Macromolecular (Pro)drugs as Potent and Efficacious Antiviral Agents

Authors

Macromolecular prodrugs (MP), or polymer–drug conjugates, are successful tools of drug delivery investigated broadly toward optimization of pharmacokinetics of drugs.¹ Specific advantage is gained when, for example, the polymer increases solubility of the drug or aids in localizing an enhanced payload at the desired site (e.g., tumor). Over the past years, our team developed MP for delivery of antiviral agents against hepatitis C virus (HCV), human immunodeficiency virus (HIV), and other viral pathogens (Fig. 1). Below, we outline the criteria of design and the milestones of development of these MP and identify potential avenues for further optimization of these agents.

Ribavirin (RBV) is a unique therapeutic agent with a broad spectrum of antiviral activity.^2,3 However, this drug has an unfortunate ability to accumulate in red blood cells, causing anemia. For this reason, despite its therapeutic effectiveness, RBV has limited utility in the clinic. In our work, we aimed to optimize pharmacokinetics of RBV, specifically through conjugation with synthetic polymers. The prime rationale for this was a well-accepted notion that polymers do not enter erythrocytes. We hypothesized that conjugation to polymers (i.e., the synthesis of MP) would eliminate the origin of the main side effect of RBV. Our prime synthetic method to prepare polymers is reversible addition-fragmentation chain transfer polymerization (RAFT),⁴ such that the polymers are made uniform in size, that is, they have predictable and well-controlled properties. To make this happen, we embarked on the search for synthetic methods to prepare appropriate monomers for the synthesis (i.e., acrylates and methacrylates). The synthesis proved most efficient when employing a nature-derived catalyst, an enzyme, which afforded a 5ʹ-derivative of RBV in high purity and yield.⁵ RBV (meth) acrylates were used to make MP based on acrylic and methacrylic acids,⁶ 2-N-hydroxypropyl methacrylamide,⁷ and N-vinyl pyrrolidone⁸ through direct copolymerization of comonomers. This approach afforded a fine control over the polymer composition and offered a high level of drug loading, up to 25 mol% of the drug-containing monomer units in the overall polymer sequence. When possible, we used high-throughput, robotic methods of polymer synthesis to screen the macromolecular parameter space and obtain MP with optimized molar mass and drug loading.^6,7 Fluorescently labelled polymer samples were used to illustrate that MP have minor if any association with the red blood cells, thus overcoming the major side effect of RBV.^5,7

Figure 1. Artist’s representation of a viral particle interacting with antiviral macromolecular prodrugs (drawn not to scale), that is, a polymer chain containing conjugated antiviral drug. Macromolecular parameter space for optimization of such prodrugs includes the chemistry of the polymer backbone and the polymer molar mass, the drug loading, and also the linkage between the drug and the carrier. Image adapted from Kock et al.12 and reproduced by permission of The Royal Society of Chemistry.

To establish a virus-free platform to screen for activity of MP in delivering RBV, we analyzed the molecular biology and intracellular activity of this drug and identified a potential connection between RBV and the synthesis of an inflammatory marker in macrophages, namely nitric oxide.⁹ Indeed, in cell culture, RBV revealed a dose-dependent anti-inflammatory activity with EC₅₀ of 7 mM, being close to 10–20 mM plasma level quoted as physiologically relevant for patients on RBV treatment. These experiments also highlighted that RBV has a dramatically narrow therapeutic window; toxicity related IC₅₀ was only marginally higher than EC50 and was established at 14 mM. Using this assay, we revealed that MP tremendously broadened the therapeutic window of RBV. However, it also became obvious that potency of (meth) acrylate-based MP was well below that of the pristine drug, highlighting a need to engineer faster and more quantitative drug release.

functionality. Yet with the use of the SIL, we were able to engineer disulfide reshuffling—a specific intracellular trigger for drug release—into successful antiviral MP for delivery of RBV¹¹ as well as azidothymidine¹² (against replication of HIV) and panobinostat¹³ (an HIV latency reversing agent). For RBV, this designed MP exhibited potent activity in both inhibiting inflammation in macrophages and inhibiting replication of the HCV RNA in hepatocytes, toward a concurrent treatment of hepatitis and against HCV.¹¹

Subsequent development of MP of RBV based on poly(methacrylic acid) led us realize that these agents may have a broad spectrum of antiviral activity, a highly sought after therapeutic characteristic (Fig. 2).¹⁴ Indeed, polyanions have a decades-long history of use as microbicides, achieved through nonspecific extracellular association with the viral particles.¹⁵ In turn, RBV is one of the few agents with documented activity against several viral pathogens,³ making the designed polyanionic MP truly unique in that both the polymer and the conjugated drug potentially exhibit antiviral effect. Furthermore, our studies revealed that these MP act as inhibitors of polymerases, owing to the anionic charge of the polymer, making up MP with triple activity against viruses.16 We put the MP to a test and successfully illustrated their ability to prevent infectivity of influenza, measles, respiratory syncytial virus, hepatitis C (replicon), and Ebola. Lead candidate MP were tested in chicken embryo to reveal successful inhibition of the influenza virus in this pre–in vivo model.¹⁶ In collaboration with several virology partners, we are now investigating the scope of these MP as broad spectrum antiviral agents.

Taken together, our efforts to date established the tools to design potent, efficacious prodrugs for delivery of diverse antiviral agents. We are now investigating the potential of these agents against diverse viral pathogens and associated diseases in animal models, and we would welcome further opportunities in collaborative projects within diverse areas of biomedicine.

Figure 2. Polyanionic macromolecular prodrugs developed in this work exhibit three modes of activity against viral pathogens: 1, extracellular inhibition of virus cell entry through interaction of the polymer with the viral particles; 2, intracellular activity through the release of the conjugated drug (e.g., inhibition of reverse transcriptase by the released azidothymidine); and 3, intracellular activity through competitive inhibition of polymerases by the polymer chain owing to high anionic charge of the polymer.

Acknowledgements

We acknowledge financial support from the Danish Council for Independent Research, Technology and Production Sciences, Denmark (ANZ; project DFF – 4184-00177).

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