Activity of Acyclic Nucleoside Analogues against Human Papillomavirus HPV

1
Activity of Acyclic Nucleoside Analogues against
Human Papillomavirus (HPV)

• Graciela Andrei
• Rega Institute for Medical Research
• K U Leuven, Belgium

Vancouver 2005
2
HPV infections

• World prevalence of HPV infection 9-13,
• 630 millions infected individuals
• Number of HPV-infected people in US 24 million
  to 40 million
• HPV is the most commonly diagnosed sexually
  transmitted disease in US and UK
• Most HPV infections are harmless and 40
  disappear without treatment

3
Symptomatic HPV diseases

• Common warts
• Palmoplantar warts verrucae plantaris
• Genital warts condylomata acuminata
• Cervical HPV infection
• Recurrent respiratory papillomatosis (RRP)
• 2,000 2,500 pediatric papillomatosis new
  cases per year in US
• 6,000 children per year are treated for RRP in
  US

4
Current HPV treatments

• Chemotherapeutic products podophyllin,
  5-fluorouracil
• Immunomodulators imiquimod, interferon
• Surgical methods cryotherapy, electro- and laser
  surgery
• Acids salicylic acid, bichloro- and
  trichlroacetic acid
• Photodynamic therapy
• Antisense oligonucleotides
• Vaccination
• Antivirals ribavirin, cidofovir
• Others indol-3-carbinol, cimetidine, mumps
  vaccine.....

5
Treatment of HPV-associated diseases with
Cidofovir
Before After

• Cutaneous warts
• Genital lesions - Condylomata accuminata
• - Premalignant lesions
• intraepithelial
  neoplasia
• Bowenoid
  papillomatosis
• - Cervical cancer
• Recurrent respiratory papillomatosis

6
Intralesional Cidofovir in the treatment of RRP

• No established protocol for intralesional
  administration of cidofovir
• is available
• Intralesional administration of cidofovir with or
  without initial debulking of the lesions in
  several small, uncontrolled case series
• - Effective and benefitial effects both in
  children and adults
• Snoeck et al., 1996, 1998 Pransky et al., 1999,
  2000, 2003 Bielamowicz et al., 2002 Akst et
  al., 2003 Chhetri and Shapiro, 2003 Naiman et
  al., 2003 Neuman et al., 2003. Lee and Rosen,
  2003 Woo et al., 2004 Mandell et al 2004
  Schraff et al 2004 Co and Woo, 2004 Schraff et
  al., 2004.
• - Low therapeutic response without side effects
• Milczuck, 2003 Shirley and Wiatrak, 2004.

7
What should be considered

• Appropriate dose 2.5 to 7. 5 mg/ml
• Volume administered
• Number of injections
• Interval between the injections uniform interval
  to obtain maximum efficacy
• Administration of additional injections at the
  previous lesion site after total involution of
  the macroscopic lesion
• Location of the papilloma best response obtained
  in supraglottic and glottic subsites

8
What should be considered

• HPV type / viral load
• Disease severity
• Age
• Combined therapy intralesional CDV and surgical
  excision
• Nephrotoxicity systemic distribution (plasma
  levels) after intralesional CDV administration is
  low, excluding the risk of systemic toxicity
  (Naiman et al., 2004 Snoeck et al., 1996 and
  unpublished results)
• Local toxicity no local sides effects reports at
  therapeutic doses

9
i.v. Cidofovir in the treatment of RRP

• 8-year-old patient with severe RRP and pulmonary
  spread
• treatment systemic cidofovir in association
  with laser treatment for tracheal lesions (Snoeck
  et al., 2001)
• 32-year-old patient with severe RRP, recurrent
  penumonias due to bronchial obstruction
• treatment systemic cidofovir in association
  with IFN-a-2b (Armbruster et al., 2001)
• 8-year old patient with severe RRP and lung
  lesions
• treatment local and intravenous cidofovir in
  association with Indole-3-Carbinol (de Bildering
  et al., 2005)

10
Acyclic nucleoside analogues with anti-HPV
activity
Cidofovir, HPMPC, Vistide PMEG
HPMPCpp PMEGpp
DNA polymerase
11
Antiproliferative effect of CDV and PMEG against
HPV positive cells
CDV
PMEG
12
Hematoxilyn-eosin staining of organotypic
epithelial raft co-cultures of PHKs and CK-1
cells (HPV-33)
CDV 50 µg/ml
CDV 20 µg/ml
Co-culture PHKCK-1 cells (ratio 11)
Treatment day 3 day 7
Untreated cultures
PMEG 2 µg/ml
PMEG 0.5 µg/ml
40x
13
Annexin V / PI uptake in SiHa cells treated with
CDV or PMEG
CDV
PMEG

• Mechanism of cell death apoptosis

14
Cell cycle analysis of SiHa cells treated with
CDV or PMEG

• Accumulation of cells in S phase and G2/M

15
Detection of p53 expression in HPV-positive cells
treated with CDV or PMEG by flow cytometry
CDV

• CDV and PMEG induce accumulation of p53

16
Activity of CDV and PMEG against SiHa cells grown
as xenografts in nu/nu mice



Folds increase in tumor size

• CDV and PMEG showed potent growth-inhibitory
  activity against SiHa (HPV-16 ) xenografts in
  nu/nu mice

17
Activity of CDV and PMEG against HeLa cells grown
as xenografts in nu/nu mice
Folds increase in tumor size

• CDV and PMEG showed potent growth-inhibitory
  activity against HeLa (HPV-18 ) xenografts in
  nu/nu mice

18
Conclusions

• CDV and PMEG inhibit the growth of HPV-positive
  cells in vitro
• CDV and PMEG showed a selective antiproliferative
  effect against HPV positive cells in organotypic
  epithelial raft cultures of normal keratinocytes
  and HPV-positive cells
• The mechanism of cell death following CDV or PMEG
  treatment appeared to be apoptosis, which was
  associated with accumulation of cells in the
  S-phase or G/M and increase in p53 levels
• In athymic-nude mice both CDV and PMEG inhibited
  the growth of human cervical carcinoma xenografts
  (which harbor integrated HPV-16 or HPV-18)

19
Acknowledgements

• Dr Robert Snoeck
• Dr Erik De Clercq
• Dr Joos Van Den Oord
• Dr Ilya Lebeau
• Anita Camps
• Lies Van Den Heurck
• Willy Zeegers
• Steven Carmans