Side effects of interferon-ribavirin combination therapy limit the sustained viral response achievable in hepatitis C virus (HCV) patients. Coupling ribavirin to macromolecular carriers that target the drug to the liver would reduce systemic complications. The aim of this study was to evaluate the efficacy of a hemoglobin-ribavirin conjugate ( C hronic hepatitis C virus (HCV) infection is a major public health problem affecting over 4 million people in the United States and more than 170 million individuals worldwide. 1,2 In addition, chronic HCV is a causative factor for approximately 50% of the cases of hepatocellular carcinoma in the United States, and is the single most common indication for orthotopic liver transplantation worldwide. 3,4 However, treatment of HCV infection remains problematic. The current standard of care is a combination of interferon alpha (IFN-␣) and ribavirin. This combination treatment is limited by severe side effects that often lead to premature cessation of therapy. 5 The major toxicity associated with ribavirin is a dose-dependent hemolytic anemia, which occurs in approximately 50% of treated individuals, resulting in a ribavirin dose reduction. 6 The development of anemia usually starts after 4 weeks of therapy and can be precipitous.Coupling of ribavirin to a carrier molecule offers the potential of a therapeutic with improved safety and efficacy by targeting drug delivery of ribavirin to key tissues infected by HCV while preventing the hemolytic anemia that is caused by exposure of red blood cells to free ribavirin. Targeting of drugs by attachment to carrier molecules for delivery to specific tissues via receptor-mediated endocytosis is a recently established method for improv-

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.