Application and removal of polyanionic microbicide compounds enhances subsequent infection by HIV-1.

BACKGROUND: Continued efforts are being directed toward the development of microbicides that will be used to reduce or eliminate the risk of HIV-1 sexual transmission. Unfortunately, clinical trials involving polyanion-containing microbicide formulations, including Carraguard (λ-carrageenan [LC]) and Ushercell (cellulose sulfate [CS]) demonstrated that these products were ineffective and may have, in some circumstances, increased the risk of HIV-1 infection. These findings prompted reassessments of the in vitro activities of these agents to determine whether variables that can affect agent safety and efficacy had been overlooked during preclinical testing. One such variable is product retention and loss following topical application. RESULTS: In the present studies involving an HIV-1-susceptible cell line and primary human immune cells, product loss was mimicked by introducing and then removing polyanionic compounds prior to HIV-1 infection. In these in vitro "washout" experiments, LC and CS significantly enhanced HIV-1 infection, despite potent antiviral activity when introduced simultaneously with the virus. The presence and magnitude of this effect were dependent on compound identity and concentration; target cell; interval between compound removal and virus challenge; and coreceptor usage. Levels of enhancement (relative to controls) were considerable, exceeding a 200% increase (CS) in P4-R5 MAGI cells and a 300% increase (LC) in human peripheral blood mononuclear cells. CONCLUSIONS: These studies, which demonstrate significant increases in HIV-1 infection subsequent to application and removal of LC and CS, support plausible explanations for the failures of microbicides formulated from these compounds. Detailed studies are now underway to determine the mechanism responsible for this enhancement effect and to assess the potential contribution of this effect to the clinical failures of these agents.

A styrene-alt-maleic acid copolymer is an effective inhibitor of R5 and X4 human immunodeficiency virus type 1 infection.

An alternating copolymer of styrene and maleic acid (alt-PSMA) differs from other polyanionic antiviral agents in that the negative charges of alt-PSMA are provided by carboxylic acid groups instead of sulfate or sulfonate moieties. We hypothesized that alt-PSMA would have activity against human immunodeficiency virus type 1 (HIV-1) comparable to other polyanions, such as the related compound, poly(sodium 4-styrene sulfonate) (PSS). In assays using cell lines and primary immune cells, alt-PSMA was characterized by low cytotoxicity and effective inhibition of infection by HIV-1 BaL and IIIB as well as clinical isolates of subtypes A, B, and C. In mechanism of action assays, in which each compound was added to cells and subsequently removed prior to HIV-1 infection ("washout" assay), alt-PSMA caused no enhancement of infection, while PSS washout increased infection 70% above control levels. These studies demonstrate that alt-PSMA is an effective HIV-1 inhibitor with properties that warrant further investigation.

Polybiguanides, particularly polyethylene hexamethylene biguanide, have activity against human immunodeficiency virus type 1.

Polyhexamethylene biguanide (PHMB) is a polybiguanide (PBG) oligomer with antimicrobial activity that is used extensively and safely as a disinfectant. The reported mechanism of PHMB antimicrobial activity, which involves interactions with cell membrane components, suggested that PHMB or other PBG-based compounds might also have antiviral or virucidal activity against the human immunodeficiency virus type 1 (HIV-1). PHMB had modest in vitro activity against both cell-free and cell-associated HIV-1, as well as the ability to interfere with viral binding and entry. However, PHMB was comparable in cytotoxicity to the spermicidal agent nonoxynol-9 (N-9), a compound that has been characterized in previous studies as generally cytotoxic and detrimental to cervicovaginal epithelial integrity. To identify structural variants of PHMB with greater anti-HIV-1 activity and/or less cytotoxicity, modified versions of PHMB incorporating length changes in the hydrocarbon linker units were synthesized and evaluated for in vitro cytotoxicity and inhibition of HIV-1 infectivity. These experiments demonstrated that the PHMB variant polyethylene hexamethylene biguanide (PEHMB) was just as active against HIV-1 as PHMB, yet was much less cytotoxic than either N-9 or PHMB, resulting in an in vitro therapeutic index (TI) approximately 114-fold greater than the TI of N-9. PEHMB, which has been identified in these studies as a promising microbicidal candidate in this family of compounds, will be the focus of further in vitro and in vivo evaluations of anti-HIV-1 activity, toxicity, and mechanisms of action.

Prolonged exposure to the candidate microbicide C31G differentially reduces cellular sensitivity to agent re-exposure.

Comparative assays of in vitro cytotoxicity using nonoxynol-9 (N-9) and the candidate microbicides C31G and sodium dodecyl sulfate (SDS) demonstrated that these agents, which are, respectively, characterized as nonionic, amphoteric, and anionic surfactants, differed in their concentration-dependent effects on cell viability, especially after prolonged exposure. We hypothesized that differences in cellular sensitivity may have been due, in part, to cellular changes induced by long-term exposure to each agent. To examine this possibility, HeLa cells were exposed to N-9, C31G, or SDS for extended periods of time and subsequently reassessed for sensitivity to each of these agents. Following 10 continuous days of C31G exposure, HeLa cells were less sensitive to a subsequent C31G exposure compared to cells that had not undergone long-term C31G treatment. Interestingly, long-term C31G exposure also changed subsequent sensitivity to N-9 but not SDS. In contrast, prolonged exposure to either N-9 or SDS did not reduce sensitivity to re-exposure. The effect of long-term C31G exposure was both concentration-dependent and transient, as treated cells reverted to pre-exposure sensitivity in a time-dependent manner following the cessation of C31G exposure. Lipid analyses of cells exposed to C31G for extended durations revealed altered phospholipid profiles relative to C31G-naïve cells. Experiments examining the individual components of C31G demonstrated the involvement of the amine oxide moiety in reductions in cellular sensitivity. These studies, which provide new information concerning the cytotoxicity of surfactant microbicides, suggest that cervicovaginal epithelial cells may have greater in vivo tolerance for products containing C31G through unique interactions between C31G and components of the cellular membranes.

Decreased cervical epithelial sensitivity to nonoxynol-9 (N-9) after four daily applications in a murine model of topical vaginal microbicide safety.

BACKGROUND: The disappointing clinical failures of five topical vaginal microbicides have provided new insights into factors that impact microbicide safety and efficacy. Specifically, the greater risk for human immunodeficiency virus type 1 (HIV-1) acquisition associated with multiple uses of a nonoxynol-9 (N-9)-containing product has highlighted the importance of application frequency as a variable during pre-clinical microbicide development, particularly in animal model studies. METHODS: To evaluate an association between application frequency and N-9 toxicity, experiments were performed using a mouse model of cervicovaginal microbicide safety. In this model system, changes in cervical and vaginal epithelial integrity, cytokine release, and immune cell infiltration were assessed after single and multiple exposures to N-9. RESULTS: After the initial application of N-9 (aqueous, 1%), considerable damage to the cervical epithelium (but not the vaginal epithelium) was observed as early as 10 min post-exposure and up to 8 h post-exposure. Subsequent daily exposures (up to 4 days) were characterized by diminished cervical toxicity relative to single exposures of like duration. Levels of pro-inflammatory cytokines released into the cervicovaginal lumen and the degree of CD14-positive immune cell infiltration proximal to the cervical epithelium were also dependent on the number of N-9 exposures. CONCLUSIONS: Rather than causing cumulative cervical epithelial damage, repeated applications of N-9 were characterized by decreased sensitivity to N-9-associated toxicity and lower levels of immune cell recruitment. These results provide new insights into the failure of N-9-based microbicides and illustrate the importance of considering multiple exposure protocols in pre-clinical microbicide development strategies.

The rise and fall of polyanionic inhibitors of the human immunodeficiency virus type 1.

Infection by the human immunodeficiency virus type 1 (HIV-1) is an ordered, multistep process involving binding and entry, reverse transcription, integration, viral gene transcription, translation, processing, and finally assembly. Numerous therapeutic and preventive compounds, which are currently available for clinical use or are under preclinical and clinical development, act on at least one of these steps. Polyanionic HIV-1 inhibitors comprise a family of compounds that are generally considered entry inhibitors. The main mechanism of anti-HIV-1 activity associated with these compounds involves electrostatic interactions with HIV-1 glycoprotein 120 that ultimately prevent binding of the virus to target cells. A number of these compounds have been considered for systemic use and for use as microbicides, which are products designed to prevent sexual HIV-1 transmission. These compounds have been studied extensively using in vitro assays of activity, cytotoxicity, and mechanism of action, ex vivo models of HIV-1 transmission, and animal models of in vivo efficacy and toxicity. Three of these polyanionic compounds - cellulose sulfate, carrageenan, and PRO 2000 - were advanced into clinical trials of microbicide safety and efficacy. Although phase I and phase II clinical trials showed these compounds to be safe and well tolerated, none of the phase III trials provided any evidence that these compounds were effective against heterosexual HIV-1 transmission. Furthermore, clinical and in vitro results suggest enhancement of HIV-1 infection in the presence of polyanionic compounds. We discuss the preclinical development of polyanionic HIV-1 inhibitors, the clinical trials of polyanionic compounds used systemically and as topical vaginal microbicides, and the prospects for the future development of these compounds as inhibitors of HIV-1 infection.

Structure-activity relationships of polybiguanides with activity against human immunodeficiency virus type 1.

Previous investigations showing that polydisperse biguanide (PDBG) molecules have activity against human immunodeficiency virus type 1 (HIV-1) also suggested a relationship between PDBG biologic activity and the lengths of hydrocarbon linkers surrounding the positively charged biguanide unit. To better define structure-activity relationships, PDBG molecules with select linker lengths were evaluated for cytotoxicity, anti-HIV-1 activity, and in vivo toxicity. Results of the in vitro experiments demonstrated that increases in linker length (and, therefore, increases in compound lipophilicity) were generally associated with increases in cytotoxicity and antiviral activity against HIV-1. However, a relationship between linker length asymmetry and in vitro therapeutic index (TI) suggested structural specificity in the mechanism of action against HIV-1. Polyethylene hexamethylene biguanide (PEHMB; biguanide units spaced between alternating ethylene and hexamethylene linkers) was found to have the highest in vitro TI (CC50/IC50) among the compounds examined. Recent improvements in PEHMB synthesis and purification have yielded preparations of PEHMB with in vitro TI values of 266 and 7000 against HIV-1 strains BaL and IIIB, respectively. The minimal toxicity of PEHMB relative to polyhexamethylene biguanide (PHMB; biguanide units alternating with hexamethylene linkers) in a murine model of cervicovaginal microbicide toxicity was consistent with considerable differences in cytotoxicity between PEHMB and PHMB observed during in vitro experiments. These structure-activity investigations increase our understanding of PDBG molecules as agents with activity against HIV-1 and provide the foundation for further preclinical studies of PEHMB and other biguanide-based compounds as antiviral and microbicidal agents.

Critical design features of phenyl carboxylate-containing polymer microbicides.

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(a)s 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.

In vitro preclinical testing of nonoxynol-9 as potential anti-human immunodeficiency virus microbicide: a retrospective analysis of results from five laboratories.

The first product to be clinically evaluated as a microbicide contained the nonionic surfactant nonoxynol-9 (nonylphenoxypolyethoxyethanol; N-9). Many laboratories have used N-9 as a control compound for microbicide assays. However, no published comparisons of the results among laboratories or attempts to establish standardized protocols for preclinical testing of microbicides have been performed. In this study, we compared results from 127 N-9 toxicity and 72 efficacy assays that were generated in five different laboratories over the last six years and were performed with 14 different cell lines or tissues. Intra-assay reproducibility was measured at two-, three-, and fivefold differences using standard deviations. Interassay reproducibility was assessed using general linear models, and interaction between variables was studied using step-wise regression. The intra-assay reproducibility within the same N-9 concentration, cell type, assay duration, and laboratory was consistent at the twofold level of standard deviations. For interassay reproducibility, cell line, duration of assay, and N-9 concentration were all significant sources of variability (P < 0.01). Half-maximal toxicity concentrations for N-9 were similar between laboratories for assays of similar exposure durations, but these similarities decreased with lower test concentrations of N-9. Results for both long (>24 h) and short (<2 h) exposures of cells to N-9 showed variability, while assays with 4 to 8 h of N-9 exposure gave results that were not significantly different. This is the first analysis to compare preclinical N-9 toxicity levels that were obtained by different laboratories using various protocols. This comparative work can be used to develop standardized microbicide testing protocols that will help advance potential microbicides to clinical trials.

Comparative safety evaluation of the candidate vaginal microbicide C31G.

C31G is currently the focus of clinical trials designed to evaluate this agent as a microbicidal and spermicidal agent. In the following studies, the in vivo safety of C31G was assessed with a Swiss Webster mouse model of cervicovaginal toxicity and correlated with results from in vitro cytotoxicity experiments and published clinical observations. A single exposure of unformulated 1% C31G resulted in mild-to-moderate epithelial disruption and inflammation at 2 and 4 h postapplication. The columnar epithelium of the cervix was the primary site of damage, while no perturbation of the vaginal mucosa was observed. In contrast, application of unformulated 1.7% C31G resulted in greater levels of inflammation in the cervical epithelium at 2 h postapplication and severe epithelial disruption that persisted to 8 h postapplication. Application of a nonionic aqueous gel formulation containing 1% C31G resulted in no apparent cervicovaginal toxicity at any time point evaluated. However, formulation of 1.7% C31G did not substantially reduce the toxicity associated with unformulated C31G at that concentration. These observations correlate with findings gathered during a recent clinical trial, in which once-daily applications resulted in no adverse events in women receiving the formulation containing 1% C31G, compared to moderate-to-severe adverse events in 30% of women receiving the 1.7% C31G formulation. The Swiss Webster mouse model was able to effectively discriminate between concentrations and formulations of C31G that produced distinct clinical effects in human trials. The Swiss Webster animal model may be a highly valuable tool for preclinical evaluation of candidate vaginal microbicides.

Comparative in vitro sensitivities of human immune cell lines, vaginal and cervical epithelial cell lines, and primary cells to candidate microbicides nonoxynol 9, C31G, and sodium dodecyl sulfate.

In experiments to assess the in vitro impact of the candidate microbicides nonoxynol 9 (N-9), C31G, and sodium dodecyl sulfate (SDS) on human immune and epithelial cell viability, cell lines and primary cell populations of lymphocytic and monocytic origin were generally shown to be equally sensitive to exposures ranging from 10 min to 48 h. However, U-937 cells were more sensitive to N-9 and C31G after 48 h than were primary monocyte-derived macrophages. Cytokine activation of monocytes and lymphocytes had no effect on cell viability following exposure to these microbicidal compounds. Primary and passaged vaginal epithelial cultures and cell lines differed in sensitivity to N-9 and C31G but not SDS. These studies provide a foundation for in vitro experiments in which cell lines of human immune and epithelial origin can be used as suitable surrogates for primary cells to further investigate the effects of microbicides on cell metabolism, membrane composition, and integrity and the effects of cell type, proliferation, and differentiation on microbicide sensitivity.

Mouse model of cervicovaginal toxicity and inflammation for preclinical evaluation of topical vaginal microbicides.

Clinical trials evaluating the efficacy of nonoxynol-9 (N-9) as a topical microbicide concluded that N-9 offers no in vivo protection against human immunodeficiency virus type 1 (HIV-1) infection, despite demonstrated in vitro inactivation of HIV-1 by N-9. These trials emphasize the need for better model systems to determine candidate microbicide effectiveness and safety in a preclinical setting. To that end, time-dependent in vitro cytotoxicity, as well as in vivo toxicity and inflammation, associated with N-9 exposure were characterized with the goal of validating a mouse model of microbicide toxicity. In vitro studies using submerged cell cultures indicated that human cervical epithelial cells were inherently more sensitive to N-9-mediated damage than human vaginal epithelial cells. These results correlated with in vivo findings obtained by using Swiss Webster mice in which intravaginal inoculation of 1% N-9 or Conceptrol gel (containing 4% N-9) resulted in selective and acute disruption of the cervical columnar epithelial cells 2 h postapplication accompanied by intense inflammatory infiltrates within the lamina propria. Although damage to the cervical epithelium was apparent out to 8 h postapplication, these tissues resembled control tissue by 24 h postapplication. In contrast, minimal damage and infiltration were associated with both short- and long-term exposure of the vaginal mucosa to either N-9 or Conceptrol. These analyses were extended to examine the relative toxicity of polyethylene hexamethylene biguanide (PEHMB), a polybiguanide compound under evaluation as a candidate topical microbicide. In similar studies, in vivo exposure to 1% PEHMB caused minimal damage and inflammation of the genital mucosa, a finding consistent with the demonstration that PEHMB was >350-fold less cytotoxic than N-9 in vitro. Collectively, these studies highlight the murine model of toxicity as a valuable tool for the preclinical assessment of toxicity and inflammation associated with exposure to candidate topical microbicides.