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.

Is O2 diffusivity within renal capillaries rate limiting for O2 delivery to hypoxic renal tubules? Equations based on diffusion theory and developed here predict that soluble hemoglobin (Hb) increases O2 diffusivity by a factor of 1 + [442 Hb%/(P50 +[Formula: see text])], where P50 is the partial pressure of O2 at which the Hb is half saturated. To examine the effect of P50 and Hb concentrations on renal function, we perfused isolated rat kidneys with Hb-P35(P50 = 35 mmHg) and Hb-P11(P50 = 11 mmHg). Venous[Formula: see text] was lower with Hb-P11 (10 ± 1 vs 16 ± 1 mmHg with arterial [Formula: see text] = 35 mmHg and 28 ± 2 vs. 40 ± 2 mmHg with arterial[Formula: see text] =140 mmHg; P < 0.001). Perfusate P50 did not influence vascular resistance, glomerular filtration rate, O2 consumption, Na reabsorption, protein excretion, or free water clearance. Percent glucose and phosphate excretion were lower with Hb-P11 than with Hb-P35( P < 0.001). Urine glucose was 0.17 mmol/l with Hb-P11 and 0.77 mmol/l with Hb-P35( P < 0.001). Hb-P35 (2%) doubled O2 delivery and lowered glucose and phosphate excretion to the level obtained with 1% Hb-P11. Thus Hb-P11 delivered O2 twice as effectively as Hb-P35 to high-affinity sodium glucose and phosphate cotransporters in the late proximal tubule (S3 segment). Hb-P11 may also have shunted O2 from the outer cortex to the outer medulla and facilitated O2 diffusion where[Formula: see text] was low. We conclude that diffusivity is a limiting factor in delivery of O2 to hypoxic tubules.

HRC 101 significantly decreased sickle-related mortality during exposure to acute hypoxic stress in transgenic mice expressing hemoglobin SAD. HRC 101 warrants further evaluation as a therapeutic modality in sickle cell disease.