transporteurs dans les études de pharmacocinétique

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-

Page 7: Use of AMS in low radioactive dose human AME studies GMP Journées des 04 et 05 Février 2004 Distribution et Transporteurs dans les études de pharmacocinétique

• 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

Page 8: Use of AMS in low radioactive dose human AME studies GMP Journées des 04 et 05 Février 2004 Distribution et Transporteurs dans les études de pharmacocinétique

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).

Page 9: Use of AMS in low radioactive dose human AME studies GMP Journées des 04 et 05 Février 2004 Distribution et Transporteurs dans les études de pharmacocinétique

Outline

• AMS Technology

• Evaluation of AMSPreclinical and clinical experiences

• Conclusions– Advantages– Disadvantages– Perspectives

Page 10: Use of AMS in low radioactive dose human AME studies GMP Journées des 04 et 05 Février 2004 Distribution et Transporteurs dans les études de pharmacocinétique

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.

Page 11: Use of AMS in low radioactive dose human AME studies GMP Journées des 04 et 05 Février 2004 Distribution et Transporteurs dans les études de pharmacocinétique

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

Page 12: Use of AMS in low radioactive dose human AME studies GMP Journées des 04 et 05 Février 2004 Distribution et Transporteurs dans les études de pharmacocinétique

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

Page 13: Use of AMS in low radioactive dose human AME studies GMP Journées des 04 et 05 Février 2004 Distribution et Transporteurs dans les études de pharmacocinétique

Evaluation of AMS [1] Dilution experiment: conclusion

Fairly good to good comparison after both 600- and 5000-fold dilution.

Page 14: Use of AMS in low radioactive dose human AME studies GMP Journées des 04 et 05 Février 2004 Distribution et Transporteurs dans les études de pharmacocinétique

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).

Page 15: Use of AMS in low radioactive dose human AME studies GMP Journées des 04 et 05 Février 2004 Distribution et Transporteurs dans les études de pharmacocinétique

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.

Page 16: Use of AMS in low radioactive dose human AME studies GMP Journées des 04 et 05 Février 2004 Distribution et Transporteurs dans les études de pharmacocinétique

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

Page 17: Use of AMS in low radioactive dose human AME studies GMP Journées des 04 et 05 Février 2004 Distribution et Transporteurs dans les études de pharmacocinétique

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).

Page 18: Use of AMS in low radioactive dose human AME studies GMP Journées des 04 et 05 Février 2004 Distribution et Transporteurs dans les études de pharmacocinétique

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

Page 19: Use of AMS in low radioactive dose human AME studies GMP Journées des 04 et 05 Février 2004 Distribution et Transporteurs dans les études de pharmacocinétique

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

Page 20: Use of AMS in low radioactive dose human AME studies GMP Journées des 04 et 05 Février 2004 Distribution et Transporteurs dans les études de pharmacocinétique

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

Page 21: Use of AMS in low radioactive dose human AME studies GMP Journées des 04 et 05 Février 2004 Distribution et Transporteurs dans les études de pharmacocinétique

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.

Page 22: Use of AMS in low radioactive dose human AME studies GMP Journées des 04 et 05 Février 2004 Distribution et Transporteurs dans les études de pharmacocinétique

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.

Page 23: Use of AMS in low radioactive dose human AME studies GMP Journées des 04 et 05 Février 2004 Distribution et Transporteurs dans les études de pharmacocinétique

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.

Page 24: Use of AMS in low radioactive dose human AME studies GMP Journées des 04 et 05 Février 2004 Distribution et Transporteurs dans les études de pharmacocinétique

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

Page 25: Use of AMS in low radioactive dose human AME studies GMP Journées des 04 et 05 Février 2004 Distribution et Transporteurs dans les études de pharmacocinétique

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.

Page 26: Use of AMS in low radioactive dose human AME studies GMP Journées des 04 et 05 Février 2004 Distribution et Transporteurs dans les études de pharmacocinétique

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.

Page 27: Use of AMS in low radioactive dose human AME studies GMP Journées des 04 et 05 Février 2004 Distribution et Transporteurs dans les études de pharmacocinétique

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.

Page 28: Use of AMS in low radioactive dose human AME studies GMP Journées des 04 et 05 Février 2004 Distribution et Transporteurs dans les études de pharmacocinétique

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

Page 29: Use of AMS in low radioactive dose human AME studies GMP Journées des 04 et 05 Février 2004 Distribution et Transporteurs dans les études de pharmacocinétique

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

Page 30: Use of AMS in low radioactive dose human AME studies GMP Journées des 04 et 05 Février 2004 Distribution et Transporteurs dans les études de pharmacocinétique

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

Page 31: Use of AMS in low radioactive dose human AME studies GMP Journées des 04 et 05 Février 2004 Distribution et Transporteurs dans les études de pharmacocinétique

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

Page 32: Use of AMS in low radioactive dose human AME studies GMP Journées des 04 et 05 Février 2004 Distribution et Transporteurs dans les études de pharmacocinétique

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

Page 33: Use of AMS in low radioactive dose human AME studies GMP Journées des 04 et 05 Février 2004 Distribution et Transporteurs dans les études de pharmacocinétique

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

Page 34: Use of AMS in low radioactive dose human AME studies GMP Journées des 04 et 05 Février 2004 Distribution et Transporteurs dans les études de pharmacocinétique

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

Page 35: Use of AMS in low radioactive dose human AME studies GMP Journées des 04 et 05 Février 2004 Distribution et Transporteurs dans les études de pharmacocinétique

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).

Page 36: Use of AMS in low radioactive dose human AME studies GMP Journées des 04 et 05 Février 2004 Distribution et Transporteurs dans les études de pharmacocinétique

Outline

• AMS Technology

• Evaluation of AMSPreclinical and clinical experiences

• Conclusions– Advantages– Disadvantages– Perspectives

Page 37: Use of AMS in low radioactive dose human AME studies GMP Journées des 04 et 05 Février 2004 Distribution et Transporteurs dans les études de pharmacocinétique

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

Page 38: Use of AMS in low radioactive dose human AME studies GMP Journées des 04 et 05 Février 2004 Distribution et Transporteurs dans les études de pharmacocinétique

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)

Page 39: Use of AMS in low radioactive dose human AME studies GMP Journées des 04 et 05 Février 2004 Distribution et Transporteurs dans les études de pharmacocinétique

AMS Interference from endogenous 14C

0.024 dpm of a plasma extract injected

Page 40: Use of AMS in low radioactive dose human AME studies GMP Journées des 04 et 05 Février 2004 Distribution et Transporteurs dans les études de pharmacocinétique

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