Candidates in this category are based on very large, stable polymers
and three of them – cellulose sulphate (CS), Carraguard and PRO 2000 –
showed sufficient promise to move into large-scale effectiveness trials. These
so-called ‘big, sticky molecules’ coat cell surfaces, thus preventing the
binding of viruses or the entry of microbes into tissue. PRO 2000 is also a sulphonated polymer, whose sulphur-containing
components have a great affinity for the receptor molecules on the surface of
the cells that HIV most readily infects within the genital tract. Thus, it is capable
of latching onto these receptors and blocking them from HIV attachment.
The Microbicide Development Programme (see above) had planned to
conduct an effectiveness trial of dextrin-2-sulfate (Emmelle) – a fourth
candidate in this category. They cancelled the plan, however, because the other
three products in this class, already entering phase III clinical trials, had
demonstrated greater potential for effectiveness in preclinical testing.
None of these entry inhibitors appeared to generate inflammatory
problems and Phase I and II clinical trials showed each to be very well
tolerated. Their molecular size also made them unlikely to be absorbed into the
bloodstream. They had the added advantage of being relatively easy and cheap to
make. Analysis of the failure of cellulose sulphate as a candidate microbicide,
however, revealed an unexpected form of tissue toxicity, as described below.
Cellulose sulfate – big sticky candidate #1
Cellulose sulfate (Ushercell)
looked like a promising candidate when it entered large-scale efficacy trials
in 2004 and 2005. Though named for its Canadian developer, George Usher, it was
more commonly referred to by the name of its active ingredient, a cotton-based
compound that had shown significant activity against HIV and other STIs in vitro.
In ten earlier human-safety studies and two contraceptive trials, cellulose
sulfate (or CS) showed no evidence
of harm. Women in the placebo arms of the CS safety trials experienced
symptoms, laboratory results and incidence of vaginal infections very similar
to those experienced by women in CS gel arms.
On the basis of these data, two Phase III trials to assess the
effectiveness of CS as a microbicide were initiated. One, conducted in Nigeria by FHI,
started enrolment in December 2004. The other, conducted by CONRAD in Benin, South
Africa, Uganda
and India,
started in July 2005.
In January 2007, CONRAD stopped its trial when the Data and Safety
Monitoring Board’s interim analysis showed that more HIV infections were
occurring among women using CS than among those using the placebo (25 versus 16, out of a total of 1425 women enrolled). FHI’s study in Nigeria
was halted at the same time as a precautionary measure, even though interim data from that study did
not suggest an increased risk of HIV infection.
Closure of the CS trials was a bigger shock to the microbicide
community than the closure of the C31G trial. As a surfactant, C31G was
automatically suspected of being cytotoxic (killing cells), so that the trial’s
failure was a disappointment, but not a surprise. No such expectation, however,
existed with regard to CS.
In its interim analysis, the CONRAD trial’s difference in
seroconversion numbers fell below the level of statistical significance,
although it suggested a trend toward harm. Stopping the trials was a wise move given
that further analysis eventually showed a total of 60 women in the CS arm (8.4%
of participants receiving CS) seroconverted, in contrast to 27 women receiving
the placebo gel (3.8% of all participants in that arm). This difference still
did not quite reach statistical significance, but certainly underscored the
possibility of a trend towards increased harm.
Hypotheses to explain the disparity include the possibility that CS
induced inflammatory reactions, localised immune dysfunction and/or that
disruption of the normal vaginal flora could have occurred with frequent CS
use. At the Microbicides 2008 Conference, investigators reported that, at the trial’s
Durban site, they observed a threefold greater-than-average risk of infection
among trial participants diagnosed with chlamydia, bacterial vaginosis or
trichomoniasis, and a greater than fourfold increase in HIV-infection risk
among participants who reported more than one new partner between the quarterly
trial visits.10
The link between CS use and the apparently increased vulnerability to
HIV was finally illuminated by Pedro Mesquita of Albert Einstein College of
Medicine in New York.
Novel imaging techniques11 allowed
Mesquita’s team to see that CS was not cytotoxic (did not kill cells), but did
cause loss of tissue integrity by destroying proteins that bind cells together.
This allowed HIV to leak into the underlying tissues. The study found that this
unexpected toxicity was also observed to a greater degree with nonoxynol-9, and
to a very limited degree with the microbicide PRO 2000, though in this case the tissue damage appeared
reversible. This effect causes neither macroscopic tissue damage that can be
seen by colposcopy, nor a significant inflammatory immune response (though
repeated application can cause inflammation). Thus, it would have been unobservable
by the standard assays for microbicide toxicity.
Carraguard - big sticky candidate #2
After the evidence that C31G and cellulose sulphate might not only be
ineffective, but actually increase risk, the results from Carraguard, the next
large Phase III study, came as a relative relief to researchers and advocates
alike. Although Carraguard did not protect women against HIV
infection, it at least appeared to be safe. There was even a hint of efficacy
at some trial sites.
Carraguard is manufactured
from carrageenan, a seaweed derivative that is well known, cheap to produce and
already used for a number of purposes, including as a commercial food additive.
The Population Council (see above) became interested in it as a potential
microbicide in the mid-1990s when data began to emerge12
indicating that it effectively prevented mice from catching the genital herpes
virus (HSV-2), an infection that is both highly contagious and lethal for mice.
In vitro, it also blocked cells from
becoming HIV infected.13
Although no animal study was done specifically demonstrating that it prevented
HIV-like infection (such as infection with SIV, SHIV or FIV, for example), it
was shown to protect mice from some other STIs.14 Numerous
safety studies had come up with a clean bill of health regarding its safety for
vaginal use.
In 1994, Carraguard became
the first novel microbicide candidate to enter clinical trials (given that
nonoxynol-9 was designed as a contraceptive, not a microbicide). A decade
later, the Populations Council started enrolment in its Carraguard Phase III trial. Completed in 2008, this was the first
large-scale efficacy trial of a new microbicide candidate to reach completion -
a notable achievement given that 6202 women participated in three sites in the South Africa
trial and each was followed for up to two years.
In total, 134 new infections occurred among women in the Carraguard arm of the trial and 151 in a
placebo arm. The difference between the two groups was not statistically
significant, nor was there any significant difference between the arms in the
incidence of side-effects, such as vaginal irritation, loss to follow-up or
withdrawal rates (participants choosing to leave the trial).
A crucial question in the Carraguard
trial was the degree of adherence to gel use. Overall, participants claimed
that they used the gel during 94% of their sex acts. But the researchers
gathered additional adherence data by applying a dye to the returned
applicators to detect the presence of vaginal mucous on them. This test showed
that only 61% of the returned applicators had actually been used. This test was
not included in the original study protocol but was added when it became
apparent that adherence was a serious concern.
By combining such objective measurements with self-reported data, the
researchers determined that the Carraguard
gel had actually been used by women in the Carraguard
arm of the trial during only 43% of sex acts. This effectively removed the
statistical power of the trial to determine the low-to-moderate efficacy. With
good adherence, the trial was powered to show effectiveness at the rate of 30%
or greater. But only a product with substantially greater efficacy than that
could have shown any measureable result when used less than half the time.
Low adherence was not the only reason the trial ended up having little
power to generate a statistically meaningful result. Although the drop-out rate
was only 13%, an additional 9% were lost to pregnancy, and the pregnant women
did not return to the study after giving birth. Circumcision rates in men were
also very different at the three trial sites, with 97% of men in the Cape Town area circumcised, 54% in Pretoria,
but only 24% in Durban.
At the Durban
site, more seroconversions actually occurred amongst the participants using Carraguard than those using the placebo
(48, or 3.3% of participants, versus 42, or 2.8%). In the other two sites, more
seroconversions occurred among the placebo users. These data are not
statistically significant, but may suggest a hint of efficacy for Carraguard when it comes to populations
with lower HIV prevalence than the extremely high rate seen at Durban and/or higher rates of male
circumcision.
Carraguard was popular:
women said it improved sex and they liked the feel. Other gel studies have
reported similar findings. In fact, participants at one site in the prematurely
terminated cellulose sulphate trial refused to hand their gel back until they
were given supplies of placebo.
Data on adherence in this trial must be considered in context.
Reinforcement of the importance of regular microbicide use took place only at
quarterly study visits and there was no supportive ongoing education in the
community. One major challenge for the field now is devising innovative ways of
addressing this issue. With closer adherence monitoring, it should be possible
to do a ‘per protocol’ analysis that looks at product effectiveness only amongst
actual product users, rather than the traditional ‘intention-to-treat’ analysis
that factors in all trial participants equally, regardless of whether or not
they completed the trial, were adherent to the protocol, etc. Researcher Elof
Johansson commented that: “using an intention-to-treat analysis in people who
do not feel sick is very difficult”, and noted that trials of blood-pressure
drugs had run into the same problems in terms of inability to show
statistically significant results.
PRO 2000 - big, sticky candidate #3
As noted above, PRO 2000 is a
compound containing sulphonated polymers, large molecules whose
sulphur-containing components are drawn to the receptor molecules on the cell surfaces
that HIV tends to target. By latching onto these receptors before HIV exposure
occurs, these molecules can block HIV attachment. Although similar to cellulose
sulfate, PRO 2000 has been shown to have
less toxicity.
PRO 2000 underwent two
large-scale efficacy trials, the first of which provided a hopeful (although
inconclusive) view of its potential and the second of which demonstrated that
it was not effective against HIV.
The first of these, called HPTN 035, was started by the HIV Prevention
Trials Network (HPTN) in January 2005 and completed in 2009 under the auspices
of the Microbicide Trials Network (MTN, see above). The MTN was spun off from
the HPTN in 2006 to serve as the NIH’s collaborative clinical-trials network, focused
specifically on preventing the sexual transmission of HIV.
In HPTN 035, participants were randomly assigned to one of four study
groups: BufferGel, PRO 2000 gel, placebo gel, and no gel.
Women assigned to the three gel groups applied gel up to one hour before sexual
intercourse using pre-filled applicators. Because all three gels and
applicators look the same, neither researchers nor participants knew who has
been assigned to use which gel for the duration of the study (although the no-gel,
or ‘condom-only’ arm was necessarily obvious).
The
no-gel arm was designed to test whether the neutral placebo gel was actually
neutral. In 2000, the results of the COL-1492 study on nonoxynol-9 caused
speculation as to whether Replens (a
vaginal moisturizer used as the comparator, or placebo, gel in that study)
might have reduced HIV risk slightly among the women in the control arm of the
study, thereby skewing the results. This led to the development of hydroxyethyl cellulose
(HEC), a gel that is now used as the ‘universal placebo’ in microbicide trials. The
absence of significant differences between the experiences of participants in
the placebo arm of HPTN 035 and those in the no-gel arm confirmed that HEC was
neutral and had no discernable effect on HIV risk.
HPTN 035 was not designed to compare the effectiveness of BufferGel to that of PRO 2000, but simply to see if their joint efficacy was more than 43.6%.
Between 2005 and 2007, the trial enrolled a total of 3100 participants at seven
sites in Malawi, South Africa, Zambia,
Zimbabwe, and the US.
Each study participant was followed for at least one year.
As noted above, this trial showed clearly that BufferGel was ineffective. Its findings regarding PRO 2000, however, were ambiguous. Women in the PRO 2000 arm had 30% fewer HIV infections
than those in the placebo or no-gel arms. While very encouraging, this result
did not reach the level of statistical significance. Given the size of the
trial and the seroconversion rate, there was a one-in-ten probability that the 30% reduction
in HIV infections among PRO 2000, versus
those who used a placebo, was due to chance (p = 0.1). In the case of women who
used no gel, this probability was one in 17 (p = 0.06). Scientifically, results
are not regarded as ‘significant’ unless the chance that they are wrong is less
than one in 20 (p = 0.05).
The
second PRO 2000 effectiveness trial
was MDP 301, a study undertaken by the Microbicides Development
Programme (MDP) of the UK Medical Research Council to compare two doses of PRO 2000 (2% and 0.5%) to placebo. With
three times the number of participants (9673 enrolled over six sites in South Africa, Zambia,
Tanzania and Uganda),
researchers and advocates fully expected that MDP 301 would be able to demonstrate PRO 2000’s efficacy conclusively if it detected a similar reduction
in the HIV-infection rate.
The hopefulness around MDP 301 was slightly dampened by the trial’s
January 2008 closure of its 2% trial arm. When launched in 2005, MDP 301 set
out to test two concentrations of PRO
2000, a 2% gel and a 0.5% gel. In January 2008, MDP announced that the 2%
trial arm was being closed due to futility (meaning that the study would not be
able to demonstrate whether or not the candidate compound had an effect). While
PRO 2000 appeared less toxic than
some other candidates, any protective effect that the 2% gel had may have been
offset by a local irritant effect caused by the higher dose.
The 0.5% gel arm continued, however, and claimed adherence to gel use
was high. The rate of reported condom use varied widely among sites, ranging
from 80% in South Africa to
17% in Zambia.
In-depth examination of condom-use rates in one South African site, however,
revealed that true
level of consistent condom use at that site was about 25%, half the rate
indicated by self-reporting.
The results of MDP 301, announced in December 2009, showed no evidence
that PRO 2000 reduced HIV risk
in trial participants. The factors potentially contributing to this surprising
result are still under examination. In addition to concern about adherence, anal sex may have
been another confounding factor. At their monthly clinic visits, only 1% of
women reported having had anal sex in the last month. But anal sex tends to be
under-reported, and condom use tends to be less common among heterosexuals when
they are having anal sex than it is during vaginal sex. Given that anal
intercourse is a highly efficient method of transmitting HIV - and that
participants were told that the gel was for vaginal use, not anal use -
unprotected anal sex may have given rise to a number of infections.
Regardless of cause, the news elicited field-wide disappointment, as
well as unanimous agreement that MDP 301 was a sound, well-conducted trial that
produced valuable information. The trial also illustrates perfectly why multiple clinical trials are needed to evaluate
novel prevention strategies. If MDP 301 had not already been underway when the HPTN
035 results came out, it would have been necessary to wait for such a confirmatory
trial to be mounted and completed. Clearly, proceeding on the assumption (based
on the HPTN 035 data) that PRO 2000
worked would have been dangerous, given PRO
2000’s now-documented lack of effectiveness.
The MDP investment in behavioural and social-science research as a
part of MDP 301 was also unprecedented in the field. Data from the trial’s
broad and sophisticated array of behavioural sub-studies are expected to provide
important insights into the ongoing challenges of acceptability and adherence
that (as illustrated above) are key to researchers’ ability to access product
effectiveness accurately.
MDP 301 is the last large
efficacy study of non-specific microbicide products for the time being. Although several non-ARV-based candidates are
being investigated by preclinical researchers - and some may prove promising
enough to advance to clinical trial eventually - virtually all of the
candidates now moving though the clinical-trial pipeline are ARV-based.