Zinc carrageenan PC710, A Novel Microbicide

1
Zinc carrageenan (PC-710), A Novel Microbicide
Robin A. Maguire1, Andrea R. Wallace1, José A.
Fernández-Romero1, Jifan Li, Mitchell Thorn1,
Kristin Sudol1, Kanani Titchen1, Robert W.
Buckheit Jr2, Tracy L. Hartman2, David M.
Phillips1 Population Council, 1230 York Ave., New
York NY, 100211, ImQuest BioSciences, Inc., 20358
Seneca Meadows Parkway, Germantown, MD 208762
ABSTRACT
Carraguard, which employs carrageenan as the
active ingredient, is currently being evaluated
in a Phase 3 trial in South Africa. In order to
improve the strength of this microbicide, zinc
(Zn) is incorporated with Carraguard (PC-710).
Because zinc salts would be readily absorbed from
the vagina, we have developed a simple rapid
method to bind zinc to carrageenan. Zn has been
proven safe for human use and has been shown to
have anti-viral activity against HIV-1,
foot-and-mouth virus, human rhinovirus, influenza
A and B, Semliki forest virus, Sindbis virus and
HSV. PC-710 has proven to be a stable and
non-toxic formulation. Stability analysis
includes measuring Zn content, pH, viscosity,
and strength against HIV infection in vitro (data
not shown). Toxicity is determined by using the
MTT assay (Figure 1). To evaluate the strength
of PC-710, the formulation is compared to
Carraguard in vitro for blocking HIV and in vivo
for preventing infection by HSV-2. Because of the
high predominance of the transmission of HIV
subtype Clade C in Sub-Saharan Africa, a panel of
Clade C clinical isolates are evaluated using a
PBMCs assay. Clinical isolates of viruses were
obtained from NIH AIDS Research and Reagent
Program (Table 1). To compare the strength of
PC-710 and Carraguard, mice were challenged with
increasing concentrations of HSV-2 (Figure
2). The longer a microbicide is retained in the
vagina and remains active is an important
property that will allow greater flexibility for
product use. The retention of PC-710 is measured
by evaluation of vaginal lavages (Figure 3).
Duration of formulation activity is determined
using the HSV-2 vaginal infection assay in mice
(Figure 4). Another important property of a
microbicide that would further increase the
flexibility of product use, and one unique to
PC-710, is the ability to prevent infection
post-viral challenge. This is evaluated by
administering the formulation to mice at
increasing time points following HSV-2 vaginal
challenge (Figure 5). There is a very high
incidence of Trichomonas vaginalis in the Phase 3
trial population in South Africa. This pathogen,
a protozoan parasite, causes mucosal lesions in
the urogenital tract, which may increase the risk
of HIV infection. An in vitro dye exclusion assay
was employed to evaluate the effects of PC-710 to
trichomonas (Figure 6).
Figure 4. Duration of activity was assayed by
vaginally applying either Carraguard or PC-710 to
mice and challenging the animals at various times
after application of the product. Carraguard
and PC-710 protected 60 and 100 of mice
respectively at 4 hours after treatment. At 12
hours after treatment, some protection was
observed in Carraguard-treated animals while
almost all mice were protected by PC-710. At 18
hours post treatment, PC-710 appeared to protect
the majority of the mice. (MC inert placebo)
Table 1. Activity of Carraguard and PC-710
against Clade C clinical isolates in blocking
infection of PBMCs. The EC50 value is given as
the concentration of carrageenan in the
formulations of PC-710 and Carraguard in order to
compare their strength. At the highest
concentration tested, 160 µg/mL, methyl cellulose
had no activity. Carraguard inhibited infection
of 9 of the 10 HIV-1 clinical isolates and PC-710
inhibited all 10. In general, the concentration
of PC-710 needed was an order of magnitude less
than the concentration of Carraguard. (MW
Malawi, TZ Tanzania, ZA South Africa)
Figure 5. Microbicides currently in clinical
trials must be applied before sexual intercourse.
One that was effective in blocking infection when
applied after intercourse could afford greater
flexibility for product use. In order to assay
this, mice are challenged with a dose of HSV-2
that infected 90 of the animals. Subsequently,
PC-710 is applied at various time intervals up to
12 hours post-viral challenge. Most animals were
protected if PC-710 were applied up to 12 hours
following viral challenge. This suggests that
PC-710 could possibly protect women if the
product was applied several hours after sexual
intercourse.
Figure 2. Efficacy of PC-710 and Carraguard in
protecting mice from infection by HSV-2,
following vaginal challenge. Carraguard protects
almost all mice at 104 pfu, a 100 infection
dose. However, PC-710 significantly protects at a
higher viral challenge dose of 107, 1,000 times
the 100 infection dose.
RESULTS
Figure 6. Effect of Carraguard and PC-710 on
viability of trichomonads over 48 hours.
Carraguard has no effect on viability, while
PC-710 kills trichomonads.
CONCLUSIONS
O.D. (450 nm)

• PC-710 is not toxic
• An order of magnitude more effective against
  clinical isolates of HIV
• Demonstrates greater strength in protecting mice
  against HSV-2 infection
• Retained in the mouse vagina for up to 18 hours
• Remains active affording significant protection
  up to 18 hours
• Affords protection post-viral challenge
• Kills trichomonads.

Figure 3. Following vaginal administration of
PC-710, vaginal lavages were collected at 1, 8,
18, 24, 30, and 40 hours. The amount of PC-710 in
the mouse vagina was determined by assaying for
zinc. The amount of PC-710 remains constant for
18 hours indicating that that PC-710 remains in
the vagina for an extended period of time.
Concentration mg/mL
Figure 1. Cytotoxicity was assayed in ME-180
cells using the MTT assay. PC-710 did not appear
toxic at any concentration tested. Higher
concentrations were not employed, because
viscosity of the formulation would adversely
affect the assay. Undiluted PC-710 contains 30
mg/mL zinc acetate.