Epigenetic drug Gar1041 (auranofin) in combination with antiretroviral therapy

(ART) reduces the proviral DNA reservoir in SIVmac251-

infected macaques

Andrea Savarino

Barriers to cure

• Latently infected T-cells

• Residual viral replication

• Anatomical reservoirs

Main latent virus reservoirs: central memory (TCM) and transitional memory (TTM) CD4+ T-cells

“Classic” strategies for eradication of virus reservoirs

• Prevent any virus spread by intensified ART– Target multiple sites of viral inhibition

• Induce latently infected cells to replicate virus– Histone deacetylase inhibitors

• Eliminate infected cells– Virus cytotopathogenicity– Immune response– Passive therapies– Drugs Van Lint et al., 2004

Hamer, 2004Archin et al., 2005Savarino et al., 2009Margolis, 2010

“Sabotage”

• Aimed at reducing the capacity of the viral reservoir to expand

• Aimed at decreasing the half-life of the latently infected reservoir persisting during antiretroviral therapy (ART)

Our novel strategy for eradication of virus reservoirs

Auranofin (Gar1041)

1) A gold-based compound,2) Orally available,3) Used for years in the treatment of rheumatoid arthritis

(“drug repositioning”),4) Capable of inducing oxidative stress (generation of

intracellular peroxide through a superoxide dismutase-mimicking reaction),

5) Adopted in the experimental treatment of certain cancers due to its pro-differentiating properties.

Auranofin (Gar1041)

Hydrogen peroxide accumulation plays a major role in cellular differentiation!

Dihydrorhodamine 123 → rhodamine 123 (FLUORESCENT)

Hydrogen peroxide ↓

ACH2 ctrl

0.6 μM auranofin

M1

Reactive oxygen species (ROS)induction

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<FITC-A>: CD27

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10.6

Auranofin induces CD27 downregulation

TCM TTM TEM

Mock treated

50nM auranofin

CD27 expression

Working hypothesis:

CD27 down-modulation in central and transitional memory CD4+ T cells may decrease the half-life of the latently infected viral reservoir and contribute to its depletion.

Response of SIVmac251-infected macaques to antiretroviral therapy

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RAL- 100mg BID, oral

PMPA - 20mg/kg, SQ

FTC – 50mg/kg, SQ

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nonhuman primates

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Lewis M. Norelli S., et al., Retrovirology 2010.

Auranofin decreases the central memory T cell compartment in vivo but does not affect total CD4 counts

A

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P < 0.05 P < 0.05

Epigenetic drug Gar1041 in combination with antiretroviral therapy (ART) transiently reduces the proviral DNA reservoir in SIVmac251-infected macaques

M. Lewis1, S. Norelli2, N. Chomont3, S. De Fonseca3, M. Sgarbanti2, M. Collins1, B. Chirullo2, J. Yalley Ogunro1, J. Greenhouse1, A.T. Palamara4, E. Garaci2, A. Savarino5

1Bioqual, Rockville, United States, 2Istituto Superiore di Sanità, Rome, Italy, 3VGTI-Florida, Port St. Lucie, United States, 4Università La Sapienza, Rome, Italy, 5Istituto Superiore di Sanita, Dept. of Infectious, Parasitic and Immune Diseases, Rome, Italy

Background: It was recently hypothesised that the lentiviral reservoir in central memory (Tcm) and transitional memory (Ttm) CD4+ cells could be restricted by new therapies targeting pathways downstream of homeostatic proliferation or pathways associated with ´stem cell-ness´, such as those developed for the treatment of leukemias (Chomont et al. Nat Med 2009). Gar1041 is one such epigenetic drug adopted in the experimental treatment of certain types of leukemia. Methods: SIVmac251-infected primates with viral loads stably suppressed by ART (tenofovir/emtricitabine/raltegravir) were administered, for two months, Gar1041 twice daily (a starting dose of 1.5 g in the first week followed by 2 g in the remaining period). ART was continued during Gar1041 treatment. Proviral DNA was quantitated using a Taqman real-time PCR.Results: The proviral DNA content of PBMCs, which had shown no significant changes during 54 days of treatment with ART alone (P > 0.05), fell below the level of detection (2 copies/106 cells) in all study subjects within one month of Gar1041 treatment [P < 0.05; Bonferroni's test following significant (P = 0.0003) repeated-measures ANOVA]. No significant changes were noticed in a control group treated with ART alone (P = 0.49). The decrease in proviral DNA was associated with a significant (P = 0.0156) decrease in the proportions of the Tcm CD4+ cell subpopulation in peripheral blood. However, both proviral DNA and the proportions of Tcm CD4+ rebound after two months of therapy.Conclusions: The present study furnishes proof of concept that pharmacological strategies may impact on the proviral DNA reservoir. Integration with other experimental approaches will be required to prevent the reconstitution of proviral DNA in peripheral blood from the as yet unidentified reservoir associated with renewal of the phenotype Tcm compartment.

SIVmac251 inhibition in CEMx174cells

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Darunavir (DRV) complexed with HIV-2 protease DRV complexed with SIVmac251 protease

Barreca ML, Norelli S, and Savarino A,unpublished

Kovalevsky et al., 2008

Darunavir as a tool for ART intensification in SIV+ macaques

iART alone

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iART/Gar1041 (average trend over time: P = 0.0207; t-test for regression). iART alone (average trend over time: P = 0.8878; t-test for regression).

Auranofin (Gar1041) significantly decreases proviral DNA in PBMC from SIV+ macaques treated with in ART intensified with ritonavir-boosted

darunavir (iART)

Stimulation of viral replication by histone deacetylase inhibitor, SAHA in SIVmac251-infected monkeys treated with intensified ART alone but not in

monkeys SIVmac251-infected monkeys treated with iART and auranofin

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therapysuspesion

Monkeys:

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intensified ART plus auranofin

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Addition of auranofin to intensified ART delayed re-appearance of viral load following treatment suspension

*

Intensified ART aloneIntensified ART plus auranofin

*P < 0.05; Gehan-Breslow-Wilcoxon test for survival

*P < 0.05; paired Student’s t-test

Upon re-appearance of viral load following therapy suspension, auranofin-treated monkeys, but not intensified ART (iART)-only controls,

acquire the ability to control viral load

iART alone

iART plus Gar1041

peakWhen viral load re-appeared in monkeys that had been treated with auranofin, an immediate peak was observed that was followed by progressive declines in viral RNA

…viral RNA eventually declined to levels lower than those presented before ART initiation

decrease

Drug free remission… eventually?

iART alone

iART plus Gar1041

Upon re-appearance of viral load following therapy suspension, auranofin-treated monkeys, but not intensified ART (iART)-only controls, maintain high CD4 counts

Conclusions

Our findings following ART intensification indicate that a drug-free remission is possible, at least for the follow up period adopted in this study, and represent a milestone towards the identification of future drugs capable of curing HIV/AIDS or inducing a drug-free remission.

Our results also highlight the need for ART intensification when adopting drugs capable of inducing HIV-1 replication.

Oxidative stress inducing agent auranofin impacts the proviral DNA reservoir in SIVmac251-infected macaques under treatment with ART, likely acting by an entirely novel mechanism (CD28 downmodulation in long-lived and proliferation-competent central and transitional memory T cells → generation of short-lived phenotypes)

AcknowledgmentsBioqualMatt CollinsJake Yalley-Ogunro University of Rome “Tor Vergata” Jack Greenhouse Enrico GaraciWendy Wagner

ICGEBIstituto Superiore di Sanità, Rome Marina LusicSandro NorelliBarbara Chirullo University of Rome, La

SapienzaMarco Sgarbanti Rossella SgarbantiAndrea Savarino Anna Teresa Palamara

VTGI, FloridaNicolas ChomontSandrina DaFonsecaRafick-Pierre Sékaly