Macromolecular prodrugs of ribavirin: towards a treatment for co-infection with HIV and HCV

Abstract

Human immunodeficiency virus (HIV) and hepatitis C virus (HCV) represent tremendous healthcare burdens with a large proportion of patients hosting the two viruses at the same time. An altered hepatic function and immunity as well as cross-interference of drugs make treatment of co-infection increasingly challenging. Herein we report the first design of macromolecular prodrugs (MP) with concurrent success in fighting HIV and alleviating hepatitis (liver inflammation). To achieve this, polymer compositions were systematically screened in a broad range of molar mass and content of ribavirin - a broad spectrum antiviral agent. For the first time, we report that ribavirin is efficacious in fighting HIV and in the form of MP, the treatment is safe, both in terms of lack of association of ribavirin with red blood cells and lack of toxicity upon cellular internalization. The lead polymer compositions were also potent in anti-inflammatory assays with relevance to viral hepatitis - thus making up formulations with potential for treatment of co-infection with HIV and HCV.

Fig. 1. (A) Schematic illustration of the synthesis of RBV methacrylate and macromolecular prodrugs for RBV based on HPMA. (B) ¹H NMR (d6-DMSO) spectra for HPMA (1), RBV methacrylate (2), and a representative MP (3) with the peaks at 5.4 and 5.7 ppm being the hydroxyls of RBV. (C) Size exclusion chromatography elution profiles for 15 representative polymers comprising polymer series with target degree of polymerization (left to right) 260, 150, and 50 (non-fluorescent series with [M]/[RAFT] 500, 250 and 50). (D): The superimposition of ¹H NMR spectra (D₂O) showing increasing RBV loading within one set of polymers with having a narrow mass range of 5–6 kDa with RBV loading increasing from 0% to 23 mol%. The signals stemming from RBV (3.9–4.4, 6.0–6.2, 8.5–8.9 ppm) are normalized to a signal from the PHPMA carrier (3.8 ppm).

Fig. 2. (A), (B): Graphic representation of M _n and content of RBV methacrylate in the polymer (mol%). Open and solid circles correspond to the polymer series synthesized with and without fluorescein methacrylate, respectively. (C) Activity of the non-labeled polymers in preventing infectivity of the HIV virus (TZM-bl cells, 0.1 g L^–1, 24 h pre-incubation prior viral challenge, 48 h incubation until luciferase read-out). (D) Activity of the non-labeled polymers in the anti-inflammatory model (RAW macrophages, 0.1 g L^–1 polymer, 24 h pre-incubation prior to LPS pro-inflammatory stimulation, 48 h until the final read-out); (E) flow cytometry data for association of the labeled polymers with macrophages and erythrocytes (0.1 g L^–1 polymer concentration, 24 h incubation). Values of mean fluorescence were normalized to reflect variation in fluorescence of the polymers solutions. No results are given for series IV as no fluorescent analogs were prepared for this series due to its low molar mass. (C–E) Results are the average ± SD of at least three independent experiments (n = 3). Statistical significance is given in relation to the negative control. *P < 0.05, **P < 0.01, ***P < 0.001.