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Prostratin:
a Potential Adjuvant Therapy for HAART Against HIV-1 Reservoirs
by
Stephen J. Brown, M.D., ARA's Director of Clinical Research
HIV reservoirs are a barrier to a cure for HIV infection.
HAART often reduces HIV viral loads below detection levels;
however, replication-competent HIV persists in latently infected
resting CD4+ T-cells (1-3). Latently infected cells are present
very early after acute HIV infection (4-6), are extremely
long-lived, and may persist as a viral reservoir for up to
60 years in patients on HAART (2,3,6,7). Furthermore latently
infected cells are "invisible" to the immune system, and are
not susceptible to available anti-viral drugs. Thus, long-lasting
viral reservoirs could represent a fundamental barrier* to
the eventual elimination of HIV infection. The intentional
activation of latent virus may be a critical strategic requirement
for future HIV therapy.
Interest
in structured treatment interruptions (STI) may partially
be fueled by wishful thinking, as patients grow ever weary
of the dreary prospect of never-ending pill-popping. With
missed doses leading to drug resistance, non-adherence is
the single most important reason for clinical failure. Growing
concerns about long-term toxicities and lack of access to
expensive regimens for millions of impoverished AIDS-sufferers
in developing nations lend a sense of urgency to finding viable
alternatives. Among the many compelling rationales for STI
research, the most basic raison d'être is simply the
need for a brief respite from unrelenting drug regimens and
intolerable side effects.
One
rationale for STIs is that the very success of HAART, ironically,
may be its flaw. Based on the observations of Bruce Walker,
HAART decreases HIV-specific cellular immunity by lowering
the amount of HIV antigen. A treatment interruption, allowing
a controlled amount of virus to be re-introduced to the immune
system might, theoretically, provide sufficient antigen stimulation
to trigger stronger HIV-specific and lympho-proliferative
responses.
The
rationale for latent virus activation strategies
The
rationale (8-10) for a latent virus activation strategy is
that, by stimulating latently infected cells to replicate
and express infectious virus, such cells will die more rapidly
and/or will present viral components (e.g., such as viral
envelope gp120) on their surfaces, making such cells more
detectable by the immune system. They might also be rendered
more susceptible to targeted destruction by immunotoxins (10)
or other potential therapeutic agents. Key to the strategy
is to administer the latent virus activating agent along with
antiretroviral drugs, to prevent the infection of uninfected
cells by the newly expressed virus.
Most
attempts or plans thus far to induce latent virus activation
focus on cytokines, such as IL-2 and tumor necrosis factor,
as the activating agent(s). (10-14) Other potential activating
agents that have been proposed include lipopolysaccharides,
bacterial superantigens and anti-CD3+ antibodies. However,
most if not all of the potential activating agents considered
thus far are macromolecular, highly toxic and/or have other
undesirable attributes. To date, few if any potential "small-molecule"
or more "drug-like" agents or compounds have been considered.
Prostratin
In
1992, National Cancer Institute (NCI) scientists reported
the purification of prostratin as the active constituent of
extracts of the tropical plant, Homalanthus nutans, used in
traditional Samoan herbal medicine. (12) Purified prostratin
demonstrates anti-viral activity against both HIV-1 and HIV-2,
as well as a high in vitro therapeutic index.(16,17) Prostratin
is a 12-deoxyphorbol ester (390 kDa). In contrast to most
other phorbol esters, prostratin is not a tumor-promoter;
indeed, it is one of the most potent known anti-tumor promoters.(18)
This agent was originally targeted for drug development; however,
it was subsequently determined that prostratin is a potent
activator of HIV expression in "latently infected" T-cell
lines (18) which, at the time, halted further development
as a potential anti-HIV therapeutic. With the subsequent advent
of HAART, an overall consensus view toward a general latent
virus activation strategy has evolved substantially within
the field of HIV therapeutics research. Indeed, many international
leaders pursuing HIV and AIDS research and therapy now see
latent virus activation as a critical strategic requirement
for future advances in therapeutics of HIV infection and AIDS.
Based
on our preliminary data shown below, prostratin appears to
be an important potential candidate as an AIDS therapeutic
in protocols in which virus activation is an inherent strategic
element. It is already known that human exposure has occurred,
although we do not know what the actual exposure in dose or
duration was. (16) Preliminary experimental studies (22) in
rodents have indicated that blood levels achievable by oral
administration of prostratin (0.4-1.4 micromolar) are well
in excess of those required for in vitro activation of HIV
expression in latently infected cells.
In
order to further the development of prostration as a potential
therapeutic, AIDS ReSearch Alliance negotiated and eventually
in-licensed prostratin from the Office of Technology Transfer.
Pending completion of the licensing application, ARA proceeded
to form important collaborations with laboratories around
the world in order to look in more detail at the activity
and potential uses of prostratin in HIV-infected individuals.
This has resulted in further elucidation of both the anti-HIV
activity as well as its ability to activate latently infected
cells, and supports the potential utility of prostratin as
an adjunctive therapy for HIV. Several of the researchers
working on prostratin presented abstracts at the recent Keystone
Symposia on AIDS Pathogenesis, and are reproduced here (see
page 4). (17-19)
Prostratin
as Anti-HIV compound
One
of ARA's first collaborations was with the leading antiviral
researcher Eric de Clercq in Belgium, with the goal of exploring
the anti-HIV mechanism of prostratin. Myriam Witvrouw in his
lab confirmed the inhibitory activity of prostratin against
the replication of HIV-1(NL4.3) in MT-4 cells and PBMCs with
an EC50 of 0.04 �g/ml. (17) This compound was not toxic for
the host cells at concentrations up to 125 �g/ml., over 3000
times higher than needed to inhibit virus. Prostratin also
proved effective against a variety of HIV-1 strains [subtype
B (IIIB, NL4.3) and subtype D (NDK)], a clinical HIV-1 isolate
L1, different strains of HIV-2(ROD and EHO) and SIV (MAC251).
Prostratin was equally active against virus strains that were
resistant towards polyanionic and polycationic types of viral
entry inhibitors as well as virus strains selected for resistance
against nucleoside and non-nucleoside reverse transcriptase
inhibitors. Time-of-addition experiments indicated that the
compound had to be present from the time of infection onwards.
However, prostratin did not inhibit the binding of HIV-1 (NL4.3)
to MT-4 cells at concentrations that inhibited the replication
of HIV-1 (NL4.3) in these cells.
Researchers
in Belgium then selected prostratin-resistant HIV-1 (NL4.3)
strains by passaging the virus in the presence of increasing
concentrations of the compound. Genotypic and phenotypic data
of these selected strains revealed mutations commonly seen
in resistance to entry inhibitors such as T-20 and AMD-3100.
This together with the Time of Addition experiments supports
the mechanism of prostratin's action as occurring at the fusion/entry
step of the viral life cycle. However, the structure of the
compound (essentially non-charged) suggests the drug should
work on a cellular target. Further experiments on the effect
of prostratin on expression of receptors and co-receptors
are underway to further clarify the antiviral mechanism of
the compound.
Prostration
as activator of latently infected cells
Of
greatest interest to ARA was the effect of prostratin on latently
infected cells. ARA developed collaborations with several
researchers who have been developing distinct experimental
models of HIV latency.
Initial
work was done in conjunction with Dr. Jerome Zack's lab at
UCLA.(18) Dr. Zack's lab has developed a model of latency
in SCID-HU-THY-THY mice. Whereas previous work by others has
concentrated on the effect of prostratin on T-cell lines,
Dr. Zack and his group assessed the effects of prostratin
on activation of highly purified primary naïve T-cells
that were DR- and CD25- (indicating "quiet" or unactivated
cells). They utilized a flow cytometric assay for cellular
RNA and DNA content to determine the activation state of the
cell population.40 These naïve T-cells do not respond
greatly to stimulation by anti-CD3 or anti-CD28 alone; rather,
they require co-stimulation through both these receptors for
proliferation. Surprisingly, prostratin alone had minimal
effects on these cells; however, it acts as a co-stimulator
in the presence of CD3 stimulation, and also acts as a primary
stimulatory signal if co-stimulation via CD28 is applied.
This
is a very unusual pattern of activity, suggesting that prostratin
might signal T-cells via a novel mechanism. Dr. Zack & colleagues
have performed extensive phenotypic characterization of the
activation response to prostratin. Naïve T-lymphocytes
stimulated with prostratin alone express CD25 (IL-2 receptor)
but not CD69. This is also very different from CD3 stimulation,
which induces CD69 but not CD25 expression. It is also different
from CD28 stimulation, which does not induce the expression
of either marker.
Using
their SCID-hu-Thy model, they next assessed whether prostratin
can activate latent HIV. From infected SCID-hu-Thy mice, they
purified human CD4+/CD8- thymocytes or peripheral blood and
splenocytes, and placed these cells in culture for three days
in the presence of protease inhibitor to prevent virus spread.
The cells were either left unstimulated, or stimulated on
day 0 with CD3/CD28 or with prostratin alone, and viral Gag
protein was measured by ELISA of the supernatant. Co-stimulation
or activation via prostratin alone induced the expression
of latent virus. These data indicate that prostratin can induce
replication of the latent pool of virus without initiating
T-cell replication.
Additional
work on the activating effect of prostratin has been done
in two other models of latency
Additional
experiments were done in the laboratory of Ivan Hirsch at
INSERM in Marseille, France. (20) Dr. Hirsch has developed
an model of latency which successively depletes cell populations
from whole blood until a very pure sample of resting infected
memory CD4+ cells remains. This allows Dr. Hirsch to test
compounds for their ability to activate viral production from
both the short-lived pre-integration state as well as the
long-lived post-integration state. At the recent Keystone
conference Dr. Hirsch shared his result with AIDS ReSearch
Alliance. In his model, prostratin caused activation of both
pre and post integration virus.
In
yet a third model of latency developed by Dr. Jose Alcami
of Spain (21), prostratin also was an effective activator
of latent virus. In Dr. Alcami's model, he takes PBMC's and
transfectes them with a full-length infectious HIV-1 provirus
in which the Luciferase gene has been cloned in the place
of Nef. This virus is fully competent for replication. Results
show that the transfected cells were activated immediately
after treatment with Prostatin at doses ranging from 0.1 to
10 micromolar. Luciferase activity in cell lysates and p24
production in viral supernatants were measured 24 hours after
transfection and showed robust activation.
Further
outside confirmation of the activity of prostratin has come
from the laboratory of Dr. Pomerantz from Jefferson University
in Philadelphia. (19) In data also presented at the recent
Keystone Conference on AIDS Pathogenesis, Dr. Pomerantz took
PBMC's from HIV infected patients and using prostratin, activated
virus from the patient samples.
Future
Development Plans
AIDS
ReSearch Alliance has formulated a scientific development
plan that encompasses future pre-clinical work through the
early phases of clinical (human) trials. It includes all steps
to ensure that patients involved in the human clinical trials
will receive a compound thoroughly tested for safety. We have
developed a very aggressive and rapid timeline for our first
year of pre-clinical work.
Important
additional studies of prostratin need to be done. In the next
phase of proposed studies, ARA and UCLA will further characterize
the effects of prostratin on various human T-cell populations
in vivo. We will determine the effects of prostratin on normal
human T-cell differentiation, and on HIV pathogenesis and
latency in vivo, using UCLA's SCID-hu-Thy mouse model. This
will be done in the presence and absence of HAART, to allow
a more complete characterization of the antiviral properties
of this agent.
Recent
evidence has established several additional potential reservoirs
and facilitators of latent infection. Replication competent
HIV has been isolated from CD8+ cell, and bone marrow epithelial
cells. AIDS ReSearch Alliance is in the process of establishing
collaborations with prominent laboratories working in these
two areas. Lastly, recent work has shown the importance of
a receptor on dendritic cells called DC-SIGN, which captures
virus and delivers it to T-cells that are subsequently infected.
Since prostratin has the effect of down-regulating several
receptors such as CD4 and CKCR-4 the effect of prostratin
on DC-SIGN in of interest. One laboratory that is currently
looking at the importance of DC-SIGN has agreed to look at
the effect of prostratin on this receptor.
We
anticipate that these studies will provide important pre-clinical
data to determine whether prostratin should proceed to clinical
trials as a means of eliminating the reservoir of latent HIV
in T-lymphocytes of infected individuals. In addition, we
hope to generate important information relevant to any similar
agent being contemplated for this type of adjunctive treatment
approach. Thus, this work may help to pave the way for strategies
to totally eliminate HIV from the human body.
In
summer of 2001, AIDS ReSearch Alliance will meet with FDA
officials to determine the required studies necessary to file
an IND for a Phase I study.
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