Creation of a long-acting rilpivirine prodrug nanoformulation

Abstract

Antiretroviral therapy requires lifelong daily dosing to attain viral suppression, restore immune function, and improve quality of life. As a treatment alternative, long-acting (LA) antiretrovirals can sustain therapeutic drug concentrations in blood for prolonged time periods. The success of recent clinical trials for LA parenteral cabotegravir and rilpivirine highlight the emergence of these new therapeutic options. Further optimization can improve dosing frequency, lower injection volumes, and facilitate drug-tissue distributions. To this end, we report the synthesis of a library of RPV prodrugs designed to sustain drug plasma concentrations and improved tissue biodistribution. The lead prodrug M3RPV was nanoformulated into the stable LA injectable NM3RPV. NM3RPV treatment led to RPV plasma concentrations above the protein-adjusted 90% inhibitory concentration for 25 weeks with substantial tissue depots after a single intramuscular injection in BALB/cJ mice. NM3RPV elicited 13- and 26-fold increases in the RPV apparent half-life and mean residence time compared to native drug formulation. Taken together, proof-of-concept is provided that nanoformulated RPV prodrugs can extend the apparent drug half-life and improve tissue biodistribution. These results warrant further development for human use.

Keywords: Antiretroviral therapy; Long-acting slow effective release antiretroviral therapy; Monocyte-derived macrophages; Nanoformulation; Prodrugs; Rilpivirine.

Figure 1.. Synthesis of RPV prodrugs.

(A) RPV was modified with variable fatty acid chain lengths (7, 12, 14 and 18 carbon) to develop a library of prodrugs (M1RPV, M2RPV, M3RPV, and M4RPV, respectively). Prodrug bioconversion is hypothesized to first proceed by enzymatic cleavage of the methylene ester, yielding N-hydroxymethyl RPV. Subsequently, hydrolysis will yield the original active compound (RPV). (B) FTIR spectroscopy produced absorption bands between 1726–1730 cm⁻¹, as well as (2852–2864 and 2918–2928 cm⁻¹), representing aliphatic chain carbonyl and alkane stretches, respectively. (C) Proton NMR spectra confirmed the synthesis of each RPV prodrug. Specifically, multiplet signals between 5.57–6.0 ppm and 1.17–1.35 ppm correspond to the methylene ester and repeating (R-CH₂-R) protons respectively. In addition, C_α and terminal methyl group protons are identified between 2.32–2.33 ppm and 0.84–0.87 ppm, respectively.

Figure 2.. M3RPV characterization.

(A) Water and (B) 1-octanol solubility of M3RPV and RPV were measured following 24 hours incubation. Data are expressed as mean ± SEM for n = 3 replicates. (**P < 0.01, ****P < 0.0001) Plasma cleavage of M3RPV was tested in numerous species (mouse, rat, rabbit, monkey, dog, and human) and (C) measured for loss of M3RPV, as well as (D) formation of RPV. Antiviral activity of M3RPV and RPV in (E) MDM and (F) CEM-CD4+ T-cells was investigated at a range of concentrations (0.1–1000 nM) and determined by HIV-1 reverse transcriptase (RT) activity after viral challenge with HIV-1_ADA at an MOI of 0.1. Data was normalized and expressed as percentage of HIV-1 control ± SEM; N=3.

Figure 3.. Stability and morphology of NM3RPV and NRPV.

(A-C) Physicochemical stability of NRPV and (D-F) NM3RPV were evaluated for size (nm), polydispersity (PDI), and zeta potential (mV) at multiple temperatures (room temperature [(RT],), 4 °C and 37 °C) across 100 days. Data is expressed at mean ± SD for n = 3 measurements. Transmission electron microscopy (TEM) provided a morphological assessment of NRPV (G) and NM3RPV (H). (I) Prodrug stability within nanoformulated M3RPV was determined by analyzing the ratio of M3PRV to RPV in NM3RPV over a period of 100 days. Data are expressed at mean ± SEM for n = 3 measurements.

Figure 4.. In vitro characterization of NM3RPV in macrophages.

Nanoformulation uptake, retention, and long-term antiretroviral efficacy was assessed in MDMs. Specifically, NM3RPV and NRPV were evaluated for uptake (A,E) and retention (B,F) of intracellular RPV and M3RPV at concentrations of 10 and 30 μM. Data is expressed as mean ± SEM with n = 3 biological replicates (* P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001 as determined by Student’s t test). Long-term antiretroviral efficacies of NM3RPV and NRPV were examined after an 8-hour drug treatment with 10 or 30 μM, followed by HIV-1_ADA (MOI = 0.1) challenge up to 30 days post treatment. Infection was characterized by HIV-1 reverse transcriptase (RT) activity (C,G) and HIV-1p24 antigen expression (D,H). HIV-1 RT activity was normalized as a percentage of HIV-1 positive control and expressed as mean ± SEM with n = 4 biological replicates. Representative images of MDMs stained for HIV-1p24 antigen expression (brown stain) are shown.

Figure 5.. Murine PK and BD.

Male BALB/cJ mice were administered a single intramuscular injection of NM3RPV or NRPV at 45, 75, or 100 RPV-eq./kg (A) RPV concentrations in plasma were determined at days 1 and 7 then weekly for 46 weeks. PA-IC₉₀ and limit of quantitation (LOQ) defined as 12 and 0.5 ng/mL respectively. Tissue biodistribution of RPV and M3RPV was assessed at 56 and 323 days after injection in the (B) spleen, (C) lymph node, (D) liver, (E) gut and (F) kidney. Data is expressed as mean ± SEM where N = 4/5 biological replicates (**** P < 0.0001, *** P < 0.001, ** P < 0.01, * P < 0.05 by Student’s t test).

Figure 6.. Rhesus macaque PK and BD.

Four rhesus macaques were administered 45 mg/kg RPV-eq. of NM3RPV by a single IM injection. (A) Plasma samples were collected and assayed for RPV and NM3RPV at day 1, 7 and weekly throughout the course of study (316 days). Additionally, rectal, lymph node, and adipose tissue biopsies were collected 204 days after drug administration and assayed for RPV (B) and NM3RPV (C) content. Both plasma and tissue drug concentrations were determined by UPLC-MS/MS. Data are expressed as mean ± SEM for n = 4.