Weekly Oral MK-8591 Protects Male Rhesus Macaques against Repeated Low Dose Intrarectal Challenge with SHIV109CP3 Martin Markowitz MD Aaron Diamond AIDS Research Center An affiliate of the Rockefeller University New York, New York USA Thanks meeting and session organizers

Conflict of Interest Research grants Advisory Board Speaker’s Bureau Merck, Gilead Sciences, GlaxoSmithKline, ViiV Advisory Board Merck, ViiV Speaker’s Bureau Gilead Sciences

Background Pre-exposure prophylaxis of HIV-1 infection with antiretroviral agents is a safe and effective addition to the HIV prevention toolbox Efficacy of PrEP is variable and has been intimately linked to adherence. Therefore, the identification of clinically relevant compounds that protect against HIV-1 infection with the potential for alternatives to daily oral dosing has become a research priority. MK-8591 (EFdA) is a novel compound of interest. Read slide

MK-8591 (4’-Ethynyl-2-Fluoro-2’-Deoxyadenosine, EFdA) Potent and long acting nucleoside reverse transcriptase translocation inhibitor (NRTTI) Multiple mechanisms of action Immediate chain termination by inhibition of primer translocation Delayed chain termination which prevents nucleotide excision (a significant mechanism of NRTI drug resistance) from occurring MK-8591-TP concentrates in both rectal and cervical tissue Read slide Michailidis et al 2009, 2014 J Biol Chem Grobler et al 2017 CROI, Seattle, WA

Single Once-Weekly 10mg Dose of MK-8591 in 6 HIV-1- infected individuals As shown in the figure on the left with days on the x axis and change in HIV RNA from baseline on the y axis a single dose of 10mg of MK-8591 orally in 6 HIV infected individuals resulted in a robust decrease in plasma viremia. This was associated with a trough level of approximately 1pmol/10^6 cells at day 7. A late breaker oral poster will be presented on Tuesday detailing additional information regarding the antiviral activity of MK-8591 at lower doses. C168hr (7d) of MK-8591-TP=0.983 pmol/106 cells *Matthews et al., “Single doses as low as 0.5 mg of the novel NRTTI MK-8591 suppress HIV for at least seven days” at Late Breaker Poster Discussion, Tuesday, 25 July, 13:00-14:00, Havana Amphitheater Friedman et al 2016 CROI, Boston, MA Grobler et al 2016. CROI, Boston, MA

MK-8591 activity in SIVmac 251-infected rhesus macaques Intracellular level of EFdA-TP of 1.1 pmol/106 cells to achieve 90% of maximum response In a previous dose ranging experiments in rhesus macaques infected with SIVmac251 animals were administered 2 weekly oral doses of MK-8591 in increasing doses as shown in this figure. Doses of 3.9mg/kg and above were associated with maximal viral load decline and an intracellular level of EFdA-TP of 1.1 pmol/10^6 cells was found to achieve a maximal EC90. These data form the basis for the dose of MK-8591 used in the challenge experiment I will describe. Grobler et al 2016, International Workshop on Clinical Pharmacology of HIV & Hepatitis Therapy, Washington, D.C.

Repeated low-dose intrarectal challenges to evaluate MK-8591 as PrEP in 16 Indian Rhesus Macaques We performed the following experiment to evaluate MK-8591 as PrEP in 16 Indian rhesus macaques. Animals were treated on day 1 and weekly thereafter with 5ml/kg of 10%Tween80 with or without MK-8591 3.9mg/kg by oral gavage up to 14 times. At baseline, day 6 and weekly thereafter until week 24 blood was drawn for viral, immune and PK evaluations On day 6 animals were challenged with 50 TCID50 of SHIV intrarectally until either infection was documented or up to a total of 12 challenges.

SHIV109CP3 Subtype C, pathogenic, R5 SHIV Constructed from a clade C HIV env clone ZM109F.PB4 then cloned into SIV and serially passaged in rhesus macaques Mucosally transmissible by the intrarectal route Establishes high peak viremia Depletes CD4+ T cells in both the periphery and GI tract Panel 1 Panel 2 Panel 3 In this particular challenge experiment we used SHIV109CP3. Read bullet point 1 and 2. As shown in panel 1 on the right, when RMs were inoculated with 5000 TCID50 of viral stock intracrectally the virus replicated to high titers. Concurrent with high plasma viremia CD4+ T cells were depleted in the peripherphery as shown in Panel 2 and the GI tract as shown in Panel 3. Ren et al 2013 J.Virol

Materials and Methods SHIV RNA in plasma Proviral DNA from PBMCs Quantified by RT-PCR Limit of detection = 40 copies/mL Systemic infection: 2 consecutive positive vRNA signals Time of infection: 1 weeks prior to the first detectable vRNA Proviral DNA from PBMCs Qualitative assay by PCR Virus-specific antibody responses Synthetic-peptide enzyme immunoassay MK-8591 concentrations in plasma (prodrug) and PBMC (-DP and –TP) HPLC-MS/MS- based Statistics and graphics performed with PRISM Read slide

SHIVC109P3 rapidly establishes infection in untreated Rhesus macaques Median: 1 challenge Mean: 2 challenges Plasma SHIV RNA Shown in this figure are the virologic data for the control animals. 6/8 became infected after one rectal challenge, one after two and the final animal after 4. This translates into a median of 1 challenge and a mean of 2 assuming an eclipse phase of 1 week.

MK-8591-treated animals remain aviremic after 168 days of study Shown is a Kaplan Meir plot summarizing the results of the experiment with the treated animals in red and the controls in black. Note that the treated animals remain aviremic through the course of the experiment. MK-8591 treated animals have a 41.5-fold lower risk of infection (95% C.I. 7.3, 237.9) P<0.0001 log rank test

Proviral DNA and Anti-SHIV Antibodies Are Not Detected in MK-8591-Treated Macaques Not detected in PBMCs in any MK-8591-treated macaques Detected in PBMCs in all placebo animals at the time plasma viremia is first detected Virus-specific antibody responses Not detected in any MK-8591-treated macaques throughout study Detectable 1 to 2 weeks after first plasma RNA detection in all placebo animals Read the slide

Mean MK-8591 levels at the times of challenge Plasma (nM) MK-8591-TP (pmol per 106 cells) IC MK-8591-TP (µM) 1 2.15 0.60 3.01 2 2.33 1.06 5.30 3 2.48 1.46 7.32 4 2.71 0.80 4.02 5 2.89 0.52 2.60 6 3.25 0.72 3.60 7 3.02 0.45 2.26 8 3.21 0.79 3.96 9 3.10 1.02 5.10 10 3.32 0.77 3.86 11 1.04 5.17 12 2.41 0.53 2.66 Mean 2.83 0.81 4.07 Standard deviation 0.40 0.29 1.45 Shown in this table is a summary of the PK data at the time of each challenge. Moving from left to right are plasma concentrations of prodrug, intracellular concentration of the triphosphate expressed in pmol/106 cells and finally the intracellular concentration of the triphosphate expressed in micromolar assuming a cell volume of 200 fentoliter/cell. Note that the concentration of prodrug is predictable orders of magnitude lower that intracellular concentrations of active drug and the mean concentration of intracellular triphosphate approximates the target concentration of 1.0 pmol per 10^6 cells.

Conclusions MK-8591, a potent NRTTI, completely protects rhesus macaques against repeated low dose intra-rectal challenge with SHIV109CP3 one week after dosing Intracellular active drug concentrations that afford protection are achieved at low dose levels in humans. These results support the potential use of MK-8591 for HIV-1 prophylaxis in individuals at high risk of acquiring infection. Read slide

The Aaron Diamond AIDS Research Center Acknowledgements The Aaron Diamond AIDS Research Center The Rockefeller University Leslie St. Bernard Chasity Andrews Cecilia Cheng-Mayer Agegnehu Gettie Hiroshi Mohri Amir Horowitz Merck Daria Hazuda Jay Grobler Li Sun Kerry Filligrove Tulane National Primate Research Center Brooke Grasperge Jim Blanchard