U.S. patent application number 11/170539 was filed with the patent office on 2005-10-27 for multifunctional molecular complexes for gene transfer to cells.
This patent application is currently assigned to Wyeth. Invention is credited to Boutin, Raymond H..
Application Number | 20050239204 11/170539 |
Document ID | / |
Family ID | 23218392 |
Filed Date | 2005-10-27 |
United States Patent
Application |
20050239204 |
Kind Code |
A1 |
Boutin, Raymond H. |
October 27, 2005 |
Multifunctional molecular complexes for gene transfer to cells
Abstract
A multifunctional molecular complex for the transfer of a
nucleic acid composition to a target cell is provided. The complex
is comprised of A) said nucleic acid composition and B) a transfer
moiety comprising 1) one or more cationic polyamines bound to said
nucleic acid compositions, 2) one or more endosome membrane
disrupting components attached to at least one nitrogen of the
polyamine and 3) one or more receptor specific binding
components.
Inventors: |
Boutin, Raymond H.;
(Thornton, PA) |
Correspondence
Address: |
HOWSON AND HOWSON
CATHY A. KODROFF
ONE SPRING HOUSE CORPORATE CENTER
BOX 457
SPRING HOUSE
PA
19477
US
|
Assignee: |
Wyeth
Madison
NJ
|
Family ID: |
23218392 |
Appl. No.: |
11/170539 |
Filed: |
June 29, 2005 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11170539 |
Jun 29, 2005 |
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10010114 |
Nov 13, 2001 |
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10010114 |
Nov 13, 2001 |
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09425597 |
Oct 22, 1999 |
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6379965 |
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09425597 |
Oct 22, 1999 |
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08809397 |
Mar 21, 1997 |
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6127170 |
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08809397 |
Mar 21, 1997 |
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PCT/US95/12502 |
Sep 28, 1995 |
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08809397 |
Mar 21, 1997 |
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08314060 |
Sep 28, 1994 |
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5837533 |
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Current U.S.
Class: |
435/455 ;
435/456; 435/458 |
Current CPC
Class: |
C12N 15/87 20130101;
A61K 48/00 20130101; A61K 47/543 20170801; A61K 47/554 20170801;
C12N 2760/16022 20130101; C07K 14/005 20130101; C12N 15/64
20130101 |
Class at
Publication: |
435/455 ;
435/456; 435/458 |
International
Class: |
C12N 015/88; C12N
015/86 |
Claims
1. A method for the transfer of a nucleic acid composition to
cells, comprising the step of introducing a multifunctional
molecular complex into cells, wherein said multifunctional
molecular complex comprises: A) a nucleic acid composition; and B)
a transfer moiety comprising (i) one or more cationic polyamine
components, wherein each cationic polyamine is non-covalently bound
to said nucleic acid composition and comprises from three to twelve
nitrogen atoms; and (ii) one or more endosome membrane disruption
promoting components attached to at least one nitrogen atom of at
least one of said polyamine components through an alkyl,
carboxamide, carbamate, thiocarbamate, or carbamoyl bridging group,
said one or more endosome membrane disruption promoting components
independently selected from (a) at least one lipophilic long chain
alkyl group or (b) a fusogenic peptide, cholic acid or cholesteryl
group or a derivative thereof; wherein said multifunctional
molecular complex transfers said nucleic acid composition to said
cells.
2. The method according to claim 1 wherein said nucleic acid
composition is a nucleic acid molecule that comprises a nucleotide
sequence that encodes a peptide or protein, or serves as a template
for a nucleic acid molecule.
3. The method according to claim 2 wherein the peptide, protein or
nucleic acid molecule is a product of industrial, commercial or
scientific value, selected from the group consisting of therapeutic
agents; vaccines; foodstuffs and nutritional supplements; compounds
of agricultural significance; herbicides and plant growth
regulants; insecticides; miticides; rodenticides; and fungicides;
compounds useful in animal health; parasiticides; nematocides.
4. The method according to claim 1 wherein the target cells are
cultures of host cells comprising microorganism cells of bacteria,
yeast, plant or mammalian cells; said cell cultures being
maintained in accordance with fermentation techniques which
maximize production of the peptide, protein or functional nucleic
acid molecule being produced.
5. The method according to claim 1 wherein the nucleic acid
composition comprises a nucleotide sequence that encodes a protein
and is operably linked to regulatory sequences.
6. The method according to claim 1 wherein the nucleic acid
composition comprises a nucleotide sequence that encodes a protein
which comprises at least one epitope that is identical or
substantially similar to an epitope of an antigen against which an
immune response is desired, said nucleotide sequence being operably
linked to regulatory sequences.
7. The method according to claim 1, wherein the transfer moiety of
said multifunctional molecular complex further comprises at least
one receptor specific binding component which is a ligand for a
receptor on a target cell.
8. The method according to claim 7, wherein the receptor specific
binding component is attached through a bridging group to either
(i) to a further nitrogen atom of at least one of said cationic
polyamine components to which said one or more endosome membrane
disruption promoting components is attached, or (ii) to a nitrogen
atom of at least one further polyamine component which does not
have attached thereto any endosome membrane disruption promoting
component.
9. The method according to claim 8, wherein the bridging group
through which the receptor specific binding component is attached
is selected from the group consisting of an alkyl, carboxamide,
carbamate, thiocarbamate, and carbamoyl bridging group.
10. The method according to claim 1, wherein the cationic polyamine
comprises the formula (1):
NR(R.sup.3)--[--(CR.sup.1R.sup.2).sub.m--N(R.s-
up.3)--].sub.n--(CR.sup.1R.sup.2).sub.m--NR(R.sup.3) (1) wherein:
R, R.sup.1 and R.sup.2 are each independently selected from the
group consisting of hydrogen and C.sub.1-6 alkyl; m in each
occurrence is independently selected from the integers 2 through 5
inclusive; n is selected from the integers 1 through 10 inclusive;
and R.sup.3 is independently selected from the group consisting of
hydrogen; C.sub.1-6 alkyl, an endosome membrane disruption
promoting component, and a receptor specific binding component, or
NR(R.sup.3) is guanidino, wherein said transfer moiety comprises at
least one endosome membrane disruption promoting component attached
to at least one nitrogen atom of at least one of said cationic
polyamine components.
11. The method according to claim 10, wherein said one or more
endosome membrane disruption promoting components are independently
selected from the group consisting of: (a)
--B--(CR.sup.1R.sup.2).sub.j--C(R).sub.3, where R is independently
selected from the group consisting of hydrogen, C.sub.1-6 alkyl, or
C(R).sub.3 is C.sub.6H.sub.5 aromatic or absent; R.sup.1 and
R.sup.2 are each independently selected from the group consisting
of hydrogen and C.sub.1-6 alkyl; j is an integer from 0 to 24
inclusive; and B is optionally absent, or is a bridging group of
the formula: (i) --(CR.sup.1R.sup.2).sub.k--C(.dbd.O)-Z-; (ii)
--(CR.sup.1R.sup.2).sub.k--N(R)--C(.dbd.O)-Z-; (iii)
--(CR.sup.1R.sup.2).sub.k--N(R)-{-C(.dbd.O)--CH.sub.2--O-[--(CH.sub.2).su-
b.2--O-].sub.1--(CH.sub.2).sub.k--N(R)}.sub.p--C(.dbd.O)-Z-; or
(iv)
--(CR.sup.1R.sup.2).sub.k--C(.dbd.O)--{--N(R)--[--(CH.sub.2).sub.2--O--].-
sub.l--CH.sub.2--C(.dbd.O)}.sub.p-Z-; where k is, independently, an
integer from 1 to 11 inclusive, 1 is an integer from 0 to 30
inclusive, and p is an integer from 1 to 3 inclusive; R is
independently defined as above or is absent, R.sup.1 and R.sup.2
are each independently selected from the group consisting of
hydrogen and C.sub.1-6 alkyl; and Z is O, OH, S, N(R), or is
absent; (b) --B--(R.sup.4)R, where R, R.sup.1 and R.sup.2 are each
independently defined as above; B cannot be absent and is a
bridging group independently selected from groups (i) through (iv)
above, and additionally from the group of the formula: (v)
--(CR.sup.1R.sup.2).sub.j.dbd.--X--, where j=is an integer from 1
to 8 inclusive; R.sup.1 and R.sup.2 are each independently defined
as above; X is O, S, N(R), or absent; and R.sup.4 is independently
selected from the group consisting of: (i) fusogenic peptides
comprising spike glycoproteins of enveloped animal viruses; (ii)
cholic acid derivatives of the formula (2): 5where: represents a
bond of unspecified stereochemistry; represents a single or double
bond, forming a saturated or unsaturated portion of the ring
system, provided that they cannot both be unsaturated at the same
time, whereby the ring system must be either .DELTA.4 or .DELTA.5;
R.sup.6 is --H, --OH, --CO.sub.2H, --C(.dbd.O)NH.sub.2,
--OC(.dbd.O)NH.sub.2, --NH.sub.2, or
--O(CH.sub.2CH.sub.2O).sub.n.dbd.H, where n=is an integer from 1 to
6 inclusive; R.sup.7 is a radical that forms the point of
attachment of the cholic acid derivative, comprising --C.sub.1-6
alkyl- or --C.sub.1-6 alkylcarbonyl-; and R.sup.8 is C.sub.1-6
alkyl; and (iii) cholesteryl derivatives of the formula (3):
6where: represents a bond of unspecified stereochemistry;
represents a single or double bond, forming a saturated or
unsaturated portion of the ring system, provided that they cannot
both be unsaturated at the same time, whereby the ring system must
be either .DELTA.4 or .DELTA.5; R.sup.6a is a radical that forms
the point of attachment of the cholesteryl derivative, comprising
--C.sub.1-6 alkyl-, --OC(.dbd.O)--, or --OCH.sub.2C(.dbd.O)--;
R.sup.7a is C.sub.1-6 alkyl; and R.sup.8a is C.sub.1-6 alkyl.
12. The method according to claim 10, wherein the cationic
polyamine has the formula
NH(R.sup.30)--(CH.sub.2).sub.3--N(R.sup.3)--(CH.sub.2).sub.4--
-N(R.sup.3)--(CH.sub.2).sub.3--NH(R.sup.30) wherein: R.sup.30 is
hydrogen or NH(R.sup.30) is guanidino; and at least one R.sup.3 is
an endosome membrane disruption promoting component of the formula
--B--(CR.sup.1R.sup.2).sub.j--C(R).sub.3.
13. The method according to claim 10, wherein said transfer moiety
comprises more than one cationic polyamine component.
14. The method according to claim 10, wherein a first cationic
polyamine component comprises an endosome membrane disruption
promoting component and a second cationic polyamine component
comprises a receptor specific binding component.
15. A method of immunization against a pathogen comprising the step
of introducing a multifunctional molecular complex; wherein said
multifunctional molecular complex comprises: A) a nucleic acid
composition; and B) a transfer moiety comprising (i) one or more
cationic polyamine components, wherein each cationic polyamine is
non-covalently bound to said nucleic acid composition and comprises
from three to twelve nitrogen atoms; and (ii) one or more endosome
membrane disruption promoting components attached to at least one
nitrogen atom of at least one of said polyamine components through
an alkyl, carboxamide, carbamate, thiocarbamate, or carbamoyl
bridging group, said one or more endosome membrane disruption
promoting components independently selected from (a) at least one
lipophilic long chain alkyl group or (b) a fusogenic peptide,
cholic acid or cholesteryl group or a derivative thereof; wherein
said multifunctional molecular complex transfers said nucleic acid
composition to said cells, wherein said nucleic acid molecule
comprises a nucleotide sequence that encodes a peptide which
comprises at least an epitope identical to, or substantially
similar to an epitope displayed on said pathogen as antigen and
wherein said nucleotide sequence is operatively linked to
regulatory sequences; and wherein said nucleic acid molecule is
capable of being expressed in the cells.
16. The method according to claim 15 wherein said nucleic acid
molecule is administered intramuscularly.
17. The method according to claim 15 wherein said pathogen is a
virus selected from the group consisting of: human immunodeficiency
virus, HIV; human T cell leukemia virus, HTLV; influenza virus;
hepatitis A virus, HAV; hepatitis B virus, HBV; hepatitis C virus,
HCV; human papilloma virus, HPV; Herpes simplex 1 virus, HSV1;
Herpes simplex 2 virus, HSV2; Cytomegalovirus, CMV; Epstein-Barr
virus, EBV; rhinovirus; and, coronavirus.
18. The method according to claim 15 wherein at least two or more
different nucleic acid molecules are administered to different
cells of said individual.
19. The method according to claim 18 wherein said different nucleic
acid molecules each comprise nucleotide sequences encoding one or
more pathogen antigens of the same pathogen.
20. A method for delivering a nucleic acid molecule to a targeted
population of cells of an individual, said method comprising the
step of delivering to the individual a multifunctional molecular
complex comprising: A) a nucleic acid molecule; and B) a transfer
moiety comprising one or more cationic polyamine components,
wherein each cationic polyamine is non-covalently bound to said
nucleic acid molecule and each independently comprises a cationic
polyamine of the formula (1):
NR(R.sup.3)--[--(CR.sup.1R.sup.2).sub.m--N(R.sup.3)--].sub.n--(CR.sup.1R.-
sup.2).sub.m--NR(R.sup.3) (1) wherein: R, R.sup.1 and R.sup.2 are
each independently selected from the group consisting of hydrogen
and C.sub.1-6 alkyl; m in each occurrence is independently selected
from the integers 2 through 5 inclusive, n is selected from the
integers 1 through 10 inclusive; R.sup.3 is independently selected
from the group consisting of hydrogen; C.sub.1-6 alkyl, and an
endosome membrane disruption promoting component, or NR(R.sup.3) is
guanidino; wherein said transfer moiety comprises at least one
endosome membrane disruption promoting component attached to at
least one nitrogen atom of at least one of said cationic polyamine
components; wherein said transfer moiety comprises at least one
receptor specific binding component attached either (i) to a
further nitrogen atom of at least one of said cationic polyamine
components to which said one or more endosome membrane disruption
promoting components is attached, or (ii) to a nitrogen atom of at
least one further polyamine component which does not have attached
thereto any endosome membrane disruption promoting component,
wherein said receptor specific binding component which is a ligand
for natural receptors of said target cells.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of U.S. patent
application Ser. No. 10/010,114, filed Nov. 13, 2001, which is a
divisional of U.S. patent application Ser. No. 09/425,597, filed
Oct. 22, 1999, now U.S. Pat. No. 6,379,965, issued Apr. 30, 2002,
which is a divisional of U.S. patent application Ser. No.
08/809,397, filed Mar. 21, 1997, now U.S. Pat. No. 6,127,170,
issued Oct. 3, 2000, which is a 35 USC .sctn.371 of PCT/US95/12502,
filed Sep. 28, 1995, which claims the benefit of the priority of
U.S. patent application Ser. No. 08/314,060, filed Sep. 28, 1994,
now U.S. Pat. No. 5,837,533, issued Nov. 17, 1998.
BACKGROUND OF THE INVENTION
[0002] The present invention is in the field of methods for the
transfer of genetic information, e.g., foreign DNA, into target
cells, especially eukaryotic cells. In particular, the present
invention relates to nonviral gene carriers comprising
multifunctional molecular conjugates which include, inter alia,
lipopolyamines of a particular configuration, a component which
promotes endosome disruption, and a receptor specific binding
component. The present application is related to U.S. patent
application Ser. No. 08/314,060 filed Sep. 28, 1994 and entitled
"Multifunctional molecular complexes for gene transfer to cells"
which is incorporated herein by reference.
[0003] Heretofore, viral vectors of various types have been
successfully utilized for the insertion of selected foreign genetic
information into a target cell, and in the case of eukaryotic
cells, for incorporation of that genetic information into the
genome of the cell. These viral vector systems have relied upon the
molecular machinery of the virus, evolved over time to surmount the
significant problems facing a virus in attempting to invade, i.e.,
infect a cell. Despite the efficiency of such viral vectors,
however, there has been continued concern regarding the safety of
using viruses, particularly from the standpoint of undesired side
effects. Thus, there has been an ongoing effort to develop
non-viral gene delivery systems that are as efficient as viral
vectors, but with an improved safety profile.
[0004] Nonviral vectors or carriers, of the type with which the
present invention is concerned, will thus have to overcome the same
obstacles as a viral vector. The problems faced by such carriers
include persistence in the biophase of the organism for a
sufficient time to reach the target cell; recognition of the target
cell and means for mediating transport of the genetic material
through the cell membrane and into the cytoplasm of the cell;
avoidance of degradation within the cell by the reticuloendothelial
system; and transport to and through the nuclear membrane into the
nucleus of the cell where transcription of the genetic material can
take place.
[0005] It is to overcoming the problems described above that the
present invention is addressed; and since the problems are several
and different, the present invention comprises a multifunctional
complex, i.e., a molecular conjugate of various ligands intended to
surmount specific obstacles.
[0006] The ultimate usefulness of gene transfer techniques is of
enormous potential benefit in a number of areas. The transfer of
genetic material into cells is the basis of a number of processes
now widely accepted in the areas of molecular biology, gene therapy
and genetic immunization. Transfer of the genetic information
encoded in DNA to cells where it expresses identified individual
proteins, has permitted investigation of the function of such
proteins on a cellular level, and of the underlying cell
physiology. Genetic material has also been transferred into cells
to introduce proteins that are absent due to an inherent genetic
flaw in the cell that expresses an inactive protein or else
prevents expression of the protein altogether. The transfer of
genetic material into cells can be used to prevent the expression
of proteins in those cells through the well-known antisense effect
of complementary DNA or RNA strands.
[0007] Exogenous, i.e., foreign genetic material can permit cells
to synthesize significant amounts of proteins that are not
available by other means in practical economic terms. These
proteins of interest can be grown in a variety of host cells such
as yeast, bacterial or mammalian cells. Genetic material can also
be used to provide protective immune responses in vivo by injection
of DNA that encodes immunogenic proteins, i.e., ones that can
stimulate the desired immune response. The in vivo introduction of
exogenous genetic material into cells also has potential utility in
applications for the alleviation, treatment or prevention of
metabolic, tumoral or infectious disorders by the same mechanisms
enumerated above.
[0008] Description of the Prior Art
[0009] It is possible to transfer genetic material into target
cells without the use of vectors or carriers. For example, genetic
material can be introduced systemically through an intravenous or
intraperitoneal injection for in vivo applications, or can be
introduced to the site of action by direct injection into that
area. For example, it has long been recognized that DNA, by itself,
injected into various tissues, will enter cells and produce a
protein that will elicit an immune response. See, e.g., P. Atanasiu
et al., Academie des Sciences (Paris) 254, 4228-30 (1962); M. A.
Israel et al., J. Virol. 29, 990-96 (1979); H. Will et al., Nature,
299, 740-42 (1982); H. Robinson, World Patent Application WO
86/00930, published Feb. 13, 1986; P. L. Felgner, J. A. Wolff, G.
H. Rhodes, R. w. Malone and D. A. Carson, World Patent Application
WO 90/11092, published Oct. 4, 1990; and R. J. Debs and N. Zhu,
World Patent Application WO 93/24640 published Dec. 9, 1993.
However, DNA by itself is hydrophilic, and the hydrophobic
character of the cellular membrane poses a significant barrier to
the transfer of DNA across it. Accordingly, it has become preferred
in the art to use facilitators that enhance the transfer of DNA
into cells on direct injection.
[0010] Another approach in the art to delivery of genetic material
to target cells is one that takes advantage of natural
receptor-mediated endocytosis pathways that exist in such cells.
Several cellular receptors have been identified heretofore as
desirable agents by means of which it is possible to achieve the
specific targeting of drugs, and especially macromolecules and
molecular conjugates serving as carriers of genetic material of the
type with which the present invention is concerned. These cellular
receptors allow for specific targeting by virtue of being localized
to a particular tissue or by having an enhanced avidity for, or
activity in a particular tissue. See, e.g., J. L. Bodmer and R. T.
Dean, Meth. Enzymol., 112, 298-306 (1985). This affords the
advantages of lower doses or significantly fewer undesirable side
effects.
[0011] One of the better known examples of a cell and tissue
selective receptor is the asialoglycoprotein receptor present in
hepatocytes. The asialoglycoprotein receptor is an extracellular
receptor with a high affinity for galactose, especially
tri-antennary oligosaccharides, i.e., those with three somewhat
extended chains or spacer arms having terminal galactose residues;
see, e.g., H. F. Lodish, TIBS, 16, 374-77 (1991). This high
affinity receptor is localized to hepatocytes and is not present in
Kupffer cells; allowing for a high degree of selectivity in
delivery to the liver.
[0012] It has also been proposed in the art of receptor-mediated
gene transfer that in order for the process to be efficient in
vivo, the assembly of the DNA complex should result in condensation
of the DNA to a size suitable for uptake via an endocytic pathway.
See, e.g., J. C. Perales, T. Ferkol, H. Beegen, O. D. Ratnoff, and
R. W. Hanson, Proc. Nat. Acad. Sci. USA, 91, 4086-4090 (1994).
[0013] An alternative method of providing cell-selective binding is
to attach an entity with an ability to bind to the cell type of
interest; commonly used in this respect are antibodies which can
bind to specific proteins present in the cellular membranes or
outer regions of the target cells. Alternative receptors have also
been recognized as useful in facilitating the transport of
macromolecules, such as biotin and folate receptors; see P. S. Low,
M. A. Horn and P. F. Heinstein, World Patent Application WO
90/12095, published Oct. 18, 1990; P. S. Low, M. A. Horn and P. F.
Heinstein, World Patent Application WO 90/12096, published Oct. 18,
1990; P. S. Low, M. A. Horn and P. F. Heinstein, U.S. Pat. No.
5,108,921, Apr. 28, 1992; C. P. Leamon and P. S. Low, Proc. Nat.
Acad. Sci. USA, 88, 5572-5576 (1991); transferrin receptors;
insulin receptors; and mannose receptors (see further below). The
enumerated receptors are merely representative, and other examples
will readily come to the mind of the artisan.
[0014] The conjugation of different functionalities on the same
molecule has also been utilized in the art. For example, in 1988 G.
Y. Wu and C. M. Wu, J. Biol. Chem., 263, 14621-14624 (1988)
described a method for cellular receptor mediated delivery of DNA
to hepatocytes. This method was further described in G. Y. Wu and
C. H. Wu, Biochem., 27, 887-892 (1988); G. Y. Wu and C. H. Wu, U.S.
Pat. No. 5,166,320, Nov. 24, 1992; and G. Y. Wu and C. H. Wu, World
Patent Application WO 92/06180, published Apr. 16, 1992. The method
consists of attaching a glycoprotein, asialoorosomucoid, to
poly-lysine to provide a hepatocyte selective DNA carrier. The
function of the poly-lysine is to bind to the DNA through ionic
interactions between the positively charged (cationic) .epsilon.
amino groups of the lysines and the negatively charged (anionic)
phosphate groups of the DNA. Orosomucoid is a glycoprotein which is
normally present in human serum. Removal of the terminal sialic
acid (N-acetyl neuraminic acid) from the branched oligosaccharides
exposes terminal galactose oligosaccharides, for which hepatocyte
receptors have a high affinity, as already described.
[0015] After binding to the asialoglycoprotein receptor on
hepatocytes, the protein is taken into the cell by endocytosis into
a pre-lysosomal endosome. The DNA, ionically bound to the
poly-lysine-asialoorosomucoid carrier, is also taken into the
endosome. Additional work using this delivery system, e.g., that
done by J. M. Wilson, M. Grossman, J. A. Cabrera, C. H. Wu and G.
Y. Wu, J. Biol. Chem, 267, 11483-11489 (1992), has found that
partial hepatectomy improves the persistence of the expression of
the DNA delivered into the hepatocytes. The transfer of the DNA
into cells by this mechanism is also significantly enhanced by the
addition of cationic lipids; see, e.g., K. D. Mack, R. Walzem and
J. B. Zeldis, Am. J. Med. Sci., 307, 138-143 (1994).
[0016] The use of a specific asialoglycoprotein is not required to
achieve binding to the asialoglycoprotein receptor; this binding
can also be accomplished with high affinity by the use of small,
synthetic molecules having a similar configuration. The
carbohydrate portion can be removed from an appropriate
glycoprotein and be conjugated to other macromolecules; see, e.g.,
S. J. Wood and R. Wetzel, Bioconj. Chem., 3, 391-396 (1992). By
this procedure the cellular receptor binding portion of the
glycoprotein is removed, and the specific portion required for
selective cellular binding can be transferred to another
molecule.
[0017] There is ample literature on the preparation of synthetic
glycosides which can be attached to macromolecules and confer on
them the ability to bind to the corresponding galactose specific
receptor. The importance of branched glycosides was recognized
early; see Y. C. Lee, Carb. Res., 67, 509-514 (1978). Further work
delineated that sugar density [K. Kawaguchi, M. Kuhlenschmidt, S.
Roseman and Y. C. Lee, J. Biol. Chem., 256, 2230-2234 (1981)] and
spacial relationships [Y. C. Lee, R. R. Townsend, M. R. Hardy, J.
Lonngren, J. Arnarp, M. Haraldsson and H. Lonn, J. Biol. Chem.,
258, 199-202 (1983)] are important determinants of binding potency.
Reductive amination of a peptide with a branched tri-lysine amino
terminus gives a ligand ending with four galactosyl residues that
can be readily coupled to poly-lysine or other macromolecules; see
C. Plank, K. Zatlouhal, M. Cotten, K. Mechtler and E. Wagner,
Bioconj. Chem., 3, 533-539(1992); and has been used to prepare DNA
constructs.
[0018] Thiopropionate and thiohexanoate glycosidic derivatives of
galactose have been prepared and linked to L-lysyl-L-lysine to form
a synthetic tri-antennary galactose derivative. A bisacridine
spermidine derivative containing this synthetic tri-antennary
galactose has been used to target DNA to hepatocytes; see F. C.
Szoka, Jr and J. Haensler, World Pat Application WO 93/19768,
published Oct. 14, 1993; and J. Haensler and F. C. Szoka, Jr.,
Bioconj. Chem., 4, 85-93 (1993).
[0019] Other means of providing cellular receptor based
facilitation of gene transfer into cells using poly-lysine as a
carrier have been described in the art. Antibodies specific for
cell surface thrombomodulin have been used with poly-lysine as a
delivery system for DNA in vitro and in vivo; see V. S. Trubetskoy,
V. P. Torchilin, S. J. Kennel and L. Huang, Bioconj. Chem., 3,
323-327 (1992). The transferrin receptor has also been used to
target DNA to erythroblasts, K562 macrophages and ML-60 leukemic
cells; see E. Wagner, M. Zenke, M. Cotten, H. Beug and M. L.
Birnstiel, Proc. Nat. Acad. Sci. USA, 87, 3410-3414 (1990); M.
Zenke, P. Steinlein, E. Wagner, M. Cotten, H. Beug and M. L.
Birnstiel, Proc. Nat. Acad. Sci. USA, 87, 3655-3659 (1990); and G.
Citro, D. Perrotti, C. Cucco, I. D'Agnano, A. Sacchi, G. Zupi and
B. Calabretta, Proc. Nat. Acad. Sci. USA, 89, 7031-7035 (1990).
These studies used both small oliogodeoxynucleotides as well as
large plasmids.
[0020] The ability of poly-lysine to facilitate DNA entry into
cells is significantly enhanced if the poly-lysine is chemically
modified with hydrophobic appendages; see X. Zhou and L. Huang,
Biochim. Biophys. Acta, 1189, 195-203 (1994); complexed with
cationic lipids; see K. D. Mack, R. Walzem and J. B. Zeldis, Am. J.
Med. Sci., 307, 138-143 (1994) or associated with viruses. Many
viruses infect specific cells by receptor mediated binding and
insertion of the viral DNA/RNA into the cell; and thus this action
of the virus is similar to the facilitated entry of DNA described
above.
[0021] Replication-incompetent adenovirus has been used to enhance
the entry of transferrin-poly-lysine complexed DNA into cells; see
D. T. Curiel, S. Agarwal, E. Wagner and M. Cotten, Proc. Nat. Acad.
Sci. USA, 88, 8850-8854 (1991); E. Wagner, K. Zatloukal, M. Cotten,
H. Kirlappos, K. Mechtler, D. T. Curiel and M. L. Birnstiel, Proc.
Nat. Acad. Sci. USA, 89, 6099-6103 (1992); M. Cotten, E. Wagner, K.
Zatloukal, S. Phillips, D. T. Curiel and M. L. Birnstiel, Proc.
Nat. Acad. Sci. USA, 89, 6094-6098 (1992); and L. Gao, E. Wagner,
M. Cotten S. Agarwal, C. Harris M Romer, L. Miller, P. C. Hu and D.
Curiel, Hum. Gene Ther., 4, 17-24 (1993). The adenovirus enhances
the entry of the poly-lysine-transferrin DNA complex when
covalently attached to the poly-lysine and when attached through an
antibody binding site. There does not need to be a direct
attachment of the adenovirus to the poly-lysine-transferrin-DNA
complex, and it can facilitate the entry of the complex when
present as a simple mixture. The poly-lysine transferrin-DNA
complex provides receptor specific binding to the cells and is
internalized into endosomes along with the DNA. Once inside the
endosomes, the adenovirus facilitates entry of the
DNA/transferrin-poly-lysine complex into the cell by disruption of
the endosomal compartment with subsequent release of the DNA into
the cytoplasm. Replication-incompetent adenovirus has also been
used to enhance the entry of uncomplexed DNA plasmids into cells
without the benefit of the cell receptor selectivity conferred by
the poly-lysine-transferrin complex; see K. Yoshimura, M. A.
Rosenfeld, P. Seth and R. G. Crystal, J. Biol. Chem., 268,
2300-2303 (1993).
[0022] Synthetic peptides such as the N-terminus region of the
influenza hemagglutinin protein are known to destabilize membranes
and are known as fusogenic peptides. Conjugates containing the
influenza fusogenic peptide coupled to poly-lysine together with a
peptide having a branched tri-lysine amino terminus ligand ending
with four galactosyl residues have been prepared as facilitators of
DNA entry into hepatocytes; see C. Plank, K. Zatlouhal, M. Cotten,
K. Mechtler and E. Wagner, Bioconj. Chem., 3, 533-539 (1992). These
conjugates combine the asialoglycoprotein receptor mediated binding
conferred by the tetra-galactose peptide, the endosomal disrupting
abilities of the influenza fusogenic peptide, and the DNA binding
of the poly-lysine. These conjugates deliver DNA into the cell by a
combination of receptor mediated uptake and internalization into
endosomes. This internalization is followed by disruption of the
endosomes by the influenza fusogenic peptide to release the DNA
into the cytoplasm. In a similar fashion, the influenza fusogenic
peptide can be attached to poly-lysine and mixed with the
transferrin-poly-lysine complex to provide a similar DNA carrier
selective for cells carrying the transferrin receptor; see E.
Wagner, C. Plank, K. Zatloukal, M. Cotten and M. L. Birnstiel,
Proc. Nat. Acad. Sci. USA, 89, 7934-7938 (1992). Synthetically
designed peptides can also be used; for example the "GALA" peptides
[N. K. Subbarao, R. A. Parente, F. C. Szoka, Jr, L. Nadasdi and K.
Pongracz, J. Biol. Chem., 26, 2964-2972 (1987)] have been coupled
to DNA carriers and an enhanced facilitated entry into cells was
observed [J. Haensler and F. C. Szoka, Jr., Bioconj. Chem., 4,
372-379 (1993)]. The cationic amphipathic peptide gramicidin S can
facilitate entry of DNA into cells [J. Y. Legendre and F. C. Szoka,
Jr., Proc. Nat. Acad. Sci. USA, 90, 893-897 (1993)], but also
requires a phospholipid to achieve significant transfer of DNA.
[0023] Poly-lysine is not unique in providing a polycationic
framework for the entry of DNA into cells. DEAE-dextran has also
been shown to be effective in promoting RNA and DNA entry into
cells; see R. Juliano and E. Mayhew, Exp. Cell. Res. 73, 3-12
(1972); and E. Mayhew and R. Juliano, Exp. Cell. Res. 77, 409-414
(1973). More recently, a dendritic cascade co-polymer of
ethylenediamine and methyl acrylate has been shown to be useful in
providing a carrier of DNA which facilitates entry into cells; see
J. Haensler and F. C. Szoka, Jr., Bioconj. Chem., 4, 372-379
(1993). An alkylated polyvinylpyridine polymer has also been used
to facilitate DNA entry into cells; see A. V. Kabanov, I. V.
Astafieva, I. V. Maksimova, E. M. Lukanidin, G. P. Georgiev and V.
A. Kabanov, Bioconj. Chem., 4, 448-454 (1993).
[0024] Positively charged liposomes have also been widely used as
carriers of DNA which facilitate entry into cells; see, e.g., F. C.
Szoka, Jr. and J. Haensler, World Pat Application WO 93/19768,
published Oct. 14, 1993; R. J. Debs and N. Zhu, World Patent
Application WO 93/24640, published Dec. 9, 1993; P. L. Felgner, R.
Kumar, C. Basava, R. C. Border and J. Y. Hwang-Felgner, World
Patent Application WO 91/16024, published Oct. 31, 1991; P. L.
Felgner and G. M. Ringold, Nature, 337, 387-388 (1989); J. K. Rose,
L. Buonocore and M. A. Whitt, BioTechniques, 10, 520-525 (1991); C.
F. Bennett, M. Y. Chiang, H. Chan, J. E. E. Schoemaker and C. K.
Mirabelli, Mol. Pharm. 41, 1023-1033 (1992); J. H. Felgner, R.
Kumar, C. N. Sridhar, C. J. Wheeler, Y. J. Tsai, R. Border, P.
Ramsey, M. Martin and P. L. Felgner, J. Biol. Chem., 269, 2550-2561
(1994); J. G. Smith, R. L. Walzem and J. B. German, Biochim.
Biophys. Acta, 1154, 327-340 (1993). These carrier compositions
have also included pH sensitive liposomes; see C. J. Chu, J.
Dijkstra, M. Z. Lai, K. Hong and F. C. Szoka, Jr., Pharm. Res., 7,
824-854 (1990); J. Y. Legendre and F. C. Szoka, Jr., Pharm. Res.,
9, 1253-1242 (1992).
[0025] A poly-cationic lipid has been prepared by coupling
dioctadecylamidoglycine and dipalmitoyl phosphatidylethanolamine to
a 5-carboxyspermine; see J. P. Behr, B. Demeniex, J. P. Loeffler
and J. Perez-Mutul, Proc. Nat. Acad. Sci. USA, 86, 6982-6986
(1989); F. Barthel, J. S. Remy, J. P. Loeffler and J. P. Behr, DNA
and Cell Biol., 12, 553-560 (1993); J. P. Loeffler and J. P. Behr,
Meth. Enzymol., 217, 599-618 (1993); J. P. Behr and J. P. Loeffler,
U.S. Pat. No. 5,171,678, Dec. 15, 1992. These lipophilic-spermines
are very active in transferring DNA through cellular membranes.
[0026] Combinations of lipids have been used to facilitate the
transfer of nucleic acids into cells. For example, in U.S. Pat. No.
5,283,185 there is disclosed such a method which utilizes a mixed
lipid dispersion of a cationic lipid with a co-lipid in a suitable
solvent. The lipid has a structure which includes a lipophilic
group derived from cholesterol, a linker bond, a linear alkyl
spacer arm, and a cationic amino group; and the co-lipid is
phosphatidylcholine or phosphatidylethanolamine.
[0027] Macrophages have receptors for both mannose and
mannose-6-phosphate which can bind to and internalize molecules
displaying these sugars. The molecules are internalized by
endocytosis into a pre-lysosomal endosome. This internalization has
been used to enhance entry of oligonucleotides into macrophages
using bovine serum albumin modified with mannose-6-phosphate and
linked to an oligodeoxynucleotide by a disulfide bridge to a
modified 3' end; see E. Bonfils, C. Depierreux, P. Midoux, N. T.
Thuong, M. Monsigny and A. C. Roche, Nucl. Acids Res. 20, 4621-4629
(1992). Similarly, oligodeoxynucleotides modified at the 3' end
with biotin were combined with mannose-modified streptavidin, and
were also found to have facilitated entry into macrophages; see E.
Bonfils, C. Mendes, A. C. Roche, M. Monsigny and P. Midoux,
Bioconj. Chem., 3, 277-284 (1992).
[0028] Various peptides and proteins, many of which are naturally
occurring, have been shown to have receptors on cell surfaces, that
once they are attached thereto, allow them to become internalized
by endocytosis. Materials bound to these receptors are delivered to
endosomal compartments inside the cell. Examples include insulin,
vasopressin, low density lipoprotein, epidermal growth factor and
others. This internalization has also been used to facilitate entry
of DNA into cells; e.g., insulin has been conjugated to polylysine
to provide facilitated DNA entry into cells possessing an insulin
receptor; see B. Huckett, M. Ariatti and A. O. Hawtrey, Biochem.
Pharmacol., 40, 253-263 (1990).
SUMMARY OF THE INVENTION
[0029] The present invention relates to a multifunctional molecular
complex for the transfer of a nucleic acid composition to a target
cell comprising in any functional combination: 1) said nucleic acid
composition; 2) one or more cationic polyamine components bound to
said nucleic acid composition, each comprising from three to twelve
nitrogen atoms; 3) one or more endosome membrane disruption
promoting components attached to at least one nitrogen atom of at
least one of said polyamine components, through an alkyl,
carboxamide, carbamate, thiocarbamate, or carbamoyl bridging group,
comprising a) at least one lipophilic long chain alkyl group, b) a
fusogenic peptide comprising spike glycoproteins of enveloped
animal viruses, or c) cholic acid or cholesteryl or derivatives;
and optionally 4) one or more receptor specific binding components
which are ligands for natural receptors of said target cell,
attached through an alkyl, carboxamide, carbamate, thiocarbamate,
or carbamoyl bridging group to either a) a further nitrogen atom of
at least one of said polyamine components to which said one or more
endosome membrane disruption promoting components is attached, or
b) a nitrogen atom of at least one further polyamine component
which does not have attached thereto any endosome membrane
disruption promoting component.
[0030] The present invention further relates to a self-assembling
delivery system for the transfer of a nucleic acid composition to a
target cell comprising the following separate components capable of
being brought together and chemically joined into a molecular
complex by simple mixing: A) said nucleic acid composition to be
transferred; and B) a delivery vehicle, referred to herein as the
"transfer moiety", comprising a) one or more cationic polyamine
components which will bind, i.e., which are capable of being bound
to said nucleic acid composition, each comprising from three to
twelve nitrogen atoms; b) one or more endosome membrane disruption
promoting components attached to at least one nitrogen atom of at
least one of said polyamine components, through an alkyl,
carboxamide, carbamate, thiocarbamate, or carbamoyl bridging group,
comprising i) at least one lipophilic long chain alkyl group, ii) a
fusogenic peptide comprising spike glycoproteins of enveloped
animal viruses, or iii) cholic acid or cholesteryl or derivatives;
and optionally iv) one or more receptor specific binding components
which are ligands for natural receptors of said target cell,
attached through an alkyl, carboxamide, carbamate, thiocarbamate,
or carbamoyl bridging group to either a) a further nitrogen atom of
at least one of said polyamine components to which said one or more
endosome membrane disruption promoting components is attached, or
b) a nitrogen atom of at least one further polyamine component
which does not have attached thereto any endosome membrane
disruption promoting component.
[0031] The present invention also includes the transfer moiety,
described in detail immediately above, as a separate composition of
matter.
[0032] The present invention also relates to a method for the
transfer of a nucleic acid composition to target cells on an in
vitro basis. The method comprises the step of contacting said
target cells with a multifunctional molecular complex which
includes said nucleic acid composition, as detailed further above,
thereby transferring to said cells, a nucleic acid molecule that
comprises a nucleotide sequence that either encodes a desired
peptide or protein, or serves as a template for functional nucleic
acid molecules. The desired protein or functional nucleic acid
molecule may be any product of industrial, commercial or scientific
interest, e.g., therapeutic agents including vaccines; foodstuffs
and nutritional supplements; compounds of agricultural significance
such as herbicides and plant growth regulants, insecticides,
miticides, rodenticides, and fungicides; compounds useful in animal
health such as parasiticides including nematocides; and so forth.
The target cells are typically cultures of host cells comprising
microoganism cells such as bacteria and yeast, but may also include
plant and mammalian cells. The cell cultures are maintained in
accordance with fermentation techniques well known in the art,
which maximize production of the desired protein or functional
nucleic acid molecule, and the fermentation products are harvested
and purified by known methods.
[0033] The present invention further relates to a method for the
transfer of a nucleic acid composition to the cells of an
individual. The method comprises the step of contacting cells of
said individual with a multifunctional molecular complex which
includes said nucleic acid composition, as detailed further above,
thereby administering to the cells, a nucleic acid molecule that
comprises a nucleotide sequence that either encodes a desired
peptide or protein, or serves as a template for functional nucleic
acid molecules. The nucleic acid molecule is administered free from
retroviral particles. The desired protein may either be a protein
which functions within the individual or serves to initiate an
immune response. The nucleic acid molecule may be administered to
the cells of said individual on either an in vivo or ex vivo basis,
i.e., the contact with the cells of the individual may take place
within the body of the individual in accordance with the procedures
which are most typically employed, or the contact with the cells of
the individual may take place outside the body of the individual by
withdrawing cells which it is desired to treat from the body of the
individual by various suitable means, followed by contacting of
said cells with said nucleic acid molecule, followed in turn by
return of said cells to the body of said individual.
[0034] The present invention also concerns a method of immunizing
an individual against a pathogen. The method comprises the step of
contacting cells of said individual with a multifunctional
molecular complex which includes a nucleic acid composition, as
detailed further above, thereby administering to the cells, a
nucleic acid molecule that comprises a nucleotide sequence that
encodes a peptide which comprises at least an epitope identical to,
or substantially similar to an epitope displayed on said pathogen
as antigen, and said nucleotide sequence is operatively linked to
regulatory sequences. The nucleic acid molecule is capable of being
expressed in the cells of the individual.
[0035] The present invention relates to methods of immunizing an
individual against a hyperproliferative disease or an autoimmune
disease. The methods comprise the step of contacting cells of said
individual with a multifunctional molecular complex which includes
a nucleic acid composition, as detailed further above, thereby
administering to cells of the individual, a nucleic acid molecule
that comprises a nucleotide sequence that encodes a peptide that
comprises at least an epitope identical to or substantially similar
to an epitope displayed on a hyperproliferative disease-associated
protein or an autoimmune disease-associated protein, respectively,
and is operatively linked to regulatory sequences. The nucleic acid
molecule being capable of being expressed in the cells.
[0036] The present invention also relates to methods of treating an
individual suffering from an autoimmune disease comprising the
steps of contacting cells of said individual with a multifunctional
molecular complex which includes a nucleic acid composition, as
detailed further above, thereby administering to cells of said
individual, a nucleic acid molecule that comprises a nucleotide
sequence which restores the activity of an absent, defective or
inhibited gene, or which encodes a protein that produces a
therapeutic effect in the individual, and is operatively linked to
regulatory sequences; the nucleic acid molecule being capable of
being expressed in said cells.
[0037] The present invention still further relates to
pharmaceutical compositions which comprise a multifunctional
molecular complex which includes a nucleic acid composition, as
detailed further above, including pharmaceutically acceptable salt
and ester forms of said molecular complex, together with a
pharmaceutically acceptable carrier. In this regard, the present
invention also relates to pharmaceutical kits which comprise a
container comprising a nucleic acid composition, and a container
comprising a transfer moiety. Optionally, there is included in such
kits excipients, carriers, preservatives and vehicles such as
solvents.
DETAILED DESCRIPTION OF THE INVENTION
[0038] In accordance with one embodiment of the present invention
there is provided a multifunctional molecular complex for the
transfer of a nucleic acid composition to a target cell, which
provides for a high level of transfection and expression of the
nucleic acid molecules in the target, i.e., host cell. This
multifunctional molecular complex comprises essentially the
combination of two key elements, (I) the nucleic acid composition
which it is desired to transfer to the target cell, and (II) the
transfer moiety, which complexes with the nucleic acid molecule,
and comprises several components whose function is i) to locate the
desired target cell within the body of an individual by means of a
receptor specific binding component responsive to a specific
receptor on the membrane surface of said target cell; ii) to
overcome the incompatibility arising from the hydrophilic nature of
the nucleic acid molecule and the lipophilic nature of the cell
membrane so that the former can pass through the latter; and iii)
to prevent degradation of the nucleic acid molecule in a lysosome
of said target cell, by disrupting the pre-lysosome, endosome
formation stage, which is accomplished by means of an endosome
membrane disrupting component which permits the multifunctional
molecular complex to escape from an endosome formed as a result of
the target cell's process of endocytosis or pinocytosis, whereby
the multifunctional molecular complex enters the target cell and is
incorporated into said endosome.
[0039] The components of the transfer moiety are as follows: A) a
cationic polyamine component bound to said nucleic acid
composition, comprising from three to twelve nitrogen atoms; B) an
endosome membrane disruption promoting component comprising at
least one lipophilic long chain alkyl group attached to a nitrogen
atom of said polyamine, or a shorter alkyl bridging group having a
terminal carboxyl, amino, hydroxyl or sulfhydryl group to which
there is attached a fusogenic peptide, or cholic acid or
cholesteryl or derivative; and optionally C) one or more receptor
specific binding components which are ligands for natural receptors
of said target cell, attached to a shorter alkyl bridging group
attached to a further nitrogen atom of said polyamine, through said
terminal group thereof.
[0040] The transfer moiety may be represented as one or more
independently selected cationic polyamine components of the formula
(1):
NR(R.sup.3)--[--(CR.sup.1R.sup.2).sub.m--N(R.sup.3)--].sub.n13
(CR.sup.1R.sup.2).sub.m--NR(R.sup.3) (1)
[0041] wherein:
[0042] R, R.sup.1 and R.sup.2 are each independently selected from
the group consisting of hydrogen and C.sub.1-6 alkyl;
[0043] m in each occurrence is independently selected from the
integers 2 through 5 inclusive; and is preferably 3 or 4;
[0044] is selected from the integers 1 through 10 inclusive; and is
preferably 1 to 6;
[0045] R.sup.3 is independently selected from the group consisting
of hydrogen; C.sub.1-6 alkyl; and one or more endosome membrane
disruption promoting components independently selected from the
group consisting of:
[0046] a) --B--(CR.sup.1R.sup.2).sub.j--C(R).sub.3, where R,
R.sup.1 and R.sup.2 are each independently defined as above; j is
an integer from 6 to 24 inclusive, preferably 8 to 18, more
preferably 8 to 12 inclusive; and B is optionally absent, or is a
bridging group of the formula:
[0047] i) --(CR.sup.1R.sup.2).sub.k--C(.dbd.O)-Z-;
[0048] ii) --(CR.sup.1R.sup.2).sub.k--N(R)--C(.dbd.O)-Z-;
[0049] iii)
--(CR.sup.1R.sup.2).sub.k--N(R)--{--C(.dbd.O)--CH.sub.2--O--[--
---(CH.sub.2).sub.2--O--].sub.l--(CH.sub.2).sub.k--N(R)}.sub.p--C(.dbd.O)--
Z-; or
[0050] iv)
--(CR.sup.1R.sup.2).sub.k--C(.dbd.O)--{--N(R)--[--(CH.sub.2).su-
b.2--O--].sub.l--CH.sub.2--C(.dbd.O)}.sub.p-Z-;
[0051] where k is, independently, an integer from 1 to 6 inclusive,
preferably 3 to 5, 1 is an integer from 0 to 30 inclusive,
preferably 4 to 9, and p is an integer from 1 to 3 inclusive,
preferably 1; R, R.sup.1 and R.sup.2 are each independently defined
as above; and Z is O, S, N(R), or is absent, i.e., a single
bond;
[0052] b) --B--(R.sup.4)R, where R, R.sup.1 and R.sup.2 are each
independently defined as above; B cannot be absent and is a
bridging group independently selected from groups i) through iv)
above, and additionally from the group of the formula:
[0053] v) --(C R.sup.1R.sup.2).sub.j, --X--, where j' is an integer
from 1 to 8 inclusive, preferably 2 to 6 inclusive, and more
preferably 5; R, R.sup.1 and R.sup.2 are each independently defined
as above;
[0054] X is O, S, N(R), or absent; and
[0055] R.sup.4 is independently selected from the group consisting
of:
[0056] i) fusogenic peptides comprising spike glycoproteins of
enveloped animal viruses;
[0057] ii) cholic acid derivatives of the formula (2): 1
[0058] where:
[0059] represents a bond of unspecified stereochemistry;
[0060] represents a single or double bond, i.e., a saturated or
unsaturated portion of the ring system, provided that they cannot
both be unsaturated at the same time, i.e., the ring system must be
either .DELTA.4 or .DELTA.5;
[0061] R.sup.6 is --H, --OH, --CO.sub.2H, --C(.dbd.O)NH.sub.2,
--OC(.dbd.O)NH.sub.2, NH.sub.2, or --O(CH.sub.2CH.sub.2O).sub.n H,
where n' is an integer from 1 to 6 inclusive;
[0062] R.sup.7 is a radical that forms the point of attachment of
the cholic acid derivative, comprising --C.sub.1-6alkyl- or
--C.sub.1-6alkylcarbonyl-; and
[0063] R.sup.8 is C.sub.1-6alkyl, especially CH.sub.3; including
the preferred cholic acid derivatives 3.alpha., 7.alpha.,
12.alpha.-trihydroxy-5.beta.-cholan-24-oic ester or amide; and
[0064] iii) cholesteryl derivatives of the formula (3): 2
[0065] where:
[0066] represents a bond of unspecified stereochemistry;
[0067] represents a single or double bond, i.e., a saturated or
unsaturated portion of the ring system, provided that they cannot
both be unsaturated at the same time, i.e., the ring system must be
either .DELTA.4 or .DELTA.5;
[0068] R.sup.6a is a radical that forms the point of attachment of
the cholesteryl derivative, comprising --C.sub.1-6alkyl-,
--OC(.dbd.O)--, or --OCH.sub.2C(.dbd.O)--;
[0069] R.sup.7a is C.sub.1-6alkyl, especially
(CH.sub.2).sub.3CH(CH.sub.3)- --.sub.2; and
[0070] R.sub.8a is C.sub.1-6alkyl, especially CH.sub.3; including
the preferred cholesteryl derivatives
cholest-5-en-3'-.beta.-carbonate, --.beta.-carbamate, or
-.beta.-methylenecarboxamide;
[0071] PROVIDED THAT R.sup.3 is one or more endosome membrane
disruption promoting components attached to at least one nitrogen
atom of at least one of said cationic polyamine components; and
[0072] OPTIONALLY, R.sup.3 may be one or more groups defined below,
attached either to a further nitrogen atom of at least one of said
cationic polyamine components to which said one or more endosome
membrane disruption promoting components is attached, or to a
nitrogen atom of at least one further polyamine component which
does not have attached thereto any endosome membrane disruption
promoting component:
[0073] c) --B--(R.sup.5)R, where B cannot be absent, and is a
bridging group independently selected from groups i) through v)
inclusive; R is independently defined as above; and
[0074] R.sup.5 is a receptor specific binding component
independently selected from the group consisting of:
[0075] i) D-biotin;
[0076] ii) .beta.-3'-propionyl
galactosyl-.beta.1-4-thioglucoside;
[0077] iii) N.sup.2,N.sup.6-bis(.beta.-3'-propionyl
galactosyl-.beta.1-4-thioglucoside)lysine;
[0078] iv) N.sup.2,N.sup.6-bis(.beta.-3'-propionyl
galactosyl-.beta.1-4-th-
ioglucoside)lysyl-N.sup.6-(.beta.-3'-propionyl
galactosyl-.beta.1-4-thiogl- ucoside)lysine;
[0079] v) 5-methyltetrahydrofolate;
[0080] vi) folic acid;
[0081] vii) folinic acid;
[0082] viii) .alpha.-3'-propionyl thiomannoside; and
[0083] ix) .alpha.-3'-propionyl thiomannoside-6-phosphate.
[0084] The Nucleic Acid Composition
[0085] The two basic components of the multifunctional molecular
complex of the present invention are the nucleic acid composition
and the transfer moiety. By "nucleic acid composition" is meant any
one or more of the group of compounds in which one or more
molecules of phosphoric acid are combined with carbohydrate, i.e.,
pentose or hexose, molecules, which are in turn combined with bases
derived from purine, e.g., adenine, and from pyrimidine, e.g.,
thymine. Particular naturally occurring nucleic acid molecules
include genomic deoxyribonucleic acid (DNA) and genomic ribonucleic
acid (RNA), as well as the several different forms of the latter,
e.g., messenger RNA (mRNA), transfer RNA (tRNA), and ribosomal RNA
(rRNA). Also included are the different DNA's which are
complementary (cDNA) to the different RNA's. Synthesized DNA or a
hybrid thereof with naturally occurring DNA, is contemplated.
[0086] The nucleic acid compositions used in the present invention
may be either single-stranded or double-stranded, may be linear or
circular, e.g., a plasmid, and are either oligo- or
polynucleotides. They may comprise as few as 15 bases or base
pairs, or may include as many as 20 thousand bases or base pairs
(20 kb). Since the transfer moiety is employed on a pro rata basis
when added to the nucleic acid composition, practical
considerations of physical transport will largely govern the upper
limit on the size of nucleic acid compositions which can be
utilized.
[0087] In addition to these naturally occurring materials, the
nucleic acid compositions used in the present invention can also
include synthetic compositions, i.e., nucleic acid analogs. These
have been found to be particularly useful in antisense methodology,
which is the complementary hybridization of relatively short
oligonucleotides to single-stranded RNA or single-stranded DNA,
such that the normal, essential functions of these intracellular
nucleic acids are disrupted. See, e.g., Cohen, Oligonucleotides:
Antisense Inhibitors of Gene Expression, CRC Press, Inc., Boca
Raton, Fla. (1989).
[0088] The size, nature and specific sequence of the nucleic acid
composition to be transferred to the target cell can be optimized
for the particular application for which it is intended, and such
optimization is well within the skill of the artisan in this field.
However, the nature of the target cells within the individual into
which it is desired to transfer a nucleic acid composition, may
have a significant bearing on the choice of the particular
multifunctional molecular complex of the present invention. For
example, where it is desired to transfer nucleic acid molecules to
target cells by injecting them intramuscularly to evoke an immune
response, it will be found that this transfer can be effected by
use of a multifunctional molecular complex of the present
invention, as defined above, comprising a cationic polyamine to
which is attached, as the endosome membrane disruption promoting
component, a lipophilic long chain alkyl group as defined above.
Where the target cells are hepatocytes, for example, transfer of
the desired nucleic acid composition is readily effected by use of
the multifunctional molecular complex of the present invention
wherein there is attached to the cationic polyamine a receptor
specific binding component which will permit discrimination among
body cells, comprising, e.g.,
N.sup.2,N.sup.6-bis(.beta.-3'-propionyl
galactosyl-.beta.1-4-thioglucosid- e)lysine, or
N.sup.2,N.sup.6-bis(.beta.-3'-propionyl
galactosyl-.beta.1-4-thioglucoside)lysyl-N.sup.6-(.beta.-3'-propionyl
galactosyl-.beta.1-4-thioglucoside)lysine.
[0089] The nucleic acid composition to be transferred to a target
cell in accordance with the present invention must have an
appropriate open reading frame and promoter to express a protein,
as well as any other regulatory sequences which may be appropriate
to expression. Nucleic acid compositions to be delivered by means
of the methods of the present invention can be designed and
constructed so as to be appropriate for the particular application
desired, all of which is well within the ordinary skill of the
artisan in this field.
[0090] The nucleic acid molecules which are delivered to cells
using the multifunctional molecular complex and methods of the
present invention may serve as: 1) genetic templates for proteins
that function as prophylactic and/or therapeutic immunizing agents;
2) replacement copies of defective, missing or non-functioning
genes; 3) genetic templates for therapeutic proteins; 4) genetic
templates for antisense molecules and as antisense molecules per
se; or 5) genetic templates for ribozymes.
[0091] In the case of nucleic acid molecules which encode proteins,
the nucleic acid molecules preferably comprise the necessary
regulatory sequences for transcription and translation in the
target cells of the individual animal to which they are
delivered.
[0092] In the case of nucleic acid molecules which serve as
templates for antisense molecules and ribozymes, such nucleic acid
molecules are preferably linked to regulatory elements necessary
for production of sufficient copies of the antisense and ribozyme
molecules encoded thereby respectively. The nucleic acid molecules
are free from retroviral particles and are preferably provided as
DNA in the form of plasmids.
[0093] The Transfer Moiety
[0094] The core, or backbone of the transfer moiety is the cationic
polyamine, containing between 3 and 12 amines. There may be more
than one of these cationic polyamine components, whose function is
to overcome the incompatibility arising from the hydrophilic nature
of the nucleic acid molecule and the lipophilic nature of the cell
membrane, although this by itself will not permit the former to
pass through the latter. The cationic groups of the polyamine bind
to the anionic groups of the nucleic acid through ionic bonding,
thus neutralizing those charges and also serving as a point of
attachment for the complex. One .mu.g of DNA contains 3.1 nanomoles
of phosphate anionic charges, assuming a mean molecular weight of
325 for a nucleotide sodium salt. The transfer moiety of the
present invention will not become effective in achieving transfer
of the nucleic acid composition until the anionic charges of said
nucleic acid are substantially neutralized by the cationic charges
of the polyamine component of the transfer moiety.
[0095] It will be appreciated that in one embodiment of the present
invention, a single cationic polyamine can be employed which,
conceptually, balances the anionic charges of the nucleic acid in a
more or less stoichiometric fashion, although it will be understood
that, as a practical matter, it will be necessary to employ amounts
of cationic polyamine which are significantly in excess of the
stoichiometric amount, because of the presence of competing binding
sites in target and other cells, whose existence is well known to
the artisan and which competitively prevent or otherwise interfere
with the binding of the polyamine to the nucleic acid as desired.
It is also contemplated that more than one such cationic polyamine
can be employed, in which case each polyamine chain or piece is
smaller than the corresponding nucleic acid to which it will become
bound. It will be understood, however, that the total size or
length of these individual cationic polyamine components should
together be substantially the same size or length as the nucleic
acid component, in order for neutralization of the anionic charges
of the nucleic acid to take place. Again, it will be understood
that for practical reasons, a significant excess of cationic
polyamine components, over the amount of nucleic acid component
present, will be necessary. Using more than one cationic polyamine
component permits flexibility with respect to the types of groups
that are attached thereto. For example, one cationic polyamine
component may carry a particular endosome membrane disruption
promoting component, while another cationic polyamine component
carries a receptor specific binding component, or perhaps a
different endosome membrane disruption promoting component. The
total number of such cationic polyamine components is variable, and
will depend not only on the size or length of the nucleic acid
component, but on the number and type of groups attached thereto as
well.
[0096] Transfer efficiency, i.e., transfection, does not become
optimum until the multifunctional molecular complex, the
combination of the transfer moiety and the nucleic acid, bears a
strong positive charge. Thus, the amount of transfer moiety must be
selected with this in mind, and the actual amount chosen be depend
on the charge density thereof, which can be calculated by means
well known in the art.
[0097] The triamine, tetraamine, pentaamine and higher polyamine
components of the transfer moiety must be cationic in order to be
functional, as explained above. This can be accomplished by the
simple expedient of making an acid addition salt, e.g., the
hydrochloride salt, where ammonium chloride units are formed. It
may also be the case that the cationic form of the polyamine is
formed under conditions of physiologic pH, in which case it is not
necessary to form the cation directly. Thus, the term "cationic
polyamine" is intended to include both of these possibilities.
[0098] It is contemplated that the number of amine groups that it
is desired to have present in the polyamine will depend to some
extent on the mode of administration of the multifunctional
molecular complex that is used. For example, it is contemplated
that for intramuscular administration, it is preferred to have from
3 to 5 amine groups in the polyamine; whereas, for systemic
injection, e.g., intravenous injection, it is preferred to have
from 5 to 8 amine goups in the polyamine. For in vitro applications
generally, it is preferred to have from 5 to 8 amine groups in the
polyamine.
[0099] The next component of the transfer moiety is the endosome
membrane disruption promoting component, which is required to be
present. This can either comprise one or more lipophilic long chain
alkyl groups attached through one or more of the nitrogen atoms of
said polyamine, or can comprise a bridging group "B", e.g., a
shorter alkyl linking moiety, optionally with a terminal amino,
hydroxyl or sulfhydryl group, through which there is attached a
fusogenic peptide, or cholic acid or cholesteryl or derivative
compound.
[0100] The lipophilic long chain alkyl group is defined by the
formula: --B--(CR.sup.1R.sup.2).sub.j--C(R).sub.3, where B is a
bridging group as defined; R, R.sup.1 and R.sup.2 are each
independently selected from the group consisting of hydrogen and
C.sub.1-6 alkyl; and j is an integer from 6 to 24 inclusive,
preferably 8 to 18, more preferably 8 to 12 inclusive.
[0101] The group "--B--" may be absent, i.e., a single bond, where
R.sup.3 is the endosome membrane disruption promoting component
comprising a lipophilic long chain alkyl group as defined under
"a)" above. The group "--B--" may also be a bridging element which
is a member independently selected from the group consisting
of:
[0102] i) --(CR.sup.1R.sup.2).sub.k--C(.dbd.O)--N(R)--;
[0103] ii) --(CR.sup.1R.sup.2).sub.k--N(R)--C(.dbd.O)--O--;
[0104] iii)
--(CR.sup.1R.sup.2).sub.k--N(R)--{--C(.dbd.O)--CH.sub.2--O--[--
---(CH.sub.2).sub.2--O--].sub.l--(CH.sub.2).sub.k--N(R)}.sub.p--C(.dbd.O)--
z-;
[0105] iv)
--(CR.sup.1R.sup.2).sub.k--C(.dbd.O)--{--N(R)--[--(CH.sub.2).su-
b.2--O--].sub.l--CH.sub.2--C(.dbd.O)}.sub.p--N(R)-z-; or
[0106] v) --(CR.sup.1R.sup.2).sub.j--X--,
[0107] where the various substituents are as defined above.
[0108] Where the endosome membrane disruption promoting component,
rather than being a lipophilic long chain alkyl group, is instead a
fusogenic peptide or a cholic acid or cholesteryl or derivative
compound, the bridging group "B" is required to be present, and
will be a member independently selected from the group i) through
v) above. This selection will be dependent upon the required or
desired type of chemical linkage to be present. For example,
members i) and iv) are carboxamide linkages, whereas members ii)
and iii) are carbamate, thiocarbamate, or carbamoyl linkages,
depending upon whether "Z" is O, S or absent, respectively. For
member v), the linkages will be oxy, thio, amino, or alkylene,
depending upon whether "X" is O, S, N(R), or absent, respectively.
The endosome membrane disruption promoting component, on the other
hand, may have a carbonyl, amino, or some other terminal group,
which can determine the choice of bridging member to be used. All
such choices, however, are well within the skill of the artisan in
this field.
[0109] Most simply, the bridging group can be an alkylene linking
moiety used primarily for steric considerations. However, the other
bridging groups may also be desirable for imparting various
physical and chemical, as well as configurational properties to the
multifunctional molecular complex of the present invention. The
polyethylene glycol group can be especially useful in this
regard.
[0110] The term "C.sub.1-6 alkyl", as used above, and throughout
the description of the present invention, refers to straight and
branched chain alkyl groups including, but not limited to methyl,
ethyl, propyl, isopropyl, n-butyl, iso-butyl, sec-butyl, t-butyl,
and n-pentyl.
[0111] In the formula for the lipophilic long chain alkyl group
above, it is preferred that R, R.sup.1 and R.sup.2 are all
hydrogen, and, as indicated that j be an integer from 8 to 18
inclusive. There must be at least one of these lipophilic long
chain alkyl groups present, but preferably there are no more than
three such groups present. It is preferred to have only one such
group. Thus, examples of preferred transfer moieties of the present
invention, where the endosome membrane disruption promoting
component is a lipophilic long chain alkyl group as described
above, are N.sup.4-octylspermidine, N.sup.4-dodecylspermidine,
N.sup.4-octadecylspermidine, N.sup.4-octylspermine,
N.sup.4-dodecylspermine, and N.sup.4-octadecylspermine.
[0112] The endosome membrane disruption promoting component can
also comprise a shorter alkyl bridging group, optionally having a
terminal amino, hydroxyl or sulfhydryl group through which there is
attached a fusogenic peptide, or cholesterol or derivative
compound. Such a component may be represented by the formula
--B--(R.sup.4)R, where the B group is
--(CR.sup.1R.sup.2).sub.j'-x-, where R, R.sup.1 and R.sup.1 are
each independently selected from the group consisting of hydrogen
and C.sub.1-6 alkyl; j' is an integer from 1 to 6 inclusive,
preferably 2 to 4 inclusive; X is O, S, N(R), or absent.
[0113] It is preferred that R, R.sup.1 and R.sup.2 each be
hydrogen, and as indicated, that j' be 2 to 4, while X is defined
as N. Thus, the shorter alkyl bridging group will preferably be
ethyl, n-propyl, or n-butyl, and will have a terminal amino group
to which is attached the fusogenic peptide, or cholic acid or
cholesteryl or derivative compound, which comprises the endosome
membrane disruption promoting component.
[0114] Alternatively, other of the members of the "B" bridging
group can be chosen. For example, member i) provides an alkyl
bridging moiety with a carboxamide linkage, the most simple
representative of which would be the group
--(CH.sub.2)--C(.dbd.O)--NH--. Member ii) provides an alkylene
bridging moiety with a carbamate type of linkage; and the most
simple representative of this member would be the group
--(CH.sub.2)--NH--C(.dbd- .O)--, which is a carbamoyl type of
linkage. A carbamate linkage would be represented by the group
--(CH.sub.2)--NH--C(.dbd.O)--O--, while a simple variant of this
group would provide a thiocarbamate linkage:
--(CH.sub.2)--NH--C(.dbd.O)--S--. Members iii) and iv) provide the
same terminal linkage variants, while adding to the alkyl bridging
moiety a polyethylene glycol bridging moiety of variable size,
i.e., number of repeating ethylene oxide monomer units, depending
upon the definitions of "1" and "p".
[0115] The fusogenic peptide which functions as an endosome
membrane disruption promoting component, comprises the spike
glycoproteins of enveloped animal viruses known in the art.
Membrane fusion, whether planar or annular, comprises the stages of
initial approach, coalescence, and separation. Fusion reactions are
rapid, highly specific, and non-leaky. The membrane proteins of
enveloped animal viruses comprise glycoproteins which span the
bilayer of the virus membrane and have the bulk of their mass
externally, and non-spanning, nonglycosylated proteins associated
with the inner bilayer surface. The glycoproteins form radial
projections on the surface of the virus membrane, and these spike
glycoproteins play a key role in virus entry into host cells. Spike
glycoproteins are among the best-characterized virus membrane
proteins. In cell entry the spike glycoproteins are responsible for
attachment of the virus particle to the cell surface, and for
penetration of the nucleocapsid into the cytosol, where, after
endocytosis of the virus particle, the spike glycoproteins play a
role in fusion with the limiting membrane of the endosome, whereby
the nucleocapsid reenters the cytosol. In some enveloped animal
viruses, the spike glycoproteins take on a specialized character,
e.g., in orthomyxoviruses, where one is a neuraminidase and another
is a haemagglutinin. All of these fusogenic peptides, in terms of
their amino acid sequences, gross morphology, role in the overall
process of fusion, and requirements for activity, have been the
subject of long term study and have been disclosed in detail in the
technical literature. See, e.g., J. White, M. Kielian and A.
Helenius, Quarterly Reviews of Biophysics, 16, 151-195 (1983),
which is incorporated herein by reference in its entirety.
[0116] Examples of such fusogenic peptides and homologues derived
from spike glycoproteins of enveloped viruses, include the
following peptide sequences reading from N-terminus to
C-terminus:
[0117] SEQ ID NO:1: KFTIVFPHNQKGNWKNVPSNYHYCP (R. Schlegel and M.
Wade, J. Biol. Chem., 259, 4691-94 (1984))-VSV;
[0118] poly (Glu-Aib-Leu-Aib) (K. Kono, H. Nishii and T. Takagishi,
Biochim. Biophys. Acta, 1164, 81-90 (1993));
[0119] SEQ ID NO:2: AVGIGALFLGFLGAAGSTMGAASMTLTVQARQ-HIV-1;
[0120] SEQ ID NO:3: EPVSLTLALLLGGLTMGGIAAGVGTGTTALVATQQ-MuLV (J. S.
Jones and R. Risser, J. Virol., 67, 67-74 (1993));
[0121] SEQ ID NO:4: AVGIGALFLGFLGAAGSTMGARS-HIV-1 (J. L. Nieva, S.
Nir, A. Muga, F. M. Goi and J. Wilschut, Biochem., 33, 3201-09
(1994));
[0122] SEQ ID NO:5: AVGAIGALFLGFLGAAG-HIV-1 (S. Soukchareun, G. W.
Tregear and J. Haralambidis, Bioconj. Chem., 6, 43-53 (1995));
[0123] ID NO:6: GLFEAIAEFIEGGWEGLIEGCA-HA-2 (P. Midoux, C. Mendes,
A. Legrand, J. Raimond, R. Mayer, M. Monsigny and A. C. Roche,
Nucl. Acids Res., 21, 871-78 (1993));
[0124] SEQ ID NO:7: GLFGAIAGFIENGWEGMIDGWYGFR-HA-2;
[0125] SEQ ID NO:8: AVGIGALFLGFLGAAGSTMGAAS-HIV-1 gp41;
[0126] SEQ ID NO:9: FAGVVIGLAALGVATAANVTAAVALVK-SV5 F1;
[0127] SEQ ID NO:10: KVYTGVYPFMWGGAYCFCD-SFV E1;
[0128] SEQ ID NO:11: KLICTGISSIPPIRALFAAINIP-PH-30 .alpha. (J. M.
White, Science, 258, 917-24 (1992));
[0129] SEQ ID NO:12: FFGAVIGTIALGVATATAAQIT-Sendai F1;
[0130] SEQ ID NO:13: FAGVVIGLAALGVATATAAQVT-SV5 F1;
[0131] SEQ ID NO:14: FIGAIIGGVALGVATATAAQIT-NDV F1;
[0132] SEQ ID NO:15: GLFGAIAGFIENGWEGMIDGWYGFRHQN-HA-2X:31;
[0133] SEQ ID NO:16: GLFGAIAGFIENGWEGLVDGWYGFRHQN-HA-2 FPV;
[0134] SEQ ID NO:17: GFFGAIAGFLEGGWEGMIAGWHGYTSHG-HA-2 B/Lee;
[0135] SEQ ID NO:18: FVAAIILGISALIAIITSFAVATTALVKEM-MMTV gp36;
[0136] SEQ ID NO:19: EPVSLTLALLLGGLTMGGIAAGIGTGTTALMATQQFQQLQAA
VQDDLREVEKS-MoMLV pISE;
[0137] SEQ ID NO:20: EPVSLTLALLLGGLTMGGIAAGVGTFTTALVATQQFQQLHAA
VQDDLKE-VEKS-F-MuLV p15E;
[0138] SEQ ID NO:21: EPVSLTLLLLGGL.TM. GGIAGVGTG-TTALVATQQFQQLQAA
MHDDLKEVEKS-AKV p15E;
[0139] SEQ ID NO:22: DYQCKVYTGVYPFMWGGAYCFCDSENT-SFV E1;
[0140] SEQ ID NO:23: DYTCKVFGGVYPFMWGGAQCFCDSENS-Sindbis E1 (J.
White, M Kielian and A. Helenius, Q. Rev. Biophys., 16, 151-95
(1983));
[0141] SEQ ID NO:24: FAGVVLAGAALGVAAAAQI-measles;
[0142] SEQ ID NO:25: FAGVVLAGAALGVATAAQI-measles (P. L. Yeagle, R.
M. Epand, C. D. Richardson and T. D. Flanagan, Biochim. Biophys.
Acta, 1065, 49-53 (1991));
[0143] SEQ ID NO:26: GLFGAIAGFIENGWEGMIDGGGC-HA-2 (E. Wagner, C.
Plank, K. Zatloukal, M. Cotten and M. L. Birnstiel, Proc. Nat.
Acad. Sci. USA, 89, 7934-38 (1992));
[0144] SEQ ID NO:27: GLFGAIAGFIENGWEGMIDG-X31F/68 HA-2;
[0145] SEQ ID NO:28: GIFGAIAGFIENGWEGMIDG-VIC/75 HA-2;
[0146] SEQ ID NO:29: GLFGAIAGFIEGGWTGMIDG-PR/8/34 HA-2;
[0147] SEQ ID NO:30: GLFGAIAGFIEGGWEGMVDG-Jap/57 HA-2;
[0148] SEQ ID NO:31: GLFGAIAGFIEGGWEGLVDG-PPV/34 HA-2; and
[0149] SEQ ID NO:32: GFFGAIAGFLEGGWEGMIAG-X31F/68 HA-2 (J. D. Lear
and W. F. DeGrado, J. Biol. Chem., 262, 6500-05 (1987)).
[0150] One of ordinary skill in the art would readily recognize
that other such fusogenic peptides could be used in the invention
including peptides functionally equivalent to those described but
having one or more amino acid additions, deletions, or
substitutions, especially those having conserved amino acid
substitutions.
[0151] The cholic acid and derivatives which function as endosome
membrane disruption promoting components, comprise compounds of the
formula (2): 3
[0152] where:
[0153] represents a bond of unspecified stereochemistry;
[0154] represents a single or double bond, i.e., a saturated or
unsaturated portion of the ring system, provided that they cannot
both be unsaturated at the same time, i.e., the ring system must be
either .DELTA.4 or .DELTA.5;
[0155] R.sup.6 is H, OH, CO.sub.2H, --C(.dbd.O)NH.sub.2,
--OC(.dbd.O)NH.sub.2, --NH.sub.2, or
--O(CH.sub.2CH.sub.2O).sub.n,H, where n' is an integer from 1 to 6
inclusive;
[0156] R.sup.7 is a radical that forms the point of attachment of
the cholic acid derivative, comprising --C.sub.1-6alkyl- or
--C.sub.1-6alkylcarbonyl-; and
[0157] R.sup.8 is C.sub.1-6alkyl, especially CH.sub.3. It is
preferred that the cholic acid and derivative compounds comprise
one or more members selected from the group consisting of 3.alpha.,
7.alpha., 12.alpha.-trihydroxy-5.beta.-cholan-24-oic ester and
amide.
[0158] The cholesteryl and derivatives which function as endosome
membrane disruption promoting components, comprise compounds of the
formula (3): 4
[0159] where:
[0160] represents a bond of unspecified stereochemistry;
[0161] represents a single or double bond, i.e., a saturated or
unsaturated portion of the ring system, provided that they cannot
both be unsaturated at the same time, i.e., the ring system must be
either .DELTA.4 or .DELTA.5;
[0162] R.sup.6a is a radical that forms the point of attachment of
the cholic acid derivative, comprising --C.sub.1-6alkyl-,
--OC(.dbd.O)--, or --OCH.sub.2C(.dbd.O)--;
[0163] R.sup.7a is C.sub.1-6alkyl, especially
(CH.sub.2).sub.3CH(CH.sub.3)- .sub.2; and
[0164] R.sup.8a is C.sub.1-6alkyl, especially CH.sub.3. It is
preferred that the cholesteryl and derivative compounds comprise
one or more members selected from the group consisting of
cholest-5-en-3'-.beta.-carb- onate, -.beta.-carbamate, and
-.beta.-methylene-carboxamide.
[0165] An optional embodiment of the present invention is to
provide for a receptor specific binding component which helps
effect transfer of nucleic acid compositions to target cells,
especially eukaryotic cells, by taking advantage of the natural
receptor-mediated endocytosis pathways which exist in those cells.
The receptor specific binding component is thus a ligand for the
natural receptor, and can thus assist in binding of the
multifunctional molecular complex to the target cell. Endocytosis
or pinocytosis will then take place whereby the entire complex is
transferred into the target cell, enclosed in an endosome.
[0166] The receptor specific binding component serves the important
function of allowing the multifunctional molecular complex of the
present invention to be targeted to specific cell populations,
e.g., hepatocytes. The binding component facilitates location of
the desired target cells within the body of the animal to which the
complex is being administered, with subsequent attachment of the
complex to the target cells.
[0167] Where the receptor specific binding component is employed,
there will also be present on the multifunctional molecular complex
an endosome membrane disruption promoting component, as defined
further above. Accordingly, once the binding component has located
the desired target cell within the individual, and attached the
complex to said cell by binding to said receptor, the complex will
be transferred into said cell by endocytosis, whereupon it will be
enclosed within an endosome. At this point, the endosome membrane
disruption promoting component assumes its important role by
disrupting said membrane, allowing escape of the complex into the
cytoplasm of said cell.
[0168] During the normal course of events in the target cell,
endosome formation is a prelude to targeting of any foreign protein
to lysosomes where degradation of the foreign protein by hydrolytic
enzymes will take place. Consequently, accumulation in lysosomal
compartments can be a major obstacle to the effectiveness of
nucleic acid delivery systems. The multifunctional molecular
complex of the present invention would suffer the same fate, were
it not for the presence of the endosome membrane disruption
promoting component. This component permits the complex to escape
from the endosome, whereupon it can migrate into the nucleus of the
target cell, and release the nucleic acid composition, whose
genetic information can then be transcribed within said nucleus.
Although the precise mechanisms which make up these steps and
pathways are not well understood, expression of the nucleic acid
molecule contained in the multifunctional molecular complex does
take place, as is demonstrated in the working examples further
below.
[0169] The receptor specific binding component may be represented
by the formula: --B--(R.sup.5)R, where R, R.sup.1 and R.sup.2 are
each independently hydrogen or C.sub.1-6 alkyl; B may be, inter
alia, --(C R.sup.1R.sup.2).sub.j'--X--, where R.sup.1 and R.sup.2
are as defined above, X is N(R), and j' is an integer from 1 to 6
inclusive, preferably 2 to 4 inclusive; and R.sup.5 is a receptor
specific binding component independently selected from the group
consisting of:
[0170] i) D-biotin;
[0171] ii) .beta.-3'-propionyl
galactosyl-.beta.1-4-thioglucoside;
[0172] iii) N.sup.2,N.sup.6-bis(.beta.-3'-propionyl
galactosyl-.beta.1-4-thioglucoside)lysine;
[0173] iv) N.sup.2,N.sup.6-bis(.beta.-3'-propionyl
galactosyl-.beta.1-4-th-
ioglucoside)lysyl-N.sup.6-(.beta.-3'-propionyl
galactosyl-.beta.1-4-thiogl- ucoside)lysine;
[0174] v) 5-methyltetrahydrofolate;
[0175] vi) folic acid;
[0176] vii) folinic acid;
[0177] viii) .alpha.-3'-propionyl thiomannoside; and
[0178] ix) .alpha.-3'-propionyl thiomannoside-6-phosphate.
[0179] It is preferred that R, R.sup.1 and R.sup.2 each be
hydrogen, and as indicated, that j' be 2 to 6. Thus, the shorter
alkyl bridging group will preferably be ethyl, n-propyl, or
n-butyl, and the receptor specific binding component is attached to
the terminal amino group.
[0180] Since the endosome membrane disruption promoting component
must also be present, examples of preferred transfer moieties of
the present invention, where the receptor specific binding
component is a galactosyl group as described above, are
[0181] N.sup.2-octyl-N-4-(5-(.beta.-3'-propionyl
galactosyl-.beta.1"-4'-th- ioglucoside)amino)pentylspermidine;
[0182] N.sup.2-dodecyl-N.sup.4-(5-(.beta.-3'-propionyl
galactosyl-.beta.1-4'-thioglucoside)amino)pentylspermidine;
[0183] N.sup.6-octadecyl-N.sup.4-(5-(.beta.-3'-propionyl
galactosyl-.beta.1"-4'-thioglucoside)amino)pentylspermidine;
[0184]
N.sup.6-octyl-N.sup.4-(5-[N.sup.2',N.sup.6'-bis(.beta.1-3'-propiony-
l
galactosyl-.beta.1-4-thioglucoside)lysyl-N.sup.6'-(.beta.-3'-propionyl
galactosyl-.beta.1-4-thioglucoside)lysyl]amino)pentyl-spermine;
[0185] N.sup.2-dodecyl-N.sup.4-(5-[N.sup.2',N.sup.6'-bis
(.beta.1-3'-propionyl
galactosyl-.beta.1-4-thioglucoside)lysyl-N.sup.6'(--
.beta.-3'-propionyl
galactosyl-.beta.1-4-thioglucoside)lysyl]amino)pentyl-- spermine;
and
[0186]
N.sup.2-octadecyl-N.sup.4-(5-[N.sup.2',N.sup.6'-bis(.beta.1-3'-prop-
-ionyl
galactosyl-.beta.1-4-thioglucoside)lysyl-N.sup.6'-(.beta.-3'-propio-
n-yl galactosyl-.beta.1'-4
thioglucoside)lysyl]amino)pentyl-spermine.
[0187] Accordingly, there has been described in detail above the
multifunctional molecular complex of the present invention for
transfer of nucleic acid compositions to a target cell. This
complex includes the transfer moiety as a separate, distinct
embodiment of the present invention. Further aspects of the present
invention relate to the methods of using the multifunctional
molecular complex.
[0188] Thus, in accordance with the present invention there is
provided a method for the transfer of a nucleic acid composition to
target cells on an in vitro basis. In this method target cells are
contacted with a multifunctional molecular complex which includes
said nucleic acid composition. In one embodiment, the target cells
have been isolated from an individual, and all of the cells are
thus of the same type, and it is not necessary, therefore, for the
complex to include a receptor specific binding component. An
especially preferred embodiment is one in which a microorganism
culture is maintained under fermentation conditions, and a protein
product is expressed by the microorganism as a result of the
transfer thereto of nucleic acid compositions using the
multifunctional molecular complex of the present invention. The
protein product is isolated and purified. Here again, a single type
of target cell is involved, so that it is not necessary that a
receptor specific binding component be present.
[0189] This method provides for transfer to target cells of a
nucleic acid molecule that comprises a nucleotide sequence that
either encodes a desired peptide or protein, or serves as a
template for functional nucleic acid molecules. The desired protein
or functional nucleic acid molecule may be any product of
industrial, commercial or scientific interest, e.g., therapeutic
agents including vaccines; foodstuffs and nutritional supplements;
compounds of agricultural significance such as herbicides and plant
growth regulants, insecticides, miticides, rodenticides, and
fungicides; compounds useful in animal health such as parasiticides
including nematocides; and so forth. The target cells are typically
cultures of host cells comprising microoganism cells such as
bacteria and yeast, but may also include plant and mammalian cells.
The cell cultures are maintained in accordance with fermentation
techniques well known in the art, which maximize production of the
desired protein or functional nucleic acid molecule, and the
fermentation products are harvested and purified by known
methods.
[0190] The present invention further relates to a method for the
transfer of a nucleic acid composition to the cells of an
individual in an in vivo manner. The method comprises the step of
contacting cells of said individual with a multifunctional
molecular complex of the present invention, which includes said
nucleic acid composition. Here again, the nucleic acid molecule
comprises a nucleotide sequence that either encodes a desired
peptide or protein, or serves as a template for functional nucleic
acid molecules. The nucleic acid molecule is administered free from
retroviral particles. The desired protein may either be a protein
which functions within the individual or serves to initiate an
immune response.
[0191] The nucleic acid molecule may be administered to the cells
of said individual on either an in vivo or ex vivo basis, i.e., the
contact with the cells of the individual may take place within the
body of the individual in accordance with the procedures which are
most typically employed, or the contact with the cells of the
individual may take place outside the body of the individual by
withdrawing cells which it is desired to treat from the body of the
individual by various suitable means, followed by contacting of
said cells with said nucleic acid molecule, followed in turn by
return of said cells to the body of said individual.
[0192] The method of transferring a nucleic acid composition to the
cells of an individual provided by the present invention, includes
particularly a method of immunizing an individual against a
pathogen. In this method, the nucleic acid composition administered
to said cells, comprises a nucleotide sequence that encodes a
peptide which comprises at least an epitope identical to, or
substantially similar to an epitope displayed on said pathogen as
antigen, and said nucleotide sequence is operatively linked to
regulatory sequences. The nucleic acid molecule must, of course, be
capable of being expressed in the cells of the individual.
[0193] The method of transferring a nucleic acid composition to the
cells of an individual provided by the present invention, further
includes methods of immunizing an individual against a
hyperproliferative disease or an autoimmune disease. In such
methods, the nucleic acid composition which is administered to the
cells of the individual comprises a nucleotide sequence that
encodes a peptide that comprises at least an epitope identical to
or substantially similar to an epitope displayed on a
hyperproliferative disease-associated protein or an autoimmune
disease-associated protein, respectively, and is operatively linked
to regulatory sequences. Here again, the nucleic acid molecule must
be capable of being expressed in the cells of the individual.
[0194] In accordance with the present invention there is also
provided methods of treating an individual suffering from an
autoimmune disease, in which the cells of said individual are
contacted with a multifunctional molecular complex including a
nucleic acid composition, thereby administering a nucleic acid
molecule that comprises a nucleotide sequence which restores the
activity of an absent, defective or inhibited gene, or which
encodes a protein that produces a therapeutic effect in said
individual, and is operatively linked to regulatory sequences; the
nucleic acid molecule being capable of being expressed in said
cells.
[0195] In order to carry out the methods described above, the
present invention provides pharmaceutical compositions which
comprise the multifunctional molecular complex including a nucleic
acid composition, as well as pharmaceutically acceptable salt and
ester forms of said molecular complex, together with a
pharmaceutically acceptable carrier. Also included are kits which
comprise a container comprising a nucleic acid composition, and a
container comprising a transfer moiety, as described herein.
Optionally, there is included in such kits excipients, carriers,
preservatives and vehicles such as solvents.
[0196] Accordingly the present invention provides compositions and
methods which prophylactically and/or therapeutically immunize an
individual against a pathogen or abnormal, disease-related cell.
The genetic material encodes a peptide or protein that shares at
least an epitope with an immunogenic protein found on the pathogen
or cells to be targeted. The genetic material is expressed by the
individual's cells and serves as an immunogenic target against
which an immune response is elicited. The resulting immune response
is broad based: in addition to a humoral immune response, both arms
of the cellular immune response are elicited. The methods of the
present invention are useful for conferring prophylactic and
therapeutic immunity. Thus, a method of immunizing includes both
methods of protecting an individual from pathogen challenge, or
occurrence or proliferation of specific cells, as well as methods
of treating an individual suffering from pathogen infection,
hyperproliferative disease or autoimmune disease. Thus, the present
invention is useful to elicit broad immune responses against a
target protein, i.e. proteins specifically associated with
pathogens or the individual's own "abnormal" cells.
[0197] The present invention is also useful in combating
hyperproliferative diseases and disorders such as cancer, by
eliciting an immune response against a target protein that is
specifically associated with the hyperproliferative cells. The
present invention is further useful in combating autoimmune
diseases and disorders by eliciting an immune response against a
target protein that is specifically associated with cells involved
in the autoimmune condition.
[0198] Other aspects of the present invention relate to gene
therapy. This involves compositions and methods for introducing
nucleic acid molecules into the cells of an individual which are
exogenous copies of genes which either correspond to defective,
missing, non-functioning or partially functioning genes in the
individual, or which encode therapeutic proteins, i.e., proteins
whose presence in the individual will eliminate a deficiency in the
individual and/or whose presence will provide a therapeutic effect
on the individual. There is thus provided a means of delivering
such a protein which is a suitable, and even preferred alternative
to direct administration of the protein to the individual.
[0199] As used herein the term "desired protein" is intended to
refer to peptides and proteins encoded by gene constructs used in
the present invention, which either act as target proteins for an
immune response, or as a therapeutic or compensating protein in
gene therapy regimens.
[0200] Using the methods and compositions of the present invention,
DNA or RNA that encodes a desired protein is introduced into the
cells of an individual where it is expressed, thus producing the
desired protein. The nucleic acid composition, e.g., DNA or RNA
encoding the desired protein is linked to regulatory elements
necessary for expression in the cells of the individual. Regulatory
elements for DNA expression include a promoter and a
polyadenylation signal. In addition, other elements, such as a
Kozak region, may also be included in the nucleic acid
composition.
[0201] As used herein, the term "nucleic acid composition" refers
to the DNA or RNA, or other nucleic acid molecule that comprises a
nucleotide sequence which encodes the desired protein, and which
includes initiation and termination signals operably linked to
regulatory elements including a promoter and polyadenylation signal
capable of directing expression in the cells of the individual to
which the construct is administered.
[0202] As used herein, the term "expressible form" refers to gene
constructs which contain the necessary regulatory elements operably
linked to a coding sequence that encodes a target protein, such
that when present in the cell of the individual, the coding
sequence will be expressed.
[0203] As used herein, the term "genetic vaccine" refers to a
pharmaceutical preparation that comprises a nucleic acid
composition that comprises a nucleotide sequence that encodes a
target protein, including pharmaceutical preparations useful to
invoke a therapeutic immune response.
[0204] As used herein, the term "genetic therapeutic" refers to a
pharmaceutical preparation that comprises a nucleic acid
composition that comprises a nucleotide sequence that encodes a
therapeutic or compensating protein. Alternatively, a genetic
therapeutic may encode antisense sequences which inhibit undesired
gene expression. Further, genetic therapeutics may encode
ribozymes.
[0205] As used herein, the term "target protein" refers to a
protein against which an immune response can be elicited. The
target protein is an immunogenic protein which shares at least an
epitope with a protein from the pathogen or undesirable cell-type,
such as a cancer cell or a cell involved in autoimmune disease,
against which immunization is required. The immune response
directed against the target protein will protect the individual
against, and treat the individual for, the specific infection or
disease with which the target protein is associated.
[0206] As used herein, the term "sharing an epitope" refers to
proteins which comprise at least one epitope that is identical to
or substantially similar to an epitope of another protein. And, the
term "substantially similar epitope" is meant to refer to an
epitope that has a structure which is not identical to an epitope
of a protein, but nonetheless invokes a cellular or humoral immune
response which cross reacts to that protein.
[0207] As used herein, the term "therapeutic protein" is meant to
refer to proteins whose presence confers a therapeutic benefit to
the individual.
[0208] As used herein, the term "compensating protein" is meant to
refer to proteins whose presence compensates for the absence of a
fully functioning endogenously produced protein, due to an absent,
defective, non-functioning or partially functioning endogenous
gene.
[0209] When taken up by a target cell, a nucleic acid composition
used in the present invention, which includes the nucleotide
sequence encoding the desired protein operably linked to regulatory
elements, may remain present in the cell as a functioning
extrachromosomal molecule, or it may integrate into the cell's
chromosomal DNA. DNA may be introduced into cells where it remains
as separate genetic material in the form of a plasmid.
Alternatively, linear DNA which can integrate into the chromosome
may be introduced into the target cell. When introducing DNA into
the cell, reagents which promote DNA integration into chromosomes
may be added. DNA sequences which are useful to promote integration
may also be included in the DNA molecule. Alternatively, RNA may be
administered to the cell. It is also contemplated to provide the
nucleic acid composition as a linear minichromosome including a
centromere, telomeres and an origin of replication. As used herein,
the terms "DNA construct", "nucleic acid composition" and
"nucleotide sequence" are meant to refer to both DNA and RNA
molecules.
[0210] The regulatory elements necessary for gene expression of a
DNA molecule include: a promoter, an initiation codon, a stop
codon, and a polyadenylation signal. In addition, enhancers are
often required for gene expression. It is necessary that these
elements be operably linked to the sequence that encodes the
desired proteins and that the regulatory elements are operable in
the individual to whom they are administered.
[0211] Initiation codons and stop codon are generally considered to
be part of a nucleotide sequence that encodes the desired protein.
However, it is necessary that these elements be functional in the
individual to whom the gene construct is administered. The
initiation and termination codons must be in frame with the coding
sequence. Promoters and polyadenylation signals used must also be
functional within the cells of the individual.
[0212] Examples of promoters useful with the nucleic acid
compositions used in the present invention, especially in the
production of a genetic vaccine for humans, include but are not
limited to, promoters from Simian Virus 40 (SV40), Mouse Mammary
Tumor Virus (MMTV) promoter, Human Immunodeficiency Virus (HIV)
such as the HIV Long Terminal Repeat (LTR) promoter, Moloney virus,
ALV, Cytomegalovirus (CMV) such as the CMV immediate early
promoter, Epstein Barr Virus (EBV), Rous Sarcoma Virus (RSV), as
well as promoters from human genes such as human Actin, human
Myosin, human Hemoglobin, human muscle creatine and human
metalothionein.
[0213] Examples of polyadenylation signals useful with the nucleic
acid compositions used in the present invention, especially in the
production of a genetic vaccine for humans, include but are not
limited to, SV40 polyadenylation signals and LTR polyadenylation
signals. In particular, the SV40 polyadenylation signal which is in
pCEP4 plasmid (Invitrogen, San Diego Calif.), referred to as the
SV40 polyadenylation signal, may be used.
[0214] In addition to the regulatory elements required for nucleic
acid molecule expression, other elements may also be included in
the DNA molecule. Such additional elements include enhancers. The
enhancer may be selected from the group including but not limited
to: human Actin, human Myosin, human Hemoglobin, human muscle
creatine, and viral enhancers such as those from CMV, RSV and
EBV.
[0215] Nucleic acid compositions can be provided with mammalian
origin of replication in order to maintain the construct
extrachromosomally and produce multiple copies of the construct in
the cell. Plasmids pCEP4 and pREP4 from Invitrogen (San Diego,
Calif.) contain the Epstein Barr virus origin of replication and
nuclear antigen EBNA-1 coding region, which produces high copy
episomal replication without integration. In aspects of the
invention relating to gene therapy, constructs with origins of
replication including the necessary antigen for activation are
preferred.
[0216] In other embodiments of the present invention relating to
immunization applications, the nucleic acid composition contains
nucleotide sequences that encode a target protein and further
include genes for proteins which enhance the immune response
against such target proteins. Examples of such genes are those
which encode cytokines and lymphokines such as a-interferon,
y-interferon, platelet derived growth factor (PDGF), GC-SF, GM-CSF,
TNF, epidermal growth factor (EGF), IL-1, IL-2, IL-4, IL-6, IL-8,
IL-10 and IL-12. In some embodiments, it will be preferred that the
gene for GM-CSF be included in nucleic acid compositions used in
immunizing compositions.
[0217] An additional element may be added to the nucleic acid
composition which serves as a target for cell destruction, if it is
desirable to eliminate the cells receiving the nucleic acid
composition for any reason. A herpes thymidine kinase (tk) gene in
an expressible form can be included in the nucleic acid
composition. The drug gangcyclovir can then be administered to the
individual and that drug will cause the selective killing of any
cell producing tk, thus providing the means for the selective
destruction of cells containing the nucleic acid composition.
[0218] In order to maximize protein production, regulatory
sequences may be selected which are well suited for gene expression
in cells into which the construct is transferred. Moreover, codons
may be selected which are most efficiently transcribed in the
target cell. One having ordinary skill in the art can readily
produce DNA constructs which are functional in the target
cells.
[0219] Nucleic acid compositions can be tested for expression
levels in vitro by using tissue culture of cells of the same type
as those to be treated. For example, if the genetic vaccine is to
be administered to human muscle cells, muscle cells grown in
culture, such as solid muscle tumors cells of rhabdomyosarcoma, may
be used as an in vitro model to measure expression level.
[0220] The nucleic acid compositions used in the present invention
are not incorporated within retroviral particles. The nucleic acid
compositions are taken up by the cell without retroviral
particle-mediated insertion, such as that which occurs when
retrovirus particles with retroviral RNA, infects a cell. As used
herein, the term "free from retroviral particles" is meant to refer
to nucleic acid compositions that are not incorporated within
retroviral particles. As used herein, "dissociated from an
infectious agent" is meant to refer to genetic material which is
not part of a viral, bacterial or eukaryotic vector, either active,
inactivated, living or dead, that is capable of infecting a
cell.
[0221] In some embodiments, the nucleic acid compositions
constitute less than a complete, replicatable viral genome such
that upon introduction into the cell, the nucleic acid composition
possesses insufficient genetic information to direct production of
infectious viral particles. As used herein, the term "incomplete
viral genome" is meant to refer to a nucleic acid composition which
contains less than a complete genome such that incorporation of
such a nucleic acid composition into a cell does not constitute
introduction of sufficient genetic information for the production
of infectious virus.
[0222] In some embodiments, an attenuated viral vaccine may be
delivered as a nucleic acid composition which contains enough
genetic material to allow for production of viral particles.
Delivery of the attenuated vaccine as a nucleic acid composition
allows production of large quantities of safe, pure, and active
immunizing product.
[0223] The present invention may be used to immunize an individual
against all pathogens such as viruses, prokaryotic and pathogenic
eukaryotic organisms such as unicellular pathogenic organisms and
multicellular parasites. The present invention is particularly
useful to immunize an individual against those pathogens which
infect cells and which are not encapsulated, such as viruses, and
prokaryotes such as Gonorrhoea, Listeria and Shigella. In addition,
the present invention is also useful for immunizing an individual
against protozoan pathogens, including any stage in their life
cycle in which they are intracellular pathogens. As used herein,
the term "intracellular pathogen" is meant to refer to a virus or
pathogenic organism that, during at least part of its reproductive
or life cycle, exists within a host cell and therein produces or
causes to be produced, pathogenic proteins. Table 1 provides a
listing of some of the viral families and genera for which vaccines
according to the present invention can be made. DNA constructs that
comprise DNA sequences which encode the peptides that comprise at
least an epitope identical or substantially similar to an epitope
displayed on a pathogen antigen, such as those antigens listed in
said table, are useful in vaccines.
[0224] Moreover, the present invention is also useful to immunize
an individual against other pathogens including prokaryotic and
eukaryotic protozoan pathogens as well as multicellular parasites
such as those listed in Table 2.
[0225] In order to produce a genetic vaccine to protect against
pathogen infection, genetic material which encodes immunogenic
proteins against which a protective immune response can be mounted,
must be included in the nucleic acid composition. Whether the
pathogen infects intracellularly, for which the present invention
is particularly useful, or extracellularly, it is unlikely that all
pathogen antigens will elicit a protective response. Because DNA
and RNA are both relatively small and can be produced relatively
easily, the present invention provides the additional advantage of
allowing for vaccination with multiple pathogen antigens. The
nucleic acid composition used in the genetic vaccine can include
genetic material which encodes many pathogen antigens. For example,
several viral genes may be included in a single construct, thereby
providing multiple targets. In addition, multiple inoculants which
can be delivered to different cells in an individual can be
prepared to collectively include, in some cases, a complete or,
more preferably, an incomplete, e.g., nearly complete set of genes
in the vaccine. For example, a complete set of viral genes may be
administered using two constructs which each contain a different
half of the genome which are administered at different sites. Thus,
an immune response may be invoked against each antigen without the
risk of an infectious virus being assembled. This allows for the
introduction of more than a single antigen target and can eliminate
the requirement that protective antigens be identified.
[0226] The ease of handling and inexpensive nature of DNA and RNA
further allow for more efficient means of screening for protective
antigens. Genes can be sorted and systematically tested much more
easily than proteins. Once the pathogenic agents and organism for
which a protective vaccine will be sought is selected, an
immunogenic protein is then identified. Tables 1 and 2 include
lists of some of the pathogenic agents and organisms for which
genetic vaccines can be prepared to protect an individual from
infection by them. The methods of immunizing an individual against
a pathogen can be directed particularly against HIV, HTLV or
HBV.
[0227] In accordance with the present invention there is also
provided a method of conferring a broad based protective immune
response against hyperproliferating cells that are characteristic
of hyperproliferative diseases, as well as a method of treating
individuals suffering from hyperproliferative diseases. As used
herein, the term "hyperproliferative diseases" is meant to refer to
those diseases and disorders characterized by hyperproliferation of
cells. Examples of hyperproliferative diseases include all forms of
cancer and psoriasis.
[0228] It has been discovered that introduction of a nucleic acid
composition that includes a nucleotide sequence which encodes an
immunogenic "hyperproliferating cell"-associated protein into the
cells of an individual, results in the production of those proteins
in the vaccinated cells of an individual. As used herein, the term
"hyperproliferative-associated protein" is meant to refer to
proteins that are associated with a hyperproliferative disease. To
immunize against hyperproliferative diseases, a nucleic acid
composition that includes a nucleotide sequence which encodes a
protein that is associated with a hyperproliferative disease is
administered to an individual.
[0229] In order for the hyperproliferative-associated protein to be
an effective immunogenic target, it must be a protein that is
produced exclusively or at higher levels in hyperproliferative
cells as compared to normal cells. Target antigens include such
proteins, fragments thereof and peptides which comprise at least an
epitope found on such proteins. In some cases, a
hyperproliferative-associated protein is the product of a mutation
of a gene that encodes a protein. The mutated gene encodes a
protein which is nearly identical to the normal protein except it
has a slightly different amino acid sequence which results in a
different epitope not found on the normal protein. Such target
proteins include those which are proteins encoded by oncogenes such
as myb, myc, fyn, and the translocation genes bcr/abl, ras, src,
P53, neu, trk and EGRF. In addition to oncogene products as target
antigens, target proteins for anti-cancer treatments and protective
regimens include variable regions of antibodies made by B cell
lymphomas, and variable regions of T cell receptors of T cell
lymphomas which, in some embodiments, are also used as target
antigens for autoimmune diseases. Other tumor-associated proteins
can be used as target proteins, such as proteins which are found at
higher levels in tumor cells, including the protein recognized by
monoclonal antibody 17-1A and folate binding proteins.
[0230] While the present invention may be used to immunize an
individual against one or more of several forms of cancer, the
present invention is particularly useful to prophylactically
immunize an individual who is predisposed to develop a particular
cancer or who has had cancer and is therefore susceptible to a
relapse. Developments in genetics and biotechnology, as well as
epidemiology, allow for the determination of probability and risk
assessment for the development of cancer in an individual. Using
genetic screening and/or family health histories, it is possible to
predict the probability that a particular individual has for
developing any one of several types of cancer.
[0231] Similarly, those individuals who have already developed
cancer and who have been treated to remove the cancer, or are
otherwise in remission, are particularly susceptible to relapse and
reoccurrence. As part of a treatment regimen, such individuals can
be immunized against the cancer that they have been diagnosed as
having had in order to combat such a recurrence. Thus, once it is
known that individuals have had a type of cancer and are at risk of
a relapse, they can be immunized in order to prepare their immune
systems to combat any future appearance of the cancer.
[0232] The present invention also provides a method of treating
individuals suffering from hyperproliferative diseases. In such
methods, the introduction of nucleic acid compositions serves as an
immunotherapeutic, directing and promoting the immune system of the
individual to combat hyperproliferative cells that produce the
target protein.
[0233] The present invention provides a method of treating
individuals suffering from autoimmune diseases and disorders by
conferring a broad based protective immune response against targets
that are associated with autoimmunity, including cell receptors and
cells which produce "self"-directed antibodies.
[0234] T cell mediated autoimmune diseases include Rheumatoid
arthritis (RA), multiple sclerosis (MS), Sjogren's syndrome,
sarcoidosis, insulin dependent diabetes mellitus (IDDM), autoimmune
thyroiditis, reactive arthritis, ankylosing spondylitis,
scleroderma, polymyositis, dermatomyositis, psoriasis, vasculitis,
Wegener's granulomatosis, Crohn's disease and ulcerative colitis.
Each of these diseases is characterized by T cell receptors that
bind to endogenous antigens and initiate the inflammatory cascade
associated with autoimmune diseases. Vaccination against the
variable region of the T cells would elicit an immune response
including CTLs to eliminate those T cells.
[0235] In RA, several specific variable regions of T cell receptors
(TCRs) which are involved in the disease have been characterized.
These TCRs include V.beta.-3, V.beta.-14, V.beta.-17 and
V.alpha.-17. Thus, vaccination with a nucleic acid composition that
encodes at least one of these proteins will elicit an immune
response that will target T cells involved in RA. See: Howell, M.
D., et al., 1991 Proc. Natl. Acad. Sci. USA 88:10921-10925;
Paliard, X., et al., 1991 Science 253:325-329; Williams, W. V., et
al., 1992 J. Clin. Invest. 90:326-333; each of which is
incorporated herein by reference.
[0236] In MS, several specific variable regions of TCRs which are
involved in the disease have been characterized. These TCRs include
V.beta.-7 and V.alpha.-10. Thus, vaccination with a nucleic acid
composition that encodes at least one of these proteins will elicit
an immune response that will target T cells involved in MS. See:
Wucherpfennig, K. W., et al., 1990 Science 248:1016-1019;
Oksenberg, J. R., et al., 1990 Nature 345:344-346; each of which is
incorporated herein by reference.
[0237] In scleroderma, several specific variable regions of TCRs
which are involved in the disease have been characterized. These
TCRs include V.beta.-6, V.beta.-8, V.beta.-14 and V.alpha.-16,
V.alpha.-3C, V.alpha.-7, V.alpha.-14, V.alpha.-15, V.alpha.-16,
V.alpha.-28 and V.alpha.-12. Thus, vaccination with a nucleic acid
composition that encodes at least one of these proteins will elicit
an immune response that will target T cells involved in
scleroderma.
[0238] In order to treat patients suffering from a T cell mediated
autoimmune disease, particularly those for which the variable
region of the TCR has yet to be characterized, a synovial biopsy
can be performed. Samples of the T cells present can be taken and
the variable region of those TCRs identified using standard
techniques. Genetic vaccines can be prepared using this
information.
[0239] B cell mediated autoimmune diseases include Lupus (SLE),
Grave's disease, myasthenia gravis, autoimmune hemolytic anemia,
autoimmune thrombocytopenia, asthma, cryoglobulinemia, primary
biliary sclerosis and pernicious anemia. Each of these diseases is
characterized by antibodies which bind to endogenous antigens and
initiate the inflammatory cascade associated with autoimmune
diseases. Vaccination against the variable region of such
antibodies would elicit an immune response including CTLs to
eliminate those B cells that produce the antibody.
[0240] In order to treat patients suffering from a B cell mediated
autoimmune disease, the variable region of the antibodies involved
in the autoimmune activity must be identified. A biopsy can be
performed and samples of the antibodies present at a site of
inflammation can be taken. The variable region of those antibodies
can be identified using standard techniques. Genetic vaccines can
be prepared using this information.
[0241] In the case of SLE, one antigen is believed to be DNA. Thus,
in patients to be immunized against SLE, their sera can be screened
for anti-DNA antibodies and a vaccine can be prepared which
includes nucleic acid compositions that encode the variable region
of such anti-DNA antibodies found in the sera.
[0242] Common structural features among the variable regions of
both TCRs and antibodies are well known. The DNA sequence encoding
a particular TCR or antibody can generally be found following well
known methods such as those described in Kabat, et al. 1987
Sequence of proteins of Immunological Interest, U.S. Department of
Health and Human Services, Bethesda Md., which is incorporated
herein by reference. In addition, a general method for cloning
functional variable regions from antibodies can be found in
Chaudhary, V. K., et al., 1990 Proc. Natl. Acad. Sci. USA 87:1066,
which is incorporated herein by reference.
[0243] In aspects of the present invention that relate to gene
therapy, the nucleic acid compositions contain either compensating
genes or genes that encode therapeutic proteins. Examples of
compensating genes include a gene which encodes dystrophin or a
functional fragment, a gene to compensate for the defective gene in
patients suffering from cystic fibrosis, a gene to compensate for
the defective gene in patients suffering from ADA, and a gene
encoding Factor VIII. Examples of genes encoding therapeutic
proteins include genes which encodes erythropoietin, interferon,
LDL receptor, GM-CSF, IL-2, IL-4 and TNF. Additionally, nucleic
acid compositions which encode single chain antibody components
which specifically bind to toxic substances can be administered. In
some preferred embodiments, the dystrophin gene is provided as part
of a mini-gene and used to treat individuals suffering from
muscular dystrophy. In some preferred embodiments, a mini-gene
which contains coding sequence for a partial dystrophin protein is
provided. Dystrophin abnormalities are responsible for both the
milder Becker's Muscular Dystrophy (BMD) and the severe Duchenne's
Muscular Dystrophy (DMD). In BMD dystrophin is made, but it is
abnormal in either size and/or amount. The patient is mild to
moderately weak. In DMD no protein is made and the patient is
wheelchair-bound by age 13 and usually dies by age 20. In some
patients, particularly those suffering from BMD, partial dystrophin
protein produced by expression of a mini-gene delivered according
to the present invention can provide improved muscle function.
[0244] In some preferred embodiments, genes encoding IL-2, IL-4,
interferon or TNF are delivered to tumor cells which are either
present or removed and then reintroduced into an individual. In
some embodiments, a gene encoding y-interferon is administered to
an individual suffering from multiple sclerosis.
[0245] Antisense molecules and ribozymes may also be delivered to
the cells of an individual by introducing a nucleic acid
composition which acts as a template for copies of such active
agents. These agents inactivate or otherwise interfere with the
expression of genes that encode proteins whose presence is
undesirable. Nucleic acid compositions which contain sequences that
encode antisense molecules can be used to inhibit or prevent
production of proteins within cells. Thus, production of proteins
such as oncogene products can be eliminated or reduced. Similarly,
ribozymes can disrupt gene expression by selectively destroying
messenger RNA before it is translated into protein. In some
embodiments, cells are treated according to the invention using
nucleic acid compositions that encode antisense or ribozymes as
part of a therapeutic regimen which involves administration of
other therapeutics and procedures. Nucleic acid compositions
encoding antisense molecules and ribozymes use similar vectors as
those which are used when protein production is desired except that
the coding sequence does not contain a start codon to initiate
translation of RNA into protein.
[0246] Ribozymes are catalytic RNAs which are capable of
self-cleavage or cleavage of another RNA molecule. Several
different types of ribozymes, such as hammerhead, hairpin,
Tetrahymena group I intron, ahead, and RNase P are known in the
art; see S. Edgington, Biotechnology (1992) 10, 256-262. Hammerhead
ribozymes have a catalytic site which has been mapped to a core of
less than 40 nucleotides. Several ribozymes in plant viroids and
satellite RNAs share a common secondary structure and certain
conserved nucleotides. Although these ribozymes naturally serve as
their own substrate, the enzyme domain can be targeted to another
RNA substrate through base-pairing with sequences flanking the
conserved cleavage site. This ability to custom design ribozymes
has allowed them to be used for sequence-specific RNA cleavage; see
G. Paolella et al., EMBO (1992), 1913-1919.) It will therefore be
within the skill of one in the art to use different catalytic
sequences from various types of ribozymes, such as the hammerhead
catalytic sequence, and design them in the manner disclosed herein.
Ribozymes can be designed against a variety of targets including
pathogen nucleotide sequences and oncogenic sequences. Preferred
embodiments include sufficient complementarity to specifically
target the abl-bcr fusion transcript while maintaining efficiency
of the cleavage reaction.
[0247] In accordance with the present invention, the
multifunctional molecular complex containing the desired nucleic
acid composition, may be administered to an individual using a
needleless injection device. In other embodiments, the
multifunctional molecular complex containing the desired nucleic
acid composition is simultaneously administered to an individual
intradermally, subcutaneously and intramuscularly using a
needleless injection device. Needleless injection devices are well
known and widely available. One having ordinary skill in the art
can, following the teachings herein, use needleless injection
devices to deliver multifunctional molecular complexes containing
the desired nucleic acid compositions to cells of an individual.
Needleless injection devices are well suited to deliver these
complexes to all of these tissues. They are particularly useful to
deliver the complexes of the present invention to skin and muscle
cells.
[0248] In some embodiments, a needleless injection device may be
used to propel the complexes of the present invention in liquid
form, that contains DNA molecules, toward the surface of the
individual's skin. The liquid is propelled at a sufficient velocity
such that upon impact with the skin, the liquid penetrates the
surface of the skin, and permeates the skin and muscle tissue
therebeneath. Thus, the nucleic acid composition is simultaneously
administered intradermally, subcutaneously and intramuscularly. In
some embodiments, a needleless injection device may be used to
deliver nucleic acid compositions to the tissue of other organs in
order to introduce a nucleic acid molecule to cells of that
organ.
[0249] According to the present invention, the multifunctional
molecular complexes containing nucleic acid compositions may be
administered directly into the individual to be immunized or ex
vivo into removed cells of the individual which are reimplanted
after administration. By either route, the genetic material is
introduced into cells which are present in the body of the
individual. Routes of administration include, but are not limited
to, intramuscular, intraperitoneal, intradermal, subcutaneous,
intravenous, intraarterially, intraoccularly and oral as well as
transdermally or by inhalation or suppository. Preferred routes of
administration include intramuscular, intraperitoneal, intradermal
and subcutaneous injection. Delivery of nucleic acid compositions
which encode target proteins can confer mucosal immunity in
individuals immunized by a mode of administration in which the
material is presented to tissues associated with mucosal immunity.
Thus, in some examples, the nucleic acid composition is delivered
by administration to the buccal cavity within the mouth of an
individual.
[0250] The multifunctional molecular complexes containing nucleic
acid compositions according to the present invention comprise
generally from about 1 nanogram to about 1000 micrograms of DNA. In
some preferred embodiments, the complexes contain about 10
nanograms to about 800 micrograms of DNA. In more preferred
embodiments, the complexes contain about 0.1 to about 500
micrograms of DNA. In still more preferred embodiments, the
complexes contain about 1 to about 350 micrograms of DNA. In yet
more preferred embodiments, the complexes contain about 25 to about
250 micrograms of DNA. In the most preferred embodiments, the
complexes contain about 100 micrograms DNA.
[0251] The multifunctional molecular complexes containing nucleic
acid compositions according to the present invention are formulated
according to the mode of administration to be used. One having
ordinary skill in the art can readily formulate a pharmaceutical
composition that comprises a nucleic acid composition. In cases
where intramuscular injection is the chosen mode of administration,
an isotonic formulation is preferably used. Generally, additives
for isotonicity can include sodium chloride, dextrose, mannitol,
sorbitol and lactose. In some cases, isotonic solutions such as
phosphate buffered saline are preferred. Stabilizers include
gelatin and albumin. In some embodiments, a vasoconstriction agent
is added to the formulation. The pharmaceutical preparations
according to the present invention are prepared so as to be sterile
and pyrogen free.
[0252] In addition to other agents which may function as
transfecting agents and/or replicating agents, there may be
co-administered with the complexes of the present invention growth
factors, cytokines and lymphokines such as .alpha.-interferon,
.gamma.-interferon, platelet derived growth factor (PDGF), GC-SF,
GM-CSF, TNF, epidermal growth factor (EGF), IL-1, IL-2, IL-4, IL-6,
IL-8, IL-10 and IL-12, as well as fibroblast growth factor, surface
active agents such as immune-stimulating complexes (ISCOMS),
Freund's incomplete adjuvant, LPS analog including monophosphoryl
Lipid A (MPL), muramyl peptides, quinone analogs and vesicles such
as squalene and hyaluronic acid may also be used, administered in
conjunction with the complexes of the present invention. In some
embodiments, combinations of these agents are administered in
conjunction with the complexes of the present invention.
[0253] The complexes of the present invention may be combined with
collagen as an emulsion and delivered parenterally. The collagen
emulsion provides a means for sustained release of DNA; 50 .mu.l to
2 ml of collagen may be used. About 100 .mu.g of DNA are combined
with 1 ml of collagen in a preferred embodiment using this
formulation. Other sustained release formulations such as those
described in Remington's Pharmaceutical Sciences, A. Osol, a
standard reference text in this field, which is incorporated herein
by reference. Such formulations include aqueous suspensions, oil
solutions and suspensions, emulsions and implants as well as
reservoirs, depots and transdermal devices. In some embodiments,
time release formulations for the complexes are preferred; where it
is desirable the complex be time released between 6-144 hours,
preferably 12-96 hours, more preferably 18-72 hours.
[0254] In some embodiments of the invention, the individual is
subject to a single vaccination to produce a full, broad immune
response. In other embodiments of the invention, the individual is
subject to a series of vaccinations to produce a full, broad immune
response. According to still other embodiments of the invention, at
least two and preferably four to five injections are given over a
period of time. The period of time between injections may be from
24 hours apart to two weeks or longer between injections,
preferably one week apart. Alternatively, at least two and up to
four separate injections are given simultaneously at different
sites.
[0255] In some embodiments of the invention, a complete vaccination
includes injection of a single inoculant which contains a nucleic
acid composition including sequences encoding one or more targeted
epitopes.
[0256] In other embodiments of the invention, a complete
vaccination includes injection of two or more different inoculants
into different sites. For example, an HIV vaccine may comprise two
different inoculants in which each one comprises a nucleic acid
composition encoding different viral proteins. This method of
vaccination allows the introduction of as much as a complete set of
viral genes into the individual without the risk of assembling an
infectious viral particle. Thus, an immune response against most or
all of the virus can be invoked in the vaccinated individual.
Injection of each inoculant is performed at different sites,
preferably at a distance to ensure that no cells receive the total
combination of nucleic acid compositions. As a further safety
precaution, some genes may be deleted or altered to further prevent
the capability of infectious viral assembly.
[0257] In accordance with the present invention there are provided
pharmaceutical compositions which facilitate delivery of the
multifunctional molecular complex, which in turn functions to
facilitate transfer of the nucleic acid composition which is
contained therein, to the target cells. The pharmaceutical
composition may be nothing more than an inert diluent and a
pharmaceutically acceptable salt or ester form of said molecular
complex. However, other pharmaceutically acceptable carriers well
known to the artisan in this field, can also be suitably employed
to provide desired properties. Thus, one or more agents may be
selected from the following recognized pharmaceutical classes of
excipients: solvents, solvent systems, and solubilizing and
dispersing agents including surfactants and emulsifying agents;
viscosity modifying agents; and stabilizing and preservative
agents, including antioxidants, UV absorbing agents, antibacterial
agents, and buffering agents.
[0258] The present invention also provides pharmaceutical kits
which comprise a container comprising a nucleic acid composition,
and a container comprising a transfer moiety. optionally, there is
included in such kits excipients, carriers, preservatives and
vehicles of the type described above with respect to pharmaceutical
compositions. The term pharmaceutical kit is also intended to
include multiple inoculants used in the methods of the present
invention. Such kits include separate containers comprising
different inoculants and transfer moieties. The pharmaceutical kits
in accordance with the present invention are also contemplated to
include a set of inoculants used in immunizing methods and/or
therapeutic methods, as described above.
[0259] The compositions and methods of the present invention are
useful in the fields of both human and veterinary medicine.
Accordingly, the present invention relates to genetic immunization
and therapeutic treatment of mammals, birds and fish. The methods
of the present invention can be particularly useful for genetic
immunization and therapeutic treatment of mammalian species
including human, bovine, ovine, porcine, equine, canine and feline
species.
DESCRIPTION OF THE PREFERRED EMBODIMENTS
[0260] The Examples set out below include representative
demonstrations of various aspects of the present invention. The
Examples are not intended to limit the scope of the invention; but
rather are merely intended to serve as illustrations thereof.
Moreover, one having ordinary skill in this art will be able
readily to appreciate additional aspects and embodiments of the
present invention, based on the foregoing detailed description
thereof. Unless otherwise indicated, all temperatures recited in
the following Examples are Celsius scale temperatures.
EXAMPLE 1
Preparation of N.sup.4-5'-Aminopentylspermidine Hydrochloride 8
N.sup.4-(4-cyanobutyl)-N.sup.1,N.sup.8-Bis(tert-butyloxycarbonyl)-S-permid-
ine (6)
[0261] A solution of N.sup.1,N.sup.8-Bis(tert-butyloxycarbonyl)
spermidine (2.92 g, 8.45 mmol, 1.0 eq) [S.Nagarajan and B. Ganem,
J. Org. Chem., 50, 5735-37 (1985)] in acetonitrile (125 mL) was
treated with N,N-diisopropylethylamine (3.534 mL, 20.0 mmol, 2.4
eq), potassium iodide (2.81 g, 16.90 mmol, 2.0 eq), and
5-chlorovaleronitrile (1.902 mL, 16.90 mmol, 2.0 eq). The resulting
homogeneous solution was heated to reflux for 2 hours. The mixture
was treated with additional N,N-diisopropylethylamine (1.767 mL;
1.2 eq), potassium iodide (1.41 g, 1.0 eq) and
5-chlorovaleronitrile (0.951 mL, 1.0 eq), and refluxed an
additional 18 hours. Thin layer chromatography (TLC) indicated no
remaining starting material. The acetonitrile was removed under
vacuum, and the residue taken up in chloroform (250 mL). This
solution was washed with water (200 mL), dried (Na.sub.2SO.sub.4),
and stripped of solvent to afford crude product as an oil. The
material was purified on silica using a gradient of 2-propanol in
chloroform plus 1% N,N-diisopropylethylamine to give an oil (3.40
g); .sup.1H NMR (CDCl.sub.3): .delta. 1.44 (s, 20.8H; should be
18H), 1.58-1.80 (m, 9.8H), 2.38-2.44 (m, 9.4H; should be 8.0H),
3.16(m, 4.1H), 4.80 (m, 0.8H), 5.38 (m, 0.8H).
N.sup.4-(5-aminopentyl)-N.sup.1,N.sup.8-Bis
(tert-butyloxycarbonyl)-Spermi- dine (7)
[0262] A solution of 6 (0.77 g, 1.81 mmol) in glacial acetic acid
(100 mL) was treated with 5% palladium on carbon (0.08 g, 10% w/w)
and placed on a Parr Hydrogenator (50 psi hydrogen gas pressure)
for 2.75 hours. The mixture was filtered through Celite.RTM. brand
diatomaceous earth filter aid (pre-rinsed with glacial acetic acid)
and the Celite.RTM. rinsed with chloroform. The filtrate and
chloroform wash were combined and sovent removed to give an oil.
The crude material was subjected to silica chromatography using a
gradient of methanol in chloroform plus 0.4% diisopropylethylamine.
The material consisted of a colorless oil (0.32 g) .sup.1H NMR
(CDCL.sub.3): .delta.1.33 (m, 3.5H; should be 2.0H), 1.44 (s,
19.1H; should be 18.0H), 1.59 (m, 10.3H), 2.37-2.45 (m, 6.2H), 2.70
(t, 1.5H), 3.14 (m, 4.0H), 4.89 (m, 0.7H), 5.57 (m, 0.8H)
N.sup.4-(5-Aminopentyl) spermidine Hydrochloride (8
[0263] Compound 7 (0.200 g, 0.46 mmol) was stirred with
trifluoroacetic acid (5 mL) at room temperature for 2 hours. The
trifluoroacetic acid was removed under vacuum, followed by three
chloroform additions and subsequent evaporations under vacuum. The
resulting crude oil was twice taken up in 0.1 N HCl (30 mL) and
lyophilized to give 8 as a hydroscopic solid (0.19 g).
EXAMPLE 2
Preparation of N.sup.4-(5-(P-3'-propionyl
galactosyl-.beta.1"-4'-thiogluco- side) aminopentyl)speridine 9
S-(Succinimidyl-.beta.-3'-propionyl)hepta-O-acetyl
galactosyl-.beta.1'4-th- ioglucoside (10)
[0264] A solution of S-.beta.-3-propionyl hepta-O-acetyl
galactosyl-.beta.1-4-thioglucoside [M. Elofsson, S. Roy, B. Walse
and J. Kihlberg, Carb. Res., 246, 89-103 (1993)] (4.60 g, 6.35
mmol) in 1:1 isopropanol/chloroform (100 mL) was treated with
N-hydroxysuccinimide (0.73 g, 6.35 mmol) and
N,N'-dicyclohexylcarbodiimide (1.31 g, 6.35 mmol). After stirring
at room temperature for 19 hours, the mixture was cooled to 40 for
1 h and filtered. The solvent was removed from the filtrate under
vacuum to give a white solid that was recrystallized from
2-propanol (3.43 g). The isolated product was 92% pure by high
performance liquid chromatography (HPLC). .sup.1H NMR (CDCL.sub.3):
.delta. 2.0-2.16 (7s, 21.0H), 2.85 (s, 3.8H), 2.85-3.1 (m, 4.2H),
3.65 (m, 0.7H), 3.78 (t, 1.0H), 3.88 (t, 1.0H), 4.11 (m, 4.0H),
4.54 (m, 2.8H), 4.97 (m, 1.8H), 5.11 (m, 1.0H), 5.23 (t, 1.0H),
5.36 (d, 0.7H).
N.sup.4(5(5-f-3'-propionyl hepta-O-acetyl
galactosyl-.beta.1"-4'-thiogluco-
side)aminopentyl)-N.sup.1,N.sup.8-bis (tert-butyloxycarbonyl)-Sperm
idine (11)
[0265] A solution of 10 (0.300 g, 0.70 mmol) in methylene chloride
(30 mL) was treated with a solution of 7 (0.57 g, 0.70 mmol) in
methylene chloride (30 mL). The mixture was stirred at room
temperature for 18 hours. The solvents were removed under vacuum.
Silica chromatography using a gradient of 2-propanol in chloroform
afforded purified product as a colorless glass (0.49 g); purity of
100% as determined by HPLC .sup.1H NMR (CDCL.sub.3): .delta. 1.33
(m, 3.0H; should be 2.0H), 1.44 (s, 20.0H; should be 18.0H), 1.54
(m, 10.7H; should be 6.0H), 1.68 (m, 2.0H), 1.97-2.16 (7 s, 27.3H;
should be 21.0H), 2.25 (m, 6.0H; should be 4H), 2.52 (m, 9.0H;
should be 8.0H), 2.84 (m, 1.0H), 3.01 (m, 1.0H), 3.1-3.27 (m,
6.7H), 3.64 (m, 1.0H), 3.80 (t, 1.0H), 3.90 (t, 1.0H), 4.11 (m,
4.0H), 4.55 (d, 2.0H), 4.70 (d, 1.0 H), 4.91-5.0 (m, 3.0H; should
be 2.0H), 5.11 (m, 1.3H), 5.21 (t, 1.0H), 5.36 (d, 1.0H), 5.44 (m,
0.7H), 6.28 (m, 0.7H). FAB Mass Spec MH.sup.+=1138.
N.sup.4-(5-(S-.beta.-3'-propionyl hepta-O-acetyl
galactosyl-.beta.1"-4' thioglucoside)aminopentyl) spermidine
trifluoroacetate (12)
[0266] Compound 11 (0.200 g, 0.18 mmol) was treated with
trifluoroacetic acid (5 mL) and stirred at room temperature for 2
hrs. The trifluoroacetic acid (TFA) was largely removed under
vacuum, and the residue subjected to three additions of chloroform,
followed by removal of solvent under vacuum. The product was
recovered as an oil (0.22 g) with trace TPA in evidence; purity of
100% as determined by HPLC. .sup.1H NMR (CDCL.sub.3): .delta.
1.25-1.53 (m, 5.5H), 1.6-2.0 (m, 5.5; should be 4H), 2.00-2.15 (7
s, 23.0H; should be 21.0H), 2.56 (m, 1.0H), 2.68 (m, 1.3H), 2.80
(m, 1.0H), 3.0-3.4 (m, 1.0H; should be 10.0H), 3.64 (m, 1.0H), 3.79
(m, 1.0H), 3.94 (m, 1.0H), 4.11 (m, 2.5H), 4.55 (m, 1.5H), 4.70 (m,
1.3H), 4.90-5.20 (broad m, 16.0H; inflated by water; should be 2.0
or 4.0H), 5.36 (m, 1.0H), 7.12 (m, 0.5H), 7.86 (m, 2.3H), 8.07 (m,
2.3H), 9.8 (m. 0.5H).
N.sup.4-(5-(.beta.-3'-propionyl
galactosyl-.beta.1"-4'-thioglucosid-e)-ami- nopentyl)spermidine
(9)
[0267] A solution of 12 (0.20 g; 0.18 mmol) in methanol (20 mL) was
treated with sodium carbonate (0.38 g; 3.08 mmol; 18 eq) and water
(35 mL) for a homogeneous solution. After 6 hrs at room
temperature, the solvents were evaporated and the residue was
desalted using Sephadex.TM. G-25 Medium gel filtration resin, and
1% glacial acetic acid as eluant. Fractions containing product were
combined and lyophilized for pure product as the triacetate salt
(0.10 g); purity of 100% as determined by HPLC. .sup.1H NMR
(DMSO-d.sub.6 D.sub.2O): 61.15 (m or broad t, 1.5H), 1.3-1.5 (m,
8.0H), 1.65 (m, 2.0H), 1.66 s, 14.0H; should be 9.0H), 2.34 (m,
6.5H), 2.60 (m, 3.0H; should be 2.0H), 2.74 (m, 6.0H), 3.00 (m,
3.0H), 3.27 (m, 3.5H), 3.40 (m, 1.5H), 3.47 (m, 2.0H), 3.50 (m,
1.0H), 3.60 (m, 1.0H), 3.71 (d, 1.0H), 4.14 (broad s, masked by
water peak), 4.27 (m, 4.0H).
EXAMPLE 3
Preparation of N.sup.4-(5-[N.sup.2,N.sup.6-bis(P-3'-propionyl
galactosyl-p 1-4-thioglucoside)lysyl-N.sup.6(.beta.-3'-propionyl
galalactosyl-.beta.1-4-thioglucoside)lysyl]-amino)pentylspermidine
Acetate Salt 18
[0268] For comparative purposes, the CAS style name of the compound
18 set out above is as follows:
4-[(N[2[N.sup.2,N.sup.6-bis(3-[4-O(.beta.-D-gala-
ctopyranosyl)-.beta.-D-glucopyranosylthio]propionyl)lysyl]N.sup.6-(3-[4-O--
(.beta.-D-galactopyranosyl)-.beta.-D-glucopyranosylthio]-propionyl)lysinam-
ido)pentyl]-1,8-diamino-4-azaoctane, Acetate Salt.
N.sup.4-(5-[N.sup.2,N.sup.6-bis(.beta.-3'-propionyl hepta-O-acetyl
galactosyl-.beta.1-4-thioglucoside)lysyl
N.sup.6-(.beta.-3'-propionyl hepta-O-acetyl
galactosyl-.beta.1-4-thioglucoside) lysine (14)
[0269] To a solution of lysyl-lysine (0.25 g, 0.64 mmol) in 1:1
water/acetonitrile (100 mLs) was added N,N-diisopropylethylamine
(0.336 mls, 1.95 mmol, 3.0 eq) and compound 10 (1.859, 2.25 mmol)
and stirred at room temperature for a few minutes until homogeneity
was achieved. The pH was closely monitored at regular intervals,
and N,N-diisopropylethylamine added as needed to maintain the pH
between 7 and 8. A total of 7 eq of base was added over 1 hour
before the pH stabilized at 7-7.5. Reverse phase HPLC was used to
follow the progress of the reaction. After 24 hrs at room
temperature, the reaction appeared to have stopped with
approximately 50% product formation. The acetonitrile was
evaporated under vacuum, and the aqueous mixture treated with
dilute HCl (pH 5). The solution was extracted into chloroform
(2.times.200 mls). The combined organic layers were dried
(Na.sub.2SO.sub.4), and the solvent removed under vacuum to afford
crude product as a glass (2.17 g). The material was subjected to
silica flash chromatography using a gradient of isopropanol in
chloroform plus 1% glacial acetic acid as eluant (1.09 g). Purity
was determined by HPLC to be approximately 96%. FAB Mass Spec:
MH.sup.+=2394.
Succinimidyl N.sup.2,N.sup.6-bis(.beta.-3'-propionyl hepta-O-acetyl
galactosyl-.beta.1-4-thioglucoside)lysyl-N.sup.6-(.beta.-3'-propionyl
hepta-O-acetyl galactosyl-.beta.1-4-thioglucoside)lysine (15)
[0270] A solution of N.sup.2, N.sup.6-bis(.beta.-3'-propionyl
hepta-O-acetyl
galactosyl-.beta.1-4-thioglucoside)lysyl-N.sup.6-(.beta.-3-
'-propionyl hepta-O-acetyl
galactosyl-.beta.1-4-thioglucoside)ly-sine (1.00 g, 0.42 mmol) in
1:1 isopropanol/chloroform (20 mL) was treated with
N-hydroxysuccinimide (48.1 mg, 0.42 mmol) and
N,N'dicyclohexylcarbodiimide (86.2 mg, 0.42 mmol). After stirring
at room temperature for 19 hours, the mixture was cooled to 40 for
1 h and filtered. Solvent was removed from the filtrate under
vacuum, and the crude product was recrystallized from 2-propanol.
The product was collected by filtration and dried under vacuum to
give a white powdery solid (0.76 g). This material was shown by
HPLC to consist of a mixture of the starting free acid and the
succinimidyl ester in a ratio of approximately 1:2 respectively.
The mixture was not subjected to further purification, but was used
as is in the next step. .sup.1H NMR (CDCl.sub.3): .delta. 1.96-2.20
(multiple s, 86.4H; should be 63.0H), 2.28 (m, 3.6H), 2.53 (m,
6.6H), 2.88 (m, 6.6H), 3.02 (m, 3.6H), 3.18-3.40 (m, 4.2H), 3.62
(m, 3.6H), 3.85 (m, 3.6H), 3.95 (m, 3.6H), 4.10 (m, 12.0H), 4.57
(m, 6.0H), 4.70 (m, 4.2H), 5.02 (m, 7.2H), 5.10 (m, 3.6H), 5.20 (t,
3.6H), 5.36 (d, 3.0H), 6.31 (t, 0.6H), 6.58 (t, 0.6H), 6.90 (d,
0.6H), 7.47 (d, 0.6H).
N.sup.4-(5-[N.sup.2,N.sup.6-bis(.beta.-3'-propionyl hepta-O-acetyl
galactosyl-.beta.1-4-thioglucoside)lysyl-N-6-(.beta.-3'-propionyl
hepta-O-acetyl
galactosyl-.beta.1-4-thioglucoside)lysyl]-aminopentyl-N.su-
p.1,N.sup.8-Bis (tert-butoxycarbonyl)spermidine (16)
[0271] A solution of 7 (87 mg, 0.20 mmol) in methylene chloride (20
mL) was treated with a solution of 15 (0.76 g of the 66% mixture,
corresponding to 0.50 g of the ester; 0.20 mmol) in methylene
chloride (20 mL). The mixture was stirred at room temperature for
18 hrs, followed by removal of the solvent under vacuum. The crude
product was purified by silica chromatography using a gradient of
isopropanol in chloroform. The resulting impure product consisted
of a 65:35 mixture of product to the free acid present as the
contaminant in the starting ester. A second silica column using the
same gradient plus 0.5% glacial acetic acid effectively isolated
pure product (0.38 g); purity of 100% as determined by HPLC.
.sup.1H NMR (CDCl.sub.3): .delta. 1.30-1.39 (m, 4.0H), 1.44 (s,
13.5H; should be 18.0H), 1.53 (m, 8.6H), 1.60-1.90 (m, 6.1H),
1.97-2.16 (multiple s, 73.8H; should be 63.0H), 2.27 (m, 3.7H),
2.49-2.70 (m, 8.0H), 2.85 (m, 2.5H), 3.00 (m, 2.5H), 3.23 (m,
6.2H), 3.65 (m, 2.5H), 3.80-3.93 (m, 5.5H), 4.12 (m, 8.0H),
4.20-4.40 (m, 1.8H), 4.57-4.69 (m, 8.0H), 4.91-5.00 (m, 5.5H), 5.09
(m, 3.1H), 5.21 (t, 3.1H), 5.36 (d, 2.5H), 6.57 (m, 0.9H), 6.95 (m,
0.6H), 7.20 (m, 0.6H).
N.sup.4-(5-[N.sup.2,N.sup.6-bis(.beta.-3'-propionyl hepta-O-acetyl
galactosyl-.beta.1-4-thioglucoside)lysyl-N.sup.6-(.beta.-3'-propionyl
hepta-O-acetyl
galactosyl-.beta.1-4-thioglucoside)lysyl]-aminopentyl)sper- midine
(17)
[0272] Compound 16 (0.170 g, 0.059 mmol) was treated with
trifluoroacetic acid (5 mL) and stirred at room temperature for 2.5
hours. The trifluoroacetic acid was largely removed under vacuum
(0.19 g). Purity was determined by HPLC to be approximately 100%.
.sup.1H NMR (CDCl.sub.3): .delta. 1.20-1.80 (m, 4.8H), 1.96-2.15
(multiple s, 84.0H; should be 63.0H), 2.54 (m, 12.0H; possibly
inflated by water peak), 3.65 (m, 3.0H), 3.81 (m, 3.0H), 3.93 (m,
4.0H), 4.12 (m, 12.0H), 4.29 (m, 2.0H), 4.55 (m, 6,0H), 4.69 (m,
4.0H), 4.89 (m, 3.0H), 5.09 (m, 6.0H), 5.19 (m, 4.0H), 5.38 (d,
3.0H), 6.99 (m, 1.0H), 7.60 (m, 0.5H), 7.91 (m, 1.0H), 8.09 (m,
1.0H).
N.sup.4-(5-[N.sup.2',N.sup.6-bis(.beta.-3'-propionyl
galactosyl-.beta.1-4-thioglucoside)lysyl-N.sup.6-(.beta.-3'-propionyl
galactosyl-.beta.1-4-thioglucoside)lysy]-aminopentyl)spermidine
acetate salt (18)
[0273] To a solution of 17 (0.17 g, 0.059 mmol) in methanol (20 mL)
was added sodium carbonate (0.37 g, 2.95 mmol), followed by water
(40 mL) until homogeneity was achieved. After stirring at room
temperature for 4 hrs, the solvents were stripped off and the crude
product eluted down a Sephadex.TM. 25 Medium column using 1 glacial
acetic acid as the eluant. Fractions containing product were
combined and lyophilized to afford pure product as an extremely
hygroscopic solid (0.07 g).
EXAMPLE 4
Preparation of N.sup.4-(5-N.sup.2-N-bis(.beta.-3'-proplonyl
galactosyl-.beta.1-4-thioglucoside)lysyl]-aminopentyl)spermidine
Acetate Salt 23
Succinimidyl N.sup.2,N.sup.6-bis(.beta.-3'-propionyl hepta-O-acetyl
galactosyl-.beta.1-4-thioglucoside)lysine (20)
[0274] A solution of N.sup.2,N.sup.6-bis(.beta.-3'propionyl
hepta-O-acetyl galactosyl-.beta.1-4-thioglucoside)lysine (1.00 g,
0.42 mmol) in 1:1 isopropanol/chloroform (20 mL) is treated with
N-hydroxysuccinimide (48.1 mg, 0.42 mmol) and
N,N'dicyclohexylcarbodiimide (86.2 mg, 0.42 mmol). After stirring
at room temperature for 19 hours, the mixture is cooled to 40 for 1
h and filtered. Solvent is removed from the filtrate under vacuum,
and the crude product recrystallized from 2-propanol. The product
is collected by filtration and dried under vacuum to give a white
powdery solid (0.76 g). This material is shown by HPLC to consist
of a mixture of the starting free acid and the succinimidyl ester
in a ratio of approximately 1:2 respectively. The mixture is not
subjected to further purification, but is used as is in the next
step.
N.sup.4-(5-[N.sup.2,N.sup.6-bis(.beta.1-3'-propionyl hepta-O-acetyl
galactosyl-.beta.1-4-thioglucoside)lysyl]aminopentyl)-N.sup.1,N.sup.8-bis-
-(tert-butoxycarbonyl)spermidine (21)
[0275] A solution of 7 (87 mg, 0.20 mmol) in methylene chloride (20
mL) is treated with a solution of 20 (0.769 of the 66% mixture,
corresponding to 0.50 g of the ester; 0.20 mmol) in methylene
chloride (20 mL). The mixture is stirred at room temperature for 18
hrs, followed by removal of the solvent under vacuum. The crude
product is purified by silica chromatography using a gradient of
isopropanol in chloroform. The resulting impure product consists of
a 65:35 mixture of product to the free acid present as the
contaminant in the starting ester. A second silica column using the
same gradient plus 0.5% glacial acetic acid effectively isolates
pure product (0.38 g); purity of 100% as determined by HPLC.
N.sup.4-(5-[N.sup.2,N.sup.6-bis(.beta.-3'-propionyl hepta-O-acetyl
galactosyl-.beta.1-4-thioglucoside)lysyl]aminopentyl)spermidine
(22)
[0276] Compound 21 (0.170 g, 0.059 mmol) is treated with
trifluoroacetic acid (5 mL) and stirred at room temperature for 2.5
hours. The trifluoroacetic acid is largely removed under vacuum
(0.19 g). Purity is determined by HPLC to be approximately
100%.
N.sup.4-(5-[N.sup.2,N.sup.6-bis(.beta.-3'-propionyl
galactosyl-.beta.1-4-thioglucoside)lysyl]aminopentyl) spermidine
Acetate Salt (23)
[0277] To a solution of 22 (0.17 g, 0.059 mmol) in methanol (20 mL)
is added sodium carbonate (0.37 g, 2.95 mmol), followed by water
(40 mL) until homogeneity is achieved. After stirring at room
temperature for 4 hrs, the solvents are stripped off and the crude
product is eluted down a Sephadex.TM. G-25 Medium column using 1%
glacial acetic acid as the eluant. Fractions containing product are
combined and lyophilized to afford pure product as an extremely
hydroscopic solid (0.07 g).
EXAMPLE 5
Preparation of N.sup.4-5-(D-biotinyl)aminopentyl Spermidine
Hydrochloride Salt 24
[0278] To a solution of 7 (0.20 g, 0.47 mmol) in 20 mL of
acetonitrile and 10 mL of water was added 160 mg of succinimidyl
D-biotin. The solution was stirred for 18 h. The volume of the
solution was reduced to 15 mL under vacuum and the remaining
solution was purified on octadecylsilyl bonded silica using a
water/acetonitrile gradient containing 0.1% trifluoroacetic acid.
Fractions containing the product were combined and solvent removed
under vacuum to give a waxy white solid (94% pure by HPLC). To the
white solid was added 10 mL of 2-propanol and 10 mL of 4 N HCl in
dioxane. The solvents were removed under vacuum. The resultant
white solid was redissolved in water and dried under vacuum to give
a hygroscopic foam (0.11 g). .sup.1H NMR (DMSO-d.sub.6) .delta.
1-31 (m, 4H), 1.44 (m, 6H), 1.64 (m, 8H), 2.09 (m, 4H), 2.63 (d,
2H), 2.91 (m, 4H), 3.06 (m, 8H), 4.18 (m, 1H), 4.36 (m, 1H).
EXAMPLE 6
Preparation of
N.sup.4-(5-cholestene-3'.beta.-oxycarbonyl)aminopentyl Spermidine
Hydrochloride Salt 25
[0279] To a solution of 7 (0.20 g, 0.47 mmol) in 20 mL of methylene
chloride was added 210 mg of cholesteryl chloroformate and 200
.mu.L of diisopropylethylamine. The solution was stirred for 24 h.
The methylene chloride was removed under vacuum and the remaining
oil was redissolved in chloroform and purified on silica using a
methanol/chloroform gradient containing 0.1% diisopropylethylamine.
Fractions containing the product were combined and solvent removed
under vacuum to give a waxy white solid (0.29 g). To the white
solid was added 10 mL of 2-propanol and 10 mL of 4 N HCl in
dioxane. The solvents were removed under vacuum. The resultant
white solid was redissolved in water and dried under vacuum to give
a waxy white solid (0.15 g).
EXAMPLE 7
Preparation of
N.sup.4-octyl-N.sup.1,N.sup.8-Bis(tert-butyloxycarbonyl) Spermidine
26
[0280] To a solution of N.sup.1,N.sup.8-Bis(tert-butyloxycarbonyl)
spermidine (1.0 g, 2.89 mmol) in acetone (100 mls) was added
N,N-diisopropylethylamine (0.605 mls, 3.47 mmol, 1.2 eq), potassium
iodide (0.489, 2.89 mmol), and 1-bromooctane (0.500 mls, 2.89
mmol). The mixture was heated to reflux for one hour, followed by
the addition of N,N-diisopropylethylamine (0.605 mls, 3.47 mmol)
and potassium iodide (0.48 g, 2.89 mmol). After an additional 3 hrs
reflux, 1-bromooctane (0.500 mls, 2.89 mmol) was added and
refluxing continued for an additional hour. The acetone was
evaporated under vacuum. The residue taken up in chloroform (125
mls) and washed with water (2.times.75 mls). The organic layer was
dried (Na.sub.2SO.sub.4), and solvent removed under vacuum to give
a liquid. The liquid was purified by silica chromatography using a
gradient of isopropanol in chloroform plus 1%
N,N-diisopropylethylamine. The pure product was recovered as a pale
orange oil (0.59 g). .sup.1H NMR (CDCl.sub.13): .delta. 0.88 (t,
3.5H), 1.27 (m, 14.2H; should be 16.0H), 1.44 (s, 18.7H), 1.58 (m,
1.6H), 1.82 (m, 1.6H), 2.37 (m, 2.4H), 2.44 (m, 2.2H), 3.19 (m,
4.0H), 4.84 (m, 0.3H), 5.62 (m, 0.3H).
[0281] The N.sup.4-octyl-N.sup.1,N.sup.8-Bis(tert-butyloxycarbonyl)
spermidine (0.15 g) was dissolved in 6 mL of 4 N HCl in dioxane and
stirred at room temperature for 1 h. The solvent was removed under
vacuum and the yellow oil suspended in chloroform and the solvent
removed under vacuum. The resultant oil was dissolved in 10 mL of
absolute ethanol and precipitated by the addition of 30 mL of
diethyl ether. The solid was isolated by decanting off the liquid
and drying under vacuum (0.10 g). .sup.1H NMR (DMSO-d.sub.6):
.delta. 0.86 (m, 3H), 1.28 (m, 10H), 1.58-1.80 (m, 6H), 2.00 (m,
2H), 2.80 (m, 2H), 2.89 (m, 2H), 3.03 (m, 4H), 3.15 (m, 2H), 8.01
and 8.13 (two m, 5H, should be 6H). Anal: Calcd for
C.sub.15H.sub.38Cl.sub.3N.sub.3 C, 49.11; H, 10.44; N, 11.45. Found
C, 48.57; H, 10.75; N, 11.29.
EXAMPLE 8
Preparation of N-dodecylspermidine trihydrochloride 27
[0282] A solution of N.sup.1,N.sup.8-Bis(tert-butyloxycarbonyl)
spermidine (0.50 g, 1.45 mmol) in acetone (50 mLs) was treated with
N,N-diisopropylethylamine (0.604 mLs, 3.48 mmol), potassium iodide
(0.48 g, 2.90 mmol), and 1-bromodedecane (0.348 mLs, 2.90 mmol).
The mixture was refluxed for 18 hrs and was treated with additional
1-bromododecane (0.348 mLs, 2.90 mmol). Refluxing was continued for
4 hours. The acetone was removed under reduced pressure, and the
residue taken up in chloroform (250 mLs). The solution was washed
with water (2.times.100 mLs). The organic layer was dried
(Na.sub.2SO.sub.4), and the solvent removed for crude product. The
material was purified by silica flash chromatography eluting with a
gradient of isopropanol in chloroform plus 0.4%
N,N-diisopropylethylamine. The pure product was recovered as an oil
of mass 0.52 g. .sup.1H NMR (CDCl.sub.3): .delta. 0.88 (t, 3.0H),
1.26 (broad s, 21.0H; should be 20.0H), 1.44 (s, 20.5; should be
18.0H), 1.64 (m, 5.8H), 2.37 (m, 3.8H), 2.46 (t, 2.5H), 3.15 (m,
4.0H), 4.84 (m, 0.5H), 5.62 (m, 0.8H).
[0283] The N.sup.1,N.sup.8-Bis
(tert-butyloxycarbonyl)-N-4-dodecyl-spermid- ine (0.52 g, 1.01
mmol) was dissolved in 2-propanol (5 mLs) and treated with 4N HCl
in dioxane (10 mLs). The homogeneous solution was stirred at room
temperature for 20 hrs, and the solvents were evaporated under
reduced pressure. The crude oil was taken up in ethanol (20 mLs)
and treated with ether (10-15 mLs) with stirring. A solid
precipitated out of solution and was collected by filtration (85
mgs). A second crop was recovered for an additional 79 mgs. Total
yield was 164 mgs. .sup.1H NMR (DMSO-d.sub.6): .delta. 0.86 (t,
2.3H), 1.25 (m, 18.0H), 1.63 (m, 3.7H), 1.76 (m, 2.3H), 1.99 (m,
1.3H), 2.79 (m, 2.0H), 2.90 (m, 2.0H), 3.02 (m, 3.7H), 3.16 (m,
2.3H), 8.03 (m, 1.7H), 8.14 (m, 2.7H).
EXAMPLE 9
Preparation of N.sup.4-hexadecylspermidine trihydrochloride 28
[0284] A solution of N.sup.1,N.sup.8-Bis(tert-butyloxycarbonyl)
spermidine (0.50 g, 1.45 mmol) in acetone (50 mLs) was treated with
N,N-diisopropylethylamine (0.302 mLs, 1.74 mmol, 1.2 eq), potassium
iodide (0.24 g, 1.45 mol) and 1-bromohexadecane (0.442 mLs, 1.45
mmol). The mixture refluxed for 20 hrs, and the solvents were
removed under vacuum. The residue was taken up in chloroform (250
mLs) and washed with water (150 mLs). The organic layer was dried
(Na.sub.2SO.sub.4), and the solvent evaporated under reduced
pressure to afford crude product as an amber oil. The material was
purified by silica flash chromatography using a gradient of
isopropanol in chloroform plus 0.4% N,N-diisopropylethylami- ne.
The pure product was recovered as a pale yellow oil, mass 0.42 g.
.sup.1H NMR (CDCl.sub.3): .delta. 0.88 (t, 2.9H), 1.26 (broad s,
30.3H; should be 28.0H), 1.44 (s, 22.9H; should be 18.0H), 1.60 (m,
4.0H), 1.72 (m, 1.7H), 2.37 (m, 3.7H), 2.46 (m, 2.9H; should be
2.0H), 3.17 (m, 4.0H), 4.83 (m, 0.6H), 5.62 (m, 0.6H).
[0285] The
N.sup.1,N.sup.8-Bis(tert-butyloxycarbonyl)-N.sup.4-hexadecyl-sp-
ermidine (0.42 g, 0.74 mmol), was dissolved in 2-propanol (5 mls)
and was treated with 4N HCl in dioxane (10 mls). The homogeneous
solution was stirred at room temperature for 1.5 hrs, followed by
evaporation of the solvents under reduced pressure. The residue was
taken up in ethanol (12 mls) and was treated with ether (10 mLs)
with stirring. A precipitate fell out of solution and was collected
by filtration; mass 125 mgs. A second crop was recovered of mass 75
mgs for a total yield of 0.200 g. .sup.1H NMR
(DMSO-d.sub.6.sup.+D.sub.2O): .delta. 0.83 (t, 2.1H; should be
3.0H), 1.22 (m, 26.6H), 1.54-1.70 (m, 6.0H), 1.96 (m, 1.7H), 2.81
(m, 1.7H), 2.89 (m, 2.1H), 3.04-3.12 (m, 6.0H).
EXAMPLE 10
Transfection of Adherent Cells in Culture Using Receptor Bearing
Cells
[0286] Human hepatocellular carcinoma HuH7 cells were grown and
seeded into a 96 well plate with 1-2.times.10.sup.4 cells in 100
.mu.L of minimal essential media .alpha., modification with 10%
serum. The plates were incubated in a 37.degree. CO.sub.2 incubator
until the cells were 60-80% confluent (approximately 24 hrs). The
media was removed and the cells washed once with Optimem.RTM.
(serum free media). Optimem.RTM. (100 .mu.L) containing 0.5 .mu.g
of pCMV .beta. plasmid (from Clonetech, No. 6177-1), 0.5 .mu.g of
N.sup.4-(5-[N.sup.2,N.sup.6-bis(.beta.-3'-propionyl
galactosyl-.beta.1-4-thioglucoside)lysyl-N.sup.6-(.beta.-3'-propionyl
galactosyl-.beta.1-4-thioglucoside)lysyl]aminopentyl)-spermidine
and 2.5 .mu.g of
N.sup.4-(5-cholestene-3'.beta.-oxycarbonyl)-aminopentyl)spermidi-
ne was added to the cells. The cells were incubated with the
mixture for 4 hrs in a 37.degree. CO.sub.2 incubator; thereafter
100 .mu.L of media containing 20% serum was added and the
incubation continued for an additional 20 hrs. Cells lysed with
0.5% NP-40 in 140 mM NaCl, 10 mM tris, 1.5 mM MgCl.sub.2 were
assayed for .beta.-galactosidase activity using o-nitrophenyl
.beta.-D-galactopyranoside, and gave A.sub.405=1.35 after 30 min.
Cells grown in 6 well plates (25 mm diameter) and treated similarly
were fixed with 2% paraformaldehyde and assayed for
.beta.-galactosidase activity using 5-bromo-4-chloro-3-indolyl
.beta.-D-galactopyranoside (J. Sambrook, et al., Molecular Cloning:
A Laboratory Manual, Cold Spring Harbor, 1989). Visual inspection
showed 5-10% of the cells were transfected as evidenced by light
microscopy. Similar results were obtained when the receptor
specific binding component of the multifunctional molecular complex
was omitted from the cell culture, while retaining the endosome
membrane disruption promoting component of the multifunctional
molecular complex.
[0287] Similar results were obtained with
N.sup.4-(5-(.beta.-3'-propionyl
galactosyl-.beta.1-4-thioglucoside)aminopentyl) spermidine; and
N.sup.4-(5-(methyltetrahydrofolyl) aminopentyl)-spermidine; with
the endosome membrane disruption promoting components included
therein. Similar results can also be obtained with
N.sup.4-octylspermidine; N.sup.4-dodecylspermidine; fusogenic
peptides acylated on the N-terminus by
N.sup.4-(5-carboxypentyl)spermidine;
N.sup.4-(5-(3.alpha.,7.alpha.,12 .alpha.-trihydroxy-5
.beta.-cholan-24-oic)aminopentyl) spermidine amide, with the
receptor specific binding components included therein.
EXAMPLE 11
Transfection of Muscle Cells In Vivo
[0288] Solutions were prepared containing 100 .mu.g of pCMV.beta.
plasmid and 100 .mu.g of either N.sup.4-octylspermidine,
N.sup.4-dodecylspermidin- e or N.sup.4-(5-cholestene-3'
.beta.-oxycarbonyl)-aminopentyl)spermidine, each in 100 .mu.L of
phosphate buffered saline. The plasmid solution (100 .mu.L) was
injected into the rear quadriceps of 6-12 week old BALB-C mice. The
mice were sacrificed approximately 96 hrs later and the entire
quadriceps muscle tissue was removed. The muscle was fixed with
formalin for 2 hrs, and assayed for .beta.-galactosidase activity
using 5-bromo-4-chloro-3-indolyl-.beta.-D-galactopyranoside (1
mg/mL in tris/EDTA pH 8.5). All three injections were scored as
positive since the blue color was more intense than the control:
injection of 100 .mu.g of pCMV.beta. plasmid injected in 100 .mu.L
of 0.25% bupivicaine hydrochloride in citrate buffer pH 6.0.
Similar results can also be obtained with fusogenic peptides
acylated on the N-terminus by N.sup.4-(5-carboxypentyl) spermidine;
and N.sup.4-(5-(3.alpha.,7.alpha.,1-
2.alpha.-trihydroxy-5-cholan-24-oic)aminopentyl) spermidine
amide.
EXAMPLE 12
Transfection of Liver Cells In Vivo
[0289] A solution is prepared containing 10 .mu.g of pHBVSA
plasmid, 5 .mu.g of
N.sup.4-(5-[N.sup.2,N.sup.6-bis(.beta.-3'-propionyl
galactosyl-.beta.1-4-thioglucoside)lysyl-N.sup.6-(.beta.3'-propionyl
galactosyl-.beta.1-4-thioglucoside)lysyl]aminopentyl)spermidine and
25 .mu.g of
N.sup.4-(5-cholestene-3'.beta.-oxycarbonyl)aminopentyl)spermidin- e
in 100 .mu.L of phosphate buffered saline. The plasmid solution
(100 .mu.L) is injected into the tail vein of 6-12 week old BALB-C
mice. The mice are sacrificed 48-120 hrs later and the serum tested
for hepatitus B surface antigen using a commercial enzyme-linked
immunoassay. The production of surface antigen is greater than the
positive control supplied with the kit at 48 hrs
post-injection.
[0290] Similar results are obtained with N.sup.4-(5-(P-3'-propionyl
galactosyl-.beta.1-4-thioglucoside)aminopentyl)spermidine;
N.sup.2,N.sup.6-(5-[bis(.beta.-3'-propionyl
galactosyl-.beta.1-4-thiogluc- oside)lysyl]aminopentyl)spermidine;
N.sup.4-(5-(methyltetrahydrofolyl) aminopentyl)spermidine;
N.sup.4-(5-(folinyl)aminopentyl)-spermidine;
N.sup.4-(5-(.alpha.-3'-propionyl thiomannoside) aminopentyl)
spermidine and N.sup.4-(5-(.alpha.-3'-propionyl
thiomannoside-6-phosphate)aminopenty- l)spermidine, with the
receptor specific binding components included therein.
[0291] Similar results can also be obtained with
N.sup.4-octylspermidine; N.sup.4-dodecylspermidine, fusogenic
peptides acylated on the N-terminus by
N.sup.4-(5-carboxypentyl)spermidine;
N.sup.4-(5-(cholest-5-en-3'-.beta-
.-carbamoyl)-aminopentyl)spermidine; and
N.sup.4-(5-(3.alpha.,7.alpha.,
12.alpha.-trihydroxy-5.beta.-cholan-24-oic)aminopentyl)-spermidine
amide, with the endosome membrane disruption promoting component
included therein.
EXAMPLE 13
A Kit for Research and Manufacturing Use
[0292] A kit for using the multifunctional molecular complexes of
the present invention in a research and manufacturing setting,
where the individual users supply their own DNA, includes a vial
containing the transfer moiety of the present invention dissolved
at 0.1 to 10 mg/mL and preferably at 1 mg/mL in a sterile buffer at
pH 6-8 and preferably at pH 6.5 to 7.5. Acceptable buffers would
include citrate, HEPES and phosphate. Use of the kit involves
removing an aliquot of transfer moiety and adding the aliquot to
the solution of DNA (at 0.05 to 2 mg/mL, and preferably 0.25 to
0.75 mg/mL), such that the final ratio of mg of transfer moiety to
mg DNA is between 0.5 and 5.0 mg/mg. Optimal ratios can be readily
determined. The DNA-transfer moiety mixture is mixed briefly and
held at 370 for 15-60 minutes. The DNA-transfer moiety mixture,
which has now formed the multifunctional molecular complex of the
present invention, is then diluted with minimal essential media
(serum free) to a concentration of 5 to 100 .mu.g/mL and added to
the cells in culture. Optimal concentrations can be readily
determined.
EXAMPLE 14
A Kit for Clinical and Veterinary Use
[0293] A kit for using the transfer moieties of the present
invention in a clinical or veterinary setting, where the individual
users supply their own DNA, includes a vial containing the transfer
moiety dissolved at 0.1 to 10 mg/mL and preferably at 1 mg/mL in a
sterile buffer at pH 6-8 and preferably at pH 6.5 to 7.5.
Acceptable buffers include citrate, HEPES and phosphate. Use of the
kit involves removing an aliquot of transfer moiety and adding the
aliquot to the solution of DNA (at 0.05 to 2 mg/mL, and preferably
0.25 to 0.75 mg/mL), such that the final ratio of mg of transfer
moiety to mg DNA is between 0.5 and 5.0 mg/mg and is preferably 1
mg/mg. Optimal ratios can be readily determined. The DNA-compound
mixture is mixed briefly and held at ambient temperature for 30-60
minutes. The DNA-transfer moiety mixture (10 to 500 .mu.g of DNA),
which has now formed the multifunctional molecular complex of the
present invention, is then injected into the patient or subject,
human or animal, as is consistent with the desired application,
e.g., i.m. injection for immunization, i.v. injection for liver
localization, etc.
EXAMPLE 15
A Kit for Clinical and Veterinary Use, Including DNA
[0294] A kit for using the multifunctional molecular complexes of
the present invention in a clinical or veterinary setting, where
the DNA is supplied as part of the kit, includes a vial containing
the compound dissolved at 0.05 to 10 mg/mL and preferably at 0.5 to
1 mg/mL in a sterile buffer at pH 6-8, and preferably at pH 6.5 to
7.5. Acceptable buffers include citrate, HEPES and phosphate. The
kit also contains DNA appropriate for the intended use (at 0.05 to
2 mg/.mu.L, and preferably 0.25 to 0.75 mg/mL), such that the final
ratio of mg of the transfer moiety component of the kit to mg of
the DNA component of the kit, is between 0.5 and 5.0 mg/mg, and is
preferably 1 mg/mg. Optimal ratios can be readily determined. The
DNA-transfer moiety mixture is held at ambient temperature for
30-60 minutes. The DNA-transfer moiety mixture (10 to 500 .mu.g of
DNA), is then injected into the patient or subject, human or
animal, as is consistent with the desired application, e.g., i.m.
injection for immunization, i.v. injection for liver localization,
etc.
EXAMPLE 16
[0295] Kit Containing Lyophilized Components
[0296] The kits described in Examples 13 through 15 above, can also
have the components thereof supplied as lyophilized powders where
the transfer moieties, buffer components and excipients are
reconstituted at the site of use by the addition of sterile water.
These lyophilized kits can also include the DNA as a lyophilized
component.
EXAMPLE 17
Preparation of N.sup.4-(benzyl 6'-hexanoyl)-spermidine
trihydrochloride
[0297] Benzyl 6-bromohexanoate was prepared by dropwise addition of
a solution of 6-bromohexanoyl chloride (20.0 mL) in 100 mL of
methylene chloride to a solution of 66.22 mL of benzyl alcohol in
60 mL of pyridine. The mixture was cooled using an ice-water bath
for 90 min, then stirred at room temperature for 18 h. The solution
was extracted with 200 mL of 1 N HCl followed by 2.times.150 mL of
sat. NaHCO.sub.3. The extracts were discarded and the solution
dried over Na.sub.2SO.sub.4. The solution was filtered and the
solvent removed in vacuo to give an oil. This oil was purified on
silica gel, eluting with a gradient of chloroform in hexanes.
Fractions containing the product (TLC Rf 0.52, CHCl.sub.3/hexanes
6:4) were combined and the solvent removed to give 24.4 g.
[0298] N.sup.4-(benzyl 6-hexanoyl)-N.sup.1,N.sup.8-bis
(tert-butyloxycarbonyl)spermidine was prepared by treating
N.sup.1,N.sup.8-bis(tert-butyloxycarbonyl)spermidine (3.0 g) with
three aliquots of benzyl 6-bromohexanoate (2.45 g each) and
K.sub.2CO.sub.3 (1.19 g each) in one hour intervals in refluxing
acetonitrile. The solvent was removed in vacuo and the residue
partitioned between 200 mL of water and 2.times.150 mL of
CHCl.sub.3. The CHCl.sub.3 layers were combined, dried over
Na.sub.2SO.sub.4, filtered and the solvent removed in vacuo to give
an oil. This oil was purified on silica gel, eluting with a
gradient of methanol in chloroform. Fractions containing the
product (TLC R.sub.f0.50, CHCl.sub.3/methanol 9:1) were combined
and the solvent removed to give 3.82 g of N.sup.4-(benzyl
6'-hexanoyl)-N',N.sup.8- -bis(tert-butyloxycarbonyl)spermidine as
an oil.
[0299] N.sup.4-(benzyl
6'-hexanoyl)-N.sup.1,N.sup.8-bis(tert-butyloxycarbo- nyl)spermidine
(0.20 g) was dissolved in 10 mL of trifluoroacetic acid and stirred
at room temperature for one hour. The trifluoroacetic acid was
removed in vacuo, and the residue dissolved twice in chloroform (50
mL) and the solvent removed in vacuo. The oily residue was
lyophilized from 10 mL of 0.1 N HCl to give 126 mg of
N.sup.4-(benzyl 6'-hexanoyl)-spermidine trihydrochloride as an oil.
NMR (DMSO-d.sub.6) .delta. 1.37 (2H), 1.77 (9.4H), 2.05 (2H), 2.42
(1.8H), 2.9 (1.7H), 3.00 (4.4H), 3.20 (1.7H), 5.12 (1.7H), 7.37
(5H), 8.19 (5H), 10.77 (1H).
EXAMPLE 18
Preparation of N-(6'-hexanoyl)-spermidine-HA-2 peptide
[0300] N.sup.4-(6'-hexanoic
acid)-N.sup.1,N.sup.8-bis(tert-butyloxycarbony- l)spermidine (1.46
g) was treated with 0.37 g of N-hydroxysuccinimide and 0.66 g of
N,N'-dicyclohexylcarbodiimide in 100 mL of tetrahydrofuran for 20
h. The precipitate was removed by filtration and the solvent
removed in vacuo to give 1.99 g of N.sup.4-(succinimidyl
6'-hexanoyl)-N.sup.1,N.s-
up.8-bis(tert-butyloxycarbonyl)spermidine. The peptide
SGSGGLFEAIAENGWEGMIDGGG was prepared using an ABI Model 431A
peptide synthesizer, preloaded FMOC-amino acid cartridges and
pre-loaded FMOC-Gly-p-alkoxy-benzyl alcohol resin. The FMOC
protecting group was removed from the N-terminal serine and the
resin dried. The peptide-resin was treated with 1.99 g of
N.sup.4-(succinimidyl 6'-hexanoyl)-N.sup.1,N.s-
up.8-bis(tert-butyloxycarbonyl)spermidine and 1.24 mL of
diisopropylethylamine in 50 mL of dimethylformamide for 24 h. The
resin was isolated by filtration and rinsed with dichloromethane.
The N.sup.4-(6'-hexanoyl)-spermidine-SGSGGLFEAIAENGWEGMIDGGGOH was
cleaved from the resin using a mixture of water (0.25 mL),
ethanedithiol (0.25 mL), thioanisole (0.50 mL) and trifluoroacetic
acid (9.5 mL). The spent resin was removed by filtration and the
crude N.sup.4-(6'-hexanoyl)-sperm- idine-SGSGGLFEAIAENGWEGMIDGGGOH
precipitated by addition of diethyl ether and isolated by
filtration. A sample of the crude N.sup.4-(6'-hexanoyl)-s-
-permidine-SGSGGLFEAIAENGWEGMIDGGG-OH (100 mg) was purified by HPLC
on a 25.times.250 mm C-18 column eluting with a gradient of 0.05 M
ammonium bicarbonate and 0.05 M ammonium bicarbonate in 80%
acetonitrile. FAB mass spectra MH.sup.+=2775.
EXAMPLE 19
Preparation of
N.sup.4-(5'-N-(3".alpha.,7".alpha.,12".alpha.-trihydroxy-5"-
-.beta.-cholanamido)pentyl)-spermidine trihydrochloride
[0301] Cholic acid (95 mg) was dissolved in 10 mL of
tetrahydrofuran and N-hydroxysuccinimide (27 mg) and
dicyclohexylcarbodiimide (48 mg) added. The mixture was stirred to
dissolve and N.sup.4-(5'-aminopentyl)-N.sup.1,-
N.sup.8-bis(tert-butyloxycarbonyl)-spermidine (100 mg) added. The
mixture was stirred at room temperature 48 h. The solvent was
removed in vacuo. The crude wax was purified on silica gel, eluting
with a gradient of methanol in chloroform. Fractions containing the
product (TLC R.sub.f 0.39, CHCl.sub.3/methanol 85:15) were combined
and the solvent removed to give 0.15 g of
N.sup.4-(5'-(3".alpha.,7".alpha.,12.alpha.-trihydroxy-5".b-
eta.-cholanamido)pentyl)-N.sup.1,N.sup.8-bis(tert-butyloxycarbonyl)spermid-
ine as a white solid. This solid was dissolved in 10 mL of
2-propanol and to this solution was added 10 mL of 4 N HCl in
dioxane. The mixture was stirred for 18 h at room temperature,
during this time
N.sup.4-(5'-(3".alpha.,7".alpha.,12".alpha.-trihydroxy-5".beta.-cholanami-
do)pentyl)-spermidine trihydrochloride precipitated as a fine white
powder. This was isolated by filtration to give 67 mg. NMR
(DMSO-d.sub.6) .delta. 0.58 (s, 3H), 0.81 (s, 3H), 0.92 (m, 5.5H),
1.28 (m, 20.8H), 1.62 (m, 16.2H), 2.05 (m, 8.8H), 2.8 (q, 2.8H),
2.9 (q, 2.8H), 3.02 (q, 8.3H), 3.18 (m, 4.4H), 7.87 (m, 1.1H), 8.13
(br s, 4H), 8.23 (br s, 4H), 10.78 (br s, 1.1H).
EXAMPLE 20
Preparation of N.sup.4-(5-N-(.alpha.-3'-propionamido
thiomannoside)pentyl)-spermidine trihydrochloride
[0302] A solution of S-.alpha.-3'-propionyl
tetra-O-acetyl-thiomannoside (prepared similarly to
S-.beta.-3'-propionyl hepta-O-acetyl-galactosyl-.b-
eta.1-4-thioglucoside, M. Elofsson, S. Roy, B. Walse and J.
Kihlberg, Carb. Res., 246, 89-103 (1993)) (0.90 g) was prepared in
50 mL of CHCl.sub.3. To the solution was added 5 mL of 2-propanol,
0.43 g of dicyclohexylcarbodiimide and 0.24 g of
N-hydroxysuccinimide. The solution was stirred at room temperature
for 1 h, then stored at 40 overnight. A precipitate was filtered
off and solvent removed from the filtrate in vacuo to give an oil.
The oil was dissolved in 30 mL of tetrahydrofuran. To this solution
was added a solution of N.sup.4-(5'-aminopentyl)-N.sup.1-
,N.sup.8-bis(tert-butyloxycarbonyl)-spermidine (0.86 g) (in 50 mL
tetrahydrofuran and 30 mL of water) and 0.55 mL of
diisopropylethylamine. The solution was stirred at room temperature
for 2 h, the resultant solids were filtered off and the solvent
removed in vacuo to give an oil. The oil was suspended in 100 mL of
sat NaHCO.sub.3 and extracted with 2.times.75 mL of CHCl.sub.3. The
solution was dried over Na.sub.2SO.sub.4, filtered and the solvent
removed to give an oil. The oil was purified on silica gel, eluting
with a gradient of methanol in chloroform. Fractions containing the
product (TLC R.sub.f 0.30, CHCl.sub.3/methanol 9:1) were combined
and the solvent removed to give 0.10 g of
N.sup.4-(5'-N-(S-.alpha.-3'-propionamido
tetra-O-acetyl-thiomannoside)pentyl)-N.sup.1,N.sup.8-bis(tert-butyloxycar-
bonyl)spermidine as a glass. This glass was dissolved in 10 mL of
trifluoroacetic acid and the mixture was stirred for 1 h at room
temperature. Solvent was removed in vacuo and the residue
redissolved and solvent removed using chloroform (3.times.20 mL).
The solid was treated with 15% NH.sub.4OH in 55% ethanol for two
hours. The product was then lyophilized (3) from 0.05 N HCL to give
N.sup.4-(5-(.alpha.-3'-propionyl
thiomannoside)aminopentyl)-spermidine trihydrochloride as a white
solid (64 mg).
EXAMPLE 21
Preparation of N.sup.4-N-CBZ-5-aminopentyl)-spermidine
trihydrochloride
[0303]
N.sup.4-(N-CBZ-5'-aminopentyl)-N.sup.1,N.sup.8-bis(tert-butyloxycar-
bonyl)spermidine was prepared similarly to N.sup.4-(benzyl
6'-hexanoyl)-N.sup.1,N.sup.8-bis(tert-butyloxycarbonyl)spermidine
by treating N.sup.1,N.sup.8-bis(tert-butyloxycarbonyl)spermidine
(0.5 g) with four aliquots of N-CBZ-5-amino-1-bromopentane (1.74 g
each) and five aliquots of K.sub.2CO.sub.3 (1.00 g each). The
resultant oil was purified on silica gel, eluting with a gradient
of methanol in chloroform (TLC R.sub.f 0.47, CHCl.sub.3/methanol
9:1+0.4% diisopropylethylamine) to give 0.72 g of
N.sup.4-(N-CBZ-5'-aminopentyl)-N.sup.1,N.sup.8-bis(tert-butylox-
ycarbonyl)spermidine as an oil.
[0304]
N.sup.4-(N-CBZ-5'-aminopentyl)-N.sup.1,N.sup.8-bis(tert-butyloxycar-
bonyl)spermidine (0.24 g) was dissolved in 10 mL of trifluoroacetic
acid and stirred in an ice-water bath under nitrogen for 2 hours.
Cold ether was added to precipitate the product as an oil. The
solvents were removed by decantation and the oil dissolved in 25 mL
of 0.1 N HCL and lyophilized to give 0.16 g of
N.sup.4-(N-CBZ-5-aminopentyl)-spermidine trihydrochloride as a
glass. NMR (DMSO-d.sub.6) .delta. 1.28 (m, 2H), 1.47 (m, 2H), 1.61
(m, 8H), 2.01 (m, 2H), 2.82, 2.90 (m, 4H), 3.02 (m, 8H), 3.15 (m,
2.7H), 5.01 (s, 2H), 7.35 (s+m, 6.7H), 8.00, 8.11 (overlapping br
m, 7.3H).
EXAMPLE 22
Preparation of N.sup.4,N.sup.9-bis(N-CBZ-5-aminopentyl)-spermine
tetrahydrochloride
[0305] N.sup.1,N.sup.12-bis(tert-butyloxycarbonyl)spermine was
prepared similarly to
N.sup.1,N.sup.8-bis(tert-butyloxycarbonyl)spermidine using the same
sequence of reactions. N.sup.4,N.sup.9-bis(N-CBZ-5'-aminopentyl)-
-N.sup.1,N.sup.8-bis(tert-butyloxycarbonyl)spermine was prepared
similarly to
N.sup.4-(N-CBZ-5'-aminopentyl)-N.sup.1,N.sup.8-bis(tert-butyloxycarbon-
yl)spermidine using a single addition of
N-CBZ-5-amino-1-bromopentane (5.10 g) and K.sub.2CO.sub.3 (4.70 g)
to 3.42 g of N.sup.1,N.sup.12-bis(tert-butyloxycarbonyl)spermine in
refluxing acetonitrile (100 mL). Workup as before gave the product
as an oil which was purified on silica gel, eluting with a gradient
of methanol in chloroform containing diisopropylethylamine (0.2%).
Fractions containing the product (TLC R.sub.f 0.43,
CHCl.sub.3/methanol 9:1+0.4% diisopropylethylamine) were combined
and the solvent removed to give 6.0 g of
N.sup.4,N.sup.9-bis(N-CBZ-5'-aminopentyl)-N.sup.1,N.sup.12-bis(tert--
butyloxycarbonyl)spermine as an oil. An aliquot (0.25 g) of this
material was deprotected an converted to the hydrochloride as
described above for N.sup.4-(N-CBZ-5-aminopentyl)-spermidine
trihydrochloride to give 0.13 g of
N.sup.4,N.sup.9-bis(N-CBZ-5-aminopentyl)-spermine
tetrahydrochloride. NMR (DMSO-d.sub.6) .delta. 1.28 (m, 4H), 1.43
(m, 4H), 1.76 (m, 8.8H), 2.01 (m, 4H), 2.90, 3.00 (overlapping m,
18.4H), 3.17 (m, 4.4H), 5.03 (s, 4H), 7.35 (s, 12H), 8.11 (br m,
5.6H).
EXAMPLE 23
Preparation of N.sup.4,N.sup.9-bis(octyl)-spermine
tetrahydrochloride
[0306] N.sup.4,N.sup.9-bis
(octyl)-N.sup.1,N.sup.12-bis(tert-butyloxycarbo- nyl)spermine was
prepared using a single addition of 1-bromooctane (0.53 g),
potassium iodide (0.45 g) and diisopropylethylamine (0.54 mL) to
0.50 g of N',N.sup.12-bis(tert-butyloxycarbonyl)spermine in acetone
(30 mL) and refluxing overnight. The acetone was removed in vacuo
and the mixture taken up in 200 mL of chloroform and washed with
100 mL of water. The chloroform solution was dried over
Na.sub.2SO.sub.4, filtered and solvent removed in vacuo to give the
product as an oil. This oil was purified on silica gel, eluting
with a gradient of methanol in chloroform. Fractions containing the
product (TLC R.sub.f 0.54, CHCl.sub.3/methanol 9:1) were combined
and the solvent removed to give 0.26 g of
N.sup.4,N.sup.9-bis(octyl)-N.sup.1,N.sup.12-bis
(tert-butyloxycarbonyl)sp- ermine as a wax. This material (0.26 g)
was treated with 10 mL of 4 N HCl in dioxane for two hours at room
temperature. The solvent was removed in vacuo to give 0.21 g of
N.sup.4,N.sup.9-bis(octyl)-spermine tetrahydrochloride as a wax.
NMR (DMSO-d.sub.6) .delta. 0.94 (t, 6H), 1.35 (m, 22H), 1.74, 1.83
(overlapping m, 8.2H), 2.10 (m, 4H), 2.97, 3.12, 3.26 (overlapping
m, 1-8H), 8.27 (br m, 5.6H).
EXAMPLE 24
Preparation of
1,12-bis-N-guanidino-N.sup.4,N.sup.9-bis(octyl)-4,9-diaza-d-
odecane tetrahydrochloride
[0307] To 0.68 g of N.sup.4,N.sup.9-bis(octyl)-spermine
tetratrifluoroacetate in 50 mL of tetrahydrofuran and 1 mL of water
was added 0.77 g of
N,N'-bis(tert-butyloxycarbonyl)-S-methylisothiourea and 0.85 mL of
diisopropylethylamine. The solution was refluxed for 1 h then
stirred at room temperature for 18 h. The solvent was removed under
a stream of nitrogen at 60.degree. the resultant solid was
suspended in 100 mL of sat. NaHCO.sub.3 and extracted with
2.times.100 mL of chloroform. The chloroform solution was dried
over Na.sub.2SO.sub.4, filtered and solvent removed in vacuo to
give an oil. This oil was purified on silica gel, eluting with a
gradient of methanol in chloroform. Fractions containing the
product (TLC R.sub.f 0.39, CHCl.sub.3/methanol 9:1) were combined
and the solvent removed to give 0.18 g of 1,12-bis-N-(N',
N"-bis-(tert-butyloxycarbonyl-guanidino))-N.sup.4,N.sup.9-bis(octyl)-4,9--
diazadodecane as a wax. This wax was dissolved in 10 mL of
trifluoroacetic acid and stirred at room temperature for 1 hour.
The solvent was removed in vacuo and the residue dissolved in
chloroform and the solvent removed (2.times.20 mL) to give an oil.
This oil was dissolved in 30 mL of 0.1 N HCl and lyophilized to
give 1,12-bis-N-guanidino-N.sup.4,N.sup.9-bis(octy-
l)-4,9-diaza-dodecane tetrahydrochloride (86 mg) as oil. NMR
(MeOH-d.sub.4) .delta. 0.82 (m, 3H), 1.23, 1.30 (overlapping m,
10H), 1.64, 1.71 (overlapping m, 4H), 1.90 (m, 2H), 3.14 (m,
6H).
EXAMPLE 25
Preparation of N-(5-N-(cholestene-3'.beta.
carbamoyl)pentyl)-spermine tetrahydrochloride
[0308]
N.sup.4-(N-CBZ-5'-aminopentyl)-N.sup.1,N.sup.12-bis(tert-butyloxyca-
rbonyl)spermine was isolated as a minor component during the
preparation of
N.sup.4,N.sup.9-bis(N-CBZ-5'-aminopentyl)-N.sup.1,N.sup.12-bis
(tert-butyloxycarbonyl)spermine. Fractions containing the
N.sup.4-(N-CBZ-5'-aminopentyl)-N.sup.1,N.sup.12-bis(tert-butyloxycarbonyl-
)spermine (TLC R.sub.f 0.26, CHCl.sub.3/methanol 9:1+0.4%
diisopropylethylamine) were combined and the solvent removed to
give 0.84 g of an oil. To this oil was added 50 mL of acetonitrile,
0.47 mL of diisopropylethylamine and 0.35 g of di-tert-butyl
dicarbonate. The mixture was stirred at room temperature for 4 h.
The solvent was removed in vacuo and the residue taken up in 100 mL
of chloroform and washed with 100 mL of water. The chloroform
solution was dried over Na.sub.2SO.sub.4, filtered and the solvent
removed in vacuo to give 0.83 g of
N.sup.4-(N-CBZ-5'-aminopentyl)-N.sup.1,N.sup.9,N.sup.12-tris(tert-butylox-
-ycarbonyl)spermine an oil, single spot by TLC (R.sub.f 0.56,
CHCl.sub.3/methanol 9:1+0.4% diisopropylethylamine). The
N.sup.4-(N-CBZ-5'-aminopentyl)-N.sup.1,N.sup.9,N.sup.12-tris(tert-butylox-
y carbonyl)spermine (0.22 g) in 20 mL of methanol was treated with
0.04 g of 10% Pd/C and 50 PSIG H.sub.2 for 2 h at room temperature.
The Pd/C was removed by filtration through diatomaceous earth and
the solvent removed in vacuo to give
N.sup.4-(5'-aminopentyl)-N.sup.1,N.sup.9,N.sup.2-tris(te-
rt-butyloxycarbonyl)spermine as an oil (0.14 g). This oil was
dissolved in methylene chloride and to the solution was added
diisopropylethylamine (0.146 mL) and cholesterol chloroformate
(0.094 g). The solution was stirred at room temperature for 24 h.
The solvent was removed in vacuo and the resultant oil was purified
on silica gel, eluting with a gradient of methanol in chloroform
containing diisopropylethylamine. Fractions containing the product
(TLC R.sub.f 0.29, CHCl.sub.3/methanol 9:1+0.4%
diisopropylethylamine) were combined and the solvent removed to
give 0.19 g of
N.sup.4-(5-N-(cholestene-3'.beta.carbamoyl)pentyl)-N.sup.1,N.sup.9,N-
.sup.2-tris(tert-butyloxycarbonyl)spermine as an oil. This oil was
dissolved in 5 mL of trifluoroacetic acid and stirred under
nitrogen in an ice-water bath for 2 h. The solvent was removed in
vacuo and the residue dissolved in chloroform and the solvent
removed (2.times.20 mL) to give an oil. This oil was dissolved in
10 mL of 0.1 N HCl and lyophilized to give a light brown oil (0.077
g). NMR (DMSO-d.sub.6) .delta. 0.58 (s, 3H), 0.78 (two s, 6H), 0.90
(overlapping m, 18H), 1.43 (overlapping m, 15H), 1.64 (overlapping
m, 8H), 1.96 (m, 6H), 2.85 (overlapping m, 16H), 3.12 (m, 4H), 5.25
(m, 1H), 7.0 (br t, 1H), 8.06 (br m, 8H), 9.19 (br m, 2H).
EXAMPLE 26
Preparation of N.sup.4,N.sup.9-bis(N,N-dimethyl
12'-dodecanamide)-spermine tetrahydrochloride
[0309] To a suspension of 7.8 g of 12-bromododecanoic acid in 100
mL of water was added 14 mL of 2 M dimethylamine in tetrahydrofuran
resulting in a clear solution. The pH was adjusted to 7 with 1 N
HCl and 5.36 g of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide was
added. The solution was stirred at room temperature for 18 h. The
resultant N,N-dimethyl 12-bromododecanamide precipitated out of
solution as a white solid during this time. This solid was filtered
off and wash with water, then dried in vacuo.
N.sup.4,N.sup.9-bis(N,N-dimethyl 12'-dodecanamide)-N.sup.1,N.sup.1-
2-bis(tert-butyloxycarbonyl)spermine was prepared similarly to
N.sup.4-(N-CBZ-5'-aminopentyl)-N.sup.1,N.sup.8-bis
(tert-butyloxycarbonyl)spermidine using a single addition of
N,N-dimethyl 12-bromododecanamide (2.34 g) and K.sub.2CO.sub.3
(1.70 g) to 0.50 g of
N.sup.1,N.sup.12-bis(tert-butyloxycarbonyl)spermine in refluxing
acetonitrile (50 mL). Workup as before gave the product as an oil
which was purified on silica gel, eluting with a gradient of
methanol in chloroform containing isopropylethylamine (0.2%).
Fractions containing the product (TLC R.sub.f 0.53,
CHCl.sub.3/methanol 8:2+0.2% diisopropylethylamine) were combined
and the solvent removed to give 0.49 g of
N.sup.4,N.sup.9-bis(N,N-dimethyl
12'-dodecanamide)-N.sup.1,N.sup.12--
bis(tert-butyloxycarbonyl)spermine as an oil. An aliquot (0.20 g)
of this material was deprotected and converted to the hydrochloride
as described above for N.sup.4-(N-CBZ-5-aminopentyl)-spermidine
trihydrochloride to give 0.19 g of N.sup.4,N.sup.9-bis(N,N-dimethyl
12'-dodecanamide)-spermin- e tetrahydrochloride. NMR (DMSO-d.sub.6)
.delta. 1.26 (m, 25.6H), 1.46 (m, 3H), 1.70, 1.80 (overlapping m,
6H), 2.04 (m, 3H), 2.79 (s, 6H), 2.94 (s, 6H), 3.0-3.2 (overlapping
m, 18H), 8.15 (br m, 5H).
EXAMPLE 27
Preparation of N.sup.4,N.sup.9-bis(benzyl 12'-dodecanoyl)-spermine
tetrahydrochloride
[0310] To a solution of 12-bromododecanoic acid (1.0 g) and benzyl
alcohol (0.74 mL) in 200 mL of toluene was added p-toluenesulfonic
acid (0.10 g). Most (ca. 180 mL) of the toluene was removed by
simple distillation at atmospheric pressure, and the remaining
solvent removed in vacuo. The residue was taken up in ethyl acetate
(200 mL) and washed with sat. NaHCO.sub.3. The ethyl acetate
solution was dried over Na.sub.2SO.sub.4, filtered and the solvent
removed in vacuo to give crude benzyl 12-bromododecanoate (1.75 g)
as a crude liquid. TLC (chloroform/hexane 85:15) showed only
product (R.sub.f 0.78) and benzyl alcohol (R.sub.f 0.08).
N.sup.4,N.sup.9-bis(benzyl 12'-dodecanoyl)-N',N.sup.12-bis(tert-bu-
tyloxycarbonyl)spermine was prepared similarly to
N.sup.4-(N-CBZ-5'-aminop-
entyl)-N.sup.1,N.sup.8-bis(tert-butyloxycarbonyl)spermidine using a
single addition of crude benzyl 12-bromododecanoate (4.6 g) and
K.sub.2CO.sub.3 (1.70 g) to 1.0 g of
N.sup.1,N.sup.12-bis(tert-butyloxycarbonyl)spermine in refluxing
acetonitrile (100 mL). Workup as before gave the product as an oil
which was purified on silica gel, eluting with a gradient of
methanol in chloroform. Fractions containing the product (TLC
R.sub.f 0.35, CHCl.sub.3/methanol 9:1) were combined and the
solvent removed to give 0.36 g of N.sup.4,N.sup.9-bis(benzyl
12'-dodecanoyl)-N.sup.1,N.sup.2-
-bis(tert-butyloxycarbonyl)spermine as an oil. An aliquot (0.27 g)
of this material was deprotected and converted to the hydrochloride
as described above for N.sup.4-(N-CBZ-5-aminopentyl-spermine
trihydrochloride to give 0.17 g of N.sup.4,N.sup.9-bis (benzyl
12'-dodecanoyl)-spermine tetrahydrochloride. NMR (DMSO-d.sub.6)
.delta. 1.24 (m, 31.4H), 1.55 (m, 4.2H), 1.70, 1.80 (overlapping m,
8.6H), 2.02 (m, 4.2H), 2.9-3.2 (overlapping m, 21.4H), 5.08 (s,
4.2H), 7.36 (s, 10H) 8.13 (brm, 5.8H).
EXAMPLE 28
Preparation of N.sup.4,N.sup.9-bis(12'-dodecanoic acid)-spermine
tetrahydrochloride
[0311] N.sup.4,N.sup.9-bis(benzyl
12'-dodenanoyl)-N.sup.1,N.sup.12-bis(ter-
t-butyloxycarbonyl)spermine (0.51 g) was dissolved in 50 mL of
methanol and treated with 0.05 g of 10 Pd/C and 50 PSIG H.sub.2 for
3 h at room temperature. The Pd/C was removed by filtration through
diatomaceous earth. The solvent was removed from the filtrate in
vacuo to give N.sup.4,N.sup.9-bis(12'-dodecanoic
acid)-N.sup.1,N.sup.12-bis(tert-butylo- xycarbonyl)spermine as an
oil. An aliquot (0.22 g) of this material was deprotected and
converted to the hydrochloride as described above for
N.sup.4-(N-CBZ-5-aminopentyl)-spermidine trihydrochloride to give
0.17 g of N.sup.4,N.sup.9-bis(12'-dodecanoic acid)-spermine
tetrahydrochloride. NMR (DMSO-d.sub.6) .delta. 1.25 (m, 28H), 1.47
(m, 5H), 1.66, 1.76 (overlapping m, 8H), 2.02 (m, 3.6H), 2.91-3.17
(overlapping m, 19H), 8.11 (br m, 5H).
EXAMPLE 29
Preparation of N.sup.4-(benzyl 12'-dodenanoyl)-spermine
tetrahydrochloride
[0312] N.sup.4-(benzyl
12'-dodenanoyl)-N.sup.1,N.sup.12-bis(tert-butyloxyc-
-arbonyl)spermine was prepared similarly to
N.sup.4-(N-CBZ-5'-aminopentyl)-
-N.sup.1,N.sup.8-bis(tert-butyloxycarbonyl)spermidine using a
single addition of benzyl 12-bromododecanoate (6.58 g) to 1.43 g of
N.sup.1,N.sup.12-bis(tert-butyloxycarbonyl)spermine in refluxing
acetonitrile (100 mL). Workup as before gave the product as an oil
which was purified on silica gel, eluting with a gradient of
methanol in chloroform containing diisopropylethylamine (0.2%).
Fractions containing the product (TLC R.sub.f 0.08,
CHCl.sub.3/methanol 8:2+0.2% diisopropylethylamine) were combined
and the solvent removed to give 0.36 g of N.sup.4-(benzyl
12'-dodenanoyl)-N.sup.1,N.sup.12-bis(tert-butyloxyca-
rbonyl)spermine as an oil. An aliquot (0.29 g) of this material was
deprotected and converted to the hydrochloride as described above
for N.sup.4-(N-CBZ-5-aminopentyl)-spermidine trihydrochloride to
give 0.19 g of N.sup.4-(benzyl 12'-dodenanoyl)-spermine
tetrahydrochloride. NMR (DMSO-d.sub.6) .delta. 1.24 (m, 12H), 1.53
(m, 1.5H), 1.69, 1.80 (overlapping m, 4H), 2.01 (m, 4H), 2.9-3.2
(overlapping m, 14H), 5.08 (s, 2H), 7.36 (s, 5H) 7.47 (m, 1H), 8.09
(br m, 5.5H), 9.20 (br m, 2H).
EXAMPLE 30
Preparation of N.sup.4-(12'-dodecanoic acid)-spermine
tetrahydrochloride
[0313] N.sup.4-(benzyl
12'-dodenanoyl)-N.sup.1N.sup.12-bis(tert-butyloxyc--
arbonyl)spermine (0.29 g) was dissolved in 30 mL of methanol and
treated with 0.03 g of 10% Pd/C and 50 PSIG H.sub.2 for 1.5 h at
room temperature. The Pd/C was removed by filtration through
diatomaceous earth. The solvent was removed from the filtrate in
vacuo to give N.sup.4-(12'-dodecanoic
acid)-N.sup.1,N.sup.12-bis(tert-butyloxycarbonyl)- -spermine as an
oil (0.19 g). This material (0.19 g) was deprotected and converted
to the hydrochloride as described above for
N.sup.4-(N-CBZ-5-aminopentyl)-spermidine trihydrochloride to give
0.18 g of N.sup.4-(12'-dodecanoic acid)-spermine
tetrahydrochloride. NMR (DMSO-d.sub.6) .delta. 1.26 (m, 16.7H),
1.48 (m, 2.7H), 1.70, 1.78 (overlapping m, 6H), 2.01 (m, 4H),
2.91-3.17 (overlapping m, 12H), 8.17 (br m, 5.3H), 9.30 (br m,
2H).
EXAMPLE 31
Preparation of N.sup.4,N.sup.9-bis(5-(.alpha.-3'-propionyl
thiomannoside)aminopentyl)-spermine tetraacetate
[0314] N.sup.4,N.sup.9-bis
(N-CBZ-5'-aminopentyl)-N.sup.1,N.sup.12-bis(ter-
t-butyloxycarbonyl)spermine (2.04 g) was dissolved in 50 mL of
methanol and treated with 0.84 g of 10% Pd/C and 50 PSIG H.sub.2
for 2.5 h at room temperature. The Pd/C was removed by filtration
through diatomaceous earth and solvent removed from the filtrate in
vacuo to give
N.sup.4,N.sup.9-bis(5'-aminopentyl)-N.sup.1,N.sup.12-bis(tert-butyloxycar-
bonyl)spermine (0.79 g) as an oil. A solution of
S-.alpha.-3'-propionyl tetra-O-acetyl-thiomannoside (2.94 g) was
prepared in 100 mL of tetrahydrofuran. To the thiomannoside
solution was added 1.39 g of dicyclohexylcarbodiimide and 0.78 g of
N-hydroxysuccinimide. The solution was stirred at room temperature
for 20 h, then stored at 4.degree. for 0.5 h. A precipitate was
filtered off and solvent removed from the filtrate in vacuo to give
succinimidyl S-.alpha.-3'-propionyl tetra-O-acetyl-thiomannoside
(3.80 g) as a white solid. The
N.sup.4,N.sup.9-bis(5'-aminopentyl)-N.sup.1,N.sup.12-bis(tert-butyloxycar-
-bonyl)spermine (0.79 g) was dissolved in tetrahydrofuran (75 mL)
and to the solution was added 0.64 mL of diisopropylethylamine and
1.32 g of succinimidyl S-.alpha.-3'-propionyl
tetra-O-acetyl-thiomannoside, and the solution stirred at room
temperature for 24 h. The solvent was removed in vacuo to give a
glass. This glass was purified on silica gel, eluting with a
gradient of methanol in chloroform containing diisopropylethylamine
(0.2%). Fractions containing the product (TLC R.sub.f 0.30,
CHCl.sub.3/methanol 9:1) were combined and the solvent removed to
give 0.69 g of N.sup.4,N.sup.9-bis(5-(.alpha.-3'-propionyl
tetra-O-acetyl
thiomannoside)aminopentyl)-N.sup.1,N.sup.12-bis(tert-butyl-
-oxycarbonyl)spermine as a glass. This glass was dissolved in 20 mL
of trifluoroacetic acid and stirred at room temperature for 1 h.
The solvent was removed in vacuo and the residue dissolved in
chloroform and the solvent removed (2.times.20 mL) to give 0.78 g
of N.sup.4,N.sup.9-bis(5-N- -(.alpha.-3'-propionamido
tetra-O-acetyl thiomannoside)pentyl)-spermine as an oil. This oil
was dissolved in 25 mL of methanol and to the solution was added 25
mL of water and 1.56 g of Na.sub.2CO.sub.3, and the solution
stirred at room temperature for 5 h. The solvents were removed in
vacuo and the residue taken up in 6 mL of 1% acetic acid and
purified in three 2 mL aliquots on three Sephadex.TM. G-25 medium
columns (12 mL each), eluting with 1% acetic acid. Fractions
containing the product were combined and lyophilized to give 0.31 g
of N.sup.4,N.sup.9-bis(5-N-(.alph- a.-3'-propionamido
thiomannoside)pentyl)-spermine tetraacetate as a white solid. NMR
(D.sub.2O) .delta. 1.42 (m, 4H), 1.59 (m, 4H), 1.79 (m, 9.5H), 1.95
(s, 38.5H), 2.14 (m, 5H),2.62 (t, 4H), 2.94 (m, 4H), 3.10 (m,
4.5H), 3.24 (m, 18H), 3.72 (m, 6H), 4.05 (m, 6.5H), 5.34 (s,
2H).
EXAMPLE 32
Preparation of N.sup.4-(5-N-(23'-N-(.alpha.-3"-propionamido
thiomannoside)-3',6',9',12'-15'-18'-hexa-oxa-tricosanamido)aminopentyl)-s-
permine tetraacetate
[0315] A solution of pentaethylene glycol (5.0 g) in 50 mL of
tetrahydrofuran was added to a rapidly stirred suspension of NaH
(0.42 g of 60%) in 40 mL of tetrahydrofuran. The reaction mixture
was kept under nitrogen and suspended in an ice-water bath and
stirred for 0.5 h. A solution of N-CBZ-5-amino-1-bromopentane (3.78
g) in 30 mL of tetrahydrofuran was added and the mixture stirred in
the ice-water bath for 1 h, then at room temperature for 18 h. The
solvent was removed in vacuo and the residue was purified on silica
gel, eluting with a gradient of 2-propanol in chloroform. Fractions
containing the product (TLC R.sub.f 0.30, CHCl.sub.3/2-propanol
95:5) were combined and the solvent removed to give 3.25 g of
N-CBZ-20-amino-3,6,9,12,15-penta-oxa-1-eicosano- l as an oil. A
solution of N-CBZ-20-amino-3,6,9,12,15-penta-oxa-1-eicosano- l
(3.25 g) in 50 mL of tetrahydrofuran was added to a rapidly stirred
suspension of NaH (0.28 g of 60%) in 25 mL of tetrahydrofuran. The
reaction mixture was kept under nitrogen and suspended in an
ice-water bath and stirred for 0.5 h. To the solution was added
tert-butyl 1-bromoacetate (1.15 mL) and the mixture stirred in the
ice-water bath for 1 h, then at room temperature for four days. The
solvent was removed in vacuo and the residue suspended in 100 mL of
chloroform and washed with water (50 mL). The chloroform solution
was dried over Na.sub.2SO.sub.4, filtered and the solvent removed
in vacuo to give an oil. This oil was purified on silica gel,
eluting with a gradient of 2-propanol in chloroform. Fractions
containing the product (TLC R.sub.f 0.45, CHCl.sub.3/2-propanol
95:5) were combined and the solvent removed to give 3.24 g of
tert-butyl N-CBZ-23-amino-3,6,9,12,15,18-hexa-oxa-1-tri- cosanoate
as a colorless oil. An aliquot of the tert-butyl
N-CBZ-23-amino-3,6,9,12,15,18-hexa-oxa-1-tricosanoate (0.49 g) was
dissolved in 10 mL of 4 N HCl in dioxane and stirred at room
temperature for 3 h. The solvents were removed in vacuo. The
resultant crude
N-CBZ-23-amino-3,6,9,12,15,18-hexa-oxa-1-tricosanoic acid was
dissolved in 15 mL of dimethylformamide. To this solution was added
N-hydroxysuccinimide (0.10 g), 1-(3-dimethylaminopropyl)-3-ethyl
carbodiimide (0.16 g) and
N.sup.4-(5'-aminopentyl)-N.sup.1,N.sup.9,N.sup.-
12-tris(tert-butyloxycarbonyl)spermine (0.50 g). The solution was
stirred at room temperature for 2.5 days. The dimethylformamide was
removed in vacuo and the residue taken up in chloroform (100 mL).
The chloroform solution was washed sequentially with 0.1 N HCL (75
mL) and sat. NaHCO.sub.3. The chloroform solution was dried over
Na.sub.2SO.sub.4, filtered and the solvent removed in vacuo to give
an oil. This oil was purified on silica gel, eluting with a
gradient of 2-propanol in chloroform. Fractions containing the
product (TLC R.sub.f 0.33, CHCl.sub.3/methanol 9:1) were combined
and the solvent removed to give 0.19 g of
N.sup.4-(5-N-(N-CBZ-23'-amino-3',6',9', 12'-15'-18'-hexa-oxa-tr-
icosanamido)aminopentyl)-N.sup.1,N.sup.9,N.sup.12-tris(tert-butyloxycarbon-
y-l)spermine as a colorless oil. This oil was dissolved in 50 mL of
ethyl acetate and treated with 0.19 g of 10% Pd/C and 50 PSIG
H.sub.2 for 2.5 h at room temperature. The Pd/C was removed by
filtration through diatomaceous earth and solvent removed from the
filtrate in vacuo to give N.sup.4-(5-N-(23'-amino-3',6',9',
12'-15'-18'-hexa-oxa-tricosanamido)
aminopentyl)-N.sup.1,N.sup.9,N.sup.12-tris(tert-butyloxycarbonyl)spermin--
e as a colorless oil (0.13 g).
N.sup.4-(5-N-(23'-amino-3',6',9',12'-15'-18-
'-hexa-oxa-tricosanamido) aminopentyl)-N.sup.1,N.sup.9,N.sup.12
tris(tert-butyloxycarbonyl)spermine (0.13 g) was dissolved in
tetrahydrofuran (20 mL) and to the solution was added 0.048 mL of
diisopropylethylamine and 0.073 g of succinimidyl
S-.alpha.-3'-propionyl tetra-O-acetyl-thiomannoside, and the
solution stirred at room temperature for 20 h. The solvent was
removed in vacuo to give a glass. This glass was purified on silica
gel, eluting with a gradient of methanol in chloroform. Fractions
containing the product (TLC R.sub.f 0.36, CHCl.sub.3/methanol 9:1)
were combined and the solvent removed to give 0.10 g of
N.sup.4-(5-N-(23'-N-(S-.alpha.-3'-propionamido
tetra-O-acetyl-thiomannoside)-3',6',9',
12'-15'-18'-hexa-oxa-triconsanami-
do)aminopentyl)-N.sup.1,N.sup.9,N.sup.12-tris
(tert-butyloxycarbonyl)sperm- ine as an oil. FAB mass spectra,
MH.sup.+=3328. The N.sup.4-(5-N-(23'-N-(S-.alpha.-3'-propionamido
tetra-O-acetyl-thiomannosi- de)-3',6',9',
12'-15'-18'-hexa-oxa-tricosanamido) aminopentyl)-N.sup.1,N.s-
up.9,N.sup.12-tris(tert-butyloxycarbonyl)spermi-ne (0.55 g) was
dissolved in 10 mL of trifluoroacetic acid and stirred at room
temperature for 2 h. The solvent was removed in vacua and the
residue dissolved in chloroform and the solvent removed (2.times.20
mL) to give 0.76 g of N-(5-N-(23'-N-(S-.alpha.-3'-propionamido
tetra-O-acetyl-thiomannoside)-3'- ,6',9',
12'-15'-18'-hexa-oxa-tricosanamido-)aminopentyl)-spermine as an
oil. This oil was dissolved in 20 mL of methanol and to the
solution was added 20 mL of water and 0.85 g of Na.sub.2CO.sub.3,
and the solution stirred at room temperature for 6.5 h. The
solvents were removed in vacua and the residue taken up in 6 mL of
1% acetic acid and purified in four 1.5 mL aliquots on three
Sephadex.TM. G-25 medium columns (12 mL each), eluting with 1
acetic acid. Fractions containing the product were combined and
lyophilized to give 0.22 g of N.sup.4-(5-N-(23'-N-(S-.alpha.-
-3'-propionamido thiomannoside)-3',6',9',
12'-15'-18'-hexa-oxa-tricosanami- do)aminopentyl)-spermine
tetraacetate as an oil. NMR (D.sub.2O) .delta. 1.35 (m, 4H), 1.55
(m, 6H), 1.75 (m, 6.4H), 1.91 (s, 14H), 2.09 (m, 4.2H), 2.58 (t,
1.7H), 2.90 (m, 1.7H), 3.09, 3.19 (overlapping m, 18.1H), 3.53 (t,
2.5H), 3.69 (m, 22H), 3.88 (m, 3.8H), 4.06 (s, 1.7H), 5.30 (s,
1H).
EXAMPLE 33
Preparation of N-(5-N-(O-(5-N-(.alpha.-3"-propionamido
thiomannoside)pentyl)-O-(2-acetamido)nonadecaethylene
glycol)pentyl)-spermine tetraacetate
[0316] N.sup.4-(5-N-(O-(5-N-(.alpha.-3"-propionamido
thiomannoside)pentyl)-O-(2-acetamido)nonadecaethylene
glycol)pentyl)-spermine tetraacetate could be prepared similarly to
N.sup.4-(5-N-(23'-N-(S-.alpha.-3'-propionamido
thiomannoside)-3',6',9',
12'-15'-18'-hexa-oxa-tricosanamido)aminopentyl)-spermine
tetraacetate by substituting 18.9 g of poly(ethylene glycol) of
average molecular weight 900 for the pentaethyleneglycol in the
procedure described in Example 32.
EXAMPLE 34
Preparation of N.sup.4-(((23-[N.sup.2,N.sup.6-bis
(.beta.-3'-propionyl
galactosyl-.beta.1-4-thioglucoside)lysyl-N.sup.6-(.beta.-3'-propionyl
galactosyl-.beta.1-4-thioglucoside)lysyl]-amino)-3',6',9',12'-15'-18'-hex-
a-oxa-tricosanamido)aminopentyl)-spermine tetraacetate
[0317] N.sup.4-(((23-[N.sup.2,N.sup.6-bis(.beta.-3'-propionyl
galactosyl-.beta.1-4-thioglucoside)lysyl-N.sup.6-(.beta.1-3'-propionyl
galactosyl-.beta.1-4-thioglucoside)lysyl]-amino)-3',6',9',12'-15'-18'-hex-
-a-oxa-tricosanamido)aminopentyl)-spermine tetraacetate was
prepared similarly to
N.sup.4-(5-N-(23'-N-(S-.alpha.-3'-propionamido
thiomannoside)-3',6',9',12'-15'-18'-hexa-oxa-tricosanamido)aminopentyl)-s-
permine tetraacetate and the procedure described in Example 32.
N.sup.4-(5-N-(23'-amino-3',6',9',12'-15'-18'-hexa-oxa-tricosanamido)amino-
-pentyl)-N.sup.1,N.sup.9,N.sup.12-tris(tert-butyloxycarbonyl)spermine
(0.14 g) was dissolved in tetrahydrofuran (25 mL) and to the
solution was added 0.052 mL of diisopropylethylamine and 0.37 g of
succinimidyl N.sup.2,N.sup.6-bis(.beta.-3'-propionyl hepta-O-acetyl
galactosyl-.beta.1-4-thioglucoside)lysyl-N.sup.6-(.beta.-3'-propionyl
hepta-O-acetyl galactosyl-.beta.1-4-thioglucoside)lysinate, and the
solution stirred at room temperature for 72 h. The solvent was
removed in vacuo to give a glass. This glass was purified on silica
gel, eluting with a gradient of methanol in chloroform. Fractions
containing the product (TLC R.sub.f0.46, CHCl.sub.3/methanol 9:1)
were combined and the solvent removed to give 0.15 g of
N.sup.4-(((5-N-(23'-N-(N.sup.2N.sup.6-b- is(.beta.-3'-propionyl
hepta-O-acetyl galactosyl-.beta.1-4-thioglucoside)l-
ysyl-N.sup.6-(.beta.-3'-propionyl hepta-O-acetyl
galactosyl-.beta.1-4-thio-
glucoside)lysinamido)-3',6',9',12'-15'-18'-hexa-oxa-tricosanamido)aminopen-
tyl)-N.sup.1,N.sup.9,N.sup.12-tris (tert-butyloxycarbonyl)spermine
as a white solid. FAB mass spectra, MH.sup.+=3328. The
N.sup.4-(((5-N-(23'-N-(- N.sup.2,N.sup.6-bis(.beta.-3'-p-ropionyl
hepta-O-acetyl
galactosyl-.beta.1-4-thioglucoside)lysyl-N.sup.6-(.beta.-3'-propionyl
hepta-O-acetyl
galactosyl-.beta.1-4-thioglucoside)lysinamido)-3',6',9',12-
'-15'-18'-hexa-oxa-tricosanamido)
aminopentyl)-N.sup.1,N.sup.9,N.sup.12-tr-
is(tert-butyloxycarbonyl)spermine (0.28 g) was dissolved in 10 mL
of trifluoroacetic acid and stirred at room temperature for 1 h.
The solvent was removed in vacuo and the residue dissolved in
chloroform and the solvent removed (2.times.20 mL) to give 0.36 g
of 4-(((5-N-(23'-N-(N.sup.- 2,N.sup.6-bis(.beta.-3'-propionyl
hepta-O-acetyl galactosyl-.beta.1-4-thio-
glucoside)lysyl-N.sup.6-(.beta.-3'-propionyl hepta-O-acetyl
galactosyl-.beta.1-4-thioglucoside)lysinamido)-3',6',9',
12'-15'-18'-hexa-oxa-tricosanamido)aminopentyl)-spermine as an oil.
This oil was dissolved in 10 mL of methanol and to the solution was
added 10 mL of water and 0.44 g of Na.sub.2CO.sub.3, and the
solution stirred at room temperature for 24 h. The solvents were
removed in vacuo and the residue taken up in 5 mL of 1% acetic acid
and 1 mL of ethanol and purified in four 1.5 mL aliquots on two
Sephadex.TM. G-25 medium columns (15 mL and 20 mL), eluting with
10% ethanol in 1% acetic acid. Fractions containing the product
were combined and lyophilized to give 0.095 g of
N.sup.4-(((23-[N.sup.2,N.sup.6-bis(.beta.-3'-propionyl
galactosyl-.beta.1-4-thioglucoside)lysyl-N.sup.6-(.beta.-3'-propionyl
galactosyl-.beta.1-4-thioglucoside)lysyl]-amino)-3',6',9,12'-15'-18'-hexa-
-oxa-tricosanamido)aminopentyl)-spernine tetraacetate.
1 TABLE 1 Picornavirus Family Genera: Rhinoviruses: (Medical)
responsible for .about. 50% cases of the common cold.
Etheroviruses: (Medical) includes polioviruses, coxsackieviruses,
echoviruses, and human enteroviruses such as hepatitis A virus.
Apthoviruses: (Veterinary) these are the foot and mouth disease
viruses. Target antigens: VP1, VP2, VP3, VP4, VPG Calcivirus Family
Genera: Norwalk Group of Viruses: (Medical) these viruses are an
important causative agent of epidemic gastroenteritis. Togavirus
Family Genera: Alphaviruses: (Medical and Veterinary) examples
include Senilis viruses, RossRiver virus and Eastern & Western
Equine encephalitis. Reovirus: (Medical) Rubella virus.
Flariviridue Family Examples include: (Medical) dengue, yellow
fever, Japanese encephalitis, St. Louis encephalitis and tick borne
encephalitis viruses. Hepatitis C Virus: (Medical) these viruses
are not placed in a family yet but are believed to be either a
togavirus or a flavivirus. Most similarity is with togavirus
family. Coronavirus Family: (Medical and Veterinary) Infectious
bronchitis virus (poultry) Porcine transmissible gastroenteric
virus (pig) Porcine hemagglutinating encephalomyelitis virus (pig)
Feline infectious peritonitis virus (cats) Feline enteric
coronavirus (cat) Canine coronavirus (dog) The human respiratory
coronaviruses cause .about. 40 cases of common cold. EX. 224E, 0C43
Note - coronaviruses may cause non-A, B or C hepatitis Target
antigens: E1 - also called M or matrix protein E2 - also called S
or Spike protein E3 - also called HE or hemagglutin-elterose
glycoprotein (not present in all coronaviruses) N - nucleocapsid
Rhabdovirus Family Genera: Vesiliovirus Lyssavirus: (medical and
veterinary) rabies Target antigen: G protein N protein Filoviridue
Family: (Medical) Hemorrhagic fever viruses such as Marburg and
Ebola virus Paramyxovirus Family: Genera: Paramyxovirus: (Medical
and Veterinary) Mumps virus, New Castle disease virus (important
pathogen in chickens) Morbillivirus: (Medical and Veterinary)
Measles, canine distemper Pneuminvirus: (Medical and Veterinary)
Respiratory syncytial virus Orthomyxovirus Family (Medical) The
Influenza virus Bungavirus Family Genera: Bungavirus: (Medical)
California encephalitis, LA Crosse Phlebovirus: (Medical) Rift
Valley Fever Hantavirus: Puremala is a hemahagin fever virus
Nairvirus (Veterinary) Nairobi sheep disease Also many unassigned
bungaviruses Arenavirus Family (Medical) LCM, Lassa fever virus
Reovirus Family Genera: Reovirus: a possible human pathogen
Rotavirus: acute gastroenteritis in children Orbiviruses: (Medical
and Veterinary) Colorado Tick fever, Lebombo (humans) equine
encephalosis, blue Tongue Retrovirus Family Sub-Family:
Oncorivirinal: (Veterinary) (Medical) feline leukemia virus, HTLVI
and HTLVII Lentivirinal: (Medical and Veterinary) HIV, feline
immunodeficiency virus, equine infections, anemia virus
Spumavirinal Papovavirus Family Sub-Family: Polyomaviruses:
(Medical) BKU and JCU viruses Sub-Family: Papillomavirus: (Medical)
many viral types associated with cancers or malignant progression
of papilloma Adenovirus (Medical) EX AD7, ARD., O.B. - cause
respiratory disease - some adenoviruses such as 275 cause enteritis
Parvovirus Family (Veterinary) Feline parvovirus: causes feline
enteritis Feline panleucopeniavirus Canine parvovirus Porcine
parvovirus Herpesvirus Family Sub-Family: alphaherpesviridue
Genera: Simplexvirus (Medical) HSVI, HSVII Varicellovirus: (Medical
- Veterinary) pseudorabies - varicella zoster Sub-Family -
betaherpesviridue Genera: Cytomegalovirus (Medical) HCMV
Muromegalovirus Sub-Family: Gammaherpesviridue Genera:
Lymphocryptovirus (Medical) EBV - (Burkitts lympho) Rhadinovirus
Poxvirus Family Sub-Family: Chordopoxviridue (Medical - Veterinary)
Genera: Variola (Smallpox) Vaccinia (Cowpox) Parapoxivirus -
Veterinary Auipoxvirus - Veterinary Capripoxvirus Leporipoxvirus
Suipoxvirus Sub-Family: Entemopoxviridue Hepadnavirus Family
Hepatitis B virus Unclassified Hepatitis delta virus
[0318]
2TABLE 2 Bacterial pathogens Pathogenic gram-positive cocci
include: pneumococcal; staphy- lococcal; and streptococcal.
Pathogenic gram-negative cocci include: meningococcal; and
gonococcal. Pathogenic enteric gram-negative bacilli include:
enterobac- teriaceae; pseudomonas, acinetobacteria and eikenella;
melioidosis; salmonella; shigellosis; hemophilus; chancroid;
brucellosis; tularemia; yersinia (pasteurella); streptobacillus
moniliformis and spirillum; listeria monocytogenes; erysipelo-
thrix rhusiopathiae; diphtheria; cholera; anthrax; donovanosis
(granuloma inguinale); and bartonellosis. Pathogenic anaerobic
bacteria include: tetanus; botulism; other clostridia;
tuberculosis; leprosy; and other mycobacteria. Pathogenic
spirochetal diseases include: syphilis; treponema- toses: yaws,
pinta and endemic syphilis; and leptospirosis. Other infections
caused by higher pathogen bacteria and patho- genic fungi include:
actinomycosis; nocardiosis; cryptococcosis, blastomycosis,
histoplasmosis and coccidioidomycosis; candidiasis, aspergillosis,
and mucormycosis; sporotrichosis; paracoccidio- domycosis,
petriellidiosis, torulopsosis, mycetoma and chromo- mycosis; and
dermatophytosis. Rickettsial infections include rickettsial and
rickettsioses. Examples of mycoplasma and chlamydial infections
include: mycoplasma pneumoniae lymphogranuloma venereum;
psittacosis; and perinatal chlamydial infections. Pathogenic
eukaryotes Pathogenic protozoans and helminths and infections
thereby include: amebiasis; malaria; leishmaniasis;
trypanosomiasis; toxoplasmosis; pneumocystis carinii; babesiosis;
giardiasis; trichinosis; filariasis; schistosomiasis; nematodes;
trematodes or flukes; and cestode (tapeworm) infections.
[0319]
Sequence CWU 1
1
32 1 25 PRT Vesicular stomatitis virus 1 Lys Phe Thr Ile Val Phe
Pro His Asn Gln Lys Gly Asn Trp Lys Asn 1 5 10 15 Val Pro Ser Asn
Tyr His Tyr Cys Pro 20 25 2 32 PRT Human immunodeficiency virus
type 1 2 Ala Val Gly Ile Gly Ala Leu Phe Leu Gly Phe Leu Gly Ala
Ala Gly 1 5 10 15 Ser Thr Met Gly Ala Ala Ser Met Thr Leu Thr Val
Gln Ala Arg Gln 20 25 30 3 35 PRT Murine leukemia virus 3 Glu Pro
Val Ser Leu Thr Leu Ala Leu Leu Leu Gly Gly Leu Thr Met 1 5 10 15
Gly Gly Ile Ala Ala Gly Val Gly Thr Gly Thr Thr Ala Leu Val Ala 20
25 30 Thr Gln Gln 35 4 23 PRT Human immunodeficiency virus type 1 4
Ala Val Gly Ile Gly Ala Leu Phe Leu Gly Phe Leu Gly Ala Ala Gly 1 5
10 15 Ser Thr Met Gly Ala Arg Ser 20 5 17 PRT Human
immunodeficiency virus type 1 5 Ala Val Gly Ala Ile Gly Ala Leu Phe
Leu Gly Phe Leu Gly Ala Ala 1 5 10 15 Gly 6 22 PRT Influenza virus
6 Gly Leu Phe Glu Ala Ile Ala Glu Phe Ile Glu Gly Gly Trp Glu Gly 1
5 10 15 Leu Ile Glu Gly Cys Ala 20 7 25 PRT Influenza virus 7 Gly
Leu Phe Gly Ala Ile Ala Gly Phe Ile Glu Asn Gly Trp Glu Gly 1 5 10
15 Met Ile Asp Gly Trp Tyr Gly Phe Arg 20 25 8 23 PRT Human
immunodeficiency virus type 1 8 Ala Val Gly Ile Gly Ala Leu Phe Leu
Gly Phe Leu Gly Ala Ala Gly 1 5 10 15 Ser Thr Met Gly Ala Ala Ser
20 9 27 PRT SV5 virus 9 Phe Ala Gly Val Val Ile Gly Leu Ala Ala Leu
Gly Val Ala Thr Ala 1 5 10 15 Ala Asn Val Thr Ala Ala Val Ala Leu
Val Lys 20 25 10 19 PRT SFV virus 10 Lys Val Tyr Thr Gly Val Tyr
Pro Phe Met Trp Gly Gly Ala Tyr Cys 1 5 10 15 Phe Cys Asp 11 23 PRT
PH-30 virus 11 Lys Leu Ile Cys Thr Gly Ile Ser Ser Ile Pro Pro Ile
Arg Ala Leu 1 5 10 15 Phe Ala Ala Ile Asn Ile Pro 20 12 22 PRT
Sendai virus 12 Phe Phe Gly Ala Val Ile Gly Thr Ile Ala Leu Gly Val
Ala Thr Ala 1 5 10 15 Thr Ala Ala Gln Ile Thr 20 13 22 PRT SV5
virus 13 Phe Ala Gly Val Val Ile Gly Leu Ala Ala Leu Gly Val Ala
Thr Ala 1 5 10 15 Thr Ala Ala Gln Val Thr 20 14 22 PRT NDV virus 14
Phe Ile Gly Ala Ile Ile Gly Gly Val Ala Leu Gly Val Ala Thr Ala 1 5
10 15 Thr Ala Ala Gln Ile Thr 20 15 28 PRT Influenza virus 15 Gly
Leu Phe Gly Ala Ile Ala Gly Phe Ile Glu Asn Gly Trp Glu Gly 1 5 10
15 Met Ile Asp Gly Trp Tyr Gly Phe Arg His Gln Asn 20 25 16 28 PRT
Influenza virus 16 Gly Leu Phe Gly Ala Ile Ala Gly Phe Ile Glu Asn
Gly Trp Glu Gly 1 5 10 15 Leu Val Asp Gly Trp Tyr Gly Phe Arg His
Gln Asn 20 25 17 28 PRT Influenza virus 17 Gly Phe Phe Gly Ala Ile
Ala Gly Phe Leu Glu Gly Gly Trp Glu Gly 1 5 10 15 Met Ile Ala Gly
Trp His Gly Tyr Thr Ser His Gly 20 25 18 30 PRT MMTV virus 18 Phe
Val Ala Ala Ile Ile Leu Gly Ile Ser Ala Leu Ile Ala Ile Ile 1 5 10
15 Thr Ser Phe Ala Val Ala Thr Thr Ala Leu Val Lys Glu Met 20 25 30
19 53 PRT MoMLV virus 19 Glu Pro Val Ser Leu Thr Leu Ala Leu Leu
Leu Gly Gly Leu Thr Met 1 5 10 15 Gly Gly Ile Ala Ala Gly Ile Gly
Thr Gly Thr Thr Ala Leu Met Ala 20 25 30 Thr Gln Gln Phe Gln Gln
Leu Gln Ala Ala Val Gln Asp Asp Leu Arg 35 40 45 Glu Val Glu Lys
Ser 50 20 53 PRT F-MuLV virus 20 Glu Pro Val Ser Leu Thr Leu Ala
Leu Leu Leu Gly Gly Leu Thr Met 1 5 10 15 Gly Gly Ile Ala Ala Gly
Val Gly Thr Gly Thr Thr Ala Leu Val Ala 20 25 30 Thr Gln Gln Phe
Gln Gln Leu His Ala Ala Val Gln Asp Asp Leu Lys 35 40 45 Glu Val
Glu Lys Ser 50 21 53 PRT AKV virus 21 Glu Pro Val Ser Leu Thr Leu
Ala Leu Leu Leu Gly Gly Leu Thr Met 1 5 10 15 Gly Gly Ile Ala Ala
Gly Val Gly Thr Gly Thr Thr Ala Leu Val Ala 20 25 30 Thr Gln Gln
Phe Gln Gln Leu Gln Ala Ala Met His Asp Asp Leu Lys 35 40 45 Glu
Val Glu Lys Ser 50 22 27 PRT SFV virus 22 Asp Tyr Gln Cys Lys Val
Tyr Thr Gly Val Tyr Pro Phe Met Trp Gly 1 5 10 15 Gly Ala Tyr Cys
Phe Cys Asp Ser Glu Asn Thr 20 25 23 27 PRT Sindbis virus 23 Asp
Tyr Thr Cys Lys Val Phe Gly Gly Val Tyr Pro Phe Met Trp Gly 1 5 10
15 Gly Ala Gln Cys Phe Cys Asp Ser Glu Asn Ser 20 25 24 19 PRT
Measles virus 24 Phe Ala Gly Val Val Leu Ala Gly Ala Ala Leu Gly
Val Ala Ala Ala 1 5 10 15 Ala Gln Ile 25 19 PRT Measles virus 25
Phe Ala Gly Val Val Leu Ala Gly Ala Ala Leu Gly Val Ala Thr Ala 1 5
10 15 Ala Gln Ile 26 23 PRT Influenza virus 26 Gly Leu Phe Gly Ala
Ile Ala Gly Phe Ile Glu Asn Gly Trp Glu Gly 1 5 10 15 Met Ile Asp
Gly Gly Gly Cys 20 27 20 PRT Influenza virus 27 Gly Leu Phe Gly Ala
Ile Ala Gly Phe Ile Glu Asn Gly Trp Glu Gly 1 5 10 15 Met Ile Asp
Gly 20 28 20 PRT Influenza virus 28 Gly Ile Phe Gly Ala Ile Ala Gly
Phe Ile Glu Asn Gly Trp Glu Gly 1 5 10 15 Met Ile Asp Gly 20 29 20
PRT Influenza virus 29 Gly Leu Phe Gly Ala Ile Ala Gly Phe Ile Glu
Gly Gly Trp Thr Gly 1 5 10 15 Met Ile Asp Gly 20 30 20 PRT
Influenza virus 30 Gly Leu Phe Gly Ala Ile Ala Gly Phe Ile Glu Gly
Gly Trp Glu Gly 1 5 10 15 Met Val Asp Gly 20 31 20 PRT Influenza
virus 31 Gly Leu Phe Gly Ala Ile Ala Gly Phe Ile Glu Gly Gly Trp
Glu Gly 1 5 10 15 Leu Val Asp Gly 20 32 20 PRT Influenza virus 32
Gly Phe Phe Gly Ala Ile Ala Gly Phe Leu Glu Gly Gly Trp Glu Gly 1 5
10 15 Met Ile Ala Gly 20
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