content_image/924701.jpg
<img src='content_image/924701.jpg'>

t AR! 77 is an effective blocker of virus binding and entry (Este J. A., et al. Mal Pharma› col.; 53(2): 340-5, 1998. §Motakis, D., and M. A. Parniak "A tight binding mode of inhibition is essential for anti› 

hwnan immunodeficiency virus type 1 virusidal activity of nonnucleoside reverse tran› scriptase inhibitors". Antimicrobial Agents and Chemotherapy 46: 1851-1856, 2002. *Catalone et al. "Mouse model of cervicovaginal toxicity and inflammation for preclinical 

evaluation of topical vaginal microbicides." Antimicrobial Agents. Chemotherapy vol 48, 2004. 

Effect of pH on the Antiviral Activity of CAP and HPMCT 

[0308] The vaginal microenvironment is hard to recapitu› late in simple tissue culture systems, but in an attempt to estimate what effects of low pH in the vaginal environment would do to anionic polymer, CAP and HPMCT were rewashed in a low pH buffer before adding the compounds to well-buffered GHOST X4 cells in the presence ofH9 cells infected with HIV-1 sKr (CD4-dependent cell-associated infection assay). This experiment mimics the effect of expo› sure to low pH followed by rapid readjustment of the pH in the vaginal lumen by the introduction of semen. The effect of test polymer and the control compound AMD 3100 on virus production was ascertained by monitoring intracellular p24 production 24 hr post-infection. 

CD4-dependent HIV Transmission Inhibition Assay 

[0309] The CD4-dependent HIV transmission inhibition assays use the CD4 positive GHOST(3) X4/R5 or the CD4 positive GHOST(3) RS cell lines. These cell lines are derived from the HOS (human osteosarcoma) cell line that is negative for HIV coreceptor and CD4 expression. The cell line is engineered to express T4 (CD4), CCR5 and/or CXCR4 via non-selectable retroviral vectors and an HIV-2 LTR hGFP construct with a hygromycin selectable marker. 

[0310] Twenty-four hours prior to the assay, cells are trypsinized, washed and seeded in 96-well flat bottom microtiter plates. On the day of the assay, effector cells (H9 cells chronically infected with the SKI clinical isolate of HIV-1, or MOLT4 cells chronically infected with the JR› CSF molecular clone) are treated with freshly made mito› mycin C (200 µg/ml) for 60 minutes at 37° C. This concen› tration of mitomycin C is sufficient to result in cell death, but 

## Example 11 

allows virus transm1ss10n to occur. After mitomycin C treatment, the effector cells are washed repeatedly with tissue culture medium. Test compounds are added to the monolayer followed by effector cells. The cells are co› cultured with effecter cells and test material for 4 hours, and the effector cells are removed by washing the monolayer repeatedly with RPMI. At 20 hours after assay initiation, the wells are again washed to ensure removal of the effecter cells, and virus replication is assessed via measurement of cell-associated HIV-1 gag p24 using an ELISA (Beckman› Coulter p24 ELISA). Compound toxicity and cell viability are assessed by MTS dye reduction. 

[0311] Compounds evaluated in the pH transition assay are set up essentially the same as described above, with the exception that compounds are prepared in medium adjusted to a pH of3.45 to 6.5 before addition to well-buffered target cells. Addition of effector cells prepared in a buffered medium results in a transition of the pH to near neutrality. All determinations are performed in triplicate with serial Log$_{10 }$ dilutions of the test materials. 

[0312] In this experiment, both compounds exhibited anti› viral activity, but the antiviral activity of CAP and HPMCT were diminished to a greater degree by the low pH treatment than was that of HPMCT (FIG. llA and 11B). In fact, the adjustment of the preincubation conditions from a standard assay condition of pH approximately 7, to a lower pH (between 4 to 6.5) seemed to slightly enhance the activity of HPMCT. In this set of experiments, the CXCR4 chemokine receptor antagonist AMD3100 was used as a positive con› trol. It is interesting to note that the activity of AMD 3100 also increased upon preincubation at low pH, as shown in table 6 (Table 6). 

