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.     

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.     

Preclinical evaluation of synthetic -2 RANTES as a candidate vaginal microbicide to target CCR5

A potential strategy that can be used to combat the worldwide AIDS epidemic is the development of a vaginal microbicide that prevents the sexual transmission of human immunodeficiency virus type 1 (HIV-1). Certain CC chemokines, including RANTES, MIP-1alpha, and MIP-1beta, might facilitate the development of such microbicides since they potently suppress HIV-1 infection by binding to CCR5, the viral coreceptor used by most sexually transmitted strains of HIV-1 to enter host cells. In this study, we evaluated whether a CCR5-specific fragment of RANTES that lacks two N-terminal residues (-2 RANTES) and possesses especially potent HIV-1 suppressive activity has toxicity profiles conducive to the advancement of testing in candidate microbicide formulations. Analyses were carried out with a synthetic version of the chemokine, which was formulated with either Novasomes 7474, a nonphospholipid liposome, or methylcellulose gel. Dialysis studies demonstrated that the formulated -2 RANTES was released from both vehicles and retained anti-HIV-1 activity. Preclinical toxicity studies carried out with Swiss Webster mouse and New Zealand White rabbit vaginal irritation models demonstrated minimal inflammation and minimal adverse changes in cervicovaginal tissue integrity after short-term (10 min) and long-term (24 h) exposure to formulations containing up to 1 mg/ml of -2 RANTES. Similarly, no toxicity was observed with formulations of bioactive murine RANTES in the Swiss Webster mouse vaginal irritation model. Overall, these preclinical studies suggest that -2 RANTES is suitable for further testing as a candidate anti-HIV-1 microbicide.     

Sodium dodecyl sulfate and C31G as microbicidal alternatives to nonoxynol 9: comparative sensitivity of primary human vaginal keratinocytes

A broad-spectrum vaginal microbicide must be effective against a variety of sexually transmitted disease pathogens and be minimally toxic to the cell types found within the vaginal epithelium, including vaginal keratinocytes. We assessed the sensitivity of primary human vaginal keratinocytes to potential topical vaginal microbicides nonoxynol-9 (N-9), C31G, and sodium dodecyl sulfate (SDS). Direct immunofluorescence and fluorescence-activated cell sorting analyses demonstrated that primary vaginal keratinocytes expressed epithelial cell-specific keratin proteins. Experiments that compared vaginal keratinocyte sensitivity to each agent during a continuous, 48-h exposure demonstrated that primary vaginal keratinocytes were almost five times more sensitive to N-9 than to either C31G or SDS. To evaluate the effect of multiple microbicide exposures on cell viability, primary vaginal keratinocytes were exposed to N-9, C31G, or SDS three times during a 78-h period. In these experiments, cells were considerably more sensitive to C31G than to N-9 or SDS at lower concentrations within the range tested. When agent concentrations were chosen to result in an endpoint of 25% viability after three daily exposures, each exposure decreased cell viability at the same constant rate. When time-dependent sensitivity during a continuous 48-h exposure was examined, exposure to C31G for 18 h resulted in losses in cell viability not caused by either N-9 or SDS until at least 24 to 48 h. Cumulatively, these results reveal important variations in time- and concentration-dependent sensitivity to N-9, C31G, or SDS within populations of primary human vaginal keratinocytes cultured in vitro. These investigations represent initial steps toward both in vitro modeling of the vaginal microenvironment and studies of factors that impact the in vivo efficacy of vaginal topical 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.     

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.     

PD 404,182 is a virocidal small molecule that disrupts hepatitis C virus and human immunodeficiency virus

We describe a virucidal small molecule, PD 404,182, that is effective against hepatitis C virus (HCV) and human immunodeficiency virus (HIV). The median 50% inhibitory concentrations (IC(50)s) for the antiviral effect of PD 404,182 against HCV and HIV in cell culture are 11 and 1 μM, respectively. The antiviral activity of PD 404,182 is due to the physical disruption of virions that is accompanied to various degrees (depending on the virus and exposure temperature/time) by the release of viral nucleic acids into the surrounding medium. PD 404,182 does not directly lyse liposomal membranes even after extended exposure, and it shows no attenuation in antiviral activity when preincubated with liposomes of various lipid compositions, suggesting that the compound inactivates viruses through interaction with a nonlipid structural component of the virus. The virucidal activity of PD 404,182 appears to be virus specific, as little to no viral inactivation was detected with the enveloped Dengue and Sindbis viruses. PD 404,182 effectively inactivates a broad range of primary isolates of HIV-1 as well as HIV-2 and simian immunodeficiency virus (SIV), and it does not exhibit significant cytotoxicity with multiple human cell lines in vitro (50% cytotoxic concentration, >300 μM). The compound is fully active in cervical fluids, although it exhibits decreased potency in the presence of human serum, retains its full antiviral potency for 8 h when in contact with cells, and is effective against both cell-free and cell-associated HIV. These qualities make PD 404,182 an attractive candidate anti-HIV microbicide for the prevention of HIV transmission through sexual intercourse.     

The microbicidal agent C31G inhibits Chlamydia trachomatis infectivity in vitro.

Safe and effective vaginal microbicidal compounds are being sought to offer women an independent method for protection against transmission of sexually acquired pathogens. The purpose of this study was to examine the efficacy of two formulations of one such compound, C31G, against Chlamydia trachomatis serovar E alone, its host epithelial cell (HEC-1B) alone, and against chlamydiae-infected HEC-1B cells. Preexposure of isolated, purified infectious chlamydial elementary bodies (EB) to C31G, at pHs 7.2 and 5.7, for 1 h at 4 degrees C resulted in reduced infectivity of EB for HEC-1B cells. Examination of the C31G-exposed 35S-EB on sodium dodecyl sulfate-polyacrylamide gel electrophoresis autoradiographs and by Western blotting revealed a C31G concentration-dependent and pH-dependent destabilization of the chlamydial envelope, resulting in the release of chlamydial lipopolysaccharide and proteins. Interestingly, when the host human genital columnar epithelial cells were infected with chlamydiae and then exposed to dilute concentrations of C31G which did not alter epithelial cell viability, chlamydial infectivity was also markedly reduced. C31G gained access to the developing chlamydial inclusion causing damage to or destruction of metabolically active reticulate bodies as well as apparent alteration of the inclusion membrane, which resulted in premature escape of chlamydial antigen to the infected epithelial surface. These studies show that the broad-spectrum antiviral and antibacterial microbicide C31G also has antichlamydial activity.     

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-na?ve 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.     

Anti-human immunodeficiency virus type 1 microbicide cellulose acetate 1,2-benzenedicarboxylate in a human in vitro model of vaginal inflammation

The sexual transmission of human immunodeficiency virus type 1 (HIV-1) is facilitated by inflammation and related epithelial barrier perturbation. Microbicides for vaginal applications are currently being developed to reduce the risk of HIV-1 transmission. However, little is known about their interference with epithelial immune function. In recent clinical trials, nonoxynol-9 (N-9), a virucide with a long history of intravaginal use as a contraceptive, failed to protect against HIV-1 possibly due to mucosal inflammatory damage. Cellulose acetate 1,2-benzenedicarboxylate, also named CAP (for "controls AIDS pandemic"), is an anti-HIV-1 microbicide selected from pharmaceutical excipients that are regarded as safe for oral administration but have not been assessed for potential effects on inflammatory factors in the vaginal environment. Here we use a sensitive human cell culture system to evaluate proinflammatory profiles of soluble CAP in reference to N-9 and known epithelial activators such as tumor necrosis factor alpha (TNF-alpha) and bacterial lysates. Within 6 h of exposure, TNF-alpha and N-9 triggered NF-kappaB and AP-1/cFos activation and upregulated interleukins and an array of chemokines by vaginal and polarized cervical epithelial cells. The induced proinflammatory status continued after removal of stimuli and was confirmed by enhanced transepithelial neutrophil migration. While sustaining stability and anti-HIV-1 activity in the epithelial environment, CAP did not increase the production of proinflammatory mediators during or after exposure, nor did it modify the epithelial resistance to leukocyte traffic. CAP attenuated some TNF-alpha-induced responses but did not interfere with epithelial cytokine responsiveness to gonococcal determinants. The described system may be useful for predicting proinflammatory side effects of other microbicide candidates for vaginal application.     

N4-acyl-modified D-2',3'-dideoxy-5-fluorocytidine nucleoside analogues with improved antiviral activity

A series of 2',3'-dideoxy (D2) and 2',3'-didehydro-2',3'-dideoxy (D4) 5-fluorocytosine nucleosides modified with substituted benzoyl, heteroaromatic carbonyl, cycloalkylcarbonyl and alkanoyl at the N4-position were synthesized and evaluated for anti-human immunodeficiency virus type 1 (HIV-1) and anti-hepatitis B virus (HBV) activity in vitro. For most D2-nucleosides, N4-substitutions improved the anti-HIV-1 activity markedly without increasing the cytotoxicity. In the D4-nucleosides series, some of the substituents at the N4-position enhanced the anti-HIV-1 activity with a modest increase in the cytotoxicity. The most potent and selective N4-modified nucleoside for the D2-series was N4-p-iodobenzoyl-D2FC, which had a 46-fold increase in anti-HIV-1 potency in MT-2 cells compared to the parent nucleoside D-D2FC. In the D4-series, N4-p-bromobenzoyl-D4FC was 12-fold more potent in MT-2 cells compared to the parent nucleoside D-D4FC. All eight N4-p-halobenzoyl-substituted D2- and D4-nucleosides evaluated against HBV in HepAD38 cells demonstrated equal or greater potency than the two parental compounds, D-D2FC and D-D4FC. The N4-modification especially in the D2-nucleoside series containing the N4-nicotinoyl, o-nitrobenzoyl and n-butyryl showed a significant reduction in mitochondrial toxicity relative to the parent nucleoside analogue. Although the 5'-triphosphate of the parent compound (D-D4FC-TP) was formed from the N4-acyl-D4FC analogues in different cells, the levels of the 5'-triphosphate nucleotide did not correlate with the cell-derived 90% effective antiviral concentrations (EC90), suggesting that a direct interaction of the triphosphates of these N4-acyl nucleosides was involved in the antiviral activity.     

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.     

C31G, a new agent for oral use with potent antimicrobial and antiadherence properties.

C31G, an equimolar mixture of alkyl dimethyl glycine and alkyl dimethyl amine oxide, was evaluated for antimicrobial and antiadherence properties. The efficacy of C31G, its two components, and several commercial mouth rinses was determined in assays measuring inhibition of glycolysis, inhibition of bacterial adherence, and MICs. Inhibition of glycolysis was determined by using a saliva sediment model, with glycolytic activity expressed as the change in pH relative to that of a control. Adherence studies were undertaken with Streptococcus sobrinus 6715 to measure inhibition of adherence to nichrome wires. MICs were determined against selected microorganisms by standard methods. C31G demonstrated broad-spectrum antimicrobial properties, with activity against both gram-positive and gram-negative organisms and Candida albicans, a yeast. C31G inhibited both glycolysis by salivary bacteria and adherence of Streptococcus strains to wire mesh. C31G was more effective in the assays conducted than any commercial formulation tested and was as effective as chlorhexidine. A synergistic effect was demonstrated between the individual components of C31G, and no loss of activity was noted when it was formulated into a mouth rinse vehicle.     

Saquinavir inhibits early events associated with establishment of HIV-1 infection: potential role for protease inhibitors in prevention

The maturation of newly formed human immunodeficiency virus type 1 (HIV-1) virions is a critical step for the establishment of productive infection. We investigated the potential of saquinavir (SQV), a protease inhibitor (PI) used in highly active antiretroviral therapy (HAART), as a candidate microbicide. SQV inhibited replication of clade B and clade C isolates in a dose-dependent manner in all cellular models tested: PM-1 CD4 T cells, peripheral blood mononuclear cells (PBMCs), monocyte-derived macrophages (MDMs), and immature monocyte-derived dendritic cells (iMDDCs). SQV also inhibited production of infectious virus in cervical, penile, and colorectal explants cocultured with T cells. Moreover, SQV demonstrated inhibitory potency against trans infection of T cells by in vitro-derived dendritic cells and by primary dendritic cells that emigrate from penile and cervical tissue explants. No cellular or tissue toxicity was detected in the presence of SQV, suggesting that this drug could be considered for development as a component of an effective microbicide, capable of blocking viral maturation and transmission of HIV-1 at mucosal surfaces.     

Evaluation of PD 404,182 as an Anti-HIV and Anti-Herpes Simplex Virus Microbicide

PD 404,182 (PD) is a synthetic compound that was found to compromise HIV integrity via interaction with a nonenvelope protein viral structural component (A. M. Chamoun et al., Antimicrob. Agents Chemother. 56:672–681, 2012). The present study evaluates the potential of PD as an anti-HIV microbicide and establishes PD''s virucidal activity toward another pathogen, herpes simplex virus (HSV). We show that the anti-HIV-1 50% inhibitory concentration (IC₅₀) of PD, when diluted in seminal plasma, is ∼1 μM, similar to the IC₅₀ determined in cell culture growth medium, and that PD retains full anti-HIV-1 activity after incubation in cervical fluid at 37°C for at least 24 h. In addition, PD is nontoxic toward vaginal commensal Lactobacillus species (50% cytotoxic concentration [CC₅₀], >300 μM), freshly activated human peripheral blood mononuclear cells (CC₅₀, ∼200 μM), and primary CD4⁺ T cells, macrophages, and dendritic cells (CC₅₀, >300 μM). PD also exhibited high stability in pH-adjusted Dulbecco''s phosphate-buffered saline with little to no activity loss after 8 weeks at pH 4 and 42°C, indicating suitability for formulation for transportation and storage in developing countries. Finally, for the first time, we show that PD inactivates herpes simplex virus 1 (HSV-1) and HSV-2 at submicromolar concentrations. Due to the prevalence of HSV infection, the ability of PD to inactivate HSV may provide an additional incentive for use as a microbicide. The ability of PD to inactivate both HIV-1 and HSV, combined with its low toxicity and high stability, warrants additional studies for the evaluation of PD''s microbicidal candidacy in animals and humans.     

Selective membrane disruption: mode of action of C16G2, a specifically targeted antimicrobial peptide

The specifically targeted antimicrobial peptide (STAMP) C16G2 was developed to target the cariogenic oral pathogen Streptococcus mutans. Because the design of this peptide was novel, we sought to better understand the mechanism through which it functioned. Compared to antimicrobial peptides (AMPs) with wide spectra of activity, the STAMP C16G2 has demonstrated specificity for S. mutans in a mixed-culture environment, resulting in the complete killing of S. mutans while having minimal effect on the other streptococci. In the current study, we sought to further confirm the selectivity of C16G2 and also compare its membrane activity to that of melittin B, a classical toxic AMP, in order to determine the STAMP's mechanism of cell killing. Disruption of S. mutans cell membranes by C16G2 was demonstrated by increased SYTOX green uptake and ATP efflux from the cells similar to those of melittin B. Treatment with C16G2 also resulted in a loss of membrane potential as measured by DiSC(3)5 fluorescence. In comparison, the individual moieties of C16G2 demonstrated no specificity and limited antimicrobial activity compared to those of the STAMP C16G2. The data suggest that C16G2 has a mechanism of action similar to that of traditional AMPs and kills S. mutans through disruption of the cell membrane, allowing small molecules to leak out of the cell, which is followed by a loss of membrane potential and cell death. Interestingly, this membrane activity is rapid and potent against S. mutans, but not other noncariogenic oral streptococci.     

The toxicity of Callilepis laureola, a South African traditional herbal medicine.

OBJECTIVES: To review the literature on the toxicity of Callilepis laureola, and to assess the cytotoxicity of C. laureola in human hepatoblastoma Hep G2 cells in vitro. DESIGN AND METHODS: Cells were incubated for up to 48 h in the presence of increasing concentrations of an aqueous extract of C. laureola (0.3-13.3 mg/mL). Cytotoxicity was quantitated spectrophotometrically by the metabolism of the tetrazolium dye MTT. Cytoviability of the control cells was considered to be 100%. RESULTS: C. laureola produced cytotoxicity in a concentration-dependent manner. Cytotoxicity was significant at all concentrations tested (0.3-2.5 mg/mL, p < 0.05 vs. controls and 3.3-13.3 mg/mL, p < 0.0001 vs. controls). After 6 h, 100% toxicity was observed at a concentration of 6.7 mg/mL. CONCLUSION: C. laureola causes significant cytotoxicity in Hep G2 cells in vitro. These findings are in accordance with the observed hepatotoxicity in clinical cases of C. laureola poisoning.     

In vitro evaluation of viability, integrity, and inflammation in genital epithelia upon exposure to pharmaceutical excipients and candidate microbicides

The use of microbicides is a promising approach for the prevention of HIV-1 transmission. Unfortunately, various candidates failed in clinical trials. In some cases, the candidate microbicide even resulted in enhanced virus transmission. Therefore, there is an urgent need to develop more predictive preclinical strategies to anticipate the in vivo efficiency/toxicity rate, including in vitro assays that evaluate effects on epithelial integrity and inflammation. The present study aims to identify potential safety issues concerning the use of microbicides and excipients commonly used in vaginal microbicide preparations. The toxicities of various active pharmaceutical ingredients (APIs; TMC-120, UC-781, tenofovir [PMPA], PRO-2000, and glycerol monolaurate [GML]) and excipients (preservatives, cosolvents, surfactants, and cyclodextrins) were evaluated using an in vitro dual-chamber model and uterine cervical explants. Epithelial viability and permeation of fluorescent virus-sized beads, as well as induction of interleukin-8 (IL-8; as a sensitive marker of an inflammatory response), were assessed. Surprisingly, cell viability and epithelial layer integrity were compromised by most excipients at concentrations near the typical concentration used in vaginal gels, and a significant increase in the production of IL-8 was observed at subtoxic concentrations. Within the APIs, TMC-120, UC-781, and PMPA showed higher selectivity indices than PRO-2000 and GML. In conclusion, identification of safety issues concerning the use of pharmaceutical excipients could help to formulate less toxic vaginal microbicide preparations.     

Influence of stereochemistry on antiviral activities and resistance profiles of dideoxycytidine nucleosides.

beta-L-2',3'-Dideoxycytidine (beta-L-ddC) and beta-L-5-fluoro-2',3'-dideoxycytidine (5-F-beta-L-ddC) were prepared and shown to have potent activity against human immunodeficiency virus type 1 (HIV-1) and hepatitis B virus (HBV). These compounds were compared with beta-D-2',3'-dideoxycytidine (beta-D-ddC) and two beta-L-oxathiolane nucleosides (beta-L-3'-thio-2',3'-dideoxycytidine and beta-L-5-fluoro-3'-thio-2',3'-dideoxycytidine) in terms of anti-HIV and anti-HBV activity, cytotoxicity, and development of HIV-1 resistance. Compared with beta-D-ddC, the beta-L-dideoxycytidine nucleosides had similar anti-HIV-1 activities, significantly greater anti-HBV activities, and decreased toxicities to a B-cell line, T-cell lines, and human bone marrow progenitor cells. HIV-1 strains resistant to beta-D-ddC were susceptible to the beta-L-ddC analogs. Compared with the beta-L-oxathiolane nucleosides, beta-L-ddC and 5-F-beta-L-ddC had similar anti-HIV-1 activities, decreased anti-HBV activities, and greater toxicities to B- and T-cell lines and bone marrow progenitor cells. There were similarities between the beta-L-ddC and beta-L-oxathiolane nucleosides in the rate of development and pattern of resistant HIV-1 selection. While the in vitro activity and cytotoxicity profiles of the beta-L-ddC nucleosides differed from those of the beta-D-ddC and beta-L-oxathiolane nucleosides, the data presented herein suggest that the sugar configuration of a dideoxynucleoside analog may play a major role in the rate of development and the pattern of HIV-1 resistance.