Genotoxicity of Antiviral Nucleoside Analogs: Mechanisms and Risk Evaluation

1. The in vitro chromosome aberration assay is very sensitive to chemical mutagens Note lack of cytotoxicity

2. Chromosomeaberrations in vitro in CHO cells With S-9 Without S-9 Numbers on top of bars are cellcountsas % control.

3. Negative genotoxicity assays of Angiotensin II receptor antagonist and analogs • Microbial mutagenicity in Salmonella and E.Coli • Alkaline elution (DNA strand breaks) in rat hepatocytes • Mammalian cell mutagenicity; hprt in V79 cells • In vivo: • Chromosome abs in M & F mice (1500 mg/kg) • Alkaline elution in F rat liver (1978 mg/kg) • Additional negative tests on analogs: • DNA adducts in calf thymus DNA • ‘phage DNA fragmentation • unscheduled DNA synthesis

4. Inhibition of DNA synthesis by AIIRAs

5. Significance of aberrations associated with cytotoxicity • Potential threshold • no structural alert • no mutation • no DNA binding • no aberrations in vivo • likely secondary to toxicity, not relevant at human exposure levels • In vitro positive at 1400 µg/ml • In vivo: mouse exposure at 500 mg/kg/d x8 • C Max 74 µg/ml, AUC 90 µg/ml.hr • Human 40mg oral: • C Max 0.2 µg/ml, AUC 0.4 µg/ml.hr

6. Mechanisms of chromosomeaberrations • Aberrations result from DNA strand breaks • Strand breaks induced by e.g., • ionizing radiation • processing of lesions/adducts/abnormal bases • but also by • inhibition of DNA synthesis • inhibition of topoisomerases • Cytotoxicity • Mis-incorporation, altered bases • nucleoside analogs; ribonucleotide reductase inhibitors; folate antagonists • DNA synthesis inhibition; chain termination; mis-pairs, gaps;

7. Mechanisms of genotoxicity that may have threshold • Disruption of cell division • Disruption of chromosome segregation • Inhibition of DNA synthesis • Inhibition of topoisomerases • Nucleotide pool imbalance • Overloading of oxidative defence mechanisms • Metabolic overload (phase II enzymes etc) • Ion chelation; disturbance of metal homeostasis • Extremes of pH/osmolality • Cytotoxicity (adapted from Henderson, Albertini & Aardema, Mutat Res 464, 123-128, 2000)

8. Possible mechanisms for chromosome aberration induction by nucleoside analogs • DNA synthesis inhibition • incorporation • chain termination • attempted repair of mispaired bases • pool imbalance • DNA synthesis inhibition • polymerase errors- attempted repair

9. Genetic Toxicology of Antivirals/Nucleosides • All are negative in AMES (no data on adenosine) Wutzler and Thust. 2001. Antiviral Research. 49, 55-74; Phillips et al, Env Molec Mutagen 18, 168-183, 2001

10. Risk evaluation for nucleoside analog • Specificity for viral vs mammalian enzyme • Is it incorporated into DNA? RNA? • If incorporated is it pro-mutagenic? (AZT) • If not incorporated: High dose effect. • Possible mechanisms • pool imbalance (try to restore) • DNA synthesis inhibition (demonstrate) • Genotoxicity may have threshold • in vivo cytogenetic assay negative • good safety margin (exposure) • May not be genotoxic risk to humans.

11. Second tier follow-up Mechanistic Studies Strategy for Genotoxicity Follow-Up Testing for Unique in vitro Positive In vitro Chromosome Aberrations + ? Indirect DNA Synthesis Inhibition Direct DNA DamageDNA adducts, or neg Ames & Alk elut Acute Mouse Bone Marrow Micronucleus Human (TK6)Cell Mutation - Measure Exposure 2nd in vivo test if exposure appropriate yes + = No Go - - Good Margin OK for single or multiple dose trials in normal volunteers.

12. Risk evaluation when mechanism is indirect • Chromosome aberrations may be induced only above a threshold dose. • Therefore if: • in vivo cytogenetic assay negative • second in vivo assay negative • good safety margin (exposure) • May not be genotoxic risk to humans.