Jun.28,2007  31  US 2007/0148124 Al  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.  [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  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.  [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  Effect of HPMCT on Bacterial Pathogens.  Example 7  Effect of HPMCT on Herpes Simplex Virus Infections. 