Antiretroviral Drug Concentrations in Lymph Nodes: A Cross-Species Comparison of the Effect of Drug Transporter Expression, Viral Infection, and Sex in Humanized Mice, Nonhuman Primates, and Humans

Abstract

In a "kick and kill" strategy for human immunodeficiency virus (HIV) eradication, protective concentrations of antiretrovirals (ARVs) in the lymph node are important to prevent vulnerable cells from further HIV infection. However, the factors responsible for drug distribution and concentration into these tissues are largely unknown. Although humanized mice and nonhuman primates (NHPs) are crucial to HIV research, ARV tissue pharmacology has not been well characterized across species. This study investigated the influence of drug transporter expression, viral infection, and sex on ARV penetration within lymph nodes of animal models and humans. Six ARVs were dosed for 10 days in humanized mice and NHPs. Plasma and lymph nodes were collected at necropsy, 24 hours after the last dose. Human lymph node tissue and plasma from deceased patients were collected from tissue banks. ARV, active metabolite, and endogenous nucleotide concentrations were measured by liquid chromatography-tandem mass spectrometry, and drug transporter expression was measured using quantitative polymerase chain reaction and quantitative targeted absolute proteomics. In NHPs and humans, lymph node ARV concentrations were greater than or equal to plasma, and tenofovir diphosphate/deoxyadenosine triphosphate concentration ratios achieved efficacy targets in lymph nodes from all three species. There was no effect of infection or sex on ARV concentrations. Low drug transporter expression existed in lymph nodes from all species, and no predictive relationships were found between transporter gene/protein expression and ARV penetration. Overall, common preclinical models of HIV infection were well suited to predict human ARV exposure in lymph nodes, and low transporter expression suggests primarily passive drug distribution in these tissues. SIGNIFICANCE STATEMENT: During human immunodeficiency virus (HIV) eradication strategies, protective concentrations of antiretrovirals (ARVs) in the lymph node prevent vulnerable cells from further HIV infection. However, ARV tissue pharmacology has not been well characterized across preclinical species used for HIV eradication research, and the influence of drug transporters, HIV infection, and sex on ARV distribution and concentration into the lymph node is largely unknown. Here we show that two animal models of HIV infection (humanized mice and nonhuman primates) were well suited to predict human ARV exposure in lymph nodes. Additionally, we found that drug transporter expression was minimal and-along with viral infection and sex-did not affect ARV penetration into lymph nodes from any species.

Fig. 1.

Cross-species differences in ARV concentrations in plasma (A) (nanograms per milliliter) and lymph nodes (B) (nanograms per gram tissue) from mice (blue boxes), NHPs (purple boxes), and humans (green boxes). Boxes are the 25th percentile, median, and 75th percentile. Whiskers are the fifth and 95th percentiles, and dots are outliers. Dashed lines indicate the lower limit of quantitation. Sample sizes for each drug in each species are given in Supplemental Table 2. Between-species differences were analyzed using Kruskal–Wallis one-way ANOVA with Dunn’s method correction for multiple comparisons. *P < 0.05.

Fig. 2.

Cross-species differences in ARV TPRs in mice (blue boxes), NHPs (purple boxes), and humans (green boxes). Boxes are the 25th percentile, median, and 75th percentile. Whiskers are the fifth and 95th percentiles, and dots are outliers. The dashed line indicates equal drug concentrations in lymph node and plasma. Sample sizes for each drug in each species are given in Supplemental Table 2. Between-species differences were analyzed using Kruskal–Wallis one-way ANOVA with Dunn’s method correction for multiple comparisons. *P < 0.05.

Fig. 3.

Cross-species differences in active metabolite and endogenous nucleotide concentrations (nanograms per gram tissue) (A) and metabolite/nucleotide ratios (B) in lymph nodes of mice (blue boxes), NHPs (purple boxes), and humans (green boxes). Boxes are the 25th percentile, median, and 75th percentile. Whiskers are the fifth and 95th percentiles, and dots are outliers. The dashed line in (A) indicates lower limits of quantitation, whereas the dashed line in (B) indicates equal metabolite and nucleotide concentrations. Sample sizes for each drug in each species are given in Supplemental Table 2. Between-species differences were analyzed using Kruskal–Wallis one-way ANOVA with Dunn’s method correction for multiple comparisons. *P < 0.05.

Fig. 4.

Differences in ARV TPRs in uninfected (gray boxes) and infected (red boxes) mouse (A) and NHP (B) lymph nodes. Boxes are the 25th percentile, median, and 75th percentile. Whiskers are the fifth and 95th percentiles, and dots are outliers. The dashed line indicates equal drug concentrations in lymph node and plasma. Sample sizes for each drug in each species are given in Supplemental Table 2. Infection differences were analyzed using Kruskal–Wallis one-way ANOVA with Dunn’s method correction for multiple comparisons.

Fig. 5.

Differences in ARV TPRs in female (pink boxes) and male (blue boxes) NHP (A) and human (B) lymph nodes. Boxes are the 25th percentile, median, and 75th percentile. Whiskers are the fifth and 95th percentiles, and dots are outliers. Dashed lines indicate equal drug concentrations in lymph node and plasma. Sample sizes for each drug in each species are given in Supplemental Table 2. Sex differences were analyzed using Kruskal–Wallis one-way ANOVA with Dunn’s method correction for multiple comparisons.

Fig. 6.

Cross-species differences in drug transporter expression in lymph nodes. (A) Gene expression (measured as the fold change over the housekeeping gene GAPDH) of eight drug transporters in the lymph nodes of mice (blue boxes) and NHPs (purple boxes). (B) Protein concentrations (picomoles per milligram protein) of eight drug transporters in the lymph nodes of NHPs (purple boxes) and humans (green boxes). Boxes are the 25th percentile, median, and 75th percentile. Whiskers are the fifth and 95th percentiles, and dots are outliers. Dashed lines in (A) and (B) represent the lower limit of quantitation and the LLOD, respectively. Sample sizes for each species are given in Supplemental Table 2. Between-species differences were analyzed using Kruskal–Wallis one-way ANOVA with Dunn’s method correction for multiple comparisons. *P < 0.05.