U.S. patent application number 11/467838 was filed with the patent office on 2006-12-28 for prophylactic and therapeutic treatment of the ductal epithelium of a mammary gland for cancer.
This patent application is currently assigned to JOHNS HOPKINS UNIVERSITY SCHOOL OF MEDICINE. Invention is credited to Saraswati Vaidyanathan Sukumar.
Application Number | 20060292113 11/467838 |
Document ID | / |
Family ID | 26704265 |
Filed Date | 2006-12-28 |
United States Patent
Application |
20060292113 |
Kind Code |
A1 |
Sukumar; Saraswati
Vaidyanathan |
December 28, 2006 |
PROPHYLACTIC AND THERAPEUTIC TREATMENT OF THE DUCTAL EPITHELIUM OF
A MAMMARY GLAND FOR CANCER
Abstract
The present invention provides prophylactic and therapeutic
methods of treating the ductal epithelium of an exocrine gland, in
particular a mammary gland, for disease, in particular cancer. The
methods comprise contacting the ductal epithelium of the exocrine
gland with an epithelium destroying agent, preferably by ductal
cannulation, so as to realize a prophylactic or therapeutic
effect.
Inventors: |
Sukumar; Saraswati
Vaidyanathan; (Columbia, MD) |
Correspondence
Address: |
BARNES & THORNBURG LLP
P.O. BOX 2786
CHICAGO
IL
60690-2786
US
|
Assignee: |
JOHNS HOPKINS UNIVERSITY SCHOOL OF
MEDICINE
100 North Charles Street 5th Floor
Baltimore
MD
|
Family ID: |
26704265 |
Appl. No.: |
11/467838 |
Filed: |
August 28, 2006 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11179322 |
Jul 12, 2005 |
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11467838 |
Aug 28, 2006 |
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09971901 |
Oct 5, 2001 |
6932777 |
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11179322 |
Jul 12, 2005 |
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09240206 |
Jan 29, 1999 |
6330472 |
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09971901 |
Oct 5, 2001 |
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09093145 |
Jun 8, 1998 |
6153184 |
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09240206 |
Jan 29, 1999 |
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08692001 |
Aug 2, 1996 |
5763415 |
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09093145 |
Jun 8, 1998 |
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60028929 |
Aug 3, 1995 |
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Current U.S.
Class: |
424/85.1 ;
514/109; 514/19.3; 514/19.4; 514/251; 514/263.3; 514/269; 514/27;
514/34; 514/410; 514/449; 514/49; 514/724; 604/500 |
Current CPC
Class: |
A61K 38/193 20130101;
A61K 31/7072 20130101; A61K 48/00 20130101; A61K 31/513 20130101;
A61K 38/193 20130101; A61K 2300/00 20130101; A61K 31/522 20130101;
C12N 2710/24132 20130101; A61K 2300/00 20130101; A61K 35/76
20130101; A61K 31/505 20130101; A61K 38/45 20130101; A61K 31/704
20130101; A61K 31/663 20130101; A61K 31/337 20130101; C12N
2710/10332 20130101; A61K 38/45 20130101; A61K 31/525 20130101 |
Class at
Publication: |
424/085.1 ;
514/002; 604/500; 514/049; 514/109; 514/251; 514/034; 514/269;
514/263.3; 514/724; 514/027; 514/449; 514/410 |
International
Class: |
A61K 38/19 20060101
A61K038/19; A61K 31/7072 20060101 A61K031/7072; A61K 31/704
20060101 A61K031/704; A61K 31/663 20060101 A61K031/663; A61K 31/525
20060101 A61K031/525; A61K 31/522 20060101 A61K031/522; A61K 31/337
20060101 A61K031/337; A61K 31/505 20060101 A61K031/505; A61K 31/513
20060101 A61K031/513 |
Goverment Interests
STATEMENT OF GOVERNMENT SUPPORT
[0002] The invention disclosed in this application was made with
government support under NIH planning grant P20 CA/ES66205,
"Gene-mediated prevention of Cancer," and grant NIH 1RO1 CA 57993,
"Genetic and hormonal factors in mammary carcinogenesis," awarded
by the National Institutes of Health, and with support of the
American Cancer Society under grant RD 388, "Targeted disruption of
breast cells: a novel strategy for cancer prevention." Therefore,
the government has certain rights in this invention.
Claims
1. A method for treating or preventing breast cancer, which method
comprises cannulating a duct of a mammary gland in need of
therapeutic or prophylactic treatment for breast cancer and locally
administering into the duct an epithelium-destroying agent in an
amount sufficient to destroy at least a portion of the ductal
epithelium, whereupon breast cancer is treated or prevented.
2. The method of claim 1, wherein the epithelium-destroying agent
selectively destroys cells of the ductal epithelium.
3. The method of claim 1, wherein the portion of the ductal
epithelium that is destroyed is up to about 70%.
4. The method of claim 1, wherein the duct is cannulated through a
nipple.
5. A method of treating or preventing breast cancer, which method
comprises identifying a duct in a nipple of a mammary gland in need
of therapeutic or prophylactic treatment, directly accessing cells
of the ductal epithelium of the duct by cannulating the duct
through the nipple, and locally administering into the duct an
epithelium-destroying agent in an amount sufficient to destroy
selectively at least a portion of the ductal epithelium
substantially without destroying other cells of the mammary gland,
whereupon breast cancer is treated or prevented.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application is a Continuation of application Ser. No.
11/179,322, filed Jul. 12, 2005, which is a Divisional of
application Ser. No. 09/971,901, filed Oct. 5, 2001 (now U.S. Pat.
No. 6,932,777), which is a Divisional of application Ser. No.
09/240,206, filed Jan. 29, 1999 (now U.S. Pat. No. 6,330,472),
which is a Continuation of application Ser. No. 09/093,145, filed
Jun. 8, 1998 (now U.S. Pat. No. 6,153,184), which is a Divisional
of application Ser. No. 08/692,001, filed Aug. 2, 1996 (now U.S.
Pat. No. 5,763,415), which claims benefit of application No.
60/028,929, filed Aug. 3, 1995, the entire contents of which are
incorporated herein by reference.
TECHNICAL FIELD OF THE INVENTION
[0003] The present invention relates to the use of an
epithelium-destroying agent to destroy the epithelium of an
exocrine gland, particularly the mammary gland, in the prophylactic
and therapeutic treatment of disease, in particular cancer.
BACKGROUND OF THE INVENTION
[0004] Exocrine glands are glands that release a secretion external
to or at the surface of an organ by means of a canal or duct.
Examples of exocrine glands include, among others, the mammary
glands, prostate, liver, gall bladder, pancreas, kidneys, sweat
glands, and salivary glands. Cancers of exocrine glands pose a
major health problem, frequently resulting in death. Currently,
cancers of the breast and prostate are among the leading causes of
death among women and men, respectively.
[0005] The mature human breast comprises from six to nine major
ducts, which emanate from the nipple, serially branch into ducts
and terminate in lobuloalveolar structures (Russo et al., Lab.
Invest. 62(3):244-278 (1990)). This branching network of ducts is
composed of epithelial cells in a supporting matrix of connective
tissue and endothelial cells.
[0006] Tissues removed from the human female breast during surgery
and autopsy have been examined in numerous studies directed to the
nature and site of origin of neoplastic growth. Subgross sampling
and histological confirmation have enabled pathological
characterization of entire breasts, leading to the postulation of
the existence of four major possible sites of origin of mammary
carcinomas, namely ducts, terminal ducts, ductules, and acini
(Russo et al., supra). Ductal origin is supported by the presence
of more extensive epithelial proliferations, which are presumed to
be preneoplastic, in surgically removed cancerous breasts as
compared to nonmalignant breasts removed during autopsies (Russo et
al., supra).
[0007] With a cumulative lifetime risk of a woman developing breast
cancer estimated to be 1 in 9, there is an urgent need to develop
therapeutic methods of treatment that are more effective, less
invasive and accompanied by fewer side effects and prophylactic
methods of treatment that are more effective than increased and
intensified physical monitoring and far less extreme than radical
mastectomy. In spite of the recent discovery of the heritable
breast cancer susceptibility loci, BRCA1 (Miki et al., Science
266:66-71 (1994)) and BRCA2, and other cancer susceptibility loci,
and the increasing ability of physicians to identify women with
elevated breast cancer risk, prophylactic methods are still
currently limited to physical monitoring and prophylactic
mastectomy.
[0008] In view of the above, it is an object of the present
invention to provide a method of locally treating an exocrine gland
prophylactically for disease. It is another object of the present
invention to provide a method of locally treating an exocrine
gland, in particular the mammary gland, prophylactically for
cancer. Another object of the present invention is to provide a
method of locally treating an exocrine gland therapeutically for
disease. Yet another object of the present invention is to provide
a method of locally treating a mammary gland therapeutically for
cancer. Still yet another object of the present invention is to
provide a method of locally treating a mammary gland both
therapeutically and prophylactically for cancer. These and other
objects and advantages, as well as additional inventive features,
will become apparent from the detailed description provided
herein.
SUMMARY OF THE INVENTION
[0009] The present invention provides prophylactic and therapeutic
methods of treating the ductal epithelium of an exocrine gland, in
particular the mammary gland, for disease, in particular cancer.
The method comprises contacting the ductal epithelium of the
exocrine gland, in particular the mammary gland, with an
epithelial-destroying agent. The ductal epithelium is preferably
contacted with the agent by ductal cannulation. The
epithelium-destroying agent is preferably a vector comprising a
thymidine kinase gene, which is used in combination with
ganciclovir (GCV), which can be systemically administered. Another
preferred epithelium-destroying agent is a vector comprising a
hypoxanthine phosphoribosyl transferase (HPRT) gene, which is used
in combination with hypoxanthine aminopterin thymidine (HAT)
nucleotide, which can be systemically administered. Also preferred
as an epithelium-destroying agent is a vector comprising a gene,
which, upon transformation of a cell of the ductal epithelium and
expression therein, induces apoptosis or death of the transformed
cell. A preferred apoptosis-inducing gene is bclxs. Other preferred
epithelial-destroying agents include a cytolytic virus, such as
Vaccinia virus, and ethanol. The preceding methods can additionally
comprise contacting the ductal epithelium with a cytokine or
hematopoietic growth factor, such as GM-CSF. Also provided by the
present invention is a method of treating the ductal epithelium of
a mammary gland both therapeutically and prophylactically for
cancer. The combined therapeutic/prophylactic method comprises
treating the mammary gland therapeutically by surgery, radiation
and/or chemotherapy and contacting the ductal epithelium of the
mammary gland, either concomitantly or subsequently, with an
epithelium-destroying agent, which does not specifically target
cancerous cells. The combined therapeutic/prophylactic method can
additionally comprise contacting the ductal epithelium with a
cytokine or hematopoietic growth factor, such as GM-CSF.
BRIEF DESCRIPTION OF THE FIGURES
[0010] FIG. 1 is a graph of the ratio of cell no. in the presence
of GCV over cell no. in the absence of GCV (Cell No.+GCV/Cell
No.-GCV) versus viral multiplicity of infection (viral MOI), which
shows the effect of the addition of 10 .mu.g/ml GCV on NMU68 and
RBA rat tumor cell lines 6 hrs after transduction with
adenoviral-Herpes simplex thymidine kinase (AdHS-tk) at titers of
0, 100, 500 and 1,000 moi. After the cells were maintained in the
presence of GCV for 3 days, they were counted using trypan blue
exclusion as a measure of cell viability and cell numbers were
normalized to the growth of cells in the absence of ganciclovir
(GCV).
DETAILED DESCRIPTION OF THE INVENTION
[0011] The present invention is based on the observation that the
overwhelming majority of breast cancers arise from epithelial
cells, particularly those epithelial cells which line the ducts of
the mammary gland and are collectively referred to as the ductal
epithelium. The present invention is also based on the exocrine
nature of the mammary gland. Given that the mammary gland is an
exocrine gland, it was further observed that the central canal or
duct could provide a means of directly accessing the ductal
epithelium for localized prophylactic and therapeutic treatment of
cancer. Based on these observations, the prophylactic and
therapeutic methods of the present invention were developed.
Prophylactic Method
[0012] The prophylactic method of the present invention is a method
of treating the ductal epithelium of an exocrine gland
prophylactically for a disease that affects the ductal epithelium
of the exocrine gland. The method comprises contacting, preferably
by ductal cannulation, the ductal epithelium of the exocrine gland
with an epithelium-destroying agent so as to destroy cells of the
ductal epithelium affected by the disease.
[0013] In one embodiment of the prophylactic method of the present
invention, the ductal epithelium of a mammary gland is treated
prophylactically for cancer so as to inhibit the formation of
cancer of ductal epithelial origin. The method comprises
contacting, preferably by ductal cannulation, the ductal epithelium
of the mammary gland with an epithelium-destroying agent. The agent
preferably is a vector comprising a thymidine kinase gene, such as
a Herpes simplex thymidine kinase gene, and ganciclovir, a vector
comprising a HPRT gene and HAT nucleotide, a cytolytic virus, such
as a Vaccinia virus, or ethanol. The method can additionally
comprise contacting the ductal epithelium with a cytokine or
hematopoietic growth factor, such as GM-CSF.
[0014] In another embodiment of the prophylactic method of the
present invention, the ductal epithelium of an exocrine gland, such
as a mammary gland, is treated prophylactically for cancer so as to
inhibit the formation of cancer of ductal epithelial origin. The
method comprises contacting, preferably by ductal cannulation, the
ductal epithelium of the exocrine gland with an
epithelium-destroying agent to destroy less than all of the ductal
epithelium so as to inhibit the formation of cancer of ductal
epithelial origin. Preferably, up to about 70%, 80%, 85%, 90% or
95% of the ductal epithelium is destroyed.
[0015] The epithelium-destroying agent is preferably a vector
comprising a thymidine kinase gene, such as that from Herpes
simplex, and ganciclovir, which can be brought into contact with
the ductal epithelium by any suitable means, preferably by
ductal-cannulation or by systemic administration, a vector
comprising a HPRT gene and HAT nucleotide, which can be brought
into contact with the ductal epithelium by any suitable means,
preferably by ductal cannulation or by systemic administration, a
vector comprising a gene, which upon transformation of a cell of
the ductal epithelium and expression therein, induces apoptosis or
death of the transformed cell, such as bclxs, ethanol, or a
cytolytic virus, such as Vaccinia virus.
[0016] The above method can additionally comprise the
administration of a cytokine or hematopoietic growth factor, such
as GM-CSF. The GM-CSF can be brought into contact with the ductal
epithelium of the mammary gland by any suitable means, such as by
ductal cannulation of GM-CSF or a vector comprising a gene encoding
GM-CSF, in which case the vector can be the same vector as the one
encoding the thymidine kinase, HPRT or apoptosis-inducing gene, or
it can be systemically administered.
[0017] This embodiment of the prophylactic method can be used to
treat any exocrine gland. However, it is particularly useful in the
treatment of the mammary gland.
[0018] The above-described prophylactic method of treating a
mammary gland is particularly useful in treating a mammary gland in
a mammal at risk for developing breast cancer. The mammary gland
can be characterized as one that has never had a tumor, one that
had a tumor previously but the tumor is no longer detectable due to
other prior therapeutic treatment, or one that has an incipient or
occult tumor, preneoplasia or ductal hyperplasia. Normally,
hyperplasias and incipient and occult tumors are not detectable by
means of physical examination or radiography. Accordingly, the
prophylactic method will find use in cases where there is reason to
take some prophylactic measures, such as when there are known
inherited factors predisposing to cancers, where there are
suspicious lesions present in a breast with the potential for
developing into a malignancy, where there has been exposure to
carcinogenic agents in the environment, where age predisposes to a
cancer, where cancer of another gland, e.g., the mammary gland of
the contralateral breast, suggests a propensity for developing
cancer, or where there is a fear or suspicion of metastasis.
Therapeutic Method
[0019] The therapeutic method of the present invention is a method
of treating the ductal epithelium of an exocrine gland
therapeutically for a disease that affects the ductal epithelium of
the exocrine gland. The method comprises contacting, preferably by
ductal cannulation, the ductal epithelium of the exocrine gland
with an epithelium-destroying agent so as to destroy cells of the
ductal epithelium affected by the disease.
[0020] In one embodiment of the therapeutic method of the present
invention, the ductal epithelium of a mammary gland is locally
treated therapeutically for cancer so as to destroy cancerous and
noncancerous cells of the ductal epithelium and inhibit the spread
of cancer. The method comprises contacting, preferably by ductal
cannulation, the ductal epithelium of the mammary gland with an
epithelium-destroying agent, which need not, and preferably does
not, specifically target cancerous cells. The agent preferably is a
vector comprising a thymidine kinase gene, such as a Herpes simplex
thymidine kinase gene, and ganciclovir, a vector comprising a HPRT
gene and HAT nucleotide, a cytolytic virus, such as a Vaccinia
virus, or ethanol. The method can additionally comprise contacting
the ductal epithelium with a cytokine or hematopoietic growth
factor, such as GM-CSF.
[0021] In the therapeutic method, the epithelial-destroying agent
should destroy all of the diseased or malignant epithelium. In
addition, the ductal epithelium immediately surrounding the
diseased/malignant epithelium also preferably should be
destroyed.
Combined Therapeutic/Prophylactic Method
[0022] The present invention also provides a method of treating the
ductal epithelium of a mammary gland both therapeutically and
prophylactically for cancer. The method comprises treating the
mammary gland therapeutically with any given therapeutic method,
such as those currently known and used in the art. Examples of such
methods include surgical removal of the cancerous tissue, radiation
therapy and chemotherapy. The method further comprises contacting,
either concomitantly with or subsequently to the therapeutic
treatment, the ductal epithelium of the mammary gland, e.g., by
ductal cannulation, with an epithelium-destroying agent, which
preferably does not specifically target cancerous cells, so as to
destroy any remaining cancerous cells and noncancerous cells and
inhibit the spread of cancer. The epithelium-destroying agent is
preferably a vector comprising a thymidine kinase gene, such as a
Herpes simplex thymidine kinase gene, combined with ganciclovir, a
vector comprising a HPRT gene combined with HAT nucleotide, a
cytolytic virus, such as a Vaccinia virus, or ethanol. The method
can additionally comprise contacting the ductal epithelium with a
cytokine or hematopoietic growth factor, such as GM-CSF.
Alternative Embodiments of Prophylactic & Therapeutic
Methods
[0023] Although preferred embodiments have been described above,
the methods of the present invention can be used to treat the
ductal epithelium of any exocrine gland. Examples of exocrine
glands, other than the mammary gland, which can be treated with the
present inventive methods include, among others, the prostate,
liver, gall bladder, pancreas, kidneys, sweat glands, and salivary
glands. The methods are especially useful in the prophylactic and
therapeutic treatment of the ductal epithelium of mammary
glands.
[0024] Similarly, the methods can be used to treat an exocrine
gland for any disease that affects the exocrine ductal epithelium.
The methods are particularly useful in the treatment of cancer,
including the stages of hyperplasia, adenoma, carcinoma in situ,
and carcinoma, of ductal epithelial origin.
[0025] Any method can be used to destroy the ductal epithelium. It
is preferred, however, that the destruction is limited to the
ductal epithelium or a part thereof.
[0026] Any method of contacting the ductal epithelium can be used
to effect local treatment. Preferably, ductal cannulation is used.
Although any duct or lobule can be cannulated, it is preferred that
the central canal or duct be cannulated. Ductal cannulation also
enables direct injection of a tumor mass, if desired.
[0027] Any epithelium-destroying agent can be used to destroy the
ductal epithelium of an exocrine gland. The agent preferably should
not destroy cells other than cells of the ductal epithelium and
preferably should not result in side effects, the adversity of
which outweigh the benefits of destruction of the ductal
epithelium. In no event should the methods be used to destroy
completely the ductal epithelium of an exocrine gland in the
prophylactic/therapeutic treatment of a given disease, wherein the
complete destruction of the ductal epithelium, in and of itself,
would result in death of the mammal so treated.
[0028] Examples of agents that can be used in the context of the
prophylactic and therapeutic methods of the present invention
include cytotoxic agents. Any cytotoxic agent known in the art and
suitable for contacting the ductal epithelium of an exocrine gland
of a mammal can be used. In addition to ethanol and GCV described
above, other examples of cytotoxic agents and their prodrugs
include genistein, okadaic acid,
1-.beta.-D-arabinofuranosyl-cytosine,
arabinofuranosyl-5-azacytosine, cisplatin, carboplatin, actinomycin
D, asparaginase, bis-chloro-ethyl-nitroso-urea, bleomycin,
chlorambucil, cyclohexyl-chloro-ethyl-nitroso-urea, cytosine
arabinoside, daunomycin, etoposide, hydroxyurea, melphalan,
mercaptopurine, mitomycin C, nitrogen mustard, procarbazine,
teniposide, thioguanine, thiotepa, vincristine, 5-fluorouracil,
5-fluorocytosine, adriamycin, cyclophosphamide, methotrexate,
vinblastine, doxorubicin, leucovorin, taxol, anti-estrogen agents
such as tamoxifen, intracellular antibodies against oncogenes, the
flavonol quercetin, Guan-mu-tong extract, retinoids such as
fenretinide, nontoxic retinoid analogues such as
N-(4-hydroxyphenyl)-retinamide (HPR), and monoterpenes such as
limonene, perillyl alcohol and sobrerol. Preferably, the agent is
locally administered, especially if administration of the agent is
accompanied by toxic side effects. Otherwise, the agent can be
administered by any suitable route, such as systemic
administration.
[0029] A cytolytic virus also can be used as an agent. Any
cytolytic virus can be used as long as the organism mounts a rapid
immunological response to it such that the virus cannot cause
disease if it escapes the ductal epithelium. Examples of cytolytic
viruses include Vaccinia viruses and Sindbis viruses, which can
also be used as vectors. Preferably, a Vaccinia virus is used. Due
to lack of mucosal immunity, Vaccinia infectious particles enter
and lyse the breast epithelial cells, yet stromal immunity destroys
the particles as soon as they leave the ductile tree of the
exocrine gland, thereby preventing cytolysis beyond the ductal
epithelium. The advantages of Vaccinia administration are that it
eliminates the need for high titer virus, the need to induce cell
division in the breast, and the need to administer a drug to effect
cell death.
[0030] A vector comprising a suicide gene also can be used as an
agent, in conjunction with an agent that destroys the ductal
epithelium of an exocrine gland. The vector comprising a suicide
gene, upon transformation of a cell of the ductal epithelium and
expression therein, renders the transformed cell sensitive to the
epithelium-destroying agent, increases the sensitivity of the
transformed cell to the agent, converts the agent from a prodrug to
an active drug, activates the conversion of the agent from a
prodrug to an active drug, enhances the effect of the agent or,
itself, produces a protein that is cytotoxic. A preferred suicide
gene for use in the present inventive methods is the one described
above, i.e., a thymidine kinase, such as the one from Herpes
simplex, which phosphorylates GCV, which, in turn, inhibits DNA
replication. Another example of a suicide gene is cytosine
deaminase, which is used in conjunction with 5-fluorocytosine. If
the vector comprising the suicide gene is administered locally to
the ducts, the cytotoxic agent or precursor can be administered
systemically, since only transfected cells will be affected. In
this regard, the bystander effect, i.e., the death of neighboring
uninfected cells, presumably due to transfer of toxic byproducts
through gap junctions between cells in the same compartment,
obviates the need for every cell in the ductal epithelium, which is
to be destroyed, to be infected. However, sufficient time must be
allowed between contacting the ductal epithelium with the suicide
gene and the prodrug, for example, to achieve efficient killing of
the breast epithelial cells.
[0031] A vector comprising an apoptosis-inducing gene also can be
used as an agent that destroys the ductal epithelium of an exocrine
gland (Vaux, Cell 76:777-779 (1994)). Examples of
apoptosis-inducing genes include ced genes, myc genes
(overexpressed), the bclxs gene, the bax gene, and the bak gene.
The apoptosis-inducing gene causes death of transfected cells,
i.e., by inducing programmed cell death. For example, the bclxs
gene, bax gene, or bak gene can be used to inhibit bcl-2 or
bcl-x.sub.L, leading to apoptosis. Where necessary, a vector
comprising an apoptosis-inducing gene can be used in combination
with an agent that inactivates apoptosis inhibitors such as bcl-z,
p35, IAP, NAIP, DAD1 and A20 proteins.
[0032] Suicide and apopotosis genes can be administered by way of a
viral vector, such as an adenoviral or retroviral vector.
Adenoviral vectors enable the generation of high titer recombinant
viruses (10.sup.11/ml) and the efficient transduction of
postmitotic cells because adenoviral DNA exists as an episome in
the nucleus (Verma, Molecular Medicine 1:2-3 (1994)).
[0033] According to one preferred embodiment, the gene can be under
the transcriptional regulation of a Rous sarcoma viral promoter.
Alternatively, it can be under the control of an epithelial
tissue-specific or cell-specific promoter.
[0034] Uptake of recombinant virus can be facilitated by
pretreatment or simultaneous treatment with polybrene or, for
example, in the case of a retrovirus, attachment of the functional
fragment of an antibody to the viral particle.
[0035] In another embodiment, the apoptosis gene or suicide gene
can be present in a recombinant microorganism, which will express
the gene. One particularly preferred microorganism for this purpose
is the bacterium Listeria monocytogenes.
[0036] Other methods known in the art for introduction of raw DNA
into cells can be used in the methods of the present invention.
Alternatively, liposomes, complexes between polypeptide ligands for
receptors on mammary ductal epithelial cells, including complexes
of antibodies and functional fragments thereof, and plasmids can be
used (Mulligan, Science 260:926-931 (1993)). Epithelial
cell-specific promoters, such as whey acidic protein (wap), can be
used to target expression of a given gene, e.g., a suicide gene, in
ductal epithelial cells. Use can also be made of wild-type tumor
suppressor genes, such as p53 or Mcs-1 (rat), homeobox genes
expressed in normal cells but not in cancerous cells, and the
maspin gene.
[0037] Additionally, the ductal epithelium can be contacted with an
agent to effect the scavenging of epithelial cells destroyed in
accordance with the present invention, e.g., a cytokine/growth
factor. Suitable cytokines/growth factors include GM-CSF, G-CSF,
IL-2, IL-4, IL-6, IL-7, hCG, TNF-.alpha., INF-.alpha. and
INF-.gamma.. Such factors can be contacted with the ductal
epithelium directly or by expression of a vector comprising a gene
encoding the factor, in which case the vector can be the same one
that comprises a suicide gene, for example. The factors stimulate a
potent, long-lasting, and specific cell immunity, requiring both
CD4 and CD8 cells. The immune response is designed to scavenge
destroyed ductal epithelial cells by generating autoimmunity
towards epithelial cell antigens.
[0038] The ductal epithelium is preferably contacted with the agent
by introduction of the agent through the central canal or duct of
the exocrine ductal epithelium, such as by ductal cannulation.
However, in the case of the mammary gland, for example, there are
6-9 major ducts that emanate from the nipple and serially branch
into other ducts, terminating in lobulo-alveolar structures (Russo
et al. (1990), supra). Accordingly, in some circumstances, such as
those in which even more localized treatment is necessary or
desired, for example, by the choice of anti-cancer agent, it may be
preferable to contact the ductal epithelium of the exocrine gland
through one of the other ducts or through a lobulo-alveolar
structure as opposed to the central canal or duct. In this regard,
ductal cannulation enables intratumoral injection.
[0039] The methods of the present invention can be combined with
other methods of prophylactic and therapeutic treatment in addition
to those cited above, such as methods that target destruction of
cancer cells, e.g., by targeting of cell-surface markers, receptor
ligands, e.g., ligands to gastrin-releasing peptide-like receptors,
tumor-associated antigens, e.g., the 57 kD cytokeratin or the
antigen recognized by the monoclonal antibody GB24, the
extracellular matrix glycoprotein tamascin, antisense oncogenes
such as c-fos, homeobox genes that are expressed in cancer cells
but not normal cells, tumor-infiltrating lymphocytes that express
cytokines, RGD-containing peptides and proteins, which are
administered following surgery, lipophilic drug-containing
liposomes to which are covalently conjugated monoclonal antibodies
for targeting to cancer cells, low fat diet, moderate physical
exercise and hormonal modulation. For prostate cancer,
anti-testosterone agents can be used as well as an inhibitor of
cell proliferation produced by prostatic stromal cells and CCAM, an
epithelial cell adhesion molecule.
[0040] The following examples are presented to illustrate the
present invention, not to limit its scope. The examples make use of
the rat mammary tumor model, which has been deemed an appropriate
experimental model for understanding breast cancer in humans
(Sukumar et al., Mutation Res. 333 (1-2):37-44 (1995); Russo et
al., supra). In fact, 90-100% of female rats develop mammary tumors
in this model when they are administered the carcinogen NMU at 55
days of age (Sukumar, Cancer Cells 4:199-204 (1990)).
EXAMPLES
Example 1
[0041] This example demonstrates the successful delivery of virus
and other agents into the mammary ductile tree by a single
injection through the teat.
[0042] ADV/CMV-.beta.-gal (from Dr. William Burns, Johns Hopkins
University) is an adenoviral 5 vector constructed with a
13-galactosidase gene controlled by a cytomegaloviral promoter. It
was delivered into the mammary gland by injection of a viral
suspension in 20 .mu.l of 0.2% trypan blue in Tris buffer through
the teat of a rat. The nipple was extruded, and the sphincter
removed by excising the nipple. In the rat, the muscle prevents
fluid from regurgitating into the breast and had to be excised in
order to visualize the ductal opening and administer the agent.
Trypan blue was used as a tracking dye to ensure correct delivery
to the ductile tree. Injection about 30 days postpartum resulted in
the mammary epithelial tree being clearly visible. Ethyl alcohol
(70%) was also successfully delivered by a single injection through
the teat.
Example 2
[0043] This example demonstrates that adenovirus can efficiently
transduce human mammary epithelial cells in vitro.
[0044] ADV/CMV-.beta.-gal was used to transduce HBLIOO mammary
epithelial cells in vitro. The .beta.-gal enzyme in this construct
contains a nuclear localization signal and results in dense nuclear
staining .HBL100 cells (10.sup.2) were plated in 24-well plates,
and transduced with virus at various doses and stained with X-gal
48 hrs later. Essentially all cells were infected at a
moi=10.sup.4.
[0045] This experiment also has been performed in human mammary
tumor cells MCF-7 (American Type Culture Collection (ATCC),
Rockville, Md.), human mammary epithelial cells MCF-10A (ATCC), and
two rat mammary cancer cell lines, RBA (from Dr. Leonard Cohen) and
37-2 (from Dr. C. Marcelo Aldaz) with the same results. More
efficient adenoviral constructs have been used, thereby achieving
100% infection at a moi=10.sup.3. These experiments demonstrate
successful infection by and expression of adenovirus carrying the
lacZ indicator gene, which permits staining the cells blue by the
expression of the enzyme .beta.-galactosidase.
Example 3
[0046] This example demonstrates that infection with an AdHS-tk
construct followed by GCV treatment effectively kills mammary tumor
cells in vitro.
[0047] RBA and NMU68 are two rat mammary tumor cell lines derived
from a DMBA- and a NMU-induced tumor, respectively
[DMBA=dimethylbenz [a] anthracene, NMU=N'-nitro N'-methylurea].
Each cell line was plated at a density of 5.times.10.sup.2 in
48-well plates (1.1 cm) and allowed to settle overnight. The next
morning, they were transduced with AdHS-tk (Chen et al., PNAS (USA)
91:3054-57 (1994); obtained from S. Woo and E. Aguilar-Cordova,
Baylor College of Medicine, Houston, Tex.) at titers of 0, 100,
500, and 1000 moi, and then, 6 hrs later, GCV (10 pg/ml) was added
to the culture media. The cells were maintained in the presence of
GCV for 3 days and then counted using trypan blue exclusion as a
measure of cell viability. The cell numbers were normalized to the
growth of cells in the absence of GCV. The results are shown in
FIG. 1. More than 80% of the cells of each cell line were killed at
a moi of 10.sup.3.
Example 4
[0048] This example demonstrates the prophylactic effect of the
method of the present invention.
[0049] The mammary glands (6 on each side) of six virgin 50 day old
Sprague Dawley rats were injected with AdHS-tk on the left side and
trypan blue on the right side or left untreated. One rat remained
completely untreated with the virus or GCV and served as a positive
control for NMU-induced tumorigenesis. On the day of surgery, rats
were given an intramuscular injection of 5 .mu.g estradiol valerate
and were anesthetized with an isofluorane/O.sub.2 mixture. The
nipples were cannulated with a 33 gauge needle. Twenty Al of
AdHS-tk diluted in trypan blue carrier (1 mM MgCl.sub.2, 20
.mu.g/ml polybrene in 10% glycerol, 0.4% trypan blue in saline) at
a concentration of 5.times.10.sup.7 particles/.mu.l were injected
into the duct. Carrier control mammary glands received 20 .mu.l of
trypan blue carrier alone. An animal was considered treated when at
least three glands were successfully injected with trypan blue and
another three glands were successfully injected with AdHS-tk. The
remaining glands were left untreated. Twelve hours later, the rats
were injected with 125 mg/kg body weight GCV twice daily for three
days. The rats were then given a second intramuscular injection of
5 .mu.g estradiol valerate and intraperitoneal injections of 100
.mu.g/kg body weight GCV once daily for three days. Five to seven
days after GCV treatment, the rats were given an intravenous
injection of NMU dissolved in 0.05% acetic acid (Ash Stevens,
Colo.; 50 mg NMU/kg body weight) and were subsequently monitored
for general health and the appearance of tumors at weekly intervals
for 8 months. The results were as follows: TABLE-US-00001 No. of
Total % of Glands Treatment No. of Tumors Glands with Tumor
Untreated 5 12 41.7 Control Trypan Blue and 5 17 29.4 Ganciclovir
No injection; 5 20 25.0 Ganciclovir AdHS-tk and 2* 35 5.7
Ganciclovir *The two tumors (size < 5 mm) in this group were
detected during necropsy at the termination of the experiment 8
months later. The difference between tumors appearing in treated
versus control glands was significant by Chi.sup.2 analysis (p <
0.01).
[0050] The above results show that the method of the present
invention inhibits the formation of cancer of ductal epithelial
origin in this rat model, in which NMU induces the formation of
mammary tumors in 90-100% of female rats of similar age.
Surprisingly and unexpectedly, this prophylactic effect was
achieved without extensive destruction of the mammary glands. These
data demonstrate that selective destruction of epithelial cells,
e.g., key stem cells, can be sufficient to provide prophylactic
protection against carcinogen-induced tumor formation in the ductal
epithelium of the mammary gland.
Example 5
[0051] This example demonstrates the efficient transfection of
mammary epithelial cells in vivo.
[0052] Lytic Vaccinia virus (10.sup.6 Vaccinia-HA, which also
carries lacZ, in 20 .mu.l 0.2% trypan blue) was injected into the
mammary glands through the teat of 45 day old virgin rats.
Contralateral control glands were injected with 0.2% trypan blue.
The glands were excised after 3 days. Frozen mammary gland sections
were stained with X-gal and counterstained with eosin. When
Vaccinia-HA was injected via the rat teat, it was able to infect
the epithelial cells. At 3 days post-infection, the X-gal staining
was confined primarily to the epithelial cells.
Example 6
[0053] This example demonstrates the cytotoxicity of 20 Vaccinia/HA
on HBL100 cells in vitro.
[0054] HBL100 cells (from ATCC, Rockville, Md.) were plated in
DME:F12 medium (50% Dulbecco's Modified Eagles Medium: 50%Ham's F12
Supplement) containing 10% fetal bovine serum and 10 .mu.g/ml
insulin at a density of 5.times.10.sup.4 cells/well and incubated
at 37.degree. C. overnight. Vaccinia/HA, at concentrations of 0
moi, 0.1 moi, or 1.0 moi, was added to the culture medium, and the
cells were incubated at 37.degree. C. for at least 3 days. More
than 90% of the cells were dead within 72 hrs of infection at 0.1
moi.
Example 7
[0055] This example shows the death of rat mammary tumor cells in
culture by infection with Vaccinia/HA.
[0056] Cells of rat mammary cancer cell line RBA were plated in
growth medium at a density of 5.times.10.sup.4 cells/well and
incubated at 37.degree. C. overnight. Vaccinia virus engineered to
express .beta.-galactosidase and hemagglutinin genes (Vaccinia/HA)
was added to the culture medium at concentrations of 0 moi, 0.1
moi, or 1.0 moi and incubated at 37.degree. C. for at least 3 days.
Up to 90% of the cells were lysed within 72 hours of injection at
1.0 moi.
Example 8
[0057] This example demonstrates the destruction of mammary
epithelium by transfection with Vaccinia/HA in vivo.
[0058] The mammary glands of 45-day old virgin rats were injected
through the teat with 1.times.10.sup.7 particles of Vaccinia/HA in
20 .mu.l 0.2% trypan blue (tracking dye). Contralateral control
glands were injected with 0.2% trypan blue. The glands were excised
after 3 days, fixed in chloroform:methanol:acetic acid and stained
in iron-hematoxylin. Branching structures of a whole-mounted
mammary gland injected with tracking dye alone were visible up to
the end buds and alveoli. Also visible as brown bodies were the
mammary lymph nodes. Examination of a whole-mounted mammary gland
of the same rat receiving Vaccinia/HA in trypan blue on the
contralateral side revealed that only about 30% of the ducts
remained. In addition, the lymph nodes were considerably enlarged,
denoting the mounting of an immune response to clear the Vaccinia
from the vicinity.
[0059] All publications, patents, and patent applications cited
herein are hereby incorporated by reference to the same extent as
if each individual document were individually and specifically
indicated to be incorporated by reference and were set forth in its
entirety herein.
[0060] While this invention has been described with emphasis upon
preferred embodiments, it will be obvious to those of ordinary
skill in the art that the preferred embodiments may be varied. It
is intended that the invention may be practiced otherwise than as
specifically described herein. Accordingly, this invention includes
all modifications encompassed within the spirit and scope of the
appended claims.
* * * * *