U.S. patent application number 10/083861 was filed with the patent office on 2003-04-17 for novel cationic lipopolymer as biocompatible gene delivery agent.
Invention is credited to Furgeson, Darin Y., Han, Sang-Oh, Mahato, Ram I..
Application Number | 20030073619 10/083861 |
Document ID | / |
Family ID | 24658024 |
Filed Date | 2003-04-17 |
United States Patent
Application |
20030073619 |
Kind Code |
A1 |
Mahato, Ram I. ; et
al. |
April 17, 2003 |
Novel cationic lipopolymer as biocompatible gene delivery agent
Abstract
A biodegradable non-toxic cationic lipopolymer comprising a
branched polyethylenimine (PEI), a lipid anchor, biocompatible
hydrophilic polymer spacer, and a biodegradable linker which
covalently links the branched PEI, the spacer and the cholesterol
derived lipid anchor. The cationic lipopolymers in the present
invention can be used in drug delivery and are especially useful
for delivery of a nucleic acid or any anionic bioactive agent to
various organs and tissues after local or systemic
administration.
Inventors: |
Mahato, Ram I.; (Memphis,
TN) ; Han, Sang-Oh; (Salt Lake City, UT) ;
Furgeson, Darin Y.; (Salt Lake City, UT) |
Correspondence
Address: |
M. Wayne Western
THORPE, NORTH & WESTERN, L.L.P.
P.O. Box 1219
Sandy
UT
84091-1219
US
|
Family ID: |
24658024 |
Appl. No.: |
10/083861 |
Filed: |
February 25, 2002 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10083861 |
Feb 25, 2002 |
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09662511 |
Sep 14, 2000 |
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Current U.S.
Class: |
514/44R ;
424/178.1; 514/1.2; 514/19.1; 514/20.9; 514/23; 514/5.4; 514/5.9;
514/53; 514/54; 514/7.7; 514/9.6; 525/54.5; 525/540 |
Current CPC
Class: |
A61K 47/645 20170801;
A61K 9/1272 20130101; A61P 29/00 20180101; A61P 35/00 20180101 |
Class at
Publication: |
514/8 ; 514/54;
514/12; 514/23; 514/53; 514/44; 424/178.1; 525/540; 525/54.5 |
International
Class: |
A61K 048/00; A61K
038/16; A61K 031/70; A61K 031/7012; A61K 031/739; A61K 039/395;
C08G 063/91 |
Claims
We claim:
1. A biodegradable non-toxic cationic lipopolymer comprising a
branched polyethylenimine (PEI), a lipid anchor, a biocompatible
hydrophilic polymer spacer, and a biodegradable linker which
covalently links the branched PEI, the spacer and the cholesterol
derived lipid anchor.
2. The cationic lipopolymer of claim 1, wherein the biodegradable
linker is an ester bond.
3. The cationic lipopolymer of claim 1, wherein the lipid anchor is
cholesterol or its derivative, a C.sub.12 to C.sub.18 fatty acid or
a derivative thereof.
4. The cationic lipopolymer of claim 1, wherein the biocompatible
hydrophilic polymer spacer is polyethylene glycol (PEG) having a
molecular weight of between 0.5 to 20K Daltons.
5. The cationic lipopolymer of claim 1, further comprising a
targeting moiety selected from the group consisting of transferrin,
asialoglycoprotein, antibodies, antibody fragments, low density
lipoproteins, interleukins, GM-C SF, G-C SF, M-CSF, stemcell
factors, erythropoietin, epidermal growth factor (EGF), insulin,
asialoorosomucoid, mannose-6-phosphate, mannose, Lewis.sup.X and
sialyl Lewis.sup.X, N-acetyllactosamine, galactose, lactose, and
thrombomodulin, fusogenic agents such as polymixin B and
hemaglutinin HA2, lysosomotrophic agents, and nucleus localization
signals (NLS).
6. The cationic lipid of claim 5 wherein the targeting moiety is
galctose or lactose.
7. The cationic lipopolymer of claim 1, wherein molar ratio of the
branched PEI to the lipid anchor is preferably within a range of
1:1 to 1:20.
8. A biodegradable non-toxic cationic lipopolymer comprising a
branched polyethylenimine (PEI) having an average molecular weight
of 600 to 1200 Daltons, a lipid anchor, biocompatible hydrophilic
polymer spacer, and a biodegradable linker which covalently links
the branched PEI, the spacer and the cholesterol derived lipid
anchor.
9. The cationic lipopolymer of claim 8, wherein the biodegradable
linker is an ester bond.
10. The cationic lipopolymer of claim 8, wherein the lipid anchor
is a cholesterol, a C.sub.12 to C.sub.18 fatty acid or a derivative
thereof.
11. The cationic lipopolymer of claim 8, wherein the biocompatible
hydrophilic polymer spacer is polyethylene glycol (PEG) having a
molecular weight of between 0.5 to 20K Daltons.
12. The cationic lipopolymer of claim 8, further comprising a
targeting moiety selected from the group consisting of transferrin,
asialoglycoprotein, antibodies, antibody fragments, low density
lipoproteins, interleukins, GM-CSF, G-CSF, M-CSF, stemcell factors,
erythropoietin, epidermal growth factor (EGF), insulin,
asialoorosomucoid, mannose-6-phosphate, mannose, Lewis.sup.X and
sialyl Lewis.sup.X, N-acetyllactosamine, galactose, lactose, and
thrombomodulin, fusogenic agents such as polymixin B and
hemaglutinin HA2, lysosomotrophic agents, and nucleus localization
signals (NLS).
13. The cationic lipid of claim 12 wherein the targeting moiety is
galctose or lactose.
14. The cationic lipopolymer of claim 8, wherein molar ratio of the
branched PEI to the lipid anchor is preferably within a range of
1:1 to 1:2.
15. A biodegradable non-toxic cationic lipopolymer comprising a
branched polyethylenimine (PEI) having an average molecular weight
of 1800 to 25000 Daltons, a lipid anchor, biocompatible hydrophilic
polymer spacer, and a biodegradable linker which covalently links
the branched PEI, the spacer and the cholesterol derived lipid
anchor.
16. The cationic lipopolymer of claim 15, wherein molar ratio of
the branched PEI to the lipid anchor is preferably within a range
of 1:1 to 1:5.
17. The cationic lipopolymer of claim 15, wherein the biodegradable
linker is an ester bond.
18. The cationic lipopolymer of claim 15, wherein the lipid anchor
is a cholesterol, a C.sub.12 to C.sub.18 fatty acid or a derivative
thereof.
19. The cationic lipopolymer of claim 15, wherein the biocompatible
hydrophilic polymer spacer is polyethylene glycol (PEG) having a
molecular weight of between 0.5 to 20K Daltons.
20. The cationic lipopolymer of claim 15, further comprising a
targeting moiety selected from the group consisting of transferrin,
asialoglycoprotein, antibodies, antibody fragments, low density
lipoproteins, interleukins, GM-CSF, G-CSF, M-CSF, stemcell factors,
erythropoietin, epidermal growth factor (EGF), insulin,
asialoorosomucoid, mannose-6-phosphate, mannose, Lewis.sup.X and
sialyl Lewis.sup.X, N-acetyllactosamine, galactose, lactose, and
thrombomodulin, fusogenic agents such as polymixin B and
hemaglutinin HA2, lysosomotrophic agents, and nucleus localization
signals (NLS).
21. The cationic lipid of claim 20 wherein the targeting moiety is
galctose or lactose.
22. A pharmaceutical composition comprising a bioactive agent and a
biodegradable non-toxic cationic lipopolymer comprising a branched
polyethylenimine (PEI), a lipid anchor, biocompatible hydrophilic
polymer spacer, and a biodegradable linker which covalently links
the branched PEI, the spacer and the cholesterol derived lipid
anchor.
23. The composition of claim 22, wherein the biodegradable linker
is an ester bond.
24. The composition of claim 22, wherein the lipid anchor is a
cholesterol, a C.sub.12 to C.sub.18 fatty acid or a derivative
thereof.
25. The composition of claim 22, wherein the biocompatible
hydrophilic polymer spacer is polyethylene glycol (PEG) having a
molecular weight of between 0.5 to 20K Daltons.
26. The composition of claim 22, further comprising a targeting
moiety selected from the group consisting of transferrin,
asialoglycoprotein, antibodies, antibody fragments, low density
lipoproteins, interleukins, GM-CSF, G-CSF, M-CSF, stemcell factors,
erythropoietin, epidermal growth factor (EGF), insulin,
asialoorosomucoid, mannose-6-phosphate, mannose, Lewis.sup.X and
sialyl Lewis.sup.X, N-acetyllactosamine, galactose, lactose, and
thrombomodulin, fusogenic agents such as polymixin B and
hemaglutinin HA2, lysosomotrophic agents, and nucleus localization
signals (NLS).
27. The composition of claim 26 wherein the targeting moiety is
galctose or lactose.
28. The composition of claim 22, wherein molar ratio of the
branched PEI to the lipid anchor is preferably within a range of
1:1 to 1:20.
29. The composition of claim 22 wherein said bioactive agent is a
nucleic acid, protein or an anionic drug.
30. The composition of claim 29, wherein the charge ratio of the
cationic lipopolymer and the nucleic acid (+/-) is within a range
of 5:1 to 1:1.
31. The composition of claim 29, further comprising a helper
lipid.
32. The composition of claim 31, wherein the helper lipid is a
member selected from the group consisting of
dioleoylphosphatidylethanolamine (DOPE),
oleoylpalmitoyl-phosphatidylethanolamin (POPE),
diphytanoylphosphatidylethanolamin (diphytanoylPE), disteroyl-,
-palmitoyl-, -myristoylphosphatidylethanolamine and 1- to 3-fold
N-methylated derivatives thereof.
33. The composition of claim 31, wherein the molar ratio of the
cationic lipopolymer and the helper lipid is within a range of 4:1
to 1:2.
34. A method of deliverying a bioactive agent into a warm blooded
animal, comprising administering a effective amount of the
composition comprising a bioactive agent and a biodegradable
non-toxic cationic lipopolymer comprising a branched
polyethylenimine (PEI), a lipid anchor, biocompatible hydrophilic
polymer spacer, and a biodegradable linker which covalently links
the branched PEI, the spacer and the cholesterol derived lipid
anchor to the animal under conditions wherein said composition
enters said cells, and the bioactive agent of said composition is
released.
35. The method of claim 34 wherein the administration is local or
systemic.
Description
[0001] This application is a continuation-in-part of pending U.S.
patent application Ser. No. 09/662,511, filed Sep. 4, 2000.
FIELD OF THE INVENTION
[0002] This invention relates to delivery of a bioactive agent.
More particularly, the invention relates to a composition and
method for delivering bioactive agents, such as DNA, RNA,
oligonucleotides, proteins, peptides, and drugs, by facilitating
their transmembrane transport or by enhancing their adhesion to
biological surfaces. It relates particularly to a novel cationic
lipopolymer comprising a branched polyethylenimine (PEI), a
cholesterol derived lipid anchor, and a biodegradable linker which
covalently links the branched PEI and cholesterol derived lipid
anchor. One example of such a novel lipolymer is poly{(ethylene
imine)-co-[N-2-aminoethyl) ethylene
imine]-co-[N-(N-cholesteryloxycabonyl-(2-aminoethyl))ethylene
imine]} (hereafter referred to as "PEACE"). The cationic
lipopolymers of the present invention can be used in drug delivery
and are especially useful for delivery of a nucleic acid or any
anionic bioactive agent.
BACKGROUND OF THE INVENTION
[0003] Gene therapy is generally considered as a promising approach
not only for the treatment of diseases with genetic defects but
also in the development of strategies for treatment and prevention
of chronic diseases such as cancer, cardiovascular disease and
rheumatoid arthritis. However, nucleic acids as well as other
polyanionic substances are rapidly degraded by nucleases and
exhibit poor cellular uptake when delivered in aqueous solutions.
Since early efforts to identify methods for delivery of nucleic
acids in tissue culture cells in the mid 1950's, steady progress
has been made towards improving delivery of functional DNA, RNA,
and antisense oligonucleotides in vitro and in vivo.
[0004] The gene carriers used so far include viral systems
(retroviruses, adenoviruses, adeno-associated viruses, or herpes
simplex viruses) or nonviral systems (liposomes, polymers,
peptides, calcium phosphate precipitation and electroporation).
Viral vectors have been shown to have high transfection efficiency
when compared to non-viral vectors, but due to several drawbacks,
such as targeting only dividing cells, random DNA insertion, their
low capacity for carrying large sized therapeutic genes, risk of
replication, and possible host immune reaction, their use in vivo
is severely limited.
[0005] Compared to viral vectors, nonviral vectors are easy to make
and less likely to produce immune reactions, and there is no
replication reaction required. There has been increasing attention
focused on the development of safe and efficient nonviral gene
transfer vectors, which are either polycationic polymers or
cationic lipids. Polycationic polymers such as poly-L-lysine,
poly-L-ornithine and polyethyleneimine (PEI), that interact with
DNA to form polyionic complexes, have been introduced for use in
gene delivery. Various cationic lipids have also been shown to form
lipoplexes with DNA and induce efficient transfection of various
eukaryotic cells. Among such kinds of synthetic vectors, cationic
lipids are widely used because it is possible to design and
synthesize numerous derivatives that are outstanding in the aspects
of transfection efficiency, biodegradability and low toxicity. Many
different cationic lipids are commercially available and several
lipids have already been used in the clinical setting. Among them,
cationic cholesterol derivatives are known to be very useful
because of their high transfection efficiency in vitro. Although
the mechanism of this transfection activity is not yet clear, it
probably involves binding of the DNA/lipid complex with the cell
surface via excess positive charges on the complex. Cell surface
bound complexes are probably internalized and the DNA released into
the cytoplasm of the cell from an endocytic compartment.
[0006] However, it is not feasible to directly extend in vitro
transfection technology to in vivo applications. Relative to in
vivo use, the biggest drawback of the diether lipids, such as
N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethyl ammonium chloride
(DOTMA) or Lipofectin is that they are not natural metabolites of
the body, and are thus not biodegradable and they are toxic to
cells. In addition, it has been reported that cationic lipid
transfection is inhibited by factors present in serum and thus it
is an ineffective means for the introduction of genetic material
into cells in vivo.
[0007] An ideal transfection reagent should exhibit a high level of
transfection activity without needing any mechanical or physical
manipulation of the cells or tissues. The reagent should be
non-toxic, or minimally toxic, at the effective dose. In order to
avoid any long-term adverse side-effects on the treated cells, it
should also be biodegradable. When gene carriers are used for
delivery of nucleic acids in vivo, it is essential that the gene
carriers themselves be nontoxic and that they degrade into
non-toxic products. To minimize the toxicity of the intact gene
carrier and its degradation products, the design of gene carriers
needs to be based on naturally occurring metabolites.
[0008] U.S. Pat. No. 5,283,185, to Epand et al. (hereafter the '185
patent), discloses a method for facilitating the transfer of
nucleic acids into cells comprising preparing a mixed lipid
dispersion of a cationic lipid
3.beta.[N-(N',N"-dimethylaminoethane)-carbamoyl]cholestero- l
(DC-cholesterol) with a co-lipid in a suitable carrier solvent. The
method disclosed in the '185 patent involves using a halogenated
solvent in preparing a liposome suspension. For pharmaceutical
applications, residues of halogenated solvents cannot be
practically removed from a preparation after having been
introduced. U.S. Pat. No. 5,753,262, (hereafter the '262 patent)
discloses using the acid salt of the lipid
3.beta.[N-(N',N"-dimethylaminoethane)-carbamoyl]cholesterol
(DC-cholesterol) and a helper lipid such as dioleoyl
phosphatidylethanolamine (DOPE) or dioleoylphosphatidylcholine
(DOPC) to produce effective transfection in vitro. In addition,
these cationic lipids have been proven less efficient in gene
transfer in vivo.
[0009] Because of their sub-cellular size, nanoparticles are
hypothesized to enhance interfacial cellular uptake, thus achieving
in a true sense a "local pharmacological drug effect." It is also
hypothesized that there would be enhanced cellular uptake of drugs
contained in nanoparticles (due to endocytosis) compared to the
corresponding free drugs. Nanoparticles have been investigated as
drug carrier systems for tumor localization of therapeutic agents
in cancer therapy, for intracellular targeting (antiviral or
antibacterial agents), for targeting to the reticuloendothelial
system (parasitic infections), as an immunological adjuvant (by
oral and subcutaneous routes), for ocular delivery with sustained
drug action, and for prolonged systemic drug therapy.
[0010] In view of the foregoing, it will be appreciated that
providing a gene carrier that is non-toxic, biodegradable, and
capable of forming nanoparticles, liposomes, or micelles for gene
therapy and drug delivery, is desired. The novel gene carrier of
the present invention comprises a novel cationic lipopolymer
comprising a branched polyethylenimine (PEI), a cholesterol derived
lipid anchor, and a biodegradable linker which covalently links the
branched PEI and cholesterol derived lipid anchor. The lipolymer of
the present invention is useful for preparing a cationic liposome,
or a cationic micelle for drug delivery, especially for delivery of
nucleic acids, other anionic bioactive molecules or both and is
readily susceptible to metabolic degradation after incorporation
into the cell.
BRIEF SUMMARY OF THE INVENTION
[0011] The present invention provides a biodegradable cationic
lipopolymer, having reduced in vivo and in vitro toxicity, for
delivery of drugs or other bioactive agents to an individual in
need thereof.
[0012] The present invention also provides a cationic lipopolymer
for delivery of nucleic acids which carries out both stable and
transient transfection of polynucleotides such as DNA and RNA into
cells more effectively.
[0013] The biodegradable, non-toxic cationic lipopolymer of the
present invention comprises a branched polyethylenimine (PEI), a
cholesterol derived lipid anchor, and a biodegradable linker which
covalently links the branched PEI and cholesterol derived lipid
anchor. Preferably, the average molecular weight of the branched
PEI is within a range of 600 to 25,000 Daltons. The branched PEI is
preferably conjugated to the cholesterol derivative by an ester
bond. The molar ratio of the branched PEI to the conjugated
cholesterol derivative is preferably within a range of 1:1 to
1:20.
[0014] By adjusting the molecular weight of the branched PEI and
the molar ratio of the branched PEI to conjugated cholesterol
derivative, the resultant lipolymer can be either water soluble or
water insoluble. For example, to obtain a water soluble
lipopolymer, the average molecular weight of the branched PEI is
preferably within a range of 1800 to 25,000, and the molar ratio of
the branched PEI to the conjugated cholesterol derivative is
preferably within a range of 1:1 to 1:5. To obtain a water
insoluble lipopolymer, the average molecular weight of the branched
PEI is preferably within a range of 600 to 1800, and the molar
ratio of the branched PEI to the conjugated cholesterol derivative
is preferably within a range of 1:1 to 1:2. Although a cholesterol
derived lipid anchor is preferred in the present invention, other
lipophilic moieties may also be used, such as C.sub.12 to C.sub.18
saturated or unsaturated fatty acids.
[0015] The biodegradable lipopolymers can be synthesized by
relatively simple and inexpensive methods. These cationic
lipopolymers invention can spontaneously form discrete
nanometer-sized particles with a nucleic acid, which can promote
more efficient gene transfection into mammalian cell lines than can
be achieved conventionally with Lipofectin and polyethyleneimine.
The lipopolymer the present invention is readily susceptible to
metabolic degradation after incorporation into animal cells.
Moreover, the water soluble cationic lipopolymer can form an
aqueous micellar solution which is particularly useful for systemic
delivery of various bioactive agents such as DNA, proteins,
hydrophobic or hydrophilic drugs. The water insoluble lipopolymer
can form cationic liposomes with a helper, which is particularly
useful for local drug delivery. Therefore, the biocompatible and
biodegradable cationic lipopolymer of this invention provides an
improved gene carrier for use as a general reagent for transfection
of mammalian cells, and for the in vivo application of gene
therapy.
[0016] The present invention further provides transfection
formulations, comprising a novel cationic lipopolymer, complexed
with a selected nucleic acid in the proper charge ratio (positive
charge of the lipopolymer/negative charge of the nucleic acid) that
is optimally effective for both in vivo and in vitro transfection.
Particularly, for systemic delivery, the charge ratio (+/-) is
preferably 5/1 to 1/1; for local delivery, the charge ratio (+/-)
is preferably 3/1 to 0.5/1.
[0017] This invention also provides for a method of transfecting,
both in vivo and in vitro, a nucleic acid into a mammalian cell.
The method comprises contacting the cell with a cationic
lipopolymer or liposome:nucleic acid complexes as described above.
In one embodiment, the method uses systemic administration of the
cationic lipopolymer or liposome:nucleic acid complexes into a warm
blooded animal. In a preferred embodiment, the method of
transfecting uses intravenous administration of the cationic
lipopolymer or liposome:nucleic acid complexes into a warm blooded
animal. In a particularly preferred embodiment, the method
comprises intravenous injection of water soluble PEACE/pDNA and
PEACE:DOPE liposomes/pDNA complexes into a warm blooded animal.
BRIEF DESCRIPTION OF THE DRAWINGS
[0018] FIG. 1 illustrates a synthetic scheme for poly{(ethylene
imine)-co-[N-2-aminoehtyl) ethylene
imine]-co-[N-(N-cholesteryloxycabonyl- -(2-aminoethyl))ethylene
imine]} ("PEACE").
[0019] FIG. 2. illustrates determination of chemical structure and
molecular weight of water insoluble PEACE 1800 by .sup.1H NMR
spectra (FIG. 2A) and MALDI-TOF mass spectra (FIG. 2B).
[0020] FIG. 3 illustrates determination of chemical structure and
molecular weight of water soluble PEACE 1800 by .sup.1H NMR spectra
(FIG. 3A) and MALDI-TOF mass spectra (FIG. 3B).
[0021] FIG. 4 illustrates gel retardation assay of water soluble
PEACE/pCMV-Luc complexes (FIG. 4A) and PEACE:DOPE liposome/pCMV-Luc
complexes (FIG. 4B).
[0022] FIG. 5 illustrates Dnase protection assay of water soluble
PEACE/pCMV-Luc complexes.
[0023] FIG. 6 illustrates viability assay of CT-26 colon
adenocarcinoma cells after being transfected by water soluble
PEACE/pCMV-Luc complexes (FIG. 6A) and PEACE:DOPE liposome/pCMV-Luc
complexes (FIG. 6B).
[0024] FIG. 7 illustrates luciferase activity assay in cultured
CT-26 colon adenocarcinoma cells after being transfected by water
soluble PEACE/pCMV-Luc complexes (FIG. 7A) and PEACE:DOPE
liposome/pCMV-Luc complexes (FIG. 7B).
[0025] FIG. 8 illustrates RT-PCR assay of CT-26 colon
adenocarcinoma cells after being transfected in vitro by PEACE:DOPE
liposome/pmlL-12 complexes.
DETAILED DESCRIPTION
[0026] Before the present composition and method for delivery of a
bioactive agent are disclosed and described, it is to be understood
that this invention is not limited to the particular
configurations, process steps, and materials disclosed herein as
such configurations, process steps, and materials may vary
somewhat. It is also to be understood that the terminology employed
herein is used for the purpose of describing particular embodiments
only and is not intended to be limiting since the scope of the
present invention will be limited only by the appended claims and
equivalents thereof.
[0027] It must be noted that, as used in this specification and the
appended claims, the singular forms "a," "an," and "the" include
plural referents unless the context clearly dictates otherwise.
Thus, for example, reference to a polymer containing "a sugar"
includes reference to two or more of such sugars, reference to "a
ligand" includes reference to one or more of such ligands, and
reference to "a drug" includes reference to two or more of such
drugs.
[0028] In describing and claiming the present invention, the
following terminology will be used in accordance with the
definitions set out below.
[0029] "Transfecting" or "transfection" shall mean transport of
nucleic acids from the environment external to a cell to the
internal cellular environment, with particular reference to the
cytoplasm and/or cell nucleus. Without being bound by any
particular theory, it is understood that nucleic acids may be
delivered to cells either in forms or after being encapsulated
within or adhering to one or more cationic lipid/nucleic acid
complexes or entrained therewith. Particular transfecting instances
deliver a nucleic acid to a cell nucleus. Nucleic acids include
both DNA and RNA as well as synthetic congeners thereof. Such
nucleic acids include missense, antisense, nonsense, as well as
protein producing nucleotides, on and off and rate regulatory
nucleotides that control protein and peptide, and nucleic acid
production. In particular, but nonlimiting, they can be genomic
DNA, cDNA, mRNA, tRNA, rRNA, hybrid sequences or synthetic or
semi-synthetic sequences and of natural or artificial origin. In
addition, the nucleic acid can be variable in size, ranging from
oligonucleotides to chromosomes. These nucleic acids may be of
human, animal, vegetable, bacterial, viral, and the like, origin.
They may be obtained by any technique known to a person skilled in
the art.
[0030] As used herein, the term "bioactive agent" or "drug" or any
other similar term means any chemical or biological material or
compound suitable for administration by the methods previously
known in the art and/or by the methods taught in the present
invention that induce a desired biological or pharmacological
effect, which may include but is not limited to (1) having a
prophylactic effect on the organism and preventing an undesired
biological effect such as preventing an infection, (2) alleviating
a condition caused by a disease, for example, alleviating pain or
inflammation caused as a result of disease, and/or (3) either
alleviating, reducing, or completely eliminating a disease from the
organism. The effect may be local, such as providing for a local
anaesthetic effect, or it may be systemic.
[0031] This invention is not drawn to novel drugs or to new classes
of bioactive agents. Rather it is limited to the compositions and
methods of delivery of genes or other bioactive agents that exist
in the state of the art or that may later be established as active
agents and that are suitable for delivery by the present invention.
Such substances include broad classes of compounds normally
delivered into the body. In general, this includes but not limited
to: nucleic acids, such as DNA, RNA, and oligonucleotides.,
antiinfectives such as antibiotics and antiviral agents; analgesics
and analgesic combinations; anorexics; antihelminthics;
antiarthritics; antiasthmatic agents; anticonvulsants;
antidepressants; antidiabetic agents; antidiarrheals;
antihistamines; antiinflammatory agents; antimigraine preparations;
antinauseants; antineoplastics; antiparkinsonism drugs;
antipruritics; antipsychotics; antipyretics; antispasmodics;
anticholinergics; sympathomimetics; xanthine derivatives;
cardiovascular preparations including potassium, calcium channel
blockers, beta-blockers, alpha-blockers, and antiarrhythmics;
antihypertensives; diuretics and antidiuretics; vasodilators
including general, coronary, peripheral and cerebral; central
nervous system stimulants; vasoconstrictors; cough and cold
preparations, including decongestants; hormones such as estradiol
and other steroids including corticosteroids; hypnotics;
immunosuppressives; muscle relaxants; parasympatholytics;
psychostimulants; sedatives; and tranquilizers. By the method of
the present invention, drugs in all forms, e.g. ionized,
nonionized, free base, acid addition salt, and the like may be
delivered, as can drugs of either high or low molecular weight.
[0032] As used herein, "effective amount" means an amount of a
nucleic acid or a bioactive agent that is nontoxic but sufficient
to provide the desired local or systemic effect and performance at
a reasonable risk/benefit ratio that would attend any medical
treatment.
[0033] As used herein, "peptide", means peptides of any length and
includes proteins. The terms "polypeptide" and "oligopeptide" are
used herein without any particular intended size limitation, unless
a particular size is otherwise stated. Typical of peptides that can
be utilized are those selected from the group consisting of
oxytocin, vasopressin, adrenocorticotrophic hormone, epidermal
growth factor, prolactin, luliberin or luteinising hormone
releasing hormone, growth hormone, growth hormone releasing factor,
insulin, somatostatin, glucagon, interferon, gastrin, tetragastrin,
pentagastrin, urogastroine, secretin, calcitonin, enkephalins,
endorphins, angiotensins, renin, bradykinin, bacitracins,
polymixins, colistins, tyrocidin, grarnicidines, and synthetic
analogues, modifications and pharmacologically active fragments
thereof, monoclonal antibodies and soluble vaccines. The only
limitation to the peptide or protein drug which may be utilized is
one of functionality.
[0034] As used herein, a "derivative" of a carbohydrate includes,
for example, an acid form of a sugar, e.g. glucuronic acid; an
amine of a sugar, e.g. galactosamine; a phosphate of a sugar, e.g.
mannose-6-phosphate; and the like.
[0035] As used herein, a "liposome" means a microscopic vesicle
composed of uni- or multilamllar bilayer or bilayers surrounding
aqueous compartments.
[0036] As used herein, "administering", and similar terms means
delivering the composition to the individual being treated such
that the composition is capable of being circulated systemically
where the composition binds to a target cell and is taken up by
endocytosis. Thus, the composition is preferably administered to
the individual systemically, typically by subcutaneous,
intramuscular, intravenous, or intraperitoneal administration.
Injectables for such use can be prepared in conventional forms,
either as a liquid solution or suspension, or in a solid form that
is suitable for preparation as a solution or suspension in a liquid
prior to injection, or as an emulsion. Suitable excipients include,
for example, water, saline, dextrose, glycerol, ethanol, and the
like; and if desired, minor amounts of auxiliary substances such as
wetting or emulsifying agents, buffers, and the like can be
added.
[0037] Fundamental to the success of gene therapy is the
development of gene delivery vehicles that are safe and efficacious
after systemic administration. Many of the cationic lipids used in
the early clinical trials, such as N[1-(2,3-dioleyloxy)propyl]-N,N,
N-trimethylammonium chloride (DOTMA) and 3-.beta.(N,
N"-dimethylaminoethane cabamoyl cholesterol) (DC-Chol) although
exhibiting efficient gene transfer in vitro, have been proven less
efficient in gene transfer in animals. See. Felgner PL et. al
Lipofection: A highly efficient, lipid-mediated DNA transfection
procedure. Proc Natl Acad Sci USA 84: 7413-7417 (1987); and Gao, X.
and Huang L. (1991) A novel cationic liposome reagent for efficient
transfection of mammalian cells. Biochem. Biophys. Res. Commun.
179: 280-285.
[0038] The general structure of a cationic lipid has three parts:
(i) a hydrophobic lipid anchor, which helps in forming liposomes
(or micellar structures) and interacts with cell membranes; (ii) a
linker group; and (iii) a positively charged head-group, which
interacts with the plasmid, leading to its condensation. Many
compounds bearing either a single tertiary or quaternary ammonium
head-group or which contain protonatable polyamines linked to
dialkyl lipid or cholesterol anchors have been used for
transfection into various cell types. The orientation of the
polyamine head-group in relation to the lipid anchor has been shown
to greatly influence the transfection efficiency. Conjugation of
spermine or spermidine head-groups to the cholesterol lipid anchor
via a carbamate linkage through a secondary amine to generate
T-shape cationic lipids has been shown to be very effective in gene
transfer to the lung. In contrast, the generation of a linear
polyamine lipid by conjugating spermine or spermidine to
cholesterol or a dialkyl lipid anchor was much less effective in
gene transfer.
[0039] A cationic lipid which contains three protonatable amines in
its head-group has been shown to be more active than
DC-Cholesterol, which contains only one protonatable amine. In
addition to the number of protonatable amines, the choice of the
linker group bridging the hydrophobic lipid anchor with the
cationic head-group has also been shown to influence gene transfer
activity. Substitution of the carbamate linker with either urea, an
amide or amine, resulted in appreciable loss of transfection
activity. PEI has been shown to be highly effective in gene
transfer, which is dependent on its molecular weight and charge
ratio. However, high molecular weight PEI is very toxic to the
cells and tissues.
[0040] The biodegradable cationic lipopolymer of the present
invention comprises a branched polyethylenimine (PEI), a
cholesterol derived lipid anchor, and a biodegradable linker which
covalently links the branched PEI and cholesterol derived lipid
anchor. Preferably, the average molecular weight of the branched
PEI is within a range of 600 to 25,000 Daltons. The branched PEI is
preferably conjugated to the cholesterol derivative by an ester
bond. One example of such a novel lipolymer is poly{(ethylene
imine)-co-[N-2-aminoehtyl) ethylene
imine]-co-[N-(N-cholesteryloxycabonyl-(2-aminoethyl))ethylene
imine]} (hereafter as "PEACE"). Primary, secondary and tertiary
amines of PEI contained in PEACE provide sufficient positive
charges for adequate DNA condensation. The linkage between the
polar head group and hydrophobic lipid anchor is biodegradable and
yet strong enough to survive in a biological environment. The ester
linkage between the cholesterol lipid anchor and polyethyleneimine
provides for the biodegradability of the lipolymer and the
relatively low molecular weight branched PEI significantly
decreases the toxicity of the lipopolymer. Although a cholesterol
derived lipid anchor is preferred in the present invention, other
lipophilic moieties may also be used, such as C.sub.12 to C.sub.18
saturated or unsaturated fatty acids.
[0041] The biodegradable cationic lipopolymer of the present
invention, such as PEACE, has amine group(s) which are
electrostatically attracted to polyanionic compounds such as
nucleic acids. The cationic lipopolymer or cationic liposome of the
present invention condenses DNA, for example, into compact
structures. Upon administration, such complexes of these cationic
lipopolymers and nucleic acids are internalized into cells through
receptor mediated endocytosis. In addition, the lipophilic group of
the lipopolymer or liposome allows the insertion of the cationic
amphiphile into the membrane or liposome of the cell and serves as
an anchor for the cationic amine group to attach to the surface of
a cell. The lipopolymers of the present invention have both highly
charged positive group(s) and hydrophilic group(s), which greatly
enhances cellular and tissue uptake in the delivery of genes,
drugs, and other bioactive agents. In addition, using relatively
low molecular weight branched PEI reduces the potential
cytotoxicity of the polymer and increases transfection
efficiency.
[0042] Stability of condensed nucleic acids in physiological
conditions is one of the major hurdles for their use in clinics.
The other major limitation to the use of condensed nucleic acids in
vivo is their tendency to interact with serum proteins, resulting
in destabilization and rapid clearance by the reticuloendothelial
cells following intravenous administration. The compatibility and
solubility of cationic lipopolymer can be improved by conjugating
with hydrophilic non-toxic polymers like poly(ethylene glycol
(PEG). PEG is an FDA-approved polymer known to inhibit the
immunogenicity of molecules to which it is attached. PEGylation
covers the condensed DNA particles with a "shell" of PEG;
stabilizes nucleic acids against aggregation in physiological salt;
decreases recognition of the cationic lipopolymer by the immune
system; and slows their breakdown by the nucleases after in vivo
administration. Therefore, another embodiment of the present
invention is PEGylated lipopolymer comprising a branched
polyethylenimine (PEI), a cholesterol derived lipid anchor, a PEG
as a spacer between the cholesterol derived lipid anchor and the
branched PEI, and a biodegradable linker which covalently links the
branched PEI, the PEG spacer, and the cholesterol derived lipid
anchor. Preferably, the average molecular weight of PEG is within a
range of 0.5 to 20K Daltons and more preferably within a range of
0.5 to 5K Daltons.
[0043] The amine groups on the branched PEI can also be conjugated
either directly to the amine groups or via spacer molecules, with
targeting ligands and the like. Preferably, only a portion of the
available amine groups are coupled to the ligand or spacer/ligand
such that the net charge of the lipopolymer is positive. The target
ligands conjugated to the lipopolymer direct the
lipopolymer-nucleic acid/drug complex to bind to specific target
cells and penetrate into such cells (tumor cells, liver cells,
heamatopoietic cells, and the like). The target ligands can also be
an intraellular targeting element, enabling the transfer of the
nucleic acid/drug to be guided towards certain favored cellular
compartments (mitochondria, nucleus, and the like). In a preferred
embodiment, the ligands can be sugar moieties coupled to the amino
groups. Such sugar moieties are preferably mono- or
oligo-saccharides, such as galactose, glucose, fucose, fructose,
lactose, sucrose, mannose, cellobiose, nytrose, triose, dextrose,
trehalose, maltose, galactosamine, glucosamine, galacturonic acid,
glucuronic acid, and gluconic acid.
[0044] The conjugation of an acid derivative of a sugar with the
cationic lipid is most preferred. In a preferred embodiment of the
present invention, lactobionic acid
(4-O-.beta.-D-galactopyranosyl-D-gluconic acid) is coupled to the
lipopolymer. The galactosyl unit of lactose provides a convenient
targeting molecule for hepatocyte cells because of the high
affinity and avidity of the galactose receptor on these cells.
[0045] Other types of ligands that can be used include peptides
such as antibodies or antibody fragments, cell receptors, growth
factor receptors, cytokine receptors, transferrin, epidermal growth
factor (EGF), insulin, asialoorosomucoid, mannose-6-phosphate
(monocytes), mannose (macrophage, some B cells), Lewis.sup.X and
sialyl Lewis.sup.X (endothelial cells), N-acetyllactosamine (T
cells), galactose (colon carcinoma cells), and thrombomodulin
(mouse lung endothelial cells), fusogenic agents such as polymixin
B and hemaglutinin HA2, lysosomotrophic agents, nucleus
localization signals (NLS) such as T-antigen, and the like.
[0046] An advantage of the present invention is that it provides a
gene carrier wherein the particle size and charge density are
easily controlled. Control of particle size is crucial for
optimization of a gene delivery system because the particle size
often governs the transfection efficiency, cytotoxicity, and tissue
targeting in vivo. In general, in order to enable its effective
penetration into tissue, the size of a gene delivery particle
should not exceed the size of a virus. In the present invention,
the particle size can be varied by using different numbers of
lysinamide linked to the cholesterol, which in turn determines the
particle size of the-nucleic acid complex.
[0047] In a preferred embodiment of the invention, the particle
sizes will range from about 80 to 200 nm depending on the cationic
lipopolymer composition and the mixing ratio of the components. It
is known that particles, nanospheres, and microspheres of different
sizes when injected accumulate in different organs of the body
depending on the size of the particles injected. For example,
particles of less than 150 nm diameter can pass through the
sinusoidal fenestrations of the liver endothelium and become
localized, in the spleen, bone marrow, and possibly tumor tissue
after systemic administration.
[0048] Intravenous, intra-arterial, or intraperitoneal injection of
particles approximately 0.1 to 2.0 .mu.m diameter leads to rapid
clearance of the particles from the blood stream by macrophages of
the reticuloendothelial system. The novel cationic lipopolymers of
the present invention can be used to manufacture dispersions of
controlled particle size, which can be organ-targeted in the manner
described herein.
[0049] It is believed that the presently claimed composition is
effective in delivering, by endocytosis, a selected nucleic acid
into hepatocytes mediated by galactosyl receptors on the surface of
hepatocyte cells. Nucleic acid transfer to other cells can be
carried out by matching a cell having a selected receptor thereof
with a selected sugar. For example, the carbohydrate-conjugated
cationic lipids of the present invention can be prepared from
mannose for transfecting macrophages, from N-acetyllactosamine for
transfecting T cells, and galactose for transfecting colon
carcinoma cells.
[0050] The cationic lipopolymer of the present invention provides a
highly positively charged cationic lipid which is biodegradable and
amphiphilic, namely hydrophilic branched PEI and hydrophobic
cholesterol derivatives, where the hydrophilic polycation PEI
complexes with negatively charged nucleic acids or other bioactive
agents and increases the cellular uptake of drug-loaded cationic
lipids. The hydrophilic group can be branched PEI where the average
molecular weight of the branched PEI is within a range of 600 to
25,000 Daltons. The hydrophobic group is preferably a cholesterol
or its derivative. The branched PEI is preferably conjugated with
the cholesterol derivative by an ester bond. The molar ratio of the
branched PEI to the conjugated cholesterol derivative is preferably
within a range of 1:1 to 1:20.
[0051] By adjusting the molecular weight of the branched PEI and
the molar ratio of the branched PEI to the conjugated cholesterol
derivative, the resultant lipolymer can be either water soluble or
water insoluble. For example, to obtain a water soluble lipopolymer
of the present invention, the average molecular weight of the
branched PEI is preferably within a range of 1800 to 25000, and the
molar ratio of the branched PEI to the conjugated cholesterol
derivative is preferably within a range of 1:1 to 1:5. To obtain a
water insoluble lipopolymer of the present invention, the average
molecular weight of the branched PEI is preferably within a range
of 600 to 1800, and the molar ratio of the branched PEI to the
conjugated cholesterol derivative is preferably within a range of
1:1 to 1:2.
[0052] The water soluable cationic lipopolymers are dispersible in
water and form cationic micelles and can therefore be used to
manufacture sustained release formulations of drugs without
requiring the use of high temperatures or extremes of pH, and, for
water-soluble drugs such as polypeptides and oligonucleotides,
without exposing of the drug to organic solvents during
formulations. Such biodegradable cationic lipopolymers are also
useful for the manufacture of sustained, continuous release
injectable formulations of drugs. They can act as very efficient
dispersing agents and can be administered by injection to give
sustained release of lipophilic drugs.
[0053] In addition, the water insoluble lipopolymers of the
invention can be used alone, or preferably, in a mixture with a
helper lipid in the form of cationic liposome formulations for gene
delivery to particular organs of the human or animal body. The use
of a neutral helper lipids is especially advantageous when the
charge ratio (amines/phosphates) is low. Preferably the helper
lipid is a member selected from the group consisting of
dioleoylphosphatidylethanolamine (DOPE),
oleoylpalmitoylphosphatidylethanolamin (POPE),
diphytanoylphosphatidyleth- anolamin (diphytanoyl PE), disteroyl-,
-palmitoyl-, and -myristoylphosphatidylethanolamine as well as
their 1- to 3-fold N-methylated derivatives. Preferably, the molar
ratio of the lipopolymer and the helper lipid is within a range of
4:1 to 1:2 and more preferably within a range of 2:1 to 1:1. To
optimaze the transfection efficiency of the present compositions,
it is preferred to use water as the excipient and diphytanoyl PE as
the helper lipid. In addition, the charge ratio (+/-) is preferably
5/1 to 1/1 for systemic delivery and 3/1 to 0.5/1 for local
delivery. This ratio may be changed by a person skilled in the art
in accordance with the polymer used, the presence of an adjuvant,
the nucleic acid, the target cell and the mode of the
administration used.
[0054] Liposomes haves been used successfully with a number of cell
types that are normally resistant to transfection by other
procedures. Liposomes have been used effectively to introduce
genes, drugs, radiotherapeutic agents, enzymes, viruses,
transcription factors, and allosteric effectors into a variety of
cultured cell lines and animals. In addition, several studies
suggest that the use of liposomes is not associated with autoimmune
responses, toxicity or gonadal localization after systemic
delivery. See, Nabel et al. Gene transfer in vivo with DNA-liposome
complexes, Human Gene Ther., 3:649-656, 1992b.
[0055] Since cationic liposomes and micelles are known to be good
for intracellular delivery of substances other than nucleic acids,
the cationic liposomes or micelles formed by the cationic
lipopolymers of the present invention can be used for the cellular
delivery of substances other than nucleic acids, such as, for
example, proteins and various pharmaceutical or bioactive agents.
The present invention therefore provides methods for treating
various disease states, so long as the treatment involves transfer
of material into cells. In particular, treating the following
disease states is included within the scope of this invention:
cancers, infectious diseases, inflammatory diseases and genetic
hereditary diseases.
[0056] The cationic lipopolymer of the present invention, having
improved cellular binding and uptake of the bioactive agent to be
delivered, is directed to overcome the problems associated with
known cationic lipids, as set forth above. For example, the
biodegradable cationic lipopolymer PEACE is easily hydrolyzed or
converted to PEI and cholesterol in the body. Due to its low
molecular weight, PEI will easily be released from circulation,
where cholesterol is naturally occurring molecule. The degradation
products are small, non-toxic molecules, that are subject to renal
excretion and are inert during the period required for gene
expression. Degradation is by simple hydrolytic and/or enzymatic
reaction. Enzymatic degradation may be significant in certain
organelles, such as lysosomes. The time needed for degradation can
vary from days to months depending on the molecular weight and
modifications made to the cationic lipids.
[0057] Furthermore, nanoparticles or microsphere complexes can be
formed from the cationic lipopolymer of the present invention and
nucleic acids or other negatively charged bioactive agents by
simple mixing. The lipophilic group (cholesterol derivative) of the
cationic lipopolymer of the present invention allows for the
insertion of the cationic amphiphile into the membrane of the cell.
It serves as an anchor for the cationic amine group to attach to
the surface of a cell, which enhances uptake of the cationic
carrier/nucleic acid complex by the cell to be transfected.
Therefore, the cationic gene carrier of the present invention
provides improved transfection efficiency both in vitro and in
vivo, compared to known cationic gene carriers.
[0058] The following examples will enable those skilled in the art
to more clearly understand how to practice the present invention.
It is to be understood that, while the invention has been described
in conjunction with the preferred specific embodiments thereof,
that which follows is intended to illustrate and not limit the
scope of the invention. Other aspects of the invention will be
apparent to those skilled in the art to which the invention
pertains.
[0059] The following is the general disclosure of the sources of
all the chemical compounds and reagents used in the
experiments.
[0060] Polyethyleneimine (PEI) of 600, 1200 and 1800 Da was
purchased from Polysciences, Inc. (Warrington, PN); cholesteryl
chloroformate was purchased from Aldrich, Inc. (Milwaukee, Wis.);
2-dioleoyl-sn-glycero-3-p- hosphoethanolamine (DOPE) was purchased
from Avanti Polar Lipids (Alabaster, Ala.). Triethylamine (TEA);
anhydrous methylene chloride; chloroform; ethyl ether, and acetone
were purchased from Sigma (St. Louis, Mo.).
EXAMPLE 1
Synthesis of Water-Insoluble PEACE
[0061] This example illustrates the preparation of water-insoluble
PEACE.
[0062] One gram of PEI (Mw:1200 Daltons) was dissolved in a mixture
of 15 mL anhydrous methylene chloride and 100 .mu.l triethylamine
(TEA). After stirring on ice for 30 minutes, 1.2 g of cholesteryl
chloroformate solution was slowly added to the PEI solution and the
mixture was stirred overnight on ice. The resulting product (PEACE)
was precipitated by adding ethyl ether followed by centrifugation
and subsequent washing with additional ethyl ether and acetone.
PEACE was dissolved in chloroform for a final concentration of 0.08
g/mL. A schematic of the above reaction is presented in FIG. 1.
Following synthesis and purification, PEACE was characterized using
MALDI-TOFF MS and .sup.1H NMR.
[0063] The NMR results of water insoluble PEACE 1200 are as
follows: as illustrated in FIG. 2A, .sup.1H NMR (200 MHz,
CDCl.sub.3), .delta. 0.6 (3 H of CH.sub.3 from cholesterol);
.delta. 2.5 (230 H of --NHCH.sub.2CH.sub.2-- from the backbone of
PED; .delta. 3.1 (72 H of .dbd.N--CH.sub.2CH.sub.2--NH.sub.2 from
the side chain of PEI); .delta. 5.3 (1 H of .dbd.C.dbd.CH--C-- from
cholesterol). Another peak appearing at .delta. 0.8, -.delta. 1.9
was cholesterol. The amount of cholesterol conjugated to PEI was
determined to be about 40%. MALDI-TOF mass spectrometric analysis
of PEACE showed its molecular weight to be approximately 1600, as
illustrated in FIG. 2B. The peak appeared from 800 to 2700 and the
majority peaks were around 1600, which is expected since PEI of
1200 Da and cholesterol of 414 (removal of chloride) were used for
synthesis. This suggests that the majority of PEACE 1200
synthesized were of 1/1 molar ratio of cholesterol and PEI,
although some were either not conjugated or conjugated at the molar
ratio of 2/1 (cholesterol/PEI).
EXAMPLE 2
Synthesis of Water Soluble PEACE
[0064] This example illustrates the preparation of water-soluble
PEACE.
[0065] Three grams of PEI (Mw: 1800 Daltons) was stirred on ice in
a mixture of 10 ml anhydrous ethylene chloride and 100 .mu.l
triethyamine for 30 minutes. One grain of cholesterol chloroformate
was dissolved in 5 ml of anhydrous ice-cold methylene chloride and
then slowly added to the PEI solution for 30 minutes. The mixture
was stirred for 12 hrs on ice and the resulting product was dried
in a rotary evaporator. The powder was dissolved in 50 ml 0.1 N
HCl. The aqueous solution was extracted with 100 mL of methylene
chloride 3 times, and then filtered through a glass microfiber
filter. The product was concentrated by solvent evaporation,
precipitated with large excess acetone, and dried under vacuum. The
product was analyzed using MALDI-TOF mass spectrophotometry and
.sup.1H NMR and then stored at -20.degree. C. until used.
[0066] The NMR results of water soluble PEACE 1800 are as
follows:as illustrated in FIG. 3A, .sup.1H NMR (500 MHz,
D.sub.2O+1,4-Dioxane-d.sub.- 6), .delta. 0.8 (2.9 H of CH.sub.3
from cholesterol); .delta. 2.7 (59.6 H of --NHCH.sub.2CH.sub.2--
from the backbone of PEI); .delta. 3.2 (80.8 H of
.dbd.N--CH.sub.2CH.sub.2--NH.sub.2 from the side chain of PEI);
.delta. 5.4 (0.4 H of .dbd.C.dbd.CH--C-- from cholesterol). Another
peak appearing at .delta. 0.8, -.delta. 1.9 was cholesterol. The
amount of cholesterol conjugated to PEI was determined to be about
47%. MALDI-TOFF mass spectrometric analysis of PEACE showed its
molecular weight to be approximately 2200. The peak appeared from
1000 to 3500 and the majority peaks were around 2200, as
illustrated in FIG. 3B. Expected position is 2400, one chloride 35
is removed from PEI 1800+cholesteryl chloroformate 449. This
suggests that the majority of PEACE 1800 synthesized were of 1/1
molar ratio of cholesterol and PEI, although some were either not
conjugated or conjugated at the molar ratio of 2/1
(cholesterol/PEI).
EXAMPLE 3
Synthesis of PEACE Using Secondary Amine Group
[0067] Fifty microliters of PEI was dissolved in 2 mL of anhydrous
methylene chloride on ice. Then, 200 .mu.L of benzyl chloroformate
was slowly added to the reaction mixture and the solution was
stirred for 4 hours on ice. Following stirring, 10 mL of methylene
chloride was added and the solution was extracted with 15 mL of
saturated NH.sub.4Cl. Water was removed from methylene chloride
phase using magnesium sulfate. The solution volume was reduced
under vacuum and the product (called CBZ protected PEI) was
precipitated with ethyl ether. Fifty micrograms of primary amine
CBZ protected PEI was dissolved in methylene chloride, 10 mg of
cholesterol chloroformate was added, and the solution was stirred
for 12 hours on ice. The product (CBZ protected PEACE) was
precipitated with ethyl ether, washed with acetone, and then
dissolved in DMF containing palladium activated carbon as catalyst
under H.sub.2 as hydrogen donor. The mixture was stirred for 15
hours at room temperature, filtered with Celite.RTM., and the
solution volume was reduced by rotatory evaporator. The product was
finally obtained from precipitation with ethyl ether.
EXAMPLE 4
Synthesis of Glycosylated PEACE
[0068] Two hundred milligrams of PEI was glycosylated using 8 mg of
a-D-glucopyranosyl phenylisothiocyanate dissolved in DMF. To
synthesize galactosylated, mannosylated and lactosylated PEACE,
.alpha.-D-galactopyranosyl phenylisothiocyanate,
.alpha.-D-mannopyranosyl phenylisothiocyanate,
.alpha.-D-lactopyranosyl phenylisothiocyanate were used,
respectively. The solution was adjusted to pH 9 by addition of 1 M
Na.sub.2CO.sub.3 and incubated for 12 hrs at room temperature. The
glucosylated PEI was dialyzed against 5 mM NaCl for 2 days. The
volume of the resulting materials were reduced under vacuum and
precipitated with acetone. The dried (under N.sub.2) mannosylated
PEI was dissolved in methylene chloride and reacted with
cholesteryl chloroformate as described in Example 2.
EXAMPLE 5
Synthesis of Folate PEACE Conjugation
[0069] Two hundred milligrams of PEI was conjugated with 10 mg of
folic acid dissolved in dimethylsulfoxide (DMSO) containing
1,3-Dicyclohexylcarbodiimide (DCC). After 12 hours with stirring,
the product (Folate-PEI) is purified with FPLC. The solution was
dialyzed against deionized water for 2 days. The volume of the
resultant materials were reduced under vacuum and precipitated with
acetone. The result materials were dried under N.sub.2. The dried
folate PEI was dissolved in methylene chloride and reacted with
cholesteryl chloroformate as described in Example 2.
EXAMPLE 6
Synthesis of RGD PEACE Conjugation
[0070] We use cyclic
NH.sub.2-Cys-Arg-Gly-Asp-Met-Phe-Gly-Cys-CO-NH.sub.2 as an RGD
peptide with an N-terminus. RGD peptide was synthesized using solid
phase peptide synthetic methods with F-moc chemistry. Cyclization
was performed using 0.01M K.sub.3[Fe(CN).sub.6] in 1 mM NH.sub.4OAc
at pH 8.0 overnight at room temperature and then purified with
HPLC. One molar N-terminal amine group of RGD peptide was reacted
with 2 mol N-succinimidyl 3 (2-pyridyldithio) propionate (SPDP) in
DMSO and precipitated with ethylether (RGD-PDP). Two hundred
milligrams of PEI was reacted with 7 milligrams of SPDP in DMSO for
2 hrs at room temperature. The resulting materials (PEI-PDP) were
treated with 0.1 M (-)1,4-Dithio-L-threitol (DTT) followed by
separation bio-spin column. RGD-PDP was dissolved in DMF and then
added to PEI-PDP solution. After 12 hrs stirring, the resulting
material (RGD-PEI) was purified by FPLC. The resulting solution was
dialyzed against deionized water for 2 days followed by reducing
volume using a rotary evaporator. The resulting materials were
precipitated with a large excess of acetone. Dried RGD-PEI was
reacted with cholesteryl chloroformate as described in Example
2.
EXAMPLE 7
Preparation of Liposomes
[0071] PEACE and DOPE were dissolved in methylene chloride at molar
ratios of 1/1, 1/2 and 2/1 and then added to a 100-mL
round-bottomed flask. The clear solution was rotated on a rotary
evaporator at 30.degree. C. for 60 min, resulting in thin
translucent lipid films. The flasks were covered with
punctured-para-film and the lipid film was dried overnight under
vacuum. The films were hydrated in 5 mL sterile water to give a
final concentration of 5 mM for PEACE. The hydrated films were
vortexed for 60 min and extruded through 0.4 .mu.m pore size
polycarbonate filters using a 10-mL extruder (Lipex Biomembranes,
Inc., Vancouver, BC).
EXAMPLE 8
Amplification and Purification of Plasmids
[0072] This example illustrates the preparation of DNA to be
complexed with the liposome prepared in Example 7. Plasmid
pCMV-Luciferase (pCMV-Luc) was used as a reporter gene and pmIL-12
(a plasmid carrying the murine interleukin-12, or mIL-12 gene) as a
therapeutic gene. The p35 and p40 sub-units of mIL-12 were
expressed from two independent transcript units, separated by an
internal ribosomal entry site (IRES), and inserted into a single
plasmid, pCAGG. This vector encodes mIL-12 under the control of the
hybrid cytomgalovirs induced enhancer (CMV-IE) and chicken
.beta.-actin promoter. All plasmids were amplified in E. coli
DH5.alpha. strain cells, and then isolated and purified by QIAGEN
EndoFree Plasmid Maxi Kits (Chatsworth, Calif.). The plasmid purity
and integrity was confirmed by 1% agarose gel electrophoresis,
followed by ethidium bromide staining, the pDNA concentration was
measured by ultraviolet (UV) absorbance at 260 nm.
EXAMPLE 9
Preparation of Water Soluble PEACE/pDNA and PEACE:DOPE/pDNA
[0073] Complexes
[0074] This example illustrates the formation of water soluble
PEACE/pCDNA and PEACE:DOPE/pDNA complexes.
[0075] The water soluble PEACE prepared in Example 2, the
PEACE:DOPE liposomes prepared in Example 7, and the pDNA prepared
in Example 8 were diluted separately with 5% glucose to a volume of
250 .mu.l each, and then the pDNA solution was added to the
liposomes under mild vortexing. Complex formation was allowed to
proceed for 30 minutes at room temperature. To study the effect of
charge ratio on gene transfer, water soluble PEACE/pcDNA and
PEACE:DOPE/pDNA complexes were prepared at charge ratios range from
1/1 to 5/1(+/-). Following complex formation, osmolality and pH of
PEACE:DOPE/pDNA complexes were measured. The results are shown in
Table 1.
[0076] The water soluble PEACE/pDNA and PEACE:DOPE liposomes/pDNA
complexes formulated at several charge ratios were diluted 5 times
in the cuvette for the measurement of particle size and .zeta.
potential of the complexes. The electrophoretic mobility of the
samples was measured at 37.degree. C., pH 7.0 and 677 nm wavelength
at a constant angle of 15.degree. with ZetaPALS (Brookhaven
Instruments Corp., Holtsville, N.Y.). The zeta potential was
calculated from the electrophoretic mobility based on
Smoluchowski's formula. Following the determination of
electrophoretic mobility, the samples were subjected to mean
particle size measurement.
[0077] The mean particle size of water soluble PEACE/pDNA complexes
was much smaller than that of PEACE:DOPE/pDNA formulated at 3/1
(+/-) ratio in 5% glucose (42 nm vs. 221 nm). Overall, these
complexes had a narrow particle size distribution. In case of
PEACE:DOPE liposome/pDNA complexes, there was decrease in particle
size with increase in the charge ratios: 430, 221 and 193 nm at
1/1, 3/1 and 5/1 (+/-) charge ratios, respectively.
[0078] [Table 1]
[0079] The zeta potential of these complexes was in the range of 8
to 47 mV, and increased with increase in charge ratios (+/-). The
osmolality of these complexes was in the range of 331-359 mOsm,
whereas that of the complexes formulated in 4% glucose was about
310 mOsm.
EXAMPLE 10
Gel Retardation and DNase Protection Assays
[0080] The ability of water soluble PEACE and water insoluble
PEACE:DOPE liposomes to condense and protect pDNA from enzymatic
degradation was evaluated in this Example. Briefly, water soluble
PEACE and PEACE:DOPE liposomes were complexed with pDNA at various
charge ratios (+/-) ranging from 0.5/1 to 5/1 in the presence of 5%
glucose (w/v) glucose to adjust the osmolality at 290-300 mOsm. The
complexes were electrophoresed on a 1% agarose gel. As illustrated
in FIG. 4A and FIG. 4B, the positively charged PEACE makes strong
complexes with the negatively charged phosphate ions on the sugar
backbone of DNA. When the charge ratio (+/-) reached 1/1, no free
DNA was seen.
[0081] The ability of water soluble PEACE and PEACE:DOPE liposomes
to protect pDNA from enzymatic degradation was assessed by a DNase
protection assay. Twenty micrograms of pDNA was complexed with
water soluble PEACE or PEACE:DOPE liposomes at various charge
ratios and incubated at ambient conditions for 30 minutes. DNase I
(273 units) were added to the formulations and the samples were
incubated at 37.degree. C. for a defined period. At 0, 5, 15, and
60 min post-incubation, 50 .mu.l samples were taken into Eppendorf
tubes and mixed with 50 .mu.l of 100 mM EDTA under mild vortexing
to inactivate DNase. Heparin (162 units/mg DNA) were added to
dissociate the pDNA from the water soluble PEACE or PEACE:DOPE
liposomes. Heparin was allowed to react with the mixtures for 20
minutes, and then the samples were loaded onto a 1% agarose gel for
electrophoresis.
[0082] Both water soluble PEACE and PEACE:DOPE liposomes could
protect plasmids from degradation by nucleases up to 60 min
post-incubation in the presence of DNase at a 3/1 (+/-) charge
ratio. FIG. 5 illustrates that water soluble PEACE could protect
DNA even after incubation at 37.degree. C. for 2 hrs. Plasmid DNA
when complexed with PEACE:DOPE liposomes at charge ratio of 3/1
(+/-), was completely condensed and formed spherical particles. The
particle size of these complexes was about 200.about.300 nm (Table
1). Even though there is limited understanding of the cellular
mechanism in the lipid-mediated gene transfer, complex formation at
the level of nanometer scale is generally considered to be a
prerequisite for entry of lipid/DNA complexes into cells.
EXAMPLE 11
Cytotoxicity
[0083] This example illustrates water soluble PEACE/pCMV-Luc and
PEACE:DOPE liposoemes/pCMV-Luc complexes being tested for
cytotoxicity using a MTT assay in CT-26 cells over a wide range of
charge ratios. MTT calorimetric assay as originally described by T.
Mosmann, Rapid Colorimetric Assay for Cellular Growth and Survival:
Application to Proliferation and Cytotoxicity Assays, 65 J.
Immunol. Methods 55-63 (1983), hereby incorporated by
reference.
[0084] CT-26 murine colon adenocarcinoma cells were grown and
maintained in RPMI 1640 medium supplemented with 10% fetal bovine
serum (FBS), 100 U/mL penicillin, 100 U/mL streptomycin, and 50
.mu.g/mL gentamycin at 37.degree. C. and humidified 5%
CO.sub.2.
[0085] CT-26 cells were seeded in a 96-well plate with RPMI (10%
FBS) at 4,000/cells per well and incubated (37.degree. C., 5%
CO.sub.2) overnight. After reaching 80% confluency, 0.64 .mu.g pDNA
were added at various water soluble PEACE/pDNA or PEACE:DOPE/pDNA
charge ratios and incubated (37.degree. C., 5% CO.sub.2) for 48
hours. Following incubation, 25 .mu.l of
3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazol- ium bromide
(MTT) stock solution in phosphate buffered saline were added to
each well with a final concentration of 0.5 mg/mL MTT per well. The
plate was incubated for an additional 4 hours. The media was
removed and 150 .mu.l DMSO were added to dissolve the formazan
crystals. The plate was spectrophotometrically read at 570 nm on an
ELISA plate reader. The relative cell (%) was calculated according
to the following equation:
Viability (%)=[OD.sub.570 (sample)/OD.sub.570
(control)].times.100
[0086] where the OD.sub.570 (control) represents the measurement
from the wells treated with PBS buffer only and the OD.sub.570
(sample) represents the measurement from the wells treated with
varying amounts of PEACE:DOPE/pDNA at various charge ratios.
[0087] Commercially available cationic liposomes
(LipofectAMINE)/pCMV-Luc complexes (5/1, w/w) and
poly(L-lysine)(PLL) were used for comparison. LipofectAMINE reagent
is a 3:1 (w/w) liposome formulation of the polycationic lipid
2,3-dioleyloxy-N-[2(sperminecarboxamido)ethyl]-N,N-dim-
ethyl-1-propanaminium trifluoroacetate (DOSPA) (MW 867) and the
neutral lipid dioleoyl phosphatidylethanolamine (DOPE) (MW 744) in
membrane filtered water. Based on its chemical structure
LipofectAMiNE has 2 primary amines, 2 secondary amines and 1
quaternary amine, totaling 5 positive charges per molecule.
Subsequent calculations show that 5/1 (w/w) corresponds to 6.823/1
(+/-) for LipofectAMINE/pDNA complexes. Following normalization by
(+/-) charge ratios, we thus confirmed that water soluble
PEACE/pDNA compleses prepared at 7/1 (+/-) charge ratio were not
toxic to cells, whereas both PEI (mw 25000 Daltons) and Superfect
were toxic to the cells, as illustrated in FIG. 6A. Similarly,
PEACE:DOPE liposomes/pCMV-Luc complexes were less toxic to the
cells when formulated at the charge ratio of 7/1 (+/-) and below.
In contrast, LipofectAMINE/pCMV-Luc complexes were very toxic to
the cells, as illustrated in FIG. 6B.
[0088] An important feature of the cationic lipopolymer of the
present invention is its relatively low toxicity toward the cells
at concentrations required for optimal transfection, for
cytotoxicity is one of the major barriers in the application of
many cationic amphiphiles. The toxicity of some of the commercially
available cationic lipids and synthetic polycationic polymers, such
as:Lipofectin and PEI, has been attributed to their non-natural,
non-biodegradable nature. The results suggest that the natural
properties and biodegradabiity of the lipopolymer of the present
invention result in the low cytotoxicity and improved
biocompatibility.
EXAMPLE 12
In Vitro Transfection
[0089] In this example, water soluble PEACE/pCMV-Luc, PEACE:DOPE
liposomes/pCMV-Luc and PEACE:DOPE liposomes/pmIL-12 complexes
formulated at different charge ratios in 5% (w/v) glucose were
evaluated for their transfection efficiency in CT-26 colon
carcinoma cell lines.
[0090] In the case of luciferase gene, CT-26 cells were seeded in
six well tissue culture plates at 4.times.10.sup.5 cells per/well
in 10% FBS containing RPMI 1640 media. Cells achieved 80%
confluency within 24 hours after which they were transfected with
water soluble PEACE/pDNA or PEACE:DOPE liposomes/DNA complexes
prepared at different charge ratios ranging from 0.5/1(+/-) to
0.5/1 to 5/1 (+/-) charge ratios. The total amount of DNA loaded
was maintained constant at 2.5 .mu.g/well and transfection was
carried out in absence of serum. The cells were incubated in the
presence of complexes for 5 hours in CO.sub.2 incubator followed by
replacement of 2 ml of RPMI 1640 containing 10% FBS and incubation
for additional 36 hours. Cells were lysed using 1.times. lysis
buffer (Promega, Madison, Wis.) after washing with cold PBS. Total
protein assays were carried out using BCA protein assay kit (Pierce
Chemical Co, Rockford, Ill.). Luciferase activity was measured in
terms of relative light units (RLU) using 96 well plate Luminometer
(Dynex Technologies Inc, Chantilly, Va.). The final values of
luciferase were reported in terms of RLU/mg total protein. Both
naked DNA and untreated cultures were used as positive and negative
controls, respectively. As illustrated in FIG. 7A, the transfection
efficiency of water soluble PEACE was higher than PEI. In case of
PEACE:DOPE liposome/pDNA complexes, the transfection efficiency was
dependent on the charge ratio and PEACE/DOPE molar ratios and was
the highest for PEACE:DOPE (2/1 mol/mol) liposomes/pCMV-Luc
complexes, as illustrated in FIG. 7B.
[0091] In case of mIL-12 gene, CT-26 cells were seeded in 75
cm.sup.2 flasks at 2.times.10.sup.6 cells/flask in 10% FBS
containing RPMI 1640. Cells achieved 80% confluency in 24 hours
after which they were transfected with PEACE:DOPE liposomes/pmIL-12
complexes prepared at different charge ratios ranging from 0.5/1
(+/-) to 5/1 (+/-). The total amount of DNA loaded was maintained
at 15 .mu.g/flask and transfection was carried out in absence of
serum. The cells were allowed to incubate in the presence of the
complexes for 5 hours in a CO.sub.2 incubator followed by
replacement of 10 ml of RPMI 1640 containing 10% FBS. Thereafter
the cells were incubated for additional 36 hours. Culture
supernatants were assayed for mIL-12 p70 and p40 using enzyme
linked immunosorbent assay (ELISA) kits as suggested by the
manufacturer. Results similar to luciferase transfection were found
when a gradient of various charge ratios was used. The mIL-12
levels for PEACE:DOPE liposomes/pmIL-12 complexes (3/1, +/-) were
substantially higher than naked pmIL-12 or non-transfected
samples.
[0092] To complement the ELISA results of in vitro transfected
samples, reverse transcriptase polymerase chain reaction (RT-PCR)
was performed to detect mRNA transcripts for mIL-12 in transfected
tumor cells. Following transfection, total RNA was isolated using
RNeasy Qiagen kit (Qiagen Inc., Valancia, Calif.). Samples were
lysed and homogenized in the presence of guanidine isothiocynate
and then reverse transcribed using Omniscript.TM. reverse
transcriptase kit (Qiagen, Valencia, Calif.). The reverse
transcribed samples were amplified by PCR technique using Taq
polymerase core kit (Qiagen, Valencia, Calif.). RT-PCR was used to
detect the p35 subunit as well as .beta.-actin promoter and pCAGGS.
The primers synthesized from 5' to 3' were as follows: For pmIL-12
(p35), 5'-GTC TCC CAA GGT CAG CGT TCC-3' upstream and 5'-CTG GTT
TGG TCC CGT GTG ATG-3' downstream. For .beta.-actin, 5'-ATG GTG GGA
ATG GGT CAG AAG-3' upstream and 5'-CAC GCA GCT CAT TGT AGA AGG-3'
downstream. For pCAGGS, 5'-GCC AAT AGG GAC TTT CCA T-3' upstream
and 5'-GGT CAT GTA CTG GGC ATA ATG-3' downstream primer,
respectively. The PCR cycling conditions were as follows:
Denaturing at 95.degree. C. for 15 secs, annealing at 56.degree. C.
for 15 secs, and extension at 72.degree. C. for 30 secs. A total of
35 cycles were run for product amplification. The PCR product was
separated by electrophoresis using 1% agarose gel. The expected
size of the PCR product from mIL-12 p35 mRNA was 297 bp and 150 bp
for .beta.-actin.
[0093] As illustrated in FIG. 8, RT-PCR results show that the
mIL-12 p35 production at mRNA level is sufficient enough to induce
the formation of mIL-12 p70 by forming disulphide linkages with
mIL-12 p40. The p-actin control confirmed that mIL-12 gene
expression was indeed from the plasmid encoding mIL-12 and not from
endogenous production of mIL-12 by the cells. The bands obtained
from RT-PCR suggest that the mIL-12 expression at protein levels
should be considerably high and mIL-12 p70 secreted from the
transfected CT-26 cells should also be very high if the relative
production of mIL-12 p35 and IL-12 p40 are close to each other.
EXAMPLE 13
In Vivo Gene Expression
[0094] Depending on the particle size and surface charge of water
soluble PEACE/pDNA and water insoluble PEACE:DOPE liposomes/pDNA
complexes can be delivered to most of the major organs, such as
lung, liver, spleen and distal tumors after systemic
administration. For effective gene delivery to the hepatocytes,
these complexes must be stable in blood and their particle size
should be below 100 nm for extravasation through the sinusoidal
hepatic endothelium and access the Space of Disse. These particles
should also have specific ligands such as galactose or lacatose to
promote binding to the hepatocyte receptors, and internalization
through receptor-mediated endocytosis. It was possible to produce
60.about.150 nm size water soluble PEACE/pDNA and PEACE:DOPE
liposome/pDNA complexes for in vivo applications.
[0095] This example illustrates in vivo gene expression using a
PEACE:DOPE cationic liposome of the present invention as a gene
carrier. It describes the transfer and expression of plasmid
pmIL-12 into the lung of mice after systemic administration at a
dose of 0.25 mg DNA/mouse using 150 .mu.L as the injection volume.
It demonstrates the especially advantageous properties of the
cationic liposome compositions according to the present invention,
in particular for gene therapy applications.
[0096] CT-26 colon adeno-carcinoma cells were injected
intravenously into BALB/c mice to generate pulmonary metastases to
assess the in vivo gene transfer efficiency of PEACE:DOPE
liposomes/pmIL-12 complexes injected intravenously. At 48 hrs after
intravenous injection of PEACE:DOPE liposomes/pmIL-12 complexes,
the lungs were harvested, chopped into small pieces, re-cultured
for 24 hours and the culture supernatants were analyzed by ELISA.
The mIL-12 production was high in the lung. One of the most
important properties of mIL-12 is its ability to induce production
of large amounts of mIFN-y. Therefore, we also measured the levels
of mIFN-.gamma. induced by mIL-12. PEACE:DOPE liposomes/pmIL-12
complexes produced much higher levels of mIFN-.gamma. compared to
naked pmIL-12 or PEACE:DOPE liposomes.
[0097] Due to the small size of water soluble PEACE/pDNA complexes,
we expect these complexes to be especially useful for intra-tumoral
gene delivery as well as for systemic delivery to the hepatocytes
and distal tumors. To minimize the plasma protein binding, small
size poly(ethyelene glycol) (PEG) can be attached to the PEI
head-group of water soluble PEACE.
EXAMPLE 14
Synthesis of Chol-PEG-PEI
[0098] This example illustrate the synthesization of a PEGylated
lipopolymer of the present invention wherein a NH2-PEG-COOH (mw
600-5,000 Da) was used as a spacer between cholesterol and PEI.
[0099] Five hundred milligrams NH2-PEG-COOH was dissolved in the
mixture of 3 ml anhydrouse methylene chloride and 50 ml TEA on ice.
After 30 min of stirring, 330 mg cholesterol chloroformate in 1 ml
ice-cold anhydrous methylene chloride was slowly added to PEG
solution and then stirred for additional 4 hrs. To remove the
non-conjugated cholesterol, the mixture was precipitated in 500 ml
ethyl ether and then washed 3 times with acetone. One hundred
milligrams of PEI (mw 600-25,000 Da), 50 mg of DCC, and 50 mg of
NHS were dissolved in 3 ml DMSO at room temperature, the mixture
was stirred for 20 min and then added to chol-PEG solution. After
stirring for 6 hrs, the mixture was precipitated in 500 ml acetone.
The product was dissolved in water and purified by FPLC.
[0100] Thus, among the various embodiments taught there has been
disclosed a composition comprising a novel cationic lipopolymer and
method of use thereof for delivering bioactive agents, such as DNA,
RNA, oligonucleotides, proteins, peptides, and drugs, by
facilitating their transmembrane transport or by enhancing their
adhesion to biological surfaces. It will be readily apparent to
those skilled in the art that various changes and modifications of
an obvious nature may be made without departing from the spirit of
the invention, and all such changes and modifications are
considered to fall within the scope of the invention as defined by
the appended claims.
* * * * *