Table 1.
The effect of anti-HIV lipid nanoparticlea on intracellular levels of lopinavir, ritonavir, and tenofovir in peripheral blood mononuclear cells and inguinal lymph nodes, as well as overall drug exposure in plasmab.
| Time (h) | Drug concentration
|
||||||||
|---|---|---|---|---|---|---|---|---|---|
| Lopinavir
|
Ritonavir
|
Tenofovir (TFV)
|
|||||||
| Free drug | LNP | LNP/free ratioc | Free drug | LNP | LNP/free ratioc | Free drug | LNP | LNP/free ratioc | |
| Intracellular drug concentration of mononuclear cells in blood (ng/106 mononuclear cells)d | |||||||||
| 0 | 0.00 ± 0.00 | 0.00 ± 0.00 | 1.0 | 0.00 ± 0.00 | 0.00 ± 0.00 | 1.0 | 0.00 ± 0.00 | 0.00 ± 0.00 | 1.0 |
| 0.5 | 0.67 ± 0.31 | 0.28 ± 0.24 | 0.4 | 0.96 ± 1.67 | 0.41 ± 0.58 | 0.4 | 5.01 ± 5.12 | 1.29 ± 1.83 | 0.3 |
| 1 | 1.93 ± 1.18 | 1.85 ± 1.11 | 1.0 | 2.92 ± 2.62 | 2.57 ± 1.04 | 0.9 | 3.55 ± 4.21 | 1.29 ± 0.93 | 0.4 |
| 3 | 0.91 ± 0.53 | 2.39 ± 1.15 | 2.6 | 2.85 ± 1.50 | 3.05 ± 1.06 | 1.1 | 2.57 ± 2.62 | 1.19 ± 0.92 | 0.5 |
| 5 | 0.32 ± 0.28 | 2.59 ± 1.44 | 8.1 | 0.90 ± 0.60 | 2.20 ± 1.43 | 2.4 | 0.27 ± 0.21 | 1.24 ± 1.16 | 4.6 |
| 8 | 0.70 ± 0.86 | 4.23 ± 2.34 | 6.0 | 0.74 ± 0.99 | 4.57 ± 3.04 | 6.2 | 0.01 ± 0.01 | 0.59 ± 0.27 | 59.0 |
| 24 | 0.16 ± 0.28 | 3.26 ± 2.25 | 20.4 | 0.00 ± 0.00 | 4.09 ± 2.90 | >409.0 | 0.01 ± 0.01 | 0.70 ± 0.14 | 70.0 |
| 48c | 0.01 ± 0.01 | 0.03 ± 0.00 | 3.0 | 0.00 ± 0.00 | 0.00 ± 0.00 | 1.0 | 0.00 ± 0.00 | 2.27 ± 0 | >227.0 |
| 120c | NA | 0.02 ± 0.00 | NA | NA | 1.37 ± 0.00 | NA | NA | 0.64 ± 0 | NA |
| 168 | 0.00 ± 0.00 | 0.08 ± 0.10 | >8.0 | 0.00 ± 0.00 | 1.32 ± 1.14 | >132.0 | 0.00 ± 0.00 | 0.25 ± 0.25 | >25.0 |
| Intracellular drug concentrations in mononuclear cells of inguinal nodes (ng/106 mononuclear cells)e | |||||||||
| 24 | 0.00 ± 0.00 | 4.85 ± 0.04 | >485.0 | 0.13 ± 0.13 | 6.57 ± 0.08 | 50.5 | 1.02 ± 0.13 | 0.76 ± 0.09 | 0.7 |
| Plasma drug exposure analysis (AUC in μgh/ml)f | |||||||||
| 0–168 | 3.83 ± 4.04 | 69.6 ± 10.7 | 18.2 | 1.39 ± 1.18 | 19.4 ± 12.2 | 14.0 | 56.6 ± 17.04 | 395.0 ± 344.5 | 7.0 |
| 0–8 (%) | 19.6 | 9.9 | 30.2 | 11.3 | 91.8 | 9.1 | |||
| 8–168 (%) | 80.4 | 90.1 | 69.8 | 88.7 | 8.2 | 90.9 | |||
LNP, lipid nanoparticle. NA, not available.
Anti-HIV LNPs composed of 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), N-(carbonyl-methoxypolyethyleneglycol-2000)-1,2-distear-oyl-sn-glycero-3-phosphoethanolamine, sodium salt (MPEG-2000-DSPE), lopinavir, ritonavir, and tenofovir (TFV) were prepared by thin film hydration method, as previously published [4]. All drugs were dried with the lipid film, rehydrated in bicarbonate-buffered saline, and size-reduced by high-pressure homogenization under aseptic conditions to produce drug-lipid nanoparticles with a mean 52 nm diameter. Free drug suspension dosages were prepared in bicarbonate-buffered saline using biocompatible solvent and surfactant to suspend the highly hydrophobic drugs.
Four primates (Macaca nemestrina) were administered anti-HIV LNPs and free drug in a cross-over study at a normalized dose of 25 mg/kg LPV, 14.3 mg/kg RTV, and 17.1 mg/kg tenofovir (TFV) subcutaneously. All experiments were done under an approved Institutional Animal Care and Use Committee (IACUC) protocol. Blood and lymph nodes were collected over 7 days at indicated time points and drug concentrations were determined with a validated liquid chromatography-tandem mass spectrometry (LC-MS/MS) published assay [11]. Data are expressed as mean ± SD. Ratios for comparative analysis are shown in bold.
LNP/free ratio is the mean anti-HIV LNP drug concentration divided by mean free drug concentration. In cases where the drug level was below detectable limits, the number was taken as ‘0.01’ to calculate ratio.
PBMCs were isolated from whole blood by density gradient method [15], and cell pellets of 2 million PBMCs each were analyzed. (n = 4/group; at 48 h and 120 h, n = 2/group).
An inguinal lymph node was collected at 24 h (n = 2/group) and mononuclear cells were isolated by pressing tissue through a 200 μm cell strainer. Pellets of 2 million cells each were analyzed.
Area under the curve (AUC) was calculated from plasma drug concentrations using the trapezoidal rule. The fractional percentage of total AUC in the early phase (0–8 h) and late phase (8–168 h) was calculated from the mean of the total AUC and the mean AUC in the indicated time range. Values are expressed as a percentage of the total AUC (n = 4/group).