March 22, 2004 David Jacobson-Kram, Ph.D. DABT Office of New Drugs

1. Positive Genetox Findings on a Candidate Pharmaceutical….Now What?Regulatory and Safety Evaluation Specialty Section March 22, 2004 David Jacobson-Kram, Ph.D. DABT Office of New Drugs Center for Drug Evaluation and Research Food and Drug Administration

2. CDER view of genotoxicity findings • Genotoxicity is seen only as a predictor of carcinogenicity prior to drug approval, most drugs will undergo carc testing • Reactions to positive findings may differ between clinical divisions • For clinical trials in patients, particularly for a serious indication, positive responses can be acceptable • For healthy volunteers, risks must be minimal

3. S2B A Standard Battery of Genotoxicity Testing for Pharmaceuticals • The 3-test battery is sufficient for compounds giving negative results • Compounds giving positive results in the standard test battery may, depending on therapeutic use, need to be tested more extensively.

4. S2A Guidance on Specific Aspects of Regulatory Genotoxicity Tests for Pharmaceuticals • Positive results • positive result in vitro is followed up by a second in vivo study--using tissue other than bone marrow. Generally, rat UDS. UDS

5. Positive results: Issues to consider regarding the product • What is the drug indication? Melanoma versus dandruff. • Who is the target population? Geriatric, pediatric, obstetric. • What is the duration of use? Single treatment, weeks, months, lifetime? • Are there other drugs already serving this medical need? What is their safety profile?

6. A review of the genotoxicity of marketed pharmaceuticals* • 1999 PDR and peer-reviewed literature • 467 marketed drugs • excluded anti-cancer, nucleosides, steroids, biologicals and peptide-based drugs • 115 of 467 had no published genetox data • acutely administered: antibiotics, antifungals, antihistamines, anesthetics • 352 had at least one standard genetox test result Snyder and Green, 2001

7. A review of the genotoxicity of marketed pharmaceuticals • 101 of 352 (29%) had at least one positive assay result • bacterial mutation 27/323 (8%) • in vitro cytogenetics 55/222 (25%) • mouse lymphoma 24/96 (25%) • in vivo cytogenetics 29/252 (12%) Snyder and Green, 2001

8. Regulatory responses to positive genetox results • In general, single dose studies in volunteers or “healthy patients” would be allowed regardless of genetox data. • Some divisions have required a negative Syrian Hamster Embryo (SHE) transformation assay or p53 carcinogenicity study before allowing repeat dose clinical studies

9. Potential alternatives to SHE or p53 • Weight of evidence (WOE) approach • Mechanism of action (MOA) • Additional supportive studies bearing on WOE

10. WOE assessment • Was the positive response observed in an in vitro or in vivo assay? • Was the positive response reproducible? • Was the response dose related? • What was the magnitude of the response? • Was the response seen at a highly cytotoxic dose? • Was a positive seen in more than one assay?

11. Mutant frequency Cytotoxicity Drug concentration

12. Mutant frequency Cytotoxicity Drug concentration

13. WOE may suggest lack of hazard: no additional testing • Positive response is statistically significant but still in the range of the historical control for the test system • High dose positive in single arm of one assay but not corroborated by data from the same or between assays • short and long term exposures in absence of S9 at comparable toxicity • complimentary arms of cytogenetics and mouse lymphoma assays

14. MOA may suggest lack of risk • High osmolarity or low pH in in vitro mammalian cell assays • Positive micronucleus test results from aneuploidy not clastogenesis, kineticore staining • Clastogenic effect results from inhibition of topoisomerase with evident threshold • Positive response is due to metabolite created by rat S9 but not seen in human

15. Equivocally positive in vitro cytogenetics assay

16. Clearly positive in vitro cytogenetics assay

17. In vitro results are positive, dose responsive and reproducible, in vivo results are negative: why is that? • drug metabolized differently, genotoxic metabolites not generated in vivo • potentially genotoxic products are metabolically inactivated • genotoxic product does not reach the target cell in vivo • most common reason: blood levels of genotoxic product can not be achieved that are comparable to concentrations giving positive results in vitro

18. Additional testing to support WOE • Genotoxicity markers from longer-term, repeat-dose studies • micronucleated normochromatic erythrocytes from mouse peripheral blood • standard or FISH metaphase analysis of cultured peripheral blood lymphocytes from rat or monkey, FISH analysis of BM

19. Why peripheral blood lymphocytes? • PBLs exist in a state of mitotic arrest with lowered capacity for DNA repair. It’s harder to kill a nondividing cell. • PBLs can accumulate damage which will be expressed when the cells are stimulated to divide in vitro. • Cells which have been repeatedly dosed for 28 to 90 days at an MTD will have a better opportunity for expressing damage.

20. What’s the evidence? • Rosselli et al.,* studied chromosome aberrations in rat PBLs after exposure to cyclophosphamide, 5-fluorouracil and adriamycin. • After single IP exposure, CA were found to persist: • CP: CA persisted 8 weeks at 6X baseline, returns to baseline after 20 weeks • 5FU: CA persisted 1 week • AM: CA persisted up to 2 weeks *Rosselli et al, Mutat. Res. 232, 107-114, 1990

21. Additional testing to support WOE • DNA adducts • Comet assay • Transgenic mutation assay • Cell transformation assays • Short-term carcinogenicity studies • Patient monitoring