An Australian research team has found that disulfiram, a long-established and safe drug used primarily as a treatment for alcohol dependency (and well-known under its brand name Antabuse) can ‘wake up’ quiescent HIV-infected ‘reservoir’ cells and thus used as the first stage of a hypothetical cure for HIV.
The researchers found that doses of disulfiram up to four times larger than the licensed dose produced modest but sustained increases in HIV RNA inside reservoir cells; the largest dose also produced a doubling of the amount of HIV RNA outside cells, in the bloodstream.
This did not produce a viral load in any study participant detectable by standard tests, but is an indication that the cells in which HIV hides are being prodded into activity and thereby revealing themselves to the immune system.
Disulfiram, by itself or with other drugs, could be the first stage in a so-called ‘kick and kill’ proposed cure strategy. This involves getting the reservoir cells, whose HIV infection is invisible to the immune system when they are quiescent, to reveal themselves. The hope then is that they can either be picked off by the immune system or, more likely, by a therapeutic vaccine that pre-sensitises the immune system to them, or an antibody-based toxin that specifically seeks them out. See this report for some of the latest research ideas.
Principal Investigator Professor Sharon Lewin from the University of Melbourne commented that “The dosage of disulfiram we used provided more of a ‘tickle’ than a kick to the virus, but this could be enough.”
In the study, Lewin’s team gave 30 people living with HIV, who had CD4 counts over 350 cells/mm3 and had had an undetectable viral load for at least three years on antiretroviral therapy (ART), three different doses of disulfiram over three days. Ten people received 500 milligrams (mg) of the drug, which is the dose licensed to treat alcohol dependency; ten received 1000mg; and ten received 2000mg.
The 30 participants were aged 54 on average (range: 26 to 67) and all but two were men (the other two were transgender women). They had an average CD4 count of 562 cells/mm3 (range: 390 to 1180). They were all virally undetectable on a range of regimens. All were on a backbone of two nucleoside (NRTI) drugs except for one person on solo lamivudine; 22 were on tenofovir/emtricitabine. For the third drug in their regimen, 15 were on non-nucleoside (NNRTI) drugs, ten on protease inhibitors, two on integrase inhibitors, one on a three-class regimen (NRTIs+raltegravir+darunavir) and one on four classes (solo lamivudine, nevirapine, darunavir and raltegravir).
Blood samples to determine levels of HIV RNA in cells and in blood plasma were drawn at a screening appointment, a few days before the first dose of disulfiram, and then immediately before the first dose. This turned out to be significant (see below).
“Blood plasma RNA was measured by two assays”, Sharon Lewin told aidsmap.com. “A standard assay that detects to 20 copies and also a single-copy assay to identify any low level changes.”
Samples were then taken two, eight and 24 hours after the first dose; 24 hours after the second (i.e. just before the third); two, eight and 24 hours after the third; and then seven and 30 days after the third. Drug levels in blood were also measured.
There were ten grade 1 (mild) side effects related to the drug in the 1000mg and 2000mg arms (none in the 500mg arm). These mainly took the form of mild nausea, headache, or light-headedness. There were no grade 2, 3 or 4 side-effects related to the drug.
There was an average 60-90% increase in so-called unspliced HIV RNA in CD4 cells during disulfiram treatment. The largest increase in unspliced RNA (90%) was in the 1000mg dose group.
This indicates activity at an early stage of viral transcription, when new viral genetic material is generated with cells, but this has to be ‘spliced’ i.e. cut up into different functioning units, in order to proceed further towards replication.
Lewin comments: “Unspliced RNA is detected in the majority of people on ART. It’s not really understood what that means, but it likely represents some low-level viral transcription in some cells. In latency reversal studies, we are looking primarily at an increase in such HIV transcription as a first step.”
What is possibly the most intriguing finding of the study, however, is that despite drug levels in the body decaying to undetectable within a week (or two weeks in the 2000mg dose), unspliced RNA levels kept increasing and were at their highest at the last measurement 30 days after the first dose; they were 110% higher (over twice as high) in the 500mg and 2000mg doses and 150% (2.5 times) higher in the 100mg dose. The researchers currently are at a loss to explain the continued accumulation of viral RNA; it could potentially be very exciting if disulfiram has a long-lasting effect.
“We haven’t measured again as the study ended at 30 days,” Lewin commented. “We are currently doing this for our vorinostat patients, who are now over two years post-study.”
In the recipients receiving 2000mg, there was also a doubling in the amount of HIV RNA in blood plasma. This is an indication that the higher dose was pushing the cells to go further down the viral replication cycle and actually produce viral particles (which might or might not be infectious). However a doubling (0.3 log) increase of HIV RNA in blood does not represent a significant enough viral load increase to indicate loss of undetectability in standard viral load tests, and certainly not infectiousness or disease progression.
There was one other unexpected finding in the study. The researchers found that higher levels of unspliced RNA were found in the blood sample collected immediately before the first disulfiram dose than in the two preceding samples. This obviously cannot be caused by the study drug, so what was happening?
The researchers have two hypotheses. Possibly the mild stress of going to a clinic, having blood drawn and having a dose of an experimental drug generated enough stress for hormonal effects to mildly stimulate the HIV-infected immune cells. Alternatively, since all the pre-dose blood samples were collected at about 9am in the morning, a natural diurnal variation in the immune system was to blame. The latter hypothesis is supported by evidence showing that proteins that regulate the daily sleep/wake cycle also have effects on immune activation; you’re more likely to be transcribing viral RNA in the morning.
Sharon Lewin told aidsmap.com that further studies were underway to establish which of these hypotheses was correct.
The effect of the unexpected pre-treatment RNA increase was that the increases in unspliced RNA on disulfiram were only modest (20%-60% increase) at day 30 if they were only compared with the immediate pre-dose levels. However the researchers did an analysis where they took the patients who had the highest levels of disulfiram in their blood and found that in these patients there was a 60% increase in unspliced RNA at all doses and on both day seven and day 30 after the first dose.
The effect seen was an order of magnitude less strong than that created by the HDAC inhibitor drug romidepsin, but greater than that seen with the drug vorinostat, which was the first HDAC inhibitor studied.
The HDAC inhibitor drugs, despite stimulating the production of HIV, did not lead to any decline in the numbers of reservoir cells harbouring the virus. This, it appears, is because they have other effects on the immune system, suppressing the production of the CD8 cells whose job it is to pick off the virus-infected cells that have revealed themselves. Lewin and colleagues hope that disulfiram will not have the same CD8-suppressing effect.
They are now going to combine disulfiram with other drugs and therapeutic vaccines designed to reverse latency (invisibility) in HIV-infected cells, especially as there is evidence of additive effects of disulfiram and HDAC inhibitors.
However what may prove to be disulfiram’s biggest selling point is its low toxicity. Professor Steven Deeks, one of the world’s other premier HIV cure researchers, commented: Most groups are seeking for a powerful weapon to shock the virus out of its hiding place. These approaches may prove to be harmful.
“I see disulfiram as a more gentle way to accomplish this same goal, particularly if we can show it works when given over a long period of time.”