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A new topical treatment for HPV-induced neoplasia

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

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A new topical treatment for HPV-induced neoplasia

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

  2. HPV-induced neoplasia • 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.

  3. Iron as a target 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 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

  4. Dihydroartemisinin Artemisia annua The structure of DHA

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

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

  7. HeLa cells treated with artemisinin and derivatives 120 100 80 Artemisinin % Cell Survival 60 Artesunate DHA 40 20 0 0 10 20 30 40 50 60 m M

  8. Normal cervical cells treated with artemisinin and derivatives 120 100 80 Artemisinin % Cell Survival 60 Artesunate DHA 40 20 0 0 10 20 30 40 50 60 m M

  9. Cell lines treated with DHA 120 100 HCX 80 HCX-E6E7 p5 HCX-E6E7 p50 % Cell Survival 60 SiHa 40 Caski HeLa 20 0 0 10 20 30 40 50 60 m M DHA

  10. Cell lines treated with artesunate 120 HCX 100 HCX-E6E7 p4 80 HCX-E6E7 p45 % Cell Survival 60 SiHa Caski 40 HeLa 20 0 0 10 20 30 40 50 60 m M Artesunate

  11. Cell killing at higher concentrations of artemether

  12. Chelation of iron inhibits killing of HeLa cells by DHA 120 100 m 0 M DFOM 80 m 25 M DFOM % Cell Survival 60 m 100 M DFOM m 200 M DFOM 40 20 0 0 25 50 75 100 125 150 175 m M DHA

  13. Measuring reactive oxygen species with DCF 488 nm Cellular esterases H2O2, OH- Non-fluorescent, reduced form Fluorescent, oxidized form Cell membrane 570 nm

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

  15. Primary cervical cells 0 uM DHA 10 uM DHA 25 uM DHA 50 uM DHA HeLa cells DHA induces apoptosis in HeLa cells but not in primary cervical cells

  16. Cleaved Caspase 3 100 + 150 DFOM 100 + 150 DFOM 0 100 0 50 100 19 kD 47 kD b-Actin 100 + 150 DFOM 100 + 150 DFOM 0 50 100 0 50 100 DHA activates caspases in the mitochondrial pathway and induces PARP cleavage in HeLa cells Cleaved Caspase 9 36 kD 47 kD b-Actin Cleaved Caspase 7 Cleaved PARP 19 kD 81 kD 47 kD 47 kD b-Actin b-Actin

  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 All tumors had regressed 5 wks 3 wks Start treatment with DHA or DMSO 24 hrs later Tumor formation started Stop treatment Canine oral papillomavirus model

  19. Dogs Challenged with COPV-1 Tumor formation started All tumors had regressed 3 wks 5 wks Start treatment with DHA or DMSO 24 hrs later Stop treatment Viral challenge + DHA Viral challenge + DMSO In vivo activity of DHA 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.

  20. Tumor formation in dogs *Tumors regressed two weeks earlier than the DMSO-treated animals

  21. Normal Dog Sera 1 2 3 4 5 6 DMSO DHA DHA does not prevent early papillomavirus infection Anti-L1 capsid protein antibody titers 0.6 0.5 0.4 OD 450 0.3 0.2 0.1 0

  22. DHA does not inhibit early viral protein expression in vitro HCX HeLa DHA mM 0 50 100 100 0 50 100 100 DFOM mM 150 150 p53 b-actin E7 b-actin

  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

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

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