Proliferation of breast cancer is treated by a combined type I
interferon/antiestrogen therapy. Preferably artiestrogen treatment
is sequential to treatment with interferon.
What is claimed is:
1. A method of inhibiting the proliferation of breast cancer cells
which comprises sequentially administering to a host in need thereof
an effective inhibiting amount of a combination of beta interferon
and temoxifen wherein the interferon is administered intramuscularly
in an amount of 2 to 10 million international units and temoxifen
is orally administered in a daily amount of about 30 milligrams.
2. The method of claim 1 in which the amount of tamoxifen is orally
administered in a daily amount of about 30 milligrams.
3. A method of inhibiting the proliferation of breast cancer cells
in a human patient which comprises administering interferon beta
and tamoxifen sequentially to said patient in which the interferon
beta is administered in an amount to increase estrogen receptor
levels in the absence of tamoxifen administration during a first
treatment period and thereafter an effective inhibiting amount of
tamoxifen is administered to the patient during a second treatment
4. The method of claim 3 in which the first treatment period is
1 to 4 weeks.
5. The method of claim 4 in which the second treatment period is
8 to 12 weeks.
6. The method of claim 5 in which the interferon beta is administered
three times per week for 2 to 4 weeks.
7. The method of claim 6 in which the tamoxifen is administered
8. The method of claim 5 in which the interferon beta and tamoxifen
are administered daily.
9. The method of claim 5 in which interferon beta is additionally
administered periodically during the second treatment period.
10. The method of claim 9 in which the interferon beta is administered
intramuscularly at about 2 to 10 million international units per
11. The method of claim 10 in which the tamoxifen is administered
orally at about 30 milligrams per day.
12. The method of claim 3 in which the interferon beta is administered
intramuscularly in an amount of about 2 to 10 million international
units per day.
13. The method of claim 3 in which the tamoxifen is administered
orally at about 30 milligrams per day.
This invention relates to the field of breast cancer therapy. More
particularly, the invention relates to the treatment of breast cancer
by administration of type I interferon in conjunction with an antiestrogen.
A preferred embodiment comprises the sequential administration
of type I interferon and antiestrogen.
Breast cancer causes the death of a quarter of a million women
worldwide each year and is estimated to be the leading cause of
death in women aged between 35 to 54, being second only to cardiovascular
diseases in women aged over 55 (LOGAN W. P. D.: Cancer of the female
breast. International mortality trends. W.H.O. Stat. Rep. 28:232,
Breast cancer accounts for 27% of all malignancies around the world.
Historically, the first to discover the role played by endocrine
treatment in breast cancer was BEATSON (1896) who observed that
breast cancer in pre-menopausal women undergoes remission after
This finding, subsequently confirmed by other scientists, supported
the evidence that at least some breast tumors are directly dependent
on hormones for their growth and created interest in the therapeutic
approach of endocrine organ ablation for the purpose of removing
the endogenous source of hormones.
As drugs specifically antagonizing the oestrogen action were discovered,
they became an attractive alternative to surgical ablation.
Several anti-oestrogen compounds have been tested in pre- and post-menopausal
women in phase I and II clinical trials. So far, Tamoxifen has proved
to be the drug best approaching the effectiveness of surgical endocrine
therapy and the one that is substantially free from serious side
A comprehensive review of the therapeutic efficacy of antiestrogens
in the treatment of breast cancer is LEGHA S. S. and CARTER S. K.:
Anti-estrogens in the treatment of breast cancer. Cancer Treat.
Rev. 3:205, 1976.
Another review more specifically related to clinical experience
with Tamoxifen is that of PATTERSON J. S., et al: A review of the
International clinical experience with Tamoxifen. Jpn. J. Cancer
clin. 11 (Suppl.): 157, 1981.
Approximately one-third of women with breast cancer respond to
antiestrogen-based hormonal therapy, while an increase up to 70%
of response is expected in patients with receptor-rich tumours.
In fact, oestrogen receptor (ER) status has been demonstrated to
be predictive of response in breast cancer patients--ALLEGRA J.
C.: Reviews on Endocrine related cancer. (Paterson A. H. G., Lees
A. W. eds) Suppl. 14:115, 1984.
Interferons are a well-known family of proteins which have been
shown to possess both antiviral and breast cell growth inhibitory
effects. Human interferons are grouped into three classes based
upon differences in biological and immunological properties as well
as molecular structures.
Interferons are also classified, in accordance with their chemical
sensitivity to acid pH, into two types:
TYPE I: Acid-stable (alpha from leukocytes, alpha from lymphoblasts,
beta from fibroblasts);
TYPE II: Acid-labile (gamma from lymphocytes).
Interferons have a wide range of cellular effects on breast cancer,
as well as on normal cells, including such effects on cell phenotype
as antigen expression, cell receptors and so on.
The mechanism by which interferons regulate human cell growth has
not been completely elucidated.
In particular, very few data are available concerning the antiproliferative
activity of interferons on human mammary neoplastic cells and little
is known about the factors determining sensitivity of these cells
to the interferon action--BORDEN E. C., et al: Comparative antiproliferative
activity in vitro of natural interferons for diploid and transformed
human cells. Cancer Res. 42:4948-4953, 1982.--STRAYDER D. R., et
al: Antiproliferative effect of natural beta interferon on fresh
tumor cells analyzed in a clonogenic assay. J. Interferon Res. 4:627-633,
Experimental evidence exists that type I IFN modifies the hormone
receptor level in breast cancer tissue cells, POUILLART T., et al,
in: "Administration of fibroblast interferon to patients with
advanced breast cancer : possible effects on skin metastasis and
on hormone receptors" (Eur. J. Cancer Clin. Oncol. 18:929-935,
1982) described the effect of human fibroblast interferon administered
to patients with metastasised breast cancer and found an increase
of the receptors for estrogens and progestogens.
DIMITROV N. V., et al, in: "Interferon as a modifier of estrogen
receptor" (Ann. clin. Lab. Sci. 14:32-39, 1984) demonstrated
that human leukocyte interferon increases estrogen receptor activity
in a cell homogenate of human breast cancer tissue.
Contradictory experimental evidence is that of MARTH Ch., et al,
in: "Effects of human Interferon Alpha-2 and Gamma on proliferation,
estrogen receptor content, and sensitivity to anti-estrogens of
cultured breast cancer cells" (The Interferon System, Ed. F.
Dianzani, G. B. Rossi, Serono Symposia Publs. from Raven Press,
Vol. 24, 1985) who were unable to demonstrate any influence by interferon
alpha on estrogen receptor content in cultured breast cancer cells.
The conclusion thus reached by the Authors is that estrogen action
is independent of interferon since the growth inhibition by anti-estrogens
is not affected by interferon treatment.
In accordance with the present invention, the combined treatment
with type I interferon and anti-estrogen has been shown to be highly
effective in treating the proliferation of the growth of breast
cancer cells. Particularly good results are obtained when the administration
of the antiestrogen is sequential to a treatment with type I interferon.
It is therefore an object of this invention to provide a method
of treating the proliferation of breast cancer by administering
type I interferon in conjunction with an anti-estrogen.
A further object of this invention is to provide a method of treating
the proliferation of breast cancer by the sequential administration
of type I interferon and anti-estrogen.
Within the meaning of "type I interferon" both alpha
and beta human interferons are included. The interferons can be
either "native", that is obtained from natural human sources
or cell lines, or "recombinant", that is obtained from
genetically engineered or otherwise modified bacterial, yeast or
Although Tamoxifen is the preferred antiestrogen, other substances
having analogous activity are within the meaning of "antiestrogen"
in accordance with the present invention. Suitable examples are
found, e.g., in the above mentioned review by Legha and Carter.
The finding on which this invention is based is that the new combined
treatment of breast cancer cells gives results that could not be
predicted from the known antiproliferative effect of interferon
or the demonstrated efficacy on an antiestrogen when used separately.
Preliminary in vitro experiments were carried out on CG-5 cells,
a variant of the MCF-7 cell line characterized by a high degree
of estrogen responsiveness and an appreciable content of estrogen,
androgen, glucocorticoid and progesterone receptors--Natoli C. et
al: Two new estrogen supersensitive variants of the MCF-7 human
breast cancer cell line--(Breast Cancer Res. Treat. 3, 23-32, 1983).
Cells were routinely cultured in Dulbecco's modified Eagle's medium
(DMEM) supplemented with 10% fetal calf serum (FCS) and antibiotics.
For cell growth experiments cells were plated out at the density
of 50,000 cells /ml in the medium described above. 24 hours later,
the medium was replaced with fresh medium containing 5% charcoal-treated
FCS (CH-FCS) plus a fixed concentration (10.sup.-7 M) of Tamoxifen
(TAM) and various concentrations (from 10 to 1000 IU/ml) of interferon
beta (IFN). Medium was renewed every 3 days.
In the experiments concerning the effects of IFN and TAM sequentially
added to CG-5 cells, cells were plated out at 50,000 cells/ml, as
described above, and 24 hours later DMEM was changed with fresh
medium containing 10 to 1000 IU/ml of IFN. For each concentration
of IFN a different number of plates were prepared in order to have,
at the end of the treatment, a sufficient number of cells to be
replaced (as IFN has an inhibitory effect itself). After 1 week
of exposure to IFN, CG-5 cells were plated in medium supplemented
with 10% FCS and antibiotics, and 24 hours later DMEM was replaced
by fresh medium supplemented with 5% CH-FCS and a fixed concentration
of TAM (10.sup.-7 M). Medium was renewed every 3 days.
In all the experiments performed cells were counted, after 3 to
6 days, with the use of an hemocytometer.
The addition to CG-5 cultures of a fixed concentration of TAM (10.sup.-7
M), combined to concentrations ranging from 10 to 1000 IU/ml of
IFN, produces an inhibition of cell proliferation which is not related
to the dose of IFN, but is higher than that induced by TAM alone,
even at the lowest concentration of IFN (about 50% with respect
to control) on the third day from the addition of the two drugs
to the culture medium.
When cells are treated for 6 days with the combination TAM-IFN,
the inhibition of cell proliferation becomes dependent on the dose
of IFN and reaches 65% with respect to controls at 1000 IU/ml of
If CG-5 cells are pretreated with different concentrations of IFN
and subsequently exposed to 10.sup.-7 M TAM, 3 days after the addition
of the antiestrogen to the culture medium, a relevant inhibition
of cell proliferation is seen (approximately 50% with respect to
control) in the cells which received the highest concentration of
IFN. On the sixth day after the addition of TAM to the culture medium,
the most pronounced inhibition of cell proliferation is obtained
in CG-5 cells pretreated with the lowest concentration of IFN (about
65% with respect to control) and it remains unmodified in cells
pretreated with increasing doses of the drug.
Results are summarized in FIG. 1, a and b, which illustrate graphically
the comparison between the different modalities (combined or sequential
treatment) used to study the effect of Tamoxifen and Interferon
on cell growth.
Graphs a and b demonstrate the effect of Interferon and Tamoxifen
added simultaneously (large dot symbol) or sequentially (triangular
symbol) to CG-5 cells on the third (a) and sixth (b) day from the
addition of the compounds to the culture medium. In the case of
sequential administration cells were pretreated with the Interferon
concentration indicated in the figure and then exposed to Tamoxifen.
The dotted line (---) represents the effect of 10.sup.-7 M Tamoxifen
alone, evaluated in parallel experiments not reported in the text.
FIG. 2 shows the effect of Interferon alone on the growth of CG-5
cells cultured in identical experimental conditions. In this case
the inhibition of cell proliferation is evident after three days
of exposure to the Interferon starting from the concentration of
100 IU/ml. After six days of treatment with Interferon, the inhibitory
effect on cell proliferation significantly increases only at the
maximum dose of 1000 IU/ml.
The comparison between FIGS. 1 and 2 clearly shows that such low
doses of Interferon as 10 IU/ml result efficacious when combined
with the anti-estrogen, whereas the same doses are practically ineffective
if Interferon is used alone.
Similar conclusions are reached if the effect of Tamoxifen alone
is compared with the combined effect of Tamoxifen with Interferon.
The efficacy of the combined therapy according to this invention
is established by clinical trials conducted in patients responding
to the following criteria:
Patients with superficial biopsiable, histologically confirmed
advanced breast cancer;
Patients in posrmenopausa;
ER/PR status: positive or unknown (provided at least 2 years of
disease free interval);
No concurrent radiotherapy or chemotherapy;
No brain metastases.
Each patient receives intramuscular injections of interferon doses
ranging from 2 to 10 million International Units and is given orally
a fixed dose of the antiestrogen corresponding to the dose known
to be optimal for breast cancer treatment. For example, in the case
of Tamoxifen, the most suitable daily dose is 30 milligrams.
When interferon alpha is used, injected doses are generally higher
than the doses of interferon beta.
Examples of suitable treatment schedules are as follows:
1) Interferon administered three times a week for two to four weeks;
Antiestrogen administered daily for 8 weeks starting immediately
after discontinuation of interferon administration.
2) Interferon administered three times a week for two to four weeks;
Antiestrogen administered daily for 12 weeks starting on the same
day the interferon treatment starts.
3) Interferon administered daily for one to four weeks;
Antiestrogen administered daily for 12 weeks starting the day after
the last interferon administration.
4) The same treatment schedule (3) above with once a week additional
administration of interferon in the period of antiestrogen therapy.
The main criterion of evaluation is the evidence of response documented
on the basis of tumor regression assessed by measurement of palpable
lesions and changes in X-Ray or computerized tomography scan or
ultrasonic echography appearances.
The increase of Estrogen receptors in the patients is also taken
into consideration as a laboratory datum supplementing clinically
The efficacy of the combined interferon antiestrogen treatment
of the proliferation of breast cancer is thus established.
Various changes and modifications can be made in the therapeutic
method of the present invention without departing from the spirit
and scope thereof. The embodiments described herein are for the
purpose of illustrating the invention but are not intended to limit