1. Mucosal safety of PHI-443 and stampidine as a combination microbicide to prevent genital transmission of HIV-1 
Osmond J. D'Cruz, Ph.D., Fatih M. Uckun, M.D., Ph.D. 
Fertility and Sterility 
Volume 88, Issue 4, Pages (October 2007)
DOI: /j.fertnstert
Copyright © 2007 American Society for Reproductive Medicine Terms and Conditions

2. Figure 1
Chemical structures of PHI-443 (N′-[2-(2-thiophene)ethyl]-N′-[2-(5-bromo pyridyl)]-thiourea) and stampidine (2′,3′-didehydro-3′-deoxythymidine 5′-(p-bromophenyl methoxyalaninyl phosphate).
D'Cruz. PHI-443/stampidine: a combination anti-HIV microbicide. Fertil Steril 2007.
Fertility and Sterility  , DOI: ( /j.fertnstert )
Copyright © 2007 American Society for Reproductive Medicine Terms and Conditions

3. Figure 2
Box-whisker plots depicting individual scores for histologic variables after daily intravaginal administration of gel with and without PHI-443, stampidine, and PHI-443 plus stampidine for 14 days. The four panels illustrate individual score (0–4) for epithelial cell damage, leukocyte infiltration, mucosal edema, and vascular congestion, respectively, in three different vaginal regions after daily intravaginal application of a gel (n = 4) or gel incorporating 1% PHI-443, 1% stampidine, or 1% PHI-443 plus stampidine for 14 consecutive days. The following individual score was used: 0 = none, 1 = minimal, 2 = mild, 3 = moderate, 4 = intense in describing the four histologic variables. Each box-whisker plot represents 25th and 75th percentile (box), and median (horizontal line dividing the box) and the range of scores (whiskers). The scores were compared with 4% N-9 gel, which was used as a positive control for vaginal irritation. STAMP = stampidine.
D'Cruz. PHI-443/stampidine: a combination anti-HIV microbicide. Fertil Steril 2007.
Fertility and Sterility  , DOI: ( /j.fertnstert )
Copyright © 2007 American Society for Reproductive Medicine Terms and Conditions

4. Figure 3
Histograms depicting total scores for histologic variables after daily intravaginal administration of gel with and without PHI-443, stampidine, and PHI-443 plus stampidine for 14 days. The total irritation score (0–16) was obtained by summing each individual score (0–4) for epithelial cell damage, leukocyte infiltration, mucosal edema, and vascular congestion in three different vaginal regions after daily intravaginal application of a gel (n = 4) or gel incorporating 1% PHI-443, 1% stampidine, or 1% PHI-443 plus stampidine for 14 consecutive days. Rabbits treated intravaginally with 4% N-9 gel were used as positive controls. The dotted horizontal line is the suggested cutoff value. Correlation to human irritation potential: total score ≤8 acceptable (mild), 9–10 marginal (moderate), and ≥11 unacceptable (severe). Data show mean ± SEM of 12 tissue sections from four rabbits per group. Comparisons by ANOVA followed by Bonferroni's post-test for multiple comparisons: ∗∗∗P<.001, gel vs. N-9; PHI-443 vs. N-9; stampidine vs. N-9; and PHI-443 plus stampidine vs. N-9. STAMP = stampidine.
D'Cruz. PHI-443/stampidine: a combination anti-HIV microbicide. Fertil Steril 2007.
Fertility and Sterility  , DOI: ( /j.fertnstert )
Copyright © 2007 American Society for Reproductive Medicine Terms and Conditions

5. Figure 4
Effect of PHI-443, stampidine, and PHI-443 plus stampidine on the cell viability of normal human vaginal epithelial cells as quantitated by MTT assay. Vaginal cell viability was tested at nine different concentrations (3.6–1,000 μM) of PHI-443, stampidine, and PHI-443 plus stampidine or N-9 (positive control). Inhibition of cell growth is expressed relative to dimethyl sulfoxide controls. Each data point represents the mean of three representative experiments.
D'Cruz. PHI-443/stampidine: a combination anti-HIV microbicide. Fertil Steril 2007.
Fertility and Sterility  , DOI: ( /j.fertnstert )
Copyright © 2007 American Society for Reproductive Medicine Terms and Conditions

6. Figure 5
Lack of membrane-damaging effect of PHI-443 and stampidine on three-dimensional human vaginal epithelia integrating Langerhans cells. Three-dimensionally reconstituted tissue of human genital squamous epithelia integrating Langerhans cells were cultured for 11 days in serum-free growth medium on 0.5-cm2 polycarbonate membrane filters at the air–liquid interface in 12-well plates. The culture inserts were then exposed for up to 72 hours in medium with and without (A) PHI-443, (B) stampidine (C), or benzalkonium chloride (BZK) (D, positive control). Direct toxicity was evaluated by histologic assessment of H & E-stained tissue-containing filter sections. Arrow points to the loss of cell integrity after co-culture in the presence of BZK (original magnification, ×400).
D'Cruz. PHI-443/stampidine: a combination anti-HIV microbicide. Fertil Steril 2007.
Fertility and Sterility  , DOI: ( /j.fertnstert )
Copyright © 2007 American Society for Reproductive Medicine Terms and Conditions

7. Figure 5
Lack of membrane-damaging effect of PHI-443 and stampidine on three-dimensional human vaginal epithelia integrating Langerhans cells. Three-dimensionally reconstituted tissue of human genital squamous epithelia integrating Langerhans cells were cultured for 11 days in serum-free growth medium on 0.5-cm2 polycarbonate membrane filters at the air–liquid interface in 12-well plates. The culture inserts were then exposed for up to 72 hours in medium with and without (A) PHI-443, (B) stampidine (C), or benzalkonium chloride (BZK) (D, positive control). Direct toxicity was evaluated by histologic assessment of H & E-stained tissue-containing filter sections. Arrow points to the loss of cell integrity after co-culture in the presence of BZK (original magnification, ×400).
D'Cruz. PHI-443/stampidine: a combination anti-HIV microbicide. Fertil Steril 2007.
Fertility and Sterility  , DOI: ( /j.fertnstert )
Copyright © 2007 American Society for Reproductive Medicine Terms and Conditions

8. Figure 5
Lack of membrane-damaging effect of PHI-443 and stampidine on three-dimensional human vaginal epithelia integrating Langerhans cells. Three-dimensionally reconstituted tissue of human genital squamous epithelia integrating Langerhans cells were cultured for 11 days in serum-free growth medium on 0.5-cm2 polycarbonate membrane filters at the air–liquid interface in 12-well plates. The culture inserts were then exposed for up to 72 hours in medium with and without (A) PHI-443, (B) stampidine (C), or benzalkonium chloride (BZK) (D, positive control). Direct toxicity was evaluated by histologic assessment of H & E-stained tissue-containing filter sections. Arrow points to the loss of cell integrity after co-culture in the presence of BZK (original magnification, ×400).
D'Cruz. PHI-443/stampidine: a combination anti-HIV microbicide. Fertil Steril 2007.
Fertility and Sterility  , DOI: ( /j.fertnstert )
Copyright © 2007 American Society for Reproductive Medicine Terms and Conditions

9. Figure 5
Lack of membrane-damaging effect of PHI-443 and stampidine on three-dimensional human vaginal epithelia integrating Langerhans cells. Three-dimensionally reconstituted tissue of human genital squamous epithelia integrating Langerhans cells were cultured for 11 days in serum-free growth medium on 0.5-cm2 polycarbonate membrane filters at the air–liquid interface in 12-well plates. The culture inserts were then exposed for up to 72 hours in medium with and without (A) PHI-443, (B) stampidine (C), or benzalkonium chloride (BZK) (D, positive control). Direct toxicity was evaluated by histologic assessment of H & E-stained tissue-containing filter sections. Arrow points to the loss of cell integrity after co-culture in the presence of BZK (original magnification, ×400).
D'Cruz. PHI-443/stampidine: a combination anti-HIV microbicide. Fertil Steril 2007.
Fertility and Sterility  , DOI: ( /j.fertnstert )
Copyright © 2007 American Society for Reproductive Medicine Terms and Conditions

10. Figure 6
Simultaneous detection of 10 human cytokines secreted by three-dimensionally reconstituted human vaginal epithelia integrating Langerhans cells in the presence and absence of PHI-443 or stampidine. Eleven-day-old three-dimensionally reconstituted human vaginal epithelia and Langerhans cells were treated with and without PHI-443 or stampidine for up to 72 hours at 37°C in a humidified 5% CO2 atmosphere. At five time points, culture supernatants were tested in duplicate for 10 cytokine levels (pg/mL) by the multiplexed chemiluminescence-based immunoassay. Combined data from five time points are shown. See text for abbreviations.
D'Cruz. PHI-443/stampidine: a combination anti-HIV microbicide. Fertil Steril 2007.
Fertility and Sterility  , DOI: ( /j.fertnstert )
Copyright © 2007 American Society for Reproductive Medicine Terms and Conditions

11. Figure 7
Simultaneous detection of six human chemokines secreted by three-dimensionally reconstituted human vaginal epithelia integrating Langerhans cells in the presence and absence of PHI-443 or stampidine. Eleven-day-old three-dimensionally reconstituted human vaginal epithelial cells and Langerhans cells were treated with and without PHI-443 or stampidine for up to 72 hours at 37°C in a humidified 5% CO2 atmosphere. At five time points, culture supernatants were tested in duplicate for six chemokine levels (pg/mL) by the multiplexed chemiluminescence-based immunoassay. Combined data from five time points are shown. See text for abbreviations.
D'Cruz. PHI-443/stampidine: a combination anti-HIV microbicide. Fertil Steril 2007.
Fertility and Sterility  , DOI: ( /j.fertnstert )
Copyright © 2007 American Society for Reproductive Medicine Terms and Conditions