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Evaluation of Oral Tenofovir Disoproxil Fumarate and Topical Tenofovir GS-7340 to Protect Infant Macaques Against Repeated Oral Challenges With Virulent Simian Immunodeficiency Virus
 
 
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JAIDS 2006
 
Van Rompay, Koen K.A. DVM, PhD*; Kearney, Brian P. PharmD†; Sexton, Jonathan J. BS*; Colón, Roxana BA*; Lawson, Jonathan R. BS*; Blackwood, Emily J. BS*; Lee, William A. PhD†; Bischofberger, Norbert PhD†; Marthas, Marta L. PhD*
 
From the *California National Primate Research Center, University of California, Davis and †Gilead Sciences, Foster City, CA.
 
"The virologic data of the infant macaques that became infected despite oral tenofovir DF administration further suggest that the drug regimen gave suboptimal systemic drug levels.
 
With these considerations in mind, the partial prophylactic efficacy of an oral tenofovir DF regimen in the current macaque study is promising. Our data suggest that for human infants a tenofovir DF regimen that can consistently provide systemic drug levels similar to or higher than those obtained by a therapeutic tenofovir regimen has the potential to reduce the HIV transmission rate through breast-feeding. These same considerations apply also to the prophylaxis trials with tenofovir DF in adults."
 
Abstract
 
Summary: Simian immunodeficiency virus (SIV) infection of infant macaques is a useful animal model of pediatric HIV infection to evaluate the potential of chemoprophylactic regimens to reduce mother-to-infant transmission of HIV. Previous studies have demonstrated that short-term subcutaneous administration of the reverse transcriptase inhibitor tenofovir was highly effective in protecting newborn macaques against infection after a single high-dose oral inoculation with virulent SIVmac251. In the current study, we mimicked HIV transmission through breast-feeding by repeatedly feeding infant macaques low doses of SIVmac251. Topical administration of a low dose of the second-generation tenofovir prodrug GS-7340 did not have detectable prophylactic efficacy. Oral administration of tenofovir disoproxil fumarate (DF; 10 mg/kg SID) lowered the infection rate at birth, but had lower efficacy against virus infection at 4 weeks of age, most likely because drug levels became suboptimal relative to those obtained with the current tenofovir DF regimen in humans. These prophylactic results further underscore the relevance of the current tenofovir DF prevention trials in pediatric and adult populations.
 
As described elsewhere, another approach to try to prevent SIV infection after oral exposure is the use of topical microbicides by administering compounds locally at the site of viral exposure in an amount too low to provide sufficient systemic drug levels.25 In a previous study, topical administration of tenofovir DF did not have detectable prophylactic efficacy against oral SIV infection.25 However, another prodrug of tenofovir, GS-7340, may be more attractive. In contrast to tenofovir DF, the GS-7340 prodrug is preferentially hydrolyzed by intracellular instead of extracellular esterases and, therefore, yields much higher intracellular levels of the active moiety tenofovir diphosphate.26 Tenofovir GS-7340 was approximately 5-fold more potent than tenofovir DF, and 100- to 1000-fold more potent than tenofovir in inhibiting HIV-1 replication in cell lines, peripheral blood mononuclear cell (PBMC), and macrophages in vitro.26 In dog studies, oral tenofovir GS-7340 administration had high oral bioavailability (>70%), targeted lymphoid tissues, and led to much higher intracellular levels of tenofovir in PBMC than equivalent oral doses of tenofovir DF.26 Accordingly, we hypothesized that oral administration of tenofovir GS-7340 at a topical dose (ie, too low to give sufficient systemic drug levels) may be more efficient in inducing intracellular accumulation of tenofovir inside the cells at the mucosal site or in the draining lymphoid tissues of the oral cavity, with the potential for enhanced prophylactic efficacy.
 
Because HIV transmission through breast-feeding involves prolonged, daily exposure to virus in breast milk, we previously developed a model in which infant macaques are repeatedly fed low doses of SIVmac251.25,27 Using this animal model, the present study demonstrates that the topical administration of tenofovir GS-7340 did not have any detectable prophylactic efficacy. In contrast, an oral tenofovir DF regimen lowered the infection rate, and the level of efficacy was associated with age-related changes in pharmacokinetics.
 
DISCUSSION
 
To mimic better the daily exposure to HIV that occurs during breast-feeding, we had previously developed an animal model in which infant macaques are fed repeatedly low doses of virulent SIVmac251.25,27 In the present study, we used this repeated low-dose exposure model to evaluate the prophylactic efficacy of 2 prodrugs of tenofovir.
 
The novel GS-7340 prodrug of tenofovir was selected for topical administration because its rapid entry into cells and high intracellular accumulation in vitro are features expected to be advantageous for a topical virucide in vivo.26 However, topical administration of a low amount of tenofovir GS-7340 was not effective in lowering infection rates, although tenofovir GS-7340 was given at a high concentration and was mixed with the relatively low virus inocula. The lower initial viremia in the GS-7340-treated animals (Fig. 2B) suggests that the "topical" administration of a low amount of tenofovir GS-7340 for 1 week may have led to some systemic intracellular levels of tenofovir, which were, as predicted, suboptimal and therefore only partially inhibited virus dissemination. In dog studies, oral tenofovir GS-7340 administration had high oral bioavailability (>70%) and led to very efficient intracellular accumulation of tenofovir (intracellular tenofovir AUC in PBMC was ~34-fold higher than that obtained with an equivalent oral dose of tenofovir DF).26 For the infant macaque studies, we predict that a higher dose of tenofovir GS-7340 may have resulted in prophylactic efficacy because of systemic intracellular levels of tenofovir, rather than a direct topical effect. Thus, in the previous and present studies, we were not able to detect topical prophylactic efficacy with either the DF or the GS-7340 prodrugs of tenofovir against oral SIV infection.25 This is in contrast to the demonstrated efficacy of a 1% tenofovir microbicide gel against intravaginal SIV infection (C. Miller, Z. Rosenberg, and N. Bischofberger, unpublished data); possible reasons for this discrepancy in efficacy include biologic differences in transmission events that occur across different mucosal surfaces and have been described in detail previously.25 In the current study, we also tested the efficacy of oral tenofovir DF to protect infant macaques against oral SIV infection. This study is highly relevant because the potential of a 2-dose oral tenofovir DF regimen (1 maternal dose and 1 infant dose) to reduce intrapartum transmission of HIV is currently being investigated.38We observed that a once-daily 10-mg/kg dosage regimen was quite effective in protecting infant macaques against infection during the first set of oral SIV feedings shortly after birth, but became less effective at 1 month of age. However, the cumulative results still demonstrated partial prophylactic efficacy of the oral tenofovir DF regimen against repeated oral SIV challenges (P = 0.04).
 
As subcutaneous tenofovir regimens have been highly effective in protecting macaques against infection,15,16,18-22 the partial efficacy observed with the oral tenofovir DF regimen in the present study was at first surprising, but made more sense when the pharmacokinetics were taken into account. Our decision of using a 10-mg/kg tenofovir DF dose was based on our prediction that this dose, if oral bioavailability would be similar to that in humans, would give plasma levels of tenofovir in infant macaques pharmacokinetically similar to those of the tenofovir DF regimen used in HIV-infected human adults and children (steady-state AUC ~3 μg·h/mL, Cmax ~300 ng/mL).24,39-41 This hypothesis proved to be true when animals were 1 week of age; however, systemic exposures were quite variable and were significantly lower 4 weeks later (Fig. 3), which may explain the reduced prophylactic efficacy of this 10-mg/kg dosage regimen at this later time point (Fig. 1). Although our infant macaque study did not evaluate intracellular levels of tenofovir diphosphate (the active form of tenofovir), a pharmacokinetic study in juvenile macaques found that an oral tenofovir DF regimen that gave similar AUC values for tenofovir levels in plasma also gave intracellular levels of tenofovir diphosphate in PBMC within the range of those seen in adults who take the once-daily 300-mg tablet (A. Ray, K. Van Rompay, and B. Lee, unpublished data); thus, it is reasonable to consider that the variable and lower plasma tenofovir exposure levels in the 4-week-old infant macaques would yield lower intracellular levels of tenofovir diphosphate.
 
The virologic data of the infant macaques that became infected despite oral tenofovir DF administration further suggest that the drug regimen gave suboptimal systemic drug levels. In previous studies, subcutaneous injection of tenofovir to SIV-infected monkeys early in infection always resulted in a strong reduction and delay of primary viremia and/or selection for the emergence of virus with reduced in vitro susceptibility and a K65R mutation in RT.16-19,30,36,42 Similarly, tenofovir DF monotherapy of HIV-infected adults induces rapid reduction of viremia.24 In contrast, in the current study, for those animals that became infected in group C2 (which received 7 weeks of daily tenofovir DF treatment), the oral tenofovir DF regimen did not delay or dampen the initial viremia, although virus had wild-type susceptibility to tenofovir. This further suggests that drug levels were suboptimal.
 
Our present observations are consistent with those of other investigators who tested the potential of oral tenofovir DF to protect adult macaques that received weekly rectal inoculations with SHIVSF162P3; tenofovir DF-treated animals required more virus inoculations than untreated animals to eventually become infected, but drug levels in plasma were variable, and once animals became infected, viremia was not reduced, and there was no detectable emergence of K65R mutants despite the continued treatment with tenofovir DF for several months.43 Together, these data suggest that the oral tenofovir DF regimens that were used in both these studies, although partially effective, were likely suboptimal to prevent infection. In vitro, tenofovir is active at lower concentrations in antigen-presenting cells (monocytes/macrophages, dendritic cells, and Langerhans cells) than in lymphocytes because of more efficient phosphorylation.44-47Considering that antigen-presenting cells may be the first cells that become infected during transmission, this may explain why a suboptimal tenofovir DF regimen may have partial prophylactic efficacy but may have no detectable therapeutic effect on reducing viremia once most virus replication occurs in lymphocytes.
 
With these considerations in mind, the partial prophylactic efficacy of an oral tenofovir DF regimen in the current macaque study is promising. Our data suggest that for human infants a tenofovir DF regimen that can consistently provide systemic drug levels similar to or higher than those obtained by a therapeutic tenofovir regimen has the potential to reduce the HIV transmission rate through breast-feeding. These same considerations apply also to the prophylaxis trials with tenofovir DF in adults. Because an efficacious HIV vaccine has so far not been identified, several ongoing clinical trials are investigating whether uninfected adult persons who engage in high-risk behavior will have a lower infection rate by taking a 300-mg tenofovir DF tablet once daily. These trials are held at several international sites and target different high-risk populations. Regardless of the ethical issues surrounding these trials (see review of the AIDS Vaccine Advocacy Coalition48), the combined data of the tenofovir studies in macaques provide further support to continue these pediatric and adult prevention trials because drug prophylaxis could be an additional strategy to reduce the risk for infection especially when other effective methods (such as abstinence, mutual monogamy, and condoms) are not an option or are not consistently followed.

 
 
 
 
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