L. C. Gunder1, H. A. Green2, A. Bilger3, H. R. Johnson1, T. H. Moyer1, E. H. Carchman1,2,4 1University Of Wisconsin, Department Of Surgery, School Of Medicine And Public Health, Madison, WI, USA 2University Of Wisconsin, Carbone Cancer Center, School Of Medicine And Public Health, Madison, WI, USA 3University Of Wisconsin, McArdle Laboratory For Cancer Research, Department Of Oncology, Madison, WI, USA 4William S. Middleton Memorial Veterans Hospital, Madison, WISCONSIN, USA
Introduction: Infection of the mouse anal tract with mouse papillomavirus (MmuPV1) is an innovative preclinical model that allows for the study of viral-mediated anal disease. Immunodeficient mice infected with papillomavirus, similar to immunosuppressed human patients, have difficulties in successfully clearing the virus. The goal of this project is to assess the effect of the topical protease inhibitor, Saquinavir, on MmuPV1 viral RNA in a mouse model of viral-mediated anal disease and its relation to tissue concentrations of the drug.
Methods: Male and female immunodeficient NOD scid gamma (NSG) mice (8 -16 weeks of age), were infected in the anus with 3×108 viral genome equivalents of MmuPV1. After 20 weeks, mice received the following topical treatments to the anal canal: 1% solution of Saquinavir (SQV) five days a week and/or the carcinogen, 0.12 μmol 7,12 dimethylbenz(a)anthracene (DMBA) weekly. After 20 weeks of treatment, anal tissue was harvested for quanitifcation of SQV by electrospray ionization mass spectroscopy. A portion of anus underwent RNA in situ hybridization (RNA-ISH) targeted to MmuPV1. SQV quantification between groups was compared by unpaired t-tests. One-way ANOVA was utilized to analyze RNA levels (measured in optical density) with Tukey’s multiple comparison tests between groups. Correlation between viral RNA and SQV levels was assessed via Pearson’s correlation coefficient.
Results: Tissue from mice treated with SQV and DMBA (n = 15) had significantly higher levels of SQV (190.1 mg/g) present than the SQV alone (85.75 mg/g; n = 15; P = 0.0144). This effect only remained statistically significant in female mice tissue (266.4 v. 96.34 mg/g; Female P = 0.0139; 123.2 v. 73.64 mg/g; Male P = 0.7711). Male tissue had significantly lower levels of SQV (P = 0.0460) when comparing tissue from males treated with SQV and DMBA to females with SQV and DMBA.
There were no significant differences in viral RNA between treated with SQV compared to respective controls with or without DMBA and regardless of sex (mean viral RNA value range for all treatment groups = 0.3014 – 0.3801 optical density; P > 0.05). There was no correlation between viral RNA and SQV levels between groups when looking at both males and females together (P > 0.05). However, in females treated with SQV alone, there was an inverse correlation between levels of SQV and viral RNA (r = -0.8596; P = 0.0282).
Conclusions: Treatment with topical SQV had a greater effect in MmuPV1-infected female mice than male mice in terms of decreased tissue viral RNA. This may be potentially related to the fact that tissue from female mice had significantly higher levels of SQV present compared to males. Increased SQV in the tissue of female mice treated with SQV only correlated with reduced viral RNA.