U.S. patent application number 09/990646 was filed with the patent office on 2002-10-24 for method of regulating the female reproductive system through angiogenesis inhibitors.
Invention is credited to D'Amato, Robert J., Demore, Nancy Klauber.
Application Number | 20020156055 09/990646 |
Document ID | / |
Family ID | 21814780 |
Filed Date | 2002-10-24 |
United States Patent
Application |
20020156055 |
Kind Code |
A1 |
D'Amato, Robert J. ; et
al. |
October 24, 2002 |
Method of regulating the female reproductive system through
angiogenesis inhibitors
Abstract
A method of inhibiting angiogenesis in a female mammal to
regulate fertility comprising administering to the female mammal an
effective amount of an angiogenesis inhibiting compound. In
addition, a method of inhibiting angiogenesis in a female mammal to
treat a disease or condition of the reproductive tissue that is
mediated by angiogenesis comprising administering to the female
mammal an effective amount of an angiogenesis inhibiting compound.
AGM-1470 is provided as such an angiogenesis inhibiting
compound.
Inventors: |
D'Amato, Robert J.;
(Cambridge, MA) ; Demore, Nancy Klauber; (Durham,
NC) |
Correspondence
Address: |
David S. Resnick
NIXON PEABODY LLP
101 Federal Street
Boston
MA
02110
US
|
Family ID: |
21814780 |
Appl. No.: |
09/990646 |
Filed: |
November 21, 2001 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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09990646 |
Nov 21, 2001 |
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09313528 |
May 17, 1999 |
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6441027 |
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09313528 |
May 17, 1999 |
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08904708 |
Aug 1, 1997 |
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6017949 |
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60023385 |
Aug 2, 1996 |
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Current U.S.
Class: |
514/169 |
Current CPC
Class: |
A61P 15/18 20180101;
A61K 31/335 20130101; A61K 31/336 20130101 |
Class at
Publication: |
514/169 |
International
Class: |
A61K 031/56 |
Claims
We claim:
1. A method of inhibiting angiogenesis in a female mammal to
regulate fertility comprising administering to the female mammal an
effective amount of a fertility regulating angiogenesis inhibiting
compound, thereby inhibiting angiogenesis in the female mammal to
regulate fertility.
2. The method of claim 1, wherein the angiogenesis inhibiting
compound is administered to prevent conception.
3. The method of claim 1, wherein the angiogenesis inhibiting
compound is administered to terminate a pregnancy.
4. The method of claim 1, wherein angiogenesis is inhibited in the
uterus, ovary, placenta or fetus.
5. The method of claim 1, wherein the angiogenesis inhibiting
compound is AGM-1470.
6. The method of claim 1, wherein the angiogenesis inhibiting
compound is an antagonist of the AGM-1470 receptor MetAP-2.
7. The method of claim 1, wherein the angiogenesis inhibiting
compound is administered in a single dose or in multiple doses.
8. The method of claim 1, wherein angiogenesis is inhibited prior
to, during, or after decidualization, placental formation, yolk sac
or fetal development.
9. The method of claim 1, wherein the angiogenesis inhibiting
compound is administered prior to intercourse, after intercourse or
after ovum fertilization.
10. A method of inhibiting angiogenesis in a female mammal to treat
a disease or condition of the reproductive tissue that is mediated
by angiogenesis comprising administering to the female mammal an
effective amount of a female reproductive tissue angiogenesis
inhibiting compound, thereby inhibiting angiogenesis in a female
mammal to treat the disease or condition of the reproductive
tissue.
11. The method of claim 10, wherein the angiogenesis mediated
disease or condition is endometriosis, adenomyosis, dysfunctional
uterine bleeding, uterine leiomyoma, an ovarian cyst,
choriocarcinoma or ectopic pregnancy.
12. The method of claim 10, wherein angiogenesis is inhibited in
the uterus, ovary, placenta, fetus or ectopic tissue originating
from the uterus or ovary.
13. The method of claim 10, wherein the angiogenesis inhibiting
compound is AGM-1470.
14. The method of claim 10, wherein the angiogenesis inhibiting
compound is an antagonist of the AGM-1470 receptor MetAP-2.
15. The method of claim 10, wherein the angiogenesis inhibiting
compound is administered in a single dose or in multiple doses.
Description
CROSS-REFERENCE TO RELATED APPLICATION
[0001] This application claims priority to U.S. Provisional
Application No. 60/023,385, filed Aug. 2, 1996.
FIELD OF THE INVENTION
[0002] The present invention relates to the use of angiogenesis
inhibiting compounds to reversibly inhibit the female mammalian
reproductive system. These angiogenesis inhibiting compounds are
capable of controlling fertility, terminating a pregnancy, as well
as controlling pathological conditions and disorders of the female
reproductive system.
BACKGROUND OF THE INVENTION
[0003] As used herein, the term "angiogenesis" means the generation
of new blood vessels into a tissue or organ. Under normal
physiological conditions, humans or animals undergo angiogenesis
only in very specific restricted situations. For example,
angiogenesis is normally observed in wound healing, the female
reproductive cycle and embryonic development..sup.1 Angiogenesis is
also critical in the progression of many disease states, such as
hemangioma, endometriosis, solid tumors and macular
degeneration.
[0004] Through angiogenesis, endothelial cells and pericytes,
surrounded by a basement membrane, form capillary blood vessels.
Angiogenesis begins with the erosion of the basement membrane by
enzymes released by endothelial cells and leukocytes. The
endothelial cells, which line the lumen of blood vessels, then
protrude through the basement membrane. Angiogenic stimulants
induce the endothelial cells to migrate through the eroded basement
membrane. The migrating cells form a "sprout" off the parent blood
vessel, where the endothelial cells undergo mitosis and
proliferate. The endothelial sprouts merge with each other to form
capillary loops, creating the new blood vessel.
[0005] Angiogenesis occurs during the cyclical changes of the
female reproductive system. In preparation for fertilization, the
theca interna of the ovaries becomes richly vascularized to support
the growth of a follicle. During ovulation, the follicle ruptures
and an egg is released to the uterus. If fertilization occurs, the
ruptured follicle then is invaded by thecal vessels and forms the
corpus luteum. The corpus luteum releases hormones that regulate
reproductive processes, including the thickening of the endometrium
for the implantation of the fertilized ovum. After the blastocyst
implants in the endometrium, the placenta develops and provides
nutrients for the growing embryo. Throughout all of these stages of
mammalian reproduction, angiogenesis is critical to further
development.
[0006] Specifically, angiogenesis occurs during follicular growth,
formation of the corpus luteum, growth of the endometrium, and
development of the placenta and embryo after
conception..sup.1,2,3,4 These angiogenic episodes are self-limiting
and presumably tightly regulated. During the menstrual cycle, the
spiral arteries undergo substantial anatomical changes. As the
endometrium thickens three to five fold during the next menstrual
cycle, the remnants of the spiral arteries must undergo substantial
growth and give rise to a new capillary bed in order to maintain
the integrity of the rapidly growing stroma. This uterine
angiogenesis provides an existing vascular supply for the
trophoblast to invade if fertilization of the ovum occurs.
Following fertilization, the first stage of implantation is the
adhesion of the blastocyst to the endometrial epithelium. This is
followed by the penetration of the trophoblast through the
epithelial lining..sup.7
[0007] Physiological changes in the ovary are especially critical
for the proper functioning of the female reproductive cycle. In the
ovary, during the course of follicular growth, the theca interna
becomes richly vascularized. Follicular maturation is associated
with increasing angiogenesis, whereas follicles undergoing atresia
are associated with decreasing vascularity..sup.8 Following
ovulation, growth factors are expressed which induce the thecal
vessels to grow and invade the ruptured follicle and form a complex
capillary network which nourishes the developing corpus
luteum..sup.9,10,11,2,12 Approximately 50% of the cells of the
mature bovine corpus luteum are endothelial cells, and in the
primate corpus luteal endothelial cells comprise 85% of
proliferating cells..sup.4
[0008] The mitotic activity of these endothelial cells is highest
during the early luteal phase, persists but declines during the
midluteal phase and is minimal during luteal regression. Corpus
luteal maintenance during pregnancy is associated with continued
angiogenesis..sup.4 This has been demonstrated in the pregnant rat
where the labeling index of endothelial cells in the corpus luteum
increases and peaks on embryonic day 14 (E14) which correlates with
further growth of the corpus luteum..sup.5 Angiogenesis and its
hormonal control in the corpus luteum of the pregnant rat..sup.13
Thus, the exponential growth of the corpus luteum is associated
with aggressive neovascularization. This vasculature plays a vital
role in providing nutrients and trophic factors to and transporting
secreted hormones from the developing follicle and corpus
luteum..sup.14
[0009] The factors controlling ovarian angiogenesis are just
beginning to be fully understood. Two growth factors demonstrated
in the ovary are basic fibroblast growth factor (bFGF) and vascular
endothelial growth factor (VEGF). Cultured granulosa cells produce
bFGF, the release of which may be modulated by heparin sulfate
proteoglycans which are produced under gonadotropin modulation.
Recent evidence suggests that vascular endothelial growth factor
(VEGF), an endothelial cell specific mitogen and permeability
factor, may play an integral role in ovarian
angiogenesis..sup.15,16,17
[0010] VEGF, a secreted homodimeric glycoprotein, is under
gonadotropin control. It is both temporally and spatially
correlated with active angiogenesis in the theca and the corpus
luteum. Thus, as is the case with other tissues throughout the
body, the ovary has multiple stimulators of angiogenesis including
VEGF and bFGF which are likely to be in balance with endogenous
inhibitors to achieve the appropriate level of blood vessel
growth..sup.18,19,20,21,22,23 In addition, the uterus undergoes
dramatic physiological changes in the course of the-female
reproductive cycle. Under hormonal regulation by estrogen and
progesterone, the uterus undergoes differentiation into a structure
which is capable of supporting the implantation of a blastocyst.
The cyclical maturation of the endometrium in the uterus is driven
by the mitogenic activity of the spiral arteries. Following
implantation, the maternal spiral arteries within the endometrium
are invaded by trophoblasts that adhere to and migrate along the
surface of the vascular endothelial cells.
[0011] Currently, biochemical female reproductive control is
primarily accomplished through regimens of hormones such as
estrogen and progesterone. Although this method of birth control is
quite effective in preventing pregnancy, there are many side
effects to the administration of additional hormones. Often such
forms of birth control cause emotional and physiological
disturbances, resulting in mood shifts or loss of libido. Health
risks such as stroke and heart problems increase for women who
smoke while using hormonal birth control. In addition, these forms
of fertility control are only potent when taken on a regularly
basis. The administration of hormones is advised to be discontinued
should fertilization occur to diminish chances of health
complications..sup.24
[0012] Many factors, chemical as well as mechanical, have been
shown to be capable of promoting or inhibiting angiogenesis in vivo
and in vitro..sup.25 Although the presence of angiogenesis
promoting factors during growth and development of the ovaries,
uterus, and placenta have been evaluated, the effects of
angiogenesis inhibiting factors on these organs have not be
elucidated. Since angiogenesis is an important component in the
female reproductive system, it might be possible to regulate
reproductive processes with angiogenesis inhibiting factors.
[0013] There are a large number of known angiogenesis inhibiting
compounds which will undoubtedly continue to grow as scientific
research continues. Some of the currently known angiogenesis
inhibiting compounds are: AGM-1470 (TNP-470) or antagonists to one
of its receptors MetAP-2; growth factor antagonists or antibodies
to growth factors (including VEGF or bFGF); growth factor receptor
antagonists or antibodies to growth factor receptors; inhibitors of
metalloproteinases including TIMP, batimastat (BB-94), and
marimastat; tyrosine kinase inhibitors including genistein and
SU5416; integrin antagonists including antagonists alphaVbeta3/5 or
antibodies to integrins; retinoids including retinoic acid or the
synthetic retinoid fenretinide; steroids
11.alpha.-epihydrocortisol, corteloxone, tetrahydrocortisone and
17.alpha.-hydoxyprogesterone; protein kinase inhibitors including
staurosporine and MDL 27032; vitamin D derivatives including
22-oxa-1 alpha, and 25-dihydroxyvitamin D3; arachidonic acid
inhibitors including indomethacin and sulindac; tetracycline
derivatives including minocycline; thalidomide derivatives;
2-methoxyestradiol; tumor necrosis factor-alpha;
interferon-gamma-inducib- le protein 10 (IP-10); interleukin 1 and
interleukin 12; interferon alpha, beta or gamma; angiostatin.TM.
protein or plasminogen fragments; endostatin.TM. protein or
collagen 18 fragments; proliferin-related protein; group B
streptococcus toxin; CM101; CAI; troponin I; squalamine; nitric
oxide synthase inhibitors including L-NAME; thrombospondin;
wortmannin; amiloride; spironolactone; ursodeoxycholic acid;
bufalin; suramin; tecogalan sodium; linoleic acid; captopril;
irsogladine; FR-118487; triterpene acids; castanospermine; leukemia
inhibitory factor; lavendustin A; platelet factor-4; herbimycin A;
diaminoantraquinone; taxol; aurintricarboxylic acid; DS-4152;
pentosan polysulphite; radicicol; fragments of human prolactin;
erbstatin; eponemycin; shark cartilage; protamine; Louisianin A, C
and D; PAF antagonist WEB 2086; auranofin; ascorbic ethers; and
sulfated polysaccharide D 4152.
[0014] AGM-1470 (O-chloroacetylcarbamoyl fumagillol) is an analog
of fumagillin with well-described angiogenesis inhibiting
activities that is highly selective, potent, and non-toxic..sup.26
AGM-1470 inhibits endothelial cell proliferation at concentrations
that are inactive for other cell types..sup.27 Fumagillin is a
chemical produced by Aspergillus fumigatus which acts as a potent
angiogenesis inhibitor in vitro and in vivo. AGM-1470 is 50 times
more active and much less toxic than the parent compound. It
potently inhibits endothelial proliferation and migration as well
as angiogenesis in the chick chorioallantoic membrane. AGM-1470 has
shown marked antineoplastic activity in animal models and is in
clinical trials for the treatment of human neoplastic tumors and
other angiogenic disorders.
[0015] Although shown to be an effective inhibitor of tumor
angiogenesis, angiogenesis inhibiting compounds have not heretofore
been shown to be effective in inhibiting angiogenesis in female
reproductive and embryonic tissues. The angiogenesis of female
reproductive tissues differs from angiogenesis in other tissues,
such as in a tumor, as they are affected by different growth
factors and under different regulatory mechanisms. Additionally,
inhibition of angiogenesis in the female reproductive system has
not heretofore been shown to disrupt or regulate critical events in
the reproductive cycle. Thus, the successful use of angiogenesis
inhibitors for contraception or treatment of disease in the tissues
of the female reproductive system has not been previously
demonstrated as possible, effective, safe, and without irreversible
effects on the female reproductive system.
[0016] Another impediment to the use of angiogenesis inhibitors as
contraceptives relates to the possible risk of inducing birth
defects if contraception fails. This concern derives from the
recent observation that thalidomide is an angiogenesis
inhibitor..sup.28 Thalidomide produces unique birth defects when
mothers ingest the drug during the first trimester. However, we
believed that thalidomide had a unique capacity to induce limb
defects by specific interaction with receptors in the limbs and
through local concentration of the drug in the limbs. Therefore, we
attempted herein to establish that other angiogenesis inhibitors
can be used as contraceptives without inducing birth defects.
[0017] It is clear that angiogenesis plays a major role in the
events encompassing reproduction. Proliferation of blood vessels is
seen coincident with ovulation, corpus luteal growth,
decidualization and placental formation. If this angiogenic
activity could be reversibly repressed or eliminated, fertility and
pathological reproductive disorders could be controlled. What is
needed, therefore, is to demonstrate that angiogenesis inhibitors
can inhibit the female reproductive system, without side-effects or
irreversibility of the physiological changes.
SUMMARY OF THE INVENTION
[0018] In accordance with the present invention, compositions and
methods are provided that are effective for modulating
angiogenesis, and inhibiting unwanted angiogenesis, especially
angiogenesis related to female reproduction and pathological
reproductive disorders. The present invention includes application
of angiogenesis inhibitors, such as AGM-1470, and other antagonists
of AGM-470 receptor MetAP-2. AGM-1470 comprises an
antibiotic-derivative (O-chloroacetylcarbamoyl fumagillol) for use
as an angiogenesis inhibiting compound. AGM-1470, also known as
TNP-470, is a highly selective, potent, and non-toxic angiogenesis
inhibitor.
[0019] The present invention is contemplated to be applicable for
administration to fertile female mammals for control of
reproduction, as well as administration to either fertile or
infertile female mammals for treatment of conditions and disorders
of the reproductive system. The compositions are administerable to
both humans and animals, as may be applicable.
[0020] The present invention provides methods and compositions for
controlling angiogenesis prior to and during decidualization,
placental formation, yolk sack development and cyclical endometrial
maturation, which are angiogenesis dependent, for the regulation of
the female reproductive processes. Applicable systems are
delineated which inhibit angiogenesis in the uterus, ovary,
placenta, or fetus.
[0021] Depending on when the angiogenesis inhibiting compound is
administered and under what regimen, the invention may function
through a number of different mechanisms for deterrence of a
pregnancy. The invention inhibits angiogenesis in the ovary,
obstructing the development of requisite vascularization, thereby
preventing ovulation or the normal release of hormones necessary
for conception, and functioning as a prophylactic to conception. In
addition, the compound inhibits angiogenesis in the uterus,
preventing the formation and engorgement of the tissue necessary
for implantation, thereby obliterating the necessary conditions for
the maturation of the blastocyst. Therefore, the administration of
the angiogenesis inhibiting compound may be before or after
intercourse and fertilization have occurred, thus providing an
effective method of birth control. Furthermore, the invention
interferes with the placental and yolk sack development, as well as
disrupting vascular development in the embryo, thereby preventing
further development of the embryo and continuation of the
pregnancy. Therefore, the invention may also function to initiate
spontaneous termination of a pregnancy.
[0022] In one embodiment, the present invention provides a
methodology for use of AGM-1470, and functional analogs thereof,
which produce no signs of toxicity to the mother and allows for
recovery of reproductive function. Thus, AGM-1470 provides for the
effective disruption of decidualization, preventing the
accompanying vascularization of the endometrium, placental
formation, and fetal development, by angiogenesis inhibition
without adverse effects to the mother.
[0023] The present invention also relates to a system for the
application of angiogenesis inhibitors, such as AGM-1470, and
functional analogs thereof, on non-pregnant female tissues for
interference of endometrial angiogenesis with reduced endometrial
glandular and stromal proliferation. The inhibitory effect of
AGM-1470, for example, on endometrial angiogenesis, stromal and
glandular proliferation, as well as placental formation, can treat
several pathological reproductive processes such as endometriosis,
adenomyosis, dysfunctional uterine bleeding, uterine leiomyoma
(fibroids), and choriocarcinoma. In addition, the inhibition of the
vascularization of the mucosa of the uterine tube interferes with
the implantation of the blastocyst in this portion of the female
reproductive tract. Thus, the application of an angiogenesis
inhibiting compound may also prevent ectopic pregnancy.
[0024] Accordingly, it is an object of the present invention to
provide methods of regulating the female reproductive system with
compositions comprising angiogenesis inhibitors, which include for
example AGM-1470, or other antagonists to the AGM-1470 receptor
MetAP-2.
[0025] It is another object of this invention is to provide a
method for controlling fertility by administering an effective
amount of an angiogenesis inhibiting compound, either in single or
multiple doses, capable of preventing conception or terminating a
pregnancy. The angiogenesis inhibiting compound may be administered
prior to intercourse, after intercourse or after ovum
fertilization.
[0026] It is yet another object of the present invention to provide
a therapy for reproductive disorders and control of reproduction
that has minimal side effects.
BRIEF DESCRIPTION OF THE FIGURES
[0027] FIGS. 1A-1I show the gross morphology of gravid mouse uteri.
Inseminated mice were treated with AGM-1470 30 mg/kg s.c. or saline
on Embryonic day 1 (E1) or E7, and mice were sacrificed at E9, 13
or 18.
[0028] FIGS. 1A, 1B, and 1C show the normal pattern of growth of
fetuses on E9, E13 and E18. When AGM-1470 mg/kg s.c. was given on
E1 there was initial growth to E9 (FIG. 1D), followed by failure of
further growth with resorption (FIGS. 1E & 1F). When AGM-1470
30 mg/kg s.c. was given on E7 there was initial growth to E9 (FIG.
1G) followed by hemorrhage around the implantation sites by E13
(FIG. 1H) and resorption by E18 (FIG. 11). Ruler at the bottom of
each panel shows mm intervals.
[0029] FIGS. 2A-2F show hematoxylin/eosin staining of mouse embryos
on E9. Inseminated mice were treated with AGM-1470 mg/kg s.c. or
saline on E1 or E7, and mice were sacrificed at E9. Uteri were
resected and stained with hematoxylin/eosin as described in the
material and methods section. FIG. 2A shows control embryo and
placenta at 25.times..
[0030] FIG. 2B shows control placenta at 100.times. demonstrating
an extension of nucleated red blood cells into the fetal placenta
(see arrows).
[0031] FIG. 2C shows AGM-1470 rejected on E1 resulted in a small,
disorganized embryo.
[0032] FIG. 2D shows the loss of invasion of blood islands and
nucleated red blood cells in the placenta.
[0033] FIG. 2E shows that AGM-1470 injected on E7 resulted in
reduced nucleated red blood cells in the embryo.
[0034] FIG. 2F shows reduced nucleated red blood cells in the fetal
placenta with reduced labyrinth structure of the placenta.
(P--placenta, D=decidua).
[0035] FIGS. 3A and 3B show ovaries and uteri from control and
AGM-1470 chronically treated mice. Randomly cycling female C57BL/6
mice were chronically treated with AGM-1470 (30 mg/kg QOD, s.c.)
for 16 days. Ovaries and uteri were removed en bloc, fixed and
stained with hemotoxylin and eosin.
[0036] FIG. 3A shows tissue from control animal at 2.5.times.
magnification.
[0037] FIG. 3B shows tissue from AGM-1470 treated animal at
2.5.times. magnification. Note the decreased size and number of
corpora lutea (small arrows) and number of endometrial glands
(large arrows) in treated mice.
[0038] FIGS. 3C and 3D show ovaries and uteri from control and
AGM-1470 chronically treated mice.
[0039] FIG. 3C shows a close up of ovary section from FIG. 1A at
10.times. magnification showing corpora lutea (large arrows) and
small antral follicles.
[0040] FIG. 3D shows a close up of ovary from FIG. 1B at 10.times.
magnification showing regressed corpora lutea and thin layer of
granulosa cells in large antral follicles (small arrows).
[0041] FIGS. 4A-4D shows the microvessel density of intersite
endometrium and the proliferation rate of the decidua on E6.
AGM-1470 30 mg/kg s.c. or saline was injected on E1 and uteri
resected on E6 as described in the material and methods
section.
[0042] FIG. 4A shows the immunohistochemical staining with
antibodies to PCNA demonstrating a high degree of positively
staining stromal cells in the control decidua (630.times., arrows
point to examples of positively stained cells).
[0043] FIG. 4B shows that the decidua from mice treated with
AGM-1470 on E1 has a decreased number of positively staining cells
(630.times.).
[0044] FIG. 4C shows the immunohistochemical staining with
antibodies to Factor VIII demonstrating a high microvessel density
in the control intersite endometrium (250.times.) versus FIG. 4D
where the specimen injected on E1 with AGM-1470 (250.times.).
[0045] FIGS. 5A-5D show anti-DC31 vascular staining of the embryo
and yolk sac on E9. The control and AGM-1470 (injected on E7)
embryos were studied by whole mount immunohistochemistry for
CD31/PECAM to stain blood vessels as described in the material and
methods section. All panels were taken at the same magnification.
The embryos treated with AGM-1470 on E7 were smaller and had a loss
of capillary density (FIG. 5A versus 5B). In some tissues such as
the heart (H) and liver bud (L) there was almost a complete loss of
CD31 labeled vessels. The yolk sac of AGM-1470 treated was also
smaller and had a decrease in density and intercapillary distance
of capillaries (arrows point to intercapillary spaces) (FIG. 5C
versus 5D).
[0046] FIGS. 6A-6E shows the effect of AGM-1470 on the uterus of
non-pregnant, cycling mice. Cycling mice were treated with AGM-1470
30 mg/kg s.c. every other day (n=8) for 16 days. a saline treated
group (n=8) served as control. Uterus and ovaries were fixed in
Carnoy's fixative and embedded in paraffin. Hematoxylin/eosin
staining and immunohistochemistry to factor VIII and PCNA were
performed as described in the material and methods section. The
mice in the AGM-1470 treated group (FIG. 6B) had 63% fewer glands
than the median of the control (FIG. 6A). (FIGS. 6A and 6B at
100.times. magnification, scale bar=160 .mu., arrows point to
glands). Immunohistochemical staining with antibodies to PCNA
revealed a 68% decrease in the proliferation rate of endometrial
stromal cells and a 67% decrease of proliferating glandular
epithelial cells in the AGM-1470 treated uteri (FIG. 6D) as
compared to control (FIG. 6C). (FIGS. 6C and 6D at 630.times.
magnification, arrows point to examples of positively staining
glandular cells). Immunohistochemical analysis with antibodies to
factor VIII demonstrated a 41% decrease in the microvessel density
in AGM-1470 treated uteri (FIG. 6F) as compared to control (FIG.
6E) (FIGS. 6E and 6F at 250.times. magnification, arrows point to
microvessels).
[0047] FIGS. 7A and 7B show the ovaries and uteri from a control
mouse or an AGM-treated mouse given a single dose of AGM-1470 on
E(-1). Group housed female C57BL/6 mice were treated with a single
injection of AGM-1470 (30 mg/kg, s.c.) and paired with male
breeders. Mice who mated on the day after treatment were sacrificed
6 days after mating. Ovaries and uteri were removed en bloc, fixed
and stained with hemotoxylin and eosin.
[0048] FIG. 7A shows tissue from control animal at 2.5.times.
magnification.
[0049] FIG. 7B. shows tissue from treated animal at 2.5.times.
magnification. Thin arrows indicate the corpora lutea. The fat
arrows indicate implantation sites in control animals not seen in
AGM-1470 treated animal.
[0050] FIG. 8 shows the effect of a single-dose of AGM-1470 on the
percentage of mated animals with viable births. Group housed female
C57BL/6 mice were treated with a single injection of AGM-1470 (30
mg/kg, s.c.) and paired with male breeders at various times after
treatment. Treatment groups are designated as the number of days
between plugging and treatment. The 6 control groups are combined
and expressed as the mean .+-.SD. The number of matings in per
group is listed below each labeled group. Note the inhibition of
viable births with treatments on E0, E-1, and E-2.
DETAILED DESCRIPTION OF THE INVENTION
[0051] The present invention includes compositions and methods for
the regulation of the female reproductive system and the treatment
of reproductive diseases and conditions that are mediated by or
associated with angiogenesis. By "female reproductive system" is
meant to include physiologic mechanisms and living tissues by which
female mammals prepare for and produce offspring. By "female
reproductive tissue" is meant to include the cells, blood vessels
and organs involved in the female reproductive system, including
the ovary, uterus, fetus or placenta, or tissues originating
therefrom (for example, ectopic endometrial tissue).
[0052] The present invention includes any of the known angiogenesis
inhibitors, and those discovered in the future, to be used as
reproductive regulators or to treat abnormal reproductive
conditions. Examples of known angiogenesis inhibitors include:
AGM-1470 (TNP-470) or antagonists to one of its receptors MetAP-2;
growth factor antagonists or antibodies to growth factors
(including VEGF or bFGF); growth factor receptor antagonists or
antibodies to growth factor receptors; inhibitors of
metalloproteinases including TIMP, batimastat (BB-94), and
marimastat; tyrosine kinase inhibitors including genistein and
SU5416; integrin antagonists including antagonists alphaVbeta3/5 or
antibodies to integrins; retinoids including retinoic acid or the
synthetic retinoid fenretinide; steroids
11.alpha.-epihydrocortisol, corteloxone, tetrahydrocortisone and
17.alpha.-hydoxyprogesterone; protein kinase inhibitors including
staurosporine and MDL 27032; vitamin D derivatives including
22-oxa-1 alpha, and 25-dihydroxyvitamin D3; arachidonic acid
inhibitors including indomethacin and sulindac; tetracycline
derivatives including minocycline; thalidomide derivatives;
2-methoxyestradiol; tumor necrosis factor-alpha;
interferon-gamma-inducible protein 10 (IP10); interleukin 1 and
interleukin 12; interferon alpha, beta or gamma; angiostatin.TM.
protein or plasminogen fragments; endostatin.TM. protein or
collagen 18 fragments; proliferin-related protein; group B
streptococcus toxin; CM101; CAI; troponin I; squalamine; nitric
oxide synthase inhibitors including L-NAME; thrombospondin;
wortmannin; amiloride; spironolactone; ursodeoxycholic acid;
bufalin; suramin; tecogalan sodium; linoleic acid; captopril;
irsogladine; FR-118487; triterpene acids; castanospermine; leukemia
inhibitory factor; lavendustin A; platelet factor-4; herbimycin A;
diaminoantraquinone; taxol; aurintricarboxylic acid; DS-4152;
pentosan polysulphite; radicicol; fragments of human prolactin;
erbstatin; eponemycin; shark cartilage; protamine; Louisianin A, C
and D; PAF antagonist WEB 2086; auranofin; ascorbic ethers; and
sulfated polysaccharide D 4152.
[0053] More particularly, the present invention relates to a method
of inhibiting angiogenesis in a female mammal to regulate fertility
comprising administering to the female mammal an effective amount
of a fertility regulating angiogenesis inhibiting compound, wherein
the compound is administered either to prevent conception or to
terminate a pregnancy. In order to accomplish either of these two
objectives, the angiogenesis inhibiting compound may be
administered in a single dose or in multiple doses. The compound
may be administered prior to intercourse, after intercourse or
after ovum fertilization, in order to inhibit angiogenesis in the
uterus, ovary, placenta or embryo.
[0054] Depending on when the compound is administered and under
what regimen, the invention functions through a number of different
modalities. The invention inhibits angiogenesis in the ovary,
obstructing the development of requisite vascularization, thereby
preventing the normal release of hormones necessary for conception,
and functioning as a prophylactic to conception. In addition, the
compound inhibits angiogenesis in the uterus, preventing the
formation and engorgement of the tissue necessary for implantation,
thereby obliterating the necessary conditions for the maturation of
the blastocyst.
[0055] The invention provides for the first time that it is
possible to use an angiogenesis inhibiting compound to regulate
fertility and treat conditions of the female reproductive tissues.
The invention provides a methodology for use of, for example
AGM-1470, which produces no signs of toxicity to the mother and
allows for recovery of reproductive function. Thus, AGM-1470 for
example prevents vascularization of the endometrium (disrupting
decidualization), placental formation, and fetal development, by
angiogenesis inhibition without adverse effects to the mother.
[0056] In addition, the present invention relates to a system for
treating a female mammal with a disease or condition of the
reproductive tissue that is mediated by angiogenesis. This method
provides for the application of an angiogenesis inhibiting
compound, such as AGM-1470, in non-pregnant female mammals for
interference of endometrial angiogenesis with reduced endometrial
glandular and stromal proliferation. The inhibitory effect of an
angiogenesis inhibiting compound, such as AGM-1470, on endometrial
angiogenesis, stromal and glandular proliferation, as well as
placental formation, can treat several pathological reproductive
processes such as endometriosis, adenomyosis, dysfunctional uterine
bleeding, uterine leiomyoma (fibroids), and choriocarcinoma. In
addition, the inhibition of the vascularization of the mucosa of
the uterine tube interferes with the implantation of the blastocyst
in this portion the female reproductive tract. Thus, the
application of an angiogenesis inhibiting compound may also prevent
ectopic pregnancy.
[0057] The present invention includes any angiagenesis inhibiting
compound which can be effectively used to regulate fertility or
treat a condition of the female reproductive tissues.
[0058] One such compound is an antibiotic-derivative designated as
AGM-1470 (O-chloroacetylcarbamoyl fumagillol)..sup.1 AGM-1470, also
known as TNP-470, is highly selective, potent, and non-toxic.
AGM-1470 is an analog of fumagillin, is a potent inhibitor of
endothelial cell migration, endothelial cell proliferation and
capillary tube formation, but has not heretofore been shown to be
an effective inhibitor of angiogenesis in the tissues of female
reproductive system. Fumagillin is a naturally secreted antibiotic
of Aspergillus Fumigatus fresenius. Fumagillin itself suppresses
angiogenesis and neovascularization but its effectiveness as a
pharmaceutical is limited because it produces side effects
including severe weight loss. Analogs of fumagillin have been
developed which retain the potent angiogenesis inhibiting activity
of fumagillin without producing side effects. AGM-1470 is 50 times
more active and much less toxic than the parent compound,
suppressing angiogenesis without the usual toxic side effects
associated with conventional chemotherapy drugs.
[0059] Fumagillin may be isolated from in vitro cultures of
Aspergillus Fumigatus fresenius, often cultured on capillary
endothelial cells. Fumagillol is then produced by alkaline
hydrolysis of fumigillan. AGM-1470 then can be isolated from
fumagillol, from which over one hundred other derivatives are
synthesized. Therefore, the invention contemplates that other
analogs of fumagillin similar to AGM-1470 would be expected to be
useful in the present invention. Furthermore, AGM-1470 is believed
to inhibit angiogenesis through its interaction with a receptor
designated MetAP-2. Therefore, the invention contemplates that
other MetAP-2 receptor antagonists or agonists would be expected to
be useful in the present invention.
[0060] Although shown to be effective in the inhibition of tumor
angiogenesis, angiogenesis inhibiting compounds, such as AGM-1470,
were not previously known to be effective to regulate female
reproductive tissues. Angiogenesis in the female reproductive
tissues differs from angiogenesis in cancerous tissues, as it is
affected by different growth factors and is under different
regulatory mechanisms. Therefore, there was formerly no expectation
for the successful use of angiogenesis inhibitors in female
reproductive tissues to inhibit angiogenesis and to effectively
regulate the reproductive system or treat diseases or conditions of
the reproductive tissues. It had not been demonstrated that these
compounds could be used safely, without irreversible effects on the
female reproductive system or without the induction of birth
defects on a living embryo. Thus, the effective use of angiogenesis
inhibiting compounds for the inhibition of the female reproductive
system was a surprising result.
[0061] AGM-1470 does not permanently impair the reproductive system
as evidenced by the recovery of the reproductive system in the
included examples. Chronic high dose administration of AGM-1470
blocks endometrial maturation and corpus luteal formation in
sexually mature cycling mice. The examples of the present invention
also show that angiogenesis inhibitors may disrupt follicular
development in ovulation. Full recovery was seen 7 weeks after the
drug was discontinued.
[0062] Single dose AGM-1470 treatment of mice one day after mating
(but three days prior to implantation in the mouse) caused a
failure of implantation induced angiogenesis and decidualization.
This latter experiment demonstrates that the female reproductive
tract can be disrupted by a single dose of angiogenesis inhibitors.
Thus, low dose or single dose treatment is effective as a
contraceptive (given before mating) without complete suppression of
menstrual cycles or other systemic side effects. Furthermore, in
the included experiments in which pregnant mice were treated with
the angiogenesis inhibitor AGM-1470, birth defects were not
documented when the drug was given during pregnancy..sup.27 High
doses of AGM-1470 given in the first half of the pregnancy did
block early placental formation resulting in a termination of the
pregnancy. However, high doses given during the last half of
pregnancy or low doses at various points throughout the pregnancy
did not produce birth defects.
[0063] The present invention encompasses AGM-1470, fragments
thereof, antisera thereof, receptor antagonists (including MetAP-2)
or receptor agonists thereof that are combined with
pharmaceutically acceptable exipients, and optionally sustained
release compounds or compositions, such as biodegradable polymers,
to form therapeutic compositions. These therapeutic compositions
may be administered either to humans or animals, as applicable.
[0064] The present invention also relates to the application of
angiogenesis inhibitors, such as AGM-1470, in a single dose or in
multiple doses, to inhibit angiogenesis in the uterus, ovary,
placenta, or fetus. In regulating angiogenesis related processes,
an effective amount of the free form, or a salt of angiogenesis
inhibitors, such as AGM-1470 can be used. Thus, angiogenesis
inhibitors, such as AGM-1470, and salts thereof can be used for
prophylaxis and/or treatment of neovascularization of
endometriosis, adenomyosis, ovarian cysts, dysfunctional uterine
bleeding, uterine leiomyoma (fibroids), choriocarcinoma and ectopic
pregnancies.
[0065] As a result of the demonstration herein of the importance
and controllable nature of angiogenesis in female reproductive
tissues, the invention also provides methods of treating
infertility disorders by promoting normal angiogenesis.
Specifically, methods of treating infertility disorders are
provided comprising administering to a female mammal an effective
amount of an angiogenesis stimulator. Examples of angiogenesis
stimulators include, vascular endothelial growth factors,
fibroblast growth factors, tumor necrosis factors, transforming
growth factors, thymidine phosphorylase, platelet derived growth
factor, scatter factor, interleukin-8, granulocyte colony
stimulating factor, angiogenin, platelet-activating factor,
proliferin, substance P, lactate, hyaluron fragment, erucamide, and
prostaglandins. The discussions of dosages, derivatives and
pharmaceutical formulations of angiogenesis inhibitors herein is
intended to apply also to angiogenesis stimulators.
[0066] It is to be understood that the present invention is
contemplated to include any derivatives of the angiogenesis
inhibitors that have endothelial inhibitory activity effective to
regulate the female reproductive system. The present invention
includes entire angiogenesis inhibiting compounds, derivatives of
angiogenesis inhibiting compounds and biologically-active fragments
of angiogenesis inhibiting compounds. These-include proteins with
angiogenesis inhibiting activity that have amino acid substitutions
or have sugars or other molecules attached to amino acid functional
groups. The present invention also includes genes that code for
angiogenesis inhibiting proteins that are expressed by those genes.
The angiogenesis inhibiting compounds described above can be
provided as isolated and substantially purified in pharmaceutically
acceptable formulations using formulation methods known to those of
ordinary skill in the art.
[0067] These formulations can be administered by standard routes.
In general, the combinations may be administered by the topical,
transdermal, intraperitoneal, oral, rectal, vaginal, intrauterine,
or parenteral (e.g., intravenous, subcutaneous or intramuscular)
route. In addition, the angiogenesis inhibitor may be incorporated
into biodegradable polymers allowing for sustained release of the
compound, the polymers being implanted in the vicinity of where
drug delivery is desired so that the angiogenesis inhibiting
compound is slowly released systemically. The biodegradable
polymers and their use are described, for example, in detail in
Brem et al., J. Neurosurg. 74:441-446 (1991), which is hereby
incorporated by reference in its entirety.
[0068] The dosage of the angiogenesis inhibiting compounds of the
present invention will depend on the type of inhibitor being
administered, disease state or condition being treated, and other
clinical factors such as weight and condition of the human or
animal and the route of administration of the compound. For
treating female mammals with AGM-1470 for example, between
approximately 0.05 mg/kg to 500 mg/kg, or preferably 0.1 to 100
mg/kg, of the angiogenesis inhibitor can be administered. For
example in human females, approximately 1 mg/kg AGM-1470 can be
administered, and in female mice approximately 30 mg/kg AGM-1470
can be administered. Depending on the use for which the drug is
given and the preferences of the doctor administering and the
patient receiving the compound, the angiogenesis inhibiting
compound can be administered between several times per day to once
a week. It is to be understood that the present invention has
application for both human and veterinary use. The methods of the
present invention contemplate single as well as multiple
administrations, given either simultaneously or over an extended
period of time.
[0069] The angiogenesis inhibiting compound formulations may
conveniently be presented in unit dosage form and may be prepared
by conventional pharmaceutical techniques. Such techniques include
the step of bringing into association the active ingredient and the
pharmaceutical carrier(s) or excipient(s). In general, the
formulations are prepared by uniformly and intimately bringing into
association the active ingredient with liquid carriers or finely
divided solid carriers or both, and then, if necessary, shaping the
product.
[0070] Formulations suitable for parenteral administration include
aqueous and non-aqueous sterile injection solutions which may
contain anti-oxidants, buffers, bacteriostats and solutes which
render the formulation isotonic with the blood of the intended
recipient; and aqueous and non-aqueous sterile suspensions which
may include suspending agents and thickening agents. The
formulations may be presented in unit-dose or multi-dose
containers, for example, sealed ampules and vials, and may be
stored in a freeze-dried (lyophilized) condition requiring only the
addition of the sterile liquid carrier, for example, water for
injections, immediately prior to use. Extemporaneous injection
solutions and suspensions may be prepared from sterile powders,
granules and tablets of the kind previously described.
[0071] Preferred unit dosage formulations are those containing a
daily dose or unit, daily sub-dose, as herein above recited, or an
appropriate fraction thereof, of the administered ingredient. It
should be understood that in addition to the ingredients,
particularly mentioned above, the formulations of the present
invention may include other agents conventional in the art having
regard to the type of formulation in question.
[0072] This invention is further illustrated by the following
examples, which are not to be construed in any way as imposing
limitations upon the scope thereof. On the contrary, it is to be
clearly understood that resort may be had to various other
embodiments, modifications, and equivalents thereof which, after
reading the description herein, may suggest themselves to those
skilled in the art without departing from the spirit of the present
invention and/or the scope of the appended claims.
EXAMPLE 1
[0073] Utilization of a Corneal Micropocket Model to Test AGM-1470
for the Ability to Inhibit Corneal Neovascularization Induced by
bFGF or VEGF
[0074] To confirm that angiogenesis was being inhibited, AGM-1470
was tested in vivo. A mouse corneal micropocket model was used
which was recently developed to model tumor angiogenesis. Pellets
of bFGF or VEGF were implanted in the mouse cornea resulting in
aggressive neovascularization. AGM-1470 inhibited the amount of
bFGF or VEGF induced neovascularization by 47% or 62% respectively
(n=10/group p<0.001).
EXAMPLE 2
[0075] Treatment of Inseminated Mice with Single Dose AGM-1470
[0076] C57BL/6 male and female mice greater than seven weeks old
were obtained from Takonic (Germantown, N.Y.). The mice were
acclimated for at least five days prior to mating and housed in a
twelve hour on and twelve hour off light schedule. The mice were
mated in the evenings and vaginal plugs were checked for the next
morning. Mating in mice is normally detected by the occurrence of
vaginal plugs, which are formed by a mixture of the secretions of
the vesicular and coagulating glands of the male and usually fill
the vagina from cervix to vulva. The day of plugging was labeled
Embryonic day 0 (E0). All animal studies were conducted according
to the protocols approved by the Animal Ethics Committee of
Children's Hospital. Plugged female mice were randomized and
received either AGM-1470 30 mg/kg s.c. or saline on E1, 7, or 14.
The dependence of female reproductive system on angiogenesis was
examined by treating sexually mature inseminated and cycling mice
with selective angiogenesis inhibitor AGM-1470. The following
results demonstrate that decidualization, placental formation, yolk
sack development, and cyclical endometrial maturation are
angiogenesis dependent. 5
[0077] Fetal Viability
[0078] One single dose of AGM-1470 resulted in 100% fetal
resorption on E18 when injected either pre-implantation on E1 (n=69
embryos, p<0.0001) or post-implantation on E7 (n=36,
p<0.0001). Since implantation in the mouse occurs on E4.5, these
treatments correspond to inhibition of angiogenesis either before
or after implantation. In contrast, normal litters were seen with
AGM-1470 treatment on E14 (n=46) and control mice (n=104). Gross
morphology of the uteri from mice treated on E1 or 7 and sacrificed
before term, illustrated the failure of fetal growth and subsequent
resorption. The pregnant mothers exhibited no signs of
toxicity.
[0079] The dosing schedule of 30 mg/kg s.c. every other day was
selected because this regiment produces no evident toxicity even
after greater than 100 days of therapy and has demonstrated
efficacy in suppressing tumors in mice. Mice were sacrificed on
either E6, 9, 13 or 18 (one day prior to term gestation). Fetal
viability was assessed and statistical analysis was performed with
a Fisher's Exact Test.
[0080] Mice given AGM-1470 on E7 showed an inhibition of placental
and fetal development. Mice given AGM-1470 on E1 (four days prior
to implantation) also had a failure of placental development. A
possible explanation was that the endometrium exposed to AGM-1470
on E1 may be permanently altered so that although implantation
occurs with trophoblast invasion, there is a failure of
decidualization and coordinated development of the placenta. This
hypothesis is supported by the observation of decreased endometrial
microvessel density and decidual proliferation rate on E6,
demonstrating a difference in endometrial response during
trophoblast invasion. This inhibition of placental development
inhibits further development of a viable fetus. See, FIG. 2.
[0081] When AGM-1470 was injected on E14 normal litters were
delivered. This may be due to the fact that at the time point
implantation, yolk sack formation, and placental development are
already complete. Alternatively, since the placental barrier has
been formed by E14, it may be capable of blocking transfer of
AGM-1470 to the fetus.
[0082] Uterine Weight
[0083] Gravid uteri and ovaries were resected enbloc and weighed.
Gravid uterine weight (grams wet weight, mean SE, n=3/day) in the
control group increased exponentially from E6 (0.20.+-.0.01), to E9
(0.53.+-.0.05), E13 (3.78.+-.0.36), and E18 (12.87.+-.0.62). Gravid
uterine weights from mice treated with AGM-1470 on E1 had no weight
increase after E9 [E6 (0.29.+-.0.01), E9 (0.63.+-.0.01), E13
(0.72.+-.0.08, p<0.05), and E18 (0.68.+-.0.02, p<0.05)].
Gravid uterine weights from mice treated with AGM-1470 on E7
increased initially but then regresses after E13 [E9
(0.71.+-.0.01), E13 (2.03.+-.0.14, p<0.05), and E18
(1.08.+-.0.06, p<0.05)].
[0084] Histology
[0085] Gravid uteri and ovaries were resected enbloc, weighed,
fixed and Carnoy's fixative, and embedded in paraffin according to
standard histological techniques. Sections were cut at 6 to 10
micrometers and stained with hematoxylin and eosin. When AGM-1470
was injected on E14, fetuses were allowed to come to term.
[0086] Fetal disorganization was seen in both AGM-1470 treated
groups on E9 which was followed by necrosis on E13 and E18. In mice
treated with AGM-1470 on E1 there was a failure of placental
development. Although blood island formation appeared normal, there
was an absence of the extension of these blood islands
(angioblasts) into the fetal placenta. There were no nucleated red
blood cells and no labyrinth structure in the placenta as compared
to control. See FIG. 2F versus FIG. 2B. In mice treated with
AGM-1470 on E7 there was a decrease in nucleated red blood cells
and labyrinth structure in the placenta (FIG. 2D), and a marked
decrease in nucleated red blood cells in the E9 embryos (See FIG.
2C). The ovaries appeared normal at all time points with the
histologic evidence of mature corpora lutea.
[0087] When one single dose of AGM-1470 was given on E7, 2.5 days
post-implantation, there was abnormal placenta and yolk
development, as well as disrupted vascular development in the
embryo. Loss of vascular development in the embryo could be due
either to a direct effect of AGM-1470 on the embryonic vasculature
or to a secondary effect caused by failure of normal placental and
yolk sack development.
[0088] Although endothelial cell proliferation has been associated
with corpora lutea maintenance during pregnancy, the fetal
resorption produced when AGM-1470 was given at both E1 or E7 could
not be accounted for by failure of the corpus luteal function.
Histologic evidence of mature corpora lutea, along with normal
serum progesterone levels provide evidence that ovarian function
was maintained.
EXAMPLE 3
[0089] Implantation in Mice Treated With Single Dose AGM-1470
Pre-Implantation
[0090] The same procedures were followed as described above in
Example 2 to administer 30 mg/kg AGM-1470 in single doses on the
specified days. The mice were sacrificed and the number of
implantation cites were counted. There was no decrease in the
number of implantation cites when AGM-1470 was given on E1 [control
mice had 8.+-.0.2 implants (n=29), mice injected with AGM-1470 on
E1 had 9.+-.0.4 implants (n=16)]. Although there was no reduction
in the number of embryos that implanted, the failure of placental
development seen histologically at E9 could be secondary to
abnormal changes in decidualization.
[0091] To test this hypothesis, mice were treated with AGM-1470 on
E1 and sacrificed at E6, a time point prior to placental formation.
The uteri of the mice were examined by antibodies to Factor VIII
and PCNA to determine if there was any difference in the degree of
endometrial angiogenesis or the proliferative response of the
decidua induced by trophoblast invasion. However, in the decidua
there was a 50% decrease in the proliferation of stromal cells in
the AGM-1470 treated mice as compared to control (PCNA
staining/X630 field in control was 48%.+-.3, n=4, versus 24%.+-.5,
n=4, P<0.005 in AGM-1470 treated mice. See FIGS. 4A and 4B).
There was also an associated 52% decrease in microvessel density in
the intersite endometrium (119.+-.13 vessels/X250 field in the
control, n=4, versus 57.+-.13 vessels/X250 field in the AGM-1470
treated mice, n=4, P<0.05. See FIGS. 4C and 4D).
[0092] These results demonstrate a reduction of endometrial
angiogenesis and the proliferative response of the decidua induced
by trophoblast invasion. Thus the inhibition of placental
development in the mice given AGM-1470 on E1 may be attributable to
alterations in the endometrium, so that although implantation
occurs with trophoblast invasion, there is a failure of
decidualization and coordinated development of the placenta.
EXAMPLE 4
[0093] Anti-CD31 Staining in Mice Treated With AGM-1470
Post-Implantation
[0094] Mice were treated with 30 mg/kg AGM-1470 following the same
protocol in Example 2. The control and AGM-1470 treated embryos
were studied on E9 by whole mount immunohistochemistry for
CD-31/PECAM to stain blood vessels. The fetus and the yolk sack in
the mice given AGM-1470 on E1 were excluded due to necrosis by E9.
The embryos treated with AGM-1470 on E7 were smaller and had a
diffuse decrease in capillary density. In the heart and liver bud,
there was an almost complete loss of CD31 labeled vessels. See FIG.
5A versus 5B. The yolk sack was also smaller and although vascular
connections between blood islands were seen, there was decreased
capillary density interconnecting the vessels. See FIG. 5C versus
5D. Loss of vascular development in the embryo could be due to
either a direct effect of AGM-1470 on the embryonic vasculature or
to a secondary effect caused by failure of normal placental and
yolk sac development.
EXAMPLE 5
[0095] Serum Progesterone Levels Measured in Mice Treated with
Single Dose AGM-1470 on Either E1 or E7
[0096] To determine whether any of the effects on the embryo and
placenta were secondarily related to actions AGM-1470 might have
had on the corpora lutea, the serum progesterone levels were
measured on E9 in control and treated mice. This time point was
chosen because histological evidence of involution was seen at E9
in both treated groups.
[0097] To determine whether corpora lutea function were intact in
AGM-1470 treated pregnant mice, blood was collected at autopsy on
E9 in control and mice treated with AGM-1470 30 mg/kg on E1 or E7.
Blood was also collected from non-pregnant cycling mice in diestrus
as a negative control. The blood was centrifuged for 10 minutes at
1000.times. g at 4.degree. Celsius and the serum was stored at
-20.degree. Celsius until use. Serum progesterone was determined
with a progesterone enzyme immunoassay test kit (Medics Biotech,
Inc.) per manufacturer's instructions. The results were compared to
a standard curve of human progesterone and data is measured a mean
percent of control E9 progesterone level.+-.s.e.m.
[0098] Serum from non-pregnant mice in diestrus served as a
negative control. There was no decrease in serum progesterone
levels in either treated group, with levels in mice treated with
AGM-1470 on E1 or E7 measuring 112%.+-.23 (n=3, NS), or 161%.+-.24
(n=3, NS) of control, respectively. Serum from non-pregnant mice
was 11%.+-.2 (n=3, P<0.001) of control. These data provide
evidence that a loss of ovarian function was not induced after a
single injection of AGM-1470 and thus not the cause of the loss of
placental development.
EXAMPLE 6
[0099] Reversibility of AGM-1470 and Recovery of the Reproductive
System
[0100] To establish long-term safety, the course of recovery of the
reproductive system after fetal resorption was studied. Six plugged
mice were injected with 30 mg/kg AGM-1470 on E1 (n=3) or E7 (n=3)
and then anesthetized on E18 in a methoxyflurane (Pittman-Moore,
Mandelein, Ill.) chamber so that a laparotomy could be performed.
The uteri were inspected to confirm the presence of resorption
cites. The incisions were closed with a running 3-0 ethelon.TM.
black monofilament nylon suture (Ethicon, Somerville, N.J.).
Animals were observed until fully recovered. After an additional
six weeks the mice were remated to determine their ability to
become inseminated and deliver a normal litter. The result was the
production of normal litters in all mice. Additionally, the uteri
of AGM-1470 treated mothers were grossly examined and appeared
normal. Therefore, there is a functional recovery of the uterus
after AGM-1470 treatment on E1 or E7.
[0101] The 30 mg/kg dose of AGM-1470 used produced no signs of
toxicity to the mother in the experiments, and recovery of
reproductive function was demonstrated by the birth of normal
litters. Thus, decidualization and placental formation can
effectively be disrupted with angiogenesis inhibitor without
adverse effects to the mother's reproductive capacity.
EXAMPLE 7
[0102] Treatment of Inseminated Mice With Single Low Dose
AGM-1470
[0103] The same protocol was followed as was performed for AGM-1470
30 mg/kg except mice were injected with only 3 mg/kg AGM-1470 on E1
or E7. The 10-fold lower dose was tested to determine if a dose in
which the fetuses come to term would produce birth defects. Mice
were sacrificed at E18 and fetal viability, number of implantation
cites, crown-rump length, and tail length were assessed.
[0104] Since AGM-1470 caused an inhibition of placental development
and fetal viability at a dose of 30 mg/kg, a 10-fold lower dose was
tested to determine what effect a non-lethal dose would have on
placenta and fetus. All fetuses at E18 were viable when AGM-1470 3
mg/kg was injected at E1 (n=21 fetuses) or E7 (n=8 fetuses). The
fetuses were alive with no birth defects, but were smaller than
normal. The crown-rump length on E18 relative to control
(20.4.+-.0.3 mm, n=13 embryos) was reduced by 36% in the AGM-1470
E1 (13.0.+-.0.1 mm, n=7 embryos, p<0.0005), and 19% in the
AGM-1470 E7 group (16.5.+-.0.5 mm, n=7 embryos, p<0.0005). Tail
lengths were similarly effected with the reduction in lengths
relative to control (11.4.+-.0.2 mm, n=13 embryos) of 43% in the
AGM-1470 E1 groups (6.5.+-.0.2 mm, n=7 embryos, p<0.0005) or 12%
in the AGM-1470 E7 group (10.0.+-.0.1 mm, n=7 embryos,
p<0.05).
EXAMPLE 8
[0105] Treatment of Cycling Mice With Chronic AGM-1470: Effect on
Estrous Cycle
[0106] Female mice were housed individually in cages and acclimated
for one week. The stage of the estrous cycles was checked by visual
inspection of the vagina according to the criteria of
Champlin.sup.28 and the examiner was blinded to the treatment
group. Determining the stage of the estrous cycle in the mouse by
the appearance of the vagina..sup.1 Animals were monitored daily
and graded for color, size of opening, striations and swelling.
Based on these observations each individual was assigned to a cycle
stage (diestrus, proestrus, estrus, or metestrus). Animals were
monitored for 10 days prior to each experiment and animals not
exhibiting a normal 4-5 day estrous cycle were excluded from the
study.
[0107] Mice were randomized and received either AGM-1470 30 mg/kg
s.c. or saline 0.1 ml s.c. every other day for 16 days
(approximately four estrous cycles). On the last of four days of
treatment mice were housed with male mice to see if successful
mating and conception would occur. On day 16 mice were weighed and
sacrificed by cervical dislocation. The ovaries were resected
enbloc, fixed in Carnoy's fixative, and embedded in paraffin
according to standard histologic techniques. Sections were cut at 6
microns and stained with hematoxylin and eosin.
[0108] During the experiment an additional morphology was observed
in some of the mice. In these mice the vaginal opening was closed
and the surrounding tissue was red, dry and non-edematous. The
first occurrence of this morphology was after 5.6.+-.3.2 days of
treatment, and it was often interspersed with an abnormal,
non-edematous diestrus-like appearance. After completion of the
study the treatment code was unmasked. It was determined that the
observation of this new morphology within 10 days of the start
treatment correctly predicted AGM-1470 treatment in 8 of 8 mice
whereas observation of at least one complete estrous cycle
correctly predicted 8 out of 8 control mice. Thus, treatment with
AGM-1470 caused an interruption of the normal estrous cycle with
the presentation of different external morphology. None of the
AGM-1470 mice were able to successfully mate as measured by no
visually evident plugging as compared to a plugging rate in the
control mice of 50% after 4 days. The changes in the external
vagina morphology that may have prevented mating were only seen
after treatment with AGM-1470 for several cycles (6 to 8 doses).
Therefore, the use of a single dose could avoid these changes,
exposing any potential contraceptive effects. However, given that
one injection of AGM-1470 on E1 in inseminated mice produced
similar changes in the endometrium (decreased angiogenesis and
decidualization), which resulted in fetal resorption, it is likely
that the suppression of angiogenesis and uterine gland and stromal
proliferation seen in mice treated with AGM-1470 would persist
after mating and also result in failure of decidualization.
[0109] Study of Immunohistochemistry
[0110] The whole-mount immunohistochemistry with monoclonal
antibody to mouse PECAM (CD31) was performed essentially as
described by Schlaeger.sup.29 Embryos were dissected from the
uterus and fixed in 4% paraformaldehyde, PBS at 4.degree. Celsius
overnight. The fixed embryos were subsequently rinsed in PBS at
room temperature and dehydrated in a methanol series. The
dehydrated embryos were bleached in 5% hydrogen peroxide in
methanol for 4 to 5 hours at room temperature and then rinsed twice
in methanol. Embryos were stored at -20.degree. Celsius until used.
The bleached embryos were rehydrated and blocked with PBSMT (3%
instant skim milk, 0.1% Triton X-100, PBS) for one hour twice at
room temperature. The embryos were then incubated with a 1:10
diluted hybridoma supernatant in PBSMT at 4.degree. Celsius
overnight. On the next day, the embryos were washed with PBSMT at
4.degree. Celsius five times (1 hour each) and then incubated with
a biotinylated secondary anti-rat IgG, mouse adsorbed (Vector
Laboratories, Burlingame, Calif.) in PBSMT at 4.degree. Celsius
overnight. On the third day, the embryos were rinsed in PBSMT at
4.degree. Celsius five times (1 hour each) and then incubated in
avidin and horse radish conjugated biotin in PBSMT (1:100 dilution)
(Vectastatin Elite Standard ABC Kit, Vector Laboratories) overnight
at 4.degree. Celsius. On the fourth day embryos were rinsed in
PBSMT at 4.degree. Celsius five time (1 hour each) and finally in
PBT (0.2% BSA, 0.1% Triton-X100, PBS) for 20 minutes at room
temperature. The peroxidase staining was performed by incubating
embryos in 0.3 mg/ml DAB (Sigma), 0.5% NiCl.sub.2 in PBT for 20
minutes followed by the H.sub.2O.sub.2 to the final concentration
of 0.03% and incubated for 10 minutes. The staining reaction was
stopped by rinsing in PBT and the PBS. The stained embryos were
post-fixed in 2% paraformaldehyde, 0.1% glutaraldehyde in PBS at
4.degree. Celsius overnight.
[0111] To determine microvessel density, Carnoy's-fixed tissue
sections were pretreated with 2 microgram/ml Proteinase K
(Boehringer Mannheim, Mannheim, Germany) at 37.degree. Celsius for
15 minutes before staining with a rabbit polyclonal antibody
against mouse factor VIII (Dako, Carpinteria, Calif.). Positive
staining was detected by incubating sequentially with a secondary
antibody against rabbit conjugated to horseradish peroxidase (Dako)
and diaminobenzidine tetrahydrochloride (DAB) (Dako) as a
chromagen. Sections were counterstained with methyl green (Schmid
& Co., Stuttgart, Germany) and mounted in Permount (Fisher,
Fair Lawn, N.J.). Microvessel density was determined by light
microscopy. Each count was expressed as the number of microvessels
identified within a selected field. Ten fields per section were
counted.
[0112] To determine the rate of cellular proliferation, staining
for proliferating cell nuclear antigen (PCNA) was performed.
Carnoy's-fixed sections were pretreated in target unmasking fluid
(Signet Laboratories, Dedham, Mass.) for 10 minutes at 90.degree.
for antigen retrieval. Immunohistochemical staining was performed
with an anti-PCNA murine monoclonal antibody (Signet). Positive
staining was detected by using horse radish peroxidase-conjugated
secondary (Dako) and tertiary (Dako) antibodies, and DAB as a
chromagen. The PCNA labeling index was determined by counting the
percentage of stained cells under light microscopy within selected
630.times. fields. A minimum of 1,000 cells were counted for each
specimen.
[0113] For staining of apoptotic cells, Carnoy's-fixed sections
were treated according to the protocol of the Apoptag manufacturer
(Oncor, Gaithersburg, Md.) except the pretreatment step with
Proteinase K was omitted. After TdT labeling of specimens, positive
staining was detected with a peroxidase-labeled antibody against
deoxyuridine peroxidase substrate. Sections were counterstained as
described above. The apoptotic index was determined by counting the
percentage of stained cells under light microscopy within selected
630.times. fields. A minimum of 1,000 cells was counted for each
specimen.
[0114] In experiments with AGM-1470 treatment of pregnant mice,
transverse sections through embryos contained within the uterus
were stained. Microvessels density was determined at the intersite
endometrium and PCNA was determined in the decidual stroma. In
experiments with AGM-1470 treatment of non-pregnant mice,
transverse sections through the endometrium were stained for factor
VIII, PCNA or apoptotic cells at the level of the endometrial lumen
at its widest diameter. Statistical analysis was performed with
ANOVA on ranked data to account for the expected variation of
endometrial angiogenesis and glandular proliferation in the control
due to different phases of the estrous cycle.
[0115] Examination of the Uterus
[0116] The uteri of treated mice were consistently thinner and
contained fewer glands than the median of the untreated control
mice. There was also a marked reduction in the proliferation of
glandular epithelial and vascular endothelial cells. The ovaries
were similarly impacted. The endometrium of the AGM-1470 30 mg/kg
treated mice has 63% fewer glands than the control (median control
had 35.+-.3 glands/X250 field, n=8, AGM-1470 treated mice had
3.+-.2 glands/X250 field, n=6, P<0.005. See FIGS. 6A and 6B). To
assess proliferation, sections of endometrium at comparable levels
through the lumen of the uterus from treated and control mice were
stained for PCNA. There was a 68% decrease in the proliferation
rate of endometrial stromal cells (5%.+-.2, n=6, P<0.05) in
AGM-1470 treated endometrium as compared to control mice (17%.+-.3,
n=5, FIGS. 6C and 6D). Similarly there was a 67% decrease in the
percentage of proliferating glandular epithelial cells (10%.+-.3,
n=6, P<0.05) in AGM-1470 treated endometrium as compared to
control mice (30%.+-.6, n=5). There was also a 71% decrease in the
number of proliferating vessels/X630 field: (5.+-.2, n=5,
P<0.001). Immunohistochemical staining using antibodies to
Factor VIII (an endothelial cell specific marker), demonstrated a
41% decrease in the microvessel density in AGM-1470 treated mice
versus control (17.+-.2, n=6). See FIGS. 6E and 6F. The apoptotic
index measured by Apo-Tag staining was low in both groups and not
significantly different.
[0117] Examination of the Ovaries
[0118] Histologic examination of the ovaries in AGM-1470 30 mg/kg
treated mice revealed the presence of Graafian follicles 20 and
corpora lutea. However, morphometric analysis revealed that the
corpora lutea were fewer and 61% smaller in treated mice (mean
cross sectional are of 0.038 mm.sup.2.+-.0.006, N=7, P<0.05)
when compared with control (0.097 mm.sup.2.+-.0.014, n=27)
demonstrating that the luteal growth was limited.
[0119] FIGS. 3A-3D show ovaries and uteri from control and AGM-1470
chronically treated mice. FIG. 3A shows tissue from control animal
at 2.5.times. magnification. FIG. 3B shows tissue from AGM-1470
treated animal at 2.5.times. magnification. Note the decreased size
and number of corpora lutea (small arrows) and number of
endometrial glands (large arrows) in treated mice. FIG. 3C shows a
close up of ovary section from FIG. 1A at 10.times. magnification
showing corpora lutea (large arrows) and small antral follicles.
FIG. 3D shows a close up of ovary from FIG. 1B at 10.times.
magnification showing regressed corpora lutea and thin layer of
granulosa cells in large antral follicles (small arrows).
[0120] Many corpora lutea in the treated mice were irregular in
shape and almost completely regressed. These regressed corpora
luteas made the remnants of ovulations occurring at the start of,
or prior to, treatment. The ovaries of AGM-1470 treated mice also
appeared to contain more antral follicles, but many of these
follicles contained fewer than expected layers of granulosa cells
suggesting that they are unhealthy.
EXAMPLE 9
[0121] Length of Time Between Application of AGM-1470 and
Insemination Allowable for Maintained Effectiveness of AGM-1470
[0122] The experiment was designed to detect how long before mating
a single dose of AGM-1470 might be effective. Sexually mature
female mice were treated with a single dose of AGM-1470 (30 mg/kg,
s.c.) up to five days prior to mating [designated E(-5), E(-4),
E(-3), E(-2), E(-1), or E0 with the date of plugging being E0]. For
treatments on E(-5) through E(-1), group-housed female mice were
injected with AGM-1470 and then individually paired with male
breeders at varying times after injection. For E0 treatment,
females were paired with males and then plugged females were
injected. Pregnancies were monitored by maternal weights and
allowed to go to term. The contraceptive efficacy of a single dose
of AGM-1470 on each of these days, calculated by the percentage of
matings resulting in any viable deliveries decreased. The
contraceptive efficacy of a single dose of AGM-1470 on each of
these days, expressed as the percentage of matings resulting in
viable deliveries is illustrated in FIG. 8.
[0123] Effect of AGM-1470 on Number of Live Births
[0124] AGM-1470 had a dramatic effect on live births at certain
time points. On E0 and E(-1), a single dose of AGM-1470 was able to
completely block live births. Treatments on E(-2) reduced the
percentage of animals with successful pregnancies by 80%. Three to
five days prior to mating the effects of AGM-1470 appeared to have
diminished with the treated mice having litters with the same
frequency as controls. Decreased percentage of viable births in
E(-4) is probably due to the small number of matings in that group.
Experiments are ongoing to increase the number of animals in that
group. Thus, a single dose of AGM-1470 appears to have its greatest
effect when given within the first two days prior to mating. This
corresponds to treatments on proestrus, estrus and metestrus. No
differences in the number of dead births relative to controls was
seen at any time point. See FIG. 8. It should be noted that female
humans cycle on a 28 day interval, in contrast to the 4.5 day cycle
of female mice, suggesting by extrapolation that human doses may be
effective even several weeks prior to mating.
[0125] In addition, the ovaries and uteri from control and single
treatment mice were examined. Group housed female C57BL/6 mice were
treated with a single injection of AGM-1470 (30 mg/kg, s.c.) and
paired with male breeders. Mice who mated on the day after
treatment were sacrificed 6 days after mating. Ovaries and uteri
were removed en bloc, fixed and stained with hemotoxylin and eosin.
The results are demonstrated in FIG. 7. FIG. 7A shows tissue from
control animal at 2.5.times. magnification. FIG. 7B shows tissue
from AGM-1470 treated animal at 2.5.times. magnification. Thin
arrows indicate the corpora lutea..sup.1 The fat arrows indicate
implantation sites in control not seen in treated animal. Thus, the
treatment with AGM-1470 prevented implantation.
[0126] These examples provide direct evidence that an angiogenesis
inhibitor can regulate the female reproductive system, and in so
doing provides a novel method for controlling fertility.
Decidualization, placental formation, yolk sac development,
cyclical endometrial maturation, ovulation and corpora lutea growth
are angiogenesis dependent and the invention provides a novel
method for controlling fertility. Additionally, the inhibitory
effects of AGM-1470 on endometrial angiogenesis, stromal and
glandular proliferation, as well as placental formation, provides
methods for treating several pathologic reproductive processes such
as endometriosis, adenomyosis, dysfunctional uterine bleeding,
choriocarcinoma, uterine leiomyoma (fibroids), and ectopic
pregnancy.
[0127] All of the references mentioned herein are hereby
incorporated by reference in their entireties.
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* * * * *