Note: Descriptions are shown in the official language in which they were submitted.
CA 02303591 2000-03-30
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METHOD AND COMPOSITIONS FOR PREVENTING OR REDUCING HIV
INFECTION
The present invention relates to a method to inhibit HIV infection by using an
antibody
to estrogen-stimulated leucine aminopeptidase (es-LAPase) or one ore more
inhibitors
of es-LAPase activity, or a combination thereof. The invention further relates
to a
method to inhibit 1~IV infection by using an antibody to es-LAPase, one ore
more
inhibitors of es-LAPase activity and an anti-estrogen compound. Novel
compositions
which inhibit HIV infection are also part of the present invention.
BACKGROUND OF THE INVENTION
In spite of having .achieved an in depth and comprehensive genetic structure
of the
human immunodeficiency virus (HIV) and of that of its several structural and
geographical clusters, a very limited success in the prevention and treatment
of AIDS
has been attained. Although HIV and its variants are well characterized as the
prime
causative agents leading to the human acquired immunodeficiency syndrome
(AIDS),
a less comprehensive identification of key cellular factors involved in the
early
molecular mechanisms leading to viral entry have been accomplished.
CD4 T-cells are the principal target of HIV infection as shown from both in
vitro and
in vivo studies. The CD4 glycoprotein at the surface of T-cells exhibits a
high affinity
for HIV virions. Other molecules, particularly from the chemokine-receptor
family,
also participate to promote viral entry into target cells.
Therapeutics modalities to thwart the progress of the disease in HIV infected
patients
relies principally on two types of medications: Reverse transcriptase
inhibitors and
protease inhibitors. Although successful at slowing the replication of the
virus, they
are limited by the high rate of mutagenesis in the virus which results in
modification
of the drugs' binding site and their concomitant lost of efficacy. One
approach to
resolve the problem has been to administer several drugs simultaneously to
lower the
CA 02303591 2000-03-30
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probability of the development of resistant strains of the virus. However,
these
multidrug regimen create serious side effects. As a result compliance may be
low.
Furthermore, current therapies for HIV infections are not specific for
particular groups
of individuals such, as women. In a recent report it was found that the number
of
women infected with HIV during 1991 and 1995 increased by 63 % , more than any
other group of people that had contracted AIDS, regardless of race or mode of
exposure to HIV virus [Centers for Disease Control and Prevention. (1996).
HIVlAIDSSurveill. Rep. Centers for Disease Control and Prevention; 8. Atlanta,
Ga.].
The increased incidence of AIDS epidemic on morbidity and mortality among
women
(ages of 25-44) has been recently confirmed by other groups [Wortley, P.M.,
and
Flemming, P.L. (1097). AIDS in Women in the United States: Recent Trends.
JAMA,
278: 911-916.]. The reasons for the increased incidence of AIDS in women are
largely
unknown. Not only women are more prone to infection than men (supra) but
studies
have shown that anti-HIV drugs work differently in women and men. For example,
toxicities, side effE:cts and blood levels have been shown to be gender
specific.
Hormonal differences between men and women are cited to explain the
differences but
no clear link has been established. In view of the above data, there is a need
to tailor
therapeutic modalities to exploit the differences in the infection process
between men
and women to improve the efficiency of treatments.
Independently of the role of CD4, the putative receptor for HIV and that of
the (3-
chemokine co-receptors (CKR-5, CCRS and CXCR-4), we have described the
important role of both membrane-bound and extracellular Leucine Aminopeptidase
in
HIV viral entry. (F'ulido-Cejudo, G. et al. (1997) Antiviral Research 36, 167-
177)
The present invention overcomes the limitation of the prior art by providing a
link
between LAPase activity and estrogen. This link has been exploited herein to
discover
new HIV viral inhibitors which can be used alone or in combination with
improved
efficacy.
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SUMMARY OF THE INVENTION
The present invention relates to a method to inhibit HIV infection by using an
antibody
to es-LAPase or an inhibitor of es-LAPase activity, or a combination thereof.
The
invention further relates to a method to inhibit HIV infection by using an
antibody to
es-LAPase, an inhibitor of es-LAPase activity and an anti-estrogen compound.
Novel
compositions which inhibit HIV infection are also part of the present
invention.
Thus according to the present invention there is provided a method for
reducing HIV
infectivity towards T lymphocytes comprising contacting said T lymphocytes
with a
sufficient amount of an inhibitor of LAPase.
In a further embodiment of the present invention there is provided a method
for
reducing HIV infc:ctivity towards T lymphocytes comprising contacting said T
lymphocytes with a sufficient amount of an antibody specific for LAPase, more
specifically a monoclonal antibody specific for es-LAPase.
This invention also provides a method for reducing HIV infectivity towards T
lymphocytes comprising contacting said T lymphocytes with a sufficient amount
of an
antibody specific for es-LAPase and an inhibitor of LAPase.
This invention also provides a method for reducing HIV infectivity towards T
lymphocytes comprising contacting said T lymphocytes with a sufficient amount
of an
antibody specific for es-LAPase, an inhibitor of LAPase and an anti-estrogen
compound.'
Further according to the present invention there is provided a method for
preventing
or reducing HIV infection comprising administering to a patient in need
thereof, a
pharmaceutically effective amount of a composition which comprises an antibody
specific to es-LAPase and a pharmaceutically acceptable carrier.
Also provided in this invention is a method for preventing or reducing HIV
infection
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comprising administering to a patient in need thereof, a pharmaceutically
effective
amount of a composition which comprises an inhibitor of LAPase and a
pharmaceutically acceptable carrier.
S This invention is also directed to a method for preventing or reducing HIV
infection
comprising administering to a patient in need thereof, a pharmaceutically
effective
amount of a composition which comprises an antibody specific to es-LAPase and
an
inhibitor of LAPasf; together with a pharmaceutically acceptable carrier.
This invention is also directed to a method for preventing or reducing HIV
infection
comprising admini<,;tering to a patient in need thereof, a pharmaceutically
effective
amount of a composition which comprises an antibody specific to es-LAPase, an
inhibitor of LAPase: and an anti-estrogen compound together with a
pharmaceutically
acceptable carrier.
Also according to the present invention there is provided a composition
comprising an
antibody specific to es-LAPase and an inhibitor of LAPase.
Also according to tree present invention there is provided a composition
comprising an
antibody specific ~:o es-LAPase, an inhibitor of LAPase and an anti-estrogen
compound.
BRIEF DESCRIPTION OF THE DRAWINGS
These and other features of the invention will become more apparent from the
following description in which reference is made to the appended drawings
wherein:
FIGURE 1 shows ~:he effect of estrogen on LAPase activity in cell supernatents
of
primary parental breast cancer cell lines.
FIGURE 2 is a SDS PAGE of crude (lane 1) and purified LAPase (lane 2) followed
by western blot with MAb 7B6.
FIGURE 3 shows the kinetics of inhibition of LAPase by Bestatin, thioredoxin
and
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glutathione.
DETAILED DESCRIPTIOiV OF PREFERRED EMBODIMENT
The present invention relates to a method to inhibit HIV infection by using an
antibody
to es-LAPase or an inhibitor of es-LAPase activity, or a combination thereof.
The
invention further relates to a method to inhibit HIV infection by using an
antibody to
es-LAPase, an inhibitor of es-IJAPase activity and an anti-estrogen compound.
Novel
compositions which inhibit HfIV infection are also part of the present
invention.
It has been observed that, independently of the role of CD4, the putative
receptor for
HIV and that of the 13-chemokine co-receptor.s (CKR-5/CCRS;CXCR-4), both
membrane-bound and extracellular leucine aminopeptidase (LAPase;EC 3.4.11.1)
can
promote HIV entry in T lymphocytes (Pulido-Cejudo et al. Antiviral Res. 36:167-
177,
1997).
In the present invention it is disclosed that LAPase activity in T lymphocytes
and in
breast cancer parental cells can be stimulated by estrogens, or estrogen
analogues. One
aspect of this invention is directed to the identification and purification of
an estrogen-
stimulated LAPase isoenzyrne, referred to herein as es-LAPase. Details of the
estrogen-stimulated LAPase isoenzyme are described in Applicants copending
Canadian patent application No. 2,303,505 entitled "A Monoclonal Antibody
against
Estrogen Stimulated Leucine Aminopeptidase".
A further aspect of the present invention is the preparation of antibodies
against the es-
LAPase isoenzyme. Antibodies can be produced by methods well known in the art.
In general, the antibody producing cells are prepared by immunizing an animal,
for
example, mouse, rat, rabbit, sheep, horse, or bovine, with an antigen. The
immunization schedule and the concentration of the antigen in suspension is
such as to
provide useful quantities of suitably primed antibody producing cells. These
antibody
producing cells can be either spleen cells, thymocytes, lymph node cells
and/or
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peripheral blood lymphocytes.
The antibody producing cells are then fused with myeloma cells, cell lines
originating
from various animals such <is mice, rats, rabbits. and humans can be used,
using a
suitable fusion promoter. N(any mouse myeloma cell lines are known and
available
generally from members of the academic community and various depositories,
such as
the American Type Culture Collection, Rockville, Maryland. The myeloma cell
line
used should preferably be medium sensitive so that unfused myeloma cells will
not
survive in a selective media, while hybrids will survive. The cell line most
conunonly
used is an 8-azaguanine resistant cell line, which lacks the enzyme
hypoxanthine-
guanine- phosphoribosyl-transferase and therefore will not be supported by HAT
(hypoxanthine-aminopterin-thymidine) medium. In general, the cell line is also
preferably a "non-secretor" type, in that it does not produce any antibody.
The
preferred fusion promoter is polyethyleneglycol having an average molecular
weight
from about 1000 to about 4000. Other fusion promoters such as
polyvinylalcohol, a
virus or an electrical field can also be used.
The immortalized cells (hybridoma) must then be screened for those which
secrete
antibody of the correct specificity. The initial screening is generally
carried out using
an enzyme-linked immunosorbent assay (ELISA). Specifically, the hybridoma
culture
supernatants are added to mic:rotitre plates which have been previously coated
with the
antigen, in this case the es-LAPase. A bound specific antibody from the
culture
supernatants can be detected using a labelled second antibody, for example,
goat
antimouse IgG labelled with peroxidase, which is commercially available.
Cultures
that are positive against the es-LAPase are then subjected to cloning by the
limiting
dilution method. Secondary hybridoma cultures are re-screened as described
above,
and further positive cultures acre then examined using the BIAcore ~M system
(Pharmacia
Biosensor AB, Uppsala, Sweden). The cultures are then evaluated as to
determine
whether or not the antibody binds the antigen and to determine the kinetic
profile of
antigen binding. Selected cultures based on these results are subject to
further cloning
until culture stability and clo:nality are obtained. Immediately after
hybridization, the
CA 02303591 2000-06-30
fusion products will have approximately 80 chromosomes, and as these cells
proceed
to divide they will randomly lose some of these chromosomes. The cloning
process
is to select those cells which still have the chromosomes coding for antibody
production. The cloning process is repeated until 100% of the sub-population
exhibits
the production of a specific antibody, which is indicative of the "stability"
of the
hybridoma. In addition, hybridoma culture wells often have multiple colonies
some
of which may be antibody non-producers. The cloning process allows the
selection of
a positive hybrid which is derived from a single cell.
In one embodiment of the present invention there is provided a monoclonal
antibody,
specific against the es-LAPase, which has been designated Mab 7B6. This
monoclonal
antibody does not inhibit the enzymatic activity of LAPase. For this reason it
will be
referred to hereinafter as a non-inhibitory monoclonal antibody. A hybridoma
cell
line producing this monoclonal antibody has been deposited with the
International
Depository Authority of Canada, Winnipeg, Manitoba on March 23, 2000
(Accession #
IDAC 230300-1 ) in compliance with the Budapest Treaty.
The present invention also encompasses MAb 7B6 and any fragments thereof
containing the active binding region of the antibody such as Fab, F(ab)2 and
Fv
fragments. These fragments can be obtained from the 7B6 antibody by using
techniques
well known to those of skills in the art (Rousseaux et al. Methods Enzymology,
121:663-69, Academic Press, 1986).
A further embodiment of the present invention encompasses antibodies or
fragments
thereof capable of binding the same antigenic determinant as the 7B6 antibody.
Including, but not limited to, antibodies possessing the same antigenic
specificity as the
7B6 antibody but originating from a different species or having a different
isotype or
exhibiting different binding affinities. It is envisioned that class and
isotype variants
of the antibody of the present invention can be prepared using recombinant
class
switching and fusion techniques that are well known to those skilled in the
art (see for
example: Thammana et al. Eur. J. Immunol, 13:614, 1983; Oi et al.,
Biotechnologies,
CA 02303591 2000-03-30
_g_
4(3):214-221, Liu et al. Proc. Nat. Acid. Sci. (USA), 84:3439-43, 1987;
Neuberger
et al., Nature 31:2:604-608, 1984 and Spira et al. J. Immunol. Meth., 74:307-
15,
1984).
The monoclonal ;antibody of the present invention can be produced either using
a
bioreactar or from ascites, both procedures of which are well known in the
art.
In one embodiment of the present invention there is provided a method of
reducing
HIV infection wherein Mab 7B6 is incubated with T lymphocytes prior to and
during
infection with HI'J-1. This incubation prevents or reduces the infectivity of
HIV-1
towards T lymph~~cytes. Even though not described in the examples (infra),
this
embodiment also encompasses incubating T lymphocytes with Mab 7B6 after they
have
been infected with HIV-1 to reduce further infection as would be obvious to
one skilled
in the art.
The monoclonal antibody can be added directly to the incubation medium or it
can be
coupled to.a solid :matrix. The solid matrix can be any matrix, as would be
known to
someone skilled in the art, onto which the antibody can be coupled as long as
this
coupling does not interfere with the binding of the antibody to its epitope on
es-
LAPase. The solid matrix can be, but is not limited to, a protein G matrix.
Without
wishing to be bound by any theory, the coupling of the antibody to the matrix
may
reduce the level of non-specific binding of the anti-body to its epitope.
HIV infection of T lymphocytes can be measured using a number of techniques.
The
three most commonly used are the quantification of viral capsid protein p24
production, cyopathycity, and the determination of proviral load by polymerise
chain
reaction (PCR)(Conway et al., Clin. Diag. Virol. 3:95-104, 1995). These
methods are
well known in the a.rt. Other methods as would be known to one skilled in the
art may
also be used to determine the levels of HIV infectivity.
According to one aspect of the present invention anti-es-LAPase antibodies are
used
CA 02303591 2000-03-30
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to reduce HIV infection. According to this embodiment of the present invention
cells
can be treated at a concentration of from about Song to about 200ng of
antibody per
ml of cells, wherein the cells are at a density of about 1.0 x 105 cells/ml.
The
preferred amount of antibody can be determined empirically, by a person of
ordinary
5 skill in the art. In one example of this embodiment the concentration of
antibody used
is about 100ng per 10 ml of cells.
The present invention also discloses that thiol containing compounds
unexpectedly
inhibit the activity of es-LAPase. By thiol containing compounds it is meant
any
10 compound having a SH group such as, but not limited to, peptides having
reduced
cysteine residues. lfn one embodiment of this aspect of the invention,
thioredoxin and
glutathione have been found to inhibit es-LAPase.
Thus, in a further embodiment of the present invention, there is provided a
method
wherein an inhibitor of es-LAPase is incubated with T lymphocytes prior to and
during
infection with HIV. According to this aspect of the invention, the inhibitor
of es-
LAPase activity cam be a competitive, an uncompetitive or non-competitive
inhibitor.
The meaning of these terms is taken in the context of the enzyme kinetic
theory, as
would be known to persons skilled in the art. In this aspect of the invention,
an
example of an uncompetitive inhibitor is thioredoxin and an example of a non-
competitive inhibitor is glutathione.
The inhibitor of LAPase can be added directly to the incubation medium or it
can be
coupled to a solid matrix. The solid matrix can be any matrix, as would be
known to
someone skilled in the art, onto which the inhibitor can be coupled as long as
this
coupling does not interfere with the binding of the inhibitor to es-LAPase.
The solid
matrix can be, but is not limited to, DPDP, an amide bonding matrix (Pierce,
Rockford,IL, USAj.
In this aspect of the present invention the inhibitors of es-LAPase activity
are used to
reduce HIV infection. According to this embodiment of the present invention
cells can
CA 02303591 2001-03-28
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be treated with the inhibitor at a concentration of from about 10-~M to about
10-5M,
wherein the cells are at a density of about 1.0 x 10' cells/ml. The preferred
amount
of inhibitor can be determined empirically, by a person of ordinary skill in
the art. In
one example of this embodirr'ent the concentration of inhibitor used is from
about 4ng
to about 8ng per ml of cells.
In another aspect of the preaent invention there is provided a method wherein
an
antibody to es-LAPase and am inhibitor of LAPase can be combined and incubated
with
T lymphocytes prior, during or after infection with HIV to reduce HIV
infectivity. In
this aspect of the present invention, the antibody and inhibitor are used
together
according to the methods already disclosed for their individual use.
In a previous publication (Mesange et al. Mol. Pharmacol. , 50:75-79, 1996) it
has been
shown that TamoxifenT"', an anti-estrogen compound can reduce HIV infectivity.
However, incomplete inhibition of HIV infection were observed at TamoxifenT~''
concentrations of 10-~M or higher. In a further aspect of the present
invention there
is provided a method wherein not only TamoxifenT~' but other anti-estrogens
can be
used together with an antibody to es-LAPase and an inhibitor of LAPase to
reduce HIV
infectivity.
In this example of the present invention the anti-estrogen compound is
selected from
the following, used alone car in combination: 4-hydroxyandrostenedione,
RaloxifeneT'~',
3~3,Sa-tetrahydro Norethisterone, Tamoxifen, Droloxifene"'' and Idoxifene'M.
In this
aspect of the present invention the anti-estrogen compound is used at a final
concentration ranging from about 106 M to about 10-~ M. The concentrations of
the
other components in this method are used as describe above.
In a further aspect of this invention a competitive inhibitor of es-LAPase
activity can
also be used together with thf: above described compounds. An example of a
suitable
competitive inhibitor is BestatinTM, but the invention also encompasses other
comptetive
inhibitors such as Amastatin rM.
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The present invention also encompasses a method for treating patients infected
with
HIV, or at risk of being infected, with an antibody to es-LAPase or one or
more
inhibitors of es-LAPase activity or a combination thereof. The present
invention also
encompasses a method for treating patients infected with HIV, or at risk of
being
infected, with an antibody to es-LAPase, one or more inhibitors of es-LAPase
activity
and an anti-estrogen. In this aspect of the invention there is provided a
pharmaceutical
composition comprising an antibody to es-LAPase. In a further aspect of this
embodiment the pharmaceutical composition comprises one or more inhibitors of
es-
LAPase activity. In a still further aspect of this embodiment the
pharmaceutical
composition comprises an antibody to es-LAPase and one or more inhibitors of
es-
LAPase activity. In a still further aspect of this embodiment the
pharmaceutical
composition comprises an antibody to es-LAPase and one or more inhibitors of
es-
LAPase activity and an anti-estrogen compound.
Pharmaceutical compositions of the present invention generally will include a
therapeutically effective amount of at least one of the compounds referred to
above
together with a pharmaceutically effective carrier. The antibody of the
present
invention may be administered by any means that enables the active agent to
reach the
agent's site of action in the body of a mammal. In the case of the antibodies
of this
invention, the primary focus is the ability to reach and bind with both
membrane-bound
and extra cellular es-LAPase. Because proteins are subject to being digested
when
administered orally, parenteral administration, i.e., intravenous,
subcutaneous,
intramuscular, would ordinarily be used to optimize absorption.
The antibodies of this invention may be administered either as individual
therapeutic
agents or in combination with other therapeutic agents, as discussed above.
They can
be administered alone, but are generally administered with a pharmaceutical
carrier
selected on the basis of the chosen route of administration and standard
pharmaceutical
practice.
The dosage administered will, of course, vary depending upon known factors
such as
CA 02303591 2000-03-30
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the pharmacodynamic characteristics of the particular agent, and its mode and
route of
administration; ag;e, health, and weight of the recipient; nature and extent
of
symptoms, kind of concurrent treatment, frequency of treatment, and the effect
desired. Usually a daily dosage of active ingredient can be about 0.1 to 100
milligrams
per kilogram of body weight. Ordinarily 0.5 to 50, and preferably 1 to 10
milligrams
per kilogram per day given in divided doses 1 to 6 times a day or in sustained
release
form is effective to obtain desired results.
Dosage forms (composition) suitable for internal administration generally
contain from
about 1 milligram to about 500 milligrams of active ingredient per unit. In
these
pharmaceutical compositions the active ingredient will ordinarily be present
in an
amount of about 0.5 - 95 % by weight based on the total weight of the
composition.
For parenteral administration, the antibody can be formulated as a solution,
suspension, emulsion or lyophilized powder in association with a
pharmaceutically
acceptable parenteral vehicle. Examples of such vehicles are water, saline,
Ringer's
solution, dextrose solution, and 5 % human serum albumin. Liposomes and
nonaqueous vehicles such as fixed oils may also be used. The vehicle or
lyophilized
powder may contain additives that maintain isotonicity (e.g., sodium chloride,
mannitol) and cherr~ical stability (e.g., buffers and preservatives). The
formulation is
sterilized by commonly used techniques.
In further embodiments of the present invention, the antibody can be
administered
together with one or more LAPase inhibitors, as discussed in detail above. In
this
embodiment, the L.APase inhibitors can be added at a dose of about 0.1 to
about 100
milligrams per kilogram of body weight.
In yet a further embodiment of the present invention, the antibody can be
administered
together with one or more anti-estrogen compound, as discussed in detail
above. In
this embodiment, the anti-estrogen compound can be added at a dose of about
0.1 to
about 100 milligrams per kilogram of body weight.
CA 02303591 2000-03-30
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In another embodiment of the present invention, the antibody can be
administered
together with one or more LAPase inhibitors and one or more anti-estrogen
compounds
as discussed in detail above. In this embodiment, both the LAPase inhibitors
and anti-
estrogen compounds can be added at doses of about 0.1 to about 100 milligrams
per
kilogram of body weight.
The antibody and the inhibitors to be included in the pharmaceutical
composition may
be coupled to a matrix as described above, as long as the matrix is non-toxic
to
mammals. Coupling of the antibody and the inhibitors to an appropriate matrix
may
also facilitate the incorporation of these compounds into liposomes.
In a further embodiment, the inhibitor of LAPase in the pharmaceutical
composition
is the uncompetitive inhibitor thioredoxin, which may be maintained in its
reduced state
by including reducing compounds in the composition. Such compounds may include
but are limited to g:lutathione and cysteine.
In order that the invention described herein may be more fully understood, the
following examples are set forth. It should be understood that these examples
are for
illustrative purposes only and are not to be construed as limiting the scope
of this
invention in any manner.
EXAMPLES
Example 1: Estrogen mediated LAPase release from HUT78 cells
CD4+ T-HUT78 cells were incubated in the presence of various concentrations of
17-~3-Estradiol and I~APase levels were determined as described by G. Pulido-
Cejudo et al. (Antiviral Research, 36:167-177, 1997).
Table 1 show that estrogen promoted the release of LAPase in HUT78 cells in a
CA 02303591 2001-03-28
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dose response manner, with a maximum stimulation observed at 10-'M.
Table 1
Extracellular LAPase activity of HUT78 cells incubated with 17-~-Estradiol
LAPase activity (U* x 10-S/ml)
[~3-Estradiol] Control 17-[3-Estradiol Stimulation
10-SM 4.2 10.8
10-6M 5 . 6 20.4
10-'M 4.8 148.6
10-gM 5.7 110.7
* One unit of LAPase is defined as the amount of enzyme required to hydrolyze
1 ~mol of L-leucine-
(~-naphthylamide to L-leucine and (3-naphthylamide per min at pEl 7.5 and
37°C.
Following estrogen stimulation, maximum release of LAPase from breast
carcinoma
parental cells was observed at an estrogen ( 17(3-Estradiol) concentration of
100 nM
following 24 hours of incubation (see Fig. 1 where LAPase activity was
determined
in supernatants of primary parental cell lines immunoprecipitated with anti-
LAPase-
SepharoseT'''' bound Mab 7B6 antibodies. Cells were incubated in the presence
of 17-
~3-Estradiol and following a :?4 hr incubation, supernatants were removed from
the
primary culture, immunoprecipitated and assayed tluorometrically for LAPasf:
activity by the method of Kuramochi et al. [1987, J. Antibiot., 40: 1605:1611]
Control cells were cultured in the absence of 17-~3-Estradiol.). During the
same
period, LAPase in cell supernatants of control cells remain unchanged. In
addition,
LAPase activity was determined in supernatants of primary parental cell lines
immunoprecipitated with anti-LAP-Protein G MAb 7B6 bound antibodies. These
results show that the extracellular LAPase activity of estrogen stimulated
cells (100
nM) was 7.7 x 10-5 U/ml in comparison to 6.4 x 10-~ U/ml detected in the
supernatant of parental cells incubated for 24 hours with cell media alone as
CA 02303591 2001-03-28
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control. In addition, there was no effect of estrogen incubation on LAPase
activity
in purified preparations of this enzyme alone. Collectively, these results
suggest that
estrogen effect on LAPase activity encompasses a cellular mediated process.
Example 2: Purification ot_es-LAPase
Primary parental breast carcinoma cells obtained from human tumour biopsies.
were
stimulated with 100nM 17-~3--Iatradiol for 24 hours or cell media alone as a
control.
The cell media was RPMI 1640 medium + 10 % FCS + 100U/ml Penicillin + 100
~,g/ml Streptomycin. Cell supernatants were collected and then dialyzed
against
PBS in seamless cellulose tubing (MW 12,400) for 12 hours at 4°C. Es-
LAPase
was subsequently purified from the dialyzed cell supernatants using HPLC-gel
permeation followed by DIRE-Cellulose and Bestatin-Sepharose affinity
Chromatography. Briefly, the cell supernatant was applied to a Bio-Sil SEC-
2:50
column (600x7.5 mm) previously equilibrated in a buffer containing 100 mM
Sodium Phosphate buffer pH fi.8, 100 mM Na~SO,, 1~,M ZnCh and 10% glycerol.
The column was washed with 300 ml of the same buffer at a flow rate of 0.5
ml/min. Protein was concentrated to 10 ml by ultrafiltration using YM5
membrane
(5000 M.W. cutoff, Amicon I)iv., Danvers, MA., USA). The concentrate was
applied to a DEAE cellulose column (2.6 cm X 28.5 cm) equilibrated and washed
with 50 mM Tris-HC1 buffer pH 7.5; 1~,M ZnCl2; and 10% (v/v) glycerol. Es-
LAP was eluted using a linear gradient (0 to 1M NaCI in Tris buffer) at a flow
rate
of 0.50 ml/min. A Bestatin-affinity column was prepared using UltralinkTM
EDC/DADPA Amide bonding matrix (Pierce, Rockford, IL U.S.A.) by reacting
100mg of pure Bestatin with the carbodiimide EDC/DADPA matrix following the
procedure provided by the manufacturer. Prior to loading the LAPase containing
eluent, the Bestatin-affinity column was equilibrated with 10 mM Tris-HC1 pH
8.0
containing 1~M ZnClz and washed with 300 ml of this binding buffer. Es-LAl?ase
was recirculated through the system using a peristaltic pump at a t7ow rate of
O.lOml/min, for 2 hours. Following this recirculation, the column was washed
with
eight column volumes of binding buffer.
CA 02303591 2000-03-30
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Bestatin-bound es-LAPase was eluted with a linear gradient (0-0.5 M NaCI)
prepared in binding buffer 10 mM Tris-HCl pH 8.0 containing 1~M ZnClz .
Elution
of bound es-LAPase was monitored by absorbance at 280 nm. Purified es-LAPase
fractions were aliquoted in 500 ~1 and stored until further use in 50 mM Tris-
HCl
pH 7.8 and SO~cM ZnClz . Es-LAPase Protein concentration was estimated by the
method of Lowry et al. [J Biol Chem 193:265 (1951)] using bovine serum albumin
as a standard. A summary of the purification fold of the 17-~3-Estradiol-
stimulated
LAPase from parental cells of human breast carcinomas is set out in Table 2.
Es-
LAPase was purified to an approximate 7000 fold after the last Bestatin-
affinity
chromatographic step.
Es-LAPase activities were determined fluorometrically using leucine-b-
naphthylamide as the substrate [Kuramochi, H., et al. (1987) J. Antibiot. ,
40,1605-
1611] .
The reaction was stopped by boiling the samples at 100°C for 10
minutes, followed
by centrifugation at 780 x g at 4°C for 10 minutes. Values obtained
represent the
average of LAPase activity determined in triplicate.
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Table 2
Summary of Purification of LAP from Human Breast Carcinoma Parental Cells
Step P:roteinlTotal Activity2Specific Activity2Fold Yield
(rng) (nmole/min)(nmole/min/mg)
Supernatant1 '.i.4 98.21 6.37 1 100
Gel Permeation4.2 82.13 19.55 3.07 83.63
Cellulose 0.72 48.25 67.01 10.51 49.13
DEAE
Bestatin- 0.005 37.11 7422 1165 37.79
Sepharose
1-the amount of protein was determined after alkaline hydrolysis and
quantitative
ninhydrin detection of hydrolyzed material as described by Pulido-Cejudo et
al. [J.
Chromatogr. B 660 (1994) 37-47)].
2-determined fluorometrically using leucine-~3-naphtylamide as a subtrate as
described by [Kuramochi, H., et al. (1987) J. Antibiot. , 40,1605-1611].
Example 3: Monoclonal Antibody Production and Purification
In producing the hvbridoma cell line 7B6 secreting the mouse monoclonal
antibody
to 17-~i-Estradiol-stimulated LAPase, protocols for antigen preparation for
immunization, preparation of spleen cells from immune animals, fusion of
spleen
cells with myeloma. cells and plating of fused cells in selective medium was
conducted following detailed guidelines described by Campbell [Burdon RH,
Knippenberg PHV (eds): Laboratory Techniques in Biochemistry and Molecular
Biology, Amsterdam, Elsevier, p219 (1984)] and by Lietzke and Unsicker
[Leitzke
R. Unsicker K: A :statistical Approach to Determine Monoclonality After
Limiting
CA 02303591 2001-03-28
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Cell Plating of a Hybridoma Clone, J Immunol Methods 76:223 (1985)].
Briefly, the primary immunization was performed with purified es-LAPase
following desalting. Boosts with purified es-LAPase were performed at days 14,
35
& 56. BALB/c mice were screened at days 24 & 45. The mice were sacrificed at
day 59 and the splenocytes from the best responder were fused with myeloma
cells.
Screening was performed by dot blot immunostaining on nitrocellulose.
The hybridoma clone 7B6 was obtained by single cell cloning using limiting
dilution. Four dilution tubes in series containing hybridoma cells with medium
supplement with 20% FBS -~ 2X OPI were prepared. 100 ~,1 of each dilution was
plated in a 96-well plate with 50 ~.l of splenocyte feeder cells in each well
and
placed inside a 37°C 5 % C.""0, incubator. At day 7, supernatants from
each well
were removed and screened by dot blot immunostaining on nitrocellulose.
Hybridoma clone 7B6 cells were transferred from the 96 well plate to 0.5 nil
medium supplemented with :?0% FBS + 1X OPI + 1X HAT in a 24 well plate.
Once the cells were dense, they were transferred into 5 mls in a 60 mm dish
and
then transferred to 10 mls in a 100 mm dish. Once in the 60 mm dish, the cells
were weened off hypoxanthine, thymidine and aminopterin. 7B6 hybridoma cells
were continued to be grown until in a log phase of growth. Anti-es-LAPase,
Nlab
7B6 was isolated from collected hybridoma 7B6 cell supernatant by affinity
chromatography using Immunopure~~'' IgG as per described by manufacturer.
Screening was performed by dot blot immunostaining on nitrocellulose.
The isotype of Mab 7B6 was determined using Sigma's Immunotype Kit~M. Briefly,
the assay involves binding of Mab 7B6 to a precoated isotyping nitrocellulose
membrane strip followed by immunodetection using a sensitive biotin-avidin-
enzyme detection system. I'he immunoglobulin isotype is revealed by self
description.
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ELISA analysis of Human Breast Carcinoma parental cell lines was conducted to
demonstrate the reactivity of Mab 7B6 against human es-LAPase. Briefly, 50 x
103
parental cells were plated per well in a 96 well plate in RPMI 1640 medium -I-
10%
FCS + 100 U/ml Penicillin -+- 100 ~.g/ml Streptomycin. The plated cells were
cultivated at 37°C 5 % CO, for 24 hours. The cell supernatants were
removed, the
cells were washed with PBS and subsequently fixed with 1 % gluteraldehyde in
PBS
for 1 hour at room temperaW re. Washing with PBS occurred prior to blocking
with
casein for 1 hour at 37°C S ~~ CO,. Following another wash with PBS,
serial
dilutions of Mab 7B6 were added to the wells and allowed to incubate for 2
hours at
37°C 5% CO~, Demonstration of the reactivity of Mab 7B6 was revealed
upon the
addition of a secondary antibody, anti-(IgG + IgM) peroxidase conjugated goat
anti-mouse IgG + IgM (H+L) followed by the substate, OPD.
Following purification of the Mab 7B6, the corresponding IgGla isotype was
subsequently immobilized to a DSS cross-linking system obtained from Pierce
(Rockford, IL, U.S.A) according to the procedures described by the
manufacturer.
As shown in Figure 2, Mab '7B6 specifically reacts with es-LAPase in both
crude
cell supernatants as well as purified es-LAPase after SDS PAGE followed by
immunoelectrotransfer. The estimated Molecular Mass of 17-13-Estradiol-
stimulated
LAPase was 315 kDa as estimated by Gel Permeation on a Bio-SiITM SEC-250
column (600x7.5 mm) using Thyroglobulin (MW 670kDa); Bovine Gamma
Globulin (MW 158 kDa); Chiken ovalbumin (MW 44 kDa) and Equine Myoglobin
(l7kDa).
Example 4: Effect of Estrosten on increased infectivitv of HUT78 cells.
Estrogen-mediated HIV-induced cytopathic effect was determined as described by
G. Pulido-Cejudo et al. [Pulido-Cejudo, G., Conway, B., Proulx, P., Brown, R.,
and Izaguirre, C.A. (1997). Anti viral Research, 36: 167-177]. The sensitivity
of
CD4+ T-HUT78 cells to HIV viral infection was determined by measuring the
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activity of reverse transcriptase in cells infected in the presence/absence of
17-~3-
Estradiol.
In light of both the; clinical observations indicating the hypersensitivity of
women to
HIV infection [Centers for Disease Control and Prevention. (1996). HIVlAIDS
Surveill. Rep. 8. Atlanta, Ga., Wortley, P.M., and Flemming, P.L. (1997).
JAMA,
278: 911-916.] as well as the role of LAPase in viral entry [Pulido-Cejudo,
G.,
Conway, B., Proul!x, P., Brown, R., and Izaguirre, C.A. (1997). Antiviral
Research, 36: 167-177], together with the direct effect of estrogen on LAPase
reported supra, the sensitivity of HUT78 cells to HIV viral infection was
determined. As shown in Table 3, at 3 days and 5 days post infection, in the
presence of estrogen, HIV viral infectivity as determined by the activity of
reverse
transcriptase (RT) was approximately 4 times higher than control cells
infected in
the absence of estrogen.
Table 3
Effect of Estrogen on increased infectivity
RT Activity (cpm x 102/10,1)
Day 0 Day 3 Day 5
(Infection) (Post Infection) (Post Infection)
Control 0.82 19.82 32.44
Estrogen 0.68 72.42 124.44
( 10-'M)
Example 5: Inhibitors of es-LAPase
Human Thioredoxin was purified from CD4+ T MP6 cells as described by Rosen et
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al. [Rosen, A. et al. (1995) lnt. Immunol. 7, 625-633 ]and compared to
purified
Thioredoxin activity from E.C'.oli. purchased from SIGMA-ALDRICH Canada
(Oakville,Ontario Canada).
Thioredoxin was covalently :linked through an amide bond to Ultralink
EDC/DADPA bonding matrix (Pierce, Rockford, IL U.S.A.) by reacting 5 mg of
purified Thioredoxin with the carbodiimide EDC/DADPA matrix following the
procedure provided by the manufacturer.
Measurements of LAPase were performed spectrophotometrically at 330nm to
measure product formation of 13-naphthylamine, using 182~eM 1-leucine-13-
naphthylamide as the substrate. The kinetic assays were performed using a
Spectronic Genesys~M ~ spec~.rophotometer from Milton Roy (Rochester, NY;).
Absorbance was recorded every sixty seconds, over a period of 30 min. Enzyme
kinetic assays were performed using 8.0 x 10-2 U of es-LAPase in a final
reaction
volume of 600.1 and triplicate samples. The reaction mixture contained the
following materials added in order and all kept on ice prior to use: es-
LAPase,
Calcium-free Hank's solution making up a 600,1 final reaction volume and
182~,M
1-leucine-li-naphthylamide. A corresponding blank without es-LAPase was used
as
baseline. Both of the cuvettes were transferred to the spectrophotometer where
the
1-leucine-13-naphthylmide solution was added last, marking time zero of the
reaction. The inhibition studies were carried out in the presence of 167~M
Bestatin,
167~,M reduced Gluthathione and 17 ~M reduced thioredoxin.
Figure 3 shows the inhibitory effect of both Bestatin and two thiol-containing
peptides, reduced thioredoxin and reduced glutathione respectively. Es-LAPase
showed significant slower mites of reaction both in the presence of
thioredoxin and
glutathione in comparison to that observed in the control es-LAPase. In
addition,
es-LAPase reaction rates were slower in the presence of 17~M thioredoxin when
compared to those observed in samples incubated with 167~,M reduced
Gluthathione. In addition, 16~7~,M Bestatin clearly inhibits es-LAPase
activity. As
CA 02303591 2000-03-30
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shown in Table 4., reduced thioredoxin is an effective inhibitor of es-LAPase
activity. The analysis of the maximum reaction rates (Vmax), Michaelis-Menten
(Km) constants and the inhibition constants (Ki) for each inhibitor as
presented in
Table 4 reveals significant differences in the inhibitory properties of each
peptide.
S In this regard, es-LAPase incubated with Bestatin shows a Vmax value very
similar
to that observed to es-LAPase alone and a significantly higher Km value. This
data
confirms the competitive nature of es-LAPase inhibition by Bestatin. In
contrast, es-
LAPase incubated with reduced thioredoxin leads to a significantly lower Vmax
and
Km with an intermediate Ki value when compared to those obtained for Bestatin
and
glutathione. Collectively these data strongly suggest that reduce thioredoxin
inhibits
es-LAPase in an uncompetitive fashion while with reduced glutahthione es-
LAPase
inhibition is noncompetitive.
Table 4
~ Bestatin, reduced thioredoxin and glutathione es-LAPase inhibition Constants
Vmax Km Ki
(10-6 M/min) (10-4 M) (10-6M)
LAPase alone 36.81.9 1.670.19 N/A
Bestatin 167uM 39.03.9 48.78.4 6.221.61
Thioredoxin l7uM 5.310.02 0.2340.006 3.900.58
Glutathione 167uM 17.21.5 1.780.25 1.530.40
Example 6: Inhibition of HIV
Twelve hours prior to infection with HTLVIIIb~ HUT78 cells were incubated in
viral culture medium alone or in the presence of Anti-es-LAPase; thioredoxin;
Bestatin and Anti-estrogens according to the combinations described below. In
the
continued presencc: of each of the factors aforementioned, the cells were
subsequently infected at a multiplicity of two infective viral particles per
cell for
two hours at 25°C with constant agitation in a final volume of 1.0 ml.
Cells were
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subsequently washed with saline phosphate buffer solution (PBS) and seeded at
a
density of 1.0 x 105 cells/ml in RPMI 1640, supplemented withl0% FBS, 100
U/ml penicillin, 1(10 mg/ml streptomycin in 96-well plates. Syncytium
formation
was evaluated by light microscopy on days 3, 5 and 7. At appropriate time
points,
the cells in selected wells were subjected to psoralen/ultraviolet light
inactivation
(Watson et al., 1990) air-dried and fixed on glass plates with cold acetone.
Indirect
immunofluorescent staining was performed using a modification of standard
procedures (Aldovini, A. and Walker, B.D. (1990) Techniques in HIV Research.
Stockton Press, New York; Johnson, G.D. and Nogueira Araujo, G.M.(1981) J.
Immunol. Methods , 43, 349-350. Plates were incubated for 10 minutes in
blocking
buffer (PBS, 5 % goat serum) and reacted with either control mouse serum or
anti-
HIV-1 gag (p24, p~~s) monoclonal antibodies (Olympus Immunochemicals), which
were diluted 1:100 in blocking buffer, for 30 minutes at room temperature.
Cells
were washed three rimes with PBS and incubated for 30 minutes with goat anti-
mouse-FITC antibo~3ies (Beckton and Dickinson). Slides were mounted in
paraphenylene-diam.ine/glycerol [Johnson, G.D. and Nogueira Araujo, G.M.(1981)
J. Immunol. Methods , 43, 349-350] and fluorescent cells were counted.
Following HIV-1 infection of HUT-78 cells, the proviral load in each
experimental
condition was determned by PCR [Conway, B., Shui-Wah Ko, D. and Cameron,
W. ( 1995) Clin. Diag.Virol. 3, 95-104].
Prior to infection, H UT78 cells were washed three times with PBS and the cell
viability was determined. Infection was performed as described above. At
indicated time intervals, 1.0 ml aliquots of were taken and p24 antigen
quantified
using a commercial ;ELISA kit (p24 Antigen Capture ELISA, ABBOTT Diagnostics
Division), following the instructions of the manufacturer.
The Effect of Mab 7:B6, thioredoxin, Bestatin and anti-estrogens on the
viability of
HUT78 cells was as:;essed as follows. HUT78 cells were seeded at a density of
1.0
x 105 cells/ml in RP:MI 1640, 10 % FBS, 100 U/ml penicillin, 100 mg/ml
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streptomycin in the presence or absence of each of the compounds
aforementioned.
The medium was changed every third day and cell viability was determined by
trypan blue dye exclusion. Thymidine incorporation was also measured [Noma, T.
,
Klein, B., Cupissol, D., Yata, J. and Serrou, B.(1984). Int. J. Immunopharmac.
,
6, 87-92.] in dupli~rate cultures on day 7. Values obtained represent the mean
~
s.d. (n=10).
Incubation of HUT 78 cells with Mab 7B6-Protein G Matrices 7 hours prior to
Infection with HIV'-1 significantly reduces the number of HIV-1 proviral
copies
when compared to control cells pre-incubated with mouse IgGI-Protein G
matrices
(see Table 5).
Table 5
Inhibition of HIV-a proviral copies by Anti-es-LAPase Mab 7B6 bound to Protein
G.
Days Post-infection
0 3 5 7
Mab 7B6 0 86~5 189~2 220~24
(100ng/lOml)
Control IgGla 0 2019~89 4002~46 5551~92
2$ (100ng/lOml)
Incubation of HUT 78 cells with thioredoxin-EDC/DADPA Matrices 7 hours prior
to Infection with HIV-1 significantly reduces the number of HIV-1 proviral
copies
when compared to control cells pre-incubated with control myoglobin IgGI-
Protein
G matrices (see Table 6).
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Table 6
Inhibition of HIV-1 Proviral copies by thioredoxin-EDC/DADPA Matrix compared
to Bestatin-HIV inlhibition
Days Post-infection
0 3 5 7
Thioredoxin 0 4~ 1 123 1228
(40ng/Sml)
Bestatin 0 7 2 29 ~ 3 457 12
(120~g/ml
Control/Myoglobin 0 24141178 4122 121 5851 ~
123
(40ng/Sml)
The independent inhibition mediated by thioredoxin and anti-LAPase 7B6
monoclonal antibodies can be synergized by their simultaneous addition to HUT-
78
cells prior to HIV-1 infection (Table 7). In this context, without wishing to
be
bound by any theory, the synergistic effect of thioredoxin and of non-
inhibitory
LAPase 7B6 monoclonal antibodies imply that both LAPase activity and Viral-
protein interaction;; may be required during HIV-1 infectivity. It equally
suggests
that the elevation o~f plasma thioredoxin levels detected in late stages of
HIV
infection as reported by Nakamura et al. (1996, Int. Immunol. 8, 603-611) may
not
only compensate for intracellular glutathione depletion but it may also be
directly
implicated in reducing HIV viral load.
CA 02303591 2000-03-30
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Table 7
Synergistic Inhibition of HIV-1 Proviral copies by thioredoxin-EDC/DADPA and
Anti-LAPase 7B6 combined Matrices
Days Post-infection
0 3 5 7
Mab 7B6/Thioredoxin 0 12~3 69~5 88~12
(100ng/40ng/lOml)
Control IgGla/Myoglobin 0 1914~55 4330~26 5110~12
(100ng/40m1)
As shown in Example 4, exposure to 17-13-estradiol increased LAPase activity,
which in turn resulted in an increase in HIV infectivity. Thus, the selective
increase in HIV infectivity of women between 1991-1995 regardless of race or
mode of exposure to HIV virus could be explained in terms of the effect of
estrogen
on LAPase.
These observations, together with the uncompetitive inhibitory effect of
thioredoxin
on LAPase raise the possibility of the use of combined therapy for the
prevention
and treatment of AIDS.
The combined anti-HIV effect of LAPase inhibition by Mab 7B6, thioredoxin and
Bestatin together 'with the administration of anti-estrogens to hamper the
effect of
estrogen on LAPase activity and hence of HIV infection can lead to a promising
therapy against AIDS. Therefore, Tamoxifen, Droloxifene, Raloxifene,
Idoxifene,
4-hydroxyandrostenedione and the 313,Sa-tetrahydro derivative of
Norethisterone,
which are all anti-estrogen compounds, were tested for their ability to
senergize the
antiviral properties of thioredoxin, Bestatin and of Mab 7B6.
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Based on the effect of anti-astrogens on the antiviral properties of combined
MAb
7B6/thioredoxin matrices, the most effective combination for the complete in
vitro
inhibition of HIV-1 infection. was evaluated according to the overall effect
on the
reduction of cytopathicity, p:?4 levels and proviral load. For simplicity in
these
experiments determinations on the various parameters herein above
aforementioned
were determined on day 5 post-infection. Prior to infection cells were
simultaneouly
incubated with thioredoxin-E;DC/DADPA and Anti-es-LAPase 7B6 Matrices. The
inhibitory effect of this anti-fatrogens is sununarized in Table 8. All anti-
estrogenic
compounds were used at a final concentration of 10-'M.
Table 8
Combined antiviral effect of Mab 7B6 and thioredoxin matrices in combination
with
anti-estrogens
1S Day 5 Post-infection
Agent P24 Cytopathicity Proviral
(nmoles/ml)(Syncytia/well)Load(copies)
Control L . 82 117 6 3777 82
(No additions) .003
4-hydroxyandrostenedione0.02.001 4.00.4 12
Raloxifene 0.01 .002 3.3 0.1 8
313,5x-tetrahydro 0 0 2
Norethisterone
Tamoxifen, 0 0 0
Droloxifene, 0.04.003 5.20.6 14
Idoxifene, 0 0 0
CA 02303591 2000-03-30
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The present invention has been defined in terms of certain examples, which are
not
to be construed as limiting. The full scope of the present invention is
defined in the
following claims.
