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 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

2. Outline AMS Technology Evaluation of AMS Preclinical and clinical experiences Conclusions –Advantages –Disadvantages –Perspectives

3. Outline AMS Technology Evaluation of AMS Preclinical and clinical experiences Conclusions –Advantages –Disadvantages –Perspectives

4. Nuclear physics technique Developed in the USA in the mid 70s 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 14 C LOQ: 0.005-0.86 dpm/ml (serum extract-faeces) upper limit of detection is 50 dpm ideally 10 dpm injected AMS Accelerator Mass Spectrometry

5. Sample preparation Graphitisation process Carbon in biological sample (+ 14 C-depleted carbon carrier) CuO 2 900°COxidation Cu CO 2 H 2 from 500°C withTiH 2 + Zn Co Catalyst Reduction H 2 O Graphite (0.5-2 mg) (inorganic carbon)

6. AMS Diagram 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. 12,13,14 C +1to+6 12,13 C 4+ 14 C 4+ 12,13,14 C -

7. not an absolute value, but 14 C/ 13 C or 14 C/ 12 C ratio (remember carbon dating) AMS data are expressed as the amount of 14 C per mg carbon or as pMC (percent modern carbon). Calibration is done with standards with known pMC values. 100 pMC = 1 14 C atom/1.18x10 12 12 C atoms or 97.6 attomole 14 C per mg carbon 100 pMC = 13.56 dpm 14 C/g C dpm per g sample is calculated after determining the carbon content with a C,H,N analyser (at 1 g/ml) (dpm 14 C/g C) x (% w/v C in sample) = dpm 14 C/ml 3-9 orders of magnitude more sensitive than LSC (atto- to zetogram for 14 C 10 -18 - 10 -21 grams). AMS Output

8. Comparing AMS to scintillation counting 6 X 10 5 atoms 14 C ß-ß- AMSscintillation counter 1 attomole 14 C 1000 counts in 2 minutes 1000 counts in 14 years (t ½ = 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 14 C decays).

9. Outline AMS Technology Evaluation of AMS Preclinical and clinical experiences Conclusions –Advantages –Disadvantages –Perspectives

10. 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 14 C-labelled compound, by LSC (without sample dilution) or by AMS after 600- (< 1.0 µSv) or 5000-fold (< 1 nCi) dilution of the samples.

11. Evaluation of AMS [1] Dilution experiment

13. Evaluation of AMS [1] Dilution experiment: conclusion Fairly good to good comparison after both 600- and 5000-fold dilution.

14. Evaluation study [2] Determination of the mass balance and plasma kinetics after single oral administration of a 14 C- 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).

15. 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.

16. Evaluation study [2] : Dosing

17. 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).

18. Evaluation study [2] : results Faeces (methanolic extracts) 0 10 20 30 40 50 60 70 80 90 100 0-24 h24-48 h48-72 h72-96 h0-24 h24-48 h48-72 h72-96 h Male rats Female rats % of dose LSC AMS

19. Evaluation study [2] : results

21. 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.

22. 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 14 C. It is possible to collect blank blood/plasma and blank urine from each individual subject, thus allowing for subtraction of the appropriate background.

23. Conclusions for mass balance and plasma kinetics studies in healthy volunteers Lessons 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.

24. 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 14 C-labelled materials. Repeated cleaning of all devices, areas and instruments that were used for activities with 14 C- labelled materials. AMS analysis of swabs taken from all relevant and potentially contaminated devices, areas and instruments, clinical unit. Clinical unit: non-radioactive area

25. 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 14 C- R115777 Garner R.C. et al. DMD 30:823-830, 2002.

26. 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.

27. 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.

28. 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) in urine : 45 individual samples faeces : 41 individual samples (extract + residue) plasma : 54 individual samples

29. 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

30. Human AMS study Total excretion in urine and faeces Garner R.C. et al. DMD 30:823-830, 2002

31. Human AMS study Total radioactivity and unchanged R115777 in plasma Garner R.C. et al. DMD 30:823-830, 2002

32. Human AMS study Metabolite profile in urine R115777- glucuronide R115777 R130525 Garner R.C. et al. DMD 30:823-830, 2002

33. Human AMS study Metabolite profile in faeces Garner R.C. et al. DMD 30:823-830, 2002

34. Human AMS study Metabolite profile in plasma Garner R.C. et al. DMD 30:823-830, 2002

35. 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 14 C (~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).

36. Outline AMS Technology Evaluation of AMS Preclinical and clinical experiences Conclusions –Advantages –Disadvantages –Perspectives

37. 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

38. AMS DISADVANTAGES OF AMS On-line metabolite profiling is not possible Synthesis of high specific activity 14 C labelled compound is still necessary Contamination !! Also senstive for endogenous 14 C (importance of predose samples)

39. AMS Interference from endogenous 14 C 0.024 dpm of a plasma extract injected

40. Future perspectives approach to the application of AMS in clinical studies Ethical considerations slow elimination : tissue retention / long half-life depot formulation Scientific 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