Synthesis and Characterization of Long-Acting Darunavir Prodrugs

Abstract

Antiretroviral therapy (ART) has improved the quality of life in patients infected with HIV-1. However, complete viral suppression within anatomical compartments remains unattainable. This is complicated by adverse side effects and poor adherence to lifelong therapy leading to the emergence of viral drug resistance. Thus, there is an immediate need for cellular and tissue-targeted long-acting (LA) ART formulations. Herein, we describe two LA prodrug formulations of darunavir (DRV), a potent antiretroviral protease inhibitor. Two classes of DRV prodrugs, M1DRV and M2DRV, were synthesized as lipophilic and hydrophobic prodrugs and stabilized into aqueous suspensions designated NM1DRV and NM2DRV. The formulations demonstrated enhanced intracellular prodrug levels with sustained drug retention and antiretroviral activities for 15 and 30 days compared to native DRV formulation in human monocyte-derived macrophages. Pharmacokinetics tests of NM1DRV and NM2DRV administered to mice demonstrated sustained drug levels in blood and tissues for 30 days. These data, taken together, support the idea that LA DRV with sustained antiretroviral responses through prodrug nanoformulations is achievable.

Keywords: LASER ART; darunavir; long acting; prodrugs; protease inhibitors.

Figure 1.. Synthesis and characterization of DRV prodrugs.

(A) Amine (M1DRV) and hydroxyl (M2DRV) modified DRV ester prodrugs were synthesized in high chemical yields. (B) Aqueous solubility demonstrated enhancement in prodrug hydrophobicity (****P<0.0001 for DRV compared to M1DRV or M2DRV, *P=0.0258 for M1DRV versus M2DRV). (C) and (D) Hydrolysis of M1DRV and M2DRV in mice and rat plasma over 48 hours at 37°C demonstrating enzymatic degradation of the prodrugs over time. Data are expressed as mean ± SEM for n=3 samples per group.

Figure 2.. Biological characterizations of NDRV, NM1DRV and NM2DRV.

Cytotoxicity of drug formulations in MDM (A) and CEM-SS CD4+ T cells (B) was assessed by MTT and Live/Dead Fixable Dead Cell Stain Kits. Antiretroviral activities against HIV-1 were determined in MDM (C) and CEM-SS CD4+ T cells (D). Data are expressed as mean ± SEM for n=3 samples per group.

Figure 3.. NDRV, NM1DRV and NM2DRV uptake in MDM

(A) and CEM-SS CD4+ T cells (B). Results demonstrate high intracellular drug levels for NM1DRV and NM2DRV treatments compared to undetectable levels for NDRV (^^^^P<0.0001 for NDRV versus NM1DRV for parent drug quantitation and ****P<0.0001 and ***P=0.0007 for NM1DRV versus NM2DRV for prodrugs quantitation). (C) TEM images of MDM loaded with native drug and prodrug nanoformulations. Data are expressed as mean ± SEM for n=3 samples per group.

Figure 4.. Drug retention and antiretroviral activities in MDM after a single 8-hour 100 μM drug treatment.

(A) NM1DRV and NM2DRV were retained in MDM for 14 and 31 days, respectively, while NDRV was washed out within hours. ****P<0.0001 for NM1DRV versus NM2DRV. (B) Consequently, NM1DRV and NM2DRV exhibited sustained release of drug into culture media. Antiretroviral activity was determined by RT activity (C) and HIV-1p24 antigen staining (D). NM2DRV formulation demonstrated long-term viral suppression for a period of 30 days compared to viral breakthrough within 15 days and one day for NM1DRV and NDRV, respectively. Data are expressed as mean ± SEM for n=3 samples per group.

Figure 5.. PK in mice.

(A) Plasma DRV levels demonstrating sustained drug release for NM1DRV and NM2DRV formulations. (B) Plasma M1DRV and M2DRV levels. (C) Blood M1DRV and M2DRV levels. (D) Tissue DRV concentrations at day 28. (E) Prodrug levels in the lymph nodes, spleen, liver, and brain demonstrating high prodrug concentrations for NM2DRV compared to NM1DRV treatment. Data are expressed as mean ± SEM for n=5 mice per group.