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In Vitro and In Vivo Measures of Microbicide Safety Differ Considerably in a Multiple Exposure Study
GENDER MEDICINE/VOL. 9, NO. 1S, 2012
Basic Science Research Poster Presentations Cationic Cell Penetrating Peptides as Potential Inhibitors of HIV-1 Infection Shawn Keogan, BS; Shendra Passic, MS; Brian Wigdahl, PhD; and Fred Krebs, PhD Department of Microbiology and Immunology, Drexel University College of Medicine, Philadelphia, Pennsylvania In the absence of microbicides or vaccines effective against human immunodeficiency virus type 1 (HIV-1), the development of new anti–HIV-1 drugs remains a priority. Our efforts in this area suggest that molecules belonging to the family of cell penetrating peptides (CPPs) may also be potent HIV-1 entry inhibitors. CPPs are short peptides that can efficiently cross the plasma membrane, which is otherwise a formidable barrier to many extracellular molecules. Because of this ability, CPPs are currently being studied as delivery vehicles for therapeutic agents and larger molecules that cannot enter the cell. A 10-amino acid peptide derived from HIV-1 Tat protein has been well studied as an attractive drug delivery agent. We have demonstrated concentration-dependent inhibition of HIV-1 IIIB (X4) infection by Tat peptide, yet minimal antiviral activity against BaL (R5). In contrast, a 9-arginine oligomeric CPP (R-9), which differs from Tat peptide by a small number of amino acid residues, was shown to have considerable activity against HIV-1 IIIB and, surprisingly, concentration-dependent activity against BaL. With consideration of previous studies, we hypothesize that (1) both Tat peptide and R-9 are CXCR4 antagonists and (2) the limited amino acid sequence differences in R-9 relative to the Tat peptide confer CCR5-specific antiviral activity. Using Tat peptide and oligoarginine as the starting points, further studies will identify sequence determinants for CPP biological activity, demonstrate mechanisms of antiviral activity, and explore the potential of these peptides as the basis for the development of novel HIV-1 inhibitors.
In Vitro Enhancement of HIV-1 Infection as a Potential Contributor to the Clinical Failures of Select Polyanionic Microbicide Candidates Fred Krebs, PhD; Vanessa Pirrone, PhD; Shendra Passic, MS; and Brian Wigdahl, PhD Department of Microbiology and Immunology, Drexel University College of Medicine, Philadelphia, Pennsylvania Increasing efforts are being directed toward the development of microbicides, which will be used to reduce or eliminate the risk of human immunodeficiency virus type 1 (HIV-1) sexual transmission. Polyanionic compounds, which interact nonspecifically with HIV-1 gp120 to block infection, were among the first agents evaluated clinically for their potential use in microbicide products. Unfortunately, Phase III clinical trials involving Carraguard, Ushercell, and PRO 2000 demonstrated that these products were ineffective and may have, in some instances, increased the risk of HIV-1 infection. These findings precipitated reassessments of the in vitro activities of these agents to determine if 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 in the female reproductive tract. By mimicking product loss using in vitro washout experiments involving either an HIV-1–susceptible cell line or primary human immune cells,
S94
we showed that several polyanionic compounds, including carrageenan and cellulose sulfate, caused enhancement of HIV-1 infection following compound removal, despite potent antiviral activity when introduced simultaneously with the viral challenge. The presence and magnitude of this effect was compound specific, dependent on the interval between compound removal and virus challenge, and dependent on HIV-1 coreceptor usage. Compounds that enhanced HIV-1 infection in this assay increased levels of HIV-1 infection up to 10-fold. More detailed studies are now under way to determine the mechanism responsible for this enhancement effect, and to determine the contributions of this effect to the clinical failures of these agents.
In Vitro and In Vivo Measures of Microbicide Safety Differ Considerably in a Multiple Exposure Study Karissa Lozenski, BS1; Tina Kish-Catalone, PhD2; Brian Wigdahl, PhD1; and Fred Krebs, PhD1 1 Department of Microbiology and Immunology, Drexel University College of Medicine, Philadelphia, Pennsylvania; and 2Department of Natural Sciences, DeSales University, Center Valley, Pennsylvania The disappointing clinical failures of 4 vaginal microbicides have provided new insights into factors that impact microbicide effectiveness, including repeated product application and its association with increased HIV-1 infection. To investigate the effects of repeated use on microbicide safety, multiple exposure experiments were performed using a mouse model of cervicovaginal toxicity. This model, which was previously used to examine the effects of single microbicide applications, was used to evaluate the effects of application frequency on 2 measures of in vivo toxicity: epithelial integrity and immune cell recruitment. In multiple exposure experiments using nonoxynol-9 (N-9), the first application of 1% N-9 caused considerable damage to the cervical epithelium. Subsequent daily exposures were characterized by diminished cervical toxicity relative to the initial exposure. Multiple daily exposures also increased the exposure duration required to elicit levels of epithelial damage similar to those seen after the first exposure. However, in vitro cytotoxicity experiments, which were conducted to explore parallels between in vitro and in vivo assays of microbicide safety, conversely demonstrated that HeLa cells became increasingly sensitive to N-9 after multiple exposures. These studies are now being expanded to include other failed microbicides, including cellulose sulfate (CS) and carrageenan (LC). Initial in vitro multiple exposure experiments demonstrated that both CS and LC were somewhat cytotoxic after the second exposure, but they caused apparent increases in cellular proliferation after additional exposures. Ongoing in vitro and in vivo studies are now investigating the mechanisms that underlie the effects of multiple exposures to agents used as microbicides.
Using the Conrad Microbicide Testing Algorithm to Screen Single Compounds and Agent Combinations for Cytotoxicity and Anti–HIV-1 Activity
Basic Science Research Poster Presentations Cationic Cell Penetrating Peptides as Potential Inhibitors of HIV-1 Infection Shawn Keogan, BS; Shendra Passic, MS; Brian Wigdahl, PhD; and Fred Krebs, PhD Department of Microbiology and Immunology, Drexel University College of Medicine, Philadelphia, Pennsylvania In the absence of microbicides or vaccines effective against human immunodeficiency virus type 1 (HIV-1), the development of new anti–HIV-1 drugs remains a priority. Our efforts in this area suggest that molecules belonging to the family of cell penetrating peptides (CPPs) may also be potent HIV-1 entry inhibitors. CPPs are short peptides that can efficiently cross the plasma membrane, which is otherwise a formidable barrier to many extracellular molecules. Because of this ability, CPPs are currently being studied as delivery vehicles for therapeutic agents and larger molecules that cannot enter the cell. A 10-amino acid peptide derived from HIV-1 Tat protein has been well studied as an attractive drug delivery agent. We have demonstrated concentration-dependent inhibition of HIV-1 IIIB (X4) infection by Tat peptide, yet minimal antiviral activity against BaL (R5). In contrast, a 9-arginine oligomeric CPP (R-9), which differs from Tat peptide by a small number of amino acid residues, was shown to have considerable activity against HIV-1 IIIB and, surprisingly, concentration-dependent activity against BaL. With consideration of previous studies, we hypothesize that (1) both Tat peptide and R-9 are CXCR4 antagonists and (2) the limited amino acid sequence differences in R-9 relative to the Tat peptide confer CCR5-specific antiviral activity. Using Tat peptide and oligoarginine as the starting points, further studies will identify sequence determinants for CPP biological activity, demonstrate mechanisms of antiviral activity, and explore the potential of these peptides as the basis for the development of novel HIV-1 inhibitors.
In Vitro Enhancement of HIV-1 Infection as a Potential Contributor to the Clinical Failures of Select Polyanionic Microbicide Candidates Fred Krebs, PhD; Vanessa Pirrone, PhD; Shendra Passic, MS; and Brian Wigdahl, PhD Department of Microbiology and Immunology, Drexel University College of Medicine, Philadelphia, Pennsylvania Increasing efforts are being directed toward the development of microbicides, which will be used to reduce or eliminate the risk of human immunodeficiency virus type 1 (HIV-1) sexual transmission. Polyanionic compounds, which interact nonspecifically with HIV-1 gp120 to block infection, were among the first agents evaluated clinically for their potential use in microbicide products. Unfortunately, Phase III clinical trials involving Carraguard, Ushercell, and PRO 2000 demonstrated that these products were ineffective and may have, in some instances, increased the risk of HIV-1 infection. These findings precipitated reassessments of the in vitro activities of these agents to determine if 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 in the female reproductive tract. By mimicking product loss using in vitro washout experiments involving either an HIV-1–susceptible cell line or primary human immune cells,
S94
we showed that several polyanionic compounds, including carrageenan and cellulose sulfate, caused enhancement of HIV-1 infection following compound removal, despite potent antiviral activity when introduced simultaneously with the viral challenge. The presence and magnitude of this effect was compound specific, dependent on the interval between compound removal and virus challenge, and dependent on HIV-1 coreceptor usage. Compounds that enhanced HIV-1 infection in this assay increased levels of HIV-1 infection up to 10-fold. More detailed studies are now under way to determine the mechanism responsible for this enhancement effect, and to determine the contributions of this effect to the clinical failures of these agents.
In Vitro and In Vivo Measures of Microbicide Safety Differ Considerably in a Multiple Exposure Study Karissa Lozenski, BS1; Tina Kish-Catalone, PhD2; Brian Wigdahl, PhD1; and Fred Krebs, PhD1 1 Department of Microbiology and Immunology, Drexel University College of Medicine, Philadelphia, Pennsylvania; and 2Department of Natural Sciences, DeSales University, Center Valley, Pennsylvania The disappointing clinical failures of 4 vaginal microbicides have provided new insights into factors that impact microbicide effectiveness, including repeated product application and its association with increased HIV-1 infection. To investigate the effects of repeated use on microbicide safety, multiple exposure experiments were performed using a mouse model of cervicovaginal toxicity. This model, which was previously used to examine the effects of single microbicide applications, was used to evaluate the effects of application frequency on 2 measures of in vivo toxicity: epithelial integrity and immune cell recruitment. In multiple exposure experiments using nonoxynol-9 (N-9), the first application of 1% N-9 caused considerable damage to the cervical epithelium. Subsequent daily exposures were characterized by diminished cervical toxicity relative to the initial exposure. Multiple daily exposures also increased the exposure duration required to elicit levels of epithelial damage similar to those seen after the first exposure. However, in vitro cytotoxicity experiments, which were conducted to explore parallels between in vitro and in vivo assays of microbicide safety, conversely demonstrated that HeLa cells became increasingly sensitive to N-9 after multiple exposures. These studies are now being expanded to include other failed microbicides, including cellulose sulfate (CS) and carrageenan (LC). Initial in vitro multiple exposure experiments demonstrated that both CS and LC were somewhat cytotoxic after the second exposure, but they caused apparent increases in cellular proliferation after additional exposures. Ongoing in vitro and in vivo studies are now investigating the mechanisms that underlie the effects of multiple exposures to agents used as microbicides.
Using the Conrad Microbicide Testing Algorithm to Screen Single Compounds and Agent Combinations for Cytotoxicity and Anti–HIV-1 Activity