CarrageenanMIV150 PC815, A Combination Microbicide

1
Carrageenan/MIV-150 (PC-815), A Combination
Microbicide José A. Fernández-Romero1, B.S.,
Mitchell Thorn1, M.S., Stuart G. Turville1,
Ph.D., Kanani Titchen1, B.S., Kristin Sudol1,
B.S., Jifan Li1, Ph.D., Todd Miller1, B.S.,
Melissa Robbiani1, Ph.D., Robin A. Maguire1,
B.S., Robert W. Buckheit Jr2, Ph.D., Tracy L.
Hartman2, M.S., David M. Phillips1, Ph.D.
Population Council, 1230 York Ave, New York, NY
100211, ImQuest BioSciences Inc., 7340 Executive
Way, Suite R, Frederick, MD 217042
ABSTRACT
The microbicide, Carraguard, which contains
carrageenan as the active ingredient, is
currently in Phase 3 clinical trials. Laboratory
studies indicate that Carraguard or carrageenan
are effective in preventing infection by HIV-1
and herpes simplex type 2 (HSV-2) in vitro, SIV
in monkeys, preventing HPV induced
transformation, and HSV-2 infection and Neisseria
gonorrhoeae infection in mice. Clinical trials
show that the formulation is safe and acceptable
in humans. Carrageenan, as well as several other
sulfated polymers, has been shown to be active in
blocking HIV infection of lymphoma cells by
T-tropic viruses in vitro. However, there is
concern and controversy over whether sulfated
polymers will be as effective in blocking the
M-tropic Clade C viruses of Southern Africa. In
order to render Carraguard more effective against
Clade C viruses, Carraguard was re-formulated to
include the non-nucleoside reverse transcriptase
inhibitor (NNRTI), MIV-150. Previous studies
have demonstrated that MIV-150 is highly
efficacious in blocking infection by various
HIV-1 strains including those resistant to other
NNRTIs and protease inhibitors. In addition,
previous in vitro studies demonstrated that
resistance to MIV-150 develops slowly. Previous
animal and human studies have shown that MIV-150
is non-toxic, and although the compound is well
tolerated, it has poor systemic absorption via
oral intake. Evidence indicates that PC-815 is
more efficacious in blocking Clade C clinical
isolates of HIV-1 in vitro than Carraguard or
MIV-150 alone. On average PC-815 is an order of
magnitude more effective against the viruses
assayed than Carraguard. In addition, MIV-150 can
inactivate free virus and is active against
HIV-2. Results also indicate that seminal fluid
had no effect on the activity of PC-815.
Figure 4 Carraguard, MIV-150, and PC-815
all display antiviral activity in blocking
infection of PBMCs with HIV-2CDC310342.
Formulations prevented infection with an EC50 of
0.001 µM (MIV-150), 10.5 µg/mL (Carraguard) and
1.5 µg/mL (PC-815). The numbers before the
parentheses represent the concentration of
Carraguard and the numbers within the parentheses
represent the concentration of MIV-150 (µM) in
the test solution or in PC-815. Each bar
represents the media of 3 wells with the standard
deviations.
Figure 3 Analysis of combined effects
using the median effect principle (Calcusyn,
Biosoft, Cambridge, UK). The dose-effect
relationships for HIV-1MN were determined for
seven concentrations (3 wells/concentration) of
MIV-150 and Carraguard alone, and for their
combination. The median-effect plot consists of
a plot of x log (D) vs y log (fa/fu). D Dose
of the drugs, fa the fraction affected by the
dose, fu the fraction unaffected, where fu
1-fa. For a given fa value, the required doses
for EC50 are drawn on the x-axis and y-axis,
respectively. If the combination data point for
an fa value falls on the diagonal, an additive
effect is indicated, if it falls on the lower
left, synergism is indicated, and if it falls on
the upper right, antagonism is indicated. An
additive effect (CI around 1) is observed when
MIV-150 and carrageenan are combined (Table 1).
RESULTS
Figure 5 Effect of seminal fluid (SF) on
anti-HIV activity of Carraguard (5a) and MIV-150
(5b). Seminal fluid was found to have no effect
on anti-HIV activity of either Carraguard or
MIV-150. Each bar represents the media of 3
wells with the standard deviations.
Table 1 The CI values are estimated using the
Calcusyn for windows software package (Biosoft,
Cambridge, UK) Dm, the median effect dose or
concentration m, a measurement of the
sigmoidicity of the dose-effect curve r, the
linear correlation coefficient of the
median-effect plot. CI, a quantitative measure
of the degree of drug interaction in terms of
additive effect (CI1), synergism (CIlt1), or
antagonism (CIgt1) is shown for a given endpoint
of the effect measurement.
CONCLUSIONS

• MIV-150 shows a good antiviral and virucidal
  activity against HIV-1MN.
• An additive effect (CI around 1) is observed when
  MIV-150 and carrageenan are combined.
• Data presented here show that Carraguard is
  effective against 9 of 10 Clade C clinical
  isolates tested at a concentration of less than
  160 µg/mL. However, when combined with MIV-150,
  PC-815 is effective against all Clade C clinical
  isolates and is effective at concentrations an
  order of magnitude less than Carraguard alone.
• Both Carraguard and MIV-150 are able to block
  HIV-2CDC310342 replication.
• In testing possible interference of SF by testing
  anti-HIV activity of MIV-150 or carrageenan in
  the presence or absence (control) of human SF, we
  found that SF had no effect.

Figure 1 Effect of MIV-150 on blocking
infection of CEM cells with HIV-1MN. In the
syncytial assay, MIV-150 prevented infection with
an EC50 below 0.0004 µg/mL (or 0.001 µM). Each
bar represents the media of 3 wells with the
standard deviations
ACKNOWLEDGMENTS
Table 2 Antiviral activity against Clade C
clinical isolates of methyl cellulose,
Carraguard, and PC-815. The EC50 value is given
as the concentration of carrageenan in the
formulations of PC-815 and Carraguard in order to
compare their strength. Carraguard inhibited
infection of 9 of the 10 HIV-1 clinical isolates,
and PC-815 inhibited all 10. (CN China, MW
Malawi, TZ Tanzania, ZA South Africa.)
We thank Bo Oberg and Disa Bottinger for sharing
their knowledge of MIV-150. The following
reagents were obtained through the NIH AIDS
Research and Reference Reagent Program, Division
of AIDS, NIAID, NIH CEM-SS from Dr. Peter L.
Nara TZMbl cells fro Dr. John C. Kappes, Dr.
Xiaoyun Wu and Tranzyme, Inc. Human rIL-2 from
Dr. Maurice Gately, Hoffmann-LaRoche Inc.
HIV-1MN, and clinical isolates 98/CN/009,
93/MW/959, 93/MN/960, 98/TZ/013, 98/TZ/017,
97/ZA/003, 97/ZA/009, 97/ZA/012, 93/MW/965, and
98/CN/006 from WHO-UNAIDS and HIV-2CDC 310342
from Dr. Mark Rayfield for Dr. Stephen Wiktor.
Figure 2 Virucidal activity of MIV-150
against HIV-1MN using the microtiter syncytial
assay. MIV-150 inactivates virus at an EC50 of
0.004 µg/mL (or 0.01 µM). Each bar represents the
media of 3 wells with the standard deviations.