Synthesis and Characterization of a Hemoglobin−Ribavirin Conjugate for Targeted Drug Delivery

Abstract

A novel conjugate of human hemoglobin (Hb) and the nucleoside analogue ribavirin (RBV) was synthesized to demonstrate the utility of Hb as a biocompatible drug carrier for improved drug delivery in the treatment of liver disease. RBV is used in combination with interferon for the treatment of hepatitis C, but its side effects can result in dose limitation or discontinuation of treatment. Targeted delivery of RBV may help to prevent or minimize its toxicity. The hemoglobin-ribavirin conjugate (Hb-RBV) was designed to release bioactive drug upon endocytosis by cells and tissues involved in extracellular Hb catabolism and clearance. Ribavirin-5'-monophosphate (RBV-P) was prepared from RBV and activated as the 5'-monophosphorimidazolide (RBV-P-Im) for reaction with carbonmonoxyhemoglobin to yield Hb-RBV consisting of multiple RBV drugs covalently attached as physiologically labile phosphoramidates via their 5'-hydroxyl groups. A molar drug ratio of six to eight RBV molecules per Hb tetramer was obtained with near complete haptoglobin (Hp) binding of the drug modified Hb maintained. The conjugate complex (Hp-Hb-RBV) was selectively taken up in vitro by cells that express the hemoglobin-haptoglobin receptor, CD163. Recovered ribavirin enzymatically cleaved from Hb-RBV showed equipotent antiproliferative activity compared to control unconjugated RBV against human HepG2 and mouse AML12 liver cell lines. Based upon the reported high level of Hb uptake in the liver, Hb-RBV may be useful in the treatment of certain liver diseases, as well as inflammatory disorders associated with CD163-positive macrophages.

Acid Denaturation Inducing Self-Assembly of Curcumin-Loaded Hemoglobin Nanoparticles

“…Therefore, the particle size of CCM-Hb-NPs can be easily controlled by altering the amount of CCM. The drug loading of CCM-Hb-NPs could be up to 20.9%, which was much better than other chemical synthesis methods [13]. …”

“…Therefore, Hb may have the potential to be a drug carrier for further application. There are several studies conjugating drugs into hemoglobin to form nanoparticles by chemical synthesis [13,14]. Complex synthesis steps and low drug loading are the main obstacles to the wide use of the chemical conjugation method.…”

“…Complex synthesis steps and low drug loading are the main obstacles to the wide use of the chemical conjugation method. For example, Brookes, et al had developed hemoglobin-ribavirin conjugates for targeting therapy purpose, but only six to eight ribavirin moieties attached per hemoglobin (less than 3% drug loading) [13]. …”

Human uridine-cytidine kinase phosphorylation of ribavirin: a convenient method for activation of ribavirin for conjugation to proteins

“…Recently, a novel hemoglobin-ribavirin conjugate (HRC) was described [44]. Ribavirin-5 0 -monophosphate was prepared by reaction of ribavirin with phosphorus oxychloride and trimethyl phosphate according to the method of Allen [45].…”

Development of a dichloroacetic acid‐hemoglobin conjugate as a potential targeted anti‐cancer therapeutic

“…Therefore, Hb could be used as a drug carrier to target the delivery of conjugated drugs to monocytes and macrophages. Hb was previously used to target the delivery of the anti-viral drug ribavirin towards monocytes and macrophages in the liver (Brookes et al, 2006).…”