Natural substrate concentrations can modulate the prophylactic efficacy

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1 JVI Accepts, published online ahead of print on 27 April 2011 J. Virol. doi: /jvi Copyright 2011, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved. Natural substrate concentrations can modulate the prophylactic efficacy of nucleotide HIV reverse transcriptase inhibitors J. Gerardo García-Lerma a,1, Wutyi Aung a, Mian-er Cong a, Qi Zheng a, Ae S. Youngpairoj a, James Mitchell a, Angela Holder a, Amy Martin a, Susan Kuklenyik b, Wei Luo a, Carol Yen-Chin Lin a, Debra L. Hanson a, Ellen Kersh a, Chou-Pong Pau a, Adrian S. Ray c, James F. Rooney c, William A. Lee c, Walid Heneine a. a Division of HIV/AIDS Prevention, National Center for HIV, Hepatitis, STD, and Prevention, and b Division of Laboratory Sciences, National Center for Environmental Health, Centers for Disease Control and Prevention, c Gilead Sciences, Foster City, CA 1 To whom correspondence should be addressed Clifton Road, Atlanta, GA 30329, USA Phone: , FAX: , GGarcia-Lerma@cdc.gov Running title: Pre-exposure prophylaxis with GS7340 tenofovir prodrug Abstract word count: 221 Text word count: 4,312 Page 1

2 ABSTRACT Pre-exposure prophylaxis (PrEP) with antiretroviral drugs is a novel HIV prevention strategy. It is generally thought that high systemic and mucosal drug levels are sufficient for protection. We evaluated if GS7340, a next-generation tenofovir (TFV) prodrug that effectively delivers tenofovir-diphosphate (TFV-DP) to lymphoid cells and tissues, could protect macaques against repeated weekly rectal SHIV exposures. Macaques received prophylactic GS7340 treatment 3 days prior to each virus exposure. At 3 days postdosing, TFV-DP concentrations in PBMCs were about 50-fold higher than those seen with TFV-disoproxil fumarate (TDF) and remained above 1,000 fmols/10 6 cells for up to 7 days. TFV-DP accumulated in lymphoid and rectal tissues, with concentrations at 3 days exceeding 500 fmols/10 6 mononuclear cells. Despite high mucosal and systemic TFV levels, GS7340 was not protective. Since TFV-DP blocks reverse transcription by competing with the natural datp substrate, we measured datp content in peripheral lymphocytes, lymphoid tissue, and rectal mononuclear cells. Compared to circulating lymphocytes and lymphoid tissue, rectal lymphocytes had 100-fold higher concentrations of datp and datp/tfv-dp ratios likely reflecting the activated status of the cells, and suggesting that TFV-DP may be less active at the rectal mucosa. Our results identify datp/tfv-dp ratios as a possible correlate of protection by TFV, and suggest that natural substrate concentrations at the mucosa will likely modulate the prophylactic efficacy of nucleotide reverse transcriptase inhibitors. Page 2

3 INTRODUCTION The HIV/AIDS pandemic remains among our greatest public health challenges. Globally, there were an estimated 33.2 million people living with HIV infection or AIDS in In that year, the annual incidence of new infections was an estimated 2.7 million, and there were an estimated 2.0 million HIV-related deaths (1). The ongoing high incidence of HIV infection and the incomplete coverage with basic HIV prevention tools underscore the need for new, highly effective biomedical HIV interventions to complement existing prevention strategies. Oral administration of antiretroviral drugs prior to and during HIV exposure (preexposure prophylaxis or PrEP) is a novel intervention to protect high-risk HIV-1 negative people from becoming infected (4, 13, 16). Drug candidates for oral PrEP have been selected from currently approved drugs for treatment of HIV-1-infected individuals. Among the available drugs, the well established potency and tolerability of tenofovir disoproxil fumarate (TDF), the approved oral prodrug of the nucleotide analog tenofovir (TFV), makes it an attractive candidate for PrEP. A recently concluded human trial with a daily combination of TDF and emtricitabine (FTC) (Truvada) among HIV-seronegative men or transgender women who have sex with men has shown a 44% reduction in the incidence of HIV-1, giving the first indication that oral PrEP may potentially provide an additive effect to current proven HIV prevention measures (15). TDF is the salt of a lipophilic and cell-permeable prodrug of TFV optimized for effective oral delivery. While oral administration of TDF improves the efficiency of lymphocyte Page 3

4 drug loading relative to subcutaneous administration of TFV(6), the effect of the prodrug is to some extent limited by instability. GS7340 is an oral prodrug of TFV whose increased stability allows for further enhancement in the delivery of TFV into cells (7, 26). As with TDF, the active intracellular metabolite of GS7340 is TFV-diphosphate (TFV-DP) which competes with the natural datp substrate for incorporation by the HIV reverse transcriptase (RT) and acts as a chain terminator. GS7340 has potent anti-hiv activity in culture, with an EC 50 value for HIV that is 1,000-fold greater than that of TFV(26). In HIV-infected patients, oral administration of 50 and 150 mg GS7340 resulted in up to 50-fold higher concentrations of TFV-DP in PBMCs and an improved antiviral response following 14 days of monotherapy relative to 300 mg of TDF (M Markowitz, A Zolopa, P Ruane, K Squires, L Zhong, B Kearney, and W Lee, GS-7340 Demonstrates Greater Declines in HIV-1 RNA than TDF during 14 Days of Monotherapy in HIV-1-infected Subjects, 18 th Conference on Retroviruses and Opportunistic Infections 2011, Boston, MA, abstract 152LB).The effective delivery of TFV into lymphoid cells and tissues by GS7340 suggests a great potential for this prodrug in PrEP. Since TFV-DP has an intracellular half-life that exceeds 4 days (19, 31), use of GS7340 prodrug might also reduce frequency of drug dosing and allow for intermittent, coitally-disassociated PrEP regimens. Because of the favorable pharmacokinetic (PK) profile and potent antiviral activity of GS7340, we hypothesized that a single weekly drug dose might be sufficient to prevent infection. We tested this hypothesis by giving prophylactic GS7340 treatment to macaques 3 days prior to exposing them rectally to SHIV. Paradoxically, we found that GS7340 was not protective despite resulting in high rectal and systemic TFV-DP levels. Page 4

5 We explain this paradox by showing that rectal mononuclear cells have a high content of the natural datp substrate that renders TFV-DP less effective. These findings demonstrate that high systemic drug levels and potent antiviral activity are not sufficient to prevent rectal SHIV transmission in macaques, and point to the importance of natural substrate concentrations at the virus point of entry for PrEP effectiveness. Page 5

6 MATERIALS AND METHODS Drug preparation and administration GS7340 was prepared in 50 mm citric acid and was given orally at 13.7 mg/kg. TDF was prepared as previously described and used orally at 22 mg/kg (12). On the bases of TFV equivalents, the TDF dose corresponds to 9.55 mg/kg of TFV and the GS7340 dose corresponds to 6.37 mg/kg of TFV, or about 3/4 of the oral TDF dose. All drugs were given by gavage to anesthetized macaques via a gastric feeding tube (12). TDF and GS7340 were provided by Gilead Sciences. Efficacy of GS7340 against rectal SHIV transmission The efficacy of GS7340 in preventing rectal SHIV transmission was evaluated using a repeat-exposure macaque model previously described (12, 30, 33). Male Indian rhesus macaques were exposed rectally once weekly to a SHIV SF162P3 chimeric virus that contains the tat, rev, and env coding regions of HIV-1 SF162 in a background of SIVmac239 (National Institutes of Health AIDS Research and Reference Reagent Program (18)). The SHIV162p3 challenge dose was 10 TCID 50 or 7.6 x 10 5 RNA copies. Virus exposures (up to 14) were done by non-traumatic inoculation of 1 ml of SHIV SF162P3 into the rectal vault via a sterile gastric feeding tube of adjusted length (12, 30, 33). Macaques were anesthetized with standard doses of ketamine hydrochloride. Anesthetized macaques remained recumbent for at least 15 min after each intra-rectal inoculation. Virus exposures were stopped when a macaque became SHIV RNA positive. Treated macaques that became infected continued receiving one weekly GS7340 dose for weeks to monitor for drug resistance emergence. The Page 6

7 Institutional Animal Care and Use Committee of the Centers for Disease Control and Prevention approved this study. Infection monitoring by molecular and serologic testing Plasma SHIV RNA was quantified using a real-time RT-PCR assay previously described (33). This assay format has a sensitivity of 50 RNA copies/ml. Detection of low-frequency K65R in plasma was performed with a sensitive allele-specific real-time PCR method previously described (20). Virus-specific serologic responses (IgG and IgM) were measured with a synthetic-peptide EIA (BioRad, Genetic Systems HIV-1/HIV- 2, Redmond, WA) assay. Animals were considered uninfected if they remained seronegative and negative for SHIV plasma RNA and SHIV DNA in PBMCs during PrEP and during the following 70 days of washout in the absence of any drug treatment (12). Separation of total mononuclear cells and cell subpopulations from blood and rectal tissues PBMCs were prepared using standard procedures. Red blood cells were lysed to minimize interferences in TFV-DP determinations (5). Lymphoid tissues were homogenized using a cell strainer followed by Ficoll separation (Lymphocyte Separation Medium (LSM, MP Biomedicals, Aurora, OH)). Rectal tissues were dissociated using an enzyme cocktail containing collagenase type II, elastase, hyaluronidase, and DNase I following procedures kindly provided by Dr. Francois Villinger. After digestion, cell suspensions were added to LSM to enrich the preparations with mononuclear cells. All Page 7

8 cells were counted using a Guava Cell counter (PBMCs) with CytoSoft Data Acquisition and Analysis Software version (Millipore, Billerica, MA). Separation of total CD4+ cells, monocytes and NK cells from blood and tissues was done by magnetic cell sorting using MACS technology according to the manufacturer s instructions (Miltenyi Biotec, Auburn, CA). CD4+ cells were purified from blood and rectal tissues by negative selection using a CD4+ T cell isolation kit. Monocytes from blood were positively selected using a monoclonal anti-cd14 antibody. The CD14- fraction was then enriched of NK cells with anti-cd16 antibodies. NK cells were included in the analysis since they are known virus reservoirs that can be persistently infected by HIV(36). After magnetic cell sorting, one fraction was used to measure intracellular TFV-DP as described below. Cell purity was evaluated by FACS analysis. Median purity was 92.8% (min, max = 64%, 97%) for blood CD4+ cells, 94.3% (min, max = 88%, 98%) for blood CD14+ cells, and 79.5% (min, max = 52%, 98%) for blood CD16+ cells. Measurement of intracellular TFV-DP and datp in mononuclear cells from blood and tissues Intracellular TFV-DP and datp concentrations were measured using an automated online weak anion exchange (WAX) solid-phase extraction (SPE) method coupled with ion-pair (IP) chromatography-tandem mass spectrometry (MS/MS) using methods recently published (23). TFV-DP and datp were monitored through and m/z fragments, respectively, with [ 13 C 5 -Adenine]-TFV-DP as internal standard. TFV-DP and datp concentrations were measured in cells from blood (total Page 8

9 mononuclear cells, CD4+ cells, monocytes, and NK cells), rectal tissues (total mononuclear cells and CD4+ cells), and lymphoid tissues (axillary, mesenteric, and inguinal lymph nodes) collected from 4 infected macaques at necropsy. Animals received orally a single oral dose of GS days prior to necropsy. Four additional macaques were used to evaluate the rate of decay in intracellular TFV-DP levels in PBMCs after a single GS7340 dose. Measurement of TFV levels in plasma and rectal secretions The kinetics of distribution of TFV in plasma and rectal secretions were evaluated by administering a single dose of GS7340 to 4 macaques followed by collection of blood and rectal secretions at 2h, 5h, and 24h. Concentrations of TFV in plasma or rectal secretions were measured by high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) as recently described (24). Isotopically labeled tenofovir- [adenine- 13 C5] ([ 13 C5]-TFV) was used as internal standard (Moravek Biochemicals, Brea, CA) (24). Rectal secretions were collected in wicks (Weck-Cel Surgical Spear, Medtronic Ophthalmic, Jacksonville, FL) using a previously established protocol (9). Briefly, dry wicks (2 consecutive wicks/animal) were inserted 5 cm into the rectum of recumbent macaques and maintained during 5 min. TFV was eluted from the wicks within 2h using a phosphate buffer solution containing 0.25% bovine serum albumin and 10% Igepal (Sigma-Aldrich, St. Louis, MO) (9). After 15 min on ice to allow for the buffer to diffuse, each wick was transferred to a PCR purification column (QIAquick PCR Purification Kit, Page 9

10 Qiagen) and centrifuged at 14,000 rpm for 5 min at 4º C to extract TFV (9). The results are expressed as ng/ml of secretion. Susceptibility of SHIV162P3 to TFV-DP and impact of datp concentrations on reverse transcription at high TFV-DP concentrations Susceptibility of wild type (WT) SHIV162p3 RT to TFV-DP was determined using the Amp-RT assay. Amp-RT detects RT activity by using a known non-retroviral heteropolymeric RNA template derived from the encephalomyocarditis virus (EMCV) genome and a complementary EMCV-specific DNA primer (10). Testing conditions included triplicate RT reactions done with the RT activity contained in approximately 10 5 particles of WT SHIV162p3 (as measured by genomic RNA levels). An isogenic TFVresistant SHIV162p3 mutant containing the K65R mutation was used as control. RT reactions were done in the absence or in the presence of TFV-DP (0.005 to 50 um) and with a fixed concentration of 5 um datp. The other three dntps were used at 20 um each (11). The RT-generated EMCV cdna was detected by real-time PCR amplification. To evaluate the impact of datp on the ability of TFV-DP to block reverse transcription, the same RT input was tested using 5 um TFV-DP and variable concentrations of datp (from 0.5 to 500 um). Statistical analysis The Cox proportional hazards model was used to estimate instantaneous risk for infection, as a hazard ratio (HR), in controls relative to treated animals, assuming constant risk at all inoculations. Graphical methods of model assessment supported the use of Cox proportional hazards regression. The Wilcoxon rank-sum statistic Page 10

11 was implemented to test for group differences in magnitude of peak virus load. TFV-DP and datp levels were compared using a mixed effects model, with a random intercept and unstructured covariance to account for within subject correlated measurements. The WinNonlin software was used to calculate AUC 24h and drug half-lives (version 5.2, Pharsight Corporation). Statistical analyses were performed with SAS software (version 9.1; SAS Institute). Page 11

12 RESULTS Pharmacokinetic profile of TFV in blood and rectal secretions The pharmacokinetic profile of GS7340 in blood and rectal secretions was evaluated at first dose in 4 macaques and was compared with that previously seen at first dose with TDF(9). GS7340 was given at 13.7 mg/kg and TDF at 22 mg/kg. On the bases of TFV equivalents, the GS7340 dose corresponds to 6.37 mg/kg of TFV and the TDF dose corresponds to 9.55 mg/kg of TFV. Figure 1 shows the levels of TFV achieved during 24h in both plasma and rectal secretions. Plasma TFV concentrations peaked at 2h with both GS7340 and TDF (Fig. 1a). Mean area under the curve (AUC 24h ) value for TFV in plasma was 3,889 ngxhr/ml (min, max = 3,117, 5,594) with GS7340 compared to 1,346 ngxhr/ml (min, max = 1,130, 1,773) with TDF. Mean plasma half-life for TFV was 6.3 h (min, max = 5.8, 6.7) after GS7340 dosing and 13h (min, max = 8, 19) after TDF dosing. In rectal secretions, TFV levels peaked at 24h with both prodrugs, with low or undetectable concentrations seen within 2-5h (Fig. 1b). Thus, the overall kinetics of distribution of TFV in plasma and rectal secretions after GS7340 dosing was similar to those seen with oral TDF, although TFV levels appear to be higher with GS7340 than with TDF. We also measured intracellular TFV-DP concentrations in PBMCs after a single GS7340 dose. Median TFV-DP concentrations were 2,581 fmol/10 6 cells at 2h, 4,845 at 5h, 3,628 at 1d, 1,597 at 3d, and 966 fmol/10 6 cells at 7d. Overall, these values are 50 Page 12

13 to 100 times those seen at first dose in macaques receiving a human-equivalent dose of TDF (20 fmol/10 6 cells at 2h, 41 at 1d, and 26 at 5d) (Fig. 1c)(9). Lack of prophylactic efficacy of GS7340 despite high TFV-DP levels in PBMCs We next explored if the high TFV-DP levels seen after oral GS7340 dosing could be sufficient to protect macaques against SHIV infection. We used a repeat low-dose rectal SHIV transmission model with weekly virus challenges. Six male Indian rhesus macaques received one weekly GS7340 dose followed by exposure to SHIV 162p3 3 days later. Infection rates were compared to those seen in 32 untreated controls; 3 were realtime controls and 29 were historical controls exposed to the same virus stock and dose under identical conditions. Fig. 2a shows the cumulative percentage of uninfected animals relative to the number of virus exposures. Despite the high intracellular TFV-DP levels achieved with GS7340 at 3 days (~1,200 fmols/10 6 cells, see below), 4/6 treated macaques became infected at challenges 2 (2 animals), 3 (1 animal), and 4 (1 animal) as did all 3 real-time untreated controls which were infected at challenges 1, 3, and 5. The challenge series in the remaining 2 treated animals were stopped at exposure 5 after an interim analysis showed absence of efficacy compared to the combined 32 realtime or historical controls (HR =1.9, p=0.23). Despite receiving only one weekly dose of GS7340, peak viremia in macaques infected during GS7340 prophylaxis (median = 5.5 log 10 ; min, max = 3.1, 5.9) was significantly lower (p = 0.01) than that seen in untreated controls (median = 7.2 log 10 ; min, max = 5.3, 8.9), a finding that is consistent with the potent antiviral activity of GS7340 and long intracellular TFV-DP persistence (Fig. 2b)(26). We also monitored for drug resistance emergence during continuous exposure Page 13

14 to one weekly dose of GS7340 for weeks. None of the 4 infected macaques developed the K65R mutation associated with TFV resistance. To confirm that breakthrough infections were not due to low TFV levels, we measured TFV-DP levels in PBMCs at each weekly virus exposure in all treated animals. Fig. 3 shows that TFV-DP concentrations at 3 days were high and similar in infected (971 fmol/10 6 cells; 95% CI=731-1,211) and uninfected (1,365, 95% CI=1,025-1,705) macaques (p=0.06). Overall, TFV-DP levels seen 3 days after GS7340 dosing were 29- fold higher than those seen at peak (24h) with oral TDF (41 fmols/10 6 cells)(9). High intracellular TFV-DP concentrations in cellular subpopulations from blood and in rectal and lymphoid tissues The failure to protect macaques with GS7340 was unexpected given the high intracellular TFV-DP concentrations seen in PBMCs 3 days after drug dosing (Figs. 1 and 3). To better understand this lack of prophylactic efficacy, we explored if GS7340 efficiently delivers TFV to different cell subpopulations and into tissues. We first measured TFV-DP in cell subpopulations from blood by administering to 4 infected male Indian rhesus macaques available from a different study a single dose of GS7340 followed by blood collection 3 days after and magnetic bead separation of CD4+, monocytes, and NK cells. Fig. 4a illustrates representative FACS results of enriched CD4+, CD14+ (monocytes) and CD14-/CD16+ (NK cells) fractions. Fig. 4b shows that median TFV-DP levels were above 500 fmols/10 6 cells in all the cell fractions evaluated. Levels were 1,172 fmols/10 6 cells in total PBMCs, 729 fmols/10 6 cells in CD4+ cells, 544 fmols/10 6 cells in monocytes, and 2,793 fmols/10 6 cells in NK cells. These findings Page 14

15 demonstrate that GS7340 effectively delivers TFV to all these cellular compartments with TFV-DP levels that remain high 3 days after drug dosing. Overall TFV-DP concentrations were also higher than those seen at 24 h in two macaques receiving oral TDF (50 and 83 fmols/10 6 in PBMCs, 35 and 34 fmols/10 6 in CD4+ cells, 48 and 38 fmols/10 6 in CD14+ cells, and 12 and 25 fmols/10 6 in CD14-/CD16+ cells). We also measured TFV-DP concentrations in lymphoid tissue (mesenteric, axillary, and inguinal lymph nodes) and rectal tissue collected at necropsy in the same 4 animals (Figure 4c). Median TFV-DP concentrations in lymphoid tissue were 590 fmols/10 6 cells (min, max = 345, 835; inguinal lymph nodes), 438 fmols/10 6 cells (min, max = 316, 828; mesenteric lymph nodes) and 542 fmols/10 6 cells (min, max = 327, 872; axillary lymph nodes). Overall, these values were about 5 to 25-fold higher than those previously seen at 2-3 days after oral TDF dosing ( fmols/10 6 cells)(9). In rectal tissues, concentrations of TFV-DP in CD4+ cells purified from 2 macaques (1,302 and 488 fmols/10 6 cells) and in total mononuclear cells from four animals (median = 377 fmols/10 6 cells; min, max = 87, 1,618) were high and similar to those previously seen in two animals 2-3 days after oral TDF dosing (1,131 and 882 fmols/10 6 cells)(9). Thus, GS7340 efficiently distributes TFV into circulating lymphocytes, lymphoid tissue, and rectal mononuclear cells, and results in high intracellular TFV-DP concentrations at 3 days. High intracellular datp content is associated with lack of prophylactic efficacy TFV-DP competes with the natural datp substrate for incorporation by the reverse transcriptase (RT). Therefore, a possible explanation for the lack of prophylactic efficacy Page 15

16 of GS7340 is that TFV-DP is not sufficiently active in rectal lymphocytes due to high intracellular datp content. To address this question we measured intracellular datp and TFV-DP concentrations and calculated datp/tfv-dp ratios in mononuclear cells obtained from rectal tissues from the same 4 infected macaques described above. Fig. 5 shows that datp levels and datp/tfv-dp ratios were about 100-fold higher in rectal mononuclear cells than in lymphoid tissues or PBMCs. These findings suggest that TFV-DP may be less active in blocking infection in rectal lymphocytes. We also measured datp levels in one uninfected, untreated macaque that was euthanized for reasons unrelated to the study. datp levels in rectal mononuclear cells (22,900 fmols/10 6 cells) from this animal were also substantially higher that those seen in PBMCs (417 fmols/10 6 cells), or lymphoid tissue (292 fmols/10 6 cells in axillary lymph nodes, 165 fmols/106 cells in inguinal lymph nodes, and 421 fmols/10 6 cells in mesenteric lymph nodes (not shown). We also compared datp levels and datp/tfv-dp ratios between the 4 PrEP breakthroughs and the 2 uninfected animals (Fig. 6). datp concentrations were measured longitudinally in PBMCs over 7 weeks during the challenge series. Mean datp levels in the 4 infected animals (63.8 fmols/10 6 cells, 95% CI = ) were significantly higher than those seen in the 2 uninfected macaques (43.0 fmols/10 6 cells, 95% CI = , p = 0.04). datp/tfv-dp ratios in the infected animals (0.074 (95% CI= ) were also significantly higher than those seen in uninfected macaques (0.037 (95% CI= , p=0.03). Fig. 6 also shows that datp levels and datp/tfv-dp ratios in infected animals were consistently higher during the complete challenge series. Page 16

17 Phenotypic reversion of SHIVp3 RT susceptibility to TFV-DP at high datp concentrations We next explored biochemically the impact of variable datp levels on the ability of TFV- DP to inhibit reverse transcription of a heteropolymeric RNA template in an in vitro assay (10, 11). A concentration of 5 um TFV-DP inhibited WT SHIV162p3 RT when the concentration of datp was also 5 um (1:1 ratio). However, 5 um TFV-DP was not sufficient to block reverse transcription when the concentration of datp was increased above 5 um (Fig. 7b). These findings demonstrate that SHIV162p3 susceptibility for TFV-DP can be diminished at high datp/tfv-dp ratios. Page 17

18 DISCUSSION We demonstrate for the first time that high mucosal and systemic antiretroviral drug concentrations in cells that are primary targets for HIV infection do not necessarily translate into high prophylactic efficacy. As expected from the high cellular permeation of GS7340, intracellular TFV-DP levels in PBMCs and lymphoid tissues were much higher with GS7340 than with TDF despite the lower GS7340 dose used (9, 26). TFV- DP concentrations in PBMCs remained high for up to 7 days with levels that exceeded by 100-fold those seen at peak with TDF. In rectal lymphocytes, TFV-DP levels at 3 days were similarly high. Despite the favorable drug PK profile and high tissue TFV-DP levels, GS7340 did not protect macaques from rectal SHIV exposures. We relate these findings to intracellular drug pharmacodynamics in the rectal mucosa by showing that rectal lymphocytes have a high datp content that likely rendered TFV-DP less effective in blocking early infection events (2). We also found that infection outcome was associated with datp/tfv-dp ratios in PBMCs. Our results identify datp/tfv-dp ratios as a negative correlate of protection with tenofovir and suggest that for nucleoside RT inhibitors, drug activity and prophylactic efficacy will depend on natural substrate concentrations at mucosal sites. Since high systemic TFV-DP levels that were sufficient to blunt acute viremias did not result in protection, our findings also suggest that active drug concentrations at the mucosa are more critical for PrEP effectiveness. The finding of high datp levels in rectal tissues was not completely surprising since immune effector sites are in a state of physiological inflammation that results in cell activation and increased dntp pools (2, 25, 27). In PBMCs, PHA stimulation increases intracellular dntp concentrations by 4 to 13-fold (8). Since most of the CD4+ cells in the Page 18

19 gut are CCR5+, activated memory CD4+ cells, and thus preferred cellular targets for HIV and SIV infection, high datp pools might have reduced the ability of TFV-DP to block reverse transcription and prevent rectal infection (3, 28, 29). In contrast, a lower datp content in PBMCs and less availability of activated target cells might increase the effectiveness of TFV against parenteral exposures. These two possible scenarios were noted in macaques receiving prophylactic treatment with oral TDF or subcutaneous TFV. While daily oral TDF did not prevent rectal infection during repeated exposures to SHIV162p3, subcutaneous TFV successfully protected macaques parenterally exposed to a high dose of SIV mne (33-35). It will be also important to explore how high datp levels are in vaginal lymphocytes and if they can be affected by inflammation associated with sexually transmitted diseases (17). Additional studies comparing TFV-DP levels achieved in vaginal and rectal tissues after oral dosing, datp content in rectal and vaginal lymphocytes, and how they all relate with efficacy in preventing infection will inform on potential pharmacodynamic and chemoprophylactic differences between these two compartments. In contrast to circulating lymphocytes and lymphoid tissues, levels of TFV-DP in rectal tissues at 3 days were similar with GS7340 and TDF dosing. In both instances, high intracellular TFV-DP concentrations were associated with high extracellular TFV levels in rectal secretions. Such high TFV levels may partially originate from degradation of GS7340 or TDF to TFV by esterases present in the intestines and/or from trapping of TFV into mucus (22, 32, 37). The lack of protection seen despite such high tissue TFV- DP concentrations is worrisome and suggests that the threshold for protection against rectal transmission by tenofovir alone may be very high. This observation may also Page 19

20 explain why the combination of TDF and FTC was more protective than TDF alone in our animal model and suggests that the additional antiviral activity provided by FTC is essential for PrEP efficacy against rectal transmission (12, 33). Several important observations can be made from the breakthrough PrEP infections. First, acute viremias were blunted in the 4 PrEP breakthrough animals despite once weekly dosing. The low viremias seen in these macaques demonstrate clearly the potent systemic antiviral activity of GS7340 and can be explained by the persistently high intracellular TFV-DP concentrations. These viologic responses are consistent with the 1.8 log 10 reduction in plasma virus loads seen in humans during GS7340 treatment (M Markowitz, A Zolopa, P Ruane, K Squires, L Zhong, B Kearney, and W Lee, GS Demonstrates Greater Declines in HIV-1 RNA than TDF during 14 Days of Monotherapy in HIV-1-infected Subjects, 18 th Conference on Retroviruses and Opportunistic Infections 2011, Boston, MA, abstract 152LB). The rapid decline in virus loads to undetectable levels within 4-8 weeks might also explain the lack of selection of the K65R mutation associated with TFV resistance. Similar blunted viremias have been noted in PrEP breakthrough infections with FTC or Truvada and have been also associated with reduced risks of resistance emergence (9, 12). Interestingly, all infections were initiated with WT viruses suggesting that initial virus replication originates from cells that are not protected by PrEP either because of suboptimal TFV- DP concentrations or, possibly, because of high intracellular datp levels in gut lymphocytes. Several important considerations have to be made to our study. First, we did not measure TFV-DP concentrations in cellular subpopulations from rectal tissues that are Page 20

21 also primary target during early infection and might be inadequately protected by GS7340, such as macrophages or dendritic cells (25). Different molecular transport pathways for GS7340 and TFV might result in variable cellular drug exposures after GS7340 or TDF dosing. Second, our analysis of datp content in rectal lymphocytes was done weeks after SHIV infection. It is not known if infection has contributed to the high datp levels seen in rectal lymphocytes from these animals although high datp concentrations are consistent with the activation status of rectal lymphocytes (2, 25, 27). Also, SHIV162p3 infections are not highly pathogenic as most animals appear to control infection (14, 21). Our analysis of datp levels in one uninfected, untreated macaque suggests that neither infection status nor drug treatment might be responsible for the high datp levels seen in our animals, although this observation needs to be further confirmed in more uninfected macaques. It will be also important to evaluate if the high datp content seen in rectal lymphocytes from rhesus macaques is also observed in the gastrointestinal tract from humans. In summary, we show that GS7340 efficiently delivers TFV into peripheral lymphocytes, lymphoid tissues, and rectal tissue. However, we demonstrate that high rectal and systemic TFV exposure was not sufficient to prevent rectal SHIV transmission in macaques. We explain this paradox by showing high datp concentrations in rectal mononuclear cells which may reduce the ability of TFV-DP to block early infection events. Our results identify datp/tfv-dp ratios as a negative correlate of protection by TFV and suggest that natural substrate concentrations in mucosal target cells may potentially modulate the prophylactic efficacy of tenofovir and have broader implications for the entire NRTI drug class. Pharmacodynamic differences between rectal, vaginal Page 21

22 and systemic tissues may possibly impact the prophylactic efficacy of antiretroviral drugs against distinct routes of HIV transmission. Page 22

23 ACKNOWLEDGEMENTS We thank Dr. Katherine Paul for serving as the attending veterinarian for this study protocol, Dr. Nelva J. Bryant for performing the necropsies on the macaques, Elizabeth D. Sweeney for performing some of the animal procedures, and Dr. Francois Villinger for sharing methods for rectal tissue dissociations. Footnotes Disclaimer: The findings and conclusions in this report are those of the authors and do not necessarily represent the views of the Centers for Disease Control and Prevention. Page 23

24 FIGURE LEGENDS Figure 1. Pharmacokinetic profiles of GS7340 and TDF at first dose in plasma (A), rectal secretions (B), and peripheral blood mononuclear cells (PBMCs) (C). Graphs show the median TFV or TFV-DP levels and the extreme values (min, max) observed in 4 animals. Levels of TFV-DP after TDF dosing were recently reported and are added for comparison (9). Figure 2. Prophylactic efficacy of one weekly GS7340 dose given 3 days prior to virus exposure. A: Lack of protection against repeated rectal SHIV exposures. Each survival curve represents the cumulative percentage of infected macaques as a function of weekly virus exposure. B: Acute viremia in macaques infected during GS7340 treatment. Red lines are GS7340 failures (n = 4), blue lines are real-time untreated controls (n = 3), and black lines are historical untreated controls (n = 22). Median peak viremia in breakthrough animals was significantly lower than that seen in untreated macaques (Wilcoxon rank sum test, p=0.01). Figure 3. Intracellular TFV-DP levels in PBMCs in infected and uninfected macaques. TFV-DP was measured during 5 weeks at virus exposure in the 4 infected (red lines) and the 2 uninfected (blue lines) animals. Mean TFV-DP levels were comparable in both groups (p =0.06). Figure 4. Intracellular TFV-DP levels in cell subpopulations from blood and tissues after a single GS7340 dose given 3 days prior to necropsy. A. Representative FACS results of enriched CD4+, CD14+ (monocytes) and CD14-/CD16+ (NK cells) fractions purified Page 24

25 from blood and used for drug level measurements. B: Distribution of GS7340 in cell subpopulations from blood. Horizontal lines denote the median value. C: TFV-DP levels in total mononuclear cells from inguinal lymph nodes (ILN), mesenteric lymph nodes (MLN), axillary lymph nodes (ALN), and rectal tissues (both total mononuclear cells and enriched CD4+ cell fraction). Horizontal lines denote the median value. Figure 5. Intracellular datp levels and datp/tfv-dp ratios in cell subpopulations from blood and tissues seen after a single GS7340 dose given 3 days prior to necropsy. A: datp levels and datp/tfv-dp ratios in cell subpopulations from blood. B: datp levels and datp/tfv-dp ratios and in ILN, MLN, ALN, and rectal tissues (both total mononuclear cells and enriched CD4+ cell fraction). Horizontal lines denote the median values. Figure 6. Intracellular datp levels (A) and datp/tfv-dp ratios (B) in PBMCs from macaques exposed to SHIV162p3 during GS7340 prophylaxis. datp and TFV-DP levels were measured during 5 weeks at virus exposure in the 4 infected (red lines) and the 2 uninfected animals (blue lines). Mean datp levels and datp/tfv-dp ratios were higher in the infected animals (p =0.04 and p = 0.03, respectively). Figure 7. Phenotypic reversion of SHIV162p3 susceptibility for TFV-DP occurs at high datp/tfv-dp ratios. A: Inhibition of WT SHIV162p3 RT by TFV-DP. A concentration of 5 um TFV-DP and 5 um datp (1:1 ratio; arrow) can efficiently block WT RT but not a TFV-resistant RT containing the K65R mutation. Data represents the mean values seen in four separate experiments done in triplicate. B: Phenotypic reversion and efficient completion of reverse transcription with TFV-DP can be achieved at high datp Page 25

26 concentrations. RT was incubated with 5 um TFV-DP and increasing concentrations of datp. Data represents the mean values seen in two separate experiments done in triplicate. Page 26

27 References 1..< UNAIDS/WHO. AIDS epidemic update: December Geneva: UNAIDS. 2. Arts EJ, Marois JP, Gu Z, Le Grice SF, and Wainberg MA Effects of 3'- deoxynucleoside 5'-triphosphate concentrations on chain termination by nucleoside analogs during human immunodeficiency virus type 1 reverse transcription of minus-strand strong-stop DNA. J Virol. 70: Brenchley JM, Schacker TW, Ruff LE, Price DA, Taylor JH, Beilman GJ, Nguyen PL, Khoruts A, Larson M, Haase AT, and D. D CD4+ T cell depletion during all stages of HIV disease occurs predominantly in the gastrointestinal tract. J Exp Med 200: Cohen MS, Gay C, Kashuba AD, Blower S, and Paxton L Narrative review: antiretroviral therapy to prevent the sexual transmission of HIV-1. Ann Intern Med. 146: Durand-Gasselin L, Da Silva D, Benech H, Pruvost A, and Grassi J Evidence and possible consequences of the phosphorylation of nucleoside reverse transcriptase inhibitors in human red blood cells. Antimicrob Agents Chemother 51: Durand-Gasselin L, Van Rompay KKA, Vela JE, Henne IN, Lee WA, Rhodes GR, and R. AS Nucleotide analogue prodrug tenofovir disoproxil enhances lymphoid cell loading following oral administration in monkeys. Molecular Pharmaceutics 6: Eisenberg EJ, He GX, and Lee WA Metabolism of GS-7340, a novel phenyl monophosphoramidate intracellular prodrug of PMAPA, in blood. Nucleosides, Nucleotides and Nucleic Acids 20: Gao WY, Shirasaka T, Johns DG, Broder S, and Mitsuya H Differential phosphorylation of azidothymidine, dideoxycytidine, and dideoxyinosine in resting and activated peripheral blood mononuclear cells. J Clin Invest. 91: García-Lerma JG, Cong M, Mitchell J, Youngpairoj AS, Zheng Q, Masciotra S, Martin A, Kuklenyik Z, Holder A, Lipscomnb J, Pau C, Barr JR, Hanson DL, Otten RA, Paxton L, Folks TM, and Heneine W Intermittent prophylaxis with oral truvada protects macques form rectal SHIV infection. Sci Transl Med 2:14ra García-Lerma JG, Heneine W Analysis of HIV-1 reverse transcriptase activity in plasma: A new tool for the detection of viral variants, virus load measurement and phenotypic drug resistance testing. AIDS Reviews 1: García-Lerma JG, Nidtha S, Heneine W Susceptibility of the human T- cell leukemia virus type 1 to reverse transcriptase inhibitors: Evidence of high level resistance to lamivudine. J Infect Dis 184: García-Lerma JG, Otten RA, Qari SH, Jackson E, Cong M, Masciotra S, Luo W, Kim C, Adams DR, Monsour M, Lipscomb J, Johnson JA, Delinsky D, Schinazi RF, Janssen R, Folks TM, and Heneine W Prevention of rectal Page 27

28 SHIV transmission in macaques by daily or intermittent prophylaxis with emtricitabine and tenofovir PLoS Med 5:e García-Lerma JG, Paxton L, Kilmarx P, and Heneine W Oral preexposure prophylaxis for HIV prevention. Trends Pharmacol Sci 31: George MD, Reay E, Sankaran S, and D. S Early antiretroviral therapy for simina immunodeficiency virus infection leads to mucosal CD4+T-cell restoration and enhanced gene expression regulating mucosal repair and regeneration. J. Virol 79: Grant RM, Lama JR, Anderson PL, McMahan V, Liu AY, Vargas L, Goicochea P, Casapía M, Guanira-Carranza JV, Ramirez-Cardich ME, Montoya-Herrera O, Fernández T, Veloso VG, Buchbinder SP, Chariyalertsak S, Schechter M, Bekker LG, Mayer KH, Kallás EG, Amico KR, Mulligan K, Bushman LR, Hance RJ, Ganoza C, Defechereux P, Postle B, Wang F, McConnell JJ, Zheng JH, Lee J, Rooney JF, Jaffe HS, Martinez AI, Burns DN, Glidden DV, and t. i. S. Team Preexposure chemoprophylaxis for HIV prevention in men who have sex with men. N Engl J Med. 16. Grant RM, Wainberg MA Chemoprophylaxis of HIV infection: moving forward with caution. J Infect Dis. 194: Haase AT Perils at the mucosal front lines for HIV and SIV and their hosts. Nature Rev Immunol 5: Harouse JM, Gettie A, Tan RC, Blanchard J, and Cheng-Mayer C Distinct pathogenic sequela in rhesus macaques infected with CCR5 or CXCR4 utilizing SHIVs. Science 284: Hawkins T, Veikley W, St Claire RL 3rd, Guyer B, Clark N, and K. BP Intracellular pharmacokinetics of tenofovir diphosphate, carbovir triphosphate, and lamivudine triphosphate in patients receiving triple-nucleoside regimens. J Acquir Immune Defic Syndr 39: Johnson JA, Rompay KK, Delwart E, and Heneine W A rapid and sensitive real-time PCR assay for the K65R drug resistance mutation in SIV reverse transcriptase. AIDS Res Hum Retroviruses 22: Kersh EN, Luo W, Adams DR, Srinivasan P, Smith JM, Promadej-Lanier N, Ellenberger D, Garcia-Lerma JG, Butera S, and Otten R Repeated rectal SHIVSF162P3 exposures do not consistently induce sustained T cell responses prior to systemic infection in the repeat-low dose preclinical macaque model. AIDS Res Hum Retroviruses. 25: Khanvilkar K, Donovan MD, and Flanagan DR Drug transfer through mucus. Adv Drug Deliv Rev 48: Kuklenyik Z, Martin A, Pau C, Holder A, Youngpairoj AS, Zheng Q, Cong M, Garcia-Lerma JG, Heneine W, Pirkle JL, and B. JR On-line coupling of anion exchange and ion pair chromatography for measurement of intracellular triphosphate metabolites of reverse transcriptase inhibitors. J Chromatogr B Analyt Technol Biomed Life Sci 877: Kuklenyik Z, Martin A, Pau CP, Garcia-Lerma JG, Heneine W, Pirkle JL, and Barr JR Effect of mobile phase ph and organic content on LC-MS Page 28

29 analysis of nucleoside and nucleotide HIV reverse transcriptase inhibitors. J Chromatogr Sci. 47: Lackner AA, Mohan M, and Veazey RS The gastrointestinal tract and AIDS pathogenesis. Gastroenterology. 136: Lee WA, He G, Eisenberg E, Cihlar T, Swaminathan S, Mulato A, and Cundy KC Selective intracellular activation of a novel prodrug of the human immunodeficiency virus reverse transcriptase inhibitor tenofovir leads to preferential distribution and accumulation in lymphatic tissue. Antimicrobial Agents and Chemotherapy 49: McGowan I, Elliott J, Fuerst M, Taing P, Boscardin J, Poles M, and Anton P Increased HIV-1 mucosal replication is associated with generalized mucosal cytokine activation. J Acquir Immune Defic Syndr. 37: Mehandru S, Poles MA, Tenner-Racz K, Horowitz A, Hurley A, Hogan C, Boden D, Racz P, and M. M Primary HIV-1 infection is associated with preferential depletion of CD4+ T lymphocytes from effector sites in the gastrointestinal tract. J Exp Med 200: Mehandru S, Poles MA, Tenner-Racz K, Manuelli V, Jean-Pierre P, Lopez P, Shet A, Low A, Mohri H, Boden D, Racz P, and Markowitz M Mechanisms of gastrointestinal CD4+ T-cell depletion during acute and early human immunodeficiency virus type 1 infection. J Virol 81: Otten RA, Adams DR, Kim CN, Jackson E, Pullium JK, Lee K, Grohskopf LA, Monsour M, Butera S, and F. TM Multiple vaginal exposures to low doses of R5 simian-human immunodeficiency virus: strategy to study HIV preclinical interventions in nonhuman primates. J Infect Dis 19: Pruvost A, Negredo E, Benech H, Theodoro F, Puig J, Grau E, Garcia E, Molto J, Grassi J, and C. B Measurement of intracellular didanosine and tenofovir phosphorylated metabolites and possible interaction of the two drugs in human immunodeficiency virus-infected patients. Antimicrob Agents Chemother 49: Shaw JP, Sueoko CM, Oliyai R, Lee WA, Arimilli MN, Kim CU, and C. KC Metabolism and pharmacokinetics of novel oral prodrugs of 9-[(R)-2- (phosphonomethoxy)propyl]adenine (PMPA) in dogs. Pharm Res. 14: Subbarao S., Otten R. A., Ramos A., Kim C., Jackson E., Monsour M., Adams D. R., Bashirian S., Johnson J., Soriano V., Rendon A., Hudgens M. G., Butera S., Janssen R., Paxton L., Greenberg A. E., and F. T. M Chemoprophylaxis with tenofovir disoproxil fumarate provided partial protection against infection with simian human immunodeficiency virus in macaques given multiple virus challenges. J Infect Dis 194: Tsai CC, Emau P, Follis KE, Beck TW, Benveniste RE, Bischofberger N, Lifson JD, and Morton WR Effectiveness of postinoculation (R)-9-(2- phosphonylmethoxypropyl) adenine treatment for prevention of persistent simian immunodeficiency virus SIVmne infection depends critically on timing of initiation and duration of treatment. J Virol 72: Tsai CC, Follis KE, Sabo A, Beck TW, Grant RF, Bischofberger N, Benveniste RE, and Black R Prevention of SIV infection in macaques by (R)-9-(2-phosphonylmethoxypropyl)adenine. Science. 270: Page 29

30 36. Valentin A, Rosati M, Patenaude DJ, Hatzakis A, Kostrikis LG, Lazanas M, Wyvill KM, Yarchoan R, and Pavlakis GN Persistent HIV-1 infection of natural killer cells in patients receiving highly active antiretroviral therapy. Proc Natl Acad Sci U S A 99: van Gelder J, Deferme S, Naesens L, De Clercq E, van den Mooter G, Kinget R, and Augustijns P Intestinal absorption enhancement of the ester prodrug tenofovir disoproxil fumarate through modulation of the biochemical barrier by defined ester mixtures. Drug Metab Dispos 30: Page 30

31 A. Plasma B. Rectal secretions 1000 Oral TDF Oral GS Oral TDF Oral GS TFV (ng/ml) 100 TFV (ng/ml) Time (hr) Time (hr) C. PBMCs Oral TDF TFV-DP (fmols/10 6 cells Oral GS Time (hr)

32 A % Uninfected macaques Controls (n = 32) GS7340 (n = 6) Number of rectal exposures B 10 Log 10 RNA copies/ml Weeks

33 10000 Infected Uninfected TFV-DP, fmols/10 6 cells Week

34 A CD4+ CD16+ B CD4 FITC CD14 APC CD CD16 PE CD14 APC CD16 PE TFV-DP, fmols/10 6 cells C TFV-DP, fmols/10 6 cells PBMC CD4+ CD14+ CD14-/CD16+ ILN MLN ALN Total MNCs CD4+ Rectal tissue

35 A B datp, fmols/10 6 cells datp, fmols/10 6 cells Total PBMC CD4+ CD14+ CD14-/CD ILN MLN ALN Total CD4+ MNC Rectal tissue datp/tfv-dp ratio datp/tfv-dp ratio Total PBMC CD4+ CD14+ CD14-/CD ILN MLN ALN Total CD4+ MNC Rectal tissue

36 A 1000 Infected Uninfected datp, fmols/10 6 cells B 1 Weeks Infected Uninfected datp/tfv-dp ratios Weeks

37 A % RT inhibition SHIV162P3-WT SHIV162P3-K65R TFV-DP, mm B 100 % RT inhibition datp, mm

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