<i>In vivo</i> study protocol in pigtailed macaques.

<p>(<b>A</b>) Two studies were performed. The first included five macaques, in which each monkey received placebo films followed six weeks later with RC-101 films. The second study included the five original macaques plus an additional monkey, in which half received RC-101 films and half received placebo films. Six weeks later, each monkey receiving the opposite film type. (<b>B</b>) Baseline measurements for colposcopy, vaginal pH, and microflora were obtained at day 0. At days 1–4, RC-101- or placebo-containing films were instilled intravaginally. At days 5 and 8, cytobrushes, blood, and pinch biopsies were obtained, and at day 8 CVL fluids were obtained from each monkey.</p

RC-101 films do not alter vaginal pH or microbiological profile of the cervicovagina.

<p>(<b>A</b>) Vaginal pH was measured prior to film instillation at days 1-4 (D1-D4) and at followup (D5 and D8). Each measurement represents one experimental condition in a pigtailed macaque. Horizontal lines represent the mean value for all 11 replicates. No differences were observed between monkeys that were receiving RC-101 films or placebo films. (<b>B</b>) represents the difference in vaginal pH between the time zero condition and 30 minutes after film instillation for each day films were applied (D1–D4). Horizontal lines represent the mean value for all 11 replicates. A significant difference was observed at Day 4 (<i>P</i> = 0.003), with the placebo films inducing a greater change in vaginal pH than the RC-101 films. (<b>C</b>) Four (of twenty) microbes are presented for both RC-101 films and placebo films. For each microbe, there are 10 vertical bars, representing the number of replicates (from a total of 11) that were positive for the microbe indicated. The ten bars for each microbe signify the following from left to right: Day 1 (D1) time zero (t0), D1, time 30 min (t30), D2 t0, D2 t30, D3 t0, D3 t30, D4 t0, D4 t30, D5 and D8.</p

RC-101 was retained in the cervicovagina up to four days post-application.

<p>RC-101 was assessed by quantitative western blot from acetic acid-extracted samples of cervical biopsies, vaginal biopsies and cytobrushes at day 5, and cytobrushes and CVL fluid at day 8 (<b>A</b>). Note that CVL fluid was subdivided into both the cellular portion and the fluid portion. A representative western blot for RC-101 in cytobrushes at day 5 is shown in (<b>B</b>), with “M1”, “M2”, and “M3” signifying three individual monkeys. Note that the same monkey received both RC-101 films and placebo films six weeks apart, and that RC-101 was absent in cytobrushes obtained from monkeys that received placebo films. The <i>P</i>-values presented were adjusted for multiple comparisons using Tukey method.</p

Colposcopy of the cervicovagina reveals no adverse effects of RC-101 films.

<p>Images from colposcopic examination of cervicovaginal mucosa of a representative pigtailed macaque obtained prior to film insertion, at the time of insertion (Time 0), and 30 min and 24 hr after film insertion. Note the absence of mucosal aberrations at all time points with either RC-101 films or placebo films.</p

Activity of formulated RC-101 is equivalent to unformulated peptide.

<p>Antiviral activities of RC-101-containing films and placebo films against HIV-1 strain BaL were compared against unformulated “pure” RC-101 peptide in TZM-bl reporter assays expressed as percent inhibition of HIV-1 infection (<b>A</b>) and p24^gag release assay in PM1 cells expressed in pg/ml of p24 released (<b>B</b>). p24^gag differed between formulated and unformulated RC-101 at days 5 and 7, <i>P</i><0.001 and <i>P</i> = 0.019, respectively. Error bars represent SEM; n = 2-5.</p