- Email: [email protected]
Targeted therapy for viral hepatitis
advanced
drug delivery reviews ELSEVIER
Advanced
Drug
Targeted
Delivery
Reviews
17 (1995)
31 l-315
therapy for viral hepatitis
Robert
Plourde”,
George
Y. WubT*
“Department of Chemistry, The Immune Response Corporation, _59.?_5Darwin Court, Carlsbad, CA 9200X. USA hDepartment of Medicine, Division of Gastroenterology-Hepatology. University of Connecticut School of Medic&. Rm AM-044. 263 Farmington Avenue, Farmington, CT 06030- 184.5, USA Accepted
June
1995
Abstract The presence of asialoglycoprotein receptors on hepatocytes has been used to target a variety hepatocytes. Chemical coupling of biologically active agents can result in specific delivery to these the use of asialoglycoprotein-based conjugates that can bind nucleic acids raises the possibility of of antisense molecules. Concepts and results of receptor-mediated targeting to hepatocytes for viral hepatitis are reviewed. Keywords:
Targeting;
Hepatocyte;
Asialoglycoprotein;
of substances to cells. In addition, targeted delivery the treatment of
Receptor mediation
Contents ......................................
1. Introduction 2. Targeting antiviral agents 2.1. Nucleoside analogs 2.2. lnterferons 2.3. Antisense oligonucleotides 3. Conclusions Acknowledgements References..........................
......................................
............ ............
............ ............
............ ............ ............
............ ............ ............
1. Introduction Because the liver is the primary site of infection and damage in viral hepatitis, specific targeting of therapeutic agents to the liver may be useful. Conventional antiviral drugs such as nucleoside analogs, which are effective inhibitors
*Corresponding 679-3159.
author.
Tel.
(203)
0169-409X/95/$29.00 @ 1995 Elsevier SSDl 0169-409X(95)00065-8
679-3158;
Fax
(203)
311 312 312 312 312 313 313 314
of viral replication in vitro, are often hampered in vivo because of broad biodistribution characteristics, and resultant extrahepatic toxicity. Selective delivery of antiviral agents to specific cell populations has been accomplished by many strategies [l]. Prodrugs, which modulate the biodistribution characteristics and pharmacokinetic properties of drugs, have been applied to improve the availability of drugs at a desired cell population. Carriers, both particulate and soluble, have been employed with the same rationale.
Science
B.V. All rights
reserved
These strategies have met with some success when applied to hepatitis virus therapy [2-41. More specific targeting has been achieved using ligands which bind specific cellular receptors. The asialoglycoprotein receptor [5] present in large numbers and high affinity only on hepatocytes, binds galactose-terminated glycoproteins and neoglycoproteins. Bound ligands are taken into the cell by receptor-mediated endocytosis. Because of its specificity, the asialoglycoprotein receptor (ASGPr) has been exploited as a means to deliver therapeutic agents to hepatocytes. Other receptors that are found largely on hepatocytes include the LDL receptor and the apoE receptor [6]. Here we review advances made toward receptor-mediated targeting of therapeutic agents to the liver with the aim of treating hepatitis.
2. Targeting antiviral agents 2.1. Nucleoside unalogs
In 1979, Fiume et al. [7] demonstrated specific delivery of a trifluorothymidine (F,T)-asialofetuin conjugate to hepatocytes. The conjugate was synthesized by coupling the N-hydroxysuccinimidyl ester of F,T glutarate with the c-amino groups of asialofetuin. It was shown to interact specifically with the ASGPr, and to selectively inhibit DNA synthesis in Ectromelia virus-infected liver cells. Subsequent studies by the same investigators have focused on adenine arabinofuranoside monophosphate (araAMP) and acyclovir monophosphate conjugates with galactosylated albumin [8-lo]. Albumin was galactosylated with lactose by reductive amination. The conjugates were synthesized with a phosphoramide bond between the drug and c-amino groups of the protein. The conjugates were shown to interact specifically with the ASGPr and release the drug within liver cells [ll]. They inhibited hepatitis B virus release in the HBV-transfected hepatoma cell line 2.2.15 [12] and in vivo, inhibited woodchuck hepatitis replication [13]. The conjugates were not immunogenic when prepared with
homologous albumin [14]. In clinical studies, administration of aruAMP galactosylated albumin conjugates resulted in a reduction in serum levels of HBV DNA comparable to that of free drug, but at doses 3-6 times lower [15,16]. No adverse effects were observed. Other systems for targeting nucleoside analogs to the liver for treatment of hepatitis have been studied. Drug conjugates with lactosylated polylysine also have been shown to target the asialoglycoprotein receptor [17,18]. Very recently, apolipoprotein E-containing chylomicrons were used to deliver iododeoxyuridine selectively to liver parenchymal cells [191. 2.2.Interferons Interferons are currently the leading choice of therapy for hepatitis B and hepatitis C virus. However, many patients do not respond to current formulations, and side effects have necessitated discontinuation of therapy in some individuals [20-221. I n an effort to specifically target y-interferon to hepatocytes, it was encapsulated in asialofetuin-tacked liposomes [23]. Liposomes with y-interferon, but no asialofetuin were prepared as controls. The AF-liposome encapsulated y-interferon interacted specifically with the ASGPr, and these liposomes were taken up by hepatocytes to a greater extent than the non-AF liposome encapsulated y-interferon. In Hep-HB107 cells the AF-liposome encapsulated y-interferon inhibited HBV DNA replication more effectively than did non-AF liposome encapsulated y-interferon.
2.3. Antisense oligonucleotides Antisense oligonucleotides are being explored extensively, as a means to inhibit replication of hepatitis viruses. A number of antisense sequences which are capable of inhibiting the replication of hepatitis B [24-261 and hepatitis C [27,28] in vitro have been identified. Efficacy has also been observed with an antisense phosphorothioate DNA in vivo [29]. However, because
R. Plourde. G.Y. Wu I Advanced
Drug Delivery Reviews 17 (199-5) 311-31.5
cellular uptake of oligo nucleotides is generally poor, and their sensitivity to degradation in plasma can be quite high, some form of targeting would be desirable for successful use of antisense DNA for therapy of viral hepatitis in vivo. We have described an asialoglycoprotein-polycation system for targeting nucleic acids to hepatocytes via the ASGPr. The plasma protein, orosomucoid, was desialylated by treatment with neuraminidase to produce asialoorosomucoid, a high-affinity ligand for the asialoglycoprotein receptor. Polylysine was then covalently attached to the asialoorosomucoid. The resulting ASORPL conjugate formed a soluble, neutral complex with DNA [30]. The conjugate not only binds DNA, but has subsequently been shown to protect complexed DNA from nuclease degradation [31]. The complex undergoes receptormediated endocytosis, and DNA is expressed within the hepatocyte. Both in vitro [32] and in vivo [33] targeting of plasmid DNA have been accomplished with this system. The same strategy was used to prepare ASORPL complexes with an antisense DNA. A 21-mer sequence complementary to the portion of the hepatitis B virus genome that contains the polyadenylation signal was prepared in a phosphorothioate linkage. Using an end-labeled species, it was determined that the oligo alone was taken up slowly, less than 0.05 pmole/hr/million cells, by the hepatoma cell lines HepG2 (ASGPr + ), or SK Hepl (ASGPr - ). However, the oligo in the form of a complex, was taken up more than 10 times more rapidly by HepG2 cells. The uptake of the complex was blocked by co-incubation with excess asialoorosomucoid. Uptake of the complex was not enhanced in the SK Hepl (ASGPr - ) cell line. To assess whether the targeted antisense was functional, the HepG2 2.2.15 cell line was used. This cell line possesses asialoglycoprotein receptors and is stably transfected with the complete HBV genome. It secretes viral antigens and infectious viral particles. Administration of complexed antisense blocked the expression of HBV surface antigen in these cells, and reduced the replication of viral DNA by about 80% compared to untreated controls. A complex containing a random sequence had no effect. The antisense oligo DNA alone decreased
313
the expression of surface antigen and viral replication by only approximately 30% [33]. An asialoglycoprotein-based DNA delivery system containing a 21-mer phosphorothioate antisense oligo DNA against the polyadenylation region and adjacent upstream sequences of Woodchuck Hepatitis Virus (WHV) was prepared. Experimental woodchucks were inoculated with the woodchuck virus (strain H7) six months prior to initiating the study, and all animals subsequently developed acute hepatitis infection, as evidenced by circulating viral DNA. Animals were injected i.v. with ASOR-poly(~lysine) complexes containing 0.4 mg antisense for 5 consecutive days, for a total dose of 2 mg antisense / animal. Levels of surface antigen did not differ significantly between treated and control animals. However, intravenous administration of antisense complex significantly decreased viral burden, as evidenced by a 5-lo-fold lower level of circulating viral DNA 25 days posttreatment. The decline lasted for at least 2 weeks, after which there was a gradual rise in DNA levels. Antisense alone or a complex containing a random oligo DNA of the same size and linkage failed to have any significant effect on viral DNA levels.
3. Conclusions Many powerful approaches are being developed to treat viral hepatitis. These include novel nucleoside analogs, inducers of viral specific nucleases, interferon, and antisense oligonucleotides. However, many of these have side effects due to toxicity in non-target tissues. Targeting through the use of receptor-mediated endocytosis may permit the realization of the potential of these new agents without the limitation of side effects.
Acknowledgments
This work was supported in part by grants from: NIH DK 42182 (G.Y.W.). G.Y.W. holds equity in the Immune Response Corp.
314
R. Plourdc.
G.Y.
Wu I Advuncrd
Drug
References ]I1 Meijer.
D.K.F., Jansen. R.W. and Molena. G. (1992) Drug targeting systems for antiviral agents: options and limitations. Antiviral Res. 18. 215-258. PI Glazier. A.. Kwong. C., Rose. J.. Buckheit. R.. Korha. B.. Abou-Donia, M. and Smith. E. (1992) Antiviral phosphate prodrugs. Antiviral Res. 17. 77. I-J1 Hostetler, K.Y.. Korba, B.E.. Sridhar. C.N. and Gardner. M.F. (1994) Antiviral activity of phosphatidyl-dideoxycytidine in hepatitis B-infected cells and enhanced hepatic uptake in mice. Antiviral Res. 24. 59967. 141 Mizushima, Y. and Hoshi. K. ( 1993) Recent advances in lipid microsphere technology for targeting prostaglandin delivery. J. Drug Targeting 1. 93-100. 151 Ashwell, G. and Morel], A.G. (1974) The role of surface carbohydrates in the hepatic recognition and transport of circulating glycoproteins. Adv. Enzymol. Rclat. Areas Mol. Biol. 41. 99-128. 161 Hoeg, J.M. and Demosky. S.J. Jr (1985) Distinct hepatic receptors for low density lipoprotein and apolipoprotein E in humans. Science 227. 759-761. A.. Balboni. P.G.. Tognon, M.. 171 Fiume, L.. Mattioli. Barbanti-Brodano. G., DeVries. J. and Wieland. ‘I’. (1979) Enhanced inhibition of virus DNA synthesis in hepatocytes hy trifluorothymidine coupled to asialofetuin. FEBS Lett. 103. 47-51. A.. Balhom. P.Ci. and IW Fiume. L.. Busi. C.. Mattioli, Barbanti-Brodano. G. (1981) Hepatocytc targeting ot adenine-9-P-n-arabinofuranoside S’-monophosphatc (ara-AMP) coupled to lactosaminated albumin, FEBS Lett. 129. 261-264. PI Fiume, L.. Bruna. B.. Busi. C’.. Mattioh, A. and Spmosa. G, (1986) Drug targeting in antiviral chemotherapy: .4 chemically stable conjugate of Y-P-o-arabinofuranosyladenine S’-monophosphate with lactosaminated albumin accomplishes a selective delivery of the drug to liver cells. Biochem. Pharmacol. 35. 967-972. 1101 Fiume, L.. Bassi. B. and Bongini. A. (1988) Conjuatcs 01 9-P-o-arabinofuranosyladenine S’-monophosphate (araAMP) with lactosaminated albumin. Characterization of the drug-carrier bonds. Pharm. Acta. Helv. 63, 137-139. 1111 Fiume. L.. Busi. C.. Spinelli. C. and Spinosa. G. (lY89) A cotrjugate of acyclovir monophosphate with lactosaminated albumin releases the phosphorylated drug in liver cells. Naturwissenschaften 76, 74-76. A., Chiabergc. E.. 1121 D’Urso, N., Actix. G.C., Ponzetto, Bonino. F. and Verme. G. (1990) Modulation of hepatitis B virus DNA release by targeted adenine arabinoside 5’.monophosphatc in HepG 2 cells that product infectious virions. In: F.B. Hollingcr. S.M. Lemon and H. Margolis (Eds). Viral Hepatitis and Liver Discasc. Williams, and Wilkins. Baltimore. p. 665. 1131 Ponzetto. A., Fiume. L.. Forzani. B.. Song. S.Y.. Busi. C.. Mattioli. A.. Spinelli, C., Marinehi, M., Smedile. A.. Chiaberge. E., Bonino, F.. Gervasi. G.B., Rapicetta, M. and Verme. G. (1991) Adenine arabinoside monophosphate and acyclovir monophosphate coupled to lac-
Delivery
Reviews
17 (199.T) ?ll-.7/S
tosaminated albumin reduce woodchuck hepatitis virus viremia at doses lower than the unconjugated drugs. Hepatology 14, 16-24.
1141Fiume, L.. Busi, Preti, P. and Spinosa,
G. (1987) Conjugates of araAMP with lactosaminated albumin: A study on their immunoenicity in mouse and rat. Cancer Drug Del. 4. 145~150.
1151Fiume.
L.. Bonino, F., Mattioh, A.. Chiaberge. E., Cerenzia, M.R.T., Busi, C., Brunetto, M.R. and Verme, G. (lY88) Inhibition of hepatitis B virus replication by vidarabine monophosphate conjugated with lactosaminated serum albumin. Lancet 2, l-15.
Ilhl Cerenzia.
M.R.T.. Fiumc. L., Busi, C., Mattioli. A.. DiStefano. G.. Gervasi. G.B., Brunetto, M.R., Piantino, P..Verme. G. and Bonino. F. (1994) Inhibitin of hepatitis B virus replication by adenine arabinoside monophosphate coupled to lactosaminated albumin. Efficacy and minimal active dose. J. Hepatol. 20. 307-309.
1171Fiume.
L.. DiStefano, G., Busi. C. and Mattioh, A. of lactosaminated poly-t.-lysine with adenine arabinoside monophosphate, administered to mice by an intramuscular route, accomplishes a selective delivery of the drug to the liver. Biochem. Pharmocol. 47, 643-650.
( 1994) A conjugate
1181Biessen.
E.A.L.. Beuting. D.M., Vietsch, H.. Bijsterhosch, M.K. and Van Berkel, T.J.C. (1994) Specific targeting of the antiviral drug S-iodo-2’-deoxyuridine to the parenchymai liver cell using lactosylated poly-I.lysine. J. Hepatol. 21, 8066815. E.C., Bij1191Rensen, P.C.N.,Van Dijk, R.C.M. Havenaar. sterbosch. M.K.. Kar Kruijt, J. and Van Berkel. T.J.C. (1995) Selective liver targeting of antivirals hy recombinant chylomicrons -a new therapeutic approach to hepatitis B. Nat. Med. 1, 221-225. I201 Hantz. 0.. Turin, F. and Trepo, C. (1993) Therapy for hepatitis, with emphasis on the role of interferons. Curr. Opin. Infect. Dis. 6. 778-783. J.H., Di Bisceglie. A.M. and Shindo, M. 1211Hoofnagle (1993) Antiviral therapy of hepatitis C-present and tuture. J. Hepatol. 17. S130-SIX 1221line. S.. Hino. K. and Yasuda. K. (1994) Current state of interferon therapy for chronic hepatitis C. Intervirol 37. 877100. [2X] Ishihara. H.. Hayashi. Y., Hara. T.. Aramaki, Y., Tsuchiya, S. and Koike, K. (1991) Specific uptake of asialofetuin-tacked liposomes encapsulating interferon-~ by human hepatoma cells and its inhibitory effect on hepatitis B virus replication. Biochem. Biophys. Res. C‘ommun. 174, X39-845. v41 Cioodarzi. Cr.. Gross. S.C.. Tewari. A. and Watenabe, A. ( 1990) Antisense oligodeoxyribonucleotides inhibit the expression of the gene for hepatitis B virus surface antigen. J. Gen. Virol. 71, 3021-302.5. WI Blum, H.E.. Gahm, E.. Von Weizsacker, F. and Wands, J. (1991) Inhibition of hepatitis B virus by antisense oligonucleotides. Lancet 337. 1230. 1261Korha. B.. Wells, F., Jones. K.. Engle. R., Buckler-White. A. and Gerin. J. (1994) Inhibition of hepatitis B virus
R. Plourde, G.Y. Wu I Advanced
Drug Delivery Reviews 17 (1995) 311-315
replication in vitro by antisense oligonucleotides. Antiviral Res. 23, 78. [27] Wakita, T., Tokushige, K., Moradpour, D. and Wands, J.R. (1994) Dual effects of antisense oligonucleotide on hepatitis C virus antigen expression. Hepatology 20, 171A. (281 Ah, M., Renz, R., Hofschneider, P.H., Paumgartner, G. and Caselmann, W.H. (1994) Specific inhibition of hepatitis C viral gene expression by antisense phosphorothioate oligodeoxynucleotides. Hepatology 20, 171A. [29] Offensperger, W.-B.. Offensperger, S., Walter, E., Teubner, K., Igloi. G.. Blum, H.E. and Gerok, W. (1993) In vivo inhibition of duck hepatitis B virus replication and gene expression by phosphorothioate modified antisense oligodeoxynucleotides. EMBO J. 12, 12571262.
315
[30] Wu, G.Y. and Wu, C.H. (1988) Evidence for targeted gene delivery to Hep G2 cells in vitro. Biochemistry 27, 887-892. [31] Chiou, H.C., Tangco, M.V., Levine, SM., Robertson, D., Kormis, K., Wu, C.H. and Wu, G.Y. (1994) Enhanced resistance to nuclease degradation of nucleic acids carriers. complexed to asialoglycoprotein-polylysine Nucl. Acids Res. 22, 5439-5446. [32] Wu, G.Y. and Wu, C.H. (1988) Receptor-mediated gene delivery and expression in vivo. J. Biol. Chem. 263, 14621-14624. [33] Wu, G.Y. and Wu, C.H. (1992) Specific inhibition of hepatitis B viral gene expression in vitro by targeted antisense oligonucleotides. J. Biol. Chem. 267, 1243612439.
drug delivery reviews ELSEVIER
Advanced
Drug
Targeted
Delivery
Reviews
17 (1995)
31 l-315
therapy for viral hepatitis
Robert
Plourde”,
George
Y. WubT*
“Department of Chemistry, The Immune Response Corporation, _59.?_5Darwin Court, Carlsbad, CA 9200X. USA hDepartment of Medicine, Division of Gastroenterology-Hepatology. University of Connecticut School of Medic&. Rm AM-044. 263 Farmington Avenue, Farmington, CT 06030- 184.5, USA Accepted
June
1995
Abstract The presence of asialoglycoprotein receptors on hepatocytes has been used to target a variety hepatocytes. Chemical coupling of biologically active agents can result in specific delivery to these the use of asialoglycoprotein-based conjugates that can bind nucleic acids raises the possibility of of antisense molecules. Concepts and results of receptor-mediated targeting to hepatocytes for viral hepatitis are reviewed. Keywords:
Targeting;
Hepatocyte;
Asialoglycoprotein;
of substances to cells. In addition, targeted delivery the treatment of
Receptor mediation
Contents ......................................
1. Introduction 2. Targeting antiviral agents 2.1. Nucleoside analogs 2.2. lnterferons 2.3. Antisense oligonucleotides 3. Conclusions Acknowledgements References..........................
......................................
............ ............
............ ............
............ ............ ............
............ ............ ............
1. Introduction Because the liver is the primary site of infection and damage in viral hepatitis, specific targeting of therapeutic agents to the liver may be useful. Conventional antiviral drugs such as nucleoside analogs, which are effective inhibitors
*Corresponding 679-3159.
author.
Tel.
(203)
0169-409X/95/$29.00 @ 1995 Elsevier SSDl 0169-409X(95)00065-8
679-3158;
Fax
(203)
311 312 312 312 312 313 313 314
of viral replication in vitro, are often hampered in vivo because of broad biodistribution characteristics, and resultant extrahepatic toxicity. Selective delivery of antiviral agents to specific cell populations has been accomplished by many strategies [l]. Prodrugs, which modulate the biodistribution characteristics and pharmacokinetic properties of drugs, have been applied to improve the availability of drugs at a desired cell population. Carriers, both particulate and soluble, have been employed with the same rationale.
Science
B.V. All rights
reserved
These strategies have met with some success when applied to hepatitis virus therapy [2-41. More specific targeting has been achieved using ligands which bind specific cellular receptors. The asialoglycoprotein receptor [5] present in large numbers and high affinity only on hepatocytes, binds galactose-terminated glycoproteins and neoglycoproteins. Bound ligands are taken into the cell by receptor-mediated endocytosis. Because of its specificity, the asialoglycoprotein receptor (ASGPr) has been exploited as a means to deliver therapeutic agents to hepatocytes. Other receptors that are found largely on hepatocytes include the LDL receptor and the apoE receptor [6]. Here we review advances made toward receptor-mediated targeting of therapeutic agents to the liver with the aim of treating hepatitis.
2. Targeting antiviral agents 2.1. Nucleoside unalogs
In 1979, Fiume et al. [7] demonstrated specific delivery of a trifluorothymidine (F,T)-asialofetuin conjugate to hepatocytes. The conjugate was synthesized by coupling the N-hydroxysuccinimidyl ester of F,T glutarate with the c-amino groups of asialofetuin. It was shown to interact specifically with the ASGPr, and to selectively inhibit DNA synthesis in Ectromelia virus-infected liver cells. Subsequent studies by the same investigators have focused on adenine arabinofuranoside monophosphate (araAMP) and acyclovir monophosphate conjugates with galactosylated albumin [8-lo]. Albumin was galactosylated with lactose by reductive amination. The conjugates were synthesized with a phosphoramide bond between the drug and c-amino groups of the protein. The conjugates were shown to interact specifically with the ASGPr and release the drug within liver cells [ll]. They inhibited hepatitis B virus release in the HBV-transfected hepatoma cell line 2.2.15 [12] and in vivo, inhibited woodchuck hepatitis replication [13]. The conjugates were not immunogenic when prepared with
homologous albumin [14]. In clinical studies, administration of aruAMP galactosylated albumin conjugates resulted in a reduction in serum levels of HBV DNA comparable to that of free drug, but at doses 3-6 times lower [15,16]. No adverse effects were observed. Other systems for targeting nucleoside analogs to the liver for treatment of hepatitis have been studied. Drug conjugates with lactosylated polylysine also have been shown to target the asialoglycoprotein receptor [17,18]. Very recently, apolipoprotein E-containing chylomicrons were used to deliver iododeoxyuridine selectively to liver parenchymal cells [191. 2.2.Interferons Interferons are currently the leading choice of therapy for hepatitis B and hepatitis C virus. However, many patients do not respond to current formulations, and side effects have necessitated discontinuation of therapy in some individuals [20-221. I n an effort to specifically target y-interferon to hepatocytes, it was encapsulated in asialofetuin-tacked liposomes [23]. Liposomes with y-interferon, but no asialofetuin were prepared as controls. The AF-liposome encapsulated y-interferon interacted specifically with the ASGPr, and these liposomes were taken up by hepatocytes to a greater extent than the non-AF liposome encapsulated y-interferon. In Hep-HB107 cells the AF-liposome encapsulated y-interferon inhibited HBV DNA replication more effectively than did non-AF liposome encapsulated y-interferon.
2.3. Antisense oligonucleotides Antisense oligonucleotides are being explored extensively, as a means to inhibit replication of hepatitis viruses. A number of antisense sequences which are capable of inhibiting the replication of hepatitis B [24-261 and hepatitis C [27,28] in vitro have been identified. Efficacy has also been observed with an antisense phosphorothioate DNA in vivo [29]. However, because
R. Plourde. G.Y. Wu I Advanced
Drug Delivery Reviews 17 (199-5) 311-31.5
cellular uptake of oligo nucleotides is generally poor, and their sensitivity to degradation in plasma can be quite high, some form of targeting would be desirable for successful use of antisense DNA for therapy of viral hepatitis in vivo. We have described an asialoglycoprotein-polycation system for targeting nucleic acids to hepatocytes via the ASGPr. The plasma protein, orosomucoid, was desialylated by treatment with neuraminidase to produce asialoorosomucoid, a high-affinity ligand for the asialoglycoprotein receptor. Polylysine was then covalently attached to the asialoorosomucoid. The resulting ASORPL conjugate formed a soluble, neutral complex with DNA [30]. The conjugate not only binds DNA, but has subsequently been shown to protect complexed DNA from nuclease degradation [31]. The complex undergoes receptormediated endocytosis, and DNA is expressed within the hepatocyte. Both in vitro [32] and in vivo [33] targeting of plasmid DNA have been accomplished with this system. The same strategy was used to prepare ASORPL complexes with an antisense DNA. A 21-mer sequence complementary to the portion of the hepatitis B virus genome that contains the polyadenylation signal was prepared in a phosphorothioate linkage. Using an end-labeled species, it was determined that the oligo alone was taken up slowly, less than 0.05 pmole/hr/million cells, by the hepatoma cell lines HepG2 (ASGPr + ), or SK Hepl (ASGPr - ). However, the oligo in the form of a complex, was taken up more than 10 times more rapidly by HepG2 cells. The uptake of the complex was blocked by co-incubation with excess asialoorosomucoid. Uptake of the complex was not enhanced in the SK Hepl (ASGPr - ) cell line. To assess whether the targeted antisense was functional, the HepG2 2.2.15 cell line was used. This cell line possesses asialoglycoprotein receptors and is stably transfected with the complete HBV genome. It secretes viral antigens and infectious viral particles. Administration of complexed antisense blocked the expression of HBV surface antigen in these cells, and reduced the replication of viral DNA by about 80% compared to untreated controls. A complex containing a random sequence had no effect. The antisense oligo DNA alone decreased
313
the expression of surface antigen and viral replication by only approximately 30% [33]. An asialoglycoprotein-based DNA delivery system containing a 21-mer phosphorothioate antisense oligo DNA against the polyadenylation region and adjacent upstream sequences of Woodchuck Hepatitis Virus (WHV) was prepared. Experimental woodchucks were inoculated with the woodchuck virus (strain H7) six months prior to initiating the study, and all animals subsequently developed acute hepatitis infection, as evidenced by circulating viral DNA. Animals were injected i.v. with ASOR-poly(~lysine) complexes containing 0.4 mg antisense for 5 consecutive days, for a total dose of 2 mg antisense / animal. Levels of surface antigen did not differ significantly between treated and control animals. However, intravenous administration of antisense complex significantly decreased viral burden, as evidenced by a 5-lo-fold lower level of circulating viral DNA 25 days posttreatment. The decline lasted for at least 2 weeks, after which there was a gradual rise in DNA levels. Antisense alone or a complex containing a random oligo DNA of the same size and linkage failed to have any significant effect on viral DNA levels.
3. Conclusions Many powerful approaches are being developed to treat viral hepatitis. These include novel nucleoside analogs, inducers of viral specific nucleases, interferon, and antisense oligonucleotides. However, many of these have side effects due to toxicity in non-target tissues. Targeting through the use of receptor-mediated endocytosis may permit the realization of the potential of these new agents without the limitation of side effects.
Acknowledgments
This work was supported in part by grants from: NIH DK 42182 (G.Y.W.). G.Y.W. holds equity in the Immune Response Corp.
314
R. Plourdc.
G.Y.
Wu I Advuncrd
Drug
References ]I1 Meijer.
D.K.F., Jansen. R.W. and Molena. G. (1992) Drug targeting systems for antiviral agents: options and limitations. Antiviral Res. 18. 215-258. PI Glazier. A.. Kwong. C., Rose. J.. Buckheit. R.. Korha. B.. Abou-Donia, M. and Smith. E. (1992) Antiviral phosphate prodrugs. Antiviral Res. 17. 77. I-J1 Hostetler, K.Y.. Korba, B.E.. Sridhar. C.N. and Gardner. M.F. (1994) Antiviral activity of phosphatidyl-dideoxycytidine in hepatitis B-infected cells and enhanced hepatic uptake in mice. Antiviral Res. 24. 59967. 141 Mizushima, Y. and Hoshi. K. ( 1993) Recent advances in lipid microsphere technology for targeting prostaglandin delivery. J. Drug Targeting 1. 93-100. 151 Ashwell, G. and Morel], A.G. (1974) The role of surface carbohydrates in the hepatic recognition and transport of circulating glycoproteins. Adv. Enzymol. Rclat. Areas Mol. Biol. 41. 99-128. 161 Hoeg, J.M. and Demosky. S.J. Jr (1985) Distinct hepatic receptors for low density lipoprotein and apolipoprotein E in humans. Science 227. 759-761. A.. Balboni. P.G.. Tognon, M.. 171 Fiume, L.. Mattioli. Barbanti-Brodano. G., DeVries. J. and Wieland. ‘I’. (1979) Enhanced inhibition of virus DNA synthesis in hepatocytes hy trifluorothymidine coupled to asialofetuin. FEBS Lett. 103. 47-51. A.. Balhom. P.Ci. and IW Fiume. L.. Busi. C.. Mattioli, Barbanti-Brodano. G. (1981) Hepatocytc targeting ot adenine-9-P-n-arabinofuranoside S’-monophosphatc (ara-AMP) coupled to lactosaminated albumin, FEBS Lett. 129. 261-264. PI Fiume, L.. Bruna. B.. Busi. C’.. Mattioh, A. and Spmosa. G, (1986) Drug targeting in antiviral chemotherapy: .4 chemically stable conjugate of Y-P-o-arabinofuranosyladenine S’-monophosphate with lactosaminated albumin accomplishes a selective delivery of the drug to liver cells. Biochem. Pharmacol. 35. 967-972. 1101 Fiume, L.. Bassi. B. and Bongini. A. (1988) Conjuatcs 01 9-P-o-arabinofuranosyladenine S’-monophosphate (araAMP) with lactosaminated albumin. Characterization of the drug-carrier bonds. Pharm. Acta. Helv. 63, 137-139. 1111 Fiume. L.. Busi. C.. Spinelli. C. and Spinosa. G. (lY89) A cotrjugate of acyclovir monophosphate with lactosaminated albumin releases the phosphorylated drug in liver cells. Naturwissenschaften 76, 74-76. A., Chiabergc. E.. 1121 D’Urso, N., Actix. G.C., Ponzetto, Bonino. F. and Verme. G. (1990) Modulation of hepatitis B virus DNA release by targeted adenine arabinoside 5’.monophosphatc in HepG 2 cells that product infectious virions. In: F.B. Hollingcr. S.M. Lemon and H. Margolis (Eds). Viral Hepatitis and Liver Discasc. Williams, and Wilkins. Baltimore. p. 665. 1131 Ponzetto. A., Fiume. L.. Forzani. B.. Song. S.Y.. Busi. C.. Mattioli. A.. Spinelli, C., Marinehi, M., Smedile. A.. Chiaberge. E., Bonino, F.. Gervasi. G.B., Rapicetta, M. and Verme. G. (1991) Adenine arabinoside monophosphate and acyclovir monophosphate coupled to lac-
Delivery
Reviews
17 (199.T) ?ll-.7/S
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