This invention concerns in vitro method for prognosticating the
progress of breast cancer, comprising detecting or quantifying the
level of 17.beta.-hydroxysteroid dehydrogenase (17HSD) type 1 enzyme
in breast tumor tissue sample, wherein the presence of said 17HSD
type 1 enzyme is indicative of severe progress of breast cancer.
Furthermore, the invention also concerns the use of a 17HSD type
1 enzyme inhibitor for prevention or treatment of breast cancer.
This invention concerns also the use of compound A in the manufacture
of a pharmaceutically acceptable preparation useful as 17HSD inhibitor.
1. A method for prognosticating the progress of human breast cancer
in vitro, comprising detecting or quantifying the level of 17.beta.-hydroxysteroid
dehydrogenase (17HSD) type 1 enzyme in breast tumor tissue sample,
characterized in that the presence of said 17HSD type 1 enzyme is
used to independently indicate the severe progress of breast cancer.
2. The method according to claim 1 characterized in that the 17HSD
type 1 enzyme is detected or quantified by nucleotide hybridization,
by immunological or immunohistochemical methods or by PCR.
3. Compound A: or a pharmaceutically acceptable salt thereof for
use as a medicament.
4. Compound A or a pharmaceutically acceptable salt thereof of
claim 3 characterized in that said medicament is for prevention
or treatment of disorders caused by the 17HSD type 1 enzyme activity.
5. Compound A or a pharmaceutically acceptable salt thereof of
claim 3 characterized in that said medicament is for prevention
or treatment of human breast cancer.
6. A pharmaceutical composition characterized in comprising compound
A: or a pharmaceutically acceptable salt thereof.
7. Pharmaceutical composition of claim 6, characterized in that
it is in the form of an oral formulation, injection, transdermal
formulation or rectal formulation.
8. The use of compound A: or a pharmaceutically acceptable salt
thereof in the manufacture of a pharmaceutically acceptable preparation
useful as 17.beta.-hydroxysteroid dehydrogenase (17HSD) type 1 inhibitor.
9. The use of claim 8, characterized in that said pharmaceutical
preparation is in the form of an oral formulation, injection, transdermal
formulation or rectal formulation.
10. The use of claim 8, characterized in that said inhibitor is
used for prevention or treatment of disorders caused by the 17HSD
type 1 enzyme activity.
11. The use of claim 10, characterized in that said disorders comprise
human breast cancer.
12. The use of compound A: or a pharmaceutically acceptable salt
thereof for prevention or treatment of disorders caused by the 17HSD
type 1 enzyme activity.
13. The use of claim 12, characterized in that said disorders comprise
human breast cancer.
14. A method for prevention or treatment of disorders caused by
the 17HSD type 1 enzyme activity on a patient, characterized in
that effective amount of compound A: is administered to said patient.
15. The method of claim 14, characterized in that said disorder
is human breast cancer.
FIELD OF THE INVENTION
 This invention relates to a method for prognosticating the
progress of breast cancer, based on the expression of 17.beta.-hydroxysteroid
dehydrogenase type 1 enzyme in the breast cancer tissue. The invention
also concerns a method for the prevention or treatment of breast
cancer by administering an effective amount of a 17.beta.-hydroxysteroid
dehydrogenase type 1 enzyme inhibitor to an individual. Further,
this invention relates also to the use of compounds known per se
as 17.beta.-hydroxysteroid dehydrogenase type 1 inhibitors, and
to pharmaceutical preparations comprising said compounds.
BACKGROUND OF THE INVENTION
 The publications and other material are used herein to illuminate
the background of the invention and they are incorporated herein
 Enzymes possessing 17.beta.-hydroxysteroid dehydrogenase
(17HSD) activity have been cloned and described in the literature.
So far at least eight types of such (17HSD types 1 to 8) human enzymes
have been discovered. (Peltoketo et al., J Mol Endocrinol (1999)
 The 17HSD enzymes control the last step in the formation
of androgens and estrogens. 17HSD type 1 is a protein of 327 amino
acids catalyzing the formation of high-activity 17.beta.-estradiol
from low-activity estrone. Type 2 17HSD converts the high-activity
17.beta.-estradiol to the low-activity estrone and high-activity
testosterone to low-activity androstenedione and dihydrotestosterone
 It has been reported that 40% of all cancers, namely breast,
prostate, ovarian and uterine cancers are sex steroid sensitive
and are thus candidates for approaches based on control of intracrine
activity. Therefore it is suggested to treat sex steroid sensitive
diseases by specific inhibitors of the 17HSD enzymes.
 Molecules able to block the enzyme activity have been screened.
Characteristics of such inhibitory molecules, which mainly have
a substrate or cofactor-like core structure, have been reported
in the literature (Penning Endocrine-Related Cancer (1996) 3, 41-56;
Tremblay and Poirier J Chem Soc Perkin Trans 1, (1996), 2765-71;
Poirier et al., J Steroid Biochem Molec Biol (1998), 64, 83-90).
Tremblay and Poirier describe an estradiol derivative, 16-[carbamoyl(bromomethyl)alkyl]-estradiol,
and tested the same in respect of its inhibition of the estradiol
formation catalyzed by the enzyme 17HSD type 1. It was found that
this compound moderately inhibited the biosynthesis of estradiol.
Poirier et al. describe 6.beta.-thiaheptan butyl methyl amide derivative
of estradiol as a potent and selective inhibitor of the 17HSD type
1 enzyme. However, the ability of these molecules to inhibit 17HSD
type 2 enzyme has not been tested.
 There are certain criteria for 17HSD type 1 inhibitors to
be used as therapeutic agents. The inhibitor shall be specific for
the enzyme in question, i.e. it shall have little or no affinity
for other 17HSDs participating in the estrogen metabolism. In particular,
it should not possess substantial affinity to 17HSD type 2 enzyme.
The inhibitors should not otherwise prevent or modulate steroidogenesis,
either. In addition, the inhibitor molecules should lack affinity
for the estrogen receptor, thus eliminating any possible agonist
 Using in situ hybridization, both 17HSD type 1 and 2 have
been detected in benign breast tissue (Miettinen et al., Breast
Cancer Res Treat (1999) 57, 175-182). The activities and mRNAs of
both enzymes are also present in several breast cancer cell lines
(Miettinen et al., Biochem J (1996) 314,839-845). Expression of
17HSD type 1 in about half of breast cancer specimens has been shown
using immunohistochemistry (Poutanen et al., Int J Cancer (1992)
 The growth-promoting influence of 17HSD type 1 has been
demonstrated in cultured MCF-7 breast cancer cell line (American
Type Culture Collection ATCC) transfected with cDNA of 17HSD type
1 (Miettinen et al., Int J Cancer (1995) 68, 600-604). In nontransfected
MCF-7 cells, estradiol induces cell proliferation but estrone does
not have any effect on the cell growth. In cells stably transfected
with 17HSD type 1, both estradiol and estrone have similar growth-promoting
effects showing that 17HSD type 1 is needed to convert estrone to
 In a review article on 17.beta.-hydroxysteroid dehydrogenases
and cancer Vihko et al (Journal of Steroid Biochemistry & Molecular
Biology, vol 83(1-5), p. 119-122, 2002) show that both type 1 and
2 17HSD enzymes are present in normal breast and in malignant breast
cells. No evidence of the roles of these enzymes in the prognostication
of breast cancer is shown.
 Gunnarsson et al (Cancer Research, vol 61, p. 8448-8451,
2001) have studied the expression of type 1 and 2 17HSD in two selected
patient groups (both 24 patients) by PCR. They found out that the
lack of type 2 enzyme combined with high content of type 1 enzyme
is a prognostic factor for breast cancer. The role of type 1 enzyme
alone as an independent prognostic factor has not been shown.
 Gunnarsson et al (Oncogene, vol 22(1), p. 34-40, 2003) have
also studied the amplification of type 1 17HSD gene in breast cancer.
They state that the amplification of HSD17B1 gene is a prognostic
factor in breast cancer. However, the material they used was not
statistically significant (p=0.052). Furthermore, the amplification
of a gene and the expression thereof are different subjects. It
is not self-evident that the levels of genes, mRNAs and proteins
correlate with each other.
 The use of 17HSD type 1 enzyme in breast tumor has not been
suggested as a marker for prognosticating the progress of breast
cancer. Moreover, there are no reports on pharmaceutical preparations
comprising 17HSD type 1 enzyme inhibitors for use in prevention
or treatment of breast cancer.
SUMMARY OF THE INVENTION
 According to one aspect, this invention concerns an in vitro
method for prognosticating the progress of breast cancer, comprising
detecting or quantifying the level of 17.beta.-hydroxysteroid dehydrogenase
(17HSD) type 1 enzyme in breast cancer tissue sample, wherein the
presence of said 17HSD type 1 enzyme is independently indicative
of severe progress of breast cancer.
 According to another aspect, the invention concerns a pharmaceutical
composition comprising compound A as disclosed in Figure I, or a
pharmaceutically acceptable salt of said compound.
 According to further aspect, this invention concerns the
use of compound A as disclosed in FIG. 1, or a pharmaceutically
acceptable salt of said compound, in the manufacture of a pharmaceutically
acceptable preparation useful as 17HSD type 1 inhibitor.
 According to still further aspect, this invention concerns
the use of said 17HSD type 1 inhibitor, compound A, for prevention
or treatment of disorders caused by the 17HSD type 1 enzyme activity,
such as human breast cancer. In this respect, postmenopausal women
form a special group since 17HSD type 1 is not expressed in the
normal breast tissue of postmenopausal patients.
 According to still further aspect, this invention concerns
a method for prevention or treatment of disorders caused by the
17HSD type 1 enzyme activity, such as human breast cancer, by administering
effective amount of compound to a patient suffering from said disorder.
BRIEF DESCRIPTION OF THE DRAWINGS
 FIG. 1 shows the structure of compound A
DETAILED DESCRIPTION OF THE INVENTION
 The inventors of the present invention have carried out
a patient study in which cancer tissue specimens from 794 breast
cancer patients were analyzed. The expression of 17HSD type 1 and
type 2 mRNAs were analyzed in cancer tissue specimens using in situ
-hybridization technique as described previously (Oduwole et al.,
Int J Cancer (2002) 97, 1-6). The immunohistochemical stainings
of 17HSD type 1 protein were made using a method described previously
(Poutanen et al., Int J Cancer (1992) 50, 266-390).
 In the study it was found that breast cancer patients with
breast tumors expressing 17HSD type 1 have significantly shorter
overall and disease-free survival than all other cases (p=0.0010,
0.0134, log rank). The probability of metastasis formation is higher
among these patients.
 Type 1 17HSD as well as type 2 are expressed in normal breast
tissue in premenopausal women. However, most women having the breast
cancer are postmenopausal and were shown not to express type 1 17HSD
in their normal breast tissue.
 In the breast cancer tissue material from 794 breast cancer
patients, multivariate Cox analysis (forward stepwise regression)
was used to determine the possible independent prognostic significance
of the following parameters: tumor size, the presence of nodal and
distant metastases, grade of the tumor, estrogen receptor (ER).alpha.,
ER.beta., progesterone receptor, 17HSD type 1, type 2, type 5, Ki67
and c-erb-b2. According to the analysis tumor size, 17HSD type 1
and ER.alpha. had independent prognostic value (Table 1). TABLE-US-00001
TABLE 1 Multivariate Cox analyses (forward stepwise regression)
for prognostic factors 95% CI for RR Step Factors Sig RR Lower Upper
Step T <0.001 2.424 1.676 3.504 1 17HSD1 <0.001 4.245 1.968
9.159 Step T <0.001 2.734 1.843 4.056 2 17HSD1 <0.001 4.186
1.927 9.096 ER.alpha. <0.013 0.408 0.201 0.829 Sig: significance;
RR: relative risk; CI: confidence interval, T: size of the tumor,
ER: estrogen receptor
 Based on these findings, the inventors suggest that the
expression of 17HSD type 1 enzyme in breast cancer tissue is indicative
of severe progress of breast cancers. Furthermore this enzyme can
be considered as an independent marker for such severe progress
of breast cancer. Such independent markers are not previously known
in the art. In postmenopausal women it is also a breast cancer marker.
The adequate level of said expression is such that it can be detected
using standard techniques well known in the art.
 The detection or quantification of the 17HSD type 1 enzyme
can be performed by any known suitable method. Such methods include
for example hybridizing techniques; PCR techniques or immunological
methods based on detection of an antibody recognizing the enzyme.
The hybridizing techniques include, for example nucleotide hybridization
and Northern blot. The detection or quantification of the antibody
can be performed according to standard immunoassay protocols, such
as label-linked immunosorbent assays, Western blot and immunohistochemical
methods. These methods are well known to a person skilled in the
 Examples of such methods for determining the level or expression
of 17HSD type 1 enzyme and other related methods are described in
Cancer Research (2004) 65, 7604-7609: "17.beta.-Hydroxysteroid
Dehydrogenase Type I Is an Independent Prognostic Marker in Breast
Cancer". This is the corresponding publication by the current
inventors and it is incorporated herein by reference.
 The inventors of the present invention have surprisingly
found a small molecule from a vast amount of compounds from a commercial
library (BioSpecs Inc. The Netherlands) which molecule is capable
of specifically inhibiting 17HSD type 1 enzyme but not 17HSD type
2 enzyme. The screening of the library compounds was performed using
two specific cell lines made by the inventors, which cell lines
stably expressed either 17HSD type 1 or 17HSD type 2 enzyme, but
not the other one.
 For the useful effect of 17HSD type 1 enzyme inhibitor it
is essential that the inhibitor is substantially specific for type
1 enzyme and not for type 2 enzyme, since these enzymes have contrary
effects. The inhibition of type 1 enzyme will have a positive effect
on the treatment of disorders caused by the 17HSD type 1 enzyme
activity, such as breast cancer and its progression. On the other
hand, the inhibition of type 2 enzyme would have a negative effect
when treating such disorders, because it is beneficial to have 17HSD
type 2 activity inactivating estradiol. To find such a molecule
from a vast amount of compounds is an extreme task and may not have
been possible without the specific cell lines mentioned above.
 An inhibitor of the invention includes the compound A of
FIG. 1, a pharmaceutically acceptable salt of said compound or a
derivative of said compound. The expression `derivative` used herein
denotes any compound derived using the original compound as the
 For the purpose of this invention, the 17HSD enzyme inhibitor
or its pharmaceutically acceptable salt or derivative can be administered
by various routes and as various pharmaceutical forms well known
in the art. The suitable administration forms include, for example,
oral formulations; topical formulations; parenteral injections including
intravenous, intramuscular, intradermal and subcutaneous injections;
and transdermal or rectal formulations.
 Suitable oral formulations include e.g. tablets, lozenges
or capsules prepared by conventional means with pharmaceutically
acceptable excipients such as binding agents (e.g. pregelatinized
maize starch, polyvinylpyrrolidone or hydroxypropyl methyl cellulose),
fillers (e.g. lactose, microcrystalline cellulose or calcium hydrogen
phosphate), lubricants (e.g. magnesium stearate, talc or silica),
disintegrants (e.g. potato starch or sodium starch glycolate) or
wetting agents (e.g. sodium lauryl sulphate). The tablets may be
coated by methods well known in the art. Liquid preparations for
oral administration may take the form of for example solutions,
syrups or suspensions, or they may be presented as a dry product
for constitution with water or other suitable vehicle before use.
Such liquid preparations may be prepared by conventional means with
pharmaceutically acceptable additives such as suspending agents
(e.g. sorbitol syrup, cellulose derivatives or hydrogenated edible
fats), emulsifying agents (e.g. lecithin or acacia), non-aqueous
vehicles (e.g. almond oil, oily esters, ethyl alcohol or fractionated
vegetable oils) and preservatives (e.g. methyl or propyl-p-hydroxybenzoates
or sorbic acid). The preparations may also contain buffer salts,
flavoring, coloring and sweetening agents as appropriate.
 Preparations for oral administration may be suitably formulated
to give controlled release of the active compound.
 Suitable formulations for parenteral administration include
e.g. bolus injection or continuous infusion. Formulations for injection
may be presented in unit dosage form, e.g. in ampoules or in multi-dose
containers, with an added preservative. The compositions may take
such forms as suspensions, solutions or emulsions in oily or aqueous
vehicles, and may contain formulatory agents such as suspending,
stabilizing and/or dispersing agents. Alternatively, the active
ingredient may be in powder form for constitution with a suitable
vehicle, e.g. sterile pyrogen-free water, before use.
 The required dosage of the 17HSD enzyme type 1 inhibitor
will vary with the particular breast cancer being treated or prevented,
the severity of the condition, the duration of the treatment, the
administration route and the specific compound being employed. Suitable
dose ranges may be calculated by those skilled in the art.
 The invention will now be illuminated by the following non-restrictive
 Compound A, which is known per se and the structure of which
is shown in FIG. 1, was purchased from BioSpecs Inc. The compound
is disclosed in a BioSpecs' database (CD disk September 1998; database
SB8I-20T.db; compound A: ID-number AH-262/33341026). The use of
this compound as a medicament has not been reported previously.
 The tested compounds (about 1000 selected different compounds)
were screened in respect of 17HSD enzyme activities according to
the following method:
 The compounds were screened in respect of 17HSD enzyme activities
in vitro on two established MCF-7 cell lines, each stably expressing
one 17HSD isoenzyme, either 17HSD type 1 or 17HSD type 2, but not
the other one. The interconversion of substrate by each isoenzyme
and the HSD-inhibiting activity of chemical compounds in these cell
lines was detected by HPLC system (Miettinen et al., Int J Cancer
(1996) 68, 600-604).
 Varying amounts of the test compounds were incubated in
the growth medium of the 17HSD expressing cells together with tritium
labeled substrate (estrone for 17HSD type 1 enzyme; 20 nM). The
medium samples were removed after exact incubation time and the
reaction was stopped by deepfreezing. The samples were analyzed
by HPLC-coupled flow scintillation analysis. The 17HSD-inhibiting
activity of the test compounds was compared to known reference compound
apigenin and to a negative control.
 For one of the most potent 17HSD type 1 inhibitors, Compound
A, the inhibition of the 17HSD type 1 enzyme was 80% at a concentration
of 1 .mu.M (average of three tests: 81%, 77% and 83%) and 95% at
a concentration of 10 .mu.M (average of two tests: 94 and 96%).
The reference compound apigenin gave an inhibition activity of 48%
at a concentration of 1 .mu.M. The tested compound did not show
any inhibition effect on the 17HSD type-2 enzyme (the reference
compound apigenin gave a 5% inhibition of the 17HSD type-2 enzyme).
Based on this test results, this compound can be considered as a
very potent and selective 17HSD-1 inhibitor.
 It will be appreciated that the methods of the present invention
can be incorporated in the form of a variety of embodiments, only
a few of which are disclosed herein. It will be apparent for the
expert skilled in the field that other embodiments exist and do
not depart from the spirit of the invention. Thus, the described
embodiments are illustrative and should not be construed as restrictive.