Changes in local tissue microenvironment in response to subcutaneous long-acting delivery of tenofovir alafenamide in rats and non-human primates

Abstract

Several implantable long-acting (LA) delivery systems have been developed for sustained subcutaneous administration of tenofovir alafenamide (TAF), a potent and effective nucleotide reverse transcriptase inhibitor used for HIV pre-exposure prophylaxis (PrEP). LA platforms aim to address the lack of adherence to oral regimens, which has impaired PrEP efficacy. Despite extensive investigations in this field, tissue response to sustained subcutaneous TAF delivery remains to be elucidated as contrasting preclinical results have been reported in the literature. To this end, here we studied the local foreign body response (FBR) to sustained subdermal delivery of three forms of TAF, namely TAF free base (TAF_fb), TAF fumarate salt (TAF_fs), and TAF_fb with urocanic acid (TAF-UA). Sustained constant drug release was achieved via titanium-silicon carbide nanofluidic implants previously shown to be bioinert. The analysis was conducted in both Sprague-Dawley (SD) rats and rhesus macaques over 1.5 and 3 months, respectively. While visual observation did not reveal abnormal adverse tissue reaction at the implantation site, histopathology and Imaging Mass Cytometry (IMC) analyses exposed a local chronic inflammatory response to TAF. In rats, UA mitigated foreign body response to TAF in a concentration-dependent manner. This was not observed in macaques where TAF_fb was better tolerated than TAF_fs and TAF-UA. Notably, the level of FBR was tightly correlated with local TAF tissue concentration. Further, regardless of the degree of FBR, the fibrotic capsule (FC) surrounding the implants did not interfere with drug diffusion and systemic delivery, as evidenced by TAF PK results and fluorescence recovery after photobleaching (FRAP).

Keywords: Foreign body response; Immune microenvironment; Long acting drug delivery; Subcutaneous implants; Tenofovir alafenamide.

Fig. 1.

Urocanic acid (UA) contribution to reduce local inflammation from TAF-releasing nanofluidic implant. Representative image of (a) control PBS, (b) UA, (c) TAF-UA_hi and (d) TAF-UA_lo nanofluidic implants in Sprague-Dawley rats at 6 weeks. (e) Schematics of subcutaneous implant illustrating the measurements of tissue thickness along the x-, y- and z-axes. Change in implant thickness $\left(\mathrm{\Delta }{\mathrm{T}}_{\mathrm{x}-\mathrm{y}}\right)$ averaged with respect to length and width (x-y plane) throughout 6 weeks in (f) PBS, (g) UA, (h) TAF-UA_hi, (i) TAF-UA_lo and (j) comparison of 4 groups. Change in implantation site thickness along the z-axis $\left(\mathrm{\Delta }{\mathrm{T}}_{\mathrm{z}}\right.$ throughout 6 weeks in (k) PBS, (l) UA, (m) TAF-UA_hi, (n) TAF-UA_lo and (o) comparison of 4 groups. With the exception of the PBS group, H&E histology of tissue surrounding the implants showed a notable difference in fibrotic capsule (FC) thickness in tissue adjacent the nanofluidic membrane, indicated as “M” if figure 2, with respect to the tissue on the opposite side of implants, denoted as “O” (Fig. 2a–i): this difference was particularly remarkable for TAF-UA_hi and TAF-UA_lo, indicating the inflammatory effect of TAF in tissues subject to sustained exposure to the drug. Thicknesses (mean ± SD, μm) of FCs in contact with membrane were: PBS (97.81 ± 23.80), UA (176.39 ± 128.64), TAF-UA_hi (1333.04 ± 1432.74) and TAF-UA_lo (1797.59 ± 1536.95). Fibrotic tissue thicknesses on the opposite side of implants were: UA (93.18 ± 21.74), TAF-UA_hi (147.59 ± 71.98) TAF-UA_lo (97.14 ± 29.17) and PBS (82.32 ± 23.07) (Fig. 2i). Statistical significance was observed for FC thickness between the membrane side and the opposite side for each group except PBS: UA (p = 0.0064), TAF-UA_hi (p = 0.0002), TAF-UA_lo (p<0.0001), and PBS (p = 0.11). Additionally, statistical significance was observed for tissue adjacent the membranes between all groups except TAF-UA_hi with TAF-UA_lo (p = 0.99).

Figure 2.

H&E staining of fibrotic capsules (FC) harvested after 6-weeks of implantation for implants loaded with (a) PBS, (b) UA, (c) TAF-UA_hi and (d) TAF-UA_lo, and corresponding magnification of rectangular areas for (e) PBS, (f) UA, (g) TAF-UA_hi and (h) TAF-UA_lo. Comparison of (i) FC thickness, (j) collagen percentage in FC area and (k) blood vessel quantity in FC for tissues adjacent the membrane (M) and on the opposite side of implants (O). (l) Evaluation of total histological scores between PBS, UA, TAF-UA_hi and TAF-UA_lo groups. (m) Assessment of average S_pair reactivity grade between UA, TAF-UA_hi and TAF-UA_lo. S_pair values: 0.0–2.9, 3.0−8.9, 9.0–15.0, and >15.1 colored as green (no reaction), yellow (slight reaction), orange (moderate reaction) and red (severe reaction). (n) Table with histopathological scoring in all 4 groups. Data presented as mean ± SD.

Fig. 3.

Pharmacokinetics of TAF from NHP implanted with subcutaneous nanofluidic devices: TAF_fs, TAF_fb and TAF-UA. (a) Comparison of intracellular TFV-DP PBMC concentrations in 3 groups throughout the study. Evaluation of plasma (b) TAF and (c) TFV concentrations between 3 groups for the duration of the study. Data presented as median ± IQR.

Fig. 4.

Comparison of safety and tolerability of TAF-releasing nanofluidic devices in NHP. Representative images of (a) PBS, (b) TAF_fs, (c) TAF_fb and (d) TAF-UA implants in NHP at 3 months. (e) Schematics of a subcutaneous implant illustrating the measurements of tissue thickness along the x-, y- and z-axes. Longitudinal changes in implant thickness $\left(\mathrm{\Delta }{\mathrm{T}}_{\mathrm{x}-\mathrm{y}}\right)$ averaged with respect to length and width (x-y plane) over 3 months for implants loaded with (f) PBS, (g) TAF_fs, (h) TAF_fb, (i) TAF-UA, and (j) comparison of 4 groups. Change in implantation site thickness along the z-axis $\left(\mathrm{\Delta }{\mathrm{T}}_{\mathrm{z}}\right)$ throughout 3 months in (k) PBS, (l) TAF_fs, (m) TAF_fb, (n) TAF-UA, and (o) comparison of the 4 groups.

Fig. 5.

H&E staining of 3-month fibrotic capsule (FC) in (a) PBS, (b) TAF_fs, (c) TAF_fb and (d) TAF-UA, and respective magnifications (e-h). (i) Evaluation of total histological scores between PBS, TAF_fs, TAF_fb and TAF-UA groups in comparison to Generation B (GB) TAF and placebo implants from Su et al. (j) Assessment of average S_pair reactivity grade between TAF_fs, TAF_fb, TAF-UA in comparison to GB TAF implants. S_pair values: 0.0–2.9 green color (no reaction), 3.0–8.9 yellow (slight reaction), 9.0–15.0 orange (moderate reaction), and >15.1 red (severe reaction). (k) Table with histopathological scoring in all 4 groups. Data presented as mean ± SD. Comparison of (l) FC thickness, (m) collagen percentage in FC area, (n) blood vessel quantity in FC and (o) FRAP diffusivity coefficient between PBS and TAF nanofluidic implant groups. Free FITC diffusivity (1.09 ± 0.06×10⁻⁶ cm²/s) is reported as reference value. (p) TFV-DP concentration in FC adjacent to membranes. Imaging Mass Cytometry (IMC) t-SNE plots for representative FC samples (q) PBS, (r) TAF_fs, (s) TAF_fb, and (t) TAF-UA.