U.S. patent application number 14/841099 was filed with the patent office on 2015-12-17 for treatment of carcinomas with a combination of egf-pathway and telomerase inhibitors.
The applicant listed for this patent is Geron Corporation. Invention is credited to Ning F. Go, Robert J. Tressler.
Application Number | 20150359883 14/841099 |
Document ID | / |
Family ID | 39760271 |
Filed Date | 2015-12-17 |
United States Patent
Application |
20150359883 |
Kind Code |
A1 |
Tressler; Robert J. ; et
al. |
December 17, 2015 |
Treatment of Carcinomas with a Combination of EGF-Pathway and
Telomerase Inhibitors
Abstract
A method and kit for inhibiting the proliferation of carcinoma
cells are disclosed, based on a combination of an EGF pathway
inhibitor and a telomerase inhibitor. When used in cancer therapy,
the two compounds in combination enhance the anti-cancer treatment
efficacy obtained with the antibody alone or the telomerase
inhibitor alone.
Inventors: |
Tressler; Robert J.;
(Capitola, CA) ; Go; Ning F.; (Palo Alto,
CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Geron Corporation |
Menlo Park |
CA |
US |
|
|
Family ID: |
39760271 |
Appl. No.: |
14/841099 |
Filed: |
August 31, 2015 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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12530014 |
Nov 30, 2009 |
9155753 |
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PCT/US2008/003001 |
Mar 6, 2008 |
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14841099 |
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60905944 |
Mar 9, 2007 |
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Current U.S.
Class: |
424/133.1 ;
424/174.1; 536/24.5 |
Current CPC
Class: |
A61K 39/39558 20130101;
A61K 45/06 20130101; A61K 2039/505 20130101; A61K 31/7088 20130101;
A61K 31/7088 20130101; A61P 35/00 20180101; A61K 39/39558 20130101;
A61K 2300/00 20130101; A61K 2300/00 20130101 |
International
Class: |
A61K 39/395 20060101
A61K039/395; A61K 31/7088 20060101 A61K031/7088 |
Claims
1. A method for inhibiting the proliferation of carcinoma cells,
comprising (a) exposing the cells to an anti-EGF receptor antibody,
and (b) either proceeding, following, or concomitantly with step
(a), exposing the cells to a telomerase inhibitor.
2. The method of claim 1, wherein the telomerase inhibitor includes
an oligonucleotide having nuclease-resistant intersubunit linkages
and an oligonucleotide sequence effective to bind by
sequence-specific hybridization to a template region of hTR.
3. The method of claim 2, wherein the internucleoside linkages in
the oligonucleotide are selected from N3'.fwdarw.P5'
phosphoramidate and N3'3.fwdarw.P5' thiophosphoramidate
linkages.
4. The method of claim 2, wherein the telomerase inhibitor includes
a lipid moiety (i) selected from the group consisting of fatty
acids, sterols, and derivatives thereof, and (ii) attached
covalently to one end of the oligonucleotide.
5. The method of claim 2, wherein the oligonucleotide is 10-20
bases in length.
6. The method of claim 5, wherein the oligonucleotide is
characterized by: (i) N3'.fwdarw.P5' thiophosphoramidate
internucleoside linkages; (ii) having the sequence identified as
SEQ ID NO: 12; and (iii) a palmitoyl (C16) moiety linked to the 5'
terminus of the oligonucleotide via a glycerol or aminoglycerol
linker.
7. The method of claim 6, wherein the telomerase inhibitor is the
compound designated herein as GRN163L.
8. The method of claim 1, for use in treating a subject having
carcinoma, wherein exposing step (a) includes administering the
anti-EGF receptor antibody to the subject in an amount effective,
when the antibody is administered alone, to inhibit proliferation
of cancer cells in the subject.
9. The method of claim 8, for use in treating a subject having
breast or ovarian cancer characterized by overexpression of HER2 in
the cancer cells, wherein the anti-EGF receptor antibody is an
anti-HER2 antibody, and exposing step (a) includes administering
the anti-HER2 antibody to the subject in an amount effective, when
the antibody is administered alone, to inhibit proliferation of
cancer cells in the subject.
10. The method of claim 8, wherein each exposing step (a) and (b)
includes administering the respective antibody and inhibitor to the
subject in an amount effective, when each compound is administered
alone, to inhibit proliferation of cancer cells in the subject.
11. The method of claim 8, wherein the telomerase inhibitor is the
compound GRN163L, and step (b) includes infusing the telomerase
inhibitor intravenously into the subject, under infusion conditions
effective to produce a blood concentration of the telomerase
inhibitor of between 1 nM and 100 uM.
12. A method for enhancing the treatment efficacy of an anti-EGF
receptor antibody in a subject with a carcinoma, comprising
administering to the subject, before, during, or after
administering the anti-EGF receptor antibody, an oligonucleotide
telomerase inhibitor of the type composed of an oligonucleotide
having nuclease-resistant intersubunit linkages and an
oligonucleotide sequence effective to bind by sequence-specific
hybridization to a template region of hTR.
13. The method of claim 12, wherein the telomerase inhibitor is
administered in an amount effective to inhibit the proliferation of
cancer cells in the subject, when the telomerase inhibitor is
administered alone.
14. The method of claim 12, wherein enhanced treatment efficacy is
evidenced by an increased survival time of the subject, inhibition
of tumor growth in the subject, or a combination thereof.
15. The method of claim 12, wherein the oligonucleotide telomerase
inhibitor is characterized by: (i) N3'.fwdarw.P5'
thiophosphoramidate internucleoside linkages; (ii) having the
sequence identified as SEQ ID NO: 12; and (iii) a palmitoyl (C16)
moiety linked to the 5' terminus of the oligonucleotide via a
glycerol or aminoglycerol linker.
16. The method of claim 15, wherein the oligonucleotide telomerase
inhibitor is GRN163L.
17. The method of claim 16, wherein said administering includes
infusing the oligonucleotide telomerase inhibitor intravenously
into the subject, under infusion conditions effective to produce a
blood concentration of the inhibitor of between 1 nM and 100
.mu.M.
18. The method of claim 12, wherein the anti-EGF receptor antibody
is an anti-HER2 antibody.
19. A kit for use in treating a carcinoma in a subject, comprising
(a) a dose of an anti-EGF receptor antibody, in an amount of the
antibody effective, when administered alone, to inhibit the
proliferation of carcinoma cells in the subject, and (b) a dose of
an oligonucleotide telomerase inhibitor having nuclease-resistant
intersubunit linkages and an oligonucleotide sequence effective to
bind by sequence-specific hybridization to a template region of
hTR.
20. The kit of claim 19, wherein the oligonucleotide is
characterized by: (i) N3'.fwdarw.P5' thiophosphoramidate
internucleoside linkages; (ii) having the sequence identified as
SEQ ID NO: 12; and (iii) a palmitoyl (C16) moiety linked to the 5'
terminus of the oligonucleotide via a glycerol or aminoglycerol
linker.
21. The kit of claim 20, wherein the telomerase inhibitor is the
compound designated herein as GRN163L.
22. The kit of claim 21, wherein the anti-EGF receptor antibody is
an anti-HER2 antibody.
23. The use of an anti-EGF receptor antibody and a telomerase
inhibitor in the manufacture of a medicament for treating a
carcinoma in a subject.
24. The use of a telomerase inhibitor in the manufacture of a
medicament for treating a carcinoma in a subject who is being
treated with an anti-EGF receptor antibody, for the purpose of
enhancing the anti-cancer efficacy of the antibody in the subject.
Description
FIELD OF THE INVENTION
[0001] The invention is directed to treatment of carcinomas by a
combination of an EGF pathway inhibitor, such as an anti-EGF
receptor antibody, and a telomerase inhibitor.
BACKGROUND
[0002] In view of the continuing high number of deaths each year
resulting from cancer, a continuing need exists to identify
effective and less toxic therapeutic regimens for use in anticancer
treatment.
[0003] For a variety of epithelial cell cancers, or carcinomas,
treatment with an epithelial growth factor (EGF) pathway inhibitor
has been proposed or demonstrated. Trastuzumab (Herceptin.RTM.), a
humanized monoclonal that specifically targets the human epidermal
growth factor 2 (HER2) receptor, inhibits the EGF signaling pathway
associated with HER2. The anti-HER2 antibody may be indicated
particularly in the treatment of cancers, such as breast and
ovarian cancers, characterized by HER2 overexpression in carcinoma
cells.
[0004] Cetuximab (Erbitux.RTM.) is a chimeric monoclonal antibody
that likewise acts as an EGF pathway inhibitors by binding to
epidermal growth factor receptor 1 (EGFR, ErbB-1 or HER1), and may
be indicated, for example, for the treatment of metastatic
colorectal cancer and head and neck cancer.
[0005] A number of small molecule anti-cancer agents that target
the EGF pathway have also been proposed in anti-cancer treatment.
Erlotinib (Tarceva.RTM.) and gefitinib (Iressa.RTM.) specifically
target the tyrosine kinase activity of EGFR, which may be highly
expressed and occasionally mutated in various forms of cancer. The
drug molecules bind in a reversible fashion to the adenosine
triphosphate (ATP) binding site of the receptor, effectively
blocking autophosphorylation of EGFR homodimers, and thus blocking
the signal cascade to the nucleus. Both compounds have shown a
survival benefit in the treatment of lung cancer in phase III
trials, and have been approved for the treatment of locally
advanced or metastatic non small cell lung cancer.
[0006] EGF pathway inhibitors are but one class of a large number
of anti-cancer agents that may be selected for treating cancer. In
addition, any selected class of anti-cancer agent may be tested
with one or more other anti-cancer agents in a combination
treatment, to determine if the two or more agents together are
capable of producing an additive therapeutic effect, or other
significant advantage, such as a reduction in undesired side
effects, due to a reduced dose of the more toxic component, and/or
a reduction in the development of drug-resistance in the cancer
being treated.
[0007] It would be desirable to provide a combined-drug cancer
therapy for the treatment carcinomas in which both drug components
are relatively specific against cancer cells, each acting through a
mechanism that involves specific binding of the drug compound to a
cellular component associated preferentially with the cancer
cells.
SUMMARY OF THE INVENTION
[0008] The invention includes a method for inhibiting the
proliferation of carcinoma cells that express an EGF receptor, by
(a) exposing the cells to an EGF pathway inhibitor, such as an
anti-EGF-receptor antibody, and (b) either proceeding, following,
or concomitantly with step (a), exposing the cells to a telomerase
inhibitor. In one embodiment, the amount of antibody to which the
cells are exposed is effective, by itself, to inhibit proliferation
of the cancer cells. In a further embodiment, the amount of both
antibody and inhibitor is effective, by itself, to inhibit
proliferation of the cancer cells. The combination may provide an
enhanced cancer-cell inhibiting effect with respect to either
component alone.
[0009] The telomerase inhibitor may include an oligonucleotide
having nuclease-resistant intersubunit linkages and an
oligonucleotide sequence effective to bind by sequence-specific
hybridization to a template region of hTR. The internucleoside
linkages in the oligonucleotide may be selected from N3'.fwdarw.P5'
phosphoramidate and N3'.fwdarw.P5' thiophosphoramidate linkages.
The telomerase inhibitor may include a lipid moiety, such as a
fatty acid, sterol, or derivative thereof, which is attached
covalently at one end of the oligonucleotide. The oligonucleotide
may be 10-20 bases in length, preferably 13-20 bases in length, and
may have the sequence identified by SEQ ID NO:12
(5'-TAGGGTTAGACAA-3'). One exemplary telomerase inhibitor is the
compound designated herein as GRN163L.
[0010] The method may be used in treating a subject having a
carcinoma, and in exemplary embodiments, for treating a subject
having breast or ovarian cancer characterized by elevated levels of
HER2 on the surface of the cancer cells, where exposing step (a)
includes administering the anti-EGF receptor antibody to the
subject in an amount effective, when administered alone, to inhibit
proliferation of cancer cells in the subject. Prior to treatment,
the subject may be confirmed to have elevated levels of EGF
receptor, such as HER2, associated with the cancer cells.
[0011] In a further embodiment, each exposing step (a) and (b)
includes administering the EGF pathway inhibitor and telomerase
inhibitor to the subject in an amount effective, when administered
alone, to inhibit proliferation of cancer cells in the subject.
Where the telomerase inhibitor is the compound GRN163L, it may be
administered to the subject by intravenous infusion, under infusion
conditions effective to produce a blood concentration of the
inhibitor of between 1 nM and 100 uM. The antibody may also be
administered to the subject in by infusion, at a dose effective to
produce a blood concentration of the antibody between about 25-500
microgram/ml.
[0012] In another aspect, the invention is directed to a method for
enhancing the efficacy of an EGF pathway inhibitor in the treatment
of a carcinoma, and in exemplary embodiments, enhancing the
efficacy of an anti-EGF receptor antibodies, such as anti-HER2
antibody, in the treatment of a carcinoma, and in exemplary
embodiments, carcinomas such as breast, ovarian cancer or
non-small-cell lung carcinomas, having elevated levels of expressed
HER2. The method includes administering to the subject, before,
during, or after administering an EGF pathway inhibitor, an
oligonucleotide telomerase inhibitor of the type composed of an
oligonucleotide having nuclease-resistant intersubunit linkages and
an oligonucleotide sequence effective to bind by sequence-specific
hybridization to a template region of hTR. Preferably, the amount
of the telomerase inhibitor is effective to inhibit the
proliferation of cancer cells in the subject when the telomerase
inhibitor is administered alone.
[0013] The two inhibitors inhibitor may be administered to the
subject as a composition containing both compounds. The enhancement
of treatment efficacy may be evidenced, for example, by an
increased survival time of the subject, or by an inhibition of
tumor growth in the subject, relative to treatment with the EGF
pathway inhibitor alone.
[0014] The oligonucleotide may be 10-20 bases in length.
Preferably, the oligonucleotide is 13-20 bases in length and
includes the sequence identified by SEQ ID NO: 12
(5'-TAGGGTTAGACAA-3'). An exemplary telomerase inhibitor is the
compound identified as GRN163L, or an analog thereof. This compound
has (i) N3'.fwdarw.P5' thiophosphoramidate internucleoside
linkages; (ii) the sequence identified as SEQ ID NO:12; and (iii) a
palmitoyl (C16) moiety linked to the 5' end of the oligonucleotide
through a glycerol or aminoglycerol linker. An exemplary anti-EGF
receptor antibody is the humanized anti-HER2 antibody
Trastuzumab.
[0015] Also disclosed is a kit for use in carcinoma therapy,
comprising (a) a dose of an EGF pathway inhibitor, such as an
anti-EGF receptor antibody, effective, when administered alone, to
inhibit the proliferation of carcinoma cells in the subject, and
(b) a dose of an oligonucleotide telomerase inhibitor having
nuclease-resistant intersubunit linkages, and an oligonucleotide
sequence effective to bind by sequence-specific hybridization to a
template region of hTR. In one embodiment, the telomerase inhibitor
is provided in an amount effective, when administered alone, to
inhibit proliferation of cancer cells in the subject.
[0016] In an exemplary embodiment, the anti-EGF receptor antibody
is an anti-HER2 antibody such as trastuzumab, and the telomerase
inhibitor is the compound identified as GRN163L, or an analog
thereof. The latter compound has (i) N3'.fwdarw.P5'
thiophosphoramidate internucleoside linkages in the
oligonucleotide; (ii) the sequence identified as SEQ ID NO: 12; and
(iii) a palmitoyl (C16) moiety linked to the 5' end of the
oligonucleotide through a glycerol or aminoglycerol linker.
[0017] Also provided is a kit comprising a telomerase inhibitor and
an anti-EGF receptor antibody, for use in treating a carcinoma.
Such therapy preferably comprises administering the antibody to a
subject, either preceding, following, or concomitantly with
administration of the telomerase inhibitor. The telomerase
inhibitor is preferably a nuclease-resistant oligonucleotide which
binds in a sequence-specific manner to the template region of
hTR.
[0018] In a related aspect, the invention provides a kit containing
a dose of an oligonucleotide telomerase inhibitor having
nuclease-resistant intersubunit linkages and an oligonucleotide
sequence effective to bind by sequence-specific hybridization to a
template region of hTR, preferably in an amount effective to
inhibit proliferation of cancer cells in a subject. The kit
preferably includes an insert with instructions for administration
of the telomerase inhibitor. The insert may provide a user with one
set of instructions for using the inhibitor in monotherapy and a
separate set of instructions for using the inhibitor in combination
with an EGF pathway inhibitor, such as an anti-EGF receptor
antibody.
[0019] The set of instructions for the combination therapy may
recommend (i) a lower dose of the telomerase inhibitor, when used
in combination with the EGF pathway inhibitor, (ii) a lower dose of
the EGF pathway inhibitor, when used in combination with the
telomerase inhibitor, and/or (iii) a different dosing regimen for
one or both inhibitors than would normally be recommended.
[0020] Also provided is the use of a telomerase inhibitor for
preparation of a medicament for use in treatment of carcinoma in a
subject, wherein the treatment comprises administering said
telomerase inhibitor to a subject in combination with an EGF
pathway inhibitor, such as an anti-EGF receptor antibody, as
exemplified by anti-HER2 antibody trastuzumab. The treatment may
comprise administering the antibody to the subject either
preceding, following, or concomitantly with the telomerase
inhibitor, which is preferably a nuclease-resistant oligonucleotide
which binds in a sequence-specific manner to the template region of
hTR.
[0021] In a related aspect, the invention provides the use of an
EGF pathway inhibitor and a telomerase inhibitor, in the
manufacture of a medicament for treating cancer in a subject.
Preferred and/or exemplary inhibitors and cancer indications are as
set out above.
[0022] Further disclosed is the use of a telomerase inhibitor in
the manufacture of a medicament for treating breast or ovarian
cancer in a subject who is being treated with an EGF pathway
inhibitor, such as an anti-EGF receptor antibody, for purposes of
enhancing the anti-cancer efficacy of the antibody in the subject.
These and other objects and features of the invention will become
more fully apparent when the following detailed description of the
invention is read in conjunction with the accompanying
drawings.
BRIEF DESCRIPTION OF THE DRAWINGS
[0023] FIGS. 1 and 2 show enhancement of telomerase inhibiting
activity of an NPS oligonucleotide hTR template inhibitor (GRN163)
by conjugation to a lipid (to produce GRN163L), in human myeloma
tumor xenografts (FIG. 1) and liver cells (FIG. 2), respectively,
in mice;
[0024] FIG. 3 illustrates the additive effect on inhibition of
tumor volume (TV) achieved by co-administration of the anti-HER2
antibody trastuzumab and the telomerase inhibitor GRN163L, in an
A549 non-small cell lung carcinoma model (see Section IV.A and
Experimental Section D below).
DETAILED DESCRIPTION OF THE INVENTION
I. Definitions
[0025] The terms below have the following meanings unless indicated
otherwise.
[0026] A "polynucleotide" or "oligonucleotide" refers to a ribose
and/or deoxyribose nucleoside subunit polymer or oligomer having
between about 2 and about 200 contiguous subunits. The nucleoside
subunits can be joined by a variety of intersubunit linkages,
including, but not limited to, phosphodiester, phosphotriester,
methylphosphonate, P3'.fwdarw.N5' phosphoramidate, N3'.fwdarw.P5'
phosphoramidate, N3'.fwdarw.'P5' thiophosphoramidate, and
phosphorothioate linkages. The term also includes such polymers or
oligomers having modifications, known to one skilled in the art, to
the sugar (e.g., 2' substitutions), the base (see the definition of
"nucleoside" below), and the 3' and 5' termini. In embodiments
where the oligonucleotide moiety includes a plurality of
intersubunit linkages, each linkage may be formed using the same
chemistry, or a mixture of linkage chemistries may be used. When an
oligonucleotide is represented by a sequence of letters, such as
"ATGUCCTG," it will be understood that the nucleotides are in 5'43'
order from left to right. Representation of the base sequence of
the oligonucleotide in this manner does not imply the use of any
particular type of internucleoside subunit in the
oligonucleotide.
[0027] The term "nucleoside" includes the natural nucleosides,
including 2'-deoxy and 2'-hydroxyl forms, e.g., as described in
Komberg and Baker, DNA Replication, 2nd Ed. (Freeman, San
Francisco, 1992), and analogs. "Analogs", in reference to
nucleosides, includes synthetic nucleosides having modified
nucleobase moieties (see definition of "nucleobase" below) and/or
modified sugar moieties, e.g., described generally by Scheit,
Nucleotide Analogs (John Wiley, New York, 1980). Such analogs
include synthetic nucleosides designed to enhance binding
properties, e.g., stability, specificity, or the like, such as
disclosed by Uhlmann and Peyman (Chemical Reviews 90:543-584,
1990). An oligonucleotide containing such nucleosides, and which
typically contains synthetic nuclease-resistant internucleoside
linkages, may itself be referred to as an "analog".
[0028] A "nucleobase" includes (i) native DNA and RNA nucleobases
(uracil, thymine, adenine, guanine, and cytosine), (ii) modified
nucleobases or nucleobase analogs (e.g., 5-methylcytosine,
5-bromouracil, or inosine) and (iii) nucleobase analogs. A
nucleobase analog is a compound whose molecular structure mimics
that of a typical DNA or RNA base.
[0029] An oligonucleotide having "nuclease-resistant linkages"
refers to one whose backbone has subunit linkages that are
substantially resistant to nuclease cleavage, in non-hybridized or
hybridized form, by common extracellular and intracellular
nucleases in the body; that is, the oligonucleotide shows little or
no nuclease cleavage under normal nuclease conditions in the body
to which the oligonucleotide is exposed. The N3'.fwdarw.P5'
phosphoramidate (NP) or N3'.fwdarw.P5' thiophosphoramidate (NPS)
linkages described below are nuclease resistant.
[0030] The term "lipid" is used broadly herein to encompass
substances that are soluble in organic solvents, but sparingly
soluble, if at all, in water. The term lipid includes, but is not
limited to, hydrocarbons, oils, fats (such as fatty acids and
glycerides), sterols, steroids and derivative forms of these
compounds. Preferred lipids are fatty acids and their derivatives,
hydrocarbons and their derivatives, and sterols, such as
cholesterol.
[0031] Fatty acids usually contain even numbers of carbon atoms in
a straight chain (commonly 12-24 carbons) and may be saturated or
unsaturated, and can contain, or be modified to contain, a variety
of substituent groups. For simplicity, the term "fatty acid" also
encompasses fatty acid derivatives, such as fatty or esters.
[0032] The term "hydrocarbon" encompasses compounds that consist
only of hydrogen and carbon, joined by covalent bonds. The term
encompasses open chain (aliphatic) hydrocarbons, including straight
chain and branched hydrocarbons, and saturated as well as mono- and
poly-unsaturated hydrocarbons. The term also encompasses
hydrocarbons containing one or more aromatic rings.
[0033] As used herein, the term "lipid" also includes amphipathic
compounds containing both lipid and hydrophilic moieties.
[0034] An "hTR template inhibitor" is a compound that blocks the
template region (the region spanning nucleotides 30-67 of SEQ ID
NO: 1 herein) of the RNA component of human telomerase, thereby
inhibiting the activity of the enzyme. The inhibitor is typically
an oligonucleotide that is able to hybridize to this region.
Preferably, the oligonucleotide includes a sequence effective to
hybridize to a more specific portion of this region, having
sequence 5'-CUAACCCUAAC-3' (SEQ ID NO: 2), spanning nucleotides
46-56 of SEQ ID NO: 1 herein.
[0035] A "carcinoma" is a malignant tumor of epithelial-cell
origin, that is, a malignant tumor that begins in the lining layer
(epithelial cells) of organs. At least 80% of all cancers are
carcinomas, and include breast cancer, both ductal and lobular
carcinomas of the breast; ovarian cancer; basal-cell carcinoma, the
most common non-melanoma skin cancer; squamous cell carcinoma, a
common form of skin cancer and the most common type of lung cancer;
hepatocellular carcinoma, the most common form of liver cancer;
renal cell carcinoma, a malignant tumor located of the kidneys; and
transitional cell carcinoma, a type of cancer that develops in the
lining of the bladder, ureter, or renal pelvis. The cancer cells
making up a carcinoma are referred to as "carcinoma cells."
[0036] A compound is said to "inhibit the proliferation of
carcinoma cells" if the proliferation of cells in the presence of
the compound is less than that observed in the absence of the
compound. That is, proliferation of the cells is either slowed or
halted in the presence of the compound. Inhibition of carcinoma
cells may be evidenced, for example, by reduction in the number of
cells or rate of expansion of cells, reduction in tumor mass or the
rate of tumor growth, or increase in survival rate of a subject
being treated.
[0037] An "epithelial growth factor (EGF) pathway inhibitor" is a
compound that inhibits cellular growth and/or division events
triggered by activation of and signaling by an EGF receptor, such
as by the binding of EGF or ATP to the receptor. EGF receptors are
members of the ErbB family receptors, a subfamily of four closely
related receptor tyrosine kinases: EGFR (ErbB-1 or HER1),
HER2/c-neu (ErbB-2 or HER2)), Her3 (ErbB-3) and Her4 (ErbB-4). EGFR
(ErbB-1) receptor exists on the cell surface and is activated by
binding of specific ligands, including epidermal growth factor and
transforming growth factor .alpha. (TGF.alpha.). The related ErbB3
and ErbB4 receptors are activated by neuregulins (NRGs). ErbB2
(HER2) has no known direct activating ligand, and may be in an
activated state constitutively.
[0038] Upon activation, EGFR undergoes a transition from an
inactive monomeric form to an active homodimer--although there is
some evidence that preformed inactive dimers may also exist before
ligand binding. In addition to forming homodimers after ligand
binding, EGFR may pair with another member of the ErbB receptor
family, such as ErbB2/Her2/neu, to create an activated heterodimer.
There is also evidence to suggest that clusters of activated EGFRs
form, although it remains unclear whether this clustering is
important for activation itself or occurs subsequent to activation
of individual dimers.
[0039] "Administration of a telomerase inhibitor to a subject is
effective to "enhance the anti-cancer treatment efficacy of an EGF
pathway inhibitor," such as an anti-EGF receptor antibody, if the
subject shows a reduced rate of tumor growth and/or an enhanced
survival rate with combined therapy over therapy with the EGF
pathway inhibitor alone.
[0040] An anti-EGF receptor antibody is an antibody that binds
specifically to an EGF receptor, i.e., EGFR (ErbB-1 or HER1),
HER2/c-neu (ErbB-2 or HER2)), Her3 (ErbB-3) or Her4 (ErbB-4), to
block signaling of the receptor related to cell growth and
division. The antibody may encompass an immunoglobulin molecule
comprised of four polypeptide chains, two heavy (H) chains and two
light (L) chains inter-connected by disulfide bonds, and various
fragments and variants thereof. For example, the antibody may lack
the Fc fragment of naturally formed antibodies, and may include (i)
an Fab fragment, which is a monovalent fragment consisting of the
V.sub.L, V.sub.H, C.sub.L and C.sub.H1 domains; (ii) a F(ab').sub.2
fragment, a bivalent fragment comprising two Fab fragments linked
by a disulfide bridge at the hinge region; (iii) an Fd fragment
consisting of the V.sub.H and C.sub.H1 domains; (iv) a Fv fragment
consisting of the V.sub.L and V.sub.H domains of a single arm of an
antibody, (v) a dAb fragment (Ward et al., (1989) Nature
341:544-546), which consists of a V.sub.H domain; and (vi) an
isolated complementarity determining region (CDR). In particular,
although the two domains of the Fv fragment, V.sub.L and V.sub.H,
are coded for by separate genes, they can be joined by recombinant
methods, by a synthetic linker that enables them to be made as a
single protein chain in which the V.sub.L and V.sub.H regions pair
to form monovalent molecules known as single chain variable
fragment or scFv antibodies; see e.g., Bird et al. (1988) Science
242:423-426; and Huston et al. (1988) Proc. Natl. Acad. Sci. USA
85:5879-5883), and the term:antibody lacking an Fc fragment also
encompasses antibodies having this scFv format.
[0041] The term human or humanized antibodies refers to an antibody
having one or more amino acid residues introduced into it from a
source which is non-human. These non-human amino acid residues are
referred to herein as "import" residues, which are typically taken
from an "import" antibody domain, particularly a variable domain.
An import residue, sequence, or antibody has a desired affinity
and/or specificity, or other desirable antibody biological activity
as discussed herein. In general, the humanized antibody will
comprise substantially all of at least one, and typically two,
variable domains (Fab, Fab', F(ab').sub.2, Fabc, Fv) in which all
or substantially all of the CDR regions correspond to those of a
non-human immunoglobulin and all or substantially all of the FR
regions are those of a human immunoglobulin consensus sequence. The
humanized antibody optimally also will comprise at least a portion
of an immunoglobulin constant region (Fc), typically that of a
human immunoglobulin. Ordinarily, the antibody will contain both
the light chain as well as at least the variable domain of a heavy
chain. The antibody also may include the CH1, hinge, CH2, CH3, and
CH4 regions of the heavy chain, humanized or human monoclonal
antibodies refers to monoclonal antibodies.
II. Treatment of Cancer with EGF Pathway Inhibitors
[0042] This section considers the treatment of carcinomas by
administration of an EGF pathway inhibitor alone, where the
inhibitor may be either an anti-EGF receptor antibody or a small
molecule inhibitor of the receptor tyrosine kinase activity.
[0043] A. Anti-EGF Receptor Antibody.
[0044] Several anti-cancer therapies are based on the binding of an
anti-EGF receptor by an anti-EGF receptor antibody. The anti-HER2
antibody trastuzumab (Herceptin.RTM.) and the anti-EGFR antibody
cetuximab (Erbitux.RTM.) have demonstrated that inhibition of EGF
signaling is an effective mechanism for treating certain solid
tumors.
[0045] Trastuzumab is a murine monoclonal antibody known as
muMAb4D5 (Fendly, B. M. et al., Cancer Res. 50:1550-1558 (1990)),
directed against the extracellular domain (ECD) of p185.sup.HER2,
specifically inhibits the growth of tumor cell lines overexpressing
p185.sup.HER2 in monolayer culture or in soft agar (Hudziak, R. M.
et al., Molec. Cell. Biol. 9:1165-1172 (1989); Lupu, R. et al.,
Science 249:1552-1555 (1990)). MuMAb4D5 also has the potential of
enhancing tumor cell sensitivity to tumor necrosis factor, an
important effector molecule in macrophage-mediated tumor cell
cytotoxicity (Hudziak, supra, 1989; Shepard, H. M. and Lewis, G. D.
J. Clinical Immunology 8:333-395 (1988)). Thus muMAb4D5 has
potential for clinical intervention in and imaging of carcinomas in
which p185.sup.HER2 is overexpressed. The muMAb4D5 and its uses are
described in PCT application WO 89/06692 published Jul. 27,
1989.
[0046] Human or humanized anti-HER2 antibodies can be obtained by
using human hybridomas (Cole et al., Monoclonal Antibodies and
Cancer Therapy, Alan R. Liss, p. 77 (1985)). Techniques developed
for the production of "chimeric antibodies" (Morrison et al., Proc.
Natl. Acad. Sci. 81, 6851 (1984); Neuberger et al., Nature 312, 604
(1984); Takeda et al., Nature 314, 452 (1985)) by splicing the
genes from a mouse antibody molecule of appropriate antigen
specificity together with genes from a human antibody molecule of
appropriate biological activity (such as ability to activate human
complement and mediate ADCC) can be used; such antibodies are
within the scope of this invention.
[0047] As noted, an anti-HER2 antibody, such as trastuzumab, is
indicated particularly for breast cancer characterized by elevated
HER2 levels on the surface of the carcinoma cells, and for ovarian
cancers with elevated levels of surface HER2 receptor. Detectable
concentrations of the circulating extracellular domain of the HER2
receptor (shed antigen) are found in the serum of some patients
with HER2 overexpressing tumors. Clinical studies in baseline serum
samples revealed that 64% (286/447) of patients had detectable shed
antigen, which ranged as high as 1880 ng/mL (median=11 ng/mL). The
Clinical Trial Assay (CTA) may be used for immunohistochemical
detection of HER2 protein overexpression. Alternatively, the DAKO
HercepTest.TM. provides another immunohistochemical test for HER2
protein overexpression, and shows strong correlation with the CTA
test. However, the antibody may be used in treating a variety of
other carcinomas, which generally have surface-bound HER2
receptor.
[0048] Trastuzumab is given every week, every two weeks, or every
three weeks by a needle placed into a vein (intravenously). In
clinical studies, short duration intravenous infusions of 10 to 500
mg once weekly demonstrated dose-dependent pharmacokinetics. Mean
half-life increased and clearance decreases with increasing dose
level. The half-life averaged 1.7 and 12 days at the 10 and 500 mg
dose levels, respectively. Trastuzumab's volume of distribution was
approximately that of serum volume (44 mL/kg). At the highest
weekly dose studied (500 mg), mean peak serum concentrations were
377 microgram/mL. In studies using a loading dose of 4 mg/kg
followed by a weekly maintenance dose of 2 mg/kg, a mean half-life
of 5.8 days (range=1 to 32 days) was observed. Between weeks 16 and
32, trastuzumab serum concentrations reached a steady-state with a
mean trough and peak concentrations of approximately 79
microgram/mL and 123 microgram/mL, respectively.
[0049] Data suggest that the disposition of trastuzumab is not
altered based on age or serum creatinine (up to 2.0 mg/dL).
Patients with higher baseline shed antigen levels were more likely
to have lower serum trough concentrations. However, with weekly
dosing, most patients with elevated shed antigen levels achieved
target serum concentrations of Trastuzumab by week 6.
[0050] Cetuximab (Erbitux), a chimeric anti-EGF receptor antibody
specific for EGFR, may be indicated, for example, for the treatment
of metastatic colorectal cancer and head and neck cancer, or for
the treatment of EGFR-expressing, metastatic colorectal carcinoma
in patients who are refractory to irinotecan-based
chemotherapy.
[0051] Pretreatment with an H.sub.1 antagonist (eg, 50 mg of
diphenhydramine IV) may be recommended in cetuximab therapy. The
recommended dose of cetuximab, in combination with radiation
therapy, is 400 mg/m.sup.2 as an initial loading dose (first
infusion) administered as a 120-minute IV infusion (maximum
infusion rate 5 mL/min) one week prior to initiation of a course of
radiation therapy. The recommended weekly maintenance dose (all
other infusions) is 250 mg/m.sup.2 infused over 60 minutes (maximum
infusion rate 5 mL/min) weekly for the duration of radiation
therapy (6-7 weeks). In clinical studies, cetuximab was
administered 1 hour prior to radiation therapy.
[0052] The recommended dosing regimen for single-agent cetuximab in
the treatment of recurrent or metastatic squamous cell carcinoma of
the head and neck is a 400-mg/m.sup.2 initial dose, (first
infusion) administered as a 120-minute IV infusion (maximum
infusion rate 5 mL/min). The recommended weekly maintenance dose
(all other infusions) is 250 mg/m.sup.2 infused over 60 minutes
(maximum infusion rate 5 mL/min).
[0053] B. Small Molecules Inhibitors of the EGF Pathway.
[0054] A number of small molecule anti-cancer agents that target
the EGF pathway have also been proposed in anti-cancer treatment.
Erlotinib (Tarceva.RTM.) and gefitinib (Iressa.RTM.) specifically
target the tyrosine kinase activity of EGFR, which may be highly
expressed and occasionally mutated in various forms of cancer. The
drug molecules bind in a reversible fashion to the adenosine
triphosphate (ATP) binding site of the receptor, effectively
blocking autophosphorylation of EGFR homodimers, and thus blocking
the signal cascade to the nucleus. Both compounds have shown a
survival benefit in the treatment of lung cancer in phase III
trials, and have been approved for the treatment of locally
advanced or metastatic non small cell lung cancer.
[0055] Erlotinib is a quinazolinamine with the chemical name
N-(3-ethynylphenyl)-6,7-bis(2-methoxyethoxy)-4-quinazolinamine and
is provided (Tarceva.RTM.) as the hydrochloride salt which has the
following structural formula:
##STR00001##
[0056] Aqueous solubility of erlotinib hydrochloride is dependent
on pH with increased solubility at a pH of less than 5 due to
protonation of the secondary amine. Over the pH range of 1.4 to
9.6, maximal solubility of approximately 0.4 mg/mL occurs at a pH
of approximately 2. Tarceva.RTM. tablets are available in three
dosage strengths containing erlotinib hydrochloride (27.3 mg, 109.3
mg and 164 mg) equivalent to 25 mg, 100 mg and 150 mg erlotinib.
Erlotinib is about 60% absorbed after oral administration and its
bioavailability is substantially increased by food to almost 100%.
Its half-life is about 36 hours and it is cleared predominantly by
CYP3A4 metabolism. Peak plasma levels occur 4 hrs after dosing.
Food increases bioavailability substantially, to almost 100%.
[0057] The drug is indicated for small non-small-cell lung cancer,
advanced pancreatic cancer, and a variety of other carcinomas,
particularly those characterized by overexpression of EGFR, such as
colorectal cancer and carcinomas of the head and neck.
[0058] Gefitinib (Iressa.RTM.) is another in the class of small
molecule drugs that inhibit the tyrosine kinase activity of the
epidermal growth factor receptor. Like erlotinib, the drug competes
with the binding of ATP to the intracellular tyrosine kinase domain
of EGFR, thereby inhibiting receptor autophosphorylation and
blocking downstream signal transduction.
[0059] Gefitinib is typically administered at a dose of 250 or 500
mg orally once daily. The drug is indicated as a monotherapy for
the treatment of patients with locally advanced or metastatic NSCLC
after failure of both platinum-based and docetaxel
chemotherapies.sup.8.
III. Treatment of Cancer with a Telomerase Inhibitor
[0060] Telomerase is a ribonucleoprotein that catalyzes the
addition of telomeric repeat sequences (having the sequence
5'-TTAGGG-3' in humans) to chromosome ends. See e.g. Blackburn,
1992, Ann. Rev. Biochem. 61:113-129. The enzyme is expressed in
most cancer cells but not in mature somatic cells. Loss of
telomeric DNA may play a role in triggering cellular senescence;
see Harley, 1991, Mutation Research 256:271-282. A variety of
cancer cells have been shown to be telomerase-positive, including
cells from cancer of the skin, connective tissue, adipose, breast,
lung, stomach, pancreas, ovary, cervix, uterus, kidney, bladder,
colon, prostate, central nervous system (CNS), retina and
hematologic tumors (such as myeloma, leukemia and lymphoma).
Targeting of telomerase can be effective in providing treatments
that discriminate between malignant and normal cells to a high
degree, avoiding many of the deleterious side effects that can
accompany chemotherapeutic regimens which target dividing cells
indiscriminately.
[0061] Inhibitors of telomerase identified to date include
oligonucleotides, preferably oligonucleotides having nuclease
resistant linkages, as well as small molecule compounds.
[0062] A. Small Molecule Compounds
[0063] Small molecule inhibitors of telomerase include, for
example, BRACO19
((9-(4-(N,N-dimethylamino)phenylamino)-3,6-bis(3-pyrrolodino
propionamido)acridine (see Mol. Pharmacol. 61(5):1154-62, 2002);
DODC (diethyloxadicarbocyanine), and telomestatin. These compounds
may act as G-quad stabilizers, which promote the formation of an
inactive G-quad configuration in the RNA component of telomerase.
Other small molecule inhibitors of telomerase include BIBR1532
(2-[(E)-3-naphthen-2-yl but-2-enoylamino]benzoic acid) (see Ward
& Autexier, Mol. Pharmacol. 68:779-786, 2005; also J. Biol.
Chem. 277(18):15566-72, 2002); AZT and other nucleoside analogs,
such as ddG and ara-G (see, for example, U.S. Pat. Nos. 5,695,932
and 6,368,789), and certain thiopyridine, benzo[b]thiophene, and
pyrido[b]thiophene derivatives, described by Gaeta et al. in U.S.
Pat. Nos. 5,767,278, 5,770,613, 5,863,936, 5,656,638 and 5,760,062.
One example is
3-chlorobenzo[b]thiophene-2-carboxy-2'-[(2,5-dichlorophenyl
amino)thia]hydrazine, described in U.S. Pat. No. 5,760,062.
[0064] B. Oligonucleotide-Based Telomerase Inhibitors: Sequence and
Composition
[0065] The genes encoding both the protein and RNA components of
human telomerase have been cloned and sequenced (see U.S. Pat. Nos.
6,261,836 and 5,583,016, respectively, both of which are
incorporated herein by reference). Oligonucleotides can be targeted
against the mRNA encoding the telomerase protein component (the
human form of which is known as human telomerase reverse
transcriptase, or hTERT) or the RNA component of the telomerase
holoenzyme (the human form of which is known as human telomerase
RNA, or hTR).
[0066] The nucleotide sequence of the RNA component of human
telomerase (hTR) is shown in the Sequence Listing below (SEQ ID NO:
1), in the 5'43' direction. The sequence is shown using the
standard abbreviations for ribonucleotides; those of skill in the
art will recognize that the sequence also represents the sequence
of the cDNA, in which the ribonucleotides are replaced by
deoxyribonucleotides, with uridine (U) being replaced by thymidine
(T). The template sequence of the RNA component is located within
the region defined by nucleotides 46-56 (5'-CUAACCCUAAC-3'), which
is complementary to a telomeric sequence composed of about
one-and-two-thirds telomeric repeat units. The template region
functions to specify the sequence of the telomeric repeats that
telomerase adds to the chromosome ends and is essential to the
activity of the telomerase enzyme (see e.g. Chen et al., Cell 100:
503-514, 2000; Kim et al., Proc. Natl. Acad. Sci. USA 98
(14):7982-7987, 2001). The design of antisense, ribozyme or small
interfering RNA (siRNA) agents to inhibit or cause the destruction
of mRNAs is well known (see, for example, Lebedeva, I, et al.
Annual Review of Pharmacology and Toxicology, Vol. 41: 403-419,
April 2001; Macejak, D, et al., Journal of Virology, Vol. 73 (9):
7745-7751, September 1999, and Zeng, Y. et al., PNAS Vol. 100 (17)
p. 9779-9784, Aug. 19, 2003) and such agents may be designed to
target the hTERT mRNA and thereby inhibit production of hTERT
protein in a target cell, such as a cancer cell (see, for example,
U.S. Pat. Nos. 6,444,650 and 6,331,399).
[0067] Oligonucleotides targeting hTR (that is, the RNA component
of the enzyme) act as inhibitors of telomerase enzyme activity by
blocking or otherwise interfering with the interaction of hTR with
the hTERT protein, which interaction is necessary for telomerase
function. See, for example, Villeponteau et al., U.S. Pat. No.
6,548,298.
[0068] A preferred target region of hTR is the template region,
spanning nucleotides 30-67 of SEQ ID NO:1. Oligonucleotides
targeting this region are referred to herein as "hTR template
inhibitors" (see e.g. Herbert et al., Oncogene 21(4):638-42
(2002).) Preferably, such an oligonucleotide includes a sequence
which is complementary or near-complementary to some portion of the
11-nucleotide region having sequence 5'-CUAACCCUAAC-3', spanning
nucleotides 46-56 of SEQ ID NO: 12.
[0069] Another preferred target region is the region spanning
nucleotides 137-179 of hTR (see Pruzan et al., Nucl. Acids
Research, 30:559-568, 2002). Within this region, the sequence
spanning 141-153 is a preferred target. PCT publication WO 98/28442
describes the use of oligonucleotides of at least 7 nucleotides in
length to inhibit telomerase, where the oligonucleotides are
designed to be complementary to accessible portions of the hTR
sequence outside of the template region, including nucleotides
137-196, 290-319, and 350-380 of hTR. Preferred hTR targeting
sequence are given below, and identified by SEQ ID NOS: 2-22.
[0070] The region of the therapeutic oligonucleotide that is
targeted to the hTR sequence is preferably exactly complementary to
the corresponding hTR sequence. While mismatches may be tolerated
in certain instances, they are expected to decrease the specificity
and activity of the resultant oligonucleotide conjugate. In
particular embodiments, the base sequence of the oligonucleotide is
thus selected to include a sequence of at least 5 nucleotides
exactly complementary to the hTR target, and enhanced telomerase
inhibition may be obtained if increasing lengths of complementary
sequence are employed, such as at least 8, at least 10, at least
12, at least 13 or at least 15 nucleotides exactly complementary to
the hTR target. In other embodiments, the sequence of the
oligonucleotide includes a sequence of from at least 5 to 20, from
at least 8 to 20, from at least 10 to 20 or from at least 10 to 15
nucleotides exactly complementary to the hTR target sequence.
[0071] Optimal telomerase inhibitory activity may be obtained when
the full length of the oligonucleotide is selected to be
complementary to the hTR target sequence. However, it is not
necessary that the full length of the oligonucleotide is exactly
complementary to the target sequence, and the oligonucleotide
sequence may include regions that are not complementary to the
target sequence. Such regions may be added, for example, to confer
other properties on the compound, such as sequences that facilitate
purification. Alternatively, an oligonucleotide may include
multiple repeats of a sequence complementary to an hTR target
sequence.
[0072] If the oligonucleotide is to include regions that are not
complementary to the target sequence, such regions are typically
positioned at one or both of the 5' or 3' termini. Exemplary
sequences targeting human telomerase RNA (hTR) include the
following:
TABLE-US-00001 Region of SEQ hTR Targeting Sequence SEQ ID NO: 1 ID
NO: ACATTTTTTGTTTGCTCTAG 160-179 2 GCTCTAGAATGAACGGTGGAAGGC 137-166
3 GGCAGG GTGGAGGCGGCAGG 137-151 4 GGAAGGCGGCAGG 137-149 5
GTGGAAGGCGGCA 139-151 6 GTGGAAGGCGG 141-151 7 CGGTGGAAGGCGG 141-153
8 ACGGTGGAAGGCG 142-154 9 AACGGTGGAAGGCGGC 143-155 10
ATGAACGGTGGAAGGCGG 144-158 11 TAGGGTTAGACAA 42-54 12 CAGTTAGGGTTAG
46-58 13 TAGGGTTAGACA 42-53 14 TAGGGTTAGAC 42-52 15 GTTAGGGTTAG
46-56 16 GTTAGGGTTAGAC 44-56 17 GTTAGGGTTAGACAA 42-56 18 GGGTTAGAC
44-52 19 CAGTTAGGG 50-58 20 CCCTTCTCAGTT 54-65 21 CGCCCTTCTCAG
56-67 22
[0073] The internucleoside linkages in the oligonucleotide may
include any of the available oligonucleotide chemistries, e.g.
phosphodiester, phosphotriester, methylphosphonate, P3'.fwdarw.N5'
phosphoramidate, N3'.fwdarw.P5' phosphoramidate, N3'.fwdarw.P5'
thiophosphoramidate, and phosphorothioate. Typically, but not
necessarily, all of the internucleoside linkages within the
oligonucleotide will be of the same type, although the
oligonucleotide component may be synthesized using a mixture of
different linkages.
[0074] In preferred embodiments, the oligonucleotide has at least
one N3'.fwdarw.P5' phosphoramidate (NP) or N3'.fwdarw.P5'
thiophosphoramidate (NPS) linkage, which linkage may be represented
by the structure: 3'-(-NH--P(.dbd.O)(--XR)--O-)-5', wherein X is O
or S and R is selected from the group consisting of hydrogen,
alkyl, and aryl; and pharmaceutically acceptable salts thereof,
when XR is OH or SH. More preferably, the oligonucleotide includes
all NP or, most preferably, all NPS linkages.
[0075] A particularly preferred sequence for an hTR template
inhibitor oligonucleotide is the sequence complementary to
nucleotides 42-54 of SEQ ID NO: 12 above. The oligonucleotide
having this sequence (TAGGGTTAGACA) and N3'.fwdarw.P5'
thiophosphoramidate (NPS) linkages is designated herein as GRN163.
See, for example, Asai et al., Cancer Research 63:3931-3939 (2003);
Gryaznov et al., Nucleosides Nucleotides Nucleic Acids
22(5-8):577-81 (2003).
[0076] As shown in Table 1 below, this oligonucleotide (first row
of table) inhibits telomerase at low concentrations in a
biochemical assay (FlashPlate.TM.; see Experimental Section). An
alternative 13-mer, having the sequence CAGTTAGGGTTAG,
complementary to nucleotides 46-58 of SEQ ID NO: 1 (fifth row of
table), showed near-equivalent activity in the FlashPlate.TM.
assay. The corresponding NP-linked oligonucleotide, and shorter
(11- and 12-mer) oligonucleotides targeting the same region
(complementary to nucleotides 42-53 and 42-42, respectively, of SEQ
ID NO: 1), showed moderate activity. The effect is clearly
sequence-specific, as shown by the mismatch and non-targeting
sequences in the table.
[0077] The oligonucleotide GRN163 administered alone has shown
inhibitory activity in vitro in cell culture, including epidermoid
carcinoma, breast epithelium, renal carcinoma, renal
adenocarcinoma, pancreatic, brain, colon, prostate, leukemia,
lymphoma, myeloma, epidermal, cervical, ovarian and liver cancer
cells.
[0078] The oligonucleotide GRN163 has also been tested and shown to
be therapeutically effective in a variety of animal tumor models,
including ovarian and lung, both small cell and non-small cell.
TABLE-US-00002 TABLE 1 Inhibition of Telomerase by NPS
Oligonucleotides: Biochemical (FlashPlate) Assay Sequence, 5' to 3'
Description 10.sub.50, nM TAGGGTTAGACAA 13-mer (GRN163) 0.045 .+-.
0.007 SEQ ID NO: 12 TAGGTGTAAGCAA Mismatch of GRN163 80 .+-. 31
(SEQ ID NO: 23) sequence TTGTCTAACCCTA Complement of GRN163 1000
.+-. 46 (SEQ ID NO: 24) sequence TAGGGTTAGACAA Duplex of GRN163 8.9
.+-. 3.0 ATCCCAATCTGTT sequence CAGTTAGGGTTAG Alternative targeting
0.049 .+-. 0.007 (SEQ ID NO: 13) 13-mer TAGGGTTAGACA 12-mer;
truncation of 0.36 .+-. 0.2 (SEQ ID NO: 14) GRN163 sequence
TAGGGTTAGAC 11-mer; truncation of 0.85 .+-. 0.35 (SEQ ID NO: 15)
GRN163 sequence GTTAGGGTTAG Alternative targeting 0.51 .+-. 0.13
(SEQ ID NO: 16) 11-mer GTTGAGTGTAG Mismatch of alternative 177 .+-.
93 (SEQ ID NO: 25) targeting 11-mer TAGGGTTAGACAA 13-mer 0.7 .+-.
0.1 (SEQ ID NO: 12) (GRN163 sequence) with NP backbone
TAGGTGTAAGCAA Mismatch of GRN163 >1000 (SEQ ID NO: 23) sequence
with NP backbone TTAGGG Telomere repeat unit >1000 (SEQ ID NO:
26) TTTTTTTTTT Oligo-T 10-mer >1000 (SEQ ID NO: 27)
[0079] C. Lipid-Oligonucleotide Conjugates
[0080] Preferably, the oligonucleotide-based enzyme inhibitor
includes at least one covalently linked lipid group (see US Pubn.
No. 2005/0113325, which is incorporated herein by reference). This
modification provides superior cellular uptake properties, such
that an equivalent biological effect may be obtained using smaller
amounts of the conjugated oligonucleotide compared to the
unmodified form. When applied to the human therapeutic setting,
this may translate to reduced toxicity risks, and cost savings.
[0081] The lipid group L is typically an aliphatic hydrocarbon or
fatty acid, including derivatives of hydrocarbons and fatty acids,
with examples being saturated straight chain compounds having 14-20
carbons, such as myristic (tetradecanoic) acid, palmitic
(hexadecanoic) acid, and stearic (octadeacanoic) acid, and their
corresponding aliphatic hydrocarbon forms, tetradecane, hexadecane
and octadecane. Examples of other suitable lipid groups that may be
employed are sterols, such as cholesterol, and substituted fatty
acids and hydrocarbons, particularly polyfluorinated forms of these
groups. The scope of the lipid group L includes derivatives such as
amine, amide, ester and carbamate derivatives. The type of
derivative is often determined by the mode of linkage to the
oligonucleotide, as exemplified below.
[0082] In one exemplary structure, the lipid moiety is palmitoyl
amide (derived from palmitic acid), conjugated through an
aminoglycerol linker to the 5' thiophosphate group of an NPS-linked
oligonucleotide. The NPS oligonucleotide having the sequence shown
for GRN163 and conjugated in this manner (as shown below) is
designated GRN163L herein. In a second exemplary structure, the
lipid, as a palmitoyl amide, is conjugated through the terminal 3'
amino group of an NPS oligonucleotide.
##STR00002##
[0083] As shown in Table 2, conjugation of a single fatty acid-type
lipid significantly increased telomerase inhibitory activity in
cell systems relative to the unconjugated oligonucleotide.
TABLE-US-00003 TABLE 2 Inhibition of Telomerase by Lipid-Conjugated
NPS Oligonucleotides (based on GRN163) Tm (.degree. C.) of duplex
IC.sub.50 in vitro, Lipid Substitution with RNA HT-3 cells, nM none
(GRN163) 70.0 1600 3'-palmitic (GRN163L) 66.5 160 3'-stearic 67.1
140 3'-(bis)stearic ~40 1960 3'-oleic 66.8 930 NH--C.sub.16
(palmitoyl) on 3.sup.rd 5' 62.6 500 residue (G) 5'-palmitic 65.5
112 3'-palmitic-5'-palmitic 61.3 ~10000 3'-trityl 66.1 3000
[0084] The effect of lipid conjugation on pharmacokinetics is
illustrated by the data shown in Table 3, below, for a 4 mg/kg dose
administered in rats. Target organ concentrations 6 hours after
administration were also more favorable for GRN163L, with approx.
4-5 .mu.M found in liver, kidney, and fat tissue, 2-3 .mu.M in bone
marrow and spleen, and about 0.5 .mu.M in lymph node. Distribution
of the unlipidated oligonucleotide, GRN163, was primarily to the
kidney (about 18 .mu.M), with only 1 .mu.M or less in the remaining
organ tissues noted above.
[0085] Table 4 presents further data directed to telomerase
inhibition in vitro by GRN163 (unconjugated) and GRN163L
(lipidated) in various cancer cell lines.
TABLE-US-00004 TABLE 3 Comparative Pharmacokinetics of Lipidated
(GRN163L) and Unlipidated (GRN163) NPS Oligonucleotide (Rat, 4
mg/kg dose) GRN163 GRN163L T.sub.1/2.alpha., min 17 20
T.sub.1/2.beta., hrs 65-86 68-72 AUC.sub.0-.infin., .mu.g-hr/g 27
120 C.sub.MAX, .mu.g/ml 16 58 % excreted in 24 h 45 13
TABLE-US-00005 TABLE 4 Comparative Telomerase Inhibitory Activity
of Lipidated (GRN163L) and Unlipidated (GRN163) NPS Oligonucleotide
in vitro GRN163 GRN163L Cell Line IC.sub.50 (.mu.M) IC.sub.50
(.mu.M) HT-3 (Cervical) 1.62 0.3 U251 (Glioblastoma) 1.75 0.17 U87
(Glioblastoma) 0.75 0.11 Hep3B (Hepatoma) 6.5 0.36 HepG2 (Hepatoma)
2.72 0.48 NCI-H522 (Lung) 2.59 0.23 RPMI 8226 (Myeloma) 2.67 0.38
Ovcar5 (Ovarian) 3.74 0.92 DU 145 (Prostate) 1.4 0.15
[0086] The conjugated oligonucleotide GRN163L had significantly
greater telomerase inhibiting activity in vivo than the
unconjugated GRN163, as demonstrated in hepatoma cell xenografts
(FIG. 1) and flank CAG myeloma tumor xenografts (FIG. 2) following
i.v. administration.
[0087] Administration of GRN163L inhibited tumor growth in mice
(A549-luc IV lung metastases model) for at least 4 weeks after i.v.
injection of cancer cells. The dosage was 1 .mu.M biweekly for 5
weeks prior to injection of cancer cells, followed by 5 mg/kg twice
weekly after injection. Controls showed substantial tumor growth,
but none was apparent in the GRN163L-treated mouse.
IV. Combination Therapy with EGF Pathway and Telomerase
Inhibitors
[0088] In accordance with the present invention, it has been
discovered that combined exposure of cancer cells to both an EGF
pathway inhibitor and a telomerase inhibitor enhances the extent to
which carcinoma cell proliferation is inhibited relative to the EGF
pathway inhibitor alone or the telomerase inhibitor alone. The
effect is seen both for inhibition of carcinoma cell growth in
vitro, where the inhibition is evidenced by a reduced rate of cell
proliferation, and for in vivo treatment of cancer in a mammalian
subject, where the inhibition is evidenced by a reduced rate of
tumor growth and/or increased survival time of the subject being
treated.
[0089] A. Combined Therapy In Vivo
[0090] In practicing the method of the invention for in vivo
treatment, the subject is to be treated for a carcinoma, such as
breast cancer, including either ductal and lobular carcinomas of
the breast; ovarian cancer; basal-cell carcinoma, squamous cell
carcinoma, non-small cell lung cancer, hepatocellular carcinoma,
renal cell carcinoma, or transitional cell carcinoma, a type of
cancer that develops in the lining of the bladder, ureter, or renal
pelvis.
[0091] In selecting the EGF pathway inhibitor for the treatment,
the subject carcinoma cells or serum may be assayed for expression
or overexpression of an EGF receptor, such as EGFR or HER2,
according to known methods, and as referenced above. The anti-HER2
antibody trastuzumab may be indicated, for example, in a patient
having breast or ovarian cancer and elevated levels of HER2
associated with the carcinoma cells. Similarly, the anti-EGFR
antibody may be indicated for non-small cell lung cancer or
carcinomas of the head and where EGFR overexpression is detected.
More generally, the carcinoma should be one characterized by
expression, and in some cases, overexpression of an EGF
receptor.
[0092] The carcinoma should also be one that is responsive to
cancer-cell inhibition by telomerase inhibition. As noted above,
oligonucleotide telomerase inhibitors, as exemplified by GRN163 and
GRN163L, have shown inhibitory activity in vitro against human
kidney, lung, pancreatic, brain, colon, prostate, breast, leukemia,
lymphoma, myeloma, epidermal, cervical, ovarian and liver cancer
cells, and in vivo, via local and systemic delivery, against human
brain, prostate, lymphoma, myeloma, cervical, lung, and liver
cancer cells. Other preferred targets include small cell lung,
esophageal, head and neck, and stomach cancers.
[0093] In an exemplary treatment method, the subject is
administered the EGF pathway inhibitor, e.g., anti-EGF receptor
antibody, in an amount that is effective at inhibiting
proliferation of carcinoma cells in the subject. The dose
administered and the dosing schedule will follow, for example,
known or recommended doses for the inhibitor employed, as
indicated, for example, in the drug product insert or published
clinical or animal-model data. One advantage of the present
invention is that lower-than-normal doses of the EGF pathway
inhibitor may be administered, if necessary, due to the
compensating enhancement effect of the telomerase inhibitor. Such a
protocol allows for a reduced dosage of the EGF pathway inhibitor,
which can have significant toxic effects at higher dosages.
[0094] Thus, a kit containing a dose of the telomerase inhibitor
could contain a product insert having one set of directions for
using the inhibitor in monotherapy, and another set of directions
for using the inhibitor in a combination therapy with an EGF
pathway inhibitor, such as trastuzumab, cetuximab, erlotinib or
gefitinib. The set of instructions for the combination therapy
could recommend (i) a lower dose of the telomerase inhibitor, when
used in combination with the EGF pathway inhibitor, (ii) a lower
dose of the EGF pathway inhibitor, when used in combination with
the telomerase inhibitor and/or (iii) a different dosing regimen
for one of both inhibitors than would normally be recommended.
[0095] The telomerase inhibitor may be administered, before,
during, or after administration of the EGF pathway inhibitor.
Typically, the two inhibitors are administered in a common dosing
regimen, as described below, and the two inhibitors themselves may
be administered in a combined-drug composition, or separately, for
example, by enteral administration of the EGF pathway inhibitor and
parenteral administration of the telomerase inhibitor. However, a
dosing regimen in which the telomerase inhibitor is administered
before or after administering the EGF pathway inhibitor is also
contemplated. For example, a person under treatment with an EGF
pathway proteasome inhibitor may be subsequently placed on a
combined therapy that includes telomerase inhibitor.
[0096] Alternatively, the patient may be initially administered the
EGF pathway inhibitor, followed one-to-several days later with the
telomerase treatment. In this regimen, the EGF pathway inhibitor
may function, in part, to sensitize the cancer cells to inhibition
by a telomerase inhibition, e.g., by synchronizing the
cell-division cycle and/or promoting apoptosis in the cells.
Preferred dose levels and dosing schedules are considered further
below.
[0097] In one exemplary method, the EGF pathway inhibitor is the
anti-HER2 antibody trastuzumab, which is administered in
combination with a telomerase-inhibitor oligonucleotide targeted
against hTR. FIG. 3, for example, shows the results of the
treatment method in which trastuzumab is administered in
combination with the telomerase inhibitor GRN163L, for the
treatment of non-small-cell lung carcinoma in a mouse xenograft
model involving A549 non-small-cell lung carcinoma cells. Details
of this study are given in Experimental Section D below. As seen
from FIG. 3, treatment with the two inhibitors, over a 21-day
treatment period (GRN163 was administered 3 times per week over the
treatment period, and trastuzumab, at the times indicated by the
arrows) limited tumor growth to an extent greater than either
inhibitor alone. For example, at day 21 of treatment, trastuzumab
alone resulted in a 38% tumor reduction, and GRN163L, in a 21%
tumor reduction, while combined therapy gave a 53% reduction in
tumor growth.
[0098] B. Administration
[0099] The therapeutic protocol for administering the two
inhibitors in the combination therapy will depend on various
factors including, but not limited to, the type of cancer, the age
and general health of the patient, the aggressiveness of disease
progression, the TRF length (terminal restriction fragment length;
see Section V below) and telomerase activity of the diseased cells
to be treated, and the ability of the patient to tolerate the
agents that comprise the combination.
[0100] In general, treatment of all carcinoma and hematological
malignancy types is contemplated. In selected embodiments, the
target disease comprises a solid tumor; in other embodiments, the
target disease comprises a hematological malignancy. An exemplary
course of treatment involves multiple doses. Sequence of
combination treatments will be determined by clinical compliance
criteria and/or preclinical or clinical data supporting dose
optimization strategies to augment efficacy or reduce toxicity of
the combination treatment. In general, various combinations of the
telomerase inhibitor and EGF pathway inhibitor may be employed,
used either sequentially or simultaneously. For multiple dosages,
the two agents may directly alternate, or two or more doses of one
agent may be alternated with a single dose of the other agent, for
example. Simultaneous administration of both agents may also be
alternated or otherwise interspersed with dosages of the individual
agents. The time between dosages may be for a period from about 1-6
hours, to about 6-12 hours, to about 12-24 hours, to about 1-2
days, to about 1-2 wk or longer following the initiation of
treatment. During a course of treatment, the need to complete the
planned dosings may be re-evaluated.
[0101] The compounds may be administered by direct injection of a
tumor or its vasculature. Alternatively, the tumor may be infused
or perfused with the therapeutic compounds using any suitable
delivery vehicle. The compounds may be administered locally to an
affected organ. Systemic administration may also be performed.
Continuous administration may be applied where appropriate; for
example, where a tumor is excised and the tumor bed is treated to
eliminate residual disease. Delivery via syringe or catheterization
is preferred. Such continuous perfusion may take place for a period
from about 1-6 hours, to about 6-12 hours, to about 12-24 hours, to
about 1-2 days, to about 1-2 weeks or longer following the
initiation of treatment. Generally, the dose of the therapeutic
composition via continuous perfusion will be equivalent to that
given by a single or multiple injections, adjusted over a period of
time during which the perfusion occurs.
[0102] The therapeutic agents are administered to a subject, such
as a human patient, in a formulation and in an amount effective to
achieve a clinically desirable result. For the treatment of cancer,
desirable results include reduction in tumor mass (as determined by
palpation or imaging; e.g., by radiography, radionucleotide scan,
CAT scan, or MRI), reduction in the rate of tumor growth, reduction
in the rate of metastasis formation (as determined e.g., by
histochemical analysis of biopsy specimens), reduction in
biochemical markers (including general markers such as ESR, and
tumor specific markers such as serum PSA), and improvement in
quality of life (as determined by clinical assessment, e.g.,
Karnofsky score), increased time to progression, disease-free
survival and overall survival.
[0103] The amount of each agent per dose and the number of doses
required to achieve such effects will vary depending on many
factors including the disease indication, characteristics of the
patient being treated and the mode of administration. Typically,
the formulation and route of administration will provide a local
concentration at the disease site of between 1 nM and 100 .mu.M of
each agent, and where the EGF pathway inhibitor is an EGF receptor
antibody, a serum concentration of between about 25-500
micrograms/ml. The physician will be able to vary the amount of the
compounds, the carrier, the dosing frequency, and the like, taking
into consideration such factors as the particular neoplastic
disease state and its severity; the overall condition of the
patient; the patient's age, sex, and weight; the mode of
administration; the suitability of concurrently administering
systemic anti-toxicity agents; monitoring of the patient's vital
organ functions; and other factors typically monitored during
cancer chemotherapy. In general, the compounds are administered at
a concentration that affords effective results without causing
excessive harmful or deleterious side effects.
[0104] In accordance with the invention, the amount of the anti-EGF
pathway inhibitor used in combination with a telomerase inhibitor
may be less than would be required for the agent used in
non-combination therapy.
[0105] C. Formulations
[0106] The pharmaceutical carrier(s) employed may be solid or
liquid. Liquid carriers can be used in the preparation of
solutions, emulsions, suspensions and pressurized compositions. The
compounds are dissolved or suspended in a pharmaceutically
acceptable liquid excipient. Suitable examples of liquid carriers
for parenteral administration include water (which may contain
additives, e.g., cellulose derivatives, preferably sodium
carboxymethyl cellulose solution), phosphate buffered saline
solution (PBS), alcohols (including monohydric alcohols and
polyhydric alcohols, e.g., glycols) and their derivatives, and oils
(e.g., fractionated coconut oil and arachis oil). The liquid
carrier can contain other suitable pharmaceutical additives
including, but not limited to, the following: solubilizers,
suspending agents, emulsifiers, buffers, thickening agents, colors,
viscosity regulators, preservatives, stabilizers and osmolarity
regulators.
[0107] For parenteral administration, the carrier can also be an
oily ester such as ethyl oleate and isopropyl myristate. Sterile
carriers are useful in sterile liquid form compositions for
parenteral administration. Sterile liquid pharmaceutical
compositions, solutions or suspensions can be utilized by, for
example, intraperitoneal injection, subcutaneous injection,
intravenously, or topically. The compositions can also be
administered intravascularly or via a vascular stent.
[0108] The liquid carrier for pressurized compositions can be a
halogenated hydrocarbon or other pharmaceutically acceptable
propellant. Such pressurized compositions may also be lipid
encapsulated for delivery via inhalation. For administration by
intranasal or intrabronchial inhalation or insufflation, the
compositions may be formulated into an aqueous or partially aqueous
solution, which can then be utilized in the form of an aerosol.
[0109] The compositions may be administered topically as a
solution, cream, or lotion, by formulation with pharmaceutically
acceptable vehicles containing the active compound. The
compositions of this invention may be orally administered in any
acceptable dosage including, but not limited to, formulations in
capsules, tablets, powders or granules, and as suspensions or
solutions in water or non-aqueous media. Pharmaceutical
compositions and/or formulations comprising the oligonucleotides of
the present invention may include carriers, lubricants, diluents,
thickeners, flavoring agents, emulsifiers, dispersing aids or
binders. In the case of tablets for oral use, carriers that are
commonly used include lactose and corn starch. Lubricating agents,
such as magnesium stearate, are also typically added. For oral
administration in a capsule form, useful diluents include lactose
and dried corn starch. When aqueous suspensions are required for
oral use, the active ingredient is combined with emulsifying and
suspending agents. If desired, certain sweetening, flavoring or
coloring agents may also be added.
[0110] Modes of administration and formulation may be dependent on
the drug and its approved mode of administration. For example, when
the chemotherapeutic agent is an anti-EGF antibody, IV infusion is
indicated, whereas oral administration may be indicated for a
small-molecule EGF pathway inhibitor. When the telomerase inhibitor
is GRN163L, formulation in 0.9% sodium chloride (normal saline) and
administration by i.v. is a preferred route, preferably via
infusion over 4-8 hours, e.g. a 6 hr infusion. While the
lipid-conjugated oligonucleotides described herein, such as
GRN163L, have superior characteristics for cellular and tissue
penetration, these and other compounds may be formulated to provide
further benefit in this area, e.g. in liposome carriers. The use of
liposomes to facilitate cellular uptake is described, for example,
in U.S. Pat. Nos. 4,897,355 and 4,394,448, and numerous
publications describe the formulation and preparation of liposomes.
Liposomal formulations can also be engineered, by attachment of
targeting ligands to the liposomal surface, to target sites of
neovascularization, such as tumor angiogenic regions. The compounds
can also be formulated with additional penetration/transport
enhancers, such as unconjugated forms of the lipid moieties
described above, including fatty acids and their derivatives.
Examples include oleic acid, lauric acid, capric acid, myristic
acid, palmitic acid, stearic acid, linoleic acid, linolenic acid,
dicaprate, tricaprate, recinleate, monoolein (a.k.a.
1-monooleoyl-rac-glycerol), dilaurin, caprylic acid, arichidonic
acid, glyceryl 1-monocaprate, 1-dodecylazacycloheptan-2-one,
acylcarnitines, acylcholines, mono- and di-glycerides and
physiologically acceptable salts thereof (i.e., oleate, laurate,
caprate, myristate, palmitate, stearate, linoleate, etc.). Other
useful adjuvants include substrates for transendothelial migration,
such as glucose uptake systems for facilitated egress from the
vascular space to the tumor microenvironment.
V. Measurement of Telomere Length, Telomerase Activity, and/or Cell
Proliferation
[0111] When employing a therapeutic regimen that involves
administration of a telomerase inhibitor, it may be useful to
determine telomere length and/or telomerase activity in a cell or
tissue sample. These parameters can be measured by assays known in
the art. Telomere length can be measured by a flow cytometry method
using fluorescence in situ hybridization, referred to as flow FISH
(see e.g. M. Hultdin et al., Nucleic Acids Res. 26(16):3651-6,
1998; N. Rufer et al., Nature Biotechnology 16:743-7, 1998). Other
methods include terminal restriction fragment (TRF) analysis, in
which genomic DNA is digested with a restriction enzyme having a
four-base recognition sequence not present in telomere repeat
sequences, and the restriction fragments are separated according to
size, e.g. by gel electrophoresis. See, for example, U.S. Pat. No.
5,489,508 (West et al.) and Harley et al., Nature 345:458, 1990.
The West et al. patent also describes methods of measuring telomere
length by a "anchored terminal primer" method and by a modified
Maxam-Gilbert reaction.
[0112] In addition, a more rapid response to a telomerase
inhibiting agent may be predicted for tumor cells having shorter
telomeric DNA, although telomerase has been shown to have other
inhibitory effects independent of telomere length. (e.g. Stewart et
al., PNAS 99:12606, 2002; Zhu et al., PNAS 93:6091, 1996; Rubaiyat
et al., Oncogene 24(8):1320, 2005); and Folini et al., Curr. Pharm.
Design 11(9):1105, 2005).
[0113] The TRAP assay (see Experimental, below) is a standard
method for measuring telomerase activity in a cell extract system
(Kim et al., Science 266:2011, 1997; Weinrich et al., Nature
Genetics 17:498, 1997). Briefly, this assay measures the amount of
nucleotides incorporated into elongation products (polynucleotides)
formed by nucleotide addition to a labeled telomerase substrate or
primer. The TRAP assay is described in detail in U.S. Pat. Nos.
5,629,154, 5,837,453 and 5,863,726, and its use in testing the
activity of telomerase inhibitory compounds is described in various
publications, including WO 01/18015. In addition, the following
kits are available commercially for research purposes for measuring
telomerase activity: TRAPeze.TM. XK Telomerase Detection Kit
(Intergen Co., Purchase N.Y.); and TeIoTAGGG Telomerase PCR ELISA
plus (Roche Diagnostics, Indianapolis Ind.).
[0114] The anticancer activity of the therapeutic combinations can
be evaluated using standard in vitro and in vivo assays. The
ability of a composition to specifically inhibit the growth of
tumor cells can be assayed using tumor cell lines in vitro, or in
xenograft animal models in vivo. A preferred protocol for such
growth curve assays is the short term cell viability assay
described in Asai et al. (2003, cited above). In established
xenograft models of human tumors, the test compound is administered
either directly to the tumor site or systemically, and the growth
of the tumor is followed by physical measurement. A preferred
example of a suitable in vivo tumor xenograft assay is also
described in Asai et al. (2003, cited above). Other examples are
described in Scorski et al., Proc. Natl. Acad. Sci. USA, 94:
3966-3971 (1997) and Damm et al., EMBO J., 20:6958-6968 (2001).
EXPERIMENTAL
[0115] A. Preparation and Lipid Conjugation of Oligonucleotide
N3'.fwdarw.P5' Phosphoramidates or N3'.fwdarw.P5'
Thiophosphoramidates
[0116] These compounds may be prepared as described, for example,
in McCurdy et al., Tetrahedron Letters 38:207-210 (1997) or
Pongracz & Gryaznov, Tetrahedron Letters 49:7661-7664 (1999).
The starting 3'-amino nucleoside monomers may be prepared as
described in Nelson et al., J. Org. Chem. 62:7278-7287 (1997) or by
the methods described in Gryaznov et al., US Appn. Pubn. No.
2006/0009636.
[0117] A variety of synthetic approaches can be used to conjugate a
lipid moiety L to the oligonucleotide, depending on the nature of
the linkage selected; see, for example, Mishra et al., Biochim. et
Biophys. Acta 1264:229-237 (1995), Shea et al., Nucleic Acids Res.
18:3777-3783 (1995), or Rump et al., Bioconj. Chem. 9:341-349
(1995). Typically, conjugation is achieved through the use of a
suitable functional groups at an oligonucleotide terminus. For
example, the 3'-amino group present at the 3'-terminus of the NP
and NPS oligonucleotides can be reacted with carboxylic acids, acid
chlorides, anhydrides and active esters, using suitable coupling
catalysts, to form an amide linkage. Thiol groups are also suitable
as functional groups (see Kupihar et al., Bioorg. Med. Chem.
9:1241-1247 (2001)). Various amino- and thiol-functionalized
modifiers of different chain lengths are commercially available for
oligonucleotide synthesis.
[0118] Specific approaches for attaching lipid groups to a terminus
of an NP or NPS oligonucleotide include those described in US Appn.
Pubn. No. 2005/0113325, which is incorporated herein by reference.
In addition to the amide linkages noted above, for example, lipids
may also be attached to the oligonucleotide chain using a
phosphoramidite derivative of the lipid, to produce a
phosphoramidate or thiophosphoramidate linkage connecting the lipid
and the oligonucleotide. The free 3'-amino group of the fully
protected support-bound oligonucleotide may also be reacted with a
suitable lipid aldehyde, followed by reduction with sodium
cyanoborohydride, which produces an amine linkage.
[0119] For attachment of a lipid to the 5' terminus, as also
described in US Appn. Pubn. No. 2005/0113325, the oligonucleotide
can be synthesized using a modified, lipid-containing solid
support. Reaction of 3-amino-1,2-propanediol with a fatty acyl
chloride (RC(O)Cl), followed by dimethoxytritylation of the primary
alcohol and succinylation of the secondary alcohol, provides an
intermediate which is then coupled, via the free succinyl carboxyl
group, to the solid support. An example of a modified support is
shown below, where S-- represents a long chain alkyl amine CPG
support, and R represents a lipid.
##STR00003##
[0120] This procedure is followed by synthesis of the
oligonucleotide in the 5' to 3' direction, as described, for
example, in Pongracz & Gryaznov (1999), starting with
deprotection and phosphitylation of the --ODMT group. This is
effective to produce, for example, the following structure, after
cleavage from the solid support:
##STR00004##
[0121] The structure above, when --R is --(CH.sub.2).sub.13CH.sub.3
(palmitoyl), is designated herein as GRN163L.
[0122] B. FlashPlate.TM. Assay
[0123] This assay was carried out essentially as described in Asai
et al., Cancer Research, 63:3931 3939 (2003). Briefly, the assay
detects and/or measures telomerase activity by measuring the
addition of TTAGGG telomeric repeats to a biotinylated telomerase
substrate primer. The biotinylated products are captured on
streptavidin-coated microtiter plates, and an oligonucleotide probe
complementary to 3.5 telomere repeats, labeled with 33P, is used
for measuring telomerase products. Unbound probe is removed by
washing, and the amount of probe annealing to the captured
telomerase products is determined by scintillation counting.
[0124] C. TRAP Assay
[0125] The ability of a compound to increase or inhibit telomerase
activity in a cell can be determined using the TRAP (Telomeric
Repeat Amplification Protocol) assay, which, is described, for
example, in Kim et al., U.S. Pat. No. 5,629,154; Harley et al.,
U.S. Pat. No. 5,891,639; and Harley et al., PCT Pubn. No. WO
2005/000245. Briefly, telomerase-expressing tumor cell lines are
incubated with test compositions, lysed, and treated with a labeled
oligonucleotide telomerase substrate, appropriate primers, and
internal standard for quantitation purposes. Depending on the
telomerase activity of the medium, telomere repeats will be added
to the substrate, to form telomerase extended products. The mixture
is incubated at room temperature, followed by multiple cycles of
PCR. The mixture is separated on a gel, and labeled extension
product is detected and quantitated via comparison with the
internal standard.
[0126] D. In vivo Antitumor Assay Employing GRN163L in combination
with trastuzumab.
[0127] A549 non-small-cell carcinoma cells (approx. 10.sup.7; TRF
length .about.4.98 Kb) were implanted subcutaneously into athymic
nude mice one day after irradiation. Treatment was initiated when
tumors reached .about.100 mm.sup.3. Groups of twelve mice each were
treated in accordance with one of three protocols: (1) GRN163L
alone, three times per week (tiw) at 15 mg/kg for four weeks,
administered IP; (2) Herceptin alone, at 2.0 mg/kg per mouse for
three weeks (4 doses), administered IV; and (3) these treatments in
combination (see FIG. 3). The days when Herceptin was administered
are indicated with arrows in FIG. 3. A fourth group received saline
buffer alone as a control. The results are discussed above.
[0128] Although the invention has been described with respect to
particular embodiments and applications, those skilled in the art
will appreciate the range of applications and methods of the
invention disclosed herein.
SEQUENCE LISTING
TABLE-US-00006 [0129] SEQ ID NO: 1: the RNA component of human
telomerase (hTR): GGGUUGCGGA GGGUGGGCCU GGGAGGGGUG GUGGCCAUUU
UUUGUCUAAC CCUAACUGAG 60 AAGGGCGUAG GCGCCGUGCU UUUGCUCCCC
GCGCGCUGUU UUUCUCGCUG ACUUUCAGCG 120 GGCGGAAAAG CCUCGGCCUG
CCGCCUUCCA CCGUUCAUUC UAGAGCAAAC AAAAAAUGUC 180 AGCUGCUGGC
CCGUUCGCCU CCCGGGGACC UGCGGCGGGU CGCCUGCCCA GCCCCCGAAC 240
CCCGCCUGGA GCCGCGGUCG GCCCGGGGCU UCUCCGGAGG CACCCACUGC CACCGCGAAG
300 AGUUGGGCUC UGUCAGCCGC GGGUCUCUCG GGGGCGAGGG CGAGGUUCAC
CGUUUCAGGC 360 CGCAGGAAGA GGAACGGAGC GAGUCCCGCC GCGGCGCGAU
UCCCUGAGCU GUGGGACGUG 420 CACCCAGGAC UCGGCUCACA CAUGCAGUUC
GCUUUCCUGU UGGUGGGGGG AACGCCGAUC 480 GUGCGCAUCC GUCACCCCUC
GCCGGCAGUG GGGGCUUGUG AACCCCCAAA CCUGACUGAC 540 UGGGCCAGUG UGCU
TABLE-US-00007 SEQ ID NOS: 2-27, nucleotide sequences of targeting
agents against SEQ ID NO: 1 ACATTTTTTGTTTGCTCTAG 2
GCTCTAGAATGAACGGTGGAAGGCGGCAGG 3 GTGGAGGCGGCAGG 4 GGAAGGCGGCAGG 5
GTGGAAGGCGGCA 6 GTGGAAGGCGG 7 CGGTGGAAGGCGG 8 ACGGTGGAAGGCG 9
AACGGTGGAAGGCGGC 10 ATGAACGGTGGAAGGCGG 11 TAGGGTTAGACAA 12
CAGTTAGGGTTAG 13 TAGGGTTAGACA 14 TAGGGTTAGAC 15 GTTAGGGTTAG 16
GTTAGGGTTAGAC 17 GTTAGGGTTAGACAA 18 GGGTTAGAC 19 CAGTTAGGG 20
CCCTTCTCAGTT 21 CGCCCTTCTCAG 22 TAGGTGTAAGCAA 23 TTGTCTAACCCTA 24
GTTGAGTGTAG 25 TTAGGG 26 TTTTTTTTTT 27
Sequence CWU 1
1
271554RNAHomo sapiens 1ggguugcgga gggugggccu gggaggggug guggccauuu
uuugucuaac ccuaacugag 60aagggcguag gcgccgugcu uuugcucccc gcgcgcuguu
uuucucgcug acuuucagcg 120ggcggaaaag ccucggccug ccgccuucca
ccguucauuc uagagcaaac aaaaaauguc 180agcugcuggc ccguucgccu
cccggggacc ugcggcgggu cgccugccca gcccccgaac 240cccgccugga
gccgcggucg gcccggggcu ucuccggagg cacccacugc caccgcgaag
300aguugggcuc ugucagccgc gggucucucg ggggcgaggg cgagguucac
cguuucaggc 360cgcaggaaga ggaacggagc gagucccgcc gcggcgcgau
ucccugagcu gugggacgug 420cacccaggac ucggcucaca caugcaguuc
gcuuuccugu uggugggggg aacgccgauc 480gugcgcaucc gucaccccuc
gccggcagug ggggcuugug aacccccaaa ccugacugac 540ugggccagug ugcu
554220DNAArtificial SequenceOligonucleoide 2acattttttg tttgctctag
20330DNAArtificial SequenceOligonucleoide 3gctctagaat gaacggtgga
aggcggcagg 30414DNAArtificial SequenceOligonucleoide 4gtggaggcgg
cagg 14513DNAArtificial SequenceOligonucleoide 5ggaaggcggc agg
13613DNAArtificial SequenceOligonucleoide 6gtggaaggcg gca
13711DNAArtificial SequenceOligonucleoide 7gtggaaggcg g
11813DNAArtificial SequenceOligonucleoide 8cggtggaagg cgg
13913DNAArtificial SequenceOligonucleoide 9acggtggaag gcg
131016DNAArtificial SequenceOligonucleoide 10aacggtggaa ggcggc
161118DNAArtificial SequenceOligonucleoide 11atgaacggtg gaaggcgg
181213DNAArtificial SequenceOligonucleoide 12tagggttaga caa
131313DNAArtificial SequenceOligonucleoide 13cagttagggt tag
131412DNAArtificial SequenceOligonucleoide 14tagggttaga ca
121511DNAArtificial SequenceOligonucleoide 15tagggttaga c
111611DNAArtificial SequenceOligonucleoide 16gttagggtta g
111713DNAArtificial SequenceOligonucleoide 17gttagggtta gac
131815DNAArtificial SequenceOligonucleoide 18gttagggtta gacaa
15199DNAArtificial SequenceOligonucleoide 19gggttagac
9209DNAArtificial SequenceOligonucleoide 20cagttaggg
92112DNAArtificial SequenceOligonucleoide 21cccttctcag tt
122212DNAArtificial SequenceOligonucleoide 22cgcccttctc ag
122313DNAArtificial SequenceOligonucleoide 23taggtgtaag caa
132413DNAArtificial SequenceOligonucleoide 24ttgtctaacc cta
132511DNAArtificial SequenceOligonucleoide 25gttgagtgta g
11266DNAArtificial SequenceOligonucleoide 26ttaggg
62710DNAArtificial SequenceOligonucleoide 27tttttttttt 10
* * * * *