Drug – bio-affecting and body treating compositions – Designated organic active ingredient containing – Carbohydrate doai
Reexamination Certificate
1998-08-03
2002-07-02
Nguyen, Dave T. (Department: 1633)
Drug, bio-affecting and body treating compositions
Designated organic active ingredient containing
Carbohydrate doai
C424S468000, C424S482000, C435S320100, C435S455000
Reexamination Certificate
active
06413941
ABSTRACT:
The present invention relates to polymers, in particular to polymers for the delivery of nucleic acid to a cell.
For many research applications in genetic manipulation and genetic engineering, it is necessary to express new or modified genes in living cells. However the uptake of DNA into cells is poor resulting in inconsistent expression. Similarly, gene therapy, antisense oligonucleotide therapy and gene vaccination require that DNA and DNA analogues can survive in a hostile biological environment, penetrate biological barriers, be taken up into cells and move to the correct subcellular compartment to exert their therapeutic effects.
The identification of defective genes responsible for disease states, either through the overproduction of key proteins, the production of defective proteins or the defective control of gene production, offers new possibilities for the treatment of disease. By controlling the defect at the genetic level a range of diseases could now be treated effectively rather than by merely treating the symptoms of these diseases. This has been achieved in some cases, or is believed to be achievable, by the expression of new competent genes, or by controlling the overproduction of unwanted gene products or by controlling the expression of genes. These processes could be achieved by the insertion of new DNA or by the administration and uptake of complementary strands of DNA or DNA analogues which inhibit the production or control the production of existing genes [1]. In both of these strategies it is necessary to deliver to the cell sufficient DNA to achieve modified cell expression. The DNA must also be delivered to the correct intracellular compartment to effect that change. While some DNA is taken up naturally into cells, the amount taken up is small and inconsistent, and expression of added DNA is poor. DNA is an inherently unstable material, particularly in a biological environment where many specific enzymes capable of degrading DNA are found [2]. Either for therapeutic purposes, or for expression of new or modified genes for research purposes, a more efficient and reliable method of delivering DNA is required and, in particular, protection of the DNA against metabolic effects is highly desirable.
A number of strategies have been proposed to achieve these aims. These include the use of liposomes [3], cationic lipids [4], which are often incorrectly referred to as ‘cationic liposomes’, and the use of cationic polymers such as polylysine [5] or polyornithine as DNA delivery agents.
Both oligonucleotides and DNA constructs, such as plasmids, have shown improved activity by condensation with polycations such as polylysine. In the former case chemical conjugation of oligonucleotide to the polymer is required, whereas in the latter case complexation of polymer with DNA also confers these effects. Poly-L-lysine (PLL) is believed to condense the DNA into a smaller volume, and by the excess positive charge of the complex, bind to negatively charged cell surfaces to facilitate interaction with the cell surface and uptake into the cell.
The effectiveness of polylysine-DNA complexes has been enhanced by coupling ligands to the polylysine which further facilitate binding and uptake into cells [6]. Membrane destabilising agents have been added to DNA preparations to facilitate exit of the DNA from the degradative endosomal compartments of the cell [7].
To date, few different cationic polymers have been used in this work, and the available polymers are deficient in a number of respects. Poly-L-lysine, the principal polymer presently used for this purpose, is known to be toxic above a small molecular weight [8], it does not interact stoichiometrically with DNA, and the resulting complex is unreliable, difficult to control and its properties strongly dependent on the ratio of DNA to polymer.
Ranucci et al [15] describes the synthesis of poly(amidoamine)s and suggests their use as polymeric drug carriers using covalent attachment of the drug molecule to the polymer.
Ranucci and Ferruti [12] describes hydrolyzable block copolymers containing poly(ethyleneglycol) (PEG) and poly(amidoamine) (PAA) or poly(amido-thioether-amine).
Haensler and Szoka [10] suggests that polyamidoamine cascade polymers (dendrimers prepared from branched chain poly(amidoamine)s) of a certain size may be useful in transfection of cells in culture and states that linear polycations in general are relatively cytotoxic and by themselves not very efficient, which limits their usefulness for transfection of cells in culture.
Duncan et al [16] describes poly(amidoamine)-Triton X-100 conjugates which may be useful for drug delivery.
Katayose and Kataoka [17] suggest that a PEG-poly(lysine) block copolymer as a potential DNA delivery system.
Attaching PEG chains to macromolecules and colloidal particles has been described for many biomedical products [11].
There remains a need for polymers which have improved properties for use in DNA delivery systems.
A first aspect of the invention provides a composition for delivering a biologically active polyanionic molecule, the composition comprising a linear polymer with a backbone comprising amido and tertiary amino groups arranged regularly on the said backbone and said biologically active polyanionic molecule bound to said polymer.
The said linear polymers are cationic and, depending on their nature, as discussed more fully below, the polymers have a range of physicochemical properties.
Conveniently, the said linear polymer comprises a poly(amidoamine) (PAA). Suitably, said linear polymer consists of a poly(amidoamnine). PAAs are degradable in water since they contain hydrolyzable amidic bonds in their main chain together with nucleophilic tertiary-aminic fuinctions in the position. The polymers can be synthesised from a wide variety of primary monoamines or secondary bisamines which enable full control to be exercised over the spacing and pKa of the cationic groups [9] for optimisation of the interaction with a suitable biologically active polyanionic molecule. Preferably, PAAs are water soluble, and thus facilitate the solubility of the complex. Further solubilisation of complexes can be achieved by use of the copolymers containing hydrophilic PEG chains. (Reference [9] is incorporated herein by reference.)
It is preferred if the pKa of the cationic groups is between 7 and 8. It has been found that PAA with a low pKa binds DNA less well than PAA with a high pKa. It is more preferred if the pKa of the PAA is around 8.
Thus, in a preferred embodiment, said linear polymer further comprises ethylene glycol or poly(ethyleneglycol). It is particularly preferred if the linear polymer is a poly(ethyleneglycol)-poly(amidoamine) block copolymer or ethyleneglycol-poly(amidoamine) block copolymer.
Preferably said linear polymer is a block copolymer with the structure [poly(amidoaniine)-(ethyleneglycol)
y
]
x
wherein x is from 1 to 50 and y is from 1 to 200, wherever it may occur.
Also preferably said linear polymer is a block copolymer with the structure (ethyleneglycol)
y
-poly(amidoamine)-(ethyleneglycol)
y
wherein each y is independently 1 to 200.
Suitably the linear polymer consists of or comprises a PAA which has the formula:
or said linear polymer comprises a PAA such as with the formula:
or (e) wherein
are replaced by
and, in any case, z is from 0 (or 1 as appropriate) to 70 and each R
1
is independently H or a linear or branched hydrocarbon chain —C
n
H
2n+1
with n=1-4 whenever it occurs; each R
2
is independently a linear or branched alkylene chain —C
n
H
2n
— with n=1-4 whenever it occurs; each R
3
is independently a linear or branched hydrocarbon chain —C
n
H
2n+1
with n=1-4 whenever it occurs; each R
4
is independently a linear or branched alkylene chain —C
n
H
2n
— with n=2-4 whenever it occurs; each R
5
is independently a linear or branched
Bignotti Fabio
Davis Stanley Stewart
Ferruti Paolo
Garnett Martin Charles
Maclaughlin Fiona Caroline
Akin Gump Strauss Hauer & Feld L.L.P.
Nguyen Dave T.
West Pharmaceutical Services Drug Delivery & Clinical Research C
LandOfFree
Polymer composition for delivering a nucleic acid or... does not yet have a rating. At this time, there are no reviews or comments for this patent.
If you have personal experience with Polymer composition for delivering a nucleic acid or..., we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Polymer composition for delivering a nucleic acid or... will most certainly appreciate the feedback.
Profile ID: LFUS-PAI-O-2889040