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

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.

Section: Discussionmentioning

confidence: 99%

In Vitro Evaluation of Viability, Integrity, and Inflammation in Genital Epithelia upon Exposure to Pharmaceutical Excipients and Candidate Microbicides

Gali

Delézay

Brouwers

et al. 2010

“…A total of 127 assays evaluating the toxicity of N-9 were provided (GH, 20 assays; SR, 8 assays; CD, 6 assays; DU, 51 assays; and CR, 42 assays). The data provided by the CR, GH, and SR laboratories were unpublished, and the data from the CD and DU laboratories were previously published (9,(19)(20)(21). The source of N-9 differed between laboratories and was not considered a covariate factor for the study.…”

Section: Methodsmentioning

confidence: 99%

In Vitro Preclinical Testing of Nonoxynol-9 as Potential Anti-Human Immunodeficiency Virus Microbicide: a Retrospective Analysis of Results from Five Laboratories

Beer

Doncel

Krebs

et al. 2006

Self Cite

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.

“…Previous studies of the impact of C31G on human cell viability indicated that the in vitro cytotoxicity of C31G is dependent on cell type, exposure duration, and compound concentration (11,12). The studies presented herein examined the in vivo safety of C31G by assessing toxicity and inflammation associated with introduction of unformulated and formulated C31G into the murine cervicovaginal environment.…”

mentioning

confidence: 99%

Comparative Safety Evaluation of the Candidate Vaginal Microbicide C31G

Catalone¹,

Kish-Catalone²,

Neely³

et al. 2005

Self Cite

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.