Drug – bio-affecting and body treating compositions – Designated organic active ingredient containing – Carbohydrate doai
Reexamination Certificate
1999-03-25
2002-02-12
O'Sullivan, Peter (Department: 1621)
Drug, bio-affecting and body treating compositions
Designated organic active ingredient containing
Carbohydrate doai
C514S546000, C514S552000, C514S642000, C514S667000, C514S669000, C514S851000, C554S108000, C554S109000, C554S110000, C560S252000, C560S253000, C564S292000, C564S503000, C564S506000
Reexamination Certificate
active
06346516
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to novel cationic amphiphiles containing novel N-hydroxyalkyl group and therapeutic formulation containing the said amphiphiles that are capable of facilitating transport of biologically active molecules into cells. The invention further provides a process for the preparation of the said thereapeutic formulation containing amphiphilic compounds. The novel cationic amphiphiles containing N-hydroxyalkyl group of this invention are potentially useful to deliver therapeutically effective amounts of biologically active molecules into the cells of mammals. The area of medical science that is likely to benefit most from the present invention is gene therapy.
BACKGROUND OF THE INVENTION
In gene therapy, patients carrying identified defective genes are supplemented with the copies of the corresponding normal genes. However, genes (DNA), the polyanionic macromolecules and the cell surfaces of the biological membranes both being negatively charged, spontaneous entry of normal copies of genes into the target cells of patients is an inefficient process (because of electrostatic repulsion). This is why the past decade has witnessed an unprecedented upsurge of global interest in developing efficient gene delivery reagents for introducing normal genes into the target cells of patients suffering from various genetic (inherited) diseases such as cystic fibrosis, Gaucher's illness, Fabry's disease etc. Many gene delivery reagents (also known as transfection vectors) including retrovirus, adenovirus, and cationic amphiphilic compounds (i.e. compounds containing both polar and non-polar functionalities) are being used as the carriers of polyanionic genes in combating hereditary diseases in gene therapy. The amphiphilic nature (presence of both polar and non-polar regions in the molecular structures) of the compounds designed to deliver therapeutically actives molecules, ensures smooth interaction of these carrier molecules with the polar and non-polar regions of plasma membranes, compartments within the cells and the biologically active molecules itself. At physiological pH, the cationic amphiphiles in the form of liposomes or micelles associate favorably with the negatively charged regions of the macromolecular polyanionic DNA enhancing the intracelluar uptake of the resulting complex between the cationic lipids and the negatively charged DNA. Reproducibility, high degree of targetability and low cellular toxicity are increasingly making the cationic amphiphiles the transfection vectors of choice in gene therapy.
Reported Developments
An impressive number of cationic lipids with varying structures have been reported for the intracellular delivery of therapeutically active molecules as exemplified by the following references:
Felgner et al.,
Proc. Natl. Acad. Sci. U.S.A.,
84, 7413-7417 (1987), reported the first use of a highly efficient cationic lipid N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethyl ammonium chloride (DOTMA) as the DNA transfection vector.
U.S. Pat. Nos. 4,897,355 and 4,946,787 (1990) reported the synthesis and use of N-[.omega..(.omega.-1)-dialkyloxy]- and N-[..omega..(.omega.-1)-dialkenyloxy]-alk-1-yl-N,N,N-tetrasubstituted ammonium amphiphiles and their pharmaceutical formulation as efficient transfection vectors.
Leventis, R. and Silvius, J. R.
Biochim. Biophys. Acta.
1023, 124-132, (1990) reported the interactions of mammalian cells with lipid dispersions containing novel metabolizable cationic amphiphiles.
U.S. Pat. No. 5,264,618 (1993) reported the synthesis and use of additional series of highly efficient cationic lipids for intracellular delivery of biologically active molecules.
Felgner et al.
J Biol. Chem.
269, 2550-2561 (1994) reported enhanced gene delivery and mechanistic studies with a novel series of cationic lipid formulations.
U.S. Pat. No. 5,283,185 (1994) reported the synthesis and use of 3&bgr;[N-(N
1
,N
1
-dimethylaminoethane)carbamoyl] cholesterol, termed as “DC-Chol” for delivery of a plasmid carrying a gene for chloramphenicol acetyl transferase into cultured mammalian cells.
U.S. Pat. No. 5,283,185 (1994) reported the use of N-[2-[[2,5-bis[(3-aminopropyl)amino]-1-Oxopentyl]aminoethyl]-N,N-dimethyl-2,3-bis-(9-Octadecenyloxy)-1-Propanaminium tetra(trifluoroacetate), one of the most widely used cationic lipids in gene delivery. The pharmaceutical formulation containing this cationic lipid is sold commercially under the trade name “Lipofectamine”.
Solodin et al.
Biochemistry
34, 13537-13544, (1995) reported a novel series of amphiphilic imidazolinium compounds for in vitro and in vivo gene delivery.
Wheeler et al.
Proc. Natl. Acad.Sci. U.S.A.
93, 11454-11459, (1996) reported a novel cationic lipid that greatly enhances plasmid DNA delivery and expression in mouse lung.
U.S. Pat No. 5,527,928 (1996) reported the synthesis and the use of N,N,N,N-tetramethyl-N,N-bis(hydroxy ethyl)-2,3-di(oleolyoxy)-1,4-butanediammonium iodide i.e. pharmaceutical formulation as transfection vector.
U.S. Pat. No. 5,698,721 (1997) reported the synthesis and use of alkyl O-phosphate esters of diacylphosphate compounds such as phosphatidylcholine or phosphatidylethanolamine for intracellular delivery of maromolecules.
U.S. Pat. Nos. 5,661,018; 5,686,620 and 5,688,958 (1997) disclosed a novel class of cationic phospholipids containing phosphotriester derivatives of phosphoglycerides and sphingolipids efficient in the lipofection of nucleic acids.
U.S. Pat. No. 5,614,503 (1997) reported the synthesis and use of an amphipathic transporter for delivery of nucleic acid into cells, comprising an essentially nontoxic, biodegradable cationic compound having a cationic polyamine head group capable of binding a nucleic acid and a cholesterol lipid tail capable of associating with a cellular membrane.
U.S. Pat. No. 5,705,693 (1998) disclosed the method of preparation and use of new cationic lipids and intermediates in their synthesis that are useful for transfecting nucleic acids or peptides into prokaryotic or eukaryotic cells. These lipids comprise one or two substituted arginine, lysine or ornithine residues, or derivatives thereof, linked to a lipophilic moiety.
U.S. Pat. No. 5,719,131 (1998) has reported the synthesis of a series of novel cationic amphiphiles that facilitate transport of genes into cells. The amphiphiles contain lipophilic groups derived from steroids, from mono or dialkylamines, alkylamines or polyalkylamines.
Although the above mentioned cationic lipids have been successfully exploited for the intracellular delivery of genes, the efficiencies for the intracellular uptake procedures are insufficient and need to be improved. The transfection activities of most of the above mentioned cationic lipids are modest and therefore substantial quantities of these cationic lipids must be consumed. The associated cellular toxicities of the lipids and the metabolites thereof are, thus naturally, issues of concern. Accordingly, demands for developing new class of cationic amphiphiles with high transfection efficiencies and low cellular toxicities continue in this field of art.
OBJECTS OF THE INVENTION
The main objective of the invention is to provide novel cationic amphiphilic compounds containing non-toxic N-hydroxyalkyl group.
Another objective of the invention is to provide novel cationic amphiphilic compounds, which are useful for delivery of therapeutically effective amounts of biologically active molecules into cells/tissues of mammals.
Yet another objective of the invention is to provide cationic amphiphilic compounds such that a hydrophobic group is either directly linked to the positively charged Nitrogen atom or is linked to the said Nitrogen atom via an ester or methylene group.
Still another objective of the invention is to provide novel cationic amphiphilic compounds with at least one hydroxyalkyl group containing 1-3 carbon atoms directly linked to the positively charged Nitrogen atom.
Yet another objective of the invention is t
Banerjee Rajkumar
Chaudhuri Arabinda
Das Prasanta Kumar
Rao Nalam Madhusudhana
Srilakshmi Gollapudi Venkata
Council of Scientific & Industrial Research
Finnegan Henderson Farabow Garrett & Dunner L.L.P.
O'Sullivan Peter
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