Chemistry: molecular biology and microbiology – Process of mutation – cell fusion – or genetic modification – Introduction of a polynucleotide molecule into or...
Reexamination Certificate
2000-01-25
2002-05-07
McGarry, Sean (Department: 1635)
Chemistry: molecular biology and microbiology
Process of mutation, cell fusion, or genetic modification
Introduction of a polynucleotide molecule into or...
C536S024500, C530S333000, C530S335000, C530S395000
Reexamination Certificate
active
06383812
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates to a novel method of synthesizing ligands for the asialoglycoprotein receptor, a recycling endocytotic receptor on the surface of mammalian hepatocytes. Novel ligands having affinity to the asialoglycoprotein receptor and a method of synthesizing thereof are also disclosed.
2. Description of the Related Art
Mammalian liver cells possess specific membrane-bound receptors, asialoglycoprotein receptors (ASGPr), which receive ligands having terminal galactose/N-acetylgalactosamine residues. Ashwell el al.,
Annu. Rev. Biochem
., 1982, 51, 531. The affinity of such a receptor to its ligand depends on the valency of the terminal galactose (Gal) or N-acetylgalactosamine (GalNAc), Lee et al.,
Biochem, Biophys. Res. Commun
., 1988, 155, 1444, as well as on the three-dimensional arrangement of the sugar residues. Lee,
FASEB
. 1992, 6, 3193-3200.
Lee et al. reported a synthetic ASGPr ligand, YEE(GalNAcAH)
3
, having a GalNAc-containing trivalents and an affinity of subnanomolar to the ASGP-r.
Glycoconjugate J
., 1987, 4, 317-328. YEE(GalNAcAH)
3
has the following structure:
The use of YEE(GalNAcAH)
3
as a vehicle for delivery of a gene or an antisense oligodeoxynucleotide to the liver has been also reported recently. Merwin et al.
Bioconjugate Chem
. 1994, 5, 612-620; Hangeland et al.,
Bioconjugate Chem
. 1995, 5, 695-701. However, synthesis of YEE(GalNAcAH)
3
suffers from some practical problems mainly due to the poor solubility of its peptide intermediates in either aqueous or organic solvents.
Therefore, it is desirable to develop a method of synthesizing YEE(GalNAcAH)
3
devoid of the solubility problems encountered by others.
SUMMARY OF THE INVENTION
Accordingly, an object of the present invention is directed to a novel process for synthesizing a general class of branched glycoside ligands having binding affinity to asialoglycoprotein receptor, and potentially useful for specific drug delivery to the liver.
Another object of the present invention is directed to a novel process for synthesizing YEE(GalNAcAH)
3
using protected GalNAc derivatives as building blocks through a stepwise solid-phase peptide synthesis (SPPS) technique. The present process of synthesizing YEE(GalNAcAH)
3
is simpler, more efficient and economical than the conventional processes and devoid of the problems of the conventional methods.
Another object of the present invention is directed to the synthesis of novel ligands of asialoglycoprotein receptors, for example YEEE(GalNAcAH)
3
, which is an analog of YEE(GalNAcAH)
3
, synthesized by a process similar to the synthesis of YEE(GalNAcAH)
3
. The analog maintains affinity to asialoglycoprotein receptors comparable to YEE(GalNAcAH)
3
. YEEE(GalNAcAH)
3
has the following structure:
Another object of the present invention is directed to the general class of branched glycoside ligands having binding affinity to asialoglycoprotein receptor (including YEE(GalNAcAH)
3
and YEEE(GalNAcAH)
3
), wherein the ligands are complexed to a therapeutically effective agent. Such therapeutically effective agents include nucleic acids, proteins, polysaccharides, lipids and radioactive isotopes. Preferred nucleic acids include DNA or RNA. A preferred protein is interferon. Such branched glycoside ligands complexed to a therapeutically effective agent may be used to transfect hepatocytes. An especially preferred complex for transfecting hepatocytes is a complex comprising YEEE(GalNAcAH)
3
and a group consisting of sequences of antisense oligonucleotides of PKC. (protein kinase C) &agr;: sequence (5′-3′) CAGCCATGGTTCCCCCCAAC. (SEQ ID NO: 2).
The various features of novelty which characterize the invention are pointed out with particularity in the claims annexed to and forming a part of the disclosure. For a better understanding of the invention, its operating advantages, and specific objects attained by its use, reference should be had to the drawing and descriptive matter in which there are illustrated and described preferred embodiments of the invention.
REFERENCES:
patent: 4859449 (1989-08-01), Mattes
patent: 5346696 (1994-09-01), Kim
patent: 5874297 (1999-02-01), Wu et al.
Ashwell, G. et al., “Carbohydrate-Specific Receptors of the Liver”,Ann. Rev. Biochem.,1982. 51:531-54 (p2, ln,11).
Lee, Reiko T. et al., “Rabbit and Rat Hepatic Lectins Have Two-Sugar-Combining Sites Per Monomeric Unit”,Biochemical and Biophysical Research Communications,1988, 155: 1444-1451 (p2, ln.14).
Lee, Y.C., “Biochemistry of Carbohydrate-Protein Interaction”,FASEB,1992, 6:3193-3200 (p2, ln.14).
Lee, Reiko T. et al., “Preparation of Cluster Glycosides of N-Acetylgalactosamine That Have Subnanomolar Binding Constants Towards the Mammalian Hepatic Gal/GalNAc-specific Receptor”,Glycoconjugate J.,1987, 4:317-328 (p2,ln.16-17).
Merwin, June Rae et al., “Targeted Delivery of DNA Using YEE/(GalNAcAAH)3, A Synthetic Glycopeptide Ligand For the Asialoglycoprotein Receptor”,Bioconjugate Chem.,1994, 5:612-620 (p3, ln.5).
Hangeland, Jon J. et al., “Cell-Type Specific and Ligand Specific Enhancement of Cellular Uptake of Oligodeoxynucleoside Methylphosphonates Covalently Linked With a Neoglycopeptide, YEE(ah-Ga1NAc)3”,Bioconjugate Chem.,1995, 5:695-701, (p3,ln.5).
“A New Procedure For The Preparation Of Oligosaccharide Oxazolines;Satoru”, Nakabayashi, et al.,Carbohydrate Research,150 (1986) pC7-C10.
“Stepwise Synthesis Of A Ga1NAc-Containing Cluster Glycoside Ligand Of The Asialoglycoprotein Receptor”; Mark A. Findeis; International Journal of Peptide & Protein Research; International Journal of Peptide & Protein Research 43. 1994, 477-485.
“New Synthetic Cluster Ligands For galactos/N-Acetylgalactosamine-Specific Lectin of Mammalian Liver”; Reiko T. Lee, et al.; Biochemistry 1984, 23, 4255-4261.
“Synthesis Of Cluster Galactosides With High Affinity For The Hepatic Asialoglycoprotein Receptor”; Erik A. L. Biessen, et al.; J. Med. Chem. 1995, 38, 1538-1546.
Chen Shui-Tein
Huang Chun-Ming
Jiaang Weir-Torn
Tseng Ping-Hui
Academia Sinica
Cohen & Pontani, Lieberman & Pavane
Epps Janet L.
McGarry Sean
LandOfFree
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