U.S. patent application number 11/063144 was filed with the patent office on 2005-06-30 for use of parthenolide to inhibit cancer.
This patent application is currently assigned to Indiana University Research and Technology Corporation. Invention is credited to Nakshatri, Harikrishna, Sweeney, Christopher J..
Application Number | 20050143451 11/063144 |
Document ID | / |
Family ID | 27390219 |
Filed Date | 2005-06-30 |
United States Patent
Application |
20050143451 |
Kind Code |
A1 |
Nakshatri, Harikrishna ; et
al. |
June 30, 2005 |
Use of parthenolide to inhibit cancer
Abstract
The invention provides a method comprising the use of
parthenolide, including its analogs, to treat cancer or conditions
characterized by abnormal angiogenesis.
Inventors: |
Nakshatri, Harikrishna;
(Indianapolis, IN) ; Sweeney, Christopher J.;
(Indianapolis, IN) |
Correspondence
Address: |
SCHWEGMAN, LUNDBERG, WOESSNER & KLUTH, P.A.
P.O. BOX 2938
MINNEAPOLIS
MN
55402-0938
US
|
Assignee: |
Indiana University Research and
Technology Corporation
|
Family ID: |
27390219 |
Appl. No.: |
11/063144 |
Filed: |
February 22, 2005 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11063144 |
Feb 22, 2005 |
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10178054 |
Jun 21, 2002 |
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6890946 |
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10178054 |
Jun 21, 2002 |
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PCT/US00/34469 |
Dec 19, 2000 |
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60173023 |
Dec 23, 1999 |
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60173024 |
Dec 23, 1999 |
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Current U.S.
Class: |
514/468 |
Current CPC
Class: |
A61K 2300/00 20130101;
A61K 31/365 20130101; A61K 31/365 20130101 |
Class at
Publication: |
514/468 |
International
Class: |
A61K 031/365 |
Claims
1-13. (canceled)
14. A therapeutical method of treating a non-tumorigenic
angiogenesis-dependent condition characterized by the abnormal
growth of blood vessels comprising administering to a mammal
afflicted with said condition an effective anti-angiogenetic amount
of parthenolide.
15. The method of claim 14 wherein the mammal is a human.
16. The method of claim 15 wherein the condition is corneal
neovascularization.
17. The method of claim 15 wherein the condition is hypertrophic
scars and keloids.
18. The method of claim 15 wherein the condition is proliferative
diabetic retinopathy.
19. The method of claim 15 wherein the condition comprises
arteriovenous malformations or atherosclerosis.
20. The method of claim 15 wherein the condition comprises delayed
wound healing.
21. The method of claim 14 wherein the parthenolide is administered
locally.
22. The method of claim 14 wherein the parthenolide is delivered in
a liquid vehicle.
23. The method of claim 14 wherein the parthenolide is delivered
from a controlled release polymeric matrix.
24. The method of claim 23 wherein the parthenolide is delivered
from a medical prosthesis.
25. The method of claim 24 wherein the parthenolide is delivered
from a shunt, a stent or a graft.
26. (canceled)
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] The present application is a continuation under 35 USC
111(a) of PCT/US00/34469 filed Dec. 19, 2000 (WO 01/45699), which
claims priority under 35 U.S.C. .sctn. 120 to U.S. provisional
patent application Nos. 60/173,023, filed Dec. 23, 1999 and
60/173,024 filed Dec. 23, 1999, which are incorporated by reference
herein.
BACKGROUND OF THE INVENTION
[0002] Fatality in cancer is generally due to metastasis and
development of resistance to chemotherapy (Fisher, 1994; Liotta et
al., 1991). Metastasis and resistance to chemotherapy are mostly
due to overexpression of pro-metastatic, pro-angiogenic, multi-drug
resistance and anti-apoptotic genes (Baldini, 1997; Fisher, 1994;
Wang et al., 1999a). The expression of a significant number of
these genes is regulated by NF-.kappa.B, Activator Protein (AP-1)
and the Ets family of transcription factors (Baeuerle & Henkel,
1994; Grumont et al., 1999; Gutman & Wasylyk, 1990; Lee et al.,
1999; Wang et al., 1999b; Wang et al., 1998; Zong et al.,
1999).
[0003] Expression of the pro-metastatic genes interleukin-6 (IL-6),
urokinase plasminogen activator, matrix metalloproteinase 9, the
pro-angiogenic gene IL-8 and the anti-apoptotic genes c-IAP1,
cIAP2, TRAF1, TRAF2, Bf1-1/A1, Bc1-X.sub.L, and Mn-SOD is induced
by NF-.kappa.B (Baeuerle & Henkel, 1994; Grumont et al., 1999;
Jones et al., 1997; Lee et al., 1999; Wang et al., 1999b, Wang et
al., 1998; Zong et al., 1999). Normally, NF-.kappa.B resides in the
cytoplasm in an inactive state bound to I.kappa.B proteins
(Baeuerie & Henkel, 1994). When cells are exposed to
TNF.alpha., IL-1 or chemotherapeutic agents, a multisubunit
I.kappa.B kinase complex (IKC) is activated, which phosphorylates
I.kappa.Bs (Zandi & Karin, 1999). NF-.kappa.B dissociates from
phosphorylated I.kappa.Bs, translocates to the nucleus and
activates target genes (Baeuerle & Henkel, 1994). The ability
of activated NF-.kappa.B to induce gene expression depends on the
cell type and the type of NF-.kappa.B inducer.
[0004] For example, in cell types that are sensitive to TNF.alpha.
and chemotherapy-induced apoptosis, NF-.kappa.B is inactivated by
caspases and the induction of NF-.kappa.B-dependent cell survival
signals is markedly reduced (Levkau et al., 1999). In contrast,
activation of NF-.kappa.B by growth factors or IL-1 can cause an
increase in anti-apoptotic gene expression and subsequent
resistance to TNF and chemotherapy (Wang et al., 1996). Inhibition
of NF-.kappa.B activation by I.kappa.B overexpression can convert
TNF- and chemotherapy-resistant cells to a sensitive phenotype (Beg
& Baltimore, 1996; Van Antwerp et al., 1996; Wang et al.,
1996).
[0005] Recent studies indicate that NF-.kappa.B is constitutively
active in a number of tumors including Hodgkin's lymphoma,
melanoma, juvenile myelomonocytic leukemia, cutaneous T cell
lymphoma, melanoma, squamous cell carcinoma and Bcr-Abl-induced
transformation (Bargou et al., 1997; Dong et al., 1999; Giri &
Aggarwal, 1998; Reuther et al., 1998; Shattuck-Brandt &
Richmond, 1997). Constitutive NF-.kappa.B activation has been
described in a subset of breast cancers (Cogswell et al., 2000;
Nakshatri et al., 1997; Sovak et al., 1997).
[0006] Although a number of drugs, including aspirin, have been
described as having some ability to prevent NF-.kappa.B activation
(Yin et al., 1998), a need exists for compounds that can potently
inhibit NF-.kappa.B activation at clinically achievable doses. Such
drugs can be used as primary or adjunct therapeutic agents in the
treatment of cancer, or in other pathologies involving NF-.kappa.B
activation.
SUMMARY OF THE INVENTION
[0007] In one aspect, the present invention provides a therapeutic
method to treat cancer, including hematological malignancies and
solid tumors, such as prostate cancer, ovarian cancer, breast
cancer, brain cancer and hepatic cancer, comprising administering
to a mammal afflicted with said cancer an amount of parthenolide,
or an analog thereof that is an NF-.kappa.B inhibitor, effective to
inhibit the viability of cancer cells of said mammal. The
parthenolide may be administered as primary therapy, or as adjunct
therapy, either following local intervention (surgery, radiation,
local chemotherapy) or in conjunction with at least one other
chemotherapeutic agent.
[0008] The present invention also provides a method of increasing
the susceptibility of human cancer cells to a chemotherapeutic
agent comprising contacting the cells with an effective sensitizing
amount of parthenolide. Thus, the invention provides a therapeutic
method for the treatment of a human or other mammal afflicted with
cancer, wherein an effective amount of parthenolide is administered
to a subject afflicted with said cancer and that may be undergoing,
or be about to undergo, treatment with a chemotherapeutic
("antineoplastic") agent. As used herein, the term "parthenolide"
includes essentially pure parthenolide, as described below, or
analogs exhibiting useful NF-.kappa.B and/or c-IAP2 inhibitory
activity that are known or apparent to the art.
[0009] Preferably, parthenolide is administered in conjunction with
one or more chemotherapeutic agents effective against the
particular cancer such as gemcitabine or 5-FU, if pancreatic cancer
is being treated, tamoxifen or paclitaxel, if breast cancer is to
be treated, leuprolide or other anti-androgens, if prostate cancer
is involved, and the like.
[0010] In another aspect, the present invention comprises a
therapeutic method comprising the administration of parthenolide to
treat non-tumorigenic angiogenesis-dependent diseases that are
characterized by the abnormal growth of blood vessels. Parthenolide
may also be utilized in surgical procedures in which
anti-angiogenesis is useful, including stent and graft placement
and in the treatment of tumor excision sites.
[0011] The present invention also provides a method to determine
whether or not a cancer patient will be amenable to treatment by a
NF-.kappa.B inhibitor, alone or in combination with one or more
other chemotherapeutic agents, comprising (a) isolating a portion
of cancer cells from said patient, and (b) determining whether said
cells comprise constitutively active NF-.kappa.B and/or express
c-IAP2; and correlating the level of NF-.kappa.B and/or c-IAP2 with
the ability of the inhibitor to inhibit NF-.kappa.B or c-IAP2 in
reference cells of said cancer that also comprise NF-.kappa.B or
c-IAP2. This method is based on the fact that a high level of
NF-.kappa.B activity and/or overexpression of c-IAP2 is indicative
of susceptibility of said cancer cells to a NF-.kappa.B inhibitor.
Thus, a cancer patient about to undergo, or undergoing, treatment
for cancer can be rapidly evaluated to see if he/she will benefit
from concurrent chemotherapy and administration of parthenolide or
an analog thereof.
[0012] The terms "high level" and "overexpression" are defined by
reference to the assays and test data set forth hereinbelow, e.g.,
a "++" or greater rating on Table 1. For example, it can readily be
determined empirically, and by in vitro tests, if a population of
cancer cells, such as a population isolated from a cancer patient,
exhibits an NF-.kappa.B level or overexpresses the c-IAP2 gene to
the extent required to render the cancer susceptible to treatment
in accord with the present invention.
BRIEF DESCRIPTION OF THE FIGURES
[0013] FIG. 1 is a graph showing the relative difference in
apoptosis among cell types LxSN11 and IxB.alpha.SR10 with or
without paclitaxel treatment. A population of 5.times.10.sup.4
LxSN11 and IxB.alpha.SR10 cells were grown overnight and incubated
with paclitaxel for 18 and 48 hrs. Apoptosis was measured by ELISA.
The rate of spontaneous apoptosis in untreated LxSN11 cells was set
as one unit.
[0014] FIG. 2 is a graph depicting the increase in sensitivity of
MD231 and HBL100 breast cancer cells to increasing concentrations
of parthenolide, in the presence and absence of 1.0 nM
paclitaxel.
[0015] FIG. 3A is a graph depicting the relative apoptosis of
HBL100 cells caused by paclitaxel and/or parthenolide.
[0016] FIG. 3B is a photocopy of a Western blot showing PARP
cleavage products in HBL100 cells treated with paclitaxel and/or
parthenolide.
[0017] FIG. 4A is a photocopy of an EMSA gel showing the effect of
parthenolide on NF-.kappa.B binding in prostate cancer cell
lines.
[0018] FIG. 4B is a graph depicting inhibition of prostate cancer
cell inhibition by parthenolide.
[0019] FIG. 5 is a graph depicting the inhibition of HUVECs by
parthenolide.
[0020] FIG. 6 is a graph depicting the inhibition of capillary
formation by HUVECs by parthenolide.
[0021] FIG. 7 is a graph depicting the effect of parthenolide on in
vivo-induced angiogenesis.
[0022] FIG. 8 is a photocopy of an electromobility gel shift assay
gel demonstrating the effect of parthenolide on NF-.kappa.B DNA
binding.
DETAILED DESCRIPTION OF THE INVENTION
[0023] Parthenolide,
([1aR-(1.alpha.R*,4E,7aS*,10.alpha.S*,-10bR*)]-2,3,6,-
7,7.alpha.,8,10.alpha.,10b-Octahydro-1.alpha.,5-dimethyl-8-methyleneoxiren-
o[9, 10]cyclodeca[1,2-b]furan-9(1.alpha.H)-one) or
4,5.alpha.-epoxy-6.beta-
.-hydroxy-germacra-1(10),11(13)-dien-12-oic acid .gamma.-lactone is
a sesquiterpene lactone found in feverfew, and in other plants. Its
formula is given below: 1
[0024] Isolation from Chrysanthemum parthenium (L.) Bernh.
Compositae and characterization has been described by V. Herout et
al., Chem. & Ind. (London), 1069 (1959); M. Soucek et al.,
Coll. Czech. Chem. Comm., 26, 803 (1961). Its isolation from
Magnolia grandiflora L., Magnoliaceae has been described by F. S.
El-Feraly, Y.-M. Chan, J. Pharm. Sci., 67, 347 (1978). The absolute
configuration was determined by A. S. Bawdekar et al., Tetrahedron
Letters, 1225 (1966). Its cytotoxicity was investigated by K.-H.
Lee et al., Cancer Res., 31, 1649 (1971) and by L. A. J. O'Neill et
al., Brit. J. Clin. Pharmacol, 23, 81 (1987).
[0025] Parthenolide is commercially available as 500 mcg tablets
from Ashbury Biologicals and is available as an herbal remedy for
migraines in Canada (Murphy et al., 1988). E. Johnson et al., in
U.S. Pat. No. 4,758,433, disclose that a parthenolide-containing
extract can be used to treat migraine, arthritis and "bronchial
complaints." D. H. Hwang et al. (U.S. Pat. No. 5,905,089) disclose
that parthenolide can be used to treat or prevent symptoms of
"severe inflammatory disorders" associated with the production of
various pro-inflammatory "cytokines, chemokines and proteins,"
including COX-2, MAPKS and NF-.kappa.B.
[0026] In toxicity studies, parthenolide has been given to rats and
dogs at dosages of from about 250-2500 mg/kg/day without
significant toxicity. In a phase I study of parthenolide in human
patients with cancer, such as incurable prostate cancer, patients
can be dosed at 1000 mcg/day, and each subsequent cohort will
receive a 30% increase in the dose, for two cycles (8 weeks) of
therapy at their assigned dose. Such dosages can also be used to
treat and develop treatments for other cancers and
angiogenesis-dependent conditions, such as those described
hereinbelow, as can doses presently used to treat migraine
headaches in humans. Dosages suitable for human administration can
be calculated from dosages effective in animal models as disclosed
in U.S. Pat. No. 5,294,430.
[0027] Cancers treatable by the present therapy include the solid
and hematological tumors discussed hereinabove, as well as the
solid tumors disclosed in U.S. Pat. No. 5,514,555. Hematological
cancers, such as the leukemias are disclosed in the Mayo Clinic
Family Health Book, D. E. Larson, ed., William Morrow, N.Y. (1990)
and include CLL, ALL, CML and the like.
[0028] Within another aspect of the present invention, methods are
provided for inhibiting angiogenesis in patients with
non-tumorigenic, angiogenesis-dependent diseases, comprising
administering a therapeutically effective amount of a composition
comprising parthenolide to a patient with a non-tumorigenic
angiogenesis-dependent disease, such that the formation of new
blood vessels is inhibited. Within other aspects, methods are
provided for inhibit reactive proliferation of endothelial cells or
capillary formation in non-tumorigenic, angiogenesis-dependent
diseases, such that the blood vessel is effectively occluded.
Within one embodiment, the anti-angiogenic composition comprising
parthenolide is delivered to a blood vessel which is actively
proliferating and nourishing a tumor.
[0029] In addition to tumors, numerous other non-tumorigenic
angiogenesis-dependent diseases, which are characterized by the
abnormal growth of blood vessels, may also be treated with the
anti-angiogenic parthenolide compositions, or anti-angiogenic
factors of the present invention. Anti-angiogenic parthenolide
compositions of the present invention can block the stimulatory
effects of angiogenesis promoters, reducing endothelial cell
division, decreasing endothelial cell migration, and impairing the
activity of the proteolytic enzymes secreted by the endothelium.
Representative examples of such non-tumorigenic
angiogenesis-dependent diseases include corneal neovasculaiization,
hypertrophic scars and keloids, proliferative diabetic retinopathy,
arteriovenous malformations, atherosclerotic plaques, delayed wound
healing, hemophilic joints, nonunion fractures, Osler-Weber
syndrome, psoriasis, pyogenic granuloma, scleroderma, trachoma,
menorrhagia, retrolental fibroplasia and vascular adhesions. The
pathology and treatment of these conditions is disclosed in detail
in published PCT application PCT/CA94/00373 (WO 95/03036), at pages
26-36. Topical or directed local administration of the present
compositions is often the preferred mode of administration of
therapeutically effective amounts of parthenolide, i.e., in depot
or other controlled release forms.
[0030] Anti-angiogenic compositions of the present invention may
also be utilized in a variety of other manners. For example, they
may be incorporated into surgical sutures in order to prevent
stitch granulomas, implanted in the uterus (in the same manner as
an IUD) for the treatment of menorrhagia or as a form of female
birth control, administered as either a peritoneal lavage fluid or
for peritoneal implantation in the treatment of endometriosis,
attached to a monoclonal antibody directed against activated
endothelial cells as a form of systemic chemotherapy, or utilized
in diagnostic imaging when attached to a radioactively labelled
monoclonal antibody which recognizes active endothelial cells.
[0031] The magnitude of a prophylactic or therapeutic dose of
parthenolide, an analog thereof or a combination thereof, in the
acute or chronic management of cancer, i.e., prostate or breast
cancer, will vary with the stage of the cancer, such as the solid
tumor to be treated, the chemotherapeutic agent(s) or other
anti-cancer therapy used, and the route of administration. The
dose, and perhaps the dose frequency, will also vary according to
the age, body weight, and response of the individual patient. In
general, the total daily dose range for parthenolide and its
analogs, for the conditions described herein, is from about 0.5 mg
to about 2500 mg, in single or divided doses. Preferably, a daily
dose range should be about 1 mg to about 100 mg, in single or
divided doses, most preferably about 5-50 mg per day. In managing
the patient, the therapy should be initiated at a lower dose and
increased depending on the patient's global response. It is further
recommended that infants, children, patients over 65 years, and
those with impaired renal or hepatic function initially receive
lower doses, and that they be titrated based on global response and
blood level. It may be necessary to use dosages outside these
ranges in some cases. Further, it is noted that the clinician or
treating physician will know how and when to interrupt, adjust or
terminate therapy in conjunction with individual patient response.
The terms "an effective amount" or "an effective sensitizing
amount" are encompassed by the above-described dosage amounts and
dose frequency schedule.
[0032] Any suitable route of administration may be employed for
providing the patient with an effective dosage of parthenolide.
While it is possible that, for use in therapy, parthenolide or its
analogs may be administered as the pure chemicals, as by inhalation
of a fine powder via an insufflator, it is preferable to present
the active ingredient as a pharmaceutical formulation. The
invention thus further provides a pharmaceutical formulation
comprising parthenolide or an analog thereof, together with one or
more pharmaceutically acceptable carriers therefor and, optionally,
other therapeutic and/or prophylactic ingredients. The carrier(s)
must be `acceptable` in the sense of being compatible with the
other ingredients of the formulation and not deleterious to the
recipient thereof, such as a human patient or domestic animal.
[0033] Pharmaceutical formulations include those suitable for oral
or parenteral (including intramuscular, subcutaneous and
intravenous) administration. Forms suitable for parenteral
administration also include forms suitable for administration by
inhalation or insufflation or for nasal, or topical (including
buccal, rectal, vaginal and sublingual) administration. The
formulations may, where appropriate, be conveniently presented in
discrete unit dosage forms and may be prepared by any of the
methods well known in the art of pharmacy. Such methods include the
step of bringing into association the active compound with liquid
carriers, solid matrices, semi-solid carriers, finely divided solid
carriers or combinations thereof, and then, if necessary, shaping
the product into the desired delivery system.
[0034] Pharmaceutical formulations suitable for oral administration
may be presented as discrete unit dosage forms such as hard or soft
gelatin capsules, cachets or tablets each containing a
predetermined amount of the active ingredient; as a powder or as
granules; as a solution, a suspension or as an emulsion; or in a
chewable base such as a synthetic resin or chicle for ingestion of
the agent from a chewing gum. The active ingredient may also be
presented as a bolus, electuary or paste. Tablets and capsules for
oral administration may contain conventional excipients such as
binding agents, fillers, lubricants, disintegrants, or wetting
agents. The tablets may be coated according to methods well known
in the art, i.e., with enteric coatings.
[0035] Oral liquid preparations may be in the form of, for example,
aqueous or oily suspensions, solutions, emulsions, syrups or
elixirs, or may be presented as a dry product for constitution with
water or other suitable vehicle before use. Such liquid
preparations may contain conventional additives such as suspending
agents, emulsifying agents, non-aqueous vehicles (which may include
edible oils), or preservatives.
[0036] The compounds according to the invention may also be
formulated for parenteral administration (e.g., by injection, for
example, bolus injection or continuous infusion) and may be
presented in unit dose form in ampules, pre-filled syringes, small
volume infusion containers or in multi-dose containers with an
added preservative. The compositions may take such forms as
suspensions, solutions, or emulsions in oily or aqueous vehicles,
and may contain formulatory agents such as suspending, stabilizing
and/or dispersing agents. Alternatively, the active ingredient may
be in powder form, obtained by aseptic isolation of sterile solid
or by lyophilization from solution, for constitution with a
suitable vehicle, e.g., sterile, pyrogen-free water, before
use.
[0037] For topical administration to the epidermis, the compounds
may be formulated as ointments, creams or lotions, or as the active
ingredient of a transdermal patch. Suitable transdermal delivery
systems are disclosed, for example, in A. Fisher et al. (U.S. Pat.
No. 4,788,603), or R. Bawa et al. (U.S. Pat. Nos. 4,931,279;
4,668,506 and 4,713,224). Ointments and creams may, for example, be
formulated with an aqueous or oily base with the addition of
suitable thickening and/or gelling agents. Lotions may be
formulated with an aqueous or oily base and will in general also
contain one or more emulsifying agents, stabilizing agents,
dispersing agents, suspending agents, thickening agents, or
coloring agents.
[0038] Formulations suitable for topical administration in the
mouth include unit dosage forms such as lozenges comprising active
ingredient in a flavored base, usually sucrose and acacia or
tragacanth; pastilles comprising the active ingredient in an inert
base such as gelatin and glycerin or sucrose and acacia;
mucoadherent gels, and mouthwashes comprising the active ingredient
in a suitable liquid carrier.
[0039] When desired, the above-described formulations can be
adapted to give sustained release of the active ingredient
employed, e.g., by combination with certain hydrophilic polymer
matrices, e.g., comprising natural gels, synthetic polymer gels or
mixtures thereof. The polymer matrix can be coated onto, or used to
form, a medical prosthesis, such as a stent, valve, shunt, graft,
or the like.
[0040] Pharmaceutical formulations suitable for rectal
administration wherein the carrier is a solid are most preferably
presented as unit dose suppositories. Suitable carriers include
cocoa butter and other materials commonly used in the art, and the
suppositories may be conveniently formed by admixture of the active
compound with the softened or melted carrier(s) followed by
chilling and shaping in molds.
[0041] Formulations suitable for vaginal administration may be
presented as pessaries, tampons, creams, gels, pastes, foams or
sprays containing, in addition to the active ingredient, such
carriers as are known in the art to be appropriate.
[0042] For administration by inhalation, the compounds according to
the invention are conveniently delivered from an insufflator,
nebulizer or a pressurized pack or other convenient means of
delivering an aerosol spray. Pressurized packs may comprise a
suitable propellant such as dichlorodifluoromethane,
trichlorofluoromethane, dichlorotetrafluoroethan- e, carbon dioxide
or other suitable gas. In the case of a pressurized aerosol, the
dosage unit may be determined by providing a valve to deliver a
metered amount.
[0043] Alternatively, for administration by inhalation or
insufflation, the compounds according to the invention may take the
form of a dry powder composition, for example, a powder mix of the
compound and a suitable powder base such as lactose or starch. The
powder composition may be presented in unit dosage form in, for
example, capsules or cartridges or, e.g., gelatin or blister packs
from which the powder may be administered with the aid of an
inhalator or insufflator.
[0044] For intra-nasal administration, the compounds of the
invention may be administered via a liquid spray, such as via a
plastic bottle atomizer. Typical of these are the Mistometer.RTM.
(Wintrop) and the Medihaler.RTM. (Riker).
[0045] For topical administration to the eye, the compounds can be
administered as drops, gels (see, S. Chrai et al., U.S. Pat. No.
4,255,415), gums (see S.-L. Lin et al., U.S. Pat. No. 4,136,177) or
via a prolonged-release ocular insert.
[0046] The invention will be further described by reference to the
following detailed examples wherein the following materials and
methods were employed:
[0047] A. Breast Cancer Cell Lines. All breast cancer cell lines
were purchased from the American Type Tissue Culture Collection
(Rockville, Md.) and grown as described previously (Bhat-Nakshatri
et al., 1998). Wild type and p65-fibroblast cells were a gift from
D. Baltimore and A. Hoffmann (Beg & Baltimore, 1996).
[0048] B. Generation of MDA-MB-231 Cells Overexpressing
I.kappa.B.alpha. Super-Repressor. I.kappa.B.alpha. super-repressor
(I.kappa.B.alpha.SR) plasmid containing S32A and S36A mutation of
I.kappa.B.alpha. was generated by PCR mediated site-directed
mutagenesis and cloned into the EcoRI site of the modified
retrovirus vector LxSN (Miller & Rosman, 1989; Wang et al.,
1996). Retrovirus and stable I.kappa.BASR expressing MDA-MB-231
clones were prepared as described previously (Newton et al.,
1999).
[0049] C. Electrophoretic Mobility Shift Assays (EMSAs). Whole cell
extracts were prepared and subjected to EMSA with NF-.kappa.B and
SP-1 probes (Promega, Madison, Wis.) (Newton et al., 1999). Nuclear
extracts were prepared as described previously (Dignam et al.,
1983). Antibodies for supershift assays were purchased from Santa
Cruz Biotechnology (CA) or Upstate Biotechnology (NY).
[0050] D. Western Blotting. Total cell lysates were prepared in
radioimmunoassay buffer and subjected to Western blotting as
described previously (Newton et al., 1999). I.kappa.B.alpha. and
Mn-SOD antibodies were purchased from Santa Cruz Biotechnology (CA)
and Upstate Biotechnology, respectively.
[0051] E. Northern Blotting and RNase Protection. Total RNA was
subjected to Northern blotting as described previously (Newton et
al., 1999). Twenty .mu.gs of RNA were subjected to RNase protection
using the hAPO-5 probe (PharMingen, San Diego, Calif.) as described
by the manufacturers.
[0052] F. Drug Treatment. Cell Death Assays, Apoptosis Assay and
PARP Cleavage. For cell death assays, 2.times.10.sup.3 cells grown
overnight on a 96-well plate were treated with paclitaxel and/or
parthenolide (dissolved in ethanol). In combination treatment,
parthenolide was added 4 h before paclitaxel treatment. Cell death
was measured 18 h after paclitaxel treatment using the MTS assay
(Promega, Madison, Wis.). Apoptosis was measured by a histone-ELISA
(Roche Diagnostics, Indianapolis, Ind.). 5.times.10.sup.4 cells
grown overnight on six well plates were treated with paclitaxel for
indicated times. Histone-DNA complexes in the supernatant were
detected by ELISA. For the PARP cleavage assay, 5.times.10.sup.5
cells were exposed to the indicated drugs for 24 h. Both adherent
and non-adherent cells were collected, lysed in urea-sodium dodecyl
sulfate buffer and subjected to Western blotting with PARP antibody
(Enzyme System Products, Livermore, Calif.) (Panvichian et al.,
1998).
[0053] G. Flow Cytometry. 5.times.10.sup.5 cells grown overnight
were treated with the indicated concentrations of paclitaxel for 18
h. Cells were collected by trypsinization, washed in
phosphate-buffered saline and resuspended in 125 .mu.l of 2
.mu.gs/ml RNase and 300 .mu.l of propidium iodide containing buffer
(50 .mu.g/ml propidium iodide, 1 mg/ml sodium citrate, 0.03% NP40).
The amount of propidium iodide incorporated was determined by
FAScan analysis and the cell cycle distribution was determined
using ModFit computer Software. Cell cycle distribution analysis
was performed in a single blinded manner. Only diploid cells were
considered while calculating the percentage of cells at each phase
of cell cycle.
[0054] H. Statistical Analysis. In synergy experiments, dose
response curves for single agents were generated first. The effect
of combined treatment was analyzed by the Isobole method
(Berenbaum, 1981).
EXAMPLE 1
Constitutive NF-.kappa.B Activation in Breast Cancer Cells
Correlates with Increased c-IAP2 and Mn-SOD Expression
[0055] Previously, it was demonstrated that constitutive
NF-.kappa.B DNA binding activity varies among breast cancer cell
lines (Nakshatri et al., 1997; Newton et al., 1999). As shown in
Table 1, the binding activity observed was
MDA-MB-436.gtoreq.HBL100>MDA-MB-231.gtoreq.MDA-MB-468.gto-
req.MDA-MB-435>SK-BR-3.gtoreq.Hs578T.gtoreq.ZR-75-1>T47-D.gtoreq.MCF-
-7 cells (Newton et al., 1999).
1TABLE 1 Summary of NF-.kappa.B DNA binding activity, C-IAP2 and
Mn- SOD expression in breast cancer cell lines. NF-.kappa.B DNA
binding activity in these cells has been described (Newton et al.,
1999). Relative expression of c-IAP2 and Mn-SOD was calculated by
densitometric scanning of autoradiograms. Cell lines NF-.kappa.B
c-IAP2 Mn-SOD MCF-7 + - + T47D + - - ZR-75-1 ++ - - MDA-MB-231 +++
+++++ +++ MDA-MB-435 +++ .+-. ++ MDA-MB-436 ++++ +++ +++++
MDA-MB-468 +++ ++ ++ SK-BR-3 ++ - + Hs578T ++ - + HBL100 ++++ ++
+++
[0056] Among these cells, MCF-7, T47D and ZR-75-1 cells are
estrogen receptor alpha (ER.alpha.)-positive (Sommers et al.,
1994). In ER.alpha.-positive breast cancer cells, transcriptional
activity but not DNA binding activity of NF-.kappa.B is inhibited
by ER.alpha. (Galien & Garcia, 1997; Nakshatri et al.,
1997).
[0057] Thus, ER.alpha.-negative breast cancer cells with higher
levels of constitutive NF-.kappa.B DNA binding activity may
overexpress NF-.kappa.B-inducible genes compared to
ER.alpha.-positive breast cancer cells. To address this
possibility, RNase protection assays, Northern analysis, cDNA
microarray and differential display methods were used to identify
NF-.kappa.B regulated genes in breast cancer cells. The RNase
protection assay was performed with hAPO-5 probe, which allows
quantitation of xIAP, TRAF1, TRAF2, CART, NIAP, c-IAP1, c-IAP2,
TRPM2 and CRAF genes. Among these genes, xIAP, TRAF1, TRAF2,
c-IAP1, c-IAP2 and NIAP are anti-apoptotic (Deveraux & Reed,
1999; Wang et al., 1998). TRPM-2 is anti-apoptotic in certain cell
types (Miyake et al., 2000). There was no significant variation in
the expression levels of xIAP, CART1 and CARF among various cell
types. In contrast, c-IAP2 expression was observed only in
MDA-MB-231, MDA-MB-436, MDA-MB-468 and HBL100 cells, all of which
contain high levels of constitutive NF-.kappa.B DNA binding
activity as shown in Table 1.
[0058] The c-IAP2 expression in these cells was further confirmed
by Northern blot analysis (data not shown). TRAF1 expression was
observed in MDA-MB-231 and MDA-MB-436 cells. Cell type-specific
variation in TRPM2 expression was observed but did not correlate
with NF-.kappa.B DNA binding activity. The expression levels of the
anti-apoptotic gene c-IAP1, Mn-SOD and survivin were measured by
Northern blotting (Jones et al., 1997; Li et al., 1998; Wang et
al., 1998). While all cell lines expressed similar levels of c-IAP1
and survivin, Mn-SOD expression was higher in ER.alpha.-negative
breast cancer cells with constitutive NF-.kappa.B DNA binding
activity. Increased expression of Mn-SOD in ER.alpha.-negative
breast cancer cells was further confirmed by Western blotting.
[0059] Differential screening of Atlas.TM. human cancer cDNA array
(Clontech, Palo Alto, Calif.) using RNA from MDA-MB-231 cells and
MDA-MB-231 cells modified to overexpress I.kappa.B.alpha.
super-repressor (I.kappa.B.alpha.SR) (Wang et al., 1996) identified
the anti-apoptotic gene DAD-1 as an NF-.kappa.B inducible gene (see
below, data not shown) (Kelleher & Gilmore, 1997). However,
DAD-1 expression did not correlate with constitutive NF-.kappa.B
DNA binding activity. Taken together, these results suggest that
constitutive activation of NF-.kappa.B leads to increased mRNA
and/or protein levels of c-IAP2 and Mn-SOD in breast cancer
cells.
EXAMPLE 2
I.kappa.B.alpha.SR Inhibits c-IAP2, Mn-SOD, TRAF1 and DAD-1
Expression in MDA-MB-231 Cells
[0060] To further investigate whether c-IAP2 and Mn-SOD expression
is dependent on NF-.kappa.B, MDA-MB-231 cells overexpressing
I.kappa.B.alpha.SR were generated. Approximately 50% of colonies
isolated using neomycin/G418 as a selection marker expressed
I.kappa.B.alpha.SR. Most of these clones lost I.kappa.B.alpha.SR
expression after continuous propagation in culture. No clones were
obtained that were completely devoid of constitutive NF-.kappa.B
DNA binding activity.
[0061] Three clones expressing I.kappa.B.alpha.SR
(I.kappa.B.alpha.SR6, 8 and 10) and a clone containing retrovirus
vector alone (LxSN11) were used for further studies. Constitutive
NF-.kappa.B DNA activity in these clones was measured by EMSA using
the general transcription factor SP-1 as an internal control.
I.kappa.B.alpha.SR6, I.kappa.B.alpha.SR8 and I.kappa.B.alpha.SR10
cells displayed 20, 10 and 40% lower NF-.kappa.B DNA binding
activity, respectively, than LxSN11 cells.
[0062] Similar results were obtained when EMSA was performed with
nuclear extracts instead of whole cell extracts. Oligonucleotide
competition studies and antibody supershift assays confirmed that
I.kappa.B.alpha.SR inhibited DNA binding of p50:p65 heterodimeric
complex of NF-.kappa.B. RNase protection assay revealed reduced
TRAF1 and c-IAP2 expression in I.kappa.B.alpha.SR cells compared to
LxSN11 cells. Furthermore, Mn-SOD and DAD-1, but neither c-IAP1 nor
survivin, expression was reduced in I.kappa.B.alpha.SR cells
compared to LxSN11 cells.
EXAMPLE 3
I.kappa.B.alpha.SR Cells Are More Sensitive to Paclitaxel than LxSN
Cells
[0063] Apoptosis by chemotherapeutic agents including paclitaxel
involves activation of caspase 9 and caspase 3 (Thomberry &
Lazebnik, 1998). Anti-apoptotic function of NF-.kappa.B is mostly
due to Mn-SOD and c-IAP2 mediated inhibition of caspase 9
activation (Deveraux & Reed, 1999; Green & Reed, 1998).
Also c-IAP2 inhibits the activity of caspase 3 (Deveraux &
Reed, 1999). Recent studies have indicated that NF-.kappa.B alters
cell cycle progression by modulating the expression of cell cycle
regulatory genes (Guttridge et al., 1999; Hinz et al., 1999). Based
on these observations, constitutively active NF-.kappa.B may
decrease the sensitivity of cancer cells to chemotherapeutic agents
whose activity is cell cycle-dependent.
[0064] After an initial survey of various chemotherapeutic drugs,
paclitaxel was chosen for further study because of a consistent
difference in response of LxSN11 and I.kappa.B.alpha.SR cells to
this drug. Paclitaxel is a microtubule-stabilizing agent whose
action is concentration dependent (Torres & Horwitz, 1998). At
<9 nM drug concentration, paclitaxel acts by retarding or
inhibiting progression through mitosis, thus altering microtubule
dynamics. At these concentrations, cells exit mitosis aberrantly
and fractionate into hypodiploid populations during cell cycle
analysis (Torres & Horwitz, 1998). At >9 nM drug
concentration, paclitaxel increases microtubule polymer mass,
terminal G2/M arrest and cell death with a concomitant decrease in
hypodiploid cells (Torres & Horwitz, 1998). At 3 nM paclitaxel
concentration, approximately 30% of all cell types were
hypodiploid. Hypodiploid population from all three cell types
formed similar numbers of colonies when grown in culture suggesting
that hypodiploid population not always represent apoptotic cells
(data not shown). Increasing paclitaxel concentration to 5 nM did
not alter the cell cycle distribution pattern of LxSN11 cells. In
contrast, a large percent of I.kappa.B.alpha.SR cells were arrested
at G2/M phase of the cell cycle. The percentage of cells at G2/M
were 28.45.+-.4.05, 70.37.+-.14.9 and 62.1.+-.13.1% for LxSN11,
I.kappa.B.alpha.SR6 and I.kappa.B.alpha.SR10 cells, respectively.
Only diploid cells were considered while calculating the percentage
of cells in different phases of the cell cycle. These results
suggest that genes activated by NF-.kappa.B reduce the ability of
paclitaxel to induce G2/M arrest.
[0065] It was demonstrated previously that rates of
paclitaxel-induced apoptosis directly correlate with number of G2/M
arrested cells rather than number of hypodiploid cells (Torres
& Horwitz, 1998). To determine whether increased G2/M arrest of
I.kappa.B.alpha.SR cells is accompanied by increased apoptosis when
compared to LxSN11 cells, a "cell death" ELISA was carried out
(Kumar et al., 1996). To avoid loss of hypodiploid or other damaged
cells during processing, the assay was performed with cell culture
supernatants. After 18 h of paclitaxel treatment, there was only a
marginal increase in apoptosis of LxSN11 cells, although a
considerable number of cells were hypodiploid (FIG. 1). In
contrast, a substantial increase in apoptotic death of
I.kappa.B.alpha.SR 0 cells was observed after paclitaxel treatment.
Similar results were obtained when cells were incubated for 48 h
with paclitaxel (FIG. 1). Note that there is an increased rate of
spontaneous apoptosis in I.kappa.B.alpha.SR10 cells compared to
LxSN11 cells, which further suggests that NF-.kappa.B activity is
required for survival of MDA-MB-231 cells. Taken together, these
results indicate that breast cancer cells with constitutively
active NF-.kappa.B require a higher concentration of paclitaxel for
G2/M arrest and possibly for apoptosis.
[0066] To determine whether lack of NF-.kappa.B in normal cells
leads to altered sensitivity to paclitaxel, we compared the cell
cycle distributions of paclitaxel-treated fibroblasts derived from
p65-/- embryos with type littermate mouse embryos. Interestingly,
paclitaxel had no effect on the cell cycle distribution of both
wild type and p65-/- fibroblasts suggesting that paclitaxel-induced
G2/M arrest is restricted to cancer cells (Table 2).
2TABLE 2 The effect of paclitaxel on cell cycle progression of wild
type and p65-/- fibroblasts. Cells were treated with indicated
concentration of paclitaxel and cell cycle distribution was
measured after 18 h of treatment. Consistent with this possibility,
paclitaxel caused G2/M arrest of several other breast cancer cell
lines (data not shown). Wild type p65-/- Paclitaxel G0/G1 S G2/M
G0/G1 S G2/M -- 69 .+-. 2 12 .+-. 5 19 .+-. 3 47 .+-. 1 49 .+-. 1 4
.+-. 2 1 nM 68 .+-. 4 20 .+-. 4 17 .+-. 8 47 .+-. 2 53 .+-. 1 -- 3
nM 70 .+-. 3 16 .+-. 1 14 .+-. 5 39 .+-. 6 61 .+-. 7 1 5 nM 71 .+-.
3 16 .+-. 4 17 .+-. 6 47 .+-. 2 46 .+-. 6 14 10 nM 73 .+-. 2 15
.+-. 2 16 .+-. 6 47 .+-. 2 48 .+-. 6 6 .+-. 4
EXAMPLE 4
Parthenolide Inhibits NF-.kappa.B DNA Binding Activity and
Increases the Sensitivity of Breast Cancer Cells to Paclitaxel
[0067] A. Curcumin, N-acetyl cysteine, pentoxyphylline,
parthenolide, epigallocatechin gallate, Bay 11-7085 or MG-132 were
evaluated for their ability to inhibit NF-.kappa.B DNA binding
activity in breast cancer cells (Biswas et al., 1993; Hehner et
al., 1998; Kumar et al., 1998; Lin et al., 1998; Pierce et al.,
1997; Yang et al., 1998). Only parthenolide, MG132 and Bay 11-7085
inhibited NF-.kappa.B DNA binding activity in MDA-MB-231 cells.
MDA-MB-231 cells were incubated with increasing concentrations (1,
2 and 5 .mu.M) of parthenolide for 3 h. Whole cell extracts or
nuclear extracts (5 .mu.M only) from untreated and treated cells
were subjected to EMSA with NF-.kappa.B or SP-1 probe. Parthenolide
also inhibited constitutive NF-.kappa.B DNA binding activity in
HBL100 cells (data not shown).
[0068] The effect of parthenolide on NF-.kappa.B DNA binding
activity in cells treated with paclitaxel was then investigated.
MDA-MB-231 cells pretreated with 5 .mu.M parthenolide for 1 h were
exposed to 50 nM paclitaxel for 1 h. EMSA was performed with whole
cell extracts using NF-.kappa.B probe or SP-1 probe. Although
paclitaxel has been shown to induce NF-.kappa.B in other cell types
(Das & White, 1997), untreated and paclitaxel-treated
MDA-MB-231 cells displayed a similar level of NF-.kappa.B DNA
binding activity. Nonetheless, parthenolide inhibited NF-.kappa.B
DNA binding activity in paclitaxel-treated cells.
[0069] Total RNA from MDA-MB-231 cells treated with 5 .mu.M
parthenolide for 0, 3, 6 or 24 hrs was subjected to Northern
blotting and probed with Mn-SOD probe. Inhibition of NF-.kappa.B
DNA binding activity by parthenolide also correlated with reduced
Mn-SOD expression in MDA-MB-231 cells.
[0070] B. MDA-MB-231 and HBL100 cells were exposed to increasing
concentrations of either paclitaxel or parthenolide and cell
survival was measured after 18 h by an MTS assay. Half-maximal
growth-inhibitory concentration (IC.sub.50) was reached at 10 nM
and 0.8 .mu.M for paclitaxel and parthenolide, respectively, in
HBL100 cells (data not shown). IC.sub.50 of >30 nM and 2 .mu.M
for paclitaxel and parthenolide, respectively, was obtained for
MDA-MB-231 cells (data not shown). For unknown reasons, only 30% of
MDA-MB-231 cells were killed when incubated with 30 nM or higher
concentrations of paclitaxel.
[0071] The effect of a combination of 1 nM paclitaxel and
increasing concentrations of parthenolide was then studied.
Parthenolide was added 4 h prior to paclitaxel addition. Cell death
was measured 18 h after paclitaxel addition by MTS assay. Percent
cell survival (average .+-. standard deviation from three or more
experiments) is shown in FIG. 2. The IC.sub.50 of parthenolide
decreased to <0.1 .mu.M and approximately 0.8 .mu.M for HBL100
and MDA-MB-231 cells, respectively (FIG. 2). As per the Isobole
method, these results translate into >3.5 fold synergism with
drug combination for HBL100 cells. The effect of combination
therapy was more than additive for MDA-MB-231 cells.
[0072] C. To investigate whether the simultaneous exposure to
paclitaxel and parthenolide leads to G2/M arrest and apoptosis, a
cell cycle distribution analysis and "cell death ELISA" of
untreated and treated HBL100 cells was carried out. Parthenolide
had no effect on the cell cycle distribution of HBL100 cells (data
not shown). Cells treated with either paclitaxel alone or in
combination with parthenolide were arrested at G2/M, although a
synergistic effect of drug combination on G2/M arrest was observed
in only some experiments (data not shown). HBL100 cells were
treated with indicated drugs and cell death was measured by ELISA
after 18 h of paclitaxel addition. In combination therapy,
parthenolide was added 4 h before paclitaxel treatment. Apoptosis
in paclitaxel treated cells was set as 10 units and relative
apoptosis in cells treated with either parthenolide alone or both
paclitaxel and parthenolide is shown. Paclitaxel alone induced
apoptosis to a certain degree whereas parthenolide was ineffective
(FIG. 3A). A synergistic increase in apoptosis was observed when
cells were exposed to a combination of parthenolide and paclitaxel
(FIG. 3A).
[0073] To further confirm that combination therapy induces
apoptosis, the cleavage of PARP from 116 kDa to 85 kDa protein by
caspases was used (Panvichian et al., 1998). Cells were lysed 24 h
after paclitaxel addition, and the lysate was examined for
uncleaved (p 116) and cleaved PARP (p85) by Western blotting (FIG.
3B). No PARP cleavage product was observed when 5.times.10.sup.5
HBL100 cells were incubated with either paclitaxel (1 nM and 5 nM)
or parthenolide (0.1 .mu.M and 0.5 .mu.M) alone (FIG. 3B). In
contrast, PARP cleavage product was detected when cells were
treated with a combination of 5 nM paclitaxel and 0.5 .mu.M
parthenolide. Note that PARP cleavage was not detected in cells
treated with low concentrations of paclitaxel and parthenolide,
although cell death at these concentrations was measurable in MTS
assays. This discrepancy is most likely due to differences in the
sensitivity of the two assays. Nevertheless, these results suggest
that inhibition of NF-.kappa.B activity by parthenolide increases
the rate of apoptotic cell death by paclitaxel.
[0074] As demonstrated by Examples 1-4, constitutive NF-.kappa.B
DNA binding activity in breast cancer cells correlates with
increased expression of the anti-apoptotic genes c-IAP2 and Mn-SOD.
Also, constitutive NF-.kappa.B DNA binding has been shown to
correlate with increased expression of the pro-metastatic genes
urokinase plasminogen activator, IL-6 and IL-8 (Newton et al.,
1999). These observations are significant because constitutive
NF-.kappa.B DNA binding has been observed in 65% of primary breast
cancers (Sovak et al., 1997).
[0075] These examples demonstrated that c-IAP2 is a major
NF-.kappa.B inducible gene in breast cancer cells. Preliminary
evidence obtained using reverse transcriptase-polymerase chain
reaction analysis of tumor RNA indicated that c-IAP2 is
overexpressed in primary breast cancers. c-IAP2 is a more potent
inhibitor of caspase 3 and caspase 7 activity than c-IAP1, and can
suppress apoptosis induced by a variety of stimuli including TNF,
Fas, menadione, staurosporine, etoposide, paclitaxel and growth
factor withdrawal (Deveraux & Reed, 1999).
[0076] Previously, it was reported that reduced expression of the
pro-apoptotic gene Bax is responsible for chemoresistance in breast
cancer (Bargou et al., 1996). Bax activates caspase-dependent and
caspase-independent apoptotic pathways (Xiang et al., 1996).
Constitutively active NF-.kappa.B through c-IAP2 may confer
chemoresistance even in tumors that express Bax, because c-IAP2 can
block the caspase-dependent apoptotic pathway (Deveraux & Reed,
1999).
[0077] Paclitaxel is a commonly used chemotherapeutic agent in both
the adjuvant and metastatic settings. Thus, c-IAP2 expression can
provide a predictor of response to this important agent and its
analogs.
[0078] Mn-SOD appears to have a dual role in cancer. Mn-SOD can
reduce oxidative stress, protect against DNA damage and prevent
initiation of cancerous mutation (Oberley & Oberley, 1997).
Consistent with this possibility, the incidence of breast cancer is
higher in premenopausal women who have inherited the polymorphic
variant of Mn-SOD with reduced biological activity (Ambrosone et
al., 1999). However, Mn-SOD may also protect cancer cells from
chemotherapy induced oxidative stress and apoptosis (Manna et al.,
1998). Indeed, overexpression of Mn-SOD alone is sufficient to
confer resistance to okadaic acid, H.sub.2O.sub.2, and paclitaxel
but not vincristine, vinblastine and daunomycin induced apoptosis
of breast cancer cells (Manna et al., 1998). These observations
raise the possibility that NF-.kappa.B may also have a dual role in
mammary epithelial cells. By upregulating Mn-SOD and other
anti-oxidant genes, NF-.kappa.B may protect normal mammary
epithelial cells from oxidative stress and DNA damage. In cancer
cells, however, these anti-oxidant gene products may protect
against chemotherapy induced oxidative stress and apoptosis.
[0079] It was reported recently that caspases active during
apoptosis cleave NF-.kappa.B and attenuate anti-apoptotic response
(Levkau et al., 1998). Therefore, although several chemotherapeutic
agents including paclitaxel induce NF-.kappa.B (Das & White,
1997), it is less likely that activated NF-.kappa.B can mount an
anti-apoptotic response. However, NF-.kappa.B can protect against
chemotherapeutic agents if it is constitutively active and, as a
consequence, cells constitutively express Mn-SOD and c-IAP2.
Therefore, I.kappa.B.alpha.SR or NF-.kappa.B inhibitors may be
useful in overcoming chemotherapeutic resistance of only those
cells that contain constitutively active NF-.kappa.B. Consistent
with this possibility, I.kappa.B.alpha.SR overexpression in HPB,
HCT116, MCF-7, and OVCAR-3 cells, all of which lack constitutively
active NF-.kappa.B, did not increase the sensitivity to paclitaxel
(Bentires-Aji et al., 1999).
[0080] A survey of a number of known inhibitors of NF-.kappa.B
revealed that parthenolide can function similarly to
I.kappa.B.alpha.SR by increasing the sensitivity of breast cancer
cells to paclitaxel. However, the degree of synergism appears to be
dependent on the cell type, as HBL100 cells were more sensitive to
the combined treatment than MDA-MB-231 cells. The cell
type-specific effect may be related to differences in the stability
of parthenolide within cells or the number of cell survival
pathways that are active in a particular cell type.
[0081] The NF-.kappa.B-mediated survival pathway is believed to be
the major cell survival pathway in HBL100 cells, whereas
NF-.kappa.B-independent survival pathways provide partial
protection to MDA-MB-231 cells. This possibility is supported by
other in vitro observations that MDA-MB-231 cells represent a more
"advanced cancer cell type" (with respect to growth in nude mice)
than HBL100 cells (Price et al., 1990; Sommers et al., 1994). In
cancer types that are dependent on NF-.kappa.B, a combination of
parthenolide and chemotherapeutic drugs may be beneficial. Such an
approach is less likely to be toxic to normal cells.
EXAMPLE 5
Parthenolide Inhibits Prostate Cancer Cell Proliferation
[0082] Two prostate cancer cell lines (hormone sensitive, LNCaP and
hormone-resistant PC-3) were cultured and treated with 0.5, 1.0,
2.5 and 5 .mu.M parthenolide. Mobility shift gel electrophoresis
assay was performed on the prostate cancer cells after treatment
with parthenolide for three hours. Proliferation assays were
performed using a 96 well plate with cell viability assesses
utilizing the MTS/PMS assay (see Example 6). Inhibition was
compared with a solvent control.
[0083] Constitutive NF-.kappa.B DNA binding was present in both
cell lines, but greater DNA binding was observed in the hormone
resistant cell line, PC-3, as shown in FIG. 4A. Parthenolide
induced a dose-dependent decrease in DNA binding as shown in FIG.
4B. There was no effect observed at 1 micromolar of parthenolide
and almost complete inhibition of DNA binding with 5 micromolar.
These results closely paralleled the observations from the
proliferation assays, as the IC.sub.50 for LNCaP and PC-3 was 3.9
.mu.M and 4.1 .mu.M respectively.
[0084] NF-.kappa.B is constitutively active in both a
hormone-sensitive and hormone-resistant prostate cancer cell line.
The greater binding in the hormone-resistant cell line suggests
that this transcription factor may be involved in the development
of hormone resistance. Inhibition of this transcription factor with
parthenolide results in an inhibition of prostate cancer cell
proliferation.
EXAMPLE 6
Anti-Angiogenic Activity of Parthenolide
[0085] A. Materials
[0086] Human umbilical venous endothelial cells (HUVECs)
(Clonetics, San Diego, Calif.) were cultured in EGM-2 media
(Clonetics, San Diego, Calif.) and harvested after having undergone
no more than five passages. Parthenolide powder (Sigma Chemical
Co., St. Louis, Mo.) was added at varying concentrations to a
proliferation and a capillary formation assay, electromobility gel
shift assay and in an in vivo assay. RhuMAb VEGF (provided by
Genentech, South San Francisco, Calif.) was used in the
electromobility gel shift assay. VEGF (Chemicon International,
Temecula, Calif.) and basic Fibroblast Growth Factor, bFGF (R&D
Systems, Minneapolis, Minn.) were added to the assays in doses that
have been shown in vitro to induce the maximal amount of HUVEC
endothelial cell proliferation. Thrombin, bovine fibrinogen and
aprotinin (all from Sigma Chemical Co., St. Louis, Mo.) were used
for the formation of a fibrin clot. Microcarrier beads consisting
of thin layer denatured collagen chemically coupled to a matrix of
cross-linked dextran (175 microns, Cytodex.TM.3, Amersham
Pharmacia, Biotech AB, Uppsale, Sweden) were employed as the base
for the capillary formation. This assay has been shown to induce
capillaries with identifiable lumens. The MTS/PMS system (Promega,
Madison, Wis.) was used to assess cell viability for the
proliferation assay.
[0087] B. Methods
[0088] 1. Proliferation
[0089] HUVECs were plated in a 96-well U-bottomed plate (Becton
Dickinson Labware, Franklin Lakes, N.J.) at a concentration of
10,000 cells per 50 microliters (.mu.L) of media and incubated in
5% CO.sub.2 at 37.degree. C. for 48 hours. Varying drug
concentrations in 50 .mu.L of media were added to the media and
this mixture was added to each well within one hour of the HUVECs
being seeded. The proliferation experiments were performed with and
without stimulation by the addition of VEGF (60 ng/mL) and bFGF (20
ng/mL). These factors in high doses were chosen to partially
simulate the tumor microenvironment. Colorimetric readings were
obtained using the MTS/PMS system and an ELISA plate reader. The
readings obtained for each concentration tested were from an
average of eight wells. Each experiment was expressed as a
percentage of the solvent control and completed at least three
times with consistent results. The results presented are an average
of at least three experiments.
[0090] 2. Endothelial Cell Capillary Formation
[0091] Two hundred milligrams of microcarrier beads suspended in
PBS were autoclaved and then added to HUVECs at a concentration of
30 HUVECs per microcarrier bead. Microcarrier beads and cells were
added to a siliconized petri dish and rocked at 37.degree. C. in 5%
CO.sub.2 for 48 hours. The HUVEC coated microcarrier beads were
transferred to a fibrin clot solution. Fibrinogen was dissolved at
2.5 mg/mL in PBS with 0.15 U/mL of aprotinin. Approximately 20
HUVEC coated microcarrier beads were added to each well of a 12
well plate and then thrombin (0.625 U/mL) was added to form a
gelatinous clot. Media (1.5 mLs) with 1% human serum and aprotinin
(0.15 U/mL) were added to the top of each clot. The addition of
VEGF (60 ng/mL) and bFGF (20 ng/mL) was required to ensure robust
capillary formation. There was minimal capillary formation without
stimulation and therefore all results reported are with
stimulation. The drugs to be tested were also added to the top
layer. Capillary formation was then quantified after four days:
every capillary greater than the radius of the bead was scored and
the average number of tubules for each bead per well was
determined. The results were expressed as a fraction of the
positive control. The experiments were repeated at least three
times and the results presented are the average of at least three
experiments.
[0092] 3. Electromobility Gel Shift Assay
[0093] HUVECs were plated on 100 mm plates and harvested in an
exponential growth phase. Drugs, antibodies and cytokines were
added three hours prior to harvesting--bFGF alone at 50 ng/mL, VEGF
alone at 100 ng/mL, rhuMAb VGEF at 10 mg/mL, bFGF plus rhuMAb VEGF;
VEGF plus rhuMAb VEGF and parthenolide at doses ranging from 2 to
10 .mu.M. Cellular extracts were made and incubated with a
radiolabelled NF-.kappa.B probe for 30 minutes at 25.degree. C.
This protein probe binds to the NF-.kappa.B DNA binding site in the
promoter region of the immunoglobulin gene. The mixture was then
electrophoresed. Specific NF-.kappa.B binding to DNA was identified
by the presence of a signal seen at autoradiography. The
protein-DNA complex was slower to migrate whereas unbound DNA and
protein migrated off the gel. The specificity of the drug
inhibiting NF-.kappa.B DNA binding was verified by the use of the
SP-1 probe as a control.
[0094] 4. Matrigel Plug Assay
[0095] Matrigel was prepared on ice and incubated with 100 ng/mL of
VEGF or 50 ng/mL of bFGF. Under light anaesthesia with isofluorane,
0.3 mL of matrigel was injected into the left and right flanks of
each mouse. The VEGF containing matrigel plug was placed on the
left and the bFGF plug was placed on the right. On the second day
after the plugs were inserted, the mice were treated daily for 14
days by oral gavage. Parthenolide was dissolved in 100% alcohol at
40 mg/mL and then diluted in PBS so that each mouse received their
assigned dose in 100 .mu.L of solution. There were 10 mice per
cohort and the treatments were (1) 20% alcohol in PBS--control; (2)
0.4 mg/mL; (3) 4.0 mg/mL and 40 mg/mL. These doses are non-toxic
when given for one month (no weight loss, no lethargy). The mice
were sacrificed and the plugs harvested on the 14.sup.th day. The
hemoglobin content was measured using the "Drabkins assay". The
weight of each plug was determined and the plugs were dissolved in
100 ml of water per mg of matrigel plug. The mixture was placed in
duplicate in a 96 well plate and incubated for 24 hours. The
optical density was determined and the average for each pair was
determined. The optical density is a measure of the hemoglobin
content and the median for each cohort was determined with the
standard error also calculated.
[0096] C. Results
[0097] 1. In Vitro
[0098] Parthenolide inhibited HUVEC proliferation. This inhibition
was not substantially altered by stimulation with survival
factors--IC.sub.50 was 7.5 .mu.M with unstimulated media (FIG. 5)
and 8.6 .mu.M with stimulated media. Capillary formation was
inhibited with a lower IC.sub.50 of 3.2 .mu.M (FIG. 6).
[0099] 2. In Vivo
[0100] The matrigel plug assay revealed that parthenolide had an
effect on in vivo angiogenesis. There was a 40% reduction in
bFGF-induced angiogenesis at the 0.4 mg/mL dose which was not
increased by the higher doses (FIG. 7). In contrast, it required 40
mg/kg of parthenolide to inhibit VEGF-induced angiogenesis by
40%.
[0101] 3. Electromobility Gel Shift Assay
[0102] As depicted in FIG. 8, bFGF induced greater DNA binding than
VEGF. The bFGF effect was not altered by rhuMAb-VEGF. RhuMAb-VEGF
did not alter NF-.kappa.B DNA binding. When controlled for loading,
VEGF did not alter baseline NF-.kappa.B DNA binding. Parthenolide
was able to markedly decrease but not eliminate the NF-.kappa.B DNA
binding at baseline at 2 .mu.M and 4 .mu.M. At doses of 6 .mu.M and
higher, there was complete inhibition of DNA binding.
[0103] C. Discussion
[0104] Angiogenesis has been found to be a critical factor in many
physiological processes such as embryonic development and wound
healing as well as in pathological processes, which include the
neovascularity that can cause blindness in diabetic retinopathy and
the induction of a new blood supply to support the growth of
cancerous growth. Inhibition of the latter process by
anti-angiogenic agents has been shown to induce tumor regression.
One such agent is rhuMAb VEGF which can induce tumor regression in
20% of patients with breast cancer.
[0105] The data presented in this example demonstrates that
actively proliferating venous endothelial-cells have constitutive
NF-.kappa.B DNA binding. This inhibition of NF-.kappa.B DNA binding
by parthenolide coincides with inhibition of endothelial cell
proliferation, capillary formation and in vivo angiogenesis as
measured by hemoglobin content in a matrigel plug. Therefore,
parthenolide may be a potent anti-angiogenic agent for the
treatment of angiogenic-based diseases.
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