48:1837-1847 (2004)) combined with HPMCT. PEHMB is a cationic polymer made up of alternating ethylene and hexamethylene units around a biguanide core. In these assays, a 1.0 % wt/vol stock solutions of HPMCT dissolved in 20 mM sodium citrate buffer pH 5.0, and a 5% PEHMB wt/vol solution made up in saline were used as stock solutions. 

[0290] Preliminary combination in vitro cytotoxicity experiments demonstrated that in assays in which the con› centration of one component (PEHMB or HPMCT) was varied while the other was kept constant, were non-cytotoxic after a two hour exposure of compounds to test cells, at the concentrations tested. This result was similar to that obtained when PEHMB and HPMCT tested alone (FIG. 1). Using a VBI assay and HIV-1 strain IIIB, HPMCT was equally or more effective when 0.01 % PEHMB was com› bined in the same assay then when using HPMCT alone (FIG. SA). Similar results were observed when the concen› tration of HPMCT was held constant at 0.0002% and the concentration of PEHMB was varied (FIG. SB). These data show that a negatively charged agent can be successfully combined with a positively charged agent. 

[0291] While logically it appears that negatively-charged polymers like HPMCT would be a poor choice for inclusion in a combination with the positively charged PEHMB, it is believed, without wishing to be bound, that the antiviral activity of PEHMB, and PEHMB-derived molecules, relies not only upon their positive charge, but also upon their three-dimensional shape. Therefore, it may be possible to obtain mixtures of polyanionic compounds with PEHMB at defined ratios which allow for the full expression of the antiviral properties of the individual components without exhibiting any deleterious effects due to their mixing. As seen in FIG. 5, at least within the concentration ranges of PEHMB and HPMCT tested, no antagonistic effects are observed when these two molecules were combined. These data strongly suggest that HPMCT can be used in combi› nation with other agents producing at least additive effects. Furthermore, and it is possible, under the appropriate con› ditions, to mix low cost polymers with completely different chemical features. 

## Effect of HPMCT on Herpes Simplex Virus Infections. 

[0292] Herpes simplex virus plaque reduction assays were performed as described by Fennewald et al. ("Inhibition of Herpes Simplex Virus in culture by oligonucleotides com› posed entirely of deoxyguanosine and thymidine."Antiviral Research 26:37-54 (1995), the contents of which are incor› porated by reference). This assay is a variation on the cytopathic effect assay described by Ehrlich et al. (Ehrlich, J., Sloan, B. J., Miller, F. A., and Machamer, H. E., "Search› ing for antiviral materials from microbial fermentations." Ann N.Y. Acad. Sci 130:5-16 (1965), the contents of which are incorporated by reference). Basically cells such as Vero or CV-1 cells are seeded onto a 96-well culture plate at approximately lxl0 4 cells/well in 0.1 ml of minimal essen› tial medium with Earle salts supplemented with 10% heat inactivated fetal bovine serum (FBS) and pennstrep (100 U/ml penicillin G, 100 ug/ml streptomycin) and incubated at 37° C. in a 5% CO$_{2 }$ atmosphere overnight. The medium was then removed, and 50 ul of medium containing 30-50 plaque 

## Example 7 

forming units (PFU) of HSVl or HSV2, diluted in test medium and various concentrations of test compound are added to the wells. The starting material for this assay was a 0.6% wt/vol stock solutions of HPMCT dissolved in 20 mM sodium citrate buffer pH 5.0. Test medium consists of MEM supplemented with 2% FBS and pennstrep. The virus was allowed to adsorb to the cells, in the presence of test compound, for 60 min at 37° C. The test medium is then removed and the cells are rinsed 3 times with fresh medium. A fmal 100 ul of test medium is added to the cells and the plates are returned to 37° C. Cytopathic effects are scored 40-48 hr post infection when control wells (no drug) showed maximum cytopathic effect. 

[0293] In these experiments HPMCT was added to HSV2 stock for ten minutes before the mixture was applied to cells for 60 min as described above. Forty to 48 hrs post removal of drug from the culture media, the control wells that received no drug treatment had over 500 plaques per well. Wells treated with 0.0001 % HPMCT for the indicated amount of time had less than 400 plaques per well, while wells treated with 0.25% HPMCT had no visible plaques, the IC50 for HPMCT in this assay system was below 0.001 % (FIG. 7). This result demonstrates the potency of HPMCT as an anti-herpes simplex virus agent. 

## Effect of HPMCT on Bacterial Pathogens. 

[0294] To test the effect of HPMCT on bacterial patho› gens, the cellulosic-based polymer was dissolved in 20 mM sodium citrate buffer pH 5.0 (0.6% final concentration of stock solution) and then mixed in equal parts with bacterial suspensions as described hereinbelow. First bacteria are sub-cultured 1-2 days prior to the assay by streaking cultures onto suitable agar plates such as Trypticase soy agar. Aseptic technique is used in all aspects of this protocol. A fresh bacterial colony is then used to inoculate 15 ml of 2xculture medium. To the first nine (9) columns of a 96 well plate, 100 µI of the inoculated 2xculture broth is transferred into the wells using a multi channel pipette. The remaining three (3) columns (usually numbered 10-12) are used as a sterility control. To these colunms, 100 µI of sterile 2xculture broth is added to each well. The culture medium in the second through eighth rows (usually designated B-H) is diluted by the addition of 80 µI of sterile water to those wells. The volume in wells B through H is at this time 180 µI. The antimicrobial solutions are diluted with water to twice the desired concentration of the uppermost starting concentra› tion. For instance, if the highest test concentration is 1 %, the solution is prepared at 2%. For some compounds, no dilution may be needed. To the first row (usually designated as "A"), 100µ1 of 2xtest solution is added to each well. The solution is thoroughly mixed by re-pipetting five times. The total volume of the well is now 200 µI. A 1: 10 serial dilution is now performed from Row A through Row G by transferring 20 µI from the higher concentration to the subsequent row using a multi channel pipette. This results in a six log reduction in the concentration of the test compound. In Row G, 20 µI is removed and discarded. No test compound is added to Row H (positive control for growth). The 96 well plate is placed on a shaker in an incubator with the tem› perature set for the organism of choice (usually 30° C. or 37° C.). After 24 hours, the optical density of the cultures is measured on a 96 well plate reader. Row H serves as a 

## Example 8 

