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Research Article
14 August 2014

Pharmacokinetics and Preliminary Safety Study of Pod-Intravaginal Rings Delivering Antiretroviral Combinations for HIV Prophylaxis in a Macaque Model

ABSTRACT

Preexposure prophylaxis using oral regimens involving the HIV nucleoside reverse transcriptase inhibitors tenofovir disoproxil fumarate (TDF) and emtricitabine (FTC) demonstrated efficacy in three clinical trials. Adherence was determined to be a key parameter for success. Incorporation of the TDF-FTC combination into intravaginal rings (IVRs) for sustained mucosal delivery could increase product adherence and efficacy compared with those of oral and vaginal gel formulations. A novel pod-IVR technology capable of delivering multiple drugs is described; this constitutes the first report of an IVR delivering TDF and FTC, as well as a triple-combination IVR delivering TDF, FTC, and the entry inhibitor maraviroc (MVC). The pharmacokinetics and preliminary local safety of the two combination pod-IVRs were evaluated in the pig-tailed macaque model. The devices exhibited sustained release at controlled rates over the 28-day study period. Median steady-state drug levels in vaginal tissues in the TDF-FTC group were 30 μg g−1 (tenofovir [TFV], in vivo hydrolysis product of TDF) and 500 μg g−1 (FTC) and in the TDF-FTC-MVC group were 10 μg g−1 (TFV), 150 μg g−1 (FTC), and 20 μg g−1 (MVC). No adverse events were observed, and there were no toxicological findings. Mild-to-moderate increases in inflammatory infiltrates were observed in the vaginal tissues of some animals in both the presence and the absence of the IVRs. The IVRs did not disturb the vaginal microbiota, and levels of proinflammatory cytokines remained stable throughout the study. Pod-IVR candidates based on the TDF-FTC combination have potential for the prevention of vaginal HIV acquisition and merit clinical investigation.

INTRODUCTION

Preexposure prophylaxis (PrEP) using FDA-approved antiretroviral (ARV) drugs is emerging as a promising strategy for the prevention of sexual HIV infection. There is growing consensus that a combination of ARV agents, analogous to highly active antiretroviral therapy (HAART), likely is essential for optimally effective PrEP (1, 2). Oral administration of tenofovir disoproxil fumarate (TDF) plus emtricitabine (FTC) (Truvada; Gilead Sciences, Inc.) is the first regimen approved by the FDA to reduce the risk of HIV infection in uninfected individuals (http://www.fda.gov/NewsEvents/Newsroom/PressAnnouncements/ucm312210.htm). Three recent clinical trials demonstrated that oral ARV regimens using the combination of TDF and FTC can be effective in susceptible men, women, and partners of HIV-infected individuals (35). However, the relative risk reduction in these trials varied widely (from 44 to 75%), and a study in which women used a daily oral TDF-FTC regimen was stopped early due to futility. A critical factor driving success in these trials appears to involve sustaining high levels of adherence to frequent dosing (6).
It is well established across different delivery methods that adherence to therapy is inversely related to the dosing period (710). Controlled topical delivery of ARV drugs using intravaginal rings (IVRs) is thought to improve adherence (11) and to provide sustained mucosal levels independent of coitus and daily dosing (12). The delivery of two or more ARV drugs from conventional IVR designs involves significant technological and manufacturing hurdles. To meet these challenges, we have developed a novel IVR technology, the pod-IVR (13), that enables rapid development of devices capable of delivering multiple agents over a wide range of target delivery rates and aqueous solubilities (1416). We recently published the design and 28-day pharmacokinetic (PK) evaluation in sheep of a five-drug pod-IVR as a proof-of-concept, advanced multipurpose prevention technology (MPT), combining three ARV drugs from different mechanistic classes (tenofovir [TFV], nevirapine, and saquinavir) with a proven estrogen-progestogen contraceptive for prevention of HIV infection and unintended pregnancy (17).
Here we present the first report of an IVR delivering TDF and FTC, as well as the first report of a triple-combination IVR delivering TDF, FTC, and maraviroc (MVC) (an entry inhibitor/antagonist of chemokine receptor 5 [CCR5]). Preliminary local safety and pharmacokinetic (PK) findings for these devices were determined in pig-tailed macaques, which are considered by many to represent the most relevant animal model for HIV vaginal PrEP studies (15, 18, 19). Steady-state drug levels for all three ARV agents in vaginal fluids were sustained over the 28-day study period, with corresponding vaginal tissue concentrations suggesting putative efficacy in preventing HIV infection.

MATERIALS AND METHODS

Materials.

Tenofovir disoproxil fumarate (TDF) and emtricitabine (FTC) were kindly provided by Gilead Sciences, Inc. (Foster City, CA), under a material transfer agreement (MTA) dated 8 August 2011. Maraviroc (MVC) (ViiV Healthcare, Brentford, Middlesex, United Kingdom) was kindly provided by the International Partnership for Microbicides, Inc. (IPM) (Silver Spring, MD) under an MTA dated 10 December 2010 and was used as an analytical reference standard. For formulation into IVRs, MVC was isolated from the commercial formulation (Pfizer, Inc., New York, NY), which consists of film-coated tablets for oral administration containing 300 mg of MVC and inactive ingredients, as described below.
The blue outer coating was removed from three MVC tablets (3.74 g) using a razor blade, and the white core containing maraviroc and inactive excipients (microcrystalline cellulose, dibasic calcium phosphate, sodium starch glycolate, and magnesium stearate) was ground to a fine powder using a pestle and mortar. The powder was placed on a sintered glass filter, treated with chloroform (5 × 5 ml) with gentle mixing using a spatula, and filtered in vacuo. The cloudy combined filtrates were filtered through a Celite pad, the chloroform was evaporated in vacuo, and the colorless oil was dried under high vacuum to afford a white foam, which was subsequently recrystallized from toluene/hexanes (2:1) to afford a white solid (0.65 g; 73%; mp, 189 to 191°C), which matched the IPM reference standard. A 1H nuclear magnetic resonance spectrum of this material closely matched published data (20) and data for the IPM reference standard, confirming purity.

Manufacture of combination pod-intravaginal rings.

Macaque-sized (21) polydimethylsiloxane (PDMS) (silicone) pod-IVRs were prepared in a multistep process that has been described in detail elsewhere (13, 15). Six pods per combination IVR were used in two different formulations, i.e., one containing three pods each of TDF and FTC and another containing two pods each of TDF, FTC, and MVC (Table 1 and Fig. 1). Each pod contained a single drug. The drug powder, admixed with 0.5% (wt/wt) magnesium stearate, was compacted into cores of 3.2-mm outer diameter in a manual tablet press (MTCM-I; Globe Pharma, New Brunswick, NJ). Drug cores were coated with polymer to yield pods (Table 1), which were placed in the corresponding IVR cavities and sealed in place by back-filling with room-temperature-cure silicone. Each pod was matched with the appropriate configuration of mechanically punched delivery channels (Table 1).
TABLE 1
TABLE 1 Physical characteristics of pod-IVRs used in pig-tailed macaque studies
Physical characteristicTDF-FTC IVRaTDF-FTC-MVC IVRa
TDF drug loading (mean ± SD) (mg)64.8 ± 2.243.9 ± 1.2
FTC drug loading (mean ± SD) (mg)67.6 ± 1.345.5 ± 0.8
MVC drug loading (mean ± SD) (mg)NA41.9 ± 0.6
Delivery channel cross-sectional area (mm2)  
    TDF1.771.77
    FTC0.790.79
    MVC 5.30b
a
There were six pods total per IVR, and each pod was coated with poly(vinyl alcohol). Values represent total drug loading in the IVR. NA, not applicable.
b
Three 1.77-mm2 channels per pod.
FIG 1
FIG 1 Photograph of a 6-pod, 3-drug macaque IVR.

In vitro studies.

All in vitro release studies were designed to mimic sink conditions, using methods reported previously (13). Briefly, the IVRs were placed in a simplified vaginal fluid simulant (22) dissolution medium (100 ml) consisting of 25 mM acetate buffer (pH 4.2) with NaCl added to achieve 220 mosM. The vessels were agitated in an orbital shaker at 25 ± 2°C and 60 rpm. Aliquots (100 μl) were removed at predetermined time points and were replaced with an equal volume of dissolution medium. Samples were stored at −30°C prior to analysis. The concentrations of TDF, its hydrolysis product tenofovir (TFV), FTC, and MVC were measured by high-performance liquid chromatography (HPLC) with UV detection (1100 Series; Agilent Technologies, Santa Clara, CA). For TDF and MVC, the mobile phase consisted of acetonitrile and phosphate buffer (20 mM, pH 2.5) in a ratio of 30:70 (vol/vol), at a flow rate of 0.2 ml min−1. A Phenomenex (Torrance, CA) Kinetex XB-C18 column (2.1 by 100 mm, 2.6 μm, 100 Å) was used as the stationary phase. For TFV and FTC, an otherwise identical method was used with a mobile phase consisting of methanol and phosphate buffer (20 mM, pH 2.5) in a ratio of 10:90 (vol/vol). The detection wavelengths were 280 nm (FTC), 260 nm (TDF and TFV), and 210 nm (MVC). The retention times were 3.7 min (TDF), 2.2 min (MVC), 4.3 min (FTC), and 1.75 min (TFV). The method run times were 5.1 min.

Nonhuman primate studies.

The pharmacokinetic (PK) and safety study was carried out at the Centers for Disease Control and Prevention (CDC) under approved CDC institutional animal care and use committee protocols and standard guidelines according to the Guide for the Care and Use of Laboratory Animals (23). The study timeline and biological sample collection points are shown in Fig. 2 and employed published protocols (15, 24). Briefly, six sexually mature female pig-tailed macaques (Macaca nemestrina) were used; three received TDF-FTC IVRs (Table 1), and three received TDF-FTC-MVC IVRs. IVRs were inserted into the posterior vagina on day 0 and were replaced on day 14 with a second set of IVRs from the same group, which remained in place for another 14 days (Fig. 2). Vaginal colposcopy was used to confirm placement and retention of the IVRs and to examine the integrity of the cervicovaginal epithelium. Induction of mucosal inflammation was monitored by measuring vaginal cytokine levels as described previously, using fluorescent multiplexed bead-based assays (Milliplex MAP; Millipore, Billerica, MA) in accordance with the manufacturer's instructions (21). Dacron swabs were placed individually in a Port-a-Cul tube (Becton, Dickinson, Franklin Lakes, NJ) and transported to Magee Women's Research Institute within 24 h after collection, for microbial analysis. The swabs were characterized for the presence of aerobic and anaerobic microorganisms. Details on the methods used to assess vaginal microbiota have been provided elsewhere (24). Histopathological assessments of vaginal biopsy samples (1 biopsy sample each collected proximal and distal to the IVR for each animal at both day 22 and day 31, for a total of 24 samples) preserved in 10% formalin were carried out at Charles River Laboratories (Pathology Associates, Frederick, MD). The samples were trimmed, processed routinely, embedded in paraffin, and stained with hematoxylin and eosin. Microscopic evaluations were conducted by a board-certified veterinary pathologist. Tissues were evaluated by light microscopy.
FIG 2
FIG 2 Macaque study timelines and biological sample collection points (n = 3 for the TDF-FTC group and n = 3 for the TDF-FTC-MVC group). Black arrows, colposcopic examination and collection of, in order, blood, pH, vaginal fluid (4 Weck-Cel samples, 2 proximal and 2 distal to the cervix/IVR), microflora (2 Dacron swabs), and cervicovaginal lavage fluid; black arrows with diamonds, collection of vaginal tissue samples (6 punch biopsy samples per time point, 3 proximal and 3 distal to the cervix/IVR).
Used IVRs were analyzed for residual drug contents at Oak Crest Institute, using published methods (14, 16). The HPLC methods were the same as those used to analyze aliquots from the in vitro studies.
Levels of TDF, TFV, FTC, and MVC in vaginal fluid, cervicovaginal lavage (CVL) fluid, vaginal tissue homogenate, and plasma samples were measured by liquid chromatography-tandem mass spectrometry (LC-MS/MS) using published methods (15, 18, 25, 26). The lower limits of quantitation (LLOQs) for these analytes in the aforementioned sample matrices are presented in Table 2.
TABLE 2
TABLE 2 Analytical lower limits of quantitation for biological samples (15, 18, 25)
Sample matrixLLOQ (ng ml−1)
TDFTFVFTCMVC
Vaginal fluid10501010
CVL fluid1511
Vaginal tissue homogenatea5551
Plasma5551
a
Assumes that vaginal tissue has a density of 1 g ml−1 (59).

Pharmacokinetic analysis.

Pharmacokinetic parameter values were determined by noncompartmental analysis (NCA) using Phoenix WinNonlin 6.3 (Pharsight Corp., Sunnyvale, CA). The NCA was run using the linear trapezoidal rule for increasing-concentration data and the logarithmic trapezoidal rule for decreasing-concentration data (linear up and log down) as the calculation method, and values below the corresponding LLOQs were treated as 0.001. Matched TDF and TFV measurements were interpreted as the sum of TDF and TFV concentrations on a molar basis, reported as total TFV mass concentration.

Statistical analysis.

Data were analyzed using GraphPad Prism (version 6.02; GraphPad Software, Inc., La Jolla, CA). Median ARV drug levels in samples were compared using a Wilcoxon matched-pairs signed-rank test (with 95% confidence intervals [CIs]). Vaginal cytokine levels below the limit of detection for the assay were assigned concentrations halfway between the LLOQ and zero. The lowest concentration of the standard curve was defined as the LLOQ. Cytokine levels measured after insertion of the IVR and after removal of the IVR were compared to baseline levels and to each other in a one-way analysis of variance (ANOVA). Cytokine levels were logarithmically transformed to normalize the data. A Bonferroni correction was implemented to avoid inference errors due to multiple comparisons. Statistical significance was defined at a P value of <0.05.

RESULTS

In vitro studies.

In vitro cumulative release profiles for the three IVR formulations exhibited linear (R2 > 0.97) sustained release of TDF, FTC, and MVC, as is typical for pod-IVRs (1316). The 14-day cumulative release rates are presented in Table 3. The difference in FTC release rates between the two formulations can be explained on the basis of IVR drug loading (Table 1), while the difference in TDF release rates is greater than expected.
TABLE 3
TABLE 3 In vitro and in vivo daily release rates and in vitro-in vivo correlation
Drug (IVR formulation)aRelease rate (mean ± SD) (mg day−1)IVIVCb
In vitroIn vivo
TDF (TDF-FTC)3.41 ± 0.330.68 ± 0.260.20
FTC (TDF-FTC)3.39 ± 0.842.50 ± 0.580.74
TDF (TDF-FTC-MVC)1.59 ± 0.290.75 ± 0.300.47
FTC (TDF-FTC-MVC)2.84 ± 0.171.05 ± 0.320.37
MVC (TDF-FTC-MVC)1.31 ± 0.170.86 ± 0.070.66
a
n = 3 for both IVR formulations.
b
In vivo release rate divided by in vitro release rate.

In vivo release rates.

The mean daily in vivo release rates for all drugs are given in Table 3. The calculation is based on the residual drug mass remaining in the used IVRs and the assumption, supported by in vitro data, that drug release is linear over the 2-week period. The TDF release rates were statistically equivalent (P = 0.695, unpaired t test with Welch's correction) with the two IVR formulations, but the FTC release rates were significantly different (P = 0.0008, unpaired t test with Welch's correction). The differences in release rates between the two formulations cannot be explained on the basis of IVR drug loading alone. It is likely that intermacaque physiological differences (e.g., vaginal fluid volumes and mucus levels) contributed significantly to these observations. Importantly, >98% of the residual TDF in the used IVR pods was present as the prodrug, i.e., no hydrolysis to TFV was observed following 2 weeks of use in vivo.

In vitro-in vivo correlation.

The use of in vitro-in vivo correlation (IVIVC) to guide in vitro experiments during the development of sustained-release formulations allows drug target levels to be achieved with a minimum number of in vivo studies. The calculated IVIVCs given in Table 3 are lower and more consistent than those reported previously (15), likely reflecting improved manufacturing of the pods. The IVIVC values suggest that the in vitro system provides an accelerated model of the in vivo release rates. This has the potential to decrease the time required for in vitro studies during product development, which is an important consideration for long-term sustained-release formulations delivering over 1 month or more, although further validation is required.

Local safety measures.

No ring expulsions or adverse events were noted by colposcopy during the course of the study. Results from cultivation of select vaginal microorganisms in the presence and absence of the IVRs are shown on a per-macaque basis in Fig. S1 and S2 in the supplemental material. No bacterial groups were systematically enriched or reduced as a result of IVR use. Figure 3 shows time-series plots of vaginal levels of lactobacilli over the course of the study, on a per-macaque basis. IVR use had no notable effects on these levels.
FIG 3
FIG 3 Enumeration of vaginal lactobacilli recovered from six M. nemestrina individuals during the course of the IVR study. Black circles, H2O2-positive lactobacilli; gray circles, H2O2-negative lactobacilli. Dotted lines, insertion (day 0) and removal (day 28) of the IVRs. (A) TDF-FTC IVR group (n = 3); (B) TDF-FTC-MVC IVR group (n = 3).
Histopathological assessments of vaginal biopsy samples from both study groups identified only the following pathological changes. On day 22 (IVRs in place), mild-to-moderate infiltration of mononuclear cells in the vaginal lamina propria (proximal to the IVR, 2 of 3 animals; distal to the IVR, 3 of 3 animals) and mild infiltration of neutrophils in the vaginal epithelium proximal to the IVR in one of three animals were observed in the TDF-FTC-MVC group. On day 31 (IVRs removed for 3 days), mild infiltration of mononuclear cells in the vaginal lamina propria was observed in the TDF-FTC pod-IVR group (proximal to the IVR location prior to removal, 1 of 3 animals; distal to the IVR location prior to removal, 2 of 3 animals). Mild-to-moderate infiltration of mononuclear cells in the vaginal lamina propria was observed in the TDF-FTC-MVC pod-IVR group (proximal to the IVR location prior to removal, 1 of 3 animals; distal to the IVR location prior to removal, 1 of 3 animals). Minimal infiltration of neutrophils in the vaginal epithelium was observed in only one animal (TDF-FTC-MVC group) at this time point.
Cytokine levels are shown in Table S1 in the supplemental material. No statistically significant differences (P < 0.05) in cytokine levels were observed between time points for all animals with the IVR in place and with the device removed.

Summary of pharmacokinetic measurements.

The PK parameters for all ARV drugs in both study groups across key anatomic compartments are summarized in Tables 4 and 5. All drug measurements in plasma were below the analytical LLOQ (Table 2). TFV, FTC, and MVC concentrations were quantifiable in all other samples, except for one CVL cell pellet MVC measurement. TDF levels in vaginal fluid samples (83% of samples were above the LLOQ) and CVL fluid samples (93% of samples were above the LLOQ) were highly variable (Fig. 4A and 5A), due to varying degrees of hydrolysis to TFV. TDF concentrations were below the LLOQ in all tissue and CVL cell pellet samples, as expected from a previous study that observed complete hydrolysis of the TDF prodrug as it partitioned into the vaginal mucosa (14).
TABLE 4
TABLE 4 Summary of ARV drug concentrations at all sampled anatomic sites in TDF-FTC pod-IVR (n = 3) and TDF-FTC-MVC pod-IVR (n = 3) groups
Analyte and matrixan% above LLOQbMedian (SD)
ProximalcDistalc
TDF-FTC pod-IVR group    
    Vaginal fluid (μg ml−1)    
        TDF308738 (3.6 × 103)37 (3.6 × 103)
        TFV30100180 (400)110 (260)
        FTC3010012 × 103 (65 × 103)15 × 103 (72 × 103)
    CVL fluid (μg ml−1)    
        TDF15930.32 (1.3)NAd
        TFV151000.23 (0.37)NA
        FTC1510025 (54)NA
    Vaginal tissue (μg g−1)    
        TDF120BLLOQBLLOQ
        TFV1210035 (79)28 (16)
        FTC12100650 (260)460 (240)
    CVL cell pellet (μg sample−1)    
        TDF150BLLOQNA
        TFV151000.13 (0.20)NA
        FTC151002.3 (2.4)NA
TDF-FTC-MVC pod-IVR group    
    Vaginal fluid (μg ml−1)    
        TDF308712 (25)13 (45)
        TFV3010061 (64)45 (54)
        FTC301001.9 × 103 (2.8 × 103)6.6 × 103 (21 × 103)
        MVC30100610 (1.7 × 103)360 (1.8 × 103)
    CVL fluid (μg ml−1)    
        TDF15930.25 (1.1)NA
        TFV151000.16 (0.19)NA
        FTC151005.2 (8.4)NA
        MVC151001.3 (3.1)NA
    Vaginal tissue (μg ml−1)    
        TDF120BLLOQBLLOQ
        TFV1210011 (9.6)9.2 (7.1)
        FTC12100160 (260)130 (67)
        MVC1210019 (28)23 (11)
    CVL cell pellet (μg sample−1)    
        TDF150BLLOQNA
        TFV151000.065 (0.11)NA
        FTC151000.39 (0.61)NA
        MVC15930.28 (0.62)NA
a
All values correspond to time points with the IVR in place. CVL fluid and CVL cell pellet measurements are integrated over the cervicovaginal tract.
b
Proportion of samples that contained quantifiable drug levels.
c
Relative to the IVR.
d
NA, not applicable; BLLOQ, below the LLOQ.
TABLE 5
TABLE 5 Summary of key pharmacokinetic parameter values for TDF-FTC and TDF-FTC-MVC pod-IVRs
Drug(s) and sample typeMedian (SD)a
Tmax (days)Cmax (μg/ml)Cmax/D (μg/ml/mg)AUC0–t (day·μg/ml)bAUC0–∞/D (day·μg/ml/mg)Vz (ml)CL (ml/day)
Vaginal fluid       
    TDF-FTC IVR       
        TFV14 (7.2)1.2E+3 (3.1)1.3E+2 (5.7)1.5E+4 (0.82)1.7E+3 (1.8)0.10 (0.76)0.62 (0.78)
        FTC14 (10)3.0E+4 (12)4.4E+2 (16)3.8E+5 (8.7)5.5E+3 (12)0.083 (0.050)0.18 (0.09)
    TDF-FTC-MVC IVR       
        TFV11 (9.5)1.9E+2 (0.62)16 (13)2.5E+3 (1.0)2.2E+2 (2.2)0.79 (0.64)4.7 (3.6)
        FTC7.0 (9.5)1.2E+4 (2.7)4.5E+2 (8.9)1.2E+5 (2.1)4.6E+3 (6.2)0.11 (0.092)0.25 (0.17)
        MVC18 (12)1.8E+3 (3.1)79 (120)2.0E+4 (1.8)8.7E+2 (6.8)1.1 (1.1)1.2 (1.3)
Vaginal tissue       
    TDF-FTC IVR       
        TFV7.0 (7.7)45 (74)6.6 (14)6.9E+2 (6.5)69 (130)8.2 (7.3)15 (13)
        FTC7.0 (7.7)6.2E+2 (8.2)8.6 (11)9.2E+3 (10)1.4E+2 (1.4)16 (18)7.7 (6.2)
    TDF-FTC-MVC IVR       
        TFV22 (7.7)18 (6.6)1.6 (1.7)2.0E+2 (0.61)19 (17)30 (14)52 (22)
        FTC7.0 (6.1)1.7E+2 (2.4)6.1 (8.3)2.9E+3 (3.2)86 (110)19 (6)12 (6)
        MVC15 (8.2)27 (24)1.1 (0.92)3.2E+2 (2.8)14 (10)71 (20)72 (33)
a
Tmax, time to Cmax; D, dose; AUC, area under the concentration-time curve; AUC0–t, AUC from the time of dosing to the time of the last observation; Vz, volume of distribution based on the terminal phase; CL, clearance.
b
The difference between AUC0–t and AUC0–∞ was less than 1%.
FIG 4
FIG 4 Distribution of ARV drug levels in undiluted vaginal fluids proximal (black symbols) and distal (gray symbols) to the TDF-FTC pod-IVRs. (A) TDF; (B) TFV; (C) FTC. Horizontal bars, means. The IVRs were removed on day 28.
FIG 5
FIG 5 Distribution of ARV drug levels in undiluted vaginal fluids proximal (black symbols) and distal (gray symbols) to the TDF-FTC-MVC pod-IVRs. (A) TDF; (B) TFV; (C) FTC; (D) MVC. Horizontal bars, means. The IVRs were removed on day 28.

ARV drug vaginal fluid levels.

Vaginal fluid drug levels are shown in Fig. 4 and 5. The TDF prodrug was largely hydrolyzed to TFV in vaginal fluids, with the mean TFV mole fractions making up 74% (standard deviation [SD], 30%) and 84% (SD, 18%) in the TDF-FTC and TDF-FTC-MVC pod-IVR groups, respectively. These estimates assume negligible hydrolysis during sample collection, storage, processing, and analysis. Median ARV drug levels in undiluted vaginal fluids proximal and distal to the pod-IVRs were compared using a Wilcoxon matched-pairs signed-rank test (95% CI), and statistically significant differences (P < 0.05) between data sets (i.e., proximal versus distal) were noted only for TFV levels in the TDF-FTC IVR group (P = 0.0026) and FTC levels in the TDF-FTC-MVC IVR group (P = 0.0043). A statistical comparison of the vaginal fluid ARV dug levels in both study groups is provided in Fig. 6. TDF (P = 0.0494), TFV (P = 0.0103), and FTC (P < 0.0001) concentrations proximal to the IVRs all were statistically different between the groups, with higher levels achieved in the TDF-FTC group (Fig. 6A). The levels measured distal to the IVRs were statistically equivalent in the two groups (Fig. 6B), as follows: TDF, P = 0.1354; TFV, P = 0.0730; FTC, P = 0.1070. The median vaginal fluid drug levels in the TDF-FTC IVR group were consistently higher than the corresponding values in the TDF-FTC-MVC IVR group (Table 4), as expected based on the number of drug pods in the IVRs.
FIG 6
FIG 6 Comparison of ARV drug levels in undiluted vaginal fluids as a function of sampling location, using the Wilcoxon matched-pairs signed-rank test. (A) Proximal to the pod-IVRs; (B) Distal to the pod-IVRs. Black symbols, TDF-FTC pod-IVRs; gray symbols, TDF-FTC-MVC pod-IVRs; horizontal bars, means. The data set means are considered to be significantly different at a P value of <0.05.

ARV drug vaginal tissue levels.

Drug levels in vaginal tissue biopsy samples are shown in Fig. 7 and 8. Median ARV drug levels in vaginal tissue biopsy samples collected proximal and distal to the IVRs were compared individually for each analyte and IVR group using a Wilcoxon matched-pairs signed-rank test (95% CI), and no statistically significant differences between data sets were noted. An analogous statistical approach was used to compare median paired ARV drug levels in vaginal tissue biopsy samples between the study groups, and no statistically significant differences between these data sets were noted.
FIG 7
FIG 7 Distribution of ARV drug levels in vaginal tissue biopsy samples obtained proximal (black symbols) or distal (gray symbols) to the TDF-FTC pod-IVRs. Horizontal bars, means. The IVRs were removed on day 28.
FIG 8
FIG 8 Distribution of ARV drug levels in vaginal tissue biopsy samples obtained proximal (black symbols) or distal (gray symbols) to the TDF-FTC-MVC pod-IVRs. Horizontal bars, means. The IVRs were removed on day 28.

DISCUSSION

Drugs and animal model.

The current study investigates for the first time the local safety and PK characteristics of IVRs delivering double and triple ARV combinations based on TDF-FTC. TDF and FTC are used in combination (Truvada, Gilead Sciences, Inc.) for the treatment and prevention of HIV infection and represent the only FDA-approved regimen for HIV PrEP (http://www.fda.gov/NewsEvents/Newsroom/PressAnnouncements/ucm312210.htm). The three drugs evaluated here are synergistic (27) and encompass multiple mechanistic classes. TDF, a prodrug of TFV, and FTC are both nucleoside reverse transcriptase inhibitors (NRTIs). MVC is an entry inhibitor/antagonist of the chemokine receptor CCR5. The implications of our findings are discussed below in the context of developing a viable HIV PrEP candidate targeted at resource-poor regions. Many people think that rigorous safety and PK testing in nonhuman primates should be a key factor for advancing HIV PrEP candidates. The pig-tailed macaque model is particularly relevant because of its similarities with respect to the human menstrual cycle, vaginal architecture, and vaginal microbiome and the ability to conduct efficacy studies with simian-human immunodeficiency virus (SHIV) (15, 18, 19, 28).

Enabling pod-IVR design.

The ability to sustain controlled vaginal mucosal delivery of multiple ARV agents with a wide range of physiochemical properties from IVRs required a novel technology platform. The pod-IVR design consists of polymer-coated solid ARV drug cores (pods) positioned in an unmedicated ring, with delivery channels in the impermeable IVR structure exposing the pods to vaginal fluids (13). We adapted our previous macaque pod-IVR design (15) to accommodate six pods per IVR (Fig. 1), enabling the configurations shown in Table 1. Human-sized pod-IVRs can accommodate up to 10 pods, each containing cores with up to 200 mg of drug substance, totaling 2 g per IVR. The two pod-IVR formulations (TDF-FTC and TDF-FTC-MVC) evaluated here exhibited the expected (13) linear, pseudo-zero-order, in vitro release of all three ARV agents. The IVR architecture has the benefit of protecting the solid drug cores from chemical degradation, as evidenced by the complete lack of residual TDF hydrolysis following 14 days in vivo. To date, there have been no reports of IVRs delivering FTC and only two accounts of TDF-releasing IVRs (14, 18), including a TDF pod-IVR in sheep (14). In the report by Smith et al. (18), the reservoir IVR required the inclusion of an osmotic agent and showed in vitro daily TDF release rates that varied over one order of magnitude (0.4 to 4 mg day−1), in a nonlinear fashion, over the 28-day test period (18).
The novel pod-IVR design enables a number of features that are important for the development of combination ARV vaginal PrEP candidates. The drug release rates are determined by the characteristics (e.g., number, geometry, and cross-sectional area) of the delivery channels in the impermeable IVR structure and by the pods' biocompatible polymer membranes (13, 29), not by the drug loading as in conventional IVR designs. These easily controlled degrees of freedom support rapid prototype development to achieve initial target rates and efficient follow-on dose-ranging studies as candidates are expeditiously advanced through the clinical pipeline (13, 15). In a recent study, we described the design and in vivo PK characteristics of a five-drug advanced multipurpose prevention technology (MPT) pod-IVR that combined three ARV drugs from different mechanistic classes with a proven estrogen-progestogen contraceptive for prevention of HIV infection and unintended pregnancy (17). Here, we show for the first time that three drugs can be delivered at independently controlled release rates from a macaque-sized pod-IVR. These complex configurations are readily achievable due to the modular nature of the IVR design. Controlled and sustained release is independent of the ring material, which acts as a scaffold to hold the pods, offering flexibility in drug and polymer choices that may be important for future large-scale production. The successful development of IVRs for the delivery of ARV combinations for HIV PrEP will require IVRs that are safe, effective, well tolerated, user-friendly, and affordable (30, 31). The modular design of the pod-IVR is key to enabling cost-effective manufacturing, especially for IVRs containing multiple drugs, as discussed in detail elsewhere (15, 32).

Local safety of combination pod-IVRs.

Innate defenses and associated inflammation in the female genital tract mucosa generally facilitate HIV infection by compromising the integrity of the mucosal barrier as well as by recruiting and activating target cells from circulation (33, 34). A safe topical product for HIV PrEP therefore needs to minimally disrupt the normal vaginal microbiota and the underlying epithelium. Overall, vaginal facultative and anaerobic bacteria were undisturbed as a result of IVR use during the course of the study (see Fig. S1 and S2 in the supplemental material), and no systematic impact on normal fluctuations within each animal was observed. The animals were not synchronized with respect to menstrual cycle, which can have an effect on vaginal microbiota and pH. Vaginal microbiomes dominated by lactobacilli are known to play a central role in maintaining vaginal health in women (3537) and are strongly associated with reduced risks of infections by sexually transmitted pathogens, including HIV-1 (38, 39) and herpes simplex virus 2 (HSV-2) (40). The pig-tailed macaque is one of the few animal models that can also have vaginal microbiomes with large proportions of lactobacilli (41). In the current study, IVR use did not perturb the vaginal lactobacilli, as shown by the per-macaque time-series analysis presented in Fig. 3.
Mild-to-moderate infiltration of mononuclear cells and neutrophils in the vaginal epithelium and underlying lamina propria was observed for some animals at day 22 (IVR in place) and day 31 (IVR removed for 3 days). While these observations warrant further investigation with greater statistical power in future studies, they could represent normal variations due to the menstrual cycles of the animals, which are independent of the IVRs, and the mucosal ARV drug levels. Along with the proinflammatory cytokine data measured in vaginal fluids (see below), the fact that these histopathological findings were similar with the IVRs in place and 3 days after removal, with nearly complete drug washout from the vaginal tissues (Fig. 7 and 8), supports the possibility that the microscopic pathology was not IVR related. Mucosal levels of the proinflammatory cytokines remained stable throughout the study period in all animals, with no statistically significant changes being observed as a result of the IVRs. This is typical and has been observed previously in pig-tailed macaques with pod-IVRs (15, 24).

Combination pod-IVR pharmacokinetics.

Pod-IVRs were formulated to deliver either TDF-FTC or TDF-FTC-MVC combinations. Both devices maintained steady-state drug levels in vaginal fluids (Fig. 4 and 5) and tissues (Fig. 7 and 8) over the 28-day period when the IVRs were in place. Systemic exposure to all three ARV drugs was below the analytical LLOQ, an advantage of topical dosing via IVR, as the risks of systemic toxicity and the emergence of drug resistance are reduced. The TDF-FTC pod-IVR (high-dose group) afforded significantly higher drug levels than the corresponding triple-combination IVR, especially in comparisons of matched data sets proximal to the IVR (Fig. 6). Paired vaginal fluid and tissue ARV drug levels proximal and distal to the IVR on day 7 and day 22 were poorly correlated, suggesting that mucosal uptake and distribution are dependent on host characteristics such as menstrual status, the structure of the vaginal epithelium, and expression of membrane transporters (42) and ARV drug-metabolizing enzymes (43) in addition to xenobiotic physicochemical characteristics. Overall, the drug distribution in vaginal fluids and tissues was homogeneous, with few statistically significant concentration gradients extending distally from the IVR. This observation agrees with previous studies (15) and has important implications for PrEP efficacy, as significant concentration troughs could lead to zones vulnerable to HIV infection (34).
Median drug levels in vaginal fluids did not follow a trend predicted based only on compound lipophilicity. The octanol-water partition coefficient (log P) values for tenofovir disoproxil, i.e., 1.25 (pH 6.5) (44), and MVC, i.e., 2.55 (pH 7 to 8) or −0.32 (pH <7) (45), are similar over the normal vaginal pH range (pH 5.0 to 8.5) for pig-tailed macaques (46), but vaginal fluid MVC concentrations were ∼10 times higher than corresponding TFV levels (Table 4), despite similar in vivo release rates (Table 3). This interesting observation agrees with results from a pod-IVR study in sheep, in which the vaginal tissue bioavailability of TDF was found to be 86 times higher than that of TFV (14), motivating many in the field to switch from TFV to TDF for vaginal delivery. In our 2012 report, we proposed a multicompartment mechanism with TDF rapidly partitioning into the vaginal mucosa, where the prodrug hydrolyzes to TFV (14). As the drug accumulates, it is possible that the tissue TFV acts as a depot and diffuses back into the lumen, which would explain the low TDF and TFV vaginal fluid levels relative to FTC and MVC observed here.

Implications for HIV prevention outcomes.

The pod-IVR HIV PrEP strategy presented here is based on the TDF-FTC combination because oral administration has demonstrated clinical efficacy in protection from HIV infection (35), and the drugs are synergistic (47) and FDA approved for PrEP (http://www.fda.gov/NewsEvents/Newsroom/PressAnnouncements/ucm312210.htm). MVC was delivered in addition to TDF-FTC in one study group because the triple combination has the theoretical benefit of multiple sites of action targeting different stages in the HIV life cycle. The CCR5 antagonist MVC prevents virus attachment to membrane receptors, likely making the extracellular vaginal mucosa the most relevant compartment for MVC. After capsid internalization, HIV reverse transcriptase catalyzes the transcription of viral RNA into DNA. Because the polyphosphorylated metabolites of the NRTIs TFV and FTP mimic nucleotides and lead to chain termination after becoming incorporated into viral DNA (4749), the site of action is thought to be the intracellular space of immune cells capable of supporting viral replication. The threshold drug levels in these key anatomic compartments required for complete protection from productive HIV infection in women largely remain unknown.
A pharmacokinetic-pharmacodynamic (PD) analysis of data from the CAPRISA 004 clinical trial, in which a 1% TFV vaginal gel was applied pericoitally, suggests that women with TFV vaginal fluid concentrations of >1 μg ml−1 were significantly protected from HIV infection (50). Follow-on PK studies in women using single- and multiple-dose 1% TFV gel administration regimens reported the following median TFV tissue levels: at 24 h, 6 to 7 μg g−1 (51); at the end-of-period visit, 0.11 μg g−1 (52). These values are lower than the median steady-state TFV concentrations observed here for both groups (Table 4). Vaginal fluid and tissue TFV levels in the current study were comparable to (TDF-FTC-MVC) or higher (TDF-FTC) than those reported in a recent study that afforded complete protection from SHIV162p3 infection in a repeated-challenge macaque vaginal model (18).
The 50% effective concentration (EC50) values for FTC were found to be in the range of 0.0013 to 0.64 μM (0.0003 to 0.158 μg ml−1) (44), i.e., 500,000 to 1,000 times lower than the median tissue levels measured in the TDF-FTC-MVC (low-dose) group (Table 4). In women, the median FTC 24-h concentration (C24h) in vaginal tissues was 0.063 μg g−1 following oral administration of a single dose of Truvada (300 mg TDF and 200 mg FTC) (53), i.e., 10,000-fold (TDF-FTC group) or 2,500-fold (TDF-FTC-MVC group) lower than the median steady-state vaginal tissue levels of FTC measured here (Table 4). Oral Truvada has been evaluated for safety and PrEP efficacy in vaginal HIV prevention in several clinical trials (54) and has shown 73% protection in heterosexual serodiscordant couples with adherence rates of 90% or more (4). Oral daily dosing of TDF-FTC was found to completely protect six pig-tailed macaques from infection using a repeated-exposure vaginal SHIV transmission model with 18 weekly exposures, spanning 4 menstrual cycles, to low doses of SHIV162p3 (55). The median maximal concentration (Cmax) values for TFV and FTC in vaginal fluid samples were found to be 1.14 μg ml−1 and 7.23 μg ml−1, respectively, several orders of magnitude lower than the steady-state concentrations reported here (Table 4). Based on these considerations and the arguments presented above, FTC alone, as delivered from both pod-IVR formulations, may be sufficient to prevent SHIV/HIV infection.
No clinical data for HIV PrEP using intravaginal MVC administration currently exist, and the levels in the pharmacologically relevant compartments required to afford protection are therefore unknown. Various MVC gel formulations were evaluated intravaginally for efficacy in preventing SHIV infection, using the high-dose, single-challenge, rhesus macaque model (56). MVC provided dose-dependent protection against SHIV162P3 challenge, with 6 of 7 macaques receiving 0.3% (6 mM) MVC gel 30 min prior to the challenge remaining uninfected. Vaginal tissue C24h values for this formulation later were estimated at ∼0.15 μg g−1 (57). Malcolm et al. recently studied the pharmacokinetics of matrix-type, silicone-elastomer IVRs delivering MVC in rhesus macaques and reported steady-state vaginal fluid concentrations of 300 μg ml−1, while vaginal tissue levels fell consistently from 20 μg g−1 (day 1) to 2 μg g−1 (day 28) (45). The median MVC vaginal tissue levels measured here (19 to 23 μg g−1) (Table 4) were equivalent to the maximal concentrations obtained from the IVRs described by Malcolm et al. (45) and 100 times higher than the protective levels obtained from vaginal gel delivery. Dorr and colleagues measured the antiviral potencies of MVC against HIV-1 primary and laboratory-adapted isolates in peripheral blood mononuclear cells (PBMCs) and reported the following ranges of inhibitory concentrations: 50% inhibitory concentrations, 0.1 to 4.5 nM; 90% inhibitory concentrations, 0.5 to 13.4 nM (58). The observed median steady-state MVC level of 20 μg g−1 (40 μM) in vaginal tissues was 3,000 times higher than the highest 90% inhibitory concentration, suggesting possible favorable pharmacodynamic outcomes. However, in a macaque model involving rectal exposure, MVC concentrations in rectal secretions that were predicted to provide prophylactic efficacy (Cmax, 10.2 μg ml−1) failed to protect macaques from SHIV infection (26), suggesting that prophylactic efficacy cannot be easily predicted based on MVC tissue levels. Additional efficacy studies in macaques with MVC alone and in combination with other ARVs are needed.
In conclusion, topical administration of ARV combinations from pod-IVRs in pig-tailed macaques demonstrated preliminary local safety and exhibited sustained controlled drug release over 28 days. The high levels of exposure to all three drugs maintained in the vaginal mucosa suggest that both pod-IVR candidates hold significant potential for the prevention of vaginal HIV acquisition and merit further investigation in a clinical setting.

ACKNOWLEDGMENTS

Research reported in this publication was supported by the National Institute of Allergy and Infectious Diseases of the National Institutes of Health, under grants R33AI079791 (development of TDF-MVC pod-IVRs), R43AI098743 and R01AI100744 (development of TDF-FTC pod-IVRs), R43HD075636 (investigational new drug studies for TDF-FTC-MVC pod-IVRs), and R44AI081552 (fabrication of TDF pod-IVRs).
The content in this article is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. 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. The use of trade names is for identification only and does not constitute endorsement by the US Department of Health and Human Services, the US Public Health Service, or the Centers for Disease Control and Prevention.
We have no commercial or other associations that might pose a conflict of interest.
We thank Martin Beliveau at Pharsight Consulting Services, Certara, for insights in the PK analysis. We gratefully acknowledge Lorna Rabe at Magee Women's Research Institute for performing the bacterial enumeration tests. We thank Lawrence Arp and Sally Jenkins at Charles River Laboratory Pathology Associates and Robyn A. Willis at Auritec Pharmaceuticals for coordinating and interpreting the histopathology study. We thank Janet McNicholl and R. Michael Hendry for editorial comments and programmatic support for the macaque studies. We acknowledge the following members of the CDC Division of HIV/AIDS Prevention, Laboratory Branch, Preclinical Evaluation Team, for their contributions to our nonhuman primate research: David Garber, James Mitchell, Leecresia Jenkins, Shanon Ellis, and Frank Deyounks.

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Information & Contributors

Information

Published In

cover image Antimicrobial Agents and Chemotherapy
Antimicrobial Agents and Chemotherapy
Volume 58Number 9September 2014
Pages: 5125 - 5135
PubMed: 24936594

History

Received: 24 March 2014
Returned for modification: 26 May 2014
Accepted: 8 June 2014
Published online: 14 August 2014

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Contributors

Authors

John A. Moss
Department of Chemistry, Oak Crest Institute of Science, Pasadena, California, USA
Priya Srinivasan
Laboratory Branch, Division of HIV/AIDS Prevention, National Center for HIV/AIDS, Viral Hepatitis, STD, and TB Prevention, Centers for Disease Control and Prevention, Atlanta, Georgia, USA
Thomas J. Smith
Department of Chemistry, Oak Crest Institute of Science, Pasadena, California, USA
Auritec Pharmaceuticals, Inc., Pasadena, California, USA
Irina Butkyavichene
Auritec Pharmaceuticals, Inc., Pasadena, California, USA
Gilbert Lopez
Auritec Pharmaceuticals, Inc., Pasadena, California, USA
Amanda A. Brooks
Department of Chemistry, Oak Crest Institute of Science, Pasadena, California, USA
Amy Martin
Laboratory Branch, Division of HIV/AIDS Prevention, National Center for HIV/AIDS, Viral Hepatitis, STD, and TB Prevention, Centers for Disease Control and Prevention, Atlanta, Georgia, USA
Chuong T. Dinh
Total Solutions, Inc., Atlanta, Georgia, USA
James M. Smith
Laboratory Branch, Division of HIV/AIDS Prevention, National Center for HIV/AIDS, Viral Hepatitis, STD, and TB Prevention, Centers for Disease Control and Prevention, Atlanta, Georgia, USA
Marc M. Baum
Department of Chemistry, Oak Crest Institute of Science, Pasadena, California, USA

Notes

Address correspondence to Marc M. Baum, [email protected].

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