Mary Lee Ferguson

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

ABSTRACT 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 pKa of the free carboxylic acid group is very important and needs careful selection. In a polymer like CAP the pKa 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 pKa. Hydroxypropyl methylcellulose trimellitate (HPMCT) is structurally similar to CAP; however, its free carboxylic acids have pKas 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.

Use of the Synthetic Copolymer PSMA as a Component in a Combination Microbicide Active Against HIV-1

The remarkable successes achieved using combination therapy to treat systemic human immunodeficiency virus type 1 (HIV-1) infection suggest that combination microbicides, which include two or more active ingredients, may also provide a particularly effective means to prevent HIV-1 transmission. We have recently identified the compound PSMA, an alternating copolymer of polystyrene (PS) and maleic anhydride (MA), as a potential partner for our candidate microbicide polyethylene hexamethylene biguanide (PEHMB), a member of the polybiguanide family of compounds. In vitro studies of PSMA demonstrated that this compound is minimally cytotoxic and highly effective against both macrophageand T celltropic strains of HIV-1. We hypothesize that the dissimilar mechanisms of action of PSMA and PEHMB may provide additive or synergistic activity against HIV-1. Experiments are now underway to identify optimal combinations of PSMA and PEHMB to be used in experiments to assess toxicity, anti-HIV-1 activity, and formulation strategies. These investigations will be used to confidently advance the preclinical development of PSMA and a combination microbicide containing both compounds toward human trials. from 2005 International Meeting of The Institute of Human Virology Baltimore, USA, 29 August – 2 September 2005

The Presence of Mucin Increases the Anti-HIV-1 Activity of the Candidate Microbicide Polyethylene Hexamethylene Biguanide (PEHMB)

Topical microbicides that reduce or eliminate the risk of human immunodeficiency virus type 1 (HIV-1) sexual transmission must function effectively within the cervicovaginal environment where multiple factors may impact the efficacy of the active agent. Factors relevant to potential changes in microbicide efficacy include the presence of mucins within the cervical mucus. We hypothesize that polycationic PEHMB molecules will interact with the anionic mucin molecules to form a lattice-like network that serves as a physical barrier to the movement of infectious virus and HIV-1-infected cells to the cervical and vaginal epithelia. In vitro experiments demonstrated that the anti-HIV-1 activity of PEHMB was increased almost two logs in the presence of mucin. In contrast, the activity of anionic dextran sulfate was unaffected. These results suggest that electrostatic interactions between PEHMB and mucin molecules may augment the inherent antiHIV-1 activity of PEHMB by facilitating the formation of a physical barrier between HIV-1 and susceptible cells. This property would be expected to increase the in vivo efficacy of PEHMB. from 2005 International Meeting of The Institute of Human Virology Baltimore, USA, 29 August – 2 September 2005

hmm.Coreceptor Perturbation as a Possible Mechanism Underlying the Immediate and Persistent Anti-HIV-1 Activity of the Microbicidal Compound PEHMB

Microbicides, which are products capable of reducing or eliminating the risk of HIV-1 sexual transmission, are urgently needed to combat the global spread of HIV-1. Our efforts in this area are focused on the preclinical development of the polybiguanide, PEHMB (polyethylene hexamethylene biguanide). PEHMB has been shown to have low cytotoxicity both in vitro and in vivo, as well as in vitro activity against both cell-free and cell-associated forms of HIV-1. Flow cytometric analyses have shown that PEHMB exposure results in epitope-specific alterations in the detection of viral co-receptors CXCR4 and CCR5, suggesting that PEHMB acts by interfering with events critical to viral binding and entry. Changes in co-receptor detection have also been observed up to 24 hr after removal of PEHMB. Consistent with the latter result was the demonstration of persistent protection from infection in experiments in which cells were challenged with HIV-1 after removal of PEHMB. Cumulatively, these results suggest that HIV-1 co-receptors may play roles in PEHMB-mediated protection from HIV-1 infection, and that products containing PEHMB may provide both shortand longterm protection against HIV-1 transmission. from 2005 International Meeting of The Institute of Human Virology Baltimore, USA, 29 August – 2 September 2005

CONRAD Testing Algorithm: Microbicidal Compounds Screened For Cytotoxicity and Activity Against HIV-1

Cytotoxicity and virucidal/antiviral testing conducted for the CONRAD Program identifies compounds that may be developed as microbicides used to reduce or eliminate the risk of HIV-1 sexual transmission. Preliminary testing begins with a cytotoxicity screen (CTS) to assess the impact of each compound on cell viability. Activity against infectious HIV-1 was measured using viral binding/entry inhibition (VBI) assays, in which each compound was evaluated for the ability to inhibit binding or entry events between target cells and HIV-1 strains IIIB (X4 phenotype) or BaL (R5 phenotype). Finally, compounds have been screened to determine their ability to interfere with cell-to-cell (CTC) HIV-1 transmission. In all assays, dextran sulfate was used as a control because of its minimal cytotoxicity and potent anti-HIV-1 activity. Out of 477 compounds tested since May 2001, 77 compounds tested using this algorithm were shown to have high selectivity indices (little or no cytotoxicity and consistently high activity in all three viral assays). These endeavors will greatly facilitate the search for compounds and formulations that can be used globally as topical microbicides. from 2005 International Meeting of The Institute of Human Virology Baltimore, USA, 29 August – 2 September 2005