Liposomal oligonucleotide compositions for modulating RAS gene expression

Pharmaceutical compositions comprising sterically stabilized liposomes containing antisense oligonucleotides are provided for the modulation of expression of the human ras gene in both the normal (wildtype) and activated (mutant) forms.

FIELD OF THE INVENTION 
This invention relates to pharmaceutical compositions comprising sterically 
stabilized liposomes containing one or more antisense oligonucleotides. 
The antisense oligonucleotides contained within the sterically stabilized 
liposomes are from about 8 to about 30 nucleotides in length, are targeted 
to a nucleic acid encoding a human wildtype or mutant ras sequence and are 
capable, individually and/or collectively, of modulating ras expression. 
In another embodiment, the sterically stabilized liposomes of the 
invention contain (a) one or more such antisense oligonucleotides and (b) 
one or more chemotherapeutic compounds which do not function by an 
antisense mechanism. 
BACKGROUND OF THE INVENTION 
Alterations in the cellular genes which directly or indirectly control 
cellular growth (proliferation) and differentiation are considered to be 
causative events leading to the development of tumors and cancers (see, 
generally, Weinberg, Sci. American 275:62, 1996). There are many families 
of genes presently implicated in human tumor formation. Members of one 
such family, the ras gene family, are frequently found to be mutated in 
human tumors. In their normal state, proteins produced by the ras genes 
are thought to be involved in normal cell growth and maturation. Mutation 
of the ras gene, causing an amino acid alteration at one of three critical 
positions in the protein product, results in conversion to a form which is 
implicated in tumor formation. A gene having such a mutation is said to be 
"activated." It is thought that such a point mutation leading to ras 
activation can be induced by carcinogens or other environmental factors. 
Over 90% of pancreatic adenocarcinomas, about 50% of adenomas and 
adenocarcinomas of the colon, about 50% of adenocarcinomas of the lung and 
carcinomas of the thyroid, and a large fraction of malignancies of the 
blood such as acute myeloid leukemia and myelodysplastic syndrome have 
been found to contain activated ras genes. Overall, some 10 to 20% of 
human tumors have a mutation in one of the three ras genes (H-ras, K-ras 
and N-ras). 
It is presently believed that inhibiting expression of activated 
cancer-associated genes in a particular tumor cell might force the cell 
back into a more normal growth habit. For example, Feramisco et al. 
(Nature, 314:639, 1985) demonstrated that cells transformed to a malignant 
state with an activated ras gene slow their rate of proliferation and 
adopt a more normal appearance when microinjected with an antibody which 
binds to the protein product of the ras gene. This has been interpreted as 
support for the involvement of the product of the activated ras gene in 
the uncontrolled growth typical of cancer cells. 
The H-ras gene has recently been implicated in a serious cardiac arrhythmia 
called long Q-T syndrome, a hereditary condition which often causes sudden 
death if treatment is not given immediately. Frequently there are no 
symptoms prior to the onset of the erratic heartbeat. Whether the H-ras 
gene is precisely responsible for long Q-T syndrome is unclear. However, 
there is an extremely high correlation between inheritance of this 
syndrome and the presence of a particular variant of the chromosome 11 
region surrounding the H-ras gene. Therefore, the H-ras gene is a useful 
indicator of increased risk of sudden cardiac death due to the long Q-T 
syndrome. 
There is a great desire to provide compositions of matter which can 
modulate the expression of the ras gene, and particularly to provide 
compositions of matter which specifically modulate the expression of the 
activated form of the ras gene. Inhibition of K-ras gene expression has 
been accomplished using retroviral vectors or plasmid vectors which 
express a 2-kilobase segment of the K-ras gene RNA in antisense 
orientation (Mukhopadhyay et al., Cancer Research 51:1744, 1991; PCT 
Patent Application PCT/US92/01852 (WO 92/15680). Georges et al., Cancer 
Research, 53:1743, 1993). 
Antisense oligonucleotide inhibition of expression has proven to be a 
useful tool in understanding the role(s) of various cancer-associated gene 
families. Antisense oligonucleotides are small oligonucleotides which are 
complementary to the "sense" (coding strand) of a given gene, and are thus 
also complementary to, and thus able to stably and specifically hybridize 
with, the mRNA transcript of the gene. Holt et al. (Mol. Cell Biol. 8:963, 
1988) state that antisense oligonucleotides designed to hybridize 
specifically with (i.e., "targeted to") mRNA transcripts of the c-myc gene 
inhibit proliferation and induce differentiation when added to cultured 
HL60 leukemic cells. Anfossi et al. (Proc. Natl Acad. Sci. 86:3379, 1989) 
state that antisense oligonucleotides targeted to the c-myb gene inhibit 
proliferation of human myeloid leukemia cell lines. Wickstrom et al. 
(Proc. Nat. Acad. Sci. 85:1028, 1988) state that expression of the protein 
product of the c-myc gene and proliferation of HL60 cultured leukemic 
cells are both inhibited by antisense oligonucleotides hybridizing 
specifically with c-myc mRNA. 
With specific regard to oligonucleotides having ras sequences, U.S. Pat. 
No. 4,871,838 to Bos et al. discloses oligonucleotides complementary to a 
mutation in codon 13 of N-ras to detect this mutation. Helene and 
co-workers have reported the selective inhibition of activated (codon 12 
G.fwdarw.T transition) H-ras mRNA expression using a 9-mer phosphodiester 
linked to an acridine intercalating agent and/or a hydrophobic tail; this 
compound displayed selective targeting of mutant ras message in both Rnase 
H and cell proliferation assays at low micromolar concentrations 
(Saison-Behmoaras et al., EMBO J. 10:1111, 1991). Chang et al. 
(Biochemistry 30:8283, 1991) disclose selective targeting of a mutant 
H-ras message, specifically, H-ras codon 61 containing an A.fwdarw.T 
transversion, with an 11-mer methylphosphonate oligonucleotide or its 
psoralen derivative. These compounds, which required concentrations of 
7.5-150 .mu.M for activity, were shown by immunoprecipitation to 
selectively inhibit mutant p21.sup.H-ras expression relative to wildtype 
p21.sup.H-ras. 
Although it has been recognized that antisense oligonucleotides have great 
therapeutic potential, there remains a long-felt need for pharmaceutical 
compositions and methods that could positively alter the in vivo 
stability, concentration, and distribution of such oligonucleotides. 
Enhanced biostability of antisense oligonucleotides in a mammal would 
generally be preferred for improved delivery of the oligonucleotide to its 
intended target tissue(s) with potentially less frequent dosing. For 
antisense oligonucleotides targeted to oncogenic molecules, enhanced 
distribution to tumor tissues would be preferred. 
OBJECTS OF THE INVENTION 
It is an object of the invention to provide sterically stabilized liposomes 
containing one or more antisense oligonucleotides and pharmaceutical 
compositions comprising such liposomes, wherein the antisense 
oligonucleotides contained within the sterically stabilized liposomes are 
from about 8 to about 30 nucleotides in length, are targeted to a nucleic 
acid encoding a human ras sequence and are capable, either individually or 
collectively, of modulating ras expression. 
It is another object of the invention to provide sterically stabilized 
liposomes containing one or more antisense oligonucleotides, and 
pharmaceutical compositions comprising such liposomes, wherein the 
antisense oligonucleotides contained within the sterically stabilized 
liposomes are from about 8 to about 30 nucleotides in length, are targeted 
to a nucleic acid encoding an activated (mutant) human ras sequence and 
are capable, either individually or collectively, of modulating the 
expression of the activated form of the ras gene. 
It is a further object of the invention to provide sterically stabilized 
liposomes containing (a) one or more antisense oligonucleotides being from 
about 8 to about 30 nucleotides in length, targeted to a nucleic acid 
encoding either a wildtype or mutant human ras sequence which are capable, 
either individually or collectively, of modulating ras expression and (b) 
one or more chemotherapeutic compounds which do not function by an 
antisense mechanism. 
An additional object of the invention is to provide liposome-based 
pharmaceutical compositions which inhibit the hyperproliferation of cells, 
including cancerous cells. Methods of inhibiting the hyperproliferation of 
cells, including cancerous cells, are also an object of this invention. 
A further object of this invention is to provide methods of treatment of, 
and liposome-based pharmaceutical compositions for, conditions arising due 
to mutation of the gene from the wildtype to a mutant, activated form of 
the ras gene. 
SUMMARY OF THE INVENTION 
In accordance with the present invention sterically stabilized liposomes 
containing one or more antisense oligonucleotides and pharmaceutical 
compositions comprising such liposomes are provided, wherein the antisense 
oligonucleotides contained within the sterically stabilized liposomes are 
from about 8 to about 30 nucleotides in length, are targeted to a nucleic 
acid encoding a human ras sequence and are capable, either individually or 
collectively, of modulating ras expression. 
Also provided are sterically stabilized liposomes containing one or more 
antisense oligonucleotides, and pharmaceutical compositions comprising 
such liposomes, wherein the antisense oligonucleotides contained within 
the sterically stabilized liposomes are from about 8 to about 30 
nucleotides in length, are targeted to a nucleic acid encoding an 
activated (mutant) human ras sequence and are capable, either individually 
or collectively, of modulating the expression of the activated form of the 
ras gene. 
Further provided are sterically stabilized liposomes containing (a) one or 
more antisense oligonucleotides being from about 8 to about 30 nucleotides 
in length, targeted to a nucleic acid encoding either a wildtype or mutant 
human ras sequence which are capable, either individually or collectively, 
of modulating ras expression and (b) one or more chemotherapeutic 
compounds which do not function by an antisense mechanism. 
Liposome-based pharmaceutical compositions which inhibit the 
hyperproliferation of cells, including cancerous cells, are provided. 
Methods of inhibiting the hyperproliferation of cells, including cancerous 
cells, are also provided. 
Methods of treatment of, and liposome-based pharmaceutical compositions 
for, conditions arising due to mutation of the gene from the wildtype to a 
mutant, activated form of the ras gene are also provided herein.

DETAILED DESCRIPTION OF THE INVENTION 
Malignant tumors develop through a series of stepwise, progressive changes 
that lead to the loss of growth control characteristic of cancer cells, 
i.e., continuous unregulated proliferation, the ability to invade 
surrounding tissues, and the ability to metastasize to different organ 
sites. Carefully controlled in vitro studies have helped define the 
factors that characterize the growth of normal and neoplastic cells and 
have led to the identification of specific proteins that control cell 
growth and differentiation. In addition, the ability to study cell 
transformation in carefully controlled, quantitative in vitro assays has 
led to the identification of specific genes capable of inducing the 
transformed cell phenotype. Such cancer-associated genes are believed to 
acquire transformation-inducing properties through mutations leading to 
changes in the regulation of expression of their protein products. In some 
cases such changes occur in non-coding DNA regulatory domains, such as 
promoters and enhancers, leading to alterations in the transcriptional 
activity of cancer associated genes, resulting in over- or 
under-expression of their gene products. In other cases, gene mutations 
occur within the coding regions of cancer associated genes, leading to the 
production of altered gene products that are inactive, overactive, or 
exhibit an activity that is different from the normal (wild-type) gene 
product. 
Many cellular cancer associated gene families have been identified and 
categorized on the basis of their subcellular location and the putative 
mechanism of action of their protein products. The ras genes are members 
of a gene family which encode related proteins that are localized to the 
inner face of the plasma membrane. Ras proteins have been shown to be 
highly conserved at the amino acid level, to bind GTP with high affinity 
and specificity, and to possess GTPase activity (for a review, see 
Downward, Trends Biochem. Sci. 15:469, 1990). Although their cellular 
function(s) is(are) unknown, the biochemical properties of the ras 
proteins, along with their significant sequence homology with a class of 
signal-transducing proteins known as GTP binding proteins, or G proteins, 
suggest that ras gene products play a fundamental role in basic cellular 
regulatory functions relating to the transduction of extracellular signals 
across plasma membranes. The ras gene product, p21.sup.ras, interacts with 
a variety of known and proposed cellular effectors (for a review, see 
Marshall, Trends Biochem. Sci. 18:250, 1993) 
Three ras genes, designated H-ras, K-ras, and N-ras, have been identified 
in the mammalian genome. Mammalian ras genes acquire 
transformation-inducing properties by single point mutations within their 
coding sequences. Mutations in naturally occurring ras genes have been 
localized to codons 12, 13, and 61. The sequences of H-ras, K-ras and 
N-ras are known (Capon et al., Nature 302:33, 1983; Kahn et al., 
Anticancer Res. 7:639, 1987; Hall and Brown, Nucleic Acids Res. 13:5255, 
1985). The most commonly detected activating ras mutation found in human 
tumors is in codon 12 of the H-ras gene in which a base change from GGC to 
GTC results in a glycine-to-valine substitution in the GTPase regulatory 
domain of the ras protein product (Tabin et al., Nature, 300:143, 1982; 
Reddy et al., Nature 300:149, 1982; Taparowsky et al., Nature 300:762, 
1982). This single amino acid change is thought to abolish normal control 
and/or function of p21.sup.H-ras, thereby converting a normally regulated 
cell protein to one that is continuously active. It is believed that such 
deregulation of normal ras protein function is responsible for the 
transformation from normal to malignant growth. 
The present invention provides pharmaceutical compositions comprising 
sterically stabilized liposomes containing one or more antisense 
oligonucleotides, wherein the antisense oligonucleotides contained within 
the sterically stabilized liposomes are from about 8 to about 30 
nucleotides in length, more preferably from about 8 to about 30 
nucleotides in length, are targeted to a nucleic acid encoding a human 
wildtype or mutant ras sequence and are capable, individually and/or 
collectively, of modulating ras expression. In another embodiment, the 
sterically stabilized liposomes of the invention contain (a) one or more 
such antisense oligonucleotides and (b) one or more chemotherapeutic 
compounds which do not function by an antisense mechanism. The remainder 
of the Detailed Description relates in more detail to (1) the 
oligonucleotides of the invention, (2) their bioequivalents, (3) 
sterically stabilized liposomes, (4) chemotherapeutic agents that can be 
combined with antisense oligonucleotides targeted to H-ras in the context 
of the liposomes of the invention and (5) administration of pharmaceutical 
compositions comprising the liposomal oligonucleotide compositions of the 
invention. 
1. Oligonucleotides: In the context of this invention, the term 
"oligonucleotide" refers to an oligomer or polymer of ribonucleic acid or 
deoxyribonucleic acid. This term includes oligonucleotides composed of 
naturally-occurring nucleobases, sugars and covalent intersugar (backbone) 
linkages as well as oligonucleotides having non-naturally-occurring 
portions which function similarly. Such modified or substituted 
oligonucleotides are often preferred over native forms because of 
desirable properties such as, for example, enhanced cellular uptake, 
enhanced binding to target and increased stability in the presence of 
nucleases. 
An oligonucleotide is a polymer of a repeating unit generically known as a 
nucleotide. The oligonucleotides in accordance with this invention 
preferably comprise from about 8 to about 30 nucleotides. An unmodified 
(naturally occurring) nucleotide has three components: (1) a 
nitrogen-containing heterocyclic base linked by one of its nitrogen atoms 
to (2) a 5-pentofuranosyl sugar and (3) a phosphate esterified to one of 
the 5' or 3' carbon atoms of the sugar. When incorporated into an 
oligonucleotide chain, the phosphate of a first nucleotide is also 
esterified to an adjacent sugar of a second, adjacent nucleotide via a 
3'-5' phosphate linkage. The "backbone" of an unmodified oligonucleotide 
consists of (2) and (3), that is, sugars linked together by phosphodiester 
linkages between the 5' carbon of the sugar of a first nucleotide and the 
3' carbon of a second, adjacent nucleotide. A "nucleoside" is the 
combination of (1) a nucleobase and (2) a sugar in the absence of (3) a 
phosphate moiety (Kornberg, A., DNA Replication, W. H. Freeman & Co., San 
Francisco, 1980, pages 4-7). The backbone of an oligonucleotide positions 
a series of bases in a specific order; the written representation of this 
series of bases, which is conventionally written in 5' to 3' order, is 
known as a nucleotide sequence. 
The oligonucleotides used in accordance with this invention may be 
conveniently and routinely made through the well-known technique of solid 
phase synthesis. Equipment for such synthesis is sold by several vendors 
including Applied Biosystems (Foster City, Calif.). Any other means for 
such synthesis may also be employed, however, the actual synthesis of the 
oligonucleotides are well within the talents of the routineer. It is also 
well known to use similar techniques to prepare other oligonucleotides 
such as the phosphorothioates and alkylated derivatives. 
Oligonucleotides may comprise nucleotide sequences sufficient in identity 
and number to effect specific hybridization with a particular nucleic 
acid. Such oligonucleotides which specifically hybridize to a portion of 
the sense strand of a gene are commonly described as "antisense." In the 
context of the invention, "hybridization" means hydrogen bonding, which 
may be Watson-Crick, Hoogsteen or reversed Hoogsteen hydrogen bonding, 
between complementary nucleotides. For example, adenine and thymine are 
complementary nucleobases which pair through the formation of hydrogen 
bonds. "Complementary," as used herein, refers to the capacity for precise 
pairing between two nucleotides. For example, if a nucleotide at a certain 
position of an oligonucleotide is capable of hydrogen bonding with a 
nucleotide at the same position of a DNA or RNA molecule, then the 
oligonucleotide and the DNA or RNA are considered to be complementary to 
each other at that position. The oligonucleotide and the DNA or RNA are 
complementary to each other when a sufficient number of corresponding 
positions in each molecule are occupied by nucleotides which can hydrogen 
bond with each other. Thus, "specifically hybridizable" and 
"complementary" are terms which are used to indicate a sufficient degree 
of complementarity or precise pairing such that stable and specific 
binding occurs between the oligonucleotide and the DNA or RNA target. An 
oligonucleotide is specifically hybridizable to its target sequence due to 
the formation of base pairs between specific partner nucleobases in the 
interior of a nucleic acid duplex. Among the naturally occurring 
nucleobases, guanine (G) binds to cytosine (C), and adenine (A) binds to 
thymine (T) or uracil (U). In addition to the equivalency of U (RNA) and T 
(DNA) as partners for A, other naturally occurring nucleobase equivalents 
are known, including 5-methylcytosine, 5-hydroxymethylcytosine (HMC), 
glycosyl HMC and gentiobiosyl HMC (C equivalents), and 
5-hydroxymethyluracil (U equivalent). Furthermore, synthetic nucleobases 
which retain partner specificity are known in the art and include, for 
example, 7-deaza-Guanine, which retains partner specificity for C. Thus, 
an oligonucleotide's capacity to specifically hybridize with its target 
sequence will not be altered by any chemical modification to a nucleobase 
in the nucleotide sequence of the oligonucleotide which does not 
significantly effect its specificity for the partner nucleobase in the 
target oligonucleotide. It is understood in the art that an 
oligonucleotide need not be 100% complementary to its target DNA sequence 
to be specifically hybridizable. An oligonucleotide is specifically 
hybridizable when there is a sufficient degree of complementarity to avoid 
non-specific binding of the oligonucleotide to non-target sequences under 
conditions in which specific binding is desired, i.e., under physiological 
conditions in the case of in vivo assays or therapeutic treatment, or in 
the case of in vitro assays, under conditions in which the assays are 
performed. The nucleotide sequences of the oligonucleotides of the 
invention are given in Example 1 and also in the Sequence Listing. 
Citations for target H-ras sequences are also presented in Example 1. 
Antisense oligonucleotides are commonly used as research reagents, 
diagnostic aids, and therapeutic agents. For example, antisense 
oligonucleotides, which are able to inhibit gene expression with exquisite 
specificity, are often used by those of ordinary skill to elucidate the 
function of particular genes, for example to distinguish between the 
functions of various members of a biological pathway. This specific 
inhibitory effect has, therefore, been harnessed by those skilled in the 
art for research uses. The specificity and sensitivity of oligonucleotides 
is also harnessed by those of skill in the art for therapeutic uses. 
A. Modified Linkages: Specific examples of some preferred modified 
oligonucleotides envisioned for this invention include those containing 
phosphorothioates, phosphotriesters, methyl phosphonates, short chain 
alkyl or cycloalkyl intersugar linkages or short chain heteroatomic or 
heterocyclic intersugar linkages. Most preferred are oligonucleotides with 
phosphorothioates and those with CH.sub.2 --NH--O--CH.sub.2, CH.sub.2 
--N(CH.sub.3)--O--CH.sub.2 [known as a methylene(methylimino) or MMI 
backbone], CH.sub.2 --O--N(CH.sub.3)--CH.sub.2, CH.sub.2 
--N(CH.sub.3)--N(CH.sub.3)--CH.sub.2 and O--N(CH.sub.3)--CH.sub.2 
--CH.sub.2 backbones, wherein the native phosphodiester backbone is 
represented as O--P--O--CH.sub.2). Also preferred are oligonucleotides 
having morpholino backbone structures (Summerton and Weller, U.S. Pat. No. 
5,034,506). Further preferred are oligonucleotides with NR--C(*)--CH.sub.2 
--CH.sub.2, CH.sub.2 --NR--C(*)--CH.sub.2, CH.sub.2 --CH.sub.2 --NR--C(*), 
C(*)--NR--CH.sub.2 --CH.sub.2 and CH.sub.2 --C(*)--NR--CH.sub.2 backbones, 
wherein "*" represents O or S (known as amide backbones; DeMesmaeker et 
al., WO 92/20823, published Nov. 26, 1992). In other preferred 
embodiments, such as the peptide nucleic acid (PNA) backbone, the 
phosphodiester backbone of the oligonucleotide is replaced with a 
polyamide backbone, the nucleobases being bound directly or indirectly to 
the aza nitrogen atoms of the polyamide backbone (Nielsen et al., Science, 
1991, 254:1497; U.S. Pat. No. 5,539,082). 
B. Modified Nucleobases: The oligonucleotides of the invention may 
additionally or alternatively include nucleobase modifications or 
substitutions. As used herein, "unmodified" or "natural" nucleobases 
include adenine (A), guanine (G), thymine (T), cytosine (C) and uracil 
(U). Modified nucleobases include nucleobases found only infrequently or 
transiently in natural nucleic acids, e.g., hypoxanthine, 6-methyladenine, 
5-methylcytosine, 5-hydroxymethylcytosine (HMC), glycosyl HMC and 
gentiobiosyl HMC, as well synthetic nucleobases, e.g., 2-aminoadenine, 
2-thiouracil, 2-thiothymine, 5-bromouracil, 5-hydroxymethyluracil, 
8-azaguanine, 7-deazaguanine, N.sup.6 (6-aminohexyl)adenine and 
2,6-diaminopurine (Kornberg, A., DNA Replication, W. H. Freeman & Co., San 
Francisco, 1980, pages 75-77; Gebeyehu, G., et al., Nucleic Acids Res., 
1987, 15, 4513). 
C. Sugar Modifications: The oligonucleotides of the invention may 
additionally or alternatively comprise substitutions of the sugar portion 
of the individual nucleotides. For example, oligonucleotides may also have 
sugar mimetics such as cyclobutyls in place of the pentofuranosyl group. 
Other preferred modified oligonucleotides may contain one or more 
substituted sugar moieties comprising one of the following at the 2' 
position: OH, SH, SCH.sub.3, F, OCN, OCH.sub.3 OCH.sub.3, OCH.sub.3 
O(CH.sub.2).sub.n CH.sub.3, O(CH.sub.2).sub.n NH.sub.2 or 
O(CH.sub.2).sub.n CH.sub.3 where n is from 1 to about 10; C.sub.1 to 
C.sub.10 lower alkyl, alkoxyalkoxy, substituted lower alkyl, alkaryl or 
aralkyl; Cl; Br; CN; CF.sub.3 ; OCF.sub.3 ; O-, S-, or N-alkyl; O-, S-, or 
N-alkenyl; SOCH.sub.3 ; SO.sub.2 CH.sub.3 ; ONO.sub.2 ; NO.sub.2 ; N.sub.3 
; NH.sub.2 ; heterocycloalkyl; heterocycloalkaryl; aminoalkylamino; 
polyalkylamino; substituted silyl; an RNA cleaving group; a reporter 
group; an intercalator; a group for improving the pharmacokinetic 
properties of an oligonucleotide; or a group for improving the 
pharmacodynamic properties of an oligonucleotide and other substituents 
having similar properties. A preferred modification includes 
2'-methoxyethoxy [2'-O--CH.sub.2 CH.sub.2 OCH.sub.3, also known as 
2'-O--(2-methoxyethyl)] (Martin et al., Helv. Chim. Acta, 1995, 78:486). 
Other preferred modifications include 2'-methoxy-(2'-O--CH.sub.3), 
2'-propoxy-(2'-OCH.sub.2 CH.sub.2 CH.sub.3) and 2'-fluoro-(2'--F). 
D. Other Modifications: Similar modifications may also be made at other 
positions on the oligonucleotide, particularly the 3' position of the 
sugar on the 3' terminal nucleotide and the 5' position of 5' terminal 
nucleotide. The 5' and 3' termini of an oligonucleotide may also be 
modified to serve as points of chemical conjugation of, e.g., lipophilic 
moieties (see immediately subsequent paragraph), intercalating agents 
(Kuyavin et al., WO 96/32496, published Oct. 17, 1996; Nguyen et al., U.S. 
Pat. No. 4,835,263, issued May 30, 1989) or hydroxyalkyl groups (Helene et 
al., WO 96/34008, published Oct. 31, 1996). 
Other positions within an oligonucleotide of the invention can be used to 
chemically link thereto one or more effector groups to form an 
oligonucleotide conjugate. An "effector group" is a chemical moiety that 
is capable of carrying out a particular chemical or biological function. 
Examples of such effector groups include, but are not limited to, an RNA 
cleaving group, a reporter group, an intercalator, a group for improving 
the pharmacokinetic properties of an oligonucleotide, or a group for 
improving the pharmacodynamic properties of an oligonucleotide and other 
substituents having similar properties. A variety of chemical linkers may 
be used to conjugate an effector group to an oligonucleotide of the 
invention. As an example, U.S. Pat. No. 5,578,718 to Cook et al. discloses 
methods of attaching an alkylthio linker, which may be further derivatized 
to include additional groups, to ribofuranosyl positions, nucleosidic base 
positions, or on internucleoside linkages. Additional methods of 
conjugating oligonucleotides to various effector groups are known in the 
art; see, e.g., Protocols for Oligonucleotide Conjugates (Methods in 
Molecular Biology, Volume 26) Agrawal, S., ed., Humana Press, Totowa, 
N.J., 1994. 
Another preferred additional or alternative modification of the 
oligonucleotides of the invention involves chemically linking to the 
oligonucleotide one or more lipophilic moieties which enhance the cellular 
uptake of the oligonucleotide. Such lipophilic moieties may be linked to 
an oligonucleotide at several different positions on the oligonucleotide. 
Some preferred positions include the 3' position of the sugar of the 3' 
terminal nucleotide, the 5' position of the sugar of the 5' terminal 
nucleotide, and the 2' position of the sugar of any nucleotide. The 
N.sup.6 position of a purine nucleobase may also be utilized to link a 
lipophilic moiety to an oligonucleotide of the invention (Gebeyehu, G., et 
al., Nucleic Acids Res., 1987, 15:4513). Such lipophilic moieties include 
but are not limited to a cholesteryl moiety (Letsinger et al., Proc. Natl. 
Acad. Sci. U.S.A., 1989, 86:6553), cholic acid (Manoharan et al., Bioorg. 
Med. Chem. Let., 1994, 4:1053), a thioether, e.g., hexyl-S-tritylthiol 
(Manoharan et al., Ann. N.Y. Acad. Sci., 1992, 660:306; Manoharan et al., 
Bioorg. Med. Chem. Let., 1993, 3:2765), a thiocholesterol (Oberhauser et 
al., Nucl. Acids Res., 1992, 20:533), an aliphatic chain, e.g., 
dodecandiol or undecyl residues (Saison-Behmoaras et al., EMBO J., 1991, 
10:111; Kabanov et al., FEBS Lett., 1990, 259:327; Svinarchuk et al., 
Biochimie, 1993, 75:49), a phospholipid, e.g., di-hexadecyl-rac-glycerol 
or triethylammonium 1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate 
(Manoharan et al., Tetrahedron Lett., 1995, 36:3651; Shea et al., Nucl. 
Acids Res., 1990, 18:3777), a polyamine or a polyethylene glycol chain 
(Manoharan et al., Nucleosides & Nucleotides, 1995, 14:969), or adamantane 
acetic acid (Manoharan et al., Tetrahedron Lett., 1995, 36:3651), a 
palmityl moiety (Mishra et al., Biochim. Biophys. Acta, 1995, 1264:229), 
or an octadecylamine or hexylaminocarbonyl-oxycholesterol moiety (Crooke 
et al., J. Pharmacol. Exp. Ther., 1996, 277:923). Oligonucleotides 
comprising lipophilic moieties, and methods for preparing such 
oligonucleotides, are disclosed in co-owned U.S. Pat. Nos. 5,138,045, 
5,218,105 and 5,459,255. 
The present invention also includes oligonucleotides that are substantially 
chirally pure with regard to particular positions within the 
oligonucleotides. Examples of substantially chirally pure oligonucleotides 
include, but are not limited to, those having phosphorothioate linkages 
that are at least 75% Sp or Rp (see co-owned U.S. Pat. No. 5,587,361 to 
Cook et al.) and those having substantially chirally pure (Sp or Rp) 
alkylphosphonate, phosphoamidate or phosphotriester linkages (see co-owned 
U.S. Pat. Nos. 5,212,295 and 5,521,302). 
E. Chimeric Oligonucleotides: The present invention also includes 
oligonucleotides which are chimeric oligonucleotides. "Chimeric" 
oligonucleotides or "chimeras," in the context of this invention, are 
oligonucleotides which contain two or more chemically distinct regions, 
each made up of at least one nucleotide. These oligonucleotides typically 
contain at least one region wherein the oligonucleotide is modified so as 
to confer upon the oligonucleotide increased resistance to nuclease 
degradation, increased cellular uptake, and/or increased binding affinity 
for the target nucleic acid. An additional region of the oligonucleotide 
may serve as a substrate for enzymes capable of cleaving RNA:DNA or 
RNA:RNA hybrids. By way of example, RNase H is a cellular endonuclease 
which cleaves the RNA strand of an RNA:DNA duplex. Activation of RNase H, 
therefore, results in cleavage of the RNA target, thereby greatly 
enhancing the efficiency of antisense inhibition of gene expression. 
Cleavage of the RNA target can be routinely detected by gel 
electrophoresis and, if necessary, associated nucleic acid hybridization 
techniques known in the art. By way of example, such "chimeras" may be 
"gapmers," i.e., oligonucleotides in which a central portion (the "gap") 
of the oligonucleotide serves as a substrate for, e.g., RNase H, and the 
5' and 3' portions (the "wings") are modified in such a fashion so as to 
have greater affinity for the target RNA molecule but are unable to 
support nuclease activity (e.g., 2'-fluoro- or 
2'-methoxyethoxy-substituted). Other chimeras include "wingmers," that is, 
oligonucleotides in which the 5' portion of the oligonucleotide serves as 
a substrate for, e.g., RNase H, whereas the 3' portion is modified in such 
a fashion so as to have greater affinity for the target RNA molecule but 
is unable to support nuclease activity (e.g., 2'-fluoro- or 
2'-methoxyethoxy-substituted), or vice-versa. 
F. Synthesis: The oligonucleotides used in accordance with this invention 
may be conveniently and routinely made through the well-known technique of 
solid phase synthesis. Equipment for such synthesis is sold by several 
vendors including, for example, Applied Biosystems (Foster City, Calif.). 
Any other means for such synthesis known in the art may additionally or 
alternatively be employed. It is also known to use similar techniques to 
prepare other oligonucleotides such as the phosphorothioates and alkylated 
derivatives. 
1. Teachings regarding the synthesis of particular modified 
oligonucleotides may be found in the following U.S. patents or pending 
patent applications, each of which is commonly assigned with this 
application and is hereby incorporated by reference: U.S. Pat. Nos. 
5,138,045 and 5,218,105, drawn to polyamine conjugated oligonucleotides; 
U.S. Pat. No. 5,212,295, drawn to monomers for the preparation of 
oligonucleotides having chiral phosphorus linkages; U.S. Pat. Nos. 
5,378,825 and 5,541,307, drawn to oligonucleotides having modified 
backbones; U.S. Pat. No. 5,386,023, drawn to backbone modified 
oligonucleotides and the preparation thereof through reductive coupling; 
U.S. Pat. No. 5,457,191, drawn to modified nucleobases based on the 
3-deazapurine ring system and methods of synthesis thereof; U.S. Pat. No. 
5,459,255, drawn to modified nucleobases based on N-2 substituted purines; 
U.S. Pat. No. 5,521,302, drawn to processes for preparing oligonucleotides 
having chiral phosphorus linkages; U.S. Pat. No. 5,539,082, drawn to 
peptide nucleic acids; U.S. Pat. No. 5,554,746, drawn to oligonucleotides 
having b-lactam backbones; U.S. Pat. No. 5,571,902, drawn to methods and 
materials for the synthesis of oligonucleotides; U.S. Pat. No. 5,578,718, 
drawn to nucleosides having alkylthio groups, wherein such groups may be 
used as linkers to other moieties attached at any of a variety of 
positions of the nucleoside; U.S. Pat. Nos. 5,587,361 and 5,599,797, drawn 
to oligonucleotides having phosphorothioate linkages of high chiral 
purity; U.S. Pat. No. 5,506,351, drawn to processes for the preparation of 
2'-O-alkyl guanosine and related compounds, including 2,6-diaminopurine 
compounds; U.S. Pat. No. 5,587,469, drawn to oligonucleotides having N-2 
substituted purines; U.S. Pat. No. 5,587,470, drawn to oligonucleotides 
having 3-deazapurines; U.S. Pat. Nos. 5,223,168, issued Jun. 29, 1993, and 
5,608,046, both drawn to conjugated 4'-desmethyl nucleoside analogs; U.S. 
Pat. Nos. 5,602,240, and 5,610,289, drawn to backbone modified 
oligonucleotide analogs; and U.S. patent application Ser. No. 08/383,666, 
filed Feb. 3, 1995, and U.S. Pat. No. 5,459,255, drawn to, inter alia, 
methods of synthesizing 2'-fluoro-oligonucleotides. In 
2'-methoxyethoxy-modified oligonucleotides, 
5-methyl-2'-methoxyethoxy-cytosine residues are used and prepared as 
described in pending application Ser. No. 08/731,199, filed Oct. 4, 1996. 
Specific methods for preparing MMI linkages are taught in U.S. Pat. Nos. 
5,378,825 (issued Jan. 3, 1995), 5,386,023 (issued Jan. 31, 1995), 
5,489,243 (issued on Feb. 6, 1996), 5,541,307 (issued on Jul. 30, 1996), 
5,618,704 (issued Apr. 8, 1997) and 5,623,070 (issued Apr. 22, 1997). MMI 
is an abbreviation for methylene(methylimino) that in turn is a shorten 
version of the more complex chemical nomenclature 
"3'-de(oxyphophinico)-3'[methylene(methylimino)]." Irrespective of 
chemical nomenclature, the linkages are as described in these patents. The 
linkages of these patents have also been described in various scientific 
publications by the inventors and their co-authors including Bhat et al. 
(J. Org. Chem. 61:8186, 1996, and references cited therein). 
2. Bioequivalents: The compounds of the invention encompass any 
pharmaceutically acceptable salts, esters, or salts of such esters, or any 
other compound which, upon administration to an animal including a human, 
is capable of providing (directly or indirectly) the biologically active 
metabolite or residue thereof. Accordingly, for example, the disclosure is 
also drawn to "prodrugs" and "pharmaceutically acceptable salts" of the 
oligonucleotides of the invention, pharmaceutically acceptable salts of 
such prodrugs, and other bioequivalents. 
A. Oligonucleotide Prodrugs: The oligonucleotides of the invention may 
additionally or alternatively be prepared to be delivered in a "prodrug" 
form. The term "prodrug" indicates a therapeutic agent that is prepared in 
an inactive form that is converted to an active form (i.e., drug) within 
the body or cells thereof by the action of endogenous enzymes or other 
chemicals and/or conditions. In particular, prodrug versions of the 
oligonucleotides of the invention are prepared as SATE 
[(S-acetyl-2-thioethyl) phosphate] derivatives according to the methods 
disclosed in WO 93/24510 to Gosselin et al., published Dec.9, 1993. 
B. Pharmaceutically Acceptable Salts: The term "pharmaceutically acceptable 
salts" refers to physiologically and pharmaceutically acceptable salts of 
the oligonucleotides of the invention: i.e., salts that retain the desired 
biological activity of the parent compound and do not impart undesired 
toxicological effects thereto. 
Pharmaceutically acceptable base addition salts are formed with metals or 
amines, such as alkali and alkaline earth metals or organic amines. 
Examples of metals used as cations are sodium, potassium, magnesium, 
calcium, and the like. Examples of suitable amines are 
N,N'-dibenzylethylenediamine, chloroprocaine, choline, diethanolamine, 
dicyclohexylamine, ethylenediamine, N-methylglucamine, and procaine (see, 
for example, Berge et al., "Pharmaceutical Salts," J. of Pharma Sci., 
1977, 66:1). The base addition salts of said acidic compounds are prepared 
by contacting the free acid form with a sufficient amount of the desired 
base to produce the salt in the conventional manner. The free acid form 
may be regenerated by contacting the salt form with an acid and isolating 
the free acid in the conventional manner. The free acid forms differ from 
their respective salt forms somewhat in certain physical properties such 
as solubility in polar solvents, but otherwise the salts are equivalent to 
their respective free acid for purposes of the present invention. As used 
herein, a "pharmaceutical addition salt" includes a pharmaceutically 
acceptable salt of an acid form of one of the components of the 
compositions of the invention. These include organic or inorganic acid 
salts of the amines. Preferred acid salts are the hydrochlorides, 
acetates, salicylates, nitrates and phosphates. Other suitable 
pharmaceutically acceptable salts are well known to those skilled in the 
art and include basic salts of a variety of inorganic and organic acids, 
such as, for example, with inorganic acids, such as for example 
hydrochloric acid, hydrobromic acid, sulfuric acid or phosphoric acid; 
with organic carboxylic, sulfonic, sulfo or phospho acids or N-substituted 
sulfamic acids, for example acetic acid, propionic acid, glycolic acid, 
succinic acid, maleic acid, hydroxymaleic acid, methylmaleic acid, fumaric 
acid, malic acid, tartaric acid, lactic acid, oxalic acid, gluconic acid, 
glucaric acid, glucuronic acid, citric acid, benzoic acid, cinnamic acid, 
mandelic acid, salicylic acid, 4-aminosalicylic acid, 2-phenoxybenzoic 
acid, 2-acetoxybenzoic acid, embonic acid, nicotinic acid or isonicotinic 
acid; and with amino acids, such as the 20 alpha-amino acids involved in 
the synthesis of proteins in nature, for example glutamic acid or aspartic 
acid, and also with phenylacetic acid, methanesulfonic acid, 
ethanesulfonic acid, 2-hydroxyethanesulfonic acid, ethane-1,2-disulfonic 
acid, benzenesulfonic acid, 4-methylbenzenesulfonic acid, 
naphthalene-2-sulfonic acid, naphthalene-1,5-disulfonic acid, 2- or 
3-phosphoglycerate, glucose-6-phosphate, N-cyclohexylsulfamic acid (with 
the formation of cyclamates), or with other acid organic compounds, such 
as ascorbic acid. Pharmaceutically acceptable salts of compounds may also 
be prepared with a pharmaceutically acceptable cation including, for 
example, alkaline, alkaline earth, ammonium and quaternary ammonium 
cations. Carbonates or hydrogen carbonates are also possible. 
For oligonucleotides, preferred examples of pharmaceutically acceptable 
salts include but are not limited to (a) salts formed with cations such as 
sodium, potassium, ammonium, magnesium, calcium, polyamines such as 
spermine and spermidine, etc.; (b) acid addition salts formed with 
inorganic acids, for example hydrochloric acid, hydrobromic acid, sulfuric 
acid, phosphoric acid, nitric acid and the like; (c) salts formed with 
organic acids such as, for example, acetic acid, oxalic acid, tartaric 
acid, succinic acid, maleic acid, fumaric acid, gluconic acid, citric 
acid, malic acid, ascorbic acid, benzoic acid, tannic acid, palmitic acid, 
alginic acid, polyglutamic acid, naphthalenesulfonic acid, methanesulfonic 
acid, p-toluenesulfonic acid, naphthalenedisulfonic acid, polygalacturonic 
acid, and the like; and (d) salts formed from elemental anions such as 
chlorine, bromine, and iodine. 
3. Sterically Stabilized Liposomes: In compositions of the invention, one 
or more antisense oligonucleotides and/or therapeutic agents are entrapped 
within sterically stabilized liposomes. Liposomes are microscopic spheres 
having an aqueous core surrounded by one or more outer layer(s) made up of 
lipids arranged in a bilayer configuration (see, generally, Chonn et al., 
Current Op. Biotech. 6:698, 1995). The therapeutic potential of liposomes 
as drug delivery agents was recognized nearly thirty years ago (Sessa et 
al., J. Lipid Res. 9:310, 1968). Liposomes include "sterically stabilized 
liposome," a term which, as used herein, refers to a liposome comprising 
one or more specialized lipids that, when incorporated into liposomes, 
result in enhanced circulation lifetimes relative to liposomes lacking 
such specialized lipids. Examples of sterically stabilized liposomes are 
those in which part of the vesicle-forming lipid portion of the liposome 
(A) comprises one or more glycolipids, such as monosialoganglioside 
G.sub.M1, or (B) is derivatized with one or more hydrophilic polymers, 
such as a polyethylene glycol (PEG) moiety. While not wishing to be bound 
by any particular theory, it is thought in the art that, at least for 
sterically stabilized liposomes containing gangliosides, sphingomyelin, or 
PEG-derivatized lipids, the enhanced circulation half-life of these 
sterically stabilized liposomes derives from a reduced uptake into cells 
of the reticuloendothelial system (RES) (Allen et al., FEBS Letters 
223:42, 1987; Wu et al., Cancer Research 53:3765, 1993). 
A. Glycolipid-comprising liposomes: Various liposomes comprising one or 
more glycolipids are known in the art. Papahadjopoulos et al. (Ann. N.Y. 
Acad. Sci., 507:64, 1987) reported the ability of monosialoganglioside 
G.sub.M1, galactocerebroside sulfate and phosphatidylinositol to improve 
blood half-lives of liposomes. These findings were expounded upon by 
Gabizon et al. (Proc. Natl. Acad. Sci. U.S.A. 85:6949, 1988). U.S. Pat. 
No. 4,837,028 and WO 88/04924, both to Allen et al., disclose liposomes 
comprising (1) sphingomyelin and (2) the ganglioside G.sub.M1 or a 
galactocerebroside sulfate ester. U.S. Pat. No. 5,543,152 (Webb et al.) 
discloses liposomes comprising sphingomyelin. Liposomes comprising 
1,2-sn-dimyristoylphosphatidylcholine are disclosed in WO 97/13499 (Lim et 
al.). 
B. Liposomes derivatized with hydrophilic polymers: Many liposomes 
comprising lipids derivatized with one or more hydrophilic polymers, and 
methods of preparation thereof, are known in the art. Sunamoto et al. 
(Bull. Chem. Soc. Jpn. 53:2778, 1980) described liposomes comprising a 
nonionic detergent, 2C.sub.12 15G, that contains a PEG moiety. Illum et 
al. (FEBS Letters 167:79, 1984) noted that hydrophilic coating of 
polystyrene particles with polymeric glycols results in significantly 
enhanced blood half-lives. Synthetic phospholipids modified by the 
attachment of carboxylic groups of polyalkylene glycols (e.g., PEG) are 
described by Sears (U.S. Pat. Nos. 4,426,330 and 4,534,899). Klibanov et 
al. (FEBS Letts. 268:235, 1990) described experiments demonstrating that 
liposomes comprising phosphatidylethanolamine (PE) derivatized with PEG or 
PEG stearate have significant increases in blood circulation half-lives. 
Plume et al. (Biochimica et Biophysica Acta 1029:91, 1990) extended such 
observations to other PEG-derivatized phospholipids, e.g., DSPE-PEG, 
formed from the combination of distearoylphosphatidylethanolamine (DSPE) 
and PEG. Liposomes having covalently bound PEG moieties on their external 
surface are described in European Patent No. 0 445 131 B1 and WO 90/04384 
to Fisher. Liposome compositions containing 1-20 mole percent of PE 
derivatized with PEG, and methods of use thereof, are described by Woodle 
et al. (U.S. Pat. Nos. 5,013,556 and 5,356,633) and Martin et al. (U.S. 
Pat. No. 5,213,804 and European Patent No. EP 0 496 813 B1). Liposomes 
comprising a number of other lipid-polymer conjugates are disclosed in WO 
91/05545 and U.S. Pat. No. 5,225,212 (both to Martin et al.) and in WO 
94/20073 (Zalipsky et al.) Liposomes comprising PEG-modified ceramide 
lipids are described in WO 96/10391 (Choi et al.). U.S. Pat. Nos. 
5,540,935 (Miyazaki et al.) and 5,556,948 (Tagawa et al.) describe 
PEG-containing liposomes that can be further derivatized with functional 
moieties on their surfaces. 
C. Liposomes comprising nucleic acids: A limited number of liposomes 
comprising nucleic acids are known in the art. WO 96/40062 to Thierry et 
al. discloses methods for encapsulating high molecular weight nucleic 
acids in liposomes. U.S. Pat. No. 5,264,221 to Tagawa et al. discloses 
protein-bonded liposomes and asserts that the contents of such liposomes 
may include an antisense RNA. U.S. Pat. No. 5,665,710 to Rahman et al. 
describes certain methods of encapsulating oligodeoxynucleotides in 
liposomes. WO 97/04787 to Love et al. discloses liposomes comprising 
antisense oligonucleotides targeted to the raf gene. 4. Chemotherapeutic 
Agents: Certain embodiments of the invention provide for sterically 
stabilized liposomes containing (a) one or more antisense oligonucleotides 
targeted to a nucleic acid encoding a ras protein and (b) one or more 
chemotherapeutic agents which do not function by an antisense mechanism. 
In a related embodiment, such chemotherapeutic agents are co-administered 
with one or more of the liposomal oligonucleotide compositions of the 
invention but are separately encapsulated in distinct liposomes or are 
administered by a non-liposomal delivery mechanism. As used herein, a 
"chemotherapeutic agent" is an anticancer agents that functions via a 
conventional (i.e., non-antisense) mode of action. Examples of such 
chemotherapeutic agents include, but are not limited to, daunorubicin, 
dactinomycin, doxorubicin, bleomycin, mitomycin, nitrogen mustard, 
chlorambucil, melphalan, cyclophosphamide, 6-mercaptopurine, 
6-thioguanine, cytarabine (CA), 5-fluorouracil (5-FU), floxuridine 
(5-FUdR), methotrexate (MTX), colchicine, vincristine, vinblastine, 
etoposide, teniposide, cisplatin and diethylstilbestrol (DES). See, 
generally, The Merck Manual of Diagnosis and Therapy, 15th Ed., Berkow et 
al., eds., 1987, Rahay, N.J., pages 1206-1228). When used with the 
liposomal oligonucleotide compositions of the invention, such 
chemotherapeutic agents may be used individually, sequentially (e.g., 5-FU 
for a period of time followed by MTX), or in combination with one or more 
other such chemotherapeutic agents (e.g., 5-FU and MTX). 
In a related embodiment, sterically stabilized liposomes containing (a) one 
or more antisense oligonucleotides targeted to a first nucleic acid 
encoding a ras sequence and (b) one or more additional antisense 
oligonucleotides targeted to a second nucleic acid encoding a cancer 
associated gene. By the term "cancer associated gene" is intended any 
cellular or viral gene the expression of which disrupts regulation of the 
cell cycle, negatively effects contact inhibition of growth, leads to 
cellular hyperproliferation, promotes pre-metastatic or metastatic events 
and/or otherwise leads to cellular hyperproliferation, tumor formation and 
the growth and spread of cancers, regardless of mechanism of action. In a 
related embodiment, such additional antisense oligonucleotides targeted to 
a second cancer-associated gene are co-administered with one or more of 
the liposomal oligonucleotide compositions of the invention but are 
separately encapsulated in distinct liposomes or are administered by a 
non-liposomal delivery mechanism. Such antisense oligonucleotides targeted 
to a second cancer associated gene include, but are not limited to, those 
directed to the following targets as disclosed in the indicated co-owned 
U.S. Patents, pending applications or published PCT applications, which 
are hereby incorporated by reference: raf (WO 96/39415, WO 95/32987 and 
U.S. Pat. Nos. 5,563,255, issued Oct. 8, 1996, and 5,656,612, issued Aug. 
12, 1997), the p120 nucleolar antigen (WO 93/17125 and U.S. Pat. No. 
5,656,743, issued Aug. 12, 1997), protein kinase C (WO 95/02069, WO 
95/03833 and WO 93/19203), multidrug resistance-associated protein (WO 
95/10938 and U.S. Pat. No. 5,510,239, issued Mar. 23, 1996), subunits of 
transcription factor AP-1 (co-pending application U.S. Ser. No. 
08/837,201, filed Apr. 14, 1997), Jun kinases (co-pending application U.S. 
Ser. No. 08/910,629, filed Aug. 13, 1997), and MDR-1 (multidrug resistance 
glycoprotein; co-pending application U.S. Ser. No. 08/731,199, filed Sep. 
30, 1997). 
5. Administration of Pharmaceutical Compositions: The formulation of 
pharmaceutical compositions comprising the liposomal oligonucleotide 
compositions of the invention and their subsequent administration is 
believed to be within the skill of those in the art. In general, for 
therapeutics, a patient in need of such therapy is administered a 
liposomal oligonucleotide composition in accordance with the invention, 
commonly in a pharmaceutically acceptable carrier, in doses ranging from 
0.01 .mu.g to 100 g per kg of body weight depending on the age of the 
patient and the severity of the disorder or disease state being treated. 
Dosing is dependent on severity and responsiveness of the disease state to 
be treated, with the course of treatment lasting from several days to 
several months, or until a cure is effected or a diminution of the disease 
state is achieved. Optimal dosing schedules can be calculated from 
measurements of drug accumulation in the body of the patient. Persons of 
ordinary skill can easily determine optimum dosages, dosing methodologies 
and repetition rates. Optimum dosages may vary depending on the relative 
potency of individual oligonucleotides, and can generally be estimated 
based on EC.sub.50 s found to be effective in in vitro and in vivo animal 
models. Further, the treatment regimen may last for a period of time which 
will vary depending upon the nature of the particular disease or disorder, 
its severity and the overall condition of the patient, and may extend from 
once daily to once every 20 years. Following treatment, the patient is 
monitored for changes in his/her condition and for alleviation of the 
symptoms of the disorder or disease state. The dosage of the 
oligonucleotide may either be increased in the event the patient does not 
respond significantly to current dosage levels, or the dose may be 
decreased if an alleviation of the symptoms of the disorder or disease 
state is observed, or if the disorder or disease state has been ablated. 
An optimal dosing schedule is used to deliver a therapeutically effective 
amount of the oligonucleotide being administered via a particular mode of 
administration. The term "therapeutically effective amount," for the 
purposes of the invention, refers to the amount of 
oligonucleotide-containing pharmaceutical composition which is effective 
to achieve an intended purpose without undesirable side effects (such as 
toxicity, irritation or allergic response). Although individual needs may 
vary, determination of optimal ranges for effective amounts of 
pharmaceutical compositions is within the skill of the art. Human doses 
can be extrapolated from animal studies (Katocs et al., Chapter 27 In: 
Remington's Pharmaceutical Sciences, 18th Ed., Gennaro, ed., Mack 
Publishing Co., Easton, Pa., 1990). Generally, the dosage required to 
provide an effective amount of a pharmaceutical composition, which can be 
adjusted by one skilled in the art, will vary depending on the age, 
health, physical condition, weight, type and extent of the disease or 
disorder of the recipient, frequency of treatment, the nature of 
concurrent therapy (if any) and the nature and scope of the desired 
effect(s) (Nies et al., Chapter 3 In: Goodman & Gilman's The 
Pharmacological Basis of Therapeutics, 9th Ed., Hardman et al., eds., 
McGraw-Hill, New York, N.Y., 1996). 
In some cases it may be more effective to treat a patient with a liposomal 
oligonucleotide composition of the invention in conjunction with other, 
traditional therapeutic modalities in order to increase the efficacy of a 
treatment regimen. In the context of the invention, the term "treatment 
regimen" is meant to encompass therapeutic, palliative and prophylactic 
modalities. Following treatment, the patient is monitored for changes in 
his/her condition and for alleviation of the symptoms of the disorder or 
disease state. The dosage of the pharmaceutical composition may either be 
increased in the event the patient does not respond significantly to 
current dosage levels, or the dose may be decreased if an alleviation of 
the symptoms of the disorder or disease state is observed, or if the 
disorder or disease state has been ablated. 
As used herein, the term "high risk individual" is meant to refer to an 
individual for whom it has been determined, via, e.g., individual or 
family history or genetic testing, that there is a significantly higher 
than normal probability of being susceptible to the onset or recurrence of 
a disease or disorder. As part of a treatment regimen for a high risk 
individual, the individual can be prophylactically treated to prevent the 
onset or recurrence of the disease or disorder. The term "prophylactically 
effective amount" is meant to refer to an amount of a pharmaceutical 
composition which produces an effect observed as the prevention of the 
onset or recurrence of a disease or disorder. Prophylactically effective 
amounts of a pharmaceutical composition are typically determined by the 
effect they have compared to the effect observed when a second 
pharmaceutical composition lacking the active agent is administered to a 
similarly situated individual. 
The pharmaceutical compositions of the present invention may be 
administered in a number of ways depending upon whether local or systemic 
treatment is desired and upon the area to be treated. Typically, 
parenteral administration is employed. The term "parenteral delivery" 
refers to the administration of an oligonucleotide of the invention to an 
animal in a manner other than through the digestive canal. Parenteral 
administration includes intravenous (i.v.) drip, subcutaneous, 
intraperitoneal (i.p.) or intramuscular injection, or intrathecal or 
intraventricular administration. Compositions for parenteral, intrathecal 
or intraventricular administration may include sterile aqueous solutions 
which may also contain buffers, diluents and other suitable additives. 
Means of preparing and administering parenteral pharmaceutical 
compositions are known in the art (see, e.g., Avis, Chapter 84 In: 
Remington's Pharmaceutical Sciences, 18th Ed., Gennaro, ed., Mack 
Publishing Co., Easton, Pa., 1990, pages 1545-1569). Parenteral means of 
delivery include, but are not limited to, the following illustrative 
examples. 
(A) Intravitreal injection, for the direct delivery of drug to the vitreous 
humor of a mammalian eye, is described in U.S. Pat. No. 5,591,720, the 
contents of which are hereby incorporated by reference. Means of preparing 
and administering ophthalmic preparations are known in the art (see, e.g., 
Mullins et al., Chapter 86 In: Remington's Pharmaceutical Sciences, 18th 
Ed., Gennaro, ed., Mack Publishing Co., Easton, Pa., 1990, pages 
1581-1595). 
(B) Intravenous administration of antisense oligonucleotides to various 
non-human mammals has been described by Iversen (Chapter 26 In: Antisense 
Research and Applications, Crooke et al., eds., CRC Press, Boca Raton, 
Fla., 1993, pages 461-469). Systemic delivery of oligonucleotides to 
non-human mammals via intraperitoneal means has also been described (Dean 
et al., Proc. Natl. Acad. Sci. U.S.A. 91:11766, 1994). 
(C) Intraluminal drug administration, for the direct delivery of drug to an 
isolated portion of a tubular organ or tissue (e.g., such as an artery, 
vein, ureter or urethra), may be desired for the treatment of patients 
with diseases or conditions afflicting the lumen of such organs or 
tissues. To effect this mode of oligonucleotide administration, a catheter 
or cannula is surgically introduced by appropriate means. For example, for 
treatment of the left common carotid artery, a cannula is inserted 
thereinto via the external carotid artery. After isolation of a portion of 
the tubular organ or tissue for which treatment is sought, a composition 
comprising the oligonucleotides of the invention is infused through the 
cannula or catheter into the isolated segment. After incubation for from 
about 1 to about 120 minutes, during which the oligonucleotide is taken up 
by cells of the interior lumen of the vessel, the infusion cannula or 
catheter is removed and flow within the tubular organ or tissue is 
restored by removal of the ligatures which effected the isolation of a 
segment thereof (Morishita et al., Proc. Natl. Acad. Sci. U.S.A. 90:8474, 
1993). Antisense oligonucleotides may also be combined with a 
biocompatible matrix, such as a hydrogel material, and applied directly to 
vascular tissue in vivo (Rosenberg et al., U.S. Pat. No. 5,593,974, issued 
Jan. 14, 1997). 
(D) Intraventricular drug administration, for the direct delivery of drug 
to the brain of a patient, may be desired for the treatment of patients 
with diseases or conditions afflicting the brain. To effect this mode of 
oligonucleotide administration, a silicon catheter is surgically 
introduced into a ventricle of the brain of a human patient, and is 
connected to a subcutaneous infusion pump (Medtronic Inc., Minneapolis, 
Minn.) that has been surgically implanted in the abdominal region (Zimm et 
al., Cancer Research 44:1698, 1984; Shaw, Cancer 72(11 Suppl.):, 3416, 
1993). The pump is used to inject the oligonucleotides and allows precise 
*dosage adjustments and variation in dosage schedules with the aid of an 
external programming device. The reservoir capacity of the pump is 18-20 
mL and infusion rates may range from 0.1 mL/h to 1 mL/h. Depending on the 
frequency of administration, ranging from daily to monthly, and the dose 
of drug to be administered, ranging from 0.01 .mu.g to 100 g per kg of 
body weight, the pump reservoir may be refilled at 3-10 week intervals. 
Refilling of the pump is accomplished by percutaneous puncture of the 
pump's self-sealing septum. 
(E) Intrathecal drug administration, for the introduction of a drug into 
the spinal column of a patient may be desired for the treatment of 
patients with diseases of the central nervous system (CNS). To effect this 
route of oligonucleotide administration, a silicon catheter is surgically 
implanted into the L3-4 lumbar spinal interspace of a human patient, and 
is connected to a subcutaneous infusion pump which has been surgically 
implanted in the upper abdominal region (Luer and Hatton, The Annals of 
Pharmacotherapy 27:912, 1993; Ettinger et al. Cancer, 41:1270, 1978; Yaida 
et al., Regul. Pept. 59:193, 1985). The pump is used to inject the 
oligonucleotides and allows precise dosage adjustments and variations in 
dose schedules with the aid of an external programming device. The 
reservoir capacity of the pump is 18-20 mL, and infusion rates may vary 
from 0.1 mL/h to 1 mL/h. Depending on the frequency of drug 
administration, ranging from daily to monthly, and dosage of drug to be 
administered, ranging from 0.01 .mu.g to 100 g per kg of body weight, the 
pump reservoir may be refilled at 3-10 week intervals. Refilling of the 
pump is accomplished by a single percutaneous puncture to the self-sealing 
septum of the pump. The distribution, stability and pharmacokinetics of 
oligonucleotides within the CNS are followed according to known methods 
(Whitesell et al., Proc. Natl. Acad. Sci. U.S.A. 90:4665, 1993). 
To effect delivery of oligonucleotides to areas other than the brain or 
spinal column via this method, the silicon catheter is configured to 
connect the subcutaneous infusion pump to, e.g., the hepatic artery, for 
delivery to the liver (Kemeny et al., Cancer 71:1964, 1993). Infusion 
pumps may also be used to effect systemic delivery of oligonucleotides 
(Ewel et al., Cancer Research 52:3005, 1992; Rubenstein et al., J. Surg. 
Oncol. 62:194, 1996). 
EXAMPLES 
The following examples illustrate the invention and are not intended to 
limit the same. Those skilled in the art will recognize, or be able to 
ascertain through routine experimentation, numerous equivalents to the 
specific substances and procedures described herein. Such equivalents are 
considered to be within the scope of the present invention. 
Example 1 
Nucleic Acid Sequences 
The oligonucleotides of this invention are designed to be complementary to, 
and thus hybridizable with, messenger RNA derived from a ras gene. Such 
hybridization, when accomplished, interferes with the normal roles of the 
messenger RNA to cause a loss of its function in the cell. The functions 
of messenger RNA to be interfered with include all vital functions such as 
translocation of the RNA to the site for protein translation, actual 
translation of protein from the RNA, splicing of the RNA to yield one or 
more mRNA species, and possibly even independent catalytic activity which 
may be engaged in by the RNA. The overall effect of such interference with 
the RNA function is to interfere with expression of the ras gene. Some 
oligonucleotides of this invention are designed to activate RNAse H 
cleavage of the ras mRNA. 
The protein products of the other mammalian ras genes, N-ras and K-ras, are 
identical to H-ras over the first 85 amino acids. However, the nucleic 
acid sequences of the three ras genes are not identical, and persons of 
ordinary skill in the art will be able to use this invention as a guide in 
preparing oligonucleotides specifically hybridizable with a particular ras 
gene. While one preferred embodiment of the invention relate to antisense 
oligonucleotides specifically hybridizable with codon 12 of the H-ras 
mRNA, the disclosure can be used by persons skilled in the art as a guide 
in preparing oligonucleotides specifically hybridizable with other point 
mutations of the H-ras gene, particularly the well defined point mutations 
at codon 12, codon 13 and codon 61 of H-ras, or point mutations within 
other ras genes. 
The nucleotide sequence of wildtype (wt) H-ras, also known as Ha-ras, has 
been described by Capon et al. (Nature 302:33, 1983), Fasano et al. (J. 
Mol. Appl. Genet. 2:173, 1983), Reddy (Science 220:1061, 1983) and Honkawa 
et al. (Mol. Cell. Biol. 7:2933, 1987). Mutant (activated) H-ras sequences 
have been reported by Tabin et al. (Nature 300:143, 1982), Taparowsky et 
al. (Nature 300:762, 1982), Yuasa et al. (Nature 303:775, 1983), Sekiya et 
al. (Proc. Natl. Acad. Sci. USA 81:4771, 1984; Jpn. J. Cancer Res. 76:787, 
1985), Kraus et al. (Proc. Natl. Acad. Sci. USA 81:5384, 1984), Stevens et 
al. (Proc. Natl. Acad. Sci. USA 85:3875), Deng et al. (Cancer Res. 
47:3195, 1987), Santos et al. (Proc. Natl. Acad. Sci. USA 80:4679, 1983), 
Tanci et al. (Nucleic Acids Res. 20:1157, 1992) and Tadokoro et al. 
(Oncogene 4:499, 1989). The sequences of wildtype and mutant H-ras genes 
may also be found in the Genbank and EMBOL databases under Accession Nos. 
J00206, J00276, J00277, K00654, K00954, M30539, M19990, M17232, M25876, 
V00574, X01227 and X16438. 
The nucleotide sequence of wildtype (wt) K-ras, also known as Ki-ras, has 
been described by McGrath et al. (Nature 304:501, 1983) and McCoy et al. 
(Mol. Cell. Biol. 4:1577, 1984). Mutant (activated) K-ras sequences have 
been reported by Shimizu et al. (Nature 304:497, 1983), Capon et al. 
(Nature 304:507, 1983), Nakano et al. (Proc. Natl. Acad. Sci. U.S.A. 
81:71, 1984), Taya et al. (EMBO J. 3:2943, 1984) and Nardeux et al. 
(Biochem. Biophys. Res. Commun. 146:395, 1987). The sequences of wildtype 
and mutant K-ras genes may also be found in Genbank under Accession Nos. 
K00652, K00653, K01519, K01520, K01912, L00045, L00049, M17087, M26261, 
M38506 and M54968. 
The nucleotide sequences of wildtype and mutant N-ras genes are known (Hall 
et al., Nucleic Acids Res. 13:5255, 1985; Taparowsky et al., Cell 34:581, 
1983; Geis et al., Biochem. Biophys. Res. Commun. 139:771, 1986; Brown et 
al., EMBO J. 3:1321, 1984). The sequences of wildtype and mutant N-ras 
genes may also be found in the Genbank and EMBOL databases under Accession 
Nos. K00082, L00043, M14307, X00645 and X02751. 
Oligonucleotides targeted to ras genes are described in U.S. Pat. Nos. 
5,576,208; 5,582,972; 5,582,986; and 5,661,134, and pending application 
Ser. No. 08/889,296, filed Jul. 8, 1997, as well as WO 94/08003, WO 
94/28720 and WO 92/22651 to Monia et al., all of which are assigned to the 
same assignee as that of the present disclosure and which are hereby 
incorporated by reference. 
The sequences and chemistries of oligonucleotides targeted to H-ras are 
detailed in Tables 1 through 7. The sequences and chemistries of 
oligonucleotides targeted to K-ras are detailed in Table 8. Sequences and 
chemistries of oligonucleotides targeted to N-ras are detailed in Table 9. 
TABLE 1 
__________________________________________________________________________ 
Phosphorothioate Antisense Oligodeoxynucleotides 
Targeted to H-ras 
__________________________________________________________________________ 
Targeted to the H-ras translation initiation codon 
ISIS # 
SEQUENCE (5'-&gt;3') SEQ ID NO: 
__________________________________________________________________________ 
2502 CTT-ATA-TTC-CGT-CAT-CGC-TC 
1 
2503 TCC-GTC-ATC-GCT-CCT-CAG-GG 
2 
2570 CCA-CAC-CGA-CGG-CGC-CC 3 
2571 CCC-ACA-CCG-ACG-GCG-CCC-A 4 
2566 GCC-CAC-ACC-GAC-GGC-GCC-CAC 
5 
2560 TGC-CCA-CAC-CGA-CGG-CGC-CCA-CC 
6 
Targeted to mutant H-ras 
ISIS # 
TARGET SEQUENCE (5'-&gt;3') 
SEQ ID NO: 
__________________________________________________________________________ 
2502 AUG CTTATATTCCGTCATCGCTC 
1 
2503 AUG TCCGTCATCGCTCCTCAGGG 
2 
6186 AUG TATTCCGTCATCGCTCCTCA 
7 
2563 CODON 12 CGACG 8 
2564 CODON 12 CCGACGG 9 
2565 CODON 12 ACCGACGGC 10 
2567 CODON 12 CACCGACGGCG 11 
2568 CODON 12 ACACCGACGGCGC 12 
2569 CODON 12 CACACCGACGGCGCC 13 
3426 CODON 12 CCACACCGACGGCGCC 14 
3427 CODON 12 CACACCGACGGCGCCC 15 
2570 CODON 12 CCACACCGACGGCGCCC 
3 
3428 CODON 12 CCCACACCGACGGCGCCC 
16 
3429 CODON 12 CCACACCGACGGCGCCCA 
17 
2571 CODON 12 CCCACACCGACGGCGCCCA 
4 
2566 CODON 12 GCCCACACCGACGGCGCCCAC 
5 
2560 CODON 12 TGCCCACACCGACGGCGCCCACC 
6 
2561 CODON 12 TTGCCCACACCGACGGCGCCCACCA 
18 
2907 CODON 12 (wt) 
CCACACCGCCGGCGCCC 
19 
__________________________________________________________________________ 
TABLE 2 
______________________________________ 
Chimeric Phosphorothioate Oligonucleotides 
Having 2'-O-Methyl Ends (Bold) and 
Central Deoxy Gap 
(Mutant 
Codon-12 Target) 
# OF DEOXY 
ISIS # RESIDUES SEQUENCE (5'-&gt;3') 
SEQ ID NO: 
______________________________________ 
4122 0 CCACACCGACGGCGCCC 
3 
3975 1 CCACACCGACGGCGCCC 
3 
3979 3 CCACACCGACGGCGCCC 
3 
4236 4 CCACACCGACGGCGCCC 
3 
4242 4 CCACACCGACGGCGCCC 
3 
3980 5 CCACACCGACGGCGCCC 
3 
3985 7 CCACACCGACGGCGCCC 
3 
3984 9 CCACACCGACGGCGCCC 
3 
2570 17 CCACACCGACGGCGCCC 
3 
______________________________________ 
TABLE 3 
______________________________________ 
Shortened Phosphorothioate Chimeric 
Oligonucleotides Derived from ISIS 3980 Having 
2'-O-Methyl Ends (Bold) and Central Deoxy Gap 
(Mutant Codon-12 Target) 
ISIS # SEQUENCE (5'-&gt;3') 
SEQ ID NO: 
______________________________________ 
3980 CCACACCGACGGCGCCC 
3 
4230 CACACCGACGGCGCC 
13 
4276 ACACCGACGGCGC 12 
4247 CACCGACGGCG 11 
3985 CCACACCGACGGCGCCC 
3 
4245 CACACCGACGGCGCC 
13 
4278 ACACCGACGGCGC 12 
4229 CACCGACGGCG 11 
______________________________________ 
TABLE 4 
______________________________________ 
Chimeric Phosphorothioate Oligonucleotides 
Having 2'-O-Methyl Ends (Bold) 
and Central Deoxy Gap 
(AUG Target) 
# OF DEOXY 
ISIS # 
RESIDUES SEQUENCE (5'-&gt;3') 
SEQ ID NO: 
______________________________________ 
2502 20 CTTATATTCCGTCATCGCTC 
1 
4998 7 CTTATATTCCGTCATCGCTC 
1 
2503 20 TCCGTCATCGCTCCTCAGGG 
2 
5122 7 TCCGTCATCGCTCCTCAGGG 
2 
______________________________________ 
TABLE 5 
__________________________________________________________________________ 
Chimeric Backbone (P = S/P = O) Oligonucleotides 
Having 2'-O-Methyl Ends (Bold) and Central Deoxy Gap 
(Backbone Linkages Indicated by "s" (P = S) or "o" (P = O) 
(Mutant Codon-12 Target) 
# OF DEOXY 
ISIS # 
RESIDUES 
SEQUENCE (5'-&gt;3') SEQ ID NO: 
__________________________________________________________________________ 
2570 
16 CsCsAsCsAsCsCsGsAsCsGsGsCsGsCsCsC 
3 
4226 
5 CoCoAoCoAoCsCsGsAsCsGoGoCoGoCoCoC 
3 
4233 
11 CsCsAsCsAsCoCoGoAoCoGsGsCsGsCsCsC 
3 
4248 
15 CsCsAsCsAsCsCsGsAoCsGsGsCsGsCsCsC 
3 
4546 
14 CsCsAsCsAsCsCsGoAoCsGsGsCsGsCsCsC 
3 
4551 
13 CsCsAsCsAsCsCsGoAoCoGsGsCsGsCsCsC 
3 
4593 
12 CsCsAsCsAsCsCoGoAoCoGsGsCsGsCsCsC 
3 
4606 
11 CsCsAsCsAsCsCoGoAoCoGoGsCsGsCsCsC 
3 
4241 
6 CsCsAsCoAoCoCoGoAoCoGoGoCoGsCsCsC 
3 
__________________________________________________________________________ 
TABLE 6 
______________________________________ 
Phosphorothioate Antisense Oligodeoxynucleotides 
Targeted to a Hairpin Structure Corresponding 
to Residues +18 to +64 of the Coding Sequence 
of Activated H-ras mRNA 
ISIS # SEQUENCE (5'-&gt;3') 
SEQ ID NO: 
______________________________________ 
3270 CACCACCACC 20 
3271 GCGCCCACCA 21 
3292 CGACGGCGCC 22 
3291 CACACCGACG 23 
3283 UUGCCCACAC 24 
3284 CACUCUUGCC 25 
______________________________________ 
TABLE 7 
______________________________________ 
2'-Modified Analogs of ISIS 2503 
(Positions with 2' Modifications are Emboldened) 
______________________________________ 
MOE Analogs (positions with 2'-MOE are emboldened) 
ISIS # Sequence (5'-&gt;3') SEQ ID NO: 
______________________________________ 
13905 TCCGTCATCGCTCCTCAGGG 
2 
13907 TCCGTCATCGCTCCTCAGGG 
2 
13909 TCCGTCATCGCTCCTCAGGG 
2 
13911 TCCGTCATCGCTCCTCAGGG 
2 
13917 TCCGTCATCGCTCCTCAGGG 
2 
13919 TCCGTCATCGCTCCTCAGGG 
2 
13920 TCCGTCATCGCTCCTCAGGG 
2 
13923 TCCGTCATCGCTCCTCAGGG 
2 
13926 TCCGTCATCGCTCCTCAGGG 
2 
13927 TCCGTCATCGCTCCTCAGGG 
2 
MMI Analogs (positions with 2'-MOE are emboldened) 
ISIS # Sequence (5'-&gt;3') SEQ ID NO: 
______________________________________ 
14896 TCCGTCATCGCTCCTCAGGG 
2 
14897 TC.sub.o CGTCATCGCTCCTCAG.sub.o GG 
2 
14898 TC.sub.s CGTCATCGCTCCTCAG.sub.s GG 
2 
14899 TC.sub.o CG.sub.o TCATCGCTCCTC.sub.o A.sub.o GGG 
2 
14900 TC.sub.s CG.sub.s TCATCGCTCCTC.sub.s AG.sub.s AG 
2 
______________________________________ 
"o" indicates a phosphodiester linkage between MMI dimers; 
"s" indicates a phosphorothioate linkage between MMI dimers. 
All unmarked linkages are phosphorothioates. 
TABLE 8 
__________________________________________________________________________ 
Phosphorothioate Antisense Oligonucleotides 
Targeted to Human K-ras 
__________________________________________________________________________ 
Oligodeoxynucleotides 
ISIS # 
SEQUENCE (5'-&gt;3') 
TARGET SEQ ID NO: 
__________________________________________________________________________ 
6958 CTGCCTCCGCCGCCGCGGCC 
5' UTR/5'-cap 
28 
6957 CAGTGCCTGCGCCGCGCTCG 
5'-UTR 29 
6956 AGGCCTCTCTCCCGCACCTG 
5'-UTR 30 
6953 TTCAGTCATTTTCAGCAGGC 
AUG 31 
6952 TTATATTCAGTCATTTTCAG 
AUG 32 
6951 CAAGTTTATATTCAGTCATT 
AUG 33 
6950 GCCTACGCCACCAGCTCCAAC 
Codon 12 (wt) 
34 
6949 CTACGCCACCAGCTCCA 
Codon 12 (wt) 
35 
7453 TACGCCAACAGCTCC 
Codon 12 (G.fwdarw.T mutant) 
36 
6948 GTACTCCTCTTGACCTGCTGT 
Codon 61 (wt) 
37 
6947 CCTGTAGGAATCCTCTATTGT 
Codon 38 38 
6946 GGTAATGCTAAAACAAATGC 
3'-UTR 39 
6945 GGAATACTGGCACTTCGAGG 
3'-UTR 40 
7679 TTTTCAGCAGGCCTCTCTCC 
5'-UTR/AUG 41 
Chimeric oligonucleotides having 2'-O-methyl ends (bold) 
ISIS # SEQUENCE (5'-&gt;3') 
SEQ ID NO: 
__________________________________________________________________________ 
6957 CAGTGCCTGCGCCGCGCTCG 
29 
7683 CAGTGCCTGCGCCGCGCTCG 
29 
7679 TTTTCAGCAGGCCTCTCTCC 
41 
7680 TTTTCAGCAGGCCTCTCTCC 
41 
__________________________________________________________________________ 
TABLE 9 
______________________________________ 
Phosphorothioate Oligodeoxynucleotides 
Targeted to Human N-ras 
Target 
ISIS # Sequence (5'-&gt;3') Region SEQ ID NO: 
______________________________________ 
14677 CCGGGTCCTAGAAGCTGCAG 
5' UTR 42 
14678 TAAATCAGTAAAAGAAACCG 
5' UTR 43 
14679 GGACACAGTAACCAGGCGGC 
5' UTR 44 
14680 AACAGAAGCTACACCAAGGG 
5' UTR 45 
14681 CAGACCCATCCATTCCCGTG 
5' UTR 46 
14682 GCCAAGAAATCAGACCCATC 
5' UTR 47 
14683 AGGGGGAAGATAAAACCGCC 
5' UTR 48 
14684 CGCTTCCATTCTTTCGCCAT 
5' UTR 49 
14685 CCGCACCCAGACCCGCCCCT 
5' UTR 50 
14686 CAGCCCCCACCAAGGAGCGG 
5' UTR 51 
14687 GTCATTTCACACCAGCAAGA 
AUG 52 
14688 CAGTCATTTCACACCAGCAA 
AUG 53 
14689 CTCAGTCATTTCACACCAGC 
AUG 54 
14690 CGTGGGCTTGTTTTGTATCA 
Coding 55 
14691 CCATACAACCCTGAGTCCCA 
3' UTR 56 
14692 CAGACAGCCAAGTGAGGAGG 
3' UTR 57 
14693 CCAGGGCAGAAAAATAACAG 
3' UTR 58 
14694 TTTGTGCTGTGGAAGAACCC 
3' UTR 59 
14695 GCTATTAAATAACAATGCAC 
3' UTR 60 
14696 ACTGATCACAGCTATTAAAT 
3' UTR 61 
______________________________________ 
Example 2 
Oligonucleotide Synthesis 
Substituted and unsubstituted deoxyoligonucleotides were synthesized on an 
automated DNA synthesizer (Applied Biosystems model 380B) using standard 
phosphoramidate chemistry with oxidation by iodine. For phosphorothioate 
oligonucleotides, the standard oxidation bottle was replaced by 0.2 M 
solution of 3H-1,2-benzodithiole-3-one 1,1-dioxide in acetonitrile for the 
stepwise thiation of the phosphite linkages. The thiation wait step was 
increased to 68 sec and was followed by the capping step. Synthesis of 
2-(amino)adenine-substituted oligonucleotides was carried out in like 
manner, with the following exception: at positions at which a 
2-(amino)adenine is desired, the standard phosphoramidite is replaced with 
a commercially available 2-aminodeoxyadenosine phosphoramidite (Chemgenes 
Corp., Waltham, Mass.). After cleavage from the CPG column and deblocking 
in concentrated ammonium hydroxide at 55.degree. C. (18 hr), the 
oligonucleotides were purified by precipitation twice out of 0.5 M NaCl 
solution with 2.5 volumes ethanol. Analytical gel electrophoresis was 
accomplished in 20% acrylamide, 8 M urea, 454 mM Tris-borate buffer, 
pH=7.0. Oligonucleotides were judged from polyacrylamide gel 
electrophoresis to be greater than 80% full-length material. 
Oligoribonucleotides were synthesized using the automated synthesizer and 
5'-dimethoxy-trityl 2'-tert-butyldimethylsilyl 3'-O-phosphoramidites 
(American Bionetics, Hayward, Calif.). The protecting group on the 
exocyclic amines of A, C and G was phenoxyacetyl (Wu et al., Nucl. Acids 
Res. 17:3501, 1989). The standard synthesis cycle was modified by 
increasing the wait step after the pulse delivery of tetrazole to 900 
seconds. Oligonucleotides were deprotected by overnight incubation at room 
temperature in methanolic ammonia. After drying in vacuo, the 2'-silyl 
group was removed by overnight incubation at room temperature in 1 M 
tetrabutylammoniumf luoride (Aldrich Chemical Co., Milwaukee, Wis.) in 
tetrahydrofuran. Oligonucleotides were purified using a C-18 Sep-Pak 
cartridge (Waters Corp., Milford, Mass.) followed by ethanol 
precipitation. Analytical denaturing polyacrylamide electrophoresis 
demonstrated the RNA oligonucleotides were greater than 90% full length 
material. 
Example 3 
Preparation of Sterically Stabilized Liposomes Comprising Antisense 
Oligonucleotides 
A. Preparation of Lipid Film 
Lipid stock solutions were prepared at 20 mg/mL in chloroform. 
Dipalmitoylphosphatidylcholine (DPPC; Avanti Polar Lipids, Inc., 
Alabaster, Ala.), cholesterol (Avanti Polar lipids, Inc. or Sigma Chemical 
Corp., St. Louis, Mo.) and N-(carbamoylmethoxypolyethylene glycol 
2000)-1,2-distearoyl-sn-glycero-3-phosphoethanolamine-(DSPE-MPEG.sub.2000 
; Avanti Polar Lipids, Inc.) were dispensed into a 30 mL round bottom 
flask as follows for 150 .mu.mol of total lipid: 
TABLE 10 
______________________________________ 
Lipid Components of DSPE-MPEG.sub.2000 Liposomes 
Comprising ISIS 2503 
Mole mg mL stock lipid 
Component ratio Mole % lipid solution 
______________________________________ 
DPPC 3 57 62.74 3.137 
Cholesterol 
2 38 22.03 1.102 
DSPE-MPEG.sub.2000 
0.265 5 20.75 1.037 
______________________________________ 
Chloroform was removed by evaporation using a rotary evaporator, heating at 
60.degree. C. with a moderate vacuum. The lipid material dried as a thin 
film on the flask wall. Evaporation was continued using high vacuum for an 
additional 30 minutes at 60.degree. C. 
B. Lipid Hydration 
Phosphorothioate oligonucleotide (ISIS 2503) was dissolved in water to 100 
mg/mL. The solution was made isotonic (80-310 mOsm) with the addition of a 
small quantity of 5M NaCl as needed. The final solution was filtered 
through a 0.22 .mu.m membrane. Then, 0.5 mL of the resultant oligo 
solution was added to the flask containing the lipid film. The flask was 
rotated at 240 rpm at 60.degree. C. for 5 minutes. The lipid suspension 
was vortexed heavily to form large multi-lamellar liposomes. 
The liposomes were frozen by immersing the flask into a dry ice/acetone 
bath for 5 minutes. Thawing of the liposomes was accomplished by immersing 
the flask into a 60.degree. C. water bath as necessary. The preceding 
freeze/thaw steps were repeated 5 times. The resulting liposome solution 
appeared "creamy". 
C. Particle Sizing 
Large multi-lamellar liposomes were converted into near-uniform unilamellar 
liposomes by either (1) physical extrusion through polycarbonate membranes 
(Avestin, Inc., Ottawa, Ontario, Canada) of defined porosity (e.g., 100 
nm) or microfluidization with a Model 110 S microfluidizer (Microfluidics 
International Corp., Newton, Mass.). Either technique produces unilamellar 
liposomes of approximately 90 to about 110 nm in diameter. 
D. Liposome Purification 
Nonencapsulated oligonucleotide material was separated from the liposomes 
by gel permeation chromatography using a Superdex-200 column (Pharmacia 
Biotech, Inc., Piscataway, N.J.) equilibrated in phosphate-buffered 
saline, pH 7.4. Encapsulation recovery was typically 25-30% and the final 
ISIS 2503 concentration in the liposomes was about 7 mg/mL. The liposome 
fractions were pooled and filter-sterilized through a 0.2 .mu.m membrane 
(Gelman Sciences, Inc., Ann Arbor, Mich.). Liposomes were stored at 
4.degree. C. 
Example 4 
Evaluation of Sterically Stabilized Liposomes Comprising Antisense 
Oligonucleotides 
A. Experimental Design and Methods 
Study Design: Thirteen rhesus monkeys (Macaca mulatta) (7 males and 6 
females) were used. The animals were pre-pubertal to young adult (in the 
age range of 3-7 years), and their body weight ranged from 3-4 kg (Table 
11). Each animal received a single intravenous infusion of ISIS 2503 
encapsulated in sterically stabilized liposomes (10 mg/kg) over 
approximately 30 minutes. Blood samples for pharmacokinetic analysis were 
collected prior to dosing and at 0, 1, 2, 6, 12, 24, 40, 60, 96, 120, 144, 
168, 192, 240, 384 and 576 hours after dosing. Animals were 
serial-sacrificed such that 2 animals (1 male and 1 female) were 
euthanized at each of the following time points from the end of infusion: 
24, 60, 120, 168, 384 and 576 hours. An additional male monkey (Animal ID 
#R4791) died of unknown causes shortly after dosing. Although samples were 
analyzed for this animal, the values were not included in the 
pharmacokinetic analysis because the animal died before the earliest study 
time point. As controls in some experiments, animals were treated in the 
same manner but with a simple saline formulation of ISIS 2503 in saline. 
TABLE 11 
______________________________________ 
Animals Assigned to Study 
Animal Body Weight Time 
ID Gender (kg) Point (hr) 
______________________________________ 
R4759 M 3.2 24 
R3524 F 4.4 24 
R4797 M 3.7 60 
R2700 F 3.5 60 
R4778 M 3.5 120 
R4784 F 3.6 120 
R4758 M 3.6 168 
R4781 F 3.6 168 
R4796 M 3.1 384 
R4782 F 3.5 384 
R4764 M 4.2 576 
R4768 F 3.5 576 
______________________________________ 
A full necropsy was conducted on all animals. The following tissues were 
collected from each animal: brain, heart, pancreas, prostate, ovaries, 
spleen, intestine, kidney cortex, kidney medulla, liver, mesenteric and 
mandibular (combined, M & M) lymph nodes, axillary and inguinal (combined, 
A & I) lymph nodes, lung, back skin, and hand skin. Whole blood and tissue 
samples were extracted and analyzed by capillary gel electrophoresis 
(CGE). 
Sample Extraction in Whole Blood: Blood samples were vortexed and an 
aliquot (100 .mu.l) was measured into a 2 mL Fastprep tube (BIO101, Inc., 
Vista, Calif.) containing approximately 1/4 inch of homogenization beads. 
Following the addition of 390 .mu.L PBS, 5 .mu.L 10% NP-40, and 5 .mu.L 
100 .mu.M T.sub.27 (a 27-mer phosphorothioate oligodeoxythimidine used as 
the internal standard), the mixture was homogenized in a Savant Tissue 
Disrupter (BIO101, Inc., Vista, Calif.). The samples were then extracted 
with phenol-chloroform to remove proteins and lipids; oligonucleotides 
remained in the aqueous phase. To enhance the separation of the aqueous 
phase from the organic phase, an aliquot of phase lock gel (Intermountain 
Scientific Corp., Kaysville, Utah) was added to the samples after adding 
phenol-chloroform. The phenol-chloroform layer was back-extracted with 500 
.mu.L of water and the aqueous phases were pooled. The aqueous phase was 
then evaporated to dryness, resuspended with 5 mL SAX loading buffer 
(containing 10 mM Tris-HCl, 0.5 M KCl, and 20% acetonitrile, at pH 9.0) in 
preparation for solid phase extraction. 
Sample Extraction in Tissue: The method for tissue sample extraction 
combined the proteinase K digestion method previously used for extraction 
of oligonucleotides from tissues (Cossum et al., J. Pharmacol. Exp. 
Therap. 269:89, 1994) with the solid phase extraction method (Leeds et 
al., Analytical Biochem. 235:36, 1996). Monkey tissues were weighed, 
homogenized in a Bio Savant, and incubated for 24 hours at 37.degree. C. 
in a 2.0 mg/mL proteinase K solution of digestion buffer consisting of 
0.5% Non-Idet P-40 (NP-40) with 20 mM Tris-HCl (pH 8.0), 20 mM EDTA, and 
100 mM NaCl. An appropriate amount of T.sub.27 ranging from 0.5 to 10 
.mu.M, was added for quantitation by capillary electrophoresis. The 
aqueous layer was then extracted with phenol-chloroform, the 
phenol-chloroform layer was back-extracted with 500 .mu.L of water and the 
aqueous phases were pooled. The aqueous layer was extracted again with 
chloroform to remove the phenol. Samples were then evaporated to dryness, 
resuspended in 200 .mu.l concentrated ammonium hydroxide and incubated at 
55.degree. C. for 12 to 24 hours. The samples were then re-evaporated to 
dryness, resuspended with 5 mL SAX loading buffer (containing 10 mM 
Tris-HCl, 0.25 M KCl, and 20% acetonitrile, at pH 9.0) in preparation for 
solid phase extraction. 
Solid Phase Extraction: After phenol-chloroform extraction, both blood and 
tissue samples were further extracted using a J&W Scientific, Inc. 
(Folson, Calif.) strong anion exchange (SAX) SPE column. For solid phase 
extraction, the column was prepared for use by wetting it with 1 ml of 
acetonitrile followed by 1 ml of distilled water. The column was then 
equilibrated with 3 ml of loading buffer prior to loading the tissue or 
blood extracts. After loading the extracts, the anion exchange SPE column 
was washed with 3 mL of the loading buffer, and the oligonucleotides were 
eluted with 3 mL of elution buffer (containing 10 mM Tris-HCl , 0.5 M KCl, 
and 1.0 M NaBr, and 30% acetonitrile, at pH 9.0). The eluted samples were 
diluted and were then desalted using a reversed-phase solid phase 
extraction column. 
The reversed-phase solid phase extraction column (Isolute, from Alltech 
Associates, Inc., Deerfield, Ill.) was pre-equilibrated with 1 mL 
acetonitrile, 1 mL distilled water, and 3 mL eluting buffer (10 mM 
Tris-HCl, 0.5 M KCl, and 1.0 M NaBr, at pH 9.0). After the diluted eluate 
from the anion exchange column was loaded onto reverse phase SPE column, 
it was washed with 5 mL of distilled water, and purified oligonucleotide 
was then eluted using 3 mL of fresh 20% acetonitrile in distilled water. 
After evaporation to dryness, the samples were resuspended in 40 .mu.l 
distilled water, and a 15 .mu.l aliquot was desalted by dialysis on a 
Millipore VS membrane (pore size 0.025 microns, Millipore Corp., Bedford, 
Mass.) floating in a 60.times.15 mm polystyrene petrie dish (Becton 
Dickinson and Co., Lincoln Park, N.J.) containing distilled water prior to 
loading into microvials for analysis by capillary electrophoresis. 
Capillary Electrophoresis: A Beckman P/ACE Model 5010 capillary 
electrophoresis instrument (Beckman Instruments, Inc., Fullerton, Calif.) 
was used for gel-filled capillary electrophoresis analysis. Samples were 
electrokinetically injected using an applied voltage between 3-10 kV for a 
duration ranging from 3-20 seconds. Length-based separation of the 
oligonucleotides was achieved by using a coated-capillary (Bio-Rad 
Laboratory, Hercules, Calif.) with Beckman eCAP ssDNA 100-R Gel. 
Separation was optimized using a constant applied voltage of 20 kV and a 
temperature of 40.degree. C. Oligonucleotide peaks were detected by UV 
absorbance at 260 nm. Beckman System Gold Software on the P/ACE instrument 
was used to determine the areas under the curve for oligonucleotide peaks 
in the resultant electropherograms. A peak area threshold of 0.01 area 
units and minimum peak width of 0.08 min were the standard integration 
parameters (Leeds et al., Analytical Biochem. 235:36, 1996). 
Quantitation: Quantitation of intact ISIS 2503 and metabolites for whole 
blood samples was based on the calibration curve with T.sub.27 as the 
internal standard. The limit of quantitation for this assay has been 
estimated to be 0.10 .mu.g/mL oligonucleotide in blood. In contrast, the 
concentrations of ISIS 2503 and metabolites in the tissue samples were 
calculated from the ratio of the absorbencies, based only on the starting 
concentration of internal standard (T.sub.27) added to the samples using 
the following equation: 
EQU C.sub.2 =C.sub.1 (E.sub.1 /E.sub.2)[(A.sub.2 /T.sub.m2)/(A.sub.1 /T.sub.m1) 
] 
Where C.sub.1 =concentration of the internal standard, C.sub.2 
=concentration of the analyte (ISIS 2503 or metabolites), E.sub.1 =molar 
extinction coefficient of the internal standard, E.sub.2 =molar extinction 
coefficient of the analyte, A.sub.1 =area of the internal standard peak, 
A.sub.2 =area of the analyte peak, T.sub.m1 =migration time of the 
internal standard peak, and T.sub.m2 =migration time of the analyte peak. 
Calculations of extinction coefficients for ISIS 2503, metabolites, and 
T.sub.27 are made using a program which calculates the sums of the 
extinction coefficients from the individual bases according to the base 
composition. For the calculation of extinction coefficients, metabolites 
are assumed, to be generated by loss of nucleotide from the 3'-end. The 
limit of quantitation for this assay has been estimated to be 0.10 .mu.g/g 
oligonucleotide in tissue. 
Pharmacokinetic Analysis: Inspection of the semi-logarithmic plots of 
intact ISIS 2503 (full length) blood level-versus time curves indicated 
that they could be described by a monoexponential equation. First order 
elimination was assumed. Initial estimates of parameters were obtained by 
linear regression of the terminal concentration time points. Nonlinear 
regression was accomplished using a one compartment model for each 
individual animal (WinNonlin 1.0, Scientific Consulting, Inc., Apex, 
N.C.). A uniform weight of 1 was used for all blood-level data. Four of 
the animals were excluded from complete individual pharmacokinetic 
analysis of blood concentrations because they were sacrificed before a 
complete blood profile could be collected (2 at 24 hours and 2 at 60 
hours). 
Tissue elimination was analyzed by noncompartmental methods using WinNonlin 
1.0. Tissue half-lives were estimated by linear regression analysis of the 
log-linear terminal phase of the tissue concentration-time curve. The area 
under the tissue concentration-time curve (AUC.sub.0.fwdarw..infin.) and 
the area under the first moment of the concentration-time curve 
(AUMC.sub.0.fwdarw..infin.) were calculated using the linear trapezoidal 
rule, up to the last measured time point, plus the extrapolated area. The 
mean residence time (MRT) was calculated as the ratio of the 
AUMC(.sub.0.fwdarw..infin.) to the AUC(.sub.0.fwdarw..infin.). 
Statistics: Statistical analysis for gender difference of kinetic 
parameters was performed by F-test (Excel 6.0, Microsoft Corp., Redmond, 
Wash.) for the analysis of variance, and t-test (Excel 6.0) for the 
analysis of mean at the p=0.05 level. Descriptive statistics were used to 
present data summaries for pharmacokinetic parameter estimates and blood 
concentration data. 
B. Results 
Blood Pharmacokinetics and Metabolism: FIGS. 6 and 7 are representative 
electropherograms of (a) liposomal and (b) saline formulations of ISIS 
2503 in blood and kidney samples, respectively, from monkeys after i.v. 
infusions of 10 mg/kg of the respective formulations. The saline 
formulated oligonucleotide samples were taken either 1 hour after 
initiation of a 2-hour infusion in the case of plasma or 48 hours after 
the last 2-hour infusion of 14 doses administered every other day (q2d). 
In contrast, the liposomal oligonucleotide formulations were evaluated at 
60 hours after an 0.5 hour infusion. Despite the longer period during 
which the liposomal oligonucleotide formulations were exposed to 
degradative processes in the tissues, the ISIS 2503 remained in a 
significantly more intact state than the saline-formulated oligonucleotide 
(as can be seen by comparing panel (a) in FIGS. 6 and 7 to panel (b)). 
The time course of the clearance of ISIS 2503 and oligonucleotide 
metabolites from blood after administration of the liposomal 
oligonucleotide composition is prolonged (see Tables 12 and 13 and FIG. 
1). Maximum blood concentration (C.sub.max) of intact ISIS 2503 was 
approximately 90 .mu.g/mL and was observed at the end of the 30-minute 
infusion. ISIS 2503 concentration did not decrease by 1 hour after 
infusion but remained at c. 90 .mu.g/mL). Concentrations in blood 
decreased slowly to approximately 10 .mu.g/mL at 144 hours after infusion. 
In these experiments, the method for quantitating ISIS 2503 concentrations 
in blood or tissues does not distinguish between free and liposome 
encapsulated oligonucleotides, and both parent compound and total 
oligonucleotide concentrations are presented because many of the 
chain-shortened metabolites retain physical and chemical properties 
similar to those of the parent compound ISIS 2503 and thus may potentially 
have some biological activity. 
Pharmacokinetic parameter estimates for males and females were averaged 
since statistical analysis indicated no significant gender differences. 
The mean blood half-life for intact ISIS 2503 was 57.2 hours (Table 14). 
The concentration of ISIS 2503 in blood generally fell below the limit of 
detection after 168 hours. The observed concentrations in blood were less 
well predicted by the model after 120 hours (FIG. 1). The values predicted 
by the model were higher than the actual values observed at the late time 
points suggesting that there were alterations in the kinetics after 
extended circulation times. This phenomenon may be a result of the loss of 
liposome integrity after prolonged circulation in blood. The average total 
body clearance and Vd.sub.ss were 1.53.+-.0.28 mL/hr/kg and 123.+-.28 
mL/kg, respectively. The volume of distribution was larger than the blood 
compartment (73.4 mL/kg) indicating some distribution into tissues, but 
also indicated a large portion of administered dose remained in the 
general circulation (Davies et al., Pharm. Res. 10:1093, 1993). 
The metabolites of ISIS 2503 in blood co-migrated on CGE with ISIS 2503 
shortened by removal of 1 or 2 bases (19-mer and 18-mer; in Tables 12, 13 
and 15 these are referred to as "n-1" and "n-2," respectively). 
Concentrations of metabolites observed were an order of magnitude lower 
than that of parent drug. The chain-shortened metabolites cumulatively 
represented approximately 5 to 20% of the total oligonucleotides in blood. 
There was only a small increase in the percentage of oligonucleotide 
metabolites with time. This pattern of very low concentrations of 
metabolites observed in blood suggests that liposomal encapsulation 
protected the oligonucleotide from blood (and tissue) nucleases that might 
otherwise rapidly metabolize the circulating oligonucleotide, and supports 
the notion that there was very little leakage of ISIS 2503 from the 
liposome. 
TABLE 12 
______________________________________ 
Concentrations (.mu.g/mL) of ISIS 2503 and All Detected 
Metabolites in Blood After 0.5 hr Intravenous Infusion 
of 10 mg/kg ISIS 2503 Encapsulated in Sterically 
Stabilized Liposomes to Rhesus Monkeys 
Time No. of Mean Concentration (.mu.g/mL) 
% Full 
(hr) Animals ISIS 2503 
n-1 n-2 Total Length 
______________________________________ 
0 12 89.0.sup.a 
4.27 nd 93.3 95.5 
(24.7).sup.b 
(3.54) (25.4) 
(3.5) 
1 12 90.4 3.44 0.28 94.1 96.1 
(18.9) (2.24) (0.96) 
(20.0) 
(2.5) 
2 12 82.1 3.27 nd 85.4 96.1 
(22.0) (2.10) (22.2) 
(2.6) 
6 12 78.3 2.92 0.31 81.5 96.0 
(20.4) (1.64) (1.06) 
(20.6) 
(2.6) 
12 12 64.6 1.35 0.28 66.2 97.8 
(18.7) (1.58) (0.96) 
(19.7) 
(2.9) 
24 12 63.3 2.08 nd 65.4 96.7 
(18.2) (1.26) (18.3) 
(2.2) 
40 10 50.1 1.45 0.06 51.9 96.9 
(15.0) (1.02) (0.17) 
(15.4) 
(1.8) 
60 10 50.6 1.37 0.29 56.7 92.2 
(9.5) (1.02) (0.91) 
(16.6) 
(14.0) 
96 8 22.6 2.65 nd 25.3 91.9 
(12.9) (2.23) (14.6) 
(7.2) 
120 7 15.7 2.61 nd 18.3 89.2 
(10.2) (1.91) (12.0) 
(8.0) 
144 4 10.6 1.07 nd 11.7 94.2 
(6.3) (2.14) (7.4) (11.6) 
168 4 4.42 0.40 nd 4.82 93.5 
(1.32) (0.69) (1.67) 
(11.3) 
192 2 3.62 0.59 nd 4.21 84.4 
(0.83) (0.60) 
(22.0) 
240 1 2.13 1.19 nd 3.32 64.2 
______________________________________ 
"% Fulllength" = percent of total detectable oligonucleotide represented 
by intact ISIS 2503. 
"nd" = not detected (detection level = 0.10 .mu.g/mL). 
.sup.a Mean value. 
.sup.b Standard deviation. 
TABLE 13 
______________________________________ 
ISIS 2503 and Total Oligonucleotide Whole Blood 
Concentrations after 0.5 hr i.v. Infusion of 10 mg/kg ISIS 2503 
Encapsulated in Sterically Stabilized Liposomes to 
Rhesus Monkeys (Average of Duplicate Analysis) 
Animal 
Gen- Time.sup.a 
.mu.g/mL % 
ID # der (hr) (ISIS 2503 
n-1 n-2 n-3 Total 
Full.sup.b 
______________________________________ 
R4759 M 0 115 1.06 nd nd 116 99.1 
R4759 M 1 120 6.58 3.32 nd 130 92.4 
R4759 M 2 93.8 6.11 nd nd 99.9 93.9 
R4759 M 6 89.8 5.71 3.66 nd 99.2 90.6 
R4759 M 12 78.7 5.30 3.33 nd 87.3 90.1 
R4759 M 24 58.9 4.09 nd nd 63.0 93.5 
R3524 F 0 131 1.11 nd nd 132 99.2 
R3524 F 1 92.2 0.33 nd nd 92.5 99.6 
R3524 F 2 92.9 0.58 nd nd 93.5 99.4 
R3524 F 6 102 0.90 nd nd 103 99.1 
R3524 F 12 98.8 0.77 nd nd 99.6 99.2 
R3524 F 24 76.4 0.11 nd nd 76.5 99.9 
R4797 M 0 84.5 6.33 nd nd 90.8 93.0 
R4797 M 1 85.1 6.42 nd nd 91.5 93.0 
R4797 M 2 71.4 6.30 nd nd 77.7 91.9 
R4797 M 6 66.1 3.65 nd nd 69.8 94.8 
R4797 M 12 68.5 nd nd nd 68.5 100 
R4797 M 24 53.8 3.17 nd nd 56.9 94.4 
R4797 N 40 40.4 0.89 nd nd 41.3 97.8 
R4797 M 60 51.4 0.49 nd nd 96.3 53.4 
R2700 F 0 75.7 6.61 nd nd 82.3 92.0 
R2700 F 1 82.1 6.62 nd nd 88.7 92.5 
R2700 F 2 64.9 6.15 nd nd 71.1 91.3 
R2700 F 6 68.8 6.03 nd nd 74.8 91.9 
R2700 F 12 45.1 nd nd nd 45.1 100 
R2700 F 24 58.7 1.48 nd nd 60.2 97.5 
R2700 F 40 44.4 1.34 0.55 nd 46.3 95.9 
R2700 F 60 61.3 0.72 nd nd 62.0 98.8 
R4778 M 0 96.3 10.3 nd nd 107 90.3 
R4778 M 1 124 1.58 nd nd 126 98.7 
R4778 M 2 133 2.59 nd nd 135 98.1 
R4778 M 6 128 2.78 nd nd 131 97.9 
R4778 M 12 94.7 2.60 nd nd 97.3 97.3 
R4778 N 24 106 2.05 nd nd 108 98.1 
R4778 M 40 44.6 1.52 nd nd 46.1 96.7 
R4778 M 60 42.3 1.03 nd nd 43.4 97.6 
R4778 M 96 5.03 nd nd nd 5.03 100 
R4784 F 0 92.9 10.5 nd nd 103 89.8 
R4784 F 1 89.6 1.94 nd nd 91.5 97.9 
R4784 F 2 95.0 1.96 nd nd 96.9 98.0 
R4784 F 6 85.0 1.34 nd nd 86.3 98.5 
R4784 F 12 45.7 nd nd nd 45.7 100 
R4784 F 24 70.9 2.05 nd nd 72.9 97.2 
R4784 F 40 48.3 nd nd nd 91.5 97.9 
R4784 F 60 53.4 0.97 nd nd 54.3 98.2 
R4784 F 96 43.5 3.99 nd nd 47.4 91.6 
R4784 F 120 23.3 3.45 nd nd 26.8 87.1 
R4758 M 0 109 4.91 nd nd 114 95.7 
R4758 M 1 78.1 3.08 nd nd 81.2 96.2 
R4758 M 2 69.0 2.60 nd nd 71.6 96.4 
R4758 M 6 75.1 2.99 nd nd 78.1 96.2 
R4758 M 12 62.4 0.87 nd nd 63.3 98.6 
R4758 M 24 85.6 3.35 nd nd 89.0 96.2 
R4758 M 40 48.1 1.22 nd nd 49.3 97.5 
R4758 M 60 52.1 0.71 nd nd 52.8 98.7 
R4758 M 96 32.2 4.78 nd nd 37.0 87.1 
R4758 M 120 18.3 2.48 nd nd 20.8 88.1 
R4758 M 144 17.3 nd nd nd 17.3 100 
R4758 M 168 11.8 nd nd nd 11.8 100 
R4781 F 0 90.6 4.07 nd nd 94.6 95.7 
R4781 F 1 96.1 4.24 nd nd 100 95.8 
R4781 F 2 105 5.26 nd nd 110 95.2 
R4781 F 6 67.1 3.12 nd nd 70.2 95.6 
R4781 F 12 43.9 nd nd nd 43.9 100 
R4781 F 24 53.4 2.66 nd nd 56.0 95.3 
R4781 F 40 51.8 1.57 nd nd 53.4 97.1 
R4781 F 60 52.9 0.85 nd nd 53.8 98.4 
R4781 F 96 26.0 4.48 nd nd 30.5 85.3 
R4781 F 120 15.5 4.28 nd nd 19.7 78.3 
R4781 F 144 3.46 nd nd nd 3.46 100 
R4781 F 168 2.93 nd nd nd 2.93 100 
R4782 M 0 30.9 0.30 nd nd 31.2 99.1 
R4782 M 1 72.4 1.42 nd nd 73.8 98.1 
R4782 M 2 60.4 1.16 nd nd 61.5 98.1 
R4782 M 6 62.1 1.32 nd nd 63.4 97.9 
R4782 M 12 58.3 1.05 nd nd 59.4 98.2 
R4782 M 24 51.8 0.89 nd nd 52.7 98.3 
R4782 M 40 37.5 2.56 nd nd 40.1 93.6 
R4782 M 60 43.8 3.84 2.89 nd 50.5 86.7 
R4782 M 96 26.8 3.50 nd nd 30.3 88.5 
R4782 M 120 19.1 3.52 nd nd 22.6 84.4 
R4782 M 144 14.1 4.27 nd nd 18.4 76.8 
R4782 M 168 5.45 nd nd nd 5.45 100 
R4782 M 192 4.64 nd nd nd 4.64 100 
R4796 F 0 76.1 2.70 nd nd 78.8 96.6 
R4796 F 1 106 4.74 nd nd 111 95.7 
R4796 F 2 64.1 2.36 nd nd 66.4 96.5 
R4796 F 6 73.4 3.23 nd nd 76.6 95.8 
R4796 F 12 63.0 1.40 nd nd 64.4 97.8 
R4796 F 24 49.1 0.89 nd nd 50.0 98.2 
R4796 F 40 43.1 2.13 nd nd 45.2 95.3 
R4796 F 60 43.7 1.21 nd nd 44.9 97.3 
R4796 F 96 21.1 4.44 nd nd 25.5 82.6 
R4796 F 120 29.3 4.58 nd nd 33.8 86.5 
R4796 F 144 7.42 nd nd nd 7.42 100 
R4796 F 168 4.89 1.19 nd nd 6.08 80.4 
R4796 F 192 2.61 1.18 nd nd 3.79 68.9 
R4796 F 240 2.13 1.19 nd nd 3.32 64.2 
R4768 F 0 80.5 1.68 nd nd 82.1 98.0 
R4768 F 1 76.4 1.35 nd nd 77.8 98.3 
R4768 F 2 72.2 1.30 nd nd 73.5 98.2 
R4768 F 6 69.6 1.46 nd nd 71.0 97.9 
R4768 F 12 69.4 1.31 nd nd 70.7 98.1 
R4768 F 24 50.4 0.84 nd nd 51.3 98.4 
R4768 F 40 90.6 3.25 nd nd 93.8 96.5 
R4768 F 60 68.7 2.44 nd nd 71.1 96.6 
R4768 F 96 5.61 nd nd nd 5.61 100 
R4768 F 120 3.24 nd nd nd 3.24 100 
R4764 M 0 85.7 1.64 nd nd 87.4 98.1 
R4764 M 1 61.8 2.99 nd nd 64.8 95.4 
R4764 M 2 63.9 2.89 nd nd 66.8 95.7 
R4764 M 6 52.8 2.57 nd nd 55.4 95.4 
R4764 M 12 46.7 2.88 nd nd 49.6 94.2 
R4764 M 24 44.5 3.35 nd nd 47.8 93.0 
R4764 M 40 52.0 nd nd nd 52.0 100 
R4764 M 60 37.0 1.40 nd nd 38.4 96.4 
R4764 M 96 20.8 nd nd nd 20.8 100 
R4764 M 120 1.16 nd nd nd 1.16 100 
______________________________________ 
"nd" = not detected 
.sup.a Time is given in hours. 
.sup.b "%Full" = % fulllength oligonucleotide detected. 
TABLE 14 
______________________________________ 
Summary of Estimated Pharmacokinetic parameters 
(n = 8) for ISIS 2503 (10 mg/kg) Encapsulated 
in Sterically Stabilized Liposomes Administered 
to Rhesus Monkeys by 0.5 hr Infusion 
Parameter Mean SD CV %.sup.b 
______________________________________ 
AUC (.mu.g.cndot.hr/mL) 
6760 1240 18.4 
K.sub.10 -t.sub.1/2 (hr) 
57.2 14.2 24.9 
Cmax (.mu.g/mL).sup.a 
90.4 23.0 26.9 
C1 (mL/hr/kg) 
1.52 0.28 18.3 
MRT (hr) 82.5 20.5 24.9 
Vd.sub.SS (mL/kg) 
123 28 22.3 
______________________________________ 
.sup.a Data obtained from 12 animals. 
.sup.b CV% = Coefficient of Variation = (Standard deviation / Mean) 
.times. 100. 
In sum, encapsulation of phosphorothioate oligonucleotide into liposomes 
greatly modified oligonucleotide pharmacokinetics. ISIS 2503 in liposomes 
was cleared slowly from the blood compared with previous experience with 
unencapsulated oligonucleotide. Phosphorothioate oligonucleotide 
concentration following intravenous infusion of unencapsulated 
oligonucleotide in monkeys decreases rapidly from circulation with an 
average distribution half-life of 36-83 minutes (Agrawal et al., Clinical 
Pharmacokinet. 28:7, 1995). In contrast, the distribution phase half-life 
of ISIS 2503 in this liposome formulation was markedly longer 
(approximately 57 hours), and resulted in an AUC that was approximately 
70-fold greater than an equivalent dose of an unencapsulated 
oligonucleotide. 
Tissue Distribution, Elimination Kinetics and Metabolism: ISIS 2503 was 
distributed widely into all the tissues analyzed. The highest tissue 
concentrations of total oligonucleotide were measured in liver, with 
slightly lower concentrations detected in spleen, followed by the lymph 
nodes, lung, hand skin, kidney cortex and medulla, heart, back skin, 
pancreas, colon, and brain (Table 15, FIGS. 2-5). It appears that the 
primary organs of ISIS 2503 distribution were the organs of the 
reticulo-endothelial system. Largest sample to sample variability was 
observed in skin where, presumably, the thickness of the skin layer 
collected varied greatly. Tissue distribution was also greatly different 
for the liposome formulation compared with unencapsulated oligonucleotides 
studied previously (Agrawal et al., Clinical Pharmacokinet. 28:7, 1995; 
Cossum et al., J. Pharmacol. Exp. Therap. 267:1181, 1993), where the 
highest concentration of oligonucleotide is consistently observed in 
kidney. 
Relatively long half-lives of ISIS 2503 were observed in all tissues 
studied (Table 16). The mean residence time (15 days) of ISIS 2503 in the 
kidney cortex was the longest among all the tissues examined. This slow 
clearance may represent slow metabolism in the kidney or, alternatively, 
the kidney may take up free oligonucleotide from the circulation as it is 
slowly released from liposomes, thus giving the appearance of prolonged 
half-life. Uptake was slow in all tissues with a time to peak 
concentration from 1 to 7 days. The concentration of ISIS 2503 in brain, 
prostate, and ovaries was still increasing up to seven days after dosing. 
However, the concentration of ISIS 2503 in these tissues was below the 
limit of quantitation for the CGE analysis by 384 hours (the next data 
point after the 7-day time point). 
TABLE 15 
__________________________________________________________________________ 
Average (n = 2) Tissue Concentrations (.mu.g/g) of ISIS 2503 
and All Detected Metabolites After 0.5 hr Intravenous 
Infusion of 10 mg/kg ISIS 2503 Encapsulated in Sterically 
Stabilized Liposomes to Rhesus Monkeys 
Time 
(.mu.g/mL) % Full 
(hr) 
2503 
n + 1 
n - 1 
n - 2 
n - 3 
n - 4 
n - 5 
n - 6 
Total 
Length 
__________________________________________________________________________ 
Kidney Cortex 
24 
0.37 
13.4 
0.73 
0.38 
0.46 
0.24 
0.11 
0.07 
15.7 
77.2 
60 
0.24 
11.2 
0.61 
0.34 
0.13 
0.09 
0.05 
0.04 
12.7 
88.7 
120 
0.12 
4.87 
0.44 
0.38 
0.38 
0.17 
0.20 
0.06 
6.66 
72.1 
168 
0.18 
6.00 
0.72 
0.22 
0.94 
0.48 
0.36 
0.16 
9.31 
66.2 
384 
0.13 
1.97 
0.36 
0.43 
0.52 
0.39 
0.22 
0.10 
4.35 
45.3 
576 
0.07 
0.90 
0.19 
0.20 
0.09 
nd nd nd 1.45 
62.1 
Kidney Medulla 
24 
0.47 
14.3 
0.33 
0.08 
0.17 
0.18 
0.07 
nd 15.6 
92.6 
60 
0.16 
13.3 
0.51 
0.15 
0.28 
0.11 
0.05 
0.09 
14.8 
91.3 
120 
0.18 
7.48 
0.53 
0.39 
0.40 
0.28 
0.29 
0.11 
10.1 
73.2 
168 
0.38 
7.17 
0.69 
0.39 
0.54 
0.38 
0.23 
0.14 
10.6 
67.8 
384 
0.06 
2.40 
0.33 
0.41 
0.17 
0.12 
0.21 
0.21 
4.05 
59.3 
576 
0.05 
0.93 
0.32 
0.26 
0.24 
0.06 
0.05 
0.06 
1.99 
46.7 
Liver 
24 
0.87 
46.7 
2.22 
0.65 
0.99 
0.44 
0.15 
0.03 
52.1 
90.0 
60 
0.22 
94.1 
4.60 
1.78 
2.73 
0.93 
0.59 
0.47 
119 
80.0 
120 
0.50 
88.9 
7.17 
3.22 
4.55 
1.71 
1.13 
1.09 
110 
80.9 
168 
0.91 
106 10.2 
4.62 
7.59 
2.76 
1.94 
1.64 
140 
72.8 
384 
0.28 
13.45 
3.27 
1.76 
3.35 
1.51 
1.15 
1.12 
28.9 
46.5 
576 
0.17 
7.49 
1.96 
0.84 
1.94 
0.81 
0.39 
0.20 
15.3 
49.0 
Spleen 
24 
0.51 
62.0 
2.92 
0.93 
0.87 
0.15 
nd nd 67.4 
92.0 
60 
0.60 
84.4 
4.17 
1.85 
2.19 
1.39 
0.62 
0.51 
96.9 
87.0 
120 
0.26 
90.0 
5.98 
2.63 
3.65 
1.61 
0.94 
1.62 
106 
83.8 
168 
0.87 
94.0 
6.20 
2.54 
3.24 
1.15 
0.63 
0.39 
109 
85.9 
384 
0.12 
26.6 
2.75 
1.48 
2.40 
1.40 
1.26 
1.20 
43.1 
61.6 
576 
0.08 
26.6 
1.78 
0.71 
1.18 
0.56 
0.42 
0.51 
32.7 
81.3 
Back Skin 
24 
0.16 
3.63 
nd nd nd nd nd nd 3.79 
94.5 
60 
0.04 
2.80 
0.07 
0.03 
0.03 
nd nd nd 2.96 
95.0 
120 
0.03 
4.63 
0.16 
0.06 
0.06 
0.02 
0.01 
nd 4.96 
93.3 
168 
0.08 
11.1 
0.53 
0.18 
0.19 
0.06 
0.04 
0.01 
12.2 
93.2 
384 
0.02 
0.24 
0.02 
nd nd nd nd nd 0.26 
88.7 
576 
0.01 
0.09 
nd nd nd nd nd nd 0.10 
89.5 
Hand Skin 
24 
0.14 
13.6 
0.23 
0.03 
0.04 
0.02 
0.02 
0.01 
14.1 
96.4 
60 
0.13 
23.9 
0.68 
0.20 
0.11 
0.09 
0.06 
0.03 
25.2 
94.2 
120 
0.17 
24.6 
0.92 
0.22 
0.29 
0.08 
0.04 
0.03 
26.4 
93.3 
168 
0.32 
25.7 
1.02 
0.27 
0.39 
0.17 
0.21 
0.07 
28.3 
92.2 
384 
0.13 
13.54 
1.11 
1.46 
0.47 
0.11 
0.05 
0.04 
17.02 
79.6 
576 
0.10 
1.10 
0.13 
0.06 
0.16 
0.03 
nd nd 1.56 
70.5 
A & I Lymph Nodes 
24 
0.26 
27.7 
0.85 
0.26 
0.17 
0.02 
nd nd 29.3 
94.6 
60 
0.05 
43.5 
2.07 
0.57 
0.96 
0.38 
0.13 
nd 47.6 
91.2 
120 
0.24 
68.2 
4.16 
1.61 
3.10 
1.08 
0.23 
0.07 
78.7 
86.6 
168 
1.03 
42.4 
4.05 
2.03 
2.99 
1.30 
0.41 
0.36 
55.4 
77.0 
384 
0.26 
29.7 
2.33 
0.99 
1.85 
0.61 
0.61 
0.46 
39.2 
75.8 
576 
0.23 
14.6 
0.79 
0.28 
0.55 
0.26 
0.26 
0.14 
18.0 
81.1 
M & M Lymph Nodes 
24 
0.21 
9.47 
0.49 
0.37 
0.11 
0.11 
0.07 
0.05 
11.0 
86.0 
60 
0.23 
14.4 
1.03 
0.82 
0.50 
0.29 
0.22 
0.16 
17.8 
81.1 
120 
0.22 
43.8 
3.18 
1.38 
2.19 
0.65 
0.31 
0.18 
52.0 
83.9 
168 
0.18 
38.3 
3.80 
1.97 
2.61 
1.02 
0.56 
0.43 
49.1 
79.1 
384 
0.10 
15.1 
1.28 
0.58 
1.08 
0.44 
0.38 
0.47 
21.8 
69.3 
576 
0.05 
8.01 
0.37 
0.18 
0.20 
0.09 
0.05 
0.07 
9.24 
88.9 
Brain 
24 
0.06 
2.21 
0.03 
nd nd nd nd nd 2.30 
96.0 
60 
0.06 
1.28 
0.01 
nd nd nd nd nd 1.35 
92.5 
20 
0.06 
0.94 
nd nd nd nd nd nd 1.00 
94.2 
168 
0.05 
2.27 
nd nd nd nd nd nd 2.32 
97.6 
Colon 
24 
0.05 
5.44 
0.13 
0.05 
0.01 
0.01 
0.01 
0.01 
5.71 
95.2 
60 
0.14 
4.02 
0.19 
0.11 
0.09 
0.03 
0.02 
nd 4.65 
86.3 
120 
0.03 
6.88 
0.65 
0.35 
0.39 
0.05 
0.01 
nd 8.35 
82.3 
168 
0.08 
6.48 
0.47 
0.55 
0.06 
0.05 
0.04 
0.01 
7.75 
86.8 
Heart 
24 
0.04 
10.3 
0.28 
nd nd nd nd nd 10.6 
97.2 
60 
0.12 
6.28 
0.20 
0.06 
0.06 
nd nd nd 6.72 
94.2 
120 
0.05 
3.72 
0.11 
nd nd nd nd nd 3.89 
95.8 
168 
0.07 
2.78 
0.10 
0.05 
nd nd nd nd 2.99 
91.3 
Lung 
24 
0.14 
22.5 
0.44 
0.20 
0.03 
nd nd nd 23.3 
96.6 
60 
0.05 
26.1 
0.22 
0.02 
nd nd nd nd 26.4 
98.5 
120 
0.10 
6.95 
0.22 
nd nd nd nd nd 7.28 
93.8 
168 
0.19 
4.45 
0.09 
0.03 
0.02 
0.01 
nd nd 4.79 
90.5 
Pancreas 
24 
0.11 
3.63 
0.14 
0.20 
0.35 
0.36 
0.72 
0.60 
9.41 
41.2 
60 
0.12 
5.53 
0.30 
0.13 
0.30 
0.19 
0.44 
0.63 
10.6 
54.6 
120 
0.09 
2.62 
0.16 
0.12 
0.27 
0.35 
0.24 
0.46 
4.75 
53.8 
168 
0.07 
1.78 
0.25 
0.33 
0.09 
0.06 
0.30 
0.55 
3.46 
53.2 
Prostate 
24 
0.11 
3.02 
0.01 
0.03 
nd nd nd nd 3.13 
96.5 
60 
0.12 
3.72 
0.17 
0.13 
nd nd nd nd 4.28 
81.5 
120 
0.09 
2.35 
0.16 
0.09 
0.42 
0.19 
0.26 
0.11 
3.75 
64.7 
168 
0.07 
6.89 
0.49 
0.21 
0.36 
0.12 
0.10 
0.14 
8.63 
80.1 
Ovary 
24 
0.09 
5.18 
0.20 
0.16 
0.13 
nd nd nd 5.77 
89.8 
60 
0.52 
6.93 
0.29 
0.17 
0.11 
0.06 
0.01 
0.03 
8.15 
85.1 
120 
0.13 
5.49 
0.29 
0.22 
0.15 
1.97 
0.08 
0.05 
8.63 
65.2 
168 
0.26 
6.58 
0.91 
0.41 
0.79 
0.30 
0.26 
0.43 
10.5 
62.7 
__________________________________________________________________________ 
"nd" = not detected (detection level = 0.10 .mu.g/mL) 
TABLE 16 
______________________________________ 
Estimated Tissue Pharmacokinetic parameters for ISIS 2503 
(10 mg/kg) Encapsulated in Sterically Stabilized Liposomes 
Administered to Rhesus Monkeys by 0.5 hr Intravenous Infusion 
T.sub.1/2 
MRT T.sub.max 
C.sub.max 
Tissue (day) (day) (day) 
(.mu.g/g) 
______________________________________ 
Kidney Cortex 11 15 1 23.3 
Kidney Medulla 5.6 8.2 1 22.6 
Liver 4.2 8.1 7 160 
A & I Lymph Node 
NA.sup.a 
18 5 97.0 
M & M Lymph Node 
7.7 13 5 57.4 
Spleen 9.7 14 5 107 
Back Skin 3.1 7.0 7 16.5 
Hand Skin 4.2 10 2 43.8 
Lung 2.0 3.4 2 32.3 
Heart 3.0 5.7 1 12.7 
Pancreas 3.2 6.4 2 9.43 
Brain NA NA 1 2.41 
Colon NA NA 7.sup.b 
8.24 
Ovary NA NA 2 6.92 
Prostate NA NA 7.sup.b 
6.89 
______________________________________ 
.sup.a "NA" = not available 
.sup.b Concentration was still increasing at the last analyzed time point 
The appearance of metabolites was low even 576 hours after infusion (Table 
15). Very low relative percentage of metabolites were observed for all the 
organs (.about.10-20%) except for the liver, the kidney cortex, and the 
pancreas (.about.30-60%). Higher concentration of oligonucleotide 
metabolites was observed as early as 24 hours after infusion in the 
pancreas. Although not wishing to be bound by any particular theory, this 
phenomenon could be related to the activity of lipases in this organ 
allowing more ISIS 2503 to escape from liposomes and be metabolized 
(McNeely et al., "Pancreas Function" In: Clinical Chemistry: Theory, 
Analysis, and Correlation, Kaplan and Pesce, eds., The C.V. Mosby Company, 
St. Louis, pp. 390-397, 1989). At later time points (.gtoreq.120 hr), 
increasing concentrations of chain-shortened oligonucleotide metabolites 
were seen in liver and kidney. Kidney and/or liver may also play a role in 
the degradation of liposomes but, alternatively, may be primary sites of 
free oligonucleotide and metabolite distribution. 
In addition to chain-shortened metabolites, there were also UV absorbing 
peaks that migrated more slowly than parent oligonucleotide. Slower 
migrating oligonucleotide peaks have been identified for other 
phosphorothioate oligonucleotides in tissue. Slower migration suggests 
that the mass to charge ratio was increased either from the addition of a 
substituent or loss of charge. These metabolites are thought to represent 
intact drugs plus an additional substituent possibly an additional 
nucleotide or two (Griffey et al., J. Mass. Spec. 32:305, 1997). Thus, 
while not wishing to be bound by any particular theory, it is possible 
that the slower migrating peak observed in these studies is such a 
lengthened metabolite, and this peak is thus referred to as "n+1" in Table 
15. 
Toxicokinetic Summary and Conclusions: In this investigation, it has been 
demonstrated that ISIS 2503 in a sterically stabilized liposome 
formulation has a markedly prolonged circulation time. Maximum 
concentration (C.sub.max) in blood is achieved at the end of infusion and 
it is approximately 90 .mu.g/mL. Pharmacokinetic modeling of ISIS 2503 
indicates a slow distribution process with a half-life of approximately 57 
hours. The half-life of ISIS 2503 in this formulation is significantly 
greater than that observed for unencapsulated oligonucleotides suggesting 
that ISIS 2503 in liposomes is slowly distributed to tissues and protected 
from metabolism in blood. Unencapsulated oligonucleotide is cleared from 
plasma by a combination of metabolism and tissue distribution. 
Unencapsulated oligonucleotide has been reported to have half-lives 
ranging from 36-83 minutes. With this formulation there appears to be 
little metabolism, and clearance from blood is slow with a half-life of 57 
hours. Clearly this formulation has altered the kinetics of circulating 
oligonucleotide. While not wishing to be bound by any particular theory, 
because tissue distribution is the primary route for both liposomal 
oligonucleotide and unencapsulated oligonucleotide clearance from 
circulation, slower kinetics of liposome uptake seen in tissue may explain 
the prolonged circulation of oligonucleotide in this study. 
Liposomal ISIS 2503 is widely distributed into all tissues tested, in 
descending maximum concentration (C.sub.max) order, liver&gt; spleen&gt; lymph 
nodes&gt; hand skin&gt; lung&gt; kidney&gt; back skin&gt; heart&gt; pancreas&gt; colon&gt; ovary&gt; 
prostate&gt; brain. Intact ISIS 2503 is the predominant oligonucleotide 
species measured indicating a slow metabolism in tissues and supporting 
the concept that liposomes remain intact in tissues. The apparent increase 
in metabolites observed in kidney, liver, and pancreas could be explained 
by digestion of the liposomes in these tissues, or preferential uptake of 
metabolites from circulation by these tissues. The high oligonucleotide 
concentrations in liver and spleen suggest that liposome formulations are 
primarily removed from blood by the reticulo-endothelial system. The 
persistence and abundance of intact ISIS 2503 in tissues is best explained 
by the protection from nucleases afforded by liposomal encapsulation. 
Example 5 
Evaluation of the Antitumor Activity of Sterically Stabilized Liposomes 
Comprising Antisense Oligonucleotides 
One advantage of some sterically stabilized liposomes is their ability to 
deliver conventional chemotherapeutic agents to tissues, particularly 
tumors, other than those of the reticuloendothelial system (RES) (Gabizon 
et al., Proc. Natl. Acad. Sci. U.S.A. 85:6949, 1988; Papahadjopoulos et 
al., Proc. Natl. Acad. Sci. U.S.A. 88:11460, 1991). In disease states 
where leaky vasculature is characteristic (e.g., inflammation, tumors), 
prolonging the circulation time via the liposomal oligonucleotide 
formulations of the invention may allow for more effective delivery of 
oligonucleotide as well as providing for a less frequent oligonucleotide 
dosing interval. In order to test the efficacy of liposomal 
oligonucleotide formulations against tumors, a human-mouse xenograft model 
was used. 
A. Experimental Design and Methods 
Liposomes: Sterically stabilized liposomes comprising DSPE-MPEG.sub.2000 in 
the lipid phase and ISIS 2503 in the aqueous phase were prepared as in 
Example 3. ISIS 2503 loaded sterically stabilized liposomes comprising the 
monosialoganglioside G.sub.M1 instead of DSPE-MPEG.sub.2000 were prepared 
in like fashion, except that monosialoganglioside G.sub.M1 (Sigma Chemical 
Co., St. Louis, Mo.) was substituted for DSPE-MPEG.sub.2000 at the same 
final molar concentration. In some experiments, ISIS 13177 was used as a 
control. This phosphorothioate oligodeoxynucleotide has the nucleotide 
sequence 5'-TCAGTAATAGCCCCACATGG (SEQ ID NO: 26). In other experiments, 
ISIS 2105 was used as a control. This oligonucleotide has the nucleotide 
sequence 5'-TTGCTTCCATCTTCCTCGTC (SEQ ID NO: 27), which is targeted to the 
E2 gene of papillomavirus HPV-11. Saline formulations of ISIS 2503 were 
also included as controls in the experiments. 
Xenografts: Xenografts of human tumor cell lines into BALB/c nude mice were 
performed essentially as described by Dean et al. (Cancer Res. 56:3499, 
1996). Cell lines NCI-H69 and MIA PaCa-2 are available from the American 
Type Culture Collection (A.T.C.C., Rockville, Md.) as accession numbers 
ATCC HTB-119 and ATCC CRL-1420, respectively. 
Dosing and Analysis: Formulations were administered intraperitoneally 
(i.p.) or intravenously (i.v.) at the indicated frequencies including 
every other day (q2d) and every third day (q3d). Tumor volume was measured 
at the indicated times by measuring perpendicular diameters and calculated 
as described Dean et al., Cancer Res. 56:3499, 1996). For distribution 
studies, mice were given two doses of 10 mg/kg of formulation and then 
sacrificed after 24 hours. Tumor tissue was removed and analyzed by 
capillary electrophoresis for the presence of various oligonucleotide 
species as described in Example 4. 
B. Results 
Distribution: Sterically stabilized liposomes comprising either 
DSPE-MPEG.sub.2000 (PEG) or monosialoganglioside G.sub.M1 (GM1) resulted 
in enhanced delivery of ISIS 2503 to H69 and Mia PaCa tumor cells (Tables 
17 and 18, respectively). The enhanced delivery was observed both in terms 
of increased concentration and total amount of oligonucleotide delivered 
to tumor tissue, and as a percentage of the total dose of oligonucleotide 
administered to each animal. Of particular significance is the fact that 
significant improvements in the percentage of intact oligonucleotide 
delivered to the tumor tissue increased from less than about 4% (saline 
formulation) to about 11% (liposomes with G.sub.M1) to over 15% (liposomes 
with PEG) (Table 18). 
TABLE 17 
______________________________________ 
Distribution of ISIS 2503 in Tumors in Mice 
with H69 Xenografts 24 Hours After Two Doses of 10 mg/kg 
Formu- Conc. (ug/g) 
Amount (ug) 
% of Dose 
lation n.sup.a 
Avg..sup.b 
SD.sup.c 
Avg. SD Avg. SD 
______________________________________ 
Saline 3 3.51 0.47 1.35 0.40 0.29 0.11 
Liposomes-PEG 
2 17.82 7.37 4.82 3.61 1.11 0.91 
Liposomes-GM1 
2 10.01 3.24 5.40 3.85 3.08 2.08 
______________________________________ 
.sup.a "n" = number of animals. 
.sup.b "Avg." = average (mean) 
"SD" = standard deviation. 
TABLE 18 
______________________________________ 
Distribution of ISIS 2503 in Tumors in Mice with MIA 
PaCa Xenografts 24 Hours After Two Doses of 10 mg/kg 
Formu- Conc. (.mu.g/g) 
Amount (.mu.g) 
% of Dose 
lation n Avg. SD Avg. SD Avg. SD 
______________________________________ 
A. Total concentration of ISIS 2503 & metabolites 
Saline 3 3.82 1.96 3.74 2.32 0.09 0.06 
Liposomes-PEG 
3 16.21 4.98 15.27 10.93 
0.36 0.25 
Liposomes-GM1 
3 15.80 8.99 10.69 2.96 0.26 0.06 
B. Concentration of full-length ISIS 2503 
Saline 3 0.87 0.12 0.09 0.06 3.74 3.74 
Liposomes-PEG 
3 9.95 2.75 0.36 0.25 15.27 
15.27 
Liposomes-GM1 
3 8.56 5.18 0.26 0.06 10.69 
10.69 
______________________________________ 
Antitumor Activity: The liposomal oligonucleotide formulations of the 
invention were evaluated for their ability to control the growth of human 
tumor cells transplanted BALB/c nude mice. One such experiment, in which 
liposomes comprising ISIS 2105 were used as a control formulation, is 
shown in Table 19. 
TABLE 19 
______________________________________ 
Antitumor Activity of Liposomal Formulations 
of ISIS 2503 Against MIA PaCa Xenografts 
Tumor Size (mm.sup.3) 
Formulation: 
Day n Mean SD Std. Error 
______________________________________ 
Saline/no oligonucleotide 
10 8 0.115 0.036 
0.013 
14 8 0.321 0.119 
0.042 
21 8 0.964 0.417 
0.148 
30 8 1.544 0.708 
0.250 
PEG-Liposome/ISIS 2503 (1 mg/kg) 
10 7 0.116 0.033 
0.012 
14 7 0.216 0.101 
0.038 
21 7 0.700 0.335 
0.127 
30 7 1.480 0.851 
0.322 
PEG-Liposome/ISIS 2503 (5 mg/kg) 
10 4 0.090 0.008 
0.004 
14 4 0.208 0.036 
0.018 
21 4 0.550 0.153 
0.077 
30 4 0.998 0.345 
0.173 
PEG-Liposome/ISIS 2503 (25 mg/kg) 
10 6 0.102 0.047 
0.019 
14 6 0.142 0.084 
0.034 
21 6 0.283 0.172 
0.070 
30 6 0.603 0.331 
0.135 
PEG-Liposome/ISIS 2105 (25 mg/kg) 
10 5 0.120 0.038 
0.017 
14 5 0.294 0.180 
0.081 
21 5 0.996 0.735 
0.329 
30 5 1.508 0.981 
0.439 
______________________________________ 
TABLE 20 
______________________________________ 
Antitumor Activity of Liposomal Formulations 
of ISIS 2503 Against NCI-H69 Xenografts 
Tumor Size (mm.sup.3) 
Formulation: 
Day n Mean SD Std. Error 
______________________________________ 
Saline.sup.a /no oligonucleotide 
21 7 0.150 0.061 
0.023 
28 7 0.513 0.493 
0.186 
35 7 0.749 0.392 
0.148 
42 7 2.106 2.277 
0.861 
Saline.sup.a /ISIS 2503 (25 mg/kg) 
21 7 0.163 0.056 
0.021 
28 7 0.334 0.205 
0.077 
35 7 0.766 0.545 
0.206 
42 7 1.021 0.751 
0.284 
PEG-Liposome.sup.b /ISIS 2503 (25 mg/kg) 
21 6 0.150 0.068 
0.028 
28 6 0.222 0.121 
0.049 
35 6 0.417 0.251 
0.102 
42 6 0.753 0.551 
0.225 
PEG-Liposome.sup.b /ISIS 13177 (25 mg/kg) 
21 7 0.163 0.043 
0.016 
28 7 0.460 0.233 
0.088 
35 7 0.956 0.410 
0.155 
42 7 1.636 1.037 
0.392 
______________________________________ 
.sup.a Saline formulations given qd. 
.sup.b liposomal formulations given q3d. 
In the experiment described in Table 19, sterically stabilized liposomes 
comprising DPSE-MPEG.sub.2000 and ISIS 2503 were given in doses of 1, 5 
and 25 mg/kg. Controls included a saline solution (0.90% NaCl) and 
sterically stabilized liposomes comprising ISIS 2105. Dosing was i.v. q3d. 
As can be seen in Table 19, treatment with sterically stabilized liposomes 
comprising ISIS 2503 resulted in a dose-dependent reduction in the rate of 
tumor growth. At day 21, tumor sizes averaged 0.964 and 0.996 mm.sup.3 for 
the animals treated with, respectively, saline and liposomal ISIS 2105. In 
contrast, animals treated with liposomal ISIS 2503 at 1, 5 and 25 mg/kg 
had tumors averaging 0.700, 0.550 and 0.283 mm.sup.3, respectively. 
A similar experiment (Table 20) shows that the liposomal oligonucleotide 
formulation is also effective against NCI-H69-derived xenografts. In this 
experiment, animals treated with 25 mg/kg of ISIS 2503 given as part of a 
liposomal formulation had tumors averaging 0.417 mm.sup.3 in size on day 
35, as compared to 0.749 and 0.766 mm.sup.3 for saline alone and saline 
formulated oligonucleotide, respectively. Treatment with a liposomal 
formulation comprising a control oligonucleotide (ISIS 13177) at 25 mg/kg 
resulted in tumors averaging 0.956 mm.sup.3 on day 35. 
The above results demonstrate that sterically stabilized liposomal 
oligonucleotide formulations have several advantages over traditional 
formulations. First, the liposomal formulations of the invention result in 
improved pharmacodynamic properties (e.g., prolonged clearance time from 
the blood, enhanced biostability in blood and kidney samples, etc.) that 
result in greater circulating concentrations and stability of full-length 
oligonucleotides. Second, the liposomal formulations of the invention 
result in enhanced delivery, relative to traditional saline formulations, 
of the oligonucleotides encompassed thereby to tumor tissues. Third, due 
at least in part to the above features, liposomal oligonucleotide 
formulations can achieve higher concentrations and greater specific 
effects attributable to antisense oligonucleotides using a less frequent 
dosing regime than seen with traditional formulations (e.g., as seen from 
the data in Table 20, 25 mg/kg of ISIS 2503 given every third day in a 
liposomal formulation was more effective than the same dose of ISIS 2503 
given daily in saline). Taken together, these properties are expected to 
result in an efficacious method for treating an animal, including a human, 
suffering from a hyperproliferative disease or disorder such as cancer. 
__________________________________________________________________________ 
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- (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - # 22: 
# 10 
- (2) INFORMATION FOR SEQ ID NO: 23: 
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- (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - # 23: 
# 10 
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- (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - # 24: 
# 10 
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- (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - # 25: 
# 10 
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- (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - # 26: 
# 20 ATGG 
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(A) LENGTH: 20 
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- (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - # 27: 
# 20 CGTC 
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(A) LENGTH: 20 
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- (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - # 28: 
# 20 GGCC 
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(A) LENGTH: 20 
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- (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - # 29: 
# 20 CTCG 
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# 20 CCTG 
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(A) LENGTH: 20 
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# 20 AGGC 
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# 20 TCAG 
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# 20 CATT 
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#21 CCAA C 
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# 17 A 
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#21 GCTG T 
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#21 ATTG T 
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# 20 ATGC 
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(A) LENGTH: 20 
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# 20 GAGG 
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# 15 
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- (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - # 41: 
# 20 CTCC 
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(A) LENGTH: 20 
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- (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - # 42: 
# 20 GCAG 
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(A) LENGTH: 20 
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- (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - # 43: 
# 20 ACCG 
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(A) LENGTH: 20 
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- (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - # 44: 
# 20 CGGC 
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(A) LENGTH: 20 
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# 20 AGGG 
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(A) LENGTH: 20 
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# 20 CGTG 
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(A) LENGTH: 20 
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# 20 CATC 
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(A) LENGTH: 20 
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# 20 CGCC 
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(A) LENGTH: 20 
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# 20 CCAT 
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(A) LENGTH: 20 
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# 20 CCCT 
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(A) LENGTH: 20 
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- (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - # 51: 
# 20 GCGG 
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- (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 20 
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- (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - # 52: 
# 20 AAGA 
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- (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 20 
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- (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - # 53: 
# 20 GCAA 
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- (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 20 
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- (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - # 54: 
# 20 CAGC 
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- (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 20 
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- (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - # 55: 
# 20 ATCA 
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- (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 20 
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- (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - # 56: 
# 20 CCCA 
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- (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 20 
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- (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - # 57: 
# 20 GAGG 
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- (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 20 
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- (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - # 58: 
# 20 ACAG 
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- (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 20 
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- (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - # 59: 
# 20 ACCC 
- (2) INFORMATION FOR SEQ ID NO: 60: 
- (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 20 
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- (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - # 60: 
# 20 GCAC 
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- (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 20 
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- (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - # 61: 
# 20 AAAT 
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