Antiretroviral Tissue Kinetics: In Vivo Imaging Using Positron Emission Tomography
ABSTRACT
Our current knowledge on the antiviral efficacy, dosing, and toxicity of available highly active antiretroviral therapy regimens is mostly derived from plasma or blood kinetics of anti-human immunodeficiency virus (anti-HIV) drugs. However, the blood comprises only 2% of the total target cells in the body. Tissue drug levels may differ substantially from corresponding plasma levels, and drug distribution processes may be characterized by high intertissue variability, leading to suboptimal target site concentrations and the potential risk for therapeutic failures. Positron emission tomography has greatly expanded the scope of the pharmacokinetic measurements that can be performed noninvasively in animal models or humans. We have prepared [18F]FPMPA, a fluorine-18-radiolabeled analogue of tenofovir, to study antiretroviral tissue kinetics in vivo noninvasively and tested the imaging probe in rats. The biodistribution of the fluorine-18 analogue closely follows that of nonfluorinated tenofovir. Compared to that in the blood, the levels of penetration of the antiretroviral drug were found to be significantly reduced in the spleen and submandibular lymph nodes (∼2-fold), in the mesenteric lymph nodes and the testes (∼4-fold), and in the brain compartment (∼25-fold). Intersubject variability of the trough drug concentration (measured at 120 min) in certain tissues, like the colon (coefficient of variation, >100%), is not reflected by the intersubject variability in the blood compartment (coefficient of variation, 24%). Positron emission tomography imaging of the fluorine-18 analogue revealed the accumulation of the antiretroviral drug in the cortex of the kidneys, a potential correlate of tenofovir-induced nephrotoxicity observed in HIV-1-infected treated patients. Thus, [18F]FPMPA is a promising radiotracer for evaluation of tenofovir biodistribution under carefully controlled drug administration protocols.
The advent of highly active antiretroviral therapy (HAART) has led to striking decreases in AIDS-related morbidity and mortality (23, 24). Most HAART-treated patients achieve viral suppression levels below 50 copies of human immunodeficiency virus type 1 (HIV-1) RNA/ml plasma within a few months of the start of antiretroviral therapy. However, these “undetectable” levels of HIV-1 RNA in the plasma do not imply that viral replication has stopped (12, 53). Whether the persistence of virus in the plasmas of viral-load-suppressed HIV-1-infected patients is the result of release of virus from a reservoir of long-lived latently infected cells or of ongoing new infections of target cells is still controversial (6, 17, 35, 38, 39, 54; S. Fiorante, S. Rodriguez-Novoa, P. Garcia-Gasco, J. Morello, F. Blanco, G. Gonzales-Pardo, A. Parra, I. Jimenez-Nacher, J. Gonzales-Lahoz, and V. Soriano, presented at the 15th Conference on Retroviruses and Opportunistic Infections, Boston, MA, 3 to 6 February 2008). The latter paradigm raises the possibility that HIV can replicate (and infect new cells) “more” undisturbed in certain sanctuary sites with suboptimal levels of drug penetration. Suboptimal concentrations of antiviral drugs in target tissues can be the result of poor patient compliance with rigid and toxic drug regimens, drug interactions, and pharmacological barriers that limit the accessibility of drugs to critical target tissues and cell reservoirs. It is known that antiretroviral compounds have differential levels of penetration in certain anatomic compartments (45, 46) and between subjects (43). While numerous examples of relationships between plasma pharmacokinetics of antiretroviral drugs and efficacy have been described (4, 22), examples of relationships between pharmacokinetics and toxicity are limited. The concentration of a drug in a specific location needs to be sufficiently high to prevent infection of new uninfected target cells, but not so high as to induce cytotoxicity of the target or nontarget cells for HIV. An improvement of the prognostic ability of drug kinetics would be expected if the drug kinetic profiles were available for tissues of specific anatomic compartments. It is currently unknown whether differential levels of penetration of drugs in certain anatomic compartments are associated with adverse toxicity events or with long-term control of viremia in HIV-1-infected treated patients, mostly because of the lack of techniques that can interrogate local drug level concentrations throughout the body noninvasively. Indeed, our current knowledge of antiretroviral drug kinetics comes primarily from studies of the blood compartment, in which only 2% of total body target cells reside (15, 51). Biopsies, for certain lymphoid organs, have frequently been applied to measure drug concentrations in tissues. However, due to the invasive nature of these procedures, it is difficult, for obvious ethical reasons, to perform longitudinal analyses of the same subject, which therefore limits our knowledge of the in vivo kinetics of antiretroviral drugs during the years of chronic treatment of HIV-1-infected patients, especially in those anatomic compartments that cannot be accessed through biopsies. A few studies of cerebrospinal fluid and seminal and extravaginal fluid kinetics have been reported, from which inferences are made regarding the central nervous system and genital tract compartments, respectively. Recently, differential levels of penetration of tritium-labeled tenofovir in subanatomic compartments of the central nervous system were reported, raising the possibility that infected microglia of deep brain sites might not be sufficiently reached by the antiretroviral drug (2).
The development of radiolabeled compounds and devices for detection of radioactivity by external imaging has expanded the use of nuclear medicine studies of drug development (13, 29, 31). Hendrix and colleagues have recently described the use of single-photon emission computed tomography for assessment of the in vivo distribution of rectal microbicidal surrogates in humans (20). In this study, we describe the biodistribution of an 18F-radiolabeled derivative of PMPA (tenofovir) (Fig. 1), a commonly used antiretroviral compound, which has low metabolism in vivo, with more than 96% of the dose recovered unchanged in urine and feces (8). Tenofovir is a nucleotide analogue that inhibits HIV reverse transcriptase and shows potent in vitro and in vivo activity against HIV (3, 9). It is converted intracellularly by cellular kinases to form the active tenofovir diphosphate necessary for antiretroviral activity. In vitro data have also shown that higher levels of intracellular concentration of tenofovir are associated with higher levels of the mono- and diphosphate moieties (30). We propose that this 18F-labeled analogue can be utilized to study antiretroviral drug distribution throughout the body noninvasively with positron emission tomography (PET) imaging (27).
MATERIALS AND METHODS
Radioligands and pharmaceuticals.
(S)-[18F]FPMPA {S-(1-(6-amino-9H-purin-9-yl)-3-[18F]fluoropropan-2-yloxy)methylphosphonic acid} was prepared as previously described (27). [14C]PMPA was purchased from Movarek Biochemicals and Radiochemicals (Brea, CA). PMPA was provided by the National Institutes of Health (NIH) AIDS Research and Reference Reagent Program.
Animals.
A total of 22 male Sprague-Dawley rats (Harlan Sprague Dawley, Indianapolis, IN) were housed in pathogen-free cages. They were acclimatized for at least 3 days in an animal facility in which temperature, humidity, and light cycle (light from 6:00 a.m. to 6:00 p.m.) were controlled, with ad libitum access to food and water. All animal studies were conducted in accordance with the principles and procedures outlined in the NIH Guide for the Care and Use of Animals and approved by the NIH Institutional Animal Care and Use Committee.
Ex vivo biodistribution studies.
Twelve male Sprague-Dawley rats (250 to 275 g) received a bolus intravenous injection through the lateral tail vein of a phosphate-buffered-saline (PBS) solution containing 5 mg/kg of body weight of unlabeled PMPA (11), 0.185 MBq of [14C]PMPA, and 7.4 MBq of (S)-[18F]FPMPA (250-μl total volume) and were left awake until sacrifice. The 12 rats were divided into four groups of 3 each, and each group was sacrificed by carbon dioxide asphyxiation at 10, 30, 60, and 120 min postinjection. Samples of the left/right submandibular, left/right popliteal, and mesenteric lymph nodes; spleen; lung; kidney (cortex); liver; jejunum; colon; Peyer's patches; brain; femur; testes; and blood were harvested and weighed into scintillation vials. The [18F]fluoride radioactivities in blood samples and various tissues were assayed by placing the vials in an automatic gamma counter (model 1480 Wallac Wizard; PerkinElmer, Turku, Finland). To determine 14C activity, the samples were solubilized for 6 days in 1.5 ml of Solvable (Packard, Meriden, CT) at room temperature. Tissues were minced in the same vials used for detection of [18F]fluoride radioactivity to aid the solubilization process. All samples were prepared further for scintillation counting by bleaching them with 300 μl of H2O2 and neutralizing them with 50 μl of 10N HCl, followed by addition of 16 ml of a scintillation cocktail (Aquasol; PerkinElmer). Samples were dark adapted and counted for 5 min each in a scintillation counter (PerkinElmer Winspectral 1414). Standards of 1:250 and 1:2,500 of the injected dose were prepared and counted along with all samples. Background counts were subtracted from reported 18F count-per-minute and 14C disintegration-per-minute values. The injected counts were determined from the standard counts, and the quantitative data, expressed as percent injected dose (%ID) per gram of tissue, were determined by the following equation: %ID/g of tissue = {sample count/[sample weight (g) × injected count]} × 100. The mean and the standard deviation (SD) for %ID/g were calculated for all the samples in each group for both 18F and 14C.
An additional group of four rats (260 to 270 g) was used to assess changes in the tissue biodistribution of (S)-[18F]FPMPA between rats left awake until sacrifice and those anesthetized with isoflurane (1.5 to 2%, vol/vol, in O2) during the PET imaging studies. These rats were coinjected intravenously through the lateral tail vein with PBS containing 5 mg/kg of unlabeled PMPA and 7.4 to 10 MBq of (S)-[18F]FPMPA in 115 to 155 μl and were sacrificed by carbon dioxide asphyxiation at 120 min postinjection. This group of four rats was gathered with the previous group of three rats analyzed 120 min after radiotracer injection in the biodistribution study to estimate the mean %ID/g at 120 min (%ID120_min/g) in the absence of anesthesia (awake group [n = 7]). Samples of spleen, lung, kidney (cortex and medulla), liver, jejunum, colon, and blood were harvested. The collected tissues were weighed into scintillation vials, and the radioactive [18F]fluoride contents of the blood samples and various tissues were assayed using an automatic gamma counter (model 1480 Wallac Wizard; PerkinElmer).
PET studies.
Six male (262 to 318 g) Sprague-Dawley rats were anesthetized using isoflurane (1.5 to 2%, vol/vol, in O2) and coinjected intravenously through the penile vein with 5 mg/kg of unlabeled PMPA and (S)-[18F]FPMPA in 100 to 380 μl of PBS. Three rats (no. 3, 5, and 6) received 7.4 to 8.4 MBq of (S)-[18F]FPMPA, and the remaining three rats (no. 1, 2, and 4) received 14.1 to 26.2 MBq of (S)-[18F]FPMPA. PET scans were performed using an Advanced Technology Laboratory Animal Scanner device (44). The scanner has a computer-controlled animal bed with a transverse field of view of 6.8 cm and an axial field of view of 2 cm. Series of whole-body acquisitions were initiated about 3 min after radiotracer injection and recorded with a 100- to 700-keV energy window. The whole-body acquisition started at the level of the ocular orbit, and then scanning continued downwards in 2-cm increments until the level of kidney was reached (seven bed positions, 4 min each). Upon completion of one scanning cycle, the rat was repositioned to the starting point, and consecutive scans were performed by repeating the same procedure. Four whole-body acquisitions were obtained per rat for a total scanning time of 130 to 140 min. At the end of the study, a cylindrical 18F source of known activity was imaged to obtain MBq of 18F per count per second (cps) for the imaging system (calibration factor).
The images were reconstructed by a two-dimensional ordered-subsets expectation maximum algorithm (52); no correction was applied for attenuation or scatter. The reconstructed spatial resolution was ∼1.8 mm. For each acquisition, regions of interest (ROI) were drawn over the major organs (brain, lung, heart, liver, spleen, kidney cortex, kidney medulla, and neck/shoulder muscle). The maximum radioactivity accumulation within the organ was obtained from the mean pixel values for the multiple ROI. The results were calculated as %ID/g by multiplying the cps per ml for each ROI by the calibration factor (MBq 18F/cps) determined for the scanner and dividing this product by the value for injected radioactivity. Tissue density was assumed to be 0.3 g/cm3 (32, 41) for the lung and 1 g/cm3 (47) for the other tissues. Then, the value for every ROI (cps per cubic centimeter) was multiplied by this factor and divided by the value for injected activity. At the end of the PET study, the rats were sacrificed, selected organs (left/right submandibular, left/right popliteal, and mesenteric lymph nodes; spleen; lung; kidney [cortex and medulla]; liver; jejunum; colon; Peyer's patches; brain; and testes) and blood were removed and weighed, and the radioactivity was determined using a gamma counter together with the standards (1/100 of the injected dose). Radioactivity was expressed as %ID/g of tissue. Whole blood was collected in heparinized sampling tubes, and the plasma was separated from the pellet in a microcentrifuge (4,000 rpm, 3 min). Data are expressed as mean ± SD. The group of rats anesthetized during the entire period of observation (anesthetized group [n = 6]) and the group of rats left awake (awake group [n = 7]) were used to study the effect of anesthesia on the mean %ID120_min/g tissue.
Cellular internalization studies.
The spleens of four rats harvested at the end of the PET imaging study (120 min) were mechanically disrupted by pressing them through a 70-μm-pore-size cell strainer (Fisher Scientific, Pittsburgh, PA). The extracted cells were suspended in 40 ml of complete RPMI medium, and the total radioactivity was determined using the gamma counter. Erythrocytes were lysed with ACK lysing buffer (BioWhittaker), and spleen cells were washed with complete RPMI medium or complete RPMI medium enriched with cold PMPA (∼1 mM). Cells were stained with trypan blue to count the number of total viable cells. After ACK lysing, the radioactivity of the collected cells resuspended in 1 ml PBS was redetermined using the gamma counter.
Statistical analyses. (i) Comparisons between the %IDs/g calculated with the two probes.
The limits of agreement between the %IDs/g calculated with (S)-[18F]FPMPA and [14C]PMPA were obtained from the 99% confidence interval (CI) for the mean log of the ratios between the two measurements (Fig. 2C). Simple linear regression analysis was performed to estimate the slope and intercept of the relationship between the %IDs/g calculated with (S)-[18F]FPMPA and [14C]PMPA. In order to account for the within-rat correlation, the 99% CI for the slope and intercept of the linear regression was obtained using a bootstrap procedure by randomly sampling with replacement the 12 rats and recomputing the slope and intercept. This procedure was repeated 10,000 times, and the distribution of slopes and intercepts was generated, from which the 99% CI was calculated.
(ii) Comparisons between AUC for the blood and various organs.
The areas under the curve (AUC) for each organ were first obtained for each probe by linear interpolation between consecutive time points for the mean %IDs/g to compare the estimates of AUC generated from each probe. The relationship between the AUC for the blood and the AUC for various organs was then analyzed by defining the mean %ID/g of tissue at each measurement as the mean value obtained from the two probes (18F and 14C labeled); the mean AUC was then calculated from the mean %ID/g of tissue. The statistical significance of the difference between the mean AUC for a given organ and the mean AUC for the blood was assessed with an exact test in which the mean %ID/g at each time point was randomly switched between the two organs of a given rat. This permutation procedure was repeated 10,000 times, and the distribution of the difference in mean AUC between the two organs was generated to determine the P value for the difference observed from the original data (Fig. 3A).
The two-sample Wilcoxon rank sum test was used to compare the %ID120_mins/g for the groups of awake and anesthetized rats at those sites for which at least four rats per group were available (blood, spleen, lungs, kidneys, liver, jejunum, and colon), followed by Bonferroni correction for multiple tests (Fig. 3B). The outlier test is based on the maximum normalized residual observed after log transformation of the data for reduction of skew when needed. The Wilcoxon signed-rank test was used to compare the %ID120_mins/g of the cortex and medulla subcompartments of the kidneys. The Spearman rank correlation test was used to assess correlations between variables. The slopes for %ID/g between 60 min and 120 min were defined as [ln(%ID120_min/g) − ln(%ID60_min/g)]/60. To account for the high number of tests performed, P values of <0.01 were considered statistically significant.
RESULTS
Synthesis of (S)-[18F]FPMPA.
The products of radiosynthesis had high radiochemical and enantiomeric purity, as demonstrated by high-performance liquid chromatography analysis (27).
Ex vivo biodistribution of (S)-[18F]FPMPA and [14C]PMPA.
In order to validate the use of the (S)-[18F]FPMPA radiotracer to image the in vivo pharmacokinetics of tenofovir, first we compared the biodistribution of tenofovir with the biodistribution of [14C]PMPA after coadministration of the two radiotracers with the equivalent of a pharmacological dose of unlabeled drug for rodent models. The data revealed a good overlapping of the biodistributions of (S)-[18F]FPMPA and [14C]PMPA in most organs (Fig. 2A and B). Figure 2C shows the mean log of the ratios between the %IDs/g calculated using (S)-[18F]FPMPA and [14C]PMPA. For the majority of organs, the two measurements were not statistically significantly different (P < 0.01). The lack of difference in uptake of (S)-[18F]FPMPA and [14C]PMPA in the femur, a tissue characterized by high avidity for unbound fluoride (19), suggests that [18F]fluoride does not significantly disassociate from the tracer in vivo. Similar estimates of AUC during the 120-min biodistribution (AUC120_min) were obtained from (S)-[18F]FPMPA and [14C]PMPA in the majority of organs (data not shown). In contrast, higher and lower retentions of (S)-[18F]FPMPA than of [14C]PMPA were observed for the kidney and lungs (Fig. 2C), respectively (P < 0.01), with the discrepancy increasing to approximately 0.5 log at later time points (Fig. 2B), probably as result of differential accumulation of metabolites in these organs or differential affinities of the two probes for transporters. The %ID/g of tissue calculated using (S)-[18F]FPMPA (0.0087% ± 0.0075% [mean ± SD]) was higher than that calculated using [14C]PMPA (0.0047% ± 0.0035% [mean ± SD]) in the brain (mean ratio, 0.25 log; P < 0.01), which appeared to be the result of suboptimal solubilization of this tissue, with the beta counter showing consequentially lower efficiency for detection of low levels of radioactivity (Fig. 2C). Finally, for one of the five lymph nodes analyzed (the left submandibular lymph node), the %ID/g of tissue calculated using [14C]PMPA was 0.14 log higher than the %ID/g of tissue calculated using (S)-[18F]FPMPA, with this difference barely reaching statistical significance (Fig. 2C). For the remaining organs, no difference was observed between the %IDs/g of tissue calculated using the two probes.
The linear regression of %ID/g calculated from [14C]PMPA data versus %ID/g calculated from (S)-[18F]FPMPA data for the 13 tissues analyzed (all except kidney, lung, and brain) revealed a high R2 value (0.99), with the estimate for the slope not statistically significantly different from 1 (slope, 0.984; 99% CI, 0.968 to 1.057) and the intercept not statistically significantly different from 0 (intercept, 0.0009; 99% CI, −0.0049 to 0.0058), thus suggesting a high degree of predictability of [14C]PMPA biodistribution on the basis of (S)-[18F]FPMPA measurements. Finally, a highly statistically significant Spearman rank correlation was found between the slopes calculated from [14C]PMPA and (S)-[18F]FPMPA probes of the %IDs/g of tissues measured between 60 and 120 min, which is our closest estimate of the mean drug clearance rate for these anatomic compartments (ρ = 0.76; P < 0.01).
Blood compartment versus various organs.
Measurements of mean AUC120_min obtained for the average population of rats revealed differential levels of penetration of tenofovir in lymphoid tissues and anatomic compartments (Fig. 3A). Using the mean %ID/g of tissues obtained from the two radiolabeled probes, we found that the lowest level of penetration was observed in the brain compartment (∼25-fold lower than that in the blood; P < 0.01) (Fig. 3A), followed by the mesenteric lymph nodes and the testes (∼4-fold; P < 0.01) and the spleen and the submandibular lymph nodes (∼2-fold; P < 0.01). Similar or increased levels of exposure of the tissues to the drug, compared to the levels for the blood, were observed in the gut compartments and popliteal lymph nodes, though the difference in mean AUC120_min between these compartments and the blood did not reach statistical significance (Fig. 3A).
Similar conclusions derive from the variability of mean %ID120_min/g for each tissue, due to highly statistically significant correlation between this variable and the mean AUC120_min (n = 16; r = 0.89; P < 0.01). The mean %ID120_min/g in the spleen, lungs, brain, femur, lymph nodes, jejunum, and colon, normalized to the level for the blood, was found to be remarkably similar to the ratio observed in the same tissues, as described by Lee and colleagues (30), calculated from the biodistribution of [14C]PMPA at 24 h following tenofovir administration in dogs (data not shown).
Effect of anesthesia.
The effect of anesthesia was assessed by comparing the %IDs/g calculated using (S)-[18F]FPMPA at 120 min postinjection (%IDs120_min/g) for different tissues for the group of seven rats left awake until the time of sacrifice (awake group) and the group of six rats sacrificed at the end of the imaging acquisition (anesthetized group). The mean ± SD %ID120_min/g in each group is shown in Fig. 3B. The anesthesia appeared to reduce the mean %ID120_min/g for the kidneys approximately 50% (11.9% ± 1.9% [anesthetized group] versus 23.3% ± 2.5% [awake group] [mean ± SD]; P < 0.05).
Intersubject variability.
To address the overall intersubject variability in %ID120_min/g for the 18F probe in different organs, we first identified the presence of outliers for each tissue. The %ID120-min/g of colon was 0.986% in 1 of the 13 analyzed rats, while the mean and SD for the remaining 12 rats were 0.041% and 0.043%, respectively; thus, the level of colon uptake in this rat was 22 SD from the mean for the remaining rats (outlier test; P < 0.01). Similarly, two other rats showed atypical uptakes in the blood (11 SD from the mean; P < 0.01) and in the jejunum (17 SD from the mean; P = 0.02). The coefficients of variation for each organ, obtained by excluding these three outliers (one for the blood, one for the jejunum, and one for the colon), are shown in Table 1. The coefficient of variation for the blood (24%) was approximately threefold lower than the mean coefficient of variation observed for the lymph nodes (77%). The highest coefficients of variation were observed in the gut subcompartments, with the colon showing a coefficient of variation greater than 100%. No significant correlations were observed between the %ID120_min/g in the blood and that in the lymphoid or extralymphoid organs analyzed (Table 1), thus suggesting that the intersubject variability in accumulation of the drug in the organs is not related to the intersubject variability observed in the blood compartment.
Intracellular retention and plasma concentration.
The %ID120_min/g of plasma samples obtained from four rats utilized in the PET studies was 0.026% ± 0.011% (mean ± SD), and the mean ratio of concentration between blood cells and plasma samples was 0.45 ± 0.09 (mean ± SD). In this group, the spleens were harvested and the intracellular retention of (S)-[18F]FPMPA was obtained after erythrocytes were lysed from the extracted spleen cells. The mean ratio of intracellular versus extracellular concentration of (S)-[18F]FPMPA was 0.34 ± 0.13 (mean ± SD).
PET imaging.
In order to better image the penetration of the antiviral drug in the lymphoid organs, including the spleen, two different radioactivity doses were used. Three rats (no. 3, 5, and 6) were coinjected with a low-radioactivity dose (7.4 to 8.4 MBq), and the remaining three rats (no. 1, 2, and 4) were coinjected with high-radioactivity dose (14.1 to 26.2 MBq) of (S)-[18F]FPMPA. Bone retention appeared at the background radioactivity level, independently of the injected radioactivity dose, confirming that free fluoride is not a major metabolite of (S)-[18F]FPMPA biodistribution in vivo. Figure 4 shows the kinetics of the antiretroviral drug during the time of imaging for each organ, expressed as %ID/g of organ. Similar to what was observed in the kinetics of the average population of rats, as inferred from the ex vivo biodistribution studies whose results are shown in Fig. 2, we observed in the PET-imaging kinetics low levels of penetration of the drug into the brain compartment, which had kinetics of drug accumulation similar to those for the heart and muscle, with the median peak of concentration observed within the first 20 min, followed by rapid decay. In the spleen, a slower accumulation of antiviral drug than in the blood, followed by a slower clearance, was observed in four out of six rats, consistent with the splenic kinetics of tenofovir obtained for the average population of rats (Fig. 2B), probably as a result of intracellular drug retention. The median peak concentration in the spleen of ∼0.14% ID/g was also similar to the Cmax inferred from the ex vivo biodistribution study.
PET imaging also confirmed the accumulation of tenofovir in the kidney, with plateau kinetics in all rats, and revealed that such accumulation is confined within the cortex (Fig. 5). This was confirmed by ex vivo determination of %ID120_min/g at the end of the imaging study. The %ID120_min/g of kidney cortex was significantly higher than the %ID120_min/g of kidney medulla (mean ratio, 16.9; P < 0.01). The %ID120_min/g of urine was determined for three rats in the imaging group and was 67.6% ± 7.35% (mean ± SD). The plateau level of %ID/g of kidneys was ∼50% lower for the three rats coinjected with high-radioactivity doses than for the three rats coinjected with low-activity doses (data not shown), consistent with the significant loss of counts due to the dead-time effect of the camera, as revealed by the higher acquired counts per unit time. In contrast, no differences in %ID/g or AUC observed in the other organs between the low- and high-radioactivity-dose groups were noted.
DISCUSSION
Using ultrasensitive assays for determination of plasma viral load in HIV-1-infected patients has revealed that the average set point for the virus 1 year following the initiation of HAART is approximately 5 HIV-RNA copies/ml of plasma (18). Given the fast clearance of viral particles in plasma (37), continuous replication of the virus from reservoirs (in which the virus is harbored in a latent form) or sanctuaries (in which suboptimal drug concentrations inefficiently curtail additional rounds of viral replication and infection) is needed to sustain this low steady-state level of viral replication in the plasma. The cumulative risk of viral load failure has been estimated to be ∼40% by 6 years from the initiation of therapy (36). Viral rebound of drug resistance strains can in principle be explained without invoking suboptimal drug therapy concentrations in sanctuary sites, as pretherapy-existing resistant strains can invade and outcompete the drug-sensitive strains (42). The number of pretherapy-existing multidrug-resistant strains in a patient is, as intuition suggests, inversely related to the number of mutations required for a given virion to be resistant to all the drugs contained in a given regimen. Thus, long-lived infected-cell reservoirs archiving replication-competent HIV-1 proviruses with drug resistance signatures can be stochastically activated even in the setting of optimally treated patients and generate new virions that can disseminate in tissues with the same rate of growth that would occur if this were happening in the absence of antiretroviral therapy. However, this theory, together with the evidence of long-lived latently infected cells, while pointing to the intrinsic persistence of the virus in the body or, equivalently, to our inability to eradicate it, does not exclude or challenge the possibility that suboptimal therapy can indeed accelerate the emergence of drug-resistant strains in treated patients. Indeed, spatial heterogeneity in the distribution of the drugs used in an antiretroviral regimen may facilitate the evolution of resistance, as the virus may be controlled locally by a less potent regimen (26). Thus, if some drugs do not penetrate in a given anatomic compartment well, the probability that a pretherapy-existing strain resistant to those drugs is selected after its random activation in the archived reservoir increases exponentially. If virions with different resistance signatures are generated from independent anatomic compartments with differential suboptimal concentrations of the drug regimens, there is a certain probability that, by recombination (1, 50), a multidrug resistance strain is generated, leading to the failure of the antiretroviral regimen. Thus, collecting as much information as we can from drug penetration in anatomic compartments may help to elucidate factors associated with the long-term control of viremia in HIV-1-infected patients, including the emergence of multidrug-resistant strains.
Most antiretroviral agents currently licensed for the treatment of HIV target two viral enzymes, HIV reverse transcriptase and HIV protease. In order to be effective, these drugs must penetrate the lipid membrane bilayer to reach their viral target in cells and be present at the active site at concentrations sufficient to prevent viral replication. The more lipid soluble the compound is, the greater is the ability of the agent to cross these barriers. The lower the drug disposition in a given anatomic compartment is, the lower is the intracellular concentration of the drug. Moreover, in addition to the emergence of resistant viral mutants, which, as explained above, might be, in principle, favored by differential drug penetration in a given compartment, cellular factors have also been suggested to play a role in declining antiviral activity (14, 48). Thus, beyond physicochemical characteristics (e.g., lipophilicity and charge) and plasma protein binding, which determine the extracellular concentrations of drugs in a given anatomic compartment, the induction of active transporters, such as P glycoprotein and the more recently discovered family of multidrug resistance proteins (MRPs) (14), can also modulate the intracellular tissue concentration. It is also unknown whether, in tissues, induction of active efflux pumps due to prolonged exposure to antiretroviral therapy may explain the waning HAART efficacy during the years of chronic antiviral therapy, thus mimicking known paradigms of drug failure in anticancer and antibacterial chemotherapy (16, 21).
In this study, we have used 18F-labeled tenofovir to image the distribution of the antiretroviral compound in vivo in Sprague-Dawley rats. We have recently shown that the labeled version of this compound retains the inhibitory activity of the unlabeled compound in vitro (27). We found that 18F-tenofovir kinetics in anatomic compartments show a good overlapping with the kinetics of 14C-tenofovir (Fig. 2), suggesting that the former agent is a candidate tracer for imaging the kinetics of this compound in vivo by using PET. In lungs and kidneys, we observed an approximately twofold deviation between the %IDs/g of tissue measured with the (S)-[18F]FPMPA- and [14C]PMPA-radiolabeled probes, probably as a result of differential accumulation of metabolites in these organs or differential affinities of the two probes for transporters. For the remaining organs, including all the lymphoid organs analyzed, the 18F-radiolabeled probe could predict the distribution in vivo of [14C]PMPA. The ex vivo biodistribution studies revealed differential levels of penetration of the drug in anatomic compartments in the average population of rats, with the lowest level of penetration observed in the brain (∼25-fold lower than that in the blood), followed by the mesenteric lymph node and testis compartments (∼4-fold lower than that in the blood) and the spleen and submandibular lymph nodes (∼2-fold lower than that in the blood). These data are consistent with the recently reported 0.55 ratio of intracellular levels of tenofovir diphosphate in lymph nodes versus those in peripheral blood mononuclear cells, observed in HIV-1-infected treated patients (C. Fletcher, T. King, L. Bushman, J. Kiser, P. Anderson, J. Brenchley, D. Douek, and T. Shacker, presented at the 15th Conference on Retroviruses and Opportunistic Infections, Boston, MA, 3 to 6 February 2008). A higher level of exposure of the tissues to the drug was observed in the gut compartments, consistent with observations derived from the biodistribution of 14C-tenofovir in dogs (30). Similar conclusions were derived from the variability of mean %ID120_min/g for each compartment. Three rats that showed high levels of uptake of the compound in isolated compartments (blood, colon, and jejunum) were identified as outliers, with high statistical significance, possibly suggesting the existence of host-specific factors affecting the distribution in vivo of the drug (28, 43). The intersubject variability in accumulation of the compound at 120 min (measured as coefficient of variation of %ID120_min/g) in the blood (24%) was approximately threefold lower than the intersubject variability for the lymph nodes (77%). The highest coefficients of variation were observed in the gut subcompartments, with the colon showing more then 100% coefficient of variation. No significant correlations were observed between the %ID120_min/g for the blood and that for the lymphoid or extralymphoid organs analyzed (Table 1), suggesting that the variability in accumulation of the drug in organs is not readily revealed by the variability observed in the blood compartment. It has been reported that approximately 15% of the total lymphocytes in the body reside in the spleen, the largest reservoir of lymphocytes (and thus of target cells for HIV-1) in the body (15, 51); thus, splenic kinetics of antiretroviral drugs are expected to be stronger predictors of drug antiviral efficacy in in vivo models of HIV-1 pathogenesis than blood kinetics. PET images of 18F-tenofovir revealed the subject-specific kinetics of the compound in those anatomic compartments, with volumes of interest sufficiently high to allow their identification in rodent models in the absence of computed tomography scans (Fig. 4).
A pattern of continuous accumulation of the drug over time was observed in the kidneys, and this pattern was not previously reported for other fluorinated compounds. For instance, the time-activity curves for 18F-fluoro-deoxy-d-glucose (FDG) in humans (10) or 18F-fluoro-d-arabinofuranosyl (FIAU) in dogs (34) show evidence of peak activity within the first 10 to 20 min after radiolabel injection, followed by rapid clearance, similar to what is observed in the blood compartment. PET image kinetics confirmed the accumulation of tenofovir in the kidney, with plateau kinetics, and revealed that such accumulation is confined within the cortex (Fig. 5), as confirmed by ex vivo determination of %IDs120_min/g of the cortex and medulla subcompartments of the kidney obtained at the end of the imaging study. Accumulation of tenofovir in the kidney cortex has not previously been reported to occur in animal models or humans, although it is known that nephrotoxicity can result from its use in HIV-infected patients. Thus, the kidney cortex appears to be in a “deep” tissue pharmacokinetic compartment that equilibrates slowly with the blood compartment. Given that the amount of drug in the kidney cortex, measured as a fraction of the dose administered, is severalfold higher than the amounts of drug in the blood and in various tissues, it is apparent that tenofovir is preferentially distributed to this tissue compartment. Active transporters of tenofovir in tubule cells have recently been characterized and may play a role in the process of drug accumulation in the kidney cortex. Human organic anion transporter 1 (hOAT1), expressed on the basolateral side of the renal proximal tubule cells, has been shown to mediate the uptake of tenofovir, adefovir, and cidofovir (7, 49), while MRP2 and MRP4, expressed on the apical side, have been proposed to mediate the efflux of tenofovir from cells (40). Recently, Kiser and colleagues showed that single-nucleotide polymorphisms for the ABCC4 gene encoding MRP4 are associated with higher intracellular levels of tenofovir diphosphate (28). It has been reported that tenofovir-containing antiretroviral regimens are associated with an increased risk of chronic renal failure in HIV-1-infected treated patients (33). The mechanisms underlying tenofovir-induced nephrotoxicity are unknown. It has been speculated that competition for MRP4, as a result of interaction with other drugs, might interfere with tenofovir exit and lead to tubular cell accumulation and toxicity (25). However, on the basis of the observed plateau kinetics of tenofovir in the kidney cortex, a possible mechanism at play is saturation of uptake transporters such that entry of the drug into cells occurs by a zero-order process over the time course of these experiments while efflux mechanisms continue to exhibit first-order kinetics for drug removal. A similar mechanism may be operational in the liver, although the tenofovir levels that we observed were an order of magnitude lower than those in the kidney cortex. This appears consistent with the observation of the higher levels of OAT1 and OAT3 (high- and low-affinity influx transporters for tenofovir) found in the kidneys than in the livers of Sprague-Dawley rats (5).
In conclusion, noninvasive in vivo total body imaging of antiretroviral compounds may provide a better understanding of the relationships between drug- and host-specific kinetics and pharmacodynamics, including antiviral efficacy and drug-induced toxicity, especially in the settings of longitudinal analyses under carefully controlled drug administration protocols.
FIG. 1.
FIG. 2.
FIG. 3.
FIG. 4.
FIG. 5.
TABLE 1.
| Site | No. of rats | CV | SR | P |
|---|---|---|---|---|
| Blood | 12 | 24.1 | ||
| Spleen | 13 | 39.7 | 0.252 | 0.215 |
| Lungs | 13 | 68.5 | 0.105 | 0.373 |
| Brain | 7 | 62.8 | 0.393 | 0.192 |
| Kidney | 0.441 | 0.076 | ||
| Awake group | 7 | 10.7 | ||
| Anesthetized group | 6 | 16.4 | ||
| Medulla | 9 | 109.5 | −0.083 | 0.416 |
| Liver | 13 | 16.3 | 0.259 | 0.208 |
| Femur | 3 | 18.7 | NA | NA |
| Left submandibular lymph node | 7 | 61.7 | −0.306 | 0.252 |
| Right submandibular lymph node | 7 | 83.5 | −0.643 | 0.060 |
| Mesenteric lymph node | 7 | 78.4 | −0.071 | 0.440 |
| Left popliteal lymph node | 6 | 76.5 | NA | NA |
| Right popliteal lymph node | 6 | 84.9 | NA | NA |
| Jejunum | 12 | 79.8 | 0.391 | 0.117 |
| Colon | 12 | 103.6 | −0.409 | 0.106 |
| Peyer's patches | 7 | 90.3 | 0.071 | 0.440 |
| Testes | 7 | 70.7 | 0.321 | 0.241 |
a
CV, coefficient of variation (SD/mean); SR, Spearman rank correlation (with P values) between the %ID120_min/g in each organ and the %ID120_min/g in the blood; NA, not applicable.
Acknowledgments
This work was supported in part by the Intramural Research Programs of the National Institute of Allergy and Infectious Diseases and the National Institute of Biomedical Imaging and Bioengineering. Additional funding has been provided by the National Cancer Institute, NIH, under contract no. HHSN261200800001E.
The content of this publication does not necessarily reflect the views or policies of the Department of Health and Human Services, nor does the mention of trade names, commercial products, or organizations imply endorsement by the U.S. Government.
We acknowledge the Positron Emission Tomography Department of the NIH Clinical Center for Radioisotope production. We thank Ruy Ribeiro, Michael Proschan, and Dean Follmann for their useful comments on the manuscript.
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Antimicrobial Agents and Chemotherapy
Volume 53 • Number 10 • October 2009
Pages: 4086 - 4095
PubMed: 19667288
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© 2009 American Society for Microbiology.
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Received: 27 March 2009
Revision received: 28 May 2009
Accepted: 31 July 2009
Published online: 1 October 2009
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