Retention Time of Carraguard in the Human Vagina

1
Retention Time of Carraguard in the Human Vagina
Jifan Li1, Lieyu (Richard) Hu1, Aaron
Teitelbaum1, Livia Wan2, Laura Guichard1, Robin
A. Maguire1, and David M. Phillips1 Population
Council, 1230 York Avenue, New York, NY 100211
Dept. of Obstetrics Gynecology, NYU School of
Medicine, 550 First Ave., New York, NY 100162.
ABSTRACT
Introduction A major concern in the microbicide
field is how long a formulation remains active in
the vagina without being absorbed and/or
inactivated. Ideally a vaginal microbicide should
remain active for an extended period of time
following application. This is imperative, as
infection might not occur at the time of
intercourse but subsequently. In addition,
prolonged activity of a microbicide may render
the product more acceptable by allowing women
more flexibility with time of product use.
Method Fourteen women inserted 4 mL of
Carraguard while being observed by the study
clinician. The clinician thoroughly flushed the
vagina with saline lavages at 1 hr, 2 hrs, 3 hrs
and 24 hrs following application, depending upon
the study regimen the participant was in. Lavage
specimens were frozen and subsequently assayed
for carrageenan content using an established
colorimetric assay. A baseline lavage sample for
each subject was used as a control. Result The
results of chemical analysis indicated that
carrageenan is detectable in all vaginal lavage
samples collected at various time points. In
addition, the amount of carrageenan in lavages
taken at the same time had little to no variation
among subjects. Interestingly, almost the same
amount of carrageenan was recovered from women
that had a second follow-up lavage as well as
lavages collected at 24 hours. In all cases, the
average amount of carrageenan collected was about
1.2 mg, which is equivalent to 1 of the 120 mg
per dose. Conclusion These findings indicate
that the lavage technique that was employed
recovered about 1 of the Carraguard. The results
also indicate that Carraguard is retained in the
human vagina for at least 24 hours. This is not
unexpected as the carrageenan is a very high
molecular weight polymer and has been shown not
to be systemically absorbed. The observation that
occurred in women that had a second follow-up
lavage collected, was that the first saline
lavage does little to change the amount of
Carraguard that is recovered in a subsequent
lavage. This suggests that Carraguard is not
easily flushed from the vagina even though it is
very water soluble. This may be a consequence of
a reservoir of carrageenan between the vagina and
ectocervix or Carraguard binding to the
cervical/vaginal epithelium.
INTRODUCTION
The Population Council has developed a
non-contraceptive microbicide called Carraguard,
that is currently in Phase 3 trials at three
sites in South Africa. The active ingredient in
Carraguard is carrageenan. Carrageenan is an
approved Food and Drug Administration (FDA)
GRAS (generally recognized as safe) compound
that has an extensive history of safety and
stability as a food additive and a cosmetic and
pharmaceutical excipient. Carrageenan belongs to
the sulfated polysaccharide class of compounds,
of which many have been shown to have anti-viral
properties. In vitro, carrageenan has been shown
to be effective in blocking HIV, and to prevent
dendritic cell mediated infection. In animal
systems, carrageenan has been shown to prevent
cell trafficking across the vaginal epithelium,
to prevent vaginal infection by herpes simplex
virus type 2 (HSV-2), to prevent infection by
Neisseria gonorrhoeae, and to prevent human
papillomavirus (HPV) infection in human
xenographs. Because Carraguard is not
systemically absorbed and remains stable in the
vaginal environment, it has been shown that
Carraguard can effectively protect against viral
infection in mice over several hours. This
evidence suggests that Carraguard may protect
women from becoming infected over a longer period
of time. Previously, we have developed a system
to test vaginal retention in a mouse, involving
the use of vaginal lavages at several time points
post gel administration. The lavage specimens
are then tested for the presence of Carraguard.
Vaginal retention studies in mice revealed that
consistent quantities of Carraguard remained
present in the vagina for six hours and small
quantities remained for as long as 18 hours
post-application. The observation that Carraguard
is retained over a long period of time is further
supported by evidence obtained from HSV-2 mouse
studies. These studies demonstrated that a
significant number of mice are protected against
infection by HSV-2 when viral challenge occurred
8 hours following formulation application. In
order to correlate the results obtained in
animals with what might happen in humans, we
designed a small probing study to investigate
vaginal retention in women. The primary objective
of the study is to see how long Carraguard is
detectable in the vagina after initial
application. Vaginal retention is defined as any
amount of detectable carrageenan in the lavage
sample. A further purpose is to investigate the
hypothesis that Carraguard may adhere to vaginal
epithelial cells or be sequestered at the
ectocervix and thus may not flush out with only
one lavage. Therefore, vaginal lavage specimens
were collected from a subject at two succeeding
hourly times. Adherence also may suggest that
Carraguard can offer protection over an extended
period of time.
Figure 1. Amount of carrageenan recovered in the
lavage from 5 women at 1 and 2 hours after
insertion of Carragard (Regimen 1). At 1 hour,
the amount of carrageenan in the lavage is
equivalent to approximately 1 (0.97 to 1.18)
of the amount that was placed in the vagina. At 2
hours, the amount of carrageenan recovered is
still approximately 1 (0.72 to 1.16) even
though subjects had a lavage one hour prior.
There is very little difference in the amount
recovered among the 5 subjects. Thus these data
indicate that although the lavage method used
recovers only a small amount of the total amount
administered in the vagina, it may be
representative of the maximum amount that can be
recovered by the lavage due to a possible
reservoir of Carraguard between the vagina and
ectocervix or Carraguard binding to the
cervical/vaginal epithelium.
n5
n9
n5
n4
METHODS
Subjects were recruited from within the Womens
Comprehensive Clinic of Bellevue Hospital and
Family Planning Division of NYU Medical Center,
New York, NY. The investigator inserted 4 mL of
Carraguard into the subjects vagina via a 5 mL
syringe. Subjects were asked to remain recumbent
for 10 minutes after the gel application. After
lavages were taken, the samples were refrigerated
and brought to the lab within 48 hours. Subjects
were asked to abstain from vaginal intercourse
for 72 hours prior to the start of the each
evaluation and 72 hours following each
evaluation. The method used to detect the
presence of carrageenan, or carrageenan-based
products, has been previously described (Helena
S. Soedjak, Colorimetric determination of
carrageenan and other anionic hydrocolloids with
methylene blue, Anal. Chem., 1994, 66,
4514-4518.) This method is based on the
principle that methylene blue solution turns from
blue to purple immediately after addition of
carrageenan due to the formation of a soluble
metachromatic complex. The absorbance of the
complex at 559 nm is proportional to the
carrageenan concentrations. Briefly, a 0.25 mL
sample is mixed with the same amount of methylene
blue water stock solution (0.41 mM) and then
diluted with 2.5 mL of distilled water, followed
by centrifugation. However, a modification has
been adapted in order to detect the presence of
Carraguard. Absorbance is measured at 540 nm
instead of 559 nm so as to obtain the absorption
maximum. A standard curve for Carraguard
quantification is constructed from a serial
dilution of a known amount of carrageenan assayed
according to the procedure described above. The
actual amount of carrageenan in each vaginal
lavage sample was determined based on the above
standard curve. Subjects were divided into three
treatment groups with different study regimens as
described in the Table 1.
Figure 2. The amount of carrageenan recovered
from the vagina at 1 hr, 2 hrs, 3 hrs and 24 hrs.
The amount recovered is similar regardless of
whether or not a previous lavage was collected.
These data suggest that a reservoir and/or
binding of Carraguard remains in the vagina for
at least a day after application. The 2 hr time
point includes 5 second lavage (Regimen 1) and 4
first lavage (Regimen 2). Error bars represent
the standard deviation. (nnumber of participants)
CONCLUSIONS

• The amount of carrageenan recovered from the
  vagina (0.86 to 1.33 mg) represents about 1 of
  the amount (120 mg) administered in the vagina.
• The amount of carrageenan recovered from the
  vagina does not vary among subjects or with time
  following insertion up to 24 hrs.
• A reservoir and/or binding of Carraguard remains
  in the vagina for at least 24 hrs following
  insertion.