Bioconjugate Chem. 1995, 6, 195-202
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Conjugation of Adenine Arabinoside 5'-Monophosphate to Arabinogalactan: Synthesis, Characterization, and Antiviral Activity Philip M. Enriquez, Chu J u n g , and Lee Josephson* Advanced Magnetics Inc., 61 Mooney Street, Cambridge, Massachusetts 02138-1038 Bud C. Tennant Department of Clinical Sciences, College of Veterinary Medicine, Cornel1 University, Ithaca, New York 14850. Received July 11, 1994@
A conjugate consisting of the antiviral nucleotide analogue adenine arabinoside 5'-monophosphate (araAMP, vidarabine monophosphate) and the naturally occurring polysaccharide arabinogalactan was synthesized. The conjugate consisted of 7.9 araAMP residues per molecule of arabinogalactan. The proposed structure of the conjugate was consistent with I3C NMR spectroscopic studies. Daily injections of the conjugate, a t a dose of 3 mg of araAMPkg, into woodchuck carriers of woodchuck hepatitis virus (WHV) decreased serum levels of WHV DNA. A dose of 3 mgkg of unconjugated araAMP was ineffective, while a higher dose of araAMP (15 mgkg, 14 days) produced a drop in WHV DNA. After cessation of dosing with the conjugate, serum viral DNA levels remained depressed for 42 days. In contrast, after cessation of dosing with araAMP, WHV DNA rapidly returned to original levels.
INTRODUCTION Hepatitis B virus (HBV) is a major cause of acute and chronic hepatitis, cirrhosis, and hepatocellular carcinoma (I, 2). The nucleotide analog araAMP has been shown to be effective in reducing HBV viremia and, in 10-20% of the cases, induces extended remission (3). Clinically however, araAMP has unacceptable neurotoxic side effects that have prevented its use in hepatitis B therapy (4).
The potency of araAMP against HBV, and its extrahepatic side effects, suggest that modifying araAMP to achieve a more hepatic specific biodistribution might provide a useful drug for the treatment of HBV. Thus, araAMP has been attached to lactosylated albumin (LHSA) for uptake by the asialoglycoprotein receptor, and this conjugate has been tested for the treatment of hepatitis B in humans (5). However, conjugates utilizing protein-based carriers to target drugs to receptors have deficiencies; see the Discussion and ref 6. Recently, we reported that a superparamagnetic iron oxide covered with arabinogalactan, a polysaccharide from the plant Larix occidentalis, was removed from blood by the asialoglycoprotein receptor of hepatocytes (7,8). The superparamagnetic iron oxide was useful as a hepatocyte-specific magnetic resonance contrast agent. This observation suggested that arabinogalactan might serve as a carrier for targeting therapeutic agents like araAMP to the liver via the asialoglycoprotein receptor. The features of arabinogalactan that are responsible for its binding to the asialoglycoprotein receptor are (i) a highly branched structure and (ii) the presence of numerous terminal galactose residues (9). In its unmodified, naturally occurring form, arabinogalactan mimics the natural ligands for this receptor. In contrast, the oli-
* Corresponding author.Tel.: (617)-497-2070,Fax: (617)-5472445). Abstract published in Advance ACS Abstracts, February 1, 1995. @
gosaccharides of glycoproteins require sialic acid removal to attain receptor binding. In this study we describe the synthesis, characterization, and antiviral activity against woodchuck hepatitis virus (WHY) of a conjugate consisting of the polysaccharide arabinogalactan and araAMP. Woodchucks (Marmota monax) infected with WHV can exhibit a viral carrier state similar to the human hepatitis B viral infection (IO). MATERIALS AND METHODS Arabinogalactan was obtained from Champion Corporation (Tacoma, WA) and purified by ultrafiltration (9). AraAMP was provided courtesy of Parke-Davis (Ann Arbor, MI). 1-Ethyl-3-(3-(dimethylaminopropyl)carbodiimide hydrochloride was obtained from Bachem California (Torrance, CA). Dextran T-10 and dextran T-25 were obtained from American Polymer Standards (Mentor, OH). Reagent grade acetone, dimethyl sulfoxide (DMSO), and 1,4-dioxane were dried over 3 A molecular sieves before use. DzO containing 1%DSS was from Wilmad (Buena, NJ). Other reagents were obtained from Aldrich Chemical Co. (Milwaukee, WI). Elemental analyses were performed by Galbraith Laboratories (Knoxville, TN). Synthesis of Reduced Arabinogalactan 1. The first step in the conjugation of araAMP to arabinogalactan, see Scheme 1, was the reduction of arabinogalactan with sodium borohydride. Sodium borohydride (10 g, 250 mmol) was added to a solution of arabinogalactan (100 g, 2.5 mmol, molecular weight of 40 kDa by light scattering) in water (250 mL). After being stirred a t room temperature for 18 h, the reaction mixture was neutralized with 6 M HC1 and dialyzed against deionized water (SpectraPor 3 dialysis membrane, Spectrum Medical Industries, Los Angeles, CA). The dialysate was lyophilized to give 100 g of reduced arabinogalactan 1 as a white crystalline solid. Synthesis of Arabinogalactan Bromide 2. Bromide was introduced into reduced arabinogalactan 1 by
1043-1802/95/2906-0195$09.00/0 0 1995 American Chemical Society
196 Bioconjugate Chem., Vol. 6,No. 2, 1995
reaction with epibromohydrin (21). A solution of zinc tetrafluoroborate hydrate (40 g, 167 mmol) in water (100 mL) and epibromohydrin (500 mL, 5.8 mol) was added to an aqueous solution of 1 (100 g, 2.5 mmol) in water (250 mL). This mixture was heated to 100 "C for 1.5 h. The product was extracted with butyl acetate and purified by ultrafiltration using a 3 kDa cutoff filter (Diaflo YM3, Amicon, Beverly, MA). Lyophilization yielded a white crystalline solid 2. Yield: 35 g (31%). Elemental analyses: 0.59 mmol of Br/g of conjugate. Synthesis of Arabinogalactan-Amine 3. A solution of 2 (35 g, 0.8 mmol) and ethylenediamine (160 g, 755 mmol) in DMF (80 mL) was stirred a t 60 "C for 4 h. Unreacted ethylenediamine was removed by vacuum distillation. The product was purified by ultrafiltration and lyophilization as described above. Yield of pale yellow crystalline solid 3: 23 g (66%). Ninhydrin assay showed a 0.17 mmol of ethylenediamine/g of conjugate incorporation. Synthesis of the Antiviral Conjugate, Arabinogalactan-ardP8 (4). 9-D-D-Arabinofuranosyladenine 5'-monophosphate (araAMP; 0.70 g, 2.0 mmol) was added to a solution of arabinogalactan-amine 3 (10 g, 0.25 mmol) in water (25 mL). After the solution pH was adjusted to 6.5,l-ethyl-3-(3-(dimethylamino)propyl)carbodiimide hydrochloride (1.8 g, 9.4 mmol) was added. The reaction mixture was stirred a t room temperature for 18 h, and the product was purified by ultrafiltration and lyophilization as described above. The product was obtained in quantitative yield. The loading was 0.18 mmol of araAMP/g as determined by U V absorbance at 260 nm using unconjugated araAMP as a reference. Anion exchange chromatography showed that 99% of the araAMP was conjugated to the arabinogalactan. On the basis of the estimate of primary amine in 3 (0.17 mmovg), essentially all of the amines were conjugated to araAMP. Arabinogalactan has a molecular weight of 40 kDa by light scattering, but a molecular size of 19 kDa by size exclusion chromatography, when run against dextran standards (9). On the basis of an approximate conjugate molecular weight of 44 kDa, there are 7.9 mol of araAMP per mole of conjugate, denoted arabinogalactan-araAMP, (4).
Synthesis of en-araAMP. To aid in the assignment of peaks from the I3CNMR spectrum of arabinogalactanaraAMPB(4), ethylenediamine was attached to the phosphate of araAMP as described (12). The resulting compound is referred to as en-araAMP. N M R Spectroscopy. Fourier transform NMR spectra were obtained on a Varian a 3 0 0 spectrometer (Varian Corp, Palo Alto, CAI. Carbon-13 NMR (75.43 MHz) spectra were acquired with 1000-4000 transients with continuous broad band lH decoupling (decoupler offset set a t -5 ppm). Digital resolution was 0.50 Hz (0.909 s acquisition time with 1 Hz digital line broadening). Sample temperatures were maintained within 19-22 "C (k0.3" during a single spectral acquisition). APT (attached proton test) I3C NMR spectra were measured to determine C-H multiplicity. All NMR measurements were performed in DzO solutions containing an internal reference standard. Carbon-13 chemical shifts were referenced to -1% DSS (sodium 2,2-dimethyl-2-silapentane sulfonate), DMSO ( 6 ~ = ~ s41.3061, acetone ( 6 ~ = s~ 32.9281, or 1,4-dioxane ( 6 ~ = s~ 69.174). Molecular Size of Arabinogalactan-araAMP8 (4). The size of 4 was determined by size exclusion chromatography using a Cellufine GCL-300M (30 x 0.78 cm ID) column (Amicon Corp., Beverly, MA) and a Knauer
Enriquez et al.
differential refractometer. The solute was eluted with 0.1% NaN3 a t 0.4 mYmin (Beckman Model llOB pump). Asialoglycoprotein Receptor Interaction. Asialoglycoprotein receptor was isolated according to the method of Hudgin (13). The tracer, an iodinated tyramine derivative of arabinogalactan, was prepared as described (9). Increasing concentrations of ligand were used to displace tracer, and the concentration inhibiting tracer binding by 50% (IC50) was determined by the logit transformation (40).The IC50 with this assay has intraassay and interassay coefficients of variation of 7.9% ( n = 5) and 27% (n = 9),respectively. The ICsO'sfor all ligands were obtained in a single assay. The nonspecific binding of the tracer, i.e., binding not displacable by tracer, was less than 5% of total counts. pH Dependence of Arabinogalactan-araAMP8 (4) Stability. A solution of (4) (120 mg/mL) was prepared in pH 7.5 water. The pH of aliquots of the stock solution was adjusted with either NaOH or HC1 as appropriate to obtain solutions varying in pH from 3 to 9. The solutions were then incubated a t 37 "C. Aliquots (100 pL) were withdrawn a t 0 , 2 , 4 , 6 , and 20 h and added to 20 mM phosphate buffer (0.95 mL; pH 7.3, 400 mM NaC1). Samples were analyzed for (41, araAMP, and araA by anion exchange HPLC using a Waters 600E system (Millipore, Marlborough MA) with a Waters Model 991 photodiode array detector. The column was an anion exchange column of Synchopak Q300, 250 x 4.6 mm, (SynChrom Inc., Lafayette, IN). The mobile phase was 0.4 M NaC1, 0.1% NaN3 in pH 7.3 water, and the flow rate was 1 mumin. Stability of Arabinogalactan-araAMPB (4) in Blood. AraAMP (50 pg/mL) and (4) (833 pg/mL) were each incubated in heparinized human blood a t 37 "C. Samples of blood were taken a t 0, 60, 90, and 120 min and centrifuged for 10 min a t lOOOg to recover the plasma. The plasma was then analyzed for araAMP (free and/or conjugated) by HPLC as described above. Antiviral Activity of Arabinogalactan-araAlMP8 (4) in WHV-InfectedWoodchucks. Woodchucks were born in captivity and infected with WHV within 1 week after birth. The carrier state was confirmed by the presence of WHV surface antigen (WHsAg)in serum over the first year of life. Woodchucks (Marmotech, Inc., Cortland, NY)were a t least 1year old, approximately 3 kg, matched for age and sex, and positive for WHsAg. Intravenous injection and blood collection of woodchucks was accomplished through vascular access ports (Model SLA-3.5, Access Technologies, Norfolk Medical Products, Inc., Skokie, IL) placed in the test animals. The catheters were implanted in the saphenous vein of anesthetized animals and the ports positioned subcutaneously on the lateral aspect of the rear limb proximal to the catheter. Test material was administered daily through the ports, which were flushed with heparinized saline following use. Withdrawal of blood from these ports was possible only for the first week, however, and blood samples for the second week were obtained from the opposite femoral artery or vein. Groups of three WHV carrier woodchucks in two separate experiments were treated with intravenous injections of 50 mg/kg/day of (41, equivalent to 3 mg/kg/ day of araAMP, for 14 consecutive days. Additional animals received either 3 or 15 mg/kg/day of araAMP (Figure 4). Finally, three animals received arabinogalactan (50 mg/kg/day), data not shown. Sera for analysis of viral DNA levels were collected a t predetermined time points during and following treatments. Blood samples for serum WHV DNA were obtained a t various times prior to and during treatment and moni-
Conjugation of araAMP to Arabinogalactan
Bioconjugate Chem., Vol. 6,No. 2, 1995 197
Scheme 1 Arabinogalactan + NaBH,
reduced arabinogalactan 1 (AG,)
Retention Time (min)
Figure 1. Size exclusion chromatography of arabinogalactanaraAMP8 (4).
tored post-treatment for 100 days. Tests for W H s A g were performed using a n ELISA assay (16), while assays for WHV DNA were performed as described (17). Standards of homologous WHV DNA were hybridized simultaneously to allow quantitation. The radioactivity of individual slots was determined using an automated P-scanner (Ambis Inc., San Diego, CAI. Single Dose Toxicity of Arabinogalactan-araAMPS(4). Mice used in this study were male CD-1 (Crl: CD-l(ICR}BR), approximately 25-30 g, obtained from Charles River Laboratories, Wilmington, MA. A limit test was conducted in male CD-1 mice according to the up-and-down method for small samples (18).The animals each were administered single doses of 5 g/kg a r a b i n o g a l a c t a n - a r a P 8 (corresponding to 300 mg araAMP/kg) by intravenous injection. In a separate study, mice were administered 5 g/kg of the conjugate but then sacrificed on day 7 for microscopic examination of the liver. The livers were evaluated for histopathological changes with hematoxylin and eosin staining. Repeat Dose Toxicity of Arabinogalactan-araAMPS (4). Rats were male CD (Crl:CD(SD)BR), approximately 200-250 g, obtained from Charles River Laboratories (Wilmington, MA). Male rats were administered either (4) a t 250 mgkglday (corresponding to 15 mg araAMP/kg/day), araAMP a t 25 mg/kg/day, or saline in single intravenous injections per day for 30 days. Animals were observed for overt clinical signs of toxicity throughout the treatment period and then sacrificed for histological examination of the liver. Blood samples from all animals were obtained at the time of sacrifice. Serum samples were obtained for the standard profile of clinical chemistry analyses, which includes liver transaminases, on a Hitachi 747 Chemistry Analyzer. Whole blood samples were analyzed on a Baker 9000 for routine hematological profile with manually performed differential counts and spun hematocrits. These analyses were performed a t the Tufts Veterinary Diagnostic Laboratory, North Grafton, MA. RESULTS
habinogalactan-araAMP8(4) was synthesized according to Scheme 1. Characterization of Arabinogalactan-araAMPS (4). Arabinogalactan-araAMP8 (4)was characterized by size exclusion chromatography as shown in Figure 1.(4) eluted as a single peak with a retention time of 28.5 min, compared to T-10 dextran and T-25 dextran standards (32.1 and 23.5 min, respectively). The starting ara-
binogalactan eluted a t 24.5 min and exhibited an elution profile similar to T-25 dextran. A small amount of a low molecular weight contaminant is present in (4) and may remain after the ultrafiltration, which is used to remove low molecular weight byproducts formed during the synthesis. The 13C NMR spectrum of (4) consists of resonances attributable to araAMP, arabinogalactan, and the linkage between araAMP and arabinogalactan, as shown in Figure 2 and summarized in Table 1. The resonances of the linking group carbons, Cl”-C5”, of (4) occurred in the range of 40-66 ppm. The peaks for adenine carbons, C2-C8, were observed between 140 and 160 ppm; they were assigned by its APT spectrum and comparison with the spectra of araAMP (Na+ salt) (19) and en-araAMP. The arabinofuranose (of araAMP) resonances were in the 65-87 ppm range. The broadened line widths (14-28 Hz) of the adenine and arabinose resonances of araAMP are consistent with the conjugation of araAMP to arabinogalactan. This line broadening is attributable to the increased translational and rotation correlation times of araAMP upon conjugation to the much larger arabinogalactan (20-24). Under similar conditions, the line widths of the resonances of free araAMP and en-araAMP were typically 1-4 Hz. The increased line widths of (4) obscured the expected small P-C couplings (-4-9 Hz), which were observed in both en-araAMP and ara-AMP. The arabinogalactan subspectrum of (4) is similar to the arabinogalactans from Larix sibirica (25) and Larix dahurica (26). These arabino-3,6-galactans are highly, branched structures consisting of a backbone of primarily ( 1-3)-P-D-galactopyranose with some (1-6)-P-D-galactopyranose groups. Branching generally occurs a t C-6 of the (1--3)-Galp backbone residues. The side chains are primarily Galp or disaccharide units of Galp-(1-6)-Gal(1-61, or Arap-(l-3)-Araf-(1-3) (27,281. The resonances of the anomeric carbons of the arabinogalactan portion of (4) at -106 ppm are characteristic of P-glycosidic linkages (29) and are assigned primarily to the backbone galactopyranosyl groups (Le., P-d-(1-6)Galp, P-d-(l43)-Galp). The anomeric carbon of the arabinose residues also occurs in this range. The major peaks a t ca. 63.7, 71.4, 73.4, 75.4, and 77.8 are assigned primarily to galactopyanosyl C6 (and C5-Araf), C2, C4, C3, C5, respectively. Other arabinogalactan P-arabinofuranosyl W a f ) resonances were observed a t 76.2 (C3) and 84.3 (C4) ppm. The former peak partially obscures
198 Bioconjugate Chem., Vol. 6,No. 2, 1995
Enriquez et al.
I
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100
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60
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Figure 2. Carbon-13 NMR spectrum (75 MHz) of arabinogalactan-araAMP8 (4) in DzO solution. Table 1. Carbon-13NMR Chemical Shifts ( 6 ~ ~ofs Antiviral ) Compounds in DzO” Na [ara-AMP] 154.94 150.94 120.56 157.78 143.71 86.40 78.24 76.37 84.56 (8.6) 65.41 (4.1)
en-ara-AMP 155.78 150.70 120.25 157.45 143.27 86.33 78.19 76.52 84.02 (8.6) 65.80 ( < 4 ) 43.58 ( 5 . 5 ) 41.18
arabinogalactan-araAMP(4) 155.33 151.42 120.64 158.04 143.67 85.97 78.15 -76b 83.77 65.23
39.93 51.7-52.2 45.45 65.72
assignment adenine C2 adenine C4 adenine C5 adenine C6 adenine C8 arabinofuranose C1’ arabinofuranose C2’ arabinofuranose C3’ arabinofuranose C4‘ arabinofuranose C5’ linking group methylene (Cl”) linking group methylenes (C2”)) linking group methylenes (Cl”, C3”) linking group methine (C4”) linking group methylenes (C5”)
a Chemical shifts in ppm downfield from internal DSS.Numbers in parentheses are P-C coupling constants (Hz). Peak partially obscured by overlapping arabinogalactan resonances.
Table 2. Asialoglycoprotein Recevtor Bindinn ligand arabinogalactan arabinogalactan-araAMP8 (4) asialofetuin galactose
ICs0 (pM) 2.0 x 10-6 1.0 x 10-6
1.3 x 10-7 >2 x 10-2
the C3’ peak of the conjugated araAMP. Since 0glycosylation generally shifts the carbon of the glycosylation site downfield by ca. 7-8 ppm (29, 301, the resonances of glycosylated carbons of (1-6I-Galp and (1-31-Galp (31) occur a t ca. 71.2 and 84.3 ppm. These two peaks are coincident with the Galp C2 and ArafC4 peaks. Asialoglycoprotein Receptor Interaction. The relative strength of the interactions of various ligands with the asialoglycoprotein receptor was examined by determining the IC50 of ligands for purified receptor (Table 2). Arabinogalactan-araAMP8 (4) bound receptor about two times less strongly than that of arabinogalactan, indicating that after modification of arabinogalactan shown in Scheme 1 receptor binding was substantially retained. Asialofetuin had a IC50 for the receptor that
was about 7 times lower than arabinogalactan, while galactose had a high I C ~ O . Stability of Arabinogalactan-araAMP8 (4) as a Function of pH. The stability of (4) in the pH range 3-9 was determined at 37 “C; see Figure 3. At pH 7 over a 24 h period, no decomposition was discernible. At pH 5 or lower, araAMP was released as the major hydrolysis product due to hydrolysis of the phosphamide bond present in (4); see Scheme 1. The rate of hydrolysis of (4) was extremely slow a t pH 7 , the approximate pH of plasma, and increased rapidly a t mildly acidic pHs, the approximate pH that might be encountered in the endosome. Stability of Arabinogalactan-araAMPs (4) in Blood. The stability of (4) in blood was determined by incubating the conjugate or the free araAh4P in whole heparinized human blood at 37 “C. After a 1 h incubation, 98% of araAMP remains conjugated in arabinogalactan-araAMP8, and 69% remains after 120 min (Table 3). By comparison, 39% of unconjugated araAMP remains intact after 1h, and less than 2% remains after a 90 min incubation. Antiviral Activity of Arabinogalactan-araAMPs
Bioconjugate Chem., Vol. 6,No. 2, 1995 199
Conjugation of araAMP to Arabinogalactan 3.0
2.5
h
1
2.0
3d
4 1.5
2
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2 1.0
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Figure 3. Hydrolysis of arabinogalactan-araAMP8 (4) as a function of pH. 10000
=i 1000;
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AG-araAMP8 (3 mg araAMPikg)
U araAMP (15 mgikg)
U araAMP (3 mglkg)
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0
20
40
60
80
100
120
Day Post Initial Injection
Figure 4. Serum WHV DNA in carrier woodchucks treated with arabinogalactan-araAMP8 (4), at a dose providing 3 mg of araAMP/ kg/day, AraAMP at 3 mg/kg/day, and araAMP at 15 mg/kg/day. Animals were injected once a day for 14 days. Error bars represent one standard deviation. Table 3. Stability Of AraAMP and Arabinogalactan-araAMP8 (4) In Heparinized Blood AraAMP in blood (% of initial concn)
starting material AraAMP arabinogalactan-arap8 (4)
Oh
l h
1.5h
2h
100 100
39 98
1.2 84
0 69
(4) in WHV-Infected Woodchucks. As shown in Figure 4, treatment of woodchucks with (4) resulted in a prompt decrease in WHV DNA, a decrease which persisted for a t least 42 days after treatment termination
in all animals. The serum WHV DNA of four of the six animals was 71-94% below pretreatment levels 3 months after treatment ended. In contrast, woodchucks treated with 15 mgkglday of araAMP also responded with a prompt decrease in viral DNA, but the decrease in serum viral DNA did not persist following treatment termination. Woodchucks treated with 3 mgkglday of araAMP or arabinogalactan showed no decrease in viral DNA. Woodchucks treated with 50 mgkglday of arabinogalactan showed no change in viral DNA (data not shown). Single Dose Toxicity of Arabinogalactan-ara. AMPS (4). Administration of single bolus intravenous
200 Bioconjugate Chem., Vol. 6, No. 2, 1995
injections of 5 g of (4) per kg in mice resulted in no mortality. A dose of 5 g k g is the upper limit based on solubility and volume constraints and corresponds to dose of 300 mg araAMPkg or 100 times the dose used in woodchuck. A second group of similarly treated animals that were sacrificed on day 7 showed no evidence of liver damage or alteration, in particular vacuolization. Repeat Dose Toxicity of Arabinogalactan-araAMPs (4). There were no significant differences in clinical chemistry or hematology in rats during or following 30 consecutive day treatments with (4)a t 250 mgl kglday, unconjugated araAMP a t 25 mgkglday, or saline vehicle (2 mL/kg). Liver histopathology 24 h after the final dose revealed no hepatic vacuoles, which would have indicated compartmentalization and storage of the conjugate in liver; however, minimal multifocal necrosis of individual hepatocytes was seen in all animals receiving (4) ( n = 3). One of three araAMP-treated animals showed a similar hepatic necrosis. Serum samples, drawn from test and control woodchucks before and immediately following the 14 day treatment with (41, revealed no differences in serum chemistry or hematology that would suggest toxicity (data not shown). DISCUSS ION
We have synthesized and characterized a conjugate of the polysaccharide arabinogalactan with the antiviral nucleotide, araAMP. Asialoglycoprotein Receptor Interaction of Arabinogalactan-araAMPs (4). The modification of arabinogalactan, Scheme I, does not reduce receptor binding (Table 2). When drugs are attached to the protein portion of asialoglycoproteins or neoglycoproteins, a s has often been accomplished ( 5 , 6 , 32), the receptor binding, oligosaccharide portion of the carrier is not modified. Arabinogalactan, a polysaccharide, does not offer amino acid side chains spatially removed from its receptor binding activity for the attachment of drug. Consequently, the receptor assay is needed to demonstrate that the receptor binding activity of arabinogalactan has not been destroyed by the chemistry employed. In spite of the fact that the IC50 of arabinogalactan is about seven times higher than that of asialofetuin (Table 2), the interaction of arabinogalactan with the receptor is sufficiently strong to permit hepatic delivery of diagnostic and therapeutic agents to the liver. Arabinogalactan has been used as a covering to deliver a magnetic resonance contrast agent, superparamagnetic iron oxide, to hepatocytes (7, 8). When a chelate of j7C0 was attached to arabinogalactan, and intravenously injected, 52%of the label showed hepatic uptake. Hepatic uptake dropped to 3.5%with the injections of asialofetuin (100 mg/kg) (9). Finally, the increase in antiviral activity produced by attaching araAMP to arabinogalactan (Figure 41, suggests arabinogalactan is achieving greater hepatic uptake of (4) in the woodchuck. Stability of Arabinogalactan-araAMP8 (4) in Blood. The conjugation of araAMP to arabinogalactan provides a form of araAMP that is stable in blood (Table 3). After intravenous injection of araAMP (20 mgkg) in the woodchuck, the nucleotide was rapidly dephosphorylated and converted to ara-hypoxanthine (33). The importance of the deamination side reaction has been shown by the fact that inhibitors of adenosine deaminase, the enzyme that deaminates araA, enhance antiviral activity of araAMP (17, 34). Stability of Arabinogalactan-araAMPs (4) in Blood and as a Function of pH. The phosphamide linkage employed between the arabinogalactan amine 3
Enriquez et al.
and araAMP affords a bond that is stable a t the pH of blood but unstable a t lower pH. AraAMP, when bound to lactosaminated serum albumin via the same type of phosphate-amide linkage, is released largely as araAMP by lysosomal action (35). Antiviral Activity in WHV-Infected Woodchucks Treated with Arabinogalactan-araAMP8 (4). WHV carrier woodchucks treated with (41, equivalent to 3 mg of araAMPkgIday, responded with a decrease in WHV DNA (Figure 4). Woodchucks that were treated with (4) in the two separate experiments responded in similar fashion, with prompt and similar reductions in viral DNA the combined data for both groups (n = 6) is shown in Figure 4. In contrast, animals treated with 3 mg of araAMPkgIday, Le., the unconjugated form of araAMP, showed no change in serum WHV DNA. This demonstrates that 3 mgkg of araAMP as (4) decreased WHV DNA, while 3 mgkg of araAMP did not. After cessation of injection of (4)(day 14), WHV DNA was still depressed a t day 56 of the study (42 days after the last injection). In contrast, treatment with araAMP produced a greater decrease in serum WHV than treatment with (41, but WHV DNA promptly returned to pretreatment levels. Such a prolonged depression in WHV DNA in response to treatment with (4) maybe due to the hepatic sequestration of (4), a r d , or araATP in the liver and slow release in amounts sufficient to inhibit viral replication. AraAMP has been conjugated to lactosaminated serum albumin (to form L-HSA-araAMP) and studied with human carriers of HBV and woodchuck carriers of WHV (33, 36). A dose of 1.5 mgkg araAMP, as L-HSAaraAMP, lowered WHV DNA, while 5 mgkg as araAMP was without effect. Drugs were administered by intravenous injection, once a day for 5 days. WHV DNA was determined by observers evaluating the intensity of a spot on an autoradiograph. When L-HSA-araAMP and L-HSA-acyclovir phosphate were administered together, a decrease in viral DNA was obtained that persisted after treatment was withdrawn. Comparison of the work of Ponzetto with the current study is difficult because of differing dosing schedules and the qualitative assessment of WHV DNA levels those workers employed. Although asialoglycoproteins or neoglycoproteins have been used to deliver diagnostic and therapeutic agents to the liver, there some advantages to using arabinogalactan for this purpose. First, arabinogalactan is cheaper than lactosylated albumins o r asialoglycoproteins, often used as carriers for targeting diagnostic or therapeutic agents to the asialoglycoprotein receptor. A highly purified arabinogalactan can be purchased for less than $1 per gram, while bovine asialofetuin, the cheapest protein-based carrier for the asialoglycoprotein receptor, costs more than $100 per gram (1994 Sigma catalog prices). In some applications, notably the targeted delivery of araAMP with repeated injections, carrier costs can be a significant element in total drug cost. For example, Fiume injected humans with L-HSA-araAMP, 35 mg conjugatekglday for 3 days, for a total dose of about 6 g of conjugate over the 3 day dosing period (70 kg man) (5). On the basis of Figure 4, 50 mg of arabinogalactankglday for 14 days, the dose for a human would be over 50 g. Second, the chemistry shown in Scheme 1 provides a method of attaching nucleotides to a receptor-recognizing carrier molecule free of some of the problems that arise when the carrier is a protein. When carbodiimide is used to attach araAMP to L-HSA, araAMP can react with different amino acid side chains. A phosphoanhydride linkage to glutamic acid occurs a t pH 5, while at pH 7.5, reaction occurs with lysine and histidine residues (36).
Conjugation of araAMP to Arabinogalactan
Another problem when carbodiimide is used to attach araAMP to L-HSA is the formation of high molecular weight aggregates, due to peptide bonds formed between two or more protein molecules. Noting these problems, Jansen and co-workers recently developed a two-step, two-pH procedure to attach araAMP and L-HSA, to minimize aggregation, and produce greater hepatocyte uptake (35). In contrast in Scheme 1, araAMP is attached to the arabinogalactan amine 3. Since the amino and hydroxyl groups differ substantially in their reactivity, the coupling of 3 with araAMP using carbodiimide proceeds exclusively with the amino group. The chemistry employed in Scheme 1 avoids the formation of high molecular weight forms of arabinogalactan by selection of the concentrations and amounts of epibromohydrin and ethylenediamine. Finally, it should be noted that the reaction of amino groups on proteins tends to make the proteins more negatively charged. Highly negatively charged proteins are recognized by scavenger receptors (35),which compete with asialoglycoprotein receptors for the injected molecule. This can lead to uptake of conjugates by scavenger receptors (37,38). Consisting solely of arabinose and galactose, the starting raw material, arabinogalactan, is a neutral polysaccharide (39). The linkage used provides a negatively charged phosphate and positively charged secondary amino group, for a net neutral charge. Therefore (4), consisting of neutral arabinogalactan and a neutral linkage, has a net neutral charge. Our results are the first time a conjugate has been synthesized consisting of a polysaccharide which binds the asialoglycoprotein receptor (arabinogalactan) and an antiviral agent (araAMP). Arabinogalactan may prove to be a satisfactory carrier for the delivery of araAMP to the liver. ACKNOWLEDGMENT
The authors wish to acknowledge with thanks Mr. John Gilmartin, Marmotech, Inc., who supervised the woodchuck studies and Brent E. Korba, Ph.D., Georgetown University School of Medicine, who performed the WHV DNA analyses. The authors also wish to thank Joseph V. Rutkowski, Ph.D., Howard Bengele, Ph.D., Jeff Bigler, B.S., James Prescott, Ph.D., Stephen A. Palmacci, B.S., Lorraine Murphy, B.S., Daniel Kolker, B.S., Debra Gaw, B.A., and Stephen Plouff, B.S., for their assistance in the preparation of this manuscript. LITERATURE CITED (1) Beasley, R. P., and Hwang, L. Y. (1984) Epidemiology of hepatocellular carcinoma. In Viral Hepatitis and Liver Disease (G. N. Vyas, J. L. Dienstag, and J. H. Hoofnagle, Eds.) pp 209-224, Grune and Stratton, New York. (2) Kolff, R. S., and Galambos, J. T. (1987) Viral hepatitis. In Diseases Of The Liver (L. Schiff and E. R. Schiff, Eds.) 6th ed., pp 457-582, J. B. Lippincott, Philadelphia. (3) Marcellin, P., Ouzan, D., Degos, F., Brechot, C., Metman, E. H., Degott, C., Chevalier, M., Berthelot, P., Trepo, C., and Benhamou, J. P. (1989) Randomized controlled trial of adenine arabinoside 5’-monophosphate in chronic active hepatitis B: comparison of the efficacy in heterosexual and homosexual patients. Hepatology 10, 328-31. (4) Lok, A. S., Wilson, L. A., and Thomas, H. C. (1984) Neurotoxicity associated with adenine arabinoside monophosphate in the treatment of chronic hepatitis B virus infection. J . Antimicrob. Chemother. 14, 93-9. (5) Fiume, L., Cerenzia, M. R., Bonino, F., Busi, C., Mattioli, A., Brunetto, M. R., Chiaberge, E., and Verme, G. (1988) Inhibition of hepatitis B virus replication by vidarabine
Bioconjugate Chem., Vol. 6, No. 2, 1995 201 monophosphate conjugated with lactosaminated serum albumin. Lancet 2, 13-5. (6) Meijer, D. F. K., Jansen, R. W., and Molema, G. (1992) Drug targeting systems for antiviral agents: options and limitations. Antiviral Res. 18, 215-258. (7) Josephson, L., Groman, E. V., Menz, E., Lewis, J. M., and Bengele, H. (1990) A functionalized superparamagnetic iron oxide colloid as a receptor directed MR contrast agent. Magn. Reson. Imag. 8, 637-46. (8) Reimer, P., Weissleder, R., Lee, A. S., Wittenberg, J., and Brady, T. J. (1990) Receptor imaging: application to MR imaging of liver cancer. Radiology 177, 729-34. (9) Groman, E. V., Enriquez, P. M., Jung, C., and Josephson, L. (1994) Arabinogalactan for hepatic drug delivery. Bioconjugate Chem. 5 , 547-56. (10) Raney, A. R., and McLachlan, A. (1991) The biology of hepatitis B virus. In Molecular Biology of Hepatitis B Virus (A. McLachlan, Eds.) pp 1-38, CRC Press, Boca Raton. (11) Molteni, L. (1985) Dextran and inulin conjugates as drug carriers. Methods Enzymol. 112, 285-298. (12) al-Deen, A. N., Cecchini, D. C., Abdel-Baky, S., Moneam, N. M., and Giese, R. W. (1990) Preparation of ethylenediaminephosphoramidates of nucleotides and derivatization with fluorescein isothiocyanate. J . Chromatogr. 512, 40914. (13) Hudgin, R. L., William E. Pricer, J.,Ashwell, G., Stockert, R. J., and Morell, A. G. (1974) The isolation and properties of a rabbit liver binding protein specific for asialoglycoproteins. J . Biol. Chem. 249, 5536-5543. (14) Kohn, J., and Wilchek, M. (1984) The use of cyanogen bromide and other novel cyanylating agents for the activation of polysaccharide resins. Appl. Biochem. Biotechnol. 9, 285305. (15) Hunter, W. M., and Greenwood, F. C. (1962) Preparation of iodine 131labelled human growth hormone of high specific activity. Nature 194, 494. (16) Korba, B. E., Brown, T. L., Wells, F. V., Baldwin, B., Cote, P. J., Steinberg, H., Tennant, B. C., and Gerin, J. L. (1990) Natural history of experimental woodchuck hepatitis virus infection: molecular virologic features of the pancreas, kidney, ovary, and testis. J. Virol. 64, 4499-506. (17) Korba, B. E., and Gerin, J. L. (1992) Use of a standardized cell culture assay to assess activities of nucleoside analogs against hepatitis B virus replication. Antiviral Res. 19, 5570. (18) Dixson, W. J. (1965) The up and down method for small samples. A m . Stat. Assoc. J . 60, 969-978. (19) Uesugi, S., Tanaka, S., and Ikehara, M. (1978) Carbon-13 nuclear magnetic resonance spectra of adenine cyclonucleosides and their phosphates. Effects of neighboring groups for elucidation of fine structure of nucleosides and nucleotides. Eur. J . Biochem. 90, 205-12. (20) Benesi, A. J., and Brant, D. A. (1985) Trends in molecular motion for a series of glucose oligomers and the corresponding polymer pullulan as measured by carbon-13 NMR relaxation. Macromolecules 18, 1109-16. (21) Dais, P., and Perlin, A. S. (1986) Chemical shifts of the methyl groups in di-0-isopropylidene furanoses and their relationship to molecular conformation and site of ring fusion. Spin-lattice relaxation measurements and motional characteristics. Carbohydr. Res. 146, 177-91. (22) Goux, W. J., Perry, C., and James, T. L. (1982) An NMR study of carbon-13-enriched galactose attached to the single carbohydrate chain of hen ovalbumin. Motions of the carbohydrates of glycoproteins. J. Biol. Chem. 257, 1829-35. (23) Matsuo, K. (1984) Carbon-13-NMR relaxation of polysaccharides: dextran and amylose in dimethyl sulfoxide and water. Macromolecules 17, 449-52. (24) Vignon, M., Michon, F., Joseleau, J . P., and Bock, K. (1983) Molecular motion of branched-chain polysaccharides studied by carbon-13 NMR spin-lattice relaxation rates. Macromolecules 16, 835-8. (25) Karacsonyi, S., Kovacik, V., Alfoldi, J., and Kubackova, M. (1984) Chemical and carbon-13 NMR studies of a n arabinogalactan from Larix sibirica L. Carbohydr. Res. 134, 26574.
Enriquez et al.
202 Bioconjugate Chem., Vol. 6,No. 2, 1995 (26) Odonmazig, P., Ebringerova, A., Machova, E., and Alfoldi, J. (1994) Structural and molecular properties of the arabinogalactan isolated from Mongolian larchwood (Lark dahurica L.). Carbohydr. Res. 252, 317-24. (27) Churms, S. C., Merrifield, E. H., and Stephen, A. M. (1978) Regularity within the molecular structure of arabinogalactan from Western Larch (Larix occidentalis). Carbohydr. Res. 64, (21422. (28) Clarke, A. E., Anderson, R. L., and Stone, B. A. (1979) Form and function of arabinogalactans and arabinogalactan proteins. Phytochemistry 18, 521-540. (29) Sarkar, A. K., Pawar, S. M., and Matta, K. L. (1991) Synthesis of two isomeric tetrasaccharides O-a-L-fucopyranosyl-(l-3) and (1-4)-0-(2-acetamido-2-deoxy-P-D-glucopyranosyl)-(1-3)-0-(P-D- galactopyranosyl)-(l-4)-D-glucopyranose and a related tetrasaccharide O-a-L-fucopyranosyl(1-3)-O-(2-acetamido-2-deoxy-~-D-glucopyranosyl-~ 1-61-0(P-D-galactopyranosy1)-( 1-4)-D-glucopyranose. J. Carbohydr. Chem. 10, 269-78. (30) Bradbury, J. H., and Jenkins, G. A. (1984) Determination of the structures of trisaccharides by carbon-13 NMR spectroscopy. Carbohydr. Res. 126, 125-56. (31) Saito, H., Miyata, E., and Sasaki, T. (1978) A carbon-13 nuclear magnetic resonance study of gel-forming (1-3)P-D-glucans: molecular-weight dependence of helical conformation and of the presence of junction zones for association of primary molecules. Macromolecules 2 1 , 1244-51. (32) Stadalnik, R. C., Vera, D. R., Woodle, E. S., Trudeau, W. L., Porter, B. A., Ward, R. E., Krohn, K. A., and O’Grady, L. F. (1985) Technetium-99m NGA functional hepatic imaging: preliminary clinical experience. J . Nucl. Med. 26, 1233-42. (33) Ponzetto, A., Fiume, L., Forzani, B., Song, S. Y., Busi, C., Mattioli, A,, Spinelli, C., Marinelli, M., Smedile, A., Chiaberge, E., and et al. (1991) Adenine arabinoside mono-
phosphate and acyclovir monophosphate coupled t o lactosaminated albumin reduce woodchuck hepatitis virus viremia at doses lower than do the unconjugated drugs. Hepatology 14, 16-24. (34) North, T. W., and Cohen, S. S. (1979) Aranucleosides and aranucleotides in viral chemotherapy. Pharmac. Ther. 4,81108. (35) Jansen, R. W., Kruijt, J. K., van Berkel, T. J., and Meijer, D. K. (1993) Coupling of the antiviral drug ara-AMP to lactosaminated albumin leads to specific uptake in rat and human hepatocytes. Hepatology 18, 146-52. (36) Fiume, L., Bassi, B., and Bongini, A. (1988) Conjugates of 9-P-D-arabinofuranosyladenine 5’-monophosphate (ara-AMP) with lactosaminated albumin. Characterization of the drugcarrier bonds. Pharm. Acta Helv. 63, 137-9. (37) Fiume, L., Mattioli, A., and Spinosa, G. (1987) Distribution of a conjugate of 9-P-D-arabinofuranosyladenine5’-mOnOphosphate (ara-AMP) with lactosaminated albumin in parenchymal and sinusoidal cells of rat liver. Cancer Drug Deliv. 4, 11-6. (38) Franssen, E. J., Jansen, R. W., Vaalburg, M., and Meijer, D. K. (1993) Hepatic and intrahepatic targeting of an antiinflammatory agent with human serum albumin and neoglycoproteins as carrier molecules. Biochem. Pharmacol. 45, 1215-26. (39) Adams, M. F., and Douglas, C. (1963) Arabinogalactan- a review of the literature. Tech. Assoc. Pulp Paper Znd. 46,544548. (40) Chard, T. (1990) An Introduction To Radioimmunoassay and Related Techniques, 4th ed., pp 166-168, Elsevier, Amsterdam. BC940106H
