Phase I Randomized Safety Study of Twice Daily Dosing
of Acidform Vaginal Gel: Candidate Antimicrobial
Marla J. Keller1,2*, Colleen A. Carpenter3, Yungtai Lo4, Mark H. Einstein2,4, Congzhou Liu6,
David N. Fredricks6, Betsy C. Herold2,3,5
1Department of Medicine, Albert Einstein College of Medicine and Montefiore Medical Center, Bronx, New York, United States of America, 2Departments of Obstetrics
and Gynecology and Women’s Health, Albert Einstein College of Medicine and Montefiore Medical Center, Bronx, New York, United States of America, 3Department of
Pediatrics, Albert Einstein College of Medicine and Montefiore Medical Center, Bronx, New York, United States of America, 4Departments of Epidemiology and Population
Health, Albert Einstein College of Medicine and Montefiore Medical Center, Bronx, New York, United States of America, 5Departments of Microbiology and Immunology,
Albert Einstein College of Medicine and Montefiore Medical Center, Bronx, New York, United States of America, 6Vaccine and Infectious Disease Division, Fred Hutchinson
Cancer Research Center, Seattle, Washington, United States of America
Background: Acidform gel, an acid-buffering product that inactivates spermatozoa, may be an effective topical non-
hormonal contraceptive. This study was designed to evaluate the safety of vaginal dosing and effects of Acidform on
mucosal immune mediators, antimicrobial properties of genital secretions, and vaginal microbiota.
Methods: Thirty-six sexually abstinent U.S. women were randomized to apply Acidform or hydroxyethylcellulose (HEC)
placebo gel twice daily for 14 consecutive days. Safety was assessed by symptoms and pelvic examination. The impact of
gel on mucosal immunity was assessed by quantifying cytokines, chemokines, antimicrobial proteins and antimicrobial
activity of genital secretions collected by cervicovaginal lavage (CVL) at screening, 2 hours after gel application, and on days
7, 14 and 21. Vaginal microbiota was characterized at enrollment and day 14 using species-specific quantitative PCR assays.
Results: The median vaginal and cervical pH was significantly lower 2 hours after application of Acidform and was
associated with an increase in the bactericidal activity of CVL against E. coli. However, 65% of women who received
Acidform had at least one local adverse event compared with 11% who received placebo (p=0.002). While there was no
increase in inflammatory cytokines or chemokines, CVL concentrations of lactoferrin and interleukin-1 receptor antagonist
(IL-1ra), an anti-inflammatory protein, were significantly lower following Acidform compared to HEC placebo gel application.
There were no significant changes in Lactobacillus crispatus or Lactobacillus jensenii in either group but there was a decrease
in Gardnerella vaginalis in the Acidform group (p=0.08).
Conclusions: Acidform gel may augment mucosal defense as evidenced by an increase in bactericidal activity of genital
secretions against E. coli and a decrease in Gardnerella vaginalis colonization. However, Acidform was associated with more
irritation than placebo and lower levels of antimicrobial (lactoferrin) and anti-inflammatory (IL-1ra) proteins. These findings
indicate the need for additional safety studies of this candidate non-hormonal contraceptive.
Trial Registration: ClinicalTrials.gov NCT00850837
Citation: Keller MJ, Carpenter CA, Lo Y, Einstein MH, Liu C, et al. (2012) Phase I Randomized Safety Study of Twice Daily Dosing of Acidform Vaginal Gel:
Candidate Antimicrobial Contraceptive. PLoS ONE 7(10): e46901. doi:10.1371/journal.pone.0046901
Editor: Rupert Kaul, University of Toronto, Canada
Received June 16, 2012; Accepted September 5, 2012; Published October 8, 2012
Copyright: ? 2012 Keller et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was supported by grants from the National Institutes of Health (U01 AI069551, UL1 RR025750, P30 AI51519, U19 AI076980). Its contents are
solely the responsibility of the authors and do not necessarily represent the official views of the NIH. The funders had no role in study design, data collection and
analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: email@example.com
Several epidemiological studies indicate that systemic hormonal
contraception, particularly progesterone-containing injectables,
may be associated with an increased risk of both HIV acquisition
and transmission [1–3]. Moreover, nonoxynol-9 (N-9), approved
in the United States as a vaginal contraceptive, provides no
protection against HIV or other sexually transmitted infections
[4,5] and frequent use was shown to be associated with an
increased risk of HIV acquisition . Thus, the development of
safe and effective alternative contraceptives is a major global
The healthy human vagina in reproductive aged women is
acidic, with a pH ranging from 3.5 to 4.5, primarily because of
lactic acid and this environment inactivates sperm . However,
following sex, the pH is neutralized to at least 6.0 by semen
PLOS ONE | www.plosone.org1 October 2012 | Volume 7 | Issue 10 | e46901
(pH 7.2–8.2) to promote sperm survival. These observations
provided the rationale for developing acid-buffering products as
candidate multi-purpose agents that could serve as topical
contraceptives and provide protection against acid-sensitive
Two acid-buffering products, BufferGelH (developed by Re-
Protect Limited Liability Company, Baltimore, MD) and Acid-
form (developed by the Program for Topical Prevention of
Conception and Disease at Rush University, Chicago, IL) were
formulated as vaginal gels. BufferGelH was safe and well tolerated
in women [8–10] and reduced the prevalence of bacterial
vaginosis (BV) when applied twice daily for 2 weeks . However,
it did not alter the risk of HIV infection in a large-scale
effectiveness trial . When combined with a diaphragm,
BufferGelH was as effective as N-9 for contraception; the 6-month
pregnancy rate per hundred women was 10.1% (95% confidence
interval [CI] 7.1–13.1%) for BufferGelH and 12.3 (95% CI 7.7–
16.9) for N-9 spermicide users . However, no reduction in
pregnancy rate was observed when BufferGelH was used alone and
dosed pericoitally .
Acidform is a bioadhesive formulation that contains lactic acid
as a primary buffering agent. In contrast, the active ingredient in
BufferGelH is the hydrogen ion, which is released from the
buffering agent Carbopol 974 . Acidform buffers twice the
volume of semen to maintain a pH of 4.45 in vitro, is spermicidal
 and is active against herpes simplex virus (HSV), Chlamydia
trachomatis and Neisseria gonorrhoeae in animal studies [12–14]. A
randomized, blinded, crossover study was conducted among 20
sexually active sterilized women to compare the spermicidal effect
of Acidform to that of a commercial 2% N-9 product. Acidform or
N-9 product administered 0–30 min precoitus or Acidform given
8–10 h precoitus significantly reduced the mean number of
progressively motile sperm compared to control cycles (0.19,
0.07, 0.75 vs. 17.94, respectively, p,0.05, Wilcoxon signed-rank
test) . Acidform has been marketed as a personal lubricant
(AmphoraTMgel; Evofem Inc., San Diego, CA), and is currently
being evaluated for contraceptive efficacy in a Phase III trial
(ClinicalTrials.gov Identifier: NCT01306331).
Prior Phase I safety studies of Acidform gel alone or in
combination with a diaphragm have been conducted. No
symptoms or irritation were reported by 6 women who used
Acidform gel once daily for 6 days . In a second study, women
were randomized to Acidform (n=44) or K-Y Jelly (n=28) and
applied gel twice daily for 14 days . Twenty-seven women in
the Acidform group (61%) compared to 8 women in the K-Y Jelly
group (29%) reported at least one symptom of genital irritation
(odds ratio=2.62, CI 1.30–5.31, p=0.009). There was a trend
towards more safety events in women who used Acidform with a
Figure 1. Trial profile.
Safety of Acidform Vaginal Gel
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diaphragm for six months compared to women who used a
diaphragm with K-Y Jelly .
The goal of the present study was to expand the safety
assessment of Acidform gel with twice daily vaginal dosing over 14
days. Outcomes included symptoms, pelvic exam findings,
concentrations of genital tract immune mediators, and quantifi-
cation of antimicrobial activity of cervicovaginal secretions.
The protocol for this trial and supporting CONSORT checklist
are available as supporting information; see Checklist S1 and
Protocol S1. The study was conducted according to the
Declaration of Helsinki and was approved by the Albert Einstein
Table 1. Demographic data of recipients of Acidform and HEC placebo gel.
Age in years (mean 6 standard deviation) 30.1567.1732.1669.42 0.48
Race (number, %)* 0.34
Black 6 (37.5%) 11 (61%)
White4 (25%) 3 (16.7%)
Asian0 1 (5.6%)
Mixed 6 (37.5%)3 (16.7%)
Hispanic6 (35.3%)5 (27.8%)
Non-Hispanic 11 (64.7%)13 (72.2%)
Level of Education0.64
Less than high school0 1 (5.6)
High school/General education
5 (29.4%)3 (16.7%)
Some college4 (23.5%) 6 (33.3%)
College 6 (35.3%)4 (22.2%)
Graduate/Professional degree 2 (11.8%)4 (22.2%)
Number lifetime sex partners (median, range) 5 (1–34) 6 (0–100)0.36
Reported history of anal sex7 (41.2%) 7 (38.9%)0.89
Current cigarette smoker 4 (23.5%)4 (22.2%) 1.0
Tampon use 10 (58.8%)12 (66.7%) 0.73
History of douching5 (29.4%) 6 (33.3%)1.0
Mean duration of menstrual cycle (days 6 standard
Mean duration of menses (days 6 standard deviation)4.560.8 4.460.98 0.78
Current contraceptive method (number, %)
Male condoms 7 (41.2%) 9 (50%)0.74
Female condoms1 (5.9%) 2 (11.1%) 1.0
Tubal ligation1 (5.9%)00.49
Intrauterine device0 2 (11.1%) 0.49
Withdrawal0 2 (11.1%) 0.49
Prior history of vaginitis (number, %)
Candida vaginitis10 (58.8%) 13 (72.2%)0.49
Bacterial vaginosis2 (11.8%) 6 (33.3%)0.23
Prior history of STI (number, %)
Trichomonas0 2 (11.1%)0.49
Chlamydia3 (17.6%)3 (16.7%)1.0
Gonorrhea1 (5.9%)0 0.49
Genital warts1 (5.9%)0 0.49
HSV seropositivity (number, %)
HSV-1 seropositive13 (76.5%) 11 (61.1%) 0.47
HSV-2 seropositive 3 (17.6%)5 (27.8%)0.47
*For one participant in the Acidform gel group, race is unknown.
Safety of Acidform Vaginal Gel
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College of Medicine Institutional Review Board (IRB) and the
NIAID Division of AIDS Prevention Science Review Committee.
All study participants provided written informed consent.
Thirty-five healthy women between the ages of 18 and 50 years
were recruited from the New York metropolitan area between
February 2009 and December 2010. Inclusion criteria included
regular menstrual cycles and willingness to abstain from sex for the
duration of the study. Participants were excluded for pregnancy,
breastfeeding, menopause, HIV infection, reproductive or urinary
tract infection, bacterial vaginosis (BV), intermenstrual bleeding,
abnormal Pap test, use of hormonal contraception during the
study or in the previous two months, and antibiotic use in the week
prior to enrollment.
At screening, participants had urine collected for microscopy,
culture and a pregnancy test. A gynecological examination was
performed for detection of BV (wet preparation with Amsel
clinical criteria), Trichomonas vaginalis (wet preparation), Candida
species (KOH prep), and semen using an antibody immunoassay
that detects p30, a glycoprotein produced by the prostate (Abacus
Diagnostics, West Hills, CA). The pH was measured using a
stainless steel sensor pH probe placed at the lateral vaginal wall,
posterior fornix and cervix (ISFET PH77 Probe Hach Company,
Loveland, CO). CVL was performed by washing the cervix and
posterior fornix with 10 mL of normal saline (pH,5.0). A Pap test
was collected, and the presence of Neisseria gonorrhoeae and Chlamydia
trachomatis infection was determined by nucleic acid amplification
testing of endocervical swabs (Gen-Probe, Inc., San Diego, CA).
Blood was collected for HIV ELISA, syphilis (rapid plasma reagin
test), pregnancy, and serotype specific antibodies for HSV-1 and
HSV-2 (HerpeSelect, Focus Diagnostics, Cypress, CA).
The enrollment visit (Day 0) was completed within 45 days of
screening and 2–6 days after cessation of menstrual bleeding to
allow ample time for dosing prior to anticipated onset of
subsequent menses. Tests for bacterial vaginosis, T. vaginalis,
Candida species, pH and semen detection were repeated. A swab of
the lateral vaginal wall was collected to assess changes in vaginal
bacterial populations. Eligible participants were then randomized
1:1 to receive Acidform or hydroxyethylcellulose (HEC) gel; the
randomization was computer generated by the pharmacist. The
gels differed in color, which precluded randomization in a double-
blind fashion. However, study participants and laboratory
personnel were not informed of the random assignments. The
first dose of Acidform or HEC was administered intravaginally by
a study clinician. Two hours post-insertion, the pH was measured
and CVL was performed. Participants were instructed to apply a
dose twice daily, preferably in the morning and at bedtime and to
not insert gel when study visits were scheduled until after the visit.
The participants were provided with a diary to record usage and
Subsequent study visits were conducted on Days 7 (range 5–9),
14 (range 14–16) and 21 (range 17–21). A speculum exam, wet
mount microscopy, semen test, pH measurement and CVL were
performed at each visit. At every visit, used and unused applicators
were counted. Adverse event (AE) data were collected at each
study visit and graded according to the NIH Division of AIDS
Table for Grading the Severity of Adverse Events . A swab of
the lateral vaginal wall was collected on Day 14.
Acidform is a viscous, off-white gel containing three acidic
compounds (lactic acid, citric acid and potassium bitartrate), a
preservative (benzoic acid), two polymer thickeners (alginic acid
and xantham gum), a humectant (glycerin), sodium hydroxide and
Table 2. Adverse events related to Acidform and HEC placebo gel.
Participant Number Gel ReceivedAdverse Event Number of Episodes Duration
1 Acidform Vaginal Burning3 1 min
4 AcidformVaginal Burning2 5 min, 20 min
5 Acidform Vulvar Itching12 20 min
5 Acidform Vulvar Itching5 5 min
5 AcidformVulvar Erythema1 3 days
9 AcidformAbdominal Cramping1 15 min
11AcidformVulvar Dryness1 2 days
13 Acidform Vulvar Burning3 30 min
13 Acidform Vulvar Burning1 10 min
13AcidformVulvar Itching1 30 min
13AcidformVulvar Erythema1 6 days
13Acidform Vulvar Abrasion1 3 days
16AcidformAbdominal Cramping1 60 min
16 Acidform Vaginal Bleeding1 13 days
17Acidform Abdominal Cramping1 6.5 hours
18Acidform Vaginitis1 4 days
22 AcidformVulvar Itching210 min, 5 min
26 AcidformVulvar Itching1 15 min
3 Placebo Vulvar Itching1 10 min
14PlaceboVaginal Itching2 5 min
Safety of Acidform Vaginal Gel
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water (pH 3.55). The placebo for this study is HEC gel, which is a
water-based formulation that contains HEC, sodium chloride,
sorbic acid and sodium hydroxide (pH 4.4) . Both Acidform
and HEC are applied with a single high-density polyethylene
(HDPE) applicator capable of administering a 5 g (equal to 5 mL)
dose of gel. Evofem, Inc. (formerly Instead Healthcare, LLC San
Diego, CA) provided Acidform lubricant, HEC gel and HDPE
applicators for this study.
CVL specimens were transported to the laboratory on ice and
were clarified by centrifugation at 700 g for 10 minutes at 4uC.
Supernatants were divided into aliquots and stored at 280uC. The
protein concentration (Pierce Micro BCA) and pH (ColorpHast,
pH 2–9, EMD Chemicals) of CVL samples were determined.
Antimicrobial Activity of CVL
The antimicrobial activity against HSV-2 and E. coli in CVL
was measured as previously described . For anti-HSV activity,
Vero cells were infected with ,50–200 pfu of HSV-2(G) mixed 1:1
with each CVL or control buffer and plaques were counted after
48 hours. All samples were tested in duplicate in two independent
experiments. To assess the bactericidal activity, E. coli (ATCC
strain 4382627) was grown overnight to stationary phase and then
3 ml of bacteria (,109cfu/ml) were mixed with 27 ml of CVL or
control genital tract buffer (20 mmol/L potassium phosphate,
60 mmol/L sodium chloride, 0.2 mg/ml albumin, pH 4.5) and
incubated at 37uC for two hours. The mixtures were further
diluted in buffer (to yield 800–1000 colonies on control plates) and
plated on agar enriched with trypticase soy broth. Colonies were
counted using ImageQuant TL v2005 after an overnight
incubation at 37uC. All samples were tested in duplicate and the
percent inhibition was calculated relative to control wells.
Measurement of Immune Mediators
Interleukin (IL)-1a, IL-1b, IL-6, IL-8, interferon (IFN)-c,
IFNa2, IL-1ra (IL-1 receptor antagonist), macrophage inflamma-
tory protein (MIP)-1a, MIP-1b, and regulated upon activation,
normal T-cell expressed and secreted (RANTES) were quantified
in each CVL sample using a multiplex proteome array with beads
from Chemicon International (Billerica, MA), measured using
Luminex100 (Austin, TX) and analyzed using StarStation
(Applied Cytometry Systems, Sacramento, CA). The levels of all
other mediators were determined using commercial ELISA kits:
secretory leukocyte protease inhibitor (SLPI) (R & D Systems),
lactoferrin (Calbiochem, San Diego, CA), human neutrophil
peptides 1–3 (HNP1–3) (HyCult Biotechnology, Uden, The
Netherlands), IgG and IgA (CygnusTechnologies, Southport,
Figure 2. The pH of the vagina and cervix was significantly lower 2 hours after application of Acidform compared to HEC placebo
gel. Box-and-whisker plots showing the pH of the posterior fornix (a), lateral vaginal wall (b), cervix (c) and CVL (d) obtained at screening (Scr), 2
hours (2 h) and at Days 7, 14 and 21 after insertion of Acidform (white) or HEC placebo gel (gray). The line indicates the median values and the circles
are outliers. The asterisks denote a significant difference between the Acidform and HEC placebo group.
Safety of Acidform Vaginal Gel
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NC) and lysozyme (Alpco Diagnostics, Salem, NH). The lower
limit of detection (LLOD) for each assay in pg/ml was: HNP1–3,
156; SLPI, 25; IgG, 100; IgA, 150; lactoferrin, 1000; lysozyme
500; IL-1a, 3.5; IL-1b, 0.4; IL-6, 0.3; IL-8, 0.2; IFN-c, 0.1; IFN-
a2, 24.5; IL-1ra, 2.9; MIP-1a, 3.5; MIP-1b, 4.5; RANTES, 1.0.
Sample values that were below the level of the lowest standard
were set at the midpoint between zero and the LLOD and then
multiplied by the dilution factor. Concentrations that were above
the highest detection concentration were repeated at higher
dilutions or, if insufficient sample was available, were assigned the
value of the highest standard multiplied by the dilution.
Dye Stain Assay (DSA) of Applicators to Assess
Each participant received 30 pre-filled individually packaged
applicators of Acidform or HEC gel and was asked to return used
and unused applicators at each study visit. Drug was dispensed
from the returned unused applicators ex vivo, and all the applicators
were then batched and stained with 0.05% FD&C Blue #1
granular food dye (Prime Ingredients INC, Saddlebrook, NJ) to
detect whether the applicators had been inserted vaginally [22,23].
Applicators inserted by study staff and unused applicators
dispensed ex vivo by staff were included as positive and negative
controls, respectively. Two independent observers scored the
applicators as exposed or unexposed to vaginal mucus, and results
were compared to subjects’ self-reports.
Quantification of Vaginal Microbiota
DNA was extracted from stored vaginal swabs as previously
described, with one extraction control for every 12 swabs. .
Human 18S rRNA gene polymerase chain reaction (PCR) was
performed on all extracted DNA samples to ensure contact with
vaginal mucosa during sampling and the presence of amplifiable
DNA; amplification control PCR targeting a jellyfish aequorin
gene was used to exclude the presence of PCR inhibitors .
Quantitative PCR (qPCR) assays utilizing primers and probes
specific to each bacterium’s 16S rRNA gene were then used to
quantify concentrations of key vaginal bacteria associated with
health (Lactobacillus crispatus and Lactobacillus jensenii) and BV
(Gardnerella vaginalis, Megasphaera-like bacterium (type 1 & type 2),
and Clostridia-like bacterial vaginosis associated bacterium 2
(BVAB2)) . Quantified bacterial levels were expressed as
copies of bacterial DNA per vaginal swab.
Figure 3. The bactericidal activity of genital tract secretions against E. coli was significantly greater 2 hours after application of
Acidform compared to HEC placebo gel. Box-and-whisker plots showing the percent inhibition of E. coli (a) and HSV-2 plaque formation (b) in
CVL samples collected at screening (Scr), 2 hours (2 h) and at Days 7, 14 and 21 after insertion of Acidform (white) or HEC placebo gel (gray). The line
indicates the median values and the circles are outliers. The asterisk denotes a significant difference between the Acidform and HEC placebo group.
Safety of Acidform Vaginal Gel
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Table 3. Summary of concentrations of immune mediators and comparison of changes between the Acidform and HEC placebo groups.
Geometric Mean for
Geometric Mean for
for Day 7
for Day 14
for Day 21
Total Protein (mg/ml)
HNP 1–3 (pg/ml)c
Abbreviations: LLOD, lower limit of detection; Q3, 3rdquartile; n/a, not applicable.
aAs there was no significant interaction between treatment group and time, p-values ,0.05 indicate that immune mediators changed over time, independent of whether participants applied Acidform or HEC placebo gel.
bTwo values were assigned the highest standard multiplied by the dilution.
cThree values were assigned the highest standard multiplied by the dilution.
dOne value was assigned the highest standard multiplied by the dilution.
Safety of Acidform Vaginal Gel
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Study Outcome and Statistical Analysis
The primary objectives of this study were to examine the effect
of Acidform or HEC placebo gel on antimicrobial activity and
mediators of mucosal immunity. Secondary objectives were to
assess the extent of acid buffering by Acidform after vaginal
application and to evaluate a candidate biomarker of adherence.
An exploratory objective was to evaluate changes in vaginal
Demographic data at baseline between the two groups were
compared using chi-squared or Fisher’s exact tests for categorical
variables and using t-tests or Wilcoxon rank-sum tests for
continuous variables. Differences in the rate of women with at
least 1 AE between the two treatment groups were compared using
Fisher’s exact test. Data on immune mediators were log
transformed, where appropriate. Linear mixed models with a
random intercept were used to examine changes in immune
mediators during the study period and the effects of treatment on
changes in immune mediators. For 3 immune mediators, (MIP-1a,
MIP-1b, and RANTES), where more than 20% of immune
mediators were below the limit of detection, immune mediators
were converted to 3-level variables: 0 if immune mediators were
undetectable, 1 if immune mediators were detectable but less than
the 3rdquartile of those with quantifiable levels, and 2 if immune
mediators were at or above the 3rdquartile. Generalized linear
mixed models with a random intercept and a cumulative logit link
function were used to examine changes in these 3 immune
mediators during the study period. An interaction between
treatment and time was included in the model to examine whether
changes in immune mediators varied with treatment. Wilcoxon
rank-sum tests were used to compare differences in pH, antimicro-
bial activity, and vaginal microbiota between the two treatment
groups. Differences in L. crispatus, L. jensenii, G. vaginalis measured at
enrollmentand 14daysaftergelusewerecomparedusing Wilcoxon
signed-rank tests. Bonferroni adjustments were applied for post hoc
comparisons between visits. Spearman’s correlation coefficients
(SCC) were estimated to assess associations between antimicrobial
activity and concentrations of immune mediators from screening
samples. Agreement between two observers for differentiating
applicators that were intravaginally inserted from those not
intravaginally inserted was assessed using the kappa statistic. A
kappa .=0.75 indicated excellent agreement between the two
observers. A kappa between 0.4 and 0.75 indicated fair to good
agreement . All statistical analyses were performed using SAS
Version 9.2 (SAS Inc., Cary, NC, USA). All P values were two-
tailed, with P,0.05 considered as statistically significant results.
A total sample size of 36 (18 subjects in each arm) was selected a
priori to allow 80% power to observe statistically significant
equivalence of means if we assume that Acidform and HEC have
no impact on anti-HSV activity and consider the mean difference
of 1 standard deviation as the equivalence limit.
Fifty-five women were assessed for eligibility, and 35 were
enrolled (Fig. 1). Study product expired prior to enrollment of the
final participant. The majority of exclusions were due to an
abnormal Pap test (n=8) or reproductive tract infection (n=5). All
35 participants completed the trial, which included 17 in the
Acidform and 18 in the placebo group. The mean age of the
enrolled women was 31.2 years and there were no differences in
race, ethnicity, education or other demographic characteristics
between participants in the Acidform compared to the placebo
group (Table 1).
Tolerance of Acidform Gel
There were 51 AEs among 18 women. Forty-two of the AEs,
which occurred among 13 women (11 in the Acidform and 2 in the
HEC group), were possibly or probably related to the study
products (Table 2). Eleven of 17 (65%) women who received
Acidform had at least 1 AE compared with 2 of 18 (11%) who
received HEC placebo gel (p=0.002). The most commonly
reported AEs were genital tract itching and burning. Two subjects
developed vulvar erythema that was temporally linked to Acidform
application. All product related AEs were graded as mild and most
occurred immediately following gel application.
Effects of Acidform on pH and on Antimicrobial Activity
of Genital Secretions
The median pH at the cervix, lateral vaginal wall, posterior fornix
compared to the placebo group (p,0.01) (Fig. 2). There were no
significant differences between the groups in pH at any location at
screening, Day 7, 14 or 21, although there was a trend towards lower
pH at all locations on days 7 and 14 in the Acidform group.
The activity of female genital secretions collected by CVL
against E. coli and HSV was measured ex vivo at each time-point.
The median percent inhibition of E. coli was 72 [interquartile
range (IQR) 36–99] and 61 [42–95] at screening in the Acidform
and HEC groups, respectively. Consistent with a lower pH 2
hours after Acidform gel application, we found that the
bactericidal activity of CVL against E. coli, which are susceptible
to lactic acid, was significantly greater and less variable 2 hours
after initial application of Acidform compared to placebo (98
[96.5–99] for Acidform vs. 47 [33–65] for HEC, p,0.01, Fig. 3).
However, bactericidal activity did not differ significantly between
the drug and placebo group at any of the other time-points, which
is consistent with the absence of any significant differences in pH
at the other time-points (Figs. 2 and 3). The median percent
inhibition of HSV-2 plaque formation was 76 [26–100] and 59
[21.5–10] at screening in the Acidform and HEC arms,
respectively. There were no statistically significant differences in
anti-HSV activity between the Acidform and placebo group after
initial or repeated gel application (Fig. 3).
Effects of Acidform on Inflammatory Mediators and Host
The concentrations of the majority of cytokines, chemokines,
and antimicrobial proteins decreased 7 and 14 days after vaginal
gel use, independent of whether participants applied Acidform or
HEC placebo gel, because there was no significant interaction
between treatment group and time (Table 3). These time-points
corresponded with menstrual cycle days 13–18 and 20–25,
respectively, and likely reflect the physiological nadir [28–30].
There was a statistically significant drug effect observed for
lactoferrin (p=0.04) and IL-1ra (p,0.01) (Table 3). In the
Acidform group, lactoferrin and IL-1ra concentrations in CVL
were significantly lower compared to the HEC group. The
estimated mean drug effects for lactoferrin and IL-1ra were
2603.9 ng/ml and -3845.3 pg/ml, respectively (Table 3). No
significant drug effect was observed for the other cytokines,
chemokines and host protective factors. However, there was a
trend towards a reduction in CVL concentrations of IgG
(p=0.06), IgA (p=0.05) and IL-8 (p=0.08) in women who
applied Acidform gel.
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Correlation of Antimicrobial Activity with Mucosal
Immune Mediators and Vaginal pH
The E. coli bactericidal activity correlated negatively with the pH
of the vaginal wall (r=-0.42, p=0.01) and positively with total
protein (r=0.67, p,0.0001), but not other immune mediators. In
contrast, the anti-HSV activity correlatedmodestly and significantly
with concentrations of IL-1a (r=0.55, p=0.0005), IL-1b (r=0.45,
p=0.007), IL-8 (r=0.52, p=0.001), HNP1–3 (r=0.42, p=0.01),
lysozyme (r=0.36, p=0.03), and IgA (r=0.34, p=0.04), but not
with vaginal pH (r=20.11, p=0.54). These correlations are
consistent with results obtained in other studies [21,31,32].
Effects of Acidform on Vaginal Microbiota
The number of women with L. crispatus or L. jensenii detected at
enrollment did not differ significantly between the two groups and
represented 51% and 57%, respectively. There were no significant
changes in the number of women with detectable L. crispatus or L.
jensenii or in the concentrations of bacteria recovered by PCR in
the Acidform or HEC group following 14 days of twice daily gel
use (Table 4). However, there was a trend towards a decrease in
the concentration of G. vaginalis following repeated application of
Acidform from a median of 1.36106to 3.66104DNA copies/
swab (p=0.083). There were no cases of BV diagnosed by Amsel
criteria throughout the study period. Five women had high levels
of either Megasphaera or BVAB2 detected at the enrollment visit
and 14 days later, suggesting that these women may have had
unrecognized BV not revealed by Amsel clinical criteria.
Measurement of Adherence by Applicator Staining
Subjects were instructed to return used and unused applicators.
The returned applicators were stained within four months along
with positive (applicators inserted by study staff) and negative
(unused applicators that had been dispensed ex vivo) controls. A
total of 1012 of 1050 (96%) polyethylene applicators were
returned, including 935 that participants reported had been
intra-vaginally applied and 77 unopened pre-filled applicators.
Four participants reported missing 1 dose, 2 reported missing 3
doses and 2 reported missing 5 doses of gel.
Both observers correctly identified 29 of the 30 applicators
inserted by study staff as positive and scored 87% and 96%,
respectively, of the returned applicators that were reported to have
been used as positive. The two observers identified 40/60 (66%)
and 58/60 (96%) of the negative controls as negative and 55/77
(71%) and 58/77 (75%), respectively, of the returned unused
applicators as negative. There was good agreement between the
observers, as demonstrated by a kappa of 0.64, 95% confidence
interval (0.58, 0.69). The sensitivity of the DSA for the two
observers was 87% and 84%, respectively and the specificity was
69% and 85%, respectively. The positive predictive value was
95% and 97%, respectively and the negative predictive value was
44% and 43%, respectively.
Acidform was found to be more irritating than HEC placebo
gel, with a greater proportion of mild genital symptoms. A similar
increased rate of mild AEs was observed in an earlier study when
Acidform was compared to K-Y Jelly . Despite the findings of
mild irritation associated with Acidform use, we observed no
increase in pro-inflammatory cytokines or chemokines, although
there was a significant decrease in the concentration of the anti-
inflammatory protein, IL-1ra in CVL obtained from participants
who applied Aciform compared to HEC gel. Whether the mild
irritation and the decrease in IL-1ra observed in this study portend
a risk for mucosal inflammation with more prolonged exposure to
Acidform requires further study.
The median pH of the cervix and vagina 2 hours after a
clinician administered Acidform gel was 3.5–3.7, which was
associated with increased E. coli bactericidal activity, and is low
enough to completely immobilize spermatozoa if the pH is
maintained following coitus. However, no significant differences in
bactericidal activity or pH at the lateral vaginal wall, posterior
fornix or cervix were observed at any other time-point, which
suggests that the acid-buffering effects do not persist. The average
time elapsed between gel dosing and CVL sampling for the Day 7
and 14 study visits was 13 and 16 hours, respectively. These findings
suggest that Acidform may be effective as a topical contraceptive
Table 4. qPCR levels of bacteria from swabs collected at baseline (Day 0) and after 14 days of twice daily application of Acidform
or HEC gel.
Gel group Bacterium
# PCR + +
% PCR + +
Median DNA copies/swab
# PCR + +
% PCR + +
Median DNA copies/
swabaat Day 14
G. vaginalis10 59 1.36106
8 47 3.66104
L. crispatus8 445.96107
L. jensenii9 506.86106
9 50 1.16107
G. vaginalis 1689 9.86105
BVAB24 22 1.26106
aMedian DNA copies/swab among participants with detectable DNA.
bDNA copies/swab for one participant.
Safety of Acidform Vaginal Gel
PLOS ONE | www.plosone.org9 October 2012 | Volume 7 | Issue 10 | e46901
and may prevent vaginal E. coli colonization, which has been
observed after sexual intercourse with and without a condom .
Werecentlydemonstrated aninverse correlationbetween vaginal E.
colicolonization and bactericidal activity of CVL, indicating that the
ex vivo activity may translate to protection against colonization .
However, the transient nature of the responses observed in the
current study suggests that the product would have to be applied
shortly prior to sex to be effective. The need to apply gel shortly
prior to intercourse, the brief duration of activity, and the potential
to develop genital irritation may limit adherence, acceptability and
efficacy of Acidform gel as a topical contraceptive.
Despite potent antiviral activity in a mouse model , there was
no significant increase in anti-HSV activity of CVL following
Acidform gel use. These findings may reflect the pH of CVL (mean
of 4 at 2 hours and .4.5 at all other visits), which was likely not
sufficient to inhibit HSV. We previously demonstrated that HSV
inactivation in vitro is rapid and substantial at pH 3.5, but less
effective at pH of 4.5 . The anti-HSV activity of CVL correlates
with concentrations of several immune mediators including
lactoferrin, IL-8 and IgA; each of these was lower following
Acidform use and this may have also contributed to the absence of
any increase in anti-HSV activity. Notably, lactoferrin, an anti-
bacterial glycoprotein produced by epithelial cells and neutrophils
has been shown to inhibit HSV in vitro . While we observed no
loss in the anti-HSV activity of CVL in this study, the lower levels of
lactoferrin and other immune mediators following Acidform gel
application suggest that more prolonged or frequent exposure could
potentially interfere with mucosal defense. Further studies are
needed to determine the clinical significance of these findings.
The healthy human vaginal microbiome is dominated by
Lactobacillus species at high concentrations, including L. crispatus
and L. jensenii , which maintain acidic vaginal pH by
producing lactic acid. BV occurs when these beneficial vaginal
lactobacilli are replaced by overgrowth of commensal vaginal
anaerobes [36,37]. In a previous Phase I study, there was no
significant increase in hydrogen peroxide-producing lactobacilli
following repeated vaginal application of Acidform gel, as
measured by semiquantitative vaginal cultures . In the present
study, qPCR was utilized to assess the impact of Acidform gel on
absolute quantities of protective lactobacilli and BV-associated
organisms. There was no increase in the concentrations of
beneficial lactobacilli following use of Acidform gel. Notably,
there was a decrease in the amount of G. vaginalis recovered
following Acidform, but not HEC gel application, on Day 14.
Gardnerella vaginalis is almost always present in the vaginas of
women with BV, but is also found in 70% of women without BV
. These findings suggest that the product may provide at least
some colonization resistance against G. vaginalis, although further
studies with sexually active women are needed to determine the
potential role Acidform may play in promoting a healthy
microbiome. Future studies should include more comprehensive
studies of the effects of Acidform on the vaginal microbiota.
This is the first study to apply the DSA to polyethylene
applicators used to insert gel more than once daily. The DSA has
been previously studied with methylcellulose, HEC, Carraguard,
and PRO 2000 gels inserted once daily from low-density
polyethylene (LDPE) applicators, with sensitivity and specificity
of .90% [22,38,39]. The sensitivity of the assay was decreased
when studied with VivaGel gel applied twice daily from
polypropylene applicators [23,39]. The difference may have been
due to gel remaining in the vagina from the previous insertion.
Alternatively, the sensitivity and specificity of the DSA may vary
with the type of applicator used (polyethylene vs. polypropylene).
In a recent study of tenofovir gel applied once daily with
polypropylene applicators , the sensitivity of the DSA was
.90% but the specificity was ,70%, suggesting that staining of
polypropylene applicators may not be as effective as that of
polyethylene. In the current study of twice daily dosing of
Acidform or HEC from polyethylene applicators, the sensitivity of
the DSA for two observers was 87% and 84%, respectively and the
specificity was 69% and 85%, respectively. These results suggest
that the DSA may not be an effective method to assess applicator
use when gel is administered more than once daily, despite the
type of applicator used. Important limitations of the DSA are the
differences in technique used by different groups and the high
degree of interobserver variability [21,23,39], indicating the need
for standardization and training to reduce subjectivity. These
findings also highlight the need for better markers of adherence. A
recent study suggests that direct inspection of polypropylene
applicators under ultraviolet light may provide a reliable
assessment of adherence .
In summary, Acidform gel was associated with a decrease in pH
and an increase in E. coli bactericidal activity 2 hours after gel
application as well as a decrease in the concentration of G. vaginalis
recovered on Day 14, suggesting that it may promote a healthier
vaginal microbial environment. However, twice daily application of
Acidform was associated with mild irritation and lower CVL levels of
several immune mediators compared to HEC placebo gel. Determi-
nation of the clinical significance of these findings requires additional
safety studies of this candidate non-hormonal contraceptive with
sexually active populations and more prolonged product exposure.
We thank Evofem for providing study product and applicators. We also
thank Kathy Anastos and Karen Beckerman for serving on the protocol
safety committee; Grace Chow for clinical operations support; Lydia Soto-
Torres, James Turpin and Barbara North for advice; Erin Diament and
Anna Lee for assisting with subject recruitment and study visits; and Sabah
Kalyoussef, N. Merna Torres and Sylvia Cho for conducting laboratory
Conceived and designed the experiments: MJK MHE BCH DNF.
Performed the experiments: CAC CL. Analyzed the data: MK MHE YL
DNF BCH. Contributed reagents/materials/analysis tools: YL. Wrote the
paper: MJK BCH.
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Safety of Acidform Vaginal Gel
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