1. A new topical treatment for HPV-induced neoplasia Georgetown University Gary Disbrow Astrid Baege Kate Kierpiec Hang Yuan Dan Hartmann Richard Schlegel

2. High-risk HPVs are the etiologic agents in 99% of cervical cancers (Walboomers 1999) and also have a role in a subset of oral, anal, esophageal, and epidermal carcinomas. Low-risk HPVs induce benign tumors at many anatomic sites, including those of mucosal and epidermal origins. To study mucosal papillomavirus infections and to evaluate potential therapies (including vaccines), we have utilized the canine oral papillomavirus model. HPV-induced neoplasia

3. Iron as a target Artemisinin is the active principle of the Chinese herb, Artemisia annua, and is currently used clinically for treating drug-resistant malaria. toxicity is dependant upon interactions with iron DHA is a metabolic intermediate of artemisinin Many HPV-expressing cells, including cervical cancer cells, overexpress the transferrin receptor Potential for higher levels of intracellular iron Distinction between cancer and normal cells

4. Dihydroartemisinin Artemisia annua The structure of DHA

5. Iron-dependent activity of DHA Endoperoxide bridge OH OH - Fe++ DNA damage Dihydroartemisinin

6. HCX controlHCX 3d 25 µM DHA HeLa controlHeLa 3d 25 µM DHA Cell morphology after treatment with DHA

7. HeLa cells treated with artemisinin and derivatives 0102030405060 0 20 40 60 80 100 120 Artemisinin Artesunate DHA  M % Cell Survival

8. Normal cervical cells treated with artemisinin and derivatives 0102030405060 0 20 40 60 80 100 120 Artemisinin Artesunate DHA  M % Cell Survival

9. Cell lines treated with DHA 0102030405060 0 20 40 60 80 100 120 HCX HCX-E6E7 p5 HCX-E6E7 p50 SiHa Caski HeLa  M DHA % Cell Survival

10. Cell lines treated with artesunate 0102030405060 0 20 40 60 80 100 120 HCX HCX-E6E7 p4 HCX-E6E7 p45 SiHa Caski HeLa  M Artesunate % Cell Survival

11. Cell killing at higher concentrations of artemether

12. Chelation of iron inhibits killing of HeLa cells by DHA 0255075100125150175 0 20 40 60 80 100 120 0  M DFOM 25  M DFOM 100  M DFOM 200  M DFOM  M DHA % Cell Survival

13. Cellular esterases Non-fluorescent, reduced form Fluorescent, oxidized form H 2 O 2, OH - Cell membrane 488 nm 570 nm Measuring reactive oxygen species with DCF with DCF

14. FITC-A Cell Count DHA DFOM + DHA Induction of reactive oxygen species in HeLa cells Untreated 0  M DHA 25  M DHA 50  M DHA Pretreated with 150  M DFOM 5  M C-DCF-DA

15. DHA induces apoptosis in HeLa cells but not in primary cervical cells Primary cervical cells HeLa cells 10 uM DHA0 uM DHA25 uM DHA50 uM DHA

16. DHA activates caspases in the mitochondrial pathway and induces PARP cleavage in HeLa cells  -Actin Cleaved Caspase 3 19 kD 47 kD  -Actin Cleaved Caspase 9 36 kD 47 kD 100 + 150 DFOM 100500 81 kD  -Actin Cleaved PARP 47 kD 19 kD  -Actin 47 kD Cleaved Caspase 7 050100 100 + 150 DFOM 100500 100 + 150 DFOM 100 0 100 + 150 DFOM

17. Canine oral papillomavirus (COPV) as a model for human disease Canine oral papillomavirus infects and induces tumors at mucosal sites, mimicking the biology of mucosal papillomavirus infections. As in human disease, tumor induction and growth is markedly affected by host immune status. In animals with persistent infection, carcinomas develop after 2 years and metastasize widely. The canine model has been used to provide “pre-clinical” data prior to phase trials of human vaccines (MedImmune and GSK).

18. Dogs Challenged with COPV-1 Start treatment with DHA or DMSO 24 hrs later 3 wks Tumor formation started All tumors had regressed 5 wks Stop treatment Canine oral papillomavirus model

19. Dogs Challenged with COPV-1 Start treatment with DHA or DMSO 24 hrs later 3 wks Tumor formation started All tumors had regressed 5 wks Stop treatment Viral challenge + DHA Viral challenge + DMSO Application method Dogs were treated daily with 100 ul of DMSO or DHA. The DHA was at a concentration of 78.13 mM (stock). Every third day, the dogs were placed under light anesthesia to ensure a more thorough treatment. In vivo activity of DHA

20. Dog ID DMSO or DHA Right Side Left Side 1DMSO++ 2 ++ 3 ++ 4DHA-- 5*5* ++ 6 -- Tumor formation in dogs * Tumors regressed two weeks earlier than the DMSO-treated animals

21. Normal Dog Sera 156342 DMSODHA OD 450 Anti-L1 capsid protein antibody titers DHA does not prevent early papillomavirus infection 0 0.1 0.2 0.3 0.4 0.5 0.6

22. 00100 50 150 DHA  M DFOM  M HCXHeLa p53  -actin E7  -actin DHA does not inhibit early viral protein expression in vitro

23. Summary DHA induces rapid, iron-dependent, p53-independent apoptosis in PV-expressing epithelial cells in vitro DHA prevents the formation of PV-induced tumors in vivo DHA has several features which make it suitable for the topical treatment of mucosal HPV infections 1. Artemisinin derivatives are currently approved in humans for the systemic treatment of malaria 2. DHA is hydrophobic and readily penetrates mucosal surfaces 3. In addition to inducing apoptosis, artemisinin derivatives have anti-angiogenic activity

26. Virions The first-generation papillomavirus vaccine: a virus-like particle (VLP) VLP