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Combination nanotherapy drastically reduces the amount of HIV inside cells in mice
Gus Cairns, 2016-02-05 08:40:00
A combination of fine-particle formulations of the drugs
atazanavir and ritonavir, plus an immune-dampener drug designed to induce cells
to keep hold of the drugs for longer, has produced a 100-fold decline in the
number of CD4 cells in the lymph nodes of mice infected with HIV. Pre-treatment
with the drug also produced infections with considerably lower viral loads,
suggesting this concept might be used in PrEP as well as treatment. The most interesting
part of this experiment is that, for the first time, a combination of two
small-molecule drugs prevented, to a large extent, the uptake of HIV into
T-lymphocyte cells and thus has considerable potential to either prevent or
suppress long-term infection.
The discovery of the potential of the drug combination was
made almost serendipitously. The experimental immune-dampening drug, URMC-099 was
being investigated as a possible suppressor of brain and nervous system
inflammation in HIV infection. It works by suppressing kinase B, a family of
proteins that act as off-switches for inflammatory genes (aspirin is also a
kinase B suppressor).
The same team was also investigating a novel drug delivery
method for the protease inhibitor drug atazanavir, boosted by ritonavir. Both
of these drugs were formulated as nanoparticles – minute particles of drug small enough to get inside cells and even cellular components.
These nanoparticles were then hitched to the common micronutrient folic acid, a
molecule also used by immune system cells, with the result that, arriving at
lymph nodes, the drug was proactively drawn into T-cells.
Adding URMC-099 to the nanoformulated boosted atazanavir (nanoATV)
induced the cells in the mice - who were genetically adapted to have human-like
immune systems – to maintain intracellular drug levels for longer, up to 10
days on a single dose, especially at sites in the cells where HIV viral
particles are constructed.
This resulted firstly in considerable declines in the
number of CD4 cells infected with HIV. In untreated mice, the CD4 count, expressed
as the percentage of T-lymphocytes that were CD4 cells, declined from 75% to
40% - as seen in most courses of HIV infection. By using the nanoATV, the CD4
percentage was maintained at 60% - within the range of uninfected mice.
However the nanoATV managed this feat by itself. The
URMC-099, while having no effect by itself, produced a considerable reduction above
and beyond the effect of the atazanavir when dosed alongside nanoATV. The viral load in untreated mice was
about 100,000 copies/ml; in mice treated just with the boosted atazanavir it
was 1600 copies/ml and with URMC-099 it was 284 copies/ml. The effect persisted
somewhat after dose withdrawal. The mice were first infected and left untreated
for a nine-week period; then nanoparticle-boosted atazanavir was injected once
a week and URMC-099 given daily, both for three weeks. CD4 counts remained
stable when measured a week later.
What was more remarkable and unprecedented, however, was
that adding URMC-099 to the atazanavir produced considerably greater declines
in the number of HIV-infected cells in general, as measured by actual viral
production by infected cells.
Untreated infected mice had 40 cells per 1000 in their
spleen actively producing HIV and 50.3 per 1000 in their lymph nodes. With nanoATV
alone, that was reduced to 4.0 and 10.8 for 1000 in spleen and lymph nodes respectively,
and to less than 0.1 and less than 2.3 respectively when URMC-099 was added.
The researchers also conducted a series of experiments in
which they infected mice with HIV after
nanoATV and URMC-099 treatment, i.e. as a form of PrEP or therapeutic
modulation. They first dosed mice with ordinary atazanavir alone, nanoATV alone
or this plus URMC-099. Then, after withdrawal of the drugs, they infected mice
The drugs, not actually present at the time of HIV infection,
did not prevent infection: but they reduced the number of cells with HIV DNA
integrated into them after infection, from 11,471 copies of DNA per 1000 cells
with ordinary atazanavir.(no better than no treatment), to 74 copies/1000 cells
with nanoATV alone and 33/1000 with URMC-099. Non-integrated viral RNA in cells
declined from 1100 copies 1000 per cells with ordinary atazanavir to 100 copies/1000cells
on nanoATV alone to 19 copies/1000 cells with added URMC-099.
Pre-infection treatment with the two drugs also produced
profound declines in the number of cells producing new viruses. NanoATV alone
produced 90.8, 94.2 and 95.7% declines in active viral expression by cells
depending on dose (1, 10 and 100 micromols of nanoATV respectively). Adding in
URMC-099 produced further declines in HIV expression by 63.8, 81.7, 97.8 and
91.3% respectively for four doses of URMC-099 (0.1, 1.0. 10 and 100 nanogram/millilitre
respectively). This means that viral production post-infection was reduced by
94.13% when the two lowest doses of nan-ATV and URMC-099 were used, and by
99.63% when the two highest doses were used or 99.29% when the two next-highest
doses were used.
How did the two drugs achieve this effect? As we said above, joining
the nanoformulated atazanavir to a molecule of folic acid enabled efficient
penetration of the drug into cells; the nanoparticles were small enough even to
enter into the organelles (cellular components) where cellular and viral
proteins are disassembled, copied and re-assembled, but were not eliminated quickly
as drug molecules alone would be. Adding in URMC-099 stopped the cells from
pressing their own auto-destruct button when stimulated by the inflammatory
proteins of HIV, so they lived longer. The drugs together suppress specific
cellular proteins that work as activators of immune-response genes that, in HIV
infection, make viral replication possible.
Measurements of the amount of HIV reverse transcriptase (RT)
enzyme in cells showed that with the two higher doses of URMC-099, RT activity
was still suppressed five days after the last dose of drug.
“This is the first combinatorial formulation known to date
to reach the lymph nodes of infected animals and reduce, by more than 90%, the
numbers of infectious viral particles,” the researchers say. They aim to
improve the targeting of nanoATV by attaching it to HIV envelope protein
components, and to conduct more experiments in human cells.