1. Positive Genetox Findings on a Candidate Pharmaceutical…. Now What
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, , 1990

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