U.S. patent application number 12/753285 was filed with the patent office on 2010-08-26 for methods of treatment of a bcl-2 disorder using bcl-2 antisense oligomers.
This patent application is currently assigned to Genta Incorporated. Invention is credited to Howard Fingert, Robert Klem, Raymond P. Warrell, JR..
Application Number | 20100216867 12/753285 |
Document ID | / |
Family ID | 27397795 |
Filed Date | 2010-08-26 |
United States Patent
Application |
20100216867 |
Kind Code |
A1 |
Warrell, JR.; Raymond P. ;
et al. |
August 26, 2010 |
Methods of treatment of a bcl-2 disorder using bcl-2 antisense
oligomers
Abstract
The present invention is directed to the use of bcl-2 antisense
oligomers to treat and prevent bcl-2 related disorders. These
disorders include cancers, tumors, carcinomas and
cell-proliferative related disorders. In one embodiment of the
invention, a bcl-2 antisense oligomer is administered at high
doses. The present invention is also directed to a method of
preventing or treating a bcl-2 related disorder, in particular
cancer, comprising administering a bcl-2 antisense oligomer for
short periods of time. The present invention is further drawn to
the use of bcl-2 antisense oligomers to increase the sensitivity of
a subject to cancer therapeutics. The present invention also
relates to pharmaceutical compositions comprising one or more bcl-2
antisense oligomers, which may comprise one or more cancer
therapeutic agents.
Inventors: |
Warrell, JR.; Raymond P.;
(Westfield, NJ) ; Fingert; Howard; (New London,
CT) ; Klem; Robert; (Rancho Santa Fe, CA) |
Correspondence
Address: |
Diehl Servilla LLC
77 Brant Avenue, Suite 210
Clark
NJ
07066
US
|
Assignee: |
Genta Incorporated
Berkeley Heights
NJ
|
Family ID: |
27397795 |
Appl. No.: |
12/753285 |
Filed: |
April 2, 2010 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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09709170 |
Nov 10, 2000 |
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12753285 |
|
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60237009 |
Sep 29, 2000 |
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60227970 |
Aug 25, 2000 |
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Current U.S.
Class: |
514/44A |
Current CPC
Class: |
C12N 15/1135 20130101;
C12N 2310/315 20130101; A61P 31/00 20180101; A61P 35/00 20180101;
A61K 38/00 20130101 |
Class at
Publication: |
514/44.A |
International
Class: |
A61K 31/7088 20060101
A61K031/7088; A61P 35/00 20060101 A61P035/00 |
Claims
1. A method for treating cancer in a human comprising administering
to a human, in which such treatment is desired, a bcl-2 antisense
oligonucleotide at a dose of 8-9 mg/kg/day, 9-10 mg/kg/day or 10-50
mg/kg/day for 1-10 days, and further comprising administering one
or more cancer therapeutics.
2. The method of claim 1, wherein a daily dose is administered by
injection.
3. The method of claim 2, wherein the daily dose is administered in
a single bolus administration.
4. The method of claim 2, wherein the daily dose is divided into
several partial administrations.
5. The method of claim 2, wherein the daily dose is administered in
multiple subcutaneous injections.
6. The method of claim 1, wherein a daily dose is administered by
intravenous infusion.
7. The method of claim 1 further comprising a treatment cycle
consisting of 2-13 days.
8. The method of claim 7 further comprising a treatment cycle
consisting of 10-13 days, 7-9 days or 5-6 days.
9. The method of claim 1, wherein the dose is administered prior
to, subsequently to or concurrently with the cancer
therapeutic.
10. The method of claim 1, wherein the dose is administered for 2
to 7 days or 3 to 4 days.
11. A composition for bolus administration to a human comprising
8-9 mg/kg, 9-10 mg/kg or 10-50 mg/kg of a bcl-2 antisense oligomer
and a pharmaceutically acceptable carrier in a single-dose
vial.
12. The composition of claim 11 which is formulated for
injection.
13. The composition of claim 11 which is formulated for intravenous
administration.
14. The composition of claim 11 wherein the composition comprises
10-50 mg/kg/day of the bcl-2 antisense oligomer.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of pending U.S.
application Ser. No. 09/709,170, filed Nov. 10, 2000, which claims
the benefit of provisional application Ser. No. 60/237,009, filed
Sep. 29, 2000, and provisional application Ser. No. 60/227,970,
filed Aug. 25, 2000.
FIELD OF THE INVENTION
[0002] The present invention is directed to the use of bcl-2
antisense oligomers to treat and prevent bcl-2 related disorders.
These disorders include cancers, tumors, carcinomas and
cell-proliferative related disorders. In one embodiment of the
invention, a bcl-2 antisense oligomer is administered at high
doses. The present invention is also directed to a method of
preventing or treating a bcl-2 related disorder, in particular
cancer, comprising administering a bcl-2 antisense oligomer for
short periods of time. The present invention is further drawn to
the use of bcl-2 antisense oligomers to increase the sensitivity of
a subject to cancer therapeutics. The present invention also
relates to pharmaceutical compositions comprising one or more bcl-2
antisense oligomers, which may comprise one or more cancer
therapeutic agents.
REFERENCE TO A SEQUENCE LISTING
[0003] The Sequence Listing submitted herewith as a 475 byte text
file named 00114918.txt, created Mar. 12, 2010, is incorporated by
reference herein.
BACKGROUND OF THE INVENTION
[0004] Traditional approaches to cancer treatment suffer from a
lack of specificity. Most drugs that have been developed are
natural products or derivatives which block enzyme pathways or
randomly interact with DNA. Moreover, most cancer treatment drugs
are accompanied by serious dose-limiting toxicities due to low
therapeutic indices. For example, the majority of anti-cancer drugs
when administered to a patient kill not only cancer cells but also
normal, non-cancerous cells. Because of these deleterious effects,
treatments that more specifically affect cancerous cells are
needed.
[0005] It has been found that a class of genes, the oncogenes, are
involved in the transformation of cells, and in the maintenance of
a cancerous state. Notably, disrupting the transcription of these
genes, or otherwise inhibiting the effects of their protein
products, can have a favorable therapeutic result. The role of
oncogenes in the etiology of many human cancers has been reviewed
in Bishop, 1987, "Cellular Oncogenes and Retroviruses," Science,
235:305-311. In many types of human cancers, a gene termed bcl-2 (B
cell lymphoma/leukemia-2) is overexpressed, and this overexpression
may be associated with tumorigenicity (Tsujimoto et al., 1985,
"Involvement of the bcl-2 gene in human follicular lymphoma",
Science 228:1440-1443). The bcl-2 gene is thought to contribute to
the pathogenesis of cancer, as well as to resistance to treatment,
primarily by prolonging cell survival rather than by accelerating
cell division.
[0006] The human bcl-2 gene is implicated in the etiology of
certain leukemias, lymphoid tumors, lymphomas, neuroblastomas, and
nasopharyngeal, prostate, breast, and colon carcinomas (Croce et
al., 1987, "Molecular Basis Of Human B and T Cell Neoplasia," in:
Advance in Viral Oncology, 7:35-51, G. Klein (ed.), New York: Raven
Press; Reed et al., 1991, "Differential expression of bcl-2
protooncogene in neuroblastoma and other human tumor cell lines of
neural origin", Cancer Res. 51:6529-38; Yunis et al., 1989, "Bcl-2
and other genomic alterations in the prognosis of large-cell
lymphomas", N. Engl. J. Med. 320:1047-54; Campos et al., 1993,
"High expression of bcl-2 protein in acute myeloid leukemia is
associated with poor response to chemotherapy", Blood 81:3091-6;
McDonnell et al., 1992, "Expression of the protooncogene bcl-2 and
its association with emergence of androgen-independent prostate
cancer", Cancer Res. 52:6940-4; Lu et al., 1993, "Bcl-2
protooncogene expression in Epstein Barr Virus-Associated
Nasopharyngeal Carcinoma", Int. J. Cancer 53:29-35; Bonner et al.,
1993, "bcl-2 protooncogene and the gastrointestinal mucosal
epithelial tumor progression model as related to proposed
morphologic and molecular sequences", Lab. Invest. 68:43A). Bcl-2
has been found to be overexpressed in a variety of tumors including
non-Hodgkin's lymphoma, lung cancer, breast cancer, colorectal
cancer, prostate cancer, renal cancer and acute and chronic
leukemias (Reed, 1995, "Regulation of apoptosis by bcl-2 family
proteins and its role in cancer and chemoresistance", Curr. Opin.
Oncol. 7:541-6).
[0007] Antisense oligonucleotides provide potential therapeutic
tools for specific disruption of oncogene function. These short
(usually less than 30 bases) single-stranded synthetic DNAs have a
sequence complementary to pre-mRNA or mRNA regions of a target
gene, and form a hybrid duplex by hydrogen-bonded base pairing.
This hybridization can disrupt expression of both the target mRNA
and the protein which it encodes, and thus can interfere with
downstream interactions and signaling. Since one mRNA molecule
gives rise to multiple protein copies, inhibition of the mRNA can
be more efficient and more specific than causing disruption at the
protein level, e.g., by inhibition of an enzyme's active site.
[0008] Synthetic oligodeoxynucleotides complementary to mRNA of the
c-myc oncogene have been used to specifically inhibit production of
c-myc protein, thereby arresting the growth of human leukemic cells
in vitro (Holt et al., 1988, Mol. Cell. Biol. 8:963-73; Wickstrom
et al., 1988, Proc. Natl. Acad. Sci. USA, 85:1028-32).
Oligodeoxynucleotides have also been employed as specific
inhibitors of retroviruses, including the human immunodeficiency
virus (Zamecnik and Stephenson, 1978, Proc. Natl. Acad. Sci. USA,
75:280-4; Zamecnik et al., 1986, Proc. Natl. Acad. Sci. USA,
83:4143-6).
[0009] The use of antisense oligonucleotides, with their ability to
target and inhibit individual cancer-related genes, has shown
promise in preclinical cancer models. These phosphorothioate
antisense oligomers have shown an ability to inhibit bcl-2
expression in vitro and to eradicate tumors in mouse models with
lymphoma xenografts. Resistance to chemotherapy of some cancers has
been linked to expression of the bcl-2 oncogene (Grover et al.,
1996, "Bcl-2 expression in malignant melanoma and its prognostic
significance", Eur. J. Surg. Oncol. 22(4):347-9). Administration of
a bcl-2 antisense oligomer can selectively reduce bcl-2 protein
levels in tumor xenografts in laboratory mice (Jansen et al., 1998,
"bcl-2 antisense therapy chemosensitizes human melanoma in SCID
mice", Nat. Med. 4(2):232-4). Moreover, administration of a bcl-2
antisense oligomer can make tumor xenografts in laboratory mice
more susceptible to chemotherapeutic agents (Jansen et al., 1998,
"bcl-2 antisense therapy chemosensitizes human melanoma in SCID
mice", Nat. Med. 4(2):232-4). In mice, systemic treatment with a
bcl-2 antisense oligomer reduced bcl-2 protein and enhanced
apoptosis. Treatment with bcl-2 antisense oligomer alone had modest
antitumor activity, but enhanced antitumor activity was observed
when combined with DTIC (also known as dacarbazine). In ten of
thirteen animals, no malignant melanoma xenografts were detectable
after administration of bcl-2 antisense oligomer in combination
with DTIC treatment. There remains a compelling need to extend
these antitumor treatments to combat cancer in humans.
[0010] The prognosis of many cancer patients is poor despite the
increasing availability of biologic, drug, and combination
therapies. For example, although DTIC is commonly used to treat
metastatic melanoma, few patients have demonstrated long-term
improvement. In fact, an extensive phase III clinical trial did not
demonstrate any better survival when DTIC was used in combination
with cisplatin, carmustine, and tamoxifen (Chapman et al., 1999,
"Phase III multicenter randomized trial of the Dartmouth regimen
versus dacarbazine in patients with metastatic melanoma", J. Clin.
Oncol. 17(9):2745-51). These serious shortcomings in cancer
treatments emphasize the need for new treatment approaches.
SUMMARY OF THE INVENTION
[0011] The present invention is directed to pharmaceutical
compositions comprising bcl-2 antisense oligomers and methods for
treating bcl-2 related disorders. The invention is based, in part,
on the Applicants' discovery that a bcl-2 antisense oligomer, when
administered to patients at high doses for the treatment of a bcl-2
related disorder, particularly cancer, results in significant
therapeutic responses, including low toxicity, high tolerance and
prolonged survival. The Applicants also discovered that bcl-2
antisense oligomers, when administered to patients at high doses
for a short period of time, i.e., less than 14 days, also resulted
in significant therapeutic responses in the treatment of cancer
patients. These therapeutic regimens further encompassed
administering the bcl-2 antisense oligomer at high doses for the
short time in combination with one or more cancer therapeutics.
Surprisingly, a reduced dose of one or more cancer therapeutics,
when given in combination with the short administration of a bcl-2
antisense oligomer, also demonstrated significant therapeutic
responses in the treatment of cancer patients. Thus, the
therapeutic regimens of the present invention provide a
therapeutically effective method of treating cancer which is of
reduced duration and toxicity, and as thus results in improved
tolerance.
[0012] In one embodiment, the present invention provides a method
for treating a bcl-2 related disorder, and a pharmaceutical
composition in dosage unit form comprising particularly high doses
of a bcl-2 antisense oligomer, such that the effective amount of
bcl-2 antisense oligomer in said pharmaceutical composition is a
dose effective to achieve a dose of about 10 to 50 mg/kg/day. In
accordance with this embodiment of the invention, the effective
amount of bcl-2 antisense oligomer of said pharmaceutical
composition is a dose effective to achieve a circulating level of
bcl-2 antisense oligomer of a minimum of 30 nM (nanomolar). In one
embodiment, the circulating level of bcl-2 antisense oligomer is 1
to 10 .mu.M (micromolar). In another embodiment, the desired
circulating level of bcl-2 antisense oligomer of at least 30 nM is
achieved about 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 hours after the
administration of the bcl-2 antisense oligomer. In another
embodiment, the circulating level of bcl-2 antisense oligomer of at
least 30 nM is achieved within about 36 to 48 hours, preferably 24
to 35 hours, more preferably in 12 to 24 hours; most preferably in
under 12 hours.
[0013] In another embodiment, the present invention provides a
method for treating a bcl-2 related disorder and a pharmaceutical
composition comprising a dose of bcl-2 antisense oligomer to be
administered for a short period of time, i.e., less than 14 days,
such that the effective amount of bcl-2 antisense oligomer to be
administered for the duration of this short treatment cycle ranges
from about 0.01 to 50 mg/kg/day. In another embodiment, the
effective amount of bcl-2 antisense oligomer to be delivered for
the duration of this short treatment cycle is a dose effective to
achieve a circulating level of bcl-2 antisense oligomer of a
minimum of 30 nM. In another embodiment, the circulating level of
bcl-2 antisense oligomer is 1 to 10 .mu.M (micromolar).
[0014] In another embodiment, the present invention provides a
method for treating a bcl-2 related disorder and a pharmaceutical
composition comprising a dose of bcl-2 antisense oligomer to be
administered for a short period of time, i.e., less than 14 days,
in combination with one or more cancer therapeutics, said cancer
therapeutics to be administered prior to, subsequent to or
concurrently with the bcl-2 antisense oligomer. The effective
amount of bcl-2 antisense oligomer to be administered for the
duration of this short treatment protocol ranges from about 0.01 to
50 mg/kg/day. The effective amount of cancer therapeutics to be
administered in combination with a bcl-2 antisense oligomer may be
administered at its standard dose, or alternatively, may be
administered at a reduced dose. In accordance with this embodiment
of the invention, the effective amount of bcl-2 antisense oligomer
of said pharmaceutical composition is a dose effective to achieve a
circulating level of bcl-2 antisense oligomer of at least 30 nM. In
a specific embodiment, the circulating level of bcl-2 antisense
oligomer is achieved within about 36 to 48 hours, preferably within
about 24 to 35 hours, most preferably under about 24 hours.
[0015] In accordance with the present invention, a bcl-2 related
disorder encompasses tumors, cancer, carcinomas, and
cell-proliferative disorders.
[0016] In accordance with the present invention, a short time
period encompasses a time period for administering the bcl-2
antisense which is less than 14 days, ranging from 2 to 13 days;
preferably ranging from 3 to 9 days, 4 to 7 days, or 5 to 6
days.
[0017] In accordance with the present invention, the dose of bcl-2
antisense oligomer to be administered for a short time period
ranges from 0.01 to 50 mg/kg/day; preferably at a dose of 4 to 9
mg/kg/day, and more preferably at a dose of 5 to 7 mg/kg/day.
[0018] The present invention also encompasses pharmaceutical
compositions comprising an effective amount of one or more bcl-2
antisense oligomers to be administered in accordance with the
methods of the present invention. Said pharmaceutical compositions
encompass a dose of bcl-2 antisense oligomer ranging from 0.01 to
50 mg/kg/day; preferably at a dose of 4 to 9 mg/kg/day, and more
preferably at a dose of 5 to 7 mg/kg/day, in combination with a
pharmaceutically acceptable carrier. In another embodiment, the
pharmaceutical compositions of the present invention also encompass
one or more additional cancer therapeutics. Said pharmaceutical
compositions are formulated to be delivered as a continuous
infusion, or in one or more bolus administrations, or in one or
more administrations during a treatment protocol.
[0019] In accordance with the present invention, pharmaceutical
compositions of the present invention comprising bcl-2 antisense
oligomer may be administered separately from pharmaceutical
compositions comprising cancer therapeutic agents.
[0020] These and other aspects of the present invention will be
better appreciated by reference to the following Figures and
Detailed Description.
DEFINITIONS
[0021] As used herein, the phrase "bcl-2 related disorder" refers
to a disease that involves regulation of the bcl-2 gene, and
includes, but is not limited to, diseases involving cells
expressing the bcl-2 gene. Such a disorder encompasses diseases
involving cells or tissues that express the bcl-2 gene or a bcl-2
related gene, or diseases involving cells or tissues that no longer
express the bcl-2 gene, but normally do. Bcl-related disorders
include, but are limited to, cell proliferative disorders and
pathologies of cells or tissues that are affected by cells that
express the bcl-2 gene or a bcl-2 related gene.
[0022] As used herein, the term "cancer" describes a disease state
in which a carcinogenic agent or agents causes the transformation
of a healthy cell into an abnormal cell, which is followed by an
invasion of adjacent tissues by these abnormal cells, and which may
be followed by lymphatic or blood-borne spread of these abnormal
cells to regional lymph nodes and/or distant sites, i.e.,
metastasis.
[0023] As used herein, the term "tumor" or "growth" means increased
tissue mass, which includes greater cell numbers as a result of
faster cell division and/or slower rates of cell death. Tumors may
be malignant or non-malignant cancers.
[0024] As used herein, the phrases "treating cancer" and "treatment
of cancer" mean to inhibit the replication of cancer cells, inhibit
the spread of cancer, decrease tumor size, lessen or reduce the
number of cancerous cells in the body, or ameliorate or alleviate
the symptoms of the disease caused by the cancer. The treatment is
considered therapeutic if there is a decrease in mortality and/or
morbidity, or a decrease in disease burden manifest by reduced
numbers of malignant cells in the body.
[0025] As used herein, the phrases "preventing cancer" and
"prevention of cancer" mean to prevent the occurrence or recurrence
of the disease state of cancer. As such, a treatment that impedes,
inhibits, or interferes with metastasis, tumor growth, or cancer
proliferation has preventive activity.
[0026] As used herein, the phrase "antisense oligomer" means an
antisense oligonucleotide or an analogue or derivative thereof, and
refers to a range of chemical species that recognize polynucleotide
target sequences through Watson-and-Crick hydrogen bonding
interactions with the nucleotide bases of the target sequences. The
target sequences may be RNA or DNA, and may be single-stranded or
double-stranded. Target molecules include, but are not limited to,
pre-mRNA, mRNA, and DNA.
[0027] As used herein, the phrase "bcl-2 gene expression" refers to
transcription of the bcl-2 gene which produces bcl-2 pre-mRNA,
bcl-2 mRNA, and/or bcl-2 protein.
[0028] As used herein, the term "derivative" refers to any
pharmaceutically acceptable homolog, analogue, or fragment
corresponding to the pharmaceutical composition of the
invention.
[0029] As used herein, the phrase "therapeutics" or "therapeutic
agents" refer to any molecules, compounds or treatments that assist
in the treatment of a disease. As such, a cancer therapeutic is a
molecule, compound or treatment protocol that aids in the treatment
of tumors or cancer. The treatment protocol includes, but is not
limited to, radiation therapy, dietary therapy, physical therapy,
and psychological therapy.
[0030] As used herein, the phrase "chemoagent" or "anti-cancer
agent" or "anti-tumor agent" or "cancer therapeutic" refers to any
molecule, compound or treatment that assists in the treatment of
tumors or cancer.
[0031] As used herein, the phrase "low dose" or "reduced dose"
refers to a dose that is below the normally administered range,
i.e., below the standard dose as suggested by the Physicians' Desk
Reference, 54.sup.th Edition (2000) or a similar reference. Such a
dose can be sufficient to inhibit cell proliferation, or
demonstrates ameliorative effects in a human, or demonstrates
efficacy with fewer side effects as compared to standard cancer
treatments. Normal dose ranges used for particular therapeutic
agents and standard cancer treatments employed for specific
diseases can be found in the Physicians' Desk Reference, 54.sup.th
Edition (2000) or in Cancer: Principles & Practice of Oncology,
DeVita, Jr., Hellman, and Rosenberg (eds.) 2nd edition,
Philadelphia, Pa.: J. B. Lippincott Co., 1985.
[0032] As used herein, the phrase "reduced toxicity" refers to the
reduced side effects and toxicities observed in connection with
administering antisense oligonucleotides and cancer therapeutics
for shorter duration and/or at lower dosages when compared to other
treatment protocols and dosage formulations, including the standard
treatment protocols and dosage formulations as described in the
Physicians' Desk Reference, 54.sup.th Edition (2000) or in Cancer:
Principles & Practice of Oncology, DeVita, Jr., Hellman, and
Rosenberg (eds.) 2nd edition, Philadelphia, Pa.: J. B. Lippincott
Co., 1985.
[0033] As used herein, the phrase "treatment cycle" or "cycle"
refers to a period during which a single therapeutic or sequence of
therapeutics is administered. In one embodiment encompassing the
use of a high dose of bcl-2 antisense oligomer, in combination with
a standard dose of a cancer therapeutic, the preferred period
length of time for one treatment cycle is less than 14 days. The
present invention contemplates at least one treatment cycle,
generally preferably more than one cycle. In some instances, one
treatment cycle may be desired, such as, for example, in the case
where a significant therapeutic effect is obtained after one
treatment cycle.
[0034] As used herein, the phrase "pharmaceutically acceptable
carrier" refers to a carrier medium that does not interfere with
the effectiveness of the biological activity of the active
ingredient. Said carrier medium is essentially chemically inert and
nontoxic.
[0035] As used herein, the phrase "pharmaceutically acceptable"
means approved by a regulatory agency of the Federal government or
a state government, or listed in the U.S. Pharmacopeia or other
generally recognized pharmacopeia for use in animals, and more
particularly for use in humans.
[0036] As used herein, the term "carrier" refers to a diluent,
adjuvant, excipient, or vehicle with which the therapeutic is
administered. Such carriers can be sterile liquids, such as saline
solutions in water, or oils, including those of petroleum, animal,
vegetable or synthetic origin, such as peanut oil, soybean oil,
mineral oil, sesame oil and the like. A saline solution is a
preferred carrier when the pharmaceutical composition is
administered intravenously. Saline solutions and aqueous dextrose
and glycerol solutions can also be employed as liquid carriers,
particularly for injectable solutions. Suitable pharmaceutical
excipients include starch, glucose, lactose, sucrose, gelatin,
malt, rice, flour, chalk, silica gel, sodium stearate, glycerol
monostearate, talc, sodium chloride, dried skim milk, glycerol,
propylene, glycol, water, ethanol and the like. The carrier, if
desired, can also contain minor amounts of wetting or emulsifying
agents, or pH buffering agents. These compositions can take the
form of solutions, suspensions, emulsion, tablets, pills, capsules,
powders, sustained-release formulations and the like. The
composition can be formulated as a suppository, with traditional
binders and carriers such as triglycerides. Examples of suitable
pharmaceutical carriers are described in Remington's Pharmaceutical
Sciences by E. W. Martin. Examples of suitable pharmaceutical
carriers are a variety of cationic lipids, including, but not
limited to N-(1(2,3-dioleyloxy)propyl)-N,N,N-trimethylammonium
chloride (DOTMA) and diolesylphosphotidylethanolamine (DOPE).
Liposomes are also suitable carriers for the antisense oligomers of
the invention. Such compositions should contain a therapeutically
effective amount of the compound, together with a suitable amount
of carrier so as to provide the form for proper administration to
the patient. The formulation should suit the mode of
administration.
[0037] As used herein, the phrase "pharmaceutically acceptable
salts" refers to salts prepared from pharmaceutically acceptable,
essentially nontoxic, acids and bases, including inorganic and
organic acids and bases. Pharmaceutically acceptable salts include
those formed with free amino groups such as those derived from
hydrochloric, phosphoric, acetic, oxalic, tartaric acids, etc., and
those formed with free carboxyl groups such as those derived from
sodium, potassium, ammonium, calcium, ferric hydroxides,
isopropylamine, triethylamine, 2-ethylamino ethanol, histidine,
procaine, etc.
BRIEF DESCRIPTION OF THE FIGURES
[0038] FIG. 1: Bcl-2 downregulation after 5 days of treatment with
Bcl-2 antisense oligomer in melanoma biopsies of patient #12.
[0039] FIG. 2: TUNEL staining of tumor biopsies of patient #12
(right leg) before treatment (a), after Bcl-2 antisense oligomer
treatment (b) and after Bcl-2 antisense oligomer plus DTIC
treatment.
[0040] FIG. 3: Skin metastases (a) and CT-scan of pelvic region (b)
of patient #12 before and after three cycles of Bcl-2 antisense
oligomer plus DTIC treatment at 6.5 mg/kg/day.
DETAILED DESCRIPTION OF THE INVENTION
[0041] The present invention provides compositions and methods for
the use of a bcl-2 antisense oligomer for preventing or treating a
bcl-2 related disorder, in particular cancer. The invention also
provides pharmaceutical compositions comprising a bcl-2 antisense
oligomer, as well as methods for their use in prophylactic and
therapeutic treatments, including drug delivery and therapeutic
regimens.
[0042] The invention is based, in part, on the discovery that short
treatment cycles of a bcl-2 antisense oligomer, alone and in
combination with other therapeutic agents, has unexpectedly potent
ameliorative effects in patients suffering from disease. This short
treatment regimen manifests additional benefits to the human
subject such as convenience, reduced psychological trauma, and a
better likelihood of compliance with the treatment protocol. Other
discoveries include: (1) short treatment cycles and reduced doses
of therapeutic agents when used in combination with a bcl-2
antisense oligomer, (2) simplified modes of delivery for the
pharmaceutical compositions comprising at least one bcl-2 antisense
oligomer with or without other therapeutic agents, and (3)
clinically significant treatment regimens for many types of
cancers. Thus, Applicants' discovery that a bcl-2 antisense
oligomer, when administered for a short treatment cycle, can
demonstrate significant therapeutic responses in a patient having a
bcl-2 related disorder, provides improved and useful pharmaceutical
compositions, treatment courses, and modes of delivery.
[0043] The invention is also based, in part, on the discovery that
high doses of bcl-2 antisense oligomer, alone and in combination
with other therapeutic agents, has reduced toxicity, including
unexpectedly few side effects as compared to most standard cancer
treatments, and has ameliorative effects in patients suffering from
disease. A treatment regimen that encompasses a high dose of bcl-2
antisense oligomer manifests additional benefits to the human
subject such as shorter treatment cycles, fewer treatments, or
improved efficacy.
[0044] In a one embodiment, a bcl-2 antisense oligomer is
administered to a human for a short treatment cycle to prevent or
treat a bcl-2 related disorder. In another embodiment, a bcl-2
antisense oligomer is administered to a human at high doses to
prevent or treat a bcl-2 related disorder. In addition to affecting
diseased tissue, the bcl-2 antisense oligomer can protect or treat
normal tissues, which include tissues containing cells that
normally express the bcl-2 gene. Additionally, the bcl-2 antisense
oligomer can protect or treat normal tissues that, although not
expressing the bcl-2 gene, are compromised by diseased tissues.
[0045] In a specific embodiment, the invention further encompasses
the use of combination therapy to prevent or treat a bcl-2 related
disorder. Such therapy includes the use of one or more molecules,
compounds or treatments that assist in the prevention or treatment
of a disease. Examples of contemplated therapeutics include
biologicals, chemicals, and therapeutic treatments (e.g.,
irradiation treatment).
[0046] In another specific embodiment, the invention provides for a
bcl-2 antisense oligomer that is administered to a human in
combination with one of more cancer therapeutic agents to prevent
or treat cancer. Such cancer therapeutics include one or more
molecules, compounds or treatments that have anti-cancer activity.
Examples of contemplated cancer therapeutics include biologicals,
chemicals, and therapeutic treatments (e.g., irradiation
treatment).
[0047] In yet another specific embodiment, the invention provides
for a bcl-2 antisense oligomer that is administered to a human, in
combination with one of more cancer therapeutic agents at reduced
doses, to prevent or treat cancer. Such treatments may involve
high, standard, or low doses of one or more bcl-2 antisense
oligomers, treatment cycles may be of long or short duration. In a
specific embodiment, the invention provides for a particularly high
dose of bcl-2 antisense oligomer that is administered to a human,
in combination with one of more cancer therapeutic agents at
greatly reduced doses for shortened treatment cycles, to prevent or
treat cancer.
Bcl-2 Antisense Oligomer
[0048] The invention contemplates use of one or more bcl-2
antisense oligomers, or its derivatives, analogues, fragments,
hybrids, mimetics, and congeners thereof. As used herein, the term
"derivative" refers to any pharmaceutically acceptable homolog,
analogue, or fragment corresponding to the pharmaceutical
composition of the invention. Antisense oligomers suitable for use
in the invention include nucleotide oligomers which range in size
from 5 to 10, 10 to 20, 20 to 50, 50 to 75, or 75 to 100 bases in
length; preferably 10 to 40 bases in length; more preferably 15 to
25 bases in length; most preferably 18 bases in length. The target
sequences may be RNA or DNA, and may be single-stranded or
double-stranded. Target molecules include, but are not limited to,
pre-mRNA, mRNA, and DNA. In a one embodiment, the target molecule
is mRNA. In a preferred embodiment, the target molecule is bcl-2
pre-mRNA or bcl-2 mRNA. In a specific embodiment, the antisense
oligomers hybridize to a portion anywhere along the bcl-2 pre-mRNA
or mRNA. The antisense oligomers are preferably selected from those
oligomers which hybridize to the translation initiation site, donor
splicing site, acceptor splicing site, sites for transportation, or
sites for degradation of the bcl-2 pre-mRNA or mRNA.
[0049] Several bcl-2 antisense oligomers have been assessed
previously with variable results (See, e.g., SEQ. ID. NOS.: 1-17 in
U.S. Pat. No. 5,831,066). Examples of bcl-2 antisense oligomers
that may be used in accordance with the present invention are
described in detail in U.S. patent application Ser. No. 08/217,082,
now U.S. Pat. No. 5,734,033; U.S. patent application Ser. No.
08/465,485, now U.S. Pat. No. 5,831,066; and U.S. patent
application Ser. No. 09/080,285, now U.S. Pat. No. 6,040,181, each
of which is incorporated herein by reference in its entirety.
[0050] In one embodiment, the bcl-2 antisense oligomer is
substantially complementary to a portion of a bcl-2 pre-mRNA or
mRNA, or to a portion of a pre-mRNA or mRNA that is related to
bcl-2. In a preferred embodiment, the bcl-2 antisense oligomer
hybridizes to a portion of the translation-initiation site of the
pre-mRNA coding strand. In a more preferred embodiment, the bcl-2
antisense oligomer hybridizes to a portion of the pre-mRNA coding
strand that comprises the translation-initiation site of the human
bcl-2 gene. More preferably, the bcl-2 antisense oligomer comprises
a TAC sequence which is complementary to the AUG initiation
sequence of the bcl-2 pre-mRNA or RNA.
[0051] In another embodiment, the bcl-2 antisense oligomer
hybridizes to a portion of the splice donor site of the pre-mRNA
coding strand for the human bcl-2 gene. Preferably, this nucleotide
comprises a CA sequence, which is complementary to the GT splice
donor sequence of the bcl-2 gene, and preferably further comprises
flanking portions of 5 to 50 bases, more preferably from about 10
to 20 bases, which hybridizes to portions of the bcl-2 gene coding
strand flanking said splice donor site.
[0052] In yet another embodiment, the bcl-2 antisense oligomer
hybridizes to a portion of the splice acceptor site of the pre-mRNA
coding strand for the human bcl-2 gene. Preferably, this nucleotide
comprises a TC sequence, which is complementary to the AG splice
acceptor sequence of the bcl-2 gene, and preferably further
comprises flanking portions of 5 to 50 bases, more preferably from
about 10 to 20 bases, which hybridizes to portions of the bcl-2
gene coding strand flanking said splice acceptor site. In another
embodiment, the bcl-2 antisense oligomer hybridizes to portions of
the pre-mRNA or mRNA involved in splicing, transport or
degradation.
[0053] One of average skill in the art can recognize that antisense
oligomers suitable for use in the invention may also be
substantially complementary to other sites along the bcl-2 pre-mRNA
or mRNA, and can form hybrids. The skilled artisan will also
appreciate that antisense oligomers, which hybridize to a portion
of the bcl-2 pre-mRNA or mRNA whose sequence does not commonly
occur in transcripts from unrelated genes are preferable so as to
maintain treatment specificity.
[0054] The design of the sequence of a bcl-2 antisense oligomer can
also be determined by empirical testing and assessment of clinical
effectiveness, regardless of its degree of sequence homology to, or
hybridization with, the bcl-2 gene, bcl-2 pre-mRNA, bcl-2 mRNA, or
bcl-2 related nucleotide sequences. One of ordinary skill in the
art will appreciate that bcl-2 antisense oligomers having, for
example, less sequence homology, greater or fewer modified
nucleotides, or longer or shorter lengths, compared to those of the
preferred embodiments, but which nevertheless demonstrate responses
in clinical treatments, are also within the scope of the
invention.
[0055] The antisense oligomers may be RNA or DNA, or derivatives
thereof. The particular form of antisense oligomer may affect the
oligomer's pharmacokinetic parameters such as bioavailability,
metabolism, half-life, etc. As such, the invention contemplates
antisense oligomer derivatives having properties that improve
cellular uptake, enhance nuclease resistance, improve binding to
the target sequence, or increase cleavage or degradation of the
target sequence. The antisense oligomers may contain bases
comprising, for example, phosphorothioates or methylphosphonates.
The antisense oligomers, instead, can be mixed oligomers containing
combinations of phosphodiesters, phosphorothioate, and/or
methylphosphonate nucleotides, among others. Such oligomers may
possess modifications which comprise, but are not limited to,
2-O'-alkyl or 2-O'-halo sugar modifications, backbone modifications
(e.g., methylphosphonate, phosphorodithioate, phosphordithioate,
formacetal, 3'-thioformacetal, sulfone, sulfamate, nitroxide
backbone, morpholino derivatives and peptide nucleic acid (PNA)
derivatives), or derivatives wherein the base moieties have been
modified (Egholm, et al., 1992, Peptide Nucleic Acids
(PNA)-Oligonucleotide Analogues With An Achiral Peptide Backbone).
In another embodiment, antisense oligomers comprise conjugates of
the oligonucleotides and derivatives thereof (Goodchild, 1990,
"Conjugates of oligonucleotides and modified oligonucleotides: a
review of their synthesis and properties", Bioconjug. Chem. 1(3):
165-87).
[0056] For in vivo therapeutic use, a phosphorothioate derivative
of the bcl-2 antisense oligomer is preferable, at least partly
because of greater resistance to degradation. In one embodiment,
the bcl-2 antisense oligomer is a hybrid oligomer containing
phosphorothioate bases. In another embodiment, the bcl-2 antisense
oligomer contains at least one phosphorothioate linkage. In another
embodiment, the bcl-2 antisense oligomer contains at least three
phosphorothioate linkages. In yet another embodiment, the bcl-2
antisense oligomer contains at least three consecutive
phosphorothioate linkages. In yet another embodiment, the bcl-2
antisense oligomer is comprised entirely of phosphorothioate
linkages. Methods for preparing oligonucleotide derivatives are
known in the art. See e.g., Stein et al., 1988, Nucl. Acids Res.,
16:3209-21 (phosphorothioate); Blake et al., 1985, Biochemistry
24:6132-38 (methylphosphonate); Morvan et al., 1986, Nucl. Acids
Res. 14:5019-32 (alphadeoxynucleotides); Monia et al., 1993,
"Evaluation of 2'-modified oligonucleotides containing 2' deoxy
gaps as antisense inhibitors of gene expression", J. Biol. Chem.
268:14514-22 (2'-O-methyl-ribonucleosides); Asseline et al., 1984,
Proc. Natl. Acad. Sci. USA 81:3297-3301 (acridine); Knorre et al.,
1985, Biochemie 67:783-9; Vlassov et al., 1986, Nucl. Acids Res.
14:4065-76 (N-2-chlorocethylamine and phenazine); Webb et al.,
1986, Nucl. Acids Res. 14:7661-74
(5-methyl-N.sup.4-N.sup.4-ethanocytosin-e); Boutorin et al., 1984,
FEBS Letters 172:43-6 (Fe-ethylenediamine tetraacetic acid (EDTA)
and analogues); Chi-Hong et al., 1986, Proc. Natl. Acad. Sci. USA
83:7147-51 (5-glycylamido-1,10-o-phenanthroline); and Chu et al.,
1985, Proc. Natl. Acad. Sci. USA 82:963-7
(diethylenetriaamine-pentaacetic acid (DTPA) derivatives).
[0057] The effective dose of bcl-2 antisense oligomer to be
administered during a treatment cycle ranges from about 0.01 to
0.1, 0.1 to 1, or 1 to 10 mg/kg/day. The dose of bcl-2 antisense
oligomer to be administered can be dependent on the mode of
administration. For example, intravenous administration of a bcl-2
antisense oligomer would likely result in a significantly higher
full body dose than a full body dose resulting from a local implant
containing a pharmaceutical composition comprising bcl-2 antisense
oligomer. In one embodiment, a bcl-2 antisense oligomer is
administered subcutaneously at a dose of 0.01 to 10 mg/kg/day; more
preferably at a dose of 4 to 9 mg/kg/day; most preferably at a dose
of 5 to 7 mg/kg/day. In another embodiment, a bcl-2 antisense
oligomer is administered intravenously at a dose of 0.01 to 10
mg/kg/day; more preferably at a dose of 4 to 9 mg/kg/day; most
preferably at a dose of 5 to 7 mg/kg/day. In yet another
embodiment, a bcl-2 antisense oligomer is administered locally at a
dose of 0.01 to 10 mg/kg/day; preferably at a dose of 0.01 to 0.1;
more preferably at a dose of 1 to 5 mg/kg/day. It will be evident
to one skilled in the art that local administrations can result in
lower total body doses. For example, local administration methods
such as intratumor administration, intraocular injection, or
implantation, can produce locally high concentrations of bcl-2
antisense oligomer, but represent a relatively low dose with
respect to total body weight. Thus, in such cases, local
administration of a bcl-2 antisense oligomer is contemplated to
result in a total body dose of about 0.01 to 5 mg/kg/day.
[0058] In another embodiment, a particularly high dose of bcl-2
antisense oligomer, which ranges from about 10 to 20, 20 to 30, or
30 to 50 mg/kg/day, is administered during a treatment cycle.
[0059] Moreover, the effective dose of a particular bcl-2 antisense
oligomer may depend on additional factors, including the type of
cancer, the disease state or stage of disease, the oligomer's
toxicity, the oligomer's rate of uptake by cancer cells, as well as
the weight, age, and health of the individual to whom the antisense
oligomer is to be administered. Because of the many factors present
in vivo that may interfere with the action or biological activity
of a bcl-2 antisense oligomer, one of ordinary skill in the art can
appreciate that an effective amount of a bcl-2 antisense oligomer
may vary for each individual.
[0060] In another embodiment, a bcl-2 antisense oligomer is at a
dose which results in circulating plasma concentrations of the
bcl-2 antisense oligomer which is at least 30 nM (nanomolar). As
will be apparent to the skilled artisan, lower or higher plasma
concentrations of the bcl-2 antisense oligomer may be preferred
depending on the mode of administration. For example, plasma
concentrations of the bcl-2 antisense oligomer of at least 30 nM
can be appropriate in connection with intravenous, subcutaneous,
intramuscular, controlled release, and oral administration methods,
to name a few. In another example, relatively low circulating
plasma levels of the bcl-2 antisense oligomer can be desirable,
however, when using local administration methods such as, for
example, intratumor administration, intraocular administration, or
implantation, which nevertheless can produce locally high,
clinically effective concentrations of bcl-2 antisense
oligomer.
[0061] In yet another embodiment, the circulating plasma
concentration of at least 30 nM (nanomolar) of the bcl-2 antisense
oligomer is achieved about 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 hours
after the administration of the bcl-2 antisense oligomer. In yet
another embodiment, the circulating plasma concentration of at
least 30 nM of the bcl-2 antisense oligomer is achieved in about 36
to 48 hours, preferably 24 to 35 hours, more preferably in 12 to 24
hours; most preferably in under 12 hours.
[0062] In a specific embodiment, the dose of a bcl-2 antisense
oligomer is a high dose. In one embodiment, the circulating plasma
concentration of the bcl-2 antisense oligomer is at least 30 nM. In
another embodiment, the circulating level of bcl-2 antisense
oligomer is 1 .mu.M to 10 .mu.M. In yet another embodiment, the
circulating level of bcl-2 antisense oligomer is 1, 2, 3, 4, 5, 6,
7, 8, 9 or 10 .mu.M. In yet another embodiment, the circulating
level of bcl-2 antisense oligomer of 1 .mu.M to 10 .mu.M is
achieved in about 36 to 48 hours, preferably 24 to 35 hours, more
preferably in 12 to 24 hours; most preferably in under 12
hours.
[0063] The high dose may be achieved by several administrations per
cycle. Alternatively, the high dose may be administered in a single
bolus administration. A single administration of a high dose may
result in circulating plasma levels of bcl-2 antisense oligomer
that are transiently much higher than 30 nM. Moreover, single
administrations of particularly high doses of a bcl-2 antisense
oligomer may result in a circulating plasma concentration of bcl-2
antisense oligomer of 1 .mu.M to 10 .mu.M in much less 12 hours,
even in less than one hour.
[0064] Additionally, the dose of a bcl-2 antisense oligomer may
vary according to the particular bcl-2 antisense oligomer used. The
dose employed is likely to reflect a balancing of considerations,
among which are stability, localization, cellular uptake, and
toxicity of the particular bcl-2 antisense oligomer. For example, a
particular chemically modified bcl-2 antisense oligomer may exhibit
greater resistance to degradation, or may exhibit higher affinity
for the target nucleic acid, or may exhibit increased uptake by the
cell or cell nucleus; all of which may permit the use of low doses.
In yet another example, a particular chemically modified bcl-2
antisense oligomer may exhibit lower toxicity than other antisense
oligomers, and therefore can be used at high doses. Thus, for a
given bcl-2 antisense oligomer, an appropriate dose to administer
can be relatively high or relatively low. Appropriate doses would
be appreciated by the skilled artisan, and the invention
contemplates the continued assessment of optimal treatment
schedules for particular species of bcl-2 antisense oligomers. The
daily dose can be administered in one or more treatments.
[0065] Other factors to be considered in determining an effective
dose of a bcl-2 antisense oligomer include whether the oligomer
will be administered in combination with other therapeutics. In
such cases, the relative toxicity of the other therapeutics may
indicate the use of a bcl-2 antisense oligomer at low doses.
Alternatively, treatment with a high dose of bcl-2 antisense
oligomer can result in combination therapies with reduced doses of
therapeutics. In a specific embodiment, treatment with a
particularly high dose of bcl-2 antisense oligomer can result in
combination therapies with greatly reduced doses of cancer
therapeutics. For example, treatment of a patient with 10, 20, 30,
40, or 50 mg/kg/day of a bcl-2 antisense oligomer can further
increase the sensitivity of a subject to cancer therapeutics. In
such cases, the particularly high dose of bcl-2 antisense oligomer
is combined with, for example, a greatly shortened radiation
therapy schedule. In another example, the particularly high dose of
a bcl-2 antisense oligomer produces significant enhancement of the
potency of cancer therapeutic agents.
[0066] Additionally, the particularly high doses of bcl-2 antisense
oligomer may further shorten the period of administration of a
therapeutically effective amount of bcl-2 antisense oligomer and/or
cancer therapeutic, such that the length of a treatment cycle is
much shorter than 14 days.
[0067] In one embodiment, an 18-base phosphorothioate bcl-2
antisense oligomer of the sequence 5'-TCTCCCAGCGTGCGCCAT-3' (SEQ ID
NO:1, G3139), which is complementary to the first six codons of the
bcl-2 mRNA and hybridizes to the respective target RNA bases, is
administered for a short treatment cycle, defined as less than two
weeks.
[0068] In one embodiment, G3139 is administered for 2 to 13 days at
a dose of 0.01 to 10 mg/kg/day. In a specific embodiment, G3139 is
administered for 2 to 3, 4 to 5, 6 to 7, 8 to 9, 10 to 11, or 12 to
13 days at a dose of 0.01 to 1, 1 to 2, 3 to 4, 5 to 6, 6 to 7, 7
to 8, or 9 to 10 mg/kg/day; more preferably at a dose of 4 to 9
mg/kg/day, and most preferably at a dose of 5 to 7 mg/kg/day. In
another embodiment, G3139 is administered at said dose for 3 to 9
days. In yet another embodiment, G3139 is administered at said dose
for 4 to 7 days. In a preferred embodiment, G3139 is administered
at said dose for 5 to 6 days. In a most preferred embodiment, G3139
is administered at a dose of 5 to 7 mg/kg/day for 5 to 6 days. The
invention contemplates other preferred treatment regimens depending
on the particular bcl-2 antisense oligomer to be used, or depending
on the particular mode of administration, or depending on whether
the bcl-2 antisense oligomer is administered as part of a
combination therapy, e.g., in combination with a cancer therapeutic
agent. The daily dose can be administered in one or more
treatments.
[0069] In another embodiment, G3139 is administered at a
particularly high dose of about 10 to 50 mg/kg/day. In a specific
embodiment, G3139 is administered at a particularly high dose of
about 10 to 15, 16 to 20, 21 to 25, 26 to 30, 31 to 35, 36 to 40,
41 to 45, or 46 to 50 mg/kg/day. In a further embodiment, G3139 is
administered at said dose for 1 to 10 days. In yet another
embodiment, G3139 is administered at said dose for 2 to 7 days. In
a yet another embodiment, G3139 is administered at said dose for 3
to 4 days. In a preferred embodiment, G3139 is administered at a
dose of 26 to 30, 31 to 35, 36 to 40, 41 to 45, or 46 to 50
mg/kg/day for a minimum of 1 day. The invention contemplates other
preferred treatment regimens depending on the particular bcl-2
antisense oligomer to be used, or depending on the particular mode
of administration, or depending on whether the bcl-2 antisense
oligomer is administered as part of a combination therapy, e.g., in
combination with a cancer therapeutic agent. The daily dose can be
administered in one or more treatments.
Cancer Therapeutics
[0070] The invention described herein encompasses a method of
preventing or treating cancer comprising a therapeutically
effective amount of a bcl-2 antisense oligomer, including but not
limited to high doses of the oligomer, to a human in need of such
therapy. The invention further encompasses the use of a short
period of administration of a bcl-2 antisense oligomer. Normal,
non-cancerous cells divide at a frequency characteristic for the
particular cell type. When a cell has been transformed into a
cancerous state, uncontrolled cell proliferation and reduced cell
death results, and therefore, promiscuous cell division or cell
growth is a hallmark of a cancerous cell type. Examples of types of
cancer, include, but are not limited to, non-Hodgkin's lymphoma,
Hodgkin's lymphoma, leukemia (e.g., acute leukemia such as acute
lymphocytic leukemia, acute myelocytic leukemia, chronic myeloid
leukemia, chronic lymphocytic leukemia, multiple myeloma), colon
carcinoma, rectal carcinoma, pancreatic cancer, breast cancer,
ovarian cancer, prostate cancer, renal cell carcinoma, hepatoma,
bile duct carcinoma, choriocarcinoma, cervical cancer, testicular
cancer, lung carcinoma, bladder carcinoma, melanoma, head and neck
cancer, brain cancer, cancers of unknown primary site, neoplasms,
cancers of the peripheral nervous system, cancers of the central
nervous system, tumors (e.g., fibrosarcoma, myxosarcoma,
liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma,
angiosarcoma, endotheliosarcoma, lymphangiosarcoma,
lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing's
tumor, leiomyosarcoma, rhabdomyosarcoma, squamous cell carcinoma,
basal cell carcinoma, adenocarcinoma, sweat gland carcinoma,
sebaceous gland carcinoma, papillary carcinoma, papillary
adenocarcinomas, cystadenocarcinoma, medullary carcinoma,
bronchogenic carcinoma, seminoma, embryonal carcinoma, Wilms'
tumor, small cell lung carcinoma, epithelial carcinoma, glioma,
astrocytoma, medulloblastoma, craniopharyngioma, ependymoma,
pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma,
meningioma, neuroblastoma, and retinoblastoma), heavy chain
disease, metastases, or any disease or disorder characterized by
uncontrolled or abnormal cell growth.
[0071] In a preferred embodiment, the invention further encompasses
the use of combination therapy to prevent or treat cancer. For
example, prostate cancer can be treated with a pharmaceutical
composition comprising a bcl-2 antisense oligomer in combination
with paclitaxel, docetaxel, mitoxantrone, and/or an androgen
receptor antagonist (e.g., flutamide). As another example, breast
cancer can be treated with a pharmaceutical composition comprising
a bcl-2 antisense oligomer in combination with docetaxel,
paclitaxel, cisplatin, 5-fluorouracil, doxorubicin, and/or VP-16
(etoposide). As another example, leukemia can be treated with a
pharmaceutical composition comprising a bcl-2 antisense oligomer in
combination with fludarabine, cytosine arabinoside, gemtuzumab
(MYLOTARG), daunorubicin, methotrexate, vincristine,
6-mercaptopurine, idarubicin, mitoxantrone, etoposide,
asparaginase, prednisone and/or cyclophosphamide. As another
example, myeloma can be treated with a pharmaceutical composition
comprising a bcl-2 antisense oligomer in combination with
dexamethasone. As another example, melanoma can be treated with a
pharmaceutical composition comprising a bcl-2 antisense oligomer in
combination with dacarbazine. As another example, colorectal cancer
can be treated with a pharmaceutical composition comprising a bcl-2
antisense oligomer in combination with irinotecan. As another
example, lung cancer can be treated with a pharmaceutical
composition comprising a bcl-2 antisense oligomer in combination
with paclitaxel, docetaxel, etoposide and/or cisplatin. As another
example, non-Hodgkin's lymphoma can be treated with a
pharmaceutical composition comprising a bcl-2 antisense oligomer in
combination with cyclophosphamide, CHOP, etoposide, bleomycin,
mitoxantrone and/or cisplatin. As another example, gastric cancer
can be treated with a pharmaceutical composition comprising a bcl-2
antisense oligomer in combination with cisplatin. As another
example, pancreatic cancer can be treated with a pharmaceutical
composition comprising a bcl-2 antisense oligomer in combination
with gemcitabine. These combination therapies can also be used to
prevent cancer or the recurrence of cancer.
[0072] Combination therapy also includes, in addition to
administration of a bcl-2 antisense oligomer, the use of one or
more molecules, compounds or treatments that aid in the prevention
or treatment of cancer, which molecules, compounds or treatments
includes, but is not limited to, chemoagents, immunotherapeutics,
cancer vaccines, anti-angiogenic agents, cytokines, hormone
therapies, gene therapies, and radiotherapies.
[0073] In one embodiment, one or more chemoagents, in addition to a
bcl-2 antisense oligomer, is administered to treat a cancer
patient. Examples of chemoagents contemplated by the present
invention include, but are not limited to, cytosine arabinoside,
taxoids (e.g., paclitaxel, docetaxel), anti-tubulin agents (e.g.,
paclitaxel, docetaxel, Epothilone B, or its analogues), cisplatin,
carboplatin, adriamycin, tenoposide, mitozantron,
2-chlorodeoxyadenosine, alkylating agents (e.g., cyclophosphamide,
mechlorethamine, thioepa, chlorambucil, melphalan, carmustine
(BSNU), lomustine (CCNU), cyclothosphamide, busulfan,
dibromomannitol, streptozotocin, mitomycin C, and
cis-dichlorodiamine platinum (II) (DDP) cisplatin, thio-tepa),
antibiotics (e.g., dactinomycin (formerly actinomycin), bleomycin,
mithramycin, anthramycin), antimetabolites (e.g., methotrexate,
6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil,
fludarabine, gemcitabine, dacarbazine, temozolamide), asparaginase,
Bacillus Calmette and Guerin, diphtheria toxin, hexamethylmelamine,
hydroxyurea, LYSODREN.RTM., nucleoside analogues, plant alkaloids
(e.g., Taxol, paclitaxel, camptothecin, topotecan, irinotecan
(CAMPTOSAR, CPT-11), vincristine, vinca alkyloids such as
vinblastine), podophyllotoxin (including derivatives such as
epipodophyllotoxin, VP-16 (etoposide), VM-26 (teniposide)),
cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin,
procarbazine, mechlorethamine, anthracyclines (e.g., daunorubicin
(formerly daunomycin), doxorubicin, doxorubicin liposomal),
dihydroxyanthracindione-, mitoxantrone, mithramycin, actinomycin D,
procaine, tetracaine, lidocaine, propranolol, puromycin,
anti-mitotic agents, abrin, ricin A, pseudomonas exotoxin, nerve
growth factor, platelet derived growth factor, tissue plasminogen
activator, aldesleukin, allutamine, anastrozle, bicalutamide,
biaomycin, busulfan, capecitabine, carboplain, chlorabusil,
cladribine, cylarabine, daclinomycin, estramusine, floxuridhe,
gamcitabine, gosereine, idarubicin, itosfamide, lauprolide acetate,
levamisole, lomusline, mechlorethamine, magestrol, acetate,
mercaptopurino, mesna, mitolanc, pegaspergase, pentoslatin,
picamycin, rituximab, campath-1, straplozocin, thioguanine,
tretinoin, vinorelbine, or any fragments, family members, or
derivatives thereof, including pharmaceutically acceptable salts
thereof. Compositions comprising one or more chemoagents (e.g.,
FLAG, CHOP) are also contemplated by the present invention. FLAG
comprises fludarabine, cytosine arabinoside (Ara-C) and G-CSF. CHOP
comprises cyclophosphamide, vincristine, doxorubicin, and
prednisone.
[0074] In one embodiment, said chemoagent is dacarbazine at a dose
ranging from 200 to 4000 mg/m.sup.2/cycle. In a preferred
embodiment, said dose ranges from 700 to 1000 mg/m.sup.2/cycle. In
another embodiment, said chemoagent is fludarabine at a dose
ranging from 25 to 50 mg/m.sup.2/cycle. In another embodiment, said
chemoagent is cytosine arabinoside (Ara-C) at a dose ranging from
200 to 2000 mg/m.sup.2/cycle. In another embodiment, said
chemoagent is docetaxel at a dose ranging from 1.5 to 7.5
mg/kg/cycle. In another embodiment, said chemoagent is paclitaxel
at a dose ranging from 5 to 15 mg/kg/cycle. In yet another
embodiment, said chemoagent is cisplatin at a dose ranging from 5
to 20 mg/kg/cycle. In yet another embodiment, said chemoagent is
5-fluorouracil at a dose ranging from 5 to 20 mg/kg/cycle. In yet
another embodiment, said chemoagent is doxorubicin at a dose
ranging from 2 to 8 mg/kg/cycle. In yet another embodiment, said
chemoagent is epipodophyllotoxin at a dose ranging from 40 to 160
mg/kg/cycle. In yet another embodiment, said chemoagent is
cyclophosphamide at a dose ranging from 50 to 200 mg/kg/cycle. In
yet another embodiment, said chemoagent is irinotecan at a dose
ranging from 50 to 75, 75 to 100, 100 to 125, or 125 to 150
mg/m.sup.2/cycle. In yet another embodiment, said chemoagent is
vinblastine at a dose ranging from 3.7 to 5.4, 5.5 to 7.4, 7.5 to
11, or 11 to 18.5 mg/m.sup.2/cycle. In yet another embodiment, said
chemoagent is vincristine at a dose ranging from 0.7 to 1.4, or 1.5
to 2 mg/m.sup.2/cycle. In yet another embodiment, said chemoagent
is methotrexate at a dose ranging from 3.3 to 5, 5 to 10, 10 to
100, or 100 to 1000 mg/m.sup.2/cycle.
[0075] In a preferred embodiment, the invention further encompasses
the use of low doses of chemoagents when administered as part of a
bcl-2 antisense oligomer treatment regimen. For example, initial
treatment with a bcl-2 antisense oligomer increases the sensitivity
of a tumor to subsequent challenge with a dose of chemoagent, which
dose is near or below the lower range of dosages when the
chemoagent is administered without a bcl-2 antisense oligomer. In
one embodiment, a bcl-2 antisense oligomer and a low dose (e.g., 6
to 60 mg/m2/day or less) of docetaxel are administered to a cancer
patient. In another embodiment, a bcl-2 antisense oligomer and a
low dose (e.g., 10 to 135 mg/m.sup.2/day or less) of paclitaxel are
administered to a cancer patient. In yet another embodiment, a
bcl-2 antisense oligomer and a low dose (e.g., 2.5 to 25
mg/m.sup.2/day or less) of fludarabine are administered to a cancer
patient. In yet another embodiment, a bcl-2 antisense oligomer and
a low dose (e.g., 0.5 to 1.5 g/m.sup.2/day or less) of cytosine
arabinoside (Ara-C) are administered to a cancer patient.
[0076] The invention, therefore, contemplates the use of one or
more bcl-2 antisense oligomers, which is administered prior to,
subsequently, or concurrently with low doses of chemoagents, for
the prevention or treatment of cancer.
[0077] In one embodiment, said chemoagent is cisplatin, e.g.,
PLATINOL or PLATINOL-AQ (Bristol Myers), at a dose ranging from 5
to 10, 10 to 20, 20 to 40, or 40 to 75 mg/m.sup.2/cycle. In another
embodiment, a dose of cisplatin ranging from 7.5 to 75
mg/m.sup.2/cycle is administered to a patient with ovarian cancer.
In another embodiment, a dose of cisplatin ranging from 5 to 50
mg/m.sup.2/cycle is administered to a patient with bladder
cancer.
[0078] In another embodiment, said chemoagent is carboplatin, e.g.,
PARAPLATIN (Bristol Myers), at a dose ranging from 2 to 4, 4 to 8,
8 to 16, 16 to 35, or to 75 mg/m.sup.2/cycle. In another
embodiment, a dose of carboplatin ranging from 7.5 to 75
mg/m.sup.2/cycle is administered to a patient with ovarian cancer.
In another embodiment, a dose of carboplatin ranging from 5 to 50
mg/m.sup.2/cycle is administered to a patient with bladder cancer.
In another embodiment, a dose of carboplatin ranging from 2 to 20
mg/m.sup.2/cycle is administered to a patient with testicular
cancer.
[0079] In another embodiment, said chemoagent is cyclophosphamide,
e.g., CYTOXAN (Bristol Myers Squibb), at a dose ranging from 0.25
to 0.5, 0.5 to 1, 1 to 2, 2 to 5, 5 to 10, 10 to 20, 20 to 40
mg/kg/cycle. In another embodiment, a dose of cyclophosphamide
ranging from 4 to 40 mg/kg/cycle is administered to a patient with
malignant cancer. In another embodiment, a dose of cyclophosphamide
ranging from 0.25 to 2.5 mg/kg/cycle is administered to a patient
with non-malignant cancer.
[0080] In one embodiment, said chemoagent is cytarabine, e.g.,
CYTOSAR-U (Pharmacia & Upjohn), at a dose ranging from 0.5 to
1, 1 to 4, 4 to 10, 10 to 25, 25 to 50, or 50 to 100
mg/m.sup.2/cycle. In another embodiment, a dose of cytarabine
ranging from 10 to 100 mg/m.sup.2/cycle is administered to a
patient with acute leukemia. In another embodiment, a dose of
cytarabine ranging from 0.5 to 5 mg/m.sup.2/cycle is administered
to a patient with meningeal leukemia. In another embodiment, a dose
of cytarabine liposome, e.g., DEPOCYT (Chiron Corp.) ranging from 5
to 50 mg/m.sup.2/cycle is administered to a patient with
cancer.
[0081] In another embodiment, said chemoagent is dacarbazine, e.g.,
DTIC or DTIC-DOME (Bayer Corp.), at a dose ranging from 15 to 250
mg/m.sup.2/cycle or ranging from 0.2 to 2 mg/kg/cycle. In another
embodiment, a dose of dacarbazine ranging from 15 to 150
mg/m.sup.2/cycle is administered to a patient with Hodgkin's
disease. In another embodiment, a dose of dacarbazine ranging from
0.2 to 2 mg/kg/cycle is administered to a patient with malignant
melanoma.
[0082] In another embodiment, said chemoagent is topotecan, e.g.,
HYCAMTIN (SmithKline Beecham), at a dose ranging from 0.1 to 0.2,
0.2 to 0.4, 0.4 to 0.8, or 0.8 to 1.5 mg/m.sup.2/cycle.
[0083] In another embodiment, said chemoagent is irinotecan, e.g.,
CAMPTOSAR (Pharmacia & Upjohn), at a dose ranging from 5 to 10,
10 to 25, or 25 to 50 mg/m.sup.2/cycle.
[0084] In another embodiment, said chemoagent is fludarabine, e.g.,
FLUDARA (Berlex Laboratories), at a dose ranging from 2.5 to 5, 5
to 10, 10 to 15, or 15 to 25 mg/m.sup.2/cycle.
[0085] In another embodiment, said chemoagent is cytosine
arabinoside (Ara-C) at a dose ranging from 200 to 2000
mg/m.sup.2/cycle.
[0086] In another embodiment, said chemoagent is docetaxel, e.g.,
TAXOTERE (Rhone Poulenc Rorer) at a dose ranging from 6 to 10, 10
to 30, or 30 to 60 mg/m.sup.2/cycle.
[0087] In another embodiment, said chemoagent is paclitaxel, e.g.,
TAXOL (Bristol Myers Squibb), at a dose ranging from 10 to 20, 20
to 40, 40 to 70, or 70 to 135 mg/kg/cycle.
[0088] In another embodiment, said chemoagent is 5-fluorouracil at
a dose ranging from 0.5 to 5 mg/kg/cycle.
[0089] In another embodiment, said chemoagent is doxorubicin, e.g.,
ADRIAMYCIN (Pharmacia & Upjohn), DOXIL (Alza), RUBEX (Bristol
Myers Squibb), at a dose ranging from 2 to 4, 4 to 8, 8 to 15, 15
to 30, or 30 to 60 mg/kg/cycle.
[0090] In another embodiment, said chemoagent is etoposide, e.g.,
VEPESID (Pharmacia & Upjohn), at a dose ranging from 3.5 to 7,
7 to 15, 15 to 25, or 25 to 50 mg/m.sup.2/cycle. In another
embodiment, a dose of etoposide ranging from 5 to 50
mg/m.sup.2/cycle is administered to a patient with testicular
cancer. In another embodiment, a dose of etoposide ranging from 3.5
to 35 mg/m.sup.2/cycle is administered to a patient with small cell
lung cancer.
[0091] In another embodiment, said chemoagent is vinblastine, e.g.,
VELBAN (Eli Lilly), at a dose ranging from 0.3 to 0.5, 0.5 to 1, 1
to 2, 2 to 3, or 3 to 3.7 mg/m.sup.2/cycle.
[0092] In another embodiment, said chemoagent is vincristine, e.g.,
ONCOVIN (Eli Lilly), at a dose ranging from 0.1, 0.2, 0.3, 0.4,
0.5, 0.6 or 0.7 mg/m.sup.2/cycle.
[0093] In another embodiment, said chemoagent is methotrexate at a
dose ranging from 0.2 to 0.9, 1 to 5, 5 to 10, 10 to 20.
[0094] In another embodiment, a bcl-2 antisense oligomer is
administered in combination with one or more immunotherapeutic
agents, such as antibodies and immunomodulators, which includes,
but is not limited to, rituxan, rituximab, campath-1, gemtuzumab,
or trastuzumab.
[0095] In another embodiment, a bcl-2 antisense oligomer is
administered in combination with one or more antiangiogenic agents,
which includes, but is not limited to, angiostatin, thalidomide,
kringle 5, endostatin, Serpin (Serine Protease Inhibitor)
anti-thrombin, 29 kDa N-terminal and a 40 kDa C-terminal
proteolytic fragments of fibronectin, 16 kDa proteolytic fragment
of prolactin, 7.8 kDa proteolytic fragment of platelet factor-4, a
13-amino acid peptide corresponding to a fragment of platelet
factor-4 (Maione et al., 1990, Cancer Res. 51:2077-2083), a
14-amino acid peptide corresponding to a fragment of collagen I
(Tolma et al., 1993, J. Cell Biol. 122:497-511), a 19 amino acid
peptide corresponding to a fragment of Thrombospondin I (Tolsma et
al., 1993, J. Cell Biol. 122:497-511), a 20-amino acid peptide
corresponding to a fragment of SPARC (Sage et al., 1995, J. Cell.
Biochem. 57:1329-1334), or any fragments, family members, or
derivatives thereof, including pharmaceutically acceptable salts
thereof.
[0096] Other peptides that inhibit angiogenesis and correspond to
fragments of laminin, fibronectin, procollagen, and EGF have also
been described (see the review by Cao, 1998, Prog. Mol. Subcell.
Biol. 20:161-176). Monoclonal antibodies and cyclic pentapeptides,
which block certain integrins that bind RGD proteins (i.e., possess
the peptide motif Arg-Gly-Asp), have been demonstrated to have
anti-vascularization activities (Brooks et al., 1994, Science
264:569-571; Hammes et al., 1996, Nature Medicine 2:529-533).
Moreover, inhibition of the urokinase plasminogen activator
receptor by receptor antagonists inhibits angiogenesis, tumor
growth and metastasis (Min et al., 1996, Cancer Res. 56: 2428-33;
Crowley et al., 1993, Proc. Natl. Acad. Sci. USA 90:5021-25). Use
of such antiangiogenic agents is also contemplated by the present
invention.
[0097] In another embodiment, a bcl-2 antisense oligomer is
administered in combination with a regimen of radiation.
[0098] In another embodiment, a bcl-2 antisense oligomer is
administered in combination with one or more cytokines, which
includes, but is not limited to, lymphokines, tumor necrosis
factors, tumor necrosis factor-like cytokines, lymphotoxin-.alpha.,
lymphotoxin-.beta., interferon-.alpha., interferon-.beta.,
macrophage inflammatory proteins, granulocyte monocyte colony
stimulating factor, interleukins (including, but not limited to,
interleukin-1, interleukin-2, interleukin-6, interleukin-12,
interleukin-15, interleukin-18), OX40, CD27, CD30, CD40 or CD137
ligands, Fas-Fas ligand, 4-1BBL, endothelial monocyte activating
protein or any fragments, family members, or derivatives thereof,
including pharmaceutically acceptable salts thereof.
[0099] In yet another embodiment, a bcl-2 antisense oligomer is
administered in combination with a cancer vaccine. Examples of
cancer vaccines include, but are not limited to, autologous cells
or tissues, non-autologous cells or tissues, carcinoembryonic
antigen, alpha-fetoprotein, human chorionic gonadotropin, BCG live
vaccine, melanocyte lineage proteins (e.g., gp100, MART-1/MelanA,
TRP-1 (gp75), tyrosinase, widely shared tumor-specific antigens
(e.g., BAGE, GAGE-1, GAGE-2, MAGE-1, MAGE-3,
N-acetylglucosaminyltransferase-V, p15), mutated antigens that are
tumor-specific.beta.-catenin, MUM-1, CDK4), nonmelanoma antigens
(e.g., HER-2/neu (breast and ovarian carcinoma), human
papillomavirus-E6, E7 (cervical carcinoma), MUC-1 (breast, ovarian
and pancreatic carcinoma)). For human tumor antigens recognized by
T cells, see generally Robbins and Kawakami, 1996, Curr. Opin.
Immunol. 8:628-36. Cancer vaccines may or may not be purified
preparations.
[0100] In yet another embodiment, a bcl-2 antisense oligomer is
used in association with a hormonal treatment. Hormonal therapeutic
treatments comprise hormonal agonists, hormonal antagonists (e.g.,
flutamide, tamoxifen, leuprolide acetate (LUPRON)), and steroids
(e.g., dexamethasone, retinoids, betamethasone, cortisol,
cortisone, prednisone, dehydrotestosterone, glucocorticoids,
mineralocorticoids, estrogen, testosterone, progestins).
[0101] In yet another embodiment, a bcl-2 antisense oligomer is
used in association with a gene therapy program in the treatment of
cancer.
[0102] In one embodiment, a bcl-2 antisense oligomer is
administered, in combination with at least one cancer therapeutic
agent, for a short treatment cycle to a cancer patient to treat
cancer. In one embodiment, said treatment cycle ranges from 2 to 13
days. In another embodiment, said treatment cycle ranges from 3 to
9 days. In another embodiment, said treatment cycle ranges from 4
to 7 days. In yet another embodiment, said treatment cycle ranges
from 5 to 6 days. The duration of treatment with the cancer
therapeutic agent may vary according to the particular cancer
therapeutic agent used. The invention also contemplates
discontinuous administration or daily doses divided into several
partial administrations. An appropriate treatment time for a
particular cancer therapeutic agent will be appreciated by the
skilled artisan, and the invention contemplates the continued
assessment of optimal treatment schedules for each cancer
therapeutic agent.
[0103] The present invention contemplates at least one cycle,
preferably more than one cycle during which a single therapeutic or
sequence of therapeutics is administered. In a preferred
embodiment, the cycle is shorter than 14 days. In one embodiment,
the length of one cycle is 10-13 days. In a preferred embodiment,
the length of one cycle is 7-9 days. In a most preferred
embodiment, the length of one cycle is 5-6 days. An appropriate
period of time for one cycle will be appreciated by the skilled
artisan, as will the total number of cycles, and the interval
between cycles. The invention contemplates the continued assessment
of optimal treatment schedules for each bcl-2 antisense oligomer
and cancer therapeutic agent.
Pharmaceutical Compositions
[0104] The present invention further provides for a pharmaceutical
composition that comprises a bcl-2 antisense oligomer and a
pharmaceutically acceptable carrier. Suitable pharmaceutically
acceptable carriers include essentially chemically inert and
nontoxic compositions that do not interfere with the effectiveness
of the biological activity of the pharmaceutical composition.
Examples of suitable pharmaceutical carriers include, but are not
limited to, saline solutions, glycerol solutions, ethanol,
N-(1(2,3-dioleyloxy)propyl)-N,N,N-trimethylammonium chloride
(DOTMA), diolesylphosphotidylethanolamine (DOPE), and liposomes.
Such compositions should contain a therapeutically effective amount
of the compound, together with a suitable amount of carrier so as
to provide the form for proper administration to the patient. The
formulation should suit the mode of administration. For example,
oral administration requires enteric coatings to protect the
antisense oligomer from degradation within the gastrointestinal
tract. In another example, the antisense oligomer may be
administered in a liposomal formulation to shield the antisense
oligomer from degradative enzymes, facilitate transport in
circulatory system, and effect delivery across cell membranes to
intracellular sites.
[0105] In another embodiment, a pharmaceutical composition
comprises a bcl-2 antisense oligomer and one or more therapeutic
agents and a pharmaceutically acceptable carrier. In a particular
embodiment, the pharmaceutical composition comprises a bcl-2
antisense oligomer and one or more cancer therapeutic agents and a
pharmaceutically acceptable carrier.
[0106] In one embodiment, a pharmaceutical composition, comprising
a bcl-2 antisense oligomer, with or without other therapeutic
agents, and a pharmaceutically acceptable carrier, is at an
effective dose.
[0107] In one embodiment, the pharmaceutical composition comprises
a bcl-2 antisense oligomer at a dose of about 0.01 to 0.1, 0.1 to
1, 1 to 5, or 6 to 10 mg/kg/day; preferably at a dose of 4 to 9
mg/kg/day; more preferably at a dose of 5 to 7 mg/kg/day; and a
pharmaceutically acceptable carrier. The actual amount of any
particular antisense oligomer administered can depend on several
factors, such as the type of cancer, the toxicity of the antisense
oligomer to normal cells of the body, the rate of uptake of the
antisense oligomer by tumor cells, and the weight and age of the
individual to whom the antisense oligomer is administered. Because
of the many factors present in vivo that may interfere with the
action or biological activity of the antisense oligomer, an
effective amount of the antisense oligomer may vary for each
individual.
[0108] In another embodiment, the pharmaceutical compositions of
the invention comprise a bcl-2 antisense oligomer at a particularly
high dose, which ranges from about 10 to 50 mg/kg/day. In a
specific embodiment a particularly high dose of bcl-2 antisense
oligomer, ranging from 11 to 15, 16 to 20, 21 to 25, 26 to 30, 31
to 35, 36 to 40, 41 to 45, or 46 to 50 mg/kg/day, is administered
during a treatment cycle.
[0109] Selection of the preferred effective dose can be determined
(e.g., via clinical trials) by a skilled artisan based upon the
consideration of several factors which will be known to one of
ordinary skill in the art. Such factors include the particular form
of antisense oligomer, the oligomer's pharmacokinetic parameters
such as bioavailability, metabolism, half-life, etc., which is
established during the development procedures typically employed in
obtaining regulatory approval of a pharmaceutical compound. Further
factors in considering the dose include the disease to be treated,
the benefit to be achieved in a patient, the patient's body mass,
the patient's immune status, the route of administration, whether
administration of the antisense oligomer or combination therapeutic
agent is acute or chronic, concomitant medications, and other
factors known by the skilled artisan to affect the efficacy of
administered pharmaceutical agents.
[0110] The compositions of the invention can be formulated as
neutral or salt forms. Pharmaceutically acceptable salts include
those formed with free amino groups such as those derived from
hydrochloric, phosphoric, acetic, oxalic, tartaric acids, etc., and
those formed with free carboxyl groups such as those derived from
sodium, potassium, ammonium, calcium, ferric hydroxides,
isopropylamine, triethylamine, 2-ethylamino ethanol, histidine,
procaine, etc.
[0111] In a preferred embodiment, the composition is formulated in
accordance with routine procedures as a pharmaceutical composition
adapted for subcutaneous injection or intravenous administration to
humans. Typically, compositions for subcutaneous injection or
intravenous administration are solutions in sterile isotonic
aqueous buffer. Where necessary, the composition may also include a
solubilizing agent and a local anesthetic such as lidocaine to ease
pain at the site of the injection. Generally, the ingredients are
supplied either separately or mixed together in unit dosage form,
for example, as a dry lyophilized powder or water-free concentrate
in a hermetically sealed container such as an ampule or sachette
indicating the quantity of active agent. Where the composition is
to be administered by infusion, it can be dispensed with an
infusion bottle, bag, or other acceptable container, containing
sterile pharmaceutical grade water, saline, or other acceptable
diluents. Where the composition is administered by injection, an
ampule of sterile water for injection or saline can be provided so
that the ingredients may be mixed prior to administration.
Modes of Administration
[0112] Administration of the pharmaceutical compositions of the
invention includes, but is not limited to, oral, intravenous
infusion, subcutaneous injection, intramuscular, topical, depo
injection, implantation, time-release mode, intracavitary,
intranasal, inhalation, intratumor, intraocular, and controlled
release. The pharmaceutical compositions of the invention also may
be introduced parenterally, transmucosally (e.g., orally), nasally,
rectally, intravaginally, sublingually, submucosally, or
transdermally. Preferably, administration is parenteral, i.e., not
through the alimentary canal but rather through some other route
via, for example, intravenous, subcutaneous, intramuscular,
intraperitoneal, intraorbital, intracapsular, intraspinal,
intrasternal, intra-arterial, or intradermal administration. The
skilled artisan can appreciate the specific advantages and
disadvantages to be considered in choosing a mode of
administration. Multiple modes of administration are encompassed by
the invention. For example, a bcl-2 antisense oligomer is
administered by subcutaneous injection, whereas a combination
therapeutic agent is administered by intravenous infusion.
Moreover, administration of one or more species of bcl-2 antisense
oligomer, with or without other therapeutic agents, may occur
simultaneously (i.e., co-administration) or sequentially. For
example, a bcl-2 antisense oligomer is first administered to
increase sensitivity of a tumor to subsequent administration of a
cancer therapeutic agent or irradiation therapy. In another
embodiment, the periods of administration of one or more species of
bcl-2 antisense oligomer, with or without other therapeutic agents
may overlap. For example, a bcl-2 antisense oligomer is
administered for 7 days, and a second therapeutic agent is
introduced beginning on the fifth day of bcl-2 antisense oligomer
treatment, and treatment with the second therapeutic agent
continues beyond the 7-day bcl-2 antisense oligomer treatment.
[0113] Pharmaceutical compositions adapted for oral administration
may be provided, for example, as capsules or tablets; as powders or
granules; as solutions, syrups or suspensions (in aqueous or
non-aqueous liquids); as edible foams or whips; or as emulsions.
Tablets or hard gelatine capsules may comprise, for example,
lactose, starch or derivatives thereof, magnesium stearate, sodium
saccharine, cellulose, magnesium carbonate, stearic acid or salts
thereof. Soft gelatine capsules may comprise, for example,
vegetable oils, waxes, fats, semi-solid, or liquid polyols etc.
Solutions and syrups may comprise, for example, water, polyols and
sugars.
[0114] An active agent intended for oral administration may be
coated with or admixed with a material (e.g., glyceryl monostearate
or glyceryl distearate) that delays disintegration or affects
absorption of the active agent in the gastrointestinal tract. Thus,
for example, the sustained release of an active agent may be
achieved over many hours and, if necessary, the active agent can be
protected from being degraded within the gastrointestinal tract
Taking advantage of the various pH and enzymatic conditions along
the gastrointestinal tract, pharmaceutical compositions for oral
administration may be formulated to facilitate release of an active
agent at a particular gastrointestinal location.
[0115] Pharmaceutical compositions adapted for parenteral
administration include, but are not limited to, aqueous and
non-aqueous sterile injectable solutions or suspensions, which may
contain antioxidants, buffers, bacteriostats and solutes that
render the compositions substantially isotonic with the blood of an
intended recipient. Other components that may be present in such
compositions include water, alcohols, polyols, glycerine and
vegetable oils, for example. Compositions adapted for parenteral
administration may be presented in unit-dose or multi-dose
containers, for example sealed ampules and vials, and may be stored
in a freeze-dried (lyophilized) condition requiring the addition of
a sterile liquid carrier, e.g., sterile saline solution for
injections, immediately prior to use. Extemporaneous injection
solutions and suspensions may be prepared from sterile powders,
granules and tablets. Such compositions should contain a
therapeutically effective amount of a bcl-2 antisense oligomer or
other therapeutic agent, together with a suitable amount of carrier
so as to provide the form for proper administration to the patient.
The formulation should suit the mode of administration.
[0116] Pharmaceutical compositions adapted for transdermal
administration may be provided as discrete patches intended to
remain in intimate contact with the epidermis for a prolonged
period of time. Pharmaceutical compositions adapted for topical
administration may be provided as, for example, ointments, creams,
suspensions, lotions, powders, solutions, pastes, gels, sprays,
aerosols or oils. A topical ointment or cream is preferably used
for topical administration to the skin, mouth, eye or other
external tissues. When formulated in an ointment, the active
ingredient may be employed with either a paraffinic or a
water-miscible ointment base. Alternatively, the active ingredient
may be formulated in a cream with an oil-in-water base or a
water-in-oil base.
[0117] Pharmaceutical compositions adapted for topical
administration to the eye include, for example, eye drops or
injectable compositions. In these compositions, the active
ingredient can be dissolved or suspended in a suitable carrier,
which includes, for example, an aqueous solvent with or without
carboxymethylcellulose. Pharmaceutical compositions adapted for
topical administration in the mouth include, for example, lozenges,
pastilles and mouthwashes.
[0118] Pharmaceutical compositions adapted for nasal administration
may comprise solid carriers such as powders (preferably having a
particle size in the range of 20 to 500 microns). Powders can be
administered in the manner in which snuff is taken, i.e., by rapid
inhalation through the nose from a container of powder held close
to the nose. Alternatively, compositions adopted for nasal
administration may comprise liquid carriers such as, for example,
nasal sprays or nasal drops. These compositions may comprise
aqueous or oil solutions of the active ingredient. Compositions for
administration by inhalation may be supplied in specially adapted
devices including, but not limited to, pressurized aerosols,
nebulizers or insufflators, which can be constructed so as to
provide predetermined dosages of the active ingredient.
[0119] Pharmaceutical compositions adapted for rectal
administration may be provided as suppositories or enemas.
Pharmaceutical compositions adapted for vaginal administration may
be provided, for example, as pessaries, tampons, creams, gels,
pastes, foams or spray formulations.
[0120] In one embodiment, a pharmaceutical composition of the
invention is delivered by a controlled-release system. For example,
the pharmaceutical composition may be administered using
intravenous infusion, an implantable osmotic pump, a transdermal
patch, liposomes, or other modes of administration. In one
embodiment, a pump may be used (See e.g., Langer, 1990, Science
249:1527-33; Sefton, 1987, CRC Crit. Ref. Biomed. Eng. 14:201;
Buchwald et al., 1980, Surgery 88:507; Saudek et al., 1989, N.
Engl. J. Med. 321:574). In another embodiment, the compound can be
delivered in a vesicle, in particular a liposome (See e.g., Langer,
Science 249:1527-33 (1990); Treat et al., 1989, in Liposomes in the
Therapy of Infectious Disease and Cancer, Lopez-Berestein and
Fidler (eds.), Liss, New York, pp. 353-65; Lopez-Berestein, ibid.,
pp. 317-27 International Patent Publication No. WO 91/04014; U.S.
Pat. No. 4,704,355). In another embodiment, polymeric materials can
be used (See e.g., Medical Applications of Controlled Release,
Langer and Wise (eds.), CRC Press: Boca Raton, Fla., 1974;
Controlled Drug Bioavailability, Drug Product Design and
Performance, Smolen and Ball (eds.), Wiley: New York (1984); Ranger
and Peppas, 1953, J. Macromol. Sci. Rev. Macromol. Chem. 23:61;
Levy et al., 1985, Science 228:190; During et al., 1989, Ann
Neurol. 25:351; Howard et al., 1989, J. Neurosurg. 71:105).
[0121] In yet another embodiment, a controlled release system can
be placed in proximity of the target. For example, a micropump may
deliver controlled doses directly into the brain, thereby requiring
only a fraction of the systemic dose (See e.g., Goodson, 1984, in
Medical Applications of Controlled Release, vol. 2, pp.
115-138).
[0122] In one embodiment, it may be desirable to administer the
pharmaceutical composition of the invention locally to the area in
need of treatment; this may be achieved, for example, and not by
way of limitation, by local infusion during surgery, topical
application (e.g., in conjunction with a wound dressing after
surgery), injection, by means of a catheter, by means of a
suppository, or by means of an implant. An implant can be of a
porous, non-porous, or gelatinous material, including membranes,
such as sialastic membranes, or fibers.
[0123] Suppositories generally contain active ingredients in the
range of 0.5% to 10% by weight. Oral formulations preferably
contain 10% to 95% active ingredient by weight.
[0124] A bcl-2 antisense oligomer can be administered before,
during, and/or after the administration of one or more therapeutic
agents. In one embodiment, a bcl-2 antisense oligomer can first be
administered to reduce the expression of bcl-2, which increases the
tumor's sensitivity to subsequent challenge with a cancer
therapeutic agent. In another embodiment, a bcl-2 antisense
oligomer can be administered after administration of a cancer
therapeutic agent to reduce tumor expression of bcl-2, which can
deter tumor resistance, and thereby prevent relapse or minimization
of response to the cancer therapeutic agent. In yet another
embodiment, there can be a period of overlap between the
administration of bcl-2 antisense oligomer and one or more
therapeutic agents.
[0125] The invention further provides a pharmaceutical kit
comprising an effective amount of a bcl-2 oligomer, in combination
with a cancer therapeutic agent, to protect from or treat a bcl-2
related disorder. In one embodiment, the effective amount of a
bcl-2 oligomer and a pharmaceutically acceptable carrier may be
packaged in a single dose vial or other container. In a specific
embodiment, the bcl-2 oligomer comprises G3139 (SEQ ID NO.:1). The
kit may comprise one or more containers filled with one or more of
the ingredients of the pharmaceutical compositions of the
invention. Optionally associated with such container(s) can be a
notice in the form prescribed by a governmental agency regulating
the manufacture, use or sale of pharmaceuticals or biological
products, which notice reflects approval by the agency of
manufacture, use or sale for human administration.
[0126] The present invention may be better understood by reference
to the following non-limiting Examples, which are provided only as
exemplary of the invention.
[0127] The following examples are presented to more fully
illustrate the preferred embodiments of the invention. They should
in no way be construed, however, as limiting the broader scope of
the invention.
Example 1
Bcl-2 Antisense Therapy Chemosensitizes Malignant Melanoma
[0128] This example demonstrates the successful use of a bcl-2
antisense oligomer for the treatment of patients with advanced
malignant melanoma. In this study, six of the patients, who were
treated with the bcl-2 antisense oligomer, were systemically
administered the oligomer at 5.3 or 6.5 mg/kg/day for seven days,
in combination with a chemoagent. The findings reported in this
Example demonstrate that, when a bcl-2 antisense oligomer is
administered in high doses for short periods of time, the treatment
exhibits low toxicity as scored by common toxicity criteria,
reduces Bcl-2 within the tumor, facilitates apoptosis, and leads to
objective tumor responses and prolonged patient survival. Included
among the responding patients were several with "treatment
resistant cancer" who had experienced progressive disease during
treatment with standard anticancer agents, where treatment with
standard agents such as dacarbazine used alone would have minimal
or no expected benefit. In contrast, the combination therapy with
bcl-2 antisense and dacarbazine led to unexpected durable responses
and prolonged survival. Moreover, a follow-up study, which used
higher doses for shorter periods in five patients, demonstrated
satisfactory tolerance when the bcl-2 antisense oligomer was
administered systemically at 7 mg/kg/day for five days. Thus, the
results indicate that administration of a bcl-2 antisense oligomer
at high doses for a short period of time is a safe and effective
therapy for melanoma. The approach outlined in this study provides
a broadly applicable strategy for treating other types of
cancer.
Materials and Methods
[0129] Fourteen patients with stage 1V metastatic melanoma were
eligible for this phase I/II dose escalation study if they had
measurable disease, and if cutaneous metastases were accessible for
biopsy and initially positive for BCL-2 expression by Western
blotting (Table 1). Patients were required to have normal renal,
hepatic, and hematopoietic function and no chemo- or immunotherapy
four weeks prior to inclusion into the study.
[0130] BCL-2 antisense oligomer sequence 5'-TCTCCCAGCGTGCGCCAT-3'
(SEQ ID NO:1) was administered as a continuous intravenous infusion
(CIV) for 14 days by an ambulatory infusion pump (Sims Deltec Inc.,
St. Paul, Minn., USA) through a central venous line. Using a
separate peripheral intravenous line, DTIC was administered at
doses of 200 mg/m.sup.2/day given by one hour infusions for 5 days
on days 5 though 9 of the 14-day BCL-2 antisense oligomer therapy.
Treatment cycles were repeated monthly. Dose escalation was started
at 0.6 mg/kg/day and continued with 1.3, 1.7, 2.1, 3.1, 4.1, 5.3
and 6.5 mg/kg/day of BCL-2 ASO. Once safety was established in a
cohort of at least 3 patients at a given dose level, new patient
cohorts were entered at the next higher dose level (Waters et al.,
2000, J. Clin. Oncol. 18(9):1812-23). Repeat 28 day cycles and
intra-patient dose escalation were permitted in stable or
responding patients after a two week observation period.
[0131] To gain clinical experience with an alternative route and
schedule, six patients in the cohorts treated with 5.3 or 6.5
mg/kg/day received their first cycle by intravenous infusion and
were then switched to subcutaneous (SC) administration of BCL-2
antisense oligomer in subsequent cycles. These patients treated by
the SC route received the same total daily dose, administered by
twice-daily SC injections on days 1 through 7, combined with DTIC
800 mg/m.sup.2 given as a one-hour infusion on day 5
[0132] Antitumor effects were assessed after every cycle of
treatment, using caliper measurement and detailed
photo-documentation of patients with skin metastases; visceral
metastases were documented and followed by computed tomography
scans. WHO criteria were used, for classification of tumor
response, requiring serial documentation lasting at least 4 weeks.
Complete response was defined as disappearance of detectable
metastases. Partial response was defined as a 50% or greater
reduction of measurable metastases. Where patients demonstrated
numerous metastases in one organ, a maximum of 5 target lesions
were documented at baseline and then followed to determine
response. An increase in measurable disease of more than 25%, or
the appearance of new, metastatic lesions, were defined as
progressive disease. In addition, a situation where target lesion
diameters regressed by less than 50% but more than 25% was
designated to be a minor response. All other situations were
defined as stable disease. Survival was assessed from the time of
first treatment on this protocol.
[0133] Toxicity was scored by common toxicity criteria, and
monitored daily during drug administration, then weekly between
cycles. Any treatment-related grade III or IV toxicity that would
not resolve in the two weeks between treatment cycles was
considered a dose limiting toxicity. Plasma samples to determine
BCL-2 antisense oligomer pharmacokinetics were collected at time 0
before treatment, then on days 2, 3, 5, 6, 10, and 14 in patients
receiving the two-week intravenous infusion of BCL-2 ASO; 12 hour
pharmacokinetic profiles were determined in patients receiving
BCL-2 antisense oligomer as subcutaneous bolus injections at the
abdominal site. BCL-2 antisense oligomer plasma levels were assayed
by Pharmanalyt, Baden, Austria, using HPLC (Chen et al., 1997, J.
Chromatogr. B. Biomed. Sci. Appl. 692:43-51).
[0134] BCL-2 expression and apoptotic rate of melanoma metastases
were assessed by Western blotting and the TUNEL method,
respectively (Jansen et al., 1998, Nat. Med. 4(2):232-4). BCL-2
reductions of less than 20% compared to baseline levels were not
considered to be significant due to technical limitations. Biopsied
tumors were selected based on size, location, and clinical
features, similar to the target lesions used for measurement of
response. Excision biopsies of cutaneous melanoma metastases were
performed at baseline and on day 5 of each BCL-2 antisense oligomer
dose level prior to DTIC administration; additional biopsies were
obtained up to cycle day 14 to document the effects of combined
BCL-2 antisense oligomer and DTIC treatment. A total of 2-4 tumor
biopsies per patient per dose level have been investigated. The
portion of the tumor biopsy used for Western blots and TUNEL assay
was also evaluated by routine histopathology to ensure consistent
tumor cell content and to limit confounding effects of non-tumor
cells in the biopsy sample.
Results
[0135] A total of 14 patients were treated with BCL-2 antisense
oligomer (0.6 to 6.5 mg/kg/day) combined with DTIC according to the
two treatment regimes (I.V. or S.C.) outlined above.
[0136] BCL-2 antisense oligomer steady-state plasma levels were
observed after one day of continuous intravenous infusion and
increased linear with the administered dose. BCL-2 antisense
oligomer doses >1.7 mg/kg/day led to consistent steady-state
plasma levels over 1 .mu.g/.mu.l, a plasma level determined to be
bioactive in animal models (Raynaud et al., 1997, J. Pharmacol.
Exp. Ther. 281:420-7). At 6.5 mg/kg/day, a mean steady state plasma
level of 6.47 .mu.g/ml+/-SD=2.51 .mu.g/ml was reached by 24 hours.
BCL-2 antisense oligomer plasma levels of SC bolus injections
administered twice daily were bell-shaped over 12 hours. A peak
concentration of 8.6 .mu.g/ml+/-SD=1.26 .mu.g/ml was observed three
to four hours after injection of the SC dose of 3.25 mg/kg
administered at 12 h intervals. More than 90% of the 12 hour period
in between subcutaneous injections, plasma levels exceeded the 1
mg/ml target plasma level, associated with biological activity. No
changes in the pharmacokinetic properties were observed in patients
receiving multiple cycles of therapy; concurrent DTIC treatment did
not affect steady-state BCL-2 antisense oligomer plasma levels.
[0137] At baseline, BCL-2 protein expression of cutaneous melanoma
metastases (Selzer et al., 1998, 8(3):197-203; Cerroni et al.,
1995, Am. J. Dermatopathol.17:7-11), was confirmed by Western
blotting in all 14 patients screened for this study; serial
biopsies of comparable lesions demonstrated reductions in BCL-2
protein levels during BCL-2 antisense oligomer administration
(Table 1). In patient 10, serial tumor specimens were not evaluable
for Western blotting due to lack of melanoma cells in the biopsy
tissue. The maximal reduction of BCL-2 protein in patients treated
by 14-day continuous infusion of BCL-2 antisense oligomer was
typically observed by day 5 with no further decrease on day 14.83%
of evaluable patients with BCL-2 antisense oligomer plasma levels
exceeding 1 .mu.g/ml (10 of 12 patients) demonstrated a clear
reduction in BCL-2 expression (Table 1). Treatment cycles with
BCL-2 antisense oligomer doses >1.7 mg/kg/day demonstrated a
median reduction of BCL-2 protein reduction of 40% by day 5.
[0138] An increase of apoptotic cells in tumor specimen following 5
days of BCL-2 antisense oligomer treatment was observed by TUNEL
staining (increase from baseline 0.85%, +/-SD=047%; to 3.17%,
+/-SD=1.16%)(FIG. 2B). However, in biopsies taken after adding the
apoptotic stimulus (DTIC), an additional dramatic enhancement in
apoptotic cell death could be observed (FIG. 2C, 19.4%+/-SD=4.2%).
The combination therapy of BCL-2 antisense oligomer and DTIC was
well tolerated up to and including 6.5 mg/kg/day of BCL-2 antisense
oligomer without dose-limiting toxicity (Table 2).
[0139] Hematological abnormalities were mild or moderate (grade
I-III, Table 2), and followed the pattern of nadir values between
treatment cycles typical for single agent DTIC. None of the
patients experienced febrile neutropenia or other major clinical
hematological toxicities. Grade II-III anemia requiring transfusion
was observed in two patients during the study, but anemia was
present at baseline in these same patients, possibly caused by
prior therapies. Grade II-III lymphopenia was observed commonly,
but without clinical sequelae such as unusual viral or fungal
infections, or other clinical evidence for immunosuppression
despite repeat cycles lasting over one year in some patients.
Transient grade II-III prolongation of partial thromboplastin time
was observed in three patients without clinical bleeding.
[0140] Non-hematological adverse events are listed in the lower
part of Table 2. BCL-2 antisense oligomer doses over 4.1 mg/kg/day
were associated with transient fever in most patients. The fever
commonly reached 38.degree. C. on days 2-3 of therapy and resolved
either spontaneously or with administration of acetaminophen and
continued antisense oligomer administration. At the dose levels
ranging from 4.1 to 6.5 mg/kg/day, transient grade II-III
elevations of transaminase and/or bilirubin were observed in 4
patients; however the causal relationship to BCL-2 antisense
oligomer was not established in all patients, since two patients
had hepatitis and alcoholism, respectively, and the transient liver
function abnormalities were observed after DTIC, which can also
lead to such laboratory changes. The liver function abnormalities
typically resolved in 1 week between treatment cycles, and were not
considered clinically significant or dose-limiting. Dermatological
adverse events included transient rashes and urticaria, grade I in
all but one patient who experienced transient grade II urticaria;
these dermatological reactions responded to antihistamines and did
not prevent subsequent therapy. No cumulative toxicities were
observed. Some patients were treated with up to 10 cycles of
therapy without requiring modifications of the planned treatment
schedules.
[0141] Even though toxicity was the main endpoint of this dose
escalation trial, antitumor activity was evident in 6 of 14
patients (43%, Table 1) with stage 1V melanoma, including responses
seen among the 12 patients who enrolled into the study after
treatment-failure of systemic melanoma therapies. 1 CR, 2 PR, and 2
MR with prolonged stabilization of disease lasting over 1 year were
noted (Table 1). Clinical antitumor activity was also observed in
two additional patients with stabilization of disease that was
clearly progressing prior to study enrollment. Patient 12, who had
bulky metastatic disease measuring over 5 cm at baseline in pelvic
lymph nodes and at the site of a prior skin graft, demonstrated
rapid response after 2 cycles and complete response after 4 cycles
(Table 1, FIG. 3). After 4 cycles of therapy, a biopsy of the
cutaneous area that had been previously positive for neoplasm
showed only fibrosis with no melanoma (pathologic complete
response). Patients 2 and 3 demonstrated partial response of target
lesions with progression-free survivals lasting over one year. At
entry to this study, patients 2 and 3 had progressive metastases
despite prior treatments with carboplatin plus interferon (patient
2) and DTIC plus IL-2 (patient 3). Patients 5 and 9 both entered
the study with progressive metastatic disease despite systemic
therapy with DTIC+interferon or interferon alone, and developed
minor responses under BCL-2 antisense plus DTIC therapy. The
estimated median survival exceeds at least one year in all
patients.
Conclusion
[0142] This report demonstrates the safety and feasibility of
treatment with an antisense drug combined with chemotherapy in
cancer patients. BCL-2 antisense oligomer treatment was well
tolerated, reduced the target protein within the tumor, facilitated
apoptosis, and led to objective tumor responses with prolonged
survival also in patients who entered the study after treatment
failure of other therapies (Table 1).
[0143] The primary aim of the present study was to determine the
toxicity of BCL-2 antisense oligomer combined with DTIC therapy.
Concerning the non-hematological side effects (Table 2), up to and
including BCL-2 antisense oligomer dose levels of 3.1 mg/kg/day, no
side effects other than those reported for single agent DTIC
therapy were noted in this study. With BCL-2 antisense oligomer
doses at and above 4.1 mg/kg/day in combination with DTIC,
transient grade II-III elevations of transaminase and/or bilirubin
were observed (Table 2). In this study, the liver function
abnormalities were not dose-limiting nor associated with adverse
clinical sequelae. Non-dose-limiting changes of.alpha.PTT were
noted at and above daily BCL-2 antisense oligomer doses of 5.3
mg/kg.
[0144] Lymphopenia was also the most frequent hematological side
effect observed in this study. The lymphopenia was not clinically
significant, and there were no unusual infections in patients
treated with cyclic therapy and followed over one year. In
contrast, some levels of thrombocytopenia have been observed with
multiple phosphorothioate antisense drugs, and this toxicity was
dose limiting in the study of BCL-2 antisense oligomer in patients
with NHL (Waters et al., 2000, J. Clin. Oncol.18(9):1812-23). Even
though this study combined BCL-2 antisense oligomer with
chemotherapy, leading to transient myelo-suppression after the
DTIC, and steady-state plasma levels exceeded those reported in the
NHL study, we did not observe dose-limiting thrombocytopenia. In
summary, neither overlapping nor cumulative dose-limiting
toxicities between DTIC and BCL-2 antisense oligomer were observed
in this patient population.
[0145] Our data demonstrate that the biologically relevant
steady-state plasma levels (>1 ug/ml) can be easily achieved
with BCL-2 antisense oligomer doses of about 2 mg/kg/day, and the
maximal tolerated dose has not been reached in combination with
DTIC chemotherapy.
[0146] In a recent follow-up to the treatment regimens investigated
in this trial, BCL-2 antisense oligomer administered intravenously
by infusion (7 mg/kg/day) over 5 days has been administered to an
expanded cohort (5 patients) prior to DTIC 1000 mg/m.sup.2 in each
21-day cycle, and demonstrated satisfactory tolerance.
[0147] The results therefore indicate that BCL-2 antisense oligomer
can be administered safely in combination with an anticancer drug
to treat cancer in the clinical setting. The results differ from
prior published data showing biologic activity and clinical
responses with a 14-day infusion given only by a continuous
subcutaneous infusion (Waters et al., 2000, J. Clin. Oncol.
18(9):1812-23), since the results described herein demonstrate that
multiple routes (intravenous infusion, multiple daily subcutaneous
injections) and shorter schedules of administration of 5-7 days can
also lead to biologic activity of G3139 and clinical responses. In
responding patients, the initial antitumor activity was seen
rapidly within 2-3 cycles. The majority of patients entered the
study with progressive metastatic disease after treatment failure
of DTIC-containing regimens or after other standard treatments for
metastatic melanoma. Nevertheless, antitumor responses were noted
in 6 of 14 patients (43%), and in two additional patients, a
stabilization of the disease was observed. The estimated median
survival of all patients exceeds 12 months. These initial results
compare favorably to negligible response rates and median survival
times of about 4 to 5 months observed in patients with advanced
melanoma after treatment failure of first-line systemic
therapy.
TABLE-US-00001 TABLE 1 Study Synopsis Date of BCL-2 Max. % Patient
First Melanoma Tumor ASO BCL-2 Survival No. Age/Sex Diagnosis
Metastases Stage Prior Therapy (mg/kg/d) Reduction Response
(Months) 1 49/F 8/95 LNN, Skin IV DTIC, IF, 0.6 0 PD 6.6 RT, HEP 2*
41/F 3/95 Skin IV CP, IFN-.alpha., 0.6-6.5 40 PR 20.5 RT, HEP 3*
69/M 6/94 LNN, Skin IV DTIC, IL-2, 0.6-6.5 40 PR 23.3.sup.+ GM-CSF
4 52/M 5/98 LNN, Skin IV DTIC, IFN-.alpha. 0.6-4.1 35 MR 13.7 5
63/F 1/92 Skin IV DTIC, IFN-.alpha. 3.1-4.1 20 MR 5.1 6 56/M 8/96
Lung, Skin IV DTIC, IFN-.alpha. 3.1-5.3 60 PD 2.4 7 61/F 5/97 Lung,
IV DTIC, FOT 4.1 20 PD 7.1 Liver, Skin 8* 60/F 3/95 Skin IV
IFN-.alpha., RT 5.3-6.5 60 Stable 15.3.sup.+ 9* 75/F 6/98 LNN, Skin
IV IFN-.alpha. 5.3-6.5 60 MR 15.3.sup.+ 10 44/F 4/86 LNN, Skin IV
IFN-.alpha. 6.5 N.A. PD 14.4.sup.+ 11 63/M 4/97 Lung, Skin IV
IFN-.alpha., CP, 6.5 0 PD 12.5.sup.+ CIS 12* 90/F 7/94 LNN, Skin IV
None 6.5 70 CR 12.5.sup.+ 13* 67/M 6/96 Lung, Skin IV None 6.5 0 PD
1.1 14 76/M 4/99 Lung, Skin IV IFN-.alpha. 6.5 40 Stable 7.8 CP =
Carboplatin; CIS = Cisplatin; FOT = Fotemustine; HEP = Hyperthermic
extremity perfusion; IFN-.alpha. = interferon-.alpha.; NA = not
applicable; RT = radiation therapy. A total number of 47 cycles of
BCL-2 ASO plus DTIC have been administered. *Patients who also
received BCL-2 ASO subcutaneously with doses of 5.3 and 6.5
mg/kg/day, administered after initial intravenous treatment cycles.
.sup.+Observation period continues.
TABLE-US-00002 TABLE 2 Adverse Events During Treatment Common
toxicitiy criteria grade No. Patients 0 1 2 3 4 Hematological
events Anemia 12 2 Leucopenia 7 2 3 2 Neutropenia 10 2 2
Lymphopenia 1 1 7 5 Thrombocytopenia 8 4 2 Coagulation 3 8 2 1
Non-hematological events Cardiovascular 14 Pulmonary 14 Renal 14
Gastrointestinal 9 5 Liver (SGOT, Bilirubin) 1 7 2 4 Neurological
(Headache 11 3 Dermatological 9 4 1 Fever 7 1 6 Events are listed
irrespective of causal relationship to BCL-2 ASO therapy.
Example 2
A Phase I, Pharmacokinetic and Biologic Correlative Study of G3139
(Bcl-2 Antisense Oligonucleotide) and Docetaxel in Patients with
Hormone-Refractory Prostate Cancer
[0148] This example demonstrates the successful use of a bcl-2
antisense oligomer for the treatment of patients with
hormone-refractory prostate cancer, which is resistant to androgen
ablation therapy and cytotoxic chemotherapy. The bcl-2 antisense
oligomer was systemically administered at 5 to 7 mg/kg/day for five
days, in combination with a chemoagent. This study reports that two
patients, who were treated with the bcl-2 antisense oligomer and a
chemoagent, demonstrated responses to the treatment. The findings
reported in this Example demonstrate that, when a bcl-2 antisense
oligomer is administered in high doses for short periods of time,
the treatment exhibits low toxicity while demonstrating objective
clinical responses. The approach outlined in this study provides a
broadly applicable strategy for treating other types of cancer.
Materials and Methods
[0149] In this study, G3139 was administered as a continuous
intravenous infusion for five days per cycle on treatment cycle
days 1-6, followed by docetaxel administered intravenously on day
6. Courses were repeated every 21 days. Eleven patients with
hormone-refractory prostate cancer received therapy at three dose
levels ranging from G3139 at 5 mg/kg/day with 60 mg/m.sup.2
docetaxel to G3139 at 7 mg/kg/day followed by 75 mg/m.sup.2
docetaxel.
Results
[0150] Major toxicities were similar to docetaxel alone. One
heavily pretreated patient had prolonged (>5 days) uncomplicated
grade 4 neutropenia. Other toxicities include grade 1 stomatitis in
three patients, and febrile neutropenia during course 2 in one
patient. Preliminary pharmacokinetic results (HPLC) demonstrate
mean G3139 steady-state plasma concentrations of 3.09 .mu.g/mL at
the 5 mg/kg/day dose level. Preliminary flow cytometric and western
blot analysis indicated >50% downregulation of Bcl-2 protein by
day 6 in peripheral blood mononuclear cells prior to docetaxel
treatment. Prostate-specific antigen and symptomatic responses were
observed in 2 of 3 evaluable taxane-naive patients, including a
nine-fold reduction in prostate-specific antigen durable for
greater than cycles.
Conclusion
[0151] G3139 can be safely administered in combination with
docetaxel, and as these results demonstrate, the combination has
significant therapeutic effects in the treatment of cancer. The
results differ from prior published data showing biologic activity
and clinical responses with a 14-day infusion given only by a
continuous subcutaneous infusion (Waters et al., 2000, J. Clin.
Oncol. 18(9):1812-23), since the results described herein
demonstrate that shorter schedules of administration (5 days) given
by an alternative route (intravenously) can also lead to biologic
activity of G3139 and clinical responses. G3139 treatment is
biologically active within five days of administration,
demonstrated by effective downregulation of Bcl-2 protein in
peripheral blood mononuclear cells, and has encouraging preliminary
antitumor activity in hormone-refractory prostate cancer
patients.
Example 3
Phase I Trial of Genasense.TM. (G3139), a Bcl-2 Antisense, in
Refractory or Relapsed Acute Leukemia
[0152] This example demonstrates the successful use of a bcl-2
antisense oligomer for the treatment of patients with acute
leukemia. The bcl-2 antisense oligomer was systemically
administered at 4 mg/kg/day for ten days, in combination with two
chemoagents. This study reports that five of ten patients, who were
treated with the bcl-2 antisense oligomer and a chemoagent,
demonstrated responses to the treatment. Moreover, responses were
also noted in patients which were administered fludarabine and
cytosine arabinoside, at doses lower than the standard doses
normally used for treatment of leukemia or other cancers. The
findings reported in this Example demonstrate that objective
clinical responses can be obtained when a bcl-2 antisense oligomer
is administered for a short period of time.
Materials and Methods
[0153] G3139 (4 mg/kg/day) was given to patients (ten patients in
total) on days 1-10, whereas fludarabine (starting at a reduced
dose of 15 mg/m.sup.2), cytosine arabinoside (Ara-C) (starting at a
reduced dose of 1000 mg/m.sup.2) and G-CSF (FLAG) are given on days
6-10 of the treatment cycle, and escalated in successive cohorts.
The normal FLAG combination regimen includes two-fold higher doses
of fludarabine and Ara-C than the doses used in this study.
TABLE-US-00003 TABLE 3 Dx & Status Time to REL Previous
Previous Disease status Pt. age/sex Pre-G3139 (m).sup.1 Regimens
HDAC.sup.2 Response (d).sup.3 69/F Primary REF NA.sup.4 1 No
CR.sup.5 NED.sup.8 (53) ALL 55/F Primary REF NA 3 Yes CR REL (142)
AML 57/F 2.sup.nd REL AML 12 2 Yes CR NED (111) 23/M 1.sup.st REL
AML 3 1 Yes PR.sup.6 REL (83) 61/F 1.sup.st REL AML 7 1 No PR NED
(76) 54/M Primary REF NA 1 No NR.sup.7 REF AML 61/F 1.sup.st REL
AML 6 2 No NR REF 73/F 2.sup.nd REL AML 8 2 Yes NR REF 39/M
2.sup.nd REL AML 3 2 Yes NR REF 55/F 2.sup.nd REL AML 6 3 Yes NR
REF .sup.1(m), months from CR; .sup.2high-dose Ara-C; .sup.3(d),
days from G3139 start; .sup.4NA, not applicable; .sup.5CR, complete
response; .sup.6PR, partial response; .sup.7NR, no response;
.sup.8NED, no evidence of disease; REF, refractory; REL,
relapsed.
Results
[0154] Results are shown in Table 3. Therapy-related fever, nausea,
emesis, hypocalcemia, hypophosphatemia, and fluid retention were
not dose-limiting. Hematologic toxicities were as expected. Steady
state G3139 plasma levels exceeding the relevant target level (1
.mu.g/ml) were achieved after 24 h. Three patients achieved
complete response and received a second course of therapy; two
continued with no evidence of disease at day 53 and day 111. Two
patients had no evidence of disease but persistent
neutropenia/thrombocytopenia at day 52 and day 55; one of them
continues with no evidence of disease at day 76. Three of five
responders had prior treatment with high-dose Ara-C, and therefore,
documenting a major response to another Ara-C combination program,
as described in this study, especially using lower doses than those
used in regimens of the prior treatments, was unexpected.
CONCLUSION
[0155] The results indicate that G3139 is feasible for addition to
multi-cycle induction regimens for acute leukemia, which in this
study demonstrated 50% response rate, including patients with
refractory acute leukemia and prior treatment with high-dose Ara-C.
Major responses were also observed using lower-than-normal dose
levels of fludarabine and Ara-C when combined with a bcl-2
antisense regimen.
[0156] All references cited herein are specifically incorporated by
reference as if fully set forth herein.
[0157] Having hereinabove disclosed exemplary embodiments of the
present invention, those skilled in the art will recognize that
this disclosure is only exemplary such that various alternatives,
adaptations, and modifications are within the scope of the
invention, and are contemplated by the Applicants. Accordingly, the
present invention is not limited to the specific embodiments as
illustrated above, but is defined by the following claims.
* * * * *