1. MGTADM 022 T 03 F 1 “microdosing” in Google: first 4 hits Gesponsorde Koppelingen U.S. Microdosing Studies www.acciumbio.comwww.acciumbio.com FDA Exploratory-IND Services; Predict failure and success now!FDA Exploratory-IND Services; Predict failure and success now! Microdosing - Wikipedia, the free encyclopedia Microdosing is a technique for studying the behaviour of compounds in vivo through the administration of doses so low they are unlikely to produce... http://en.wikipedia.org/wiki/Microdosing - 19k Xceleron - Microdosing Human microdosing (Human Phase 0) is a new concept which relies on the ultrasensitivity of accelerator mass spectrometry (AMS).... http://www.xceleron.co.uk/index.pl?id=2188 - 20k Human microdosing proves its value in drug R&D Human microdosing proves its value in drug R&D Xceleron has announced the long-awaited results of the CREAM trial into human microdosing in drug development... http://www.drugresearcher.com/news/ng.asp?n=58575-human-microdosing-proves - 46k

2. MGTADM 022 T 03 F 2 What is Microdosing ? Microdosing From Wikipedia, the free encyclopedia Microdosing is a technique for studying the behaviour of compounds in vivo through the administration of doses so low they are unlikely to produce whole-body effects..... This allows us to see the Pharmacokinetics of the drug with almost no risk of side effects. This is called a Phase 0 study and is usually done before testing on animals to predict whether a drug is viable for the next phase of testing. This is lowering the cost spent on non viable drugs and the amount of testing done on animals.......

3. MGTADM 022 T 03 F Conducting a human Phase 0 microdose study workshop AGAH 19 April 2008 Berend Oosterhuis Scientific Director EDS NL PRA International Experiences at PRA EDS in The Netherlands

4. MGTADM 022 T 03 F 4 Contents Selecting the human dose Supporting toxicity studies CMC aspects and IMP “manufacturing” Particulars about the clinical study Bioanalysis and role of AMS Concluding remarks

5. MGTADM 022 T 03 F 5 Selecting the human dose (1) “In the current context, the term ‘microdose’ would be less then 1/100th of the dose calculated to yield a pharmacological effect of the test substance based on primary pharmacodynamic data obtained in vitro and in vivo (typically doses in or below the low microgram range) and at a maximum dose of  100 microgram.” EMEA / FDA definition of microdose:

6. MGTADM 022 T 03 F 6 Selecting the human dose (2) Difficulties with EMEA / FDA definition –100 microgram as upper limit is arbitrary –calculation/prediction of human pharmacological dose often uncertain Guiding suggestions: –aim for (free) plasma concentrations ≤ 2 x EC50 or scaled dose ≤ 2x ED50 in most reliable pharmacological test/animal model –include in pharmacology tests known related agonists as “bench mark” –bottom line: any dose acceptable if supported by “clean” toxicology, including safety factor single dose tox study

7. MGTADM 022 T 03 F 7 EMEA position paperFDA exploratory IND single dose toxicity study in one species at one dose-level with 1000x safety factor relative to human microdose; 14 days extended observation with interim sacrifice on day 2; intended human route of administration and i.v. route single dose toxicity study in one species at one dose-level with 100x safety factor relative to human microdose; 14 days extended observation with interim sacrifice on day 2; intended human route of administration only genotoxity according to ICH M3 or abridged no genotoxity studies required comparative in vitro metabolism data comparative in vitro pharmacodynamic data Supporting toxicity studies(1)

8. MGTADM 022 T 03 F 8 Single dose 100 x human microdose in rats with 8 days observation –iv and intended human route (n=6 per route) –biochemistry on days 2 and 8 –animals sacrificed on day 8 –gross necropsy, limited histopathology Abridged genotoxicity (optional) Comparative in vitro metabolism; microsomes or hepatocytes Single i.v. dose CV safety in dogs (100 x microdose) –48h observation cardiovascular parameters Microdose tox program agreed between PRA and Ethics Committee (late 2003) Supporting toxicity studies(2)

9. MGTADM 022 T 03 F 9 Single dose 100 x human microdose in rats with 8 days observation –iv and intended human route (n=6 per route) –biochemistry on days 2 and 8 –animals sacrificed on day 8 –gross necropsy, limited histopathology Abridged genotoxicity (optional) Comparative in vitro metabolism; microsomes or hepatocytes Single i.v. dose CV safety in dogs (100 x microdose) –48h observation cardiovascular parameters Microdose tox program agreed between PRA and Ethics Committee (late 2003) Supporting toxicity studies(2) Human dose ( µ g) Safety factor 200 300 400 …. 1000 >10001000

10. MGTADM 022 T 03 F 10 Varations accepted by Ethics Committee, e.g. –14 days observation with interim sacrifices on day 2 (doubling the number of animals) –1000x safety factor and hERG instead of CV safety in dogs What is a ‘clean’ tox study ? –slight ‘no adverse’ effects in single dose tox study acceptable, especially if 1000x safety factor Microdose-toxicology programs (GLP) often outsourced by Sponsor –lack of internal flexibility? –most programs were conducted at NOTOX, Netherlands Supporting toxicity studies(3)

11. MGTADM 022 T 03 F 11 Varations accepted by Ethics Committee, e.g. –14 days observation with interim sacrifices on day 2 (doubling the number of animals) –1000x safety factor and hERG instead of CV safety in dogs What is a ‘clean’ tox study ? –slight ‘no adverse’ effects in single dose tox study acceptable, especially if 1000x safety factor Microdose-toxicology programs (GLP) often outsourced by Sponsor –lack of internal flexibility? –most programs were conducted at NOTOX, Netherlands Supporting toxicity studies(3) total time for tox program: 10-12 weeks from receipt test substance/documentation to draft reports ~7 gram of test substance required per compound

12. MGTADM 022 T 03 F 12 CMC aspects and IMP “manufacturing”(1) Should drug substance (API) for human microdose be manufactured under GMP? –not addressed in EMEA position paper –FDA exploratory IND allows same batch as in toxicology studies –MHRA (UK) allows “GLP quality” –radiolabelled substance (for AMS): not drug substance but (novel) excipient (CoA and some other data needed) –don’t mix drug substance and radiolabelled substance before manufacturing of IMP

13. MGTADM 022 T 03 F 13 CMC aspects and IMP “manufacturing”(1) Should drug substance (API) for human microdose be manufactured under GMP? –not addressed in EMEA position paper –FDA exploratory IND allows same batch as in toxicology studies –MHRA (UK) allows “GLP quality” –-> no GMP but concise CMC description needed in IMPD –radiolabelled substance (for AMS): not drug substance but (novel) excipient (CoA and some other data needed) –don’t mix drug substance and radiolabelled substance before shipment to PRA (ship separately) amounts needed for entire study: “cold” compound 50-100 mg labelled compound corresponding with 20-30 µCi

14. MGTADM 022 T 03 F 14 Manufacturing of “drug product” on site ! –manufacturing under GMP: manufacturing licence and QP release at clinical site –on the morning or day before dosing to subjects –high risk of adsorption losses during preparation and dosing –always run test preparations and mock administrations –analytical testing of at least “hot” dose by LSC –select best composition, procedures and materials for vessels, syringes, infusion lines –keep samples to assess actually administered doses during study CMC aspects and IMP “manufacturing”(2)

15. MGTADM 022 T 03 F 15 Keep the IMPD (and IB) lean and functional –IMP documentation in CTA can be IB + “IMPD-Addendum” containing the CMC data –Drug Product section to describe how “product” will be manufactured, control parameters for test batch, which specifications should be met –test batch preparation usually after CTA submission –-> Drug Product section may need amendment based on outcome of test preparation results CMC aspects and IMP “manufacturing”(3)

16. MGTADM 022 T 03 F 16 Keep the IMPD (and IB) lean and functional –IMP documentation in CTA can be IB + “IMPD-Addendum” containing the CMC data –Drug Product section to describe how “product” will be manufactured, control parameters for test batch, which specifications should be met –test batch preparation usually after CTA submission –-> Drug Product section may need amendment based on outcome of test preparation results CMC aspects and IMP “manufacturing”(3) we have seen considerable delays from internal SOPs and requirements by Sponsor’s regulatory group

17. MGTADM 022 T 03 F 17 For radioactive doses ≤ 1 μCi: –“trivial” radiation burden ICRP-62 (<< 0.1 mSv) –no (animal studies to support) dosimetry required Conduct study outside area for normal radiolabel studies –to avoid contamination of subjects and samples Screening of subjects for background radioactivity ? Particulars about the clinical study

18. MGTADM 022 T 03 F 18 LC-MS/MS –>100 x more sensitive than ‘classical’ HPLC: picogram/mL range –can provide parent vs. metabolite/structure information –no 14 C radiolabeled compound required Accelerator Mass Spectrometry (AMS) –ultrasensitive: femtograms (10 -15 g/mL) and below – 14 C radiolabeled compound required (50-200 nanoCurie) Only a few providers for AMS worldwide –Xceleron, York UK and Gaithersburg USA –Accium Biosciences, Seattle, USA –Vitalea Sciences, USA –IAA, Tokyo, Japan Bioanalysis and role of AMS(1)

19. MGTADM 022 T 03 F 19 Bioanalysis and role of AMS (2) AMS “counts” 14 C-atoms – 14 C radiolabeled compound required (50-200 nanoCurie per dose) Samples converted to graphite before AMS –direct AMS only gives total 14 C content –off-line HPLC separation of parent compound and metabolites Verification of HPLC separation by “fractionation” –need certainty about full separation of parent !

20. MGTADM 022 T 03 F 20 LC-AMS parent vs. metabolite separation of diazepam Bioanalysis and role of AMS (3)

21. MGTADM 022 T 03 F 21 Profile 14 C Parent Diazepam IV microdose diazepam: total radioactivity and parent concentration-time profile Bioanalysis and role of AMS (4)

22. MGTADM 022 T 03 F 22 Concluding remarks planned human microdose supported primarily by ‘clean’ tox study with safety factor (100-1000x) single dose in one species no clear rationale for 100 microgram upper limit pharmaceutical procedures to administer the right dose are critical AMS is unique analytical tool; off-line separation of parent is essential

23. MGTADM 022 T 03 F 23 Concluding remarks Conducting a human Phase 0 microdose study Time-critical activitiesParallel activitiesMinimal time Amount of substance Reduced toxicity programRadiosynthesis CMC and IMPD 12 weeks7 gram Compilation CTA dossierWriting IB, protocol, subject information 3 weeks CTA approvalTest batch IMP and QC2 weeks Clinical Study + recruitment/screening First samples to AMS lab; setup LC-AMS 5 weeks< 0.5 gram 20-30 µCi 14 C LC-AMS and assay of samples Initiate PK evaluation6 weeks< 0.1 gram < 5 µCi 14 C Draft PK report1 week Total29 weeks~ 7.5 gram 25-35 µCi 14 C

24. MGTADM 022 T 03 F 24 Concluding remarks Conducting a human Phase 0 microdose study Time-critical activitiesParallel activitiesMinimal time Amount of substance Reduced toxicity programRadiosynthesis CMC and IMPD 12 weeks7 gram Compilation CTA dossierWriting IB, protocol, subject information 3 weeks CTA approvalTest batch IMP and QC2 weeks Clinical Study + screeningFirst samples to AMS lab; setup LC-AMS 5 weeks< 0.5 gram 20-30 µCi 14 C LC-AMS and assay of samples Initiate PK evaluation4 weeks< 0.1 gram < 5 µCi 14 C Draft PK report1 week Total27 weeks~ 7.5 gram 25-35 µCi 14 C Do you consider microdosing? KISS ! (keep it small and simple)