The invention provides compositions for targeting methylated promoters
in breast cancer and pre-cancer patients. The invention also provides
methods for intraductal treatment of breast cancer and pre-cancer
patients by delivering intraductally an agent that targets methylated
promosters of silenced genes.
12. A method for inhibiting DNA methylation of breast cancer genes
in a patient having premalignant or malignant breast duct epithelial
cells in a breast duct, the method comprising: delivering a composition
into the breast duct of the patient, said composition comprising
an inhibitor of DNA methylation and a biocompatible solution suitable
as a vehicle for delivering the inhibitor of DNA methylation into
the breast duct of the patient; thereby inhibiting DNA methylation
of breast cancer-related genes within said premalignant or malignant
breast duct epithelial cells.
14. The method of claim 12 wherein the breast cancer genes are
selected from the group consisting of cyclin D2, RARbeta2, twist,
BRCA1, maspin, estrogen receptor, progesterone receptor, e-cadherin,
p16 (INK4a), P15 (INK4b), P14 (ARF), death associated protein (DAP),
retinoblastoma Rb, and vonHippel-Lindaur (VHL) gene.
17. The method of claim 1 12 wherein the DNA methylation inhibitor
competitively binds methyl groups and prevents methylation at cytosines.
18. The method of claim 1 12 wherein the DNA methylation inhibitor
is an oligonucleotide directed against a CpG island region of a
promoter of a breast cancer related gene.
21. The method of claim 12 wherein the biocompatible solution is
selected from the group consisting of saline, viscous material,
and gel material.
 This application claims the benefit of U.S. Provisional
Application 60/279,762, David Hung, filed Mar. 30, 2001.
FIELD OF THE INVENTION
 The field of this invention is methods and compositions
to intraductally treat breast cancer and precancer by targeting
methylated promoters of breast cancer related genes.
 Some genes that have lower expression in breast cancer than
normal tissue are silenced by hypermethylation of promoter sequences
of the gene.
 Hypermethylation, the creation of 5-methylacytosines, occurs
in the CpG rich regions (called CpG islands) of the promoter. The
CpG island sequences consist minimally of a CCGG sequence. Hypermethylation
of these regions appears to suppress transcription and is associated
with cancer. The methylation specific PCR (MSP) detects the 5-methyl
cytosine moieties the presence of which indicates hypermethylation.
 Transcription of the downstream gene is inactivated by promoter
hypermethylation. Thus, in a cancer context, a gene controlled by
a hypermethylated promoter becomes silent in the cancer or malignant
state, and silenced to a lesser degree in an atypical or precancer
 Expression of these genes or silencing of them is evidenced
by the methylation specific PCR of ductal epithelial cells from
which these genes can be expressed in a normal healthy individual.
Transcription of a hypermethylated gene can be restored upon demethylation
of the CpG islands in the gene's promoter.
SUMMARY OF THE INVENTION
 An aspect of the invention is a composition to reduce or
eliminate hypermethylation of promoters controlling breast cancer-related
genes. Accordingly is provided, a composition for intraductal administration
to a patient having abnormal breast ductal epithelial cells including
atypical or malignant cells comprising a demethylating agent selected
from the group consisting of an inhibitor of DNA methylation, a
demethylating agent, an antagonist of DNA methyl transferase activity,
and a deliverable amount of a biocompatible solution suitable as
a vehicle for the agent for delivery intraductally to the patient
in order to contact breast duct epithelial cells therein.
 An aspect of the invention is a method of treating a patient
having locally identified hypermethylation of promoters controlling
breast cancer-related genes. Accordingly, is provided a method of
treating a patient having a breast duct comprising atypical or malignant
breast duct epithelial cells comprising, delivering intraductally
to the breast duct an amount of an agent comprising an agent selected
from the group consisting of an inhibitor of DNA methylation, a
demethylating agent, and an antagonist of DNA methyl transferase
activity sufficient to inhibit or reverse DNA methylation of genes
transcribed within said breast duct epithelial cells.
DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS OF THE INVENTION
 The following preferred embodiments and examples are offered
by way of illustration and not by way of limitation.
 The invention is a novel composition and treatment method
for treating patients having breast cancer and precancer conditions
that have been identified in a specific breast duct or ducts of
 The composition comprises one or more of a demethylating
agent (to remove existing hypermethylations), an inhibitor of DNA
methylation (e.g. an agent comprising a moiety that competitively
binds methyl groups and/or prevents methylation at cytosines) or
an antagonist/inhibitor of DNA methyl transferase (the enzyme) or
its activity leading to methylation of cytosines. DNA methyl transferase
(MeTase or DMT) is the enzyme that catalyzes the methylation reaction
at cytosines. One DMT is e.g. 5-aza-2-deoxycytidine (5-aza-CdR).
Antisense oligonucleotides against the region of the promoter comprising
a CpG island may also be used as an inhibitor of DNA methylation.
The composition may comprise one or more or all or several of these
classes of agents that relate to and/or affect methylation or demethylation
at CpG sites on promoters for breast cancer-related genes. Antagonists
or inhibitors can be any molecule capable of antagonizing or inhibiting
the target bio-activity. Thus, for example, antagonists or inhibitors
can be for example small organic molecules, proteins, polypeptides,
peptides, oligonucleotides, lipids, carbohydrates, polymers and
 The composition also necessarily requires an agent or medium
that makes the active agent deliverable intraductally to a breast
duct. The composition comprising the agent or medium for delivery
to a breast duct and comprising one or more of the classes of active
agents listed need be biocompatible for humans, optimally provides
an optimal delivery window of the active agent to resident ductal
epithelial cells in the breast duct. Thus, also the composition
can comprise a solution that comprises saline or other common safe
deliverable solutions or agents or mediums; e.g. the composition
having an agent or medium to aid the intraductal delivery can optimally
comprise a viscous or gel material or other such medium that may
provide the active agent carried by the medium a longer residence
and/or activity in the duct to which is it delivered.
 Breast cancer genes that are known to be vulnerable to hypermethylation
and subsequent degrees of gene transcription and expression silencing
include, e.g. cyclin D2, RARbeta2, twist, BRCA1, maspin, estrogen
receptor, progesterone receptor, and e-cadherin. There are others
not listed here. Other genes having promoters that can be methylated
but that are not necessarily present in a breast context include
e.g. p16 (INK4a), P 15 (INK4b), P 14 (ARF), death associated protein
(DAP), retinoblastoma Rb, and von-Hippel-Lindaur (VHL) gene.
 The treatment method comprises delivering the claimed composition
intraductally to a breast duct in a patient. Preferably the duct
has been previously identified as having premalignant (e.g. hyperplastic
and/or atypical) or malignant (carcinoma) cells and thus been identified
as a target for the local treatment protocol proposed in the method.
 The treatment method comprises delivering intraductally
to a breast duct (e.g. a target duct previously identified as having
atypical or malignant cells) an amount of an agent such as an inhibition
of DNA methylation, a demethylating agent, and/or an antagonist
of DNA methyl-transferase. The delivery to the duct can be accomplished
by accessing a breast duct with a delivery tool (e.g. a catheter,
cannula, or the like) and infusing the agent (in a suitable medium
or solution for delivery of the active agent) into the duct to contact
target ductal epithelial cells lining the duct. The delivery can
also be accomplished e.g. by pump delivery, time-release capsule
placed in the duct, and the like.
 The amount of agent can vary, but in any event optimally
will be an amount sufficient to target all atypical or malignant
cells in the duct. Estimates of the quantity of target cells can
be made upon the initial identification of the target duct (e.g.
by cytological evaluation of ductal epithelial cells retrieved from
the duct. The amount may vary depending on the agent's potency and
other mitigating factors such as the extent of any time delay of
delivery of the agent once inside the duct (e.g. with a time release
formulation). Other factors such as whether the ductal epithelial
cells are atypical or malignant (e.g. greater therapeutic activity
may be needed for malignant cells), and/or how many genes might
be affected by the methylation activity can also affect a determination
of the amount of active agent to deliver to any given duct. The
agent should be delivered in a sufficient amount to inhibit or reverse
DNA methylation on promoters controlling genes transcribed and/or
expressed in ductal epithelial cells of the target breast duct.
Preferably the status of ductal markers and of the ductal epithelial
cells will be evaluated prior to intraductal delivery of the demethylating
and/or antimethylating agent(s), e.g. the evaluation can comprise
MSP of the methylated genes (e.g to identify them and/or to quantify
the amount of methylation) and/or cytological evaluation of the
ductal epithelial cells (e.g. identify hyperplastic, atypical, or
 A breast duct on the right breast of a patient is identified
as having atypical cells that are suspicious for malignancy. Four
genes are tested in ductal epithelial cells retrieved from a breast
duct by methylation specific PCR (MSP) to further establish a methylated
state of some promoters of some genes transcribed and/or expressed
in the ductal environment. The information quantified as a degree
of methylation in addition helps to determine an amount or specific
activity required of the agent for effective treatment. It is found
that RARbeta2, twist, maspin, and cyclin D2 are all genes expressed
in the ductal epithelium that show some percentage of methylation
on the promoter CpG islands as indicated by MSP. A viscous composition
of mixture of a demethylating agent, an antisense oligonucleotide
against CpG regions on the various promoters of the various target
genes, and an inhibitor of DNA methyl transferase (5-azaCdR) activity
are administered. The formula provides a week-long time-release
of the active agents present in the composition. Ductal fluid is
again retrieved and analyzed a month following the procedure in
order to assess a need for follow-up 2nd dose intraductal administration
of agent by conducting cytological analysis of retrieved ductal
epithelial cells, and by MSP of the genes studied and targeted in
the first administration.
 All publications and patent applications cited in this specification
are herein incorporated by reference as if each individual publication
or patent application were specifically and individually indicated
to be incorporated by reference. Although the foregoing invention
has been described in some detail by way of illustration and example
for purposes of clarity of understanding, it will be readily apparent
to those of ordinary skill in the art in light of the teachings
of this invention that certain changes and modifications may be
made thereto without departing from the spirit or scope of the appended