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Antimicrobial Agents and Chemotherapy, September 2006, p. 3081-3089, Vol. 50, No. 9
0066-4804/06/$08.00+0     doi:10.1128/AAC.01609-05
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

Critical Design Features of Phenyl Carboxylate-Containing Polymer Microbicides

Novaflux Biosciences, Inc., Princeton, New Jersey 08540,¹ Shin Etsu Chemical Company Limited, Cellulose & Pharmaceutical Excipients Department, Chiyoda-ku, Tokyo 100-0004, Japan,² Southern Research Institute, Frederick, Maryland 21701,³ Department of Microbiology and Immunology and Center for Molecular Therapeutics and Resistance, Institute for Molecular Medicine and Infectious Disease, Drexel University College of Medicine, Philadelphia, Pennsylvania 19129⁴

Received 19 December 2005/ Returned for modification 4 April 2006/ Accepted 10 July 2006

	ABSTRACT

Top Abstract Introduction Materials and Methods Results Discussion References

 
Recent studies of cellulose-based polymers substituted with carboxylic acids like cellulose acetate phthalate (CAP) have demonstrated the utility of using carboxylic acid groups instead of the more common sulfate or sulfonate moieties. However, the pK_a of the free carboxylic acid group is very important and needs careful selection. In a polymer like CAP the pK_a is approximately 5.28. This means that under the low pH conditions found in the vaginal lumen, CAP would be only minimally soluble and the carboxylic acid would not be fully dissociated. These issues can be overcome by substitution of the cellulose backbone with a moiety whose free carboxylic acid group(s) has a lower pK_a. Hydroxypropyl methylcellulose trimellitate (HPMCT) is structurally similar to CAP; however, its free carboxylic acids have pK_as of 3.84 and 5.2. HPMCT, therefore, remains soluble and molecularly dispersed at a much lower pH than CAP. In this study, we measured the difference in solubility and dissociation between CAP and HPMCT and the effect these parameters might have on antiviral efficacy. Further experiments revealed that the degree of acid substitution of the cellulose backbone can significantly impact the overall efficacy of the polymer, thereby demonstrating the need to optimize any prospective polymer microbicide with respect to pH considerations and the degree of acid substitution. In addition, we have found HPMCT to be a potent inhibitor of CXCR4, CCR5, and dual tropic strains of human immunodeficiency virus in peripheral blood mononuclear cells. Therefore, the data presented herein strongly support further evaluation of an optimized HPMCT variant as a candidate microbicide.

	INTRODUCTION

Top Abstract Introduction Materials and Methods Results Discussion References

 
In developing countries, heterosexual transmission is responsible for the majority of new human immunodeficiency virus type 1 (HIV-1) infections (33, 35). In addition, other sexually transmitted disease (STD) pathogens have been shown to facilitate HIV-1 infection (41). In the absence of inexpensive antiviral systemic agents and effective prophylactic vaccines against HIV-1 and other STD pathogens, simple methods to control these infectious agents must be sought, especially for the areas suffering the most from the HIV-1 pandemic.

One of the first compounds to enter clinical trials as a microbicidal candidate was nonoxynol-9 (N-9), a nonionic surfactant originally shown to inactivate enveloped viruses, such as HIV-1, in 1985 (17). N-9 was already available in over-the-counter spermicidal and lubricant formulations and therefore was fairly easy to obtain and test in clinical trails. However, despite its in vitro antiviral activity, N-9 has been shown to be damaging to tissues both in vivo and in vitro (5, 9), and results from clinical, safety, and efficacy trials undertaken to date illustrate the need for efficacious, nontoxic agents (1, 34, 38).

The clinical experience obtained from the N-9 studies highlights the need for better understanding of the safety and efficacy of microbicidal candidates within a vaginal microenvironment before they enter clinical trials. This is especially true today as a number of potential candidate microbicides are moving down the development pathway or are already in clinical trials. Many of these candidates fall into the broad class of agents known as negatively charged polymers, or polyanions. Within this class, most of the reports to date have centered on sulfated or sulfonated polymers that, presumably, work through similar mechanisms of action. Included in this list are polystyrene 4-sulfonate, cellulose sulfate, polymethylene hydroquinone sulfonate, carrageenin, and PRO 2000, a naphthalene sulfonic acid polymer (7, 9, 10, 23, 40, 43). Carboxylic acid-containing polymers such as cellulose acetate phthalate (CAP) or the sulfuric acid modified mandelic acid (SAMMA) polymer have also joined the list of anionic polymers under investigation (16, 22, 26-31).

Most of the anionic polymer candidates have been shown, under simple laboratory conditions, to have similar profiles against herpes simplex virus (6, 15) and HIV-1 (31, 40). In a comparative efficacy analysis of several sulfate and sulfonated polymers, Scordi-Bello et al. (40) reported that they could find little to differentiate the candidate molecules. In fact, these investigators report data supporting the widely held belief that this class of polymer has a similar mechanism of action, that is, tight binding affinities to both CXCR4 and CCR5 tropic viral gp120. In addition, when mixed with cervicovaginal lavage fluid, the sulfated and sulfonated polymers maintain their inhibitory activity at concentrations achievable in formulated products (40).

The plethora of candidate polyanions, all of which have similar in vitro antiviral profiles against HIV-1 and herpes simplex virus type 2, suggests that more stringent criteria should be applied to separate out those candidates with the greatest potential for clinical success. For example, the approach taken by Dezzutti et al. (9), in which formulated samples of different classes of microbicidal candidates were tested, was able to discern some subtle differences in the toxicity and efficacy profiles among CAP, PRO 2000, and carrageenin. In the Dezzutti study, CAP performed as well as, or better than, PRO 2000 in head-to-head comparisons under conditions that would make sense from a dosing perspective.

With that said, the proposed use of CAP highlights another technical hurdle for the development of microbicides, the possibility for change in efficacy or toxicity in the low-pH microenvironment found in a healthy vaginal lumen (2). Therefore, the analysis of pH effects on compound efficacy should be built into assay systems when attempting to identify potential candidate compounds. For example, CAP may be extremely vulnerable in this regard since the pK_a of its free carboxylic acid moiety is in the range of 5.3, and this may be the reason why CAP is formulated as a micronized particle (22). To ensure that the electrostatic anionic charge is maintained under the low-vaginal-pH conditions, if a carboxylic acid-containing polymer is proposed, one must be careful in the selection of the acid group used to append to the backbone of choice. For example, the replacement of a phthalate group with a trimellityl moiety results in a polymer that remains substantially dissociated and molecularly dispersed at much lower pH, such as that found in the vaginal microenvironment. Additional attributes of a candidate polymer microbicide that need careful evaluation include the average molecular mass of the polymer and the degree of anionic moiety substitution.

In the present study, we provide data demonstrating (i) that a trimellitate-containing cellulose polymer (hydroxypropyl methylcellulose trimellitate [HPMCT]) is at least as effective as CAP at inhibiting HIV-1 in vitro at neutral pH, (ii) that HPMCT remains dissociated in solution and is molecularly dispersed at low pH relative to phthalate-containing polymers, and (iii) that HPMCT retains all of its antiviral activity even after being exposed to a pH as low as 4.0. We also show how the size of the polymer and the degree of trimellitic substitution can be varied to produce polymers with better activity against CCR5 tropic strains of HIV-1. Both an understanding of the behavior of the putative microbicide at low pH and the ability to manipulate the degree of backbone substitution to enhance activity against CCR5 tropic HIV-1 are design features that are critical to the overall success of any anti-HIV microbicide candidate.

	MATERIALS AND METHODS

Top Abstract Introduction Materials and Methods Results Discussion References

 
Cells and viruses. HIV-1 strains IIIB, JRCSF, BaL CMU06, and BR/92/014, and the HeLa CD4 long terminal repeat (LTR) ß-galactosidase (ß-Gal), MAGI-R5, GHOST X4/R5, GHOST R5, MOLT4, and HL2/3 cell lines were obtained from the NIH AIDS Research and Reference Reagent Program (Germantown, MD). ME 180 cells, Lactobacillus jensenii, and Lactobacillus crispatus were obtained from the American Type Culture Collection (Manassas, VA). All cell lines were maintained as recommended by the suppliers. Human blood was obtained commercially from Biological Specialty Corporation (Colmar, PA). The HIV coreceptor inhibitor controls, AMD3100 (CXCR4) and TAK779 (CCR5), were obtained from the NIH AIDS Research and Reference Program (Germantown, MD). Zidovudine (AZT) was purchased from Sigma (St. Louis, MO).

Test materials. HPMCT is produced from HPMC (hydroxypropyl methylcellulose), as described previously by Kokubo et al. (18). For the synthesis of HPMCT, 700 g of HMPC is dissolved in 2,100 g of acetic acid (reagent grade) in a 5-liter kneader at 70°C. Then, an appropriate amount of trimellitic anhydride (Wako Pure Chemical Industries) to obtain the desired degree of substitution and 275 g of sodium acetate (reagent grade) to act as a catalyst are added, and the reaction is allowed to proceed at 85 to 90°C for 5 h. After the reactions, 1,200 g of purified water are poured into the reaction mixture, and the resultant mixture is poured into an excess amount of purified water to precipitate the polymer. The crude polymer is washed well with water and then dried. For the described studies, the average molecular mass of the HPMCT polymers used was 50 kDa except for HPMCT-37, which was approximately 30 kDa.

CAP, cellulose acetate trimellitate (CAT), and HPMC phthalate (HPMCP) were purchased from Sigma-Aldrich (St. Louis, MO). The carboxylic acid substitution patterns for these three polymers are between 32 and 35% of molecular mass, and their average molecular mass distributions are in the range of 50 kDa. Dextran sulfate (DS) was also purchased from Sigma-Aldrich and had an average molecular mass of 500 kDa. All polyanions used in these studies were suspended in 50 mM sodium citrate buffer (pH 7.0) at concentrations ranging from 2 to 5% and were stored at 4°C. If polymer compounds stored in this fashion were not assayed within 3 weeks, freshly prepared material was provided.

CD4-dependent HIV transmission inhibition assay. The CD4-dependent HIV transmission inhibition assays use the CD4-positive GHOST (3) cell line expressing either CCR5 or both CXCR4 and CCR5 (24). Twenty-four hours prior to the assay, cells are trypsinized, washed, and seeded in 96-well flat-bottomed microtiter plates. On the day of the assay, effector cells (H9 cells chronically infected with the SK1 clinical isolate of HIV-1, H9/HIV-1_SK1) are treated with freshly made mitomycin C (200 µg/ml) for 60 min at 37°C. This concentration of mitomycin C is insufficient to result in cell death and allows virus transmission to occur. After mitomycin C treatment, the effector cells are washed repeatedly with tissue culture medium. Test compounds and then effector cells are added to the monolayer. The cells are cocultured with effector cells and test material for 4 h, and the effector cells are removed by washing the monolayer repeatedly with RPMI medium. At 20 h after assay initiation, the wells are again washed to ensure removal of the effector cells, and virus replication is assessed via measurement of cell-associated HIV-1 gag p24 using a p24 enzyme-linked immunosorbent assay (ELISA; Beckman-Coulter). Compound toxicity and cell viability are assessed by MTS [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)- 2H-tetrazolium] dye reduction.

Compounds evaluated in the pH transition assay were set up in essentially the same way as described above, with the exception that compounds were prepared in medium adjusted to a pH of 3.45 to 6.5 before addition to buffered target cells. Addition of effector cells prepared in a buffered medium results in a transition of the pH to near neutrality. In this fashion the cells are exposed to low-pH conditions for the short time it takes for the pH to adjust. All determinations were performed in triplicate with serial log₁₀ dilutions of the test materials.

CD4-independent HIV transmission inhibition assay. ME 180 target cells were adapted to increasingly acidic conditions over three to four successive passages. The resultant cell line was able to survive a pH of 4.0 during an overnight incubation with no apparent distress (data not shown). In the described assays, the conditions were not as severe as the selection process, in that 100 µl of drug containing medium with a pH of 4.0 was placed over 50 µl of buffered medium (Dulbecco's minimal essential medium), followed quickly by the challenge cells also in buffered medium. The challenge cells (H9/HIV-1_SK1) helped to buffer the entire mixture back into the neutral range. Target cells were washed after 4 h and again at 24 and 48 h postinfection, and the culture was maintained for 6 days, at which time the culture supernatants were collected and assayed by ELISA for the presence of HIV-1 gag p24 antigen.

Viral p24 antigen ELISA. ELISA kits were purchased from Coulter, and detection of supernatant or cell-associated p24 antigen was performed according to the manufacturer's instructions or as previously described (8, 14). Data were obtained by spectrophotometric analysis at 450 nm using a Molecular Devices Vmax or SpectraMaxPlus plate reader. Final concentrations were calculated from linear regression analysis of the optical density values and expressed in picograms of p24 antigen per milliliter.

Viral reverse transcriptase activity assay. A microtiter plate-based reverse transcriptase (RT) reaction was carried out as previously described by Buckheit and Swanstrom (4), Gu et al. (14), and Ojwang et al. (32). Tritiated dTTP (³H-TTP; 80 Ci/mmol) was purchased from Perkin Elmer (Boston, MA) and was received in 1:1 dH₂O:ethanol at 1 mCi/ml. The RT reaction buffer was prepared fresh on the day of use. The enzyme reaction mixture was incubated at 37°C for 60 min. Following incubation, the entire reaction volume was spotted on DE81 filter mats (Wallac), washed extensively, and then dried. Incorporated radioactivity was quantified using standard liquid scintillation techniques.

HIV-1 infection and replication in PBMCs. Fresh human peripheral blood mononuclear cells (PBMCs), seronegative for HIV and hepatitis B virus, were isolated from screened donors using lymphocyte separation medium (density, 1.078 ± 0.002 g/ml; Cellgro; Mediatech, Inc.) as previously described (14, 32, 37, 42).

Phytohemagglutinin-P stimulated cells from at least two normal donors were pooled, diluted in fresh medium, and plated in a 96-well round-bottomed microtiter plate. Test compound dilutions were prepared and added to the cells, and then a predetermined dilution of virus stock was placed in each test well at a final multiplicity of infection of approximately 0.1. The PBMC cultures were maintained for 7 days following infection (37°C, 5% CO₂), after which time cell-free supernatant samples were obtained and tested for HIV-1 RT activity.

HIV-1 infection inhibition assay. Compound activities at neutral pH were evaluated in assays that measure inhibition of HIV-1 infection of susceptible indicator cells (plated at 8 x 10⁴ cells per well in 12-well culture plates). A known titer of HIV-1_IIIB or HIV-1_BaL was added to MAGI-R5 or HeLa CD4 LTR ß-Gal cells (see Tables 3 and 4), and the incubation was continued for 2 to 4 h as previously described (32). Alternatively, HIV-1_IIIB was added to P4-CCR5 indicator cells and incubated for 2 h (Table 1) . At the end of the incubation, the wells were washed, and the culture was continued for 40 to 48 h. At the termination of the assay, ß-Gal enzyme expression was determined by chemiluminescence using the Galacto-Star ß-galactosidase reporter gene assay system for mammalian cells (Applied Biosystems, Bedford, MA). Compound toxicity was monitored on a duplicate plate using the MTS assay (see Tables 3 and 4) or in an independent MTT [3-(4,5-dimethyl-2-thiazolyl)-2, 5-diphenyltetrazolium bromide] assay (Table 1). Dextran sulfate (Dextralip 50; Sigma-Aldrich, St. Louis, MO) served as a positive control for antiviral activity.

Solubility and dissociation measurements. UV absorbance spectra were obtained for both CAP and HPMCT, and standard concentration-versus-absorbance curves were generated. The wavelengths used to monitor CAP and HPMCT in solution were 282 and 288 nm, respectively. To stay in the linear absorbance range, the starting concentrations used in the solubility study were 0.052% for CAP and 0.038% for HPMCT.

In this experiment diluted polymers were suspended in 1 mM sodium citrate buffer pH 7.0, and 0.5 N HCl was slowly added to each solution. The resulting pH was monitored at various points, resulting in a pH titration curve. Polymer samples were centrifuged briefly (2,000 rpm for 5 minutes in a Sorvall RT6000B centrifuge) at each measurement point, and the concentration of polymer remaining in the supernatant was assessed by absorbance spectroscopy.

The degree of dissociation of CAP was determined using acid-base titration curves performed as previously described by Kokubo et al. (18). The initial pH of the CAP suspension was in the range of 2.5 to 3.0, and the titration was completed at pH 6.5. The percent dissociation at any pH was computed from the ratio of milliequivalents of NaOH consumed to that used to achieve full neutralization of the polymer carboxylic groups.

Since CAP precipitates as the pH is decreased, the remaining dissociated carboxylic groups present in the form of soluble polymer can be estimated by the amount of CAP that precipitates under the different pH conditions. In order to calculate the remaining fraction of dissociated soluble CAP available, we multiplied the degree of dissociation obtained from the titration by the fraction of polymer remaining in solution at the different pH points.

Similarly, the degree of dissociation of HPMCT is based on the titration data provided previously by Kokubo et al. (18). To compute the fraction of dissociated soluble polymer at different pH conditions, we followed the calculation methods described above.

CellTiter96 staining for cell viability and compound cytotoxicity. Cell viability data were derived using a commercially available, soluble, tetrazolium-based MTS reagent (CellTiter96; Promega). At the termination of each assay, MTS reagent was added to cells and incubated for 2 to 4 h at 37°C in 5% CO₂. Adhesive plate sealers were applied, the sealed plate was inverted several times to mix the soluble formazan product, and the optical density was determined for each well at 490/650 nm with a Molecular Devices Vmax or SpectraMaxPlus plate reader.

Lactobacillus assay. Lactobacillus crispatus and Lactobacillus jensenii were grown in Lactobacilli MRS broth (Difco/Fisher Scientific, Pittsburgh, PA). To assess the effect of compounds on growth of L. crispatus and L. jensenii, 10 ml of MRS media was inoculated with a stab from the glycerol bacterial stock. and the culture was incubated for 24 h at 37°C, at which time the bacterial density was adjusted to an optical density of 0.06 at a wavelength of 670 nm. Compounds were diluted and dispensed into 96-well round-bottomed plates, and the diluted Lactobacillus spp. were added. Commercially available penicillin/streptomycin solution (1.25 U/ml and 1.25 µg/ml, respectively), was used as the positive control. The plates were further incubated for 24 h at 37°C in a Gas Pak CO₂ bag, and bacterial growth was determined by measurement of the optical density at 490 nm using a 96-well Molecular Devices Vmax plate reader. All determinations were performed with six 0.5-log dilutions from a high test concentration in triplicate.

Statistical analysis. The calculations for mean, standard deviation (SD), and the Student's t test for statistical significance were made using the functions provided within Microsoft Excel spreadsheets.

	RESULTS

Top Abstract Introduction Materials and Methods Results Discussion References

 
Antiviral activity of phenyl carboxylate-containing cellulose polymers. Phthalic and trimellitic acid can be covalently linked to a cellulose backbone by reacting the latter with the corresponding anhydride intermediate. The resulting polymer then takes on some of the physical characteristics of the remaining free carboxylic acid groups. In the case of phthalic acid, the pK_a of the free carboxylic acid group is approximately 5.3 (3, 20). This free carboxylic acid then becomes the driving force in the electrostatic interactions of the substituted cellulose polymer (Fig. 1).

View larger version (9K): [in this window] [in a new window]   FIG. 1. Physical representation of phthalic and trimellitic acids. The pKa values shown were obtained from Brown et al. (3) for HPMCT or the CRC Handbook (20) for CAP.

As with most polyanions, under neutral conditions in tissue culture experiments, both phthalate- and trimellitate-containing cellulose-based polymers are effective inhibitors of enveloped viruses such as HIV-1. To demonstrate this point, we used CAP, CAT, HPMCP and HPMCT to treat reporter cells in culture for 2 h in the presence of HIV-1_IIIB (HIV-1 infection inhibition assay). In this experiment, all test polymers were approximately 35% (wt) phthallic or trimellitic acid. The results from this experiment show that, as expected, all four polyanions were effective inhibitors of HIV-1_IIIB (Table 1). The well-studied polyanion DS, which is a prototypical blocker of HIV-1 binding and entry (11, 12, 29, 36, 39), was used as a positive control in these experiments.

The same reporter cells were assayed for cell viability to assess the differential between effective antiviral activity and cellular toxicity. In this experiment, cells were initially treated for 2 h to parallel the exposure time in the efficacy experiment. Because there was very limited toxicity at the highest concentration of polymers tested, we increased the exposure time to 24 h (Table 1). After a 24-h treatment, a slight increase in toxicity was observed, with all polymers tested yielding similar results. The in vitro therapeutic indices (TIs) (50% inhibitory concentration [IC₅₀]/50% cytotoxicity concentration [CC₅₀]) show that all four polymers are effective inhibitors of HIV-1_IIIB (Table 1) with 50% inhibition doses 3 orders of magnitude, or more, below the CC₅₀. It should be noted that for each polymer tested the actual CC₅₀ determined after a 2-hour exposure was between 1 and 2% (data not shown). Therefore, the estimated TIs calculated for this data set were within a factor of 2 of their actual value.

CAP and HPMCT solubility and dissociation measurements. As shown in Table 1, under neutral conditions all polymers tested were effective inhibitors of the CXCR4 tropic strain of HIV-1 used; however, the biochemical information presented in Fig. 1 predicts that trimellitate-containing polymers will remain dissociated and molecularly dispersed in a microenvironment that would likely be encountered in a healthy vaginal lumen (e.g., pH ranges of 3.8 to 8 or higher [2]). To support this prediction, we performed experiments designed to monitor the solubility and dissociation of the phthalate- and trimellitate-containing polymers.

To measure solubility, we slowly added HCl to buffered polymer solutions of HPMCT or CAP. At each titration point the samples were centrifuged briefly, and the polymer remaining in the supernatant was monitored. The results of this experiment showed that as the pH is lowered, CAP falls out of solution at a faster rate than HPMCT (data not shown). In a similar fashion, we monitored the amount of carboxylic acid remaining dissociated as a function of pH. The results were similar to those observed in the solubility experiment in that the residual remaining dissociated carboxylic acid as a function of pH clearly fell in line with the pK_as of the different carboxylic acid groups (data not shown).

Because the degree of available polymer and its functional carboxylic acid groups is predicated on both the amount of polymer in solution and the degree of carboxylic acid group dissociation, we combined the two studies to graphically represent the differences between phthalic- and trimellitic-acid-containing polymers (Fig. 2).

View larger version (22K): [in this window] [in a new window]   FIG. 2. pH effects solubility and dissociation of phthalic and trimellitic acid-containing cellulose polymers. HCl was slowly added to buffered polymer solutions of HPMCT or CAP. At each titration point the samples were centrifuged briefly, and the polymer remaining in the supernatant was monitored as was the amount of carboxylic acid remaining dissociated. The data sets were combined to visualize the effect of pH on these two parameters.

The adjustment from the conditions of a standard assay (pH 7.4) to conditions in which a preincubation at a lower pH was used (pH 4.0 and 5.85) significantly enhanced the activity of HPMCT (Table 2). At the same time, however, the antiviral activity of CAP was significantly diminished by all three low-pH treatments (Table 2). In this set of experiments, the CXCR4 chemokine receptor antagonist AMD3100 was used as a positive control (39). It is interesting to note that the activity of AMD3100 also increased upon preincubation at low pH (Table 2), though the trend was not significant. In addition, the increase in antiviral activity of HPMCT did not correspond to a change of a similar magnitude in the cytotoxicity of this compound; therefore, the overall effect of the preincubation was a net increase in therapeutic indices (Table 2).

In this assay CAP, HPMCT, and DS were preincubated at pH 3.5, 4.0, or 5.2. The results showed that the IC₅₀s calculated for all three polyanions were negatively affected when the pH of the preincubation buffer was below 4.0 (Table 2). The results for CAP and HPMCT were significantly lower following a preincubation at pH 3.45. However, at pH 4.0 or 5.2, there was no detectable change in the activity of HPMCT compared to the standard assay conditions. This is readily apparent when comparing the calculated IC₅₀s or by observation of the dose-response values obtained (Table 2; Fig. 3A). Under these same conditions, while there was an observable decrease in the antiviral activity of CAP, and surprisingly and to a lesser extent in that of DS, the changes were not significant. Uninfected target cells did not appear to be affected by the addition of test compound under these same conditions (Fig. 3B).

View larger version (35K): [in this window] [in a new window]   FIG. 3. pH effect on cell viability and antiviral efficacy of HPMCT. HPMCT-35 was serially diluted and then placed in low-pH conditions for a brief time before being rapidly neutralized by addition to well-buffered target cells (ME 180). The target cells were then infected by adding H9/HIV-1SK1 cells to the medium. The effect of HPMCT (A) on HIV-1 in this system was determined by monitoring intracellular p24 production 6 days postinfection. To assess the effect of this protocol on cell viability, ME 180 cells were treated as described above but not infected. Cell viability (B) was monitored 6 days after removal of the test compound.

In this assay, all test compounds, including the polyanion control, DS, were less active against HIV-1_BaL than against HIV-1_IIIB (Table 3). However, the ability of HPMCT to inhibit either CXCR4 or CCR5 tropic virus in this assay system increased with increasing trimellityl content. It is interesting that HPMCT-37 (37% trimellityl substitution), which has an average molecular mass of 30 kDa (compare to the average molecular mass of 50 kDa for the other HMCT polymers tested), had mixed results. In some experiments HPMCT-37 seemed to follow the trend equating better activity with higher trimellityl content (Table 3), but this was not always the case (Table 4).

HPMCT variants were further tested for their ability to retard cell-associated HIV-1 transmission. In these experiments, CD4-positive GHOST cells were used as target cells and were incubated with cell-associated virus as described in Materials and Methods. The results show that as in the virus inhibition assay, all of the polymers tested were less effective against the CCR5 tropic strain of virus than against the CXCR4 tropic strain (Table 4). In addition, as seen in the virus inhibition assay, the most heavily substituted variant of HPMCT tested (HPMCT-49) was the polymer observed to be most efficacious against both CXCR4 and CCR5 tropic strains of virus.

Effect of HPMCT on HIV-1 infection and replication PBMCs. The ability of HPMCT to interfere with HIV infection or replication was next assessed using PBMCs infected with either a CXCR4 tropic strain (CMU06), a CCR5 tropic strain (JRCSF), or a dual tropic strain (BR/92/014) of HIV-1. In these experiments, the virus was added to cells in the presence of test compound (HPMCT-35 or AZT) for 7 days, and the results show how, over this extended period of exposure, the efficacy of HPMCT was the same or better relative to that observed in the shorter-duration exposures (Table 5). The toxicity of the compounds also increases with the increased exposure time, but the resulting therapeutic indices obtained in all cases were >5,000.

The results from this experiment are presented in Table 6 and clearly show that HPMCT, like CAP, is capable of interfering with binding or fusion events. In addition, as seen in the viral inhibition studies, the degree of trimellityl substitution directly correlates to the degree of fusion inhibition.

	DISCUSSION

Top Abstract Introduction Materials and Methods Results Discussion References

 
CAP and HPMCP were first developed for use as excipients in enteric coating to help protect pharmaceutical preparations from degradation by the low pH of gastric juices and to simultaneously protect the gastric mucosa from irritation by the drug. One desirable attribute of these coatings was the ability to not dissolve until the drug substance reached the intestines, where the pH is mostly neutral or alkaline. Indeed, most commercially available enteric coating agents of both cellulose and acrylic polymers are designed to be soluble in the pH range from 5.0 to 7.0 (18, 19). This lack of solubility at low pH (3.5 to 5.5) was a desired goal in the selection of HPMCP and CAP for use as enteric coatings.

In 1997, Kokubo and colleagues at Shin-Etsu Chemical Company demonstrated that by careful selection of the carboxylic acid-containing moiety used, the overall pK_a of the polymer could be tailored to fit specific needs (18). The Kokubo study specifically outlined the solubility and dissolution characteristics of HPMCT, which was designed to begin to dissolve in the stomach. HPMCT remains dissociated and molecularly dispersed at low pH. It is also similar in structure to HPMCP and CAP. Therefore, we hypothesized that HPMCT would be an effective anti-HIV-1 agent and retain its activity even in vaginal microenvironments in which the overall pH is below 5.0.

Our data illustrate, in a variety of assay formats, that HPMCT is quite effective at inhibiting HIV-1 and that the extent of inhibition can be modulated by the degree of trimellityl substitution on the cellulose backbone. What differentiates HPMCT from similar cellulose-based polymers (CAP and HPMCP) is its ability to remain dissociated in solution and molecularly dispersed even after exposure to a low-pH environment. For example, while all four cellulose-based polymers tested were effective inhibitors of HIV-1_IIB under neutral conditions (Table 1), the exposure of CAP to a low-pH environment for even a brief period of time dramatically lowered its antiviral effectiveness (Table 2). The effect of low pH on phthalate-containing polymers was further visualized by monitoring the solubility and dissociation of CAP under a wide range of pH conditions (Fig. 2). By monitoring the combined effect of both solubility and dissociation on CAP, we determined that less than 10% of the original polymer is available when the pH drops to 4.0. The fact that more CAP is not available once the pH has been rapidly neutralized under these assay conditions is simply due to the long dissolution time of this type of polymer once it has fallen out of solution (18).

A number of reports have suggested that sulfated polymers, such as dextran sulfate, bind the V3 loops of CXCR4 viruses more readily than they bind the V3 loops of CCR5 viruses (25) and hence have reduced activity against CCR5 tropic virus (21). However, Scordi-Bello et al. (40) report very little difference in actual binding affinity of DS to different strains of HIV-1 gp120. In fact, in their analysis, Scordi-Bello et al. (40) show that there were no discernible differences between binding to CXCR4 or CCR5 gp120 by polystyrene sulfate, cellulose sulfate, polymethylene hydroquinone sulfonate, and PRO 2000. Additionally, all these polyanionic compounds were potent inhibitors of CCR5 virus in tissue culture assays. This is an important observation since newly infected individuals harbor predominantly M-tropic virus which utilizes the CCR5 coreceptor and readily infects cervical tissues (13).

Experiments performed with HPMCT have been conducted in three independent laboratories, and while there is some variation in results depending upon the laboratory, for the most part there is a great deal of consistency given the number of assay systems used and internal controls employed. With that said, some of our observations differ from other reports. For example, in the current studies, we show that there was a measurable differential between activity against HIV-1_IIIB (CXCR4) and HIV-1_BaL (CCR5) when compounds were tested using a virus infection assay (Table 3). In these experiments, DS was clearly able to inhibit HIV-1_BaL—albeit at a reduced level as was the case for all compounds tested against HIV-1_BaL—which is contrary to the observations of others (21). Additional differences in compound efficacy were observed when the compounds were tested in a cell-associated transmission assay (Table 4). It should be noted that the degree of change in either experiment for trimellityl-containing polymers roughly correlated with the degree of carboxylic acid substitution. It is apparent from the comparison of the different HPMCT lots that minor variations in the degree of trimellityl substitution had a dramatic impact on the antiviral efficacy of the polymer, especially with respect to its activity against CCR5 virus (Tables 3, 4, and 6). In all cases, the degree of enhanced activity observed as a result of increased trimellitate substitution was greater than any negative change in cytotoxicity, resulting, in general, in higher TIs.

In addition to differences in the degree of anionic substitution, the overall average molecular mass of the polymer can also play a role in its antiviral efficacy, as noted for HPMCT-37 (average molecular mass, 30 kDa), which was synthesized to this specification with the intent to compare it with the standard 50-kDa average molecular mass of the other HPMCT polymers used in this study. As with the degree of acid substitution, differences in the average molecular mass of the polymer under investigation (which can arise from lot-to-lot variations) can account for the variations in activity observed for those compounds in different studies.

In the current study, we defined three criteria that can help in the overall design of polymer microbicides. The first of these criteria is to design the polymer so that the overall pK_a of the free acid group(s) is low enough to allow the molecule to remain dissociated and molecularly dispersed within the pH range found in the vaginal lumen. The second criterion is to account for the potential effects of variations in the average molecular mass of the product polymer. The third consideration is to monitor the degree of acid substitution, since this can have dramatic effects on the efficacy of the resultant polymer both in different assay systems and against differing strains of virus. With these criteria in mind, we have used HPMCT as a prototype carboxylic acid-containing microbicide that remains in solution at low pH, can be synthesized with variations in its average molecular mass to produce an optimized size distribution, and can have its acid substitution pattern varied to produce a polymer that is effective against CCR5 tropic virus in a cell-associated virus infection assay.

HPMCT is a derivative of HPMC, which is widely used as a thickener in the pharmaceutical and cosmetic industries. HPMCT itself is also viscous in solution. Therefore, the polymer can act as a thickening agent for gel formulations. Since HPMCT dissolves in a wide range of pHs it would be easy to prepare a topical gel formulation in which the polymer is well dispersed. All of these factors, taken together, strongly suggest that HPMCT is a solid candidate for further development as a topical microbicide.

	ACKNOWLEDGMENTS

 
We thank A. Labib (Novaflux Biosciences, Inc.) for help in editing the manuscript and B. Snyder and K. Luckenbaugh (Southern Research Institute) and L. Schlipf and V. Pirrone (Drexel University College of Medicine) for expert technical assistance.

We also thank Shin-Etsu Chemical Company, Ltd., for their generous contribution in support of this research.

	FOOTNOTES

 
* Corresponding author. Mailing address: Novaflux Biosciences, Inc., 1 Wall Street, Princeton, New Jersey 08540. Phone: (609) 683-0215. Fax: (609) 683-5003. E-mail: robertrando{at}patmedia.net.

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