Combination treatment with intralesional cidofovir and viral-DNA vaccination cures large cottontail rabbit papillomavirus-induced papillomas and reduces recurrences.

We used the cottontail rabbit papillomavirus (CRPV) New Zealand White rabbit model to test a combination treatment of large established papillomas with intralesional cidofovir and DNA vaccination to cure sites and reduce recurrences. Intralesional 1% (wt/vol) (0.036 M) cidofovir treatment of rabbit papillomas led to elimination, or "cure," of the papillomas over a 6- to 8-week treatment period (N. D. Christenson, M. D. Pickel, L. R. Budgeon, and J. W. Kreider, Antivir. Res. 48:131-142, 2000). However, recurrences at periods from 1 to 8 weeks after treatment cessation were observed at approximately 50% of cured sites. DNA vaccinations with CRPV E1, E2, E6, and E7 were initiated either after or at the time of intralesional treatments, and the recurrence rates were observed. When DNA vaccinations were started after intralesional cures, recurrence rates were similar to those of vector-vaccinated rabbits. A small proportion of recurrent sites subsequently regressed (4 out of 10, or 40%) in the vaccinated group versus no regression of recurrences in the vector-immunized group (0 out of 19, or 0%), indicating partial effectiveness. In contrast, when DNA vaccinations were conducted during intralesional treatments, a significant reduction of recurrences (from 10 out of 19, or 53%, of sites in vector-immunized rabbits to 3 out of 20, or 15%, of sites in viral-DNA-immunized rabbits) was observed. DNA vaccination without intralesional treatments had a minimal effect on preexisting papillomas. These data indicated that treatment with a combination of antiviral compounds and specific immune stimulation may lead to long-term cures of lesions without the ensuing problem of papilloma recurrence.

In vivo anti-papillomavirus activity of nucleoside analogues including cidofovir on CRPV-induced rabbit papillomas.

A series of nucleoside analogues were tested for in vivo anti-papillomavirus activity using the cottontail rabbit papillomavirus (CRPV) domestic rabbit model. Compounds were delivered either topically, injected into growing papillomas, or delivered subcutaneously at a site remote from the papillomas. Compounds tested included cidofovir [(S)-1-(3-hydroxy-2-phosphonylmethoxypropyl)cytosine] (HPMPC); cyclic HPMPC (cHPMPC); cyclopentenylcytosine (CPE-C); lobucavir [1R(1alpha,2beta,3alpha)]-9-[2, 3-bis(hydroxymethyl)cyclobutyl]guanine; 9-((2-phosphonylmethoxy)propyl)adenine (PMPA); adefovir 9-((2-phosphonylmethoxy)ethyl)adenine(PMEA) and cyclopropyl 9-(2-phosphonylmethoxyethyl)-2,6-diaminopurine (cyclopropylPMEDAP). Dose response curves and time-course treatments were included for most compounds tested. Strong anti-viral activity was detected using cidofovir and cHPMPC when delivered either topically or by the intralesional route. Complete cures were obtained using 1% (w/v) topical cidofovir at dosing schedules of twice daily for 8 weeks beginning at 4 weeks after CRPV infection, which represents a time when papillomas were clearly visible. Complete cures of large established papillomas were obtained by intralesional injection of 1% cidofovir three times per week for 8 weeks. Topical treatments with adefovir had strong anti-viral activity, cyclopropyl PMEDAP had moderate anti-viral activity, and CPE-C, PMPA and lobucavir showed no effects. These data indicate that certain nucleoside analogues have strong in vivo anti-papillomavirus activity and that the CRPV/rabbit model is a good model for assessing clinical responses of anti-viral treatments for patients with HPV disease.

A broad-spectrum microbicide with virucidal activity against sexually transmitted viruses.

Sodium dodecyl sulfate (SDS), an alkyl sulfate surfactant derived from an organic alcohol, possesses surfactant properties but also denatures and unfolds both monomeric and subunit proteins. In preliminary experiments, we demonstrated that SDS is a potent inactivator of herpes simplex virus type 2 and human immunodeficiency virus type 1 at concentrations comparable to those used for the surfactant nonoxynol-9. We hypothesized that SDS might be capable of denaturing the capsid proteins of nonenveloped viruses. In this report, we demonstrate inactivation of rabbit, bovine, and human papillomaviruses after brief treatment with dilute solutions of SDS. Effective concentrations were nontoxic to rabbit skin and to split-thickness grafts of human foreskin epithelium. This is the first report of a microbicidal surfactant that will inactivate papillomaviruses. We propose that SDS is now a candidate microbicide for formulation and testing with humans.

Association of tumor necrosis factor-alpha gene expression and apoptotic cell death with regression of Shope papillomas.

The objective of this study was to test the hypothesis that spontaneous regression of Shope papillomas involves tumor necrosis factor-alpha and apoptotic cell death of the papilloma cells. In situ hybridization using RNA probes of rabbit tumor necrosis factor-alpha revealed tumor necrosis factor-alpha mRNA in most of the numerous mononuclear cells infiltrating the upper dermis of regressing papillomas and at the dermoepidermal junction. Such cells in progressing papillomas were much fewer in number and were located in the deeper dermis. In situ terminal deoxynucleotidyl transferase assay demonstrated DNA strand breaks in many scattered epidermal keratinocytes of regressing papillomas but in only a few thin layers just beneath the horny layer in progressing papillomas. Electron microscopy demonstrated that regressing papillomas contained many apoptotic bodies and keratinocytes showing apoptotic changes such as chromatin condensation, degradation of condensed nuclei, surface protuberances, and a filamentous degeneration, as well as infiltrating lymphocytes and macrophages. We propose that tumor necrosis factor-alpha produced by infiltrating mononuclear cells probably plays a role in the papilloma regression.

Podofilox-induced regression of Shope papillomas may be independent of host immunity.

We tested the hypothesis that infiltrating leukocytes might contribute to papilloma destruction following podofilox treatment. New Zealand White (NZW) rabbits were inoculated with cottontail rabbit papillomavirus (CRPV) onto abraded areas of the dorsal skin. At 21 d after viral inoculation, 5.0% podofilox solution was applied to some papillomas, whereas others were used as controls. Three rabbits were sacrificed at each of three different periods after treatment initiation (1, 4, and 7 d). Four monoclonal antibodies (MoAbs), RG-16 (for B cells), L11/135 (specific for T cells), 2C4 (specific for class II antigen), and Ki67 (specific for proliferating cells), were used in an immunohistochemical study. All positive cells and total cells in the field were counted with an ocular grid. After 1 d of treatment, proliferation of papilloma cells was strongly suppressed in treated papillomas, but leukocytic infiltration was not altered. At 4 d and 7 d of treatment, there were substantial increases (about two to three times) in the numbers of B and T cells and class II-expressing leukocytes. The upper layers of the papillomas were highly necrotic and cell proliferation was absent in all layers. These data support the view that podofilox has a direct toxic effect on papilloma tissue. Leukocyte infiltration is not strongly associated with papilloma tissue and may not contribute to papilloma destruction.

Influence of schedule and mode of administration on effectiveness of podofilox treatment of papillomas.

We investigated the timing of podofilox delivery to see how it correlated with papilloma size. We looked at times ranging from once per week (morning and afternoon) to five times per week (morning and afternoon). We also looked at delivery systems that might enhance the effectiveness of the drug by increasing penetration of the overlying cutaneous horn. These included soaking prior to drug administration, the use of a grooved needle subsequent to drug administration, and the various possible combinations of these techniques. We found that the timing of treatments had relatively little effect on the size of the papillomas. For example, at the end of ten weeks, the geometric mean diameter (GMD) (mm) of the papillomas treated five times per week and induced by a 10(-1) dilution of the virus (group B) was 18. Likewise the GMD (mm) of papillomas treated once per week was 18 (group D). On the other hand, we found that soaking plus the use of a grooved needle plus podofilox resulted in the curing of all lesions induced by a 10(-2) virus dilution and of most induced by the 10(-1) dilution, whereas soak plus podofilox or podofilox alone were not as effective in curing the lesions. Podofilox plus needle approached the soak plus podofilox plus needle in effectiveness. Treatment schedule was not a critical determinant of podofilox effectiveness. Therapeutic benefits were enhanced by hydration of the overlying cutaneous horn and penetration with a grooved needle.

Preclinical system for evaluating topical podofilox treatment of papillomas: dose-response and duration of growth prior to treatment.

The objective of the present study was to assess the utility of Shope rabbit papillomas as an animal model system for studying topical podofilox treatment and to evaluate dose-response relations and influence of duration of papilloma growth prior to treatment. New Zealand White rabbits received inoculations of cottontail rabbit papillomavirus (CRPV) virions of two dilutions at four sites total on the dorsum. Two papillomas on the left side were treated with podofilox (Oclassen Pharmaceuticals, Inc., San Rafael, CA). The drug was given topically twice each day, 5 d per week, for 21 d. We evaluated the effects of drug dose and the duration of papilloma growth prior to treatment. Results indicated that treatment beginning on day 28 with both 0.5 and 2.5% (w/v) podofilox inhibited papilloma growth, but 5.0% was more effective. In a separate experiment, papillomas were treated at 7, 21, or 60 d after virus inoculation. At 7 d, the untreated lesions were latent (not visible). At 21 d after infection, they were about 2.5 mm in diameter. At 60 d, papillomas were about 25 mm. Treatment with 5.0% podofilox beginning on any of those days strongly inhibited papilloma growth. Neither Southern blots nor PCR detected CRPV DNA in cured sites of previous virus infection. Antibody production to CRPV virion was not affected by drug treatment. 5.0% podofilox irritated normal skin adjacent to papillomas as evidenced by inflammation, induration, and superficial erosion. However, healing was satisfactory and no scarring resulted. We concluded that the Shope papilloma was a good model system for studying podofilox treatment because the lesions responded to drug across a broad range of drug concentrations and papilloma sizes.