U.S. patent application number 10/408723 was filed with the patent office on 2004-02-05 for method for enhancing the effectiveness of therapies of hyperproliferative diseases.
Invention is credited to Chang, Yan, Sasak, Vodek.
Application Number | 20040023925 10/408723 |
Document ID | / |
Family ID | 26872020 |
Filed Date | 2004-02-05 |
United States Patent
Application |
20040023925 |
Kind Code |
A1 |
Chang, Yan ; et al. |
February 5, 2004 |
Method for enhancing the effectiveness of therapies of
hyperproliferative diseases
Abstract
The efficacy of conventional cancer therapies such as surgery,
chemotherapy and radiation is enhanced by the use of a therapeutic
material which binds to and interacts with galectins. The
therapeutic material can enhance apoptosis thereby increasing the
effectiveness of oncolytic agents. It can also inhibit angiogenesis
thereby moderating tumor growth and/or metastasis.
Inventors: |
Chang, Yan; (Ashland,
MA) ; Sasak, Vodek; (Northboro, MA) |
Correspondence
Address: |
ROPES & GRAY LLP
ONE INTERNATIONAL PLACE
BOSTON
MA
02110-2624
US
|
Family ID: |
26872020 |
Appl. No.: |
10/408723 |
Filed: |
April 7, 2003 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10408723 |
Apr 7, 2003 |
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10176235 |
Jun 20, 2002 |
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60299991 |
Jun 21, 2001 |
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Current U.S.
Class: |
514/54 |
Current CPC
Class: |
A61K 31/732 20130101;
A61P 43/00 20180101; A61P 33/10 20180101; A61K 31/00 20130101; A61K
2300/00 20130101; A61K 45/06 20130101; A61K 31/732 20130101; A61P
35/00 20180101 |
Class at
Publication: |
514/54 |
International
Class: |
A61K 031/732 |
Claims
We claim:
1. A method for enhancing the efficacy of a therapeutic treatment
for cancer and unwanted proliferation of cells in a patient, said
therapeutic treatment one which the degree of cytotoxicity is
influenced by the status of an anti-apoptotic Bcl-2 protein of the
tumor cell, said method further comprising the conjoint
administration to said patient of a therapeutically effective
amount of a partially depolymerized pectin having a galacturan
backbone which may be disrupted by rhamnose and low branched
neutral sugars dependent from said backbone.
2. A method for enhancing the pro-apoptotic effect of a
chemotherapeutic agent that interferes with DNA replication
fidelity or cell-cycle progression of cells undergoing unwanted
proliferation in a patient, comprising the conjoint administration
to said patient of a therapeutically effective amount of a
partially depolymerized pectin having a galacturan backbone which
may be disrupted by rhamnose and low branched neutral sugars
dependent from said backbone.
3. A method for treating chemoresistant tumors in a patient,
wherein said tumors have elevated levels of anti-apoptotic Bcl-2
proteins (relative to wild-type), comprising the conjoint
administration to said patient of (i) a therapeutically effective
amount of a partially depolymerized pectin having a galacturan
backbone which may be disrupted by rhamnose and low branched
neutral sugars dependent from said backbone, and (ii) a
chemotherapeutic agent that induces apoptosis in a manner
influenced by the anti-apoptotic Bcl-2 protein status of the tumor
cell.
4. A method for enhancing the cytotoxicity of a chemotherapeutic
for killing cells undergoing unwanted proliferation in a patient,
comprising the conjoint administration to said patient of a
therapeutically effective amount of a partially depolymerized
pectin having a galacturan backbone which may be disrupted by
rhamnose and low branched neutral sugars dependent from said
backbone.
5. A method for rendering a cytostatic agent cytotoxic for cells
undergoing unwanted proliferation, wherein said agent's
cytotoxicity is influenced by the status of an anti-apoptotic Bcl-2
protein, comprising the conjoint administration of a
therapeutically effective amount of a partially depolymerized
pectin having a galacturan backbone which may be disrupted by
rhamnose and low branched neutral sugars dependent from said
backbone with said cytostatic agent.
6. The method of claim 2, 3 or 4, wherein the chemotherapeutic
agent induces mitochondrial dysfunction and/or caspase
activation.
7. The method of claim 2, 3 or 4, wherein the chemotherapeutic
agent induces cell cycle arrest at G2/M in the absence of said
partially depolymerized pectin.
8. The method of claim 2, 3 or 4, wherein said chemotherapeutic is
an inhibitor of chromatin function.
9. The method of claim 8, wherein said chemotherapeutic is a
topoisomerase inhibitor.
10. The method of claim 9, wherein said topoisomerase inhibitor is
selected from the group consisting of adriamycin, amsacrine,
camptothecin, daunorubicin, dactinomycin, doxorubicin, eniposide,
epirubicin, etoposide, idarubicin and mitoxantrone.
11. The method of claim 8, wherein said inhibitor is a microtubule
targeting drug.
12. The method of claim 11, wherein said microtubule targeting drug
is a taxane.
13. The method of claim 11, wherein said microtubule targeting drug
is selected from the group consisting of paclitaxel, docetaxel,
vincristin, vinblastin, nocodazole, epothilones and navelbine.
14. The method of claim 2, 3 or 4, wherein said chemotherapeutic is
a DNA damaging agent.
15. The method of claim 14, wherein said DNA damaging agent is
selected from the group consisting of actinomycin, amsacrine,
anthracyclines, bleomycin, busulfan, camptothecin, carboplatin,
chlorambucil, cisplatin, cyclophosphamide, cytoxan, dactinomycin,
daunorubicin, docetaxel, doxorubicin, epirubicin,
hexamethylmelamineoxaliplatin, iphosphamide, melphalan,
merchlorehtamine, mitomycin, mitoxantrone, nitrosourea, paclitaxel,
plicamycin, procarbazine, teniposide, triethylenethiophosphor-
amide and etoposide (VP16).
16. The method of claim 2, 3 or 4, wherein said chemotherapeutic is
an antimetabolite.
17. The method of claim 16, wherein said antimetabolite is selected
from the group consisting of folate antagonists, pyrimidine
analogs, purine analogs, and sugar-modified analogs.
18. The method of claim 2, 3 or 4, wherein said chemotherapeutic is
a DNA synthesis inhibitor.
19. The method of claim 18, wherein said DNA synthesis inhibitor is
a thymidilate synthase inhibitors, such as 5-fluorouracil.
20. The method of claim 18, wherein said DNA synthesis inhibitor is
a dihydrofolate reductase inhibitor, such as methoxtrexate.
21. The method of claim 18, wherein said DNA synthesis inhibitor is
a DNA polymerase inhibitor, such as fludarabine.
22. The method of claim 2, 3 or 4, wherein said chemotherapeutic is
a DNA binding agent.
23. The method of claim 22, wherein said DNA binding agent is an
intercalating agent.
24. The method of claim 2, 3 or 4, wherein said chemotherapeutic is
a DNA repair inhibitor.
25. The method of any of claims 2-5, wherein said chemotherapeutic
is a corticosteroid.
26. The method of claim 25, wherein said corticosteroid is selected
from the group consisting of cortisone, dexamethasone,
hydrocortisone, methylprednisolone, prednisone, and
prenisolone.
27. The method of any of claims 2-5, wherein said chemotherapeutic
is a combinatorial therapy selected from the group consisting of
ABV, ABVD, AC (Breast), AC (Sarcoma), AC (Neuroblastoma), ACE, ACe,
AD, AP, ARAC-DNR, B-CAVe, BCVPP, BEACOPP, BEP, BIP, BOMP, CA, CABO,
CAF, CAL-G, CAMP, CAP, CaT, CAV, CAVE ADD, CA-VP16, CC, CDDP/VP-16,
CEF, CEPP(B), CEV, CF, CHAP, ChlVPP, CHOP, CHOP-BLEO, CISCA,
CLD-BOMP, CMF, CMFP, CMFVP, CMV, CNF, CNOP, COB, CODE, COMLA, COMP,
Cooper Regimen, COP, COPE, COPP, CP--Chronic Lymphocytic Leukemia,
CP--Ovarian Cancer, CT, CVD, CVI, CVP, CVPP, CYVADIC, DA, DAT, DAV,
DCT, DHAP, DI, DTIC/Tamoxifen, DVP, EAP, EC, EFP, ELF, EMA 86, EP,
EVA, FAC, FAM, FAMTX, FAP, F-CL, FEC, FED, FL, FZ, HDMTX, Hexa-CAF,
ICE-T, IDMTX/6-MP, IE, IfoVP, IPA, M-2, MAC-III, MACC, MACOP-B,
MAID, m-BACOD, MBC, MC, MF, MICE, MINE, mini-BEAM, MOBP, MOP, MOPP,
MOPP/ABV, MP--multiple myeloma, MP--prostate cancer, MTX/6-MO,
MTX/6-MP/VP, MTX-CDDPAdr, MV--breast cancer, MV--acute myelocytic
leukemia, M-VAC Methotrexate, MVP Mitomycin, MVPP, NFL, NOVP, OPA,
OPPA, PAC, PAC-I, PA-CI, PC, PCV, PE, PFL, POC, ProMACE,
ProMACE/cytaBOM, PRoMACE/MOPP, Pt/VM, PVA, PVB, PVDA, SMF, TAD,
TCF, TIP, TTT, Topo/CTX, VAB-6, VAC, VACAdr, VAD, VATH, VBAP,
VBCMP, VC, VCAP, VD, VelP, VIP, VM, VMCP, VP, V-TAD, 5+2, 7+3, "8
in 1".
28. The method of claim 1, wherein said therapeutic treatment
includes ionizing radiation.
29. The method of claim 5, wherein cytostatic agent is selected
from the group consisting of tamoxifen,
4-(3-chloro-4-fluorophenylamino)-7-methoxy-
-6-(3-(4-morpholinyl)propoxy)quinazoline,
4-(3-ethynylphenylamino)-6,7-bis- (2-metho-xyethoxy)quinazoline,
hormone, steroids, steroid synthetic analogs, 17a-Ethinylestradiol,
Diethylstilbestrol, Testosterone, Prednisone, Fluoxymesterone,
Dromostanolone propionate, Testolactone, Megestrolacetate,
Methylprednisolone, Methyl-testosterone, Prednisolone,
Triamcinolone, chlorotrianisene, Hydroxyprogesterone,
Aminoglutethimide, Estramustine, Medroxyprogesteroneacetate,
Leuprolide, Flutamide, Toremifene, Zoladex, antiangiogenics, matrix
metalloproteinase inhibitors, VEGF inhibitors, ZD6474, SU6668,
SU11248, anti-Her2 antibodies (ZD1839 and OS1774), EGFR inhibitors,
EKB-569, Imclone antibody C225, src inhibitors, bicalutamide,
epidermal growth factor inhibitors, Her-2 inhibitors, MEK-1 kinase
inhibitors, MAPK kinase inhibitors, P13 inhibitors, PDGF
inhibitors, combretastatins, MET kinase inhibitors, MAP kinase
inhibitors, inhibitors of non-receptor and receptor tyrosine
kinases (imatinib), inhibitors of integrin signaling, and
inhibitors of insulin-like growth factor receptors.
30. The method of any of claims 2-4, wherein the chemotherapeutic
is selected from the group consisting of aminoglutethimide,
amsacrine, anastrozole, asparaginase, bcg, bicalutamide, bleomycin,
buserelin, busulfan, campothecin, capecitabine, carboplatin,
carmustine, chlorambucil, cisplatin, cladribine, clodronate,
colchicine, cyclophosphamide, cyproterone, cytarabine, dacarbazine,
dactinomycin, daunorubicin, dienestrol, diethylstilbestrol,
docetaxel, doxorubicin, epirubicin, estradiol, estramustine,
etoposide, exemestane, filgrastim, fludarabine, fludrocortisone,
fluorouracil, fluoxymesterone, flutamide, gemcitabine, genistein,
goserelin, hydroxyurea, idarubicin, ifosfamide, imatinib,
interferon, irinotecan, ironotecan, letrozole, leucovorin,
leuprolide, levamisole, lomustine, mechlorethamine,
medroxyprogesterone, megestrol, melphalan, mercaptopurine, mesna,
methotrexate, mitomycin, mitotane, mitoxantrone, nilutamide,
nocodazole, octreotide, oxaliplatin, paclitaxel, pamidronate,
pentostatin, plicamycin, porfimer, procarbazine, raltitrexed,
rituximab, streptozocin, suramin, tamoxifen, temozolomide,
teniposide, testosterone, thioguanine, thiotepa, titanocene
dichloride, topotecan, trastuzumab, tretinoin, vinblastine,
vincristine, vindesine, and vinorelbine
31. The method of claim 1, used to inhibit growth of a tumor cell
selected from the group consisting of a pancreatic tumor cell, a
lung tumor cell, a prostate tumor cell, a breast tumor cell, a
colon tumor cell, a liver tumor cell, a brain tumor cell, a kidney
tumor cell, a skin tumor cell and an ovarian tumor cell.
32. The method of claim 1, wherein said tumor cell is selected from
the group consisting of a squamous cell carcinoma, a non-squamous
cell carcinoma, a glioblastoma, a sarcoma, an adenocarcinoma, a
myeloma, a melanoma, a papilloma, a neuroblastoma and a leukemia
cell.
33. The method of claim 1, used in the treatment of a proliferative
disorder selected from the group consisting of a pancreatic cancer,
renal cell cancer, Kaposi's sarcoma, chronic leukemia, breast
cancer, sarcoma, ovarian carcinoma, rectal cancer, throat cancer,
melanoma, colon cancer, bladder cancer, lymphoma, mastocytoma, lung
cancer, mammary adenocarcinoma, pharyngeal squamous cell carcinoma,
gastrointestinal cancer, stomach cancer, myeloma, or prostate
cancer.
34. The method of claim 1, wherein said partially depolymerized
pectin is a substantially demethoxylated polygalacturonic acid
which is interrupted with rhamnose residues.
35. The method of claim 1, wherein said partially depolymerized
pectin comprises a pH modified pectin, an enzymatically modified
pectin, and/or a thermally modified pectin.
36. The method of claim 35, wherein said modified pectin comprises
a modified citrus pectin.
37. The method of claim 1, wherein said partially depolymerized
pectin is administered simultaneously with said therapeutic
treatment, chemotherapeutic or cytostatic agent.
38. The method of claim 1, wherein said partially depolymerized
pectin is administered before treatment with said therapeutic
treatment, chemotherapeutic or cytostatic agent.
39. The method of claim 1, wherein said partially depolymerized
pectin is administered after treatment with said therapeutic
treatment, chemotherapeutic or cytostatic agent.
40. The method of claim 1, wherein said partially depolymerized
pectin is administered by intravenous infusion.
41. The method of claim 1, wherein said partially depolymerized
pectin is administered orally.
42. The method of claim 1, wherein said partially depolymerized
pectin is administered by intramuscular or intraperitoneal
injection or infusion.
43. The method of claim 1, wherein said partially depolymerized
pectin is administered by inhalation.
44. The method of claim 1, wherein said partially depolymerized
pectin is administered by topically.
45. The method of claim 1, wherein said partially depolymerized
pectin is administered by subcutaneous injection.
46. The method of claim 1, wherein the effective dose (ED50) for
said therapeutic treatment, chemotherapeutic or cytostatic agent
used in combination with said partially depolymerized pectin is at
least 5 fold less than the ED50 for said therapeutic treatment,
chemotherapeutic or cytostatic agent alone.
47. The method of claim 1, wherein the therapeutic index (TI) for
said therapeutic treatment, chemotherapeutic or cytostatic agent
used in combination with said partially depolymerized pectin is at
least 5 fold greater than the TI for said therapeutic treatment,
chemotherapeutic or cytostatic agent alone.
48. A kit comprising (i) a chemotherapeutic agent that interferes
with DNA replication fidelity or cell-cycle progression of cells
undergoing unwanted proliferation, (ii) a therapeutically effective
amount of a partially depolymerized pectin having a galacturan
backbone which may be disrupted by rhamnose and low branched
neutral sugars dependent from said backbone; and (iii) instructions
and/or a label for conjoint administration of the chemotherapeutic
agent and the partially depolymerized pectin.
49. A packaged pharmaceutical comprising (i) a therapeutically
effective amount of a partially depolymerized pectin having a
galacturan backbone which may be disrupted by rhamnose and low
branched neutral sugars dependent from said backbone; and (ii)
instructions and/or a label for conjoint administration of the
partially depolymerized pectin with a chemotherapeutic agent.
50. A method for treating a tumor comprising: (i) obtaining a
sample of tumor cells from a patient; (ii) ascertaining the
anti-apoptotic Bcl-2 protein status of the tumor cell sample; and
(iii) for patients having a wild-type or elevated level or
anti-apoptotic Bcl-2 proteins, treating the patient with a regimen
including administering a therapeutically effective amount of a
partially depolymerized pectin having a galacturan backbone which
may be disrupted by rhamnose and low branched neutral sugars
dependent from said backbone.
51. The method of claim 50, wherein said treatment regimen further
includes treating the patient with a chemotherapeutic agent that
interferes with DNA replication fidelity or cell-cycle progression
of cells undergoing unwanted proliferation.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] The present application is a continuation-in-part of U.S.
Ser. No. 10/176,235 filed Jun. 20, 2002, which claims the benefit
of U.S. Provisional Application No. 60/299,991, filed Jun. 21,
2001, and entitled Method for Enhancing the Effectiveness of Cancer
Therapies; the specifications of each of which are hereby
incorporated by reference in their entirety.
BACKGROUND OF THE INVENTION
[0002] Galectins comprise a family of proteins which are expressed
by plant and animal cells and which bind .beta.-galactoside sugars.
These proteins can be found on cell surfaces, in cytoplasm, in the
nucleus, and in extracellular fluids. The two most studied
galectins, galectin-1 and galectin-3, have a molecular weight in
the general range of 13-16 kDa and 29-35 kD, respectively; they
have an affinity for .beta.-galactoside containing materials, and
have been found to play a number of important roles in biological
processes including cell migration, cell-cell adhesion,
angiogenesis, cell fusion and other cell-cell interactions, as well
as immune-based reactions and apoptosis. As such, the role of
galectins is very strongly tied to cancer and other proliferative
diseases. While there are a large number of galectins which
manifest the foregoing activities, galectin-3 and galectin-1 have
been strongly implicated in connection with cellular processes
involving cancers.
[0003] Galectin-3 is a carbohydrate binding protein having a
molecular weight of approximately 30,000. It is composed of two
distinct structural motifs, an amino-terminal portion containing
Gly-X-Y tandem repeats which are characteristic of collagens, and a
carboxyl-terminal portion containing a carbohydrate binding site.
Galectin-3 is found in almost all tumors, and has a binding
affinity for .beta.-galactoside-containing glyco-conjugates.
Galectin-3 is believed to play a role in mediating cell-cell
interactions and thereby fostering metastasis. It has been found
that cells which have high expressions of galectin-3 are more prone
to metastasis and are more resistant to apoptosis induced by
chemotherapy or radiation. It has also been reported in the
literature that galectin-3 plays a role in promoting
angiogenesis.
[0004] Galectin-1 is a highly conserved homodimer of 14-15 kD and
is one of the most abundant of the galectins. It binds to laminin
which has been found to exert strong regulatory effects on cellular
interactions such as adhesion, proliferation, migration and
differentiation. In this regard, galectin-1 has been found to
strongly influence these processes in various cells. It is believed
to be implicated in the secretion of a number of cellular growth
factors and interleukins. Galectin-1 has been found to be expressed
at very high levels in many cancer cells and is strongly implicated
in metastasis.
[0005] It has been shown that galectin-3 shares the "death
suppression motif" of Bcl-2, a protein involved in the regulation
of apoptosis, or programmed cell death. Bcl-2 is a member of a
family of proteins regulating apoptosis. Some members of the family
promote apoptosis, whereas others, including Bcl-2 and Bcl-xL,
counterbalance by preventing it. The latter group is called herein
"anti-apoptotic Bcl-2 protein." In chemoresistant cells, changes in
the activities of Bcl family of proteins by changes in the
expression levels, phosphorylation state, or intracellular
localization, that prevent the induction of apoptosis are often
implicated as the mechanism of such resistance. Inhibition of
anti-apoptotic Bcl-2 protein, in combination with the
administration of cytotoxic or cytostatic chemotherapeutic agents,
may overcome chemoresistance and restore or enhance the efficacy of
cytotoxic chemotherapeutic agents or may potentiate the effect of
cytotoxic or cytostatic chemotherapeutic agents. Overabundance of
anti-apoptotic Bcl-2 protein, which is seen in some cancerous
cells, correlates with the lack of cellular response to apoptosis
inducers. Galectin-3 has the ability to form a heterodimer with
Bcl-2, and, through this interaction, perhaps participate in the
anti-apoptotic effect of Bcl-2. There is also evidence that the
signal transduction pathway for galectin-3 may share some
commonality with the Bcl-2 pathway.
[0006] The Bcl-2 pathway is a target of many cancer treatment
regimens. Neoplasts that develop or possess resistance to
antineoplastic agents often have elevated levels of anti-apoptotic
Bcl-2 proteins and are resistant to apoptosis induction by these
agents. In such instances, combination of antineoplastic agents
with therapeutic agents that abolish the anti-apoptotic effect
mediated by the anti-apoptotic Bcl-2 proteins is an effective
treatment for those patients that fail to respond to the
antineoplastic agents alone.
[0007] Conventional treatment for cancers and other diseases
involving unwanted cellular proliferation involves the use of
chemotherapeutic agents, radiation, and surgery, either alone or in
combination. The medical arts have developed a number of treatments
based upon the foregoing therapies.
BRIEF SUMMARY OF THE INVENTION
[0008] Disclosed herein is a method for enhancing the efficacy of a
therapeutic treatment for cancer and other hyperproliferative
disorders involving unwanted cellular proliferation, such as
psoriasis or rheumatoid arthritis, in a patient by conjointly
administering conventional therapy and specific materials disclosed
herein. The treatment being enhanced may comprise chemotherapy,
radiation therapy, surgery and combinations thereof. The specific
material to be conjointly administered is a therapeutically
effective amount of a compound which binds to a galectin. This
compound may be administered prior to, after, or concomitant with
the other treatment.
[0009] A preferred class of therapeutic treatment for cancer in a
patient is chemotherapy. Chemotherapy may be carried out using
various classes of therapeutic agents, including but not limited
to: anti-proliferative agents, anti-angiogenic agents, antimitotic
agents, antimicrotubule agents, antimetabolites, anti-migratory
agents, differentiation modulators, growth factor inhibitors, cell
cycle inhibitors, hormone analogs, apoptosis inducers,
poly(ADP-ribose) polymerase inhibitors, DNA topoisomerase
inhibitors, retinoic acid receptor alpha/beta selective agonists,
and antibiotics.
[0010] A class of reagent of such chemotherapy is chemotherapeutic
agents that interfere with DNA replication fidelity or cell-cycle
progression of neoplastic cells. Alternatively, merely to
illustrate, the chemotherapeutic can be an inhibitor of chromatin
function, a topoisomerase inhibitor, a microtubule inhibiting drug,
a DNA damaging agent, an antimetabolite (such as folate
antagonists, pyrimidine analogs, purine analogs, and sugar-modified
analogs), a DNA synthesis inhibitor, a DNA interactive agent (such
as an intercalating agent), and/or a DNA repair inhibitor. In
addition to conventional chemotherapeutics, the agent of the
subject method can also be antisense RNA, RNAi or other
polynucleotides to inhibit the expression of the cellular
components that contribute to unwanted cellular proliferation that
are targets of conventional chemotherapy.
[0011] In other embodiments, the subject method combines an agent
that binds to a galectin and reduces its biological activities
related to cell proliferation, angiogenesis, and anti-apoptosis
("galectin inhibitor") with a corticosteroid, such as cortisone,
dexamethasone, hydrocortisone, methylprednisolone, prednisone, and
prenisolone.
[0012] Another preferred class of therapeutic treatment is
radiation therapy, and more specifically radioimmunotherapy or
ionizing radiation therapy.
[0013] Another aspect of the invention provides a method for
reducing the rate of growth of cells undergoing unwanted
proliferation which express anti-apoptotic Bcl-2 proteins
comprising, (i) obtaining a sample of unwanted proliferating cells
from a patient; (ii) ascertaining the status of the anti-apoptotic
Bcl-2 proteins of such cell sample; and (iii) for patients having
cells that express the anti-apoptotic Bcl-2 proteins, administering
treatment regimen including (a) a chemotherapeutic agent whose
cytotoxicity is influenced by the status of the anti-apoptotic
Bcl-2 proteins of the proliferating cell for cytotoxicity, (b) a
galectin inhibitor.
[0014] Another aspect of the invention provides a kit that includes
(i) a chemotherapeutic agent that interferes with DNA replication
fidelity or cell-cycle progression of cells undergoing unwanted
proliferation, (ii) a therapeutically effective amount of a
galectin inhibitor; and (iii) instructions and/or a label for
conjoint administration of the chemotherapeutic agent and the
galectin inhibitor.
[0015] It has been found that certain therapeutic materials can
bind to galectins thereby inactivating them toward interaction with
other carbohydrate materials and/or cells. Specifically, it has
been found that treatment of galectin-bearing cells with the
therapeutic materials of this invention can inhibit the interaction
of those cells with other cells and/or biomolecules and thereby
inhibit angiogenesis and enhance the efficacy of apoptosis-inducing
therapies such as chemotherapy or radiation. Furthermore, these
materials can inhibit cell-cell interactions and thereby enhance
the effectiveness of surgical therapies by inhibiting metastases,
which are often initiated by surgical dislodgement of cells.
[0016] As will be explained in detail herein below, the materials
employed in the present invention are generally comprised of
natural or synthetic polymers and oligomers. They are very low in
toxicity and interact synergistically with heretofore employed
cancer therapies so as to increase the effectiveness thereof.
Through the method of the present invention, the dosages of
potentially toxic therapies such as chemotherapies and radiation
may be reduced. Likewise, the effectiveness of surgical therapies
is enhanced by the method of the present invention. For example,
since the methodology of the present invention acts to inhibit the
post-surgery metastatic process, use of this invention allows a
surgeon to implement more aggressive surgical therapies without
being limited by the possibility of precipitating metastatic
events.
[0017] A preferred class of therapeutic materials of the present
invention comprises a polymeric backbone having side chains
dependent therefrom. The side chains are terminated by a galactose,
rhamnose, xylose, or arabinose unit. This material may be
synthetic, natural, or semi-synthetic. In one particular
embodiment, the therapeutic compound comprises a substantially
demethoxylated polygalacturonic acid backbone which is interrupted
with rhamnose residues. Such compounds may be prepared from
naturally occurring pectin, and are referred to as partially
depolymerized pectin or modified pectin.
[0018] The method of present invention may be administering such
materials orally, transdermally, by injection, by pulmonary
inhalation, by subcutaneous implantation, or by topical
application, depending upon the specific type of cancer or
hyperproliferative disorder being treated, and the adjunct
therapy.
BRIEF DESCRIPTION OF THE FIGURES
[0019] FIGS. 1A-1C depict the promotion of apoptosis in vitro by
formulations comprising modified pectin GCS-100 in a dose- and
time-dependent manner.
DETAILED DESCRIPTION OF THE INVENTION
[0020] I. Overview
[0021] Conventional treatment for cancers and other diseases
involving unwanted cellular proliferation involves the use of
chemotherapeutic agents, radiation, and surgery, either alone or in
combination. The medical arts have developed a number of treatments
based upon the foregoing therapies. The present invention
recognizes that the effectiveness of conventional therapies for
cancer and other unwanted cellular proliferation, such as
chemotherapy, surgery and radiation can be enhanced through the use
of a therapeutic material which interacts with galectins.
[0022] Although chemotherapy has been effective in treating various
types of malignancies, many antineoplastic compounds induce
undesirable side effects. It has been shown that when two or more
different treatments are combined, the treatments may work
synergistically and allow reduction of dosage of each of the
treatments, thereby reducing the detrimental side effects exerted
by each compound at higher dosages. In other instances,
malignancies that are refractory to a treatment may respond to a
combination therapy of two or more different treatments.
[0023] A salient feature of certain aspects of the present
invention relies on a relationship between anti-apoptotic Bcl-2
proteins and galectin-3 in regulating cell death, particularly that
galectin-3 has a positive effect on the anti-apoptotic activity of
these proteins. To further illustrate, galectin-3 expression has
been implicated in sensitivity of tumor cells to certain
chemotherapeutic agents, such as cisplatin and genistein. For
instance, it has been observed that genistein effectively induces
apoptosis in BT549 cells, a human breast epithelial cell line that
does not express detectable levels of galectin-3. When galectin-3
transfected BT549 cells are treated with genistein, cell cycle
arrest at the G(2)/M phase takes place without apoptosis induction.
However, treatment of those cells with a galectin-3 inhibitor is
sufficient to restore chemotherapeutic sensitivity.
[0024] The present invention is directed to methods and
compositions for augmenting treatment of cancers and other
hyperproliferative disorders such as psoriasis, rheumatoid
arthritis, lamellar ichthyosis, epidermolytic hyperkeratosis,
restenosis, endometriosis, or abnormal wound healing. In particular
embodiments, the invention combines the administration of a
galectin inhibitor with a chemotherapeutic agent so as to
potentiate the cytotoxicity of the chemotherapeutic agent. In
certain preferred embodiments, the conjoint therapies of the
present invention can be used to improve the efficacy of those
chemotherapeutic agents whose cytotoxicity is influenced by the
status of an anti-apoptotic Bcl-2 protein for the treated cell. For
instance, galectin inhibitors can be administered in combination
with a chemotherapeutic agent that interferes with DNA replication
fidelity or cell-cycle progression of cells undergoing unwanted
proliferation.
[0025] Another aspect of the invention relies on the observation
that galectins are involved in promoting angiogenesis. In order for
a solid tumor to grow or metastasize the tumor must be
vascularized. Galectin-3 in particular has been demonstrated to
affect chemotaxis and morphology, and to stimulate angiogenesis in
vivo. In accord with the present invention, a galectin inhibitor is
administered to a patient in combination with conventional
chemotherapy.
[0026] Depending on the nature of the cancer and the therapy, the
galectin inhibitor may be administered prior to, contemporaneously
with and/or after other therapies. When administration
contemporaneously with other drugs, the galectin inhibitor may be
formulated separately from, or co-formulated with, one or more of
the other drugs.
[0027] II. Definitions
[0028] The terms "apoptosis" or "programmed cell death," refers to
the physiological process by which unwanted or useless cells are
eliminated during development and other normal biological
processes. Apoptosis is a mode of cell death that occurs under
normal physiological conditions and the cell is an active
participant in its own demise ("cellular suicide"). It is most
often found during normal cell turnover and tissue homeostasis,
embryogenesis, induction and maintenance of immune tolerance,
development of the nervous system and endocrine-dependent tissue
atrophy. Cells undergoing apoptosis show characteristic
morphological and biochemical features. These features include
chromatin aggregation, nuclear and cytoplasmic condensation,
partition of cytoplasm and nucleus into membrane bound vesicles
(apoptotic bodies) which contain ribosomes, morphologically intact
mitochondria and nuclear material. Cytochrome C release from
mitochondria is seen as an indication of mitochondrial dysfunction
accompanying apoptosis. In vivo, these apoptotic bodies are rapidly
recognized and phagocytized by either macrophages or adjacent
epithelial cells. Due to this efficient mechanism for the removal
of apoptotic cells in vivo no inflammatory response is elicited. In
vitro, the apoptotic bodies as well as the remaining cell fragments
ultimately swell and finally lyse. This terminal phase of in vitro
cell death has been termed "secondary necrosis."
[0029] The term "anti-apoptotic Bcl-2 protein" refers to a family
of proteins related to the Bcl-2 protein and which are antagonists
of cellular apoptosis. This family includes Bcl-2, Bcl-xL, Bcl-w,
Mcl-1 and A-1. See, for example, Hockenbery et al., 1990, Nature
348:334-336; Boise et al., 1993, Cell 74:597-608; Gibson et al.,
1996, Oncogene 13:665-675; Zhou et al., 1997, Blood 89 :630-643;
and Lin et al., 1993, J. Inmunol. 151:1979-1988. This family of
proteins shares four homology regions, termed Bcl homology (BH)
domains, namely BH1, BH2, BH3, and BH4. A representative sequence
for a human Bcl-2 coding sequence and protein are provided in
GenBank Accession NM.sub.--000657 (GI 4557356). A representative
sequence for a human Bcl-xL coding sequence and protein are
provided in GenBank Accession Z23115 (GI 510900). Exemplary
anti-apoptotic Bcl-2 proteins are those which are at least 90
percent identical to the protein sequences set forth in GenBank
Accessions NM.sub.--000657 or Z23115, and/or which can be encoded
by a nucleic acid sequence that hybridizes under stringent wash
conditions of 0.2.times.SSC at 65 C. to a coding sequence set forth
in GenBank Accessions NM.sub.--000657 or Z23115.
[0030] The term "status of anti-apoptotic Bcl-2 proteins" includes
within its meaning such quantitative measures as: the level of mRNA
encoding an anti-apoptotic Bcl-2 protein; the level of the protein;
the number and location of, or the absence of, phosphorylated
residues or other posttranslational modifications; the
intracellular localization of the protein; the status of
association of anti-apoptotic Bcl-2 proteins with each other or
with other proteins; and/or any other surrogate or direct
measurement of anti-apoptotic activity due to an anti-apoptotic
Bcl-2 protein.
[0031] More specifically, the term "status of anti-apoptotic Bcl-2
protein levels" means the amount of an anti-apoptotic Bcl-2 protein
in a cell, such as may be detected by immunohistochemistry using
antibodies specific to an anti-apoptotic Bcl-2 protein.
[0032] As used herein the term "animal" refers to mammals,
preferably mammals such as humans. Likewise, a "patient" or
"subject" to be treated by the method of the invention can mean
either a human or non-human animal.
[0033] The term "antibody" as used herein, unless indicated
otherwise, is used broadly to refer to both antibody molecules and
a variety of antibody-derived molecules. Such antibody derived
molecules comprise at least one variable region (either a heavy
chain of light chain variable region), as well as individual
antibody light chains, individual antibody heavy chains, chimeric
fusions between antibody chains and other molecules, and the like.
Functional immunoglobulin fragments according to the present
invention may be Fv, scFv, disulfide-linked Fv, Fab, and
F(ab')2.
[0034] As used herein, the term "cancer" refers to any neoplastic
disorder, including such cellular disorders as, for example, renal
cell cancer, Kaposi's sarcoma, chronic leukemia, prostate cancer,
breast cancer, sarcoma, pancreatic cancer, ovarian carcinoma,
rectal cancer, throat cancer, melanoma, colon cancer, bladder
cancer, lymphoma, mastocytoma, lung cancer, mammary adenocarcinoma,
pharyngeal squamous cell carcinoma, and gastrointestinal or stomach
cancer. Preferably, the cancer which is treated in the present
invention is melanoma, myeloma, lung cancer, breast cancer,
pancreatic cancer, prostate cancer, colon cancer, or ovarian
cancer.
[0035] The "growth state" of a cell refers to the rate of
proliferation of the cell and the state of differentiation of the
cell.
[0036] As used herein, "hyperproliferative disease" or
"hyperproliferative disorder" refers to any disorder which is
caused by or is manifested by unwanted proliferation of cells in a
patient. Hyperproliferative disorders include but are not limited
to cancer, psoriasis, rheumatoid arthritis, lamellar ichthyosis,
epidermolytic hyperkeratosis, restenosis, endometriosis, and
abnormal wound healing.
[0037] As used herein, "proliferating" and "proliferation" refer to
cells undergoing mitosis.
[0038] As used herein, "unwanted proliferation" means cell division
and growth that is not part of normal cellular turnover,
metabolism, growth, or propagation of the whole organism. Unwanted
proliferation of cells is seen in tumors and other pathological
proliferation of cells, does not serve normal function, and for the
most part will continue unbridled at a growth rate exceeding that
of cells of a normal tissue in the absence of outside intervention.
A pathological state that ensues because of the unwanted
proliferation of cells is referred herein as a "hyperproliferative
disease" or "hyperproliferative disorder."
[0039] As used herein, "transformed cells" refers to cells that
have spontaneously converted to a state of unrestrained growth,
i.e., they have acquired the ability to grow through an indefinite
number of divisions in culture. Transformed cells may be
characterized by such terms as neoplastic, anaplastic and/or
hyperplastic, with respect to their loss of growth control. For
purposes of this invention, the terms "transformed phenotype of
malignant mammalian cells" and "transformed phenotype" are intended
to encompass, but not be limited to, any of the following
phenotypic traits associated with cellular transformation of
mammalian cells: immortalization, morphological or growth
transformation, and tumorigenicity, as detected by prolonged growth
in cell culture, growth in semi-solid media, or tumorigenic growth
in immuno-incompetent or syngeneic animals.
[0040] III. Exemplary Embodiments
[0041] A. Galectin Inhibitors
[0042] While galectins are known to bind galactose and other such
simple sugars in vitro, those simple sugars are not therapeutically
effective in moderating galectin mediated cellular processes in
vivo. Preferred materials for the practice of the present invention
generally comprise molecules which contain an active galectin
binding sugar site, but which have somewhat higher molecular
weights than simple sugars.
[0043] One group of materials falling within this general class
comprises a substantially demethoxylated polygalacturonic acid
backbone having rhamnose, galactose, arabinose, or other sugar
residues pendent therefrom. It is believed that in materials of
this type, the terminal galactose or arabinose units pendent from
the backbone bind to galectin proteins. The remaining bulk of the
molecule potentiates the compound's action in moderating immune
system response. Materials of this general type are described by
formulas I and II below, and it is to be understood that yet other
variants of this general compound may be prepared and utilized in
accord with the principles of the present invention.
[0044] 1. Homogalacturonan
-[.alpha.-Galpa-(1.fwdarw.4)-.alpha.-GalpA].sub.n- (I)
[0045] 1
[0046] (Where m, n, o.gtoreq.1, X can be either .alpha.-GalpA or
.alpha.-Rhap; Y can be any of the following sugars (and can
represent different sugars within a branch) such as .alpha.-Galp,
.beta.-Galp, .beta.-Apif, .beta.-Rhap, .alpha.-Rhap, .alpha.-Fucp,
.beta.-GlcpA, .alpha.-GalpA, .beta.-GalpA, .beta.-DhapA, Kdop,
.beta.-Acef, .alpha.-Galp, .alpha.-Arap, .beta.-Araf, .alpha.-Xylp,
not limited to these sugars. The abbreviated monomer names each
corresponds to the following: GalA: galacturonic acid, Rha:
rhamnose, Gal: galactose, Api: erythro-apiose, Fuc: fucose, GlcA:
glucuronic acid, DhaA: 3-deoxy-D-lyxo-heptulosaric acid, Kdo:
3-deoxy-D-manno-2-octulosonic acid, Ace: aceric acid
(3C-carboxy-5-deoxy-L-lyxose), Ara: arabinose. Italicized p stands
for pyranose and italicized f stands for furanose.)
[0047] Pectin is a complex carbohydrate having a highly branched
structure comprised of a polygalacturonic backbone with numerous
branching side chains dependent therefrom. The branching creates
regions which are characterized as being "smooth" and "hairy." It
has been found that pectin can be modified by various chemical,
enzymatic or physical treatments to break the molecule into smaller
portions having a more linearized, substantially demethoxylated,
polygalacturonic backbone with pendant side chains of rhamnose
residues having decreased branching. The resulting partially
depolymerized pectin is known in the art as modified pectin, and
its efficacy in treating cancer has been established; although
galectin blocker materials of this type have not been used in
conjunction with surgery, chemotherapy or radiation.
[0048] U.S. Pat. No. 5,895,784, the disclosure of which is
incorporated herein by reference, describes modified pectin
materials, techniques for their preparation, and use of the
material as a treatment for various cancers. The material of the
'784 patent is described as being prepared by a pH based
modification procedure in which the pectin is put into solution and
exposed to a series of programmed changes in pH which results in
the breakdown of the molecule to yield therapeutically effective
modified pectin. The material in the '784 patent is most preferably
prepared from citrus pectin; although, it is to be understood that
modified pectins may be prepared from pectin starting material
obtained from other sources, such as apple pectin and the like.
Also, modification processes may be accomplished by enzymatic
treatment of the pectin, or by physical processes such as heating.
Further disclosure of modified pectins and techniques for their
preparation and use are also disclosed in U.S. Pat. No. 5,834,442
and U.S. patent application Ser. No. 08/024,487, the disclosures of
which are incorporated herein by reference. Modified pectins of
this type generally have molecular weights less than 100
kilodalton. A group of such materials has an average molecular
weight of less than 3 kilodalton. Another group has an average
molecular weight in the range of 1-15 kilodalton, with a specific
group of materials having a molecular weight of about 10
kilodalton.
[0049] In certain embodiments, the modified pectin preparation is a
substantially ethanol-free product suitable for parenteral
administration. By substantially free of ethanol, it is meant that
the compositions of the invention contain less than 5% ethanol by
weight. In preferred embodiments the compositions contain less than
2%, and more preferably less than 0.5% ethanol by weight. In
certain embodiments, the compositions further comprise one or more
pharmaceutically acceptable excipients. Such compositions include
aqueous solutions of the modified pectin of the invention. In
certain embodiments of such aqueous solutions, the pectin
modification occurs at a concentration of at least 7 mg/mL, and
preferably at least 10 or even 15 or more mg/ml. Any of such
compositions are also substantially free of organic solvents other
than ethanol.
[0050] The apoptosis-promoting activity of a modified pectin
material is illustrated in Example 1, below.
[0051] As disclosed in the prior art, such modified pectin
materials have therapeutic efficacy against a variety of cancers
and other hyperproliferative disorders. These materials interact
with galectins, including galectin-1 and galectin-3, and in that
regard also have efficacy against immune-based diseases. In accord
with the present invention, the effect of conventional therapies
for cancer and other hyperproliferative disorders is enhanced by
use of pectin materials and other materials which interact with
galectins. These materials may be administered orally; or by
intravenous injection; or by injection directly into an affected
tissue, as for example by injection into a tumor site. In some
instances the materials may be applied topically at the time
surgery is carried out. Also, other techniques such as transdermal
delivery systems, inhalation, intramuscular injection, or
subcutaneous implantation may be employed.
[0052] B. Chemotherapeutic Agents
[0053] Pharmaceutical agents that may be used for the subject
combination chemotherapy include, merely to illustrate:
aminoglutethimide, amsacrine, anastrozole, asparaginase, bcg,
bicalutamide, bleomycin, buserelin, busulfan, campothecin,
capecitabine, carboplatin, carmustine, chlorambucil, cisplatin,
cladribine, clodronate, colchicine, cyclophosphamide, cyproterone,
cytarabine, dacarbazine, dactinomycin, daunorubicin, dienestrol,
diethylstilbestrol, docetaxel, doxorubicin, epirubicin, estradiol,
estramustine, etoposide, exemestane, filgrastim, fludarabine,
fludrocortisone, fluorouracil, fluoxymesterone, flutamide,
gemcitabine, genistein, goserelin, hydroxyurea, idarubicin,
ifosfamide, imatinib, interferon, irinotecan, ironotecan,
letrozole, leucovorin, leuprolide, levamisole, lomustine,
mechlorethamine, medroxyprogesterone, megestrol, melphalan,
mercaptopurine, mesna, methotrexate, mitomycin, mitotane,
mitoxantrone, nilutamide, nocodazole, octreotide, oxaliplatin,
paclitaxel, pamidronate, pentostatin, plicamycin, porfimer,
procarbazine, raltitrexed, rituximab, streptozocin, suramin,
tamoxifen, temozolomide, teniposide, testosterone, thioguanine,
thiotepa, titanocene dichloride, topotecan, trastuzumab, tretinoin,
vinblastine, vincristine, vindesine, and vinorelbine.
[0054] These chemotherapeutic agents may be categorized by their
mechanism of action into, for example, following groups:
anti-metabolites/anti-canc- er agents, such as pyrimidine analogs
(5-fluorouracil, floxuridine, capecitabine, gemcitabine and
cytarabine) and purine analogs, folate antagonists and related
inhibitors (mercaptopurine, thioguanine, pentostatin and
2-chlorodeoxyadenosine (cladribine)); antiproliferative/antimitotic
agents including natural products such as vinca alkaloids
(vinblastine, vincristine, and vinorelbine), microtubule disruptors
such as taxane (paclitaxel, docetaxel), vincristin, vinblastin,
nocodazole, epothilones and navelbine, epidipodophyllotoxins
(teniposide), DNA damaging agents (actinomycin, amsacrine,
anthracyclines, bleomycin, busulfan, camptothecin, carboplatin,
chlorambucil, cisplatin, cyclophosphamide, cytoxan, dactinomycin,
daunorubicin, doxorubicin, epirubicin,
hexamethylmelamineoxaliplatin, iphosphamide, melphalan,
merchlorehtamine, mitomycin, mitoxantrone, nitrosourea, plicamycin,
procarbazine, teniposide, triethylenethiophosphoramide and
etoposide (VP16)); antibiotics such as dactinomycin (actinomycin
D), daunorubicin, doxorubicin (adriamycin), idarubicin,
anthracyclines, mitoxantrone, bleomycins, plicamycin (mithramycin)
and mitomycin; enzymes (L-asparaginase which systemically
metabolizes L-asparagine and deprives cells which do not have the
capacity to synthesize their own asparagine); antiplatelet agents;
antiproliferative/antimitotic alkylating agents such as nitrogen
mustards (mechlorethamine, cyclophosphamide and analogs, melphalan,
chlorambucil), ethylenimines and methylmelamines
(hexamethylmelamine and thiotepa), alkyl sulfonates-busulfan,
nitrosoureas (carmustine (BCNU) and analogs, streptozocin),
trazenes-dacarbazinine (DTIC); antiproliferative/antimitot- ic
antimetabolites such as folic acid analogs (methotrexate); platinum
coordination complexes (cisplatin, carboplatin), procarbazine,
hydroxyurea, mitotane, aminoglutethimide; hormones, hormone analogs
(estrogen, tamoxifen, goserelin, bicalutamide, nilutamide) and
aromatase inhibitors (letrozole, anastrozole); anticoagulants
(heparin, synthetic heparin salts and other inhibitors of
thrombin); fibrinolytic agents (such as tissue plasminogen
activator, streptokinase and urokinase), aspirin, COX-2 inhibitors,
dipyridamole, ticlopidine, clopidogrel, abciximab; antimigratory
agents; antisecretory agents (breveldin); immunosuppressives
(cyclosporine, tacrolimus (FK-506), sirolimus (rapamycin),
azathioprine, mycophenolate mofetil); anti-angiogenic compounds
(TNP-470, genistein) and growth factor inhibitors (vascular
endothelial growth factor (VEGF) inhibitors, fibroblast growth
factor (FGF) inhibitors); angiotensin receptor blocker; nitric
oxide donors; anti-sense oligonucleotides; antibodies
(trastuzumab); cell cycle inhibitors and differentiation inducers
(tretinoin); mTOR inhibitors, topoisomerase inhibitors (doxorubicin
(adriamycin), amsacrine, camptothecin, daunorubicin, dactinomycin,
eniposide, epirubicin, etoposide, idarubicin and mitoxantrone,
topotecan, irinotecan), corticosteroids (cortisone, dexamethasone,
hydrocortisone, methylpednisolone, prednisone, and prenisolone);
growth factor signal transduction kinase inhibitors; mitochondrial
dysfunction inducers and caspase activators; chromatin
disruptors.
1TABLE 1 Exemplary conventional combination cancer chemotherapy
Name Therapeutic agents ABV Doxorubicin, Bleomycin, Vinblastine
ABVD Doxorubicin, Bleomycin, Vinblastine, Dacarbazine AC (Breast)
Doxorubicin, Cyclophosphamide AC (Sarcoma) Doxorubicin, Cisplatin
AC Cyclophosphamide, Doxorubicin (Neuro- blastoma) ACE
Cyclophosphamide, Doxorubicin, Etoposide ACe Cyclophosphamide,
Doxorubicin AD Doxorubicin, Dacarbazine AP Doxorubicin, Cisplatin
ARAC-DNR Cytarabine, Daunorubicin B-CAVe Bleomycin, Lomustine,
Doxorubicin, Vinblastine BCVPP Carmustine, Cyclophosphamide,
Vinblastine, Procarbazine, Prednisone BEACOPP Bleomycin, Etoposide,
Doxorubicin, Cyclophosphamide, Vincristine, Procarbazine,
Prednisone, Filgrastim BEP Bleomycin, Etoposide, Cisplatin BIP
Bleomycin, Cisplatin, Ifosfamide, Mesna BOMP Bleomycin,
Vincristine, Cisplatin, Mitomycin CA Cytarabine, Asparaginase CABO
Cisplatin, Methotrexate, Bleomycin, Vincristine CAF
Cyclophosphamide, Doxorubicin, Fluorouracil CAL-G Cyclophosphamide,
Daunorubicin, Vincristine, Prednisone, Asparaginase CAMP
Cyclophosphamide, Doxorubicin, Methotrexate, Procarbazine CAP
Cyclophosphamide, Doxorubicin, Cisplatin CaT Carboplatin,
Paclitaxel CAV Cyclophosphamide, Doxorubicin, Vincristine CAVE ADD
CAV and Etoposide CA-VP16 Cyclophosphamide, Doxorubicin, Etoposide
CC Cyclophosphamide, Carboplatin CDDP/VP-16 Cisplatin, Etoposide
CEF Cyclophosphamide, Epirubicin, Fluorouracil CEPP(B)
Cyclophosphamide, Etoposide, Prednisone, with or without/Bleomycin
CEV Cyclophosphamide, Etoposide, Vincristine CF Cisplatin,
Fluorouracil or Carboplatin Fluorouracil CHAP Cyclophosphamide or
Cyclophosphamide, Altretamine, Doxorubicin, Cisplatin ChlVPP
Chlorambucil, Vinblastine, Procarbazine, Prednisone CHOP
Cyclophosphamide, Doxorubicin, Vincristine, Prednisone CHOP-BLEO
Add Bleomycin to CHOP CISCA Cyclophosphamide, Doxorubicin,
Cisplatin CLD-BOMP Bleomycin, Cisplatin, Vincristine, Mitomycin CMF
Methotrexate, Fluorouracil Cyclophosphamide CMFP Cyclophosphamide,
Methotrexate, Fluorouracil, Prednisone CMFVP Cyclophosphamide,
Methotrexate, Fluorouracil, Vincristine, Prednisone CMV Cisplatin,
Methotrexate, Vinblastine CNF Cyclophosphamide, Mitoxantrone,
Fluorouracil CNOP Cyclophosphamide, Mitoxantrone, Vincristine,
Prednisone COB Cisplatin, Vincristine, Bleomycin CODE Cisplatin,
Vincristine, Doxorubicin, Etoposide COMLA Cyclophosphamide,
Vincristine, Methotrexate, Leucovorin, Cytarabine COMP
Cyclophosphamide, Vincristine, Methotrexate, Prednisone Cooper
Cyclophosphamide, Methotrexate, Fluorouracil, Regimen Vincristine,
Prednisone COP Cyclophosphamide, Vincristine, Prednisone COPE
Cyclophosphamide, Vincristine, Cisplatin, Etoposide COPP
Cyclophosphamide, Vincristine, Procarbazine, Prednisone CP
Chlorambucil, Prednisone (Chronic Lymphocytic Leukemia) CP (Ovarian
Cyclophosphamide, Cisplatin Cancer) CT Cisplatin, Paclitaxel CVD
Cisplatin, Vinblastine, Dacarbazine CVI Carboplatin, Etoposide,
Ifosfamide, Mesna CVP Cyclophosphamide, Vincristine, Prednisome
CVPP Lomustine, Procarbazine, Prednisone CYVADIC Cyclophosphamide,
Vincristine, Doxorubicin, Dacarbazine DA Daunorubicin, Cytarabine
DAT Daunorubicin, Cytarabine, Thioguanine DAV Daunorubicin,
Cytarabine, Etoposide DCT Daunorubicin, Cytarabine, Thioguanine
DHAP Cisplatin, Cytarabine, Dexamethasone DI Doxorubicin,
Ifosfamide DTIC/ Dacarbazine, Tamoxifen Tamoxifen DVP Daunorubicin,
Vincristine, Prednisone EAP Etoposide, Doxorubicin, Cisplatin EC
Etoposide, Carboplatin EFP Etoposie, Fluorouracil, Cisplatin ELF
Etoposide, Leucovorin, Fluorouracil EMA 86 Mitoxantrone, Etoposide,
Cytarabine EP Etoposide, Cisplatin EVA Etoposide, Vinblastine FAC
Fluorouracil, Doxorubicin, Cyclophosphamide FAM Fluorouracil,
Doxorubicin, Mitomycin FAMTX Methotrexate, Leucovorin, Doxorubicin
FAP Fluorouracil, Doxorubicin, Cisplatin F-CL Fluorouracil,
Leucovorin FEC Fluorouracil, Cyclophosphamide, Epirubicin FED
Fluorouracil, Etoposide, Cisplatin FL Flutamide, Leuprolide FZ
Flutamide, Goserelin acetate implant HDMTX Methotrexate, Leucovorin
Hexa-CAF Altretamine, Cyclophosphamide, Methotrexate, Fluorouracil
ICE-T Ifosfamide, Carboplatin, Etoposide, Paclitaxel, Mesna
IDMTX/6-MP Methotrexate, Mercaptopurine, Leucovorin IE Ifosfamide,
Etoposie, Mesna IfoVP Ifosfamide, Etoposide, Mesna IPA Ifosfamide,
Cisplatin, Doxorubicin M-2 Vincristine, Carmustine,
Cyclophosphamide, Prednisone, Melphalan MAC-III Methotrexate,
Leucovorin, Dactinomycin, Cyclophosphamide MACC Methotrexate,
Doxorubicin, Cyclophosphamide, Lomustine MACOP-B Methotrexate,
Leucovorin, Doxorubicin, Cyclophosphamide, Vincristine, Bleomycin,
Prednisone MAID Mesna, Doxorubicin, Ifosfamide, Dacarbazine m-BACOD
Bleomycin, Doxorubicin, Cyclophosphamide, Vincristine,
Dexamethasone, Methotrexate, Leucovorin MBC Methotrexate,
Bleomycin, Cisplatin MC Mitoxantrone, Cytarabine MF Methotrexate,
Fluorouracil, Leucovorin MICE Ifosfamide, Carboplatin, Etoposide,
Mesna MINE Mesna, Ifosfamide, Mitoxantrone, Etoposide mini-BEAM
Carmustine, Etoposide, Cytarabine, Melphalan MOBP Bleomycin,
Vincristine, Cisplatin, Mitomycin MOP Mechlorethamine, Vincristine,
Procarbazine MOPP Mechlorethamine, Vincristine, Procarbazine,
Prednisone MOPP/ABV Mechlorethamine, Vincristine, Procarbazine,
Prednisone, Doxorubicin, Bleomycin, Vinblastine MP (multiple
Melphalan, Prednisone myeloma) MP (prostate Mitoxantrone,
Prednisone cancer) MTX/6-MO Methotrexate, Mercaptopurine
MTX/6-MP/VP Methotrexate, Mercaptopurine, Vincristine, Prednisone
MTX- Methotrexate, Leucovorin, Cisplatin, Doxorubicin CDDPAdr MV
(breast Mitomycin, Vinblastine cancer) MV (acute Mitoxantrone,
Etoposide myelocytic leukemia) M-VAC Vinblastine, Doxorubicin,
Cisplatin Methotrexate MVP Vinblastine, Cisplatin Mitomycin MVPP
Mechlorethamine, Vinblastine, Procarbazine, Prednisone NFL
Mitoxantrone, Fluorouracil, Leucovorin NOVP Mitoxantrone,
Vinblastine, Vincristine OPA Vincristine, Prednisone, Doxorubicin
OPPA Add Procarbazine to OPA. PAC Cisplatin, Doxorubicin PAC-I
Cisplatin, Doxorubicin, Cyclophosphamide PA-CI Cisplatin,
Doxorubicin PC Paclitaxel, Carboplatin or Paclitaxel, Cisplatin PCV
Lomustine, Procarbazine, Vincristine PE Paclitaxel, Estramustine
PFL Cisplatin, Fluorouracil, Leucovorin POC Prednisone,
Vincristine, Lomustine ProMACE Prednisone, Methotrexate, Leucovonn,
Doxorubicin, Cyclophosphamide, Etoposide ProMACE/ Prednisone,
Doxorubicin, Cyclophosphamide, Etoposide, cytaBOM Cytarabine,
Bleomycin, Vincristine, Methotrexate, Leucovorin, Cotrimoxazole
PRoMACE/ Prednisone, Doxorubicin, Cyclophosphamide, Etoposide, MOPP
Mechlorethamine, Vincristine, Procarbazine, Methotrexate,
Leucovorin Pt/VM Cisplatin, Teniposide PVA Prednisone, Vincristine,
Asparaginase PVB Cisplatin, Vinblastine, Bleomycin PVDA Prednisone,
Vincristine, Daunorubicin, Asparaginase SMF Streptozocin,
Mitomycin, Fluorouracil TAD Mechlorethamine, Doxorubicin,
Vinblastine, Vincristine, Bleomycin, Etoposide, Prednisone TCF
Paclitaxel, Cisplatin, Fluorouracil TIP Paclitaxel, Ifosfamide,
Mesna, Cisplatin TTT Methotrexate, Cytarabine, Hydrocortisone
Topo/CTX Cyclophosphamide, Topotecan, Mesna VAB-6 Cyclophosphamide,
Dactinomycin, Vinblastine, Cisplatin, Bleomycin VAC Vincristine,
Dactinomycin, Cyclophosphamide VACAdr Vincristine,
Cyclophosphamide, Doxorubicin, Dactinomycin, Vincristine VAD
Vincristine, Doxorubicin, Dexamethasone VATH Vinblastine,
Doxorubicin, Thiotepa, Flouxymesterone VBAP Vincristine,
Carmustine, Doxorubicin, Prednisone VBCMP Vincristine, Carmustine,
Melphalan, Cyclophosphamide, Prednisone VC Vinorelbine, Cisplatin
VCAP Vincristine, Cyclophosphamide, Doxorubicin, Prednisone VD
Vinorelbine, Doxorubicin VelP Vinblastine, Cisplatin, Ifosfamide,
Mesna VIP Etoposide, Cisplatin, Ifosfamide, Mesna VM Mitomycin,
Vinblastine VMCP Vincristine, Melphalan, Cyclophosphamide,
Prednisone VP Etoposide, Cisplatin V-TAD Etoposide, Thioguanine,
Daunorubicin, Cytarabine 5 + 2 Cytarabine, Daunorubicin,
Mitoxantrone 7 + 3 Cytarabine with/, Daunorubicin or Idarubicin or
Mitoxantrone "8 in 1" Methylprednisolone, Vincristine, Lomustine,
Procarbazine, Hydroxyurea, Cisplatin, Cytarabine, Dacarbazine
[0055] In addition to conventional chemotherapeutics, the agent of
the subject method can also be compounds and antisense RNA, RNAi or
other polynucleotides to inhibit the expression of the cellular
components that contribute to unwanted cellular proliferation that
are targets of conventional chemotherapy. Such targets are, merely
to illustrate, growth factors, growth factor receptors, cell cycle
regulatory proteins, transcription factors, or signal transduction
kinases.
[0056] An antisense nucleic acid can be delivered, for example, as
an expression plasmid which, when transcribed in the cell, produces
RNA which is complementary to at least a unique portion of the
cellular mRNA which encodes a target gene. Alternatively, the
construct is an oligonucleotide which is generated ex vivo and
which, when introduced into the cell causes inhibition of
expression by hybridizing with the mRNA and/or genomic sequences
encoding a target gene. Such oligonucleotide are optionally
modified oligonucleotide which are resistant to endogenous
nucleases, e.g., exonucleases and/or endonucleases, and is
therefore stable in vivo. Exemplary nucleic acid molecules for use
as antisense oligonucleotides are phosphoramidate, phosphothioate
and methylphosphonate analogs of DNA (see also U.S. Pat. Nos.
5,176,996; 5,264,564; and 5,256,775). Additionally, general
approaches to constructing oligomers useful in nucleic acid therapy
have been reviewed, for example, by van der Krol et al., 1988,
Biotechniques 6:958-976; and Stein et al., 1988, Cancer Res.
48:2659-2668.
[0057] In another embodiment, the invention relates to the use of
RNA interference (RNAi) to effect knockdown of expression of a
target gene. RNAi constructs comprise double stranded RNA that can
specifically block expression of a target gene. "RNA interference"
or "RNAi" is a term initially applied to a phenomenon observed in
plants and worms where double-stranded RNA (dsRNA) blocks gene
expression in a specific and post-transcriptional manner. RNAi
provides a useful method of inhibiting gene expression in vitro or
in vivo. As used herein, the term "RNAi construct" is a generic
term including small interfering RNAs (siRNAs), hairpin RNAs, and
other RNA species which can be cleaved in vivo to form siRNAs. RNAi
constructs herein also include expression vectors (also referred to
as RNAi expression vectors) capable of giving rise to transcripts
which form dsRNAs or hairpin RNAs in cells, and/or transcripts
which can produce siRNAs in vivo.
[0058] RNAi constructs can comprise either long stretches of dsRNA
identical or substantially identical to the target nucleic acid
sequence or short stretches of dsRNA identical to substantially
identical to only a region of the target nucleic acid sequence.
[0059] Optionally, the RNAi constructs contain a nucleotide
sequence that hybridizes under physiologic conditions of the cell
to the nucleotide sequence of at least a portion of the mRNA
transcript for the gene to be inhibited (i.e., the "target" gene).
The double-stranded RNA need only be sufficiently similar to
natural RNA that it has the ability to mediate RNAi. Thus, the
invention has the advantage of being able to tolerate sequence
variations that might be expected due to genetic mutation, strain
polymorphism or evolutionary divergence. The number of tolerated
nucleotide mismatches between the target sequence and the RNAi
construct sequence is no more than 1 in 5 basepairs, or 1 in 10
basepairs, or 1 in 20 basepairs, or 1 in 50 basepairs. Mismatches
in the center of the siRNA duplex are most critical and may
essentially abolish cleavage of the target RNA. In contrast,
nucleotides at the 3' end of the siRNA strand that is complementary
to the target RNA do not significantly contribute to specificity of
the target recognition. Sequence identity may be optimized by
sequence comparison and alignment algorithms known in the art (see
Gribskov and Devereux, Sequence Analysis Primer, Stockton Press,
1991, and references cited therein) and calculating the percent
difference between the nucleotide sequences by, for example, the
Smith-Waterman algorithm as implemented in the BESTFIT software
program using default parameters (e.g., University of Wisconsin
Genetic Computing Group). Greater than 90% sequence identity, or
even 100% sequence identity, between the inhibitory RNA and the
portion of the target gene is preferred. Alternatively, the duplex
region of the RNA may be defined functionally as a nucleotide
sequence that is capable of hybridizing with a portion of the
target gene transcript (e.g., 400 mM NaCl, 40 mM PIPES pH 6.4, 1 mM
EDTA, 50.degree. C. or 70.degree. C. hybridization for 12-16 hours;
followed by washing).
[0060] The double-stranded structure may be formed by a single
self-complementary RNA strand or two complementary RNA strands. RNA
duplex formation may be initiated either inside or outside the
cell. The RNA may be introduced in an amount which allows delivery
of at least one copy per cell. Higher doses (e.g., at least 5, 10,
100, 500 or 1000 copies per cell) of double-stranded material may
yield more effective inhibition, while lower doses may also be
useful for specific applications. Inhibition is sequence-specific
in that nucleotide sequences corresponding to the duplex region of
the RNA are targeted for genetic inhibition.
[0061] The subject RNAi constructs can be "small interfering RNAs"
or "siRNAs." These nucleic acids are around 19-30 nucleotides in
length, and even more preferably 21-23 nucleotides in length. The
siRNAs are understood to recruit nuclease complexes and guide the
complexes to the target mRNA by pairing to the specific sequences.
As a result, the target mRNA is degraded by the nucleases in the
protein complex. In a particular embodiment, the 21-23 nucleotides
siRNA molecules comprise a 3' hydroxyl group. In certain
embodiments, the siRNA constructs can be generated by processing of
longer double-stranded RNAs, for example, in the presence of the
enzyme dicer. In one embodiment, the Drosophila in vitro system is
used. In this embodiment, dsRNA is combined with a soluble extract
derived from Drosophila embryo, thereby producing a combination.
The combination is maintained under conditions in which the dsRNA
is processed to RNA molecules of about 21 to about 23 nucleotides.
The siRNA molecules can be purified using a number of techniques
known to those of skill in the art. For example, gel
electrophoresis can be used to purify siRNAs. Alternatively,
non-denaturing methods, such as non-denaturing column
chromatography, can be used to purify the siRNA. In addition,
chromatography (e.g., size exclusion chromatography), glycerol
gradient centrifugation, affinity purification with antibody can be
used to purify siRNAs.
[0062] Production of RNAi constructs can be carried out by chemical
synthetic methods or by recombinant nucleic acid techniques.
Endogenous RNA polymerase of the treated cell may mediate
transcription in vivo, or cloned RNA polymerase can be used for
transcription in vitro. The RNAi constructs may include
modifications to either the phosphate-sugar backbone or the
nucleoside, e.g., to reduce susceptibility to cellular nucleases,
improve bioavailability, improve formulation characteristics,
and/or change other pharmacokinetic properties. For example, the
phosphodiester linkages of natural RNA may be modified to include
at least one of an nitrogen or sulfur heteroatom. Modifications in
RNA structure may be tailored to allow specific genetic inhibition
while avoiding a general response to dsRNA. Likewise, bases may be
modified to block the activity of adenosine deaminase. The RNAi
construct may be produced enzymatically or by partial/total organic
synthesis, any modified ribonucleotide can be introduced by in
vitro enzymatic or organic synthesis. Methods of chemically
modifying RNA molecules can be adapted for modifying RNAi
constructs (see, e.g., Heidenreich et al., 1997, Nucleic Acids Res.
25:776-780; Wilson et al., 1994, J. Mol. Recog. 7:89-98; Chen et
al., 1995, Nucleic Acids Res. 23:2661-2668; Hirschbein et al.,
1997, Antisense Nucleic Acid Drug Dev. 7:55-61). Merely to
illustrate, the backbone of an RNAi construct can be modified with
pbosphorothioates, phosphoramidate, phosphodithioates, chimeric
methylphosphonate-phosphodiesters, peptide nucleic acids,
5-propynyl-pyrimidine containing oligomers or sugar modifications
(e.g., 2'-substituted ribonucleosides, a-configuration).
[0063] In some cases, at least one strand of the siRNA molecules
has a 3' overhang from about 1 to about 6 nucleotides in length,
though may be from 2 to 4 nucleotides in length. More preferably,
the 3' overhangs are 1-3 nucleotides in length. In certain
embodiments, one strand having a 3' overhang and the other strand
being blunt-ended or also having an overhang. The length of the
overhangs may be the same or different for each strand. In order to
further enhance the stability of the siRNA, the 3' overhangs can be
stabilized against degradation. In one embodiment, the RNA is
stabilized by including purine nucleotides, such as adenosine or
guanosine nucleotides. Alternatively, substitution of pyrimidine
nucleotides by modified analogues, e.g., substitution of uridine
nucleotide 3' overhangs by 2'-deoxythyinidine is tolerated and does
not affect the efficiency of RNAi. The absence of a 2' hydroxyl
significantly enhances the nuclease resistance of the overhang in
tissue culture medium and may be beneficial in vivo.
[0064] The RNAi construct can also be in the form of a long
double-stranded RNA. In certain embodiments, the RNAi construct is
at least 25, 50, 100, 200, 300 or 400 bases. In certain
embodiments, the RNAi construct is 400-800 bases in length. The
double-stranded RNAs are digested intracellularly, e.g., to produce
siRNA sequences in the cell. However, use of long double-stranded
RNAs in vivo is not always practical, presumably because of
deleterious effects which may be caused by the sequence-independent
dsRNA response. In such embodiments, the use of local delivery
systems and/or agents which reduce the effects of interferon or PKR
are preferred.
[0065] Alternatively, the RNAi construct is in the form of a
hairpin structure (named as hairpin RNA). The hairpin RNAs can be
synthesized exogenously or can be formed by transcribing from RNA
polymerase III promoters in vivo. Examples of making and using such
hairpin RNAs for gene silencing in mammalian cells are described
in, for example, Paddison et al., 2002, Genes Dev. 16:948-58;
McCaffrey et al., 2002, Nature 418:38-9; McManus et al., 2002, RNA
8:842-50; Yu et al., 2002, Proc. Nat'l Acad. Sci. USA 99:6047-52).
Preferably, such hairpin RNAs are engineered in cells or in an
animal to ensure continuous and stable suppression of a desired
gene. It is known in the art that siRNAs can be produced by
processing a hairpin RNA in the cell.
[0066] PCT application WO 01/77350 describes an exemplary vector
for bi-directional transcription of a transgene to yield both sense
and antisense RNA transcripts of the same transgene in a eukaryotic
cell. Accordingly, in certain embodiments, the present invention
provides a recombinant vector having the following unique
characteristics: it comprises a viral replicon having two
overlapping transcription units arranged in an opposing orientation
and flanking a transgene for an RNAi construct of interest, wherein
the two overlapping transcription units yield both sense and
antisense RNA transcripts from the same transgene fragment in a
host cell.
[0067] In another embodiment, the invention relates to the use of
ribozyme molecules designed to catalytically cleave a target mRNA
transcripts to prevent translation of the mRNA (see, e.g., PCT
International Publication WO90/11364, published Oct. 4, 1990;
Sarver et al., 1990, Science 247:1222-1225; and U.S. Pat. No.
5,093,246). While ribozymes that cleave mRNA at site-specific
recognition sequences can be used to destroy particular mRNAs, the
use of hammerhead ribozymes is preferred. Hammerhead ribozymes
cleave mRNAs at locations dictated by flanking regions that form
complementary base pairs with the target mRNA. The sole requirement
is that the target mRNA have the following sequence of two bases:
5'-UG-3'. The construction and production of hammerhead ribozymes
is well known in the art and is described more fully in Haseloff
and Gerlach, 1988, Nature 334:585-591. The ribozymes of the present
invention also include RNA endoribonucleases (hereinafter
"Cech-type ribozymes") such as the one which occurs naturally in
Tetrahymena thermophila (known as the IVS or L-19 IVS RNA) and
which has been extensively described (see, e.g., Zaug et al., 1984,
Science 224:574-578; Zaug and Cech, 1986, Science, 231:470-475;
Zaug et al., 1986, Nature 324:429-433; published International
patent application No. WO88/04300 by University Patents Inc.; Been
and Cech, 1986, Cell 47:207-216).
[0068] In a further embodiment, the invention relates to the use of
DNA enzymes to inhibit expression of a target gene which encodes a
cellular component relevant to cellular proliferation. DNA enzymes
incorporate some of the mechanistic features of both antisense and
ribozyme technologies. DNA enzymes are designed so that they
recognize a particular target nucleic acid sequence, much like an
antisense oligonucleotide, however much like a ribozyme they are
catalytic and specifically cleave the target nucleic acid. Briefly,
to design an ideal DNA enzyme that specifically recognizes and
cleaves a target nucleic acid, one of skill in the art must first
identify the unique target sequence. Preferably, the unique or
substantially sequence is a G/C rich of approximately 18 to 22
nucleotides. High G/C content helps insure a stronger interaction
between the DNA enzyme and the target sequence. When synthesizing
the DNA enzyme, the specific antisense recognition sequence that
will target the enzyme to the message is divided so that it
comprises the two arms of the DNA enzyme, and the DNA enzyme loop
is placed between the two specific arms. Methods of making and
administering DNA enzymes can be found, for example, in U.S. Pat.
No. 6,110,462.
[0069] The method of present invention is advantageous over
combination therapies known in the art because it allows
conventional chemotherapeutic agent to exert greater effect at
lower dosage. In preferred embodiment of the present invention, the
effective dose (ED50) for a chemotherapeutic agent or combination
of conventional chemotherapeutic agents when used in combination
with the compound of present invention is at least 5 fold less than
the ED50 for the chemotherapeutic agent alone. Conversely, the
therapeutic index (TI) for such chemotherapeutic agent or
combination of such chemotherapeutic agent when used in combination
with the compound of present invention is at least 5 fold greater
than the TI for conventional chemotherapeutic regimen alone.
[0070] C. Radiation Therapy
[0071] Radiation therapy, which includes gamma radiation as well as
particle beams, and chemotherapeutic agents, for cancer and other
hyperproliferative disorders, are cytotoxic, and their
effectiveness in treating a disorder is based upon the fact that
unwanted proliferating cells are generally more sensitive to such
cytotoxic therapies than are normal cells either because of their
rapid metabolism, or because they employ biochemical pathways not
employed by normal cells. It is believed that these therapies exert
their cytotoxic effects by activating programmed cell death, also
referred to as apoptosis. Cells undergo apoptosis when they undergo
a critical level of damage. A balance between the activities of
apoptotic and anti-apoptotic intracellular signal transduction
pathways is important toward a cell's decision of whether to
undergo apoptosis or to attempt internal repair. It has been
demonstrated that galectins, and specifically galectin-3, are
involved in both apoptosis resistance and tumor progression.
[0072] D. Administration
[0073] The therapeutic materials of the present invention may be
administered orally, by injection, transdermally, by pulmonary
inhalation, by subcutaneous implantation, or by topical
application, depending upon the specific type of cancer being
treated, and the adjunct therapy.
[0074] In accord with the present invention, a galectin binding
therapeutic material is administered to a patient, in combination
with conventional therapies such as surgery, radiation or
chemotherapy. The material is most preferably administered prior to
the administration of the conventional therapy, so as to allow it
sufficient time to interact with and bind to galectins in the tumor
or in non-cancerous cells. Depending on the nature of the cancer
and the therapy, administration of the galectin binding therapeutic
material may be continued while the other therapy is being
administered and/or thereafter. Administration of the galectin
binding material may be made in a single dose, or in multiple
doses. In some instances, administration of the therapeutic
material is commenced at least several days prior to the
conventional therapy, while in other instances, administration is
begun either immediately before or at the time of the
administration of the conventional therapy.
[0075] In some instances, particularly with regard to surgical
therapies, the carbohydrate material may be advantageously
administered. both before, during and after the therapy.
[0076] E. Exemplary Targets for Treatment
[0077] The method of present invention is effective in treatment of
various types of cancers, including but not limited to: pancreatic
cancer, renal cell cancer, Kaposi's sarcoma, chronic leukemia,
breast cancer, sarcoma, ovarian carcinoma, rectal cancer, throat
cancer, melanoma, colon cancer, bladder cancer, lymphoma,
mastocytoma, lung cancer, mammary adenocarcinoma, pharyngeal
squamous cell carcinoma, gastrointestinal cancer, stomach cancer,
myeloma or prostate cancer.
[0078] The present invention is also effective against other
diseases related to unwanted cell proliferation. Such
hyperproliferative diseases include but are not limited to:
psoriasis, rheumatoid arthritis, lamellar ichthyosis, epidermolytic
hyperkeratosis, restenosis, endometriosis, or abnormal wound
healing.
[0079] Because galectin-3 and Bcl-2 or Bclx interact and because
the compounds of present invention are especially useful to treat
cells with elevated anti-apoptotic Bcl-2 protein activities, it is
beneficial to determine the level of active anti-apoptotic Bcl-2
proteins in a tumor or in leukemic cells in a patient. The presence
of elevated levels of anti-apoptotic Bcl-2 proteins in a tumor can
be determined by immunodetection using antibodies specific to each
of these proteins, either through enzyme-linked immunosolvent
assays, or immunohistochemistry of solid tumor samples. The
immunohistochemistry will also allow determination of the
intracellular localization of anti-apoptotic Bcl-2 protein in a
tumor sample. By using monoclonal antibodies specific to
phosphorylated proteins, the phosphorylation state of
anti-apoptotic Bcl-2 proteins can also be determined by the same
techniques. The expression of anti-apoptotic Bcl-2 proteins can be
determined by detecting the levels of mRNA in Southern blots, using
probes specific to the nucleotide sequence of each of the
anti-apoptotic Bcl-2 proteins. Alternatively, quantitative
polymerase chain reaction may be done, using a pair of primers
specific to each of the anti-apoptotic Bcl-2 protein. Once the
expression levels and the status of the anti-apoptotic Bcl-2
protein are determined, a patient with cancerous growth which have
elevated levels of anti-apoptotic Bcl-2 protein activities are
treated with the compounds of present invention along with other
anti-cancer therapies as necessary.
F. EXAMPLES
Example 1
[0080] Promotion of Apoptosis by a Modified Pectin
[0081] Experiments were performed to demonstrate the ability of a
modified pectin to promote apoptosis in a cell line with high Bcl-2
expression and chemoresistance.
[0082] Cell line DoHH2 is a spontaneously growing EBV-negative
B-cell line, established from the pleural fluid cells of a patient
with centroblastic/centrocytic non-Hodgkin's lymphoma, that had
transformed into an immunoblastic lymphoma. Kluin-Nelemans et al.,
"A new non-Hodgkin's B-cell line (DoHH2) with a chromosomal
translocation t(14;18)(q32;q21)," Leukemia Mar. 5, 1991 (3):221-4.
The expression of Bcl-2 is upregulated in DoHH2 due to chromosomal
translocation, and the cell line is known to have high
chemoresistance that is dependent on the status of Bcl-2. When
treated with a Bcl-2 antisense polynucleotide, DoHH2 proceeds to
apoptosis, indicating the overexpression of Bcl-2 is a cause of
lack of apoptosis.
[0083] DoHH2 cells were exposed to modified pectin GCS-100 in three
different formulations, V1, V2, and V3. Formulation VI contained
12.6% ethanol, V2 contained 15% ethanol, and V3 contained 0.2%
ethanol. In vitro apoptosis was quantitated by DioC6(3) stain as a
measure of mitochondrial depolarization at 4, 24, 48, and 72 hours
after 0, 40, 80, 160, or 320 .mu.g /ml of each formulation was
added to cell culture. See FIGS. 1A-1C. All samples demonstrated
increased apoptosis over time, but the addition of GCS-100
increased the number of cells undergoing apoptosis in a
dose-dependent manner. The three formulations performed similarly
at the highest dose of 320 .mu.g/ml, but at lower dosages of 40,
80, or 160 .mu.g /ml, formulation V3, which contained the least
amount of ethanol, was more effective in inducing apoptosis at
earlier time points compared to formulation V1 or V2.
[0084] The foregoing discussion has been primary directed toward
modified pectin materials and materials which interact with
galectins-1 and 3; however, it is to be understood that other
galectins are also known to be involved in the progress of various
cancers, and both the modified pectin material as well as the other
therapeutic materials discussed hereinabove interact with
galectins. Therefore, other materials and methods may be employed
in the practice of the present invention. The foregoing discussion
and description is illustrative of specific embodiments, but is not
meant to be a limitation upon the practice thereof. It is the
following claims, including all equivalents, which define the scope
of the invention.
* * * * *