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 and an even greater reduction in the amount of new virus produced by the cells. Pre-treatment with the drug also produced infections with considerably lower viral loads, suggesting this concept might be used in pre-exposure prophylaxis (PrEP) as well as treatment. The combination of 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 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 on its own, 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 nanoATV 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 unprecedented, however, was that adding URMC-099 to the atazanavir produced considerably greater declines in the number of HIV-infected T-lymphocyte cells and macrophages in the spleen and lymph nodes, and especially 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 with HIV.
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 94.1% when the two lowest doses of nanoATV and URMC-099 were used, by 99.6% when the two highest doses were used, and 99.3% 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.
Zhang G et al. The mixed lineage kinase-3 inhibitor URMC-099 improves therapeutic outcomes for long-acting antiretroviral therapy. Nanomedicine 12, 109-122. 2016.