Bioconjugate Chem. 2006, 17, 530−537
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Synthesis and Characterization of a Hemoglobin-Ribavirin Conjugate for Targeted Drug Delivery Steve Brookes, Pieter Biessels, Nancy F. L. Ng, Caroline Woods, David N. Bell,* and Gord Adamson Hemosol Corporation, 2585 Meadowpine Boulevard, Mississauga, Ontario, Canada L5N 8H9. Received November 17, 2005; Revised Manuscript Received January 26, 2006
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.
INTRODUCTION Many therapeutic drugs do not achieve optimal efficacy due to systemic toxicity and side effects that require dose reduction, or due to inherent limitations in drug stability, half-life, and solubility (1). Targeted drug delivery to diseased tissues is one strategy for improving drug efficacy and reducing systemic side effects. One common targeting strategy employs chemical conjugation of drugs to a ligand capable of binding to specific cell surface receptors. The premise is that, following internalization and metabolism of the ligand-drug conjugate, the attached drug is released intracellularly to exert its therapeutic effect (2). Examples of ligands that have been used for targeted drug delivery include transferrin, hormones, growth factors, lectins, folic acid, carbohydrates, asialoglycoproteins, glycoproteins, and antibodies (1-3). In addition, natural and non-natural agents such as liposomes, polymers, and modified carbohydrates (46) have been used to target drugs through tissue-specific clearance pathways for the particular macromolecular carrier. We have developed a novel drug targeting strategy using hemoglobin (Hb) as a natural carrier to target the delivery of drugs to the liver by taking advantage of the natural Hb clearance pathways. Hemoglobin is the most abundant blood protein and consists of two R and two β chains (R2β2) which are noncovalently associated within erythrocytes (red blood cells) as a 64.5 kDa tetramer (7, 8). The main function of Hb is to bind oxygen in the lungs for delivery to respiring tissues (7). Extracellular Hb released as a result of red blood cell deterioration and senescence dissociates into two 32 kDa Rβ dimers (8) which are rapidly and essentially irreversibly bound (KD < 10-15 M) by the plasma protein haptoglobin (Hp) (912). Three major organs are known to take up and degrade Hb* Corresponding author. Telephone: (905) 286-6207. Fax: (905) 286-0603. E-mail address:
[email protected].
Hp complexes: liver, spleen, and bone marrow, with the liver being the principal (>70%) site of Hb catabolism (9, 13, 14). The liver was confirmed recently as the principal site of Hb uptake; radiolabeled Hb was found primarily in the liver following Hb dosing at near Hp-saturating levels in rats (15). Multiple mechanisms exist for liver uptake of Hb. Hepatocyte receptors for Hb-Hp complexes have been reported (16, 17) to account for the majority of liver uptake. Liver macrophages (Kupffer cells) specifically recognize and internalize Hb-Hp complexes via Hb-Hp scavenger receptors such as CD163 (18, 19). This receptor is expressed by cells of the monocyte/ macrophage lineage, with Kupffer cells expressing the highest levels of CD163 mRNA (20). Cellular expression of CD163 is also elevated in association with certain liver diseases and inflammatory disorders (19, 21). In addition to the specific receptor-mediated uptake by hepatocytes and Kupffer cells, Hb is taken up by the liver through Hp-independent mechanisms (22) and via uptake into transendothelially located hepatocytes through open fenestrations in the sinusoidal endothelial cells (23). By using Hb as a drug carrier, the natural mechanisms of Hb clearance can be utilized to selectively transport therapeutic drugs to the liver for the treatment of both liver and macrophageassociated diseases. Ribavirin (RBV) is a nucleoside analogue currently approved for use in combination with interferon-R for the treatment of chronic hepatitis C virus (HCV) infection, and most recently in combination with PEGylated forms of interferon-R (24, 25). The major toxic side effect of RBV therapy is a dose-dependent hemolytic anemia in approximately 10% of the treatment-eligible population which results from high drug accumulation in red blood cells (26). Accordingly, RBV is contraindicated for certain subpopulations of HCV patients with concurrent medical disease. We have selected RBV as a model drug for conjugation to Hb to demonstrate the feasibility of Hb-based drug delivery.
10.1021/bc0503317 CCC: $33.50 © 2006 American Chemical Society Published on Web 02/21/2006
A Hemoglobin−Ribavirin Conjugate for Drug Delivery
RBV is both an antiviral agent and an immunomodulator (27), making it an ideal choice for targeted delivery to cells of the liver for the treatment of viral infections and inflammatory disorders. Increased drug potency may result from increased RBV bioavailability through targeted delivery. An additional benefit may be realized through a reduction in side effects such as hemolytic anemia in susceptible patients. Fiume and coworkers have demonstrated reduced toxicity of RBV through coupling of the drug to lactosaminated poly-L-lysine, a carrier polymer that is specific for asialoglycoprotein receptors on hepatocytes (28, 29). We describe the synthesis, characterization, and physical properties of a novel Hb-ribavirin conjugate designed for targeted delivery and release of bioactive drug within cells capable of taking up and internalizing the drug conjugate. We have chosen phosphoramidate conjugation chemistry (30, 31) because of the labile nature of this linkage within the endosomal compartment, thereby allowing release of free drug following endocytosis.
EXPERIMENTAL PROCEDURES Materials. All chemicals, proteins, and enzymes used were purchased from Sigma-Aldrich (Oakville, ON, Canada) unless otherwise indicated. Ribavirin (1-β-D-ribofuranosyl-1,2,4-triazole-3-carboxamide) was obtained from Inter-Chemical Ltd. (Shenzhen, China). Pure human Hb was derived from expired whole blood according to established procedures at Hemosol Corp. (32) and used in the carbonmonoxy form (COHb). Human Hp (mixed phenotype) was from Athens Research and Technology, Inc. (Athens, GA). Human plasma was obtained from informed donors, and mouse plasma was obtained from Balb/c mice (Charles River, St. Constant, QC, Canada). Alexa Fluor 488 was purchased from Molecular Probes (Cedarlane Laboratories, Ltd., Hornby, ON, Canada). Phosphate buffered saline (PBS), AIMV, fetal bovine serum (FBS), Dulbecco’s modified eagle medium (DMEM), insulin transferrin selenium supplement (ITSS), and Ham’s F12 culture media were all purchased from Invitrogen (Burlington, ON, Canada). Lactated Ringer’s solution (Ringer’s Lactate) was purchased from Abbott Laboratories (Montreal, QC, Canada). Chinese hamster ovary (CHO), human hepatoma HepG2, and mouse hepatocyte AML12 cells were from the American Type Culture Collection (ATCC, Manassas, VA). The bromodeoxyuridine (BrdU) cell proliferation enzymelinked immunosorption assay (ELISA) kit was from Roche (Mississauga, ON, Canada). Electrospray ionization (EI) and matrix-assisted laser desorption ionization (MALDI) mass spectral analyses were performed at the Proteomic and Mass Spectrometry Centre, Faculty of Medicine, University of Toronto (Toronto, ON, Canada). 31P NMR (121.5 Hz) analyses were performed at the Department of Chemistry, University of Toronto. Cell Lines and Culture Conditions. HepG2 (ATCC HB8065) human hepatoma and mouse AML12 (CRL-2254) hepatocyte cells were cultured in DMEM/Ham’s F12 supplemented with 10% FBS, ITSS, and dexamethasone. Chinese hampster ovary (CHO) cells expressing human CD163 (variant 1, CHO/ CD163) were cultured in Ham’s F12 supplemented with 10% FBS. HPLC Analyses. All high performance liquid chromatography (HPLC) analyses were performed using a Beckman HPLC Gold system. Anion exchange chromatography was performed on a Poros H/HQ (4.6 mm × 100 mm) column, using a pH gradient of 8.3-6.3 over 10 min (25 mM Tris pH 8.3 buffer, 25 mM bisTris pH 6.3 buffer) at a flow rate of 4 mL/min, with absorbance monitoring at 280 nm (to detect total protein) and 414 nm (to detect only heme protein). C4 reverse-phase HPLC was performed on a Vydac C4 column (4.6 mm × 250 mm)
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using a gradient of 20-60% acetonitrile in water containing 0.1% trifluoroacetic acid (TFA) over 90 min, at a flow rate of 0.9 mL/min, and absorbance was measured at 220 nm (33). C18 reverse-phase HPLC was performed on a C18 Phenomenex Luna column (4.6 mm × 250 mm) eluted with water (0.1% TFA), pH 2.9, at a flow rate of 1 mL/min, and absorbance was measured at 210 and 254 nm. Size-exclusion chromatography (SEC) was performed using a Pharmacia Superdex 200 column (10 mm × 310 mm) eluted with 0.5 M magnesium chloride in 25 mM Tris buffer, pH 7.2, at a flow rate of 0.4 mL/min, and absorbance was measured at 280 nm (to detect total protein) and 414 nm (to detect only heme protein). Synthesis of Ribavirin-5′-monophosphate. Ribavirin-5′monophosphate (RBV-P) was prepared by reaction of RBV with phosphorus oxychloride (POCl3) and trimethyl phosphate (TMP) according to the method of Allen (34), with progress of the reaction monitored by C18 reverse-phase HPLC. RBV (2 mmol) was reacted with POCl3 (8 mmol) and purified water (2 mmol) in 8.3 mL of TMP. Following completion of the reaction (5 h), the product was poured over 20 g of ice and 2 N sodium hydroxide solution was added to bring the pH up to 3. The product was allowed to hydrolyze overnight at room temperature. The hydrolyzed product was extracted with 2 × 20 mL portions of chloroform. The product RBV-P in chloroform was mixed with 10 g of fine charcoal (100-400 mesh). The reaction mixture/charcoal slurry was centrifuged at 2000g for 15 min, and the supernatant was recovered. The wash steps were repeated until no inorganic phosphate (Pi) could be detected in the supernatant by C18 reversed-phase HPLC or by the Ames method (35). The charcoal was extracted three times with ethanol/water/ammonium hydroxide (10:10:1), and the pooled extract was evaporated to dryness. The resulting RBV-P ammonium salt was converted to the free acid by ion exchange using BioRAD AG 50W-X2 (H form) resin and elution of product with water according to the method by Streeter (36). The isolated yield after purification was 70%. The RBV-P was characterized using two assays: C18 reverse-phase HPLC quantification of the RBV released by enzymatic cleavage using acid phosphatase (acid phosphatase assay, described below), and quantification of total inorganic phosphate (Pi) by the Ames method (35). EI mass spectral analysis: m/z ) 323.2 (calculated 324). 31P NMR (D2O): δ 0.95 ppm. Synthesis of Ribavirin-5′-phosphorimidazolide. Purified RBV-P was converted to ribavirin-5′-monophosphorimidazolide (RBV-P-Im) according to the procedure of Fiume (30, 31) with slight modifications. The reaction was performed under dry nitrogen using anhydrous solvents. RBV-P (324 mg, 1 mmol) was dissolved in 10 mL of N,N′-dimethylformamide (DMF). Carbonyldiimidazole (CDI, 5 mmol) dissolved in 5 mL of DMF was added to the RBV-P solution with stirring, followed by addition of 5 mmol of imidazole freshly predissolved in 5 mL of DMF. The reaction mixture was stirred at room temperature for 45 min followed by removal of the DMF by evaporation. The resulting waxy solid was dissolved in 2 mL of ethanol, followed by precipitation of the RBV-P-Im product by slow addition of 20 mL of ether. The precipitate was washed twice with ether, and residual ether was evaporated using a gentle stream of dry nitrogen. The RBV-P-Im was isolated in >90% yield and used immediately for conjugation to Hb. EI mass spectral analysis: m/z ) 373.2 (calculated 374). 31P NMR (D2O) analysis: δ 20.3 ppm, trace Pi (3.3 ppm), and pyrophosphate (-10.3 ppm). Conjugation of Ribavirin-5′-phosphorimidazolide to Hemoglobin. COHb (6.67 µmol, 430 mg) (4.3 mL of a 100 mg/ mL solution in purified water) was mixed with 1 mmol of RBVP-Im dissolved in 8.6 mL of 0.1 M sodium bicarbonate, pH 9.5, buffer (150:1 ratio of RBV-P-Im to Hb). The pH of the
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reaction mixture was maintained at pH 9.5-9.6 over the first hour by addition of 0.2 M sodium carbonate solution as required. After the pH of the reaction mixture had stabilized, the reaction vessel was charged with carbon monoxide (CO) for 15 min and the reaction was allowed to continue under positive CO pressure with mixing at 37 °C for 96 h. The degree of Hb modification was monitored by anion exchange HPLC. The final reaction mixture was purified by dialysis (10 kDa molecular weight cutoff) against Ringer’s Lactate (3 × 0.5 L exchanges), sterile filtered (0.2 µm filter), charged with CO, and stored at -80 °C. The final Hb concentration following the reaction was 15 mg/mL. Determination of Molar Drug Ratio (MDR). The RBV/ Hb molar drug ratio (MDR) in the hemoglobin-ribavirin conjugate (Hb-RBV) was determined by C18 reverse-phase HPLC quantification of RBV released by enzymatic cleavage using the acid phosphatase assay (described below), and determination of total inorganic phosphate (Pi) using the Ames inorganic phosphate assay (described below). The molar concentration of Hb was determined using the Drabkin’s assay (Sigma Diagnostics). The MDR was also assessed by MALDI mass spectrometry. Hb-RBV MALDI mass spectra were analyzed in the m/z range corresponding to 15-20 kDa to identify [R globin + n] or [β globin + n] peaks, where the mass of the R globin chains was measured as 15145 g/mol, β globin chains were measured as 15869 g/mol, and n is the calculated number of attached RBV-P (307 g/mol). Acid Phosphatase Assay. RBV-P analysis: 20 µL of 30 µmol/mL RBV-P (0.6 µmol) was diluted into 0.3 mL of 1 mM sodium acetate buffer, pH 4.8, containing 3 units of acid phosphatase (type IV-S, potato). Conjugate analysis: 20 µL of 15 mg/mL Hb-RBV in Ringer’s Lactate solution (0.3 mg, 5 nmol) was diluted into 0.3 mL of 1 mM sodium acetate buffer, pH 4.8, containing 3 units of acid phosphatase. The mixtures were incubated at 37 °C for 2 h. Precipitates were removed by centrifugation through a Centricon concentrator (Amicon, 10 kDa molecular weight cutoff), and RBV in the supernatant was quantified by C18 reverse-phase HPLC against standard calibration curves of RBV and Hb + RBV admixture solutions also treated with acid phosphatase. Inorganic Phosphate Assay. Total inorganic phosphate (Pi) was determined using the Ames method (35) using potassium phosphate solutions as standards. 80 µL of 10% magnesium nitrate in ethanol was used for analysis of 5 nmol of the HbRBV sample (25 µL of ∼15 mg/mL Hb-RBV solution). Samples were ashed over an open flame in a test tube and then hydrolyzed with 0.4 mL of 0.5 M hydrochloric acid solution in a 100 °C water bath for 2 h. The samples were filtered to remove precipitates and adjusted back to 0.4 mL with water. 1.0 mL of the Ames color reagent (prepared by mixing 1 part 10% ascorbic acid solution with 6 parts of 0.42% ammonium molybdate in 1.0 N sulfuric acid solution) was added and incubated at 37 °C for 45 min. The absorbances of the samples were measured at 820 nm, and the Pi concentration was determined using a standard curve generated from the phosphate standards. Haptoglobin-Binding Assay. Hb-RBV (500 µL of a 1 mg/ mL solution in PBS, 7.7 nmol) was mixed with a 10% molar excess of human Hp (85 µL of a 10 mg/mL solution in PBS, 8.5 nmol), at room temperature for 1 h to allow complex formation of Hb-RBV with the added Hp. The extent of HbRBV binding to Hp was determined by SEC analysis of the mixture. Hb-containing species were detected at 414 nm. Hpbound complexes appear as high molecular weight early-eluting species, predominantly 164 kDa, but up to as high as 400 kDa due to the heterogeneous molecular weight of the mixed phenotype polymerized Hp (10, 11). Noncomplexed Hb species appear as late-eluting low molecular weight species (∼32 kDa)
Brookes et al.
due to dissociation of the tetrameric R2β2 Hb into Rβ dimers under the high salt elution conditions. The Hb-Hp complex, however, is stable under the dissociating elution conditions. The extent of Hb-RBV binding to Hp was determined by comparing the amount of Hp-bound early-eluting Hb species to the amount of nonbound late-eluting Hb species remaining in the Hpcontaining incubation mixture injected. Stability Assay. Approximately 15 nmol of Hb-RBV (1 mg in 75 µL of PBS) was mixed with an equal volume of freshly prepared plasma (human and mouse) and adjusted to a final volume of 0.2 mL with PBS. Samples were removed for analysis after incubation at 37 °C for 0.5, 1, and 2 h. Samples were centrifuged through a Centricon concentrator (Amicon, 10 kDa molecular weight cutoff), and the filtrate was analyzed for RBV by C18 reverse-phase HPLC. Plasma samples were prepared with known amounts of Hb and RBV as standards to achieve a RBV standard curve against which released RBV in the incubated samples could be measured by comparing HPLC peak areas. The standards were processed and analyzed identically and in parallel to account for any loss of RBV due to plasma protein binding and removal from the supernatant. Percent release was estimated by dividing the measured RBV concentration determined at each time point by the initial conjugated RBV concentration (calculated by multiplying the amount of HbRBV treated (15 nmol) by the MDR determined from the acid phosphatase and Ames’ assays, and dividing by the volume of the sample, 0.2 mL). The amount of RBV released in plasma over time was compared with the known MDR to assess conjugate stability. Labeling Haptoglobin with Alexa Fluor 488. Hp was fluorescently labeled (fHp) with Alexa Fluor 488 (a dye containing an amine-reactive carboxylic acid tetrafluorophenolate ester tethering functionality) to allow characterization of cell binding and uptake of Hp-Hb-RBV in Vitro. Human Hp (500 µL of a 2 mg/mL solution in PBS) was labeled according to the Alexa Fluor 488 kit procedure, and the extent of the labeling was determined using the formula below:
moles of fluor/moles of Hp ) (A495fHp - A495Hp)/F495 × d/CHp where F495 is the published extinction coefficient for the fluor (71 000 cm-1‚M-1, Molecular Probes, Inc., Eugene, OR), d is the assay dilution factor, CHp is the concentration of the labeled Hp sample, and A495fHp and A495Hp are the measured absorbance values for fHp and Hp, respectively. Typically, 2-3 fluors were attached per Hp to give fluorescently labeled Hp (fHp). Hp-Hb Cell Internalization Assay. To evaluate uptake of Hp-Hb-RBV by cells expressing a known Hb-Hp receptor (CD163), internalization assays were performed using wild type CHO cells (CHO/WT) and proprietary CHO cells stably transfected with CD163 variant 1 (CHO/CD163). Both cell types were incubated with Alexa Fluor 488-labeled Hp (fHp) complexed with either Hb or Hb-RBV. The flow cytometry analysis procedure was adapted from the method published by Schaer et al. (37). CHO/WT and CHO/CD163 cells were plated in media overnight in a 24-well plate at 1 × 105 cells/well. fHpHb and fHp-Hb-RBV complexes were prepared fresh for the assay by combining fHp with Hb and Hb-RBV at a 1.1:1 molar ratio and incubated for 1 h at room temperature in the dark prior to dilution to the appropriate concentrations (spanning a Hb concentration range from 0 to 100 µg/mL) with AIMV culture medium. The cell culture medium was removed, and the cells were rinsed with PBS. 200 µL aliquots of fHp, fHpHb, and fHp-Hb-RBV in AIMV were added to the wells and incubated for 1 h at 37 °C. The labeling medium was removed from the cells, and the cells were washed with PBS. The cells were removed from the wells using trypsin and transferred to a
A Hemoglobin−Ribavirin Conjugate for Drug Delivery
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Scheme 1. Hemoglobin-Ribavirin Conjugate Synthesis
Figure 1. Overlaid C18 chromatography profiles of RBV, Pi byproduct, and RBV-P, illustrating how reaction progress was monitored based on elution time.
5 mL polypropylene flow tube. PBS was combined with the cells in the tube, and the cells were pelleted at 1500 rpm at 4 °C for 10 min. The supernatant was removed, and the cells were resuspended in 500 µL of PBS in flow tubes. Fluorescent intensity was measured by flow cytometry. FL-1% is the percentage of cells within the sampled population fluorescing above baseline, and X-mean is the mean relative fluorescence of the cell population on a logarithmic scale expressed as relative fluorescence units (RFU). A high X-mean indicates that the cells are highly fluorescent and is related to the amount of fluor taken up in the form of fHp. Recovery of Ribavirin from Hb-RBV. RBV was cleaved from Hb-RBV by enzymatic hydrolysis and isolated for evaluation of bioactivity. Hb-RBV (500 µL of a 15 mg/mL solution in PBS, ∼120 nmol, 7.5 mg) was treated with 100 U of acid phosphatase in a final volume of 2 mL of 0.2 M sodium acetate buffer, pH 4.8, at 37 °C for 3 h. The reaction mixture was centrifuged through a Centricon concentrator (Amicon, 10 kDa molecular weight cutoff) to remove precipitated Hb, and the supernatant was analyzed by C18 HPLC (according to the acid phosphatase assay) to quantify the amount of RBV and then was lyophilized and reconstituted in PBS prior to in Vitro activity evaluation. In Vitro Ribavirin Bioactivity Assay. RBV recovered from acid phosphatase cleavage of Hb-RBV was evaluated for bioactivity in an in Vitro cell proliferation assay using a BrdU incorporartion cell proliferation ELISA. Human HepG2 and mouse AML12 cells were plated at a density of 4 × 104 cells/ well in flat bottom 96-well plates. The cells were allowed to grow for 24 h, at which time they were treated in quadruplicate with RBV or RBV cleaved from Hb-RBV. Cells were incubated for 6 h at 37 °C over a dose range of 0-200 µM RBV. Following incubation, the medium was removed, fresh medium containing BrdU was added to the wells, and the incubation was continued for 18 h to allow for incorporation of BrdU into the DNA. The extent of BrdU incorporation is a measure of cell proliferation and was determined by ELISA using an antibody directed to BrdU according to the kit protocol. The growth inhibitory effect of RBV with respect to dose was expressed as a percent of control with untreated cells set as 100% proliferation.
RESULTS Synthesis of Hb-RBV. The preparation of Hb-RBV is shown in Scheme 1. The synthesis of RBV-P was carried out using POCl3 in TMP to phosphorylate the 5′-hydroxyl group of RBV according to the procedure by Fiume (30, 31). More than 95% of RBV was modified according to C18 reverse-phase
Figure 2. Anion exchange chromatographic profile of Hb-RBV showing the conjugate eluting later than native Hb (concentrationequivalent injection) due to a greater net negative charge on the Hb imparted by attached RBV.
HPLC, where RBV-P had an earlier-eluting peak than RBV. Figure 1 is a normalized overlay of the C18 chromatograms of purified RBV-P, RBV, and inorganic phosphate (Pi) byproduct, illustrating the different retention times. Instead of using a charcoal column to separate RBV-P from inorganic phosphate, the reaction solution was mixed with charcoal to adsorb RBVP, and then the charcoal containing RBV-P was washed/ centrifuged several times. This resulted in sufficient Pi removal compared to product loss. Washing was stopped when Pi was no longer detected in the supernatants using the indicator from the Ames inorganic phosphate assay (35) or by C18 HPLC. The ammonium salt of RBV-P was extracted from the charcoal with ethanol/water/ammonium hydroxide, converted to its free acid form, and lyophilized. Analysis of the recovered RBV-P showed it to contain less than 5% of unmodified RBV. The product was also evaluated using acid phosphatase to convert the RBV-P to RBV, which was quantified by C18 HPLC analysis. This assay indicated more than 90% of the purified product RBV-P could be cleaved into RBV and Pi. The Ames inorganic phosphate assay was used to determine the purity of the RBV-P as measured by free Pi liberated upon pyrolysis. The measured phosphate accounted for >95% of the theoretical phosphate content. The overall yield from RBV after charcoal purification was 60%. This product was used in the next step to form the 5′-monophosphorimidazolide, RBV-P-Im. Synthesis of the intermediate RBV-P-Im was achieved by activating RBV-P with CDI and imidazole followed by evaporation of DMF solvent. Numerous precipitations from ethanol with ether yielded an off-white powder after drying. RBV-P-Im was conjugated to Hb immediately and without further purification. Reacting RBV-P-Im in a 150-fold molar excess over Hb (COHb) with the pH held constant at 9.5 was found to provide the highest ratio of RBV to Hb in the final conjugate. Anion exchange chromatographic analysis (Figure 2) indicated a lowering and broadening of the Hb isoelectric point, consistent
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Figure 3. C4 reverse-phase HPLC profile of Hb-RBV showing the shift in retention time of the individual globin chains due to conjugation of RBV to native Hb chains.
Figure 4. MALDI mass spectral analysis of the Hb-RBV conjugate showing several [R globin + n] and [β globin + n] peaks, where n is the calculated number of RBV-P attached. The average MDR estimated from this distribution is between 4 and 12.
Figure 5. Overlaid SEC profiles of Hb-RBV and Hp-Hb-RBV showing a shift of all of the 32 kDa Hb-RBV to higher molecular weight upon Hp-binding, but only some of the 64 kDa Hb-RBV.
Figure 6. Release of RBV from Hb-RBV in human and mouse plasma after 2 h (mean ( std dev).
Table 1. Hb-RBV Molar Drug Ratio (MDR)
acid phosphatase assay inorganic phosphate assay
RBV conc
Hb conc
MDR
100 µM 2.36 mM
16 µM 0.29 mM
6 8
with addition of multiple negatively charged RBV-P moieties to the protein resulting in increased retention time. Greater than 95% of the starting Hb was modified in the reaction. C4 reversephase HPLC analysis (Figure 3) showed modification of both the R and β chains, as indicated by a shift of the peaks to slightly longer retention times and by peak broadening. The molar drug ratio (MDR) of the product mixture was calculated by dividing either the acid phosphatase-cleaved RBV concentration (acid phosphatase assay) or the Pi concentration (Ames inorganic phosphate assay) by the Hb concentration (Drabkin’s assay), taking into account any dilutions. The MDR was found to be between 6 and 8 RBV moieties attached per hemoglobin tetramer (Table 1). MALDI mass spectral analysis (Figure 4) indicated the attachment of 1-3 RBV drugs per R or β globin chain (the [R + n] and [β + n] peaks were within 0.2% of the expected values based on 307 g/mol RBV-P incremental mass additions). This corresponds to a MDR distribution of 4-12 if the R and β monomers were recombined in the Hb R2β2 tetrameric form and is consistent with the average 6-8 MDR determined by the acid phosphatase and Ames assays, which give the ratio per R2β2 tetramer. SEC analysis of Hb-RBV under high magnesium chloride salt conditions that dissociate 64.5 kDa R2β2 Hb into 32 kDa Rβ dimers revealed the conjugate mixture to contain predominantly 32 kDa material (82%) eluting at 41 min, as well as some 64.5 kDa material (18%) that appeared as an earlier-eluting 38.5 min shoulder on the 32 kDa 41 min peak (Figure 5). Binding of Hb-RBV to Hp. Approximately 90% of the Hb-RBV mixture bound to human Hp (mixed phenotype), which accounted for all of the 32 kDa Hb-RBV species (Figure 5) but only some of the 64.5 kDa Hb-RBV species. The lower molecular weight Hb-RBV species moved completely to an
earlier elution time by SEC analysis, indicating Hp-binding, whereas most of the higher molecular weight Hb-RBV species did not change elution time, indicating no Hp-binding. Incubation overnight did not increase binding of this species to Hp. The non-Hp-bound, high molecular weight Hb-RBV can be removed by preparative SEC due to the 10 min difference in elution times; however, this was not done for any of the MDR or in Vitro analyses described in this report. Stability of Hb-RBV in Plasma. The stability of Hb-RBV in fresh human and mouse plasma was tested in Vitro to provide an estimate of its expected stability in circulation in ViVo. Incubation of the Hb-RBV in plasma resulted in increasing levels of free RBV with the same elution time as that of RBV control by C18 reversed-phase HPLC. There was no evidence of release of RBV-P from the conjugate. Hb-RBV was more stable in human plasma in this experiment, with release of only 6 ( 1.3% of the conjugated RBV load compared to 12 ( 3.4% release in the mouse plasma after 2 h (Figure 6). CD163+ Cell-Specific Uptake of Hb-RBV. Fluorescent labeling of Hp for cell uptake studies did not interfere with Hbbinding when analyzed by SEC (data not shown). At 50 µg/ mL (Hb) incubation concentration, a 100% FL-1 (percent of cells fluorescing) was measured for the CHO/CD163 cells with Hb and Hb-RBV, indicative of binding of fluor (in the form of fHp-bound Hb complexes) to the transfected cells. Much lower FL-1 values were observed for all the CHO/WT cell samples and the CHO/CD163 cells incubated with fHp, indicative of minimal binding. X-mean (relative brightness of the fluorescent cells as measured by RFU) increased in a linear fashion over time for the CHO/CD163 cells, indicating binding and uptake (data not shown), in contrast to the case of CHO/ WT cells, which showed only low nonspecific binding and no uptake. Figure 7 shows the results of uptake after 1 h by CHO/ WT and CHO/CD163 cells incubated with 50 µg/mL fHp-bound Hb, 50 µg/mL fHp-bound Hb-RBV, or fHp control (80 µg/ mL; the concentration of fHp bound to Hb and Hb-RBV in the 50 µg/mL Hb samples). CHO/WT cells did not take up fHp,
A Hemoglobin−Ribavirin Conjugate for Drug Delivery
Figure 7. Relative fluorescence uptake (RFU) of fHp, fHp-Hb, and fHp-Hb-RBV into CHO/WT and CHO/CD163 at 50 µg/mL Hb (80 µg/mL Hp) after 1 h.
Figure 8. Antiproliferative activity of RBV control and RBV enzymatically cleaved from Hb-RBV against human HepG2 cells. Determined by measuring the extent of BrdU incorporation relative to control untreated cells (mean ( std dev).
fHp-Hb, or fHp-Hb-RBV complexes above the background level. CHO/CD163 transfectants bound and internalized fHpHb (85 RFU) and fHp-Hb-RBV (60 RFU) but did not internalize fHp, indicating that binding was specific for HpHb complexes and occurred only in cells expressing the HpHb receptor (CD163). Bioactivity of Hb-RBV. The bioactivity of RBV released from Hb-RBV by enzymatic hydrolysis was assessed using the known antiproliferative activity of RBV (27, 38) in an in Vitro cell proliferation assay based on BrdU incorporation into DNA by actively dividing HepG2 cells. Proliferation of HepG2 cells was decreased by RBV in a concentration-dependent manner (Figure 8). RBV posthydrolysis and control unmodified RBV had similar dose-response curves, demonstrating that the conjugated RBV could be released in active form through enzymatic action. The antiproliferative activities of the control and cleaved RBV were also assessed in mouse AML12 cells, yielding similar dose-response curves (Figure 9).
DISCUSSION A novel conjugate of human Hb and RBV was prepared to demonstrate the utility of Hb as a biocompatible carrier for improved drug delivery for the treatment of liver disease and diseases affecting Hb-Hp receptor bearing cells. Ribavirin is a drug currently approved for use in the treatment of HCV, but it suffers from dose-limiting toxicity associated with its systemic biodistribution. A Hb-RBV conjugate was prepared with releasable bioactive drug through stepwise synthesis from purified human Hb and RBV. RBV was first phosphorylated at its 5′-hydroxyl and subsequently activated to a phosphorimidazolide and reacted with COHb to yield a phosphoramidate
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Figure 9. Antiproliferative activity of RBV control and RBV enzymatically cleaved from Hb-RBV against mouse AML12 cells. Determined by measuring the extent of BrdU incorporation relative to control untreated cells (mean ( std dev).
attachment of ribarivin to amino groups on hemoglobin. The attachment chemistry is similar to that developed by Baddiley (39), Staab (40), and Fiume (30, 31). Hb was maintained in the carbonmonoxy ligation state instead of the oxygenated or deoxygenated state to prevent heme oxidation, methemoglobin formation, and protein precipitation under the reaction conditions. Sites of drug attachment include some of the approximately 40 surface lysine residues of Hb and possibly terminal amino groups of the globin chains. Several lysine residues on the R and β globin chains of Hb, as well as the β globin valine (Val1) amino termini, have been shown to be reactive toward a range of amine-directed Hb modifiers (41), supporting the selection of an amino group-directed chemistry to achieve a high molar drug ratio. The MDR of the Hb-RBV conjugate was determined to be 6-8 RBVs per Hb tetramer with little unmodified hemoglobin remaining after 96 h of reaction. It is important that all Hb molecules contain some drug to minimize competition of the unmodified Hb for binding to Hp and cell receptors. Native Hb-Hp may be a more effective competitor for cell receptors than the Hp-Hb-RBV complex and may explain the difference in CHO/CD163 cell binding and uptake for these two species (see CHO/CD163 cell uptake data and explanation below). The Hb-RBV formulation consists of two species: (1) a Hb that dissociates into two 32 kDa Hb-RBV Rβ dimers under dissociating conditions (magnesium chloride containing buffer) and binds Hp despite the extensive modification by RBV, and (2) an ∼64 kDa Hb-RBV species that does not dissociate and which essentially does not bind Hp. The sequence homology of haptoglobin is highly conserved across species (42), and cross-species compatibility of Hb-Hp binding has been demonstrated in our lab using rat Hp and human Hb (unpublished data). In addition, Hp has been shown to bind a range of modified Hbs (43, 44), providing evidence for the ability of Hp to accommodate a variety of Hb surface features. However, Hp does not bind Hb cross-linked between the R globin chains but does bind Hb cross-linked between the β globin chains (44, 45). The high molecular weight Hb component that does not bind Hp as observed on SEC under dissociating conditions is most likely cross-linked across the R chains, while the fraction that binds Hp is most likely cross-linked across the β chains. It is possible that Hb subunit cross-linking can occur through the secondary 3′ and 5′ hydroxyl groups of RBV, a consequence of ribavirin-3′,5′-diphosphate (RBV-P2) and ribavirin-3′,5′diphosphorimidazolide (RBV-[P-Im]2) formation during the activation of RBV which is carried through to the conjugation step. Supporting this concept is the observation of a lone contaminant with m/z ) 403 identified in the MALDI mass spectrum of RBV-P, corresponding to RBV-P2, with a calculated
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mass of 404 g/mol. The R chains are easily cross-linked by molecules of this size (44), which explains why most of the fraction did not bind Hp. Retention of Hp-binding by the Hbdrug conjugates is important for CD163-mediated cellular uptake since this receptor has been reported to bind and internalize complexes of Hb and Hp. However, multiple mechanisms including direct uptake of free Hb (22) and cross-linked Hb (15) appear to be necessary to account for the high capacity of the liver for Hb uptake. For subsequent in Vitro and in ViVo studies, residual cross-linked Hb-RBV was readily removed from the Hp-Hb-RBV complex using preparative SEC based upon the ∼100 kDa size difference. SEC purification was not performed on the initial formulations used in the characterization experiments presented in this report. However for in ViVo studies, it is important that all Hb-RBV is bound to Hp to minimize rapid renal clearance of unbound 32 kDa Hb (46, 47). Aside from eliminating renal clearance, Hp complexation helps to selectively target Hb-Hp receptor bearing cells such as liver macrophages and aids in overall liver targeting (9-12). The Hb-RBV conjugate was sufficiently stable in plasma to allow for efficient liver clearance, which is expected to occur within minutes of administration (half-life of clearance on the order of 9-12 min)(9-12). It is expected that most of the conjugate would be rapidly taken up by liver cells and metabolized before significant release of free drug into plasma could occur as a result of conjugate degradation. To investigate receptor binding and uptake of Hb-drug conjugates, CHO cells expressing CD163 (CHO/CD163) were constructed. The Hp-Hb-RBV conjugate complex was taken up by CHO/CD163 cells but not by wild type cells lacking CD163 (CHO/WT). Fluorescent labeling of Hp did not impair Hb-binding and did not appear to prevent recognition and uptake by CD163. On the other hand, CD163 did not internalize fluorescently labeled Hp as an individual entity. These data indicate that Hb-RBV is specifically bound and internalized by Hb-Hp receptor bearing cells and provide evidence for the receptor-mediated uptake of Hp-Hb-RBV by cells that express CD163, primarily cells of the monocyte/macrophage lineage (48). The CD163 membrane protein has been identified as the macrophage scavenger receptor for Hb-Hp complexes, as part of a Hb uptake and iron recovery pathway (18, 21). A similar receptor or process may mediate the uptake of Hb-Hp by hepatocytes; however, a related receptor has not yet been identified. Tissue macrophages are ideal targets for Hb-based drug delivery, particularly with conjugated RBV due to the known presence of CD163 on such cells. Several studies have shown that RBV alters the Th1/Th2 cytokine expression ratio during an immune response (27). In the design of the Hb-RBV conjugate, a linkage was used that would be cleavable by lysosomal enzymes. A phosphoramidate linkage was employed due to the lability of phosphoramidates in ViVo (30, 31, 39, 40). As a model of cellular metabolism, RBV was enzymatically hydrolyzed and cleaved from the Hb-RBV conjugate using acid phosphatase in Vitro. Released RBV was shown to retain bioactivity similar to that of unmodified RBV against HepG2 and AML12 cells, indicating active drug would be released following exposure of the HbRBV to hydrolytic enzymes in the lysosome following endocytosis (49). Also, nonphosphorylated RBV was detected by HPLC in this simulation of lysosomal release of drug. However, drug activity is expected from release of either RBV or RBV-P (phosphorylated form). One mechanism by which RBV exerts its antiviral effect is through conversion to RBV-P, which inhibits inosine monophosphate dehydrogenase, resulting in reduced guanosine triphosphate (GTP) levels, thereby allowing RBV to ultimately compete against GTP in the host cell for incorporation into
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growing viral RNA (38, 50, 51). Successive phosphorylation steps ultimately lead to the RBV-5′-triphosphate nucleotide (52), which exerts antiviral activity through inhibition of viral RNA polymerase and causes mutations through error-prone replication when incorporated into growing viral RNA in place of GTP (51). Presumably, it is this form of RBV that is responsible for the antiproliferative effect on HepG2 and AML12 cells reported here. Due to the dual immunomodulatory and antiviral mode of action of RBV, a Hb-RBV conjugate is expected to be suitable for the treatment of HCV infection, a viral disease affecting both hepatocytes and macrophages. Recent studies have already demonstrated the enhanced activity of the drug conjugate in a model of acute viral hepatitis, in which significantly lower amounts of conjugated RBV provided improved outcomes relative to the case of free RBV (53). As a protein-drug conjugate, it is anticipated that Hb-RBV would be administered intravenously. As an injectable form of targeted RBV, Hb-RBV may serve as a more potent source of RBV that is particularly suited to acute stages of viral infection or the prevention of HCV recurrence following liver transplant, when free RBV cannot be used due to unacceptably high toxicity. RBV conjugated to Hb is in essence sequestered and prevented from entering red blood cells, thus minimizing the associated toxicities of systemic administration of the drug while targeting the drug to the cells of the liver where therapeutic benefit may be greatest. Thus, treatment of hepatitis C patients with Hb-RBV may reduce the dose-limiting toxicity of hemolytic anemia (26, 54, 55). Further work is being conducted to determine the clinical potential of this novel conjugate.
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