Jun.28,2007  35  US 2007/0148124 Al  TABLE 5-continued  Example 11  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  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).  Classes of agents approved or under consideration for use in hwnan therapy.  Mechanism of Action  Drug or drug class  tetracyclines  Arninoglycosides  Bacterial  ribosomes/translation  Bacterial  ribosomes/translation  Streptomycin and variations  macrolides  Fungal  Erythromycin and  variations  Polyenes  Disrupt fungal cell wall  Arnphotericin B, Nystatin  causing electrolyte leakage  Azoles  Inhibit ergosterol  Fluconazole, Ketoconazole  biosynthesis by blocking 14- alpha-demethylase  Allylames  Disrupt ergosteral synthesis  Terbinafine  Anti-metabolites  Substrate for fungal DNA  flucytosine  polymerase  Glucan synthesis Inhibitors  Glucan is a key component in caspofungin  fungal cell wall 