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Ciclopirox and Deferiprone Inhibit HIV Gene Expression and Restore Apoptosis

A pair of drugs approved for other uses were shown to inhibit HIV replication and trigger death of HIV-infected cells in laboratory cell cultures, researchers reported in the September 23, 2013, edition of the open-access journal PLoS ONE. These findings suggest a potential new approach for eradicating the virus from cells, but it likely will be years before it can be used as part of a practical cure strategy.

Many different strategies for curing HIV are currently under study, including activating hidden virus in resting T-cells and using gene therapy to protect cells from infection. Most researchers expect that a combination approach will be necessary, such as a "shock and kill" strategy in which dormant HIV genes are activated ("shocked") and the virus or the immune system then kills infected cells.

Hartmut Hanauske-Abel from Rutgers New Jersey Medical School and colleagues tested 2 related drugs, ciclopirox and deferiprone, in laboratory cell cultures; ciclopirox was also evaluated in mice. The report also briefly describes a small human clinical trial of deferiprone, but those findings have not yet been published.

For more than a decade, Hanauske-Abel's team has been studying a novel strategy for blocking HIV replication and its detrimental effects on cells and the immune system. Their latest findings explain how this multi-pronged approach works and how it might one day contribute to viral eradication in people with HIV.

HIV employs several tricks to evade the immune system. Normally cells infected by viruses undergo apoptosis, or "cell suicide." But HIV neutralizes this natural defense mechanism, allowing activated T-cells to continue producing infectious virus and enabling establishment of a reservoir of latent proviral HIV DNA (genetic material) in long-lived resting cells. Existing antiretroviral drugs can block HIV replication but cannot reach this hidden virus, which is why HIV is so difficult to eradicate and why people with HIV must remain on antiretroviral therapy for life.

The researchers tested ciclopirox and deferiprone in H9 human embryonic stem cell cultures infected with HIV obtained from 2 patients, one with advanced and one with moderate immune suppression. Ciclopirox was also evaluated in peripheral blood mononuclear cells (PBMCs). Healthy, uninfected PBMCs were incubated together with PBMCs from one of the HIV patients, allowing the virus to spread. They looked at models of both acute infection (with ciclopirox added at the time of HIV exposure or 12 hours later) and persistent infection (with ciclopirox maintained at a steady level of 30 mcM).

Ciclopirox is an ingredient in various products used to treat fungal infections such as athlete's foot and vaginal candidiasis (yeast infection). Deferiprone (Ferriprox) is an oral chelating agent used to manage iron overload in people with beta thalassemia, a genetic condition that causes reduced hemoglobin production.

Ciclopirox and deferiprone appear to work by interfering with the enzyme deoxyhypusyl hydroxylase (DOHH), which is responsible for hydroxylation of the amino acid hypusine. This process is required for proper functioning of the host cell protein eukaryotic initiation factor 5A (eIF-5A), which plays a role in maintaining HIV infection. Although both drugs act as iron chelators, this is not the key to their antiretroviral activity.

To evaluate safety and cytotoxicity, the researchers administered 1% ciclopirox cream (Batrafen Vaginalcrème) or a saline solution to female mice that were then exposed to herpes simplex virus type 2 (HSV-2) -- as mice cannot normally be infected with HIV -- and viral shedding was measured.

Results

• Both ciclopirox and deferiprone blocked HIV gene expression by DNA fragmentation, preventing production of new virus particles.
• In H9 cell cultures, ciclopirox and deferiprone decreased HIV expression by 40% to 50%.
• The drugs significantly reduced levels of HIV proteins including p24 and Tat, though Vpr levels rose.
• In acutely infected PBMCs, ciclopirox inhibited HIV RNA production and prevented establishment of productive infection.
• In PBMCs with already established infection, adding ciclopirox reduced virus to undetectable levels.
• Viral breakthrough did not occur during 30 days or more of ciclopirox administration.
• Even 12 weeks after stopping the drug, viral replication did not resume.
• Blocking HIV gene expression reduced immune activation and inflammatory responses associated with HIV infection, as indicated by lower levels of interferon-gamma and interleukin-10.
• Ciclopirox and deferiprone halted viral control over apoptosis of HIV-infected cells and restored the normal cell suicide response by disrupting mitochondria, the energy-producing structures within cells -- a mechanism the researchers dubbed "therapeutic reclamation of apoptotic proficiency," or TRAP.
• In H9 cell cultures, the drugs caused selective death of HIV-infected cells, causing them to shrink and eventually die; cell survival decreased by about 2-fold at 24 hours and by about 5-fold at 48 hours after drug administration.
• In PBMCs, 72% of HIV-exposed cells treated with ciclopirox underwent apoptosis.
• Untreated infected PBMCs displayed marked HIV expression early on and minimal apoptosis later, while ciclopirox-treated PBMCs showed minimal HIV expression early but extensive apoptosis later.
• Healthy uninfected cells were minimally affected by the drugs.
• In mice, ciclopirox cream administered for 4 days at concentrations high enough to inhibit HIV did not damage vaginal epithelium or increase susceptibility to HSV-2.

"Two structurally distinct drugs, the antifungal ciclopirox and the chelator deferiprone, inhibit HIV-1 gene expression and activate the intrinsic pathway of apoptosis preferentially in infected cells," the study authors concluded. "In contrast to current antiretrovirals, these medications therefore terminate the infection by HIV-1 of human lymphocytes in culture."

The researchers likened this two-step process to the natural activity of neutralizing antibodies and cytotoxic T-lymphocytes (killer T-cells). They suggested that infected cells undergoing apoptosis might trigger HIV-specific cellular and humoral immune responses, acting as a "vaccineless vaccination."

In their discussion the researchers wrote, "We conclude that the apparent functional sterilization of HIV-infected primary cultures treated with ciclopirox or deferiprone correlates with the preferential apoptotic ablation of HIV-infected cells, and thus the destruction of the proviral reservoir, by each of these drugs."

This study looked at ciclopirox and deferiprone in productively infected cells, or active cells that support viral replication. Although the researchers infer that prolonged post-treatment viral suppression indicates elimination of the HIV reservoir, this has yet to be demonstrated directly in resting cells.

"The real question is how these drugs function in the setting of HIV latency," cure expert Sharon Lewin from Monash University in Melbourne told HIVandHepatitis.com. "This will be key as virus production and apoptotic signals in resting cells are likely to be quite different from productively infected cells."

"Drugs that don't just 'shock' but also 'kill' latently infected cells are what the field is currently searching for," she added. "So these drugs could be promising, but there's plenty more work to do."

Because ciclopirox and deferiprone are already approved for human use, the researchers suggested, they might be able to move through the drug evaluation process more quickly.

Coauthor Deepti Saxena and colleagues have already performed a proof-of-concept Phase 1 clinical trial of deferiprone in people with HIV in South Africa. According to the PLoS ONE article, a 1-week course of deferiprone reduced HIV viral load as much as zidovudine (AZT or Retrovir) and the effect persisted through 7 weeks of post-treatment follow-up.

The next step, they proposed, could be testing the drugs' antiretroviral activity in HIV positive people who are taking them for approved indications, for example people taking deferiprone for beta thalassemia. Up to one-quarter of people with beta thalassemia in resource-limited countries are infected with HIV, they noted. Another example might be testing whether HIV positive women using ciclopirox cream for vaginal candidiasis have less virus in their genital fluid, which could have implications for sexual transmission.

"The team have uncovered a novel therapeutic approach which has promised," said Steven Deeks from the University of California at San Francisco, co-chair of the International AIDS Society's HIV Cure Scientific Working Group. "The approach will unlikely work alone, however, and it will likely take years to determine if this work might one day contribute to a cure."

9/24/13

Reference

HM Hanauske-Abel, D Saxena, PE Palumbo, MB Mathews, et al. Drug-Induced Reactivation of Apoptosis Abrogates HIV-1 Infection. PLoS ONE 8(9):e74414. Septeber 23, 2013.

Other Source

Rutgers New Jersey Medical School.Drug Is Found to Eradicate HIV Permanently from Infected Cells. Press release. September 24, 2013.