1. The HDAC inhibitor romidepsin is safe and effectively reverses HIV-1 latency in vivo as measured by standard clinical assays
Thank the organizers
Clinical with romidepsin to activate viral transcription and release from latently infected cells.
Ole Schmeltz Søgaard, MD PhD Department of Infectious Diseases, Aarhus University Hospital, Denmark

2. The “kick and kill” approach to cure HIV
Reactivate latent viral expression
HDAC inhibitors, PKC activators, BET bromodomain inhibitors, etc.
Kick
cART
Kill
Put the trial in an hiv cure context
Follows the kick and kill strategy
Deeks S. Nature 2012

3. HDAC inhibitors
Induce HIV mRNA tran-scription in latently infected resting CD4 cells in vivo
Considerable variability in potency between HDACis
Ultimately, HDACis should release viral particles in to plasma to allow for immune-mediated killing of infected cells
Acetylation of the lysine residues on the histone tails relaxes the chromatin structure and allows for gene expression, whereas deacetylation of the histone tails leads to condensation of the chromatin structure and silencing of gene expression.
In accordance with this mechanism, HDACis have been shown to induce HIV transcription in latently infected cells
Many HDACis, considerable difference in their effect on HIV gene expression
Archin Nature 2012; Lewin CROI 2013; Rasmussen HVIT 2013

4. Romidepsin Licensed in the US for treatment of PTCL and CTCL
Increase extracellular RNA release from memory and resting CD4+ T cells in patients on cART ex vivo
EC50 for RMD approx. 4.5 nM compared with 3,950 nM for SAHA in a primary T cell model
RMD (IV) T½~4 hrs
However, ex vivo viral outgrowth data question the ability of HDACi (incl. RMD) to reverse latency in vivo
In the present study, we have used the HDACi, romidepsin
Much credit for the advancement of this drug in HIV latency should be given to Romas Geleziunas, who together with his team at Gilead and external collaborators have done a tremendous amount of in vitro/ex vivo work with RMD
Wei PLoS Path 2014, Bullen Nature Med 2014

5. Trial design Non-randomized interventional trial
Romidepsin (5 mg/m2) IV day 0, 7, and 14
Primary endpoints: Safety as well as activation of HIV-1 from latency as determined by plasma HIV-1 RNA and cell-associated unspliced HIV-1 RNA in total CD4+ cells
Secondary endpoints: H3 acetylation, HIV-1 DNA, T cell activation
HIV-1 patients on cART
Age >18 years
CD4 >500 cells/µL
VL <50 copies/mL for >1 year
No HBV/HCV infection
No significant cardiac disease
- Emphasize 3 doses of romidepsin at a dosing which equals 1/3 the standard dosing used in cancer treatment

6. Patient characteristics
N=6, 5 males and 1 female (caucasian)
Median
CD4+ cell count: 760 (range )
age: 54 years (range 37-60)
duration of cART: 9.5 years (range )
None started cART during PHI
ART regimens: PI+2NRTIs (n=3), NNRTI+2NRTIs (n=2), INT+2NRTIs (n=1)
- Just read

7. Self-reported AEs
40 adverse events (AE) were registered ̶ 36 AEs were considered to be related to the study drug
Most AEs were mild (grade 1, n=38) and resolved spontaneously within a few days
Two AEs were grade 2 (fatigue and fever in one individual after the 2nd infusion)
The number of AEs reported by each study participant during follow-up ranged from 1 to 13 (up to day 21)
The most common AEs were abdominal symptoms such as nausea (n=12), borborygmia (n=4), abdominal pain (n=2), diarrhea (n=1), and vomiting (n=1) and fatigue (n=5).
- first: the self-reported safety data

8. Biochemistry
Romidepsin and other HDACis are known to affect the white blood cell compartment, often resulting in thrombocytopenia and neutropenia
Initial minor reductions in most leucocyte compartments most pronounced in neutrophils.
7 days following the last romidepsin infusion the levels of the various populations of leucocytes had more or less returned to pre-therapy levels

9. Lymphocyte histone H3 acetylation
The degree of histone acetylation is a direct measure of the pharmacodynamic effect of an HDACi on cells. Thus, to closely describe the chain of events leading from RMD administration to activation of HIV-1 transcription, we determined lymphocyte histone H3 acetylation at each study visit.
This graft shows the mean H3 acetylation for the 6 patients as a group
Emphasize dosing and sampling schedule
- The lasting effect of RMD compared to other HDACi may be related to its unique intracellular pharmacology and interaction with HDAC enzymes. RMD acts as an intracellular prodrug that undergoes reduction of its intramolecular disulfide bond upon entering cells. The released free sulfhydryl groups tightly interact with the Zn2+ ion in the active site of various target HDAC isoforms, a mechanism of inhibition that does not apply to VOR or PNB.

10. Cell-associated unspliced HIV-1 RNA
We determined intracellular HIV RNA levels in total CD4+ cells by ddPCR as a measure of HIV transcriptional activity
Top graft shows the actual changes in HIV RNA for each individual and the bottom graft shows fold-changes from pre-therapy level for the whole group
We observed significant increases in CA US HIV RNA in all 6 patients
Highest levels observed after the 2nd and 3rd dosing (mean 3.5 fold increase)

11. Plasma HIV-1 RNA
Plasma viral RNA was determined using the standard COBAS taqman assay
”Take 1 infusion at a time”
5 of 6 patients reached quantifiable plasma RNA levels during the 3 infusions
Using the same qualitative transcription mediated assay that blood bank use for screening of donor blood for early signs of HIV infections, we noted that 50% of the pre-infusion samples were positive for plasma HIV RNA, whereas more than 90% were positive 3 days post-infusion
Collectively, these data may be represent convincing evidence of a reactivating agent that ”kicks” the latently infected cells out of latency
Viral load: COBAS® TaqMan® HIV-1 Test, v2.0
TMA: Qualitative NAT screening system (PROCLEIX ULTRIO Plus, Genprobe)

12. T cell subsets, activation and PD-1 expr.
- RMD induced immediately changes in the composition of the peripheral CD4+ and CD8+ compartment
Shift towards larger proportions of naive cells and fever effector and central memory cells. However, changes had almost reversed by day 10
CD4 T cell activation as measured by CD69 increased primarily in the effector memory and term diff subsets which is consistant with ex vivo findings
Expression of the exhaustion marker PD1 decreased initially in both CD4+ and CD8+
Collectively, these changes may impact measurements of the HIV reservoir and the chance of clearing reactivated cells

13. Total HIV-1 DNA in CD4+ T cells
Individual HIV DNA levels in total CD4+ cells measured by qPCR shown left, fold changes from pre-therapy level shown to the right
One person had an 80% decrease in HIV DNA but on a group level there was no change from baseline to day 21

14. Conclusions
RMD safely activated latently infected cells and induced transient quantifiable plasma viremia
Phenotypic changes occurred in the T cell compartment during RMD treatment
The HIV-1 reservoir was not significantly reduced by RMD (as measured by HIV-1 DNA)
A clinical trial combining a therapeutic HIV vaccine (Vacc-4x) and RMD is ongoing

15. Acknowledgments THE STUDY PARTICIPANTS
Department of Infectious Diseases, Aarhus University Hospital
Martin Tolstrup
Thomas Aagaard Rasmussen
Lars Østergaard
Steffen Leth
Mette Graversen
Paul Denton
Christel Rothe Brinkmann
Rikke Olesen
Anne Sofie Kjær
Sara Konstantin Nissen
Lene Svinth Jøhnke
Erik Hagen Nielsen
Department of Clinical Immunology, Aarhus University Hospital
Christian Erikstrup
Bionor Pharma (SPONSOR)
Maja Sommerfelt
Anker Lundemose
Potential cART and RMD: Tested in a TZM-bl assay. Using a range of concentrations of PI, NRTIs, NNRTIs and INT, we did not see any see reduced sensitivity to the antiviral effect of these drugs when co-administered with HDACis including RMD and PAN
- Potential RMD and PI interaction: Romidepsin is metabolized by CYP3A4. In recently conducted drug interaction clinical trial evaluating the strong CYP3A4 inhibitor, ketoconazole, showed co-administration of 8mg/m2 romidepsin (4-hour infusion) with ketoconazole, resulted in an overall increase in romidepsin exposure by approximately 25% and 10% for AUC0-∞ and Cmax, respectively, compared to romidepsin. As a comparison, ritonavir has been shown to have an inhibitory potency on CYP3A4 slightly less than that of ketoconazole (von Moltke, Greenblatt et al. 1998). Given the lower dosing of romidepsin in the current study and the estimated <25% increase in romidepsin (AUC0-∞) with ritonavir regimens, subjects on ritonavir boosted protease inhibitors will be allowed in the current study but will be monitored closely for toxicity related to increased romidepsin exposure. BUT PHARMACOKINETICS VS PHARMACODYNAMICS (H3 acetylation). Highest VL blip in a NNRTI treated pt, least and most side effects observed in two PI patients.